Secondary Metabolites from Gorgonian Corals of the Genus Eunicella: Structural Characterizations, Biological Activities, and Synthetic Approaches

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Review Secondary Metabolites from Gorgonian Corals of the Genus Eunicella: Structural Characterizations, Biological Activities, and Synthetic Approaches

Dario Matulja 1, Maria Kolympadi Markovic 2, Gabriela Ambrožić 2, Sylvain Laclef 3, Sandra KraljevićPavelić 1,* and Dean Marković 1,*

1 Department of Biotechnology, University of Rijeka, Radmile Matejˇci´c2, 51000 Rijeka, Croatia; [email protected] 2 Department of Physics and Centre for Micro- and Nanosciences and Technologies, University of Rijeka, Radmile Matejˇci´c2, 51000 Rijeka, Croatia; [email protected] (M.K.M.); [email protected] (G.A.) 3 Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources (LG2A) UMR CNRS 7378—Institut de Chimie de Picardie FR 3085, Université de Picardie Jules Verne, 33 rue Saint Leu, FR-80039 Amiens CEDEX, France; [email protected] * Correspondence: [email protected] (S.K.P.); [email protected] (D.M.); Tel.: +385-51-584-550 (S.K.P.); +385-51-584-816 (D.M.)

Academic Editor: Paula B. Andrade Received: 9 December 2019; Accepted: 26 December 2019; Published: 28 December 2019

Abstract: Gorgonian corals, which belong to the genus Eunicella, are known as natural sources of diverse compounds with unique structural characteristics and interesting bioactivities both in vitro and in vivo. This review is focused primarily on the secondary metabolites isolated from various Eunicella species. The chemical structures of 64 compounds were divided into three main groups and comprehensively presented: a) terpenoids, b) sterols, and c) alkaloids and nucleosides. The observed biological activities of depicted metabolites with an impact on cytotoxic, anti-inﬂammatory, and antimicrobial activities were reviewed. The most promising biological activities of certain metabolites point to potential candidates for further development in pharmaceutical, cosmetic, and other industries, and are highlighted. Total synthesis or the synthetic approaches towards the desired skeletons or natural products are also summarized.

Keywords: Eunicella genus; eunicellin-type diterpenes; sterols; nitrogenated compounds; cytotoxic; anti-inﬂammatory; antimicrobial; synthetic approaches

1. Introduction Oceans and seas cover more than 70% of the surface of our planet and, therefore, are an extensive and rich source of biotechnological potential [1]. Terrestrial organisms, mainly plants, have been widely explored and utilized to obtain novel compounds with the purpose to develop novel drugs for improved treatment for various diseases [2]. In contrast, species living in the marine environment have traditionally been used as food, contributing to human health as valuable sources of proteins, polysaccharides, amino acids, vitamins, minerals, and other micronutrients [1,3]. More than 30,000 compounds have been isolated from marine organisms since biochemical research began in the second half of the last century [4,5]. Numerous sea bioprospecting programs have been performed with the aim to search for new compounds as target leads in the drug discovery processes, emphasizing on anticancer products with improved performance and/or less severe adverse eﬀects [2,6]. Consequently, since 1969 and the approval of cytarabine, the ﬁrst marine-derived product for the treatment of leukemia, six more

Molecules 2020, 25, 129; doi:10.3390/molecules25010129 www.mdpi.com/journal/molecules Molecules 2019, 24, x 2 of 26 Molecules 2020, 25, 129 2 of 26 the treatment of leukemia, six more compounds have been approved by the Food and Drug Administration (FDA) for clinical use, while dozens of others are currently in different stages of compoundspreclinical and have clinical been approvedtrials [7]. byBesides the Food anticancer and Drug activity, Administration marine natural (FDA) compounds for clinical use,have while also dozensdemonstrated of others neuroprotective, are currently in dianticardiovascular,ﬀerent stages of preclinical antimicrobial, and clinical anti-inflammatory, trials [7]. Besides antioxidant, anticancer activity,antifouling, marine and other natural interesting compounds biological have alsoactivities demonstrated [1]. neuroprotective, anticardiovascular, antimicrobial,Marine sponges anti-inﬂammatory, from the phylum antioxidant, Porifera antifouling, are recognized and other among interesting the most biologicalinteresting activities organisms [1]. in bioprospectingMarine sponges as sources from the of phylum the largest Porifera number are recognizedof so far characterized among the mostmarine interesting metabolites. organisms Other ininvertebrates bioprospecting like ascnidarians sources of include the largest more number than of11,000 so far species, characterized some marineof which metabolites. are poorly Other or invertebratescompletely unexplored like cnidarians [4]. Moreover, include more corals than are 11,000 interesting species, sedentary some of which organisms are poorly that ormay completely survive unexploredharsh internal [4]. and Moreover, external corals marine are environmenta interesting sedentaryl conditions. organisms The main that mayreason survive why harshthey produce internal andvarious external secondary marine environmentalmetabolites is conditions.to protect The themselves main reason from why predatory they produce species various [4,8]. secondary These metabolitescharacteristics is to may protect be themselvesexploited fromin search predatory of novel species bioactives [4,8]. These with characteristics still unexplored may be biological exploited inmechanisms search of novel of action. bioactives with still unexplored biological mechanisms of action. Eunicella coral species belong to the orderorder Alcyonacea,Alcyonacea, familyfamily Gorgoniidae.Gorgoniidae. Gorgonian corals, also known as sea fans and soft corals, are characterizedcharacterized by their branched, fan-like structure which can bebe foundfound on on both both hard hard and and soft soft sea sea bottoms bottoms in the in Mediterranean, the Mediterranean, tropical, tropical, and subtropical and subtropical marine areasmarine [8 ,areas9]. Due [8,9]. to their Due tree-like to their morphological tree-like morp features,hological they features, provide they nutrients provide and nutrients protection and to otherprotection marine to invertebratesother marine and invertebrates vertebrates and [10]. vert Forebrates example, [10]. bacteria For example, of the genus bacteriaEndozoicomonas of the genusare keyEndozoicomonas endosymbionts are withinkey endosymbionts colonies of Eunicella within cavolinicolonies(Figure of Eunicella1) and maycavolini contribute (Figure to 1) the and coral’s may adaptationcontribute to to the the coral’s environment adaptation or to to its the metabolism environment [9,11 or]. to its metabolism [9,11].

Figure 1.1.Eunicella Eunicella cavolini cavolinifrom from the Adriaticthe Adriatic Sea (Rtina, Sea Paška(Rtina, vrata, Pašk Croatia),a vrata, geographical Croatia), geographical coordinates:

Geographicalcoordinates: Geographical longitude 44 ◦longitude190140’, geographical 44°19′14′’, geographical latitude 15◦55 latitude0420’. 15°55′42′’.

Recently, RaimundoRaimundo etet al.al. have analyzed genomes of 15 bacteria from 12 genera, cultivated from Eunicella sp.,sp., andand identified identified gene gene clusters clusters responsible responsible for for the the biosynthesis biosynthesis ofterpenes, of terpenes, polyketides, polyketides, and peptidesand peptides [12]. [12]. The The microorganisms microorganisms might might be also be also involved involved in symbiotic in symbiotic biosyntheses biosyntheses producing producing the ditheff erentdifferent oxygenation oxygenation and unsaturationand unsaturation patterns patter of thens naturalof the productsnatural products originating originating from Eunicella from species.Eunicella Furthermore, species. Furthermore, those symbiotic those organisms symbiotic can organisms be analyzed can by be a metagenomicanalyzed by approach,a metagenomic which allowsapproach, the which bioprospecting allows the of bioprospecting genes encoding of enzymes genes encoding or discovering enzymes of or biocatalysts discovering for of the biocatalysts synthesis offor bioactive the synthesis secondary of bioactive metabolites secondary [13–15 metabolites]. [13–15]. Eunicella cavolini (yellow gorgonian, Koch, 1887) 1887) and and EunicellaEunicella singularis singularis (white(white gorgonian, gorgonian, Esper, Esper, 1791) are thethe mostmost abundantabundant gorgoniangorgonian coralscorals inin thethe MediterraneanMediterranean marinemarine areaarea [[10].10]. The first first is recognized byby itsits orange-coloredorange-colored colonies colonies and and is is widespread widespread from from the the Tunisian Tunisian coast coast to the to Aegeanthe Aegean Sea. ItSea. can It be can found be found up to 150 up m to where 150 m it canwhere form it larger,can fo morerm larger, arborescent more coloniesarborescent due tocolonies the environmental due to the stabilityenvironmental [10,16]. stability Other Eunicella [10,16]. membersOther Eunicella mentioned members in this mentioned review are ingranulata this review(Grassho are granulataff, 1992), verrucosa(Grasshoff,(Pallas, 1992), 1766), verrucosa and labiata(Pallas,(Thomson, 1766), and 1927) labiata [17 (Thomson,]. In addition 1927) to their[17]. potentIn addition biological to their activities, potent highbiological susceptibility activities, to high climate susceptibility change, anthropogenic to climate change, influences, anthropogenic and other influences, abiotic and and biotic other factors,abiotic togetherand biotic with factors, their together slow growth with and their recovery slow growth rates, makeand recovery the Eunicella rates,sp. make A very the interestingEunicella sp. subject A very in diinterestingfferent research subject areas. in different research areas.

Molecules 2019, 24, x 3 of 26

Two previous reviews cover the isolation of bioactive metabolites from gorgonian species between 2009 and 2012 [8,18]. Herein, we summarize advances in isolation and characterization of all known compounds from Eunicella sp. during the last three decades with a focus on the research conducted from 2013 to present. The presented compounds were divided into three groups, i.e., (1) terpenoids, (2) sterols, and (3) alkaloids and nucleosides. Biogenetic pathways involved in the formation of terpenoids from geranylgeranyl diphosphate are also described. In connection to the diversity and complexity of isolated structures, their biological effects are discussed, highlighting cytotoxic, anti-inflammatory, and antimicrobial activities. The most interesting or promising compounds and their biological activities are summarized in Tables 1–3. Moreover, synthetic approaches and total syntheses of isolated metabolites are schematically depicted. Molecules 2020, 25, 129 3 of 26 2. Terpenoids

