A Review of Fungal Protoilludane Sesquiterpenoid Natural Products

antibiotics

Review A Review of Fungal Protoilludane Sesquiterpenoid Natural Products

Melissa M. Cadelis 1,2,* , Brent R. Copp 1 and Siouxsie Wiles 2

1 School of Chemical Sciences, University of Auckland, 23 Symonds Street, Auckland 1010, New Zealand; [email protected] 2 Bioluminescent Superbugs Lab, School of Medical Sciences, University of Auckland, 85 Park Road, Grafton, Auckland 1023, New Zealand; [email protected] * Correspondence: [email protected]

Received: 1 December 2020; Accepted: 18 December 2020; Published: 19 December 2020

Abstract: Natural products have been a great source for drug leads, due to a vast majority possessing unique chemical structures. Such an example is the protoilludane class of natural products which contain an annulated 5/6/4-ring system and are almost exclusively produced by fungi. They have been reported to possess a diverse range of bioactivities, including antimicrobial, antifungal and cytotoxic properties. In this review, we discuss the isolation, structure elucidation and any reported bioactivities of this compound class, including establishment of stereochemistry and any total syntheses of these natural products. A total of 180 protoilludane natural products, isolated in the last 70 years, from fungi, plant and marine sources are covered, highlighting their structural diversity and potential in drug discovery.

Keywords: sesquiterpenoid; natural products; protoilludanes

1. Introduction Fungi remain an untapped source of bioactive natural products with an estimate of over 1.5 million species of fungi worldwide, of which only approximately 7% have been chemically investigated [1–6]. Over 90% of the recorded species are higher fungi which are considered one of the largest biodiverse resources on Earth. With the development of fungal natural product isolation techniques and a better understanding of biosynthetic pathways, higher fungi have become an abundant source for drug discovery. Ascomycota, the largest phylum of fungi, is one of the two divisions of higher fungi along with Basidiomycota. Both divisions of higher fungi, having undergone evolution to survive harsh and unfavourable environments, are well known producers of an array of structurally diverse natural products that possess a variety of bioactivities. These natural products can be divided into several groups by their chemical structures, one of which are the terpenoids. Representative terpenoid scaffolds in higher fungi include sesquiterpenoids, diterpenoids and triterpenoids which are all derived from common precursors [1–6]. Fungal terpenoid natural products are derived from isoprene units (C5) and are classified by the number of isoprene units in the structure [7–11]. Higher terpenoids such as monoterpenoids (C10), sesquiterpenoids (C15) and diterpenoids (C20) are synthesised from two isoprene intermediates, dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), in the mevalonate pathway. The mevalonate pathway begins with the phosphorylation of mevalonic acid, derived from acetyl-CoA, by two molecules of adenosine triphosphate (ATP). Subsequent decarboxylation forms IPP, the stereospecific isomerisation of which affords DMAPP (Scheme1). Loss of diphosphate from DMAPP forms an allylic cation which undergoes nucleophilic attack by IPP to afford geranyl diphosphate. Reaction of geranyl diphosphate with another unit of IPP gives farnesyl diphosphate

Antibiotics 2020, 9, 928; doi:10.3390/antibiotics9120928 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, 928 2 of 29

Antibiotics 2020, 9, 928 2 of 29 from which sesquiterpenoids (C15) are derived. Repetition of this process results in the formation of terpenoidfrom which chains sesquiterpenoids of different (C length.15) are derived. Linear terpenoidsRepetition of can this undergo process results cyclisation in the formation by a number of of terpenoid chains of different length. Linear terpenoids can undergo cyclisation by a number of different terpene cyclases which can generate numerous terpenoid scaffolds [7–11]. different terpene cyclases which can generate numerous terpenoid scaffolds [7–11].

SchemeScheme 1. Biosynthesis 1. Biosynthesis of sesquiterpenoidsof sesquiterpenoids from from isopentenylisopentenyl pyrophosphate pyrophosphate (IPP) (IPP) and and dimethylallyl dimethylallyl pyrophosphate (DMAPP) through the mevalonate pathway. pyrophosphate (DMAPP) through the mevalonate pathway. Cyclisation of farnesyl diphosphate can produce a wide range of natural products, many of Cyclisation of farnesyl diphosphate can produce a wide range of natural products, many of which are biosynthesised through a cyclisation pathway involving a humulyl cation [12–15]. The which are biosynthesised through a cyclisation pathway involving a humulyl cation [12–15]. humulene pathway proceeds through 1,11-cyclisation of 2E,6E-farnesyl diphosphate to afford the The humulenetrans-humulyl pathway cation proceedswhich can throughthen undergo 1,11-cyclisation rearrangement of to 2E produce,6E-farnesyl a number diphosphate of different to classes afford the trans-humulylof sesquiterpenoid cation which carbon can skeletons then undergo including rearrangement α-humulene and to produce (E)-β-caryophyllene. a number of Of di ffinteresterent classesis of sesquiterpenoidthe 2,9 and 3,6-cyclisation carbon skeletons product, protoilludyl including α cation,-humulene the precursor and (E to)- βvarious-caryophyllene. protoilludane Of natural interest is the 2,9products and 3,6-cyclisation [12–15]. product, protoilludyl cation, the precursor to various protoilludane natural products [Sesquiterpenoid12–15]. protoilludane natural products contain a characteristic annulated 5/6/4-ring Sesquiterpenoidsystem and have protoilludanebeen reported naturalto possess products a diverse contain range aof characteristic bioactivities. annulatedThey have 5been/6/4-ring systempredominantly and have been isolated reported from to higher possess fungi a diverse (96%) with range very of bioactivities. few examples Theyfrom marine have been (1%) predominantly and plant (3%) sources (Figure 1). A total of 180 examples of this class have been isolated over a span of 70 years isolated from higher fungi (96%) with very few examples from marine (1%) and plant (3%) sources with almost half of these natural products being isolated in the last 20 years (Figure 2). (Figure1). A total of 180 examples of this class have been isolated over a span of 70 years with almost The purpose of this review is to give an overview of sesquiterpenoid protoilludane natural half ofproducts these natural bearing products an annulated being 5/6/4-ring isolated system in the and last does 20 years not cover (Figure the 2structurally). related illudins Thewhich purpose are derived of this from review protoilludane is to give precursors. an overview In this review, of sesquiterpenoid we will first discuss protoilludane the isolation, natural productsstructure bearing elucidation, an annulated establishment 5/6/4-ring of stereo systemchemistry and does and notsummarise cover thethe structurallybioactivities relatedof illudinssesquiterpenoid which are derivedprotoilludan frome natural protoilludane products isolated precursors. to date Infrom this fungal review, origins we beginning will first with discuss the isolation,those isolated structure from elucidation, Basidiomycota establishment followed ofby stereochemistry Ascomycota. We and will summarise then briefly the bioactivities cover of sesquiterpenoidprotoilludanes protoilludaneisolated from other natural sources. products We isolatedwill acknowledge to date from any fungalknown originssyntheses beginning of these with thosenatural isolated products from Basidiomycota but will not elaborate followed on bythese, Ascomycota. as a comprehensive We will overview then briefly of synthetic cover protoilludanes attempts towards this class of natural products has been reported previously [14]. A short discussion on the isolated from other sources. We will acknowledge any known syntheses of these natural products but biosynthetic studies conducted on these natural products has also been included. will not elaborate on these, as a comprehensive overview of synthetic attempts towards this class of natural products has been reported previously [14]. A short discussion on the biosynthetic studies conducted on these natural products has also been included. Antibiotics 2020, 9, 928 3 of 29 AntibioticsAntibiotics 2020 2020, 9,, 9928, 928 3 of3 of29 29

Figure 1. Isolation sources of protoilludane natural products. FigureFigure 1. 1.Isolation Isolation sources sources of of protoilludane protoilludane natural natural products. products.

FigureFigureFigure 2. Timeline2. 2.Timeline Timeline of of of thethe the discovery discovery discovery of of ofprotoilludane protoilludane protoilludane na naturaltural natural products products products from from marine, marine, from plantmarine, plant and and fungal plant fungal and fungalsources. sources.

2. Basidiomycota2.2. Basidiomycota Basidiomycota The BasidiomycotaTheThe Basidiomycota Basidiomycota account account account for for for approximately approximately approximately 30% 30% of of of the the the described describeddescribed species species species of of higher of higher higher fungi fungi fungi and and and includeincludeinclude the smuts, the the smuts, smuts, rusts, rusts, rusts, jelly jelly jelly fungi, fungi, fungi, bracket bracket bracket fungi,fungi, fungi, stinkhorns, stinkhorns, fairy fairyfairy clubs, clubs, clubs, bird’s bird’s bird’s nest nest nest fungi, fungi, fungi, puffballs, puffballs, puﬀballs, earthstars, toadstools and mushrooms [12–14]. This group of fungi are well-known producers of a earthstars,earthstars, toadstools toadstools and and mushrooms mushroom [12s [12–14].–14]. This This group group ofof fungifungi are are well-known well-known producers producers of a of a wide range of terpenoid natural products, including protoilludane sesquiterpenoids [12–14]. Indeed, wide rangewide range of terpenoid of terpenoid natural natural products, products, including including protoilludane protoilludane sesquiterpenoids [12–14]. [12–14 Indeed,]. Indeed, BasidiomycotaBasidiomycota account account for for over over 82% 82% of of the the protoi protoilludaneslludanes produced produced by by higher higher fungi fungi (Figure (Figure 1). 1). Basidiomycota account for over 82% of the protoilludanes produced by higher fungi (Figure1). 2.1.2.1. Protoilludane Protoilludane Sesquiterpenes Sesquiterpenes 2.1. Protoilludane Sesquiterpenes TheThe earliest earliest reported reported protoillud protoilludaneane sesquiterpenoid, sesquiterpenoid, with with the the fused fused 5/6/4-ring 5/6/4-ring system, system, was was Theilludolilludol earliest (1 ()1 (Figure) (Figure reported 3) 3) [16]. [16]. protoilludane It Itwas was is olatedisolated sesquiterpenoid, from from the the mushroom mushroom with Omphalotus theOmphalotus fused 5olearius,/ 6olearius,/4-ring formerly formerly system, Clitocybe Clitocybe was illudol (1) (Figureilludensilludens3)[, in, 16in 1950 ].1950 It and was and reported isolatedreported as fromas an an unknown theunknown mushroom natural natural Omphalotusproduct product at at the the olearius, time. time. Illudol formerlyIlludol (1 ()1 was) Clitocybewas tested tested illudensfor for , in 1950activityactivity and reported against against several as several an unknown bacterial bacterial naturalspecies species including product including at Bacillus theBacillus time. mycoides mycoides Illudol, Bacillus, (Bacillus1) was subtilis testedsubtilis, Escherichia, for Escherichia activity coli againstcoli, , severalKlebsiellaKlebsiella bacterial pneumoniae pneumoniae species including, Mycobacterium, MycobacteriumBacillus smegmatis smegmatis mycoides, ,Mycobacterium BacillusMycobacterium subtilis tuberculosis ,tuberculosisEscherichia, Pseudomonas, Pseudomonas coli, Klebsiella aeruginosa aeruginosa pneumoniae , Mycobacterium smegmatis, Mycobacterium tuberculosis, Pseudomonas aeruginosa and Staphylococcus aureus but was found to be inactive [16]. The planar structure of illudol (1) was not reported until 1967, Antibiotics 2020, 9, 928 4 of 29 and Staphylococcus aureus but was found to be inactive [16]. The planar structure of illudol (1) was not reported until 1967, while the absolute stereochemistry was only established in 1971 based on X-ray crystallography and was then successfully synthesised as a racemic mixture in the same year [17–19]. Three other protoilludanes (Figure 3) have since been isolated from the Clitocybe genus including neoilludol (2) from C. illudens and 3-epi-illudol (3) and 1-O-acetyl-3-epi-illudol (4) from C. candicans [20,21]. Determination of the relative stereochemistry of neoilludol (2) was achieved through nuclear Overhauser effect spectroscopy (NOESY) analysis and like illudol (1), the natural product exhibited no antimicrobial activity against either S. aureus NBRC13276 or P. aeruginosa ATCC15442 at a loading of 100 µg per disk [20,22]. The absolute stereochemistry of 3-epi-illudol (3) was established using the partial resolution method of Horeau on an acetonide derivative and analysis of the electronic circular

Antibioticsdichroism2020 ,(ECD)9, 928 spectrum of 1-O-pivaloyl-3,6-dinitrobenzoyl derivative [21]. The relative4 of 29 stereochemistry of 1-O-acetyl-3-epi-illudol (4) was then established by consideration of shared biogenetic origin with 3 [21]. The latter was reported to exhibit antimicrobial activity against E. coli whileATCC25922 the absolute with stereochemistrya minimum inhibitory was only concentration established in (MIC) 1971 basedof 32 onµg/mL X-ray and crystallography a total synthesis and wastowards then the successfully racemic mixture synthesised of the as natural a racemic product mixture was in reported the same in year 1997 [17 [23,24].–19].

FigureFigure 3.3. StructuresStructures ofof protoilludanesprotoilludanes 11––66 isolatedisolated fromfrom thethe ClitocybeClitocybeand and OmphalotusOmphalotusgenera genera ofof fungi.fungi.

Three other protoilludanes (Figure3) have since been isolated from the Clitocybe genus Another protoilludane, illudiolone (5) was isolated from a mushroom, Omphalotus illudens [25]. including neoilludol (2) from C. illudens and 3-epi-illudol (3) and 1-O-acetyl-3-epi-illudol (4) from X-ray crystallographic analysis of 5 allowed assignment of the structure and absolute configuration C. candicans [20,21]. Determination of the relative stereochemistry of neoilludol (2) was achieved [25]. A related protoilludane, 5-O-acetyl-7,14-dihydroxy-protoilludanol (6), was isolated from through nuclear Overhauser effect spectroscopy (NOESY) analysis and like illudol (1), the natural Conocybe siliginea, the structure and relative configuration of which were assigned using rotating- S. aureus P. aeruginosa productframe nuclear exhibited Overhauser no antimicrobial effect spectroscopy activity against (ROESY) either spectroscopic dataNBRC13276 [26]. The orcytotoxicity of 6 ATCC15442 at a loading of 100 µg per disk [20,22]. The absolute stereochemistry of 3-epi-illudol (3) was investigated against several human cancer cell lines including SK-BR-3, SMMC-7721, HL-60, was established using the partial resolution method of Horeau on an acetonide derivative and analysis PANC-1 and A-549 and was found to be non-cytotoxic [26]. O of theTwo electronic examples circular of less dichroism oxygenated (ECD) protoilludanes spectrum of 1- (Figure-pivaloyl-3,6-dinitrobenzoyl 4) were isolated from derivativethe mycelia [21 of]. The relative stereochemistry of 1-O-acetyl-3-epi-illudol (4) was then established by consideration of Fomitopsis insularis, Δ6-protoilludene (7) and Δ7-protoilludene-6-ol (8), of which the former has also shared biogenetic origin with 3 [21]. The latter was reported to exhibit antimicrobial activity against been isolated from O. olearius [27,28]. In the isolation reports of Δ6-protoilludene, the sesquiterpenoid E. coli ATCC25922 with a minimum inhibitory concentration (MIC) of 32 µg/mL and a total synthesis was shown to undergo isomerisation upon reaction with palladium on carbon to afford Δ7- towards the racemic mixture of the natural product was reported in 1997 [23,24]. protoilludene (9) [27]. Though Δ7-protoilludene (9) was only a semi-synthetic derivative at the time, 5 Omphalotus illudens it wasAnother the first protoilludane, of the three protoilludanes illudiolone ( ) wasto be isolated synthesised, from aalbeit mushroom, racemically [29]. Soon after,[ 25the]. X-ray crystallographic analysis of 5 allowed assignment of the structure and absolute synthesis of racemic Δ6-protoilludene (7) was achieved which allowed the relative stereochemistry of O 6 configurationthe natural product [25]. A to related be established protoilludane, [28]. 5-Recent-acetyl-7,14-dihydroxy-protoilludanol reports finally identified Δ7-protoilludene ( ), was isolated (9) as a Conocybe siliginea fromnatural product, isolated, thefrom structure slime mold and Dictyostelium relative configuration discoideum [30]. of which The relative were assignedstereochemistry using rotating-frameof 9 was established nuclear Overhauser by NOESY eff ectspectroscopic spectroscopy analysis (ROESY) spectroscopicwith absolute data stereochemistry [26]. The cytotoxicity being of 6 was investigated against several human cancer cell lines including SK-BR-3, SMMC-7721, HL-60, established by conversion of chirally pure deuterium labelled farnesyl diphosphate to Δ7- PANC-1protoilludene and A-549 (9) [30]. and wasAnother found slime to be mold non-cytotoxic from the[ 26Dictyostelium]. genus, D. mucoroides afforded mucoroidiolTwo examples (10), the of relative less oxygenated configuration protoilludanes of which was (Figure assigned4) were by analysis isolated of from NOESY the mycelia data, while of Fomitopsis insularis, ∆6-protoilludene (7) and ∆7-protoilludene-6-ol (8), of which the former has also been isolated from O. olearius [27,28]. In the isolation reports of ∆6-protoilludene, the sesquiterpenoid was shown to undergo isomerisation upon reaction with palladium on carbon to afford ∆7-protoilludene (9)[27]. Though ∆7-protoilludene (9) was only a semi-synthetic derivative at the time, it was the first of the three protoilludanes to be synthesised, albeit racemically [29]. Soon after, the synthesis of racemic ∆6-protoilludene (7) was achieved which allowed the relative stereochemistry of the natural product to be established [28]. Recent reports finally identified ∆7-protoilludene (9) as a natural product, isolated from slime mold Dictyostelium discoideum [30]. The relative stereochemistry of 9 was established by NOESY spectroscopic analysis with absolute stereochemistry being established by conversion of chirally pure deuterium labelled farnesyl diphosphate to ∆7-protoilludene (9)[30]. Another slime mold from the Dictyostelium genus, D. mucoroides afforded mucoroidiol (10), the relative Antibiotics 2020, 9, 928 5 of 29

