Design, Synthesis, and Anti-RNA Virus Activity of 6′-Fluorinated-Aristeromycin Analogues
Identifieur interne : 000132 ( Pmc/Corpus ); précédent : 000131; suivant : 000133Design, Synthesis, and Anti-RNA Virus Activity of 6′-Fluorinated-Aristeromycin Analogues
Auteurs : Ji-Seong Yoon ; Gyudong Kim ; Dnyandev B. Jarhad ; Hong-Rae Kim ; Young-Sup Shin ; Shuhao Qu ; Pramod K. Sahu ; Hea Ok Kim ; Hyuk Woo Lee ; Su Bin Wang ; Yun Jeong Kong ; Tong-Shin Chang ; Natacha S. Ogando ; Kristina Kovacikova ; Eric J. Snijder ; Clara C. Posthuma ; Martijn J. Van Hemert ; Lak Shin JeongSource :
- Journal of Medicinal Chemistry [ 0022-2623 ] ; 2019.
Abstract
The
6′-fluorinated aristeromycins were designed as dual-target
antiviral compounds aimed at inhibiting both the viral RNA-dependent
RNA polymerase (RdRp) and the host cell
Url:
DOI: 10.1021/acs.jmedchem.9b00781
PubMed: 31244113
PubMed Central: 7075649
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Design, Synthesis,
and Anti-RNA Virus Activity of
6′-Fluorinated-Aristeromycin Analogues</title><author><name sortKey="Yoon, Ji Seong" sort="Yoon, Ji Seong" uniqKey="Yoon J" first="Ji-Seong" last="Yoon">Ji-Seong Yoon</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kim, Gyudong" sort="Kim, Gyudong" uniqKey="Kim G" first="Gyudong" last="Kim">Gyudong Kim</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">College of Pharmacy and Research Institute of Drug Development,<institution>Chonnam National University</institution>, Gwangju 500-757,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Jarhad, Dnyandev B" sort="Jarhad, Dnyandev B" uniqKey="Jarhad D" first="Dnyandev B." last="Jarhad">Dnyandev B. Jarhad</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kim, Hong Rae" sort="Kim, Hong Rae" uniqKey="Kim H" first="Hong-Rae" last="Kim">Hong-Rae Kim</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Shin, Young Sup" sort="Shin, Young Sup" uniqKey="Shin Y" first="Young-Sup" last="Shin">Young-Sup Shin</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Qu, Shuhao" sort="Qu, Shuhao" uniqKey="Qu S" first="Shuhao" last="Qu">Shuhao Qu</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff6">College of Pharmaceutical Engineering,<institution>Henan University of Animal Husbandry and Economy</institution>, Zhengzhou, 450046,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Sahu, Pramod K" sort="Sahu, Pramod K" uniqKey="Sahu P" first="Pramod K." last="Sahu">Pramod K. Sahu</name><affiliation><nlm:aff id="aff3"><institution>Future Medicine Co., Ltd.</institution>, Seoul 06665,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kim, Hea Ok" sort="Kim, Hea Ok" uniqKey="Kim H" first="Hea Ok" last="Kim">Hea Ok Kim</name><affiliation><nlm:aff id="aff3"><institution>Future Medicine Co., Ltd.</institution>, Seoul 06665,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Lee, Hyuk Woo" sort="Lee, Hyuk Woo" uniqKey="Lee H" first="Hyuk Woo" last="Lee">Hyuk Woo Lee</name><affiliation><nlm:aff id="aff3"><institution>Future Medicine Co., Ltd.</institution>, Seoul 06665,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Wang, Su Bin" sort="Wang, Su Bin" uniqKey="Wang S" first="Su Bin" last="Wang">Su Bin Wang</name><affiliation><nlm:aff id="aff4">College of Pharmacy,<institution>Ewha Womans University</institution>, Seoul 120-750,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kong, Yun Jeong" sort="Kong, Yun Jeong" uniqKey="Kong Y" first="Yun Jeong" last="Kong">Yun Jeong Kong</name><affiliation><nlm:aff id="aff4">College of Pharmacy,<institution>Ewha Womans University</institution>, Seoul 120-750,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Chang, Tong Shin" sort="Chang, Tong Shin" uniqKey="Chang T" first="Tong-Shin" last="Chang">Tong-Shin Chang</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff4">College of Pharmacy,<institution>Ewha Womans University</institution>, Seoul 120-750,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Ogando, Natacha S" sort="Ogando, Natacha S" uniqKey="Ogando N" first="Natacha S." last="Ogando">Natacha S. Ogando</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Kovacikova, Kristina" sort="Kovacikova, Kristina" uniqKey="Kovacikova K" first="Kristina" last="Kovacikova">Kristina Kovacikova</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J." last="Snijder">Eric J. Snijder</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Posthuma, Clara C" sort="Posthuma, Clara C" uniqKey="Posthuma C" first="Clara C." last="Posthuma">Clara C. Posthuma</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Van Hemert, Martijn J" sort="Van Hemert, Martijn J" uniqKey="Van Hemert M" first="Martijn J." last="Van Hemert">Martijn J. Van Hemert</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Jeong, Lak Shin" sort="Jeong, Lak Shin" uniqKey="Jeong L" first="Lak Shin" last="Jeong">Lak Shin Jeong</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31244113</idno><idno type="pmc">7075649</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075649</idno><idno type="RBID">PMC:7075649</idno><idno type="doi">10.1021/acs.jmedchem.9b00781</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000132</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000132</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Design, Synthesis,
and Anti-RNA Virus Activity of
6′-Fluorinated-Aristeromycin Analogues</title><author><name sortKey="Yoon, Ji Seong" sort="Yoon, Ji Seong" uniqKey="Yoon J" first="Ji-Seong" last="Yoon">Ji-Seong Yoon</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kim, Gyudong" sort="Kim, Gyudong" uniqKey="Kim G" first="Gyudong" last="Kim">Gyudong Kim</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">College of Pharmacy and Research Institute of Drug Development,<institution>Chonnam National University</institution>, Gwangju 500-757,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Jarhad, Dnyandev B" sort="Jarhad, Dnyandev B" uniqKey="Jarhad D" first="Dnyandev B." last="Jarhad">Dnyandev B. Jarhad</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kim, Hong Rae" sort="Kim, Hong Rae" uniqKey="Kim H" first="Hong-Rae" last="Kim">Hong-Rae Kim</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Shin, Young Sup" sort="Shin, Young Sup" uniqKey="Shin Y" first="Young-Sup" last="Shin">Young-Sup Shin</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Qu, Shuhao" sort="Qu, Shuhao" uniqKey="Qu S" first="Shuhao" last="Qu">Shuhao Qu</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff6">College of Pharmaceutical Engineering,<institution>Henan University of Animal Husbandry and Economy</institution>, Zhengzhou, 450046,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Sahu, Pramod K" sort="Sahu, Pramod K" uniqKey="Sahu P" first="Pramod K." last="Sahu">Pramod K. Sahu</name><affiliation><nlm:aff id="aff3"><institution>Future Medicine Co., Ltd.</institution>, Seoul 06665,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kim, Hea Ok" sort="Kim, Hea Ok" uniqKey="Kim H" first="Hea Ok" last="Kim">Hea Ok Kim</name><affiliation><nlm:aff id="aff3"><institution>Future Medicine Co., Ltd.</institution>, Seoul 06665,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Lee, Hyuk Woo" sort="Lee, Hyuk Woo" uniqKey="Lee H" first="Hyuk Woo" last="Lee">Hyuk Woo Lee</name><affiliation><nlm:aff id="aff3"><institution>Future Medicine Co., Ltd.</institution>, Seoul 06665,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Wang, Su Bin" sort="Wang, Su Bin" uniqKey="Wang S" first="Su Bin" last="Wang">Su Bin Wang</name><affiliation><nlm:aff id="aff4">College of Pharmacy,<institution>Ewha Womans University</institution>, Seoul 120-750,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kong, Yun Jeong" sort="Kong, Yun Jeong" uniqKey="Kong Y" first="Yun Jeong" last="Kong">Yun Jeong Kong</name><affiliation><nlm:aff id="aff4">College of Pharmacy,<institution>Ewha Womans University</institution>, Seoul 120-750,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Chang, Tong Shin" sort="Chang, Tong Shin" uniqKey="Chang T" first="Tong-Shin" last="Chang">Tong-Shin Chang</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff4">College of Pharmacy,<institution>Ewha Womans University</institution>, Seoul 120-750,<country>Korea</country></nlm:aff></affiliation></author><author><name sortKey="Ogando, Natacha S" sort="Ogando, Natacha S" uniqKey="Ogando N" first="Natacha S." last="Ogando">Natacha S. Ogando</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Kovacikova, Kristina" sort="Kovacikova, Kristina" uniqKey="Kovacikova K" first="Kristina" last="Kovacikova">Kristina Kovacikova</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J." last="Snijder">Eric J. Snijder</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Posthuma, Clara C" sort="Posthuma, Clara C" uniqKey="Posthuma C" first="Clara C." last="Posthuma">Clara C. Posthuma</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Van Hemert, Martijn J" sort="Van Hemert, Martijn J" uniqKey="Van Hemert M" first="Martijn J." last="Van Hemert">Martijn J. Van Hemert</name><affiliation><nlm:aff id="aff5">Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></nlm:aff></affiliation></author><author><name sortKey="Jeong, Lak Shin" sort="Jeong, Lak Shin" uniqKey="Jeong L" first="Lak Shin" last="Jeong">Lak Shin Jeong</name><affiliation><nlm:aff id="aff1">Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Medicinal Chemistry</title><idno type="ISSN">0022-2623</idno><idno type="eISSN">1520-4804</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="jm9b00781_0010" id="ab-tgr1"></graphic></p><p>The
6′-fluorinated aristeromycins were designed as dual-target
antiviral compounds aimed at inhibiting both the viral RNA-dependent
RNA polymerase (RdRp) and the host cell <italic>S</italic>-adenosyl-<sc>l</sc>-homocysteine (SAH) hydrolase, which would indirectly target
capping of viral RNA. The introduction of a fluorine at the 6′-position
enhanced the inhibition of SAH hydrolase and the activity against
RNA viruses. The adenosine and <italic>N</italic><sup>6</sup>-methyladenosine
analogues <bold>2a–e</bold> showed potent inhibition against
SAH hydrolase, while only the adenosine derivatives <bold>2a–c</bold> exhibited potent antiviral activity against all tested RNA viruses
such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe
acute respiratory syndrome-coronavirus, chikungunya virus, and/or
Zika virus. 6′,6′-Difluoroaristeromycin (<bold>2c</bold>) showed the strongest antiviral effect for MERS-CoV, with a ∼2.5
log reduction in infectious progeny titer in viral load reduction
assay. The phosphoramidate prodrug <bold>3a</bold> also demonstrated
potent broad-spectrum antiviral activity, possibly by inhibiting the
viral RdRp. This study shows that 6′-fluorinated aristeromycins
can serve as starting points for the development of broad-spectrum
antiviral agents that target RNA viruses.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Baltimore, D" uniqKey="Baltimore D">D. Baltimore</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thiel, V" uniqKey="Thiel V">V. Thiel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zumla, A" uniqKey="Zumla A">A. Zumla</name></author><author><name sortKey="Hui, D S" uniqKey="Hui D">D. S. Hui</name></author><author><name sortKey="Perlman, S" uniqKey="Perlman S">S. Perlman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Caglioti, C" uniqKey="Caglioti C">C. Caglioti</name></author><author><name sortKey="Lalle, E" uniqKey="Lalle E">E. Lalle</name></author><author><name sortKey="Castilletti, C" uniqKey="Castilletti C">C. Castilletti</name></author><author><name sortKey="Carletti, F" uniqKey="Carletti F">F. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Med Chem</journal-id><journal-id journal-id-type="iso-abbrev">J. Med. Chem</journal-id><journal-id journal-id-type="publisher-id">jm</journal-id><journal-id journal-id-type="coden">jmcmar</journal-id><journal-title-group><journal-title>Journal of Medicinal Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0022-2623</issn><issn pub-type="epub">1520-4804</issn><publisher><publisher-name>American Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31244113</article-id><article-id pub-id-type="pmc">7075649</article-id><article-id pub-id-type="doi">10.1021/acs.jmedchem.9b00781</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>Design, Synthesis,
and Anti-RNA Virus Activity of
6′-Fluorinated-Aristeromycin Analogues</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Yoon</surname><given-names>Ji-seong</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Kim</surname><given-names>Gyudong</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Jarhad</surname><given-names>Dnyandev B.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath4"><name><surname>Kim</surname><given-names>Hong-Rae</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath5"><name><surname>Shin</surname><given-names>Young-Sup</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath6"><name><surname>Qu</surname><given-names>Shuhao</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff6" ref-type="aff">#</xref></contrib><contrib contrib-type="author" id="ath7"><name><surname>Sahu</surname><given-names>Pramod K.</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath8"><name><surname>Kim</surname><given-names>Hea Ok</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath9"><name><surname>Lee</surname><given-names>Hyuk Woo</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath10"><name><surname>Wang</surname><given-names>Su Bin</given-names></name><xref rid="aff4" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath11"><name><surname>Kong</surname><given-names>Yun Jeong</given-names></name><xref rid="aff4" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath12"><name><surname>Chang</surname><given-names>Tong-Shin</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff4" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath13"><name><surname>Ogando</surname><given-names>Natacha S.</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath14"><name><surname>Kovacikova</surname><given-names>Kristina</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath15"><name><surname>Snijder</surname><given-names>Eric J.</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath16"><name><surname>Posthuma</surname><given-names>Clara C.</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath17"><name><surname>van Hemert</surname><given-names>Martijn J.</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath18"><name><surname>Jeong</surname><given-names>Lak Shin</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref></contrib><aff id="aff1"><label>†</label>Research Institute of Pharmaceutical Sciences, College of Pharmacy,<institution>Seoul National University</institution>, Seoul 151-742,<country>Korea</country></aff><aff id="aff2"><label>‡</label>College of Pharmacy and Research Institute of Drug Development,<institution>Chonnam National University</institution>, Gwangju 500-757,<country>Korea</country></aff><aff id="aff3"><label>§</label><institution>Future Medicine Co., Ltd.</institution>, Seoul 06665,<country>Korea</country></aff><aff id="aff4"><label>∥</label>College of Pharmacy,<institution>Ewha Womans University</institution>, Seoul 120-750,<country>Korea</country></aff><aff id="aff5"><label>⊥</label>Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, Albinusdreef 2, 2333ZA Leiden,<country>The Netherlands</country></aff><aff id="aff6"><label>#</label>College of Pharmaceutical Engineering,<institution>Henan University of Animal Husbandry and Economy</institution>, Zhengzhou, 450046,<country>China</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>E-mail: <email>lakjeong@snu.ac.kr</email>. Phone: <phone>82-2-880-7850</phone>.</corresp></author-notes><pub-date pub-type="epub"><day>07</day><month>06</month><year>2019</year></pub-date><pub-date pub-type="ppub"><day>11</day><month>07</month><year>2019</year></pub-date><volume>62</volume><issue>13</issue><fpage>6346</fpage><lpage>6362</lpage><history><date date-type="received"><day>13</day><month>05</month><year>2019</year></date></history><permissions><copyright-statement>Copyright © 2019 American Chemical Society</copyright-statement><copyright-year>2019</copyright-year><copyright-holder>American Chemical Society</copyright-holder><license license-type="open-access"><license-p>This article is made available via the PMC Open Access Subset 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 the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="jm9b00781_0010" id="ab-tgr1"></graphic></p><p>The
6′-fluorinated aristeromycins were designed as dual-target
antiviral compounds aimed at inhibiting both the viral RNA-dependent
RNA polymerase (RdRp) and the host cell <italic>S</italic>-adenosyl-<sc>l</sc>-homocysteine (SAH) hydrolase, which would indirectly target
capping of viral RNA. The introduction of a fluorine at the 6′-position
enhanced the inhibition of SAH hydrolase and the activity against
RNA viruses. The adenosine and <italic>N</italic><sup>6</sup>-methyladenosine
analogues <bold>2a–e</bold> showed potent inhibition against
SAH hydrolase, while only the adenosine derivatives <bold>2a–c</bold> exhibited potent antiviral activity against all tested RNA viruses
such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe
acute respiratory syndrome-coronavirus, chikungunya virus, and/or
Zika virus. 6′,6′-Difluoroaristeromycin (<bold>2c</bold>) showed the strongest antiviral effect for MERS-CoV, with a ∼2.5
log reduction in infectious progeny titer in viral load reduction
assay. The phosphoramidate prodrug <bold>3a</bold> also demonstrated
potent broad-spectrum antiviral activity, possibly by inhibiting the
viral RdRp. This study shows that 6′-fluorinated aristeromycins
can serve as starting points for the development of broad-spectrum
antiviral agents that target RNA viruses.</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>jm9b00781</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>jm9b00781</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta><notes id="notes-d1e21-autogenerated"><fn-group><fn fn-type="" id="d30e308"><p>This article is made available for a limited time sponsored by ACS under the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/freetoread/index.html">ACS Free to Read License</ext-link>, which permits copying and redistribution of the article for non-commercial scholarly purposes.</p></fn></fn-group></notes></front><body><sec id="sec1"><title>Introduction</title><p>Over
the past 15 years, outbreaks of a number of emerging positive-stranded
RNA (+RNA) viruses,<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> such as the severe
acute respiratory syndrome coronavirus (SARS-CoV),<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> Middle East respiratory syndrome coronavirus (MERS-CoV),<sup><xref ref-type="bibr" rid="ref3">3</xref></sup> chikungunya virus (CHIKV),<sup><xref ref-type="bibr" rid="ref4">4</xref></sup> and Zika virus (ZIKV)<sup><xref ref-type="bibr" rid="ref5">5</xref></sup> have
seriously threatened human health and have had a substantial socio-economic
impact. SARS-CoV and MERS-CoV cause serious respiratory diseases<sup><xref ref-type="bibr" rid="ref6">6</xref></sup> that can be fatal in approximately 10 and 35%
of cases, respectively. CHIKV is transmitted by mosquitoes and causes
a painful arthritis that can persist for months.<sup><xref ref-type="bibr" rid="ref7">7</xref></sup> ZIKV is also transmitted by mosquitoes,<sup><xref ref-type="bibr" rid="ref8">8</xref></sup> although sexual transmission<sup><xref ref-type="bibr" rid="ref8">8</xref></sup> occurs as well. This virus usually causes mild disease, but can
cause neurological complications in adults and fetal death or severe
complications, including microcephaly in infants when women are infected
during pregnancy.<sup><xref ref-type="bibr" rid="ref9">9</xref></sup> CHIKV and ZIKV have
caused massive outbreaks, totaling millions of infections over the
past decade. Currently, there are no effective chemotherapeutic agents
or vaccines that can prevent or cure infections of any of these four
serious pathogens.</p><p>The aforementioned viruses belong to the
+RNA virus group (Baltimore
class IV),<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> which indicates that their
genomic RNA has the same polarity as mRNA and can be directly translated
by host ribosomes upon release into the cytoplasm of a host cell.
After infection, the genomes of these viruses are translated into
polyproteins that are subsequently cleaved into individual proteins
by viral and/or host proteases. The nonstructural proteins (nsps)
of these viruses harbor a variety of enzymatic activities that are
required for the replication of the viral RNA and invariably include
a RNA-dependent RNA polymerase (RdRp),<sup><xref ref-type="bibr" rid="ref10">10</xref></sup> an enzyme which is not present in uninfected cells. The RdRp transcribes
the genomic RNA into a complementary negative-stranded RNA that subsequently
serves as the template for the synthesis of new positive-stranded
RNA.</p><p>Many +RNA viruses (including coronaviruses, CHIKV, and
ZIKV) also
encode methyltransferases (MTases)<sup><xref ref-type="bibr" rid="ref11">11</xref></sup> that
are required for methylations of viral mRNA cap structures.<sup><xref ref-type="bibr" rid="ref12">12</xref></sup> Because this capping is crucial for stability
and translation of the viral RNA, and evasion of the host innate immune
response, the viral MTases are considered promising targets for the
development of antiviral therapy.<sup><xref ref-type="bibr" rid="ref12">12</xref></sup> Inhibition
of MTases can be indirectly achieved by the inhibition of <italic>S</italic>-adenosyl-<sc>l</sc>-homocysteine (SAH) hydrolase.<sup><xref ref-type="bibr" rid="ref13">13</xref></sup> The SAH hydrolase catalyzes the interconversion
of SAH into adenosine and <sc>l</sc>-homocysteine. Inhibition of this
enzyme leads to the accumulation of SAH in the cell, which in turn
inhibits <italic>S</italic>-adenosyl-<sc>l</sc>-methionine (SAM)-dependent
transmethylase reactions by feedback inhibition.<sup><xref ref-type="bibr" rid="ref13">13</xref>,<xref ref-type="bibr" rid="ref14">14</xref></sup> Most of the viral MTases are dependent on SAM as the only methyl
donor. Compounds that target cellular proteins might exhibit a broader
spectrum of activity, are less likely to lead to drug-resistance,
but have a higher likelihood of toxicity. Compounds that are specifically
aimed at viral proteins are expected to be less cytotoxic, but might
have a narrower spectrum of antiviral activity and might have a lower
barrier antiviral drug-resistance<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Thus,
the approach of targeting cellular proteins such as SAH hydrolase
can be considered as a promising strategy for the development of broad-spectrum
antiviral agents.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> A number of compounds
have been reported to act as SAH hydrolase inhibitors.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Type I inhibitors act through inactivation of
the NAD<sup>+</sup> cofactor, and their inhibitory effect on the catalytic
activity of the enzyme can be reversed by the addition of excess NAD<sup>+</sup>.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Type II inhibitors are irreversible
inhibitors of the SAH hydrolase that form covalent bonds with amino
acid residues in the active site of the enzyme. This irreversible
inhibition cannot be reversed by the addition of NAD<sup>+</sup> or
adenosine or by dialysis.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup></p><p>Because
both the viral RdRp and host SAH hydrolase are critical
for virus replication, we aimed to design broad-spectrum nucleoside
analogue inhibitors that could directly target RdRp activity and/or
indirectly inhibit the methylation of viral RNA through their effect
on the host SAH hydrolase. Modified nucleosides are usually taken
up by the cell via nucleoside transporters and can be successively
converted into mono-, di-, and triphosphates by cellular kinases.<sup><xref ref-type="bibr" rid="ref15">15</xref></sup> Then, these modified nucleoside triphosphates
(NTPs) can compete with natural NTPs during RNA synthesis or can be
incorporated into the nascent viral RNA, leading to chain termination
or detrimental mutations.<sup><xref ref-type="bibr" rid="ref15">15</xref></sup></p><p>(−)-Aristeromycin
(<bold>1</bold>) is a naturally occurring
carbocyclic nucleoside that was originally identified as a metabolite
of <italic>Streptomyces citricolor</italic> in 1967.<sup><xref ref-type="bibr" rid="cit16a">16a</xref></sup> The first synthesis of <bold>1</bold> as racemate
was reported by Clayton and his co-worker,<sup><xref ref-type="bibr" rid="cit16b">16b</xref>−<xref ref-type="bibr" rid="cit16d">16d</xref></sup> and its asymmetric syntheses have since been reported.<sup><xref ref-type="bibr" rid="cit16e">16e</xref>−<xref ref-type="bibr" rid="cit16h">16h</xref></sup> It is a type I SAH hydrolase inhibitor and exhibits potent antiviral
activity against many viruses.<sup><xref ref-type="bibr" rid="cit14a">14a</xref></sup> However,
it could not be further advanced into clinical development because
of its cytotoxicity.<sup><xref ref-type="bibr" rid="ref17">17</xref></sup> Compound <bold>1</bold> was found to be toxic at low concentrations in both adenosine kinase-positive
(AK<sup>+</sup>) and AK<sup>–</sup> cells. AK<sup>+</sup> cells
were presumably killed by the 5′-phosphorylated form of <bold>1</bold>, while the toxicity in AK<sup>–</sup> cells was caused
by <bold>1</bold> itself.<sup><xref ref-type="bibr" rid="ref17">17</xref></sup> However, this
compound is also metabolized into a triphosphate form and has been
observed to exert a variety of metabolic effects.<sup><xref ref-type="bibr" rid="ref17">17</xref></sup> We aimed to use <bold>1</bold> as a prototype for the design
of dual-target compounds intended at directly inhibiting the viral
RdRp and indirectly inhibiting the capping process through targeting
of cellular SAH hydrolase.</p><p>Since the introduction of a fluorine
at the 6′-position
of carbocyclic nucleosides has been known to affect biological activities
to a significant extent,<sup><xref ref-type="bibr" rid="ref18">18</xref></sup> we aimed to
synthesize the 6′-fluorinated-aristeromycin analogues <bold>2</bold> by introducing fluorine at the 6′-position of <bold>1</bold> (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>). Prisbe and his co-workers<sup><xref ref-type="bibr" rid="cit18a">18a</xref></sup> have reported
the synthesis of (±)-6′-α- and (±)-6′-β-fluorinated
aristeromycins and their inhibitory activity on SAH hydrolase, but
the synthesis and biological activity of (±)-6′,6′-difluoroaristeromycin
was not reported, despite the fact that the structure was claimed
in the patent.<sup><xref ref-type="bibr" rid="cit18b">18b</xref></sup> Thus, we set out to synthesize
the 6′-fluorinated-aristeromycin analogues <bold>2</bold> in
the optically pure <sc>d</sc>-forms because biological activity can
generally be attributed to one enantiomer, the <sc>d</sc>-isomer.
