Bioorganic&MedicinalChemistryLettersjournalhomepage:www.elsevier.com/locate/bmclTranskarbamsastransdermalpermeationenhancers:Effectsofesterpositionandammoniumcarbamateformation
ˇinaVávrová*´,AlexandrHrabálek,BarboraJanu´,Kater˚šová,JakubNovotnyMichalNovotny
CentreforNewAntiviralsandAntineoplastics,CharlesUniversityinPrague,FacultyofPharmacy,Heyrovského1203,50005HradecKrálové,CzechRepublicarticleinfoabstract
Transkarbam12,anammoniumcarbamateformedbythereactionofdodecyl6-aminohexanoatewithcarbondioxide,isahighlyactive,broad-spectrum,nontoxic,andnonirritanttransdermalpermeationenhancer.Itprobablyactsbyadualmechanism:apartofitsactivityisassociatedwiththecarbamicacidsaltand/oritsdecompositionintheacidicstratumcorneum.Theammoniumestertherebyreleasedisanactiveenhancerspeciesaswell,anditsactivityhighlydependsonthepositionoftheestergroup.Ó2010ElsevierLtd.Allrightsreserved.Articlehistory:Received10February2010Revised11March2010Accepted19March2010Availableonline25March2010Keywords:TransdermaldrugdeliveryPermeationenhancersAmmoniumcarbamateStructure–activityrelationshipsTransdermaldeliveryofdrugsthroughtheskintothesystemiccirculationoffersnumerousadvantagesovertheconventionalroutesofdrugadministration.1Adisadvantagetodevelopmenthowever,stemsfromthefactthattheskinisaremarkablyefficientbarrier,designedtokeep‘ourinsidesinandtheoutsidesout’.2Toovercomethislimitation,substancestemporarilydiminishingthebarrierpropertiesoftheskinknownaspermeationenhancersorpenetration/absorptionpromotershavebeenused.2–8Thesechem-icalsinteractwithconstituentsofthemajorskinbarrier,stratumcorneum,topromotedrugflux.Althoughhundredsofcompoundshavebeenevaluatedaspermeationenhancers,to-datetheiruseintransdermalformulationsislimitedsincethemechanismsofac-tionoftheseagentsareseldomdefined.2Transkarbam12(Fig.1)isanammoniumcarbamateformedbythereactionof6-aminohexanoicaciddodecylesterwithcarbondioxide.9Thisamphiphiliccompoundisahighlypotentbroad-spectrumenhancerwithlowtoxicityandminimalskinirrita-tion.9,10Inourefforttoobtainenhancerswithhigheractivityand/ortoelucidatethemechanismofactionofTranskarbam12,numeroussyntheticmodificationshavebeenstudied.ThestructuralrequirementsaregiveninFigure1.Thehydro-phobicchain(s)shouldbe10–12carbonslongandlinearbecausebothbranchingandcyclizationhadnegativeeffectsontheenhanc-ingactivity.11–13Thelinkingchainbetweenthenitrogenandestercarbonylshouldbeflexible.14Theestergroupisimportantaswell;itsreplacementbycarbonate,carbamate,amide,ketone,andmethylenegroupledtoamarkeddecreaseorlossofactivity.15,16*Correspondingauthor.Tel.:+420495067497;fax:+420495067166.E-mailaddress:katerina.vavrova@faf.cuni.cz(K.Vávrová).0960-894X/$-seefrontmatterÓ2010ElsevierLtd.Allrightsreserved.doi:10.1016/j.bmcl.2010.03.077Themostunusualstructuralfeatureofthisenhanceristheammo-niumcarbamatepolarhead.Itwassuggestedtobeessentialbe-causetheparentaminoesterwasinactive.9However,itsimportanceforthemechanismofactionoftranskarbamsisstillunknown.Ingeneral,saltsofcarbamicacidsareunstableinanacidicenvironmentreleasingcarbondioxideandtherespectiveammoniumsalt17(Fig.1).Itwassuggestedthatsuchcarbondiox-idereleasecantakeplaceintheacidicstratumcorneum,andmaybeconnectedwiththemechanismofactionoftranskarbams.9,14Inaccordancewiththishypothesis,stablecarbondioxidederivativesofsimilarstructurewereinactive.18ThepurposeofthisstudywastoevaluatethemostimportantstructuralfeaturesofTranskarbam12further.Sincetheestergroupwasfoundnecessaryfortheactivityofthisenhancer,weaimedtoprepareandstudyaseriesofTranskarbam12isomerswithvaryingdistanceoftheestergroupfromthecarbamatepolarFigure1.Transkarbam12,itsdecompositioninanacidicenvironment,andthestructuralfeaturesimportantforitstransdermalpermeation-enhancingactivity.