Synthesis and screening of potential antimicrobial compounds

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SYNTHESIS AND SCREENING OF POTENTIAL
ANTIMICROBIAL COMPOUNDS
PREETI PRAMOD GUNTHEY
Doctor of Philosophy
ASTON UNIVERSITY
December 2013
(cid:13)c PreetiGunthey, 2013 asserts her moralright to be identified as the authorof this thesis.
This copy of the thesis has been supplied on condition that anyone who consults it is un-
derstood to recognise that its copyright rests with its author and that no quotation from the
thesis and no information derived from it may be published without appropriate permission or
acknowledgement.
1
ASTONUNIVERSITY
SYNTHESISANDSCREENINGOFPOTENTIALANTIMICROBIALCOMPOUNDS
AthesissubmittedbyPreetiGunthey,forthedegreeofDoctorofPhilosophy
December2013
Abstract
Tuberculosis (TB), an infection caused by human pathogen Mycobacterium tuberculosis, continues to
kill millions each year and is as prevalent as it was in the pre-antimicrobial era. With the emergence
of continuously-evolving multi-drug resistant strains (MDR) and the implications of the HIV epidemic,
it is crucial that new drugs with better efficacy and affordable cost are developed to treat TB. With
this in mind, the first part of this thesis discusses the synthesis of libraries of derivatives of pyridine
carboxamidrazones,alongwithcyclised(1,2,4-triazoleand1,2,4-oxadiazole)andfluorinatedanalogues.
MicrobiologicalscreeningagainstM.tuberculosiswascarriedoutattheTAACF,NIAIDandIDRI(USA).
This confirmed theearlier findings that 2-pyridyl-substituted carboxamidrazones were more active than
the 4-pyridyl-substituted carboxamidrazones. Another important observation was that upon cyclisation
of these carboxamidrazones, a small number of the triazoles retained their activity while in most of the
remaining compounds the activity was diminished. This might be attributed to the significant increase
inlogPvaluecausedbycyclisationoftheselinearcarboxamidrazones,resultinginhighlipophilicityand
decreased permeability. Another reason might be that the rigidity conferred upon the compound due
to cyclisation, results in failure of the compound to fit into the active site of the putative target enzyme.
Inordertoinvestigatethepotentialchangetothecompounds’ metabolism intheorganism and/or host,
themostactive compoundswereselected andafluorineatom wasintroducedinthepyridinering. The
microbiological results shows adrasticimprovement intheactivity ofthefluorinatedcarboxamidrazone
amidesascomparedtotheirnonfluorinatedcounterpart. Thisimprovementintheactivitycouldpossibly
betheresultoftheincreasedcellpermeabilitycausedbythefluorine.
In a subsidiary strand, a selection of long-chain α, β-unsaturated carboxylic esters, α-keto, β-hydroxy
carboxylic esters and β-keto, α-hydroxy carboxylic esters, structurally similar to mycolic acids, were
synthesised. Themicrobiologicaldatarevealedthatoneoftheopenchaincompoundwasactiveagainst
the Mycobacterium tuberculosis H37Rv strain and some resistant isolates. The possible compound
activity could be its potential to disrupt mycobacterial cell wall synthesis by interfering with the FAS-II
2
pathway.
Keywords : Mycobacteriumtuberculosis,carboxamidrazone,cyclisation,fluorination,Horner-Wadsworth-
EmmonsmodificationtoWittig’sreaction.
3
Acknowledgements
Theworkpresentedinthisthesiswouldnothavebeenpossiblewithoutmycloseassociationwithmany
people who were always there when I needed them the most. I take this opportunity to acknowledge
themandextendmysinceregratitudeforhelpingmemakethisPhDthesisapossibility.
Firstandforemost,IwouldliketothankmysupervisorDr. DanielRathbonefor allhisfriendlyguidance
andpatience. Iamindebtedtohimforarrangingseveralmechanismssessionsthathelpedmetogaina
betterunderstandingofchemistry. Iamgratefultohimforcarryingoutapainstakingtaskofproofreading
thisthesis.
I would like to thank the School of Life and Health Sciences and The Sir Richard Stapley Educational
Trust for providing me financial support. I am grateful to EPSRC National Mass Spectrometry Service
Centre (SwanseaUniversity) for providing the massspectrometry data for the compoundssynthesised
inthisthesis.
This work was supported by National Institutes of Health and the National Institute of Allergy and
Infectious Diseases (NIAID), Contract No. HHSN272201100009I, Tuberculosis Antimicrobial Acqui-
sition and Coordinating Facility (TAACF) through a research and development contract with the NI-
AID, Contract No. HHSN27200001 and Infectious Disease Research Institute (IDRI), Contract No.
HHSN272201100009I/HHSN27200001.
