Bioactive Secondary Metabolites from Fungi of the Genus Cytospora Ehrenb. (Ascomycota)

Cytospora is a genus of fungi belonging to the Cytosporaceae family (Sordariomycetes, Ascomycota) considered as a prolific source of specialized metabolites due to their ability to produce diverse secondary metabolites with a broad range of biological activities. Since the first chemical investigation of this genus in the 1980s, further studies have led to the isolation and structural elucidation of several bioactive compounds including cytosporones, nonanolides, macrocyclic dilactones, and terpenoids. This review summarizes, for the first time, the chemical diversity of bioactive secondary metabolites from the genus Cytospora and highlights its potential as an alternative source of secondary metabolites for pharmacological studies. Moreover, this review will serve as a basis for future investigations of compounds of this genus.


Introduction
Significant progress has been made in the control of many diseases caused by infectious microorganisms. Recurrent epidemics due to drug-resistant microorganisms and the appearance of new microbial pathogenic strains mandate the discovery of new antibiotics [1]. Fungi are growing tremendously in terms of their repertoire of biosynthetic and pharmaceutical chemistry as they greatly enrich structurally novel secondary metabolites with promising bioactivities. In their natural environment, fungi must compete for resources and it has been hypothesized that this competition likely induces the biosynthesis of secondary metabolites for defence. Only about 7% of fungi have been studied for the chemistry of secondary metabolites [2]. Among the studied fungi species, those of the genus Cytospora are also reputed to be a prolific source for the generation of structurally versatile and biologically significant metabolites. They are particularly known for the production of potent antimicrobial compounds [3,4].
To date, approximately 688 species epithets of Cytospora have been listed in Index Fungorum [8]. However, most of these species names were considered synonyms of previously
The naphthalenone derivative (36) and the dihydroisocoumarin congener (3R)-5methylmellein (37) are among the secondary metabolites obtained from Cytospora sp. isolated from the plant Ilex canariensis [47]. (3R)-5-methylmellein (37) was discovered for the first time in Fusicoccum amygdale as the main phytotoxic metabolite [48] and later widely reported from endophytic fungi, pathogenic fungi, marine-derived mangrove fungi, and the heartwood of the Fijian species Euphorbia fidjiana [47,49]. The chemical investigation of the fermentation broth of the filamentous fungus Cytospora sp. (MF 6608) isolated from leaf litter of Manilkara bidentata collected from Puerto Rico led to the discovery of cytosporic acid (38). which inhibited the strand transfer reaction of HIV-1 integrase with an IC 50 of 20 µM [3]. Cytosporacin (39), a novel antibacterial polyketide containing naphthopyranone and isochromandione moieties, was isolated from the fermentation broth of the fungus Cytospora rhizophorae obtained from the roots of two species of the mangrove tree Rhizophora mangle and R. racemosae growing in Florida by He and collaborators [50]. This compound showed moderate activity in vitro against Gram-positive bacteria. The minimum inhibitory concentrations obtained by the broth dilution method were 32 µg/mL for Staphylococcus aureus (two strains, including a methicillin-resistant strain), 16 µg/mL for Enterococcus faecium, and 32 µg/mL for Bacillus subtilis [50]. A biosynthetic 13 C-labeling experiment indicated that cytosporacin was derived from acetate origin. Two bisanthraquinones, namely cytoskyrins A (40) and B (41), were isolated from fermentation broths of the fungus Cy-tospora sp. CR200 which was obtained from a branch of Conocarpus erecta in the Guanacaste National Park, Costa Rica [21]. Cytoskyrin A exhibited potent in vitro antibacterial against Gram-positive bacteria with MICs ranging from 0.03 to 0.25 µg/mL and DNA-damaging activities (10 ng/spot), whereas cytoskyrin B was inactive in these assays. Some compounds related to cytoskyrins A and B include (+)-epicytoskyrin produced by the endophytic fungus Diaporthe sp. isolated from a tea plant [51], as well as luteoskyrin and rugulosin obtained from Penicillium islandicum and Penicillium rugulosum [52].

