Xanthine-based acyclic nucleoside phosphonates with potent antiviral activity against varicella-zoster virus and human cytomegalovirus
Identifieur interne : 000809 ( Pmc/Corpus ); précédent : 000808; suivant : 000810Xanthine-based acyclic nucleoside phosphonates with potent antiviral activity against varicella-zoster virus and human cytomegalovirus
Auteurs : Ond Ej Baszczy Ski ; Martin Maxmilian Kaiser ; Michal Esnek ; Petra B Ehová ; Petr Jansa ; Eliška Procházková ; Martin Dra Nsk ; Robert Snoeck ; Graciela Andrei ; Zlatko JanebaSource :
- Antiviral Chemistry & Chemotherapy [ 0956-3202 ] ; 2018.
Abstract
While noncanonic xanthine nucleotides XMP/dXMP play an important role in balancing and maintaining intracellular purine nucleotide pool as well as in potential mutagenesis, surprisingly, acyclic nucleoside phosphonates bearing a xanthine nucleobase have not been studied so far for their antiviral properties. Herein, we report the synthesis of a series of xanthine-based acyclic nucleoside phosphonates and evaluation of their activity against a wide range of DNA and RNA viruses. Two acyclic nucleoside phosphonates within the series, namely 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX) and 9-[3-hydroxy-2-(phosphonomethoxy)propyl]xanthine (HPMPX), were shown to possess activity against several human herpesviruses. The most potent compound was PMEX, a xanthine analogue of adefovir (PMEA). PMEX exhibited a single digit µM activity against VZV (EC50 = 2.6 µM, TK+ Oka strain) and HCMV (EC50 = 8.5 µM, Davis strain), while its hexadecyloxypropyl monoester derivative was active against HSV-1 and HSV-2 (EC50 values between 1.8 and 4.0 µM). In contrast to acyclovir, PMEX remained active against the TK– VZV 07–1 strain with EC50 = 4.58 µM. PMEX was suggested to act as an inhibitor of viral DNA polymerase and represents the first reported xanthine-based acyclic nucleoside phosphonate with potent antiviral properties.
Url:
DOI: 10.1177/2040206618813050
PubMed: 30497281
PubMed Central: 6287304
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Xanthine-based acyclic nucleoside phosphonates with potent antiviral activity against varicella-zoster virus and human cytomegalovirus</title><author><name sortKey="Baszczy Ski, Ond Ej" sort="Baszczy Ski, Ond Ej" uniqKey="Baszczy Ski O" first="Ond Ej" last="Baszczy Ski">Ond Ej Baszczy Ski</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Kaiser, Martin Maxmilian" sort="Kaiser, Martin Maxmilian" uniqKey="Kaiser M" first="Martin Maxmilian" last="Kaiser">Martin Maxmilian Kaiser</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey=" Esnek, Michal" sort=" Esnek, Michal" uniqKey=" Esnek M" first="Michal" last=" Esnek">Michal Esnek</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="B Ehova, Petra" sort="B Ehova, Petra" uniqKey="B Ehova P" first="Petra" last="B Ehová">Petra B Ehová</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Jansa, Petr" sort="Jansa, Petr" uniqKey="Jansa P" first="Petr" last="Jansa">Petr Jansa</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Prochazkova, Eliska" sort="Prochazkova, Eliska" uniqKey="Prochazkova E" first="Eliška" last="Procházková">Eliška Procházková</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Dra Nsk, Martin" sort="Dra Nsk, Martin" uniqKey="Dra Nsk M" first="Martin" last="Dra Nsk">Martin Dra Nsk</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Snoeck, Robert" sort="Snoeck, Robert" uniqKey="Snoeck R" first="Robert" last="Snoeck">Robert Snoeck</name><affiliation><nlm:aff id="aff2-2040206618813050">Laboratory of Virology and Chemotheraphy, Rega Institute, Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="Andrei, Graciela" sort="Andrei, Graciela" uniqKey="Andrei G" first="Graciela" last="Andrei">Graciela Andrei</name><affiliation><nlm:aff id="aff2-2040206618813050">Laboratory of Virology and Chemotheraphy, Rega Institute, Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="Janeba, Zlatko" sort="Janeba, Zlatko" uniqKey="Janeba Z" first="Zlatko" last="Janeba">Zlatko Janeba</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">30497281</idno><idno type="pmc">6287304</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287304</idno><idno type="RBID">PMC:6287304</idno><idno type="doi">10.1177/2040206618813050</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000809</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000809</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Xanthine-based acyclic nucleoside phosphonates with potent antiviral activity against varicella-zoster virus and human cytomegalovirus</title><author><name sortKey="Baszczy Ski, Ond Ej" sort="Baszczy Ski, Ond Ej" uniqKey="Baszczy Ski O" first="Ond Ej" last="Baszczy Ski">Ond Ej Baszczy Ski</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Kaiser, Martin Maxmilian" sort="Kaiser, Martin Maxmilian" uniqKey="Kaiser M" first="Martin Maxmilian" last="Kaiser">Martin Maxmilian Kaiser</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey=" Esnek, Michal" sort=" Esnek, Michal" uniqKey=" Esnek M" first="Michal" last=" Esnek">Michal Esnek</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="B Ehova, Petra" sort="B Ehova, Petra" uniqKey="B Ehova P" first="Petra" last="B Ehová">Petra B Ehová</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Jansa, Petr" sort="Jansa, Petr" uniqKey="Jansa P" first="Petr" last="Jansa">Petr Jansa</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Prochazkova, Eliska" sort="Prochazkova, Eliska" uniqKey="Prochazkova E" first="Eliška" last="Procházková">Eliška Procházková</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Dra Nsk, Martin" sort="Dra Nsk, Martin" uniqKey="Dra Nsk M" first="Martin" last="Dra Nsk">Martin Dra Nsk</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author><author><name sortKey="Snoeck, Robert" sort="Snoeck, Robert" uniqKey="Snoeck R" first="Robert" last="Snoeck">Robert Snoeck</name><affiliation><nlm:aff id="aff2-2040206618813050">Laboratory of Virology and Chemotheraphy, Rega Institute, Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="Andrei, Graciela" sort="Andrei, Graciela" uniqKey="Andrei G" first="Graciela" last="Andrei">Graciela Andrei</name><affiliation><nlm:aff id="aff2-2040206618813050">Laboratory of Virology and Chemotheraphy, Rega Institute, Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="Janeba, Zlatko" sort="Janeba, Zlatko" uniqKey="Janeba Z" first="Zlatko" last="Janeba">Zlatko Janeba</name><affiliation><nlm:aff id="aff1-2040206618813050">Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</nlm:aff></affiliation></author></analytic><series><title level="j">Antiviral Chemistry & Chemotherapy</title><idno type="ISSN">0956-3202</idno><idno type="eISSN">2040-2066</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>While noncanonic xanthine nucleotides XMP/dXMP play an important role in balancing and maintaining intracellular purine nucleotide pool as well as in potential mutagenesis, surprisingly, acyclic nucleoside phosphonates bearing a xanthine nucleobase have not been studied so far for their antiviral properties. Herein, we report the synthesis of a series of xanthine-based acyclic nucleoside phosphonates and evaluation of their activity against a wide range of DNA and RNA viruses. Two acyclic nucleoside phosphonates within the series, namely 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX) and 9-[3-hydroxy-2-(phosphonomethoxy)propyl]xanthine (HPMPX), were shown to possess activity against several human herpesviruses. The most potent compound was PMEX, a xanthine analogue of adefovir (PMEA). PMEX exhibited a single digit µM activity against VZV (EC<sub>50</sub> = 2.6 µM, TK<sup>+</sup> Oka strain) and HCMV (EC<sub>50</sub> = 8.5 µM, Davis strain), while its hexadecyloxypropyl monoester derivative was active against HSV-1 and HSV-2 (EC<sub>50</sub> values between 1.8 and 4.0 µM). In contrast to acyclovir, PMEX remained active against the TK<sup>–</sup> VZV 07–1 strain with EC<sub>50</sub> = 4.58 µM. PMEX was suggested to act as an inhibitor of viral DNA polymerase and represents the first reported xanthine-based acyclic nucleoside phosphonate with potent antiviral properties.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bennett, Bd" uniqKey="Bennett B">BD Bennett</name></author><author><name sortKey="Kimball, Eh" uniqKey="Kimball E">EH Kimball</name></author><author><name sortKey="Gao, M" uniqKey="Gao M">M Gao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wheeler, Lj" uniqKey="Wheeler L">LJ Wheeler</name></author><author><name sortKey="Rajagopal, I" uniqKey="Rajagopal I">I Rajagopal</name></author><author><name sortKey="Mathews, Ck" uniqKey="Mathews C">CK. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Antivir Chem Chemother</journal-id><journal-id journal-id-type="iso-abbrev">Antivir. Chem. Chemother</journal-id><journal-id journal-id-type="publisher-id">AVC</journal-id><journal-id journal-id-type="hwp">spavc</journal-id><journal-title-group><journal-title>Antiviral Chemistry & Chemotherapy</journal-title></journal-title-group><issn pub-type="ppub">0956-3202</issn><issn pub-type="epub">2040-2066</issn><publisher><publisher-name>SAGE Publications</publisher-name><publisher-loc>Sage UK: London, England</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">30497281</article-id><article-id pub-id-type="pmc">6287304</article-id><article-id pub-id-type="doi">10.1177/2040206618813050</article-id><article-id pub-id-type="publisher-id">10.1177_2040206618813050</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Xanthine-based acyclic nucleoside phosphonates with potent antiviral activity against varicella-zoster virus and human cytomegalovirus</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Baszczyňski</surname><given-names>Ondřej</given-names></name><xref ref-type="aff" rid="aff1-2040206618813050">1</xref></contrib><contrib contrib-type="author"><name><surname>Kaiser</surname><given-names>Martin Maxmilian</given-names></name><xref ref-type="aff" rid="aff1-2040206618813050">1</xref></contrib><contrib contrib-type="author"><name><surname>Česnek</surname><given-names>Michal</given-names></name><xref ref-type="aff" rid="aff1-2040206618813050">1</xref></contrib><contrib contrib-type="author"><name><surname>Břehová</surname><given-names>Petra</given-names></name><xref ref-type="aff" rid="aff1-2040206618813050">1</xref></contrib><contrib contrib-type="author"><name><surname>Jansa</surname><given-names>Petr</given-names></name><xref ref-type="aff" rid="aff1-2040206618813050">1</xref></contrib><contrib contrib-type="author"><name><surname>Procházková</surname><given-names>Eliška</given-names></name><xref ref-type="aff" rid="aff1-2040206618813050">1</xref></contrib><contrib contrib-type="author"><name><surname>Dračínský</surname><given-names>Martin</given-names></name><xref ref-type="aff" rid="aff1-2040206618813050">1</xref></contrib><contrib contrib-type="author"><name><surname>Snoeck</surname><given-names>Robert</given-names></name><xref ref-type="aff" rid="aff2-2040206618813050">2</xref></contrib><contrib contrib-type="author"><name><surname>Andrei</surname><given-names>Graciela</given-names></name><xref ref-type="aff" rid="aff2-2040206618813050">2</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="false">https://orcid.org/0000-0003-4654-679X</contrib-id><name><surname>Janeba</surname><given-names>Zlatko</given-names></name><xref ref-type="aff" rid="aff1-2040206618813050">1</xref><xref ref-type="corresp" rid="corresp1-2040206618813050"></xref></contrib><aff id="aff1-2040206618813050"><label>1</label>Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic</aff><aff id="aff2-2040206618813050"><label>2</label>Laboratory of Virology and Chemotheraphy, Rega Institute, Leuven, Belgium</aff></contrib-group><author-notes><corresp id="corresp1-2040206618813050">Zlatko Janeba, Institute of Organic Chemistry and Biochemistry CAS, Flemingovo náměstí 542/2, Prague 16610, Czech Republic. Email: <email>janeba@uochb.cas.cz</email></corresp></author-notes><pub-date pub-type="epub"><day>29</day><month>11</month><year>2018</year></pub-date><pub-date pub-type="collection"><year>2018</year></pub-date><volume>26</volume><elocation-id>2040206618813050</elocation-id><history><date date-type="received"><day>7</day><month>8</month><year>2018</year></date><date date-type="accepted"><day>15</day><month>10</month><year>2018</year></date></history><permissions><copyright-statement>© The Author(s) 2018</copyright-statement><copyright-year>2018</copyright-year><copyright-holder content-type="sage">SAGE Publications</copyright-holder><license license-type="creative-commons" xlink:href="http://creativecommons.org/licenses/by-nc/4.0/"><license-p>Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (<ext-link ext-link-type="uri" xlink:href="http://www.creativecommons.org/licenses/by-nc/4.0/">http://www.creativecommons.org/licenses/by-nc/4.0/</ext-link>) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (<ext-link ext-link-type="uri" xlink:href="https://us.sagepub.com/en-us/nam/open-access-at-sage">https://us.sagepub.com/en-us/nam/open-access-at-sage</ext-link>).</license-p></license></permissions><abstract abstract-type="short"><p>While noncanonic xanthine nucleotides XMP/dXMP play an important role in balancing and maintaining intracellular purine nucleotide pool as well as in potential mutagenesis, surprisingly, acyclic nucleoside phosphonates bearing a xanthine nucleobase have not been studied so far for their antiviral properties. Herein, we report the synthesis of a series of xanthine-based acyclic nucleoside phosphonates and evaluation of their activity against a wide range of DNA and RNA viruses. Two acyclic nucleoside phosphonates within the series, namely 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX) and 9-[3-hydroxy-2-(phosphonomethoxy)propyl]xanthine (HPMPX), were shown to possess activity against several human herpesviruses. The most potent compound was PMEX, a xanthine analogue of adefovir (PMEA). PMEX exhibited a single digit µM activity against VZV (EC<sub>50</sub> = 2.6 µM, TK<sup>+</sup> Oka strain) and HCMV (EC<sub>50</sub> = 8.5 µM, Davis strain), while its hexadecyloxypropyl monoester derivative was active against HSV-1 and HSV-2 (EC<sub>50</sub> values between 1.8 and 4.0 µM). In contrast to acyclovir, PMEX remained active against the TK<sup>–</sup> VZV 07–1 strain with EC<sub>50</sub> = 4.58 µM. PMEX was suggested to act as an inhibitor of viral DNA polymerase and represents the first reported xanthine-based acyclic nucleoside phosphonate with potent antiviral properties.</p></abstract><kwd-group><kwd>Acyclic nucleoside phosphonates</kwd><kwd>xanthine</kwd><kwd>PMEX</kwd><kwd>antiviral</kwd><kwd>HCMV</kwd><kwd>VZV</kwd></kwd-group><funding-group><award-group id="award1-2040206618813050"><funding-source id="funding1-2040206618813050"><institution-wrap><institution>Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences</institution><institution-id institution-id-type="FundRef"></institution-id></institution-wrap></funding-source><award-id rid="funding1-2040206618813050">RVO61388963</award-id></award-group></funding-group><custom-meta-group><custom-meta><meta-name>cover-date</meta-name><meta-value>January-December 2018</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="sec1-2040206618813050"><title>Introduction</title><p>The concentration and ratio of purine nucleotides and deoxynucleotides in the nucleotide pool is highly regulated in order to maintain the proper function and genetic stability of mammalian cells.<sup><xref rid="bibr1-2040206618813050" ref-type="bibr">1</xref></sup> Imbalances in (deoxy)nucleotide pool may have mutagenic consequences<sup><xref rid="bibr2-2040206618813050" ref-type="bibr">2</xref></sup> and may lead to various diseases, such as combined immunodeficiency (loss of purine nucleoside phosphorylase (PNP) function),<sup><xref rid="bibr3-2040206618813050" ref-type="bibr">3</xref></sup> hyperuricemia (loss of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) function)<sup><xref rid="bibr4-2040206618813050" ref-type="bibr">4</xref></sup> or cancer (uncontrollable activity of inosine-5′-monophosphate dehydrogenase, IMPDH).<sup><xref rid="bibr5-2040206618813050" ref-type="bibr">5</xref></sup></p><p>Xanthosine monophosphate (XMP, <bold>1</bold>, <xref ref-type="fig" rid="fig1-2040206618813050">Figure 1</xref>) is an important intermediate in the <italic>de novo</italic> synthesis of guanine nucleotides and its concentration is essential for the maintenance of guanine nucleotide pool,<sup><xref rid="bibr6-2040206618813050" ref-type="bibr">6</xref></sup> where XMP serves as a substrate for guanosine monophosphate synthase,<sup><xref rid="bibr7-2040206618813050" ref-type="bibr">7</xref></sup> that produces guanosine monophosphate (GMP). XMP is formed either from inosine monophosphate (IMP) by IMPDH or <italic>via</italic> salvage pathway using hypoxanthine or xanthine phosphoribosyltransferase. The XMP level is regulated by 5′-nucleotidase that hydrolyzes XMP to xanthosine.</p><fig id="fig1-2040206618813050" orientation="portrait" position="float"><label>Figure 1.</label><caption><p>(a) Xanthine-based nucleotides; (b) cidofovir (an example of acyclic nucleoside phosphonate, ANP); (c) target xanthine-based ANPs.</p></caption><graphic xlink:href="10.1177_2040206618813050-fig1"></graphic></fig><p>In contrast, the corresponding deoxyribonucleotide analogues, dXMP (<bold>2</bold>, <xref ref-type="fig" rid="fig1-2040206618813050">Figure 1</xref>) and dXTP, are catabolic products of dGMP and dGTP enzymatic hydrolysis, or can be formed by defective purine nucleotide metabolism (involving deaminase enzymes),<sup><xref rid="bibr8-2040206618813050" ref-type="bibr">8</xref></sup> or by chemical hydrolysis<sup><xref rid="bibr9-2040206618813050" ref-type="bibr">9</xref></sup> of dGMP/dGTP <italic>via</italic> NO<sub>x</sub>-mediated nitrosative stress.<sup><xref rid="bibr10-2040206618813050" ref-type="bibr">10</xref></sup> These processes can lead to a substantial incorporation of xanthine nucleotides into DNA and/or RNA,<sup><xref rid="bibr8-2040206618813050" ref-type="bibr">8</xref></sup> and subsequently to RNA miscoding and mutagenesis.<sup><xref rid="bibr11-2040206618813050" ref-type="bibr">11</xref></sup> Moreover, deaminated nucleotides can interfere with RNA editing<sup><xref rid="bibr12-2040206618813050" ref-type="bibr">12</xref></sup> and with functions of noncoding RNAs.<sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup></p><p>Under cell physiological homeostasis, the concentration and ratio of potentially mutagenic nucleotide intermediates, such as (d)IDP/(d)ITP/(d)XTP, is maintained by housekeeping enzymes,<sup><xref rid="bibr14-2040206618813050" ref-type="bibr">14</xref></sup> especially those from nudix family such as ITPases/XTPpases,<sup><xref rid="bibr15-2040206618813050" ref-type="bibr">15</xref>,<xref rid="bibr16-2040206618813050" ref-type="bibr">16</xref></sup> NUDT<sup><xref rid="bibr16-2040206618813050" ref-type="bibr">16</xref></sup> or ITPA,<sup><xref rid="bibr17-2040206618813050" ref-type="bibr">17</xref></sup> that can hydrolyze corresponding nucleoside di- or triphosphates. The main function of housekeeping enzymes is to prevent or minimize the incorporation of noncanonical nucleotides into DNA/RNA. Unfortunately, the literature on housekeeping enzymes hydrolyzing dXDP/dXTP has been quite rare up to date.