molecules Article Enhanced Antibacterial Activity of Ent-Labdane Derivatives of Salvic Acid (7α-Hydroxy-8(17)- ent-Labden-15-Oic Acid): Effect of Lipophilicity and the Hydrogen Bonding Role in Bacterial Membrane Interaction JavierEcheverría1,* ,AlejandroUrzúa1,LoretoSanhueza2andMarcelaWilkens1 1 FacultaddeQuímicayBiología,UniversidaddeSantiagodeChile,Casilla40,Correo33, Santiago9170022,Chile;[email protected](A.U.);[email protected](M.W.) 2 NúcleodeQuímicayBioquímica,FacultaddeCiencias,UniversidadMayor,Santiago8580745,Chile; [email protected] * Correspondence:[email protected];Tel.:+56-2-2718-1154 Received:12May2017;Accepted:19June2017;Published:23June2017 Abstract: Inthepresentstudy,theantibacterialactivityofseveralent-labdanederivativesofsalvic acid (7α-hydroxy-8(17)-ent-labden-15-oic acid) was evaluated invitro against the Gram-negative bacteriumEscherichiacoliandtheGram-positivebacteriaStaphylococcusaureusandBacilluscereus. For all of the compounds, the antibacterial activity was expressed as the minimum inhibitory concentration(MIC)inliquidmediaandminimuminhibitoryamount(MIA)insolidmedia.Structure activity relationships (SAR) were employed to correlate the effect of the calculated lipophilicity parameters (logP ) on the inhibitory activity. Employing a phospholipidic bilayer (POPG) as a ow bacterialmembranemodel,ent-labdane-membraneinteractionsweresimulatedutilizingdocking studies. Theresultsindicatethat(i)thepresenceofacarboxylicacidintheC-15position,whichacted as a hydrogen-bond donor (HBD), was essential for the antibacterial activity of the ent-labdanes; (ii) an increase in the length of the acylated chain at the C-7 position improved the antibacterial activityuntilanoptimumlengthoffivecarbonatomswasreached; (iii)anincreaseinthelength of the acylated chain by more than five carbon atoms resulted in a dramatic decrease in activity, whichcompletelydisappearedinacylchainsofmorethanninecarbonatoms;and(iv)thestructural factorsdescribedabove,includingoneHBDatC-15andahexanoyloximoietyatC-7,hadagoodfit toaspecificlipophilicrangeandantibacterialactivity. Thelipophilicityparameterhasapredictive characteristicfeatureontheantibacterialactivityofthisclassofcompounds,tobeconsideredinthe designofnewbiologicallyactivemolecules. Keywords: salvicacid; antibacteriallabdane-typediterpene; lipophilicacyl-labdanesderivatives; membranedisruption 1. Introduction Manytherapeuticsmallmoleculesusedtodayasdrugshavetheiroriginsinnaturalproducts (secondary metabolites), providing or inspiring the development of between 50% and 70% of all chemotherapeuticagents[1]. Antibioticsareoneclearexampleandnowthereisparticularconcern over the ability of natural product investigations to yield new classes of antibacterial agents [2,3]. Althoughallearlyantibioticswerederivedfromnaturalsources,therehavebeennonewclinically approved,product-basedantibioticsdiscoveredforover30years[4,5]. Withinsecondarymetabolitesofaterpenicnature,thelabdanenucleusisausefulstructurefor themolecularexplorationanddevelopmentofnewpharmaceuticalcompounds. Thesynthesisof Molecules2017,22,1039;doi:10.3390/molecules22071039 www.mdpi.com/journal/molecules Molecules2017,22,1039 2of19 Molecules 2017, 22, 1039 2 of 19 labdanederivativeshasreceivedsignificantattentionbecauselabdanehasproventobeimportant, labdane derivatives has received significant attention because labdane has proven to be important, astheypossesspharmaceuticalproperties,includingantibacterial(againstdifferentstrainsofboth as they possess pharmaceutical properties, including antibacterial (against different strains of both Gram-positiveandGram-negativebacteria)andantifungalactivity,amongotherbiologicaleffects[6–8]. Gram-positive and Gram-negative bacteria) and antifungal activity, among other biological effects [6–8]. Thesuccessofthisgroupofmoleculeshasstimulatedthesearchfornewbiologicallyactivederivatives The success of this group of molecules has stimulated the search for new biologically active derivatives andresearchontheroleandinfluenceofchemicalstructureinbiologicalactivity. Progressintheuse and research on the role and influence of chemical structure in biological activity. Progress in the use ofstructure-activityrelationship(SAR)methodshasshowntheimportanceofthehydrophobicityor of structure-activity relationship (SAR) methods has shown the importance of the hydrophobicity or lipophilicityofbiologicallyactivemolecules. Lipophilicitymodifiestheabilityofbioactivemolecules lipophilicity of biologically active molecules. Lipophilicity modifies the ability of bioactive molecules topenetratethroughapolarpartsofcellmembranes. Thispropertyisusuallycharacterizedbythe to penetrate through apolar parts of