Design, synthesis, antiviral and cytotoxic evaluation of novel acyclic phosphonate nucleotide analogues with a 5,6-dihydro-1H-[1,2,3]triazolo[4,5-d]pyridazine-4,7-dione system
Identifieur interne : 000308 ( Pmc/Corpus ); précédent : 000307; suivant : 000309Design, synthesis, antiviral and cytotoxic evaluation of novel acyclic phosphonate nucleotide analogues with a 5,6-dihydro-1H-[1,2,3]triazolo[4,5-d]pyridazine-4,7-dione system
Auteurs : Emilia Bankowska ; Jan Balzarini ; Iwona E. Głowacka ; Andrzej E. Wr BlewskiSource :
- Monatshefte Fur Chemie [ 0026-9247 ] ; 2014.
Abstract
A series of diethyl 2-(4,5-dimethoxycarbonyl-1
The online version of this article (doi:10.1007/s00706-013-1137-x) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1007/s00706-013-1137-x
PubMed: 26166892
PubMed Central: 4494773
Links to Exploration step
PMC:4494773Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Design, synthesis, antiviral and cytotoxic evaluation of novel acyclic phosphonate nucleotide analogues with a 5,6-dihydro-1<italic>H</italic>-[1,2,3]triazolo[4,5-<italic>d</italic>]pyridazine-4,7-dione system</title><author><name sortKey="Bankowska, Emilia" sort="Bankowska, Emilia" uniqKey="Bankowska E" first="Emilia" last="Bankowska">Emilia Bankowska</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.8267.b</institution-id><institution>Bioorganic Chemistry Laboratory, Faculty of Pharmacy,</institution><institution>Medical University of Łódź,</institution></institution-wrap>Muszyńskiego 1, 90-151 Lodz, Poland</nlm:aff></affiliation></author><author><name sortKey="Balzarini, Jan" sort="Balzarini, Jan" uniqKey="Balzarini J" first="Jan" last="Balzarini">Jan Balzarini</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.5596.f</institution-id><institution-id institution-id-type="ISNI">0000000106687884</institution-id><institution>Rega Institute for Medical Research,</institution><institution>KU Leuven,</institution></institution-wrap>Minderbroedersstraat 10, 3000 Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="Glowacka, Iwona E" sort="Glowacka, Iwona E" uniqKey="Glowacka I" first="Iwona E." last="Głowacka">Iwona E. Głowacka</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.8267.b</institution-id><institution>Bioorganic Chemistry Laboratory, Faculty of Pharmacy,</institution><institution>Medical University of Łódź,</institution></institution-wrap>Muszyńskiego 1, 90-151 Lodz, Poland</nlm:aff></affiliation></author><author><name sortKey="Wr Blewski, Andrzej E" sort="Wr Blewski, Andrzej E" uniqKey="Wr Blewski A" first="Andrzej E." last="Wr Blewski">Andrzej E. Wr Blewski</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.8267.b</institution-id><institution>Bioorganic Chemistry Laboratory, Faculty of Pharmacy,</institution><institution>Medical University of Łódź,</institution></institution-wrap>Muszyńskiego 1, 90-151 Lodz, Poland</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26166892</idno><idno type="pmc">4494773</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494773</idno><idno type="RBID">PMC:4494773</idno><idno type="doi">10.1007/s00706-013-1137-x</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000308</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000308</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Design, synthesis, antiviral and cytotoxic evaluation of novel acyclic phosphonate nucleotide analogues with a 5,6-dihydro-1<italic>H</italic>-[1,2,3]triazolo[4,5-<italic>d</italic>]pyridazine-4,7-dione system</title><author><name sortKey="Bankowska, Emilia" sort="Bankowska, Emilia" uniqKey="Bankowska E" first="Emilia" last="Bankowska">Emilia Bankowska</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.8267.b</institution-id><institution>Bioorganic Chemistry Laboratory, Faculty of Pharmacy,</institution><institution>Medical University of Łódź,</institution></institution-wrap>Muszyńskiego 1, 90-151 Lodz, Poland</nlm:aff></affiliation></author><author><name sortKey="Balzarini, Jan" sort="Balzarini, Jan" uniqKey="Balzarini J" first="Jan" last="Balzarini">Jan Balzarini</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.5596.f</institution-id><institution-id institution-id-type="ISNI">0000000106687884</institution-id><institution>Rega Institute for Medical Research,</institution><institution>KU Leuven,</institution></institution-wrap>Minderbroedersstraat 10, 3000 Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="Glowacka, Iwona E" sort="Glowacka, Iwona E" uniqKey="Glowacka I" first="Iwona E." last="Głowacka">Iwona E. Głowacka</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.8267.b</institution-id><institution>Bioorganic Chemistry Laboratory, Faculty of Pharmacy,</institution><institution>Medical University of Łódź,</institution></institution-wrap>Muszyńskiego 1, 90-151 Lodz, Poland</nlm:aff></affiliation></author><author><name sortKey="Wr Blewski, Andrzej E" sort="Wr Blewski, Andrzej E" uniqKey="Wr Blewski A" first="Andrzej E." last="Wr Blewski">Andrzej E. Wr Blewski</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.8267.b</institution-id><institution>Bioorganic Chemistry Laboratory, Faculty of Pharmacy,</institution><institution>Medical University of Łódź,</institution></institution-wrap>Muszyńskiego 1, 90-151 Lodz, Poland</nlm:aff></affiliation></author></analytic><series><title level="j">Monatshefte Fur Chemie</title><idno type="ISSN">0026-9247</idno><idno type="eISSN">1434-4475</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Abstract</title><p>A series of diethyl 2-(4,5-dimethoxycarbonyl-1<italic>H</italic>-1,2,3-triazol-1-yl)alkylphosphonates was synthesised from <italic>ω</italic>-azidoalkylphosphonates and dimethyl acetylenedicarboxylate and was further transformed into the respective diamides, dihydrazides, and 5,6-dihydro-1<italic>H</italic>-[1,2,3]triazolo[4,5-<italic>d</italic>]pyridazine-4,7-diones as phosphonate analogues of acyclic nucleosides having nucleobases replaced with substituted 1,2,3-triazoles. All compounds containing P–C–C–triazole or P–C–C–CH<sub>2</sub>–triazole moieties exist in single conformations in which the diethoxyphosphoryl and substituted 1,2,3-triazolyl or substituted (1,2,3-triazolyl)methyl groups are oriented <italic>anti</italic>. All phosphonates were evaluated in vitro for activity against a variety of DNA and RNA viruses. None of the compounds were endowed with antiviral activity. They were not cytostatic at 100 μM.</p></sec><sec><title>Graphical abstract</title><p><graphic position="anchor" xlink:href="706_2013_1137_Figa_HTML" id="MO1"></graphic></p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1007/s00706-013-1137-x) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="De Clercq, E" uniqKey="De Clercq E">E De Clercq</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Clercq, E" uniqKey="De Clercq E">E De Clercq</name></author><author><name sortKey="Hol, A" uniqKey="Hol A">A Holý</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Naesens, L" uniqKey="Naesens L">L Naesens</name></author><author><name sortKey="Snoeck, R" uniqKey="Snoeck R">R Snoeck</name></author><author><name sortKey="Andrei, G" 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Monatsh Chem</journal-id><journal-id journal-id-type="iso-abbrev">Monatsh. Chem</journal-id><journal-title-group><journal-title>Monatshefte Fur Chemie</journal-title></journal-title-group><issn pub-type="ppub">0026-9247</issn><issn pub-type="epub">1434-4475</issn><publisher><publisher-name>Springer Vienna</publisher-name><publisher-loc>Vienna</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26166892</article-id><article-id pub-id-type="pmc">4494773</article-id><article-id pub-id-type="publisher-id">1137</article-id><article-id pub-id-type="doi">10.1007/s00706-013-1137-x</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Paper</subject></subj-group></article-categories><title-group><article-title>Design, synthesis, antiviral and cytotoxic evaluation of novel acyclic phosphonate nucleotide analogues with a 5,6-dihydro-1<italic>H</italic>-[1,2,3]triazolo[4,5-<italic>d</italic>]pyridazine-4,7-dione system</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bankowska</surname><given-names>Emilia</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Balzarini</surname><given-names>Jan</given-names></name><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Głowacka</surname><given-names>Iwona E.