Coumarin Antifungal Lead Compounds from Millettia thonningii and Their Predicted Mechanism of

13 Pages·2016·2.1 MB·English

molecules
Article
Coumarin Antifungal Lead Compounds from
Millettia thonningii and Their Predicted Mechanism
of Action
DanielM.Ayine-Tora1,2,RobertKingsford-Adaboh2,WilliamA.Asomaning2,
JerryJ.E.K.Harrison2,FelixC.Mills-Robertson3,YahayaBukari4,PatrickO.Sakyi2,
SylvesterKaminta5andJóhannesReynisson1,*
1 SchoolofChemicalSciences,UniversityofAuckland,23SymondsStreet,1142Auckland,NewZealand;
[email protected]
2 DepartmentofChemistry,UniversityofGhana,LG56,Legon-Accra,Ghana;
[email protected](R.K.-A.);[email protected](W.A.A.);[email protected](J.J.E.K.H.);
[email protected](P.O.S.)
3 DepartmentofBiochemistryandBiotechnology,KwameNkrumahUniversityofScienceandTechnology,
Kumasi,Ghana;[email protected]
4 BotanyDepartment,UniversityofGhana,LG55,Legon-Accra,Ghana;[email protected]
5 CentreforPlantMedicineResearch,73,Mampong-Akuapem,Ghana;[email protected]
* Correspondence:[email protected];Tel.:[phone](ext.83746)
AcademicEditor:ThomasJ.Schmidt
Received:14July2016;Accepted:7October2016;Published:15October2016
Abstract: Fungal pathogens continue to pose challenges to humans and plants despite efforts to
controlthem. Twocoumarins, robusticacidandthonningine-CisolatedfromMillettiathonningii,
showpromisingactivityagainstthefungusCandidaalbicanswithminimumfungicidalconcentration
of1.0and0.5mg/mL,respectively. Molecularmodellingagainsttheputativebio-moleculartarget,
lanosterol14α-demethylase(CYP51),revealedaplausiblebindingmodefortheactivecompounds,
inwhichthehydroxylgroupbindswithamethioninebackbonecarboxylicgroupblockingaccessto
theironcatalyticsite. Thisbindingdisruptsthesynthesisofseveralimportantsterolsforthesurvival
offungi.
Keywords: naturalproducts;Candidaalbicans;molecularmodelling;CYP51;Sclorotium;isoflavone
1. Introduction
Candidaalbicansisoneofthemostcommonfungalpathogenscausinginfectiondespitemajor
efforts to control it [1,2]. C. albicans has emerged as one of the main causes of morbidity and
mortality in immunocompromised patients suffering from diseases such as cancer or AIDS [3–6].
Candidiasisinfectioncanaffecttheskin,oralcavity,oesophagus,gastrointestinaltract,vaginaand
vascular system of humans [7–9]. There are a number of drugs such as flucoconazole, nystatin,
voriconazole,terbinafineandechinocandintoaddressfungalinfections[10]. However,drugresistance,
restricted systemic usage due to dose-related toxicity and the emergence of new strains of fungal
infectionsallunderminetheefficacyofthesedrugs[11–15]. Hence,itisnecessarytodevelopnew
antifungaltreatmentstoaddresstheseemergingchallenges.
Milletia thonningii (Schum-Thonn) Baker is a deciduous tree that grows in tropical climates.
It belongs to the family Papilionaceae and is indigenous to West and Central Africa [16–18].
Preparations from this tree are used in traditional medicine for alleviating several diseases and
disorderssuchasdysentery,intestinalpain,worms,tuberculoidleprosy,menstrualdisorder,measles
andchickenpox[16,18]. Inaddition,theseedsshowmolluscicideactivity[19]andthejuicefromthe
leavesislethaltoBulinussnails,thehostforschistosomiasis[20].
Molecules2016,21,1369;doi:10.3390/molecules21101369 www.mdpi.com/journal/molecules
Molecules2016,21,1369 2of13
Molecules 2016, 21, 1369 2 of 12
TThhee mmaannyy uusseess ooffM M..t hthoonnnnininggiiiia assa at rtardaditiitoinoanlasl osuorucrecoe forfe rmemedeidesiehsa hvaevaet tartatcrtaecdterde sreeasrecahrcinht oinittos
citosn csotnitsuteitnutesn.tFso. Froinr sintasntacnec,es,o smomeies oisfloaflvaovnoenseiss iosloaltaetdedf rformomt hthisisp plalannttw weerreer reeppoorrtteedd ttoo ddeemmoonnssttrraattee
lleetthhaall ttooxxiicciittyy ttoo bbrriinnee sshhrriimmpp [[2211]] aass wweellll aass ttoo iinnhhiibbiitt aaccttiivvaattiioonn ooff hhyyppooxxiiaa--iinndduucciibbllee ffaaccttoorr--11 ((HHIIFF--11))
iinn hhuummaann bbrreeaasstt ttuummoouurr TT4477DD cceellllss [[2222]].. IInntteerreessttiinnggllyy,, ccoouummaarriinn ddeerriivvaattiivveess hhaavvee bbeeeenn sshhoowwnn ttoo
ddeemmoonnssttrraattee aannttiiffuunnggaall aaccttiivviittyy [[2233––2266]]..
