Alcyonacea: A Potential Source for Production of Nitrogen-Containing Metabolites

Abstract

Alcyonacea (soft corals and gorgonia) are well known for their production of a wide array of unprecedented architecture of bioactive metabolites. This diversity of compounds reported from Alcyonacea confirms its productivity as a source of drug leads and, consequently, indicates requirement of further chemo-biological investigation. This review can be considered a roadmap to investigate the Alcyonacea, particularly those produce nitrogen-containing metabolites. It covers the era from the beginning of marine nitrogen-containing terpenoids isolation from Alcyonacea up to December 2018. One hundred twenty-one compounds with nitrogenous moiety are published from fifteen genera. Their prominent biological activity is evident in their antiproliferative effect, which makes them interesting as potential leads for antitumor agents. For instance, eleutherobin and sarcodictyins are in preclinical or clinical stages.

1. Introduction

The marine biota is characterized by living under harsh environmental conditions (e.g., high salinity, variable pressures, hydrothermal vents and variable nutrient accessibility) [1,2]. The marine invertebrates, particularly soft corals, suffer from absence of mechanical defenses [3,4,5]. They produce chemical agents to maintain their life. These defenses can be represented by production of secondary metabolites that possess ecological functions including anti-predatory protection, thus several natural metabolites were discovered from marine sources [6]. Most of these substances have unprecedented structures with diversity of pharmacological application [7,8,9,10,11,12,13]. Alcyonacea comprises marine invertebrates that live ubiquitously in tropical sea waters, particularly intertidal zones or inner reefs below the stony corals, and are less prone to damage from collecting or shipping than the stony corals [14]. They are animals, provide stinging cells in the form of toxic stinging nematocysts with absence of the rigid protective skeleton of scleractinians, and possess allelopathic capabilities of chemical production [15,16]. This enables the sessile corals to strive and reduce their palatability, by reducing fouling effect through producing chemical substances, mucus or terpenoids, aiming at protecting them against predators [17,18,19]. Alcyonacea is well known for the production of terpenoids, while the occurrence of nitrogenous terpenoidal derivatives is rare. The bio-synthetic pathway of the nitrogenous moiety is unclear [6,20,21]. Conclusively, Alcyonacea has conceivable therapeutics, which includes immunomodulator, anticancer and useful antifouling agents.

Amazingly, 40% of the chemical frameworks that appear in different databases are natural compounds. Approximately, half of new drugs recently reported are of natural origin or constructed based on natural architectures [22]. A comparative analysis study indicates that marine products are superior to earthly metabolites in terms of chemical uniqueness [23]. An investigation has led to establishing that 70% of metabolites that appear in Dictionary of Marine Natural Products (DMNP) are completely exploited by marine invertebrates. Additionally, marine drugs have successfully launched in the market, and others are still in different phases of clinical trials. A recent review reports the marine pharmaceutical products [24,25,26].

In 1969, FDA approved cytarabine as an anticancer agent, while vidarabine was permitted in 1976 as an antiviral agent. Around fourteen years later, ziconotide was approval by FDA for treatment of severe chronic pain. Trabectedin was approved from FDA in 2010 for metastatic breast cancer [25]. On their way to the market, another twelve marine-derived drugs are being clinically investigated [26]; for instance, the bryostatin and dolastatin derivatives, soblidotin and synthadotin, respectively. A tricyclic diterpenoid, eleutherobin, has been reported from Eleutherobia sp., with potent inducer of tubulin polymerization in vitro, which mimics taxol-like effect [25,26,27,28].

In the current review, marine nitrogen-containing metabolites isolated from Alcyonacea are presented. These compounds show significant effects toward certain diseases and/or have a role in drug discovery. It is interesting to discuss the future perspectives of the structure–activity relationship of these metabolites. An extensive bibliographic literature survey was conducted employing different scientific databases including Scopus, Pubmed, SciFinder, Google scholar and Web of Science.

2. Chemical Constituents

2.1. N-Containing Sesquiterpenes

Sesquiterpenes are a class of widely distributed metabolites among marine soft corals. Although nitrogen-containing sesquiterpenoids are rare, they are produced by certain soft corals (e.g., Alcyonium sp.) [20]. Illudalane-type sesquiterpenoids (

Table 1. Bioactivity of nitrogen containing metabolites from Alcyonacea.

