Lys162nano6

1Structure-ActivityInvestigationsandOptimisationsofNon-metaboliteAgonistsfortheSuccinateReceptor1ElisabethRexenUlven1,MetteTrauelsen2,MatjazBrvar1,MichaelLückmann3,Line.
Bielefeldt1,LisaK.
I.
Jensen1,ThueW.
Schwartz2,3&ThomasM.
Frimurer2Thesuccinatereceptor1(SUCNR1)isareceptorforthemetabolitesuccinate,whichfunctionsasametabolicstresssignalintheliver,kidney,adiposetissueandtheretina.
However,potentnon-metabolitetoolcompoundsareneededtorevealthephysiologicalroleandpharmacologicalpotentialofSUCNR1.
Recently,wepublishedthediscoveryofacomputationallyreceptor-structurederivednon-metaboliteSUCNR1agonistserieswithhightargetselectivity.
Weherereportourstructure-activityexplorationandoptimisationthathasresultedinthedevelopmentofagonistswithnanomolarpotencyandexcellentsolubilityandstabilitypropertiesinanumberofinvitroassays.
Ligand-guidedreceptormodelswithhighdiscriminativepowerbetweenbindingofactiveandinactivecompoundsweredevelopedfordesignofnovelchemotypes.
The7-transmembraneG-proteincoupledreceptorSUCNR1wasfirstidentifiedin2001andfoundtohaveclosehomologywiththepurinergicreceptorP2Y1.
Thereceptorwasthereforeinitiallybelievedtobeactivatedbypurinergiccompounds1.
However,in2004,SUCNR1wasdeorphanisedandfoundtobeactivatedbythecitricacidcycleintermediatesuccinateatmicromolarconcentrations2.
Theothercitricacidcycleintermediatesoxalo-acetateandα-ketoglutaratewerealsofoundtoactivatethereceptor,albeitwithreducedpotency3,4.
SUCNR1ishighlyexpressedinliver,kidney,andadiposetissue,buthasalsobeenreportedinretina,heart,andimmunecells2,5–7.
DuringoxidativestresssuccinatecanaccumulateandreachlocalconcentrationssufficientlyhightoactivateSUCNR18,leadingtoavarietyofunwantedphysiologicaleffectssuchashypertension2,hypertrophyoftheheart7,inflammation9,inhibitionoflipolysisinthewhiteadiposetissue5,activationofhepaticstellatecells10,andvasculargrowthintheretina6.
AlthoughmoststudiessofarindicatethatantagonistsforSUCNR1mightbeoptimalfromatherapeuticperspectivethishasyettobeexperimentallyconfirmedandismainlybasedonstudiesusingthenaturalagonistsuccinate,acompoundthatbesideitsrelativelyweakpotencyalsoisanintermediateinthecitricacidcycleandexertseffectsunrelatedtoSUCNR1.
In2011,thefirstandsofaronlyantagonistsforSUCNR1werereported,con-sistingofaseriesofnaphthyridinesthatdemonstratedgoodpharmacokineticpropertiesinrats11.
Smallsyntheticsuccinateanalogueshaverecentlybeenreportedasfullagonistsandalthoughthecompoundsshowimprovedagonisticpotency,thebestcompound,cis-epoxysuccinicacid,hasanEC50ofonly2.
7μM3.
BasedonthecharacterisationofthebindingsiteforsuccinateinSUCNR1andidentificationofanemptyside-pocketinthereceptor,wehaverecentlypublishedthediscoveryofnoveldrug-likeSUCNR1agonistswhichinaccordancewithbothloss-andgain-of-functionmutationaldataexploitedthispocket4.
Herewereportthefurtherdevelopmentandstructure-activityinvestigationsofthisagonistseries.
ThegeneratedlibraryofactiveandinactivecompoundstogetherwiththeoriginalreceptormodelbasedontheX-raystructureoftheP2Y1receptorallowedforgenerationofnewSUCNR1modelswhichcoulddiscriminatebetweenactiveandinactive1DepartmentofPhysics,ChemistryandPharmacy,UniversityofSouthernDenmark,Campusvej55,5230,OdenseM,Denmark.
2NNFCenterforBasicMetabolicResearch,SectionforMetabolicReceptology,FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Blegdamsvej3,2200,Copenhagen,Denmark.
3LaboratoryforMolecularPharmacology,DepartmentofBiomedicalResearch,FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Blegdamsvej3,2200,Copenhagen,Denmark.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoE.
R.
U.
(email:elc@sdu.
dk)Received:18April2018Accepted:19June2018Published:xxxxxxxxOPEN2compoundswithhighdiscriminativepower.
Althoughthehumanandmousereceptororthologuesarecloselyrelatedtheyarenotidenticalandsuccinateshowsasmallincreaseinpotencyonthemousereceptor2.
Allcom-poundshavethereforebeenstudiedonboththehumanandmouseorthologuesinordertodeveloptoolcom-poundsforfurtherinvestigationsofSUCNR1.
ResultsandDiscussionSynthesis.
Alltestcompoundsweresynthesisedbythesameoverallroute,startingfromL-asparticacid(1)thatwasconvertedtothecorrespondingdimethylesterhydrochloride2byanacidcatalysedesterification(Fig.
1).
Couplingof2tovariouscarboxylicacidswasefficientlyachievedviathecorrespondingacylfluoridegeneratedinsitubyfluoro-N,N,N',N'-bis(tetramethylene)foramidiniumhexafluorophosphate(BTFFH)12.
HalogenatedarylandheteroarylcompoundswerecoupledwithvariousarylboronicacidsbyaSuzukireactionusingthe4thgen-erationPd-XPhosprecatalyst.
Thephenoliccompoundswereafterwardsalkylatedbyalkylhalidesor–tosylates.
Finally,basepromotedhydrolysisusingLiOHgavethedesireddicarboxylicacidtestcompounds.
Explorationofstructureactivityrelationship(SAR).
Inordertooptimisetheactivityoftheleadcom-poundCmpd(S)130(3),westartedbyexploringlargermodificationsofthearomaticpartofthemolecule(selectedexamplesareincludedinTable1).
Initially,thearomaticpartwastruncatedbyexchangingthefluoro-phenylwithabromine(4),whichresultedinmorethanoneorderofmagnitudedecreaseinpotency.
Exchangingthebromofuranewiththehalogenatedphenyls5–7revealedweakactivityonbothreceptororthologuesoftheortho-chlorineandpara-bromineanalogs.
Onlythelargermeta-iodinesubstitutedcompound6showedmicro-molaractivityonbothreceptororthologues.
Insertingamethylene-linkerbetweentheamideandthebromophe-nyl(8)resultedinaninactivecompoundandexchangingthebromineforthefluorophenyl(9)onlygaveaweaklyactivecompound,farlesspotentthanthelead3.
Sincethesmallercompoundsdidnotshowsufficientactivitywemovedthefocustoexplorationsontheterminalphenylring(Table2).
Initially,theoriginal4-fluorosubstituentwasremoved(10).
Thisonlyaffectedthepotencymarginallyonbothreceptororthologues,butexposesapotentialmetabolicallylabilesite.
Next,amethylscanoftheringwasperformedtoinvestigatethebindingsiteforadditionalspace(11–13).
Whereasboththeortho-andmeta-methylweretoleratedonlythepara-methyl13ledtoamorepotentcompoundonbothhumanandmouseSUCNR1.
Attemptstopickuphydrogenbondinteractionsrevealedthat2-methoxy(16)wasequivalenttothe2-methylinpotencyandthat3-hydroxymethyl(14)waslesstoleratedthanthe3-methyl(12)witha3-folddecreaseinpotency.
Again,thepara-positionwasfavouredwiththe4-hydroxy(15)beingalmostequipotentwiththe4-methylcompound.
Furtherexplorationofthepara-positionindicatedthatthispartofthebindingpocketisabletoaccommodatepolarsubstituentssuchasmethoxy(17)andnitrile(19)aswellasnon-polarsubstituentssuchastrifluoromethyl(18),allshowingsimilarpotenciesinthesubmicromolarrangeonthehumanreceptororthologueandwith17slightlyfavouredonthemouseorthologue.
Tofurtherexplorethe4-methoxycompound,themostpotentanalogueonthemousereceptor,asubseriesofpara-alkoxyanalogueswereinvestigated(Table3).
Thelargerandmoreelectronegativetrifluoromethoxy(20)showeda2-foldincreaseonhSUCNR1butwasaccompaniedby>4-folddecreaseonmSUCNR1,renderingthecompoundapproximatelyequipotentonbothorthologues.
Expandingtoeitherethoxy(21)ori-propoxy(22)resultedinasmallincreaseinpotency,indicatingthatthebindingpocketcanaccommodatemoreelongatedandbulkysubstituents.
Toexplorewhetherornotthebindingsitecouldalsoaccommodatelargerhydrophilicsub-stituentstheoxatane23wasinvestigatedandfoundtolerable,butledto>2-foldreductionofpotency.
Finally,themesylpropoxyanalogue24wasexplored.
Thisappendage,whichlowersthelipophilicityanorderofmagnitude,haspreviouslybeenappliedtolowerlipophilicityofligandsforthefreefattyacidreceptorFFA1andhasproventobeametabolicallystablesubstituent13,14.
LipophilicityofthiscompoundisinthelowendofthedesiredrangeFigure1.
Generalsyntheticroute.
Reagentsandconditions:(a)SOCl2,MeOH,0°C→reflux.
(b)BTFFH,DIPEA,ArCOOH,DCM,80°C.
(c)ArB(OH)2,Pd-XPhos-G4,0.
5MK3PO4aq,THF,roomtemp.
(d)ForR"=OH:K2CO3,alkylhalide/tosylate,MeCN,50–55°C.
(e)0.
6MLiOHaq,THF,roomtemp.
3butitisinterestingtonotethatthecompoundexhibitedaligandlipophilicityefficiency(LLE)>5basedonClogPandalmostsustainedpotencyonbothmSUCNR1andhSUCNR1.
Subsequently,theattentionwasdirectedtowardsthecentralaromaticring(Table4).
Replacingthefuraneof3withthe1,3-substitutedphenyl25improvedthepotencysomewhatonthehumanorthologuewhereasthepotencyonthemouseorthologuedecreased10-fold.
The1,4-substitutedphenyl26clearlyledtoanunfavouredgeometrywithonlytraceactivityonSUCNR1.
Thecorresponding2,6-substitutedpyridine27sustainedthepotencyonhSUCNR1andalmostregainedthepotencyonmSUCNR1.
Next,asmallselectionofcompoundswassynthesisedtocombinetheobservedSARofthealkoxy-substituentsandalterationsofthecentralring(Table5).
The4-methoxyand4-trifluoromethoxysubstituents,beingthemostpotentalkoxy-substituentsonthemouseandhumanreceptororthologues,respectively,wereattachedtocom-poundsbearingthe1,3-substitutedphenylandthe2,6-substitutedpyridineasthecentralring.
Noimprovementwasobservedforthemethoxyanalogues(28–29).
Incontrast,thetrifluoromethoxyanalogues(30–31)werefoundtobethemostpotentcompoundsonhSUCNR1,butunfortunatelywithouthighpotencyonmSUCNR1,especiallyfor30.
Theelongatedtrifluoroethoxyanalogue32showedimprovedpotencyonthemouseortho-logue.
Still,31remainedthemostpotentagonistonhSUCNR1.
Finally,the4-propoxyanalogue33wasfoundtobeapproximatelytwiceaspotentasthecorrespondingmethoxyanalogue29andalmostequipotentonbothorthologues.
TogetabetteroverviewoftheobservedSARonthehumanandmousereceptororthologuesascatterplotofallactivecompoundswasmade(Fig.
2a).
Thecompoundswerecolor-codedaccordingtothecentralorter-minalring,whichclearlyindicatedthatcompoundswithacentralmeta-substitutedphenylwerebettertoler-atedonhSUCNR1.
The2,6-substitutedpyridineswereingeneralmorepotentandespecially31wasfavouredonhSUCNR1.
Compoundswithafuraneascentralringwereingeneralequipotentonthetworeceptorort-hologues,butwithadditionofaphenalkoxymoiety,thecompoundsbecamemorepotentonmSUCNR1with21beingthemostpotent.
Furthermore,dose-responsecurvesofsuccinate,3,21,and31clearlyshowedthatthenon-metabolitecompoundswerepartialagonists,withclearspeciesdifferencesonpotencybutnotefficacy(Fig.
2b,c).
Thepartialagonismwasobservedforallagonisticcompounds(Tables1–5,SupplementaryFig.
S1).
MolecularmodellingandunbiasedligandguidedrefinementofSUCNR1receptor-ligandcom-plexes.
Todescribethedetailedmolecularmechanismofactionandtorationalisethestructure-activitydata,RhSUCNR1mSUCNR1ClogPbpEC50(efficacy,%)a35.
75±0.
08(72.
8±2.
6)6.
46±0.
06(79.
3±1.
6)1.
6644.
53±0.
13(78.
1±7.
5)4.
93±0.
05(95.
8±2.
9)0.
44522%@104M13%@104M0.
28650%@104M64%@104M1.
5278%@104M15%@104M1.
2685%@104M11%@104M1.
28925%@104M15%@104M2.
44Table1.
Investigationofmiscellaneousamideanalogues.
apEC50valuesweredeterminedfromdose-responsecurvesofinductionofIP3turnoverinSUCNR1transfectedHEKcells(N=3),efficacyisdeterminedrelativetosuccinate(100%).
bCalculatedbyBioByte'salgorithmasimplementedinChemDrawProfessional16.
0.
1.
4(ClogPoption).
4wedockedthecompoundsinTables1–5toamodelofSUCNR1incomplexwiththeleadcompound(3)sup-portedbyadozenofsitedirectedmutagenesisdataaspresentedearlier4.
Despitethatweobservedbindingcon-formationssimilartotheleadandwithfavourablescores,themodel-asperhapscouldbeexpected-wasunabletorankthecompoundsinreasonableagreementwiththeactivitydata.
