A Review of Diterpenes from Marine-Derived Fungi: 2009–2021

Marine-derived fungi are important sources of novel compounds and pharmacologically active metabolites. As an important class of natural products, diterpenes show various biological activities, such as antiviral, antibacterial, anti-inflammatory, antimalarial, and cytotoxic activities. Developments of equipment for the deep-sea sample collection allow discoveries of more marine-derived fungi with increasing diversity, and much progress has been made in the identification of diterpenes with novel structures and bioactivities from marine fungi in the past decade. The present review article summarized the chemical structures, producing organisms and biological activities of 237 diterpenes which were isolated from various marine-derived fungi over the period from 2009 to 2021. This review is beneficial for the exploration of marine-derived fungi as promising sources of bioactive diterpenes.


Introduction
As the largest part of the Earth's surface area, the ocean contains resources worthy of in-depth exploration. Marine-derived fungi, which are rich sources of secondary metabolites, have great potential for the discovery of bioactive compounds. The number of new compounds derived from marine fungi is increasing every year, from 287 in 2012 [1] to 724 in 2019 [2]. The contribution of marine fungi in marine-derived compounds is also increasing, from 27.7% in 2012 to 48.6% in 2019 [3][4][5][6][7][8] (Figure 1).
The marine fungi-derived compounds are of very diverse types, which include alkaloids [9], terpenes [10], polyketones [11], peptides [12], etc. As a major class of secondary metabolites of marine fungi, terpenes show many excellent activities [13]. Diterpenes are a group of terpenes with various bioactivities and rich structural diversity [10].
There are quite a few review articles on the isolation, structure elucidation, and biological activities of diterpenes. Hanson James R. has been publishing reviews of new diterpenoids discovered every year since 1984 [14] and the latest report was published in 2009 [15]. After that, Hanson began to limit the scope of the reviews to terrestrial diterpenoids and the first report was published in 2011 [16]. His serial review articles were then published almost every year and the latest one was published in 2019 [17]. These review articles offer important information for newly found diterpenoids of terrestrial origin. In contrast, no such systematic and up-to-date review articles are available for marine-derived diterpenes. In recent years, more and more research articles have reported works on the discovery of new diterpenes from marine-derived fungi. Expectedly, a review of these works will help to better understand recent discoveries and advances in this field. Herein, we summarize the structures and activities of newly discovered diterpenes derived from marine fungi in the past 13 years from 2009 to 2021.

Characteristics of Diterpenes from Marine-Derived Fungi
From 2009 to 2021, 237 new diterpenes were isolated from 47 strains of marine fungi that belong to 15 genera (Actinomadura, Arthrinium, Aspergillus, Botryotinia, Curvularia, Eupenicillium, Eutypella, Epicoccum, Micromonospora, Mucor, Neosartorya, Penicillium, Stachybotrys, Talaromyces, and Trichoderma). The pie chart in Figure 2A shows the distribution of the genera of the fungi covered in the 59 articles that reported newly discovered diterpenes. In these articles, Penicillium (25%), Aspergillus (20%), and Trichoderma (19%) are the most frequently studied genus. A total of 38 articles reported diterpenes from fungi of these three genera. Regarding the number of compounds, Botryotinia (34%), Penicillium (19%), and Aspergillus (16%) are the most productive, producing 80, 45, and 38 of new diterpenes, respectively ( Figure 2B). In recent years, more and more research articles have reported works on the discovery of new diterpenes from marine-derived fungi. Expectedly, a review of these works will help to better understand recent discoveries and advances in this field. Herein, we summarize the structures and activities of newly discovered diterpenes derived from marine fungi in the past 13 years from 2009 to 2021.