cell membranes. This property is usually characterized by the octanol-waterpartitioncoefficient(logP ),whichisessentiallydeterminedfromdistributionstudies ow octanol-water partition coefficient (logPow), which is essentially determined from distribution studies ofacompoundbetweenanimmisciblepolarandnon-polarsolventpair. Thisquantitativedescriptor of a compound between an immiscible polar and non-polar solvent pair. This quantitative descriptor oflipophilicityisoneofthekeydeterminantsofpharmacokineticproperties[9]. Iftheexactvaluesfor of lipophilicity is one of the key determinants of pharmacokinetic properties [9]. If the exact values thisparameterareknown,theinhibitoryactivityofarelatedgroupofcompoundscanbepredicted. for this parameter are known, the inhibitory activity of a related group of compounds can be In this context, the partition coefficient (logP), which is an index of lipophilicity, is an important predicted. In this context, the partition coefficient (logP), which is an index of lipophilicity, is an physicochemicalparameterinthedevelopmentofantibacterialagents,becauseitiscloselyrelatedto important physicochemical parameter in the development of antibacterial agents, because it is closely thepermeationofthesecompoundsthroughthestructureofthebacteriallipidlayer. related to the permeation of these compounds through the structure of the bacterial lipid layer. Dockingstudiesprovidevaluableinformationontheinteractionofactivecompoundswiththeir Docking studies provide valuable information on the interaction of active compounds with their biologicaltargets[10,11]. Recently,dockingstudiessuggestedthatditerpenespromotebacteriallysis biological targets [10,11]. Recently, docking studies suggested that diterpenes promote bacterial lysis duetotheirinsertionintothelipophiliccellmembraneanditsconsequentdisruption[12]. According due to their insertion into the lipophilic cell membrane and its consequent disruption [12]. According totheseauthors,thestructuralfeaturesrelatedwiththeefficientantibacterialeffectsofthesenatural to these authors, the structural features related with the efficient antibacterial effects of these natural compoundsincludealipophilicdecalinringsystem,whichenablesinsertionintoalipophilicregion, compounds include a lipophilic decalin ring system, which enables insertion into a lipophilic region, andastrategicallypositionedhydrogen-bonddonorgroup(HBD;hydrophilicgroup),whichpromotes and a strategically positioned hydrogen-bond donor group (HBD; hydrophilic group), which interactionswithmembranephosphorylatedgroups. Moreover,inthisstudy,asecondHBDinthe promotes interactions with membrane phosphorylated groups. Moreover, in this study, a second decalinringsystemledtoareductionorsuppressionofactivity. HBD in the decalin ring system led to a reduction or suppression of activity. Inaddition,andfollowingourprograminstudyoftheSARofantibacterialsecondarymetabolites In addition, and following our program in study of the SAR of antibacterial secondary metabolites of Chilean flora [13,14], lipophilic 7-O-esters with linear, branched, and unsaturated (C1 to C12) of Chilean flora [13,14], lipophilic 7-O-esters with linear, branched, and unsaturated (C1 to C12) chains,7-alkoxy,15-esterlinearchainderivatives,15-methylester,and7,15-diolderivativesofsalvic chains, 7-alkoxy,15-ester linear chain derivatives, 15-methylester, and 7,15-diol derivatives of salvic acid1(Figure1)weresynthesized;wetestedtheantimicrobialactivityofthesecompoundsagainst acid 1 (Figure 1) were synthesized; we tested the antimicrobial activity of these compounds against twoGram-positive(StaphylococcusaureusandBacilluscereus)andoneGram-negative(Escherichiacoli) two Gram-positive (Staphylococcus aureus and Bacillus cereus) and one Gram-negative (Escherichia coli) bacteriatosystematicallyevaluatetheroleoftheHBDandtheeffectoflipophilicityonantibacterial bacteria to systematically evaluate the role of the HBD and the effect of lipophilicity on antibacterial activity. Additionally,weexaminedtheinteractionof7-O-acylsalvicacidderivativeswiththelipid activity. Additionally, we examined the interaction of 7-O-acyl salvic acid derivatives with the lipid bilayer (POPG), as an artificial model bacterial membrane. The model allowed us to correlate the bilayer (POPG), as an artificial model bacterial membrane. The model allowed us to correlate the antibacterialactivityofthe7-O-acylderivativeswiththeirlogP . ow antibacterial activity of the 7-O-acyl derivatives with their logPow. Figure 1. Structure of salvic acid. Figure1.Structureofsalvicacid. Molecules2017,22,1039 3of19 Molecules 2017, 22, 1039 3 of 19 22.. RReessuullttss aanndd DDiissccuussssiioonn TThhee