Natural Product Repertoire of the Genus Amphimedon

Marine sponges are a very attractive and rich source in the production of novel bioactive compounds. The sponges exhibit a wide range of pharmacological activities. The genus Amphimedon consists of various species, such as viridis, compressa, complanata, and terpenensis, along with a handful of undescribed species. The Amphimedon genus is a rich source of secondary metabolites containing diverse chemical classes, including alkaloids, ceramides, cerebrososides, and terpenes, with various valuable biological activities. This review covers the literature from January 1983 until January 2018 and provides a complete survey of all the compounds isolated from the genus Amphimedon and the associated microbiota, along with their corresponding biological activities, whenever applicable.


Introduction
The broadness in the discovery of natural marine products have increased over the last two decades with the number of new compounds discovered per year similarly increasing [1]. One of the richest sources of marine natural products, among all the marine organisms investigated in literature, are the marine sponges. Nearly thirty percent of all marine natural products discovered in history come from marine sponges [2][3][4]. Sponges have been considered an important source of new bioactive natural products [5], although the role of microbial symbionts as the producers of sponge metabolites needs more further research in the future. The number of metabolites reported from sponges varies from year to year, e.g., in 2014, 283 new structures were described from the phylum Porifera. By 2015, the number of metabolites (291) remained similar to 2014 and constant in comparison to previous years [6]. In 2016, 224 new compounds were reported from sponges. This reduction may represent the focus changing from studying sponges to studying microorganisms. However, sponges remain one of the important groups in the discovery of new bioactive compounds [7]. Wide ranges of microbes are associated with sponges, these include heterotrophic bacteria, cyanobacteria, dinoflagellates, and diatoms [8]. These microbes all play several roles in the sponges and affect sponge health, growth rates, and the ability to defend against predators [9]. The interaction between sponges and the associated microorganism could help in the nutrition of the sponge [10], or in the fixation of nitrogen [11]. The most important role, however, is the production of secondary metabolites as antifungals and antibiotics [12]. The genus Amphimedon (Kingdom: Animalia; phylum: Porifera; class: Demospongiae; sub class: Heteroscleromorpha; order: Haplosclerida; family: Niphatidae) is reported to be a very rich source of bioactive metabolites. Amphitoxin [26] was isolated from different marine sponges belonging to the order Haplosclerida, which is found in relatively high quantities in extracts of Amphimedon compressa, collected near the coast of San Salvador Island. Amphitoxin is a polymeric compound containing 3alkyl-and 3-alkenyl pyridinium units and contains a mixture of high-molecular-weight pyridinium salts. It is similar in structure to a polymeric pyridinium alkaloid, which has recently been isolated from a sponge belonging to the genus Callyspongia (C. fibrosa). However, the amphitoxin of Amphimedon compressa lacks the carbon-carbon double bond in the side-chain and its molecular weight has not yet been established precisely. The amphitoxin showed ichthyotoxic activity towards Xiphophorus variatus (moon fish) and insecticidal activity against Cylas formicarius elegantulus (sweet potato weevil) in concentrations of 0.2, 0.8, and 0.4 ppm and corresponding mortality rates of 30, 20 and 10%, respectively, with a life span of 72 h. The toxicity towards the moon fish was evidenced by the high mortality rates. In amphitoxin concentrations of 1000 and 100 ppm, the lifespan of the moon fish is <30 min and <60 min, respectively, thus demonstrating a mortality rate of 100%. [27].
Amphitoxin [26] was isolated from different marine sponges belonging to the order Haplosclerida, which is found in relatively high quantities in extracts of Amphimedon compressa, collected near the coast of San Salvador Island. Amphitoxin is a polymeric compound containing 3-alkyl-and 3-alkenyl pyridinium units and contains a mixture of high-molecular-weight pyridinium salts. It is similar in structure to a polymeric pyridinium alkaloid, which has recently been isolated from a sponge belonging to the genus Callyspongia (C. fibrosa). However, the amphitoxin of Amphimedon compressa lacks the carbon-carbon double bond in the side-chain and its molecular weight has not yet been established precisely. The amphitoxin showed ichthyotoxic activity towards Xiphophorus variatus (moon fish) and insecticidal activity against Cylas formicarius elegantulus (sweet potato weevil) in concentrations of 0.2, 0.8, and 0.4 ppm and corresponding mortality rates of 30, 20 and 10%, respectively, with a life span of 72 h. The toxicity towards the moon fish was evidenced by the high mortality rates. In amphitoxin concentrations of 1000 and 100 ppm, the lifespan of the moon fish is <30 min and <60 min, respectively, thus demonstrating a mortality rate of 100%. [27].