Cpd. No.	Species	Bioactivity	Ref. No.
1–8	Alcyonium paessleri	Cytotoxic	[20]
9	Cladiella sp.	Not published data	[21]
10–12	Cespitularia taeniata	Cytotoxic	[31,32]
13–15	Cespitularia taeniata	Cytotoxic	[33]
16	Eleutherobia sp.	Cytotoxic	[34]
17–22	Erythropodium caribaeorum	Cytotoxic	[35,36,37,38,39]
23–28	Sarcodictyon roseum	Cytotoxic	[40,41,42]
29–33	Alcyonium valdivae	Anti-inflammatory	[43]
34–35	Eleutherobia aurea	Cytotoxic	[44]
36–37	Erythropodium caribaeorum	Cytotoxic	[36]
38–50	Cespitularia taeniata	Cytotoxic and Antimicrobial	[45,46,47]
51	Pseudopterogorgia elisabethae	Cytotoxic	[48]
52–54	Pseudopterogorgia elisabethae	Cytotoxic	[49,50]
55	Pseudopterogorgia elisabethae	Cytotoxic	[51,52,53,54,55,56,57]
56–58	Sinularia sp.	Antiproliferative	[58,59]
59	Lobophytum sp.	Cytotoxic	[60,61]
		HIV inhibitory
60	Pseudopterogorgia acerosa	Cytotoxic	[62,63]
61	Pseudopterogorgia bipinnata	Antimalarial	[64]
62	Pseudopterogorgia acerosa	-	[65]
63	Pseudopterogorgia acerosa	-	[66]
64	Pseudopterogorgia spp.	Cytotoxic	[67,68]
65	Lobophytum sp.	Cytotoxic	[69,70,71]
66–70	Echinogorgia pseudossapo	Antiviral	[72]
71–72	Sinularia sp.	Inhibit LPS-induced NO release	[73,74]
73–86	Euplexaura nuttingi and Leptogorgia gilchristi	Cytotoxic	[75,76,77]
87–88	Sarcophyton infundibuliforme	Immunomodulator	[78]
89	Sinularia verruca	-	[79]
90–92	Sinularia flexibilis	Cytotoxic	[80]
93–94	Sinularia crassa	-	[81,82,83,84,85]
95–96	Sinularia leptoclados	Antibacterial	[86]
97–98	Lobophytum sp.	-	[87]
99	Sarcophyton auritum	Anxiolytic effect	[88]
100–102	Pseudopterogorgia australiensis	Antimicrobial	[89]
103–108	Sarcophyton ehrenbergi	Anti-inflammatory	[90]
109–112	Lobophytum sp.	Cytotoxic	[91]
113	Litophyton arboreum	HIV-1 PR inhibitory	[92]
114	Sinularia sp.	Anti-inflammatory	[93,94]
115–116	Sinularia brongersmai	Cytotoxic	[95]
117	Sinularia sp.	Gastric H, K-ATPase and cytotoxic	[96]
118–119	Sinularia sp.	Cytotoxic	[97]
120	Sinularia sp.	Cytotoxic	[98]
121–123	Clavularia viridis	Cytotoxic	[99,100]
124	Sinularia flexibilis	Cytotoxic	[101]
125–126	Eunicella granulate	Cytotoxic	[102]

and Figure 1), have limited distribution in nature and metabolites of this class are exclusively reported from ferns and fungi [29,30]. Congeners of illudalane-type sesquiterpenoids identified as alcyopterosins (B, C, E, F, G, H, J and M) (1–8) are reported from the sub-Antarctic globular spongy pink coral Alcyonium paessleri, gathered around South Georgia Islands. The combined extracts (EtOH and EtOAc) purified by employing different chromatographic techniques led to isolation of the congeners (1–8), which numerated as the first report of nitrogen-containing illudalane type sesquiterpenoids, as well as a first report of nitrate ester to be isolated from marine soft corals [20]. These metabolites represent a new trend in biosynthetic pathway of sesquiterpenoids. All reported sesquiterpenes may have sulfate, phosphate, or halogenic moieties, but finding nitrogen-containing sesquiterpenes is infrequent. Alcyopterosins (1–8) were evaluated for their cytotoxicity towards Human larynx carcinoma (Hep-2) and Human colon carcinoma (HT-29). Alcyopterosins E (5) showed cytotoxicity against Hep-2 with IC₅₀ value of 13.5 µM, while Alcyopterosins C (2) and H (6) showed cytotoxicity against HT-29 with IC₅₀ value of 10 µM [20]. Although nitrate ester derivatives are very rare, some synthetic nitrate esters are published. An acceptable speculation is that the hydrolysis of esters leads to the corresponding alcohols, which play roles in their cytotoxic effects [20].

Cladioxazole (9) represents the first oxazole-derived terpenoid, isolated from Cladiella sp. which, collected from Andaman Island [21]. Unfortunately, no information about the biological activity of 9 has been published. Three sesquiterpene lactams, taenialactams A (10), B (11) and atractylenolactam (12), were isolated from Cespitularia taeniata collected from Taiwan [31]. The methanolic extract of C. taeniata has been partitioned between water and ethyl acetate. The EtOAc-soluble portion has been purified on silica gel columns and HPLC to yield 10–12. Atractylenolactam (12) was previously isolated from the rhizome of a terrestrial plant, Atractylodes macrocephala, a Chinese medicinal plant employed for the treatment of indigestion and anorexia [32]. Compounds 10–12 were evaluated towards KB, WiDr, and Daoy tumor cells, no effects were observed [31]. Fractionation of the organic extract of Taiwanese soft coral Cespitularia taeniata, by employing different chromatographic techniques including HPLC led to identification of ten compounds, three of which are nitrogenous sesquiterpenoids, cespilamides C–E (13–15, Figure 1). Cespilamide E (15) exhibited cytotoxicity against Hela, Daoy, WiDr and MCF-7 cells with IC₅₀ of 24.7, 22.3, 34.1 and 17.5 μM, respectively. Cespilamide E (13) exhibited cytotoxicity against Hela, Daoy, WiDr and MCF-7 cells with IC₅₀ of 30.9, 34.8, 49.5 and 30.6 μM, respectively, while cespilamide D (14) was inactive (>40 μM) [31,33].