hheemmiissyynntthheessiiss ooff ssaallvviicc aacciidd ddeerriivvaattiivveess ((SScchheemmee 11)) iinnvvoollvveedd rreedduuccttiioonn,, mmeetthhyyllaattiioonn,, aaccyyllaattiioonn,, aanndd aallkkooxxyyllaattiioonn--eesstteerriifificcaattiioonn rreeaaccttiioonnss.. TThheessee rreeaaccttiioonnss wweerree ppeerrffoorrmmeedd ttoo iinnccrreeaassee tthhee lliippoopphhiilliicciittyy aanndd ttoo aasssseessss tthhee ssiiggnniiffiiccaannccee ooff hhyyddrrooggeenn--bboonndd ddoonnoorr ((HHBBDD)) ggrroouuppss iinn aannttiibbaacctteerriiaall aaccttiivviittyy.. FFoorr tthhiiss ppuurrppoossee,, aa sseett ooff ssaallvviicc aacciidd ddeerriivvaattiivveess wwaass oobbttaaiinneedd,, iinncclluuddiinngg 77,,1155--ddiiooll 22 aanndd 1155--mmeetthhyylleesstteerr 33 aannaallooggss,, ffoouurrtteeeenn 77--OO--aaccyyll ddeerriivvaattiivveess 44––1177,, aanndd ffoouurr 77--aallkkooxxyy,,1155--eesstteerr ddeerriivvaattiivveess 1188––2211.. TThhee aabbssoolluuttee ccoonnffiigguurraattiioonn ooff ssaallvviicc aacciidd,, wwhhiicchh hhaass bbeeeenn rreecceennttllyy aassssiiggnneedd,, aalloonngg wwiitthh iinnffoorrmmaattiioonn oobbttaaiinneedd vviiaa XX--rraayy ddiiffffrraaccttiioonn aanndd vviibbrraattiioonnaall cciirrccuullaarr ddiicchhrrooiissmm aannaallyyssiiss [[1155]],, wwaass uusseedd ttoo vveerriiffyy tthhee sstteerreeoocchheemmiissttrryy ooff tthhee hheemmiissyynntthheettiicc ddeerriivvaattiivveess.. Scheme 1. Structure of the hemisynthetic derivatives of salvic acid. Scheme1.Structureofthehemisyntheticderivativesofsalvicacid. 2.1. Antibacterial Activity of the Salvic Acid Derivatives 2.1. AntibacterialActivityoftheSalvicAcidDerivatives The obtained MIC and MIA values are shown in Table 1. The MIC values in liquid media were not TheobtainedMICandMIAvaluesareshowninTable1. TheMICvaluesinliquidmediawere significantly different compared to the MIA values in solid media, although the latter showed greater notsignificantlydifferentcomparedtotheMIAvaluesinsolidmedia,althoughthelattershowed differentiation in detailed SAR analysis. greaterdifferentiationindetailedSARanalysis. Table 1. Minimum inhibitory concentration (MIC), minimum inhibitory amount (MIA), and lipophilicity Table1.Minimuminhibitoryconcentration(MIC),minimuminhibitoryamount(MIA),andlipophilicity of salvic acid and its derivatives. ofsalvicacidanditsderivatives. Compound S. aureus B. cereus E. coli logP 3 SCaolmvipco aucnidd (1) 5S0..0a0u r1e us 20.00 2 50B.0.c0e r1 eus 22.50 2 >100.0E0.c 1o li>10.00 2 lo4g.P903 Salvicac2id (1) 50.>010010.00 12 0.0>0225.00 2 50.>001010.00 1 22.>52052.00 2 >1>0100.000.010 1 >>1100.0.0002 2 45.9.008 23 >100.2050.100 1 >25.030.200 2 >100.5000.100 1 >25.020.020 2 >1>0100.000.010 1 >>1100.0.0002 2 55.0.283 3 25.001 3.002 50.001 2.002 >100.001 >10.002 5.23 4 50.00 1 4.50 2 25.00 1 4.00 2 >100.00 1 >10.00 2 5.47 4 50.001 4.502 25.001 4.002 >100.001 >10.002 5.47 55 25.02051.00 1 2.5022.50 2 12.51021.50 1 1.5012.50 2 >1>0100.000.010 1 >>1100.0.0002 2 55.9.944 66 6.2561.25 1 1.5012.50 2 3.1331.13 1 1.2512.25 2 >1>0100.000.010 1 >>1100.0.0002 2 66.3.300 77 6.2561.25 1 1.2512.25 2 6.2561.25 1 1.0012.00 2 >1>0100.000.010 1 >>1100.0.0002 2 66.5.522 8 3.131 0.752 3.131 0.502 >100.001 >10.002 6.84 8 3.13 1 0.75 2 3.13 1 0.50 2 >100.00 1 >10.00 2 6.84 9 3.131 0.502 3.131 0.302 >100.001 >10.002 6.74 109 3.1331.13 1 0.7502.50 2 3.1331.13 1 0.4002.30 2 >1>0100.000.010 1 >>1100.0.0002 2 66.9.724 1110 3.1331.13 1 0.3502.75 2 3.1331.13 1 0.2502.40 2 >1>0100.000.010 1 >>1100.0.0002 2 76.3.982 1211 3.1331.13 1 1.0002.35 2 3.1331.13 1 0.7502.25 2 >1>0100.000.010 1 >>1100.0.0002 2 77.5.358 13 6.251 1.252 6.251 1.252 >100.001 >10.002 7.13 12 3.13 1 1.00 2 3.13 1 0.75 2 >100.00 1 >10.00 2 7.55 14 3.131 0.402 3.131 0.452 >100.001 >10.002 8.47 1513 12.506.125 1 3.0012.25 2 6.2561.25 1 2.5012.25 2 >1>0100.000.010 1 >>1100.0.0002 2 97.0.113 1614 >100.030.113 1 >10.000.240 2 25.0031.13 1 >10.000.425 2 >1>0100.000.010 1 >>1100.0.0002 2 98.5.457 1715 >100.1020.150 1 >10.030.200 2 >100.060.215 1 >10.020.520 2 >1>0100.000.010 1 >>1100.0.0002 2 190..6031 16 >100.00 1 >10.00 2 25.00 1 >10.00 2 >100.00 1 >10.00 2 9.55 17 >100.00 1 >10.00 2 >100.00 1 >10.00 2 >100.00 1 >10.00 2 10.63 Molecules2017,22,1039 4of19 Table1.Cont. Compound S.aureus B.cereus E.coli logP3 18 >100.001 >10.002 >100.001 >10.002 >100.001 >10.002 5.76 19 >100.001 >10.002 >100.001 >10.002 >100.001 >10.002 6.50 20 >100.001 >10.002 >100.001 >10.002 >100.001 >10.002 7.55 21 >100.001 >10.002 >100.001 >10.002 >100.001 >10.002 8.26 Penicillin 0.031 1.232 2.501 5.002 n/t n/t - Ciprofloxacin 0.251 2.502 0.131 2.502 n/t n/t - Kanamycin 1.001 2.502 1.001 5.002 n/t n/t - Tetracycline 1.001 1.232 5.001 5.002 n/t n/t - Chloramphenicol 2.001 1.502 0.251 0.252 n/t n/t - Methanol i1 i2 i1 i2 i1 i2 - 1MICinliquidmedia(µg/mL);2MIAinsolidmedia(µg);3XlogP3,n/t:nottestedi:inactive. Theresultsshowedaselectiveinhibitoryeffectthatent-labdanetypediterpenesdisplayingeneral onthegrowthofGram-positivebacteria.NoneoftestedcompoundswereactiveagainstGram-negative bacteriaE.coli(MICupto10µg/mLandMIAupto10µg). Compounds8–12and14inliquidmediaand5insolidmedia,showedthestrongestantibacterial activityagainstS.aureus. Compounds6,8–11,and12inliquidmediaand11insolidmediawerethe mostactiveagainstB.cereus. TheresultsrevealedthatcompoundswithoneHBDgroup,suchasmethylester3andsomeacyl derivatives,suchas7-O-hexanoylsalvicacid11,werethemostactivesones. MICandMIAvaluesof severalacylderivativesofsalvicacidweresimilartothoseofreferenceantibiotics,suggestingfurther studiesforpossibleclinicaluse. 2.2. LipophilicityandStructure-ActivityRelationshipofAntibacterialofSalvicAcidandItsDerivatives Lipophilicity,whichiswellcorrelatedwiththebioactivityofachemicalcompound,isanimportant molecular descriptor, and the lipophilic behavior of a compound plays a significant role in their mechanismofbiologicalactivity.Allofthecompoundssynthesizedhereinshowedgreaterlipophilicity valuesthansalvicacid1. SARanalysisofacylderivatives,particularlybetweenpairs6–7and8–9, suggestedthattheramificationofthehydrocarbonchainoftheacylgroupofC-7causedasignificant decrease in the MIC values in both Gram-positive bacteria. The observed decrease in MIC values wascloselyrelatedtoanincreaseinlipophilicityvalues. Incontrasttotheresultsdescribedabove, SARanalysisofacylatedderivatives9,10,12,and13showedthatthepresenceofinsaturationsin thehydrocarbonchainoftheacylgroupatC-7causedanincreaseinMICvalues,whichinturnwas relatedtotheobservedreductioninlipophilicityvalues. Thelipophilicityvalues(logP )ofhemisyntheticderivatives(Table1)ofcompoundswithacyl ow groups,includingcompounds4–17,exhibitedagradualincreaseasthechainlengthoftheacylgroup increased;thesederivativesshowedgreaterlipophilicitycomparedtosalvicacid1. Theantibacterial activityofthisseriesincreasedthroughouttheseriesuptoamaximum,correspondingtocompound11, thendecreasedsharplytocompound15,disappearingcompletelyinacylderivativeswithgreater chain lengths, such as compounds 16–17. These results show that the linear relationship between lipophilicityandactivitydidnotcontinueadinfinitum. Therefore,acompleteregressionanalysiswas applied,includinglinear,quadratic,andcubicrelationships. Thedatashowthatthefittingequations improvedwhenhigher-order(second-orthird-order)polynomialswereemployed[16]. Careful observation of the results depicted in Table 1 shows that the reduction of the CO H 2 group of 1 to yield diol 2 increased the lipophilicity, but resulted in a loss of antibacterial activity (MICupto10µg/mLandMIAupto10µg),suggestingthatthepresenceofacarboxylicacidgroup strategicallylocatedatC-15isimportantfortheantimicrobialactivityofthelabdanes. Esterificationof 1toyieldmethylester3increasedboththelipophilicityandantibacterialactivity. Theseresultsshow Molecules2017,22,1039 5of19 thatthepresenceofonlyoneHBDsignificantlyimprovedtheantimicrobialeffectofthelabdane-type diterpenescomparedtothelabdaneswithtwoHBDgroups(salvicacid1anddiol2). TheimportanceofonlyoneHBDwasprovenintheseriesof7-alkoxy,15-esterderivatives,inwhich blockingthetwoHBDgroupsofsalvicacid1wasperformedthroughalkoxylationandesterification reactions to yield compounds 18–21; this synthetic modification caused an absolute inhibition of antibacterialactivity,despitethelargeincreaseinlogP values. However,thepresenceoftheanchor ow HBDgroupallowedthesecompoundstointeractwiththesurfaceofthephospholipidbilayer,and because the lipophilicity of the decalin moiety is enhanced by the presence of the C-7 acyl group, the penetration of microorganisms through the lipid layer or disruption of the components of the bacterial membrane easily occurs [17]. These results were in agreement with our recent research, inwhichwedemonstratedthatalipophilicdecalinringsystemwithastrategicallypositionedHBD (hydrophilicgroup)isimportantfortheantimicrobialactivityofditerpenes. Moreover,inthesame study,asecondHBDintroducedinthedecalinringsystemledtoareductioninorsuppressionofthe activity. Wearguedthattherewerebasicallytwomechanismsforthereducedantibacterialactivityof diterpenoidscontainingtwoHBDgroups: (i)thepresenceoftwoHBDsdecreasesthelipophilicity ofthehydrophobicmoiety,hinderingitsinteractionwiththebacterialmembrane;(ii)intramolecular HBDgroupinteractionscompetewithintermolecularhydrogenbondsbetweeneachHBDandthe cell membrane [12]. This model has been successfully applied to other labdanes [18,19] and other