2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure–Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization
Identifieur interne : 000030 ( Pmc/Corpus ); précédent : 000029; suivant : 0000312-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure–Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization
Auteurs : Dinakaran Murugesan ; Peter C. Ray ; Tracy Bayliss ; Gareth A. Prosser ; Justin R. Harrison ; Kirsteen Green ; Candice Soares De Melo ; Tzu-Shean Feng ; Leslie J. Street ; Kelly Chibale ; Digby F. Warner ; Valerie Mizrahi ; Ola Epemolu ; Paul Scullion ; Lucy Ellis ; Jennifer Riley ; Yoko Shishikura ; Liam Ferguson ; Maria Osuna-Cabello ; Kevin D. Read ; Simon R. Green ; Dirk A. Lamprecht ; Peter M. Finin ; Adrie J. C. Steyn ; Thomas R. Ioerger ; Jim Sacchettini ; Kyu Y. Rhee ; Kriti Arora ; Clifton E. Barry ; Paul G. Wyatt ; Helena I. M. BoshoffSource :
- ACS Infectious Diseases [ 2373-8227 ] ; 2018.
Abstract
Url:
DOI: 10.1021/acsinfecdis.7b00275
PubMed: 29522317
PubMed Central: 5996347
Links to Exploration step
PMC:5996347Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and <italic>Mycobacterium tuberculosis</italic>: Structure–Activity Relationships, Mechanism of Action and
Absorption, Distribution, Metabolism, and Excretion Characterization</title><author><name sortKey="Murugesan, Dinakaran" sort="Murugesan, Dinakaran" uniqKey="Murugesan D" first="Dinakaran" last="Murugesan">Dinakaran Murugesan</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Ray, Peter C" sort="Ray, Peter C" uniqKey="Ray P" first="Peter C." last="Ray">Peter C. Ray</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Bayliss, Tracy" sort="Bayliss, Tracy" uniqKey="Bayliss T" first="Tracy" last="Bayliss">Tracy Bayliss</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Prosser, Gareth A" sort="Prosser, Gareth A" uniqKey="Prosser G" first="Gareth A." last="Prosser">Gareth A. Prosser</name><affiliation><nlm:aff id="aff2">Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Harrison, Justin R" sort="Harrison, Justin R" uniqKey="Harrison J" first="Justin R." last="Harrison">Justin R. Harrison</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Green, Kirsteen" sort="Green, Kirsteen" uniqKey="Green K" first="Kirsteen" last="Green">Kirsteen Green</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Soares De Melo, Candice" sort="Soares De Melo, Candice" uniqKey="Soares De Melo C" first="Candice" last="Soares De Melo">Candice Soares De Melo</name><affiliation><nlm:aff id="aff4">Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Feng, Tzu Shean" sort="Feng, Tzu Shean" uniqKey="Feng T" first="Tzu-Shean" last="Feng">Tzu-Shean Feng</name><affiliation><nlm:aff id="aff4">Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Street, Leslie J" sort="Street, Leslie J" uniqKey="Street L" first="Leslie J." last="Street">Leslie J. Street</name><affiliation><nlm:aff id="aff4">Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Chibale, Kelly" sort="Chibale, Kelly" uniqKey="Chibale K" first="Kelly" last="Chibale">Kelly Chibale</name><affiliation><nlm:aff id="aff4">Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff5">South African Medical Research Council Drug Discovery and Development Research Unit, Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Warner, Digby F" sort="Warner, Digby F" uniqKey="Warner D" first="Digby F." last="Warner">Digby F. Warner</name><affiliation><nlm:aff id="aff6">SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Mizrahi, Valerie" sort="Mizrahi, Valerie" uniqKey="Mizrahi V" first="Valerie" last="Mizrahi">Valerie Mizrahi</name><affiliation><nlm:aff id="aff6">SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Epemolu, Ola" sort="Epemolu, Ola" uniqKey="Epemolu O" first="Ola" last="Epemolu">Ola Epemolu</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Scullion, Paul" sort="Scullion, Paul" uniqKey="Scullion P" first="Paul" last="Scullion">Paul Scullion</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Ellis, Lucy" sort="Ellis, Lucy" uniqKey="Ellis L" first="Lucy" last="Ellis">Lucy Ellis</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Riley, Jennifer" sort="Riley, Jennifer" uniqKey="Riley J" first="Jennifer" last="Riley">Jennifer Riley</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Shishikura, Yoko" sort="Shishikura, Yoko" uniqKey="Shishikura Y" first="Yoko" last="Shishikura">Yoko Shishikura</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Ferguson, Liam" sort="Ferguson, Liam" uniqKey="Ferguson L" first="Liam" last="Ferguson">Liam Ferguson</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Osuna Cabello, Maria" sort="Osuna Cabello, Maria" uniqKey="Osuna Cabello M" first="Maria" last="Osuna-Cabello">Maria Osuna-Cabello</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Read, Kevin D" sort="Read, Kevin D" uniqKey="Read K" first="Kevin D." last="Read">Kevin D. Read</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Green, Simon R" sort="Green, Simon R" uniqKey="Green S" first="Simon R." last="Green">Simon R. Green</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Lamprecht, Dirk A" sort="Lamprecht, Dirk A" uniqKey="Lamprecht D" first="Dirk A." last="Lamprecht">Dirk A. Lamprecht</name><affiliation><nlm:aff id="aff7"><institution>Africa Health Research Institute (AHRI)</institution>, K-RITH Tower Building Level 3, 719 Umbilo Road, Durban, 4001,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Finin, Peter M" sort="Finin, Peter M" uniqKey="Finin P" first="Peter M." last="Finin">Peter M. Finin</name><affiliation><nlm:aff id="aff7"><institution>Africa Health Research Institute (AHRI)</institution>, K-RITH Tower Building Level 3, 719 Umbilo Road, Durban, 4001,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Steyn, Adrie J C" sort="Steyn, Adrie J C" uniqKey="Steyn A" first="Adrie J. C." last="Steyn">Adrie J. C. Steyn</name><affiliation><nlm:aff id="aff7"><institution>Africa Health Research Institute (AHRI)</institution>, K-RITH Tower Building Level 3, 719 Umbilo Road, Durban, 4001,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff8">Department of Microbiology,<institution>University of Alabama at Birmingham</institution>, 1720 Second Avenue South, Birmingham, Alabama 35294-2170,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Ioerger, Thomas R" sort="Ioerger, Thomas R" uniqKey="Ioerger T" first="Thomas R." last="Ioerger">Thomas R. Ioerger</name><affiliation><nlm:aff id="aff9">Department of Computer Science and Engineering,<institution>Texas A&M University</institution>, College Station, Texas 77843,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Sacchettini, Jim" sort="Sacchettini, Jim" uniqKey="Sacchettini J" first="Jim" last="Sacchettini">Jim Sacchettini</name><affiliation><nlm:aff id="aff9">Department of Computer Science and Engineering,<institution>Texas A&M University</institution>, College Station, Texas 77843,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Rhee, Kyu Y" sort="Rhee, Kyu Y" uniqKey="Rhee K" first="Kyu Y." last="Rhee">Kyu Y. Rhee</name><affiliation><nlm:aff id="aff10">Division of Infectious Diseases, Weill Department of Medicine,<institution>Weill Cornell Medical College</institution>, New York, New York 10065,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Arora, Kriti" sort="Arora, Kriti" uniqKey="Arora K" first="Kriti" last="Arora">Kriti Arora</name><affiliation><nlm:aff id="aff2">Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Barry, Clifton E" sort="Barry, Clifton E" uniqKey="Barry C" first="Clifton E." last="Barry">Clifton E. Barry</name><affiliation><nlm:aff id="aff2">Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Wyatt, Paul G" sort="Wyatt, Paul G" uniqKey="Wyatt P" first="Paul G." last="Wyatt">Paul G. Wyatt</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Boshoff, Helena I M" sort="Boshoff, Helena I M" uniqKey="Boshoff H" first="Helena I. M." last="Boshoff">Helena I. M. Boshoff</name><affiliation><nlm:aff id="aff2">Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29522317</idno><idno type="pmc">5996347</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996347</idno><idno type="RBID">PMC:5996347</idno><idno type="doi">10.1021/acsinfecdis.7b00275</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000030</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000030</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and <italic>Mycobacterium tuberculosis</italic>: Structure–Activity Relationships, Mechanism of Action and
Absorption, Distribution, Metabolism, and Excretion Characterization</title><author><name sortKey="Murugesan, Dinakaran" sort="Murugesan, Dinakaran" uniqKey="Murugesan D" first="Dinakaran" last="Murugesan">Dinakaran Murugesan</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Ray, Peter C" sort="Ray, Peter C" uniqKey="Ray P" first="Peter C." last="Ray">Peter C. Ray</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Bayliss, Tracy" sort="Bayliss, Tracy" uniqKey="Bayliss T" first="Tracy" last="Bayliss">Tracy Bayliss</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Prosser, Gareth A" sort="Prosser, Gareth A" uniqKey="Prosser G" first="Gareth A." last="Prosser">Gareth A. Prosser</name><affiliation><nlm:aff id="aff2">Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Harrison, Justin R" sort="Harrison, Justin R" uniqKey="Harrison J" first="Justin R." last="Harrison">Justin R. Harrison</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Green, Kirsteen" sort="Green, Kirsteen" uniqKey="Green K" first="Kirsteen" last="Green">Kirsteen Green</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Soares De Melo, Candice" sort="Soares De Melo, Candice" uniqKey="Soares De Melo C" first="Candice" last="Soares De Melo">Candice Soares De Melo</name><affiliation><nlm:aff id="aff4">Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Feng, Tzu Shean" sort="Feng, Tzu Shean" uniqKey="Feng T" first="Tzu-Shean" last="Feng">Tzu-Shean Feng</name><affiliation><nlm:aff id="aff4">Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Street, Leslie J" sort="Street, Leslie J" uniqKey="Street L" first="Leslie J." last="Street">Leslie J. Street</name><affiliation><nlm:aff id="aff4">Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Chibale, Kelly" sort="Chibale, Kelly" uniqKey="Chibale K" first="Kelly" last="Chibale">Kelly Chibale</name><affiliation><nlm:aff id="aff4">Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff5">South African Medical Research Council Drug Discovery and Development Research Unit, Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Warner, Digby F" sort="Warner, Digby F" uniqKey="Warner D" first="Digby F." last="Warner">Digby F. Warner</name><affiliation><nlm:aff id="aff6">SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Mizrahi, Valerie" sort="Mizrahi, Valerie" uniqKey="Mizrahi V" first="Valerie" last="Mizrahi">Valerie Mizrahi</name><affiliation><nlm:aff id="aff6">SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Epemolu, Ola" sort="Epemolu, Ola" uniqKey="Epemolu O" first="Ola" last="Epemolu">Ola Epemolu</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Scullion, Paul" sort="Scullion, Paul" uniqKey="Scullion P" first="Paul" last="Scullion">Paul Scullion</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Ellis, Lucy" sort="Ellis, Lucy" uniqKey="Ellis L" first="Lucy" last="Ellis">Lucy Ellis</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Riley, Jennifer" sort="Riley, Jennifer" uniqKey="Riley J" first="Jennifer" last="Riley">Jennifer Riley</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Shishikura, Yoko" sort="Shishikura, Yoko" uniqKey="Shishikura Y" first="Yoko" last="Shishikura">Yoko Shishikura</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Ferguson, Liam" sort="Ferguson, Liam" uniqKey="Ferguson L" first="Liam" last="Ferguson">Liam Ferguson</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Osuna Cabello, Maria" sort="Osuna Cabello, Maria" uniqKey="Osuna Cabello M" first="Maria" last="Osuna-Cabello">Maria Osuna-Cabello</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Read, Kevin D" sort="Read, Kevin D" uniqKey="Read K" first="Kevin D." last="Read">Kevin D. Read</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Green, Simon R" sort="Green, Simon R" uniqKey="Green S" first="Simon R." last="Green">Simon R. Green</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Lamprecht, Dirk A" sort="Lamprecht, Dirk A" uniqKey="Lamprecht D" first="Dirk A." last="Lamprecht">Dirk A. Lamprecht</name><affiliation><nlm:aff id="aff7"><institution>Africa Health Research Institute (AHRI)</institution>, K-RITH Tower Building Level 3, 719 Umbilo Road, Durban, 4001,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Finin, Peter M" sort="Finin, Peter M" uniqKey="Finin P" first="Peter M." last="Finin">Peter M. Finin</name><affiliation><nlm:aff id="aff7"><institution>Africa Health Research Institute (AHRI)</institution>, K-RITH Tower Building Level 3, 719 Umbilo Road, Durban, 4001,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Steyn, Adrie J C" sort="Steyn, Adrie J C" uniqKey="Steyn A" first="Adrie J. C." last="Steyn">Adrie J. C. Steyn</name><affiliation><nlm:aff id="aff7"><institution>Africa Health Research Institute (AHRI)</institution>, K-RITH Tower Building Level 3, 719 Umbilo Road, Durban, 4001,<country>South Africa</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff8">Department of Microbiology,<institution>University of Alabama at Birmingham</institution>, 1720 Second Avenue South, Birmingham, Alabama 35294-2170,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Ioerger, Thomas R" sort="Ioerger, Thomas R" uniqKey="Ioerger T" first="Thomas R." last="Ioerger">Thomas R. Ioerger</name><affiliation><nlm:aff id="aff9">Department of Computer Science and Engineering,<institution>Texas A&M University</institution>, College Station, Texas 77843,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Sacchettini, Jim" sort="Sacchettini, Jim" uniqKey="Sacchettini J" first="Jim" last="Sacchettini">Jim Sacchettini</name><affiliation><nlm:aff id="aff9">Department of Computer Science and Engineering,<institution>Texas A&M University</institution>, College Station, Texas 77843,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Rhee, Kyu Y" sort="Rhee, Kyu Y" uniqKey="Rhee K" first="Kyu Y." last="Rhee">Kyu Y. Rhee</name><affiliation><nlm:aff id="aff10">Division of Infectious Diseases, Weill Department of Medicine,<institution>Weill Cornell Medical College</institution>, New York, New York 10065,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Arora, Kriti" sort="Arora, Kriti" uniqKey="Arora K" first="Kriti" last="Arora">Kriti Arora</name><affiliation><nlm:aff id="aff2">Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Barry, Clifton E" sort="Barry, Clifton E" uniqKey="Barry C" first="Clifton E." last="Barry">Clifton E. Barry</name><affiliation><nlm:aff id="aff2">Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></nlm:aff></affiliation></author><author><name sortKey="Wyatt, Paul G" sort="Wyatt, Paul G" uniqKey="Wyatt P" first="Paul G." last="Wyatt">Paul G. Wyatt</name><affiliation><nlm:aff id="aff1">Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Boshoff, Helena I M" sort="Boshoff, Helena I M" uniqKey="Boshoff H" first="Helena I. M." last="Boshoff">Helena I. M. Boshoff</name><affiliation><nlm:aff id="aff2">Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></nlm:aff></affiliation></author></analytic><series><title level="j">ACS Infectious Diseases</title><idno type="eISSN">2373-8227</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="id-2017-002759_0014" id="ab-tgr1"></graphic></p><p><italic>Mycobacterium tuberculosis</italic> (<italic>MTb</italic>) possesses
two nonproton pumping type II NADH dehydrogenase (NDH-2)
enzymes which are predicted to be jointly essential for respiratory
metabolism. Furthermore, the structure of a closely related bacterial
NDH-2 has been reported recently, allowing for the structure-based
design of small-molecule inhibitors. Herein, we disclose <italic>MTb</italic> whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the <italic>ndh</italic> encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in <italic>MTb</italic> was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by <italic>ndhA</italic> in <italic>MTb</italic>. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial <italic>MTb</italic> SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Horsburgh, C R J" uniqKey="Horsburgh C">C. R. J. Horsburgh</name></author><author><name sortKey="Barry, C E I" uniqKey="Barry C">C. E. I. Barry</name></author><author><name sortKey="Lange, C" uniqKey="Lange C">C. Lange</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Iseman, M D" uniqKey="Iseman M">M. D. Iseman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Migliori, G B" uniqKey="Migliori G">G. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">ACS Infect Dis</journal-id><journal-id journal-id-type="iso-abbrev">ACS Infect Dis</journal-id><journal-id journal-id-type="publisher-id">id</journal-id><journal-id journal-id-type="coden">aidcbc</journal-id><journal-title-group><journal-title>ACS Infectious Diseases</journal-title></journal-title-group><issn pub-type="epub">2373-8227</issn><publisher><publisher-name>American Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29522317</article-id><article-id pub-id-type="pmc">5996347</article-id><article-id pub-id-type="doi">10.1021/acsinfecdis.7b00275</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and <italic>Mycobacterium tuberculosis</italic>: Structure–Activity Relationships, Mechanism of Action and