Amphimedon viridis
1,3-Dimethylisoguanine 15 [32] is a purine-based alkaloid, as shown in Figure 4, which was isolated from Amphimedon viridis. The alkaloid induces contractions when obtained by a transmural electrical stimulation in a guinea pigs' longitudinal muscle/myenteric plexus in a dose-dependent manner [33]. Acetamidoglucosyl ceramide 14, as shown in Figure 3, was isolated from Amphimedon compressa collected from the coast of Key Largo, Florida [31].

Amphimedon viridis
1,3-Dimethylisoguanine 15 [32] is a purine-based alkaloid, as shown in Figure 4, which was isolated from Amphimedon viridis. The alkaloid induces contractions when obtained by a transmural electrical stimulation in a guinea pigs' longitudinal muscle/myenteric plexus in a dose-dependent manner [33].  Theophylline 16 [32] is another purine-based alkaloid isolated from Amphimedon viridis, which was previously isolated from the plant source Camellia sinensis [34]. Furthermore, cerebrosoides amphicerebroside B-F 17-21, as shown in Figure 5, [35] were obtained from this sponge and were collected from the Red Sea.

Amphimedon complanata
Amphimedon complanata is a species of sponge rich in fatty acids, such as 11,l5-icosadienoic acid 22, 7-methyl-6-hexadecenoic acid 23, and 6,11-icosadienoic acid 24 [36], and was isolated from a sponge collected near the shelf edge of La Parguera at a depth of 80 ft, as shown in Figure 6. Theophylline 16 [32] is another purine-based alkaloid isolated from Amphimedon viridis, which was previously isolated from the plant source Camellia sinensis [34]. Furthermore, cerebrosoides amphicerebroside B-F 17-21, as shown in Figure 5, [35] were obtained from this sponge and were collected from the Red Sea.  Theophylline 16 [32] is another purine-based alkaloid isolated from Amphimedon viridis, which was previously isolated from the plant source Camellia sinensis [34]. Furthermore, cerebrosoides amphicerebroside B-F 17-21, as shown in Figure 5, [35] were obtained from this sponge and were collected from the Red Sea.

Amphimedon complanata
Amphimedon complanata is a species of sponge rich in fatty acids, such as 11,l5-icosadienoic acid 22, 7-methyl-6-hexadecenoic acid 23, and 6,11-icosadienoic acid 24 [36], and was isolated from a sponge collected near the shelf edge of La Parguera at a depth of 80 ft, as shown in Figure 6.

Amphimedon complanata
Amphimedon complanata is a species of sponge rich in fatty acids, such as 11,l5-icosadienoic acid 22, 7-methyl-6-hexadecenoic acid 23, and 6,11-icosadienoic acid 24 [36], and was isolated from a sponge collected near the shelf edge of La Parguera at a depth of 80 ft, as shown in Figure 6.