</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Wróblewski</surname><given-names>Andrzej E.</given-names></name><address><email>andrzej.wroblewski@umed.lodz.pl</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="GRID">grid.8267.b</institution-id><institution>Bioorganic Chemistry Laboratory, Faculty of Pharmacy,</institution><institution>Medical University of Łódź,</institution></institution-wrap>Muszyńskiego 1, 90-151 Lodz, Poland</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="GRID">grid.5596.f</institution-id><institution-id institution-id-type="ISNI">0000000106687884</institution-id><institution>Rega Institute for Medical Research,</institution><institution>KU Leuven,</institution></institution-wrap>Minderbroedersstraat 10, 3000 Leuven, Belgium</aff></contrib-group><pub-date pub-type="epub"><day>25</day><month>1</month><year>2014</year></pub-date><pub-date pub-type="pmc-release"><day>25</day><month>1</month><year>2014</year></pub-date><pub-date pub-type="ppub"><year>2014</year></pub-date><volume>145</volume><issue>4</issue><fpage>663</fpage><lpage>673</lpage><history><date date-type="received"><day>30</day><month>9</month><year>2013</year></date><date date-type="accepted"><day>16</day><month>12</month><year>2013</year></date></history><permissions><copyright-statement>© The Author(s) 2014</copyright-statement><license license-type="OpenAccess"><license-p><bold>Open Access</bold>This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.</license-p></license></permissions><abstract id="Abs1"><sec><title>Abstract</title><p>A series of diethyl 2-(4,5-dimethoxycarbonyl-1<italic>H</italic>-1,2,3-triazol-1-yl)alkylphosphonates was synthesised from <italic>ω</italic>-azidoalkylphosphonates and dimethyl acetylenedicarboxylate and was further transformed into the respective diamides, dihydrazides, and 5,6-dihydro-1<italic>H</italic>-[1,2,3]triazolo[4,5-<italic>d</italic>]pyridazine-4,7-diones as phosphonate analogues of acyclic nucleosides having nucleobases replaced with substituted 1,2,3-triazoles. All compounds containing P–C–C–triazole or P–C–C–CH<sub>2</sub>–triazole moieties exist in single conformations in which the diethoxyphosphoryl and substituted 1,2,3-triazolyl or substituted (1,2,3-triazolyl)methyl groups are oriented <italic>anti</italic>. All phosphonates were evaluated in vitro for activity against a variety of DNA and RNA viruses. None of the compounds were endowed with antiviral activity. They were not cytostatic at 100 μM.</p></sec><sec><title>Graphical abstract</title><p><graphic position="anchor" xlink:href="706_2013_1137_Figa_HTML" id="MO1"></graphic></p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1007/s00706-013-1137-x) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>Cycloadditions</kwd><kwd>Cyclizations</kwd><kwd>Heterocycles</kwd><kwd>NMR spectroscopy</kwd><kwd>Conformation</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© Springer-Verlag Wien 2014</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1" sec-type="introduction"><title>Introduction</title><p>In the past two decades acyclic nucleoside phosphonates (ANPs) have become one of the most important classes of antiviral drugs [<xref ref-type="bibr" rid="CR1">1</xref>]. Three of them (adefovir, cidofovir, tenofovir; Fig. <xref rid="Fig1" ref-type="fig">1</xref>) have been marketed for treatment of viral infection caused by HIV, HBV, HSV and other DNA viruses [<xref ref-type="bibr" rid="CR2">2</xref>–<xref ref-type="bibr" rid="CR5">5</xref>]. The concept of acyclic nucleosides is based on the assumption that an acyclic moiety most often bearing an oxygen atom mimics the furanose ring at least partially. Acyclic nucleoside phosphonates require conversion in vivo to their triphosphate metabolites to become active [<xref ref-type="bibr" rid="CR2">2</xref>, <xref ref-type="bibr" rid="CR6">6</xref>]. The replacement of the natural phosphate moiety by a phosphonate group makes analogues less susceptible to enzymatic hydrolysis [<xref ref-type="bibr" rid="CR7">7</xref>, <xref ref-type="bibr" rid="CR8">8</xref>].<fig id="Fig1"><label>Fig. 1</label><caption><p>ANPs marketed for treatment of viral infections</p></caption><graphic xlink:href="706_2013_1137_Fig1_HTML" id="MO2"></graphic></fig></p><p>Further studies in this field brought a new generation of nucleotide/nucleoside analogues in which natural nucleobases were modified as exemplified by the 2,4-diaminopyrimidine framework present in antiviral HPMPO-DAPy and PMPO-DAPy [<xref ref-type="bibr" rid="CR9">9</xref>–<xref ref-type="bibr" rid="CR11">11</xref>] and the 1,2,4-triazole ring in ribavirin (Fig. <xref rid="Fig2" ref-type="fig">2</xref>) [<xref ref-type="bibr" rid="CR12">12</xref>].<fig id="Fig2"><label>Fig. 2</label><caption><p>Structures of HPMPO-DAPy, PMPO-DAPy and ribavirin</p></caption><graphic xlink:href="706_2013_1137_Fig2_HTML" id="MO3"></graphic></fig></p><p>The antiviral activity of ribavirin stimulated interest in replacing the 1,2,4-triazole system with an isomeric 1,2,3-triazole ring because several 1,2,3-triazoles exhibit antibacterial [<xref ref-type="bibr" rid="CR13">13</xref>–<xref ref-type="bibr" rid="CR15">15</xref>], antifungal [<xref ref-type="bibr" rid="CR15">15</xref>–<xref ref-type="bibr" rid="CR17">17</xref>], anticancer [<xref ref-type="bibr" rid="CR18">18</xref>–<xref ref-type="bibr" rid="CR20">20</xref>], anti-inflammatory [<xref ref-type="bibr" rid="CR21">21</xref>, <xref ref-type="bibr" rid="CR22">22</xref>] and antiviral [<xref ref-type="bibr" rid="CR23">23</xref>–<xref ref-type="bibr" rid="CR26">26</xref>] properties. It was found that carbocyclic analogues <bold>1</bold> and phosphonocarbocyclic analogues <bold>2</bold> of ribavirin displayed antiviral activity against HIV-1 (Fig. <xref rid="Fig3" ref-type="fig">3</xref>) [<xref ref-type="bibr" rid="CR27">27</xref>].<fig id="Fig3"><label>Fig. 3</label><caption><p>Antiviral analogues of ribavirin having the 1,2,3-triazole ring</p></caption><graphic xlink:href="706_2013_1137_Fig3_HTML" id="MO4"></graphic></fig></p><p>Furthermore, preliminary structure–activity relationship evaluation of 1,2,3-triazole nucleoside phosphonates <bold>3</bold> and <bold>4</bold> suggested that this scaffold could be further optimised to afford selective inhibitors of HCV replication (Fig. <xref rid="Fig4" ref-type="fig">4</xref>) [<xref ref-type="bibr" rid="CR28">28</xref>].<fig id="Fig4"><label>Fig. 4</label><caption><p>1,2,3-Triazole nucleoside phosphonates as potential inhibitors of HCV replication</p></caption><graphic xlink:href="706_2013_1137_Fig4_HTML" id="MO5"></graphic></fig></p><p>On the other hand, it was reported that nucleoside analogues <bold>5</bold> and <bold>6</bold> containing the 5,6-dihydro-1<italic>H</italic>-imidazo[4,5-<italic>d</italic>]pyridazine-4,7-dione ring instead of the natural purine framework exhibited antiviral activity by inhibition of a viral helicase from West Nile Virus (WNV) and HCV (Fig. <xref rid="Fig5" ref-type="fig">5</xref>). These observations may be useful in designing a lead structure for the development of new classes of antiviral agents [<xref ref-type="bibr" rid="CR29">29</xref>].