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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: Structure–activity relationship study</title>
<author><name sortKey="Thanigaimalai, Pillaiyar" sort="Thanigaimalai, Pillaiyar" uniqKey="Thanigaimalai P" first="Pillaiyar" last="Thanigaimalai">Pillaiyar Thanigaimalai</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Konno, Sho" sort="Konno, Sho" uniqKey="Konno S" first="Sho" last="Konno">Sho Konno</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yamamoto, Takehito" sort="Yamamoto, Takehito" uniqKey="Yamamoto T" first="Takehito" last="Yamamoto">Takehito Yamamoto</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Koiwai, Yuji" sort="Koiwai, Yuji" uniqKey="Koiwai Y" first="Yuji" last="Koiwai">Yuji Koiwai</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Taguchi, Akihiro" sort="Taguchi, Akihiro" uniqKey="Taguchi A" first="Akihiro" last="Taguchi">Akihiro Taguchi</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Takayama, Kentaro" sort="Takayama, Kentaro" uniqKey="Takayama K" first="Kentaro" last="Takayama">Kentaro Takayama</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yakushiji, Fumika" sort="Yakushiji, Fumika" uniqKey="Yakushiji F" first="Fumika" last="Yakushiji">Fumika Yakushiji</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Akaji, Kenichi" sort="Akaji, Kenichi" uniqKey="Akaji K" first="Kenichi" last="Akaji">Kenichi Akaji</name>
<affiliation><nlm:aff id="aff2">Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kiso, Yoshiaki" sort="Kiso, Yoshiaki" uniqKey="Kiso Y" first="Yoshiaki" last="Kiso">Yoshiaki Kiso</name>
<affiliation><nlm:aff id="aff2">Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kawasaki, Yuko" sort="Kawasaki, Yuko" uniqKey="Kawasaki Y" first="Yuko" last="Kawasaki">Yuko Kawasaki</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chen, Shen En" sort="Chen, Shen En" uniqKey="Chen S" first="Shen-En" last="Chen">Shen-En Chen</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Naser Tavakolian, Aurash" sort="Naser Tavakolian, Aurash" uniqKey="Naser Tavakolian A" first="Aurash" last="Naser-Tavakolian">Aurash Naser-Tavakolian</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Schon, Arne" sort="Schon, Arne" uniqKey="Schon A" first="Arne" last="Schön">Arne Schön</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Freire, Ernesto" sort="Freire, Ernesto" uniqKey="Freire E" first="Ernesto" last="Freire">Ernesto Freire</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Hayashi, Yoshio" sort="Hayashi, Yoshio" uniqKey="Hayashi Y" first="Yoshio" last="Hayashi">Yoshio Hayashi</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">23747811</idno>
<idno type="pmc">7115367</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115367</idno>
<idno type="RBID">PMC:7115367</idno>
<idno type="doi">10.1016/j.ejmech.2013.05.005</idno>
<date when="2013">2013</date>
<idno type="wicri:Area/Pmc/Corpus">000F91</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000F91</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: Structure–activity relationship study</title>
<author><name sortKey="Thanigaimalai, Pillaiyar" sort="Thanigaimalai, Pillaiyar" uniqKey="Thanigaimalai P" first="Pillaiyar" last="Thanigaimalai">Pillaiyar Thanigaimalai</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Konno, Sho" sort="Konno, Sho" uniqKey="Konno S" first="Sho" last="Konno">Sho Konno</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yamamoto, Takehito" sort="Yamamoto, Takehito" uniqKey="Yamamoto T" first="Takehito" last="Yamamoto">Takehito Yamamoto</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Koiwai, Yuji" sort="Koiwai, Yuji" uniqKey="Koiwai Y" first="Yuji" last="Koiwai">Yuji Koiwai</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Taguchi, Akihiro" sort="Taguchi, Akihiro" uniqKey="Taguchi A" first="Akihiro" last="Taguchi">Akihiro Taguchi</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Takayama, Kentaro" sort="Takayama, Kentaro" uniqKey="Takayama K" first="Kentaro" last="Takayama">Kentaro Takayama</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yakushiji, Fumika" sort="Yakushiji, Fumika" uniqKey="Yakushiji F" first="Fumika" last="Yakushiji">Fumika Yakushiji</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Akaji, Kenichi" sort="Akaji, Kenichi" uniqKey="Akaji K" first="Kenichi" last="Akaji">Kenichi Akaji</name>
<affiliation><nlm:aff id="aff2">Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kiso, Yoshiaki" sort="Kiso, Yoshiaki" uniqKey="Kiso Y" first="Yoshiaki" last="Kiso">Yoshiaki Kiso</name>
<affiliation><nlm:aff id="aff2">Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kawasaki, Yuko" sort="Kawasaki, Yuko" uniqKey="Kawasaki Y" first="Yuko" last="Kawasaki">Yuko Kawasaki</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chen, Shen En" sort="Chen, Shen En" uniqKey="Chen S" first="Shen-En" last="Chen">Shen-En Chen</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Naser Tavakolian, Aurash" sort="Naser Tavakolian, Aurash" uniqKey="Naser Tavakolian A" first="Aurash" last="Naser-Tavakolian">Aurash Naser-Tavakolian</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Schon, Arne" sort="Schon, Arne" uniqKey="Schon A" first="Arne" last="Schön">Arne Schön</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Freire, Ernesto" sort="Freire, Ernesto" uniqKey="Freire E" first="Ernesto" last="Freire">Ernesto Freire</name>
<affiliation><nlm:aff id="aff3">Department of Biology, Johns Hopkins University, Baltimore, MD, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Hayashi, Yoshio" sort="Hayashi, Yoshio" uniqKey="Hayashi Y" first="Yoshio" last="Hayashi">Yoshio Hayashi</name>
<affiliation><nlm:aff id="aff1">Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</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="2013">2013</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><p>This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (<bold>8</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL<sup>pro</sup>
. In particular, compounds <bold>26m</bold>
and <bold>26n</bold>
exhibited good inhibitory activities with <italic>K</italic>
<sub><italic>i</italic>
</sub>
values of 0.39 and 0.33 μM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound <bold>26m</bold>
with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1′ position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the <italic>N</italic>
-arylglycine unit and a backbone hydrogen bond donor at the S3-position.</p>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<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">23747811</article-id>
<article-id pub-id-type="pmc">7115367</article-id>
<article-id pub-id-type="publisher-id">S0223-5234(13)00305-X</article-id>
<article-id pub-id-type="doi">10.1016/j.ejmech.2013.05.005</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: Structure–activity relationship study</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="au1"><name><surname>Thanigaimalai</surname>
<given-names>Pillaiyar</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au2"><name><surname>Konno</surname>
<given-names>Sho</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au3"><name><surname>Yamamoto</surname>
<given-names>Takehito</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au4"><name><surname>Koiwai</surname>
<given-names>Yuji</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au5"><name><surname>Taguchi</surname>
<given-names>Akihiro</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au6"><name><surname>Takayama</surname>
<given-names>Kentaro</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au7"><name><surname>Yakushiji</surname>
<given-names>Fumika</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au8"><name><surname>Akaji</surname>
<given-names>Kenichi</given-names>
</name>
<xref rid="aff2" ref-type="aff">b</xref>
</contrib>
<contrib contrib-type="author" id="au9"><name><surname>Kiso</surname>
<given-names>Yoshiaki</given-names>
</name>
<xref rid="aff2" ref-type="aff">b</xref>
</contrib>
<contrib contrib-type="author" id="au10"><name><surname>Kawasaki</surname>
<given-names>Yuko</given-names>
</name>
<xref rid="aff3" ref-type="aff">c</xref>
</contrib>
<contrib contrib-type="author" id="au11"><name><surname>Chen</surname>
<given-names>Shen-En</given-names>
</name>
<xref rid="aff3" ref-type="aff">c</xref>
</contrib>
<contrib contrib-type="author" id="au12"><name><surname>Naser-Tavakolian</surname>
<given-names>Aurash</given-names>
</name>
<xref rid="aff3" ref-type="aff">c</xref>
</contrib>
<contrib contrib-type="author" id="au13"><name><surname>Schön</surname>
<given-names>Arne</given-names>
</name>
<xref rid="aff3" ref-type="aff">c</xref>
</contrib>
<contrib contrib-type="author" id="au14"><name><surname>Freire</surname>
<given-names>Ernesto</given-names>
</name>
<xref rid="aff3" ref-type="aff">c</xref>
</contrib>
<contrib contrib-type="author" id="au15"><name><surname>Hayashi</surname>
<given-names>Yoshio</given-names>
</name>
<email>yhayashi@toyaku.ac.jp</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>
Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan</aff>
<aff id="aff2"><label>b</label>
Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan</aff>
<aff id="aff3"><label>c</label>
Department of Biology, Johns Hopkins University, Baltimore, MD, USA</aff>
<author-notes><corresp id="cor1"><label>∗</label>
Corresponding author. Tel./fax: +81 42 676 3275. <email>yhayashi@toyaku.ac.jp</email>
</corresp>
</author-notes>
<pub-date pub-type="pmc-release"><day>20</day>
<month>5</month>
<year>2013</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"><month>7</month>
<year>2013</year>
</pub-date>
<pub-date pub-type="epub"><day>20</day>
<month>5</month>
<year>2013</year>
</pub-date>
<volume>65</volume>
<fpage>436</fpage>
<lpage>447</lpage>
<history><date date-type="received"><day>11</day>
<month>3</month>
<year>2013</year>
</date>
<date date-type="rev-recd"><day>20</day>
<month>4</month>
<year>2013</year>
</date>
<date date-type="accepted"><day>7</day>
<month>5</month>
<year>2013</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2013 Elsevier Masson SAS. All rights reserved.</copyright-statement>
<copyright-year>2013</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>This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (<bold>8</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL<sup>pro</sup>
. In particular, compounds <bold>26m</bold>
and <bold>26n</bold>
exhibited good inhibitory activities with <italic>K</italic>
<sub><italic>i</italic>
</sub>
values of 0.39 and 0.33 μM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound <bold>26m</bold>
with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1′ position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the <italic>N</italic>
-arylglycine unit and a backbone hydrogen bond donor at the S3-position.</p>
</abstract>
<abstract abstract-type="graphical" id="abs0015"><title>Graphical abstract</title>
<fig id="undfig1" position="anchor"><graphic xlink:href="fx1"></graphic>
</fig>
</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">A series of dipeptide-type SARS CoV 3CL<sup>pro</sup>
inhibitors were synthesized.</p>
</list-item>
<list-item id="u0015"><label>•</label>
<p id="p0015">Compounds have shown significant inhibitory activity.</p>
</list-item>
<list-item id="u0020"><label>•</label>
<p id="p0020">A potent compound was discovered by changing the P3 position.</p>
</list-item>
<list-item id="u0025"><label>•</label>
<p id="p0025">Compounds <bold>26m</bold>
and <bold>26n</bold>
displayed potent inhibitory activities.</p>
</list-item>
</list>
</p>
</abstract>
<kwd-group id="kwrds0010"><title>Keywords</title>
<kwd>SARS</kwd>
<kwd>SARS-CoV 3CL protease</kwd>
<kwd>Peptidomimetics</kwd>
<kwd>Dipeptide</kwd>
<kwd>Cysteine protease inhibitors</kwd>
<kwd>Docking study</kwd>
</kwd-group>
