Biodiversidad y Conservación de Recursos Fitogenéticos. Las Amarillidáceas como Fuente de Productos Bioactivos

Pedro Miguel Pérez Guerrero

Biodiversidad y Conservación de Recursos Fitogenéticos. Las Amarillidáceas como Fuente de Productos Bioactivos Natalia Belén Pigni Aquesta tesi doctoral està subjecta a la llicència ReconeixementCompartirIgual 3.0. Espanya de Creative Commons. NoComercial – Esta tesis doctoral está sujeta a la licencia Reconocimiento - NoComercial – CompartirIgual 3.0. España de Creative Commons. This doctoral thesis is licensed under the Creative Commons Attribution-NonCommercialShareAlike 3.0. Spain License. !"# $%&"'"# "&"(!" ()%!*%!# +#&" "(&,&+(''+-&+ # (" %!. $!&"&' " -(+%" +-&+ "%'" ! -&+ "&"(!" ()%!*%! /0"1 "$&!'" %*+( 'Z/D/EdK^ ! " # $ " % " & & ' ( #) ) * % " ) + , $ - ) # %- # . * & / 0 1 + & 2 -) 3 4 5 2 % % % - ( %6 % * 5 ) 4 % % * # ) 7 . % 3 ) 0 4 + 0 && 89: ! % !4 #;1&94-' + ;345' # ' < % ' < 4 ' 4 % 4 ;345*&< % % 4 % 4 "* 2= )4 * * ;* % < $ 0 2 %" ! % ' $ + ! > % 1 ' % 8 $ / ! :5-$;% < # ' & ;& < 3 3& ;4" < ! %* !* + ;% -+< #+ " 1 * 5 %4 ! 1 ) ! ? '*: ' ) # # % > # #7@13%&=%-A ÍNDICE 1.INTRODUCCIÓN ......................................................................................................................... 1 1.1.ProductosNaturalesyDesarrollodeFármacos ................................................................. 3 1.2.PlantasdelaFamiliaAmaryllidaceaeysusAlcaloides ....................................................... 4 1.2.1.QuímicayBiosíntesis................................................................................................... 5 1.2.2.ActividadBiológica .................................................................................................... 10 1.3.GC9MSyRMN:TécnicasClaveenelEstudiodeAlcaloidesdeAmaryllidaceae............... 14 1.3.1.CromatografíadeGasesEspectrometríadeMasas(GCMS) ................................. 14 1.3.1.1.Tipolicorina........................................................................................................ 16 1.3.1.2.Tipohomolicorina .............................................................................................. 17 1.3.1.3.Tiposcrininayhemantamina ............................................................................. 18 1.3.1.4.Tipotazetina....................................................................................................... 19 1.3.1.5.Tipomontanina .................................................................................................. 20 1.3.1.6.Tipogalantamina................................................................................................ 20 1.3.2.ResonanciaMagnéticaNuclear(RMN) ..................................................................... 22 1.3.2.1.Tipolicorina........................................................................................................ 23 1.3.2.2.Tipohomolicorina .............................................................................................. 23 1.3.2.3.Tiposcrininayhemantamina ............................................................................. 24 1.3.2.4.Tipotazetina....................................................................................................... 25 1.3.2.5.Tipomontanina .................................................................................................. 25 1.3.2.6.Tipogalantamina................................................................................................ 25 2.OBJETIVOS ............................................................................................................................... 27 2.1.Objectives......................................................................................................................... 30 3.RESULTADOS ........................................................................................................................... 31 3.1.Artículo1:TwonewalkaloidsfromNarcissusserotinusL. .............................................. 35 3.2.Artículo2:AlkaloidsfromNarcissusserotinus ................................................................. 45 3.3.Artículo3:WilddaffodilsofthesectionGanymedesfromtheIberianPeninsulaasa sourceofmesembranealkaloids ............................................................................................ 53 3.4.Artículo4:WildArgentinianAmaryllidaceae,anewrenewablesourceof acetylcholinesteraseinhibitorgalanthamineandotheralkaloids.......................................... 99 4.DISCUSIÓN............................................................................................................................. 111 4.1.AlcaloidesdeNarcissusserotinusL. ............................................................................... 113 4.1.1.Narseronina............................................................................................................. 115 4.1.2.1O(3´acetoxibutanoil)licorina .............................................................................. 116 4.1.3.DerivadosdeNarcisidina......................................................................................... 116 4.1.4.2Metoxipratosina................................................................................................... 117 4.1.5.11Hidroxigalantina................................................................................................. 118 4.1.6.2OMetilclivonina................................................................................................... 118 4.2.AlcaloidesdeNarcisosdelaSecciónGanymedes .......................................................... 119 4.2.1.AlcaloidesdeN.triandrusL. .................................................................................... 120 4.2.1.1.29Oxomesembrenona ...................................................................................... 121 4.2.1.2.7,7a9Dehidromesembrenona ........................................................................... 121 4.2.1.3.29Oxoepimesembranol..................................................................................... 121 4.2.2.AnálisisdelContenidodeAlcaloidesdeEspeciesdelaSecciónGanymedes........... 122 4.3.AmaryllidaceaeArgentinascomoFuentedeAlcaloidesBioactivos............................... 124 5.CONCLUSIONES ..................................................................................................................... 127 5.1.Conclusions..................................................................................................................... 131 6.BIBLIOGRAFÍA ........................................................................................................................ 133 7.ANEXOS ................................................................................................................................. 143 7.1.AnexoI:CapítulodeRevisión.ChemicalandbiologicalaspectsofAmaryllidaceae alkaloids................................................................................................................................. 145 7.2.AnexoII........................................................................................................................... 183 7.2.1.Narseronina............................................................................................................. 184 7.2.2.1O(3´Acetoxibutanoil)licorina.............................................................................. 186 7.2.3.3OMetilnarcisidina................................................................................................ 188 7.2.4.1OAcetil3Ometilnarcisidina............................................................................... 190 7.2.5.1OAcetil3Ometil6oxonarcisidina .................................................................... 192 7.2.6.2Metoxipratosina................................................................................................... 194 7.2.7.11Hidroxigalantina................................................................................................. 196 7.2.8.2OMetilclivonina................................................................................................... 198 7.2.9.2Oxomesembrenona .............................................................................................. 200 7.2.10.7,7aDehidromesembrenona ................................................................................ 202 7.2.11.2Oxoepimesembranol .......................................................................................... 204 1.INTRODUCCIÓN Introducción 1.INTRODUCCIÓN 1.1.ProductosNaturalesyDesarrollodeFármacos Alolargodelahistoria,elhombrehatenidoquevalersedeunagranvariedadde recursos naturales para sobrevivir. En el campo de la medicina tradicional, por ejemplo,elusodehierbasmedicinalesparaeltratamientodediversasafeccionesdela salud se remonta a miles de años atrás, con registros que datan de la antigua Babilonia, Egipto, India y China. En la industria farmacéutica moderna, a pesar de la gran variedad de moléculas derivada de los progresos en el ámbito de la química combinatoria, los productos naturales continúan desempeñando un papel fundamental en el desarrollo de fármacos (Ngo et al., 2013). Tal como señalan Newman y Cragg (2012) en su última revisión sobre las fuentes de nuevos fármacos aprobados durante el período de 30 años comprendido entre 1981 y 2010, la contribucióndeestaáreadeinvestigaciónhasidomuysignificativa.Pormencionarun ejemplo, de todas las pequeñas moléculas aprobadas como fármacos, cerca del 35% correspondeacompuestosdeorigennaturalyderivadossemisintéticos,mientrasque el 30% son moléculas sintéticas inspiradas en productos naturales o con un farmacóforodesarrolladoapartirdeuncompuestonatural. Entre los recursos naturales disponibles, los vegetales representan una fuente importantedemoléculasestereoespecíficasconestructurascomplejas.Estacapacidad para producir metabolitos secundarios con diversas actividades biológicas suele explicarse como parte de una estrategia de supervivencia contra el ataque de herbívoros, o bien para facilitar una adecuada dispersión. La gran influencia de las plantas en el desarrollo de fármacos se ejemplifica adecuadamente con el conocido alcaloide morfina, aislado por primera vez a principios del siglo XIX de Papaver somniferumytodavíaampliamenteaplicadoenlamedicinaactualparaeltratamiento deldolor(Houghton,2001). A pesar del enorme desarrollo en el campo de la síntesis orgánica, aún hoy existencasosdefármacosqueseobtienendirectamenteapartirdelmaterialvegetal, loqueresultaenunanecesidadcrecientedegenerarfuentesrenovablesypromoverla proteccióndelabiodiversidadvegetal(LubbeyVerpoorte,2011).Elusoracionaldelos recursosnaturalesesesencialparaevitarunposibledeterioroirreversible. 3 Introducción 1.2.PlantasdelaFamiliaAmaryllidaceaeysusAlcaloides La familia botánica Amaryllidaceae, un grupo de monocotiledóneas pertenecientes al orden Asparagales, ha sido objeto de debate taxonómico durante mucho tiempo. Según la última clasificación actualizada del APG (Angiosperm PhylogenyGroup),sustentadaporvariosanálisismolecularesymorfológicos(Meerow etal.,1999;MeerowySnijman,2006),lafamiliaAmaryllidaceaeJ.St.9Hil.comprende tres subfamilias: Agapanthoideae, Allioideae y Amaryllidoideae las cuales, a su vez, habíansidopreviamenteconsideradascomoAgapanthaceae(comúnmenteconocidos comoagapantos),Alliaceae(incluyendoalajoylasespeciesafines)yAmaryllidaceae (Chase et al., 2009; APG III, 2009). Cabe señalar que, a pesar de que la estrecha relación entre estos tres grupos está generalmente aceptada, la modificación de la nomenclaturapuededarlugaramalentendidos,yaqueelconceptomásampliamente utilizadorelacionadoconeltérmino"Amaryllidaceae"implicasólounasubfamiliadel taxónactual. LasplantasdelasubfamiliaAmaryllidoideae,objetodeesteestudio,sonhierbas perennes y bulbosas que suelen presentar flores llamativas, lo que les proporciona valor ornamental. Se han reconocido 59 géneros, tales como Crinum, Hippeastrum, Zephyranthes,Narcissus,Galanthus,entreotros,yalrededorde800especies(Stevens, 2012).Sudistribucióngeográficaescosmopolita,incluyendoprincipalmenteregiones tropicales y subtropicales, pero también es común encontrar algunos géneros en el áreadelMediterráneoyenzonastempladasdeAsia(Figura1.1).Estudiosfilogenéticos apuntan a Sudáfrica y América del Sur como centros de diversificación primaria y secundaria,respectivamente(Itoetal.,1999).Dadoquenumerosasespeciesdeeste grupo son endémicas y muy vulnerables, existe la necesidad de promover su conservaciónymejorarelconocimientoexistentesobrelasmismas. UnadelascaracterísticasmásinteresantesdelasplantasdeAmaryllidoideaees la presencia de un grupo de alcaloides exclusivo, los cuales han sido objeto de investigacióndurantemásde150años(Bastidaetal.,2006).Teniendoencuentalos recientescambiosensuclasificacióntaxonómicayamencionados,esimportantehacer hincapié en que estos compuestos no son típicos de las subfamilias Allioideae y Agapanthoideae,aunquecontinúansiendoampliamenteconocidoscomoalcaloidesde 4 Introducción "Amaryllidaceae". Estos alcaloides se han aislado a partir de todos los géneros de la subfamiliaAmaryllidoideae,ypuedenserconsideradoscomomarcadoresquímicos. Figura1.1:DistribuciónmundialdelafamiliaAmaryllidaceae(subfam.Amaryllidoideae). Imagentomadadewww.thecompositaehut.com. ElgraninterésenelestudiodelosalcaloidesdeAmaryllidaceae,especialmente enelámbitodelafarmacología,sedebeasuampliorangodeactividadesbiológicas, que incluyen, entre otras, propiedades antivirales, antitumorales y antiparasitarias (Bastida et al., 2011). De hecho, estas plantas han sido utilizadas como hierbas medicinalesdurantemilesdeaños.EnelsigloIVa.C.,elaceitedeNarcissuspoeticusL. ya era conocido por Hipócrates de Cos por ser adecuado para el tratamiento de tumores,mientrasqueenelsigloId.C.,sehabíaestablecidoparaestefinenOriente Medio y en el Imperio Romano (Pettit et al., 1986). En la actualidad, uno de los compuestos más interesantes del grupo es la galantamina, un inhibidor potente, reversible y competitivo de la enzima acetilcolinesterasa (AChE), aprobado y comercializado como estrategia para el tratamiento paliativo de la enfermedad de Alzheimer(Reminyl®,Razadyne®). 1.2.1.QuímicayBiosíntesis Desde el aislamiento de licorina a partir de Narcissus pseudonarcissus en 1877 hastaelpresente,sehancaracterizadomásde400alcaloidesdelasAmaryllidoideae (Jin, 2009), y todos ellos están relacionados a nivel biosintético. En general, se clasifican en nueve grupos diferentes representados por: norbelladina, licorina, 5 Introducción homolicorina, crinina, hemantamina, narciclasina, tazetina, montanina y galantamina (Figura 1.2) (Bastida et al., 2011). Sus particularidades químicas más notables se resumenacontinuación(BastidayViladomat,2002): 1. Una estructura base C69C19N9C29C6, en la que la porción C69C1 deriva del aminoácidoL9fenilalaninayelfragmentoN9C29C6provienedeL9tirosina. 2. SonbasesmoderadamentedébilesconpKaentre6y9. 3. La mayoría contiene un solo átomo de nitrógeno, el cual puede ser secundario, terciario o incluso cuaternario. Típicamente, el número de carbonos varía entre 16 y 20, según los sustituyentes del sistema policíclico. 12 11 OH 3 HO HO 4 HO 2 10 5' 6' 4' 5 1 E 6 HO NH 1' 3' 2' D O 9 O H 10b 10a 6a 8 7 norbelladina 4 3 3 H E' MeN 2 1 4 MeO H 10 N 2 1 H 12 MeO 6 licorina 6a 8 H 10b 10a 9 11 4a 4a O 6 7 O homolicorina OH 2 1 2 OH 3 11 11 10 10 O 10a 9 H O 4a 10b 8 6a 7 N 4 O 10a 9 10b H 12 O 8 6 6a 4 4a 2 OMe OH 3 1 1 9 10b O 8 6a OH hemantamina crinina 4a OH NH 6 7 6 7 4 10a 12 N OH H 10 O 3 O narciclasina OMe 2 10 O O 4 H NMe 4a 1 10a 9 11 10b 8 O 6a 7 OH 2 3 6 tazetina 12 1 3 10a 9 4a OH O 12 8 6a 7 OH 3 1 O 2 11 11a 10 O OMe MeO 4 10b 10 10a 9 4a 11 12 4 N H 6 montanina N 6a 8 7 6 Me galantamina Figura 1.2: Alcaloides de Amaryllidaceae representativos de los 9 grupos. La numeración correspondealapropuestaporGhosaletal.(1985). 6 Introducción La ruta biosintética que origina esta variedad de estructuras sigue un esquema general de cuatro etapas que comienza con la preparación enzimática de los precursoresapartirdelosaminoácidosL9fenilalanina(L9phe)yL9tirosina(L9tyr).Enlos alcaloidesdeamarillidáceas,L9phesirvecomoprecursorprimariodelfragmentoC69C1, que corresponde al anillo A y la posición bencílica (C96), mientras que L9tyr es precursordelanilloC,lacadenalateraldedoscarbonos(C911yC912)yelnitrógeno, C69C29N.ParaformarlaporciónC69C1,L9pheesconvertidaenaldehídoprotocatéquico a través de la vía de ácidos cinámicos, involucrando la participación de la enzima fenilalanina amonio liasa (PAL). Por otro lado, L9tyr es mínimamente modificada medianteunpasodedescarboxilaciónantesdeserincorporada(Figura1.3)(Bastidaet al.,2011). Lasegundaetapadelabiosíntesisimplicalafusiónentretiraminayelaldehído protocatéquico,quedalugaranorbelladinapormediodelaformacióndeunabasede Schiff.Estareacciónclaverepresentalaentradadelosmetabolitosprimariosaunavía metabólicasecundaria.Laposteriormetilacióndenorbelladinaenlaposiciónorto(4´) delanilloAseconsideraeltercerpaso. NH2 HO COOH COOH L-Tyr L-Phe Tyr-descarboxilasa PAL R1 HO HO R2 COOH HO CHO ácido trans-cinámico, R1=R2=H aldehído protocatéquico ácido para-cumárico, R1=OH, R2=H HO ácido cafeico, R1=R2=OH base de Schiff H2N H 2N tiramina HO HO HO HO (estructuras isoméricas en solución) N HO H NH O N H HO HO OH HO HO NH norbelladina Figura1.3:Rutabiosintéticahastanorbelladina. 7 Introducción Finalmente, la última etapa incluye una serie de reacciones secuenciales que resultan en la diversificación hacia los ocho esqueletos restantes mostrados en la Figura 1.2. El paso inicial consiste en una ciclación secundaria producida por el acoplamiento oxidativo de O9metilnorbelladina, el cual puede transcurrir a través de tres vías diferentes para dar lugar a las distintas estructuras. El acoplamiento fenol oxidativoortopara´resultaenlaformacióndelesqueletotipolicorina,apartirdelcual seoriginanloscompuestosdetipohomolicorina.Porotrolado,laciclaciónsecundaria parapara´ produce los alcaloides con estructura base 5,10b9etanofenantridina (tipos crininayhemantamina),loscualespuedensufrirciertasmodificacionesparagenerar las estructuras de tipo tazetina, narciclasina y montanina. De manera similar, los alcaloidesdetipogalantamina,consunúcleodibenzofuranocaracterístico,derivande unacoplamientofenoloxidativoparaorto´(Figura1.4). OH MeO 4' NH HO O-metilnorbelladina para-para' orto-para' para-orto' OH OH HO MeO MeO N HO tipos O MeO N HO NH tipos tipos licorina crinina homolicorina hemantamina galantamina tazetina narciclasina montanina Figura1.4:Víasalternativasdeacoplamientofenoloxidativo. Recientemente,apartirdelaislamientodenuevosalcaloidesdeespeciesdelos géneros Cyrtanthus, Narcissus y Galanthus (Brine et al., 2002; de Andrade et al., 2012a; Ünver et al., 1999), se han propuesto algunos subgrupos adicionales de estructuras, tales como las gracilinas, que incorporan un esqueleto 10b,4a9 8 Introducción etanoiminodibenzo[b,d]pirano;lasplicaminas,compuestosdinitrogenadosenlos que el oxígeno del anillo Bde tazetinaestá reemplazado por un nitrógeno que, a su vez, presenta un sustituyente de tipo hidroxifenetil; y el galantindol (Figura 1.5) (Ünver, 2007).Conrespectoasubiosíntesis,lasgracilinasposiblementeseoriginanapartirde alcaloides de tipo hemantamina, mientras que las plicaminas proceden muy probablementedeltipotazetina(deAndradeetal.,2012b). OMe C O NMe O O O A B O H NMe D O NH O O O HN OH OH gracilina plicamina galantindol Figura1.5: Estructurasrepresentativasdelossubgruposadicionalespropuestos. Además, se han encontrado otras estructuras inusuales en plantas de la subfamilia Amaryllidoideae, tales como (9)9capnoidina y (+)9bulbocapnina, ambos consideradosalcaloidesisoquinolínicostípicos,quehansidoidentificadosenlaespecie Galanthusnivalissubsp.cilicicusdeTurquía(Kayaetal.,2004).Sinembargo,hastala fecha, los compuestos inusuales siempre se han hallado en compañía de alcaloides típicosdeAmaryllidaceae. Otro caso curioso corresponde a los alcaloides de tipo mesembrano, característicosdelgéneroSceletium(Aizoaceae)deSudáfrica(Figura1.6)(Smithetal., 1996). Estos compuestos han sido encontrados en algunas especies de Amaryllidoideae, incluyendo Narcissus pallidulus, Crinum oliganthum, y Narcissus triandrus (Bastida et al., 1989; Döpke y Sewerin, 1981; Seijas et al., 2004). En un principio, su similitud estructural con los alcaloides de tipo crinano sugirió la posibilidad de una ruta biosintética en común con los alcaloides de Amaryllidaceae (Jeffsetal.,1971a),peroestudiosposterioresrevelaronprocesosfundamentalmente diferentes, aunque con la participación de los mismos aminoácidos precursores (Gaffney,2008). 9 Introducción OMe OMe OH OMe OMe N O mesembrina N joubertiamina N N O sceletium A4 Figura1.6: Estructurasrepresentativasdelosalcaloidesdetipomesembrano. 1.2.2.ActividadBiológica Tal como ocurre con muchos grupos de plantas, el estudio detallado de los componentesactivosdelasespeciesdeAmaryllidoideaehasidoocasionadograciasa laobservacióndesuusotradicionalenlamedicinapopular,yelgéneroNarcissusesun buen ejemplo. Como ya se hamencionado, algunas especies se han utilizado para el tratamientodetumoresdesdehacemásdedosmilaños,perotambiénsehadescrito, entre otros, su uso en aplicaciones locales para heridas, tratamiento de trastornos respiratorios y como antieméticos (Bastida et al., 2006). Si bien un gran número de extractosvegetaleshansidoensayadosdemostrandounampliorangodeactividades biológicas, el aislamiento de sus alcaloides puros, junto con su síntesis en algunos casos,asícomolosresultadosobtenidosdeestudiosderelaciónestructura9actividad (SAR), han permitido establecer ciertas actividades para los diversos tipos de estructuras. Elalcaloidehalladoconmásfrecuenciadentrodeestegrupoeslicorina,quefue elprimeroenseraislado.Sehareportadosuactividadcomopotenteinhibidordela biosíntesis de ácido ascórbico, del crecimiento y la división celular, y de la organogénesis en plantas superiores, algas y levaduras. En animales, ha demostrado una importante actividad antitumoral, siendo considerado un agente quimioterapéutico prometedor debido a su efecto antiproliferativo selectivo, más marcado en células cancerosas que en células normales (Lamoral9Theys et al., 2009; McNultyetal.,2009).Otraactividaddestacabledelosalcaloidesdetipolicorinaessu potente efecto inhibidor sobre parásitos, habiéndose reportado que su actividad frenteaTrichomonasvaginalistranscurreatravésdelamuertecelularmediadaporun 10 Introducción mecanismoconocidocomoparapoptosis(Giordanietal.,2011),ademásdenumerosos ensayos que señalan la inhibición de Plasmodium falciparum y Trypanosoma brucei (Toriizukaetal.,2008).Licorinaypseudolicorinatambiénhandemostrado,entreotras propiedades,efectosantivirales,asícomolacapacidaddeinteraccionarconelADN.Al igualqueotrostiposdealcaloidesdelgrupo,licorinaysusderivadospresentanefectos analgésicosehipotensores(Bastidaetal.,2011). Algunos compuestos de tipo homolicorina muestran actividad citotóxica e hipotensora, mientras que la propia homolicorina también ha demostrado actividad antirretroviral.Lahipeastrina,alcaloideactivofrenteaHerpessimplextipoI,muestra propiedadesantifúngicasfrenteaCandidaalbicansyposeeunadébilaccióndisuasoria dealimentacióneninsectos(Bastidaetal.,2011).Porotraparte,candiminapresenta actividadfrenteaT.vaginalis.Sinembargo,labioactividaddeestetipodealcaloides sedesconoceengranmedida(deAndradeetal.,2012b). Los compuestos de la serie hemantamina han demostrado efectos inhibidores significativos del crecimiento de una gran variedad de células tumorales, siendo potentes inductores de apoptosis a concentraciones micromolares (McNulty et al., 2007). Entre otras actividades destacadas descritas para este tipo de estructuras, los efectos antimaláricos son particularmente notables, y se ha propuesto que la presencia del grupo metilendioxi, junto con el nitrógeno terciario no metilado, contribuyenaunaumentodelaactividad(Osorioetal.,2008).Porotrolado,vitatina ha demostrado actividad antibacteriana frente a organismos Gram9positivos, Staphylococcusaureus,yGram9negativos,E.coli(Evidenteetal.,2004).Conrespectoa los alcaloides tipo crinina, con su característica configuración del puente 5,10b9 etano, también han mostrado efectos antiproliferativos sobre células tumorales humanas(Berkovetal.,2011c),peroserequierenestudiosadicionalesparacorroborar laspropiedadesdeesteesqueletotipo. Tantonarciclasinacomopancratistatinahansidoobjetodenumerososestudios debidoasuprometedoraactividadantitumoralyotrosefectosbiológicos(Bastidaet al., 2011). McNulty et al. (2010) reportan la inhibición potente y selectiva del citocromo humano P450 3A4 por análogos sintéticos de pancratistatina. Asimismo, a pesar de que algunos ensayos preclínicos con narciclasina resultaron desalentadores debidoasutoxicidad,recientementesehapropuestolaaplicaciónpotencialdeeste 11 Introducción alcaloide y estructuras relacionadas para el tratamiento de tumores cerebrales (Van Goietsenovenetal.,2013). Elalcaloidetazetinahademostradoserunartefactodelaislamientooriginadoa partir de pretazetina, un compuesto químicamente lábil (de Andrade et al., 2012a). Aunqueamboshanmostradopropiedadescitotóxicas,pretazetinaresultamuchomás interesantedadasuactividadcomoantiviralyantitumoral(Bastidaetal.,2011). Unodelosesqueletosmásrelevantesdebidoasuamplioespectrodeacciónes la montanina. Su actividad antioxidante se ha evaluado mediante ensayos con el radical DPPH (2,29difenil919picril9hidracilo), reportándose su actividad inhibidora del crecimientodealgunosmicroorganismos,comoStaphylococcusaureus,Pseudomonas aeruginosa y E. coli (Castilhos et al., 2007). Otros estudios también han indicado efectos de tipo ansiolítico, antidepresivo y anticonvulsivante en ratones, así como citotoxicidadeinhibicióndelaAChE(deAndradeetal.,2012b).Además,pancracinaha demostradounaampliagamadeactividadesantibacterianas. Desde el punto de vista farmacológico, el alcaloide galantamina merece un capítulo exclusivo, dado que es el único de este grupo que ha sido aprobado y que actualmente se comercializa para el tratamiento sintomático de la enfermedad de Alzheimer,untipodedemenciaconunenormeycrecienteimpactosobrelapoblación mundial. Este interesante compuesto se descubrió durante la década de 1950 en la especie Galanthus woronowii, y rápidamente atrajo la atención de la industria farmacéutica (Berkov et al., 2009a). En la actualidad, se comercializa como sal de hidrobromuro con la denominación de Razadyne® (o Reminyl®). Aunque su actividad inhibidora de la enzima AChE es ampliamente conocida, se ha propuesto un mecanismodeaccióndualqueimplicasuparticipaciónenlamodulaciónalostéricade los receptores nicotínicos (Farlow, 2003). Es interesante citar que se ha evaluado la actividadinhibidoradeAChEdevarioscompuestosestructuralmenterelacionadoscon el alcaloide galantamina, como algunos derivados Nalquilados y sanguinina (con un hidroxilo en posición 9), y resultaron ser unas diez veces más activos que dicho compuesto(Berkovetal.,2008a). A pesar de que la síntesis química de galantamina se ha logrado de manera exitosa, las plantas continúan siendo la fuente principal de este producto. Mientras queenEuropaCentralyOccidentalseobtienemayoritariamenteapartirdecultivares 12 Introducción deNarcissus,enEuropadelEsteesobtenidaapartirdeLeucojumaestivum,yenChina se extrae de Lycoris radiata. Además, se ha descrito su presencia en plantas de diversos géneros, incluyendo Hippeastrum, Hymenocallis, Zephyranthes, Ungernia y Haemanthus(Berkovetal.,2009a).Debidoalacontinuademandadeestealcaloide, existe un gran interés en la búsqueda de nuevas fuentes que sean altamente productorasdegalantamina. Entre las estructuras inusuales encontradas en las plantas de la subfamilia Amaryllidoideae, los alcaloides de tipo mesembrano han demostrado propiedades biológicasdestacables.Estoscompuestossoncaracterísticosdelasplantasdelgénero Sceletium N.E.Br. (anteriormente Mesembryanthemun L.), de Sudáfrica, y su descubrimiento fue impulsado gracias al interés en un preparado comúnmente utilizadoporgruposétnicosdelaregión,conocidocomo“Kanna”,otambiénllamado “Channa” o “Kougoed” (Popelak y Lettenbauer, 1967; Smith et al., 1996). Se han llevadoacabonumerososestudiossobrelaquímicaylasaplicacionesdelosalcaloides de tipo mesembrano, mostrando una marcada actividad como inhibidores de la recaptación de serotonina y, consecuentemente, con un considerable potencial para ser usados como antidepresivos (Gericke y Viljoen, 2008; Harvey et al., 2011). En efecto,existeunapatentedesarrolladaenEstadosUnidosparaelusodepreparados farmacéuticos que contienen mesembrina y compuestos relacionados en el tratamiento de estados depresivos y otros trastornos tales como ansiedad o drogodependencia(GerickeyVanWyk,2001). Talcomoyasehamencionado,losalcaloidesdeamarillidáceassonmarcadores químicos característicos sintetizados exclusivamente por plantas de la subfamilia Amaryllidoideae. Esta cualidad, unida a sus actividades biológicas distintivas, ha conducido a un interesante estudio que reveló una correlación significativa entre filogenia,variabilidaddealcaloidesyensayosdeactividadbiológicarelacionadoscon elSistemaNerviosoCentral(SNC),paraestegrupodeplantas(Rønstedetal.,2012).En dicho trabajo se utilizaron más de un centenar de especies de Amaryllidaceae, combinando el análisis de secuencias de ADN nuclear, plastídico y mitocondrial, con ensayosinvitrodeinhibicióndeAChEydeuniónaltransportadorderecaptaciónde serotonina,asícomoconunanálisisdelcontenidodealcaloides.Apesardequeestos resultados no son extrapolables a otros sistemas, poseen una importante aplicación 13 Introducción potencial, por ejemplo, en la selección de taxones candidatos para el desarrollo de fármacos. 1.3.GCMSyRMN:TécnicasClaveenelEstudiodeAlcaloidesdeAmaryllidaceae El análisis de la composición de alcaloides de extractos vegetales, así como la identificacióndenuevoscompuestosdeespeciesdeAmaryllidoideae,hasidoposible en gran medida gracias al desarrollo de dos técnicas de estudio fundamentales que han pasado a formar parte de los procedimientos de aplicación rutinaria: la cromatografíadegasesacopladaaespectrometríademasas(GC9MS)ylaresonancia magnéticanuclear(RMN).Laextensainvestigaciónllevadaacabodurantelosúltimos 50añosenelámbitodealcaloidesdeamarillidáceashadadolugaralacaracterización de ciertos patrones para los diversos tipos estructurales, posibilitando la rápida identificacióndeloscompuestosyaconocidosylaelucidaciónestructuraldetalladaen elcasodelosproductosaisladosdenovo. 1.3.1.CromatografíadeGasesEspectrometríadeMasas(GCMS) La cromatografía de gases (GC), introducida en los años 50, es una conocida técnica con la capacidad de separar componentes de una mezcla, que implica la volatilizacióndelamuestramediantesucalentamiento.Elequipoincluyeunacolumna conlafaseestacionaria,ungasportadorinerte,yundetector.Sólolasmoléculasque pueden ser vaporizadas sin experimentar descomposición son adecuadas para este análisis.Porotraparte,unespectrómetrodemasases,básicamente,uninstrumento que mide la relación masa9carga (m/z) de iones en fase gaseosa, proporcionando informaciónsobrelaabundanciadecadaespecieiónica,yqueofrecelaposibilidadde ser acoplado como detector (Kitson et al., 1996). Generalmente, los compuestos orgánicos presentan patrones de fragmentación característicos después de ser ionizados, lo que permite su identificación mediante comparación con los datos obtenidos previamente. La combinación de ambas técnicas es una poderosa herramientacomúnmenteconocidacomoGC9MS,lacualtieneuncostorelativamente bajo,unidoaunaaltaresoluciónyeficiencia. 14 Introducción Los extractos de plantas Amaryllidoideae suelen ser mezclas complejas con un número elevado de compuestos. La técnica de GC9MS, ya sea en modo de impacto electrónico(EI)odeionizaciónquímica(CI),hademostradosermuyútilparalarápida separaciónydeteccióndesuscomponentes.Losalcaloidesdeamarillidáceaspueden seranalizadossinnecesidaddeunaderivatizaciónpreviayaqueretienensuspatrones defragmentaciónparticularesbajolascondicionesdeGC,permitiendolaidentificación de compuestos ya caracterizados, o la obtención de información estructural valiosa cuandosetratadenuevasmoléculas(Berkovetal.,2005).Especialmentesignificativa resulta la observación de que pequeños cambios en la estereoquímica de estos alcaloidessuelensersuficientesparacausarapreciablesdiferenciasenelespectrode masasdelosestereoisómeros(Berkovetal.,2012;Duffieldetal.,1965). Al abordar el estudio del contenido de alcaloides de una especie vegetal o variedad particular, es de gran utilidad obtener un perfil general mediante GC9MS, tanto del extracto crudo como de las fracciones derivadas del mismo. Además de proporcionarinformaciónsobrelapresenciadealcaloidesconocidosyposiblesnuevas estructuras, permite el análisis de rendimientos y potenciales pérdidas que pueden surgirdurantelaaplicacióndelasdiferentestécnicasdeaislamiento.Recientemente, sehareportadolavalidacióndeGC9MScomométododeelecciónparaelcontrolde calidaddemateriasprimasvegetalesusadasenlaproduccióndegalantamina(Berkov etal.,2011b),loquedemuestralasventajasdesuutilizaciónenelanálisiscualitativoy cuantitativodeestasplantas,inclusoencomparaciónconotrasmetodologías(Gottiet al.,2006). Durante los años 60 y 70 se llevaron a cabo numerosos estudios de espectrometría de masas de impacto electrónico (EIMS) de los alcaloides de Amaryllidaceae, permitiendo establecer patrones de fragmentación característicos para varios esqueletos tipo (Bastida et al., 2006). Además, el posterior desarrollo de nuevasmetodologías,asícomolacaracterizacióndeotrasestructuras,hadadolugara la generación de información bien documentada con valor diagnóstico considerable paralaidentificacióndeestegrupodealcaloides.Porello,valelapenarealizaralgunos comentarios sobre los casos más representativos. Los ejemplos que se describirán a continuacióndemuestranelgranvalordelametodologíadeGC9MSenlaidentificación de alcaloides de amarillidáceas, aunque cabe destacar que no todas las estructuras 15 Introducción pertenecientesaestegrupopuedenserasignadasinequívocamente,comoocurreen elcasodelosderivadosNóxidos. 1.3.1.1.Tipolicorina El patrón de fragmentación de este esqueleto tipo se mantiene encondiciones de GC. El pico molecular aparece con una intensidad apreciable y suele experimentarlapérdidadeagua,asícomodeC91yC92,juntoconlosrespectivos sustituyentes,pormediodeunafragmentacióndetiporetro9Diels9Alder(Figura 1.7).Curiosamente,lapérdidadeaguaapartirdeliónmoleculardependedela estereoquímica del grupo hidroxilo en C92, y no ocurre en derivados acetilados (Bastida et al., 2006). En estructuras con dos grupos metoxilo en el anillo A en lugar del grupo metilendioxi de licorina (como es el caso de galantina) el pico basedelespectroes16unidadesmayorquem/z226,apareciendoam/z242. OH HO 10 O 9 OH 2 1 HO 3 10b 10a 4 O N 6a 8 7 - C 2H 4 O 2 O 11 4a 12 N O 6 O N O m/z 287 m/z 227 -H - H2 O O HO HO O N O N O O -H m/z 226 N O m/z 268 m/z 269 226 100 O O 50 65 41 51 77 HO 91 119 82 97 60 147 135 HO 154 167 0 40 60 80 100 120 140 N 160 211 180 191 180 200 238 220 Figura1.7: Patróndefragmentacióndelicorina. 