Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9

Article abstract of DOI:10.1039/c6md00159a

Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 μM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM).

Full text of DOI:10.1039/c6md00159a

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Chessum, S. Lamont, K. G. Pike, P. F. Faulder, J. Demeritt, P. D. Kemmitt, J. Tucker, L. Zani, M.
Cheeseman, R. Isaac, L. Goodwin, J. Boros, F. Raynaud, A. Hayes, A. Henley, E. de Billy, C. Lynch, S. Sharp,
R. te Poele, L. O Fee, K. Foote, S. Green, P. Workman and K. Jones, Med. Chem. Commun., 2016, DOI:
1
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JournalꢀNameꢀ
ꢀ
ARTICLEꢀ
Discoveryꢀofꢀ4,6-disubstitutedꢀpyrimidinesꢀasꢀpotentꢀinhibitorsꢀofꢀ
theꢀHeatꢀShockꢀFactorꢀ1ꢀ(HSF1)ꢀstressꢀpathwayꢀandꢀCDK9.ꢀ
Receivedꢀ00thꢀJanuaryꢀ20xx,ꢀ
Acceptedꢀ00thꢀJanuaryꢀ20xxꢀ
a§
a§
b
b
b
b
CarlꢀS.ꢀRye, ꢀNicolaꢀE.ꢀA.ꢀChessum, ꢀScottꢀLamont, ꢀKurtꢀG.ꢀPike, ꢀPaulꢀFaulder, ꢀJulieꢀDemeritt, ꢀ
b
b,d
a,c
a
b
PaulꢀKemmitt, ꢀJulieꢀTucker, ꢀLorenzoꢀZani, ꢀMatthewꢀD.ꢀCheeseman, ꢀRosieꢀIsaac, ꢀLouiseꢀ
DOI:ꢀ10.1039/x0xx00000xꢀ
b
b
a
a
a
ꢀa
Goodwin, ꢀJoannaꢀBoros, ꢀFlorenceꢀRaynaud, ꢀAngelaꢀHayes, ꢀAlanꢀT.ꢀHenley, ꢀEmmanuelꢀdeꢀBilly, ꢀ
www.rsc.org/ꢀ
a
a
a
aꢀ
b
ChristopherꢀJ.ꢀLynch, ꢀSweeꢀY.ꢀSharp, ꢀRobertꢀteꢀPoele, ꢀLisaꢀO’ꢀFee, KevinꢀM.ꢀFoote, ꢀStephenꢀ
b
a
a
Green, ꢀPaulꢀWorkman, *ꢀandꢀKeithꢀJones *ꢀ
Heatꢀshockꢀfactorꢀ1ꢀ(HSF1)ꢀisꢀaꢀtranscriptionꢀfactorꢀthatꢀplaysꢀkeyꢀrolesꢀinꢀcancer,ꢀincludingꢀprovidingꢀaꢀmechanismꢀforꢀcellꢀ
survivalꢀunderꢀproteotoxicꢀstress.ꢀTherefore,ꢀꢀinhibitionꢀofꢀtheꢀHSF1-stressꢀꢀpathwayꢀrepresentsꢀanꢀexcitingꢀnewꢀ
opportunityꢀinꢀꢀcancerꢀtreatment.ꢀWeꢀemployedꢀanꢀunbiasedꢀphenotypicꢀscreenꢀtoꢀꢀdiscoverꢀinhibitorsꢀofꢀtheꢀHSF1-stressꢀ
pathway.ꢀUsingꢀthisꢀapproachꢀweꢀidentifiedꢀanꢀinitialꢀhitꢀ(1)ꢀbasedꢀonꢀaꢀ4,6-pyrimidineꢀscaffoldꢀ(2.00ꢀµM).ꢀOptimisationꢀofꢀ
cellularꢀSARꢀledꢀtoꢀanꢀinhibitorꢀwithꢀimprovedꢀpotencyꢀ(25,ꢀ15ꢀnM)ꢀꢀinꢀtheꢀHSF1ꢀphenotypicꢀassay.ꢀTheꢀ4,6-pyrimidineꢀ25ꢀ
wasꢀalsoꢀshownꢀtoꢀhaveꢀhighꢀpotencyꢀagainstꢀtheꢀCDK9ꢀenzymeꢀ(3ꢀnM).ꢀ
stronglyꢀassociatedꢀwithꢀmetastasisꢀandꢀpoorꢀsurvivalꢀinꢀcancerꢀ
9
patients. ꢀThereꢀareꢀmultipleꢀmechanismsꢀbyꢀwhichꢀHSF1ꢀhasꢀ
1)ꢀIntroductionꢀꢀ
beenꢀ proposedꢀ toꢀ facilitateꢀ oncogenesis.ꢀ HSF1ꢀ upregulatesꢀ
proteinsꢀinvolvedꢀinꢀdiverseꢀbiologicalꢀprocessesꢀwhichꢀincludeꢀ
cellꢀ cycleꢀ progression,ꢀ survival,ꢀ glucoseꢀ metabolism,ꢀ DNAꢀ
Heatꢀshockꢀfactorꢀ1ꢀ(HSF1)ꢀisꢀaꢀtranscriptionꢀfactorꢀthatꢀisꢀtheꢀ
masterꢀ regulatorꢀ ofꢀ theꢀ canonicalꢀ heatꢀ shockꢀ response,ꢀ
modulatingꢀtheꢀexpressionꢀofꢀhundredsꢀofꢀgenesꢀcriticalꢀtoꢀtheꢀ
4,ꢀ 10
1-3
repairꢀ andꢀ chromatinꢀ re-modelling.
ꢀ Furthermore,ꢀ HSF1ꢀ
survivalꢀ ofꢀ theꢀ cell. ꢀ HSF1ꢀ isꢀ implicatedꢀ inꢀ theꢀ cellularꢀ
responseꢀ toꢀ aꢀ varietyꢀ ofꢀ stressorsꢀ andꢀ playsꢀ aꢀ keyꢀ roleꢀ inꢀ
oncogenesisꢀandꢀmalignantꢀprogression,ꢀamongꢀotherꢀbenefitsꢀ
enablingꢀtheꢀcellꢀtoꢀcopeꢀwithꢀtheꢀproteotoxicꢀstressꢀresultingꢀ
supportsꢀ malignantꢀ progressionꢀ byꢀ promotingꢀ tumourꢀ
1
1-13
invasion,ꢀangiogenesisꢀandꢀmetastasis,
re-programmingꢀ ofꢀ stromalꢀ cellsꢀ withinꢀ theꢀ tumourꢀ
ꢀwhichꢀincludesꢀtheꢀ
14
4,ꢀ5
ꢀ
microenvironment. ꢀ
fromꢀmalignantꢀtransformation.
Inꢀadditionꢀtoꢀitsꢀtransientꢀactivationꢀinꢀtheꢀclassicalꢀheatꢀshockꢀ
Aꢀ keyꢀ featureꢀ inꢀ theꢀ HSF1-mediatedꢀ responseꢀ toꢀ proteotoxicꢀ
,ꢀ6- stressꢀ isꢀ theꢀ upregulationꢀ ofꢀ heatꢀ shockꢀ proteinsꢀ (HSPs)ꢀ
4
response,ꢀHSF1ꢀisꢀfrequentlyꢀupregulatedꢀinꢀhumanꢀcancers.
