Fragment-Based Discovery of a Selective and Cell-Active Benzodiazepinone CBP/EP300 Bromodomain Inhibitor (CPI-637)

Article abstract of DOI:10.1021/acsmedchemlett.6b00075

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

Full text of DOI:10.1021/acsmedchemlett.6b00075

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Letter
Fragment-based discovery of a selective and cell-active
benzodiaze-pinone CBP/EP300 bromodomain inhibitor (CPI-637)
Alexander M. Taylor, Alexandre Côté, Michael C. Hewitt, Richard Pastor, Yves Leblanc, Christopher G.
Nasveschuk, F. Anthony Romero, Terry D. Crawford, Nico Cantone, Hariharan Jayaram, Jeremy W. Setser,
Jeremy M. Murray, Maureen H. Beresini, Gladys de Leon Boenig, Zhongguo Chen, Andrew R. Conery,
Richard T. Cummings, Les A. Dakin, E. Megan Flynn, Oscar W. Huang, Susan Kaufman, Patricia J. Keller,
James R. Kiefer, Tommy Lai, Yingjie Li, Jiangpeng Liao, Wenfeng Liu, Eneida Pardo, Vickie Tsui, Jian Wang,
Yongyun Wang, Zhaowu Xu, Fen Yan, Dong Yu, Laura E. Zawadzke, Xiaoqin Zhu, Xiaoyu Zhu, Robert J.
Sims, Andrea G. Cochran, Steven F. Bellon, James E. Audia, Steven R. Magnuson, and Brian K. Albrecht
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00075 • Publication Date (Web): 15 Mar 2016
Downloaded from http://pubs.acs.org on March 16, 2016
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Cochran, Andrea; Genentech, Inc., Department of Protein Engineering
Bellon, Steven; Constellation Pharmaceuticals, Medicinal Chemistry
Audia, James; Constellation Pharmaceuticals
Magnuson, Steven; Genentech, Discovery Chemistry
Albrecht, Brian; Amgen, Chemistry Research and Discovery
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Fragment-based discovery of a selective and cell-active benzodiaze-
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AlexanderꢀM.ꢀTaylor†*,ꢀAlexandreꢀCôté†,ꢀMichaelꢀC.ꢀHewitt†,ꢀRichardꢀPastor‡,ꢀYvesꢀLeblancǁ,ꢀChris-
topherꢀG.ꢀNasveschukǁ,ꢀF.ꢀAnthonyꢀRomero‡,ꢀTerryꢀD.ꢀCrawford‡,ꢀNicoꢀCantone†,ꢀHariharanꢀ
Jayaramǁ,ꢀJeremyꢀSetser†,ꢀJeremyꢀMurray‡,ꢀMaureenꢀH.ꢀBeresini‡,ꢀGladysꢀdeꢀLeonꢀBoenig‡,ꢀ
ZhongguoꢀChen§,ꢀAndrewꢀR.ꢀConery†,ꢀRichardꢀT.ꢀCummings†,ꢀLeslieꢀA.ꢀDakinǁ,ꢀE.ꢀMeganꢀFlynn‡,ꢀ
OscarꢀW.ꢀHuang‡,ꢀSusanꢀKaufman‡,ꢀPatriciaꢀJ.ꢀKeller†,ꢀJamesꢀR.ꢀKiefer‡,ꢀTommyꢀLai§,ꢀYingjieꢀLi§,ꢀ
JiangpengꢀLiao§,ꢀWenfengꢀLiu§,ꢀHenryꢀLu§,ꢀEneidaꢀPardoǁ,ꢀVickieꢀTsui‡,ꢀJianꢀWang§,ꢀYongyunꢀ
Wang§,ꢀZhaowuꢀXu§,ꢀFenꢀYan§,ꢀDongꢀYu§,ꢀLauraꢀZawadzkeǁ,ꢀXiaoqinꢀZhu§,ꢀXiaoyuꢀZhu§,ꢀRobertꢀJ.ꢀ
Sims,ꢀ3rd†,ꢀAndreaꢀG.ꢀCochran‡,ꢀSteveꢀBellon†,ꢀJamesꢀE.ꢀAudia†,ꢀStevenꢀMagnuson‡,ꢀBrianꢀK.ꢀAl-
brechtǁ.ꢀ
†Constellation Pharmaceuticals, 215 First Street, Suite 200, Cambridge, Massachusetts 02142, United
States.
‡
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States.
§
WuxiꢀAppTecꢀCo.,ꢀLtd.,ꢀ288ꢀFuteꢀZhongꢀRoad,ꢀWaigaoqiaoꢀFreeꢀTradeꢀZone,ꢀShanghaiꢀ200131,ꢀPeople’sꢀRepublicꢀofꢀ
China.ꢀ
Keywords: bromodomain, CREBBP, CBP, EP300, benzodiazepinone
ꢀ
ABSTRACT:ꢀCBPꢀandꢀEP300ꢀareꢀhighlyꢀhomologous,ꢀbromodomain-containingꢀtranscriptionꢀcoactivatorsꢀinvolvedꢀinꢀnu-
merousꢀcellularꢀpathwaysꢀrelevantꢀtoꢀoncology.ꢀAsꢀpartꢀofꢀourꢀeffortꢀtoꢀexploreꢀtheꢀpotentialꢀtherapeuticꢀimplicationsꢀofꢀse-
lectivelyꢀtargetingꢀbromodomains,ꢀweꢀsetꢀoutꢀtoꢀidentifyꢀaꢀCBP/EP300ꢀbromodomainꢀinhibitorꢀthatꢀwasꢀpotentꢀbothꢀin vitroꢀ
andꢀinꢀcellularꢀtargetꢀengagementꢀassaysꢀandꢀwasꢀselectiveꢀoverꢀtheꢀotherꢀmembersꢀofꢀtheꢀbromodomainꢀfamily.ꢀReportedꢀ
hereꢀisꢀaꢀseriesꢀofꢀcell-potentꢀandꢀselectiveꢀprobesꢀofꢀtheꢀCBP/EP300ꢀbromodomains,ꢀderivedꢀfromꢀtheꢀfragmentꢀscreeningꢀ
hitꢀ4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.ꢀ
Interestꢀ inꢀ exploringꢀ theꢀ biologicalꢀ functionꢀ andꢀ thera-
peuticꢀrelevanceꢀofꢀbromodomainꢀinhibitionꢀhasꢀincreasedꢀ
inꢀ recentꢀ years,ꢀ especiallyꢀ followingꢀ theꢀ progressionꢀ ofꢀ
erozygousꢀlossꢀofꢀCBP/EP300ꢀhaveꢀbeenꢀlinkedꢀtoꢀtheꢀneu-
10
rologicalꢀdisorderꢀRubenstein-Taybiꢀsyndrome ꢀasꢀwellꢀasꢀ
11,12
lateꢀonsetꢀlymphomasꢀinꢀmice.