2.1. BiogenesisTwo previous and Structural reviews cover Analysis the isolationof Eunicellins of bioactive metabolites from gorgonian species between 2009 and 2012 [8,18]. Herein, we summarize advances in isolation and characterization of all known Among all metabolites isolated from gorgonian soft corals, those belonging to the class of compounds from Eunicella sp. during the last three decades with a focus on the research conducted terpenoids exhibited the most significant activities (IC50 values lower than 5 μg/mL) when assayed in from 2013 to present. The presented compounds were divided into three groups, i.e., (1) terpenoids, (2) vitro against tumor cell lines [4]. Diterpenes make up to 65% of all published metabolites from sterols, and (3) alkaloids and nucleosides. Biogenetic pathways involved in the formation of terpenoids gorgonian natural resources [19]. Eunicella sp. are producers of eunicellin-type diterpenoids, i.e., from geranylgeranyl diphosphate are also described. In connection to the diversity and complexity of eunicellin-based diterpenoids from the gorgonian Eunicella verrucosa [20] or from Eunicella cavolini isolated structures, their biological effects are discussed, highlighting cytotoxic, anti-inflammatory, and [21] and Eunicella labiata [22]. These compounds have the cladiellane skeleton and contain four antimicrobialisoprene units activities. linked head The mostto tail interesting (Figure 2). or According promising to compounds Welford et and al., their cladiellins biological are activitiesformed by are summarizedcyclization of in Tablesgeranylgeranyl 1–3. Moreover, diphosphate, synthetic C2–C11 approaches cyclization, and total and syntheses C3–C10 of isolatedtetrahydrofuranyl metabolites areformation schematically from cembrane depicted. scaffold intermediate [23]. Eunicellin (1), shown in Figure 3, possess a tricyclic 6-5-9 membered structure with etheral 2. Terpenoids bridge. The skeleton can be divided into the more oxidized north edge, and the more hydrophobic 2.1.south Biogenesis edge that and communicate Structural Analysis through of a Eunicellins tricyclic structure. Whereas the west part of the molecule is more rigid due to a bicyclic cyclohexyl-tetrahydrofuranyl system, the eastern part is more flexible as it containsAmong an all oxonane metabolites structure. isolated A cyclohexyl from gorgonian ring is fused soft to corals, a tetrahydrofuranyl those belonging ring to through the class C1 of µ terpenoidsand C10 pointing exhibited hydrogens the most syn significant to each other activities and anti (IC50 tovalues the H-atoms lower thanof the 5 etherealg/mL) whenO-C2 and assayed O- inC9 vitro bonds.against Examples tumor of cell deviations lines [4]. of Diterpenes this common make oxygen up tobridge 65% ofcan all also published be found, metabolites from no ether from gorgonianbridge, 2,6-epoxy natural bridge, resources to compounds [19]. Eunicella havingsp. two are epoxy producers bridges. of Angular eunicellin-type methyl groups diterpenoids, at C3 and i.e., eunicellin-basedC11 are anti-periplanar diterpenoids to the from isopropyl the gorgonian group atEunicella C14, whereas verrucosa at [C720] is or placed from Eunicella methylene cavolini group.[21 ] andEunicellinEunicella (1) labiata possess[22]. Thesenine sterocenters. compounds haveC1, theC2, cladiellaneC9, and skeletonC10 form and the contain central four bridging isoprene unitstetrahydrofuranyl linked head to system, tail (Figure and 2still). According C3, C6, C11, to WelfordC12, and et C14 al., cladiellinsare chiral. areFour formed acetoxy by groups cyclization are ofplaced geranylgeranyl on C3, C6, C11, diphosphate, and C12. Eunicellins C2–C11 cyclization, also bear different and C3–C10 oxygenation tetrahydrofuranyl and unsaturation formation patterns from cembranewith one or sca moreffold common intermediate acetoxyl [23]. moieties [22,24–28].

FigureFigure 2.2. Biosynthetic pathway to to cembranoid cembranoid and and cladiellin cladiellin cores cores [23]. [23].

Eunicellin (1), shown in Figure3, possess a tricyclic 6-5-9 membered structure with etheral bridge. The skeleton can be divided into the more oxidized north edge, and the more hydrophobic south edge that communicate through a tricyclic structure. Whereas the west part of the molecule is more rigid due to a bicyclic cyclohexyl-tetrahydrofuranyl system, the eastern part is more ﬂexible as it contains an oxonane structure. A cyclohexyl ring is fused to a tetrahydrofuranyl ring through C1 and C10 pointing hydrogens syn to each other and anti to the H-atoms of the ethereal O-C2 and O-C9 bonds. Examples of deviations of this common oxygen bridge can also be found, from no ether bridge, 2,6-epoxy bridge, to compounds having two epoxy bridges. Angular methyl groups at C3 and C11 are anti-periplanar to the isopropyl group at C14, whereas at C7 is placed methylene group. Eunicellin (1) possess nine sterocenters. C1, C2, C9, and C10 form the central bridging tetrahydrofuranyl system, and still C3, C6, C11, C12, and C14 are chiral. Four acetoxy groups are placed on C3, C6, C11, and Molecules 2020, 25, 129 4 of 26

C12.Molecules Eunicellins 2019, 24, x also bear diﬀerent oxygenation and unsaturation patterns with one or more common4 of 26 Moleculesacetoxyl 2019 moieties, 24, x [22,24–28]. 4 of 26

Figure 3. Eunicellin (1). Figure 3. EunicellinEunicellin ( (11).). 2.2. Structural Diversity of Terpenoids from Eunicella sp. 2.2.2.2. Structural Structural Diversity Diversity of of Terpenoids Terpenoids from Eunicella sp. More than 40 eunicellan-type diterpenes have been isolated from gorgonians so far. However, few MorestudiesMore than have 4040 alsoeunicellan-typeeunicellan-type included evaluation diterpenes diterpenes of have havethe beenbiological been isolated isolated activity from from gorgoniansof gorgoniathese compounds sons far.so far. However, [8,19].However, fewThe fewstudiesfirst studies such have metabolite have also also included was included isolated evaluation evaluation in 1968 of theby of biologicalKennardthe biological et activity al., activitynamed of these eunicellinof th compoundsese compounds (1), having [8,19 [8,19].].four The acetyl The first firstsuchgroups such metabolite attached metabolite was to cladiellane isolatedwas isolated in skeleton 1968 in by1968 Kennard (Figure by Kennard 3) et [2 al.,5]. namedet In al., 1993, named eunicellin Ortega eunicellin et (1 ),al. having reported (1), having four the acetyl fourisolation groupsacetyl of groupsattachedfive new attached toditerpenoids cladiellane to cladiellane 2 skeleton–6 from skeleton (Figurethe methanolic3 (Figure)[ 25]. Inextract3) 1993,[25]. ofIn Ortega Eunicella 1993, et Ortega al. verrucosa reported et al. collected reported the isolation near the isolationthe of five Spanish new of fivediterpenoidscoastal new village diterpenoids2– of6 fromPalmones 2 the–6 from methanolic and the named methanolic extract palmonines of extractEunicella A–Eof Eunicella verrucosa(2–6), after verrucosacollected the sampling collected near the sitenear Spanish of the gorgonian Spanish coastal coastalvillage(Figure ofvillage 4). Palmones The of latter Palmones and diterpenoid named and palmoninesnamed 6 formed palmonines A–E white (2– 6 crystals,A–E), after (2– the 6while), samplingafter the the rest sampling site of of the gorgonian sitecompounds of gorgonian (Figure were4). (FigureTheisolated latter 4). as diterpenoid Thecolorless latter oils.diterpenoid 6 formed Apart fromwhite 6 formed the crystals, acetate white while grcrystals,oups, the they restwhile ofalso the differ compoundsrest ofin thesome compoundswere other isolated structural were as isolatedcolorlessfeatures. as oils.For colorless example, Apart oils. from palmonine Apart the acetate from D ( groups,5the) and acetate E they (6) grare alsooups, two di ff theyisomerser in also some bearing differ other in a structural conjugatedsome other features. ketostructural group For features.example,with an exocyclicFor palmonine example, or D endocyclic (palmonine5) and E ( 6double )D are (5) two andbond, isomers E (respectively6) are bearing two isomers a [20]. conjugated The bearing same keto aauthors groupconjugated withisolated anketo exocyclic another group withoreunicellin-type endocyclic an exocyclic double diterpenoid or endocyclic bond, respectivelyfrom double the samebond, [20 ].octocoral, respectively The same palmonine authors[20]. The isolated Fsame (7), whichauthors another is isolated eunicellin-typea derivative another of eunicellin-typediterpenoidpalmonine B from (3 )diterpenoid but the displaying same octocoral,from one th lesse palmoninesame of acetoxyl octocoral, F (NMR7), whichpalmonine signals. is a derivativeThey F (7 ),also which performed of palmonine is a derivative oxidation B (3) but ofof palmoninedisplayingthe secondary oneB (3 hydroxyl less) but of displaying acetoxyl group NMR ofone palmonine less signals. of acetoxyl They F (7) alsoby NMR Jones performed signals. reagent, oxidation They which also led of performedthe to the secondary semisynthesis oxidation hydroxyl of of thegrouppalmonine secondary of palmonine D (hydroxyl5). F (7 group) by Jones of palmonine reagent, which F (7) by led Jones to the reagent, semisynthesis which led of palmonineto the semisynthesis D (5). of palmonine D (5).

FigureFigure 4.4. Palmonines A–F (22––77)) isolatedisolated byby Ortega’sOrtega’s group.group. Figure 4. Palmonines A–F (2–7) isolated by Ortega’s group. FromFrom 2013 to 2017, Deghrigue’s Deghrigue’s group group and and Io Ioannou’sannou’s group group conducted conducted thorough thorough studies studies on on E. E.singularis singularisFrom and2013and Eunicella toEunicella 2017, cavoliniDeghrigue’s cavolini comprisingcomprising group andpreliminary preliminary Ioannou’s biological biological group conducted ev evaluationaluation thorough of of the organic studies extracts on E. singularisandand fractions.fractions. and Eunicella It was found cavolini that comprising the polar fractionsfraction preliminarys show biological anti-inﬂammatoryanti-inflammatory evaluation and of the analgesic organic potentials, extracts andasas wellwell fractions. asas anti-proliferativeanti-proliferative It was found that eeffectsﬀ ectsthe polar on breast fraction cancercancers show cells anti-inflammatory MCF-7 and prostate and analgesiccancer cells potentials, LNCaP, asindicating well as anti-proliferativethe presence of novel effects bioactive on breast compou cancernds cells [19,29–32]. MCF-7 and The prostate organic cancer extracts cells of theLNCaP, coral indicatingwere prepared the presence by maceration of novel and bioactive were examined compou fornds the [19,29–32]. presence ofThe several organic class extracts of compounds, of the coral i.e., werepolyphenols, prepared alkaloids,by maceration saponins, and were steroids, examined terpenoids, for the presence and glycosides of several byclass qualitative of compounds, chemical i.e., polyphenols, alkaloids, saponins, steroids, terpenoids, and glycosides by qualitative chemical

Molecules 2019, 24, x 5 of 26

Moleculesscreening2020 tests, 25, 129 based on visible chemical change [29,31]. Final purification of fractions resulted5 ofin 26 isolation of 2 terpenoids, palmonines D (5) and F (7), and nine steroids of various chemical structures indicating(Section 2.3., the vide presence infra) [30,32]. of novel Moreover, bioactive Rosa compounds et al. first [ 19isolated,29–32 diterpenoid]. The organic 8 bearing extracts two of acetoxyl the coral weregroups, prepared from the by E. maceration cavolini acetone and wereextract examined (Figure 5) for [21]. the presence of several class of compounds, Molecules 2019, 24, x 5 of 26 i.e., polyphenols, alkaloids, saponins, steroids, terpenoids, and glycosides by qualitative chemical screening tests based on visiblevisible chemicalchemical changechange [[29,29,3131].]. Final purification puriﬁcation of fractions resulted in isolation of 2 terpenoids, palmonines D ( 5) and F ( 7), and nine steroids steroids of va variousrious chemical structures (Section 2.32.3.,., vide infra) [[30,32].30,32]. Moreover, Moreover, Rosa et al. first ﬁrst isolated isolated diterpenoid diterpenoid 8 bearing two acetoxyl groups, from the E. cavolini acetone extract (Figure(Figure5 5))[ 21[21].].

