Fungal phthalimidines-chemodiversity, bioactivity and biosynthesis of a unique class of natural products

Abstract

A wide range of natural products important for the engineering and drug design of pharmaceuticals comprise largely of nitrogen-based heterocycles. Fungal natural products have proven to be a rich source of the industrially-important molecules, many of which are promising drug leads. Although, natural products containing a phthalimidine core tends not to be given distant classification, but compounds containing these structures exhibit antimicrobial, anthelmintic, antimalarial and insecticidal activities, and are among the potential target for discovering new drug candidates. Intriguingly, these are primarily isolated from fungal sources and to a very lesser extent from plants or bacteria. This review surveys fungal-derived phthalimidine metabolites published until the end of 2022, isolated from both terrestrial and aquatic or marine sources with emphasis on their unique chemistry, bioactivities, biogenesis and taxonomic classification. Their unique chemistry and diverse bioactivities (including antiviral, antiproliferative, antioxidant and antimicrobial) provide a chemical library with high medicinal potential, representing a treasure trove for synthetic chemists.

Graphical Abstract

Introduction

A vast number of nitrogen-containing heterocyclic compounds are known to exhibit wide range of pharmacological activities including anticancer, anti-HIV, antimalarial, anti-tubercular, anti-microbial and anti-diabetic (Chaudhari et al. 2016; Kerru et al. 2020; Ma et al. 2018). Natural N-heterocyclic moieties are part of many biologically important molecules, including vitamins, nucleic acids, pharmaceuticals, antibiotics, dyes and agrochemicals. The reason behind the variety of biological activities of these compounds is that the electron-rich nitrogen atom readily accepts or donates a proton and can easily establish diverse weak interactions with other molecules, which allow N-heterocyclic compounds to bind with active sites of enzymes and receptors in biological targets. FDA databases discloses that about 75% of unique small-molecule drugs contain a nitrogen-based heterocycle, which are important in drug design and engineering of pharmaceuticals (Kerru et al. 2020).

Among such heterocycles, phthalimidines got fame in several capacities including discovery of new drug candidates (Tok et al. 2023) and exhibit antimicrobial, anthelmintic, antimalarial and insecticidal activities (Chi et al. 2022). In the synthetic chemistry field, phthalimides are reduced to phthalimidines (1,3-dihydro-2H-isoindole-1-one, A), which are a rare class of N-heterocyclic natural products. These compounds are characterized by a bicyclic nucleus derived from fusion of a benzene ring and a γ-lactam. They are a growing class of compounds attracting the interest of synthetic chemists due to their wide applications in material sciences, serving as pigments or biochemical fluorescent markers (Azumaya et al. 1991; Iqbal et al. 2009; Tsuruta and Inoue 1998). Furthermore, they are valuable building blocks for the synthesis of medicinally relevant compounds due to their diverse biological activities. Pharmaceutically important compounds with a phthalimidine skeleton include the thiazide diuretic, chlorthalidone (B); the cyclooxygenase (COX)-inhibitor, indobufen (C); and the thalidomide derivative, lenalidomide (D) (Fig. 1). Their wide spectrum of biological activities is mainly due to their diverse structural functionalisation, making them an interesting class of compounds to be explored further.

Fig. 1

Pharmaceuticals containing a phthalimidine core structure

Interestingly, phthalimidine core is also found in nature as part of a variety of secondary metabolites, however less abundant in nature compared to their oxygen containing analogues known as phthalides. They are reported primarily from fungi and to a lesser extent from bacteria and plants. The fungal kingdom is therefore regarded as a prolific producer of phthalimidine-containing metabolites, and this review aims to illustrate the diversity of this rare and unique class of fungal metabolites. Molecules containing the phthalimidine-core tend not to be given a distinct classification and are often labeled as polyketides, meroterpenoids, alkaloids or nitrogen-containing compounds/heterocycles. To date, phthalimidines have been reported from the fungal sub-kingdom Dikayra (higher fungi), comprising the fungal phyla Ascomycota and Basidiomycota, both of which have been well-studied for their secondary metabolite (SM) production. Here fungal-derived phthalimidines from both terrestrial and aquatic marine and non-marine sources are surveyed till the end of 2022, highlighting characteristic chemical features, interesting bioactivities and insights into their biosynthesis. Grou** these compounds according to their chemical structure (e.g. simple phthalimidines, prenylated phthalimidines (meroterpenoids) or hybrid phthalimidines) proved challenging, as several fungal species were found to simultaneously produce several phthalimidines with various functionalities. Thus, we opted for a taxonomic classification, as specific classes of SMs have been widely used in fungal chemotaxonomic profiling (Frisvad et al. 2008; Helaly et al. 2018; Wang et al. 2015a).

Chemodiversity and biological activity of fungal phthalimidines

Phthalimidines from Ascomycete

Phthalimidines from Aspergillus species (phylum Ascomycota, order Eurotiales)

The fungal genus Aspergillus of Ascomycetes has been intensively investigated for its bioactive natural products. This genus has also been identified as producer of various phthalimidines-derivatives. For example the first phthalimidine, duricaulic acid (1) was isolated in 1985 from Aspergillus duricaulis (Achenbach et al. 1985). Although phthalide analogues of compound 1 from the same fungus were identified as antimicrobial, however, no such activity was reported for compound 1. Similarly 6-hydroxy-4-methoxy-5-methylphthalimidine (2) was isolated from Aspergillus silvaticus (Ageta and Ageta 1984; kawahara et al. 1988), and later it was re-isolated from Alternaria cichorii, and was named cichorine (2) (Stierle et al. 1993), but again no activity has been reported for this compound. Compounds 1 and 2 isolated as new natural products, but were never tested for their medicinal potential, however, newer analogues 8-methoxycichorine (3), 8-epi-methoxycichorine (4), and N-(4’-carboxybutyl) cichorine (5) (Liao et al. 2019), separated through genetic inactivation of cichorine biosynthetic pathway in Aspergillus nidulans strain LO8030, were identified as phytotoxic and could be candidates for herbicide development. A genetic dereplication strategy in the fungus A. nidulans also produced a hybrid metabolite aspercryptin (6) (Chiang et al. 2016), which is a cichorine-derived hexapeptide with 2-aminododecanol and 2-aminocaprylic acid residues. Other phthalimidines obtained due to epigenetic modification through histone deacetylase inhibition (HDACi) and MS/MS networking were characterized as aspercryptin analogues 7–9 (Henke et al. 2016). Unfortunately, these metabolites were also not studied for their bioactivity.