Absorption, Distribution, Metabolism, and Excretion Characterization</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Murugesan</surname><given-names>Dinakaran</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="notes-2" ref-type="notes">α</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Ray</surname><given-names>Peter C.</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="notes-2" ref-type="notes">α</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Bayliss</surname><given-names>Tracy</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath4"><name><surname>Prosser</surname><given-names>Gareth A.</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath5"><name><surname>Harrison</surname><given-names>Justin R.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath6"><name><surname>Green</surname><given-names>Kirsteen</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath7"><name><surname>Soares de Melo</surname><given-names>Candice</given-names></name><xref rid="aff4" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath8"><name><surname>Feng</surname><given-names>Tzu-Shean</given-names></name><xref rid="aff4" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath9"><name><surname>Street</surname><given-names>Leslie J.</given-names></name><xref rid="aff4" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath10"><name><surname>Chibale</surname><given-names>Kelly</given-names></name><xref rid="aff4" ref-type="aff">∥</xref><xref rid="aff3" ref-type="aff">§</xref><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath11"><name><surname>Warner</surname><given-names>Digby F.</given-names></name><xref rid="aff6" ref-type="aff">#</xref><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath12"><name><surname>Mizrahi</surname><given-names>Valerie</given-names></name><xref rid="aff6" ref-type="aff">#</xref><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath13"><name><surname>Epemolu</surname><given-names>Ola</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath14"><name><surname>Scullion</surname><given-names>Paul</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath15"><name><surname>Ellis</surname><given-names>Lucy</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath16"><name><surname>Riley</surname><given-names>Jennifer</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath17"><name><surname>Shishikura</surname><given-names>Yoko</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath18"><name><surname>Ferguson</surname><given-names>Liam</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath19"><name><surname>Osuna-Cabello</surname><given-names>Maria</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath20"><name><surname>Read</surname><given-names>Kevin D.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath21"><name><surname>Green</surname><given-names>Simon R.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath22"><name><surname>Lamprecht</surname><given-names>Dirk A.</given-names></name><xref rid="aff7" ref-type="aff">¶</xref></contrib><contrib contrib-type="author" id="ath23"><name><surname>Finin</surname><given-names>Peter M.</given-names></name><xref rid="aff7" ref-type="aff">¶</xref><xref rid="notes-1" ref-type="notes">¤</xref></contrib><contrib contrib-type="author" id="ath24"><name><surname>Steyn</surname><given-names>Adrie J. C.</given-names></name><xref rid="aff7" ref-type="aff">¶</xref><xref rid="aff8" ref-type="aff">○</xref></contrib><contrib contrib-type="author" id="ath25"><name><surname>Ioerger</surname><given-names>Thomas R.</given-names></name><xref rid="aff9" ref-type="aff">◆</xref></contrib><contrib contrib-type="author" id="ath26"><name><surname>Sacchettini</surname><given-names>Jim</given-names></name><xref rid="aff9" ref-type="aff">◆</xref></contrib><contrib contrib-type="author" id="ath27"><name><surname>Rhee</surname><given-names>Kyu Y.</given-names></name><xref rid="aff10" ref-type="aff">●</xref></contrib><contrib contrib-type="author" id="ath28"><name><surname>Arora</surname><given-names>Kriti</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath29"><name><surname>Barry</surname><given-names>Clifton E.</given-names><suffix>III</suffix></name><xref rid="aff2" ref-type="aff">‡</xref><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath30"><name><surname>Wyatt</surname><given-names>Paul G.</given-names></name><xref rid="cor2" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath31"><name><surname>Boshoff</surname><given-names>Helena I. M.</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff2" ref-type="aff">‡</xref></contrib><aff id="aff1"><label>†</label>Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences,<institution>University of Dundee</institution>, Sir James Black Centre, Dundee, DD1 5EH,<country>United Kingdom</country></aff><aff id="aff2"><label>‡</label>Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease,<institution>National Institutes of Health</institution>, 9000 Rockville Pike, Bethesda, Maryland 20892,<country>United States</country></aff><aff id="aff3"><label>§</label>Institute of Infectious Disease and Molecular Medicine,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></aff><aff id="aff4"><label>∥</label>Drug Discovery and Development Centre (H3D), Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></aff><aff id="aff5"><label>⊥</label>South African Medical Research Council Drug Discovery and Development Research Unit, Department of Chemistry,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></aff><aff id="aff6"><label>#</label>SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences,<institution>University of Cape Town</institution>, Rondebosch, 7701,<country>South Africa</country></aff><aff id="aff7"><label>¶</label><institution>Africa Health Research Institute (AHRI)</institution>, K-RITH Tower Building Level 3, 719 Umbilo Road, Durban, 4001,<country>South Africa</country></aff><aff id="aff8"><label>○</label>Department of Microbiology,<institution>University of Alabama at Birmingham</institution>, 1720 Second Avenue South, Birmingham, Alabama 35294-2170,<country>United States</country></aff><aff id="aff9"><label>◆</label>Department of Computer Science and Engineering,<institution>Texas A&M University</institution>, College Station, Texas 77843,<country>United States</country></aff><aff id="aff10"><label>●</label>Division of Infectious Diseases, Weill Department of Medicine,<institution>Weill Cornell Medical College</institution>, New York, New York 10065,<country>United States</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>E-mail: <email>hboshoff@niaid.nih.gov</email>.</corresp><corresp id="cor2"><label>*</label>E-mail: <email>P.G.Wyatt@dundee.ac.uk</email>.</corresp></author-notes><pub-date pub-type="epub"><day>09</day><month>03</month><year>2018</year></pub-date><pub-date pub-type="ppub"><day>08</day><month>06</month><year>2018</year></pub-date><volume>4</volume><issue>6</issue><fpage>954</fpage><lpage>969</lpage><history><date date-type="received"><day>21</day><month>12</month><year>2017</year></date></history><permissions><copyright-statement>Copyright © 2018 American Chemical Society</copyright-statement><copyright-year>2018</copyright-year><copyright-holder>American Chemical Society</copyright-holder><license><license-p>This is an open access article published under a Creative Commons Attribution (CC-BY) <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html">License</ext-link>, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="id-2017-002759_0014" id="ab-tgr1"></graphic></p><p><italic>Mycobacterium tuberculosis</italic> (<italic>MTb</italic>) possesses
two nonproton pumping type II NADH dehydrogenase (NDH-2)
enzymes which are predicted to be jointly essential for respiratory
metabolism. Furthermore, the structure of a closely related bacterial
NDH-2 has been reported recently, allowing for the structure-based
design of small-molecule inhibitors. Herein, we disclose <italic>MTb</italic> whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the <italic>ndh</italic> encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in <italic>MTb</italic> was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by <italic>ndhA</italic> in <italic>MTb</italic>. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial <italic>MTb</italic> SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.</p></abstract><kwd-group><kwd><italic>Mycobacterium tuberculosis</italic></kwd><kwd>mercapto-quinazolinones</kwd><kwd>structure−activity relationship</kwd><kwd>type II NADH
dehydrogenase</kwd><kwd>small molecule NDH-2 inhibitors</kwd><kwd>respiration</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>id7b00275</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>id-2017-002759</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta></front><body><p id="sec1">Tuberculosis (TB) is a major
global health problem, resulting in significant morbidity each year.
Although mortality has fallen dramatically since 1990, TB now ranks
alongside HIV as a leading cause of death worldwide. While HIV-related
deaths have been declining largely as a result of improved access
to, and availability of, better HIV treatments, this has not been
the case for TB,<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> the treatment of which
requires 6 months of chemotherapy with a combination of four agents
(isoniazid, rifampicin, pyrazinamide, and ethambutol) to achieve durable
cure of drug-sensitive TB.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> The large number
of TB patients, coupled with the chemotherapeutic burden, often leads
to poor patient adherence and suboptimal treatment outcomes in the
developing world, as well as the emergence of multidrug resistant
TB (MDR-TB, defined as resistance to isoniazid (INH) and rifampicin
(RIF)) and extensively drug-resistant tuberculosis (XDR-TB, defined
as MDR-TB plus resistance to any fluoroquinolone and one of three
second-line injectable drugs, capreomycin, kanamycin (Kan), and amikacin).
To address this global TB health problem, an improved treatment regimen
is needed which will reduce treatment duration and prevent relapse
and the development of TB drug resistance.<sup><xref ref-type="bibr" rid="ref3">3</xref>−<xref ref-type="bibr" rid="ref8">8</xref></sup> However, achieving this goal will require discovery of multiple
novel and mechanistically distinct antimycobacterial agents possessing
reduced liabilities for investigation of new drug regimens that might
shorten the duration of treatment and simplify management of the disease
by improving adherence and reducing costs.<sup><xref ref-type="bibr" rid="ref9">9</xref>−<xref ref-type="bibr" rid="ref12">12</xref></sup></p><p>Identification of novel
drug targets that will lead to treatment
shortening is challenging. Targets of drugs currently in use or phase
3 clinical evaluation for TB chemotherapy include cell wall biosynthesis,
translation, transcription, folate synthesis, ATP generation, and
maintenance of DNA topology as broad categories, although the nitroimidazoles
delaminid and pretomanid generate reactive nitrogen intermediates
that inhibit several essential processes.<sup><xref ref-type="bibr" rid="ref13">13</xref></sup> Mechanistically novel drugs would conceptually target distinct processes
from the above, and to date, successes in the field have all emerged
from target identification of hits discovered in <italic>Mycobacterium
tuberculosis</italic> (<italic>MTb</italic>) whole cell screens.<sup><xref ref-type="bibr" rid="ref13">13</xref></sup></p><p>In this work, we describe the identification
of a 2-mercapto-quinazoline
scaffold identified from a <italic>MTb</italic> whole cell screen,
which had been previously reported to inhibit the mycobacterial type
II NADH dehydrogenase,<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> providing further
evidence for its inhibition of the <italic>MTb</italic> type II NADH
dehydrogenase (NDH-2). <italic>MTb</italic> encodes two NDH-2 genes
of which the one encoded by <italic>ndh</italic> plays a critical
role for growth both <italic>in vitro</italic> and <italic>in vivo</italic>.<sup><xref ref-type="bibr" rid="ref15">15</xref>−<xref ref-type="bibr" rid="ref17">17</xref></sup> NDH-2 catalyzes the transfer of electrons from NADH into the mycobacterial
respiratory pathway and has been proposed to be targeted by a number
of early stage inhibitors.<sup><xref ref-type="bibr" rid="ref16">16</xref>,<xref ref-type="bibr" rid="ref18">18</xref>−<xref ref-type="bibr" rid="ref20">20</xref></sup> In contrast, the proton pumping type I NADH dehydrogenase can be
deleted without apparent effects on growth both <italic>in vitro</italic> or <italic>in vivo</italic>.<sup><xref ref-type="bibr" rid="ref15">15</xref>−<xref ref-type="bibr" rid="ref17">17</xref></sup> As the current series was considered
to have a promising drug-like profile (<xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>), a focused optimization program was initiated.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><title>Profiling
of Confirmed Hits <bold>1</bold>, <bold>2</bold>, and <bold>3</bold></title></caption><graphic xlink:href="id-2017-002759_0009" id="fx1" position="float"></graphic><table-wrap-foot><fn id="t1fn1"><label>a</label><p>Calculated using StarDrop (<uri xlink:href="http://www.optibrium.com">http://www.optibrium.com</uri>).</p></fn><fn id="t1fn2"><label>b</label><p>Kinetic aqueous solubility
was measured
using laser nephelometry of compounds in 2.5% DMSO.</p></fn></table-wrap-foot></table-wrap><sec id="sec2"><title>Results
and Discussion</title><p>Screening of a commercial diversity library,
details of which will
be published elsewhere, afforded a 2-mercapto-quinazolinone cluster
of hits (<bold>1</bold>, <bold>2</bold>, and <bold>3</bold>), with
compounds <bold>1</bold> and <bold>2</bold> showing potent <italic>MTb</italic> whole-cell minimum inhibitory concentration (MIC) in
two distinct growth media. The compounds were assessed in a series
of early stage biology profiling assays to understand better the mechanism
of action (MoA). These included screening the compounds against a <italic>cyd</italic> knockout strain which is known to be hyper-susceptible
to inhibitors of the cytochrome <italic>bc</italic><sub>1</sub> complex,<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> which suggested that the series was unlikely
to target QcrB. Profiling also investigated the extent of upregulation
of the promoter of the <italic>iniBAC</italic> gene cluster, known
to be induced by inhibitors of cell wall biosynthesis, such as isoniazid,
ethionamide, SQ109, and ethambutol,<sup><xref ref-type="bibr" rid="ref22">22</xref>,<xref ref-type="bibr" rid="ref23">23</xref></sup> which suggested <bold>1</bold> and <bold>2</bold> did not have an effect on cell wall biosynthesis.
These biology profiling data were considered promising, especially
in conjunction with a recent report<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> in
which mutations in <italic>MTb</italic> mutants spontaneously resistant
to compound <bold>1</bold> mapped to <italic>ndhA</italic>, the gene
encoding the nonessential type-II NADH dehydrogenase that is involved
in NADH reoxidation in the mycobacterial oxidative phosphorylation
pathway.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> There are two closely related
nonproton pumping type II NADH dehydrogenases in the <italic>MTb</italic> genome, of which only one (encoded by <italic>ndh</italic>, Rv1854c)
is essential.<sup><xref ref-type="bibr" rid="ref24">24</xref>−<xref ref-type="bibr" rid="ref27">27</xref></sup> Ioerger et al.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> identified promoter
mutations in <italic>ndhA</italic> which resulted in >40-fold upregulation
of gene expression, likely compensating for compound <bold>1</bold> inhibiting the essential NDH-2 homologue.</p><p>Compound <bold>1</bold> had a promising <italic>MTb</italic> MIC-derived
ligand-lipophilicity efficiency (LLE) drug-likeness profile, suggestive
of a quality starting point for medicinal chemistry optimization.<sup><xref ref-type="bibr" rid="ref28">28</xref>,<xref ref-type="bibr" rid="ref29">29</xref></sup> Compound <bold>1</bold> also showed no noticeable cytotoxicity in
a mammalian cell line (HepG2). Compounds <bold>1</bold> and <bold>2</bold> also had moderate kinetic solubility and reasonable mouse
hepatic microsomal stability, with <bold>1</bold> having excellent
human microsomal stability (<xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>). Herein, we report on the development of the structure–activity
relationship (SAR) for <bold>1</bold>, as well as extended absorption,
distribution, metabolism, and excretion (ADME) characterization of
key compounds.</p><sec id="sec2.1"><title>Synthetic Chemistry</title><p>Quinazolinone amides reported herein
were synthesized utilizing known procedures, which are detailed in <xref rid="sch1" ref-type="scheme">Scheme <xref rid="sch1" ref-type="scheme">1</xref></xref>. Commercially available
anthranilic acids (<bold>28</bold>) were cyclized with thiourea, and
the resulting 2-mercapto quinazoline-4-diones (<bold>29</bold>) or
commercially available 2-mercapto-4(3<italic>H</italic>)-quinazolinone
was reacted with 2-bromo acetic acid to form the 2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic
acids (<bold>30</bold>). Primary and secondary amines (<bold>31</bold>) were coupled using standard coupling reagents to afford compounds <bold>1</bold>, <bold>2</bold>, <bold>4</bold>–<bold>11</bold>,
and <bold>14</bold>.</p><fig id="sch1" position="float"><label>Scheme 1</label><caption><title>General Synthetic Route for Synthesis of
Quinazolinone Amides</title><p id="sch1-fn1">Reagents and conditions:
General
synthetic approach to quinazolinones <bold>1</bold>, <bold>2</bold>, <bold>4</bold>–<bold>11</bold>, and <bold>14</bold>: (a)
neat thiourea at 180 °C, 3 h; (b) 2-bromoacetic acid, triethylamine,
DMF, 80 °C, 12 h; (c) primary and/or secondary amine <bold>31</bold>, EDC·HCl, HOAT, <italic>N</italic>,<italic>N</italic>-diisopropylethylamine,
DMF/ACN (1:1), room temperature, 12 h or primary and/or secondary
amine <bold>31</bold>, HATU, <italic>N</italic>,<italic>N</italic>-diisopropylethylamine, DCM, room temperature, 12 h.</p></caption><graphic xlink:href="id-2017-002759_0008" id="gr1" position="float"></graphic></fig></sec><sec id="sec2.3"><title>MTb Whole-Cell SAR</title><p>The initial medicinal chemistry
plan focused on developing the SAR with the aim of better understanding
the pharmacophore. Initial efforts were guided by mouse microsomal
metabolite identification (met-ID) studies on <bold>1</bold>, which
revealed significant oxidation of the cyclohexyl and quinazolin-4(3<italic>H</italic>)-one rings as well as cleavage of the amide bond (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Figure S1</ext-link>). To understand the scope, cycloalkyls <bold>4</bold>, <bold>5</bold>, and <bold>6</bold> were prepared (<xref rid="tbl2" ref-type="other">Table <xref rid="tbl2" ref-type="other">2</xref></xref>). The large bulky
lipophilic cyclohexyl <bold>1</bold> and cycloheptyl <bold>4</bold> were both equally favored, with good whole-cell MIC potency. In
contrast, the smaller ring-contracted cyclopentyl <bold>5</bold> and
cyclobutyl <bold>6</bold> were slightly less potent (<xref rid="tbl2" ref-type="other">Table <xref rid="tbl2" ref-type="other">2</xref></xref>), and the cyclopropyl and ring
deletion NHMe analogues (data not shown) resulted in a complete loss
of whole-cell activity, suggesting that a bulky hydrophobic group
was required to obtain good whole-cell potency. As mouse met-ID of <bold>1</bold> showed significant oxidation of the cyclohexyl group, the
2-, 3-, and 4-hydroxylated cyclohexyl derivatives were prepared with
the aim of improving solubility as well as microsomal stability. Improvements
in both kinetic solubility and mouse microsomal stability were indeed
achieved, but no whole-cell activity was observed (data not shown).
