Metabolites from marine invertebrates and their symbiotic microorganisms: molecular diversity discovery, mining, and application

The marine environment has been proven to be a rich source of new bioactive natural products for drug discovery. Coral reefs are among the most productive ecosystems and are a source of a large group of structurally unique biosynthetic products. To date, more than 40, 000 marine natural products (MNPs) have been identified from various marine organisms, such as sponges, cnidarians, tunicates, molluscs, echinoderms, bryozoans, red algae, brown algae, green algae, and microorganisms (Carroll et al. 2019; Deshmukh et al. 2017; Jiménez 2018; Leal et al. 2016; Newman and Cragg 2016b). The upward trend in the discovery of new MNPs sourced from marine microorganisms continues unabated and now represents 57% of the total new MNPs reported in 2017 (Carroll et al. 2019). Based upon the putative biogenetic origins, these MNPs can be classified as polyketides, terpenoids, alkaloids, steroids, lactones, peptides, phenols, and lipids. Also, a large proportion of MNPs display interesting pharmaceutical activities, such as cytotoxic, antimicrobial, hypolipidemic, anti-inflammatory, antimalarial, analgesic, and antiasthmatic activities (According to the following websites: http://marinepharmacology.midwestern.edu; Jiménez 2018). Hence, MNPs are considered as an excellent and potentially valuable source for new chemical entities with novel structures and distinct mechanisms of action. To date, there have been 13 approved therapeutic agents that could be considered derivatives of MNPs (Altmann 2017; Jiménez 2018; Newman 2019; Pereira et al. 2019). Moreover, more than 30 MNP derivatives constitute the global marine pharmaceutical clinical pipeline in Phases Ⅲ, Ⅱ or Ⅰ of drug development (According to the following websites: http://marinepharmacology.midwestern.edu; http://pharma.id.informa.com (accessed on August 6, 2019); Jiménez 2018; Newman 2019; Pereira et al. 2019). The significant potential for new drug development based on MNPs in all disease areas has been previously discussed (Newman and Cragg 2016a).

Marine invertebrates have proven to be a primary source of bioactive MNPs, as many serve as chemical defense tools against predators, competitors and other ecological pressures. It has been demonstrated that the true origin of most MNPs appears to be the microorganisms living in concert with invertebrates. Most invertebrates are sessile, soft-bodied and move slowly, and are thus subject to potential parasite predation and detrimental microbial colonization. Therefore, they require a complex arsenal of secondary metabolites produced by their symbiotic microorganisms to facilitate a form of chemical defense (Jiménez 2018; Wang et al. 2008). This is likely the reason why MNPs from marine invertebrates and their symbiotic microorganisms are a rich sources of diverse and bioactive secondary metabolites (Martins et al. 2014; Mayer et al. 2010; Newman and Cragg 2016b). The chemical ecology underlying invertebrate-microorganism interactions provides a great opportunity for natural product chemists to mine for novel drug discovery. Therefore, the invertebrates and the abundant microorganisms in their ecosystems have attracted widespread attention for producing novel structural metabolites with potential bioactive applications (Blunt et al. 2018).

In the recent decade, the China Sea, especially the South China Sea, has become a hot spot in searching for novel bioactive MNPs. The invertebrates including sponges, soft corals, gorgonians and tunicates are prolific in the coral reefs in the South China Sea, and the microorganisms associated with these invertebrates have been demonstrated as a distinctive source for new bioactive MNPs.

In recent years, we have initiated a research program to find biological active MNPs based on marine chemical ecology (Figs. 1, 2) (Hou et al. 2015, 2019a; Wang et al. 2008). A total of 709 MNPs including 307 new compounds have been obtained from marine invertebrates and their symbiotic microorganisms collected from the South China Sea. In this review, we summarize the representative 287 MNPs (Table 1) obtained by our group, highlighting multiple structural types of compounds and demonstrating discovery, diversity, compound mining, and bioactive application. The goal is to provide further inspiration for the discovery of bioactive MNPs and subsequent drugs development.

Figure 1.  Collection positions for marine invertebrates and microorganisms in the South China Sea. The red points represent the sampling sites

Figure 2.  The coral reef ecosystem and the chemical defensive metabolites with chemoecological effects

Table 1.  Representative compounds and their original organisms

Macrocyclic lactones derived from marine organisms have attracted much attention on account of their multiple potent biological activities including antitumor (Hirata and Uemura 1986; Isaka et al. 2002, 2009; Qi and Ma 2011; Suo et al. 2018), antifungal (Shier et al. 2001), and antimalarial activities (Isaka et al. 2009). For instance, caniferolides A and B, two glycosylated 36-membered polyol macrolides from marine-derived Streptomyces sp., showed pronounced antifungal activity (Perez-Victoria et al. 2019). Peloruside E, a macrolide from the New Zealand marine sponge Mycale hentscheli, displayed potent antiproliferative activity (Hong et al. 2018). As part of our current research, 88 macrolides with unique structures and significant biological properties have been obtained from marine invertebrates and their symbiotic microorganisms (Liu et al. 2014; Shao et al. 2011, 2015a, 2018). To obtain more valuable undiscovered natural compounds, multiple strategies and methods, including OSMAC and chemical epigenetic manipulation, were applied to microorganisms. Structural modification and chemical synthesis were performed to provide more derivatives, and the structure-activity relationships were discussed (Zhang et al. 2017). The molecular mechanisms of compounds with strong activities were also investigated (Wang et al. 2016).

Three new 14-membered resorcylic acid lactones (RALs), including compounds 1 and 2 (cochliomycins A and B) with a rare natural acetonide group and 3 (cochliomycin C) with a 5-chloro substituted lactone (Fig. 3), were isolated from the culture broth of Cochliobolus lunatus (M351) isolated from the gorgonian Dichotella gemmacea collected from the South China Sea (Shao et al. 2011). In an antifouling assay, compound 1 (cochliomycin A) showed significant antifouling activity against the barnacle Amphibalanus (= Balanus) amphitrite at nontoxic concentrations (EC₅₀ = 1.2 µg/mL). Thus, compound 1 merits further investigation as a molecular model for the discovery of new antifouling molecules (Shao et al. 2011).

Figure 3.  14-Membered resorcylic acid lactones from the gorgonian-derived fungus C. lunatus (M351) (1‒3) and the sea anemone-derived fungus C. lunatus (TA26-46) (4‒13)

The molecular mechanism underlying antifouling activity of 1 against the cyprids of barnacle A. amphitrite has been investigated using the isobaric tags for the relative or absolute quantification (iTRAQ) labeling proteomic method (Wang et al. 2016). Differentially expressed proteins were examined by analyzing the changes in the proteome of A. amphitrite cyprids in response to 1 treatment. The results suggested that compound 1 affected the cytochrome P450, glutathione S-transferase (GST) and NO/cGMP pathways. The results of real-time PCR further demonstrated that the NO/cGMP pathway was activated in response to 1. Larval settlement assays suggested that S-methylisothiourea sulfate (SMIS) and 1H-(1, 2, 4)oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ) rescued cyprids from 1 (cochliomycin A)-induced inhibition of larval settlement. These findings supported the hypothesis that 1 inhibited barnacle larval settlement through stimulating the NO/cGMP pathway (Wang et al. 2016).

However, further research on compound 1 and its analogues was hindered by strain-specific variation of the strain C. lunatus (M351), especially altering metabolite profiles when re-cultured. Fortunately, another fungal strain C. lunatus (TA26-46) derived from the sea anemone Palythoa haddoni was also found to produce 14-membered RALs. Three new 14-membered RALs, 4-6 (cochliomycins D-F) together with eight known analogues 1 (cochliomycin A), 7 ((7′E)-6′-oxozeaenol), 8 (deoxy-aigialomycin C), 9 (LL-Z1640-2), 10 (zeaenol), 11 (LL-Z1640-1), 12 (paecilomycin F) and 13 (aigialomycin B) (Fig. 3) were obtained (Liu et al. 2014). Compounds 4, 6-9, and the acetonide derivative 9a displayed strong anti-larval settlement activity against B. amphitrite (EC₅₀ = 1.82 to 22.5 μg/mL; LC₅₀/EC₅₀ > 50), suggesting that they might be promising environmentally benign antifouling agents (Liu et al. 2014).