In addition to epigenetic modification techniques, the one-strain many compounds (OSMAC) approach is another powerful tool to investigate microorganisms secondary metabolome through alteration of cultivation parameters (Bode et al. 2002). This approach led to the isolation of the unique prenylated phthalimidines, aspernidine A (10) and B (11) (Scherlach et al. 2010) and F–H (12–14) from the fungus A. nidulans (Li et al. 2020), which showed moderate antiproliferative activity against various tumour cell lines, especially compounds 13 and 14 exhibited significant IC₅₀ values i.e. in the range of 4.77–33.03 μM against HL-60, A-549, SMMC-7721, MCF-7, and SW- 480 cancer cell lines. Further, compounds 10 and 11 were re-isolated from a mangrove endophytic Aspergillus sp. (HK-ZJ). In addition, HK-ZJ species has also been reported to produce emerimidines A (15) and B (16) and the O-prenylated phthalimidines emeriphenolicin A-D (17–20) (Zhang et al. 2011). Compound 10–20 are prenylated analogues except compounds 15 and 16; these compounds exhibited moderate in-vitro inhibitory activity against anti-influenza A virus (H1N1) replication in MDCK cells, with IC₅₀ values of 201 and 296.8 μM, respectively. It can be predicted that the absence or presence of a smaller prenyl moiety may be significant in antiviral activity. Compound 15 also showed antimicrobial activity against Bacillus subtilis, Candida albicans, Salmonella typhi and methicillin-resistant Staphylococcus aureus (MRSA), with a MIC value of 59.9 μM and Aspergillus fumigatus, Klebsiella pneumoniae and Pseudomonas aeruginosa with a MIC value of 59.8 μM (Yashavantha Rao et al. 2017). This compound has also been identified as metabolite of an endophytic fungus Aspergillus variecolor CLB38. Another endophytic Aspergillus sp., A. nidulans HDN12-249 produces N-farnesyl dicarboxylate derivatives of phthalimidines; emeriphenolicins E–G (21–23), and macrolide derivatives; emericellolides A-C (24–26), composed of an unprecedented L-glutamate fragment and a sesquiterpene moiety. All these isolates were screened for cytotoxic activity but only compound 21 showed selective activity against three human cancer cell lines; HeLa, A549, and HCT-16, with IC₅₀ values of 4.77, 12.04, and 33.05 μM, respectively (Zhou et al. 2016). A review of their structure–activity relationship (SAR) revealed that the macrolide moiety eliminates cytotoxic activity of these compounds.

Enantiomers of ( ±)-asperglactam (27) were isolated from the mangrove endophytic fungus Aspergillus versicolor SYSU-SKS025, using a chiral HPLC column, and were identified due to their opposite optical rotations and opposite Cotton effects at 210 nm in their circular dichroism (CD) spectra. Both enantiomers were nearly equally active (IC₅₀ value of 50.5 and 60.1 μM, respectively) against the enzyme α-glucosidase, and although the investigators did not comment on the SAR, it is speculated that a chiral center is not necessary for enzyme inhibition activity, rather the phthalimidines core is essential (Cui et al. 2018b). The phthalimidines isolated from various Aspergillus species are presented in Fig. 2.

Fig. 2

Phthalimidines isolated from Aspergillus species

Phthalimidines from Alternaria species (phylum Ascomycota, order Pleosporales)

Alternaria fungi are plant pathogens, since they produce phytotoxins (Logrieco et al. 2009), however, they are producing other worthy metabolites, for example mycotoxin cichorine (2), O-prenylated analogues, zinnimidine (28) and Z-hydroxyzinnimidine (29), which were isolated from the phytopathogenic fungus A. cichorii (Fig. 3). Unlike cichorine (2), the zinnimidines (28 and 29) are non-phytotoxic compounds. This indicates that prenylation could have masked the phytotoxicity by converting the phenolic function to an ether group; however, later the analogues of 28 as N-substituted compounds porritoxin (30) and porritoxin sulfonic acid (31) (Fig. 4), isolated from A. porri were also found phytotoxic. These variations in activity could be attributed to the experimental design and not the structural diversity. The fungus A. porri is known as the causal fungus of black spot disease in stone-leek and onion (Horiuchi et al. 2003; Suemitsu et al. 1995). The phytotoxic compound 30 was first reported in 1992 as a benzoxazocine derivative (Suemitsu et al. 1992), whose structure was revised a decade later through detailed 2D NMR as 2-(2′-hydroxyethyl)-4-methoxy-5-methyl-6-(3′′-methyl-2′′-butenyloxy)-2,3-dihydro-1H-isoindol-1-one (Horiuchi et al. 2002) and subsequently confirmed through total synthesis (Cornella and Kelly 2004). Compound 30 inhibited seedling growth at concentrations as low as 32.7 μM, while compound 31 exhibited weaker phytotoxic activity compared to compound 30, possibly due to sulfonation of the terminal hydroxyl group (Horiuchi et al. 2003). Porritoxin (30) belongs to the group of non-host specific phytotoxins and has the potential to be developed as a natural broad spectrum herbicide (Xu et al. 2021). In cytotoxicity assays, 28 was not active against both HeLa and KB cells (Phuwapraisirisan et al. 2009), however, its N-substituted analogue 30 showed 96% inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by 1000 mol ratio of 12-O-tetradecanoylphorbol-13-acetate (TPA), without remarkable cytotoxicity on the viability of Raji cells. This is superior to the 91.9% inhibition of EBV-EA activation of the known anti-tumor promoter β-carotene, which makes it a potential anti-tumor agent (Horiuchi et al. 2006).

Fig. 3

Phthalimidines isolated from Alternaria species

Fig. 4

Phthalimidine-type triprenyl phenols isolated from Stachybotrys spp

Phthalimidines from Stachybotrys species (phylum Ascomycota, order Hypocreales)

Stachybotrys spp. are prolific producers of SMs and their metabolites were comprehensively reviewed till the year 2014 by Wang et al. (Wang et al. 2015a). An important class of Stachybotrys metabolites are the triprenyl phenols (TPPs) consisting of a multitude of compounds, of which those incorporating a phthalimidine nucleus will be discussed here in detail.

The first phthalimidine-type TPPs, stachybotrins A (32) and B (33), were reported in culture of Stachybotrys sp. (CS-710–1) collected from brackish water in Florida (Xu et al. 1992). Later stachybotrin C (34) was isolated from the liquid culture broth of Stachybotrys parvispora F4708 (Nozawa et al. 1997a, 1997b). Compounds 32–34 possess a unique pyrano-isoindolinone ring system, with compound 34 having an additional ethyl phenol moiety. Compounds 32 and 33 showed antibacterial activity against B. subtilis (ATCC 6051) and antifungal activity against Ascobolus furfuraceus (NRRL 6460) and Sordaria fimicola (NRRL 6459) (Xu et al. 1992). Interestingly, compound 34 demonstrated promising neuritogenic activity, where it induced significant neurite outgrowths in PC12 cells and showed cell survival activity in the primary culture of cerebral cortical neurons. This protective effect on neuronal cell damage assumes it functions as a neurotrophic factor in cerebral neurons, and is thus expected to prevent hypoxic neuronal injury caused by ischemia and could therefore be a strong candidate for the treatment of neurodegenerative diseases (Nozawa et al. 1997b). Later, the total synthesis of compound 34 and its four stereoisomers helped to establish the absolute configurations of the stereocenters C-8 and C-9, with the relative configuration of 34 initially assigned as (8S*, 9S*) based on NOESY analysis. This was revised to (8S, 9R), and was further confirmed through X-ray diffraction analysis of its 4-bromobenzyl ether derivative (Jacolot et al. 2013; Kuroda et al. 2018).

