Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C

22 Pages·2017·3.11 MB·English

RESEARCHARTICLE
Biotransformation of a potent anabolic
steroid, mibolerone, with Cunninghamella
blakesleeana, C. echinulata, and Macrophomina
phaseolina, and biological activity evaluation
of its metabolites
MahwishSiddiqui1,MalikShoaibAhmad1,Atia-tul-Wahab2,SammerYousuf1,
NarjisFatima2,NimraNaveedShaikh1,Atta-ur-Rahman1,M.IqbalChoudhary1,2,3*
a1111111111
a1111111111
1 H.E.J.ResearchInstituteofChemistry,InternationalCenterforChemicalandBiologicalSciences,
a1111111111 UniversityofKarachi,Karachi,Pakistan,2 Dr.PanjwaniCenterforMolecularMedicineandDrugResearch,
a1111111111 InternationalCenterforChemicalandBiologicalSciences,UniversityofKarachi,Karachi,Pakistan,
a1111111111 3 DepartmentofBiochemistry,FacultyofScience,KingAbdulazizUniversity,Jeddah,SaudiArabia
*[email protected]
Abstract
OPENACCESS
Citation:SiddiquiM,AhmadMS,WahabA-t, Sevenmetaboliteswereobtainedfromthemicrobialtransformationofanabolic-androgenicste-
YousufS,FatimaN,NaveedShaikhN,etal.(2017) roidmibolerone(1)withCunninghamellablakesleeana,C.echinulata,andMacrophominapha-
Biotransformationofapotentanabolicsteroid,
seolina.Theirstructuresweredeterminedas10β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-
mibolerone,withCunninghamellablakesleeana,C.
one(2),6β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one(3),6β,10β,17β-trihydroxy-7α,17α-
echinulata,andMacrophominaphaseolina,and
biologicalactivityevaluationofitsmetabolites. dimethylestr-4-en-3-one(4),11β,17β-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-
PLoSONE12(2):e0171476.doi:10.1371/journal. 3-one(5),1α,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one(6),1α,11β,17β-trihydroxy-7α,17α-
pone.0171476
dimethylestr-4-en-3-one(7),and11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one(8),onthe
Editor:MohammadShahid,AligarhMuslim basisofspectroscopicstudies.Allmetabolites,except8,wereidentifiedasnewcompounds.
University,INDIA
ThisstudyindicatesthatC.blakesleeana,andC.echinulataareabletocatalyzehydroxylation
Received:October7,2016 atallylicpositions,whileM.phaseolinacancatalyzehydroxylationofCH andCH groupsof
2 3
Accepted:January20,2017 substrate1.Mibolerone(1)wasfoundtobeamoderateinhibitorofβ-glucuronidaseenzyme
(IC =42.98±1.24μM)duringrandombiologicalscreening,whileitsmetabolites2–4,and8
Published:February24,2017 50
werefoundtobeinactive.Mibolerone(1)wasalsofoundtobesignificantlyactiveagainstLeish-
Copyright:©2017Siddiquietal.Thisisanopen
maniamajorpromastigotes(IC =29.64±0.88μM).Itstransformedproducts3(IC =79.09±
accessarticledistributedunderthetermsofthe 50 50
CreativeCommonsAttributionLicense,which 0.06μM),and8(IC50=70.09±0.05μM)showedaweakleishmanicidalactivity,while2and4
permitsunrestricteduse,distribution,and werefoundtobeinactive.Inaddition,substrate1(IC =35.7±4.46μM),anditsmetabolite8
50
reproductioninanymedium,providedtheoriginal
(IC =34.16±5.3μM)exhibitedpotentcytotoxicityagainstHeLacancercellline(humancervi-
authorandsourcearecredited. 50
calcarcinoma).Metabolite2(IC =46.5±5.4μM)alsoshowedasignificantcytotoxicity,while
50
DataAvailabilityStatement:Allrelevantdataisin
3(IC =107.8±4.0μM)and4(IC =152.5±2.15μM)showedweakcytotoxicityagainst
thepaperandsupportinginformationfiles. 50 50
HeLacancercellline.Compound1(IC =46.3±11.7μM),anditstransformedproducts2
50
Funding:OPCWfinanciallysupportthewhole
(IC =43.3±7.7μM),3(IC =65.6±2.5μM),and4(IC =89.4±2.7μM)werealsofoundto
project,througharesearchprojectentitled, 50 50 50
bemoderatelytoxicto3T3cellline(mousefibroblast).Interestingly,metabolite8showedno
“Structuralandbiologicalstudiesonnewanabolic
steroidsobtainedbyBiotransformation”. cytotoxicityagainst3T3cellline.Compounds1–4,and8werealsoevaluatedforinhibitionof
tyrosinase,carbonicanhydrase,andα-glucosidaseenzymes,andallwerefoundtobeinactive.