An additional focused set of polar saturated 4-, 5-, and 6-membered
oxygen-containing saturated heterocycles was prepared; however, while
having good kinetic solubility and mouse microsomal stability, they
too lost all whole-cell activity (data not shown). There were also
attempts to improve solubility with differentially functionalized
4-substituted piperidines (NAc, NSO<sub>2</sub>Me, NMe, and NBn),
but once again, there was a loss of whole-cell activity (data not
shown). As the cyclohexyl group within <bold>1</bold> did not appear
to tolerate polar solubilizing groups, we turned our focus to introducing
fluorine in an attempt to improve microsomal stability.<sup><xref ref-type="bibr" rid="ref30">30</xref>,<xref ref-type="bibr" rid="ref31">31</xref></sup> We were encouraged to find that the 4,4-difluorocyclohexan <bold>7</bold> retained good whole-cell potency and also had slightly improved
mouse microsomal stability versus <bold>1</bold>. In comparison to <bold>1</bold>, mouse microsomal met-ID on <bold>7</bold> showed a small
amount of amide bond cleavage as well as oxidation of the quinazolin-4(3<italic>H</italic>)-one ring (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Figure S2</ext-link>). The
2,2- and 3,3-difluorocyclohexan compounds <bold>8</bold> and <bold>9</bold> had moderate to poor whole-cell potency. Met-ID for both <bold>1</bold> and <bold>7</bold> revealed cleavage of the amide bond,
so attempts to hinder the amide through formation of the 1-methyl-,
1-methanol-, and 1-cyano-substituted cyclohexane were explored, all
of which resulted in a complete loss of MIC potency as well as increased
mouse microsomal instability (data not shown). Addition of a methylene
bridge in <bold>1</bold> and <bold>7</bold> to afford <bold>10</bold> and <bold>11</bold> improved MIC potency, further emphasizing the
requirement for bulky lipophilic groups.</p><table-wrap id="tbl2" position="float"><label>Table 2</label><caption><title>Evaluation
R1 (Cyclohexyl) SAR</title></caption><graphic xlink:href="id-2017-002759_0010" id="fx2" position="float"></graphic><graphic xlink:href="id-2017-002759_0011" id="fx3" position="float"></graphic><table-wrap-foot><fn id="t2fn1"><label>a</label><p>Minimum
inhibitory concentration
(MIC) is the minimum concentration required to inhibit >99% growth
of <italic>M. tuberculosis</italic> in liquid culture. Isoniazid
was included as an internal control reference compound (MIC of 0.2
± 0.1 μM).</p></fn><fn id="t2fn2"><label>b</label><p>Intrinsic
clearance (Cli) using
CD1 mouse liver microsomes.</p></fn><fn id="t2fn3"><label>c</label><p>Kinetic aqueous solubility was measured
using laser nephelometry of compounds in 2.5% DMSO.</p></fn></table-wrap-foot></table-wrap><p>There were concerns over the S-linker,
based on previous experience
from whole-cell screening where confirmed hits with similar S-linker
compounds were found to react with glutathione (GSH) both with and
without microsomal activation. GSH trapping on <bold>7</bold>, with
and without human liver microsomes (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Figure S3</ext-link>), showed GSH adducts <bold>12</bold> and <bold>13</bold>, without
microsomal activation. It is presumed that GSH results in cleavage
of the sulfur-quinazolinone <bold>7</bold> linker, to afford <bold>12</bold>, with GSH coupling to the displaced S-linker to afford <bold>13</bold>. Human microsomal oxidation of the quinazolinone ring of <bold>7</bold> was also observed (see <xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>).</p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Metabolite identification of <bold>7</bold> in
a GSH trapping experiment.</p></caption><graphic xlink:href="id-2017-002759_0001" id="gr2" position="float"></graphic></fig><p>While the level of GSH adduct formation for <bold>7</bold> was
relatively low and no HepG2 cytotoxicity was observed, this was considered
a liability of the series as the reactivity did not require microsomal
activation and the ability to predict and quantify the risk of idiosyncratic
adverse drug reactions is limited.<sup><xref ref-type="bibr" rid="ref32">32</xref>,<xref ref-type="bibr" rid="ref33">33</xref></sup> We attempted
to reduce this liability by modifying the linker. <italic>N</italic>-Methylation of the amide and/or the methylene linker to afford <bold>14</bold>, <bold>15</bold>, and <bold>16</bold>, was not tolerated.
The NH-, O-, and CH<sub>2</sub>-linkers were readily prepared to afford <bold>17</bold>, <bold>18</bold>, and <bold>19</bold>. However, all resulted
in a loss of MIC potency (see <xref rid="tbl3" ref-type="other">Table <xref rid="tbl3" ref-type="other">3</xref></xref>). Oxidation of S-atom in compound <bold>1</bold> was
attempted using a variety of conditions and oxidation reagents (for
example, 3-chlorobenzoic acid, potassium permanganate, and Oxone).
However, all attempts failed to deliver the desired sulfone, possibly
as a result of increased reactivity.</p><table-wrap id="tbl3" position="float"><label>Table 3</label><caption><title>Evaluation
of S-Linker SAR</title></caption><graphic xlink:href="id-2017-002759_0012" id="fx4" position="float"></graphic><table-wrap-foot><fn id="t3fn1"><label>a</label><p>Minimum inhibitory concentration
(MIC) is the minimum concentration required to inhibit >99% growth
of <italic>M. tuberculosis</italic> in liquid culture. Isoniazid
was included as an internal control reference compound (MIC of 0.2
± 0.1 μM).</p></fn><fn id="t3fn2"><label>b</label><p>Intrinsic
clearance (Cli) using
CD1 mouse liver microsomes.</p></fn><fn id="t3fn3"><label>c</label><p>Kinetic aqueous solubility was measured
using laser nephelometry of compounds in 2.5% DMSO.</p></fn></table-wrap-foot></table-wrap><p>Changes to the quinazolinone ring
were then explored, starting
with <italic>N</italic>-methylation to afford <bold>20</bold>, which
was not tolerated. Removal of the carbonyl, by synthesis of quinazoline <bold>21</bold> and quinazolin-4-amine <bold>22</bold>, was also not tolerated.
Saturation of the quinazolinone core phenyl afforded <bold>23</bold>, which retained good MIC potency and also resulted in improved solubility,
albeit microsomal stability was poor. The related 5-membered saturated
analogue <bold>24</bold> showed similar MIC potency but with improved
microsomal stability as well as solubility. However, the dimethyl
analogue <bold>25</bold> was less well tolerated, suggesting that
a bulky hydrophobic ring was preferred. The above SAR suggested that
the pyrimidinone core was a key part of the pharmacophore. As human-metID
showed oxidation of the quinazolinone ring of <bold>7</bold>, we synthesized
the presumably more stable fluorine analogue <bold>26</bold> which,
while not as potent as <bold>7</bold>, retained good MIC potency.
However, the solubility was poor, and the mouse microsomal stability
was moderate. In an attempt to improve solubility as well as microsomal
stability, the pyridopyrimidinone <bold>27</bold> was prepared; while
solubility and microsomal stability were improved, the compound had
moderate to weak MIC potency. A summary of the overall whole-cell
SAR for <bold>1</bold>, as well as the effects on both kinetic solubility
and microsomal stability, is shown in <xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref> and in <xref rid="tbl2" ref-type="other">Tables <xref rid="tbl2" ref-type="other">2</xref></xref>–<xref rid="tbl4" ref-type="other">4</xref>.</p><table-wrap id="tbl4" position="float"><label>Table 4</label><caption><title>Evaluation of Quinazolinone Core</title></caption><graphic xlink:href="id-2017-002759_0013" id="fx5" position="float"></graphic><table-wrap-foot><fn id="t4fn1"><label>a</label><p>Minimum
inhibitory concentration
(MIC) is the minimum concentration required to inhibit >99% growth
of <italic>M. tuberculosis</italic> in liquid culture. Isoniazid
was included as an internal control reference compound (MIC of 0.2
± 0.1 μM).</p></fn><fn id="t4fn2"><label>b</label><p>Intrinsic
clearance (Cli) using
CD1 mouse liver microsomes.</p></fn><fn id="t4fn3"><label>c</label><p>Kinetic aqueous solubility was measured
using laser nephelometry of compounds in 2.5% DMSO.</p></fn></table-wrap-foot></table-wrap><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Overview of structure–activity and property
relationships
of the mercapto-quinazolinones.</p></caption><graphic xlink:href="id-2017-002759_0002" id="gr3" position="float"></graphic></fig><p>Pharmacokinetic studies were initiated in order to assess
the potential
for <italic>in vivo</italic> efficacy studies of the 2-mercapto-quinazolinones.
Compound <bold>1</bold>, when dosed as the free base, had reasonable
bioavailability, consistent with its moderate Cli and solubility,
and good permeability (<xref rid="tbl5" ref-type="other">Table <xref rid="tbl5" ref-type="other">5</xref></xref>). Compound <bold>7</bold> showed a similar bioavailability
and exposure profile to <bold>1</bold> (<xref rid="tbl5" ref-type="other">Table <xref rid="tbl5" ref-type="other">5</xref></xref>).</p><table-wrap id="tbl5" position="float"><label>Table 5</label><caption><title>Pharmacokinetic Profiling
of Compounds <bold>1</bold>, <bold>7</bold>, and <bold>11</bold></title></caption><table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col></colgroup><thead><tr><th style="border:none;" align="center"> </th><th style="border:none;" align="center"><bold>1</bold></th><th style="border:none;" align="center"><bold>7</bold></th><th style="border:none;" align="center"><bold>11</bold></th></tr></thead><tbody><tr><td style="border:none;" align="left">GAST MIC
(μM)</td><td style="border:none;" align="left">0.3 (95 ng/mL)</td><td style="border:none;" align="left">0.4 (141 ng/mL)</td><td style="border:none;" align="left">0.8 (294 ng/mL)</td></tr><tr><td style="border:none;" align="left">7H9-ADC MIC (μM)</td><td style="border:none;" align="left">0.6 (190 ng/mL)</td><td style="border:none;" align="left">0.4 (141 ng/mL)</td><td style="border:none;" align="left">0.8 (294 ng/mL)</td></tr><tr><td style="border:none;" align="left">HepG2 IC<sub>50</sub> (μM)</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">>50</td></tr><tr><td style="border:none;" align="left">measured CHI-LogD</td><td style="border:none;" align="left">1.8</td><td style="border:none;" align="left">1.5</td><td style="border:none;" align="left">1.6</td></tr><tr><td rowspan="2" style="border:none;" align="left">microsomal
clearance (mL/min/g)</td><td style="border:none;" align="left">mouse 2.3</td><td rowspan="2" style="border:none;" align="left">mouse 1.4</td><td rowspan="2" style="border:none;" align="left">mouse 4.8</td></tr><tr><td style="border:none;" align="left">human <0.5</td></tr><tr><td style="border:none;" align="left">MW<xref rid="t5fn1" ref-type="table-fn">a</xref></td><td style="border:none;" align="left">317</td><td style="border:none;" align="left">353</td><td style="border:none;" align="left">367</td></tr><tr><td style="border:none;" align="left">cLogP<xref rid="t5fn1" ref-type="table-fn">a</xref>/cLogD<xref rid="t5fn1" ref-type="table-fn">a</xref></td><td style="border:none;" align="left">2.3/2.3</td><td style="border:none;" align="left">2.3/2.3</td><td style="border:none;" align="left">2.7/2.7</td></tr><tr><td style="border:none;" align="left">TPSA<xref rid="t5fn1" ref-type="table-fn">a</xref></td><td style="border:none;" align="left">75</td><td style="border:none;" align="left">75</td><td style="border:none;" align="left">75</td></tr><tr><td style="border:none;" align="left">PAMPA (nm/s)</td><td style="border:none;" align="left">83</td><td style="border:none;" align="left">64</td><td style="border:none;" align="left">65</td></tr><tr><td style="border:none;" align="left">kinetic solubility (μM)<xref rid="t5fn2" ref-type="table-fn">b</xref></td><td style="border:none;" align="left">83 (free base)</td><td style="border:none;" align="left">111 (HCl
salt)</td><td style="border:none;" align="left">>250</td></tr><tr><td style="border:none;" align="left">C57 mouse PK
at 3 iv and 10 po (mg/kg)</td><td style="border:none;" align="left">free base</td><td style="border:none;" align="left">HCl salt</td><td style="border:none;" align="left">free
base</td></tr><tr><td style="border:none;" align="left"><italic>C</italic><sub>max</sub> (ng/mL)</td><td style="border:none;" align="left">748</td><td style="border:none;" align="left">399</td><td style="border:none;" align="left">1112</td></tr><tr><td style="border:none;" align="left"><italic>T</italic><sub>1/2</sub> (h)</td><td style="border:none;" align="left">1.5</td><td style="border:none;" align="left">1.3</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">AUC<sub>0–8h</sub> (ng·min/mL)</td><td style="border:none;" align="left">156 800</td><td style="border:none;" align="left">67 085</td><td style="border:none;" align="left">128 309</td></tr><tr><td style="border:none;" align="left">Cl<sub>b</sub> (mL/min/kg)</td><td style="border:none;" align="left">23</td><td style="border:none;" align="left">39</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">Vd<sub>ss</sub> (L/kg)</td><td style="border:none;" align="left">0.8</td><td style="border:none;" align="left">1</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">% F</td><td style="border:none;" align="left">46</td><td style="border:none;" align="left">29</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">PPB (% unbound)</td><td style="border:none;" align="left">14</td><td style="border:none;" align="left">13</td><td style="border:none;" align="left">21</td></tr></tbody></table><table-wrap-foot><fn id="t5fn1"><label>a</label><p>Calculated using StarDrop (<uri xlink:href="http://www.optibrium.com">http://www.optibrium.com</uri>).</p></fn><fn id="t5fn2"><label>b</label><p>Kinetic aqueous solubility was measured
using laser nephelometry of compounds in 2.5% DMSO.</p></fn></table-wrap-foot></table-wrap><p>As compound <bold>7</bold> had comparable
MIC potency, mouse microsomal
stability, and pharmacokinetic profile to <bold>1</bold>, a dose linearity
study was conducted to evaluate if exposures above the MIC could be
achieved. However, no dose linearity was observed between the 30 and
100 mg/kg doses, determined by the area under the curve (AUC) comparison
(<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Figure S4</ext-link>). This may be a consequence
of solubility limited absorption at the highest dose. Compound <bold>11</bold> demonstrated better kinetic solubility and comparable MIC
to compounds <bold>1</bold> and <bold>7</bold>, yet despite an improved <italic>C</italic><sub>max</sub> and <italic>T</italic><sub>max</sub> compared
to compound <bold>1</bold>, likely driven by its improved kinetic
solubility, it had a lower exposure (AUC) over time (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Figure S5</ext-link> and <xref rid="tbl5" ref-type="other">Table <xref rid="tbl5" ref-type="other">5</xref></xref>), likely a consequence of it is higher clearance (mouse Cli