A specialwordofthanksfor Dr. Mike Davis, KarenFarrowandTomHinton for thehelpandcontinuous
technicalassistanceduringmyPhD.IoweasincerethankstoGillPilfold(Seniorresearchadministrator)
and my colleagues Ren, Soumick, Alex, Matt, Pranav, Kshama, Cornelius and Shibu for always being
thereandbearingwithmethegoodandbadtimesduringmywonderfuldaysofPhD.
Finally,Iwouldliketothankmymom(Shubhada),dad(Pramod)andlittlesister(Supriya)forbeingvery
supportiveandpatientwithme. Tomyuncle(Avinash)andaunt(Neelima)andmyin-laws,thank-youfor
alwaysbeingsowonderfulandencouraging. AndlastbutnottheleastIwouldliketothankmyhusband
Ganesh. Hewasalwaystherecheeringmeupandhasbeenmyrockthroughout.
4
Contents
1 Introduction 22
1.1 Evolutionaryhistoryoftuberculosisanditsdiscovery . . . . . . . . . . . . . . . . . . . . 23
1.2 TBepidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
1.3 Mechanismsofdiseaseandimmunologicalresponse . . . . . . . . . . . . . . . . . . . . 27
1.3.1 OverviewofTBdisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
1.3.2 DiagnosingTB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.3.3 M.tuberculosis infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.3.4 Othermycobacteriacausingthedisease . . . . . . . . . . . . . . . . . . . . . . . 29
1.4 Themycobacterialcellwall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
1.4.1 Generalstructureandfunctions . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
1.4.2 Peptidoglycan-arabinogalactan . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
1.4.3 Mycolicacidsandtheirbiosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . 32
1.4.4 Intercalationoffree-outerlipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
1.5 ControlofTB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
1.5.1 Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
1.5.2 WHOinitiatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
1.5.3 Currently-useddrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
1.6 Treatmentregimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
1.7 Drugresistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
1.8 Projectaims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2 Chemistryandsynthesisofheteroarylamidrazonecompounds 54
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.2 Structureactivityrelationshipstudies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5
2.2.1 Imines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.2.2 Ureas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.2.3 SulphonamidesandN-oxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.2.4 Amides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.2.5 Generalcarboxamidrazoneamidestructure . . . . . . . . . . . . . . . . . . . . . 61
2.3 LeadOptimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.3.1 Intendedvariations ofthebuildingblocksinsynthesisofcarboxamidrazone . . . . 63
2.3.2 COMPOUNDA(Carboxamidrazoneamide) . . . . . . . . . . . . . . . . . . . . . 65
2.3.3 COMPOUNDB(N1-[(alkyl)oxy]pyridylcarboximidamide) . . . . . . . . . . . . . . 66
2.3.4 COMPOUNDC(Bis-amideversionofcarboxamidrazone) . . . . . . . . . . . . . 66
2.4 ResultsandDiscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
2.4.1 COMPOUNDA(Carboxamidrazoneamide) . . . . . . . . . . . . . . . . . . . . . 67
2.4.2 COMPOUNDB(N1-[(alkyl)oxy]pyridylcarboximidamide) . . . . . . . . . . . . . . 72
2.4.3 COMPOUNDC(Bis-amidecarboxamidrazone) . . . . . . . . . . . . . . . . . . . 75
2.4.4 Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
2.4.5 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2.4.6 NMRData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2.4.7 Meltingpointdata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.4.8 Microbiologicalresults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.4.9 Structureactivityrelationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.5 SummaryandConclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.5.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.5.2 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
2.6 EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
2.6.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
2.6.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
2.6.3 Preparationofpyridine-2-carboxamidrazone(85) . . . . . . . . . . . . . . . . . . 97
2.6.4 Preparationofpyridine-4-carboxamidrazone(86) . . . . . . . . . . . . . . . . . . 97
2.6.5 Preparationofpyridine-2-amidoxime(87) . . . . . . . . . . . . . . . . . . . . . . 98
2.6.6 Preparationofpyridine-4-amidoxime(88) . . . . . . . . . . . . . . . . . . . . . . 99
2.6.7 Generalprocedureforcarboxamidrazonesynthesisfromacidchlorides . . . . . . 99
2.6.8 Generalprocedureforsynthesisofcarboxamidrazonefromacids . . . . . . . . . 100
6
2.6.9 GeneralmethodforpreparationofLithocholicacidderivatives . . . . . . . . . . . 143
3 Cyclisationofcarboxamidrazoneamides 146
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
3.2 Leadoptimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
3.2.1 Synthesisoftheintendedtriazolesandoxadiazoles . . . . . . . . . . . . . . . . 149
