Discovery of unsymmetrical aromatic disulfides as novel inhibitors of SARS-CoV main protease: Chemical synthesis, biological evaluation, molecular docking and 3D-QSAR study
Identifieur interne : 000F92 ( Pmc/Corpus ); précédent : 000F91; suivant : 000F93Discovery of unsymmetrical aromatic disulfides as novel inhibitors of SARS-CoV main protease: Chemical synthesis, biological evaluation, molecular docking and 3D-QSAR study
Auteurs : Li Wang ; Bo-Bo Bao ; Guo-Qing Song ; Cheng Chen ; Xu-Meng Zhang ; Wei Lu ; Zefang Wang ; Yan Cai ; Shuang Li ; Sheng Fu ; Fu-Hang Song ; Haitao Yang ; Jian-Guo WangSource :
- European Journal of Medicinal Chemistry [ 0223-5234 ] ; 2017.
Abstract
The worldwide outbreak of severe acute respiratory syndrome (SARS) in 2003 had caused a high rate of mortality. Main protease (Mpro) of SARS-associated coronavirus (SARS-CoV) is an important target to discover pharmaceutical compounds for the therapy of this life-threatening disease. During the course of screening new anti-SARS agents, we have identified that a series of unsymmetrical aromatic disulfides inhibited SARS-CoV Mpro significantly for the first time. Herein, 40 novel unsymmetrical aromatic disulfides were synthesized chemically and their biological activities were evaluated
Url:
DOI: 10.1016/j.ejmech.2017.05.045
PubMed: 28624700
PubMed Central: 7115414
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PMC:7115414Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Discovery of unsymmetrical aromatic disulfides as novel inhibitors of SARS-CoV main protease: Chemical synthesis, biological evaluation, molecular docking and 3D-QSAR study</title><author><name sortKey="Wang, Li" sort="Wang, Li" uniqKey="Wang L" first="Li" last="Wang">Li Wang</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author><author><name sortKey="Bao, Bo Bo" sort="Bao, Bo Bo" uniqKey="Bao B" first="Bo-Bo" last="Bao">Bo-Bo Bao</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Song, Guo Qing" sort="Song, Guo Qing" uniqKey="Song G" first="Guo-Qing" last="Song">Guo-Qing Song</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author><author><name sortKey="Chen, Cheng" sort="Chen, Cheng" uniqKey="Chen C" first="Cheng" last="Chen">Cheng Chen</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Zhang, Xu Meng" sort="Zhang, Xu Meng" uniqKey="Zhang X" first="Xu-Meng" last="Zhang">Xu-Meng Zhang</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author><author><name sortKey="Lu, Wei" sort="Lu, Wei" uniqKey="Lu W" first="Wei" last="Lu">Wei Lu</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Zefang" sort="Wang, Zefang" uniqKey="Wang Z" first="Zefang" last="Wang">Zefang Wang</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Cai, Yan" sort="Cai, Yan" uniqKey="Cai Y" first="Yan" last="Cai">Yan Cai</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Li, Shuang" sort="Li, Shuang" uniqKey="Li S" first="Shuang" last="Li">Shuang Li</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Fu, Sheng" sort="Fu, Sheng" uniqKey="Fu S" first="Sheng" last="Fu">Sheng Fu</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Song, Fu Hang" sort="Song, Fu Hang" uniqKey="Song F" first="Fu-Hang" last="Song">Fu-Hang Song</name><affiliation><nlm:aff id="aff4">CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China</nlm:aff></affiliation></author><author><name sortKey="Yang, Haitao" sort="Yang, Haitao" uniqKey="Yang H" first="Haitao" last="Yang">Haitao Yang</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Jian Guo" sort="Wang, Jian Guo" uniqKey="Wang J" first="Jian-Guo" last="Wang">Jian-Guo Wang</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28624700</idno><idno type="pmc">7115414</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115414</idno><idno type="RBID">PMC:7115414</idno><idno type="doi">10.1016/j.ejmech.2017.05.045</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000F92</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000F92</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Discovery of unsymmetrical aromatic disulfides as novel inhibitors of SARS-CoV main protease: Chemical synthesis, biological evaluation, molecular docking and 3D-QSAR study</title><author><name sortKey="Wang, Li" sort="Wang, Li" uniqKey="Wang L" first="Li" last="Wang">Li Wang</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author><author><name sortKey="Bao, Bo Bo" sort="Bao, Bo Bo" uniqKey="Bao B" first="Bo-Bo" last="Bao">Bo-Bo Bao</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Song, Guo Qing" sort="Song, Guo Qing" uniqKey="Song G" first="Guo-Qing" last="Song">Guo-Qing Song</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author><author><name sortKey="Chen, Cheng" sort="Chen, Cheng" uniqKey="Chen C" first="Cheng" last="Chen">Cheng Chen</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Zhang, Xu Meng" sort="Zhang, Xu Meng" uniqKey="Zhang X" first="Xu-Meng" last="Zhang">Xu-Meng Zhang</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author><author><name sortKey="Lu, Wei" sort="Lu, Wei" uniqKey="Lu W" first="Wei" last="Lu">Wei Lu</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Zefang" sort="Wang, Zefang" uniqKey="Wang Z" first="Zefang" last="Wang">Zefang Wang</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Cai, Yan" sort="Cai, Yan" uniqKey="Cai Y" first="Yan" last="Cai">Yan Cai</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Li, Shuang" sort="Li, Shuang" uniqKey="Li S" first="Shuang" last="Li">Shuang Li</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Fu, Sheng" sort="Fu, Sheng" uniqKey="Fu S" first="Sheng" last="Fu">Sheng Fu</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Song, Fu Hang" sort="Song, Fu Hang" uniqKey="Song F" first="Fu-Hang" last="Song">Fu-Hang Song</name><affiliation><nlm:aff id="aff4">CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China</nlm:aff></affiliation></author><author><name sortKey="Yang, Haitao" sort="Yang, Haitao" uniqKey="Yang H" first="Haitao" last="Yang">Haitao Yang</name><affiliation><nlm:aff id="aff2">School of Life Sciences, Tianjin University, Tianjin 300072, China</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Jian Guo" sort="Wang, Jian Guo" uniqKey="Wang J" first="Jian-Guo" last="Wang">Jian-Guo Wang</name><affiliation><nlm:aff id="aff1">State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</nlm:aff></affiliation></author></analytic><series><title level="j">European Journal of Medicinal Chemistry</title><idno type="ISSN">0223-5234</idno><idno type="eISSN">1768-3254</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The worldwide outbreak of severe acute respiratory syndrome (SARS) in 2003 had caused a high rate of mortality. Main protease (M<sup>pro</sup>) of SARS-associated coronavirus (SARS-CoV) is an important target to discover pharmaceutical compounds for the therapy of this life-threatening disease. During the course of screening new anti-SARS agents, we have identified that a series of unsymmetrical aromatic disulfides inhibited SARS-CoV M<sup>pro</sup> significantly for the first time. Herein, 40 novel unsymmetrical aromatic disulfides were synthesized chemically and their biological activities were evaluated <italic>in vitro</italic> against SARS-CoV M<sup>pro</sup>. These novel compounds displayed excellent <italic>IC</italic><sub>50</sub> data in the range of 0.516–5.954 μM. Preliminary studies indicated that these disulfides are reversible and mpetitive inhibitors. A possible binding mode was generated via molecular docking simulation and a comparative field analysis (CoMFA) model was constructed to understand the structure-activity relationships. The present research therefore has provided some meaningful guidance to design and identify anti-SARS drugs with totally new chemical structures.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="World Health Organization" uniqKey="World Health Organization">World Health Organization</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goetz, D H" uniqKey="Goetz D">D.H. Goetz</name></author><author><name sortKey="Choe, Y" uniqKey="Choe Y">Y. Choe</name></author><author><name sortKey="Hansell, E" uniqKey="Hansell E">E. Hansell</name></author><author><name sortKey="Chen, Y T" uniqKey="Chen Y">Y.T. Chen</name></author><author><name sortKey="Mcdowell, M" uniqKey="Mcdowell M">M. McDowell</name></author><author><name sortKey="Jonsson, C B" uniqKey="Jonsson C">C.B. Jonsson</name></author><author><name sortKey="Roush, W R" uniqKey="Roush W">W.R. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Eur J Med Chem</journal-id><journal-id journal-id-type="iso-abbrev">Eur J Med Chem</journal-id><journal-title-group><journal-title>European Journal of Medicinal Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0223-5234</issn><issn pub-type="epub">1768-3254</issn><publisher><publisher-name>Elsevier Masson SAS.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28624700</article-id><article-id pub-id-type="pmc">7115414</article-id><article-id pub-id-type="publisher-id">S0223-5234(17)30406-3</article-id><article-id pub-id-type="doi">10.1016/j.ejmech.2017.05.045</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Discovery of unsymmetrical aromatic disulfides as novel inhibitors of SARS-CoV main protease: Chemical synthesis, biological evaluation, molecular docking and 3D-QSAR study</article-title></title-group><contrib-group><contrib contrib-type="author" id="au1"><name><surname>Wang</surname><given-names>Li</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Bao</surname><given-names>Bo-Bo</given-names></name><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff3" ref-type="aff">c</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>Song</surname><given-names>Guo-Qing</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Chen</surname><given-names>Cheng</given-names></name><email>chengchen@tju.edu.cn</email><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff3" ref-type="aff">c</xref><xref rid="cor2" ref-type="corresp">∗∗</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Zhang</surname><given-names>Xu-Meng</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Lu</surname><given-names>Wei</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au7"><name><surname>Wang</surname><given-names>Zefang</given-names></name><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au8"><name><surname>Cai</surname><given-names>Yan</given-names></name><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au9"><name><surname>Li</surname><given-names>Shuang</given-names></name><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au10"><name><surname>Fu</surname><given-names>Sheng</given-names></name><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au11"><name><surname>Song</surname><given-names>Fu-Hang</given-names></name><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author" id="au12"><name><surname>Yang</surname><given-names>Haitao</given-names></name><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au13"><name><surname>Wang</surname><given-names>Jian-Guo</given-names></name><email>nkwjg@nankai.edu.cn</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib></contrib-group><aff id="aff1"><label>a</label>State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin 300071, China</aff><aff id="aff2"><label>b</label>School of Life Sciences, Tianjin University, Tianjin 300072, China</aff><aff id="aff3"><label>c</label>Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China</aff><aff id="aff4"><label>d</label>CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author. <email>nkwjg@nankai.edu.cn</email></corresp><corresp id="cor2"><label>∗∗</label>Corresponding author. School of Life Sciences, Tianjin University, Tianjin 300072, China. <email>chengchen@tju.edu.cn</email></corresp><fn id="fn1"><label>1</label><p id="ntpara0010">These two authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>9</day><month>6</month><year>2017</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>8</day><month>9</month><year>2017</year></pub-date><pub-date pub-type="epub"><day>9</day><month>6</month><year>2017</year></pub-date><volume>137</volume><fpage>450</fpage><lpage>461</lpage><history><date date-type="received"><day>27</day><month>2</month><year>2017</year></date><date date-type="rev-recd"><day>17</day><month>5</month><year>2017</year></date><date date-type="accepted"><day>21</day><month>5</month><year>2017</year></date></history><permissions><copyright-statement>© 2017 Elsevier Masson SAS. All rights reserved.