Characteristics of Diterpenes from Marine-Derived Fungi
From 2009 to 2021, 237 new diterpenes were isolated from 47 strains of marine fungi that belong to 15 genera (Actinomadura, Arthrinium, Aspergillus, Botryotinia, Curvularia, Eupenicillium, Eutypella, Epicoccum, Micromonospora, Mucor, Neosartorya, Penicillium, Stachybotrys, Talaromyces, and Trichoderma). The pie chart in Figure 2A shows the distribution of the genera of the fungi covered in the 59 articles that reported newly discovered diterpenes. In these articles, Penicillium (25%), Aspergillus (20%), and Trichoderma (19%) are the most frequently studied genus. A total of 38 articles reported diterpenes from fungi of these three genera. Regarding the number of compounds, Botryotinia (34%), Penicillium (19%), and Aspergillus (16%) are the most productive, producing 80, 45, and 38 of new diterpenes, respectively ( Figure 2B). In recent years, more and more research articles have reported works on the discovery of new diterpenes from marine-derived fungi. Expectedly, a review of these works will help to better understand recent discoveries and advances in this field. Herein, we summarize the structures and activities of newly discovered diterpenes derived from marine fungi in the past 13 years from 2009 to 2021.

Characteristics of Diterpenes from Marine-Derived Fungi
From 2009 to 2021, 237 new diterpenes were isolated from 47 strains of marine fungi that belong to 15 genera (Actinomadura, Arthrinium, Aspergillus, Botryotinia, Curvularia, Eupenicillium, Eutypella, Epicoccum, Micromonospora, Mucor, Neosartorya, Penicillium, Stachybotrys, Talaromyces, and Trichoderma). The pie chart in Figure 2A shows the distribution of the genera of the fungi covered in the 59 articles that reported newly discovered diterpenes. In these articles, Penicillium (25%), Aspergillus (20%), and Trichoderma (19%) are the most frequently studied genus. A total of 38 articles reported diterpenes from fungi of these three genera. Regarding the number of compounds, Botryotinia (34%), Penicillium (19%), and Aspergillus (16%) are the most productive, producing 80, 45, and 38 of new diterpenes, respectively ( Figure 2B).   Of the 237 new compounds, 68 compounds were reported to possess various bioactivities (Table 1). A total of 70 pieces of activity data are available since both compounds 38 and 202 were reported to possess two kinds of bioactivities. Cytotoxic activity is the most reported bioactivity, with 25 of 70 compounds (compounds with more than one kind of activity were also counted more than once) (36%, Figure 3) being active. Antibacterial activity (20%), inhibition of enzymes (14%), antiviral activity (7%), and inhibition of the germination of seeds (7%) were the next, with the number of active compounds being 14, 10, 5, and 5, respectively.  Of the 237 new compounds, 68 compounds were reported to possess various bioactivities (Table 1). A total of 70 pieces of activity data are available since both compounds 38 and 202 were reported to possess two kinds of bioactivities. Cytotoxic activity is the most reported bioactivity, with 25 of 70 compounds (compounds with more than one kind of activity were also counted more than once) (36%, Figure 3) being active. Antibacterial activity (20%), inhibition of enzymes (14%), antiviral activity (7%), and inhibition of the germination of seeds (7%) were the next, with the number of active compounds being 14, 10, 5, and 5, respectively. Anti-allergic effect on immunoglobulin E (IgE)-mediated rat mast RBL-2H3 cells with 18% inhibition at 20 µg/mL [23] 202 Harzianone Trichoderma longibrachiatum 82.6% of lethality to brine shrimp (Artemia salina L.) larvae at 100 µg/mL 1 Compounds 139−141 were reported as compounds 1−3 in the reference [25], where only compound 139 (numbered as compound 1 in the reference) was named as aspergilone A, while the names for compounds 140 and 141 (numbered as compounds 2 and 3, respectively, in the reference) were not provided. The name aspergilone B was used to represent compound 140 in this article.

Actinomadura
Only one new diterpene was reported to be produced by the genus Actinomadura since 2009 (1, Figure 4). Compound JBIR-65 (1) was obtained from the sponge-derived fungus Actinomadura sp. SpB081030SC-15 [49]. This is the first report of a diterpene isolated from the genus Actinomadura. This work found that compound JBIR-65 possessed an ability to protect neuronal hybridoma N18-RE-105 cells from L-glutamate toxicity with an EC50 value of 31 μM (Table 1).