Noticeably, besides significant antifouling activity, compound 9 (LL-Z1640-2) produced by C. lunatus (TA26-46) exhibited promising inhibitory activity against phytopathogenic fungal strain Pestalotia calabae, with a minimum inhibitory concentration (MIC) value of 0.39 μmol/L, 25-fold stronger than that of the positive control, ketoconazole (MIC = 10 μmol/L) (Liu et al. 2014). A fungicide whole plant assay suggested that 9 exhibited pronounced activity on the phytopathogenic fungus Plasmopara viticola preventative test at 6 × 10^-6 and concentration-dependent activity on the phytopathogenic fungus Phytophthora infestans preventative application at 200, 60, and 20 × 10^-6. Compound 9 could be considered as a promising new natural fungicide lead (Liu et al. 2014).

Further optimization of fermentation conditions of fungus C. lunatus (TA26-46) led to the isolation of two major natural products 9 (LL-Z1640-2) and 10 (zeaenol) (Fig. 3) with multi-gram quantities (Zhang et al. 2017). By one or two steps, we semi-synthesized six trace natural compounds 1 (cochliomycin A), 3 (cochliomycin C) (Fig. 3), 4 (cochliomycin D), 5 (cochliomycin E), 6 (cochliomycin F) and 7 ((7′E)-6′-oxozeaenol) (Fig. 3), and a series of derivatives 14-29 (Fig. 4) from compounds 9 and 10 with high yields (65-95%) (Zhang et al. 2017). Compounds 14-16 displayed potent antiplasmodial activity against P. falciparum (IC₅₀ = 1.84, 8.36, and 6.95 μmol/L, respectively). The acetoxy groups and acetonide functionality might contribute to the antiplasmodial activity (Fig. 5). However, the chlorine atom has a negative effect on the activity. Very importantly, 14 and 15 were non-toxic with very safety and high therapeutic indices (CC₅₀/IC₅₀ > 180), and thus represent potential promising leads for antiplasmodial drug discovery. In addition, 2-OH was found to be an important functionality for reducing toxicity. Only 14 showed antileishmanial activity against Leishmania donovani (IC₅₀ = 9.22 μmol/L). Most of the enone RALs showed high antileishmanial efficacy but poor therapeutic indices, suggesting that the cis or trans-functionality has a contribution to toxicity, and their antileishmanial activities may well be related to their toxicity (Fig. 5). Furthermore, 14 and 15 also displayed antiplasmodial activity against the neglected Chagas' disease causing Trypanosoma cruzi (IC₅₀ = 11.9 and 17.2 μmol/L, respectively) (Zhang et al. 2017).

Figure 4.  Semisynthesized resorcylic acid lactone derivatives

Figure 5.  The structure-activity relationships of resorcylic acid lactones

It should be noted that many gene clusters in marine-derived fungi for the production of undiscovered secondary metabolites generally remain unexpressed under common laboratory culture conditions. In order to obtain uncharacterized metabolites, the chemical epigenetic perturbation method was applied to C. lunatus (TA26-46) to manipulate the silent fungal genes. Two new 14-membered RALs characterized with bromine substitution, 30 (5-bromozeaenol) and 31 (3, 5-dibromozeaenol) (Fig. 6), were obtained from the culture treated with histone deacetylase inhibitor, sodium butyrate (Zhang et al. 2014). Compounds 30 and 31 represent the first examples of naturally occurring brominated RALs.

Figure 6.  Brominated resorcylic acid lactones from the sea anemone-derived fungus C. lunatus (TA26-46) by chemical epigenetic manipulation

Facing the challenges of discovery repetition and silent biosynthetic pathways, only limited mining approaches were applied in our work to date. As well known, microorganisms are easily manipulated on the genetic level. With gene sequencing technology blooming, genetic techniques and bioinformatics algorithms will continue to provide more opportunities for disclosing the novel pathways within marine microorganisms to then enable exploration of the untapped valuable bioactive molecules encoded within.

Furthermore, co-culturing as an effective method to induce production of novel secondary metabolites from two interacting microbial strains was applied to mine macrocyclic lactones. In single strain cultivation, the sponge-derived actinomycete Streptomyces rochei MB037 could produce two 18-membered macrolides, 32 (borrelidin) and 33 (borrelidin F) (Yu et al. 2019). Interestingly, in the co-culture of S. rochei MB037 and the gorgonian-derived fungus Rhinocladiella similis 35, besides macrolides 32 and 33, S. rochei MB037 was induced to produce two new fatty acids with a rare nitrile group, 34 (borrelidin J) and 35 (borrelidin K) (Fig. 7) (Yu et al. 2019), which are structurally related to borrelidins. Compounds 34 and 35 were derived from the actinomycete S. rochei MB037, suggesting that the silent hydrolytic enzyme genes for hydrolyzing lactone in the borrelidin macrolides could be activated by the co-culture approach. Notably, both 34 and 35, obtained only in co-culture, exhibited stronger antibacterial activities against methicillin-resistant S. aureus than 32 and 33.

Figure 7.  18-Membered macrolides and structurally related fatty acids from the co-culture of the sponge-derived actinomycete S. rochei MB037 and the gorgonian-derived fungus Rhinocladiella similis 35

In addition to marine invertebrates and their symbiotic microorganisms, macrolides were also obtained from marine-derived bacteria. Compound 36 (bastimolide A) (Fig. 8), a polyhydroxy macrolide with a 40-membered ring, was obtained from a new genus of the tropical marine cyanobacterium Okeania hirsute (Shao et al. 2015a). Its complete structure and absolute configuration were unambiguously identified by X-ray diffraction analysis of the nona-p-nitrobenzoate derivative 36a (Fig. 8). Compound 36 is a complex 40-membered macrolactone with repeating 1, 5-diol and 1, 3-diol groups as well as a t-butyl group moiety which is quite rare among natural products. Compound 36 showed strong antimalarial activity against four resistant strains of P. falciparum (IC₅₀ = 80 to 270 nmol/L), including TM90-C2A (chloroquine, mefloquine, and pyrimethamine resistant), TM90-C2B (chloroquine, mefloquine, pyrimethamine, and atovaquone resistant), W2 (chloroquine and pyrimethamine resistant), and TM91-C235 (chloroquine, mefloquine, and pyrimethamine resistant). Although this compound displayed toxicity to the host Vero cells (IC₅₀ = 2.1 μmol/L), it still represents a potentially promising lead for antimalarial drug discovery (Shao et al. 2015a).

Figure 8.  Polyhydroxy macrolides from the cyanobacterium O. hirsute

After discovery of the potent antimalarial 40-membered macrolide, continued investigation of the biologically-active and structurally-complex polyketides from O. hirsuta led to the isolation of a novel analogue 37 (bastimolide B) (Fig. 9), a 24-membered macrolide characterized by a long aliphatic chain with polyhydroxy groups and bearing a unique terminal tert-butyl group (Shao et al. 2018). A methanolysis mechanism for 36 is proposed and one unexpected isomerization product of the C₂-C₃ double bond, 38 (2-(E)-bastimolide A) (Fig. 9), was also obtained. The natural product 37 showed pronounced antimalarial activity against chloroquine-sensitive P. falciparum strain HB3 (EC₅₀ = 5.72 ± 0.65 μmol/L). Compound 38 showed even stronger antimalarial potency with an EC₅₀ value of 1.41 ± 0.47 μmol/L. Thus, the bastimolides show considerable antimalarial activity and represent an intriguing new class of antimalarial agents (Shao et al. 2018).

