Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors
Identifieur interne : 000834 ( Pmc/Corpus ); précédent : 000833; suivant : 000835Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors
Auteurs : Arun K. Ghosh ; Gangli Gong ; Valerie Grum-Tokars ; Debbie C. Mulhearn ; Susan C. Baker ; Melissa Coughlin ; Bellur S. Prabhakar ; Katrina Sleeman ; Michael E. Johnson ; Andrew D. MesecarSource :
- Bioorganic & Medicinal Chemistry Letters [ 0960-894X ] ; 2008.
Abstract
Design, synthesis and biological evaluation of a series of chloropyridyl ester-derived SARS-CoV 3CLpro Inhibitors are described Inhibitors
Url:
DOI: 10.1016/j.bmcl.2008.08.082
PubMed: 18796354
PubMed Central: 2745596
Links to Exploration step
PMC:2745596Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors</title><author><name sortKey="Ghosh, Arun K" sort="Ghosh, Arun K" uniqKey="Ghosh A" first="Arun K." last="Ghosh">Arun K. Ghosh</name><affiliation><nlm:aff id="aff1">Departments of Chemistry and Medicinal Chemistry, Purdue University, 560 Oval drive, West Lafayette, IN 47907, USA</nlm:aff></affiliation></author><author><name sortKey="Gong, Gangli" sort="Gong, Gangli" uniqKey="Gong G" first="Gangli" last="Gong">Gangli Gong</name><affiliation><nlm:aff id="aff1">Departments of Chemistry and Medicinal Chemistry, Purdue University, 560 Oval drive, West Lafayette, IN 47907, USA</nlm:aff></affiliation></author><author><name sortKey="Grum Tokars, Valerie" sort="Grum Tokars, Valerie" uniqKey="Grum Tokars V" first="Valerie" last="Grum-Tokars">Valerie Grum-Tokars</name><affiliation><nlm:aff id="aff2">Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Mulhearn, Debbie C" sort="Mulhearn, Debbie C" uniqKey="Mulhearn D" first="Debbie C." last="Mulhearn">Debbie C. Mulhearn</name><affiliation><nlm:aff id="aff2">Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Baker, Susan C" sort="Baker, Susan C" uniqKey="Baker S" first="Susan C." last="Baker">Susan C. Baker</name><affiliation><nlm:aff id="aff3">Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL, USA</nlm:aff></affiliation></author><author><name sortKey="Coughlin, Melissa" sort="Coughlin, Melissa" uniqKey="Coughlin M" first="Melissa" last="Coughlin">Melissa Coughlin</name><affiliation><nlm:aff id="aff4">Department of Microbiology and Immunology, University of Illinois at Chicago, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Prabhakar, Bellur S" sort="Prabhakar, Bellur S" uniqKey="Prabhakar B" first="Bellur S." last="Prabhakar">Bellur S. Prabhakar</name><affiliation><nlm:aff id="aff4">Department of Microbiology and Immunology, University of Illinois at Chicago, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Sleeman, Katrina" sort="Sleeman, Katrina" uniqKey="Sleeman K" first="Katrina" last="Sleeman">Katrina Sleeman</name><affiliation><nlm:aff id="aff3">Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL, USA</nlm:aff></affiliation></author><author><name sortKey="Johnson, Michael E" sort="Johnson, Michael E" uniqKey="Johnson M" first="Michael E." last="Johnson">Michael E. Johnson</name><affiliation><nlm:aff id="aff2">Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D." last="Mesecar">Andrew D. Mesecar</name><affiliation><nlm:aff id="aff2">Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">18796354</idno><idno type="pmc">2745596</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2745596</idno><idno type="RBID">PMC:2745596</idno><idno type="doi">10.1016/j.bmcl.2008.08.082</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">000834</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000834</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors</title><author><name sortKey="Ghosh, Arun K" sort="Ghosh, Arun K" uniqKey="Ghosh A" first="Arun K." last="Ghosh">Arun K. Ghosh</name><affiliation><nlm:aff id="aff1">Departments of Chemistry and Medicinal Chemistry, Purdue University, 560 Oval drive, West Lafayette, IN 47907, USA</nlm:aff></affiliation></author><author><name sortKey="Gong, Gangli" sort="Gong, Gangli" uniqKey="Gong G" first="Gangli" last="Gong">Gangli Gong</name><affiliation><nlm:aff id="aff1">Departments of Chemistry and Medicinal Chemistry, Purdue University, 560 Oval drive, West Lafayette, IN 47907, USA</nlm:aff></affiliation></author><author><name