typesofditerpenes, includingpimaranesandabietanes[20–25]andpseudopteranediterpene[26]. Furthermembrane-diterpeneinteractionstudieshavebeenperformed,specificallyinlabdanes,e.g., sclareol, labd-7,13-dien-15-ol, and labd-13-ene-8α,15-diol, [27,28], (8R,13S)-labdane-8,15-diol and (8R,13R)-labdane-8,15-diol[29],myriadenolide[30],aswellasantibacterialditerpenesthatinteract withbacterialmembranemodels,e.g.,(+)-totarol,[31,32]andabieticacid[33,34],demonstratingthat lipophilicityplaysanimportantroleinadditiontostructuralfactors. Also,amongnaturalpentacyclic triterpenes,manyreportshavebeenpublishedconcerningtheinteractionbetweenthelipidmembrane andlupanetriterpenoids[35–40].Thecommonfunctionalityinthesecompoundsisthehydroxylgroup atC3. Inaddition,ursolicacidhasanadditionalcarboxylicacidfunctionatC17. Literaturereports haveshownthattheacidmoietyatC17andtheesterificationofC3-OHareessentialforpentacyclic triterpeneswithenhancedpharmacologicalactivities. Basedontheseobservations,theC3-OHcanbe derivatizedwithlipophilicesterchains. 2.3. MolecularModeling: DockingStudiesofLabdane-MembraneInteractions Amoleculardockingstudywasperformedusingoptimizedstructuresofthelabdanederivatives andaphospholipidicbilayer(POPG)asabacterialmembranemodel. Thisstudywasperformedto corroborateourhypothesisandtoexaminethestructuralandfunctionalfactorsthatinfluencethemode ofactioninantibacterialditerpenes. TheobservedbonddistancesbetweentheHBDgroupsofthe diterpenesandtheHBA(hydrogenbondacceptors)atomsofthephospholipidsinPOPGmembranes, as shown in Figure 2, indicate that this type of interaction may be a significant contributor to the association,integration,andinteractionoflabdanesinthebacterialmembranemodel. Interactionsbetweensalvicacid1andthePOPGmodelmembrane,asshowninFigure2,account forthestrongbonding,whichwasstabilizedbytheformationofthreehydrogenbridgebonds.Thefirst interactionoccurredbetweenthehydrogenatomofthecarboxylicacidgroupandtheO13oxygen atomofthephosphatidylglycerolphosphategroups,withaC15(O)OH—O=Pbonddistanceof1.825Å. Inthesameway,thehydrogenatomofthehydroxylgroupformedahydrogenbondwiththeO13 atomofanotherphospholipid,withaC7-OH—O=Pbonddistanceof1.891Å.Finally,weobservedthat theoxygenatomofthehydroxylgroupofthecarboxylicacidactedasanacceptor,andthehydrogen bondsformedbytheH16hydrogenatomandglycerolhydroxylofanotherphospholipidexhibiteda C7-HO—HObonddistanceof1.758Å.Theconformationadoptedbysalvicacidonthesurfaceofthe bacterialmembranemodelwasprimarilylocatedinthepolarareaofthebilayerPOPG,withasmall portionofthedecalinskeletonintroducedintothehydrophobiczoneofthebilayer. Theseinteractions Molecules2017,22,1039 6of19 explain the low antibacterial activity of salvic acid 1, against both Gram-positive bacteria and the correlationwiththeirlipophilicityvalue. Molecules 2017, 22, 1039 6 of 19 Figure 2. (A) Cross-sectional view of the spatial conformation that adopts compound 1 by interacting with Figure2.(A)Cross-sectionalviewofthespatialconformationthatadoptscompound1byinteracting the phospholipid bilayer (POPG); (B,C) Interactions by the formation of hydrogen bonds between withthephospholipidbilayer(POPG);(B,C)Interactionsbytheformationofhydrogenbondsbetween compound 1 and POPG. compound1andPOPG. In the same way, the interpretation given to the interaction of salvic acid 1 with the POPG model meImntbhreansea mcaen wbea ya,ptphleieidn tteor tphree toatthieorn dgeirvivenatitvoetsh weiitnht etwraoc tHioBnDo gfrsoaulvpisc, ainccidlu1diwngit hditohl edPerOivPaGtivme o2d, el meams bsrhaonwenc ainn bFeigauprpe l3ie. dInto ththise coatshee, rtdhee rdiveartivivaetisvwe iitnhtetrwacoteHdB wDitghr otuhep sP,OinPcGlu dbiinlagyedri otlhdroeurigvha ttihvee 2, formation of hydrogen bonds with the two hydrophilic groups, C7-OH and C15-OH. Both groups as shown in Figure 3. In this case, the derivative interacted with the POPG bilayer through the were formed through an interaction with the oxygen atoms of the phosphate groups of the two formationofhydrogenbondswiththetwohydrophilicgroups,C7-OHandC15-OH.Bothgroups phospholipids, the first bond having a C7-OH---O=P distance of 1.849 Å and the second bond with a were formed through an interaction with the oxygen atoms of the phosphate groups of the two C15(O)-OH---O=P bond distance of 2.178 Å. phospholipids,thefirstbondhavingaC7-OH—O=Pdistanceof1.849Åandthesecondbondwitha In contrast, the methyl ester derivative 3 showed an increase in both lipophilicity and antibacterial C15(O)-OH—O=Pbonddistanceof2.178Å. activity, reflected in the conformation that it adopts when interacting with the phospholipid bilayer, Incontrast,themethylesterderivative3showedanincreaseinbothlipophilicityandantibacterial showing that the decalin is