Figure 9.  Bastimolide B and isomerization product of bastimolide A from the cyanobacterium O. hirsute

The diverse activities of anthraquinones isolated from marine-derived fungi with cytotoxic (Chen et al. 2013a; Xie et al. 2010; Zhu et al. 2012), antiviral (Huang et al. 2017), antioxidant (Li et al. 2016) and other activities have attracted many interests for drug discovery. For example, SZ-685C, a hydroanthraquinone isolated from the marine-derived Halorosellinia fungus, exhibited potent cytotoxic activity (Chen et al. 2013a; Xie et al. 2010; Zhu et al. 2012). In our investigation of bioactive anthraquinone derivatives from marine-derived fungi (Yang et al. 2012; Zheng et al. 2012), a total of 54 anthraquinone monomers and dimers have been isolated from Nigrospora sp., Alternaria sp. and other fungal genera. Analysis of anthraquinones with various chemical structures possessing interesting biological activities revealed the key structural features required for their activities.

Nine anthraquinone derivatives were obtained from a sea anemone-derived fungus Nigrospora sp. ZJ-2010006, including two new hydroanthraquinone analogues, 39 (4a-epi-9α-methoxydihydrodeoxybostrycin) and 40 (10-deoxybostrycin), together with seven known anthraquinone derivatives 41 (nigrosporin B), 42 (9α-hydroxydihydrodesoxybostrycin), 43 (9α-hydroxyhalorosellinia A), 44 (4-deoxybostrycin), 45 (bostrycin), 46 (3, 5, 8-trihydroxy-7-methoxy-2-methylanthracene-9, 10-dione), 47 (austrocortirubin) (Fig. 10). Ten acetyl derivatives (44a, 45a, 46a-46g, 47a) (Fig. 10) were prepared by structural modification (Yang et al. 2012). Compound 39 represents the first case of a hydroanthraquinone with a 9-OMe group. Notably, 40 displayed significant antibacterial activity against Bacillus subtilis (MIC = 625 nmol/L). Compound 41 exhibited promising antibacterial activity against B. subtilis and B. cereus (MICs = 312 nmol/L). Additionally, the derivative 44a showed pronounced inhibitory activity against B. cereus (MIC = 48.8 nmol/L), 25-fold stronger than that of ciprofloxacin (MIC = 1250 nmol/L). These results demonstrated that hydroanthraquinone derivatives might be a promising source for antimicrobial agent discovery.

Figure 10.  Anthraquinone derivatives from the sea anemone-derived fungus Nigrospora sp. ZJ-2010006

Structure-activity relationship analysis revealed that the introduction of the 4-OH/9-OH/10-OH groups had little effect on antibacterial activity (Fig. 11). Interestingly, transforming from a hydroxy group at C-3 to an acetoxy group significantly increased selective antibacterial activity. The cycloaliphatic ring has a positive contribution to the antibacterial activity. Additionally, an aromatic B ring may be necessary for the antibacterial activity (Yang et al. 2012).

Figure 11.  Antibacterial structure-activity relationships for hydroanthraquinone derivatives

Ten anthraquinone derivatives were isolated from the culture of Sarcophyton soft coral-derived fungus Alternaria sp. ZJ-2008003 collected from the South China Sea, including five new hydroanthraquinone derivatives, 48-51 (tetrahydroaltersolanols C-F) and 52 (dihydroaltersolanol A), and five new alterporriol-type anthranoid dimers, 53-57 (alterporriols N-R) (Fig. 12) (Zheng et al. 2012). Compound 54 (alterporriol O) was the first isolated alterporriol dimer with a C-4-C-4′ linkage. Compound 48 (tetrahydroaltersolanol C) and 56 (alterporriol Q) showed antiviral activity against the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) (IC₅₀ = 65 and 22 μmol/L, respectively). Compound 55 (alterporriol P) exhibited cytotoxic activity against the PC-3 and HCT-116 cell lines (IC₅₀ = 6.4 and 8.6 μmol/L, respectively) (Zheng et al. 2012).

Figure 12.  Anthraquinone monomers and dimers from the soft coral-derived fungus Alternaria sp. ZJ-2008003

The azaphilone molecules are a structurally variable family of fungal polyketide metabolites with a highly oxygenated pyranoquinone bicyclic core and exhibiting multiple bioactivities such as cytotoxic (Yamada et al. 2008), antimicrobial (Che et al. 2002), antiviral (Wang et al. 2011a), and anti-inflammatory (Yasukawa et al. 2008) activities. For instance, sclerketide C, an azaphilone analogous isolated from gorgonian-derived fungus Penicillium sclerotiorin CHNSCLM-0013, exhibited significant anti-inflammatory activity (Liu et al. 2019). Pleosporalone B from the culture of marine-derived fungus Pleosporales sp. CF09-1 and penicilazaphilone C from Penicillium sclerotiorum M-22 displayed potent antimicrobial activities (Cao et al. 2019; Zhou et al. 2016). Azaphilones have attracted much attention due to their fascinating structural features and distinguished bioactivities (Wei et al. 2017). Our previous work on metabolites produced by symbiotic microorganisms of marine invertebrates found 48 azaphilones, including 37 new compounds, many of which were reported to exhibit antifouling and antiviral activities (Wang et al. 2018; Wei et al. 2017; Zhao et al. 2015a). Desiring promising antifouling molecules, a one-step semisynthetic method was applied to discover more new azaphilonoids derivatives.

A series of azaphilone derivatives were isolated from the gorgonian-derived fungal strain, Penicillium pinophilum XS-20090E18, collected from the Xisha Islands coral reef, including three new azaphilone derivatives 58-60 (pinophilins D-F), together with six known azaphilone derivatives 61 (Sch 1385568), 62 (pinophilin B), 63 (Sch 725680), 64 ((-)-mitorubrin), 65 ((-)-mitorubrinol) and 66 ((-)-mitorubrinic acid) (Fig. 13) (Zhao et al. 2015a). However, none of the obtained azaphilone derivatives showed antifouling, cytotoxic, or topoisomerase I (Topo I) inhibitory activity (Zhao et al. 2015a).

Figure 13.  Azaphilone derivatives from the gorgonian-derived fungus P. pinophilum XS-20090E18

Six azaphilone derivatives were isolated from the sponge-derived fungus Penicillium sclerotiorum, including two new azaphilones 67 and 68 (penicilazaphilones D and E), together with four known analogs, 69 ((+)-sclerotiorin), 70 (geumsanol C), 71 (WB (CAS No. 1701443-52-8, 6H-2-benzopyran-6-one, 5-chloro-3-[(1E, 3R, 4R, 5S)-3, 4-dihydroxy-3, 5-dimethyl-1-hepten-1-yl]-1, 7, 8, 8a-tetrahydro-7, 8-dihydroxy-7-methyl-, (7R, 8R, 8aS)) and 72 (geumsanol G) (Fig. 14) (Wang et al. 2018). Compound 67 was the second reported azaphilone with a C4 aliphatic side chain, and 68 was the first azaphilone with a tetrahydrofuran ring at C-3. Compound 69 demonstrated antiviral activity against HSV (IC₅₀ = 19.5 µmol/L) and EV71 (IC₅₀ = 132 μmol/L), 16- and 3-fold stronger than the positive control ribavirin, respectively (Wang et al. 2018).

Figure 14.  Azaphilone derivatives from the sponge-derived fungus P. sclerotiorum

On account of the significant bioactivities of 69 ((+)-sclerotiorin), 30 sclerotioramine derivatives 73-102 has been semi-synthesized from 69 by a one-step reaction (Fig. 15) (Wei et al. 2017). Most of them except 77, 78, 79, 83, and 99 showed strong anti-barnacle settlement activity against B. amphitrite. The aromatic amino-derivatives 84-88, 90-92, 94, 96-98, and 100-102 demonstrated potent antifouling activity; while only two aliphatic amino-derivatives 76 and 101 displayed antifouling activity. It should be noted that 76 and 101 showed promising activity (EC₅₀ = 0.94 and 0.47 μg/mL, respectively) stronger than that of the commercial biocide Sea-Nine 211™ (EC₅₀ = 1.2 μg/mL). Meanwhile, compounds 76 and 101 possess high therapeutic ratios (LC₅₀/EC₅₀ 53.2 and 106.4, respectively), indicating that they may be antifouling candidates with low-toxicity for the development of new environmentally benign antifoulants (Wei et al. 2017).