</p><p>Herpesviruses<sup><xref rid="bibr18-2040206618813050" ref-type="bibr">18</xref></sup> are DNA-containing enveloped viruses from large <italic>Herpesviridae</italic> family and include herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). Although current anti-herpetic therapy uses powerful antivirotics such as nucleoside analogues (acyclovir (ACV), penciclovir, vidarabine, and ganciclovir (GCV)), acyclic nucleoside phosphonate (ANP) cidofovir (CDV) (<bold>3</bold>, <xref ref-type="fig" rid="fig1-2040206618813050">Figure 1</xref>),<sup><xref rid="bibr19-2040206618813050" ref-type="bibr">19</xref></sup> or diphosphate mimic foscarnet,<sup><xref rid="bibr20-2040206618813050" ref-type="bibr">20</xref></sup> many drug insensitive viruses have been identified in the clinics. The origin of virus resistance for HSV, VZV or CMV comes mostly from treatment using DNA polymerase inhibitors, such as ACV and GCV, where various alterations in the viral thymidine kinase gene [<italic>UL23</italic> (HSV) and <italic>ORF36</italic> (VZV)], protein kinase [<italic>UL97</italic> (CMV)] and/or viral DNA polymerase gene [<italic>UL30 (HSV), ORF28</italic> (VZV) and <italic>UL54</italic> (CMV)] may occur.<sup><xref rid="bibr21-2040206618813050" ref-type="bibr">21</xref><xref rid="bibr22-2040206618813050" ref-type="bibr"></xref>–<xref rid="bibr23-2040206618813050" ref-type="bibr">23</xref></sup> As recent literature has shown,<sup><xref rid="bibr24-2040206618813050" ref-type="bibr">24</xref></sup> the presence of resistant herpesviruses should be considered seriously not only in the case of immunocompromised individuals. Evidently, there is an urgent need for novel potent anti-herpetic agents with high barrier of resistance development.</p><p>ANPs,<sup><xref rid="bibr25-2040206618813050" ref-type="bibr">25</xref></sup> mimics of natural nucleotides (avoiding the first phosphorylation step), represent a potent group of antiviral agents. ANPs are converted inside the cells to their diphosphates (ANPpp) that target DNA polymerase – viral and/or cellular.<sup><xref rid="bibr19-2040206618813050" ref-type="bibr">19</xref></sup> These nucleoside triphosphate analogues act as competitive inhibitors and/or alternative substrates of the respective enzymes, in the later case leading to termination of DNA chain elongation.<sup><xref rid="bibr19-2040206618813050" ref-type="bibr">19</xref></sup> Although some ANPs derived from xanthine were studied before as potential antiviral agents (namely the 9-[3-fluoro-2-(phosphonomethoxy)propyl] derivative, FPMPX),<sup><xref rid="bibr26-2040206618813050" ref-type="bibr">26</xref></sup> the general lack of interest in such compounds was probably caused by their relatively complicated synthesis, since simple alkylation of xanthine base was expected to give a mixture of several regioisomers as well as polyalkylated products. Recently, we have reported<sup><xref rid="bibr27-2040206618813050" ref-type="bibr">27</xref></sup> a simple and high-yielding synthesis of xanthine ANPs exploiting the MW-assisted hydrolysis of the corresponding 2,6-dichloropurine derivatives. Here, we report the synthesis and antiviral evaluation of a series of xanthine-based ANPs (compounds <bold>4</bold>–<bold>9</bold>, <xref ref-type="fig" rid="fig1-2040206618813050">Figure 1</xref>), designed as non-hydrolyzable analogues of dXMP/XMP.</p></sec><sec id="sec2-2040206618813050"><title>Chemistry</title><p>The synthesis of 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX, <bold>4</bold>, <xref ref-type="fig" rid="fig3-2040206618813050">Scheme 1</xref>), a xanthine analogue of the well-known antiviral agent adefovir (PMEA),<sup><xref rid="bibr28-2040206618813050" ref-type="bibr">28</xref></sup> has been reported by our group earlier.<sup><xref rid="bibr27-2040206618813050" ref-type="bibr">27</xref></sup> The microwave-assisted hydrolysis of 2,6-dichloropurine derivative <bold>10</bold><sup><xref rid="bibr29-2040206618813050" ref-type="bibr">29</xref></sup> in aqueous HCl afforded the desired xanthine compound <bold>4</bold> in a 85% yield.</p><fig fig-type="fig" id="fig3-2040206618813050" orientation="portrait" position="float"><label>Scheme 1.</label><caption><p>Preparation of PMEX (<bold>4</bold>). Reaction conditions: (a) 1 M aq. HCl, MW-assisted heating, 140 °C, 20 min.</p></caption><graphic xlink:href="10.1177_2040206618813050-fig3"></graphic></fig><p>For the synthesis of other target ANPs, compounds <bold>5</bold>–<bold>9</bold> (<xref ref-type="fig" rid="fig4-2040206618813050">Scheme 2</xref>), previously reported<sup><xref rid="bibr26-2040206618813050" ref-type="bibr">26</xref>,<xref rid="bibr30-2040206618813050" ref-type="bibr">30</xref><xref rid="bibr31-2040206618813050" ref-type="bibr"></xref><xref rid="bibr32-2040206618813050" ref-type="bibr"></xref><xref rid="bibr33-2040206618813050" ref-type="bibr"></xref>–<xref rid="bibr34-2040206618813050" ref-type="bibr">34</xref></sup> guanine containing ANPs <bold>11</bold>–<bold>15</bold>, have been exploited as a starting material. Standard diazotization of compounds <bold>11</bold>–<bold>15</bold> followed by 2-hydroxy-dediazoniation afforded the desired xanthine-based ANPs <bold>5</bold>–<bold>9</bold> in moderate to good yields (36–82%).</p><fig fig-type="fig" id="fig4-2040206618813050" orientation="portrait" position="float"><label>Scheme 2.</label><caption><p>Preparation of xanthine-based ANPs <bold>5</bold>–<bold>9</bold>. Reaction conditions: (a) isoamylnitrite, 80% aq. AcOH, 25 °C, 16 h.</p></caption><graphic xlink:href="10.1177_2040206618813050-fig4"></graphic></fig><p>Since PMEX (<bold>4</bold>) exhibited promising antiviral properties, we decided to prepare several PMEX prodrugs in order to improve the compound permeability which might be limited for this negatively charged compound. At first, <italic>N</italic><sup>6</sup>-cyclopropylaminopurine derivative <bold>17</bold> (<xref ref-type="fig" rid="fig5-2040206618813050">Scheme 3</xref>) was prepared in a 75% yield from the corresponding <italic>N</italic><sup>6</sup>-cyclopropyl-2,6-diaminopurine derivative <bold>16</bold>,<sup><xref rid="bibr35-2040206618813050" ref-type="bibr">35</xref></sup> using the above mentioned diazotization/2-hydroxy-dediazoniation procedure. Compound <bold>17</bold> was expected to be enzymatically converted (deaminated) to PMEX in an analogy to compound GS-9219 (an acyclic nucleotide analogue with potent antineoplastic activity),<sup><xref rid="bibr36-2040206618813050" ref-type="bibr">36</xref></sup> and abacavir (a carbocyclic nucleoside used for the treatment of HIV infection).<sup><xref rid="bibr37-2040206618813050" ref-type="bibr">37</xref></sup></p><fig fig-type="fig" id="fig5-2040206618813050" orientation="portrait" position="float"><label>Scheme 3.</label><caption><p>Synthesis of compound <bold>17</bold>. Reaction conditions: (a) isoamylnitrite, 80% aq. AcOH, 25 °C, 16 h.</p></caption><graphic xlink:href="10.1177_2040206618813050-fig5"></graphic></fig><p>Next, PMEX hexadecyloxypropyl (HDP) monoester <bold>18</bold> (<xref ref-type="fig" rid="fig6-2040206618813050">Scheme 4</xref>), a prodrug approach developed by Hostetler <italic>et al.</italic> as a mimic of natural lipids,<sup><xref rid="bibr38-2040206618813050" ref-type="bibr">38</xref></sup> was prepared from PMEX (<bold>4</bold>) and hexadecyloxypropyl alcohol <italic>via</italic> DCC-mediated coupling in a 23% yield. Similarly, phosphonate ester <bold>19</bold> (<xref ref-type="fig" rid="fig6-2040206618813050">Scheme 4</xref>) bearing a perfluorinated-C12 chain was prepared by the same procedure in a 30% yield. Finally, the bisamidate prodrug <bold>20</bold> (<xref ref-type="fig" rid="fig6-2040206618813050">Scheme 4</xref>) was obtained in a 13% yield starting from PMEX (<bold>4</bold>) and isopropyl ester of L-phenylalanine using the previously described procedure developed in our lab.<sup><xref rid="bibr39-2040206618813050" ref-type="bibr">39</xref></sup></p><fig fig-type="fig" id="fig6-2040206618813050" orientation="portrait" position="float"><label>Scheme 4.</label><caption><p>Synthesis of PMEX prodrugs <bold>18</bold>–<bold>20</bold>. Reaction conditions: (a) corresponding alcohol, pyridine, DCC, 100 °C, 16 h; (b) TMSBr, 25 °C, 16 h; (c) <italic>i</italic>Pr-L-PheAla.HCl, pyridine, Et<sub>3</sub>N, Aldrithiol-2, triphenylphosphine, 70 °C, 72 h.</p></caption><graphic xlink:href="10.1177_2040206618813050-fig6"></graphic></fig><p>In order to confirm the expected mode of action of PMEX (<bold>4</bold>), i.e. viral DNA polymerase inhibition, the corresponding phosphonodiphosphate <bold>21</bold> (<xref ref-type="fig" rid="fig7-2040206618813050">Scheme 5</xref>), as an analogue of natural nucleoside triphosphate, was also prepared. The two-step synthesis <italic>via</italic> a morpholidate intermediate<sup><xref rid="bibr40-2040206618813050" ref-type="bibr">40</xref></sup> afforded, after the HPLC purification, the desired triphosphate mimic <bold>21</bold> in a low (3%) yield.</p><fig fig-type="fig" id="fig7-2040206618813050" orientation="portrait" position="float"><label>Scheme 5.</label><caption><p>Synthesis of PMEX diphosphate <bold>21</bold>. Reaction conditions: (a) morpholine, DCC, <italic>t</italic>-BuOH, H<sub>2</sub>O, 105 °C, 16 h; (b) pyrophosphate (0.5 M in DMF), DMSO, 25 °C.