8
15
includingꢀ HSP72ꢀ andꢀ HSP90. ꢀ Theꢀ HSPsꢀ areꢀ chaperoneꢀ
proteinsꢀ criticalꢀ forꢀ properꢀ proteinꢀ folding,ꢀ preventingꢀ self-
association,ꢀ maintainingꢀ activeꢀ multi-proteinꢀ complexesꢀ andꢀ
ꢀ
Anꢀ HSF1-regulatedꢀ transcriptionalꢀ programꢀ hasꢀ beenꢀ
identifiedꢀthatꢀisꢀspecificꢀtoꢀhighlyꢀmalignantꢀcells,ꢀoverlappingꢀ
withꢀ butꢀ distinctꢀ fromꢀ theꢀ heatꢀ shockꢀ response,ꢀ whichꢀ isꢀ
16,ꢀ 17
directingꢀmisfoldedꢀproteinsꢀtoꢀbeꢀdegraded.
ꢀInꢀaddition,ꢀ
depletionꢀ ofꢀ HSF1ꢀ destabilizesꢀ ribosomalꢀ subunitꢀ proteins,ꢀ
whichꢀ revealsꢀ aꢀ linkꢀ betweenꢀ cellularꢀ chaperoningꢀ andꢀ
18
translationalꢀ capacity. ꢀ Importantlyꢀ thereꢀ isꢀ aꢀ positiveꢀ
correlationꢀ betweenꢀ increasedꢀ expressionꢀ ofꢀ nuclearꢀ
(
includingꢀpoorꢀprognosisꢀinꢀmanyꢀbreastꢀcancers.
activated)ꢀ HSF1ꢀ andꢀ HSPsꢀ andꢀ poorꢀ patientꢀ outcome,ꢀ
6,ꢀ9
ꢀ
Takenꢀ together,ꢀ theꢀ aboveꢀ resultsꢀ supportꢀ theꢀ excitingꢀ
possibilityꢀ thatꢀ inhibitingꢀ theꢀ HSF1-stressꢀ pathwayꢀ couldꢀ
representꢀ aꢀ novelꢀ therapeuticꢀ strategyꢀ thatꢀ wouldꢀ deliverꢀ
strongꢀselectiveꢀeffectsꢀagainstꢀcancerꢀcells.ꢀThisꢀisꢀsupportedꢀ
byꢀ targetꢀ validationꢀ studiesꢀ usingꢀ knockdownꢀ ofꢀ HSF1ꢀ byꢀ
4,ꢀ19
geneticꢀmeans.
ꢀ
Aꢀ numberꢀ ofꢀ structurallyꢀ diverseꢀ compoundsꢀ haveꢀ beenꢀ
reportedꢀ toꢀ actꢀ asꢀ inhibitorsꢀ ofꢀ HSF1ꢀ orꢀ theꢀ HSF1-stressꢀ
8,ꢀ 20
pathway,ꢀviaꢀaꢀvarietyꢀofꢀproposedꢀmechanismsꢀofꢀaction. ꢀ
Moreover,ꢀHSF1ꢀisꢀaꢀligand-lessꢀtranscriptionꢀfactorꢀwithꢀpoorꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ1ꢀꢀ
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JournalꢀNameꢀ
ꢀARTICLEꢀ
predictedꢀdruggabilityꢀandꢀasꢀsuchꢀisꢀdifficultꢀtoꢀinhibitꢀdirectlyꢀ toꢀ CDK2,ꢀ butꢀ withoutꢀ success.ꢀ Fortunately,ꢀ addinewgꢀAranꢀ extlinraꢀ
Vi ticle On e
usingꢀaꢀsmallꢀmoleculeꢀapproach.ꢀConsequently,ꢀweꢀdecidedꢀtoꢀ methyleneꢀtoꢀtheꢀsideꢀchainꢀ(4)ꢀafford eD dO ꢀI a: n1 0ꢀ .a1 0n 3a 9l o/ Cg 6uMe Dꢀ w0 0i 1t 5h 9ꢀ Aa ꢀ
conductꢀanꢀunbiasedꢀcell-basedꢀphenotypicꢀscreenꢀtoꢀidentifyꢀ modestꢀimprovementꢀinꢀaffinityꢀforꢀCDK2ꢀwhichꢀcouldꢀbeꢀusedꢀ
inhibitorsꢀofꢀtheꢀHSF1-stressꢀpathway.ꢀꢀ
toꢀ generateꢀ aꢀ crystalꢀ structureꢀ (Figꢀ 2).ꢀ Theꢀ CDK2ꢀ usedꢀ forꢀ
crystallisationꢀ wasꢀ expressed,ꢀ purifiedꢀ andꢀ crystallisedꢀ inꢀ theꢀ
absenceꢀ ofꢀ cyclin.ꢀ Analysisꢀ ofꢀ theꢀ crystalꢀ structureꢀ revealedꢀ
thatꢀ theꢀ pyrimidineꢀ N1ꢀ andꢀ 6-NHꢀ groupꢀ formꢀ aꢀ typicalꢀ
2
)ꢀResultsꢀandꢀDiscussionꢀ
23
hydrogenꢀ bondꢀ pairꢀ interactionꢀ withꢀ Leu83 ꢀ inꢀ theꢀ hingeꢀ
regionꢀofꢀCDK2.ꢀTheꢀbenzimidazoleꢀN3ꢀisꢀobservedꢀtoꢀformꢀaꢀ
water-mediatedꢀ hydrogenꢀ bondꢀ toꢀ theꢀ side-chainꢀ ofꢀ Asp145ꢀ
andꢀ theꢀ etherꢀ oxygenꢀ isꢀ involvedꢀ inꢀ aꢀ water-mediatedꢀ
hydrogenꢀ bondꢀ toꢀ Asp86.ꢀ Theꢀ benzimidazoleꢀ ringꢀ systemꢀ
stacksꢀ betweenꢀ threeꢀ lipophilicꢀ residuesꢀ Phe80,ꢀ Val18ꢀ andꢀ
Ala144.ꢀ
2
.1ꢀHitꢀIdentificationꢀ
Toꢀ discoverꢀ inhibitorsꢀ ofꢀ theꢀ HSF1-stressꢀ pathway,ꢀ weꢀ
employedꢀanꢀautomatedꢀcellularꢀimagingꢀandꢀanalysisꢀmethodꢀ
(
ArrayScan™)ꢀ thatꢀ quantifiesꢀ theꢀ abilityꢀ ofꢀ aꢀ compoundꢀ toꢀ
suppressꢀtheꢀexpressionꢀofꢀtheꢀHSF1-mediatedꢀinducibleꢀHSP70ꢀ
isoform,ꢀHSP72.ꢀCancerꢀcellsꢀwereꢀtreatedꢀwithꢀ17-allylamino-
1
knownꢀ toꢀ stimulateꢀ anꢀ HSF1-mediatedꢀ response
7-demethyoxygeldanamycinꢀ (17-AAG)ꢀ anꢀ HSP90ꢀ inhibitorꢀ
ꢀ andꢀ
ꢀ
ꢀ
21,ꢀ 22
compoundsꢀ thatꢀ blockedꢀ expressionꢀ ofꢀ HSP72ꢀ wereꢀ therebyꢀ
definedꢀasꢀinhibitorsꢀofꢀtheꢀHSF1-stressꢀpathway.ꢀ
Approximatelyꢀ200,000ꢀsmallꢀmoleculesꢀ(consistingꢀofꢀ35,000ꢀ
kinase-directedꢀ compoundsꢀ andꢀ aꢀ diversityꢀ setꢀ ofꢀ 165,000ꢀ
compoundsꢀ fromꢀ theꢀ AstraZenecaꢀ collection)ꢀ wereꢀ screenedꢀ
usingꢀthisꢀapproachꢀinꢀtheꢀU2OSꢀhumanꢀosteosarcomaꢀtumourꢀ
cellꢀline.ꢀOneꢀofꢀtheꢀhitsꢀselectedꢀforꢀprogressionꢀwasꢀtheꢀ4,6-
disubstitutedꢀ pyrimidineꢀ 1ꢀ which,ꢀ followingꢀ re-synthesis,ꢀ wasꢀ
confirmedꢀ asꢀ activeꢀ withꢀ aꢀ cellularꢀ IC50ꢀ valueꢀ ofꢀ 2.00ꢀ µMꢀ forꢀ
HSF1-stressꢀpathwayꢀinhibition.ꢀ
ꢀ
ꢀ
Fig.ꢀ2ꢀX-rayꢀco-crystalꢀstructureꢀofꢀ4ꢀboundꢀtoꢀCDK2.ꢀMeasurementsꢀinꢀ
