Dolabellane diterpenes from the Caribbean soft corals Eunicea laciniata and Eunicea asperula and determination of their anti HSV-1 activity

Resumo =olabellane diterpenes have considerable antiviral activityy but most studies have been focused towards compounds isolated from Dictyota brown algaeT %lthough soft corals are also a significant source of these diterpenesy their antiviral potential has not been studied in detailT With the aim of assessing the biological activity of marine sourcesy we evaluated the dolabellane content in the soft corals Eunicea laciniata and E. asperula collected in Santa Martay μolombian μaribbeanT =olabellanes 657 were isolated from E. laciniata while compounds Ky : and 0 were isolated from E. asperulaT %ll compounds were identified by NMRy éμA9OMSy optical rotation and comparison with previously reported dolabellanesT éμA9OMS analyses showed that dolabellatrienone JKz transforms into compounds : and 0 as oxidation products upon prolonged storage) howevery those compounds were also naturally present in the extract of the studied organismsT Pure dolabellanes were tested in vitro in antiviral assays against qSVA5T μompound 7 inhibited virus replication in infected cells JW:TWI of inhibition at f4 μMz without cytotoxic effect Jμμf4 ; EfEzy showing similar activity to the positive control %cyclovir®T Thusy compound 7 is an interesting candidate for further studies of dolabellanes as antiviralsT 8os dolabellanos son diterpenos con actividad antiviraly la mayor parte de los estudios se han realizado con compuestos aislados de algas pardas del género DictyotaT 8os corales blandos son también una importante fuente de dolabellanosy pero el potencial antiviral de éstos ha sido muy poco estudiadoT Se llevó a cabo el estudio químico de los dolabellanos presentes en los octocorales Eunicea laciniata y Eunicea asperulay recolectados en Santa Martay μaribe colombianoT 8os dolabellanos 6A7 fueron aislados del octocoral E. laciniata mientras que en E. asperula se encontraron los compuestos Ky : y 0T 8a elucidación estructural se llevó a cabo mediante RMNy espectrometría de masasy rotación óptica y comparación con reportes previosT 9l análisis por μéA9M evidenció que la dolabellatrienona JKz se puede transformar en los compuestos : y 0 como producto del almacenamiento prolongadoy no obstantey tales compuestos también estuvieron presentes en los extractos de los organismos estudiadosT 9l compuesto 7 inhibió la replicación del VqSA5 JW:yWI de inhibición en células infectadas a una concentración de f4 μMz sin efecto citotóxico Jμμf4 ; EfEzy mostrando una citotoxicidad similar al %ciclovir®y un control positivoy por lo cual es un candidato para la realización de estudios adicionales sobre el potencial antiviral de los dolabellanosT Os dolabellanos são diterpenos que têm mostrado atividade antiviraly os estudos neste campo estão centrados nos compostos isolados de algas do gênero DictyotaT Os octocorais também são uma fonte importante de dolabellanosy mas não tem sido estudadosT ®oirealizado o estudo químico dos octocorais Eunicea laciniata e Eunicea asperulay coletados em Santa Martay μaribe μolombianoT O estudo químico dos dois organismos permitiu o isolamento dos dolabellanos 6A7 de E. laciniatay enquanto que para E. aspérula foram identificados os compostos Ky : e 0T % elucidação estrutural foi realizada mediante RMNy espectrometria de massasy rotação óptica e comparação com os dados da literaturaT % análise por éμAMS evidenciou que a dolabelatrienona JKz pode gerar os compostos : e 0 como produto de degradaçãoy a partir de um armazenamento prolongadoT No entantoy os compostos também estavam presentes nos extratos dos organismos estudadosT O composto 7 mostrou uma citotoxicidade similar ao %ciclovir®y um controle positivoy numa porcentagem de inibição da replicação do qVSA5 JW:yWI de inibição em células infectadas na concentração de f4 μMz sem efeito citotóxico Jμμf4 ; EfEzy o quetorna esse composto um candidato para o desenvolvimento de antiviraisT


Abstract Resumo