Figure 5. Diterpenoid (8) isolated by Rosa et al. from E. cavolini.

Another Senegalese, deep-water gorgonian coral, E. labiate, was used to extract five diterpenoids with unique structural characteristics from the brown organic extract obtained by maceration of the natural material with dichloromethane and methanol (9:1). Roussis et al. reported the structures of labiatamides A (9) and B (10), as well as labiatins A–C (11–13 ) (Figure 6). These compounds have an isopropyl group andFigure a large 5. Diterpenoidnumber of (8acetoxyl) isolated groups, by Rosa eta al.characteristic from from E. cavolini which. is also found in palmonines, mentioned previously. Labiatamides A (9) and B (10) possess a N-methyl acetamide Another Senegalese, deep-water gorgonian coral, E. labiate, was used to extract ﬁve diterpenoids moiety,Another while Senegalese, labiatin A ( deep-water11) contains gorgonianan unusual coral, ether E. bridge labiate, between was used C2 toand extract C6 [26]. five Furthermore, diterpenoids with unique structural characteristics from the brown organic extract obtained by maceration of the withKakonikos unique et structural al. also isolated characteristics two new labiatinsfrom the D br (own14) and organic E (15) extractfrom E. obtained labiata organic by maceration extract (Figure of the natural material with dichloromethane and methanol (9:1). Roussis et al. reported the structures of natural6). An eunicellin-typematerial with dichloromethanediterpenoid, labiatin and D methanol (14) is a colorless (9:1). Roussis oil and et showed al. reported similar the spectroscopic structures of labiatamidescharacteristics A A as( (99 )palmonine) and and B B ( (1010), E), as ( as6 )well wellwith as asone labiatins labiatins more acetyl A–C A–C (carbonyl11 (11–13–13) (Figure) (Figuremoiety. 6).6 Labiatin). These These compounds E compounds (15), a yellowish have have an anisopropyloil, isopropyl also has group 3 group acetyl and and groups a large a large and number numberspectroscopically ofof acetoxyl acetoxyl isgroups, groups,identical a a tocharacteristic characteristic previously isolatedwhich which is islabiatin also also found C (13 ), in palmonines,with the only mentioned difference beingpreviously. the stereochemistry Labiatamides Labiatamides ofA A hydroxyl ( (99)) and and B group ( 10) possessat C6 position a NN-methyl-methyl [22]. acetamideacetamide moiety,So whilewhilefar, all labiatin mentioned A (11)) containsditerpenoids an unusual contain ether the bridgecladiellane between carbon C2 and skeleton C6 [26]. [26 ].(vide Furthermore, Furthermore, supra). KakonikosHowever, etthere et al. al. also is also an isolated example isolated two of two new a metabolite new labiatins labiatins Dhavi (14 Dng) and ( 14the) E andsame (15) E fromstructure (15) E. from labiata withoutE. organic labiata an organicethereal extract (Figure extractO-C2 (Figure6).and An O-C9 eunicellin-type6). Anbridge. eunicellin-type The diterpenoid, 12,13-Diacetoxycladiella-2,6-dien-11-ol diterpenoid, labiatin D labiatin(14) is a Dcolorless ( 14) is (oil16 a )and colorless was showed isolated oil similar and from showed spectroscopicE. labiata similar by spectroscopiccharacteristicsOrtega et al. in characteristics as 1997 palmonine (Figure as7).E ( palmonineThis6) with secondary one E more (6) metabolite with acetyl one carbonyl more 16 possesses acetyl moiety. carbonyl two Labiatin acetoxyl moiety. E groups(15 Labiatin), a yellowish and E one (15 ), aoil,hydroxyl yellowish also has group. oil, 3 acetyl also The has groups endocyclic 3 acetyl and groups double spectroscopically and bond spectroscopically at C2 positionis identical has is identicalto cis previously stereochemistry, to previously isolated whereas isolatedlabiatin the labiatinC one(13), C(withat 13C6 ),the position with only the difference is only trans di [24].ﬀ erencebeing the being stereochemistry the stereochemistry of hydroxyl of hydroxyl group groupat C6 position at C6 position [22]. [22]. So far, all mentioned diterpenoids contain the cladiellane carbon skeleton (vide supra). However, there is an example of a metabolite having the same structure without an ethereal O-C2 and O-C9 bridge. The 12,13-Diacetoxycladiella-2,6-dien-11-ol (16) was isolated from E. labiata by Ortega et al. in 1997 (Figure 7). This secondary metabolite 16 possesses two acetoxyl groups and one hydroxyl group. The endocyclic double bond at C2 position has cis stereochemistry, whereas the one at C6 position is trans [24].

FigureFigure 6.6. Labiatamides A ( 9) and B B ( (10)) and and labiatins labiatins A–E A–E ( (1111––1515)) isolated isolated from from E.E. labiata labiata. .

So far, all mentioned diterpenoids contain the cladiellane carbon skeleton (vide supra). However, there is an example of a metabolite having the same structure without an ethereal O-C2 and O-C9 bridge. The 12,13-Diacetoxycladiella-2,6-dien-11-ol (16) was isolated from E. labiata by Ortega et al. in 1997 (Figure7). This secondary metabolite 16 possesses two acetoxyl groups and one hydroxyl group.

The endocyclic double bond at C2 position has cis stereochemistry, whereas the one at C6 position is trans [24].Figure 6. Labiatamides A (9) and B (10) and labiatins A–E (11–15) isolated from E. labiata.

Molecules 2020, 25, 129 6 of 26 Molecules 2019, 24, x 6 of 26 Molecules 2019, 24, x 6 of 26

Figure 7. The 12,13-diacetoxycladiella-2,6-dien-11-ol (16) isolated from E. labiata.. Figure 7. The 12,13-diacetoxycladiella-2,6-dien-11-ol (16) isolated from E. labiata. Two gorgonians, E. cavolini and E. singularis, were collected near Marseille and extracted with TwoTwo gorgonians,gorgonians, E.E. cavolinicavolini and E. singularis,, werewere collectedcollected nearnear MarseilleMarseille and and extracted extracted with with ethanol by Mancini et al., leading to the isolation of, in total, 20 compounds (Figure8). Some of them, ethanolethanol by by Mancini Mancini etet al.,al., leadingleading to the isolation of,of, inin total,total, 2020 compoundscompounds (Figure (Figure 8). 8). Some Some of of them, them, like palmonines D (5) and E (6), labiatins B–D (12–14) and eunicellin (1) were already characterized by likelike palmonines palmonines DD ((55)) andand EE ((6), labiatins B–D ((12––1414)) andand eunicellineunicellin ((11)) were were already already characterized characterized other scientiﬁc groups and discussed above (vide supra). Additionally, various oxidation numbers at byby other other scientific scientific groupsgroups andand discussed above (vide supra).supra). Additionally,Additionally, various various oxidation oxidation numbers numbers the 10-membered ring and a second epoxy bridge or a diﬀerently located double bond can be observed atat thethe 10-membered10-membered ringring andand a second epoxy bridge oror aa differentlydifferently locatedlocated doubledouble bond bond can can be be in massileunicellins A–C (17–19)[27,28]. The isolation, conformations, and reactivities of diterpenoids observedobserved inin massileunicellinsmassileunicellins A–C (17–19) [27,28]. TheThe isolation,isolation, conforconformations,mations, and and reactivities reactivities of of 20diterpenoidsditerpenoids–26 isolated 2020 from––2626 E. isolatedisolated cavolini ,fromfrom and diterpenoidsE. cavolini,, andand27 and diterpenoidsditerpenoids28 from E. singularis2727 andand 2828are fromfrom discussed E.E. singularissingularis by Mancini are are etdiscusseddiscussed al. emphasizing byby ManciniMancini the et variouset al.al. emphasizing oxygenation the patterns variouss ofoxygenationoxygenation cyclohexane patternspatterns ring [28 of of]. cyclohexane cyclohexane ring ring [28]. [28].

FigureFigure 8.8. MassileunicellinsMassileunicellins A–C ( 17–19) and diterpenoids diterpenoids (( 2020––2626)) isolated isolated from from E. E. cavolini cavolini, ,as as well wellwell as asas ((27(27,, ,2828 28))) isolatedisolated isolated fromfromfrom E. E.E. singularis singularis.. 2.3. Steroids 2.3.2.3. Steroids Steroids ThereThere have have been been several several reports reports on isolation,on isolation, characterization, characterization, and biologicaland biological evaluation evaluation of various of There have been several reports on isolation, characterization, and biological evaluation of steroidsvarious fromsteroids members from ofmembers the Eunicella of thegenus Eunicella with uniquegenus structuralwith unique features. structural These features. molecules These diﬀer various steroids from members of the Eunicella genus with unique structural features. These inmolecules their degrees differ of in oxygenation,their degrees asof welloxygenation, as side chain as well patterns, as side whichchain patterns, could be which the reason could for be theirthe molecules differ in their degrees of oxygenation, as well as side chain patterns, which could be the miscellaneousreason for their activity, miscellaneous both in vitroactivity, and both in vivo in vitro [19,30 and–35 in]. vivo [19,30–35]. reason for their miscellaneous activity, both in vitro and in vivo [19,30–35]. TheThe acetone acetone extract extract of ofE. E. cavolini cavoliniwas was used used to to isolate isolate a novela novel steroid, steroid, pregna-4,20-dien-11 pregna-4,20-dien-11α-ol-3-oneα-ol-3- The acetone extract of E. cavolini was used to isolate a novel steroid, pregna-4,20-dien-11α-ol-3- acetateone acetate (29), ( a29 pregnane), a pregnane derivative derivative containing containing vinyl vinyl group. group. Besides Besides spectroscopicspectroscopic elucidation, elucidation, the the one acetate (29), a pregnane derivative containing vinyl group. Besides spectroscopic elucidation, the authorsauthors also also presented presented a a chemical chemical synthesis synthesis of of29 29starting starting from from a progesterone a progesterone derivative derivative (Figure (Figure9)[ 359) ]. authors also presented a chemical synthesis of 29 starting from a progesterone derivative (Figure 9) [35]. It should also be mentioned that Kashman et al. extracted verrucoside (30), a pregnane glycoside, [35]. It should also be mentioned that Kashman et al. extracted verrucoside (30), a pregnane glycoside,

Molecules 2020, 25, 129 7 of 26

MoleculesMolecules 2019 2019,, 2424,, xx 7 7of of 26 26 It should also be mentioned that Kashman et al. extracted verrucoside (30), a pregnane glycoside, after studyingafterafter studyingstudying the cytotoxic thethe cytotoxiccytotoxic activity activity of the of DCM the DCM/MeOH/DCM/MeOHMeOH extract extractextract of another ofof anotheranother gorgonian, gorgonian, gorgonian,E. verrucosa E. E. verrucosa verrucosa. This. . 20 compoundThisThis compoundcompound is known isis knownknown for having for having a 6-deoxy a 6-deoxy hexose hexosehexose derivative derivativederivative as a sugar as as a unit,a sugar sugar and unit, unit,∆ and pregnaneand Δ Δ2020 pregnane pregnane skeleton asskeletonskeleton aglycone asas aglyconeaglycone [36]. [36].

