Chapter8| SummaryandFutureProspects
Isoquinolinesulfonamidebasedkinaseinhibitors
Anincreasingnumberofbacterialstrainsdevelopresistanceagainstmultipleantibiotics,
1
includingsomeofthesocalledlastresortdrugs. TheseMultidrugresistantbacterialstrains
poseaserioushealththreat.Inordertoremainabletotreatfutureoutbreaksseveral,steps
must be taken. First, besides the optimization of existing antibiotics, new classes of
antibiotics must be developed. Second, these new classes of drugs should preferentially
target biochemical pathways that are currently unexploited. Chapter 2 describes the
discoveryofanewdruggabletargetforantibiotics.InhibitorsofproteinkinaseB(PKB/Akt1)
are currently pursued as potential antitumor agents. The chapter describes the discovery
thatthisproteinisrequiredforintracellularsurvivalofseveralstrainsofpathogenicbacteria,
including Salmonella. Targeting host cell kinases is a promising new strategy to treat
pathogenbacterialinfections.Chapter3describesthesynthesisoftwoseriesofcompounds
aimed at optimizing the inhibition profile towards PKB/Akt1. Starting point is the known
protein kinase A inhibitor, H89. Several structural modifications of the H89 structure
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Chapter8
resulted in the identification of two positions on the H89 skeleton amenable for
optimization towards more potent and selective inhibitors. Application of the reduction
transimination reduction sequence led to the construction of a library of derivatives with
increased structural variation, namely phenyl substitutions, double bond substitutions and
double bond configurations. Chapter 4 describes the parallel synthesis of a 63member
compound library based on Suzuki coupling of the bromine moiety in three suitably
protectedH89derivatives,bearingthebromineontheortho,metaandparaposition,with
21differentboronicacids.
Figure1;NovelH89analoguesdescribedinChapters3and4
ModificationsoftheH89skeletonarebynomeanslimitedtothepositionsdescribedin
chapters 3 and 4. In the next section, several strategies are discussed that allow the
modificationoftheH89skeletoninthe3regionsdepictedinFigure2.
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SummaryandFutureProspects
Figure2;ThestructureofH89.Highlightedarethethree domainsthatarediscussedinthetextbelow.
ModifyingregionA
Thisregionofthepharmacophoreengagesinhydrogenbondingwiththesocalledhinge
region which is located at the bottom of the ATP binding pocket of the kinase. The
isoquinoline nitrogen atom serves as a hydrogen bond acceptor in the active site of the
kinase. Replacing the isoquinoline moiety with an aminonaphtyl group would make this
group a hydrogen bond donating moiety. A synthetic strategy towards this class of
compoundsisexemplifiedbythesynthesisof6aminonaphtylderivative11,(inScheme1).
Amineprotectionin6aminonaphtalenesulfonicacidusingBoc2OandtriethylamineinMeOH
shouldgiveprotected2.Transformationofthesulfonicacidintothecorrespondingsulfonyl
chloride requires 6 as mild chlorinating agent. Acylation of 8 followed by Staudinger
reduction of the azide in 9 should yield amine 10. Submitting known 4 and 10 to the
reduction transimination reduction protocol as described in chapter 3, followed by acidic
deprotectionwouldgive11asnovelH89analogue.
Scheme1;Synthesisofnapthylaminobasedinhibitors.Reagentsandconditions:(i)Et3N,Boc2O,MeOH.(ii)
NaH, pbromobenzaldehyde, DMF, 0°C. (iii) SOCl2, DCM. (iv) 2, DCM. (v) DiPEA, DCM. (vi) PMe3, THF/H2O.
(vii)a:4,DiBAlH,Et2O,78°C.b:MeOH,100°C.c:9.d:NaBH4.(viii)TFA/DCM.
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Chapter8
ModifyinglinkerregionB
2
The linker region in H89 serves two purposes. It contains a crucial amino functionality
which is involved in hydrogen bonding and it enables proper alignment of the
isoquinolinesulfonamide moiety and the styrene end group. Reducing the number of
rotatable bond by rigidifying this region using a variety of cyclic diamine spacers neither
3
improved potency nor selectivity. Incorporating small substituents on the diamine spacer
maybeamoresubtlewaytoreducerotatablefreedomwithoutlockingthestructure.The
assemblyofbuildingblocks4,12and13usingchemistrydescribedinchapter2enablesthe
synthesisof15and17asdepictedinScheme2.