Stachybotrys microspora IFO30018 has been reported to produce plasminogen activators, staplabin (35) and its analogues Stachybotrys microspora triprenyl phenols (SMTPs) (36–43). These natural products were named after the producing organisms S. microspora TPP (Hasumi et al. 1998; Hu et al. 2000; Kohyama et al. 1997; Shinohara et al. 1996). The staplabin/SMTP molecule consists of a tricyclic γ-lactam moiety, an isoprene side chain and an N-linked side chain, which only differs among congeners. Compounds 35–41 are single-unit congeners with an amino acid as an N-linked side chain, while compounds 42 and 43 are two-unit congeners with two core SMTP structures bridged by ornithine and lysine, respectively. The two-unit congeners 42 and 43 have been reported as the most potent in enhancing plasminogen-fibrin binding, urokinase-catalyzed activation of plasminogen, and urokinase- and plasminogen-mediated fibrin degradation (Hu et al. 2000). In particular the efficacy of 42 (orniplabin) in treating several types of ischemic strokes (including thrombotic and embolic strokes) in rodents and primates models was studied (Sawada et al. 2014; Suzuki et al. 2018). Another marine fungus Stachybotrys longispora FG216 was found to produce fungi fibrinolytic compound 1 (FGFC1), with the same structural features to that of 42 and likewise showed in-vitro and in-vivo fibrinolytic activity (Wang et al. 2015b). Fungi fibrinolytic compound 2 (FGFC2) (44) was later identified from the same fungal strain and being a one-unit congener, displayed moderate in-vitro fibrinolytic activity (Guo et al. 2016).

In addition to epigenetic modification, biotransformation (culture supplements) is another potential tool to obtain diverse fungal metabolites. Based on this technique, the supply of amino compounds to S. microspora and S. longispora FG216 cultures provided more than 50 SMTP congeners, which led to establish a detailed insight into their Severe Acute Respiratory Syndrome (SARs) (Hasegawa et al. 2010; Hasumi and Suzuki 2021; Koide et al. 2012; Matsumoto et al. 2015; Yin et al. 2017). This revealed that the N-linked side chain is crucial for their plasminogen-modulating activity. In fact, SMTP-0, the most basic congener lacking the N-linked side chain, showed no plasminogen modulator activity (Hasumi et al. 2007). Thus, SMTPs are regarded as prospective candidates for the development of a drug useful for ischemic diseases that are associated with inflammation, such as stroke. Stachybotrys sp. phthalimidine-type TPPs are illustrated in Fig. 4.

Phenylspirodrimanes (spirocyclic drimanes) are another important group of structurally diverse Stachybotrys TPPs, featuring a drimane skeleton-containing sesquiterpene with a benzene ring attached through a spirofuran. In accordance with the focus of this review, only phthalimidine-type phenylspirodrimanes have been reviewed and included. The first member of this class has been reported to be stachybotramide (45) from the aspen-tree fungus Stachybotrys cylindrospora (Ayer and Miao 1993). It was also found in the culture of the black toxic mold Stachybotrys chartarum S-17. The same fungus and other species of this group, S. chartarum MRC 1422 and Egypt 1, were found to produce 45 and the structural analogues; stachybotrylactam (46), stachybotrylactam acetate (47) and 2α-acetoxy stachybotrylactam (48) (Jarvis et al. 1995). Although no activity has been reported for these metabolites, due to their structural diversity, they are important targets for a medicinal chemist.

In 1997, Kamalov et al. isolated a nitrogen-containing compound from cultures of S. chartarum, established the structure and named as stachybotrin (Kamalov et al. 1997), which in fact was incorrectly assigned, however, a year later the structure was revised and confirmed by single X-ray crystallography, revealing it possessed the same structure and stereochemistry as compound 45 (Kamalov et al. 1998). The same fungus also produces a methylated analogue of 45, which was coined stachybotrin A (49), not to be confused with the precedent stachybotrin A (32) (Kamalov et al. 1999). In another study on the Stachybotrys strain F-1839 (closely related to S. chartarum), seven more congeners designated as F1839-A, -B, -C, -D, -E, -F, and -J (50–56) were isolated. They exhibited moderate pancreatic cholesterol esterase activity (Sakai et al. 1995).

The stachybocins A-C (57–59) are phenylspirodrimane dimers connected by a lysine residue and only differing in the hydroxyl group substitution in the drimane skeleton. They are produced by the soil-derived Stachybotrys sp. M6222 taxonomically related to S. chartarum (Nakamura et al. 1995; Ogawa et al. 1995). These compounds are reported as endothelin (ET) receptor antagonists with no in-vivo or in-vitro toxicity, making them potential candidates for the treatment of some cardiovascular diseases. They are commercialized as dual enzyme inhibitors inhibiting the binding of ET-1 to human ET-subtype A and ET-subtype B receptors in ligand binding assays.

Spirodihydrobenzofuranlactams I (46), II (45), III (60), IV (61) and VI (57) were identified from cultures of two different Stachybotrys strains, DSM 8767 and F11402. Similar to the stachybocins (57–59), they showed antagonistic effects in the ET receptor binding assay and additionally inhibited HIV-1 protease. These are regioisomers to the already reported phenylspirodrimanes. Intrigued by the similar ET receptor antagonist activity to stachybocins (57–59) (Roggo et al. 1996a, 1996b) and to assign the absolute configuration, however, their structures were revised through synthesis (Deng et al. 2003; Kende et al. 2003). In fact, spirodihydrobenzofuranlactams I has the same structure as compound 46, spirodihydrobenzofuranlactams II has the same structure as compound 45, and spirodihydrobenzofuranlactams VI has the same structure as compound 57. The most active of these compounds was spirodihydrobenzofuranlactams VI (57), with IC₅₀ values of 1.5 μM in the ET-subtype A receptor binding assay and 11 μM in the HIV-1 protease inhibition assay (ROGGO et al. 1996b).

Tyrosine kinase receptors with immunoglobulin and epidermal growth factor (EGF) homology domains (Tie2) and their ligands are important in angiogenesis and small molecule inhibitors of Tie2 activity are of therapeutic interest. Vázquez et al. found that extracts of a soil-derived isolate S. chartarum inhibited the Tie2 kinase receptor, from which seven K-76 phenylspirodrimane derivatives (62–68) differing in the N-substituted side chain were isolated (Vázquez et al. 2004). All the seven compounds 62–68 reproducibly inhibited Tie2 kinase receptor, with 65 showing the highest inhibition (IC₅₀ = 25 μM). The reported inhibitory potential of each compound and structural features are not helpful to comment on SAR.