ꢀAdditionally,ꢀbothꢀCBPꢀ
1
severalꢀ BETꢀ bromodomainꢀ inhibitorsꢀ intoꢀ clinicalꢀ trials. ꢀ
andꢀEP300ꢀareꢀinvolvedꢀinꢀtheꢀregulationꢀofꢀcriticalꢀsurveil-
lanceꢀ mechanismsꢀ thatꢀ ensureꢀ appropriateꢀ cellꢀ prolifera-
Amongꢀ otherꢀ bromodomain-containingꢀ proteinsꢀ implicat-
edꢀ inꢀ diseaseꢀ pathways,ꢀ cyclic-AMPꢀ responseꢀ elementꢀ
bindingꢀproteinꢀ(CBP)ꢀandꢀadenoviralꢀE1Aꢀbindingꢀproteinꢀ
13
tionꢀandꢀtissueꢀgrowth, ꢀareꢀpartꢀofꢀoncogenicꢀfusionꢀpro-
teinsꢀinꢀrareꢀformsꢀofꢀleukemia,ꢀandꢀareꢀalsoꢀrequiredꢀforꢀ
2
3
ofꢀ300ꢀkDaꢀ(EP300),ꢀaꢀhighlyꢀhomologous ꢀpairꢀofꢀbromo-
theꢀoncogenicꢀactivityꢀofꢀadditionalꢀfusionꢀproteins. ꢀOver-
3
domain-containingꢀ transcriptionalꢀ coactivators, ꢀ wereꢀ ofꢀ
expressionꢀofꢀCBPꢀandꢀEP300ꢀhasꢀbeenꢀidentifiedꢀinꢀseveralꢀ
tumorꢀtypesꢀandꢀhasꢀbeenꢀdirectlyꢀcorrelatedꢀwithꢀaggres-
interestꢀ toꢀ usꢀ asꢀ potentialꢀ drugꢀ targetsꢀ becauseꢀ ofꢀ theirꢀ
reportedꢀinvolvementꢀinꢀaꢀnumberꢀofꢀdiseaseꢀstates.ꢀBothꢀ
areꢀmultidomainꢀpolypeptidesꢀwithꢀintrinsicꢀproteinꢀlysineꢀ
acetyltransferaseꢀ activityꢀ andꢀ haveꢀ beenꢀ shownꢀ toꢀ acety-
lateꢀhistoneꢀtailsꢀasꢀwellꢀasꢀaꢀnumberꢀofꢀoncology-relevantꢀ
transcriptionꢀ factors,ꢀ includingꢀ p53,ꢀ c-MYC,ꢀ andꢀ c-
1
4,15
siveness.
ꢀ Finally,ꢀ recentꢀ sequencingꢀ effortsꢀ haveꢀ
demonstratedꢀrecurrentꢀloss-of-functionꢀmutationsꢀinꢀbothꢀ
CBPꢀandꢀEP300ꢀinꢀaꢀnumberꢀofꢀtumorꢀtypes. 6,17,18,19,20ꢀ
1
Asꢀ partꢀ ofꢀ ourꢀ bromodomainꢀ inhibitionꢀ platform,ꢀ weꢀ
soughtꢀtoꢀdesignꢀaꢀselectiveꢀandꢀcell-potentꢀsmallꢀmoleculeꢀ
probeꢀofꢀtheꢀCBP/EP300ꢀbromodomainꢀinꢀorderꢀtoꢀcharac-
terizeꢀ theꢀ pharmacologicalꢀ impactꢀ ofꢀ disruptingꢀ acetyl-
lysineꢀ recognitionꢀ withoutꢀ perturbingꢀ theꢀ scaffoldingꢀ orꢀ
enzymaticꢀ functionsꢀ ofꢀ theꢀ proteins.ꢀ Specifically,ꢀ weꢀ tar-
getedꢀ aꢀ compoundꢀ withꢀ sub-micromolarꢀ potencyꢀ inꢀ aꢀ
CBP/EP300ꢀcellularꢀtargetꢀengagementꢀassayꢀandꢀ100-foldꢀ
4
,5,6,7,8
MYB.