</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>Elsevier Masson SAS</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 id="abs0010"><p>The worldwide outbreak of severe acute respiratory syndrome (SARS) in 2003 had caused a high rate of mortality. Main protease (M<sup>pro</sup>) of SARS-associated coronavirus (SARS-CoV) is an important target to discover pharmaceutical compounds for the therapy of this life-threatening disease. During the course of screening new anti-SARS agents, we have identified that a series of unsymmetrical aromatic disulfides inhibited SARS-CoV M<sup>pro</sup> significantly for the first time. Herein, 40 novel unsymmetrical aromatic disulfides were synthesized chemically and their biological activities were evaluated <italic>in vitro</italic> against SARS-CoV M<sup>pro</sup>. These novel compounds displayed excellent <italic>IC</italic><sub>50</sub> data in the range of 0.516–5.954 μM. Preliminary studies indicated that these disulfides are reversible and mpetitive inhibitors. A possible binding mode was generated via molecular docking simulation and a comparative field analysis (CoMFA) model was constructed to understand the structure-activity relationships. The present research therefore has provided some meaningful guidance to design and identify anti-SARS drugs with totally new chemical structures.</p></abstract><abstract abstract-type="graphical" id="abs0015"><title>Graphical abstract</title><p><fig id="undfig1" position="anchor"><alt-text id="alttext0010">Image 1</alt-text><graphic xlink:href="fx1_lrg"></graphic></fig></p></abstract><abstract abstract-type="author-highlights" id="abs0020"><title>Highlights</title><p><list list-type="simple" id="ulist0010"><list-item id="u0010"><label>•</label><p id="p0010">40 novel unsymmetrical aromatic disulfides were synthesized.</p></list-item><list-item id="u0015"><label>•</label><p id="p0015">The synthesized disulfide compounds are potent inhibitors of SARS main protease.</p></list-item><list-item id="u0020"><label>•</label><p id="p0020">Possible binding mode and structure-activity relationships of the compounds were established.</p></list-item></list></p></abstract><kwd-group id="kwrds0010"><title>Keywords</title><kwd>SARS-CoV M<sup>pro</sup></kwd><kwd>Aromatic disulfide</kwd><kwd>Molecular docking</kwd><kwd><italic>In vitro</italic> activity</kwd></kwd-group></article-meta></front><body><sec id="sec1"><label>1</label><title>Introduction</title><p id="p0025">Severe acute respiratory syndrome (SARS) is a highly infective respiratory disease caused by SARS coronavirus (SARS-CoV). Its sudden emergence and rapid outbreak during 2002–2003 had resulted in ∼800 deaths among >8000 reported individual cases worldwide <xref rid="bib1" ref-type="bibr">[1]</xref>. Although the SARS epidemic had been under control for years, reemergence of this threatening illness is still a possible risk and potentially new strains of SARS can be more dangerous than the previous ones. A number of important targets have been recognized to take part in the biological events critical to SARS-CoV replication, among which a papain-like protease (PL<sup>pro</sup>) and a chymotrypsin-like protease (3CL<sup>pro</sup>) are of significant importance to design anti-SARS inhibitors <xref rid="bib2" ref-type="bibr">[2]</xref>. The 3CL<sup>pro</sup>, also known as the main protease (M<sup>pro</sup>), has attracted much attention, which could be revealed from numerous publications about novel inhibitor discovery. Crystal structures of SARS-CoV M<sup>pro</sup>, either free enzyme alone or in complex with an inhibitor, had been determined to facilitate the structural and functional investigation of this protease <xref rid="bib3" ref-type="bibr">[3]</xref>, <xref rid="bib4" ref-type="bibr">[4]</xref>. The active site of SARS-CoV M<sup>pro</sup> contains Cys145 and His41 to constitute a catalytic dyad, in which cysteine functions as the common nucleophile in the proteolytic process.</p><p id="p0030">Biological active inhibitors against SARS-CoV M<sup>pro</sup> have been reported mainly from two different approaches: one is screening large library to identify new active compounds using high-throughput technique, the other is novel inhibitor design based on the substrate structure or active site properties rationally <xref rid="bib5" ref-type="bibr">[5]</xref>. The inhibitory activities of these compounds were then validated by <italic>in vitro</italic> protease assays. In most cases the kinetic study indicated that the inhibitor is involved in an irreversible process by forming a covalent bond with Cys145, while in some other cases the inhibition is actually a reversible behavior. The reported SARS-CoV M<sup>pro</sup> inhibitors covered a variety of different chemical scaffolds, which contain peptidomimetic compounds, 3-quinoline carboxylic acid derivatives, thiophene-2-carboxylate derivatives, zinc-conjugated compounds, cinanserin, calmodulin, keto-glutamine analogues, anilide, bifunctional boronic acid compounds, isatin derivatives, etacrynic acid derivatives, serine derivatives, trifluoromethyl ketones, acetamides, pyrazolone and quercertins <xref rid="bib5" ref-type="bibr">[5]</xref>, <xref rid="bib6" ref-type="bibr">[6]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>, <xref rid="bib8" ref-type="bibr">[8]</xref>, <xref rid="bib9" ref-type="bibr">[9]</xref>, <xref rid="bib10" ref-type="bibr">[10]</xref>, <xref rid="bib11" ref-type="bibr">[11]</xref>, <xref rid="bib12" ref-type="bibr">[12]</xref>. It is a pity that research on drugs and vaccines towards SARS or SARS-like coronavirus has not brought any candidate for clinical use. Hence there still exists an urgent need to discover and identify new SARS-CoV M<sup>pro</sup> agents, especially those compounds from totally new chemical families, to develop effective therapy against this fatal viral infection.</p><p id="p0035">Disulfide bonds play essential roles for bioactive proteins to keep correct folding <xref rid="bib13" ref-type="bibr">[13]</xref>. There are a few cases that simple disulfides such as diallyl disulfide and dimethyl disulfide exhibit hypochlorous acid scavenging activity and tyrosinase inhibitory activity (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A) <xref rid="bib14" ref-type="bibr">[14]</xref>, <xref rid="bib15" ref-type="bibr">[15]</xref>. The unsymmetrical disulfide compounds are useful tools in the research of dynamic combinatorial chemistry <xref rid="bib16" ref-type="bibr">[16]</xref>. These compounds have also been reported to display a variety of biological activities. For examples, Turos et al. reported that some unsymmetrical aryl-alkyl disulfides were inhibitors of methicillin-resistant <italic>Staphylococcus aureus</italic> and <italic>Bacillus anthracis</italic> (<xref rid="fig1" ref-type="fig">Fig. 1</xref>B) <xref rid="bib17" ref-type="bibr">[17]</xref>, while Khosla and co-workers published some unsymmetrical disulfides that could selectively inhibit extracellular thioredoxin (<xref rid="fig1" ref-type="fig">Fig. 1</xref>C) <xref rid="bib18" ref-type="bibr">[18]</xref>. Yoon et al. showed that some unsymmetrical disulfide compounds were inhibitors of <italic>Mycobacterium tuberculosis</italic> and <italic>Haemophilus influenzae</italic>, by interfering with acetohydroxyacid synthase (AHAS), a key enzyme in the biosynthesis pathway of branched chain amino-acids (<xref rid="fig1" ref-type="fig">Fig. 1</xref>D) <xref rid="bib19" ref-type="bibr">[19]</xref>, <xref rid="bib20" ref-type="bibr">[20]</xref>. In the past of our research, we found that some unsymmetrical aromatic disulfides could inhibit plant AHAS and was useful for herbicide research (<xref rid="fig1" ref-type="fig">Fig. 1</xref>E) <xref rid="bib21" ref-type="bibr">[21]</xref>, <xref rid="bib22" ref-type="bibr">[22]</xref>.<fig id="fig1"><label>Fig. 1</label><caption><p>Different disulfide compounds with various biological activities from literature.</p></caption><alt-text id="alttext0020">Fig. 1</alt-text><graphic xlink:href="gr1_lrg"></graphic></fig></p><p id="p0040">There are indeed a few reports that some aromatic disulfides exhibit antiviral activity. The virucidal activity of NSC4492 was due to targeting of arenavirus RNA synthesis (<xref rid="fig1" ref-type="fig">Fig. 1</xref>F) <xref rid="bib23" ref-type="bibr">[23]</xref>, while 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) had antiviral propierties against T-tropic human immunodeficiency virus type 1 (HIV-1) (<xref rid="fig1" ref-type="fig">Fig. 1</xref>G) <xref rid="bib24" ref-type="bibr">[24]</xref>. An antiviral disulfide NSC20625 compound could block interaction between arenavirus Z protein and cellular promyelocytic leukemia protein (<xref rid="fig1" ref-type="fig">Fig. 1</xref>H) <xref rid="bib25" ref-type="bibr">[25]</xref>. However, the antiviral activities against arenavirus might be different from that against coronavirus. There is no evidence that the reported antiviral activities of aromatic disulfides have any direct relationships with SARS-CoV M<sup>pro</sup> inhibition.