Figure 15.  Semisynthetic azaphilone derivatives from the natural compound (+)-sclerotiorin (69)

Marine-derived alkaloids represent a class of compounds with particularly privileged structures, which have been frequently encountered in a vast number of pharmaceuticals as well as agrochemicals (Bandini and Eichholzer 2009; Hibino and Choshi 2001; Humphrey and Kuethe 2006; Kochanowska-Karamyan and Hamann 2010; Lounasmaa and Tolvanen 2000). For example, raistrickindole A, an indole diketopiperazine alkaloid obtained from the marine-derived fungus Penicillium raistrickii, displayed antiviral activity against the hepatitis C virus (Li et al. 2019a). Agelastatin A, a bromopyrrole marine alkaloid isolated from the Mexican sponge, Agelas sp., exhibited strong antineoplastic activity (Pettit et al. 2005). Our group has isolated more than 100 alkaloids with promising biological activities from diverse fungal strains derived from marine invertebrates (Chen et al. 2014a; Jia et al. 2015; Shao et al. 2015b; Wang et al. 2015a). To obtain significant antibacterial compounds, efficient and environmental friendly methods for synthesis of unsymmetrical bisindolylmethanes or triarylmethane were developed (Wen et al. 2015).

Compound 103 ((+)- and (-)-pestaloxazine A) (Fig. 16), a pair of new enantiomeric alkaloid dimers with an unprecedented symmetric spiro[oxazinane-piperazinedione] skeleton, were isolated from a soft coral-derived fungus Pestalotiopsis sp. (Jia et al. 2015). A plausible biosynthetic pathway for 103 was proposed starting from two molecules of _L-ornithine (Fig. 17). The piperazinedione intermediate 103a is produced by double dehydration. The oxidation results in the presence of the N-oxide 103b, and the key racemic intermediates (±)-103c are achieved by intermolecular nucleophilic attack at the same face. The condensation between 103c and 103d gives the final product (±)-103. The epimer (2R, 2′S or 2S, 2′R) of (±)-103, a mesomer, was not observed, indicating that the production of 103 in the fungus was regulated by enzymes with selective catalytic functions. Compound 103 exhibited potent and selective antiviral activity against Enterovirus 71 (EV71) (IC₅₀ = 14.2 ± 1.3 μmol/L), 18-fold more potent than the positive control ribavirin (IC₅₀ = 256.1 ± 15.1 μmol/L). Therefore, 103 could be considered as a promising antiviral agent (Jia et al. 2015).

Figure 16.  Enantiomeric alkaloid dimers with an symmetric spiro[oxazinane-piperazinedione] skeleton from the soft coral-derived fungus Pestalotiopsis sp.

Figure 17.  Plausible biosynthetic pathway to 103

From the gorgonian coral-derived fungus Scopulariopsis sp., six dihydroquinolin-2-one-containing alkaloids were isolated, including three 4-phenyl-3, 4-dihydroquinolin-2(1H)-one alkaloids containing a monoterpenoid moiety, 104 (aniduquinolone A), 105 (aflaquinolone A), 106 (aflaquinolone D), and three 4-phenyl-3, 4-dihydroquinolin-2(1H)-one alkaloids, 107 (6-deoxyaflaquinolone E), 108 (aflaquinolone F), 109 (aflaquinolone G) (Fig. 18) (Shao et al. 2015b). These dihydroquinolin-2-one-containing alkaloids except 109 exhibited significant anti-larval settlement activity against B. amphitrite. This is the first report on the antifouling activity for quinolin alkaloids. In particular, compound 104 showed highly potent antifouling activity against B. amphitrit at a picomolar level (EC₅₀ = 17.5 pmol/L), 249, 000-fold stronger than that of Sea-Nine 211™ (EC₅₀ 4.36 μmol/L). Particularly, 104 displayed a high therapeutic ratio (LC₅₀/EC₅₀ = 1200). Therefore, it represents a structural class for discovery of effective, non-toxic, environmentally friendly antifouling agents (Shao et al. 2015b).

Figure 18.  Dihydroquinolin-2-one-containing alkaloids from the gorgonian coral-derived fungus Scopulariopsis sp.

From the mycelia of a gorgonian-derived Aspergillus sp. fungus, two new prenylated dihydroquinolone derivatives, 110 (22-O-(N-Me-L-valyl)-aflaquinolone B) and 111 (22-O-(N-Me-L-valyl)-21-epi-aflaquinolone B), and two known analogues, 105 and 106 (aflaquinolones A and D) (Fig. 19), were obtained (Chen et al. 2014a). Compounds 110 and 111 possess an unusual esterification of N-Me-L-Val to the side chain prenyl group. It is worth noting that compound 111 exhibited outstanding anti-RSV (Human respiratory syncytial virus (RSV)) activity (IC₅₀ = 42 nmol/L), 500-fold more potent than that of ribavirin (IC₅₀ = 20 μmol/L) and showed a comparatively higher therapeutic ratio (TC₅₀/IC₅₀ = 520). This is the first report of prenylated dihydroquinolone derivatives with potent antiviral activity (Chen et al. 2014a).

Figure 19.  Prenylated dihydroquinolone derivatives from the gorgonian-derived fungus Aspergillus sp.

Eight indol alkaloids were isolated from the bacterium Pseudovibrio denitrificans strain UST4-50 derived from an unidentified ascidian, including a diindol-3-ylmethane (DIM), 112 (di(1H-indol-3-yl)methane), and several analogues, 113 (vibrindole A), 114 (3, 3′-di-1H-indol-3-yl-1, 2-propandiol), 115 (tri(1H-indol-3-yl)methane), 116 (1, 2, 2-tri(1H-indol-3-yl) ethanone), 117 (arsindoline A), 118 (3, 3′-(phenylmethylene) bis-1H-indole), and 119 (4-(di(1H-indol-3-yl)methyl) phenol (DIM-Ph-4-OH)) (Fig. 20) (Wang et al. 2015a). All DIMs showed low-toxic anti-larval settlement activity against B. amphitrite (EC₅₀ = 18.57 to 1.86 μmol/L; LC₅₀/EC₅₀ > 15). Structure-activity relationship analysis revealed that 3, 3′-diindolylmethylene and Ph-C1′′′ phenolic hydroxyl were necessary for the activity. A field test of 112 over a period of 5 months further confirmed its antifouling activity comparable to Sea-Nine 211™ (Wang et al. 2015a). DIMs could be considered promising candidates as environmentally friendly antifouling agents owing to their simple structures, excellent activities, and low toxicities against marine target organisms.

Figure 20.  Diindol-3-ylmethanes from the ascidian-derived bacterium P. denitrificans UST4-50

The bisindole derivatives manifest significant biological activities, while the synthesis method for unsymmetrical bisindolylmethanes or triarylmethanes still remains a great challenge (Abe et al. 2013; Fu et al. 2013; Ma and Yu 2005; Yu and Yu 2009; Zhu et al. 2002). In our study, an efficient S_N1-type reaction was developed for 3-indolylmethanols with miscellaneous nucleophiles, featuring catalyst-free, low cost, wide substrate scope and mild reaction conditions (Figs. 21, 22). This approach provides an efficient and environmental friendly method for synthesis of diverse 3-substituted indolyl derivatives as well as unsymmetrical bisindolylmethanes and triarylmethanes (Wen et al. 2015).

Figure 21.  Reaction of 3-indolylmethanol with diverse nucleophiles

Figure 22.  The active methylene compounds used as nucleophiles

As part of our continuous work in this area, versatile 3-substitued indolyl derivatives were synthesized in high yields, including unsymmetrical diarylmethanes and triarylmethanes. Among them, compounds 120 (2-(phenyl(2-phenyl-1H-indol-3-yl)methyl)malononitrile), 121 (4-hydroxy-3-(phenyl(2-phenyl-1H-indol-3-yl)methyl)-2H-chromen-2-one), 122 (4-(phenyl(2-phenyl-1H-indol-3-yl)methyl)phenol) and 123 (2-phenyl-3-(phenyl(2, 4, 6-trimethoxyphenyl)methyl)-1H-indole) (Fig. 23) exhibited antibacterial activities against B. megaterium and M. lysodeikticus. In particular, 121 and 122 displayed antibacterial activities against B. megaterium (MIC = 13.5 and 8.36 μmol/L, respectively). In addition, 120, 121 and 122 exhibited potent antibacterial activities against M. lysodeikticus (MIC = 2.10, 3.35 and 4.18 μmol/L, respectively). These synthetic compounds could be considered as promising lead compounds for further investigation and application (Wen et al. 2015).