Actinomadura
Only one new diterpene was reported to be produced by the genus Actinomadura since 2009 (1, Figure 4). Compound JBIR-65 (1) was obtained from the sponge-derived fungus Actinomadura sp. SpB081030SC-15 [49]. This is the first report of a diterpene isolated from the genus Actinomadura. This work found that compound JBIR-65 possessed an ability to protect neuronal hybridoma N18-RE-105 cells from L-glutamate toxicity with an EC 50 value of 31 µM (Table 1).

Actinomadura
Only one new diterpene was reported to be produced by the genus Actinomadura since 2009 (1, Figure 4). Compound JBIR-65 (1) was obtained from the sponge-derived fungus Actinomadura sp. SpB081030SC-15 [49]. This is the first report of a diterpene isolated from the genus Actinomadura. This work found that compound JBIR-65 possessed an ability to protect neuronal hybridoma N18-RE-105 cells from L-glutamate toxicity with an EC50 value of 31 μM (Table 1).
In addition, in another fungus, Arthrinium sacchari, which was isolated from the sponge surface, researchers obtained three new diterpenes: myrocin D (7), libertellenone E (8), and libertellenone F (9) [55]. Antitumoral potentials of compounds 7-9 were tested in an in vitro angiogenesis assay against human umbilical vascular endothelial cell (HUVEC) sprouting induced by VEGF-A, but no positive result was obtained.
It is worth noticing that both compounds 6 and 7 were named myrocin D. They were identified from different strains of Arthrinium by different researchers. The coincidence may be explained by the close timing of submission and acceptance of the two articles, which were published in different journals [50,55].

Aspergillus
From 2009 to 2021, 12 articles reported the discovery of 38 new diterpenes (10-47, Figure 5) from marine Aspergillus, accounting for over one-fifth of the total articles. Aspergillus of marine origin is an important source of active compounds. More than 170 of 232 compounds isolated from marine Aspergillus from 2006 to 2016 showed cytotoxic and antimicrobial activities [56].
Li et al. isolated the fungus Aspergillus wentii SD-310 from deep-sea sediments. Further investigation of its products led to the isolation of 18 new diterpenes (10-27), including two new tetranorlabdane diterpenes, asperolides D (10) and E (11) [33]. Compound 10 had moderate inhibitory activities against Edwardsiella tarda, with an MIC value of 16 µg/mL (Table 1). Chloramphenicol and ampicillin were used as positive controls, with MIC values being 8.0 and 2.0 µg/mL, respectively.

Botryotinia
Botryotinia has previously been studied more as a plant pathogenic fungus than as a natural product producer [62]. The investigation of a marine Botryotinia strain, Botryotinia fuckeliana MCCC 3A00494, led to the isolation of 80 new diterpenes (48-127, Figure 6).
Botryotinia fuckeliana MCCC 3A00494 was isolated from the deep sea at −5572 m. A new pimarane diterpenoid with a Δ 9(11) double bond, botryopimarene A (48), which was rarely discovered in the pimarane family, was obtained from its fermentation [63].
New diterpenes were also found from the fermentation of the fungus Aspergillus wentii na-3, which was isolated from the surface of Sargassum alagl [51]. A chemical epigenetic manipulation strategy was used to turn on the silent metabolic pathways. A histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), was added to the medium, and three new norditerpenes (35)(36)(37) were obtained [51].
In addition, the inhibitory effects of compounds 35-36 on the growth of one marine zooplankton (Artemia salina) and three marine phytoplankton species (Chattonella marina, Heterosigma akashiwo, and Alexandrium sp.) were evaluated. The results showed that compound 36 inhibited the growth of Artemia salina with an LC 50

Botryotinia
Botryotinia has previously been studied more as a plant pathogenic fungus than as a natural product producer [62]. The investigation of a marine Botryotinia strain, Botryotinia fuckeliana MCCC 3A00494, led to the isolation of 80 new diterpenes (48-127, Figure 6).
Botryotinia fuckeliana MCCC 3A00494 was isolated from the deep sea at −5572 m. A new pimarane diterpenoid with a ∆ 9(11) double bond, botryopimarene A (48), which was rarely discovered in the pimarane family, was obtained from its fermentation [63].
showed anti-allergic activity with an IC50 value of 0.2 mM (loratadine as the positive control with an IC50 of 0.1 mM) [53].