</p><p id="p0045">In an effort to discover novel inhibitors of SARS-CoV M<sup>pro</sup>, we have synthesized a series of novel unsymmetrical aromatic disulfides and evaluated their biological activities in this study. The target compounds could inhibit main protease of SARS-CoV remarkably, with the best <italic>IC</italic><sub>50</sub> value of 0.516 μM. Subsequent enzymatic kinetics study indicated that the aromatic disulfides acted as reversible and non-competitive inhibitors. Therefore we have demonstrated that unsymmetrical disulfide compounds with aromatic rings are novel inhibitors of SARS-CoV M<sup>pro</sup> from a totally new chemical family, which will provide helpful information for further drug discovery.</p></sec><sec id="sec2"><label>2</label><title>Results and discussion</title><sec id="sec2.1"><label>2.1</label><title>Chemistry of the target compounds</title><p id="p0050">The target unsymmetrical disulfides were synthesized by the reaction of various substituted 2-mercapto- [1,3,4]oxadiazole, substituted 2-mercapto-thiazole, substituted 2-mercapto- 1<italic>H</italic>-imidazole or substituted 2-mercapto-pyrimidine with substituted arenesulfenyl chloride in ethyl ether under very mild condition as reported previously <xref rid="bib21" ref-type="bibr">[21]</xref>, <xref rid="bib22" ref-type="bibr">[22]</xref>. It is a quite straightforward nucleophilic substitution, in which the thiol group in the mercapto compound serves as a nucleophilic reagent and attacks the sulfur atom in the arenesulfenyl chloride. Most yields for the reactions were satisfactory, showing that this is a simple and easy procedure to produce unsymmetrical aromatic disulfides, superior to the synthetic route developed by Hahn et al. <xref rid="bib26" ref-type="bibr">[26]</xref>. In Hahn's paper, moist tetrahydrofuran gave better yields than dried benzene as the solvent for the reaction, so the authors added 5–10 M equivalent of water to the reaction mixture and observed satisfactory yields. Thus, the presence of water was thought to be preferable for the reaction by Hahn et al. <xref rid="bib26" ref-type="bibr">[26]</xref>. In our synthesis experiment, only ethyl ether was used as the solvent and no additional water was added to the reaction, nevertheless, we observed very high yields for most of the target compounds.</p><p id="p0055">The —S-S- bond had been confirmed in our previous paper <xref rid="bib21" ref-type="bibr">[21]</xref>. Compound <bold>3-4</bold>, <bold>3-6</bold>, <bold>3-7</bold>, <bold>3-8</bold>, <bold>3-9</bold>, <bold>3-10</bold> and <bold>3-11</bold> were further acetylated from corresponding parent compounds that had been published by us <xref rid="bib21" ref-type="bibr">[21]</xref>. The molecular structures of the compounds are listed in <xref rid="tbl1" ref-type="table">Table 1</xref>. The title compounds were fully characterized by <sup>1</sup>H NMR, <sup>13</sup>C NMR and HRMS (<xref rid="appsec1" ref-type="sec">Part 1 of the supplementary data for the original figures</xref>).<table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>The novel unsymmetrical aromatic disulfide compounds and their SARS-CoV M<sup>pro</sup> inhibitory activities (<italic>IC</italic><sub>50</sub>).</p></caption><alt-text id="alttext0055">Table 1</alt-text><table frame="hsides" rules="groups"><thead><tr><th>Entry no.</th><th>Chemical structure</th><th><italic>IC</italic><sub>50</sub> (μM)</th><th>Entry no.</th><th>Chemical structure</th><th><italic>IC</italic><sub>50</sub>(μM)</th></tr></thead><tbody><tr><td><bold>3-1</bold></td><td><inline-graphic xlink:href="fx2_lrg.gif"><alt-text id="alttext0060">Image 2</alt-text></inline-graphic></td><td>1.871 ± 0.071</td><td><bold>3-21</bold></td><td><inline-graphic xlink:href="fx3_lrg.gif"><alt-text id="alttext0065">Image 3</alt-text></inline-graphic></td><td>1.250 ± 0.023</td></tr><tr><td><bold>3-2</bold></td><td><inline-graphic xlink:href="fx4_lrg.gif"><alt-text id="alttext0070">Image 4</alt-text></inline-graphic></td><td>2.803 ± 0.052</td><td><bold>3-22</bold></td><td><inline-graphic xlink:href="fx5_lrg.gif"><alt-text id="alttext0075">Image 5</alt-text></inline-graphic></td><td>2.211 ± 0.152</td></tr><tr><td><bold>3-3</bold></td><td><inline-graphic xlink:href="fx6_lrg.gif"><alt-text id="alttext0080">Image 6</alt-text></inline-graphic></td><td>3.675 ± 0.193</td><td><bold>3-23</bold></td><td><inline-graphic xlink:href="fx7_lrg.gif"><alt-text id="alttext0085">Image 7</alt-text></inline-graphic></td><td>3.321 ± 0.068</td></tr><tr><td><bold>3-4</bold></td><td><inline-graphic xlink:href="fx8_lrg.gif"><alt-text id="alttext0090">Image 8</alt-text></inline-graphic></td><td>3.130 ± 0.052</td><td><bold>3-24</bold></td><td><inline-graphic xlink:href="fx9_lrg.gif"><alt-text id="alttext0095">Image 9</alt-text></inline-graphic></td><td>2.555 ± 0.270</td></tr><tr><td><bold>3-5</bold></td><td><inline-graphic xlink:href="fx10_lrg.gif"><alt-text id="alttext0100">Image 10</alt-text></inline-graphic></td><td>1.506 ± 0.184</td><td><bold>3-25</bold></td><td><inline-graphic xlink:href="fx11_lrg.gif"><alt-text id="alttext0105">Image 11</alt-text></inline-graphic></td><td>2.452 ± 0.126</td></tr><tr><td><bold>3-6</bold></td><td><inline-graphic xlink:href="fx12_lrg.gif"><alt-text id="alttext0110">Image 12</alt-text></inline-graphic></td><td>4.344 ± 0.538</td><td><bold>3-26</bold></td><td><inline-graphic xlink:href="fx13_lrg.gif"><alt-text id="alttext0115">Image 13</alt-text></inline-graphic></td><td>1.679 ± 0.042</td></tr><tr><td><bold>3-7</bold></td><td><inline-graphic xlink:href="fx14_lrg.gif"><alt-text id="alttext0120">Image 14</alt-text></inline-graphic></td><td>4.100 ± 0.832</td><td><bold>3-27</bold></td><td><inline-graphic xlink:href="fx15_lrg.gif"><alt-text id="alttext0125">Image 15</alt-text></inline-graphic></td><td>1.557 ± 0.116</td></tr><tr><td><bold>3-8</bold></td><td><inline-graphic xlink:href="fx16_lrg.gif"><alt-text id="alttext0130">Image 16</alt-text></inline-graphic></td><td>1.762 ± 0.044</td><td><bold>3-28</bold></td><td><inline-graphic xlink:href="fx17_lrg.gif"><alt-text id="alttext0135">Image 17</alt-text></inline-graphic></td><td>1.713 ± 0.052</td></tr><tr><td><bold>3-9</bold></td><td><inline-graphic xlink:href="fx18_lrg.gif"><alt-text id="alttext0140">Image 18</alt-text></inline-graphic></td><td>5.654 ± 0.259</td><td><bold>3-29</bold></td><td><inline-graphic xlink:href="fx19_lrg.gif"><alt-text id="alttext0145">Image 19</alt-text></inline-graphic></td><td>1.118 ± 0.132</td></tr><tr><td><bold>3-10</bold></td><td><inline-graphic xlink:href="fx20_lrg.gif"><alt-text id="alttext0150">Image 20</alt-text></inline-graphic></td><td>4.511 ± 0.105</td><td><bold>3-30</bold></td><td><inline-graphic xlink:href="fx21_lrg.gif"><alt-text id="alttext0155">Image 21</alt-text></inline-graphic></td><td>1.264 ± 0.033</td></tr><tr><td><bold>3-11</bold></td><td><inline-graphic xlink:href="fx22_lrg.gif"><alt-text id="alttext0160">Image 22</alt-text></inline-graphic></td><td>5.794 ± 0.050</td><td><bold>3-31</bold></td><td><inline-graphic xlink:href="fx23_lrg.gif"><alt-text id="alttext0165">Image 23</alt-text></inline-graphic></td><td>0.516 ± 0.060</td></tr><tr><td><bold>3-12</bold></td><td><inline-graphic xlink:href="fx24_lrg.gif"><alt-text id="alttext0170">Image 24</alt-text></inline-graphic></td><td>2.626 ± 0.082</td><td><bold>3-32</bold></td><td><inline-graphic xlink:href="fx25_lrg.gif"><alt-text id="alttext0175">Image 25</alt-text></inline-graphic></td><td>0.921 ± 0.060</td></tr><tr><td><bold>3-13</bold></td><td><inline-graphic xlink:href="fx26_lrg.gif"><alt-text id="alttext0180">Image 26</alt-text></inline-graphic></td><td>1.651 ± 0.048</td><td><bold>3-33</bold></td><td><inline-graphic xlink:href="fx27_lrg.gif"><alt-text id="alttext0185">Image 27</alt-text></inline-graphic></td><td>1.437 ± 0.053</td></tr><tr><td><bold>3-14</bold></td><td><inline-graphic xlink:href="fx28_lrg.gif"><alt-text id="alttext0190">Image 28</alt-text></inline-graphic></td><td>2.075 ± 0.016</td><td><bold>3-34</bold></td><td><inline-graphic xlink:href="fx29_lrg.gif"><alt-text id="alttext0195">Image 29</alt-text></inline-graphic></td><td>1.121 ± 0.060</td></tr><tr><td><bold>3-15</bold></td><td><inline-graphic xlink:href="fx30_lrg.gif"><alt-text id="alttext0200">Image 30</alt-text></inline-graphic></td><td>5.954 ± 0.363</td><td><bold>3-35</bold></td><td><inline-graphic xlink:href="fx31_lrg.gif"><alt-text id="alttext0205">Image 31</alt-text></inline-graphic></td><td>1.991 ± 0.086</td></tr><tr><td><bold>3-16</bold></td><td><inline-graphic xlink:href="fx32_lrg.gif"><alt-text id="alttext0210">Image 32</alt-text></inline-graphic></td><td>3.957 ± 0.190</td><td><bold>3-36</bold></td><td><inline-graphic xlink:href="fx33_lrg.gif"><alt-text id="alttext0215">Image 33</alt-text></inline-graphic></td><td>1.495 ± 0.055</td></tr><tr><td><bold>3-17</bold></td><td><inline-graphic xlink:href="fx34_lrg.gif"><alt-text id="alttext0220">Image 34</alt-text></inline-graphic></td><td>4.126 ± 0.094</td><td><bold>3-37</bold></td><td><inline-graphic xlink:href="fx35_lrg.gif"><alt-text id="alttext0225">Image 35</alt-text></inline-graphic></td><td>0.883 ± 0.028</td></tr><tr><td><bold>3-18</bold></td><td><inline-graphic xlink:href="fx36_lrg.gif"><alt-text id="alttext0230">Image 36</alt-text></inline-graphic></td><td>2.565 ± 0.075</td><td><bold>3-38</bold></td><td><inline-graphic xlink:href="fx37_lrg.gif"><alt-text id="alttext0235">Image 37</alt-text></inline-graphic></td><td>0.684 ± 0.012</td></tr><tr><td><bold>3-19</bold></td><td><inline-graphic xlink:href="fx38_lrg.gif"><alt-text id="alttext0240">Image 38</alt-text></inline-graphic></td><td>1.947 ± 0.508</td><td><bold>3-39</bold></td><td><inline-graphic xlink:href="fx39_lrg.gif"><alt-text id="alttext0245">Image 39</alt-text></inline-graphic></td><td>0.697 ± 0.053</td></tr><tr><td><bold>3-20</bold></td><td><inline-graphic xlink:href="fx40_lrg.gif"><alt-text id="alttext0250">Image 40</alt-text></inline-graphic></td><td>2.029 ± 0.488</td><td><bold>3-40</bold></td><td><inline-graphic xlink:href="fx41_lrg.gif"><alt-text id="alttext0255">Image 41</alt-text></inline-graphic></td><td>1.522 ± 0.214</td></tr></tbody></table></table-wrap></p></sec><sec id="sec2.2"><label>2.2</label><title>In vitro inhibitory activity of SARS-CoV M<sup>pro</sup></title><p id="p0060">All the synthesized unsymmetrical aromatic disulfides were subjected to the <italic>in vitro</italic> assay of SARS-CoV M<sup>pro</sup>. The results are also illustrated in <xref rid="tbl1" ref-type="table">Table 1</xref>, expressed by <italic>IC</italic><sub>50</sub> values. It could be seen that the target compounds exhibited encouraging biological potency, with excellent <italic>IC</italic><sub>50</sub> values ranging from 0.516 μM to 5.954 μM (The inhibition curves of all target compounds can be found in <xref rid="appsec1" ref-type="sec">part 2 of the supplementary data</xref>). This was surprising due to the fact that not any research group had ever identified the disulfide compounds as inhibitors of SARS-CoV M<sup>pro</sup>, not to mention such strong inhibition.