TheinterpretationofSARandstruc-turebaseddesignreachesitsmaximumpotentialwhenthereceptordisplaysthestructuralchangesneededforligandbinding,asithaspreviouslybeenshowninablindpredictionassessmentofadenosineA2areceptorcom-plexes(GPCRDock2008)15–18.
WethereforeappliedaniterativeAutomatedLigand-guidedBackboneEnsembleReceptorOptimisationprotocol(ALiBERO)19,whichsamplesfullreceptorandligandflexibilityguidedbytheligandinformationgainedinthisstudytovalidateandbuildconfidenceinthemodel.
Inbrief,startingfromthehomologymodelsupportedbymutagenesisdata4,ALiBEROintroducedreceptorflexibilityviaNormalModeAnalysisandMonteCarlosampling,togenerateasmallsubsetofreceptormod-els(pockets).
Allcompoundstestedinthisstudyweregroupedintoanactive(EC50≤10M)andadecoyset(EC50>10M)consistingof25compoundseach.
Foralistofallcompoundsusedintheoptimisationprotocol,seeTableS1.
ReceptorstructureswerethenchosenbasedontheirabilitytodiscriminateactivesfrominactivesRingAhSUCNR1mSUCNR1ClogPbpEC50(efficacy,%)a3c5.
75±0.
08(72.
8±2.
6)6.
46±0.
06(79.
3±1.
6)1.
66105.
29±0.
19(81.
6±8.
4)6.
16±0.
11(81.
3±3.
6)1.
47115.
27±0.
18(94.
7±8.
7)6.
13±0.
10(76.
7±3.
1)1.
67125.
02±0.
10(91.
9±5.
3)6.
07±0.
09(81.
6±3.
0)1.
97136.
33±0.
14(69.
1±3.
9)6.
83±0.
11(73.
2±2.
7)1.
97144.
81±0.
20(74.
2±11.
8)5.
71±0.
09(79.
6±3.
7)0.
43156.
13±0.
14(71.
1±4.
2)6.
82±0.
08(74.
8±2.
0)0.
87165.
29±0.
15(103.
8±7.
9)6.
14±0.
10(78.
1±3.
1)0.
89176.
41±0.
14(85.
3±4.
5)7.
39±0.
07(84.
2±1.
8)1.
45186.
55±0.
25(81.
0±7.
2)6.
81±0.
17(80.
6±4.
6)2.
43196.
39±0.
11(72.
1±3.
2)6.
79±0.
06(76.
0±1.
5)1.
00Table2.
InvestigationofringA.
apEC50valuesweredeterminedfromdose-responsecurvesofinductionofIP3turnoverinSUCNR1transfectedHEKcells(N=3),efficacyisdeterminedrelativetosuccinate(100%).
bCalculatedbyBioByte'salgorithmasimplementedinChemDrawProfessional16.
0.
1.
4(ClogPoption).
cDuplicationofdatafromTable1.
5inaretrospectivevirtualscreeningusingthedockingprotocolandscoringfunctioninICM(MolsoftL.
L.
C.
,SanDiego,CA,USA)20,asmeasuredbythenormalisedsquarerootareaunderthecurve(NSQ_AUC).
Thebest-performingstructuresfromthefirstgenerationwereconsequentlyselectedforthenextgenerationandthestepswererepeatedinaniterativefashionuntilmaximumdockingperformanceofreceptorstructurestoenrichactivecompoundswasreached.
TheALiBERO-optimisedreceptorensemblewassubsequentlyvalidatedinavirtualligandscreeningusinganexternaltestsetwithahigheractive:decoyratio(~1:50).
Notably,theligand-guidedALiBERO-basedmSUCNR1modelsdemonstratedadramaticimprovementinret-rospectivevirtualscreeningperformanceofthedevelopedcompoundscomparedwiththeinitialhomologymod-elsandprovedsuccessfulinseparatingthemajorityofactivefrominactiveligandsindockingscreens(Fig.
3a,SupplementaryFig.
S2).
Itisinterestingtonotethattheactiveligandsbindtoanextendedbindingcavityinaveryconsistentposecomparedtotheleadcompound(3)supportedbybothloss-of-functionbutalsogain-of-functionmutagenesisdataaswehavereportedpreviously4.
Forexample,thereceptormutantsR251:6.
58LandR276:7.
39Fshowedapotencydecreaseofmorethan100-fold.
ThebindingcavityofSUCNR1ischaracterisedbyapolarnetworkconsistingofresiduesinTM-II(Y79:2.
64),TM-III(R95:3.
29),TM-VI(R248:6.
55,R251:6.
58),TM-VII(K269:7.
32,Y272:7.
35,R276:7.
39),ECL2(D174)aswellasarelativelyhydrophobicsubpocketspanningbetweenTM-I,-II,ECL1and2uptowardstheextracellularsurfaceofthereceptor(residuenumberingaccordingtoRhSUCNR1mSUCNR1ClogPbpEC50(efficacy,%)a17cMe6.
41±0.
14(85.
3±4.
5)7.
39±0.
07(84.
2±1.
8)1.
4520F3C6.
99±0.
15(61.
7±3.
6)6.
72±0.
11(69.
7±2.
8)3.
1721Et6.
59±0.
14(56.
3±3.
1)7.
44±0.
09(70.
2±2.
0)1.
9822iPr6.
84±0.
14(69.
6±4.
3)7.
29±0.
07(75.
6±1.
7)2.
29236.
27±0.
11(76.
5±3.
1)6.
97±0.
07(82.
4±1.
9)1.
81246.
10±0.
09(63.
9±2.
4)7.
33±0.
09(74.
6±2.
1)0.
53Table3.
Alkoxyanalogues.
apEC50valuesweredeterminedfromdose-responsecurvesofinductionofIP3turnoverinSUCNR1transfectedHEKcells(N=3),efficacyisdeterminedrelativetosuccinate(100%).
bCalculatedbyBioByte'salgorithmasimplementedinChemDrawProfessional16.
0.
1.
4(ClogPoption).
cDuplicationofdatafromTable2.
RingBhSUCNR1mSUCNR1ClogPbpEC50(efficacy,%)a3c5.
75±0.
08(72.
8±2.
6)6.
46±0.
06(79.
3±1.
6)1.
66255.
91±0.
11(49.
7±3.
9)5.
40±0.
13(58.
0±4.
5)2.
27264%@104M7%@104M2.
27276.
03±0.
25(78.
2±8.
0)6.
10±0.
17(89.
3±6.
1)2.
05Table4.
InvestigationofringB.
apEC50valuesweredeterminedfromdose-responsecurvesofinductionofIP3turnoverinSUCNR1transfectedHEKcells(N=3),efficacyisdeterminedrelativetosuccinate(100%).
bCalculatedbyBioByte'salgorithmasimplementedinChemDrawProfessional16.
0.
1.
4(ClogPoption).
cDuplicationofdatafromTable1.
6gpcrdb.
org).
Theoptimisedreceptorensemble(Fig.
3b)accommodatestheactivecompoundsinacommonbind-ingmodeinwhichtheleft-handsidecarboxylicacidsmakehydrogenbondinteractionswithR95:3.
29,R251:6.
58andR276:7.
39andwheretheright-handsideoftheligandsadoptsanangledconformationthatisdefinedbythelinkerbetweenringAandBingoodagreementwiththeobservedstructure-activityrelationship.
DuetotheRingARingBhSUCNR1mSUCNR1ClogPbpEC50(efficacy,%)a286.
79±0.
12(58.
0±2.
7)5.
92±0.
09(64.
0±2.
5)2.
01296.
85±0.
19(52.
6±3.
9)6.
74±0.
13(71.
2±3.
2)1.
07307.
23±0.
14(49.
2±2.
7)5.
84±0.
16(45.
3±3.
4)3.
11317.
64±0.
13(61.
7±2.
5)6.
75±0.
09(69.
9±2.
2)2.
18327.
23±0.
23(88.
7±6.
1)7.
00±0.
09(68.
1±2.
5)1.
86337.
23±0.
37(66.
4±7.
2)7.
29±0.
18(61.
5±3.
8)2.
13Table5.
Combinedanalogues.
apEC50valuesweredeterminedfromdose-responsecurvesofinductionofIP3turnoverinSUCNR1transfectedHEKcells(N=3),efficacyisdeterminedrelativetosuccinate(100%).
bCalculatedbyBioByte'salgorithmasimplementedinChemDrawProfessional16.
0.
1.
4(ClogPoption).
Figure2.
Summaryofinvitroagonistpotencies.
(a)ScatterplotofinvitroagonistpotenciesbyIP3accumulationinSUCNR1transfectedHEK-293cellsofthecompoundsinTables1–5onthehSUCNR1andmSUCNR1receptor.
(b,c)Dose-responsecurvesforsuccinateandselectedcompounds(3,21and31)onhSUCNR1andmSUCNR1.
7shapeanddirectionofthesubpocketaccommodatingringA,compoundswithabentconformationbetweentheamideandtheterminalring,e.
g.
compoundshaving2,5-substituedfuranormeta-substitutedphenylascentralring,arestericallybettertoleratedthane.
g.
thepara-substitutedphenyl26andelongatedpara-substitutedphenyl9.
Furthermore,compoundswithapyridine(e.
g.
27,29,31),orfuranascentralring,canstabilisethefavouredbentconformationbyintramolecularhydrogenbondingbetweenthepyridinenitrogenorfuranoxygenandtheamideN-H,therebyinducinganoptimallowenergyconformation.
ThesubpocketthatisoccupiedbyringA,containsW10:1.
42whichallowsforparalleldisplacedaromaticstackinginteractionsthatarepresentinallbindingposesfortheactiveligands.
AsthispocketspansallthewaytotheextracellulartipsofTM-Iand-II,itisabletoaccomodatelongersubstituents,suchasthemesylate24(Fig.
3b),whichlikelyinteractswithsolventwatermoleculesontheextracellularsurfaceofthereceptorcavity.
CompoundsthatcontainO-alkyl,hydroxyl,ornitrilegroupsinthepara-positiononringA,suchas15,17,19–24and28–31,canformhydrogenbondinteractionswithK83(Asn87inhSUCNR1)andbackboneNHofGlu170inECL1and2b(Fig.
3c).
Duetothegeometryofthisinteraction,ortho-andmeta-subtitutions(14+16)arelessfavorable.
WhilethepolarresiduesthatcoordinatethedicarbocylicacidmoietyarehighlyconservedbetweenmouseandhumanSUCNR1,thehydrophobicsubpocketaccommodatingringAismorediversewithseveraldiffer-encesinECL1(Lys83/Asn87),ECL2(Lys162/Thr166,Glu163/Asp166,Glu164/Asn166,Asn167/Thr171)andTM-VII(Lys269/Asn274)betweenmSUCNR1andhSUCNR1,respectively.
TheLys83/Asn87differencebetweenmSUCNR1andhSUCNR1mightexplainthehigherpotenciesofthetrifluoromethoxy-substitutedcompounds30and31onhSUCNR1,asthesebulkymoitieslikelyformmorefavorableinteractionswiththeshorterAsnside-chain(Fig.
3c).
Inconclusion,thedevelopedligand-guidedSUCNR1modelsareconsistentwiththeSAR,andtheyaresuffi-cientlyaccuratetoseparateactivesfromdecoys,suggestingthatthemodelswillbevaluableinprospectivestudiestosupportstructure-baseddrugdesignofadditionalchemotypesdirectlyrelatedtodrugdiscoveryapplications.
Physicochemicalandinvitrostabilityproperties.
BasedontheSARexploration,theoriginallead(3)andthemostpotentagonistsonhSUCNR1(31)andmSUCNR1(21)wereselectedforfurtherpropertyinvestiga-tions.
Allcompoundsshowedexcellentchemicalstability(>98%after70hours)andkineticsolubility(>200μM)in10mMPBS7.
4.
However,thehighaqueoussolubilitymadelogD7.
4determinationsverychallengingandonlythemostlipophiliccompound31couldbequantified(logD7.
4=2.
13).
ThestabilityofthecompoundswasfurtherFigure3.
IterativeSUCNR1receptoroptimisationbasedonligandinformationgainedinthisstudy.
(a)Small-scalevirtualligandscreening(VLS)resultsatdifferentstagesoftheoptimisationprocess(generationsGEN_1-GEN_4).
Receiveroperatingcharacteristic(ROC)curvesandnormalisedsquarerootareaunderthecurve(NSQ_AUC)valuesareshownforeachgenerationofreceptorpockets.
ThediagonalcorrespondstoarandomVLSperformance.
(b)Best-performingreceptorpocketensemble(GEN_4),consistingofthreereceptorstructuresshownwiththebest-scoreddockingposesofall25activecompoundsinvestigatedinthisstudy(darkgreenlines).
Best-scoreddockingposesofcompound24(b),31(c),3(d)and21(e)areshowningreensticks.
Polarreceptor-ligandinteractionsareindicatedbyyellowspheres.
Homologymodelsarebasedonthex-raycrystalstructureoftheP2Y1receptor(PDB4XNW)whichsharesseveralkeyside-chainsinthebindingsitewithSUCNR1,i.
e.
Y79:2.
64,D174andR276:7.
39.
FigurewasmadeusingPyMol.
8examinedinaselectionofsimulatedgastrointestinalfluids(FaSSGF,FaSSIFandFeSSIF)andallcompoundswerefoundtobestablefor2hours,withexceptionof31inFeSSIF,whichcouldnotbedeterminedduetooverlappingUV-absorptionofthecompoundandmedia.
Finally,thestabilityinmouselivermicrosomeswasinvestigatedandallcompoundswerefoundtobestablefor1hour,possiblypartlyduetothehydrophilicnatureofthecompounds.
ConclusionWeherereportthestructure-activityinvestigationsofaseriesofnon-metaboliteSUCNR1agoniststhatwasorig-inallyidentifiedfromacomputational,receptor-structurederivedagonisticlead.
ALiBEROoptimisedhomologymodelsofthemouseSUCNR1weredevelopedbasedonthex-raystructureofthecloselyrelatedP2Y1recep-torandfoundtobesufficientlyaccuratetodiscriminatebetweenactivesandinactivesandcouldexplainthemajorityoftheSARobservations.