<fig id="Fig5"><label>Fig. 5</label><caption><p>Cyclic nucleoside analogues based on the 5,6-dihydro-1<italic>H</italic>-imidazo[4,5-<italic>d</italic>]pyridazine-4,7-dione framework</p></caption><graphic xlink:href="706_2013_1137_Fig5_HTML" id="MO6"></graphic></fig></p><p>Based on the active compounds already discussed, a novel series of phosphonate analogues <bold>11</bold> having the 5,6-dihydro-1<italic>H</italic>-[1,2,3]triazolo[4,5-<italic>d</italic>]pyridazine-4,7-dione system was designed as potential antiviral agents (Scheme <xref rid="Sch1" ref-type="fig">1</xref>). Furthermore, because their immediate precursor dihydrazides <bold>10</bold> as well as diamides <bold>9</bold> share several common structural features with ribavirin, they also may show antiviral activity. The key step of our synthetic plan involves the 1,3-dipolar cycloaddition of dimethyl acetylenedicarboxylate and <italic>ω</italic>-azidoalkylphosphonates <bold>7</bold> which contain structurally diversified alkyl chains to provide the intermediate diesters <bold>8</bold>.<fig id="Sch1"><graphic xlink:href="706_2013_1137_Sch1_HTML" id="MO7"></graphic></fig></p></sec><sec id="Sec2" sec-type="results"><title>Results and discussion</title><sec id="Sec3"><title>Chemistry</title><p>The 1,2,3-triazoles <bold>8a</bold>–<bold>8d</bold> and <bold>8f</bold> were synthesised in 64–98 % yield employing the Huisgen 1,3-dipolar cycloaddition of the corresponding azidophosphonates <bold>7</bold> and dimethyl acetylenedicarboxylate at 110 °C in the same way as the known compounds <bold>8e</bold> and <bold>8g</bold> [<xref ref-type="bibr" rid="CR30">30</xref>, <xref ref-type="bibr" rid="CR31">31</xref>]. They were finally purified either by chromatography on a silica gel column or by crystallisation (Scheme <xref rid="Sch1" ref-type="fig">1</xref>).</p><p>The required azidoalkylphosphonates <bold>7a</bold>–<bold>7e</bold> and <bold>7g</bold> have already been described in the literature [<xref ref-type="bibr" rid="CR30">30</xref>, <xref ref-type="bibr" rid="CR32">32</xref>–<xref ref-type="bibr" rid="CR36">36</xref>]. Azidophosphonate <bold>7f</bold> was obtained in the Abramov reaction [<xref ref-type="bibr" rid="CR37">37</xref>, <xref ref-type="bibr" rid="CR38">38</xref>] from 3-azidopropanal [<xref ref-type="bibr" rid="CR39">39</xref>] and diethyl phosphite in 34 % yield (Scheme <xref rid="Sch2" ref-type="fig">2</xref>). It was found that 3-azidopropanal is unstable in the presence of triethylamine used as a catalyst in this reaction. For this reason only 0.4 equiv. of diethyl phosphite was applied to avoid tedious separation of <bold>7f</bold> from the reaction mixture.<fig id="Sch2"><graphic xlink:href="706_2013_1137_Sch2_HTML" id="MO8"></graphic></fig></p><p>The diamides <bold>9a</bold>–<bold>9g</bold> were obtained from diesters <bold>8a</bold>–<bold>8g</bold> by ammonolysis according to a standard protocol (Scheme <xref rid="Sch1" ref-type="fig">1</xref>) [<xref ref-type="bibr" rid="CR6">6</xref>, <xref ref-type="bibr" rid="CR40">40</xref>]. The crude products were subjected to column chromatography on silica gel and finally purified by crystallisation to give <bold>9a</bold>–<bold>9g</bold> in 37–61 % yields. The <sup>1</sup>H NMR spectra of diamides <bold>9a</bold>–<bold>9g</bold> in chloroform-<italic>d</italic> confirmed the nonequivalence of protons from the carbamoyl groups because in the 6.0–10.8 ppm region four broad singlets were always observed.</p><p>The diesters <bold>8a</bold>–<bold>8g</bold> were also converted to the corresponding dihydrazides <bold>10a</bold>–<bold>10g</bold> with hydrazine hydrate (Scheme <xref rid="Sch1" ref-type="fig">1</xref>). Preliminary attempts at synthesising dihydrazide <bold>10a</bold> followed the literature procedure [<xref ref-type="bibr" rid="CR41">41</xref>] and showed that refluxing phosphonate <bold>8a</bold> and hydrazine hydrate in ethanolic solution for 5 h led to the formation of several products. The <sup>31</sup>P NMR spectrum of the reaction mixture revealed the presence of the expected phosphonate <bold>10a</bold> (76 %), bicyclic 1,2,3-triazolopyridazinedione <bold>11a</bold> (11 %) and their monodealkylated counterparts <bold>12a</bold> and <bold>13a</bold> (10 and 3 %, respectively) (Fig. <xref rid="Fig6" ref-type="fig">6</xref>). When hydrazinolysis of the diester <bold>8a</bold> was conducted for 2 h only phosphonates <bold>10a</bold> (79 %) and <bold>11a</bold> (21 %) were produced. For this reason syntheses of dihydrazides <bold>10b</bold>–<bold>10g</bold> were performed under the same conditions. However, it appeared that for compounds <bold>8c</bold> and <bold>8g</bold> significant dealkylation still occurred. In both cases the respective crude reaction mixtures contained almost 50 % of by-products. Purifications on silica gel columns and crystallisations gave dihydrazides <bold>10a</bold>–<bold>10g</bold> in 28–66 % yields.<fig id="Fig6"><label>Fig. 6</label><caption><p>Compounds <bold>12a</bold> and <bold>13a</bold></p></caption><graphic xlink:href="706_2013_1137_Fig6_HTML" id="MO9"></graphic></fig></p><p>Finally, heating dihydrazides <bold>10a</bold>–<bold>10g</bold> with 10 % hydrochloric acid for 2.5 h gave [1,2,3]triazolo[4,5-<italic>d</italic>]pyridazine-4,7-diones <bold>11a</bold>–<bold>11g</bold> (Scheme <xref rid="Sch1" ref-type="fig">1</xref>) in 30–66 % yields [<xref ref-type="bibr" rid="CR41">41</xref>].</p></sec><sec id="Sec4"><title>Conformational analysis</title><p>Detailed analyses of <sup>1</sup>H and <sup>13</sup>C NMR spectral data revealed conformational preferences of phosphonates described in this paper. Compounds <bold>8a</bold>–<bold>11a</bold> contain an 1,2-substituted ethylene fragment which does not freely rotate around a C–C bond because their <sup>1</sup>H NMR spectra display AA′XX′P patterns. A similar spectrum was also noticed for 2-azidoethylphosphonate [<xref ref-type="bibr" rid="CR31">31</xref>]. Antiperiplanar disposition of the diethoxyphosphoryl groups and substituted 1,2,3-triazoles <bold>14a</bold> (Fig. <xref rid="Fig7" ref-type="fig">7</xref>) was proved by the presence of two identical <sup>3</sup><italic>J</italic>(P–H<sub><italic>X</italic></sub>) = 10.5 Hz couplings which were calculated from the <sup>1</sup>H{<sup>31</sup>P} NMR spectrum of <bold>8a</bold>.<fig id="Fig7"><label>Fig. 7</label><caption><p>Preferred conformations of the phosphonates described in this study</p></caption><graphic xlink:href="706_2013_1137_Fig7_HTML" id="MO10"></graphic></fig></p><p>In the <sup>1</sup>H NMR spectra of compounds <bold>8g</bold>, <bold>10g</bold>, and <bold>11g</bold> three hydrogen atoms attached to the two-carbon linker appeared as deceptively simple but very similar ABXP spectral patterns. However, the relevant <sup>3</sup><italic>J</italic>(H1–H2a) and <sup>3</sup><italic>J</italic>(H1–H2b) coupling constants were precisely calculated (10.2 and 3.6 Hz, respectively) from the <sup>1</sup>H NMR spectrum of diamide <bold>9g</bold>. These values [<xref ref-type="bibr" rid="CR42">42</xref>] together with small couplings for <sup>3</sup><italic>J</italic>(P–H2a) and <sup>3</sup><italic>J</italic>(P–H2b) [<xref ref-type="bibr" rid="CR43">43</xref>, <xref ref-type="bibr" rid="CR44">44</xref>] allowed us to unequivocally establish <bold>14g</bold> (Fig. <xref rid="Fig7" ref-type="fig">7</xref>) as the preferred conformation of phosphonate <bold>9g</bold> although other phosphonates from this series very likely adopt the same <italic>anti</italic> conformation.