Scheme2;Introducingrotationalconstraintsinthelinker.Reagentsandconditions:(i)DiPEA,DCM.
(ii)TFA/DCM.(iii)a:DiBAlH,Et2O,78°C.b:MeOH,100°C.c:13ordeprotected14.d:NaBH4.
Another way to functionalize the linker is depicted in Scheme 3. Acylation of 2
aminoacetonitrilewithisoquinolinesulfonylchloride12givesnitrile18.Submittingnitrile18,
cynnamic aldehyde 19 (chapter 3) and diethyl methylphosphonate to nbutyllithium under
HornerEmmons conditions according to the method developed by Orru et al. gives
4
unsaturatedimine20. Thisintermediatecanbeprocessedinatransiminationreactionwith
avarietyofaminestogeneratederivativesof21.
146
SummaryandFutureProspects
Scheme3;Linkermodifications:Reagentsandconditions:(i)2aminoacetonitril,DMAP,pyridine.(ii)nBuLi,THF.
(iii)R1CH2NH2,MeOH.
ModificationofregionC
The reductiontransiminationreduction procedure described in chapter 3 can be
5
modified to introduce substuted amines (see Scheme 4). Treatment of 4 with Grignard
reagentsinrefluxingEt2O,followedbymethanolysisoftheintermediatemagnesiumspecies
affordssecondaryimine22,whichaftertransiminationwith12andNaBH4reductionaffords
isoquinolinesulfonamides23asracemates.
Scheme4;Grignardtransiminationreductionstrategy.Reagentsandconditions:(i)RMgBr,Et2O,reflux.(ii)
MeOH,100°C.(iii)12.(iv)NaBH4
Asisdescribedinchapter3,increasingthesizeofthedoublebondsubstituentincreases
selectivity towards PKB/Akt1. Several strategies can be envisaged to elaborate on these
results(Scheme5).HornerWittigreactionbetweentheanionofsubstitutedacetonitriles24
and diphenylphosphinyl chloride 25 yields anion 26. Addition to, for instance, p
bromobenzaldehyde should result in the formation of isomeric mixtures of substituted
cynnamic aldehydes 27. These can be used to extend the range of substituents in 28.
Condensingbromoacetonitrileto25usingLiHMDSfollowedbyadditionofbenzaldehyde
147
Chapter8
Scheme5;Noveldoublebondsubstitutions.(i)LiHMDS,THF.(ii)LiHMDS,pbromobenzaldehyde,THF.(iii))
a:DiBAlH,Et2O,78°C.b:MeOH,100°C.c:N(2aminoethyl)isoquinoline5sulfonamide.d:NaBH4.(iv)2eq,
LiHMDS,bromoacetonitril,THF,78°C.(v)K2CO3,phenylboronicacid,Pd(PPh3)4,toluene/H2O.(vi)KOH.(vii)
Br2,CHCl3,reflux.
should yield bromocynnamic aldehyde 29. Transformation into 30 yields a precursor
that,afternitrogenprotection,allowsforSuzukitype crosscouplingsleadingtoavarietyof
arylsubstitutedanalogueslike31.Similaranalogueswithanadditionalarylsubstituentcan
be obtained starting from a Knoevenagel condensation between benzophenone 32 and
acetonitrile33.Thethusobtained,unsaturatednitrile34canbebrominatedleadingto
148
SummaryandFutureProspects
35assingleisomer,whichcanbetransformedintovinylbromide36that,afterprotectionof
the secondary amine group, may serve as precursor for palladium based crosscoupling
reactionstowards37.
The restriction of flexibility in the styrene moiety is exemplified by the synthesis of 41
(Scheme6).Herecomerciallyavailable6bromo2napthoicacid38isreducedto39andre
oxidized towards the aldehyde 40 followed by reductive amination with the appropiate
aminetoaffordnaphtylatedisoquinolinesulfonamide41.
Scheme6;constraintanalogues;Reagentsandconditions:(i)LiAlH4,Et2O,0°C.(ii)DessMartinperiodane.(ii)a:
DiBAlH,Et2O,78°C.b:MeOH,100°C.c:N(2aminoethyl)isoquinoline5sulfonamide.d:NaBH4.