Figure 9. Pregna-4,20-dien-11α-ol-3-one acetate (29) and verrucoside (30). Figure 9. Pregna-4,20-dien-11α-ol-3-one acetate (29) and verrucoside ((30).). More pregnanes were extracted from E. cavolini within a comprehensive study by Ioannou et al. More pregnanespregnanes werewere extracted extracted from fromE. E. cavolini cavoliniwithin within a comprehensivea comprehensive study study by by Ioannou Ioannou et al.et al. to to identify new natural compounds from Greek marine organisms in 2008 (Figure 10). Seven identifyto identify new new natural natural compounds compounds from Greekfrom marineGreek marine organisms organisms in 2008 (Figurein 2008 10 (Figure). Seven 10). pregnanes Seven pregnanes 31–37 were reported whose absolute stereochemistries were determined by Mosher’s 31pregnanes–37 were 31 reported–37 were whose reported absolute whose stereochemistries absolute stereochemistries were determined were by determined Mosher’s method. by Mosher’s Their method. Their structures differ in moieties at C3 and C11 positions of pregn-20-ene skeleton having structuresmethod. Their diﬀer structures in moieties differ at C3 in and moieties C11 positions at C3 and of pregn-20-eneC11 positions skeletonof pregn- having20-ene acetoxy, skeleton methoxy, having acetoxy, methoxy, or hydroxy groups. The authors also pointed to the possibility of 34 being the oracetoxy, hydroxy methoxy, groups. or The hydroxy authors groups. also pointed The toau thethors possibility also pointed of 34 tobeing the thepossibility product of reaction34 being with the product of reaction with methanol, a solvent which was used for both extraction and purification methanol,product of a reaction solvent whichwith methanol, was used fora solvent both extraction which was and used puriﬁcation for both [extraction37]. and purification [37]. [37].

Figure 10. StructuresStructures of of pregnanes pregnanes ( (3131––3737).). Figure 10. Structures of pregnanes (31–37). TheThe samesame authors went went on on the the isolation isolation of ofster steroidsoids with with more more comple complexx structures. structures. Six 9,11- Six 9,11-secosterolssecosterolsThe same 38– 43authors38 –and43 and eight went eight 5 onα,8 5 αthe,8-epidioxysterolsα isolation-epidioxysterols of ster 44–oids4451– 51were withwere reportedmore reported comple from from xthe structures. the same same gorgonian gorgonian Six 9,11- speciessecosterolsspecies conservingconserving 38–43 and thethe steroidaleight 5α ,8carbon α-epidioxysterols skeleton skeleton but but having44 having–51 weredifferent diﬀerent reported aliphatic aliphatic from side side the chains chains same (Figure (Figuregorgonian 11). 11 ). ThespeciesThe ﬁrstfirst conserving group of of compounds the compounds steroidal showed carbon showed C9–C11skeleton C9–C11 bond but bond having cleavage cleavage different in tetracyclic in aliphatic tetracyclic nucleus, side nucleus, chains while (Figure the while latter 11). the latterTheone firsthad one highergroup had higherofoxygenation compounds oxygenation levels showed due levels toC9–C11 the due presen bond to thece cleavageof presence an endoperoxide in oftetracyclic an endoperoxide bond. nucleus, Those while characteristic bond. the Those latter characteristiconefeatures had higherare often features oxygenation found are in often levelsmarine found due invertebrates, to in the marine presen invertebrates, particularlyce of an endoperoxide soft particularly corals bond.[33,34,38,39]. soft Those corals characteristic [The33,34 5,α38,8,α39- ]. Thefeaturesendoperoxides 5α,8 αare-endoperoxides often 44 –found51 are inoxidized44 marine–51 are sterols invertebrates, oxidized which sterols can particularly be which formed can softduring be formedcorals photooxidation [33,34,38,39]. during photooxidation reaction The 5α ,8byα - reactionendoperoxidesaddition by of addition an oxygen44–51 of are anmolecule oxygenoxidized to molecule sterolsa conjugated which to a conjugated 5,7-dienecan be formed 5,7-dienesystem during in system the photooxidationprecursor in the precursor molecule reaction molecule which by whichadditioncontains contains ofthe an steroidal oxygen the steroidal moleculecore core[40–43]. [to40 –a 43 Thisconjugated]. This 5α,8 5αα-endoperoxide,8 5,7-dieneα-endoperoxide system bond bondin isthe exactly is precursor exactly responsible responsible molecule for forwhich the the observedcontainsobserved the biologicalbiological steroidal activitiesactivities core [40–43]. [40]. [40]. Sterol Sterol This 48 485α has has,8α -endoperoxidea a structure structure almost almost bond identical identical is exactly to to ergosterol ergosterolresponsible peroxide, peroxide, for the observedone of the biological most characteristic activities [40]. epidioxysteroidal Sterol 48 has a derivatives structure almost found identicalin both marineto ergosterol and terrestrial peroxide, oneorganisms. of the mostThe only characteristic difference epidioxysteroidalbetween 39 and ergosterol derivatives peroxide found is in the both C24 marine stereogenic and centerterrestrial of organisms. The only difference between 39 and ergosterol peroxide is the C24 stereogenic center of

Molecules 2020, 25, 129 8 of 26 one of the most characteristic epidioxysteroidal derivatives found in both marine and terrestrial Molecules 2019, 24, x 8 of 26 organisms. The only diﬀerence between 39 and ergosterol peroxide is the C24 stereogenic center ofS-S and- and R-configuration,R-conﬁguration, respectively respectively [42,44–46]. [42,44–46 Gorgonian]. Gorgonian VerrucellaVerrucella umbraculum umbraculum fromfrom the the South South ChinaChina SeaSea isis alsoalso a natural source source of of sterols sterols 4848, ,4949, ,and and 5252 [47]. [47] .Interestingly, Interestingly, 4444 standsstands out out from from the the restrest ofof epidioxysterolsepidioxysterols found in in EunicellaEunicella sp.,sp., due due to to the the presence presence of of a acyclopropyl cyclopropyl moiety moiety at atthe the side side chainchain [[33,34].33,34].

FigureFigure 11. StructuresStructures of of nine nine 11-secosterols 11-secosterols (38 (38––4343) )and and 5α 5α,8,8α-epidioxysterolsα-epidioxysterols (44 (44–51–51). ).

AsAs mentionedmentioned previously in in Section Section 2.1.2.1 .(vide (vide supra), supra), together together with with various various classes classes of ofnatural natural products,products, Deghrigue et et al. al. and and Lajili Lajili et al. et extrac al. extractedted nine mono- nine mono- or polyhydroxylated or polyhydroxylated steroids steroids from the Tunisian gorgonian coral E. singularis, 48 (Figure 11) and 52–59 (Figure 12), of which two were epidioxysterols. Besides the number of hydroxyl groups and oxygenation of steroidal core, these products also differ in saturation patterns of side chain which was confirmed by comparison of 1D

Molecules 2020, 25, 129 9 of 26 from the Tunisian gorgonian coral E. singularis, 48 (Figure 11) and 52–59 (Figure 12), of which two were epidioxysterols. Besides the number of hydroxyl groups and oxygenation of steroidal core, Molecules 2019, 24, x 9 of 26 these products also differ in saturation patterns of side chain which was confirmed by comparison ofand 1D 2D and NMR 2D data NMR with data the with literature the literature [19,30,32]. [19, 30Steroid,32]. Steroid56 was 56alsowas found also in found other inmarine other species, marine species,soft coral soft Dendronephthya coral Dendronephthya gigantean gigantean and andmarine marine bryozoans, bryozoans, BiflustraBiflustra grandicella, grandicella, andand Cryptosula pallasianapallasiana [[48].48]. Furthermore, compounds 51 and 55 were also isolatedisolated fromfrom thethe tunicatetunicate DidemnumDidemnum salarysalary byby BensemhounBensemhoun etet al.al. [[49].49].

Figure 12. Structures of steroids (52–59) isolated from the Tunisian gorgoniangorgonian coralcoral E.E. singularissingularis..

2.4.2.4. Alkaloids and Nucleosides About 2% of metabolites isolated from all gorgonian species contain an nitrogen atom including alkaloids,alkaloids, nucleosides,nucleosides, amide,amide, andand tryptamine derivativesderivatives [[8,50–52].8,50–52]. As with steroids and terpenoids, nitrogennitrogen containingcontaining compounds contribute to organismsorganisms by acting as aa defensedefense againstagainst negativenegative externalexternal influences, inﬂuences, increasing increasing the the biodiversity biodiversity of of marine marine habitat habitat [50,51]. [50,51 Particularly,]. Particularly, xanthines xanthines and their methylated derivatives are often found in marine organisms included in nucleotide metabolism [50]. They have already been isolated from other corals, particularly of the genus Sinularia, exhibiting antibacterial and cardiotonic activities [53,54]. Samori et al. reported the concentration of N- heterocyclic compounds to be differently obtained following extraction of several gorgonian species with CH2Cl2/MeOH mixtures or acetonitrile during optimization of the extraction protocol for more