T Thheea aimimo offt hthisisw woorrkkw waasst otoi ninvveessttigigaatteet thhee aaccttiivviittyy ooff tthhee ccoonnssttiittuueenntt ooff MM.. tthhoonnnniinnggiiii aaggaaiinnsstt
CC.. aallbbiiccaannss aanndd ininvvesetsitgigataet ea ammecehcahnaisnmis magaaignasitn lsatnloasnteorsotel r1o4lα1-d4αem-deetmhyeltahsyel a(CseYP(C51Y)P, 5a 1c)o,mamcoomn manotni-
afunntig-faul ntagragletat.r Tgehti.sT ehnizsyemnzey cmonevceorntvs elartnsolsatneorostle troo letrogoesrtgeorsotle, rtohle, tdhiesrduisprtuiopnti oonf tohfisth einszeynmzyem reesruelstus litns
isntrustcrtuucrtaul rcahlacnhgaensg oefs polfaspmlaas mmaemmbermanberas,n aens,da cnadusceasu as ecshaancghea ning ethien ctohnecceonntrcaetniotrnast ioofn vsaorifovuasr 1io4uαs-
1m4eαt-hmyle tshteyrloslste, rwohlsi,chw hhaicsh cyhtaosstcaytitco satnadti ccyatnodtoxcyict octoonxsiecqcuoennsceeqsu [e2n7–c2es9][. 2I7t –is2 9h]o.pIetdi sthhaotp thede ftihnadtinthges
fiwnildl ipnrgosvwidiell nperowv ildeaedn ecwomlepaoduncodms paso uwnedlls aass wuseelfluals iunssiegfhutlsi ninsitgoh tthsei nrtaotitohnearl adtieosniganl doefs tihgen aocfttihvee
acoctmivpeocuonmdps oouf nMd.s tohfonMn.inthgoiin fnoirn gainitfiofurnagnatilf utrnegatamlterneat.t ment.
22.. RReessuultlsts aanndd DDiissccuussssiioonn
22..11.. IIssoollaattiioonn aanndd SSyynntthheessiiss
IInn oorrddeerr ttoo iinnvveesstitgigaatete ththe eacatcitviivtiyt yofo tfhteh ceocmopmopuonudnsd fsrofmro mM. Mth.onthnoinnngiiin aggiiaaingsati nCs.t aClb.icaalnbsic, atnhse,
tchoensctiotnusetnittus eonft sthoef steheedss eoefd Ms .o tfhoMnn.itnhgoini nwinegriei iwsoelraetedis.o Tlahteed i.solaTthioen isyoilealdtieodn ryoibeuldsetidc arocibdu s(t1i)c,
athcoidnn(i1n)g,inteh-oCn ni(n2g),i nea-lCpin(u2)m, isaolpflianvuomneis ofl(3a)v, onOe,O(-3d),imOet,hOy-dlailmpientuhmyliaslopfilnauvmonies ofl(a4v) onaend( 4)4a’-nOd-
4m’-eOth-myleatlhpyinlaulmpiinsuofmlaivsooflnae v(o5)n. eT(h5e).seT hcoemsepcooumnpdosu anred skanroewknn ocownnstciotunesntittsu oenf ttsheo fptlhaentp [l2a0n,t30[2–03,23]0.– T3h2e].
Tyiheeldy oiefl dcoomfpcooumndp o5u nwdas5 lowwa sbulot wmobruet wmaos resuwccaessssfuulclcye sssyfnutlhlyesissyendt hveisai sdeedmveitahydlaetmioent hoyfl a4t ifoonr
obfiol4ogfiocarl btieosltoinggic.a lCotemstpinogu.nd C5o mwpaso uanldso 5acwetayslataeldso toac eftoyrlmat eda ntoewf ordmerivaatniveew, adceertiyvla-4ti-vOe-,
amceetthyly-l4a-lOp-imnuemthiysloafllpaivnounme i(s6o)fl, taov ohnelep( 6u)n,dtoerhsetlapndu nthdee rssttrauncdtutrhee asctrtiuvcittuyr reealacttiiovnitsyhrieplsa toiof nthseh icphseomfitchael
cshereimesi.c Talhsee mrieosl.ecTuhlearm sotrlueccutularress tarruec dtuisrpeslaayreedd iinsp Fliagyuerde i1n. Figure1.

Figure 1. Structures of isolated coumarins (1, 2) and isoflavanones (3, 4, 5) from M. thonningii and a
Figure1. Structuresofisolatedcoumarins(1,2)andisoflavanones(3,4,5)fromM.thonningiianda
synthesised derivative (6).
synthesisedderivative(6).
2.2. Investigation into the Selective Demethylation of Isoflavone 4
2.2. InvestigationintotheSelectiveDemethylationofIsoflavone4
Previous studies focused on determination of reagents to give optimum yields for demethylation
Previousstudiesfocusedondeterminationofreagentstogiveoptimumyieldsfordemethylation
of compounds such as visnagen and khellin. According to these investigations, a combination of
of compounds such as visnagen and khellin. According to these investigations, a combination of
formic acid and KI gives the optimal yield. Replacing KI with NaBr, KBr, and NaI did not improve
formicacidandKIgivestheoptimalyield. ReplacingKIwithNaBr,KBr,andNaIdidnotimprovethe
the yield of this reaction. In addition, using glacial acetic acid instead of formic acid resulted in a
yieldofthisreaction. Inaddition,usingglacialaceticacidinsteadofformicacidresultedinalower
lower yield. Therefore, formic acid and KI were used for the demethylation of isoflavone 4 to
synthesise derivative 5 [33].
Molecules2016,21,1369 3of13
Myioelelcdu.leTs 2h0e1r6e, 2fo1,r 1e3,6f9o rmicacidandKIwereusedforthedemethylationofisoflavone4tosynth3e osfi s1e2
derivative5[33].