Yin and co-workers<sup><xref ref-type="bibr" rid="cit18c">18c</xref></sup> reported the elegant
synthesis of optically pure (−)-6′-β-fluoro-aristeromycin,
but its biological activity was not reported. Their synthetic route
involved the 6-β-fluoroazide as the key intermediate, which
was synthesized by employing S<sub>N</sub>2 fluorination of the 6-α-triflic
azide with tris(dimethylamino)sulfur(trimethylsilyl)difluoride, whereas
our current approach<sup><xref ref-type="bibr" rid="ref19">19</xref></sup> included the stereoselective
electrophilic fluorination of silyl enol ether with Selectfluor as
the fluorine source. In addition to the adenosine analogues, aimed
at inhibiting SAH hydrolase and/or RdRp, we have also synthesized
6′-fluorinated purine and pyrimidine nucleosides (changes in
B of the structures in <xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>), which could interfere with viral RNA synthesis by targeting
the viral RdRp after their phosphorylation by cellular kinases.<sup><xref ref-type="bibr" rid="ref15">15</xref></sup> To bypass the first and rate-limiting 5′-monophosphorylation
step, we have also synthesized a phosphoramidate prodrug <bold>3</bold> of nucleoside <bold>2</bold>, using the McGuigan ProTides.<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> Herein, we report the synthesis of the 6′-fluoro-aristeromycin
analogues <bold>2</bold> and <bold>3</bold> and a preliminary characterization
of their effect on several +RNA viruses, which provided insight into
structure–activity relationships (SARs).</p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Rationale for the design
of the target nucleosides <bold>2</bold> and <bold>3</bold>.</p></caption><graphic xlink:href="jm9b00781_0001" id="gr1" position="float"></graphic></fig></sec><sec id="sec2"><title>Results and Discussion</title><sec id="sec2.1"><title>Chemistry</title><p>For
the synthesis of the target nucleosides <bold>2</bold>, the key fluorosugars <bold>8a–c</bold> were synthesized
from <sc>d</sc>-ribose via electrophilic fluorination, as shown in <xref rid="sch1" ref-type="scheme">Scheme <xref rid="sch1" ref-type="scheme">1</xref></xref>.</p><fig id="sch1" position="float"><label>Scheme 1</label><caption><title>Synthesis of 6-β-Fluoro-,
6-α-Fluoro-, and 6-Difluorosugar <bold>8a–c</bold></title><p id="s1fn1">Reagents and conditions: (a)
LiCu(CH<sub>2</sub>O<italic>t</italic>-Bu)<sub>2</sub>; (b) TESCl,
LiHMDS, THF, −78 °C, 10 min; (c) Selectfluor, DMF, 0 °C,
12 h; (d) NaBH<sub>4</sub>, MeOH, 0 °C, 30 min. (e) LiBH<sub>4</sub>, MeOH, 0 °C, 30 min.</p></caption><graphic xlink:href="jm9b00781_0004" id="gr4" position="float"></graphic></fig><p><sc>d</sc>-Ribose was converted to <sc>d</sc>-cyclopentenone <bold>4</bold> according to our previously published procedure.<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> The 1,4-conjugated addition of <bold>4</bold> with Gilman
reagent yielded the <sc>d</sc>-cyclopentanone derivative <bold>5</bold>.<sup><xref ref-type="bibr" rid="ref19">19</xref>,<xref ref-type="bibr" rid="ref22">22</xref></sup> Treatment of <bold>5</bold> with
lithium hexamethyldisilazide (LiHMDS) followed by trapping with triethylsilyl
chloride (TESCl) gave silylenol ether <bold>6</bold>, which was treated
with (1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate):
Selectfluor) in dimethylformamide (DMF) at 0 °C to yield a 5:1
ratio of 6-β-fluorosugar <bold>7a</bold> to 6-α-fluorosugar <bold>7b</bold>.<sup><xref ref-type="bibr" rid="ref19">19</xref></sup> The stereochemistry of the
fluorine in <bold>7a</bold> and <bold>7b</bold> was confirmed by <sup>1</sup>H NOE experiments. Irradiation of 6-H of <bold>7b</bold> gave
NOE effects on its 2-H and 5-H, indicating the 6-α-fluoro configuration,
but no NOE effects were observed on the same experiment in the case
of <bold>7a</bold>, confirming the 6-β-fluoro configuration.
The configuration of the fluorine in <bold>7b</bold> was further confirmed
by the X-ray crystal structure obtained after it was converted to
the final uracil derivative <bold>2g</bold> (<xref rid="sch5" ref-type="scheme">Scheme <xref rid="sch5" ref-type="scheme">5</xref></xref>). Further electrophilic fluorination of
6-β-fluorosugar <bold>7a</bold> or 6-α-fluorosugar <bold>7b</bold> under the same conditions yielded the 6,6-difluorosugar <bold>7c</bold>, which was equilibrated to form a geminal diol because
of the presence of electronegative fluorine atoms. Electrophilic fluorinations
with other electrophilic fluorines such as <italic>N</italic>-fluorobenzenesulfonimide
(NFSI) or <italic>N</italic>-fluoro-<italic>O</italic>-benzenedisulfonimide
(NFOBS) were problematic, resulting in low yields with many side spots.
The reduction of <bold>7a–c</bold> with sodium borohydride
(NaBH<sub>4</sub>) or lithium borohydride (LiBH<sub>4</sub>) in MeOH
resulted in the production of the 1-hydroxyl derivatives <bold>8a–c</bold>.</p><p>As the α-fluoro derivative <bold>8b</bold> was obtained
as
the minor isomer, as shown in <xref rid="sch1" ref-type="scheme">Scheme <xref rid="sch1" ref-type="scheme">1</xref></xref>, we wanted to improve the stereoselective synthesis
of <bold>8b</bold>, by using Rubottom<sup><xref ref-type="bibr" rid="ref23">23</xref></sup> oxidation as the key step, as illustrated in <xref rid="sch2" ref-type="scheme">Scheme <xref rid="sch2" ref-type="scheme">2</xref></xref>. Rubottom oxidation of silylenol ether <bold>6</bold> with osmium tetroxide (OsO<sub>4</sub>) and <italic>N</italic>-methylmorpholine-<italic>N</italic>-oxide (NMO) followed by trapping
with <italic>t</italic>-butyldimethylsilyl chloride (TBSCl) produced
6-β-alkoxyketone <bold>9</bold> as a single stereoisomer in
53% yield. The reduction of ketone <bold>9</bold> with NaBH<sub>4</sub> gave alcohol <bold>10</bold>, which was protected with a benzyl
group to give <bold>11</bold>. Removal of the <italic>t</italic>-butyldimethylsilyl
(TBS) group in <bold>11</bold> with tetra-<italic>n</italic>-butylammonium
fluoride (TBAF) yielded the 6-β-alcohol <bold>12</bold>. To
our disappointment, the treatment of <bold>12</bold> with <italic>N</italic>,<italic>N</italic>-diethylaminosulfur trifluoride (DAST)
gave the desired product, 6-α-fluoride <bold>13a</bold>, but
also the undesired product 1-β-fluoride <bold>13b</bold> at
a 1:1 ratio. The formation of <bold>13a</bold> (route I) resulted
from the direct S<sub>N</sub>2 reaction of <bold>12a</bold> with fluoride,
while <bold>12a</bold> was readily converted into the oxonium ion <bold>12b</bold> (route II) via its participation of the neighboring benzyl
group, which was attacked exclusively by the fluoride at the less
sterically hindered 1-position to yield the undesired product <bold>13b</bold> (route III). However, the product via route IV was not
formed because of the steric effect of the <italic>t</italic>-butyloxymethyl
substituent.</p><fig id="sch2" position="float"><label>Scheme 2</label><caption><title>Synthetic Approach to 6-α-Fluorosugar <bold>8b</bold> via Rubottom
Oxidation</title><p id="s2fn1">Reagents and conditions: (a)
(i) OsO<sub>4</sub>, NMO·H<sub>2</sub>O, THF, rt, 1 h, then NaHCO<sub>3</sub>, MeOH, rt, 3 h; (ii) TBSCl, imidazole, DMF, rt, 3 h; (b)
NaBH<sub>4</sub>, MeOH, rt, 1 h; (c) BnBr, NaH, DMF, 0 °C to
rt, 12 h; (d) TBAF, THF, rt, 12 h; (e) DAST, toluene, 0 °C to
rt, 2 h.</p></caption><graphic xlink:href="jm9b00781_0005" id="gr5" position="float"></graphic></fig><p>To avoid the participation of the
neighboring group, we considered
using a cyclic sulfate substrate with electron-withdrawing property
and conformational restraint to be the best choice. Furthermore, cyclic
sulfate has the advantage that it can be utilized as a surrogate for
epoxide during nucleobase condensation, as shown in <xref rid="sch3" ref-type="scheme">Scheme <xref rid="sch3" ref-type="scheme">3</xref></xref>. The regioselective cleavage
of the 2,3-acetonide in <bold>10</bold> with trimethylaluminum (AlMe<sub>3</sub>) followed by treatment of the resulting diol with thionyl
chloride (SOCl<sub>2</sub>) yielded the 6-β-hydroxyl cyclic
sulfite <bold>14</bold> after the removal of the TBS group. The treatment
of <bold>14</bold> with DAST yielded the desired 6-α-fluoro
cyclic sulfite <bold>15</bold> as a single stereoisomer. The cyclic
sulfite <bold>15</bold> was oxidized to form cyclic sulfate <bold>16</bold>, which was subsequently condensed with 6-chloropurine anions;
however, this resulted in decomposition.<sup><xref ref-type="bibr" rid="ref19">19</xref></sup> Thus, we decided to synthesize the 6-α-fluoro derivative <bold>8b</bold> according to <xref rid="sch1" ref-type="scheme">Scheme <xref rid="sch1" ref-type="scheme">1</xref></xref>.</p><fig id="sch3" position="float"><label>Scheme 3</label><caption><title>Synthetic Approach to <bold>2b</bold> via Cyclic Sulfate</title><p id="s3fn1">Reagents and conditions: (a)
AlMe<sub>3</sub>, CH<sub>2</sub>Cl<sub>2</sub>, −78 °C
to rt, 12 h; (b) SOCl<sub>2</sub>, Et<sub>3</sub>N, CH<sub>2</sub>Cl<sub>2</sub>, 0 °C, 10 min; (c) TBAF, AcOH, THF, rt, 12 h;
(d) DAST, CH<sub>2</sub>Cl<sub>2</sub>, 0 °C to rt, 4 h; (e)
RuCl<sub>3</sub>, NaIO<sub>4</sub>, CCl<sub>4</sub>/CH<sub>3</sub>CN/H<sub>2</sub>O (1/1/1.5), rt, 20 min; (f) (i) 6-chloropurine,
18-crown-6, NaH, THF, 65 °C, 15 h; (ii) 20% H<sub>2</sub>SO<sub>4</sub>, rt, 1 h.</p></caption><graphic xlink:href="jm9b00781_0006" id="gr6" position="float"></graphic></fig><p><xref rid="sch4" ref-type="scheme">Scheme <xref rid="sch4" ref-type="scheme">4</xref></xref> depicts
the synthesis of the aristeromycin analogues <bold>2a–e</bold> from the 6-β-fluoro-, 6-α-fluoro-, and 6,6-difluorosugars <bold>8a–c</bold>.<sup><xref ref-type="bibr" rid="ref19">19</xref></sup> Compounds <bold>8a–c</bold> were treated with triflic anhydride (Tf<sub>2</sub>O) followed by treatment with sodium azide to give azido derivatives <bold>18a–c</bold>. The catalytic hydrogenation of <bold>18a–c</bold> yielded the amino derivatives <bold>19a–c</bold>, respectively,
which are starting compounds for the base-building process. The treatment
of <bold>19a–c</bold> with 5-amino-4,6-dichloropyrimidine<sup><xref ref-type="bibr" rid="cit18a">18a</xref>−<xref ref-type="bibr" rid="cit18c">18c</xref>,<xref ref-type="bibr" rid="ref24">24</xref></sup> in the presence of <italic>N</italic>,<italic>N</italic>-diisopropylethylamine (DIPEA) under microwave
radiation conditions yielded <bold>20a–c</bold>, which were
cyclized with diethoxymethyl acetate<sup><xref ref-type="bibr" rid="cit18a">18a</xref>−<xref ref-type="bibr" rid="cit18c">18c</xref>,<xref ref-type="bibr" rid="ref24">24</xref></sup> in the presence of microwave radiation to produce the 6-chloropurine
derivatives <bold>21a–c</bold>. The treatment of <bold>21a–c</bold> with <italic>t</italic>-butanolic ammonia followed by the removal
of protective groups under acidic conditions yielded the 6′-β-fluoro-,
6′-α-fluoro-, and 6′,6′-difluoroaristeromycins <bold>2a–c</bold>, respectively. The structure of compound <bold>2c</bold> was confirmed by a single-crystal X-ray analysis (see the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b00781/suppl_file/jm9b00781_si_001.pdf">Supporting Information</ext-link>).<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> The treatment of <bold>21a</bold> and <bold>21c</bold> with 40%
aqueous methylamine followed by aqueous trifluoroacetic acid (TFA)
resulted in <italic>N</italic><sup>6</sup>-methyl-aristeromycin analogues <bold>2d</bold> and <bold>2e</bold>, respectively.</p><fig id="sch4" position="float"><label>Scheme 4</label><caption><title>Synthesis of β-Fluoro-,
α-Fluoro-, and Difluoro-Aristeromycin
Analogues <bold>2a–e</bold></title><p id="s4fn1">Reagents
and conditions: (a)
(i) Tf<sub>2</sub>O, pyridine, 0 °C, 30 min; (ii) NaN<sub>3</sub>, DMF, 60–100 °C, 4–15 h; (b) Pd/C, H<sub>2</sub>, MeOH, rt, 18 h; (c) 5-amino-4,6-dichloropyrimidine, DIPEA, <italic>n</italic>-BuOH, 170–200 °C, 4–7 h, MW; (d) CH<sub>3</sub>C(O)OCH(OEt)<sub>2</sub>, 140 °C, 3 h, MW; (e) NH<sub>3</sub>/<italic>t</italic>-BuOH, 120 °C, 15 h; (f) NH<sub>2</sub>Me/H<sub>2</sub>O, (40 wt %), EtOH, 30 °C, 2 h; (g) 67% aq TFA,
50 °C, 15 h.</p></caption><graphic xlink:href="jm9b00781_0007" id="gr7" position="float"></graphic></fig><p>The amino derivatives <bold>19a–c</bold> were also converted
into the pyrimidine nucleoside derivatives <bold>2f–j</bold>, as shown in <xref rid="sch5" ref-type="scheme">Scheme <xref rid="sch5" ref-type="scheme">5</xref></xref>. Treatment of <bold>19a–c</bold> with
(<italic>E</italic>)-3-methoxy-2-propenoyl isocyanate, which was prepared
by reacting 3-methoxyacryloyl chloride with silver cyanate,<sup><xref ref-type="bibr" rid="ref26">26</xref></sup> in benzene produced <bold>22a–c</bold>, respectively, which were cyclized with 2 M H<sub>2</sub>SO<sub>4</sub> to yield the uridine derivatives <bold>2f–h</bold>, respectively. The structures of <bold>2g</bold> and <bold>2h</bold> were confirmed by the X-ray crystallography (see the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b00781/suppl_file/jm9b00781_si_001.pdf">Supporting Information</ext-link>) (<xref rid="sch5" ref-type="scheme">Scheme <xref rid="sch5" ref-type="scheme">5</xref></xref>).<sup><xref ref-type="bibr" rid="ref27">27</xref></sup> To synthesize
the cytidine derivatives <bold>2i</bold> and <bold>2j</bold>, compounds <bold>2f</bold> and <bold>2h</bold> were benzoylated to give <bold>23a</bold> and <bold>23b</bold>, respectively, which were converted to the
cytidine derivatives <bold>2i</bold> and <bold>2j</bold> using conventional
three-step procedures.<sup><xref ref-type="bibr" rid="ref28">28</xref></sup></p><fig id="sch5" position="float"><label>Scheme 5</label><caption><title>Synthesis of Fluorinated
Pyrimidine Nucleoside Analogues <bold>2f–j</bold></title><p id="s5fn1">Reagents and conditions: (a)
(<italic>E</italic>)-3-methoxy-2-propenoyl isocyanate, benzene, 4
Å-MS, DMF, −20 °C to rt, 15 h; (b) 2 M H<sub>2</sub>SO<sub>4</sub>, dioxane, reflux, 1.5 h; (c) BzCl, pyridine, CH<sub>2</sub>Cl<sub>2</sub>, rt, 15 h; (d) (i) 1,2,4-triazole, POCl<sub>3</sub>, Et<sub>3</sub>N, CH<sub>3</sub>CN, rt, 15 h. (ii) NH<sub>4</sub>OH, dioxane, rt, 15 h. (iii) NH<sub>3</sub>/MeOH, rt, 15 h.</p></caption><graphic xlink:href="jm9b00781_0008" id="gr8" position="float"></graphic></fig><p>The uracil phosphoramidate analogue Sofosbuvir<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> is used in the clinic as a powerful anti-hepatitis
C virus
agent. Therefore, we have also synthesized the uracil phosphoramidate
prodrugs <bold>3b–c</bold> and the adenine phosphoramidate
prodrug <bold>3a</bold> derived from the purine and pyrimidine nucleoside
analogues <bold>2a–j</bold> by using McGuigan’s ProTide
prodrug methodology,<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> as shown in <xref rid="sch6" ref-type="scheme">Scheme <xref rid="sch6" ref-type="scheme">6</xref></xref>. 6′,6′-Difluoro-aristeromycin
(<bold>2c</bold>) was treated with acetone under acidic conditions
to give 2,3-acetonide <bold>24</bold>. The treatment of <bold>24</bold> with di-<italic>tert</italic>-butyl dicarbonate (Boc<sub>2</sub>O) yielded a mixture of <bold>25a</bold> and <bold>25b</bold> in
a 2:1 ratio, which was converted to the phosphoramidate prodrug <bold>26</bold> by treating with phosphoramiditing reagent (<bold>A</bold>)<sup><xref ref-type="bibr" rid="ref29">29</xref></sup> in the presence of <italic>t</italic>-butylmagnesium chloride. The treatment of <bold>26</bold> with 50%
formic acid produced the final product, prodrug <bold>3a</bold>. The
monofluoro- and difluoropyrimidine derivatives <bold>2f</bold> and <bold>2h</bold> were similarly converted to the final prodrugs <bold>3b</bold> and <bold>3c</bold>.</p><fig id="sch6" position="float"><label>Scheme 6</label><caption><title>Synthesis of Phosphoramidate Prodrugs <bold>3a–c</bold></title><p id="s6fn1">Reagents and conditions: (a)
cH<sub>2</sub>SO<sub>4</sub>, acetone, rt, 4 h; (b) (i) TMSOTf, DMAP,
HMDS, 75 °C, 2 h; (ii) Boc<sub>2</sub>O, THF, rt, 4 h; (iii)
MeOH/Et<sub>3</sub>N (5:1), 55 °C, 16 h; (c) <bold>A</bold>, <italic>t</italic>-BuMgCl, 4 Å-MS, THF, 0 °C to rt, 36 h; (d) 50%
HCOOH, rt, 8 h.</p></caption><graphic xlink:href="jm9b00781_0009" id="gr9" position="float"></graphic></fig></sec><sec id="sec2.2"><title>Inhibition of SAH Hydrolase</title><p>All compounds <bold>1</bold>, <bold>2a–j</bold>, and <bold>3a–c</bold> were assayed
for their ability to inhibit recombinant human SAH hydrolase protein,
expressed in <italic>Escherichia coli</italic> JM109,
using a 5,5′-dithiobis-2-nitrobenzoate (DTNB) coupled assay
as described by Lozada-Ramírez et al.<sup><xref ref-type="bibr" rid="ref30">30</xref></sup> As expected, all adenosine derivatives <bold>2a–e</bold> potently
inhibited SAH hydrolase, but none of the pyrimidine analogues <bold>2f–j</bold> showed any inhibitory activity at concentrations
up to 100 μM. None of the prodrugs <bold>3a–c</bold> exhibited
inhibitory activity at concentrations up to 100 μM. This result
is not surprising because adenosine is the substrate for SAH hydrolase.
Among the adenosine analogues, 6′-β-fluoroaristeromycin
(<bold>2a</bold>) exhibited the most potent inhibitory activity (IC<sub>50</sub> = 0.37 μM), which was 3.6-fold more potent than the
control <bold>1</bold> (IC<sub>50</sub> = 1.32 μM). However,
6′-α-fluoroaristeromycin (<bold>2b</bold>, IC<sub>50</sub> = 9.70 μM) was 26-fold less potent than the corresponding
6′-β-fluoro analogue <bold>2a</bold> and 7.4-fold less
active than the 6′-unsubstituted compound <bold>1</bold>. This
indicates that the stereochemistry at the 6′-position is important
for inhibitory activity. Interestingly, the introduction of two fluorines
at the 6′-position resulted in <bold>2c</bold> (IC<sub>50</sub> = 1.06 μM), which was slightly more potent than the control <bold>1</bold>. The inhibitory activity of the 6′-fluoro-aristeromycin
series can be ranked in the following order: 6′-β-F >
6′,6′-F,F > 6′-H > 6′-α-F.
The introduction
of a methyl group at the <italic>N</italic><sup>6</sup>-amino group
of <bold>2a</bold>, resulting in <bold>2d</bold>, decreased the inhibitory
activity (IC<sub>50</sub> = 4.39 μM) by 11.9-fold, while the
addition of a methyl group to the <italic>N</italic><sup>6</sup>-amino
group of <bold>2c</bold>, resulting in <bold>2e</bold>, increased
the inhibitory activity (IC<sub>50</sub> = 0.76 μM) by 1.7-fold.
These results demonstrate that the <italic>N</italic><sup>6</sup>-methyladenine
and the adenine moieties do not lead to a decrease in inhibitory activity.</p></sec><sec id="sec2.3"><title>Antiviral Activity</title><p>The novel 6′-fluoro-aristeromycin
analogues <bold>2a–j</bold> and <bold>3a–c</bold> were
screened for antiviral activity against a variety of +RNA viruses.