2.2. N-Containing Diterpenes

Eleutherobin (16) is unique nitrogen-containing diterpene glycoside, isolated from an Australian soft coral, identified as Eleutherobia sp. (Figure 2). The non-sugar moiety of eleutherobin was escalated by presence of eunicellane nucleus, a diterpene framework that is first reported from the gorgonian octocoral Eunicella stricta [34]. It possesses N-methylurocanic acid ester at the C-8 position, while the sugar moiety is acetyl-arabinose [26,34,35]. Eleutherobin possessed significant cytotoxic effect towards various cancer cells with IC₅₀ range of 10–15 nM. National Cancer Institute measured the selectivity of Eleutherobin (16) on 60 diverse panels of cancer cells. Its potency was estimated as 100-fold more against selected types of cancer cell lines than paclitaxel [36,37,38,39]. Investigation of a Caribbean octocoral, namely Erythropodium caribaeorum, collected from shallow reefs near Dominica, led to isolation of seven compounds: eleutherobin (16), desmethyleleutherobin (17), desacetyleleutherobin (18), isoeleutherobin A (19), Z-eleutherobin (20), caribaeoside (21), and caribaeolin (22) (Figure 2) [36]. The reported pharmacological mechanism of eleutherobin is mimetic to the mode of action observed with paclitaxel [37,38,39]. This appeared through stabilization of microtubules and confirmed by employing the electron microscopy technique [37,38,39].

This class of compounds (nitrogen-containing diterpenes, 16–22), was examined as pharmacophore models for microtubule stabilizing agents. Desacetyleleutherobin (18) possesses the arabinose, instead of 2′acetyl substituent, while isoeleutherobin A (19) has an acetyl group at the 3′ instead of the 2′. Z-eleutherobin (20) is a geometric isomer of eleutherobin at the C-2′ to C-3′ double bond of the C-8 N-(6′)-methylurocanic acid ester side chain. Desmethyleleutherobin (17) differs from eleutherobin (16) by the occurrence of a hydroxyl function instead of an OMe at C-4. Caribaeoside (21) differs from eleutherobin by the presence of a hydroxyl at C-11 of the tricyclic core, and a double bond at C-12 to C-13 instead of C-11 to C-12, significantly altering the cyclohexene ring. Caribaeolin (22) differs from caribaeoside only by the occurrence of a –CH₂OCO-CH₃ substituent in the C-3 terminal chain. Eleutherobin (16) had an IC₅₀ of 100 nM. The activity of Z-eleutherobin (20) was close, with an IC₅₀ of 250 nM. Desmethyleleutherobin (17) and isoeleutherobin A (19) were more potent than eleutherobin, with IC₅₀ of 20 and 50 nM, respectively, while Desacetyleleutherobin (18) was less potent, with an IC₅₀ of 400 nM [35,36,37,38,39].

Sarcodictyins are congeners of eleutherobin and belong to a family of marine-derived diterpenoids with potent antitumor effects. They possess a rigid oxygen-bridged bi­ cyclo[8.4.0]tetradecatriene skeleton [40]. Sarcodictyins A and B (23–24) were isolated from stoloniferan coral Sarcodictyon roseum [41]. A year later, sarcodictyin C–F (25–28) were isolated from the same soft coral [42]. These metabolites showed their cytotoxic effects through tubulin binding in a mechanism similar to Taxol. Sarcodictyins A (23), B (24), C (25) and E (27) were proven to be more potent cytotoxic than the other sarcodictyins. Pharmacia-Upjohn′s researcher (1997) reported that sarcodictyins A–C (23–25) and E (27) exhibited cytotoxicity against Ll 210 murine leukemia (IC₅₀ ranged between 408.5 ± 21.3 and 911.7 ± 393.5 nM; compare with taxol: IC₅₀ = 16.6 ± 5.2 nM). The dose required to promote 90% tubulin polymerization ranged 1.7–6.6 µM, while 4.4 µM for taxol [40]. Five non-nitrogenous sarcodyctins have been isolated from Alcyonium valdivae: valdivone A (29), valdivone B (30), 4-O-Methyl valdivone A (31) 4-O-Methyl valdivone B (32), and dihydrovaldivone A (33) [43]. Compounds 29–30 were shown to inhibit the chemical induced anti-inflammatory. Sarcodictyin A (23) showed lower activity, with an IC₅₀ of 2 μM, while caribaeoside (21) and caribaeolin (22) were considerably less potent, with an IC₅₀ of 20 µM for both compounds [35,36,37,38,39].

Eleuthosides A (34) and B (35), along with sarcodictyin A (23) were reported from Eleutherobia aurea, gathered around Kwazulu-Natal Coast, South Africa [44]. Both 34 and 35 are novel diterpenoidal glycosides, and were proven to have potent microtubule stabilizing effect. The detailed information is registered in a patent with publication Number WO1999021862 A1.

Investigation of another Erythropodium species, E. caribaeorum, led to identification of two unprecedented natural metabolites, namely caribaeorane (36) and 15-hydroxycaribaeorane (37), which are characterized by the presence of C-4 methylketal (Figure 3) [36].