268 250 240 260 287 280 300 16 Introducción 1.3.1.2.Tipohomolicorina El fragmento dominante en el espectro de masas de los compuestos de tipo homolicorina surge de la rotura de los enlaces lábiles del anillo C por una reacciónretro9Diels9Alderquegeneradosfragmentos(Figura1.8),siendoelmás característicoyabundanteelcorrespondientealanillodepirrolidina,unidoalos sustituyentesdelaposición2(Bastidaetal.,2006).Porlotanto,enelcasode homolicorina el pico base se observa a m/z 109, mientrasque hipeastrina (con un grupo hidroxilo en C92) lo presenta a m/z 125. Los alcaloides de esta serie suelen ser difíciles de diferenciar debido a la reducida abundancia del ión molecularylosdemásfragmentos. MeN - CH=CH2 m/z 82 MeN 12 -H 11 MeN 4 MeN m/z 109 3 4a MeO 10 m/z 108 10b 10a 9 2 1 MeO 8 6a 6 MeO O - CO MeO 7 m/z 315 O O MeO m/z 206 O MeO O m/z 178 109 100 N O 50 O O 42 53 0 40 65 60 82 80 O 94 100 115 120 135 140 178 150 160 180 206 200 220 240 260 Figura1.8: Patróndefragmentacióndehomolicorina. 280 300 320 17 Introducción 1.3.1.3.Tiposcrininayhemantamina La fragmentación de este tipo de alcaloides ha sido estudiada en detalle para varias estructuras. En la mayoría de los casos, el ión molecular corresponde al picobase,elanilloaromáticotieneunpapelfundamentalenlaestabilizaciónde los fragmentos, mientras que el átomo de nitrógeno suele perderse, y el paso inicialenlosmecanismosdefragmentaciónimplicalaaperturadelpuentededos carbonos 11912. Se han descrito varios patrones característicos teniendo en cuenta la presencia de sustituyentes en diversas posiciones, la saturación del anilloC,ylainfluenciadelaestereoquímica(Bastidaetal.,2006;Longevialleet al., 1973). Sin embargo, con respecto a la aplicación de la metodología de GC9 MS, es interesante destacar que hemantamina es susceptible a la descomposición térmica, lo quemodifica el espectro observado en condiciones de GC, en comparación con el obtenido mediante inyección directa en un espectrómetro de masas (Figura 1.9). Kreh et al. (1995) han propuesto un mecanismo para explicar los iones producidos por el efecto de una alta temperatura, involucrando la ruptura del puente 5,10b9etano, seguida de una roturaen. 100 301 A O O 227 HO 50 181 43 O 56 63 N 77 91 128 102 257 269 211 114 141 153 187 173 240 200 286 0 40 100 60 80 100 120 140 160 180 200 220 240 260 280 300 272 B 50 181 45 0 40 56 63 60 77 80 115 91 102 100 120 128 141 153 140 160 186 173 180 199 200 211 225 220 240 240 301 257 260 280 300 Figura1.9:MSdehemantamina, medianteinyeccióndirecta(A)yencondicionesdeGC(B). 18 Introducción 1.3.1.4.Tipotazetina El esqueleto de tipo tazetina es un buen ejemplo para ilustrar cómo pequeños cambios en la estereoquímica pueden verse reflejados en los patrones de fragmentación. Tazetina y criwellina difieren sólo en la configuración del grupo metoxiloenC93,peroelloessuficienteparaproducirvariacionesnotablesensus espectros de masas. La reacción principal involucra una fragmentación de tipo retro9Diels9Alder, la cual en criwellina está precedida por la pérdida del grupo metoxilo,dadoquesuconfiguraciónlafavorece,mientrasqueentazetinaocurre tras una simple reorganización de protones (Figura 1.10). Además, ambas estructuras experimentan la pérdida sucesiva de un radical metilo y agua, resultando en la formación de los iones a m/z 316 y m/z 298, así como posterioresfragmentaciones(Duffieldetal.,1965). H OMe OMe 3 OMe 4 10 O O 4a 1 10a 9 NMe 11 O 6a O OH O O 6 7 NMe O 12 10b 8 NMe - OH O O OH m/z 247 m/z 331 247 100 O 331 50 45 O 55 82 0 40 60 H 2C O 100 OH 121 141 159 120 140 160 181 201 180 200 298 227 220 4 4a 10a 9 NMe 11 O 6a - CH2O NMe 260 316 281 280 300 320 340 - NMe O OH O O 6 7 240 260 O 12 10b 8 242 H 3 1 O O 102 80 H O N O 71 OH m/z 301 m/z 331 O O OH m/z 247 71 100 O 331 301 50 45 N O O OH O 57 77 181 260 199 229 141 153 95 247 316 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 Figura1.10:Patronesdefragmentacióndetazetina(A)ycriwellina(B). 340 19 Introducción 1.3.1.5.Tipomontanina El patrón de fragmentación de alcaloides con el núcleo 5,119 metanomorfantridinadependeengranmedidadelanaturalezayconfiguración de los sustituyentes en C92 y C93. Las estructuras que contienen un grupo metoxilo dan lugar a un fragmento 31 unidades menor que el ión molecular (Figura1.11).Porotraparte,laconfiguracióndelsustituyenteenC92tieneuna considerable influencia en la medida en como transcurre la fragmentación de tiporetro9Diels9Alder,queseveaumentadacuandoelsustituyenteseencuentra enposición(Bastidaetal.,2006). OMe 1 10 O 2 11 3 10a 9 4a O 4 12 8 N H 6a 7 O OH - OMe O OH O O N 6 m/z 301 m/z 223 m/z 270 301 100 270 50 185 45 55 63 115 77 128 91 102 141 153 174 199 257 226 212 252 242 286 0 40 60 80 100 120 140 160 180 200 220 240 260 280 Figura1.11: Patróndefragmentacióndemontanina. 300 1.3.1.6.Tipogalantamina Las estructuras de esta serie son probablemente las más estudiadas entre los alcaloides de Amaryllidaceae. Durante la década de 1970, se propuso que la fragmentación de algunos compuestos de este grupo incluía tres pasos principalesquecomprendenlaeliminacióndelsustituyenteenC93,delanilloC,y del átomo de nitrógeno (Figura 1.12). Más recientemente, se ha utilizado la metodologíadeGC9MSparaelanálisisdetalladodelcomportamientodevarios esqueletos de tipo galantamina, demostrando que sus patrones de fragmentación se mantienen en dichas condiciones. Esto ha permitido su establecimiento como una técnica de rutina para el estudio de extractos vegetalesquecontienenestetipodealcaloides(Berkovetal.,2012). 20 Introducción Enlacitadareferenciasereportaronademásnumerososfactoresqueinfluyenen la fragmentación, como por ejemplo la posición de diversos sustituyentes. Una vez más, es interesante mencionar cómo una modificación estereoquímica puede afectar el espectro de masas de un compuesto, tal es el caso de galantaminaysuepímeroenposición3.Ambospresentandiferentestiemposde retención bajo las mismas condiciones cromatográficas, junto a una sutil, pero significativa,diferenciaensusespectros:laabundanciadelfragmentoam/z216, lacualseexplicaporlapresenciadeunpuentedehidrógenointramolecularque estabilizaeliónmoleculardegalantamina(Figura1.13). OH 2 O MeO 10a 8 6a O 4 10b 10 9 OH 3 1 O MeO 4a 11 MeO - C 4 H6 O 12 N 6 7 N Me N Me Me m/z 217 m/z 287 - NH5C2 -H O O MeO MeO N m/z 174 Me m/z 216 Figura 1.12: Fragmentacióndegalantamina. 287 100 A OH O 42 O 50 55 65 77 91 128 103 141 150 216 174 N 115 244 165 187 270 230 201 211 256 0 40 100 60 80 100 120 140 160 180 200 220 240 260 280 286 216 42 B OH 50 174 115 55 51 300 O O N 77 128 91 65 70 94 103 150 141 244 165 211 187 201 230 226 270 258 0 40 60 80 100 120 140 160 180 200 220 240 260 Figura1.13:Espectrodemasasdegalantamina(A) y39epigalantamina(B). 280 300 21 Introducción 1.3.2.ResonanciaMagnéticaNuclear(RMN) La resonancia magnética nuclear (RMN) es un tipo de espectroscopia de absorción, tal como la de infrarrojo (IR) o ultravioleta (UV). Básicamente, bajo la influencia de un campo magnético y en las condiciones apropiadas, una muestra es capaz de absorber radiación electromagnética en la región de las radiofrecuencias dependiendo de sus características particulares. La absorción es una función que dependedeciertosnúcleospresentesenlamolécula(SilversteinyWebster,1998).La técnicadeRMNseaplicaprincipalmenteenlaidentificacióndecompuestosorgánicos puros, pero durante los últimos años también se ha extendido al análisis de mezclas complejasenelámbitodelametabolómica,siendoutilizadaennumerososestudiosde extractosvegetales,incluyendoelanálisisdeespeciesyvariedadesdeamarillidáceas (Kimetal.,2010;Lubbeetal.,2010). Además de los datos obtenidos a partir de IR, UV, dicroísmo circular (CD) y espectrometría de masas de alta resolución (HRMS), entre otras metodologías, la elucidaciónestructuraldeuncompuestodesconocidoselogrageneralmentemediante la combinación de diversas técnicas de RMN complementarias. Con respecto a los alcaloides de Amaryllidaceae, la espectroscopia de 1H9RMN otorga información fundamentalsobrelosdistintostiposestructurales,mientrasquesucombinacióncon la espectroscopia de 13C9RMN y las técnicas de RMN bidimensional (2D9RMN), suele permitir la identificación inequívoca de la molécula en estudio, así como el establecimientodesuestereoquímicaenmuchoscasos. Lascaracterísticasmássignificativasdelosespectrosde1H9RMNdelosalcaloides de amarillidáceas han sido esbozadas, detallando las claves para su identificación (Bastida et al., 2011). En general, la región aromática ( 6.598.5 ppm) contribuye a definireltipodeesqueleto,mientrasquelasustitucióndelanilloaromáticosueleser evidente por la observación de las señales correspondientes a uno o más grupos metoxilo alrededor de 3.694.0 ppm, o la presencia de la señal típica de un grupo metilendioxisobre6.0ppm.Enmuchasestructurasenlasquelaposiciónbencílica enC96está saturada,comolicorina,hemantaminaygalantamina,lapresenciadeun sistema AB, es característica de dichos protones. Curiosamente, su desplazamiento químicoseveinfluenciadoporlaorientacióndelpardeelectroneslibredelátomode 22 Introducción nitrógeno. Además de estos rasgos comunes, vale la pena mencionar algunas particularidadesparacadatipodealcaloide. 1.3.2.1.Tipolicorina Entre las características principales del espectro de 1H9RMN de licorina y sus derivados, se encuentran los dos singuletes de los protones aromáticos en orientación para, junto con un único protón olefínico, y los dobletes correspondientes a la posición bencílica 6. El desapantallamiento observado en las señales de los protones de las posiciones 6 y 12, en relación a sus homólogosen,sedebealefectodelpardeelectroneslibreencisdelátomode nitrógeno. Generalmente, los alcaloides aislados del género Narcissus muestran una configuración trans en la unión de los anillos B/C, con una constante de acoplamientoentrelosprotones4a910bdeunos11Hz.Sólokirkinapresentauna configuracióncis,conunaconstantedeacoplamientomenor(8Hz). 1.3.2.2.Tipohomolicorina Estosalcaloidesincluyenungrupocaracterísticoquepuedeserunalactona,un hemiacetalounétercíclico.Ensuespectrode 1H9RMNsesuelenobservardos singuletes correspondientes a los protones aromáticos en para, siendo la señal de H97 la que usualmente se encuentra más desapantallada debido al grupo carboniloenperi. Lamayoríadeestoscompuestospertenecenaunaúnicaserieenantioméricacon launióncisentrelosanillosB/C,locualescongruenteconelreducidovalordela constantedeacoplamientoentrelosprotones1910b.EnelgéneroNarcissusno sehaencontradoningunaexcepciónaestaregla.Porotraparte,elelevadovalor delaconstanteentreH94ayH910b(J~10Hz)sóloescompatibleconunarelación trans9diaxial. Por lo general, el anillo C presenta un protón vinílico. Si la posición 2 se encuentrasustituidaporungrupohidroxilo,metoxilooacetilo,siempremuestra una disposición . El grupo N9metilo suele hallarse en el intervalo de 2.092.2 ppm, pero en el caso de alcaloides con el anillo C saturado, se han descrito 23 Introducción algunascorrelacionesempíricasparalaestereoquímicadelasunionesentrelos anillosB/CyC/D,enlasquesereportanseñalesmásdesapantalladas(Jeffsetal., 1988). 1.3.2.3.Tiposcrininayhemantamina La configuración absoluta deestos alcaloides sedeterminamediante dicroísmo circular (CD). Los alcaloides del género Narcissus son exclusivamente del tipo hemantamina,mientrasqueenlosgéneroscomoBrunsvigiayBoophane,entre otros,losalcaloidesdetipocrininasonpredominantes.Además,esimportante mencionar que los alcaloides aislados del género Narcissus no muestran sustituciones adicionales en el anillo aromático, aparte de las de C98 y C99, mientras que en los géneros dominados por los esqueletos del tipo crinina es bastantecomúnlapresenciadecompuestosconunsustituyentemetoxiloenC9 7. UtilizandoCDCl3comosolvente,lamagnituddelasconstantesdeacoplamiento entre H93 y cada uno de los protones olefínicos (H91, H92) ofrece información sobrelaconfiguracióndelsustituyenteenC93.Enaquellosalcaloidesenlosque elpuentededoscarbonos(C911yC912)sehallaenconfiguracióncisrespectoal sustituyenteenC93,H91presentaunacoplamientoalílicoconH93(J1,3~192Hz)y H92 muestra una constante más pequeña con H93 (J2,3 ~091.5 Hz), tal como ocurreencrinamina.Porelcontrario,enlaserieepiméricadelahemantamina, se observa una constante de acoplamiento mayor entre H92 y H93 (J2,3 = 5 Hz), mientras que el acoplamiento entre H91 y H93 no es detectable. Esta regla tambiénseaplicaalosalcaloidestipocrinina. Los compuestos con un sustituyente hidroxilo en C96, como papiramina/69 epipapiraminaohemantidina/69epihemantidina,aparecencomounamezclade epímerosquenopuedenserseparadosnisiquieramedianteHPLC. 24 Introducción 1.3.2.4.Tipotazetina La presencia de un grupo Nmetilo ( 2.492.5 ppm) distingue a este tipo de alcaloides de los tipos hemantamina o crinina, a partir de los que proceden biosintéticamente. Por lo demás, el espectro de 1H9RMN siempre muestra la señalcorrespondientealgrupometilendioxi. 1.3.2.5.Tipomontanina Laconfiguraciónabsolutadelosalcaloidesdetipomontaninadebedeterminarse mediante CD. Su espectro de 1H9RMN es muy similar a los alcaloides con esqueletodetipolicorina,aunquelasestructurasdetipomontaninapuedenser distinguidasatravésdelanálisisdelespectroCOSY.Lasseñalesatribuidasalos protones H94 (las más apantalladas) muestran correlación con las correspondientesaH93yH94a,mientrasqueenelespectrodeunesqueletode tipolicorina,lasseñalesmásapantalladascorrespondenalosdosprotonesdela posición11yalprotón12. 1.3.2.6.Tipogalantamina Entre los alcaloides de Amaryllidaceae, sólo los de tipo galantamina muestran unaconstantedeacoplamientoenorto(~8Hz)entrelosprotonesaromáticosdel anilloA. LaasignacióndelaestereoquímicadelsustituyenteenC93serealizaenbasea lasconstantesdeacoplamientodelprotónolefínicoH94.CuandolaconstanteJ3,4 tieneunvalordealrededorde5Hz,elsustituyenteespseudo9axial,mientrasque si el valor es próximo a 0 Hz indica que el sustituyente en C93 es pseudo9 ecuatorial. EstetipodealcaloidessuelemostrarlapresenciadeungrupoN9metiloaunque, ocasionalmente, también se ha reportado la existencia de gruposN9formilo. La presenciadelanillofuranoprovocaunefectodedesapantallamientosobreH91. 25 Introducción Con respecto a la espectroscopia de 13 C9RMN, la misma se ha utilizado ampliamente en la determinación del esqueleto carbonado de estos alcaloides. En líneas generales, el espectro de 13C9RMN de los alcaloides de amarillidáceas puede dividirseendosregionesprincipales:acamposmásbajos(>90ppm)seobservanlas señales correspondientes a grupos carbonilo, carbonos olefínicos y aromáticos, así como la señales del grupo metilendioxi; mientras que las señales de los carbonos alifáticos se encuentran a campos más altos, siendo la señal del grupo N9metilo la únicafácilmentereconocible,entre40946ppm. Elefectodelsustituyente(OH,OMe, OAc)enlasresonanciasdecarbonoesdegranimportanciaparalalocalizacióndela posicióndelosgruposfuncionales. Finalmente, tal como ya se ha mencionado, los experimentos bidimensionales sondeimportanciasignificativapararealizarunacorrectaasignacióndelasseñalesde 1 H9RMN y 13C9RMN, especialmente en el caso de estructuras desconocidas. Entre las técnicasde2D9RMNqueseutilizanmásampliamente,puedencitarselassiguientes: 9 1 H1H COSY (COrrelated SpectroscopY), en la cual las correlaciones observadas corresponden a acoplamientos directos entre los protones involucrados, siendo de gran utilidad en la asignación de acoplamientos geminalesyvecinales. 9 1 H1H NOESY (Nuclear Overhauser Effect SpectroscopY), de gran valor paraobtenerinformaciónsobrelaproximidadespacialentreprotonesy,porlo tanto,sobrelaestereoquímica. 9 1 H13CHSQC(HeteronuclearSingleQuantumCorrelation),quemuestra lascorrelacionesentre 1H913Cdirectamenteenlazados,permitiendolaadecuada asignacióndetodosloscarbonos,aexcepcióndeloscuaternarios. 9 1 H13CHMBC(HeteronuclearMultipleBondCorrelation),muyútilenla determinación de correlaciones a larga distancia entre 1H913C. Permite la identificación de los carbonos cuaternarios a través de la observación de su correlaciónconprotonessituadosatresenlacesdedistancia. 26 2.OBJETIVOS Objetivos 2.OBJETIVOS El objetivo general planteado para este proyecto de tesis ha sido la bioprospección de la diversidad vegetal de especies de la familia Amaryllidaceae (subfam. Amaryllidoideae) del área mediterránea e iberoamericana, analizando sus alcaloides como marcadores químicos, con el fin último de utilizar esta información para el aprovechamiento de estos recursos en la obtención de productos farmacológicamenteactivos. ObjetivosEspecíficos: • Estudio del contenido de alcaloides de especies de la familia Amaryllidaceae, incluyendo la determinación de la composición de extractos mediantecromatografíadegasesacopladaaespectrometríademasas(GC9MS), asícomoelaislamientodesuscomponentesenloscasosenquesedispongade material suficiente, para la posterior identificación y caracterización estructural aplicando diversas técnicas espectroscópicas, como resonancia magnética nuclear(RMN). • Determinacióndelaactividadbiológicadelosextractosvegetalesyde sus alcaloides, considerando ensayos de inhibición de la enzima acetilcolinesterasa(AChE)ydeactividadantiparasitaria,entreotros. • Identificacióndeespeciesdepotencialinterésfarmacéuticoporsualto contenidoencompuestoscondestacableactividadbiológica. • Contribución a la revisión taxonómica de las especies estudiadas basándoseenlapresenciadeciertosalcaloidescomomarcadoresquímicos. 29 Objetivos 2.1.Objectives Thegeneralaimofthepresentworkhasbeenbioprospectingamongthediverse plant species of the family Amaryllidaceae (subfam. Amaryllidoideae) found in the Mediterranean and Iberoamerican areas by analyzing alkaloids as chemical markers. Theultimategoalistomakeuseofthisinformationtofavourtherationalexploitation oftheseresourcesfortheproductionofpharmacologicallyactivecompounds. SpecificObjectives: • Study the alkaloid content of species belonging to the family Amaryllidaceae,includingthedeterminationofcompositionofplantextractsby gas chromatography coupled to mass spectrometry (GC9MS), as well as the isolation of their components, if sufficient material were available, for their subsequent identification and structural elucidation using a combination of spectroscopicmethodologiessuchasnuclearmagneticresonance(NMR). • Determinethebiologicalactivitiesofplantextractsandtheiralkaloids, considering assays of acetylcholinesterase (AChE) inhibition and antiparasitic activity,amongothers. • Identify species with potential pharmaceutical interest due to a high contentofcompoundsshowingremarkablebioactivity. • Contributetothetaxonomicalrevisionofthespeciesunderstudy,based onthepresenceofcertaintypesofalkaloidsaschemicalmarkers. 30 3.RESULTADOS Resultados 3.RESULTADOS Los resultados de la presente tesis están reflejados en los siguientes artículos científicos,loscualessepresentanacontinuaciónacompañadosporunbreveresumen encastellano: Artículo 1. Pigni, N.B., Berkov, S., Elamrani, A., Benaissa, M., Viladomat, F., Codina,C.,Bastida,J.(2010).TwonewalkaloidsfromNarcissusserotinusL.Molecules, 15,708397089. Artículo2.Pigni,N.B.,Ríos9Ruiz,S.,Martínez9Francés,V.,Nair,J.J.,Viladomat,F., Codina, C., Bastida, J. (2012). Alkaloids from Narcissus serotinus. Journal of Natural Products,75(9),164391647. Artículo3.Pigni,N.B.,Ríos9Ruiz,S.,Luque,F.J.,Viladomat,F.,Codina,C.,Bastida, J. (2013). Wild daffodils of the section Ganymedes from the Iberian Peninsula as a sourceofmesembranealkaloids.EnviadoparasupublicaciónenPhytochemistry. Artículo4.Ortiz,J.E.,Berkov,S.,Pigni,N.B.,Theoduloz,C.,Roitman,G.,Tapia,A., Bastida, J., Feresin, G.E. (2012). Wild Argentinian Amaryllidaceae, a new renewable sourceofacetylcholinesteraseinhibitorgalanthamineandotheralkaloids.Molecules, 17,13473913482. 33 Resultados 3.1.Artículo1 TwonewalkaloidsfromNarcissusserotinusL. Molecules,15,708397089(2010) Narcissus serotinus L. es una especie perteneciente a la familia botánica Amaryllidaceaequepresentaunadistribucióngeográficalocalizadaprincipalmenteen zonas costeras del Mediterráneo. En el presente artículo se reporta el aislamiento y elucidación estructural de dos nuevos alcaloides a partir de ejemplares de dicha especierecolectadoscercadeCasablanca(Marruecos). A partir de 350 g de material vegetal fresco consistente en planta entera, se aplicaron técnicas estandarizadas de extracción y separativas, incluyendo cromatografía líquida de vacío (VLC) y cromatografía en capa fina semi9preparativa (pTLC), con la finalidad de obtener fracciones y compuestos purificados adecuados parasuanálisismedianteGC9MSyRMN. El análisis del extracto utilizando GC9MS permitió la identificación de cinco alcaloidesconocidosporcomparacióncon lospatronesdefragmentaciónreportados en la literatura: licorina, galantina, 19O9(3´9hidroxibutanoil)licorina, asoanina e hipeastrina. Además, se describe el aislamiento y elucidación estructural de dos nuevosalcaloides:19O9(3´9acetoxibutanoil)licorinaynarseronina. Narseronina, alcaloide mayoritario del extracto, es el primero de la serie homolicorina que presenta un doble enlace en la unión entre los anillos B y C, en posición 1910b. Curiosamente, corresponde al mismo compuesto previamente reportado por Vrondeli et al. (2005) caracterizado como un isómero de 39 epimacronina,unaasignaciónquenoconcuerdaconlosresultadosaquíexpuestos. 35 Molecules 2010, 15, 7083-7089; doi:10.3390/molecules15107083 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.org/journal/molecules Article Two New Alkaloids from Narcissus serotinus L. Natalia B. Pigni 1, Strahil Berkov 1, Abdelaziz Elamrani 2, Mohammed Benaissa 2, Francesc Viladomat 1, Carles Codina 1 and Jaume Bastida 1,* 1 2 Department of Natural Products, Plant Biology and Soil Science, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain Department of Chemistry, Faculty of Sciences Ain Chock, University Hassan II, Casablanca, Morocco * Author to whom correspondence should be addressed; E-Mail: jaumebastida@ub.edu; Tel.: +34 934020268. Received: 15 September 2010; in revised form: 5 October 2010 / Accepted: 11 October 2010 / Published: 14 October 2010 Abstract: The Amaryllidaceae family is well known for the presence of an exclusive group of alkaloids with a wide range of biological activities. Narcissus serotinus L. is a plant belonging to this family and its geographical distribution is mainly located along the Mediterranean coast. In the present work, specimens collected near Casablanca (Morocco) were used to study the alkaloid content of this species. Starting with 350 g of the whole plant we used standard extraction and purification procedures to obtain fractions and compounds for GC-MS and NMR analysis. As well as five known alkaloids, we isolated two new compounds: 1-O-(3´-acetoxybutanoyl)lycorine and narseronine. The latter has been previously published, but with an erroneous structure. Keywords: Narcissus serotinus; Amaryllidaceae; alkaloids; narseronine; 1-O-(3´-acetoxybutanoyl)lycorine 1. Introduction Plants belonging to the Amaryllidaceae family are well known for the presence of an exclusive group of alkaloids with a wide range of biological activities [1]. Within this group, the genus Narcissus has been extensively used in traditional medicine to treat a variety of health problems. Molecules 2010, 15 7084 Antiviral, antifungal and antitumoral activities are just some of the phamacological effects that have been proven for these alkaloids. Narcissus serotinus L. is an autumn flowering species and the only member of the monotypic section Serotini. It grows mostly in calcareous sandy soil or maquis in dry coastal areas, and its geographical distribution extends over the coastal southern Mediterranean region, including southern Portugal, southern and eastern Spain, western and eastern Italy, Croatia, much of Greece and Israel, almost all the Mediterranean islands, north-west Morocco, Algeria, Tunisia and Libya [2,3]. The aim of this work is to investigate the alkaloid content of this species through the analysis of specimens collected in Morocco. In a previously published article, Vrondeli et al. [4] described the isolation of a new alkaloid from this species, suggesting a 3-epimacronine isomer. Based on the results reported herein, we propose an alternative structure, which also represents a new compound within the Amaryllidaceae alkaloid family. 2. Results and Discussion The MeOH extract of the fresh aerial parts and bulbs of N. serotinus L. was fractioned according to the methodology described in the experimental section. The GC-MS analysis of fraction B revealed the presence of lycorine. The analysis of fraction A showed a more complex mixture: in addition to lycorine [1,5] we determined the presence of galanthine [1,6], 1-O-(3´-hydroxybutanoyl)lycorine [7], assoanine [8] and hippeastrine [9] together with two new alkaloids (Figure 1). Identification of known compounds and structural elucidation of the new ones were achieved through the combined use of GC-MS, HRMS and one and two-dimensional NMR techniques. The HRMS of 1 suggested a molecular formula C22H26NO7 for [MH]+ with a parent ion at m/z 416.1702 (calc. 416.1704). The EIMS showed a molecular ion [M]+ at m/z 415 (18%) with a base peak at m/z 226. It is interesting to note that the isomer 2-O-(3´-acetoxybutanoyl)lycorine, isolated from Galanthus nivalis [10], shows a very similar fragmentation pattern but with a base peak at m/z 250. However, the pattern observed for 1 shows the base peak at m/z 226 [7]. These empirical cases prove that the GC-MS technique is useful for differentiating between isomers with substituents at position 1 or 2. The 1H-NMR spectral data of compound 1 and the isomer, 2-O-(3´-acetoxybutanoyl)lycorine, are very similar too, showing the major difference in proton shielding at positions 1 and 2: in 1 H-1 is more deshielded ( 5.68) than the same proton in the isomer ( 4.51) and the inverse situation occurs for H-2, which appears at 4.23 in the spectrum of 1 and at 5.31 for the isomer. Considering its coupling constant values, we assume that the configuration of 1 is the same as that proposed for 2-O(3´-acetoxybutanoyl)lycorine and 1-O-(3´-hydroxybutanoyl)lycorine. The high coupling constant (10.4) observed between H-4a and H-10b suggests a trans-diaxial configuration. Protons 6 and 12 are more deshielded than 6 and 12, respectively, because of the cis-lone pair of the nitrogen atom. The combined data suggested for compound 1 the structure of 1-O-(3´-acetoxybutanoyl)lycorine. The 1 H-NMR, COSY and HSQC data are recorded in Table 1. Molecules 2010, 15 7085 Figure 1. New alkaloids isolated from N. serotinus L. 1-O-(3´-acetoxybutanoyl)lycorine (1) and narseronine (2). 2´´ O 1´´ O 4´ 3´ 12 O 2´ 1´ O 10 O 9 O 8 1 10b 10a H 6a 7 2 10 3 4a N 6 1 H MeN OH H 11 O 4a H 11 O 2 1 4 12 3 10b 10a 9 4 8 6 6a OMe O 7 O 2 Table 1. 1H-NMR, COSY and HSQC data of 1-O-(3´-acetoxybutanoyl)lycorine (1). Position 1 2 3 4a 6 6 7 10 10b 11 (2H) 12 12 OCH2O 2´A 2´B 3´ 4´ AcO (2´´) 1 H į (J in Hz) 5.68 s 4.23 dt (3.3, 1.7) 5.56 m 2.76 d (10.4) 3.54 d (14.1) 4.16 d (14.1) 6.58 s 6.72 s 2.91 d (10.4) 2.65 m 2.42 dd (9.3, 5.0) 3.38 dt (9.2, 4.8) 5.92 s 2.43 dd (15.5, 5.4) 2.53 dd (15.5, 7.8) 5.10 m 1.14 d (6.3) 1.95 s COSY H-2, H-10b H-1, H-3, H-11 H-2, H-11 H-10b H-6 H-6 H-1, H-4a H-2, H-3, H-12, H-12 H-11, H-12 H-11, H-12 H-2´B, H-3´ H-2´A, H-3´ H-2´A, H-2´B, H-4´ H-3´ HSQC 72.5 d 69.4 d 116.9 d 61.9 d 56.6 t 56.6 t 107.3 d 104.8 d 38.8 d 28.4 t 53.4 t 53.4 t 100.8 t 40.5 t 40.5 t 66.9 d 19.3 q 20.7 q The HRMS analysis of narseronine (2) suggested a molecular formula C18H20NO5 for the parent ion [M+H]+ at m/z 330.1340 (calc. 330.1336). This indicates a molecular formula C18H19NO5, in accordance with a molecular weight of 329. The EIMS showed a molecular ion [M]+ at m/z 329 (20%). The mass spectral fragmentation pattern is not similar to those commonly shown by the homolycorine type compounds, because of the absence of a double bond between C-3 and C-4. The unusual occurence of a double bond at position C-1/C-10b probably drastically changes this pattern. Its 1H NMR spectrum exhibited two singlets at 7.66 and 7.29 for the para-oriented aromatic prontons H-7 and H-10, respectively, with H-7 more deshielded due to the peri-carbonyl group [1]. Also, the NOESY experiment showed the spatial proximity between H-10 and the N-methyl group. Two doublets appeared at 6.10 and 6.12 for the protons of the methylendioxy group. A triplet at 4.22 was assigned to H-2, coupled to H-3 with a J = 6.1 Hz, suggesting an equatorial orientation with a similar dihedral angle between H-2 and the two H-3 protons; this is consistent with the position of the methoxy group at C-2 and with the NOESY correlation of this substituent with H-11. A doublet at Molecules 2010, 15 7086 3.94, was undoubtedly assigned to H-4a for a 3JC-H HMBC correlation with the N-methyl group; COSY experiment showed its only correlation with H-4, with a J = 6.4 Hz suggesting a cis- C/D ring fusion [11,12]. The spectrum also showed, between the most significant signals, a singlet integrating for 3 protons at 3.57 assigned to the methoxy group at C-2, a doublet of triplets at 3.05, assigned to H-12, more deshielded than H-12 because of the cis-lone pair of the nitrogen atom [1], a singlet corresponding to the N-methyl group at 2.41, also supporting the cis-C/D ring junction if we consider the empirical correlations of N-methyl chemical shifts with stereochemical assignments suggested by Jeffs et al. [11]; and a doublet of triplets at 2.01 assigned to H-3, showing spatial proximity with the O-CH3 group in the NOESY experiment. The NMR spectral data is shown in Table 2. Narseronine was previously isolated by Vrondeli et al. [4] but published with an erroneous structure. They suggested a 3-epimacronine isomer, a tazettine type alkaloid but their mass spectral fragmentation proposal does not explain the occurrence of the most abundant peaks of the mass spectrum, such as m/z 240 or 241. Also, the 1H-NMR assignment is not adequate, including, for instance, the protons in an -position to the N-methyl group (H-6 in their numbering system) at 2.30-1.80 ppm, a more shielded displacement than can be expected for a proton in such an electronic environment. Table 2. 1H-NMR, COSY, NOESY, 13C-NMR (HSQC) and HMBC data of narseronine (2). Position 1 1 H į (J in Hz) - COSY - NOESY - 13 Cį 152.9 s HMBC - 2 4.22 t (6.1) H-3, H-3 H-3, H-3, OCH3 74.9 d 3 H-2, H-3, H-4 4a 3.94 d (6.4) H-3, H-3, H-4a, H-11, H-11 H-4 H-2, H-3, H-4, OCH3 H-2, H-3, H-4, OCH3 H-3, H-3, H-4a, H-11, H-11 H-4, H-10, NCH3 31.4 t 4 2.01 dt (13.5, 5.5) 2.22 - 2.13 m (overlapped) 2.64 m C-1, C-3, C-4, C-10b, OCH3 C-1, C-2, C-4, C-4a, C-11 C-1, C-2, C-4, C-4a, C-11 C-12 6 - - - 161.5 s 6a - - - 116.4 s - 7 7.66 s - - 107.8 d C-6, C-8, C-9, C-10a 3 H-2, H-3, H-4 31.4 t 35.1 d 61.6 d C-1, C-3, C-4, C-10a, C10b, C-11, C-12, NCH3 - 8 - - - 148.4 s - 9 - - - 153.8 s - 10 7.29 s - H-4a, NCH3 103.3 d C-6a, C-8, C-9, C-10b 10a - - - 135.1 s - 10b - - - 110.8 s - 11 2.22 - 2.13 m (overlapped) 1.90 ddd (12.6, 8.3, 4.2) 3.05 dt (11.0, 7.6) 2.81 m H-4, H-11, H-12, H-12 H-4, H-11, H-12, H-12 H-11, H-11, H-12 H-11, H-11, H-12 - H-4, H-11, H-12, H-12, OCH3 H-4, H-11, H-12, H-12, OCH3 H-11, H-11, H-12, NCH3 H-11, H-11, H-12, NCH3 - 29.6 t C-3, C-4a 29.6 t C-3, C-4a 54.3 t C-4, C-4a, C-11, NCH3 54.3 t C-4, C-4a, C-11, NCH3 102.4 t C-8, C-9 - H-2, H-3, H-3, H-11, H-11 H-2, H-3, H-3, H-11, H-11 58.3 q C-2 41.8 q C-4a, C-12 11 12 12 OCH2O OCH3 6.10 d (1.2) 6.12 d (1.2) 3.57 s NCH3 2.41 s - Molecules 2010, 15 7087 3. Experimental 3.1. General NMR spectra were recorded in a Mercury 400 MHz or a Varian VXR 500 MHz, instrument using CDCl3 as the solvent and TMS as the internal standard. Chemical shifts were reported in units (ppm) and coupling constants (J) in Hz. EIMS were obtained on a GC-MS Agilent 6890 + MSD 5975 operating in EI mode at 70 eV. A HP-5 MS column (30 m × 0.25 mm × 0.25 m) was used. The temperature program was: 100-180 ºC at 15 ºC min-1, 1 min hold at 180 ºC, 180-300 ºC at 5 ºC min-1 and 1 min hold at 300 ºC. Injector temperature was 280 ºC. The flow rate of carrier gas (Helium) was 0.8 mL min-1. In most cases the split ratio was 1:20, but with more diluted samples a split ratio of 1:5 was applied. UV spectra were obtained on a DINKO UV2310 instrument and IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrophotometer. 3.2. Plant material Whole plants of Narcissus serotinus L. (Amaryllidaceae) were collected in October 2009 during the flowering period in Ben Slimane, near Casablanca (Morocco), and identified by Professor El Ghazi. A voucher sample (MB-026/2009) was deposited at the Herbarium of the Faculty of Sciences Ain Chock, University Hassan II. 3.3. Extraction and isolation of alkaloids The fresh whole plant (350 g) was crushed and extracted with methanol (1 × 800 mL, 24 h; 1 × 800 mL, 72 h; and 2 × 400 mL, 48 h each). The extract was evaporated under reduced pressure to yield 5.5 g. This crude extract was dissolved in 100 mL of H2SO4 1% (v/v) and neutral material was removed with Et2O (6 × 100 mL). The acidic soln. was basified with 25% ammonia up to pH 9-10 and extracted with EtOAc (3 × 100 mL) to give extract A (149.4 mg). Another extraction with EtOAc (2 × 100 ml) gave extract B (23 mg). Both fractions were dried with anhydrous Na2SO4, filtered and completely dried under reduced pressure. Referred to the fresh weight, the sum of these two extracts represents approximately 0.05%. After dissolving A and B in MeOH, lycorine crystallized directly. Extract A was subjected to a vacuum liquid chromatography (VLC) [13] using a silica gel 60 A (6-35 ) column with a diameter of 1 cm and a height of 4 cm. Alkaloids were eluted using hexane gradually enriched with EtOAc, and then EtOAc gradually enriched with MeOH. Fractions of 10 mL were collected (105 in total) monitored by TLC (Dragendorff´s reagent, UV 254 nm) and combined according to their profiles. Five main fractions were obtained and subjected to preparative TLC (20 cm × 20 cm × 0.25 mm, silica gel 60F254). Narseronine (2, 4.5 mg) and 1-O-(3´-acetoxybutanoyl)lycorine (1, 1.5 mg) were obtained in major quantities from fractions 32-38 (eluted from VLC with hexaneEtOAc, 30:70 to 20:80) through preparative TLC (EtOAc-hexane 4:1 + 25% ammonia). 1-O-(3´-Acetoxybutanoyl)lycorine (1). UV (MeOH) max nm: 368.0, 260.0. IR (CHCl3) max cm-1: 2959, 2924, 2853, 1735, 1461, 1371, 1244, 1170, 1038, 776. 