4.7 mL/min/g versus 2.3 or 1.4 for compounds <bold>1</bold> and <bold>7</bold>, respectively). The modest compound exposures upon oral
dosing, combined with the lack of <italic>ex vivo</italic> intramacrophage
efficacy (<italic>vide infra</italic>), suggested that efficacy studies
of these compounds in infected mice were unlikely to validate the
target/drug candidate pair in an animal model where the disease is
macrophage-based. As such, an efficacy experiment was not performed.</p></sec><sec id="sec2.4"><title>Biological Characterization of the Mercapto-Quinazolinones</title><p>Previous work on a mercapto-quinazolinone had suggested that compound <bold>1</bold> might target NDH-2 as evidenced by promoter mutations in
the nonessential <italic>ndhA</italic> gene encoding an orthologue
of the type II NADH dehydrogenase.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> We
similarly identified promoter mutations for <italic>ndhA</italic> but
were not able to identify polymorphisms in the apparently essential <italic>ndh</italic> (Rv1854c) (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Table S1</ext-link>) suggesting
either that mutations were deleterious for enzyme function or that
single amino acid mutations alone might not sufficiently decrease
affinity of this putative inhibitor. The upregulation of <italic>ndhA</italic> could serve to compensate for loss of NDH-2 function or could serve
to bind excess inhibitor in the cell. It is intriguing that <italic>ndh</italic> promoter mutations were not identified possibly because
this gene is not readily upregulated by single nucleotide substitutions
in its promoter. We also identified mutations in <italic>Rv0678</italic> (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Table S1</ext-link>) encoding a transcriptional
repressor that has previously been demonstrated to control expression
of the MmpS5-MmpL5 transporter and has been implicated in resistance
to bedaquiline and clofazimine.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> In accordance
with the predicted essential role of NDH-2 as complex I in oxidative
phosphorylation,<sup><xref ref-type="bibr" rid="ref16">16</xref></sup> treatment of <italic>MTb</italic> with compound <bold>1</bold> resulted in depletion of
cellular ATP levels, a phenomenon also observed with bedaquiline (TM207),
an inhibitor of the ATP synthase, as well as EU306, an inhibitor of
the cytochrome <italic>bc</italic><sub>1</sub> complex<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> but not with inhibitors of macromolecular biosynthesis
including INH (cell wall), Rif (RNA polymerase), and Kan (protein
synthesis) (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>).</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Mercapto-quinazolinones deplete cellular ATP levels in <italic>MTb</italic>. ATP was measured using the BacTiter Glo assay after
24 h of drug exposure and expressed as a fraction of the drug-free
vehicle control levels.</p></caption><graphic xlink:href="id-2017-002759_0003" id="gr4" position="float"></graphic></fig><p>We predicted that inhibition of NDH-2 would result in a more
severe
phenotype on a mutant lacking the second copy of the type II NADH
dehydrogenase. Indeed, while treatment of wild-type <italic>MTb</italic> H37Rv at MIC concentrations could not fully suppress growth of cells
over 7 days of treatment, similar concentrations of compound resulted
in an almost 2-log kill of a Δ<italic>ndhA</italic> mutant (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>). Despite the higher
vulnerability of the <italic>ndhA</italic> mutant to killing by compound <bold>1</bold>, the MIC to this strain, as well as a <italic>nuoG</italic> deletion mutant lacking a functional type I NADH dehydrogenase,
was indistinguishable from WT cells (results not shown). Inhibition
of anaerobic nonreplicating cells with compound <bold>1</bold> did
not affect viability (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Figure S6</ext-link>) which
contrasts with the anaerobic cidal activity of other NDH-2 inhibitors
including the phenothiazines<sup><xref ref-type="bibr" rid="ref16">16</xref></sup> and a recently
described quinolone scaffold,<sup><xref ref-type="bibr" rid="ref19">19</xref></sup> possibly
due to differences in compound access to the target under anaerobic
conditions or to growth-dependent differences in compound modification
by <italic>MTb</italic>. In addition, this compound lacked activity
against <italic>MTb</italic> growing in infected macrophages (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">Figure S7</ext-link>) possibly due GSH-catalyzed compound
inactivation or inability to access the mycobacterial phagosome.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p><italic>Mtb</italic> lacking the alternative type II NADH dehydrogenase
encoded by <italic>ndhA</italic> was more susceptible to compound <bold>1</bold>. Colony-forming units (CFU) after 7 days of treatment as
compared to CFU at start of drug treatment (inoculum). Unpaired <italic>t</italic> test comparison between WT and Δ<italic>ndhA</italic> at 0.5 μM compound <bold>1</bold>: two-tailed <italic>P</italic>-value = 0.0023.</p></caption><graphic xlink:href="id-2017-002759_0004" id="gr5" position="float"></graphic></fig><p>The effect of the mercapto-quinazolinones
on <italic>MTb</italic> bioenergetics in whole cells was further demonstrated
by analysis
of the mycobacterial oxygen consumption rate (OCR) (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>). Addition of compounds <bold>1</bold> and <bold>7</bold> had a minimal effect on basal <italic>MTb</italic> OCR levels over ∼45 min. The same results were
obtained in the presence of glucose or palmitate as carbon sources
(<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>A,B). Uncoupling
of <italic>MTb</italic> oxidative phosphorylation (OXPHOS) with carbonyl
cyanide <italic>m</italic>-chlorophenyl hydrazone (CCCP) significantly
diminished maximal respiration in the presence of the two inhibitors
compared to the untreated control. These findings suggest an energy
generation pathway common to both fatty acid and glucose oxidation
as the target of the compounds. Previous studies<sup><xref ref-type="bibr" rid="ref21">21</xref>,<xref ref-type="bibr" rid="ref36">36</xref></sup> have shown that <italic>MTb</italic> can rapidly reroute electron
flux to overcome inhibition of cytochrome <italic>bc</italic><sub>1</sub>–<italic>aa</italic><sub>3</sub>, cytochrome <italic>bd</italic>, or ATP synthase, resulting in enhanced respiration.
Therefore, we speculated that the target of compounds <bold>1</bold> and <bold>7</bold> was at the point of entry into the electron transport
chain (ETC), that is, Complex 1 (NADH dehydrogenase) or Complex II
(succinate dehydrogenase). To test this prediction, ATP production
was measured in <italic>MTb</italic> inverted membrane vesicles (IMVs)
in the presence of the mercapto-quinazolinones (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>C,D). Notably, ATP production was significantly
inhibited when NADH was provided as electron donor but not when succinate
was the source of reducing equivalents, suggesting that the site of
inhibition was NADH dehydrogenase. The mechanism by which the compounds
interfere with NADH dehydrogenase function differs from clofazimine
and the quinolinequinones, which have been proposed to interfere with
electron transport by activating NDH-2 resulting in production of
reactive oxygen species.<sup><xref ref-type="bibr" rid="ref18">18</xref>,<xref ref-type="bibr" rid="ref20">20</xref></sup></p><fig id="fig5" position="float"><label>Figure 5</label><caption><p>Mercapto-quinazolinones
target Complex I of the MTb ETC. At the
times indicated by the dotted vertical lines, either glucose (A) or
palmitate (B) was added to MTb as carbon source (CS), followed by
the mercapto-quinazolinones (Compound) and, last, the uncoupler CCCP
to induce maximal respiration. The mercapto-quinazolinones diminished
MTb’s uncoupling capacity significantly compared to that of
the untreated (UT) control. The oxygen consumption rate (OCR) is reported
as a percentage of baseline values. Mercapto-quinazolinones inhibit
ATP production in the presence of NADH but not succinate. (C, D) ATP
production in inverted membrane vesicles (IMVs) in the presence of
NADH or succinate as electron donors after 30 min (C) and over 60
min (D). <italic>P</italic>-values were calculated by one-way ANOVA
using GraphPad Prism 7.02.</p></caption><graphic xlink:href="id-2017-002759_0005" id="gr6" position="float"></graphic></fig><p>To verify that NDH-2 was indeed the target for this class
of mercapto-quinazolinones,
we expressed <italic>MTb</italic> NDH-2 (MtNdh) encoded by the predicted
essential <italic>ndh</italic> in <italic>E. coli</italic> as
a recombinant MBP-fusion protein and purified the protein to near
homogeneity via amylose-resin affinity chromatography and gel filtration.
The recombinant enzyme was highly active with coenzyme Q2 (ubiquinone
Q-2) and NADH as substrates, delivering steady-state kinetic parameters
similar to previously published results.<sup><xref ref-type="bibr" rid="ref37">37</xref></sup> Quinazolinones <bold>1</bold>–<bold>3</bold>, <bold>7</bold>, and <bold>11</bold> were found to have submicromolar IC<sub>50</sub> values against the MtNdh (<xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>). Compounds <bold>1</bold>, <bold>7</bold>, and <bold>11</bold> showed the highest potency, with IC<sub>50</sub> values
from 7 to 26 nM, values 3-orders of magnitude lower than that for
the previously characterized NDH-2 inhibitor chlorpromazine (CPZ;
10–25 μM; <xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>)<sup><xref ref-type="bibr" rid="ref24">24</xref></sup> but superior to the quinolinyl
pyrimidines discovered in a target-based screening effort against
the <italic>MTb</italic> NDH-2.<sup><xref ref-type="bibr" rid="ref38">38</xref></sup> No time-dependent
inhibition was observed for these compounds. The mode of inhibition
against MtNdh was further investigated using compound <bold>1</bold>: double reciprocal plots of enzyme activity under varying concentrations
of <bold>1</bold> and either Q2 or NADH suggested patterns indicative
of a noncompetitive mode of inhibition with both substrates (<italic>K</italic><sub>i</sub> values of 30–40 nM), although the effect
on slope was less evident when varying Q2 (<xref rid="fig7" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>). This is similar to previous studies with
a thiophenazine-based NDH-2 inhibitor<sup><xref ref-type="bibr" rid="ref39">39</xref></sup> and suggests that inhibition occurs through binding of the compound
at an allosteric site, independent of the main substrate binding pockets.</p><fig id="fig6" position="float"><label>Figure 6</label><caption><p>Inhibition
of recombinant MBP-MtNdh by select quinazolinones. Steady-state
enzyme activity was measured by monitoring absorbance changes at 340
nm due to NADH oxidation, as described in the <xref rid="sec3" ref-type="other">Experimental
Section</xref>. NADH and Q2 were fixed at 250 and 40 μM, respectively.
Each data point is the mean ± SD of at least 3 independent measurements.
Inset table shows calculated IC<sub>50</sub> values.</p></caption><graphic xlink:href="id-2017-002759_0006" id="gr7" position="float"></graphic></fig><fig id="fig7" position="float"><label>Figure 7</label><caption><p>Double-reciprocal plot of inhibition kinetics of MBP-MtNdh
by <bold>1</bold>. NADH (left) or Q2 (right) concentrations were varied,
in
the presence of fixed concentrations of 40 μM Q2 (A) or 250
μM NADH (B). Data points are the average of at least 3 independent
measurements. Solid lines represent best-fit values of the data to
the general Michaelis–Menten reversible inhibition equation,
using nonlinear regression analysis.</p></caption><graphic xlink:href="id-2017-002759_0007" id="gr8" position="float"></graphic></fig><p>In conclusion, the nonproton pumping type II NADH dehydrogenase
(NDH-2) has been shown to play a critical role in the respiratory
metabolism of bacteria. The 2-mercapto-quinazolinone scaffold is a
potent inhibitor of the mycobacterial NDH-2, and although in this
study <italic>MTb</italic> whole-cell SAR only obtained limited improvement
in potency, this information, together with the recently disclosed
structural information on the enzyme from the bacterium <italic>Caldalkalibacillus
thermarum</italic>,<sup><xref ref-type="bibr" rid="ref40">40</xref></sup> will aid in the future
rational structure-based development of new and improved NDH-2 inhibitors.
Critical for any structure-guided inhibitor design is an understanding
of the ligand-binding pocket of the NDH-2 protein. The low amino acid
identity between the <italic>C. thermarum</italic> and <italic>MTb</italic> NDH-2 proteins (30%) combined with the finding that
this inhibitor likely binds to an allosteric site with no allosteric
ligand-binding sites identified on the existing bacterial NDH-2 structure<sup><xref ref-type="bibr" rid="ref40">40</xref></sup> suggests that cocrystallization of the compound
with the <italic>MTb</italic> NDH-2 protein will be essential to facilitate
rational drug design. During revision of this paper, the SAR of a
similar mercapto-quinazolinone scaffold<sup><xref ref-type="bibr" rid="ref41">41</xref></sup> was reported with a complementary paper describing the importance
of the mycobacterial type II NADH dehydrogenase despite the nonessentiality
of the individual <italic>ndh</italic> and <italic>ndhA</italic> genes.<sup><xref ref-type="bibr" rid="ref42">42</xref></sup></p></sec></sec><sec id="sec3"><title>Experimental Section</title><sec id="sec3.1"><title>Chemistry Summary</title><p>All commercially available reagents,
solvents, and starting materials were purchased from Aldrich Chemical
Co. (UK). Where necessary, a Biotage FLASH 25+ column chromatography
system was used to purify mixtures; reagent-grade solvents used for
chromatography were purchased from Fisher Scientific (UK), and flash
column chromatography silica cartridges were obtained from Biotage
(UK). Analytical thin-layer chromatography (TLC) was performed on
precoated TLC plates (layer 0.20 mm silica gel 60 with fluorescent
indicator UV254, from Merck). Developed plates were air-dried and
analyzed under a UV lamp (UV 254/365 nm). Microwave irradiation was
conducted using a BIOTAGE INITIATOR unit. The machine consists of
a continuous focused microwave power delivery system with operator-selectable
power output (0–400 W at 2.45 GHz). All <sup>1</sup>H and <sup>13</sup>C NMR spectra were recorded on a Bruker ARX-500 spectrometer
(500 and 300 MHz for <sup>1</sup>H and <sup>13</sup>C NMR, respectively).
Chemical shifts (δ) are reported in ppm relative to the residual
solvent peak or internal standard (tetramethylsilane), and coupling
constants (<italic>J</italic>) are reported in hertz (Hz). Data are
reported as follows: chemical shift, multiplicity (br = broad, s =
singlet, d = doublet, t = triplet, m = multiplet), and integration.