Eleutherobin has potent inducing in vitro tubulin polymerization effect, although its cytotoxicity is less than those obtained from paclitaxel [35,36,37,38,39,44]. P-glycoprotein is a target substance of Eleutherobin, similar to paclitaxel. Both showed cross-resistance in MDR1-expressing lines. Sarcodictyins are reported from Sarcodictyon roseum and seemed to be more promising than eleutherobin, despite their lower effects. Eleutherobins and sarcodictyins have been extensively modified by employing conventional and combinatorial chemistry techniques, which have also allowed the formation of hybrid molecules of the two base structures. These investigations indicated their SARS (Figure 4) [36,44].

The side chain and the imidazole ring are important.

Both OH and OCH₃ groups are tolerated, with little difference in effects.

Elimination or alteration of the aglycone moiety of eleutherobin changes the cytotoxicity and resistance pattern.

Sarcodictyin esters are more potent than amides.

Cespitulactams A–C (38–40, Figure 5) are reported from Cespitularia taeniata, collected around Taiwan [45]. The obtained metabolites are nitrogenous tricyclic derivative, close to taxane diterpenes, particularly 3, 8-seco-taxoids and an infrequent N-phenylethyl-butyrolactamyl moiety. Four cancer cell lines, WiDr and Daoy, KB and Hepa59T/VGH, were tested. Cespitulactam A (38) exhibited potent toxicity against Widr and Daoy cancer cells (IC₅₀ = 2.72 and 6.34 μg/mL, respectively) [46]. Further investigation of the organic extract of Cespitularia taeniata led to identification of eight more nitrogenous verticillene diterpenoids, cespitulactams (D–K) (41–48) [47]. These substances were evaluated against KB and murine L1210 leukemia. Compound 48 was active towards both cancer cells at 8.49 and 11.7 µM, respectively, and no effect was observed with the rest. Diterpene alkaloids (41–48) have been assayed for antimicrobial effects towards several microorganisms. Cespitulactam G (44) showed potent effect towards Trichophyton mentagrophytes (IFM45110) with MIC value of 11.56 µM. Compounds 41, 47, and 48 displayed significant effect towards Micrococcus luteus (IFM2066) and Cryptococcus neoformans (IFM46914) (46, 47 and 48) with MIC values of 12.53, 9.06 and 9.54 µM, respectively, and T. mentagrophytes (42 and 47) with MIC values of 11.62 and 9.06 µM. Unfortunately, all isolated Cespitulactams (41–48) were inactive against Staphylococcus aureus (209P) [46]. Another investigation on Cespitularia taeniata led to identification of two nitrogenous verticillene (Figure 5), cespilamides A and B (49 and 50). Neither compound exhibited cytotoxicity against Hela, Daoy, WiDr, or MCF-7 at concentration >40 μM [34,47].

Diterpenoidal alkaloid, elisabethamine (51), has a serrulatane nucleus with methyl amino functionality, isolated from Pseudopterogorgia elisabethae [48]. Elisabethamine (51) had significant effect against LNCap and Calu cancer cell lines (IC₅₀ = 10.35 and 20 μg/mL, respectively) [48]. Further investigation of the organic extract of Pseudopterogorgia elisabethae afforded pseudopteroxazole (52) and seco-pseudopteroxazole (53), having amphilectane skeleton and the uncommon benzoxazole function [49]. One more amphilectane derivative homopseudopteroxazole (54) was isolated from Pseudopetrospongia elisabethae collected from the Western Atlantic Ocean [50]. Pseudopteroxazole (52) showed potent inhibitory effect (97%) against Mycobacterium tuberculosis H37Rv at 40.45 µM, whereas seco-pseudopteroxazole (53) inhibited 66% of mycobacterial growth. Therefore, the potency effect of 52 might be attributed to the benzoxazole function. Remarkably, biological screening of 52 against 60 cancer cell lines designated potent in vitro cytotoxicity. Several approaches for syntheses of pseudopteroxazole are published [51,52,53,54]. Another diterpene, ileabethoxazole (55), was obtained as a light yellow oil from Pseudopterogorgia elisabethae collected near Providencia Island (Figure 5). It represents novel diterpenes carbon skeleton (Ileabethane), which appears to be biosynthetically related to the serrulatane skeleton. It displayed strong inhibitory effect against M. tuberculosis H37Rv [55,56,57].

Three new nitrogen containing cembranolide diterpenes, sinularamine I (56), sinularamine II (57), and sinularamine III (58), each possessing a dimethylamino group, were isolated from the Okinawan soft coral species of Sinularia genus and their structures were elucidated by spectroscopic analyses and chemical transformations [58]. Compounds 56 and 58 inhibited the proliferation of KB cells at concentrations of 2.0 and 1.65 µg/mL, respectively [58,59]. Modified structures of sinularamines I–III were isolated from Okinawan soft coral of Sinularia species [59,60].

Investigation on Lobophytum sp., collected from Philippine Islands, led to identification of a cembranoid diterpene (17-dimethylaminolobohedleolide, 59), which blocked the cytopathic effect of in vitro HIV-1 infection in a cell-based assay with EC₅₀ = 8.78 µM [61].

