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Endophytic fungi: A treasure trove of novel anticancer compounds

Cancer is a multifactorial disease with a convoluted genesis and progression. The emergence of multidrug resistance to presently be offered drug and relapse is by far, the most critical concern to tackle this deteriorating disease. Henceforth, there is undeniably an inflated necessity for safe, promising, and less harmful new anticancer drugs. Natural compounds from various sources like plants, animals, and microorganisms have occupied a center stage in drug discovery due to their tremendous chemical diversity and potential as therapeutic agents. Endophytic microbes are symbiotically associated with plants and have been proven to produce novel or analogues of host bioactive metabolites exhibiting a variety of biological activities including anticancer activity. This review emphasizes on structurally diverse unprecedented anticancer natural compounds that have been reported exclusively from endophytic fungi from 2016 to 2020. It covers chemical nature of metabolites, its fungal source associated with terrestrial, as well as marine plants and anticancer activity based on their cytotoxicity profile against various cancer cell lines. Many of these fungal metabolites with promising anticancer activity can be used as lead molecules for in silico experiments and deserve special attention from scientists for further in vitro and clinical research.


Introduction
Cancer is a global health problem that affects all population, regardless of age, gender, ethnicity, wealth, or socioeconomic status and therefore, it is undeniably the biggest obstacle in the twenty-first century that is jeopardizing medical system and research community. Cancer is the second leading cause of death worldwide, accounting for about 10 million deaths per year and this number is still rising (GLOBOCAN, 2020). A variety of clinical methods, including surgery, chemotherapy, and/or radiation are the key treatment for cancer (Subramaniam et al., 2019). Currently present chemotherapeutic drugs in cancer treatment offer temporary relief to the patients and extend their longevity, but with this come other disadvantages which includes side effects, lack of selectivity and overpriced that not only impairs the quality of life but is also unaffordable by millions of patients living in developing nations (Reis-Mendes et al., 2018). Additionally, the emergence of multidrug resistance and relapse of malignancy is by far, the most critical concern in the treatment of this multifactorial condition with a convoluted genesis and progression (Cree and Charlton, 2017;Patel et al., 2018). To tackle this deteriorating disease, there is certainly an unmet need for new promising, safe, and less harmful medicines produced from natural compounds. Research findings suggest that phytochemicals possess antitumor ability, and many of them are currently being used to treat this high-profile disease (Patel et al., 2018;Bhadresha et al., 2021;Shah et al., 2015). Despite the fact that phytochemicals are recognized as the most significant source of potential drugs, their use in drug research has recently seen a decline in interest due to some remarkable challenges with the plant as a drug molecule source, such as its unusual habitat, slow growth rate, limited yield and non-reproducibility of the desired phytochemicals and an unwavering threat by the civilization (Kala et al., 2006;Garcia-Oliveira et al., 2021). On the other side, microorganisms have sparked a lot more interest as a source of therapeutic agents because of their abundance, high biodiversity, and unique scaffold of produced bioactive components. They are often rudimentary to culture in a fermenter under controlled environments, and consistently deliver the desired molecules. (Abdel-Razek et al., 2020).
The great discovery of the billion-dollar chemotherapeutic drug Paclitaxel originally known to be produced only by plant Taxus longifolia (discovered in 1971), was later identified to be produced by an endophytic fungus Taxomyces andreanae (discovered in 1993), from the same plant, Taxus longifolia has led scientists to appreciate plants as reservoirs for an infinite range of microorganisms, normally alluded as the endophytes (Stierle et al., 1995). Endophytes are microorganisms that live within plant tissues with mutualistic association for at least part of their life cycle without causing any apparent disease manifestation (Arora et al., 2016). Some endophytes have evolved the ability to synthesize bioactive compounds in a de novo manner that are identical or related to those produced by their host plants during mutualistic association . For instance, endophytic fungi isolated from Taxus chinensis var. mairei, Podophyllum hexandrum, Camptotheca acuminata and Catharanthus roseus can produce anticancer drug taxol, podophyllotoxin, camptothecin and vincristine, respectively (Palem et al., 2015;Nadeem, 2012;Qiao et al., 2017;Ran et al., 2017). Moreover, these endophytes also produce novel secondary metabolites with unprecedented scaffolds (Pang et al., 2018) and sometimes hybrid chemical skeletons . The most widely found endophyte fungal species belong to the genus of Aspergillus, Chaetomium, Penicillium, Fusarium and Pestalotiopsis.
Research on endophytic fungi have evinced that, they are an auspicious resource of bioactive compounds and to access this valuable source, the diversity of endophytic fungi and their species richness in different parts of world has been explored.
Large number of secondary metabolites, especially from fungal endophytes are extracted, isolated, and characterized, of which, some of these have been discussed in earlier reviews in detail (Bedi et al., 2018a;Deshmukh et al., 2014;Kharwar et al., 2011;Guo et al., 2008). Most of these fungal metabolites are bioactive and classified under various chemical classes like, alkaloids, terpenoids, polyketides, flavonoids, steroids, lactones, lignans, depsipeptides, quinones, etc. In the past three decades, around 180 natural molecules with significant cytotoxicity towards selected tumor cell lines are reported from the endophytic fungi harbored in various plants (Bedi et al., 2018a;Kharwar et al., 2011). This review summarizes 205 novel compounds produced by terrestrial and marine plant-associated endophytic fungi from 2016 to 2020, including detailed chemical structures, structural type classifications, and cytotoxicity towards particular cancer cell lines.