</p><p>Large values (16.9–19.2 Hz) of <sup>3</sup><italic>J</italic>(P–CC–C3) [<xref ref-type="bibr" rid="CR44">44</xref>–<xref ref-type="bibr" rid="CR46">46</xref>] observed in the <sup>13</sup>C NMR spectra of phosphonates <bold>8</bold>–<bold>11</bold> containing a three-carbon fragment between the phosphorus atom and the 1,2,3-triazole ring (series <bold>b</bold>, <bold>e</bold>, and <bold>f</bold>) evidenced the preference of antiperiplanar conformations <bold>14b</bold>, <bold>14e</bold>, and <bold>14f</bold> (Fig. <xref rid="Fig7" ref-type="fig">7</xref>) for these compounds. This conclusion was further supported by vicinal H1–H2 couplings calculated for 2- and 1-hydroxyphosphonates (series <bold>e</bold> and <bold>f</bold>) which clearly indicated <italic>gauche</italic> (3.0–3.6 Hz) and <italic>anti</italic> (9.6–10.8 Hz) arrangements of the respective H–C1C2–H protons.</p><p>Although values (9.3–12.1 Hz) of <sup>3</sup><italic>J</italic>(P–C–O–C) were easily extracted from the <sup>13</sup>C NMR spectra of phosphonates <bold>8</bold>–<bold>11</bold> (series <bold>c</bold>) they could not be unequivocally applied in the estimation of the extent to which rotation around the PC–OC bond is hindered because the angular dependence of <sup>3</sup><italic>J</italic>(P–C–O–C) has not been established so far. However, the rotation around the OC–CN bond is not restricted because vicinal H<sub>2</sub>C–CH<sub>2</sub> coupling constants observed for phosphonates <bold>8c</bold>–<bold>11c</bold> fall in the 4.8–5.2 Hz range. On the other hand, on the basis of the values of <sup>3</sup><italic>J</italic>(HC–CH) found for PH<sub>2</sub>C–CH<sub>2</sub>O and OH<sub>2</sub>C–CH<sub>2</sub>N units (7.2–7.8 and 5.1–5.7 Hz, respectively), full conformational freedom within a five-atom linker in phosphonates <bold>8</bold>–<bold>11</bold> (series <bold>d</bold>) is anticipated.</p></sec><sec id="Sec5"><title>Antiviral activity evaluation</title><p>The synthesised compounds <bold>9a</bold>–<bold>9g</bold>, <bold>10a</bold>–<bold>10g</bold>, and <bold>11a</bold>–<bold>11g</bold> were evaluated for their antiviral activities against a wide variety of DNA and RNA viruses using the following cell-based assays: (a) human embryonic lung (HEL) cell: herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), herpes simplex virus-1 (TK<sup>−</sup> ACV<sup>r</sup> KOS), vaccinia virus, vesicular stomatitis virus, varicella-zoster virus (TK<sup>+</sup> VZV strain OKA and TK<sup>¯</sup> VZV strain 07-1) and cytomegalovirus (CMV) (strain AD-169 and Davis); (b) CEM cell cultures: human immunodeficiency virus-1 (HIV-1) and HIV-2; (c) Vero cell cultures: para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus; (d) HeLa cell cultures: vesicular stomatitis virus, Coxackie virus B4 and respiratory syncytial virus; (e) Crandel-Rees feline kidney (CRFK) cell cultures: feline corona virus (FIPV) and feline herpes virus (FHV) and (f) Madin Darby Canine kidney (MDCK) cell culture: influenza A virus H1N1 subtype A/PR/8, influenza H3N2 subtype A/HK/7/87 and influenza B virus (B/HK/5/72). Ganciclovir, cidofovir, acyclovir, brivudin, (<italic>S</italic>)-9-(2,3-dihydroxypropyl)adenine [(<italic>S</italic>)-DHPA], ribavirin, oseltamivir carboxylate, amantadine and rimantadine were used as the reference compounds. The antiviral activity was expressed as the EC<sub>50</sub>: the compound concentration required to reduce virus plaques formation (VZV, CMV) by 50 % or to reduce virus-induced cytopathogenicity by 50 % (other viruses). None of the compounds showed appreciable antiviral activity at subtoxic concentrations.</p></sec><sec id="Sec6"><title>Evaluation of cytotoxicity</title><p>The cytotoxicity of the tested compounds towards the uninfected host cells was defined as the minimum cytotoxic concentration (MMC) that causes a microscopically detectable alteration of normal cell morphology. The 50 % cytotoxic concentration (CC<sub>50</sub>), i.e. causing a 50 % decrease in cell viability, was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2<italic>H</italic>-tetrazolium (MTS) assay system. None of the tested compounds affected the cell morphology of Vero, HEL, HeLa or CRFK cells (MCC or CC<sub>50</sub>) at compound concentrations up to 100 μM. They were also not cytostatic against murine leukemia and human CEM and HeLa cells at 100 μM.</p></sec></sec><sec id="Sec7" sec-type="conclusion"><title>Conclusions</title><p>Phosphonylated 1,2,3-triazoles <bold>8a</bold>–<bold>8g</bold> were obtained in good to excellent yields by 1,3-dipolar cycloaddition between <italic>ω</italic>-azidophosphonates <bold>7a</bold>–<bold>7g</bold> and dimethyl acetylenedicarboxylate. New series of diamides <bold>9a</bold>–<bold>9g</bold>, dihydrazides <bold>10a</bold>–<bold>10g</bold> and 1,2,3-triazolopyridazinediones <bold>11a</bold>–<bold>11g</bold> were efficiently synthesised as phosphonate analogues of acyclic nucleosides in which nucleobases were replaced by substituted 1,2,3-triazoles.</p><p>Compounds <bold>8</bold>–<bold>11</bold> (series <bold>a</bold>, <bold>b</bold>, <bold>e</bold>, <bold>f</bold>, <bold>g</bold>) exist in single conformations in which diethoxyphosphoryl and substituted 1,2,3-triazolyl (series <bold>a</bold> and <bold>b</bold>) or (1,2,3-triazolyl)methyl groups (series <bold>e</bold>, <bold>f</bold>, <bold>g</bold>) prefer the <italic>anti</italic> orientation.</p><p>All synthesised compounds were evaluated for their antiviral activity against DNA and RNA viruses and were inactive. None of the compounds were cytotoxic (Vero, HEL, HeLa) or cytostatic (L1210, CEM, HeLa) at a concentration up to 100 μM.</p></sec><sec id="Sec8" sec-type="materials|methods"><title>Experimental</title><p>The <sup>1</sup>H NMR spectra were recorded in CDCl<sub>3</sub>, CD<sub>3</sub>OD or D<sub>2</sub>O on the following spectrometers: Varian Mercury-300 and Bruker Avance III (600 MHz) with TMS as an internal standard; chemical shifts <italic>δ</italic> in ppm with respect to TMS; coupling constants <italic>J</italic> in Hz. The <sup>13</sup>C NMR spectra were recorded for CDCl<sub>3</sub>, CD<sub>3</sub>OD or D<sub>2</sub>O solutions on Varian Mercury-300 and Bruker Avance III (600 MHz) machines at 75.5 and 150.5 MHz, respectively. The <sup>31</sup>P NMR spectra were recorded in CDCl<sub>3</sub>, CD<sub>3</sub>OD or D<sub>2</sub>O on Varian Mercury-300 and Bruker Avance III (600 MHz) spectrometers at 121.5 and 243 MHz, respectively.</p><p>IR spectra were measured on an Infinity MI-60 FT-IR spectrometer. Melting points were determined on the Boetius apparatus. Elemental analyses were performed by the microanalytical laboratory of the host institution on Perkin Elmer PE 2400 CHNS analyzer and the results were found to be in good agreement (±0.3 %) with the calculated values.</p><p>The following absorbents were used: column chromatography, Merck silica gel 60 (70–230 mesh); analytical TLC, Merck TLC plastic sheets, silica gel 60 F<sub>254</sub>. TLC plates were developed in chloroform–methanol solvent systems. Visualization of spots was effected with iodine vapours. All solvents were dried according to standard literature methods.