Antibiotics 2020, 9, 928 5 of 29 conﬁguration of which was assigned by analysis of NOESY data, while the absolute was suggested Antibiotics 2020, 9, 928 5 of 29 by considerationthe absolute was of shared suggested biogenetic by consideration origins with of shared9 [31 ].biogenetic Mucoroidiol origins (10 with) exhibited 9 [31]. Mucoroidiol no cytotoxicity (10) exhibited no cytotoxicity (HeLa cells, IC50 > 40 µM) or antibacterial activity (S. aureus and E. coli) (HeLa cells, IC > 40 µM) or antibacterial activity (S. aureus and E. coli)[31]. the[31]. absolute 50 was suggested by consideration of shared biogenetic origins with 9 [31]. Mucoroidiol (10) exhibited no cytotoxicity (HeLa cells, IC50 > 40 µM) or antibacterial activity (S. aureus and E. coli) [31].

Figure 4. Structures of protoilludanes 7–10 isolated from the Fomitopsis and Dictyostelium genera. Figure 4. Structures of protoilludanes 7–10 isolated from the Fomitopsis and Dictyostelium genera. FourFour protoilludanesFigure protoilludanes 4. Structures were of wereprotoilludanes isolated isolated from 7 –from10 the isolated mushroomthe mushroomfrom theLaurilia Fomitopsis Laurilia sulcate and sulcate Dictyostelium(Figure (Figure5), namelygenera. 5), namely sulcatine A(11sulcatine) and B (A12 (11),) armillol and B (12 (),13 armillol) and 5- (13epi) -armilloland 5-epi-armillol (14)[32 (,1433)]. [32,33]. The relativeThe relative configurations configurations of of11 –14 11–14Four were protoilludanes determined by were analysis isolated of NOESY from NMRthe mushroom data. The absolute Laurilia configurationsulcate (Figure of 5),sulcatine namely A were determined by analysis of NOESY NMR data. The absolute configuration of sulcatine A sulcatine(11) was confirmedA (11) and throughB (12), armillol analysis (13 of) andan ECD 5-epi -armillolspectrum ( 14of) a[32,33]. dibenzoate The relative derivative, configurations while that ofof (11) was confirmed through analysis of an ECD spectrum of a dibenzoate derivative, while that of 11sulcatine–14 were B (determined12) and armillol by analysis (13) were of NOESYestablished NMR by data.biogenetic The absolute consideration configuration [32,33]. ofBoth sulcatine sulcatine A sulcatine(A11 )( 11was B) (12and confirmed) andB (12 armillol) throughhave (been13 analysis) were shown establishedof toan exhibitECD spectr by moderate biogeneticum of aantimicrobial dibenzoate consideration derivative, activity [32 ,33against while]. Both thatE. sulcatinecoli of A(11sulcatineATCC25922) and B B ( 12 (MIC)) and have armillol32 beenµg/mL) (13 shown ) withwere established11 to also exhibit exhibiting by moderate biogenetic activity antimicrobialconsideration against Mannheimia [32,33]. activity Both againsthaemolytica sulcatineE. coli ATCC25922AATCC14003 (11) (MICand B(MIC32 (12µ )g32 /havemL) µg/mL) withbeen [34]. 11shownalso In addition, exhibitingto exhibit 12 activity andmoderate 14 have against antimicrobial beenMannheimia reported activity to haemolytica exhibit against antifungal ATCC14003E. coli (MICATCC25922activity 32 µg/ mL)against [(MIC34 Cladiosporium]. In32 addition, µg/mL) cucumerinum 12withand 1114 also andhave exhibitingCandida been reportedalbicans activity atto a againstloading exhibit Mannheimiaof antifungal 50 µg per activitydiskhaemolytica [33]. against CladiosporiumATCC14003 cucumerinum (MIC 32 µg/mL)and Candida[34]. In addition, albicans at12 aand loading 14 have of 50beenµg reported per disk to [33 exhibit]. antifungal activity against Cladiosporium cucumerinum and Candida albicans at a loading of 50 µg per disk [33].

Figure 5. Structures of protoilludanes 11–14 isolated from Laurilia sulcate.

Another FiguremushroomFigure 5. Structures5. Structuresfrom the of of Laurilia protoilludanes protoilludanes genus, 11 11L.–– 1414tsugicola isolatedisolated, fromafforded from LauriliaLaurilia tsugicolines sulcate. sulcate. A–E (15–19) [35,36]. The relative configurations of tsugicolines A–E (15–19) were established by NOESY AnotherspectroscopicAnother mushroom mushroomanalysis from [35]. from theThe Lauriliatheabsolute Lauriliagenus, configuration genus,L.tsugicola L. tsugicola of tsugicoline, aff,orded afforded A tsugicolines (15 tsugicolines) was determined A–E A–E (15 (– 15by19– )[19the35) ,36]. The relative[35,36]partial. resolutionThe configurations relative method configurations of of tsugicolines Horeau, of while tsugicolines A–E that (15 of–19 tsugicolineA–E) were (15– established19 E) (were19) was established by determined NOESY by spectroscopic byNOESY X-ray spectroscopic analysis [35]. The absolute configuration of tsugicoline A (15) was determined by the analysiscrystallographic [35]. The absolute analysis configuration [35,36]. Tsugicolines of tsugicoline A (15), AC ( 1715)) and was E determined (19) have been by theshown partial to exhibit resolution partial resolution method of Horeau, while that of tsugicoline E (19) was determined by X-ray methodmoderate of Horeau, antimicrobial while that activity of tsugicoline against E. E coli (19 )ATCC25922 was determined (MIC 32 by µg/mL) X-ray crystallographicwith 15 and 17 also analysis crystallographicexhibiting activity analysis against [35,36]. the Gram-negative Tsugicolines bacterium, A (15), C (M.17) haemolytica and E (19) ATCC14003have been shown (MIC 32to µg/mL)exhibit [35,36]. Tsugicolines A (15), C (17) and E (19) have been shown to exhibit moderate antimicrobial moderate[34]. Tsugicoline antimicrobial A (15) activityhas also against been shown E. coli toATCC25922 exhibit allelopathic (MIC 32 activityµg/mL) againstwith 15 garden and 17 cress, also activity against E. coli ATCC25922 (MIC 32 µg/mL) with 15 and 17 also exhibiting activity against the exhibitingLepidium sativum activity [35] against. the Gram-negative bacterium, M. haemolytica ATCC14003 (MIC 32 µg/mL) Gram-negative[34]. AnotherTsugicoline bacterium, closely A ( 15related)M. has haemolytica coalsompound, been shownATCC14003 tsugicoline to exhibit M (MIC (20 allelopathic), 32 wasµg isolated/mL) activity [34 from]. Tsugicoline againstthe edible garden mushroom A (15 cress,) has also beenLepidiumClavicorona shown tosativum exhibitpyxidate, [35] allelopathic .the relative activityconfiguration against of gardenwhich was cress, assignedLepidium utilising sativum NOESY[35]. NMR data Another[37]. AnotherTsugicoline closely closely M related ( 20related) was compound, coinvestigatedmpound, tsugicolinetsugicoline for antimicrobial M M (20 (20), activity), was was isolated isolatedagainst from E. from coli the, B. theedible subtilis edible mushroom, S. mushroomaureus ClavicoronaClavicoronaand Candida pyxidate, pyxidate, albicans,the relativetheat a relativeconcentration configuration configuration of 50 µg/mL, of of which which and was cytotoxicitywas assigned assigned against utilising utilising HeLa NOESYNOESY cells, at NMR 20 µg/mL data data [37]. [37]. Tsugicoline M (20) was investigated for antimicrobial activity against E. coli, B. subtilis, S. aureus Tsugicolineand was M found (20) was inactive investigated in all assays for antimicrobial[37]. The same activity mushroom against alsoE. afforded coli, B. subtilistwo other, S. aureusnovel and and Candida albicans, at a concentration of 50 µg/mL, and cytotoxicity against HeLa cells, at 20 µg/mL Candidaprotoilludanes albicans, at (Figure a concentration 6), pyxidatol of 50A (µ21g)/ andmL, B and (22), cytotoxicity along with the against previously HeLa reported cells, at tsugicoline 20 µg/mL and and was found inactive in all assays [37]. The same mushroom also afforded two other novel was foundE (19) inactive[38]. The in relative all assays configurations [37]. The same of pyxidatol mushroom A ( also21) and afforded B (22 two) were other resolved novel by protoilludanes ROESY protoilludanesanalysis with the (Figure absolute 6), pyxidatol configurations A (21) andof 21 B (and22), along22 being with assigned the previously by consideration reported tsugicoline of shared (Figure6), pyxidatol A ( 21) and B (22), along with the previously reported tsugicoline E (19)[38]. Ebiogenetic (19) [38]. origins The relative with 19 configurations [37,38]. of pyxidatol A (21) and B (22) were resolved by ROESY The relativeanalysis configurationswith the absolute of configurations pyxidatol A ( 21of )21 and and B 22 (22 being) were assigned resolved by by consideration ROESY analysis of shared with the absolutebiogenetic configurations origins with of 2119 [37,38].and 22 being assigned by consideration of shared biogenetic origins with 19 [37,38]. Antibiotics 2020, 9, 928 6 of 29

Antibiotics 2020, 9, 928 6 of 29

Figure 6. Structures of tsugicolines 15–20 and pyxidatols A (21) and B (22) isolated from the Laurilia Figure 6. Structures of tsugicolines 15–20 and pyxidatols A (21) and B (22) isolated from the Laurilia and Clavicoronaand Clavicoronagenera genera of fungi.of fungi. Figure 6. Structures of tsugicolines 15–20 and pyxidatols A (21) and B (22) isolated from the Laurilia and Clavicorona genera of fungi. The injuredThe injured fruiting fruiting bodies bodies of of the the mushroom mushroomRussula Russula delicadelica affordedafforded five five protoilludanes protoilludanes (Figure (Figure 7), 7), plorantinone A–D (23–26) and epi-plorantinone B (27), while the intact fruiting bodies gave the plorantinone A–D (23–26) and epi-plorantinone B (27), while the intact fruiting bodies gave the first Theexamples injured of fruiting fatty ac bodiesid ester of protoilludanes, the mushroom stearoyl Russula plorantinonedelica afforded B five (28 )protoilludanes and stearoyl delicone (Figure first examples7),(29 )plorantinone [39–41]. of The fatty A–D relative acid (23– esterconfigurations26) and protoilludanes, epi-plorantinone of plorantinone stearoyl B (27), A–C while plorantinone (23 the–25 intact), stearoyl B fruiting (28 )plorantinone and bodies stearoyl gave B delicone(the28) (29)[firstand39–41 examplesstearoyl]. The relative deliconeof fatty configurations ac(29id) esterwere protoilludanes, determined of plorantinone from stearoyl NOESY A–C plorantinone (23data.–25 ),The stearoyl B absolute(28) and plorantinone stearoylconfiguration delicone B (28of ) and stearoyl(plorantinone29) delicone[39–41]. The (B29 ( )24relative were) was determined configurationsestablished from by of analysis NOESYplorantinone of data. ECD A–C The spectra, ( absolute23–25 ),conformational stearoyl configuration plorantinone analysis of plorantinone B (and28) B(24andmolecular) was stearoyl established mechanics delicone by (MM3) analysis(29) werecalculations of determined ECD [39–41]. spectra, from Subsequently, conformational NOESY data. the absolute analysisThe absolute configurations and molecularconfiguration of 23 mechanics, 25of, (MM3)plorantinone28 and calculations 29 were B determined(24 [39) –was41]. established by Subsequently, comparison by analysisof the optical absolute of rotation ECD configurations spectra,with plorantinone conformational of 23B (,2425) ,[39–41].analysis28 and and29 were determinedmolecular by mechanics comparison (MM3) of optical calculations rotation [39–41]. with Subsequently, plorantinone the B absolute (24)[39 configurations–41]. of 23, 25, 28 and 29 were determined by comparison of optical rotation with plorantinone B (24) [39–41].