ꢀ Theꢀ singleꢀ bromodomainꢀ ofꢀ bothꢀ CBPꢀ andꢀ
EP300ꢀ(locatedꢀN-terminalꢀrelativeꢀtoꢀtheꢀacetyltransferaseꢀ
domain)ꢀisꢀknownꢀtoꢀbeꢀrequiredꢀforꢀtheꢀacetylationꢀofꢀnu-
9
cleosomalꢀhistonesꢀandꢀtransactivationꢀfunction. ꢀ
BothꢀCBPꢀandꢀEP300ꢀplayꢀaꢀkeyꢀroleꢀinꢀseveralꢀdiseaseꢀ
pathways.ꢀForꢀinstance,ꢀlossꢀofꢀfunctionꢀmutationsꢀorꢀhet-
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biochemicalꢀselectivityꢀoverꢀotherꢀbromodomains. ꢀAtꢀtheꢀ
outsetꢀ ofꢀ ourꢀ investigations,ꢀ noꢀ inhibitorsꢀ thatꢀ metꢀ thoseꢀ
criteriaꢀwereꢀavailable.ꢀSinceꢀourꢀprobeꢀoptimization,ꢀthreeꢀ
potentꢀ CBPꢀ bromodomainꢀ inhibitorsꢀ ofꢀ noteꢀ haveꢀ beenꢀ
reported. 2,23,24ꢀ
12.0ꢀ
(2.7)ꢀ
1
1
2
CH
2
ꢀ
-ꢀ
60ꢀ
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-
1
NHꢀ
Oꢀ
4.1ꢀ(2.4)ꢀ
1.9ꢀ(4.2)ꢀ
0.53ꢀ
8.5ꢀ
2
ꢀ
ꢀ
7
6
-
-
1
3
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3.50ꢀ
>20ꢀ
(
R)-Meꢀ
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NHꢀ
(
4.1)ꢀ
ꢀ
aTime-resolvedꢀ fluorescenceꢀ resonanceꢀ energyꢀ transferꢀ (TR-
FRET)ꢀassayꢀwithꢀtheꢀisolatedꢀCBPꢀbromodomain.ꢀNꢀ=ꢀ2ꢀorꢀ3.ꢀ
LLEꢀforꢀkeyꢀcompoundsꢀ=ꢀpIC50ꢀ-ꢀclogP.ꢀ TR-FRETꢀassayꢀwithꢀ
b
c
theꢀisolatedꢀBRD4ꢀBD-1ꢀbromodomain.ꢀNꢀ=ꢀ2ꢀorꢀ3.ꢀ
Ourꢀ effortꢀ beganꢀ byꢀ screeningꢀ aꢀ fragmentꢀ collectionꢀ ofꢀ
roughlyꢀ2000ꢀlowꢀmolecularꢀweightꢀcompoundsꢀinꢀaꢀther-
malꢀshiftꢀassay. ꢀCompoundsꢀthatꢀincreasedꢀtheꢀCBPꢀbro-
modomainꢀ meltingꢀ temperatureꢀ byꢀ atꢀ leastꢀ oneꢀ degreeꢀ
Celsiusꢀwereꢀfurtherꢀcharacterizedꢀbyꢀdoseꢀresponseꢀinꢀaꢀ
time-resolvedꢀfluorescenceꢀresonanceꢀenergyꢀtransferꢀ(TR-
FRET)ꢀ assay,ꢀ andꢀ aꢀ subsetꢀ ofꢀ thoseꢀ thatꢀ showedꢀ aꢀ dose-
responseꢀ wereꢀ evaluatedꢀ byꢀ Nꢀ HSQCꢀ NMR,ꢀ isothermalꢀ
titrationꢀ calorimetryꢀ (ITC),ꢀ andꢀ x-rayꢀ crystallography.ꢀ
Whileꢀpotencyꢀwasꢀofꢀobviousꢀconsiderationꢀinꢀevaluatingꢀ
hits,ꢀ ofꢀ equalꢀ importanceꢀ wasꢀ selectivityꢀ overꢀ off-targetꢀ
bromodomains,ꢀ especiallyꢀ theꢀ BETꢀ familyꢀ (usingꢀ BRD4ꢀ
BD1ꢀ asꢀ aꢀ surrogate)ꢀ becauseꢀ ofꢀ theꢀ pronouncedꢀ cellularꢀ
2
5
ꢀ
1
5
Figure 1.ꢀAꢀco-crystalꢀstructureꢀofꢀ1ꢀboundꢀtoꢀtheꢀCBPꢀbromo-
domain.ꢀ (1.6ꢀ Åꢀ resolution)ꢀ PDBꢀ code:ꢀ 4YK0.ꢀ Hydrogenꢀ bondꢀ
interactionsꢀ betweenꢀ 1,ꢀ Asn1168,ꢀ water1,ꢀ andꢀ Tyr1125ꢀ areꢀ
highlightedꢀ byꢀ dashedꢀ linesꢀ (distancesꢀ inꢀ Å).ꢀ Theꢀ compoundꢀ
makesꢀ hydrophobicꢀ interactionsꢀ withꢀ Leu1120,ꢀ Ile1122,ꢀ
Val1174ꢀ (notꢀ depictedꢀ forꢀ clarity).ꢀ Theꢀ BRD4ꢀ BD-1ꢀ bindingꢀ
siteꢀ differsꢀ primarilyꢀ atꢀ residuesꢀ Leu1109ꢀ andꢀ Arg1173ꢀ
(Trp81ꢀandꢀAsp145,ꢀrespectively,ꢀinꢀBRD4ꢀBD-1).ꢀ
26
phenotypesꢀ associatedꢀ withꢀ BETꢀ inhibition. ꢀ Outꢀ ofꢀ thisꢀ
effort,ꢀ 4-methyl-1,3,4,5-tetrahydro-2H-
benzo[b][1,4]diazepin-2-oneꢀ (Figureꢀ 1,ꢀ Tableꢀ 1,ꢀ 1)ꢀ
emergedꢀasꢀaꢀpromisingꢀvalidatedꢀhit,ꢀdemonstratingꢀaꢀTR-
FRETꢀ IC50ꢀ ofꢀ 32ꢀ ꢀM,ꢀ whichꢀ correspondedꢀ toꢀ aꢀ lipophilicꢀ
Table 1. Impact of various modifications on 1 CBP po-
tency
2
7,28
ligandꢀ efficiencyꢀ ofꢀ 3.4.
