Chemical Diversity and Biological Activity of Secondary Metabolites Isolated from Indonesian Marine Invertebrates

Marine invertebrates have been reported to be an excellent resource of many novel bioactive compounds. Studies reported that Indonesia has remarkable yet underexplored marine natural products, with a high chemical diversity and a broad spectrum of biological activities. This review discusses recent updates on the exploration of marine natural products from Indonesian marine invertebrates (i.e., sponges, tunicates, and soft corals) throughout 2007–2020. This paper summarizes the structural diversity and biological function of the bioactive compounds isolated from Indonesian marine invertebrates as antimicrobial, antifungal, anticancer, and antiviral, while also presenting the opportunity for further investigation of novel compounds derived from Indonesian marine invertebrates.


Introduction
A wide range of natural products (NPs) has been isolated from various marine organisms, especially marine invertebrates such as sponges, tunicates, soft corals, bryozoans, and nudibranchs. These marine invertebrates are excellent sources of NPs with vast chemical structures and potential biological activities [1][2][3]. During 2012-2017, no less than 550-700 new compounds have been reported from marine invertebrates [4], in which half of these compounds were isolated from marine sponges [5]. Among those, 4% and 22% of the compounds were identified in 2017 and 2016, respectively [4,5]. Between 1998 to 2018, one hundred and fourteen secondary metabolites were isolated from the marine sponges of the genus Suberea [6]. Meanwhile, a hundred and seventy compounds were isolated from soft corals of the genus Dendronephthya alone throughout 1999-2019 [7]. Soft corals belonging to the genus Xenia are rich in terpenoids, with 199 compounds isolated from 1977-2019 [8]. To date, approximately 30,000-40,000 marine natural products (MNPs) have been identified, with the majority of the compounds exhibiting cytotoxic and anticancer properties [4,5,9].
The biological potential of MNPs from marine invertebrates has been proven to be a valuable source for drug discovery and development. Most of the approved commercial marine-based drugs are of marine invertebrate origin. Eight marine drugs have been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). The first approved marine drug is Ziconotide (Prialt ® ) discovered in the marine snail Conus magus. This peptide-derived marine drug is commonly used as an analgesic drug for the management of severe chronic pain through the intrathecal route. The second one is Omega-3-acid-ethyl esters (Lovaza ® ) derived from fish oil and used as an anti-hypertriglyceridemia drug. The third is Vidarabine (Vira-A ® ) derived from the Table 1. Summary of the marine natural products isolated from the marine sponges from Indonesian oceans.
A novel 3-alkylpiperidine alkaloid (8) was also isolated from A. ingens collected from Wakatobi Marine National Park in Southeast Sulawesi [20]. Recently, the alkaloid compounds 9-19 were also isolated from A. ingens collected from South Sulawesi along with compound 8 [21]. Moreover, compound 8 was reported to have specific antimicrobial activity against E. coli and showed an inhibitory effect on amyloid β-42 production induced by aftin-5 without cytotoxicity at 26 µM. Compounds 8 and 11 exhibited antibacterial activity at 100 µg/disc against E. coli and S. aureus. In addition to that, compound 12 was reported to have selective inhibitory activity against the protein kinase CK1 δ/ε with an IC 50 value of 6 µM, while the diketopiperazine compound 13 showed a selective kinase inhibitory activity against CDK2/cyclin A with an IC 50 value of 1 µM. However, no bioactivity was reported from compounds 14-19 [21].
A novel pyridoacridine alkaloid, sagitol C (38), was isolated from the ethyl acetate fraction of Oceaniapia sp. collected in Ambon, Maluku Islands [30]. Alkaloid 38 was found to exhibit cytotoxic activity against the mouse lymphoma L5187Y cancer cell line, HeLa cell, and the rat pheochromocytoma PC12 cell lines with ED 50 values of 0.7, 0.9, and 2.3 µM, respectively [30].
Seven novel anti-angiogenic steroidal alkaloids ( Figure 4) were isolated from the sponge Corticium simplex collected in Flores Island, East Nusa Tenggara [31,32] Compound 37 was found to inhibit the chymotrypsin-like activity of the proteasome and Ubc13 (E2)-Uev1A interaction with IC50 values of 16.5 and 20.6 µM, respectively. In contrast, compound 36 showed a weak effect on proteasome. This result suggested that the sulfate group in variabines decreases chymotrypsin-like activity [29]. A novel pyridoacridine alkaloid, sagitol C (38), was isolated from the ethyl acetate fraction of Oceaniapia sp. collected in Ambon, Maluku Islands [30]. Alkaloid 38 was found to exhibit cytotoxic activity against the mouse lymphoma L5187Y cancer cell line, HeLa cell, and the rat pheochromocytoma PC12 cell lines with ED50 values of 0.7, 0.9, and 2.3 µM, respectively [30].