sortKey="Grum Tokars, Valerie" sort="Grum Tokars, Valerie" uniqKey="Grum Tokars V" first="Valerie" last="Grum-Tokars">Valerie Grum-Tokars</name><affiliation><nlm:aff id="aff2">Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Mulhearn, Debbie C" sort="Mulhearn, Debbie C" uniqKey="Mulhearn D" first="Debbie C." last="Mulhearn">Debbie C. Mulhearn</name><affiliation><nlm:aff id="aff2">Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Baker, Susan C" sort="Baker, Susan C" uniqKey="Baker S" first="Susan C." last="Baker">Susan C. Baker</name><affiliation><nlm:aff id="aff3">Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL, USA</nlm:aff></affiliation></author><author><name sortKey="Coughlin, Melissa" sort="Coughlin, Melissa" uniqKey="Coughlin M" first="Melissa" last="Coughlin">Melissa Coughlin</name><affiliation><nlm:aff id="aff4">Department of Microbiology and Immunology, University of Illinois at Chicago, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Prabhakar, Bellur S" sort="Prabhakar, Bellur S" uniqKey="Prabhakar B" first="Bellur S." last="Prabhakar">Bellur S. Prabhakar</name><affiliation><nlm:aff id="aff4">Department of Microbiology and Immunology, University of Illinois at Chicago, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Sleeman, Katrina" sort="Sleeman, Katrina" uniqKey="Sleeman K" first="Katrina" last="Sleeman">Katrina Sleeman</name><affiliation><nlm:aff id="aff3">Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL, USA</nlm:aff></affiliation></author><author><name sortKey="Johnson, Michael E" sort="Johnson, Michael E" uniqKey="Johnson M" first="Michael E." last="Johnson">Michael E. Johnson</name><affiliation><nlm:aff id="aff2">Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</nlm:aff></affiliation></author><author><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D." last="Mesecar">Andrew D. Mesecar</name><affiliation><nlm:aff id="aff2">Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</nlm:aff></affiliation></author></analytic><series><title level="j">Bioorganic & Medicinal Chemistry Letters</title><idno type="ISSN">0960-894X</idno><idno type="eISSN">1464-3405</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Graphical abstract</title><p>Design, synthesis and biological evaluation of a series of chloropyridyl ester-derived SARS-CoV 3CLpro Inhibitors are described Inhibitors <bold>10</bold> has shown potent activity in both enzyme inhibitory and antiviral assays</p><fig id="d32e1103" position="anchor"><graphic xlink:href="fx1"></graphic></fig></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="He, J F" uniqKey="He J">J.-F. He</name></author><author><name sortKey="Peng, G W" uniqKey="Peng G">G.-W. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Bioorg Med Chem Lett</journal-id><journal-id journal-id-type="iso-abbrev">Bioorg. Med. Chem. Lett</journal-id><journal-title-group><journal-title>Bioorganic & Medicinal Chemistry Letters</journal-title></journal-title-group><issn pub-type="ppub">0960-894X</issn><issn pub-type="epub">1464-3405</issn><publisher><publisher-name>Elsevier Ltd.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">18796354</article-id><article-id pub-id-type="pmc">2745596</article-id><article-id pub-id-type="publisher-id">S0960-894X(08)00986-4</article-id><article-id pub-id-type="doi">10.1016/j.bmcl.2008.08.082</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Ghosh</surname><given-names>Arun K.</given-names></name><email>akghosh@purdue.edu</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib><contrib contrib-type="author"><name><surname>Gong</surname><given-names>Gangli</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Grum-Tokars</surname><given-names>Valerie</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Mulhearn</surname><given-names>Debbie C.</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Baker</surname><given-names>Susan C.</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Coughlin</surname><given-names>Melissa</given-names></name><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Prabhakar</surname><given-names>Bellur S.</given-names></name><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Sleeman</surname><given-names>Katrina</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Johnson</surname><given-names>Michael E.