Å.ꢀPDBꢀIDꢀ4BZD.ꢀ
ꢀ
ꢀ
Fig.ꢀ1ꢀHigh-throughputꢀscreeningꢀhitꢀpyrimidineꢀ1ꢀandꢀdimethylamino-
Withꢀ theꢀ crystalꢀ structureꢀ inꢀ hand,ꢀ weꢀ wereꢀ inꢀ aꢀ positionꢀ toꢀ
exploreꢀtheꢀhypothesisꢀthatꢀinhibitionꢀofꢀCDK2ꢀformsꢀtheꢀbasisꢀ
ofꢀ theꢀ observedꢀ HSF1-stressꢀ pathwayꢀ inhibition.ꢀ Usingꢀ singleꢀ
structuralꢀchangesꢀwithꢀrespectꢀtoꢀ4,6-disubstitutedꢀpyrimidineꢀ
containingꢀanalogueꢀ2.ꢀꢀ
ꢀ
In-houseꢀ dataꢀ revealedꢀ thatꢀ 4,6-pyrimidineꢀ 1ꢀ alsoꢀ possessedꢀ
modestꢀ CDK2ꢀ activityꢀ withꢀ anꢀ IC50ꢀ valueꢀ ofꢀ 1.14ꢀ µMꢀ inꢀ aꢀ
biochemicalꢀassay,ꢀthoughꢀitꢀwasꢀunclearꢀatꢀthisꢀstageꢀwhetherꢀ
thisꢀ kinaseꢀ activityꢀ wasꢀ importantꢀ forꢀ theꢀ observedꢀ HSF1ꢀ
cellularꢀphenotype.ꢀ
3
ꢀandꢀcomparingꢀtheꢀactivityꢀofꢀtheꢀcompoundsꢀgeneratedꢀweꢀ
aimedꢀtoꢀdistinguishꢀwhichꢀfeaturesꢀofꢀtheꢀSARꢀgaveꢀpotencyꢀ
andꢀselectivityꢀforꢀeachꢀassay.ꢀ
ꢀ
2
.2ꢀRationalꢀanalogueꢀdesign.ꢀ
Priorꢀtoꢀinvestigatingꢀtheꢀstructureꢀactivityꢀrelationshipꢀ(SAR)ꢀitꢀ
wasꢀ necessaryꢀ toꢀ improveꢀ theꢀ solubilityꢀ ofꢀ alcoholꢀ 1.ꢀ Toꢀ Weꢀbeganꢀbyꢀexaminingꢀtheꢀroleꢀofꢀtheꢀcentralꢀpyrimidineꢀring.ꢀ
achieveꢀthis,ꢀtheꢀphenethylꢀalcoholꢀchainꢀwasꢀreplacedꢀwithꢀanꢀ IntroductionꢀofꢀaꢀmethylꢀgroupꢀatꢀC-5ꢀtoꢀaffordꢀpyrimidineꢀ(5)ꢀ
oxygen-linkedꢀ dimethylaminoꢀ sideꢀ chainꢀ toꢀ giveꢀ 2.ꢀ Thisꢀ wasꢀ predictedꢀ toꢀ causeꢀ aꢀ stericꢀ clashꢀ withꢀ theꢀ otherꢀ
modificationꢀ retainedꢀ potencyꢀ inꢀ theꢀ HSF1-stressꢀ pathwayꢀ substituentsꢀ onꢀ theꢀ ring,ꢀ disfavouringꢀ theꢀ planarꢀ bindingꢀ
assayꢀ(1.35ꢀµM),ꢀbutꢀwasꢀlessꢀpotentꢀagainstꢀCDK2ꢀ(20.0ꢀµM).ꢀ conformationꢀandꢀdisruptingꢀCDK2ꢀbinding.ꢀInꢀbothꢀtheꢀCDK2ꢀ
PreliminaryꢀexplorationsꢀofꢀtheꢀSARꢀ(Tableꢀ1)ꢀwereꢀinitiatedꢀtoꢀ andꢀtheꢀHSF1-stressꢀpathwayꢀassaysꢀaꢀlargeꢀdropꢀinꢀactivityꢀwasꢀ
assessꢀtheꢀeffectꢀthatꢀstructuralꢀchangesꢀwouldꢀhaveꢀonꢀbothꢀ measuredꢀ (>100-ꢀ andꢀ >300-foldꢀ respectively).ꢀ Next,ꢀ weꢀ
theꢀ HSF1-stressꢀ pathwayꢀ activityꢀ andꢀ biochemicalꢀ CDK2ꢀ examinedꢀtheꢀcontributionꢀofꢀtheꢀbenzimidazoleꢀheadꢀgroup.ꢀ
activity,ꢀusingꢀtheꢀdimethylamino-containingꢀcompoundꢀ2ꢀasꢀaꢀ Compoundsꢀ wereꢀ designedꢀ thatꢀ wouldꢀ interrogateꢀ whetherꢀ
startingꢀpoint.ꢀSubstitutionꢀofꢀtheꢀphenylꢀringꢀforꢀaꢀ2-pyridineꢀ theꢀwater-mediatedꢀhydrogenꢀbondꢀtoꢀAsp145,ꢀandꢀtheꢀabilityꢀ
ringꢀ(3)ꢀaffordedꢀaꢀcompoundꢀwhichꢀwasꢀapproximatelyꢀ15-foldꢀ ofꢀtheꢀgroupꢀtoꢀbeꢀinvolvedꢀinꢀπ-stacking,ꢀwereꢀimportantꢀforꢀ
moreꢀ potentꢀ inꢀ theꢀ HSF1-stressꢀ pathwayꢀ assayꢀ andꢀ 35-foldꢀ CDK2ꢀ activityꢀ andꢀ whetherꢀ thisꢀ wouldꢀ alsoꢀ impactꢀ theꢀ HSF1-
moreꢀ activeꢀ againstꢀ CDK2ꢀ whenꢀ comparedꢀ withꢀ phenylꢀ stressꢀpathwayꢀactivity.ꢀꢀ
compoundꢀ 2.ꢀ Toꢀ facilitateꢀ progressionꢀ ofꢀ thisꢀ seriesꢀ weꢀ
attemptedꢀtoꢀobtainꢀaꢀcrystalꢀstructureꢀofꢀ2-pyridineꢀ3ꢀboundꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ2ꢀ
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JournalꢀNameꢀ
Tableꢀ1ꢀInvestigationꢀofꢀSARꢀsurroundingꢀ4,6-pyrimidinesꢀforꢀHSF1-ꢀstressꢀpathwayꢀandꢀCDK2ꢀassays.ꢀ
ꢀARTICLEꢀ
View Article Online
DOI: 10.1039/C6MD00159A
ꢀ
ꢀꢀꢀꢀꢀꢀGeneralꢀStructureꢀforꢀTableꢀ1ꢀ
ꢀ
HSF1ꢀIC50ꢀꢀ
CDK2ꢀIC50ꢀ
Compoundꢀ
1
R ꢀ
2
R ꢀ
3
R ꢀ
a
a
(
µM) ꢀ
(µM) ꢀ
Hꢀ
0.080ꢀ
0.580ꢀ
3
4
5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Hꢀ
0.165ꢀ
>30ꢀ
0.368ꢀ
65.8ꢀ
ꢀ
Meꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
6
7
8
9
ꢀ
ꢀ
ꢀ
ꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
6.15ꢀ
>30ꢀ
7.10ꢀ
29.8ꢀ
4.43ꢀ
>100ꢀ
1.60ꢀ
ꢀ
ꢀ
ꢀ
2.33ꢀ
17.6ꢀ
0.760ꢀ
ꢀ
1
1
0ꢀ
1ꢀ
ꢀ
ꢀ
Hꢀ
Hꢀ
Hꢀ
0.028ꢀ
0.039ꢀ
4.28ꢀ
0.161ꢀ
0.121ꢀ
4.61ꢀ
ꢀ
ꢀ
1
1
2ꢀ
3ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1
1
1
1
1
1
2
2
2
2
4ꢀ
5ꢀ
6ꢀ
7ꢀ
8ꢀ
9ꢀ
0ꢀ
1ꢀ
2ꢀ
3ꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
Hꢀ
0.842ꢀ
0.198ꢀ
0.055ꢀ
0.045ꢀ
0.295ꢀ
0.057ꢀ
0.884ꢀ
0.450ꢀ
1.38ꢀ
0.059ꢀ
0.690ꢀ
0.535ꢀ
0.492ꢀ
0.554ꢀ
0.358ꢀ
1.24ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0.643ꢀ
0.836ꢀ
0.776ꢀ
ꢀ
ꢀ
0.610ꢀ
ꢀ
aꢀ
Potencyꢀdataꢀareꢀreportedꢀasꢀtheꢀaverageꢀofꢀtwoꢀdeterminations.
ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ3ꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

Page 5 of 8
Pleas eM ꢀ de od ꢀCn ho te ꢀ ma d Cj uo s mt ꢀ mm arginsꢀ
JournalꢀNameꢀ
ꢀARTICLEꢀ
Explorationꢀ commencedꢀ withꢀ imidazoleꢀ 6,ꢀ aꢀ truncatedꢀ 2.3ꢀCombiningꢀSelectedꢀSAR.ꢀ
View Article Online
DOI: 10.1039/C6MD00159A
compoundꢀ thatꢀ isꢀ ableꢀ toꢀ formꢀ theꢀ requiredꢀ hydrogenꢀ bond,ꢀ
butꢀcannotꢀbeꢀinvolvedꢀinꢀπ-stacking.ꢀAꢀdropꢀinꢀpotencyꢀwasꢀ
observedꢀ withꢀ imidazoleꢀ 6ꢀ inꢀ theꢀ HSF1-stressꢀ pathwayꢀ assayꢀ
Toꢀ assessꢀ whetherꢀ theꢀ SARꢀ featuresꢀ describedꢀ aboveꢀ wereꢀ
additive,ꢀ theꢀ combinationꢀ compoundsꢀ 24ꢀ andꢀ 25ꢀ wereꢀ
synthesisedꢀ(Tableꢀ2).ꢀꢀ
(
(
77-fold)ꢀ andꢀ toꢀ aꢀ lesserꢀ extentꢀ theꢀ CDK2ꢀ biochemicalꢀ assayꢀ
12-fold).ꢀ Indazoleꢀ 7ꢀ andꢀ indoleꢀ 8ꢀ areꢀ unableꢀ toꢀ formꢀ aꢀ
Modificationꢀ ofꢀ theꢀ benzimidazoleꢀ headꢀ groupꢀ toꢀ affordꢀ aꢀ4-
azabenzimidazoleꢀinꢀcombinationꢀwithꢀselectedꢀcyclisedꢀamineꢀ
tailsꢀ affordedꢀ twoꢀ potentꢀ compounds,ꢀ methylpyrrolidineꢀ 24ꢀ
andꢀpiperidineꢀ25.ꢀBothꢀanaloguesꢀdisplayedꢀexcellentꢀactivityꢀ
inꢀ theꢀ HSF1-stressꢀ pathwayꢀ assayꢀ (14ꢀ nMꢀ andꢀ 15ꢀ nMꢀ
respectively).ꢀ Piperidineꢀ 25ꢀ wasꢀ alsoꢀ ourꢀ mostꢀ activeꢀ CDK2ꢀ
inhibitorꢀ(55ꢀnM).ꢀꢀ
hydrogenꢀbondꢀwithꢀAsp145ꢀandꢀshowedꢀaꢀdropꢀinꢀpotencyꢀinꢀ
bothꢀ assays.ꢀ Theꢀ correspondingꢀ quinolineꢀ 9ꢀ isꢀ capableꢀ ofꢀ
formingꢀaꢀweakꢀhydrogenꢀbondꢀwhilstꢀmaintainingꢀtheꢀabilityꢀ
forꢀπ-stacking.ꢀHowever,ꢀthisꢀcompoundꢀwasꢀalsoꢀlessꢀpotentꢀ
(
~200ꢀ foldꢀ forꢀ theꢀ HSF1-stressꢀ pathwayꢀ assayꢀ andꢀ inactiveꢀ inꢀ
theꢀ CDK2ꢀ assay)ꢀ asꢀ comparedꢀ toꢀ benzimidazoleꢀ 3.ꢀ 7-
Azabenzimidazoleꢀ10ꢀhasꢀtheꢀpotentialꢀforꢀH-bondꢀformation,ꢀ
butꢀaꢀpolarꢀgroupꢀhasꢀbeenꢀintroducedꢀontoꢀtheꢀaromaticꢀringꢀ
andꢀaꢀlossꢀinꢀpotencyꢀisꢀobservedꢀinꢀbothꢀassaysꢀcomparedꢀtoꢀ
benzimidazoleꢀ 3.ꢀ Surprisinglyꢀ theꢀ introductionꢀ ofꢀ aꢀ nitrogenꢀ
atomꢀ atꢀ C-4ꢀ ofꢀ theꢀ benzimidazoleꢀ ring,ꢀ toꢀ affordꢀ 4-
azabenzimidazoleꢀ 11ꢀ ledꢀ toꢀ aꢀ 3-foldꢀ increaseꢀ inꢀ HSF1-stressꢀ
pathwayꢀ inhibitionꢀ andꢀ aꢀ 5-foldꢀ improvementꢀ inꢀ CDK2ꢀ
inhibitionꢀ comparedꢀ withꢀ benzimidazoleꢀ 3.ꢀ Finally,ꢀ theꢀ co-
crystalꢀ structureꢀ ofꢀ 4ꢀ withꢀ CDK2ꢀ suggestedꢀ aꢀ smallꢀ lipophilicꢀ
substituentꢀ atꢀ C-6ꢀ ofꢀ theꢀ benzimidazoleꢀ couldꢀ beꢀ
accommodated.ꢀ Thisꢀ hypothesisꢀ wasꢀ confirmedꢀ withꢀ 6-
methylbenzimidazoleꢀ 12ꢀ andꢀ thisꢀ compoundꢀ showedꢀ aꢀ smallꢀ
increaseꢀinꢀpotencyꢀinꢀbothꢀassays.ꢀInꢀcontrast,ꢀsubstitutionꢀatꢀ
C-2ꢀofꢀtheꢀbenzimidazoleꢀwasꢀpredictedꢀtoꢀcreateꢀaꢀstericꢀclashꢀ
withꢀ theꢀ phenylalanineꢀ gatekeeperꢀ ofꢀ CDK2.ꢀ Thisꢀ hypothesisꢀ
wasꢀ confirmedꢀ withꢀ 2-methylbenzimidazoleꢀ 13,ꢀ whereꢀ aꢀ >7-
foldꢀlossꢀinꢀactivityꢀwasꢀobservedꢀinꢀbothꢀassays.ꢀInꢀsummary,ꢀ
modificationsꢀ toꢀ theꢀ benzimidazoleꢀ ringꢀ showedꢀ thatꢀ theꢀ 4-
azabenzimidazoleꢀ wasꢀ theꢀ mostꢀ potentꢀ substituentꢀ atꢀ thisꢀ
position.ꢀ
ꢀ
Tableꢀ2ꢀAnaloguesꢀwhichꢀcombineꢀbestꢀSARꢀfeaturesꢀforꢀHSF1ꢀ
pathwayꢀinhibition.ꢀ
ꢀ
HSF1ꢀIC50ꢀ CDK2ꢀIC50ꢀ
Compoundꢀ
Structureꢀ
a
nM) ꢀ
a
(nM) ꢀ
(
2
2
4ꢀ
5ꢀ
14ꢀ
15ꢀ
146ꢀ
55ꢀ
ꢀ
ꢀ
Potencyꢀdataꢀareꢀreportedꢀasꢀtheꢀaverageꢀtwoꢀdeterminations.ꢀ
aꢀ
ꢀ
3
)ꢀDiscussionꢀ
ꢀ
Withꢀaꢀrangeꢀofꢀpyrimidinesꢀinꢀhand,ꢀweꢀwereꢀableꢀtoꢀanalyseꢀ
theꢀ roleꢀ ofꢀ CDK2ꢀ inhibitionꢀ inꢀ theꢀ HSF1ꢀ cellularꢀ phenotype.ꢀꢀ
Despiteꢀ aꢀ varietyꢀ ofꢀ structuralꢀ changesꢀ weꢀ observedꢀ someꢀ
2
correlationꢀ (R ꢀ =ꢀ 0.56)ꢀ betweenꢀ CDK2ꢀ activityꢀ andꢀ HSF1ꢀ
pathwayꢀ inhibitionꢀ (Fig.ꢀ 3).ꢀ However,ꢀ thereꢀ wereꢀ analoguesꢀ
whichꢀseemedꢀtoꢀindicateꢀthatꢀCDK2ꢀactivityꢀandꢀHSF1ꢀactivityꢀ
couldꢀbeꢀuncoupled,ꢀforꢀexampleꢀphenylꢀpyrimidineꢀ2ꢀdisplaysꢀ