Molecules 2020, 25, 129 10 of 26 and their methylated derivatives are often found in marine organisms included in nucleotide metabolismMolecules 2019, [2450, ].x They have already been isolated from other corals, particularly of the genus Sinularia10 of 26, exhibiting antibacterial and cardiotonic activities [53,54]. Samori et al. reported the concentration of polar metabolites. Furthermore, they observed no significant difference in the presence of nitrogen N-heterocyclic compounds to be differently obtained following extraction of several gorgonian species containingMolecules 2019 metabolites, 24, x in three Eunicella species (verrucosa, cavolini, and singularis). They extracted10 of 26 with CH2Cl2/MeOH mixtures or acetonitrile during optimization of the extraction protocol for more hypoxanthine, guanine, and adenosine, the latter being absent in E. verrucosa and guanine being the polarpolar metabolites. metabolites. Furthermore,Furthermore, they observed no si significantgnificant difference difference in in the the presence presence of of nitrogen nitrogen most abundant [50]. containingcontaining metabolitesmetabolites inin threethree EunicellaEunicella speciesspecies ((verrucosaverrucosa, cavolini, and singularis).). They They extracted extracted Gorgonian E. granulata collected in Senegal was the subject of the study led by Reyes et al. to hypoxanthine,hypoxanthine, guanine,guanine, andand adenosine,adenosine, the latter being absent in in E.E. verrucosa verrucosa andand guanine guanine being being the the find new compounds with antitumor activity (Figure 13). The 2-propanol coral extract afforded mostmost abundant abundant [ 50[50].]. granulatamides A (60) and B (61) after semipreparative, reverse-phased HPLC. Both novel GorgonianGorgonian E.E. granulatagranulata collectedcollected inin SenegalSenegal was the subject of of the the study study led led by by Reyes Reyes et et al. al. to to compounds contained a tryptamine moiety connected to a fatty acid and differ in the number of findfind newnew compoundscompounds withwith antitumorantitumor activityactivity (Figure(Figure 1313).). The The 2-propanol 2-propanol coral coral extract extract afforded afforded double bonds in the aliphatic chain. These compounds exerted only a moderate effect in vitro against granulatamidesgranulatamides A A (60 ()60 and) and B (61 B) after(61) semipreparative,after semipreparative, reverse-phased reverse-phased HPLC. Both HPLC. novel Both compounds novel different tumor cell lines. [51]. containedcompounds a tryptamine contained moietya tryptamine connected moiety to aconnected fatty acid to and a difattyffer acid in the and number differ ofin doublethe number bonds of in thedouble aliphatic bonds chain. in the These aliphatic compounds chain. These exerted compounds only a moderate exerted only effect a moderatein vitro against effect in di ffvitroerent against tumor celldifferent lines [ 51tumor]. cell lines. [51].

Figure 13. Structures of granulatamides A (60) and B (61) isolated from E. granulata collected in Senegal. FigureFigure 13. 13.Structures Structures of of granulatamides granulatamides A (60A )( and60) and B (61 B) isolated(61) isolated from E.from granulata E. granulatacollected collected in Senegal. in TheSenegal. n-butanolic extract of Eunicella cavolini was found to be a natural source of the polar nucleosides:The n-butanolic 9-β-D-arabinofuranosyla extract of Eunicella cavolinidenine was(araA) found ( to62), be aits natural 3′-O-acetyl source ofderivative the polar nucleosides: (63) and 9-spongouridineβ-d-arabinofuranosyladenineThe n-butanolic (araU) extract(64) (Figure of (araA)Eunicella 14). (62 The),cavolini its 3latter0-O was-acetyl had found alread derivative to ybe been a ( 63natural )isolated and spongouridinesource from of the the sponge (araU)polar (Cryptothetia64nucleosides:) (Figure 14 cripta).9- Theβ-.D -arabinofuranosyla latterHowever, had alreadylow solubility beendenine isolated (araA)and fromsusceptibility (62 the), spongeits 3 ′-toOCryptothetia -acetyldeamination derivative cripta .of However, the(63 )natural and low solubilitycompoundsspongouridine and led susceptibility to(araU) the synthesis (64) to(Figure deamination of several 14). The acyl of thelatterderiva natural hadtives compoundsalread that showedy been led betterisolated to the properties synthesisfrom the in of termssponge several of acyldrugCryptothetia derivatives administration cripta that. showedHowever,[52]. better lowproperties solubility inand terms susceptibility of drug administration to deamination [52]. of the natural compounds led to the synthesis of several acyl derivatives that showed better properties in terms of drug administration [52].

Figure 14.14. StructuresStructures of nucleosides 9-9-ββ-dD-arabinofuranosyladenine (araA) ((62), itsits 330′--OO-acetyl-acetyl

derivative (6363),), andand spongouridinespongouridine (araU)(araU) ((6464)) isolatedisolated fromfrom EunicellaEunicella cavolinicavolini.. Figure 14. Structures of nucleosides 9-β-D-arabinofuranosyladenine (araA) (62), its 3′-O-acetyl 3. Biological Activities 3. Biologicalderivative Activities (63), and spongouridine (araU) (64) isolated from Eunicella cavolini. 3.1. Cytotoxic Activity 3.1.3. Biological Cytotoxic ActivityActivities Cnidarians, and, therefore, members of the genus Eunicella, are prominent sources of steroids and terpenoids3.1. CytotoxicCnidarians, which Activity and, demonstrated therefore, antitumor members activitiesof the genus [4]. All Eunicella palmonines, are prominent (2–7) mentioned sources in Sectionof steroids 2.1. and terpenoids which demonstrated antitumor activities [4]. All palmonines (2–7) mentioned in Cnidarians, and, therefore, members of the genus Eunicella, are prominent sources of steroids Section 2.1. (vide supra) were screened for their cytotoxic effect against several human or murine and terpenoids which demonstrated antitumor activities [4]. All palmonines (2–7) mentioned in cancer cell lines: A549, HT29, P-388, and MEL28. The last two, mice lymphoma and human melanoma Section 2.1. (vide supra) were screened for their cytotoxic effect against several human or murine lines, respectively, were the most affected by 3, exhibiting IC50 values of 5 μg/mL [55]. Similar results cancer cell lines: A549, HT29, P-388, and MEL28. The last two, mice lymphoma and human melanoma were observed with excavatolides O and Q, diterpenoids with a briarane-type skeleton extracted lines, respectively, were the most affected by 3, exhibiting IC50 values of 5 μg/mL [55]. Similar results

were observed with excavatolides O and Q, diterpenoids with a briarane-type skeleton extracted

Molecules 2020, 25, 129 11 of 26

(vide supra) were screened for their cytotoxic effect against several human or murine cancer cell lines: A549, HT29, P-388, and MEL28. The last two, mice lymphoma and human melanoma lines, respectively, were the most affected by 3, exhibiting IC50 values of 5 µg/mL [55]. Similar results were observed with excavatolides O and Q, diterpenoids with a briarane-type skeleton extracted from the gorgonian Briareum excavatum [8]. The authors hypothesized that the activity of those metabolites could arise from synergistic effects and interactions with other compounds. Since palmonine B (3) has the highest number of acetoxyl groups, that could also play a role in the observed cytotoxic activity [55]. Furthermore, significant anticancer potential of palmonines F (7) and D (5) was observed by inducing apoptosis of 92% and 93% of cells, respectively, in a breast cancer cell model (MCF-7 monolayer) at concentration of 200 µg/mL. The authors determined EC50 values from the concentration-response curves for 7 and 5, which were 13 µg/mL and 49 µg/mL, respectively. Lajili et al. also investigated the effect of two previously mentioned palmonines on MCF-7 mammospheres and reported reduction in spheroid size, which was also concentration-dependent and visible 2 days after incubation [19]. Labiatins B, C. and E (12, 13, and 15) were assayed for their cytotoxic effect against human cancer cell lines [22,26]. Labiatin B (12) was tested against colon cancer line (HCT-116) and the authors reported an IC50 value of 0.85 µg/mL [26]. The last two compounds showed a cytostatic effect against non-small cell lung cancer line (NSCLC-N6) with half inhibitory concentrations of 35 µg/mL and 7.7 µg/mL for labiatin E (15) and C (13), respectively, indicating that the stereochemistry influences the biological activity of these secondary metabolites [22]. Massileunicellins A–C (17–19) were additionally assayed for antitumor and antiviral activity against KB and L1210 doxorubicin-resistant tumor cells, and Dengue virus, respectively. After observing only low cytotoxic effects of massileunicellins, Mancini et al. screened labiatin B (12) and palmonine D(5) against the same cells which also resulted in poor activity. Comparing those results with the significant cytotoxic activity reported by Ortega et al. [55] and Roussis et al. [26], they hypothesized that diterpenoids might be selective for specific tumor cells. Furthermore, they did not observed any stabilization effect of 5 and 12 on tubulin, a feature characteristic for other types of 2,11-cyclized cembranoids, i.e., sarcodictyns and eleutherobin [27]. The most promising cytotoxic diterpenoids isolated and studied from the genus Eunicella are shown in Table1.

Table 1. The most promising diterpenoids with remarkable cytotoxic activity isolated and studied from corals of the genus Eunicella.

Tumor Cell Compound Name Source IC /(µg/mL) Reference Line 50 P-388 5 3 Palmonine B E. verrucosa [55] MEL28 5 E. verrucosa 5 Palmonine D MCF-7 49 [19] E. singularis E. verrucosa 7 Palmonine F MCF-7 13 [19] E. singularis 12 Labiatin B E. labiata HCT-116 0.85 [26] 13 Labiatin C E. labiata NSCLC-N6 7.7 [22] 15 Labiatin E E. labiata NSCLC-N6 35 [22]