IInntteerreessttiinnggllyy,, tthhee ddeemmeetthhyyllaattiioonn ooff iissooffllaavvoonnee 44 oonnllyy ooccccuurrrreedd aatt tthhee mmeetthhooxxyy ggrroouupp oonn tthhee
bbeennzzooppyyrroonnee mmooiieettyy ((XX)) bbuutt nnoott oonn tthhee pphheennyyll ggrroouupp ((YY)) ((sseeee FFiigguurree 22)).. TThhiiss iiss iinn lliinnee wwiitthh aa pprreevviioouuss
iinnvveessttiiggaattiioonn,, wwhhiicchh sshhoowweedd tthhaatt mmeetthhooxxyy ggrroouuppss oorrtthhoo ttoo aaccyyll mmooiieettiieess aarree hhiigghhllyy rreeaaccttiivvee aanndd aacciidd
sseennssiittiivvee ccoommppaarreedd ttoo aarroommaattiicc mmeetthhooxxyy ggrroouuppss [[3344]].. IInn oorrddeerr ttoo sshheedd lliigghhtt oonn tthhiiss pphheennoommeennoonn,,
tthheerrmmoocchheemmiiccaall ddeennssiittyy ffuunnccttiioonnaall tthheeoorryy ((DDFFTT)) ccaallccuullaattiioonnss wweerree ppeerrffoorrmmeedd ttoo ddeetteerrmmiinnee bboonndd
ddiissssoocciiaattiioonn eenneerrggiieess ((BBDDEE)) ffoorr tthhee bboonnddss XX aanndd YY,, aanndd pprroottoonn aaffffiinniittiieess ((PPAA)) ffoorr ddeerriivvaattiivvee 44.. IItt hhaass
bbeeeenn sshhoowwnn tthhaatt BBDDEEss aanndd PPAAss ccaann bbee rreelliiaabbllyy ccaallccuullaatteedd uussiinngg tthhee DDFFTT mmeetthhoodd [[3355––3388]].. PPAAss ooff tthhee
ccaarrbboonnyyll aanndd mmeetthhooxxyy ooxxyyggeenn aattoommss wweerree ccaallccuullaatteedd ttoo ddeetteerrmmiinnee tthhee pprreeffeerrrreedd pprroottoonnaattiioonn ssiittee dduuee
ttoo tthhee aacciiddiicc nnaattuurree ooff tthhee rreeaaccttiioonn mmeeddiiuumm.. TThhee PPAA vvaalluueess sshhooww tthhaatt tthhee ccaarrbboonnyyll ooxxyyggeenn iiss tthhee
pprreeffeerrrreedd pprroottoonnaattiioonn ssiittee ((sseeee Ttaabbllee SS11 iinn tthhee SSuupppplleemmeettaarryy IInnffoorrmmaattiioonn)).. TThhee BBDDEEss ooff nneeuuttrraall aanndd
pprroottoonnaatteedd ffoorrmmss ooff 44 wweerree ddeetteerrmmiinneedd ffoorr bbootthh hhoommoollyyttiicc aanndd hheetteerroollyyttiicc cclleeaavvaaggeess ooff tthhee OO--CC bboonndd
aanndd tthhee rreessuullttss aarree sshhoowwnn iinn TTaabbllee 11..

FFiigguurree 22.. SSttrruuccttuurree ooff OO,,OO--ddiimmeetthhyyllaallppiinnuummiissooffllaavvoonnee ((44)) aanndd tthhee ttwwoo ccaarrbboonn--ooxxyyggeenn bboonnddss uunnddeerr
iinnvveessttiiggaattiioonn XX aanndd YY..
Table 1. Bond dissociation energies (BDEs) in kcal/mol.
Table1.Bonddissociationenergies(BDEs)inkcal/mol.
Bond Dissociation X Y Difference
BondDissociation X Y Difference
Homolytic 51.2 54.0 2.8
HeterolyHtiocm olytic 2165.10. 2 542.027.8 2.8 11.8
Heterolytic 216.0 227.8 11.8
Homolytic-protonated 42.5 62.7 20.2
Homolytic-protonated 42.5 62.7 20.2
Heterolytic-protonated 111.4 167.6 56.2
Heterolytic-protonated 111.4 167.6 56.2
The results in Table 1 show that demethylation of bond X is more thermochemically favourable
TheresultsinTable1showthatdemethylationofbondXismorethermochemicallyfavourable
for both protonated and non-protonated forms of 4. Furthermore, the difference in BDE values for
forbothprotonatedandnon-protonatedformsof4. Furthermore,thedifferenceinBDEvaluesfor
non-protonated 4 are small, which can result in a mixture of demethylated products, in particular for
non-protonated4aresmall,whichcanresultinamixtureofdemethylatedproducts,inparticularfor
the homolytic cleavage. Thus, it can be argued that protonation of the carbonyl oxygen is necessary
thehomolyticcleavage. Thus,itcanbearguedthatprotonationofthecarbonyloxygenisnecessaryfor
for the selective demethylation to proceed. After protonation, the reaction is kinetically driven by KI
theselectivedemethylationtoproceed. Afterprotonation,thereactioniskineticallydrivenbyKIto
to the desired product.
thedesiredproduct.
2.3. Biological Testing
2.3. BiologicalTesting
The compounds were tested against the wild C. albicans strain and a reference strain ATCC18804
The compounds were tested against the wild C. albicans strain and a reference strain
[39–41]. The agar-well diffusion method was used to evaluate the zones of inhibition (ZOI). This was
ATCC18804[39–41].Theagar-welldiffusionmethodwasusedtoevaluatethezonesofinhibition(ZOI).
followed by the determination of minimum inhibitory concentrations (MIC) and minimum
Thiswasfollowedbythedeterminationofminimuminhibitoryconcentrations(MIC)andminimum
fungicidal concentrations (MFC) for the active compounds. The results are shown in Table 2.
fungicidalconcentrations(MFC)fortheactivecompounds. TheresultsareshowninTable2.
As can be seen in Table 2, the ZOIs show that compounds 1, 2 and 3 are active against C. albicans
AscanbeseeninTable2,theZOIsshowthatcompounds1,2and3areactiveagainstC.albicans
and the reference strain, but no activity was observed for derivatives 4, 5 and 6. The antifungal drug
andthereferencestrain,butnoactivitywasobservedforderivatives4,5and6. Theantifungaldrug
clotrimazole was used as a reference, and it gave ~27% larger ZOI for the wild type strain and 16%
clotrimazolewasusedasareference,anditgave~27%largerZOIforthewildtypestrainand16%for
for the reference strain 18804. All the compounds were tested at 2 mg/mL concentration. Considering
thereferencestrain18804. Allthecompoundsweretestedat2mg/mLconcentration. Considering
that clotrimazole is an optimised drug in clinical use, it can be stated that compounds 1–3 are quite
thatclotrimazoleisanoptimiseddruginclinicaluse,itcanbestatedthatcompounds1–3arequite
active. According to the MIC results, compound 2 is more active than 1 and 3, and, finally, the MFC
active. AccordingtotheMICresults,compound2ismoreactivethan1and3,and,finally,theMFC
measurements revealed that coumarins 1 and 2 are fungicidal with 1.0 and 0.5 mg/mL, respectively.