=olabellane diterpenes have considerable antiviral activityy but most studies have been focused towards compounds isolated from Dictyota brown algaeT %lthough soft corals are also a significant source of these diterpenesy their antiviral potential has not been studied in detailT With the aim of assessing the biological activity of marine sourcesy we evaluated the dolabellane content in the soft corals Eunicea laciniata and E. asperula collected in Santa Martay µolombian µaribbeanT =olabellanes 657 were isolated from E. laciniata while compounds Ky : and 0 were isolated from E. asperulaT %ll compounds were identified by NMRy éµA9OMSy optical rotation and comparison with previously reported dolabellanesT éµA9OMS analyses showed that dolabellatrienone JKz transforms into compounds : and 0 as oxidation products upon prolonged storage) howevery those compounds were also naturally present in the extract of the studied organismsT Pure dolabellanes were tested in vitro in antiviral assays against qSVA5T µompound 7 inhibited virus replication in infected cells JW:TWI of inhibition at f4 µMz without cytotoxic effect Jµµ f4 ; EfEzy showing similar activity to the positive control %cyclovir ® T Thusy compound 7 is an interesting candidate for further studies of dolabellanes as antiviralsT 8os dolabellanos son diterpenos con actividad antiviraly la mayor parte de los estudios se han realizado con compuestos aislados de algas pardas del género DictyotaT 8os corales blandos son también una importante fuente de dolabellanosy pero el potencial antiviral de éstos ha sido muy poco estudiadoT Se llevó a cabo el estudio químico de los dolabellanos presentes en los octocorales Eunicea laciniata y Eunicea asperulay recolectados en Santa Martay µaribe colombianoT 8os dolabellanos 6A7 fueron aislados del octocoral E. laciniata mientras que en E. asperula se encontraron los compuestos Ky : y 0T 8a elucidación estructural se llevó a cabo mediante RMNy espectrometría de masasy rotación óptica y comparación con reportes previosT 9l análisis por µéA9M evidenció que la dolabellatrienona JKz se puede transformar en los compuestos : y 0 como producto del almacenamiento prolongadoy no obstantey tales compuestos también estuvieron presentes en los extractos de los organismos estudiadosT 9l compuesto 7 inhibió la replicación del VqSA5 JW:yWI de inhibición en células infectadas a una concentración de f4 µMz sin efecto citotóxico Jµµ f4 ; EfEzy mostrando una citotoxicidad similar al %ciclovir ® y un control positivoy por lo cual es un candidato para la realización de estudios adicionales sobre el potencial antiviral de

General
™n some of our previous studiesC the soft coral Eunicea laciniata collected in Santa MartaC yielded the dolabellane diterpenes β3 araneosene 01T and dolabellatrienone 02T as major compounds in the organic extract 024T´RecentlyC we reported the anti3-™V activity of those compounds and some semisynthetic derivatives 025T´Natural compounds showed low antiviral activity and they did not have considerable cytotoxic activity´On the other handC semisynthetic derivatives were kPP times more active compared with their precursors inhibiting -™V3k replication without a considerable increase in their cytotoxicity´ThusC dolabellane diterpenes are very interesting candidates for the development of new antiviralsF s a part of our ongoing research on antivirals from marine sourcesC herein we show the characterization of six dolabellane diterpenes from the soft corals Eunicea laciniata and Eunicea asperulaC collected at Santa MartaC /olombian /aribbean as well as the evaluation of their antiviral activity assessed as the inhibition of -SV3k replication and cytotoxic activityÍ ntroduction Marine organisms are a prolific source of structurally diverse and bioactive compounds 01T´™nterestinglyC during the qPPq3qPkk periodC k®q new compounds of marine origin with anti3-™V activity on different stages of the replication cycle were reported 02T´Viral diseases are hard to treat and vaccine development against these diseases is still a major challenge and continues to elude the majority of drug discovery programs´-ighly active antiretroviral therapy 0-FFRTT is considered the best alternative of treatment against viral infectious diseases 03, 4T´-FFRT´s main components for treating F™:S for instanceC are nucleoside reverse transcriptase inhibitors 0NRT™sTC non3nucleoside