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0171476 February24,2017 1/22
Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
Introduction
Mibolerone(7α,17α-dimethyl-19-nortestosterone)(1)isapotentsyntheticanabolicand
androgenicsteroid,marketedbytheUpjohnCompanyunderthebrandnameofCheck
Drops,forthetreatmentofestrous(heat)infemaledogs.Itisstabletometabolicconversion
intheratventralprostate.Becauseofitsstabilityandhighaffinitybinding,ithasbeenusedas
aligandforthecharacterizationandquantitationofandrogeninprostate,liver,andcultured
cells.Inaddition,mibolerone(1)ismorereceptor-selectiveforandrogenicreceptorthan
methyltrienolone.Thebindinginteractionofcompound1withtestosterone-estradiolbinding
globulinofhumanserumisweakerthan5α-dihydrotestosterone.Mibolerone(1)alsoacts
throughtheprogesteronereceptor(PR)asiteliminatesprogesteronereceptorexpressionat
lowerdoses(1nM),incontrastto5α-dehydrotestosterone(10–100nM),whichreducesPRto
basallevels.Inbreastcancercells,mibolerone(1)hasshownadualaction,i.e.,androgenicand
progestagenic[1–4].
Theregio-,chemo-,andstereo-selectivesynthesisoforganiccompoundshasbeenanarea
ofactiveresearchsinceseveraldecades.Manyoftheseconversionsaredifficulttoachieve
throughconventionalsyntheticmethodologies.However,biocatalystscancarryoutthesereac-
tionseffectively.Biocatalysishasseveraladvantagesoverchemicalsynthesis,suchasselectivity,
mildreactionconditions,andtheireco-friendlynature.Variousbiocatalysts,suchaspure
enzymesandwhole-cellsystems,arebeingusedforthetransformationoforganiccompounds.
However,whole-cellbiocatalysis,especiallywithfungi,isanefficientchoiceforregio-andste-
reo-selectivetransformations[5–9],astheyhaveP450cytochromeenzymesystems,whichcat-
alyzehydroxylationatvariouspositionsofsteroids[10–12].
Incontinuationofourresearchonthefungaltransformationofbioactivesteroids[13–17],
weincubatedmibolerone(1)(C H O ),withCunninghamella blakesleeana,C.echinulata,
20 30 2
andMacrophomina phaseolina.Compounds2–4(Fig1)wereobtainedonincubationofsub-
strate1withC.blakesleeana,andC.echinulata,whereasmetabolites5–8(Fig2)wereobtained
onthetransformationofsubstrate1withM.phaseolina.Metabolites2–7werefoundtobe
new,whereasmetabolite8wasidentifiedasaknowncompound.Interestingly,substrate1was
foundtobeactiveagainstβ-glucuronidaseenzyme,Leishmaniamajorpromastigotes,and
HeLa(cervicalcancer)and3T3(normal)celllinesinpreliminaryassays.
β-Glucuronidaseisaninducibleexoglycosidaseenzyme.Itsincreasedlevelinbloodcan
createproblemsinthedetoxificationprocessofvarioustoxicsubstances.Toxiccarcinogenic
substances,alongwithendogenouslyproducedtoxicmetabolitessuchassteroids,aremetabo-
lizedintheliver.Beforetheirexcretionintothesmallintestineviathebile,thesesubstances
undergoconjugationwithglucuronicacid.β-Glucuronidase,producedbyintestinalbacteria,
catalyzesthehydrolysisoftheseconjugatesinthecolon.Increasedactivityofβ-glucuronidase
isoneofthekeyobservationsincoloncancer.Hence,β-glucuronidaseplaysakeyroleinthe
etiologyofcoloncancer[18–22].
Leishmaniasis,aneglectedtropicaldisease(NTD),isamajorvectorbornediseaseofproto-
zoalorigin.Nearly350millionpeoplein88countriesareatriskofthedisease.Orallyavailable
drugsforthetreatmentofleishmaniasisareveryfewandoftenlesseffective.Therefore,devel-
opmentofsafeandeffectivenewtherapeuticagentsforleishmaniasisisurgentlyneededto
reducetheburdenofthedisease[23–25].
Afterbreastcancer,cervicalcanceristhesecondmostprevalentcancerinwomenacross
theworld.Inthiscancer,malignantcellsareformedintissuesofthecervix.Currently,radio-
therapy,surgery,andmostcommonly,cisplatinbasedchemotherapicagentsareusedforthe
treatmentofcancers.However,theresponseratetochemotherapyisoftenverylowduetofre-
quentdevelopmentofresistanceofcancercellsagainstchemotherapeuticagents.HeLacell
PLOSONE|DOI:10.1371/journal.pone.0171476 February24,2017 2/22
Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
Fig1.Compounds2–4obtainedbythebiotransformationofmibolerone(1)withCunninghamellablakesleeanaandC.echinulata.
doi:10.1371/journal.pone.0171476.g001
line,obtainedfromhumancervicalcancer,provideausefulmodeltoevaluatethecytotoxic
potentialofchemicalcompoundsagainstcervicalcancerinvitro[26–29].