ꢀMostꢀ importantly,ꢀ however,ꢀ 1ꢀ
hadꢀaꢀBRD4ꢀBD-1ꢀTR-FRETꢀIC50ꢀofꢀ220ꢀꢀM,ꢀrepresentingꢀaꢀ
7-foldꢀselectivityꢀforꢀCBP,ꢀwhichꢀfiguredꢀprominentlyꢀinꢀitsꢀ
choiceꢀasꢀaꢀstartingꢀpointꢀforꢀfurtherꢀoptimization.ꢀ
Theꢀbindingꢀmodeꢀofꢀthisꢀseries wasꢀestablishedꢀwithꢀaꢀ
1.6ꢀÅꢀresolutionꢀco-crystalꢀstructureꢀofꢀ1ꢀboundꢀtoꢀtheꢀiso-
latedꢀ CBPꢀ bromodomain.ꢀ Asꢀ seenꢀ inꢀ Figureꢀ 1,ꢀ 1ꢀ isꢀ posi-
tionedꢀinꢀtheꢀacetylꢀlysineꢀbindingꢀpocket,ꢀwhereꢀtheꢀben-
zodiazepinoneꢀ carbonylꢀ satisfiesꢀ theꢀ criticalꢀ hydrogenꢀ
bondingꢀinteractionꢀwithꢀAsn1168ꢀobservedꢀinꢀtheꢀcrystalꢀ
structuresꢀ ofꢀ allꢀ reportedꢀ bromodomainꢀ inhibitors.ꢀ Aꢀ se-
cond,ꢀ solvent-mediatedꢀ interactionꢀ betweenꢀ theꢀ carbonylꢀ
ofꢀ1,ꢀwaterꢀ1ꢀ(labelledꢀaccordingꢀtoꢀHewings,ꢀet al. ),ꢀandꢀ
Tyr1125ꢀwasꢀalsoꢀobserved.ꢀTheꢀlactamꢀNHꢀofꢀ 1ꢀcontrib-
utesꢀ aꢀ furtherꢀ H-bondingꢀ interactionꢀ withꢀ Asn1168,ꢀ andꢀ
theꢀ4-methylꢀgroupꢀisꢀpositionedꢀinꢀtheꢀpocketꢀwhereꢀtheꢀ
acetylꢀmethylꢀofꢀtheꢀnaturalꢀsubstrateꢀbinds,ꢀmakingꢀfavor-
ableꢀ Vanꢀ derꢀ Waalsꢀ interactionsꢀ withꢀ Val1174.ꢀ Theꢀ ben-
zeneꢀringꢀofꢀ1 fillsꢀsomeꢀofꢀtheꢀremainingꢀspaceꢀinꢀtheꢀbind-
ingꢀ site,ꢀ makingꢀ hydrophobicꢀ contactsꢀ withꢀ theꢀ Leu1120ꢀ
andꢀIle1122ꢀsidechainsꢀ(notꢀdepictedꢀinꢀFigureꢀ1ꢀforꢀclari-
ty).ꢀFinally,ꢀsinceꢀonlyꢀtheꢀRꢀenantiomerꢀofꢀ1 wasꢀobservedꢀ
inꢀ theꢀ electronꢀ density,ꢀ theꢀ individualꢀ enantiomersꢀ ofꢀ 1ꢀ
wereꢀ separatedꢀ andꢀ tested.ꢀ Consistentꢀ withꢀ theꢀ bindingꢀ
model,ꢀ theꢀ Rꢀ enantiomerꢀ wasꢀ >50-foldꢀ moreꢀ potentꢀ thanꢀ
CBP
IC50
BRD4
a
R1
IC50c
cmpd
X
R
(
ꢀM)
(ꢀM)
(
LLE)b
1
2
3
4
5
6
7
8
(±)-Meꢀ
(R)-Meꢀ
(S)-Meꢀ
Hꢀ
-ꢀ
-ꢀ
-ꢀ
-ꢀ
-ꢀ
-ꢀ
-ꢀ
-ꢀ
32ꢀ(3.4)ꢀ
27ꢀ(3.5)ꢀ
>2000ꢀ
>200ꢀ
>200ꢀ
>200ꢀ
120ꢀ
220ꢀ
260ꢀ
2
9
3700ꢀ
>200ꢀ
>200ꢀ
>200ꢀ
>200ꢀ
>200ꢀ
NHꢀ
(±)-3-Meꢀ
4,4’-di-Meꢀ
(R)-Etꢀ
(S)-CH
2
OHꢀ
130ꢀ
10.0ꢀ
9
Oꢀ
Sꢀ
-ꢀ
-ꢀ
80ꢀ
43ꢀ
(
3.3)ꢀ
(±)-Meꢀ
10
7.0ꢀ(3.0)ꢀ
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theꢀSꢀanalogꢀ(Tableꢀ1,ꢀ2ꢀandꢀ3),ꢀaꢀtrendꢀthatꢀheldꢀtrueꢀforꢀ
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elaboratedꢀanalogs,ꢀasꢀwellꢀ(dataꢀnotꢀshown).ꢀ
25
26
27
0.077ꢀ(5.7)ꢀ
0.25ꢀ(4.5)ꢀ
0.033ꢀ(4.3)ꢀ
1.9ꢀ
7ꢀ
13.0ꢀ
>20ꢀ
8.9ꢀ
Asꢀ expected,ꢀ manyꢀ featuresꢀ ofꢀ theꢀ startingꢀ fragmentꢀ
wereꢀessentialꢀtoꢀbinding.ꢀForꢀexample,ꢀdisruptionꢀofꢀeitherꢀ
theꢀH-bondingꢀinteractionꢀwithꢀAsn1168ꢀthroughꢀsubstitu-
tionꢀatꢀtheꢀlactamꢀNHꢀ(dataꢀnotꢀshown)ꢀorꢀalterationꢀofꢀtheꢀ
VanꢀderꢀWaalsꢀinteractionꢀbyꢀremovalꢀofꢀtheꢀmethylꢀgroupꢀ
ꢀ
ꢀ
ꢀ
(Tableꢀ 1,ꢀ 4)ꢀ causedꢀ aꢀ drasticꢀ lossꢀ inꢀ potency.ꢀ Likewise,ꢀ
3ꢀ
shiftingꢀtheꢀ4-methylꢀgroupꢀtoꢀtheꢀ3-positionꢀ(5)ꢀorꢀgemi-
nalꢀdimethylꢀsubstitutionꢀatꢀtheꢀ4-positionꢀ(6)ꢀresultedꢀinꢀaꢀ
dramaticꢀ lossꢀ inꢀ bindingꢀ affinity.ꢀ Modificationsꢀ thatꢀ ex-
tendedꢀ(7)ꢀtheꢀ4-methylꢀgroupꢀorꢀattemptedꢀtoꢀengageꢀtheꢀ
buriedꢀwatersꢀatꢀthatꢀsiteꢀ(8)ꢀwereꢀsomewhatꢀtoleratedꢀbutꢀ
wereꢀnotꢀadvantageous.ꢀVariousꢀreplacementsꢀforꢀtheꢀNHꢀ
linkerꢀresultedꢀinꢀanaloguesꢀthatꢀboundꢀtheꢀbromodomainꢀ
withꢀpotencyꢀandꢀselectivityꢀcomparableꢀtoꢀ 1 (9,ꢀ10,ꢀ11)ꢀ
butꢀwhichꢀdidꢀnotꢀrepresentꢀanꢀimprovementꢀinꢀlipophilicꢀ