</article-meta>
</front>
<body><sec id="sec1"><label>1</label>
<title>Introduction</title>
<p id="p0030">Since its first appearance in Southern China in late 2002, severe acute respiratory syndrome (SARS) has been recognized as a global threat <xref rid="bib1" ref-type="bibr">[1]</xref>
, <xref rid="bib2" ref-type="bibr">[2]</xref>
. Its rapid and unexpected spread to 32 countries has affected more than 8000 individuals and caused nearly 800 (∼10%) fatalities worldwide within a few months <xref rid="bib1" ref-type="bibr">[1]</xref>
, <xref rid="bib2" ref-type="bibr">[2]</xref>
, <xref rid="bib3" ref-type="bibr">[3]</xref>
. The causative SARS pathogen is a novel coronavirus, SARS-CoV <xref rid="bib4" ref-type="bibr">[4]</xref>
, <xref rid="bib5" ref-type="bibr">[5]</xref>
. SARS-CoV is a positive-strand RNA virus with a genome sequence that is only moderately homologous to other known coronaviruses <xref rid="bib6" ref-type="bibr">[6]</xref>
, <xref rid="bib7" ref-type="bibr">[7]</xref>
. SARS-CoV encodes a chymotrypsin-like protease (3CL<sup>pro</sup>
), also referred to as the main protease (M<sup>pro</sup>
), which plays a pivotal role in processing viral polyproteins and controlling replicase complex activity <xref rid="bib8" ref-type="bibr">[8]</xref>
. This enzyme is indispensable for viral replication and infection processes, making it an ideal target for the design of antiviral therapies. The 3CL<sup>pro</sup>
active site contains a catalytic dyad in which a cysteine residue (Cys145) acts as a nucleophile and a histidine (His41) residue acts as a general acid or base <xref rid="bib9" ref-type="bibr">[9]</xref>
, <xref rid="bib10" ref-type="bibr">[10]</xref>
. The SARS epidemic was successfully controlled in 2003; however, the potential reemergence of pandemic SARS-CoV continues to pose a risk, and new strains of SARS could potentially be more virulent than the strains that contributed to the 2003 outbreak. Since 2003, two additional human coronaviruses, NL63 and HKU1, have been identified in patients around the world <xref rid="bib11" ref-type="bibr">[11]</xref>
, <xref rid="bib12" ref-type="bibr">[12]</xref>
. Recently, a new SARS-like virus, HCoV-EMC, was identified in at least two individuals, one of whom died <xref rid="bib13" ref-type="bibr">[13]</xref>
. Very recently, the first case of a fatal respiratory illness similar to the deadly SARS was confirmed in Britain <xref rid="bib14" ref-type="bibr">[14]</xref>
. The World Health Organization (WHO) has announced that it is closely monitoring the situation and is working to “ensure a high degree of preparedness, should the new virus be found to be sufficiently transmissible to cause a community outbreak”. There is a significant need to develop anti-SARS agents that are capable of treating this potentially fatal respiratory illness. Several reports of crystalline forms of the SARS-CoV 3CL<sup>pro</sup>
protein bound to hexapeptidyl chloromethyl ketone inhibitors have been reported <xref rid="bib6" ref-type="bibr">[6]</xref>
, <xref rid="bib9" ref-type="bibr">[9]</xref>
, and numerous peptidic structures have been reported in the context of targeted antiviral drug design <xref rid="bib15" ref-type="bibr">[15]</xref>
, <xref rid="bib16" ref-type="bibr">[16]</xref>
, <xref rid="bib17" ref-type="bibr">[17]</xref>
, <xref rid="bib18" ref-type="bibr">[18]</xref>
, <xref rid="bib19" ref-type="bibr">[19]</xref>
, <xref rid="bib20" ref-type="bibr">[20]</xref>
, <xref rid="bib21" ref-type="bibr">[21]</xref>
, <xref rid="bib22" ref-type="bibr">[22]</xref>
. The reported protease inhibitors are generally peptidic in nature, often five to three residues in length, and bear a reactive warhead group at the <italic>C</italic>
-terminus which forms an interaction with the protease catalytic Cys145 (<xref rid="fig1" ref-type="fig">Fig. 1</xref>
, <bold>1</bold>
–<bold>9</bold>
). Two of these compounds (<bold>8</bold>
and <bold>9</bold>
), recently described in a separate report from our group <xref rid="bib22" ref-type="bibr">[22]</xref>
, exhibited excellent potent inhibitory activities with <italic>K</italic>
<sub><italic>i</italic>
</sub>
values of 4.1 and 3.1 nM, respectively. These peptidic inhibitors provided valuable insight into the design constraints for this system and quickly led to the development of non-peptidic small molecule inhibitors (<xref rid="fig1" ref-type="fig">Fig. 1</xref>
, <bold>10</bold>
–<bold>17</bold>
) <xref rid="bib23" ref-type="bibr">[23]</xref>
, <xref rid="bib24" ref-type="bibr">[24]</xref>
, <xref rid="bib25" ref-type="bibr">[25]</xref>
, <xref rid="bib26" ref-type="bibr">[26]</xref>
, <xref rid="bib27" ref-type="bibr">[27]</xref>
, <xref rid="bib28" ref-type="bibr">[28]</xref>
, <xref rid="bib29" ref-type="bibr">[29]</xref>
, <xref rid="bib30" ref-type="bibr">[30]</xref>
. These small molecular inhibitors generally showed moderate to good activities.<fig id="fig1"><label>Fig. 1</label>
<caption><p>Representative peptidomimetics (<bold>1</bold>
–<bold>9</bold>
) and small molecular (<bold>10</bold>
–<bold>17</bold>
) 3CL<sup>pro</sup>
inhibitors highlighting reactive warhead groups (red).</p>
</caption>
<graphic xlink:href="gr1"></graphic>
</fig>
</p>
<p id="p0035">Recently, we performed a structure–activity relationship study based on the lead compound, Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-thiazole (<bold>7</bold>
) <xref rid="bib21" ref-type="bibr">[21]</xref>
. This study led to the discovery of the potent compounds <bold>8</bold>
and <bold>9</bold>
, with <italic>K</italic>
<sub><italic>i</italic>
</sub>
values in the low nanomolar range <xref rid="bib22" ref-type="bibr">[22]</xref>
.</p>
<p id="p0040">Extending our studies toward the development of new anti-SARS agents, we now report the design, synthesis, and evaluation of a series of low-molecular weight dipeptide-type compounds in which the P3 valine unit is removed from the previous lead Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole compound (<bold>8</bold>
, <xref rid="fig1" ref-type="fig">Fig. 1</xref>
). A preliminary SAR study led to the identification of inhibitors with moderate to good inhibitory activities. In particular, compounds <bold>26m</bold>
and <bold>26n</bold>
exhibited potent inhibitory activities with <italic>K</italic>
<sub><italic>i</italic>
</sub>
values of 0.39 and 0.33 μM, respectively. The binding interactions of <bold>26m</bold>
were predicted using molecular modeling studies. We describe the results of these extensive studies in detail, including the design, synthesis, molecular modeling, and biological evaluation of a series of SARS-CoV 3CL<sup>pro</sup>
inhibitors.</p>
</sec>
<sec id="sec2"><label>2</label>
<title>Results and discussion</title>
<sec id="sec2.1"><label>2.1</label>
<title>Synthesis</title>
<p id="p0045">The synthesis of the title inhibitors was achieved through a coupling reaction involving two key fragments, as shown in <xref rid="sch1" ref-type="fig">Scheme 1</xref>
. One of the key fragment intermediates (<bold>19</bold>
) was synthesized from the amino acid esters <bold>18</bold>
with either corresponding carboxylic acids <italic>via</italic>
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride–1-hydroxybenzotriazole (EDC·HCl–HOBt) mediated coupling in the presence of triethylamine (TEA) in DMF or acid chlorides in the presence of TEA in dichloromethane (CH<sub>2</sub>
Cl<sub>2</sub>
). The resulting <italic>N</italic>
-protected amino acid esters (<bold>19</bold>
) were consequently deprotected in the presence of TFA or lithium hydroxide·water (LiOH·H<sub>2</sub>
O) to afford the <italic>N</italic>
-protected amino acids (<bold>20</bold>
), which were used directly in the subsequent step.<fig id="sch1"><label>Scheme 1</label>
<caption><p>Reagents and conditions: i) EDC·HCl, HOBt·H<sub>2</sub>
O, TEA/DMF (if reactant is a carboxylic acid) or Et<sub>3</sub>
N/CH<sub>2</sub>
Cl<sub>2</sub>
(if reactant is a benzyloxycarbonyl chloride), 0 °C–rt; ii) TFA/H<sub>2</sub>
O, CH<sub>2</sub>
Cl<sub>2</sub>
(if R<sub>1</sub>
= <italic>tert</italic>
-butyl unit), or LiOH·H<sub>2</sub>
O, THF/H<sub>2</sub>
O, (if R<sub>1</sub>
= methyl unit); iii) 4M NaOH, MeOH; iv) HN(OMe)Me·HCl, EDC·HCl, HOBt·H<sub>2</sub>
O, Et<sub>3</sub>
N/DMF, 0 °C–rt; v) <italic>n</italic>
-BuLi, THF, −78 °C (if R<sub>3</sub>
= benzothiazole) or LDA, THF, −78 °C (if R<sub>3</sub>
= 5-arylated thiazoles); vi) TFA/H<sub>2</sub>
O (10:1), CH<sub>2</sub>
Cl<sub>2</sub>
; vii) <bold>20</bold>
, HBTU, DIPEA/DMF, 0 °C–rt followed by HPLC purification. Note: The substituents R–R<sub>3</sub>
are indicated in <xref rid="tbl1" ref-type="table">Table 1</xref>
, <xref rid="tbl2" ref-type="table">Table 2</xref>
, <xref rid="tbl3" ref-type="table">Table 3</xref>
</p>
</caption>
<graphic xlink:href="sc1"></graphic>
</fig>
</p>
<p id="p0050">The γ-lactam-thiazoles (<bold>24</bold>
) were synthesized using an approach similar to the syntheses reported previously <xref rid="bib21" ref-type="bibr">[21]</xref>
, <xref rid="bib22" ref-type="bibr">[22]</xref>
, <xref rid="bib31" ref-type="bibr">[31]</xref>
. Accordingly, the optically pure <sc>l</sc>
-glutamic acid ester <bold>21</bold>
was converted to the γ-lactam ester <bold>22</bold>
by treatment with bromoacetonitrile, followed by reduction with PtO<sub>2</sub>
(5%) and cyclization. The resulting ester <bold>22</bold>
was hydrolyzed in the presence of 4 M NaOH in methanol to yield the corresponding acid, which was coupled to <italic>N</italic>
,<italic>O</italic>
-dimethylhydroxylamine <italic>via</italic>
the EDC–HOBt method to afford the Weinreb amide <bold>23</bold>
. The Weinreb amide <bold>23</bold>
was then coupled to the appropriate thiazoles in the presence of <italic>n</italic>
-butyl lithium (<italic>n</italic>
-BuLi) or lithium diisopropylamide (LDA) at −78 °C to afford the γ-lactam-thiazoles <bold>24</bold>
. The Boc protecting group was then removed, and the resulting intermediate was subsequently reacted with the above-mentioned <italic>N</italic>
-protected amino acids <bold>20</bold>
in the presence of <italic>O</italic>
-benzotriazole-<italic>N</italic>
,<italic>N</italic>
,<italic>N</italic>
′,<italic>N</italic>
′-tetramethyluroniumhexafluoro phosphate (HBTU) and DIPEA in DMF to afford the title compounds <bold>25</bold>
–<bold>27</bold>
. All compounds were purified by reverse phase HPLC and characterized by <sup>1</sup>
H and <sup>13</sup>
C NMR, and mass spectrometry analysis. The purity of each compound exceeded 90–95%.</p>
</sec>
<sec id="sec2.2"><label>2.2</label>
<title>Biological activity</title>
<p id="p0055">The compounds were subjected to a fluorometric protease inhibitory assay against SARS-CoV 3CL<sup>pro</sup>
, as described previously <xref rid="bib32" ref-type="bibr">[32]</xref>
, <xref rid="bib33" ref-type="bibr">[33]</xref>
. Briefly, the kinetic parameters were determined at a constant substrate concentration, and the inhibitor concentrations were varied to assess the <italic>K</italic>
<sub><italic>i</italic>
</sub>
values <xref rid="bib22" ref-type="bibr">[22]</xref>
. The IC<sub>50</sub>
values were determined only for certain potent inhibitors, based on the apparent decrease in the substrate concentration (H-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-NH<sub>2</sub>
) upon digestion by R188I SARS 3CL<sup>pro</sup>
, as described previously <xref rid="bib19" ref-type="bibr">[19]</xref>
, <xref rid="bib34" ref-type="bibr">[34]</xref>
. The cleavage reaction was monitored by analytical HPLC, and the cleavage rates were calculated from the decrease in the substrate peak area. <xref rid="tbl1" ref-type="table">Table 1</xref>
, <xref rid="tbl2" ref-type="table">Table 2</xref>
, <xref rid="tbl3" ref-type="table">Table 3</xref>
, <xref rid="tbl4" ref-type="table">Table 4</xref>
report the <italic>K</italic>
<sub><italic>i</italic>
</sub>
or IC<sub>50</sub>