aꢀ 15-foldꢀ differenceꢀ inꢀ activityꢀ betweenꢀ theꢀ CDK2ꢀ andꢀ HSF1-
stressꢀ pathwayꢀ assays.ꢀ Thisꢀ suggestsꢀ thatꢀ eitherꢀ inhibitionꢀ ofꢀ
CDK2ꢀ activityꢀ isꢀ notꢀ theꢀ keyꢀ determinantꢀ ofꢀ HSF1-stressꢀ
pathwayꢀ antagonism,ꢀ orꢀ alternativelyꢀ thatꢀ itꢀ isꢀ necessaryꢀ toꢀ
inhibitꢀmultipleꢀkinasesꢀ(polypharmacology)ꢀtoꢀobserveꢀHSF1-
Inꢀ theꢀ CDK2ꢀ co-crystalꢀ structureꢀ theꢀ amineꢀ tailꢀ residesꢀ inꢀ aꢀ
solventꢀ channel.ꢀ Choꢀ etꢀ al.ꢀ haveꢀ demonstratedꢀ thatꢀ bulkyꢀ
amineꢀ groupsꢀ atꢀ theꢀ peripheryꢀ ofꢀ theꢀ ATPꢀ bindingꢀ siteꢀ clashꢀ
withꢀLys89ꢀofꢀCDK2ꢀandꢀhelpꢀimproveꢀselectivityꢀtowardsꢀotherꢀ
24
kinaseꢀfamilyꢀmembers,ꢀsuchꢀasꢀCDK4ꢀandꢀCDK6. ꢀTherefore,ꢀ
inꢀ anꢀ attemptꢀ toꢀ examineꢀ whetherꢀ CDK2ꢀ andꢀ HSF1-stressꢀ
pathwayꢀ inhibitionꢀ correlated,ꢀ severalꢀ analoguesꢀ wereꢀ
synthesisedꢀtoꢀexploreꢀtheꢀimpactꢀofꢀvariationꢀatꢀthisꢀvectorꢀonꢀ
CDK2ꢀandꢀHSF1-stressꢀpathwayꢀactivity.ꢀ
25
stressꢀpathwayꢀinhibition. ꢀꢀ
Itꢀ isꢀ wellꢀ establishedꢀ thatꢀ thereꢀ isꢀ aꢀ highꢀ levelꢀ ofꢀ sequenceꢀ
homology,ꢀ particularlyꢀ atꢀ theꢀ catalyticꢀ bindingꢀ site,ꢀ betweenꢀ
26,ꢀ 27
Removalꢀofꢀtheꢀsolventꢀchannelꢀsubstituentꢀtoꢀaffordꢀpyridineꢀ
membersꢀ ofꢀ theꢀ CDKꢀ proteinꢀ family.
ꢀ Consequently,ꢀ
4ꢀ showedꢀ aꢀ 10-foldꢀ increaseꢀ inꢀ CDK2ꢀ activityꢀ withꢀ aꢀ
developingꢀ selectiveꢀ inhibitorsꢀ withinꢀ thisꢀ proteinꢀ familyꢀ isꢀ
1
26
concomitantꢀ 10-foldꢀ lossꢀ ofꢀ HSF1-stressꢀ pathwayꢀ activity.ꢀ Toꢀ
probeꢀ whetherꢀ theꢀ water-mediatedꢀ hydrogenꢀ bondꢀ byꢀ theꢀ
oxygen-linkedꢀ tailꢀ (observedꢀ inꢀ theꢀ co-crystalꢀ structure)ꢀ wasꢀ
essential,ꢀ propyl-linkedꢀ 15ꢀ wasꢀ synthesised.ꢀ However,ꢀ littleꢀ
changeꢀ inꢀ potencyꢀ wasꢀ observedꢀ againstꢀ eitherꢀ CDK2ꢀ orꢀ theꢀ
HSF1-stressꢀ pathway.ꢀ Compoundsꢀ 16-23ꢀ wereꢀ synthesisedꢀ toꢀ
investigateꢀtheꢀeffectsꢀonꢀactivityꢀinꢀbothꢀassaysꢀofꢀincreasedꢀ
bulkꢀ aroundꢀ theꢀ amineꢀ tail.ꢀ Bothꢀ ofꢀ theꢀ N-methylpyrrolidineꢀ
enantiomersꢀ (16ꢀ andꢀ 17)ꢀ andꢀ theꢀ piperidineꢀ 19ꢀ displayedꢀ
modestꢀ 2-foldꢀ improvementꢀ inꢀ HSF1-stressꢀ pathwayꢀ activityꢀ
butꢀmaintainedꢀsimilarꢀpotencyꢀagainstꢀCDK2,ꢀcomparedꢀwithꢀ
theꢀdimethylamino-solubilisingꢀtailꢀ3.ꢀꢀ
challenging. ꢀ Ifꢀ CDK2ꢀ isꢀ notꢀ responsibleꢀ orꢀ isꢀ onlyꢀ partiallyꢀ
responsibleꢀ forꢀ theꢀ observedꢀ HSF1-stressꢀ pathwayꢀ inhibition,ꢀ
weꢀ hypothesisedꢀ thatꢀ theꢀ directꢀ targetꢀ mustꢀ shareꢀ
considerableꢀstructuralꢀsimilarityꢀinꢀtheꢀbindingꢀsiteꢀtoꢀCDK2.ꢀꢀ
Thereforeꢀ4,6-pyrimidineꢀ3ꢀwasꢀsubmittedꢀtoꢀaꢀEMDꢀMilliporeꢀ
CDKꢀ panelꢀ toꢀ assessꢀ potencyꢀ acrossꢀ theꢀ selectedꢀ CDKꢀ familyꢀ
membersꢀ (Tableꢀ 3).ꢀ Modestꢀ activityꢀ wasꢀ observedꢀ againstꢀ
severalꢀ CDKs;ꢀ however,ꢀ thisꢀ compoundꢀ wasꢀ highlyꢀ potentꢀ
againstꢀCDK9ꢀ(14ꢀnM).ꢀFollowingꢀthisꢀresult,ꢀweꢀexpandedꢀourꢀ
screeningꢀandꢀseveralꢀ4,6-disubstitutedꢀpyrimidinesꢀwereꢀalsoꢀ
selectedꢀforꢀtestingꢀagainstꢀCDK9.ꢀInꢀeachꢀcaseꢀtheꢀcompoundsꢀ
wereꢀ potentꢀ againstꢀ CDK9ꢀ (Tableꢀ 4),ꢀ withꢀ theꢀ exceptionꢀ ofꢀ
ꢀ
ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ4ꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

Pleas eM ꢀ de od ꢀCn ho te ꢀ ma d Cj uo s mt ꢀ mm arginsꢀ
Page 6 of 8
JournalꢀNameꢀ
ꢀARTICLEꢀ
a
Tableꢀ3ꢀTheꢀactivityꢀofꢀCompoundꢀ3ꢀinꢀaꢀCDKꢀfamilyꢀscreeningꢀpanel. ꢀ
View Article Online
ꢀ
DOI: 10.1039/C6MD00159A
ꢀ
CDK1/CyclinBꢀ CDK2/CyclinEꢀ CDK3/CyclinEꢀ CDK5/p35ꢀ CDK6/CyclinD3ꢀ CDK7/CyclinH/MAT1ꢀ CDK9/CyclinT1ꢀ
1800ꢀ 2800ꢀ 3500ꢀ 610ꢀ 1300ꢀ 1100ꢀ 14ꢀ
IC50ꢀ(nM)ꢀ
ꢀ
a
DataꢀobtainedꢀfromꢀEMDꢀMilliporeꢀscreeningꢀpanelꢀ(N=1),ꢀsinceꢀcarryingꢀoutꢀthisꢀscreenꢀthisꢀCDKꢀassayꢀisꢀnowꢀrunꢀbyꢀEurofinsꢀ
http://www.eurofins.com/en.aspx).ꢀForꢀassayꢀconditionsꢀseeꢀsupportingꢀinformation.