Cytotoxic secondary metabolites from endophytic fungi
Endophytic fungi are valuable source of natural bioactive compounds in drug discovery and have a wide range of medical applications. Secondary metabolites from endophytic fungi have diverse biological activities, such as cytotoxic, antifungal, antibacterial, antiviral, antioxidant, anti-inflammatory, phytotoxic, antimalarial, antialgae, antimigratory, and pro-apoptotic activity. In past thirty years, researchers have been driven for the use of novel and promising cytotoxic compounds in pharmaceuticals. Herein, 205 new natural compounds synthesized by terrestrial as well as marine plant associated 95 endophytic fungi from 4 phyla and 41 genera, collected from 16 countries during the period of 2016-2020 are described (Fig. 1). In the subsequent sections, we discuss about the cytotoxic profile of compounds which are majorly classified as polyketides, terpenoids, sterols, macrolides, lactones, azaphilones, alkaloids, preussomerins, p-terphenyls, hybrid structures and miscellaneous compounds based on their chemical nature. List of secondary metabolites from endophytic fungi, with their plant host and cytotoxic activities towards specific tumor cell lines are provided in Table 1.

Chromones
Chromones are phenolic derivatives of chromone and isomers of coumarin that have been confirmed to have anti-tumor, anti-viral, antimicrobial, anti-inflammatory, and antioxidant properties (di Duan et al., 2019). A chromone derivative, Botryochromone (1) was isolated from unidentified strain (BCC 54265) of Botryosphaeriaceae family, which was obtained from a leaf of the Vietnamese coriander, Polygonum odoratum and exhibited weak cytotoxicity to epidermoid (KB) and human small cell lung cancer (NCI-H187) cell lines with IC 50 values of 43 and 18 μg/mL, while remain inactive against MCF-7, a human breast cancer cell line (Isaka et al., 2018). Rhytidchromones A-D (2-5) are highly oxygenated chromones, which were separated from the culture broth of fungus Rhytidhysteron rufulum, an endophyte associated with marine Thai Bruguiera gymnorrhiza. All compounds were tested for cytotoxic activity against four tumor cell lines human breast (MCF-7), liver (HepG2), gastric (KATO-3) and cervical (CaSki), in which they remain inactive against HepG2 and CaSki cells with IC 50 s of >25 μM. Compound Rhytidchromone A and Rhytidchromone D were active against MCF-7 and KATO-3 cells with IC 50 value of 19.3 AE 2.5, 23.3 AE 1.1 μM and 17.7 AE 3.7, 16.0 AE 1.9 μM, respectively, while compound Rhytidchromone B and Rhytidchromone C were cytotoxic towards only KATO-3 cells with IC 50 s of 21.4 AE 2.3 and 16.8 AE 3.6 μM, respectively (Chokpaiboon et al., 2016).

Isocoumarins
Isocoumarins are a large group of natural substances that belong to the well-known polyketide family. They are the structural isomer of coumarin and have a wide range of chemical configurations and therapeutic effects (Saddiqa et al., 2017). Four new isocoumarin derivatives, Oryzaeins A-D (18-21) were extracted from solid cultures of fungus Aspergillus oryzae, an endophyte associated with Paris polyphylla var. yunnanensis. All compounds showed antiproliferative activity towards human tumor cell lines NB4 (human leukemia), A-549, SHSY5Y (human neuroblastoma), PC-3, and/or MCF-7 with IC 50 s in the range between 2.8 and 8.8 μM . Aspergisocoumrin A (22) and Aspergisocoumrin B (23) are new cytotoxic isocoumarin derivatives, which were isolated from the fungus Aspergillus sp. HN15-5D, an endophyte harbored in the leaves of the mangrove plant Acanthus ilicifolius. inactive against other tested cell lines ). An unprecedented halogenated dihydroisocoumarin, Palmaerone E (24) was                  separated from SAHA (histone deacetylase inhibitor) exposed fermented culture of fungus Lachnum palmae, an endophyte associated with Przewalskia tangutica. It exhibited weak cytotoxic activity towards only HepG2 cell line with the IC 50 value of 42.8 μM, while remain inactive (IC 50 > 50 μM) against HL-60 and SGC-7901 (human gastric carcinoma) cells (Zhao et al., 2018a). The structures of isocoumarins (18-24) are shown in Fig. 2.
Because Xanthoquinodin B9 does have a lower IC 50 value towards NCI-H187 cells than the Vero cells, it requires exploratory research into discovering its mechanism of action. Chryxanthone A (27) and Chryxanthone B (28) were separated from a culture of endophytic fungus Penicillium chrysogenum AD-1540, which was harbored in the marine red alga Grateloupia turuturu. Chryxanthone A possess cytotoxic ability towards A-549, BT-549 (Triple negative breast  respectively (Zhao et al., 2018b). The mangrove plant Bruguiera gymnorrhiza derived endophytic fungus Peniophora incarnata Z4 was detected with a new cytotoxic xanthone derivative named Incarxanthone B (29). It exhibited cytotoxicity towards A375 (human melanoma), MCF-7, and HL-60 tumor cell lines with IC 50 s in the range of 4.9-8.6 μM ). An endophytic fungus Paecilamyces sp. TE-540 was isolated from the leaves of Nicotiana tabacum L., which was collected from China in August 2016. One novel compound with xanthone moiety, Chryxanthone C (30) was separated from the EtOAc crude extract of isolated fungus, which possess cytotoxic ability against only HeLa cell line with IC 50 value of 23.6 AE 2.1 μM while remain inactive (IC 50 s > 100 μM) against other tested A-549, BT-549, HepG2, and MCF-7 cell lines . The structures of xanthones (25-30) are shown in Fig. 3.