</p></caption><graphic xlink:href="10.1177_2040206618813050-fig7"></graphic></fig></sec><sec id="sec3-2040206618813050"><title>Biology</title><p>The synthesized xanthine-based ANPs (compounds <bold>4</bold>–<bold>9</bold>) were evaluated for inhibitory activity against a wide range of DNA and RNA viruses: in human embryonic lung (HEL) cells (herpes simplex virus-1 (KOS strain), herpes simplex virus-2 (G strain), thymidine kinase deficient (ACV resistant) herpes simplex virus-1 (TK<sup>–</sup> KOS ACV<sup>r</sup>), vaccinia virus, vesicular stomatitis virus, human cytomegalovirus (HCMV) (AD-169 strain and Davis strains), VZV (TK<sup>+</sup> VZV strain and TK<sup>–</sup> VZV strains)), in HeLa cell cultures (vesicular stomatitis virus, Coxsackie virus B4 and respiratory syncytial virus (RSV)), in Vero cell cultures (para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, yellow fever virus), in CrFK cell cultures (feline corona virus (FIPV)), and in MDCK cell cultures (influenza A virus (H1N1 and H3N2 subtypes) and influenza B virus). GCV, CDV, ACV, brivudin (BVDU), zalcitabine, zanamivir, alovudine, amantadine, rimantadine, ribavirin, dextran sulfate (molecular weight 10000, DS-10000), mycophenolic acid, Hippeastrum hybrid agglutinin (HHA), and Urtica dioica agglutinin (UDA) were used as the reference compounds. The antiviral activity was expressed as the EC<sub>50</sub>, i.e. compound concentration required to decrease virus plaque formation (VZV) or virus-induced cytopathogenicity (other viruses) by 50%. While none of the compounds showed any activity against RNA viruses, compounds <bold>4</bold>, <bold>5</bold> and <bold>6</bold> were able to inhibit the replication of herpesviruses (<xref rid="table1-2040206618813050" ref-type="table">Tables 1</xref> and <xref rid="table2-2040206618813050" ref-type="table">2</xref>). PMEX (<bold>4</bold>) emerged as the most active compound against VZV and HCMV, being as active as the reference drug ACV against the TK<sup>+</sup> Oka strain (EC<sub>50</sub> = 2.62 µM (PMEX) <italic>versus</italic> 3.42 µM (ACV)). In contrast to ACV, compound <bold>4</bold> remained active against the TK<sup>–</sup> VZV 07–1 strain (EC<sub>50</sub> = 4.58 µM). PMEX also inhibited the replication of HCMV with EC<sub>50</sub> values of the same order of magnitude as the reference anti-HCMV drug GCV, while PMEX had a 50% cytostatic concentration of 111 µM for HEL cells. Thus, compound <bold>4</bold> was not only potent but also selective as the calculated selectivity indices (ratio CC<sub>50</sub>/EC<sub>50</sub>) were of 24 and 42 (VZV 07–1 and Oka strains, respectively) and of 11 and 13 (HCMV AD-169 and Davis strains, respectively). Compounds <bold>5</bold> and <bold>6</bold> were, respectively, 6 to 12 folds and 3 to 8 folds less active than PMEX against VZV and HCMV. However, compounds <bold>4</bold>, <bold>5</bold> and <bold>6</bold> inhibited the replication of HSV-1, HSV-2 and TK-HSV-1 at equivalent EC<sub>50</sub> values in the range of 16 to 39 µM. PMEX (<bold>4</bold>) lacked activity against vaccinia virus, while HPMPX compounds (both <italic>S</italic>-isomer <bold>6</bold> and racemic mixture <bold>5</bold>) were weak inhibitors of this poxvirus (EC<sub>50</sub> = 39 µM, <xref rid="table2-2040206618813050" ref-type="table">Table 2</xref>).</p><table-wrap id="table1-2040206618813050" orientation="portrait" position="float"><label>Table 1.</label><caption><p>Activity of compounds <bold>4</bold>–<bold>9</bold> against varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) in human embryonic lung (HEL) cells.</p></caption><alternatives><graphic specific-use="table1-2040206618813050" xlink:href="10.1177_2040206618813050-table1"></graphic><table frame="hsides" rules="groups"><thead valign="top"><tr><th rowspan="3" colspan="1">Compound</th><th colspan="4" rowspan="1">Antiviral activity EC<sub>50</sub> (µM)<sup><xref ref-type="table-fn" rid="table-fn1-2040206618813050">a</xref></sup><hr></hr></th><th colspan="2" rowspan="1">Cytotoxicity (μM)<hr></hr></th></tr><tr><th rowspan="1" colspan="1">TK<sup>+</sup> VZV<hr></hr></th><th rowspan="1" colspan="1">TK<sup>–</sup> VZV<hr></hr></th><th colspan="2" rowspan="1">HCMV<hr></hr></th><th rowspan="2" colspan="1">Cell morphology (MCC)<sup><xref ref-type="table-fn" rid="table-fn2-2040206618813050">b</xref></sup></th><th rowspan="2" colspan="1">Cell growth (CC<sub>50</sub>)<sup><xref ref-type="table-fn" rid="table-fn3-2040206618813050">c</xref></sup></th></tr><tr><th rowspan="1" colspan="1">OKA strain</th><th rowspan="1" colspan="1">07-1 strain</th><th rowspan="1" colspan="1">AD-169 strain</th><th rowspan="1" colspan="1">Davis strain</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"><bold>4</bold> PMEX</td><td rowspan="1" colspan="1">2.62 ± 1.19</td><td rowspan="1" colspan="1">4.58 ± 2.59</td><td rowspan="1" colspan="1">10.5 ± 3.9</td><td rowspan="1" colspan="1">8.5 ± 2.0</td><td rowspan="1" colspan="1">>345</td><td rowspan="1" colspan="1">111 ± 71</td></tr><tr><td rowspan="1" colspan="1"><bold>5</bold> (<italic>RS</italic>)-HPMPX</td><td rowspan="1" colspan="1">30.9 ± 12.3</td><td rowspan="1" colspan="1">27.1 ± 9.8</td><td rowspan="1" colspan="1">86.9 ± 21.1</td><td rowspan="1" colspan="1">48.6 ± 24.2</td><td rowspan="1" colspan="1">≥300</td><td rowspan="1" colspan="1">≥300</td></tr><tr><td rowspan="1" colspan="1"><bold>6</bold> (<italic>S</italic>)-HPMPX</td><td rowspan="1" colspan="1">22.7 ± 6.8</td><td rowspan="1" colspan="1">17.1 ± 6.4</td><td rowspan="1" colspan="1">80.2 ± 36.0</td><td rowspan="1" colspan="1">43.0 ± 33.9</td><td rowspan="1" colspan="1">>300</td><td rowspan="1" colspan="1">>300</td></tr><tr><td rowspan="1" colspan="1"><bold>7</bold> (<italic>R</italic>)-FPMPX</td><td rowspan="1" colspan="1">>313</td><td rowspan="1" colspan="1">>313</td><td rowspan="1" colspan="1">>313</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">>313</td><td rowspan="1" colspan="1">ND<sup><xref ref-type="table-fn" rid="table-fn4-2040206618813050">d</xref></sup></td></tr><tr><td rowspan="1" colspan="1"><bold>8</bold> (<italic>S</italic>)-FPMPX</td><td rowspan="1" colspan="1">>329</td><td rowspan="1" colspan="1">>329</td><td rowspan="1" colspan="1">>329</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">>329</td><td rowspan="1" colspan="1">ND</td></tr><tr><td rowspan="1" colspan="1"><bold>9</bold> (<italic>R</italic>)-PMPX</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">ND</td></tr><tr><td rowspan="1" colspan="1">Acyclovir</td><td rowspan="1" colspan="1">3.42 ± 2.25</td><td rowspan="1" colspan="1">115 ± 68</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">>440</td><td rowspan="1" colspan="1">>440</td></tr><tr><td rowspan="1" colspan="1">Brivudin</td><td rowspan="1" colspan="1">0.019 ± 0.013</td><td rowspan="1" colspan="1">116 ± 57</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">>300</td><td rowspan="1" colspan="1">309 ± 213</td></tr><tr><td rowspan="1" colspan="1">Ganciclovir</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">6.13 ± 2.38</td><td rowspan="1" colspan="1">4.83 ± 1.88</td><td rowspan="1" colspan="1">>350</td><td rowspan="1" colspan="1">≥445 ± 204</td></tr><tr><td rowspan="1" colspan="1">Cidofovir</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">0.86 ± 0.37</td><td rowspan="1" colspan="1">0.92 ± 0.34</td><td rowspan="1" colspan="1">>300</td><td rowspan="1" colspan="1">263 ± 171</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="table-fn1-2040206618813050"><p><sup>a</sup>Effective concentration required to reduce virus plaque formation (VZV) or viral-induced cytopathic effect (HCMV) by 50%. Virus input was 20 (VZV) or 100 PFU (HCMV) plaque forming units (PFU).</p></fn><fn id="table-fn2-2040206618813050"><p><sup>b</sup>Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.</p></fn><fn id="table-fn3-2040206618813050"><p><sup>c</sup>Cytostatic concentration required reducing cell growth by 50%.</p></fn><fn id="table-fn4-2040206618813050"><p><sup>d</sup>Not determined.</p></fn></table-wrap-foot></table-wrap><table-wrap id="table2-2040206618813050" orientation="portrait" position="float"><label>Table 2.</label><caption><p>Activity of compounds <bold>4</bold>–<bold>9</bold> against herpes simplex virus 1 and 2 (HSV-1 and HSV-2), thymidine kinase deficient (TK<sup>–</sup>) HSV-1 and vaccinia virus in human embryonic lung (HEL) cells.</p></caption><alternatives><graphic specific-use="table2-2040206618813050" xlink:href="10.1177_2040206618813050-table2"></graphic><table frame="hsides" rules="groups"><thead valign="top"><tr><th rowspan="2" colspan="1">Compound</th><th colspan="4" rowspan="1">Antiviral activity EC<sub>50</sub> (μM)<sup><xref ref-type="table-fn" rid="table-fn5-2040206618813050">a</xref></sup><hr></hr></th><th rowspan="1" colspan="1">Cytotoxicity (μM)<hr></hr></th></tr><tr><th rowspan="1" colspan="1">HSV-1</th><th rowspan="1" colspan="1">HSV-2</th><th rowspan="1" colspan="1">HSV-1 TK<sup>–</sup></th><th rowspan="1" colspan="1">Vaccinia virus</th><th rowspan="1" colspan="1">Cell morphology (MCC)<sup><xref ref-type="table-fn" rid="table-fn6-2040206618813050">b</xref></sup></th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"><bold>4</bold> PMEX</td><td rowspan="1" colspan="1">21.5 ± 7.6</td><td rowspan="1" colspan="1">25.6 ± 14.8</td><td rowspan="1" colspan="1">23.7 ± 14.0</td><td rowspan="1" colspan="1">>345 ± 0</td><td rowspan="1" colspan="1">>345</td></tr><tr><td rowspan="1" colspan="1"><bold>5</bold> (<italic>RS</italic>)-HPMPX</td><td rowspan="1" colspan="1">39 ± 27</td><td rowspan="1" colspan="1">20 ± 0</td><td rowspan="1" colspan="1">38 ± 0</td><td rowspan="1" colspan="1">39 ± 27</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1"><bold>6</bold> (<italic>S</italic>)-HPMPX</td><td rowspan="1" colspan="1">33 ± 18</td><td rowspan="1" colspan="1">16 ± 6</td><td rowspan="1" colspan="1">20 ± 0</td><td rowspan="1" colspan="1">39 ± 27</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1"><bold>7</bold> (<italic>R</italic>)-FPMPX</td><td rowspan="1" colspan="1">>313</td><td rowspan="1" colspan="1">>313</td><td rowspan="1" colspan="1">>313</td><td rowspan="1" colspan="1">>313</td><td rowspan="1" colspan="1">>313</td></tr><tr><td rowspan="1" colspan="1"><bold>8</bold> (<italic>S</italic>)-FPMPX</td><td rowspan="1" colspan="1">>329</td><td rowspan="1" colspan="1">>329</td><td rowspan="1" colspan="1">>329</td><td rowspan="1" colspan="1">>329</td><td rowspan="1" colspan="1">>329</td></tr><tr><td rowspan="1" colspan="1"><bold>9</bold> (<italic>R</italic>)-PMPX</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1">Acyclovir</td><td rowspan="1" colspan="1">0.27 ± 0.12</td><td rowspan="1" colspan="1">0.14 ± 0.10</td><td rowspan="1" colspan="1">10 ± 0</td><td rowspan="1" colspan="1">>250</td><td rowspan="1" colspan="1">>250</td></tr><tr><td rowspan="1" colspan="1">Brivudin</td><td rowspan="1" colspan="1">0.031 ± 0.020</td><td rowspan="1" colspan="1">112 ± 37</td><td rowspan="1" colspan="1">32.4 ± 24.1</td><td rowspan="1" colspan="1">8.26 ± 9.62</td><td rowspan="1" colspan="1">>250</td></tr><tr><td rowspan="1" colspan="1">Ganciclovir</td><td rowspan="1" colspan="1">0.028 ± 0.015</td><td rowspan="1" colspan="1">0.032 ± 0.004</td><td rowspan="1" colspan="1">1.47 ± 1.60</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1">Cidofovir</td><td rowspan="1" colspan="1">1.50 ± 0.50</td><td rowspan="1" colspan="1">1.54 ± 1.46</td><td rowspan="1" colspan="1">1.92 ± 1.31</td><td rowspan="1" colspan="1">22.0 ± 19.9</td><td rowspan="1" colspan="1">>250</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="table-fn5-2040206618813050"><p><sup>a</sup>Effective concentration required to reduce virus plaque formation (VZV) or viral-induced cytopathic effect (HCMV) by 50%. Virus input was 20 (VZV) or 100 PFU (HCMV) plaque forming units (PFU).