Experimental
Chemicalsandreagents
Mibolerone(1)waspurchasedfromtheBettersynCo.,Ltd(China).Mediaingredientswere
purchasedfromDaejungChemicalsandMetalsCo.,Ltd.(Korea),OxoidLtd.(England),and
Sigma-Aldrich(Germany).
Chromatographicprotocols
Thepurityofcompound1andthedegreeofitstransformationswereanalyzedbyTLC(Thin
layerchromatography)(silicagel,20×20,0.25mmthick,PF Merck,Germany),whilesilica
254,
gel(70–230mesh,Merck,Germany)wasusedforcolumnchromatography.Compoundswere
finallypurifiedonarecyclingHPLC(JAILC-908W,Japan),equippedwithYMCL-80(4–
5μm,20−50mmi.d.).Cericsulphatereagentwasusedforvisualizingthecompoundson
TLCs.Allsolventsusedforchromatographywereofanalyticalgrade.
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Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
Fig2.Compounds5–8obtainedbythebiotransformationofmibolerone(1)withMacrophominaphaseolina.
doi:10.1371/journal.pone.0171476.g002
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Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
Instrumentalanalysis
1H-(400,500,and600MHz),and13C-NMR(100,125,and150MHz)and2D-NMRspectra
wererecordedonBrukerAvance-NMRspectrometers(France)inCD OD,CD COCD or
3 3 3
DMSO-d.MeltingpointswererecordedonBuchiM-560apparatus(Japan).EI-andHREI-MS
6
wererecordedonJEOLJMS-600H(Japan).Opticalrotationsofallisolatedcompoundswere
measuredonJASCOP-2000polarimeter(Japan)inchloroformormethanol.IRanalyseswere
performedonBrukerVector22FT-IRspectrometer(France).Evolution300UV-visiblespectro-
photometerwasusedtorecordtheUVspectra(ThermoScientific,England).Single-crystalX-ray
diffractiondatawascollectedonBrukerAPEXIID8Venturediffractometer,fittedwithPHO-
TON100detector(CMOStechnology),andfine-focussealedtubehavingX-raysource[CuKα
radiationα=1.54178Å].ReflectionintensitieswereintegratedusingSAINTsoftware.Absorp-
tioncorrectionwasdoneonM-multi-scan.StructuresweresolvedonSHELXTL[30–31].
Crystallographicdataforcompounds1,2,4,and8weredepositedwiththeCambridge
CrystallographicDataCenterandcanbeobtainedfreeofchargefromtheCambridgeCrystal-
lographicDataCenterviawww.ccdc.cam.ac.uk/data_request/cif.
Fungalcultures
FungalstrainsofCunninghamella blakesleeana(ATCC8688A),andCunninghamellaechinulata
(ATCC9244)wereobtainedfromtheAmericanTypeCultureCollection(ATCC),whereas
Macrophomina phaseolina(KUCC730)wasobtainedfromtheKarachiUniversityCultureCol-
lection(KUCC).AllcultureswerestoredonSabourauddextroseagar(SDA)at4˚C.
Generalfermentationprotocol
Theingredientsusedfor1Lculturemediumwerecomprised10gglucose,5gpeptone,5g
KH PO ,5gyeastextract,5gNaCl,and10mLglycerolin1Lofdistilledwater.
2 4
Theaforementionedingredientswereusedtopreparetheculturemediumforthegrowth
ofC.blakesleeana,C.echinulata, andM.phaseolina.Theexperimentswerecarriedouton
twoscales,i.e.,theexperimentalandthepreparativescales.Intheexperimentalscale,600
mLmediawaspreparedforeachfungus,transferredto6flasksof250mL,andautoclaved.
Twoflasksservedaspositive(fungalmedia+substrate)andnegative(fullygrownfungusin
media)controls,whereastheremainingfourflaskswereusedastestflasks.Thefungiwere
grownintestflasksandnegativecontrolbytransferringitsspores.Aftermaturegrowthof
thefungi,20mgofthesubstratewasdissolvedin0.5mLofmethanolandincubatedineach
culture-containingtestflask.Onetestflaskwasharvestedevery4thday,followedbyfiltra-
tionofthemycelia,andextractionwithdichloromethane(DCM).Basedonthesmallscale
screening,substrate1wassubjectedtopreparativescaletransformation.
Fourlitersofliquidmediawaspreparedforeachfungus,anddistributedequallyin40flasks
of250mLeach.Themediawasthenautoclavedandinoculatedwithsporesoftheappropriate
fungusat22˚C.After4daysofinoculation,funguscultureswerefoundtobefullymatured.
Substrate1(600mg)wasdissolvedin20mLmethanol,anddispensedequally(0.5mLineach)
inallflasks.Theseflaskswerethenplacedonarotatoryshakerfor12daysat22˚C.
Extractionofmetabolites
Onthe12thday,thecontentofallflasksofeachfunguswerecombinedandfilteredtoremove
themycelia.TheaqueouslayerwasextractedthricewithCH Cl (24L).Thecrudeextractwas
2 2
mademoisturefreebyaddingsodiumsulphate,filtered,andconcentratedonarotaryevapora-
tor,whichyieldedathickbrowngumlikematerial.