reverse transcriptase inhibitors 0NNRT™sTC and protease inhibitors 0P™sTC although new targets are constantly explored 05T´This kind of therapy has turned F™:S from being a mortal disease to becoming a treatable chronic conditionC improving life quality and expectationÓ n the other sideC Fcyclovir ® and its related compounds constitute the first alternative for treating genital and oral herpes infections caused by -erpes Simplex Viruses k and q due their unsurpassed capacity to attenuate the symptoms and manifestations of these infections 06T´Fs mentioned earlierC although -FFRT is not a definite cure for viral infectionsC it allows management of viral diseases and improves the life quality of patients and is widely used for treating F™:S´-oweverC the emergence of resistant strains and the various side effects reported for antivirals used in -FFRT together with lack of efficacy of some of these substancesC constitute a major challenge to overcome in order to improve this therapy 07, 8T´: olabellanes are diterpenes that have been isolated from natural sources such as plants 09-12TC fungi 013TC and several marine organismsC mainly brown algaeC cnidariansC and sponges 014TŚ tructurallyC they are produced by the ring formation between positions kCkk and kPCk4 in the geranyl3geraniol pyrophosphate to constitute a bicyclic [+´®´P] fused ring´:iterpenes usually exhibit a very interesting range of biological activity including antitumoral 014TC cytotoxic 015,16T antiparasitary 017TC besides a remarkable antiviral activity´:ifferent dolabellanes with antiviral properties have been isolated from brown algae from the genus Dictyota 018, 19T´:olabellanetriol isolated from Dictyota friabilis 0as D. pfaffiiT is the most studied so farC due to its potent inhibition of the human immunodeficiency virus 0-™V3kT and the herpes simplex virus 0-SV3kT 018, 20T´Studies concerning the mechanism of action of dolabellanetriol concluded that the molecule acts as a non3 competitive -™V3k reverse transcriptase inhibitor and classifies it into the non3nucleoside reverse transcriptase inhibitors category 0NNRT™sT 021T´-oweverC despite the significant evidence on the antiviral activity of dolabellane diterpenesC very little has been reported on the antiviral properties of dolabellanes isolated from marine sources other than brown algaeĹ rown algae are an important source of dolabellane diterpenes of marine origin 014TC but soft corals also constitute a prolific source of such compoundsC and in several cases significant ecological roles have been attributed to those 022T´Most dolabellane diterpenes have been isolated from the Flcyonacean genus Clavularia and the Octocorallian genus Eunicea 014T´The /olombian /aribbean harbors are large and possess diverse communities of Eunicea soft corals 023T´O ptical rotation was measured on a F:P44PzC Lellingham zStanley polarimeter´k-and k® /3NMR spectra were recorded on a Lruker FVFN/G 4PP 04PP M-zT and Lruker FVFN/G 0®PP M-zT spectrometers as dilute solutions in /:/l ® 0δ -]´q9 δ / ]]´PT at room temperatureC unless stated otherwise´/hemical shields are quoted in parts per million 0ppmT relative to residual solvent peaks and coupling constants are quoted in -ertz´Kovats index determination and mass spectra analyses were performed on an Fgilent Technologies ]B+PL chromatograph equipped with a -P3[MS column 0®P m x P´q [ mm x P´q[ µmT and an Fgilent [+]]F MS: mass detector using electronic impact ionization 0]P eVT at q+P °/Ḿ ass detection range was selected between m/z qP to BPP= or each analysisC k µL of a k mgDmL solution in acetone was injected in split mode 0kE®PT at an injector temperature of q9P °/elium [´P grade was used as carrier gas maintaining a k mLDmin flux´Temperature program started at 4P °/ maintaining this temperature for kminC followed by a temperature ramp as followsE 9 °/Dmin until kPP °/C 4 °/Dmin until q9P °/C kP °/Dmin until q+P °/C maintaining at q+P °/ for k[ min´Kovats indexes werecalculated based on a mixture of paraffins from / kP to / q[ 0:r GhrenstorferC FugsburgC%ermanyTT hin layer chromatography was developed on aluminum plates precoated with silica gel 9P% = q[4 from Merck´Plates were visualized under ultraviolet light 0λ K q[4 nmT and [V cerium ammonium sulfate with kPV sulfuric acid in methanol followed by heating´Silica gel 0P´P4®-P´P9P mmC MerckTC Sephadex L-3qP 0%G -ealthcare Life SiencesTC and -yperSep ™ :iol cartridges 0Thermo Scientific ™ T were employed for column chromatography separationsŚ olvents of analytical quality were employed and they were acquired from Merck´