Tinto et al. (1991) reported seven pseudopteranoids isolated from Pseudopterogorgia acerosa, including the rare bisditerpenoid amine bis(gorgiacerol)amine (60). It showed selective growth inhibition activity against cancer cell lines (HCT116 and HeLa) [62,63].

Caucanolide B (61), a possible pesudopterane-type diterpenoid successor through oxidation cleavage at C-2/C-3 together with five rare diterpenes, was isolated from Pseudoptergorgia bipinnata collected near the Colombian Southwestern Caribbean Sea. These caucanolides were evaluated against the malaria parasite, Plasmodium falciparum. Unfortunately, Caucanolide B showed no significant activity, although it is the only example from nature of a secondary metabolite possessing the N1,N1-dimethyl-N2- acylformamidine functionality [64].

Fenical and Clardy (1982) examined the constituents of Floridian specimens of Pseudopterogorgia acerosa, leading to the isolation of pseudopterolide, a remarkable metabolite based on the 12-membered carbocyclic pseudopterane skeleton [64]. Further examination of extracts of several Pseudopterogorgia spp. has since resulted in the isolation of other pseudopterane metabolites, many of which possess chemically unique structural features (Figure 6). Tobagolide (62), one such metabolite, is a rare nitrogen-containing diterpenoid isolated from a Trinidadian specimen of Pseudoptergorgia acerosa [65]. Another examination of Puerto Rican specimens of Pseudoptergorgia acerosa led to isolation and structural determination of alanolide (63), a novel tetracyclic norditerpene, which appears to be biogenetically related to tobagolide [66]. The structure of aceropterine (64), the first pseudopterane with a transposed lactone moiety, is closely related to that of tobagolide, which was isolated from a Trinidadian specimen of Pseudoptergorgia acerosa [67,68].

2.3. Terpenoidal Alkaloids

Zoanthamine-type alkaloid (Lobozoantamine, 65) was identified from an Indonesian coral, Lobophytum sp. It belongs to a unique class of alkaloids, the precursor type of which is still ambiguous, whether it is a triterpenoid or a polyketide [68]. The first member of the zoanthamine-type alkaloid was isolated from Zoanthus sp. [69,70], followed by a series of analogs isolated from the genus Zoanthus, with the single exception of zooxanthellamine, which was isolated from the unicellular dinoflagellate Symbiodinium sp. [71]. Lobozoanthamine (65) was evaluated against AGS and C6, and showed cytotoxic effect with IC₅₀ value > 50 μM on both cell lines.

Five zoanthoxanthin alkaloids (Figure 7) were reported from Chinese Sea gorgonian Echinogorgia pseudossapo, pseudozoanthoxanthins III (66), pseudozoanthoxanthins IV (67), Zoanthoxanthin (68), Paragracine (69) and Zoanthoxanthin (70) [72]. In vitro antiviral activity of 66–69 against HSV-1 was evaluated using plaque reduction assay. The completely non-toxic concentration of 66–69 and positive control ACV on Vero cells were tested to be 270.3, 523.6, 185.2, 195.3, and >7500 μM by MTT assay, respectively. Further antiviral studies, employing concentrations lower than the tested non-toxic concentration values. Compounds 66–69 exhibited anti-HSV-1 activity with EC₅₀ values of 108.1, 471.2, 70.4, 117.2, and 6.08 μM, respectively. The results suggest that the side chain at the nitrogen N(3) in 66–69 could affect their antiviral activity. Although 66 and 68 showed mild anti-HSV-1 activity, their effects were less than that obtained with the positive control [72].

Sinulasulfoxide (71) and sinulasulfone (72), two alkaloids with sulfur moiety, were reported from Indonesian Sinularia sp. [73]. Their skeleton is characterized by presence of a phytanic acid moiety connected to sulfated diterpene through an amide bond. Among marine metabolites, the 2-methylsulfinyl ethanamine moiety infrequently occurs in nature; however, it was isolated by Piper [74]. Another example, psammaplin N, was isolated from sponge Aplysinella rhax [72]. An analog, 3-methylsulfinyl propanamine (decarboxylated methionine sulfoxide), was also reported from the same sponge [74]. Both compounds were evaluated for anti-inflammatory activity. Sinulasulfoxide (71) showed moderate inhibition of NO₂ production (22.6%) [72].

Nuttingins A–F (tetraprenylated purine alkaloids, 73–78) and malonganenones D–H (82–86) were reported from Euplexaura nuttingi [75], while malonganenones, A–C (79–81) were previously reported from Leptogorgia gilchristi [76]. Nuttingins A–E (73–77) and malonganenones D–G (82–84) showed inhibitory effect towards K562 and UT7 cancer cells (Figure 6).