1H-NMR, COSY, HSQC (400 MHz, 500 MHz, CDCl3) see Table 1. EIMS 70eV (rel. int.): 416 (4), 415 (18), 354 (1), 269 (12), 268 (37), 250 Molecules 2010, 15 7088 (27), 227 (75), 226 (100), 192 (4), 147 (5), 96 (4), 69 (15), 43 (27). HRMS of [M+H]+ m/z 416.1702 (Calc. 416.1704 for C22H26NO7). Narseronine (2). UV (MeOH) max nm: 322.5, 286.0, 241.5. IR (CHCl3) max cm-1: 2928, 1718, 1503, 1482, 1415, 1283, 1256, 1162, 1106, 1035, 935, 754. 1H-NMR, COSY, NOESY, HSQC, HMBC and 13 C-NMR (500 MHz, CDCl3) see Table 2. EI-MS 70eV (rel. int.): 329 (20), 328 (21), 314 (2), 299 (28), 272 (38), 271 (18), 256 (46), 255 (16), 254 (30), 242 (34), 241 (98), 240 (100), 228 (13), 213 (18), 212 (13), 59 (42), 57 (60), 44 (37). HRMS of [M+H]+ m/z 330.1340 (Calc. 330.1336 for C18H20NO5). 4. Conclusions These results lead us to conclude that N. serotinus L. is an interesting source of alkaloids with potential pharmacological activities. Lycorine type alkaloids have shown notable properties as potent antimalarial and antitrypanosomal agents [7]. Recent investigations, including structure-activity studies, have also demonstrated they are potent inducers of apoptosis with good antitumoral activities [5,14]. In this sense, 1-O-(3´-acetoxybutanoyl)lycorine (1) is an attractive candidate for research in these areas. The isolation of narseronine (2) is also promising; this is the first report of a double bond between C-1 and C-10b in a homolycorine type structure, a feature that confers rigidity to the portion of the molecule formed by A-B rings, and also has a stabilising effect due to the extended conjugated system. This could be an interesting characteristic for potential biological activities related with pharmocophores with such requirements. In other respects, previous reports of antifungal activity of homolycorine-related structures such as hippeastrine [15], suggest narseronine has potential activity as an antifungal agent. Acknowledgements The authors are grateful for the collaboration of SCT-UB technicians. N. Pigni thanks the Spanish Ministerio de Educación for a FPU fellowship. This work was performed within the framework of project AECID A/019023/08. The authors also thank Generalitat de Catalunya (2009-SGR1060) for financial support. References and Notes 1. 2. 3. 4. Bastida, J.; Lavilla, R.; Viladomat, F. Chemical and biological aspects of Narcissus alkaloids. In The Alkaloids; Cordell, G.A., Ed.; Elsevier Scientific Publishing: Amsterdam, The Netherlands, 2006; Volume 63, pp. 87-179. Blanchard, J.W. Narcissus. A Guide to Wild Daffodils; Alpine Garden Society: Surrey, UK, 1990; pp. 40-42. Díaz Lifante, Z.; Andrés Camacho, C. Morphological variation of Narcissus serotinus L. s.l. (Amaryllidaceae) in the Iberian Peninsula. Bot. J. Linn. Soc. 2007, 154, 237-257. Vrondeli, A.; Kefalas, P.; Kokkalou, E. A new alkaloid from Narcissus serotinus L. Pharmazie 2005, 60, 559-560. Molecules 2010, 15 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 7089 Lamoral-Theys, D.; Andolfi, A.; Van Goietsenoven, G.; Cimmino, A.; Le Calvé, B.; Wauthoz, N.; Mégalizzi, V.; Gras, T.; Bruyère, C.; Dubois, J.; Mathieu, V.; Kornienko, A.; Kiss, R.; Evidente, A. Lycorine, the main phenanthridine Amaryllidaceae alkaloid, exhibits significant antitumor activity in cancer cells that display resistance to proapoptotic stimuli: an investigation of structure-activity relationship and mechanistic insight. J. Med. Chem. 2009, 52, 6244-6256. Berkov, S.; Bastida, J.; Sidjimova, B.; Viladomat, F.; Codina, C. Phytochemical differentiation of Galanthus nivalis and Galanthus elwesii (Amaryllidaceae): A case study. Biochem. Syst. Ecol. 2008, 36, 638-645. Toriizuka, Y.; Kinoshita, E.; Kogure, N.; Kitajima, M.; Ishiyama, A.; Otoguro, K.; Yamada, H.; Omura, S.; Takayama, H. New lycorine-type alkaloid from Lycoris traubii and evaluation of antitrypanosomal and antimalarial activities of lycorine derivatives. Bioorg. Med. Chem. 2008, 16, 10182-10189. Llabrés, J.M.; Viladomat, F.; Bastida, J.; Codina, C.; Rubiralta, M. Phenantridine alkaloids from Narcissus assoanus. Phytochemistry 1986, 25, 2637-2638. Almanza, G.R.; Fernández, J.M.; Wakori, E.W.T.; Viladomat, F.; Codina, C.; Bastida, J. Alkaloids from Narcissus cv. Salome. Phytochemistry 1996, 43, 1375-1378. Berkov, S.; Codina, C.; Viladomat, F.; Bastida, J. Alkaloids from Galanthus nivalis. Phytochemistry 2007, 68, 1791-1798. Jeffs, P.W.; Mueller, L.; Abou-Donia, A.H.; Seif El-Din, A.A.; Campau, D. Nobilisine, a new alkaloid from Clivia nobilis. J. Nat. Prod. 1988, 51, 549-554. Evidente, A.; Abou-Donia, A.H.; Darwish, F.A.; Amer, M.E.; Kassem, F.F.; Hammoda, H.A.M.; Motta, A. Nobilisitine A and B, two masanane-type alkaloids from Clivia nobilis. Phytochemistry 1999, 51, 1151-1155. Coll, J.C.; Bowden, B.F. The application of vacuum liquid chromatography to the separation of terpene mixtures. J. Nat. Prod. 1986, 49, 934-936. McNulty, J.; Nair, J.J.; Bastida, J.; Pandey, S.; Griffin, C. Structure-activity studies on the lycorine pharmacophore: A potent inducer of apoptosis in human leukemia cells. Phytochemistry 2009, 70, 913-919. Evidente, A.; Andolfi, A.; Abou-Donia, A.H.; Touema, S.M.; Hammoda, H.M.; Shawky, E.; Motta, A. (-)-Amarbellisine, a lycorine-type alkaloid from Amaryllis belladonna L. growing in Egypt. Phytochemistry 2004, 65, 2113-2118. Sample Availability: Not available. © 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). Resultados 3.2.Artículo2 AlkaloidsfromNarcissusserotinus JournalofNaturalProducts,75(9),164391647(2012) Elobjetivodedeestetrabajoconsistióenidentificarlosalcaloidespresentesen ejemplaresdelaespecieNarcissusserotinusrecolectadoscercadeVinarós(Castellón, España).Traslaextracciónyfraccionamientode2.43kgdematerialvegetalfresco,el análisisdeGC9MSpermitiólaidentificacióndecincoalcaloides:narseronina,galantina, incartina, masonina e hipeastrina. Conjuntamente, se reporta el aislamiento y elucidación de seis nuevas estructuras dentro del grupo de alcaloides típicos de amarillidáceas, cuya caracterización se ha logrado mediante la combinación de diversastécnicasespectroscópicas,incluyendoHRMS,GC9MSyRMN. Losprincipalescomponentesdelextractocorrespondenadosnuevosalcaloides derivados de narcisidina: 39O9metilnarcisidina y 19O9acetil939O9metilnarcisidina. Además,seaislóuntercerderivadopresenteencantidadesminoritarias,19O9acetil939 O9metil929oxonarcisidina. La estereoquímica de estos tres compuestos ha sido determinada con la ayuda de las correlaciones observadas en el espectro de RMN bidimensionalNOESY. Delmismomodo,sereportalacaracterizacióndetalladadeotrostresalcaloides con estructuras novedosas: 29metoxipratosina, 119hidroxigalantina y 29O9metil9 clivonina. Todos los componentes identificados en el extracto de N. serotinus correspondenaalcaloidesdelasserieslicorinayhomolicorina. 45 Note pubs.acs.org/jnp Alkaloids from Narcissus serotinus Natalia B. Pigni,† Segundo Ríos-Ruiz,‡ Vanessa Martínez-Francés,‡ Jerald J. Nair,§ Francesc Viladomat,† Carles Codina,† and Jaume Bastida*,† † Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Avinguda Diagonal 643, 08028 Barcelona, Spain ‡ Estación Biológica Torretes, Instituto Universitario de Biodiversidad CIBIO, Universidad de Alicante, Ctra. de San Vicente del Raspeig, s/n 03690 Alicante, Spain § Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa S Supporting Information * ABSTRACT: Narcissus serotinus belongs to the Amaryllidaceae family, a group well known for an exclusive variety of alkaloids with interesting biological activities. This study was aimed at identifying the alkaloid constituents of N. serotinus collected in the Spanish region of Valencia, using a combination of chromatographic, spectroscopic, and spectrometric methods, including GC-MS and 2D NMR techniques. GC-MS analysis allowed for the direct identiﬁcation of ﬁve known compounds. In addition, the isolation and structure elucidation of six new Amaryllidaceae alkaloids are described. The MeOH extract of whole N. serotinus L. plants was fractioned according to the sequence described in the Experimental Section. GC-MS analysis of extracts H1 and A1 revealed the presence of 11 compounds with typical fragmentation patterns of Amaryllidaceae alkaloids of the lycorine and homolycorine series (Table 1). Identiﬁcation of the known compounds narseronine, galanthine, incartine, masonine, and hippeastrine was made by comparison of their MS data with those gleaned from the literature.8,9 In addition, six new alkaloids of the Amaryllidaceae group (1−5 and 11) were identiﬁed. The main components of the extracts correspond to two new structures related to narcissidine: 3-O-methylnarcissidine (1) and 1-O-acetyl-3-O-methylnarcissidine (2). The HRMS data of 1 suggested the molecular formula C19H26NO5 for the parent ion [M + H]+ at m/z 348.1808 (calcd 348.1805), while in 2, a parent ion at m/z 390.1913 indicated the formula C21H28NO6 for its [M + H]+ (calcd 390.1911). The EIMS data of 1, with a base peak at m/z 284, showed a fragmentation pattern similar to that of narcissidine, with the only diﬀerence of the [M]+ peak being 14 mass units higher at m/z 347 (8%), suggesting the presence of an additional methyl group.10 The MS fragmentation observed for 2, with a molecular ion [M]+ at m/z 389 (3%), does not coincide with the typical narcissidine pattern, probably due to the presence of an acetyl substituent at C-1. Narcissus serotinus L. is an autumn ﬂowering species belonging to the Amaryllidaceae (subgen. Hermione, sect. Serotini), whose morphological and genetic variability (2n = 10, 2n = 20, and 2n = 30) has stirred debate about the precise standing of this taxon as separate from other closely allied taxa such as N. obsoletus (Haw.) Steud., N. deficiens Herbert,1,2 and N. miniatus Koop., Donnison-Morgan, Zonn.3,4 This plant family is well known for the presence of an exclusive array of alkaloids with a wide range of biological activities. Galanthamine is the standout example of these alkaloids, having received FDA approval in 2001 due to its marked ability to selectively and reversibly inhibit the enzyme acetylcholinesterase of signiﬁcance in the progression of neurodegeneration associated with Alzheimer's disease (AD).5,6 At present, under the commercial names Razadyne and Reminyl, it is prescribed for the treatment of AD in its mild to moderate stages. Apart from this, alkaloids of the genus Narcissus have been shown to possess antifungal, antitumoral, and antiparasitic activities.7 We previously described the isolation of two new alkaloids from whole plants of N. serotinus L. collected in Morocco.8 During this investigation,8 GC-MS analysis indicated the presence of several unknown structures with MS fragmentation patterns characteristic of Amaryllidaceae alkaloids. However, due to sample quantity limitations, we were unable to isolate these compounds. Thus, for the present study, a larger collection was made in the Spanish region of Valencia in order to identify these targets of interest and to compare the chemical constituents of the Moroccan and Spanish populations. © 2012 American Chemical Society and American Society of Pharmacognosy Received: May 22, 2012 Published: August 23, 2012 1643 dx.doi.org/10.1021/np3003595 | J. Nat. Prod. 2012, 75, 1643−1647 Journal of Natural Products Note Table 1. GC-MS Analysis of the Alkaloid Content of N. serotinus L. Extracts alkaloid % A1 M+ MS 0.26 <0.20 57.22 8.78 299 (−) 317 (22) 389 (3) <0.20 9.94 1.16 31.98 333 (42) 347 (8) <0.20 5.09 315 (−) 333 (16) 190 (1), 162 (2), 134 (2), 109 (100), 108 (25), 94 (3), 82 (3) 316 (15), 298 (10), 268 (18), 243 (96), 242 (100), 228 (8) 388 (5), 357 (50), 326 (98), 314 (3), 298 (35), 294 (20), 284 (9), 272 (19), 266 (100), 258 (31), 228 (40) 332 (100), 315 (25), 259 (73), 258 (97), 244 (17), 214 (9), 172 (5) 348 (2), 346 (16), 315 (47), 298 (6), 284 (100), 266 (35), 258 (22), 242 (8), 230 (38), 228 (30) 162 (4), 134 (3), 125 (100), 96 (36), 82 (3) 332 (15), 316 (11), 314 (11), 302 (5), 284 (13), 266 (8), 259 (100), 258 (89), 240 (13), 228 (4), 162 (11), 141 (12) 316 (6), 162 (3), 134 (2), 126 (2), 115 (2), 96 (39), 83 (100) 328 (24), 314 (2), 299 (31), 272 (42), 256 (47), 241 (100), 240 (97), 228 (12), 213 (17), 59 (50), 57 (70), 44 (45) 310 (20), 294 (16), 278 (2), 266 (22), 251 (12), 236 (7), 222 (5), 208 (7), 193 (4), 164 (4), 125 (2) 371 (10), 340 (19), 312 (4), 298 (17), 280 (100), 272 (8), 255 (10) RI % H1 masonine (8) galanthine (6) 1-O-acetyl-3-Omethylnarcissidine (2) incartine (7) 3-O-methylnarcissidine (1) 2670 2678 2718 2731 2800 hippeastrine (9) 11-hydroxygalanthine (5) 2859 2870 2-O-methylclivonine (11) narseronine (10) 2886 2909 0.41 13.86 2-methoxypratosine (4) 3025 0.25 1-O-acetyl-3-O-methyl-6oxonarcissidine (3) 3055 0.40 331 (13) 329 (23) <0.20 309 (100) 403 (1) pair of N is also β. However, for compounds 1 and 2, the spectroscopic data prompted us to reconsider this assumption, bearing in mind the NOESY correlation between the β-oriented H-10b with the less deshielded H-6. Thus, an α orientation has been assigned to the more deshielded proton and, therefore, to the lone pair of the nitrogen atom. The HRMS data of compound 3 suggested the molecular formula C21H26NO7 for [M + H]+ with a parent ion at 404.1706 (calcd 404.1704). The NMR data (Table 2) revealed that this structure is consistent with 1-O-acetyl-3-O-methyl-6oxonarcissidine. Comparing the 1H NMR spectra of 3 and 2, the absence of H-6 is evident and H-7 is strongly deshielded due to the eﬀect of the peri-carbonyl group. The EIMS data showed a molecular ion [M]+ at m/z 403 (1%) and a base peak at m/z 280, a fragment that could be attributed to an ion stabilized by a conjugated system. Compound 4 has been identiﬁed as the novel 2methoxypratosine, a derivative of pratosine ﬁrst isolated from Crinum latifolium.13 The HRMS analysis suggested a molecular formula of C18H16NO4 for [M + H]+ with a parent ion at 310.1074 (calcd 310.1073). The EIMS data exhibited a base peak coincident with the molecular ion [M]+ at m/z 309 as well as a low degree of fragmentation, characteristic of an ion stabilized by an extended conjugated system. The MS pattern is in accordance with the reported data,13,14 showing similar relative intensities of the fragments, each peak being 30 mass units higher than those of pratosine, which could be explained by the presence of an additional methoxy group. The 1H NMR data (Table 3) reveal the presence of two sets of aromatic protons [δ 8.03 d (3.5) to 6.84 d (3.5) and δ 7.55 d (2.0) to 7.30 d (2.0)] assigned to H-12/H-11 and H-1/H-3, respectively, as well as two aromatic singlets (δ 8.01 for H-7 and δ 7.59 for H-10) and three signals corresponding to methoxy groups attached to aromatic rings. The occurrence of this alkaloid is presumably a byproduct of the other lycorinetype structures found in the extract. However, in a recently published article describing the isolation of two analogous compounds named lycoranines from Lycoris radiata, the authors propose a diﬀerent biosynthetic pathway for Amaryllidaceae alkaloids related with these structures.15 The MS fragmentation pattern observed for compound 5 is similar to that of galanthine with a diﬀerence of 16 units for the main peaks.9,16 A molecular ion [M]+ at m/z 333 (16%) (m/z 317 for galanthine) as well as a pair of peaks (with the highest The 1H NMR spectra of 1 and 2 (Table 2) showed the H10b double doublet resonance between δ 2.70 and 3.00 (J ≈ 11.0, 2.0), characteristic of narcissidine and its derivatives. The spectrum of 1 is in accordance with the data published for narcissidine and 3-O-acetylnarcissidine.11,12 The assignment of the methoxy substituent at C-3 is supported by its spatial correlation with H-11 (NOESY, see Figure 1). Interestingly, the COSY correlation between H-1 and H-3 suggests the existence of “W-coupling” between these protons. The 1H NMR data of 2 revealed signiﬁcant diﬀerences compared to 1, including the presence of a singlet for the acetyl group at δ 1.99, the deshielding of H-1 from δ 4.71 to 5.81, and the shielding of H10 from δ 6.98 to 6.57. The designation α/β of H-6 and H-12 relates to the orientation of the electron lone pair on the nitrogen atom, through which the vicinal cis protons are markedly deshielded.7 Although this is not always deﬁned for the narcissidine-type structures, in the majority of cases, the more deshielded proton is assigned to H-β, assuming that the orientation of the lone 1644 dx.doi.org/10.1021/np3003595 | J. Nat. Prod. 2012, 75, 1643−1647 Journal of Natural Products Note Table 2. NMR Data for Compounds 1−3 1a position 1 2 3 4 4a 6α 6β 6a 7 8 9 10 10a 10b 11 12α 12β OMe (2) OMe (3) OMe (8) OMe (9) OCOMe OCOMe a 2a δH mult (J in Hz) 4.71, br s 3.80, t (2.9) 4.29, br d (2.0) 3.87, m (overlapped) 4.17, d (12.8) 3.67, d (12.4) 6.75, s 6.98, s 2.81, 5.87, 4.21, 3.65, 3.47, 3.27, 3.86, 3.91, dd (11.2, 1.7) q (1.8) br d (14.7) ddd (14.5, 6.0, 2.0) s s s s δH mult (J in Hz) δC 68.1 80.5 77.8 137.2 62.6 54.9 54.9 128.8 111.0 147.2 148.3 107.9 130.1 41.6 125.9 62.5 62.5 58.4 56.4 56.3 56.3 3b CH CH CH qC CH CH2 CH2 qC CH qC qC CH qC CH CH CH2 CH2 CH3 CH3 CH3 CH3 5.81, br t (2.6) 3.73, dd (2.8, 2.0) 4.08, br d (1.7) 3.92, m 4.21, d (13.1) 3.70, d (13.0) 6.74, s 6.57, s 2.98, 5.87, 4.18, 3.66, 3.52, 3.24, 3.86, 3.81, 1.99, dd (11.0, 2.0) q (1.8) m (overlapped) ddd (14.4, 5.7, 2.1) s s s s s δH mult (J in Hz) δC 68.2 79.1 76.6 137.6 62.7 54.5 54.5 128.4 110.8 147.4 148.2 107.0 128.5 39.8 125.9 62.1 62.1 58.7 56.3 56.1 56.1 21.2 171.3 CH CH CH qC CH CH2 CH2 qC CH qC qC CH qC CH CH CH2 CH2 CH3 CH3 CH3 CH3 CH3 qC 5.78, br t (2.7) 3.77, dd (2.9, 2.0) 4.06, br d (1.9) 4.72, m 7.57, s 6.58, d (0.8) 3.30, 5.97, 4.40, 4.68, 3.51, 3.27, 3.93, 3.86, 2.01, ddd (12.8, 2.5, 0.9) q (1.8) ddd (16.0, 3.2, 1.6) ddd (16.1, 5.1, 2.0) s s s s s δC 66.8 79.1 75.8 136.1 60.1 162.6 CH CH CH qC CH qC 130.8 111.4 148.0 151.9 105.7 124.6 41.6 125.4 52.6 52.6 58.9 56.5 56.2 56.2 21.1 171.1 qC CH qC qC CH qC CH CH CH2 CH2 CH3 CH3 CH3 CH3 CH3 qC 500 MHz for 1H, 125 MHz for 13C. b400 MHz for 1H, 100 MHz for 13C; CDCl3. fragmentation of clivonine.19 The molecular ion [M]+ at m/z 331, which is 14 units higher than that observed for clivonine, as well as the result of the HRMS analysis indicated a molecular formula of C18H22NO5 for [M + H]+ with the parent ion at m/z 332.1491 (calcd 332.1492), suggesting the presence of an additional methyl group. A structure similarity search on SciFinder (accessed on July 2011) identiﬁed “dihydroungerine”, a hydrogenation product of the alkaloid ungerine (isolated from the genus Ungernia).20 The conﬁguration described for ungerine includes a cis-B/C ring junction. However, the 1H NMR spectrum of 11 showed the signal of the N-methyl group near δ 2.5 ppm, a position that suggests a trans-B/C ring fusion if we consider the correlations between stereochemical assignments and N-methyl shifts.21 Additionally, the CD spectrum of 11 had the same shape as that reported for clivonine,22,23 with observed negative and positive Cotton eﬀects supporting a trans-B/C anti, cis-C/D conﬁguration for the ring junctions. With the diﬀerences expected for substitution with a methoxy rather than hydroxy group, the 1 H and 13C NMR data recorded in Table 4 is analogous to that reported for synthetic clivonine.24 As such, our ﬁnding is the ﬁrst for 2-O-methylclivonine from a natural source. In conclusion, N. serotinus L. is an interesting source of Amaryllidaceae alkaloids. Six of the components isolated are reported for the ﬁrst time, ﬁve of which (1, 2, 3, 4, and 5) are structurally related to lycorine, while 11 belongs to the homolycorine series. Additionally, ﬁve known alkaloids have been identiﬁed. By comparison with the analysis of N. serotinus L. plants obtained from Morocco,8 it is worth mentioning the absence of lycorine and its derivative 1-O-(3′-acetoxybutanoyl)lycorine in the Spanish population. The presence of narseronine, an unusual homolycorine-type structure reported for the ﬁrst time in the Moroccan plants, is also conﬁrmed in the Spanish Figure 1. Key NOESY correlations of compounds 1 and 2. relative abundance of the spectrum) at m/z 259 and 258 (m/z 243 and 242 for galanthine) suggested the presence of an additional oxygen atom. The HRMS data conﬁrmed the expected molecular formula C18H24NO5 for [M + H]+ with a parent ion at m/z 334.1652 (calcd 334.1649). NMR data analysis (Table 3) allowed the unambiguous identiﬁcation of this component as 11-hydroxygalanthine. Comparatively, the reported 1H and 13C NMR data of galanthine support the structural assignment.17 The α orientation of the hydroxy group is supported by the allylic coupling observed for H-3 and H-11. The J values are correlated to the values of the dihedral angle (φ): angles of 30° usually correspond to small coupling constants (1.0−1.5), whereas larger angles (60−90°) are associated with larger J values (2.2−2.8).18 A Dreiding model of 5 showed that the angle deﬁned by H-11 and the plane formed by C-3/C-4/C-11 is about 30° if we consider a β orientation for H-11, while it is around 80° in the opposite situation. Thus, a J11,3 of 1.5 Hz conﬁrms the β orientation of H-11 and the α position of the substituent. The EIMS data of compound 11 revealed the characteristic fragmentation of a homolycorine-type structure. A base peak at m/z 83 and a peak at m/z 96 (39%) were similar to the MS 1645 dx.doi.org/10.1021/np3003595 | J. Nat. Prod. 2012, 75, 1643−1647 Journal of Natural Products Note species, being among the three most abundant alkaloids of the extract. The genetic and morphological variability of Narcissus serotinus L. s.l. has been interpreted diﬀerently by some authors. Since Fernandes ﬁrst recognized variations in the genetic endowment (2n = 10, 2n = 20, and 2n = 30) inside the taxon,25 subsequent studies considered that the diploid plants (2n = 10), one-ﬂowered with a six-lobed yellow crown, correspond to N. serotinus L. s.s.,1,2 while 2n = 30 plants, with (1) 2−3 (4) ﬂowers per scape and an orange three-lobed crown, correspond to N. def iciens Herbert. According to these authors, and due to its geographical distribution and morphological characteristics, the material studied here (N. serotinus L. s.l.) would correspond to N. deficiens Herbert s.s., while the plants from Morocco can be classiﬁed as N. serotinus L. s.s. This taxonomic division could explain the chemical diﬀerences observed within the N. serotinus L. s.l. group. Table 3. NMR Data for Compounds 4 and 5 4a position δH mult (J in Hz) 1 7.55, d (2.0) 5a δH mult (J in Hz) δC δC 106.2 CH 4.68, br s 69.0 CH 157.7 qC 80.9 CH 106.9 CH 3.88, ddd (3.0, 3.0, 1.5) 5.94, m 119.4 CH 4 4a 129.1 126.6 qC qC 146.0 59.8 qC CH 6α 158.3 qC 56.1 CH2 6β 6a 7 8 9 10 10a 10b 121.2 110.4 149.9 153.7 104.1 129.3 117.1 qC CH qC qC CH qC qC 56.1 129.4 111.0 148.0 148.1 107.5 125.9 41.7 CH2 qC CH qC qC CH qC CH 110.7 CH 71.6 CH 124.2 CH 63.2 CH2 63.2 CH2 58.1 CH3 2 3 7.30, d (2.0) 8.01, s 7.59, s 11 12α a 6.64, s 6.85, s 3.98, s 56.5 CH3 2.65, br d (10.6) 4.89, br ddd (6.5, 1.5) 2.35, dd (9.2, 6.7) 3.68, dd (9.2, 6.5) 3.56, s 4.07, s 56.4 CH3 3.86, s 56.1 CH3 4.12, s 56.4 CH3 3.90, s 56.3 CH3 6.84, d (3.5) 8.03, d (3.5) 12β OCH3 (2) OCH3 (8) OCH3 (9) 3.03, dd (10.5, 1.4) 3.60, br d (14.0) 4.07, d (13.9) ■ General Experimental Procedures. Optical rotations were measured in CHCl3 at 22 °C using a Perkin-Elmer 241 polarimeter. UV spectra were obtained on a Dinko UV2310 instrument. The CD spectrum was recorded at room temperature at a concentration of 1 mg/mL on a JASCO J-810 spectropolarimeter using HPLC grade MeOH as the solvent. IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrophotometer. NMR spectra were recorded on a Gemini 300 MHz, a Varian VNMRS 400 MHz, or a Varian VNMRS 500 MHz, using CDCl3 or methanol-d4 as solvent and TMS as the internal standard. Chemical shifts are reported in δ units (ppm) and coupling constants (J) in Hz. EIMS were obtained on a GC-MS Agilent 6890 + MSD 5975 operating in EI mode at 70 eV. A DB-5 MS column (30 m × 0.25 mm × 0.25 μm) was used. The temperature program was 100−180 at 15 °C min−1, 1 min hold at 180 °C, 180− 300 at 5 °C min−1, and 1 min hold at 300 °C. The injector temperature was 280 °C. The ﬂow rate of He carrier gas was 0.8 mL min−1. In most cases the split ratio was 1:20, but with more diluted samples a split ratio of 1:5 was applied. A hydrocarbon mixture (C9− C36, Restek, cat no. 31614) was used for performing the RI calibration. GC-MS results were analyzed using AMDIS 2.64 software (NIST). The proportion of each compound in the alkaloid fractions was expressed as a percentage of the total alkaloids (Table 1). These data do not express a real quantiﬁcation, although they can be used to compare the relative quantities of each component. HRESIMS data were obtained on an LC/MSD-TOF (Agilent 2006). Plant Material. Whole plants of N. serotinus L. were collected in October 2010 during the ﬂowering period from a population located near Vinarós, Castellón Province (Spain), and identiﬁed by S.R.-R. and E.L. This population, growing in an industrial area close to a road, is under threat. It has survived because it receives extra water from nearby crops, which favors an extraordinary growth unusual in the other Valencian populations. Voucher specimens (BCN-83312) have been deposited in the Herbarium of Barcelona University (CeDocBiV). Some live plants have been included in the Iberian Narcissus Collection of the Field Station of Torretes (Ibi, Spain) for conservation and further studies. Extraction and Isolation. The fresh whole plant (2.43 kg) was crushed and extracted with MeOH (1 × 4.5 L, 36 h; 1 × 2.5 L, 48 h; and 1 × 2.5 L, 96 h). The extract was evaporated under reduced pressure and freeze-dried to yield 63.5 g. This crude extract was dissolved in 400 mL of H2SO4 2% (v/v), and neutral material was removed with Et2O (9 × 400 mL). The acidic solution was basiﬁed with 25% ammonia up to pH 9−10 and extracted with n-hexane (15 × 400 mL) to give extract H1 (694 mg). Another extraction with EtOAc (12 × 400 mL) gave extract A1 (1.02 g). Both fractions were dried over anhydrous Na2SO4, ﬁltered, and completely dried under reduced pressure. Referred to as fraction weight, the sum of these two extracts represents approximately 0.07%. 500 MHz for 1H, 125 MHz for 13C; CDCl3. Table 4. NMR Data for Compound 11a position 1 2 3α 3β 4 4a 6 6a 7 8 9 10 10a 10b 11α 11β 12α 12β OCH3 (2) NCH3 OCH2O a δH mult (J in Hz) 4.09, dd (12.6, 2.6) 3.78, q (2.9) 2.37, ddd (15.5, 2.8, 1.6) 1.61, ddd (15.4, 6.5, 3.1) 2.62−2.46, m (overlapped) 2.87, dd (9.8, 6.7) 7.50, s 7.86, br s 3.27, dd (12.8, 9.5) 2.29−2.15, m 2.15−2.02, m 3.35−3.21, m (overlapped) 2.62−2.46, m (overlapped) 3.49, s 2.54, s 6.02, d (1.3) to 6.03, d (1.3) δC 81.4 76.8 26.3 26.3 33.6 70.3 164.9 118.9 109.4 146.8 152.6 107.4 141.1 34.2 30.5 30.5 53.1 53.1 58.2 45.5 101.9 EXPERIMENTAL SECTION CH CH CH2 CH2 CH CH qC qC CH qC qC CH qC CH CH2 CH2 CH2 CH2 CH3 CH3 CH2 300 MHz for 1H, 125 MHz for 13C; CDCl3. 1646 dx.doi.org/10.1021/np3003595 | J. Nat. Prod. 2012, 75, 1643−1647 Journal of Natural Products Note The extracts were subjected to a combination of chromatographic techniques, including vacuum liquid chromatography (VLC)26 and semipreparative TLC. The general VLC procedure consisted in the use of a silica gel 60 A (6−35 μm) column with a height of 4 cm and a variable diameter according to the amount of sample (2.5 cm for 400− 1000 mg; 1.5 cm for 150−400 mg). Alkaloids were eluted using nhexane gradually enriched with EtOAc and then EtOAc gradually enriched with MeOH (reaching a maximum concentration of 20%). Fractions of 15 mL were collected, monitored by TLC (UV 254 nm, Dragendorﬀ's reagent), and combined according to their proﬁles. For semipreparative TLC, silica gel 60F254 was used (20 cm × 20 cm × 0.25 mm) together with diﬀerent solvent mixtures depending on each particular sample, always using an environment saturated with ammonia. The puriﬁcation of the new reported compounds is described in detail in the Supporting Information. 3-O-Methylnarcissidine (1): [α]22D −7 (c 0.09, CHCl3); UV (MeOH) λmax (log ε) 279 (3.44), 224 (3.81) nm; IR (CHCl3) νmax 3476, 2924, 2854, 1732, 1670, 1515, 1464, 1374, 1262, 1100, 932 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) see Table 2; EIMS data shown in Table 1; HREIMS of [M + H]+ m/z 348.1808 (calcd for C19H26NO5, 348.1805). 1-O-Acetyl-3-O-methylnarcissidine (2): [α]22D −30 (c 0.18, CHCl3); UV (MeOH) λmax (log ε) 272 (3.83), 221 (4.05) nm; IR (CHCl3) νmax 2924, 2854, 1732 1610, 1516, 1464, 1373, 1242, 1101, 1047, 941, 757 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) see Table 2; EIMS data shown in Table 1; HREIMS of [M + H]+ m/z 390.1913 (calcd for C21H28NO6, 390.1911). 1-O-Acetyl-3-O-methyl-6-oxonarcissidine (3): [α]22D −123 (c 0.26, CHCl3); UV (MeOH) λmax (log ε) 298 (3.54), 263 (3.59), 221 (4.25) nm; IR (CHCl3) νmax 3454, 2928, 1735, 1666, 1644, 1604, 1512, 1457, 1432, 1371, 1284, 1241, 1215, 1100, 1053, 1008, 941, 756 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) see Table 2; EIMS data shown in Table 1; HREIMS of [M + H]+ m/z 404.1706 (calcd for C21H26NO7, 404.1704). 2-Methoxypratosine (4): [α]22D −4 (c 0.27, CHCl3); UV (MeOH) λmax (log ε) 369 (2.79), 296 (3.36), 253 (3.60), 225 (3.62) nm; IR (CHCl3) νmax 2923, 2854, 1733, 1672, 1604, 1509, 1463, 1376, 1309, 1267, 1145, 1100 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) see Table 3; EIMS data shown in Table 1; HREIMS of [M + H]+ m/z 310.1074 (calcd for C18H16NO7, 310.1073). 11-Hydroxygalanthine (5): [α]22D +47 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 282 (3.62), 225 (3.92) nm; IR (CHCl3) νmax 3330, 2926, 1611, 1515, 1465, 1353, 1258, 1214, 1091, 998, 960, 850, 756 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) see Table 3; EIMS data shown in Table 1; HREIMS of [M + H]+ m/z 334.1652 (calcd for C18H24NO5, 334.1649). 2-O-Methylclivonine (11): [α]22D +14 (c 0.13, CHCl3); UV (MeOH) λmax (log ε) 297 (3.51), 254 (3.64), 225 (3.99) nm; CD (MeOH, [θ]λ) [θ]302 −195, [θ]291 0, [θ]273.5 +1761, [θ]261.5 0, [θ]251 −1242, [θ]239.3 0, [θ]233.5 +1255; IR (CHCl3) νmax 2924, 2854, 1714, 1476, 1274, 1036 cm−1; 1H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl3, 125 MHz) see Table 4; EIMS data shown in Table 1; HREIMS of [M + H]+ m/z 332.1491 (calcd for C18H22NO5, 332.1492). ■ Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS ■ REFERENCES The authors thank E. Laguna (CIEF, Generalitat Valenciana), A. Robledo (ISLAYA Consultoriá Ambiental s.l.), J. Pérez (Servicio de Protección de Especies, Generalitat Valenciana), and J. Juan (CIBIO, Universidad de Alicante) for their kind participation during the collection of plant material. Also, the collaboration of SCT-UB technicians has been valuable for the completion of the study. N.B.P. thanks the Spanish Ministerio de Educación for an FPU fellowship. This work was performed within the framework of project 2009-SGR-1060 (Generalitat de Catalunya). (1) Díaz-Lifante, Z.; Andrés-Camacho, C. Bot. J. Linn. Soc. 2007, 154, 237−257. (2) Fernández-Casas, J. Fontqueria 2008, 55 (67), 843−872. (3) Donnison-Morgan, D.; Koopowitz, H.; Zonneveld, B. J. M.; Howe, M. Daffodil, Snowdrop Tulip Yearb. 2005, 2005−2006, 19−25. (4) Zonneveld, B. M. Plant Syst. Evol. 2008, 275 (1−2), 109−132. (5) Berkov, S.; Bastida, J.; Viladomat, F.; Codina, C. 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(15) Wang, L.; Zhang, X.; Yin, Z.; Wang, Y.; Ye, W. Chem. Pharm. Bull. 2009, 57, 610−611. (16) Kobayashi, S.; Ishikawa, H.; Kihara, M.; Shingu, T.; Hashimoto, T. Chem. Pharm. Bull. 1977, 25, 2244−2248. (17) Bastida, J.; Codina, C.; Viladomat, F.; Rubiralta, M.; Quirion, J. C.; Husson, H. P.; Ma, G. J. Nat. Prod. 1990, 53, 1456−1462. (18) Barfield, M.; Spear, R. J.; Sternhell, S. Chem. Rev. 1976, 76, 593−624. (19) Ali, A. A.; Ross, S. A.; El-Moghazy, A. M.; El-Moghazy, S. A. J. Nat. Prod. 1983, 46, 350−352. (20) Yunusov, S. Yu.; Abduazimov, Kh. A. Zh. Obshch. Khim 1957, 27, 3357−3361. (21) Jeffs, P. W.; Mueller, L.; Abou-Donia, A. H.; Seif El-Din, A. A.; Campau, D. J. Nat. Prod. 1988, 51, 549−554. (22) Jeffs, P. W.; Abou-Donia, A.; Campau, D.; Staiger, D. J. Org. Chem. 1985, 50, 1732−1737. (23) Wagner, J.; Pham, H. L.; Döpke, W. Tetrahedron 1996, 52, 6591−6600. (24) Haning, H.; Giró-Mañas, C.; Paddock, V. L.; Bochet, C. G.; White, A. J. P.; Bernardinelli, G.; Mann, I.; Oppolzer, W.; Spivey, A. C. Org. Biomol. Chem. 2011, 9, 2809−2820. (25) Fernandes, A. Anal. Inst. Bot. Cavanilles 1975, 32 (2), 843−872. (26) Coll, J. C.; Bowden, B. F. J. Nat. Prod. 1986, 49, 934−936. ASSOCIATED CONTENT * Supporting Information S A detailed description of the isolation procedure, as well as tables with complete COSY, NOESY, and HMBC data, and 1H and 13C NMR spectra of alkaloids 1−5 and 11 are available free of charge via the Internet at http://pubs.acs.org. ■ ■ AUTHOR INFORMATION Corresponding Author *E-mail: jaumebastida@ub.edu. Tel: +34 934020268. Fax: +34 934029043. 1647 dx.doi.org/10.1021/np3003595 | J. Nat. Prod. 2012, 75, 1643−1647 Resultados 3.3.Artículo3 WilddaffodilsofthesectionGanymedesfromtheIberianPeninsulaasasourceof mesembranealkaloids EnviadoaPhytochemistry(2013) El presente trabajo se realizó con la finalidad de llevar a cabo un estudio detalladodelosalcaloidesdelaespecieNarcissustriandrusL.,asícomounanálisisdel perfil de alcaloides de 18 poblaciones silvestres incluyendo muestras de todos los taxones de la sección Ganymedes. Las especies pertenecientes a esta sección presentan una distribución geográfica que abarca la Península Ibérica y las islas Glenan, en Francia. Estudios previos de N. triandrus y N. pallidulus han reportado la presenciadealcaloidesdetipomesembrano,típicosdelgéneroSceletium(Sudáfrica)e inusualesenplantasdelafamiliaAmaryllidaceae. Partiendode600gdematerialvegetalfrescodeN.triandrus,recolectadocerca deProaza(Asturias,España),seabordóelfraccionamientoypurificacióndelextracto con el objeto de estudiar su composición de alcaloides. En el análisis de GC9MS se detectó la presencia de ocho componentes, tres de los cuales se identificaron rápidamente por comparación de sus patrones de fragmentación con los datos de la literarura: 4´9O9demetilmesembrenona, mesembrina y mesembrenona, siendo este últimoelcomponentemayoritario.Delmismomodo,conlaayudadelosdatosde 1H9 RMN para la determinación correcta de la estereoquímica, se identificaron 69 epimesembrenol y 69epimesembranol. Los tres componentes minoritarios restantes detectadosmedianteGC9MS,permanecieronsinidentificar,aunquesusespectrosde masassugierenquesetratadeestructurasdelmismotipo. Porotraparte,selogróelaislamientodetresalcaloidesobtenidosporprimera vezdeunafuentenatural,cuyapresencianosedetectóenelanálisisdeGC9MS.Así, los datos complementarios de HRMS, GC9MS y RMN, permitieron la elucidación estructural de 29oxomesembrenona, 7,7a9dehidromesembrenona y 29oxoepi9 mesembranol, representando este trabajo una contribución actualizada a la química deestosalcaloides. El análisis mediante GC9MS de ejemplares de 18 poblaciones silvestres de narcisosdelasecciónGanymedes,recolectadosendiversaslocalidadesdelaPenínsula 53 Resultados Ibérica, ha permitido confirmar la presencia de alcaloides de tipo mesembrano en todos los taxones descritos, sin detectarse trazas de alcaloides habituales de plantas de la familia Amaryllidaceae. En todas las poblaciones analizadas el componente mayoritarioseidentificócomomesembrenonay,sibienlavariabilidadobservadano demostró tendencias marcadas para proponer agrupamientos definidos entre las muestras estudiadas, la especie N. iohannis destacó por presentar una baja abundancia de alcaloides en comparación con las demás. Asimismo, se observó una reducidaproporcióndealcaloidesenlasmuestrasdeN.pallidulus(Segovia),diferencia quepuedeatribuirseaunestadoprematuroeneldesarrollo,dadoquedichasplantas noestabanenfloraciónenelmomentodelarecolección. Mediante los resultados de este estudio se confirma que los narcisos de la sección Ganymedes son una fuente alternativa de alcaloides de tipo mesembrano, conocidos inhibidores de la recaptación de serotonina y con potencial aplicación farmacológica en el tratamiento de trastornos psíquicos tales como depresiones, ansiedadodrogodependencia. 