(
ꢀ
imidazoleꢀ 6,ꢀ activityꢀ inꢀ theꢀ HSF1ꢀ assayꢀ andꢀ CDK9ꢀ assayꢀ wereꢀ CDK2ꢀactivityꢀ(1.14ꢀµM).ꢀThroughꢀtheꢀapplicationꢀofꢀSARꢀandꢀ
rationalꢀ designꢀ againstꢀ aꢀ CDK2ꢀ co-crystalꢀ structure,ꢀ weꢀ
demonstratedꢀ thatꢀ althoughꢀ thereꢀ wasꢀ someꢀ correlationꢀ
betweenꢀHSF1-stressꢀpathwayꢀinhibitionꢀandꢀCDK2ꢀinhibition,ꢀ
itꢀ wasꢀ possibleꢀ toꢀ generateꢀ analoguesꢀ whereꢀ theꢀ twoꢀ
measuredꢀactivitiesꢀwereꢀuncoupled.ꢀ
comparable.ꢀꢀAlthoughꢀ6ꢀisꢀtheꢀleastꢀpotentꢀcompoundꢀinꢀtheꢀ
CDK9ꢀ assayꢀ theꢀ fallꢀ offꢀ inꢀ cellularꢀ potencyꢀ isꢀ greaterꢀ thanꢀ
expected.ꢀꢀꢀ
CDK9ꢀisꢀknownꢀtoꢀbeꢀaꢀcomponentꢀofꢀtheꢀpositiveꢀtranscriptionꢀ
elongationꢀ factorꢀ bꢀ (P-TEFb).
28,ꢀ 29
ꢀ Thisꢀ enzymeꢀ isꢀ vitalꢀ asꢀ itꢀ
ꢀ
stimulatesꢀtheꢀtranscriptionꢀelongationꢀofꢀmostꢀproteinꢀcodingꢀ
genesꢀ (whichꢀ includeꢀ keyꢀ developmentalꢀ andꢀ stimulusꢀ
responsiveꢀgenes)ꢀbyꢀphosphorylationꢀofꢀtheꢀcarboxy-terminalꢀ
30,ꢀ31
domainꢀofꢀRNAꢀpolymeraseꢀIIꢀ(RNAPII).
ꢀInhibitionꢀofꢀCDK9ꢀꢀ
hasꢀ beenꢀ shownꢀ toꢀ haveꢀ particularꢀ impactꢀ onꢀ theꢀ levelsꢀ ofꢀ
mRNAsꢀ withꢀ shortꢀ half-lives,ꢀ typifiedꢀ byꢀ transcriptionallyꢀ
32
inducibleꢀmRNAs. ꢀItꢀhasꢀalsoꢀbeenꢀdemonstratedꢀthatꢀP-TEFbꢀ
associatesꢀwithꢀHSF1ꢀandꢀplaysꢀaꢀfunctionalꢀroleꢀinꢀtheꢀstress-
33
inducedꢀexpressionꢀofꢀHSP72. ꢀ
Indeed,ꢀ Aquavivaꢀ etꢀ al.ꢀ recentlyꢀ publishedꢀ aꢀ screenꢀ ofꢀ drugsꢀ
andꢀ lateꢀ stageꢀ compoundsꢀ toꢀ discoverꢀ HSF1ꢀ transcriptionꢀ
inhibitorsꢀ (definedꢀ byꢀ theꢀ abilityꢀ ofꢀ aꢀ compoundꢀ toꢀ blockꢀ
inductionꢀ ofꢀ HSP72ꢀ followingꢀ treatmentꢀ withꢀ anꢀ HSP90ꢀ
34
inhibitor,ꢀ ganetespib). ꢀ ꢀ Amongstꢀ theꢀ compoundsꢀ theyꢀ
identifiedꢀ wasꢀ SNS-032ꢀ 26,ꢀ aꢀ knownꢀ inhibitorꢀ ofꢀ CDK2,ꢀ CDK7ꢀ
3
5-37
ꢀ
andꢀ CDK9,
CDK9ꢀinꢀtheꢀHSF1ꢀinhibitionꢀphenotype.ꢀTheꢀstructureꢀofꢀSNS-
ꢀ whichꢀ isꢀ consistentꢀ withꢀ theꢀ apparentꢀ roleꢀ ofꢀ
ꢀ
Fig.ꢀ3ꢀGraphꢀshowingꢀtheꢀcorrelationꢀbetweenꢀHSF1ꢀpathwayꢀinhibitionꢀ
TM
ArrayScan )ꢀ pIC50ꢀ andꢀ CDK2ꢀ inhibitionꢀ pIC50ꢀ forꢀ compoundsꢀ inꢀ thisꢀ
0
32ꢀ26ꢀderivesꢀfromꢀaꢀcompletelyꢀdifferentꢀchemotypeꢀtoꢀourꢀ
,6-pyrimidineꢀ seriesꢀ thereforeꢀ itꢀ isꢀ lessꢀ likelyꢀ thatꢀ bothꢀ
(
4
compoundsꢀhitꢀaꢀlargeꢀnumberꢀofꢀsimilarꢀproteinꢀtargets.
38,ꢀ 39
paper.ꢀꢀ
ꢀ
ꢀ
However,ꢀtoꢀincreaseꢀconfidenceꢀinꢀtheꢀroleꢀofꢀCDK9ꢀinhibitionꢀ
inꢀ ourꢀ HSF1ꢀ pathwayꢀ inhibitionꢀ phenotype,ꢀ weꢀ purchasedꢀ aꢀ
ꢀ
Tableꢀ4ꢀActivitiesꢀofꢀselectedꢀ4,6-pyrimidinesꢀagainstꢀCDK9ꢀ
ꢀ
40,ꢀ41
secondꢀstructurallyꢀdistinctꢀCDK9ꢀinhibitor,ꢀdinaciclibꢀ27.