Compounds 73–77 and 82–86 also induced apoptosis in transformed mammalian cells at 1.25 µg/mL [5,76]. Nuttingin A (73) is the first 3, 7-disubstituted hypoxanthine, while Nuttingin B (74) and Nuttingin C (75) represent the first formamides identified from soft corals. The anti-esophageal cancer effect of Nuttingins A–C (73–75) was compared with that of rietone, which was previously identified from L. gilchristi. Nuttingins A–C (73–75) were assessed towards several tumor cells (WHCO1, WHCO5, WHCO6, KYSE70, KYSE180, and KYSE520). Compounds 73–75 showed moderate cytotoxicity against seven cancer cells. Remarkably, Nuttingin A (73) exhibited toxicity against WHCO1 cells comparable with cisplatin (IC₅₀ =15 μM). The anti-microbial effect of Nuttingins A (73) was evaluated against Staphylococcus aureus, Escherichia coli, and Aspergillus niger (20 and 100 mg/disk). Nuttingins A–E (73–77) showed no effect against E. coli and A. niger, while showed mild effect against S. aureus (100 mg/disk) [5,77].

Two unprecedented nitrogenous diterpenoidal metabolites, sarinfacetamides A (87) and B (88), were reported from the Chinese Sarcophyton infundibuliforme [78]. These compounds are members of uncommon tricycle-dodecane scaffold with an acetamide group (Figure 6). Compounds 87 and 88 were evaluated for different biological effects, including cytotoxicity and immunomodulatory effects. Unfortunately, when evaluated against HL-60, K562, MGC-803, BEL-7402, SH-SY5Y, HCT-116, MDAMB-231, A549, MCF-7/ADM, HO8910, U87, and NCIH1975 cell lines, they showed no effects at 10 μM. Interestingly, in the immunological assay, compound 87 was found to moderately promote the ConA-induced T lymphocytes at 10 μM with proliferation rate of 36.18% [78].

A rare pyrroloindoline alkaloid verrupyrroloindoline (89) was isolated from Sinularia verruca. It showed no protection towards the cytopathic effects of HIV-1 infection and no effect was observed against LPS-induced NO production [79].

4-(2-aminoethy1)-2-methoxyphenol (90), 5-(2-aminoethy1)-2-methoxyphenol (91) and (2)-3-methyldodec-2-enoic acid (92), three tyramine derivatives, were isolated from the soft coral Sinularia flexibilis (Figure 7). It is worth mentioning that total synthesis was the main tool for structural elucidation of these amides [80]. Compound 90 displayed potent anti-inflammatory effect in the inhibition of superoxide generation and elastase release in fMLP/CB-induced human neutrophils [80].

2.4. Ceramides and Cerebrosides

Cerebrosides are a group of metabolites holding a ceramide moiety and sugar units [81]. They have been published from different invertebrates including, sponges [82], sea anemones, [83], ascidians [84] and soft corals [85,86,87,88,89,90,91,92,93,94].

Investigation of Sinularia crassa led to identification of two new sphingosine derivatives, N-hexadecanoyl-1,3-dihydroxy-2-amino-4,8-octadecadiene (93) and N-heneicosanoyl-1,3,4-trihydroxy-2-aminotetradecane (94) [85]. Two sphingolipids, (2S,3S,4R)-1,3,4-trihydroxy-2-[((R)-2′-hydroxytetradecanoyl) amino] tricosane (95) and (2S,3S,4R)-1,3,4-triacetoxy-2-l((R)-2′–acetoxy octadecanoyl) amino] octadecane (96), were isolated from Sinularia leptoclados (Figure 8). They exhibited mild antibacterial effect against Gram-negative bacteria, while no activity was found against Gram-positive bacteria tested [86].

Investigations of Lobophytum sp. yielded a ceramide: (2S,3S,4R)-2-[(R)-2′-hy-droxytricosanoyl amino]-1,3,4-tridecanetriol (97). The length of the sphingosine chain of the fatty acid was determined by methanolysis, followed by acetylation of sphingamines to give 98 [87].

Investigation of Sarcophyton auritum led to identification ceramide N-((2S,3R,4E,6E)-1,3-dihydroxyhenicosa-4,6-dien-2-yl) tridecanamide (99), which showed antagonized effect the lethality of pentylenetetrazole in mice. In addition to this effect, compound 99 showed significant anxiolytic effect as well as CNS depressing activity, possibly through GABA and serotonin receptor modulation [88].

Three sphingosines, (2S,3R)-2-(docosanoyl amino)nonadecane-1,3-diol (100) and (2S,3S,4R)-2-[(29R)-29-hydroxynonadecanoylamino]nonadecane-1,3,4-triol (101), along with the known (2S,3R,4E)-2- (heptadecanoylamino)octadec-4-ene-1,3-diol (102), were isolated from Pseudopterogorgia australiensis collected from the Tuticorin Coast, India (Figure 7) [89]. Compounds 100–102 showed moderate antibacterial activity against Gram-positive bacteria Bacillus pumilis, B. subtilis, and S. aureus and Gram-negative bacteria E. coli, Proteus vulgaris, and Pseudomonas aeruginosa. Unfortunately, no significant effect was observed towards Candida albicans or Aspergillus niger [89].