LC–MS analyses were performed with either an Agilent HPLC 1100
series connected to a Bruker Daltonics MicrOTOF or an Agilent Technologies
1200 series HPLC connected to an Agilent Technologies 6130 quadrupole
spectrometer, where both instruments were connected to an Agilent
diode array detector. LC–MS chromatographic separations were
conducted with a Waters X bridge C18 column, 50 mm × 2.1 mm,
3.5 μm particle size; mobile phase of water/acetonitrile + 0.1%
HCOOH or water/acetonitrile + 0.1% NH<sub>3</sub>; linear gradient
from 80:20 to 5:95 over 3.5 min and then held for 1.5 min; flow rate
of 0.5 mL·min<sup>–1</sup>. All assay compounds had a
measured purity of ≥95% (by TIC and UV) as determined using
this analytical LC–MS system. High resolution electrospray
measurements were performed on a Bruker Daltonics MicrOTOF mass spectrometer.</p><sec id="sec3.1.1"><title>General Method for the Preparation of Amide
Coupling (<bold>1</bold>, <bold>4</bold>–<bold>11</bold>, <bold>14</bold>–<bold>16</bold>, <bold>19</bold>–<bold>27</bold>)</title><p>To a
solution of 2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic acid
(1.0 equiv) in a 1:1 ratio of acetonitrile (1.5 mL) and DMF (1.5 mL)
were added EDC·HCl (1.0 equiv), HOAT (1.2 equiv), or HATU (1.0
equiv) and primary/secondary amine (1.0 equiv), and the mixture was
stirred for 30 min; DIPEA (2.0 equiv) was added to the reaction mixture
and stirred at room temperature overnight. If a precipitate had formed
this was filtered off, washed with acetonitrile, and air-dried. If
the reaction mixture had remained homogeneous, the solution was purified
directly by preparative HPLC under acidic conditions, using 0.1% HCOOH
in water/acetonitrile (5–95%) gradient elution to afford the
product.</p><sec id="sec3.1.1.1"><title><italic>N</italic>-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>1</bold>)</title><p>To a solution of acid of 2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic
acid (<bold>30</bold>) (500 mg, 2.1 mmol, 1.0 equiv) in 1:1 ratio
of acetonitrile (1.5 mL) and DMF (1.5 mL) were added EDC·HCl
(1.2 equiv), HOAT (1.2 equiv), or HATU (804 mg, 2.1 mmol, 1.0 equiv)
in DCM (3 mL) and cyclohexylamine (209 mg, 2.1 mmol, 1.0 equiv), and
the mixture was stirred for 30 min; DIPEA (0.73 mL, 2.1 mmol, 2.0
equiv) was added to the reaction mixture and stirred at room temperature
overnight. The reaction mixture was purified by preparative HPLC under
acidic conditions, using 0.1% HCOOH in water/acetonitrile (5–95%)
gradient elution to afford a white solid of <italic>N</italic>-cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
as a white powder (450 mg, 66% yield), C<sub>16</sub>H<sub>19</sub>N<sub>3</sub>O<sub>2</sub>S, MW 317.41; <sup>1</sup>H NMR (500 MHz,
DMSO) δ 12.66 (brs, 1H), 8.14 (d, <italic>J</italic> = 7.9 Hz,
1H), 8.04 (dd, <italic>J</italic> = 1.3, 7.9 Hz, 1H), 7.80–7.76
(m, 1H), 7.50 (d, <italic>J</italic> = 7.9 Hz, 1H), 7.45–7.41
(m, 1H), 3.92 (s, 2H), 3.59–3.51 (m, 1H), 1.76–1.64
(m, 4H), 1.55 (dd, <italic>J</italic> = 3.5, 9.1 Hz, 1H), 1.30–1.12
(m, 5H). LCMS <italic>m</italic>/<italic>z</italic> 318.12 [M + 1]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M +
H]<sup>+</sup> calcd for C<sub>16</sub>H<sub>20</sub>N<sub>3</sub>O<sub>2</sub>S, 318.1270; found, 318.1284.</p></sec><sec id="sec3.1.1.2"><title><italic>N</italic>-Cycloheptyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>4</bold>)</title><p>White powder (21 mg, 23% yield), C<sub>17</sub>H<sub>21</sub>N<sub>3</sub>O<sub>2</sub>S, MW 331.43; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.65 (brs, 1H), 8.26 (d, <italic>J</italic> = 7.8 Hz, 1H), 8.02 (dd, <italic>J</italic> = 1.3, 7.9 Hz, 1H),
7.77–7.72 (m, 1H), 7.48–7.37 (m, 2H), 3.88 (s, 2H),
3.73 (ddd, <italic>J</italic> = 4.2, 8.6, 16.9 Hz, 1H), 1.60–1.34
(m, 12H). LCMS <italic>m</italic>/<italic>z</italic> 332.14 [M + 1]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M +
H]<sup>+</sup> calcd for C<sub>17</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub>S, 332.1427; found, 332.1439.</p></sec><sec id="sec3.1.1.3"><title><italic>N</italic>-Cyclopentyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>5</bold>)</title><p>White powder (39 mg, 24% yield), C<sub>15</sub>H<sub>17</sub>N<sub>3</sub>O<sub>2</sub>S, MW 303.38; <sup>1</sup>H NMR (500 MHz, DMSO) δ 8.23 (d, <italic>J</italic> = 7.1 Hz,
1H), 8.04 (dd, <italic>J</italic> = 1.3, 8.0 Hz, 1H), 7.79–7.75
(m, 1H), 7.49 (d, <italic>J</italic> = 8.0 Hz, 1H), 7.45–7.41
(m, 1H), 4.03–3.98 (m, 1H), 3.91 (s, 2H), 1.82–1.76
(m, 2H), 1.67–1.62 (m, 2H), 1.53–1.36 (m, 4H). LCMS <italic>m</italic>/<italic>z</italic> 304.12 [M + H]<sup>+</sup> 100%. HRMS
(<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd
for C<sub>15</sub>H<sub>18</sub>N<sub>3</sub>O<sub>2</sub>S, 304.1114;
found, 304.1107.</p></sec><sec id="sec3.1.1.4"><title><italic>N</italic>-Cyclobutyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>6</bold>)</title><p>White powder (76 mg, 66% yield), C<sub>14</sub>H<sub>15</sub>N<sub>3</sub>O<sub>2</sub>S, MW 289.35; <sup>1</sup>H NMR (400 MHz, DMSO) δ 12.67 (s, 1H), 8.54 (d, <italic>J</italic> = 7.7 Hz, 1H), 8.05–8.02 (m, 1H), 7.81–7.75 (m, 1H),
7.51 (d, <italic>J</italic> = 8.1 Hz, 1H), 7.43 (t, <italic>J</italic> = 7.5 Hz, 1H), 4.19 (dd, <italic>J</italic> = 8.1, 16.1 Hz, 1H),
3.91 (s, 2H), 2.20–2.10 (m, 2H), 1.97–1.85 (m, 2H),
1.67–1.57 (m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 290.04 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd for C<sub>14</sub>H<sub>16</sub>N<sub>3</sub>O<sub>2</sub>S, 290.0957; found, 290.0962.</p></sec><sec id="sec3.1.1.5"><title><italic>N</italic>-(4,4-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>7</bold>)</title><p>To a solution of 2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic
acid <bold>30</bold> (108 mg, 0.4 mmol, 1.0 equiv) in DCM (3 mL) were
added HATU (160 mg, 0.4 mmol, 1.0 equiv) and 4,4-difluorocyclohexan-1-amine
(57 mg, 0.4 mmol, 1.0 equiv) and the mixture was stirred for 30 min;
DIPEA (0.074 mL, 0.4 mmol, 2.0 equiv) was added to the reaction mixture
and stirred at room temperature overnight. The reaction mixture was
purified by preparative HPLC under acidic conditions, using 0.1% HCOOH
in water/acetonitrile (5–95%) gradient elution to afford a
white solid of <italic>N</italic>-(4,4-difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
as a white powder <bold>7</bold> (115 mg, 75% yield), C<sub>16</sub>H<sub>17</sub>F<sub>2</sub>N<sub>3</sub>O<sub>2</sub>S, MW 353.39; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.64 (s, 1H), 8.39 (d, <italic>J</italic> = 6.9 Hz, 1H), 8.02 (dd, <italic>J</italic> = 1.2, 7.9
Hz, 1H), 7.77–7.72 (m, 1H), 7.46 (d, <italic>J</italic> = 7.9
Hz, 1H), 7.40 (t, <italic>J</italic> = 7.5 Hz, 1H), 3.90 (s, 2H),
3.77 (t, <italic>J</italic> = 4.0 Hz, 1H), 2.04–1.75 (m, 6H),
1.56–1.47 (m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 354.14 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd for C<sub>16</sub>H<sub>18</sub>F<sub>2</sub>N<sub>3</sub>O<sub>2</sub>S, 354.1082; found, 354.1091.</p></sec><sec id="sec3.1.1.6"><title><italic>N</italic>-(4,4-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
HCl Salt Preparation (<bold>7</bold>)</title><p><italic>N</italic>-(4,4-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(60 mg, 0.17 mmol, 1.0 equiv) in ethyl acetate (10 mL) and ethanol
(3 mL) was heated up to 80 °C, until dissolved (90%). The solution
was cooled immediately, followed by the addition of 2 M HCl in diethyl
ether (1 mL, 747 mg, 20.49 mmol, 120 equiv) and stirred for a further
15 min, where white precipitates were formed. The precipitate was
filtered and dried to afford the <italic>N</italic>-(4,4-difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
HCl salt (50 mg, 75% yield), C<sub>16</sub>H<sub>17</sub>F<sub>2</sub>N<sub>3</sub>O<sub>2</sub>S·HCl, MW 389.85; <sup>1</sup>H NMR
(500 MHz, DMSO) δ 8.32 (d, <italic>J</italic> = 7.6 Hz, 1H),
8.04 (dd, <italic>J</italic> = 1.2, 7.9 Hz, 1H), 7.80–7.76
(m, 1H), 7.49 (d, <italic>J</italic> = 7.8 Hz, 1H), 7.45–7.41
(m, 1H), 3.95 (s, 2H), 2.52–2.50 (m, 1H), 2.05–1.99
(m, 2H), 1.97–1.76 (m, 4H), 1.57–1.48 (m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 354.14 [M + H]<sup>+</sup> 100%. HRMS
(<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd
for C<sub>16</sub>H<sub>18</sub>F<sub>2</sub>N<sub>3</sub>O<sub>2</sub>S, 354.1082; found, 354.1091.</p></sec><sec id="sec3.1.1.7"><title><italic>N</italic>-(2,2-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>8</bold>)</title><p>White powder (93 mg, 62% yield), C<sub>16</sub>H<sub>17</sub>F<sub>2</sub>N<sub>3</sub>O<sub>3</sub>S<sub>2</sub>, MW 353.39; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.64 (s,
1H), 8.46 (d, <italic>J</italic> = 9.0 Hz, 1H), 8.04 (d, <italic>J</italic> = 7.9 Hz, 1H), 7.79–7.75 (m, 1H), 7.50 (d, <italic>J</italic> = 8.2 Hz, 1H), 7.43 (dd, <italic>J</italic> = 7.5 Hz, 1H), 4.03–4.01
(m, 3H), 2.08–2.03 (m, 1H), 1.86–1.63 (m, 4H), 1.49–1.38
(m, 3H). LCMS <italic>m</italic>/<italic>z</italic> 354.10 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M +
H]<sup>+</sup> calcd for C<sub>16</sub>H<sub>18</sub>F<sub>2</sub>N<sub>3</sub>O<sub>3</sub>S<sub>2</sub>, 354.1082; found, 354.1094.</p></sec><sec id="sec3.1.1.8"><title><italic>N</italic>-(3,3-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>9</bold>)</title><p>White powder (83 mg, 55% yield), C<sub>16</sub>H<sub>17</sub>F<sub>2</sub>N<sub>3</sub>O<sub>3</sub>S<sub>2</sub>, MW 353.39; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.61 (brs,
1H), 8.31 (d, <italic>J</italic> = 7.9 Hz, 1H), 8.04 (dd, <italic>J</italic> = 1.3, 7.9 Hz, 1H), 7.80–7.76 (m, 1H), 7.50 (d, <italic>J</italic> = 8.2 Hz, 1H), 7.43 (t, <italic>J</italic> = 7.5 Hz, 1H),
3.94 (s, 2H), 3.91–3.75 (m, 1H), 2.21–2.15 (m, 1H),
1.98–1.95 (m, 1H), 1.81–1.68 (m, 4H), 1.45–1.24
(m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 354.10 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M +
H]<sup>+</sup> calcd for C<sub>16</sub>H<sub>18</sub>F<sub>2</sub>N<sub>3</sub>O<sub>3</sub>S<sub>2</sub>, 354.1082; found, 354.1095.</p></sec><sec id="sec3.1.1.9"><title><italic>N</italic>-(Cyclohexylmethyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>10</bold>)</title><p>White powder (34 mg, 25% yield), C<sub>17</sub>H<sub>21</sub>N<sub>3</sub>O<sub>2</sub>S, MW 331.43; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.64 (s, 1H), 8.16 (s, 1H),
8.05–8.02 (m, 1H), 7.79–7.75 (m, 1H), 7.50 (dd, <italic>J</italic> = 3.1, 8.1 Hz, 1H), 7.44–7.40 (m, 1H), 3.93 (d, <italic>J</italic> = 2.5 Hz, 2H), 2.95–2.92 (m, 2H), 1.67–1.58
(m, 5H), 1.38–1.34 (m, 1H), 1.12–1.07 (m, 3H), 0.87–0.79
(m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 332.21 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M +
H]<sup>+</sup> calcd for C<sub>17</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub>S, 332.1427; found, 332.1431.</p></sec><sec id="sec3.1.1.10"><title><italic>N</italic>-((4,4-Difluorocyclohexyl)methyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>11</bold>)</title><p>White powder (120 mg, 77% yield), C<sub>17</sub>H<sub>19</sub>N<sub>3</sub>F<sub>2</sub>O<sub>2</sub>S, MW
367.41; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.64 (s, 1H), 8.27
(t, <italic>J</italic> = 5.8 Hz, 1H), 8.04 (dd, <italic>J</italic> = 1.3, 7.9 Hz, 1H), 7.80–7.76 (m, 1H), 7.50 (d, <italic>J</italic> = 8.0 Hz, 1H), 7.45–7.42 (m, 1H), 3.95 (s, 2H), 3.02 (t, <italic>J</italic> = 6.4 Hz, 2H), 1.96–1.87 (m, 2H), 1.74–1.52
(m, 5H), 1.17–1.08 (m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 368.01 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd for C<sub>17</sub>H<sub>20</sub>N<sub>3</sub>F<sub>2</sub>O<sub>2</sub>S, 368.1238;
found, 368.1255.</p></sec><sec id="sec3.1.1.11"><title><italic>N</italic>-Cyclohexyl-<italic>N</italic>-methyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>14</bold>)</title><p>White powder (35 mg, 25% yield), C<sub>17</sub>H<sub>21</sub>N<sub>3</sub>O<sub>2</sub>S, MW 331.43; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.60 (brs, 1H), 8.04 (dd, <italic>J</italic> = 1.6, 7.9 Hz, 1H), 7.80–7.74 (m, 1H), 7.49–7.40
(m, 2H), 4.34 (s, 1H), 4.23 (s, 1H), 3.80–3.76 (m, 1H), 3.01
(s, 2H), 2.74 (s, 1H), 1.76 (t, <italic>J</italic> = 12.0 Hz, 3H),
1.62–1.52 (m, 2H), 1.52–1.43 (m, 2H), 1.38–1.23
(m, 2H), 1.15–1.05 (m, 1H). LCMS <italic>m</italic>/<italic>z</italic> 332.15 [M + H]<sup>+</sup>. HRMS (<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd for C<sub>17</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub>S, 332.1427; found, 332.1440.</p></sec><sec id="sec3.1.1.12"><title>2-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)propanoic
Acid (<bold>56</bold>)</title><p>To a solution of 2-thioxo-2,3-dihydroquinazolin-4(1<italic>H</italic>)-one <bold>29</bold> (500 mg, 2.8 mmol, 1.0 equiv) were
added 2-bromopropanoic acid (557 mg, 3.6 mmol, 1.3 equiv) and triethylamine
(2.3 mL, 16.8 mmol, 6.0 equiv) in DMF (5 mL), and the mixture was
stirred for 12 h at 75 °C. Once the reaction has completed, it
was poured onto crushed ice and acidified with 1 N HCl, where the
product precipitated out of the solution. The precipitate was filtered
and dried to yield 2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)propanoic
acid <bold>56</bold> (548 mg, 78% yield), C<sub>11</sub>H<sub>10</sub>N<sub>2</sub>O<sub>3</sub>S, MW 250.27; <sup>1</sup>H NMR (500 MHz,
DMSO) δ 12.66 (s, 1H), 8.04 (q, <italic>J</italic> = 3.10 Hz,
1H), 7.70 (m, 1H), 7.50 (d, <italic>J</italic> = 8.00 Hz, 1H), 7.44
(m, 1H), 4.57 (q, <italic>J</italic> = 7.3 Hz, 2H), 1.55 (d, <italic>J</italic> = 7.30 Hz, 3H). LCMS <italic>m</italic>/<italic>z</italic> 251.00 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.13"><title><italic>N</italic>-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)propanamide
(<bold>15</bold>)</title><p>White powder (45 mg, 35% yield), C<sub>17</sub>H<sub>21</sub>N<sub>3</sub>O<sub>2</sub>S, MW 331.43; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.59 (brs, 1H), 8.17 (d, <italic>J</italic> = 7.7 Hz, 1H), 8.04 (dd, <italic>J</italic> = 1.4, 7.9
Hz, 1H), 7.81–7.77 (m, 1H), 7.53 (d, <italic>J</italic> = 8.2
Hz, 1H), 7.45–7.42 (m, 1H), 4.54 (q, <italic>J</italic> = 7.0
Hz, 1H), 3.56–3.48 (m, 1H), 1.76–1.61 (m, 4H), 1.53–1.50
(m, 4H), 1.29–1.10 (m, 5H). LCMS <italic>m</italic>/<italic>z</italic> 332.10 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd for C<sub>17</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub>S, 332.1427; found, 332.1432.</p></sec><sec id="sec3.1.1.14"><title><italic>N</italic>-Cyclohexyl-<italic>N</italic>-methyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)propanamide
(<bold>16</bold>)</title><p>White powder (40 mg, 29% yield), C<sub>18</sub>H<sub>23</sub>N<sub>3</sub>O<sub>2</sub>S, MW 345.46; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.60 (brs, 1H), 8.07–8.02
(m, 1H), 7.82–7.75 (m, 1H), 7.50–7.41 (m, 2H), 4.24–4.18
(m, 1H), 3.05 (s, 2H), 2.75 (s, 1H), 1.80–1.70 (m, 3H), 1.64–1.54
(m, 1H), 1.54–1.47 (m, 6H), 1.36–1.20 (m, 2H), 1.14–1.02
(m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 346.10 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M +
H]<sup>+</sup> calcd for C<sub>18</sub>H<sub>24</sub>N<sub>3</sub>O<sub>2</sub>S, 346.1584; found, 346.1608.</p></sec><sec id="sec3.1.1.15"><title><italic>N</italic>-Cyclohexylglycine (<bold>53</bold>)</title><p>Cyclohexylamine <bold>51</bold> (100 mg, 1.0 mmol, 1.0 equiv) and
ethyl 2-hydroxyacetate <bold>52</bold> (104.9 mg, 1.0 mmol, 61.0 equiv)
were stirred neat in a round-bottom flask for 12 h. The solvent of
the crude mixture was evaporated completely, and the residue was purified
via flash column chromatography eluting with 10% DCM in methanol to
afford <italic>N</italic>-Cyclohexylglycine <bold>53</bold> (100 mg,
63% yield), C<sub>8</sub>H<sub>15</sub>NO<sub>2</sub>, MW 157.21; <sup>1</sup>H NMR (500 MHz, DMSO) δ 6.34 (brs, 1H), 4.05–4.04
(m, 2H), 3.82–3.77 (m, 1H), 3.77–3.69 (m, 1H), 1.86–1.83
(q, <italic>J</italic> = 5.28 Hz, 2H), 1.67–1.63 (m, 2H), 1.57–1.53
(m, 1H), 1.35–1.26 (m, 2H), 1.15–1.07 (m, 3H). LCMS <italic>m</italic>/<italic>z</italic> 158.21 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.16"><title>2-(Methylthio)quinazolin-4(3<italic>H</italic>)-one (<bold>54</bold>)</title><p>A solution of iodomethane (398 mg, 2.8 mmol, 1.0 equiv)
in methanol (8 mL) was added slowly to a closed vessel containing
a solution of 2-thioxo-2,3-dihydroquinazolin-4(1<italic>H</italic>)-one <bold>29</bold> (500 mg, 2.8 mmol, 1.0 equiv) in 1% aqueous
NaOH (8 mL) under N<sub>2</sub> atmosphere and stirred for 1 h at
room temperature. The reaction mixture was then cooled to 0 °C,
and the pH was adjusted to 7 with an aqueous solution of 1 N HCl.