54 Elsevier Editorial System(tm) for Phytochemistry Manuscript Draft Manuscript Number: Title: Wild daffodils of the section Ganymedes from the Iberian Peninsula as a source of mesembrane alkaloids Article Type: Full Length Article Section/Category: Chemistry Keywords: Narcissus triandrus; section Ganymedes; Amaryllidaceae; GC-MS; NMR; alkaloid profile; 2oxomesembrenone; 7,7a-dehydromesembrenone; 2-oxoepimesembranol. Corresponding Author: Prof. Jaume Bastida, Corresponding Author's Institution: University of Barcelona, Faculty of Pharmacy First Author: Natalia B Pigni Order of Authors: Natalia B Pigni; Segundo Ríos-Ruíz; F. Javier Luque; Francesc Viladomat; Carles Codina; Jaume Bastida Abstract: The aim of this work was to perform a detailed study of the alkaloid content of Narcissus triandrus, as well as a metabolomic analysis of the alkaloid profile of 18 wild populations, comprising all the taxa of the section Ganymedes. Through the application of a combination of spectroscopic and chromatographic methods, the isolation and structural elucidation of 3 compounds are reported for the first time from a natural source (2-oxomesembrenone, 7,7a-dehydromesembrenone and 2oxoepimesembranol), together with the identification of 5 major common mesembrane alkaloids. Additionally, the GC-MS analysis of the alkaloid profile demonstrated the regular presence of mesembranes in all the studied plants, showing mesembrenone as the predominant compound without any typical Amaryllidaceae alkaloid being detected. *Graphical Abstract (for review) Graphical abstract Wild daffodils of the section Ganymedes from the Iberian Peninsula as a source of mesembrane alkaloids Natalia B. Pigni, Segundo Ríos-Ruiz, F. Javier Luque, Francesc Viladomat, Carles Codina, Jaume Bastida* Narcissus triandrus L. and other species from the section Ganymedes are atypical members of the Amaryllidaceae family, being characterized by the presence of mesembrane alkaloids. *Highlights (for review) Highlights Wild daffodils of the section Ganymedes from the Iberian Peninsula as a source of mesembrane alkaloids Natalia B. Pigni, Segundo Ríos-Ruiz, F. Javier Luque, Francesc Viladomat, Carles Codina, Jaume Bastida* x x x x x The first detailed reporting of the alkaloid composition of N. triandrus. Structural elucidation of 3 new mesembrane alkaloids (with GC-MS and NMR data). Conformational analysis of 2-oxoepimesembranol. GC-MS of the alkaloid profile of 18 wild populations (Narcissus sp., Ganymedes). Confirmation of the occurrence of mesembrane alkaloids in all samples analyzed. *Manuscript Click here to view linked References Graphical abstract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Wild daffodils of the section Ganymedes from the Iberian Peninsula as a source of mesembrane alkaloids Natalia B. Pigni, Segundo Ríos-Ruiz, F. Javier Luque, Francesc Viladomat, Carles Codina, Jaume Bastida* Narcissus triandrus L. and other species from the section Ganymedes are atypical members of the Amaryllidaceae family, being characterized by the presence of mesembrane alkaloids. 1 Wild daffodils of the section Ganymedes from the Iberian Peninsula as a source of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 mesembrane alkaloids Natalia B. Pigni1, Segundo Ríos-Ruiz2, F. Javier Luque3, Francesc Viladomat1, Carles Codina1, Jaume Bastida1* 1 Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona. Av. Diagonal 643, 08028 Barcelona, Spain. 2 Estación Biológica-Jardín Botánico Torretes. Instituto Universitario de Biodiversidad CIBIO. Universidad de Alicante. Ctra. de San Viçent del Raspeig s/n, 03690 Alicante, Spain. 3 Departament de Fisicoquímica i Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Avda Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain. * Corresponding author. Tel.: +34 934020268; fax: +34 934029043. E-mail address: jaumebastida@ub.edu 2 Abstract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 The aim of this work was to perform a detailed study of the alkaloid content of Narcissus triandrus, as well as a metabolomic analysis of the alkaloid profile of 18 wild populations, comprising all the taxa of the section Ganymedes. Through the application of a combination of spectroscopic and chromatographic methods, the isolation and structural elucidation of 3 compounds are reported for the first time from a natural source (2-oxomesembrenone, 7,7a-dehydromesembrenone and 2-oxoepimesembranol), together with the identification of 5 major common mesembrane alkaloids. Additionally, the GC-MS analysis of the alkaloid profile demonstrated the regular presence of mesembranes in all the studied plants, showing mesembrenone as the predominant compound without any typical Amaryllidaceae alkaloid being detected. Keywords Narcissus triandrus; section Ganymedes; Amaryllidaceae; GC-MS; NMR; alkaloid profile; 2-oxomesembrenone; 7,7a-dehydromesembrenone; 2-oxoepimesembranol. 3 1. Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Within the Amaryllidaceae family (subfam. Amaryllidoideae), the genus Narcissus L. comprises around a hundred wild species with a center of diversity in the Iberian Peninsula and North Africa (Fernandes, 1968; Meerow et al., 1999; Ríos et al., 1999). The great majority of these species are characterized by the presence of a group of alkaloids virtually exclusive to the family, such as lycorine, galanthamine and homolycorine (Bastida et al., 2006). However, other types of alkaloids have also been reported in some Amaryllidaceae species, albeit infrequently, as in the case of Narcissus pallidulus and N. triandrus (section Ganymedes), from which some mesembrane alkaloids have been identified (Bastida et al., 1989; Berkov et al., in preparation; Seijas et al., 2004). Many bioactive secondary metabolites have been discovered by observing traditional uses of their sources. The mesembrane alkaloids were found due to the interest on a drug preparation named “Kanna” (also known as “Channa” or “Kougoed”), commonly used by ethnic groups in South Africa and prepared from plants belonging to the genus Sceletium N.E.Br. (formerly Mesembryanthemun L.) from the Aizoaceae family (Popelak and Lettenbauer, 1967; Smith et al., 1996). Several studies have focused on the chemistry and application of these species, revealing a marked pharmacological activity of their alkaloids, notably as serotonin-uptake inhibitors, which gives them considerable potential as antidepressants (Gericke and Viljoen, 2008; Harvey et al., 2011). In fact, a US Patent has been developed for the use of pharmaceutical preparations containing mesembrine and related compounds for the treatment of depressive states and other disorders like anxiety or drug dependence (Gericke and Van Wyk, 2001). These alkaloids are usually divided into three different skeleton types represented by 4 mesembrine, joubertiamine and sceltium A4 (Fig. 1) (Gaffney, 2008). Their structural 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 similarity with crinane-type alkaloids of Amaryllidaceae plants originally led researchers to think of a common biosynthetic route, involving tyrosine and phenylalanine (Jeffs et al., 1971a), but subsequent studies revealed a different pathway, although with the same amino acids as precursors (Gaffney, 2008; Jeffs et al., 1978). The section Ganymedes (Narcissus sp.) has been the subject of considerable debate among taxonomists. Whereas some authors consider that it includes only a single species (N. triandrus L.) divided into 3 subspecies and some varieties (Barra Lázaro, 2000), studies based on molecular data and genome size support the idea of three different species: N. triandrus L., N. pallidulus Graells, and N. lusitanicus Dorda & Fern. Casas (Santos-Gally et al., 2011; Vives et al., 2010; Zonneveld, 2008); and, more recently, a fourth one has been added, N. iohannis Fern. Casas (Fernández Casas, 2011). Geographical distribution of these species is known to comprise the Iberian Peninsula and the Iles Glenans (France). Only a few studies on the alkaloid composition of two of these species have been reported, describing the isolation of mesembrenone and roserine from N. pallidulus (Bastida et al., 1989, 1992), a GC-MS analysis of some populations of N. pallidulus demonstrating the presence of more than 95% of Sceletium alkaloids (Berkov et al., in preparation), as well as a brief summary of the identification of mesembrine, mesembrenol and mesembrenone from N. triandrus (Seijas et al., 2004). The application of GC-MS for the detection of these compounds has been previously reported (Shikanga et al., 2012; Smith et al., 1998). In addition, the analysis of metabolic patterns by GC-MS applied to the field of Amaryllidaceae alkaloids with phytochemical differentiation purposes has been described, as in the case of Galanthus elwesii and G. nivalis (Berkov et al., 2008, 2011). Although the production of specialized metabolites in plants could be influenced by many factors, not only genetic 5 but also ontogenic and environmental, Rønsted et al. (2012) have demonstrated a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 significant correlation between phylogenetic and chemical diversity in the Amaryllidoideae subfamily. The aim of this work was to perform a detailed study on the alkaloid content of N. triandrus through the application of a combination of spectroscopic and chromatographic methods, including GC-MS and NMR. The identification of 5 major mesembrane alkaloids has been achieved, together with the isolation and structural elucidation of 3 minor compounds reported for the first time from a natural source: 2oxomesembrenone (6), 7,7a-dehydromesembrenone (7) and 2-oxoepimesembranol (8) (Fig. 2). Additionally, a metabolomic approach was developed to obtain the GC-MS alkaloid profile of 18 wild populations, comprising all the varieties of the section, with the purpose of studying chemical differences and to confirm the regular presence of mesembrane alkaloids. 2. Results and discussion 2.1. Alkaloids from N. triandrus L. The alkaloid fractions “H” and “A” from N. triandrus, obtained by the extraction procedure described in section 4.3, were analyzed by GC-MS, which allowed the detection of 8 compounds (Table 1). The most abundant alkaloid in both samples was mesembrenone (5), which was identified by comparison with its characteristic mass fragmentation pattern, together with 4´-O-demethylmesembrenone (2) and mesembrine (4) (Fig. 3) (Bastida et al., 1989; Jeffs et al., 1974; Martin et al., 1976; Shikanga et al., 2012). On the other hand, although the MS of compounds 1 and 3 were similar to previously reported data for mesembrenol and mesembranol, respectively, their 1H NMR spectra suggested an α orientation for the hydroxyl substituent, typical of the 6 corresponding epimers. Consequently, compound 1 was identified as 6-epimesembrenol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 after observing the diagnostic pattern for the two olefinic protons, a ddd at δ6.15 ppm (H5; J = 9.8, 5.5, 0.6 Hz) coupled to a dd at δ5.76 ppm (H4; J = 9.8, 1.2 Hz), in complete agreement with the data reported by Jeffs et al. (1970). Compound 3 was recognized as 6-epimesembranol by the complete assignment of its 1H NMR spectrum, together with 2D NMR, in which the signal corresponding to H6 appears as a broad singlet at δ 3.95 ppm, suggesting an equatorial orientation. The 1H NMR and NOESY data obtained (Table 2) are consistent with the conformation proposed by Jeffs et al. (1969), involving the six-membered saturated ring in a ground-state chair conformation, with the dimethoxyphenyl substituent occupying an axial position in favour of the formation of an intramolecular hydrogen bond between the proton of the hydroxyl group and the nitrogen atom. Additionally, three minority compounds were detected in low abundance in fraction A (M1, M2 and M3). The mass spectra observed for M1 and M2, both showing the same peaks but with different abundances, were very similar to the data described for 4´-Odemethylmesembranol (Jeffs et al., 1970), suggesting that one of them could correspond to this alkaloid, whereas the other could be an isomer. On the other hand, the mass spectrum of M3 remains unclear: although some of its most notable fragments are described for mesembrane alkaloids, such as m/z 244 and m/z 256, which are reported for some joubertiamine derivatives (Martin et al., 1976), the base peak at m/z 60 is unusual. Thus, with these data it was not possible to propose a structure for M3. After the application of a combination of chromatographic methods, such as VLC and TLC, three additional compounds not detected by GC-MS were isolated. This led to the elucidation of 2-oxomesembrenone (6), which can be considered a new alkaloid, 7,7adehydromesembrenone (7) and 2-oxoepimesembranol (8), both of them reported for the 7 first time from a natural source. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 The HRESIMS of 6 suggested the molecular formula of C17H20NO4 for the parent ion [M+H]+ at m/z 302.1382 (calcd. 302.1387). The mass fragmentation pattern showed a base peak coincident with the molecular ion at m/z 301, together with less abundant fragments not previously described for mesembrane alkaloids, possibly owing to the uncommon carbonyl moiety at C2 (Fig. 4). The 1H NMR spectrum showed 3 aromatic protons with characteristic ortho (8.4 Hz) and meta (2.2 Hz) J value couplings, two methoxyl groups in an aromatic environment (δ3.88 ppm), a singlet at δ2.81 ppm corresponding to an N-methyl group, and a pair of coupled olefinic protons (δ6.71, 6.24 ppm). The α/β orientation of H3 was determined through the observation of NOESY correlations between H3β and the aromatic protons H2´and H6´. The carbonyl groups appear in the 13C NMR spectrum as deshielded signals at δ171.9 and 195.3 ppm. All the data were in accordance with the proposed structure (Table 3). Compound 7 was first obtained through the oxidation of 5 with diethyl azodicarboxylate as reported by Jeffs et al. (1971b). The reported 1H NMR data were in accordance with our results, as well as its mass fragmentation pattern (Martin et al., 1976). HRESIMS data support the formula C17H20NO3 for the parent ion [M+H]+ at m/z 286.1431 (calcd. 286.1438). In order to complete the characterization, the whole structure was confirmed with 2D NMR techniques, and complementary 13C NMR data is reported (Table 4). The HRESIMS of 8 indicated the molecular formula C17H24NO4 for the parent ion [M+H]+ at m/z 306.1691 (calcd. 306.1700). Ishibashi et al. (1991) developed a stereoselective synthesis to obtain mesembranol, which involved a mixture of 8 and its 6-epimer in a ratio of 1 to 3.7, respectively, as intermediary compounds. The 1H NMR data reported for the epimer with the hydroxyl substituent in β orientation shows a signal at δ3.72 as a double triplet (J = 11.0, 4.0 Hz) corresponding to H6, where the 8 high coupling value indicates an axial position for this proton. In contrast, our results 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 revealed smaller J values for H6 (δ3.95, tt, J = 6.1, 3.6 Hz), suggesting a different situation. Moreover, the evidence of spatial correlations in the NOESY experiment pointed to the presence of more than one conformer, such as the correlation observed between H3β and H7a, which supported a chair conformation for the six-membered Cring with the dimethoxyphenyl group in an axial position similar to that proposed for mesembranol by Jeffs et al. (1969). There was also evidence of spatial proximity among H3α, H5α and H7α, which could only be explained by a chair conformation with the dimethoxyphenyl in an equatorial position (Fig. 5). In order to confirm our proposal that there are at least two conformers in equilibrium, the conformational preferences of compound 8 in chloroform were determined by means of high-level quantum mechanical calculations. To this end, the population of conformers was estimated by combining the relative stabilities in the gas phase determined at the MP2/aug-cc-pVDZ level, with the solvation free energy in chloroform determined by using both MST and SMD solvation continuum models (see section 4.5). Calculations were done for a model compound in which the methoxy groups in the phenyl ring were replaced by protons. This is justified by the lack of direct contact between the methoxy groups with the rest of the molecule and by the concomitant saving in the cost of computations. Besides the conformational flexibility of the sixmembered ring (chair versus boat), the conformational study also explored the optimal orientations of the phenyl ring, which was found to adopt two main orientations, and of the hydroxyl group, which may form an intramolecular hydrogen bond with the lone pair of the amide nitrogen. Finally, additional calculations were done for a compound in which the phenyl ring was replaced by hydrogen with the aim of examining the intrinsic conformational preferences of the bicyclic ring. 9 The free energy differences between conformers of the two model compounds are 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 reported as Supplementary Information (see Tables S1 and S2). The results confirmed the energetic destabilization of the conformations where the six-membered ring adopts a boat structure (Table 5). Furthermore, the six-membered ring adopts two main chair conformations, where the phenyl ring (or the hydrogen atom) adopts an equatorial or axial position (Fig. 5). The axial arrangement is clearly predominant in the gas phase due to the stabilization afforded by the intramolecular hydrogen bond formed between the hydroxyl group and the lone pair of the amide nitrogen. However, both axial and equatorial arrangements are similarly populated upon solvation in chloroform. Previous studies have shown that the measured coupling constants can reflect the population-weighted average of the J values determined for individual conformers of a given compound (Arnó et al., 2000). Therefore, we have performed similar calculations for the theoretical J values obtained for each conformation of the model compound with a phenyl substituent using the graphical tool MestRe-J (Navarro-Vázquez et al., 2004), based on the values of the dihedral angles and the HLA generalization of the Karplus equation (Haasnoot et al., 1980). As noted in Table 6 (see also Fig. 5), these calculations reveal a close correspondence between the calculated and observed J values for each vicinal proton of the six-membered C-ring. Overall, the existence of two major chair conformations of compound 8 allows us to explain the observed NOESY correlations, as well as the intermediate values of the coupling constants. Finally, the fragments observed in the mass spectrum of 8 coincide with those reported for an isomer of this compound, obtained through a synthetic procedure (Keck and Webb, 1982). Interestingly, the main alkaloids from N. triandrus with a hydroxyl substituent at position 6 identified in this work showed the same stereochemistry. 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2.2. Section Ganymedes: alkaloid profile analysis The alkaloid extracts of plants from 18 wild populations were analyzed by GC-MS. The sample distribution comprised a wide geographical range with locations from 3 NW Spanish Communities (Castilla y León, Galicia and Asturias) and N of Portugal. The analysis included plants belonging to all the taxa described in the section Ganymedes (Fernández Casas, 2011). Three repetitions were performed for each population, except for NT-B, NI-B and NL-P6, which were analyzed using 2 samples for each one. The chromatograms were manually analyzed, recording the peak area. The mass spectra of 8 different compounds were detected (1-7 and the unidentified M3). The data obtained were normalized to the area of the internal standard (codeine) and to the dry wt (g) of plant material. Finally, the mean and s.d. of the repetitions were calculated for each population. In order to focus on the most significant information, minority components with values lower than 1 (6, 7 and M3) were discarded from the graphical representation of the data. The results are summarized in Fig. 6. All samples revealed the presence of mesembrane alkaloids, and no typical Amaryllidaceae alkaloids were detected. Mesembrenone (5) was predominant in all populations, present in notably higher amounts than the second most abundant component: the relation between the mean values calculated for 5 and the second compound oscillated from 3 for NT15 to 89 for NI-B. Although the variability within populations was considerable, reflected by the high s.d., some grouping trends are noticeable. N. pallidulus (NP1, NP2) and N. iohannis (NI-B) showed markedly lower alkaloid contents than the other groups. In the case of N. pallidulus from Segovia, this could be attributed to the early developmental stage of the plants, which were not flowering at the time of collection; however, other factors such 11 as location or taxonomic divisions cannot be discarded. On the other hand, N. iohannis, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 which belongs to a taxon previously reported as N. triandrus var. alejandrei by Barra Lázaro (2000), but recently classified as a new species with a putative hybrid origin between N. triandrus and N. pallidulus (Fernández Casas, 2011), showed the lowest quantity of alkaloid content, which together with its distinct habitat and morphology could support its independence as a species. In the remaining populations, the alkaloid profile and abundance were comparatively similar, with a subtle variation in the populations of N. triandrus (Galicia-Asturias and Burgos) and one population from Portugal (NP-P2), which showed a higher contribution of the minority compounds. 3. Concluding remarks The analysis of the alkaloid content of N. triandrus led to the identification of eight mesembrane alkaloids, three of which were isolated from a natural source for the first time. The GC-MS data of all the compounds are reported, as well as the NMR data of the alkaloids 6-epimesembranol (3), 2-oxomesembrenone (6), 7,7adehydromesembrenone (7) and 2-oxoepimesembranol (8), which represents a detailed and updated contribution to the structural chemistry of these alkaloids. These results confirm for the first time the presence of mesembrane alkaloids in all the Narcissus taxa of the section Ganymedes, without any trace of typical alkaloids of the Amaryllidaceae group. However, the studied populations were not definitely clustered by their alkaloid patterns, neither in accordance with taxonomic groups, nor with geographical locations, except for N. iohannis, which is endemic to a very small area and the only one growing on alkaline substrates. This study, together with a previous analysis of N. pallidulus and some hybrids (Berkov 12 et al., in preparation), reveals an important aspect of the phylogeny of Narcissus: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Ganymedes is the only section within the Amaryllidaceae with an atypical alkaloid biosynthetic pathway. According to Santos-Gally et al. (2011), this group has a relatively recent onset (about 4 million years ago) and, although it shows a high morphological variability, its alkaloid chemistry seems to be relatively conservative. Finally, it is interesting to note that the presence of mesembrane alkaloids is a chemical feature shared by this group of Mediterranean daffodils with the very distantly related dicotyledonous plants of the genus Sceletium from South Africa. Thus, Narcissus taxa belonging to the section Ganymedes represent an alternative source of these compounds, whose potential therapeutic applications have already been demonstrated. 4. Experimental and Computational Section 4.1. General experimental procedures Optical rotations were measured in CHCl3 at 22 °C using a Perkin-Elmer 241 polarimeter. UV spectra were obtained on a Dinko UV2310 instrument. IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrophotometer. NMR spectra were recorded on a Varian VNMRS 500 MHz, using CDCl3 as the solvent and TMS as the internal standard. Chemical shifts are reported in δ units (ppm) and coupling constants (J) in Hz. EIMS were obtained on a GC-MS Agilent 6890 + MSD 5975 operating in EI mode at 70 eV. A HP-5 MS column (30 m × 0.25 mm × 0.25 μm) was used. The temperature program was 100−180 at 15 °C min−1, 1 min hold at 180 °C, 180−300 at 5 °C min−1, and 1 min hold at 300 °C. The injector temperature was 280 °C. The flow rate of He carrier gas was 0.8 mL min−1. A hydrocarbon mixture (C9−C36, Restek, cat no. 31614) was used for performing the RI calibration. GC-MS results were analyzed using AMDIS 2.64 software (NIST). The proportion of each compound in the alkaloid 13 fractions is reported as a percentage of the total alkaloids (Table 1). These data do not 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 express a real quantification, although they can be used to compare the relative quantities of each component. HRESIMS data were obtained on an LC/MSD-TOF (Agilent 2006). 4.2. Plant Material Plant material was collected in March 2012 by S.R.-R. and N.B.P., according to the flowering period reported for the target populations. Beginning in Segovia, the expedition covered Castilla y León (Spain), the center and North of Portugal, Galicia and Asturias (Spain). As the flowering of wild populations depends on the specific climatic conditions of each year, the search for some reference locations, mainly in the interior, was unsuccessful. Samples from Burgos (Spain) were collected in April 2012 by Rafa Díez, as a kind collaboration to our work. Data regarding location, labeling and additional information are detailed in Table 7. An abundant and disperse population of Narcissus triandrus L. located near Proaza, Asturias (Spain), allowed the collection of an adequate quantity of material for the alkaloid isolation, without threatening its survival. Voucher specimens (BCN-102933) have been deposited in the Herbarium of Barcelona University (CeDocBiV), as well as specimens of the majority of the populations analyzed (BCN-102921-BCN-102932). Live plants have been included in the Iberian Narcissus Collection of the Field Station of Torretes (Ibi, Spain) for conservation and further studies. 4.3. Extraction and isolation of alkaloids from N. triandrus The fresh whole plant (600 g) was crushed and extracted with MeOH (1 × 1.5 l, 72 h; 1 × 1.5 l, 48 h; and 1 × 1.5 l, 48 h). The extract was evaporated under reduced pressure to 14 yield 27.5 g. This crude extract was dissolved in 200 ml of H2SO4 2% (v/v) and neutral 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 material was removed with Et2O (5 × 200 ml). The acidic solution was then basified with 25% ammonia up to pH 9−10 and extracted with n-hexane (10 × 200 ml) to give extract H (350 mg). Another extraction with EtOAc (7 × 200 ml) gave extract A (118 mg). Both fractions were dried over dry Na2SO4, filtered, and completely dried under reduced pressure. Referred to fraction wt, the sum of these two extracts represents approximately 0.08%. The extracts were subjected to a combination of chromatographic techniques, including vacuum liquid chromatography (VLC) (Coll and Bowden, 1986) and semiprep. TLC. The general VLC procedure consisted of the use of a silica gel 60 A (6−35 μm) column with a height of 4 cm and a variable diameter according to the amount of sample (2.5 cm for 400−1000 mg; 1.5 cm for 150−400 mg). Alkaloids were eluted using n-hexane gradually enriched with EtOAc, and then EtOAc gradually enriched with MeOH (reaching a maximum concentration of 20%). Fractions of 10-15 ml were collected, monitored by TLC (UV 254 nm, Dragendorff's reagent), and combined according to their profiles. For semiprep. TLC, silica gel 60F254 was used (20 cm × 20 cm × 0.25 mm) together with different solvent mixtures depending on each particular sample, always using an environment saturated with ammonia. 4.3.1. 2-Oxomesembrenone (6) Amorphous solid; [α]D22 +3.6 (c 0.24, CHCl3); UV (MeOH) λmax (log ε): 279.0 (2.88), 219.0 (3.67), 209.5 (3.78) nm; IR (CHCl3) υmax: 1693, 1519, 1464, 1257, 1148, 1025, 757 cm-1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table 3; EIMS data shown in Table 1; ESI-TOF-MS m/z 302.1382 [M+H]+ (calcd. for C17H20NO4, 302.1387). 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4.3.2. 7,7a-Dehydromesembrenone (7) Amorphous solid; [α]D22 -6.7 (c 0.07, CHCl3); UV (MeOH) λmax (log ε): 342.5 (2.89), 284.5 (2.95), 223.0 (3.77) nm; IR (CHCl3) υmax: 1733, 1637, 1566, 1515, 1464, 1256, 1145, 1025, 761 cm-1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table 4; EIMS data shown in Table 1; ESI-TOF-MS m/z 286.1431 [M+H]+ (calcd. for C17H20NO3, 286.1438). 4.3.3. 2-Oxoepimesembranol (8) Amorphous solid; [α]D22 -9.7 (c 0.15, CHCl3); UV (MeOH) λmax (log ε): 278.0 (3.29), 227.5 (3.71) nm; IR (CHCl3) υmax: 3399, 1673, 1520, 1464, 1255, 1149, 1026, 758 cm-1; 1 H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table 4; EIMS data shown in Table 1; ESI-TOF-MS m/z 306.1691 [M+H]+ (calcd. for C17H24NO4, 306.1700). Supplementary information file contains complete NMR data (Table S4). 4.4. Section Ganymedes: alkaloid profile analysis Fresh plant material from one or two individual whole plants was macerated with 10 ml MeOH in 15 ml glass vials at the time of collection, with 3 repetitions per population. After 7 days, the solvent was transferred to a new vial and dried on a hot plate at 60 ºC. The crude extracts were dissolved in 4 ml of H2SO4 2% (v/v), and 1 ml of codeine in MeOH (0.1 mg/ml) was added as an internal standard. The solution was defatted with Et2O (3 x 5 ml) and the aq. layer was basified with 450 μl of 32% ammonia to extract the alkaloids with EtOAc (3 x 5 ml). After evaporation of the org. solvent, the dried alkaloid fractions were dissolved in 1 ml of CHCl3 from which 500 μl were transferred to a GC-MS vial. The split ratio was 1:10 and the methodology was the same as 16 previously described in this section 4.1. The design of the assay included the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 randomization of the order of samples for both the extraction procedure and the GC-MS analysis. 4.5. Quantum mechanical calculations. Geometry optimizations were carried out at the MP2 level with the 6-31+G(d) basis (Clark et al., 1983). The minimum energy nature of the stationary points was confirmed by inspection of the vibrational frequencies, which were positive in all cases. The relative stabilities in the gas phase were determined from single-point calculations at the MP2 level with the aug-cc-pVDZ basis (Dunning, 1989), which has been shown to predict well the conformational preferences of flexible compounds (Riley et al., 2007; Forti et al., 2012). Zero-point energy, thermal and entropic effects (at 298 K) were estimated by using the harmonic oscillator-rigid rotor formalism. Finally, the relative stability in chloroform was determined by adding the solvation free energy estimated by using the B3LYP/6-31G(d) parametrized versions of both MST (Curutchet et al., 2001; Soteras et al., 2005) and SMD (Marenich et al., 2009) continuum solvation models. All calculations were performed using Gaussian 03 (Frisch et al., 2009). Acknowledgments The authors thank Rafa Díez for his kind participation in the collection of plant material. This gratitude is extended to SCT-UB personnel for their valuable collaboration, Dr. M. Feliz, Dr. Ma.A. Molins and Dr. A. Linares from the NMR area, as well as Dr. A. Marín from the GC-MS unit. N.B.P. also thanks the Spanish Ministerio . This work was performed within the framework of projects 2009-SGR-1060 and 2009-SGR-298 (Generalitat de Catalunya), 17 and SAF2011-27642 (DGICYT). Finally, the Centre de Serveis Científics i Acadèmics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 (CESCA) is acknowledged for computational facilities. 18 Figures and Legends 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fig. 1. Representative structures of mesembrane alkaloids. OMe OMe OMe 5´ 6´ 2´ 4 3 5 3a 2 N OMe OH 6 7a N O 7 Mesembrine N O Joubertiamine N Sceletium A4 Figure 2. Alkaloids identified in N. triandrus extracts. OMe OR OMe N N OH H OMe OMe H N O 2 (R=H) 1 OMe OMe OMe N OH H 3 H O 4 5 (R=Me) OMe OMe OMe OMe OMe O OMe O N H 6 O N O 7 N H OH 8 19 Figure 3. MS of the alkaloids identified in N. triandrus extracts. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 OMe 100 OMe 70 1 219 50 N H OH 42 58 40 OH 77 86 94 103 115 128 0 100 OMe 60 80 100 120 141 151 140 172 160 180 232 204 187 200 220 245 289 272 240 260 280 300 70 2 50 273 N 42 H O 55 63 0 40 OMe 100 OMe 77 60 91 80 103 115 100 128 120 149 159 140 172 160 188 180 205 200 216 244 230 220 240 219 274 70 H 57 41 99 82 109 153 132 232 175 100 OMe 60 80 100 120 140 160 190 180 258 200 220 H 289 232 115 77 151 161 131 174 246 187 0 OMe 60 80 100 120 140 160 180 200 220 240 258 260 274 280 H O 300 70 5 287 50 N 300 204 55 100 280 96 70 40 OMe 260 4 42 O 240 218 50 N 248 0 40 OMe 280 3 204 OH 260 290 50 N 258 42 51 0 40 63 60 77 80 91 103 100 115 128 120 219 144 157 140 160 172 185 180 230 202 200 220 244 240 258 260 272 280 300 20 Figure 4. MS of the 3 minority compounds isolated from N. triandrus. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 OMe 100 OMe 301 6 50 163 O N H O 51 58 65 0 40 OMe 100 OMe 60 77 84 91 80 103 100 115 138 120 140 217 173 185 201 180 200 160 259 244 230 220 270 240 260 286 280 300 285 7 242 50 257 N O 51 91 102 119 147 132 156 170 199 184 270 214 226 0 50 OMe 77 63 100 OMe 70 90 110 130 150 170 190 210 230 250 270 290 305 8 50 233 O N H OH 57 65 0 40 60 77 80 123 91 100 120 149 167 178 190 138 140 160 180 204 200 218 220 246 240 258 260 272 280 290 300 320 21 Figure 5. Two representative conformers of compound 8 showing key NOESY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 correlations (note that the methoxyl groups have been removed for the sake of simplicity). Fig. 6. Alkaloid profile analysis. The approximate locations of populations analyzed are indicated as colored dots. The larger fuchsia point corresponds to N. triandrus used for alkaloid isolation. Colored bars represent the mean of the measurements of each population (s.d. in black). Data are the peak area (GC-MS analysis) normalized to the area of the internal standard and to the dry wt (g) of plant material. 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 80 80 N. triandrus (Galicia-Asturias) 80 N. triandrus (Burgos) 60 60 40 40 40 20 20 20 0 0 NT13 80 60 NT14 NT15 0 NT-B NT16 NI-B 80 N. triandrus var. capax (Galicia) 60 60 40 40 0 20 N. iohannis (Burgos) N. pallidulus (Segovia) 20 0 0 NT10-Cap 80 NT11-Cap NP1 NT12-Cap 80 N. lusitanicus (Portugal) 60 60 40 40 20 20 0 NP2 N. pallidulus var. paivae (Portugal) 0 NL-P6 NL-P7 NL-P8 6-Epimesembrenol (1) NL-P9 4´-O-Demethylmesembrenone (2) NP-P1 6-Epimesembranol (3) Mesembrine (4) NP-P2 NP-P3 Mesembrenone (5) 23 Tables 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Table 1. Components of the alkaloid extracts of N. triandrus. %A M+ MS 2317 3.96 ND 289 (7) 232 (8), 219 (55), 204 (5), 115 (4), 70 (100) M1 2325 ND <5 277 (74) 276 (100), 260 (41), 218 (27), 205 (96), 153 (21), 44 (58) 4´-O-Demethylmesembrenone (2) 2343 ND 9.28 273 (29) 258 (1), 244 (3), 205 (9), 115 (6), 70 (100), 42 (13) 6-Epimesembranol (3) 2350 17.74 ND 291 (64) 290 (100), 274 (29), 248 (15), 232 (17), 219 (46), 204 (35), 70 (24) Mesembrine (4) 2354 7.07 289 (72) 274 (8), 232 (22), 218 (100), 204 (38), 96 (74), 70 (70) Mesembrenone (5) 2375 70.49 69.92 287 (45) 258 (5), 219 (12), 115 (7), 70 (100) M2 2382 ND <5 277 (58) 276 (100), 260 (26), 205 (40), 190 (35), 44 (22) M3 2440 ND <5 303 (31) 256 (10), 244 (85), 232 (10), 213 (26), 151 (31), 115(21), 60(100) 2-Oxomesembrenone (6) 2675 ND ND 301 (100) 259 (26), 244 (14), 217 (30), 163 (32), 115 (10) 7,7a-Dehydromesembrenone (7) 2697 ND ND 285 (100) 270 (20), 257 (20), 242 (55), 147 (11), 119 (9) 2-Oxoepimesembranol (8) 2755 ND ND 305 (100) 290 (6), 246 (8), 233 (28), 218 (11), 167 (14), 149 (12) Compound RI 6-Epimesembrenol (1) %H ND 24 Table 2. NMR data of 3 (500 MHz, CDCl3) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Position 2α 2β 3α 3β 4α 4β 5α 5β 6 7α 7β 7a 2´ 5´ 6´ NMe OMe (3´, 4´) 3 δH (mult., J in Hz) 3.44 (br s) 2.41 (m) 1.95 (m) 1.86 (td, 12.0, 6.7) 2.33 (td, 14.3, 3.7) 1.95 (m) 1.73 (m) 1.42 (tdd, 14.0, 3.7, 2.3) 3.95 (br s) 2.19 (br d, 15.0) 1.65 (dt, 15.0, 3.0) 2.94 (br s) 6.87 (d, 2.2) 6.81 (d, 8.3) 6.88 (dd, 8.3, 2.2) 2.52 (s) 3.87, 3.89 (s) COSY H2α, H3α/β H2α, H3α/β H3β, H2α/β H3α, H2α/β H4β, H5α/β H4α, H5α/β H5β, H4α/β, H6 H5α, H4α/β, H6 H5α/β, H7α/β H7β, H6, H7a H7α, H6, H7a H7α/β H6´ H6´ H2´, H5´ - NOESY H2β, H3α/β, H4α, NMe H2α, H3α/β, H7a H2α/β, H3β, H4α, H2´, H6´ H2α/β, H3α, H7a, H2´, H6´ H2α, H3α, H4β, H5α H4α, H5α/β, H2´, H6´ H4α/β, H5β, H6 H4β, H5α, H6, H7β, H2´, H6´ H5α/β, H7α/β H6, H7β, H7a, NMe H5β, H6, H7α, H7a, H2´, H6´ H2β, H3β, H7α/β, H2´, H6´, NMe H3α/β, H4β, H5β, H7β, H7a H3α/β, H4β, H5β, H7β, H7a H2α, H7α, H7a - Table 3. NMR data of 6 (500 MHz, CDCl3) Position 2 3α 3β 3a 4 5 6 7 (2H) 7a 1´ 2´ 3´ 4´ 5´ 6´ NMe OMe (3´, 4´) 6 δH (mult., J in Hz) 2.66 (d, 17.1) 3.18 (d, 17.1) 6.71 (dd, 10.2, 1.6) 6.24 (d, 10.2) 2.72 (d, 3.8) 4.06 (td, 3.8, 1.6) 6.85 (d, 2.2) 6.87 (d, 8.4) 6.92 (dd, 8.4, 2.2) 2.81 (s) 3.88 (s) δC 171.9 44.2 44.2 45.9 150.7 129.0 195.3 36.5 65.7 131.6 109.9 149.7 149.1 111.6 119.4 27.4 56.1, 56.2 COSY H3β H3α H5, H7a H4 H7a H7, H4 H6´ H6´ H2´, H5´ - NOESY H3β, H4 H3α, H7a, H2´, H6´ H3α, H5, H2´, H6´ H4 H7a H3β, H7, H2´, H6´, NMe H3β, H4, H7a OMe H3β, H4, H7a H7a H5´ HMBC C2, C3a, C4, C7a, C1´ C2, C3a, C4, C1´ C3, C3a, C6, C7a, C1´ C3a, C7 C3a, C5, C6, C7a C3a, C4, C6, C1´ C3a, C4´, C6´ C1´, C3´ C3a, C2´, C4´ C2, C7a C3´, C4´ 25 Table 4. NMR data of 7 and 8 (500 MHz, CDCl3) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Position 2α 2β 3α 3β 3a 4α 4β 5α 5β 6 7α 7β 7a 1´ 2´ 3´ 4´ 5´ 6´ NMe OMe (3´) OMe (4´) 7 δH (mult., J in Hz) 3.31 (dd, 10.3, 8.1) 3.41 (ddd, 10.6, 10.3, 5.0) 2.27 (ddd, 11.7, 10.8, 8.1) 2.56 (dd, 11.9, 5.0) - δC 52.7 52.7 35.9 35.9 53.5 6.77 (d, 9.6) 142.8 6.03 (dd, 9.6, 1.4) 128.8 - 185.6 5.46 (br s) 93.7 6.84 (d, 2.2) 6.78 (d, 8.3) 6.86 (dd, 8.3, 2.3) 2.98 (s) 3.84 (s) 3.85 (s) 171.5 133.2 109.9 149.1 148.6 111.3 118.4 33.1 56.2 56.1 8 δH (mult., J in Hz) δC - 173.8 2.65 (d, 16.4) 2.54 (d, 16.4) 2.14 (ddd, 15.0, 10.0, 3.6) 1.85 (ddd, 14.5, 7.4, 3.6) 1.60 (m) 1.73 (ddtd, 14.0, 10.0, 3.6, 1.0) 3.95 (tt, 6.1, 3.6) 1.96 (dddd, 14.6, 6.0, 5.5, 1.0) 2.14 (dddd, 14.7, 5.0, 3.6, 1.0) 3.91 (dd, 5.5, 5.0) 6.81 (d, 2.4) 6.82 (d, 8.4) 6.86 (dd, 8.4, 2.3) 2.90 (s) 3.88 (s) 3.87 (s) 45.7 45.7 42.9 30.0 30.0 30.1 30.1 66.1 32.8 32.8 62.3 137.4 110.0 149.2 148.0 111.2 118.2 28.1 56.2 56.1 Table 5. Conformer distribution (in percentage) of the model compounds (obtained by replacing the dimethoxyphenyl ring by either hydrogen or phenyl) used to examine the conformational preferences of 8. Conformer Gas boat_axial boat-equatorial chair_axial chair_equatorial 0.4 0.7 79.1 19.8 boat_axial boat-equatorial chair_axial chair_equatorial 0.5 0.5 86.0 13.0 MST R=H 0.8 0.7 56.7 41.8 R=phenyl 0.9 0.8 67.4 31.0 Chloroform SMD Average 0.8 0.5 43.3 55.4 0.8 0.6 50.0 48.6 1.0 0.6 50.3 48.1 0.9 0.7 58.9 39.6 26 Table 6. Coupling constants of protons of the six-membered C-ring of compound 8. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Vicinal protons 6 - 5β 6 - 5α 6 - 7β 6 - 7α 7a - 7β 7a - 7α 4α - 5β 4α - 5α 4β - 5β 4β - 5α J (Hz) calc.a 2.7 5.9 3.2 5.5 4.7 4.8 9.7 3.2 3.1 6.4 J (Hz) obs. 3.6 6.1 3.6 6.1 5.0 5.5 10.0 3.6 3.6 7.4 Differenceb 0.9 0.2 0.4 0.6 0.3 0.7 0.3 0.4 0.5 1.0 a For details see Table S3 in Supplementary Information. b Difference = J calc. - J obs. 27 Table 7. Plant material information. P: Portugal, S: Spain. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Label NP1 NP2 NP-P1 NP-P2 NP-P3 NL-P6 NL-P7 NL-P8 NL-P9 NT10-Cap NT11-Cap NT12-Cap NT13 NT14 NT15 NT16 NT-B NI-B a Species N. pallidulus Graells var. pallidulus N. pallidulus Graells var. pallidulus N. pallidulus Graells var. paivae Barra N. pallidulus Graells var. paivae Barra N. pallidulus Graells var. paivae Barra N. lusitanicus Dorda & Fern. Casas N. lusitanicus Dorda & Fern. Casas N. lusitanicus Dorda & Fern. Casas N. lusitanicus Dorda & Fern. Casas N. triandrus L. var. capax (Salisbury) Barra & G. Lópeza N. triandrus L. var. capax (Salisbury) Barra & G. Lópeza N. triandrus L. var. capax (Salisbury) Barra & G. Lópeza N. triandrus L. var. triandrus N. triandrus L. var. triandrus N. triandrus L. var. triandrus N. triandrus L. var. triandrus N. triandrus L. var. triandrus N. iohannis Fern. Casasb Near Location (Country) El Espinar - Segovia (S) El Espinar - Segovia (S) Prado (P) Casas do soeiro (P) Fiais da Beira (P) Figueiró dos Vinhos (P) Ferreira do Zêzere (P) Ferreira do Zêzere (P) Albergaria-a-Velha (P) Baiona, Galicia (S) Marín, Galicia (S) Marín, Galicia (S) Viveiro, Galicia (S) Luarca, Asturias (S) Trevías, Asturias (S) Peñaflor, Asturias (S) Montorio, Burgos (S) Peñahorada, Burgos (S) n 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 2 2 N. loiseleuri Rouy b N. triandrus L. subsp. triandrus var. alejandrei Barra 28 References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Arnó, M., González, M.A., Marin, M.L., Zaragozá, R.J., 2000. 1H and 13C NMR assignments and conformational analysis of some podocarpene derivatives. Magn. Reson. Chem. 38, 1019-1022. Barra Lázaro, A., 2000. Dos nuevas variedades de Narcissus triandrus L. (Amaryllidaceae). 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Biol. 36, 173-179. Soteras, I., Curutchet, C., Bidon-Chanal, A., Orozco, M., Luque, F.J., 2005. Extension of the MST model to the IEF formalism: HF and B3LYP parametrizations. J. Mol. Struct. (THEOCHEM) 727, 29-40. Vives, P., Montserrat-Martí, J.M., Fernández-Casas, F.J., 2010. Contenido de ADN en Narcissi (Amaryllidaceae). Adumbrationes ad Summæ Editionem 39, 1-26. Zonneveld, B.J.M., 2008. The systematic value of nuclear DNA content for all species of Narcissus L. (Amaryllidaceae). Plant Syst. Evol. 275, 109-132. 34 !"#$% &'#"!%' (%))"&&')%*'&!+*,!%' " $)*&#'!+-#%" % *""*"*'#,'& *-#" "."'%)* "&"(!" /!*%! *1%'+,+$Z1![/0".! 1\1 "%-$-!("'+"&" ($ +'!%"0"1 "$&!'"] "- // % *^ '!__ %-$" `j-"(q+(z {&|% -+%_+ $ +_ " +'( -+#+1%' !% |}!-} &} '!&}+~^#}%^( 1%!& # $%& !% 0 }"$ {% #("-' {^ " }^' +*%"&+/ +%_+ { Z{ ' !%* {+"&"~ Z/ {+"&"~ / {+"&"~ / {+"&\ Z/ {+"&\ / {+"&\ Z/ -}"! "~ Z/ -}"! "~ / -}"! 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Al mismo tiempo, se ha evaluado la capacidad inhibidora de la enzima AChE de dichos extractos. Los extractos clorofórmicos de bulbo se analizaron mediante GC9MS, con el objetivo de estudiar la composición de alcaloides de cada especie. Los resultados se expresaronenvaloresporcentualesreferidosaláreatotaldelcromatograma(%TIC), útiles para la comparación entre diversas muestras. Todos demostraron la presencia de galantamina en cantidades variables, desde 0.6 a 17.8% del total del contenido alcaloídico, siendo Z. filifolia la especie más destacada en cuanto a la abundancia de dicho alcaloide. Además, se detectó la presencia de tazetina, licorina, galantina, licoramina y algunos alcaloides de tipo hemantamina, entre los componentes mayoritariosdelasdiversasespeciesestudiadas. LosresultadosdelensayodeinhibicióndeAChEvariaronenunrangodevalores deIC50entre1y2g/mL.LosextractosmásprometedoresfueroneldeZ.filifolia(San Juan)yeldeH.jamesonii(Mendoza),quemostraronvaloresdeIC50(1.0±0.08y1.0± 0.01g/mL,respectivamente)alrededorde3vecesmenosactivosencomparacióncon elcontrolpositivo,galantamina(0.29±0.07g/mL). Este estudio demuestra el potencial de especies silvestres argentinas de la familia Amaryllidaceae procedentes de la región andina como fuente renovable de alcaloidesbioactivos,comogalantamina. 99 Molecules 2012, 17, 13473-13482; doi:10.3390/molecules171113473 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Wild Argentinian Amaryllidaceae, a New Renewable Source of the Acetylcholinesterase Inhibitor Galanthamine and Other Alkaloids Javier E. Ortiz 1, Strahil Berkov 2, Natalia B. Pigni 2, Cristina Theoduloz 3, German Roitman 4, Alejandro Tapia 1, Jaume Bastida 2 and Gabriela E. Feresin 1,* 1 2 3 4 Instituto de Biotecnología-Instituto de Ciencias Básicas, Universidad Nacional de San Juan, Av. Libertador General San Martín 1109 (O), CP 5400, San Juan, Argentina; E-Mails: jortiz@unsj.edu.ar (J.E.O.); atapia@unsj.edu.ar (A.T.) Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Catalunya, Spain; E-Mails: berkov_str@yahoo.com (S.B.); npigni@ub.edu (N.B.P.); jaumebastida@ub.edu (J.B.) Facultad de Ciencias de la Salud, Universidad de Talca, Casilla 747, Talca, Chile; E-Mail: ctheodul@utalca.cl Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, 1417, Buenos Aires, Argentina; E-Mail: roitman@agro.uba.ar * Author to whom correspondence should be addressed: E-Mail: gferesin@unsj.edu.ar; Tel.: +54-264-421-1700 (ext. 410/294); Fax: +54-264-420-0289. Received: 8 October 2012; in revised form: 2 November 2012 / Accepted: 9 November 2012 / Published: 13 November 2012 Abstract: The Amaryllidaceae family is well known for its pharmacologically active alkaloids. An important approach to treat Alzheimer’s disease involves the inhibition of the enzyme acetylcholinesterase (AChE). Galanthamine, an Amaryllidaceae alkaloid, is an effective, selective, reversible, and competitive AChE inhibitor. This work was aimed at studying the alkaloid composition of four wild Argentinian Amarillydaceae species for the first time, as well as analyzing their inhibitory activity on acetylcholinesterase. Alkaloid content was characterized by means of GC-MS analysis. Chloroform basic extracts from Habranthus jamesonii, Phycella herbertiana, Rhodophiala mendocina and Zephyranthes filifolia collected in the Argentinian Andean region all contained galanthamine, and showed a strong AChE inhibitory activity (IC50 between 1.2 and 2 μg/mL). To our knowledge, no previous reports on alkaloid profiles and AChEIs activity of wild Argentinian Amarillydaceae species have been publisihed. The demand for renewable Molecules 2012, 17 13474 sources of industrial products like galanthamine and the need to protect plant biodiversity creates an opportunity for Argentinian farmers to produce such crops. Keywords: Argentinian Amaryllidaceae wild; alkaloids; galanthamine; lycorine; tazettine; acetylcholinesterase inhibitors 1. Introduction Many species of medicinal and aromatic plants are cultivated for such industrial uses, but most are still collected in the wild. The demand for renewable sources of industrial products and the need to protect plant biodiversity create an opportunity for farmers to produce such plants as crops. More than 25% of the pharmaceutical drugs used in the World today are derived from plant natural products [1]. In the conventional pharmaceutical industry, pharmaceutical companies produce drugs from compounds extracted from plant material, or use plant derived compounds as starting material to produce drugs semi-synthetically [2]. Examples of the former include the anti-cancer alkaloid paclitaxel from Pacific yew (Taxus brevifolia), vinblastine from the Madagascar periwinkle (Cataranthus roseus), and digoxin from the foxglove (Digitalis lanata) [1]. The alkaloids of the Amaryllidaceae family are extensively studied for their biological activities in several pharmaceutical areas, for example, Alzheimer’s disease (AD), a neurodegenerative problem of enormous economic and social impact (15 million people, mainly in developed countries, suffer from the symptoms of this disease). The treatment is based on drugs that increase levels of acetylcholine. Galanthamine is a long-acting, selective, reversible and competitive inhibitor of acetylcholinesterase (AChE) and an allosteric modulator of the neuronal nicotinic receptor for acetylcholine. AChE is responsible for the degradation of acetylcholine at the neuromuscular junction, in peripheral and central cholinergic synapses. Galanthamine has the ability to cross the blood-brain barrier and to act within the central nervous system [3,4]. According to data presented by the Alzheimer’s Association in 2007, the prevalence of Alzheimer’s disease will quadruple by 2050. Galanthamine hydrobromide has superior pharmacological profiles and higher tolerance as compared to the original AChE inhibitors, physostigmine or tacrine [5]. This alkaloid galanthamine (biosynthesized exclusively by species of Amaryllidaceae family) is the treatment for mild and moderate stages of the AD. Galanthamine, approved in 2001 by FDA (Razadyne®), was originally isolated from Galanthus woronowii. While several total syntheses of the alkaloid galanthamine are available [6–10], current marketing is done mainly by the limited extraction of natural populations of Leucojum aestivum from Turkey (of varying quality and low content of active principle), or from small plantations of this species in Bulgaria, which are insufficient to meet current pharmaceutical company demand. The worldwide production of galanthamine is about 250 kg per year. Around 61 species of the Amaryllidaceae family grow in Argentina, covering a wide variety of genera (Chlidanthus, Crinum, Habranthus, Haylockia, Hieronymiella, Hippeastrum, Phycella, Rhodophiala, Stenomesson and Zephyranthes) [11]. To our knowledge, there are no reports on the chemistry and biological activity of Argentinian species belonging to the Amaryllidaceae group. Molecules 2012, 17 13475 Our search for plant raw materials for medicinal products is now aimed at investigating the acetylcholinesterase inhibitory activity (AChE) of basic chloroform extracts (BCE) obtained from Habranthus jamesonii, Phycella herbertiana, Rhodophiala mendocina, and Zephyranthes filifolia (Amaryllidaceae species that grow in Argentine) to find new sources of production of galanthamine, and other potential alkaloids for treating AD. AChE inhibitory activity was determined by the spectrophotometric method by Ellman et al. [12]. Alkaloid profiles were analyzed by gas chromatography-mass spectrometry (GC–MS). 2. Results and Discussion The AChE inhibitory activity of the BCE from Habranthus jamesonii, Phycella herbertiana, Rodophiala mendocina and Zephyranthes filifolia species, collected from the Andean region of San Juan (SJ), Mendoza (MDZ), and Neuquén (NQN) provinces (Argentine), were tested according to the methodology developed by Ellman et al. [12] with some modifications [13]. Galanthamine was used as a positive control. The results, expressed as IC50 values (μg/mL) are shown in Table 1. BCE showed the highest acetylcholinesterase inhibitory activity, with IC50 values ranging from 1 to 2 μg/mL (reference compound: galanthamine 0.29 ± 0.07 μg/mL). The BCE-Z. filifolia MZA and BCE-H. jamesonii SJ displayed the highest inhibition towards AChE with similar values (IC50 1 ± 0.01 and 1 ± 0.08 μg/mL respectively) only three times higher than that of galanthamine. BCE-P. herbertiana SJ was found to have the second highest inhibition on AChE (IC50 values 1.2 ± 0.12 μg/mL). Acetylcholinesterase inhibition was similar to that of specie R. mendocina regardless of collection site (IC50 values 2 ± 0.15, 2 ± 0.20 μg/mL). BCE-H. jamesonii SJ showed a similar AChE inhibitory activity (IC50 values 2 ± 0.11 μg/mL). The yield percentages of the basic chloroform extract (BCE) (g/100 g dry bulbs) are reported in Table 1. BCE-Zephyranthes filifolia SJ had the lowest percentage at 0.21%, whereas BCE-Rhodophiala mendocina SJ gave the highest one at 0.38%. Table 1. Acetylcholinesterase Enzyme Inhibition of Wild Argentinian Amaryllidaceae extracts expressed as IC50 [g/mL]. Samples (voucher number) BCE a Yield [%] b 0.34 0.25 0.38 0.21 0.27 0.26 IC50 [ȝg/mL] Phycella herbertiana SJ (IBT-Arg1) 1.2 ± 0.12 Habranthus jamesonii SJ (IBT-Arg2) 2.0 ± 0.11 Rhodophiala mendocina SJ (IBT-Arg3) 2.0 ± 0.15 Zephyranthes filifolia SJ (IBT-Arg4 ) 1.0 ± 0.08 Habranthus jamesonii MZA (IBT-Arg5) 1.0 ± 0.01 Rhodophiala mendocina NQN (IBT-Arg6) 2.0 ± 0.20 c Galanthamine 0.29 ± 0.07 a b c Basic chloroform extract, Percentage yield BCE [w/w], Reference compound. The alkaloids detected by GC-MS in the BCE-H. jamesonii MZA, BCE-H. jamesonii SJ, BCE-P. herbertiana SJ, BCE-R. mendocina NQN, BCE-R. mendocina SJ and BCE-Z. filifolia SJ are listed in Table 2. Molecules 2012, 17 13476 Table 2. Alkaloid composition of four Amaryllidaceae plants. Compound Trisphaeridine (1) Ismine (2) 5,6-Dihydrobicolorine (3) Galanthamine (4) Lycoramine (5) Lycoraminone (6) Vittatine (7) Narwedine (8) Anhydrolycorine (9) A-289 (10) A-315 (11) A-249 (12) A-319 (13) Montanine (14) Haemanthamine/Crinamine(15) b Tazettine (16) A-301 (17) Pancracine (18) 11-Hydroxyvittatine (19) Galanthine (20) Lycorine (21) Incartine (22) Methylpseudolycorine (23) Epimacronine (24) 8-O-Demethylhomolycorine (25) Homolycorine type (26) 2-O-Acetyllycorine (27) A-345 (28) Tazettamide (29) m/z 109 (Homolycorine type) (30) Sanguinine (31) m/z 83 (285) (32) m/z 297(33) m/z 281 (34) m/z 283 (35) N-Demethylgalanthamine (36) 2-O-Methylpancracine (37) N-Formylnorgalanthamine (38) Total Alkaloids identified a H. jamesonii a SJ MZA 0.7 1.4 1.9 4.3 13.2 2.5 1.1 0.8 1.5 P. herbertiana a SJ 4.2 27.4 0.5 0.1 0.2 0.4 R. mendocina a SJ NQN 0.1 0.6 3.2 0.8 Z. filifolia a SJ 1.2 0.7 1.7 17.8 1.2 0.2 0.4 0.9 0.9 0.4 1.3 1.1 5.7 2.9 28.1 2.0 0.8 18.7 4.9 8.2 3.1 1.8 2.5 5.4 9.1 31.2 32.9 6.8 69.7 0.3 43.6 4.6 17.2 33.2 1.1 0.2 13.3 20.4 0.6 0.6 0.2 3.9 2.6 2.7 3.5 7.7 0.5 1.1 11.8 0.9 2.3 40.0 84.6 99.1 99.9 0.2 3.8 0.1 32.7 92.0 Values are expressed as GC-MS area %, b Cannot be distinguished by GC-MS. Galanthamine (4) was found in all the species and it ranged from 0.6 to 17.8% of total ion current (TIC). Zephyranthes filifolia presented the highest galanthamine (4) content (17.8% TIC, Figure 1, whereas R. mendocina showed the lowest one (0.6% TIC). The highest AChE inhibitory activity of these species (IC50 1.0 ± 0.08 μg/mL) belonged to BCE-Z. filifolia SJ a fact that could be related to the high content of galanthamine (4). Molecules 2012, 17 13477 Figure 1. Representative GC-MS Chromatogram of Wild Argentinian Amaryllidaceae BCE-Z. filifolia SJ. Peaks: 1: Trisphaeridine; 2: Ismine; 3: 5,6-Dihydrobicolorine; 4: Galanthamine 8: Narwedine; 16: Tazettine. 16 4 12 3 8 Galanthamine (4) content was collection site dependent: the BCE-H. jamesonii SJ sample presented 1.4% galanthamine TIC, while the BCE-H. jamesonii MZA with 4.3% TIC was four times higher. The differences in alkaloid content, depending on geographical distribution of H. jamesonii populations, coincides with a previous report on the European species. Berkov et al. [14], reported an intraspecies diversity in alkaloid profiles in Galanthus elwesii and G. nivalis populations collected in different locations in Bulgaria. They presented galanthamine TIC between 0 and 46%. The main alkaloid types (chemotypes) showed a wide variation in the number of compounds comprising their alkaloid mixture. Genetic and environmental factors and their interaction play a role in determining alkaloid profiles. Additionally, sanguinine (31), identified in BCE-H. jamesonii MZA (0.5% TIC), has a hydroxyl group at C9 instead of a methoxyl group, and is around 10 times more active than galanthamine (4). Although the differential content could indicate that some environmental parameters might be influencing galanthamine (4) production, this specie could be considered for the sustainable production of galanthamine. A similar galanthamine (4) content (4.2% TIC) has been found in BCE-P. herbertiana SJ. BCE-R. mendocina SJ and BCE-R. mendocina NQN, showed a similar galanthamine content (<1% TIC). Narwedine (8), another AChE inhibitor [15] was found in all the populations studied (with the exception of the H. jamesonii and R. mendocina collected in San Juan province), but this compound comprised no more than 1% TIC. Other main alkaloids characterized by GC-MS in the BCE-P. herbertiana SJ were lycorine (21) (33%), lycoramine (5) (27%) and tazettine (16) (5.4%). The occurrence of trisphaeridine (1), galanthamine (4), lycoramine (5), vittatine (7), anhydrolycorine (9), montanine (14), haemanthamine/crinamine (15), tazettine (16), 11-hydroxyvittatine (19), galanthine (20), lycorine (21) and tazettamide (29) are reported for the first time in Habranthus jamesonii from Argentina. According to the literature, haemanthamine (15) and galanthine (20), have been reported previously in Habranthus brachyandrus, a specie of the genus [16]. At the same time, thirteen alkaloids were characterized in BCE-R. mendocina SJ, whereas five of them montanine (14), vittatine (7), haemanthamine (15), tazettine (16) and lycorine (21) have been previously reported as constituents Molecules 2012, 17 13478 of Rhodophiala bifida [17]. The main alkaloids identified in native Amaryllidaceae species from San Juan, Mendoza and Neuquén (Argentina) are shown in Figure 2. Figure 2. Main alkaloids in Wild Amaryllidaceae Species from Argentina. OH OR1 HO O 4 MeO H R3O 4a H NMe galanthamine (4) lycoramine: 4,4a dihidro (5) R2O N lycorine: R1=H, R2+R3=CH2 (21) galanthine: R1=R2=R3=Me (20) OMe R1 H NMe O R2 R3 O O O OH H O tazettine (16) N vittatine: R1=OH, R2=R3=H (7) haemanthamine: R1=OMe, R2=H, R3=OH (15) crinamine: R1=H, R2=OMe, R3=OH (15) 11-hydroxyvittatine: R1=OH, R2=H, R3=OH (19) The presence of main alkaloids identified by GCMS (lycorine (21) and tazettine (16) as percentage of the total ion current (TIC) is consistent with that observed in TLC alkaloid profiles of chloroform basic extract (Figure 3). Figure 3. TLC Analysis of Argentinian Amaryllidaceae (BCE). 1: H. jamesonii, 2: P. herbertiana, 3: R. mendocina, 4: Z. filifolia, 5: Galanthamine, 6: Lycorine, 7: Tazettine. 1 2 3 4 5 6 7 The AChE inhibitory activity of these species can be explained by the presence of other AChE inhibitors in the alkaloid mixtures. Montanine (4) has shown significant AChE inhibitory activity [18], Molecules 2012, 17 13479 while a weak activity has been reported for lycorine (21) and haemanthamine (15) [19]. The other major alkaloids, lycoramine (5) and tazettine (16) have no AChE inhibitory activity [13] while, to the best of our knowledge, no AChE inhibitory activity assays have been performed for galanthine (20), 11-hydroxyvittatine, 2-O-acetyllycorine (27). The alkaloids found in the Argentinian species studied possess other interesting biological properties besides their AChE inhibitory activity. Haemanthamine (15) is a potent inducer of apoptosis [20], and has antimalarial activity [3]. Vittatine (7) has shown cytotoxic activity [3]. Antibacterial activity has been reported for vittatine (7) and 11-hydroxyvittatine (19) [21]. Lycorine (21) exhibits citotoxic, apoptotic, antiviral, antifungal, anti-protozoan [22], and anti-inflammatory activities [23]. It is a good candidate for a therapeutic agent against leukemia [24].Analgesic and hypotensive effects have been reported for galanthine (20) [3]. Moderate cytotoxic activity has been reported for tazettine (16) [25] which is an isolation artefact of chemically labile pretazettine. This compound, which is indeed present in plants, has shown remarkable cytotoxicity against a number of tumor cell lines [3]. In addition to the alkaloids identified in the species studied, other unknown compounds (10, 11, 12, 13, 17, 26, 28, 30, 32, 33, 34, and 35) were detected in minor quantities, showing mass spectral patterns that also suggest structures related to the Amaryllidaceae alkaloids. Isolation studies are currently being developed. 3. Experimental 3.1. Plant Material Wild plants of the species Habranthus jamesonii (BAK) Rav, Phycella herbertiana LINDL, Rhodophiala mendocina (PHIL.) Rav., and Zephyranthes filifolia (HERB.) ex Kraenzlin (Amaryllidaceae) were collected in the Andean regions of San Juan (SJ), Mendoza (MZA), and Neuquén (NQN) provinces (Argentina), during the flowering period between October and March 2009-2010 and then transferred to flowerpots and kept under greenhouse conditions. The species collected and identified, and voucher numbers are shown in Table 1. Figure 4 shows a map of the collection area. All plant species were authenticated by MCS German Roitman when they were collected. Voucher specimens were deposited at the Instituto de Biotecnología (UNSJ) with the codes: IBT-UNSJ- Arg1-6. 3.2. Alkaloid Extraction Dried bulbs (100 g per each plant) were extracted under reflux three times with MeOH (300 mL) for 1 h each. The solvent was evaporated under reduced pressure to give the methanolic crude extracts (MCEs). MCEs were dissolved in H2SO4 (2% v/v) and neutral material was removed with CHCl3 (200 mL). Then, the aqueous solutions were basified with 25% NaOH up to pH 10–12 and the alkaloids were extracted with CHCl3 (3 u 500 mL) to obtain the basic chloroform extract (BCE). After evaporation of the organic solvent, the dry alkaloid fractions were dissolved in MeOH for GC/MS analysis. The BCE were named as BCE-H. jamesonii MZA, BCE-H. jamesonii SJ, BCE-P. herbertiana SJ, BCE-R. mendocina NQN, BCE-R. mendocina SJ and BCE-Z. filifolia SJ after their origin. Molecules 2012, 17 13480 Figure 4. Collection areas of Argentinian wild Amaryllidaceae. San Juan Mendoza Neuquén Phycella herbertiana Zephyranthes filifolia Habranthus jamesonii Rhodophiala mendocina 3.3. Gas Chromatography-Mass Spectroscopy Analyses GC-MS analyses were performed on a Hewlett Packard 6890/MSD 5975 instrument (Hewlett Packard, Palo Alto, CA, USA) operating in EI mode at 70 eV. A DB-5 MS column (30 m u 0.25 mm u 0.25 m) was used. The temperature program was: 100–180 °C at 15 °C min1, 1 min hold at 180 °C, 180–300 °C at 5 °C min1, and 1 min hold at 300 °C. Injector temperature was 280 °C. The flow rate of carrier gas (He) was 0.8 mL min1. The split ratio was 1:20. The results obtained were analyzed using AMDIS 2.64 software (NIST). Compounds were identified through the comparison of their mass spectral patterns and retention indexes, with the data recorded in literature. 3.4. Microplate Assay for Acetylcholinesterase Activity AChE activity was assayed as described by Ellman et al. [12] with some modifications [13]. Fifty L of AChE in buffer phosphate (8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, 0.05% Tween 20, pH 7.6) and 50 L of the sample dissolved in the same buffer were added to the wells. The plates were incubated for 30 minutes at room temperature before the addition of 100 L of the substrate solution (0.1 M Na2HPO4, 0.5 M DTNB, 0.6 mM ATCI in Millipore water, pH 7.5). The absorbance was read in a Labsystems microplate reader (Helsinki, Finland) at 405 nm after three minutes. Enzyme activity was calculated as a percentage compared to an assay using a buffer without any inhibitor. The AChE inhibitory data were analyzed with the software package Prism (Graph Pad Inc., San Diego, CA, USA). IC50 values are means ± SD of three individual determinations each performed in triplicate. Molecules 2012, 17 13481 3.5. TLC Analysis of BCE TLC was carried out on Merck Silica gel 60 F254 plates, using chloroform-methanol-ammonia (99:9:1) mixtures as mobile phase. TLC plates were sprayed with Dragendorff’s reagent; main alkaloids gave orange spots. 4. Conclusions The findings of the present study demonstrate the potential of wild Argentinian Amaryllidaceae species collected in the central Andean region, as a new renewable source of galanthamine. The most promising species seen to be H. jamesonii MDZ and Z. filifolia SJ. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 4.DISCUSIÓN Discusión 4.DISCUSIÓN Los resultados expuestos en el presente trabajo comprenden el estudio del contenidodealcaloidesdeespeciessilvestresdeplantasdelafamiliaAmaryllidaceae (subfam.Amaryllidoideae)deláreaMediterráneaydeArgentina.Enlíneasgenerales, se pueden considerar tres secciones diferentes: por un lado, el aislamiento y caracterizacióndenuevosalcaloidesdelaespecieNarcissusserotinusL.;ensegundo lugar,elestudiodelosalcaloidesinusualesdelosnarcisospertenecientesalasección Ganymedes,comoN.triandrusL.;y,finalmente,elanálisisdelcontenidodealcaloides deespeciessilvestresargentinascomofuentedecompuestosbioactivos. 4.1.AlcaloidesdeNarcissusserotinusL. Narcissus serotinus L. es una especie de floración otoñal perteneciente a la secciónmonotípicaSerotini,conunadistribucióngeográficaqueseextiendealolargo del área Mediterránea, incluyendo el sur y este de la Península Ibérica, regiones costeras de Italia, Croacia, Grecia, Israel y el norte de África, así como casi todas las islasdelMediterráneo(Blanchard,1990;DíazLifanteetal.,2007).Apesardequese han llevado a cabo numerosos estudios analizando la variabilidad genética y morfológica de esta especie, sólo existía una publicación disponible en la literatura haciendo referencia a su contenido de alcaloides (Vrondeli et al., 2005), previa al presente trabajo. Curiosamente, en dicho artículo se reporta el aislamiento de un nuevo alcaloide al que se le asigna una estructura del tipo tazetina, una asignación que,enbaseanuestrosresultados,hemosconsideradoincorrecta. Entérminosgenerales,enlosartículos1y2incluidosenlaseccióndeResultados se describe la identificación y elucidación estructural de ocho alcaloides nuevos aisladosdeN.serotinus.Paraelprimerestudio(Pignietal.,2010)seutilizaron350g dematerialvegetalfrescorecolectadoenMarruecosduranteOctubrede2009,loque permitió la caracterización de narseronina y 19O9(3´9acetoxibutanoil)licorina. De maneraadicional,enelanálisisdeGC9MSsedetectólapresenciadelicorina,galantina, 19O9(3´9hidroxibutanoil)licorina, asoanina e hipeastrina, así como de algunos compuestos no identificados que presentaron patrones de fragmentación típicos de alcaloides de Amaryllidaceae. Consecuentemente, se planificó una segunda 113 Discusión recolección de material vegetal durante Octubre de 2010 en localidades de la ComunidadValenciana(España),conelobjetivodeidentificardichoscomponentesy,a lavez,obtenerunaideageneraldelasposiblesdiferenciasenelperfildealcaloidesde poblaciones con diferente ubicación geográfica. El hallazgo de una población abundante de N. serotinus cerca de Vinarós (Castellón) permitió obtener 2.43 kg de material vegetal, que resultaron en el aislamiento y elucidación estructural de seis nuevosalcaloides:39O9metilnarcisidina,19O9acetil939O9metilnarcisidina,19O9acetil939O9 metil969oxonarcisidina, 29metoxipratosina, 119hidroxigalantina, y 29O9metilclivonina, además de la identificación de los ya conocidos narseronina, galantina, incartina, masonina e hipeastrina mediante GC9MS (Pigni et al., 2012). Todos los alcaloides detectadosenN.serotinuspertenecenalasserieslicorinayhomolicorina(Figura4.1). 2´´ O 1´´ O 4´ 3´ 12 11 H MeN 10 O O H 10a 9 4a 3 1 8 6a O 2 10b 6 O 2´ OH 1´ 4 OMe O 10 O 9 2 1 3 H 10b 10a H 7 O O narseronina 8 1-O-(3´-acetoxibutanoil)licorina OMe OMe MeO H MeO R2 H MeO N MeO N R3 O 3-O-metilnarcisidina (R1=R2=R3=H) 1-O-acetil-3-O-metilnarcisidina (R1=Ac; R2=R3=H) 1-O-acetil-3-O-metil-6-oxonarcisidina (R1=Ac; R2+R3=O) 2-metoxipratosina H MeN OMe H HO H MeO H MeO 12 6 OMe R1O 11 N 6a 7 4 4a H O OH H O N 11-hidroxigalantina OMe O O 2-O-metilclivonina Figura4.1:NuevosalcaloidesaisladosdeN.serotinus. 