values as the mean of 3 independent experiments.<table-wrap position="float" id="tbl1"><label>Table 1</label>
<caption><p>SARS-CoV 3CL<sup>pro</sup>
inhibitory activities (<italic>K</italic>
<sub><italic>i</italic>
</sub>
) of <bold>25a</bold>
–<bold>h</bold>
.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>Entry no.</th>
<th align="center">Inhibitors</th>
<th><italic>K</italic>
<sub><italic>i</italic>
</sub>
(μM)</th>
<th>Entry no.</th>
<th align="center">Inhibitors</th>
<th><italic>K</italic>
<sub><italic>i</italic>
</sub>
(μM)</th>
</tr>
</thead>
<tbody><tr><td><bold>25a</bold>
</td>
<td><inline-graphic xlink:href="fx2.gif"></inline-graphic>
</td>
<td align="char">5.90</td>
<td><bold>25e</bold>
</td>
<td><inline-graphic xlink:href="fx3.gif"></inline-graphic>
</td>
<td align="char">1.71</td>
</tr>
<tr><td><bold>25b</bold>
</td>
<td><inline-graphic xlink:href="fx4.gif"></inline-graphic>
</td>
<td align="char">23.0</td>
<td><bold>25f</bold>
</td>
<td><inline-graphic xlink:href="fx5.gif"></inline-graphic>
</td>
<td align="char">29.0</td>
</tr>
<tr><td><bold>25c</bold>
</td>
<td><inline-graphic xlink:href="fx6.gif"></inline-graphic>
</td>
<td align="char">0.46</td>
<td><bold>25g</bold>
</td>
<td><inline-graphic xlink:href="fx7.gif"></inline-graphic>
</td>
<td align="char">9.40</td>
</tr>
<tr><td><bold>25d</bold>
</td>
<td><inline-graphic xlink:href="fx8.gif"></inline-graphic>
</td>
<td align="char">1.60</td>
<td><bold>25h</bold>
</td>
<td><inline-graphic xlink:href="fx9.gif"></inline-graphic>
</td>
<td align="char">1.20</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap position="float" id="tbl2"><label>Table 2</label>
<caption><p>SARS-CoV 3CL<sup>pro</sup>
inhibitory activities (<italic>K</italic>
<sub><italic>i</italic>
</sub>
) of <bold>26a</bold>
–<bold>n</bold>
.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>Entry No.</th>
<th align="center">Inhibitors</th>
<th><italic>K</italic>
<sub><italic>i</italic>
</sub>
(μM)</th>
<th>Entry No.</th>
<th align="center">Inhibitors</th>
<th><italic>K</italic>
<sub><italic>i</italic>
</sub>
(μM)</th>
</tr>
</thead>
<tbody><tr><td><bold>26a</bold>
</td>
<td><inline-graphic xlink:href="fx10.gif"></inline-graphic>
</td>
<td align="char">3.20</td>
<td><bold>26h</bold>
</td>
<td><inline-graphic xlink:href="fx11.gif"></inline-graphic>
</td>
<td align="char">0.56</td>
</tr>
<tr><td><bold>26b</bold>
</td>
<td><inline-graphic xlink:href="fx12.gif"></inline-graphic>
</td>
<td align="char">0.42</td>
<td><bold>26i</bold>
</td>
<td><inline-graphic xlink:href="fx13.gif"></inline-graphic>
</td>
<td align="char">1.56</td>
</tr>
<tr><td><bold>26c</bold>
</td>
<td><inline-graphic xlink:href="fx14.gif"></inline-graphic>
</td>
<td align="char">0.61</td>
<td><bold>26j</bold>
</td>
<td><inline-graphic xlink:href="fx15.gif"></inline-graphic>
</td>
<td align="char">8.4</td>
</tr>
<tr><td><bold>26d</bold>
</td>
<td><inline-graphic xlink:href="fx16.gif"></inline-graphic>
</td>
<td align="char">7.4</td>
<td><bold>26k</bold>
</td>
<td><inline-graphic xlink:href="fx17.gif"></inline-graphic>
</td>
<td align="char">0.84</td>
</tr>
<tr><td><bold>26e</bold>
</td>
<td><inline-graphic xlink:href="fx18.gif"></inline-graphic>
</td>
<td align="char">0.69</td>
<td><bold>26l</bold>
</td>
<td><inline-graphic xlink:href="fx19.gif"></inline-graphic>
</td>
<td align="char">3.20</td>
</tr>
<tr><td><bold>26f</bold>
</td>
<td><inline-graphic xlink:href="fx20.gif"></inline-graphic>
</td>
<td align="char">0.70</td>
<td><bold>26m</bold>
</td>
<td><inline-graphic xlink:href="fx21.gif"></inline-graphic>
</td>
<td align="char">0.39</td>
</tr>
<tr><td><bold>26g</bold>
</td>
<td><inline-graphic xlink:href="fx22.gif"></inline-graphic>
</td>
<td align="char">1.30</td>
<td><bold>26n</bold>
</td>
<td><inline-graphic xlink:href="fx23.gif"></inline-graphic>
</td>
<td align="char">0.33</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap position="float" id="tbl3"><label>Table 3</label>
<caption><p>SARS-CoV 3CL<sup>pro</sup>
inhibitory activities (<italic>K</italic>
<sub><italic>i</italic>
</sub>
) of <bold>27a</bold>
–<bold>d</bold>
.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>Entry no.</th>
<th align="center">Inhibitors</th>
<th><italic>K</italic>
<sub><italic>i</italic>
</sub>
(μM)</th>
</tr>
</thead>
<tbody><tr><td><bold>27a</bold>
</td>
<td><inline-graphic xlink:href="fx24.gif"></inline-graphic>
</td>
<td align="char">0.66</td>
</tr>
<tr><td><bold>27b</bold>
</td>
<td><inline-graphic xlink:href="fx25.gif"></inline-graphic>
</td>
<td align="char">37.0</td>
</tr>
<tr><td><bold>27c</bold>
</td>
<td><inline-graphic xlink:href="fx26.gif"></inline-graphic>
</td>
<td align="char">52.0</td>
</tr>
<tr><td><bold>27d</bold>
</td>
<td><inline-graphic xlink:href="fx27.gif"></inline-graphic>
</td>
<td align="char">2.50</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap position="float" id="tbl4"><label>Table 4</label>
<caption><p>Inhibitory activities (IC<sub>50</sub>
) of selected inhibitors.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>Entry no.</th>
<th>IC<sub>50</sub>
(μM)</th>
</tr>
</thead>
<tbody><tr><td><bold>25c</bold>
</td>
<td align="char">21.0</td>
</tr>
<tr><td><bold>26b</bold>
</td>
<td align="char">43.0</td>
</tr>
<tr><td><bold>26h</bold>
</td>
<td align="char">24.0</td>
</tr>
<tr><td><bold>26m</bold>
</td>
<td align="char">10.0</td>
</tr>
<tr><td><bold>26n</bold>
</td>
<td align="char">14.0</td>
</tr>
</tbody>
</table>
</table-wrap>
</p>
<p id="p0060">In an effort to develop low-molecular weight peptidic SARS-CoV 3CL<sup>pro</sup>
inhibitors, we designed a series of dipeptide-type inhibitors based on the previous potent lead compound <bold>8</bold>
(see <xref rid="fig1" ref-type="fig">Fig. 1</xref>
). In a first attempt, the P3 valine region was removed and the P4-benzyloxycarbonyl (Cbz) in <bold>8</bold>
was replaced with a series of small moieties with sizes similar to that of the P3 valine group in <bold>8</bold>
. The inhibitory activities of the resulting dipeptidic compounds bearing a valine mimic, isopentanoyl (<bold>25a</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 5.9 μM) or <italic>tert</italic>
-butoxy carbonyl (<bold>25b</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 23 μM), were dramatically reduced compared to the activity of <bold>8</bold>
(<italic>K</italic>
<sub><italic>i</italic>
</sub>
= 0.0041 μM) <xref rid="bib22" ref-type="bibr">[22]</xref>
; however, the introduction of Cbz (<bold>25c</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
and IC<sub>50</sub>
= 0.46 and 21.0 μM) as a P3 moiety resulted in a 12-fold or 50-fold activity increase for <bold>25a</bold>
or <bold>25b</bold>
, respectively, although the potency was reduced relative to the value for the tripeptidic lead <bold>8</bold>
. This result suggested that the Cbz group, which was introduced in place of the P3 scaffold in the dipeptidic <bold>25c</bold>
, conveyed appreciable activity; therefore, compound <bold>25c</bold>
could serve as a lead for further optimization steps. By retaining the P3 Cbz moiety in <bold>25c</bold>
, we examined the relevance of the leucine residue (or isobutyl unit) for P2 substrate selectivity in comparison with a variety of its congeners. Accordingly, a series of isosteres was introduced, including <italic>n</italic>
-butyl (<bold>25d</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 1.60 μM), isopropyl (<bold>25e</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 1.71 μM), <italic>sec</italic>
-butyl (<bold>25f</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 29.0 μM), 3-methyl(thio)ethyl (<bold>25g</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 9.40 μM), and benzyl (<bold>25h</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 1.20 μM). The compounds bearing <italic>n</italic>
-butyl (<bold>25d</bold>
), isopropyl (<bold>25e</bold>
), or benzyl (<bold>25h</bold>
) groups exhibited reasonable inhibitory activities, although the potencies were reduced by a factor of 3–4 relative to the value for <bold>25c</bold>
. The inhibitory activity of the compound bearing <italic>sec</italic>
-butyl (<bold>25f</bold>
) or 3-methyl(thio)ethyl (<bold>25g</bold>
) was severely reduced compared to the activity of <bold>25c</bold>
. Therefore, the leucine residue (or isobutyl group) was more selective and appropriate as a P2 group in <bold>25c</bold>
, providing enhanced inhibitory activity.</p>
<p id="p0065">The P3 moiety of the lead compound <bold>25c</bold>
was examined by introducing a wide variety of substituents, such as aryl (or heteroaryl) acetyls and propionyls, arylacrylyls, aryloxyacetyls, and <italic>N</italic>
-arylglycyls. Initially, the aryl (or heteroaryl) acetyls and propionyls were introduced, including phenylacetyl (<bold>26a</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 3.20 μM), 4-methoxyphenylacetyl (<bold>26b</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
and IC<sub>50</sub>
= 0.42 and 43 μM), 4-methoxyphenylpropionyl (<bold>26c</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 0.62 μM), and pyridine-3-propionyl (<bold>26d</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 7.4 μM). Among the compounds, a 4-methoxyphenylacetyl derivative (<bold>26b</bold>
) exhibited potent inhibitory activity; the phenylacetyl (<bold>26a</bold>
) and pyridine-3-propionyl (<bold>26d</bold>
) derivatives in particular displayed low inhibitory activities. Therefore, the 4-methoxyphenylacetyl moiety provided a good alternative to the P3 Cbz group in <bold>25c</bold>
. We next introduced the arylacrylyls, including cinnamoyl (<bold>26e</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 0.69 μM), 4-methoxycinnamoyl (<bold>26f</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 0.70 μM), and 3,4-dimethoxycinnamoyl (<bold>26g</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 1.30 μM). These compounds showed moderate inhibitory activities relative to the lead <bold>25c</bold>
; thus, the acrylyls were not considered further as P3 moieties in the context of <bold>25c</bold>
.</p>
<p id="p0070">A previous report describing a SAR study of the tripeptidomimetic <bold>7</bold>
(see <xref rid="fig1" ref-type="fig">Fig. 1</xref>
) as a SARS inhibitor revealed that the introduction of an aryloxyacetyl at the <italic>N</italic>
-terminal position (the P4 position) appeared to significantly enhance the inhibitory activity against SARS-CoV 3CL<sup>pro</sup>
<xref rid="bib22" ref-type="bibr">[22]</xref>
. Therefore, we introduced a series of aryloxyacetyls, including phenoxyacetyl (<bold>26h</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
and IC<sub>50</sub>
= 0.56 and 24 μM), 4-methoxyphenoxyacetyl (<bold>26i</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 1.56 μM), 4-hydroxyphenoxyacetyl (<bold>26j</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 8.4 μM), and 3-dimethylaminophenoxyacetyl (<bold>26k</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 0.84 μM) as P3 groups in place of the Cbz group in <bold>25c</bold>
. Compounds <bold>26h</bold>
and <bold>26k</bold>
exhibited comparable activities to <bold>25c</bold>
; however, the other derivatives (<bold>26i</bold>
and <bold>26j</bold>
) displayed reduced inhibitory activities relative to the lead compound <bold>25c</bold>
. These results suggested that the introduction of an aryloxyacetyl P3 group in the dipeptidic <bold>25c</bold>
did not appreciably improve the inhibitory activity against 3CL<sup>pro</sup>
. We also introduced several <italic>N</italic>
-arylglycyls, including <italic>N</italic>
-(4-methoxyphenyl)glycyl (<bold>26l</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 3.20 μM), <italic>N</italic>
-(3-methoxyphenyl)glycyl (<bold>26m</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
and IC<sub>50</sub>
= 0.39 and 10.0 μM), and <italic>N</italic>
-(2-methoxyphenyl)glycyl (<bold>26n</bold>