</p><sec id="d29e1187"><title><italic>Diethyl 3</italic>-<italic>azido</italic>-<italic>1</italic>-<italic>hydroxypropylphosphonate</italic> (<bold>7f</bold>, C<sub>7</sub>H<sub>16</sub>N<sub>3</sub>O<sub>4</sub>P)</title><p>A mixture of 5.77 g 3-azidopropanal [<xref ref-type="bibr" rid="CR39">39</xref>] (0.0582 mol), 3.0 cm<sup>3</sup> diethyl phosphite (0.023 mol) and 0.81 cm<sup>3</sup> triethylamine (0.0058 mol) was stirred at 5 °C for 24 h. The crude product was subjected to chromatography on silica gel with chloroform/methanol (100:1 and 50:1, v/v) to give a yellow oil (4.671 g, 34 %). IR (film): <inline-formula id="IEq1"><alternatives><tex-math id="M1">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M2"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq1.gif"></inline-graphic></alternatives></inline-formula> = 3,272, 2,985, 2,934, 2,911, 2,874, 2,102, 1,227, 1,028 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.35 (t, <italic>J</italic> = 7.1 Hz, 6H, 2 × POCH<sub>2</sub>C<italic>H</italic><sub>3</sub>), 1.87–2.06 (m, 3H, CH<sub>2</sub>, OH), 3.55 (t, <italic>J</italic> = 6.3 Hz, 2H, C<italic>H</italic><sub>2</sub>N<sub>3</sub>), 4.01 (dt, <italic>J</italic> = 9.8, 4.6 Hz, 1H, H-1), 4.12–4.24 (m, 4H, 2 × POC<italic>H</italic><sub>2</sub>CH<sub>3</sub>) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75.5 MHz): <italic>δ</italic> = 16.6 and 16.7 (2 d, <italic>J</italic> = 5.4 Hz, POC<italic>C</italic>), 30.8 (d, <italic>J</italic> = 2.6 Hz, C-2), 47.7 (d, <italic>J</italic> = 15.5 Hz, C-3), 62.9 and 63.2 (2 d, <italic>J</italic> = 7.2 Hz, POC), 64.6 (d, <italic>J</italic> = 164.6 Hz, C-1) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121.5 MHz): <italic>δ</italic> = 25.28 ppm.</p></sec><sec id="d29e1335"><title>Synthesis of 1,2,3-triazoles <bold>8a</bold>–<bold>8g</bold> (general procedure)</title><p>A solution of azidophosphonate <bold>7a</bold>–<bold>7g</bold> (1.00 mmol) and dimethyl acetylenedicarboxylate <bold>13</bold> (1.00 mmol) in 4 cm<sup>3</sup> toluene was refluxed for 4 h. The reaction mixtures were concentrated to dryness to leave yellow oils which were purified on silica gel columns with chloroform/methanol (100:1, v/v) or were crystallised to give 1,2,3-triazoles <bold>8a</bold>–<bold>8g</bold>.</p></sec><sec id="d29e1365"><title><italic>Diethyl 2</italic>-<italic>(4,5</italic>-<italic>dimethoxycarbonyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)ethylphosphonate</italic> (<bold>8a</bold>, C<sub>12</sub>H<sub>20</sub>N<sub>3</sub>O<sub>7</sub>P)</title><p>From 0.450 g azidophosphonate <bold>7a</bold> (2.17 mmol) and 0.309 g dimethyl acetylenedicarboxylate <bold>13</bold> (2.17 mmol) phosphonate <bold>8a</bold> was obtained as a yellowish oil (0.674 g, 89 %) after chromatography on a silica gel column with chloroform/methanol (100:1, v/v). IR (film): <inline-formula id="IEq2"><alternatives><tex-math id="M3">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M4"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq2.gif"></inline-graphic></alternatives></inline-formula> = 3,462, 2,985, 2,958, 1,736, 1,468, 1,447, 1,225, 1,140, 1,060, 1,025, 957 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.33 (t, <italic>J</italic> = 7.1 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.40–2.52 (m, 2H, PCH<sub>2</sub>), 3.98 (s, 3H, OCH<sub>3</sub>), 4.02 (s, 3H, OCH<sub>3</sub>), 4.08–4.18 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.82–4.91 (m, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.6 (d, <italic>J</italic> = 5.7 Hz, <italic>C</italic>COP), 27.2 (d, <italic>J</italic> = 140.6 Hz, PC), 45.4 (s, PC<italic>C</italic>N), 52.9 (s, OCH<sub>3</sub>), 53.7 (s, OCH<sub>3</sub>), 62.4 (d, <italic>J</italic> = 6.3 Hz, C<italic>C</italic>OP), 129.8 and 140.2 (2 s, C=C), 158.6 and 160.4 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 25.9 ppm.</p></sec><sec id="d29e1526"><title><italic>Diethyl 3</italic>-<italic>(4,5</italic>-<italic>dimethoxycarbonyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)propylphosphonate</italic> (<bold>8b</bold>, C<sub>13</sub>H<sub>22</sub>N<sub>3</sub>O<sub>7</sub>P)</title><p>From 0.785 g azidophosphonate <bold>7b</bold> (3.565 mmol) and 0.507 g dimethyl acetylenedicarboxylate <bold>13</bold> (3.565 mmol) phosphonate <bold>8b</bold> was obtained as a yellowish oil (1.067 g, 83 %) after chromatography on a silica gel column with chloroform/methanol (100:1, v/v). IR (film): <inline-formula id="IEq3"><alternatives><tex-math id="M5">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M6"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq3.gif"></inline-graphic></alternatives></inline-formula> = 2,953, 2,836, 1,736, 1,463, 1,226, 1,030 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.34 (t, <italic>J</italic> = 7.1 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 1.72–1.84 (m, 2H, PCH<sub>2</sub>C<italic>H</italic><sub>2</sub>), 2.16–2.31 (m, 2H, PC<italic>H</italic><sub>2</sub>), 4.00 (s, 3H, OCH<sub>3</sub>), 4.03 (s, 3H, OCH<sub>3</sub>), 4.05–4.18 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.71 (t, <italic>J</italic> = 7.1 Hz, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.5 (d, <italic>J</italic> = 6.0 Hz, POC<italic>C</italic>), 22.6 (d, <italic>J</italic> = 143.1 Hz, PC), 23.5 (d, <italic>J</italic> = 4.3 Hz, PC<italic>C</italic>), 50.4 (d, <italic>J</italic> = 16.9 Hz, PCC<italic>C</italic>), 52.7 (s, OCH<sub>3</sub>), 53.2 (s, OCH<sub>3</sub>), 61.8 (d, <italic>J</italic> = 6.3 Hz, POC), 129.7 and 139.8 (2 s, C=C), 158.6 and 160.3 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 30.7 ppm.</p></sec><sec id="d29e1709"><title><italic>Diethyl 2</italic>-<italic>(4,5</italic>-<italic>dimethoxycarbonyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)ethoxymethylphosphonate</italic> (<bold>8c</bold>, C<sub>13</sub>H<sub>22</sub>N<sub>3</sub>O<sub>8</sub>P)</title><p>From 1.027 g azidophosphonate <bold>7c</bold> (4.330 mmol) and 0.615 g dimethyl acetylenedicarboxylate <bold>13</bold> (4.330 mmol) phosphonate <bold>8c</bold> was obtained as a yellowish oil (1.538 g, 93 %) after chromatography on a silica gel column with chloroform/methanol (100:1, v/v). IR (film): <inline-formula id="IEq4"><alternatives><tex-math id="M7">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M8"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq4.gif"></inline-graphic></alternatives></inline-formula> = 3,459, 2,983, 2,957, 2,908, 1,732, 1,462, 1,225, 1,117, 1,060, 1,027, 960 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 600 MHz): <italic>δ</italic> = 1.31 (t, <italic>J</italic> = 7.0 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 3.74 (d, <italic>J</italic> = 7.9 Hz, 2H, PCH<sub>2</sub>), 3.98 (s, 3H, OCH<sub>3</sub>), 4.02 (s, 3H, OCH<sub>3</sub>), 4.02 (t, <italic>J</italic> = 5.2 Hz, 2H, OC<italic>H</italic><sub>2</sub>CH<sub>2</sub>N), 4.08–4.13 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.86 (t, <italic>J</italic> = 5.2 Hz, 2H, OCH<sub>2</sub>C<italic>H</italic><sub>2</sub>N) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.5 (d, <italic>J</italic> = 5.7 Hz, POC<italic>C</italic>), 49.8 (s, CN), 52.8 (s, OCH<sub>3</sub>), 53.5 (s, OCH<sub>3</sub>), 62.6 (d, <italic>J</italic> = 6.5 Hz, PO<italic>C</italic>C), 65.2 (d, <italic>J</italic> = 164.7 Hz, PC), 71.0 (d, <italic>J</italic> = 9.3 Hz, PCO<italic>C</italic>), 131.0 and 139.6 (2 s, C=C), 158.8 and 160.3 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 243 MHz): <italic>δ</italic> = 20.04 ppm.