Theexplorationledtodevelopmentofpotentdrug-like,non-metabolitetoolcompoundswithnanomolarpotencyonboththehumanandmurinereceptororthologuesandexcellentphysic-ochemicalandinvitrostabilityproperties.
WebelievethesecompoundswillbeusefulforfurtherinvestigationsofSUCNR1asapotentialtherapeutictargetandpharmacokineticstudiesinmicearecurrentlyongoingandwillrevealifabsorptionofthecompoundsmightbechallengedbytheirhydrophilicnatureandiftheexcellentstabil-itypropertiesareconservedinvivo.
MethodsGeneral.
Commercialstartingmaterialsandsolventswereusedwithoutfurtherpurification,unlessotherwisestated.
THFwasfreshlydistilledfromsodium/benzophenone.
DCMwasdistilledandstoredover3sieves.
MeCNandN,N-diisopropylethylamineweredriedover3sieves.
K2CO3wasdriedandstoredinanoven.
TLCwasperformedonTLCsilicagel60F254platesandvisualisedat254or365nmorbystainingwithphosphomo-lybdicacid,ninhydrin,orKMnO4stains.
Purificationbyflashchromatographywascarriedoutusingsilicagel60(0.
040–0.
063mm,Merck).
1Hand13CNMRspectrawererecordedat400and101MHz,respectively,onaBrukerAvanceIII400at300K.
SpectrawerecalibratedrelativetotheinternalstandardTMSorresidualsolventpeak:CDCl3(δC=77.
16ppm,δH=7.
26ppm),DMSO-d6(δC=39.
52ppm,δH=2.
50ppm)andacetone-d6(δC=29.
84ppm,δH=2.
05ppm).
HPLCanalysiswasperformedusingaGeminiC18column(5μm,4.
6*150mm);flow:1mL/min;10%MeCNinwater(0–1min),10–100%MeCNinwater(1–10min),100%MeCN(11–15min),withbothsolventscon-taining0.
1%HCOOHasmodifier;UVdetectionat254nm(or280nm,304nmand315nmdependingontestcompoundabsorptionmaximumforsolubilityandstabilitystudies).
UPLCanalysiswasperformedusinga100C18column(1.
7μm,2.
1*100mm);flow:0.
3mL/min;70%MeOHinwater(0–6min),thewatercontaining0.
1%HCOOHasmodifier;UVdetectionat254nm,280nm,304nmor315nmdependingontestcompoundabsorptionmaximum.
High-resolutionmassspectra(HRMS)wereobtainedonaBrukermicrOTOF-QII(ESI).
OpticalrotationwasmeasuredonanAntonPaarMCP100Polarimeter(AntonPaarCell100mm,CL.
0.
01,5mm).
PuritywasdeterminedbyHPLC(254nm)andconfirmedbyinspectionofNMRspectra.
Thepurityofalltestcompoundswere>95%.
Amidecoupling.
Anovendriedmicrowavevialunderargonatmospherewaschargedwiththeacid(1.
3equiv),dryDCM(2mL/mmol),N,N-diisopropylethylamine(5.
5equiv)andBTFFH(1.
5equiv).
Thereactionmixturewasstirredatrtfor30minbeforetheHClsaltoftheamine(1equiv)wasadded.
Afteraddition,thevialwascappedandheatedto80°Covernight.
ThereactionwascooledtortanddilutedwithwaterandextractedwithEtOAc(x3).
Theorganicphaseswerecombined,washedwithbrine,driedoverNa2SO4andconcentratedinvacuo.
Theresiduewaspurifiedbyflashcolumnchromatography(SiO2,EtOAc:petroleumether).
Suzukicoupling.
Aschlenckflaskunderargonwaschargedwithboronicacid(1.
1equiv),aryl/pyridylhalide(1equiv)andPd-XPhos-G4(2mol%).
Theflaskwasevacuatedandbackfilledwithargon(x3).
THF(5mL/mmol)andaqueous0.
5MK3PO4(2equiv)wasadded,andthereactionwasstirredatrt.
Aftercompletion,thereactionmixturewasdilutedwithwaterandextractedwithEtOAc(x3).
Theorganicphaseswerecombined,washedwithbrine,driedoverNa2SO4andconcentratedinvacuo.
Theresiduewaspurifiedbyflashcolumnchromatography(SiO2,EtOAc:petroleumether).
Alkylation.
Thephenol(1equiv)wasdissolvedindryMeCN(~6mL/mmol)inadryvialunderargonatmos-phere.
Thealkylhalide/tosylate(2–7equiv)anddryK2CO3(2equiv)wereaddedandthereactionwasstirredat50–55°CuntilconsumptionofthephenolasmonitoredbyTLC.
Aftercompletion,thereactionwasdilutedwithwaterandextractedwithEtOAc(x3).
Theorganicphaseswerecombined,washedwithbrine,driedoverNa2SO4,andconcentratedinvacuo.
Theresiduewaspurifiedbyflashcolumnchromatography(SiO2,EtOAc:petroleumether).
Esterhydrolysis.
Theester(1equiv)wasdissolvedinTHF(~6mL/mmol),andaqueous0.
6MLiOH(3equiv)wasadded.
ThereactionwasstirredatrtuntilconsumptionoftheesterasmonitoredbyTLC.
Aftercom-pletion,thereactionwasdilutedwithwater,acidifiedwithaqueous1MHClandextractedwithEtOAc(x3).
Theorganicphaseswerecombined,washedwithbrine,anddriedoverNa2SO4.
Theresiduewasconcentratedinvacuotogivethepuretitlecompounds.
(5-Bromofuran-2-carbonyl)-L-asparticacid(4).
Dimethyl(5-bromofuran-2-carbonyl)-L-aspartate(4e)wassynthesisedfrom5-bromofuroicacid(126mg,0.
66mmol)and2(100mg,0.
51mmol)accordingtothegeneralamidecouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:2)gave136mg(81%)ofayellowoil:Rf=0.
30(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
29(d,9J=7.
9Hz,1H),7.
09(d,J=2.
5Hz,1H),6.
45(d,J=3.
5Hz,1H),5.
02(dt,J=8.
6,4.
5Hz,1H),3.
80(s,3H),3.
73(s,3H),3.
13(dd,J=17.
3,4.
5Hz,1H),2.
94(dd,J=17.
3,4.
5Hz,1H);13CNMR(101MHz,CDCl3)δ171.
4,170.
8,156.
8,148.
9,125.
1,117.
3,114.
2,53.
0,52.
1,48.
3,36.
1;ESI-HRMS:calcdforC11H12BrNNaO6(M+Na)+355.
9740,found355.
9757;[α]20D16.
5°(c0.
2,MeOH).
4wassynthesisedfrom4e(35mg,0.
10mmol)accordingtothegeneralesterhydrolysisproceduretogive28mg(88%)ofawhitesolid:tR=7.
68min(HPLC);1HNMR(400MHz,Acetone-d6)δ7.
91(d,J=7.
9Hz,1H),7.
13(d,J=3.
5Hz,1H),6.
67(d,J=3.
5Hz,1H),5.
02–4.
94(m,1H),3.
02(d,J=5.
8Hz,2H);13CNMR(101MHz,Acetone-d6)δ172.
4,172.
3,157.
4,150.
7,125.
2,117.
4,115.
1,49.
4,36.
4;ESI-HRMScalcdforC8H9BrNNaO6(M+Na)+327.
9427,found327.
9431;[α]20D+7.
2°(c0.
2,MeOH).
(2-Chlorobenzoyl)-L-asparticacid(5).
Dimethyl(2-chlorobenzoyl)-L-aspartate(5e)wassynthesisedfrom2-chlorobenzoicacid(103mg,0.
66mmol)and2(99mg,0.
50mmol)accordingtothegeneralamidecouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave121mg(80%)ofapaleyellowsolid:Rf=0.
65(EtOAc);1HNMR(400MHz,CDCl3)δ7.
70–7.
66(m,1H),7.
44–7.
30(m,3H),7.
25–7.
23(m,1H),5.
11–5.
05(m,1H),3.
80(s,3H),3.
70(s,3H),3.
19–2.
98(m,2H);13CNMR(101MHz,CDCl3)δ171.
6,171.
0,166.
2,134.
4,131.
8,131.
2,130.
5,130.
4,127.
2,53.
1,52.
2,49.
3,36.
2;ESI-HRMScalcdforC13H14ClNNaO5(M+Na)+322.
0453,found322.
0461;[α]20D22.
5°(c0.
2,MeOH).
5wassynthesisedfrom5e(25mg,0.
08mmol)accordingtothegeneralesterhydrolysisproceduretogive22mg(97%)ofawhitesolid:tR=7.
34min(HPLC);1HNMR(400MHz,Acetone-d6)δ10.
80(brs,2H),7.
88–7.
80(m,1H),7.
60–7.
55(m,1H),7.
50–7.
44(m,2H),7.
44–7.
36(m,1H),5.
08–4.
98(m,1H),3.
09–2.
96(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
3,172.
2,166.
9,136.
8,132.
1,131.
6,130.
8,130.
4,127.
9,50.
0,36.
4;ESI-HRMScalcdforC11H9ClNO5(MH)270.
0175,found270.
0177;[α]20D+2.
0°(c0.
2,MeOH).
(3-Iodobenzoyl)-L-asparticacid(6).
Dimethyl(3-iodobenzoyl)-L-aspartate(6e)wassynthesisedfrom3-iodobenzoicacid(161mg,0.
65mmol)and2(99mg,0.
50mmol)accordingtothegeneralamidecouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave164mg(84%)ofapaleyellowstickyoil:Rf=0.
33(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ8.
17–8.
12(m,1H),7.
88–7.
83(m,1H),7.
78–7.
72(m,1H),7.
22–7.
14(m,2H),5.
08–4.
98(m,1H),3.
80(s,3H),3.
71(s,3H),3.
20–2.
91(m,2H);13CNMR(101MHz,CDCl3)δ171.
8,171.
2,165.
5,141.
0,136.
4,135.
8,130.
4,126.
4,94.
4,53.
1,52.
3,49.
2,36.
1;ESI-HRMScalcdforC13H14INNaO5(M+Na)+413.
9809,found413.
9801;[α]20D22.
0°(c0.
2,MeOH).
6wassynthesisedfrom6e(27mg,0.
07mmol)accordingtothegeneralesterhydrolysisproceduretogive24mg(97%)ofawhitesolid:tR=8.
93min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
25(t,J=1.
7Hz,1H),8.
13–8.
07(m,1H),7.
94–7.
89(m,2H),7.
30(t,J=7.
8Hz,1H),5.
06–4.
99(m,1H),3.
09–2.
93(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
3,172.
2,165.
9,141.
2,137.
3,137.
1,131.
4,127.
6,94.
4,50.
3,36.
4;ESI-HRMScalcdforC11H9INO5(MH)361.
9531,found361.
9531;[α]20D+4.
0°(c0.
2,MeOH).
(4-Bromobenzoyl)-L-asparticacid(7).
Dimethyl(4-bromobenzoyl)-L-aspartate(7e)wassynthesisedfrom4-bromobenzoicacid(133mg,0.
66mmol)and2(99mg,0.
50mmol)accordingtothegeneralamidecou-plingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave137mg(80%)ofabeigesolid:Rf=0.
35(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
71–7.
66(m,2H),7.
61–7.
56(m,2H),7.
23–7.
17(m,1H),5.
07–5.
00(m,1H),3.
80(s,3H),3.
71(s,3H),3.
19–2.
93(m,2H);13CNMR(101MHz,CDCl3)δ171.
9,171.
2,166.
1,132.
7,132.
0,128.
9,126.
8,53.
1,52.
3,49.
1,36.
1;ESI-HRMScalcdforC13H14BrNNaO5(M+Na)+365.
9948,found365.
9937;[α]20D22.
5°(c0.
2,MeOH).
7wassynthesisedfrom7e(25mg,0.
07mmol)accordingtothegeneralesterhydrolysisproceduretogive21mg(93%)ofawhitesolid:tR=8.
73min(HPLC);1HNMR(400MHz,Acetone-d6)δ10.
69(brs,2H),8.
09–8.
01(m,1H),7.
89–7.
82(m,2H),7.
72–7.
65(m,2H),5.
07–4.
99(m,1H),3.
09–2.
93(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
4,172.
3,166.
5,134.
3,132.
5,130.
2,126.
5,50.
3,36.
4;ESI-HRMScalcdforC11H9BrNO5(MH)313.
9670,found313.
9674;[α]20D+4.
0°(c0.
2,MeOH).
(2-(4-Bromophenyl)acetyl)-L-asparticacid(8).
Adryflaskunderanargonatmospherewaschargedwith4-bromophenylaceticacid(300mg,1.
4mmol),2(276mg,1.
4mmol),DMF(5mL),N,N-diisopropylethylamine(0.
85mL),1-hydroxybenzotriazole(256mg,1.
67mmol),andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride(321mg,1.
67mmol).
Thereactionwasstirredovernightatrt.
Aftercompletion,thesolventwasevaporatedandtheresiduewasextractedwithEtOAc(x3).
Theorganicphaseswerecombined,washedwithaqueous1MHCl,saturatedaqueousNaHCO3,brine,anddriedoverNa2SO4andconcentratedinvacuo.
Theresiduewaspurifiedbyflashchromatography(EtOAc:petroleumether,1:1)togive317mg(63%)ofdimethyl(2-(4-bromophenyl)acetyl)-L-aspartate(8e)asawhitesolid:Rf=0.
14(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
50–7.
44(m,2H),7.
19–7.
13(m,2H),6.
47(d,J=7.
5Hz,1H),4.
86–4.
79(m,1H),3.
74(s,3H),3.
64(s,3H),3.
54(s,2H),3.
04–2.
96(m,1H),2.
86–2.
78(m,1H);13CNMR(101MHz,CDCl3)δ171.
3,170.
9,170.
0,133.
4,132.
0,131.