</p></fn><fn id="table-fn6-2040206618813050"><p><sup>b</sup>Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.</p></fn></table-wrap-foot></table-wrap><p>ANPs, including PMEX, are polar compounds, showing severely limited bioavailability. To increase the likelihood of good cell wall permeability, several different prodrug approaches were tested for PMEX (<xref rid="table3-2040206618813050" ref-type="table">Tables 3</xref> and <xref rid="table4-2040206618813050" ref-type="table">4</xref>). Compound <bold>17</bold> was completely inactive in all assays. Phosphonate ester <bold>19</bold> and biamidate prodrug <bold>20</bold> did not display any potent anti-herpesvirus activity which might be explained by insufficient prodrug activation in these cells. The HDP-PMEX prodrug <bold>18</bold> proved to be 7 to 26 folds (VZV), 52 to 170 folds (HCMV) and 6 to 12 folds (HSV) more active compound than the parent compound <bold>4</bold>, although a concomitant increase in its cytostatic activity of 11-fold was also observed indicating successful increase in the cell uptake.</p><table-wrap id="table3-2040206618813050" orientation="portrait" position="float"><label>Table 3.</label><caption><p>Evaluation of prodrug compounds <bold>18</bold>–<bold>20</bold> against VZV and CMV in HEL cells.</p></caption><alternatives><graphic specific-use="table3-2040206618813050" xlink:href="10.1177_2040206618813050-table3"></graphic><table frame="hsides" rules="groups"><thead valign="top"><tr><th rowspan="1" colspan="1"></th><th colspan="4" rowspan="1">Antiviral activity EC<sub>50</sub> (μM)<sup><xref ref-type="table-fn" rid="table-fn7-2040206618813050">a</xref></sup><hr></hr></th><th colspan="2" rowspan="1">Cytotoxicity (μM)<hr></hr></th></tr><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1">TK<sup>+</sup> VZV<hr></hr></th><th rowspan="1" colspan="1">TK<sup>–</sup> VZV<hr></hr></th><th colspan="2" rowspan="1">HCMV<hr></hr></th><th rowspan="2" colspan="1">Cell morphology (MCC)<sup><xref ref-type="table-fn" rid="table-fn8-2040206618813050">b</xref></sup></th><th rowspan="2" colspan="1">Cell growth (CC<sub>50</sub>)<sup><xref ref-type="table-fn" rid="table-fn9-2040206618813050">c</xref></sup></th></tr><tr><th rowspan="1" colspan="1">Compound</th><th rowspan="1" colspan="1">OKA strain</th><th rowspan="1" colspan="1">07-1 strain</th><th rowspan="1" colspan="1">AD-169 strain</th><th rowspan="1" colspan="1">Davis strain</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1">17</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">ND<sup><xref ref-type="table-fn" rid="table-fn10-2040206618813050">d</xref></sup></td></tr><tr><td rowspan="1" colspan="1">18</td><td rowspan="1" colspan="1">0.10 ± 0.05</td><td rowspan="1" colspan="1">0.64 ± 0.77</td><td rowspan="1" colspan="1">0.20 ± 0.28</td><td rowspan="1" colspan="1">0.05 ± 0.05</td><td rowspan="1" colspan="1">100</td><td rowspan="1" colspan="1">10 ± 0</td></tr><tr><td rowspan="1" colspan="1">19</td><td rowspan="1" colspan="1">>20</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">63</td><td rowspan="1" colspan="1">55</td><td rowspan="1" colspan="1">≥100</td><td rowspan="1" colspan="1">ND</td></tr><tr><td rowspan="1" colspan="1">20</td><td rowspan="1" colspan="1">22</td><td rowspan="1" colspan="1">26.5</td><td rowspan="1" colspan="1">63 ± 19</td><td rowspan="1" colspan="1">6.2 ± 6.8</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">100 ± 0</td></tr><tr><td rowspan="1" colspan="1">Acyclovir</td><td rowspan="1" colspan="1">2.23 ± 2.16</td><td rowspan="1" colspan="1">98 ± 61</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">>440</td><td rowspan="1" colspan="1">>440</td></tr><tr><td rowspan="1" colspan="1">Brivudin</td><td rowspan="1" colspan="1">0.019 ± 0.010</td><td rowspan="1" colspan="1">19.6 ± 23.0</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">≥300</td><td rowspan="1" colspan="1">168 ± 41</td></tr><tr><td rowspan="1" colspan="1">Ganciclovir</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">10.0 ± 10.2</td><td rowspan="1" colspan="1">5.5 ± 4.4</td><td rowspan="1" colspan="1">>350</td><td rowspan="1" colspan="1">≥317 ± 98</td></tr><tr><td rowspan="1" colspan="1">Cidofovir</td><td rowspan="1" colspan="1">0.92 ± 0.38</td><td rowspan="1" colspan="1">0.78 ± 0.32</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">>300</td><td rowspan="1" colspan="1">170 ± 61</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="table-fn7-2040206618813050"><p><sup>a</sup>Effective concentration required to reduce virus plaque formation (VZV) or viral-induced cytopathic effect (HCMV) by 50%. Virus input was 20 (VZV) or 100 PFU (HCMV) plaque forming units (PFU).</p></fn><fn id="table-fn8-2040206618813050"><p><sup>b</sup>Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.</p></fn><fn id="table-fn9-2040206618813050"><p><sup>c</sup>Cytostatic concentration required reducing cell growth by 50%.</p></fn><fn id="table-fn10-2040206618813050"><p><sup>d</sup>Not determined.</p></fn></table-wrap-foot></table-wrap><table-wrap id="table4-2040206618813050" orientation="portrait" position="float"><label>Table 4.</label><caption><p>Evaluation of prodrug compounds <bold>18</bold>–<bold>20</bold> on HSV and vaccinia virus in HEL cells.</p></caption><alternatives><graphic specific-use="table4-2040206618813050" xlink:href="10.1177_2040206618813050-table4"></graphic><table frame="hsides" rules="groups"><thead valign="top"><tr><th rowspan="2" colspan="1">Compound</th><th colspan="4" rowspan="1">Antiviral activity EC<sub>50</sub> (μM)<sup><xref ref-type="table-fn" rid="table-fn11-2040206618813050">a</xref></sup><hr></hr></th><th rowspan="1" colspan="1">Cytotoxicity (μM)<hr></hr></th></tr><tr><th rowspan="1" colspan="1">HSV-1</th><th rowspan="1" colspan="1">HSV-2</th><th rowspan="1" colspan="1">HSV-1 TK<sup>–</sup></th><th rowspan="1" colspan="1">Vaccinia virus</th><th rowspan="1" colspan="1">Cell morphology (MCC)<sup><xref ref-type="table-fn" rid="table-fn12-2040206618813050">b</xref></sup></th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1">17</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1">18</td><td rowspan="1" colspan="1">1.8</td><td rowspan="1" colspan="1">4.0</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1">19</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1">20</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1">Acyclovir</td><td rowspan="1" colspan="1">0.50 ± 0.14</td><td rowspan="1" colspan="1">0.50 ± 0.14</td><td rowspan="1" colspan="1">3.25 ± 1.77</td><td rowspan="1" colspan="1">>250</td><td rowspan="1" colspan="1">>250</td></tr><tr><td rowspan="1" colspan="1">Brivudin</td><td rowspan="1" colspan="1">0.05 ± 0.05</td><td rowspan="1" colspan="1">≥250 ± 0</td><td rowspan="1" colspan="1">50</td><td rowspan="1" colspan="1">16.4 ± 17.8</td><td rowspan="1" colspan="1">>250</td></tr><tr><td rowspan="1" colspan="1">Ganciclovir</td><td rowspan="1" colspan="1">0.020 ± 0.014</td><td rowspan="1" colspan="1">0.045 ± 0.049</td><td rowspan="1" colspan="1">0.90 ± 0.14</td><td rowspan="1" colspan="1">>100 ± 0</td><td rowspan="1" colspan="1">>100</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="table-fn11-2040206618813050"><p><sup>a</sup>Effective concentration required to reduce virus plaque formation (VZV) or viral-induced cytopathic effect (HCMV) by 50%. Virus input was 20 (VZV) or 100 PFU (HCMV) plaque forming units (PFU).</p></fn><fn id="table-fn12-2040206618813050"><p><sup>b</sup>Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.</p></fn></table-wrap-foot></table-wrap><p>In order to determine whether the DNA polymerase was the actual target of action of PMEX (<bold>4</bold>), the compound was evaluated against well-characterized HSV-1 mutant viruses. Alike all ANPs, PMEX remained active against viruses bearing mutations in the viral TK (<xref ref-type="fig" rid="fig2-2040206618813050">Figure 2</xref>). Importantly, an increase in the EC<sub>50</sub> of PMEX of the same magnitude as that measured for the ANP adefovir<sup><xref rid="bibr28-2040206618813050" ref-type="bibr">28</xref></sup> was found for DNA polymerase mutant viruses indicating that the target of action of the active form of compound <bold>4</bold> (i.e. PMEXpp) is the herpesvirus DNA polymerase.</p><fig id="fig2-2040206618813050" orientation="portrait" position="float"><label>Figure 2.</label><caption><p>Activity of compound <bold>4</bold> against several thymidine kinase and DNA polymerase HSV-1 mutants in HEL cells. Fold-resistance was calculated as the ratio EC<sub>50</sub> mutant virus/EC<sub>50</sub> wild-type Kos strain.</p></caption><graphic xlink:href="10.1177_2040206618813050-fig2"></graphic></fig><p>The inhibitory activity of the diphosphate form of PMEX (PMEXpp, <bold>21</bold>) was evaluated in an enzymatic assay against herpes (VZV and HCMV) DNA polymerases compared to cellular (α and β) DNA polymerases (<xref rid="table5-2040206618813050" ref-type="table">Table 5</xref>). The inhibition of ACV triphosphate and the pyrophosphate analogue of foscarnet (PFA) were determined in this study for comparison. Compound <bold>21</bold> was not inhibitory towards cellular DNA polymerases, while it inhibited VZV DNA polymerase when dGTP was used as the competitive radiolabeled substrate (IC<sub>50</sub> = 7.4 µM). However, no activity at the highest concentration of <bold>21</bold> (100 µM) could be detected against HCMV DNA polymerase when either dGTP or dTTP were used as the competitive radiolabeled substrates. These data suggest that compound <bold>21</bold> is a poor inhibitor of HCMV DNA polymerase or that the compound may require the HCMV DNA polymerase interact with other proteins of the replication complex to be active. It is also possible that PMEX or its metabolite targets another viral enzyme. To confirm that the compound targets the viral DNA polymerase, PMEX-resistant herpesviruses should be selected under the pressure of PMEX and characterized both genotypically and phenotypically.