ꢀInꢀ
ourꢀ hands,ꢀ SNS-032ꢀ 26ꢀ andꢀ dinaciclibꢀ 27ꢀ displayedꢀ CDK9ꢀ
inhibitionꢀ valuesꢀ ofꢀ 6ꢀ andꢀ 4ꢀ nMꢀ respectively.ꢀ Bothꢀ publishedꢀ
CDK9ꢀ inhibitorsꢀ andꢀ ourꢀ optimisedꢀ 4,6-pyrimidineꢀ 25ꢀ (CDK9ꢀ
IC50ꢀ=ꢀ3ꢀnM)ꢀwereꢀthenꢀsubmittedꢀtoꢀaꢀU2OSꢀcell-ELISAꢀassay,ꢀ
whichꢀ asꢀ withꢀ theꢀ ArrayScan™ꢀ assayꢀ format,ꢀ quantifiesꢀ theꢀ
abilityꢀ ofꢀ aꢀ compoundꢀ toꢀ blockꢀ theꢀ inductionꢀ ofꢀ HSP72ꢀ
expressionꢀuponꢀcellꢀtreatmentꢀwithꢀ17-AAG.ꢀAsꢀillustratedꢀinꢀ
Figureꢀ 4,ꢀ allꢀ threeꢀ potentꢀ CDK9ꢀ inhibitors,ꢀ fromꢀ distinctꢀ
chemotypes,ꢀareꢀeffectiveꢀinhibitorsꢀofꢀtheꢀHSF1ꢀpathway.ꢀInꢀ
addition,ꢀ whenꢀ theseꢀ compoundsꢀ wereꢀ testedꢀ inꢀ aꢀ
sulforhodamineꢀ Bꢀ (SRB)ꢀ cellularꢀ growthꢀ inhibitionꢀ assayꢀ asꢀ
singleꢀagents,ꢀallꢀthreeꢀwereꢀobservedꢀtoꢀpotentlyꢀabrogateꢀcellꢀ
growth.ꢀ
HSF1ꢀIC50ꢀ CDK2ꢀIC50ꢀ
CDK9/ꢀ
CyclinꢀT1ꢀ(nM) ꢀ
Compoundꢀ
b
a
µM) ꢀ
a
(µM) ꢀ
(
3
6
ꢀ
ꢀ
0.080ꢀ
6.15ꢀ
0.580ꢀ
7.10ꢀ
14ꢀ
88ꢀ
8ꢀ
1
1
2
7ꢀ
9ꢀ
1ꢀ
0.045ꢀ
0.057ꢀ
0.450ꢀ
0.492ꢀ
0.358ꢀ
0.643ꢀ
8ꢀ
22ꢀ
ꢀ
a
Potencyꢀ dataꢀ areꢀ reportedꢀ asꢀ theꢀ averageꢀ ofꢀ twoꢀ determinations.ꢀ
b
Dataꢀ obtainedꢀ fromꢀ EMDꢀ Milliporeꢀ Screeningꢀ Panelꢀ (N=1),ꢀ sinceꢀ
carryingꢀ outꢀ thisꢀ screenꢀ thisꢀ CDKꢀ assayꢀ isꢀ nowꢀ runꢀ byꢀ Eurofinsꢀ
(
http://www.eurofins.com/en.aspx).ꢀ Forꢀ assayꢀ conditionsꢀ seeꢀ
supportingꢀinformation.ꢀ
ꢀ
Owingꢀtoꢀtheꢀhighꢀhomologyꢀbetweenꢀtheꢀactiveꢀsitesꢀofꢀtheꢀ
CDKꢀfamilyꢀmembers,ꢀ4,6-pyrimidineꢀ3ꢀwasꢀscreenedꢀinꢀaꢀCDKꢀ
panelꢀ andꢀ foundꢀ toꢀ beꢀ aꢀ highlyꢀ potentꢀ inhibitorꢀ ofꢀ CDK9.ꢀ Toꢀ
testꢀ theꢀ hypothesisꢀ thatꢀ CDK9ꢀ inhibitionꢀ isꢀ involvedꢀ inꢀ HSF1-
stressꢀ pathwayꢀ inhibitionꢀ weꢀ purchasedꢀ twoꢀ validatedꢀ CDK9ꢀ
Conclusionsꢀ
Aꢀ high-throughputꢀ phenotypicꢀ screenꢀ wasꢀ conductedꢀ toꢀ findꢀ
inhibitorsꢀofꢀtheꢀHSF1-stressꢀpathwayꢀinꢀcancerꢀcellsꢀandꢀaꢀhitꢀ
compoundꢀ basedꢀ onꢀ aꢀ di-substitutedꢀ 4,6-pyrimidineꢀ scaffoldꢀ
wasꢀdiscovered.ꢀThisꢀcompoundꢀwasꢀknownꢀtoꢀexhibitꢀmodestꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ5ꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

Page 7 of 8
Pleas eM ꢀ de od ꢀCn ho te ꢀ ma d Cj uo s mt ꢀ mm arginsꢀ
JournalꢀNameꢀ ꢀARTICLEꢀ
View Article Online
inhibitorsꢀ withꢀ entirelyꢀ distinctꢀ chemicalꢀ structuresꢀ andꢀ theꢀ preparationꢀ ofꢀ theꢀ CDK2ꢀ proteinꢀ andꢀ crystals,ꢀ andꢀ T.ꢀ
submittedꢀ themꢀ toꢀ anꢀ assayꢀ thatꢀ quantifiesꢀ HSF1-stressꢀ ChaudaryꢀandꢀN.ꢀMcGuinessꢀforꢀtheꢀCD KD 2O ꢀI a: 1s 0s a. 1 y0 ꢀ3 a9 n/ Cd 6ꢀ Md aD t0a0 . 1ꢀ 5W9 Ae ꢀ
pathwayꢀ inhibition.ꢀ Bothꢀ validatedꢀ CDK9ꢀ inhibitorsꢀ wereꢀ thankꢀ Y.ꢀ Mokꢀ forꢀ assistanceꢀ withꢀ productionꢀ ofꢀ theꢀ
potentꢀ inhibitorsꢀ ofꢀ theꢀ HSF1-stressꢀ pathway.ꢀ Theꢀ rationalꢀ crystallographyꢀfigureꢀforꢀthisꢀpaper.ꢀThisꢀworkꢀwasꢀsupportedꢀ
designꢀofꢀhighlyꢀselectiveꢀinhibitorsꢀtoꢀinvestigateꢀtheꢀfunctionꢀ byꢀ Cancerꢀ Researchꢀ UKꢀ grantꢀ numbersꢀ C309/A8274ꢀ andꢀ
andꢀ synergismꢀ ofꢀ differentꢀ CDKsꢀ isꢀ extremelyꢀ challenging.ꢀ C309/A11566,ꢀ andꢀ byꢀ Theꢀ Instituteꢀ ofꢀ Cancerꢀ Research.ꢀ Paulꢀ
Moreꢀ workꢀ isꢀ neededꢀ toꢀ confirmꢀ CDK9ꢀ asꢀ theꢀ keyꢀ molecularꢀ WorkmanꢀisꢀaꢀCancerꢀResearchꢀLifeꢀFellowꢀ(C309/A8992).ꢀ
targetꢀ thatꢀ deliversꢀ theꢀ HSF1-stressꢀ pathwayꢀ inhibitionꢀ
phenotypeꢀandꢀtoꢀestablishꢀwhetherꢀkinaseꢀpolypharmocologyꢀ
isꢀalsoꢀrequired.ꢀTheꢀ4,6-pyrimidineꢀ25ꢀisꢀaꢀusefulꢀadditionꢀasꢀaꢀ
structurallyꢀdistinctꢀinꢀvitroꢀchemicalꢀtoolꢀtoꢀstudyꢀtheꢀroleꢀofꢀ
Notesꢀandꢀreferencesꢀ
ꢀ