Figure 7. Structures of protoilludanes 23–33 isolated from the Russula genus of fungi. Figure 7. Structures of protoilludanes 23–33 isolated from the Russula genus of fungi. The fruitingFigure 7.bodies Structures of ofanother protoilludanes mushroom 23–33 fromisolated the from Russula the Russula genus, genus R. of japonica, fungi. afforded Therussujaponols fruiting bodies A–D ( of30 another–33) (Figure mushroom 7) [42]. fromAssignment the Russula of thegenus, relativeR. japonica,configurationafforded of 30 russujaponols–33 was The fruiting bodies of another mushroom from the Russula genus, R. japonica, afforded A–Dachieved (30–33) (Figurethrough7)[ analysis42]. Assignment of ROESY ofand the NOESY relative NMR configuration data. The of absolute30–33 was configuration achieved throughof russujaponols A–D (30–33) (Figure 7) [42]. Assignment of the relative configuration of 30–33 was analysisachieved of ROESY through and analysis NOESY of NMRROESY data. and The NOESY absolute NMR configuration data. The absolute of russujaponol configuration A( 30of) was assigned by X-ray crystallographic analysis of a benzoate derivative which in turn allowed the Antibiotics 2020, 9, 928 7 of 29 Antibiotics 2020, 9, 928 7 of 29 absoluterussujaponol configuration A (30) was of russujaponols assigned by X-ray B–D crystallographic (31–33) to be analysis ascertained of a benzoate by biogenetic derivative consideration. which The cytotoxicityin turn allowed of 30 the, 31 absoluteand 33 wasconfiguration investigated of russujaponols and all three B–D natural (31–33 products) to be ascertained were found byto be non-cytotoxicbiogenetic againstconsideration. 39 human The cytotoxicity tumour cell of lines. 30, 31Russujaponol and 33 was investigated A (30) was and found all three to suppress natural the invasionproducts of human were found fibrosarcoma to be non-cytotoxic cells (HT1080) agains intot 39 Matrigel human tumour with 63% cell inhibitionlines. Russujaponol at 3.73 µ AM[ (3042) ]. Investigationwas found to suppress of the fruiting the invasion bodies of ofhuman a mushroom, fibrosarcomaLactarius cells (HT1080) atlanticus into, led Matrigel to the with discovery 63% of atlanticonesinhibition A–D at 3.73 (34 –µM37) [42]. (Figure 8)[ 43]. As with the plorantinones, the intact fruiting bodies afforded Investigation of the fruiting bodies of a mushroom, Lactarius atlanticus, led to the discovery of fatty acid sesquiterpenoids, atlanticones A (34) and B (35) which were isolated as a mixture of fatty acid atlanticones A–D (34–37) (Figure 8) [43]. As with the plorantinones, the intact fruiting bodies afforded estersfatty (ca. acid 90% sesquiterpenoids, oleic acid and ca. atlanticones 10% linoleic A (34 acid),) and while B (35) atlanticones which were isolated C (36) and as a Dmixture (37) were of fatty isolated fromacid injured esters fruiting (ca. 90% bodies. oleic acid Both and atlanticones ca. 10% linoleic A (34 acid),) and while B (35 atlanticones) were investigated C (36) and for D antimicrobial(37) were activityisolated against fromS. aureusinjuredand fruitingE. coli bodies.but were Both found atlanticones to be inactive. A (34) Theand relativeB (35) were configurations investigated of for all four naturalantimicrobial products wereactivity assigned against by S. NOESY, aureus and while E. thecoli absolutebut were configuration found to be inactive. of atlanticone The relative A (34) was determinedconfigurations by a combination of all four ofnatural PM3 calculationsproducts were and assigned ECD spectral by NOESY, comparison whilewith the plorantinoneabsolute B(24)[configuration43]. Both a of racemic atlanticone synthesis A (34) was and determined enantioselective by a combination total synthesis of PM3 of calculations atlanticone and C (ECD36) have beenspectral reported comparison [44,45]. with plorantinone B (24) [43]. Both a racemic synthesis and enantioselective total synthesis of atlanticone C (36) have been reported [44,45].

FigureFigure 8. Structures 8. Structures of of protoilludanes protoilludanes34 34––38 isolated from from the the LactariusLactarius genusgenus of fungi. of fungi.

RepraesentinRepraesentin A (38 A) (Figure(38) (Figure8), a closely8), a closely related related natural natural product product to ∆ 6to-protoilludene Δ6-protoilludene (7), ( was7), was isolated fromisolated another from mushroom another from mushroom the Lactarius from genus,the LactariusL. repraesentaneus genus, L. repraesentaneus[46]. The relative [46]. The configuration relative of configuration of the natural product was assigned utilising NOESY spectroscopic data, while the the natural product was assigned utilising NOESY spectroscopic data, while the absolute configuration absolute configuration was assigned by consideration of common biosynthetic origins with 7. was assigned by consideration of common biosynthetic origins with 7. Repraesentin A (38) was shown Repraesentin A (38) was shown to promote radicle elongation of lettuce seedlings [46]. to promoteThe radicle mushroom elongation Agrocybe of lettuceaegerita seedlingsafforded three [46]. α,β-unsaturated carboxylic acid containing Theprotoilludanes mushroom (FigureAgrocybe 9), pasteurestins aegerita a ffA–Corded (39– three41) [47,48].α,β-unsaturated Structure elucidation carboxylic of all three acid natural containing protoilludanesproducts was (Figure achieved9), pasteurestins by spectroscopic A–C analysis ( 39– 41[47,48].)[47, 48The]. st Structureereoselective elucidation syntheses of of allboth three 39 and natural products40 have was been achieved reported by [49,50]. spectroscopic This allowed analysis assignment [47,48]. of The the stereoselectiverelative configurations syntheses of 39of and both 40 by39 and 40 haveNOESY been data reported and absolute [49,50]. configurations This allowed assignmentby comparison of theof optical relative rotations configurations with the ofrespective39 and 40 by NOESYnatural data products and absolute [49,50]. configurations The relative configuration by comparison of 41 of was optical assigned rotations by analysis with the ofrespective ROESY data, natural productswhile [49 the,50 absolute]. The relativeconfiguration configuration was suggested of 41 bywas biogenetic assigned consideration by analysis [48]. of ROESY The antimicrobial data, while the activities of pasteurestins A (39) and B (40) have been investigated against Pasteurella multocida N791 absolute configuration was suggested by biogenetic consideration [48]. The antimicrobial activities of BBP 0101 (MIC 0.89 and 0.78 µg/mL, respectively) and Mannheimia haemolytica N811 BBP 0102 (MIC pasteurestins A (39) and B (40) have been investigated against Pasteurella multocida N791 BBP 0101 1.56 and 0.78 µg/mL, respectively) where 40 was found to be a more potent inhibitor than 39 [34]. (MIC 0.89Another and 0.78 exampleµg/mL, of respectively)an α,β-unsaturated and Mannheimiacarboxylic acid haemolytica containing N811protoilludane, BBP 0102 epicoterpene (MIC 1.56 and 0.78 µAg /(mL,42), was respectively) isolated from where a co-culture40 was of found the honey to be fungus a more Armillaria potent inhibitor sp. and the than endophytic39 [34]. fungus Epicoccum sp. [51]. This co-culture also afforded three other protoilludanes, epicoterpene B (43), C (44) and E (45) (Figure 9). The relative configurations of all four epicoterpenes were assigned by Antibiotics 2020, 9, 928 8 of 29

analysis of ROESY data, while the absolute configurations were determined by comparison of experimental ECD spectra with time-dependent density functional theory (TDDFT) calculated ECD spectra. None of the four compounds exhibited cytotoxicity (IC50 > 40 µM) against HL-60, A-549, AntibioticsSMMC-7721,2020, 9, 928 MCF7, SW480 cell lines or acetylcholinesterase inhibitory activity at a concentration of8 of 29 50 µM [51].

FigureFigure 9. 9.Structures Structures of protoilludanes 3939–47.–47 .

AnotherThe exampleLentinellus of genus an α ,βafforded-unsaturated two protoilludanes, carboxylic acid lentinellic containing acid protoilludane,(46) and lentinellone epicoterpene (47) A (42), was[52,53]. isolated Lentinellic from acid a co-culture(46), isolated of from the Lentinellus honey fungus ursinusArmillaria and L. omphalodessp. and, contains the endophytic a lactone and fungus an α,β-unsaturated carboxylic acid. Structure elucidation and assignment of the relative Epicoccum sp. [51]. This co-culture also afforded three other protoilludanes, epicoterpene B (43), C (44) configuration of 46 was achieved by X-ray crystallography, while that of 47 was achieved by NOESY and E (45) (Figure9). The relative configurations of all four epicoterpenes were assigned by analysis spectroscopic analysis. The absolute configuration of 47 was tentatively assigned by consideration of of ROESYshared data, biogenetic while origins. the absolute Both natural configurations products were were investigated determined for by their comparison antimicrobial of experimental activity ECDagainst spectra a with variety time-dependent of strains including; density Bacillus functional brevis, theory B. subtilis, (TDDFT) Coryne calculatedbacterium ECDinsidiosum, spectra. E. coli, None of the fourMicrococcus compounds luteus, exhibited Mycobacterium cytotoxicity phlei and (IC Streptomyces50 > 40 µM) sp., against where lentinellone HL-60, A-549, (47) SMMC-7721,was found to be MCF7, SW480inactive. cell lines Alternately, or acetylcholinesterase lentinellic acid (46 inhibitory) exhibited activity potent atantimicrobial a concentration activity of (MIC 50 µM[ 1–551 µg/mL)]. TheagainstLentinellus Aerobactergenus aerogenes afforded, B. twobrevis protoilludanes,, C. insidiosum lentinellicand moderate acid activity (46) and against lentinellone Acinetobacter (47)[52 ,53]. Lentinellicaerogenes acid (MIC (46 20–50), isolated µg/mL), from B. subtilisLentinellus (MIC ursinus20–50 µg/mL),and L. M. omphalodes luteus (MIC, contains 10–20 µg/mL), a lactone Proteus and an α,β-unsaturatedvulgaris (MIC carboxylic 20–50 µg/mL), acid. S. Structureaureus (MIC elucidation 20–50 µg/mL) and and assignment Streptomyces of the sp. relative(MIC 10–20 configuration µg/mL). of In addition, 46 also exhibited antifungal activity against Absidia glauca and Nematospora coryli at a 46 was achieved by X-ray crystallography, while that of 47 was achieved by NOESY spectroscopic loading of 100 µg per disk. The cytotoxicity of 47 was investigated against various cancer cell lines analysis. The absolute configuration of 47 was tentatively assigned by consideration of shared biogenetic where it exhibited moderate cytotoxicity against HL-60 (IC50 20 µg/mL) [52,53]. origins. BothThree naturalprotoilludanes, products radudiol were (48 investigated), and radulone for A their (49) and antimicrobial B (50), were activityisolated from against the crust a variety of strainsfungus, including; RadulomycesBacillus confluens brevis, (Figure B. subtilis, 10) [54]. Corynebacterium Radudiol (48) is insidiosum, another example E. coli, Micrococcusof a saturated luteus, Mycobacteriumprotoilludane, phlei whileand Streptomycesradulone A (49sp.,) has where four lentinellonerings, one of (which47) was contains found an to beoxygen inactive. bridge. Alternately, The lentinellicrelative acid configurations (46) exhibited of all potent three antimicrobial natural produc activityts were assigned (MIC 1–5 byµ ganalysis/mL) against of NOESYAerobacter NMR data. aerogenes , B. brevisThe, cytotoxicityC. insidiosum of 48and–50 was moderate investigated activity against against HeLaAcinetobacter S3, HL-60 and aerogenes L-1210 cell(MIC lines 20–50 whereµ 49g/ mL), B. subtilis(IC50 16,(MIC 2, and 20–50 1 µM,µg /respectively)mL), M. luteus exhibited(MIC potent 10–20 cytotoxicityµg/mL), Proteus against vulgaris all three(MIC cell lines, 20–50 whileµg/ mL), S. aureusradudiol(MIC (48 20–50) andµ gradulone/mL) and B Streptomyces(50) were non-cytotoxic.sp. (MIC 10–20 Raduloneµg/mL). A In(49 addition,) also exhibited46 also weak exhibited antimicrobial activity against Corynebacterium insiduodum, Micrococcus luteus, Nematospora coryli and antifungal activity against Absidia glauca and Nematospora coryli at a loading of 100 µg per disk. Ustilago nuda with an MIC of 20 µM as well as Streptomyces bikiniensis, Saccharomyces cerevisiae and The cytotoxicity of 47 was investigated against various cancer cell lines where it exhibited moderate Mucor miehei with an MIC of 41 µM. In addition, 49 was found to be a potent inhibitor of human (IC50 cytotoxicity against HL-60 (IC 20 µg/mL) [52,53]. 2 µM) and bovine (IC50 12 µM)50 platelet aggregation induced by adenosine diphosphate (conc 25 µM) Three[54]. The protoilludanes, stereoselective synthesis radudiol of ( 48the), non-natural and radulone enantiomer A (49) of and radudiol B (50 ),(48 were) has been isolated reported from the crustallowing fungus, assignmentRadulomyces of confluensthe absolute(Figure configuration 10)[54]. of Radudiol the natural (48 product) is another by comparison example of aoptical saturated protoilludane,rotations [55]. while radulone A (49) has four rings, one of which contains an oxygen bridge. The relative configurations of all three natural products were assigned by analysis of NOESY NMR data. The cytotoxicity of 48–50 was investigated against HeLa S3, HL-60 and L-1210 cell lines where 49 (IC50 16, 2, and 1 µM, respectively) exhibited potent cytotoxicity against all three cell lines, while radudiol (48) and radulone B (50) were non-cytotoxic. Radulone A (49) also exhibited weak antimicrobial activity against Corynebacterium insiduodum, Micrococcus luteus, Nematospora coryli and Ustilago nuda with an MIC of 20 µM as well as Streptomyces bikiniensis, Saccharomyces cerevisiae and Mucor miehei with an MIC of 41 µM. In addition, 49 was found to be a potent inhibitor of human (IC50 2 µM) and bovine (IC50 12 µM) platelet aggregation induced by adenosine diphosphate (conc 25 µM) [54]. Antibiotics 2020, 9, 928 9 of 29

The stereoselective synthesis of the non-natural enantiomer of radudiol (48) has been reported allowing assignment of the absolute conﬁguration of the natural product by comparison of optical rotations [55]. Antibiotics 2020, 9, 928 9 of 29

Antibiotics 2020, 9, 928 9 of 29

FigureFigure 10. 10. StructuresStructures of of protoilludanes protoilludanes 4848––5050 isolatedisolated from from RadulomycesRadulomyces confluens. conﬂuens.

6 TwoTwo examplesexamplesFigure of of oxygen-bridged 10.oxygen-bridged Structures of protoilludanes protoilludanes protoilludanes 48–50 (Figure isolated (Figure 11 from), coprinolone Radulomyces 11), coprinolone confluens. (51) and ∆(51-coprinolone) and Δ6- coprinolone(52), were isolated (52), were from isolatedCoprinus from psychromorbidus Coprinus psychromorbidus[56,57]. The structures [56,57]. The of both structures natural of products both natural were productsassigned bywereTwo spectroscopic assignedexamples byof analysis spectroscopicoxygen-bridged and the relativeanalysis protoilludanes stereochemistry and the (Figure relative of11), coprinolonestereochemistry coprinolone (51 )(51 wasof) coprinoloneand determined Δ6- (by51) analysis wascoprinolone determined of NOESY (52), wereby data analysis isolated of the from naturalof NOESY Coprinus product data psychromorbidus andof the its derivednatural [56,57]. isomersproduct The structures [56and,57 its]. of Furtherderived both natural examples isomers products were assigned by spectroscopic analysis and the relative stereochemistry of coprinolone [56,57].of oxygen-bridged Further examples protoilludanes, of oxygen-bridged including pr 2-hydroxycoprinoloneotoilludanes, including ( 532-hydroxycoprinolone), 2a-hydroxycoprinolone (53), (51) was determined by analysis of NOESY data of the natural product and its derived isomers 2a-hydroxycoprinolone(54), 3-hydroxycoprinolone[56,57]. Further examples (54), ( 553-hydroxycoprinoloneof )oxygen-bridged and coprinolone protoilludanes, diol (55 B) (and56) wereincludingcoprinolone isolated 2-hydroxycoprinolone diol from B the(56 plant) were pathogen (isolated53), fromGranulobasidium the2a-hydroxycoprinolone plant pathogen vellereum [Granulobasidium58 (,5459),]. 3-hydroxycoprinolone Structure vellereum elucidation [58,59]. ( and55) and establishment Structure coprinolone elucid of diol theation B absolute (56 and) were establishment conﬁgurations isolated ofof the 2-hydroxycoprinolonefrom absolute the plant configurations pathogen (53 Granulobasidium), of 2a-hydroxycoprinolone 2-hydroxycoprinolone vellereum [58,59]. ( 54( 53Structure)), and 2a-hydroxycoprinolone 3-hydroxycoprinolone elucidation and establishment (54 (55) and) were 3- hydroxycoprinoloneachievedof the using absolute a ROESY configurations(55) spectroscopicwere achieved of 2-hydroxycoprinolone data using and a analysis ROESY of (spectroscopic53 ECD), 2a-hydroxycoprinolone spectra. data The absoluteand analysis (54 conﬁguration) and of 3- ECD spectra.of coprinolonehydroxycoprinolone The absolute diol B (configuration56 ()55 was) were established achieved of coprinolone byusing consideration a ROESY diol B spectroscopic(56 of) sharedwas established biogenetic data and byanalysis origin consideration withof ECD that of of sharedcoprinolonespectra. biogenetic diol,The absolute origin a semi-synthetic with configuration that of derivative coprinolone of coprinolone from diol, coprinolonediol a semi-syntheticB (56) was (51 established). The derivative antimicrobial by consideration from coprinolone activities of of (5351–).56 Thesharedwere antimicrobial investigatedbiogenetic origin againstactivities with that several of of 53 coprinolone species–56 were including diol,investigated a semi-syntheticPenicillium against canescensderivative several, fromFusarium species coprinolone oxysporumincluding Penicilliumand Heterobasidion(51). Thecanescens antimicrobial occidentale, Fusarium activitiesat oxysporum concentrations of 53 and–56 wereHeterobasidion up toinvestigated 100 µg/ mLoccidentale against and all severalat four concentrations natural species products including up to were 100 Penicillium canescens, Fusarium oxysporum and Heterobasidion occidentale at concentrations up to 100 µg/mLfound toand be all inactive four natural [58,59]. products were found to be inactive [58,59]. µg/mL and all four natural products were found to be inactive [58,59].