ligandꢀefficiencyꢀ(LLE).ꢀAlthoughꢀourꢀprimaryꢀaimꢀwasꢀtoꢀ
generateꢀ usefulꢀ probesꢀ forꢀ cellularꢀ studies,ꢀ weꢀ aimedꢀ toꢀ
keepꢀtheꢀphysicochemicalꢀpropertiesꢀofꢀcompoundsꢀinꢀthisꢀ
seriesꢀ(asꢀcapturedꢀbyꢀLLE)ꢀwithinꢀaꢀrangeꢀthatꢀwouldꢀbeꢀ
consistentꢀwithꢀmoderateꢀclearanceꢀinꢀanimals,ꢀinꢀorderꢀtoꢀ
beꢀableꢀtoꢀprogressꢀcompoundsꢀasꢀ in vivoꢀtools,ꢀshouldꢀaꢀ
meaningfulꢀphenotypeꢀbeꢀdiscovered.ꢀ
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a
Time-resolvedꢀ fluorescenceꢀ resonanceꢀ energyꢀ transferꢀ (TR-
b
FRET)ꢀassayꢀwithꢀtheꢀisolatedꢀCBPꢀbromodomain.ꢀNꢀ=ꢀ2.ꢀ LLEꢀ
=
cellularꢀtargetꢀengagementꢀassay.ꢀNꢀ=ꢀ3.ꢀ TR-FRETꢀassayꢀwithꢀ
theꢀisolatedꢀBRD4ꢀBD-1ꢀbromodomain.ꢀNꢀ=ꢀ3.ꢀ
c
ꢀ pIC50ꢀ -ꢀ clogP.ꢀ Bioluminesenceꢀ resonanceꢀ energyꢀ transferꢀ
d
Next,ꢀweꢀsubstitutedꢀtheꢀbenzeneꢀringꢀtoꢀmodulateꢀpo-
tencyꢀandꢀphysicochemicalꢀpropertiesꢀandꢀdeterminedꢀthatꢀ
theseꢀsubstituentsꢀhadꢀanꢀimpactꢀnotꢀonlyꢀonꢀpotencyꢀbutꢀ
alsoꢀ onꢀ selectivity.ꢀ Groupsꢀ atꢀ theꢀ 8-positionꢀ didꢀ notꢀ im-
proveꢀCBPꢀpotencyꢀandꢀdramaticallyꢀdecreasedꢀselectivityꢀ
overꢀ BRD4,ꢀ whileꢀ onlyꢀ veryꢀ smallꢀ substituentsꢀ atꢀ theꢀ 9-
positionꢀwereꢀtoleratedꢀandꢀdidꢀnotꢀimproveꢀpotencyꢀ(dataꢀ
notꢀ shown).ꢀ Aꢀ varietyꢀ ofꢀ substitutionsꢀ atꢀ theꢀ 7-positionꢀ
(e.g.ꢀ 12ꢀ andꢀ 13)ꢀ improvedꢀ affinityꢀ substantiallyꢀ butꢀ re-
ducedꢀ selectivityꢀ overꢀ BRD4.ꢀ Aꢀ co-crystalꢀ structureꢀ withꢀ
theꢀ CBPꢀ bromodomainꢀ (Figureꢀ 2A)ꢀ showedꢀ thatꢀ theꢀ α-
methylbenzylꢀ substituentꢀ ofꢀ 12ꢀ positionedꢀ theꢀ pendantꢀ
phenylꢀgroupꢀintoꢀaꢀpocketꢀformedꢀbetweenꢀPro1110ꢀandꢀ
theꢀArg1173ꢀsidechain.ꢀTheꢀbindingꢀmodeꢀofꢀtheꢀcoreꢀben-
zodiazepinoneꢀwasꢀotherwiseꢀunchangedꢀfromꢀtheꢀparentꢀ
compound.ꢀ Basedꢀ onꢀ anꢀ analysisꢀ ofꢀ thisꢀ andꢀ otherꢀ co-
crystalꢀstructures,ꢀweꢀhypothesizedꢀthatꢀshiftingꢀsubstitu-
tionꢀtoꢀtheꢀ6-positionꢀwouldꢀorientꢀgroupsꢀalongꢀtheꢀLPFꢀ
shelfꢀ (residuesꢀ L1109-P1110-F1111),ꢀ increasingꢀ bindingꢀ
affinityꢀ throughꢀ closeꢀ contactsꢀ withꢀ theꢀ proteinꢀ surfaceꢀ
whileꢀ alsoꢀ introducingꢀ potentialꢀ stericꢀ clashesꢀ withꢀ theꢀ
bromodomainsꢀofꢀtheꢀBETꢀfamily.ꢀToꢀthatꢀend,ꢀcompoundꢀ
14,ꢀwhichꢀdemonstratedꢀsub-micromolarꢀpotencyꢀagainstꢀ
CBP,ꢀ improvedꢀ selectivityꢀ overꢀ BRD4ꢀ BD-1,ꢀ andꢀ higherꢀ
lipophilicꢀligandꢀefficiency,ꢀprovidedꢀanꢀearlyꢀvalidationꢀforꢀ
thisꢀapproach,ꢀwhichꢀbecameꢀtheꢀfocusꢀofꢀourꢀsubsequentꢀ
efforts.ꢀAꢀco-crystalꢀstructureꢀofꢀ14ꢀandꢀtheꢀbromodomainꢀ
ofꢀ CBPꢀ (Figureꢀ 2B)ꢀ highlightedꢀ theꢀ proximityꢀ ofꢀ theꢀ N-
benzylꢀcarboxamideꢀmoietyꢀtoꢀtheꢀLPFꢀshelfꢀandꢀtheꢀ pro-
pensityꢀforꢀlipophlicꢀsubstituentsꢀtoꢀcoverꢀPro1110.ꢀInter-
estingly,ꢀ theꢀ amideꢀ carbonylꢀ ofꢀ theꢀ sideꢀ chainꢀ wasꢀ posi-
tionedꢀ 2.7ꢀ Åꢀ awayꢀ fromꢀ theꢀ NHꢀ ofꢀ theꢀ diazepinoneꢀ ring,ꢀ
suggestingꢀthatꢀtheꢀintramolecularꢀhydrogenꢀbondꢀstabiliz-
esꢀ theꢀ observedꢀ conformation.ꢀ Inꢀ orderꢀ toꢀ determineꢀ
whetherꢀ theꢀ improvementsꢀ inꢀ CBPꢀ bromodomainꢀ inhibi-
tionꢀ thatꢀ weꢀ observedꢀ inꢀ ourꢀ biochemicalꢀ assayꢀ wouldꢀ
translateꢀ intoꢀ aꢀ cellularꢀ context,ꢀ aꢀ cell-based,ꢀ target-
Table 2. Structure-activity relationship of 6-
substituted benzodiazepinones.