Undescribed Marine Sponges of the Genus Amphimedon
One of the classes repeatedly isolated from Amphimedon sp. is the manzamine alkaloids. In 1986, Higa and coworkers isolated the manzamine A prototype for this group, a novel cytotoxic β-carboline alkaloid from marine sponge Haliclona sp. collected from Manzamo, Okinawa [38]. The isolation of manzamines (B-D), from the marine sponge Haliclona sp subsequently followed [39], as well as ircinals (A-B) from an ircinia sponge [40]. In the last two decades more than 80 β -carboline-containing manzamine alkaloids and manzamine-related alkaloids have been isolated from marine sponges

Undescribed Marine Sponges of the Genus Amphimedon
One of the classes repeatedly isolated from Amphimedon sp. is the manzamine alkaloids. In 1986, Higa and coworkers isolated the manzamine A prototype for this group, a novel cytotoxic β-carboline alkaloid from marine sponge Haliclona sp. collected from Manzamo, Okinawa [38]. The isolation of manzamines (B-D), from the marine sponge Haliclona sp subsequently followed [39], as well as ircinals (A-B) from an ircinia sponge [40]. In the last two decades more than 80 β -carboline-containing manzamine alkaloids and manzamine-related alkaloids have been isolated from marine sponges

Undescribed Marine Sponges of the Genus Amphimedon
One of the classes repeatedly isolated from Amphimedon sp. is the manzamine alkaloids. In 1986, Higa and coworkers isolated the manzamine A prototype for this group, a novel cytotoxic β-carboline alkaloid from marine sponge Haliclona sp. collected from Manzamo, Okinawa [38]. The isolation of manzamines (B-D), from the marine sponge Haliclona sp. subsequently followed [39], as well as ircinals (A-B) from an ircinia sponge [40]. In the last two decades more than 80 β -carboline-containing manzamine alkaloids and manzamine-related alkaloids have been isolated from marine sponges following manzamine A discovery [41]. Manzamines are very attractive bioactive natural metabolites due to their wide range of biological applications as well as insecticidal [42], cytotoxic [43], and anti-inflammatory properties [44].

Bacteria Associated with the Genus Amphimedon
Marine sponges contain a huge diversity of fatty acids which are hypothesized to be composed from bacterial origin. Regarding Amphimedon terpenensis, the brominated long-chain fatty acids 29 and 30, were more distributed in the sponge and its associated bacterial symbionts [61]. However, the role of these fatty acids in marine sponges is unclear, but they may be involved in the relationship between the sponge and their associated bacteria and in protecting the bacterial symbionts from sponge phagocytosis. Amphitoxin [62] is a pyridinium alkaloid isolated from Amphimedon chloros, which was previously described as Amphimedon viridis. The amphitoxin exhibited broad-spectrum activity against bacteria derived from seawater. Furthermore, the sponge-associated bacteria alpha proteobacteria were resistant against this compound, as well as six different bacterial strains associated with the sponge A. viridis [63]. This inhibition may explain the ability of the bacteria to live in close association with their host sponge while having a chemical defense against other microbial pathogens. Moreover, actinomycetes such as Kocuria, Microbacterium, and Micrococcus were cultivated from the Amphimedon sp. that had been collected offshore of the Red Sea, Egypt, but were never chemically investigated [64].
Several types of mycobacteria, including a strain closely related to M. tuberculosis, have been isolated from Amphimedon queenslandica. The strain Salinispora arenicola was also isolated from Amphimedon queenslandica. This strain can synthesize antimycobacterial compounds [65].
As the polyketide class is well-known for their antibiotic activities, the polyketide synthetase genes of the sponge-derived mycobacteria were examined [66] and found to catalyze the synthesis of mycobacterial outer membrane lipids.
The diversity in the regions in which Amphimedon and its different species were collected, play a role in the production of manzamine alkaloids 30-50. They are widely believed to be produced by a symbiotic relationship between the sponge and common or closely related microorganism(s), which may account for the generation of the manzamine alkaloids [67]. Furthermore, there is evidence that sponge-associated microbes have a significant role in the bioconversion of manzamines to the growing number of alkaloids found in sponges producing manzamines and is provided by the biotransformation of 8-hydroxymanzamine A and its enantiomer to manzamine A and ent-12,34oxamazamine F, respectively [41].
Classes of chemical compounds, such as fatty acids, were used as markers for the the chemodiversity in the genus Amphimedon. Several unique fatty acids were isolated from this genus as 17, 18, and 19-hexacosenoic acids. Commonly, there were two biosynthetic pathways that were reported in marine sponges such as 18: ln-7 to 26: ln-7, and the 18: ln-9 to 26: ln-9 routes. While the 18: ln-8 to 26: ln-8 pathway has only recently existed in these invertebrates [28,68] the long chain monounsaturated fatty acids were rare in nature due to their existence in small amounts, especially in microgram quantities [28]. The biosynthesis of the long chain brominated fatty acids in Amphimedon terpenensis has previously been explained in past literature. The substituent of bromine was introduced following chain elongation and desaturation of a 16:0 precursor. The formation of 24:2 brominated and 26:2 brominated acids might have been predicted, however, the isolation of a 25:2 brominated acid suggests that sponges may additionally have the capacity to extend chains, desaturate, and functionalize an odd carbon chain length fatty acid. Odd carbon chain fatty acids,