FigureFigure 11. Structures 11. Structures of coprinolonesof coprinolones51 51––5656 isolated from from the the CoprinusCoprinus andand GranulobasidiumGranulobasidium generagenera of Figureof fungi.fungi. 11. Structures of coprinolones 51–56 isolated from the Coprinus and Granulobasidium genera of fungi. OtherOther protoilludanes protoilludanes isolated isolated from Granulobasidium from Granulobasidium vellereum include vellereum 8-deoxy-4a-hydroxytsugicoline include 8-deoxy-4a- hydroxytsugicoline A (57) and B (58), 8-deoxydihydrotsugicoline (59), granulodienes A (60) and B A(57Other) and B (protoilludanes58), 8-deoxydihydrotsugicoline isolated from ( 59Granulobasidium), granulodienes Avellereum (60) and B (include61), granulones 8-deoxy-4a- A (62) (61), granulones A (62) and B (63) and demethylgranulone (64) (Figure 12) [58,59]. Determination of hydroxytsugicolineand B (63) and demethylgranulone A (57) and B ( (5864),) (Figure8-deoxydihydrotsugicoline 12)[58,59]. Determination (59), granulodienes of the relative conﬁgurationsA (60) and B the relative configurations of 57–64 was achieved by ROESY spectroscopic analysis. The absolute (61), granulones A (62) and B (63) and demethylgranulone (64) (Figure 12) [58,59]. Determination of of 57–configurations64 was achieved 59–61 by was ROESY established spectroscopic by ECD spectral analysis. analysis, The while absolute those of conﬁgurations 57, 58, and 62–6459 were–61 was the relative configurations of 57–64 was achieved by ROESY spectroscopic analysis. The absolute establishedassigned by upon ECD biosynthetic spectral analysis, consideration while those with other of 57 ,protoilludanes.58, and 62–64 Thewere antifungal assigned activities upon biosynthetic of 57– configurationsconsideration64 were investigated with59–61 other was against protoilludanes.established Penicillium by ECD The canescens antifungalspectral, Fusarium analysis, activities oxysporum while of 57 thoseand–64 Heterobasidion wereof 57, investigated 58, and occidentale 62–64 against were assignedPenicilliumat concentration upon canescens biosynthetic, Fusariumup to 100 consideration oxysporumµg/mL andand none withHeterobasidion of other the naturalprotoilludanes. occidentale products at exhibitedThe concentration antifungal activity activities up[58,59]. to 100 The µofg /57mL– 64and were nonestereoselective investigated of the natural synthesis against products of Penicillium the non-natural exhibited canescens enantiomer activity, Fusarium [58 of, 598-deoxydihydrotsugicoline oxysporum]. The stereoselective and Heterobasidion ( synthesis59) has occidentale been of the atnon-natural concentrationreported enantiomer [55]. up to 100 of 8-deoxydihydrotsugicolineµg/mL and none of the natural (59) has products been reported exhibited [55 activity]. [58,59]. The stereoselective synthesis of the non-natural enantiomer of 8-deoxydihydrotsugicoline (59) has been reported [55]. AntibioticsAntibiotics 20202020,, 99,, 928928 10 of 29

Antibiotics 2020, 9, 928 10 of 29

FigureFigure 12. 12. Structures Structures of of protoilludanes protoilludanes 57 57––6464 isolated isolated from from Granulobasidium Granulobasidium vellereum. vellereum.

The crust fungus, Echinodontium tsugicola, afforded four protoilludanes named echinocidins A– The crust fungus, fungus, EchinodontiumEchinodontium tsugicola tsugicola, afforded, afforded four four protoilludanes protoilludanes named named echinocidins echinocidins A– D (65–68) (Figure 13) [60,61]. The relative configurations of 65–68 was assigned by NOESY A–DD (65 (–6568–)68 (Figure) (Figure 13) 13 )[[60,61].60,61 ].The The relative relative configurations configurations of of 6565––6868 waswas assigned assigned by NOESY spectroscopicspectroscopic analysis analysis where where65 65 and 6767 werewere based based on onthe thenatural natural products, products, while 66 while and 6866 wereand on68 were spectroscopica diacetyl analysisand acetonide where derivative, 65 and 67 respectively.were based onThe the absolute natural configurations products, while of these66 and natural 68 were on on a diacetyl and acetonide derivative, respectively. The absolute configurations of these natural a diacetylproducts and were acetonide ascertained derivative, by consideration respectively. of shared biogeneticThe absolute origins configurations with tsugicoline of A these(15) and natural productsproductsB (16). werewere The ascertainedascertainedantimicrobial byby activities consideration consideration of echinocidin of of shared shared A biogenetic( 65biogenetic) and B ( origins66 origins) were with withinvestigated tsugicoline tsugicoline against A A (15 (C.15) and) and B (B16 ().16albicans The). The antimicrobial ATCC2019, antimicrobial S. activities aureus activities NBRC13276 of echinocidin of echinocidin and P. A aeruginosa (65 A) and(65 ATCC15442) B and (66) B were (66 at) investigated awere loading investigated concentration against againstC. of albicans C. µ ATCC2019,albicans100 µgATCC2019, perS. aureusdisk and S.NBRC13276 bothaureus natural NBRC13276 and productsP. aeruginosa andwere P. found aeruginosaATCC15442 to be inactive ATCC15442 at a [60,61]. loading atAllconcentration a four loading natural concentration products of 100 g per of disk100 µg65 and– per68 both were disk natural investigated and both products natural in a lettuce were products foundseedling were to bioassay be found inactive at to a [concentrationbe60 ,inactive61]. All four[60,61]. of 100 natural ppm All four where products natural they65 were products–68 were observed to accelerate primary root growth to 117%, 180%, 190% and 179% of controls, respectively investigated65–68 were investigated in a lettuce in seedling a lettuce bioassay seedling at bioassay a concentration at a concentration of 100 ppm of where 100 ppm they where were they observed were [60,61]. The total syntheses of racemic echinocidin B (66) and D (68) have been reported [62]. toobserved accelerate to accelerate primary rootprimary growth root to growth 117%, to 180%, 117%, 190% 180%, and 190% 179% and of 179% controls, of controls, respectively respectively [60,61]. The[60,61]. total The syntheses total syntheses of racemic of racemic echinocidin echinocidin B (66) and B ( D66 ()68 and) have D (68 been) have reported been reported [62]. [62].

Figure 13. Structures of echinocidins A–D (65–68) from Echinodontium tsugicola.

2.2. Protoilludane Sesquiterpene Aryl Esters Figure 13. Structures of echinocidins A–D (65–68) from Echinodontium tsugicola. The genus Armillaria has been one of the most extensive producers of aryl ester containing protoilludanes. These protoilludanes can be divided into three types of which the first two are 2.2. Protoilludane Sesquiterpene Aryl Esters classified based on the position of the double bond in the protoilludane skeleton, i.e., Δ2,4- protoilludeneThe genus Armillaria (e.g., Δ6-protoilludene) has been one and ofΔ2,3 thethe-protoilludene mostmost extensiveextensive (e.g., Δ producers7producers-protoilludene) ofof arylaryl skeletons, esterester containingwhilecontaining protoilludanes.protoilludanes.the third is classified TheseThese protoilludanes protoilludanesby the absence canof candouble be dividedbe bondsdivided into in the threeinto 5/6/4-ring three types typesofsystem. which of thewhich first the two first are classifiedtwo are 2,4 basedclassified onAryl thebased positionsesquiterpenoids on the of theposition doubleof the fiof bondrst the type, indoub theof which protoilludanele bond only ina fewthe skeleton, examplesprotoilludane i.e., exist,∆ include -protoilludeneskeleton, armillyl i.e., (e.g.,Δ2,4- 6 orsellinate (69) [63], judeol2,3 (70) [64], armillyl everninate7 (71) [65], arnamiol (72) [65], armillaribin (73) ∆protoilludene-protoilludene) (e.g., and Δ6-protoilludene)∆ -protoilludene and (e.g., Δ2,3-protoilludene∆ -protoilludene) (e.g., skeletons, Δ7-protoilludene) while the thirdskeletons, is classified while [66], armillaricin (74) [67], arnamial (75) [68], dehydroarmillyl orsellinate (76) [69] and 6’- bythe the third absence is classified of double by the bonds absence in the of 5 double/6/4-ring bonds system. in the 5/6/4-ring system. dechloroarnamial (77) [70], all isolated from the honey fungus Armillaria mellea (Figure 14). Structure Aryl sesquiterpenoids of the first type, of which only a few examples exist, include armillyl elucidationAryl sesquiterpenoids and determination of the offi rstthe type, absolute of which configurations only a few of armillylexamples orsellinate exist, include (69) and armillyl orsellinatearmillaricin ((6969)[) 63([63],74],) judeolwere judeol achieved (70 (70)[)64 [64], ],utilising armillyl armillyl a everninatecombination everninate (71 of ()[ 71spectroscopic65) [65],], arnamiol arnamiol and (72 X-ray)[ (7265)], [65], crystallographic armillaribin armillaribin (73 )[ (6673],) armillaricin[66], armillaricin (74)[67 (],74 arnamial) [67], arnamial (75)[68], dehydroarmillyl(75) [68], dehydroarmillyl orsellinate (76 orsellinate)[69] and 6’-dechloroarnamial(76) [69] and 6’- (dechloroarnamial77)[70], all isolated (77) from[70], all the isolated honey fr fungusom theArmillaria honey fungus mellea Armillaria(Figure mellea14). Structure(Figure 14). elucidation Structure andelucidation determination and determination of the absolute of configurationsthe absolute configurations of armillyl orsellinate of armillyl (69) andorsellinate armillaricin (69) and (74) armillaricin (74) were achieved utilising a combination of spectroscopic and X-ray crystallographic Antibiotics 2020, 9, 928 11 of 29 were achieved utilising a combination of spectroscopic and X-ray crystallographic analysis [63,67,71]. Antibiotics 2020, 9, 928 11 of 29 Subsequently, the relative configurations of arnamial (75) and 6’-dechloroarnamial (77) were ascertained by biosyntheticanalysis [63,67,71]. consideration Subsequently, [68,70]. the Both relative armillyl configurations orsellinate (of69 )arnamial and judeol (75) (70and) exhibited 6’- antimicrobialdechloroarnamial activity ( against77) wereB. ascertained subtilis ATCC6633 by biosynthetic (minimum consideration loading of [68,70]. 5.6 and Both 8.7 µarmillylg per disk, respectively)orsellinate and (69S.) aureusand judeolATCC53156 (70) exhibited (minimum antimicrobial loading of activity 5.6 and 8.7againstµg per B. disk,subtilis respectively) ATCC6633 with 69 also(minimum exhibiting loading activity of 5.6 against and 8.7E. coliµg per(MIC disk, 1.0 respectively)µg) and C. albicansand S. aureus(MIC ATCC53156 1.0 µg) on bioautographic(minimum assaysloading [63,64 of,72 5.6]. Arnamialand 8.7 µg (per75), disk, dehydroarmillyl respectively) with orsellinate 69 also exhibiting (76) and 6’-dechloroarnamialactivity against E. coli (MIC (77) were also investigated1.0 µg) and for C. theiralbicans antimicrobial (MIC 1.0 propertiesµg) on bioautographic and were reported assays to moderately[63,64,72]. Arnamial inhibit the (75 growth), dehydroarmillyl orsellinate (76) and 6’-dechloroarnamial (77) were also investigated for their of Penicillium notatum, Aspergillus flavus and Aspergillus nidulans [70]. In addition, 75 also exhibited antimicrobial properties and were reported to moderately inhibit the growth of Penicillium notatum, Tapinella panuoides Pleurotus ostreatus Omphalotus illudens Fomitopsis pinicola potentAspergillus activity againstflavus and Aspergillus nidulans, [70]. In addition, 75, also exhibited potent, activity against , P. oxalicumTapinella, A. panuoides flavus and, PleurotusStreptomyces ostreatus scabies, Omphalotus[73]. Fourilludens of, theseFomitopsis natural pinicola products, P. oxalicum (74–77, A.) haveflavus been shownand to Streptomyces exhibit cytotoxicity scabies [73]. against Fourseveral of these human natural cell products lines [ 68(74––7077,74) have]. Armillaricin been shown (74) to wasexhibit shown to becytotoxicity potently cytotoxic against se towardsveral human H460, cell MCF7, lines HT-29[68–70,74]. and Armillaricin CEM cell lines (74) (ICwas50 shown5.5, 4.8, to 4.6,be potently and 5.8 µM, respectively).cytotoxic towards Meanwhile, H460,75 MCF7,–77 exhibited HT-29 and moderate CEM cell tolines potent (IC50 5.5, cytotoxicity 4.8, 4.6, and towards 5.8 µM,HUVEC respectively). (GI50 2.0, 5.3, 5.1Meanwhile,µM) MCF7 75–77 (GI exhibited50 15.4, 8.0,moderate 4.1 µ M),to potent K-562 cytotoxicity (GI50 2.3, towards 5.0, 4.1 HUVECµM) and (GI HeLa50 2.0, (CC5.3, 505.1HeLa µM) 4.9, 15.2, 12.3MCF7µ M)(GI cell50 15.4, lines. 8.0, Additionally,4.1 µM), K-56275 (GIwas50 2.3, also 5.0, found 4.1 µM) to and becytotoxic HeLa (CC towards50 HeLa 4.9, HCT-116, 15.2, 12.3 Jurkat µM) and cell lines. Additionally, 75 was also found to be cytotoxic towards HCT-116, Jurkat and CCRF-CEM CCRF-CEM (IC50 10.69, 3.9, and 8.91 µM, respectively) cell lines, while 76 exhibited cytotoxicity (IC50 10.69, 3.9, and 8.91 µM, respectively) cell lines, while 76 exhibited cytotoxicity towards Jurkat towards Jurkat (IC50 16.9 µM) cells [68–70,74]. (IC50 16.9 µM) cells [68–70,74].

R O OH O O O R H HO O H HO Armillyl everninate (71)R=H H Arnamiol (72)R=Cl OH OH H OH OH Armillyl orsellinate (69)R=H Judeol (70)R=OH R2 R O OR1 O O O H HO O H HO

H OH O O Arnamial (75)R1 =CH3,R2 =Cl Armillaribin (73)R=H Dehydroarmillylorsellinate (76)R1 =R2 =H Armillaricin (74)R=Cl 6'-Dechloroarnamial (77)R =CH ,R =H 1 3 2 FigureFigure 14. Structures14. Structures of of∆2,4 Δ-protoilludene-containing2,4-protoilludene-containing aryl aryl sesquiterpenoids sesquiterpenoids 69–7769 –isolated77 isolated from from ArmillariaArmillaria mellea. mellea.