; <italic>K</italic>
<sub><italic>i</italic>
</sub>
and IC<sub>50</sub>
= 0.33 and 14.0 μM). The results of these studies revealed that compounds <bold>26m</bold>
and <bold>26n</bold>
displayed relatively potent inhibitory activities compared to the lead <bold>25c</bold>
. The compound bearing an <italic>N</italic>
-(2-methoxyphenyl)glycyl (<bold>26n</bold>
) was identified as the most potent inhibitor in the present study. This result suggested that the hydrogen bonding properties of the amino group on the <italic>N</italic>
-arylglycyl moiety might have contributed to the improvement in activity (see <xref rid="fig2" ref-type="fig">Fig. 2</xref>
B).<fig id="fig2"><label>Fig. 2</label>
<caption><p>Molecular dynamics stimulated pose of compound <bold>26m</bold>
(orange stick) bound to SARS-CoV 3CL<sup>pro</sup>
(PDB ID: <ext-link ext-link-type="uri" xlink:href="pdb:1WOF" id="intref0035">1WOF</ext-link>
(black stick)). (A) Overlapped view of <bold>26m</bold>
with an original vinyl ester (black stick) and lead <bold>9</bold>
(light blue stick); (B) compound <bold>26m</bold>
was shown as an orange stick. Dotted black lines represent the hydrogen bonding interaction. A notable hydrogen bonding interaction between the amino group (–NH) of <italic>N</italic>
-(3-methoxyphenyl)glycyl of <bold>26m</bold>
with a backbone amino acid residue Glu166 of 3CL<sup>pro</sup>
.</p>
</caption>
<graphic xlink:href="gr2"></graphic>
</fig>
</p>
<p id="p0075">The P1′ moiety was examined next by varying the 5-substituted thiazoles (<bold>27a</bold>
–<bold>d</bold>
). The inhibitory activities of compounds <bold>27a</bold>
–<bold>d</bold>
are illustrated in <xref rid="tbl3" ref-type="table">Table 3</xref>
. Inhibitor <bold>27a</bold>
exhibited an inhibitory activity comparable to that of <bold>25c</bold>
. The other 5-arlylated thiazoles (<bold>27b</bold>
–<bold>d</bold>
) generally exhibited very low inhibitory activities compared to <bold>25c</bold>
. These studies confirmed that the benzothiazole unit was more suitable as a warhead group on the P1′ moiety in <bold>25c</bold>
.</p>
</sec>
<sec id="sec2.3"><label>2.3</label>
<title>Molecular docking study</title>
<p id="p0080">The binding mode of compound <bold>26m</bold>
with 3CL<sup>pro</sup>
was simulated using a molecular docking program as described previously <xref rid="bib22" ref-type="bibr">[22]</xref>
. We examined the molecular docking of the potent active compound <bold>26m</bold>
in comparison with docking of the tripeptidic <bold>9</bold>
and a structurally similar ligand, the docking structure of which has been elucidated previously by X-ray crystallography (PDB ID: <ext-link ext-link-type="uri" xlink:href="pdb:1WOF" id="intref0010">1WOF</ext-link>
, <italic>K</italic>
<sub><italic>i</italic>
</sub>
= 10.7 μM) (<xref rid="fig2" ref-type="fig">Fig. 2</xref>
) <xref rid="bib35" ref-type="bibr">[35]</xref>
. Several minimization processes were performed using the MMFF94X force field to model the solvation environment surrounding the inhibitor. A molecular simulation was then performed. As shown in <xref rid="fig2" ref-type="fig">Fig. 2</xref>
A, the P1′–P2 moieties in <bold>26m</bold>
, in the tripeptidic compound <bold>9</bold>
, and in the original ligand interacted with the same region of the protease. Interestingly, the aminoacetyl group on the <italic>N</italic>
-(3-methoxyphenyl)glycyl group in <bold>26m</bold>
mimicked the P3 valine moieties of the tripeptidomimetic <bold>9</bold>
and the reported ligand. The interactions of compound <bold>26m</bold>
with the protein are described in detail in <xref rid="fig2" ref-type="fig">Fig. 2</xref>
B. Particularly, a notable hydrogen bonding involved between the amino group (–NH) of P3 <italic>N</italic>
-(3-methoxyphenyl)glycine with a backbone amino acid residue Glu166 of 3CL<sup>pro</sup>
was observed to have relatively potent inhibition than other inhibitors presented in the study against SARS-CoV 3CL<sup>pro</sup>
. The essential nature of this hydrogen bond will be explored in further optimization studies of dipeptide-type SARS-CoV 3CL<sup>pro</sup>
inhibitors.</p>
</sec>
</sec>
<sec id="sec3"><label>3</label>
<title>Conclusion</title>
<p id="p0085">In an effort to develop low-molecular weight peptidic anti-SARS agents, we designed, synthesized, and evaluated a series of dipeptide-type inhibitors in which the P3 valine unit in our potent tripeptidomimetic lead compound <bold>8</bold>
was replaced with a number of distinct functionalities. In a preliminary study, the compound <bold>25c</bold>
, bearing a benzyloxycarbonyl (Cbz) P3 moiety, was identified as a lead compound. The P3 moiety in <bold>25c</bold>
was systematically modified with a view to improve the biological activity. This study led to the identification of a compound bearing an <italic>N</italic>
-arylglycyl as a P3 moiety as having higher inhibitory activity due to the presence of a hydrogen bond with the backbone amino acid residue Glu166 of 3CL<sup>pro</sup>
. Leucine and benzothiazole units were identified as appropriate P2 and P1′ moieties, respectively, in <bold>25c</bold>
. Accordingly, compounds <bold>26m</bold>
and <bold>26n</bold>
were recognized as potent inhibitors in the present study. These potent dipeptidic inhibitors provide attractive leads, which are undergoing further structural modifications in an effort to improve the pharmaceutical profiles. These processes are underway and will be reported in the near future.</p>
</sec>
<sec id="sec4"><label>4</label>
<title>Experimental section</title>
<sec id="sec4.1"><label>4.1</label>
<title>Materials and methods</title>
<p id="p0090">Reagents and solvents were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan), and Aldrich Chemical Co. Inc. (Milwaukee, WI) and were used without further purification. Analytical thin-layer chromatography (TLC) was performed on Merck Silica Gel 60F254 pre-coated plates. Preparative HPLC was performed using a C18 reverse-phase column (19 × 150 mm; Sun-Fire Prep C18 OBD™, 5 μm) with a binary solvent system: a linear gradient of CH<sub>3</sub>
CN in 0.1% aqueous TFA at a flow rate of 6 mL/min, detected at UV 254 and 230 nm. All solvents used for HPLC were HPLC-grade. All other chemicals were of analytical grade or better. <sup>1</sup>
H and <sup>13</sup>
C NMR spectra were obtained using a JEOL 400 MHz spectrometer, a Varian Mercury 300 spectrometer (300 MHz), or a BRUKER AV600 spectrometer (600 MHz) with tetramethylsilane as an internal standard. High-resolution mass spectra (ESI or EI) were recorded on a micromass Q-Tof Ultima API or a JEOL JMS-GCmate BU-20 spectrometer. Mass spectra (ESI) were recorded on an LCMS-2010EV (SHIMADZU).</p>
</sec>
<sec id="sec4.2"><label>4.2</label>
<title>Synthetic procedures</title>
<sec id="sec4.2.1"><label>4.2.1</label>
<title>Synthetic procedures for the preparation of (<italic>S</italic>
)-<italic>tert</italic>
-butyl 2-(3-methylbutanamido)-4-methylpentanoate (<bold>19a</bold>
)</title>
<p id="p0095">To a solution of the commercially available <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester <bold>18a</bold>
(0.200 g, 0.89 mmol) in DMF (15 mL) was added an isovaleric acid (0.100 g, 0.98 mmol), HOBt·H<sub>2</sub>
O (0.151 g, 0.98 mmol), and EDC·HCl (0.189 g, 0.98 mmol). The resulting solution was cooled to 0 °C under ice bath conditions, and TEA was then added dropwise. After 5 min, the ice bath was removed and the mixture was allowed to stir for 2 h at ambient temperature. DMF was removed under high vacuum, and the resulting residue was dissolved in EtOAc (30 mL). The organic layer was washed with 5% citric acid (20 mL × 2), 5% NaHCO<sub>3</sub>
(20 mL × 2), and brine (20 mL). The solution was dried over Na<sub>2</sub>
SO<sub>4</sub>
, filtered, and evaporated under reduced pressure to give compound <bold>19a</bold>
. The resulting crude compound was purified by silica gel column chromatography using hexane–EtOAc as eluents. Yield 65%; white solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
4.20 (t, <italic>J</italic>
= 7.6 Hz, 1H), 2.03–1.90 (m, 3H), 1.57–1.42 (m, 3H), 1.34 (s, 9H), 0.86–0.79 (m, 12H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>15</sub>
H<sub>30</sub>
NO<sub>3</sub>
[M + H]<sup>+</sup>
272.2226, found 272.2230.</p>
<p id="p0100">The intermediates <bold>19h</bold>
–<bold>u</bold>
were prepared from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester <bold>18a</bold>
and various commercially available carboxylic acids according to the procedure described for the synthesis of <bold>19a</bold>
.</p>
</sec>
<sec id="sec4.2.2"><label>4.2.2</label>
<title>Synthesis of benzyl (<italic>S</italic>
)-1-(<italic>tert</italic>
-butoxycarbonyl)-3-methylbutylcarbamate (<bold>19b</bold>
)</title>
<p id="p0105">To a solution of the commercially available <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester <bold>18a</bold>
(0.500 g, 2.20 mmol) in CH<sub>2</sub>
Cl<sub>2</sub>
(20 mL) was added benzyloxycarbonyl chloride (0.410 mL, 2.5 mmol). The resulting solution was cooled to 0 °C and TEA (0.380 mL, 2.5 mmol) was then added dropwise. After 5 min, the ice bath was removed and the mixture was allowed to stir for 2 h at room temperature. The mixture was washed with H<sub>2</sub>
O (20 mL) and brine (10 mL). The resulting solution was dried over Na<sub>2</sub>
SO<sub>4</sub>
, filtered, and evaporated under reduced pressure to give compound <bold>19b</bold>
, which was purified by silica gel column chromatography using hexane–EtOAc as eluents. Yield 85%; colorless oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.35–7.28 (m, 5H, merged with CDCl<sub>3</sub>
), 5.10 (s, 2H), 4.29–4.23 (m, 1H), 1.74–1.67 (m, 2H), 1.62–1.58 (m, 1H), 1.44 (s, 9H), 0.95–0.93 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>18</sub>
H<sub>27</sub>
NO<sub>4</sub>
Na [M + Na]<sup>+</sup>
344.1838, found 344.1848.</p>
<p id="p0110">The intermediates <bold>19c</bold>
–<bold>g</bold>
were prepared from benzyloxycarbonyl chloride and various commercially available amino acid esters <bold>18b</bold>
–<bold>f</bold>
according to the procedure described for the synthesis of <bold>19c</bold>
.</p>
</sec>
<sec id="sec4.2.3"><label>4.2.3</label>
<title>Benzyl (<italic>S</italic>
)-1-(methoxycarbonyl)pentylcarbamate (<bold>19c</bold>
)</title>
<p id="p0115">Yield 67% from <sc>l</sc>
-norleucine methyl ester (<bold>18b</bold>
); colorless oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.36–7.29 (m, 5H), 5.11 (s, 2H), 4.39–4.34 (q, <italic>J</italic>
= 5.6 Hz, 1H), 3.73 (s, 3H), 1.88–1.79 (m, 1H), 1.66–1.63 (m, 1H), 1.38–1.33 (m, 4H), 0.90–0.87 (t, <italic>J</italic>
= 6.4 Hz, 3H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>15</sub>
H<sub>22</sub>
NO<sub>4</sub>
[M + H]<sup>+</sup>
280.1549, found 280.1545.</p>
</sec>
<sec id="sec4.2.4"><label>4.2.4</label>
<title>Benzyl (<italic>S</italic>
)-1-(<italic>tert</italic>
-butoxycarbonyl)-2-methylpropylcarbamate (<bold>19d</bold>
)</title>
<p id="p0120">Yield 71% from <sc>l</sc>
-valine <italic>tert</italic>
-butyl ester (<bold>18c</bold>
); colorless oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.39–7.31 (m, 5H), 5.10 (s, 2H), 4.20–4.17 (m, 1H), 2.15–2.12 (m, 1H), 1.46 (s, 9H), 0.96–0.87 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>17</sub>
H<sub>25</sub>
NO<sub>4</sub>
Na [M + Na]<sup>+</sup>
330.1681, found 330.1683.</p>
</sec>
<sec id="sec4.2.5"><label>4.2.5</label>
<title>Benzyl (1<italic>S</italic>
,2<italic>R</italic>
)-1-(<italic>tert</italic>
-butoxycarbonyl)-2-methylbutylcarbamate (<bold>19e</bold>
)</title>
<p id="p0125">Yield 66% from <sc>l</sc>
-isoleucine <italic>tert</italic>
-butyl ester (<bold>18d</bold>
); colorless oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.36–7.29 (m, 5H), 5.30–5.28 (m, NH, 1H), 5.17 (s, 2H), 4.25–4.20 (m, 1H), 1.85–1.80 (m, 1H), 1.44 (s, 9H), 1.21–1.01 (m, 2H), 0.94–0.91 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>18</sub>