Steroid compounds of natural origin have recently attracted considerable attention among the scientific community due to their interference with molecular pathways of MCF-7 cells. It has been shown that steroid derivatives inhibit estrogen-dependent proliferation and survival of those cells binding to estrogen receptor, thus disrupting the interaction with transmembrane growth factor receptor [33,34,37]. Furthermore, according to Chen and his group, secosteroids and polyhydroxylated steroids could be potential inhibitors of protein-tyrosine phosphatase 1B (PTP-1B), which is involved in metabolism of glucose via hydrolyzation of insulin receptor [39,56]. However, PTP-1B was also found Molecules 2020, 25, 129 12 of 26 to have a contradictory role in cancer biology. Its deficiency has led to the growth of B lymphoma in mice while it had no role in breast tumorigenesis [57]. Nevertheless, it presents a novel target in both cancer and metabolism treatment. Ioannou et al. assayed the growth inhibitory potential of sterols isolated from E. cavolini (31–39, 41, 42, 44–51) against MCF-7 cells cultured with and without a physiological concentration of 17β-estradiol (1 nM) [33,34,37]. When compared to the effect of positive control (ICI 182,780, Faslodex®, a commercially available estrogen receptor down-regulator), compounds 31–35 inhibited cell growth for 40 to 49%, while the last two pregnanes (36, 37) were almost inactive. According to the authors, the presence of acetoxyl moiety at C11, observed in metabolites with significant inhibitory effect, could play a role in mediating estrogen-dependent growth of MCF-7. Another possible molecular target is the nuclear pregnane X receptor, a target of several marine-derived compounds involved in drugs and xenobiotics metabolism, oxidative stress, cancer formation, and other physiological processes which yet need to be examined [37,58–60]. Secosterols 38 and 39 exhibited IC50 values of 7.6 and 7.4 µM against MCF-7 cells, respectively. On the other side, growth inhibition by all compounds was significantly enhanced in MCF-7 cells cultivated in the absence of growth factors and endogenous estrogen. Since 38 was also found to be effective against proliferation of K562 and HeLa cells, it is assumed that secosterols’ antiproliferative activity is selective towards specific tumor cells, as well as the applied growth conditions. Furthermore, since tested compounds differ only in the side chain structure, the better biological activities of 38 and 39 could also be attributed to the double bond at C22 position with E geometry [34]. The gorgonian coral, Subergorgia suberosa, collected from the South China Sea, was used to extract several secosterols bearing a hydroxyl group at C11, including 38 and 43. Zhang et al. reported IC50 values of 28.1 and 23.3 µM against HeLa cells for 38 and 43, respectively, and hypothesized that the presence of a free -OH group at C11 does not contribute to the observed cytotoxic effects of the tested compounds [38]. That is also consistent with the conclusion reported by Kongkathip et al. who have synthesized and evaluated the cytotoxicity of 9,11-secosterol derivatives against KB, HeLa, and MCF-7 cells, as well as non-transformed Vero cells. According to the authors, the key to the observed biological activity is in the presence of a cholesterol-like side chain and a keto group at C9 position [61]. Epidioxysterols 44–51 also displayed significant inhibition activity against growth of MCF-7 cells in the growth factor and endogenous steroids depleted medium. At a concentration of 10 µM, 44, 45, and 46 displayed almost 60% of the effect of positive control, ICI 182,780 (Faslodex®). Considering that 44 was particularly effective against MCF-7 cultured in both cell growth medium conditions, the authors assume that the cyclopropyl group at the side chain of 44 was responsible for the observed effects [33]. Luo et al. also isolated 45 from another marine organism, sponge Topsentia sp., and observed its moderate cytotoxic activity against different tumor cell lines of human origin: A549, SK-OV-3, SK-MEL-2, XF498, and HCT15 (IC50 values: 30.0, 19.4, 25.9, >30.0, 21.4 µg/mL, respectively) [62]. Another marine sponge, Monanchora sp., was used in the study conducted by Mun et al. in 2015 who isolated epidioxysterols 45 and 47. The authors evaluated the activities of these two products against renal, pancreatic, and colorectal cancer cell lines and suggested that the C24 (28) double bond, which is a feature of 47, might be responsible for its increased cytotoxic activities. At a concentration of 23 µM, 47 induced 50% inhibition of renal A-498 cells. It should be emphasized that 47 exhibited higher cytotoxicity than the positive controls, temsirolimus and 5-fluorouracil, used in the clinical treatment of the above mentioned cancers [63]. The 48 showed significant antiproliferative activity against A549, H460, and HGC27, displaying IC50 values of 10.9, 10.9, and 11.6 µg/mL, respectively, as well as moderate cytotoxicity towards human ovarian cells (A2780) with an IC50 value of 16 µg/mL [64,65]. Raslan et al. isolated epidioxysterols 49 and 51 from the marine sponge Monanchora clathrate but observed only weak cytotoxicity against MES-SA, MCF-7, and HK-2 cancer cell lines with IC50 values higher than 25 µg/mL [66]. Deghrigue et al. and Lajili et al. also investigated the cytotoxic activity of steroids isolated from E. singularis after observing promising antiproliferative, antioxidant, and other pharmacological activities Molecules 2020, 25, 129 13 of 26 of its organic extract and fractions [19,30,32]. Metabolites 52, 54, and 59 were evaluated for their anticancer activity against a monolayer and a spheroid model of MCF-7 using AnnexinV- fluorescein isothiocyanate/propidium iodide (FITC/PI) flow cytometry and spheroid size analysis, respectively. Cholesta-5,22-diene-3β-ol (59) displayed an EC50 value of 30 µg/mL against monolayer cells causing apoptosis in 91% of cancer cells at 200 µg/mL. However, steroids were more effective against the spheric model than diterpenoids (5 and 7) isolated from the same organism. The reported growth rates for 52, 54, and 59 were 1.17, 1.25, and 1.15, respectively, at a concentration of 200 µg/mL and comparable to the control, taxol at 100 µM[19]. On the contrary, according to Tian et al., 52 was inactive in vitro against HL-60, HepG2, and SGC7901 cell lines [67]. Finally, Pan et al. investigated the proapoptotic activity of the ethyl acetate extract of the hard clam Meretrix lusoria and consequently isolated a mixture of epidioxysterols containing 55. The authors observed 35% and 73% apoptotic HL-60 cells after treatment with 25 and 50 µg/mL of this mixture, respectively [68]. The antitumor activities of sterols are summarized in Table2.

Table 2. The most promising sterols with remarkable cytotoxic activity isolated and studied from the genus Eunicella.

Tumor Cell Compound Class Source IC * Reference Line 50 MCF-7 7.6 a [34] 38 Secosterol E. cavolini HeLa 28.1 a [38] 39 Secosterol E. cavolini MCF-7 7.4 a [34] E. cavolini 43 Secosterol HeLa 23.3 a [38] S. suberosa A549 30.0 b E. cavolini SK-OV-3 19.4 b 45 Epidioxysterol [62] Topsentia sp. SK-MEL-2 25.9 b HCT15 21.4 b E. cavolini 47 Epidioxysterol A-498 23 a [63] Monanchora sp. E. cavolini A549 10.9 b E. singularis H460 10.9 b 48 Epidioxysterol [64,65] M. azedarach HGC27 11.6 b H. sphaerocarpa A2780 16 b 59 Sterol E. singularis MCF-7 30 b [19]

* Values for IC50 are expressed as either µM (a) or µg/mL (b).

Granulatamides A (60) and B (61) were both evaluated for their cytotoxic activities against 16 diﬀerent human cancer cell lines by the use of sulphorhodamine B colorimetric assay (Table3), exhibiting low GI50 (growth inhibition concentration) values of 1.7 and 3.5 µM against two human prostate cancer cell lines, DU-145 and LN-caP,respectively [51]. Another nitrogen containing compound, nucleoside 63 was assayed for cytotoxic activity against KB cells displaying a low IC50 value of 5 µg/mL [52].

Table 3. Growth inhibitory activity of granulatamides A (60) and B (61) against various tumor cell lines [51].

IC50/µM Compound Du-145 LN-caP SK-BR3 HT29 IGROV A549 K-562 60 1.7 4.7 2.7 2.2 6.7 6.7 6.8 61 7.7 3.5 6.0 >10 8.2 8.9 4.3 Molecules 2020, 25, 129 14 of 26

3.2. Anti-Inflammatory Activity Discovery of novel compounds that may be used to target inflammatory processes is highly needed since the use of many existing nonsteroidal anti-inflammatory drugs have been linked with various severe adverse effects [30,32]. In this context, Deghrigue et al. have reported that the organic extract and fractions obtained by further purification with ethanol, acetone, and dichloromethane/methanol have exhibited anti-inflammatory activity in a dose-dependent manner regarding the inhibition of edema in rats induced by carrageenan. At a dose of 25 mg/kg of the ethanolic semipurified fraction, the edema was inhibited by 66% after 3 h, which is 10% higher than observed with a reference drug, acetylsalicylaze-lysine (ASL). Final purification of the ethanolic fraction led to isolation of owp palmonines, 5 and 7, and nine sterols, 48, 52–59, which could contribute to the investigated biological activity [30,32]. Anti-inflammatory activity of epidioxysterols 48 and 55 was further investigated by other scientific groups [68–70]. Just recently, Huynh et al. studied the influence of 48 on expression of pro-inflammatory proteins, iNOS and COX-2, released from macrophages. They observed significant suppression of iNOS by 48 at 10 µM. However, dendronesterone D bearing acetoxyl moiety at C11 showed the highest suppression of inflammation [70]. Inhibition of NO production by lipopolysaccharide (LPS)-stimulated macrophages was used to assess anti-inflammatory activity of 55 in a mixture of epidioxysterols by Pan et al. NO production did not arise from the cytotoxic activity and was inhibited by 38%, 39%, 99%, and 100% at concentrations of 5, 10, 25, and 50 µg/mL, respectively [68]. Immunosuppressive activity of 47 and 48 against T and B lymphocyte cells proliferation was also examined by Yang et al. in 2019. Both epidioxysterols were extracted from a soft coral Sinularia sp. Reported IC50 values of 47 and 48 were 57.54 and 59.54 µM for T cells, and 47.57 and 19.30 µM for B lymphocytes, respectively. Thus, they showed weaker anti-inflammatory activity than cyclosporin A (IC50 = 0.04 and 0.40 µM for T and B cells, respectively), however they were less cytotoxic against murine splenocytes [69].

3.3. Antimicrobial Activity Bioactive secondary metabolites from Eunicella sp., as well as compounds from other soft corals, showed interesting antibacterial and antiviral activities [4,52,71,72]. According to Gauvin et al., epidioxysterols are the first natural metabolites to display antiviral activity against lymphoma causer, HTLV-I retrovirus. A mixture of sterols, including 49, 51, and 55, reduced by 50% β-galactosidase activity which is correlated to virus activity at a concentration of 0.3 mg/mL. However, they were inactive against another RNA virus, HIV [71]. Polar nucleosides from E. cavolini, 9-β-d-arabinofuranosyladenine (spongoadenosine, araA) (62) and spongouridine (araU) (64), showed promising antiviral activity against DNA viruses [52]. Pharmacological properties and biosynthesis of araA (53) have been already reviewed by Huang et al. [73]. It is worth mentioning that 62 was the first drug to treat herpes encephalitis infections and diseases caused by other herpes viruses in patients with impaired immune system [73]. Synthetic O-acyl derivatives of 62 showed also promising antiviral effects but its 30-O-acetyl derivative (63) yet needs to be examined in order to complete the structure activity relationship studies [52]. We would also like to mention that Liang et al. observed an antibacterial activity of epidioxysterols 47 with MIC50 value of 500 µM against Staphylococcus aureus Newman strain. This activity is considered significant because the MIC50 value is lower than 1 mM [72].

4. Synthetic Approaches One of the major problems during the preclinical and clinical development of naturally derived drugs is the insufficient quantity of the isolated natural product precursors [2,4,74]. As the consequent market application by the pharmaceutical and cosmetic industries entails the desired metabolite to be available often on the ‘kilogram scale’, it is not surprising that some compounds which demonstrated promising biological activities have never reached the market. In order to obtain sufficient amounts of a natural product or its derivatives, either the extraction of the natural row material should be Molecules 2020, 25, 129 15 of 26 repeatedly performed or the natural product should be synthetized [75]. The extraction pathway may be problematic in terms of the percentage of the desired secondary metabolite in the row material and/or ecological and ethical principles. For example, in the case of Eunicella species, mass mortality events have emerged in the past few years mainly hitting the gorgonian corals in the Mediterranean Sea [76–80]. Furthermore, collecting the same species in various geographical areas or during different timeMolecules seasons 2019, 24 might, x result in a different chemical composition, thereby reducing or increasing15 of 26 the yield of the desired product [75,81]. Finally, several characteristics of marine natural products, suchcharacteristics as halogenation, of marine oxygenation natural products, substitution such as patterns, halogenation, different oxygenation degrees substitution of carbonframework patterns, functionalization,different degrees andof carbon stereochemical framework diversity function makealization, chemical and synthesis stereochemical even more diversity complex make and challengingchemical synthesis for organic even chemists. more complex The fulfilment and challeng of theing principlesfor organic of chemists. green chemistry The fulfilment including of the the applicationprinciples of of green atom-economical chemistry including and high-yield the applicat reactions,ion of atom-economical the use of affordable and high-yield and nontoxic reactions, starting materialsthe use of and affordable solvents, and or nontoxic the design starting of protecting materials group-free and solvents, chemical or the synthesis design of are protecting additional group- issues tofree also chemical consider synthesis [2,75,82 ,83are]. additional Thus, novel issues synthetic to also strategies consider have[2,75,82,83]. been developed Thus, novel recently, synthetic with newstrategies scientific have and been technological developed recently, progress with and new insights, scientific and and are technological oriented toward progress total and syntheses insights, of naturaland are products oriented and toward their total stereochemical syntheses of congeners, natural products at the same and timetheir complying stereochemical with congeners, green chemical at approachthe same [time2]. complying with green chemical approach [2].

