Naturally Occurring Isocoumarins Derivatives from Endophytic Fungi: Sources, Isolation, Structural Characterization, Biosynthesis, and Biological Activities

Recently, the metabolites separated from endophytes have attracted significant attention, as many of them have a unique structure and appealing pharmacological and biological potentials. Isocoumarins represent one of the most interesting classes of metabolites, which are coumarins isomers with a reversed lactone moiety. They are produced by plants, microbes, marine organisms, bacteria, insects, liverworts, and fungi and possessed a wide array of bioactivities. This review gives an overview of isocoumarins derivatives from endophytic fungi and their source, isolation, structural characterization, biosynthesis, and bioactivities, concentrating on the period from 2000 to 2019. Overall, 307 metabolites and more than 120 references are conferred. This is the first review on these multi-facetted metabolites from endophytic fungi.


Introduction
The search for new metabolites for the agrochemical and pharmaceutical industries is an on-going work that needs continual optimization. Fungi are eukaryotic microorganisms that reside in almost all environmental types in nature where they have key roles in preserving the ecological balance [1,2]. Endophytes primarily inhabit their hosts without causing any harm to the hosts [3][4][5][6]. These endophytic fungi have played pivotal roles in their host's survival through supplying nutrients and producing plenty of bioactive metabolites to prevent the danger of phytopathogenic bacteria on the host [7,8]. Endophytic fungi have gained loads of attention in natural products chemistry field due to their sustainability to biosynthesize structurally diverse and bioactive molecules, some of which are important agrochemicals and pharmaceuticals [9,10]. Isocoumarins (1H-2-benzopyran-1-ones or isochromene derivatives) are a class of biosynthetically, structurally, and pharmacologically intriguing natural products, which chemistry field due to their sustainability to biosynthesize structurally diverse and bioactive molecules, some of which are important agrochemicals and pharmaceuticals [9,10]. Isocoumarins (1H-2-benzopyran-1-ones or isochromene derivatives) are a class of biosynthetically, structurally, and pharmacologically intriguing natural products, which are coumarins isomers with a reversed lactone moiety that could possess 6,8-dioxygenated pattern, 3-(un)substituted phenyl ring or 3-alkyl chain (C1-C17) [11,12]. The oxygenation could exist at one or more of the six free positions of the isocoumarin skeleton. The oxygen atoms may be in the form of ethereal, phenolic, or glycosidic functionalities. Additionally, C-3 substituents are found more commonly on both natural and synthetic isocoumarins derivatives. Substituents that exist on the isocoumarin ring may involve alkyl, halogen, heterocyclic, aryl, or other groups [13]. Furthermore, the saturation of C-3/C-4 in isocoumarins will give 3,4-dihydroisocoumarins (DHICs) analogs ( Figure 1). Moreover, isocoumarins and DHICs possess a close relation with isochromans, they are known as isochromen-1-one and isochroman-1-ones, respectively, since the C-1 active methylene in isochromans can be easily oxidized to the related isocoumarins derivatives. Most of the natural isocoumarins and DHICs are given trivial names, which are derived mainly from the name of the species or genus of the host organisms. They have been reported from a broad scope of natural sources, including plants, microbes, marine organisms, bacteria, insects, liverworts, and fungi (e.g., soil, endophytic, and marine fungi) [14,15]. Isocoumarins are considered as important intermediates in the synthesis of a wide range of carbo-and heterocyclic compounds such as isoquinolines, isochromenes, and different aromatic compounds [16]. Thus, isocoumarin framework has been explored in various areas, including drug discovery, pharmaceutical and medicinal chemistry, and organic synthesis [13]. It has been reported that these metabolites possess various bioactivities: antimicrobial, cytotoxic, algicidal, antiallergic, immunomodulatory, antimalarial, plant growth regulatory, and acetylcholinesterase and protease inhibitors [11,[17][18][19][20]. This review aims to give a highlight on the naturally occurring isocoumarins derivatives reported from endophytic fungi, focusing on the period from 2000 to July 2019. Herein, 307 naturally occurring isocoumarins derivatives have been listed most of them are reported from Aspergillus and Penicillium genera (Figure 2). Moreover, isocoumarins and DHICs possess a close relation with isochromans, they are known as isochromen-1-one and isochroman-1-ones, respectively, since the C-1 active methylene in isochromans can be easily oxidized to the related isocoumarins derivatives. Most of the natural isocoumarins and DHICs are