Bacteria Associated with the Genus Amphimedon
Marine sponges contain a huge diversity of fatty acids which are hypothesized to be composed from bacterial origin. Regarding Amphimedon terpenensis, the brominated long-chain fatty acids 29 and 30, were more distributed in the sponge and its associated bacterial symbionts [61]. However, the role of these fatty acids in marine sponges is unclear, but they may be involved in the relationship between the sponge and their associated bacteria and in protecting the bacterial symbionts from sponge phagocytosis. Amphitoxin [62] is a pyridinium alkaloid isolated from Amphimedon chloros, which was previously described as Amphimedon viridis. The amphitoxin exhibited broad-spectrum activity against bacteria derived from seawater. Furthermore, the sponge-associated bacteria alpha proteobacteria were resistant against this compound, as well as six different bacterial strains associated with the sponge A. viridis [63]. This inhibition may explain the ability of the bacteria to live in close association with their host sponge while having a chemical defense against other microbial pathogens. Moreover, actinomycetes such as Kocuria, Microbacterium, and Micrococcus were cultivated from the Amphimedon sp. that had been collected offshore of the Red Sea, Egypt, but were never chemically investigated [64].
Several types of mycobacteria, including a strain closely related to M. tuberculosis, have been isolated from Amphimedon queenslandica. The strain Salinispora arenicola was also isolated from Amphimedon queenslandica. This strain can synthesize antimycobacterial compounds [65].
As the polyketide class is well-known for their antibiotic activities, the polyketide synthetase genes of the sponge-derived mycobacteria were examined [66] and found to catalyze the synthesis of mycobacterial outer membrane lipids.
The diversity in the regions in which Amphimedon and its different species were collected, play a role in the production of manzamine alkaloids 30-50. They are widely believed to be produced by a symbiotic relationship between the sponge and common or closely related microorganism(s), which may account for the generation of the manzamine alkaloids [67]. Furthermore, there is evidence that sponge-associated microbes have a significant role in the bioconversion of manzamines to the growing number of alkaloids found in sponges producing manzamines and is provided by the biotransformation of 8-hydroxymanzamine A and its enantiomer to manzamine A and ent-12,34-oxamazamine F, respectively [41].
Classes of chemical compounds, such as fatty acids, were used as markers for the the chemodiversity in the genus Amphimedon. Several unique fatty acids were isolated from this genus as 17, 18, and 19-hexacosenoic acids. Commonly, there were two biosynthetic pathways that were reported in marine sponges such as 18: ln-7 to 26: ln-7, and the 18: ln-9 to 26: ln-9 routes. While the 18: ln-8 to 26: ln-8 pathway has only recently existed in these invertebrates [28,68] the long chain monounsaturated fatty acids were rare in nature due to their existence in small amounts, especially in microgram quantities [28]. The biosynthesis of the long chain brominated fatty acids in Amphimedon terpenensis has previously been explained in past literature. The substituent of bromine was introduced following chain elongation and desaturation of a 16:0 precursor. The formation of 24:2 brominated and 26:2 brominated acids might have been predicted, however, the isolation of a 25:2 brominated acid suggests that sponges may additionally have the capacity to extend chains, desaturate, and functionalize an odd carbon chain length fatty acid. Odd carbon chain fatty acids, such as 15:0 and 17:0, generally have a branched structure rather than straight chain structure which were usually found in bacteria. Amphimedon terpenensis contains both cyanobacterial and bacterial symbionts and thus an additional consideration is which of the three cell types, sponge, bacteria, or cyanobacteria contains the brominated fatty acids [69].
Terpenes biosynthesis is under investigation as a rearranged diterpenoid diisocyanoadociane and was isolated from the Amphimedon sp. that was collected at the Great Barrier Reef in Australia [60]. Terpenes biosynthesis could be explained by the sponge-associated microorganisms which enable the enzymes to synthesize isoprenoids or alternatively, from the modification in a sponge mediated cell of a symbiont-produced precursor such as isoagelaxanthin [70]. Cyanide incorporation by the Amphimedon sp. was first established by Karuso and Scheuer [71] and demonstrated that these sponges utilize carbon and nitrogen from the cyanide precursor. The role of the symbionts may be incorporated in the biosynthesis of isonitriles as the production of diisocyanoadociane from Amphimedon sp. in high rates is due to the cyanide production by the symbionts utilization in the sponges or, a metabolic process which can proceed without symbionts.
An important class of metabolites that were reported from the genus Amphimedon are manzamine alkaloids owing to their fascinating biological activities. Manzamine alkaloids were biogenetically synthesized from the condensation of ircinals A or B from the icrinia sponge [48]. Keramaphidin B, isolated from Amphimedon sp., might be the biogenetic precursor of the ircinals and may be produced by hydrolysis of the N2/C3 bond of the 2, 3-imino form of keramaphidin B. In the continuous search for biogenetic precursors of manzamine alkaloids keramaphidin C and keramamine C might be plausible biogenetic precursors of manzamine C, especially considering that keramaphidin C is an important intermediate in the biogenetic pathway of manzamine C generation.
Several Amphimedon metabolites were targets for chemical synthesis such as nakadomarin A, owing to its interesting biological applications including antibacterial, cytotoxic and antimicrobial properties [72]. Synthesis was carried out by using a thioamide based strategy [73]. Marine diterpenoid diisocyanoadociane is another example that was synthesized by using intramolecular Michael reaction due to its potent antimalarial activity [74].