Interestingly, the isoflavone 3 is only fungistatic. This activity of the coumarins makes them promising
lead compounds for further development for antifungal treatment. Furthermore, compounds 1, 2 and 3
Molecules2016,21,1369 4of13
measurementsrevealedthatcoumarins1and2arefungicidalwith1.0and0.5mg/mL,respectively.
Interestingly,theisoflavone3isonlyfungistatic. Thisactivityofthecoumarinsmakesthempromising
leadcompoundsforfurtherdevelopmentforantifungaltreatment. Furthermore,compounds1,2and
3alsoshowedactivityagainsttheSclerotiumfungalstrainqualitativelyconfirmingtheirefficacy(data
notshown).
Table2.Zonesofinhibition(ZOI),minimuminhibitoryconcentrations(MIC)andminimumfungicidal
concentrations(MFC).
ZOI(mm) MIC(mg/mL) MFC(mg/mL)
Compound
WILD 18804 WILD 18804 WILD 18804
Robusticacid(1) 10.3 14.7 0.25 1.00 1.0 1.0
Thonningine-C(2) 10.3 14.7 0.13 0.25 0.5 0.5
Alpinumisoflavone(3) 10.7 15.0 0.25 0.50 F F
O,O-dimethylalpinumisoflavone(4) 0 0 X X X X
4-O-methylalpinumisoflavone(5) 0 0 X X X X
Acetyl-4-O-methylalpinumisoflavone(6) 0 0 X X X X
Clotrimazole 14.4 17.6 X X X X
DMSO(20%) 0 0 X X X X
Zonesofinhibition=ZOI;minimuminhibitoryconcentrations=MIC;minimumfungicidalconcentrations=
MFC;WILD=wildC.albicansstrain;18804=referenceATCC18804strain;X=Nodata;F=Fungistatic.
2.4. MolecularModelling
Itisestablishedthatmicroorganismsproduceauniqueclassofsterolssuchasergosterolnecessary
for their growth and survival [42]. The absence of these sterols in mammalian host cells makes
these pathways an excellent target for therapeutic antifungal design [43,44]. In this light, some
clinicallyemployedantifungalssuchastheazoleshavebeendesignedtodisruptCYP51,acytochrome
P450enzymeresponsibleforthesynthesisofthesterols[28,45,46]. Coumarinantifungalderivatives
designedinsilicoagainsttheCYP51enzymehavealsoshowedpromisingpreliminaryresults[47,48].
ItcanthereforebehypothesisedthatCYP51istheputativetargetoftheactivecompounds1–3from
M.thonningii.
ThecoumarinsandisoflavonesweredockedtothecrystalstructureofCYP51(Proteindatabank
ID:4ZE1)[49]. Theco-crystallisedposaconazolewasremovedandredocked. Theroot-mean-square
deviation(RMSD)betweentheheavyatomsoftheco-crystallisedligandanditsdockedcounterparts
was1.33Åand1.51ÅusingChemScore(CS)andGoldScore(GS)respectively,verifyingtheprediction
powerofthemethod.
Itispredictedthatthehydroxylgroupofthecoumarinsbindtothebackbonecarboxylicmoiety
ofmethionine(MET509),whichenablesthemtoadoptaplausiblebindingconfigurationwithmethyl
groupsblockingtheironcatalyticbindingsiteasshowninFigure3A,B.Theisoflavones3and5,which
havehydroxylgroups,alsobindtothemethioninebuthavetheirphenylmoietiesinsertedintothe
bindingsite;whereastheisoflavones4and6withoutthehydroxylgroupshavesimilarconfigurations
tothecoumarinsbutdonotformhydrogenbondswiththemethionine, asshowninFigure4A,B,
respectively. Thisisapossibleexplanationwhythecoumarinsareactivebutnottheisoflavones.
Robusticacid(1)hadthehighestpredictedaffinitybyCSandthonningine-C(2)fortheGSresults.
Both of the coumarins gave better scores for both functions when compared to clotrimazole (see
TableS2intheSupplementaryMaterials). ThesepredictionspointtowardCYP51asthebio-molecular
targetofthesecompoundsbutwiththeveryimportantcaveatthatexperimentalverificationisneeded.
Nevertheless,themolecularmodellingworksupportsaplausiblehypothesisandpointsthewayin
whichassaysthesecompoundsshouldbetestedfirst.
Molecules2016,21,1369 5of13
Molecules 2016, 21, 1369 5 of 12
Molecules 2016, 21, 1369 5 of 12
A B
A B
Figure 3. (A) the docked configuration of robustic acid (1) in the binding site of lanosterol 14α-
Figure 3. (A) the docked configuration of robustic acid (1) in the binding site of lanosterol
d1Fe4imgαu-edrtehe my3l.ea ts(heAy )(l CatshYeeP (5dC1o)Yc; Pka5end1d) ; c(oaBnn)f ditgh(ueBr )adttoihocekne dodof c crkooenbdfuigcsuotinrca fitagicouindr ao(t1fio )t nhinoo nftnhtiehn ogbniinnnedin-iCngg in( 2es)i- tCien ( o2tfh) elia nnboitnhsdteeirnboginl ds1ii4tneαg.-
Hsdiyetemd.reHotghyeydnlra obsgoee nn(dCbsY oaPnr5ed1 ss)h;a oraewnsdnh ao(Bsw )gn rtehaeesn gd droeocetknteedddo lctitnoeendsf.il ginuersa.tion of thonningine-C (2) in the binding site.
Hydrogen bonds are shown as green dotted lines.