The solvent was removed under reduced pressure, and the solid was
filtered, washed with water/methanol, and dried to afford 2-(methylthio)quinazolin-4(3<italic>H</italic>)-one <bold>54</bold> (532 mg, 98% yield), C<sub>9</sub>H<sub>8</sub>N<sub>2</sub>OS, MW 192.24; <sup>1</sup>H NMR (500 MHz,
DMSO) δ 12.62 (s, 1H), 8.09 (q, <italic>J</italic> = 3.13 Hz,
1H), 7.81 (m, 1H), 7.59 (d, <italic>J</italic> = 8.10 Hz, 1H), 7.47
(m, 1H), 2.63 (s, 3H). LCMS <italic>m</italic>/<italic>z</italic> 193.18
[M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.17"><title><italic>N</italic>-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)amino)acetamide
(<bold>17</bold>)</title><p>2-Amino-<italic>N</italic>-cyclohexyl-acetamide <bold>55</bold> (40.63 mg, 0.26 mmol, 1.0 equiv), 2-(methylthio)quinazolin-4(3<italic>H</italic>)-one <bold>54</bold> (50 mg, 0.26 mmol, 1.0 equiv) were
suspended in ethyl acetate (2 mL) in a sealed microwave vial and irradiated
(0–400 W at 2.65 GHz) at 140 °C for 2 h. Upon completion,
the reaction was filtered and the filtrate was extracted with diethyl
ether to afford <italic>N</italic>-cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)amino)acetamide
as a white powder <bold>17</bold> (4.4 mg, 4.4% yield), C<sub>16</sub>H<sub>20</sub>N<sub>4</sub>O<sub>2</sub>, MW 300.36; <sup>1</sup>H NMR (500 MHz, DMSO) δ 8.10 (d, <italic>J</italic> = 7.7 Hz,
1H), 7.58–7.56 (m, 1H), 7.33 (d, <italic>J</italic> = 8.7 Hz,
1H), 7.22–7.18 (m, 1H), 3.95 (s, 2H), 1.76–1.66 (m,
4H), 1.56–1.53 (m, 1H), 1.31–1.07 (m, 6H). LCMS <italic>m</italic>/<italic>z</italic> 301.0 [M + H]<sup>+</sup> 100%. HRMS
(<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd
for C<sub>16</sub>H<sub>21</sub>N<sub>4</sub>O<sub>2</sub>, 301.1659;
found, 301.1665.</p></sec><sec id="sec3.1.1.18"><title><italic>N</italic>-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)oxy)acetamide
(<bold>18</bold>)</title><p>2-(Methylthio)quinazolin-4(3<italic>H</italic>)-one <bold>54</bold> (50 mg, 0.26 mmol, 1.0 equiv) and <italic>N</italic>-cyclohexylglycine <bold>53</bold> (21.7 mg, 0.12 mmol, 1.0 equiv)
were dissolved in THF (2 mL) and heated to 80 °C. Upon reaction
completion, the crude product was directly purified via prep-HPLC
Gilson purification using 0.1% ammonia in water/acetonitrile 5–95%
gradient elution to afford the product as a white powder <bold>18</bold> (10 mg, 24% yield), C<sub>16</sub>H<sub>19</sub>N<sub>3</sub>O<sub>3</sub>, MW 301.34; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.36
(brs, 1H), 8.03 (d, <italic>J</italic> = 7.7 Hz, 1H), 7.9 (d, 1H),
7.73 (d, <italic>J</italic> = 8.7 Hz, 1H), 7.40–7.33 (m, 1H),
3.77 (s, 2H), 3.60–3.56 (m, 1H), 1.76–1.66 (m, 4H),
1.57–1.53 (m, 1H), 1.31–1.10 (m, 6H). LCMS <italic>m</italic>/<italic>z</italic> 302.1 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd for C<sub>16</sub>H<sub>20</sub>N<sub>3</sub>O<sub>32</sub>, 302.1499; found,
302.1487.</p></sec><sec id="sec3.1.1.19"><title><italic>N</italic>-Cyclohexyl-3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanamide
(<bold>19</bold>)</title><p>White powder (110 mg, 80% yield), C<sub>17</sub>H<sub>21</sub>N<sub>3</sub>O<sub>2</sub>, MW 299.37; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.18 (s, 1H), 8.08 (dd, <italic>J</italic> = 1.3, 7.9 Hz, 1H), 7.85–7.76 (m, 2H), 7.56 (d, <italic>J</italic> = 8.0 Hz, 1H), 7.48–7.45 (m, 1H), 3.55–3.48
(m, 1H), 2.83 (t, <italic>J</italic> = 7.4 Hz, 2H), 2.58 (t, <italic>J</italic> = 7.4 Hz, 2H), 1.73–1.64 (m, 4H), 1.55–1.52
(m, 1H), 1.28–1.09 (m, 5H). LCMS <italic>m</italic>/<italic>z</italic> 300.10 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd for C<sub>17</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub>, 300.1707; found, 300.1721.</p></sec><sec id="sec3.1.1.20"><title>2-((3-Methyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic Acid
(<bold>50</bold>)</title><p>2-Mercapto-3-methylquinazolin-4(3<italic>H</italic>)-one <bold>49</bold> (220 mg, 1.14 mmol, 1.0 equiv), 2-bromoacetic
acid (206 mg, 1.48 mmol, 1.3 equiv), and triethylamine (0.95 mL, 6.8
mmol, 6.0 equiv) were stirred in DMF (5 mL) for 12 h at 75 °C.
Upon reaction completion, the reaction mixture was poured onto crushed
ice and acidified with 1 N HCl. The precipitate formed was collected
and dried to afford the product 2-((3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic
acid <bold>50</bold> (250 mg, 87% yield), as a white powder, C<sub>11</sub>H<sub>10</sub>N<sub>2</sub>O<sub>3</sub>S, MW 250.27; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.89 (s, 1H), 8.08 (q, <italic>J</italic> = 3.05 Hz, 1H), 7.79 (m, 1H), 7.46 (m, 2H), 4.11 (s, 2H),
3.55 (s, 3H). LCMS <italic>m</italic>/<italic>z</italic> 251.04 [M
+ H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.21"><title><italic>N</italic>-Cyclohexyl-2-((3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>20</bold>)</title><p>White powder (20 mg, 15% yield), C<sub>17</sub>H<sub>21</sub>N<sub>3</sub>O<sub>2</sub>S, MW 331.42; <sup>1</sup>H NMR (500 MHz, DMSO) δ 8.15 (d, <italic>J</italic> =
7.9 Hz, 1H), 8.08 (d, <italic>J</italic> = 7.9 Hz, 1H), 7.82–7.78
(m, 1H), 7.52 (d, <italic>J</italic> = 8.2 Hz, 1H), 7.47–7.43
(m, 1H), 3.98 (s, 2H), 3.54 (s, 4H), 1.76–1.65 (m, 4H), 1.55
(d, <italic>J</italic> = 12.8 Hz, 1H), 1.28–1.11 (m, 5H). LCMS <italic>m</italic>/<italic>z</italic> 332.15 [M + H]<sup>+</sup> 100%. HRMS
(<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd
for C<sub>17</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub>S, 332.1427;
found, 332.1436.</p></sec><sec id="sec3.1.1.22"><title>2-(Quinazolin-2-ylthio)acetic Acid (<bold>44</bold>)</title><p>2-Chloroquinazoline <bold>43</bold> (0.2 g, 1.22
mmol) and sodium
thioglycolic acid <bold>39b</bold> (1 equiv) were dissolved in DMF,
and triethylamine (6.0 equiv) was added. The reaction mixture was
heated to 100 °C overnight. The resulting precipitate was filtered
and used without any further purification to afford the product 2-(quinazolin-2-ylthio)acetic
acid <bold>44</bold> (0.153 g, 57% yield) as a white powder, C<sub>10</sub>H<sub>8</sub>N<sub>2</sub>O<sub>2</sub>S, MW 220.25; <sup>1</sup>H NMR (500 MHz, DMSO) δ 9.41 (d, <italic>J</italic> =
1.5 Hz, 1H), 8.09 (m, 1H), 7.95–7.93 (m, 1H), 7.79–7.77
(m, 1H), 7.66–7.61 (m, 1H), 4.05 (s, 2H). LCMS <italic>m</italic>/<italic>z</italic> 221.1 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.23"><title><italic>N</italic>-Cyclohexyl-2-(quinazolin-2-ylthio)acetamide
(<bold>21</bold>)</title><p>White powder (21 mg, 10% yield), C<sub>16</sub>H<sub>19</sub>N<sub>3</sub>OS, MW 301.41; <sup>1</sup>H NMR
(300 MHz, DMSO) δ 9.36 (m, 1H), 8.05–8.0 (m, 2H), 7.79–7.76
(m, 1H), 3.95 (s, 2H), 3.52–3.48 (m, 1H), 2.49–2.48
(m, 2H), 1.72–1.32 (m, 5H), 1.25–1.21 (m, 3H). LCMS <italic>m</italic>/<italic>z</italic> 302.1 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.24"><title>2-((4-Aminoquinazolin-2-yl)thio)acetic
Acid (<bold>46</bold>)</title><p>2-Chloroquinazolin-4-amine <bold>45</bold> (0.2 g, 1.11
mmol, 1.0 equiv), sodium thioglycolic acid <bold>39b</bold> (0.103
g, 1.11 mmol, 1.0 equiv), and triethylamine were dissolved in DMF
(2 mL) (0.93 mL, 6.68 mmol, 6.0 equiv). The reaction mixture was heated
to 100 °C overnight. The resulting precipitate was filtered to
afford a white powder (0.2 g, 76% yield) and used without further
purification. LCMS (ESI) <italic>m</italic>/<italic>z</italic> 236.1
[M + H]<sup>+</sup></p></sec><sec id="sec3.1.1.25"><title>2-((4-Aminoquinazolin-2-yl)thio)-<italic>N</italic>-cyclohexylacetamide
(<bold>22</bold>)</title><p>White powder (21 mg, 10% yield), C<sub>16</sub>H<sub>20</sub>N<sub>4</sub>OS, MW 316.42; <sup>1</sup>H NMR
(300 MHz, DMSO) δ 8.13 (dd, <italic>J</italic> = 3.0, 6.0 Hz,
1H), 7.97 (d, <italic>J</italic> = 6 Hz, 1H), 7.87 (brs, 2H), 7.77–7.69
(m, 1H), 7.51–7.49 (m, 1H), 7.38–7.34 (m, 1H), 3.75
(s, 2H), 3.55–3.45 (m, 1H), 1.76–1.47 (m, 5H), 1.23–1.09
(m, 5H). LCMS <italic>m</italic>/<italic>z</italic> 317.1 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.26"><title>2-((4-Oxo-3,4,5,6,7,8-hexahydroquinazolin-2-yl)thio)acetic
Acid
(<bold>40</bold>)</title><p>2-Sulfanyl-5,6,7,8-tetrahydro-4-quinazolinol <bold>38</bold> (0.1 g, 5.49 mmol, 1.0 equiv), chloroacetic acid <bold>39a</bold> (1.3 equiv), and triethylamine (6 equiv) were stirred
in DMF at room temperature overnight. Once the reaction was complete,
the reaction mixture was poured over crushed ice and acidified with
1 N HCl. The white precipitate was collected by filtration and washed
with water to afford the product as a white powder (750 mg, 58% yield)
which was used without further purification, C<sub>10</sub>H<sub>12</sub>N<sub>2</sub>O<sub>3</sub>S, MW 240.28; <sup>1</sup>H NMR (300 MHz,
DMSO) δ 12.59 (brs, 1H), 3.93 (s, 2H), 2.45 (t, <italic>J</italic> = 3.0 Hz, 2H), 2.29 (t, <italic>J</italic> = 3.0 Hz, 2H), 1.70–1.63
(m, 4H). LCMS (ESI) <italic>m</italic>/<italic>z</italic> 239.1 [M
– H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.27"><title><italic>N</italic>-Cyclohexyl-2-((4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-yl)thio)acetamide
(<bold>23</bold>)</title><p>White powder (43 mg, 17% yield), C<sub>16</sub>H<sub>23</sub>N<sub>3</sub>O<sub>2</sub>S, MW 321.44; <sup>1</sup>H NMR (300 MHz, DMSO) δ 12.47 (brs, 1H), 8.00 (brs,
1H), 3.80 (s, 2H), 3.58–3.49 (m, 1H), 2.47 (t, <italic>J</italic> = 3.0 Hz, 2H), 2.31 (t, <italic>J</italic> = 3.0 Hz, 2H), 1.75–1.54
(m, 9H), 1.29–1.13 (m, 5H). LCMS <italic>m</italic>/<italic>z</italic> 320.1 [M – H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.28"><title>2-((4-Oxo-4,5,6,7-tetrahydro-3<italic>H</italic>-cyclopenta[d]pyrimidin-2-yl)thio)acetic
acid (<bold>42</bold>)</title><p>2-Thioxo-1,2,3,5,6,7-hexahydro-4<italic>H</italic>-cyclopenta[d]pyrimidin-4-one <bold>41</bold> (1.0 g, 5.94
mmol, 1.0 equiv), chloroacetic acid <bold>39a</bold> (1.3 equiv),
and triethylamine (6 equiv) were stirred in DMF (2 mL) at room temperature
overnight. Once the reaction was complete, it was poured over crushed
ice and acidified with 1 N HCl. The white precipitate was collected
by filtration and washed with water to afford the product as a white
powder (800 mg, 59% yield), which was used without further purification,
C<sub>9</sub>H<sub>10</sub>N<sub>2</sub>O<sub>3</sub>S, MW 226.05; <sup>1</sup>H NMR (300 MHz, DMSO) δ 12.65 (brs, 1H), 3.96 (s, 2H),
2.71 (t, <italic>J</italic> = 3.0 Hz, 2H), 2.58 (t, <italic>J</italic> = 3.0 Hz, 2H), 1.97–1.93 (m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 227.1 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.29"><title><italic>N</italic>-Cyclohexyl-2-((4-oxo-4,5,6,7-tetrahydro-3<italic>H</italic>-cyclopenta[d]pyrimidin-2-yl)thio)acetamide(<bold>24</bold>)</title><p>White powder (59 mg, 22% yield), C<sub>15</sub>H<sub>23</sub>N<sub>3</sub>O<sub>2</sub>S, MW 307.41; <sup>1</sup>H NMR (300 MHz,
DMSO) δ 12.59 (brs, 1H), 8.03 (s, 1H), 3.84 (s, 2H), 3.58–3.49
(m, 1H), 2.71 (t, <italic>J</italic> = 3.0 Hz, 2H), 2.60 (t, <italic>J</italic> = 3.0 Hz, 2H), 2.0–1.92 (m, 2H), 1.75–1.52
(m, 5H), 1.29–1.16 (m, 5H). LCMS (ESI) <italic>m</italic>/<italic>z</italic> 308.1 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.30"><title>2-((4,5-Dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio)acetic
Acid (<bold>48</bold>)</title><p>5,6-Dimethyl-2-thiouracil <bold>47</bold> (0.5 g, 3.20 mmol), chloroacetic acid <bold>39a</bold> (0.39 g,
4.16 mmol), and triethylamine (2.62 mL, 1.94 mmol) were stirred in