Benzophenones
Cytosporins A-D (46-49) are hemiterpene-conjugated phenolics with an unprecedented benzo[b] [1,5]dioxocane skeleton produced by the fungus Cytospora rhizophorae A761 derived from Morinda officinalis How (Rubiaceae) [23]. They represent the first examples of natural meroterpenoids which bear a benzo[b] [1,5]dioxocane framework embodying hemiterpene and benzophenone moieties [23]. As discussed by Liu and collaborators [23], cytosporins A-D are generated through the elaboration of a formally unified precursor that might be envisaged from the well-known fungal metabolite monodictyphenone by the installation of the densely functionalized hemiterpene nucleus through chemoselective prenylation and stereoselective dihydroxylation. They also demonstrated the intramolecular lactonization of the precursor that would directly transform it into cytosporin B, whereas cytosporin A might be accessible from the carboxylic acid reduction followed by etherification. Moreover, cytosporins C and D could be readily deduced to be generated from cytosporin A by a simple carbonyl reduction (Scheme 1) [23]. These compounds were shown inactive against the bacteria Escherichia coli and Staphylococcus aureus even at a concentration of 250 μg/mL [23].

Benzophenones
Cytosporins A-D (46-49) are hemiterpene-conjugated phenolics with an unprecedented benzo[b] [1,5]dioxocane skeleton produced by the fungus Cytospora rhizophorae A761 derived from Morinda officinalis How (Rubiaceae) [23]. They represent the first examples of natural meroterpenoids which bear a benzo[b] [1,5]dioxocane framework embodying hemiterpene and benzophenone moieties [23]. As discussed by Liu and collaborators [23], cytosporins A-D are generated through the elaboration of a formally unified precursor that might be envisaged from the well-known fungal metabolite monodictyphenone by the installation of the densely functionalized hemiterpene nucleus through chemoselective prenylation and stereoselective dihydroxylation. They also demonstrated the intramolecular lactonization of the precursor that would directly transform it into cytosporin B, whereas cytosporin A might be accessible from the carboxylic acid reduction followed by etherification. Moreover, cytosporins C and D could be readily deduced to be generated from cytosporin A by a simple carbonyl reduction (Scheme 1) [23]. These compounds were shown inactive against the bacteria Escherichia coli and Staphylococcus aureus even at a concentration of 250 µg/mL [23].
Further investigation of Cytospora rhizophorae A761 led to the discovery of four novel polyketide heterodimers sharing an unprecedented 6/6/5/6/8 or 6/6/5/6/7 pentacyclic ring system and fused as a fascinating cage-like skeleton, embodying a polyoxygenated isopentyl unit and a highly structure-combined benzophenone scaffold trivially named cytorhizins A-D (50-53) [24]. Cytorhizins B and D showed weak cytotoxic activity against HepG-2, MCF-7, SF-268, and A549 cell lines with IC 50 values ranging from 29.4 to 68.6 µM [24]. However, cytorhizins A-D were found to be devoid of any significant antimicrobial activity even at a concentration of 100 µM [24]. Inspired by their structural characteristics, a hypothetical biogenetic pathway embracing intriguing aldol condensation and dihydroxylation/selective ketalization cascade reaction as key biosynthetic steps was proposed as shown in Scheme 2 [24]. Further investigation of Cytospora rhizophorae A761 led to the discovery of four novel polyketide heterodimers sharing an unprecedented 6/6/5/6/8 or 6/6/5/6/7 pentacyclic ring system and fused as a fascinating cage-like skeleton, embodying a polyoxygenated isopentyl unit and a highly structure-combined benzophenone scaffold trivially named cytorhizins A-D (50-53) [24]. Cytorhizins B and D showed weak cytotoxic activity against HepG-2, MCF-7, SF-268, and A549 cell lines with IC50 values ranging from 29.4 to 68.6 μM [24]. However, cytorhizins A-D were found to be devoid of any significant antimicrobial activity even at a concentration of 100 μM [24]. Inspired by their structural characteristics, a hypothetical biogenetic pathway embracing intriguing aldol condensation and dihydroxylation/selective ketalization cascade reaction as key biosynthetic steps was proposed as shown in Scheme 2 [24].