114 Discusión 4.1.1.Narseronina Estealcaloidepresentaunaestructuradetipohomolicorinainusualdebidoala presenciadeundobleenlaceenlaposición1910b,unacaracterísticaquenosehabía reportadoanteriormente.Losdatosespectralesobtenidosindicaronquesetratabadel mismo compuesto aislado de ejemplares griegos de N. serotinus por Vrondeli et al. (2005),identificadoerróneamentecomounisómerode39epimacronina. La fórmula molecular C18H19NO5 se confirmó mediante HRMS. Su espectro de masas no mostraba los rasgos típicos de una estructura del tipo homolicorina, probablemente debido que la existencia del doble enlace 1910b afecta el patrón de fragmentación. Su espectro de 1H9RMN demostró la presencia de dos protones aromáticos en posición para, un grupo metilendioxi y un sustituyente metoxilo. La observacióndeungrupoN9metilorelativamentedesapantallado(2.41ppm)sugirió una posible configuración cis para la unión de los anillos C/D (Jeffs et al., 1988), tambiénapoyadaporelvalordelaconstantedeacoplamientoentrelosprotones494a de6.4Hz,nolosuficientementeelevadacomoparaindicarunarelacióntrans9diaxial. EltripleteasignadoaH92(J=6.1Hz)apuntóaunaorientaciónpseudo9ecuatorialpara dichoprotón,deacuerdoconlaorientacióndegrupometoxilo. La presencia de un doble enlace en la unión entre los anillos B/C es una característicaestructuralqueconfiererigidezalaporcióndelamoléculaformadapor losanillosA9B,conunefectoestabilizanteadicionaldebidoalaextensióndelsistema conjugado. Esta particularidad podría resultar interesante para farmacóforos con dichos requerimientos. Resultados preliminares de ensayos de actividad biológica no demostraronqueestealcaloidetuviesepropiedadesdestacablescomoinhibidordela enzima acetilcolinesterasa, ni como antiparasitario. Sin embargo, es importante destacar que después de nuestra publicación, un grupo de investigación australiano desarrolló un proceso de obtención de narseronina mediante síntesis quimio9 enzimática,confirmandolaestructuraconanálisisderayosX,yfacilitandoqueenel futuropuedanensayarseotrotipodebioactividades(Schwartzetal.,2011). 115 Discusión 4.1.2.1O(3´acetoxibutanoil)licorina Laobservacióndelespectrodemasasdeestealcaloideindicóquesetratabade underivadodelicorina,conelpicobasecaracterístico(m/z226)yunpesomolecular de415unidades.Suisómeroconelsustituyenteenposición2,aisladodeGalanthus nivalis(Berkovetal.,2007),presentaunpatróndefragmentaciónmuysimilarconla diferencia de que el ión más abundante de su MS es m/z 250. Esta particularidad también se ha observado en isómeros del mismo tipo con otros grupos funcionales, comoacetilo3´9hidroxibutanoil(Berkovetal.,2009b;Cedrónetal.,2010;deAndrade et al., 2012a; Toriizuka et al., 2008), demostrando que es posible diferenciar varios derivadosdelicorinaconenlacestipoésterenlasposiciones1o2medianteGC9MS. El espectro de 1H9RMN es similar al reportado para el isómero en posición 2, exceptoporlaesperablediferenciaenlosdesplazamientosquímicosdeH91yH92,ya que el protón de la posición sustituida aparece más desapantallado: 5.68 y 4.23 ppm,paralosprotonesrespectivosdelderivadoenposición1;y,4.51y5.31ppm, para el isómero en 2. La relación trans9diaxial entre los protones 4a y 10b, fue confirmadaporelelevadovalordesuconstantedeacoplamiento(10.4Hz). 4.1.3.DerivadosdeNarcisidina LosdoscomponentesmásabundantesdelextractodeN.serotinusrecolectado enlaComunidadValencianaseidentificaroncomo39O9metilnarcisidinay19O9acetil939 O9metilnarcisidina, ambos derivados del alcaloide narcisidina, una estructura de tipo licorinaconundobleenlaceenposición4911delanilloD(Kiharaetal.,1995).Además, se aisló un tercer derivado, 19O9acetil939O9metil969oxonarcisidina, hallado en cantidadesmuyminoritarias. El espectro de masas de 39O9metilnarcisidina mostró un patrón muy similar al reportado para narcisidina, aunque con un pico molecular 14 unidades mayor (m/z 347) indicando la presencia del grupo metilo adicional. Contrariamente, las fragmentaciones de los otros dos derivados no se correspondían con el patrón característico de narcisidina, probablemente a causa de la sustitución en C91 y el carboniloenC96. 116 Discusión Los espectros de 1 H9NMR de 39O9metilnarcisidina y 19O9acetil939O9 metilnarcisidinapresentabaneldobledobletecaracterísticodeestetipodealcaloides asignadoaH910bentre2.70y3.00ppm,conconstantesdeacoplamientocercanas a11.0y2.0Hz.ElelevadovalordeJ,asignadoalacoplamientoentreH910byH94a,es un indicio de la configuración transdiaxial. En ambos casos, la información obtenida del espectro bidimensional NOESY, ha sido de gran utilidad para resolver la configuración y posición de los grupos funcionales, como lo demuestra el caso del grupometiloen3,queevidencióunaproximidadespacialconH911.Losdosderivados conelgrupoacetiloenC91,presentaronelsinguletecorrespondienteen2.00ppm. Es interesante mencionar también que en el espectro de 1H9RMN de 19O9acetil939O9 metil969oxonarcisidina, el protón aromático en posición 7 se encuentra fuertemente desapantallado ( 7.57 ppm) debido al efecto del grupo carbonilo en peri, tal como ocurreenlosalcaloidesdelactónicosdetipohomolicorina. A nivel biosintético, ya en los años 70 se propuso que las estructuras de tipo narcisidina derivan de galantina, involucrando la participación del alcaloide incartina comocompuestointermediarioepoxidado(Fugantietal.,1974).Precisamente,ambos alcaloidessehandetectadoenelextractodeN.serotinus. 4.1.4.2Metoxipratosina Los datos espectrales de este alcaloide indicaron la presencia de un sistema establededoblesenlacesconjugados.Porunlado,suespectrodemasaspresentaba unpicobasecoincidenteconelpicomolecular(m/z309),asícomounreducidogrado de fragmentación. Por otra parte, en el espectro de 1H9RMN se observaron las 6 señales correspondientes a los protones aromáticos, con sus respectivos acoplamientos,ylos3gruposmetoxilomostrandoeldesplazamientocaracterísticode unentornoaromático(3.9094.20ppm). Sibienlapresenciadeestecomponentepuedeatribuirseaunsubproductode licorina y derivados, recientemente se ha propuesto que estructuras de este tipo podríanserpartedeunarutabiosintéticaalternativadealcaloidesdeamarillidáceas, quetranscurriríasinladescarboxilaxiónpreviadeL9tirosina(Wangetal.,2009). 117 Discusión 4.1.5.11Hidroxigalantina El patrón de fragmentación de este compuesto es muy similar al de galantina (Bastidaetal.,1990;Kobayashietal.,1977),conlanotablediferenciade16unidades entre los iones mayoritarios de ambos compuestos, sugiriendo la presencia de un átomo de oxígeno adicional. Los datos de RMN concuerdan con los equivalentes reportadosparagalantina.Laorientacióndelgrupohidroxiloseasignóteniendoen cuentaelvalordelaconstantedeacoplamientoalílicoentreH93yH911,quedepende delángulodiedrodefinidoporH911yelplanoformadoporC94/C93/C911.Laconstante observadade1.5HzconcuerdaconlaorientacióndeH911. 4.1.6.2OMetilclivonina El espectro de masas de este alcaloide mostró los dos iones mayoritarios característicosdeclivonina(Alietal.,1983),m/z83(100%)ym/z96(39%),conunpico molecular 14 unidades mayor (m/z 331) indicando la presencia de un grupo metilo adicional. Con respecto a la estereoquímica, tanto los datos de RMN como los del espectro de dicroísmo circular, sostuvieron una configuración trans9B/C anti, cis9C/D paralasunionesdelosanillos,encoincidenciaconlosdatosreportadosparaclivonina (Haningetal.,2011;Wagneretal.,1996). Un aspecto destacable del espectro de 1H9RMN de este alcaloide es el desplazamiento químico inusualmente desapantallado de los protones aromáticos, especialmentedeH910,cuyaseñalapareceacamposmásbajosqueladeH97,apesar de la presencia del grupo carbonilo en peri. Jeffs et al. (1971c) han propuesto que dichodesplazamiento,tambiénobservadoenotrasestructurassimilares,sedebeala proximidadespacialentreH910yelátomodenitrógenodelanilloD. En conjunto, estos resultados permiten concluir que la especie N. serotinus es una fuente interesante de alcaloides típicos de Amaryllidaceae, siendo los componentesmayoritariosdelosextractosestructurasnovedosas.Agrandesrasgos,si secomparaelanálisisdelasplantasdeMarruecosconlasdelaComunidadValenciana es destacable mencionar la ausencia de licorina y 19O9(3´9acetoxibutanoil)licorina en 118 Discusión estasúltimas.Noobstante,elalcaloidenarseroninaseconfirmócomounodelosmás abundantes en ambos casos. Es importante comentar que, teniendo en cuenta la variabilidadmorfológicaygenética,asícomosudistribucióngeográfica,losejemplares recolectados en la Comunidad Valenciana (N. serotinus L. s.l.) corresponderían a N. deficiensHerberts.s.segúnalgunosautores(DíazLifanteetal.,2007;FernándezCasas, 2008),mientrasquelasplantasmarroquíesseclasificancomoN.serotinusL.s.s.Esta separación taxonómica podría explicar la variación del contenido de alcaloides, sin embargo,dadoquelaproduccióndemetabolitossecundariosenunorganismovegetal sueleestarcondicionadaporungrannúmerodefactores,noesconvenienterealizar unageneralizacióndeestecasoenparticularapartirdenuestrosdatos. 4.2.AlcaloidesdeNarcisosdelaSecciónGanymedes La clasificación taxonómica de los narcisos pertenecientes a la sección Ganymedeshasidoobjetodedebate;así,mientrasalgunosautoresconsideranquese trata de una única especie (N. triandrus L.) junto con tres subespecies y algunas variedades (Barra Lázaro, 2000), estudios moleculares sostienen la idea de tres especiesdiferentes:N.triandrusL.,N.pallidulusGraells,yN.lusitanicusDorda&Fern. Casas (Santos9Gally et al., 2011; Vives et al., 2010; Zonneveld, 2008), a las que recientementeseagregóunacuarta,N.iohannisFern.Casas(FernándezCasas,2011). La distribución geográfica de estas especies abarca la Península Ibérica y las islas Glenan (Francia), siendo su época de floración habitual entre los meses de Marzo y Abril. Estudios previos sobre su contenido de alcaloides reportan la presencia de estructuras de tipo mesembrano, inusuales entre las plantas de la familia Amaryllidaceae,enN.pallidulus(Bastidaetal.,1989;Berkovetal.,enpreparación)y N.triandrus(Seijasetal.,2004). EneltercerartículopresentadoenlaseccióndeResultados,seabordaelestudio del contenido de alcaloides de especies de la sección Ganymedes, incluyendo la identificación y aislamiento de alcaloides de la especie N. triandrus, así como un análisisporGC9MSdelextractodealcaloidesdeplantasrecolectadasen18localidades delaPenínsulaIbérica,quecomprendenmuestrasdetodoslostaxonesdescritospara lasección. 119 Discusión 4.2.1.AlcaloidesdeN.triandrusL. El acceso a una población silvestre abundante de N. triandrus cercana a la localidad de Proaza (Asturias, España) permitió la recolección de 600 g de material vegetalfrescoqueresultaronenlaidentificaciónde8alcaloidesdetipomesembrano (Figura4.2). OMe OR OMe N OMe N OH H OMe 6-epimesembrenol OMe N O H 6-epimesembranol OMe OMe O H O 2-oxomesembrenona N O H mesembrine OMe OMe OMe O 2 N OMe OH H 4´-O-demetilmesembrenona (R=H) mesembrenona (R=Me) OMe OMe N O 7,7a-dehidromesembrenona N H OH 2-oxoepimesembranol Figura4.2: Alcaloidesidentificadosen N.triandrus. Los compuestos mesembrenona, 4´9O9demetilmesembrenona y mesembrina se identificaron directamente mediante el análisis de GC9MS, por comparación con los patrones de fragmentación previamente reportados (Bastida et al., 1989; Jeffs et al., 1974;Shikangaetal.,2012).Sinembargo,enelcasodeloscompuestoshidroxilados enlaposición6(69epimesembrenoly69epimesembranol),elespectrodemasasnofue suficiente para definir la estereoquímica, siendo necesaria la información complementariade1H9RMN.Lasseñalescorrespondientesalosprotonesolefínicosde 69epimesembrenol,asícomoelanálisisdelasconstantesdeacoplamientodeH96enel caso de 69epimesembranol, permitieron la asignación del grupo hidroxilo en , en acuerdoconlosdatospreviosreportados(Jeffsetal.,1969,1970).Conjuntamente,en elanálisisdeGC9MSsedetectarontrescomponentesminoritariosquenofueposible identificar. 120 Discusión Porotraparte,losotrostresalcaloidesrestantescaracterizadosnosedetectaron por GC9MS y corresponden a estructuras reportadas por primera vez a partir de una fuente natural. Su aislamiento permitió la identificación y elucidación estructural mediantelacombinacióndelastécnicasespectroscópicasderutina. 4.2.1.1.29Oxomesembrenona Estealcaloidepresentaunaestructuranovedosaconuncarboniloenlaposición 2yunsistemaconjugadodeinsaturacionesenelanillodeseismiembros.Esta particularidadsevereflejadaensuespectrodemasasporlaestabilidaddelión molecular(m/z301)yelreducidogradodefragmentación.Aligualquetodoslos alcaloidesidentificadosenN.triandrus,laregiónaromáticadesuespectrode1H9 RMNescaracterística,presentandotresprotonesaromáticosconacoplamientos típicosdedisposicionesortoymeta(J=8.4,2.2Hz,respectivamente).Además, seobservanseñalescorrespondientesadosmetoxilosaromáticos,ungrupo N9 metilo ( 2.81 ppm) y un par de protones olefínicos. El espectro de 13C9RMN presentalasdosseñalesdesapantalladasdeloscarbonilos(171.9,195.3ppm), quemuestranlascorrelacionesrespectivasdelargadistanciaconprotonesados ytresenlaces(HMBC). 4.2.1.2.7,7a9Dehidromesembrenona Esta estructura ha sido previamente reportada como producto de la oxidación sintética de mesembrenona con azodicarboxilato de dietilo (DEAD) (Jeffs et al., 1971b).Suespectro 1H9RMNysufragmentaciónconcuerdanconlosdatosdela literatura. La asignación ha sido confirmada con técnicas de RMN bidimensionales, además del espectro de 13C9RMN. Es interesante mencionar que el carbono metínico de la posición olefínica 7, se observa inusualmente apantallado(93.7ppm). 4.2.1.3.29Oxoepimesembranol Este alcaloide se había obtenido previamente por procedimientos sintéticos (Ishibashi et al., 1991), pero los datos espectrales reportados no estaban 121 Discusión completos. Su espectro de masas presenta un abundante pico molecular (m/z 305)yunreducidogradodefragmentación. Al analizar los datos de los espectros de RMN de este compuesto surgieron algunos aspectos controvertidos sobre la elucidación estructural, como la asignación de la disposición del sustituyente hidroxilo, o la observación de correlacionesdeproximidadespacialqueapuntabanalapresenciade,almenos, dos confórmeros diferentes para el anillo saturado de seis miembros. Además, losvaloresdelasconstantesdeacoplamientodeH96(tt,J=6.1,3.6Hz)noeran lo suficientemente elevados como para indicar una posición axial, ni se justificabancompletamenteconunaposiciónecuatorial. Consecuentemente,seplanteólarealizacióndeunestudiodeestabilidaddelos posiblesconfórmerosmediantecálculosdemecánicacuántica,cuyosresultados indicaronlacontribucióndedosconformacionesdesillaprincipales:unaconel grupo dimetoxifenilo en posición axial (en concordancia con estudios conformacionales reportados por Jeffs et al., 1969) y otra con dicho grupo en posiciónecuatorial.Asimismo,dadoqueestudiosprevioshanmostradoquelas constantesdeacoplamientoobservadaspuedenreflejarlamediaponderadade losvaloresdeJdeterminadosparacadaconfórmeroindividualdeuncompuesto en particular (Arnó et al., 2000), se realizaron cálculos similares que revelaron mínimas diferencias entre los valores de J calculados y los observados, permitiendoconfirmarlapresenciadedichasconformacionesenequilibrio. 4.2.2.AnálisisdelContenidodeAlcaloidesdeEspeciesdelaSecciónGanymedes Se recolectaron plantas de 18 poblaciones silvestres de especies de la sección GanymedesconelobjetivodellevaracabounanálisismedianteGC9MSparainvestigar lacomposicióndesusextractosdealcaloides.Lasmuestrasincluyeronejemplaresde todos los taxones descritos para la sección, procedentes de diversas ubicaciones geográficas,comprendiendotresComunidadesAutónomasdeEspaña(CastillayLeón, GaliciayAsturias)ylamitadnortedePortugal. Loscromatogramasobtenidosseanalizaronmanualmenteregistrandoeláreade cada pico. Los datos fueron normalizados respecto al área del estándar interno 122 Discusión agregado(codeína)yalpesosecodematerialvegetal(g),conelobjetodeposibilitar unacomparaciónadecuada.Losresultadosserepresentarongráficamentetomandola media y desviación estándar de los datos obtenidos de los individuos de cada población. En todas las muestras analizadas se detectó la presencia de alcaloides de tipo mesembrano,sinobservarindiciosdeestructurastípicasdeamarillidáceas.Todaslas poblaciones analizadas presentaron el mismo alcaloide mayoritario, mesembrenona, conunagrandiferenciarespectodelosdemáscomponentes.Enlíneasgenerales,los perfiles de alcaloides resultaron similares entre la mayoría de las poblaciones estudiadas. A pesar de la alta variabilidad, reflejada en los elevados valores de desviación estándardelosdatos,sedestacaronalgunastendenciassutilesdeagrupamientocon respecto a la cantidad de alcaloides. Tanto las muestras de N. pallidulus de Segovia, comoladeN.iohannisdeBurgos,mostraronunareducidaabundanciadealcaloides en comparación con las demás. Enel primercaso, la diferencia puedeatribuirse ala variabilidadontogénica,dadoquedichasplantaseranlasúnicasdelmuestreoqueno estaban en la etapa de floración durante la recolección, aunque tampoco se pueden descartar otros factores de variabilidad. En el caso de N. iohannis, la diferencia observadapodríaestarrelacionadaconelhechodequeestaespecieseconsideraun caso aislado, por ser endémica de una pequeña región, por sus diferencias morfológicas y por las particularidades ambientales de su hábitat, que llevaron a considerarsuclasificaciónindependiente(FernándezCasas,2011). Para las demás poblaciones, el perfil y abundancia de alcaloides resultaron comparativamentesimilares,exceptuandounaligeradiferenciaenpoblacionesdeN. triandrus(Galicia9AsturiasyBurgos)yunapoblaciónN.pallidulusvar.paivae(NP9P2, Portugal),quepresentaronunacontribuciónunpocomáselevadadeloscomponentes minoritarios. Este estudio confirma por primera vez la presencia de alcaloides de tipo mesembranoentodoslostaxonesdelasecciónGanymedes,sindetectarestructuras típicas de Amaryllidaceae. En conjunto con un análisis previo de ejemplares de N. pallidulus, que también reveló la presencia de mesembranos (Berkov et al., en preparación),demuestraunprocesointeresanteenlafilogeniadelgéneroNarcissus, 123 Discusión dado que esta sección es la única que presenta exclusivamente este tipo inusual de alcaloides. Curiosamente, esta es una característica fitoquímica compartida con el distantegrupodedicotiledóneassudafricanasdelgéneroSceletium. 4.3.AmaryllidaceaeArgentinascomoFuentedeAlcaloidesBioactivos Elcuartoartículoincluidoenelpresentetrabajoformapartedeunconjuntode proyectos dirigidos al estudio de la composición de alcaloides de amarillidáceas latinoamericanas,conelobjetivodeinvestigarfuentesdecompuestosbioactivoscon potencial aplicación farmacológica. El grupo de investigación del Departamento de Productos Naturales, Biología Vegetal y Edafología de la Facultad de Farmacia (UB), mantiene numerosas colaboraciones con países latinoamericanos como Argentina, Brasil,Colombia,CostaRicayMéxico,quepermiteneldesarrollodeinvestigacionesde especies no estudiadas, favoreciendo el conocimiento de los recursos disponibles y promoviendosuconservación. EnArgentinahaydescritas61especiesdeplantasdelafamiliaAmaryllidaceae, incluyendounagranvariedaddegéneros.Hastaelmomentodelapublicacióndeeste artículo(Ortizetal.,2012),noseconocíanreportesdeestudiossobrelacomposición de alcaloides y actividad biológica de dichas especies. Dada la potencialidad ya conocida de estas plantas para la producción de alcaloides bioactivos, junto a la creciente demanda de fuentes renovables y altamente productoras de compuestos medicinales,talescomogalantamina,seplanteóabordarelestudiodelcontenidode alcaloides y la capacidad inhibidora de la enzima AChE de cuatro especies silvestres andinas: Habranthus jamesonii, Phycella herbertiana, Rhodophiala mendocina y Zephyranthesfilifolia. En el estudio se incluyeron muestras de las cuatro especies recolectadas en la provinciadeSanJuan(SJ),asícomounamuestraadicionaldeH.jamesoniideMendoza (MZA)yunadeR.mendocinadeNeuquén(NQN). LaactividadinhibidoradeAChEseevaluómedianteunamodificacióndelmétodo colorimétrico de Ellman et al. (1961) (López et al., 2002). Los datos obtenidos se expresaron en valores de concentración inhibitoria del 50% (IC50) y se utilizó galantamina como control positivo. Los resultados de inhibición de los extractos 124 Discusión clorofórmicosmostraronvaloresdeIC50enelrangode1a2g/mL.Losextractosmás destacables, con valores que demostraron una actividad sólo tres veces menor que galantamina(IC50=0.29±0.07g/mL),fueronlosdeZ.filifolia(IC50=1.0±0.08g/mL) yH.jamesoniiMZA(IC50=1.0±0.01g/mL). El contenido de alcaloides de los extractos se analizó mediante GC9MS y los resultadosseexpresaronenvaloresporcentualesreferidosaláreadelcromatograma (% TIC, Total Ion Current), los cuales no se corresponden exactamente con una cuantificación real pero pueden ser utilizados con fines comparativos (Berkov et al., 2008b).Entodaslasmuestrassedetectóelalcaloidegalantamina,conunavariación entre0.6y17.8%deltotaldealcaloides.ElextractodeZ.filifoliamostróelvalormás alto,coincidiendoconunadelasmayoresactividadesinhibidorasdeAChE. En las dos muestras de H. jamesonii, tanto el contenido de alcaloides como la actividad biológica ensayada, presentaron diferencias importantes según el lugar de recolección. La mayor capacidad de inhibición de AChE del extracto de H. jamesonii MZA,podríaexplicarseporlapresenciadeunmayorcontenidodegalantamina,4.3% (frente a 1.4% de H. jamesonii SJ), sumada a la de otros ya conocidos inhibidores, como sanguinina (10 veces más activo que galantamina), narwedina, y licorina. Las variaciones en el perfil de alcaloides dependientes de la ubicación geográfica de especiessilvestreshansidopreviamenteestudiadas,comoenelcasodepoblaciones búlgarasdeGalanthusnivalisyG.elwesiienlasquesedetectóunaampliavariabilidad intraespecífica en el contenido de alcaloides, ya que la interacción entre factores genéticosyambientesdiversospuedeejercerunagraninfluenciaenlaproducciónde alcaloidesdeunaespecie(Berkovetal.,2011a). La técnica de GC9MS presenta algunas limitaciones para la identificación inequívocadealgunostiposdealcaloidesdeamarillidáceas,talcomoocurreenelcaso deestructurasdetipocrinano,paralacualesnosiempreesposiblediferenciarentre compuestos que tienen el puente 5910b en (hemantamina) y los de orientación (crinina).Sinembargo,enmuchoscasos,losvaloresdeRIdecompuestospatrónyla informacióndeinvestigacionespreviassobreelgénerobajoestudio,puedenpermitir proponerloscompuestosmásprobables. Teniendo en cuenta lo expuesto, dado que estudios previos de especies de los génerosHabranthusyRhodophialahanreportadolapresenciadealcaloidesdelaserie 125 Discusión hemantamina (Jitsuno et al., 2009; Wildman et al., 1967), se ha propuesto que los alcaloides detectados en las muestras estudiadas corresponden a dicha serie. Sin embargo,enelcasodehemantaminaycrinamina,epímerosenlaposición3,losdatos disponiblesnopermitenafirmarquesetratedeunouotro.Tantovitatinacomo119 hidroxivitatina,sedetectaronencantidadesabundantesenlamuestradeH.jamesonii SJ (13.2% y 18.7%, respectivamente), mientras que hemantamina/crinamina resultó serunodelosdoscomponentesmayoritariosdelextractodeR.mendocinaSJ(31.2%). Ademásdegalantamina,elalcaloidemásabundanteenelextractodeZ.filifolia corresponde a tazetina (~70%), que también demostró una participación importante en los extractos de H. jamesonii SJ (28.1%) y R. mendocina SJ (32.9%). Licorina se detectóenabundanciavariable(8.2943.6%)entodaslasmuestras,exceptoZ.filifolia. EnelcasodeP.herbertiana,loscomponentesmayoritariosfuerongalantina(17.2%)y licoramina (27.4%), además de licorina (33.2%). En adición a los compuestos identificados, se detectó la presencia de componentes minoritarios con patrones de fragmentación que sugieren estructuras relacionadas con los esqueletos típicos de alcaloidesdeamarillidáceas. Este estudio demuestra el potencial de especies silvestres argentinas de la familiaAmaryllidaceaeprocedentesdelaregiónandina,unodeloscentrosprincipales de diversificación de este grupo de plantas, como fuente de alcaloides bioactivos. Segúnelpresenteanálisis,lasespeciesmásprometedorasencuantoalaproducción degalantamina,resultaronserZ.filifoliaSJyH.jamesoniiMZA.Lademandadefuentes renovables y la necesidad de proteger la biodiversidad vegetal de estas especies representan una oportunidad potencialmente productiva para los agricultores argentinosdelaregión. 126 5.CONCLUSIONES Conclusiones 5.CONCLUSIONES 1. ElestudiodelacomposicióndealcaloidesdelaespeciemediterráneaNarcissus serotinus L. ha demostrado la presencia de componentes cuyas estructuras pueden clasificarse dentro de las series licorina y homolicorina del grupo de alcaloides característicos de la familia Amaryllidaceae. Se ha reportado el aislamientoyelucidaciónestructuralde8compuestosnuevos:narseronina,19O9 (3´9acetoxibutanoil)licorina,39O9metilnarcisidina,19O9acetil939O9metilnarcisidina, 19O9acetil939O9metil969oxonarcisidina, 29metoxipratosina, 119hidroxigalantina, y 29O9metilclivonina, indicando que esta especie representa una fuente interesantedealcaloidesnovedososparaserensayadosenestudiosdeactividad biológica. Al mismo tiempo, la caracterización del alcaloide narseronina ha planteado la reconsideración de una estructura previamente publicada de maneraerróneacomounisómerode39epimacronina. 2. ElestudiodealcaloidesdeespeciesdelasecciónGanymedeshaconfirmadola presencia de alcaloides de tipo mesembrano, estructuras típicas de las dicotiledóneassudafricanasdelgéneroSceletiumeinusualesdentrodelgrupode las amarillidáceas, en todos los taxones descritos para la sección. La conocida capacidaddeestosalcaloidescomoinhibidoresdelarecaptacióndeserotonina, posibilitasuaplicacióneneltratamientofarmacológicodeestadosdepresivosy otrostrastornostalescomoansiedadydrogodependencia. 3. LacaracterizacióndeloscompuestosaisladosdelaespecieNarcissustriandrusL. representauna contribución detallada y actualizada a la químicaestructural de estos alcaloides, en la que se aportan datos de GC9MS y RMN. Se destaca el aislamiento de 29oxomesembrenona, 7,7a9dehidromesembrenona y 29 oxoepimesembranol,reportadosporprimeravezapartirdeunafuentenatural. 4. Elanálisisdeespeciessilvestresdeamarillidáceasargentinasdelaregiónandina representa el primer reporte en el que se aborda el estudio del contenido de alcaloides de Habranthus jamesonii, Phycella herbertiana, Rhodophiala 129 Conclusiones mendocina y Zephyranthes filifolia. Los resultados del ensayo de la actividad inhibidoradelaenzimaAChEdelosextractosclorofórmicosdedichasespecies, unidos a la caracterización de su composición de alcaloides mediante GC9MS, han demostrado su potencial como fuentes renovables de compuestos activos talescomogalantamina.Enestesentido,lasmuestrasdeH.jamesoniiyZ.filifolia recolectadas en las provincias de Mendoza y San Juan, respectivamente, han resultadoespecialmenteprometedoras. Enlíneasgenerales,esteestudioensuconjuntodemuestraunavezmáselgran potencial de las especies vegetales de la familia Amaryllidaceae (subfam. Amaryllidoideae) como fuente de productos bioactivos y alcaloides con estructuras novedosas, confirmando la importancia de promover la continua caracterización de especiesqueaúnnohansidoexploradas. 130 Conclusiones 5.1.Conclusions 1. The study of the alkaloid composition of the Mediterranean species Narcissus serotinusL.hasshowntheoccurrenceofcomponentswhosestructurescanbe classified within the lycorine and homolycorine series, among the typical alkaloidsoftheAmaryllidaceaeplants.Theisolationandstructuralelucidationof 8 new compounds acetoxybutanoyl)lycorine, have been reported: 39O9methylnarcissidine, narseronine, 19O9(3'9 19O9acetyl939O9methyl9 narcissidine, 19O9acetyl939O9methyl969oxonarcissidine, 29methoxypratosine, 119 hydroxygalanthine, and 29O9methylclivonine, indicating that this species represents an interesting source of novel alkaloids to be assayed in further studies of biological activities. At the same time, the characterization of the alkaloidnarseroninehaspromptedareconsiderationofitspreviouslypublished structure,whichwasincorrectlyassignedasanisomerof39epimacronine. 2. ThestudyofalkaloidsfromspeciesofthesectionGanymedeshasconfirmedthe presence of mesembrane alkaloids, typical structures of the dicotyledonous South African genus Sceletium but unusual within the Amaryllidaceae group, in all taxa described for the section. The known activity of these alkaloids as inhibitors of serotonin reuptake indicates their potential application in the pharmacological treatment of depressive states and other disorders, such as anxietyanddrugdependence. 3. The characterization of compounds isolated from Narcissus triandrus L. represents a detailed and updated contribution to the structural chemistry of these alkaloids, providing complete GC9MS and NMR data. The isolation of 29 oxomesembrenone, 7,7a9dehydromesembrenone and 29oxoepimesembranol is reportedforthefirsttimefromanaturalsource. 4. The analysis of wild Argentinian Amaryllidaceae from the Andean region constitutesthefirstreportedstudyofalkaloidcontentofHabranthusjamesonii, Phycella herbertiana, Rhodophiala mendocina and Zephyranthes filifolia. The 131 Conclusiones resultsoftheactivityassayofAChEenzymeinhibitionofthechloroformextracts from these species, together with the characterization of the alkaloid compositionbyGC9MS,havedemonstratedtheirpotentialasrenewablesources of active compounds such as galanthamine. In this regard, samples of H. jamesonii and Z. filifolia collected in the provinces of Mendoza and San Juan, respectively,havebeenparticularlypromising. This study as a whole demonstrates once again the great potential of plant species of the Amaryllidaceae family (subfam. 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(Amaryllidaceae).PlantSystematicsandEvolution,275,1099132. 142 7.ANEXOS AnexoI 7.ANEXOS 7.1.AnexoI:CapítulodeRevisión ChemicalandbiologicalaspectsofAmaryllidaceaealkaloids JaumeBastida,StrahilBerkov,LauraTorras,NataliaBelénPigni,JeanPaulode Andrade,VanessaMartínez,CarlesCodinayFrancescViladomat En:RecentAdvancesinPharmaceuticalSciences,3,659100(2011) TransworldResearchNetwork.Ed.DiegoMuñoz9Torrero ISBN:97898197895952895 145 T Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India Recent Advances in Pharmaceutical Sciences, 2011: 65-100 ISBN: 978-81-7895-528-5 Editor: Diego Muñoz-Torrero 3. Chemical and biological aspects of Amaryllidaceae alkaloids Jaume Bastida, Strahil Berkov, Laura Torras, Natalia Belén Pigni Jean Paulo de Andrade, Vanessa Martínez, Carles Codina and Francesc Viladomat Department of Natural Products, Plant Biology and Soil Science, Faculty of Pharmacy University of Barcelona, 08028 Barcelona, Spain Abstract. The Amaryllidaceae alkaloids represent a large (over 300 alkaloids have been isolated) and still expanding group of biogenetically related isoquinoline alkaloids that are found exclusively in plants belonging to this family. In spite of their great variety of pharmacological and/or biological properties, only galanthamine is used therapeutically. First isolated from Galanthus species, this alkaloid is a long-acting, selective, reversible and competitive inhibitor of acetylcholinesterase, and is used for the treatment of Alzheimer’s disease. Other Amaryllidaceae alkaloids of pharmacological interest will also be described in this chapter. Introduction The Amaryllidaceae are richly represented in the tropics and have pronounced centers of diversity in South-Africa and the Andean region. Correspondence/Reprint request: Dr. Jaume Bastida, Department of Natural Products, Plant Biology and Soil Science, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain. E-mail: jaumebastida@ub.edu 66 Jaume Bastida et al. Some genera are also found in the Mediterranean area and temperate regions of Asia. A particular characteristic of Amaryllidaceae is a consistent presence of an exclusive group of alkaloids, which have been isolated from the plants of all the genera of this family. The Amaryllidaceae alkaloids represent a large and still expanding group of isoquinoline alkaloids, the majority of which are not known to occur in any other family of plants. Since the isolation of the first alkaloid, lycorine, from Narcissus pseudonarcissus in 1877, substantial progress has been made in examining the Amaryllidaceae plants, although they still remain a relatively untapped phytochemical source [1]. At present, over 300 alkaloids have been isolated from plants of this family [2] and, although their structures vary considerably, these alkaloids are considered to be biogenetically related. The large number of structurally diverse Amaryllidaceae alkaloids are classified mainly into nine skeleton types, for which the representative alkaloids are: norbelladine, lycorine, homolycorine, crinine, haemanthamine, narciclasine, tazettine, montanine and galanthamine (Fig. 1). With the aim of unifying the numbering system of the different skeleton types, Ghosal’s model will be used in this review [3]. As the alkaloids of the Amaryllidaceae family species fall mainly into one of these subgroups, they can serve as a classifying tool for including genera and species in this family. Recently, Unver and Jin have proposed subgroups for some skeleton types, according to the structures of new alkaloids isolated from Galanthus species [4,5]. Furthermore, although it is unusual to find other types of alkaloids in this family, if present, they are always accompanied by typical Amaryllidaceae alkaloids. The classical example is the reported presence of the mesembrane (Sceletium) alkaloids, generally found in the Aizoaceae family [6,7], in a few species of Amaryllidaceae such as Hymenocallis arenicola, Crinum oliganthum, Narcissus pallidulus and Narcissus triandrus [8-10]. In turn, the unexpected isolation of ()-capnoidine and (+)-bulbocapnine from Galanthus nivalis subsp. cilicicus is the first report of the occurrence of classical isoquinoline alkaloids in a typical member of the Amaryllidaceae [11]. Plants of the Amaryllidaceae family have been used for thousands of years as herbal remedies. The alkaloids from their extracts have been the object of active chemical investigation for nearly 200 years. Over the past three decades many have been isolated, screened for different biological activities, and synthesized by a number of research groups. The structural elucidation of the Amaryllidaceae alkaloids and their biological profiles, as well as their synthesis, have been summarized in the last few years [12-14], which, together with the regular publications of the journal Amaryllidaceae alkaloids 67 12 11 OH 3 HO HO 4 HO 2 5' 10 6' 4' 5 1 E 6 HO NH 1' 3' 2' D E' O 9 H 10b 10a 6a 8 7 norbelladine 4 3 3 H O MeN 2 1 4 MeO 10 9 N H 10b 10a 11 4a 4a H 2 1 H 12 MeO 6 lycorine 8 6a O 6 7 homolycorine O OH 2 OH 3 1 2 11 11 10 10 O 10a 9 10b H O 8 6a 7 4a N O 4 10a 9 10b H 12 O 6a 8 7 6 4 4a 2 OMe OH 3 1 1 O O 10b 8 4a 4 OH 6a NH 6 7 6 OH haemanthamine crinine OH 10a 9 12 N 3 H 10 O narciclasine OMe 2 10 O O 4 1 11 10b 8 O 6a 7 1 H NMe 4a 10a 9 OH 2 3 12 4a OH O 6 12 8 6a 7 tazettine 3 10a 9 OH 3 1 O 2 11 11a 10 O OMe MeO 10 4 10b 10a 9 4a 11 12 4 N H 6 montanine N 6a 8 7 6 Me galanthamine Figure 1. Amaryllidaceae alkaloid types. Natural Products Reports [5,15-17] over the last decade, represents a valuable source of information. The present review provides coverage of the biosynthesis, NMR spectroscopy and biological activity of the Amaryllidaceae alkaloids up to the end of 2010. 1. Biosynthetic pathways Most of the biosynthetic research done on Amaryllidaceae alkaloids was carried out in the sixties and early seventies. Since then, the only noteworthy study has been the biosynthesis of galanthamine and related alkaloids [18]. As in most alkaloid biosyntheses, that of the Amaryllidaceae follows a pattern made up of certain steps. 