The compounds were tested for antiviral activity in cytopathic effect
(CPE) reduction assays at 4 concentrations, that is, 150, 50, 16.7,
and 5.6 μM by preparing 3-fold serial dilutions. Compounds that
demonstrated antiviral activity in this primary screen were further
tested more extensively in dose response experiments at 8 different
concentrations to determine the EC<sub>50</sub>. Cytotoxicity (CC<sub>50</sub>) was determined in parallel in uninfected cells (<xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>).</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><title>Inhibition of SAH Hydrolase and the
Replication of Several +RNA Viruses by All Final Nucleoside Analogues <bold>2a–j</bold> and <bold>3a–c</bold><xref rid="t1fn1" ref-type="table-fn">a</xref><sup>,</sup><xref rid="t1fn2" ref-type="table-fn">b</xref><sup>,</sup><xref rid="t1fn3" ref-type="table-fn">c</xref><sup>,</sup><xref rid="t1fn4" ref-type="table-fn">d</xref></title></caption><table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col></colgroup><thead><tr><th style="border:none;" align="center"> </th><th style="border:none;" align="center"> </th><th colspan="3" align="center">MERS-CoV<hr></hr></th><th colspan="3" align="center">SARS-CoV<hr></hr></th><th colspan="3" align="center">ZIKV<hr></hr></th><th colspan="3" align="center">CHIKV<hr></hr></th></tr><tr><th style="border:none;" align="center">compound
no.</th><th style="border:none;" align="center">SAH hydrolase
IC<sub>50</sub> (μM)</th><th style="border:none;" align="center">EC<sub>50</sub> (μM)</th><th style="border:none;" align="center">CC<sub>50</sub> (μM)</th><th style="border:none;" align="center">SI</th><th style="border:none;" align="center">EC<sub>50</sub> (μM)</th><th style="border:none;" align="center">CC<sub>50</sub> (μM)</th><th style="border:none;" align="center">SI</th><th style="border:none;" align="center">EC<sub>50</sub> (μM)</th><th style="border:none;" align="center">CC<sub>50</sub> (μM)</th><th style="border:none;" align="center">SI</th><th style="border:none;" align="center">EC<sub>50</sub> (μM)</th><th style="border:none;" align="center">CC<sub>50</sub> (μM)</th><th style="border:none;" align="center">SI</th></tr></thead><tbody><tr><td style="border:none;" align="left">1</td><td style="border:none;" align="left">1.32</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">2</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">>5</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">0.64</td><td style="border:none;" align="left">2.4</td><td style="border:none;" align="left">3.8</td><td style="border:none;" align="left">0.8</td><td style="border:none;" align="left">6.3</td><td style="border:none;" align="left">7.9</td></tr><tr><td style="border:none;" align="left">2a</td><td style="border:none;" align="left">0.37</td><td style="border:none;" align="left">0.20</td><td style="border:none;" align="left">0.60</td><td style="border:none;" align="left">3</td><td style="border:none;" align="left">ND</td><td style="border:none;" align="left">ND</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">ND</td><td style="border:none;" align="left">ND</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">2b</td><td style="border:none;" align="left">9.70</td><td style="border:none;" align="left">ND</td><td style="border:none;" align="left">ND</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">ND</td><td style="border:none;" align="left">ND</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">2.54</td><td style="border:none;" align="left">3.97</td><td style="border:none;" align="left">1.56</td><td style="border:none;" align="left">0.53</td><td style="border:none;" align="left">1.32</td><td style="border:none;" align="left">2.49</td></tr><tr><td style="border:none;" align="left">2c</td><td style="border:none;" align="left">1.06</td><td style="border:none;" align="left">0.2</td><td style="border:none;" align="left">3.2</td><td style="border:none;" align="left">16</td><td style="border:none;" align="left">0.5</td><td style="border:none;" align="left">5.9</td><td style="border:none;" align="left">11.8</td><td style="border:none;" align="left">0.26</td><td style="border:none;" align="left">>2.5</td><td style="border:none;" align="left">>9.6</td><td style="border:none;" align="left">0.13</td><td style="border:none;" align="left">>1.25</td><td style="border:none;" align="left">>9.6</td></tr><tr><td style="border:none;" align="left">2d</td><td style="border:none;" align="left">4.39</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">2e</td><td style="border:none;" align="left">0.76</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">12.5</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">2f</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">2g</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">2h</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">2i</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">2j</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">3a</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">9.3</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">6.8</td><td style="border:none;" align="left">>25</td><td style="border:none;" align="left">>3.7</td><td style="border:none;" align="left">1.75</td><td style="border:none;" align="left">>25</td><td style="border:none;" align="left">>14.3</td><td style="border:none;" align="left">1.95</td><td style="border:none;" align="left">>12.5</td><td style="border:none;" align="left">>6.4</td></tr><tr><td style="border:none;" align="left">3b</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">3c</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left">>50</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left">>100</td><td style="border:none;" align="left"> </td></tr></tbody></table><table-wrap-foot><fn id="t1fn1"><label>a</label><p>ND: not determined;
SI = CC<sub>50</sub>/EC<sub>50</sub>.</p></fn><fn id="t1fn2"><label>b</label><p>EC<sub>50</sub>: effective concentration
to inhibit the replication of the virus by 50%.</p></fn><fn id="t1fn3"><label>c</label><p>CC<sub>50</sub>: cytotoxic concentration
to inhibit the replication of normal cells by 50%.</p></fn><fn id="t1fn4"><label>d</label><p>EC<sub>50</sub> > 100 indicates
that no antiviral activity was observed at the highest concentration
tested because either there was no protection or the compound was
toxic.</p></fn></table-wrap-foot></table-wrap><p>As shown in <xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>, only the adenosine
derivatives <bold>2a–c</bold> exhibited
potent antiviral activities against +RNA viruses, while the other
purine <italic>N</italic><sup>6</sup>-methyladenine derivatives <bold>2d</bold> and <bold>2e</bold> and pyrimidine derivatives <bold>2f–j</bold> did not show significant antiviral activities, not even at 100 μM.
This result suggests that the antiviral activity might be due to an
(indirect) effect on viral MTase activity through the inhibition of
host SAH hydrolase. Inhibition of the viral RdRp appears not to be
important. The mechanism of action of these compounds has been studied
in more detail and results will be published elsewhere.</p><p>Compound <bold>2a</bold> inhibited MERS-CoV replication with an
EC<sub>50</sub> of 0.20 μM; however, it was also rather cytotoxic,
resulting in a selectivity index (SI) of 3. Replacement of the remaining
6′-H in <bold>2a</bold> with F resulted in compound <bold>2c</bold>, which exhibited a > 5-fold reduction in cytotoxicity, while
its
antiviral activity remained unchanged, with an EC<sub>50</sub> of
∼0.20 μM and an SI of 15 for MERS-CoV. This compound
was also active against SARS-CoV with an SI of 12.5, suggesting that
it may be a broad-spectrum coronavirus inhibitor. In addition, it
also inhibited ZIKV replication with an EC<sub>50</sub> of 0.26 μM
(SI > 10) and was active against CHIKV with an EC<sub>50</sub> of
0.13 μM. Compound <bold>2b</bold> showed some inhibitory effects
on CHIKV and ZIKV replication, but this was likely due to pleiotropic
cytotoxic effects, as the SI was <3. Among the phosphoramidate
prodrugs <bold>3a–c</bold>, only the adenosine prodrug <bold>3a</bold> exhibited significant broad-spectrum antiviral activities,
demonstrating that it may inhibit the RdRp of RNA viruses after conversion
into the triphosphate form, although it remains to be determined in
biochemical assays whether the triphosphate form affects RdRp activity.<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> Compound <bold>3a</bold> had an EC<sub>50</sub> of 9.3 μM for MERS-CoV and 6.8 μM for SARS-CoV, but
it also had an SI < 10, and it was therefore not considered a potent
inhibitor of coronavirus replication. However, for CHIKV and ZIKV, <bold>3a</bold> had EC<sub>50</sub> values of 1.95 and 1.75 μM, respectively,
with good selectivity indices. Interestingly, the prodrug <bold>3a</bold> was less potent, but also much less cytotoxic than the parent compound <bold>2c</bold>, which is unusual as regularly the phosphoramidate is more
potent than the parent drug.<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> The phosphoramidate <bold>3a</bold> might be slowly hydrolyzed to the 5′-monophosphate
by metabolic enzymes, or to the parent drug <bold>2c</bold> by a phosphatase,
which could inhibit SAH hydrolase, explaining the observed antiviral
effect. Viral load reduction assays were performed with compound <bold>2c</bold> by infecting cells with CHIKV, ZIKV, SARS-CoV, and MERS-CoV,
followed by treatment with different concentrations of <bold>2c</bold>. At 30 hpi (CHIKV) or 48 hpi (ZIKV, SARS- and MERS-CoV), infectious
progeny titers in the medium were determined by plaque assay (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>). Treatment with
concentrations higher than 1 μM of <bold>2c</bold> reduced infectious
CHIKV titers by more than 2 log. The effect on ZIKV infectious progeny
titers was limited and showed a ∼1 log reduction. For SARS-CoV,
the reduction in infectious progeny titer was ∼1.5 log at 2c
concentrations above 0.3 μM. The strongest antiviral effect
was observed for MERS-CoV, with a ∼2.5 log reduction in infectious
progeny titers when infected cells were treated with 2c concentrations
above 0.3 μM. Follow-up studies to gain more insights into the
mode of action of <bold>2c</bold> and <bold>3a</bold> and related
compounds are currently ongoing, and results will be published elsewhere.</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Effect
of <bold>2c</bold> on the infectious progeny of CHIKV, ZIKV,
SARS-CoV, and MERS-CoV. Cells were infected with the virus indicated
on the y-axis of the graph in medium with various concentrations of <bold>2c</bold>. Infectious progeny titers were determined by plaque assay
(<italic>n</italic> = 4) and viability of noninfected cells was monitored
using the CellTiter 96AQueous Non-Radioactive Cell Proliferation Assay
(Promega). Significant differences are indicated by *: *, <italic>p</italic> < 0.05; **, <italic>p</italic> < 0.01; ***, <italic>p</italic> < 0.001; ****, <italic>p</italic> < 0.0001.</p></caption><graphic xlink:href="jm9b00781_0002" id="gr2" position="float"></graphic></fig><p>Finally, we measured the log <italic>P</italic> of the most active
compound <bold>2c</bold> by the pH-metric method, using a T3 Sirius
instrument, because the lipophilicity is a major determinant for compound
absorption, distribution in the body, penetration across biological
barriers, metabolism, and excretion. The measured log <italic>P</italic> was 0.02, indicating that it is almost equally partitioned between
the lipid and aqueous phases. The relatively low log <italic>P</italic> of <bold>2c</bold> is expected to be overcome by converting it to
the phosphoramidate <bold>3a</bold>.</p></sec></sec><sec id="sec3"><title>Conclusions</title><p>We
have synthesized the 6′-fluorinated aristeromycin analogues <bold>2a–j</bold>, which were designed as dual-target antiviral
compounds aimed at inhibiting both the viral RdRp and the host SAH
hydrolase. The electrophilic fluorination of silyl enol ether with
Selectfluor was the key step in the synthesis. We have also synthesized
the phosphoramidate prodrugs <bold>3a–c</bold> to determine
whether these would inhibit virus replication through an effect on
the viral RNA polymerase. <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref> depicts the summarized SAR of the synthesized 6′-fluorinated
final nucleoside analogues <bold>2a–j</bold> and <bold>3a–c</bold> concerning the inhibition of human SAH hydrolase and the inhibition
of the replication of various +RNA viruses with capped genomes. It
was discovered that the introduction of fluorine at the 6′-position
increases the inhibitory activity on SAH hydrolase and the replication
of selected +RNA viruses. Compared to the 6′-unsubstituted
compound <bold>1</bold>, the 6′-fluorinated aristeromycin analogues <bold>2a</bold> and <bold>2c</bold> more potently inhibited SAH hydrolase
activity and the replication of MERS-CoV, SARS-CoV, ZIKV, and CHIKV.
Among these compounds, 6′-β-fluoroaristeromycin (<bold>2a</bold>) was the most potent with an IC<sub>50</sub> of 0.37 μM
for SAH hydrolase activity and an EC<sub>50</sub> of 0.20 μM
for MERS-CoV replication. There was a correlation between the inhibition
of SAH hydrolase and the antiviral activity of the compounds, suggesting
that the latter was mainly due to indirect targeting of viral methylation
reactions. The SAR studies and a lack of antiviral effects of several
purine and pyrimidine analogues suggest that the antiviral effect
of <bold>1</bold>, <bold>2a</bold>, and <bold>2c</bold> is unlikely
due to targeting of the viral RdRp. Compound <bold>2c</bold> appears
to be an interesting compound for further development and evaluation
as a broad-spectrum antiviral agent, as it inhibited several coronaviruses,
CHIKV, and ZIKV. More detailed biological studies on the efficacy
of these compounds in virus-infected cells and into their mode of
action are currently ongoing and will be published elsewhere.</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Summarized
SAR of 6′-fluorinated aristeromycin analogues <bold>2</bold> and <bold>3</bold>.</p></caption><graphic xlink:href="jm9b00781_0003" id="gr3" position="float"></graphic></fig></sec><sec id="sec4"><title>Experimental Section</title><sec id="sec4.1"><title>Chemical Synthesis</title><sec id="sec4.1.1"><title>General
Methods</title><p>Proton (<sup>1</sup>H) and carbon (<sup>13</sup>C)
NMR spectra were obtained on a Bruker AV 400 (400/100
MHz), Bruker AMX 500 (500/125 MHz), JEOL JNM-ECA600 (600/150 MHz),
or Bruker AVANCE III 800 (800/200 MHz) spectrometer. Chemical shifts
are reported as parts per million (δ) relative to the solvent
peak. Coupling constants (<italic>J</italic>) are reported in hertz.
Mass spectra were recorded on a Thermo LCQ XP instrument. Optical
rotations were determined on Jasco III in appropriate solvent. UV
spectra were recorded on U-3000 made by Hitachi in methanol or water.
Infrared spectra were recorded on FT-IR (FTS-135) made by Bio-Rad.
Melting points were determined on a Buchan B-540 instrument and are
uncorrected. The crude compounds were purified by column chromatography
on a silica gel (Kieselgel 60, 70–230 mesh, Merck). Elemental
analyses (C, H, and N) were used to determine the purity of all synthesized
compounds, and the results were within ±0.4% of the calculated
values, confirming ≥95% purity.</p></sec><sec id="sec4.1.2"><title>(((3a<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-2,2-dimethyl-6,6a-dihydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)oxy)triethylsilane
(<bold>6</bold>)</title><p>To a cooled (−78 °C) solution
of <bold>5</bold> (1568.0 mg, 6.470 mmol) in anhydrous tetrahydrofuran
(THF; 32.0 mL, 0.2 M) was dropwise added chlorotriethylsilane (5.4
mL, 32.355 mmol), followed by addition of LiHMDS (19.0 mL, 1.0 M solution
in THF, 19.0 mmol) under N<sub>2</sub>. After being stirred at the
same temperature for 10 min, the reaction mixture was quenched with
saturated aqueous NH<sub>4</sub>Cl (80 mL). The layers were separated,
and the aqueous layer was extracted with ethyl acetate (EtOAc; 150
mL). The combined organic layers were washed successively with H<sub>2</sub>O and saturated brine, dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified by column chromatography
(silica gel, hexanes/EtOAc, 100/1 to 30/1) to give <bold>6</bold> (2267.0
mg, 98%) as colorless oil: [α]<sub>D</sub><sup arrange="stack">20</sup> = +36.48 (<italic>c</italic> 1.23, CHCl<sub>3</sub>); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>): δ
4.73 (dd, <italic>J</italic> = 1.1, 6.0 Hz, 1H), 4.58 (d, <italic>J</italic> = 2.1 Hz, 1H), 4.36 (d, <italic>J</italic> = 6.1 Hz, 1H),
3.27 (dd, <italic>J</italic> = 5.6, 8.6 Hz, 1H), 3.15 (dd, <italic>J</italic> = 6.6, 8.6 Hz, 1H), 2.72 (dd, <italic>J</italic> = 5.9,
5.9 Hz, 1H), 1.42 (s, 3H), 1.32 (s, 3H), 1.12 (s, 9H), 0.96 (t, <italic>J</italic> = 8.0 Hz, 9H), 0.66–0.72 (m, 6H); <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>): δ 154.1, 110.3, 104.4, 82.8,
79.7, 72.5, 63.9, 47.9, 27.4 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.3, 25.8, 6.5 (3 × triethylsilyl), 4.6 (3 ×
triethylsilyl); IR (neat): 2973, 1648, 1363, 1262, 1204, 1056, 851,
748 cm<sup>–1</sup>; HRMS (FAB): found, 356.2388 [calcd for
C<sub>19</sub>H<sub>36</sub>O<sub>4</sub>Si<sup>+</sup> (M + H)<sup>+</sup>, 356.2383].</p></sec><sec id="sec4.1.3"><title>(3a<italic>R</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyldihydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4(5<italic>H</italic>)-one (<bold>7a</bold>) and (3a<italic>R</italic>,5<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyldihydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4(5<italic>H</italic>)-one (<bold>7b</bold>)</title><p>To a cooled (0 °C) solution
of silyl enol ether <bold>6</bold> (8.75 g, 24.548 mmol) in anhydrous
DMF (123.0 mL, 0.20 M)
was added 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane
bis(tetrafluoroborate) (13.04 g, 36.824 mmol, Selectfluor) in one
portion under N<sub>2</sub>. After being stirred at the same temperature
for 12 h, the reaction mixture was quenched with saturated aqueous
NH<sub>4</sub>Cl (130 mL), diluted with EtOAc (130 mL). The layers
were separated and the aqueous layer was extracted with EtOAc (2 ×
100 mL). The combined organic layers were washed successively with
H<sub>2</sub>O and saturated brine, dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified by column
chromatography (silica gel, hexanes/EtOAc, 40/1 to 20/1) to give <bold>7a</bold> and <bold>7b</bold> (5.80 g, 91%, total yield, <bold>7a</bold>/<bold>7b</bold> = 5.2:1 by <sup>1</sup>H NMR analysis).</p><sec id="sec4.1.3.1"><title>Compound <bold>7a</bold></title><p>It was obtained as a white
solid; [α]<sub>D</sub><sup arrange="stack">25</sup> = −156.69 (<italic>c</italic> 0.735, CHCl<sub>3</sub>); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>): δ 5.29 (dd, <italic>J</italic> = 8.2, 49.5 Hz, 1H), 4.70 (t, <italic>J</italic> = 5.7
Hz, 1H), 4.20 (dd, <italic>J</italic> = 2.4, 6.1 Hz, 1H), 3.61 (dd, <italic>J</italic> = 1.6, 8.6 Hz, 1H) 3.38–3.41 (m, 1H), 2.75 (d, <italic>J</italic> = 8.2 Hz, 1H), 1.41 (s, 3H), 1.30 (s, 3H), 1.06 (s, 9H); <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>): δ 203.0 (d, <italic>J</italic> = 12.9 Hz), 111.4, 88.5 (d, <italic>J</italic> = 201.5
Hz), 78.2 (d, <italic>J</italic> = 6.9 Hz), 75.0 (d, <italic>J</italic> = 3.1 Hz), 74.3, 56.6 (d, <italic>J</italic> = 6.6 Hz), 40.5 (d, <italic>J</italic> = 15.5 Hz), 26.8 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.2, 23.6; <sup>19</sup>F NMR (376 MHz, CDCl<sub>3</sub>): δ −220.60 to –221.14 (m); LRMS (ESI<sup>+</sup>): found, 283.13 [calcd for C<sub>13</sub>H<sub>21</sub>FO<sub>4</sub>Na<sup>+</sup> (M + Na)<sup>+</sup>, 283.1322]; Anal. Calcd for C<sub>13</sub>H<sub>21</sub>FO<sub>4</sub>: C, 59.98; H, 8.13. Found: C,
59.99; H, 8.53.</p></sec><sec id="sec4.1.3.2"><title>Compound <bold>7b</bold></title><p>It was
obtained as a white
solid; [α]<sub>D</sub><sup arrange="stack">25</sup> = −83.72 (<italic>c</italic> 0.495, CHCl<sub>3</sub>); <sup>1</sup>H NMR (600 MHz, CDCl<sub>3</sub>): δ 5.21–5.36
(ddd, <italic>J</italic> = 1.3, 4.5, 50.8 Hz, 1H), 4.55 (d, <italic>J</italic> = 5.9 Hz, 1H), 4.50 (d, <italic>J</italic> = 5.9 Hz, 1H),
3.63 (d, <italic>J</italic> = 2.2 Hz, 2H), 2.52–2.58 (m, 1H),
1.41 (s, 3H), 1.33 (s, 3H), 1.13 (s, 9H); <sup>13</sup>C NMR (150
MHz, CDCl<sub>3</sub>): δ 207.8 (d, <italic>J</italic> = 12.9
Hz), 112.2, 91.9 (d, <italic>J</italic> = 192.4 Hz), 78.78 (d, <italic>J</italic> = 3.5 Hz), 78.74, 73.6, 60.5 (d, <italic>J</italic> =
4.3 Hz), 45.0 (d, <italic>J</italic> = 17.9 Hz), 27.2 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.8, 25.2; <sup>19</sup>F
NMR (376 MHz, CDCl<sub>3</sub>): δ −196.0 to –196.2
(m); HRMS (FAB): found, 262.1679 [calcd for C<sub>13</sub>H<sub>22</sub>FO<sub>4</sub><sup>+</sup> (M + H)<sup>+</sup>, 261.1505]; Anal.
Calcd for C<sub>13</sub>H<sub>21</sub>FO<sub>4</sub>: C, 59.98; H,
8.13. Found: C, 59.77; H, 8.45.</p></sec></sec><sec id="sec4.1.4"><title>(3a<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5,5-difluoro-2,2-dimethyldihydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4(5<italic>H</italic>)-one (<bold>7c</bold>)</title><p>It was obtained in 70% yield (mixture
of <bold>7c</bold> and <bold>7d</bold>) as a white solid; [α]<sub>D</sub><sup arrange="stack">25</sup> = −4.34
(<italic>c</italic> 0.21, MeOH); <sup>1</sup>H NMR (<bold>7c</bold> and <bold>7d</bold> mixture, 400 MHz, CDCl<sub>3</sub>; <bold>7c</bold> and <bold>7d</bold> mixture): δ 4.82 (s, 1H), 4.72 (t, <italic>J</italic> = 6.1 Hz, 1H), 4.52–4.57 (m, 1H), 4.35–4.41
(m, 1H), 4.25 (dd, <italic>J</italic> = 8.0, 4.0 Hz, 1H), 3.74 (s,
1H), 3.69 (d, <italic>J</italic> = 8.0 Hz, 1H), 3.67–3.60 (m,
1H), 3.54–3.59 (m, 1H), 3.46 (d, <italic>J</italic> = 8.3 Hz,
1H), 2.68 (d, <italic>J</italic> = 17.4 Hz, 1H), 2.53–2.62
(m, 1H), 1.48 (s, 3H), 1.44 (s, 3H), 1.34 (s, 3H), 1.32 (s, 3H), 1.21
(s, 9H), 1.06 (s, 9H).</p></sec><sec id="sec4.1.5"><title>General Procedure for the Synthesis of <bold>8a–c</bold></title><p>To a cooled (0 °C) solution of <bold>7a–c</bold> (1 equiv) in MeOH (0.18 M), sodium borohydride
or lithium borohydride
was added in a single portion in a N<sub>2</sub> atmosphere. After
stirring for 30 min at the same temperature, the reaction mixture
was neutralized with acetic acid (2 mL) and evaporated. The residue
was diluted with saturated aqueous NH<sub>4</sub>Cl, and the aqueous
layer was extracted with EtOAc (2 × 100 mL). The combined organic
layers were dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated.
The residue was purified by column chromatography (silica gel, hexanes/EtOAc,
20/1) to give <bold>8a–c</bold>.</p><sec id="sec4.1.5.1"><title>(3a<italic>S</italic>,4<italic>R</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-ol (<bold>8a</bold>)</title><p>It was obtained in 71% yield as a colorless syrup;
[α]<sub>D</sub><sup arrange="stack">25</sup> = −47.46
(<italic>c</italic> 0.395, CHCl<sub>3</sub>); <sup>1</sup>H NMR (400
MHz, CDCl<sub>3</sub>): δ 4.91 (td, <italic>J</italic> = 6.6,
52.5 Hz, 1H), 4.51–4.52 (m, 1H), 4.47 (ddd, <italic>J</italic> = 1.6, 6.3, 7.8 Hz, 1H), 4.26–4.34 (m, 1H), 3.52 (dd, <italic>J</italic> = 3.3, 8.8 Hz, 1H), 3.36–3.39 (m, 1H), 2.67 (d, <italic>J</italic> = 7.9 Hz, 1H), 2.46 (br s, 1H), 1.45 (s, 3H), 1.32 (s,
3H), 1.14 (s, 9H); <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>):
δ 111.1, 99.5 (d, <italic>J</italic> = 185.9 Hz), 81.2 (d, <italic>J</italic> = 4.4 Hz), 76.3 (d, <italic>J</italic> = 9.0 Hz), 74.0
(d, <italic>J</italic> = 23.4 Hz), 73.0, 56.8 (d, <italic>J</italic> = 8.2 Hz), 44.6 (d, <italic>J</italic> = 18.1 Hz), 27.3 (3 ×
CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.1, 24.1; <sup>19</sup>F NMR (376 MHz, CDCl<sub>3</sub>) −211.0 to −211.21
(m); HRMS (FAB): found, 263.1662 [calcd for C<sub>13</sub>H<sub>24</sub>FO<sub>4</sub><sup>+</sup> (M + H)<sup>+</sup>, 263.1659]; Anal.