Phenalenone
Phenalenones are a special class of fungal metabolites with three-ring systems of hydroxyl-perinaphthenones that have a wide range of biological activities, including anti-HIV, antimicrobial, anti-malarial, and cytotoxic activity (Gombodorj et al., 2017). A new phenalenone analogue, Hispidulone B (31) with naphthalene backbone was isolated from dessert plant associated endophytic fungus Chaetosphaeronema hispidulum. It possess significant cytotoxic ability towards A-549, human hepatoma cell line (Huh7), and HeLa cell lines with IC 50 s of 2.71 AE 0.08, 22.93 AE 1.61, and 23.94 AE 0.33 μM, respectively, compared to the positive control cis-platinum which had an IC 50 s of 8.73 AE 1.77, 5.89 AE 0.15, and 14.68 AE 0.10 μM, respectively . As the compound had a better cytotoxic ability against particular cancer cell lines than the positive control, it should be investigated further to determine its molecular mechanism to develop it as a new anti-cancer therapeutic agent after thorough pharmacological evaluation. The structure of phenalenone (31) is shown in Fig. 3.

Diphenyl ethers
Prenylated diphenyl ethers have been shown to have antimicrobial, cytotoxic, antioxidant, and antiviral effects on a number of cases . developed as an anticancer agent. The structures of diphenyl ethers (32-35) are shown in Fig. 3.
Six new bicyclic polyketides, Preussilides A¡F (45-50), were obtained from the fermented culture of endophytic fungi Preussia similis harbored in medicinal plant Globularia alypum, which was collected in Batna, Algeria. All isolated preussilides exhibited antiproliferative activity against either some or all tested cell lines L929, KB3.1, A431, A-549, SKOV-3 (ovarian cancer), PC-3, MCF-7 and U2OS with IC 50 values in the range of 2.5-80 μM (Noumeur et al., 2017). Selective cytotoxicity of Preussilide A and Preussilide C and influence on the morphology of human osteosarcoma cells (U2OS) suggest that these two compounds might affects the cell division process and more research is needed to determine the molecular mechanism. The structures of unclassified polyketides (36-50) are shown in Fig. 4.

Terpenoids
Terpenoids are a class of natural bioactive substances which have isoprene as a scaffold and are found in a wide range of organisms. Many terpenoids are studied for anticancer activity, antimicrobial activities, lethal toxicity, anti-inflammatory action, enzyme inhibitor activity, and other biological activities (Jiang et al., 2020). Herein, 40 terpenoids are divided into four categories: meroterpenoids, sesquiterpenoids, diterpenoid, and triterpenoids.
Two novel meroterpenoids, Isopenicin A (67) and Isopenicin B (68) with unprecedented hybrid skeleton of polyketide-terpenoid moiety were obtained from fermented culture of endophytic fungus Penicillium   . Detailed analysis of both compounds on human embryonic kidney cell line (HEK293), SW620, and HCT116 cells revealed that they selectively suppress the Wnt pathway in colon cancer cells. However, more research and in vivo experiments are essential before it could be termed an effective anticancer compound. The structures of meroterpenoid (51-68) are shown in Fig. 5.   Fig. 9. Chemical structures of alkaloids (119-140).

Triterpenoids
Triterpenoids are a group of chemical compounds composed of three terpene units that have a versatile chemistry and pharmacology and contain several pentacyclic motifs. Three new tetracyclic triterpenoids, including Integracide H (89) and Integracide J (90) were separated from the fermented culture of endophytic fungus Fusarium sp. associated with Mentha longifolia L. Both the compounds showed strong cytotoxicity towards BT-549, SKOV-3 and KB cell lines with IC 50 s of 1.82, 1.32, 0.18 μM and 2.46, 3.01, 2.54 μM, respectively, compared to positive control doxorubicin (2.78, 0.16 and 0.41 μM). However, both the compounds remain inactive against SKOV-3 cell line, while other compound remain inactive against all tested cell lines (Ibrahim et al., 2016a). The structures of triterpenoids (89-90) are shown in Fig. 6.