</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Mesecar</surname><given-names>Andrew D.</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Departments of Chemistry and Medicinal Chemistry, Purdue University, 560 Oval drive, West Lafayette, IN 47907, USA</aff><aff id="aff2"><label>b</label>Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA</aff><aff id="aff3"><label>c</label>Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL, USA</aff><aff id="aff4"><label>d</label>Department of Microbiology and Immunology, University of Illinois at Chicago, IL 60607, USA</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Tel.: +1 765 494 5323; fax: +1 765 496 1612. <email>akghosh@purdue.edu</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>28</day><month>8</month><year>2008</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><day>15</day><month>10</month><year>2008</year></pub-date><pub-date pub-type="epub"><day>28</day><month>8</month><year>2008</year></pub-date><volume>18</volume><issue>20</issue><fpage>5684</fpage><lpage>5688</lpage><history><date date-type="received"><day>15</day><month>7</month><year>2008</year></date><date date-type="rev-recd"><day>12</day><month>8</month><year>2008</year></date><date date-type="accepted"><day>15</day><month>8</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © 2008 Elsevier Ltd. All rights reserved.</copyright-statement><copyright-year>2008</copyright-year><copyright-holder>Elsevier Ltd</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract abstract-type="graphical"><title>Graphical abstract</title><p>Design, synthesis and biological evaluation of a series of chloropyridyl ester-derived SARS-CoV 3CLpro Inhibitors are described Inhibitors <bold>10</bold> has shown potent activity in both enzyme inhibitory and antiviral assays</p><fig id="d32e1103" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract><p>Design, synthesis and biological evaluation of a series of 5-chloropyridine ester-derived severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. Position of the carboxylate functionality is critical to potency. Inhibitor <bold>10</bold> with a 5-chloropyridinyl ester at position 4 of the indole ring is the most potent inhibitor with a SARS-CoV 3CLpro IC<sub>50</sub> value of 30 nM and an antiviral EC<sub>50</sub> value of 6.9 μM. Molecular docking studies have provided possible binding modes of these inhibitors.</p></abstract><kwd-group><title>Keywords</title><kwd>Synthesis</kwd><kwd>SARS 3CLpro</kwd><kwd>Ester</kwd><kwd>Antiviral</kwd><kwd>Inhibitor</kwd></kwd-group></article-meta></front><body><p>Since its first appearance in southern China in late 2002, severe acute respiratory syndrome (SARS) has been recognized as a global threat.<xref rid="bib1" ref-type="bibr"><sup>1</sup></xref> It has affected more than 8000 individuals in 32 countries and caused nearly 800 fatalities worldwide within a few months.<xref rid="bib2" ref-type="bibr"><sup>2</sup></xref> Its causative pathogen is a novel coronavirus and termed as SARS-CoV.<xref rid="bib3" ref-type="bibr">3</xref>, <xref rid="bib4" ref-type="bibr">4</xref> While SARS is contained in the world and no more cases have been reported since April 2004, there is expectation that this epidemic will strike again in an even more severe form. Furthermore, the nature of its unpredictable outbreak is a potential threat to the global economy and public health. To date, no effective therapy exists for this viral illness.</p><p>The SARS coronavirus is a positive-strand RNA virus. The 5′ two-thirds of the genome encodes two overlapping polyproteins, pp1a and pp1ab, which are processed to generate the viral replication complex. During viral replication, the replicase polyprotein undergoes extensive processing by two viral proteases namely, chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro).<xref rid="bib5" ref-type="bibr">5</xref>, <xref rid="bib6" ref-type="bibr">6</xref> Because of their essential roles in viral replication, both proteases are recognized as attractive targets for development of anti-SARS therapeutics.