3.2.2 COMPOUNDD(1,2,4-triazolecompounds) . . . . . . . . . . . . . . . . . . . . . 151
3.2.3 COMPOUNDE(1,2,4-oxadiazolecompounds) . . . . . . . . . . . . . . . . . . . 152
3.2.4 Cyclisationofureacompounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
3.2.5 CyclisationofN-oxidecompounds . . . . . . . . . . . . . . . . . . . . . . . . . . 153
3.3 ResultsandDiscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
3.3.1 COMPOUNDD(1,2,4-triazolecompounds) . . . . . . . . . . . . . . . . . . . . . 153
3.3.2 COMPOUNDE(1,2,4-oxadiazolecompounds) . . . . . . . . . . . . . . . . . . . 158
3.3.3 Attemptedcyclisation ofcarboxamidrazoneureacompounds . . . . . . . . . . . . 162
3.3.4 CyclisationofN-oxidecompounds . . . . . . . . . . . . . . . . . . . . . . . . . . 163
3.3.5 Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
3.3.6 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
3.3.7 NMRData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
3.3.8 Meltingpointdata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
3.3.9 Microbiologicalresults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
3.3.10 Structureactivityrelationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
3.4 SummaryandConclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
3.5 EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
3.5.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
3.5.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
3.5.3 Generalprocedureforsynthesisof1,2,4-triazolefromcarboxamidrazoneamide . 171
3.5.4 Generalprocedureforsynthesisof1,2,4-oxadiazolefromaldoxime . . . . . . . . 202
4 FluorinatedCarboxamidrazoneDerivatives 220
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
4.2 Leadoptimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
4.3 ResultsandDiscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
4.3.1 TrifluoromethylationofheteroarylamidrazonecompoundsusingLangloisreagent 223
7
4.3.2 Trifluoromethylationofheteroarylamidrazonecompoundsbyphotoredoxcatalysis
method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
4.3.3 TrifluoromethylationofheteroarylamidrazonecompoundsusingtheTognireagent 231
4.3.4 Synthesisoffluorinatedcarboxamidrazoneamides . . . . . . . . . . . . . . . . . 232
4.3.5 Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
4.3.6 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
4.3.7 Microbiologicalresults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
4.4 SummaryandConclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4.5 Experimental. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
4.5.1 Preparationofpyridine(2-fluoro)-4-carboxamidrazone(167) . . . . . . . . . . . . 238
4.5.2 Preparationofpyridine(5-fluoro)-2-carboxamidrazone(168) . . . . . . . . . . . . 238
4.5.3 Preparationofpyridine(3-fluoro)-2-carboxamidrazone(169) . . . . . . . . . . . . 239
4.5.4 General procedure for fluorinated carboxamidrazone amide synthesis from acid
chlorides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
4.5.5 Generalprocedureforsynthesisoffluorinatedcarboxamidrazoneamidefromacids240
4.5.6 General procedure for trifluoromethylation of heteroaryl amidrazone compounds
usingLangloisreagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
4.5.7 General procedure for trifluoromethylation of heteroaryl amidrazone compounds
byphotoredoxcatalysismethod . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
4.5.8 General procedure for trifluoromethylation of heteroaryl amidrazone compounds
usingtheTognireagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
5 Analoguesofmycolicacidsaspotentialinhibitorsofmycobacterialcellwallsynthesis 246
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
5.2 Leadoptimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
5.3 ResultsandDiscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
5.3.1 Synthesisofα,β-unsaturatedesters(alkene)byHornerWadsworthEmmonsmod-
ificationtoWittig’sreaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
5.3.2 SynthesisoflongchainaldehydesusingSwernoxidation . . . . . . . . . . . . . . 252
5.3.3 Synthesisofβ-hydroxyestersbyReformatskyreaction . . . . . . . . . . . . . . . 254
5.3.4 Synthesisof2-alkyl-2-hydroxy-3-oxocarboxylic esters . . . . . . . . . . . . . . . . 256
5.3.5 Attemptedtertiaryketol rearrangementof 2-hydroxy-3-oxocarboxylic esters by di-
n-butyltinoxidecatalystmethod . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
8
5.3.6 Microbiologicalresults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
5.4 SummaryandConclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
5.5 Experimental. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
5.5.1 Generalpreparationofα,β-unsaturatedesters . . . . . . . . . . . . . . . . . . . 261
5.5.2 GeneralprocedureforsynthesisofaldehydesusingSwernoxidation . . . . . . . 264
5.5.3 Generalprocedureforoxidationofα,β-unsaturatedcarboxylicesters . . . . . . . 265
5.5.4 GeneralprocedureusedforReformatskyreaction . . . . . . . . . . . . . . . . . . 270
5.5.5 Generalprocedurefortertiaryketolrearrangementusingdi-n-butyltinoxidecatalyst272
9