PLOSONE|DOI:10.1371/journal.pone.0171476 February24,2017 5/22
Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
Isolationofmetabolitesofmibolerone(1)fromtheincubationof
mibolerone(1)withCunninghamellablakesleeana,andC.echinulata
Incubationofsubstrate1withC.blakesleeana,andC.echinulatayieldedabout2gofcrude
extracts.Theextractsweresubjectedtosilicagelcolumnchromatography.Themobilephase
comprisedhexanes-acetonemixtures.Thepolarityofthemobilephasewasincreasedbyincreas-
ingtheconcentrationofacetone(5–100%gradientofacetone).500mLofsolventsystemateach
concentrationwaspassedthroughthecolumn.ThefractionsobtainedwereanalyzedbyTLC
analysis,andthefractionsofsimilarcompositionwerepooledtogether.Threemainfractions
(1–3)wereobtained,whichwerethenpurifiedonareversephaserecyclingHPLC.Metabolite2
(R =26min,MeOH:H O;70:30)wasobtainedfromfraction1.Fraction2yieldedcompound
T 2
3(R =23min,MeOH:H O;70:30),andcompound4(R =22min,MeOH:H O;70:30)was
T 2 T 2
obtainedfromfraction3onpurificationwithreversephaserecyclingHPLC.
17β-Hydroxy-7α,17α-dimethylestr-4-en-3-one(1). Single-crystalX-raydata:empirical
formulaC H O ;formulaweight302.44;crystalsystemorthorhombic,spacegroupP2 2 2 ,
20 30 2 1 1 1
unitcell,a=8.2316(5)Å,b=9.8785(6)Å,c=20.6304(12)Å,α=β=γ=90˚,volume1677.58(17)
A3,Z=4,calculateddensity1.197mg/m3,F(000)664,crystalsize0.32x0.24x0.12mm,Ɵrange
fordatacollection4.29to79.81˚,reflectionscollected20,227,uniquereflections3,597(R =
int
0.0976),goodness-of-fitonF21.097,finalRindices[I>2δ(I)]R =0.0661,wR2=0.2004,Rindi-
1
cesR1=0.0695,wR2=0.2074foralldata,largestdiff.peakandhole0.599and-0.620e.A-3.
10β,17β-Dihydroxy-7α,17α-dimethylestr-4-en-3-one(2). Whitecrystalline;yield(per-
centageyield):40mg(4%);meltingpoint200–201˚C;UVλ (logε):235(4.38);½a(cid:138)25=–22.5
max D
(c0.004,CH OH);IR(CHCl )υ ;3360(O-H),1654(α,β-unsaturatedketone);EI-MSm/z(rel.
3 3 max
int.,%):318.2[M+](59),300.0(63),290.2(23),230.2(47),229.1(64),43.9(100);HREI-MSm/z:
318.2192[M+](mol.formulaC H O ,calcd.318.2195);1H-NMR(CD OD,400MHz):Table1;
20 30 3 3
13C-NMR(CD OD,125MHz):Table1.Single-crystalX-raydata:empiricalformulaC H O ;
3 20 30 3
formulaweight318.44;crystalsystemorthorhombicspacegroupP2 2 2 ,unitcell,a=8.338(2)
1 1 1
Å,b=9.733(2)Å,c=20.949(5)Å,α=β=γ=90˚,volume1700.1(7)A3,Z=4,calculateddensity
1.244mg/m3,F(000)696,crystalsize0.31x0.11x0.07mm,Ɵrangefordatacollection4.22to
43.50˚,reflectionscollected3,259,uniquereflections770(R =0.0465),goodness-of-fitonF2
int
1.119,finalRindices[I>2δ(I)]R1=0.0418,wR2=0.1008,RindicesR1=0.0458,wR2=0.1035
foralldata,largestdiff.peakandhole0.294and-0.200e.A-3.
6β,17β-Dihydroxy-7α,17α-dimethylestr-4-en-3-one(3). Whitesolid;yield(percentage
yield):20mg(2%);meltingpoint219–221˚C;UVλ (logε):230(4.27);½a(cid:138)25=–134.2(c
max D
0.0014,CH OH);IR(CHCl )υ ;3431(O-H),1659(α,β-unsaturatedketone);EI-MSm/z
3 3 max
(rel.int.,%):318.3[M+](16),300.3(100),242.2(39),229.2(98),174.1(41),147.0(27),136.0
(26),95.0(21),42.9(54);HRESI-MSm/z:318.2196[M+](mol.formulaC H O ,calcd.
20 30 3
318.2195);1H-NMR(CD OD,500MHz):Table1;13C-NMR(CD OD,125MHz):Table1.
