Enhanced Antibacterial Activity of Ent-Labdane Derivatives of Salvic Acid

19 Pages·2017·5.71 MB·English

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.:[phone]
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
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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.
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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
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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