Figure 1: Examples of compounds covered in this review categorized in six sub-classes (see text).

These compounds will be strictly organized due to their structures and not due a possible concurrent isolation or even to common names (e.g., graphislactones A, C, E, and G will be treated in different sub-chapters, although graphislactones A and C were isolated together and all of them share a common name).

Figure 2: Examples of compounds not covered in this review.

Figure 3: Wrongly assigned and thus obsolete structures (details will be discussed in the respective chapters).

Occasionally, natural products have been reported more than one time as new compounds and were given several names. Only the first given name should be used and further names are thus obsolete: Verrulactone D is erroneously given in the SciFinder database as synonym for altertenuol, but is in fact a different natural product. The originally proposed structure of altenuisol turned out to be wrong; it is in fact identical with the structure of altertenuol and the name ‘altenuisol’ is thus obsolete (although it is still frequently used in the community) . The occasionally used designations ‘graphislactone S1–S4’ were used in the initial reports , but have not been proposed as names for these compounds. The names ‘graphislactones A–D’ were used in the following reports by the authors and by most of the community. The name ‘talaroflavone’ has once erroneously been assigned to 1-deoxyrubralacton (116) . Unfortunately, this mistake has been adopted in a later report and the names ‘deoxytalaroflavone’ (for 106) and ‘7-hydroxy-deoxytalaroflavone’ (for 107) were proposed , although these compounds have no structural relationship with talaroflavone (122) and furthermore the name ‘deoxytalaroflavone’ had already previously been assigned to a different compound 110 . The name ‘7-hydroxy-deoxytalaroflavone’ is misleading and thus should not be used.

The most prominent and most frequently referenced members of the herein discussed class of natural products are the parent alternariol (1, 1099 publications given in the SciFinder database at 04/2024), 9-O-methylalternariol (2, 578 entries), altenuene (54, 320), altenusin (47, 130), dehydroaltenusin (74, 55), altertenuol (31, 45), and isoaltenuene (55, 24), where the most abundant Alternaria toxins are usually abbreviated: Alternariol (AOH), 9-O-methylalternariol (AME), altenuene (ALT), and altenusin (ALS) have commonly accepted abbreviations, while further abbreviations are used inconsistently (e.g., iALT or isoALT for isoaltenuene).

Alternaria metabolites (which not only consist of the herein discussed resorcylic lactones) are predominantly, but by far not exclusively, isolated from Alternaria spp., especially from Alternaria alternata. The genus Alternaria in the family Pleosporaceae (Pleosporales, Dothideomycetes, Ascomycota) belongs to the fungi imperfecti and all species are known as plant pathogens . It should be noted that Alternaria alternata is a species complex and many producer strains reported in the literature were not correctly identified . The rather low toxicity of alternariol and most of the further Alternaria toxins (especially those with resorcylic lactone structure) discouraged a thorough investigation of their biological activities, especially of their toxicities. The European Food Safety Authority (EFSA) stated that “at present (2011), the knowledge on the possible effects of Alternaria toxins on farm and companion animals as well as the database describing the occurrence of these mycotoxins in feedstuffs are scarce and are not sufficient to assess the risk regarding Alternaria toxins for animal health,” and encouraged further investigations on Alternaria toxins in many areas.

Figure 4: Alternariol with the correct IUPAC numbering and an occasionally used numbering based on the biphenyl substructure.

The ubiquity and easy availability of this toxin by microbiological and synthetic methods (5 g of the compound were once synthesized in the group of the author) allowed for numerous biological investigations, which can hardly be summarized comprehensively in this overview, but can be further studied in numerous reviews on this and related toxins .

Already in the first reports on alternariol, an antibacterial activity against Staphylococcus aureus (at 25 ppm) and Escherichia coli (at 50 ppm) was mentioned , which was later corrected to an activity against the Gram-positive S. aureus and Corynebacterium betae, but not against Gram-negative E. coli . It was further noted that AOH showed neither fungicidal (possibly due to its low solubility in the standard tests, performed in aqueous media) nor phytotoxic activity .