CBP IC50a
(ꢀM)
CBP
EC50
BRD4
IC50
c
d
cmpd
R
LLE)b
(ꢀM)
(ꢀM)
(
1
5 (R)
0.9ꢀ(3.5)ꢀ
5.0ꢀ(2.8)ꢀ
>10ꢀ
>10ꢀ
44ꢀ
1
6 (S)
>20ꢀ
ꢀ
1
7
6.5ꢀ(5.3)ꢀ
>10ꢀ
>10ꢀ
>20ꢀ
>20ꢀ
ꢀ
18
19
0.62ꢀ(3.2)ꢀ
ꢀ
0.67ꢀ(4.1)ꢀ
0.039ꢀ(5.4)ꢀ
0.13ꢀ(4.2)ꢀ
>10ꢀ
1.20ꢀ
5ꢀ
8ꢀ
8ꢀ
ꢀ
2
0
1
ꢀ
2
12ꢀ
ꢀ
ꢀ
3
0
engagementꢀassayꢀwasꢀemployed. ꢀSpecifically,ꢀaꢀbiolumi-
nescenceꢀresonanceꢀenergyꢀtransferꢀ(BRET,ꢀPromega)ꢀ as-
sayꢀwasꢀused,ꢀinꢀwhichꢀaꢀsmallꢀmoleculeꢀCBP/EP300ꢀbro-
modomainꢀ inhibitorꢀ disruptedꢀ theꢀ interactionꢀ betweenꢀ aꢀ
HaloTag-labelledꢀhistoneꢀandꢀtheꢀbromodomainꢀconjugat-
edꢀtoꢀNanoLucꢀluciferase.ꢀMostꢀcompoundsꢀdemonstratedꢀaꢀ
22
0.160ꢀ(3.3)ꢀ
3.4ꢀ
16ꢀ
2
3
4
0.11ꢀ(2.2)ꢀ
0.24ꢀ(4.1)ꢀ
>10ꢀ
8.1ꢀ
7ꢀ
ꢀ
>
10-foldꢀ shiftꢀ inꢀ potencyꢀ betweenꢀ theꢀ biochemical andꢀ
cellularꢀassays,ꢀwhichꢀledꢀusꢀtoꢀtargetꢀbromodomainꢀinhibi-
torsꢀwithꢀbiochemicalꢀpotencies wellꢀbelowꢀ0.1ꢀꢀM.ꢀ
2
>20ꢀ
ꢀ
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ACS Medicinal Chemistry Letters
Ourꢀexplorationꢀofꢀ6-substitutionꢀfocusedꢀonꢀimprovingꢀ
CBP
TR-FRET IC50aꢀ
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0.03ꢀ±ꢀ0.01ꢀꢀMꢀ
.051ꢀ±ꢀ0.004ꢀꢀMꢀ
3.4ꢀ±ꢀ0.4ꢀꢀMꢀ
theꢀinteractionꢀbetweenꢀtheꢀcompoundsꢀandꢀtheꢀresiduesꢀ
ofꢀ theꢀ LPFꢀ shelfꢀ ofꢀ CBPꢀ andꢀ onꢀ introducingꢀ clashesꢀ withꢀ
non-conservedꢀ residuesꢀ inꢀ BRD4.ꢀ Toꢀ thatꢀ end,ꢀ α-methylꢀ
benzylꢀcompoundsꢀ15ꢀandꢀ16ꢀwereꢀpreparedꢀtoꢀintroduceꢀ
aꢀ conformationalꢀ constraintꢀ toꢀ 14,ꢀ whichꢀ reducedꢀ BRD4ꢀ
activityꢀinꢀbothꢀdiastereomersꢀbutꢀfailedꢀtoꢀimproveꢀpoten-
cyꢀagainstꢀCBP.ꢀSubsequentꢀeffortsꢀtoꢀmodifyꢀtheꢀN-benzylꢀ
ringꢀ(17),ꢀextendꢀtheꢀαꢀsubstituentꢀ(18),ꢀorꢀcyclizeꢀtheꢀam-
ideꢀsubstituentꢀ(19)ꢀdidꢀnotꢀimproveꢀCBPꢀaffinity,ꢀreducedꢀ
selectivityꢀoverꢀBRD4,ꢀorꢀboth.ꢀAnilideꢀderivativesꢀsuchꢀasꢀ
EP300
TR-FRET IC50bꢀ
0
-ꢀ
CBP ITC K c
d
ꢀ
0.03ꢀ±ꢀ0.01ꢀꢀMꢀ
-ꢀ
-ꢀ
LLEdꢀ
5.0ꢀ
CBP
BRET EC50eꢀ
0
.3ꢀ±ꢀ0.1ꢀꢀMꢀ
>10ꢀꢀMꢀ
>20ꢀꢀMꢀ
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BRD4 BD-1
TR-FRET IC50fꢀ
2
0ꢀdemonstratedꢀtheꢀpossibilityꢀofꢀidentifyingꢀpotentꢀCBPꢀ
inhibitors,ꢀbutꢀthisꢀsubseriesꢀwasꢀnotꢀpursuedꢀbecauseꢀofꢀ
concernꢀaboutꢀextendedꢀconjugationꢀandꢀlowerꢀsolubility.ꢀ
11.0ꢀ±ꢀ0.6ꢀꢀMꢀ
CECR2:ꢀ7.7ꢀꢀMꢀ
Off-target
bromo IC50sgꢀ
-
ꢀ
BRD9:ꢀ0.73ꢀ±ꢀ0.04ꢀꢀMꢀ
a
Time-resolvedꢀfluorescenceꢀenergyꢀtransferꢀ(TR-FRET)ꢀassayꢀ
withꢀ theꢀ isolatedꢀ CBPꢀ bromodomain.ꢀ Nꢀ >ꢀ3.ꢀ TR-FRETꢀ assayꢀ
withꢀtheꢀisolatedꢀEP300ꢀbromodomain.ꢀNꢀ=ꢀ3.ꢀ K ꢀ=ꢀ0.031ꢀ±ꢀ
0.012ꢀ µM,ꢀ N=0.968,ꢀ ꢁHꢀ =ꢀ 7007ꢀ ±ꢀ 115.5ꢀ cal/mol,ꢀ ꢁSꢀ =ꢀ 10.9ꢀ