</p><p id="p0065">Inhibition type of the disulfides was determined by means of enzymatic kinetic study, for which <bold>3-31</bold> and <bold>3-39</bold> were used. From <xref rid="fig2" ref-type="fig">Fig. 2</xref>it can be seen that in the plot of enzyme concentration versus reaction velocity, the lines represent different inhibitor concentrations intersect at a same point, suggesting that the inhibition is actually a reversible action. We then measured the enzymatic velocity of SARS-CoV M<sup>pro</sup> versus substrate concentrations in the presence of either <bold>3-31</bold> or <bold>3-39</bold> (<xref rid="appsec1" ref-type="sec">Part 3 of the supplementary data</xref>). The lines displayed in reciprocal plots intersect at a same point, indicating that both inhibitors serve as a non-competitive inhibitor with α < 1 (<xref rid="appsec1" ref-type="sec">Part 3 of the supplementary data</xref>) <xref rid="bib27" ref-type="bibr">[27]</xref>. On this basis, the kinetic parameters (α<italic>K</italic><sub>i</sub> = 0.20 μM, <italic>K</italic><sub>i</sub> = 0.24 μM) of <bold>3-31</bold> were determined <xref rid="bib28" ref-type="bibr">[28]</xref> (<xref rid="fig3" ref-type="fig">Fig. 3</xref>), which clearly proved that, the non-competitive inhibitor <bold>3-31</bold> is characterized by smaller equilibrium-binding constant compared to some known inhibitors such as N3 (<italic>K</italic><sub>i</sub> = 9.0 μM) and N9 (<italic>K</italic><sub>i</sub> = 6.7 μM) <xref rid="bib3" ref-type="bibr">[3]</xref>.<fig id="fig2"><label>Fig. 2</label><caption><p>Plot of enzyme concentration versus reaction velocity for enzymatic kinetic study of <bold>3-31</bold> (A) and <bold>3-39</bold> (B).</p></caption><alt-text id="alttext0025">Fig. 2</alt-text><graphic xlink:href="gr2_lrg"></graphic></fig><fig id="fig3"><label>Fig. 3</label><caption><p>Secondary plots for the determination of the kinetic constants (<italic>K</italic><sub><italic>i</italic></sub> and <italic>αK</italic><sub><italic>i</italic></sub>) of inhibitor <bold>3-31</bold> as a non-competitive inhibitor. The values of <italic>αK</italic><sub><italic>i</italic></sub> (A)and <italic>K</italic><sub><italic>i</italic></sub> (B) are calculated from the <italic>x</italic> intercept.</p></caption><alt-text id="alttext0030">Fig. 3</alt-text><graphic xlink:href="gr3_lrg"></graphic></fig></p><p id="p0070">Since there is a cysteine in the active site of SARS-CoV M<sup>pro</sup> (Cys145), which plays an essential role for the biological activity of this protease, it is possible that the disulfide compound reacts with Cys145 to form a new —S-S- bond and results in a loss of enzyme activity. It is known that, if a disulfide reacts with another thiol to give a new disulfide, the thiols that are parts of the old disulfide can also react directly with this thiol to form the same new disulfide <xref rid="bib29" ref-type="bibr">[29]</xref>. We tested the biological activities of different aryl thiols derived from our disulfides, and no inhibition of SARS-CoV M<sup>pro</sup> could be detected for any of them even at very high concentration. Accordingly it seemed unlikely that Cys145 formed a —S-S- bond by reacting with the target disulfide compounds. Another method to rule out this possibility is to determine the change of the molecular weight of the protein before and after inhibition <xref rid="bib30" ref-type="bibr">[30]</xref>, <xref rid="bib31" ref-type="bibr">[31]</xref>. If the disulfide compound reacts with Cys145, the molecular weight would have a shift after SARS-CoV M<sup>pro</sup> is inhibited, and this is approximately the mass of half moiety of the unsymmetrical disulfide. Bearing this in mind, we measured the molecular weight of protease before and after inhibition by three disulfide compounds with significant structure difference (<bold>3-8</bold>, <bold>3-31</bold> and <bold>3-39,</bold> data shown in <xref rid="appsec1" ref-type="sec">part 4 of the supplementary data</xref>). However, no such assumed shift in the molecular weight was observed to support this idea.</p><p id="p0075">For another possibility, Khosla et al. had reported selective inhibition of extracellular thioredoxin by unsymmetrical disulfides <xref rid="bib18" ref-type="bibr">[18]</xref>, in which two cysteine residues in a close distance form an intramolecular disulfide bond. After careful analysis of the SARS-CoV M<sup>pro</sup> crystal structure (pdb entry <ext-link ext-link-type="uri" xlink:href="pdb:2AMD" id="intref0010">2AMD</ext-link>) <xref rid="bib3" ref-type="bibr">[3]</xref>, no other cysteine residue was found to be in a nearby space of Cys145. In fact not any two cysteine residues are in a reasonable distance to form possible intramolecular disulfide bond. Thus we also denied this probable inhibitory mechanism.</p></sec><sec id="sec2.3"><label>2.3</label><title>Molecular docking and three dimensional structure-activity relationships</title><p id="p0080">Since the unsymmetrical aromatic disulfides do not react with the residues of the SARS-CoV M<sup>pro</sup>, it means that these compounds act as intact molecules when inhibiting this protease. Therefore, in silico molecular docking technique was utilized to predict possible binding modes of the disulfide compounds with SARS-CoV M<sup>pro</sup>. In our previous study, we had docked a small library of isatin compounds to the active site of AHAS and probable binding modes were predicted by FlexX <xref rid="bib32" ref-type="bibr">[32]</xref>, <xref rid="bib33" ref-type="bibr">[33]</xref>, <xref rid="bib34" ref-type="bibr">[34]</xref>, <xref rid="bib35" ref-type="bibr">[35]</xref>, <xref rid="bib36" ref-type="bibr">[36]</xref>, <xref rid="bib37" ref-type="bibr">[37]</xref>. Here similar strategy was adopted to carry out database docking. After investigation of the resulting docked conformations, nineteen compounds were found to overlay one another quite well (<xref rid="appsec1" ref-type="sec">Part 5 of the supplementary data</xref>), while all the other twenty-one compounds were in unreasonable binding space, and they failed to overlay well with one another. Thus, the docked conformations of the nineteen compounds were thought to be the possible binding conformations in this study. Compound <bold>3-31</bold> was chosen to depict the binding mode of the disulfide inhibitors. <xref rid="fig4" ref-type="fig">Fig. 4</xref>is a two-dimensional illustration of the interactions between the inhibitor and the surrounding residues of SARS-CoV M<sup>pro</sup> drawn by LIGPLOT <xref rid="bib38" ref-type="bibr">[38]</xref>. The location of <bold>3-31</bold> has some overlap with the inhibitor N9, the inhibitor in the original pdb file. The compound binds with SARS-CoV M<sup>pro</sup> via multiple hydrogen bonding contacts and hydrophobic contacts. Phe140, Leu141, His163, Met165, Glu166 and His172 form hydrophobic interactions with the small molecule, while Asn142, Gly143 and Cys145 form intermolecular H-bond with the inhibitor. The predicted binding mode therefore provides a useful clue to understand the possible molecular basis of these inhibitors.<fig id="fig4"><label>Fig. 4</label><caption><p>LIGPLOT 2D representation of <bold>3-31</bold> bound with SARS CoV Mpro from FlexX docking. The hydrogen bonds between the enzyme and the inhibitor are shown as green dashed lines, and distances are in Å units. Amino acid residues that are within van der Waals contact of the inhibitor are shown as red arcs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</p></caption><alt-text id="alttext0035">Fig. 4</alt-text><graphic xlink:href="gr4_lrg"></graphic></fig></p><p id="p0085">Comparative field analysis (CoMFA) is a tool to generate 3D contour models to quantitatively analyze the structure-activity relationships of bioactive compounds by steric and electrostatic contributions <xref rid="bib39" ref-type="bibr">[39]</xref>, <xref rid="bib40" ref-type="bibr">[40]</xref>, <xref rid="bib41" ref-type="bibr">[41]</xref>. On the basis of the docked conformation, the molecules in the database were aligned to construct a CoMFA model. Compounds <bold>3-8</bold>, <bold>3-23</bold> and <bold>3-40</bold> were excluded from the database because they were statistical outliers in the training set, that is, the inclusion of any of these molecules did not yield a satisfactory leave-one-out <italic>q</italic><sup>2</sup>. The training set without the outliers gave a leave-one-out <italic>q</italic><sup>2</sup> of 0.681 when the optimum components was 6, and the non-crossvalidated <italic>r</italic><sup>2</sup> was 0.916, with a standard error of estimate of 0.088 and F values of 37.968. The steric and electrostatic contributions were 43.6% and 56.4%, respectively.</p><p id="p0090">Compound <bold>3-31</bold> was used to illustrate the steric and electrostatic contour maps, together with the neighboring residues in the docked binding pocket (<xref rid="fig5" ref-type="fig">Fig. 5</xref>). For the steric contour map, a bulky group is favorable for better inhibition in the green contour region and such a group is likely to decrease the activity in the yellow contour space. The green maps are mostly in three bulks: one formed by Ser1and Leu141, one formed by Asn142 and Gly143 and the last one formed by Ser144 and Cys145; whereas the yellow maps are just located in a space nearby Gly143. For the electrostatic contour map, in the blue contour region, an increase in the positive charge will lead to an increase of activity, yet in the red contour region, negative charge is favorable to enhance the activity. The blue map is in a region surrounded by Leu27, Cly143, Ser144 and Cys145, whereas the red maps are in three cavities: one formed by Leu141 and Asn142, one formed by Ser144, Cys145 and Met165 and the last one formed by Asn142 and Gly143. The 3D CoMFA maps have afforded important structural features of the unsymmetrical aromatic disulfides from steric and electrostatic views, which is valuable for further design and discovery of more potent inhibitors.<fig id="fig5"><label>Fig. 5</label><caption><p>Steric contour map (A) and electrostatic contour map (B) for the CoMFA model. Sterically favored and disfavored regions are shown in green and yellow in map A. Electrostatic favored and disfavored regions are shown in blue and red in map B. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</p></caption><alt-text id="alttext0040">Fig. 5</alt-text><graphic xlink:href="gr5_lrg"></graphic></fig></p></sec></sec><sec id="sec3"><label>3</label><title>Conclusion</title><p id="p0095">The lack of effective anti-SARS agents makes it a possible danger when SARS breaks out sometime in the future, numerous people will be killed again. Therefore it is still an urgent demand to discover novel anti-SARS inhibitors to combat this deadly disease. SARS CoV M<sup>pro</sup> is an important target for the design of therapeutically useful drugs. In the present study, in an effort to develop non-peptidic anti-SARS inhibitors, we have identified for the first time, that some unsymmetrical aromatic disulfides are excellent inhibitors of SARS CoV M<sup>pro</sup>, the mechanism of which seems distinct as they are reversible and mpetitive inhibitors. This suggests that the unsymmetrical disulfides are promising lead compounds identification and development of a new family of biologically active anti-SARS agents. A possible binding model of the disulfide inhibitor was built by molecular docking, and a CoMFA model was constructed subsequently to point out the structural features of these novel inhibitors of SARS CoV M<sup>pro</sup>. Based on this information, further structural modifications are ongoing for better pharmaceutical compounds. The Lipinski rules will also be utilized to help to develop compounds with a final <italic>in vivo</italic> activity <xref rid="bib42" ref-type="bibr">[42]</xref>. We are also trying to co-crystallize the protease and the best inhibitor, to gain insight into a real binding mode and explain the molecular basis of these compounds.</p></sec><sec id="sec4"><label>4</label><title>Experimental section</title><sec id="sec4.1"><label>4.1</label><title>General synthesis and instruments</title><p id="p0100">Various heterocyclic aromatic thiols <bold>1</bold> were commercial procured from 5A Pharmatech (China), Apichemical (China), Aldrich and ACES pharma, which were all >95% purity grade. All solvents and liquid reagents were dried in advance using standard methods and distilled before use. Substituted arenesulfenyl chlorides <bold>2</bold> were synthesized as described in our previous publications <xref rid="bib21" ref-type="bibr">[21]</xref>, <xref rid="bib22" ref-type="bibr">[22]</xref>. Synthetic methods for compounds <bold>3′</bold> had been reported and these parent compounds for <bold>3-4</bold>, <bold>3-6</bold>. <bold>3-7</bold>, <bold>3-8</bold>, <bold>3-9</bold>, <bold>3-10</bold> and <bold>3-11</bold> had also been fully characterized before <xref rid="bib21" ref-type="bibr">[21]</xref>. Melting points were determined using an X-4 melting apparatus and were uncorrected. <sup>1</sup>H NMR spectra and <sup>13</sup>C NMR were obtained using a 400 MHz Varian Mercury Plus 400 spectrometer. The chemical shift values (d) for the NMR spectra were reported as parts per million (ppm), using deuterated chloroform (CDCl<sub>3</sub>) or dimethyl sulfoxide (DMSO-<italic>d</italic><sub>6</sub>) as the solvent and tetramethylsilane (TMS) as an internal reference standard. Mass spectra were recorded on a Thermo Finnigan LCQ Advantage LC/mass detector instrument.</p></sec><sec id="sec4.2"><label>4.2</label><title>Synthesis of the target compounds (<xref rid="sch1" ref-type="fig">Scheme 1</xref>)</title><p id="p0105">Heterocyclic aromatic thiols (<bold>1</bold>, 5 mmol) was added to a solution of freshly prepared arenesulfenyl chlorides (<bold>2</bold>, 5 mmol) in 25 mL of anhydrous ethyl ether at room temperature. The mixture was then stirred for 5 h at the same temperature, after that the solvent was removed under reduced pressure. Products <bold>3</bold> (<bold>3′</bold> for <bold>3-4</bold>, <bold>3-6</bold>. <bold>3-7</bold>, <bold>3-8</bold>, <bold>3-9</bold>, <bold>3-10</bold> and <bold>3-11)</bold> were purified by column chromatography in 75–95% yields.<fig id="sch1"><label>Scheme 1</label><caption><p>Synthesis route of the target unsymmetrical aromatic disulfide compounds.</p></caption><alt-text id="alttext0045">Scheme 1</alt-text><graphic xlink:href="sc1_lrg"></graphic></fig></p></sec><sec id="sec4.3"><label>4.3</label><title>Synthesis of the compounds <bold>3-4</bold>, <bold>3-6</bold>. <bold>3-7</bold>, <bold>3-8</bold>, <bold>3-9</bold>, <bold>3-10</bold> and <bold>3-11</bold> (<xref rid="sch2" ref-type="fig">Scheme 2</xref>)</title><p id="p0110">Unsymmetrical aromatic disulfide (<bold>3′</bold>, 5 mmol) was added to 10 mL of acetic anhydride. The reaction mixture was stirred for 0.5 h at 60 °C and then 200 mL water was added to the mixture. The pH value was adjusted by sodium bicarbonate to 7–8. The product was extracted by ethyl acetate and the solvent was removed under reduced pressure. The target compounds were finally purified by column chromatography in 87–95% yields.<fig id="sch2"><label>Scheme 2</label><caption><p>Synthesis route of the target unsymmetrical aromatic disulfide compounds (<bold>3-4, 3-6, 3-7, 3-8, 3-9, 3-10</bold> and <bold>3-11</bold>).</p></caption><alt-text id="alttext0050">Scheme 2</alt-text><graphic xlink:href="sc2_lrg"></graphic></fig></p></sec><sec id="sec4.4"><label>4.4</label><title>Characterization of the target compounds</title><sec id="sec4.4.1"><label>4.4.1</label><title>2-((4-chlorophenyl)disulfanyl)thiazole (<bold>3-1</bold>)</title><p id="p0115">Yield 89%; m.p.: 114–116 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 7.62 (d, <italic>J</italic> = 8.5 Hz, 2H, ArH), 7.47 (d, <italic>J</italic> = 8.5 Hz, 2H, ArH), 7.31 (s, 1H, NCH), 6.99 (d, <italic>J</italic> = 4.3 Hz, 1H, SCH); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 134.8, 132.2, 129.3, 121.9, 114.20, 99.7; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>9</sub>H<sub>6</sub>ClNS<sub>3</sub> 259.9423, found 259.9420 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.2"><label>4.4.2</label><title>N-(2-(<italic>p</italic>-tolyldisulfanyl)thiazol-5-yl)acetamide (<bold>3-2</bold>)</title><p id="p0120">Yield 91%; m.p.: 113–115 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 11.70 (s, 1H, NH), 7.50 (d, <italic>J</italic> = 6.5 Hz, 2H, ArH), 7.47 (s, 1H, CH), 7.26 (d, <italic>J</italic> = 7.8 Hz, 2H, ArH), 2.32 (s, 3H, CH<sub>3</sub>), 2.10 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), 167.6, 154.1, 138.9, 132.3, 130.6, 130.0, 128.4, 22.6, 21.1; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>12</sub>H<sub>12</sub>N<sub>2</sub>OS<sub>3</sub> 297.0185, found 297.0185 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.3"><label>4.4.3</label><title>Ethyl 2-((4-chlorophenyl)disulfanyl)-1H-imidazole-4-carboxylate (<bold>3-3</bold>)</title><p id="p0125">Yield 87%; m.p.: 100–102 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 7.98 (s, 1H), 7.64 (d, <italic>J</italic> = 8.6 Hz, 2H, ArH), 7.49 (d, <italic>J</italic> = 8.6 Hz, 2H, ArH), 4.24 (q, <italic>J</italic> = 7.0 Hz, 2H, CH<sub>2</sub>CH<sub>3</sub>), 1.27 (t, <italic>J</italic> = 7.1 Hz, 3H, CH<sub>2</sub>CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 161.1, 132.9, 131.7, 128.7, 117.1, 116.9, 60.5, 14.7; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>12</sub>H<sub>11</sub>ClN<sub>2</sub>O<sub>2</sub>S<sub>2</sub> 315.0022, found 315.0021 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.4"><label>4.4.4</label><title>1-(5-Methyl-3-((2-nitrophenyl)disulfanyl)-1H-1,2,4-triazol-1-yl)ethanone (<bold>3-4</bold>)</title><p id="p0130">Yield 90%; m.p.: 157–159 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.31 (d, <italic>J</italic> = 8.2 Hz, 1H), 8.24 (d, <italic>J</italic> = 8.2 Hz, 1H), 7.91 (t, <italic>J</italic> = 7.7 Hz, 1H), 7.58 (t, <italic>J</italic> = 7.8 Hz, 1H), 2.59 (s, 3H), 2.55 (s, 3H); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 169.3, 157.8, 157.3, 144.9, 135.6, 134.3, 127.9, 126.6, 23.5, 15.6; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>10</sub>N<sub>4</sub>O<sub>3</sub>S<sub>2</sub> 311.0273, found 311.0261 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.5"><label>4.4.5</label><title>N-(2-(phenyldisulfanyl)thiazol-5-yl)acetamide (<bold>3-5</bold>)</title><p id="p0135">Yield 89%; m.p.: 108–110 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 11.79 (s, 1H, NH), 7.64 (t, <italic>J</italic> = 8.6 Hz, 2H, ArH), 7.53 (d, <italic>J</italic> = 8.5 Hz, 1H, ArH), 7.51–7.42 (m, 2H, ArH), 7.39 (d, <italic>J</italic> = 7.1 Hz, 1H, ArH), 2.10 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.6, 139.4, 137.2, 130.8, 130.0, 129.9, 129.0, 128.8, 128.42, 22.6; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>10</sub>N<sub>2</sub>OS<sub>3</sub> 283.0029, found 283.0031 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.6"><label>4.4.6</label><title>1-(5-Phenyl-3-(<italic>p</italic>-tolyldisulfanyl)-1H-1,2,4-triazol-1-yl)ethanone (<bold>3-6</bold>)</title><p id="p0140">Yield 92%; m.p.: 112–114 °C; white solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.35 (d, <italic>J</italic> = 17.6 Hz, 1H), 8.00 (d, <italic>J</italic> = 8.1 Hz, 2H), 7.79 (d, <italic>J</italic> = 7.6 Hz, 3H), 7.56 (t, <italic>J</italic> = 7.6 Hz, 1H), 7.27 (s, 2H), 2.72 (s, 3H), 2.33 (s, 3H); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 135.4, 129.4, 128.9, 128.1, 126.2, 125.8, 129.5, 22.2, 20.8; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>17</sub>H<sub>15</sub>N<sub>3</sub>OS<sub>2</sub> 342.0729, found 342.0734 [M+ H]<sup>+</sup>.</p></sec><sec id="sec4.4.7"><label>4.4.7</label><title>1-(3-((4-methoxyphenyl)disulfanyl)-5-phenyl-1H-1,2,4-triazol-1-yl)ethanone (<bold>3-7</bold>)</title><p id="p0145">Yield 91%; m.p.: 133–135 °C; white solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.11 (s, 2H), 7.70 (d, <italic>J</italic> = 7.3 Hz, 2H), 7.38 (s, 3), 6.73 (d, <italic>J</italic> = 7.2 Hz, 2H), 3.65 (s, 3H), 2.58 (s, 3H); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 169.3, 161.9, 161.2, 158.9, 134.8, 129.9, 129.3, 128.5, 127.1, 124.8, 115.0, 55.2, 22.5; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>17</sub>H<sub>15</sub>N<sub>3</sub>O<sub>2</sub>S<sub>2</sub> 358.0683, found 358.0685 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.8"><label>4.4.8</label><title>1-(3-((2-nitrophenyl)disulfanyl)-5-(pyridin-3-yl)-1H-1,2,4-triazol-1-yl)ethanone (<bold>3-8</bold>)</title><p id="p0150">Yield 89%; m.p.: 186–188 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>), <italic>δ</italic> 8.64 (d, <italic>J</italic> = 4.6 Hz, 1H), 7.95 (d, <italic>J</italic> = 4.8 Hz, 1H), 7.70–7.42 (m, 1H), 7.33 (d, <italic>J</italic> = 8.4 Hz, 2H), 7.21 (d, <italic>J</italic> = 8.7 Hz, 3H), 3.06 (s, 3H); <sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>), <italic>δ</italic> 174.9, 150.1, 135.3, 129.9, 129.5, 129.3, 127.5, 120.7, 46.0, 29.7; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>15</sub>H<sub>11</sub>N<sub>5</sub>O<sub>3</sub>S<sub>2</sub> 374.0381, found 374.0380 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.9"><label>4.4.9</label><title>Ethyl 2-((1-acetyl-5-(pyridin-3-yl)-1H-1,2,4-triazol-3-yl)disulfanyl)benzoate(<bold>3-9</bold>)</title><p id="p0155">Yield 92%; m.p.: 128–130 °C; white solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 9.10 (s,1H), 8.66 (s, 1H), 8.26 (d, <italic>J</italic> = 7.8 Hz, 2H), 8.00 (d, <italic>J</italic> = 7.5 Hz, 1H), 7.75 (s, 1H), 7.57 (d, <italic>J</italic> = 7.2 Hz, 1H), 7.41 (s, 1H), 4.22 (q, 2H), 2.50 (s, 3H), 1.33 (t, <italic>J</italic> = 7.1 Hz, 3H); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 150.1, 135.1, 133.6, 130.9, 129.5, 129.3, 120.7, 45.9, 29.7, 22.5, 8.7; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>18</sub>H<sub>16</sub>N<sub>4</sub>O<sub>3</sub>S<sub>2</sub> 