0,121.
452.
8,52.
0,48.
7,42.
8,35.
9;ESI-HRMScalcdforC14H17BrNO5(M+H)+358.
0285,found358.
0295;[α]20D6.
0°(c0.
2,MeCN).
8wassynthesisedfrom8e(50mg,0.
14mmol)accordingtothegeneralesterhydrolysisproceduretogive45mg(98%)ofawhitesolid:tR=8.
88min(HPLC);1HNMR(400MHz,DMSO-d6)δ8.
44(d,J=7.
9Hz,1H),7.
54–7.
41(m,2H),7.
21(d,J=8.
4Hz,2H),4.
55–4.
45(m,1H),3.
45(s,2H),2.
73–2.
64(m,1H),2.
62–2.
53(m,1H);13CNMR(101MHz,DMSO-d6)δ177.
5,176.
8,174.
8,140.
9,136.
5,136.
2,124.
7,54.
0,46.
3,41.
3;ESI-HRMScalcdforC12H12BrNNaO5(M+Na)+351.
9791,found351.
9775;[α]20D+3.
0°(c0.
2,MeOH).
10(2-(4′-Fluoro-[1,1′-biphenyl]-4-yl)acetyl)-L-asparticacid(9).
Dimethyl(2-(4′-fluoro-[1,1′-biphenyl]4-yl)acetyl)-L-aspartate(9e)wassynthesisedfrom8e(100mg,0.
28mmol)and4-fluorophenylboronicacid(43mg,0.
31mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatogra-phy(EtOAc:petroleumether,1:4)gave80mg(77%)ofawhitesolid:Rf=0.
22(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
57–7.
49(m,4H),7.
34(d,J=8.
4Hz,2H),7.
15–7.
08(m,2H),6.
51(d,J=7.
6Hz,1H),4.
89–4.
82(m,1H),3.
73(s,3H),3.
65–2.
60(m,5H),3.
04–2.
96(m,1H),2.
88–2.
80(m,1H);13CNMR(101MHz,CDCl3)δ171.
3,171.
0,170.
5,160.
7(d,J=292.
3Hz),139.
3,136.
8(d,J=3.
3Hz),133.
5,129.
8,128.
6(d,J=8.
0Hz),127.
5,115.
7(d,J=21.
5Hz),52.
8,52.
0,48.
7,43.
1,36.
0;ESI-HRMScalcdforC20H20FNNaO5(M+Na)+396.
1218,found396.
1224;[α]20D5.
0°(c0.
2,MeCN).
9wassynthesisedfrom9e(73mg,0.
20mmol)accordingtothegeneralesterhydrolysisproceduretogive65mg(96%)ofawhitesolid:tR=10.
25min(HPLC);1HNMR(400MHz,Acetone-d6)δ7.
71–7.
65(m,2H),7.
58–7.
54(m,2H),7.
42(d,J=8.
1Hz,2H),7.
25–7.
18(m,2H),4.
85–4.
79(m,1H),3.
65(s,2H),2.
90–2.
86(m,2H);13CNMR(101MHz,Acetone-d6)δ176.
6,176.
5,175.
6,167.
6(d,J=244.
4Hz),143.
4,142.
3(d,J=3.
2Hz),140.
4,135.
0,133.
8(d,J=8.
1Hz),131.
9,120.
7(d,J=21.
6Hz),53.
9,47.
1,40.
8;ESI-HRMScalcdforC18H15FNO5(M+H)+344.
0940,found344.
0949;[α]20D+14.
6°(c0.
2,MeCN).
(5-Phenylfuran-2-carbonyl)-L-asparticacid(10).
Dimethyl(5-phenylfuran-2-carbonyl)-L-aspartate(10e)wassynthesisedfrom4e(100mg,0.
30mmol)andphenylboronicacid(40mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatography(EtOAc:petroleumether,1:3)gave67mg(67%)ofaclearoil:Rf=0.
25(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
76–7.
72(m,2H),7.
46–7.
33(m,4H),7.
22(d,J=3.
6Hz,1H),6.
75(d,J=3.
6Hz,1H),5.
10–5.
04(m,1H),3.
82(s,3H),3.
73(s,3H),3.
20–3.
12(m1H),3.
03–2.
95(m,1H);13CNMR(101MHz,CDCl3)δ171.
6,171.
1,158.
0,156.
0,146.
3,129.
6,128.
9,128.
8,124.
6,117.
2,107.
3,53.
0,52.
1,48.
3,36.
3;ESI-HRMScalcdforC17H18NO6(M+H)+332.
1129,found332.
1118;[α]20D7.
5°(c0.
2,MeCN).
10wassynthesisedfrom10e(50mg,0.
15mmol)accordingtothegeneralesterhydrolysisproceduretogive44mg(96%)ofawhitesolid:tR=9.
38min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
08(d,J=8.
1Hz,1H),7.
87–7.
80(m,2H),7.
49–7.
42(m,2H),7.
40–7.
33(m,1H),7.
21(d,J=3.
6Hz,1H),7.
01(d,J=3.
6Hz,1H),5.
09–5.
01(m,1H),3.
12–2.
97(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
4,172.
3,158.
5,156.
3,148.
0,130.
7,129.
8,129.
5,125.
2,117.
1,108.
2,49.
4,36.
5;ESI-HRMScalcdforC15H13NNaO6(M+Na)+326.
0635,found326.
0645;[α]20D+3.
9°(c0.
2,MeCN).
(5-(o-Tolyl)furan-2-carbonyl)-L-asparticacid(11).
Dimethyl(5-(o-tolyl)furan-2-carbonyl)-L-aspartate(11e)wassynthesisedfrom4e(100mg,0.
30mmol)ando-tolylboronicacid(45mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatography(EtOAc:petroleumether,1:3)gave80mg(77%)ofaclearoil:Rf=0.
29(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
71–7.
76(m,1H),7.
39(d,J=8.
1Hz,1H),7.
32–7.
27(m,3H),7.
25(d,J=3.
6Hz,1H),6.
64(d,J=3.
6Hz),5.
09–5.
03(m,1H),3.
80(s,3H),3.
71(s,3H),3.
19–3.
11(m,1H),3.
02–3.
94(m,1H),2.
54(s,3H);13CNMR(101MHz,CDCl3)δ171.
5,171.
1,158.
0,155.
8,146.
0,135.
4,131.
4,129.
0,128.
8,127.
8,126.
2,116.
6,110.
6,52.
9,52.
1,48.
2,36.
3,21.
7;ESI-HRMScalcdforC18H19NNaO6(M+Na)+368.
1105,found368.
1120;[α]20D+23.
7°(c0.
2,DCM).
11wassynthesisedfrom11e(50mg,0.
14mmol)accordingtothegeneralesterhydrolysisproceduretogive45mg(98%)ofapaleyellowsolid:tR=9.
85min(HPLC);1HNMR(400MHz,Acetone-d6)δ7.
99(d,J=8.
3Hz,1H),7.
77–7.
73(m,1H),7.
34–7.
26(m,3H),7.
23(d,J=3.
6Hz,1H),6.
83(d,J=3.
6Hz,1H),5.
08–5.
01(m,1H),3.
05(d,J=5.
7Hz,2H),2.
52(s,3H);13CNMR(101MHz,Acetone-d6)δ172.
5,172.
3,158.
5,156.
1,147.
7,136.
2,132.
2,130.
0,129.
5,128.
4,127.
1,116.
7,111.
6,49.
2,36.
4,21.
9;ESI-HRMScalcdforC16H14NO6(M+H)+316.
0827,found316.
0816;[α]20D+30.
5°(c0.
2,MeCN).
(5-(m-Tolyl)furan-2-carbonyl)-L-asparticacid(12).
Dimethyl(5-(m-tolyl)furan-2-carbonyl)-L-aspartate(12e)wassynthesisedfrom4e(100mg,0.
30mmol)andm-tolylboronicacid(45mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatography(EtOAc:petroleumether,1:3)gave70mg(68%)ofaclearoil:Rf=0.
23(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
56–7.
52(m,2H),7.
37(d,J=8.
1Hz,1H),7.
32(t,J=7.
9Hz,1H),7.
22(d,J=3.
6Hz,1H),7.
17(d,J=7.
6Hz,1H),6.
73(d,J=3.
6Hz,1H),5.
11–5.
04(m,1H),3.
82(s,3H),3.
73(s,3H),3.
20–3.
12(m,1H),3.
04–2.
96(m,1H),2.
42(s,3H);13CNMR(101MHz,CDCl3)δ171.
6,171.
1,158.
0,156.
2,146.
2,138.
6,129.
7,129.
5,128.
8,125.
2,121.
9,117.
2,107.
2,53.
0,52.
1,48.
3,36.
3,21.
5;ESI-HRMScalcdforC18H19NaNO6(M+Na)+368.
1105,found368.
1103;[α]20D+19.
3°(c0.
2,MeCN).
12wassynthesisedfrom12e(50mg,0.
14mmol)accordingtothegeneralesterhydrolysisproceduretogive46mg(quant.
)ofawhitesolid:tR=9.
88min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
04(d,J=8.
1Hz,1H),7.
68–7.
61(m,2H),7.
34(t,J=7.
7Hz,1H),7.
22–7.
17(m,2H),6.
99(d,J=3.
6Hz,1H),5.
08–5.
00(m,1H),3.
10–2.
96(m,2H),2.
37(s,3H);13CNMR(101MHz,Acetone-d6)δ172.
34,172.
27,156.
5,154.
2,148.
0,139.
4,130.
7,130.
3,129.
7,125.
8,122.
5,117.
0,108.
1,49.
4,36.
43,21.
4;ESI-HRMScalcdforC16H14NO6(M+H)+316.
0827,found316.
0826;[α]20D+7.
0°(c0.
2,MeCN).
(5-(p-Tolyl)furan-2-carbonyl)-L-asparticacid(13).
Dimethyl(5-(p-tolyl)furan-2-carbonyl)-L-aspartate(13e)wassynthesisedfrom4e(100mg,0.
30mmol)andp-tolylboronicacid(45mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatography(EtOAc:petroleumether,1:3)gave80mg(77%)ofaclearoil:Rf=0.
26(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
66–7.
59(m,2H),7.
37(d,J=8.
1Hz,1H),7.
23(d,J=7.
9Hz,2H),7.
21(d,J=3.
6Hz,1H),6.
69(d,J=3.
6Hz,1H),5.
10–5.
03(m,1H),3.
81(s,3H),3.
71(s,3H),3.
19–3.
11(m,1H),3.
03–2.
95(m,1H),2.
39(s,3H);13CNMR(101MHz,11CDCl3)δ171.
6,171.
1,158.
0,156.
2,145.
9,138.
9,129.
6,126.
9,124.
6,117.
2,106.
6,52.
9,52.
1,48.
3,36.
3,21.
4;ESI-HRMScalcdforC18H19NaNO6(M+Na)+368.
1105,found368.
1104;[α]20D+15.
5°(c0.
2,DCM).
13wassynthesisedfrom13e(50mg,0.
14mmol)accordingtothegeneralesterhydrolysisproceduretogive45mg(98%)ofawhitesolid:tR=9.
89min(HPLC);1HNMR(400MHz,Acetone-d6)δ7.
74–7.
69(m,2H),7.
27(d,J=8.
0Hz,2H),7.
18(d,J=3.
6Hz,1H),6.
94(d,J=3.
6Hz,1H),5.
03(t,J=5.
9Hz,1H),3.
11–2.
96(m,2H),2.
35(s,3H);13CNMR(101MHz,Acetone-d6)δ172.
48,172.
45,158.
5,156.
6,147.
7,139.
6,130.
4,128.
0,125.
3,117.
2,107.
5,49.
3,36.
6,21.
3;ESI-HRMScalcdforC14H16NO6(M+H)+316.
0827,found316.
0821;[α]20D+10.
5°(c0.
2,MeCN).
(5-(3-(Hydroxymethyl)phenyl)furan-2-carbonyl)-L-asparticacid(14).
Dimethyl(5-(3-(hydrox-ymethyl)phenyl)furan-2-carbonyl)-L-aspartate(14e)wassynthesisedfrom4e(107mg,0.
32mmol)and3-hydroxymethylphenylboronicacid(54mg,0.
35mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatography(EtOAc:petroleumether,2:1)gave55mg(47%)ofaclearoil:Rf=0.
29(EtOAc:petroleumether,2:1);1HNMR(400MHz,CDCl3)δ7.
73(s,1H),7.
66(d,J=7.
7Hz,1H),7.
46–7.
33(m,3H),7.
21(d,J=3.
6Hz,1H),6.
76(d,J=3.
6Hz,1H),5.
09–5.
03(m,1H),4.
76(s,2H),3.
81(s,3H),3.
73(s,3H),3.
19–3.
11(m,1H),3.
03–2.
95(m,1H),1.
99(s,1H);13CNMR(101MHz,CDCl3)δ171.
6,171.
1,157.
9,155.
8,146.
3,141.
7,129.
8,129.
1,127.
3,123.
8,123.
0,117.
2,107.
5,65.
0,52.
9,52.
1,48.
3,36.
2;ESI-HRMScalcdforC18H20NO7(M+H)+362.
1234,found362.
1246;[α]20D8.
0°(c0.
2,MeCN).
14wassynthesisedfrom14e(50mg,0.
14mmol)accordingtothegeneralesterhydrolysisproceduretogive44mg(95%)ofawhitesolid:tR=8.
04min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
11(d,J=8.
3Hz,1H),7.
82(s,1H),7.
70(d,J=7.
5Hz,1H),7.
45–7.
33(m,2H),7.
21(d,J=3.
6Hz,1H),7.
00(d,J=3.
6Hz,1H),5.
09–5.
01(m,1H),4.
68(s,2H),3.
10–2.
97(m,2H),1.
31–1.
25(m,1H);13CNMR(101MHz,Acetone-d6)δ172.
5,172.
4,158.
6,156.
6,147.
9,144.
2,130.
6,129.
7,127.
8,123.
8,123.
4,117.
2,108.
2,64.
5,49.