</p><table-wrap id="table5-2040206618813050" orientation="portrait" position="float"><label>Table 5.</label><caption><p>Inhibition of viral and cellular DNA polymerases (pol) by compound <bold>21</bold> (PMEXpp) compared to the triphosphate form of the nucleoside analogue acyclovir (ACV-TP) and the pyrophosphate analogue of foscarnet (PFA).</p></caption><alternatives><graphic specific-use="table5-2040206618813050" xlink:href="10.1177_2040206618813050-table5"></graphic><table frame="hsides" rules="groups"><thead valign="top"><tr><th rowspan="2" colspan="1"></th><th colspan="7" rowspan="1">IC<sub>50</sub> (μM)<sup><xref ref-type="table-fn" rid="table-fn13-2040206618813050">a</xref></sup><hr></hr></th></tr><tr><th colspan="2" rowspan="1">HCMV DNA pol<hr></hr></th><th rowspan="1" colspan="1">VZV DNA pol<hr></hr></th><th colspan="2" rowspan="1">DNA pol α<hr></hr></th><th colspan="2" rowspan="1">DNA pol β<hr></hr></th></tr><tr><th rowspan="1" colspan="1">Compound</th><th rowspan="1" colspan="1">dGTP<sup><xref ref-type="table-fn" rid="table-fn14-2040206618813050">b</xref></sup></th><th rowspan="1" colspan="1">dTTP<sup><xref ref-type="table-fn" rid="table-fn14-2040206618813050">b</xref></sup></th><th rowspan="1" colspan="1">dTTP<sup><xref ref-type="table-fn" rid="table-fn14-2040206618813050">b</xref></sup></th><th rowspan="1" colspan="1">dGTP<sup><xref ref-type="table-fn" rid="table-fn14-2040206618813050">b</xref></sup></th><th rowspan="1" colspan="1">dTTP<sup><xref ref-type="table-fn" rid="table-fn14-2040206618813050">b</xref></sup></th><th rowspan="1" colspan="1">dGTP<sup><xref ref-type="table-fn" rid="table-fn14-2040206618813050">b</xref></sup></th><th rowspan="1" colspan="1">dTTP<sup><xref ref-type="table-fn" rid="table-fn14-2040206618813050">b</xref></sup></th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"><bold>21</bold> (PMEXpp)</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">7.4 ± 2.7</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">>100</td></tr><tr><td rowspan="1" colspan="1">ACV-TP</td><td rowspan="1" colspan="1">0.77 ± 0.04</td><td rowspan="1" colspan="1">82 ± 26</td><td rowspan="1" colspan="1">ND<sup><xref ref-type="table-fn" rid="table-fn15-2040206618813050">c</xref></sup></td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">ND</td></tr><tr><td rowspan="1" colspan="1">PFA pyrophosphate</td><td rowspan="1" colspan="1">7.0 ± 0.7</td><td rowspan="1" colspan="1">9.4 ± 6.0</td><td rowspan="1" colspan="1">0.18 ± 0.01</td><td rowspan="1" colspan="1">56.0 ± 12.0</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">>100</td><td rowspan="1" colspan="1">ND</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="table-fn13-2040206618813050"><p><sup>a</sup>50% inhibitory concentration or compound concentration required to inhibit the polymerase-catalyzed DNA synthesis by 50%.</p></fn><fn id="table-fn14-2040206618813050"><p><sup>b</sup>Enzyme reaction in the presence of calf thymus DNA and radiolabeled [<sup>3</sup>H]dGTP or [<sup>3</sup>H]dTTP.</p></fn><fn id="table-fn15-2040206618813050"><p><sup>c</sup>Not determined.</p></fn></table-wrap-foot></table-wrap></sec><sec id="sec4-2040206618813050"><title>Experimental part</title><sec id="sec5-2040206618813050"><title>Methods</title><p>Starting compounds and other chemicals were purchased from commercial suppliers or prepared according to the published procedures. Solvents were dried by standard procedures. Solvents were evaporated at 40 °C/2 kPa. Analytical TLC was performed on plates of Kieselgel 60 F 254 (Merck). Column chromatography was performed on silica gel 230–400 mesh, 60 Å (Merck). Reverse phase HPLC separation was performed on a Waters Delta 600 instrument with a Waters 486 Tunable Absorbance Detector using column Phenomenex Gemini C18 (10 μm, 250 × 21.2 mm, flow 10 ml/min preparative column). NMR spectra were recorded on Bruker Avance 500 (<sup>1</sup>H at 500 MHz, <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C at 125.8 MHz, <sup><xref rid="bibr31-2040206618813050" ref-type="bibr">31</xref></sup>P at 202.4 MHz,) spectrometer with TMS or 1,4-dioxane (3.75 ppm for <sup>1</sup>H, 67.19 ppm for <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR) as internal standard or referenced to the residual solvent signal. HR MS spectra were taken on a LTQ Orbitrap XL spectrometer. The purity of the tested compounds was determined by HPLC (H<sub>2</sub>O-CH<sub>3</sub>CN, linear gradient) and was higher than 95%.</p></sec><sec id="sec6-2040206618813050"><title>Method A. General procedure for diazotization/2-hydroxy-dediazoniation of guanine-based starting compounds</title><p>Guanine-based phosphonate (0.5 mmol) was dissolved in 80% AcOH (20 mL) and excess of isoamylnitrite (2.0 mL) was added. The reaction mixture was stirred at 20 °C for 16 h. Volatiles were evaporated, and the residue was co-evaporated with water (3 × 10 mL) and evaporated to dryness. The crude product was dissolved in a small amount of water and purified by preparative HPLC in 0.1 M TEAB buffer using gradient water/methanol (from 98/2 to 20/80).</p><sec><title>((2–(2,6-Dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)ethoxy)methyl)phosphonic acid (4), PMEX</title><p>Synthesis of compound <bold>4</bold> was performed from compound <bold>10</bold> (2.0 g, 4.86 mmol), using previously described procedure.<sup><xref rid="bibr27-2040206618813050" ref-type="bibr">27</xref></sup> Microwave-assisted heating (130 °C, 20 min) of compound <bold>10</bold> in aqueous HCl (1.0 M), followed by solvent removal and precipitation form a water/methanol mixture gave <bold>4</bold> (1.2 g, 85%) as a white solid. The analytical data are in an agreement with the published data.<sup><xref rid="bibr27-2040206618813050" ref-type="bibr">27</xref></sup><inline-graphic xlink:href="10.1177_2040206618813050-img1.jpg"></inline-graphic></p></sec><sec><title>(<italic>RS</italic>)-(((1–(2,6-Dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)-3-hydroxypropan-2-yl)oxy)methyl)phosphonic acid (5), (<italic>RS</italic>)-HPMPX</title><p>Treatment of <bold>11</bold> (223 mg, 0.70 mmol) by Method A afforded <bold>5</bold> (160 mg, 71%) as a white solid. <sup>1</sup>H NMR (D<sub>2</sub>O + NaOD) <italic>δ</italic>: 7.84 (s, 1H, H-8), 4.21 (dd, <italic>J</italic><sub>gem</sub> = 14.7 Hz, <italic>J</italic><sub>1′a-2′</sub> = 4.9 Hz, 1H, H-1′a), 4.15 (dd, <italic>J</italic><sub>gem</sub> = 14.7 Hz, <italic>J</italic><sub>1′b-2′</sub> = 6.1 Hz, 1H, H-1′b), 3.80 (m, 1H, H-2′), 3.71 (dd, <italic>J</italic><sub>gem</sub> = 12.5 Hz, <italic>J</italic><sub>3′a-2′</sub> = 3.7 Hz, 1H, H-3′a), 3.55 (dd, <italic>J</italic><sub>gem</sub> = 12.3 Hz, <italic>J</italic><sub>4′a-P</sub> = 8.8 Hz, 1H, H-4′a), 3.49 (dd, <italic>J</italic><sub>gem</sub> = 12.3 Hz, <italic>J</italic><sub>4′b-P</sub> = 9.6 Hz, 1H, H-4′b), 3.47 (dd, <italic>J</italic><sub>gem</sub> = 12.5 Hz, <italic>J</italic><sub>3′b-2′</sub> = 5.4 Hz, 1H, H-3′b). <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR (D<sub>2</sub>O + NaOD): <italic>δ</italic> 161.88 (C-6), 160.57 (C-2), 154.36 (C-4), 140.89 (C-8), 114.98 (C-5), 80.60 (d, <italic>J</italic><sub>C-O-C-P</sub> = 10.8 Hz, C-2′), 68.66 (d, <italic>J</italic><sub>C-P</sub> = 150.7 Hz, C-4′), 60.98 (C-3′), 43.59 (C-1′). MS-ESI<sup>–</sup><italic>m</italic>/<italic>z</italic> (%): 319 (100, M-H<sup>+</sup>). HRMS(ESI<sup>–</sup>) m/z (C<sub>9</sub>H<sub>13</sub>N<sub>4</sub>O<sub>7</sub>P) [M-H]<sup>–</sup>: calcd 319.0449; found 319.0447.<inline-graphic xlink:href="10.1177_2040206618813050-img2.jpg"></inline-graphic></p></sec><sec><title>(<italic>S</italic>)-(((1–(2,6-Dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)-3-hydroxypropan-2-yl)oxy)methyl) phosphonic acid (6), (<italic>S</italic>)-HPMPX</title><p>Treatment of <bold>12</bold> (154 mg, 0.48 mmol) by Method A gave <bold>6</bold> (55 mg, 36%) as a white solid. <sup>1</sup>H NMR and <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR spectra are identical to those of compound <bold>5</bold>. MS-ESI<sup>–</sup><italic>m/z</italic> (%): 319 (100, M-H<sup>+</sup>). HRMS(ESI<sup>–</sup>) m/z (C<sub>9</sub>H<sub>13</sub>N<sub>4</sub>O<sub>7</sub>P) [M-H]<sup>–</sup>: calcd 319.0449; found 319.0448. [α]<sup><xref rid="bibr20-2040206618813050" ref-type="bibr">20</xref></sup><sub>D</sub> = –4.7 (c = 0.254 g/100 ml, H<sub>2</sub>O).<inline-graphic xlink:href="10.1177_2040206618813050-img3.jpg"></inline-graphic></p></sec><sec><title>(<italic>R</italic>)-(((1–(2,6-Dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)-3-fluoropropan-2-yl)oxy)methyl)phosphonic acid (7), (<italic>R</italic>)-FPMPX</title><p>Treatment of <bold>13</bold> (150 mg, 0.47 mmol) by Method A gave <bold>7</bold> (123 mg, 82%) as a white solid. <sup>1</sup>H NMR (DMSO-<italic>d</italic><sub>6</sub>): <italic>δ</italic> 10.84 (bs, 1H, 1 or 3), 7.72 (s, 1H, 8), 4.62 (ddd, <italic>J</italic><sub>3′b-F</sub> = 47.4 Hz, <italic>J</italic><sub>gem</sub> = 10.5 Hz, <italic>J</italic><sub>3′b-2′</sub> = 3.0 Hz, H-3′b), 4.42 (ddd, <italic>J</italic><sub>3′a-F</sub> = 47.2 Hz, <italic>J</italic><sub>gem</sub> = 10.5 Hz, <italic>J</italic><sub>3′a-2′</sub> = 4.2 Hz, H-3′a), 4.27 (dd, <italic>J</italic><sub>gem</sub> = 14.9 Hz, <italic>J</italic><sub>1′b-2′</sub> = 4.3 Hz, H-1′b), 4.20 (dd, <italic>J</italic><sub>gem</sub> = 14.8 Hz, <italic>J</italic><sub>1′a-2′</sub> = 7.4 Hz, H-1′a), 3.95 (bddtd, 1H, <italic>J</italic><sub>2′-F</sub> = 24.1 Hz, <italic>J</italic><sub>2′-1′a</sub> = 7.4 Hz, <italic>J</italic><sub>2′-1′b</sub> = <italic>J</italic><sub>2′-3′a</sub> = 4.2 Hz, <italic>J</italic><sub>2′-3′b</sub> = 3.0 Hz, H-2′), 3.67 (dd, <italic>J</italic><sub>gem</sub> = 13.5 Hz, <italic>J</italic><sub>4′b-P</sub> = 9.0 Hz, H-4′b), 3.57 (dd, <italic>J</italic><sub>gem</sub> = 13.5 Hz, <italic>J</italic><sub>4′a-P</sub> = 9.0 Hz, H-4′a); <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR (DMSO-<italic>d</italic><sub>6</sub>): <italic>δ</italic> 157.95 (C-6), 151.00 (C-2), 140.80 (C-4), 137.75 (C-8), 114.89 (C-5), 82.26 (d, <italic>J</italic><sub>3′-F</sub> = 169.9 Hz, C-3′), 77.77 (dd, <italic>J</italic><sub>2′-F</sub> = 18.4 Hz, <italic>J</italic><sub>2</sub>′<sub>-P</sub> = 10.3 Hz, C-2′), 65.96 (d, <italic>J</italic><sub>4′-P</sub> = 160.3 Hz, C-4′), 44.38 (d, <italic>J</italic><sub>1′-F</sub> = 8.4 Hz, C-1′). HRMS(ESI<sup>–</sup>) m/z (C<sub>9</sub>H<sub>12</sub>FN<sub>4</sub>O<sub>6</sub>P) [M-H]<sup>–</sup>: calcd 321.0478; found 321.0472. [α]<sup><xref rid="bibr20-2040206618813050" ref-type="bibr">20</xref></sup><sub>D</sub> = + 6.2 (c = 0.194 g/100 ml).