<xref rid="bib7" ref-type="bibr"><sup>7</sup></xref> The structure and activity of the active sites of both SARS-CoV 3CLpro and SARS-CoV PLpro have been elucidated. Thus far, inhibitor design efforts are mostly limited to SARS-CoV 3CLpro and numerous covalent and noncovalent inhibitors have been reported.<xref rid="bib7" ref-type="bibr"><sup>7</sup></xref> In our continuing interest in the design and development of SARS-CoV 3CLpro inhibitors, we recently reported structure-based design of a number of potent peptidomimetic SARS-CoV 3CLpro inhibitors (<bold>1</bold> and <bold>2</bold>).<xref rid="bib8" ref-type="bibr"><sup>8</sup></xref> The SARS-CoV 3CLpro active site contains a catalytic dyad where a cysteine residue acts as a nucleophile and a histidine residue acts as the general acid base.<xref rid="bib9" ref-type="bibr"><sup>9</sup></xref> The inhibitors bind to SARS-CoV 3CLpro through a covalent bond with the active site Cys-145 residue. These inhibitors contain peptidomimetic scaffolds and lacked adequate potency, particularly antiviral activity suitable for drug-development. Recently, Wong and co-workers reported a new class of potent small molecule benzotriazole ester-based 3CLpro inhibitors. Compound <bold>3</bold> is the most potent inhibitor among the benzotriazole esters.<xref rid="bib10" ref-type="bibr"><sup>10</sup></xref> The mode of action involves acylation of the active site Cys-145 assisted by the catalytic dyad. This irreversible enzyme acylation was verified by electrospray ionization mass spectrometry of the inhibited enzyme. While these inhibitors have shown very impressive SARS-CoV 3CLpro enzyme inhibitory activity, their antiviral activity required improvement.<xref rid="bib11" ref-type="bibr"><sup>11</sup></xref> It seems the indole-5-carboxylate moiety plays an important role in binding with the enzyme active site. Another class of hetereoaromatic ester inhibitors was also identified and studied.<xref rid="bib12" ref-type="bibr">12</xref>, <xref rid="bib13" ref-type="bibr">13</xref> The 5-chloropyridine moiety in <bold>4</bold> proved to be the key unit for the activity against 3CLpro. The report however lacked antiviral data. We report herein the development of 3-chloropyridyl ester-based SARS-CoV 3CLpro inhibitors that exhibit potent enzyme inhibitory activity as well as very good SARS-CoV antiviral activity in cell culture assays. We have also carried out molecular docking studies to obtain the potential binding mode of these inhibitors.</p><p>The general synthetic method for 5-chloropyridyl ester inhibitors is outlined in <xref rid="sch1" ref-type="fig">Scheme 1</xref>. Various chloro-3-pyridinyl esters <bold>5</bold>, <bold>9</bold>, <bold>10</bold>, <bold>12</bold>–<bold>14</bold> (<xref rid="tbl1" ref-type="table">Table 1</xref>) were synthesized by esterification of 5-chloro-3-pyridinol and the corresponding carboxylic acids<xref rid="bib14" ref-type="bibr"><sup>14</sup></xref> mediated by DCC and DMAP at 23 °C in CH<sub>2</sub>Cl<sub>2</sub>. The synthesis of 1-acetylindolecarboxylate inhibitors were carried out by acetylation of indole <bold>5</bold> and <bold>10</bold> with acetic anhydride and pyridine under reflux to provide amide <bold>6</bold> and <bold>11</bold>, respectively, in excellent yields.<fig id="sch1"><label>Scheme 1</label><caption><p>Synthesis of inhibitors <bold>5</bold>, <bold>6</bold>, <bold>9</bold>–<bold>14</bold>.</p></caption><graphic xlink:href="gr4"></graphic></fig><table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>Structures and activity of inhibitors</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Compound structure</th><th>SARS 3CLpro IC<sub>50</sub> (μM)</th><th>SARS-CoV EC<sub>50</sub> (μM)<xref rid="tblfn1" ref-type="table-fn">a</xref></th></tr></thead><tbody><tr><td><inline-graphic xlink:href="fx2.gif"></inline-graphic></td><td>0.2</td><td>NT<xref rid="tblfn2" ref-type="table-fn">b</xref></td></tr><tr><td><inline-graphic xlink:href="fx3.gif"></inline-graphic></td><td>0.31 ± 0.05</td><td>24 ± 0.9</td></tr><tr><td><inline-graphic xlink:href="fx4.gif"></inline-graphic></td><td>0.40 ± 0.06</td><td>NI<xref rid="tblfn3" ref-type="table-fn">c</xref></td></tr><tr><td><inline-graphic xlink:href="fx5.gif"></inline-graphic></td><td>0.37 ± 0.06</td><td>NT</td></tr><tr><td><inline-graphic