given trivial names, which are derived mainly from the name of the species or genus of the host organisms. They have been reported from a broad scope of natural sources, including plants, microbes, marine organisms, bacteria, insects, liverworts, and fungi (e.g., soil, endophytic, and marine fungi) [14,15]. Isocoumarins are considered as important intermediates in the synthesis of a wide range of carbo-and heterocyclic compounds such as isoquinolines, isochromenes, and different aromatic compounds [16]. Thus, isocoumarin framework has been explored in various areas, including drug discovery, pharmaceutical and medicinal chemistry, and organic synthesis [13]. It has been reported that these metabolites possess various bioactivities: antimicrobial, cytotoxic, algicidal, antiallergic, immunomodulatory, antimalarial, plant growth regulatory, and acetylcholinesterase and protease inhibitors [11,[17][18][19][20]. This review aims to give a highlight on the naturally occurring isocoumarins derivatives reported from endophytic fungi, focusing on the period from 2000 to July 2019. Herein, It is hoped that by using these figures in conjunction with the trivial name, fungal source, host, and place (Table 1) the readers will be able to locate key references in the literature and gain much understanding of the fascinating chemistry of these metabolites. Many of these derivatives have substituents at C-3, which could be one carbon or more. The majority of them have an oxygen atom at C-8 and some have the C-6 oxygen. Further alkylation or oxygenation may occur at the remaining positions of the isocoumarin skeleton. Isocoumarins with 3,4-, 4,5-, 5,6-, 6,7-, and 7,8fused carbocyclic rings are reported. Some of the reported derivatives have chlorine (e.g., 9, 12, 22, and 28-31) or bromine (e.g., 23, 27, 32, and 33) atom at C-5 and/or C-7. Some show sugar moieties such as glucose (e.g., 15, 77-79, and 151) and ribose moiety (e.g., 78 and 79). In addition, some isocoumarins dimers are reported (e.g., 259, 260, and 266-268). Moreover, some linked to other moieties such as anthraquinone and indole diketopiperazine (e.g., 285 and 296) or contain sulphur (e.g., 278 and 279) or nitrogen (e.g., 269-271) substituents. This review also mentions briefly their isolation, structural characterization, biosynthesis, and bioactivities ( Figures 31-35, Tables 2 and 3). Strengthening of their bioactivities may draw the attention of medicinal and synthetic chemists for designing new agents using the known isocoumarins derivatives as raw materials and the discovery of new therapeutic properties not yet attributed to known compounds. The published literature search was conducted over various databases: Web of Science, PubMed, Google Scholar, Scopus, SpringerLink, ACS Publications, Wiley, Taylor and Francis, and Sci-Finder using the keywords (isocoumarin, endophytes, and biological activities).

Biosynthesis
Isocoumarin was originated of the acetate-malonate or the polyketide synthase (PKS) pathway [21,22]. Kurosaki et al. stated that 11 is biosynthesized from malonyl-CoA and acetyl-CoA through a It is hoped that by using these figures in conjunction with the trivial name, fungal source, host, and place (Table 1) the readers will be able to locate key references in the literature and gain much understanding of the fascinating chemistry of these metabolites. Many of these derivatives have substituents at C-3, which could be one carbon or more. The majority of them have an oxygen atom at C-8 and some have the C-6 oxygen. Further alkylation or oxygenation may occur at the remaining positions of the isocoumarin skeleton. Isocoumarins with 3,4-, 4,5-, 5,6-, 6,7-, and 7,8-fused carbocyclic rings are reported. Some of the reported derivatives have chlorine (e.g., 9, 12, 22, and 28-31) or bromine (e.g., 23, 27, 32, and 33) atom at C-5 and/or C-7. Some show sugar moieties such as glucose (e.g., 15, 77-79, and 151) and ribose moiety (e.g., 78 and 79). In addition, some isocoumarins dimers are reported (e.g., 259, 260, and 266-268). Moreover, some linked to other moieties such as anthraquinone and indole diketopiperazine (e.g., 285 and 296) or contain sulphur (e.g., 278 and 279) or nitrogen (e.g., 269-271) substituents. This review also mentions briefly their isolation, structural characterization, biosynthesis, and bioactivities (Figures 31-35, Tables 2 and 3). Strengthening of their bioactivities may draw the attention of medicinal and synthetic chemists for designing new agents using the known isocoumarins derivatives as raw materials and the discovery of new therapeutic properties not yet attributed to known compounds. The published literature search was conducted over various databases: Web of Science, PubMed, Google Scholar, Scopus, SpringerLink, ACS Publications, Wiley, Taylor and Francis, and Sci-Finder using the keywords (isocoumarin, endophytes, and biological activities).