Steroid
Steroids are a class of fungal metabolites with a wide range of chemical structures and biological functions. Many researchers are currently looking for steroidal metabolites to use as potential lead compounds in drug development (Ur Rahman et al., 2017). A new compound, Demethylincisterol A5 (91) with sterol moiety was separated from the rice fermented culture of endophytic fungus Aspergillus tubingensis YP-2, which was isolated from traditional Chinese medicinal plant Taxus yunnanensis. It showed weak cytotoxicity towards A-549 and HepG2 cells with the IC 50 s of 11.05 and 19.15 μM, respectively, compared to positive control Doxorubicin, which had an IC 50 s of 0.94 and 1.16 μM, respectively . The structure of steroid (91) is shown in Fig. 7.

Macrolides
The macrolides are a group of natural products that are made up of a large macrocyclic lactone ring with one or more deoxy sugars attached and have a wide range of biological activities, including antiparasitic, antifungal, and antimalarial properties (Karpi nski, 2019). Myrothecines D-G (92-95), 16-hydroxymytoxin B (96), and 14′-dehydrovertisporin (97) are six unprecedented macrolides, which were obtained from endophytic fungus Myrothecium roridum, associated with Trachelospermum jasminoides (Lindl.) Lem. All compounds possess cytotoxic ability against two tumor cell lines K562 (chronic myeloid leukemia) and SW1116 (colorectal carcinoma) between 0.056 and 15.98 μM of Fig. 12. Chemical structures of other hybrids (168-174).
IC 50 values. A three-dimensional quantitative structure activity relationship (3D QSAR) analysis suggested that the cytotoxicity of these trichothecene macrolides at nanomolar to micromolar level is dependent on steric, electrostatic, hydrophobic, and H-bond accepting pharmacophore group present in them (Shen et al., 2019). Moreover, additional research could be useful in developing these fungal metabolites as anticancer agents with specific molecular mechanism. A macrolide molecule consists of sesquiterpenoid with a diacyl residue anchored at the 4β and 15 positions, named Epiroridin acid (98) was separated from fermented culture of fungus Myrothecium roridum A553, an endophyte harbored in the medicinal plant Pogostemon cablin. It exhibited strong cytotoxic capability against SF-268, MCF-7, NCI-H460, and HepG2 cell lines with IC 50 s of 0.751 AE 0.03, 0.170 AE 0.01, 0.360 AE 0.05 and 0.380 AE 0.03 μM, respectively, compared to positive control cisplatin, which showed activity with IC 50 s of 4.060 AE 0.16, 2.850 AE 0.45, 2.850 AE 0.15 and 2.470 AE 0.17 μM, respectively (Liu et al., 2016b). The fact that Epiroridin acid has lower IC 50 values than the positive control signals that more in vitro investigations such as apoptosis, cell cycle-based studies, and so on, is needed to determine its mechanism of action. Two new trichothecene macrolides, named Myrothecine H (99) and Myrothecine I (100) were extracted from the culture of endophytic fungus Paramyrothecium roridum, associated with the medicinal plant Morinda officinalis. Both the compounds showed strong cytotoxicity towards SF-268 and HepG2 cell lines with IC 50 s of 6.75 AE 0.22, 6.72 AE 0.27 μM and 0.20 AE 0.01, 0.20 AE 0.03 μM, respectively . 7-O-Methylnigrosporolide (101) and Pestalotioprolide C-H (102-107) are 14-membered macrolides, which were separated from the culture of Pestalotiopsis microspora, an endophytic fungus associated with mangrove Drepanocarpus lunatus. All compounds possess cytotoxic ability against murine lymphoma cell line L5178Y and/or A2780 with IC 50 s between 1.2 and 39 μM (Liu et al., 2016c). The structures of macrolides (92-107) are shown in Fig. 7.

Lactones
Lactones are cyclic esters, which consist of a ring of two or more carbon atoms and a single oxygen atom with a ketone group next to one of the carbons. Lactones are used in the flavors and fragrances industry, and they also possess antioxidant, antimicrobial, and anticancer properties (Robinson et al., 2019). Two new compounds, Asperlactone G (108) and Asperlactone H (109) were separated from ethyl acetate extract of an endophytic fungus Aspergillus sp., associated with Pinellia ternate. Both the compounds showed weak cytotoxicity towards A-549 cells with IC 50 s of 65.3 AE 1.06 and 23.3 AE 2.01 μM, respectively, while remain inactive against MCF-7/DOX cell line at IC 50 of >100 μM (Xin et al., 2019). Four new compounds including a pair of butanolide derivatives, (¡)-asperteretone F (110) and (þ)-asperteretone F (111), were obtained from the endophytic fungus Aspergillus terreus, associated with Hypericum perforatum. Compounds (110) and (111) exhibited cytotoxic activity against human pancreatic cancer cells AsPC-1, SW1990 and PANC-1 cell line with IC 50 values of 9.5, 11.7, 9.8 μM and 9.9, 10.3, 15.6 μM, respectively. Both the compounds remain inactive against HeLa and SW480 cancer cell lines, while other compounds were inactive against all tested cell lines . Hispidulactone F (112), a novel metabolite was separated from the EtOAc extract of dessert plant associated endophytic fungus Chaetosphaeronema hispidulum (TS-8-1) and showed antiproliferative activity against HepG2 cells with IC 50 of 61.05 μM by the cell counting kit-8 (CCK-8) assay (Zheng et al., 2020).