more introduced in the lipophilic zone of the membrane. activity,reflectedintheconformationthatitadoptswheninteractingwiththephospholipidbilayer, This finding suggests a greater effect on membrane disruption, due to the formation of a hydrogen showingthatthedecalinismoreintroducedinthelipophiliczoneofthemembrane. bond bridge between the C7-OH---O=P group, with a distance of 2.226 Å, favoring the anchoring of Thisfindingsuggestsagreatereffectonmembranedisruption,duetotheformationofahydrogen this compound to the phospholipid matrix (Figure 4). bondbridgebetweentheC7-OH—O=Pgroup,withadistanceof2.226Å,favoringtheanchoringof thiscompoundtothephospholipidmatrix(Figure4). Molecules2017,22,1039 7of19 Molecules 2017, 22, 1039 7 of 19 Molecules 2017, 22, 1039 7 of 19 Figure 3. (A) Cross-sectional view of the spatial conformation that compound 2 a dopts by interacting Figurwei3th. (tAhe) pChroosspsh-soelciptiiodn baillavyieerw POoPfGth; e(Bs,pCa) tIinatlecroacntfioonrm bya ttihoen fothrmatactioomn opfo huynddro2geand obpontsdsb ybeitnwteeeranc ting Figure 3. (A) Cross-sectional view of the spatial conformation that compound 2 adopts by interacting withtchoempphouonspdh 2o alnipdi dtheb inlaeayreerstP tOwPo Gph;o(Bsp,Cho)lIinptider uancittiso onf bthyet PhOePfoGr mbilaatyieorn. ofhydrogenbondsbetween with the phospholipid bilayer POPG; (B,C) Interaction by the formation of hydrogen bonds between compound2andthenearesttwophospholipidunitsofthePOPGbilayer. compound 2 and the nearest two phospholipid units of the POPG bilayer. Figure 4. (A) Cross-sectional view of the spatial conformation that compound 3 adopts by interacting with the phospholipid bilayer POPG; (B,C) Interaction by the formation of hydrogen bonds between Figure 4. (A) Cross-sectional view of the spatial conformation that compound 3 adopts by interacting the compound 3 and one POPG. Figurweit4h. t(hAe) pChroosspsh-osleicptiido nbialalyveire wPOoPfGth; (eBs,Cp)a Itniatelrcaocntifoonr mbya tthioe nfotrhmaattcioonm opf ohuyndrdog3eand boopntdssb byetiwnteeerna cting w ithtthhee cpomhopsopuhnodl i3p ainddb oilnaey PeOrPPGO.P G;(B,C)Interactionbytheformationofhydrogenbondsbetween thecompound3andonePOPG. Molecules2017,22,1039 8of19 In the conformations of the 7-O-acyl labdane series, a sequential increase was observed in Molecules 2017, 22, 1039 8 of 19 the penetration of the acyl chain into the POPG membrane, as shown in Figure 5. This degree ofpenetraInti othne wcoansfocromrraetliaontesd ofw tihteh 7t-hOe-amcyalx liambduamnea snetriibesa,c at esreiqauleenfftieacl tinocfr7e-aOse- hweaxsa onbosyerlvdeedr iinv atthiev e11, thempoesnteatrcatitvioenc oofm tphoe uancdyl acghaaiinns tinbtoot hthGe rPaOmP-Gp omsietimvebrbaancet, earsia s.hTohwense inco Fnifgourrme a5t.i oTnhsisw deergersetea boifl ized penetration was correlated with the maximum antibacterial effect of 7-O-hexanoyl derivative 11, the by the formation of two hydrogen bridge bonds, the first of which was stabilized by a donation most active compound against both Gram-positive bacteria. These conformations were stabilized by betweenC15(O)-OH—O=P,withadistanceof1.860Å,allowingthecompoundtoactasananchor the formation of two hydrogen bridge bonds, the first of which was stabilized by a donation between inthephospholipidmatrix. Thesecondoxygenatomofthecarbonylgroupoftheacylmoietyacted C15(O)-OH---O=P, with a distance of 1.860 Å, allowing the compound to act as an anchor in the asahydrogenbondacceptortooneofthehydroxylgroupsofthephospholipidglycerolphosphate, phospholipid matrix. The second oxygen atom of the carbonyl group of the acyl moiety acted as a withaC7–OCOR —HObonddistanceof1.869Å.Forexample,inderivative17,althoughahydrogen hydrogen bon2d acceptor to one of the hydroxyl groups of the phospholipid glycerolphosphate, with bondah Ca7d–OfoCrOmRe2d---iHnOC b1o5n-(dO d)OistHan—ceO o=f P1,.8t6h9e Åin. tFeorra cetxiaomnpwlea, sinv deeryrivwateivaek 1b7e, caaltuhsoeugthhe ab hoynddrodgiesnt ance was 3b.o2n2d3 hÅa.dS fourbmseedq uine nCt15in-(cOr)eOaHse-s--Oin=Pth, tehea ilnktyelracchtiaoinn woafst vheerya wcyelakg rboeucapusoev tehre bthoends idxisctaanrbceo wnaast oms hinde3r.e22d3 tÅh.e Spuebnseeqturaentito inncorefalsaebsd ina nthee ianlktyhle chmaienm obf trhaen aec,ylli kgerolyupd ouveerto theel esicxt rcoarsbtaotni cataonmds hhiynddreorepdh obic repulsthioen psebneettrwateieonn tohfe lahbyddarnoec ianr bthoen mcheaminbsraonfet,h liekaeclyy ldgureo tuop ealencdtrothsteatpico laanrdg hroyudprospohfotbhiec prehpouslpsihoonlsi pids; between the hydrocarbon chains of the acyl group and the polar groups of the phospholipids; in inadditiontostericeffectsassociatedwiththeincreasedvolumeandlengthofthechain. Asaresult, addition to steric effects associated with the increased volume and length of the chain. As a result, a a drastic decrease in activity was observed until the antibacterial effect was completely inhibited, drastic decrease in activity was observed until the antibacterial effect was completely inhibited, as asexemplifiedin7-O-lauriloxiderivative17,duetoitsinteractionwithPOPG(Figure4). exemplified in 7-O-lauriloxi derivative 17, due to its interaction with POPG (Figure 4). Figure 5. (A) Cross-sectional view of the spatial conformation that compound 11 adopts by interacting Figure5.