Animal material
The octocorals Eunicea laciniata and Eunicea asperula were collected in reef flats along the coast of Santa MartaF =olombian =aribbean by scuba diving in /ecemberF -IUUL at an approximate depth of UI mó The Ministerio de ]mbienteF Vivienda y /esarrollo Territorial granted permission for research on marine organisms N=ontratos de ]cceso a Recurso Genético No 88F UII and UI89ó ]ccording to the permissionsF soft corals were cut carefully and the collected quantities were not enough to cause an ecological impactó Small fragments were cut out of large colonies with sharp scissorsF leaving the remaining colony intactó Fresh material was frozen immediately after collection and remained frozen until extractionó =ollected organisms were identified by Mónica Puyana and voucher specimens were deposited at the invertebrate collection of I=N NInstituto de =iencias NaturalesF Universidad Nacional de =olombia9F coded as I=NzMHNz=RzUI7 and POI-7kF respectivelyó

Organism extraction and compound isolation
The sample of Eunicea laciniata was cut in small pieces and extracted with dichloromethane N/=M9 four timesó The extract was filtered and concentrated under vacuum yielding a dark green oil NUHó3 g9ó The extract was separated by flash column chromatography N-I cm x 7 cm9 eluting with OII mL mobile phases of increasing polarity Nhexane5toluene 8I5UI to OI5OIF tolueneF toluene5ethyl acetate OI5OIF ethyl acetateF ethyl acetate5methanol OI5OIF methanol9 to yield nine fractions NFLU to FL89 according to their TL= profileó The fractions were monitored by U HzNMR looking for signals assignable to methyl groups and double bound protonsF which are common for diterpenesó This analysis allowed us identifying fractions FLU and FLO as those with a high diterpene contentó FLU N378 mgF hex5tol 8I5UI9 was a yellow oiló Its U HzNMR and ]PT spectra showed it was constituted by the dolabellane βz araneosene N19ó Fraction FLO Nk6II mgF tol and hex5tol OI5OI9 was further purified by column chromatography eluting with hex5YtO]c NUII5I to OI5OI9 to yield six fractions NFLOóU to FLOó79ó Subfraction FLOóH corresponded to pure dolabellatrienone N29 NUk3I mg9ó Subfraction FLOóO NO--mg9 was separated through a /iol cartridge with hex5YtO]c mixtures N3I5-I to I5UII9 to yield seven fractions NFLOóOóU to FLOóOók9ó Subfraction FLOóOó-was separated using column chromatography to yield eight fractions NFLOóOó-óU to FLOóOó-ó39ó Subfraction FLOóOó-óH NHOó6 mg9 was finally purified by a Sephadex LHz-I column eluting with MeOH to yield pure compound 3 NU3ó6 mg9ó Subfraction FLOóOó-óO corresponded to pure compound 4 NH6ó3 mg9ó Subfraction FLOóOó-ók was purified by a Sephadex LHz-I column eluting with MeOH to yield pure compound 5 NU3ó3 mg9ó Subfraction FLOó7 was fractionated using a /iol cartridge eluting withóhex5YtO]c mixtures NkI5HI to I5UII9 to obtain seven subfractions NFLOó7óU to FLOó7ók9ó Subfraction FLOó7óH NH-ók mg9 was separated through silica gel column chromatography eluting with hex5YtO]c NkI5HI9 to obtain pure compound 6 NkóU mg9ó The sample of Eunicea asperula NUk6 g dry weight9 was extracted and fractionated using the same methodology previously explained for E. laciniataó The crude extract NHó-g9 was obtained as a dark green oiló Silica gel flash chromatography yielded nine fractions NF]U to F]89 based on their TL= profilesó Fraction F]-N-6Hó8 mgF toluene9 was separated through column chromatography eluting with hex5YtO]c N8I5UI to 7I56I9 to obtain pure compound 2 N3I mg9ó Fraction F]6 NU63I mgF tol5YtO]c OI5OI9 was fractionated with a /IOL cartridge eluting with hex5YtO]c N8I5UI to OI5OI9 yielding five subfractions NF]6óU to F]6óO9ó Fraction F]6ó-corresponded to pure compound 4 N33I mg9ó Fraction F]6ó6 was separated through column chromatography eluting with a gradient of hex5YtO]c N8I5UI to kI5HI9 to obtain