A general but not an acute toxicity of AOH has already been mentioned in late 1970ies, e.g., an activity against Bacillus mycoides at 60 μg/disc and a toxicity to HeLa cells with an ID50 value of 6 μg/mL . It turned out that induced cytotoxicity is mediated by activation of the mitochondrial pathway of apoptosis in human colon carcinoma cells and that cytotoxicity on HCT116 cells is increased, when AOH is combined with 9-O-methylalternariol (2, AME) . AOH further showed to have a detrimental effect on initial embryo development . Further reports on its mutagenicity and genotoxicity have been published in the due course , but it seems as if an unambiguous mutagenic activity has not been proven before 2006, when AOH was found to cause mutations in cultured Chinese hamster V79 cells and in mouse lymphoma cells even at non-toxic or moderately cytotoxic concentrations . AOH was further shown to cause strand-breaking in mammalian cell lines V79, HepG2, and HT-29 . It was a significant finding by Marko et al. that AOH acts as a topoisomerase poison, preferentially affecting the IIα isoform , where on the other hand the damage turned out to be repaired in less than two hours . Its induction of oxidative stress leading to DNA damage and its causing cell cycle arrest, apoptosis, and changing the cell morphology further contribute to the detrimental effects of AOH . AOH furthermore activated the nuclear translocation of the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid 2-related factor 2 (Nrf2) . The possible anticancer effects of AOH through its cytotoxic, antiinflammatory, genotoxic, mutagenic, apoptotic, and anti-proliferative effect and its influence on immune response , cell cycle, and autophagy have been comprehensively summarized only recently .

AOH has been found to be a weak estrogenic mycotoxin that also has the ability to interfere with the steroidogenesis pathway , to have a negative effect on progesterone synthesis in porcine granulosa cells , and to be an androgen antagonist with an EC50 value of 269 μM .

Phase I and phase II metabolization of AOH has been thoroughly investigated and it turned out that AOH is not metabolized by human fecal microbiota .

Figure 5: Alternariol O-methyl ethers.

3-O-Methylalternariol (3) was isolated as a natural product only once from a non-specified Alternaria sp. and its structure was confirmed by comparison with an intermediate obtained during the total synthesis of alternariol .

3,9-O,O-Dimethylalternariol (4) was first isolated from an unidentified endophytic fungus and later from colletotrichum sp. , Lachnum abnorme , Diaporthe phragmitis and a further D. sp. , and deviantly from the flower buds of the banana species Musa nana . Its structure was determined by NMR spectroscopy and unambiguously confirmed by independent synthesis . It showed inhibitory activity against Pseudomonas syringae pv. actinidae with an MIC value of 25 μg/mL .

O-Glucosides, O-acetates, and O-sulfates of alternariol: As for all natural products bearing hydroxy groups, especially of phenolic hydroxy groups , it is quite likely that alternariol and its derivatives are partly present in the organisms as O-glucosides, acetates, or sulfates , where these substituents are most generally cleaved during the isolation and work-up processes. These derivatives (glucosides and sulfates) are furthermore built during phase II metabolization in the body , where the respective metabolites are not comprehensively covered in this overview.

Figure 6: Alternariol O-glycosides.

Lysilactones A–C (7–9) bearing β-glucopyranoside moieties were first isolated from Lysimachia clethroides, where lysilactone B is mentioned in this review only due to its structural analogy to the other lysilactones; it is not a resorcylic lactone and has not been mentioned after the initial report . The structures of these compounds were determined by NMR-spectroscopic methods and constitution and configuration of lysilactone A was further unambiguously confirmed by total syntheses .

β-ᴅ-Galactopyranoside 10 was isolated from Penicillium sp. , where it turned out to be cytotoxic against A559 cells (IC50: 6.8 μg/mL) but not against further tested cell lines.

Alternariol-derived glucopyranoside 6 was identified together with lysilactone A in LC–MS/MS analyses of invested cereals based on comparison with synthesized samples , but seems to have never been isolated or further investigated.

Figure 7: Alternariol O-acetates and O-sulfates.

A number of sulfate conjugates have been synthesized and used as standards for mass-spectrometric or HPLC analyses . In this course it turned out that 3-O-sulfates of alternariol and 9-O-methylalternariol and the alternariol-9-O-sulfate (13–15) are conjugates repeatedly present in fungal sources and the 3,9-O,O-disulfate of alternariol (16) has similarly been isolated . Due to the instability of these sulfates towards hydrolysis they were typically not isolated. Nevertheless, alternariol sulfates were isolated from Alternaria sp. and alternariol-9-O-sulfate (14) turned out to be active against 23 protein kinases with IC50 values ranging from 0.22 to 8.4 µg/mL and to be cytotoxic against L5178Y cells (EC50: 4.5 µg/mL) . The sulfated 9-O-methylalternariol derivative 15 was isolated from A. alternata and showed inhibitory activity against HCV NS3/4A protease (IC50: 52.0 μg/mL), cytotoxic activity against HEP-G2 cancer cells, and turned out to be antibacterial against Bacillus cereus, B. megaterium, and Escherichia coli with inhibition zones of 17, 12, and 10 mm, respectively, at 50 μg/disk . There is some evidence that not only alternariol and its 9-O-methyl ether can be present as sulfated conjugates, but that further toxins could similarly be sulfated in the organisms, e.g., altenusin and dehydroaltenusin .