3 3
6β,10β,17β-Trihydroxy-7α,17α-dimethylestr-4-en-3-one(4). Whitecrystalline;yield
(percentageyield):300mg(30%);meltingpoint228–229˚C;UVλ (logε):230(5.20);½a(cid:138)25=
max D
–22.9(c0.0041,CH OH);IR(CHCl )υ ;3412(O-H),1664(α,β-unsaturatedketone);
3 3 max
EI-MSm/z(rel.int.,%):334.2[M+](100),298.2(24),240.1(11),227.1(64),172.1(19),43.0
(16);HREI-MSm/z:334.2159[M+](mol.formulaC H O ,calc.for334.2144);1H-NMR
20 30 4
(CD OD,400MHz):Table1;13C-NMR(CD OD,125MHz):Table1.Single-crystalX-raydata:
3 3
empiricalformulaC H O ;formulaweight352.46;crystalsystemorthorhombic,spacegroup
20 32 5
P2 2 2 ,unitcell,a=7.6451(10)Å,b=10.734(2)Å,c=23.051(3)Å,α=β=γ=90˚,Volume
1 1 1
1891.6(5)A3,Z=4,calculateddensity1.238mg/m3,F(000)768,crystalsize0.30x0.25x0.20
mm,Ɵrangefordatacollection7.11to70.13˚,reflectionscollected15,121,uniquereflections
2040(R =0.0240),goodness-of-fitonF21.079,finalRindices[I>2δ(I)]R =0.0300,
int 1
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Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
4 (JinHz)δH 1.84,overlap2.16,m 2.26,m;2.63,m ─ 5.83,s ─ 4.08,s 2.01,m 2.30,m 1.35,m ─ 1.67,overlap;1.78,m 1.32,overlap;1.54,m ─ 1.45,m 1.35,m;1.57,m 1.69,m;1.88,m ─ 0.93,s 0.75,d(J=7.2)19/7β 1.19,s
δC 34.1 34.7 202.3 127.9 160.6 80.1 38.6 33.6 46.6 72.4 20.8 32.1 46.9 46.6 23.5 39.0 82.1 14.3 10.9 26.1
4)
=
J8a/7e
5,
1.
1
=
J8a/14a 5)
3 (JinHz)δH 1.32,m;1.61,m 2.32,m;2.40,m ─ 5.86,d(J=2)4/6 ─ 3.97,d(J=3)6/7 1.95,m 2.14,ddd(J=8a/9a 1.14,m 2.50,m 1.44,m;1.54,m 1.30,m;1.52,m ─ 1.42,m 1.89,m;2.30,m 1.67,m;1.87,m ─ 0.94,m 0.74,d(J=7.19/7β 1.18,s
δC 27.3 37.2 202.9 127.4 167.3 78.7 38.4 37.4 43.3 39.8 23.2 32.5 47.1 46.7 27.6 39.1 82.1 14.5 11.1 26.1
16.8) 3.6)
= =
J1/3 J6/7
nds1–4(ppm).δ 2 (JinHz)δH 1.92,m;2.22,m 2.27,m;2.56,ddd(J=4.8,1/2 ─ 5.72,s ─ 2.10,m;2.90,dd(J=13.2,6/6 2.02,m 1.99,m 1.41,m ─ 1.70,m;1.73,m 1.29,overlap;1.56,overlap ─ 1.32,overlap 1.30,overlap;1.57,overlap 1.67,m;1.84,m ─ 0.92,s 0.79,d(J=6.8)19/7β 1.18,s
u
o
comp δC 34.5 34.5 202.0 126.7 166.7 41.0 39.5 32.1 47.4 70.8 21.0 32.3 46.7 46.6 23.6 39.1 82.1 14.3 12.6 26.1
of
z)
JinH =4.8)
a( J6/7 Hz) Hz)
at 4, M M
d 1
H-NMRchemicalshift 1 (JinHz)δH 1.94,m;2.35,overlap 1.31,overlap;1.51,overlap ─ 5.79,s ─ 2.33,overlap;2.56,dd(J=6/6 1.99,m 1.66,overlap 1.83,overlap 2.15,m 1.57,overlap;1.86,overlap 1.69,overlap;1.86,overlap ─ 1.42,overlap 1.34,overlap;1.53,overlap 2.32,overlap;2.36,overlap ─ 0.92,s 0.79,d(J=7.2)19/7β 1.29,s 13Hz),andC-NMR(12513Hz),andC-NMR(125 ne.0171476.t001
1 M M o
13C-and δC 27.8 32.5 202.5 126.6 169.4 44.5 32.1 44.6 43.6 44.2 23.3 39.1 46.9 47.4 27.7 37.4 82.1 14.5 13.1 26.1 MR(400 MR(500 1/journal.p
e1. on H-N H-N 0.137
Tabl Positi 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1a=1b= doi:1
PLOSONE|DOI:10.1371/journal.pone.0171476 February24,2017 7/22
Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
wR2=0.0799,RindicesR1=0.0302,wR2=0.0802foralldata,largestdiff.peakandhole0.243
and-0.175e.A-3.