Stachybotrys spp. derived from marine sources further enriched the growing array of phthalimidine-type phenylspirodrimanes, since the endophytic fungus S. chartarum MXH-X73 associated with the sponge Xestospongia testudinaris, produced stachybotrins D-F (69–71), the phenylspirodrimane dimers stachybocins E (72) and F (73) and compound 62 (Ma et al. 2013). In antiviral assays against HIV-1, only compound 69 displayed inhibitory effects on wild-type and five non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI)-resistant HIV-1 strains with no cytotoxicity. EC₅₀ values for inhibitory effects on HIV-1 replication were between 6.2 and 23.8 μM. Compound 69 also inhibited HIV-1 RT RNA-dependent DNA polymerase activity in a dose-dependent manner with an EC₅₀ of 50 μM, providing a new chemical class of novel NNRTIs (Minagawa et al. 2002c). SAR could suggest that the nature of the N-alkyl chain is important, which revealed a keto-function and overall hydrophobicity could be the activity determining factors. Ma et al. characterized stachybotrin G (74) (Ma et al. 2015), which does not belong to the isoindolinones but is rather a cyclised isoindolinimine with farnesylated decahydropyrrolo[1,2-α][1,3]diazocine skeleton. A year earlier, Zhou et al. isolated compound 45 and a derivative stachybotrin G (75) with a 2-piperidinone as the N-substituent, from the soil-derived fungus S. parvispora HS-FG-843 (Zhou et al. 2008).

Fig. 12

Miscellaneous phthalimidines isolated from other Basidiomycetes

The sterenin class has recently seen an expansion with the addition of three new members, namely sterenin K-M (204–206). In addition to sterenin A-C, these compounds were isolated from S. hirsutum cultivated on potato dextrose agar slants. The six sterenins demonstrated inhibitory effects on α-glucosidase, an enzyme responsible for catalyzing carbohydrate digestion. They also exhibited notable 11β-HSD1 inhibitory activity, consistent with observations for sterenin A-D. These findings suggest the potential for develo** these compounds into novel hypoglycemic drugs (Wang et al. 2014). Sterenin D was additionally identified in a liquid mycelial fermentation process of S. hirsutum from Chile. It exhibited antifungal activity, inhibiting both the sporogenesis and mycelial growth of Botrytis cinerea. This suggests its potential use as a biofungicide (Aqueveque et al. 2017).

Aldose reductase (AR) is another key enzyme involved in the complications of DM; thus inhibitors of this enzyme represent a potential pharmacological avenue in the treatment of side effects of this metabolic disorder (Tang et al. 2012). The salfredins are a family of AR inhibitors produced by Crucibulum sp. and are divided into A-, B- and C-type, based on structural features. Of relevance to this review are the A-type salfredins, A₃ (207), A₄ (208) and A₇ (209) with a furo-phthalimidine skeleton and an attached propionic acid residue (Fig. 12). The N atom in compounds 207–209 originates from glutamic acid, glycine and alanine, respectively, and addition of 1.0% glycine to the fermentation media markedly enhanced the production of compound 208 verifying this assumption. The relative stereostructure of compound 208 was confirmed by X-ray crystallographic analysis and implied for compounds 207 and 209. In AR inhibitory assays on rat lens, compound 208 was found to be the most active (Matsumoto et al. 1995). Another salfredin type A metabolite (210) was isolated from cultures of Crucibulum laeve DSM 1653 and DSM 8519 (Fig. 12). Compound 210 afforded an N-ethylphenyl substituent (Neumann et al. 1999).

The natural antioxidant clitocybin A (211) was isolated from the culture broth of the mushroom Hygrophoropsis aurantiaca (Fig. 12) (Kim et al. 2008). It is a 4,6-dihydroxy-isoindolinone with a p-hyrdroxybenzene moiety attached to the N-atom. Its antioxidant activity was evaluated by measuring the free radical scavenging effects in three different assays. It exhibited potent superoxide radical scavenging activity and significant ABTS [2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation scavenging effects with an IC₅₀ value of 10.3 and 6.4 μM, respectively, but displayed no scavenging activity against DPPH radical. Furthermore, it had a protective effect against cellular DNA damage induced by oxidative stress (Süssmuth and Mainz 2017). Synthetic modifications in compound 211 provided further derivatives, clitocybin B (212) and C (213) (Fig. 12) (Moon et al. 2009). Compounds 211–213 inhibited intracellular reactive oxygen species (ROS) and H₂O₂-induced cell death mediated by the reduction of caspase 3 and 9 activation, cytochrome c release from mitochondria and NF-KB activation. Recently the anti-wrinkle capacity of compound 211, supported through elastase inhibitory activity, stimulation of procollagen synthesis and inhibition of matrix metalloproteinase-1 (MM-1), all suggests its use in anti-wrinkle cosmetic products (Lee et al. 2017). In fact, the extract of H. aurantiaca and clitocybin derivatives have been patented as an active ingredient in anti-aging preparations (Yoo et al. 2010). However, clinical trials are crucially required before its commercial usage. Extended studies on bioactivities of compounds 211 and 212 revealed that the natural analogue 211 was found to block the platelet-derived growth factor (PDGF)-BB-induced proliferation through G1 phase arrest by regulating the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in vascular smooth muscle cells (VSMCs) (Yoo et al. 2012a). However, compound 211 did not change the expression levels of extracellular signal-related kinase (ERK) 1/2, phospholipase C-γ1 (PLC-γ1), and PDGF-Rβ phosphorylation. While the synthetic derivative 212 significantly inhibited the phosphorylation of Akt, ERK 1/2, PLC-γ1, and PDGF-Rβ phosphorylation in the PDGF-BB signaling pathway (Yoo et al. 2012b). Altogether, these anti-proliferative activities on VSMCs make them a potential therapeutic agent for preventing or treating vascular disease such as atherosclerosis, hypertension and restenosis.

The story of compound 211 continued with its re-isolation along with its non-nitrogen substituted analogue, 4,6-dihydroxy-2,3-dihydro-1H-isoindol-1-one (214) from Langermannia fenzlii, a fungus used in TCM as a remedy for bleeding (Fig. 12). Both compounds were devoid of cytotoxic activity when tested against A549, PC-3, U87, and HeLa tumor cells in the concentration range of 12.5–100 μM (Lü et al. 2013).

Phthalimidines from Basidiomycete and Ascomycete co-culture

From a co-culture of the two endophytic fungi, Irpex lacteus and Phaeosphaeria oryzae of the fungal phyla Basidiomycota and Ascomycota, respectively, irpexine (215) was isolated, which was not identified in monocultures (Fig. 13). This expanded the array of the so far known 3-aryl substituted phthalimidines, and due to hindered rotation around the C8–C9 bond also seen in pestalachloride A (138); compound 215 was also isolated as a mixture of atropisomers. However, this mixture was resolved on chiral-phase column and the absolute configuration determined using ECD of a simplified model with a methyl group replacing the prenyl group. At a concentration of 50 μM, compound 215 was inactive in cytotoxicity and antimicrobial assays (Sadahiro et al. 2020).