4,36.
5;ESI-HRMScalcdforC16H14NO7(M+H)+332.
0776,found332.
0760;[α]20D+14.
5°(c0.
2,MeCN).
(5-(4-Hydroxyphenyl)furan-2-carbonyl)-L-asparticacid(15).
Dimethyl(5-(4-hydroxyphenyl)furan-2-carbonyl)-L-aspartate(15e)wassynthesisedfrom4e(100mg,0.
30mmol)and4-hydroxyphenylboronicacid(45mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatog-raphy(EtOAc:petroleumether,1:2)gave30mg(29%)ofaclearoil:Rf=0.
23(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
64–7.
56(m,2H),7.
37(d,J=8.
1Hz,1H),7.
21(d,J=3.
6Hz,1H),6.
94–6.
87(m,2H),6.
59(d,J=3.
6Hz,1H),5.
79(s,1H),5.
10–5.
04(m,1H),3.
82(s,3H),3.
73(s,3H),3.
20–3.
12(m,1H),3.
03–2.
95(m,1H);13CNMR(101MHz,CDCl3)δ171.
7,171.
2,158.
2,156.
6,156.
3,145.
5,126.
4,122.
5,117.
5,115.
9,105.
8,53.
0,52.
1,48.
3,36.
3;ESI-HRMScalcdforC17H18NO7(M+H)+348.
1078,found348.
1083;[α]20D3.
5°(c0.
2,MeCN).
15wassynthesisedfrom15e(27mg,0.
08mmol)accordingtothegeneralesterhydrolysisproceduretogive24mg(97%)ofapalebrownsolid:tR=8.
15min(HPLC);1HNMR(400MHz,Acetone-d6)δ7.
99(d,J=8.
2Hz,1H),7.
71–7.
64(m,2H),7.
16(d,J=3.
6Hz,1H),6.
94–6.
88(m,2H),6.
80(d,J=3.
6Hz,1H),5.
07–4.
99(m,1H),3.
09–2.
97(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
41,172.
36,159.
0,158.
6,156.
9,147.
1,127.
0,122.
5,117.
2,116.
7,106.
1,49.
3,36.
5;ESI-HRMScalcdforC15H12NO7(M+H)+318.
0619,found318.
0615;[α]20D+5.
4°(c0.
2,MeCN).
(5-(2-Methoxyphenyl)furan-2-carbonyl)-L-asparticacid(16).
Dimethyl(5-(2-methoxyphenyl)furan-2-carbonyl)-L-aspartate(16e)wassynthesisedfrom4e(100mg,0.
30mmol)ando-methoxyphenylboronicacid(40mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatog-raphy(EtOAc:petroleumether,1:3)gave61mg(56%)ofaclearoil:Rf=0.
26(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
89(dd,J=7.
8,1.
7Hz,1H),7.
39(d,J=8.
1Hz,1H),7.
33(ddd,J=8.
4,7.
4,1.
7Hz,1H),7.
23(d,J=3.
6Hz,1H),7.
07(td,J=7.
7,1.
0Hz,1H),7.
03(d,J=3.
6Hz,1H),6.
99(d,J=8.
3Hz,1H),5.
12–5.
01(m,1H),3.
96(s,3H),3.
81(s,3H),3.
73(s,3H),3.
20–3.
12(m,1H),3.
03–2.
95(m,1H);13CNMR(101MHz,CDCl3)δ171.
6,171.
2,158.
1,156.
3,152.
5,145.
2,129.
6,126.
6,120.
9,118.
6,117.
2,112.
1,111.
2,55.
5,52.
9,52.
1,48.
3,36.
3;ESI-HRMScalcdforC18H20NO7(M+H)+362.
1234,found362.
1249;[α]20D5.
9°(c0.
2,MeCN).
16wassynthesisedfrom16e(49mg,0.
13mmol)accordingtothegeneralesterhydrolysisproceduretogive40mg(89%)ofawhitesolid:tR=9.
59min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
07(d,J=8.
3Hz,1H),7.
93(dd,J=7.
8,1.
7Hz,1H),7.
35(ddd,J=8.
4,7.
4,1.
7Hz,1H),7.
19(d,J=3.
6Hz,1H),7.
14(d,J=8.
4,0.
8Hz,1H),7.
08(d,J=3.
6Hz,1H),7.
04(td,J=7.
7,1.
1Hz,1H),5.
08–5.
02(m,1H),3.
99(s,3H),3.
10–2.
98(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
5,172.
4,158.
6,157.
3,152.
9,146.
9,130.
5,127.
0,121.
5,119.
4,117.
1,112.
8,112.
5,55.
9,49.
4,36.
5;ESI-HRMScalcdforC16H15NNaO7(M+Na)+356.
0741,found356.
0743;[α]20D+3.
0°(c0.
2,MeCN).
(5-(4-Methoxyphenyl)furan-2-carbonyl)-L-asparticacid(17).
Dimethyl(5-(4-methoxyphenyl)furan-2-carbonyl)-L-aspartate(17e)wassynthesisedfrom4e(100mg,0.
30mmol)andp-methoxyphenylboronicacid(40mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatog-raphy(EtOAc:petroleumether,1:3)gave89mg(82%)ofaclearoil:Rf=0.
20(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
69–7.
64(m,2H),7.
35(d,J=8.
0Hz,1H),7.
20(d,J=3.
60Hz,1H),6.
99–6.
92(m,2H),6.
61(d,J=3.
6Hz,1H),5.
09–5.
03(m,1H),3.
86(s,3H),3.
81(s,3H),3.
71(s,3H),3.
19–3.
11(m,1H),3.
02–2.
94(m,1H);13CNMR(101MHz,CDCl3)δ171.
6,171.
2,160.
2,158.
0,156.
1,145.
7,126.
2,122.
5,117.
3,114.
4,105.
8,55.
4,52.
9,52.
1,48.
2,36.
3;ESI-HRMScalcdforC18H20NO7(M+H)+362.
1234,found362.
1249,[α]20D6.
0°(c0.
2,MeCN).
1217wassynthesisedfrom17e(75mg,0.
21mmol)accordingtothegeneralesterhydrolysisproceduretogive66mg(96%)ofawhitesolid:tR=9.
48min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
05(d,J=8.
2Hz,1H),7.
75(d,J=8.
8Hz,2H),7.
19(d,J=3.
6Hz,1H),7.
00(d,J=8.
8Hz,2H),6.
84(d,J=3.
6Hz,1H),5.
09–5.
00(m,1H),3.
83(s,3H),3.
11–2.
98(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
5,172.
4,161.
2,158.
7,156.
7,147.
3,126.
9,123.
5,117.
4,115.
3,106.
6,55.
8,49.
4,36.
5;ESI-HRMScalcdforC16H15NNaO7(M+Na)+356.
0741,found356.
0755;[α]20D+2.
5°(c0.
2,MeCN).
(5-(4-(Trifluoromethyl)phenyl)furan-2-carbonyl)-L-asparticacid(18).
Dimethyl(5-(4-(trif-luoromethyl)phenyl)furan-2-carbonyl)-L-aspartate(18e)wassynthesisedfrom4e(100mg,0.
30mmol)and4-trifluoromethylphenylboronicacid(63mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatography(EtOAc:petroleumether,1:3)gave67mg(56%)ofaclearoil:Rf=0.
59(EtOAc:petroleumether,2:1);1HNMR(400MHz,CDCl3)δ7.
83(d,J=8.
6Hz,2H),7.
68(d,J=8.
6Hz,2H),7.
44(d,J=8.
1Hz,1H),7.
25(d,J=3.
6Hz,1H),6.
86(d,J=3.
6Hz,1H),5.
09–5.
03(m,1H),3.
82(s,3H),3.
74(s,3H),3.
21–3.
13(m,1H),3.
03–2.
95(m,1H);13CNMR(101MHz,CDCl3)δ171.
6,171.
0,157.
7,154.
2,147.
2,132.
7,130.
5(q,J=32.
7Hz),126.
0(q,J=3.
8Hz),125.
3,124.
7,117.
1,109.
1,53.
0,52.
2,48.
3,36.
2;ESI-HRMScalcdforC18H16F3NaNO6(M+Na)+422.
0822,found422.
0832;[α]20D5.
0°(c0.
2,MeCN).
18wassynthesisedfrom18e(50mg,0.
13mmol)accordingtothegeneralesterhydrolysisproceduretogive46mg(99%)ofawhitesolid:tR=10.
73min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
19(d,J=8.
2Hz,1H),8.
02(t,J=9.
7Hz,2H),7.
78(d,J=8.
3Hz,2H),7.
26(d,J=3.
6Hz,1H),7.
21(d,J=3.
6Hz,1H),5.
10–5.
01(m,1H),3.
08–3.
00(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
4,172.
3,158.
4,154.
6,148.
9,134.
2,130.
4(q,J=32.
3Hz),126.
8(q,J=3.
8Hz),125.
7,117.
2,110.
5,49.
5,36.
5;ESI-HRMScalcdforC16H11F3NO6(M+H)+370.
0544,found370.
0528;[α]20D+3.
5°(c0.
2,MeCN).
(5-(4-Cyanophenyl)furan-2-carbonyl)-L-asparticacid(19).
Dimethyl(5-(4-cyanophenyl)furan-2-carbonyl)-L-aspartate(19e)wassynthesisedfrom4e(100mg,0.
30mmol)and4-cyanophenylboronicacid(48mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatog-raphy(EtOAc:petroleumether,1:3)gave70mg(66%)ofaclearoil:Rf=0.
21(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
85–7.
79(m,2H),7.
75–7.
69(m,2H),7.
44(d,J=8.
0Hz,1H),7.
25(d,J=3.
7Hz,1H),6.
90(d,J=3.
7Hz,1H),5.
08–5.
02(m,1H),3.
82(s,3H),3.
74(s,3H),3.
21–3.
13(m,1H),3.
03–2.
95(m,1H);13CNMR(101MHz,CDCl3)δ171.
6,170.
9,157.
5,153.
6,147.
6,133.
3,132.
8,124.
9,118.
5,117.
1,112.
0,110.
0,53.
0,52.
2,48.
3,36.
1;ESI-HRMScalcdforC18H16N2NaO6(M+Na)+379.
0901,found379.
0892;[α]D20–9.
5°(c0.
2,MeCN).
19wassynthesisedfrom19e(57mg,0.
16mmol)accordingtothegeneralesterhydrolysisproceduretogive50mg(95%)ofapaleyellowsolid:tR=9.
16min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
18(d,J=8.
2Hz,1H),8.
06–8.
12(m,2H),7.
89–7.
85(m,2H),7.
27(d,J=3.
6Hz,1H),7.
25(d,J=3.
6Hz,1H),5.
06–5.
00(m,1H),3.
07–2.
98(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
3,172.
2,158.
2,154.
2,145.
9,134.
5,133.
7,125.
7,119.
1,117.
2,112.
4,111.
2,49.
5,36.
4;ESI-HRMScalcdforC16H11N2O6(M+H)+327.
0623,found327.
0628;[α]D20+15.
8°(c0.
2,MeCN).
(5-(4-(Trifluoromethoxy)phenyl)furan-2-carbonyl)-L-asparticacid(20).
Dimethyl(5-(4-(trif-luoromethoxy)phenyl)furan-2-carbonyl)-L-aspartate(20e)wassynthesisedfrom4e(100mg,0.
30mmol)and4-trifluoromethoxyphenylboronicacid(68mg,0.
33mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashchromatography(EtOAc:petroleumether,1:3)gave54mg(43%)ofaclearoil:Rf=0.
32(EtOAc:petroleumether,1:1);1HNMR(400MHz,CDCl3)δ7.
78–7.
73(m,2H),7.
40(d,J=8.
0Hz,1H),7.
28(d,J=8.
5Hz,2H),7.
23(d,J=3.
4Hz,1H),6.
75(d,J=3.
6Hz,1H),5.
09–5.
03(m,1H),3.
82(s,3H),3.
73(s,3H),3.
20–3.
12(m,1H),3.
02–2.
94(m,1H);13CNMR(101MHz,CDCl3)δ171.
6,171.
1,157.
8,154.
6,149.
4,146.
7,135.
5,128.
3,126.
1,121.
4,117.
2,107.
9,53.
0,52.
1,48.
3,36.
2;ESI-HRMScalcdforC18H16F3NNaO7(M+Na)+438.
0771,found438.
0782;[α]20D8.
0°(c0.
2,MeCN).
20wassynthesisedfrom20e(46mg,0.
11mmol)accordingtothegeneralesterhydrolysisproceduretogive41mg(95%)ofawhitesolid:tR=10.
88min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
00–7.
92(m,2H),7.
46–7.
38(m,2H),7.
22(d,J=3.
6Hz,1H),7.
08(d,J=3.
6Hz,1H),5.
03(t,J=5.
9Hz,1H),3.
07–2.
99(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
39,172.
36,158.
4,154.
8,149.
8,148.
5,129.
9,127.
1,122.
4,117.
2,109.
1,49.
4,36.
5;ESI-HRMScalcdforC16H11F3NO7(M+H)+386.
0493,found386.
050;[α]20D+6.
5°(c0.
2,MeCN).
(5-(4-Ethoxyphenyl)furan-2-carbonyl)-L-asparticacid(21).
Dimethyl(5-(4-ethoxyphenyl)furan-2-carbonyl)-L-aspartate(21e)wassynthesisedfrom15e(61mg,0.
18mmol)andethyliodide(50L,0.
62mmol)accordingtothegeneralalkylationprocedure.
Afteronedayofstirring,additionalethyliodide(50L,0.
62mmol)wasadded.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave55mg(95%)ofapaleyellowsolid:Rf=0.
22(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ8.
09–8.
02(m,1H),7.
78–7.
73(m,2H),7.
16(d,J=3.
6Hz,1H),7.
03–6.
98(m,2H),6.
85(d,J=3.
6Hz,1H),5.
07–4.
99(m,1H),4.
10(q,J=7.
0Hz,2H),3.
71(s,3H),3.
67(s,3H),3.
09–2.