</p></sec><sec id="d29e1899"><title><italic>Diethyl 2</italic>-<italic>[2</italic>-<italic>(4,5</italic>-<italic>dimethoxycarbonyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)ethoxy]ethylphosphonate</italic> (<bold>8d</bold>, C<sub>14</sub>H<sub>24</sub>N<sub>3</sub>O<sub>8</sub>P)</title><p>From 0.647 g azidophosphonate <bold>7d</bold> (2.68 mmol) and 0.382 g dimethyl acetylenedicarboxylate <bold>13</bold> (2.68 mmol) phosphonate <bold>8d</bold> (1.030 g, 98 %) was obtained as a yellowish oil. The crude product was sufficiently pure and was used in the next step without further purification. IR (film): <inline-formula id="IEq5"><alternatives><tex-math id="M9">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M10"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq5.gif"></inline-graphic></alternatives></inline-formula> = 3,459, 2,983, 2,957, 2,909, 1,736, 1,466, 1,229, 1,118, 1,063, 1,027, 963 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 600 MHz): <italic>δ</italic> = 1.32 (t, <italic>J</italic> = 7.1 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.00 (dt, <italic>J</italic> = 15.3, 7.7 Hz, 2H, PCH<sub>2</sub>), 3.63 (dt, <italic>J</italic> = 10.4, 7.7 Hz, 2H, PCH<sub>2</sub>C<italic>H</italic><sub>2</sub>O), 3.82 (t, <italic>J</italic> = 5.2 Hz, 2H, OC<italic>H</italic><sub>2</sub>CH<sub>2</sub>N), 4.00 (s, 3H, OCH<sub>3</sub>), 4.01 (s, 3H, OCH<sub>3</sub>), 4.05–4.12 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.82 (t, <italic>J</italic> = 5.2 Hz, 2H, OCH<sub>2</sub>C<italic>H</italic><sub>2</sub>N) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 150 MHz): <italic>δ</italic> = 16.3 (d, <italic>J</italic> = 5.7 Hz, POC<italic>C</italic>), 26.7 (d, <italic>J</italic> = 139.3 Hz, PC), 49.8 (s, OC<italic>C</italic>N), 52.6 (s, OCH<sub>3</sub>), 53.3 (s, OCH<sub>3</sub>), 61.6 (d, <italic>J</italic> = 6.4 Hz, C<italic>C</italic>OP), 65.3 (s, PC<italic>C</italic>), 68.7 (s, O<italic>C</italic>CN), 131.4 and 139.5 (2 s, C=C), 159.1 and 160.3 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 243 MHz): <italic>δ</italic> = 27.58 ppm.</p></sec><sec id="d29e2107"><title><italic>Diethyl 1</italic>-<italic>hydroxy</italic>-<italic>3</italic>-<italic>(4,5</italic>-<italic>dimethoxycarbonyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)propylphosphonate</italic> (<bold>8f</bold>, C<sub>13</sub>H<sub>22</sub>N<sub>3</sub>O<sub>8</sub>P)</title><p>From 0.560 g azidophosphonate <bold>7f</bold> (2.36 mmol) and 0.335 g dimethyl acetylenedicarboxylate <bold>13</bold> (2.36 mmol) phosphonate <bold>8f</bold> (0.850 g, 95 %) was obtained as a white amorphous solid after chromatography on a silica gel column with chloroform/methanol (100:1, v/v). M.p.: 107–109 °C; IR (film): <inline-formula id="IEq6"><alternatives><tex-math id="M11">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M12"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq6.gif"></inline-graphic></alternatives></inline-formula> = 3,420, 3,218, 3,081, 2,985, 2,878, 1,675, 1,451, 1,280, 1,223, 1,030, 970 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.33 and 1.34 (2 t, <italic>J</italic> = 7.2 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.28–2.47 (m, 2H, PCC<italic>H</italic><sub>2</sub>), 3.70 (dd, <italic>J</italic> = 6.0, 5.1 Hz, 1H, OH), 3.84 (dddd, <italic>J</italic> = 10.5, 6.3, 6.0, 3.3 Hz, 1H, PCH), 3.98 (s, OCH<sub>3</sub>), 4.00 (s, OCH<sub>3</sub>), 4.10–4.22 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.75–4.90 (m, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.6 and 16.7 (2 d, <italic>J</italic> = 5.4 Hz, <italic>C</italic>COP), 31.9 (d, <italic>J</italic> = 3.2 Hz, PC<italic>C</italic>), 47.2 (d, <italic>J</italic> = 16.3 Hz, PCC<italic>C</italic>), 52.8 (s, OCH<sub>3</sub>), 53.6 (s, OCH<sub>3</sub>), 63.0 and 63.3 (2 d, <italic>J</italic> = 7.3 Hz, C<italic>C</italic>OP), 64.6 (d, <italic>J</italic> = 164.2 Hz, PC), 130.3 and 139.7 (2 s, C=C), 158.8 and 160.4 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 23.5 ppm.</p></sec><sec id="Sec9"><title>Synthesis of diamides <bold>9a</bold>–<bold>9g</bold> (general procedure)</title><p>To a solution of the diester <bold>8a</bold>–<bold>8g</bold> (1.00 mmol) in 14 cm<sup>3</sup> ethanol 16 cm<sup>3</sup> was added aqueous ammonia. The reaction mixtures were stirred at room temperature for 24 h. Ethanol and excess ammonia were evaporated in vacuo. Diamides <bold>9a</bold>–<bold>9g</bold> were purified on silica gel columns with chloroform/methanol or by crystallisation.</p><sec id="d29e2324"><title><italic>Diethyl 2</italic>-<italic>(4,5</italic>-<italic>dicarbamoyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)ethylphosphonate</italic> (<bold>9a</bold>, C<sub>10</sub>H<sub>18</sub>N<sub>5</sub>O<sub>5</sub>P)</title><p>From 0.165 g diester <bold>8a</bold> (0.472 mmol) diamide <bold>9a</bold> was obtained as a white amorphous solid (0.065 g, 42 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, v/v) followed by crystallisation from ethanol/diethyl ether. M.p.: 172–173 °C; IR (KBr): <inline-formula id="IEq7"><alternatives><tex-math id="M13">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M14"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq7.gif"></inline-graphic></alternatives></inline-formula> = 3,428, 3,228, 3,113, 2,986, 2,932, 1,687, 1,451, 1,221, 1,023 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.34 (t, <italic>J</italic> = 6.9 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.41–2.53 (m, 2H, PCH<sub>2</sub>), 4.09–4.21 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 5.08–5.17 (m, 2H, CH<sub>2</sub>N), 6.01 (s, 2H, NH<sub>2</sub>), 7.58 (s, 1H, NH), 10.77 (s, 1H, NH) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 75 MHz): <italic>δ</italic> = 16.8 (d, <italic>J</italic> = 6.0 Hz, <italic>C</italic>COP), 27.3 (d, <italic>J</italic> = 140.3 Hz, PC), 47.2 (s, PC<italic>C</italic>N), 63.8 (d, <italic>J</italic> = 6.6 Hz, C<italic>C</italic>OP), 132.1 and 140.4 (2 s, C=C), 160.3 and 165.2 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 26.5 ppm.</p></sec><sec id="d29e2472"><title><italic>Diethyl 3</italic>-<italic>(4,5</italic>-<italic>dicarbamoyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)propylphosphonate</italic> (<bold>9b</bold>, C<sub>11</sub>H<sub>20</sub>N<sub>5</sub>O<sub>5</sub>P)</title><p>From 0.114 g diester <bold>8b</bold> (0.314 mmol) diamide <bold>9b</bold> was obtained as a white amorphous solid (0.070 g, 61 %) after chromatography on a silica gel column with chloroform/methanol (50:1, v/v) and crystallisation from ethanol. M.p.: 143–144 °C; IR (KBr): <inline-formula id="IEq8"><alternatives><tex-math id="M15">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M16"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq8.gif"></inline-graphic></alternatives></inline-formula> = 3,455, 3,270, 2,984, 2,909, 1,694, 1,629, 1,592, 1,454, 1,205, 1,018, 961 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.31 (t, <italic>J</italic> = 6.9 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 1.74–1.86 (m, 2H, PCH<sub>2</sub>C<italic>H</italic><sub>2</sub>), 2.18–2.32 (m, 2H, PCH<sub>2</sub>), 4.04–4.16 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.98 (t, <italic>J</italic> = 6.9 Hz, 2H, CH<sub>2</sub>N), 6.13 (s, 1H, NH), 6.25 (s, 1H, NH), 7.62 (s, 1H, NH), 10.79 (s, 1H, NH) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.7 (d, <italic>J</italic> = 6.6 Hz, <italic>C</italic>COP), 23.0 (d, <italic>J</italic> = 141.9 Hz, PC), 23.8 (d, <italic>J</italic> = 4.3 Hz, PC<italic>C</italic>), 51.8 (d, <italic>J</italic> = 19.2 Hz, PCC<italic>C</italic>), 62.0 (d, <italic>J</italic> = 6.6 Hz, C<italic>C</italic>OP), 130.7 and 138.7 (2 s, C=C), 158.6 and 163.7 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 31.4 ppm.