4.1.4.1. SyntheticSynthetic ApproachesApproaches toward the Synthesis of of Terpenoids Terpenoids Isolated Isolated from from Eunicella Eunicella Species Species SeveralSeveral studiesstudies havehave beenbeen conducted over over the the pa pastst two two decades decades with with the the aim aim of of developing developing a a syntheticsynthetic approachapproach toto obtainobtain highlyhighly oxidized eu eunicellin-typenicellin-type diterpenes diterpenes [84–89]. [84–89 ].Two Two major major issues issues hadhad to to be be overcome overcome inin constructingconstructing the tricyclic skeleton skeleton of of eunicellins. eunicellins. The The first first was was stereoselective stereoselective formationformation of of hydroisobenzofuranhydroisobenzofuran core, which posses possessesses four four stereogenic stereogenic centers centers and and the the second second was was formationformation ofof thethe nine-memberednine-membered ring. Approaches, Approaches, including including a afirst first synthesis synthesis of of the the isobenzofuran isobenzofuran part followed by the cyclization of the nine-membered ring or vice versa, have been developed [88]. part followed by the cyclization of the nine-membered ring or vice versa, have been developed [88]. The first successful total synthesis of cladiellin, (−)-7-deacetoxyalcyonine (67), starting from (S)- The first successful total synthesis of cladiellin, ( )-7-deacetoxyalcyonine (67), starting from dihydrocarvone 65, was accomplished in 1995 by −MacMillian and Overman. Prins-pinacol (S)-dihydrocarvone 65, was accomplished in 1995 by MacMillian and Overman. Prins-pinacol rearrangement was employed to achieve hydroisobenzofuran 66 as a single diastereomer, followed rearrangement was employed to achieve hydroisobenzofuran 66 as a single diastereomer, followed by by Nozaki–Hiyama–Kishi coupling reaction, resulting in the final tricyclic product (Scheme 1) [90]. Nozaki–Hiyama–Kishi coupling reaction, resulting in the final tricyclic product (Scheme1)[ 90]. Since Since that, numerous total or partial syntheses of eunicellins (cladiellins) and other types of C2–C11 that, numerous total or partial syntheses of eunicellins (cladiellins) and other types of C2–C11 cyclized cyclized cembranoids were conducted, which had been previously reviewed by Ellis et al. in 2008 cembranoids were conducted, which had been previously reviewed by Ellis et al. in 2008 [89]. [89].

SchemeScheme 1.1. MacMillian’s and Overman’s Overman’s total total synthesis synthesis of of cladiellin cladiellin (67 (67) [90].)[90 ].

SyntheticSynthetic access access to to eunicellin-type eunicellin-type metabolites metabolites produced produced by by members members of theof genusthe genusEunicella Eunicellaneeds stillneeds to bestill developed. to be developed. Up to now,Up to profound now, profound investigations investigations were performedwere performed by the by McIntosh the McIntosh group togroup evolve to the evolve complete the hydroisobenzofurancomplete hydroisobenzofuran core from core less oxidizedfrom less compounds, oxidized compounds, thus obtaining thus the moreobtaining complex the stereochemistrymore complex of stereochemistry massileunicellins of [ 86massileunicellins,91]. They synthesized [86,91]. hydroisobenzofuran They synthesized of massileunicellinshydroisobenzofuran containing of massileunicel eight oflins a total containing of nine eight stereocenters of a total inof annine overall stereocenters 12 steps in with an overall a single temporary12 steps with trimethylsilylation a single temporary protection trimethylsilylati of hydroxylon group.protection As shownof hydroxyl in Scheme group.2,( AsS)-( shown+)-carvone in (65Scheme) was reacted2, (S)-(+)-carvone with methacrolein (65) was inreacted aldol reaction,with methacrolein followed byin Williamsonaldol reaction, etheriﬁcation followed by and cycloaldolizationWilliamson etherification of the formed and cycloaldolization glycolate intermediate of the formed to aﬀord glycolate allylic alcoholintermediate68 in 85%to afford yield allylic [87,91 ]. alcohol 68 in 85% yield [87,91]. The intermediate 68 is converted to epoxy ketone with Collins’ reagent, which in subsequent treatment with potassium bis(trimethylsilyl)amide/trimethylsilyl chloride (KHMDS/TMSCl) yielded silylated enolate 69. Rubottom oxidation by using dioxirane produced keto diol 70 in 83% yield from intermediate 68. Evans–Saksena reduction of product 70 by using borohydride reagent led to the corresponding triol, which was subsequently esterified to diacetate 71 in high yield as a single diastereomer. To obtain the desired hydroisobenzofuran product 73, 71 was subjected to dihydroxylation followed by oxidative cleavage with NaIO4, giving rise to

Molecules 2020, 25, 129 16 of 26

The intermediate 68 is converted to epoxy ketone with Collins’ reagent, which in subsequent treatment with potassium bis(trimethylsilyl)amide/trimethylsilyl chloride (KHMDS/TMSCl) yielded silylated enolate 69. Rubottom oxidation by using dioxirane produced keto diol 70 in 83% yield from intermediate 68. Evans–Saksena reduction of product 70 by using borohydride reagent led to the corresponding triol, which was subsequently esteriﬁed to diacetate 71 in high yield as a single diastereomer. To obtain the desired hydroisobenzofuran product 73, 71 was subjected to dihydroxylation followed by Molecules 2019, 24, x 16 of 26 oxidative cleavage with NaIO4, giving rise to methyl ketone 72. Finally, hydrogenation of a double bondmethyl of ketone72 was 72 conducted. Finally, withhydrogenation Crabtree’s of catalyst a double atmoderate bond of 72 H 2waspressure conducted to yield with quantitively Crabtree’s the hydroisobenzofuran core 73 [87]. catalyst at moderate H2 pressure to yield quantitively the hydroisobenzofuran core 73 [87].

SchemeScheme 2.2. McIntosh’sMcIntosh’s synthetic synthetic approach approach toward toward the the hydroisobenzofuran hydroisobenzofuran core core (73)( 73of) the of the massileunicellinsmassileunicellins [85,86]. [85,86].

Clark’sClark’s groupgroup previously previously developed developed synthetic synthetic approaches approaches to synthesizeto synthesize the the tricyclic tricyclic core core of labiatin of A,labiatin which A, is anwhich unusual is an eunicellinunusual eunicellin (cladiellin) (cladiellin) possessing possessing a C2–C6 a ether C2–C6 bridge ether as bridge discussed as discussed above (vide infra)above [ (vide84,85 ,infra)92,93]. [84,85,92,93]. The authors The firstly authors synthesized firstly synthesized bromide 74bromidein five 74 steps in five and steps coupled and coupled it through Williams’sit through etherificationWilliams’s ethe withrification alcohol with75 that alcohol was easily75 that prepared was easily from preparedd-mannitol from (Scheme D-mannitol3). The obtained(Scheme 3). ether The76 obtained(87%) wasether deprotected 76 (87%) was with deprotected pyridinium with ppyridinium-toluensulfonate p-toluensulfonate (PPTS) to cleave(PPTS) the to cleave the acetonide moiety, and the produced syn-diol was oxidatively cleaved with NaIO4 to acetonide moiety, and the produced syn-diol was oxidatively cleaved with NaIO4 to form a terminal form a terminal aldehyde which was further oxidized to carboxylic acid 77 with NaClO2. A mixed aldehyde which was further oxidized to carboxylic acid 77 with NaClO . A mixed anhydride formation anhydride formation of acid 77 and chloroformate followed by 2subsequent treatment with of acid 77 and chloroformate followed by subsequent treatment with diazomethane gave the diazo diazomethane gave the diazo ketone 78 in 79% yield from 76. Finally, cyclization of diazo compound ketone 78 in 79% yield from 76. Finally, cyclization of diazo compound 78 was performed by using 78 was performed by using rhodium (II) tifluoroacetamide as a catalyst and produced rhodium (II) tifluoroacetamide as a catalyst and produced dihydrofuranone 79 in 66% yield [85]. dihydrofuranone 79 in 66% yield [85].

Molecules 2020, 25, 129 17 of 26 Molecules 2019, 24, x 17 of 26

Scheme 3. Clark’s synthesis of dihydrofuranone ( 79) via intramolecular dihydrofuranonedihydrofuranone formation through diazo ketone (78)[) [85].85].

Dihydrofuranone 79 was reacted with methyl lithium giving rise to tert-alcohol 8080 (85%) (Scheme 44).). Acetylation Acetylation and andremoval removal of p-methoxybenzyl of p-methoxybenzyl protecting protecting group (PMB) group with 2,3-dichloro- (PMB) with 5,6-dicyano-1,4-benzoquinone2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) of 80 (DDQ) produced of 80 produced primary primaryalcohol alcohol81 (56%).81 (56%).The latter The latterwas wasconverted converted to carboxylic to carboxylic acid acid82 (89%)82 (89%) after after Dess–Martin Dess–Martin and and Pinick Pinick type type oxidation oxidation reactions. reactions. The The 82 was82 was transformed transformed to tothe the corresponding corresponding acyl acyl chloride chloride with with oxalyl oxalyl chloride, chloride, and, and, after sequential treatment withwith diazomethane,diazomethane, resulted resulted in in the the formation formation of diazoof diazo ketone ketone83 (89%)83 (89%) required required for the for final the finalkeystep. key step. Intramolecular Intramolecular cyclization cyclization of the of electrophilic the electrophilic carbenoid carbenoid species species of intermediate of intermediate83 with 83 withcopper copper (II) hexafluoroacetylacetonate (II) hexafluoroacetylacetonate gave gave tricyclic tricyclic ketone ketone84 (76%). 84 (76%). Finally, Finally, after 21after steps 21 steps in total, in total,tertiary tertiary alcohol alcohol85 was 85 formed was formed by the by reaction the reaction with MeLi with leadingMeLi leading to the to carbon the carbon core of core labiatin of labiatin A (11) Aas ( a11 single) as a isomer.single isomer. Its structure Its structure was confirmed was confirmed by X-ray by crystallography X-ray crystallography [85]. [85].