Fig. 13

Phthalimidines reported from Basidiomycete and Ascomycete co-culture

Clinical development of fungal phthalimidines

SMTP-7 (42) is a novel thrombolytic agent that demonstrates effective improvement in ischemic stroke in rodent models (Ito et al. 2014). To enhance the translation of animal findings to humans and ensure the safety and efficacy for drug development, studies in higher-order species are crucial. In evaluating the therapeutic potential of 42, a monkey occlusion model with thrombotic middle cerebral artery (MCA) was employed. The findings demonstrated its effectiveness in treating thrombotic stroke in monkeys, with the added relevance of their cerebral vascular anatomy closely resembling that of humans. This observation aligns with earlier results obtained in rodent models (Sawada et al. 2014). In addition, 42 was identified as having a protective effect against renal damage in a mouse model of acute kidney injury (AKI) (Shibata et al. 2021). As a member of the SMTP family of compounds, TMS-007 was subsequently progressed into clinical studies. In a phase 1 randomized, double-blind, placebo-controlled, dose-escalation study, registered as JapicCTI-142 654 at the Japan Pharmaceutical Information Center Clinical Trials Information, involving healthy 40 male volunteers in Japan, TMS-007 exhibited favorable tolerability. Administered intravenously at doses up to 6 mg/kg, the compound showed no indication of serious adverse reactions, including any signs or symptoms of bleeding (Moritoyo et al. 2023). Subsequently, a phase 2a study involving 90 acute ischemic stroke (AIS) patients in Japan was conducted. This study, registered as JapicCTI-183 842 at the Japan Pharmaceutical Information Center Clinical Trials Information, involved the administration of a single intravenous infusion approximately 9 h after the last known normal (LKN) (Nishimura et al. 2022). Biogen Inc., a leading biotechnology company focused on neuroscience, acquired TMS-007 (renamed BIIB131) as an investigational drug for AIS 6, building upon encouraging results from phase 2 study. The current gold standard treatment for AIS is tissue-type plasminogen activator (t-PA) therapy, which activates the thrombolytic enzyme plasmin by cleaving plasminogen. Nevertheless, the restricted time frame for administering t-PA within 3–4.5 h limits its applicability. Moreover, the use of t-PA benefits only a limited percentage (3%–9%) of AIS patients, primarily because of the elevated risk of fatal hemorrhagic transformation (HT) (dela Peña et al. 2017). The distinctive mode of action of TMS-007, which encompasses profibrinolytic, antioxidative, and anti-inflammatory activities, holds the potential to extend the time window for AIS treatment. These characteristic underscores the importance of develo** TMS-007 into the next generation of thrombolytic agents. Such advancements are urgently required to address the critical need for reducing mortality and morbidity in stroke patients. Additionally, there is a need to clinically investigate the use of TMS-007 in other thromboembolic and thrombotic diseases, such as pulmonary embolism and myocardial infarction.

The initial assessment of physicochemical properties through absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling is essential. This preliminary assessment assists in forecasting the drug-like properties of potential drug candidates, thereby mitigating the risk of expensive failures in the later stages of drug development. Stachyflin (109), while exhibiting promising in-vitro anti-viral activity, displayed diminished effectiveness when orally administered to mice infected with the influenza virus. The lipophilic nature and high water insolubility of 109 explain its limited gastrointestinal bioavailability. Various vehicles, including soybean oil, polyethylene glycol (PEG), and surfactants, were experimented with to enhance solubility and oral absorption. Positive in-vivo activity was observed when the substance was administered as an aqueous solution of a phosphate ester prodrug or as a solution in PEG4000 (Yagi et al. 1999; Yoshimoto et al. 2000). While the intraperitoneal injection of 109 in mice did not induce toxicity, its antiviral activity in mice was comparable to clinically used antiviral neuraminidase inhibitors, such as oseltamivir. Notably, this effect was achieved at a significantly higher dose of 100 mg/kg/day, as opposed to the 20 mg/kg/day used for oseltamivir (Motohashi et al. 2013). In a recent computational screening study aimed at discovering new inhibitors for Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), 109 exhibited a positive ADMET profile. Further investigations through molecular dynamics (MD) simulations and molecular docking demonstrated a strong binding affinity between 109 and the amino acid residues of the M^pro active site, a critical protease involved in viral replication. However, the MD simulation of the M^pro–Stachyflin docking complex suggested a less stable interaction (Bharadwaj et al. 2021). Currently, the prospect of advancing 109 into clinical development does not appear promising. However, in a separate in-silico investigation utilizing docking and MD simulations, sterenin M (206) emerged as a potential SARS-CoV-2-M^pro inhibitor within the Medicinal Fungi Secondary Metabolites and Therapeutics (MeFSAT) database. All ADME parameters analyzed fell within recommended ranges, indicating that sterenin M holds promise as a ligand against SARS-CoV-2. Nevertheless, further in vitro and in vivo studies are warranted for comprehensive evaluation (Prajapati et al. 2021).

Culinary medicinal mushrooms, exemplified by H. erinaceus, offer a rich source of bioactive phthalimidine compounds, presenting potential for future advancements. Despite indications of their neuro-health benefits, derived from the combined and synergistic effects of numerous bioactive compounds in the fruiting bodies or cultured mycelium, the availability of clinical trials is currently limited (Brandalise et al. 2023). The primary reason for the limited availability of clinical trials is that naturally occurring compounds in their original state may lack patentability, making them less appealing for pharmaceutical companies to invest in.

Cultivating medicinal mushrooms submerged in bioreactors and fine-tuning fermentation conditions is essential for achieving maximum biomass production. This approach helps overcome challenges related to unpredictable metabolite profiles caused by seasonal variations. In order to generate substantial quantities of TMS-007 for clinical studies, a large-scale fermentation strategy involving precursor amine feeding was implemented. Although an alternative method involving the total synthesis of an SMTP congener was devised, it consisted of a 10-step process with relatively modest production yields (Hasumi and Suzuki 2021). Likewise, there was a need for optimizing the fermentation process on a large scale to produce clitocybin A (211) from C. aurantiaca for mass production. This was pursued with the goal of utilizing clitocybin A as a novel anti-wrinkle agent in cosmetics (Kim et al. 2014).

In summary, fungal phthalimidine metabolites boast diverse scaffolds and intricate structural complexity. Despite these advantages, few pharmaceutical industries are actively exploring drug leads from natural sources. On the other hand, several synthetic small molecule ligands, featuring a phthalimidine core, such as iberdomide, JM-1232, and CC-885—show promise as clinical candidates for treating multiple myeloma (Charliński et al. 2021). Conversely, obstacles in supplying adequate quantities for drug development, particularly in cases where chemical synthesis is impractical, significantly impede the progress of drug leads derived from natural sources. Another prevalent challenge lies in the limited dissolution/solubility and permeation of hydrophobic natural compounds. An integrated approach, which merges in-silico ADMET analysis with MD simulation studies, and is complemented by in-vivo studies, is crucial for propelling fungal phthalimidines into clinical development or validating them as leads for drug targets.

Table 1 below summarizes the fungal phthalimidines and phthalimidine-containing metabolites outlined in this review, including their origin and biological activity.