Figure 23.  3-Substitued indolyl derivatives synthesized from di(1H-indol-3-yl)methane

Terpenoids constitute a class of broadly active natural products isolated from a diverse range of marine organisms. For example, 11R-methoxy-5, 9, 13-proharzitrien3-ol, obtained from an endophytic fungus Trichoderma harzianum X-5 derived from the marine brown alga Laminaria japonica, displayed growth inhibition of some marine phytoplankton species (Song et al. 2018). Nakijinol G, a meroterpenoid obtained from a sponge Hyrtios sp. collected from the South China Sea, showed protein tyrosine phosphatase (PTP1B) inhibitory activity (Wang et al. 2017). Trichodermanin C, a diterpenes obtained from a fungal strain Trichoderma harzianum OUPS-111D-4 derived from sponge Halichondria okadai, exhibited significant cytotoxic activity (Yamada et al. 2017). In our ongoing research on the marine invertebrates and their symboitic microorgnisms, 101 terpenoids including 43 new compounds with novel structures were isolated, which exhibited antibacterial, cytotoxic and antifouling activities (Cao et al. 2015, 2017; Li et al. 2012a).

Four new bisabolane-type sesquiterpenoids were separated from the culture of Aspergillus sp. (ZJ-2008004) derived from the sponge Xestospongia testudinaria, including 124 (aspergiterpenoid A), 125 ((-)-sydonol), 126 ((-)-sydonic acid), and 127 ((-)-5-(hydroxymethyl)-2-(2′, 6′, 6′-trimethyltetrahydro-2H-pyran-2-yl)phenol) (Fig. 24) (Li et al. 2012a). All are optically active compounds. Compound 125 displayed potent inhibitory activity against S. albus and M. tetragenus (MIC = 5.00 and 1.25 μmol/L, respectively), and 127 on S. albus and B. subtilis (MIC = 5.00 and 2.50 μmol/L, respectively). Compound 126 showed strong inhibiting activity against four pathogenic bacteria B. subtilis, Sarcina lutea (MIC = 2.50 μmol/L), E. coli and M. tetragenus and uniquely against two marine bacteria (V. parahaemolyticus and V. anguillarum) (Li et al. 2012a).

Figure 24.  Bisabolane-type sesquiterpenoids from the sponge-derived fungus Aspergillus sp.

Eleven new scalarane sesterterpenoids were isolated from the sponge Carteriospongia foliascens, including three 20, 24-bishomo-25-norscalaranes, 128-130 (carteriofenones A-C), and eight 20, 24-bishomoscalaranes, 131-138 (carteriofenones D-K) (Fig. 25) (Cao et al. 2015). Scalarane sesterterpenoids with 4-methylpentanate or pentanoate substituents at the C-12 position were reported for the first time. Compounds 132-135 (carteriofenones E-H) represented rare naturally occurring scalarane sesterterpenoids with a cyclobutane ring. Specifically, the 18-carboxylic scalarane sesterterpenoid 131 displayed strong cytotoxicity against P388, HT-29, and A549 cell lines (IC₅₀ = 0.96, 1.43 and 3.72 μmol/L, respectively). Whereas, 130 without the 18-carboxy was inactive, indicating that the carboxyl group at C-18 might be an indispensable functional group for activity. Additionally, 131 was also found to display brine shrimp lethality towards Artemia salina (LC₅₀ = 5.80 μmol/L) and anti-larval settlement activity against B. amphitrite (EC₅₀ = 2.50 μmol/L) (Cao et al. 2015).

Figure 25.  Scalarane sesterterpenoids from the sponge C. foliascens

From the gorgonian Euplexaura sp. GXWZ-05, three new serrulatane-type diterpenoids, 139-141 (euplexaurenes A-C), and a known metabolite, 142 (anthogorgiene P) (Fig. 26), were isolated (Cao et al. 2017). The absolute configurations of C-11 in 139-142 were difficult to determine by common methods due to the high conformational flexibility of the eight-carbon aliphatic chain attached at C-4. By vibrational circular dichroism (VCD), their absolute configurations were determined. Compounds 139-142 displayed selective cytotoxic activities against the human laryngeal carcinoma (Hep-2) cell line (IC₅₀ = 1.95, 7.80, 13.6 and 5.85 μmol/L, respectively) (Cao et al. 2017).

Figure 26.  Serrulatane-type diterpenoids from the gorgonian Euplexaura sp. GXWZ-05

Six sesquiterpenoids were obtained from the gorgonian Anthogorgia ochracea, including two new guaiazulene-based analogues, 143 and 144 (ochracenoids A and B), along with four known analogues 145 (1-formylguaiazulene), 146 (1-formyl-4-methyl-7-isopropylazulene), 147 (ketolactone), and 148 (3, 8-dimethyl-5-isopropyl-6-formylindenone) (Fig. 27) (Zheng et al. 2014). Compound 143 is a rare guaiazulene-based analogue possessing a unique C₁₆ skeleton. Compound 145 exhibited strong ichthyootoxicity with the antiproliferative effects on several aspects of embryo development in zebrafish Danio rerio, including coagulated eggs (48 h), notochord malformation (72 h), and embryo death (72 h) with the EC₅₀ values of 3.98, 6.50, and 7.39 μmol/L, respectively (Zheng et al. 2014).

Figure 27.  Guaiazulene-based analogues from the gorgonian A. ochracea

Six diterpenoids were found from the soft coral Sinularia compacta, including two new prenylgermacrane type diterpenoids, 149-150 (lobophytumins A-B), two new prenyleudesmane type diterpenoids, 151-152 (lobophytumins C-D), and two new spatane type diterpenoids, 153-154 (lobophytumins E-F) (Fig. 28) (Li et al. 2011). Although structures of 149-154 are formally quite different, they are biogenetically related to each other. Sesquiterpene compound 155 ((-)-germacrene D) is considered as their common precursor (Faulkner 1984). A plausible biogenetic pathway for 149-154 was proposed (Fig. 29). It is worth noting that prenylgermacrane and prenyleudesmane diterpenes are quite rare in soft coral. This is the first report of spatane type diterpenoids from a soft coral source (Li et al. 2011).

Figure 28.  Diterpenoids from the soft coral S. compacta

Figure 29.  Plausible biosynthetic correlations between three extended sesquiterpenoid skeleton diterpenoids

Additionally, multiple known diterpenoids were obtained and showed various bioactivities. From the soft coral Sarcophyton infundibuliforme, four known cembrene diterpenoids were obtained, including 156 (sarcophytol-A), 157 (sarcophytol-A acetate), 158 (sarcophytol-H) and 159 (sarcophytonolid-J) (Wang et al. 2011b) (Fig. 30). These compounds exhibited strong anti-larval settlement activity against B. amphitrite (EC₅₀ = 2.25, 1.75, 8.13 and 7.50 μg/mL, respectively) (Wang et al. 2011b). From a soft coral Sarcophyton sp., a cembranoid diterpene 160 (sarcophytol B) was isolated. This compound displayed antibacterial activity against B. cereus, S. albus and V. parahaemolyticus (MIC = 3.13, 1.56, and 0.50 μmol/L, respectively) (Cao et al. 2013). Briarane type diterpenoids 161 (juncin P) and 162 (junceellolide D) isolated from the gorgonian Dichotella fragilis (Ridleg) exhibited potent antifouling activity (EC₅₀ = 0.80 and 0.77 μg/mL, respectively) (Zhou et al. 2011).