</p></sec><sec id="d29e2639"><title><italic>Diethyl [2</italic>-<italic>(4,5</italic>-<italic>dicarbamoyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)ethoxy]methylphosphonate</italic> (<bold>9c</bold>, C<sub>11</sub>H<sub>20</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.280 g diester <bold>8c</bold> (0.738 mmol) diamide <bold>9c</bold> was obtained as a white amorphous solid (0.139 g, 54 %) after chromatography on a silica gel column with chloroform/methanol (50:1, v/v). M.p.: 100–101 °C; IR (KBr): <inline-formula id="IEq9"><alternatives><tex-math id="M17">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M18"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq9.gif"></inline-graphic></alternatives></inline-formula> = 3,361, 3,259, 3,110, 2,990, 2,959, 2,901, 1,689, 1,613, 1,454, 1,303, 1,219, 1,118, 1,048, 1,019, 967, 943 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 600 MHz): <italic>δ</italic> = 1.29 (t, <italic>J</italic> = 7.1 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 3.89 (d, <italic>J</italic> = 8.5 Hz, 2H, PCH<sub>2</sub>), 4.06 (t, <italic>J</italic> = 5.2 Hz, 2H, OC<italic>H</italic><sub>2</sub>CH<sub>2</sub>N), 4.06–4.10 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 5.14 (t, <italic>J</italic> = 5.2 Hz, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 150 MHz): <italic>δ</italic> = 15.3 (d, <italic>J</italic> = 5.9 Hz, <italic>C</italic>COP), 50.3 (s, CN), 62.8 (d, <italic>J</italic> = 6.5 Hz, C<italic>C</italic>OP), 64.0 (d, <italic>J</italic> = 164.7 Hz, PC), 71.1 (d, <italic>J</italic> = 11.9 Hz, PCO<italic>C</italic>), 131.0 and 139.0 (2 s, C=C), 159.1 and 163.9 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 243 MHz): <italic>δ</italic> = 21.3 ppm.</p></sec><sec id="d29e2806"><title><italic>Diethyl 2</italic>-<italic>[2</italic>-<italic>(4,5</italic>-<italic>dicarbamoyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)ethoxy]ethylphosphonate</italic> (<bold>9d</bold>, C<sub>12</sub>H<sub>22</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.264 g diester <bold>8d</bold> (0.671 mmol) diamide <bold>9d</bold> was obtained as a white amorphous solid (0.090 g, 37 %) after chromatography on a silica gel column with chloroform/methanol (50:1, v/v) and crystallisation from ethyl acetate. M.p.: 102–103 °C; IR (KBr): <inline-formula id="IEq10"><alternatives><tex-math id="M19">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M20"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq10.gif"></inline-graphic></alternatives></inline-formula> = 3,400, 3,306, 3,214, 2,986, 2,906, 1,672, 1,608, 1,453, 1,245, 1,109, 1,024, 961 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.31 (t, <italic>J</italic> = 6.9 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.05 (dt, <italic>J</italic> = 15.3, 7.8 Hz, 2H, PCH<sub>2</sub>), 3.69 (dt, <italic>J</italic> = 11.1, 7.8 Hz, 2H, PCH<sub>2</sub>C<italic>H</italic><sub>2</sub>O), 3.91 (t, <italic>J</italic> = 5.4 Hz, 2H, OC<italic>H</italic><sub>2</sub>CH<sub>2</sub>N), 4.01–4.12 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 5.11 (t, <italic>J</italic> = 5.4 Hz, 2H, OCH<sub>2</sub>C<italic>H</italic><sub>2</sub>N), 6.00 (s, 1H, NH), 6.07 (s, 1H, NH), 7.60 (s, 1H, NH), 10.77 (s, 1H, NH) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.6 (d, <italic>J</italic> = 6.0 Hz, CCOP), 26.9 (d, <italic>J</italic> = 139.1 Hz, PC), 51.0 (s, OC<italic>C</italic>N), 61.9 (d, <italic>J</italic> = 6.3 Hz, C<italic>C</italic>OP), 65.0 (s, PC<italic>C</italic>O), 69.1 (s, O<italic>C</italic>CN), 130.9 and 139.7 (2 s, C=C), 158.7 and 163.7 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 29.2 ppm.</p></sec><sec id="d29e2995"><title><italic>Diethyl 3</italic>-<italic>(4,5</italic>-<italic>dicarbamoyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)</italic>-<italic>2</italic>-<italic>hydroxypropylphosphonate</italic> (<bold>9e</bold>, C<sub>11</sub>H<sub>20</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.145 g diester <bold>8e</bold> (0.382 mmol) diamide <bold>9e</bold> was obtained as a white amorphous solid (0.060 g, 45 %) after chromatography on a silica gel column with chloroform/methanol (50:1, v/v) and crystallisation from methanol. M.p.: 142–143 °C; IR (KBr): <inline-formula id="IEq11"><alternatives><tex-math id="M21">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M22"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq11.gif"></inline-graphic></alternatives></inline-formula> = 3,428, 3,308, 3,034, 2,988, 1,686, 1,667, 1,599, 1,443, 1,375, 1,293, 1,113, 1,083, 1,055, 1,029, 960 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 300 MHz): <italic>δ</italic> = 1.32 (t, <italic>J</italic> = 7.2 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.06 (ddd, <italic>J</italic> = 22.8, 15.3, 7.8 Hz, 1H, PC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 2.18 (ddd, <italic>J</italic> = 20.4, 15.3, 5.1 Hz, 1H, PCH<sub>a</sub><italic>H</italic><sub>b</sub>), 4.07–4.19 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.37–4.50 (m, 1H, PCC<italic>H</italic>), 4.99 (dd, <italic>J</italic> = 13.5, 8.1 Hz, 1H, PCCC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 5.02 (dd, <italic>J</italic> = 13.5, 4.2 Hz, 1H, PCCCH<sub>a</sub><italic>H</italic><sub>b</sub>) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 75 MHz): <italic>δ</italic> = 16.9 (d, <italic>J</italic> = 6.3 Hz, <italic>C</italic>COP), 32.3 (d, <italic>J</italic> = 141.7 Hz, PC), 58.1 (d, <italic>J</italic> = 18.0 Hz, PCC<italic>C</italic>), 63.4 and 63.7 (2 d, <italic>J</italic> = 6.6 Hz, C<italic>C</italic>OP), 67.0 (d, <italic>J</italic> = 4.0 Hz, PC<italic>C</italic>), 132.6 and 140.4 (2 s, C=C), 160.6 and 165.3 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 121 MHz): <italic>δ</italic> = 30.1 ppm.</p></sec><sec id="d29e3203"><title><italic>Diethyl 3</italic>-<italic>(4,5</italic>-<italic>dicarbamoyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)</italic>-<italic>1</italic>-<italic>hydroxypropylphosphonate</italic> (<bold>9f</bold>, C<sub>11</sub>H<sub>20</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.180 g diester <bold>8f</bold> (0.475 mmol) diamide <bold>9f</bold> was obtained as a white amorphous solid (0.093 g, 56 %) after crystallisation from methanol. M.p.: 179–180 °C; IR (KBr): <inline-formula id="IEq12"><alternatives><tex-math id="M23">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M24"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq12.gif"></inline-graphic></alternatives></inline-formula> = 3,433, 3,319, 3,212, 2,974, 2,930, 2,874, 1,668, 1,605, 1,455, 1,399, 1,221, 1,167, 1,066, 1,014, 953 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 300 MHz): <italic>δ</italic> = 1.32 (t, <italic>J</italic> = 7.2 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.13–2.28 (m, 1H, PCC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 2.31–2.44 (m, 1H, PCCH<sub>a</sub><italic>H</italic><sub>b</sub>), 3.89 (ddd, <italic>J</italic> = 10.5, 7.2, 3.0 Hz, 1H, PCH), 4.10–4.21 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.95–5.14 (m, 2H, CH<sub>2</sub>N) ppm; solubility of <bold>9f</bold> in D<sub>2</sub>O or CD<sub>3</sub>OD was not sufficient to measure the <sup>13</sup>C NMR spectrum; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 121 MHz): <italic>δ</italic> = 25.4 ppm.</p></sec><sec id="d29e3357"><title><italic>Diethyl 2</italic>-<italic>(4,5</italic>-<italic>dicarbamoyl</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl)</italic>-<italic>1</italic>-<italic>hydroxyethylphosphonate</italic> (<bold>9g</bold>, C<sub>10</sub>H<sub>18</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.230 g diester <bold>8g</bold> (0.630 mmol) diamide <bold>9g</bold> was obtained as a white amorphous solid (0.112 g, 53 %) after chromatography on a silica gel column with chloroform/methanol (20:1, v/v) and crystallisation from methanol. M.p.: 200–202 °C; IR (KBr): <inline-formula id="IEq13"><alternatives><tex-math id="M25">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M26"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq13.gif"></inline-graphic></alternatives></inline-formula> = 3,431, 3,223, 3,081, 2,990, 2,847, 1,671, 1,599, 1,452, 1,393, 1,289, 1,225, 1,031, 979 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 600 MHz): <italic>δ</italic> = 1.39 and 1.40 (2 t, <italic>J</italic> = 7.2 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 4.22-4.29 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.55 (ddd, <italic>J</italic> = 10.2, 9.9 Hz, 3.6 Hz, 1H, PCH), 5.10 (ddd, <italic>J</italic> = 13.5, 10.2, 8.7 Hz, 1H, PCC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 5.22 (ddd, <italic>J</italic> = 13.5, 3.6, 3.1 Hz, 1H, PCCH<sub>a</sub><italic>H</italic><sub>b</sub>) ppm; solubility of <bold>9g</bold> in D<sub>2</sub>O and CD<sub>3</sub>OD was not sufficient to measure the <sup>13</sup>C NMR spectrum; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 243 MHz): <italic>δ</italic> = 21.2 ppm.