TheThe second second type type of arylof aryl ester ester sesquiterpenoids, sesquiterpenoids, containing the the Δ2,3∆-protoilludene2,3-protoilludene skeleton, skeleton, constituteconstitute the largestthe largest portion portion of of this this protoilludane protoilludane class.class. Melleolides Melleolides A–N A–N (78 (–7891–)91 [74–78],)[74– 78P (],92 P) [79] (92)[ 79] and T (93) (Figure 15) [79] were isolated from Armillaria mellea with the exception of melleolide I (86) and T (93) (Figure 15)[79] were isolated from Armillaria mellea with the exception of melleolide I [80] which was first isolated from another mushroom A. novae-zelandiae along with melleolide J (87) 86 A novae-zelandiae ( )[80(also] which named was armillarikin) first isolated [81]. Of from these another natural mushroomproducts, melleolide. N (91) is thealong only example with melleolide with J(87)esterification (also named at armillarikin) the C-1 hydroxyl [81]. group Of these [74]. naturalThe structure products, and the melleolide absolute configuration N (91) is the of only the examplefirst withmelleolide esterification (78) atwas the solved C-1 hydroxyl by X-ray groupcrystallographic [74]. The analysis structure [75]. and Assignment the absolute of the configuration relative of theconfigurations first melleolide of melleolides (78) was solvedB (79), byC (80 X-ray), E–K crystallographic (82–88) and N (91 analysis) were achieved [75]. Assignment by extensive of the relativeNOESY configurations spectroscopic of analysis melleolides [74,76–78 B (,80],79), while C (80 ),that E–K of melleolide (82–88) and P ( N92), ( 91also) were known achieved as 6’- by extensive NOESY spectroscopic analysis [74,76–78,80], while that of melleolide P (92), also known as Antibiotics 2020, 9, 928 12 of 29

6’-chloromelleolideAntibiotics 2020, 9, 928 F, was ascertained by consideration of common biogenetic origins with melleolide12 of 29 F (83)[70,79]. The absolute stereochemistry of melleolide L (89) was established by X-ray crystallographic analysischloromelleolide conducted on F, awas 2,4-dinitrophenylhydrazone ascertained by consideration derivativeof common [ 78biogenetic]. On the origins other with hand, melleolide the absolute conﬁgurationsF (83) [70,79]. of melleolide The absolute D (81stereochemistry) and M (90) wereof melleolide determined L (89 by) was semi-synthesis established ofby theX-ray natural crystallographic analysis conducted on a 2,4-dinitrophenylhydrazone derivative [78]. On the other products and subsequent comparison of optical rotations [78,82]. Finally, the absolute conﬁguration hand, the absolute configurations of melleolide D (81) and M (90) were determined by semi-synthesis of melleolide T (93), also named 13-hydroxymelleolide K, was assigned by comparison of ECD of the natural products and subsequent comparison of optical rotations [78,82]. Finally, the absolute spectraconfiguration with melleolide of melleolide D (81)[ T 79(93,83), also]. Melleolides named 13-hydroxymelleolide A (78) (IC50 HepG2 K, was 4.95 assignedµg/mL, by L02 comparison 16.05 µg/ mL), F(83of) (ICECD50 spectraMCF7 with 8.3 melleolideµM, H460 D 5.1(81)µ [79,83].M), I alsoMelleolides known A as(78) 5’-chloromelleolide (IC50 HepG2 4.95 µg/mL, (86 )L02 (IC 16.0550 A-549 20.11µg/mL),µM, MCF7 F (83 30.06) (IC50 µMCF7M), J 8.3 (87 µM,) (IC H46050 MCF7 5.1 µM), 4.4 µI M,also H460 known 5.7 asµ 5’-chloromelleolideM, HL-60 14.11 µ M,(86) A-549 (IC50 A-549 14.73 µM, SMMC-772120.11 µM, 17.41 MCF7µM), 30.06 N µM), (91) J (IC (8750) (ICHT-2950 MCF7 7.1 4.4µM, µM, H460 H460 5.5 5.7µ M,µM, CEM HL-60 5.4 14.11µM) µM, and A-549 P (92 14.73) (IC µM,50 MCF7 4.8 µM,SMMC-7721 H460 4.5 17.41µM, µM), CEM N 28.8(91) (ICµM)50 HT-29 have 7.1 been µM, reported H460 5.5to µM, be CEM cytotoxic 5.4 µM) against and P ( human92) (IC50 cancerMCF7 cell lines4.8 [70 ,µM,72,74 H460,84,85 4.5]. µM, The CEM antimicrobial 28.8 µM) activityhave been of reported some of to the be melleolides cytotoxic against have alsohuman been cancer investigated. cell Melleolidelines [70,72,74,84,85]. A (78) was reported The toantimicrobial exhibit antimicrobial activity of activity some againstof the B.melleolides subtilis ATCC6633 have also (MIC been 0.5 µg), investigated. Melleolide A (78) was reported to exhibit antimicrobial activity against B. subtilis E. coli (MIC 1.0 µg), C. cucumerinum (MIC 1.0 µg) and C. albicans (MIC 1.0 µg) in bioautographic assays, ATCC6633 (MIC 0.5 µg), E. coli (MIC 1.0 µg), C. cucumerinum (MIC 1.0 µg) and C. albicans (MIC 1.0 while melleolides B–D (79–81) were reported to exhibit antibacterial activity against B. subtilis ATCC6633, µg) in bioautographic assays, while melleolides B–D (79–81) were reported to exhibit antibacterial B. cereusactivityATCC10702 against B. and subtilisE. coli ATCC6633,ATCC10536 B. atcereus a minimum ATCC10702 loading and E. of coli 10 µATCC10536g per disk [72at ,a76 minimum]. In addition, melleolidesloadingK–M of 10 ( 88µg– 90per) were disk investigated[72,76]. In addition, for antimicrobial melleolides activity K–M ( against88–90) were several investigated species, where for 89 and 90antimicrobialwere found activity to be inactive against [ 78several]. Melleolide species, Kwhere (88) exhibited89 and 90 moderate were found activity to be against inactiveMicrococcus [78]. luteusMelleolideIF03333 (MIC K (88 12.5) exhibitedµg/mL), moderateB. subtilis activityPCI219 against (MIC Micrococcus 6.25 µg/mL), luteusCorynebacterium IF03333 (MIC 12.5 bovis µg/mL),1810 (MIC 25 µgB./mL), subtilisSaccharomyces PCI219 (MIC cerevisiae 6.25 µg/mL),(MIC Corynebacterium 25 µg/mL), Trichophyton bovis 1810 (MIC rubrum 25 IFO9185µg/mL), Saccharomyces (MIC 25 µg/ mL), S. aureuscerevisiae(FDA209P (MIC 25 (MICµg/mL), 6.25 Trichophytonµg/mL), Smithrubrum (MICIFO9185 12.5 (MICµg /25mL), µg/mL), MS9610 S. aureus (MIC (FDA209P 12.5 µg/ mL))(MIC and MRSA6.25 (MS16526 µg/mL), Smith (MIC (MIC 12.5 µ 12.5g/mL), µg/mL), TY-04282 MS9610 (MIC (MIC 12.5 12.5µ gµg/mL))/mL)) [ 78and]. TheMRSA total (MS16526 syntheses (MIC of 12.5 racemic µg/mL), TY-04282 (MIC 12.5 µg/mL)) [78]. The total syntheses of racemic melleolide A (78) and F (83) melleolide A (78) and F (83) were reported recently [62]. were reported recently [62].

FigureFigure 15. 15.Structures Structures of of melleolides melleolides 7878–93 fromfrom the the ArmillariaArmillaria genusgenus of fungi. of fungi. Antibiotics 2020, 9, 928 13 of 29

Other melleolides isolated from A. mellea include 4-O-methylmelleolide (94)[64], Antibiotics 2020, 9, 928 13 of 29 dihydromelleolide (95)[86], 13-hydroxydihydromelleolide (96)[87], 10α-hydroxymelleolide (97)[87Other], 10melleolidesα-hydroxydihydromelleolide isolated from A. mellea (98 )[include87], 4-O 13-hydroxy-4-methoxymelleolide-methylmelleolide (94) [64], (99)[dihydromelleolide88], 4-dehydromelleolide (95) [86], 13-hydroxydihydromelleolide (100)[86], 6’-chloro-10 (96α) [87],-hydroxymelleolide 10α-hydroxymelleolide (101 ()[97) [87],72] and 6’-chloro-13-hydroxy-dihydromelleolide10α-hydroxydihydromelleolide (98) ([87],102 )[6913-hydroxy-4-methoxymelleolide]. Structure elucidation and conﬁrmation(99) [88], 4- of the absolutedehydromelleolide stereochemistry (100 of) 4- [86],O-methylmelleolide 6’-chloro-10α-hydroxymelleolide (94) was achieved (101 utilising) [72] and X-ray 6’-chloro-13-hydroxy- crystallography [64]. dihydromelleolide (102) [69]. Structure elucidation and confirmation of the absolute stereochemistry The relative conﬁgurations of 13-hydroxydihydromelleolide (96), 13-hydroxy-4-methoxymelleolide of 4-O-methylmelleolide (94) was achieved utilising X-ray crystallography [64]. The relative (99) and 6’-chloro-10α-hydroxymelleolide (101) (Figure 16) were determined using NOESY configurations of 13-hydroxydihydromelleolide (96), 13-hydroxy-4-methoxymelleolide (99) and 6’- α spectroscopicchloro-10α-hydroxymelleolide data [72,87,88]. ( Unlike101) (Figure the 16) other were melleolides, determined using 10 -hydroxymelleolide NOESY spectroscopic data (97) and 6’-chloro-13-hydroxy-dihydromelleolide[72,87,88]. Unlike the other melleolides, ( 10210α)-hydroxymelleolide were found to be(97 weakly) and 6’-chloro-13-hydroxy- cytotoxic and against CCRF-CEMdihydromelleolide (IC50 63.23 (102µM)) were and found Jurkat to (IC be 50weakly46.6 µ cytotoxicM) cells, and respectively against CCRF-CEM [68,69]. Upon(IC50 63.23 investigation µM) of theand antibacterial Jurkat (IC50 46.6 properties µM) cells, of respectively some of these [68,69]. compounds, Upon investigation94 exhibited of the antibacterial activity against propertiesB. subtilis ATCC6633of some and of S.these aureus compounds,ATCC53156 94 exhibited at a minimum activity loadingagainst B. of subtilis 5.6 µ gATCC6633 per disk, and while S. 97aureusand 101 exhibitedATCC53156 activity at against a minimumB. subtilis loadingATCC6633 of 5.6 µg per (MIC disk, 0.5 whileµg) and 97 andE. coli 101(MIC exhibited 1.0 µ activityg) on bioautographic against B. assayssubtilis [64,72 ATCC6633]. In addition, (MIC the0.5 µg) latter and two E. coli also (MIC exhibited 1.0 µg) antifungalon bioautographic activity assays against [64,72].C. cucumerinum In addition, and the latter two also exhibited antifungal activity against C. cucumerinum and C. albicans with MICs of C. albicans with MICs of 1.0 µg on bioautographic assays [72]. 1.0 µg on bioautographic assays [72].

FigureFigure 16. 16.Structures Structures of of melleolides melleolides 94––102102 isolatedisolated from from ArmillariaArmillaria mellea. mellea.

Of theOf the melledonals melledonals (Figure (Figure 17),17), a agroup group of α of,β-unsaturatedα,β-unsaturated aldehydes, aldehydes, melledonals melledonals A–C (103– A–C (103–105))[ [82,89],82,89], 15-hydroxy-5’- 15-hydroxy-5’-O-methylmelledonalO-methylmelledonal (106 ()106 [90])[ 90and] and 5’-O 5’--methylmelledonalO-methylmelledonal (107) (107[90])[ 90] were isolated from A. mellea, while melledonals D (108) and E (109) were isolated from Clitocybe were isolated from A. mellea, while melledonals D (108) and E (109) were isolated from Clitocybe elegans [77]. The relative configurations of 105 and 108 were assigned by NOESY spectroscopic AntibioticsAntibiotics2020 2020,,9 9,, 928 928 1414 ofof 2929 elegans [77]. The relative configurations of 105 and 108 were assigned by NOESY spectroscopic analysis of the natural products, while for 103 and 106, analysis was conducted on a diacetate derivative analysis of the natural products, while for 103 and 106, analysis was conducted on a diacetate and a dibenzoate derivative, respectively [77,82,89,90]. On the other hand, the relative configurations derivative and a dibenzoate derivative, respectively [77,82,89,90]. On the other hand, the relative of 104, 107 and 109 were assigned by biogenetic consideration [77,82,89,90]. The absolute configuration configurations of 104, 107 and 109 were assigned by biogenetic consideration [77,82,89,90]. The of melledonal C (105) was established by X-ray crystallographic analysis [82]. Of the melledonals, absolute configuration of melledonal C (105) was established by X-ray crystallographic analysis [82]. only 105 was found to exhibit cytotoxicity against human cancer cell lines, albeit weakly (CEM IC Of the melledonals, only 105 was found to exhibit cytotoxicity against human cancer cell lines, albeit50 49.6 µM, HT-29 IC50 85.6 µM) [74]. Melledonal (103) and melledonal C (105) were investigated for weakly (CEM IC50 49.6 µM, HT-29 IC50 85.6 µM) [74]. Melledonal (103) and melledonal C (105) were antifungal activity; only 103 exhibited weak activity (MIC 50 µg/mL) against T. panuoides, O. illudens investigated for antifungal activity; only 103 exhibited weak activity (MIC 50 µg/mL) against T. and F. pinicola [73]. In addition, melledonals A–C (103–105) were found to exhibit antimicrobial panuoides, O. illudens and F. pinicola [73]. In addition, melledonals A–C (103–105) were found to exhibit activity against B. cereus ATCC10702 and B. subtilis ATCC6633 at a loading of 100 µg per disk with antimicrobial activity against B. cereus ATCC10702 and B. subtilis ATCC6633 at a loading of 100 µg 105 also exhibiting activity against E. coli (MIC 1.0 µg) and C. albicans (MIC 3.0 µg) in bioautographic per disk with 105 also exhibiting activity against E. coli (MIC 1.0 µg) and C. albicans (MIC 3.0 µg) in assays [72,82]. A dihydro derivative of melledonal, melledonol (110) was also isolated from A. mellea [89]. bioautographic assays [72,82]. A dihydro derivative of melledonal, melledonol (110) was also isolated Structure elucidation was conducted by NMR spectroscopic analysis and the relative configuration from A. mellea [89]. Structure elucidation was conducted by NMR spectroscopic analysis and the assigned by biosynthetic consideration [89]. relative configuration assigned by biosynthetic consideration [89].

FigureFigure 17.17. StructuresStructures ofof melledonalsmelledonals 103103––109109 andand melledonolmelledonol ((110110)) isolatedisolated fromfrom thethe ArmillariaArmillaria andand ClitocybeClitocybegenera genera ofof fungi.fungi.