Molecules 2020, 25 pentaketide [23]. 3,4-Dihydro-6-hydroxymellein (III) is considered as an intermediate which would be transformed to 11 by O-methyltransferase which methylates the 6-OH group of the isocoumarin [23]. The loss of the OH group at C-6 gives rise to mellein [24]. A heptaketide II, a longer polyketone chain is implicated in 165 biosynthesis [25] (Figure 31). Krohn et al. reported that the existence of a biosynthetic relationship between 56 and 125 [27]. They assumed that the open-chain precursor A can be directly closed to a six-membered lactone (pathway I) or cyclized after the side chain rotation through the acetyl enol tautomer to produce 56 (pathway II) [27] (Figure 32).  Krohn et al. reported that the existence of a biosynthetic relationship between 56 and 125 [27]. They assumed that the open-chain precursor A can be directly closed to a six-membered lactone (pathway I) or cyclized after the side chain rotation through the acetyl enol tautomer to produce 56 (pathway II) [27] (Figure 32). pentaketide [23]. 3,4-Dihydro-6-hydroxymellein (III) is considered as an intermediate which would be transformed to 11 by O-methyltransferase which methylates the 6-OH group of the isocoumarin [23]. The loss of the OH group at C-6 gives rise to mellein [24]. A heptaketide II, a longer polyketone chain is implicated in 165 biosynthesis [25] (Figure 31). Krohn et al. reported that the existence of a biosynthetic relationship between 56 and 125 [27]. They assumed that the open-chain precursor A can be directly closed to a six-membered lactone (pathway I) or cyclized after the side chain rotation through the acetyl enol tautomer to produce 56 (pathway II) [27] (Figure 32).  [27]. Figure 32. Proposed biosynthetic pathway of 56 and 125 [27].
It was postulated that 273 is also derived from the malonate-acetate pathway [28]. The pentaketide (I) cyclization and enolization produce 88. A Claisen condensation occurs between 88 and tetraketide (II) to yield III. The side chain enolization, along with the hemiketal formation by the side chain ketone carbonyl and C-6 phenolic OH of the isocoumarin nucleus, forms a hemiketal IV. Then, the ketal formation and methylation in the side chain by S-adenosyl methionine (SAM) yield V and finally 273 [28] (Figure 33).