Table 1 Fungal phthalimidines and phthalimidine-containing metabolites

Biosynthesis of fungal phthalimidines

Fungal phthalimidines have shown diversity in their structural features and bioactivities, therefore, we have discussed here biosynthetic routes of these metabolites established or proposed in different fungi. Fungal phthalimidines belong to the class of fungal aromatic polyketides, synthesized by the non-reducing group of iterative polyketide synthases (NR-PKSs), which utilise simple carboxylic acid precursors to assemble the polyketide backbone (Crawford and Townsend 2010). Canonical PKS domains include a ketosynthase domain (KS) for decarboxylative Claisen-like condensation of the extender unit, an acyltransferase domain (AT) for extender unit selection and transfer and an acyl-carrier protein (ACP) for extender unit loading. Fungal NR-PKS feature two additional types of dedicated domains, a starter unit ACP transacylase (SAT) domain for starter unit selection and a product template (PT) domain involved in chain-length determination and cyclisation (Crawford et al. 2008; Herbst et al. 2018). For product release from the PKS, a thioesterase (TE) domain, a thioesterase/Claisen cyclase (TE/CLC) domain or a reductase (R) domain is tethered to the C-terminus, although some NR-PKS have an enzymatic domain that acts in trans to release the nascent polyketide chain, or may not require any releasing domain enzyme (Chiang et al. 2010).

Biosynthetic labelling studies in Stachylidium sp.

Biosynthetic examples of phthalimidines and phthalides have been reported from fungal secondary metabolism, and several fungal strains simultaneously produce both types of metabolites. For example, the marine-derived Stachylidium strain produces both marilones (phthalides) and marilines (phthalimidines) (Almeida et al. 2012, 2011a). Feeding experiments using ¹³C-labeled acetate, propionate and methionine established the tetraketide nature of the phthalide/phthalimidine core skeleton and the mevalonate origin of the attached geranyl side chain in both marilone A (216) and mariline B (149) (Fig. 14) (El Maddah et al. 2019). It is well-established that fungi exclusively use the mevalonate pathway (MVA) for the synthesis of their terpenoid units (Lombard and Moreira 2011). In addition to confirming the assumption of an analogous biosynthetic origin for phthalide/phthalimidine frameworks, an important conclusion from these feeding studies was the involvement of a methylated acetate starter unit for initiation of the biosynthesis, which is considered unique for fungal metabolites (El Maddah et al. 2019).

Fig. 14

Observed labeling pattern in marilone A (216) and mariline B (149) after feeding [1-¹³C]sodium acetate, [2-¹³C]sodium acetate, and L-[Me-¹³C]methionine. Smaller bullets (■) indicates indirect incorporation

The mariline analogue (148) has a unique O-prenylated tri-substituted benzene ring attached to the nitrogen of the phthalimidine backbone putatively tyrosine-derived. Interestingly, similar motifs; the stachylines were independently isolated from the same fungal strain (Almeida et al. 2011b) and it could be speculated that 148 is a product resulting from crosstalk between the phthalimidine and the stachyline pathways. However, this needs further exploration for determination of the BGC for each building block and the enzyme(s) catalyzing the coupling, and several dimeric fungal natural products provide excellent examples for such biosynthetic crosstalks, e.g. aspercryptin (6) (Dai et al. 2022). As for the N-substituent in 149 this is likely provided from amino acid metabolism, e.g. ethanolamine from serine.

Biosynthetic studies in Aspergillus nidulans

As an established molecular genetic system, A. nidulans has successfully served as a model fungus for heterologous expression of fungal SMs (Chiang et al. 2013; Yaegashi et al. 2014). The group of Clay Wang has developed a targeted gene deletion strategy employing the genome of A. nidulans. By directly replacing the promoters of the NR-PKS genes as well as other genes required for compound production or release, they were successful in determining NR-PKS genes required for the synthesis of the simple phthalimidine cichorine (2), prenylated phthalimidine aspernidine A (10) and the hybrid metabolite aspercryptin (6). This strategy helped to increase the titre of the target metabolite of the biosynthetic gene cluster (BGC) (when the promoters of all genes in the cluster are replaced) as well as pathway intermediates (when some of the genes in the cluster are replaced), which allowed them to propose a biosynthetic pathway for the above metabolites (Ahuja et al. 2012).

For cichorine (2), the BGC in A. nidulans includes a PKS gene AN6448 (cicF), a transcriptional activator (regulatory) gene (cicD), a transporter gene (cicA), and four tailoring genes (cicB, cicC, cicE and cicH) (Fig. 15A). The ΔcicB and the ΔcicC knockout strains produced the biosynthetic intermediates 3-methylorsellinic acid (217) and nidulol (218), respectively. The cichorine analogs 3–5 are also products of the cicF, which provides the basic phthalimidine backbone (Liao et al. 2019). However, 3 and 4 feature an additional methoxy group at C-8, probably introduced at a later stage mediated by tailoring enzymes, e.g. hydroxylase, methyltransferase. As for the N-carboxybutyl substituent in 5, this is postulated to be derived from amino acid metabolism, e.g. 5-aminovaleric acid from proline, and an amino acid transporter gene (AN11921) has been annotated upstream of cichorine BGC. A proposed biosynthetic scheme for cichorine (2) is outlined in Fig. 15B (Sanchez et al. 2012).

Fig. 15

A Organization of genes surrounding the PKS cicF involved in cichorine (2) biosynthesis. B Proposed biosynthesis of cichorine (2) and related analogues (3–5) in Aspergillus nidulans (metabolites in boxes have been analytically identified). Abbreviations: SAT, starter unit ACP transacylase; KS, ketosynthase; AT, acyltransferase; PT, product template; ACP, acyl carrier protein; C-MeT, C-methyltransferase; TE; thioesterase; SAM, S-adenosyl-L-methionine

Manipulating the expression of kinases, which play a role in aspects of regulation and signal transduction, is a novel approach for activating cryptic gene clusters (De Souza et al. 2013). This approach was employed for elucidating the genetic basis for the biosynthesis of aspernidine A (10). Through screening a genome-wide kinase knockout (KO) library of A. nidulans (a total of 98 kinase KO strains), one strain with a mitogen-activated protein kinase gene knockout (ΔmpkA) was detected. Sequential targeted gene deletions in this strain allowed the characterization of aspernidine A (10) BGC and further related metabolites (aspernidine C-E) (219–221) probably involved in the biosynthesis. In silico analysis using the Aspergillus Genome Database (AspGD, http://www.aspgd.org/) correlated well with results of targeted gene deletion predicting the aspernidine BGC to contain 6 genes pkfA-pkfF (Fig. 16A), although other gene involved in the biosynthesis cannot be excluded. For example, no gene accounting for the methylation was annotated in the aspernidine BGC. Based on gene deletion data and isolated intermediates a biosynthetic scheme for aspernidine A (10) biosynthesis was proposed starting with the production of orsellinaldehyde (222) by the PKS PkfA (Fig. 16B) (Yaegashi et al. 2013).