DMF (2 mL) at room temperature overnight. Once the reaction was complete,
it was poured over crushed ice and acidified with 1 N HCl. The white
precipitate was collected by filtration and washed with water to afford
the product as a white powder (0.36 g, 54% yield) which was used without
further purification, C<sub>8</sub>H<sub>10</sub>N<sub>2</sub>O<sub>3</sub>S, MW 214.24; <sup>1</sup>H NMR (300 MHz, DMSO) δ 12.56
(brs, 1H), 3.89 (s, 2H), 2.14 (s, 3H), 1.85 (s, 3H). LCMS (ESI) <italic>m</italic>/<italic>z</italic> 213.1 [M – H]; HPLC purity 98%.</p></sec><sec id="sec3.1.1.31"><title><italic>N</italic>-Cyclohexyl-2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio)acetamide
(<bold>25</bold>)</title><p>White powder (0.047 g, 17% yield), C<sub>14</sub>H<sub>21</sub>N<sub>3</sub>O<sub>2</sub>S, MW 295.40; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>) δ
7.99 (dd, <italic>J</italic> = 20.3, 8.1 Hz, 1H), 3.80 (s, 2H), 3.11–2.97
(m, 1H), 2.20 (s, 3H), 1.89 (s, 3H), 1.78–1.64 (m, 5H), 1.33–1.12
(m, 5H). LCMS (ESI) <italic>m</italic>/<italic>z</italic> 296.1 [M
+ H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.32"><title>6-Fluoro-2-thioxo-2,3-dihydroquinazolin-4(1<italic>H</italic>)-one (<bold>33</bold>)</title><p>2-Amino-5-fluorobenzoic
acid <bold>32</bold> (1 g, 6.5 mmol, 1.0 equiv) and thiourea (2.1
g, 25.8 mmol,
6.0 equiv) were heated neat in a round-bottom flask to 180 °C
for 3 h with stirring. The reaction was cooled, and 25 mL of water
was added. After stirring for 10 min, the formed precipitate was filtered
off, washed with cold water, and dried to afford 6-fluoro-2-thioxo-2,3-dihydroquinazolin-4(1<italic>H</italic>)-one <bold>33</bold> (0.45 g, 35.9% yield) as a yellow
powder, C<sub>8</sub>H<sub>5</sub>FN<sub>2</sub>OS, MW 196.2; <sup>1</sup>H NMR (500 MHz, DMSO) δ 9.72 (s, 1H), 7.51–7.39
(m, <italic>J</italic> = 8.5 Hz, 1H), 7.37–7.30 (m, <italic>J</italic> = 8.6 Hz, 1H), 7.19–7.16 (m, <italic>J</italic> = 8.85 Hz, 1H). LCMS <italic>m</italic>/<italic>z</italic> 197.78
[M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.33"><title>2-((6-Fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic
Acid
(<bold>34</bold>)</title><p>6-Fluoro-2-thioxo-2,3-dihydroquinazolin-4(1<italic>H</italic>)-one <bold>33</bold> (414 mg, 2.1 mmol, 1.0 equiv), 2-bromoacetic
acid (586.39 mg, 4.2 mmol, 2.0 equiv), and triethylamine (1.76 mL,
12.6 mmol, 6.0 equiv) were stirred in DMF (5 mL) at 75 °C for
12 h. Upon reaction completion, the reaction mixture was poured over
crushed ice and acidified with 1 N HCl. The precipitate was filtered
and dried to afford 2-((6-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic
acid <bold>34</bold> (150 mg, 27.9% yield), C<sub>10</sub>H<sub>7</sub>FN<sub>2</sub>O<sub>3</sub>S, MW 254.24; <sup>1</sup>H NMR (500 MHz,
DMSO) δ 12.88 (s, 1H), 7.72 (q, <italic>J</italic> = 3.87 Hz,
1H), 7.65 (m, 1H), 7.54 (q, <italic>J</italic> = 4.62 Hz, 1H), 4.03
(s, 2H). LCMS <italic>m</italic>/<italic>z</italic> 254.10 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.34"><title><italic>N</italic>-(4,4-Difluorocyclohexyl)-2-((6-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(<bold>26</bold>)</title><p>To a solution of 2-((6-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetic
acid <bold>34</bold> (100 mg, 0.39 mmol, 1.0 equiv) in a 1:1 ratio
of acetonitrile (1.5 mL) and DMF (1.5 mL) were added EDC·HCl
(90 mg, 0.74 mmol, 1.2 equiv), HOAT (64 mg, 0.47 mmol, 1.2 equiv),
and 4,4-difluorocyclohexan-1-amine (67 mg, 0.39 mmol, 1.0 equiv),
and the mixture was stirred for 30 min. DIPEA (0.13 mL, 0.78 mmol,
2.0 equiv) was added, and the resulting mixture was stirred at room
temperature overnight. The crude mixture was purified by preparative
HPLC under acidic conditions, using 0.1% HCOOH in water/acetonitrile
(5–95%) gradient elution to afford <italic>N</italic>-(4,4-difluorocyclohexyl)-2-((6-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
as a white powder <bold>26</bold> (12 mg, 8.2% yield), C<sub>16</sub>H<sub>16</sub>N<sub>3</sub>F<sub>3</sub>O<sub>2</sub>S, MW 371.86; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.76 (brs, 1H), 8.39–8.36
(m, 1H), 7.71–7.60 (m, 2H), 7.52 (dd, <italic>J</italic> =
5.0, 8.9 Hz, 1H), 3.88 (s, 2H), 3.77 (d, <italic>J</italic> = 8.5
Hz, 1H), 2.08–1.77 (m, 6H), 1.55–1.47 (m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 372.3 [M + H]<sup>+</sup> 100%. HRMS
(<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd
for C<sub>16</sub>H<sub>17</sub>N<sub>3</sub>F<sub>3</sub>O<sub>2</sub>S, 372.0988; found, 372.0987.</p></sec><sec id="sec3.1.1.35"><title>2-Thioxo-2,3-dihydropyrido[3,4-<italic>d</italic>]pyrimidin-4(1<italic>H</italic>)-one (<bold>36</bold>)</title><p>2-Aminopyridine-4-carboxylic
acid <bold>35</bold> (1 g, 7.2 mmol, 1.0 equiv) and thiourea (2.8
g, 36.2 mmol, 5.0 equiv) were heated neat in a round-bottom flask
to 180 °C for 3 h with stirring. The reaction was cooled, and
25 mL of water was added. After stirring for 10 min, the formed precipitate
was filtered off, washed with cold water, and dried to afford 2-thioxo-2,3-dihydropyrido[3,4-<italic>d</italic>]pyrimidin-4(1<italic>H</italic>)-one <bold>36</bold> (1.11
g, 85% yield) as a yellow powder, C<sub>7</sub>H<sub>5</sub>FN<sub>2</sub>OS, MW 179.2; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.89
(brs, 1H), 12.70 (brs, 1H), 8.73 (s, 1H), 8.49 (d, <italic>J</italic> = 4.85 Hz, 1H), 7.79 (d, <italic>J</italic> = 0.80 Hz, 1H). LCMS <italic>m</italic>/<italic>z</italic> 180.11 [M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.36"><title>2-((4-Oxo-3,4-dihydropyrido[3,4-<italic>d</italic>]pyrimidin-2-yl)thio)acetic
Acid (<bold>37</bold>)</title><p>2-Thioxo-2,3-dihydropyrido[3,4-<italic>d</italic>]pyrimidin-4(1<italic>H</italic>)-one <bold>36</bold> (500
mg, 2.79 mmol, 1.0 equiv), 2-bromoacetic acid (775.39 mg, 5.5 mmol,
2.0 equiv), and triethylamine (2.33 mL, 16.7 mmol, 6.0 equiv) were
stirred in DMF (5 mL) at 75 °C for 12 h. Upon reaction completion,
the reaction mixture was poured over crushed ice and acidified with
1 N HCl, and the precipitate was collected and dried to afford 2-((4-oxo-3,4-dihydropyrido[3,4-<italic>d</italic>]pyrimidin-2-yl)thio)acetic acid <bold>37</bold> (600 mg,
90.6% yield), C<sub>9</sub>H<sub>7</sub>N<sub>3</sub>O<sub>3</sub>S, MW 237.24; <sup>1</sup>H NMR (500 MHz, DMSO) δ 12.89 (s,
1H), 8.85 (d, <italic>J</italic> = 0.65 Hz, 1H), 8.57 (d, <italic>J</italic> = 5.00 Hz, 1H), 7.88 (d, <italic>J</italic> = 0.80 Hz,
1H), 4.08 (s, 2H). LCMS <italic>m</italic>/<italic>z</italic> 238.15
[M + H]<sup>+</sup> 100%.</p></sec><sec id="sec3.1.1.37"><title><italic>N</italic>-(4,4-Difluorocyclohexyl)-2-((4-oxo-3,4-dihydropyrido[3,4-<italic>d</italic>]pyrimidin-2-yl)thio)acetamide (<bold>27</bold>)</title><p>White powder (64 mg, 42% yield), C<sub>15</sub>H<sub>16</sub>N<sub>4</sub>O<sub>2</sub>F<sub>2</sub>S, MW 354.38; <sup>1</sup>H NMR
(500 MHz, DMSO) δ 13.02 (s, 1H), 8.86 (s, 1H), 8.57 (d, <italic>J</italic> = 5.2 Hz, 1H), 8.29 (d, <italic>J</italic> = 7.4 Hz, 1H),
7.87 (d, <italic>J</italic> = 5.0 Hz, 1H), 3.98 (s, 2H), 3.31 (s,
1H), 2.09–1.99 (m, 2H), 1.98–1.86 (m, 2H), 1.86–1.77
(m, 2H), 1.58–1.48 (m, 2H). LCMS <italic>m</italic>/<italic>z</italic> 355.22 [M + H]<sup>+</sup> 100%. HRMS (<italic>m</italic>/<italic>z</italic>): [M + H]<sup>+</sup> calcd for C<sub>15</sub>H<sub>17</sub>N<sub>4</sub>O<sub>2</sub>F<sub>2</sub>S, 355.1034;
found, 355.1033.</p></sec></sec></sec><sec id="sec3.1.2"><title>Intrinsic Clearance (Cli) Experiments</title><p>Test compound
(0.5 μM) was incubated with female CD1 mouse liver microsomes
(Xenotech LLC; 0.5 mg/mL, 50 mM potassium phosphate buffer, pH 7.4),
and the reaction started with addition of excess NADPH (8 mg/mL, 50
mM potassium phosphate buffer, pH 7.4). Immediately, at time zero,
and then at 3, 6, 9, 15, and 30 min, an aliquot (50 μL) of the
incubation mixture was removed and mixed with acetonitrile (100 μL)
to stop the reaction. Internal standard was added to all samples;
the samples were centrifuged to sediment precipitated protein, and
the plates were then sealed prior to UPLC-MS/MS analysis using a Quattro
Premier XE (Waters Corporation, USA). XLfit (IDBS, UK) was used to
calculate the exponential decay and consequently the rate constant
(<italic>k</italic>) from the ratio of peak area of test compound
to internal standard at each time point. The rate of intrinsic clearance
(Cli) of each test compound was then calculated using the following
calculation.<disp-formula id="ueq1"><graphic xlink:href="id-2017-002759_m001" position="anchor"></graphic></disp-formula>where <italic>V</italic> (mL/mg protein) is
the incubation volume/mg protein added and microsomal protein yield
is taken as 52.5 mg protein/g liver. Verapamil (0.5 μM) was
used as a positive control to confirm acceptable assay performance.</p></sec><sec id="sec3.1.3"><title>Aqueous Solubility</title><p>The aqueous solubility of the test
compounds was measured using laser nephelometry. Compounds were subject
to serial dilution from 10 to 0.5 mM in DMSO. An aliquot was then
mixed with Milli-Q water to obtain an aqueous dilution plate with
a final concentration range of 250–12 μM, with a final
DMSO concentration of 2.5%. Triplicate aliquots were transferred to
a flat bottomed polystyrene plate which was immediately read on the
NEPHELOstar (BMG Lab Technologies). The amount of laser scatter caused
by insoluble particulates (relative nephelometry units, RNU) was plotted
against compound concentration using a segmental regression fit, with
the point of inflection being quoted as the compounds aqueous solubility
(μM).</p></sec><sec id="sec3.1.4"><title>CHI LogD Determination</title><p>The chromatographic
hydrophobicity
index (CHI) was determined according the method previously described.<sup><xref ref-type="bibr" rid="ref43">43</xref>,<xref ref-type="bibr" rid="ref44">44</xref></sup> Briefly, test compounds were prepared as 0.5 mM solutions in 50:50
acetonitrile/water and analyzed by reversed-phase HPLC-UV (wavelength
254 nm) using a Phenomenex Luna C18 100 Å, 150 × 4.6 mm,
5 μm column with a gradient of aqueous phase (50 mM ammonium
acetate (pH 7.4)) and mobile phase (acetonitrile). By plotting the
retention time of a set of reference compounds against known CHI values,
the CHI value of test compounds was calculated according to their
retention time.</p></sec><sec id="sec3.1.5"><title>Plasma Protein Binding Experiments</title><p>In brief, a 96-well
equilibrium dialysis apparatus was used to determine the free fraction
in plasma for each compound (HT Dialysis LLC, Gales Ferry, CT). Membranes
(12–14 kDaA cutoff) were conditioned in deionized water for
60 min, followed by conditioning in 80:20 deionized water/ethanol
overnight and then rinsed in water and isotonic buffer before use.
Mouse plasma from appropriate species was removed from the freezer
and allowed to thaw on the day of experiment. Thawed plasma was then
centrifuged (Allegra X12-R, Beckman Coulter, USA) and spiked with
test compound (final concentration 10 μg/mL), and 150 μL
aliquots (<italic>n</italic> = 3 replicate determinations) were loaded
into the 96-well equilibrium dialysis plate. Dialysis against isotonic
buffer (150 μL) was carried out for 5 h in a temperature controlled
incubator at ca. 37 °C (Barworld scientific Ltd., UK) using an
orbital microplate shaker at 100 rpm (Barworld scientific Ltd., UK).