Other compounds isolated from Sulawesi included nakijiquinone V (83), isolated from Dactylospongia elegans from Tahuna, Sangihe Islands, North Sulawesi [39]. It has three methyl groups attached to a decalin system with an exocyclic double bond [39] (Figure 6). Another was a new meroditerpene-namely, Halioxepine (84), which was isolated from an Indonesian marine sponge from the genus Haliclona collected in Baubau, Southeast Sulawesi [40]. This compound showed moderate cytotoxicity against the rat bladder tumour NBT-T2 cells with IC50 of 11.6 µM and antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 of 7.7 µM [40]. Recently, two new terpenoids, melophluosides A (85) and B (86), were successfully isolated from Melophlus sarassinorum collected in Siladen, North Sulawesi. These new compounds belong to the triterpene galactosides of the pouoside class, with compound 85 as the first without an oxygenated group Two new cycoldepsipeptide jaspamide derivatives, jaspamide Q (87) and jaspamide R (88), were identified from the marine sponge J. splendens collected in neighbouring islands near East Kalimantan [42]. They exhibited potent cytotoxic activity against the mouse lymphoma L5187Y cancer cell line with IC 50 values of <126.8 µM and <203 µM, respectively [42].
Novel tridecapeptides of the theonellapeptolide family were isolated from T. swinhoei collected from Bunaken Marine Park in Manado, North Sulawesi-namely, sulfinyltheonellapeptolide (89) and theonellapeptolide If (90) [43]. These compounds differ in the abrine moiety. Compound 89 is a theonellapeptolide with a methylsulfinylacetyl group at the N-terminus, while compound 90 was the first theonellapeptolide with four valine residues ( Figure 7). Both compounds showed significant antiproliferative activity against human liver HepG2 cancer cells with the same IC 50 value of 3 µM [43].
S. mirabilis is also one of the rich sources of diverse secondary metabolites, as exemplified by the isolation of six new depsipeptides with two different structural classes named celebesides A-C (91-93) from the species collected in Sulawesi [44]. Compounds 91-93 are cyclic depsipeptides with a polyketide moiety and five amino acid residues (Figure 7). The celebesides A and B (91, 92) possess a 3-carbamoyl threonine and a phosphoserine residue, which is quite uncommon. S. mirabilis also produced the peptides theopapuamides B-D (94-96), which are undecapeptides with an N-terminal fatty acid moiety, with theopapuamide D (96) containing a rare homoisoleucine residue (Figure 7). These compounds exhibited several bioactivities. Theopapuamides B and C (94, 95) were found to have relatively strong antifungal activity against amphotericin B-resistant C. albicans [44]. Additionally, celebeside A (91) and theopapuamide B (94) neutralized HIV-1 in a single-round infectivity assay with an IC 50 value of 2.1 ± 0.4 µM and 499.7 ± 0.3 µg/mL [44]. Celebeside 91 and theopapuamides 94 and 95 also demonstrated cytotoxicity against human colon carcinoma cells with IC 50 values 9.9 µM, 1.3 µM, and 2.5 µM, respectively [44]. Although they were potent against human colon HCT116 carcinoma cells, celebesides A, B, and C (91-93) were also found to be cytotoxic for healthy cell lines [44].