Scheme 1. Proposed biogenetic pathway of cytosporins A-D [23].
Two axially chiral benzophenones featuring an epoxy isopentyl unit and a propionyl moiety, rhizophols A (54) and B (55), were among the metabolites produced by Cytospora rhizophorae A761 [25]. Rhizophol B was disclosed as a pair of inseparable atropisomers (4:3, 1 H NMR integration) in acetone-d 6 . Moreover, temperature-dependent NMR experiments, crystal X-ray diffraction, and quantum molecular mechanical calculation were conducted to illustrate the existence of the rapidly interconverting diastereoisomers and the absolute structure [25]. Rhizophol A was proved to be a promising lead compound for novel antioxidant drug development with an IC 50 of 13.07 ± 0.94 µM and no cytotoxicity at 100 µM (DPPH radical scavenging) [25].
A pair of novel enantiomeric benzophenone-hemiterpene adducts, (+)-cytorhizophin A (56a) and (−)-cytorhizophin A (56b), together with cytorhizophin B (57) and their biosynthetically related precursor cytorhizophin C (58), were obtained from Cytospora rhizophorae A761 [26] (Figure 5). These compounds were evaluated for their antimicrobial activities against the bacteria Escherichia coli and Staphylococcus aureus and were found to be devoid of significant activity even at a concentration of 100 µg/mL [26]. 9  Two axially chiral benzophenones featuring an epoxy isopentyl unit and a propionyl moiety, rhizophols A (54) and B (55), were among the metabolites produced by Cytospora rhizophorae A761 [25]. Rhizophol B was disclosed as a pair of inseparable atropisomers (4:3, 1 H NMR integration) in acetone-d6. Moreover, temperature-dependent NMR experiments, crystal X-ray diffraction, and quantum molecular mechanical calculation were conducted to illustrate the existence of the rapidly interconverting diastereoisomers and the absolute structure [25]. Rhizophol A was proved to be a promising lead compound for novel antioxidant drug development with an IC50 of 13.07 ± 0.94 μM and no cytotoxicity at 100 μM (DPPH radical scavenging) [25].

Nonanolides and Macrocyclic Dilactones
Naturally occurring nonanolides are a large class of secondary metabolites with an interesting 10-membered macrolide subunit. Metabolites of the nonanolide family can be roughly divided into two groups according to the structural features of the side chain: simple nonanolides with a methyl group at C-9 and nonanolides with extended alkyl chains at C-9. Structurally complex nonanolides with additional rings are sometimes also

Nonanolides and Macrocyclic Dilactones
Naturally occurring nonanolides are a large class of secondary metabolites with an interesting 10-membered macrolide subunit. Metabolites of the nonanolide family can be roughly divided into two groups according to the structural features of the side chain: simple nonanolides with a methyl group at C-9 and nonanolides with extended alkyl chains at C-9. Structurally complex nonanolides with additional rings are sometimes also included in the family [27].
Cytospolides A-E (59-63) possessing an unprecedented 15-carbon skeleton with the unique chemical feature of a C-2 methyl group were isolated from Cytospora sp., obtained from Ilex canariensis [27]. Their structures were elucidated by spectroscopic analysis, chemical interconversion, and single-crystal XRD. The structure of the solution and solid state conformers were compared by experimental methods (X-ray, NMR), as well as by solvent and gas phase DFT calculations [27]. The results of the cytotoxicity activity revealed that the 8-O-monoacetate and the absolute configuration at C-2 clearly play important roles in the growth inhibition of the tumour line. Further chemical investigation of this fungus led to the discovery of fourteen nonanolide derivatives named cytospolides F-Q (64-75) and decytospolides A and B (76, 77) [53]. Furan containing nonanolides 71, 72, 73, and 75 showed considerably cytotoxic activity against A-549 cells [53]. The free hydroxyl groups at C-3 and C-4 seem to play an important role in the cytotoxic activity since the inhibition remarkably decreased in 73, lacking the two secondary hydroxyl groups. The activity of decytospolide A (76) increased dramatically when the 8-OH was acetylated [53].
Grahamimycins A (78), Al (79), and B (80) are three macrocyclic dilactones obtained in crystalline form from Cytospora sp. Ehrenb isolated from Pinus contorta var. Latifolia Egelm ( Figure 6). Grahamimycin A exhibited activity against thirty-six species of bacteria, eight species of blue-green algae, and two species of green algae, as well as antifungal activity against five fungi [54]. Grahamimycin A1 was also among the macrodiolides isolated from the marine fungus Varicosporina ramulosa [55]. Grahamimycins A (78), Al (79), and B (80) are three macrocyclic dilactones obtained in crystalline form from Cytospora sp. Ehrenb isolated from Pinus contorta var. Latifolia Egelm ( Figure 6). Grahamimycin A exhibited activity against thirty-six species of bacteria, eight species of blue-green algae, and two species of green algae, as well as antifungal activity against five fungi [54]. Grahamimycin A1 was also among the macrodiolides isolated from the marine fungus Varicosporina ramulosa [55].