pure compound 5 N-IóH mg9ó Compound 1 was isolated as a yellowish oil and was the major component in fraction FLz obtained from E. laciniata, GC(MS analysis indicated that compound 1 was pure enough and was identified without further purification steps, The analysis showed a major peak with a molecular ion at m/z E )A) corresponding to [M] R and consistent with a C )8 H 9) molecular formula, Compound 1 z H( NMR spectrum had signals in δ H 6,)6 HzH5 dd5 J E zz,85 v,P Hz5 H(93 and v,q8 HzH5 br d5 J E z8,v Hz5 H(A3 corresponding to olefinic protons and five additional signals were observed in δ H z,O6 H9H5 s5 H(zA35 z,O9 H9H5 s5 H(zq35 z,O8 H9H5 s5 H()835 z,v6 H9H5 s5 H(zO3 and z,z9 H9H5 s5 H(z63 corresponding to methyl groups, On the other hand5 the APT spectrum of 1 showed twenty signals5 which were assigned as five methyls5 seven methylenes Hall sp935 three methines Hone sp 9 and two sp ) 35 and five quaternary carbons Hone sp 9 and four sp ) 3, All this information suggested that compound z was a dolabellane diterpene with two trisubstituted and one tetrasubstituted double bonds, Comparison of spectral data were consistent with those reported for the known dolabellane β( araneosene obtained by total synthesis H263 and allowed us to identify compound 1, The stereochemistry at the ring fusion was defined as zS5 zzR based on the similar value of optical rotation comparable to that obtained by total synthesis, Compound 2 was isolated as a yellow oil, z H(NMR and APT spectra were quite similar to those obtained for compound 1, However5 minimal differences suggested the presence of additional functional groups, z H(NMR spectrum had five methyl signals but two of them were in δ H ),)8 H9H5 br s5 H()83 and z,Pz H9H5 s5 H(zq3 while the APT spectrum showed a signal in δ C )8A,) HC(z93 together with a deshielded methylene in δ C 6v,P HCH ) (zv3, These observations were consistent with the presence of a conjugated ketone carbonyl and justified the observed signals, The spectroscopic properties and experimental optical rotation H Rz6,8 Hc 8,q CHCl 9 33 were consistent with those reported in dolabellatrienone H23 H R9),8 Hc 8,zv CHCl 9 335 a dolabellane previously isolated from several Eunicea species and also obtained by total synthesis H273, Compound 3 was isolated as a colorless oil, zH(NMR signals in δ H 6,zz HzH5 d5 J E q,z Hz3 and ),A) HzH5 t5 J E9,v Hz3 corresponded with an olefinic proton and an oxygenated methine5 respectively, Additionally5 five methyl signals were also identified in δ H z,A8 H9H5 s35 z,)) H9H5 s35 z,zv HzH5 d5 J E A,8 Hz35 8,qA HzH5 d5 J E A,8 Hz35 8,PP H9H5 s3, The APT spectrum showed signals for five methyls Hδ C zO,P5 zA,v5 zP,z5 )z,9 and )),P35 six methylenes5 H)6,95 )O,95 99,P5 9P,z5 v8,95 6P,O35 five methines5 one olefinic5 one oxygenated Hδ C )A,q5 v),85 O8,)5 O8,v and z)6,P35 and four quaternary carbons5 one oxygenated Hδ C 6q,P35 one olefinic Hδ C z96,z35 and a ketone carbonyl Hδ C )zq,)3, We inferred that compound 3 was a dolabellane diterpene with a trisubstituted double bond and an epoxide group5 and the structural elucidation process was conducted by )D(NMR spectroscopy, However5 compound 3 decomposed during the experiments suggesting an unexpected lability and a low probability of application as an antiviral compound, Despite this5 we proposed a planar structure for compound 3 based on information deduced from zD( NMR spectra and comparison with other dolabellanes, We observed two doublet methyls with chemical shifts and coupling constants quite similar to a previous reported dolabellane without a double bound between positions C(z) and C(zP H313, Rev. Colomb.Quim.20175 46 Hz35 6(z), Moreover5 signals at δ C )zq,) and O8,) indicated the presence of a ketone with and an alfa methine5 and they were also consistent with those reported in the known compound, The position of the double bond position was deduced taking advantage of the high degree of conservation in the chemical shifts for double bonds in dolabellanes, We have observed in many dolabellanes that the chemical shift for carbon atoms in a double bond between C(9 and C(v differs from that showed by a double bond between C(A and C(P H17, 313, Compound 9 had the expected