91(m,3H),1.
38(t,J=7.
0Hz,3H);13CNMR(101MHz,Acetone-d6)δ172.
0,171.
7,160.
6,158.
4,156.
7,147.
3,126.
9,123.
4,117.
3,115.
8,106.
5,64.
3,52.
8,52.
1,49.
6,36.
7,15.
0;ESI-HRMScalcdforC19H21NNaO7(M+Na)+398.
1210,found398.
1223;[α]20D4.
5°(c0.
2,MeOH).
21wassynthesisedfrom21e(44mg,0.
12mmol)accordingtothegeneralesterhydrolysisproceduretogive37mg(89%)ofapaleyellowfoam:tR=10.
01min(HPLC);1HNMR(400MHz,Acetone-d6)δ10.
93(brs,2H),8.
05–7.
97(m,1H),7.
80–7.
71(m,2H),7.
16(d,J=3.
6Hz,1H),7.
04–6.
97(m,2H),6.
85(d,J=3.
6Hz,1H),5.
08–5.
00(m,1H),4.
10(q,J=7.
0Hz,2H),3.
12–2.
96(m,2H),1.
38(t,J=7.
0Hz,3H);13CNMR(101MHz,Acetone-d6)13δ172.
42,172.
38,160.
6,158.
6,156.
6,147.
4,126.
9,123.
4,117.
2,115.
8,106.
5,64.
3,49.
4,36.
5,15.
1;ESI-HRMScalcdforC17H16NO7(MH)346.
0932,found346.
0945;[α]20D+6.
5°(c0.
2,MeOH).
(5-(4-Isopropoxyphenyl)furan-2-carbonyl)-L-asparticacid(22).
Dimethyl(5-(4-isopropoxyphenyl)furan-2-carbonyl)-L-aspartate(22e)wassynthesisedfrom15e(60mg,0.
17mmol)and2-bromopropane(50L,0.
53mmol)accordingtothegeneralalkylationprocedure.
Inaddition,KI(6mg,0.
03mmol)wasaddedtopromotethereaction.
Afteronedayofstirring,additional2-bromopropane(50L,0.
53mmol)wasadded.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave11mg(16%)ofapaleyellowsolid:Rf=0.
25(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ8.
08–8.
00(m,1H),7.
77–7.
71(m,2H),7.
16(d,J=3.
6Hz,1H),7.
03–6.
96(m,2H),6.
85(d,J=3.
6Hz,1H),5.
08–4.
98(m,1H),4.
74–4.
64(m,1H),3.
71(s,3H),3.
67(s,3H),3.
09–2.
91(m,2H),1.
32(d,J=6.
0Hz,6H);13CNMR(101MHz,Acetone-d6)δ172.
0,171.
7,159.
5,158.
5,156.
7,147.
2,126.
9,123.
2,117.
3,116.
9,106.
5,70.
6,52.
8,52.
1,49.
6,36.
7,22.
3;ESI-HRMScalcdforC20H23NNaO7(M+Na)+412.
1367,found412.
1387.
22wassynthesisedfrom22e(11mg,0.
03mmol)accordingtothegeneralesterhydrolysisproceduretogive9mg(86%)ofapaleyellowsolid:tR=10.
50min(HPLC);1HNMR(400MHz,Acetone-d6)δ11.
19(brs,2H),8.
02–7.
97(m,1H),7.
78–7.
71(m,2H),7.
16(d,J=3.
6Hz,1H),7.
03–6.
96(m,2H),6.
85(d,J=3.
6Hz,1H),5.
09–4.
98(m,1H),4.
74–4.
63(m,1H),3.
10–2.
95(m,2H),1.
32(d,J=6.
0Hz,6H);13CNMR(101MHz,Acetone-d6)δ172.
41,172.
38,159.
5,158.
6,156.
7,147.
4,126.
9,123.
3,117.
2,116.
9,106.
5,70.
6,49.
4,36.
5,22.
3;ESI-HRMScalcdforC18H18NO7(MH)360.
1089,found360.
1103;[α]20D+57.
0°(c0.
2,MeOH).
(5-(4-((3-Methyloxetan-3-yl)methoxy)phenyl)furan-2-carbonyl)-L-asparticacid(23).
Dimethyl(5-(4-((3-methyloxetan-3-yl)methoxy)phenyl)furan-2-carbonyl)-L-aspartate(23e)wassynthesisedfrom15e(55mg,0.
35mmol)and(3-methyloxetan-3-yl)methyl4-methylbenzenesulfonate(89mg,0.
53mmol)accord-ingtothegeneralalkylationprocedure.
Inaddition,KI(10mg,0.
06mmol)wasaddedtopromotethereaction.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave38mg(55%)ofayellowsolid:Rf=0.
17(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ8.
10–8.
03(m,1H),7.
82–7.
75(m,2H),7.
17(d,J=3.
6Hz,1H),7.
12–7.
06(m,2H),6.
88(d,J=3.
6Hz,1H),5.
09–4.
98(m,1H),4.
55(d,J=5.
8Hz,2H),4.
35(d,J=5.
8Hz,2H),4.
16(s,2H),3.
71(s,3H),3.
67(s,3H),3.
09–2.
89(m,2H),1.
43(s,3H);13CNMR(101MHz,Acetone-d6)δ171.
9,171.
7,160.
8,158.
4,156.
6,147.
4,126.
9,123.
8,117.
3,116.
0,106.
7,79.
7,73.
9,52.
8,52.
1,49.
6,40.
4,36.
7,21.
5;ESI-HRMScalcdforC22H26NO8(M+H)+432.
1653,found432.
1668;[α]20D+0.
5°(c0.
2,MeOH).
23wassynthesisedfrom23e(30mg,0.
07mmol)accordingtothegeneralesterhydrolysisproceduretogive26mg(92%)ofapaleyellowfoam:tR=9.
55min(HPLC);1HNMR(400MHz,Acetone-d6)δ10.
93(brs,2H),8.
06–7.
98(m,1H),7.
83–7.
75(m,2H),7.
17(d,J=3.
6Hz,1H),7.
13–7.
05(m,2H),6.
88(d,J=3.
6Hz,1H),5.
07–5.
00(m,1H),4.
56(d,J=5.
8Hz,2H),4.
35(d,J=5.
8Hz,2H),4.
15(s,2H),3.
12–2.
97(m,2H),1.
43(s,3H);13CNMR(101MHz,Acetone-d6)δ172.
41,172.
38,160.
8,158.
6,156.
5,147.
5,126.
9,123.
8,117.
2,116.
0,106.
7,79.
7,73.
8,49.
4,40.
4,36.
5,21.
5;ESI-HRMScalcdforC20H20NO8(MH)402.
1194,found402.
1197;[α]20D+10.
0°(c0.
2,MeOH).
(5-(4-(3-(Methylsulfonyl)propoxy)phenyl)furan-2-carbonyl)-L-asparticacid(24).
Dimethyl(5-(4-(3-(methylsulfonyl)propoxy)phenyl)furan-2-carbonyl)-L-aspartate(24e)wassynthesisedfrom15e(40mg,0.
12mmol)and3-(methylsulfonyl)propyl4-methylbenzenesulfonate(69mg,0.
24mmol)accordingtothegen-eralalkylationprocedure.
Inaddition,KI(16mg,0.
09mmol)wasaddedtopromotethereaction.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,2:1)gave35mg(65%)ofawhitesolid:Rf=0.
11(EtOAc:petroleumether,2:1);1HNMR(400MHz,Acetone-d6)δ8.
09–8.
01(m,1H),7.
80–7.
74(m,2H),7.
16(d,J=3.
6Hz,1H),7.
08–7.
02(m,2H),6.
87(d,J=3.
6Hz,1H),5.
07–4.
99(m,1H),4.
23(t,J=6.
1Hz,2H),3.
71(s,3H),3.
67(s,3H),3.
35–3.
27(m,2H),3.
07–2.
92(m,2H),2.
99(s,3H),2.
35–2.
25(m,2H);13CNMR(101MHz,Acetone-d6)δ171.
9,171.
7,160.
2,158.
4,156.
5,147.
4,126.
9,123.
8,117.
3,115.
9,106.
7,66.
9,52.
8,52.
1,51.
9,49.
6,40.
8,36.
7,23.
5;ESI-HRMScalcdforC21H26NO9S(M+H)+468.
1323,found468.
1339;[α]20D9.
0°(c0.
2,MeOH).
24wassynthesisedfrom24e(25mg,0.
05mmol)accordingtothegeneralesterhydrolysisproceduretogive24mg(99%)ofawhitesolid:tR=8.
76min(HPLC);1HNMR(400MHz,DMSO-d6)δ12.
66(brs,2H),8.
72–8.
64(m,1H),7.
90–7.
81(m,2H),7.
18(d,J=3.
6Hz,1H),7.
05(d,J=8.
9Hz,2H),6.
96(d,J=3.
6Hz,1H),4.
82–4.
73(m,1H),4.
15(t,J=6.
2Hz,2H),3.
38–3.
25(m,2H),3.
03(s,3H),2.
94–2.
68(m,2H),2.
24–2.
11(m,2H);13CNMR(101MHz,DMSO-d6)δ172.
3,171.
8,158.
6,157.
4,154.
8,145.
9126.
0,122.
3,116.
3,114.
9,106.
0,65.
8,50.
5,48.
5,40.
2,35.
8,21.
9;ESI-HRMScalcdforC19H20NO9S(MH)438.
0864,found438.
0871;[α]20D+15.
0°(c0.
2,MeOH).
(4′-Fluoro-[1,1′-biphenyl]-3-carbonyl)-L-asparticacid(25).
Dimethyl(4′-fluoro-[1,1′-biphenyl]-3-carbonyl)-L-aspartate(25e)wassynthesisedfrom(4-fluorophenyl)boronicacid(36mg,0.
26mmol)and6e(79mg,0.
20mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave25mg(35%)ofapaleyellowstickyoil:Rf=0.
36(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ8.
22–8.
15(m,1H),8.
15–8.
12(m,1H),7.
91–7.
86(m,1H),7.
84–7.
80(m,1H),7.
77–7.
70(m,2H),7.
57(t,J=7.
8Hz,1H),7.
30–7.
22(m,2H),5.
10–5.
01(m,1H),3.
71(s,3H),3.
66(s,3H),3.
08–2.
90(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
0,171.
6,167.
2,164.
9,141.
1,137.
5(d,J=3.
4Hz),135.
8,130.
8,130.
0,129.
9(d,J=8.
2Hz),127.
2,126.
6,116.
6(d,J=21.
6Hz),52.
7,52.
1,50.
5,36.
7;ESI-HRMScalcdforC19H18FNNaO5(M+Na)+382.
1061,found382.
1053.
25wassynthesisedfrom25e(25mg,0.
07mmol)accordingtothegeneralesterhydrolysisproceduretogive20mg(87%)ofawhitefoam:tR=10.
03min(HPLC);1HNMR(400MHz,Acetone-d6)δ8.
18–8.
10(m,2H),7.
8914(dd,J=7.
7,1.
1Hz,1H),7.
85–7.
79(m,1H),7.
78–7.
71(m,2H),7.
61–7.
54(m,1H),7.
31–7.
20(m,1H),5.
11–5.
02(m,1H),3.
10–2.
94(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
5,172.
3,167.
3,163.
6(d,J=245.
2Hz),141.
1,137.
5(d,J=3.
1Hz),135.
9,130.
8,130.
0,129.
9(d,J=8.
2Hz),127.
2,126.
6,116.
6(d,J=21.
7Hz),50.
3,36.
5;ESI-HRMScalcdforC17H13FNO5(MH)330.
0783,found330.
0796;[α]20D+9.
0°(c0.
2,MeOH).
(4′-Fluoro-[1,1′-biphenyl]-4-carbonyl)-L-asparticacid(26).
Dimethyl(4′-fluoro-[1,1′-biphenyl]-4-carbonyl)-L-aspartate(26e)wassynthesisedfrom(4-fluorophenyl)boronicacid(37mg,0.
26mmol)and7e(81mg,0.
34mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave71mg(84%)ofawhitesolid:Rf=0.
31(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ8.
10–8.
04(m,1H),8.
00–7.
95(m,2H),7.
80–7.
72(m,4H),7.
30–7.
22(m,2H),5.
09–5.
02(m,1H),3.
72(s,3H),3.
67(s,3H),3.
08–2.
93(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
1,171.
7,166.
9,163.
8(d,J=245.
6Hz),143.
9,137.
2(d,J=3.
1Hz),133.
9,129.
9(d,J=8.
2Hz),128.
9,127.
7,116.
6(d,J=21.
7Hz),52.
7,52.
1,50.
4,36.
7;ESI-HRMScalcdforC19H18FNNaO5(M+Na)+382.
1061,found382.
1056;[α]20D27.
0°(c0.
2,MeOH).
26wassynthesisedfrom26e(56mg,0.
15mmol)accordingtothegeneralesterhydrolysisproceduretogive48mg(94%)ofawhitesolid:tR=10.
02min(HPLC);1HNMR(400MHz,DMSO-d6)δ12.
58(brs,2H),8.
83–8.
75(m,1H),8.
02–7.
92(m,2H),7.
85–7.
75(m,4H),7.
39–7.
28(m,2H),4.
83–4.
74(m,1H),2.
93–2.
69(m,2H);13CNMR(101MHz,DMSO-d6)δ172.
5,171.
7,165.
6,161.
0,141.
8,135.
6(d,J=3.
1Hz),132.
6,128.
9(d,J=8.
3Hz),128.
0,126.
4,115.
8(d,J=21.
4Hz),49.
4,35.
8;ESI-HRMScalcdforC17H13FNO5(MH)330.
0783,found330.
0768;[α]20D+1.
0°(c0.
2,MeOH).
(6-(4-Fluorophenyl)picolinoyl)-L-asparticacid(27).
Dimethyl(6-(4-fluorophenyl)picolinoyl)-L-aspartate(27e)wassynthesisedfrom(4-fluorophenyl)boronicacid(40mg,0.