xlink:href="fx6.gif"></inline-graphic></td><td>0.089 ± 0.014</td><td>NT</td></tr><tr><td><inline-graphic xlink:href="fx7.gif"></inline-graphic></td><td>0.23 ± 0.04</td><td>>25</td></tr><tr><td><inline-graphic xlink:href="fx8.gif"></inline-graphic></td><td>0.03 ± 0.01</td><td>6.9 ± 0.9</td></tr><tr><td><inline-graphic xlink:href="fx9.gif"></inline-graphic></td><td>1.08 ± 0.24</td><td>NI</td></tr><tr><td><inline-graphic xlink:href="fx10.gif"></inline-graphic></td><td>0.08 ± 0.02</td><td>12.1 ± 1.6</td></tr><tr><td><inline-graphic xlink:href="fx11.gif"></inline-graphic></td><td>>100</td><td>NT</td></tr><tr><td><inline-graphic xlink:href="fx12.gif"></inline-graphic></td><td>0.14 ± 0.017</td><td>NT</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>a</label><p>For assay protocol, see Ref. <xref rid="bib18" ref-type="bibr">18</xref>.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tblfn2"><label>b</label><p>NI = no inhibtion.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tblfn3"><label>c</label><p>NT = not tested.</p></fn></table-wrap-foot></table-wrap></p><p>The synthesis of 1-sulfonylindolecarboxylate inhibitors is outlined in <xref rid="sch2" ref-type="fig">Scheme 2</xref>. Direct sulfonamidation of the indole under regular TsCl/DMAP condition at 23 °C or higher temperatures could not provide the desired product. To increase its reactivity, the indole <bold>15</bold> was reduced to indoline <bold>16</bold> by sodium cyanoborohydride in excellent yield.<xref rid="bib15" ref-type="bibr"><sup>15</sup></xref> The resulting indoline readily reacted with tosyl chloride or 3-nitrobenzenesulfonyl chloride to give sulfonamides <bold>17</bold> or <bold>18</bold> in good yields. Oxidation of indolines <bold>17</bold> and <bold>18</bold> to their corresponding indoles <bold>19</bold> and <bold>20</bold>, respectively, was achieved using manganese dioxide at high temperature.<xref rid="bib16" ref-type="bibr"><sup>16</sup></xref> Hydrolysis of the methyl esters to the corresponding acids <bold>21</bold> or <bold>22</bold> using sodium hydroxide followed by the general esterification method described in <xref rid="sch1" ref-type="fig">Scheme 1</xref> afforded the target compounds <bold>7</bold> or <bold>8</bold>.<fig id="sch2"><label>Scheme 2</label><caption><p>Synthesis of inhibitors <bold>7</bold> and <bold>8</bold>.</p></caption><graphic xlink:href="gr5"></graphic></fig></p><p>The structure and activity of inhibitors are shown in <xref rid="tbl1" ref-type="table">Table 1</xref>. The enzyme inhibitory activity of the active esters against SARS-CoV-3CLpro was determined using the full-length, authentic version of the enzyme in a FRET-based, microplate assay described by Grum-Tokars and co-workers.<xref rid="bib8" ref-type="bibr">8</xref>, <xref rid="bib17" ref-type="bibr">17</xref> The assays were performed in 96-well microplates using a reaction volume of 100 μL which contained 50 mM HEPES, pH 7.5, 100 nM authentic SARS-CoV-3CLpro enzyme, 1 mM DTT, 0.01 mg/mL BSA and varying concentrations of inhibitors. The reaction components, with the exception of substrate, were incubated for 20 min and the reaction was initiated by the addition of FRET-substrate HiLyte Fluor™ 488-Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Arg-Lys-Ala-Lys(QXL520™)-NH<sub>2</sub>, giving a final substrate concentration of 2 μM as described.<xref rid="bib8" ref-type="bibr">8</xref>, <xref rid="bib17" ref-type="bibr">17</xref> The IC<sub>50</sub> values for inhibitors were determined by measuring the rates of reaction with increasing inhibitor concentrations.</p><p>As shown in <xref rid="tbl1" ref-type="table">Table 1</xref>, the known<xref rid="bib10" ref-type="bibr"><sup>10</sup></xref> benzotriazole ester inhibitor <bold>3</bold> was evaluated in our assay as a control. In inhibitor <bold>5</bold>, the benzotriazole unit was replaced by a 5-chloropyridine unit. Inhibitor <bold>5</bold> has shown comparable enzymatic inhibitory potency (IC<sub>50</sub> 0.31 μM) as that of <bold>3</bold>. However, inhibitor <bold>3</bold> did not exhibit any antiviral activity while 5-chloropyridyl ester <bold>5</bold> exhibited antiviral activity with an EC<sub>50</sub> value of 24 μM.