CDK9ꢀ inhibitionꢀ andꢀ HSF1-stressꢀ pathwayꢀ inhibition.ꢀ Theꢀ 1.ꢀ
broaderꢀkinaseꢀselectivityꢀprofileꢀofꢀ4,6-pyrimidineꢀ25ꢀandꢀtheꢀ
R.ꢀ I.ꢀ Morimoto,ꢀ Coldꢀ Springꢀ Harborꢀ Symp.ꢀ Quant.ꢀ Biol.,ꢀ
2011,ꢀ76,ꢀ91-99.ꢀ
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J.ꢀAnckarꢀandꢀL.ꢀSistonen,ꢀAnn.ꢀRev.ꢀBiochem.,ꢀ2011,ꢀ80,ꢀ
effectꢀonꢀtheꢀHSF1ꢀpathwayꢀofꢀaꢀnumberꢀofꢀCDK9ꢀinhibitors,ꢀ
possessingꢀ differentꢀ kinaseꢀ selectivityꢀ profiles,ꢀ areꢀ currentlyꢀ
underꢀinvestigationꢀ(aꢀtableꢀshowingꢀsomeꢀCDKꢀselectivityꢀdataꢀ
canꢀbeꢀfoundꢀinꢀtheꢀsupplementaryꢀinformation).ꢀ
1
089-1115.ꢀ
A.ꢀVihervaaraꢀandꢀL.ꢀSistonen,ꢀJ.ꢀCellꢀSci.,ꢀ2014,ꢀ127,ꢀ261-
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007,ꢀ130,ꢀ1005-1018.ꢀ
P.ꢀ Workmanꢀ andꢀ E.ꢀ deꢀ Billy,ꢀ Nat.ꢀ Med.,ꢀ 2007,ꢀ 13,ꢀ 1415-
417.ꢀ
2
Whilstꢀ theꢀ disubstitutedꢀ 4,6-pyrimidineꢀ seriesꢀ hasꢀ shownꢀ
potencyꢀagainstꢀCDK9ꢀandꢀtheꢀHSF1-stressꢀpathway,ꢀthisꢀseriesꢀ
showedꢀhighꢀclearanceꢀinꢀmouseꢀpharmacokineticꢀexperimentsꢀ
2
1
makingꢀ theseꢀ compoundsꢀ unsuitableꢀ forꢀ progressionꢀ intoꢀ 6.ꢀ
animalꢀmodels.ꢀHowever,ꢀotherꢀchemicalꢀseriesꢀdevelopedꢀasꢀ
inhibitorsꢀ ofꢀ theꢀ HSF1-stressꢀ pathwayꢀ haveꢀ shownꢀ moreꢀ
promiseꢀandꢀwillꢀbeꢀpublishedꢀinꢀdueꢀcourse.ꢀ
S.ꢀSantagata,ꢀR.ꢀHu,ꢀN.ꢀU.ꢀLin,ꢀM.ꢀL.ꢀMendillo,ꢀL.ꢀC.ꢀCollins,ꢀ
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8.ꢀ
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M.ꢀL.ꢀMendillo,ꢀS.ꢀSantagata,ꢀM.ꢀKoeva,ꢀG.ꢀW.ꢀBell,ꢀR.ꢀHu,ꢀ
R.ꢀM.ꢀTamimi,ꢀE.ꢀFraenkel,ꢀT.ꢀA.ꢀInce,ꢀL.ꢀWhitesellꢀandꢀS.ꢀ
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VandeꢀWoude,ꢀ S.ꢀR.ꢀ Granter,ꢀ M.ꢀ Bosenberg,ꢀ G.ꢀC.ꢀ Chu,ꢀ
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Fig.ꢀ4ꢀPublishedꢀCDK9ꢀinhibitorsꢀSNS-032ꢀandꢀdinaciclib.ꢀResultsꢀfromꢀ 15.ꢀ
cell-ELISAꢀ assayꢀ forꢀ HSF1-mediatedꢀ HSP72ꢀ inductionꢀ inhibition,ꢀ CDK9ꢀ
dataꢀfromꢀCaliper®ꢀbiochemicalꢀassayꢀandꢀsulforhodamineꢀBꢀ(SRB)ꢀcellꢀ 16.ꢀ
K.ꢀR.ꢀBrandvoldꢀandꢀR.ꢀI.ꢀMorimoto,ꢀJ.ꢀMol.ꢀꢀBiol.,ꢀ2015,ꢀ
427,ꢀ2931-2947.ꢀ
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Biol.,ꢀ2015,ꢀ40,ꢀ142-152.ꢀ
growthꢀinhibitionꢀassay.ꢀAllꢀpotencyꢀdataꢀareꢀreportedꢀasꢀtheꢀaverageꢀ
1
1
1
7.ꢀ
8.ꢀ
9.ꢀ
S.ꢀM.ꢀDoyle,ꢀO.ꢀGenestꢀandꢀS.ꢀWickner,ꢀNat.ꢀRev.ꢀMol.ꢀCellꢀ
Biol.,ꢀ2013,ꢀ14,ꢀ617-629.ꢀ
ofꢀtwoꢀdeterminations.ꢀ*Atꢀlimitꢀofꢀassayꢀsensitivity.ꢀ
Z.ꢀ Tang,ꢀ S.ꢀ Dai,ꢀ Y.ꢀ He,ꢀ R.ꢀA.ꢀ Doty,ꢀ L.ꢀD.ꢀ Shultz,ꢀ S.ꢀB.ꢀ
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Y.ꢀChen,ꢀJ.ꢀChen,ꢀA.ꢀLoo,ꢀS.ꢀJaeger,ꢀL.ꢀBagdasarian,ꢀJ.ꢀYu,ꢀF.ꢀ
Chung,ꢀ J.ꢀ Korn,ꢀ D.ꢀ Ruddy,ꢀ R.ꢀ Guo,ꢀ M.ꢀ E.ꢀ McLaughlin,ꢀ F.ꢀ
Feng,ꢀP.ꢀZhu,ꢀF.ꢀStegmeier,ꢀR.ꢀPagliarini,ꢀD.ꢀPorterꢀandꢀW.ꢀ
Zhou,ꢀOncotarget,ꢀ2013,ꢀ4,ꢀ816-829.ꢀ
Acknowledgementsꢀ
WeꢀthankꢀA.ꢀMirza,ꢀM.ꢀRichards,ꢀandꢀM.ꢀLiuꢀforꢀassistanceꢀinꢀ
theꢀ spectroscopicꢀ characterizationꢀ ofꢀ testꢀ compounds.ꢀ Weꢀ
thankꢀ A.ꢀ Thomasꢀ forꢀ chemistryꢀ insightꢀ andꢀ discussions.ꢀ Weꢀ
thankꢀJ.ꢀStanway,ꢀA.ꢀValentine,ꢀandꢀC.ꢀBrassingtonꢀforꢀhelpꢀwithꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ6ꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

Pleas eM ꢀ de od ꢀCn ho te ꢀ ma d Cj uo s mt ꢀ mm arginsꢀ
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Page 9 of 8
MedChemComm
View Article Online
DOI: 10.1039/C6MD00159A
Graphical Abstract
A series of 4,6-disubstituted pyrimidines from a phenotypic screen targeting the
HSF1 pathway are also potent inhibitors of CDK9.