1.1. Enzymatic preparation of the precursors Although L-phenylalanine (L-phe) and L-tyrosine (L-tyr) are closely related in chemical structure, they are not interchangeable in plants. In the Amaryllidaceae alkaloids, L-phe serves as a primary precursor of the C6-C1 Jaume Bastida et al. 68 fragment, corresponding to ring A and the benzylic position (C-6), and L-tyr is the precursor of ring C, the two-carbon side chain (C-11 and C-12) and nitrogen, C6-C2-N. The conversion of L-phe to the C6-C1 unit requires the loss of two carbon atoms from the side chain as well as the introduction of at least two oxygenated substituents into the aromatic ring, which is performed via cinnamic acids. The presence of the enzyme phenylalanine ammonia lyase (PAL) has been demonstrated in Amaryllidaceae plants [19] and the elimination of ammonia mediated by this enzyme is known to occur in an antiperiplanar manner to give trans-cinnamic acid, with loss of the E-pro-S hydrogen [20]. Thus, it may be expected that L-phe would be incorporated into Amaryllidaceae alkaloids with retention of the E-pro-R hydrogen. However, feeding experiments in Narcissus ‘King Alfred’ showed that tritium originally present at C-E of L-phe, whatever the configuration, was lost in the formation of several haemanthamine and homolycorine type alkaloids, which led to the conclusion that fragmentation of the cinnamic acids involves oxidation of C-E to ketone or acid level, the final product being protocatechuic aldehyde or its derivatives (Fig. 2). On the other hand, L-tyr is degraded no further than tyramine before incorporation into the Amaryllidaceae alkaloids. NH2 COOH HO COOH L-Tyr L-Phe Tyr-decarboxylase PAL R1 H2N HO COOH R2 trans-cinnamic acid, R1=R2=H para-coumaric acid, R1=OH, R2=H caffeic acid, R1=R2=OH Schiff's base HO HO CHO protocatechuic aldehyde H2N tyramine HO HO HO HO HO (isomeric structures in solution) N HO H NH O N H HO HO OH HO HO NH norbelladine Figure 2. Biosynthetic pathway to norbelladine. Amaryllidaceae alkaloids 69 1.2. Primary cyclization mechanisms Tyramine and protocatechuic aldehyde or its derivatives are logical components for the biosynthesis of the precursor norbelladine. This pivotal reaction represents the entry of primary metabolites into a secondary metabolic pathway. The junction of the amine and the aldehyde results in a Schiff’s base, two of which have been isolated up to now from several Crinum species: craugsodine [21] and isocraugsodine [22]. The existence of Schiff’s bases in nature as well as their easy conversion into the different ring-systems of the Amaryllidaceae alkaloids suggest that the initial hypothesis about this biosynthetic pathway was correct. 1.3. Enzymatic preparation of intermediates In 1957, Barton and Cohen [23] proposed that norbelladine or related compounds could undergo oxidative coupling in Amaryllidaceae plants, once ring A had been suitably protected by methylation, resulting in the different skeletons of the Amaryllidaceae alkaloids (Fig. 3). The key intermediate in most of cases is O-methylnorbelladine. OH MeO 4' NH HO O-methylnorbelladine para-para' ortho-para' para-ortho' OH OH HO MeO MeO N HO types lycorine homolycorine O MeO N HO types crinine haemanthamine tazettine narciclasine montanine Figure 3. Phenol oxidative coupling in Amaryllidaceae. NH types galanthamine Jaume Bastida et al. 70 1.4. Secondary cyclization, diversification and restructuring Secondary cyclization is produced by an oxidative coupling of O-methylnorbelladine. 1.4.1. Lycorine and homolycorine types The alkaloids of this group are derivatives of the pyrrolo[de]phenanthridine (lycorine type) and the 2-benzopirano-[3,4-g]indole (homolycorine type) skeletons, and both types originate from an ortho-para’ phenol oxidative coupling (Fig. 4). The biological conversion of cinnamic acid via hydroxylated cinnamic acids into the C6-C1 unit of norpluviine has been used in a study of hydroxylation mechanisms in higher plants [24]. When [3-3H, E-14C] cinnamic acid was fed to Narcissus ‘Texas’ a tritium retention in norpluviine of 28% was observed, which is very close to the predicted value resulting from parahydroxylation with hydrogen migration and retention. In the conversion of O-methylnorbelladine into lycorine, the labelling position [3-3H] on the aromatic ring of L-tyr afterwards appears at C-2 of norpluviine, which is formed as an intermediate, the configuration of the tritium apparently being E [25]. This tritium is retained in subsequently formed OH HO MeO H MeO NH HO H N MeO O-methylnorbelladine OMe HO HO HO pluviine H MeO H N MeO H MeO H N MeO 9-O-methylpseudolycorine galanthine o-p' OH O MeO NH HO H H N 8 O MeO HO H N kirkine MeO MeO H O H N H N O caranine MeN H H O norpluviine HO HO H MeO O HO HO lycorine MeN H H O OH lycorenine MeO MeO H H H O O homolycorine Figure 4. Alkaloids proceeding from an ortho-para’ coupling. Amaryllidaceae alkaloids 71 lycorine, which means that hydroxylation at C-2 proceeds with an inversion of configuration [26] by a mechanism involving an epoxide, with ring opening followed by allylic rearrangement of the resulting alcohol (Fig. 5). Supporting evidence comes from the incorporation of [2E-3H]caranine into lycorine in Zephyranthes candida [27]. However, an hydroxylation of caranine in Clivia miniata occuring with retention of configuration was also observed [28]. Further, [2D-3H; 11-14C]caranine was incorporated into lycorine with high retention of tritium at C-2, indicating that no 2-oxo-compound can be implicated as an intermediate. The conversion of the O-methoxyphenol to the methylenedioxy group may occur late in the biosynthetic pathway. Tritiated norpluviine is converted to tritiated lycorine by Narcissus ‘Deanna Durbin’, which not only demonstrates the previously mentioned conversion but also indicates that the C-2 hydroxyl group of lycorine is derived by allylic oxidation of either norpluviine or caranine [29]. Regarding the conversion of [2E-3H, 8-OMe-14C]pluviine into galanthine, in Narcissus ‘King Alfred’, the retention of 79% of the tritium label confirms that hydroxylation of C-2 may occur with inversion of configuration [30]. It was considered [31] that another analogous epoxide could give narcissidine in the way shown by loss of the pro-S hydrogen from C-11, galanthine being a suitable substrate for epoxidation. Labelled [D-14C, E-3H]O-methylnorbelladine, when fed to Narcissus ‘Sempre Avanti’ afforded galanthine (98% of tritium retention) and narcissidine (46% tritium retention). Loss of hydrogen from C-11 of galanthine was therefore stereospecific. In the nineties, Kihara et al. [32] isolated a new alkaloid, incartine, from flowers of Lycoris incarnata, which could be considered as the biosynthetic intermediate of this pathway (Fig. 6). The biological conversion of protocatechuic aldehyde into lycorenine, which proceeds via O-methylnorbelladine and norpluviine, first involves a reduction of the aldehyde carbonyl, and afterwards, in the generation of lycorenine, oxidation of this same carbon atom. The absolute stereochemistry T HO HO H MeO H HO T N norpluviine H H N H OH T HO O H O H O N lycorine Figure 5. Biosynthesis of lycorine with inversion of the configuration. Jaume Bastida et al. 72 OMe OMe OMe HO HO HO H MeO H MeO H N O H MeO MeO N HS HR OH H MeO H MeO N narcissidine galanthine Figure 6. Conversion of galanthine to narcissidine via epoxide. HO HO H MeO H HO [O] HO H MeO H N HO norpluviine MeO N CH HO OH NH O rotation MeO HN MeN MeN H H H MeO [O] O O homolycorine MeO H H H MeO MeO methylations O OH HO H H OH CH O lycorenine Figure 7. Conversion of norpluviine to homolycorine type alkaloids. of these processes has been elucidated in subsequent experiments [33], and the results show that hydrogen addition and removal take place on the re-face of the molecules concerned [34], the initially introduced hydrogen being the one later removed [35]. It is noteworthy that norpluviine, unlike pluviine, is converted in Narcissus ‘King Alfred’ primarily to alkaloids of the homolycorine type. Benzylic oxidation at position 6 followed by a ring opening forms an amino aldehyde; the formation of hemiacetal and subsequent methylation provides lycorenine [30], which after oxidation gives homolycorine, as shown in Fig. 7. 1.4.2. Crinine, haemanthamine, tazettine, narciclasine and montanine types This group includes the alkaloids derived from 5,10b-ethanophenanthridine (crinine and haemanthamine types), 2-benzopyrano[3,4-c]indole (tazettine type), phenanthridine (narciclasine type) and 5,11- Amaryllidaceae alkaloids 73 methanomorphanthridine (montanine type) skeletons, originating from a parapara’ phenol oxidative coupling (Fig. 8). Results of experiments with labelled crinine, and less conclusively with oxovittatine, indicate that the two naturally occurring enantiomeric series, represented in Fig. 8 by crinine and vittatine, are not interchangeable in Nerine bowdenii [36]. Incorporation of O-methylnorbelladine, labelled in the methoxy carbon and also in positions [3,5-3H], into the alkaloid haemanthamine was without loss of tritium, half of which was at C-2. Consideration of the possible mechanisms involved in relation to tritium retention led to the suggestion that the tritium which is expected at C-4 of haemanthamine might not be stereospecific [37]. The conversion of O-methylnorbelladine into haemanthamine involves loss of the pro-R hydrogen from the C-E of the tyramine moiety, as well as a further entry of a hydroxyl group at this site [38]. The subsequent benzylic oxidation results in an epimeric mixture that even HPLC cannot separate. The epimeric forms were proposed to be interchangeable. The biosynthetic conversion of the 5,10b-ethanophenanthridine alkaloids to the 2-benzopyrano[3,4-c]indole was demonstrated by feeding tritium-labelled alkaloids to Sprekelia formosissima. It was shown that this plant converts haemanthamine to haemanthidine/epihaemanthamine and subsequently to pretazettine in an essentially irreversible manner [39]. This transformation was considered to proceed through an intermediate but it has never been detected by spectral methods [40] (Fig. 9). OH O H O N OMe OH crinine OH H NMe H OH O O OH NH O MeO O OH O NH HO NH p-p' O O H OH narciclasine pretazettine O-methylnorbelladine OH O O H O N H O vittatine O OH N H montanine N haemanthamine OMe O OMe OH OMe OH O H O N OH haemanthidine/ 6-epihaemanthidine MeHN O O CH2OH ismine Figure 8. Alkaloids proceeding from a para-para’ coupling. Jaume Bastida et al. 74 O O O H O H N OH OMe OH OMe OH OMe OH O CH H NH O H O haemanthidine epihaemanthidine OMe OMe NH O O CH N OH methylation OH O H NMe O O OH OH pretazettine Figure 9. Biosynthesis of pretazettine. It has also been proved that the alkaloid narciclasine proceeds from the pathway of the biosynthesis of crinine and haemanthamine type alkaloids and not through norpluviine and lycorine derivatives. In fact, in view of its structural affinity to both haemanthamine and lycorine, narciclasine could be derived by either pathway. When O-methylnorbelladine labelled in the methoxy carbon and in both protons of position 3 and 5 of the tyramine aromatic ring, was administered to Narcissus plants, all four alkaloids incorporated activity. The isotopic ratio [3H:14C] for norpluviine and lycorine was, as expected, 50% that of the precursor, because of its ortho-para' coupling. On the contrary, in haemanthamine the ratio was unchanged. These results show clearly that the methoxy group of O-methylnorbelladine is completely retained in the alkaloids mentioned, providing a satisfactory internal standard and also, the degree of tritium retention is a reliable guide to the direction of phenol coupling. Narciclasine showed an isotopic ratio (75%) higher than that of lycorine or norpluviine though lower than that of haemanthamine. However, the fact that more than 50% of tritium is retained suggests that O-methylnorbelladine is incorporated into narciclasine via para-para' phenol oxidative coupling. O-methylnorbelladine and vittatine are implicated as intermediates in the biosynthesis of narciclasine [41-43], and the loss of the ethane bridge from the latter could occur by a retro-Prins reaction on 11-hydroxyvittatine. Strong support for this pathway was obtained by labelling studies. 11-Hydroxyvittatine has also been proposed as an intermediate in the biosynthesis of haemanthamine and montanine (a 5,11-methanomorphanthridine alkaloid) following the observed specific incorporation of vittatine into the two alkaloids in Rhodophiala bifida [36] (Fig. 10). Amaryllidaceae alkaloids 75 OMe OH OMe O O H O H N O N haemanthamine OH OH OH O O H O H N O N 11-hydroxyvittatine vittatine OMe O O OH N H montanine OH O O OH N H pancracine Figure 10. Proposed biosynthetic pathways to haemanthamine and montanine. Fuganti and Mazza [42,43] concluded that in the late stages of narciclasine biosynthesis, the two-carbon bridge is lost from the oxocrinine skeleton, passing through intermediates bearing a pseudoaxial hydroxy-group at C-3 position and further hydrogen removal from this position does not occur. Noroxomaritidine was also implicated in the biosynthesis of narciclasine and further experiments [44] showed that it is also a precursor for ismine. The alkaloid ismine has also been shown [45] to be a transformation product of the crinine-haemanthamine series. The precursor, oxocrinine labelled with tritium in the positions 2 and 4, was administered to Sprekelia formosissima plants and the radiactive ismine isolated was shown to be specifically labelled at the expected positions. 1.4.3. Galanthamine type This type of alkaloids have a dibenzofuran nucleus (galanthamine type) and are obtained from a para-ortho’ phenol oxidative coupling. Jaume Bastida et al. 76 The initial studies of this pathway suggested that the para-ortho’ coupling does not proceed from O-methylnorbelladine but from N,O-dimethylnorbelladine to finally give galanthamine [46]. N,O-dimethylnorbelladine was first isolated from Pancratium maritimum [47] a species that also contains galanthamine. However, the most recent study seems to contradict the evidence set forth here. Experiments carried out with application of 13C-labelled O-methylnorbelladine to organs of field grown Leucojum aestivum have shown that the biosynthesis of galanthamine involves the phenol oxidative coupling of O-methylnorbelladine to a postulated dienone, which undergoes spontaneous closure of the ether bridge to yield N-demethylnarwedine, giving norgalanthamine after stereoselective reduction. Furthermore, it was shown that norgalanthamine is N-methylated to galanthamine in the final step of biosynthesis [18] (Fig. 11). In contrast with the literature, N,Odimethylnorbelladine was metabolized to a lesser extent in L. aestivum and incorporated into galanthamine as well as norgalanthamine at about 1/3 of the rate of O-methylnorbelladine. According to Eichhorn et al. [18], narwedine is not the direct precursor of galanthamine, and could possibly exist in equilibrium with galanthamine, a reaction catalyzed by a hypothetically reversible oxido-reductase. Chlidanthine, by analogy with the known conversion of codeine to morphine, might be expected to arise from galanthamine by O-demethylation. This was shown to be true when both galanthamine and narwedine, with tritium labels, were incorporated into chlidanthine [48]. HO O O H O OH HO MeO MeO MeO p-o' H O-methylnorbelladine spontaneous N N H N-demethylnarwedine OH O H O MeO N Me narwedine H O MeO MeO N galanthamine Me OH OH H O H O MeO N H N H norgalanthamine Figure 11. Biosynthesis of galanthamine and derivatives. N norlycoramine H Amaryllidaceae alkaloids 77 2. NMR studies In a discussion of Proton Nuclear Magnetic Resonance (1H NMR) and Carbon Nuclear Magnetic Resonance (13C NMR), the most significant characteristics of each of Amaryllidaceae alkaloid-type are outlined, indicating the keys for their identification. 2.1. Proton nuclear magnetic resonance 1 H NMR spectroscopy gives the most extensive and important information about the different types of Amaryllidaceae alkaloids. In the last 25 years, the routine use of 2D NMR techniques has facilitated the structural assignments and the settling of their stereochemistry. 2.1.1. Lycorine type This group has been subjected to several 1H NMR studies and lycorine, as well as its main derivatives, has been completely assigned. The general characteristics of the 1H NMR spectra are: a. Two singlets for the para-oriented aromatic protons, together with a unique olefinic proton. b. Two doublets as an AB system corresponding to the benzylic protons of C- 6. The deshielding observed in the E-protons of positions 6 and 12 in relation to their D-homologues is due to the effect of the cis-lone pair of the nitrogen atom. c. Like almost all other lycorine type examples, the alkaloids isolated from the Narcissus genus show a trans B/C ring junction, the coupling constant being J4a,10b~11 Hz. Only kirkine shows a cis B/C ring junction, with a smaller coupling constant J4a,10b 8 Hz. In the plant, the alkaloid lycorine is particularly vulnerable to oxidation processes, giving several ring-C aromatized products. 2.1.2. Homolycorine type This group includes lactone, hemiacetal or the more unusual cyclic ether alkaloids. The general traits for this type of compounds could be summarized as follows: 78 Jaume Bastida et al. a. Two singlets for the para-oriented aromatic protons. In lactone alkaloids, the deshielding of H-7 is caused by the peri-carbonyl group. b. The hemiacetal alkaloids always show the substituent at C-6 in D-disposition. c. The majority of compounds belong to a single enantiomeric series containing a cis B/C ring junction, which is congruent with the small size of the coupling constant J1,10b. In the Narcissus genus no exception to this rule has been observed. d. The large coupling constant between H-4a and H-10b (J4a,10b~10 Hz) is only consistent with a trans-diaxial relationship. e. In general, ring C presents a vinylic proton. If position 2 is substituted by an OH, OMe or OAc group, it always displays an D-disposition. f. The singlet corresponding to the N-methyl group is in the range ofG 2.02.2 ppm, its absence being very unusual. g. The H-12D is more deshielded than H-12E as a consequence of the cislone pair of the nitrogen atom. Homolycorine type alkaloids with a saturated ring C have been studied by Jeff and co-workers [49]. They describe empirical correlations of N-methyl chemical shifts with stereochemical assignments of the B/C and C/D ring junction. 2.1.3. Haemanthamine and crinine types The absolute configuration of these alkaloids is determined through the circular dichroism spectrum. The alkaloids of the Narcissus genus are exclusively of the haemanthamine type, while in genera such as Brunsvigia, Boophane etc., the crinine type alkaloids are predominant. It is also noteworthy that the alkaloids isolated from the Narcissus genus do not show additional substitutions in the aromatic ring apart from those of C-8 and C-9. On the contrary, in the genera where crinine type alkaloids predominate, the presence of compounds with a methoxy substituent at C-7 is quite common. Thus, haemanthamine type alkaloids show the following characteristics: a. Two singlets for the para-oriented aromatic protons, although of course only one for crinane type alkaloids substituted at C-7. b. Using CDCl3 as the solvent, the magnitude of the coupling constants between each olefinic proton (H-1 and H-2) and H-3 gives information about the configuration of the C-3 substituent. Thus, in those alkaloids in which the two-carbon bridge (C-11 and C-12) is cis to the substituent at Amaryllidaceae alkaloids 79 C-3, H-1 shows an allylic coupling with H-3 (J1,3~1-2 Hz) and H-2 shows a smaller coupling with H-3 (J2,3~0-1.5 Hz), as occurs in crinamine. On the contrary, in the corresponding C-3 epimeric series, e.g. haemanthamine, a larger coupling between H-2 and H-3 (J2,3 5 Hz) is shown, the coupling between H-1 and H-3 not being detectable. This rule is also applicable to the crinane type alklaoids. c. In the haemanthamine series there is frequently an additional W coupling of H-2 with the equatorial H-4E, while the proton H-4D shows a large coupling with H-4a (J4D,4a~13 Hz) due to their trans-diaxial disposition. The same is applicable in the crinane series. d. Two doublets for an AB system corresponding to the benzylic protons of position C-6. e. The pairs of alkaloids with a hydroxy substituent at C-6, like papyramine/ 6-epipapyramine, haemanthidine/6-epihaemanthidine etc, appear as a mixture of epimers not separable even by HPLC. f. Also in relation with position C-6, it is interesting to note that ismine, a catabolic product from the haemanthamine series, shows a restricted rotation around the biarylic bond, which makes the methylenic protons at the benzylic position magnetically non-equivalent. 2.1.4. Tazettine type Although tazettine is one of the most widely reported alkaloids in the Amaryllidaceae family, it was found to be an extraction artifact from pretazettine [50]. The presence of an N-methyl group (2.4-2.5 ppm) in tazettine type alkaloids immediately distinguishes them from the haemanthamine or crinine types, from which they proceed biosynthetically. Moreover, the 1H NMR spectrum always shows the signal corresponding to the methylenedioxy group. 2.1.5. Montanine type The absolute configuration of Montanine-type alkaloids is determined through the circular dichroism spectrum. Their 1H NMR data are very similar to those of alkaloids with a lycorine skeleton, but Montanine-type alkaloids can be distinguished by the analysis of a COSY spectrum. The signals attributable to the H-4 hydrogens (the most upfield signals) show correlation with those corresponding to H-3 and H-4a, while in a lycorine skeleton the most upfield signals correspond to the H-11 hydrogens. Jaume Bastida et al. 80 2.1.6. Narciclasine type The narciclasine-type alkaloids present the highest degree of oxidation. The absolute configuration of the most studied alkaloid of this group pancratistatin, was determined by X-ray diffraction [51]. The main 1H NMR characteristics of the narciclasine-type alkaloids are: a. The only aromatic hydrogen appears as a singlet with a chemical shift higher than 7 ppm. b. Those alkaloids with a hydrogenated double bond C-1/C-10b possess a trans stereochemistry for the B-C ring junction and, consequently, a large coupling constant value for J4a-10b. c. The hydrogen attached to the nitrogen atom appears as a broad singlet with a chemical shift around 5 ppm, which disappears on the addition of D2O. 2.1.7. Galanthamine type Among the Amaryllidaceae alkaloids, only the galanthamine type shows an ortho-coupling constant between both aromatic protons of ring A. The general characteristics of their 1H NMR spectra are: a. Two doublets for the two ortho-oriented aromatic protons with a coupling constant of J7,8~8 Hz. b. The assignment of the substituent stereochemistry at C-3 is made in relation with the coupling constants of the olefinic protons H-4 and H-4a. When coupling constant J3,4 is about 5 Hz, the substituent is pseudoaxial, while if it is ~0 Hz this indicates that the substituent at C-3 is pseudoequatorial. c. Two doublets corresponding to the AB system of the C-6 benzylic protons. d. The existence of the furan ring results in a deshielding effect in H-1. e. This type of alkaloids often shows an N-methyl group but occasionally an N-formyl group has been reported. 2.2. Carbon13 nuclear magnetic resonance 13 C NMR spectroscopy has been extensively used for determining the carbon framework of Amaryllidaceae alkaloids, and there are several major contributions [52-54]. The assignments are made on the basis of chemical shifts and multiplicities of the signals (by DEPT experiment). The use of 2D Amaryllidaceae alkaloids 81 NMR techniques such as HMQC and HMBC allow the assignments to be corroborated. The 13C NMR spectra of Amaryllidaceae alkaloids can be divided in two regions. The low-field region (>90 ppm) contains signals of the carbonyl group, the olefinic and aromatic carbons as well as that of the methylenedioxy group. The other signals corresponding to the saturated carbon resonances are found in the high-field region, the N-methyl being the only characteristic group, easily recognizable by a quartet signal between 40-46 ppm. The effect of the substituent (OH, OMe, OAc) on the carbon resonances is of considerable importance in localizing the position of the functional groups. The analysis of the spectra allows conclusions to be drawn about the following aspects: - The number of methine olefinic carbons. The presence and nature of the nitrogen substituent. The existence of a lactonic carbonyl group. The presence of a quaternary carbon signal assignable to C-10b in the chemical shift range of 42-50 ppm. 3. Biological and pharmacological activities This section covers the pharmacological and/or biological properties of the most representative Amaryllidaceae alkaloids. Until now only galathamine is being marketed, but the significant activities of other alkaloids in the family demonstrated in recent years could favour their therapeutic use in the near future. 3.1. Lycorine type The most characteristic and common Amaryllidaceae alkaloid is lycorine, reported to be a powerful inhibitor of ascorbic acid (L-Asc) biosynthesis [55,56], and thus a useful tool in studying Asc-dependent metabolic reactions in L-Asc-synthesising organisms [57,58]. Specifically, lycorine is a powerful inhibitor of the activity of L-galactono-J-lactone dehydrogenase, the terminal enzyme of L-Asc biosynthesis [59-62], which is thought to be localised in the mitochondrial membrane [63,64]. Galanthine also has a high capacity to inhibit ascorbic acid biosynthesis [56]. Lycorine is a powerful inhibitor of cell growth, cell division and organogenesis in higher plants, algae, and yeasts, inhibiting the cell cycle during interphase, which seems to be related with the L-Asc levels [57,65-69]. In plants, it also inhibits cyanide-insensitive respiration, peroxidase activity 82 Jaume Bastida et al. and protein synthesis [70-72]. The effects of lycorine on L-Asc biosynthesis have been reported to occur at concentrations below those at which protein synthesis is affected, but it seems difficult to completely rule out non-specific effects of this alkaloid since it has been reported that, at least in yeasts, lycorine is able to interact directly with mitocondrial DNA. Thus, differing sensitivity to the alkaloid among cells devoid of mitochondrial DNA (rho0) and cells with mitochondrial DNA either rho+ or rho- has been found in yeasts [59,67,73,74], rho0 cells being resistant to high concentrations of the drug [69,75-77]. Some strains can even adapt to the presence of lycorine, because they are able to degrade the alkaloid and use its biotransformation products as growth stimulating factors [77]. In contrast, lycorine-1-O-E-D-glucoside promotes cell growth, seed germination, and rate of development of root and root hairs in higher plants. The glucosyloxy derivatives of lycorine and pseudolycorine and their aglicones form stable complexes with phytosterols and also with divalent metal ions and are able to translocate them from the rhizosphere to the aerial part [78]. Palmilycorine and some acylglucosyloxy conjugates of lycorine, in turn, are frequently encountered among the phytosterols exhibiting membrane-stabilizing action. Plants also use lycorine1-O--D-glucoside and acylglucosyloxy conjugates of lycorine to recognize and reject microorganisms and parasites [79]. The antitumor activity of lycorine in animals [80,81] has been demonstrated by the inhibition of in vivo and in vitro growth of diverse tumor cells, such as BL6 mouse melanoma, Lewis lung carcinoma, murine ascite or HeLa cells [3,79,82-86]. It induces flat morphology in K-ras-NRK cells (transformed fibroblasts) [87], and reduces the cellular activity in femoral bone marrow tissue that results in granulocytic leucopenia and a decrease in the number of erythrocytes. This alkaloid’s mechanism of action is thought to be through inhibition of protein synthesis at the ribosomal level, even though the cytotoxic effects of calprotectin can also be suppressed using lycorine [80,81,88-90]. Lycorine also inhibits murine macrophage production of Tumor Necrosis Factor alpha (TNF-) [91], and shows inhibitory effects on nitric oxide production and induction of inducible nitric oxide synthase (NOS) in lipopolysaccharide-activated macrophages [92]. The molecular mechanism of lycorine against leukaemia (human cell line HL-60) shows that it can suppress cell growth and reduce cell survival by arresting the cell cycle at the G2/M phase and inducing apoptosis of tumor cells [93]. Recent studies show that the TNF-D signal transduction pathway and p21-mediated cell-cycle inhibition are involved in the apoptosis of HL-60 cells induced by lycorine [94]. The effects of lycorine on the human multiple myeloma cell line KM3, and the possible mechanisms of these effects have also been studied [95]. The growth rates of the KM3 cells exposed to lycorine clearly slowed down. Cell Amaryllidaceae alkaloids 83 fluorescent apoptotic morphological changes, DNA degradation fragments, and a sub-G1 peak were detected, indicating the occurrence of cell apoptosis after lycorine treatment. Furthermore, the release of mitochondrial cytochrome c, the augmentation of Bas with the attenuation of Bcl-2, and the activation of Caspase-9, -8, and -3 were also observed, suggesting that the mitochondrial pathway and the death acceptor pathway were involved. The results also showed that lycorine was able to block the cell cycle at the G0/G1 phase through the downregulation of both cyclin D1 and CDK4. In short, lycorine can suppress the proliferation of KM3 cells and cell survival by arresting cell cycle progression as well as inducing cell apoptosis [96]. A recent paper describes the preparation of a mini-library comprised of synthetic and natural lycorane alkaloids and the investigation of apoptosis-inducing activity in human leukemia (Jurkat) cells. Further insights into the nature of this apoptosis-inducing pharmacophore are described, including the requirement of both free hydroxyl groups in ring-C [97]. Another recent study describes the induction of apoptosis in human leukemia cells by lycorine via an intrinsic mitochondria pathway, causing a rapid turnover of protein level of Mcl-1 before Caspases activation. Pronounced apoptosis accompanied by the down-regulation of Mcl-1 was also observed in blasts from patients with acute myeloid leukemia. Lycorine also displays pronounced cell growth inhibitory activities against both parental and multidrug resistant L5178 mouse lymphoma cell lines, but is almost inactive in inhibiting the glycoprotein responsible for the efflux-pump activity of tumor cells. Assays for interactions with tRNA revealed that the antiproliferative effects of lycorine result from their complex formation with tRNA [98]. Interaction of lycorine, pseudolycorine and 2-O-acetylpseudo-lycorine with DNA has been observed [99,100]. Most of the alkaloids that showed promising antiproliferative activities have also proved to be efficient apoptosis inducers [14]. Some other alkaloids of this series, such as caranine, galanthine, pseudolycorine and 2-O-acetylpseudolycorine, are also active against a variety of tumor cells [84,101,102]. Pseudolycorine inhibits the protein synthesis in tumor cells at the step of peptide bond formation, but it has a different binding site than lycorine [89,103]. Ungeremine, a natural metabolite of lycorine, is responsible, at least partially, for the growth-inhibitory and cytotoxic effects of lycorine, being active against leukemia [104,105]. Lycorine-1-O-E-Dglucoside, in turn, has the reverse effect of lycorine, and may produce mitogenic activity in animal cells [106]. A mini-panel of semi-synthetic analogs of lycorine was screened for cytochrome P450 3A4 (CYP3A4) inhibitory activity, the most potent of which (1-O-acetyl-2-O-tert-butyldimethylsilyllycorine) exhibited inhibition at a concentration as low as 0.21 PM. Elements of this unraveled novel 84 Jaume Bastida et al. pharmacophore include bulky lipophilic substitution at C-2 in conjunction with a small hydrogen donor/acceptor bond at C-1, or bulky electron-rich substitution at C-1 in conjunction with a vicinal hydrogen donor/acceptor bond [107]. Two semisynthetic silylated lycorane analogs, accesed via a chemoselective silylation strategy from lycorine exhibited low micromolar activities [108]. Lycorine and pseudolycorine exert antiviral effects on several RNA and DNA-containing viruses [109]. Antiviral activity has been observed in tests with flaviviruses, and to a slightly lesser degree, bunyaviruses. Lycorine and pseudolycorine also show inhibitory activity against the Punta Toro and Rift Valley fever viruses, but with low selectivity [110,111]. Lycorine, in turn, acts as an anti-SARS-CoV (Severe Acute Respiratory Syndrome-associated Coronavirus) and shows pronounced activity against poliomyelitis, coxsackie and herpes type 1 [3,112]. It possesses high antiretroviral activity accompanied by low therapeutic indices [113]. The relationship between its structure and the mechanism of activity has been studied in the Herpes simplex virus, suggesting that alkaloids that may eventually prove to be antiviral agents have a hexahydroindole ring with two functional hydroxyl groups [114]. The activity was found to be due to the inhibition of multiplication, and not to the direct inactivation of extracellular viruses, and the mechanism of the antiviral effect was partially explained as a blocking of viral DNA polymerase activity [109,115-117]. Lycorine has appreciable inhibitory activity against acetylcholinesterase [118]. Cholinesterase activity appears to be associated with the two free hydroxyl groups present in some of the alkaloids of this structural type [119]. The higher acetylcholinesterase inhibitory activity of assoanine and oxoassoanine with respect to the other lycorine-type alkaloids could be explained by an aromatic ring C, which gives a certain planarity to those molecules [120]. Another alkaloid, galanthine, exhibits powerful cholinergic activity and has therefore attracted much interest in the treatment of myasthenia gravis, myopathy and diseases of the central nervous system [121]. Caranine, pseudolycorine, ungiminorine, and in particular, ungeremine, also show an inhibitory effect on acetylcholinesterase [120,122,123]. Recently, the synthesis of differentially functionalized analogs of lycorine, accessed via a concise chemoselective silylation strategy, has allowed two of the most potent inhibitors of acetylcholinesterase to be described. Important elements of this novel pharmacophore were elucidated through SAR studies [94]. Lycorine is analgesic, more so than aspirin, and hypotensive [124,125], as are caranine and galanthine. The analgesic activity exhibited by the Amaryllidaceae alkaloids is attributed to their similarity with the morphine Amaryllidaceae alkaloids 85 and codeine skeletons. Lycorine also has antiarrhythmic action, and lycorine hydrochloride is a strong broncholytic [126]. In fact, lycorine shows a relaxant effect on an isolated epinephrine-precontracted pulmonary artery and increases contractility and the rate of an isolated perfused heart. These effects are mediated by stimulation of -adrenergic receptors [127]. Lycorine also has a strong inhibitory effect on parasite (Encephalitozoon intestinalis) development [128] and antifungal activity against Candida albicans [129]. Recently, several lycorine derivatives were examined for their activity against Trypanosoma brucei and Plasmodium falciparum. Among them, 2-O-acetyllycorine showed the most potent activity against parasitic T. brucei, while 1-O-(3R)hydroxybutanoyllycorine, 1,2-di-O-butanoyllycorine, and 1-O-propanoyllycorine showed significant activity against P. falciparum in an in vitro experiment [130], although the antimalarial activity of lycorine was already known [131-133]. Galanthine, in turn, shows mild in vitro activity against Tripanosoma brucei rhodesiense and Plasmodium falciparum [134]. Additionally, lycorine has antifeedant [135], emetic [136], anti-inflammatory [137], antiplatelet [138] as well as antifertility [125] activities. 