Fig. 16

A Organization of genes surrounding the PKS pkfA involved in aspernidine A (10) biosynthesis. B Proposed biosynthesis of aspernidine A (10) in Aspergillus nidulans (metabolites in boxes have been analytically identified). Abbreviations: SAT, starter unit ACP transacylase; KS, ketosynthase; AT, acyltransferase; PT, product template; ACP, acyl carrier protein; C-MeT, C-methyltransferase; R, reductase

For the production of cichorine (2) and aspernidine A (10) in A. nidulans, the CicF and PkfA, respectively, provide the phthalimidine core structure. Both CicF and PkfA belong to the NR-PKS in subclade III, distinguished by the C-methyl transferase (C-MeT) domain, that adds one or more S-adenosylmethionine(SAM)-derived methyl groups at the α-position of the polyketide chain (Herbst et al. 2018). Additionally, C-MeT acts as a gatekeeper, where α-methyl groups function as check-point tags for KS, thus controlling chain elongation cycles and polyketide formation by iterative PKS (Storm et al. 2017). Subclade III is further sub-divided into group VI with the domain architecture SAT-KS-AT-PT-ACP-(ACP)-CMeT-TE and group VII with the domain architecture SAT-KS-AT-PT-ACP-(CMeT)-R. Phylogenetic analysis revealed that CicF belongs to group VI while PkfA belongs to group VII (Ahuja et al. 2012). Both groups VI and VII NR-PKS produce monocyclic aromatics with a C2-C7 regioselective cyclisation under the control of the PT domain. However, group VI terminates with a TE that cleaves the thioester bond and releases the acid product, e.g. 3-methylorsellinic acid (217), and group VII has a NADPH-dependent R domain to release the product typically as an aldehyde, e.g. orsellinaldehyde (222).

Aspercryptin (6) is the product of two distinct BGCs physically separated in the genome of A. nidulans strain LO8030. The NR-PKS containing AN6448 (cicF) cluster and a sole non-ribosomal peptide synthetase (NRPS), AN7884 (atnA). Twelve additional genes (atnB-atnM) are involved in the biosynthesis of aspercryptin (6) (Fig. 17) (Chiang et al. 2016). Analogous to PKS, NRPS are multifunctional mega-enzymes organised structurally into modules, in which each module fulfils a cycle of peptide elongation. A minimal NRPS module consists of 3 core catalytic domains; an adenylation (A) domain for amino acid selection and activation, which is then tethered onto a thiolation (T)/peptidyl carrier protein (PCP) domain through the phosphopantetheine arm, which is then shuttled to the condensation (C) domain for peptide bond formation (Süssmuth and Mainz 2017; Vassaux et al. 2019). The final peptide is then released with the help of a TE domain, a R domain, a terminal C domain or with no obvious release domain (Du and Lou 2010).

Fig. 17

Organization of genes surrounding the NRPS atnA involved in aspercryptin (6) biosynthesis in Aspergillus nidulans

The 6 module NRPS AtnA is a type A linear NRPS, with each module responsible for the incorporation of a single amino acid residue (Chiang et al. 2016). The second module harbors an epimerise (E) domain which is responsible for generating the d-allo-threonine residue, while modules 4 and 6 incorporate the unusual amino acids 2-aminocaprylic acid and 2-aminododecanoic acid, respectively. These are believed to be the products of the fatty acid synthases (atnF and atnM), dehydrogenase (atnD), cytochrome P450 (atnE) and the amino acid transferases (atnJ and atnH) genes. Similar enzymes have earlier been proposed for the introduction of non-canonical amino acids in cyclosporine (Bushley et al. 2013), apicidin (Niehaus et al. 2014) and HC-toxin (Cheng et al. 1999) and d-amino acids and non-canonical amino acids are common in fungal NRP (Eisfeld 2009). Similar to aspernidine A (10), a C-terminal R domain in module 6 of the the NRP_S would be involved in the reductive release of the aldehyde hexapeptide (Chiang et al. 2016). A proposed biosynthetic pathway for aspercryptin (6) is depicted in Fig. 18 using cichorine-serine as a precursor.

Fig. 18

Proposed biosynthetic pathway for aspercryptin (6) in Aspergillus nidulans. Abbreviations: A, adenylation domain; T, thiolation domain; C, condensation domain; E, epimerization domain; R, reductase

The proposed biosynthetic pathways (Figs. 15B, 16B and 18) for the A. nidulans phthalimidines cichorine (2), aspernidine A (10) and aspercryptin (6) were based on the analysis of deletion mutants and isolated metabolites, and further work would be essential to confirm those pathways especially that other genes expected to be involved in the biosynthesis where so far not found within the cluster. For example, genes required for the conversion of the lactone nidulol (218) to a lactam in cichorine (2) and genes involved in the biosynthesis of the terpenoid moiety of apsernidine A (10). The O-methyltransferase cicE is believed to play a role in adding the methyl group to cichorine (10). However, a gene with a similar methyl functionality was not identified in the aspernidine (2) BGC. It is proposed that methylation could be carried out by a separate, standalone methyltransferase located outside of the gene cluster (Chen et al. 2019a). It would also be relevant to search for homologous cichorine (2) biosynthetic genes in A. cichorii the first source from which cichorine (2) was isolated; however, whole genome sequencing of A. cichorii was so far not undertaken. The predicted core genes involved in cichorine (2), aspernidine A (10) and aspercryptin (6) biosynthesis are summarized in Table 2.

Table 2 Summary of putative function of genes within the cichorine (2), aspernidine A (10) and aspercryptin (6) BGCs in Aspergillus nidulans

Biosynthetic studies in Stachybotrys spp.

Genome mining of the sequenced Stachybotrys strains, S. chartarum and Stachybotrys chlorohalonata genome was undertaken by Zhang et al. to identify the BGC involved in the synthesis of the structurally related Stachybotrys phthalimidines. A cluster containing 10 genes (idlA-I, R) was identified in both genomes (Fig. 19A) (Yin et al. 2017). Genes homologous to the stb cluster, earlier reported in Stachybotrys bisbyi PYH05-7 genome, were identified (Table 3). Heterologous expression of the stb genes in Aspergillus oryzae probed the functionality of the biosynthetic enzymes, revealing that the stb cluster has a NR-PKS (StbA), an NRPS-like enzyme (StbB), and a UbiA-like prenyltransferase (PTase) (StbC) involved in the biosynthesis of ilicicolin B (LL-Z1272β) (223) (Li et al. 2016). Ilicicolin B (223) is a prenylated aryl-aldehyde, considered to be a common precursor in the biosynthesis of Stachybotrys phthalimidines. The NR-PKS (StbA) provides the orsellinic acid starter unit (224), which is then farnesylated by the StbC using farnesyl pyrophosphate (FPP) as a prenyl donor to give grifolic acid (225). Here, the aldehyde moiety in ilicicolin B (223) is not generated by the R domain covalently appended at the end of the NR-PKS as seen in aspernidine A (10) biosynthesis, but is instead catalysed by the subtype II NRPS-like enzyme (StbB), considered as the first NRPS-like enzyme which activates a prenylated acid (225) (Fig. 19B) (Tao and Abe 2021).