Figure 30.  Cembrene diterpenoids and briarane diterpenoids from the soft coral S. infundibuliforme and Sarcophyton sp. and gorgonian D. fragilis

Steroid derivatives from marine organisms are noted for diverse unusual structures with multiple potent biological properties. For instance, petasitosterone B, a steroid isolated from a Formosan marine soft coral Umbellulifera petasites exhibited promising anti-inflammatory activity (Huang et al. 2016). Two 9, 11-secosteroidal glycosides, sinularosides A and B, isolated from the South China Sea soft coral Sinularia humilis, exhibited potent antimicrobial activity (Sun et al. 2012). In our previous reports, 86 steroidal compounds were obtained from marine invertebrates and their symbiotic fungi, which exhibited cytotoxic, antiviral and antibacterial activities (Cao et al. 2014; Sun et al. 2015; Zhao et al. 2013).

From the gorgonian Echinogorgia rebekka, four new steroids with an acetoxy linked at the end of the side chain, 163-166 (echrebsteroids A-D) (Fig. 31) were isolated (Cao et al. 2014). Among them, 164 and 165 were a pair of new C-25 epimers of 26-acetoxy steroids, representing the first reported separation of C-25 epimers of 26-acetoxy steroids. Compound 165 exhibited pronounced antiviral activity against RSV (IC₅₀ = 0.19 μmol/L) and a comparatively higher therapeutic ratio (TC₅₀/IC₅₀ = 128), suggesting that it could be considered as a potential antiviral agent (Cao et al. 2014).

Figure 31.  Steroids with 26-acetoxy from the gorgonian E. rebekka

From a gorgonian Carijoa sp., four pregnane steroid were isolated, including a new pregnane steroid, 167 (15β-hydroxypregna-1, 4, 20-trien-3-one), and three known analogues 168 (15β-acetoxypregna1, 4, 20-trien-3-one), 169 (18-acetoxypregna-1, 4, 20-trien-3-one), and 170 (pregna-1, 4, 20-trien3-one) (Fig. 32), all with rare 3-one dienones (Zhao et al. 2013). Compounds 167, 169 and 170 displayed cytotoxicity against human hepatoma cell line Bel-7402 (IC₅₀ = 9.33, 11.02 and 18.68 µmol/L, respectively). Additionally, 167 exhibited potent antibacterial activity against S. aureus, S. albus, E. coli, V. parahaemolyticus and Nocardia brasiliensis (MIC = 0.063, 1.00, 1.00, 4.00, and 0.500 μmol/L, respectively), and displayed excellent inhibitory activity against Pseudomonas putida (MIC = 31 nmol/L), fivefold stronger than that of ciprofloxacin (MIC = 156 nmol/L). Compound 168 showed very strong antibacterial activity against pathogenic bacteria Tetragenococcus halophilus (MIC = 312 nmol/L). While 169 demonstrated strong antibacterial activity against B. cereus, S. aureus and T. halophilus (MIC = 2.50, 0.156, and 1.25 μmol/L, respectively) (Zhao et al. 2013).

Figure 32.  Pregnane steroids from the gorgonian E. rebekka

From the gorgonian Subergorgia rubra, sixteen secosteroids were obtained, including twelve new 9, 10-secosteroids 171-182 (subergorgiaols A-L), along with four known analogues 183 (astrogorgiadiol), 184 (calicoferol C), 185 (calicoferol F), 186 (calicoferol A) (Fig. 33) (Sun et al. 2015). Among them, compounds 171-182, with a series of different aliphatic side chains, represent the first examples of 9, 10-secosteroids bearing a hydroxy group at C-8, which are 8-OH derivatives of astrogorgiadiols/calicoferols. Compound 174 showed selective strong cytotoxicity against the cervical carcinoma cell line (CaSki) (IC₅₀ = 2.4 μmol/L). Additionally, 176 displayed toxicity toward brine shrimp A. salina (LC₅₀ = 2.0 μmol/L) (Sun et al. 2015).

Figure 33.  9, 10-Secosteroids from the gorgonian S. rubra

Four polyoxygenated sterols were separated from a soft coral Sinularia sp., including 187 ((3S, 23R, 24S)-ergost-5-ene-3β, 23α, 25-triol), 188 ((24S)-ergostane-6-acetate-3β, 5α, 6β, 25-tetraol), 189 ((24S)-ergostane-6-acetate-3β, 6β, 12β, 25-tetraol) and 190 (24-methylenecholestane-3β, 5α, 6β-triol-6-monoacetate) (Fig. 34) (Li et al. 2012b). Compound 190 showed moderate cytotoxicity against the K562 cell line (IC₅₀ = 3.18 μmol/L) and also demonstrated potent lethality toward brine shrimp A. salina (LC₅₀ = 0.96 μmol/L).

Figure 34.  Polyoxygenated sterols from the soft coral Sinularia sp.

Chemical investigation of sponge Topsentia sp. led to the isolation of three novel polyhydroxylated sterol derivatives, 191-193 (topsensterols A-C), with novel 2β, 3α, 4β, 6α-tetrahydroxy-14α-methyl Δ⁹⁽¹¹⁾ steroidal core and rare side chains (Fig. 35) (Chen et al. 2016). The differences between them were mainly existed in the respective aliphatic side chains. Compound 192 demonstrated significant cytotoxicity against human gastric carcinoma cell line SGC-7901 (IC₅₀ = 8.0 μmol/L). While 193 showed cytotoxicity against human erythroleukemia cell line K562 (IC₅₀ = 6.0 μmol/L) (Chen et al. 2016).

Figure 35.  Polyhydroxylated sterol derivatives from the sponge Topsentia sp.

Six steroids and one steroidal glycoside were isolated from the fungal strain Cladosporium sp. WZ-2008-0042 separated from the gorgonian Dichotella gemmacea, including a new pregnane, 194 (3α-hydroxy-7-ene-6, 20-dione), five known steroids 195 (5α, 8α-epidioxy-ergosta-6, 9, 22E-triene3β-ol), 196 (5α, 8α-epidioxy-ergosta-6, 22E-dien-3β-ol), 197 (ergosta-7, 22E-diene-3β, 5α, 6β-triol), 198 (3β, 5α-dihydroxy-6β-methoxyergosta7, 22-diene), 199 (ergosterol peroxide), along with one known steroidal glycoside 200 (stigma-5-en-3-O-β-glucopyranoside) (Fig. 36) (Yu et al. 2018). Compounds 194-196 and 198 exhibited potential antiviral activity against RSV virus (IC₅₀ = 0.11 to 0.17 mmol/L; TC₅₀/IC₅₀ = 5.18 to 9.92).

Figure 36.  Steroids and steroidal glycoside from the gorgonian-derived fungus Cladosporium sp. WZ-2008-0042

Phenylpropanoids contain many chemical structure types, such as chromone, coumarin, flavone and lignin, which possess various biological activities including antioxidant, cytotoxic, antimicrobial, and enzyme inhibitory activities. For example, arthone C, a chromone derivative isolated from a deep-sea-derived fungus Arthrinium sp., exhibited potent antioxidant activity (Bao et al. 2018). Two tetracyclic coumarin derivatives and two coumarin dimers isolated from the marine-derived fungus Eurotium rubrum showed significant tyrosinase inhibitory activity (Kamauchi et al. 2018). In our previous studies, 80 phenylpropanoid compounds with 54 new compounds were obtained, which showed antifouling, antibacterial and cytotoxic activities (Qi et al. 2013; Zhao et al. 2015b). These findings provide further insight into the chemical diversity and biological activities of this class of MNPs.

Ten isocoumarins were isolated from the sponge-derived fungus Penicillium sp. MWZ14-4, including three new hydroisocoumarins, 201-203 (penicimarins A-C), three new isocoumarins, 206-208 (penicimarins D-F), together with four known isocoumarin derivatives 204 (aspergillumarin B), 205 (aspergillumarin A), 209 (sescandelin B), and 210 (5, 6, 8-trihydroxy-4-(1′-hydroxyethyl)isocoumarin) (Fig. 37) (Qi et al. 2013). Compound 201 represents a rare naturally occurring isocoumarin derivative with 4-substitution but no substituent at the 3-position.