Azaphilones
A pyranoquinone oxabicyclic skeleton with an oxygenated non protonated carbon at C-7 distinguishes azaphilones, which can be classified into various groups. Azaphilones have a wide variety of biological activities, including antimicrobial, cytotoxic, anticancer, antiviral, and anti-inflammatory effects (Luo et al., 2018). Chaephilone C (113) is an unprecedented azaphilone, which was obtained from EtOAc extract of endophytic fungi Chaetomium globosum TY-2 isolated from the plant Polygonatum sibiricum. It showed moderate antiproliferative activity towards HepG2 tumor cell line with IC 50 value of 38.6 μM . Isochromophilone A (114), Isochromophilone D (115), Isochromophilone F (116) and Isochromophilone C (117) are cytotoxic chloroazaphilone derivatives, which were separated from the fermented culture of Diaporthe sp. SCSIO 41011, an endophyte associated with mangrove plant Rhizophora stylosa. Compounds (114-116) showed cytotoxic activity against ACHN, 786-O and OS-RC-2 cell lines of renal carcinoma with IC 50 s between 8.9 and 38 μM, while compound (117) was active against only 786-O and OS-RC-2 cells with IC 50 s of 38 and 44 μM, respectively. Additionally, effect of Isochromophilone D on apoptosis and cell cycle study showed that it induces apoptosis in time and dose dependent manner, while do not have any effect on cell proliferation (Luo et al., 2018). Fungus Phomopsis sp. CGMCC No.5416, an endophyte was isolated from the stems of Achyranthes bidentata, which was collected from Nan Ling County, Anhui Province of China. The fermented culture of isolated endophyte leads to separation of three novel azaphilones, including Phomopsone C (118), which possess cytotoxic activity against three cancer cell lines A-549, MDA-MB-231 and PANC-1 with CC 50 s of 8.9, 3.2 and 17.3 μM, respectively, while other compounds remain inactive at CC 50 of >100 μM (Yang et al., 2020a). The structures of azaphilones (113-118) are shown in Fig. 8.

Alkaloids
Alkaloids are a class of basic organic substances that originate from plants, bacteria and fungi and contain at least one nitrogen atom in a ring structure. Vinblastine and vincristine are two well-recognized indole alkaloids which possess anticancer activity (Uzma et al., 2018). Six new alkaloids, Asperchalasins A-F (119-124) were separated from ethyl acetate extract of an endophytic fungus Aspergillus sp., associated with Pinellia ternate. All compounds showed weak cytotoxicity towards A-549 cells with IC 50 s of 55.5 AE 1.87, 54.2 AE 1.22, 47.2 AE 0.92, 40.6 AE 1.30, 55.2 AE 1.85, and 70.2 AE 1.76, respectively, while remain inactive against MCF-7/DOX cell line at IC 50 of >100 μM (Xin et al., 2019). Pchaeglobosal B (125), a novel rearranged cytochalasan with 13-aza-21-oxa-tetracyclo-[10.6.1.2 17,19 .0 15,19 ]henicosane core was separated from the culture of endophytic fungus Chaetomium globosum P2-2-2, which was isolated from Ptychomitrium plant. It showed anticancer activity against MCF-7, HepG2, CT26, HT29, and A-549 with IC 50 s of 8.59 AE 0.40, 7.09 AE 0.11, 1.04 AE 0.04, 9.90 AE 0.68 and 6.92 AE 0.21 μM, respectively (Peng et al., 2020a). Detailed research revealed that it halted the cell cycle progression of CT26 cells in the S phase and HT29 cells in the G0/G1 phase, as well as inducing apoptosis in both cell lines, which makes this compound substantial for additional pharmacokinetic study to foster it as strong chemotherapeutic drug. A rare natural metabolite, Chaetomiamide A (126) with the 13-membered ring skeleton was discovered from endophytic fungus Chaetomium sp., which was harbored in the roots of Cymbidium goeringii. respectively (Meng et al., 2016). The greater cytotoxicity of these molecules prompts us to explore their mechanism of action. Two new cytotoxic chaetoglobosin, Penochalasin I (132) and Penochalasin J (133) were separated from the fermented culture of Penicillium chrysogenum V11, an endophyte associated with mangrove Myoporum bontioides. Both the compounds showed cytotoxic activity against MDA-MB-435, SGC-7901 and A-549 human cancer cell lines with IC 50 s in the range of 7.32-37.70 μM . Penochalasin K (134), a new chaetoglobosin was extracted from the culture of fungus Penicillium chrysogenum V11, an endophyte harbored in Myoporum bontioides and  (Zhu et al., 2017a). An unprecedented epipolythiodioxopiperazine (ETP) alkaloids, Penicisulfuranols A-C (135-137) were separated from the fermented culture of Penicillium janthinellum HDN13-309, an endophyte associated with mangrove Sonneratia caseolaris. All compounds showed cytotoxic ability towards HeLa and HL-60 cell lines with IC 50 s of 0.5, 0.1 μM and 3.9, 1.6 μM and 0.3, 1.2 μM, respectively (Zhu et al., 2017b). A new compound, Chromenopyridin A (138) with N-methoxy-1-pyridone alkaloid moiety was separated from the culture of fungus Penicillium nothofagi P-6, an endophyte harbored in the bark of the critically endangered conifer Abies beshanzuensis. It possesses cytotoxic activity towards A-549 and HeLa tumor cell lines with IC 50 s of 14.7 and 11.3 μM, respectively . 2′-aminodechloromaldoxin (139) and 2′-aminodechlorogeodoxin (140) are two novel spiroketal derivatives with an amino group moiety, which were obtained from the Cinnamomum camphora endophytic fungus Pestalotiopsis flavidula. Both the new compounds exhibited cytotoxicity against four cancer cell lines NCI-H460, SF-268, MCF-7 and PC-3 with IC 50 s in the range of 16.47-23.53 μM (Rao et al., 2019). The structures of alkaloids (119-140) are shown in Fig. 9.
Lower IC 50 values against cancer cell lines suggest further research for this compound to be developed as a therapeutic molecule. The structures of p-terphenyls (143-150) are shown in Fig. 10.