Molecules 2020, 25, x 30 of 109 It was postulated that 273 is also derived from the malonate-acetate pathway ] 28 [ . The pentaketide (I) cyclization and enolization produce 88. A Claisen condensation occurs between 88 and tetraketide (II) to yield III. The side chain enolization, along with the hemiketal formation by the side chain ketone carbonyl and C-6 phenolic OH of the isocoumarin nucleus, forms a hemiketal IV. Then, the ketal formation and methylation in the side chain by S-adenosyl methionine (SAM) yield V and finally 273 [28] (Figure 33). Moreover, Song et al. reported that an intramolecular cyclization occurs of a polyketide chain (Path A, Figure 6) [22]. The C-4 substituted derivatives have been resulted from the participation of an additional carbon unit in the cyclization (Path B, Figure 34). Therefore, the rare isocoumarin derivatives, 179 and 180 biosynthesis differs from those of 70, 71, and 138, in which a carbon moiety (CH2OH) from formate or serine took part in the cyclization. Additionally, the 3-unsubstituted derivatives couldn't be yielded in the biosynthesis of compounds Moreover, Song et al. reported that an intramolecular cyclization occurs of a polyketide chain (Path A, Figure 6) [22]. The C-4 substituted derivatives have been resulted from the participation of an additional carbon unit in the cyclization (Path B, Figure 34). It was postulated that 273 is also derived from the malonate-acetate pathway ] 28 [ . The pentaketide (I) cyclization and enolization produce 88. A Claisen condensation occurs between 88 and tetraketide (II) to yield III. The side chain enolization, along with the hemiketal formation by the side chain ketone carbonyl and C-6 phenolic OH of the isocoumarin nucleus, forms a hemiketal IV. Then, the ketal formation and methylation in the side chain by S-adenosyl methionine (SAM) yield V and finally 273 [28] (Figure 33). Moreover, Song et al. reported that an intramolecular cyclization occurs of a polyketide chain (Path A, Figure 6) [22]. The C-4 substituted derivatives have been resulted from the participation of an additional carbon unit in the cyclization (Path B, Figure 34). Therefore, the rare isocoumarin derivatives, 179 and 180 biosynthesis differs from those of 70, 71, and 138, in which a carbon moiety (CH2OH) from formate or serine took part in the cyclization. Additionally, the 3-unsubstituted derivatives couldn't be yielded in the biosynthesis of compounds Therefore, the rare isocoumarin derivatives, 179 and 180 biosynthesis differs from those of 70, 71, and 138, in which a carbon moiety (CH 2 OH) from formate or serine took part in the cyclization. Additionally, the 3-unsubstituted derivatives couldn't be yielded in the biosynthesis of compounds 138 and 70; due to the C-11 oxidation is usually taking place after the polyketide chain cyclization [22]. Chen et al. postulated the biosynthetic origin of 296, an isocoumarin-indole diketopiperazine alkaloid ( Figure 35) [29].

Structural Characterization of Isocoumarins Derivatives
Isocoumarins can be characterized by different spectral techniques such as 1D ( 1 H, 13 C, and NOE) and 2D NMR techniques (COSY, HSQC, HMBC, ROESY, and NOESY) combined with other usual methods (chemical synthesis, UV, IR, MS, etc.). However, their spectral data cannot be generalized as the data differ to a wide range relying on the type, position, number, and nature of substituents connected to the core skeleton. Furthermore, these data vary basically due to the variation of the core ring. In the compounds having isocoumarins framework, the lactone carbonyl frequency generally appears in the region 1745-1700 cm −1 in the IR. In 1 H NMR, the C-3 vinylic proton appears at 6.2-7.0 ppm as a singlet or doublet for C3-substituted and unsubstituted derivatives, respectively. In 13 C NMR, the lactone C=O appears in the range from 164 ppm to 168 ppm. In the 3-substituted derivatives, C-4 vinylic proton appears at 6.11-6.7 ppm as a singlet. 3,4-Dihydroisocoumarins derivatives have relatively more complicated 1 H NMR spectra than isocoumarins due to C-4 and C-3 vicinal coupling and/or C-4 diastereotopic protons geminal coupling. In both derivatives, the 8-OH group appears at 10.0-12.0 ppm due to the hydrogen bonding to the C-1 carbonyl. 6,8-Dihydroxy-3-(2-oxopropyl)-1H-isochromen-1-one (I) was originated from the PKS pathway. It was then chlorinated and O-methylated to produce 3-(3-chloro-2-oxopropyl)-6,8-dimethoxy-1H-isochromen-1-one (II) by the catalytic effect of a bifunctional hybrid enzyme (BFHEnz). The methyl-carbonyl group of II undergoes chlorination and reduction leading to the formation of 235 and 236, respectively. Then, the hybridization of the diketopiperazine and isocoumarin units by a free radical mechanism, which could be catalyzed by cytochrome P450 giving 296 [29].