Cationicantimicrobialpeptides
Killingbacteriabydisruptingtheircellwallmembranewithcationicantimicrobialpeptides(CAPs)
is currently being explored as a new approach in the treatment of multidrug resistant bacterial
infections.Theagentsusedintheseapproachesareusuallyverypotent,buttheirlackofselectivity
6
hamperstheirclinicalapplicationtotopicaluse. Chapter5describesresultspreviouslyobtainedwith
the cationic peptide Gramicidin S 38 and analogues thereof (39a and 39b, Figure 3). Structural
analysisofanalogues4042revealedasimilarsheetstructurecomparedtoGSwith,however,an
alteredbackboneorientationaroundtheSAAamidegroupand anincreased twistangle. Biological
evaluationoftheseanaloguesrevealedalossinactivityfor39a.Analogue39bwasshowntobeas
potentasGSinboththeantimicrobialandthehemolyticalassay.Chapter5describesthesynthesis
ofthreenovelGSanalogues(40–42)andtheirstructuralandbiologicalevaluation.Thestructureof
40, containing a furanoid SAA, adopted a similar sheet structure with reoriented backbone
conformationinsolutioncomparedto39b.Thesolutionstructureofanalogues41and42resemble
theoverallstructureofGSitselfandthebackbonereorientationcouldnotbeobserved.Theresults
obtainedfromtheantimicrobialandhemolyticalassaysshowasimilarprofilefor40comparedtoGS
and analogue 39b. Whereas the antimicrobial potencies of 41 and 42 are comparable to GS, both
showareducedtoxicityforerythrocytesinthehemolyticalassaywith41abouthalfastoxicasGS.
TheintroductionofcyclicfuranoidSAAsapparentlyaltersthebackboneconformationbutdoesnot
149
Chapter8
influence the biological profile. Linear dipeptide isosters 41 and 42 did not disrupt the backbone
conformationandshowedanimprovedbiologicalprofile.
Figure3;GS,knownderivatives39aandbandnewlydevelopedderivative40 42
Introduction of a second copy of AAA 44 might shed light on the tolerance for less
conformationallyrestricteddipeptideisosterswithrespecttoantimicrobialandhemolyticalactivity.
Figure4;GSanaloguewithincreasedflexibility
150
SummaryandFutureProspects
InChapter6,asimilarstrategy,theuseofdipeptideisosters,isappliedonLoloatinCand
thestructuralandbiologicalconsequencesoftheincorporationoffuranoidSAA46replacing
three different motifs (highlighted in Figure 5). Structural analysis by means of NMR
indicated that the solution structure of each analogue differed from the dumbbelllike
conformation of Loloatin C. Also, all analogues containing SAA 46 were shown to be less
potentintheantimicrobialandthehemolyticalassaythentheparentcompound,LoloatinC.
Possibly, incorporating AAA 48 into the Loloatin C allows the compound to adopt the
dumbbelllike conformation but still profit from the benefits of nonnatural amino acid
replacements.
Figure5;StructuresofSAA46andLoloatinC,47.HighlightedmotifhavebeenreplacedbySAA46.
Biotinylatedmethylfluorphosphonateasbiochemicalprobe
In Chapter 7, the synthesis of biotinylated methylfluorophosphonate 50 has been
described.Thecompoundswereappliedtostudythebiologicaleffectsofintoxicationwith
toxins like sarin 51 and the isolation of butyrylcholinesterase from human plasma.
Competitionexperimentsbetween50and51inmonkeyliverhomogenates,intactandlysed
A549lungcellsidentified8proteinsthatwereinhibitedby51inacompetitivemanner.The
synthesis of 50 from precursor 49 allows the synthesis of organophosphonate bearing a
differentleavinggroup(52)oradifferentreportergroup(53).In52,thefluorideisreplaced
bya2(diisopropylamino)ethanethiolmoietythatisalsofoundinthenerveagentVXwhich
enables a detailed study on the inhibition profile of the agent. The introduction of a solid
supportintotheconstructmayopenthewayforanalternativeisolationprocedure.
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Chapter8
Scheme7;Structuresandproposedsynthesisofbiotinylatedmethylfluorophosphonate50andanaloguebearinga
differentleavinggroup(52)ortag(53).Reagentandconditions:(i)TBAT,THF.(ii)TFA/DCM.(iii)biotinOSu,DiPEA,
DMF.(iv)2(diisopropylamino)ethanethiol,DBU,THF.(v)SepharoseOSu,DiPEA,THF
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