3.2. Homolycorine type It is reported that some alkaloids of this series, such as homolycorine, 8-O-demethylhomolycorine, dubiusine, 9-O-demethyl-2D-hydroxyhomoly-corine, hippeastrine, lycorenine or O-methyllycorenine present cytotoxic effects against non-tumoral fibroblastic LMTK cells [84], also being moderately active in inhibiting the in vivo and in vitro growth of a variety of tumor cells, such as Molt 4 lymphoma, HepG2 human hepatoma, LNCaP human prostate cancer or HT [84,125,139]. Dubiusine, lycorenine, 8-O-demethylhomolycorine and 9-Odemethyl-2D-hydroxyhomolycorine also show DNA binding activity comparable to that of vinblastine [99]. Homolycorine possesses high antiretroviral activity, accompanied by low therapeutic indices [113]. Hippeastrine, in turn, displays antiviral activity against Herpes simplex type 1 [114]. Dubiusine, homolycorine, 8-O-demethylhomolycorine and lycorenine have a hypotensive effect on the arterial pressure of normatensive rats [140]. Lycorenine also shows a vasodepressor action ascribed to the maintenance of its D-adrenergic blocking action, and produces bradycardia by modifying vagal activity [141]. Another feature of lycorenine is its analgesic activity [3]. Homolycorine and masonine are other inductors of delayed hypersensitivity in animals [142]. Hippeastrine, in turn, shows antifungal activity against Candida albicans and it also possesses a weak insect antifeedant activity [129]. 86 Jaume Bastida et al. 3.3. Haemanthamine and crinine types Haemanthamine, haemanthidine, crinamine, maritidine and papyramine display pronounced cell growth inhibitory activities against a variety of tumor cells, such as Rauscher viral leukaemia, Molt 4 lymphoma, BL6 mouse melanoma, HepG2 human hepatoma, HeLa, LNCaP human prostate cancer or HT [82-84,88,139,143,144]. Some of these alkaloids, namely crinamine, haemanthamine and papyramine, also present a cytotoxic effect against nontumoral fibroblastic LMTK cells [84]. The mechanism of action of haemanthamine is thought to be through inhibition of protein synthesis, blocking the peptide bond formation step on the peptidyl transferase centre of the 60S ribosomal subunit [89,103]. Haemanthamine and haemanthidine also display the same pronounced cell growth inhibitory activities against both parental and multidrug resistant L5178 mouse lymphoma cell lines as described above for lycorine [98]. Crinamine, in turn, shows inhibitory effects on nitric oxide (NO) production and induction of inducible nitric oxide synthase (NOS) in lipopolysaccharide-activated macrophages [92]. Crinamine and haemanthamine are potent inducers of apoptosis in tumor cells at micromolecular concentrations [145]. The pharmacophoric elements are the alpha-C-2 bridge as well as a small substituent (H, or OH) at C-11. Studies have also shown that D- or E-methoxy or the hydroxyl H-bond acceptor are all tolerated at C-3, and that a C-1/C-2 double bond modulates, but is not a requirement, for apoptosis-inducing activity [146]. The antimalarial activity against strains of chloroquine-sensitive Plasmodium falciparum observed in haemanthamine and haemanthidine can be attributed to the methylenedioxybenzene part of the molecule and the terciary nitrogen without methyl [131]. Crinamine also exhibits moderate antimalarial activity [132,147]. Haemanthidine also works in vitro against Trypanosoma brucei rhodesiense and to a lesser extend against Trypanosoma cruzi [134]. Vittatine has antibacterial activity against the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli [129], and the alkaloid crinamine shows strong activity against Bacillus subtilis and Staphylococcus aureus [148]. Like lycorine, haemanthidine has stronger analgesic and anti-inflammatory activity than aspirin [118,137], and vittatine has been found to potentiate the analgesic effect of morphine [149]. Moreover, some alkaloids of this series, such as haemanthamine or papyramine have a hypotensive effect [140,150], and haemanthamine strong antiretroviral activity [113]. Amaryllidaceae alkaloids 87 3.4.Tazettine type Tazettine is mildly active against certain tumor cell lines [88,139,151], with a slight cytotoxicity when tested on fibroblastic LMTK cell lines [84]. Tazettine also displays weak hypotensive and antimalarial activities and interacts with DNA [99,138,140]. Its chemically labile precursor, pretazettine, is far more interesting owing to its antiviral and anticancer activities. In fact, when pretazettine is stereochemically rearranged to tazettine, the biological activity of the precursor is to a large extent inactivated [152,153]. Pretazettine shows cytotoxicity against fibroblastic LMTK cell lines and inhibits HeLa cell growth, being therapeutically effective against advanced Rauscher leukaemia, Ehrlich ascites carcinoma, spontaneous AKR lymphocytic leukaemia and Lewis lung carcinoma [151,154-159]. It is one of the most active of the Amaryllidaceae alkaloids against Molt4 lymphoid cells [84], and is used in combination with DNA-binding and alkylating agents in treating the Rauscher leukaemia virus [151,154]. In fact, pretazettine strongly inhibits the activity of reverse transcriptase from various oncogenic viruses by binding to the enzyme [3]. It inhibits both the growth of the Rauscher virus and cellular protein synthesis in eukaryotic cells by a mechanism that does not affect DNA and RNA synthesis, even though it has a pronounced DNA binding activity [88,89,99,101,111,156,160]. Pretazettine on human MDR1gene-transfected L5158 mouse lymphoma significantly increased the intracellular concentration of Rh-123 and enhanced the antiproliferative activity of doxorubicin in the L5178 MDR cell line [161]. This alkaloid has also been shown to be active against selected RNA-containing flavoviruses (Japanese encephalitis, yellow fewer and dengue) and bunyaviruses (Punta Toro and Rift Valley fever) in organ culture [111]. It also possesses pronounced activity against Herpes simplex type 1 virus [114]. This activity may reflect a general ability to inhibit protein synthesis during viral replication [162]. 3.5. Narciclasine type Narciclasine, an antimitotic and antitumoral alkaloid [163], affects cell division at the metaphase stage and inhibits protein synthesis in eukaryotic ribosomes by directly interacting with the 60s subunit and inhibiting peptide bond formation by preventing binding of the 3' terminal end of the donor substrate to the peptidyl transferase center [89,103,164-166]. It also retards DNA synthesis [167] and inhibits calprotectin-induced cytotoxicity at a more than 10-fold lower concentration than lycorine [90]. The peculiar effects of 88 Jaume Bastida et al. narciclasine seem to arise from the functional groups and conformational freedom of its C-ring [168], with the 7-hydroxyl group believed to be important in its biological activity [169]. This alkaloid, related to pancratistatin [167], is one of the most important antineoplastic Amaryllidaceae alkaloids [80] and shows some promise as an anticancer agent. It inhibits HeLa cell growth, has antileukaemic properties and is active against a variety of tumor cells, such as human and murine lymphocytic leukaemia, larynx and cervix carcinomas and Ehrlich tumor cells [115,167,170-172]. One hemisynthetic derivative of narciclasine demonstrated higher in vivo antitumor activity in human orthotopic glioma models in mice than narciclasine in nontoxic doses [173], by both the i.v and oral routes. No effect has been observed on solid tumors. Narciclasine-4-O-D-glucopiranoside shows a very similar cytotoxic and antitumoral activity to narciclasine [174]. The anticancer activity and preclinical studies of narciclasine and its congeners has been gathered by Kornienko and Evidente in a recent review [175]. Melanomas display poor response rates to adjuvant therapies because of their intrinsic resistance to proapoptotic stimuli. Such resistance can be overcome, at least partly, through the targeting of the eEF1A elongation factor with narciclasine [176]. This alkaloid directly binds to human recombinant and yeast-purified eEF1A in a nanomolar range, but not to actin or elongation factor 2. Thus, eEF1A is a potential target to combat melanomas regardless of their apoptosis-sensitivity, which has renewed interest in the pleiotropic cytostatic activity of narciclasine. Apoptosis in Jurkat cells was triggered by narciclasine, narciclasine tetraacetate, C-10b-Rhydroxypancratistatin, cis-dihydronarciclasine and trans-dihydronarciclasine [177]. The effect of pancratistatin treatment on cancerous and normal cells has also been reported [178]. The results indicated that pancratistatin selectively induced apoptosis in cancer cells, and the mitochondria may be the site of action. To further explore the structure-activity relationship of pancratistatinrelated compounds, the anticancer efficacy and specificity of two related natural alkaloids were investigated. Both of these compounds lack the polyhydroxylated lycorane element of pancratistatin, instead having a methoxy-substituted crinane skeleton. These results indicated that the phenanthridone skeleton in natural Amaryllidaceae alkaloids may be a significant common element for selectivity against cancer cells. The synergy of pancratistatin and tamoxifen on breast cancer cells in inducing apoptosis by targeting mitochondria has been also reported [179]. The 3,4-O-cyclic phosphate salt of pancratistatin is a novel, water soluble synthetic derivative of pancratistatin that in vivo caused statistically significant tumor growth delays at its maximum-tolerated dose. Significant vascular shutdown and tumor necrosis were also observed [180], Amaryllidaceae alkaloids 89 offering a way forward for improved clinical treatment by greatly enhancing solubility without loss of antitumor activity. Narciclasine has a prophylactic effect on the adjuvant arthritis model in rats, significantly suppressing the degree of swelling of adjuvant-treated as well as untreated feet [90]. This alkaloid is also active against Corynebacterium fascians, inhibits the pathogenic yeast Cryptococcus neoformans, and modifications like 2,3,4,7-tetra-O-acetylnarciclasine inhibit the growth of the pathogenic bacterium Neisseria gonorrhoeae [181]. Antiviral activity has been observed against RNA-containing flaviviruses and bunyaviruses [111]. At the plant level, narciclasine is a potent inhibitor, showing a broad range of effects, including the ability to inhibit seed germination and seedling growth of some plants in a dose-dependent manner, interacting with hormones in some physiological responses [182]. Thus, indole-3-acetic acid cannot overcome the inhibition of elongation of wheat coleoptile sections caused by narciclasine. Additionally, narciclasine suppresses the gibberellin-induced D-amylase production in barley seeds and cytokinin-induced expansion and greening of excised radish cotyledons [183]. Like lycorine, narciclasine also inhibits ascorbic acid biosynthesis [184]. Narciclasine, present in daffodil mucilage, can delay tepal senescence in cut Iris flowers by attenuation of protease activity, which, in turn, is apparently related with the inhibition of the protein synthesis involved in senescence [185]. At the organelle level, narciclasine inhibits both isocitrate lyase (ICL) activity in glyoxysomes and hydroxypyruvate reductase (HPR) activity in peroxisomes. It also blocks the formation of chloroplasts, markedly reducing the chlorophyll content of lightgrown wheat seedlings, probably due to the inhibition of the formation of 5-aminolevulinic acid, an essential chlorophyll precursor [186]. The formation of light harvesting chlorophyll a/b binding protein (LHCP) is also inhibited by this alkaloid [187]. Some alkaloids of this series, such as trisphaeridine, possess high antiretroviral activities, accompanied by low therapeutic indices [113]. Ismine, in turn, shows a significant hypotensive effect on the arterial pressure of normotensive rats [140] and is cytotoxic against Molt 4 lymphoid and LMTK fibroblastic cell lines [84]. 3.6. Montanine type There is little information about the montanine type alkaloids, only some data about pancracine, which shows antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa [129], as well as weak activity against Trypanosoma brucei rhodesiense, T. cruzi and Plasmodium falciparum [188]. Montanine inhibited, in a dose-dependent manner, more 90 Jaume Bastida et al. than 50% of the enzyme acetylcholinesterase at 1 mM concentration. With the concentrations 500 PM and 100 PM, 30-45% of inhibition was detected [189]. 3.7. Galanthamine type Galanthamine, originally isolated from Galanthus nivalis L. in the 1940s, is a long-acting, selective, reversible and competitive inhibitor of acetylcholinesterase. This enzyme is responsible for the degradation of acetylcholine at the neuromuscular junction, in peripheral and central cholinergic synapses and in parasympathetic target organs [190-192]. Galanthamine has the ability to cross the blood-brain barrier and act within the central nervous system [193,194]. It binds at the base of the active site gorge of acetylcholinesterase, interacting with both the choline-binding site and the acyl-binding pocket, having a number of moderate-to-weak interactions with the protein [195-197]. In addition, galanthamine stimulates pre- and postsynaptic nicotinic receptors which can, in turn, increase the release of neurotransmitters, thus directly stimulating neuronal function [192,198]. It is also suggested that the stimulation of nicotinic receptors protects against apoptosis induced by -amyloid toxicity [192,199,200]. Its dual mode of action [195], coupled with the evidence that galanthamine has reduced side effects, make it a promising candidate for the treatment of nervous diseases, paralysis syndrome, schizophrenia and other forms of dementia, as well as Alzheimer's disease [192,195,196]. Other significant pharmacological actions of Galanthamine include an ability to amplify the nerve-muscle transfer [3], affecting membrane ionic processes [201]. It is also known to cause bradycardia or atrioventricular conduction disturbances [150], has long been used as a reversal agent in anaesthetic practice [18], inhibits traumatic shock and has been patented for use in the treatment of nicotine dependence. Besides this, galanthamine acts as a mild analeptic, shows an analgesic power as strong as morphine, compensates for the effects of opiates on respiration, relieves jet lag, fatigue syndrome, male impotence and alcohol dependence, and when applied in eye drops, reduces the intraocular pressure [3,202-204]. It also acts as a hypotensive and has a weak antimalarial activity [138,140]. At present, Alzheimer's disease cannot be prevented or cured, so the symptomatic relief offered by AChEI therapy is the only approved therapeutic option. Due to the relative lack of alternative treatment, galanthamine is a reasonable approximation of the ideal concept of symptomatic Alzheimer's disease therapy [191,205]. Galanthamine hydrobromide (a third-generation cholinesterase inhibitor used against Alzheimer’s disease) offers superior pharmacological profiles and increased tolerance compared to the original Amaryllidaceae alkaloids 91 acetylcholinesterase inhibitors, physostigmine or tacrine [193,206-209]. Galanthamine is effective and well tolerated, resulting in short-term improvements in cognition, function and daily life activities in patients with mild to moderate symptoms [198,210,211]. However, there is doubt about its long-term benefits [212] since persistent elevation of acetylcholine beyond 6 months may lead to over-stimulation of both nicotinic and muscarinic acetylcholine receptors, the former causing receptor desensitisation and the latter potentially causing an increased frequency of cholinergic side effects [192,198,213]. The safety profile of galanthamine as well as its clinical effectiveness will only be demonstrated after large-scale clinical trials [213-215]. The development of galanthamine into a widely used Alzheimer’s drug can be divided into three main periods: 1- the early development in Eastern Europe for its use in the treatment of poliomyelitis; 2- the pre-clinical development in the 1980s; 3- the clinical development in the 1990s [213]. Galanthamine hydrobromide was first used by Bulgarian and Russian researchers in the 1950s and exploited for a variety of clinical purposes. It has been used clinically for postsurgery reversal of tubocurarine-induced muscle relaxation and for treating post-polio paralysis, myasthenia gravis and other neuromuscular diseases, as well as traumatic brain injuries [216,217]. As early as 1972, Soviet researchers demonstrated that galanthamine could reverse scopolamine-induced amnesia in mice, a finding that was demonstrated in man 4 years later. However, this compound was not applied to Alzheimer's disease until 1986, long after the widely accepted cholinergic hypothesis had been first postulated, when researchers in Western Europe switched their attention to galanthamine due to its ability to penetrate the blood-brain barrier and specifically to augment the central cholinergic function [213,218]. This led to clinical trials of galanthamine in the treatment of Alzheimer's disease. In 1996, Sanochemia Pharmazeutika in Austria first launched galanthamine as ‘Nivalin®’, but its strictly limited availability meant the international pharmaceutical community adopted a cautions approach [18,194], until Sanochemia Pharmazeutika developed a method to synthetically produce the compound in 1997 [219]. Later, galanthamine was co-developed by Shire Pharmaceuticals (Great Britain) and the Janssen Research Foundation (Belgium), who have launched galanthamine as ‘Reminyl®’ in many countries [192,213]. This renewed interest is reflected in the increasing number of scientific reviews dealing exclusively with galanthamine and its derivatives [220-223]. Sanguinine has a more potent acetylcholinesterase inhibitory activity than galanthamine due to an extra hydroxyl group available for potential interaction with acetylcholinesterase [120]. Sanguinine, in turn, is 10-fold Jaume Bastida et al. 92 more selective than galanthamine for acetylcholinesterase (AChE) vs. butyrylcholinesterase (BuChE) [224]. The lack of AChE inhibitory activity of lycoramine and epinorlycoramine could be due to the occurrence of a double bond in ring C, which does not allow these compounds to have the same spatial configuration as the active alkaloids of this series [120]. Narwedine, the biogenic precursor of galanthamine, has been studied as a respiratory stimulator. It increases the amplitude and decreases the frequency of cardiac contractions and would therefore be of value in reducing blood loss during surgery [150]. It also inhibits the action of narcotics and hypnotics, and increases the analgesic effect of morphine [149], as well as the pharmacological effects of caffeine, carbazole, arecoline and nicotine [126]. 3.8. Other alkaloids Cherylline is a 4-arylisoquinoline derivative, a group with several potential medicinal properties [188], including a weak acetylcholinesterase inhibitory activity [118]. Mesembrenone, in turn, is mildly active against Molt 4 lymphoid and non-tumoral fibroblastic LMTK cells [84], has a moderate hypotensive effect on arterial pressure and interacts slightly with DNA [99,140]. Acknowledgements The authors are grateful to Ms Lucy Brzoska for language corrections. The authors also thank Generalitat de Catalunya (2009-SGR1060) for financial support. References 1. 2. 3. 4. 5. 6. 7. 8. Meerow, A. W., Snijman, D. 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Neurother. 4, 153. 221. Dengiz, A. N., Kershaw, P. 2004, CNS Spectr. 9, 377. 222. Marco-Contelles, J., Perez-Mayoral, E., Van Nhien, A. N., Postel, D. 2007, Targets Heterocycl. Syst. 11, 365. 223. Heinrich, M. 2010, in “The Alkaloids: Chemistry and Biology” (G. A. Cordell, ed.), Vol 68, p. 157. Elsevier Inc. Amsterdam. 224. Bachus, R., Bickel, U., Thomsen, T., Roots, I., Kewitz, H. 1999, Pharmacogenetics 9, 661. AnexoII 7.2.AnexoII AcontinuaciónseadjuntanlastablascondatosdeRMN,espectrosdemasasy espectrosde1H9RMNdeloscompuestosnuevoscaracterizadosenelpresentetrabajo. 183 AnexoII 7.2.1.Narseronina Posición 1 COSY NOESY 1 9 9 9 2 4.22t(6.1) H3/ H3/,OMe 74.9 d C1,C3,C4,C10b,OMe 3 2.01dt(13.5,5.5) H2,H3,H4 H2,H3,H4,OMe 31.4 t C1,C2,C4,C4a,C11 3 2.2292.13m(solap.) H2,H3,H4 H2,H3,H4,OMe 31.4 t C1,C2,C4,C4a,C11 4 2.64m H3/,H4a,H11/ H3/,H4a,H11/ 35.1 d C12 4a 3.94d(6.4) H4 H4,H10,NMe 61.6 d C1,C3,C4,C10a,C10b,C11,C12,NMe 6 9 9 9 161.5 s 9 6a 9 9 9 116.4 s 9 7 7.66s 9 9 107.8 d C6,C8,C9,C10a 8 9 9 9 148.4 s 9 9 9 9 9 153.8 s 9 H(JinHz) 13 C HMBC 152.9 s 9 10 7.29s 9 H4a,NMe 103.3 d C6a,C8,C9,C10b 10a 9 9 9 135.1 s 9 10b 9 9 9 110.8 s 9 11 2.2292.13m(solap.) H4,H11,H12/ H4,H11,H12/,OMe 29.6 t C3,C4a 11 1.90ddd(12.6,8.3,4.2) H4,H11,H12/ H4,H11,H12/,OMe 29.6 t C3,C4a 12 3.05dt(11.0,7.6) H11/,H12 H11/,H12,NMe 54.3 t C4,C4a,C11,NMe 12 2.81m H11/,H12 H11/,H12,NMe 54.3 t C4,C4a,C11,NMe OCH2O 6.10d(1.2),6.12d(1.2) 9 9 OMe 3.57s 9 H2,H3/,H11/ 58.3 q C2 NMe 2.41s 9 H4a,H10,H12/ 41.8 q C4a,C12 102.4 t 12 11 H MeN 10 O 9 O 8 4 4a H 3 2 10b 10a OMe 1 6a C8,C9 6 O 7 O EspectrodeMasas 240 100 57 50 256 272 44 82 63 115 96 139 155 167 127 213 185 200 299 267 228 282 314 0 40 60 80 100 120 140 160 180 200 220 240 260 280 328 300 320 340 184 AnexoII 185 AnexoII 7.2.2.1O(3´Acetoxibutanoil)licorina Posición 1 COSY 1 5.68s H2,H10b 2 4.23dt(3.3,1.7) H1,H3,H11 3 5.56m H2,H11 4a 2.76d(10.4) H10b 61.9 d 6 3.54d(14.1) H6 56.6 t 6 4.16d(14.1) H6 56.6 t 7 6.58s 9 107.3 d 10 6.72s 9 104.8 d 10b 2.91d(10.4) H1,H4a 38.8 d 11(2H) 2.65m H2,H3,H12/ 28.4 t 12 2.42dd(9.3,5.0) H11,H12 53.4 t H(JinHz) 13 C 72.5 d 69.4 d 116.9 d 12 3.38dt(9.2,4.8) H11,H12 OCH2O 5.92s 9 2´A 2.43dd(15.5,5.4) H2´B,H3´ 40.5 t 2´B 2.53dd(15.5,7.8) H2´A,H3´ 40.5 t 3´ 5.10m H92´A,H92´B,H94´ 66.9 d 4´ 1.14d(6.3) H3´ 19.3 q 9 20.7 q OAc(2´´) 1.95s 2´´ 53.4 t 100.8 t O 1´´ O 4´ 3´ O 2´ OH 1´ O 10 O 9 O 8 2 1 3 H 10b 10a 11 4a H N 6a 7 4 12 6 EspectrodeMasas 226 100 50 268 43 250 69 51 61 0 40 77 70 96 100 115 134 147 130 167 160 192 190 211 220 415 240 286 250 280 354 310 340 370 400 430 186 AnexoII 187 AnexoII 7.2.3.3OMetilnarcisidina Posición 1 COSY NOESY 1 4.71brs H2,H3,H10b H2,H10,H10b,OMe(2) 68.1 d 2 3.80t(2.9) H1,H3 H1,H3,OMe(2/3) 80.5 d C1,C3,C4,C10b,OMe(2) 3 4.29brd(2.0) H1,H2 H2,H11,OMe(2/3) 77.8 d C1,C2,C4a,C11,OMe(3) 4 9 9 9 137.2 s 4a 3.87m(solap.) H10b,H11,H12 9 62.6 d C12 6 4.17d(12.8) H6,H7 H6,H7 54.9 t C4a,C6a,C7,C10a,C12 6 3.67d(12.4) H6,H7 H6,H7,H10b 54.9 t C4a,C6a,C7,C10a,C12 6a 9 9 9 128.8 s 9 7 6.75s H6/,OMe(8) H6/,OMe(8) 111.0 d C6,C9,C10a 8 9 9 9 147.2 s 9 9 9 9 9 148.3 s 9 10 6.98s H10b,OMe(9) H1,H10b,OMe(9) 107.9 d C6a,C8,C10b 10a 9 9 9 130.1 s 9 10b 2.81dd(11.2,1.7) H1,H4a,H10 H1,H6,H10,OMe(2) 11 5.87q(1.8) H4a,H12/ H3,H12/,OMe(3) 12 4.21brd(14.7) H11,H12 H11,H12 62.5 t C4,C4a,C6,C11 12 3.65ddd(14.5,6.0,2.0) H4a,H11,H12 H11,H12 62.5 t C4,C4a,C6,C11 OMe(2) 3.47s 9 H1,H2,H3,H10b 58.4 q C2 OMe(3) 3.27s 9 H2,H3,H11 56.4 q C3 OMe(8) 3.86s H7 H7 56.3 q C8 OMe(9) 3.91s H10 H10 56.3 q C9 H(JinHz) 13 C HMBC 41.6 d 125.9 d C2,C3,C4a 9 C1,C4a,C10,C10a C3,C4,C4a OMe 2 HO MeO 10 9 OMe 1 10b 10a 3 H 4 11 4a H MeO 6a 8 N 12 6 7 EspectrodeMasas 284 100 315 50 230 266 258 65 0 40 60 80 94 105 80 100 121 133 120 140 151 162 160 191 180 200 214 220 242 240 346 272 260 280 298 300 320 340 360 188 AnexoII 189 AnexoII 7.2.4.1OAcetil3Ometilnarcisidina Posición 1 COSY NOESY 1 5.81brt(2.6) H2,H3,H10b H2,H10,H10b,OMe(2),OCOMe 2 3.73dd(2.8,2.0) H1,H3 H1,H3,OMe(2/3),OCOMe 79.1 d C1,C3,C4,C10b,OMe(2) 3 4.08brd(1.7) H1,H2,H11 H2,H11,OMe(2/3) 76.6 d C1,C2,C4,C4a,C11,OMe(3) 4 9 9 9 4a 3.92m H10b,H11,H12 6 4.21d(13.1) 6 H(JinHz) 13 C HMBC 68.2 d C2,C3,C4a,OCOMe 137.6 s 9 OMe(3) 62.7 d 9 H6,H7 H6,H7 54.5 t C4a,C7,C10a,C12 3.70d(13.0) H6,H7 H6,H7,H10b 54.5 t C4a,C7,C10a,C12 6a 9 9 9 128.4 s 9 7 6.74s H6/,OMe(8) H6/,OMe(8) 110.8 d C6,C9,C10,C10a,C10b 8 9 9 9 147.4 s 9 9 9 9 9 148.2 s 9 10 6.57s H10b,OMe(9) H1,H10b,OMe(9),OCOMe 107.0 d C6,C6a,C7,C8,C10b 10a 9 9 9 128.5 s 9 10b 2.98dd(11.0,2.0) H1,H4a,H10 H1,H6,H10,OMe(2) 11 5.87q(1.8) H3,H4a,H12/ H3,H12/,OMe(3) 12 4.18m(solap.) H11,H12 12 3.66ddd(14.4,5.7,2.1) OMe(2) 39.8 d C4,C4a,C6a,C8,C10,C10a 125.9 d C3,C4,C4a,C12 H11,H12 62.1 t C4,C6,C10b,C11 H4a,H11,H12 H11,H12 62.1 t C4,C6,C10b,C11 3.52s 9 H1,H2,H3,H10b 58.7 q C2 OMe(3) 3.24s 9 H2,H3,H4a,H11,OCOMe 56.3 q C3 OMe(8) 3.86s H7 H7 56.1 q C8 OMe(9) 3.81s H10 H10,OCOMe 56.1 q C9 OCOMe 1.99s 9 H1,H2,H10,OMe(3/9) OCOMe 9 9 9 21.2 q 171.3 s C1,OCOMe 9 OMe AcO MeO 10 2 H 10b 10a 9 4a H MeO N 6a 8 OMe 1 3 4 11 12 6 7 EspectrodeMasas 326 266 100 357 50 228 298 258 235 55 0 40 60 75 80 89 110 100 120 133 140 151 160 178 191 180 200 284 212 220 240 260 280 388 314 300 320 340 360 380 400 190 AnexoII 191 AnexoII 7.2.5.1OAcetil3Ometil6oxonarcisidina Posición 1 COSY NOESY 1 5.78brt(2.7) H2,H3,H10b H2,H10,H10b,OMe(2),OCOMe 2 3.77dd(2.9,2.0) H1,H3 3 4.06brd(1.9) H1,H2 4 9 9 9 4a 4.72m H10b,H11,H12/ H10b,H12,OMe(3),OCOMe 6 9 9 9 162.6 s 9 6a 9 9 9 130.8 s 9 7 7.57s OMe(8) OMe(8) 111.4 d C6,C6a,C8,C9,C10a 8 9 9 9 148.0 s 9 9 9 9 9 151.9 s 9 10 6.58d(0.8) H10b,OMe(9) H1,H10b,OMe(9),OCOMe 105.7 d C6,C8,C9,C10a,C10b 10a 9 9 9 124.6 s 9 10b 3.30ddd(12.8,2.5,0.9) H1,H4a,H10 H1,H4a,H10,OMe(2) 11 5.97q(1.8) H4a,H12/ H3,H12/,OMe(3) 12 4.40ddd(16.0,3.2,1.6) H4a,H11,H12 H4a,H11,H12 52.6 t C4,C11 12 4.68ddd(16.1,5.1,2.0) H4a,H11,H12 H11,H12 52.6 t C4,C11 OMe(2) 3.51s 9 H1,H2,H3,H10b,OCOMe 58.9 q C2 OMe(3) 3.27s 9 H2,H3,H4a,H11,OCOMe 56.5 q C3 OMe(8) 3.93s H7 H7 56.2 q C8 OMe(9) 3.86s H10 H10,OCOMe 56.2 q C9 OCOMe 2.01s 9 H1,H2,H4a,H10,OMe(2/3/9) 21.1 q OCOMe OCOMe 9 9 9 H(JinHz) 13 C HMBC 66.8 d C2,C3,C4a,OCOMe H1,H3,OMe(2/3),OCOMe 79.1 d C1,C3,C4,C10b,OMe(2) H2,H11,OMe(2/3) 75.8 d C1,C2,C4a,C11,OMe(3) 136.1 s 60.1 d 41.6 d 125.4 d 171.1 s 9 C4,C11 C4a,C6a C3,C4a,C12 9 OMe AcO MeO 10 9 2 OMe 1 H 10b 10a 3 4 11 4a H MeO N 6a 8 12 6 7 O EspectrodeMasas 280 100 50 43 80 94 0 40 60 80 110 100 120 242 149 165 178 191 207 140 160 180 200 220 240 255 272 260 280 340 298 371 312 300 320 340 360 380 400 192 AnexoII 193 AnexoII 7.2.6.2Metoxipratosina Posición 1 COSY NOESY 1 7.55d(2.0) H3 OMe(2) 2 9 9 3 7.30d(2.0) H1 4 9 4a H(JinHz) 13 C HMBC 106.2 d C2,C3,C4a,C10a 9 157.7 s 9 H11,OMe(2) 106.9 d C1,C2,C4a,C11 9 9 129.1 s 9 9 9 9 126.6 s 9 6 9 9 9 158.3 s 9 6a 9 9 9 121.2 s 9 7 8.01s OMe(8) OMe(8) 110.4 d C6,C6a,C8,C9,C10a 8 9 9 9 149.9 s 9 9 9 9 9 153.7 s 9 10 7.59s OMe(9) OMe(9) 104.1 d C6a,C8,C9,C10b 10a 9 9 9 129.3 s 9 10b 9 9 9 117.1 s 9 11 6.84d(3.5) H12 H3,H12 110.7 d C4,C4a 12 8.03d(3.5) H11 H11 124.2 d 9 OMe(2) 3.98s 9 H1,H3 56.5 q C2 OMe(8) 4.07s H7 H7 56.4 q C8 OMe(9) 4.12s H10 H10 56.4 q C9 OMe 2 1 MeO 10 9 3 10b 10a 4 11 4a MeO 8 6a 7 N 12 6 O EspectrodeMasas 309 100 50 266 125 137 75 0 50 70 90 110 130 152 164 150 177 170 193 190 208 210 222 236 230 294 251 278 250 270 290 310 194 AnexoII 195 AnexoII 7.2.7.11Hidroxigalantina Posición 1 COSY NOESY 1 4.68brs H2,H3,H10b H2,H10,H10b,OMe(2) 2 3.88ddd(3.0,3.0,1.5) H1,H3,H4a,H11 H1,H3,OMe(2) 80.9 d 3 5.94m H1,H2,H4a,H11 H2,H11,OMe(2) 119.4 d C4a 4 9 9 9 146.0 s 9 4a 3.03dd(10.5,1.4) H2,H3,H10b,H11 H6,H12 59.8 d C4 6 3.60brd(14.0) H6,H7,H10b H4a,H6,H7,H12 56.1 t C4a,C6a,C10a,C12 6 4.07d(13.9) H6,H7,H10b H6,H7,H10b,H12 56.1 t C4a,C6a,C7,C10a 6a 9 9 9 129.4 s 9 7 6.64s H6/,OMe(8) H6/,OMe(8) 111.0 d C6,C9,C10a 8 9 9 9 148.0 s 9 9 9 9 9 148.1 s 9 H(JinHz) 13 C HMBC 69.0 d C2,C3,C4a C1,C4,OMe(2) 10 6.85s H10b,OMe(9) H1,H10b,OMe(9) 107.5 d C6a,C8,C10b 10a 9 9 9 125.9 s 9 10b 2.65brd(10.6) H1,H4a,H6/,H10 H1,H6,H10 41.7 d C4a,C10a 11 4.89brddd(6.5,1.5) H2,H3,H4a,H12/ H3,H12/ 71.6 d C3,C4 12 2.35dd(9.2,6.7) H11,H12 H4a,H6,H11,H12 63.2 t C6,C11 12 3.68dd(9.2,6.5) H11,H12 H6,H11,H12 63.2 t C4,C4a,C11 OMe(2) 3.56s 9 H1,H2,H3 58.1 q C2 OMe(8) 3.86s H7 H7 56.1 q C8 OMe(9) 3.90s H10 H10 56.3 q C9 OMe 2 HO MeO 10 9 1 10b 10a H MeO 8 6a 3 H 4a N 4 OH 11 12 6 7 EspectrodeMasas 259 100 50 0 45 53 40 65 60 77 80 91 103 115 126 100 120 141 140 151 240 162 178 160 180 193 200 212 226 220 240 266 260 284 302 280 300 314 320 333 340 196 AnexoII 197 AnexoII 7.2.8.2OMetilclivonina Posición 1 COSY NOESY 1 4.09dd(12.6,2.6) H2,H10b H2,H3,H4a 2 3.78q(2.9) H1,H3/ H1,H3/,OMe(2) 76.8 d C1,C4,OMe(2) 3 2.37ddd(15.5,2.8,1.6) H2,H3,H4 H2,H3,OMe(2) 26.3 t C1,C2,C4,C4a,C11 3 1.61ddd(15.4,6.5,3.1) H2,H3,H4 H1,H2,H3,H4 26.3 t C4,C11 4 2.6292.46m(solap.) H3/ H3,H94a,H11 33.6 d 9 4a 2.87dd(9.8,6.7) H10b H1,H4,H10,NMe 70.3 d 9 6 9 9 9 164.9 s 9 6a 9 9 9 118.9 s 9 7 7.50s 9 9 109.4 d C6,C6a,C8,C9,C10a 8 9 9 9 146.8 s 9 9 9 9 9 152.6 s 9 10 7.86brs 9 H4a,NMe 107.4 d 9 10a 9 9 9 141.1 s 9 10b 3.27dd(12.8,9.5) H1,H4a H11,OMe(2) 34.2 d 9 11 2.2992.15m H11,H12/ H10b,H11,H12,OMe(2) 30.5 t 9 11 2.1592.02m H11,H12/ H4,H11,H12 30.5 t 9 12 3.3593.21m(solap.) H11/,H12 H11,H12 53.1 t 9 12 2.6292.46m(solap.) H11/,H12 H11,H12 53.1 t 9 OMe(2) 3.49s 9 H2,H3,H10b,H11 58.2 q C2 NMe 2.54s 9 H4a,H10 45.5 q 9 OCH2O 6.02d(1.3)96.03d(1.3) 9 9 H(JinHz) 13 C 81.4 d 101.9 t 12 O 4 3 H 10b 2 4a H 1 H O 8 C8,C9 H 10a 9 9 11 MeN 10 HMBC OMe O 6a 6 7 O EspectrodeMasas 83 100 50 96 331 42 0 40 55 60 67 76 80 115 89 100 120 133 140 162 173 160 180 316 228 200 220 240 260 280 300 320 340 198 AnexoII 199 AnexoII 7.2.9.2Oxomesembrenona Posición 1 COSY NOESY 13 HMBC 2 9 9 9 171.9 s 9 H(JinHz) C 3 2.66d(17.1) H3 H3,H4 44.2 t C2,C3a,C4,C7a,C1´ 3 3.18d(17.1) H3 H3,H7a,H2´,H6´ 44.2 t C2,C3a,C4,C1´ 3a 9 9 9 45.9 s 9 4 6.71dd(10.2,1.6) H5,H7a H3,H5,H2´,H6´ 150.7 d C3,C3a,C6,C7a,C1´ 5 6.24d(10.2) H4 H4 129.0 d C3a,C7 6 9 9 9 195.3 s 9 7(2H) 2.72d(3.8) H7a H7a 36.5 t C3a,C5,C6,C7a 7a 4.06td(3.8,1.6) H7,H4 H3,H7,H2´,H6´,NMe 65.7 d C3a,C4,C6,C1´ 1´ 9 9 9 131.6 s 9 2´ 6.85d(2.2) H6´ H3,H4,H7a 109.9 d C3a,C4´,C6´ 3´ 9 9 9 149.7 s 9 4´ 9 9 9 149.1 s 9 5´ 6.87d(8.4) H6´ OMe 111.6 d C1´,C3´ 6´ 6.92dd(8.4,2.2) H2´,H5´ H3,H4,H7a 119.4 d C3a,C2´,C4´ NMe 2.81s 9 H7a 27.4 q C2,C7a OMe(3´,4´) 3.88s 9 H5´ 56.1,56.2 q C3´,C4´ OMe 4´ 5´ OMe 3´ 2´ 4 6´ 3 1´ 2 5 3a O 7a N 6 H 7 O EspectrodeMasas 301 100 50 163 51 58 65 0 40 60 115 77 84 91 80 100 120 138 140 160 217 173 185 201 180 200 259 230 220 244 270 240 260 286 280 300 200 AnexoII 201 AnexoII 7.2.10.7,7aDehidromesembrenona Posición 1 COSY 2 3.31dd(10.3,8.1) H2,H3 52.7 t C3,C3a 2 3.41ddd(10.6,10.3,5.0) H2,H3/ 52.7 t 9 3 2.27ddd(11.7,10.8,8.1) H2/,H3 35.9 t C3a,C1´ 3 2.56dd(11.9,5.0) H2,H3 35.9 t C3a,C7a,C1´ 3a 9 9 53.5 s 9 4 6.77d(9.6) H5 142.8 d C3a,C6,C7a,C1´ 5 6.03dd(9.6,1.4) H4 128.8 d C3a 6 9 9 185.6 s 9 7 5.46brs 9 93.7 d 9 7a 9 9 171.5 s 9 1´ 9 9 133.2 s 9 2´ 6.84d(2.2) H6´ 109.9 d C3a,C4´,C6´ 3´ 9 9 149.1 s 9 4´ 9 9 148.6 s 9 5´ 6.78d(8.3) H6´ 111.3 d C1´,C3´ 6´ 6.86dd(8.3,2.3) H2´,H5´ 118.4 d C3a,C2´,C4´ NMe 2.98s 9 33.1 q C2,C7a OMe(3´) 3.84s 9 56.2 q C3´ OMe(4´) 3.85s 9 56.1 q C4´ H(JinHz) 13 C HMBC OMe 4´ 5´ OMe 3´ 2´ 4 6´ 3 1´ 5 3a 2 N 7a 6 7 O EspectrodeMasas 285 100 242 50 257 51 77 63 91 119 102 132 147 156 170 184 199 270 214 226 0 50 70 90 110 130 150 170 190 210 230 250 270 290 202 AnexoII 203 AnexoII 7.2.11.2Oxoepimesembranol Posición 1 COSY NOESY 2 9 9 9 H(JinHz) 13 C HMBC 173.8 s 9 3 2.65d(16.4) H3 H3,H4/,H5,H7,H2´,H6´ 45.7 t C2,C3a,C4,C7a,C1´ 3 2.54d(16.4) H3 H3,H4,H7a,H2´,H6´ 45.7 t C2,C3a,C4,C7a,C1´ 3a 9 9 9 42.9 s 9 4 2.14ddd(15.0,10.0,3.6) H4,H5/ H3/,H4,H5/,H2´,H6´ 30.0 t C3,C3a,C5,C6,C7a,C1´ 4 1.85ddd(14.5,7.4,3.6) H4,H5/ H3,H4,H5/,H6,H2´,H6´ 30.0 t C3,C3a,C5,C6,C7a,C1´ 5 1.60m(solap.) H4/,H5,H6 H3,H4/,H5,H6 30.1 t C3a,C6,C7 5 1.73ddtd(14.0,10.0,3.6,1.0) H4/,H5,H6 H4/,H5,H6,H2´,H6´ 30.1 t C3a,C6,C7 6 3.95tt(6.1,3.6) H5/,H7/ H4,H5/,H7/ 66.1 d 9 7 1.96dddd(14.6,6.0,5.5,1.0) H6,H7,H7a H3,H6,H7,H7a,NMe 32.8 t C3a,C4,C5,C6,C7a 7 2.14dddd(14.7,5.0,3.6,1.0) H6,H7,H7a H6,H7,H7a,H2´,H6´,NMe 32.8 t C3a,C4,C5,C6,C7a 7a 3.91dd(5.5,5.0) H7/ H3,H7/,H2´,H6´,NMe 62.3 d C2,C3a,C4,C6,C1´ 1´ 9 9 9 137.4 s 9 2´ 6.81d(2.4) H6´ H3/,H4/,H5,H7,H7a 110.0 d C3a,C4´,C6´ 3´ 9 9 9 149.2 s 9 4´ 9 9 9 148.0 s 9 5´ 6.82d(8.4) H6´ 9 111.2 d C1´,C3´ 6´ 6.86dd(8.4,2.3) H2´,H5´ H3/,H4/,H5,H7,H7a 118.2 d C3a,C2´,C4´ NMe 2.90s 9 H7/,H7a 28.1 q C2,C7a OMe(3´) 3.88s 9 9 56.2 q C3´ OMe(4´) 3.87s 9 9 56.1 q C4´ OMe 4´ OMe 5´ 3´ 2´ 6´ 3 1´ O 2 4 5 3a 7a N 6 H 7 OH EspectrodeMasas 305 100 50 233 57 65 0 40 60 77 80 123 91 100 120 149 167 138 140 160 178 190 180 204 200 218 220 246 240 258 260 272 280 290 300 320 204 AnexoII 205

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Biodiversidad y Conservación de Recursos Fitogenéticos. Las Amarillidáceas como Fuente de Productos Bioactivos

Biodiversidad y Conservación de Recursos Fitogenéticos. Las Amarillidáceas como Fuente de Productos Bioactivos

Biodiversidad y Conservación de Recursos Fitogenéticos. Las Amarillidáceas como Fuente de Productos Bioactivos

Biodiversidad y Conservación de Recursos Fitogenéticos. Las Amarillidáceas como Fuente de Productos Bioactivos

Biodiversidad y Conservación de Recursos Fitogenéticos. Las Amarillidáceas como Fuente de Productos Bioactivos

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