Calcd for C<sub>13</sub>H<sub>23</sub>FO<sub>4</sub>: C, 59.52; H,
8.84. Found: C, 59.32; H, 9.15.</p></sec><sec id="sec4.1.5.2"><title>(3a<italic>S</italic>,4<italic>R</italic>,5<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-ol (<bold>8b</bold>)</title><p>It was obtained in 67% yield as a colorless syrup;
[α]<sub>D</sub><sup arrange="stack">25</sup> = −40.42
(<italic>c</italic> 0.22, MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 4.68 (dd, <italic>J</italic> = 4.1, 52.4 Hz, 1H),
4.46–4.53 (m, 2H), 4.13–4.24 (m, 1H), 3.33–3.40
(m, 1H), 2.81 (d, <italic>J</italic> = 11.4 Hz, 1H), 2.50 (dt, <italic>J</italic> = 2.9, 22.9 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.08
(s, 9H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): δ 111.4,
98.4 (d, <italic>J</italic> = 181.5 Hz), 82.8, 79.3, 73.8 (d, <italic>J</italic> = 16.3 Hz), 73.0, 60.6 (d, <italic>J</italic> = 12.1 Hz),
49.2 (d, <italic>J</italic> = 18.3 Hz), 27.1 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.2, 24.2; HRMS (ESI<sup>+</sup>):
found, 285.1480 [calcd for C<sub>13</sub>H<sub>23</sub>FNaO<sub>4</sub><sup>+</sup> (M + Na)<sup>+</sup>, 285.1478]; Anal. Calcd for C<sub>13</sub>H<sub>23</sub>FO<sub>4</sub>: C, 55.70; H, 7.91. Found: C,
55.40; H, 7.75.</p></sec><sec id="sec4.1.5.3"><title>(3a<italic>S</italic>,4<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-ol (<bold>8c</bold>)</title><p>It was obtained in 74% yield as a colorless syrup;
[α]<sub>D</sub><sup arrange="stack">25</sup> = 22.37 (<italic>c</italic> 0.28, MeOH); <sup>1</sup>H NMR (500
MHz, CDCl<sub>3</sub>): δ 4.53 (t, <italic>J</italic> = 5.7
Hz, 1H), 4.44 (ddd, <italic>J</italic> = 2.6, 6.4, 8.9 Hz, 1H), 4.20–4.29
(m, 1H), 3.55 (d, <italic>J</italic> = 8.7 Hz, 1H), 3.39 (d, <italic>J</italic> = 8.8 Hz, 1H), 2.76 (d, <italic>J</italic> = 11.5 Hz,
1H), 2.43 (d, <italic>J</italic> = 17.2 Hz, 1H), 1.46 (s, 3H), 1.31
(s, 3H), 1.12 (s, 9H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): δ 126.9 (dd, <italic>J</italic> = 252.3, 260.3 Hz), 110.9,
79.6 (d, <italic>J</italic> = 5.9 Hz), 75.5 (d, <italic>J</italic> = 11.3 Hz), 73.7 (dd, <italic>J</italic> = 18.5, 25.8 Hz), 73.4,
57.6 (dd, <italic>J</italic> = 4.6, 8.5 Hz), 48.7 (t, <italic>J</italic> = 20.8 Hz), 27.2 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl),
25.9, 24.2; HRMS (ESI<sup>+</sup>): found, 298.1834 [calcd for C<sub>13</sub>H<sub>26</sub>F<sub>2</sub>NO<sub>4</sub><sup>+</sup> (M
+ NH<sub>4</sub>)<sup>+</sup>, 298.1830 ]; Anal. Calcd for C<sub>13</sub>H<sub>22</sub>F<sub>2</sub>O<sub>4</sub>: C, 55.70; H, 7.91. Found:
C, 55.45; H, 7.56.</p></sec></sec><sec id="sec4.1.6"><title>(3a<italic>R</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-((<italic>tert</italic>-butyldimethylsilyl)oxy)-2,2-dimethyldihydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4(5<italic>H</italic>)-one (<bold>9</bold>)</title><p>To a cooled (0 °C) solution
of <bold>6</bold> (1275 mg, 3.57 mmol) in anhydrous THF (12 mL, 0.3
M) were added 4-methylmorpholine <italic>N</italic>-oxide monohydrate
(967 mg, 7.15 mmol, 2 equiv) and osmium tetroxide (1000 mg, 3.93 mmol,
1.1 equiv) under a N<sub>2</sub> atmosphere. After stirring for 30
min, to the reaction mixture were added sodium thiosulfate pentahydrate
(300 mg), sodium sulfite (300 mg), and acetone (30 mL) and stirred
for additional 1 h at the same temperature. The layers were separated,
and the aqueous layer was extracted with EtOAc (100 mL). The combined
organic layers were washed with H<sub>2</sub>O followed by saturated
brine, dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated.
The residue was used for the next step without further purification.
To a solution of above generated intermediate in anhydrous DMF (18
mL, 0.19 M) were added TBSCl (1614 mg, 10.71 mmol) and imidazole (729
mg, 10.71 mmol) under a N<sub>2</sub> atmosphere. After stirring for
3 h at room temperature, the reaction mixture was quenched with saturated
aqueous NH<sub>4</sub>Cl (50 mL) and diluted with EtOAc (50 mL). The
layers were separated, and the aqueous layer was extracted with EtOAc
(2 × 50 mL). The combined organic layers were washed successively
with H<sub>2</sub>O and saturated brine, dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified by column
chromatography (silica gel, hexanes/EtOAc, 40/1 to 20/1) to give <bold>9</bold> (705 mg, 53%) as a colorless syrup: [α]<sub>D</sub><sup arrange="stack">25</sup> = −103.19
(<italic>c</italic> 0.30, MeOH); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>): δ 4.65 (d, <italic>J</italic> = 6.4 Hz, 1H), 4.53
(d, <italic>J</italic> = 8.0 Hz, 1H), 4.11 (d, <italic>J</italic> =
6.3 Hz, 1H), 3.61 (dd, <italic>J</italic> = 1.6, 8.0 Hz, 1H), 3.30
(dd, <italic>J</italic> = 2.4, 8.1 Hz, 1H), 2.41–2.46 (m, 1H),
1.42 (s, 3H), 1.30 (s, 3H), 1.03 (s, 9H), 0.88 (s, 9H), 0.13 (s, 3H),
0.05 (s, 3H); <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>): δ
207.2, 110.9, 78.1, 75.8, 73.7, 71.3, 56.9, 42.3, 27.0 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.4, 25.7 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 23.8, 18.3, −4.4, −5.6;
HRMS (FAB<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found,
373.2398 [calcd for C<sub>19</sub>H<sub>37</sub>O<sub>5</sub>Si<sup>+</sup> (M + H)<sup>+</sup>, 373.2410]; Anal. Calcd for C<sub>19</sub>H<sub>36</sub>O<sub>5</sub>Si: C, 61.25; H, 9.74. Found: C, 61.26;
H, 9.75.</p></sec><sec id="sec4.1.7"><title>(3a<italic>S</italic>,4<italic>R</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-((<italic>tert</italic>-butyldimethylsilyl)oxy)-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-ol (<bold>10</bold>)</title><p>To a cooled (0 °C) solution of <bold>9</bold> (471 mg, 1.26 mmol) in methanol (6.3 mL, 0.2 M) was added sodium
borohydride (144 mg, 3.79 mmol, 3 equiv) under a N<sub>2</sub> atmosphere.
After being stirred at the same temperature for 1 h, the reaction
mixture was diluted with H<sub>2</sub>O (20 mL) and EtOAc (20 mL).
The layers were separated, and the aqueous layer was extracted with
EtOAc (3 × 50 mL). The combined organic layers were washed successively
with H<sub>2</sub>O and saturated brine, dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified by column
chromatography (silica gel, hexanes/EtOAc, 30/1 to 20/1) to give <bold>10</bold> (415 mg, 88%) as a colorless syrup: [α]<sub>D</sub><sup arrange="stack">25</sup> = −40.39
(<italic>c</italic> 0.32, MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 4.49 (d, <italic>J</italic> = 6.1 Hz, 1H), 4.41
(t, <italic>J</italic> = 6.2 Hz, 1H), 4.07 (t, <italic>J</italic> =
6.9 Hz, 1H), 3.95 (dd, <italic>J</italic> = 6.8, 14.7 Hz, 1H), 3.48
(dd, <italic>J</italic> = 3.9, 8.5 Hz, 1H), 3.32 (dd, <italic>J</italic> = 4.6, 8.5 Hz, 1H), 2.43 (d, <italic>J</italic> = 8.4 Hz, 1H), 2.12–2.18
(m, 1H), 1.45 (s, 3H), 1.32 (s, 3H), 1.12 (s, 9H), 0.87 (s, 9H), 0.09
(s, 3H), 0.05 (s, 3H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): δ 110.4, 81.0, 78.8, 77.0, 76.1, 72.6, 57.3, 46.0, 27.4
(3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.2, 25.8
(3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 24.0, 18.1,
−4.5, −5.1; HRMS (FAB<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 375.2584 [calcd for C<sub>19</sub>H<sub>39</sub>O<sub>5</sub>Si<sup>+</sup> (M + H)<sup>+</sup>, 375.2567];
Anal. Calcd for C<sub>19</sub>H<sub>38</sub>O<sub>5</sub>Si: C, 60.92;
H, 10.23. Found: C, 60.91; H, 10.25.</p></sec><sec id="sec4.1.8"><title>(((3a<italic>R</italic>,4<italic>R</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-4-(Benzyloxy)-6-(<italic>tert</italic>-butoxymethyl)-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-5-yl)oxy)(<italic>tert</italic>-butyl)dimethylsilane (<bold>11</bold>)</title><p>To
a cooled (0 °C)
solution of <bold>10</bold> (193 mg, 0.515 mmol) in DMF (5.2 mL, 0.1
M) was added benzyl chloride (0.12 mL, 1.030 mmol, 2.0 equiv) and
sodium hydride (41 mg, 1.030 mmol, 2.0 equiv) under a N<sub>2</sub> atmosphere. After being stirred at room temperature for 12 h, the
reaction mixture was diluted with H<sub>2</sub>O (20 mL) and EtOAc
(20 mL). The layers were separated, and the aqueous layer was extracted
with EtOAc (3 × 50 mL). The combined organic layers were washed
successively with H<sub>2</sub>O and saturated brine, dried over anhydrous
MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified
by column chromatography (silica gel, hexanes/EtOAc, 50/1) to give <bold>11</bold> (204 mg, 85%) as a colorless syrup: [α]<sub>D</sub><sup arrange="stack">25</sup> = −46.64
(<italic>c</italic> 0.66, MeOH); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>): δ 7.22–7.39 (m, 5H), 4.76 (d, <italic>J</italic> = 12.4 Hz, 1H), 4.59 (d, <italic>J</italic> = 12.4 Hz, 1H), 4.45
(d, <italic>J</italic> = 6.0 Hz, 1H), 4.33–4.37 (m, 2H), 3.83
(dd, <italic>J</italic> = 5.6, 8.8 Hz, 1H), 3.39 (dd, <italic>J</italic> = 4.4, 8.8 Hz, 1H), 3.32 (dd, <italic>J</italic> = 4.0, 8.4 Hz,
1H), 2.05–2.11 (m, 1H), 1.48 (s, 3H), 1.29 (s, 3H), 1.03 (s,
9H), 0.88 (s, 9H), 0.09 (s, 3H), 0.05 (s, 3H); <sup>13</sup>C NMR
(200 MHz, CDCl<sub>3</sub>): δ 138.9, 128.4, 128.1, 127.9, 127.7,
127.2, 110.0, 82.1, 80.2, 76.0, 75.6, 72.4, 71.7, 57.5, 45.7, 27.3
(3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.4, 25.8
(3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 24.2, −4.7,
−4.9; HRMS (FAB<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 465.3001 [calcd for C<sub>26</sub>H<sub>45</sub>O<sub>5</sub>Si<sup>+</sup> (M + H)<sup>+</sup>, 465.3029]; Anal. Calcd for C<sub>26</sub>H<sub>44</sub>O<sub>5</sub>Si: C, 67.20; H, 9.54. Found:
C, 67.22; H, 9.55.</p></sec><sec id="sec4.1.9"><title>(3a<italic>R</italic>,4<italic>S</italic>,5<italic>R</italic>,6<italic>S</italic>,6a<italic>R</italic>)-4-(Benzyloxy)-6-(<italic>tert</italic>-butoxymethyl)-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-5-ol (<bold>12</bold>)</title><p>To a cooled (0 °C) solution of <bold>11</bold> (179 mg, 0.385
mmol) in anhydrous THF (3.8 mL, 0.1 M) was added TBAF solution (1.2
mL, 1.0 M solution in THF, 1.2 mmol, 3.0 equiv) under a N<sub>2</sub> atmosphere. After being stirred at room temperature for 12 h, the
reaction mixture was diluted with H<sub>2</sub>O (30 mL) and EtOAc
(30 mL). The layers were separated, and the aqueous layer was extracted
with EtOAc (3 × 50 mL). The combined organic layers were washed
successively with H<sub>2</sub>O and saturated brine, dried over anhydrous
MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified
by column chromatography (silica gel, hexanes/EtOAc, 8/1) to give <bold>12</bold> (129 mg, 88%) as a colorless syrup: [α]<sub>D</sub><sup arrange="stack">25</sup> = −49.04
(<italic>c</italic> 0.28, MeOH); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>): δ 7.39 (d, <italic>J</italic> = 7.2 Hz, 2H), 7.29–7.35
(m, 2H), 7.23–7.28 (m, 1H), 4.85 (d, <italic>J</italic> = 12.4
Hz, 1H), 4.62 (d, <italic>J</italic> = 12.4 Hz, 1H), 4.51 (t, <italic>J</italic> = 6.0 Hz, 1H), 4.40–4.45 (m, 2H), 3.81 (dd, <italic>J</italic> = 4.8, 7.2 Hz, 1H), 3.58 (dd, <italic>J</italic> = 3.6,
8.8 Hz, 1H), 3.44 (dd, <italic>J</italic> = 4.4, 8.8 Hz, 1H), 2.70
(br s, 1H), 2.26–2.32 (m, 1H), 1.48 (s, 3H), 1.31 (s, 3H),
1.08 (s, 9H); <sup>13</sup>C NMR (200 MHz, CDCl<sub>3</sub>): δ
138.5, 128.3 (2 × CH-benzene), 128.0 (2 × CH-benzene), 127.5,
111.1, 82.7, 80.6, 77.2, 76.7, 73.4, 71.9, 59.3, 45.4, 27.2 (3 ×
CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.5, 24.6; Anal. Calcd
for C<sub>20</sub>H<sub>30</sub>O<sub>5</sub>: C, 68.54; H, 8.63.
Found: C, 68.52; H, 8.64.</p></sec><sec id="sec4.1.10"><title>(3a<italic>R</italic>,4<italic>R</italic>,5<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-4-(Benzyloxy)-6-(<italic>tert</italic>-butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxole (<bold>13a</bold>)</title><p>To a cooled (0 °C) solution of <bold>12</bold> (20
mg, 0.052 mmol) in anhydrous toluene (2.0 mL, 0.026 M) was dropwise
added diethylaminosulfur trifluoride (30 μL, 0.210 mmol, 4.0
equiv) under a N<sub>2</sub> atmosphere. After being stirred at room
temperature for 2 h, the reaction mixture was quenched with saturated
aqueous NH<sub>4</sub>Cl (30 mL) and EtOAc (30 mL). The layers were
separated, and the aqueous layer was extracted with EtOAc (3 ×
50 mL). The combined organic layers were washed successively with
H<sub>2</sub>O and saturated brine, dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified by column
chromatography (silica gel, hexanes/EtOAc, 30/1) to give <bold>13a</bold> (5.6 mg, 30%) and <bold>13b</bold> (5.6 mg, 30%) as a colorless
syrup.</p><sec id="sec4.1.10.1"><title>Compound <bold>13a</bold></title><p>[α]<sub>D</sub><sup arrange="stack">25</sup> = −26.59 (<italic>c</italic> 0.22, MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ
7.25–7.34 (m, 5H), 4.96 (ddd, <italic>J</italic> = 2.6, 6.8,
52.7 Hz, 1H), 4.72 (dd, <italic>J</italic> = 0.8, 11.6 Hz, 1H), 4.54
(d, <italic>J</italic> = 11.6 Hz, 1H), 4.44–4.52 (m, 2H), 4.02–4.09
(m, 1H), 3.41–3.47 (m, 2H), 2.15–2.18 (m, 1H), 1.47
(s, 3H), 1.28 (s, 3H), 1.12 (s, 9H); <sup>13</sup>C NMR (200 MHz,
CDCl<sub>3</sub>): δ 137.8, 128.3 (2 × CH-benzyl), 128.1
(2 × CH-benzyl), 127.8, 111.8, 96.0 (d, <italic>J</italic> =
187.1 Hz), 81.6, 79.3, 78.2 (d, <italic>J</italic> = 15.7 Hz), 72.6,
71.8, 60.6 (d, <italic>J</italic> = 11.0 Hz), 50.2 (d, <italic>J</italic> = 18.7 Hz), 27.0 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl),
26.6, 24.4; HRMS (FAB<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 353.2121 [calcd for C<sub>20</sub>H<sub>30</sub>FO<sub>4</sub><sup>+</sup> (M + H)<sup>+</sup>, 353.2128]; Anal. Calcd for
C<sub>20</sub>H<sub>29</sub>FO<sub>4</sub>: C, 68.16; H, 8.29. Found:
C, 68.13; H, 8.27.</p></sec><sec id="sec4.1.10.2"><title>Compound <bold>13b</bold></title><p>[α]<sub>D</sub><sup arrange="stack">25</sup> = −61.72
(<italic>c</italic> 0.42, MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ
7.38 (t, <italic>J</italic> = 7.3 Hz, 2H), 7.31 (t, <italic>J</italic> = 7.2 Hz, 2H), 7.25 (d, <italic>J</italic> = 7.2 Hz, 1H), 5.18 (dt, <italic>J</italic> = 7.8, 53.7 Hz, 1H), 4.76 (d, <italic>J</italic> = 12.2
Hz, 1H), 4.66 (d, <italic>J</italic> = 12.2 Hz, 1H), 4.45–4.49
(m, 1H), 4.41–4.44 (m, 1H), 4.19 (ddd, <italic>J</italic> =
5.9, 7.7, 16.5 Hz, 1H), 3.45 (dd, <italic>J</italic> = 3.0, 8.8 Hz,
1H), 3.31–3.34 (m, 1H), 2.37–2.43 (m, 1H), 1.47 (s,
3H), 1.28 (s, 3H), 1.01 (s, 9H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): δ 138.0, 128.3, 127.9 (2 × CH-benzyl), 127.7
(2 × CH-benzyl), 112.2, 103.5, 102.1, 81.5 (d, <italic>J</italic> = 27.5 Hz), 81.1 (d, <italic>J</italic> = 20.0 Hz), 72.6, 72.4,
57.6, 48.8 (d, <italic>J</italic> = 6.2 Hz), 27.4 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.1, 25.0; HRMS (FAB<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 353.2131 [calcd
for C<sub>20</sub>H<sub>30</sub>FO<sub>4</sub><sup>+</sup> (M + H)<sup>+</sup>, 353.2128]; Anal. Calcd for C<sub>20</sub>H<sub>29</sub>FO<sub>4</sub>: C, 68.16; H, 8.29. Found: C, 68.13; H, 8.27.</p></sec></sec><sec id="sec4.1.11"><title>(3a<italic>R</italic>,4<italic>R</italic>,5<italic>S</italic>,6<italic>R</italic>,6a<italic>S</italic>)-4-(<italic>tert</italic>-Butoxy)-5-(<italic>tert</italic>-butoxymethyl)-6-hydroxytetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3,2]dioxathiole 2-Oxide
(<bold>14</bold>)</title><p>To perform regioselective cleavage, to
a cooled (−78 °C) solution of <bold>10</bold> (420 mg,
1.121 mmol) in anhydrous CH<sub>2</sub>Cl<sub>2</sub> (5.6 mL, 0.2
M) was dropwise added trimethylaluminum (3.4 mL, 2.0 M solution in
hexane, 6.727 mmol, 6.0 equiv) under a N<sub>2</sub> atmosphere. After
being stirred at room temperature for 12 h, the reaction mixture was
quenched with saturated aqueous NH<sub>4</sub>Cl (30 mL) and EtOAc
(30 mL). The layers were separated, and the aqueous layer was extracted
with EtOAc (3 × 50 mL). The combined organic layers were washed
successively with H<sub>2</sub>O and saturated brine, dried over anhydrous
MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified
by column chromatography (silica gel, hexanes/EtOAc, 10/1) to give
diol intermediate (245 mg, 56%) 10a as a colorless syrup. For the
introduction of cyclic sulfite, to a cooled (0 °C) solution of
diol intermediate <bold>10a</bold> (250 mg, 0.639 mmol) in anhydrous
CH<sub>2</sub>Cl<sub>2</sub> (6.4 mL, 0.1 M) was dropwise added triethylamine
(0.3 mL, 2.239 mmol, 3.5 equiv) followed by thionyl chloride (70 μL,
0.959 mmol) under a N<sub>2</sub> atmosphere. After being stirred
at room temperature for 30 min, the reaction mixture was quenched
with saturated aqueous NH<sub>4</sub>Cl (30 mL) and diluted with EtOAc
(30 mL). The layers were separated, and the aqueous layer was extracted
with EtOAc (3 × 50 mL). The combined organic layers were washed
successively with H<sub>2</sub>O and saturated brine, dried over anhydrous
MgSO<sub>4</sub>, filtered, and evaporated. The residue was purified
by flash column chromatography (silica gel, hexanes/EtOAc, 10/1) to
give cyclic sulfite intermediate <bold>10b</bold> (249 mg, 89%) as
a colorless syrup. For TBS deprotection, to a cooled (0 °C) solution
of <bold>10b</bold> (286 mg, 0.654 mmol) in anhydrous THF (6.5 mL,
0.1 M) was added acetic acid (0.13 mL, 0.131 mmol, 0.2 equiv) followed
by TBAF solution (2.6 mL, 1.0 M solution in THF, 2.6 mmol, 4.0 equiv)
under a N<sub>2</sub> atmosphere. After being stirred at room temperature
for 12 h, the reaction mixture was quenched with H<sub>2</sub>O (30
mL) and diluted with EtOAc (30 mL). The layers were separated, and
the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined
organic layers were washed successively with H<sub>2</sub>O and saturated
brine, dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated.
The residue was purified by column chromatography (silica gel, hexanes/EtOAc,
6/1) to give <bold>14</bold> (202 mg, 96%, two diastereomers <bold>A</bold> and <bold>B</bold> were generated from sulfoxide stereogenic
center) as a colorless syrup: for <bold>A</bold>: <sup>1</sup>H NMR
(400 MHz, CDCl<sub>3</sub>): δ 5.27 (t, <italic>J</italic> =
5.4 Hz, 1H), 5.02 (d, <italic>J</italic> = 5.9 Hz, 1H), 4.79 (s, 1H),
4.44 (dd, <italic>J</italic> = 4.8, 11.4 Hz, 1H), 4.19 (d, <italic>J</italic> = 3.9 Hz, 1H), 3.80 (dd, <italic>J</italic> = 2.6, 9.3
Hz, 1H), 1.90–1.94 (m, 1H), 1.27 (s, 9H), 1.21 (s, 9H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): δ 86.9, 82.6, 74.9,
74.5, 74.1, 69.4, 58.2, 43.6, 28.3 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.2 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl); HRMS (FAB<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 323.1530 [calcd for C<sub>14</sub>H<sub>27</sub>O<sub>6</sub>S<sup>+</sup> (M + H)<sup>+</sup>, 323.1528]; for <bold>B</bold>: <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 4.98–5.07
(m, 2H), 4.79 (d, <italic>J</italic> = 6.4 Hz, 1H), 4.36 (dd, <italic>J</italic> = 4.6, 11.5 Hz, 1H), 4.31 (d, <italic>J</italic> = 4.1
Hz, 1H), 3.84 (d, <italic>J</italic> = 9.2 Hz, 1H), 3.77 (d, <italic>J</italic> = 9.3 Hz, 1H), 2.65 (d, <italic>J</italic> = 10.1 Hz,
1H), 1.25 (s, 9H), 1.21 (s, 9H).</p></sec><sec id="sec4.1.12"><title>(3a<italic>R</italic>,4<italic>R</italic>,5<italic>R</italic>,6<italic>S</italic>,6a<italic>R</italic>)-4-(<italic>tert</italic>-Butoxy)-5-(<italic>tert</italic>-butoxymethyl)-6-fluorotetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3,2]dioxathiole 2-Oxide
(<bold>15</bold>)</title><p>To a cooled (0 °C) solution of <bold>14</bold> (33 mg, 0.102 mmol) in anhydrous CH<sub>2</sub>Cl<sub>2</sub> (1.5 mL, 0.068 M) was dropwise added diethylaminosulfur trifluoride
(60 μL, 0.434 mmol, 4.0 equiv) under a N<sub>2</sub> atmosphere.