signals for a double bond between C(9 and C(v while the epoxide group was located between C(A and C(P, Figure ) shows the planar structure of compound 3 and some related structures that were useful to our elucidation purposes, Compound 4 was isolated as a white solid, The EIMS spectrum showed a molecular ion corresponding to [M] R in m/z E 98), z H and z9 C(NMR Hz88 MHz5 CDCl 9 3 spectra showed similarities with dolabellatrienone H23 but there were differences that indicated the presence of an additional functional group in 4, It was observed one signal at δ H ),q8 HzH5 br d5 J E P,A Hz5 H(A3 together with signals at  HzR59R5vR5AR5PR5zzS3(di(95vxA5P(epoxy(z9(keto(dolabell(z)HzP3(ene5 a dolabellane previously isolated from an unidentified Eunicea species and whose absolute stereochemistry was determined previously H173, Compounds 1(6 were isolated from Eunicea laciniata whereas only compounds 25 45 and 5 were isolated from E. asperula5 where 4 was the major compound5 and 2 and 5 were minor components, Previous works on the chemistry of this particular species5 showed that E. asperula collected in Tobago yielded cembrane and asperketal diterpenes H333, Although variations in chemical profiles is not unusual between organisms collected at different places5 the diterpene core usually remains the same H345 353, Interestingly5 our sample yielded dolabellane diterpenes suggesting it might be a different chemotype5 assuming5 in both studies5 that taxonomic identity is correct, Previous studies with E. laciniata have shown the high dolabellane content of this species and the considerable number of compounds with variable oxygenation gradej Samples collected in Puerto RicoE BahamasE TobagoE HondurasE and Colombia have reported the isolation of dolabellanes βMaraneosene N1F and dolabellatrienone N2F N14, 24Fj In this work we report the isolation of compounds 3M6 from E. laciniata which were not previously described from this speciesj NeverthelessE it was found that following purificationE compound 2 experiments an oxidation process to yield a mixture of compounds 4 and 5j Figure :a shows a GCMEIMS analysis for compound 2 Ninitially pureF and after several weeks of storagej The chromatogram shows a major peak corresponding to compound 2 and two minor peaks with Kovats index and EIMS coincident with 4 and 5j InterestinglyE compound 5 has been reported as a natural product only from a Clavularia soft coral collected in Japan N36Fj Considering thisE the isolation of compounds 4 and 5 in this study suggested that these could be artifacts produced during storage of the extract and fractionsj AlternativelyE the presence of those compounds in a particular sample could reflect a different chemotypej In order to establish if compounds 4 and 5 were storage artifactsE a new GCM EIMS analysis was conducted after extraction and fractionation of a fresh specimenj As an additional precautionE fractions were kept under argon atmosphere and frozen at temperature below zeroE dissolving them just before the chromatographic analysisj In E. laciniataE compounds 2E 4 and 5 were identified in the fraction that eluted with hex®EtOAc WJ®:J NFigure :cFj Compound 6 was not found in this fraction nor in those of greater polarityj  We conclude that although minimal quantities of compounds 4 and 5 are present in the extracts from E. laciniataE the absence of compound 6 suggests that both monoepoxides are mainly oxidation products of N2F in this soft coralj On the other handE compound 2 and the major compound 4 were found in E. asperula but compound 5 was not NFigure :bFE suggesting that compound 5 is the only oxidation product of N2F in E. asperulaj Vero cells have been used to evaluate antiMHSVMA activity of a wide range of natural productsE including dolastane and dolabellane diterpenes N20Fj Cytotoxic activity in Vero cells was determined for compounds 2E 4E 5E 6 with Acyclovir ® as a positive controlj We did not test compound 1 since it was not pure enough to run the assaysj Compounds 2 NCC 7J 6 AO0 µMF and 5 NCC 7J 6 O0W µMF showed a greater cytotoxic effect as compared to Acyclovir ® NCC 7J 6 V0J µMF while compounds 4 NCC 7J 6 7z0 µMF and 6 NCC 7J 6 V7V µMF had comparable valuesj An important