**1. Introduction**

Nature has substantially participated in the discovery of drugs for human remedial treatments since the beginning of mankind [1]. The marine environment, with more than 70% of the surface of the Earth, represents the largest ecosystem and is characterized by quite variable physicochemical parameters (e.g., limited light access, low temperature, high pressure, and high salinity) [2]. Among the various marine microbes, fungi are a superabundant and ecologically substantial component of marine microbiota [3]. Fungi are one of nature's treasures that inhabit various environments on the earth's surface, including the marine environment [4–7]. They play a growing relevant role in drug development and biomedicine research, either directly as drugs or indirectly as lead structures for bio-inspired drug synthesis [8–12]. In the last decades, natural product chemists and pharmacologists have turned their research interests to marine-derived fungi, which are renowned as a vast unexploited reservoir of metabolic diverseness and found to have the capability to produce structurally unique bio-metabolites [6,7,12–16]. Furthermore, research on fungiderived metabolites has tremendously increased because of the need for compounds with potential economical values and pharmaceutical applications. Sesquiterpenes belonging to various classes, including hirsutane, alliacane, tremulane, bergamotane, drimane, etc.,

**Citation:** Khayat, M.T.; Mohammad, K.A.; Omar, A.M.; Mohamed, G.A.; Ibrahim, S.R.M. Fungal Bergamotane Sesquiterpenoids—Potential Metabolites: Sources, Bioactivities, and Biosynthesis. *Mar. Drugs* **2022**, *20*, 771. https://doi.org/10.3390/ md20120771

Academic Editor: Dehai Li

Received: 5 November 2022 Accepted: 5 December 2022 Published: 8 December 2022

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**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/).

are reported from fungi [17–19]. The biosynthesis of their C15 skeleton from FPP (farnesyl pyrophosphate) was catalyzed by sesquiterpene synthases [19,20].

Among these metabolites, the bergamotane family represents an uncommon class of natural sesquiterpenes that includes bi-, tri-, or tetracyclic derivatives [19]. Bergamotane sesquiterpenoids having a bridged 6/4 bicyclic skeleton involved in an isopentyl unit are biosynthesized by fungi and plants [21,22]. Interestingly, polyoxygenated derivatives featuring a 6/4/5/5 tetracyclic framework represent a rare class of natural metabolites, and all polycyclic bergamotanes are mainly encountered in fungi [23–26]. Bergamotane sesquiterpenoids have been reported from various marine sources such as sponges, sea mud, deep-sea hydrothermal sulfide deposits, and sea sediments. These metabolites could gain the interest of chemists and biologists because of their unusual structural features and diversified activities, such as phytotoxicity, plant growth regulation, antimicrobial, anti-HIV, cytotoxic, pancreatic lipase inhibition, immunosuppressive, antidiabetic, and anti-inflammatory properties. It is noteworthy that no available work has addressed this class of sesquiterpenes in term of their sources, bioactivities, and biosynthesis. In the current work, the reported fungal bergamotane sesquiterpenoids ranging from 1958 to June 2022 have been listed. They have been classified according to their ring system, i.e., into bi-, tri-, or tetracyclic derivatives (Table 1). Additionally, their fungal sources, structural characterization, biosynthesis, and biological relevance have been provided. Moreover, some of their reported structural characteristics and methods of separation and characterization, as well as their structure–activity relation, are discussed.

**Table 1.** Naturally occurring fungal bergamotane sesquiterpenoids (name, source, extract/fraction, molecular weights and formulae, and location).