Novel tridecapeptides of the theonellapeptolide family were isolated from T. swinhoei collected from Bunaken Marine Park in Manado, North Sulawesi-namely, sulfinyltheonellapeptolide (89) and theonellapeptolide If (90) [43]. These compounds differ in the abrine moiety. Compound 89 is a theonellapeptolide with a methylsulfinylacetyl group at the N-terminus, while compound 90 was the first theonellapeptolide with four valine residues (Figure 7). Both compounds showed significant antiproliferative activity against human liver HepG2 cancer cells with the same IC50 value of 3 µM [43]. S. mirabilis is also one of the rich sources of diverse secondary metabolites, as exemplified by the isolation of six new depsipeptides with two different structural classes named celebesides A-C (91-93) from the species collected in Sulawesi [44]. Compounds 91-93 are cyclic depsipeptides with a polyketide moiety and five amino acid residues (Figure 7). The celebesides A and B (91, 92) possess a 3-carbamoyl threonine and a phosphoserine residue, which is quite uncommon. S. mirabilis also produced the peptides theopapuamides B-D (94-96), which are undecapeptides with an N-terminal fatty acid moiety, with theopapuamide D (96) containing a rare homoisoleucine residue (Figure 7). These compounds exhibited several bioactivities. Theopapuamides B and C (94, 95) were found to have relatively strong antifungal activity against amphotericin B-resistant C. albicans Haloirciniamide A (97) and seribunamide A (98), new polyhalogenated peptides, have been isolated from Ircinia sp. collected from the coast of Thousand Islands. Compound 97 was the first dibromopyrrole cyclopeptide with a chlorohistidine ring, while compound 98 possess a rare tribromopyrrole ring. Unfortunately, both compounds did not show significant cytotoxicity against four human tumour cell lines [45].
Four new endoperoxyketal polyketides, manadoperoxides A-D (99-102), were isolated from the sponge Plakortis cfr. simplex obtained from the Bunaken Marine Park of Manado, North Sulawesi [46]. In these compounds, the methoxy group at C-6 is replaced by either a methyl or ethyl group instead of a peroxyketal-type (Figure 8), making them slightly different from those previously isolated from the same species in the Caribbean. All compounds isolated from that sponge Plakortis cfr. simplex showed moderate antimalarial activity against D10 and W2 strains of Plasmodium falciparum [46].  C (91-93) were also found to be cytotoxic for healthy cell lines [44]. Haloirciniamide A (97) and seribunamide A (98), new polyhalogenated peptide have been isolated from Ircinia sp. collected from the coast of Thousand Islands. Com pound 97 was the first dibromopyrrole cyclopeptide with a chlorohistidine ring, whil compound 98 possess a rare tribromopyrrole ring. Unfortunately, both compounds di not show significant cytotoxicity against four human tumour cell lines [45].
Four new endoperoxyketal polyketides, manadoperoxides A-D (99-102), were iso lated from the sponge Plakortis cfr. simplex obtained from the Bunaken Marine Park o Manado, North Sulawesi [46]. In these compounds, the methoxy group at C-6 is replace by either a methyl or ethyl group instead of a peroxyketal-type (Figure 8), making them slightly different from those previously isolated from the same species in the Caribbean All compounds isolated from that sponge Plakortis cfr. simplex showed moderate antima larial activity against D10 and W2 strains of Plasmodium falciparum [46]. A novel cytotoxic macrolide, namely Callyspongiolide (103), was isolated from th Indonesian marine sponge Calyspongia sp [47]. This compound has a notable feature of conjugated diene-ynic sidechain ending at a benzene ring with bromine, which was no found in the previously isolated marine macrolides (Figure 9). Its cytotoxicity against hu man acute T cell leukaemia Jurkat J16 T and human Burkitt's lymphoma Ramos B lym phocytes showed remarkable IC50 values of 70 and 60 nM, respectively, as well as EC A novel cytotoxic macrolide, namely Callyspongiolide (103), was isolated from the Indonesian marine sponge Calyspongia sp [47]. This compound has a notable feature of a conjugated diene-ynic sidechain ending at a benzene ring with bromine, which was not found in the previously isolated marine macrolides (Figure 9). Its cytotoxicity against human acute T cell leukaemia Jurkat J16 T and human Burkitt's lymphoma Ramos B lymphocytes showed remarkable IC 50 values of 70 and 60 nM, respectively, as well as EC 50 values of 80 and 50 nM, respectively [47]. A novel cytotoxic macrolide, namely Callyspongiolide (103), was isolated from the Indonesian marine sponge Calyspongia sp [47]. This compound has a notable feature of a conjugated diene-ynic sidechain ending at a benzene ring with bromine, which was not found in the previously isolated marine macrolides (Figure 9). Its cytotoxicity against human acute T cell leukaemia Jurkat J16 T and human Burkitt's lymphoma Ramos B lymphocytes showed remarkable IC50 values of 70 and 60 nM, respectively, as well as EC50 values of 80 and 50 nM, respectively [47]. A novel A-nor steroid, namely clathruhoate (104), was isolated from a marine sponge Clathria sp., collected from Bintang Samudra Marine Education Park, Southeast Sulawesi A novel A-nor steroid, namely clathruhoate (104), was isolated from a marine sponge Clathria sp., collected from Bintang Samudra Marine Education Park, Southeast Sulawesi [48]. Moreover, a new nortriterpenoid saponin, namely sarasinoside S (105), was identified from the marine sponge Petrosia sp. obtained in North Sulawesi [49]. Compound 105 was the first among the nortriterpenes in the saranoside family with a degraded side chain (Figure 9).