cal/mol/deg.ꢀ LLEꢀ=ꢀlipophilicꢀligandꢀefficiencyꢀ=ꢀpIC50ꢀ-ꢀclogP.ꢀ
Bioluminescenceꢀresonanceꢀenergyꢀtransferꢀcellularꢀassay.ꢀNꢀ
ꢀ 2.ꢀ TR-FRETꢀ assayꢀ withꢀ theꢀ isolatedꢀ BRD4ꢀ BD-1ꢀ bromo-
domain.ꢀNꢀ>ꢀ3.ꢀ TR-FRETꢀassaysꢀwithꢀtheꢀrespectiveꢀisolatedꢀ
bromodomains;ꢀinhibitionꢀisꢀconsideredꢀoff-targetꢀifꢀIC50ꢀ<10ꢀ
M.ꢀSeeꢀsupportingꢀinformationꢀforꢀdetails.ꢀ
b
c
d
d
e
f
=
g
ꢀ
ꢀ
ꢀ
Figure 2. Co-crystalꢀ structuresꢀ ofꢀ benzodiazepinonesꢀ boundꢀ
toꢀ theꢀ CBPꢀ bromodomain.ꢀ A)ꢀ 12ꢀ (1.4ꢀ Åꢀ resolution).ꢀ Theꢀ 7-
benzyloxyꢀsideꢀchainꢀisꢀorientedꢀtowardꢀsolventꢀandꢀadoptsꢀaꢀ
favorableꢀ conformationꢀ thatꢀ positionsꢀ theꢀ phenylꢀ groupꢀ intoꢀ
theꢀcleftꢀbetweenꢀPro1110ꢀandꢀArg1173.ꢀPDBꢀcode:ꢀ5I83.ꢀB)ꢀ
ꢀ
Figure 3.ꢀ Compoundꢀ 28ꢀ inhibitsꢀ MYCꢀ expressionꢀ inꢀ AMO-1ꢀ
cellsꢀ(EC50ꢀ=ꢀ0.60ꢀꢀM).ꢀItsꢀenantiomerꢀdisplaysꢀanꢀEC50ꢀ>ꢀ10ꢀ
ꢀM.ꢀGeneꢀexpressionꢀnormalizedꢀtoꢀGAPDH/PPIB.ꢀ
1
4ꢀ(1.1ꢀÅꢀresolution).ꢀAꢀsubstituentꢀatꢀtheꢀ6-positionꢀisꢀproxi-
malꢀtoꢀtheꢀLPFꢀshelfꢀandꢀstillꢀprovidesꢀaꢀvectorꢀtoꢀtheꢀPro/Argꢀ
cleft.ꢀPDBꢀcode:ꢀ5I86.ꢀC)ꢀ23ꢀ(2.3ꢀÅꢀresolution).ꢀTheꢀsubstitutedꢀ
phenylꢀ sideꢀ chainꢀ fillsꢀ spaceꢀ overꢀ Pro1110ꢀ andꢀ positionsꢀ itsꢀ
benzyloxyꢀgroupꢀintoꢀtheꢀPro/Argꢀcleft.ꢀPDBꢀcode:ꢀ5I8B.ꢀD)ꢀ27ꢀ
1.1ꢀ Åꢀ resolution).ꢀ Indazolesꢀ orientꢀ 3-positionꢀ substituentsꢀ
intoꢀtheꢀcleftꢀbetweenꢀPro1110ꢀandꢀArg1173.ꢀPDBꢀcode:ꢀ5I89.ꢀ
Aꢀ breakthroughꢀ cameꢀ withꢀ theꢀ discoveryꢀ thatꢀ potencyꢀ
andꢀselectivityꢀcouldꢀalsoꢀbeꢀachievedꢀwithꢀaꢀvarietyꢀofꢀdi-
rectly-linkedꢀ 6-arylꢀ groups,ꢀ asꢀ evidencedꢀ byꢀ 21,ꢀ 22,ꢀ andꢀ
(
2
3.ꢀAꢀcrystalꢀstructureꢀofꢀ23ꢀ(Figureꢀ2C)ꢀshowedꢀthatꢀtheꢀ
directly-linkedꢀ phenylꢀ groupꢀ stackedꢀ onꢀ topꢀ ofꢀ Pro1110,ꢀ
withꢀtheꢀbenzyloxyꢀsideꢀchainꢀsittingꢀinꢀtheꢀPro/Argꢀcleft.ꢀ
Encouragedꢀ byꢀ thisꢀ finding,ꢀ weꢀ preparedꢀ severalꢀ 3-
substitutedꢀ5-linkedꢀindazoles,ꢀwhichꢀweꢀintendedꢀtoꢀori-
entꢀsubstituentsꢀintoꢀtheꢀsameꢀspaceꢀwhileꢀmaintainingꢀaꢀ
moreꢀfavorableꢀclogP. ꢀAꢀrangeꢀofꢀgroupsꢀatꢀtheꢀindazoleꢀ
-positionꢀledꢀtoꢀmeaningfulꢀimprovementsꢀinꢀCBPꢀinhibi-
tionꢀ relativeꢀ toꢀ unsubstitutedꢀ parentꢀ indazoleꢀ 24ꢀ (i.e.ꢀ 25ꢀ
andꢀ27).ꢀMostꢀimportantly,ꢀtheꢀbiochemicalꢀpotencyꢀofꢀthe-
seꢀ indazolesꢀ translatedꢀ intoꢀ low-micromolarꢀ cellularꢀ po-
tency.ꢀ Aꢀ co-crystalꢀ structureꢀ ofꢀ 27ꢀ inꢀ theꢀ CBPꢀ bromo-
domainꢀ (Figureꢀ 2D)ꢀ confirmedꢀ thatꢀ theꢀ indazolesꢀ filledꢀ
Table 3. Profile of CBP/EP300 probe compound 28
(CPI-637) and its enantiomer.