Eutypella
Seven diterpene compounds were produced by the genus Eutypella since 2009 (132-138, Figure 7). Sun et al. isolated five new oxygenated pimarane diterpenes from a marine sediment-derived fungus Eutypella scoparia FS26, which were named scopararanes C-G (132-136). The biological activities of these compounds were evaluated on three human cell lines, including SF-268 (human glioma cell line), MCF-7 (human breast adenocarcinoma cell line), and NCI-H460 (human non-small cell lung cancer cell line) [65]. The results showed that compounds 132 and 133 exhibited weak cytotoxicity against the MCF-

Eutypella
Seven diterpene compounds were produced by the genus Eutypella since 2009 (132-138, Figure 7). Sun et al. isolated five new oxygenated pimarane diterpenes from a marine sediment-derived fungus Eutypella scoparia FS26, which were named scopararanes C-G (132-136). The biological activities of these compounds were evaluated on three human cell lines, including SF-268 (human glioma cell line), MCF-7 (human breast adenocarcinoma cell line), and NCI-H460 (human non-small cell lung cancer cell line) [65]. The results showed that compounds 132 and 133 exhibited weak cytotoxicity against the MCF-7 cell line with IC 50 values of 35.9 and 25.6 µM, respectively.

Epicoccum
The genus Epicoccum generated four diterpenes (139-142, Figure 7). Xia et al. isolated three new pimarane-type diterpenes, compounds 139-141, from a marine-derived fungus Epicoccum sp. HS-1. All isolated compounds were tested for cytotoxicity against KB (human epidermis carcinoma cell line) and KBv200 (a classic multidrug-resistant cell line) cells [25]. Xia et al. also isolated another new isopimarane diterpene from the same strain, naming it isopimarane diterpene (142) [43]. In the bioactivity assay, compound 142 exhibited α-glucosidase inhibitory activity with an IC 50 value of 4.6 µM. Isopimarane diterpenes were reported to have biological activities such as antiviral, cytotoxic, etc. This is the first report on the α-glucosidase inhibition activity of isopimarane diterpenes. Compound 142 might be applied for the treatment of type 2 diabetes.

Micromonospora
The genus Micromonospora produced three new diterpene compounds (143-145, Figure 7). Mullowney et al. isolated a novel ∆ 8,9 -pimarane diterpene, named isopimara-2-one-3-ol-8,15diene (143), from a sediment-derived fungus Micromonospora sp. [66]. Micromonospora sp. WMMC-218 is a fungus derived from the marine ascidian Symlegma brakenhielmi. LC-MS-based metabolomics was used and showed that the secondary metabolite profile of the strain is unique. Further investigation of the fermentation led to the isolation of two new halimane-type diterpenoid micromonohalimanes A (144) and B (145) [37]. This is the first time that halimane-type diterpenes isolated from the genus Micromonospora. In terms of activity, compound 145 displayed an inhibitory effect on the methicillin-resistant Staphylococcus aureus with an MIC value of 40 µg/mL, compared with the MIC value of 1 µg/mL for the positive control, vancomycin.
Activities of compounds 146-151 were assessed on human A-549 and HL-60 cancer cell lines . Compounds 146, 147, and 151 showed biological activities against the A-549 cancer cell line with IC 50 values of 11.5, 6.3, and 9.2 µM, respectively, compared with 0.30 µM of adriamycin as the positive control. Compounds 146 and 147 were active against the HL-60 cancer cell line with IC 50 values of 9.6 and 5.0 µM, respectively, compared with 0.067 µM of adriamycin.

Neosartorya
Only one diterpene was produced by the genus Neosartorya since 2009 (152, Figure 8). The new compound, a meroditerpene, sartorypyrone C (152), was obtained from a rare sponge-derived fungus Neosartorya paulistensis. The antibacterial activity of sartorypyrone C against four reference strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa) was tested, but no significant activity was observed [67].

Penicillium
As an important source of bioactive secondary metabolites, Penicillium produced many diterpenes with novel structures [10].  (159 and 160) from a red alga-derived fungus Penicillium chrysogenum QEN-24S [68]. However, the two compounds did not show biological activity in the antimicrobial test.