Soft Corals
Terpenoids were often isolated from soft corals, with varying degrees of bioactivities (Table 3). Sarcofuranocembrenolides A (120) and B (121) were isolated from the soft coral Sarcophyton sp. collected in North Sulawesi [55]. Cembranoid 120 is a bisnorcembrenolide featuring a unique carbon skeleton of 8,19-bisnorfuranocembrenolide ( Figure 12). On the other hand, cembranoid 121 is a furanocembrenolide with a C 1 unit (C-20) attached to C-10. In the ordinary cembrenolides, the C 1 unit is attached to C-12 [55]. Table 3. Summary of the marine natural products isolated from the marine soft corals from Indonesian oceans.

Soft Corals
Terpenoids were often isolated from soft corals, with varying degrees of bioactivities (Table 3). Sarcofuranocembrenolides A (120) and B (121) were isolated from the soft coral Sarcophyton sp. collected in North Sulawesi [55]. Cembranoid 120 is a bisnorcembrenolide featuring a unique carbon skeleton of 8,19-bisnorfuranocembrenolide ( Figure 12). On the other hand, cembranoid 121 is a furanocembrenolide with a C1 unit (C-20) attached to C-10. In the ordinary cembrenolides, the C1 unit is attached to C-12 [55]. Three C-4 norcembranoids-type macrocyclic diterpenoids, namely chloroscabrolide A (122), chloroscabrolide B (123), and prescarbolide (124), were isolated from Sinularia sp. collected from Bunaken Marine Park, Manado, North Sulawesi [56]. Compounds 122 and 123 are two of very few chlorinated compounds from soft coral metabolites and the second example within the class of cembranoids (Figure 12). These two compounds also feature an oxygen bridge connecting C-13 and C-15, which is quite unusual. Meanwhile, the terpenoid prescarbolide (124) is believed to be the precursor of the scabrolide/leptocladolide family of cembranoids [56].
Two new isomeric eunicellin-type diterpenoids were isolated from an Indonesian oc- Three C-4 norcembranoids-type macrocyclic diterpenoids, namely chloroscabrolide A (122), chloroscabrolide B (123), and prescarbolide (124), were isolated from Sinularia sp. collected from Bunaken Marine Park, Manado, North Sulawesi [56]. Compounds 122 and 123 are two of very few chlorinated compounds from soft coral metabolites and the second example within the class of cembranoids ( Figure 12). These two compounds also feature an oxygen bridge connecting C-13 and C-15, which is quite unusual. Meanwhile, the terpenoid prescarbolide (124) is believed to be the precursor of the scabrolide/leptocladolide family of cembranoids [56].
The latest finding on terpenoids from soft coral was discovered from Anthelia sp. collected at Banten, West Java, which successfully isolated two new dolabellane diterpenoids, sangiangol A (128) and sangiangol B (129). These two compounds were found to show weak toxicity against NBT-T2 rat bladder epithelial cells (BRC-1370) at 18 and 28.2 µM, respectively [59]. The first reported zoanthamine-type alkaloid from a marine invertebrate other than zoanthids was named lobozoanthamine (130), isolated from the Indonesian soft coral Lobophytum sp. collected from the Bunaken Marine Park, Manado, North Sulawesi [60]. However, there is no report on the bioactivity of the compounds 120-125 and 130 (Table 3).  A terpenoid compound, 3,4-epoxy-nephtenol (127), was isolated from the soft coral Nephthea sp. found in Seribu Islands, DKI Jakarta [58]. Compound 127 showed weak inhibitory growth against three human tumour cell lines, i.e., human glioblastoma SF268, human breast MCF-7, and human non-small cell lung H460 cancer cells [58].
The latest finding on terpenoids from soft coral was discovered from Anthelia sp. collected at Banten, West Java, which successfully isolated two new dolabellane diterpenoids, sangiangol A (128) and sangiangol B (129). These two compounds were found to show weak toxicity against NBT-T2 rat bladder epithelial cells (BRC-1370) at 18 and 28.2 µM, respectively [59]. The first reported zoanthamine-type alkaloid from a marine invertebrate other than zoanthids was named lobozoanthamine (130), isolated from the Indonesian soft coral Lobophytum sp. collected from the Bunaken Marine Park, Manado, North Sulawesi [60]. However, there is no report on the bioactivity of the compounds 120-125 and 130 (Table 3).