H<sub>28</sub>
NO<sub>4</sub>
[M + H]<sup>+</sup>
322.2018, found 322.2010.</p>
</sec>
<sec id="sec4.2.6"><label>4.2.6</label>
<title>Benzyl (<italic>S</italic>
)-1-(<italic>tert</italic>
-butoxycarbonyl)-3-(methylthio)propylcarbamate (<bold>19f</bold>
)</title>
<p id="p0130">Yield 72% from <sc>l</sc>
-methionine <italic>tert</italic>
-butyl ester (<bold>18e</bold>
); colorless oil; <italic>δ</italic>
7.36–7.28 (m, 5H), 5.10 (s, 2H), 4.36 (q, <italic>J</italic>
= 6.0 Hz, 1H), 2.54–2.46 (m, 2H), 2.08 (m, 5H), 1.94–1.92 (m, 2H), 1.46 (s, 9H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>17</sub>
H<sub>26</sub>
NO<sub>4</sub>
S [M + H]<sup>+</sup>
340.1583, found 340.1580.</p>
</sec>
<sec id="sec4.2.7"><label>4.2.7</label>
<title>Benzyl (<italic>S</italic>
)-1-(methoxycarbonyl)-2-phenylethylcarbamate (<bold>19g</bold>
)</title>
<p id="p0135">Yield 82% from <sc>l</sc>
-phenylalanine methyl ester (<bold>18f</bold>
): colorless solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.36–7.31 (m, 4H), 7.29–7.21 (m, 3H), 7.10–7.07 (m, 3H), 5.09 (s, 2H), 4.68–4.63 (m, 1H), 3.70 (s, 3H), 3.16–3.04 (m, 2H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>18</sub>
H<sub>19</sub>
NO<sub>4</sub>
Na [M + Na]<sup>+</sup>
336.1212, found 336.1224.</p>
</sec>
<sec id="sec4.2.8"><label>4.2.8</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-phenylacetamido)-4-methylpentanoate (<bold>19h</bold>
)</title>
<p id="p0140">Yield 59% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); colorless solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.35 (t, <italic>J</italic>
= 7.6 Hz, 2H), 7.31–7.28 (m, 3H, merged with CDCl<sub>3</sub>
), 4.52–4.47 (m, 1H), 3.59 (s, 2H), 1.57–1.52 (m, 3H), 1.42 (s, 9H), 0.88 (d, <italic>J</italic>
= 4.8 Hz, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>18</sub>
H<sub>28</sub>
NO<sub>3</sub>
[M + H]<sup>+</sup>
306.2069, found 306.2065.</p>
</sec>
<sec id="sec4.2.9"><label>4.2.9</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-(4-methoxyphenyl)acetamido)-4-methylpentanoate (<bold>19i</bold>
)</title>
<p id="p0145">Yield 69% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); colorless solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.17 (d, <italic>J</italic>
= 8.4 Hz, 2H), 6.87 (d, <italic>J</italic>
= 8.0 Hz, 2H), 4.51–4.46 (m, 1H), 3.79 (s, 3H), 3.53 (s, 2H), 1.57–1.52 (m, 3H), 1.42 (s, 9H), 0.88–0.87 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>19</sub>
H<sub>27</sub>
NO<sub>4</sub>
[M + H]<sup>+</sup>
336.2175, found 336.2173.</p>
</sec>
<sec id="sec4.2.10"><label>4.2.10</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(3-(4-methoxyphenyl)propanamido)-4-methylpentanoate (<bold>19j</bold>
)</title>
<p id="p0150">Yield 79% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); colorless oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.13 (d, <italic>J</italic>
= 8.8 Hz, 2H), 6.83 (d, <italic>J</italic>
= 8.0 Hz, 2H), 4.54–4.48 (m, 1H), 3.79 (s, 3H), 2.94–2.90 (m, 2H), 2.55–2.44 (m, 2H), 1.56–1.44 (m, 3H), 1.44 (s, 9H), 0.90–0.88 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>20</sub>
H<sub>31</sub>
NO<sub>4</sub>
Na [M + Na]<sup>+</sup>
372.2151, found 372.2145.</p>
</sec>
<sec id="sec4.2.11"><label>4.2.11</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(3-(pyridin-3-yl)propanamido)-4-methylpentanoate (<bold>19k</bold>
)</title>
<p id="p0155">Yield 56% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); yellow oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.52 (s, 1H), 8.46 (d, <italic>J</italic>
= 3.6 Hz, 1H), 7.74–7.69 (t, <italic>J</italic>
= 8.0 Hz, 1H), 7.32–7.28 (m, 1H), 4.52–4.47 (m, 1H), 3.05–3.01 (m, 2H), 2.62–2.52 (m, 2H), 1.58–1.48 (m, 3H), 1.46 (s, 9H), 0.91–0.89 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>18</sub>
H<sub>29</sub>
N<sub>2</sub>
O<sub>3</sub>
[M + H]<sup>+</sup>
321.2178, found 321.2188.</p>
</sec>
<sec id="sec4.2.12"><label>4.2.12</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(cinnamamido)-4-methylpentanoate (<bold>19l</bold>
)</title>
<p id="p0160">Yield 79% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); colorless oil; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
7.44 (d, <italic>J</italic>
= 21.0 Hz, 1H), 7.41 (d, <italic>J</italic>
= 7.4 Hz, 2H), 7.29–7.26 (m, 5H), 6.58 (d, <italic>J</italic>
= 21.0 Hz, 1H), 4.40–4.30 (m, 1H), 1.86–1.50 (m, 3H), 1.36 (s, 9H), 0.95–0.79 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>19</sub>
H<sub>28</sub>
NO<sub>3</sub>
[M + H]<sup>+</sup>
318.2069, found 318.2077.</p>
</sec>
<sec id="sec4.2.13"><label>4.2.13</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-((<italic>E</italic>
)-3-(4-methoxyphenyl)acrylamido)-4-methylpentanoate (<bold>19m</bold>
)</title>
<p id="p0165">Yield 76% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); colorless oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.58 (d, <italic>J</italic>
= 15.6 Hz, 1H), 7.44 (d, <italic>J</italic>
= 8.6 Hz, 2H), 6.88 (d, <italic>J</italic>
= 8.6 Hz, 1H), 6.29 (d, <italic>J</italic>
= 15.6 Hz, 1H), 4.70–4.65 (m, 1H), 3.89 (s, 3H), 1.73–1.48 (m, 3H), 1.43 (s, 9H), 0.98–0.95 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>20</sub>
H<sub>31</sub>
NO<sub>4</sub>
[M + H]<sup>+</sup>
348.2175, found 348.2183.</p>
</sec>
<sec id="sec4.2.14"><label>4.2.14</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-((<italic>E</italic>
)-3-(3,4-dimethoxyphenyl)acrylamido)-4-methylpentanoate (<bold>19n</bold>
)</title>
<p id="p0170">Yield 69% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); colorless oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.53 (d, <italic>J</italic>
= 15.6 Hz, 1H), 7.04 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.00 (s, 1H), 6.84 (d, <italic>J</italic>
= 8.0 Hz, 1H), 6.30 (d, <italic>J</italic>
= 15.6 Hz, 1H), 4.71–4.65 (m, 1H), 3.90 (s, 6H), 1.74–1.49 (m, 3H), 1.43 (s, 9H), 0.98–0.95 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>21</sub>
H<sub>33</sub>
NO<sub>5</sub>
[M + H]<sup>+</sup>
378.2280, found 378.2282.</p>
</sec>
<sec id="sec4.2.15"><label>4.2.15</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-phenoxyacetamido)-4-methylpentanoate (<bold>19o</bold>
)</title>
<p id="p0175">Yield 77% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); colorless oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.33 (t, <italic>J</italic>
= 8.4 Hz, 2H), 7.03 (t, <italic>J</italic>
= 8.0 Hz, 1H), 6.96 (d, <italic>J</italic>
= 8.0 Hz, 2H), 4.63–4.60 (m, 1H), 4.52 (s, 2H), 1.68–1.54 (m, 3H), 1.47 (s, 9H), 0.96–0.94 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>18</sub>
H<sub>27</sub>
NO<sub>4</sub>
Na [M + Na]<sup>+</sup>
344.1838, found 344.1835.</p>
</sec>
<sec id="sec4.2.16"><label>4.2.16</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-(4-methoxyphenoxy)acetamido)-4-methylpentanoate (<bold>19p</bold>
)</title>
<p id="p0180">Yield 79% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); yellow oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
6.90–6.84 (m, 4H), 4.62–4.59 (m, 1H), 4.46 (s, 2H), 3.78 (s, 3H), 1.68–1.54 (m, 3H), 1.47 (s, 9H), 0.96–0.94 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>18</sub>
H<sub>29</sub>
NO<sub>4</sub>
Na [M + Na]<sup>+</sup>
374.1943, found 374.1953.</p>
</sec>
<sec id="sec4.2.17"><label>4.2.17</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-(4-hydroxyphenoxy)acetamido)-4-methylpentanoate (<bold>19q</bold>
)</title>
<p id="p0185">Yield 63% from <sc>l</sc>
-leucine <italic>tert-</italic>
butyl ester (<bold>18a</bold>
); colorless solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
6.75–6.72 (m, 2H), 6.63–6.60 (m, 2H), 4.36 (s, 2H), 4.32–4.29 (m, 1H), 1.56–1.50 (m, 3H), 1.35 (s, 9H), 0.84–0.78 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>18</sub>
H<sub>27</sub>
NO<sub>5</sub>
[M + H]<sup>+</sup>
338.1967, found 338.1961.</p>
</sec>
<sec id="sec4.2.18"><label>4.2.18</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-(3-(dimethylamino)phenoxy)acetamido)-4-methylpentanoate (<bold>19r</bold>
)</title>
<p id="p0190">Yield 62% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); yellow solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.17 (t, <italic>J</italic>
= 8.0 Hz, 1H), 6.97 (d, <italic>J</italic>
= 8.0 Hz, 1H), 6.43 (d, <italic>J</italic>
= 8.0 Hz, 1H), 6.32 (s, 1H), 4.66–4.61 (m, 1H), 4.52 (s, 2H), 2.85 (s, 6H), 1.68–1.55 (m, 3H), 1.46 (s, 9H), 0.97–0.94 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>20</sub>
H<sub>33</sub>
N<sub>2</sub>
O<sub>4</sub>
[M + H]<sup>+</sup>
365.2440, found 365.2430.</p>
</sec>
<sec id="sec4.2.19"><label>4.2.19</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-((4-methoxyphenyl)amino)acetamido)-4-methylpentanoate (<bold>19s</bold>
)</title>
<p id="p0195">Yield 54% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); brown viscous oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
6.78 (d, <italic>J</italic>
= 8.0 Hz, 2H), 6.58 (d, <italic>J</italic>
= 8.0 Hz, 1H), 4.56–4.49 (m, 1H), 3.77–3.71 (m, 5H), 1.55–1.38 (m, 12H), 0.96–0.86 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>19</sub>
H<sub>30</sub>
N<sub>2</sub>
O<sub>4</sub>
[M + H]<sup>+</sup>
351.2284, found 351.2279.</p>
</sec>
<sec id="sec4.2.20"><label>4.2.20</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-((3-methoxyphenyl)amino)acetamido)-4-methylpentanoate (<bold>19t</bold>
)</title>
<p id="p0200">Yield 57% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); brown viscous oil; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
7.00 (d, <italic>J</italic>
= 8.0 Hz, 1H), 6.87–6.85 (m, 2H), 6.77 (s, 1H), 4.58–4.53 (m, 1H), 3.89–3.71 (m, 5H), 1.57–1.51 (m, 2H), 1.48–1.46 (m, 1H), 1.43 (s, 9H), 0.95–0.91 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>19</sub>
H<sub>30</sub>
N<sub>2</sub>
O<sub>4</sub>
[M + H]<sup>+</sup>
351.2284, found 351.2297.</p>
</sec>
<sec id="sec4.2.21"><label>4.2.21</label>
<title>(<italic>S</italic>
)-<italic>tert</italic>
-Butyl 2-(2-((2-methoxyphenyl)amino)acetamido)-4-methylpentanoate (<bold>19u</bold>
)</title>
<p id="p0205">Yield 60% from <sc>l</sc>
-leucine <italic>tert</italic>
-butyl ester (<bold>18a</bold>
); light pink solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
6.87–6.74 (m, 3H), 6.51 (d, <italic>J</italic>
= 8.0 Hz, 1H), 4.58–4.53 (m, 1H), 3.87–3.75 (s, 5H), 1.57–1.52 (m, 2H), 1.48–1.46 (m, 1H), 1.43 (s, 9H), 0.92–0.87 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>19</sub>
H<sub>30</sub>
N<sub>2</sub>
O<sub>4</sub>
[M + H]<sup>+</sup>
351.2284, found 351.2277.</p>
</sec>
</sec>
<sec id="sec4.3"><label>4.3</label>
<title>Synthetic procedure for the preparation of <bold>20</bold>
</title>
<p id="p0210"><italic>Procedure A</italic>
: To a solution of the corresponding <italic>tert</italic>
-butyl ester <bold>19</bold>
(0.110 g, 0.31 mmol) in CH<sub>2</sub>
Cl<sub>2</sub>
(2 mL) at 0 °C was added TFA/H<sub>2</sub>
O (10:1, 3 mL). After 5 min stirring, the reaction mixture was allowed to stir at room temperature for 1 h. The solvent was completely evaporated under reduced pressure to give the corresponding acids <bold>20</bold>
, which were used directly in the subsequent step without further characterization.</p>
<p id="p0215"><italic>Procedure B</italic>
: To a solution of the corresponding methyl ester <bold>19</bold>
(0.100 g, 0.35 mmol) in THF (3 mL) at room temperature was added LiOH in water (0.102 g, 2.45 mmol). After 3 h stirring, the solvent was completely evaporated under reduced pressure and the resulting residue was neutralized with 2 M HCl. The solution was extracted with EtOAc (20 mL × 2), dried over Na<sub>2</sub>
SO<sub>4</sub>
, filtered and evaporated under reduced pressure to give the corresponding acids <bold>20</bold>
, which were directly used for next step without further characterizations.</p>
</sec>
<sec id="sec4.4"><label>4.4</label>