A B
A B
Figure 4. (A) the docked configuration of alpinumisoflavone (3) in the binding site of CYP51; and (B)
tFFhiiegg duuorreeck 44e.. d((AA co)) ntthhfieeg dduooracctkkieoeddn ccooof nnOffii,Ogguu-drraaimttiiooennth ooyffl aaalllpppiiinnnuuummmiiisssoooffflllaaavvvooonnneee (((433))) iiinnn ttthhheee bbbiiinnndddiiinnnggg sssiiittteee. ooHff yCCdYYroPPg55e11n;; a abnnoddn (d(BBs) )
attrhheee s ddhoooccwkkneedd a csc oognnrfifeieggnuu rrdaaottitiootennd oo lffi nOOe,,sOO. --ddiimmeetthhyyllaallppiinnuummiissooflflaavvoonnee ((44)) iinn tthhee bbiinnddiinngg ssiittee.. HHyyddrrooggeenn bboonnddss
aarree sshhoowwnn aass ggrreeeenn ddootttteedd lliinneess..
2.5. Chemical Space
2.5. Chemical Space
2.5. ChemicalSpace
The calculated molecular descriptors molecular weight (MW), log P, hydrogen bond donors
(HD),TT hhhyeed ccraaollcgcuuelnala ttebedodnm dmo oaleclcecuceulpaltraordr sed se(cHsrciAprit)po ptrosormlsa orm lseouclureflcaaucrleaw rae rwiegahe it(gP(hMStA W(M) ),aWlnod)g, rPloo,tgha ytPad,b rlhoegy ebdnoronbgdoesnn d( RbdBoon)n dgo rivdseo(nHn oDinr)s ,
Th(HaybdDlre)o ,S gh3e ynindb rtoohngede Snua cpbcopenlpedtmo arescnc(teHaprAyto) Mrps oa(tlHaerrAisa)ul srp.f oaAlcalelr o asrfu etarhfe(aP cmSeA oal)reeacaun dl(ePsrS oaAtrae)t awabniltdeh birnoo nttahdtesa bd(RrleuB g)b-oglinikvede csnh (ieRnmBTi)ac abglli evsepSna3 ciienn
(tTfhoaerb Sdleue pSfi3pn liientmi othnene o tSaf urlyepaMpdl-ealimtkeere,i nadtlrsau.rgyA- lMlilkoeaf taetnhridea mlKs.on Aloelwclu onlfe Dsthareur emgw Soipltehacicunel etrhsee gadiroerun wgs,-i ltsihkeeienc rhtehefem. [di5cr0ua]lg sa-lpnikadec ecTh(afebomlredi cSea4fil) n.s ipTtiahocene
mo(ffoollree adcdue-llfiaiknre i,twidorenuig goh-lfti kloeeafa dtn-hldiek Kel,i ngdoarwungdn-slD ikisre u bagentSdwp Keaecneno rw3e3gn6i o.D3n rsau,ngsd eS e4pr3ae8cf..e4 [ r5ge0·gm]iaoonnld−s1, Tasanebdel ertehSfe4. )[l.5oT0gh] Pean mvdao lTlueacebusl lelai reS w4in)e. iTtghhheet
romafnotglheeec uoliflga 3ar .n6wd easingidsh tb4 eo.6tfw. tTheehen elsi3ge3a 6vn.ad3lsua einssd bd4ee3tmw8.oe4engns·t mr3a3ot6el.− 3t 1haaantnd dt h4t3eh8 ec.4ol omgg·pmPoouvln−a1d lausn eadsr eltih eieni n loatg hf aePv rvoauanlrguaeebslo elf iea3 r.i6enaa tnohdfe
c4rha.6en.mgTeic haoelf s se3p.va6c aealu nfeodsr 4fdu.e6rm.t hToehnre sdsterea vvteealoltuhpeamst etdnhetem. coonmstproauten dthsaat rtehien caomfapvoouunradbsl earaer eian oaf cfahveomuircaabllsep aarceeaf oorf
chemical space for further development.
furtherdevelopment.
3. Materials and Methods
33.. MMaatteerriiaallss aanndd MMeetthhooddss
3.1. Isolation
33..11.. IIssoollaattiioonn
3.1.1. Plant Material
33..11..11.. PPllaanntt MMaatteerriiaall
The seeds of Milletia thonningii were collected from the University of Ghana campus (Legon-Accra,
GhanTTa)hh.e eA ss eeveeoddussc oohffe MMr siiplllleeettciiiaam tthheoonnn wnniinnasgg iiiiid wweneerrteeif icceoodllll eebccytt eeMdd rff.rr oDomm. K tthh. Aee UbUbnniiiwvvee arrnssiidttyy t hoofef nGG lhhaaatnneraa Mccaarmm. Jpp.Yuu.ss A((LLmeeggpooonnn--sAAacchcc raraat ,,
tGGhehh aaGnnhaaa)).n. AAa Nvvooauuticcohhneearrl ssHppeeerccbiimmareeiunnm ww,aa Dss eiiddpeeannrtttmiiffiieeenddt bboyyf BMMorrt..a DDny.. KK, U.. AAnibbvbbeiirwwsi taaynn oddf t tGhheehnna nllaaatt.ee Trr hMMe rrv.. oJJ..uYYc.. hAAemmr spppooenncssimaahhe anatt
(tchaeta Glohgaunea NNOat: i1o4n8a3l 2H) ewrbasa rdiuepmo,s Diteepda artt mtheen ht eorfb Baorituamny. , TUhnei vseeersdisty w oefr Ge hshaandae. Tdhreie vdo fuocrh tehrr esep ewciemekesn
a(ncadt atlhoegnu ep NulOve: r1i4se8d32. )A wlla ss odlevpeonstsit ewde arte thane ahleyrtbicaarli ugmra.d Teh. eT sheiend-lsa wyeerr ec hshroamdea tdorgieradp fhoyr t(hTrLeeC )w weeakss
and then pulverised. All solvents were analytical grade. Thin-layer chromatography (TLC) was
Molecules2016,21,1369 6of13
theGhanaNationalHerbarium,DepartmentofBotany,UniversityofGhana. Thevoucherspecimen
(catalogueNO:14832)wasdepositedattheherbarium.Theseedswereshadedriedforthreeweeksand
thenpulverised. Allsolventswereanalyticalgrade. Thin-layerchromatography(TLC)wasperformed
onaluminiumfoilslidespre-coatedgel(thickness0.2mm,typeKiesegel60F Merck,Darmstadt,
254
Germany)usingpetrol/ethylacetate(10%,15%,20%,25%,30%and35%);detectionwasperformed
usingUVlightandiodinevapour. Columnchromatographywascarriedoutonaluminiumoxide
(Fluka,Seelze,Germany).