Figure 37.  Isocoumarins from the sponge-derived fungus Penicillium sp. MWZ14-4

Twelve new chromone derivatives, 211-222 (corynechromones A-L) (Fig. 38), were obtained from the sponge-derived fungus Corynespora cassiicola XS-200900I7 (Zhao et al. 2015b). These are the first chromone derivatives reported from the genus Corynespora. The absolute configurations of 211-220 were determined by the modified Mosher's method and TDDFT ECD calculations along with comparison of their CD spectra. Interestingly, 211/212, 213/214, 215/216, and 217/218 were pairs of epimers at C-2. A biogenetic pathway of the isolated chromone derivatives was proposed (Fig. 39). The different cyclization pathways from an uncyclized α, β-unsaturated ketone precursor may lead to production of the octalactones (Ebrahim et al. 2012, 2013). Alternatively, the decalactones may be produced by ester formation with the distal hydroxy group. A possible non-enzymatic cyclization route may be involved to form the epimeric pairs. A Michael reaction can occur with the nucleophile attacking from either face, leading to the formation of the epimeric pairs (Zhao et al. 2015b).

Figure 38.  Chromone derivatives from the sponge-derived fungus C. cassiicola XS-200900I7

Figure 39.  Proposed biogenetic pathway for corynechromones, octalactones and decalactones

From a gorgonian-derived fungus Eurotium sp. XS-200900E6, five pairs of new dihydroisocoumarin enantiomers, 223-232 ((±)-eurotiumides A-E), together with two related racemates, 233 and 234 ((±)-eurotiumides F and G), were isolated, all of them are rare dihydroisocoumarin derivatives with a methoxy at C-4 (Fig. 40) (Chen et al. 2014b). The (+)- and (-)-eurotiumides B and D with cis configurations of H-3/H-4 demonstrated significant anti-larval settlement activity against B. amphitrite (EC₅₀ = 0.7 to 2.3 μg/mL; LC₅₀/EC₅₀ > 15), indicating that they could be considered as promising environmentally benign antifouling agents. Additionally, 223, 224, 225, and 226 displayed significant antibacterial activities against S. epidermidis (MICs = 0.39 to 0.78 μmol/L), and 223 and 224 showed remarkable activities against B. cereus (MICs = 0.39 and 0.78 μmol/L, respectively). All of the tested compounds, especially 223 and 224, exhibited strong inhibitory activities against two marine bacteria, V. anguillarum and V. parahaemolyticus (Chen et al. 2014b).

Figure 40.  Dihydroisocoumarin enantiomers from the gorgonian-derived fungus Eurotium sp. XS-200900E6

Peptides isolated from many marine species present various biological activities, such as antimicrobial, antiviral, antitumor, antioxidative and cardioprotective activities. For instance, mirabamide A, a cyclic depsipeptide obtained from the sponge Siliquariaspongia mirabilis, showed potent inhibitory activity on HIV-1 fusion (Plaza et al. 2007). Reniochalistatin E, an octapeptide isolated and characterized from the marine sponge Reniochalina stalagmitis, displayed significant cytotoxic activity (Zhan et al. 2014). In our studies, a total of 34 peptides were identified, of which more than half are cyclopeptides involved in 18 new peptides (Chen et al. 2014c; Hou et al. 2019b, 2019c). Particularly, LC-MS/MS-dependent molecular networking and ¹H NMR techniques were applied in order to identify new peptides.

The strategy of molecular networking based on MS/MS cheminformatics could shed light on structural relationships and accelerate the dereplication of molecules in crude extracts to mine new molecules. Based on integrating LC-MS/MS-dependent molecular networking and ¹H NMR techniques, the targeted identification of ten cyclohexadepsipeptides were achieved from the gorgonian coral-derived fungus Penicillium chrysogenum (TA01-16) (Fig. 41), including the new compounds 235-237 (chrysogeamides A-C) and 240-244 (hrysogeamides D-H), as well as the known compounds 238 and 239 (scopularides A and B) (Fig. 42) (Hou et al. 2019b). These cyclohexadepsipeptides contain the same D-Leu fragment, and 235 and 242 are reported for the first time featuring a 3-hydroxy-4-methylhexanoic acid (HMHA) moiety of cyclodepsipeptides. Interestingly, isotope labelling feeding experiments demonstrated that ¹³C₁-L-Leu was transformed into ¹³C₁-D-Leu moiety in 238 and 239 indicating that D-Leu in these molecules may be epimerized from L-Leu by a leucine racemase. Compounds 235-239 displayed selective activity in promoting angiogenesis toward the Tg (Flk1: EGFP) transgenic zebra fish D. rerio embryo (Hou et al. 2019b).

Figure 41.  The conceptual framework of LC-MS/MS-dependent molecular networking to target the isolation of new compounds

Figure 42.  Cyclohexadepsipeptides from the gorgonian coral-derived fungus P. chrysogenum (TA01-16)

Applying molecular networking techniques, three new cycloheptapeptides, 245-247 (asperversiamides A-C) (Fig. 43), were isolated from the coral-derived fungus Aspergillus versicolor (CHNSCLM-0063) (Hou et al. 2019c). Their complete structures including configuration were confirmed by total synthesis. These compounds showed strong inhibitory activity against Mycobacterium marinum (MICs = 23.4, 81.2, 87.5 μmol/L, respectively), demonstrating valuable application potential in treating M. marinum infection (Hou et al. 2019c).

Figure 43.  Cycloheptapeptides from the coral-derived fungus A. versicolor (CHNSCLM-0063)

Until now, the molecular networking approach has only been applied to mine the undiscovered cycloheptapeptides from marine-derived fungus in our research on MNPs. Undoubtedly, the molecular networking strategy based on LC-MS/MS and relevant data libraries should be considered an effectively targeted isolation technique to speed the discovery of new cryptic secondary metabolites. It is worth applying the molecular networking approach to exploit novel molecules from the pools of MNPs in extracts from marine organisms.

From the gorgonian-derived fungus Aspergillus sp. XS-20090B15, four lumazine peptides, 248 (penilumamide) and 249-251 (penilumamides B-D), together with a cyclic pentapeptide, 252 (asperpeptide A) (Fig. 44), were isolated (Chen et al. 2014c). Penilumamide B (249) was obtained from a feeding culture experiment with _L-methionine. Compounds 249-251 are rare lumazine peptides, of which 248 and 250 are formed from 249 by oxidation of the _L-methionine residue. The cyclic pentapeptide (252) exhibited antibacterial activity against B. cereus and S. epidermidis (MICs = 12.5 μmol/L) (Chen et al. 2014c).

Figure 44.  Lumazine peptides and cyclic pentapeptide from gorgonian-derived fungus Aspergillus sp. XS-20090B15

An increasing number of marine-derived phenyl ether derivatives with novel structures have been reported with diverse biological activities, including antimicrobial, antitumor, cardiotonic, antidiabetic, antiinflammatory and antiviral activities. For instance, 4-carbglyceryl-3, 30-dihydroxy-5, 50-dimethyldiphenyl ether, a diphenyl ether isolated from the deep-sea-derived fungus Aspergillus versicolor SCSIO 41502 showed potent antifouling activity (Huang et al. 2017). A series of antibacterial polybrominated diphenyl ethers were isolated from the Indonesian sponge Lamellodysidea herbacea (Hanif et al. 2007). In our studies, 35 phenyl ether derivatives were obtained in all, among them 9 compounds were first reported, showing antibacterial, antifouling and cytotoxic activities (Chen et al. 2013b; Shi et al. 2017). To clarify the structure-activity relationships, chemical synthesis was performed (Chen et al. 2013b).