<title>Synthesis of <italic>tert</italic>
-butyl (<italic>S</italic>
)-1-(<italic>N</italic>
-methoxy-<italic>N</italic>
-methylcarbamoyl)-2-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)ethylcarbamate (<bold>23</bold>
)</title>
<p id="p0220">Compound <bold>22</bold>
was prepared through sequential reactions from the well-known intermediate <bold>21</bold>
, as reported previously <xref rid="bib20" ref-type="bibr">[20]</xref>
, <xref rid="bib21" ref-type="bibr">[21]</xref>
, <xref rid="bib22" ref-type="bibr">[22]</xref>
, <xref rid="bib31" ref-type="bibr">[31]</xref>
.</p>
<p id="p0225">To a solution of <bold>22</bold>
(3.27 g, 8.0 mmol) in methanol (5 mL) was added a 4 M NaOH solution (0.04 mol) at room temperature. The mixture was stirred for 4 h and the methanol was evaporated. The residue was neutralized with 2 M HCl and extracted with EtOAc (20 mL × 4). The organic layer was washed with brine, dried over Na<sub>2</sub>
SO<sub>4</sub>
, and evaporated under reduced pressure to yield the corresponding acid. To a solution of the acid (12.0 mmol) in DMF (30 mL) were added EDC·HCl (0.540 g, 10.0 mmol), HOBt·H<sub>2</sub>
O (0.330 g, 11.0 mmol), and <italic>N</italic>
,<italic>O</italic>
-dimethylhydroxylamine (0.213 g, 11.0 mmol) at ambient temperature. The solution was cooled to 0 °C, and TEA (0.240 mL, 11.0 mmol) was added slowly. After 2 h, the DMF was evaporated under high vacuum, and the resulting residue was dissolved in EtOAc (100 mL). The resulting solution was subsequently washed with 5% citric acid (20 mL × 2), 5% NaHCO<sub>3</sub>
(20 mL × 2), and brine (50 mL). The organic layer was then dried over Na<sub>2</sub>
SO<sub>4</sub>
and concentrated under reduced pressure to yield the Weinreb amide derivative <bold>23</bold>
, which was purified by column chromatography (EtOAc/MeOH = 9.5:0.5).</p>
<p id="p0230">The data for compounds <bold>22</bold>
and <bold>23</bold>
were reported previously <xref rid="bib20" ref-type="bibr">[20]</xref>
, <xref rid="bib21" ref-type="bibr">[21]</xref>
.</p>
<sec id="sec4.4.1"><label>4.4.1</label>
<title><italic>tert</italic>
-Butyl ((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (<bold>24a</bold>
)</title>
<p id="p0235">To a solution of benzothiazole (1.37 g, 10.0 mmol) in THF at −78 °C was added <italic>n</italic>
-BuLi (2.0 M in THF, 1.67 mL) dropwise over 20 min. After 1 h stirring, the Weinreb amide <bold>23</bold>
(0.064 g, 2.0 mmol) in THF was slowly added dropwise over 20 min and then the solution was stirred for 3 h at same temperature. The reaction was quenched with sat. NH<sub>4</sub>
Cl and allowed to stir at 0 °C for 20 min. The mixture was evaporated and dissolved in EtOAc. This solution was washed with water (100 mL) and brine (50 mL), and then dried over Na<sub>2</sub>
SO<sub>4</sub>
. After filtration, the organic layer was concentrated under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/MeOH = 9:1) to obtain the pure compound <bold>24a</bold>
. The data for the compound <bold>24a</bold>
has been reported in a previous article <xref rid="bib21" ref-type="bibr">[21]</xref>
.</p>
</sec>
<sec id="sec4.4.2"><label>4.4.2</label>
<title><italic>tert</italic>
-Butyl ((<italic>S</italic>
)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-2-yl)carbamate (<bold>24b</bold>
)</title>
<p id="p0240">To a cooled solution of the commercially available 5-phenylthiazole (0.175 g, 10 mmol) in dry THF at −78 °C was slowly added a solution of LDA (1.0 mL, 14 mmol). After 1 h, a pre-cooled solution containing the Weinreb amide <bold>23</bold>
(0.329 g, 10 mmol) in anhydrous THF was slowly added, and the reaction was stirred at −78 °C over 2 h. The solution was allowed to warm to room temperature, was quenched by the addition of water (35 mL), and then extracted with EtOAc (3 × 50 mL). The combined organic layers were dried over anhydrous Na<sub>2</sub>
SO<sub>4</sub>
, filtered, and evaporated in vacuo. The crude mixture was then purified using flash chromatography (<italic>n</italic>
-hexane/EtOAc = 3:7) to furnish <bold>24b</bold>
. The data for the compound <bold>24b</bold>
were reported previously <xref rid="bib22" ref-type="bibr">[22]</xref>
.</p>
<p id="p0245">Compounds <bold>24c</bold>
–<bold>e</bold>
were prepared from <bold>23</bold>
according to the procedure described for the synthesis of <bold>24b</bold>
.</p>
</sec>
<sec id="sec4.4.3"><label>4.4.3</label>
<title><italic>tert</italic>
-Butyl ((<italic>S</italic>
)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)-1-(5-(<italic>p</italic>
-tolyl)thiazol-2-yl)propan-2-yl)carbamate (<bold>24c</bold>
)</title>
<p id="p0250">The data for the compound <bold>24c</bold>
has been reported in a previous article <xref rid="bib22" ref-type="bibr">[22]</xref>
.</p>
</sec>
<sec id="sec4.4.4"><label>4.4.4</label>
<title><italic>tert</italic>
-Butyl ((<italic>S</italic>
)-1-(5-(4-methoxyphenyl)thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (<bold>24d</bold>
)</title>
<p id="p0255">The data for the compound <bold>24d</bold>
has been reported in a previous article <xref rid="bib22" ref-type="bibr">[22]</xref>
.</p>
</sec>
<sec id="sec4.4.5"><label>4.4.5</label>
<title><italic>tert</italic>
-Butyl ((<italic>S</italic>
)-1-(5-(4-methoxyphenyl)thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (<bold>24e</bold>
)</title>
<p id="p0260">The data for the compound <bold>24e</bold>
has been reported in a previous article <xref rid="bib22" ref-type="bibr">[22]</xref>
.</p>
</sec>
</sec>
<sec id="sec4.5"><label>4.5</label>
<title>Synthesis of (<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-(3-methylbutanamido)pentanamide (<bold>25a</bold>
)</title>
<p id="p0265">To a solution of <bold>24a</bold>
(0.200 g, 0.5 mmol) in CH<sub>2</sub>
Cl<sub>2</sub>
(3 mL) at 0 °C was added TFA/H<sub>2</sub>
O (10:1, 5 mL), and the solution was stirred for 1 h. After evaporating the solvent under reduced pressure, the corresponding deprotected lactam residue (0.100 g, 0.53 mmol) was coupled to the carboxylic acid <bold>20a</bold>
(0.136 g, 0.38 mmol) using the coupling agent HBTU (0.147 g, 0.38 mmol) in the presence of diisopropylethylamine (0.050 mL, 0.38 mmol) in DMF (3 mL) at 0 °C. The reaction mixture was allowed to stir for 2–3 h under ambient conditions. The solvent was then evaporated under a high vacuum, and the residue was dissolved in EtOAc (50 mL). The organic layer was washed with 5% citric acid (20 mL × 2), 5% NaHCO<sub>3</sub>
(20 mL × 2), and brine (25 mL). This solution was dried over Na<sub>2</sub>
SO<sub>4</sub>
, filtered, and evaporated under reduced pressure to give compound <bold>25a</bold>
. Yield 35%; light yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.21 (d, <italic>J</italic>
= 8.0 Hz, 1H), 8.11 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.65–7.51 (m, 2H), 5.72–5.69 (m, 1H), 4.61–4.42 (m, 1H), 3.38–3.29 (m, 4H, merged with CD<sub>3</sub>
OD), 2.73–2.66 (m, 1H), 2.46–2.40 (m, 1H), 2.22–2.00 (m, 4H), 1.67–1.53 (m, 3H), 1.28–1.21 (m, 2H), 0.95–0.86 (m, 12H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.3, 181.8, 175.8, 165.5, 154.8, 138.4, 129.3, 126.5, 123.7, 55.3, 53.1, 52.9, 46.1, 45.9, 41.8, 41.5, 40.0, 39.8, 29.4, 28.8, 27.5, 25.8, 23.4, 22.8, 21.9. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>25</sub>
H<sub>35</sub>
N<sub>4</sub>
O<sub>4</sub>
S [M + H]<sup>+</sup>
487.2379 found 487.2373.</p>
<p id="p0270">Compounds <bold>25b</bold>
–<bold>h</bold>
were prepared from <bold>24a</bold>
with <bold>20a</bold>
–<bold>h</bold>
using a procedure similar to that described for the synthesis of <bold>25a</bold>
.</p>
<sec id="sec4.5.1"><label>4.5.1</label>
<title><italic>tert</italic>
-Butyl ((<italic>S</italic>
)-1-(((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (<bold>25b</bold>
)</title>
<p id="p0275">Yield 37% from <bold>24a</bold>
; colorless solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.18 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.99 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.60–7.52 (m, 2H), 5.75–5.66 (m, 1H), 4.24–4.04 (m, 1H), 3.40–3.37 (m, 2H), 2.66–2.45 (m, 2H), 2.17–1.95 (m, 2H), 1.94–1.72 (m, 1H), 1.70–1.53 (m, 3H), 1.44 (s, 9H), 0.96–0.88 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>25</sub>
H<sub>35</sub>
N<sub>4</sub>
O<sub>5</sub>
SNa [M + Na]<sup>+</sup>
525.2148, found 525.2145.</p>
</sec>
<sec id="sec4.5.2"><label>4.5.2</label>
<title>Benzyl ((<italic>S</italic>
)-1-(((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (<bold>25c</bold>
)</title>
<p id="p0280">Yield 43% from <bold>24a</bold>
; colorless solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.16 (d, <italic>J</italic>
= 8.4 Hz, 1H), 7.97 (d, <italic>J</italic>
= 8.4 Hz, 1H), 7.58–7.51 (m, 2H), 7.32–7.27 (m, 5H, merged with CDCl<sub>3</sub>
), 5.70–5.58 (m, 1H), 5.12–5.08 (m, 2H), 4.39–4.14 (m, 1H), 3.30–3.27 (m, 2H), 2.64–2.49 (m, 2H), 2.24–2.10 (m, 2H), 2.09–1.89 (m, 1H), 1.86–1.67 (m, 2H), 1.66–1.48 (m, 1H), 0.93–0.85 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.5, 181.8, 175.8, 165.5, 158.4, 154.8, 138.4, 129.4, 128.7, 127.3, 126.5, 123.7, 67.7, 67.5, 56.0, 55.1, 54.9, 42.0, 41.9, 39.9, 33.9, 32.7, 29.3, 28.7, 25.8, 23.7, 22.0, 21.8. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>28</sub>
H<sub>33</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
537.2172 found 537.2153.</p>
</sec>
<sec id="sec4.5.3"><label>4.5.3</label>
<title>Benzyl ((<italic>S</italic>
)-1-(((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-1-oxohexan-2-yl)carbamate (<bold>25d</bold>
)</title>
<p id="p0285">Yield 45% from <bold>24a</bold>
; light yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.37–8.28 (m, 2H), 7.69–7.64 (m, 2H), 7.40–7.30 (m, 5H), 5.56–5.47 (m, 1H), 5.04–4.99 (m, 2H), 4.07–4.01 (m, 1H), 3.22–3.11 (m, 2H, merged with CD<sub>3</sub>
OD), 2.51–2.11 (m, 3H), 1.97–1.75 (m, 2H), 1.67–1.49 (m, 2H), 1.21–1.08 (m, 4H), 0.85–0.72 (m, 3H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
202.2, 192.8, 178.0, 172.4, 164.3, 155.8, 152.8, 137.0, 136.3, 77.2, 65.2, 64.2, 42.1, 41.9, 41.5, 37.8, 36.6, 32.0, 31.6, 31.5, 28.1, 27.4, 25.9, 23.6, 21.7, 21.5, 13.6. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>28</sub>
H<sub>33</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
537.2172 found 537.2166.</p>
</sec>
<sec id="sec4.5.4"><label>4.5.4</label>
<title>Benzyl ((<italic>S</italic>
)-1-(((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (<bold>25e</bold>
)</title>
<p id="p0290">Yield 39% from <bold>24a</bold>
; yellow solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.18 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.98 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.59–7.51 (m, 2H), 7.39–7.30 (m, 5H), 5.74–5.65 (m, 1H), 5.15–5.09 (m, 2H), 4.40–4.20 (m, 1H), 3.39–3.36 (m, 2H), 2.65–2.52 (m, 2H), 2.30–2.16 (m, 3H), 2.03–1.96 (m, 1H), 1.10–0.89 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.5, 181.8, 174.6, 165.4, 158.5, 154.7, 138.4, 129.5, 129.0, 128.7, 126.6, 123.7, 79.5, 78.9, 67.6, 61.1, 55.3, 41.6, 40.0, 38.2, 38.1, 33.9, 33.2, 32.7, 28.7, 25.9, 23.7. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>27</sub>
H<sub>31</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
523.2015 found 523.2003.</p>
</sec>
<sec id="sec4.5.5"><label>4.5.5</label>
<title>Benzyl ((2<italic>S</italic>
,3<italic>R</italic>
)-1-(((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-3-methyl-1-oxopentan-2-yl)carbamate (<bold>25f</bold>
)</title>
<p id="p0295">Yield 42% from <bold>24a</bold>
; light yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.19 (d, <italic>J</italic>
= 8.4 Hz, 1H), 8.08 (d, <italic>J</italic>
= 8.4 Hz, 1H), 7.63–7.56 (m, 2H), 7.33–7.27 (m, 5H), 5.75–5.73 (m, 1H), 5.11–5.03 (s, 2H), 4.12–4.05 (m, 1H), 3.35–3.25 (m, 2H, merged with CD<sub>3</sub>
OD), 2.70–2.50 (m, 1H), 2.49–2.39 (m, 1H), 2.38–2.01 (m, 2H), 2.00–1.85 (m, 1H), 1.80–1.75 (m, 1H), 1.67–1.49 (m, 2H), 1.08–0.86 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>28</sub>