OtherOther arylaryl esterester sesquiterpenoidssesquiterpenoids containingcontaining∆ Δ2,32,3-protoilludene-protoilludene skeletonsskeletons isolatedisolated fromfrom A.A. melleamellea includeinclude armillarinarmillarin ((111111)[) [91],91], armillaridinarmillaridin ((112112)[) [91],91], armillariginarmillarigin ((113113)[) [81],81], armillarilinarmillarilin ((114)[) [92],92], armillarininarmillarinin ( (115115))[ [92],92 ],armillaripin armillaripin (116 ()116 [93],)[ armillaritin93], armillaritin (117) [94], (117 armillarivin)[94], armillarivin (118) [94], ( 118armillasin)[94], armillasin(119) [95], (119armillatin)[95], armillatin (120) [95], (120 armillaric)[95], armillaric acid (121 acid) [96], (121 )[1096α-hydroxyarmillarin], 10α-hydroxyarmillarin (122) ([97],122)[ 4-97O],- 4-methylarmillaridinO-methylarmillaridin (123) ([97],123)[ 5’-methoxy-armillasin97], 5’-methoxy-armillasin (124) [84] (124 and)[ 5-hydroxyl-armillarivin84] and 5-hydroxyl-armillarivin (125) [84]. (Armillasin125)[84]. Armillasin(119), armillatin (119), armillatin (120) and (120 )5’-methoxy-armillasin and 5’-methoxy-armillasin (124 ()124 are) are examples examples ofof norsesquiterpenoids,norsesquiterpenoids, whilewhile armillaricarmillaric acidacid ((121121)) isis thethe ﬁrstfirst exampleexample ofof ananα α,β,β-unsaturated-unsaturated carboxyliccarboxylic acid-containingacid-containing aryl aryl sesquiterpenoid sesquiterpenoid (Figure (Figure 18)[84 ,9518),98 ].[84,95,98]. Assignment Assignment of the absolute of stereochemistrythe absolute ofstereochemistry111 was achieved of 111 by was X-ray achieved crystallographic by X-ray crystallographic analysis, while thatanalysis, of 112 whilewas that ascertained of 112 was by considerationascertained by of consideration shared biogenetic of shared origins biogenetic [91]. The relativeorigins [91]. conﬁgurations The relative of configurations119, 121, 124 and of125 119were, 121, established124 and 125 by were NOESY established spectroscopic by NOESY analysis, spectr whileoscopic that of analysis,120, 122 andwhile123 thatwere of assigned120, 122 byand biogenetic 123 were considerationassigned by biogenetic [84,95,97,98 consideration]. Several of these [84,95,97,98 natural products]. Several have of these been reportednatural products to exhibit have moderate been cytotoxicityreported to exhibit towards moderate human cancercytotoxicity cell lines towards [69,70 hu,74man,84,85 cancer]. Armillarin cell lines ( 111[69,70,74,84,85].) and armillaridin Armillarin (112) (111) and armillaridin (112) have been shown to be moderately cytotoxic towards HUVEC (GI50 9.9, 7.8 µM), K-562 (GI50 9.9, 8.9 µM), MCF7 (GI50 11.6, 7.8 µM) and HeLa (CC50 16.7, 9.2 µM) cell lines Antibiotics 2020, 9, 928 15 of 29

have been shown to be moderately cytotoxic towards HUVEC (GI50 9.9, 7.8 µM), K-562 (GI50 9.9, Antibiotics 2020, 9, 928 15 of 29 8.9 µM), MCF7 (GI50 11.6, 7.8 µM) and HeLa (CC50 16.7, 9.2 µM) cell lines with the latter also exhibiting cytotoxicity towards H460, CEM, HepG2 and L02 (IC50 4.5 µM, 5.1 µM, 13.37 µg/mL and 12.15 µg/mL) with the latter also exhibiting cytotoxicity towards H460, CEM, HepG2 and L02 (IC50 4.5 µM, 5.1 µM, 112 117 119 cell lines.13.37 Meanwhile, µg/mL and 12.15 armillarilin µg/mL) (cell lines.), armillaritin Meanwhile, ( armillarilin), armillasin (112 ( ), armillaritin) and 5-hydroxyl-armillarivin (117), armillasin 125 µ ( ) exhibited(119) and 5-hydroxyl-armillarivin cytotoxicity towards HepG2(125) exhibited (IC50 13.25, cytotoxicity 12.26, towards 15.63, 18.03 HepG2g (IC/mL)50 13.25, and 12.26, L02 (IC 15.63,50 18.00, 31.95,18.03 14.38, µg/mL) 22.70 µ andg/mL) L02 cells. (IC50 In18.00, addition, 31.95, the14.38, cytotoxicity 22.70 µg/mL) ofarmillarigin cells. In addition, (113) (ICthe 50cytotoxicityHL-60 14.27 of µM, SMMC-7721armillarigin 19.51 (113µM,) (IC A-54650 HL-60 10.01 14.27µM, µM, MCF7 SMMC-7721 10.67 µM, 19.51 SW480 µM, 19.19 A-546µM) 10.01 and µM, 4-O MCF7-methylarmillaridin 10.67 µM, (123) (ICSW48050 MCF7 19.19 µM) 6.7 µandM, 4- JurkatO-methylarmillaridin 4.1 µM, HeLa ( 18.8123) µ(ICM)50 MCF7 have also6.7 µM, been Jurkat investigated 4.1 µM, HeLa [69 18.8,70,74 µM),84 ,85]. On thehave other also hand,been investigated armillaric [69,70,74,84,85]. acid (121) has On been the shownother hand, to exhibitarmillaric antimicrobial acid (121) has activitybeen shown against S. aureusto exhibit209PJC antimicrobial (19.8 mm), activityM. lutaus againstATCC9341 S. aureus (8.8 209PJC mm) and(19.8C. mm), albicans M. lutausNHL4019 ATCC9341 (10.1 (8.8 mm) mm) in zone of inhibitionand C. albicans assays NHL4019 at a loading (10.1 of mm) 250 inµg zone/mL of per inhibition disk [96 assays,98]. at a loading of 250 µg/mL per disk [96,98].

2,3 FigureFigure 18. Structures 18. Structures of ∆of2,3 Δ-protoilludene-containing-protoilludene-containing aryl aryl sesquiterpenoids sesquiterpenoids 111111–125–125 isolatedisolated from from ArmillariaArmillaria mellea. mellea.

ArmellidesArmellides A (126 A ()126 and) and B ( B127 (127), isolated), isolated from fromArmillaria Armillaria novae-zelandiae novae-zelandiae, were, were the the first first examples examples of aryl ester protoilludanes esterified at the C-1 hydroxyl group (Figure 19) [80]. Two related of aryl ester protoilludanes esterified at the C-1 hydroxyl group (Figure 19)[80]. Two related armellides, 13-deoxyarmellides A (128) and B (129) were later reported from A. mellea [70]. The armellides,relative 13-deoxyarmellides configurations of armellides A (128) andA (126 B (129) and) were B (127 later) were reported determined from A.by melleaNOESY[70 NMR]. The data, relative configurations of armellides A (126) and B (127) were determined by NOESY NMR data, while those Antibiotics 2020, 9, 928 16 of 29

Antibiotics 2020, 9, 928 16 of 29 of 13-deoxyarmellides A (128) and B (129) were assigned by consideration of shared biogenetic while those of 13-deoxyarmellides A (128) and B (129) were assigned by consideration of shared origins [70,80]. The cytotoxicity of 127–129 have been investigated and only 129 was found to be biogeneticAntibiotics origins 2020, 9, [70,80].928 The cytotoxicity of 127–129 have been investigated and only 129 16was of 29 found cytotoxic, albeit weakly against HUVEC (GI50 91.0 µM), K-562 (GI50 91.8 µM), MCF7 (GI50 90.8 µM) to be cytotoxic, albeit weakly against HUVEC (GI50 91.0 µM), K-562 (GI50 91.8 µM), MCF7 (GI50 90.8 and HeLawhile (CCthose50 of89.5 13-deoxyarmellidesµM) cell lines [69 A,70 (128]. ) and B (129) were assigned by consideration of shared µM) and HeLa (CC50 89.5 µM) cell lines [69,70]. biogenetic origins [70,80]. The cytotoxicity of 127–129 have been investigated and only 129 was found to be cytotoxic, albeit weakly against HUVEC (GI50 91.0 µM), K-562 (GI50 91.8 µM), MCF7 (GI50 90.8 µM) and HeLa (CC50 89.5 µM) cell lines [69,70].

Figure 19. StructuresStructures of armellides 126–129 isolated from the Armillaria genus of fungi. Figure 19. Structures of armellides 126–129 isolated from the Armillaria genus of fungi. Another mushroom from the Armillaria genus, A. tabescens, affordedaﬀorded four melleolides including 5β,10,10α-dihydroxy-1-orsellinate-dihydromelleolide-dihydroxy-1-orsellinate-dihydromelleolideAnother mushroom from the Armillaria genus, A.( 130 tabescens,), 4-dehydro-14-hydroxydihydromelleolide4-dehydro-14-hydr afforded four melleolidesoxydihydromelleolide including (131),5 β 4-dehydrodihydromelleolide,10α-dihydroxy-1-orsellinate-dihydromelleolide ( (132132)) and and 14-hydroxydihydromelleolide (130), 4-dehydro-14-hydroxydihydromelleolide ( (133133)) (Figure (Figure 20)20)[ [88].88]. Assignment(131), 4-dehydrodihydromelleolide of the relative conﬁgurationconfiguration (132 of) and all four14-hydroxydihydromelleolide natural products was achieved (133) (Figure utilising 20) [88]. NOESY Assignment of the relative configuration of all four natural products was achieved utilising NOESY spectroscopic data [[88].88]. spectroscopic data [88].

Figure 20. Structures of melleolides 130–133 isolated from Armillaria tabescens. Figure 20. Structures of melleolides 130–133 isolated from Armillaria tabescens. MoreMore examples examplesFigure of 20.∆ of 2,3Structures Δ-protoilludene-type2,3-protoilludene-type of melleolides sesquiterpenoid130–133 isolated aryl from aryl esters Armillaria esters were were isolatedtabescens. isolated from from several several undeterminedundeterminedArmillaria Armillariaspp. spp (Figure. (Figure 21 21)).. TheseThese included 10-hydroxydihydromelleolide 10-hydroxydihydromelleolide (134) ([86],134)[ 86], MoreA52a ( examples135) [99], A52b of Δ (2,3136-protoilludene-type) [99], 1-O-formyl-10-dehydroxy-melleolide sesquiterpenoid aryl estersB (137 )were [85], 10-oxo-melleolideisolated from several A52a (135)[99], A52b (136)[99], 1-O-formyl-10-dehydroxy-melleolide B (137)[85], 10-oxo-melleolide undeterminedB (138) [85], Armillaria 10-dehydroxymelleolide spp. (Figure 21)D (.139 These) [83] includedand armilliphatic 10-hydroxydihydromelleolide A (140). Structure elucidation (134 of) [86], B(138)[85], 10-dehydroxymelleolide D (139)[83] and armilliphatic A (140). Structure elucidation of A52aall (135 seven) [99], natural A52b products (136) [99], was 1- achievedO-formyl-10-dehydroxy-melleolide by NMR spectroscopic analysis [51,83,85,86,99].B (137) [85], 10-oxo-melleolide The relative all seven natural products was achieved by NMR spectroscopic analysis [51,83,85,86,99]. The relative B (138configurations) [85], 10-dehydroxymelleolide of A52b (136) (also named D (139 10-dehydroxy-melleolide) [83] and armilliphatic B) andA (140 armilliphatic). Structure A (elucidation140) were of conﬁgurationsdetermined of by A52b analysis (136 of) (alsoROESY named data, while 10-dehydroxy-melleolide those of 1-O-formyl-10-dehydroxy-melleolide B) and armilliphatic A B ( 140(137)) were all seven natural products was achieved by NMR spectroscopic analysis [51,83,85,86,99]. The relative determinedand 10-oxo-melleolide by analysis of B ROESY (138) were data, assigned while thoseby biogenetic of 1-O-formyl-10-dehydroxy-melleolide consideration [51,85,99]. The absolute B (137) configurations of A52b (136) (also named 10-dehydroxy-melleolide B) and armilliphatic A (140) were and 10-oxo-melleolideconfigurations of 139 B and (138 140) were were assigned established by by biogenetic comparison consideration of experimental [51 ,ECD85,99 spectra]. The with absolute determined by analysis of ROESY data, while those of 1-O-formyl-10-dehydroxy-melleolide B (137) and 10-oxo-melleolide B (138) were assigned by biogenetic consideration [51,85,99]. The absolute configurations of 139 and 140 were established by comparison of experimental ECD spectra with Antibiotics 2020, 9, 928 17 of 29 conﬁgurations of 139 and 140 were established by comparison of experimental ECD spectra with thoseAntibiotics of melleolide 2020, 9, 928 D (81) and TDDFT calculated data, respectively [51,83]. A52a (135), A52b17 of 29 ( 136), 1-O-formyl-10-dehydroxy-melleolide B (137), 10-oxo-melleolide B (138) and armilliphatic A (140) werethose investigated of melleolide forcytotoxicity D (81) and TDDFT against calculated several data, cell linesrespectively where [51,83].138 was A52a found (135 to), A52b be non-cytotoxic (136), 1- O-formyl-10-dehydroxy-melleolide B (137), 10-oxo-melleolide B (138) and armilliphatic A (140) were (IC50 > 40 µM)[51,69,85]. Aryl esters 135–137 and 140 exhibited moderate cytotoxicity against HL-60 investigated for cytotoxicity against several cell lines where 138 was found to be non-cytotoxic (IC50 (IC50 17.06, 17.79, 14.50, 15.80 µM), SMMC-7721 (IC50 17.77, 20.90, 23.16, 19.42 µM), A-549 (IC50 15.89, > 40 µM) [51,69,85]. Aryl esters 135–137 and 140 exhibited moderate cytotoxicity against HL-60 (IC50 16.79, 18.41, 15.93 µM), MCF7 (IC 14.10, 16.49, 5.34, 19.22 µM) and SW480 (IC 15.70, 17.44, 10.77, 17.06, 17.79, 14.50, 15.80 µM), SMMC-772150 (IC50 17.77, 20.90, 23.16, 19.42 µM), A-549 50(IC50 15.89, 16.79, µ 23.0318.41,M) cell15.93 lines µM), [ 51MCF7,69, 85(IC].50 In 14.10, addition, 16.49, 5.34,135 was19.22 also µM) investigated and SW480 (IC for50 15.70, cytotoxicity 17.44, 10.77, against 23.03 Jurkat (IC50 µM)10.4 cellµM), lines HeLa [51,69,85]. (IC50 40.0 In µaddition,M) and 135 K-562 was (IC also50 38.9 investigatedµM) cell for lines cytotoxicity [69]. The against antimicrobial Jurkat (IC activities50 of 13510.4and µM),136 HeLawere (IC investigated50 40.0 µM) and against K-562Heterobasidion (IC50 38.9 µM) cell annosum lines [69]., Gloeophyllum The antimicrobial abietinum activities, E. coli of and S. aureus135 onand disks. 136 were However, investigated neither against were Heterobasidion found to be active annosum [99, ].Gloeophyllum Armilliphatic abietinum A (140, E.)[ 51coli], and which S. was isolatedaureus from on adisks. co-culture However, with neitherEpicoccum were sp.,found was to foundbe active to [99]. exhibit Armilliphatic weak acetylcholinesterase A (140) [51], which inhibitory was isolated from a co-culture with Epicoccum sp., was found to exhibit weak acetylcholinesterase activity (IC50 23.85 0.20 µM) [51]. Another aryl ester, the 13,14-dihydroxylated derivative of A52a inhibitory activity± (IC50 23.85 ± 0.20 µM) [51]. Another aryl ester, the 13,14-dihydroxylated derivative (141), was isolated from A. mellea and was found to be non-cytotoxic against MCF7, Jurkat, HeLa and of A52a (141), was isolated from A. mellea and was found to be non-cytotoxic against MCF7, Jurkat, K-562 cell lines (IC50 > 100 µM) [69]. HeLa and K-562 cell lines (IC50 > 100 µM) [69].

FigureFigure 21. Structures 21. Structures of ∆of2,3 Δ2,3-protoilludene-containing-protoilludene-containing aryl aryl sesquiterpenoids sesquiterpenoids 134134–141– 141isolatedisolated from fromthe the ArmillariaArmillariagenus genus of fungi. of fungi.