Isolationofmetabolitesofmibolerone(1)fromtheincubationof
mibolerone(1)withM.phaseolina
Incubationofsubstrate1withM.phaseolinaafforded2.3gcrudeextract.Silicagelcolumn
chromatographyofthecrudeextractyieldedfourmainfractions(4–7),whichwerefurther
purifiedonreversephaserecyclingHPLC.Metabolite5(R =21min,6mg)wasobtained
T
fromfraction4throughpurificationonreversephaseHPLC(MeOH:H O;70:30).Metabolite
2
6(R =22min,7mg)fromfraction5,compound7(R =24min,6mg)fromfraction6,and
T T
compound8(R =23min,6mg)fromfraction7wereobtained,onpurificationwithreverse
T
phaserecyclingHPLC(MeOH:H O;70:30).
2
11β,17β-Dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one(5). White
solid;yield(percentageyield):2.9mg(0.4%);meltingpoint228–230˚C;UVλ (logε):248
max
(3.82);½a(cid:138)25=+70.0(c0.0019,CHOH);IR(CHCl )υ ;3412(O-H),1643(α,β-unsaturated
D 3 3 max
ketone);EI-MSm/z(rel.int.,%):334.2[M+](25),317.2(31),286.1(32),285.1(80),267.1(51),242.1
(50),229.1(100)133.1(18),55.0(11);HREI-MSm/z:334.2146[M+](mol.formulaC H O,calc.
20 30 4
for334.2144);1H-NMR(CD OD,600MHz):Table2;13C-NMR(CD OD,150MHz):Table2.
3 3
1α,17β-Dihydroxy-7α,17α-dimethylestr-4-en-3-one(6). Whitesolid;yield(percentage
yield):2.4mg(0.24%);meltingpoint170–174˚;UVλ (logε):247(2.99);½a(cid:138)25=–41.4(c
max D
0.0049,CH OH);(CHCl );υ 3433(O-H),1651(α,β-unsaturatedketone);EI-MSm/z(rel.
3 3 max
int.,%):318.9[M+](28),301.9(35),300.9(100),299.9(96),261.9(61),228.8(67),226.9(49)
173.9(53),135.0(33);HREI-MSm/z:318.2193[M+](mol.formulaC H O ,calc.for
20 30 3
318.2195);1H-NMR(CD OD,400MHz):Table2;13C-NMR(CD OD,150MHz):Table2.
3 3
1α,11β,17β-Trihydroxy-7α,17α-dimethylestr-4-en-3-one(7). Whitesolid;yield(percent-
ageyield):2.4mg(0.34%);meltingpoint280–282˚C;UVλ (logε):247(4.12);½a(cid:138)25=–94.2(c
max D
0.0019,CH OH);IR(CHCl )υ ;3418(O-H),1661(α,β-unsaturatedketone);EI-MSm/z(rel.
3 3 max
int.,%):334.2[M+](2.6),318.1(41),317.1(90),259.1(68),241.1(49),228.1(100),214.1(81)201.1
(47),43.0(36);HREI-MSm/z334.2140[M+](mol.formulaC H O ,calc.for334.2144);1H-NMR
20 30 4
(CD OD,600MHz):Table2;13C-NMR(CD OD,150MHz):Table2.
3 3
11β,17β-Dihydroxy-7α,17α-dimethylestr-4-en-3-one(8). Whitecrystalline;yield(per-
centageyield):21.1mg(3%);meltingpoint249–252˚C;UVλ (logε):229(3.88);½a(cid:138)25=
max D
+104.3(c0.0021,IRCH OH);(CHCl )υ ;3410(O-H),1651(α,β-unsaturatedketone);
3 3 max
EI-MSm/z(rel.int.,%):334.2[M+](2.6),318.1(41),317.1(90),259.1(68),241.1(49),228.1
(100),214.1(81)201.1(47),43.0(36);HREI-MSm/z:318.2211[M+](mol.formulaC H O ,
20 30 3
calcd.for318.2195);1H-NMR(CD OD,400MHz):Table2;13C-NMR(CD OD,100MHz):
3 3
Table2.Single-crystalX-raydata:empiricalformulaC H O ;formulaweight318.44;crystal
20 30 3
systemmonoclinic,spacegroupP2 ,unitcell,a=5.9750(11)Å,b=11.550(3)Å,c=12.642(2)
1
Å,β=99.728(14˚,volume859.9(3)A3,Z=2,calculateddensity1.230mg/m3,F(000)348,crys-
talsize0.32x0.28x0.18mm,Ɵrangefordatacollection7.52to72.00˚,reflectionscollected
13,571,uniquereflections3,334(R =0.0236),goodness-of-fitonF21.037,finalRindices
int
[I>2δ(I)]R1=0.0270,wR2=0.0734,RindicesR1=0.0272,wR2=0.0736foralldata,largest
diff.peakandhole0.219and-0.160e.A-3.