401.0736, found 401.0746 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.10"><label>4.4.10</label><title>Ethyl 2-((1-acetyl-5-(pyridin-4-yl)-1H-1,2,4-triazol-3-yl)disulfanyl)benzoate (<bold>3-10</bold>)</title><p id="p0160">Yield 95%; m.p.: 130–132 °C; white solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 9.10 (s, 1H), 8.66 (s,1H), 8.26 (d, <italic>J</italic> = 7.8 Hz, 2H), 8.00 (d, <italic>J</italic> = 7.5 Hz,1H), 7.75 (t, <italic>J</italic> = 7.6 Hz,1H), 7.54 (s, 1H), 7.41 (t, <italic>J</italic> = 7.2 Hz, 1H), 4.34 (q, <italic>J</italic> = 7.1 Hz, 2H), 2.50 (s, 3H), 1.33 (t, <italic>J</italic> = 7.1 Hz, 3H); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.9, 150.1, 135.1, 133.6, 130.9, 129.5, 129.3, 120.7, 45.9, 29.7, 8.6; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>18</sub>H<sub>16</sub>N<sub>4</sub>O<sub>3</sub>S<sub>2</sub> 401.0737, found 401.0746 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.11"><label>4.4.11</label><title>1-(3-((4-methoxyphenyl)disulfanyl)-5-(pyridin-3-yl)-1H-1,2,4-triazol-1-yl)ethanone (<bold>3-11</bold>)</title><p id="p0165">Yield 87%; m.p.: 113–115 °C; white solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 9.32 (d, <italic>J</italic> = 37.6 Hz, 1H), 8.53 (d, <italic>J</italic> = 5.9 Hz, 2H), 7.72 (d, <italic>J</italic> = 8.5 Hz, 1H), 7.54–7.27 (m, 2H), 6.76 (d, <italic>J</italic> = 8.8 Hz, 2H), 3.70 (s, 3H), 2.64 (s, 3H); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 161.0, 160.3, 150.9, 148.3, 135.8, 134.0, 123.7, 115.0, 114.7, 55.4, 29.7; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>16</sub>H<sub>14</sub>N<sub>4</sub>O<sub>2</sub>S<sub>2</sub> 359.0630, found 359.0639 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.12"><label>4.4.12</label><title>N-(2-((4-chlorophenyl)disulfanyl)thiazol-5-yl)acetamide (<bold>3-12</bold>)</title><p id="p0170">Yield 94%; m.p.: 127–129 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 11.73 (s, 1H, NH), 7.65 (d, <italic>J</italic> = 8.6 Hz, 2H, ArH), 7.52 (d, <italic>J</italic> = 8.5 Hz, 2H, ArH), 7.48 (s, 1H, CH), 2.10 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.6, 153.1, 152.0, 139.3, 134.4, 133.6, 130.8, 129.9, 128.5, 22.6; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>9</sub>ClN<sub>2</sub>OS<sub>3</sub> 316.9639, found 316.9641 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.13"><label>4.4.13</label><title>N-(2-((4-bromophenyl)disulfanyl)thiazol-5-yl)acetamide (<bold>3-13</bold>)</title><p id="p0175">Yield 93%; m.p.: 123–125 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 11.72 (s, 1H, NH), 7.65 (d, <italic>J</italic> = 8.6 Hz, 2H, ArH), 7.58 (d, <italic>J</italic> = 8.6 Hz, 2H, ArH), 7.48 (s, 1H, CH), 2.10 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.5, 167.0, 152.0, 139.6, 132.4, 130.5, 128.0, 121.6, 22.2; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>9</sub>BrN<sub>2</sub>OS<sub>3</sub> 360.9132, found 360.9128 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.14"><label>4.4.14</label><title>Methyl 2-((2-nitrophenyl)disulfanyl)-1H-imidazole-4-carboxylate (<bold>3-14</bold>)</title><p id="p0180">Yield 88%; m.p.: 160–163 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.51 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 8.31 (dd, <italic>J</italic> = 8.2, 1.1 Hz, 1H, ArH), 7.98 (s, 1H), 7.96 (s, 1H, NHCH), 7.58 (dd, <italic>J</italic> = 11.4, 4.1 Hz, 1H, ArH), 3.74 (s, 3H, OCH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 158.9, 151.1, 139.8, 137.7, 131.2, 130.5, 129.4, 125.2, 124.4, 122.2, 53.0; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>9</sub>N<sub>3</sub>O<sub>4</sub>S<sub>2</sub> 312.0107, found 312.0112 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.15"><label>4.4.15</label><title>Methyl 2-((2-(ethoxycarbonyl)phenyl)disulfanyl)-1H-imidazole-4-carboxylate (<bold>3-15</bold>)</title><p id="p0185">Yield 92%; m.p.: 93–95 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.34 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 8.03–7.96 (m, 1H, ArH), 7.96 (s, 1H, NHCH), 7.79–7.66 (m, 1H, ArH), 7.41 (t, <italic>J</italic> = 7.5 Hz, 1H, ArH), 4.32 (q, <italic>J</italic> = 7.1 Hz, 2H, CH<sub>2</sub>CH<sub>3</sub>), 3.74 (s, 3H, OCH<sub>3</sub>), 1.32 (t, <italic>J</italic> = 7.1 Hz, 3H, CH<sub>2</sub>CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 166.1, 161.8, 157.8, 141.0, 139.9, 134.1, 131.5, 126.9, 126.8, 126.5, 121.9, 61.9, 51.8, 14.5; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>14</sub>H<sub>14</sub>N<sub>2</sub>O<sub>4</sub>S<sub>2</sub> 339.0468, found 339.0475 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.16"><label>4.4.16</label><title>Methyl 2-((2-(methoxycarbonyl)phenyl)disulfanyl)-1H-imidazole-4-carboxylate (<bold>3-16</bold>)</title><p id="p0190">Yield 89%; m.p.: 114–116 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.35 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 8.00 (d, <italic>J</italic> = 7.8 Hz, 1H, ArH), 7.95 (s, 1H, NHCH), 7.76 (t, <italic>J</italic> = 7.8 Hz, 1H, ArH), 7.41 (t, <italic>J</italic> = 7.5 Hz, 1H, ArH), 3.87 (s, 3H, OCH<sub>3</sub>), 3.74 (s, 3H, OCH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 158.9, 137.8, 134.2, 131.9, 129.6, 128.9, 127.8, 126.9, 125.2, 124.4, 53.2, 52.4; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>13</sub>H<sub>12</sub>N<sub>2</sub>O<sub>4</sub>S<sub>2</sub> 325.0311, found 325.0314 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.17"><label>4.4.17</label><title>Methyl 2-((4-chlorophenyl)disulfanyl)-1H-imidazole-4-carboxylate (<bold>3-17</bold>)</title><p id="p0195">Yield 89%; m.p.: 138–140 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.00 (s, 1H, NHCH), 7.64 (d, <italic>J</italic> = 8.6 Hz, 2H, ArH), 7.49 (d, <italic>J</italic> = 8.7 Hz, 2H, ArH), 3.76 (s, 3H, OCH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 158.7, 147.5, 137.8, 133.5, 129.9, 128.2, 127.9, 125.2, 124.4, 53.2; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>9</sub>ClN<sub>2</sub>O<sub>2</sub>S<sub>2</sub> 300.9867, found 300.9894 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.18"><label>4.4.18</label><title>N-(2-((4-fluorophenyl)disulfanyl)thiazol-5-yl)acetamide (<bold>3-18</bold>)</title><p id="p0200">Yield 95%; m.p.: 119–122 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 11.78 (s, 1H, NH), 7.68 (d, <italic>J</italic> = 8.6 Hz 2H, ArH), 7.50 (s, 1H, CH), 7.32 (t, <italic>J</italic> = 8.3 Hz, 2H, ArH), 2.11 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.4, 153.8, 139.4, 132.6, 132.5, 131.5, 128.5, 117.3, 117.1, 22.6; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>9</sub>FN<sub>2</sub>OS<sub>3</sub> 300.9934, found 300.9934 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.19"><label>4.4.19</label><title>N-(2-((2-nitrophenyl)disulfanyl)thiazol-5-yl)acetamide (<bold>3-19</bold>)</title><p id="p0205">Yield 93%; m.p.: 130–133 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 11.76 (s, 1H, NH), 8.41 (d, <italic>J</italic> = 7.9 Hz, 1H, ArH), 8.35 (d, <italic>J</italic> = 8.5 Hz, 1H, ArH), 7.96 (d, <italic>J</italic> = 7.4 Hz, 1H, ArH), 7.61 (t, <italic>J</italic> = 7.4 Hz, 1H, ArH), 7.49 (s, 1H CH), 2.09 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.6, 151.4, 145.3, 139.4, 135.8, 134.8, 128.4, 128.3, 127.8, 126.8, 22.5; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>9</sub>N<sub>3</sub>O<sub>3</sub>S<sub>3</sub> 327.9879, found 327.9876 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.20"><label>4.4.20</label><title>2-((2-nitrophenyl)disulfanyl)thiazole (<bold>3-20</bold>)</title><p id="p0210">Yield 90%; m.p.: 105–107 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.37 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 8.23 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 7.93 (t, <italic>J</italic> = 7.7 Hz, 1H, ArH), 7.87–7.74 (m, 2H, CH), 7.62 (t, <italic>J</italic> = 7.7 Hz, 1H, ArH); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 163.8, 145.6, 144.9, 136.2, 133.9, 128.7, 127.6, 126.9, 124.5; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>9</sub>H<sub>6</sub>N<sub>2</sub>O<sub>2</sub>S<sub>3</sub> 270.9664, found 270.9663 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.21"><label>4.4.21</label><title>2-(<italic>p</italic>-tolyldisulfanyl)thiazole (<bold>3-21</bold>)</title><p id="p0215">Yield 75%; m.p.: 60–62 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>), <italic>δ</italic> 7.80 (d, <italic>J</italic> = 6.1 Hz, 2H, ArH), 7.53 (d, <italic>J</italic> = 7.9 Hz, 2H, ArH), 7.25 (d, <italic>J</italic> = 7.7 Hz, 2H, ArH), 2.30 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>), <italic>δ</italic> 136.5, 136.3, 130.2, 129.3, 127.6, 124.9, 21.5; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>10</sub>H<sub>9</sub>NS<sub>3</sub> 239.9969, found 239.9970 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.22"><label>4.4.22</label><title>2-((4-fluorophenyl)disulfanyl)thiazole (<bold>3-22</bold>)</title><p id="p0220">Yield 82%; m.p.: 89–91 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.37 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 8.23 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), δ 7.66 (d, <italic>J</italic> = 8.7 Hz, 2H, ArH), 7.52 (d, <italic>J</italic> = 8.7 Hz, 2H, ArH); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 144.9, 132.7, 132.6, 130.6, 125.0, 123.9, 117.3, 117.1, 40.1, 39.9, 39.7; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>9</sub>H<sub>6</sub>FNS<sub>3</sub> 243.9719, found 243.9721 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.23"><label>4.4.23</label><title>2-((4-bromophenyl)disulfanyl)thiazole (<bold>3-23</bold>)</title><p id="p0225">Yield 80%; m.p.: 92–94 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 7.61 (d, <italic>J</italic> = 8.5 Hz, 2H, ArH), 7.48 (d, <italic>J</italic> = 8.5 Hz, 2H, ArH), 7.30 (s, 1H, NCH), 6.99 (d, <italic>J</italic> = 4.3 Hz, 1H, SCH); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 134.9, 132.3, 129.2, 121.9, 114.2, 99.4; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>9</sub>H<sub>6</sub>BrNS<sub>3</sub> 303.8918, found 303.8914 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.24"><label>4.4.24</label><title>4-Methyl-2-((2-nitrophenyl)disulfanyl)thiazole (<bold>3-24</bold>)</title><p id="p0230">Yield 92%; m.p.: 61–63 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>), <italic>δ</italic> 8.36 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 8.20 (d, <italic>J</italic> = 8.2Hz, 1H, ArH), 8.01–7.84 (m, 1H, ArH), 7.64–7.53 (m, 1H, ArH), 7.33 (s, 1H, CH), 2.33 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>), <italic>δ</italic> 162.8, 154.6, 145.7, 136.0, 133.9, 128.7, 127.6, 126.9, 118.4, 17.3; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>10</sub>H<sub>8</sub>N<sub>2</sub>O<sub>2</sub>S<sub>3</sub> 284.9821, found 284.9823 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.25"><label>4.4.25</label><title>Ethyl 