Figure 8: 2-Hydroxy- and 4-hydroxy-substituted alternariol and its O-methyl ethers.

Biosynthetic metabolization of alternariol and its 9-O-methyl ether is predominantly started with a hydroxylation in 4-position (c.f., chapter on biosynthesis, vide infra), which leads to a variety of further metabolites. 4-Hydroxyalternariol (19) was first identified as intermediate of the human, rat, and porcine metabolism , but was later similarly found to be a fungal natural product in Alternaria sp. , A. tenuissima , and Trichoderma (Hypocrea) sp. . It is quite astonishing that this assumed main intermediate in the biosynthetic downstream of alternariol was identified as natural product only recently (2021) and that no biological properties were established.

The respective 4-hydroxy derivative of 9-O-methylalternariol (20) was again initially identified as a product of metabolization , but was similarly found to be a natural product in Alternaria alternata , in further A. spp. , and in Nigrospora and Phialophora sp. . It showed various biological activities: 20 turned out to be antibacterially active, inter alia, against Pseudomonas syringae pv. lachrymans, Staphylococcus haemolyticus, and Xanthomonas vesicatoria (IC50 values of 7.3, 10.8, and 10.1 µM, respectively) , X. oryzae pv. oryzae, X. o. pv. oryzicola, and Ralstonia solanacearum (MIC: 13.8, 1.7, and 1.7 μM, respectively) , and further bacteria and showed antimicrobial activity against Trypanosoma brucei rhodesiense, Leishmania donovani, and Plasmodium falciparum (IC50: 13.6, 7.5, 28.3 µM) . It was cytotoxic against soybean cell cultures with an EC50 value of 0.63 µM and against cell line 293 (IC50: 15.5 µM) , showed hydroxyl radical scavenging activity (EC50: 68.3 µM) , and turned out to be active against 24 tested protein kinases with IC50 values ranging from 0.35 to 5.7 µg/mL .

Graphislactones A and B (21 and 22) were first isolated from the lichens Graphis scripta var. pulverulenta, G. prunicola, and G. cognata . Their structures were proposed based on NMR-spectroscopic investigations and were unambiguously confirmed by total syntheses . Graphislactone A had already been obtained previously from the synthetic reduction of botrallin (77; vide infra) . While no further report on graphislactone B was published, graphislactone A was furthermore isolated from various fungi: from Microsphaeropsis olivacea , Cephalosporium acremonium , and a further C. sp. , from Coniothyrium sp. , from Rhizopycnis vagum , Hyalodendriella sp. , Paraconiothyrium sporulosum , Pestalotiopsis uvicola , and from Talaromyces amestolkiae . Graphislactone A showed moderate activity against acetylcholinesterase (AChE) with an IC50 value of 8.1 μg/mL and is a scavenger for the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH; IC50: 9.6 μM) and hydroxyl radicals (scavenging activity of 70% and 91% at 0.05 and 0.27 μg/mL, respectively) . Furthermore, it turned out to be cytotoxic against SW1116 cells (IC50: 9.5 μg/mL) .

The trimethyl ether of 4-hydroxyalternariol was named ‘graphislactone H’ (23). This might suggest a close relationship to the graphislactones A–F (partly discussed in later sections), which is quite misleading, since 23 is not obtained from the genus Graphis and is not a lichen metabolite. It was isolated from Cephalosporium acremonium , and its structure was determined by NMR spectroscopy and unambiguously confirmed by total syntheses . It showed cytotoxic activity against SW1116 cells with an IC50 value of 12 μg/mL .

Figure 9: Chloro- and amino-substituted alternariol and its O-methyl ethers.

The 4- and 2-chlorinated derivatives of 9-O-methylalternariol, palmariols A and B (25 and 26), were first isolated from Lachnum palmae and their proposed structures were later unambiguously confirmed by total syntheses . Palmariol B was further found in Hyalodendriella sp. and Rhizopycnis vagum . Palmariol A and B showed weak antifungal activity against Mucor racemosus (8 mm inhibition zone at 20 μM/disc) and palmariol A was furthermore active against Bacillus subtilis (9 mm) . Palmariol B was found to show a higher antimicrobial activity than 9-O-methylalternariol, obviously due to the additional chlorine substituent. It was active against Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas lachrymans, Ralstonia solanacearum, and Xanthomonas vesicatoria (IC50 values from 16.7 to 18.1 μg/mL) and disclosed antinematodal activity against Caenorhabditis elegans (IC50: 56.2 μg/mL) .