#### **Compound Name Fungal Source/Host Extract/Fraction Mol. Wt. Mol. Formula Location Ref.** Brasilamide B (**46**) *Paraconiothyrium brasiliense* Verkley (M3–3341) (Leptosphaeriaceae)/ *Acer truncatum* Bunge (branches, Sapindaceae) Acetone extract 265 C15H23NO3 Dongling Mountain, Beijing, China [43] Brasilamide C (**47**) *Paraconiothyrium brasiliense* Verkley (M3–3341) (Leptosphaeriaceae)/ *Acer truncatum* Bunge (branches, Sapindaceae) Acetone extract 279 C15H21NO4 Dongling Mountain, Beijing, China [43,44] Brasilamide D (**48**) *Paraconiothyrium brasiliense* Verkley (M3–3341) (Leptosphaeriaceae)/ *Acer truncatum* Bunge (branches, Sapindaceae) Acetone extract 321 C17H23NO5 Dongling Mountain, Beijing, China [43] Brasilamide K (**49**) *Paraconiothyrium brasiliense* Verkley (M3–3341) (Leptosphaeriaceae)/ *Acer truncatum* Bunge (branches, Sapindaceae) EtOAc extract 279 C15H21NO4 Dongling Mountain, Beijing, China [44] Brasilamide L (**50**) *Paraconiothyrium brasiliense* Verkley (M3–3341) (Leptosphaeriaceae)/ *Acer truncatum* Bunge (branches, Sapindaceae) EtOAc extract 265 C15H23NO3 Dongling Mountain, Beijing, China [44] Brasilamide M (**51**) *Paraconiothyrium brasiliense* Verkley (M3–3341) (Leptosphaeriaceae)/ *Acer truncatum* Bunge (branches, Sapindaceae) EtOAc extract 293 C15H19NO5 Dongling Mountain, Beijing, China, [44] Brasilamide N (**52**) *Paraconiothyrium brasiliense* Verkley (M3–3341) (Leptosphaeriaceae)/ *Acer truncatum* Bunge (branches, Sapindaceae) EtOAc extract 279 C15H21NO4 Dongling Mountain, Beijing, China [44] Craterodoratin I (**53**) *Craterellus odoratus* (Cantharellaceae) EtOAc extract 250 C15H22O3 Southern part of the Gaoligong-Mountains, Yunnan, China [30] Craterodoratin J (**54**) *Craterellus odoratus* (Cantharellaceae) EtOAc extract 282 C15H22O5 Southern part of the Gaoligong-Mountains, Yunnan, China [30] Craterodoratin K (**55**) *Craterellus odoratus* (Cantharellaceae) EtOAc extract 282 C15H22O5 Southern part of the Gaoligong-Mountains, Yunnan, China [30] Craterodoratin L (**56**) *Craterellus odoratus* (Cantharellaceae) EtOAc extract 278 C15H18O5 Southern part of the Gaoligong-Mountains, Yunnan, China [30]





Surveying their bioactivities may open a new research area for the synthesis of new agents from these metabolites by synthetic and medicinal chemists. The literature search for the reported data was performed using diverse databases and publishers, including Web of Science, Google Scholar, PubMed, Scopus, SciFinder, Wiley, SpringerLink, and ACS Publications, using specific keywords (bergamotane, marine, fungi, biosynthesis, and biological activities).

#### **2. Structural Assignment and Stereochemistry Determination**

A total of 97 metabolites have been separated from various fungal source extracts using different chromatographic techniques and characterized by NMR, MS, and IR spectral analyses as well as chemical derivatization. The relative configuration of these metabolites was established using NOESY or ROESY spectral analyses. Various studies reported the assigning of their absolute stereochemistry using total synthesis [53,54], Mosher's method [26], X-ray diffraction, chemical conversion [34,43,55], and ECD analyses [31]. The reported metabolites have been categorized into bi-, tri-, and tetracyclic derivatives.

#### **3. Biological Activities of Bergamotane Sesquiterpenoids**

Various reported studies revealed the assessment of bergamotane sesquiterpenoids for diverse bioactivities, including plant growth regulation, phototoxic, antimicrobial, anti-HIV, cytotoxic, pancreatic lipase inhibition, antidiabetic, anti-inflammatory, and immunosuppressive, which were summarized in this work (Table 2). Additionally, the reported structure–activity relation was included.