2-((4-methylthiazol-2-yl)disulfanyl)benzoate (<bold>3-25</bold>)</title><p id="p0235">Yield 91%; m.p.: 103–105 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.09–8.03 (m, 1H, ArH), 8.01 (d, <italic>J</italic> = 7.9 Hz, 1H, ArH), 7.74 (t, <italic>J</italic> = 7.7 Hz, 1H, ArH), 7.45 (t, <italic>J</italic> = 7.5 Hz, 1H, ArH), 7.28 (s, 1H), 4.41 (s, 2H, CH<sub>2</sub>CH<sub>3</sub>), 2.32 (s, 3H, CH3), 1.36 (t, <italic>J</italic> = 7.1 Hz, 3H, CH<sub>2</sub>CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 166.2, 156.3, 154.5, 147.4, 138.4, 134.4, 131.8, 127.4, 125.9, 117.6, 62.2, 17.4, 14.5; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>13</sub>H<sub>13</sub>NO<sub>2</sub>S<sub>3</sub> 312.0181, found 312.0188 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.26"><label>4.4.26</label><title>Methyl 2-((5-methyl-1,3,4-oxadiazol-2-yl)disulfanyl)benzoate (<bold>3-26</bold>)</title><p id="p0240">Yield 93%; m.p.: 80–82 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.14 (d, <italic>J</italic> = 7.4 Hz, 1H, ArH), 8.03 (d, <italic>J</italic> = 6.7 Hz, 1H, ArH), 7.78 (s, 1H, ArH), 7.47 (d, <italic>J</italic> = 6.3 Hz, 1H, ArH), 3.89 (s, 3H, OCH<sub>3</sub>), 2.49 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.3, 166.7, 161.7, 138.7, 134.4, 131.6, 127.4, 127.1, 126.5, 53.3, 11.3; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>10</sub>N<sub>2</sub>O<sub>3</sub>S<sub>2</sub> 283.0206, found 283.0209 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.27"><label>4.4.27</label><title>Ethyl 2-((5-methyl-1,3,4-oxadiazol-2-yl)disulfanyl)benzoate (<bold>3-27</bold>)</title><p id="p0245">Yield 88%; m.p.: 73–74 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.14 (d, <italic>J</italic> = 8.0 Hz, 1H, ArH), 8.04 (d, <italic>J</italic> = 7.7 Hz, 1H, ArH), 7.78 (t, <italic>J</italic> = 7.8 Hz,1H, ArH), 7.46 (t, <italic>J</italic> = 7.5 Hz, 1H, ArH), 4.35 (q, <italic>J</italic> = 7.1 Hz, 2H, CH<sub>2</sub>CH<sub>3</sub>), 2.50 (s, 3H,CH<sub>3</sub>), 1.34 (t, <italic>J</italic> = 7.1 Hz, 3H, CH<sub>2</sub>CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.3, 166.3, 161.7, 138.6, 134.3, 131.6, 127.4, 126.5, 62.2, 14.5, 11.3; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>12</sub>H<sub>12</sub>N<sub>2</sub>O<sub>3</sub>S<sub>2</sub> 297.0362, found 297.0365 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.28"><label>4.4.28</label><title>2-Methyl-5-((2-nitrophenyl)disulfanyl)-1,3,4-oxadiazole (<bold>3-28</bold>)</title><p id="p0250">Yield 92%; m.p.: 91–93 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.34 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 8.30 (d, <italic>J</italic> = 8.2 Hz, 1H, ArH), 7.97 (t, <italic>J</italic> = 7.7 Hz, 1H, ArH), 7.63 (t, <italic>J</italic> = 7.7 Hz, 1H, ArH), 2.51 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 167.5, 161.2, 145.4, 135.9, 134.0, 128.7, 128.1, 126.8, 11.3; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>9</sub>H<sub>7</sub>N<sub>3</sub>O<sub>3</sub>S<sub>2</sub> 270.0002, found 270.0000 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.29"><label>4.4.29</label><title>Methyl 2-((1,3,4-oxadiazol-2-yl)disulfanyl)benzoate (<bold>3-29</bold>)</title><p id="p0255">Yield 87%; m.p.: 99–101 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 9.37 (s, 1H, CH), 8.14 (d, <italic>J</italic> = 8.2, Hz, 1H, ArH), 8.05 (d, <italic>J</italic> = 7.8, Hz, 1H, ArH), 7.87 (t, <italic>J</italic> = 7.8, Hz, 1H, ArH), 7.48 (t, <italic>J</italic> = 7.7, Hz, 1H, ArH), 3.91 (s, 3H, OCH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 166.8, 162.3, 157.5, 138.5, 134.5, 131.7, 127.5, 127.2, 126.6, 53.3; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>10</sub>H<sub>8</sub>N<sub>2</sub>O<sub>3</sub>S<sub>2</sub> 290.9869, found 290.9868 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.30"><label>4.4.30</label><title>Methyl 2-((4-methyloxazol-2-yl)disulfanyl)benzoate (<bold>3-30</bold>)</title><p id="p0260">Yield 90%; m.p.: 68–70 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.16 (d, <italic>J</italic> = 8.0 Hz, 1H, ArH), 8.04 (d, <italic>J</italic> = 7.7, Hz, 1H, ArH), 7.97 (s, 1H, CH), 7.88 (t, <italic>J</italic> = 7.2 Hz, 1H, ArH), 7.45 (t, <italic>J</italic> = 7.2 Hz, 1H, ArH), 3.90 (s, 3H, OCH<sub>3</sub>), 2.07 (s 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 166.7, 139.6, 139.3, 138.8, 134.3, 131.6, 127.2, 126.9, 126.4, 53.2, 11.7; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>12</sub>H<sub>11</sub>NO<sub>3</sub>S<sub>2</sub> 282.0253, found 282.0257 [M+H]<sup>+</sup>.</p></sec><sec id="sec4.4.31"><label>4.4.31</label><title>2-((4-chlorophenyl)disulfanyl)-1,3,4-oxadiazole (<bold>3-31</bold>)</title><p id="p0265">Yield 90%; m.p.: 69–72 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 9.46 (s, 1H, CH), 7.71 (d, <italic>J</italic> = 8.5 Hz, 2H, ArH), 7.54 (d, <italic>J</italic> = 8.3 Hz, 2H, ArH); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 157.7, 134.7, 133.8, 132.5, 131.4, 130.1; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>8</sub>H<sub>5</sub>ClN<sub>2</sub>OS<sub>2</sub> 261.9871, found 261.9872 [M + NH<sub>4</sub>] <sup>+</sup>.</p></sec><sec id="sec4.4.32"><label>4.4.32</label><title>4,6-Dimethyl-2-((2-nitrophenyl)disulfanyl)pyrimidine (<bold>3-32</bold>)</title><p id="p0270">Yield 94%; m.p.: 151–153 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.34 (d, 6.5 Hz, 1H, ArH), 7.99–7.69 (m, 2H, ArH), 7.55 (d, <italic>J</italic> = 8.4 Hz, 1H, ArH), 7.12 (d, <italic>J</italic> = 25.8 Hz, 1H, ArH), 2.35 (s, 6H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 168.7, 168.3, 135.5, 128.5, 128.0, 127.4, 127.0, 126.3, 118.7, 118.1, 23.8; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>12</sub>H<sub>11</sub>N<sub>3</sub>O<sub>2</sub>S<sub>2</sub> 294.0365, found 294.0364 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.33"><label>4.4.33</label><title>2-((4-chlorophenyl)disulfanyl)-4,6-dimethylpyrimidine (<bold>3-33</bold>)</title><p id="p0275">Yield 80%; m.p.: 84–86 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 7.60 (d, <italic>J</italic> = 8.6 Hz, 2H, ArH), 7.44 (d, <italic>J</italic> = 8.6 Hz, 2H, ArH), 7.14 (s, 1H, ArH), 2.40 (s, 6H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 168.6, 167.8, 135.6, 133.1, 130.9, 129.6, 118.4, 23.8; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>12</sub>H<sub>11</sub>ClN<sub>2</sub>S<sub>2</sub> 283.0125, found 283.0130 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.34"><label>4.4.34</label><title>2-((4-bromophenyl)disulfanyl)-4,6-dimethylpyrimidine (<bold>3-34</bold>)</title><p id="p0280">Yield 84%; m.p.: 76–79 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 7.53 (dd, <italic>J</italic> = 15.6, 7.2 Hz, 4H, ArH), 7.15 (s, 1H, ArH), 2.38 (s, 6H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 168.6, 167.7, 136.1, 132.5, 131.0, 121.5, 118.4, 23.8; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>12</sub>H<sub>11</sub>BrN<sub>2</sub>S<sub>2</sub> 326.9620, found 326.9622 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.35"><label>4.4.35</label><title>4,6-Dimethyl-2-(phenyldisulfanyl)pyrimidine (<bold>3-35</bold>)</title><p id="p0285">Yield 81%; m.p.: 61–63 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 7.67 (t, <italic>J</italic> = 8.0 Hz, 2H, ArH), 7.60–7.31 (m, 3H, ArH), 7.22 (s, 1H, ArH), 2.48 (s, 6H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 168.6, 130.9, 129.7, 129.6, 129.2, 128.3, 118.4, 23.8; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>12</sub>H<sub>12</sub>N<sub>2</sub>S<sub>2</sub> 249.0515, found 249.0515 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.36"><label>4.4.36</label><title>4,6-Dimethyl-2-(p-tolyldisulfanyl)pyrimidine (<bold>3-36</bold>)</title><p id="p0290">Yield 84%; m.p.: 72–75 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 7.50 (d, <italic>J</italic> = 8.2 Hz, 2H, ArH), 7.18 (d, <italic>J</italic> = 8.0 Hz, 2H, ArH), 7.12 (s, 1H, ArH), 2.40 (s, 6H, CH<sub>3</sub>), 2.28 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 168.4, 138.4, 133.1, 131.6, 130.3, 130.2, 118.2, 23.86, 21.1; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>13</sub>H<sub>14</sub>N<sub>2</sub>S<sub>2</sub> 263.0671, found 263.0674 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.37"><label>4.4.37</label><title>2-((2-nitrophenyl)disulfanyl)pyrimidine (<bold>3-37</bold>)</title><p id="p0295">Yield 86%; m.p.: 130–132 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.63 (s, 2H, ArH), 8.31 (d, <italic>J</italic> = 8.1 Hz, 2H, ArH), 7.96 (s, 1H, ArH), 7.59 (s, 1H, ArH), 7.38 (s, 1H, ArH), 7.17 (s, 1H, ArH); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 169.4, 158.6, 136.3, 134.3, 128.1, 126.7, 125.9, 118.9, 99.9; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>10</sub>H<sub>7</sub>N<sub>3</sub>O<sub>2</sub>S<sub>2</sub> 265.0052, found 266.0053 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.38"><label>4.4.38</label><title>2-((4-chlorophenyl)disulfanyl)pyrimidine (<bold>3-38</bold>)</title><p id="p0300">Yield 80%; m.p.: 79–80 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.66–8.47 (m, 2H), 7.47 (t, <italic>J</italic> = 9.0 Hz, 2H), 7.29–7.16 (m, 2H), 7.07 (d, <italic>J</italic> = 4.9 Hz, 1H); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 158.0, 134.9, 133.8, 130.2, 129.3, 129.1, 118.3; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>10</sub>H<sub>7</sub>ClN<sub>2</sub>S<sub>2</sub> 254.9812, found 254.9816 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.39"><label>4.4.39</label><title>2-((4-bromophenyl)disulfanyl)pyrimidine (<bold>3-39</bold>)</title><p id="p0305">Yield 83%; m.p.: 75–78 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.63 (dd, <italic>J</italic> = 22.9, 4.7 Hz, 2H), 7.43 (dd, <italic>J</italic> = 20.0, 12.8 Hz, 4H), 7.10 (d, <italic>J</italic> = 4.0 Hz, 1H); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 158.1, 135.5, 132.0, 130.3, 128.9, 121.8, 118.3; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>10</sub>H<sub>7</sub>BrN<sub>2</sub>S<sub>2</sub> 298.9307, found 298.9307 [M + H] <sup>+</sup>.</p></sec><sec id="sec4.4.40"><label>4.4.40</label><title>2-(<italic>p</italic>-tolyldisulfanyl)pyrimidine (<bold>3-40</bold>)</title><p id="p0310">Yield 86%; m.p.: 45–48 °C; yellow solid; <sup>1</sup>H NMR (400 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 8.56 (d, <italic>J</italic> = 4.9 Hz, 2H), 7.44 (d, <italic>J</italic> = 8.2 Hz, 2H), 7.13–6.90 (m, 3H), 2.24 (s, 3H, CH<sub>3</sub>); <sup>13</sup>C NMR (101 MHz, DMSO-<italic>d</italic><sub>6</sub>), <italic>δ</italic> 171.2, 157.9, 138.1, 132.9, 129.8, 129.7, 118.0, 21.1; HRMS(MALDI) <italic>m</italic>/<italic>z</italic>: calculated for C<sub>11</sub>H<sub>10</sub>N<sub>2</sub>S<sub>2</sub> 235.0358, found 235.0361 [M + H] <sup>+</sup>.