The lastThe last of the of the three three types types of of aryl aryl ester ester protoilludaneprotoilludane structures structures can can be beidentified identified by absence by absence of of double bonds in the 5/6/4-ring system. The first example of this type of aryl ester sesquiterpenoid, double bonds in the 5/6/4-ring system. The first example of this type of aryl ester sesquiterpenoid, armillane (142) also known as armillarizin, was isolated from A. mellea (Figure 22) [97]. Other armillane (142) also known as armillarizin, was isolated from A. mellea (Figure 22)[97]. Other examples of examples of aryl esters with saturated protoilludane skeletons from A. mellea include 5’-methoxy-6’- aryl esterschloroarmillane with saturated (143) [62], protoilludane 10-hydroxy-5’-methoxy-6’-chloroarmillane skeletons from A. mellea include (144 5’-methoxy-6’-chloroarmillane) [70] and melleolides Q–S (143)[(14562],–147 10-hydroxy-5’-methoxy-6’-chloroarmillane) [74,79]. The relative configurations of 142 (144, 145)[ 70and] and 146 melleolideswere determined Q–S ( 145by –NOESY147)[74 ,79]. The relativespectroscopic configurations analysis, while of 142 those, 145 ofand 143146 andwere 144 determinedwere assigned by by NOESY consideration spectroscopic of common analysis, whilebiogenetic those of 143originsand [69,70,74].144 were Melleolide assigned R by (146 consideration) is another example of common of an aryl biogenetic ester sesquiterpenoid origins [69,70 ,74]. Melleolidewith esterification R (146) is another at the examplehydroxyl ofof anC-1 aryl [74]. ester Both sesquiterpenoid 145 and 146 were with shown esterification to exhibit at moderate the hydroxyl of C-1cytotoxicity [74]. Both against145 and several146 were human shown cancer to exhibitcell lines moderate including cytotoxicity MCF7 (IC50 against 1.5 and several 3.7 µM, human respectively) and CEM (IC50 10.3 and 3.4 µM, respectively) [74]. In addition, 143 and 144 were also cancer cell lines including MCF7 (IC50 1.5 and 3.7 µM, respectively) and CEM (IC50 10.3 and 3.4 µM, Antibiotics 2020, 9, 928 18 of 29

Antibiotics 2020, 9, 928 18 of 29 respectively) [74]. In addition, 143 and 144 were also found to be cytotoxic albeit weakly (IC50 > 20 µM found to be cytotoxic albeit weakly (IC50 > 20 µM and > 40 µM, respectively) against HUVEC, K-562 and >40 µM, respectively) against HUVEC, K-562 and MCF7 cell lines [69,70]. and MCF7 cell lines [69,70].

FigureFigure 22. Structures22. Structures of saturatedof saturated protoilludane-containing protoilludane-containing aryl aryl sesquiterpenoids sesquiterpenoids 142–142147– 147isolatedisolated from fromArmillaria Armillaria mellea. mellea.

3. Ascomycota3. Ascomycota Ascomycota,Ascomycota, which which include include cupcup andand flaskflask fungi,fungi, moulds moulds and and most most yeasts, yeasts, account account for for approximatelyapproximately 60% 60% of theof the described described species species ofof higher fungi fungi and and are are well well known known for forproducing producing polyketides, non-ribosomal peptides and indole-derived natural products [12]. Fewer polyketides, non-ribosomal peptides and indole-derived natural products [12]. Fewer sesquiterpenoids sesquiterpenoids have been reported from Ascomycota in comparison to Basidiomycota with only a havefew been species reported of fungi from to Ascomycota date found to in have comparison produced to protoilludanes Basidiomycota bearing with the only annulated a few species 5/6/4-ring of fungi to datesystem. found The to havefirst protoilludane produced protoilludanes to be isolated bearingfrom an theAscomycete, annulated Ceratocystis 5/6/4-ring piceae system., was TheΔ6- first 6 protoilludaneprotoilludene to be (7 isolated) which fromis a anknown Ascomycete, natural productCeratocystis prev piceaeiously, wasreported∆ -protoilludene from two strains (7) which of is a knownBasidiomycota natural product [100]. previously reported from two strains of Basidiomycota [100]. IsomericIsomeric allylic allylic alcohols, alcohols, punctaporonins punctaporonins A ( 148),), D–F D–F (149 (149–151–151) (also) (also known known as punctatins), as punctatins), werewere isolated isolated from from the the dung dung fungus fungusPoronia Poronia punctatapunctata (Figure 23)23 )[[101–103].101–103 ].The The structures structures and and absoluteabsolute configurations configurations of punctaporonins of punctaporonins A, D andA, ED (148and–150 E )( were148–150 solved) were by X-raysolved crystallographic by X-ray analysescrystallographic of the natural analyses products of the or natu in theral products case of 150or in, onthe ancase acetonide of 150, on derivative. an acetonide For derivative. punctaporonin For punctaporonin F (151), a combination of derivatisation and NMR spectroscopic data comparison with F(151), a combination of derivatisation and NMR spectroscopic data comparison with the other the other punctaporonins was required to assign the structure, while the relative stereochemistry was punctaporoninsassigned by wasbiogenetic required consideration. to assign theOnly structure, punctaporonins while theD (149 relative) and E stereochemistry (150) have been shown was assigned to by biogeneticexhibit any consideration. bioactivity. In Onlyaddition, punctaporonins 149 was shown D to (149 inhibit) and a Emycelial (150) have form beenof Candida shown albicans to exhibit at 1 any bioactivity.ppm and In addition,150 inhibited149 wasTrichomonas shown tovaginalis inhibit at a mycelial100 ppm formin vitro of Candida[101–103]. albicans The enantiospecificat 1 ppm and 150 inhibitedsynthesesTrichomonas of both punctaporonins vaginalis at 100 A ppm(148) andin vitro D (149[101) have–103 been]. The reported enantiospecific [104]. syntheses of both punctaporonins A (148) and D (149) have been reported [104]. Antibiotics 2020, 9, 928 19 of 29 Antibiotics 2020, 9, 928 19 of 29 Antibiotics 2020, 9, 928 19 of 29

Figure 23. Structures of punctaporonins 148–153 isolated from the Poronia and Pestalotiopsis genera of Figure 23. StructuresStructures of of punctaporonins punctaporonins 148148––153153 isolatedisolated from from the the PoroniaPoronia andand PestalotiopsisPestalotiopsis generagenera of fungi. offungi. fungi.

Two protoilludanes,protoilludanes, 6-hydroxypunctaporonin 6-hydroxypunctaporonin A A (152 (152) and)) andand E ( 153EE (()153 (Figure)) (Figure(Figure 23), were23),23), werewere isolated isolatedisolated from thefromfrom plant thethe pathogenplantplant pathogenpathogenPestalotiopsis Pestalotiopsis disseminate disseminate[105]. The [105][105] absolute.. The configurationabsolute configuration of 153 was of resolved 153 was by X-rayresolved crystallographic by X-ray crystallographic analysis of a mono-bromobenzoate analysis of a mono-bromobenzoate derivative, while derivative, that of 152 whilewas assigned that of 152 by considerationwas assigned ofby sharedconsideration biogenetic of shared origins. biogenetic Both 152 and origins.153 wereBoth investigated152 andand 153 forwere their investigated antimicrobial for activitiestheirtheir antimicrobialantimicrobial against B. activitiesactivities subtilis ATCC6051,againstagainst B. subtilisS. aureus ATCC6051,ATCC29213, S. aureusE. coli ATCC29213,ATCC25922 E.coli and ATCC25922ATCC25922C. albicans ATCC14053and C. albicans and ATCC14053ATCC14053153 was found andand to 153 exhibit waswas foundfound weak toto activity exhibitexhibit against weakweak activi theactivi twotyty Gram-positiveagainstagainst thethe twotwo bacterial Gram-positiveGram-positive strains µ inbacterial disk di ffstrainsusion assaysin disk at diffusion a loading assays of 100 atg pera lo diskading [105 of]. 100 In addition, µg per disk both [105]. compounds In addition,152 and both153 werecompounds found to152 be and non-cytotoxic 153 were found against to HeLabe non-cytotoxic cells with IC against>100 HeLaµM[ 106cells]. with IC50 >100 µM [106]. compounds 152 and 153 were found to be non-cytotoxic against50 HeLa cells with IC50 >100 µM [106]. Punctaporonins L ( 154)) and and M M ( (155)) (Figure(Figure 24)2424)) were were isolatedisolated from from the the sponge-derived sponge-derived fungus, fungus, HansfordiaHansfordia sinuosaesinuosae [[107][107]107]... The relative configurations configurations of both natural products were determineddetermined by analysis ofof NOESY NOESY spectroscopic spectroscopic data data with thewith absolute the absolute stereochemistry stereochemistry of 154 assigned of 154 by assigned comparison by ofcomparison optical rotations of optical with 153 rotations. The cytotoxicity with 153 and.. The antibacterialThe cytotoxicitycytotoxicity activity andand of punctaporoninsantibacterialantibacterial activityactivity L (154) and ofof µ M(punctaporonins155) were investigated. L (154)) andand Both MM ((154155and)) werewere155 investigated.investigated.exhibited no BothBoth antibacterial 154 andand activity 155 exhibitedexhibited (MIC > 125nono antibacterialantibacterialM) against µ E.activity coli, S. (MIC aureus >125, Bacillus µM) against thuringensis E. colior,, S.B. aureus subtilis,, Bacillusand were thuringensis weakly cytotoxic oror B. subtilis (IC50 andand>10 werewereM) againstweaklyweakly HCT-8,cytotoxic Bel7402, (IC50 >10 BGC823, µM) against A549, HCT-8, and A2780 Bel7402, cell lines BGC823, [107]. A549, and A2780 cell lines [107]. cytotoxic (IC50 >10 µM) against HCT-8, Bel7402, BGC823, A549, and A2780 cell lines [107].

Figure 24. Structures of punctaporonins 154–160 isolated from the Hansfordia and Pestalotiopsis genera Figure 24. Structures of punctaporonins 154–160 isolated from the Hansfordia and Pestalotiopsis genera of fungi. of fungi.

Five protoilludanes, punctaporonins N N ( 156)) and and O O ( (157),), 6-hydroxypunctaporonin 6-hydroxypunctaporonin DD (( 158),), 6,13-dihydroxypunctaporonin A A ( 159)) and and 6,13-dihydroxypunctaporonin6,13-dihydroxypunctaporonin EE ( (160)) (Figure (Figure 24)2424)) werewere isolatedisolated fromfrom anotheranother speciesspecies ininin thethe PestalotiopsisPestalotiopsis genus,genus, PestalotiopsisPestalotiopsis sp.sp.. [[108].[108].108]. NOESY NOESY spectroscopic spectroscopic analysis ledled to to assignment assignment of relative of relative configurations configurations of all the of natural all the products natural except products for punctaporonin except for N(punctaporonin156). The relativeN (156).). and TheThe absolute relativerelative configurationsandand absoluteabsolute configurationsconfigurations of 156 were assignedofof 156 were based assigned on biosynthetic based on considerationbiosynthetic consideration upon successful upon conversion successful of conversion 6-hydroxypunctaporonin of 6-hydroxypunctaporonin A (152) to 156 A. The(152 absolute)) toto 156.. configurationsThe absolute configurations of 158 and 159 ofwere 158 ascertained andand 159 werewere by considerationascertainedascertained byby of considerationconsideration shared biogenetic ofof sharedshared origins biogeneticbiogenetic with 152. Subsequently,origins with 152 the.. absoluteSubsequently,Subsequently, configurations thethe absoluteabsolute of 157 configurationsconfigurationsand 160 wereassigned ofof 157 andand on biogenetic 160 werewere consideration assignedassigned onon ofbiogenetic158 and 153consideration, respectively. of 158 All and five 153 compounds,, respectively.respectively.156– 160AllAll fivewerefive compoundscompounds investigated 156 for–160 antimicrobial werewere investigatedinvestigated activity againstforfor antimicrobialantimicrobialA. flavus, Fusarium activityactivity againstagainst verticillioides, A. flavus, S. aureus Fusarium, B. subtilis verticillioides,, E. coli and S. C.aureus albicans,, B. subtilisat a loading,, E. coli of and 100and µ C.g peralbicans disk. atat However, aa loadingloadingnone ofof 100100 of µgµg the perper compounds disk.disk. However,However, were found nonenone ofof active thethe compoundscompounds [108]. werewere foundfound activeactive [108].[108]. A further five protoilludanes, punctaporonins O–S (161–165),), werewere isolatedisolated fromfrom thethe plantplant pathogen, Cytospora sp.sp. (Figure(Figure 25)25) [106].[106]. DeterminationDetermination ofof thethe absoluteabsolute configurationconfiguration ofof punctaporonin O (161)) waswas achievedachieved byby comparisoncomparison ofof opticaloptical rotationsrotations withwith 6-6- Antibiotics 2020, 9, 928 20 of 29

A further ﬁve protoilludanes, punctaporonins O–S (161–165), were isolated from the plant pathogen, Cytospora sp. (Figure 25)[106]. Determination of the absolute conﬁguration of punctaporonin Antibiotics 2020, 9, 928 20 of 29 O(161) was achieved by comparison of optical rotations with 6-hydroxypunctaporonin A (152) which led to assignment of the remaining natural products by consideration of shared biogenetic origins. hydroxypunctaporonin A (152) which led to assignment of the remaining natural products by The cytotoxicity of puctaporonins O–S (161–165) were investigated against HeLa cells, where 161, considerationAntibiotics 2020 of, 9shared, 928 biogenetic origins. The cytotoxicity of puctaporonins O–S (161–20165 of )29 were 164 and 165 exhibited moderate cytotoxicity (IC50 16.6, 10.4 and 47.4 µM, respectively), while 162 and investigated against HeLa cells, where 161, 164 and 165 exhibited moderate cytotoxicity (IC50 16.6, 163 werehydroxypunctaporonin non-cytotoxic (IC 50A >(100152) µwhichM) [106 led]. to assignment of the remaining natural products by 10.4 and 47.4 µM, respectively), while 162 and 163 were non-cytotoxic (IC50 >100 µM) [106]. consideration of shared biogenetic origins. The cytotoxicity of puctaporonins O–S (161–165) were investigated against HeLa cells, where 161, 164 and 165 exhibited moderate cytotoxicity (IC50 16.6, 10.4 and 47.4 µM, respectively), while 162 and 163 were non-cytotoxic (IC50 >100 µM) [106].

Figure 25. Structures of punctaporonins O–S (161–165) isolated from Cytospora sp. Figure 25. Structures of punctaporonins O–S (161–165) isolated from Cytospora sp. Figure 25. Structures of punctaporonins O–S (161–165) isolated from Cytospora sp. The plant-derived fungus, fungus, PhomopsisPhomopsis sp.,sp., afforded afforded eight eight protoilludanes protoilludanes named named phomophyllin phomophyllin B– B–I (166–173) (Figure 26)[109]. Assignment of the relative configurations of all phomophyllins I (166–173The) (Figure plant-derived 26) [109]. fungus, Assignment Phomopsis of sp., the afforded relative eight configurations protoilludanes of namedall phomophyllins phomophyllin 166 B– –173 166was– 173achievedI (166was–173 achieved ) by(Figure NOESY 26) by [109]. NOESY spectroscopic Assignment spectroscopic analysof the relative analysis.is. X-ray configurations X-raycrystallographic crystallographic of all phomophyllins analysis analysis of 166 166 of –and173166 and173 173allowedallowedwas assignmentachieved assignment by ofNOESY the of the absolutespectroscopic absolute configurations configurations analysis. X-ray of of crystallographicthese these two two natural natural analysis products. of 166 andA A modifiedmodified 173 Mosher’sallowed method assignment was utilisedof the absoluteto establish configurations the absolute of these configurations configurations two natural of products. 170 and A 171 modified, while the absoluteMosher’s configurationsconfigurations method was of theutilised remaining to establish phomophyllins the absolute ( 167configurations–169,, 172) were of 170 assigned and 171 ,by while comparison the of experimentalabsolute configurations ECD spectra of the with remaining TDDFT phomophyllins calculatedcalculated ECD (167 spectra. –169, 172 All All) were the the assignedphomophyllins phomophyllins by comparison ( (166166–– 173173),), exceptof forexperimental 172, were ECD investigatedinvestigated spectra with forfor TDDFT inhibitioninhibition calc ulated ofof β-site ECD amyloid spectra. precursorAll the phomophyllins protein-cleaving (166–173 enzyme), except for 172, were investigated for inhibition of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) which is a target for Alzheimer’s disease. They exhibited inhibiti inhibitionon rates ranging between 1 (BACE1) which is a target for Alzheimer’s disease. They exhibited inhibition rates ranging between 19–40% at a concentration of 40 µµM,M, whilewhile exhibiting no hepatoxicity to L-02 liver cells at the same 19–40% at a concentration of 40 µM, while exhibiting no hepatoxicity to L-02 liver cells at the same concentration [109]. concentrationconcentration [109]. [109].