Investigation of Sarcophyton ehrenbergi collected from water around Taiwan led to cerebrosides sarcoehrenoside A (103), sarcoehrenoside B (104), and known ceramide (105), along with three known cerebrosides (106–108) [90]. Sarcoehrenoside A (103) has a characteristic β-glucose moiety, which differs from previous reported marine cerebrosides (Figure 8). Compounds 103–108 were evaluated toward their antimicrobial effects including Enterobacter aerogenes, Serratia marcescens, Salmonella enteritidis, Yersinia enterocolitica and Shigella sonnei. Unfortunately, they showed no antimicrobial effect (100 μg/disk). However, in-vitro anti-inflammatory effects of 103–108 were estimated [90]. Both 103 and 108 reduced the levels of iNOS to 46.9 ± 9.7% and 20.3 ± 6.8%, respectively, and of COX-2 to 77.2 ± 9.9% and 64.3 ±8.6%, respectively. Compounds 104, 105, and107 reduced iNOS protein expression to be ranged 25.8- 47.3%, did not inhibit COX-2 protein expression [90].

Four sphingolipids were isolated from Lobophytum sp. (Figure 9), identified as (2S,3S,4R)-2- nonadecanoylamino-octadecane-1,3,4-triol (109), (2S,3R,4E,8E)-[(2′R)-2′-hydroxyhepta decanoylamino]-4,8-octadecadiene-1,3-diol (110), 1-O-(β-d-glucopyranosyl)-(2S,3R,4E,8E)-2-[(2′R)-2′-hydroxynonadecanoyl amino]-9-methyl-4,8-octadecadiene-1,3-diol (111), and (2S,3R,4E,8E)-2-hexadecanoyl amino-4,8-octadecadien-1,3-diol (112). All these sphingolipids were evaluated for toxicity against human Peripheral Blood Mononuclear Cells (PBMC) [91].

Bioactivity directed purification of the Litophyton arboreum extract led to the identification of erythro-N-dodecanoyl-docosasphinga-(4E,8E)-dienine (113) and showed inhibitory effect against HIV-1 PR (IC₅₀ = 4.80 ± 0.92 μM). Compound 113 showed potent HIV-1 PR inhibitory effect in the absence of cytotoxicity against the tested cell lines [92]. Compound 113, which was previously isolated from Anemonia sulcata, showed cytotoxicity (ED₅₀ of 37.31 μM [83].

Anti-inflammatory cerebroside (114) was isolated from Indonesian Sinularia sp. [93]. The ESI-MS spectrum of 114 indicated it to be a mixture of homologs (Figure 8). Micro-scale chemical degradation of 114 allowed indicating the nature of the fatty acid moieties present [93,94]

2.5. Miscellaneous Nitrogen-Containing Metabolites

Spermidine is a polyamine found in ribosomes and living tissues. It has various metabolic functions within organisms and was isolated originally from semen. Spermidine is commonly used for in vitro molecular biology reactions, particularly in vitro transcription by Phage RNA polymerases, in vitro transcription by human RNA polymerase II, and in vitro translation. Spermidine increases specificity and reproducibility of Taq-mediated PCR by neutralizing and stabilizing the negative charge on DNA phosphate backbone. Spermidine is, at physiological pH, a polycationic reagent that aids in enzyme digestion by forcing apart DNA molecules. Two cytotoxic spermidine derivatives (115 and 116) (Figure 9) were reported from Pacific Sinularia brongersmai [95]. Sinulamide (117), a tetraprenylatedspermine derivative, was identified from Japanese Sinularia sp. Sinulamide (117) not only inhibits H, K-ATPase with an IC₅₀ value of 5.5 µM, but also is cytotoxic against L1210 and P388 with IC₅₀ values of 3.1 and 4.5 µg/mL, respectively [96].

Two N-methylated spermidine amides, 118 and 119, were isolated from Sinularia sp. [97]. An acylated spermidine (120) was identified from Sinularia sp. and was found to be cytotoxic against P-388 cells (ED50 0.04 µg/mL) [98].

A pyrazine congener, clavulazine (121), was first isolated from isolated from Okanawan Clavularia viridis [99], along with two marine pyrazine derivatives, Clavulazols A (122) and B (123) [100].

A unique cinnamide dimer (124) was isolated from Sinularia flexibilis [101]. Granulatamides A (125) and B (126) and a tryptamine derivative (Figure 10) were isolated from the Eunicella granulate (Figure 10). The cytotoxic effects of 125 and 126 were evaluated against certain cancer cell and GI₅₀ values were in ranges 1.7–12.7 and 3.5–13.8 µM, respectively [102].

3. Summary and Conclusions

Natural products possess a characteristic chemical spatial orientation. This enables them to interact with their biological targets, which validates initial points for drug discovery. Recently, half of new drugs reported are naturally occurring or constructed on the basis of natural chemical frame. Forty percent of the bioactive compounds are natural metabolites and appear in the Dictionary of Natural Products. Chemical novelty of marine products is superior to terrestrial metabolites. Approximately 70% of the molecular skeletons that appear in databases are produced by marine organisms. Additionally, marine drugs have successfully been purchased and others are in different clinical phases.

Alcyonacea will be considered as potential source of bioactive nitrogen containing metabolites. The engagement of different approaches played a significant role in the facilitation of the forthcoming drug discovery process. Noteworthy, many marine metabolites displaying fascinating molecular structures with diverse pharmacological effects have been reported from Alcyonacea during the last four decades (1978–2018). Of the 121 distinctive structures accounted for in this review, 61 (50.4%) are nitrogen-containing terpenoidal metabolites. Figure 11 illustrates the number of nitrogenous metabolites, as reported from 44 species. These species belong to six genera, as illustrated in Figure 12.