Assayprotocolforβ-glucuronidaseinhibition
Theinhibitionofβ-glucuronidaseenzyme(E.C.3.2.1.31,bovineliver)bytestcompoundswas
determinedonaspectrophotometerbymeasuringtheabsorbanceofp-nitrophenolat405nm,
PLOSONE|DOI:10.1371/journal.pone.0171476 February24,2017 8/22
Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
Table2. 13C-and1H-NMRchemicalshiftdata(JinHz)ofcompounds5–8(δppm).
Position 5 6 7 8
δ δ (JinHz) δ δ (JinHz) δ δ (JinHz) δ δ (JinHz)
C H C H C H C H
1 27.2 2.29,overlap;2.34,overlap 66.3 4.40,d(J=2.8) 66.3 4.38,t(J=3) 27.3 1.57,m;2.33,m
2 37.5 2.30,overlap;2.35,overlap 47.1 2.41,overlap;2.45, 48.9 2.55,overlap;2.59, 37.5 2.34,m2(H)
overlap overlap
3 202.6 - 200.8 - 200.9 - 202.6 -
4 126.8 5.83,s 126.4 5.83,s 126.5 5.86,s 126.8 5.79,s
5 170.8 - 164.9 - 166.4 - 170.6 -
6 44.3 2.31,overlap;2.54,dd(J 44.3 2.37,d(J=14.8); 44.2 2.52,dd(J = 44.3 2.31,m;2.53,dd(J =13.6,J =
6/6 6/6 6/6 6/7
=13.5,J =4.5) 2.53,overlap 14.4,J =4.2) 4.4)
6/7 6/7
7 32.1 2.18,m 31.4 1.95,m 31.4 2.02,m 32.1 2.07,m
8 38.9 1.96,m 36.4 1.68,overlap 38.9 1.96,m 39.3 1.94,m
9 49.2 1.23,m 44.2 1.69,overlap 41.2 1.81,overlap 48.3 1.24,m
10 39.1 2.65,overlap- 48.5 2.27,m 43.3 2.85,overlap; 39.0 2.64,m
11 67.4 4.19,d(J =7.2) 27.0 1.32,m;1.99,m 67.2 4.25,d(J = 67.5 4.21,d(J =3.2)
11/9,12 11/9,12 11/9,12
2.4)
12 39.9 1.87,dd(J =13.8,J 32.4 1.37,m;1.54,m 39.8 1.49,m;1.82, 39.9 1.46,dd;(J =14,J =3.2);
12/12 12/11 12/12 12/11
=2.4);1.49,overlap overlap 1.82,dd(J =14,J =2.8)
12/12 12/11
13 46.4 - 46.9 - 47.1 - 46.5 -
14 48.2 1.48,m 47.6 1.31,overlap 46.8 1.58,m 48.4 1.38,overlap
15 23.6 1.35,m;1.61,m 23.2 1.21,m;1.45,m 23.3 1.41,m;1.61,m 26.4 1.35,m;1.61,m
16 33.3 1.67,m;1.88,m 39.0 1.69,overlap;1.89, 48.7 1.45,m;1.59,m 39.0 1.67,m;1.88,m
overlap
17 84.5 - 82.2 - 82.5 - 82.4 -
18 17.4 1.15,s 14.4 0.93,s 17.0 1.13,s 17.1 1.12,s
19 13.3 0.79,d(J =7.2) 13.0 0.81,d(J =7.2) 12.9 0.82,d(J = 13.0 0.79,d(J =6.8)
19/7β 19/7β 19/7β 19/7β
7.2)
20 67.7 3.42,d(J =11.4);3.57, 26.1 1.21,s 26.4 1.18,s 23.3 1.18,s
20/20
d(J =11.4)
20/20
c=1H-NMR(600MHz),and13C-NMR(150MHz)
d=1H-NMR(400MHz),and13C-NMR(150MHz)
e=1H-NMR(400MHz),and13C-NMR(100MHz)
doi:10.1371/journal.pone.0171476.t002
producedfromthesubstrate.Thereactionmixturecomprised185μLof0.1Macetatebuffer,
and5μLoftestcompoundsolution,and10μLof(1U)enzymesolutionina96-wellplate.The
mixturewasincubatedat37˚Cfor30min.ThetestcompoundsweresolubilizedinDMSO
(100%),and5μLvolumewasaddedineachwell(2%oftotalvolume).Similarconditionswere
usedforthestandard(D-saccharicacid1,4-lactone).Theplateswerereadonamultiplate
reader(SpectraMaxplus384)at405nmand37˚C,afteradditionof50μLof0.4mMp-nitro-
phenyl-β-D-glucuronide.Allassayswereperformedintriplicate.IC valueswerecalculated
50
throughEZ-Fitsoftware(PerrellaScientificInc.,Amherst,MA,USA).