Hybrid compounds
Secondary metabolite biosynthesis occurs in microorganisms through a variety of metabolic pathways. As a result, several hybrid secondary metabolites are generated by the simultaneous action of enzymes, building blocks, and cofactors from various pathways (Goyal et al., 2017).

Other hybrids
Two novel indolyl diketopiperazines, Gartryprostatin A (168) and Gartryprostatin B (169) were separated from the EtOAc extracts of a solid culture of endophytic fungus Aspergillus sp. GZWMJZ-258, which was isolated from the medicinal and edible plant Garcinia multiflora.  peptide synthetases and polyketide synthases, which were produced by the fungus Chaetomium globosum, an endophyte associated with plant Pinellia ternate. Both the compounds showed moderate cytotoxicity towards MDA-MB-231 breast cancer cells with IC 50 s of 7.6 AE 0.5 and 10.8 AE 0.64 μM, respectively, compared to the positive control doxorubicin which showed activity at IC 50 value of 1.0 AE 0.10 μM ). An endophytic fungus Cytospora rhizophorae A761 associated with Morinda officinalis was detected for novel polyketide heterodimers, Cytorhizin B (172) and Cytorhizin C (173) with benzophenone moiety. Both the compounds possess antiproliferative activity against cancer cell lines, HepG2, MCF-7, SF-268 and NCI-H460 with IC 50 values between 29.4 and 68.6 μM . A cyclopentenoneÀpyrone hybrid skeleton containing unique polyketide, Trichoderpyrone (174)   . The structures of other hybrids (168-174) are shown in Fig. 12.