<inline-graphic xlink:href="10.1177_2040206618813050-img4.jpg"></inline-graphic></p></sec><sec><title>(<italic>S</italic>)-(((1–(2,6-Dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)-3-fluoropropan-2-yl)oxy)methyl)phosphonic acid (8), (<italic>S</italic>)-FPMPX</title><p>Treatment of <bold>14</bold> (150 mg, 0.47 mmol) by Method A gave <bold>8</bold> (73 mg, 49%) as a white solid. The analytical data are identical to compound <bold>7</bold>. [α]<sup><xref rid="bibr20-2040206618813050" ref-type="bibr">20</xref></sup><sub>D</sub> =- 4.2 (c = 0.0238 g/100 mL).</p></sec><sec><title>(<italic>R</italic>)-(((1–(2,6-Dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)propan-2-yl)oxy)methyl)phosphonic acid (9), (<italic>R</italic>)-PMPX</title><p>Treatment of <bold>15</bold> (250 mg, 0.82 mmol) by Method A gave <bold>9</bold> (129 mg, 51%) as a white solid. <sup>1</sup>H NMR (DMSO-<italic>d</italic><sub>6</sub>): <italic>δ</italic> 10.65 (bs, 1H, 1 or 3), 7.61 (s, 1H, 8), 4.27 (dd, <italic>J</italic><sub>gem</sub> = 14.5 Hz, <italic>J</italic><sub>1′a-2′</sub> = 4.6 Hz, H-1′b), 4.13 (dd, <italic>J</italic><sub>gem</sub> = 14.5 Hz, <italic>J</italic><sub>1′b-2′</sub> = 4.5 Hz, H-1′a), 3.86 (m, 1H, H-2′), 3.54 (dd, <italic>J</italic><sub>gem</sub> = 13.5 Hz, <italic>J</italic><sub>4′b-P</sub> = 9.0 Hz, H-4′b), 3.45 (dd, <italic>J</italic><sub>gem</sub> = 13.6 Hz, <italic>J</italic><sub>4′a-P</sub> = 6.8 Hz, H-4′a), 0.99 (d, 3H, <italic>J</italic><sub>3′-2′</sub> = 6.4 Hz, H-3′). <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR (DMSO-<italic>d</italic><sub>6</sub>): <italic>δ</italic> 158.24 (C-6), 151.22 (C-2), 141.57 (C-4), 138.04 (C-8), 115.38 (C-5), 74.64 (d, <italic>J</italic><sub>2′-P</sub> = 6.7 Hz, C-2′), 48.64 (C-1′), 17.45 (C-3′), C-4′- not observed. HRMS(ESI<sup>–</sup>) m/z (C<sub>9</sub>H<sub>13</sub>N<sub>4</sub>O<sub>6</sub>P) [M-H]<sup>–</sup>: calcd 303.0573; found 303.0499. [α]<sup><xref rid="bibr20-2040206618813050" ref-type="bibr">20</xref></sup><sub>D</sub> = + 4.7 (c = 0.189 g/100 ml).<inline-graphic xlink:href="10.1177_2040206618813050-img5.jpg"></inline-graphic></p></sec><sec><title>((2–(6-(Cyclopropylamino)-2-oxo-2,3-dihydro-9<italic>H</italic>-purin-9-yl)ethoxy)methyl)phosphonic acid (17)</title><p>Treatment of <bold>16</bold> (200 mg, 0.61 mmol) by Method A gave <bold>17</bold> (149 mg, 75%) as a white solid. <sup>1</sup>H NMR (DMSO-<italic>d</italic><sub>6</sub>): <italic>δ</italic> 8.67 (bs, 1H, N<italic>H</italic>-CH), 7.80 (s, 1H, H-8), 4.15 (t, 2H, <italic>J</italic><sub>1′-2′</sub> = 5.2 Hz, H-1′), 3.78 (t, 2H, <italic>J</italic><sub>2′-1′</sub> = 5.2 Hz, H-2′), 3.56 (d, 2H, <italic>J</italic><sub>3′-P</sub> = 8.4 Hz, H-3′), 2.98 (bs, 1H, NH-C<italic>H</italic>), 0.77 (m, 2H, CH<sub>2</sub>-cPr), 0.66 (m, 2H, CH<sub>2</sub>-cPr). <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR (DMSO-<italic>d</italic><sub>6</sub>): <italic>δ</italic> 155.73 (C-6), 153.45 (C-2), 149.9 (C-4), 139.40 (C-8), 110.37 (C-5), 70.30 (d, <italic>J</italic><sub>2′-P</sub> = 9.7 Hz, C-2′), 67.0 (d, <italic>J</italic><sub>3′-P</sub> = 158.8 Hz, C-3′), 42.76 (C-1′), 24.2 (CH-NH), 7.02 (CH<sub>2</sub>-cypr). HRMS(ESI<sup>–</sup>) m/z (C<sub>11</sub>H<sub>16</sub>N<sub>5</sub>O<sub>5</sub>P) [M-H]<sup>–</sup>: calcd. 328.0889; found 328.0816.<inline-graphic xlink:href="10.1177_2040206618813050-img6.jpg"></inline-graphic></p></sec><sec><title>3-(Hexadecyloxy)propyl hydrogen ((2–(2,6-dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)ethoxy)methyl)phosphonate (18)</title><p>A suspension of <bold>4</bold> (100 mg, 0.31 mmol) and 3-(hexadecyloxy)propan-1-ol (124 mg, 0.41 mmol) in anhydrous pyridine (10 mL) was preheated to 100 °C. Dicyclohexylcarbodiimide (142 mg, 0.69 mmol) in anhydrous pyridine (3 ml) was added and the reaction mixture was stirred at 100 °C for 16 h. Solvent was evaporated and the crude product was purified using silica gel chromatography (CHCl<sub>3</sub>/MeOH, 0–50%) to give <bold>18</bold> (46 mg, 23%) as a white amorphous solid. <sup>1</sup>H NMR (CD<sub>3</sub>OD): <italic>δ</italic> 7.69 (s, 1H, H-8), 4.18 (m, 2H, H-1′), 3.91 (q, 2H, <italic>J</italic><sub>4′- 5′</sub> = <italic>J</italic><sub>4′-P</sub> = 6.4 Hz, H-4′), 3.80 (m, 2H, H-2′), 3.61 (d, 2H, <italic>J</italic><sub>3′-P</sub> = 8.9 Hz, H-3′), 3.46 (t, 2H, <italic>J</italic><sub>6′-5′</sub> = 6.5 Hz, H-6′), 3.36 (t, 2H, <italic>J</italic><sub>7′-8′</sub> = 6.6 Hz, H-7′), 1.79 (p, 2H, <italic>J</italic><sub>5′-4′</sub> = <italic>J</italic><sub>5′-6′</sub> = 6.4 Hz, H-5′), 1.52 (m, 2H, H-8′), 1.34–1.27 (m, 26H, H-9′–21′), 0.90 (t, 3H, <italic>J</italic><sub>22′-21′</sub> = 7.0 Hz, H-22′). <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR (CD<sub>3</sub>OD): <italic>δ</italic> 161.79 (C-6), 154.0 (C-4), 139.66 (C-8), 115.98 (C-5), 72.24 (d, <italic>J</italic><sub>2′-P</sub> = 12.2 Hz, C-2′), 72.04 (C-7′), 68.48 (C-6′), 68.02 (d, <italic>J</italic><sub>3′-P</sub> = 158.5 Hz, C-3′), 63.10 (d, <italic>J</italic><sub>4′-P</sub> = 5.6 Hz, C-4′), 44.35 (C-1′), 33.07 (C-20′), 32.40 (d, <italic>J</italic><sub>5′-P</sub> = 6.2 Hz, C-5′), 30.82–30.46 (m, C-8′, 10′–19′), 27.30 (C-9′), 23.73 (C-21′), 14.43 (C-22′). HRMS(ESI<sup>+</sup>) m/z (C<sub>27</sub>H<sub>49</sub>N<sub>4</sub>NaO<sub>7</sub>P) [M+Na]<sup>+</sup>: calcd. 595.3237; found 595.32314.<inline-graphic xlink:href="10.1177_2040206618813050-img7.jpg"></inline-graphic></p></sec><sec><title>4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluoroundecyl hydrogen ((2–(2,6-dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)ethoxy)methyl)phosphonate (19)</title><p>A suspension of <bold>4</bold> (100 mg, 0.31 mmol) and 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecan-1-ol (198 mg, 0.41 mmol) in anhydrous pyridine (10 mL) was preheated to 100 °C. Dicyclohexylcarbodiimide (142 mg, 0.69 mmol) in anhydrous pyridine (3 ml) was added and the reaction mixture was stirred at 100 °C for 16 h. Solvent was evaporated and the crude product was purified using silica gel chromatography (CHCl<sub>3</sub>/MeOH, 0–50%) to give <bold>19</bold> (78 mg, 30%) as a white amorphous solid. <sup>1</sup>H NMR (CD<sub>3</sub>OD): <italic>δ</italic> 7.69 (s, H-8), 4.19 (m, 2H, H-1′), 3.91 (q, 2H, <italic>J</italic><sub>4′-5′</sub> = <italic>J</italic><sub>4′-P</sub> = 6.1 Hz, H-4′), 3.81 (m, 2H, H-2′), 3.63 (d, 2H, <italic>J</italic><sub>3′-P</sub> = 8.9 Hz, H-3′), 2.28 (m, 2H, H-6′), 1.84 (m, 2H, H-5′). <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR (CD<sub>3</sub>OD): <italic>δ</italic> 161.73 (C-6), 153.1 (C-4), 139.58 (C-8), 116.02 (C-5), 72.28 (d, <italic>J</italic><sub>2′-P</sub> = 12.2 Hz, C-2′), 68.21 (d, <italic>J</italic><sub>3′-P</sub> = 158.8 Hz, C-3′), 64.60 (d, <italic>J</italic><sub>4′-P</sub> = 5.5 Hz, C-4′), 44.40 (C-1′), 28.68 (t, <italic>J</italic><sub>6′-F</sub> = 21.8 Hz, C-6′), 23.20 (C-5′). <sup><xref rid="bibr19-2040206618813050" ref-type="bibr">19</xref></sup>F NMR (CD<sub>3</sub>OD, ref. C<sub>6</sub>F<sub>6</sub> –163 ppm): <italic>δ</italic> –123.7 (m, 2F), –120.83 (m, 2F), –120.17 (m, 2F), –119.34 (m, 2F), –119.13 (m, 2F), –111.75 (m, 2F), –78.81 (m, 3F). HRMS(ESI<sup>+</sup>) m/z (C<sub>19</sub>H<sub>16</sub>F<sub>17</sub>N<sub>4</sub>NaO<sub>6</sub>P) [M+Na]<sup>+</sup>: calcd. 773,0434; found 773.0429.<inline-graphic xlink:href="10.1177_2040206618813050-img8.jpg"></inline-graphic></p></sec><sec><title>Bis(L-phenylalanine isopropyl ester) ((2–(2,6-dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)ethoxy)methyl)phosphonate (20)</title><p>TMSBr (330 µL) was added to the mixture of <bold>4</bold> (117 mg, 0.33 mmol) in dry pyridine (4 ml) and DMF (1 ml). The reaction mixture was stirred at 25 °C for 16 h. Volatiles were removed and the moisture-sensitive product was permanently kept under argon. Solid isopropyl ester L-phenylalanine hydrochloride (330 mg, 1.4 mmol) was added to the silylated intermediate under argon, followed by dry pyridine (5 ml) and dry Et<sub>3</sub>N (660 µl, 4.7 mmol). The mixture was preheated to 70 °C and freshly prepared solution of aldrithiol-2 (0.45 g, 2.0 mmol) and triphenylphosphine (0.54 g, 2.0 mmol) in pyridine (4 ml) was added. The resulting mixture was stirred at 70 °C for 72 h. Reaction mixture was evaporated <italic>in vacuo</italic> and the residue was purified by column chromatography (0–100% MeOH in a mixture of Hexane:EtOAc, 6:4) followed by C18 reversed phase column chromatography (0–100% MeOH in water) to give <bold>20</bold> (30 mg, 13%) as an amorphous white solid. <sup>1</sup>H NMR (DMSO<italic>-d<sub>6</sub></italic>): <italic>δ</italic> 9.93 (bs, 1H, NH), 7.50 (s, 1H, H-8), 7.27–7.09 (m, 10H, H-2'',3'',4''), 4.86–4.74 (m, 2H, CH-<italic>i</italic>Pr), 4.45 (m, 1H, CH-<italic>NH</italic>), 4.19 (m, 1H, CH-<italic>NH</italic>), 4.03 (m, 2H, H-1'), 3.95 (m, 1H, <italic>CH</italic>-NH), 3.88 (m, 1H, <italic>CH</italic>-NH), 3.57 (m, 2H, H-2'), 3.30–3.20 (m, 2H, H-3’), 2.90–2.73 (m, 4H, CH<sub>2</sub>Ph); 1.16, 1.11, 1.06 a 1.01 (4 × d, 12H, <italic>J</italic><sub>CH3,CH</sub> = 6.3 Hz, CH<sub>3</sub>). <sup><xref rid="bibr13-2040206618813050" ref-type="bibr">13</xref></sup>C NMR (DMSO-<italic>d</italic><sub>6</sub>): <italic>δ</italic> 172.49 and 172.35 (m, COO), 158.90 (C-6), 154.20 (C-2), 146.20 (C-4), 137.31 and 137.24 (C-1''), 136.63 (C-8), 129.66 (C-2''), 128.28 and 128.24 (C-3''), 126.66 and 126.61 (C-4''), 115.03 (C-5), 70.65 (d, <italic>J</italic><sub>2'-P</sub><italic>=</italic> 10.6 Hz, C-2'), 68.15 and 68.01 (CH-<italic>i</italic>Pr), 67.61 (d, <italic>J</italic><sub>C-P</sub><italic>=</italic> 134.0 Hz, C-3′), 54.25 and 54.04 (NH-<italic>CH</italic>), 43.10 (C-1'), 39.90 (CH<sub>2</sub>Ph), 21.69, 21.63, 21.56 and 21.49 (CH<sub>3</sub>-<italic>i</italic>Pr). HRMS(ESI<sup>+</sup>) m/z (C<sub>32</sub>H<sub>42</sub>N<sub>6</sub>O<sub>8</sub>P) [M + H]<sup>+</sup>: calcd. 