Hyalodendriol C (27) was isolated from Hyalodendriella sp. and the proposed structure could be confirmed by total synthesis . It proved to be moderately active against Bacillus subtilis and Xanthomonas vesicatoria with MIC values of 50 and 25 mM, respectively, and showed some larvicidal activity against Aedes aegypti.

The 2-chlorinated dimethylated alternariol derivative, graphislactone G (28), was (similar as graphislactone H) not isolated from the lichen genus Graphis but was obtained from the fungus Cephalosporium acremonium . Its structure was confirmed by total syntheses . Graphislactone G showed a significant cytotoxic activity against SW1116 cells with an IC50 value of 21 μg/mL .

Penicilliumolide D (29), the 2-chlorinated derivative of graphislactone A (vide supra), was first isolated from Penicillium chermesinum and later additionally from Rhizopycnis vagu and Hyalodendriella sp. . It had already previously been obtained as intermediate in a TMC-264 total synthesis . Penicilliumolide D showed some antibacterial activity, was cytotoxic against A549 and HTC116 cell lines with IC50 values of 25.5 and 37.3 μM, respectively , and exhibited significant larvicidal activity against Aedes aegypti (LC50: 7.2 μg/mL) .

Pestauvicolactone A (30) bearing an amino group in position C-4 of dimethylated alternariol was isolated from Pestalotiopsis uvicola . It was tested for cytotoxicity, but turned out to be inactive.

Figure 10: Presumed alternariol derivatives with non-canonical substitution pattern.

Figure 11: Alternariol derivatives with the 1-methyl group hydroxylated.

Graphislactones C and D (39 and 40) were isolated from the lichen Graphis scripta var. pulverulenta , graphislactone C was later additionally isolated from G. prunicola and G. cognata, and graphislactone D was obtained from G. cognata . Their structures were elucidated by NMR-spectroscopic methods and unambiguously confirmed by total syntheses . No biological activities were tested for these compounds.

Alterlactone (41) is structurally related with graphislactone D, where two methoxy groups are demethylated to hydroxy groups. It was first isolated from Alternaria sp. and later similarly from Ulocladium sp. , A. alternata , and further A. sp. . The proposed structure could be confirmed by total syntheses . Alterlactone showed antimicrobial activity against Bacillus subtilis (IC50 value of 41 µM) , Candida albicans, Trichophyton rubrum (MIC80: 17, 36 µg/mL) , Staphylococcus aureus (MIC: 31 µg/mL) , Trypanosoma brucei rhodesiense and Leishmania donovani (IC50: 7.1, 11.7 µM) , and against Xanthomonas oryzae pv. oryzae (MIC: 16 μg/mL) . It turned out to be a scavenger of DPPH (IC50: 99 µM) and showed neuroprotective effects against oxidative injuries .

Ulocladol (42), a further natural product bearing a seven-membered ring was isolated from Ulocladium botrytis and Microsphaeropsis olivacea . Its structure was unambiguously confirmed by NMR spectroscopy and by total syntheses . Ulocladol is a tyrosine kinase (p56lck) inhibitor leading to a reduction of enzyme activity to 7% at 0.02 µg/µL) .

Figure 12: Verrulactones: pseudo-dimeric derivatives of altertenuol and related compounds.

Figure 13: Biaryls formed by reductive lactone opening and/or by decarboxylation.