3.1.2. ExtractionandIsolation
Thepulverisedplantmaterial(4.5kg)wasextractedusingcoldpercolationwithpetroleumether
(2L)forfivedaysat27◦C.Theextractwasfilteredandconcentratedusingrotaryevaporatortogive
a brown solid (18 g) and stored in a refrigerator. In addition, 15 g of this solid were dissolved
in chloroform, mixed with aluminium oxide and dried at 50 ◦C. This was placed on a column
(50mm×1200mm) pre-packed with aluminium oxide. The column was first eluted with petrol
(500mL),andthenwitha5%incrementofethylacetateinpetrol,and500mLforeachincrement.At45%
ofethylacetateinpetrol,TLCprofilingshowedtwodominantfractionsF1aandF1b. Thesefractions
werepurifiedusingcolumnchromatography(CC)(20mm×600mm)withaluminiumoxideandan
increasingamountethylacetateinpetrol. Afterwards,thedefattedplantmaterialwasalsoextracted
with ethyl acetate (2 L) for a week at 27 ◦C. A yellow mixture (20 g) was obtained after solvent
removalandalsostoredinarefrigerator. CCof15goftheyellowmixturewasconductedasabove.
At80%ofethylacetateinpetrol,TLCprofilingshowedfourdominantfractions—F2a,F2b,F2cand
F2d. These fractions were also purified on CC. The compounds were characterised by TLC, mass
spectrometry,IR,1Hand13C-NMRspectraandcomparedtopublisheddata. F1aandF2bwerefound
to be the same compound and identified as isoflavone (4) (4.8 g). F1b and F2c were identified as
isoflavones(3)(1.2g)and(5)(23mg),respectively. F2aandF2dwerealsoidentifiedascoumarin(1)
(1.4g)and(2)(642mg),respectively.
Robustic Acid (1): Colourless block crystal, melting point (mp.) 207–209 ◦C. IR ν : 3381, 1701,
max
1612 cm−1; Gas chromatography tandem Mass spectrometry (GC-MS) m/z 380, 365; 1H-NMR
(400MHz,CDCl )δ: 9.91(1H,s),7.49(2H,d,J=8Hz),6.98(2H,d,J=8Hz),6.64(1H,s),6.53(1H,
3
d,J=10Hz),5.80(1H,d,J=10Hz),3.98(3H,s),3.83(3H,s),1.49(6H,s);13C-NMR(100MHz,CDCl )
3
δ: 162.2,159.7,158.6,156.8,153.5,151.8,131.1,131.4,123.0,113.2,114.7,110.3,103.6,101.5,101.4,98.0,
64.0,54.9,and27.6. Thisisinagreementwithpublisheddata[30,31].
Thonningine-C(2):Yellowgranules;mp.200–203◦C;IRν :3288,3093,1716,1658,1620cm−1;GC-MS
max
m/z437; 1H-NMR(400MHz, CDCl )δ: 10.23(1H,s), 7.48(1H,s), 7.45(1H,d, J =8Hz), 7.00(1H,
3
s),6.99(1H,d,J=8Hz),6.90(1H,s),6.89(1H,m),3.94(1H,s),3.91(1H,s),3.90(1H,s),3.85(1H,s),
2.23(1H,s);13C-NMR(100MHz,CDCl )δ: 165.0,160.1,158.8,158.5,158.3,143.8,131.1,122.1,113.0,
3
110.4,103.9,102.1,95.8,64.7,54.7,and19.9. Thisisinagreementwithpublisheddata[32].
Alpinumisoflavone(3): Yellowneedle-likecrystal;mp. 204–207◦C;IRν : 3454,1654,1614,and1573
max
cm−1; GC-MSm/z: 336, 321; 1H-NMR(400MHz, CDCl )δ: 13.11(1H,s),7.82(1H,s), 7.39(2H,d,
3
J=8Hz),6.88(2H,d,J=8Hz)6.71(1H,d,J=10Hz),6.33(1H,s),5.64(1H,d,J=10Hz),5.25(1H,s),
4.25(1H,s),3.93(1H,s),1.47(6H,s);13C-NMR(100MHz,CDCl )δ: 180.4,159.0,156.8,156.3,155.4,
3
152.0, 129.8, 127.6, 123.0, 122.5, 115.1, 114.9, 105.6, 105.1, 94.3, and 27.8. This is in agreement with
publisheddata[30,51].
O,O-dimethylalpinumisoflavone(4): Colourlessgranules(8.9g);mp. 137–139◦C;IRν : 1636,1605,
max
1512,1389,1362,1244,1067cm−1,GC-MSm/z: 364,349;1H-NMR(400MHz,CDCl )δ: 7.76(1H,s),
3
7.47(2H,d,J=8Hz),6.97(2H,d,J=8Hz),6.76(1H,d,J=10Hz),6.60(1H,s),5.74(1H,d,J=10Hz),
3.83(3H,s),3.90(3H,s),1.47(6H,s);13C-NMR(100MHz,CDCl )δ: 173.3,157.8,157.0,156.3,154.1,
3
Molecules2016,21,1369 7of13
148.6,129.0,128.6,123.9,122.6,114.4,112.2,111.6,111.5,98.9,61.0,53.6,and26.6. Thisisinagreement
withpublisheddata[30,31].