(A)Cross-sectionalviewofthespatialconformationthatcompound11adoptsbyinteracting with the phospholipid bilayer POPG; (B,C) Interaction by the formation of hydrogen bonds between withthephospholipidbilayerPOPG;(B,C)Interactionbytheformationofhydrogenbondsbetween the compound 11 and one of the phospholipid bilayer POPGs. thecompound11andoneofthephospholipidbilayerPOPGs. Finally, we evaluated the conformations of the 7-alkoxy, 15-ester derivatives, which have significant structural characteristics, such as an absence of HBD groups, resulting in a low affinity for the POPG Finally,weevaluatedtheconformationsofthe7-alkoxy,15-esterderivatives,whichhavesignificant bilayer (Figure 6). Although we did not observe the formation of any hydrogen bridge bonds, the structuralcharacteristics,suchasanabsenceofHBDgroups,resultinginalowaffinityforthePOPG bilaye r(Figure6). Althoughwedidnotobservetheformationofanyhydrogenbridgebonds,the Molecules2017,22,1039 9of19 Molecules 2017, 22, 1039 9 of 19 hydrophobicfractionwaspositionedtowardtheinteriorofthebilayerbutwasnotfirmlybonded, hydrophobic fraction was positioned toward the interior of the bilayer but was not firmly bonded, indicatingthatthisconformationwastransient,whichexplainstheabsenceofactivity. indicating that this conformation was transient, which explains the absence of activity. Figure 6. Cross-sectional view of the spatial conformation which compound 19 adopts by interacting Figure6.Cross-sectionalviewofthespatialconformationwhichcompound19adoptsbyinteracting with the phospholipid bilayer POPG, where the formation of hydrogen bonds is not observed. withthephospholipidbilayerPOPG,wheretheformationofhydrogenbondsisnotobserved. The observations described above were based on experimental studies of labdane incorporation in theT hliepoosbosmerevsa otifo pnhsodsepshcartibideydlcahboolivnee wanerde dbeamseodnostnraetxe ptehraitm laebndtaalnsetsu pdoiessseosfsilnagb daa snienginlec oHrpBoDr awtieorne ibnetttheer liinpcoosropmoreastoefdp ihnotos plihpaotsidoymlcehso tlhinane alanbdddaenmeso wnsittrha ttewtoh aHtBlaDbsd [a2n8e,s41p]o. sTsheess pinrgesaensicneg olef tHwBoD HwBeDres breedttuecreidn ciotsr pinocroartepdorianttioonli pinotsoo mthees mtheamnblraabndea,n weshiwlei tthhetw acoeHtyBlaDtiso[n2 8o,f4 t1h].esTeh HepBrDe sgernocueposf rtewsuolHteBdD ins rinedcruecaesdedit isnicnocroproproartaiotino.n intothemembrane,whiletheacetylationoftheseHBDgroupsresultedin increFasoerd opinticmorapl ionrtaetriaocnti.on with amphipathic membranes, the presence of a polar substituent is required, prefeFraobrloyp at icmaarbloinxtyelrica catcioidn gwroituhpa, mwphhicihp aatchtisc ams eam HbBraDn, eisn, athsseopciraetsieonnc ewoitfh aa plioplaorphsuilbics tsiktueelenttonis, rperqimuiarreidly, plorceafeteradb ilny tahcea lrabbodxaynliec saicdied cghraoiunp. ,Twheh ipcrhesaecntscea soaf aH sBeDco,nind aHssBoDc iagtrioounpw init hthae lmipoolpehcuilliec srekdeluecteosn t,hper liimpoaprihlyililcoictya toefd thine hthyedrlaobpdhaonbeic smidoeiecthya, iwnh.iTchh ehipnrdeesersn icneteorfaactsioencos nwditHh BthDe hgyroduroppihnotbhiec mregoiloecnu olfe trheed buaccetsertihael mlipeompbhrialniceit. yInotefrtehsetihnygdlyr,o tphhe oobbiscermvoedie rtye,dwuchtiicohn hinin adcteirvsitiyn tdeurae cttoi othnes pwrietshenthcee hofy dar ohpyhdorobpichrieligci ognrooufpth ien baa cltaebrdiaalnme-etmypber adniete.rIpnetenree sitsi nngolyt ,ltihmeitoebds etrov ethder ecdoumcptioounnidnsa cptrievvitiyoudsulye tsotutdhieedp r[e1s2e]n. ceofahydrophilicgroupinalabdane-typediterpeneisnotlimitedtothecompounds previouslystudied[12]. 3. Materials and Methods 3. MaterialsandMethods 3.1. General 3.1. General NMR spectra were obtained on a Bruker DPX 400 spectrometer (400 MHz for 1H and 100 MHz NMRspectrawereobtainedonaBrukerDPX400spectrometer(400MHzfor1Hand100MHz for 13C). Samples were dissolved in CDCl3, and the spectra were calibrated using TMS signals. The for 13C). Samples were dissolved in CDCl , and the spectra were calibrated using TMS signals. chemical shifts are given in ppm. The carbo3n atoms in the alkyl chains of the acyloxy groups were Thechemicalshiftsaregiveninppm. Thecarbonatomsinthealkylchainsoftheacyloxygroups numbered by labelling the carbon atom of the carbonyl group as number one and subsequently werenumberedbylabellingthecarbonatomofthecarbonylgroupasnumberoneandsubsequently increasing towards the methyl terminus of the acyl chain. All 1H-NMR spectra were been added in increasingtowardsthemethylterminusoftheacylchain. All1H-NMRspectrawerebeenaddedin Supplementary Materials. SupplementaryMaterials. 