H<sub>33</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
537.2178 found 537.2178.</p>
</sec>
<sec id="sec4.5.6"><label>4.5.6</label>
<title>Benzyl ((<italic>S</italic>
)-1-(((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-(methylthio)-1-oxobutan-2-yl)carbamate (<bold>25g</bold>
)</title>
<p id="p0300">Yield 45% from <bold>24a</bold>
; white solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.19 (d, <italic>J</italic>
= 8.4 Hz, 1H), 8.00 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.59–7.52 (m, 2H), 7.35–7.25 (m, 5H), 5.78–5.61 (m, 1H), 5.20–5.10 (m, 2H), 4.60–4.44 (m, 1H), 3.40–3.34 (m, 2H), 2.69–2.50 (m, 1H), 2.49–2.38 (m, 2H), 2.38–2.09 (m, 2H), 2.08–2.00 (m, 5H), 1.99–1.89 (m, 2H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.5, 181.8, 174.8, 165.7, 165.4, 158.4, 154.7, 138.4, 129.4, 129.3, 128.8, 127.3, 126.5, 123.7, 67.8, 56.0, 41.5, 40.0, 33.8, 32.8, 31.7, 31.0, 30.7, 29.3, 28.7, 24.1, 15.2. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>27</sub>
H<sub>31</sub>
N<sub>4</sub>
O<sub>5</sub>
S<sub>2</sub>
[M + H]<sup>+</sup>
555.1736 found 555.1743.</p>
</sec>
<sec id="sec4.5.7"><label>4.5.7</label>
<title>Benzyl ((<italic>S</italic>
)-1-(((<italic>S</italic>
)-1-(benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (<bold>25h</bold>
)</title>
<p id="p0305">Yield 43% from <bold>24a</bold>
; colorless solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.19 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.99 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.61–7.55 (m, 2H), 7.51–7.17 (m, 10H, merged with CDCl<sub>3</sub>
), 5.82–5.79 (m, 1H), 5.11–5.08 (m, 2H), 4.43–4.30 (m, 1H), 3.39–3.31 (m, 2H), 3.20–3.15 (m, 2H), 2.65–2.45 (m, 2H), 2.39–2.14 (m, 3H). <sup>13</sup>
C NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
192.9, 181.2, 174.3, 165.6, 163.8, 161.8, 154.8, 141.9, 139.2, 129.3, 128.9, 127.4, 127.0, 126.2, 125.9, 125.5, 123.3, 117.9, 114.1, 63.1, 55.2, 54.4, 41.6, 44.3, 41.4, 39.7, 38.2, 33.0, 27.8, 24.7, 22.8. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>31</sub>
H<sub>32</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
571.2015 found 571.2003.</p>
</sec>
</sec>
<sec id="sec4.6"><label>4.6</label>
<title>Synthesis of <bold>26</bold>
</title>
<p id="p0310">Compounds <bold>26a</bold>
–<bold>r</bold>
were prepared from <bold>24a</bold>
with <bold>20i</bold>
–<bold>u</bold>
using a procedure similar to that described for the synthesis of <bold>25a</bold>
.</p>
<sec id="sec4.6.1"><label>4.6.1</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-(2-phenylacetamido)pentanamide (<bold>26a</bold>
)</title>
<p id="p0315">Yield 47% from <bold>24a</bold>
; white solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.09 (d, <italic>J</italic>
= 8.0 Hz, 1H), 8.01 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.55–7.47 (m, 2H), 7.19–7.11 (m, 5H), 5.61–5.57 (m, 1H), 4.35–4.32 (m, 1H), 3.44 (s, 2H), 3.40–3.36 (m, 1H), 3.19–3.07 (m, 1H), 2.57–2.52 (m, 1H), 2.39–2.20 (m, 2H), 2.12–1.88 (m, 2H), 1.79–1.48 (m, 3H), 0.88–0.76 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.5, 181.8, 175.1, 165.5, 154.8, 138.4, 136.9, 130.2, 129.5, 128.4, 127.8, 126.5, 124.2, 123.7, 55.9, 55.2, 53.3, 43.7, 41.8, 39.9, 39.2, 33.7, 32.8, 29.4, 28.7, 25.8, 23.7, 22.1. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>28</sub>
H<sub>33</sub>
N<sub>4</sub>
O<sub>4</sub>
S [M + H]<sup>+</sup>
521.2223 found 521.2216.</p>
</sec>
<sec id="sec4.6.2"><label>4.6.2</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-(2-(4-methoxyphenyl)acetamido)-4-methylpentanamide (<bold>26b</bold>
)</title>
<p id="p0320">Yield 37% from <bold>24a</bold>
; yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.20 (d, <italic>J</italic>
= 8.0 Hz, 1H), 8.11 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.64–7.58 (m, 2H), 7.20 (d, <italic>J</italic>
= 8.0 Hz, 2H), 6.84 (d, <italic>J</italic>
= 8.4 Hz, 2H), 5.69–5.67 (m, 1H), 4.45–4.41 (m, 1H), 3.73 (s, 3H), 3.51–3.21 (m, 4H, merged with CD<sub>3</sub>
OD), 2.63–2.52 (m, 1H), 2.51–2.21 (m, 2H), 2.10–1.88 (m, 2H), 1.86–1.55 (m, 3H), 0.92–0.81 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.5, 181.8, 175.1, 174.4, 165.5, 160.1, 154.8, 138.4, 131.4, 129.3, 128.4, 126.5, 123.7, 114.9, 55.6, 55.2, 42.8, 42.6, 41.6, 41.5, 39.9, 33.7, 29.4, 28.7, 25.8, 25.4, 24.2, 23.2, 22.1. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>29</sub>
H<sub>34</sub>
N<sub>4</sub>
O<sub>5</sub>
SNa [M + Na]<sup>+</sup>
573.2148 found 573.2148.</p>
</sec>
<sec id="sec4.6.3"><label>4.6.3</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-(3-(4-methoxyphenyl)propanamido)-4-methylpentanamide (<bold>26c</bold>
)</title>
<p id="p0325">Yield 43% from <bold>24a</bold>
; colorless solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.17 (d, <italic>J</italic>
= 8.4 Hz, 1H), 7.98 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.60–7.51 (m, 2H), 7.10 (d, <italic>J</italic>
= 8.4 Hz, 2H), 6.81 (d, <italic>J</italic>
= 8.4 Hz, 2H), 5.67–5.55 (m, 1H), 4.59–4.50 (m, 1H), 3.79 (s, 3H), 3.40–3.37 (m, 2H), 2.93–2.89 (m, 2H), 2.66–2.43 (m, 4H), 2.22–1.99 (m, 3H), 1.77–1.57 (m, 2H), 1.46–1.40 (m, 1H), 0.91–0.86 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
192.2, 180.3, 172.2, 162.9, 162.5, 158.0, 155.7, 155.4, 147.6, 140.6, 132.7, 129.7, 129.3, 127.3, 113.8, 55.2, 54.8, 50.0, 41.5, 40.6, 38.6, 38.4, 34.8, 33.4, 30.0, 28.7, 27.9, 24.7, 22.8, 21.9. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>30</sub>
H<sub>36</sub>
N<sub>4</sub>
O<sub>5</sub>
SNa [M + Na]<sup>+</sup>
587.2034 found 587.2032.</p>
</sec>
<sec id="sec4.6.4"><label>4.6.4</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-(3-(pyridin-3-yl)propanamido)pentanamide (<bold>26d</bold>
)</title>
<p id="p0330">Yield 27% from <bold>24a</bold>
; brown solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.30–8.28 (m, 2H), 7.88–7.86 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.66–7.64 (m, 2H), 7.51–7.26 (m, 2H), 7.20–7.16 (m, 1H), 5.61–5.57 (m, 1H), 4.49–4.45 (m, 1H), 3.35–3.29 (m, 2H), 3.01–2.97 (m, 2H), 2.83–2.80 (m, 2H), 2.71–2.69 (m, 2H), 2.58–2.42 (m, 3H), 2.41–2.25 (m, 2H), 1.90–1.84 (m, 1H), 1.73–1.53 (m, 2H), 0.96–0.85 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>28</sub>
H<sub>34</sub>
N<sub>5</sub>
O<sub>4</sub>
S [M + H]<sup>+</sup>
536.2332 found 536.2327.</p>
</sec>
<sec id="sec4.6.5"><label>4.6.5</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-cinnamamido-4-methylpentanamide (<bold>26e</bold>
)</title>
<p id="p0335">Yield 41% from <bold>24a</bold>
; yellow solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.17–8.15 (m, <italic>J</italic>
= 8.0 Hz, 1H), 7.97–7.93 (m, 1H), 7.70–7.50 (m, 5H), 7.49–7.45 (m, 2H), 6.47–6.43 (m, 1H), 5.70–5.60 (m, 1H), 4.90–4.72 (m, 1H), 3.40–3.27 (m, 2H), 2.60–2.45 (m, 2H), 2.40–1.99 (m, 2H), 1.95–1.80 (m, 1H), 1.76–1.42 (m, 3H), 0.96–0.83 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.5, 181.2, 176.3, 165.7, 154.7, 142.4, 138.3, 136.2, 130.9, 129.9, 128.4, 126.5, 123.7, 121.3, 56.1, 55.6, 54.4, 53.7, 42.1, 41.6, 40.1, 33.8, 30.7, 29.52, 28.9, 26.0, 25.9, 23.3, 22.1. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>29</sub>
H<sub>33</sub>
N<sub>4</sub>
O<sub>4</sub>
S [M + H]<sup>+</sup>
533.2223 found 533.2243.</p>
</sec>
<sec id="sec4.6.6"><label>4.6.6</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-((<italic>E</italic>
)-3-(4-methoxyphenyl)acrylamido)-4-methylpentanamide (<bold>26f</bold>
)</title>
<p id="p0340">Yield 33% from <bold>24a</bold>
; yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.26 (d, <italic>J</italic>
= 7.0 Hz, 1H), 8.18–8.10 (m, 1H), 7.70–7.55 (m, 2H), 7.54–7.42 (m, 3H), 6.95 (d, <italic>J</italic>
= 8.8 Hz, 2H), 6.90–6.88 (m, 1H), 6.61–6.50 (m, 2H), 5.80–5.62 (m, 1H), 4.62–4.50 (m, 1H), 3.84 (s, 3H), 3.40–3.29 (m, 2H, merged with CD<sub>3</sub>
OD), 2.80–2.65 (m, 1H), 2.63–2.45 (m, 1H), 2.43–2.20 (m, 1H), 2.19–1.99 (m, 1H), 1.92–1.78 (m, 1H), 1.77–1.50 (m, 3H), 1.05–0.88 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
191.9, 180.2, 173.3, 166.6, 163.7, 160.8, 153.2, 140.9, 137.1, 129.3, 127.9, 127.4, 127.0, 125.5, 122.3, 117.9, 117.7, 114.1, 55.2, 54.4, 51.6, 51.4, 41.6, 40.4, 38.7, 33.0, 27.6, 24.6, 22.7, 21.6. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>30</sub>
H<sub>34</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
563.2328 found 563.2328.</p>
</sec>
<sec id="sec4.6.7"><label>4.6.7</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-((<italic>E</italic>
)-3-(3,4-dimethoxyphenyl)acrylamido)-4-methylpentanamide (<bold>26g</bold>
)</title>
<p id="p0345">Yield 42% from <bold>24a</bold>
; yellow solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.20–8.14 (m, 1H), 8.00–7.94 (m, 1H), 7.60–7.50 (m, 3H), 7.10–6.99 (m, 2H), 6.84 (d, <italic>J</italic>
= 8.0 Hz, 1H), 6.30 (d, <italic>J</italic>
= 16.0 Hz, 1H), 5.85–5.60 (m, 1H), 4.78–4.66 (m, 1H), 3.90 (s, 6H), 3.42–3.30 (m, 2H), 2.70–2.45 (m, 2H), 2.40–2.25 (m, 2H), 2.20–1.85 (m, 2H), 1.80–1.64 (m, 2H), 1.10–0.88 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.5, 182.4, 176.2, 169.7, 155.5, 152.3, 149.5, 142.8, 138.3, 135.3, 131.9, 129.3, 127.3, 125.7, 123.5, 119.3, 112.7, 117.7, 56.5, 54.2, 53.6, 42.3, 41.6, 40.0, 33.8, 30.7, 29.3, 28.8, 25.8, 23.6, 22.1. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>31</sub>
H<sub>36</sub>
N<sub>4</sub>
O<sub>6</sub>
SNa [M + Na]<sup>+</sup>
615.2253 found 615.2253.</p>
</sec>
<sec id="sec4.6.8"><label>4.6.8</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-(2-phenoxyacetamido)pentanamide (<bold>26h</bold>
)</title>
<p id="p0350">Yield 45% from <bold>24a</bold>
; colorless solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.17 (d, <italic>J</italic>
= 8.0 Hz, 1H), 8.00–7.97 (m, 1H), 7.60–7.51 (m, 2H), 7.31–7.27 (m, 2H, merged with CDCl<sub>3</sub>
), 7.10–6.85 (m, 3H), 5.70–5.65 (m, 1H), 4.76–4.66 (m, 1H), 4.55–4.50 (m, 2H), 3.40–3.21 (m, 2H), 2.70–2.50 (m, 2H), 2.35–2.19 (m, 1H), 2.34–2.20 (m, 2H), 2.19–1.85 (m, 1H), 1.80–1.65 (m, 2H), 1.00–0.88 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.6, 181.8, 174.8, 174.5, 171.5, 165.5, 159.2, 154.7, 138.4, 130.6, 129.3, 126.7, 123.7, 115.8, 68.1, 55.9, 52.9, 41.9, 40.7, 39.8, 33.7, 29.2, 28.8, 25.7, 23.5, 22.0, 21.7, 21.2. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>28</sub>
H<sub>33</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
537.2174 found 537.2174.</p>
</sec>
<sec id="sec4.6.9"><label>4.6.9</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-(2-(4-methoxyphenoxy)acetamido)-4-methylpentanamide (<bold>26i</bold>
)</title>
<p id="p0355">Yield 45% from <bold>24a</bold>
; yellow solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.18 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.99 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.60–7.52 (m, 2H), 6.90–6.80 (m, 4H), 5.81–5.65 (m, 1H), 4.70–4.61 (m, 1H), 4.60–4.45 (m, 2H), 3.76 (s, 3H), 3.40–3.28 (m, 2H), 2.75–2.40 (m, 2H), 2.39–1.90 (m, 3H), 1.89–1.58 (m, 3H), 1.00–0.89 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
192.5, 180.2, 172.4, 169.4, 168.7, 163.8, 154.7, 151.5, 145.3, 137.2, 128.0, 127.1, 125.6, 122.4, 115.9, 114.7, 68.1, 55.6, 53.8, 41.7, 40.6, 38.0, 37.8, 32.9, 28.8, 25.8, 23.5, 22.0, 21.8. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>29</sub>
H<sub>35</sub>
N<sub>4</sub>
O<sub>6</sub>
S [M + H]<sup>+</sup>
567.2277, found 567.2268.</p>
</sec>
<sec id="sec4.6.10"><label>4.6.10</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-(2-(4-hydroxyphenoxy)acetamido)-4-methylpentanamide (<bold>26j</bold>
)</title>
<p id="p0360">Yield 40% from <bold>24a</bold>
; yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.21 (d, <italic>J</italic>
= 8.1 Hz, 1H), 8.11 (d, <italic>J</italic>