´etal./Bioorg.Med.Chem.Lett.20(2010)2726–2728M.Novotny2727Scheme1.SynthesisofTranskarbam12(2d)anditsisomers2a–cand2e–h.Reagentsandconditions:(i)HCl,rt,1h;(ii)(1)SOCl2,rt,1h,(2)CH3(CH2)yOH,CHCl3,60°C,2h;(iii)(1)potassiumphthalimide,DMF,40°C,4h,(2)N2H4.H2O,EtOH,reflux,4h,(3)HCl(g),Et2O,30min;(iv)(1)Et3N,H2O/Et2O,15min,(2)CO2,rt,15min.head2a–h(Scheme1).Moreover,theactivitiesoftheseammo-niumcarbamatesaltswerecomparedtothoseoftherespectiveammoniumchlorides1a–htoconfirmthesuggestionthatthecar-bamatepolarheadwasessentialfortheactionofTranskarbam12.Thetargetammoniumcarbamates2a–hwerepreparedfromtherespectiveaminoacidsorbromoacid(Scheme1).Theaminoacidswereprotonizedandconvertedtotheiracylchlorides,whichimmediatelyreactedwiththecorrespondingalcoholstoformtheesters1a–fand1h.13,1910-Aminodecanoicacidesterwaspreparedfromabromoacidviaacylchloride.Thebromoesterwasthensub-jectedtotheGabrielsynthesiswithsubsequenthydrazinolysis20toyieldtheaminoester,whichwasstoredandcharacterizedasitshydrochloride1g.Theammoniumchlorides1a–hwerealkalizedandreactedwithcarbondioxidetoyieldtheammoniumcarba-mates2a–h.13,19TheeffectsofthepreparedenhancersonthepermeationofamodeldrugtheophyllinethroughporcineearskinwereevaluatedinvitrousingFranzdiffusioncells.Thedonorsamplescontained5%(w/v)theophyllinewith/without1%(w/v)enhancerinpropyleneglycol/water6:4(v/v).Atthisconcentration,maximumthermody-namicactivityofthedrugwasmaintainedthroughouttheFigure2.Effectsofammoniumchlorides1a–handthecorrespondingammoniumcarbamates2a–hontheinvitrotransdermalfluxoftheophylline.Mean±SEM,n=5–20.*Indicatesstatisticallysignificantdifferenceagainstcontrolsample(i.e.,withoutenhancer),+indicatesstatisticallysignificantdifferenceagainstthecorre-spondingammoniumchloride.experimenteitherwithorwithouttheenhancers.Forfurtherexperimentaldetails,seeourpreviousreports.21,22Thetransdermalfluxvaluesoftheophyllineeitherwithout(controlsample)orwiththerespectiveenhancersareshowninFigure2.Ammoniumcarbamatesversusammoniumchlorides.Thefirstaimofthisstudywastocomparetheactivitiesoftheammoniumchlo-rides1andtranskarbams2,thatis,thecorrespondingtwo-chainammoniumcarbamates.Similarexperimentwasalreadyperformed;however,itwasacomparisonofTranskarbam12(2d)withafreebaseof1d,nottheammoniumsalt.Inthatstudy,thefreebaseshowednoactivity.9However,whenstudyingtheef-fectsof2donadefovirpermeation,themaximumactivitywasfoundatpH4.10AtsuchpHvalue,only1dbutnotthecarbamate2dcouldbepresentinthedonorsample.Thus,weaimedatrevis-itingtheactivityof1dbyperformingadirectcomparisonwith2d.Figure2showsthatthecarbamates2weremoreactiveenhanc-ersthantheammoniumchlorides1.Nevertheless,compounds1d–fwerenotinactiveasassumedbefore.Becauseallthedonorsam-plescontainingcompounds1werepreparedundernitrogenandthedonorcompartmentoftheFranzcellwasfilledwithnitrogen,thereactionwithcarbondioxideandpartialformationofthecar-bamates2inthedonorsamplesof1wasexcluded.Thus,theammoniumsaltformedupondecompositionofthecarbamateisanactiveenhancerspecies.Thepreviouslackofactivityofthefreebaseof1dmaybeexplainedbyanintramolecularaminolysisoftheesterbytheprimaryaminogroupyieldingcaprolactamanddodecanol.Toexcludethepossibilitythatthehigheractivitiesofthecarba-mates2comparedto1werecausedonlybyadifferentpHofthedonorsamplesandthatitmaybedrug-specific,anotherexperi-mentwasperformed.Twodrugs,theophyllineandhydrocortisone,wereused,andthedonorsamplescontaining1dor2dwerecare-fullyadjustedatpH7.0.TheeffectsonthefluxofthesetwodrugsareshowninFigure3.Theseresultsconfirmedthattheammoniumchlorides1pos-sessenhancingactivity,buttheydonotreachthatofthecarba-mates2.Thus,apartofthepermeation-enhancingpotencyofcarbamates2canbeattributedtotheammoniumcarbamatepolarheadandaparttothesimpleammoniumesterthatisprobablyreleasedfromthecarbamateinthestratumcorneum.Itshouldbenotedthatalthoughtheammoniumcarbamatesaredecomposedinanacidicenvironmentandatelevatedtempera-Figure3.Effectsoftheammoniumchloride1dandthecorrespondingammoniumcarbamate2dontheinvitrotransdermalfluxoftheophylline(A)andhydrocor-tisone(B)atpH7.0.Mean±SEM,n=5–14.*Indicatesstatisticallysignificantdifferenceagainstcontrolsample(i.e.,withoutenhancer),+indicatesstatisticallysignificantdifferenceagainst1d.2728´etal./Bioorg.Med.Chem.Lett.20(2010)2726–2728M.NovotnyFigure4.Effectsoftheammoniumchloride1dandthecorrespondingammoniumcarbamate2dontheinvitropermeationprofilesoftheophylline(A)andhydrocortisone(B)atpH7.0.tures,theyarerelativelystableatneutralorslightlyalkalinepHor,moreprecisely,equilibriumbetweencarbamate,bicarbonateandcarbonateisformedinaqueoussolutions.17Thus,theammoniumcarbamateconcentrationinthedonorsampledidnotsignificantlychangeovertimeasreflectedbystablefluxvalues(andtheratiosoffluxvaluesofthecarbamatesandammoniumsalts)duringtheassay.Toillustratethis,Figure4showsthepermeationprofilesoftheophylline(Fig.4A)andhydrocortisone(Fig.4B)inthepres-enceof1dand2d.Esterposition.Fig.2showsaroughlybell-shapedrelationshipbetweentheesterpositionandtheenhancingactivityofbothser-iesofthestudiedenhancers.Withineachseries,thecompoundsarepositionalisomers;thatis,theyhavethesamemolecularfor-mulabutdifferinthepositionoftheestergroup.Compounds1a–cand2a–bhavingtheestergroupclosesttothenitrogenandlongesthydrophobicchainwereinactive.Whentheestergroup‘moved’towardstheendofthechain,theenhancingactivityin-creaseduptocompoundse–fwith6-to7-carbonlinkeranddecyltoundecylchainanddecreasedthereafter.Thereasonforsuchmarkeddifferencesbetweentheseisomericcompoundsisunknownatpresent.Themostlikelyexplanationisthatthemostpotentisomers1d–fand2d–fpossess10–12carbonchains,whichistheidealchainlengthfoundinmanyamphiphilicpermeationenhancers(forareview,seeRefs.8,23).Suchamphi-philicenhancerscaninsertintothestratumcorneumlipidmem-braneswiththeirpolarheadandhydrophobictailintothepolarandhydrophobicregions,respectively.Theshorterenhancerchains(approximatelyhalfoftheacylchainlengthofceramidesandfattyacidspresentinthestratumcorneum)thendisturbtherigidlipidpackingandincreasetheskinpermeabilityfordrugs.24Thiswouldimplythattheestergroupisactuallylocatedwithinthepolarregionofthestratumcorneumlipidmembranesaswellastheammoniumsalt.Thelinkerchainbetweenthesetwogroupsmaybeeitherpositionedwithinthepolarheadregionorformaloopprotrudingbetweenthelipidchainspreventingtheirtightpacking.Thiswouldrequirecertainflexibilityofthelinker,whichisinagoodagreementwithourpreviousworkshowingconsider-ablyloweractivityofthetranexamicacidderivativeshavinglessflexiblecyclohexan-1,4-diyllinker.14Thishypothesisthatboththepolargroupsarepositionedwith-inthepolarlayerofthelipidmembranesandprobablyclosetoeachotheragreeswellthepreviouslysuggestedformationofanintramolecularhydrogenbondbetweentheestercarbonylandN–Hoftranskarbams.15,16Suchinteractionwouldleadtoa‘cyclic’polarheadsimilartoAzone,whichisawelldescribedpermeationenhancerhavingadodecylchainattachedtoaseven-memberedazepan-2-onering.25,26Anotherpossibleexplanationoftheseresults,thatis,thattrans-karbamsareonlypro-enhancersreleasingtherespectivefattyalco-hols,wasexcludedpreviously.10,13Inconclusion,transkarbams,whicharehighlypotentammo-niumcarbamatetransdermalpermeationenhancers,probablyactbyadualmechanism.First,apartoftheiractivityisassociatedwiththecarbamicacidsaltand/orwithitsdecompositionintheacidicstratumcorneum.Consequently,theammoniumesterstherebyreleasedpossesstransdermalpermeation-enhancingactivityaswell.Furtherinvestigationonthemechanismofactionoftheseunusualenhancersisinprogress.AcknowledgementsThisworkwassupportedbytheMinistryofEducationoftheCzechRepublic(CentreforNewAntiviralsandAntineoplastics1M0508andaResearchProjectMSM0021620822).Referencesandnotes1.Prausnitz,M.R.;Langer,R.Nat.Biotechnol.2008,26,1261.2.Williams,A.C.;Barry,B.W.Adv.DrugDeliv.Rev.2004,56,603.3.Barry,B.W.Eur.J.Pharm.Sci.2001,14,101.4.Benson,H.A.Curr.DrugDeliv.2005,2,23.5.Kaushik,D.;Batheja,P.;Kilfoyle,B.;Rai,V.;Michniak-Kohn,B.ExpertOpin.DrugDeliv.2008,5,517.6.Thong,H.Y.;Zhai,H.;Maibach,H.I.SkinPharmacol.Physiol.2007,20,272.7.Trommer,H.;Neubert,R.H.SkinPharmacol.Physiol.2006,19,106.8.Vavrova,K.;Zbytovska,J.;Hrabalek,A.Curr.Med.Chem.2005,12,2273.9.Hrabalek,A.;Dolezal,P.;Vavrova,K.;Zbytovska,J.;Holas,T.;Klimentova,J.;Novotny,J.Pharm.Res.2006,23,912.10.Vavrova,K.;Lorencova,K.;Klimentova,J.;Novotny,J.;Holy,A.N.;Hrabalek,A.Eur.J.Pharm.Biopharm.2008,69,597.11.Hrabalek,A.;Dolezal,P.;Roman,M.;Machacek,M.;Sklubalova,Z.Pharmazie1994,49,325.12.Hrabalek,A.;Vavrova,K.;Dolezal,P.;Machacek,M.J.Pharm.Sci.2005,94,1494.13.Klimentova,J.;Kosak,P.;Vavrova,K.;Holas,T.;Novotny,J.;Hrabalek,A.Bioorg.Med.Chem.Lett.2008,18,1712.14.Vavrova,K.;Hrabalek,A.;Dolezal,P.;Holas,T.;Klimentova,J.J.Control.Release2005,104,41.15.Holas,T.;Vavrova,K.;Klimentova,J.;Hrabalek,A.Bioorg.Med.Chem.2006,14,2896.16.Holas,T.;Vavrova,K.;Sima,M.;Klimentova,J.;Hrabalek,A.Bioorg.Med.Chem.2006,14,7671.17.Dell’Amico,D.B.;Calderazzo,F.;Labella,L.;Marchetti,F.;Pampaloni,G.Chem.Rev.2003,103,3857.18.Klimentova,J.;Hrabalek,A.;Vavrova,K.;Holas,T.;Kroutil,A.Bioorg.Med.Chem.Lett.2006,16,1981.19.Dolezal,P.;Hrabalek,A.;Semecky,V.Pharm.Res.1993,10,1015.20.Sheehan,J.C.;Bolhofer,W.A.J.Am.Chem.Soc.1950,72,2786.21.Novotny,J.;Kovarikova,P.;Novotny,M.;Janusova,B.;Hrabalek,A.;Vavrova,K.Pharm.Res.2009,26,811.22.Novotny,M.;Hrabalek,A.;Janusova,B.;Novotny,J.;Vavrova,K.Bioorg.Med.Chem.Lett.2009,19,344.23.Kanikkannan,N.;Kandimalla,K.;Lamba,S.S.;Singh,M.Curr.Med.Chem.2000,7,593.24.Suhonen,T.M.;Bouwstra,J.A.;Urtti,A.J.Control.Release1999,59,149.25.Stoughton,R.B.Arch.Dermatol.1982,118,474.26.Sugibayashi,K.;Nakayama,S.;Seki,T.;Hosoya,K.;Morimoto,Y.J.Pharm.Sci.1992,81,58.
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