Molecules 2020, 25, 129 18 of 26 Molecules 2019, 24, x 18 of 26

Scheme 4. SynthesisSynthesis of of the the tricyclic tricyclic core core ( 8585)) of of labiatin labiatin A A ( (11).).

4.2. Synthesis Synthesis of of Gr Granulatamideanulatamide Alkaloids Alkaloids InIn order order to to obtain obtain the thekinetically kinetically favored favored Z-isomerZ-isomer of cytotoxic of cytotoxic alkaloids, alkaloids, Pakhare and Pakhare Kusurkar and reportedKusurkar the reported first total the ﬁrstsynthesis total synthesisof granulatamide of granulatamide A (60) from A (60tryptamine) from tryptamine (86) via Horner– (86) via Wadsworth–EmmonsHorner–Wadsworth–Emmons olefination oleﬁnation reaction reaction (Scheme (Scheme 5). In5 ).brief, In brief, tryptamine tryptamine (86) ( was86) was converted converted to bromoacetamideto bromoacetamide 87 87whichwhich was was treated treated with with diphenyl diphenyl phosphite phosphite under under basic basic conditions conditions to to provide provide phosphonate 8888 inin 90%90% yield.yield. TheThedesired desired natural natural product product granulatamide granulatamide A A (60 (60) and) and its itsE-congener E-congener89 89were were obtained obtained by theby reactionthe reaction of phosphonate of phosphonate88 with 88 2-undecanonewith 2-undecanone in E/Z in ratio E/Z= ratio9/1 and = 9/1 80% and overall 80% overallyield. [ 94yield.]. [94].

Molecules 2020, 25, 129 19 of 26 Molecules 2019, 24, x 19 of 26 Molecules 2019, 24, x 19 of 26

Scheme 5. Pakhare’s and Kusurkar’s synthesis of granulatamide A (60) via Z-favored Horner– SchemeScheme 5.5. Pakhare’sPakhare’s and and Kusurkar’s Kusurkar’s synthesis synthesis of granulatamide of granulatamide A (60) Avia (60 Z)-favored via Z-favored Horner– Wadsworth–Emmons olefination [94]. Horner–Wadsworth–EmmonsWadsworth–Emmons olefination oleﬁnation [94]. [94]. Sun and Fürstner developed a highly yielded three-step total synthesis of the cytotoxic SunSun and and Fürstner Fürstner developed developed a highly a highly yielded yielded three-step three-step total synthesis total synthesis of the cytotoxic of the tryptamine cytotoxic tryptamine derivative granulatamide B (61) by employing their ring-opening/cross-coupling reaction derivativetryptamine granulatamide derivative granulatamide B (61) by employing B (61) by their employing ring-opening their /ring-opening/cross-couplingcross-coupling reaction of 2-pyrones reaction of 2-pyrones methodology (Scheme 6). Pyrone 91 was obtained in multigram amounts by the reaction methodologyof 2-pyrones methodology (Scheme6). Pyrone (Scheme91 6).was Pyrone obtained 91 was in obtained multigram in multigram amounts amounts by the reaction by the reaction of the of the commercial crotonate 90 and octanoyl chloride. The iron catalyzed ring-opening/cross- commercialof the commercial crotonate crotonate90 and octanoyl 90 andchloride. octanoyl Thechloride. iron catalyzed The iron ring-opening catalyzed ring-opening/cross-/cross-coupling of coupling of pyrone 91 with methyl Grignard reagent smoothly afforded the acid92 as a single isomer pyronecoupling91 ofwith pyrone methyl 91 with Grignard methyl reagent Grignard smoothly reagent asmoothlyﬀorded the afforded acid92 the as acid92 a single as isomera single in isomer 83% in 83% yield. The amide bond formation was performed under standard conditions by using 1- yield.in 83% The yield. amide The bond amide formation bond formation was performed was performed under standard under standard conditions conditions by using by 1-hydroxy using 1- hydroxy benzotriazole/1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride benzotriazolehydroxy /1-ethyl-3-[3-dimethylaminopropyl]benzotriazole/1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride carbodiimide (HOBt/EDCA) hydrochloride coupling (HOBt/EDCA) coupling protocol to furnish granulatamide B (61) [95]. protocol(HOBt/EDCA) to furnish coupling granulatamide protocol Bto (furnish61)[95]. granulatamide B (61) [95].

Scheme 6. Sun’s and Fürstner’s Fe-catalyzed synthesis of granulatamide B (61) via formal ring- SchemeScheme 6.6. Sun’sSun’s and and Fürstner’s Fürstner’s Fe-catalyzed Fe-catalyzed synthesis synthesis of granulatamide of granulatamide B (61 B) (via61) formal via formal ring- opening/cross-coupling reaction of 2-pyrones [95] ring-openingopening/cross-coupling/cross-coupling reaction reaction of 2-pyrones of 2-pyrones [95] [ 95]. 5. Conclusions 5.5. Conclusions Conclusions Natural products and their semisynthetic derivatives have been traditionally among the most NaturalNatural productsproducts and their their semisynthetic semisynthetic derivatives derivatives have have been been traditionally traditionally among among the most the important sources in the creation of novel products, especially in the pharmaceutical and cosmetic mostimportant important sources sources in the in creation the creation of novel of products novel products,, especially especially in the pharmaceutical in the pharmaceutical and cosmetic and industries. cosmeticindustries. industries. As shown in this review, the interdisciplinary studies linked to gorgonian soft corals have As shown in this review, the interdisciplinary studies linked to gorgonian soft corals have contributed to the discovery of new marine natural products. More than 60 metabolites were isolated contributed to the discovery of new marine natural products. More than 60 metabolites were isolated

Molecules 2020, 25, 129 20 of 26

As shown in this review, the interdisciplinary studies linked to gorgonian soft corals have contributed to the discovery of new marine natural products. More than 60 metabolites were isolated from the Eunicella species in the past three decades, belonging to the class of terpenoids, sterols, alkaloids and nucleosides. These metabolites exhibited anticancer, anti-inflammatory, antiviral, analgesic, and other activities. Their full therapeutic spectra remain to be examined in detail. Already, seven compounds displayed very high cytotoxic activities against cancer cell lines in vitro, namely palmonine B(3), labiatin C (12), labiatin D (13), secosterols 38 and 39, and granualtamides A (60) and B (61) with IC50 values less than 10 µM or 10 µg/mL. Epidioxysterol 48 showed significant immunosuppressive activity, while nucleosides 62 and 64 showed relevant antiviral activity against DNA viruses. The interesting biological activities of Eunicella’s secondary metabolites have attracted a broad attention of the scientific community with the purpose to isolate novel compounds, develop new and innovative synthetic methodologies, or conduct their partial or total synthesis. By committing studies in the direction of their synthesis, essential stereochemical and mechanistic issues were encountered and solved. The partial or total synthesis of these natural products is expected to guide advancements of new chemical approaches with wider synthetic applications. In conclusion, molecules isolated from the genus Eunicella, from their first discovery to date, present an interesting case which has stimulated research advancements in diverse scientific fields, attaining new valuable experimental and theoretical knowledge for future development in medicinal applications.

Author Contributions: D.M. (Dean Markovic) devised the concept of the manuscript. D.M. (Dario Matulja), M.K.M., G.A. and S.L. wrote parts of the manuscript. D.M. (Dario Matulja), M.K.M., G.A. and S.L. performed literature searches. S.K.P. and D.M. (Dean Markovic) participated in the manuscript writing, performed literature searches, wrote and discussed parts relevant for synthesis and biological data, finalized the paper concept, and performed the final manuscript revision. All authors have read and agreed to the published version of the manuscript. Funding: This research is funded by European Regional Development Fund (the Competitiveness and Cohesion Operational Program, KK.01.1.1.01) through project Bioprospecting of the Adriatic Sea (KK.01.1.1.01.0002) granted to The Scientific Centre of Excellence for Marine Bioprospecting (BioProCro). Furthermore, financial support is provided by the project, Research Infrastructure for Campus-based Laboratories at University of Rijeka, co-financed by European Regional Development Fund (ERDF) and the University of Rijeka research grants, UNIRI-biomed-18-133 (1277) and UNIRI-prirod-18-102. Acknowledgments: We would like to thank the Croatian Science Foundation project, Career Development of Young Researchers, Training of New PhDs, for funding PhD of D. Matulja. M. Kolympadi Markovic and G. Ambrožićare grateful to the Croatian Science Foundation for funding the postdoctoral position of M.K.M. through the project IP-2016-06-3568. Conflicts of Interest: The authors declare no conflict of interest.

List of Abbreviations

A2780 human ovarian carcinoma cell line A498 human renal epithelial cancer cell line A549 human lung carcinoma cell line ASL acetylsalicylate of lysine COX-2 cyclooxygenase-2 DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DHQD)2PHAL hydroquinidine 1,4-phthalazinediyl diether DIPEA N,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF dimethylformamide Du145 human prostate cancer cell line EDTA ethylenediaminetetraacetic acid H460 human large cell lung cancer cell line HCT-115 human colorectal adenocarcinoma cell line Molecules 2020, 25, 129 21 of 26

HCT-116 human colon cancer cell line HeLa human cervical cancer cell line HepG2 human hepatocellular carcinoma cell line Hfacac hexaﬂuoroacetylacetonat HGC-27 human gastric cancer cell line HK-2 human papillomavirus 16 (HPV-16) transformed renal cell line HL-60 human leukemia cell line HT29 human colon adenocarcinoma cell line HTLV-1 human T-lymphotropic virus 1 IC50 half maximal inhibitory concentration IGROV human ovarian cancer cell line iNOS inducible nitric oxide synthase K562 human myelogenous leukemia cell line KB keratin-forming tumor cell line HeLa KHMDS potassium bis(trimethylsilyl)amide KtOBu potassium t-butoxide L1210 murine lymphocytic leukemia cell line LDA lithium diisopropylamide LNcaP androgen-sensitive human prostate adenocarcinoma cell line LPS lipopolysaccharide MCF-7 human breast adenocarcinoma cell line MES-SA human uterine carcinoma cell line MIC50 minimum inhibitory concentration required to inhibit the growth of 50% or organisms NO nitric oxide NSCLC-N6 human non-small cell lung cancer cell line P-388 menogaril-resistant leukemia cell line PPTS pyridinium p-toluensulfonate SGC-7901 human gastric cancer cell line SK-BR3 human breast cancer cell line SK-MEL-2 human melanoma cell line derived from metastasis on skin of thigh SK-OV-3 human ovarian carcinoma cell line THF tetrahydrofuran TMSCl trimethylsilyl chloride Vero epithelial cell line derived from kidney of Cercopithecus aethiops XF498 human glioblastoma cell line

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Sample Availability: Samples of the compounds are not available from the authors.

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