After being stirred at room temperature for 4 h, the reaction mixture
was quenched with saturated aqueous NH<sub>4</sub>Cl (30 mL) and diluted
with EtOAc (30 mL). The layers were separated, and the aqueous layer
was extracted with EtOAc (3 × 50 mL). The combined organic layers
were washed successively with H<sub>2</sub>O and saturated brine,
dried over anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The
residue was purified by flash column chromatography (silica gel, hexanes/EtOAc,
15/1) to give <bold>15</bold> (12 mg, 37%) as a colorless syrup: <sup>1</sup>H NMR (600 MHz, CDCl<sub>3</sub>): δ 5.17 (ddd, <italic>J</italic> = 4.6, 7.8, 52.7 Hz, 1H), 5.03 (t, <italic>J</italic> =
8.2 Hz, 1H), 4.92 (ddd, <italic>J</italic> = 5.0, 8.7, 17.8 Hz, 1H),
4.06 (ddd, <italic>J</italic> = 7.8, 11.0, 16.5 Hz, 1H), 3.53 (ddd, <italic>J</italic> = 2.7, 2.7, 6.8 Hz, 1H), 3.44 (dd, <italic>J</italic> =
2.2, 9.1 Hz, 1H), 2.54–2.58 (m, 1H), 1.17 (s, 18H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): δ 102.1 (d, <italic>J</italic> = 191.2 Hz), 87.2 (d, <italic>J</italic> = 28.2 Hz), 81.9 (d, <italic>J</italic> = 5.8 Hz), 74.5, 72.8, 72.4 (d, <italic>J</italic> = 19.2
Hz), 55.5, 50.4 (d, <italic>J</italic> = 6.5 Hz), 28.6 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.5 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl).</p></sec><sec id="sec4.1.13"><title>(3a<italic>R</italic>,4<italic>R</italic>,5<italic>R</italic>,6<italic>S</italic>,6a<italic>R</italic>)-4-(<italic>tert</italic>-Butoxy)-5-(<italic>tert</italic>-butoxymethyl)-6-fluorotetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3,2]dioxathiole 2,2-Dioxide
(<bold>16</bold>)</title><p>To a solution of cyclic sulfite <bold>15</bold> (13 mg, 0.040 mmol) in CCl<sub>4</sub>/CH<sub>3</sub>CN/H<sub>2</sub>O (1:1:1.5, total 1.75 mL, 0.14 M) was added in one portion sodium
periodate (26 mg, 0.120 mmol), followed by ruthenium(III) chloride
trihydrate (2 mg, 0.008 mmol) at room temperature under a N<sub>2</sub> atmosphere. After being stirred at the same temperature for 20 min,
the reaction mixture was quenched with H<sub>2</sub>O (20 mL) and
diluted with CH<sub>2</sub>Cl<sub>2</sub> (20 mL). The layers were
separated, and the aqueous layer was extracted with CH<sub>2</sub>Cl<sub>2</sub> (2 × 50 mL). The combined organic layers were
washed successively with H<sub>2</sub>O and saturated brine, dried
over anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The crude
product <bold>16</bold> was used for the next step without further
purification.</p></sec><sec id="sec4.1.14"><title>General Procedure for the Synthesis of <bold>18a–c</bold></title><sec id="sec4.1.14.1"><title>Triflation</title><p>To a cooled (0 °C)
solution of <bold>8a–c</bold> (1 equiv) in anhydrous pyridine
(0.32 M), trifluoromethanesulfonic
anhydride (2 equiv) was added dropwise in a N<sub>2</sub> atmosphere.
After stirring at the same temperature for 30 min, the reaction mixture
was quenched with H<sub>2</sub>O (50 mL) and diluted with EtOAc (30
mL). The layers were separated, and the aqueous layer was extracted
with EtOAc (2 × 30 mL). The combined organic layers were washed
with saturated aqueous CuSO<sub>4</sub> followed by water, dried over
anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The residue
was used for the next step without further purification.</p></sec><sec id="sec4.1.14.2"><title>Azidation</title><p>To a solution of triflate intermediate (1
equiv) in anhydrous DMF (0.19 M), sodium azide (3 equiv) was added
in a single portion at room temperature. After being heated to 60–100
°C and stirred for 4–15 h, the reaction mixture was cooled
to room temperature, quenched with H<sub>2</sub>O (50 mL), and diluted
with EtOAc (50 mL). The layers were separated, and the aqueous layer
was extracted with EtOAc (2 × 50 mL). The combined organic layers
were washed with H<sub>2</sub>O followed by saturated brine, dried
over anhydrous MgSO<sub>4</sub>, filtered, and evaporated. The residue
was purified by column chromatography (silica gel, hexanes/EtOAc,
10/1) to give <bold>18a–c</bold>.</p></sec><sec id="sec4.1.14.3"><title>(3a<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-4-Azido-6-(<italic>tert</italic>-butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxole (<bold>18a</bold>)</title><p>It was obtained in 45% yield as a colorless syrup; [α]<sub>D</sub><sup arrange="stack">25</sup> = −24.42
(<italic>c</italic> 0.016, CH<sub>2</sub>Cl<sub>2</sub>); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 5.16 (td, <italic>J</italic> = 52.4, 3.1 Hz, 1H), 4.66 (t, <italic>J</italic> = 6.0 Hz, 1H),
4.41 (t, <italic>J</italic> = 6.5 Hz, 1H), 3.62–3.69 (m, 1H),
3.54 (s, 1H), 3.50 (s, 1H), 2.27–2.36 (m, 1H), 1.47 (s, 3H),
1.29 (s, 3H), 1.16 (s, 9H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): δ 114.1, 96.9 (d, <italic>J</italic> = 182.6 Hz),
82.0, 80.2, 73.1, 67.9 (d, <italic>J</italic> = 15.7 Hz), 57.8 (d, <italic>J</italic> = 7.2 Hz), 49.4 (d, <italic>J</italic> = 17.6 Hz), 27.3
(3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.1, 24.6; <sup>19</sup>F NMR (376 MHz, CDCl<sub>3</sub>) −206.9 to –207.2
(m); IR (neat): 2108 cm<sup>–1</sup>; LR-MS (ESI<sup>+</sup>): 310.15 [calcd for C<sub>13</sub>H<sub>22</sub>FN<sub>2</sub>NaO<sub>3</sub><sup>+</sup> (M + Na)<sup>+</sup>, 310.1543]; Anal. Calcd
for C<sub>13</sub>H<sub>22</sub>FN<sub>3</sub>O<sub>3</sub>: C, 54.34;
H, 7.72; N, 14.62. Found: C, 54.35; H, 7.45; N, 14.23.</p></sec><sec id="sec4.1.14.4"><title>(3a<italic>S</italic>,4<italic>S</italic>,5<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-4-Azido-6-(<italic>tert</italic>-butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxole (<bold>18b</bold>)</title><p>It was obtained in 88% yield as a colorless syrup; [α]<sub>D</sub><sup arrange="stack">25</sup> = 9.66 (<italic>c</italic> 0.51, MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 4.75 (dt, <italic>J</italic> = 7.7, 53.0 Hz, 1H), 4.41
(dd, <italic>J</italic> = 4.5, 6.7 Hz, 1H), 4.22 (t, <italic>J</italic> = 5.7 Hz, 1H), 4.00 (ddd, <italic>J</italic> = 5.5, 7.4, 16.6 Hz,
1H), 3.43–3.50 (m, 2H), 2.33–2.44 (m, 1H), 1.50 (s,
3H), 1.27 (s, 3H), 1.15 (s, 9H); <sup>13</sup>C NMR (150 MHz, CDCl<sub>3</sub>): δ 112.7, 95.8 (d, <italic>J</italic> = 188.9 Hz),
81.0 (d, <italic>J</italic> = 8.6 Hz), 77.8 (d, <italic>J</italic> = 7.2 Hz), 73.0, 70.9 (d, <italic>J</italic> = 20.1 Hz), 57.9, 49.1
(d, <italic>J</italic> = 18.7 Hz), 27.3 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.2, 25.0; IR (neat): 2111 cm<sup>–1</sup>; Anal. Calcd for C<sub>13</sub>H<sub>22</sub>FN<sub>3</sub>O<sub>3</sub>: C, 54.34; H, 7.72; N, 14.62. Found: C, 54.12; H, 7.94; N,
14.33.</p></sec><sec id="sec4.1.14.5"><title>(3a<italic>S</italic>,4<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-4-Azido-6-(<italic>tert</italic>-butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-3a<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxole (<bold>18c</bold>)</title><p>It was obtained in 75% yield as a colorless syrup; [α]<sub>D</sub><sup arrange="stack">25</sup> = −43.39
(<italic>c</italic> 0.36, MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 4.40–4.44 (m, 1H), 4.34–4.39 (m,
1H), 3.87–3.95 (m, 1H), 3.61 (dd, <italic>J</italic> = 6.5,
9.3 Hz, 1H), 3.48 (t, <italic>J</italic> = 7.6 Hz, 1H), 2.54–2.66
(m, 1H), 1.49 (s, 3H), 1.28 (s, 3H), 1.17 (s, 9H); <sup>13</sup>C
NMR (125 MHz, CDCl<sub>3</sub>): δ 127.1 (dd, <italic>J</italic> = 255.9, 260.9 Hz), 113.0, 80.0 (d, <italic>J</italic> = 5.9 Hz),
78.4 (d, <italic>J</italic> = 5.6 Hz), 73.4, 69.1 (dd, <italic>J</italic> = 18.8, 25.1 Hz), 57.2 (d, <italic>J</italic> = 6.4 Hz), 50.8 (t, <italic>J</italic> = 20.0 Hz), 27.3 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 26.9, 24.7; IR (neat): 2116 cm<sup>–1</sup>; Anal.
Calcd for C<sub>13</sub>H<sub>21</sub>F<sub>2</sub>N<sub>3</sub>O<sub>3</sub>: C, 51.14; H, 6.93; N, 13.76. Found: C, 51.45; H, 7.21; N,
14.10.</p></sec></sec><sec id="sec4.1.15"><title>General Procedure for the Synthesis of <bold>19a–c</bold></title><p>To a suspension of <bold>18a–c</bold> (1 equiv)
in methanol (0.2 M), 10% palladium on activated carbon (0.03 equiv)
was added and stirred overnight at room temperature in a H<sub>2</sub> atmosphere. After filtration, the solvent was removed, and the residue
was used for the next step without further purification.</p></sec><sec id="sec4.1.16"><title>General
Procedure for the Synthesis of <bold>20a–c</bold></title><p>To a solution of <bold>19a–c</bold> (1 equiv) in <italic>n</italic>-butanol (0.38 M), 5-amino-4,6-dichloro pyrimidine (3–10
equiv) and diisopropylamine (10 equiv) were added. The reaction mixture
was placed under microwave irradiation at 170–200 °C for
4–7 h. The solvent was co-evaporated with MeOH, and the residue
was purified with column chromatography (silica gel, hexane/EtOAc,
4/1) to give <bold>20a–c</bold>, respectively.</p><sec id="sec4.1.16.1"><title><italic>N</italic><sup>4</sup>-((3a<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)-6-chloropyrimidine-4,5-diamine
(<bold>20a</bold>)</title><p>It was obtained in 66% yield from <bold>18a</bold>; yellow foam; [α]<sub>D</sub><sup arrange="stack">25</sup> = −53.8 (<italic>c</italic> 0.10,
CH<sub>2</sub>Cl<sub>2</sub>); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 8.08 (s, 1H), 5.27–5.33 (br s, 1H), 5.24
(td, <italic>J</italic> = 3.5, 52.9 Hz, 1H), 4.71–4.81 (m,
1H), 4.57 (t, <italic>J</italic> = 6.1 Hz, 1H), 4.44 (t, <italic>J</italic> = 6.3 Hz, 1H), 3.58–3.63 (m, 1H), 3.53 (t, <italic>J</italic> = 9.2 Hz, 1H), 3.39 (br s, 2H), 2.42–2.55 (m, 1H), 1.52 (s,
3H), 1.30 (s, 3H), 1.18 (s, 9H); <sup>13</sup>C NMR (200 MHz, CDCl<sub>3</sub>): δ 154.4, 149.0, 122.4, 113.8, 95.9 (d, <italic>J</italic> = 178.7 Hz), 84.2, 80.1, 77.1, 73.3, 59.8 (d, <italic>J</italic> = 15.9 Hz), 58.0 (d, <italic>J</italic> = 7.0 Hz), 49.4 (d, <italic>J</italic> = 17.6 Hz), 27.4 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.2, 24.8; <sup>19</sup>F NMR (376 MHz, CDCl<sub>3</sub>) −212.8 to –213.1 (m); UV (CH<sub>2</sub>Cl<sub>2</sub>): λ<sub>max</sub> 287 nm; LRMS (ESI<sup>+</sup>): found, 388.17
[calcd for C<sub>17</sub>H<sub>27</sub>ClFN<sub>4</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 389.1756]; Anal. Calcd for C<sub>17</sub>H<sub>26</sub>ClFN<sub>4</sub>O<sub>3</sub>: C, 52.51; H,
6.50; N, 14.45. Found: C, 52.45; H, 6.13; N, 14.15.</p></sec><sec id="sec4.1.16.2"><title><italic>N</italic><sup>4</sup>-((3a<italic>S</italic>,4<italic>S</italic>,5<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)-6-chloropyrimidine-4,5-diamine
(<bold>20b</bold>)</title><p>It was obtained in 47% yield from <bold>18b</bold>; yellow foam; [α]<sub>D</sub><sup arrange="stack">25</sup> = −11.79 (<italic>c</italic> 0.36,
MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 8.10
(s, 1H), 5.56 (d, <italic>J</italic> = 9.2 Hz, 1H), 4.89 (dt, <italic>J</italic> = 3.1, 51.0 Hz, 1H), 4.77 (dd, <italic>J</italic> = 9.1,
21.2 Hz, 1H), 4.61 (dd, <italic>J</italic> = 2.5, 5.0 Hz, 1H), 4.51
(dd, <italic>J</italic> = 2.4, 6.0 Hz, 1H), 3.60 (dd, <italic>J</italic> = 2.6, 9.2 Hz, 1H), 3.55 (dd, <italic>J</italic> = 2.5, 9.3 Hz,
1H), 3.39 (br s, 2H), 2.60 (d, <italic>J</italic> = 23.5 Hz, 1H),
1.54 (s, 3H), 1.29 (s, 3H), 1.21 (s, 9H); <sup>13</sup>C NMR (125
MHz, CDCl<sub>3</sub>): δ 154.2, 149.6, 143.4, 122.4, 111.7,
101.3 (d, <italic>J</italic> = 185.1 Hz), 85.5 (d, <italic>J</italic> = 3.3 Hz), 82.0 (d, <italic>J</italic> = 2.6 Hz), 74.0, 63.7 (d, <italic>J</italic> = 26.6 Hz), 60.6 (d, <italic>J</italic> = 7.1 Hz), 51.3
(d, <italic>J</italic> = 20.5 Hz), 27.5 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.1, 24.9; UV (MeOH): λ<sub>max</sub> 297.60, 265.07 nm; HRMS (ESI<sup>+</sup>): found, 389.1762 [calcd
for C<sub>17</sub>H<sub>27</sub>ClFN<sub>4</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 389.1756]; Anal. Calcd for C<sub>17</sub>H<sub>26</sub>lFN<sub>4</sub>O<sub>3</sub>: C, 52.51; H, 6.50; N,
14.45. Found: C, 52.56; H, 6.51; N, 14.43.</p></sec><sec id="sec4.1.16.3"><title><italic>N</italic><sup>4</sup>-((3a<italic>S</italic>,4<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)-6-chloropyrimidine-4,5-diamine
(<bold>20c</bold>)</title><p>It was obtained in 67% yield from <bold>18c</bold>; yellow foam; [α]<sub>D</sub><sup arrange="stack">25</sup> = −61.76 (<italic>c</italic> 0.23,
MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 8.11
(s, 1H), 5.71 (d, <italic>J</italic> = 10.1 Hz, 1H), 5.03 (t, <italic>J</italic> = 12.7 Hz, 1H), 4.56 (t, <italic>J</italic> = 4.6 Hz,
1H), 4.40–4.45 (m, 1H), 3.69 (dd, <italic>J</italic> = 2.6,
9.5 Hz, 1H), 3.57 (dd, <italic>J</italic> = 4.4, 9.4 Hz, 1H), 3.38
(br s, 2H), 2.72 (d, <italic>J</italic> = 14.7 Hz, 1H), 1.53 (s, 3H),
1.44 (s, 3H), 1.25 (s, 9H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): δ 154.5, 149.6, 143.9, 128.0 (dd, <italic>J</italic> = 257.3, 260.0 Hz), 122.3, 111.7, 84.5, 79.7 (d, <italic>J</italic> = 4.1 Hz), 74.5, 61.7 (dd, <italic>J</italic> = 18.1, 31.9 Hz),
58.3 (t, <italic>J</italic> = 5.8 Hz), 51.6 (t, <italic>J</italic> = 22.6 Hz), 27.5 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl),
26.7, 24.6; UV (MeOH): λ<sub>max</sub> 297.39, 263.29 nm; HRMS
(ESI<sup>+</sup>): found, 407.1658 [calcd for C<sub>17</sub>H<sub>26</sub>ClF<sub>2</sub>N<sub>4</sub>O<sub>3</sub><sup>+</sup> (M
+ H)<sup>+</sup>, 407.1661]; Anal. Calcd for C<sub>17</sub>H<sub>25</sub>ClF<sub>2</sub>N<sub>4</sub>O<sub>3</sub>: C, 50.19; H, 6.19; N,
13.77. Found: C, 50.11; H, 6.23; N, 13.65.</p></sec></sec><sec id="sec4.1.17"><title>General
Procedure for the Synthesis of <bold>21a–c</bold></title><p>A solution of <bold>20a–c</bold> in diethoxymethyl
acetate (0.15 M) was placed under microwave irradiation at 140 °C
for 3 h. The mixture was then co-evaporated with MeOH three times,
and the resulting residue was purified with column chromatography
(silica gel, hexane/EtOAc, 7/1) to give <bold>21a–c</bold>.</p><sec id="sec4.1.17.1"><title>9-((3a<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)-6-chloro-9<italic>H</italic>-purine (<bold>21a</bold>)</title><p>It was obtained in
96% yield as yellow foam; [α]<sub>D</sub><sup arrange="stack">25</sup> = −29.2 (<italic>c</italic> 0.17,
CH<sub>2</sub>Cl<sub>2</sub>); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>): δ 8.74 (s, 1H), 8.34 (d, <italic>J</italic> = 2.4
Hz, 1H), 5.28–5.43 (td, <italic>J</italic> = 2.8, 52.8 Hz,
1H), 5.12–5.23 (m, 2H), 4.61 (t, <italic>J</italic> = 5.0 Hz,
1H), 3.65–3.69 (m, 1H), 3.61 (t, <italic>J</italic> = 9.2 Hz,
1H), 2.56–2.71 (m, 1H), 1.56 (s, 3H), 1.32 (s, 3H), 1.17 (s,
9H); <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>): δ 152.3,
151.4, 144.2, 144.1, 131.4, 115.4, 97.7–95.9 (d, <italic>J</italic> = 181.2 Hz), 82.9, 80.1, 73.5, 63.1 (d, <italic>J</italic> = 16.1
Hz), 58.0 (d, <italic>J</italic> = 7.4 Hz), 50.0 (d, <italic>J</italic> = 17.5 Hz), 27.6 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl),
27.5, 25.1; <sup>19</sup>F NMR (376 MHz, CDCl<sub>3</sub>) −202.6–202.9
(m); UV (CH<sub>2</sub>Cl<sub>2</sub>): λ<sub>max</sub> 271
nm; LRMS (ESI<sup>+</sup>): found, 399.16 [calcd for C<sub>18</sub>H<sub>25</sub>ClFN<sub>4</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 399.1599]; Anal. Calcd for C<sub>18</sub>H<sub>24</sub>ClFN<sub>4</sub>O<sub>3</sub>: C, 54.20; H, 6.06; N, 14.05. Found: C, 54.12;
H, 6.34; N, 14.23.</p></sec><sec id="sec4.1.17.2"><title>9-((3a<italic>S</italic>,4<italic>S</italic>,5<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)-6-chloro-9<italic>H</italic>-purine (<bold>21b</bold>)</title><p>It was obtained in
76% yield as yellow foam; [α]<sub>D</sub><sup arrange="stack">25</sup> = −31.54 (<italic>c</italic> 0.54,
MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 8.67
(s, 1H), 8.15 (s, 1H), 5.55 (dt, <italic>J</italic> = 8.4, 53.6 Hz,
1H), 5.02 (t, <italic>J</italic> = 6.4 Hz, 1H), 4.84–4.94 (m,
1H), 4.65 (t, <italic>J</italic> = 5.1 Hz, 1H), 3.53–3.63 (m,
2H), 2.47–2.57 (m, 1H), 1.54 (s, 3H), 1.25 (s, 3H), 1.17 (s,
9H); <sup>13</sup>C NMR (150 MHz, CDCl<sub>3</sub>): δ 151.7,
151.5, 151.3, 144.8, 132.3, 113.1, 93.9 (d, <italic>J</italic> = 191.0
Hz), 79.1 (d, <italic>J</italic> = 7.9 Hz), 77.6 (d, <italic>J</italic> = 7.9 Hz), 73.1, 67.8 (d, <italic>J</italic> = 20.8 Hz), 58.1, 48.7
(d, <italic>J</italic> = 18.7 Hz) 27.5 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.3, 25.0; UV (MeOH): λ<sub>max</sub> 264.36 nm; HRMS (ESI<sup>+</sup>): found, 399.1589 [calcd for C<sub>18</sub>H<sub>25</sub>ClFN<sub>4</sub>O<sub>3</sub><sup>+</sup> (M
+ H)<sup>+</sup>, 399.1599]; Anal. Calcd for C<sub>18</sub>H<sub>24</sub>ClFN<sub>4</sub>O<sub>3</sub>: C, 54.20; H, 6.06; N, 14.05. Found:
C, 54.34; H, 6.46; N, 13.99.</p></sec><sec id="sec4.1.17.3"><title>9-((3a<italic>S</italic>,4<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)-6-chloro-9<italic>H</italic>-purine (<bold>21c</bold>)</title><p>It was obtained in
92% yield as yellow foam; [α]<sub>D</sub><sup arrange="stack">25</sup> = −46.05 (<italic>c</italic> 0.43,
MeOH); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 8.73
(s, 1H), 8.28 (d, <italic>J</italic> = 2.1 Hz, 1H), 5.30 (dt, <italic>J</italic> = 6.9, 20.1 Hz, 1H), 5.10 (t, <italic>J</italic> = 6.7
Hz, 1H), 4.57–4.62 (m, 1H), 3.63–3.73 (m, 2H), 2.81–2.93
(m, 1H), 1.56 (s, 3H), 1.30 (s, 3H), 1.18 (s, 9H); <sup>13</sup>C
NMR (125 MHz, CDCl<sub>3</sub>): δ 152.4, 152.4, 151.3, 143.9
(d, <italic>J</italic> = 4.0 Hz), 131.2, 125.6 (dd, <italic>J</italic> = 253.4, 264.6 Hz), 114.0, 79.5 (d, <italic>J</italic> = 7.7 Hz),
77.9 (d, <italic>J</italic> = 7.5 Hz), 73.7, 64.6 (dd, <italic>J</italic> = 19.3, 24.3 Hz), 57.1 (d, <italic>J</italic> = 7.1 Hz), 50.3 (t, <italic>J</italic> = 19.8 Hz), 27.3 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.2, 25.0; UV (MeOH): λ<sub>max</sub> 263.74 nm;
HRMS (ESI<sup>+</sup>): found, 417.1500 [calcd for C<sub>18</sub>H<sub>24</sub>ClF<sub>2</sub>N<sub>4</sub>O<sub>3</sub><sup>+</sup> (M
+ H)<sup>+</sup>, 417.1505]; Anal. Calcd for C<sub>18</sub>H<sub>23</sub>ClF<sub>2</sub>N<sub>4</sub>O<sub>3</sub>: C, 51.86; H, 5.56; N,
13.44. Found: C, 51.56; H, 5.96; N, 13.13.</p></sec></sec><sec id="sec4.1.18"><title>General
Procedure for the Synthesis of <bold>2a–c</bold></title><p>To
a solution of <bold>21a–c</bold> in <italic>tert</italic>-butanol
(2 mL, 0.27 M) contained in a stainless steel bomb reactor,
saturated ammonia in <italic>tert</italic>-butanol (15 mL) was added
and the reactor was locked. After being heated to 120 °C with
stirring for 15 h, the mixture was cooled to room temperature and
co-evaporated with MeOH. Without purification, the residue was added
to a TFA/H<sub>2</sub>O solution (2:1, v/v, total 15 mL) and heated
to 50 °C with stirring for 15 h. After the reaction mixture was
evaporated, the residue was purified by column chromatography (silica
gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 9/1) to give <bold>2a–c</bold>.</p><sec id="sec4.1.18.1"><title>(1<italic>R</italic>,2<italic>S</italic>,3<italic>S</italic>,4<italic>R</italic>,5<italic>R</italic>)-3-(6-Amino-9<italic>H</italic>-purin-9-yl)-4-fluoro-5-(hydroxymethyl)cyclopentane-1,2-diol
(<bold>2a</bold>)</title><p>It was obtained in 43% yield as a white
solid;
mp 172–177 °C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −64.49 (<italic>c</italic> 0.22,
MeOH); <sup>1</sup>H NMR (800 MHz, CD<sub>3</sub>OD-<italic>d</italic><sub>6</sub>): δ 8.26 (d, <italic>J</italic> = 2.0 Hz, 1H),
8.21 (s, 1H), 5.21 (dt, <italic>J</italic> = 4.0, 54.6, 1H), 4.99
(ddd, <italic>J</italic> = 3.4, 10.8, 29.5 Hz, 1H), 4.75 (dd, <italic>J</italic> = 6.7, 9.4 Hz, 1H), 4.02 (dd, <italic>J</italic> = 4.8,
6.4 Hz, 1H), 3.79–3.85 (m, 2H), 2.42–2.51 (m, 1H); <sup>13</sup>C NMR (200 MHz, CD<sub>3</sub>OD): δ 158.1, 154.6,
152.2, 142.4 (d, <italic>J</italic> = 3.3 Hz), 120.5, 92.8 (d, <italic>J</italic> = 180.7 Hz), 74.3, 71.8, 64.0 (d, <italic>J</italic> =
17.0 Hz), 60.6 (d, <italic>J</italic> = 10.7 Hz), 54.3 (d, <italic>J</italic> = 17.9 Hz); <sup>19</sup>F NMR (376 MHz, CD<sub>3</sub>OD): δ −204.7 to −205.4 (m); UV (MeOH): λ<sub>max</sub> 259.90 nm; HRMS (ESI<sup>+</sup>): found, 284.1161 [calcd
for C<sub>11</sub>H<sub>15</sub>FN<sub>5</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 284.1159]; Anal. Calcd for C<sub>11</sub>H<sub>14</sub>FN<sub>5</sub>O<sub>3</sub>: C, 46.64; H, 4.98; N, 24.72.
Found: C, 46.65; H, 5.38; N, 25.10.</p></sec><sec id="sec4.1.18.2"><title>(1<italic>R</italic>,2<italic>S</italic>,3<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>)-3-(6-Amino-9<italic>H</italic>-purin-9-yl)-4-fluoro-5-(hydroxymethyl)cyclopentane-1,2-diol
(<bold>2b</bold>)</title><p>It was obtained in 71% yield as a white
solid;
mp 182–186 °C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −11.85 (<italic>c</italic> 0.26,
MeOH); <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ 8.19
(s, 1H), 8.18 (s, 1H), 5.40 (ddd, <italic>J</italic> = 5.2, 7.3, 54.4
Hz, 1H), 5.03 (ddd, <italic>J</italic> = 7.5, 9.8, 20.7 Hz, 1H), 4.60
(dd, <italic>J</italic> = 5.1, 9.9 Hz, 1H), 4.05–4.09 (m, 1H),
3.80 (d, <italic>J</italic> = 5.8 Hz, 2H), 2.28–2.40 (m, 1H); <sup>13</sup>C NMR (125 MHz, CD<sub>3</sub>OD): δ 158.0, 154.3,
151.9, 143.4, 121.6, 95.8 (d, <italic>J</italic> = 186.4 Hz), 74.2
(d, <italic>J</italic> = 7.4 Hz), 73.2 (d, <italic>J</italic> = 3.3
Hz), 68.6 (d, <italic>J</italic> = 21.1 Hz), 62.6, 54.6 (d, <italic>J</italic> = 19.0 Hz); <sup>19</sup>F NMR (378 MHz, CD<sub>3</sub>OD): δ −185.244 (dt, <italic>J</italic> = 23.8, 53.7
Hz); UV (MeOH): λ<sub>max</sub> 260.88 nm; HRMS (ESI<sup>+</sup>): found, 284.1155 [calcd for C<sub>11</sub>H<sub>15</sub>FN<sub>5</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 284.1159];
Anal. Calcd for C<sub>11</sub>H<sub>14</sub>FN<sub>5</sub>O<sub>3</sub>: C, 46.64; H, 4.98; N, 24.72. Found: C, 46.38; H, 5.12; N, 24.33.</p></sec><sec id="sec4.1.18.3"><title>(1<italic>R</italic>,2<italic>S</italic>,3<italic>S</italic>,5<italic>R</italic>)-3-(6-Amino-9<italic>H</italic>-purin-9-yl)-4,4-difluoro-5-(hydroxymethyl)cyclopentane-1,2-diol
(<bold>2c</bold>)</title><p>It was obtained in 61% yield as a white
solid; mp 180–185 °C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −56.51 (<italic>c</italic> 0.30, MeOH); <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ
8.26 (d, <italic>J</italic> = 19.5 Hz, 1H), 8.20 (s, 1H), 5.33 (dt, <italic>J</italic> = 10.0, 17.0 Hz, 1H), 4.79 (dd, <italic>J</italic> = 5.2,
10.6 Hz, 1H, merged with solvent peak), 4.13–4.17 (m, 1H),
3.79–3.91 (m, 2H), 2.60–2.71 (m, 1H); <sup>13</sup>C
NMR (200 MHz, CD<sub>3</sub>OD): δ 158.2, 154.8, 152.6, 142.7
(d, <italic>J</italic> = 2.4 Hz), 125.9 (dd, <italic>J</italic> =
252.3, 258.4 Hz), 120.6, 73.7 (d, <italic>J</italic> = 7.3 Hz), 71.8
(d, <italic>J</italic> = 3.3 Hz), 64.8 (dd, <italic>J</italic> = 19.4,
23.8 Hz), 59.6 (d, <italic>J</italic> = 10.8 Hz), 56.4 (t, <italic>J</italic> = 19.9 Hz); <sup>19</sup>F NMR (378 MHz, CD<sub>3</sub>OD): δ −97.5 (d, <italic>J</italic> = 238.5 Hz), −115.4
(dt, <italic>J</italic> = 15.9, 238.9 Hz); UV (MeOH): λ<sub>max</sub> 259.92 nm; HRMS (ESI<sup>+</sup>): found, 302.1066 [calcd
for C<sub>11</sub>H<sub>14</sub>F<sub>2</sub>N<sub>5</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 302.1065]; Anal. Calcd for C<sub>11</sub>H<sub>13</sub>F<sub>2</sub>N<sub>5</sub>O<sub>3</sub>: C,
43.86; H, 4.35; N, 23.25. Found: C, 44.17; H, 4.14; N, 23.05.</p></sec></sec><sec id="sec4.1.19"><title>General Procedure for the Synthesis of <bold>2d</bold> and <bold>2e</bold></title><p>To a solution of <bold>21a</bold> and <bold>21c</bold> (0.283 mmol) in EtOH (1.5 mL, 0.19 M) in a sealed glass tube, methylamine
(40 wt % in H<sub>2</sub>O, 10 mL) was added. After being stirred
at room temperature for 2 h, the mixture was concentrated and added
to a TFA/H<sub>2</sub>O solution (2:1, v/v, total 15 mL) without purification.
After being heated to 50 °C with stirring for 15 h, the reaction
mixture was evaporated. The residue was purified by column chromatography
(silica gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 9/1) to give <bold>2d</bold> and <bold>2e</bold>.</p><sec id="sec4.1.19.1"><title>(1S,2<italic>R</italic>,3<italic>R</italic>,4<italic>R</italic>,5<italic>S</italic>)-4-Fluoro-3-(hydroxymethyl)-5-(6-(methylamino)-9<italic>H</italic>-purin-9-yl)cyclopentane-1,2-diol (<bold>2d</bold>)</title><p>It was obtained in 67% yield as a white solid; mp 197–201
°C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −61.46 (<italic>c</italic> 0.40, MeOH); <sup>1</sup>H NMR (800 MHz, CD<sub>3</sub>OD): δ 8.27 (s, 1H), 8.20 (d, <italic>J</italic> = 18.4 Hz, 1H), 5.21 (dt, <italic>J</italic> = 4.0, 54.6
Hz, 1H), 4.98 (ddd, <italic>J</italic> = 3.4, 10.0, 29.6 Hz, 1H),
4.74 (dd, <italic>J</italic> = 6.7, 9.4 Hz, 1H), 4.01 (dd, <italic>J</italic> = 4.9, 6.4 Hz, 1H), 3.79–3.85 (m, 2H), 3.11 (br
s, 3H), 2.42–2.51 (m, 1H); <sup>13</sup>C NMR (200 MHz, CD<sub>3</sub>OD): δ 157.5, 154.6, 151.1, 141.8 (d, <italic>J</italic> = 3.7 Hz), 121.1, 92.9 (d, <italic>J</italic> = 180.8 Hz), 74.3,
71.8, 64.0 (d, <italic>J</italic> = 17.0 Hz), 60.6 (d, <italic>J</italic> = 10.5 Hz), 54.3 (d, <italic>J</italic> = 18.0 Hz), 28.5; <sup>19</sup>F NMR (376 MHz, CD<sub>3</sub>OD): δ −206.3 (dt, <italic>J</italic> = 29.7, 53.4 Hz); UV (MeOH): λ<sub>max</sub> 266.89
nm; HRMS (ESI<sup>+</sup>): found, 298.1317 [calcd for C<sub>12</sub>H<sub>17</sub>FN<sub>5</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 298.1315]; Anal. Calcd for C<sub>12</sub>H<sub>16</sub>FN<sub>5</sub>O<sub>3</sub>: C, 48.48; H, 5.42; N, 23.56. Found: C, 48.50;
H, 5.22; N, 23.93.</p></sec><sec id="sec4.1.19.2"><title>(1S,2<italic>R</italic>,3<italic>R</italic>,5<italic>S</italic>)-4,4-Difluoro-3-(hydroxymethyl)-5-(6-(methylamino)-9<italic>H</italic>-purin-9-yl)cyclopentane-1,2-diol (<bold>2e</bold>)</title><p>It was
obtained in 76% yield as a white solid; mp 125–129 °C;
[α]<sub>D</sub><sup arrange="stack">25</sup> = −48.62 (<italic>c</italic> 0.25, MeOH); <sup>1</sup>H NMR
(500 MHz, CD<sub>3</sub>OD): δ 8.24 (s, 1H), 8.20 (s, 1H), 5.33
(dt, <italic>J</italic> = 9.9, 18.4 Hz, 1H), 4.79 (dd, <italic>J</italic> = 10.3, 10.2 Hz, 1H), 4.17 (s, 1H), 3.81–3.90 (m, 2H), 3.10
(br s, 3H), 2.67 (m, 1H); <sup>13</sup>C NMR (125 MHz, CD<sub>3</sub>OD): δ 157.5, 154.7, 151.5, 142.1, 125.9 (dd, <italic>J</italic> = 252.4, 258.1 Hz), 121.1, 73.7 (d, <italic>J</italic> = 7.25 Hz),
71.9 (d, <italic>J</italic> = 3.1 Hz), 64.7 (dd, <italic>J</italic> = 20.0, 24.3 Hz), 59.6 (d, <italic>J</italic> = 10.8 Hz), 56.4 (t, <italic>J</italic> = 19.9 Hz), 28.6; <sup>19</sup>F NMR (378 MHz, CD<sub>3</sub>OD): δ −97.4 (d, <italic>J</italic> = 238.5 Hz),
−115.3 (d, <italic>J</italic> = 238.9 Hz); UV (MeOH): λ<sub>max</sub> 263.72 nm; HRMS (ESI<sup>+</sup>): found, 316.1227 [calcd
for C<sub>12</sub>H<sub>16</sub>F<sub>2</sub>N<sub>5</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 316.1221]; Anal. Calcd for C<sub>12</sub>H<sub>15</sub>F<sub>2</sub>N<sub>5</sub>O<sub>3</sub>: C,
45.71; H, 4.80; N, 22.21. Found: C, 45.99; H, 4.47; N, 22.02.</p></sec></sec><sec id="sec4.1.20"><title>General Procedure for the Synthesis of <bold>22a–c</bold></title><p>To a cooled (−20 °C) solution of <bold>19a–c</bold> (1 equiv) in DMF (0.2 M), 3-methoxyacryloyl isocyanate (2 equiv)
in benzene was added dropwise in a N<sub>2</sub> atmosphere. After
the reaction mixture was slowly warmed to room temperature for 15
h with stirring, the reaction mixture was filtered with CH<sub>2</sub>Cl<sub>2</sub> and co-evaporated with toluene and ethanol. The residue
was purified by column chromatography (silica gel, hexane/EtOAc, 1.5/1)
to give <bold>22a–c</bold>.</p><sec id="sec4.1.20.1"><title>(<italic>E</italic>)-<italic>N</italic>-(((3a<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)carbamoyl)-3-methoxyacrylamide
(<bold>22a</bold>)</title><p>It was obtained in 76% yield as a colorless
syrup; [α]<sub>D</sub><sup arrange="stack">25</sup> = −19.41 (<italic>c</italic> 0.37, MeOH); <sup>1</sup>H NMR
(600 MHz, CDCl<sub>3</sub>): δ 10.24 (s, 1H), 9.16 (d, <italic>J</italic> = 8.2 Hz, 1H), 7.61 (d, <italic>J</italic> = 12.4 Hz,
1H), 5.35 (d, <italic>J</italic> = 12.4 Hz, 1H), 5.06 (dt, <italic>J</italic> = 3.2, 52.7 Hz, 1H), 4.51 (t, <italic>J</italic> = 6.6
Hz, 1H), 4.29–4.38 (m, 2H), 3.64 (s, 3H), 3.45–3.52
(m, 2H), 2.21–2.31 (m, 1H), 1.41 (s, 3H), 1.21 (s, 3H), 1.10
(s, 9H); <sup>13</sup>C NMR (150 MHz, CDCl<sub>3</sub>): δ 168.0,
163.3, 155.4, 113.7, 97.5, 96.7 (d, <italic>J</italic> = 178.8 Hz),
84.4, 80.1, 72.9, 58.6 (d, <italic>J</italic> = 15.8 Hz), 57.8 (d, <italic>J</italic> = 6.5 Hz), 57.4, 49.8 (d, <italic>J</italic> = 17.2 Hz),
27.2 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.1,
24.6; UV (MeOH): λ<sub>max</sub> 243.14 nm; HRMS (ESI<sup>+</sup>): found, 389.2088 [calcd for C<sub>18</sub>H<sub>30</sub>FN<sub>2</sub>O<sub>6</sub><sup>+</sup> (M + H)<sup>+</sup>, 389.2088].</p></sec><sec id="sec4.1.20.2"><title>(E)-<italic>N</italic>-(((3a<italic>S</italic>,4<italic>S</italic>,5<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)carbamoyl)-3-methoxyacrylamide
(<bold>22b</bold>)</title><p>It was obtained in 88% yield as a colorless
syrup; [α]<sub>D</sub><sup arrange="stack">25</sup> = −20.47 (<italic>c</italic> 0.34, MeOH); <sup>1</sup>H NMR
(500 MHz, CDCl<sub>3</sub>): δ 10.33 (s, 1H), 8.96 (d, <italic>J</italic> = 7.4 Hz, 1H), 7.63 (d, <italic>J</italic> = 12.3 Hz,
1H), 5.39 (d, <italic>J</italic> = 12.3 Hz, 1H), 4.80 (dt, <italic>J</italic> = 6.4, 52.5 Hz, 1H), 4.44 (t, <italic>J</italic> = 5.5
Hz, 1H), 4.33–4.41 (m, 2H), 3.67 (s, 3H), 3.46 (d, <italic>J</italic> = 32.5 Hz, 2H), 2.33–2.42 (m, 1H), 1.46 (s, 3H),
1.24 (s, 3H), 1.13 (s, 9H); <sup>13</sup>C NMR (150 MHz, CDCl<sub>3</sub>): δ 168.1, 163.2, 155.5, 111.9, 97.9 (d, <italic>J</italic> = 187.4 Hz), 97.5, 83.3 (d, <italic>J</italic> = 7.2 Hz), 79.0 (d, <italic>J</italic> = 6.5 Hz), 73.1, 61.9 (d, <italic>J</italic> = 23.7 Hz),
58.6 (d, <italic>J</italic> = 2.1 Hz), 57.4, 49.9 (d, <italic>J</italic> = 19.4 Hz), 27.3 (3 × CH<sub>3</sub>-<italic>tert</italic>-butyl),
27.2, 25.0; UV (MeOH): λ<sub>max</sub> 242.93 nm; HRMS (ESI<sup>+</sup>): found, 389.2098 [calcd for C<sub>18</sub>H<sub>30</sub>FN<sub>2</sub>O<sub>6</sub><sup>+</sup> (M + H)<sup>+</sup>, 389.2088].</p></sec><sec id="sec4.1.20.3"><title>(<italic>E</italic>)-<italic>N</italic>-(((3a<italic>S</italic>,4<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-6-(<italic>tert</italic>-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)carbamoyl)-3-methoxyacrylamide
(<bold>22c</bold>)</title><p>It was obtained in 90% yield as a colorless
syrup; [α]<sub>D</sub><sup arrange="stack">25</sup> = −40.41 (<italic>c</italic> 0.52, MeOH); <sup>1</sup>H NMR
(500 MHz, CDCl<sub>3</sub>): δ 10.26 (s, 1 Η), 9.11 (d, <italic>J</italic> = 8.7 Hz, 1H), 7.65 (d, <italic>J</italic> = 12.3 Hz,
1H), 5.37 (d, <italic>J</italic> = 12.4 Hz, 1H), 4.52–4.62
(m, 1H), 4.39 (s, 2H), 3.67 (s, 3H), 3.53–3.60 (m, 2H), 2.57–2.68
(m, 1H), 1.47 (s, 3H), 1.27 (s, 3H), 1.16 (s, 9H); <sup>13</sup>C
NMR (125 MHz, CDCl<sub>3</sub>): δ 167.9, 163.4, 155.7, 126.9
(dd, <italic>J</italic> = 252.9, 261.3 Hz), 112.4, 97.4, 82.5 (d, <italic>J</italic> = 6.9 Hz), 78.6 (d, <italic>J</italic> = 4.9 Hz), 73.5,
60.6 (dd, <italic>J</italic> = 19.4, 29.2 Hz), 57.4 (d, <italic>J</italic> = 6.1 Hz), 57.3, 50.8 (t, <italic>J</italic> = 20.8 Hz), 27.1 (3
× CH<sub>3</sub>-<italic>tert</italic>-butyl), 27.0, 24.9; UV
(MeOH): λ<sub>max</sub> 242.22 nm; HRMS (ESI<sup>+</sup>): found,
407.1991 [calcd for C<sub>18</sub>H<sub>29</sub>F<sub>2</sub>N<sub>2</sub>O<sub>6</sub><sup>+</sup> (M + H)<sup>+</sup>, 407.1994].</p></sec></sec><sec id="sec4.1.21"><title>General Procedure for the Synthesis of <bold>2f–h</bold></title><p>To a stirred solution of <bold>22a–c</bold> in 1,4-dioxane
(3 mL, 2.5 M), 2 M sulfuric acid (0.3 mL) was dropwise added. After
refluxing with stirring for 1 h, the reaction mixture was cooled to
room temperature and neutralized with DOWEX 66 ion-exchange resin.
The mixture was filtered and evaporated. The residue was purified
by column chromatography (silica gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 9/1) to give <bold>2f–h</bold>.</p><sec id="sec4.1.21.1"><title>1-((1<italic>S</italic>,2<italic>R</italic>,3<italic>R</italic>,4<italic>R</italic>,5<italic>S</italic>)-2-Fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1<italic>H</italic>,3<italic>H</italic>)-dione (<bold>2f</bold>)</title><p>It was obtained in 56% yield as a white solid; mp 112–118
°C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −77.11 (<italic>c</italic> 0.20, MeOH); <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ 7.70 (dd, <italic>J</italic> = 1.1, 8.1 Hz, 1H), 5.69 (d, <italic>J</italic> = 8.0 Hz, 1H), 5.10
(dt, <italic>J</italic> = 4.1, 55.3 Hz, 1H), 4.91 (dd, <italic>J</italic> = 3.4, 10.2 Hz, 1H, merged with solvent peak), 4.46 (dd, <italic>J</italic> = 6.6, 10.1 Hz, 1H), 3.93 (t, <italic>J</italic> = 4.8
Hz, 1H), 3.70–3.80 (m, 2H), 3.69 (s, 1H), 2.29–2.41
(m, 1H); <sup>13</sup>C NMR (125 MHz, CD<sub>3</sub>OD): δ 166.9,
154.2, 145.5 (d, <italic>J</italic> = 3.8 Hz), 102.7, 93.0 (d, <italic>J</italic> = 180.1 Hz), 72.4, 71.7, 64.4 (d, <italic>J</italic> =
16.6 Hz), 60.6 (d, <italic>J</italic> = 11.4 Hz), 53.8 (d, <italic>J</italic> = 17.9 Hz); <sup>19</sup>F NMR (378 MHz, CD<sub>3</sub>OD): δ −208.9 (dt, <italic>J</italic> = 29.9, 59.7 Hz);
UV (MeOH): λ<sub>max</sub> 264.11 nm; HRMS (ESI<sup>+</sup>):
found, 261.0886 [calcd for C<sub>10</sub>H<sub>14</sub>FN<sub>2</sub>O<sub>5</sub><sup>+</sup> (M + H)<sup>+</sup>, 261.0887]; Anal. Calcd
for C<sub>10</sub>H<sub>13</sub>FN<sub>2</sub>O<sub>5</sub>: C, 46.16;
H, 5.04; N, 10.77. Found: C, 45.98; H, 5.44; N, 10.98.</p></sec><sec id="sec4.1.21.2"><title>1-((1<italic>S</italic>,2<italic>S</italic>,3<italic>R</italic>,4<italic>R</italic>,5<italic>S</italic>)-2-Fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1<italic>H</italic>,3<italic>H</italic>)-dione (<bold>2g</bold>)</title><p>It was obtained in 53% yield as a white solid; mp 195–200
°C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −16.89 (<italic>c</italic> 0.35, MeOH); <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ 7.60 (d, <italic>J</italic> = 7.9 Hz, 1H), 5.69 (d, <italic>J</italic> = 7.9 Hz, 1H), 5.07–5.21
(ddd, <italic>J</italic> = 5.10, 6.8, 55.2 Hz, 1H), 4.61–4.69
(ddd, <italic>J</italic> = 7.3, 8.7, 22.6 Hz, 1H), 4.32 (dd, <italic>J</italic> = 5.25, 9.0 Hz, 1H), 3.98 (t, <italic>J</italic> = 3.7
Hz, 1H), 3.70 (m, 2H), 2.24 (m, 1H); <sup>13</sup>C NMR (125 MHz,
CDCl<sub>3</sub>): δ 167.1, 153.6, 147.4, 103.5, 94.8 (d, <italic>J</italic> = 183.9 Hz), 73.4 (d, <italic>J</italic> = 7.3 Hz), 73.1
(d, <italic>J</italic> = 22.0 Hz), 72.7 (d, <italic>J</italic> = 3.5
Hz), 62.3 (d, <italic>J</italic> = 1.8 Hz), 54.1 (d, <italic>J</italic> = 18.9 Hz); <sup>19</sup>F NMR (378 MHz, CD<sub>3</sub>OD): δ
−184.3 (dt, <italic>J</italic> = 23.8, 53.7 Hz); UV (MeOH):
λ<sub>max</sub> 265.33 nm; HRMS (ESI<sup>+</sup>): found, 261.0894
[calcd for C<sub>10</sub>H<sub>14</sub>FN<sub>2</sub>O<sub>5</sub><sup>+</sup> (M + H)<sup>+</sup>, 261.0887]; Anal. Calcd for C<sub>10</sub>H<sub>13</sub>FN<sub>2</sub>O<sub>5</sub>: C, 46.16; H, 5.04;
N, 10.77. Found: C, 46.24; H, 5.23; N, 10.78.</p></sec><sec id="sec4.1.21.3"><title>1-((1<italic>S</italic>,3<italic>R</italic>,4<italic>R</italic>,5<italic>S</italic>)-2,2-Difluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1<italic>H</italic>,3<italic>H</italic>)-dione (<bold>2h</bold>)</title><p>It was obtained in 52% yield as a white solid; mp 164–169
°C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −31.06 (<italic>c</italic> 0.30, MeOH); <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ 7.67 (dd, <italic>J</italic> = 2.3, 8.1 Hz, 1H) 5.71 (d, <italic>J</italic> = 8.0 Hz, 1H), 5.36
(dt, <italic>J</italic> = 10.3, 17.7 Hz, 1H), 4.41 (dd, <italic>J</italic> = 5.15, 10.7 Hz, 1H), 4.07 (m, 1H), 3.73–3.82 (m, 2H), 2.53
(m, 1H); <sup>13</sup>C NMR (150 MHz, CD<sub>3</sub>OD): δ 166.6,
154.1, 145.3 (d, <italic>J</italic> = 4.3 Hz), 126.8 (dd, <italic>J</italic> = 252.8, 258.5 Hz), 103.4, 72.5 (d, <italic>J</italic> = 7.9 Hz), 71.8 (d, <italic>J</italic> = 2.9 Hz), 64.4 (dd, <italic>J</italic> = 18.7, 25.1 Hz), 59.5 (d, <italic>J</italic> = 11.5 Hz),
56.3 (t, <italic>J</italic> = 20.1 Hz); <sup>19</sup>F NMR (378 MHz,
CD<sub>3</sub>OD): δ −96.6 (d, <italic>J</italic> = 238.9
Hz), −116.9 (dt, <italic>J</italic> = 15.1, 238.5 Hz); UV (MeOH):
λ<sub>max</sub> 262.41 nm; HRMS (ESI<sup>+</sup>): found, 279.0801
[calcd for C<sub>10</sub>H<sub>13</sub>F<sub>2</sub>N<sub>2</sub>O<sub>5</sub><sup>+</sup> (M + H)<sup>+</sup>, 279.0793]; Anal. Calcd for
C<sub>10</sub>H<sub>12</sub>F<sub>2</sub>N<sub>2</sub>O<sub>5</sub>: C, 43.17; H, 4.35; N, 10.07. Found: C, 43.34; H, 4.67; N, 9.94.</p></sec></sec><sec id="sec4.1.22"><title>General Procedure for the Synthesis of <bold>2i</bold> and <bold>2j</bold></title><sec id="sec4.1.22.1"><title>Benzoylation</title><p>To a cooled (0 °C)
solution of <bold>2f</bold> or <bold>2h</bold> (1 equiv) in CH<sub>2</sub>Cl<sub>2</sub> (0.07 M), benzoyl chloride (6 equiv) and pyridine
(6.7 equiv) were
added in a N<sub>2</sub> atmosphere. After being stirred for 15 h
at room temperature, the reaction mixture was quenched with H<sub>2</sub>O and extracted with CH<sub>2</sub>Cl<sub>2</sub>. The organic
layers were combined and washed with H<sub>2</sub>O followed by brine,
dried over MgSO<sub>4</sub>, filtered, and evaporated. The residue
was purified with column chromatography (silica gel, hexane/EtOAc,
1/1) to give the benzoylated intermediate.</p></sec><sec id="sec4.1.22.2"><title>Introduction
of Triazole</title><p>To a cooled (0 °C) suspension
of 1,2,4-triazole (10 equiv) in anhydrous MeCN (0.6 M), phosphoryl
chloride (10 equiv) was added dropwise in a N<sub>2</sub> atmosphere.