Altenusin (47) is biosynthetically obtained through reductive cleavage of 4-hydroxyalternariol (19) and it was already mentioned in one of the first reports on Alternaria metabolites when it was isolated from Alternaria tenuis (synonymous to A. alternata) . Its structure was proposed based on NMR-spectroscopic investigations and unambiguously confirmed after total syntheses and comparison of NMR data . Altenusin was further isolated from A. longipes and further non-specified A. spp. , from Talaromyces flavus , T. pinophilus , Penicillium verruculosum , P. simplicissimum , and a further unidentified P. sp. , from Streptomyces verticillus , Coleophoma sp. , Ulocladium sp. , Botryosphaeria dothidea , and from a Pleosporales sp. . As one of the most important and highly abundant Alternaria toxins, altenusin was repeatedly investigated with respect of various biological activities. It showed antimicrobial activity against MRSA, Streptococcus pneumonia, Enterococcus faecium, and Aspergillus faecalis (MIC values of 31.3, 31.3, 62.5, 62.5 µg/mL, respectively) , against various strains of Paracoccidioides brasiliensis (MIC: 1.9–31.2 µg/mL), against Schizosaccharomyces pombe (MIC: 62.5 µg/mL) , and Bacillus subtilis (IC50: 39 µM) . It was antifungal against Botrytis cinereal (inhibitory efficacy of 56.7% at 200 μg/mL) , Aspergillus fumigatus, A. niger, and Candida albicans (zones of inhibition: 16, 15, 12 mm, respectively, at 200 µg/disc) , and against Alternaria alternata (MIC: 1 µg/mL) . It irreversibly inactivated Escherichia coli biotin protein ligase opening a potential application as antimicrobial or biocide . It furthermore turned out to be an inhibitor of trypanothione reductase (IC50: 4.3 μM) giving rise to an activity against Trypanosoma and Leishmania sp. and inhibited Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) (but not the human orthologue) with an IC50 value of 5.9 µM . Altenusin was cytotoxic against Pf3D7 and MRC-5 cells (IC50: 60.2, 24.8 µM, respectively) , L5178Y cells (IC50: 6.8 µg/mL) , and against the human colorectal HCT 166 cell line (IC50: 28.9 µM) . It inhibited 18 different kinases with IC50 values ranging from 1.1 to 9.8 µg/mL) and was especially active against the kinase pp60c-Src (IC50: 20 nM) . Altenusin turned out to be an inhibitor of α-glucosidase and pancreatic lipase (IC50: 46.1, 21.5 µM, respectively) , and of neutral sphingomyelinase (nSMase) with an IC50 value of 28 µM (but not of aSMase) . It inhibited tau aggregation, attracting interest for the treatment of Alzheimer’s disease , was a selective agonist of the farnesoid X receptor FXR (EC50: 3.2 µM) , and displayed remarkable neuroprotective effects against oxidative injuries by acting as potent activator of nuclear factor erythroid-derived 2-like 2 (NRF2) in PC12 cells . Altenusin finally showed radical scavenging activity against DPPH (IC50 values in the range of 10.7 and 53 were determined) .

Desmethylaltenusin (48), the 9-O-demethylated derivative of altenusin has first been obtained from a fungal endophyte Alternaria sp. and later from Talaromyces sp. . Its structure was elucidated by NMR spectroscopy and later unambiguously confirmed by total synthesis . It was cytotoxic against L5178Y mouse lymphoma cells (EC50 = 6.2 µg/mL) and against THLE and HBE cell lines (IC50 = 44 and 41 µg/mL, respectively) , showed inhibitory potential against various protein kinases with IC50 values of 1.5–9.7 µg/mL , and displayed significant scavenging activities against nitrite .

Biosynthetic decarboxylation of desmethylaltenusin affords biaryl 49, which was isolated from Penicillium sp. and from Talaromyces sp. . It showed significant α-glucosidase inhibition with an IC50 value of 2.2 μM and was a potent scavenger of DPPH and of nitrite .

Decarboxyaltenusin (50) was reported in 1974 to be obtained by chemical decarboxylation of altenusin (47) and by reduction of dehydroaltenusin (74) , but was identified as natural product when isolated from Ulocladium sp. . It was in the due course additionally obtained from Nigrospora, Alternaria, and Phialophora spp. , from further A. spp. , from A. alternata , Botryosphaeria dothidea , and from Pleosporales sp. . The structure of decarboxyaltenusin was elucidated by chemical and NMR-spectroscopic methods and was later unambiguously confirmed by total synthesis . This compound turned out to be cytotoxic against the human colorectal HCT116 cell line (IC50: 73 µM) , showed moderate DPPH free radical-scavenging activities (IC50: 18.7 µM) , exhibited COX-2 inhibitory activity (IC50: 9.5 µM) , and proved to be antiparasitic against Trypanosoma brucei rhodesiense and Leishmania donovani (IC50 values of 8.3 and 21.5 µM, respectively) .

Altenusin B (51), the methyl ester of desmethylaltenusin, was isolated from Alternaria alternata; it showed neuroprotective effects against oxidative stress-mediated damages in PC12 cells .

Two further biaryl derivatives are most likely derived from alternariol and their biosynthesis similarly seems to include a reductive bond cleavage and further transformations. The isomeric altenusinoides A and B (52 and 53) were isolated from Alternaria sp., where no biological activities were determined so far for these compounds .