FigureFigure 26. 26.Structures Structures of of phomophyllins phomophyllins B–I ( 166166––173173) )isolated isolated from from PhomopsisPhomopsis sp. sp.

4. Protoilludanes4. ProtoilludanesFigure from 26. from Structures Other Other Sources Sources of phomophyllins B–I (166–173) isolated from Phomopsis sp.

4. ProtoilludanesA fewA examplesfew examples from of Other protoilludanesof protoilludanes Sources have have beenbeen isolat isolateded from from sources sources other other than than fungi fungi (Figure (Figure 27). 27). TwoTwo marine marine natural natural products, products, paesslerins paesslerins AA ((174174) and B B (175 (175) )were were isolated isolated from from the thesoft softcoral coral AAlcyonium few examples paessleri of [110]. protoilludanes The relative have configurations been isolat edof 174from and sources 175 were other assigned than fungi by NOESY(Figure 27). Alcyonium paessleri [110]. The relative conﬁgurations of 174 and 175 were assigned by NOESY Two spectroscopicmarine natural analysis prod [110].ucts, Bothpaesslerins natural productsA (174) exhibitedand B (175 modera) werete cytotoxicityisolated from in preliminary the soft coral spectroscopic analysis [110]. Both natural products exhibited moderate cytotoxicity in preliminary Alcyoniumstudies paessleri [110]. A stereoselective[110]. The relative synthesis configurations for the proposed of structure 174 and of 175174 waswere reported assigned a few by years NOESY studies [110]. A stereoselective synthesis for the proposed structure of 174 was reported a few years spectroscopicafter its isolation. analysis However, [110]. Both the spectroscopicnatural products data was exhibited not in agreement moderate resulting cytotoxicity in the inconclusion preliminary studiesthat [110]. a structure A stereoselective correction was synt requiredhesis [111].for the More proposed recently, structure another total of 174 synthesis was reported of the suspected a few years structure of 174 was reported for which the spectroscopic data was in agreement with the natural after its isolation. However, the spectroscopic data was not in agreement resulting in the conclusion product resulting in revision of the natural product structure [112]. that a structure correction was required [111]. More recently, another total synthesis of the suspected structure of 174 was reported for which the spectroscopic data was in agreement with the natural product resulting in revision of the natural product structure [112]. Antibiotics 2020, 9, 928 21 of 29 after its isolation. However, the spectroscopic data was not in agreement resulting in the conclusion that a structure correction was required [111]. More recently, another total synthesis of the suspected structure of 174 was reported for which the spectroscopic data was in agreement with the natural product resulting in revision of the natural product structure [112]. Antibiotics 2020, 9, 928 21 of 29

Figure 27. Structures of protoilludanes 174–180 isolated from marine andand plantplant sources.sources.

The firstfirst example of aa protoilludaneprotoilludane sesquiterpenesesquiterpene glucoside, pteridanoside (176), waswas isolatedisolated from thethe brackenbracken fern fernPteridium Pteridium aquilinum aquilinumvar. var.caudatum caudatumalong along with with its aglycon its aglycon pteridanone pteridanone (177)[ (113177].) Determination[113]. Determination of the of relative the relative configuration configuration of 177 ofwas 177 achieved was achieved utilising utilisin NOESYg NOESY spectroscopic spectroscopic data. Thedata. absolute The absolute configuration configuration of pteridanone of pteridanone was determined was determined by a combination by a combination of ECD spectralof ECD analysisspectral andanalysis conformational and conformational calculations calc uponulations conversion upon conversion of 177 to 176 of .177 Pteridanoside to 176. Pteridanoside (176), unlike (176 its), aglycon unlike 177its ,aglycon exhibited 177 weak, exhibited toxicity weak against toxicity brine shrimp,against Artemiabrine shrimp, salina (LC Artemia50 = 250 salina and (LC 62.550µ =g /250mL, and 24 h 62.5 and 48µg/mL, h) [113 24]. h and 48 h) [113]. Xanthocerapene ((178178)) waswas isolated isolated from from the the wood wood of ofXanthoceras Xanthoceras sorbifolia sorbifoliawith with assignment assignment of the of relativethe relative stereochemistry stereochemistry of the of naturalthe natural product prod achieveduct achieved utilising utilising NOESY NO spectroscopicESY spectroscopic data [114data]. Another[114]. Another protoilludane protoilludane from a plant source,from 2,2,4a,7a-tetramethyldecahydro-1a plant source, 2,2,4a,7a-tetramethyldecahydro-1H-cyclobuta[e]inden-5-olH- (cyclobuta[179), was firste]inden-5-ol detected (179 by gas), was chromatography first detected by mass gas spectrometry chromatography (GC–MS) mass fromspectrometry 4-year-old (GC–MS)Radix et Rhizomafrom 4-year-old ginseng [ 115Radix]. Moreet Rhizoma recently, ginseng it has [115]. also been More detected recently, by GC–MSit has also from been other detected species by of ginseng.GC–MS However,from otherthe species relative of ginseng. stereochemistry However, of the the relati naturalve stereochemistry product remains of unknownthe natural [ 116product–118]. remains Lastly, (3unknownS,4aS,6R ,8aS[116–118].,8bR)-6-hydroxy-3,6,8a-trimethyloctahydro-3,8 Lastly, (3S,4aS,6R,8aS,8bR)-6-hydroxy-3,6,8a-trimethyloctahydro-3,8b-methanocyclobuta[h]chromene-5,9(6Hb)-- dionemethanocyclobuta[ (180) was isolatedh]chromene-5,9(6 from the rootsH)-dione of Lindera (180) strychnifolia was isolated[119 from]. The the roots relative of Lindera stereochemistry strychnifolia of 180[119]was. The assigned relative bystereochemistry analysis of NOESY of 180 was data, assigned while the by absoluteanalysis of configuration NOESY data, was while assigned the absolute upon comparisonconfiguration of was experimental assigned ECDupon data comparison with calculated of experimental data. The ECD natural data product with calculated has been reported data. The to improvenatural product the cell has viability been ofreported human to umbilical improve veinthe ce endothelialll viability cellsof human injured umbilical by oxidised vein low-density endothelial lipoproteincells injured [ 119by ].oxidised low-density lipoprotein [119]. All 180 protoilludanes were analysed on Datawarrior [[120]120] to calculate descriptors such as lipophilicity (cLogP),(cLogP), aqueousaqueous solubilitysolubility (cLogS)(cLogS) andand druglikeness.druglikeness. A total of 173 compounds showed cLogP values of less than 5, which is considered ideal for good absorption across cell membranes, while 139 139 were were considered considered to to have have good good solubility solubility with with cLogS cLogS values values greater greater than than-4, which4, whichare values are − valuesthat more that than more 80% than of80% the current of the current drugs drugson the onmarket the market have (Figure have (Figure 28). Lastly 28). Lastly the compounds the compounds were wereevaluated evaluated for their fortheir druglikeness druglikeness which which is a is descriptor a descriptor indicating indicating the the frequency frequency of of occurrence occurrence of fragments foundfound in in commercial commercial drugs drugs where where a positive a positive value statesvalue higherstates amountshigher amounts of these fragments of these infragments the compound. in the compound. Only 64 protoilludanes Only 64 protoilludanes showed positive showed values positive for the values druglikeness for the rangingdruglikeness from ranging from 0 to 2.8. Overall, 51 protoilludanes were found to have good lipophilicity, aqueous solubility and druglikeness properties. Antibiotics 2020, 9, 928 22 of 29

0 to 2.8. Overall, 51 protoilludanes were found to have good lipophilicity, aqueous solubility and druglikeness properties. Antibiotics 2020, 9, 928 22 of 29

Antibiotics 2020, 9, 928 22 of 29

Figure 28. Comparison of calculated descriptors from Datawarrior for protoilludanes (red) with current Figure 28. Comparison of calculated descriptors from Datawarrior for protoilludanes (red) with FDA-approvedcurrent FDA-approved drugs (grey). drugs (grey). Figure 28. Comparison of calculated descriptors from Datawarrior for protoilludanes (red) with 5. Biosynthetic5. Biosyntheticcurrent StudiesFDA-approved Studies drugs (grey).

The5. Biosynthetic biosynthesisThe biosynthesis Studies of two of two protoilludane protoilludane natural natural products, products, coprinolone coprinolone ( 51(51)) and and∆ Δ66-coprinolone-coprinolone (52), has been(52), investigatedhas been investigated using sodium using [1,2–sodium13C] [1,2– acetate13C] labellingacetate labelling experiments experiments [56,57 ].[56,57]. In both In instances,both The biosynthesis of two protoilludane natural products, coprinolone (51) and Δ6-coprinolone instances, successful incorporation13 of [1,2–13C] acetate was confirmed by the presence of enriched successful(52), has incorporation been investigated of [1,2– usingC] acetatesodium was [1,2– conﬁrmed13C] acetate by labelling the presence experiments of enriched [56,57]. carbon In both signals carbon13 signals in the 13C NMR spectrum.13 13Subsequently, 13C–13C connectivities13 from a 13C-COSY in theinstances,C NMR successful spectrum. incorporation Subsequently, of [1,2–C– 13C]C connectivitiesacetate was confirmed from a byC-COSY the presence experiment of enriched identiﬁed experiment identified the intact acetate units (Figure 29) which were in accordance with the labelling the intactcarbon acetate signals units in the (Figure 13C NMR 29) whichspectrum. were Subsequently, in accordance 13C– with13C connectivities the labelling from pattern a 13 expectedC-COSY for pattern expected for compounds derived from farnesyl diphosphate (Scheme 1) [56,57]. A similar compoundsexperiment derived identified from the farnesyl intact acetate diphosphate units (Figur (Schemee 29) which1)[ were56,57 in]. accordance A similar with feeding the labelling experiment feeding experiment was conducted on Armillaria mellea, to investigate the origin of the orsellinic acid pattern expected for compounds derived from farnesyl diphosphate (Scheme 1) [56,57]. A similar was conductedmoiety in arnamial on Armillaria (75) using mellea sodium, to investigate [1,2–13C] acetate the origin [68]. of Successful the orsellinic incorporation acid moiety of [1,2– in arnamial13C] feeding experiment was13 conducted on Armillaria mellea, to investigate the origin13 of the orsellinic acid (75) usingacetate sodium was observed [1,2– forC] orsellinic acetate [acid68]. ( Successful181), a common incorporation biosynthetic of intermediate [1,2– C] acetatein Armillaria was, observedwith moiety in arnamial (75) using sodium [1,2–13C] acetate [68]. Successful incorporation of [1,2–13C] for orsellinic13C-COSY acid couplings (181), aidentifying common intact biosynthetic acetate units. intermediate This experiment in Armillaria supported, with the13 hypothesisC-COSY couplings that acetate was observed for orsellinic acid (181), a common biosynthetic intermediate in Armillaria, with identifyingorsellinic intact acid ( acetate181) and units. in turn This arnamial experiment (75) were supported of polyketide the origin hypothesis [68]. that orsellinic acid (181) 13C-COSY couplings identifying intact acetate units. This experiment supported the hypothesis that and in turn arnamial (75) were of polyketide origin [68]. orsellinic acid (181) and in turn arnamial (75) were of polyketide originCl [68].

O Cl O Cl HO O HO OO HO O Cl OH HO HO O O O OH HO HOO HO O O O O OH O HO HO 51 52 75 181 O O OH O 13 75 181 Figure51 29. Structures C labelled 52 protoilludanes, illustrating the incorporation of intact acetate units (bold). FigureFigure 29. Structures 29. Structures13C 13 labelledC labelled protoilludanes, protoilludanes, illustrating the the incorporation incorporation of intact of intact acetate acetate units units (bold). (bold).

Antibiotics 2020, 9, 928 23 of 29 Antibiotics 2020, 9, 928 23 of 29

6.6. ConclusionsConclusions FungiFungi areare aa prolificprolific sourcesource ofof sesquiterpenoid sesquiterpenoid protoilludaneprotoilludane naturalnatural products,products, whichwhich bearbear aa uniqueunique 55/6/4/6/4 fused-ringfused-ring system,system, withwith 96%96% ofof describeddescribed compoundscompounds isolatedisolated fromfrom higherhigher fungifungi ofof whichwhich Basidiomycota Basidiomycota was was the the major major producer. producer. The The total total synthesis synthesis of of only only 16 16 protoilludanes protoilludanes has has been been undertakenundertaken so so far, far, where where two two were were of of the the non-natural non-natural enantiomer, enantiomer, due due to theto the structural structural complexity complexity of theof tricyclicthe tricyclic core. core. Overall, Overall, 78% of 78% the isolatedof the isolat protoilludanesed protoilludanes have been have investigated been investigated for their bioactivity, for their predominantlybioactivity, predominantly for antimicrobial for antimicrobial and/or cytotoxic and/or properties cytotoxic (89%). properties Of the (89%). 180 examples Of the 180 reported examples so farreported (Table S1),so far 18% (Table were S1), identified 18% were as antimicrobials, identified as antimicrobials, of which 6% exhibited of which modest 6% exhibited to potent modest activity, to againstpotent aactivity, range of against bacterial a range and fungal of bacterial pathogens, and whilefungal 23% pathogens, were identified while 23% as cytotoxic, were identified with 16% as exhibitingcytotoxic, modestwith 16% to exhibiting potent cytotoxicity, modest to againstpotent cy humantotoxicity, cancer against cell lines human (Figure cancer 30 ).cell The lines diversity (Figure of30). chemical The diversity structures of chemical in the protoilludane structures in classthe protoilludane of natural products class of combinednatural products with their combined interesting with bioactivitiestheir interesting make bioactivities this scaffold make worthy this of scaffold further worthy exploration. of further exploration.

FigureFigure 30.30.Summary Summary ofof protoilludanesprotoilludanes isolatedisolated toto datedate with with their their bioactivity. bioactivity.

SupplementarySupplementary Materials: Materials:The The followingfollowing areare availableavailable onlineonline atat http: www.mdpi.com/2079-6382/9/12/928/s1,//www.mdpi.com/2079-6382/9/12/928 Table/s1, TableS1: A S1: review A review of fungal of fungal protoilludan protoilludanee sesquiterpenoid sesquiterpenoid natural natural products. products.

AuthorAuthor Contributions: Contributions:Investigation, Investigation, formal formal analysis, analys datais, curation,data curation, writing—original writing—or draftiginal preparation, draft preparation, M.M.C.; writing—review and editing, B.R.C. and S.W. All authors have read and agreed to the published version of theM.M.C.; manuscript. writing—review and editing, B.R.C. and S.W. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conflicts of Interest: The authors declare no conflict of interest. References References 1. Blackwell, M. The Fungi: 1, 2, 3 ... 5.1 million species? Am. J. Bot. 2011, 98, 426–438. [CrossRef][PubMed] 2.1. Zhong,Blackwell, J.-J.; M. Bai, The F.-W.; Fungi: Zhang, 1, 2,W. 3 … (Eds.) 5.1 millionBiotechnology species? in Am. China J. Bot. I; Springer: 2011, 98 Berlin, 426–438,/Heidelberg, doi:10.3732/ajb.1000298. Germany, 2009; 2. ISBNZhong, 978-3-540-88414-9. J.-J.; Bai, F.-W.; Zhang, W.; Eds. Biotechnology in China I; Springer: Berlin/Heidelberg, Germany, 2009; 3. Schrader,ISBN 978-3-540-88414-9. J.; Bohlmann, J. (Eds.) Biotechnology of Isoprenoids; Advances in Biochemical Engineering/Biotechnology; 3. SpringerSchrader, International J.; Bohlmann, Publishing: J.; Cham,Eds. Germany,Biotechnology 2015; Volumeof Isoprenoids 148, ISBN; 978-3-319-20106-1.Advances in Biochemical Engineering/Biotechnology; Springer International Publishing: Cham, Germany, 2015; Volume 148; ISBN 978-3-319-20106-1. Antibiotics 2020, 9, 928 24 of 29

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