28mmol)anddimethyl(6-bromopicolinoyl)-L-aspartate(88mg,0.
26mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave81mg(88%)ofapaleyellowstickyoil:Rf=0.
62(EtOAc);1HNMR(400MHz,Acetone-d6)δ9.
18–9.
11(m,1H),8.
29–8.
23(m,2H),8.
18–8.
13(m,1H),8.
12–8.
07(m,2H),7.
35–7.
26(m,2H),5.
13–5.
04(m,1H),3.
74(s,3H),3.
69(s,3H),3.
11–3.
04(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
0,171.
9,164.
7(d,J=247.
5Hz),164.
5,155.
7,150.
5,139.
8,135.
5(d,J=3.
1Hz),130.
0(d,J=8.
5Hz),123.
8,121.
4,116.
5(d,J=21.
9Hz),52.
9,52.
2,49.
7,36.
8;ESI-HRMScalcdforC18H17FN2NaO5(M+Na)+383.
1014,found383.
1011;[α]20D+4.
5°(c0.
2,MeOH).
27wassynthesisedfrom27e(70mg,0.
19mmol)accordingtothegeneralesterhydrolysisproceduretogive57mg(88%)ofapaleyellowsolid:tR=9.
77min(HPLC);1HNMR(400MHz,Acetone-d6)δ11.
25(brs,2H),9.
27–9.
16(m,1H),8.
35–8.
21(m,2H),8.
21–8.
07(m,3H),7.
34–7.
22(m,2H),5.
13–5.
03(m,1H),3.
19–3.
01(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
6,172.
3,164.
7(d,J=247.
3Hz),164.
5,155.
6,150.
6,139.
7,135.
5(d,J=3.
2Hz),129.
9(d,J=8.
5Hz),123.
7,121.
4,116.
5(d,J=21.
8Hz),49.
5,36.
5;ESI-HRMScalcdforC16H12FN2O5(MH)331.
0736,found331.
0745;[α]20D+19.
0°(c0.
2,MeOH).
(4′-Methoxy-[1,1′-biphenyl]-3-carbonyl)-L-asparticacid(28).
Dimethyl(4′-methoxy-[1,1′-biphenyl]-3-carbonyl)-L-aspartate(28a)wassynthesisedfrom(4-methoxyphenyl)boronicacid(43mg,0.
28mmol)and7e(88mg,0.
26mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave72mg(75%)ofapaleyellowstickyoil:Rf=0.
29(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ8.
20–8.
14(m,1H),8.
13–8.
09(m,1H),7.
85–7.
76(m,2H),7.
67–7.
60(m,2H),7.
57–7.
50(m,1H),7.
08–7.
02(m,2H),5.
11–5.
01(m,1H),3.
85(s,3H),3.
71(s,3H),3.
66(s,3H),3.
09–2.
90(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
1,171.
6,167.
3,160.
7,141.
9,135.
7,133.
4,130.
4,129.
9,129.
0,126.
5,126.
2,115.
3,55.
7,52.
7,52.
1,50.
5,36.
7;ESI-HRMScalcdforC20H21NNaO6(M+Na)+394.
1261,found394.
1268;[α]20D23.
5°(c0.
2,MeOH).
28wassynthesisedfrom28e(64mg,0.
17mmol)accordingtothegeneralesterhydrolysisproceduretogive57mg(97%)ofapaleyellowsolid:tR=9.
92min(HPLC);1HNMR(400MHz,Acetone-d6)δ11.
15(brs,2H),8.
16–8.
08(m,2H),7.
87–7.
82(m,1H),7.
82–7.
76(m,1H),7.
68–7.
61(m,2H),7.
56–7.
50(m,1H),7.
09–7.
01(m,2H),5.
12–5.
02(m,1H),3.
85(s,3H),3.
13–2.
95(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
5,172.
3,167.
5,160.
7,141.
9,135.
8,133.
4,130.
3,129.
9,129.
0,126.
4,126.
2,115.
3,55.
7,50.
3,36.
5;ESI-HRMScalcdforC18H16NO6(MH)342.
0983,found342.
0989;[α]20D1.
5°(c0.
2,MeOH).
(6-(4-Methoxyphenyl)picolinoyl)-L-asparticacid(29).
Dimethyl(6-(4-methoxyphenyl)picol-inoyl)-L-aspartate(29e)wassynthesisedfrom(4-methoxyphenyl)boronicacid(42mg,0.
28mmol)anddime-thyl(6-bromopicolinoyl)-L-aspartate(86mg,0.
25mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:1)gave63mg(67%)ofamilkystickyoil:Rf=0.
25(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ9.
20–9.
11(m,1H),8.
20–8.
13(m,2H),8.
11–7.
98(m,3H),7.
12–7.
04(m,2H),5.
12–5.
04(m,1H),3.
89(s,3H),3.
74(s,3H),3.
69(s,3H),3.
13–3.
01(m,2H);13CNMR(101MHz,Acetone-d6)δ171.
94,171.
93,164.
6,162.
2,156.
5,150.
3,139.
4,131.
4,129.
2,123.
1,120.
6,115.
1,55.
8,52.
8,52.
2,49.
7,36.
8;ESI-HRMScalcdforC19H20N2NaO6(M+Na)+395.
1214,found395.
1217;[α]20D+4.
0°(c0.
2,MeOH).
29wassynthesisedfrom29e(54mg,0.
14mmol)accordingtothegeneralesterhydrolysisproceduretogive48mg(96%)ofapaleyellowfoam:tR=9.
69min(HPLC);1HNMR(400MHz,Acetone-d6)δ11.
26(brs,2H),9.
29–9.
16(m,1H),8.
22–8.
13(m,2H),8.
11–7.
99(m,3H),7.
12–7.
00(m,2H),5.
13–5.
01(m,1H),3.
88(s,3H),3.
24–3.
01(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
7,172.
4,164.
7,162.
1,156.
4,150.
4,139.
4,131.
5,129.
1,123.
0,120.
6,115.
1,55.
8,49.
4,36.
6;ESI-HRMScalcdforC17H15N2O6(MH)343.
0936,found343.
0932;[α]20D+18.
0°(c0.
2,MeOH).
15(4′-(Trifluoromethoxy)-[1,1′-biphenyl]-3-carbonyl)-L-asparticacid(30).
Dimethyl(4′-(trifluo-romethoxy)-[1,1′-biphenyl]-3-carbonyl)-L-aspartate(30e)wassynthesisedfrom(4-(trifluoromethoxy)phenyl)boronicacid(58mg,0.
28mmol)and7e(87mg,0.
25mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,2:3)gave77mg(72%)ofapaleyellowsolid:Rf=0.
40(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ8.
23–8.
18(m,1H),8.
18–8.
15(m,1H),7.
94–7.
90(m,1H),7.
89–7.
85(m,1H),7.
85–7.
80(m,2H),7.
63–7.
57(m,1H),7.
49–7.
43(m,2H),5.
11–5.
02(m,1H),3.
72(s,3H),3.
66(s,3H),3.
09–2.
91(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
0,171.
6,167.
1,140.
7,140.
4,135.
9,131.
0,130.
1,129.
7,127.
6,126.
8,122.
4,52.
7,52.
1,50.
5,36.
7;ESI-HRMScalcdforC20H18F3NNaO6(M+Na)+448.
0978,found448.
0981;[α]20D22.
5°(c0.
2,MeOH).
30wassynthesisedfrom30e(68mg,0.
16mmol)accordingtothegeneralesterhydrolysisproceduretogive60mg(94%)ofawhitesolid:tR=11.
38min(HPLC);1HNMR(400MHz,Acetone-d6)δ11.
21(brs,2H),8.
21–8.
18(m,1H),8.
18–8.
11(m,1H),7.
97–7.
90(m,1H),7.
90–7.
80(m,3H),7.
64–7.
57(m,1H),7.
49–7.
42(m,2H),5.
12–5.
02(m,1H),3.
11–2.
94(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
5,172.
3,167.
2,149.
7,140.
7,140.
4,136.
1,130.
9,130.
1,129.
7,127.
6,126.
8,122.
4,50.
4,36.
5;ESI-HRMScalcdforC18H13F3NO6(MH)396.
0700,found396.
0716;[α]20D1.
0°(c0.
2,MeOH).
(6-(4-(Trifluoromethoxy)phenyl)picolinoyl)-L-asparticacid(31).
Dimethyl(6-(4-(trifluorometh-oxy)phenyl)picolinoyl)-L-aspartate(31e)wassynthesisedfrom(4-(trifluoromethoxy)phenyl)boronicacid(60mg,0.
29mmol)anddimethyl(6-bromopicolinoyl)-L-aspartate(86mg,0.
25mmol)accordingtothegeneralSuzukicouplingprocedure.
Purificationbyflashcolumnchromatography(EtOAc:petroleumether,1:2)gave76mg(72%)ofamilkystickyoil:Rf=0.
38(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ9.
19–9.
11(m,1H),8.
36–8.
31(m,2H),8.
23–8.
18(m,1H),8.
15–8.
11(m,2H),7.
53–7.
47(m,2H),5.
13–5.
06(m,1H),3.
74(s,3H),3.
69(s,3H),3.
10–3.
04(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
0,171.
9,164.
4,155.
3,151.
0,150.
6,139.
9,138.
2,129.
7,124.
2,122.
2,121.
9,52.
9,52.
2,49.
8,36.
7;ESI-HRMScalcdforC19H17F3N2NaO6(M+Na)+449.
0931,found449.
0949;[α]20D+2.
0°(c0.
2,MeOH).
31wassynthesisedfrom31e(65mg,0.
15mmol)accordingtothegeneralesterhydrolysisproceduretogive58mg(95%)ofawhitesolid:tR=10.
98min(HPLC);1HNMR(400MHz,Acetone-d6)δ11.
14(brs,2H),9.
25–9.
18(m,1H),8.
37–8.
30(m,2H),8.
24–8.
17(m,1H),8.
17–8.
10(m,2H),7.
51–7.
45(m,2H),5.
12–5.
03(m,1H),3.
21–3.
00(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
6,172.
3,164.
4,155.
2,151.
0,150.
7,139.
9,138.
2,129.
7,124.
0,122.
1,121.
8,49.
5,49.
4,36.
5;ESI-HRMScalcdforC17H12F3N2O6(MH)397.
0653,found397.
0665;[α]20D+14.
0°(c0.
2,MeOH).
(6-(4-(2,2,2-Trifluoroethoxy)phenyl)picolinoyl)-L-asparticacid(32).
Diethyl(6-chloropicolinoyl)-L-aspartate(32a)wassynthesisedfrom6-chloropicolinicacid(615mg,3.
90mmol)anddiethylL-aspartatehydro-chloride(679mg,3.
01mmol)accordingtothegeneralamidecouplingprocedure.
Purificationbyflashcolumnchro-matography(EtOAc:petroleumether,1:3)gave840mg(85%)ofanorangeoil:Rf=0.
31(EtOAc:petroleumether,1:1);1HNMR(400MHz,Acetone-d6)δ8.
73(d,J=7.
5Hz,1H),8.
13–8.
05(m,2H),7.
70(dd,J=7.
4,1.
4Hz,1H),5.
07–4.
99(m,1H),4.
28–4.
07(m,4H),3.
12–2.
96(m,2H),1.
24(t,J=7.
1Hz,3H),1.
23(t,J=7.
1Hz,3H);13CNMR(101MHz,Acetone-d6)δ171.
2,171.
1,163.
1,151.
2,150.
7,141.
9,128.
3,122.
1,62.
1,61.
3,49.
9,36.
9,14.
5,14.
4.
ASchlenckflaskunderanargonatmospherewaschargedwithPd-XPhos-G4(8mg,0.
01mmol),XPhos(9mg,0.
02mmol),diboronicacid(181mg,2.
02mmol),andKOAc(198mg,2.
02mmol).
Theflaskwasevacu-atedandbackfilledwithargon,beforeadditionofdegassedEtOH(7mL)and1-chloro-4-(2,2,2-trifluoroethoxy)benzene(141mg,0.
67mmol,preparedaccordingto21).
Theflaskwasevacuatedandbackfilledwithargonandstirredat80°Cuntilthereactionturnedyellow(30min).
DegassedaqueousK2CO3(1.
8M,1.
1mL)and32a(222mg,0.
67mmol)dissolvedindegassedEtOH(1mL)wereadded,andtheflaskwasonceagainevacuatedandbackfilledwithargonandstirredat80°Cfor16hours.
Aftercompletion,thereactionwascooledtort,dilutedwithwaterandextractedwithEtOAc(x3).
Theorganicphaseswerecombined,washedwithbrine,driedoverNa2SO4andconcentratedinvacuo.
Theresiduewaspurifiedbyflashcolumnchromatography(EtOAc:petroleumether:AcOH,1:2:0→99:0:1)togive73mg(25%)of(S)-4-ethoxy-4-oxo-2-(6-(4-(2,2,2-trifluoroethoxy)phenyl)picolinamido)butanoicacid(32b)asabrownstickysolid:Rf=0.
22(EtOAc+1%AcOH);1HNMR(400MHz,Acetone-d6)δ9.
16(d,J=8.
5Hz,1H),8.
25–8.
20(m,2H),8.
15–8.
09(m,1H),8.
09–8.
03(m,2H),7.
25–7.
17(m,2H),5.
06(dt,J=8.
5,5.
3Hz,1H),4.
77(q,J=8.
6Hz,2H),4.
15(q,J=7.
1Hz,2H),3.
07(qd,J=16.
7,5.
3Hz,2H),1.
22(t,J=7.
1Hz,3H);13CNMR(101MHz,Acetone-d6)δ172.
4,172.
0,171.
5,164.
6,159.
6,156.
0,150.
4,139.
6,133.
2,129.
3,122.
1(d,J=238.
1Hz),116.
1,66.
2(d,J=35.
1Hz),61.
3,49.
6,37.
1,14.
5;ESI-MSm/z439.
1(MH);[α]20D0.
5°(c0.
2,MeOH).
32wassynthesisedfrom32b(40mg,0.