Miscellaneous compounds
A new natural compound, Alternate C (175) was separated from endophytic fungus Alternaria alternata associated with root of Paeonia lactiflora. Alternate C exhibited cytotoxic activity towards two cancer cell lines MDA-MB-231 and MCF-7 with 20.1 μM and 32.2 μM of IC 50 values, respectively . Nigronapthaphenyl (176), a new cytotoxic metabolite was separated from the fermented culture of endophytic fungus Nigrospora sphaerica, associated with a mangrove plant Bruguiera gymnorrhyza. It possess cytotoxic ability towards colon cancer cell line HCT 116 with IC 50 value of 9.62 AE 0.5 μM (Ukwatta et al., 2019). Varioloid A (177), a new cytotoxic fungal metabolite was extracted from fungus Paecilomyces variotii EN-291, an endophyte associated with the marine red alga Grateloupia turuturu and showed cytotoxic activity towards A-549, HCT116, and HepG2 cell lines, with IC 50 values of 3.5, 6.4, and 2.5 μg/mL, respectively . A new compound Altertoxin IV (178) was separated from the ethyl acetate extract of culture of fungus Alternaria sp. G7, an endophyte harbored in the leaves of Broussonetia papyrifera (L.) Vent. It showed cytotoxic activity against MG-63 and SMMC-7721 tumor cell lines with IC 50 s of 14.81 and 22.87 μg/mL, while remain inactive against A-549 cell lines with IC 50 of >50 μg/mL . A new aflatoxin, Asperxin A (179) was isolated from the fungus Aspergillus sp. Y-2, an endophyte harbored in the needles of conifer Abies beshanzuensis. It showed significant cytotoxic activity towards A-549 and HeLa cells, with IC 50 s of 11.72 and 16.78 μg/mL, respectively, compared to the positive control doxorubicin which showed activity at 1.7 AE 0.5 and 0.6 AE 0.3 μg/mL, respectively . A novel metabolite, 6-formamide-chetomin (180) was separated from fermented culture of fungus Chaetomium sp. M336, which was isolated from the traditional Chinese medicinal plant Huperzia serrata Trev. Compound (180) exhibited strong cytotoxic activity towards HeLa, SGC-7901 and A-549 cells with IC 50 s of 21.6, 23.0 and 27.1 nM, respectively . 6-Formamide-chetomin exhibits cytotoxic effects in the nanomolar range, however its mechanism of action has yet to be discovered. After thorough pre-experimental and clinical studies, such fungal metabolites with low IC 50 values could be valuable for therapeutic treatment. Chaetominin A (181) was obtained from fungus Chaetomium subaffine L01, an endophyte obtained from healthy potato tissue, which was collected from Lincang county of Yunnan province, China. It possess cytotoxicity against two human tumor cell lines Hep-2 and HepG2 with the IC 50 values of 0.23 and 0.38 μM, respectively (Han et al., 2019). An endophytic fungus Diaporthe sp. was isolated from Datura inoxia, a traditional Indian medicinal plant. Addition of the valproic acid in the fermentation culture of fungus, induce synthesis of novel compounds, including Xylarolide A (182) and Diportharine A (183), which showed weak antiproliferative activity against cancer cell lines MIA-PA-CA-2, MCF-7 and PC-3 with IC 50 s of 20 AE 0.17, 30 AE 1.02, 14 AE 0.12 μM and 34 AE 1.32, >50, 22 AE 0.83 μM, respectively, compared to known anticancer drug paclitaxel which is active at nanomolar level for tested cell lines (Sharma et al., 2018b). Five new hormonemate derivatives, Hormonemates A-E (184-188) were separated from fermented culture of fungus Dothiora sp., an endophyte associated with the endemic plant Launaea arborescens. All compounds showed moderate cytotoxic ability towards HepG2, MCF-7 and/or MiaPaca-2 tumor cell lines with IC 50 s between 11.1 and 36.4 μM, compared to positive control doxorubicin, which had an IC 50 values of 0.2, 1.4 and 1.9 μM, respectively (P erez- Bonilla et al., 2017). A new macrolactone, (13R,14S,15R)-13-hydroxy-14-deoxyoxacyclododecindione (189) was obtained from endophytic fungus Exserohilum rostratum, associated with Gymnadenia conopsea and exhibited weak antiproliferative activity against A-549 lung cell line with an IC 50 value of 9.2 μM compared to paclitaxel, which gave IC 50 value of 0.11 μM . A new aminobenzamide derivative named Fusarithioamide B (190), was extracted from EtOAc extract of an endophytic fungi Fusarium chlamydosporium, which was isolated from Anvillea garcinii  (Chowdhury et al., 2017). An endophytic fungus Penicillium sp. T2-11 was isolated from the rhizomes of the Gastrodia elata. A new citrinin dimer, Penctrimertone (195) was separated from the EtOAc crude extract of isolated fungus, which showed cytotoxicity against five cancer cell lines HL-60, SMMC-7721, A-549, MCF-7 and SW480 with IC 50 s of 16.77, 23.63, 28.62, 21.53 and 39.32 μM, respectively compared to positive control cisplatin, which showed activity at 3.50, 23.03, 23.90, 23.64 and 9.26 μM, respectively (Li et al., 2020e). Two new natural metabolites, Cosmochlorin D (196) and Cosmochlorin E (197), were isolated from Phomopsis sp. N-125, an endophytic fungus harbored in Ficus ampelasa. Both the compound showed selective cytotoxic ability towards HL60 cells with IC 50 values of 6.1 and 1.8 μM, respectively, while remain inactive (IC 50 > 100 μM) against HeLa cell line (Shiono et al., 2017). Two endophytic fungi, Pyrenochaetopsis sp. FVE-001 and Pyrenochaetopsis sp. FVE-087 were isolated from the marine brown alga Fucus vesiculosus. Three new metabolites, Pyrenosetin A (198), Pyrenosetin B (199) and Pyrenosetin C (200) possessing cytotoxic activity against A-375 and non-cancerous keratinocyte cell line (HaCaT) with IC 50 s between 2.8 and 142.9 μM were separated from the culture of Pyrenochaetopsis sp. FVE-001 (Fan et al., 2020a). Pyrenosetin D (201) was separated from the culture of Pyrenochaetopsis sp. FVE-087 and exhibited cytotoxic activity against A-375 and HaCaT cell lines with IC 50 s of 77.5 and 39.3 μM, respectively (Fan et al., 2020b). Two new spirobisnaphthalenes, Rhytidenone G (202) and Rhytidenone H (203) were separated from the broth culture of Rhytidhysteron rufulum AS21B, an endophytic fungus harbored in leaves of mangrove plant Azima sarmentosa. Both the compound showed anticancer activity against human lymphoma Ramos cells and NSCLC H1975 cells with IC 50 s in the range of 0.018-17.98 μM (Siridechakorn et al., 2017). A new compound, 3-demethyl-3-(2-hydroxypropyl)-skyrin (204) with unsymmetrical dimeric anthraquinone moiety was separated from fermented culture of fungal strain Talaromyces sp. YE 3016, an endophyte harbored in the roots of Aconitum carmichaeli and showed cytotoxic activity against MCF-7 cell line with IC 50 of 20.76 AE 3.41 μg/mL (Xie et al., 2016).
Terezine E (205) is a prenylated tryptophan analogue isolated from the fungus Mucor sp., an endophyte of the medicinal plant Centaurea stoebe. It showed potent antiproliferative activity against human umbilical vein endothelial cells (HUVEC), multipotential leukemia cell line (K-562) and HeLa cancer cell lines with CC 50 values of 28.02, 27.31, and 60.43 mg/mL respectively (Abdou et al., 2020). The structures of miscellaneous compounds are shown in Fig. 13 (175-190) and .