3.2. Synthesis
3.2.1. Synthesisof4(cid:48)-O-methylalpinumisoflavone(5)
ToasolutionofO,O-dimethylalpinumisoflavone(4)(1.00g,2.75mmol)in30mLofethylacetate,
KI(5.10g,30.73mmol)wasadded,followedbyformicacid(30mL)asdescribedbyMustafaetal. for
thedemethylationofkhellinandvisnagen[33]. Thismixturewasrefluxedfor110min. Afterthereflux,
50mLofwaterwasaddedandtheproductprecipitated. Theproductwaspurifiedbychromatography
(ethylacetate/petroleumether,1:5)toyield780mgof4(cid:48)-O-methylalpinumisoflavone(5)aspaleyellow
needle-likecrystals. mp.: 136–138◦C;IRν : 3423,1651,1614,1512cm−1; GC-MSm/z: 350,335;
max
1H-NMR(400MHz,CDCl )δ: 13.16(1H,s),7.82(1H,s),7.47(2H,d,J=8Hz),7.00(2H,d,J=8Hz),
3
6.75(1H,d,J=10Hz),6.34(1H,s),5.64(1H,d,J=10Hz),3.85(3H,s),1.48(6H,s);13C-NMR(100MHz,
CDCl )δ: 179.5,158.4,158.1,155.9,155.5,151.0,128.7,126.7,122.1,121.6,114.1,112.7,104.7,104.2,93.4,
3
53.9,and26.9. Thisisinagreementwithpublisheddata[30].
3.2.2. SynthesisofAcetyl-4(cid:48)-methylalpinumisoflavone(6)
Asolutionof4(cid:48)-O-methylalpinumisoflavone(5)(253mg,0.72mmol)in20mLofaceticanhydride
was refluxed for 120 min. After the reflux, a solution of sodium bicarbonate (10 g, 119 mmol) in
100 mL of water was added to the mixture to neutralise the acetic acid produced, which resulted
in the precipitation of the product. The product was filtered and purified by chromatography
(ethyl acetate/petroleum ether, 1:5) to afford 146 mg of acetyl-4(cid:48)-methylalpinumisoflavone (6) as
acolourlessblockcrystal. mp.: 204–206◦C;IRν : 1766,1635,1608,1514cm−1;GC-MSm/z: 393,
max
335;1H-NMR(400MHz,CDCl )δ: 7.75(1H,s),7.41(2H,d,J=8Hz),6.96(2H,d,J=8Hz),6.71(1H,s),
3
6.51(1H,d,J=10Hz),5.78(1H,d,J=10Hz),2.45(3H,s),3.83(3H,s),1.49(6H,s);13C-NMR(100MHz,
CDCl )δ:175.0,169.3,159.6,158.1,157.6,150.8,145.0,132.0,130.4,125.7,124.0,115.2,114.0,113.0,102.2,
3
94.0,55.3,28.4,and21.1.
3.3. BioactivityStudies
C.albicanswereisolatedandidentifiedattheKomfoAnokyeTeachingHospitalatKumasi,Ghana.
TheorganismswerebroughttotheMicrobiologyDepartmentoftheCentreforPlantMedicineResearch
(CPMR)atMampong-Akuapem,Ghana.
3.3.1. PreparationofSolutionsandMedia
20 mg of all the compounds were dissolved in DMSO (dimethylsulphoxide, 10 mL of 20%,
(Sigma-Aldrich,D5879,Münich,Germany)),and100µg/mLsolutionofclotrimazolewasalsoprepared
usingDMSO(20%). Nutrientagar,bacteriologicalpeptone(Sigma-Aldrich,P0556)andSabouraud4%
glucoseagar(FlukaBiocheika84088,Sigma-Aldrich,Bangalore,India)werepreparedaccordingtothe
manufacturer’sinstructions. ThestockculturesofC.albicansweresubculturedontofreshNutrient
agarplatesandthenincubatedfor24hoursandstoredinarefrigeratorovernight.
3.3.2. DeterminationofthePotencyoftheCompounds
The agar-well diffusion method was used to investigate the antimicrobial properties of the
isoflavonesandthecoumarins[52,53].
Aboutthreetofivecoloniesofthesamemorphologicaltypeofthemicrobesweresuspended
in test tubes containing 5 mL of sterilised bacteriological peptone and incubated at 37 ◦C for 16 h.
Afterthe16hours,themicroorganismsweresubculturedinsterilisedbacteriologicalpeptonewaterand
incubatedagainforanothertwohours. Theturbiditywasadjustedtoa0.5McFarlandstandard[52].
ThedriedSabouraud4%glucoseagarplateswerethenfloodedwiththefungiandthesurfacewas
Molecules2016,21,1369 8of13
allowedtodry. Asterilisedcockborerofaninternaldiameterof6mmwasusedtopunchwellsinthe
medium. Thecompoundswerethendispensedintotheirrespectivelabelledwellswithconcentrations
of2mg/mLforallthecompoundstested;andDMSO(20%)wasdispensedintoitslabelledwellas
anegativecontrolwhilstclotrimazole(2mg/mL)wasdispensedasapositivecontrol. Triplicatesof
eachplatewiththesamelabelledcompoundsweremadeforeachfungus. Theplateswerekeptin
therefrigeratorovernightforcompletediffusion. Theplateswerethenincubatedat35◦Cfor48h.
Aftertheincubationperiod,thediameterofeachinhibitionzonewasmeasuredinmillimeters(mm)
withametrerule.
3.3.3. DeterminationofMinimumInhibitoryConcentrationandtheFungicidalorFungistaticEffectof
theActiveCompounds
TheMICsoftheactivecompoundsweredeterminedusingthemicroplatedilutionmethodas
describedbyEloff[54]. Ahundredmicroliters(100µL)of2mg/mLsolutionofanactivecompound
wasplacedinthefirstwellofthesecondrowofthe96-wellmicroplate. About100µLofthesame
activecompoundwasaddedto100µLofsterilisedbacteriologicalpeptonewaterinthenextwellofthe
secondcolumn. Thiswasmixedwithamicropipetteand100µLofthisdilutionwasaddedtothewell
inthenextcolumn,whichcontained100µLofsterilisedbacteriologicalpeptonewater. Thisprocess
wasrepeatedtoproducetwo-foldserialdilutionsintheoriginalconcentration. About100µLofDMSO
(20%)wasplacedinthelastcolumn.Thisprocesswasrepeatedforthreerowsonthe96-wellmicroplate.
Inaddition,100µLofclotrimazolewasplacedinthelastrow. Furthermore,100µLofsubcultured
fungiwereplacedinallthewellsexceptthefirstrow,whichcontainsonlypeptonewater. Theplates
werethencoveredandincubatedat35◦Cfor48h,afterwhich40µLofiodonitrotetrazoliumchloride
(INT,0.2mg/mL)wasaddedtoeachwellandexaminedafter120minofincubation. Fungigrowth
was indicated by a red colour of formazan, which is the reduced form of the INT. The minimum
concentrationatwhichtherewasnocolourwastakenastheminimuminhibitoryconcentration.
Samplesfromthewellswithnocolour,and,therefore,wellswithnoobservablefungigrowth
wereincubatedat35◦Cfor48honfreshSabouraud4%glucoseagarplatesusinginoculationpins.
Theminimumconcentrationatwhichnogrowthofmicrobeswasseenwastakenastheminimum
fungicidalconcentration.
3.4. ThermochemicalCalculations
ThecalculationsweredoneusingGaussian09softwaresuite[55](GaussianInc.,Wallingford,
CT,USA)employingarestrictednon-localB3LYPfunctionalhybridmethod[56–58]. Thestandard
6-31+G(d,p)[59,60]basissetwasusedforthegeometryoptimisationandfrequencyanalysiswhile
thelarger6-311+G(2df,p)basissetwasemployedtoperformtheenergycalculations. Thezero-point
vibrationalenergies(ZPE)werescaledusingWong’svalueof0.9804[61].Inallcases,thenormalmodes
revealednoimaginaryfrequencies,whichindicatethattheyrepresenttheminimaonthepotential
energysurface. ThePAsandBDEswerecalculatedasdescribedbyForesmanandFrisch[62].
3.5. MolecularModelling
Compounds1–6weredockedintothecrystalstructureofCYP51(ProteinDataBankID:4ZE1
resolution2.05Å)[49],whichwasobtainedfromProteinDataBank(PDB)[63,64]. TheScigressversion
FJ2.6program[65](FijitsuLimited,Kanagawa,Japan)wasusedtopreparethecrystalstructurefor
docking, hydrogenatomswereadded, theco-crystallisedligandaswellascrystallographicwater
moleculeswereremoved. TheScigresssoftwaresuitewasalsousedtobuildtheinhibitorsandthe
MM2 [66] force field was used for structural optimisation. The centre of the binding was defined
as the position the carbonyl oxygen of methionine 509 (x = 4.024, y = 15.088, z = 99.736) with a
10 Å radius. Hundred docking runs were allowed for each ligand with default search efficiency
(100%). The GoldScore [67], and ChemScore [68,69], scoring functions were implemented using
the parameter file for cytochromes P450 and heme-containing proteins [70] developed to predict
Molecules2016,21,1369 9of13
binding modes and relative binding energies of the ligands using the GOLD v5.4 software suite
(CambridgeCrystallographicDataCentre,Cambridge,UK).Thisheme-containingproteinsparameter
file was developed using data from the Cambridge Structural Database (CSD) and Protein Data
Bank. The root square mean deviation (RSMD) of the co-crystallised ligand for CSD-ChemScore,
CSD-GoldScore,PDB-ChemScore,andPDB-GoldScorewere1.333Å,1.710Å,1.595Åand1.509Å,
respectively. TheCSD-Chemscore,whichhadthebestRSMDvalue,wasusedforfurtheranalysis.
ThedockingscoresofthecompoundsaregiveninTableS1inthesupplementarymaterials.
The QikProp 3.2 [71] software package (Schrödinger LLC, New York, NY, USA) was used to
calculate the molecular descriptors of the molecules. The reliability has been established for the
calculateddescriptors[72].
4. Conclusions
Previous studies have focused on azoles, polyenes, griseofulvin, flucytosine and allylamine
molecularscaffoldsforantifungaldesign,e.g.,theclinicallyuseddrugsvoriconazole,ketoconazole
andfluconazolearederivedfromazolecontainingmoleculesandterbinafinefromallylamine[10].
However,notmuchattentionhasbeenpaidtothedevelopmentofcoumarinsasantifungals.Thisstudy
demonstratestheirpotentialforantifungaldrugdevelopment.
Robusticacid(1)andthonnigine-C(2)wereactiveagainstthefungalstrainstested. Thepredicted
mechanismofactionshowedthatthehydroxylgroupbindstothemethioninebackboneofCYP51
helping the coumarins to adopt a pose that blocks access to the catalytic site of the enzyme.
Interestingly,thecoumarinsunderstudyappeartobenon-toxicinmammaliansystemsduetothe
numeroustraditionalmedicinaluseoftheM.thonningii.Thisnon-toxicnaturepresentsthesecoumarins
asgoodantifungalleads,whichmightbedevelopedfurtherasantifungaldrugsandfoodpreservatives.
Thisstudyextendsthenumberofcoumarinswithantifungalpropertiesreported[23–26]inaddition
toprovidingdirectionforrationaldesignofcoumarinsasantifungalagents.
SupplementaryMaterials:Supplementarymaterialscanbeaccessedat:http://www.mdpi.com/1420-3049/21/
10/1369/s1.TableS1:Bonddissociationenergies,TableS2:Resultsofthescoringfunctionfortheligands,TableS3:
Thecalculatedmoleculardescriptorsfortheligands,TableS4:Definitionoflead-like,drug-likeandknowndrug
space(KDS)intermsofmoleculardescriptors.
Acknowledgments: Government of Ghana Graduate Studies Allowance and Department of Chemistry, UG
GraduateAssistantshiptoDMA-Tisdulyacknowledged.
AuthorContributions:RobertKingsford-Adaboh,WilliamA.AsomaningandJerryJ.E.K.Harrisondesignedthe
research;DanielM.Ayine-ToraconductedtheresearchwithsupportofJerryJ.E.K.HarrisonandPatrickO.Sakyi;
FelixC.Mills-Robertson,SylvesterKamintaandYahayaBukariconductedbiologicalstudies;JóhannesReynisson
andDanielM.Ayine-Toradesignedandconductedthemodellingandcomputationalstudiesandwrotethepaper.
Alltheauthorsreadthepaper.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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