After stirring, the benzoylated intermediate (1 equiv) in MeCN (0.14
M) followed by trimethylamine (10 equiv) were added to the reaction
mixture. After additional stirring at room temperature for 15 h, the
reaction mixture was evaporated. The reaction mixture was diluted
with CH<sub>2</sub>Cl<sub>2</sub> and H<sub>2</sub>O. The layers were
separated, and the organic layers were washed with H<sub>2</sub>O,
dried over MgSO<sub>4</sub>, filtered, and evaporated.</p></sec><sec id="sec4.1.22.3"><title>Amination</title><p>In the sealed glass tube, the above-generated
intermediate in 1,4-dioxane (0.06 M) was added to excess saturated
aqueous ammonia at room temperature. After being stirred at the same
temperature for 2 h, the reaction mixture was evaporated and purified
with flash chromatography (silica gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 7/1) to give the benzoyl protected cytosine intermediate.</p></sec><sec id="sec4.1.22.4"><title>Benzoyl Deprotection</title><p>In a sealed glass tube, the above-generated
benzoyl-protected cytosine intermediate in MeOH (0.2 M) was added
to saturated ammonia in MeOH (0.2 M). After being stirred at the same
temperature for 2 d, the reaction mixture was evaporated and diluted
with H<sub>2</sub>O and CH<sub>2</sub>Cl<sub>2</sub>. The layers were
separated, and the H<sub>2</sub>O layers were washed with CH<sub>2</sub>Cl<sub>2</sub> 10 times and evaporated to give <bold>2i</bold> and <bold>2j</bold>, respectively.</p></sec><sec id="sec4.1.22.5"><title>4-Amino-1-((1<italic>S</italic>,2<italic>R</italic>,3<italic>R</italic>,4<italic>R</italic>,5<italic>S</italic>)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopentyl)pyrimidin-2(1<italic>H</italic>)-one (<bold>2i</bold>)</title><p>It was obtained in 17%
yield as a white solid; mp 230–233 °C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −84.26
(<italic>c</italic> 0.20, MeOH); <sup>1</sup>H NMR (800 MHz, CD<sub>3</sub>OD): δ 7.67 (dd, <italic>J</italic> = 1.3, 7.5 Hz, 1H),
5.88 (d, <italic>J</italic> = 7.4 Hz, 1H), 5.23 (dt, <italic>J</italic> = 3.7, 55.4 Hz, 1H), 4.93 (ddd, <italic>J</italic> = 3.4, 10.3,
30.4 Hz, 1H), 4.44 (dd, <italic>J</italic> = 6.6, 10.3 Hz, 1H), 3.92
(dd, <italic>J</italic> = 4.5, 6.3 Hz, 1H), 3.71–3.78 (m, 2H),
2.31–2.40 (m, 1H); <sup>13</sup>C NMR (200 MHz, CDCl<sub>3</sub>): δ 168.3, 160.3, 145.7 (d, <italic>J</italic> = 3.1 Hz),
96.2, 93.0 (d, <italic>J</italic> = 179.9 Hz), 72.5, 71.8, 65.3 (d, <italic>J</italic> = 16.6 Hz), 60.7 (d, <italic>J</italic> = 11.3 Hz), 53.9
(d, <italic>J</italic> = 17.9 Hz); <sup>19</sup>F NMR (376 MHz, CD<sub>3</sub>OD): δ −209.4 (dt, <italic>J</italic> = 29.3,
53.4 Hz); UV (MeOH): λ<sub>max</sub> 274.67 nm; HRMS (ESI<sup>+</sup>): found, 260.1041 [calcd for C<sub>10</sub>H<sub>15</sub>FN<sub>3</sub>O<sub>4</sub><sup>+</sup> (M + H)<sup>+</sup>, 260.1047];
Anal. Calcd for C<sub>10</sub>H<sub>14</sub>FN<sub>3</sub>O<sub>4</sub>: C, 46.33; H, 5.44; N, 16.21. Found: C, 46.71; H, 5.12; N, 15.99.</p></sec><sec id="sec4.1.22.6"><title>4-Amino-1-((1<italic>S</italic>,3<italic>R</italic>,4<italic>R</italic>,5<italic>S</italic>)-2,2-difluoro-4,5-dihydroxy-3 (hydroxymethyl)cyclopentyl)pyrimidin-2(1<italic>H</italic>)-one (<bold>2j</bold>)</title><p>It was obtained in 20%
yield as a white solid; mp 242–245 °C; [α]<sub>D</sub><sup arrange="stack">25</sup> = −39.85
(<italic>c</italic> 0.30, MeOH); <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ 7.62 (dd, <italic>J</italic> = 7.45, 2.35 Hz,
1H), 5.90 (d, <italic>J</italic> = 7.40 Hz, 1H), 5.51 (dt, <italic>J</italic> = 18.2, 10.0 Hz, 1H), 4.37 (dd, <italic>J</italic> = 10.6,
5.25 Hz, 1H), 4.06 (m, 1H), 3.73–3.83 (m, 2H), 2.54 (m, 1H); <sup>13</sup>C NMR (150 MHz, CD<sub>3</sub>OD): δ 168.2, 160.1,
145.7 (d, <italic>J</italic> = 3.6 Hz), 126.9 (dd, <italic>J</italic> = 252.1, 259.2 Hz), 96.8, 72.9 (d, <italic>J</italic> = 8.6 Hz),
71.7 (d, <italic>J</italic> = 3.6 Hz), 65.1 (dd, <italic>J</italic> = 18.7, 23.0 Hz), 59.6 (d, <italic>J</italic> = 10.8 Hz), 56.3 (t, <italic>J</italic> = 20.1 Hz); <sup>19</sup>F NMR (378 MHz, CD<sub>3</sub>OD): δ −97.4 (d, <italic>J</italic> = 235.9 Hz), −117.4
(dt, <italic>J</italic> = 14.7, 238.9 Hz); UV (MeOH): λ<sub>max</sub> 272.27, 237.93 nm; HRMS (ESI<sup>+</sup>): found, 278.0954
[calcd for C<sub>10</sub>H<sub>14</sub>F<sub>2</sub>N<sub>3</sub>O<sub>4</sub><sup>+</sup> (M + H)<sup>+</sup>, 278.0952]; Anal. Calcd for
C<sub>10</sub>H<sub>13</sub>F<sub>2</sub>N<sub>3</sub>O<sub>4</sub>: C, 43.32; H, 4.73; N, 15.16. Found: C, 43.56; H, 4.56; N, 15.44.</p></sec></sec><sec id="sec4.1.23"><title>General Procedure for the Synthesis of <bold>24</bold>, <bold>27a</bold> and <bold>27b</bold></title><p>To a cooled (0 °C)
suspension of <bold>2c</bold>, <bold>2f</bold>, and <bold>2h</bold> (1 equiv) in acetone (0.005 M) were added 1–2 drops of cH<sub>2</sub>SO<sub>4</sub> in N<sub>2</sub> (g). After being stirred at
room temperature for 4 h, the reaction mixture was neutralized with
solid NaHCO<sub>3</sub>, filtered, and evaporated under reduced pressure.
The residue was further purified by silica gel column chromatography
to give <bold>24</bold>, <bold>27a</bold>, and <bold>27b</bold>, respectively.</p><sec id="sec4.1.23.1"><title>((3a<italic>R</italic>,4<italic>R</italic>,6<italic>S</italic>,6a<italic>S</italic>)-6-(6-Amino-9<italic>H</italic>-purin-9-yl)-5,5-difluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)methanol
(<bold>24</bold>)</title><p>It was obtained in 96% yield as a colorless
syrup; <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ 8.31
(s, 1H), 8.21 (s, 1H), 5.30–5.40 (m, 2H), 4.70 (br s, 1H),
3.94 (dd, <italic>J</italic> = 6.8, 11.4 Hz, 1H), 3.86 (dd, <italic>J</italic> = 6.8, 11.4 Hz, 1H), 2.81–2.90 (m, 1H), 1.58 (s,
3H), 1.35 (s, 3H); <sup>13</sup>C NMR (125 MHz, CD<sub>3</sub>OD):
δ 163.5 (dd, <italic>J</italic> = 33.1, 69.2 Hz), 156.5, 152.2,
152.0, 143.4, 128.0 (dd, <italic>J</italic> = 251.7, 263.6 Hz), 116.0,
80.7 (d, <italic>J</italic> = 7.3 Hz), 79.6 (d, <italic>J</italic> = 8.3 Hz), 66.1 (dd, <italic>J</italic> = 19.2, 22.8 Hz), 59.2 (d, <italic>J</italic> = 8.0 Hz), 53.8 (t, <italic>J</italic> = 19.4 Hz), 28.2,
25.9; HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 342.1370 [calcd for C<sub>14</sub>H<sub>18</sub>F<sub>2</sub>N<sub>5</sub>O<sub>3</sub><sup>+</sup> (M + H)<sup>+</sup>, 342.1372];
Anal. Calcd for C<sub>14</sub>H<sub>17</sub>F<sub>2</sub>N<sub>5</sub>O<sub>3</sub>: C, 49.27; H, 5.02; N, 20.52. Found: C, 49.28; H, 4.98;
N, 20.91.</p></sec><sec id="sec4.1.23.2"><title>1-((3a<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>,6<italic>R</italic>,6a<italic>R</italic>)-5-Fluoro-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)pyrimidine-2,4(1<italic>H</italic>,3<italic>H</italic>)-dione (<bold>27a</bold>)</title><p>It was obtained in 98% yield as a colorless syrup; <sup>1</sup>H
NMR (500 MHz, CD<sub>3</sub>OD): δ 7.75 (dd, <italic>J</italic> = 1.4, 8.1 Hz, 1H), 5.70 (d, <italic>J</italic> = 8.1 Hz, 1H), 5.20
(dt, <italic>J</italic> = 3.1, 54.1 Hz, 1H), 5.01–5.13 (m,
2H), 4.58 (d, <italic>J</italic> = 6.3 Hz, 1H), 3.73–3.83 (m,
2H), 2.42–2.56 (m, 1H), 1.50 (s, 3H), 1.32 (s, 3H); <sup>13</sup>C NMR (125 MHz, CD<sub>3</sub>OD): δ 166.7, 153.7, 145.3 (d, <italic>J</italic> = 5.9 Hz), 116.5, 103.2, 99.2 (d, <italic>J</italic> =
180.2 Hz), 82.2, 82.0, 65.0 (d, <italic>J</italic> = 15.7 Hz), 60.3
(d, <italic>J</italic> = 8.7 Hz), 53.2 (d, <italic>J</italic> = 17.7
Hz), 28.4, 25.9; HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 301.1185 [calcd for C<sub>13</sub>H<sub>18</sub>FN<sub>2</sub>O<sub>5</sub><sup>+</sup> (M + H)<sup>+</sup>, 301.1194];
Anal. Calcd for C<sub>13</sub>H<sub>17</sub>FN<sub>2</sub>O<sub>5</sub>: C, 52.00; H, 5.71; N, 9.33. Found: C, 52.15; H, 5.47; N, 9.15.</p></sec><sec id="sec4.1.23.3"><title>1-((3a<italic>S</italic>,4<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-5,5-Difluoro-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)pyrimidine-2,4(1<italic>H</italic>,3<italic>H</italic>)-dione (<bold>27b</bold>)</title><p>It was obtained in 97% yield; <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ 7.71 (dd, <italic>J</italic> = 2.0, 8.1 Hz, 1H),
5.73 (d, <italic>J</italic> = 8.1 Hz, 1H), 5.33 (dt, <italic>J</italic> = 6.8, 21.3 Hz, 1H), 4.94 (d, <italic>J</italic> = 6.8 Hz, 1H),
4.57–4.63 (m, 1H), 3.88 (dd, <italic>J</italic> = 6.7, 11.4
Hz, 1H), 3.81 (dd, <italic>J</italic> = 6.7, 11.4 Hz, 1H), 2.68–2.79
(m, 1H), 1.54 (s, 3H), 1.34 (s, 3H); HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 319.1104 [calcd for C<sub>13</sub>H<sub>17</sub>F<sub>2</sub>N<sub>2</sub>O<sub>5</sub><sup>+</sup> (M + H)<sup>+</sup>, 319.1100]; Anal. Calcd for C<sub>13</sub>H<sub>16</sub>F<sub>2</sub>N<sub>2</sub>O<sub>5</sub>: C, 49.06;
H, 5.07; N, 8.80. Found: C, 49.43; H, 5.47; N, 8.43.</p></sec></sec><sec id="sec4.1.24"><title>Synthesis
of <italic>tert</italic>-Butyl-(9-((3a<italic>S</italic>,4<italic>S</italic>,6<italic>R</italic>,6a<italic>R</italic>)-5,5-difluoro-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)-9<italic>H</italic>-purin-6-yl)carbamate (<bold>25a</bold>) and Its <italic>N</italic><sup>6</sup>-Di-Boc derivative (<bold>25b</bold>)</title><p>To a suspension of <bold>24</bold> (20 mg, 0.058 mmol) and 4-dimethylaminopyridine
(1 mg, 0.0058 mmol) in hexamethyldisilazane (3 mL), trimethylsilyl
trifluoromethanesulfonate (TMSOTf; 5 μL) was added dropwise
at room temperature in a N<sub>2</sub> atmosphere (g). After being
heated to 75 °C with stirring for 2 h, the reaction mixture was
evaporated, and anhydrous THF (7 mL) was added. To a cooled (0 °C)
reaction mixture, di-<italic>t</italic>-butyl dicarbonate (63 mg,
0.29 mmol) was added. After stirring for 4 h at room temperature,
the reaction mixture was evaporated, and the residue was added to
MeOH/trimethylamine (6 mL, 5:1 (v/v)). After heating to 55 °C
with stirring for 16 h, the reaction mixture was evaporated, and the
residue was purified with column chromatography (silica gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 50/1) to give <bold>25a</bold> (13 mg,
52%) and <bold>25b</bold> (8 mg, 25%) as a colorless syrup.</p><sec id="sec4.1.24.1"><title>Compound <bold>25a</bold></title><p><sup>1</sup>H NMR (500 MHz,
CD<sub>3</sub>OD): δ 8.59 (s, 1H), 8.49 (s, 1H), 5.36–5.50
(m, 2H), 4.72 (d, <italic>J</italic> = 5.6 Hz, 1H), 3.95 (dd, <italic>J</italic> = 6.8, 11.4 Hz, 1H), 3.87 (dd, <italic>J</italic> = 6.8,
11.4 Hz, 1H), 2.83–2.95 (m, 1H), 1.57 (s, 12H), 1.34 (s, 3H);
HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found,
442.1899 [calcd for C<sub>19</sub>H<sub>26</sub>F<sub>2</sub>N<sub>5</sub>O<sub>5</sub><sup>+</sup> (M + H)<sup>+</sup>, 442.1897].</p></sec><sec id="sec4.1.24.2"><title>Compound <bold>25b</bold></title><p><sup>1</sup>H NMR (500 MHz,
CD<sub>3</sub>OD): δ 8.87 (s, 1H), 8.73 (d, <italic>J</italic> = 1.8 Hz, 1H), 5.46–5.57 (m, 2H), 4.75 (d, <italic>J</italic> = 5.4 Hz, 1H), 3.95 (dd, <italic>J</italic> = 6.8, 11.4 Hz, 1H),
3.88 (dd, <italic>J</italic> = 6.8, 11.4 Hz, 1H), 2.84–2.95
(m, 1H), 1.59 (s, 3H), 1.37 (s, 21H); <sup>13</sup>C NMR (125 MHz,
CD<sub>3</sub>OD): δ 156.2, 154.2, 152.2, 152.1 (2 × C(O)-Boc
protection group), 147.8 (d, <italic>J</italic> = 2.4 Hz), 130.6,
128.1 (dd, <italic>J</italic> = 251.8, 263.3 Hz), 116.0, 86.1, 80.4
(d, <italic>J</italic> = 7.4 Hz), 79.7 (d, <italic>J</italic> = 8.2
Hz), 72.7, 66.5 (dd, <italic>J</italic> = 19.1, 23.1 Hz), 59.2 (d, <italic>J</italic> = 8.0 Hz), 53.8 (t, <italic>J</italic> = 19.2 Hz), 28.7
(6 × CH<sub>3</sub>-<italic>tert</italic>-butyl), 28.3, 25.9;
HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found,
542.2411 [calcd for C<sub>24</sub>H<sub>34</sub>F<sub>2</sub>N<sub>5</sub>O<sub>7</sub><sup>+</sup> (M + H)<sup>+</sup>, 542.2421].</p></sec></sec><sec id="sec4.1.25"><title>Iso-propyl ((<italic>S</italic>)-(((3a<italic>R</italic>,4<italic>R</italic>,6<italic>S</italic>,6a<italic>S</italic>)-6-(6-((<italic>tert</italic>-Butoxycarbonyl)amino)-9<italic>H</italic>-purin-9-yl)-5,5-difluoro-2,2-dimethyltetrahydro-4<italic>H</italic>-cyclopenta[<italic>d</italic>][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-<sc>l</sc>-alaninate (<bold>26</bold>)</title><p>To a stirred suspension
of <bold>25a</bold> (16 mg, 0.036 mmol), <bold>25b</bold> (7 mg, 0.012
mmol), and powdered molecular sieves (4 Å, 62 mg) in anhydrous
THF (20 mL), <italic>tert</italic>-butylmagnesium chloride solution
(0.26 mL, 1.0 M in THF, 0.26 mmol) was added at 0 °C in a nitrogen
atmosphere. After 10 min, a solution of pentafluorophosphoramidate
reagent <bold>A</bold> (47 mg, 0.10 mmol) in THF (12 mL) was slowly
added, and the reaction mixture was stirred at room temperature for
36 h. Then, it was quenched by the dropwise addition of methanol (10
mL), filtered, and evaporated. The residue was purified by column
chromatography (silica gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 9/1)
to give the phosphoramidate <bold>26</bold> as a colorless liquid
(12 mg, 33%): <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ
8.59 (s, 1H), 8.45 (s, 1H), 7.37 (d, <italic>J</italic> = 7.8 Hz,
2H), 7.25 (d, <italic>J</italic> = 8.1 Hz, 2H), 7.19 (d, <italic>J</italic> = 7.5 Hz, 1H), 5.50 (dt, <italic>J</italic> = 5.9, 22.3 Hz, 1H),
5.40–5.45 (m, 1H), 4.92–4.99 (m, 1H), 4.73–4.80
(m, 1H), 4.36–4.50 (m, 2H), 3.86–3.98 (m, 1H), 3.07–3.19
(m, 1H), 1.58 (s, 12H), 1.34 (s, 6H), 1.21 (d, <italic>J</italic> =
6.2 Hz, 3H), 1.17 (d, <italic>J</italic> = 6.2 Hz, 3H); HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 711.2716
[calcd for C<sub>31</sub>H<sub>42</sub>F<sub>2</sub>N<sub>6</sub>O<sub>9</sub>P<sup>+</sup> (M + H)<sup>+</sup>, 711.2713].</p></sec><sec id="sec4.1.26"><title>Iso-propyl
((<italic>S</italic>)-(((1<italic>R</italic>,3<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>)-3-(6-Amino-9<italic>H</italic>-purin-9-yl)-2,2-difluoro-4,5-dihydroxycyclopentyl)methoxy)(phenoxy)phosphoryl)-<sc>l</sc>-alaninate (<bold>3a</bold>)</title><p>A solution of <bold>26</bold> (15 mg, 0.021 mmol) in 10 mL of formic acid/H<sub>2</sub>O (1:1,
v/v) was stirred at room temperature for 8 h. After evaporation, the
crude product was purified by column chromatography (silica gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 6/1) to give <bold>3a</bold> (9.9 mg,
82%) as a colorless solid: mp 95–100 °C; UV (MeOH): λ<sub>max</sub> 259.6 nm; [α]<sub>D</sub><sup arrange="stack">25</sup> = −38.06 (<italic>c</italic> 0.1,
MeOH); <sup>1</sup>H NMR (400 MHz, CD<sub>3</sub>OD): δ 8.18
(s, 1H), 8.17 (d, <italic>J</italic> = 1.6 Hz, 1H), 7.35 (d, <italic>J</italic> = 8.4 Hz, 2H), 7.23 (d, <italic>J</italic> = 8.6 Hz, 2H),
7.18 (d, <italic>J</italic> = 8.0 Hz, 1H), 5.26–5.38 (m, 1H),
4.81–4.98 (m, merged with H<sub>2</sub>O peak, 1H), 4.74 (dd, <italic>J</italic> = 4.8, 10.0 Hz, 1H), 4.29–4.43 (m, 2H), 7.17 (br
s, 1H), 3.82–3.93 (m, 1H), 2.79–2.94 (m, 1H), 1.32 (d, <italic>J</italic> = 6.8 Hz, 3H), 1.19 (d, <italic>J</italic> = 6.2 Hz, 3H),
1.14 (d, <italic>J</italic> = 6.2 Hz, 3H); <sup>13</sup>C NMR (150
MHz, CD<sub>3</sub>OD): δ 175.2 (d, <italic>J</italic> = 5.7
Hz), 158.1, 154.7, 153.0, 152.9, 152.6, 142.6, 131.6 (2 × CH-phenyl),
127.0, 124.4 (dd, <italic>J</italic> = 253.5, 260.6 Hz), 122.2 (d, <italic>J</italic> = 4.3 Hz), 120.6 (2 × CH-phenyl), 73.3 (d, <italic>J</italic> = 7.1 Hz), 71.2 (d, <italic>J</italic> = 5.0 Hz), 70.9,
64.6, 64.1 (dd, <italic>J</italic> = 5.0, 10.7 Hz), 52.4, 22.6 (d, <italic>J</italic> = 2.9 Hz, 2 × CH<sub>3</sub>), 21.2 (d, <italic>J</italic> = 6.5 Hz); <sup>19</sup>F NMR (376 MHz, CD<sub>3</sub>OD): δ
−98.71 (d, <italic>J</italic> = 238.4 Hz), −115.13 (dt, <italic>J</italic> = 14.9, 236.4 Hz); HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 571.1889 [calcd for C<sub>23</sub>H<sub>30</sub>F<sub>2</sub>N<sub>6</sub>O<sub>7</sub>P<sup>+</sup> (M +
H)<sup>+</sup>, 571.1876]; Anal. Calcd for C<sub>23</sub>H<sub>29</sub>F<sub>2</sub>N<sub>6</sub>O<sub>7</sub>P: C, 48.42; H, 5.12; N, 14.73.