Penicillium
As an important source of bioactive secondary metabolites, Penicillium produced many diterpenes with novel structures [10].  (159 and 160) from a red alga-derived fungus Penicillium chrysogenum QEN-24S [68]. However, the two compounds did not show biological activity in the antimicrobial test.
An indole diterpene, named penicindopene A (192), was obtained from the fungus Penicillium sp. YPCMAC1, collected at a depth of −4500 m in the western Pacific Ocean [29]. This is the first report of indole diterpenes containing a 3-hydroxy-2-indolone moiety. Penicindopene A also showed moderate cytotoxic activities against A-549 and HeLa cell lines with IC 50 values of 15.2 and 20.5 µM, respectively.
In addition, Penicillium thomii YPGA3, Penicillium sp. YPGA11, and Penicillium sp. YPCMAC1 were all derived from the deep-sea water at a depth of −4500 m in the Yap Trench (West Pacific Ocean). A rare 19-nor labdane-type diterpenoid, named penitholabene (193), was isolated from Penicillium thomii YPGA3 [46]. This represents the first 19-nor labdane-type diterpenoid found in nature. It showed an inhibitory effect against α-glucosidase with an IC 50 value of 282 µM, being more active than the positive control, acarbose (1330 µM).
Three unreported cyclopiane diterpenes (195)(196)(197) were obtained from the deep-sea sediment fungus Penicillium sp. TJ403-2 [73]. The anti-inflammatory activities of these compounds were evaluated. Compound 195 could significantly reduce LPS-induced NO production with an IC 50 value of 2.19 mM, which was only one-third of that of the positive control, indomethacin.

Talaromyces
Only one new diterpene was produced by the genus Talaromyces (201, Figure 9). The compound, roussoellol C (201), was obtained from the fungus Talaromyces purpurogenus PP-414 isolated from a beach in Qinhuangdao, Hebei Province [74]. It was cytotoxic to MCF-7 cells with an IC50 of 6.5 μM.

Trichoderma
Marine-derived fungi of the Trichoderma genus have produced many structurally novel natural products with diverse bioactivities [75]. From 2009 to 2021, 11 articles reported the discovery of 27 new diterpenes (202-228, Figure 9) from marine Trichoderma.
Xie et al. detected unusual signals in the 13 C NMR spectra recorded on the fractions of the fungus Trichoderma erinaceum [76], and thereafter identified a new diterpene trichodermaerin (203) in the subsequent fermentation and isolation.
A novel diterpene trichocitrin (204) was isolated from the culture of the fungus Trichoderma citrinoviride cf-27 isolated from the seaweed surface. This represents both the first report of the isolation of a fusicoccane diterpene from Trichoderma, and the first discovery of a furan-bearing fusicoccane diterpene. At 20 μg/disk, trichocitrin formed an 8.0 mm inhibition zone against Escherichia coli [41]. Later, a fungus, Trichoderma asperellum cf44-2, was isolated from the alga collected in the same batch. Additionally, an unreported diterpene, named 11-hydroxy-9-harzien-3-one (205), was isolated from the fermentation of this fungus [77].

Talaromyces
Only one new diterpene was produced by the genus Talaromyces (201, Figure 9). The compound, roussoellol C (201), was obtained from the fungus Talaromyces purpurogenus PP-414 isolated from a beach in Qinhuangdao, Hebei Province [74]. It was cytotoxic to MCF-7 cells with an IC 50 of 6.5 µM.

Trichoderma
Marine-derived fungi of the Trichoderma genus have produced many structurally novel natural products with diverse bioactivities [75]. From 2009 to 2021, 11 articles reported the discovery of 27 new diterpenes (202-228, Figure 9) from marine Trichoderma.
Xie et al. detected unusual signals in the 13 C NMR spectra recorded on the fractions of the fungus Trichoderma erinaceum [76], and thereafter identified a new diterpene trichodermaerin (203) in the subsequent fermentation and isolation.
A novel diterpene trichocitrin (204) was isolated from the culture of the fungus Trichoderma citrinoviride cf-27 isolated from the seaweed surface. This represents both the first report of the isolation of a fusicoccane diterpene from Trichoderma, and the first discovery of a furan-bearing fusicoccane diterpene. At 20 µg/disk, trichocitrin formed an 8.0 mm inhibition zone against Escherichia coli [41]. Later, a fungus, Trichoderma asperellum cf44-2, was isolated from the alga collected in the same batch. Additionally, an unreported diterpene, named 11-hydroxy-9-harzien-3-one (205), was isolated from the fermentation of this fungus [77].