669.2798; found 669.2799.<inline-graphic xlink:href="10.1177_2040206618813050-img9.jpg"></inline-graphic></p></sec><sec><title>((2–(2,6-Dioxo-1,2,3,6-tetrahydro-9<italic>H</italic>-purin-9-yl)ethoxy)methyl)phosphonic diphosphoric anhydride (21)</title><p>PMEX morpholidate: Morpholine (0.35 mL, 4.0 mmol) was added to a mixture of <bold>4</bold> (360 mg, 1.0 mmol) in <italic>t</italic>-BuOH/H<sub>2</sub>O (35 mL, 2.5/1, v/v) preheated to 100 °C. Then, a solution of dicyclohexylcarbodiimide (825 mg, 4.0 mmol) in <italic>t</italic>-BuOH/H<sub>2</sub>O (48 mL, 5/1, v/v) was added dropwise to the boiling reaction mixture over a period of 1 h. The mixture was heated to 100 °C overnight. After cooling down, solids were filtered off, and the mixture was concentrated up to half of the volume, and diluted with water (200 ml). The aqueous solution was extracted with diethyl ether (3 × 10 mL) and the organic layer was dried (Na<sub>2</sub>SO<sub>4</sub>) and evaporated to dryness. Crude morpholidate was used directly for the pyrophosphate coupling.<inline-graphic xlink:href="10.1177_2040206618813050-img10.jpg"></inline-graphic></p><p>Pyrophosphate coupling: Prepared morpholidate (0.2 mmol) was carefully dried over P<sub>2</sub>O<sub>5</sub> and treated with (NHBu<sub>3</sub>)<sub>2</sub>H<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (0.5 M solution in DMF, 3 ml) at room temperature for 48 h. The product was precipitated with diethyl ether (10 ml) and the solid was washed with diethyl ether (10 ml). The precipitated product was dissolved in 0.05 M TEAB (4 ml) and purified on a column packed with POROS® 50 HQ (50 ml) with use of a gradient of TEAB in water (0.05–0.5 M). The product was co-evaporated several times with water and converted into a sodium salt form (Dowex 50 in Na<sup>+</sup> cycle). Lyophylisation afforded <bold>21</bold> (5 mg, 3%) as a white amorphous solid. <sup>1</sup>H NMR (D<sub>2</sub>O): <italic>δ</italic> 7.86 (s, 1H, H-8), 4.26 (t, 2H, <italic>J</italic><sub>1′-2′</sub> = 5.1 Hz, H-1′), 3.96 (t, 2H, <italic>J</italic><sub>2′-1′</sub> = 5.1 Hz, H-2′), 3.85 (d, 2H, <italic>J</italic><sub>3′-P</sub> = 8.3 Hz, H-3′), 3.19 (q, 20H, <italic>J</italic> = 7.3 Hz, Et<sub>3</sub>N), l.27 (t, 28H, <italic>J</italic> = 7.3 Hz, Et<sub>3</sub>N). <sup><xref rid="bibr31-2040206618813050" ref-type="bibr">31</xref></sup>P NMR (D<sub>2</sub>O): <italic>δ</italic> 10.73 (d, <italic>J</italic> = 26.2 Hz, P<sub>α</sub>), –6.96 (dm, <italic>J</italic> = 20.0 Hz, P<sub>γ</sub>), –20.62 (dd, <italic>J</italic> = 26.0 Hz, <italic>J</italic> = 20.3 Hz, P<sub>β</sub>). HRMS(ESI<sup>–</sup>) m/z (C<sub>8</sub>H<sub>11</sub>O<sub>12</sub>N<sub>4</sub>NaP<sub>3</sub>) [M-H]<sup>–</sup>: calcd. 470.9489; found 470.9488.</p></sec><sec><title>Biological assays</title><p>The compounds were evaluated against different herpesviruses, including herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK–) HSV-1 KOS strain resistant to ACV (ACV<sup>r</sup>), herpes simplex virus type 2 (HSV-2) strain G, VZV strain Oka, TK– VZV strain 07–1, human cytomegalovirus (HCMV) strains AD-169 and Davis as well as vaccinia virus, adeno virus-2, vesicular stomatitis virus, para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, RSV, FIPV and influenza A virus subtypes H1N1 (A/PR/8), H3N2 (A/HK/7/87) and influenza B virus (B/HK/5/72). The antiviral assays were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey kidney cells (Vero), human epithelial cervix carcinoma cells (HeLa), Crandell-Rees feline kidney cells (CRFK), or Madin Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID<sub>50</sub> of virus (1 CCID<sub>50</sub> being the virus dose to infect 50% of the cell cultures) or with 20 PFU, and the cell cultures were incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation (VZV) was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC<sub>50</sub> or compound concentration required reducing virus-induced cytopathicity or viral plaque formation by 50%.</p><p>Compound <bold>4</bold> was evaluated against several TK and DNA polymerase mutants derived from the reference Kos strain by CPE reduction assay using as reference drugs ACV, GCV, BVDU, foscavir, CDV and adefovir (ADV).</p><p>Cytotoxicity of the tested compounds was expressed as the minimum cytotoxic concentration or the compound concentration that caused a microscopically detectable alteration of cell morphology. Alternatively, the cytostatic activity of the test compounds was measured based on the inhibition of cell growth. HEL cells were seeded at a rate of 5 × 10<sup>3</sup> cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After three days of incubation at 37°C, the cell number was determined with a Coulter counter. The cytostatic concentration was calculated as the CC<sub>50</sub>, or the compound concentration required reducing cell proliferation by 50% relative to the number of cells in the untreated controls.</p><p>The inhibitory effects of PMEXpp on human (α and β) and viral (VZV and HCMV) DNA polymerases were determined as previously described using activated calf thymus DNA, 100 µM of each of the three unlabeled dNTPs, and 0.5 µM of the rate limiting tritium-labeled dNTP, and serial dilutions of PMEXpp (<bold>21</bold>). Foscarnet pyrophosphate and acyclovir triphosphate (ACV–TP) were included as the reference compound. The 50% inhibitory concentration or compound concentration required to inhibit the polymerase-catalyzed DNA synthesis by 50% was then determined.</p></sec></sec></sec><sec sec-type="conclusions" id="sec8-2040206618813050"><title>Conclusions</title><p>A series of novel ANPs bearing xanthine as a nucleobase was prepared and evaluated for their potential antiviral properties. Two synthetic approaches were exploited for the synthesis of the target compounds: (a) recently developed MW-assisted hydrolysis of 2,6-dichloropurine derivatives and (b) well-established diazotization/2-hydroxy-dediazoniation of the corresponding guanine analogues. All prepared ANPs were tested against a wide range of DNA and RNA viruses. Two compounds exhibited antiviral activity. PMEX (<bold>4</bold>) was active against VZV, HCMV, HSV-1, and HSV-2 (with EC<sub>50</sub> values between 2.6 and 25.6 µM), while HPMPX (both as <italic>S</italic>-isomer <bold>6</bold> and as a racemic mixture <bold>5</bold>) exhibited moderate to weak activity (EC<sub>50</sub> in the range of 17–43 µM) against VZV, HCMV, HSV-1, HSV-2, and Vaccinia virus. PMEX (<bold>4</bold>) was the most active compound against VZV in the series (EC<sub>50</sub> = 2.62 µM, TK<sup>+</sup> Oka strain) and was equipotent to the reference drug ACV (EC<sub>50</sub> = 3.42 µM). In contrast to ACV, PMEX (<bold>4</bold>), as ANP which activity is independent of the first phosphorylation step, remained active against the TK<sup>–</sup> VZV 07–1 strain with EC<sub>50</sub> = 4.58 µM. The hexadecyloxypropyl monoester derivative of PMEX, compound <bold>18</bold>, slightly improved the anti-HSV potency of the parent compound (EC<sub>50</sub> values between 1.8 and 4.0 µM). Further studies of PMEX (<bold>4</bold>) and of its diphosphate analogue PMEXpp (<bold>21</bold>) suggested that the compound acts as the inhibitor of herpesvirus DNA polymerases (HSV-1 and VZV). This study represents the first report of xanthine containing ANPs with potent antiviral properties and urges further studies of various xanthine nucleotide analogues as potential antiviral agents.</p></sec></body><back><ack><title>Acknowledgements</title><p>This study is a part of the research project RVO61388963 of the Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences. The authors thank the Mass Spectrometry team (Dr. Josef Cvačka) for measurement of HRMS spectra. The authors wish to express their gratitude to Leentje Persoons, Ellen De Waegenaere, Mrs Lizette van Berckelaer Mrs Bianca Stals, Mrs, Kirsten Lepaige, Mr Niels Willems and Mrs. Nathalie Van Winkel for excellent technical assistance.</p></ack><sec id="sec9-2040206618813050"><title>Declaration of conflicting interests</title><p>The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.</p></sec><sec id="sec10-2040206618813050"><title>Funding</title><p>The author(s) disclosed receipt of the following finacial support for the research, authorship, and/or publication of this article: This article received funding from the Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences (RVO61388963).</p></sec><ref-list content-type="numbered"><title>References</title><ref id="bibr1-2040206618813050"><label>1</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bennett</surname><given-names>BD</given-names></name><name><surname>Kimball</surname><given-names>EH</given-names></name><name><surname>Gao</surname><given-names>M</given-names></name><etal>et al</etal></person-group><article-title>Absolute metabolite concentrations and implied enzyme active site occupancy in <italic>Escherichia coli</italic></article-title>. <source>Nat Chem Biol</source>. <year>2009</year>;
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