feature for any antiviral compound is that it must not be toxic and must be selective to infected cellsj For this reasonE we decided to evaluate antiviral activity only for compounds 4 and 6 in an HSVMA inhibition assay using concentration of 7J µM for each compoundj Compound 4 showed a Ozj0C of inhibition while compound 0 exhibited a W:jWC of inhibitionj Considering that 6 also has a remarkable in vitro antiMHIVMA activity N25FE it is a potential candidate to perform further studies to assess its full potential as an antiviral agent as well as understanding its mechanism of action against viruses including HSVMAj 4f is considered that variations in the family of isolated diterpenes do not depend on locationA either this work studied a different chemotype or there were problems with taxonomical characterization2 8oweverA further studies should be developed in order to confirm this hypothesis2 39®;46á analyses proved that prolonged storage of pure dolabellatrienone 125 induces a spontaneous oxidation to yield compounds 4 and 52 This finding evidences a need to improve the manipulation and storage conditions of extracts and fractions obtained from those organisms in order to minimize the artifact formation and increase the reproducibility of chemical studies2 àiological assays showed that compound 6 inhibited 8áV®í replication at a concentration of "N µ6 and was not cytotoxic2 çlthough the remaining compounds were more toxic than çcyclovir ® A their antiviral activity should be determined to calculate selectivity indexes and establish their potential as antivirals2 These results encourage us to continue the study of dolabellanes of marine origin and evaluating their antiviral activity2 Revf Colombf Quimf 2017A 46 1í5A "®íó2

References Conclusions
áix dolabellane diterpenes from the soft corals Eunicea laciniata and Ef asperulaA collected in áanta 6artaA 9olombian 9aribbean áeaA were isolated in this work2 9ompounds 1A 2A 4®6 were identified by spectroscopic means and compared with previously reported dolabellanes2 9ompound 3 was not fully characterized due to its decompositionA suggesting it had a high degree of reactivity2 Wolabellatrienone 125 and 1íRAJEAjRA"RAííS5®jA"®epoxy®íJ®keto® dolabella®JAíó1í"5®diene 145 were the major compounds isolated from Ef laciniata and Ef asperulaA respectively2 To the best of our knowledgeA this is the first study of Ef asperula collected in the 9olombian 9aribbean and dolabellanes were found as the major diterpenes2 Figure 15 Dolabellanes isolated from soft corals E. laciniata (146) E. asperula (2, 445) in this study.
δ C O6,P HCH(A3 and O8,O HC(P3 which corresponded to an epoxide functionality, As previously explained5 the double bond was located between C(9 and C(v due to the observed chemical shifts for the trisubstituted double bond in δ C z)v,P HCH(93 and z9A,O HC(v3, The experimental optical rotation and spectroscopic properties were consistent with those reported for the compound HzR59E5AR5PR5zzS3(A5P(epoxy(z9(keto(dolabella(95z)HzP3(diene and allowed us identifying compound 4 H323, Compound 5 exhibited z H(NMR5 APT5 and EIMS spectral data almost identical to compound 45 with minimal differences that indicated they were structural isomers, The observed signals at δ H ),qA HzH5 dd5 J E zz,85 ),q Hz5 H(935 δ C Ov,8 HCH(935 Oz,O HC(v3 allowed confirming the presence of an epoxide group while signals at δ C z99,z HC(P3 and z)P,v HCH(A3 located the double bond between C(A and C(P, Compound 5 was identified by comparison with the spectroscopic properties and optical rotation values reported in the total synthesis of the dolabellane claenone H273, Compound 6 showed an EIMS with a molecular ion at m/z E 9zP5 which was consistent with a C )8 H 98 O 9 molecular formula, The z H(NMR spectrum showed signals at 9,86 HzH5 dd5 J E zz,z5 ),A Hz5 H(93 and ),qO HzH5 d5 J E A,P Hz5 H(A3 while the APT spectrum showed four signals for oxygenated carbons at δ C O9,A HC(A35 O9,8 HCH(935 O8,O HCH(P3 and O8,9 HC(v3, This information confirmed the presence of two epoxide groups and indicated that compound 6 was a diepoxide analogue of 4 and 5, The remaining signals and experimental optical rotation allowed us to identify compound 6 as