Conclusions
Marine sponges contain treasures of undiscovered natural metabolites by having a wide spectrum of pharmacological activities. Among the several Amphimedon species discovered, A. viridis, A. compressa, A. complanata, and A. terpenensis are the richest producers of natural products with various pharmaceutically relevant bioactivities. The research on this genus is interesting and has made variable progression over the years. However, in recent years there is increasing interest in studying sponge-associated microorganisms in order to explain this complex relationship and in order to reveal the facts of whether metabolites originate mainly from the sponge or from their associated microorganism(s). One hundred and four natural products from various marine sponges belonging to the genus Amphimedon were reported in literature as well as in the Marinlit databases until 2018. Undescribed species of the sponge represent the highest source of secondary metabolites and thus, the discovery of new species of this sponge indicate that there are still possibilities in isolating new secondary metabolites with predicted strong pharmacological activities. The genus Amphimedon and its associated microorganism(s) showed their potential to produce great diversity of chemical leads from the different chemical classes. Alkaloids showed the highest distribution among the different chemical classes followed by fatty acids and then ceramides and cerebrosoides and finally, the lowest percent were the macrocyclic lactones/lactams, as shown in Figure 26. Among the various types of alkaloids pyridine type alkaloids and manzamine alkaloids were the highest followed by purine alkaloids and finally 3-alkyl pyridine glycosides, as shown in Figure 27. These chemical leads exhibited a huge diversity of bioactivities such as antibacterial, antifungal, and anticancer activities.   Amphimedon and its associated microorganisms have a broad range of pharmacological activities and, therefore, we must carry out further investigations for the genus and their associated microorganisms as they play an important role in the discovery of new natural products.