3.2. Plant Material 3.2. PlantMaterial Aerial portions of resinous Eupatorium salvia Colla were collected from Cuesta Lo Prado (Región AerialportionsofresinousEupatoriumsalviaCollawerecollectedfromCuestaLoPrado(Región Metropolitana, Chile, 31°28′ S, 71°27′ W) at an altitude of 785 m above the average sea level during Metropolitana,Chile,31◦28(cid:48) S,71◦27(cid:48) W)atanaltitudeof785mabovetheaveragesealevelduring the flowering season in March of 2008. Voucher specimens were deposited in the Herbarium of the National Museum of Natural History, Santiago, Chile (SGO 108833). Molecules2017,22,1039 10of19 thefloweringseasoninMarchof2008. VoucherspecimensweredepositedintheHerbariumofthe NationalMuseumofNaturalHistory,Santiago,Chile(SGO108833). 3.3. ExtractionandIsolationofSalvicAcid(1) Aerial portions of E. salvia (1.8 kg) were extracted by dipping fresh plant material into 5 L of CH Cl at room temperature for 30 s. This procedure was repeated twice to ensure the total and 2 2 selective extraction of epicuticular components. The combined CH Cl extracts were evaporated, 2 2 and the residue (133 g) was fractionated by column chromatography on silica gel (CC) using pentane/CH Cl andCH Cl /CH OHstepgradients.ThefractionswereelutedwithCH Cl /CH OH 2 2 2 2 3 2 2 3 (99:1) and spontaneously crystallized to yield 40 g of salvic acid 1, which was identified by direct comparisonwithanauthenticsample[12,42]. 3.4. ReductionofSalvicAcid: Diol(2) Toasolutionof300mg(0.93mmol)ofsalvicacid1indryethylether,asuspensionoflithium aluminumhydride(200mg,5.3mmol)inanhydrousethylether(20mL)wascarefullyaddeddropwise underanitrogenatmosphere. Thereactionmixturewasrefluxedfor1handwashedsuccessivelywith asolutionof5%hydrochloricacidanddistilledwater. Theresultingorganicphasewasdriedover anhydroussodiumsulfate,andthesolventwasremovedonarotaryevaporatortoyield282.8mg (0.92mmol,99%)ofawhitecrystallinecompound. Thepartialspectraldataof2wereinagreement withpublishedresults[43];therefore,thefullNMRassignmentispresented. 7β,15-dihydroxy-ent-lab-8(17)-ene(2)(yield99%,282.8mg): 1H-NMR(CDCl ,400MHz)δ5.04(1H,t, 3 J=1.3Hz,H-17β),4.63(1H,t,J=1.6Hz,H-17α),4.38(1H,d,J=2.9Hz,H-7),3.68(1H,m,H-15α),3.66 (1H,m,H-15β),2.05(1H,m,H-9),1.78(1H,d,J=10.2Hz,H-1α),1.66(1H,dd,J=7.5;2,1Hz,H-14α), 1.59(1H,s,H-5),1.58(1H,s,H-11α),1.57(2H,d,J=3.0Hz,H-6),1.56(1H,d,J=3.0Hz,H-2α),1.54 (1H,m,H-13),1.50(1H,m,H-2β),1.44(1H,dd,J=7.5;3.8Hz),1.42(1H,m,H-3α),1.37(1H,m,H-14β), 1.26(1H,dd,J=9.0;3.8Hz,H-11β),1.24(1H,dd,J=12.8;5,2Hz,H-3β),1.09(1H,td,J=12.3;4.1Hz, H-1β),0.98(1H,dd,J=11.3;6.3Hz,H-12β),0.91(3H,d,J=6.5Hz,H-16),0.88(3H,s,H-18),0.80(3H, s,H-19),0.65(3H,s,H-20). 13C-NMR(CDCl ,101MHz)δ: 149.82(C=CH ,C-8),109.72(C=CH ,C-17), 3 2 2 74.16(CH,C-7),61.23(CH ,C-15),51.40(CH,C-9),47.65(CH,C-5),42.07(CH ,C-3),39.88(C,C-10), 2 2 39.59(CH ,C-14),38.78(CH ,C-1),35.91(CH ,C-12),33.30(CH ,C-18),33.09(C,C-4),30.88(CH , 2 2 2 3 2 C-6),30.20(CH,C-13),21.51(CH ,C-19),20.43(CH ,C-11),19.83(CH ,C-16),19.34(CH ,C-2),13.39 3 2 3 2 (CH ,C-20). 3 3.5. MethylationofSalvicAcid: Methylsalvate(3) Asolutionof200mg(0.62mmol)ofsalvicacid1indryethylether(15mL)wastreatedwith a solution of diazomethane in ethyl ether, which was obtained from the treatment of N-methyl- N-nitroso-p-toluene sulfonamide (Diazald) with a concentrated solution of potassium hydroxide. The aqueous phase was discarded, and the organic phase was successively dried with potassium hydroxidebeadsinanErlenmeyerflask. Thesolventwasremovedusingarotaryevaporator,yielding 208.7mg(0.62mmol,100%)ofmethylsalvate3. 3.6. AcylationofSalvicAcid: 7-O-AcylDerivatives(4–17) Toasolutionof200mg(0.62mmol)ofsalvicacid1indichloromethane(20mL),theappropriatechoice ofacylchlorideoracidanhydride(0.7mL)wasadded,followedby4-N,N-dimethylaminopyridine (DMAP)(80mg,0.7mmol). Thereactionmixturewasstirredatroomtemperaturefor24handwas successivelywashedwitha5%solutionofhydrochloricacid,a6%solutionofsodiumbicarbonate,and distilledwater. Theresultingorganicphasewasdriedoveranhydroussodiumsulfate,andthesolvent wasremovedusingarotaryevaporatortogivethecorrespondingacylderivative.Thereactionproduct waspurifiedbyCCwithaCH Cl –CH OHgradienttoyieldtherespectivederivatives. ToidentifyC 2 2 3