Six phenyl ether derivatives were obtained from Aspergillus sp. XS-20090066 isolated from the gorgonian Dichotella gemmacea, including two new phenyl ether derivatives, 253 and 257 (cordyols D and E), together with 4 known analogue compounds 254 (3, 3′-O-dimethyl-violaceol-I), 255 (cordyol C), 256 (4-methoxycarbonyl-diorcinol), and 258 (diorcinol) (Fig. 45) (Chen et al. 2013b). Among them, 256 displayed significant antibacterial activity against S. epidermidis (MIC = 2.71 μmol/L). A series of derivatives (256a, 258a-258g) were designed and synthesized for the study of structure-activity relationships, suggesting that one free hydroxy plays a critical role for antibacterial activity. In particular, 258g exhibited the strongest antibacterial activity toward S. epidermidis (MIC = 0.556 μmol/L). These results revealed that the ester functionality or bromination could increase antibacterial activity, but the simultaneous presence of both the ester and brominated benzene ring caused a loss of activity (Chen et al. 2013b).

Figure 45.  Phenyl ether derivatives from the gorgonian-derived fungus Aspergillus sp. XS-20090066

Two new diphenyl glycosides, 259—260 (phomaethers A-B), one new diphenyl ether derivative, 261 (phomaether C), and one known diphenyl ether analog, 262 (2, 3′-dihydroxy-4-methoxy-5′, 6-dimethyl diphenyl ether) (Fig. 46), were isolated from a gorgonian-derived fungus Phoma sp. (TA07-1) (Shi et al. 2017). It was the first report to find diphenyl glycoside derivatives from coral-derived fungi. Compounds 259, 261, 262 exhibited potent antibacterial activities, indicating that they might be developed as promising antibacterial agents. Specifically, 259 showed remarkable antibacterial activity against S. albus, S. aureus, E. coli and V. parahaemolyticus (MICs = 0.312 to 0.625 μmol/L; minimum bactericidal concentrations (MBCs) = 0.625 to 2.50 μmol/L). Compound 261 displayed significant antibacterial activity to S. albus, S. aureus and E. coli (MICs = 0.312 to 1.25 μmol/L; MBCs = 0.625 to 5.00 μmol/L). Notably, 262 demonstrated strong antibacterial activity against all the tested pathogenic bacteria (MICs and MBCs = 0.156 to 5.00 μmol/L) (Shi et al. 2017).

Figure 46.  Diphenyl glycosides and diphenyl ether derivatives from the gorgonian-derived fungus Phoma sp. (TA07-1)

Besides those indicated above, other phenyl ether derivatives, obtained from various fungi including Penicillium pinophilum, Talaromyces sp. and Aspergillus elegans isolated from gorgonian and soft coral, also exhibited anti-larval settlement activity against B. amphitrite and cytotoxic activity towards the tested human cell lines (Chen et al. 2015; Zhao et al. 2015a). For instance, diphenyl ether derivatives 263 (talaromycin C), 264 (purpactin C′), 265 (pencillide) and 266 (purpactin A) (Fig. 47) displayed strong anti-larval settlement activity against B. amphitrite (EC₅₀ = 2.2-4.8 μg/mL). Compound 267 (tenellic acid A methyl ester) (Fig. 47) showed remarkable cytotoxicity towards human cell lines HepG2, Hep3B, MCF-7/ADR, PC-3, and HCT-116 (IC₅₀ = 4.3-9.8 μmol/L) (Chen et al. 2015).

Figure 47.  Diphenyl ether derivatives from the gorgonian-derived fungus Talaromyces sp.

In summary, our research on the discovery of MNPs resulted in the discovery of diverse structures from soft corals, gorgonians, sponges, tunicates, anemones and their symbiotic microorganisms collected from the South China Sea. Based on our findings, the statistical analysis of 709 MNPs including 307 new compounds demonstrated that the proportions of terpenoids, alkaloids, steroids, and macrocyclic lactones are larger than that of other structures (Fig. 48). It is also revealed that macrocyclic lactones, azaphilones and phenylpropanoids are the most potential classes for the discovery of novel MNPs (Fig. 48). In terms of biological activities, more than half of the obtained MNPs displayed tested biological activities, with the most frequent findings as antibacterial, antifouling, and cytotoxic activities (Fig. 49). Interestingly, many of the active MNPs exhibited multiple bioactivities, for example, MNPs with antiviral activities usually displayed cytotoxic activities, while MNPs with antibacterial activities commonly showed cytotoxic and antifouling activities. It is worth noting that more than 40 compounds were found to exhibit potent activities even stronger than the positive controls. Particularly, the macrocyclic lactone cochliomycin A (1), the azaphilone sclerotioramine derivative (101), and the alkaloid aniduquinolone A (104) may be candidates for efficient antifouling agents, and the macrocyclic lactone bastimolide A (36), the anthraquinones nigrosporin B (41), and the steroid echrebsteroid C (165) might be promising lead compounds for further development of antimalarial, antibiotic, and antiviral drugs.

Figure 48.  The distribution of MNPs obtained from marine invertebrates and their symbiotic microorganisms

Figure 49.  The frequency of MNPs based on diverse biological activities

The South China Sea possesses rich and unique species resources, with more than 95% of the invertebrates mainly existing in the coral reefs in this sea area in China (Zhang et al. 2006). Not surprisingly, many investigations on marine invertebrates and their symbiotic microorganisms target this "biodiversity hotspot" as the center of sample collection. It should be mentioned that the diverse abundance of MNPs derived from marine invertebrates and their symbiotic microorganisms from the South China Sea were discovered and studied. In recent decades, research on the diversity of MNPs discovered from the South China Sea by other Chinese researchers has been well underway. These groundbreaking research efforts contributed to the discovery of a great number of novel and promising bioactive molecules usually from such sources as sponges (Gui et al. 2019; Jiao et al. 2019; Wang et al. 2015b), corals (Li et al. 2019b; Wu et al. 2019), bryozoans (Yu et al. 2015), and associated microorganisms (Cheng et al. 2016; Nong et al. 2016). Regarding the abundance of MNPs with diverse structures and a wealth of biological activities, we believe that only the proverbial "tip of the iceberg" has been explored from the South China Sea, and the resulting novel active metabolites with potential pharmacology applications are worthy of further exploration.

In this review, we exemplified nine types of structurally unique MNPs including macrocyclic lactones, anthraquinones, azaphilones, alkaloids, terpenoids, steroids, phenylpropanoids, peptides, and phenyl ether derivatives obtained from marine invertebrates and their symbiotic microorganisms. Our research provides several typical representative MNPs from marine invertebrates, especially sponges, soft corals, gorgonian corals, and their symbiotic microorganisms (mainly fungi). These MNPs display various potent bioactivities involved in not only chemoecological effects such as antifouling, ichthyootoxic, and brine shrimp lethal activities but also pharmaceutical activities including antibacterial, antiviral, fungicidal, cytotoxic, and antimalarial activities. Our studies demonstrate that MNPs derived from marine invertebrates and their symbiotic microorganisms in the South China Sea are a prolific resource for the discovery of bioactive MNPs. It could be expected that the symbiotic microorganisms associated with marine invertebrates have great potential as a significant source of structurally interesting molecules. It should be noted that the application of multiple discovery strategies and methods could effectively promote the exploitation of novel MNPs with diverse structures. In our study, different methodological approaches, including single culture, OSMAC, chemical epigenetic manipulation, co-culture, structural modification and chemical synthesis, have been applied in the search for new MNPs. Among them, co-culture, structural modification and chemical synthesis were found to be effective approaches to obtain potential bioactive MNPs. Particularly, LC-MS/MS-dependent molecular networking has been applied as a promising approach to dereplicate complex natural product mixtures, contributing to the targeted isolation of a series of new compounds. It is anticipated that future genetic techniques and bioinformatics tools, especially metagenomic approaches, genome mining, and heterologue biosynthesis, could accelerate the exploration and accessibility of remaining undiscovered MNPs with novel structures and promising bioactivities from marine microorganisms.

This work is supported by the National Natural Science Foundation of China (nos. 41830535; 81673350; U1706210), the Fundamental Research Funds for the Central Universities of China (no. 201962002), and the Taishan Scholars Program, China.

LL and YYZ collected the data and wrote the paper; CLS and CYW designed, directed and revised the manuscript.

The authors declare that they have no conflict of interest.

This article does not contain any studies with human participants or animals performed by any of the authors.

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