Found: C, 48.74; H, 4.98; N, 14.54.</p></sec><sec id="sec4.1.27"><title>Iso-propyl ((<italic>S</italic>)-(((1<italic>R</italic>,2<italic>R</italic>,3<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>)-3-(2,4-Dioxo-3,4-dihydropyrimidin-1(2<italic>H</italic>)-yl)-2-fluoro-4,5-dihydroxycyclopentyl)methoxy)(phenoxy)phosphoryl)-<sc>l</sc>-alaninate (<bold>3b</bold>)</title><sec id="sec4.1.27.1"><title>Introduction of Phosphoramidate</title><p>To a cooled (0 °C)
suspension of <bold>27a</bold> (21 mg, 0.069 mmol) and molecular sieves
(4 Å, 35 mg) in anhydrous THF (15 mL, 0.005 M), <italic>tert</italic>-butylmagnesium chloride solution (0.34 mL, 1.0 M in THF, 0.34 mmol)
was added dropwise in a N<sub>2</sub> atmosphere (g). After being
stirred for 5 min, a solution of the phosphoramidate reagent <bold>A</bold> (31 mg, 0.069 mmol) in anhydrous THF (7 mL) was added dropwise,
and the reaction mixture was stirred at room temperature for 36 h,
quenched with MeOH (5 mL), filtered, and evaporated, and the residue
was purified by column chromatograph (silica gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 24/1) to give phosphoramidate as a colorless liquid
(13 mg, 33%): <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ
7.73 (dd, <italic>J</italic> = 1.3, 8.1 Hz, 1H), 7.36 (d, <italic>J</italic> = 7.8 Hz, 2H), 7.24 (d, <italic>J</italic> = 7.8 Hz, 2H),
7.19 (d, <italic>J</italic> = 7.4 Hz, 1H), 5.70 (d, <italic>J</italic> = 8.1 Hz, 1H), 5.02–5.22 (m, 3H), 4.93–5.01 (m, 1H),
4.66 (d, <italic>J</italic> = 6.3 Hz, 1H), 4.29 (d, <italic>J</italic> = 7.6 Hz, 2H), 3.87–3.95 (m, 1H), 2.62–2.73 (m, 1H),
1.51 (s, 3H), 1.34 (d, <italic>J</italic> = 7.7 Hz, 3H), 1.32 (s,
3H), 1.22 (d, <italic>J</italic> = 6.2 Hz, 6H); HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 570.2003 [calcd
for C<sub>25</sub>H<sub>34</sub>FN<sub>3</sub>O<sub>9</sub>P<sup>+</sup> (M + H)<sup>+</sup>, 570.2011].</p></sec><sec id="sec4.1.27.2"><title>Hydrolysis</title><p>A solution
of phosphoramidate (13 mg, 0.022
mmol) in a formic acid/H<sub>2</sub>O solution (1:1, v/v, 7 mL total)
was stirred at room temperature for 8 h. The reaction mixture was
evaporated and the residue was purified by column chromatography (silica
gel, CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 7/1) to give the phosphoramidate
prodrug <bold>3b</bold> (10.8 mg, 90%) as a white solid: mp 107–110
°C; UV (MeOH): λ<sub>max</sub> 262.8 nm; [α]<sub>D</sub><sup arrange="stack">25</sup> = −59.40
(<italic>c</italic> 0.1, MeOH); <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ 7.64 (d, <italic>J</italic> = 8.1 Hz, 1H), 7.36
(d, <italic>J</italic> = 7.9 Hz, 2H), 7.23 (d, <italic>J</italic> =
7.9 Hz, 2H), 7.19 (d, <italic>J</italic> = 7.4 Hz, 1H), 5.68 (d, <italic>J</italic> = 8.1 Hz, 1H), 5.04 (dt, <italic>J</italic> = 4.1, 55.4
Hz, 1H), 4.87–4.98 (m, merged with H<sub>2</sub>O peak, 2H),
4.45 (dd, <italic>J</italic> = 6.6, 9.7 Hz, 1H), 4.26 (d, <italic>J</italic> = 7.1 Hz, 2H), 3.99 (d, <italic>J</italic> = 5.4 Hz, 1H),
3.85–3.93 (m, 1H), 2.49–2.60 (m, 1H), 1.33 (d, <italic>J</italic> = 7.0 Hz, 3H), 1.21 (d, <italic>J</italic> = 6.1 Hz, 6H); <sup>13</sup>C NMR (125 MHz, CD<sub>3</sub>OD): δ 175.2, 166.9,
154.0, 151.1, 145.4, 131.5 (2 × CH-phenyl), 126.9 (2 × CH-phenyl),
122.2 (d, <italic>J</italic> = 4.6 Hz), 102.8, 93.3 (d, <italic>J</italic> = 184.5 Hz), 80.3, 79.9 (d, <italic>J</italic> = 32.5 Hz), 72.3,
71.2, 70.9, 64.2 (d, <italic>J</italic> = 16.0 Hz), 52.4, 22.7 (d, <italic>J</italic> = 9.2 Hz, 2 × CH<sub>3</sub>), 21.2 (d, <italic>J</italic> = 6.8 Hz); <sup>19</sup>F NMR (376 MHz, CD<sub>3</sub>OD): δ
−208.27 (dt, <italic>J</italic> = 29.7, 59.4 Hz); HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 530.1685
[calcd for C<sub>22</sub>H<sub>30</sub>FN<sub>3</sub>O<sub>9</sub>P<sup>+</sup> (M + H)<sup>+</sup>, 530.1698]; Anal. Calcd for C<sub>22</sub>H<sub>29</sub>FN<sub>3</sub>O<sub>9</sub>P: C, 49.91; H,
5.52; N, 7.94. Found: C, 50.03; H, 5.32; N, 7.54.</p></sec></sec><sec id="sec4.1.28"><title>Iso-propyl
((<italic>S</italic>)-(((1<italic>R</italic>,3<italic>S</italic>,4<italic>S</italic>,5<italic>R</italic>)-3-(2,4-Dioxo-3,4-dihydropyrimidin-1(2<italic>H</italic>)-yl)-2,2-difluoro-4,5-dihydroxycyclopentyl)methoxy)(phenoxy)phosphoryl)-<sc>l</sc>-alaninate (<bold>3c</bold>)</title><p>Compound <bold>3c</bold> was synthesized according the same procedure used in the preparation
of <bold>3b</bold>: yield = 30%; white solid; mp 174 °C (decomp);
UV (MeOH): λ<sub>max</sub> 262.8 nm; [α]<sub>D</sub><sup arrange="stack">25</sup> = −19.40 (<italic>c</italic> 0.1, MeOH); <sup>1</sup>H NMR (500 MHz, CD<sub>3</sub>OD): δ
7.53 (dd, <italic>J</italic> = 2.1, 8.1 Hz, 1H), 7.36 (d, <italic>J</italic> = 7.8 Hz, 2H), 7.25 (d, <italic>J</italic> = 7.8 Hz, 2H),
7.20 (d, <italic>J</italic> = 7.6 Hz, 1H), 5.70 (d, <italic>J</italic> = 8.1 Hz, 1H), 5.29–5.39 (m, 1H), 4.93–5.02 (m, 1H),
4.30–4.39 (m, 2H), 4.23–4.29 (m, 1H), 4.08 (br s, 1H),
3.84–3.92 (m, 1H), 2.69–2.80 (m, 1H), 1.33 (d, <italic>J</italic> = 7.1 Hz, 3H), 1.22 (d, <italic>J</italic> = 6.2 Hz, 6H); <sup>19</sup>F NMR (376 MHz, CD<sub>3</sub>OD): δ −98.47
(d, <italic>J</italic> = 237.2 Hz), −116.91 (dt, <italic>J</italic> = 17.6, 237.2 Hz); HRMS (ESI<sup>+</sup>) (<italic>m</italic>/<italic>z</italic>): found, 548.1619 [calcd for C<sub>22</sub>H<sub>29</sub>F<sub>2</sub>N<sub>3</sub>O<sub>9</sub>P<sup>+</sup> (M + H)<sup>+</sup>, 548.1604]; Anal. Calcd for C<sub>22</sub>H<sub>28</sub>F<sub>2</sub>N<sub>3</sub>O<sub>9</sub>P: C, 48.27; H, 5.16; N, 7.68. Found:
C, 48.12; H, 4.98; 8.01.</p></sec></sec><sec id="sec4.2"><title>SAH Hydrolase Assay<sup><xref ref-type="bibr" rid="cit18e">18e</xref>−<xref ref-type="bibr" rid="cit18g">18g</xref>,<xref ref-type="bibr" rid="ref30">30</xref></sup></title><p>The gene encoding human
placental SAH hydrolase was cloned
into expression plasmid pPROKcd20. Recombinant SAH hydrolase protein
was produced in <italic>E. coli</italic> JM109 in 50
mM Tris-HCl (pH 7.5) containing 2 mM ethylenediaminetetraacetic acid
and was purified by DEAE-cellulose column (2.8 cm × 6 cm), ammonium
sulfate fractionation (35–60%), Sephacryl S-300HR (1.0 cm ×
105 cm), and DEAE cellulose (2.8 cm × 24 cm). The protein homogeneity
was confirmed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis.
The protein concentration was determined by using the Bradford method.
Bovine serum albumin was a standard material for protein assay. Enzyme
activity was determined in reaction mixtures (250 μL) that contain
50 mM sodium phosphate (pH 8.0), 2 μM SAH hydrolase (0.5 μM
tetrameric form), and varying concentrations of compounds. The reaction
mixtures were first preincubated with the compounds for 10 min at
37 °C, after which the reaction was initiated by adding 100 μM
SAH. The reaction was allowed to proceed for 20 min, followed by the
addition of 5,5′-dithiobis-2-nitrobenzoate (DNTB) to a final
concentration of 200 μM. The absorbance of the product 5-thio-2-nitrobenzoic
acid (TNB) was measured at 412 nm using a spectrophotometer (Varian,
Cary 100). The molar extinction coefficient for TNB (ε<sub>412</sub> = 13 700 M<sup>–1</sup> cm<sup>–1</sup>) was
used in calculations to quantify TNB formation.</p></sec><sec id="sec4.3"><title>Cells, Viruses,
and Compounds</title><p>Vero E6 and Vero CCL81
cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM; Lonza), supplemented with 8% fetal calf serum (FCS;
PAA), 2 mM <sc>l</sc>-glutamine, 100 IU/mL of penicillin and 100 μg/mL
of streptomycin, and were grown at 37 °C in a humidified incubator
with 5% CO<sub>2</sub>. Vero cells were maintained in Eagle’s
minimum essential medium (EMEM; Lonza), supplemented with 8% FCS (FCS;
PAA), 100 IU/mL of penicillin and 100 μg/mL of streptomycin,
and were grown at 37 °C in a humidified incubator with 5% CO<sub>2</sub>. Infections were performed in EMEM with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (Lonza) supplemented with 2% FCS, <sc>l</sc>-glutamine, and antibiotics.
Infectious clone-derived CHIKV(CHIKV-LS3) was generated as described
by Scholte et al.<sup><xref ref-type="bibr" rid="ref31">31</xref></sup> The ZIKV strain SL0612
was isolated from an infected traveler returning from Suriname as
described by van Boheemen et al.<sup><xref ref-type="bibr" rid="ref32">32</xref></sup> The
Sindbis virus (SINV) strain HR and Semliki forest virus (SFV) strain
SFV4 are part of the LUMC virus collection. The MERS-CoV strain EMC/2012
was isolated from patient material in the Dr. Soliman Fakeeh Hospital,
Jeddah, Saudi Arabia, and was obtained from Erasmus Medical Center,
Rotterdam.<sup><xref ref-type="bibr" rid="ref33">33</xref></sup> The SARS-CoV strain Frankfurt
1 was provided by H. F. Rabenau and H. W. Doerr (Johann Wolfgang Goethe-Universität,
Frankfurt am Main, Germany).<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> The compounds
were dissolved in dimethylsulfoxide to obtain 20 mM stock solutions.
All work with infectious CHIKV, MERS-CoV, SARS-CoV, and ZIKV was performed
inside biosafety cabinets in the BSL-3 facilities of the Leiden University
Medical Center.</p></sec><sec id="sec4.4"><title>Antiviral CPE-Reduction Assays</title><p>VeroE6
cells were seeded
at a density of 5000 cells/well (CHIKV) and 10 000 cells/well
(SARS-CoV, SFV and SINV) in a total volume of 100 μL per well
in 96-well plates. Vero cells were seeded at a density of 20 000
cells/well when used for MERS-CoV infections, and Vero CCL81 cells
were seeded at a density of 5000 cells/well for ZIKA infections under
the same conditions as described for Vero E6. The following day, compound
dilutions with concentrations of 150, 50, 16.7, and 5.6 μM were
prepared in the infection medium by 3-fold serial dilution of the
150 μM solution. After replacing the culture medium with the
respective dilutions of the compound, the cells were infected with
CHIKV (MOI 0.005), SFV (MOI 0.025), SINV (MOI 0.025), ZIKV (MOI 0.05),
MERS-CoV (MOI 0.005), or SARS-CoV (MOI 0.01). Viability assays were
conducted in parallel. Each compound was tested at each concentration
in quadruplicate (4 biological replicates per concentration). An MTS
colorimetric assay was conducted 40 hpi for SFV, 76 hpi for SINV,
72 h hpi for MERS- and SARS-CoV, and 96 hpi for CHIKV and ZIKV by
adding 20 μL/well of the CellTiter 96 AQueous One Solution Cell
Proliferation Assay (MTS) reagent (Promega). The assay was stopped
after 2–2.5 h by fixing the cells with 37% formaldehyde. The
absorbance was measured at 495 nm in a Berthold Mithras LB 940 plate
reader, and the values were expressed relative to uninfected (infection)
or untreated (viability) samples. The results represent the average
of quadruplicate samples expressed as the mean ± SD. Compounds
that were found to be protective were further evaluated in CPE reduction
assays by testing 8 different concentrations to determine the EC<sub>50</sub> as previously described.<sup><xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="ref34">34</xref></sup> The cytotoxicity
(CC<sub>50</sub>) of the compounds was determined in parallel, and
all experiments were performed in quadruplicate. Graph-Pad Prism 8.0.1
was used for EC<sub>50</sub> and CC<sub>50</sub> determination by
nonlinear regression.</p></sec><sec id="sec4.5"><title>Viral Load Reduction Assays</title><p>VeroE6
(CHIKV, ZIKV) cells
were seeded at a density of 7.5 × 10<sup>4</sup> cells/well in
0.5 mL DMEM/8%FCS in 24-well cell culture plates and allowed to adhere
overnight. For MERS-CoV and SARS-CoV, a cell density of or 6.0 ×
10<sup>4</sup> cells/well of Vero E6 and Vero cells was used, respectively,
under the same conditions as described above. The next day, compound
dilutions (0–1.5 μM) were prepared in EMEM/2%FCS to which
virus was added to yield inocula for infecting the cells with an MOI
of 0.1 for CHIKV, MOI of 1 for ZIKV, and MOI of 0.01 for SARS- and
MERS-CoV. Cells were incubated at 37 °C with 250 μL/well
of the inoculum for 1 h (CHIKV and SARS- and MERS-CoV) or 2 h (ZIKV).
After the infection, the cells were washed twice with 1 mL/well warm
phosphate-buffered saline and 0.5 mL/well fresh EMEM/2%FCS with different
concentrations of compound <bold>2c</bold> (0–1.5 μM)
was added. The cells were incubated for 30 h (CHIKV) or 48 h (ZIKV,
SARS- and MERS-CoV) at 37 °C, after which supernatants were harvested
and stored at −80 °C for determination of the infectious
virus titer by plaque assay. Viability assays were conducted in parallel
as described in the previous paragraph. Plaque assays with CHIKV and
SARS-CoV on VeroE6 cells, MERS-CoV on Vero cells, and ZIKV on Vero
CCL81 cells were performed as described previously.<sup><xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="cit34a">34a</xref>,<xref ref-type="bibr" rid="ref35">35</xref></sup> Compound <bold>2c</bold> was tested at each
concentration in duplicate in two independent experiments (<italic>n</italic> = 4). Graph-Pad Prism 8.0.1 was used for statistical analysis
with a one-way ANOVA multiple comparison test.</p></sec></sec></body><back><notes id="notes1" notes-type="si"><title>Supporting Information Available</title><p>The Supporting Information is
available free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications website</ext-link> at DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.9b00781">10.1021/acs.jmedchem.9b00781</ext-link>.<list id="silist" list-type="simple"><list-item><p><sup>1</sup>H
and <sup>13</sup>C NMR copies of all final
compounds <bold>2a–j</bold> and <bold>3a–c</bold> (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b00781/suppl_file/jm9b00781_si_001.pdf">PDF</ext-link>)</p></list-item><list-item><p>Crystallographic
data for <bold>2c</bold>, <bold>2g</bold>, and <bold>2h</bold> (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b00781/suppl_file/jm9b00781_si_002.zip">ZIP</ext-link>)</p></list-item><list-item><p>Molecular formula
strings (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b00781/suppl_file/jm9b00781_si_003.csv">CSV</ext-link>)</p></list-item></list></p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="jm9b00781_si_001.pdf"><caption><p>jm9b00781_si_001.pdf</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="sifile2"><media xlink:href="jm9b00781_si_002.zip"><caption><p>jm9b00781_si_002.zip</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="sifile3"><media xlink:href="jm9b00781_si_003.csv"><caption><p>jm9b00781_si_003.csv</p></caption></media></supplementary-material></sec><notes notes-type="" id="notes2"><title>Author Contributions</title><p>J.-s.Y.
and
G.K. contributed equally to this work. All authors have contributed
to the manuscript and given approval to the final version of the manuscript.</p></notes><notes notes-type="COI-statement" id="NOTES-d7e9107-autogenerated"><p>The authors declare no
competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>This research was supported by grants from
Mid-career
Research Program (2016R1A2B3010164) and the Ministry of Science, ICT
& Future Planning (2017M3A9A8032086) of the National Research
Foundation (NRF), Korea. The work in Leiden (N.S.O. and K.K.) was
supported by the EU Marie Skłodowska-Curie ETN “ANTIVIRALS”
(grant agreement no. 642434).</p></ack><glossary id="dl1"><def-list><title>Abbreviations</title><def-item><term>RdRp</term><def><p>RNA-dependent RNA polymerase</p></def></def-item><def-item><term>SAH</term><def><p><italic>S</italic>-adenosyl-<sc>l</sc>-homocysteine</p></def></def-item><def-item><term>SARS-CoV</term><def><p>severe acute respiratory syndrome coronavirus</p></def></def-item><def-item><term>CHIKV</term><def><p>chikungunya virus</p></def></def-item><def-item><term>ZIKV</term><def><p>Zika virus</p></def></def-item><def-item><term>nsps</term><def><p>nonstructural proteins</p></def></def-item><def-item><term>MTase</term><def><p>methyltransferase</p></def></def-item><def-item><term>NTP</term><def><p>nucleoside triphosphate</p></def></def-item><def-item><term>SAM</term><def><p><italic>S</italic>-adenosyl-<sc>l</sc>-methionine</p></def></def-item><def-item><term>AK</term><def><p>adenosine kinase</p></def></def-item><def-item><term>LiHMDS</term><def><p>lithium hexamethyldisilazide</p></def></def-item><def-item><term>TESCl</term><def><p>triethylsilyl chloride</p></def></def-item><def-item><term>NFSI</term><def><p><italic>N</italic>-fluorobenzenesulfonimide</p></def></def-item><def-item><term>NFOBS</term><def><p><italic>N</italic>-fluoro-<italic>O</italic>-benzenedisulfonimide</p></def></def-item><def-item><term>NaBH<sub>4</sub></term><def><p>sodium borohydride</p></def></def-item><def-item><term>LiBH<sub>4</sub></term><def><p>lithium
borohydride</p></def></def-item><def-item><term>NMO</term><def><p><italic>N</italic>-methylmorpholine-<italic>N</italic>-oxide</p></def></def-item><def-item><term>TBS</term><def><p><italic>t</italic>-butyldimethylsilyl</p></def></def-item><def-item><term>TBAF</term><def><p>tetra-<italic>n</italic>-butylammonium fluoride</p></def></def-item><def-item><term>DAST</term><def><p><italic>N</italic>,<italic>N</italic>-diethylaminosulfur
trifluoride</p></def></def-item><def-item><term>AlMe<sub>3</sub></term><def><p>trimethylaluminum</p></def></def-item><def-item><term>SOCl<sub>2</sub></term><def><p>thionyl chloride</p></def></def-item><def-item><term>DIPEA</term><def><p><italic>N</italic>,<italic>N</italic>-diisopropylethylamine</p></def></def-item><def-item><term>TFA</term><def><p>trifluoroacetic
acid</p></def></def-item><def-item><term>Boc<sub>2</sub>O</term><def><p>di-<italic>tert</italic>-butyl dicarbonate</p></def></def-item><def-item><term>DNTB</term><def><p>5,5′-dithiobis-2-nitrobenzoate</p></def></def-item><def-item><term>CPE</term><def><p>cytopathic effect</p></def></def-item><def-item><term>TMSOTf</term><def><p>trimethylsilyl
trifluoromethanesulfonate</p></def></def-item><def-item><term>DMEM</term><def><p>Dulbecco’s modified Eagle’s medium</p></def></def-item><def-item><term>FCS</term><def><p>fetal calf serum</p></def></def-item><def-item><term>NEAA</term><def><p>non-essential amino
acid</p></def></def-item><def-item><term>EMEM</term><def><p>Eagle’s
minimum essential medium</p></def></def-item><def-item><term>SINV</term><def><p>Sindbis virus</p></def></def-item><def-item><term>SFV</term><def><p>Semliki forest virus</p></def></def-item></def-list></glossary><ref-list><title>References</title><ref id="ref1"><mixed-citation publication-type="journal" id="cit1a"><label>a</label><name><surname>Baltimore</surname><given-names>D.</given-names></name><article-title>Expression of animal virus genomes</article-title>. <source>Bacteriol. Rev.</source><year>1971</year>, <volume>35</volume>, <fpage>235</fpage>–<lpage>241</lpage>.<pub-id pub-id-type="pmid">4329869</pub-id></mixed-citation><mixed-citation publication-type="book" id="cit1b"><label>b</label><person-group person-group-type="allauthors"><name><surname>Modrow</surname><given-names>S.</given-names></name>; <name><surname>Falke</surname><given-names>D.</given-names></name>; <name><surname>Truyen</surname><given-names>U.</given-names></name>; <name><surname>Schätzl</surname><given-names>H.</given-names></name></person-group><source>Viruses with Single-Stranded, Positive-Sense
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