ANewSynthe+cSpiroketal:StudiesonAn+tumorAc+vityonMurineMelanomaModelInVivoandMechanismofAc+onInVitro
MariaPiaFugge+a*1,PietroSpanu*2,FaustaUlgheri2,FrancescoDeligia2,GiovanniLoriga2,PaolaCarta2,AlbertoMannu2,VeronicaTro+a3,RosannaDeCicco3,AdrianoBarra3,EnricaZona3,FrancoMorelli*3
1Is$tutodiFarmacologiaTraslazionale-ConsiglioNazionaledelleRicerche,Roma,Italy
2Is$tutodiChimicaBiomolecolare-ConsiglioNazionaledelleRicerche,Sassari,Italy3Is$tutodiGene$caeBiofisicaA.Buzza$Traverso-ConsiglioNazionaledelleRicerche,Napoli,Italy
• INTRODUCTION:InspiredbybioacHvenaturalspiroketals,spirocyclicrigidifiedstructureshavedemonstratedtobe privileged scaffolds for the design of new tool compounds endowed with diversifiedbiological acHviHes. We synthesised the natural-like spiroketal, 2-hydroxy-8-methyl-1,7-dioxaspiro[5.5]undec-3-en-5-one(5)thatshowedapotentanHcanceracHvityagainsthumancancercellsofdifferentnatureandhistotype.1InordertoconfirmthetherapeuHcpotenHalofthismoleculeweverifiedinvitroandinvivo,inasyngenicmurinemelanomamodel,thatourproductisabletoinducecancerregressionandgrowthinhibiHon.
Modulator of tubulin cytoskeleton integrityPhosphatase inhibitors
Cytotoxic against cancer cellsmicrotuble assembly inhibitor Proapoptotic activity
O
OMe
N2
OMe
MeO
OO
O
O
O
HNO
O O
COOH
O
HO
OH
HO OHO
N
OO
O
Me
2 3
4 5 6
7 8
OO
MeMe
OTBS
AcO
OO
OH
HOO
O
OBn
Me1
Me
Cytotoxic agents towardsprimary CLL cells
Telomerase inhibitor proapototic activity antitumor activity
SGLT2 inhibitor
Modulator of the RXRα gene tanscription factor
anti-Helicobacter pylori activity
OH
• SYNTHESISOFTHESPIROKETAL5:Thesynthesisof thespiroketalic stereoisomericmixture5a,bwascarriedoutbyusinga twosteps procedurebasedon2-furyl ketoneoxidaHon-rearrangementmethod. Because, in ourprevious experiments, �the inhibiHon acHvity of the enanHomers was comparable, thesteroisomericmixture5a,bwasusedassuchforinvitroandinvivoexperiments.
• CONCLUSION:We have idenHfied the spiroketal5a,b as a new promising anHcancer agentwith in vivo acHvity on amurinemelanomamodel.Compound5a,bshowedpotentdose-dependentanHcancerefficacyinsyngenicmurinemodel(C57Blackmice)ofmelanoma,suppressingcancergrowthbyanaverageof90%atadoseof5mg/kgbyoneintra-peritoneumadministraHonatalternatedaysfor15days.ItalsodisplayedhighanHcanceracHvityintheB16cellsinvitrowithnanomolarIC50value.InaddiHontotheproapoptoHcandtelomerase inhibiHon acHvity previously observed, the compound 5a,bhas also shown to inhibit cellmigraHonandthedeterioraHonoftheacHncytoskeleton.MoreoverourspiroketalstronglyreducestheHIF1αexpressionthatisconsideredasregulatorofmulHplecellularfuncHonsrelatedtotheprogressionfrom primary to metastaHc disease. Therefore, although the full mechanism of acHon has yet to becompletelyelucidated,wecanconcludethatspiroketal5a,bisapromisinganHcancerdrugcandidatefortheclinicaltreatmentofmelanoma.
O OO+ O
OH
ONBS
THF/H20BuLi
TMEDA, THF**
9 10 11
OO
O5a
O
OO
5b
OO
O HOOH HO+
12
• INVITROACTIVITY:Inthefirstsetofexperiments,theeffectofspiroketal5a,bwasevaluated invitroonB16cellgrowthinordertoconfirmonthismurinemodel,thepreviouslyreportedefficacyonhumancell lines.Moreover, to acquire addiHonal informaHonon theorigin of the anHproliferaHveacHvity,wehavealsostudiedtheroleofcellcyclemodificaHon,theapoptosisinducHon,themigraHoncharacterisHcsofB16culturedcells, thegeneexpressionof thehypoxia induciblefactor1α (HIF1α) inB16murinemelanomacellsandthemodificaHonof theacHnstructureandcytoskeletonconformaHoninducedby5a,b.
• INVIVOACTIVITY:TheassessmentoftheinvivoacHvityofthespiroketal5a,binawell-knownB16/C57BL/6Jsyngenicmodelofmurinemelanomawas performed. The effect of this compound on cancer growth reported in theFigures,showedatumorvolumereducHonwithrespecttothecontrol.
Control24h 48h
ac.n
HIF1α
PCRexpressionanalysisofHIF1αinpresenceofspiroketal5a,b
Cell migra*on analysis.Culture of untreatedcontrol cells (le6 plate)and treated with 10 µg/mL of5a,ba6er 24 hours(rightplate).
Proapopto'cinVitroAc'vity
0
50
100
control 5ab0.62μg/ml5ab1.25μg/ml5ab2.5μg/ml
Luminescence
An#prolifera#veeffectof5a,bonB16tumorcellline
Cellgrowthinhibi#onevaluatedbyMTTtest
CellProlifera.onInhibi.onAssay
0
200
400
600
800
0h 24h 48h 72h liv
e ce
ll nu
mbe
r x
100
0 B16 CT
0,781
3,125
12,5
25
50
200
0
20
40
60
0,39
0,78
1,56
3,125
6,25
12,5
25
50
100
200
Percen
tofg
rowth
inhibi.o
n
Reorganiza*onofthecytoskeletoninresponseto5a,b(B16Cells)
TumorGrowthInhibi/oninSpiroketalTreatedMice
• Acknowledgements:ThisstudywassupportedbyRegioneAutonomadellaSardegna-Italy(researchgrant Dr. Paola Carta). We thank Dr. Maria CrisHna Porcu and Mr. Salvatore Baiano for technicalassistanceandMr.AntonioGuicciardiforanimalexperiments.
• References:1)Fugge+a,M.P.;DeMico,A.;Co+arelli,A.;Morelli,F.;Zonfrillo,M.;Ulgheri,F.;Peluso,P.;Mannu,A.; Deligia, F.; Marchei, M.; Roviello, G.; Reyes Romero, A.; Dömling, A.; Spanu, P. Synthesis and EnanHomericSeparaHon of a Novel Spiroketal DerivaHve: A Potent Human Telomerase Inhibitor with High in Vitro AnHcancerAcHvity.J.Med.Chem.2016,59,9140−9149.