</p></sec></sec><sec id="sec4.5"><label>4.5</label><title>In vitro enzyme inhibition assay</title><p id="p0315">The expression and purification of SARS CoV M<sup>pro</sup> was described by Rao et al. <xref rid="bib43" ref-type="bibr">[43]</xref>. Basically, the sequence of SARS-CoV M<sup>pro</sup> cloned into the pGEX-6P-1 vector was transformed into <italic>E. coli</italic> BL21 (DE3) cells. The GST fusion protein, GST-SARS-CoV M<sup>pro</sup>, was purified by GST-glutathione affinity chromatography, cleaved with PreScission protease, and the recombinant SARS-CoV M<sup>pro</sup> was further purified by using anion-exchange chromatography. Eventually purified protein was of high purity (>95%) as judged by SDSPAGE analysis and the concentration is 0.5 μM, and the buffer contains 50 mM Tris-HCl, pH 7.3 and 1 mM EDTA. The fluorogenic substrate with consensus sequence of CoV M<sup>pro</sup>, MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 (95% purity), was synthesized in Shanghai Biological Engineering Company. The substrate was dissolved in DMSO in 0.8 mM liquid storage for use.</p><p id="p0320">The inhibition assay was similar to Yang's procedure <xref rid="bib11" ref-type="bibr">[11]</xref>. The SARS CoV M<sup>pro</sup> inhibition assays were conducted by fluorescence resonance energy transfer (FRET). The natural substrate amino acid sequence (AVLQSGFRKK) of SARS-CoV M<sup>pro</sup> started with the MCA fluorescent group and connected the Dnp fluorescence quenching group with penultimate K. The tested compounds were dissolved by sterilized DMSO and diluted to various concentrations. The settled concentrations of proteins, compounds and substrate were preheated at 37 °C and oscillated. The excitation/emission light was 320/405 nm, and the test was carried out every 5 s for 200 times. Drawing curves, the maximum value of the negative control curve slope is <italic>V</italic><sub>0</sub>, and the largest compound curve slope is <italic>V</italic>1. The inhibition ratio can be defined (1−<italic>V</italic><sub>1</sub>/<italic>V</italic><sub>0</sub>). And the <italic>IC</italic><sub>50</sub> value was calculated by equation <xref rid="fd1" ref-type="disp-formula">(1)</xref> using CraphPad Prism5:<disp-formula id="fd1"><label>(1)</label><italic>V</italic><sub>0</sub>/<italic>V</italic>=1+[I]/<italic>IC</italic><sub>50</sub></disp-formula></p><p id="p0325"><italic>V</italic><sub>0</sub> shows the initial rate of the reaction without inhibitor, <italic>V</italic> means the initial rate of the reaction with the inhibitor at various concentrations and [I] indicates the concentration of the inhibitor.</p><p id="p0330">The determination of the inhibitor as a valent inhibitor employs the above methods as well, albeit with two modifications. Firstly, the inhibitor concentration was set to 2 or 4 μM. And for each inhibitor concentration, we measured the enzymatic activity of M<sup>pro</sup> whose concentration spans 0–2 μM. Secondly, M<sup>pro</sup> and inhibitor were first incubated for 20 min to ensure a thorough ‘M<sup>pro</sup>-inhibitor’ reaction and then the inhibition assay was initiated by adding substrate and characterized by fluorescence monitoring.</p><p id="p0335">The further characterization of the inhibitor as a non-competitive inhibitor employs the methods described in earlier work <xref rid="bib28" ref-type="bibr">[28]</xref>. Basically, the enzymatic velocity of SARS-CoV M<sup>pro</sup> versus substrate concentrations with presence of inhibitors is depicted by equation <xref rid="fd2" ref-type="disp-formula">(2)</xref><xref rid="bib27" ref-type="bibr">[27]</xref>, where <italic>K</italic><sub>i</sub> is the dissociation constant for the SARS-CoV M<sup>pro</sup> complexed with inhibitor <bold>3-31</bold>; factor α reflects the effect of inhibitor <bold>3-31</bold> on the affinity of the enzyme for its substrate; <italic>V</italic><sub>max</sub> and <italic>K</italic><sub>m</sub> represent the maximum velocity and Michaelis-Menten constant, respectively.<disp-formula id="fd2"><label>(2)</label><mml:math id="M1" display="block" altimg="si1.gif" overflow="scroll"><mml:mrow><mml:mi>v</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>V</mml:mi><mml:mrow><mml:mi>max</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>[</mml:mo><mml:mi>S</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>S</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:mo>[</mml:mo><mml:mi>I</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mrow><mml:mi>α</mml:mi><mml:msub><mml:mi>K</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:mo>[</mml:mo><mml:mi>I</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mrow><mml:msub><mml:mi>K</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula></p><p id="p0340">The values of <italic>V</italic><sub>max</sub> and <italic>K</italic><sub>m</sub> at different inhibitor concentrations were apparent <italic>V</italic><sub>max</sub> and <italic>K</italic><sub>m</sub>, called <inline-formula><mml:math id="M2" altimg="si2.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>V</mml:mi><mml:mrow><mml:mi>max</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula>and<inline-formula><mml:math id="M3" altimg="si3.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>K</mml:mi><mml:mi>m</mml:mi><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula>, respectively. According to equation <xref rid="fd2" ref-type="disp-formula">(2)</xref>, <inline-formula><mml:math id="M4" altimg="si2.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>V</mml:mi><mml:mrow><mml:mi>max</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> and<inline-formula><mml:math id="M5" altimg="si3.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>K</mml:mi><mml:mi>m</mml:mi><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> can be calculated by equation <xref rid="fd3" ref-type="disp-formula">(3)</xref>.<disp-formula id="fd3"><label>(3)</label><mml:math id="M6" display="block" altimg="si4.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>V</mml:mi><mml:mrow><mml:mi>max</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>V</mml:mi><mml:mrow><mml:mi>max</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:mo>[</mml:mo><mml:mi>I</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mrow><mml:mi>α</mml:mi><mml:msub><mml:mi>K</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:mfrac><mml:mo>,</mml:mo><mml:msubsup><mml:mi>K</mml:mi><mml:mi>m</mml:mi><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>K</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:mo>[</mml:mo><mml:mi>I</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mrow><mml:msub><mml:mi>K</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:mo>[</mml:mo><mml:mi>I</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mrow><mml:mi>α</mml:mi><mml:msub><mml:mi>K</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula></p><p id="p0345">The kinetic parameters of <inline-formula><mml:math id="M7" altimg="si2.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>V</mml:mi><mml:mrow><mml:mi>max</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> and<inline-formula><mml:math id="M8" altimg="si3.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>K</mml:mi><mml:mi>m</mml:mi><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula>, were determined by adding 1 μM SARS-CoV M<sup>pro</sup> to 20 μM substrate containing varying concentrations of inhibitor 3-31 (0–3 μM). The value of <italic>αK</italic><sub>i</sub> was then calculated from plots of 1/<inline-formula><mml:math id="M9" altimg="si2.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>V</mml:mi><mml:mrow><mml:mi>max</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> versus 1/[I]. Similarly, the value of <italic>K</italic><sub>i</sub> was calculated from plots of <inline-formula><mml:math id="M10" altimg="si2.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>V</mml:mi><mml:mrow><mml:mi>max</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> and<inline-formula><mml:math id="M11" altimg="si3.gif" overflow="scroll"><mml:mrow><mml:msubsup><mml:mi>K</mml:mi><mml:mi>m</mml:mi><mml:mrow><mml:mi>a</mml:mi><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> versus 1/[I].</p><p id="p0350">Mass spectra were recorded on Waters Xevo G2-XS Q-TOF mass spectrometry. Mass spectra were acquired in positive ion mode using a capillary voltage of 3 kV, a sampling cone voltage of 40 V and a source offset voltage of 80 V. The cone gas flow was set up to 50 L/h and desolvation gas flow was 800 L/h. Desolvation temperature and source temperature were set to 400 °C and 100 °C, respectively. The mass of intact protein was obtained by deconvolution of the raw data using MaxEnt1 tool. The samples were prepared at the similar condition with the <italic>IC</italic><sub>50</sub> determination, except that the concentration of the inhibitors was 20 times of the concentration of SARS M<sup>pro</sup>.</p></sec><sec id="sec4.6"><label>4.6</label><title>Molecular docking and comparative field analysis</title><p id="p0355">Chemical structures of the compounds were built within Sybyl 7.3 (Tripos Inc., St Louis, MO). All the molecules were assigned Gasteiger-Hückel charges and minimized by the Tripos force field when convergence reached 0.001 kcal/mol/Å.</p><p id="p0360">Molecular docking of the unsymmetrical aromatic disulfides to the active site of SARS-CoV M<sup>pro</sup> was performed by FlexX. The crystal structure of SARS-CoV M<sup>pro</sup> in complex with inhibitor (pdb code <ext-link ext-link-type="uri" xlink:href="pdb:2AMD" id="intref0015">2AMD</ext-link>) was retrieved from the pdb databank. All water molecules were removed, and hydrogen atoms were added in the standard geometry. Any amino acid residue within 6.5 Å of the location of the original inhibitor N9 was considered to be in the binding pocket. Cscore calculation was enabled and set to serial mode. Database docking and subsequent scoring procedures were performed using the default parameters in the program.</p><p id="p0365">For CoMFA, The molecules were superimposed using <bold>3-31</bold> from the molecular docking result as the template. All the parameters were used the default value within CoMFA module and the column filtering was set to 2.0 kcal/mol. The “leave-one-out” (LOO) cross validation method was applied to determine the optimum number of partial least squares (PLS) components. 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We appreciate Professor Chuanzheng Zhou in Nankai University for his kind assistance for the mass spectrometry of SARS CoV main protease.</p></ack><fn-group><fn id="appsec2" fn-type="supplementary-material"><label>Appendix A</label><p id="p0380">Supplementary data related to this article can be found at <ext-link ext-link-type="doi" xlink:href="10.1016/j.ejmech.2017.05.045" id="intref0020">http://dx.doi.org/10.1016/j.ejmech.2017.05.045</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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