= 8.1 Hz, 1H), 7.65–7.58 (m, 2H), 6.82–6.78 (m, 2H), 6.70–6.65 (m, 2H), 5.78–5.66 (m, 1H), 4.55–4.50 (m, 1H), 4.60–4.45 (m, 2H), 3.35–3.18 (m, 2H, merged with CD<sub>3</sub>
OD) 2.75–2.45 (m, 1H), 2.44–2.20 (m, 2H), 2.19–1.89 (m, 2H), 1.88–1.61 (m, 1H), 1.60–1.35 (m, 2H), 0.99–0.97 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
192.9, 181.5, 173.2, 165.3, 154.7, 138.4, 132.3, 128.5, 127.2, 126.5, 124.3, 123.7, 116.8, 113.1, 69.2, 56.0, 54.2, 42.0, 40.0, 39.8, 34.0, 32.6, 29.2, 28.8, 25.7, 23.4, 22.0, 21.7. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>28</sub>
H<sub>32</sub>
N<sub>4</sub>
O<sub>6</sub>
SNa [M + Na]<sup>+</sup>
575.1940 found 575.1926.</p>
</sec>
<sec id="sec4.6.11"><label>4.6.11</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-(2-(3-(dimethylamino)phenoxy)acetamido)-4-methylpentanamide (<bold>26k</bold>
)</title>
<p id="p0365">Yield 35% from <bold>24a</bold>
; light green solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.18 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.99–7.97 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.60–7.52 (m, 2H), 7.29–7.22 (m, 1H, merged with CDCl<sub>3</sub>
), 7.11 (d, <italic>J</italic>
= 8.4 Hz, 1H), 6.77–6.70 (m, 1H), 6.56 (dd, 8.1, 8.4 Hz, 2H), 5.70–5.66 (m, 1H), 4.79–4.67 (m, 1H), 4.66–4.50 (m, 2H), 3.41–3.28 (m, 2H), 3.04 (s, 3H), 3.01 (s, 3H), 2.75–2.60 (m, 1H), 2.59–2.49 (m, 1H), 2.48–2.35 (m, 1H), 2.34–2.10 (m, 2H), 2.05–1.85 (m, 1H), 1.79–1.57 (m, 2H), 0.99–0.90 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.6, 181.9, 178.0, 172.3, 171.6, 171.3, 165.7, 142.6, 138.4, 131.2, 129.3, 128.5, 126.8, 123.7, 121.9, 90.7, 77.2, 66.7, 57.4, 52.8, 41.5, 40.0, 33.7, 28.8, 25.7, 23.4, 22.0, 21.7. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>30</sub>
H<sub>38</sub>
N<sub>5</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
580.2594 found 580.2563.</p>
</sec>
<sec id="sec4.6.12"><label>4.6.12</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-(2-((4-methoxyphenyl)amino)acetamido)-4-methylpentanamide (<bold>26l</bold>
)</title>
<p id="p0370">Yield 45% from <bold>24a</bold>
; light yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.24 (d, <italic>J</italic>
= 8.0 Hz, 1H), 8.14 (d, <italic>J</italic>
= 8.0 Hz, 1H), 7.68–7.60 (m, 2H), 7.06 (t, <italic>J</italic>
= 8.4 Hz, 1H), 6.30–6.17 (m, 3H), 5.72–5.68 (m, 1H), 4.53–4.50 (m, 1H), 3.84–3.65 (m, 2H), 3.82 (s, 3H), 3.37–3.28 (m, 2H, merged with CD<sub>3</sub>
OD), 2.72–2.55 (m, 1H), 2.54–2.29 (m, 1H), 2.28–1.83 (m, 3H), 1.62–1.41 (m, 3H), 0.90–0.83 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.4, 181.8, 175.4, 165.6, 164.0, 155.8, 154.7, 138.3, 132.0, 129.3, 129.2, 128.4, 127.1, 126.4, 124.2, 122.9, 116.7, 113.9, 105.2, 103.2, 56.2, 53.4, 41.8, 40.0, 39.4, 33.9, 29.3, 28.8, 26.1, 22.1, 21.9. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>29</sub>
H<sub>36</sub>
N<sub>5</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
566.2437 found 566.2428.</p>
</sec>
<sec id="sec4.6.13"><label>4.6.13</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-(2-((3-methoxyphenyl)amino)acetamido)-4-methylpentanamide (<bold>26m</bold>
)</title>
<p id="p0375">Yield 41% from <bold>24a</bold>
; light yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.21 (d, <italic>J</italic>
= 7.6 Hz, 1H), 8.12 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.66–7.58 (m, 2H), 7.10–6.89 (m, 4H), 5.80–5.65 (m, 1H), 4.60–4.45 (m, 1H), 3.94–3.72 (m, 2H), 3.76 (s, 3H), 3.36–3.29 (m, 2H, merged with CD<sub>3</sub>
OD), 2.75–2.55 (m, 1H), 2.49–2.25 (m, 1H), 2.20–1.80 (m, 3H), 1.70–1.45 (m, 3H), 0.99–0.84 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.4, 181.8, 177.4, 162.3, 160.2, 157.8, 141.2, 138.4, 133.9, 129.3, 128.4, 126.5, 123.7, 119.3, 116.4, 109.9, 66.0, 55.6, 55.4, 53.0, 42.0, 41.7, 41.5, 34.3, 28.8, 25.7, 23.3, 21.9, 21.5. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>29</sub>
H<sub>36</sub>
N<sub>5</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
566.2437 found 566.2415.</p>
</sec>
<sec id="sec4.6.14"><label>4.6.14</label>
<title>(<italic>S</italic>
)-<italic>N</italic>
-((<italic>S</italic>
)-1-(Benzo[<italic>d</italic>
]thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)-2-(2-((2-methoxyphenyl)amino)acetamido)-4-methylpentanamide (<bold>26n</bold>
)</title>
<p id="p0380">Yield 45% from <bold>24a</bold>
; light yellow solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.11 (d, <italic>J</italic>
= 7.6 Hz, 1H), 8.02 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.56–7.48 (m, 2H), 6.85–6.82 (m, 1H), 6.79–6.87 (m, 2H), 6.56–6.54 (m, 1H), 5.65–5.56 (m, 1H), 4.43–4.39 (m, 1H), 3.81–3.73 (m, 5H), 3.31–3.25 (m, 2H, merged with CD<sub>3</sub>
OD), 2.58–2.45 (m, 2H), 2.44–2.10 (m, 1H), 2.09–1.68 (m, 3H), 1.40–1.38 (m, 3H), 0.80–0.71 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
193.4, 181.8, 174.9, 173.3, 165.5, 159.8, 154.4, 138.4, 133.3, 130.2, 129.3, 128.0, 126.9, 123.7, 123.2, 116.3, 65.4, 56.1, 55.4, 53.0, 42.0, 41.6, 40.1, 34.3, 33.7, 28.8, 25.7, 23.3, 21.9, 21.2. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>29</sub>
H<sub>36</sub>
N<sub>5</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
566.2437 found 566.2426.</p>
</sec>
</sec>
<sec id="sec4.7"><label>4.7</label>
<title>Synthesis of <bold>27</bold>
</title>
<p id="p0385">Compounds <bold>27a</bold>
–<bold>d</bold>
were prepared from <bold>24b</bold>
–<bold>d</bold>
with <bold>20a</bold>
using a similar procedure to that described for the synthesis of <bold>25a</bold>
.</p>
<sec id="sec4.7.1"><label>4.7.1</label>
<title>Benzyl ((<italic>S</italic>
)-4-methyl-1-oxo-1-(((<italic>S</italic>
)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-2-yl)amino)pentan-2-yl)carbamate (<bold>27a</bold>
)</title>
<p id="p0390">Yield 55% from <bold>24b</bold>
; colorless solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.32–8.30 (m, 1H), 7.72 (d, <italic>J</italic>
= 7.20 Hz, 2H), 7.48–7.40 (m, 3H), 7.37–7.24 (m, 5H), 5.65–5.61 (m, 1H), 5.15–5.03 (m, 2H), 4.30–4.20 (m, 1H), 3.40–3.23 (m, 2H, merged with CD<sub>3</sub>
OD), 2.75–2.65 (m, 1H), 2.64–2.06 (m, 2H), 2.05–1.80 (m, 2H), 1.79–1.60 (m, 1H), 1.59–1.52 (m, 2H), 0.99–0.89 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
192.0, 181.8, 175.8, 164.2, 158.4, 148.9, 141.9, 131.5, 130.9, 130.7, 129.4, 128.9, 128.7, 128.3, 67.7, 61.5, 55.7, 54.8, 42.4, 41.5, 40.1, 38.9, 34.2, 29.3, 28.6, 25.9, 24.2, 23.4, 22.1, 21.8. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>30</sub>
H<sub>35</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
563.2328 found 563.2308.</p>
</sec>
<sec id="sec4.7.2"><label>4.7.2</label>
<title>Benzyl ((<italic>S</italic>
)-4-methyl-1-oxo-1-(((<italic>S</italic>
)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)-1-(5-(<italic>p</italic>
-tolyl)thiazol-2-yl)propan-2-yl)amino)pentan-2-yl)carbamate (<bold>27b</bold>
)</title>
<p id="p0395">Yield 51% from <bold>24c</bold>
; colorless solid; <sup>1</sup>
H NMR (400 MHz, CD<sub>3</sub>
OD): <italic>δ</italic>
8.32–8.29 (m, 1H), 7.62 (d, <italic>J</italic>
= 8.4 Hz, 2H), 7.40–7.20 (m, 7H), 5.70–5.57 (m, 1H), 5.15–5.01 (m, 2H), 4.30–4.18 (m, 1H), 3.40–3.25 (m, 2H, merged with CD<sub>3</sub>
OD), 2.75–2.40 (m, 1H), 2.50–2.30 (m, 2H), 2.38 (s, 3H), 2.29–2.09 (m, 1H), 2.08–1.75 (m, 2H), 1.74–1.65 (m, 1H), 1.63–1.49 (m, 2H), 0.97–0.90 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>31</sub>
H<sub>37</sub>
N<sub>4</sub>
O<sub>5</sub>
S [M + H]<sup>+</sup>
577.2485 found 577.2484.</p>
</sec>
<sec id="sec4.7.3"><label>4.7.3</label>
<title>Benzyl ((<italic>S</italic>
)-1-(((<italic>S</italic>
)-1-(5-(4-methoxyphenyl)thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (<bold>27c</bold>
)</title>
<p id="p0400">Yield 47% from <bold>24d</bold>
; light yellow solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.05–8.03 (m, 1H), 7.60–7.52 (m, 2H), 7.60–7.27 (m, 5H), 7.00–6.95 (m, 2H), 5.64–5.56 (m, 1H), 5.20–5.08 (m, 1H), 4.40–4.25 (m, 1H), 3.86 (s, 3H), 3.35–3.34 (m, 2H), 2.67–2.42 (m, 2H), 2.22–2.04 (m, 2H), 2.02–1.96 (m, 1H), 1.80–1.52 (m, 3H), 0.97–0.86 (m, 6H). HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>31</sub>
H<sub>37</sub>
N<sub>4</sub>
O<sub>6</sub>
S [M + H]<sup>+</sup>
593.2434 found 593.2414.</p>
</sec>
<sec id="sec4.7.4"><label>4.7.4</label>
<title>Benzyl ((<italic>S</italic>
)-1-(((<italic>S</italic>
)-1-(5-(2-methoxyphenyl)thiazol-2-yl)-1-oxo-3-((<italic>S</italic>
)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (<bold>27d</bold>
)</title>
<p id="p0405">Yield 51% from <bold>24e</bold>
; light yellow solid; <sup>1</sup>
H NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
8.12–8.10 (m, 1H), 7.37–7.27 (m, 6H), 7.19 (d, <italic>J</italic>
= 7.6 Hz, 1H), 7.13–7.11 (m, 1H), 6.95 (dd, 1.8, 8.2 Hz, 1H), 5.70–5.53 (m, 1H), 5.15–5.08 (m, 2H), 4.40–4.30 (m, 1H), 3.85 (s, 3H), 3.50–3.25 (m, 2H), 2.70–2.37 (m, 2H), 2.35–2.05 (m, 2H), 2.04–1.80 (m, 1H), 1.79–1.60 (m, 2H), 1.59–1.45 (m, 1H), 0.99–0.80 (m, 6H). <sup>13</sup>
C NMR (400 MHz, CDCl<sub>3</sub>
): <italic>δ</italic>
190.8, 180.3, 172.8, 162.9, 160.1, 156.1, 147.4, 140.6, 136.4, 131.5, 130.4, 128.4, 127.9, 119.7, 115.1, 113.1, 66.8, 55.4, 53.4, 42.2, 41.7, 40.7, 38.9, 33.5, 31.9, 28.2, 24.7, 24.6, 23.0, 22.9, 22.0. HRMS (ESI): <italic>m</italic>
/<italic>z</italic>
calcd for C<sub>31</sub>
H<sub>37</sub>
N<sub>4</sub>
O<sub>6</sub>
S [M + H]<sup>+</sup>
593.2434 found 593.2427.</p>
</sec>
</sec>
<sec id="sec4.8"><label>4.8</label>
<title>Molecular docking studies</title>
<p id="p0410">The crystal structure of the SARS-CoV 3CL protease in complex with a substrate analog inhibitor (coded <ext-link ext-link-type="uri" xlink:href="pdb:1WOF" id="intref0015">1WOF</ext-link>
) <xref rid="bib35" ref-type="bibr">[35]</xref>
was obtained from the Protein Data Bank (PDB; <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/home/home.do" id="intref0020">http://www.rcsb.org/pdb/home/home.do</ext-link>
). Initially, a binding model of <bold>26m</bold>
with 3CL<sup>pro</sup>
was simulated to form the basis of a comparison with our previous potent lead tripeptidomimetic <bold>9</bold>
and a substrate analog, using molecular operating environment (MOE) software. Several minimization processes were performed using the MMFF94X force field to model the solvation environment surrounding the inhibitor. Structures having a relatively low binding free energy and a high number of cluster members were selected for the subsequent docking conformation optimization step. The minimized energies of <bold>26m</bold>
, obtained from the docking study, were −40.67 and −35.52 kcal/mol.</p>
</sec>
</sec>
</body>
<back><ref-list id="cebib0010"><title>References</title>
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<sec id="appsec1" sec-type="supplementary-material"><label>Appendix A</label>
<title>Supplementary data</title>
<p id="p0420"><supplementary-material content-type="local-data" id="ec1"><media xlink:href="mmc1.pdf"></media>
</supplementary-material>
</p>
</sec>
<ack id="ack0010"><title>Acknowledgments</title>
<p>This research was supported by Grants from the <funding-source id="gs1">Ministry of Education, Culture, Sports, Science and Technology</funding-source>
(MEXT) of Japan, including a Grant-in-aid for Young scientist (<funding-source id="gs2">Tokubetsu Kenkyuin Shorei-hi</funding-source>
) 23·01104 and a Grant-in-aid for <funding-source id="gs3">Scientific Research</funding-source>
23659059 and 23390029. E.F. acknowledges support from the <funding-source id="gs4">National Institutes of Health</funding-source>
(grant GM57144).</p>
</ack>
<fn-group><fn id="appsec2" fn-type="supplementary-material"><label>Appendix A</label>
<p id="p0425">Supplementary data related to this article can be found at <ext-link ext-link-type="doi" xlink:href="10.1016/j.ejmech.2013.05.005" id="intref0025">http://dx.doi.org/10.1016/j.ejmech.2013.05.005</ext-link>
.</p>
</fn>
</fn-group>
</back>
</pmc>
</record>
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