Fig. 19

A Predicted BGC for phthalimidine derivatives in Stachybotrys chlorohalonata IBT 40285. B Proposed biosynthetic pathway of the putative intermediate ilicicolin B (223) (metabolites in boxes have been analytically identified). Abbreviations: SAT, starter unit ACP transacylase; KS, ketosynthase; AT, acyltransferase; PT, product template; ACP, acyl carrier protein; C-MeT, C-methyltransferase; TE; thioesterase; R, reductase

Table 3 Putative function of genes for the Stachybotrys phthalimidine BGCs in Stachybotrys chartarum IBT 7711 and Stachybotrys chlorohalonata IBT 40285, and their homologues in Stachybotrys bisbyi PYH05-7

Accordingly, a biosynthetic scheme was proposed for Stachybotrys phthalimidines, starting with the common precursor 223. They suggested two modes of cyclisation of the farnesyl side chain, initiated by an acid-catalysed epoxidation on different double bonds of the farnesyl group to provide different classes of Stachybotrys phthalimidine derivatives (Fig. 20) (Yin et al. 2017). Stereospecific olefin epoxidation, usually catalysed by a flavin-dependent monooxygenase (FMO) or P450 monooxygenase, precedes cyclisation by a terpene cyclase (TC) (Matsuda and Abe 2016; Quan et al. 2019). However, the detailed mechanism and enzymes involved in this catalysis still remain unclear. These scaffolds are then modified by tailoring enzymes in versatile manners to provide the huge structural diversity observed for Stachybotrys phthalimidines.

Fig. 20

Proposed biosynthetic pathway for phthalimidine derivatives in Stachybotrys sp. starting with ilicicolin B (223)

Additionally, homologous genes of a NR-PKS, an NRPS-like enzyme and two putative short-chain dehydrogenases seen in cichorine BGC in A. nidulans, and homologous PTase genes seen in the austinols and terretonin BGC in A. nidulans and A. terreus, respectively, were found in the Stachybotrys genome. The authors postulated that the nitrogen in the phthalimidine core skeleton is probably introduced non-enzymatically by the reaction of the phthalic aldehyde precursor (226 and 227) and ammonium ions or amino compounds (Yin et al. 2017). This was already proposed for the biosynthesis of the pyridine ring in rubrolone tropolone alkaloids, where absence of candidate genes in the rub cluster suggested the capture of free amines from solution by a reactive 1,5-dione moiety (Yan et al. 2016). In an earlier study, focusing on the production of SMTP congeners by feeding amine precursors to S. microspora cultures, significant amounts of 223 and 226 were found to accumulate during the ‘starvation phase’ before the addition of amines to the culture, and were rapidly consumed following the addition of amines, which strongly supports their involvement in the biosynthesis of SMTPs (Nishimura et al. 2012). They also suggested that the nitrate reductase (NR) gene in the Stachybotrys cluster is responsible for the balance of the ammonium ion concentration, where NR is the rate limiting enzyme for nitrate reduction prior to the formation of ammonium ions (Yin et al. 2017).

Biosynthetic studies in Hericium sp.

In contrast to Ascomycetes, genes and enzymes involved in the biosynthesis of phthalimidines in Basdiomycetes remain obscure. Initial work focused on the characterisation of two NR-PKSs and two PTases in the genome of a stereaceous Basidiomycete BY1. Both PKSs were found to produce orsellinic acid, a key intermediate involved in the biosynthesis of fungal phthalimidines (Braesel et al. 2017). Furthermore, the genome of Hericium sp. (H. erinaceus and H. coralloides) have been recently sequenced and published (Chen et al.

Abbreviations

PLC-γ1:

1/2, Phospholipase C-γ1

11β-HSD1:

11β-Hydroxysteroid dehydrogenase type 1

ADMET:

Absorption, distribution, metabolism, excretion, and toxicity

ABTS:

2,2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)

AIS:

Acute ischemic stroke

AKI:

Acute kidney injury

ACP:

Acyl-carrier protein

AT:

Acyltransferase domain

A:

Adenylation

AR:

Aldose reductase

BGC:

Biosynthetic gene cluster

BDNF:

Brain-derived neurotrophic factor

COPD:

Chronic obstructive pulmonary disease

C-MeT:

C-methyl transferase

C:

Condensation

COX:

Cyclooxygenase

DM:

Diabetes mellitus

DPPH:

2,2-Diphenyl-1-picrylhydrazyl

ECD:

Electron Capture Detector

CBL38:

Endophytic fungus Aspergillus variecolor CLB38

ET:

Endothelin

EGF:

Epidermal growth factor

ERK:

Extracellular signal-related kinase

FPP:

Farnesyl pyrophosphate

FMO:

Flavin-dependent monooxygenase

FGFC1:

Fungi fibrinolytic compound 1

FGFC2:

Fungi fibrinolytic compound 2

G6PT:

Glucose-6-phosphate translocase

SERCA2:

Sarco(endo)plasmic reticulum Ca2 + ATPase

HT:

Hemorrhagic transformation

HPLC:

High-Performance Liquid Chromatography

Tie2:

Homology domains

HLE:

Human leukocyte elastase

TG:

Intracellular triglyceride

KS:

Ketosynthase domain

LKN:

Last known normal

HK-ZJ:

Mangrove endophytic Aspergillus sp.

MM-1:

Matrix metalloproteinase-1

MeFSAT:

Medicinal Fungi Secondary Metabolites and Therapeutics

MRSA:

Methicillin-resistant Staphylococcus aureus

MVA:

Mevalonate pathway

MCA:

Middle cerebral artery

MIC:

Minimal inhibitory concentration

MD:

Molecular dynamics

MptpB:

Mycobacterium tuberculosis protein tyrosine phosphatase B

NGF:

Nerve growth factor

NR:

Nitrate reductase

NNRTI:

Non-nucleoside reverse transcriptase inhibitors

NR-PKSs:

Non-reducing group of iterative polyketide synthases

NRPS:

Non-ribosomal peptide synthetase

NMR:

Nuclear Magnetic Resonance

NO:

Nitric oxide

OSMAC:

One-strain many compounds

PCP:

Peptidyl carrier protein

PI3K:

Phosphatidylinositol-3-kinase

PDGF:

Platelet-derived growth factor s

PEG:

Polyethylene glycol

PKS:

Polyketide synthase

PTase:

Prenyltransferase

PT:

Product template

PTP1B:

Protein tyrosine phosphatase-1B

CD:

Quantum-chemical

ROS:

Reactive oxygen species

R:

Reductase

RT:

Reverse transcriptase

SAM:

S-Adenosylmethionine

SM:

Secondary metabolite

SMASH:

Secondary Metabolite Analysis Shell

SARs:

Severe Acute Respiratory Syndrome

SARS-CoV:

Severe Acute Respiratory Syndrome Coronavirus

SSF:

Solid-state fermentation

SMTPs:

Stachybotrys microspora Triprenyl phenols

SAR:

Structure–activity relationship

SAT:

Starter unit ACP transacylase

SAHA:

Suberoylanilide hydroxamic acid

TC:

Terpene cyclase

TE:

Thioesterase

CLC:

Claisen cyclase

T:

Thiolation

TD-DFT:

Time-dependent density functional theory

t-PA:

Tissue-type plasminogen activator

TC:

Total cholesterol

TCM:

Traditional Chinese Medicine

TPPs:

Triprenyl phenols

VSMCs:

Vascular smooth muscle cells