Conclusion and future aspects
The discoveries included in this review presents the cytotoxicity profile of 205 small compounds with novel skeletons, that have been isolated from 95 fungal species harbored in both terrestrial and marine plants, collected across 16 countries. From these 95 strains, a total of five endophyte genera, namely Aspergillus, Chaetomium, Penicillium, Fusarium and Pestalotiopsis were most frequently explored in the targeted time frame of our current study. In terms of chemical diversity, polyketides make up the majority of the chemical population, accounting for a total of 24%, followed by terpenoids, which account for 20% of the total. These findings reveal that metabolites of endophytic fungi are extremely valuable, and more novel skeletons with a variety of biological activities should indeed be hunted out. Remarkably, in the last few years, there are more findings on endophytic fungi metabolites than ever before, indicating that endophytic fungi are becoming progressively more valuable sources for discovering novel natural drug like molecules. However, as seen in Fig. 15, China dominates the study on endophytic fungi. There are many biodiversity hotspots on the Earth that are still untapped and therefore need to be investigated for new endophytes to inspect their metabolites.
Advances in the domain of structural biology has allowed us to identify new promising drug targets for tumors, as well as acquire better understanding of the molecular basis of tumor resistance for existing available chemotherapeutic agents, this scenario has led investigators to reevaluate their strategy for exploring new molecules to combat various types of cancer. The cytotoxic effect of the fungal metabolites under consideration is widespread, with inhibitory concentrations varying from low to high micromolar towards particular tumor cell lines. Some of these metabolites had a strong cytotoxic effect for particular cell lines, that was at par or even better than some of the frequently used chemotherapeutic agents. Irrefutably, such endophytic metabolites which are screened as leads have not rigorously been investigated to determine the molecular mechanism that induces cytotoxic effects. Evolving computational tools are the hallmark for modern pharmaceutical research and is perceived as a useful aspect in drug development (Parmar et al., 2020;Prajapati et al., 2021). This might aid in determining anticancer effects by interacting with different protein targets responsible for specific cancer, and it could also speed up the drug development process by limiting in vitro assays. A complete exploration of this area has potentials for causing quantum leap in the traditional drug development process. On the other hand, many concurrent efforts are indispensable to develop and sustain endophyte metabolite as a drug molecule. The creation of a computational database or library of bioactive chemically diverse compounds, as well as the establishment of regional and global amenities as an endophytic microbe culture reservoir, are two such requirements. Endophytic culture reservoir will aid not only in preserving these valuable cultures, which are, either collected from endangered plant species, or plants located in remote and challenging areas where human access is difficult, but also allowing their extended reach to diverse community of researchers. Additionally, a dedicated computational database featuring details on both biological activity and chemical structure of endophytic metabolites would allow us to assist in determining the credible qualities of such metabolites, along with the efforts to comprehend their mechanism of action. Researchers now must stay one step ahead of cancer and be equipped with the several therapeutic agents in the form of bullets to strike the tumor specific target in the bullseye.
Endophytic fungi are a gold mine of novel bioactive molecules with enormous potential in the healthcare industries. Getting to the bottom of interactions of these endophytic fungal metabolites with precise cellular targets that are responsible for their biological activity will succeed in unlocking their ultimate value. Advanced pharmacological tools, as well as evolving bioinformatic methods, combined with sophisticated metagenomic approaches, will strive to improve effective exploitation of secondary metabolite production by valuable fungal endophytic species, and even its development as a potential anticancer drug molecule.

Funding sources
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Additional information
Library for the 3D structures of reviewed fungal metabolites will be available upon request. Request for the library should be addressed to J.P.
CRediT authorship contribution statement Jignesh Prajapati: Conceptualization, Writingoriginal draft, Drew the chemical structures of the compounds and prepared the library of 3D compounds. Dweipayan Goswami: Conceptualization, Writingoriginal draft. Rakesh M. Rawal: Reviewed the manuscript, Supervision, All authors discussed the manuscript critically for important intellectual content and approved the final version of the manuscript.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.