Figure 13 illustrates nitrogenous metabolites produced by 15 genera. The five most productive genera are: Cespitularia, Expeuxaura, Lobophytum, Pseudopterogorgonia and Sinularia, with 19 (15.7%), 11 (9%), and 10 (8%), 13 (9.9%) and 21 (17.3%), respectively.

A remarkable 121 metabolites reported from Alcyonacea are discussed in the current review. Of these distinctive structures, 121 (100%) are nitrogen-containing terpenes. The major classes of nitrogen-containing metabolites produced by Alcyonacea are sesquiterpenes, diterpenes and ceramides. The estimated analysis per class is as follows: 46 (38%) are diterpenes, 15 (12.4%) are sesquiterpenes, 24 (19.8%) are alkaloids and the remaining 36 (28.8%) are spermidins, cerebrosides, and ceramides, as appeared in

Table 2. Distribution of nitrogen-containing metabolites isolated from soft corals by skeletal class.

Organism	Compounds	Chemical Class
Alcyonium paessleri	1–8	Illudalane-sesquiterpenoid
Cladiella sp.	9	Oxazole-derived sesquiterpene
Cespitularia taeniata	10–15	Eudesmane-sesquiterpenoid
	38–50	Verticillane-diterpenoid
Eleutherobia sp. and Eleutherobia aurea	16,34, 35	Eunicellane-diterpenoid
Erythropodium caribaeorum	17–22, 36, 37	Eunicellane-diterpenoid
Sarcodictyon roseum	23–28	Eunicellane-diterpenoid
Pseudopterogorgia elisabethae	51	Serrulatane-diterpenoid
	52–54	Amphilectane-diterpenoid
	55	Ileabethane-diterpenoid
Pseudopterogorgia acerosa	60	Pseudopteranoids bisditerpenoids
Pseudopterogorgia bipinnata	63–64, 61	Pesudopterane-diterpenoid
Pseudopterogorgia sp.	62	Pesudopterane-diterpenoid
Pseudopterogorgia australiensis	100–102	Ceramides
Sinularia sp.	56–58	Cembranoid-diterpenoid
	71–72	Geranylgeraniane-diterpenoid
Sinularia verruca	89	Pyrroloindoline alkaloid
Sinularia crassa, Sinularia leptoclados, and Sinularia sp.	93–94,114	Ceramides and cerebrosides
Sinularia brongersmai and Sinularia sp.	115–120	Spermidines
Sinularia flexibilis	90–92	Tyramine derivatives
Lobophytum sp.	59	Cembranoid-diterpenoid
	65	Zoanthamine-type alkaloid
	88–112	Ceramides and cerebrosides
Litophyton arboreum	113	Ceramides
Echinogorgia pseudossapo	66–70	Zoanthoxanthin alkaloids
Euplexaura nuttingi	73–78	Hypoxanthine
Leptogorgia gilchristi	79–86	Hypoxanthine and Seco-Hypoxanthine
Sarcophyton infundibuliforme	87–88	Tricyclic dodeca-diterpenoid
Sarcophyton auritum	99	Ceramides
Sarcophyton ehrenbergi	103–108	Ceramides and Cerebrosides
Clavularia viridis	124	Spermidines
Eunicella granulate	125–126	Spermidines

.

Diterpenoids, the major division, are in turn further analyzed for each Alcyonacean species, and the distribution by skeleton classes of compounds in this group is shown in

Table 3. Distribution of nitrogen-containing metabolites isolated from soft corals by genus and chemical classes.

Genus	Family	Common N-Containing Chemical Class
Alcyonium paessleri Cladiella sp. Eleutherobia Sinularia Lobophytum Sarcophyton	Alcyoniidae	Illudalane-type sesquiterpenes Oxazole-derived sesquiterpene Eunicellane- diterpenoid Cembranoid-diterpenoid Geranylgeraniane-diterpenoid Pyrroloindoline alkaloid Zoanthamine-type alkaloid Tricyclic dodeca-diterpenoid Ceramides and cerebrosides Spermidines
Pseudopterogorgia Leptogorgia Eunicella	Gorgoniidae	Serrulatane- diterpenoid Amphilectane- diterpenoid Ileabethane- diterpenoid Pseudopteranoids-bisditerpenoids Hypoxanthine and seco- Hypoxanthine Spermidines
Cespitularia	Xeniidae	Eudesmane- sesquiterpenoid Verticillane- diterpenoid
Erythropodium	Anthothelidae	Eunicellane- diterpenoid
Sarcodictyon Clavularia	Clavulariidae	Eunicellane- diterpenoid Spermidines
Euplexaura	Plexauridae	Hypoxanthine

, which summarizes the impressive structural variety of terpenoid carbon skeletons found in these animals. The diterpene skeleton is most frequently elaborated by the genera Pseudopterogorgia, Erythropodium, Sarcodictyon, Lobophytom and Sinularia. These diterpenes have highly functionalized moieties with potent cytotoxicity that could be mimetic the mode of action of taxol. Some of them are currently in clinical trials.