Assayprotocolforleishmanicidalactivity
Leishmaniamajor(DESTO,Pakistan)wasculturedinamixtureofNNN-biphasicmedium,
andnormalphysiologicalsalinesolution.L.majorpromastigotesweregrownonRPMI1640
medium,supplementedwithfetalbovineserum(FBS)(10%heatinactivated).Parasiteswere
centrifuged(atlogphase)for10minat2,000rpmandwashedthricewithsaline.Thefinal
densityoffreshculture(106cells/mL)wasacquiredthroughdilutionofparasites.Sample
PLOSONE|DOI:10.1371/journal.pone.0171476 February24,2017 9/22
Fungalbiotransformationofmibolerone,andbiologicalactivityevaluationofitsmetabolites
solutionof1mgoftestcompoundswaspreparedinamixture50μLofDMSOand950μLof
RPMImedia.Fullydissolvedcompoundsweretransferredto96-wellplate.Thefirstrowof
96-wellplatereceived180μLofthemedium,whiletheremainingwellsreceived100μL
medium.Testcompounds(20μLeach)wereaddedintothemediumcontainingwells,fol-
lowedbyserialdilution.Theparasiteculture(100μL)wasthentransferredtoeachwell.Nega-
tivecontrolscomprisedofonlythegrowthmedium,whilestandardleishmanicidaldrugs
(amphotericinorpentamidine)wereusedaspositivecontrols.Theplatewasthenincubated
for72hat21–22˚C.Theinhibitoryeffectoftestcompoundsontheculturewasanalyzed
microscopicallyonanimprovedNeubauercountingchamber.IC valueswerecalculated
50
throughEzfit5.03PerellaScientificsoftware(USA).
Assayprotocolforcytotoxicity
MTT(3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide)assaywasemployedto
studythecytotoxicactivityagainstHeLa(humancervicalcarcinoma,providedbyProf.Dr.
AnwarAliSiddiquifromAgaKhanUniversity,Karachi,Pakistan),andmousefibroblast3T3
(ATCC1CRL-1658,purchasedfromAmericanTypeCultureCollection,ATCC,Virginia,
USA)celllines.Thecells(1×105/well)wereplatedin0.2mLofDMEMhighglucose
medium/wellin96-wellplates.Thecellsweretreatedfor24hourswithtestcompoundsinthe
rangeof25,50,75,100,and200μMconcentrations,respectively.FortheMTTassay,the
mediumfromthewellswasremovedcarefullyafter24hourstreatment.Eachwellwaswashed
with1XPBSfor2–3times,and200μLofMTT(5mg/mL)wasaddedintomediacontaining
wells(1:10).Theplateswereincubatedfor4hoursat37˚C,ina5%CO incubator.Afterincu-
2
bation,MTTwasremovedand0.1mLofDMSOwasaddedtoeachwellandmixedbykeeping
onastirrer.Thepresenceofviablecellswasvisualizedbythedevelopmentofpurplecolordue
toformationoftheformazancrystals.Theplateswereobservedunderspectrophotometer
(Spectramax)andabsorbancewastakenat545and570nmforcancerandnormalcelllines,
respectively.Measurementswereperformedandtheconcentrationrequiredfora50%inhibi-
tionofviability(IC )wasdeterminedgraphically.
50
Resultsanddiscussion
Structureelucidationofcompounds1–8
Compound1isawhitesolidwiththe[M+]atm/z302.2.Theidentityofsubstrate1wascon-
firmedby1H-,13C-NMRand2D-NMRtechniques(Table1)[1–4].Single-crystalX-raydif-
fractionanalysisofcompound1showedfourfusedrings,i.e.,A(C1-C5/C10),B(C5-C10),C
(C8-C9/C11-C14),andD(C13-C17).RingAexistsinahalfchairconformationandcontains
α-βunsaturatedcarbonylmoiety,whereastrans-fusedringsB,andCwereinchairconforma-
tions.FivememberedringDattainsanenvelopeconformation.Aβ-orientedhydroxylgroup
atC-17isinapseudoequatorialorientation(Fig3).Single-crystalX-raydataof1wassubmit-
tedtotheCambridgeCrystallographicDataCentre(CCDC1483437).
Metabolite2wasisolatedaswhitecrystals.Itdisplayedthe[M+]inHREI-MSatm/z
318.2192(C H O ,calcd.318.2195),whichsuggestedtheadditionofanoxygeninsubstrate
20 30 3
1(m/z302.45).TheIRabsorbancesat1654and3360cm-1wereduetothepresenceofα,β-
unsaturatedketone,andhydroxylfunctionalities,respectively.The1H-NMRspectrumwas
distinctlysimilartosubstrate1(Table1).Anadditionaloxygenatedquaternarycarbonsignal
(δ70.8)wasobservedinthe13C-NMRspectrumofcompound2,whichsuggestedthehydrox-
ylationofsubstrate1(Table1).ThisOHgroupwasplacedatC-10,basedontheHMBCcorre-
lationsofH-4(δ5.72,s)andH -1(δ1.92,m;2.22,m)withC-10(δ70.8)(Fig4).TheOH(δ
2
4.93)(DMSO-d )atC-10wasdeducedtobeβ-orientedbasedonitsNOEcorrelationwiththe
6
PLOSONE|DOI:10.1371/journal.pone.0171476 February24,2017 10/22