Conclusions
Indonesian marine biodiversity holds immense potential for drug discovery and bioprospecting, supporting the continuous exploration and investigation of Indonesian MNPs from marine invertebrates such as sponges, tunicates, and soft corals. This is further justified by no less than a hundred and thirty novel compounds reported in this review from 43 publications throughout 2007-2020, the majority of which were isolated from the Indonesian marine invertebrates collected from the North region of Sulawesi, Indonesia ( Figure 13).
Indonesian sponges have been known as a major source of many novel MNPs. In this review, sponges were found to contribute to 105 novel compounds reported between 2007-2020, the majority being alkaloids (Figure 14). Most of the sponge alkaloids were isolated from the genera Aplysinella and Acanthostrongulophora. The next large groups of metabolites isolated from Indonesian sponges were terpenes and peptides. Nearly all the sponge terpenoids were isolated from the order Dictyoceratida, in which the dominant genera were Lamellodysidea and Carteriospongia.
MNPs from marine invertebrates such as sponges, tunicates, and soft corals. This is further justified by no less than a hundred and thirty novel compounds reported in this review from 43 publications throughout 2007-2020, the majority of which were isolated from the Indonesian marine invertebrates collected from the North region of Sulawesi, Indonesia ( Figure 13).
Indonesian sponges have been known as a major source of many novel MNPs. In this review, sponges were found to contribute to 105 novel compounds reported between 2007-2020, the majority being alkaloids (Figure 14). Most of the sponge alkaloids were isolated from the genera Aplysinella and Acanthostrongulophora. The next large groups of metabolites isolated from Indonesian sponges were terpenes and peptides. Nearly all the sponge terpenoids were isolated from the order Dictyoceratida, in which the dominant genera were Lamellodysidea and Carteriospongia.  Meanwhile, all the peptides, with the exception of two peptides from Ircinia sp., were isolated from the order Tetractinellida. On the other hand, macrolides, steroids, and saponins were the least frequently isolated metabolites from Indonesian sponges. The sponge Plakortis simplex was the sole contributor of the polyketides isolated from Indonesia (Table 1).
During 2007-2020, ten alkaloids were reported from Indonesian tunicates, mainly from the family Didemnidae and Styelidae. Soft corals are also a good source of novel metabolites and are well-known as a producer of terpenoids, particularly the group of terpenes and cembranoid diterpenes. Soft coral-derived terpenoids have received significant attention in Indonesian MNPs research (Table 3). Compared to other invertebrates with alkaloids as the most abundant secondary metabolites, only one alkaloid was found in Indonesian soft coral species in this review.
Indonesian soft corals and tunicates yielded far lower novel secondary metabolites than sponges reported throughout 2007-2020. Therefore, this review highlighted the opportunity to explore further the chemical diversity and the biological activity of tunicates from Indonesian waters.
Diverse biological activities were shown by fifty per cent of the compounds isolated from the three Indonesian marine invertebrates discussed ( Figure 14). The majority of compounds in this review were reported to possess cytotoxic or antiproliferative activity against various cancer cell lines. Four of these compounds showed a remarkable cytotoxic activity with IC 50 values of less than 1 µM, namely compounds 30, 38, 103 and 112. Furthermore, four compounds showed IC 50 values between 1-4 µM, and another eight showed moderate cytotoxic with an IC 50 value of less than 10 µM. Most of these compounds belong to the alkaloid group, followed by peptides and terpenoids. It is, therefore, evident that the exploration of potential anticancer drugs from Indonesian marine resources warrants further investigation.
The bioactivity of compounds isolated from the Indonesian marine invertebrates (sponges, tunicates, and soft corals) as antimicrobial, antifungal, or antiviral was also described in this review. Unfortunately, little or no such activities were yet to be found from many compounds derived from the three Indonesian marine invertebrates, demonstrating the gap in the knowledge of their beneficial biological activity other than their cytotoxic activity. This knowledge gap opens up the opportunity for further research focusing on exploring and harnessing the potential of Indonesian marine invertebrates as sources of compounds with antimicrobial, antifungal, or antiviral activities, as well as further exploration of Indonesian marine biodiversity for the discovery of novel bioactive compounds.