Terpenoids
Terpenoids represent a large and diverse bioactive class of secondary metabolites produced by various plants and fungi. Accumulating evidence on their broad spectrum of biological activities coupled with a tolerable toxicity profile has sparked renewed interest with regard to their application, especially in cancer treatment [60]. Several compounds belonging to this class were reported from Cytospora species.

Terpenoids
Terpenoids represent a large and diverse bioactive class of secondary metabolites produced by various plants and fungi. Accumulating evidence on their broad spectrum of biological activities coupled with a tolerable toxicity profile has sparked renewed interest with regard to their application, especially in cancer treatment [60]. Several compounds belonging to this class were reported from Cytospora species.
Nine caryophyllene sesquiterpenoids named punctaporonins N-S (111-116) and 6hydroxypunctaporonins B (117) [63]. Five other caryophyllene sesquiterpenoids named cytosporinols A-C (120-122) were previously isolated from solid cultures of the same fungal strain by Li and collaborators [28] (Figure 8). The structures of cytosporinols A-C were elucidated by NMR spectroscopy, and the absolute configurations of the C-11 secondary alcohol in cytosporinol A and the 6,8-diol moiety in cytosporinol C were deduced using the modified Mosher and Snatzke's method [28]. Furthermore, the configuration of cytosporinol C was confirmed by a single crystal X-ray crystallographic analysis. These compounds were evaluated for their cytotoxic activities. Cytosporinols B (121) and C (122) showed moderate cytotoxicity against HeLa (cervical epithelium) cells, with IC 50 values of 16.5 and 21.1 µM, respectively, while the positive control cisplatin showed an IC 50 value of 7.6 µM [28].
Although the compounds described in this review possessed very interesting biological properties, they have been isolated in very small amounts. However, the development of drugs requires large quantities of lead compounds, which can be obtained by fermentation. Nevertheless, some secondary metabolites isolated from fungi of the genus Cytospora have already been synthesized by organic chemists, as summarized in Table 1 below. Table 1. Synthesis of some secondary metabolites produced by fungi of the genus Cytospora.
Although the compounds described in this review possessed very interesting biological properties, they have been isolated in very small amounts. However, the development of drugs requires large quantities of lead compounds, which can be obtained by fermentation. Nevertheless, some secondary metabolites isolated from fungi of the genus Cytospora have already been synthesized by organic chemists, as summarized in Table 1 below.

Conclusions and Recommendations
The present review indicates 122 compounds consisting mainly of xanthones, quinones, coumarins, benzophenones, macrolides, and terpenoids isolated from fungi of the genus Cytospora. These secondary metabolites were evaluated in most cases for their antimicrobial and cytotoxic activities, and several of these compounds were found to be very effective. Hence, these fungi represent an important source of bioactive compounds that could be used as leads in the development of antibiotics and anticancer drugs. Surprisingly, most of the studied Cytospora species were not fully identified. Taking into consideration their diversity and richness in the production of bioactive secondary metabolites, it is recommended that mycologists pay more attention to the taxonomy of species of this genus, which will certainly lead to the discovery of several new strains and to the efficient exploration of their biotechnological potentials. Moreover, a more diligent search on the occurrence of the reported compounds in other plant pathogenic fungi should be conducted as this may give a more realistic picture on the ecological function of these compounds. Additional studies should also be carried out on this genus using omics and co-culturing strategies to shed more light on the bioactive compounds species that this genus could produce.

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