09mmol)accordingtothegeneralesterhydrolysisproceduretogive29mg(77%)ofawhitesolid:tR=10.
84min(HPLC);1HNMR(400MHz,Acetone-d6)δ11.
24(brs,2H),9.
21(d,J=8.
4Hz,1H),8.
26–8.
18(m,2H),8.
11(dd,J=8.
7,4.
2Hz,1H),8.
09–8.
02(m,2H),7.
26–7.
14(m,2H),5.
11–5.
01(m,1H),4.
77(q,J=8.
6Hz,2H),3.
21–3.
00(m,2H);13CNMR(101MHz,Acetone-d6)δ172.
6,172.
4,164.
6,159.
6,156.
0,150.
5,139.
5,133.
2,129.
4,124.
9(q,J=276.
5Hz),123.
3,121.
0,116.
1,66.
2(q,J=35.
1Hz),49.
5,36.
6;ESI-HRMScalcdforC18H14F3N2O6(MH)411.
0809,found411.
0812;[α]20D+29.
9°(c0.
2,MeOH).
(6-(4-Propoxyphenyl)picolinoyl)-L-asparticacid(33).
1-Bromo-4-propoxybenzene(33a)wassynthe-sisedfrom4-bromophenol(250mg,1.
44mmol)and1-iodopropane(0.
45mL,4.
61mmol)accordingtothegen-eralalkylationprocedure.
Purificationbyflashcolumnchromatography(petroleumether)gave248mg(80%)ofaclearoil:Rf=0.
62(EtOAc:petroleumether,1:6);1HNMR(400MHz,Acetone-d6)δ7.
44–7.
38(m,2H),6.
92–6.
86(m,2H),3.
94(t,J=6.
5Hz,2H),1.
83–1.
70(m,2H),1.
01(t,J=7.
4Hz,3H);13CNMR(101MHz,Acetone-d6)δ159.
5,133.
0,117.
5,112.
8,70.
4,23.
2,10.
7.
16ASchlenckflaskunderanargonatmospherewaschargedwithPd-XPhos-G4(4mg,1mol%),XPhos(6mg,2mol%),diboronicacid(135mg,1.
50mmol),andKOAc(147mg,1.
50mmol).
Theflaskwasevacuatedandbackfilledwithargon,beforeadditionofdegassedEtOH(2.
5mL)and33a(114mg,0.
53mmol).
Theflaskwasevacuatedandbackfilledwithargonandstirredat80°Cuntilthereactionturnedorange(80min).
DegassedaqueousK2CO3(1.
8M,0.
85mL)anddimethyl(6-chloropicolinoyl)-L-aspartate(151mg,0.
50mmol)dissolvedindegassedEtOH(0.
25mL)wereadded,andtheflaskwasonceagainevacuatedandbackfilledwithargonandstirredat80°Cfor3hours.
Aftercompletion,thereactionwascooledtort,dilutedwithwaterandextractedwithEtOAc(x3).
Theorganicphaseswerecombined,washedwithbrine,driedoverNa2SO4,andconcentratedinvacuo.
Theresiduewaspurifiedbyflashcolumnchromatographytogiveamixtureofmethylandethylestersof(6-(4-propoxyphenyl)picolinoyl)-L-asparticacidaftertrans-esterificationwiththereactionsolvent.
Thecrudewasthereforeuseddirectlyinthehydrolysisfollowingthegeneralproceduretogive27mg(15%overtwosteps)of33asastickypaleyellowfoam:tR=11.
09min(HPLC);1HNMR(400MHz,Acetone-d6)δ10.
89(brs,2H),9.
22(d,J=8.
4Hz,1H),8.
19–8.
10(m,2H),8.
10–7.
99(m,3H),7.
09–7.
01(m,2H),5.
06(dt,J=8.
6,5.
1Hz,1H),4.
04(t,J=6.
5Hz,2H),3.
20–2.
97(m,2H),1.
88–1.
72(m,2H),1.
04(t,J=7.
4Hz,3H);13CNMR(101MHz,Acetone-d6)δ172.
6,172.
4,164.
7,161.
6,156.
5,150.
4,139.
4,131.
3,129.
1,123.
0,120.
5,115.
6,70.
3,49.
4,36.
6,23.
3,10.
7;ESI-HRMScalcdforC19H19N2O6(MH)371.
1249,found371.
1240;[α]20D+49.
5°(c0.
2,MeOH).
Kineticaqueoussolubility.
Duplicatesofa200μMsolutionofthedicarboxylicacidswerepreparedfrom0.
01MPBS7.
4anda10mMstocksolutioninDMSO.
ThesampleswereincubatedinanEppendorfThermomixer(25°C,800rpm)for24h.
Afterwards,thesampleswerecentrifugedfor5minat11,000rpmandthesuperna-tantwasfiltered(0.
45μmPTFEmembrane)beforeanalysisbyHPLC.
Thesolubilitywascalculatedbasedonaconcentration-absorptioncurve.
Chemicalstability.
Triplicatesofa50μMsolutionofthetestcompoundswerepreparedfrom0.
01MPBS7.
4anda10mMstocksolutioninDMSO.
ThesampleswereincubatedinanEppendorfThermomixer(37°C,650rpm).
Thesampleswerebrieflyvortexedand50μLaliquotswerewithdrawnatthetimepoints0h,24h,48hetc.
andanalysedimmediatelyonUPLC.
ThechemicalstabilityinPBS7.
4wasdeterminedateverytimepointinpercentagerelativetothe0htimepoint.
Stabilityinsimulatedgastricandintestinalfluids.
FaSSIF,FeSSGFandFaSSGFwerepreparedinaccordancetothemanufacturersprocedure,Biorelevant.
com.
Triplicatesofa50μMsolutionofthetestcom-poundswerepreparedfroma10mMstocksolutioninDMSOdilutedwithFaSSGF/FaSSIF/FeSSIF.
ThesampleswereincubatedinanEppendorfThermomixer(37°C,650rpm).
Sampleswerewithdrawnat0minand120min,centrifuged(10minat10,000rpm)andthesupernatantanalysedbyHPLC/UPLC.
Thestabilitywascalculatedbasedonpeakareaofa0pointsample.
logD7.
4determination.
Aglassvialwithscrewcap(8mL)waschargedwithtestcompound(40μL,10mMinDMSO),PBS7.
4(0.
01M,1980μL),and1-octanol(1980μL).
Thevialwascappedandsealedwithparafilmandshakenat700rpmusinganIKAKS125basicshakerfor24hatroomtemperature.
Theparafilmwasremovedandthesamplewasallowedtoequilibratefor1hbeforeanalysis.
100μLoftheoctanolphasewaswithdrawnanddiluted1:10withMeOH(+0.
1%formicacid)/MilliQwater(4:1,v/v)andanalysedbyHPLC/UPLC.
TheinterfacewasremovedandthePBS7.
4phaseanalyseddirectlybyHPLC/UPLC.
AllanalysiswasperformedinduplicatesandlogDvalueswerecalculatedfromthepeakareas(mAU*min)andadjustedfordifferenceininjectionvolumeandconcentration-absorptioneffectsfromthesolvents,usingtwocalibrationpointspercompoundpersolvent,anddilutionoftheoctanolphase.
Allcompoundswereanalysedintriplicates.
Metabolicstability.
Microsomalstabilitywasstudiedinmouselivermicrosomes(0.
5mg/mL)atafinaltestcompoundconcentrationof1μMandperformedintriplicatesinaccordancetothepublishedprotocol22.
Inshort:Prewarmed(37°C)0.
1MPBS7.
4,10mMNADPHinPBS7.
4andtestcompound(1mMinDMSO)wereaddedtoanEppendorfTube.
Thesampleswereincubatedfor5minat37°Cbeforeadditionofnewlythawnedmicro-somes.
Thesamplesweremixedbygentlevortexingandincubatedfor1hat37°C,300rpminanEppendorfThermomixer.
Sampleswerequenchedbyadditionofice-coldMeOH/MeCN(1:1)andcentrifugedfor5minat10,000g.
ThesupernatantwastransferredtoHPLCvialsandstoredinthefreezeruntilanalysisbyHPLC/UPLC.
Themetabolicstabilitywascalculatedbasedona0minsample.
Allcompoundswereanalysedintriplicates.
Molecularbiology,cellculture,andtransfection.
ReceptorconstructsformSUCNR1andhSUCNR1wereboughtfromOrigeneandclonedintotheeukaryoticexpressionvectorpCMV-Tag(2B)(Stratagene).
HEK-293cellswereculturedinDulbecco'smodifiedEagle'smedium1885(DMEM)supplementedwith10%fetalcalfserum,100units/mLpenicillin,and100μg/mLstreptomycin.
TransienttransfectionoftheHEK-293cellswasdonewithLipofectamine-2000accordingtomanufacturer'sprotocol.
Cellsweresupplementedwithfreshmediumafter5h.
IP3turnoverassay.
96-wellplateswerecoatedwithpoly-D-lysineandHEK-293cellswereplated(35.
000cells/well).
Cellsweretransfectedfor5hthefollowingdayandsubsequentlyincubatedO/Nwith0.
5μCi/mLmyo[3H]inositol(PerkinElmer)in100μLgrowthmedium.
Onday3cellswerewashedwith200LHBSS/well(Gibco,LifeTechnologies)followedbyapre-incubation(30min,37°C)with100LHBSSsupplementedwith10mMLiCl.
Cellswerestimulatedwithligand(120min,37°C)andlysedwith50L10mMformicacid(30minonice).
Inawhite96wellplate20Lcellextractsand80L1:8dilutedYSiscintillationbeads(PerkinElmer)weremixed.
Theplatewasspundown,andaPackardTopCountNXTcounterrecordedlightemission(scintillation)afteran8hdelay.
17AutomatedLigand-guidedBackboneEnsembleReceptorOptimizationprotocol(ALiBERO).
ALiBEROisaniterativesampling-selectionprotocolforreceptoroptimisationthatreliesontheuseofligandinfor-mationforselectingthebest-performingreceptorconformations19.
HomologymodelsofthemouseSUCNR1receptorwereconstructedaccordingtoTrauelsenetal.
4andloadedintoICM(MolsoftL.
L.
C.
,SanDiego,CA,USA).
ThestructurewasconvertedintoanICMobject,therebyassigningproteinatomtypes,optimisinghydrogensandHis,Pro,Asn,GlyandCysside-chainconformations.
Theexploredchemicalcompoundswereusedasatrainingsetbydividingallcompoundsintoanactive(EC50≤10M)andaninactive(EC50>10M)group,consistingof25compoundseach.
Foralistofallcompoundsusedintheoptimisationprotocol,seeTableS1.
ALiBEROwasper-formedusingthepreparedreceptorstructureandligandtrainingsetasinput.
BindingsiteresiduesweremanuallyselectedbasedonproximitytothepositionofMRS2500inthesuperimposedstructureoftheP2Y1receptor(PDB4XNW).
100elasticnetworknormalmodeanalysisderivedconformerswerebuiltinordertorecreatebackboneandside-chainflexibility(T=300K).
Next,aflexible-ligandstatic-receptorsmall-scalevirtualscreeningwasperformedoneachofthereceptorconformers,fromwhichseveralpocketswereselectedforthefollowinggeneration.
TheligandanddecoymoleculesweredockedintomSUCNR1,representedaspre-calculatedpotentialgridsandthensortedaccordingtotheirICMVLSscores.
Themaximumnumberofcomplementarypocketsforeachgenerationwassetto5withamaximumof10generations.
Receptormodelswereselectedbasedontheircombinedscreeningperformance,asdeterminedbythenormalisedsquarerootareaunderthecurve,NSQ_AUC.
Aftereachroundofvirtualscreening,anall-atomMonteCarloside-chainrefinementwasperformedtoaccountforinduced-by-ligandchanges.
NSQ_AUCvalueswerecalculatedaccordingtoKatritchetal.
16.
Theoptimisedmodelensemblewasvalidatedinavirtualligandscreeningwithanexternaltestset,con-sistingofthe25activecompoundsusedintheALiBEROoptimisationand1247decoymoleculesthatwereselectedinasimilaritysearchwithaDaylight-typefingerprintthresholdofTc>0.
6withtheactivecompound3asquerymoleculeandsubsequentstructuralclusteringwithaTcthresholdof0.
15.
Thetestsetwasdockedintothebest-performingreceptorensemblefromgeneration10oftheALiBEROrefinement,usingICM4Ddocking.
TheROC-plotforthecombinedperformanceoftheoptimisedreceptorensemblewascomputedbytakingthebestICLVLSdockingscoreofthereceptormodelsforeachdockedcompound.
Dataavailability.
Thedatasetsgeneratedandanalysedduringthecurrentstudyareavailablefromthecor-respondingauthoronreasonablerequest.
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18AcknowledgementsERUissupportedbyafellowshipfromtheLundbeckFoundation(R181–2014–3247).
TheNNFCenterforBasicMetabolicResearchissupportedbyanunconditionalgrant(NNF10CC1016515)fromtheNovoNordiskFoundationtoUniversityofCopenhagen.
TheworkonmetabolitereceptorsincludingSUCNR1isfurthersupportedbyanImmunometabolismgrantNNF15CC0018346fromtheNovoNordiskFoundationtoOxfordUniversity,UniversityofCopenhagenandKarolinskaInstituttet,Stockholm,Sweden.
AuthorContributionsE.
R.
U.
,M.
B.
andL.
K.
I.
J.
synthesizedthecompounds.
L.
.
B.
performedstabilitystudies.
M.
T.
performedinvitroassays.
M.
L.
andT.
M.
F.
performedmolecularmodeling.
E.
R.
U.
,T.
W.
S.
andT.
M.
F.
designedtheoverallproject.
E.
R.
U.
andT.
M.
F.
wrotethemanuscript.
Allauthorsreviewedthemanuscript.
AdditionalInformationSupplementaryinformationaccompaniesthispaperathttps://doi.
org/10.
1038/s41598-018-28263-7.
CompetingInterests:Theauthorsdeclarenocompetinginterests.
Publisher'snote:SpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
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