<xref rid="bib18" ref-type="bibr"><sup>18</sup></xref> When the indole nitrogen was acetylated, the resulting compound <bold>6</bold> remained quite potent (IC<sub>50</sub> of 0.40 μM) as did the tosylated indole <bold>7</bold> (IC<sub>50</sub> of 0.37 μM). Interestingly, its nitrobenzenesulfonamide analog <bold>8</bold> shows an improved IC<sub>50</sub> value (89 nM). We then investigated the importance of the carboxylic position on the benzene ring of indole. Accordingly, carboxylate substitution on indole rings at 5, 6, 4 and 7 positions resulted in chloropyridinyl esters <bold>5</bold>, <bold>9</bold>, <bold>10</bold> and <bold>12</bold>, respectively. These inhibitors were evaluated and as it turned out, inhibitor <bold>10</bold>, with a carboxylate at the 4-position, was the most potent inhibitor with an IC<sub>50</sub> of 30 nM, a 10-fold potency enhancement over <bold>5</bold> containing a carboxylate at the 5-position. Compound <bold>10</bold> also shows the best SARS-CoV antiviral activity with an EC<sub>50</sub> value of 6.9 μM. Acylation of <bold>10</bold>–<bold>11</bold> resulted in a 30-fold loss of potency. Penicillin-derived chloropyridine <bold>13</bold> did not show any appreciable activity. Tetrahydroisoquinoline derivative <bold>14</bold>, however, exhibited an enzyme IC<sub>50</sub> value of 0.14 μM. In general, an indole with a free nitrogen is more potent than its corresponding protected analogue.</p><p>To confirm that 3CLpro is covalently modified by <bold>10</bold>, we determined enzyme modification using MALDI-TOF.<xref rid="bib19" ref-type="bibr"><sup>19</sup></xref> Authentic SARS-CoV 3CLpro was incubated with compound <bold>10</bold> for 20 min and then analyzed in comparison with untreated enzyme. A shift of approximately 217 Da was observed after treatment of SARS-CoV 3CLpro with the inhibitor confirming covalent modification. Covalent modification by similar reactive esters has also been reported.<xref rid="bib13a" ref-type="bibr">(a)</xref>, <xref rid="bib20" ref-type="bibr">20</xref></p><p>To obtain molecular insight into the binding properties of these active ester-based inhibitors, we conducted docking studies in the 3CLpro active site. GOLD3.2<xref rid="bib21" ref-type="bibr"><sup>21</sup></xref> was used to dock our most active compound, <bold>10</bold> (GRL-0496), into the active site of the authentic SARS-CoV 3CLpro structure (PDBID: 2HOB).<xref rid="bib22" ref-type="bibr"><sup>22</sup></xref> In search of obtaining a model of the associated complex between the unreacted ester and protein, (i.e. prior to nucleophilic attack by Cys-145), the distance between the carbonyl carbon atom of <bold>10</bold> and the sulfur atom of Cys-145 was constrained to be in the range of 2.5–3.5 Å. This pre-reaction or ‘collision complex’ is shown in <xref rid="fig1" ref-type="fig">Figures 1</xref>and <xref rid="fig2" ref-type="fig">2</xref>, and resulted in a distance of 2.8 Å between the carbonyl carbon of <bold>10</bold> and the sulfur of Cys-145. This orientation of the ligand has the chloropyridyl group situated in the S1 pocket, with the chloro group pointing towards the surface of the protein. The nitrogen of the chloropyridinyl leaving group is in close proximity (2.4 Å) to the imidazole nitrogen of His-163. The carbonyl oxygen is situated between three backbone nitrogens, forming three hydrogen bonds. As shown in <xref rid="fig2" ref-type="fig">Figure 2</xref>: the first hydrogen bond is from Cys-145(NH) (2.3 Å), the second is from Ser-144(NH) (2.4 Å), and the third is from Gly-143(NH) (2.8 Å). This suggests that a fairly strong hydrogen bonding network is present within the active site which likely aids in positioning and stabilizing the carbonyl group of the ester for nucleophilic attack by the Cys-145. The indole group of <bold>10</bold> is positioned near the more hydrophobic S2 pocket, with the indole nitrogen likely interacting with the imidazole group of His-41, see <xref rid="fig2" ref-type="fig">Figure 2</xref>.<fig id="fig1"><label>Figure 1</label><caption><p>Structures of SARS-CoV 3CLpro inhibitors.</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="fig2"><label>Figure 2</label><caption><p>Relaxed stereoview of the GOLD conformation of an associated complex between <bold>10</bold> (green) and 3CLpro (PDBID: 2HOB), with residues shown in magenta.