Others
In addition to the 15 genera mentioned above, there are also some marine fungi whose taxonomic status has not been determined, but their secondary metabolites have been obtained and studied. Nine new diterpenoids were reported to be produced by unidentified marine fungi (229-237, Figure 10). They were named phomactin I (229), 13-epiphomactin I (230), phomactin J (231), phomactins K-M (232-234), and phomactins N−P (235-237). They were isolated by Masahiro Ishino et al. from a fungus of unknown red algal origin [80][81][82]. HUVECs, NHDF (normal human dermal fibroblasts) cells, and HeLa cells were used to test the cytotoxicity of these compounds. However, they did not show any observable cytotoxic effect.

Conclusions
This review provides a comprehensive overview of the structures and activities of 237 new diterpenes discovered from 47 strains of marine-derived fungi from 2009 to 2021. The articles reporting Penicillium, Aspergillus, and Trichoderma accounted for the majority (64%) of all the relevant publications. The numbers of diterpenes isolated from the four genera Botryotinia (80), Penicillium (45), Aspergillus (38), and Trichoderma (27) are the top four. It is noteworthy that 80 new diterpenes were isolated from a single strain of the genus Botryotinia, 71 of which are aphidicolin congeners. After aphidicolanes, indole-type diterpenes (46) are the most numerous diterpenes, followed by pimarane-type (29), harziane-type (16), and cyclopiane-type (9) diterpenes. Among the bioactive compounds, the compounds with cytotoxic activity were the most, accounting for 36%, followed by compounds with antibacterial effects, accounting for 20%. The compound with the most notable cytotoxicity is conidiogenone C (154), which showed cytotoxic activities in HL-60 and BEL-7402 cell lines, with IC50 values of 0.038 and 0.97 μM, respectively. The compound with the most promising antimicrobial activity is aspewentin D (12). It showed inhibitory activity against Edwardsiella tarda and Vibrio harveyi with MIC values of 2.0 and 4.0 μg/mL, respectively. These marine-derived diterpenes show rich structural diversities and bioactivities. The reported compounds partially uncovered the untapped potential of marine fungi as diterpene producers.

Conclusions
This review provides a comprehensive overview of the structures and activities of 237 new diterpenes discovered from 47 strains of marine-derived fungi from 2009 to 2021. The articles reporting Penicillium, Aspergillus, and Trichoderma accounted for the majority (64%) of all the relevant publications. The numbers of diterpenes isolated from the four genera Botryotinia (80), Penicillium (45), Aspergillus (38), and Trichoderma (27) are the top four. It is noteworthy that 80 new diterpenes were isolated from a single strain of the genus Botryotinia, 71 of which are aphidicolin congeners. After aphidicolanes, indoletype diterpenes (46) are the most numerous diterpenes, followed by pimarane-type (29), harziane-type (16), and cyclopiane-type (9) diterpenes. Among the bioactive compounds, the compounds with cytotoxic activity were the most, accounting for 36%, followed by compounds with antibacterial effects, accounting for 20%. The compound with the most notable cytotoxicity is conidiogenone C (154), which showed cytotoxic activities in HL-60 and BEL-7402 cell lines, with IC 50 values of 0.038 and 0.97 µM, respectively. The compound with the most promising antimicrobial activity is aspewentin D (12). It showed inhibitory activity against Edwardsiella tarda and Vibrio harveyi with MIC values of 2.0 and 4.0 µg/mL, respectively. These marine-derived diterpenes show rich structural diversities and bioactivities. The reported compounds partially uncovered the untapped potential of marine fungi as diterpene producers.