Structural Characterization of Isocoumarins Derivatives
Isocoumarins can be characterized by different spectral techniques such as 1D ( 1 H, 13 C, and NOE) and 2D NMR techniques (COSY, HSQC, HMBC, ROESY, and NOESY) combined with other usual methods (chemical synthesis, UV, IR, MS, etc.). However, their spectral data cannot be generalized as the data differ to a wide range relying on the type, position, number, and nature of substituents connected to the core skeleton. Furthermore, these data vary basically due to the variation of the core ring. In the compounds having isocoumarins framework, the lactone carbonyl frequency generally appears in the region 1745-1700 cm −1 in the IR. In 1 H NMR, the C-3 vinylic proton appears at 6.2-7.0 ppm as a singlet or doublet for C 3 -substituted and unsubstituted derivatives, respectively. In 13 C NMR, the lactone C=O appears in the range from 164 ppm to 168 ppm. In the 3-substituted derivatives, C-4 vinylic proton appears at 6.11-6.7 ppm as a singlet. 3,4-Dihydroisocoumarins derivatives have relatively more complicated 1 H NMR spectra than isocoumarins due to C-4 and C-3 vicinal coupling and/or C-4 diastereotopic protons geminal coupling. In both derivatives, the 8-OH group appears at 10.0-12.0 ppm due to the hydrogen bonding to the C-1 carbonyl.
Mass spectroscopy is a helpful tool for the identification of these metabolites. The existence of sulfur was evident by the intensity of [M + 2] + ion peak (∼4.5% of the molecular ion peak) [30]. Moreover, the chlorine atom in the structure was characterized by two ion peaks [M + H] + and [M+2H] + in a ratio 3:1 [31,32]. The relative configuration was determined by NOE, NOESY, and ROESY. The circular dichroism (CD) is usually utilized to assess the absolute configuration by comparison of the theoretical and experimental CD spectra [30,31,33]. Besides, the total synthesis provides important information and an additional confirmation for characterization of these metabolites structures. Furthermore, it allows the synthesis of analogs with improved biological efficiencies [11,34,35]. The X-ray structure crystallographic analysis of the crystalline derivatives is another tool for the absolute configuration determination. This technique could not be applied in many cases since the crystals with the required qualifications are not available because most of these metabolites do not crystallize conveniently [20,27,36]. Finally, the assignment of the absolute configuration could be done using Mosher's method and the differences in chemical shift between the (R)-and (S)-MTPA were analyzed [33,37].

Cytotoxic Activity
The cytotoxic activities of isocoumarins have been assessed towards various cancer cell lines using various assays and the most active compounds have been listed in Table 3.
Acetylcholinesterase (AChE), an enzyme that catalyzes acetylcholine (ACh) hydrolysis leading to a decrease in the levels of ACh in the brain [105]. Thus, appears to be a critical element in the development of neurodegenerative diseases such as Alzheimer's disease (AD) and dementia. The most suitable therapeutic approach for treating AD and other forms of dementia is to restore ACh levels by inhibiting AChE [106]. Compounds 3, 6, and 8 were evaluated for their AChE inhibitory activities. Compound 6 had a moderate AChE inhibitory potential with a limit of detection 30.0 µg, whereas 3 and 8 were inactive (limit of detection over 100 µg) [18]. Compounds 34, 36, and 37 showed weak inhibition of AChE with a limit of detection 10 µg in a TLC-based AChE inhibition assay [17]. Only 34 displayed moderate AChE inhibitory activity (limit of detection 3.0 µg) compared to galantamine (MICs 1.0 µg) [17].
Protein kinases are enzymes that catalyze the transfer of a phosphate group from a high energy molecule such as adenine triphosphate (ATP) to a specific amino acid. They play important roles in regulating many cellular functions, including survival, proliferation, motility, apoptosis, as well as DNA damage repair and metabolism [107]. Some of them are commonly activated in cancer cells and known to play roles in tumorigenesis [108]. Protein kinases inhibitors are anticipated to be a source of potential therapeutic targets for treating various human disorders such as neoplastic and neuroinflammatory diseases [107,108]. The isolated alternariol derivatives from Alternaria sp. were assessed for their inhibitory activities against 24 protein kinases. Interestingly, alternariol (188) and its derivatives 189-191 prohibited protein kinases: Aurora A, ARK5, Aurora B, IGF1-R, b-RAF, VEGF-R2, FLT3, VEGF-R3, SAK, and PDGF-Rbeta with IC 50 below 1 × 10 −6 g/mL. Moreover, 193 exhibited activity with an IC 50 1 × 10 −5 g/mL or less towards the various tested kinases [40].

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