At the end of the incubation period, 50 μL aliquots of plasma
or buffer were transferred into a 96-well deep plate and the composition
in each well was balanced with control fluid (50 μL), such that
the volume of buffer to plasma is the same. Sample extraction was
performed by the addition of 200 μL of acetonitrile containing
an appropriate internal standard. Samples were allowed to mix for
1 min and then centrifuged at 3000 rpm in 96-well blocks for 15 min
(Allegra X12-R, Beckman Coulter, USA) after which 150 μL of
supernatant was removed to 50 μL of water. All samples were
analyzed by UPLC-MS/MS on a Quattro Premier XE Mass Spectrometer (Waters
Corporation, USA). The unbound fraction was determined as the ratio
of the peak area in buffer to that in plasma.</p></sec><sec id="sec3.1.6"><title>PAMPA Assay</title><p>The permeability was performed using a
96-well precoated BD Gentest PAMPA plate (BD Biosciences, U.K.). Each
well was divided into two chambers: donor and acceptor, separated
by a lipid-oil-lipid trilayer constructed in a porous filter. The
effective permeability, Pe, of the compound was measured at pH 7.4.
Stock solutions (5 mM) of the compound were prepared in DMSO. The
compound was then further diluted to 10 μM in phosphate buffered
saline at pH 7.4. The final DMSO concentration did not exceed 5% v/v.
The compound dissolved in phosphate buffered saline was then added
to the donor side of the membrane, and phosphate buffered saline without
compound was added to the acceptor side. The PAMPA plate was left
at room temperature for 5 h; after which time, an aliquot (100 μL)
was then removed from both acceptor and donor compartments and mixed
with acetonitrile (80 μL) containing an internal standard: donepezil
at 50 ng/mL). The samples were centrifuged (10 min, 5 °C, 3270<italic>g</italic>) to sediment precipitated protein and sealed prior to
UPLC-MS/MS analysis using a Quattro Premier XE (Waters Corp, USA).
Pe was calculated as shown in the below equation.<disp-formula id="ueq2"><graphic xlink:href="id-2017-002759_m002" position="anchor"></graphic></disp-formula>where CA(<italic>t</italic>) = peak area of
compound present in acceptor well at time (<italic>t</italic>) = 18 000
s; Cequi = [CD(<italic>t</italic>) ×
VD + CA(<italic>t</italic>) × VA]/(VD + VA); <italic>A</italic> = filter area; VD = donor well volume; VA = acceptor well volume; <italic>t</italic> = incubation time; CD(<italic>t</italic>) = peak area
of compound present in donor well at time (<italic>t</italic>) = 18 000
s. Recovery of compound from donor and acceptor wells was calculated,
and data was only accepted when recovery exceeded 70%.</p></sec><sec id="sec3.1.7"><title>Animal Care
Assurance</title><p>All mouse studies performed at
the University of Dundee were performed under the authority of a project
license (PPL70/8346) issued by the Home Office under the Animals (Scientific
Procedures) Act 1986, as amended in 2012 (and in compliance with EU
Directive EU/2010/63). License applications are approved by the University’s
Ethical Review Committee (ERC) before submission to the Home Office.
The ERC develops and oversees policy on all aspects of the use of
animals on University premises and is a subcommittee of the University
Court, its highest governing body.</p></sec><sec id="sec3.1.8"><title>Mouse Pharmacokinetics</title><p>Test compound was dosed as a
bolus solution intravenously at 3 mg free base/kg (dose volume: 5
mL/kg; dose vehicle: 10% DMSO; 40% PEG400; 50% saline) to female C57Bl/6
mice (<italic>n</italic> = 3) or dosed orally by gavage as a suspension
at 10 or at 5, 30, or 100 mg free base/kg (dose volume: 10 mL/kg;
dose vehicle: 1.0% carboxy methyl cellulose) to female C57Bl/6 mice
(<italic>n</italic> = 3/dose level). Blood samples were taken from
each mouse tail vein at predetermined time points postdose, mixed
with nine volumes of distilled water, and stored frozen until UPLC-MS/MS
analysis. Pharmacokinetic parameters were derived from the blood concentration
time curve using PK Solutions software v 2.0 (Summit Research Services,
USA).</p></sec><sec id="sec3.1.9"><title>Strains and Media</title><p><italic>Mycobacterium tuberculosis</italic> H37Rv (ATCC 27294) was used for all work with this organism unless
otherwise indicated. The activity of the compound on cell wall synthesis
and the <italic>bc</italic><sub>1</sub> complex was measured using
the <italic>piniBAC</italic> reporter strain and the <italic>cydC</italic>::<italic>aph</italic> strains, respectively, using previously described
methods.<sup><xref ref-type="bibr" rid="ref35">35</xref>,<xref ref-type="bibr" rid="ref45">45</xref></sup> Broth-based medium (7H9/ADC/Tw) for <italic>MTb</italic> consisted of Middlebrook 7H9 (Becton Dickinson) supplemented
with 10% ADC [albumin (50 g/L)/dextrose (20 g/L)/NaCl (8.1 g/L)], 0.2%
glycerol] and 0.05% Tween 80. Solid growth medium for <italic>MTb</italic> was Middlebrook 7H11 (Becton Dickinson) supplemented with OADC [ADC
with 0.06% oleic acid]. Potencies of compounds by MIC determination
were performed as previously described.<sup><xref ref-type="bibr" rid="ref45">45</xref></sup> The Δ<italic>ndhA</italic> strain was generated by insertion
of the <italic>aph</italic> gene into the native <italic>Bam</italic>HI restriction site 873 nucleotides downstream of the start codon
using the previously described suicide vector method<sup><xref ref-type="bibr" rid="ref46">46</xref></sup> to effect legitimate recombination in <italic>MTb</italic>.</p></sec><sec id="sec3.1.10"><title>Measurement of Intracellular ATP Levels</title><p><italic>MTb</italic> H37Rv was grown to early log phase (OD650 nm of 0.2) and diluted
20-fold in the same medium (7H9/ADC/Tw). 100 μL aliquots of
cells were added to each well of sterile white polystyrene 96-well
plates (Corning Inc., Corning, NY) containing 2 μL drug dilutions
in DMSO in triplicate. ATP was measured at different time points as
indicated by a bioluminescent assay using the BacTiter-Glo reagent
(Promega, Madison, WI) as recommended by the manufacturer.</p></sec><sec id="sec3.1.11"><title>Bioenergetics</title><p>The extracellular flux analysis assays
were performed as described previously.<sup><xref ref-type="bibr" rid="ref21">21</xref>,<xref ref-type="bibr" rid="ref36">36</xref></sup> In short, <italic>M. tuberculosis</italic> H37Rv were grown in 7H9 (10% OADC,
0.01% tyloxapol) to an OD of 0.8. Bacilli were pelleted, suspended
in unbuffered carbon-source-free XF 7H9 media, and seeded into the
XF culture plate, at a density of 2 × 10<sup>6</sup> bacilli/well.
During the XF screen, the oxygen consumption rate (OCR) was measured,
noninvasively and in real time. OCR data points are representative
of the average OCR during 3 min of continuous measurement in the transient
microchamber, with the error being calculated from the OCR measurements
taken from eight replicate wells by the Wave Desktop 2.3 software
(Agilent). During the assay carbon sources (glucose and palmitate
at a final concentration of 0.2%), the mercapto-quinazolinones (Compounds <bold>1</bold> and <bold>7</bold> at final concentrations of 39 and 45
μM, respectively) and CCCP (final concentration of 3 μM)
were added to the assay media at the indicated time.</p></sec><sec id="sec3.1.12"><title>Inverted
Membrane Vesicle (IMV)</title><p>IMVs were isolated,
and the ATP generation assay was performed as described previously.<sup><xref ref-type="bibr" rid="ref36">36</xref></sup> In brief, <italic>MTb</italic> mc<sup>2</sup>6230 (cultured in 7H9, 0.01% tyloxapol, 10% OADC, 0.2% casamino acids,
and 24 μg/mL pantothenic acid) were collected via centrifugation,
incubated with lysozyme, and lysed via bead beating in a buffer containing
10 mM HEPES, 50 mM KCl, 5 mM MgCl<sub>2</sub>, and 10% glycerol. IMVs
were isolated by differential centrifugation. The ATP synthesis assay
was performed using a Roche Bioluminescence Assay Kit CL II. Membrane
vesicles were provided either 250 μM NADH or 1 mM succinate
as an electron donor, 50 μM ADP, and 5 mM phosphate in the above
buffer containing the luciferase/luciferin reagents. Luminescence
was measured in a 384-well plate using a BioTek Synergy H4 plate reader.</p></sec><sec id="sec3.1.13"><title>Recombinant Rv1854c Purification</title><p>The ORF corresponding
to Rv1854c was codon optimized for <italic>E. coli</italic>,
synthesized (ThermoFisher), and cloned between the NcoI and <italic>Hin</italic>dIII sites of pMALc5x, affording a vector for the IPTG-inducible
overexpression of MtNdh with an N-terminal maltose-binding protein
(MBP) fusion tag. This vector was transformed into <italic>E. coli</italic> BL21 (DE3) pLysS, and protein expression was achieved in 1 L of
media (ZYM + 0.1% glucose, 0.1% glycerol, 2 mM MgSO<sub>4</sub>, 34
μg/mL chloramphenicol, 50 μg/mL carbenicillin) following
induction with 0.3 mM IPTG at 37 °C for 4 h. Cells were harvested,
resuspended in 40 mL of buffer A (50 mM HEPES, 100 mM KPO<sub>4</sub>, pH 7.1, 5% (w/v) glycerol) with added 5 U/mL DNase, complete protease
inhibitor, 5 μM FAD, 10 mM MgCl2, and 0.25% CHAPS, and sonicated
on ice to lyse. Following centrifugation at 20 000<italic>g</italic> for 30 min, the soluble fraction was incubated with 2 mL of settled
volume of pre-equilibrated (buffer A + 0.25% CHAPS) amylose resin
for 30 min at 4 °C and then applied to a gravity flow column.
The resin was washed with 15 CV buffer A + 0.25% CHAPS and then 15
CV buffer A without CHAPS. Bound protein was eluted with 10 mL of
buffer A + 20 mM maltose. The same purification procedure was repeated
twice on the initial resin flow-throughs; all eluates were combined
and concentrated using spin columns. The concentrated prep was then
passed through a Superdex 75 10/300 column (running buffer was buffer
A), and relevant fractions were collected and concentrated. Aliquots
of the resulting weakly yellow solution were flash frozen and stored
at −80 °C until needed. Protein concentration was measured
by spectrophotometric flavin (FAD) analysis, as described in ref (<xref ref-type="bibr" rid="ref47">47</xref>).</p></sec><sec id="sec3.1.14"><title>Ndh Activity
Assays</title><p>MtNdh catalytic activity was measured
by monitoring NADH oxidation at 340 nm (ε = 6220 M<sup>–1</sup> cm<sup>–1</sup>) in a UV–vis spectrophotometer at
RT. Reactions (1 mL) consisted of 0.1 M HEPES, pH 7.0, 10% DMSO, 0.1–0.5
nM recombinant MBP-MtNdh, and varying concentrations of Q2 and NADH.
Reactions were started by addition of NADH, following ∼1 min
preincubation of all other reaction components. Kinetic data was taken
at steady state within the first 1 min of the reaction. Background
NADH oxidation in the absence of Q2 was measured (typically <5%
of rate with Q2 present) and subtracted from all subsequent measurements,
as required. All kinetic data was plotted and analyzed using GraphPad
Prism software.</p></sec></sec></body><back><notes id="notes-3" notes-type="si"><title>Supporting Information Available</title><p>The Supporting Information
is available free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications website</ext-link> at DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acsinfecdis.7b00275">10.1021/acsinfecdis.7b00275</ext-link>.<list id="silist" list-type="simple"><list-item><p>Compound syntheses
and characterization, Met-ID studies
of compounds <bold>1</bold> and <bold>7</bold> with microsomes with
and without GSH, graphs showing metabolic transformations in Met-ID
studies, pharmacokinetic studies, anaerobic cidal and intramacrophage
activity of compound <bold>1</bold>, and whole genome sequencing methods
and data table (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.7b00275/suppl_file/id7b00275_si_001.pdf">PDF</ext-link>)</p></list-item></list></p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="id7b00275_si_001.pdf"><caption><p>id7b00275_si_001.pdf</p></caption></media></supplementary-material></sec><notes notes-type="" id="notes-1"><title>Author Present Address</title><p><sup>¤</sup> P.M.F.:
Department of Internal Medicine, University of Pittsburgh,
1218 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261,
USA.</p></notes><notes notes-type="" id="notes-2"><title>Author Contributions</title><p><sup>α</sup> D.M. and P.C.R. contributed equally.</p></notes><notes notes-type="" id="notes-4"><title>Author Contributions</title><p>H.I.M.B.,
D.F.W., K.A., and K.Y.R. performed biological experiments on <italic>MTb</italic>. T.R.I. and J.S. performed sequencing analysis of resistant
mutants. G.A.P. performed enzyme assays. D.A.L., P.M.F., and A.J.C.S.
performed and analyzed bioenergetics. D.M., P.C.R., T.B., J.R.H.,
K.G., C.S.M., T.-S.F., L.J.S., and K.C. synthesized compounds. P.S.,
L.E., J.R., Y.S., L.F., M.O.-C., O.E., and K.D.R. performed ADME/PK
analysis. H.I.M.B., S.R.G., C.E.B., P.C.R., P.G.W., and V.M. helped
with data interpretation. P.C.R., H.I.M.B., and S.R.G. wrote the manuscript.
D.F.W., T.-S.F., C.S.M., C.E.B., and D.M. edited the manuscript which
was reviewed by all authors.</p></notes><notes notes-type="COI-statement" id="notes-5"><p>The authors
declare no competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>This work was funded in part by the Intramural
Research Program
of NIAID (AI000693-25), by grants from the Foundation for the National
Institutes of Health (BARRY11HTB0) with support from the Bill &
Melinda Gates Foundation (OPP1024021), and by the South African Medical
Research Council (SAMRC) with funds received from Strategic Health
Innovation Partnerships (SHIP) unit of the SAMRC, by the National
Research Foundation of South Africa, and OPP1066891 “A Centre
of Excellence for Lead Optimization for Diseases of the Developing
World”, a joint award from the Wellcome Trust and the Bill
& Melinda Gates Foundation. A.J.C.S. is a
Burroughs Welcome Investigator in the Pathogenesis of Infectious Diseases.
P.M.F. was a Howard Hughes Medical Institute Medical Research Fellow.
We thank David Gray, James Roberts, and Alastair Pate for support
with data analysis, compound handling, and data management.</p></ack><glossary id="dl1"><def-list><title>Abbrevations</title><def-item><term>NDH-2</term><def><p>type II NADH
dehydrogenase</p></def></def-item><def-item><term>MTb</term><def><p><italic>Mycobacterium
tuberculosis</italic></p></def></def-item><def-item><term>MtNdh</term><def><p><italic>MTb</italic> NDH-2</p></def></def-item><def-item><term>SAR</term><def><p>structure–activity relationship</p></def></def-item><def-item><term>Kan</term><def><p>kanamycin</p></def></def-item><def-item><term>INH</term><def><p>isoniazid</p></def></def-item><def-item><term>RIF</term><def><p>rifampicin</p></def></def-item><def-item><term>MDR-TB</term><def><p>multidrug resistant
TB</p></def></def-item><def-item><term>XDR-TB</term><def><p>extensively
drug-resistant tuberculosis</p></def></def-item><def-item><term>MoA</term><def><p>mechanism of action</p></def></def-item><def-item><term>MIC</term><def><p>minimum inhibitory concentration</p></def></def-item><def-item><term>AUC</term><def><p>area under the curve</p></def></def-item><def-item><term>IMVs</term><def><p>inverted membrane vesicles</p></def></def-item><def-item><term>OCR</term><def><p>oxygen consumption rate</p></def></def-item><def-item><term>LLE</term><def><p>ligand-lipophilicity
efficiency</p></def></def-item><def-item><term>ADME</term><def><p>absorption, distribution, metabolism, and excretion</p></def></def-item><def-item><term>met-ID</term><def><p>metabolite identification</p></def></def-item><def-item><term>cLogP</term><def><p>calculated logP</p></def></def-item><def-item><term>Cli</term><def><p>intrinsic clearance</p></def></def-item><def-item><term>CFU</term><def><p>colony-forming
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