### *3.1. Anti-Inflammatory Activity*

NO (nitric oxide) is a substantial pro-inflammatory mediator, and its excessive production is accompanied with various inflammatory illnesses; therefore, it possesses a remarkable role for regulating immune responses and inflammation [56]. NO production inhibitors may represent the potential capacity for treating various inflammatory disorders. Thus, further research for fungal metabolites must be conducted to discover novel anti-inflammation agents.

The epigenetic chemical manipulation of *Eutypella* sp. derived from deep-sea hydrothermal sulfide deposit by co-treatment with SBHA (histonedeacetylase inhibitor, suberohydroxamic acid) and 5-Aza (DNA methyltransferase inhibitor, 5-azacytidine) was shown to activate a biosynthetic sesquiterpene-linked gene cluster [35]. From elicitortreated cultures EtOAc extract, eutypeterpenes A–F (**18**–**22** and **28**) along with xylariterpenoids A (**16**) and B (**17**) were purified using SiO2/RP-18/HPLC that were identified by spectral analyses, as well as by using chemical conversion, X-ray diffraction, ECD, and calculated NMR for configuration assignments.

Eutypeterpene A (**28**) is the first bergamotene sesquiterpene incorporating a dioxolanone moiety. These metabolites were assessed for their NO production inhibitory capacity induced by LPS-(lipopolysaccharide) in RAW 264.7 macrophages [35]. The results indicated thatcompound **18** and **19** (IC50 13.4 and 16.8 μM, respectively) displayed more effectiveness than quercetin (IC50 of 17.0 μM), whereas other metabolites had noticeable potentials (IC50 values ranged from 18.7 to 24.3 μM) with weak cytotoxic capacities (IC50 > 100 μM). A structure–activity study revealed that the analog with a triol unit (**18**) at the side chain was more effective than compound **16**, **17**, and **19** with a diol unit, which were more potent than compound **20**, **21**, and **28** with one hydroxy group. Furthermore, the α,β-unsaturated ketone unit (as in compound **21** and **22**) and the OH-linked carbon configuration also affected the activities (**16** versus **17**) [35] (Figure 1).

**Figure 1.** Structures of bicyclic bergamotane sesquiterpenoids (**1**–**17**).

Biogenetically, compounds **18**–**22** are derived from FPP that performs a 1,6-cyclization to produce bisabolane (**A**). The 4,7-cyclization of **A** generates bergamotane (**B**), which further generates **18**–**22** via diverse oxidation and reduction processes. Additionally, compound **28** is formed from **18** by carbonate incorporation [35] (Scheme 1).

The deep-sea-isolated *Graphostroma* sp. MCCC3A00421 associated with the Atlantic Ocean hydrothermal sulfide deposits biosynthesized new bergamotane sesquiterpenoids: (10S)-xylariterpenoid A (**23**), (10R)-xylariterpenoid B (**24**), xylariterpenoid E (**25**), xylariterpenoid F (**26**), and xylariterpenoid G (**27**), which were purified using SiO2/OSD/Sephadex LH-20/RP-18 CC and preparative TLC. They were characterized by extensive spectral data, and their absolute configuration was established by ECD, Cu-Ka-single-crystal X-ray diffraction, and modified Mosher's method analyses. Compound **25** is trinor-bergamotane. Compounds **23**, **26**, and **27** revealed moderate inhibition potentials (IC50s of 86, 85, and

85 μM, respectively) of NO production in LPS-stimulated RAW264.7 macrophages compared with aminoguanidine (IC50 of 23 μM). It was noted that bergamotane moiety's 10S configuration obviously boosted the activity as in compound **23** (10S, IC50 of 85 μM) versus compound **24** (10R, of IC50 230 μM) (Figure 2) [34].

**Scheme 1.** Biosynthetic pathway of eutypeterpenes A–F (compounds **18**–**22** and **28**) [35].