31
3
ꢀ
ꢀ
Enantiomer
28ꢀ
of 28ꢀ
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ACS Medicinal Chemistry Letters
Page 6 of 7
spaceꢀ overꢀ Pro1110ꢀ asꢀ expectedꢀ andꢀ orientedꢀ theirꢀ 3-
substituentꢀintoꢀtheꢀPro/Argꢀcleft.ꢀ
ꢀMꢀ andꢀ 2.7ꢀ ꢀM,ꢀ respectively.ꢀ Theꢀ inactiveꢀ enantiomerꢀ ofꢀ
28ꢀ displayedꢀ anꢀ EC ꢀ inꢀ theꢀ sameꢀ assayꢀ ofꢀ >10ꢀ ꢀM.ꢀ Inꢀ
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summary,ꢀ28ꢀmetꢀourꢀcriteriaꢀforꢀbiochemicalꢀpotencyꢀandꢀ
off-targetꢀbromodomainꢀselectivityꢀandꢀcellularꢀtargetꢀen-
gagement.ꢀ Togetherꢀ withꢀ itsꢀ enantiomer,ꢀ thisꢀ compound
representsꢀaꢀusefulꢀprobeꢀforꢀtheꢀinvestigationꢀofꢀtheꢀbio-
logicalꢀ implicationsꢀ ofꢀ CBP/EP300ꢀ bromodomainꢀ inhibi-
tion.ꢀFurtherꢀdevelopmentsꢀofꢀtheꢀchemicalꢀbiologyꢀappli-
cationꢀofꢀtheseꢀandꢀotherꢀbromodomainꢀinhibitorsꢀwillꢀbeꢀ
reportedꢀinꢀdueꢀcourse.ꢀ
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ASSOCIATED CONTENT
Supporting Information.ꢀDescriptionsꢀofꢀtheꢀbiochemicalꢀandꢀ
cellularꢀ assaysꢀ andꢀ results;ꢀ syntheticꢀ andꢀ characterizationꢀ
dataꢀ forꢀ theꢀ compoundsꢀ reported;ꢀ crystallographicꢀ methodsꢀ
andꢀ data;ꢀ off-targetꢀ bromodomainꢀ potencyꢀ dataꢀforꢀ 28.ꢀ Thisꢀ
materialꢀ isꢀ availableꢀ freeꢀ ofꢀ chargeꢀ viaꢀ theꢀ Internetꢀ atꢀ
http://pubs.acs.org.ꢀꢀ
AUTHOR INFORMATION
Corresponding Author
*AlexanderꢀM.ꢀTaylor:ꢀalex.taylor@constellationpharma.com.ꢀ
Present Addresses
SeeꢀSupportingꢀInformation.ꢀ
Author Contributions
Theꢀmanuscriptꢀwasꢀwrittenꢀthroughꢀcontributionsꢀofꢀallꢀau-
thors.ꢀAllꢀauthorsꢀhaveꢀgivenꢀapprovalꢀtoꢀtheꢀfinalꢀversionꢀofꢀ
theꢀmanuscript.ꢀ
Figure 4.ꢀ Aꢀ co-crystalꢀ structureꢀ ofꢀ 28ꢀ inꢀ theꢀ CBPꢀ bromo-
domain.ꢀ(1.1ꢀÅꢀresolution)ꢀPDBꢀcode:ꢀ5I8G.ꢀTheꢀbenzodiaze-
pinoneꢀcoreꢀrecapitulatesꢀtheꢀkeyꢀhydrogenꢀbondingꢀinterac-
tionsꢀ observedꢀ withꢀ theꢀ startingꢀ fragment,ꢀ 1,ꢀ whileꢀ theꢀ 3-
pyrazolylindazoleꢀ substituentꢀ improvesꢀ potencyꢀ byꢀ fillingꢀ
spaceꢀoverꢀPro1110ꢀandꢀtheꢀPro/Argꢀcleft.ꢀ
ACKNOWLEDGMENT
WeꢀareꢀgratefulꢀtoꢀTedꢀPetersꢀandꢀChristinaꢀLeeꢀinꢀ theꢀLeadꢀ
Discoveryꢀgroupꢀforꢀplatingꢀcompoundsꢀusedꢀinꢀtheseꢀstudiesꢀ
andꢀtoꢀCustomꢀNMRꢀServicesꢀforꢀcompoundꢀcharacterization.ꢀ
Furtherꢀexplorationꢀofꢀtheꢀindazoleꢀsubstituentsꢀresultedꢀ
inꢀ28ꢀ(CPI-637),ꢀwhichꢀdemonstratedꢀsubstantialꢀbiochem-
icalꢀ potencyꢀ thatꢀ wasꢀ confirmedꢀ byꢀ isothermalꢀ titrationꢀ
calorimetry.ꢀ Aꢀ co-crystalꢀ structureꢀ ofꢀ 28ꢀ inꢀ theꢀ CBPꢀ bro-
modomainꢀindicatedꢀthatꢀtheꢀcompoundꢀrecapitulatedꢀtheꢀ
keyꢀhydrogenꢀbondingꢀinteractionsꢀobservedꢀwithꢀtheꢀpar-
entꢀ compound,ꢀ 1,ꢀ withꢀ theꢀ substitutedꢀ indazoleꢀ fillingꢀ
spaceꢀaboveꢀPro1110ꢀandꢀtheꢀPro/Argꢀcleft.ꢀAsꢀexpected,ꢀ
28ꢀwasꢀalsoꢀpotentꢀagainstꢀEP300ꢀ(Tableꢀ3),ꢀandꢀitsꢀoppo-
siteꢀenantiomerꢀdisplayedꢀaꢀ>200-foldꢀlossꢀinꢀpotency.ꢀTheꢀ
biochemicalꢀpotencyꢀofꢀ28ꢀtranslatedꢀwellꢀintoꢀcellsꢀ(CBPꢀ
BRETꢀEC50ꢀ=ꢀ0.3ꢀꢀM),ꢀandꢀtheꢀcompoundꢀdemonstratedꢀaꢀ
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