</p></caption><graphic xlink:href="gr2"></graphic></fig></p><p>Next, we analyzed the interaction between <bold>10</bold> and 3CLpro in the ‘post-reaction’ or covalently modified state. The product of the reaction of <bold>10</bold> with 3CLpro was docked using a more recently released 3CLpro crystal structure, (PDBID: 2V6N).<xref rid="bib20" ref-type="bibr"><sup>20</sup></xref> This crystal structure contains a benzotriazole ester molecule which has reacted with the thiol of Cys-145, forming a covalently bound ligand similar to the compounds presented in this paper. We have used this crystal structure for the post-reaction complex due to the notable movement of the His-41 in 2V6N, which flips and is able to π stack with the aromatic moiety of the smaller covalently bound ligand.<xref rid="bib20" ref-type="bibr"><sup>20</sup></xref> GOLD3.2<xref rid="bib21" ref-type="bibr"><sup>21</sup></xref> was chosen again for generating this model, as it has the capability for docking covalently bound ligands.</p><p><xref rid="fig3" ref-type="fig">Figure 3</xref>is a docked model of <bold>10</bold> covalently attached to the Cys-145 sulfur atom. The model suggests that the indole group of <bold>10</bold> shifts and positions itself where the leaving group was in the complex, more towards the S1 pocket. This positioning of inhibitor <bold>10</bold> in the complex is not surprising as a similar orientation has been proposed before by James’ group for similar esters.<xref rid="bib23" ref-type="bibr">23</xref>, <xref rid="bib24" ref-type="bibr">24</xref> More importantly though is the obvious π–π stacking of the indolyl of <bold>10</bold> with the imidazole ring of His-41, which is also seen in the benzotriazole group of the referenced crystal structure (2V6N).<xref rid="bib20" ref-type="bibr"><sup>20</sup></xref> There is approximately 4 Å between the aromatic rings of the indolyl and the imidazole of His-41. This interaction clearly determines the position of the indolyl group of our compounds. Other residues shown in <xref rid="fig3" ref-type="fig">Figure 3</xref>, such as Asp-187 and Gln-189 are more than 5 Å away from the indolyl, but might come into play with larger substituents, such as in compound <bold>11</bold>.<fig id="fig3"><label>Figure 3</label><caption><p>GOLD docked conformation of <bold>10</bold> (green), covalently linked to Cys-145 of 3CLpro based on the 2V6N 3CLpro structure.<xref rid="bib20" ref-type="bibr"><sup>20</sup></xref></p></caption><graphic xlink:href="gr3"></graphic></fig></p><p>The results of the docking studies presented here suggest that the indole group of <bold>10</bold> can potentially occupy two different binding pockets during the course of the reaction. Dynamic enzymatic rearrangement in the vicinity of Cys-145 have recently been suggested from the X-ray structure of SARS-CoV 3CLpro that had been reacted with 1-(4-dimethylaminobenzoyloxyl)-benzotriazole,<xref rid="bib20" ref-type="bibr"><sup>20</sup></xref> and suggests that after covalently linking to Cys-145, the indolyl of <bold>10</bold> shifts towards the S1 pocket and stacks with the shifted imidazole ring of His-41, locking the orientation, with implications of where substitutions on the indolyl ring would be most beneficial. By having both of these models now available, structural modifications of the indole group can be tailored to enhance interactions with the S2 pocket for the complex formation to readily occur, but at the same time, consider that the ligand must be mobile enough to then occupy the S1 pocket post-reaction.</p><p>In conclusion, our design strategies by combining the key parts of two mechanism-based inhibitors led to a series of 5-chloropyridinyl indolecarboxylate inhibitors with enzymatic potency at submicromolar levels. The position of the carboxylic acid ester is critical to its potency. Indolecarboxylate <bold>10</bold> with a carboxylate functionality at the 4-position is the most potent inhibitor with an enzyme inhibitory activity against SARS-CoV 3CLpro with an IC<sub>50</sub> of 30 nM and antiviral potency with an EC<sub>50</sub> value of 6.9 μM. 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