</p></sec></sec><sec id="Sec10"><title>Synthesis of dihydrazides <bold>10a</bold>–<bold>10g</bold> (general procedure)</title><p>A solution of the diester <bold>8a</bold>–<bold>8g</bold> (0.73 mmol) and 0.530 cm<sup>3</sup> hydrazine hydrate (10.8 mmol) in 6 cm<sup>3</sup> ethanol was refluxed for 2 h. The reaction mixtures were concentrated to give yellow oils or solids which were subjected to chromatography on a silica gel column with chloroform/methanol or crystallisation to obtain dihydrazides <bold>10a</bold>–<bold>10g</bold>.</p><sec id="d29e3545"><title><italic>Diethyl 2</italic>-<italic>[4,5</italic>-<italic>bis(hydrazinocarbonyl)</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl]ethylphosphonate</italic> (<bold>10a</bold>, C<sub>10</sub>H<sub>20</sub>N<sub>7</sub>O<sub>5</sub>P)</title><p>From 0.115 g diester <bold>8a</bold> (0.330 mmol) dihydrazide <bold>10a</bold> was obtained (0.070 g, 61 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, 10:1, v/v) as a white amorphous solid. M.p.: 84–86 °C; IR (KBr): <inline-formula id="IEq14"><alternatives><tex-math id="M27">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M28"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq14.gif"></inline-graphic></alternatives></inline-formula> = 3,335, 3,282, 2,984, 2,932, 1,661, 1,606, 1,551, 1,491, 1,262, 1,223, 1,056, 1,020, 963 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 300 MHz): <italic>δ</italic> = 1.32 (t, <italic>J</italic> = 6.6 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>O), 2.48–2.59 (m, 2H, PCH<sub>2</sub>), 4.07–4.18 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>O), 5.03–5.14 (m, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 75 MHz): <italic>δ</italic> = 16.9 (d, <italic>J</italic> = 6.0 Hz, <italic>C</italic>COP), 27.4 (d, <italic>J</italic> = 140.0 Hz, PC), 47.1 (s, PC<italic>C</italic>N), 63.8 (d, <italic>J</italic> = 6.3 Hz, C<italic>C</italic>OP × 2), 130.8 and 139.3 (2 s, C=C), 157.6 and 161.8 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 121 MHz): <italic>δ</italic> = 28.2 ppm.</p></sec><sec id="d29e3690"><title><italic>Diethyl 3</italic>-<italic>[4,5</italic>-<italic>bis(hydrazinocarbonyl)</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl]propylphosphonate</italic> (<bold>10b</bold>, C<sub>11</sub>H<sub>22</sub>N<sub>7</sub>O<sub>5</sub>P)</title><p>From 0.170 g diester <bold>8b</bold> (0.468 mmol) dihydrazide <bold>10b</bold> was obtained as a white amorphous solid (0.113 g, 66 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, 10:1, v/v). M.p.: 73–75 °C; IR (KBr): <inline-formula id="IEq15"><alternatives><tex-math id="M29">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M30"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq15.gif"></inline-graphic></alternatives></inline-formula> = 3,300, 3,192, 2,983, 2,931, 1,656, 1,551, 1,209, 1,024, 969 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.28 (t, <italic>J</italic> = 6.9 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 1.71–1.82 (m, 2H, PCH<sub>2</sub>C<italic>H</italic><sub>2</sub>), 2.13–2.27 (m, 2H, PCH<sub>2</sub>), 4.00–4.13 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.94 (t, <italic>J</italic> = 7.2 Hz, 2H, CH<sub>2</sub>N), 5.15–6.03 (brs, 6H, NHNH<sub>2</sub>) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.7 (d, <italic>J</italic> = 6.0 Hz, <italic>C</italic>COP), 22.9 (d, <italic>J</italic> = 139.9 Hz, PC), 23.9 (d, <italic>J</italic> = 7.4 Hz, PC<italic>C</italic>), 51.8 (d, <italic>J</italic> = 18.9 Hz, PCC<italic>C</italic>), 62.0 (d, <italic>J</italic> = 6.6 Hz, C<italic>C</italic>OP), 129.5 and 137.6 (2 s, C=C), 156.3 and 161.3 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 31.5 ppm.</p></sec><sec id="d29e3861"><title><italic>Diethyl [2</italic>-<italic>[4,5</italic>-<italic>bis(hydrazinocarbonyl)</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl]ethoxy]methylphosphonate</italic> (<bold>10c</bold>, C<sub>11</sub>H<sub>22</sub>N<sub>7</sub>O<sub>6</sub>P)</title><p>From 0.430 g diester <bold>8c</bold> (1.13 mmol) dihydrazide <bold>10c</bold> was obtained as a colourless oil (0.139 g, 32 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, 10:1, v/v). IR (film): <inline-formula id="IEq16"><alternatives><tex-math id="M31">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M32"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq16.gif"></inline-graphic></alternatives></inline-formula> = 3,326, 3,248, 2,985, 2,918, 2,890, 1,666, 1,544, 1,440, 1,300, 1,238, 1,117, 1,024, 968 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 600 MHz): <italic>δ</italic> = 1.28 (t, <italic>J</italic> = 7.0 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 3.89 (d, <italic>J</italic> = 8.5 Hz, 2H, PCH<sub>2</sub>), 4.05–4.10 (m, 6H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP and OC<italic>H</italic><sub>2</sub>CH<sub>2</sub>N), 5.14 (t, <italic>J</italic> = 4.8 Hz, 2H, OCH<sub>2</sub>C<italic>H</italic><sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 150 MHz): <italic>δ</italic> = 15.3 (d, <italic>J</italic> = 5.5 Hz, <italic>C</italic>COP), 50.2 (s, CN), 62.8 (d, <italic>J</italic> = 6.5 Hz, C<italic>C</italic>OP), 64.0 (d, <italic>J</italic> = 164.9 Hz, PC), 71.1 (d, <italic>J</italic> = 11.9 Hz, PCO<italic>C</italic>), 129.9 and 137.9 (2 s, C=C), 156.5 and 160.5 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 243 MHz): <italic>δ</italic> = 21.4 ppm.</p></sec><sec id="d29e4032"><title><italic>Diethyl 2</italic>-<italic>[2</italic>-<italic>[4,5</italic>-<italic>bis(hydrazinocarbonyl)</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl]ethoxy]ethylphosphonate</italic> (<bold>10d</bold>, C<sub>12</sub>H<sub>24</sub>N<sub>7</sub>O<sub>6</sub>P)</title><p>From 0.216 g diester <bold>8d</bold> (0.549 mmol) dihydrazide <bold>10d</bold> was obtained as a white amorphous solid (0.128 g, 59 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, v/v) and crystallisation from ethyl acetate. M.p: 95–96 °C; IR (KBr): <inline-formula id="IEq17"><alternatives><tex-math id="M33">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M34"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq17.gif"></inline-graphic></alternatives></inline-formula> = 3,300, 3,232, 2,983, 2,913, 2,827, 1,667, 1,545, 1,550, 1,252, 1,023, 966, 918 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.31 (t, <italic>J</italic> = 7.2 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.03 (dt, <italic>J</italic> = 15.0, 7.5 Hz, 2H, PCH<sub>2</sub>), 3.69 (dt, <italic>J</italic> = 11.1, 7.5 Hz, 2H, PCH<sub>2</sub>C<italic>H</italic><sub>2</sub>O), 3.91 (t, <italic>J</italic> = 5.7 Hz, 2H, OC<italic>H</italic><sub>2</sub>CH<sub>2</sub>N), 4.01–4.13 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.20 (brs, 4H, NH<sub>2</sub>), 5.13 (t, <italic>J</italic> = 5.7 Hz, 2H, OCH<sub>2</sub>C<italic>H</italic><sub>2</sub>N), 8.81 (s, 1H, NH), 12.0 (s, 1H, NH) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.6 (d, <italic>J</italic> = 6.0 Hz, <italic>C</italic>COP), 27.0 (d, <italic>J</italic> = 138.5 Hz, PC), 51.0 (s, OC<italic>C</italic>N), 61.9 (d, <italic>J</italic> = 6.3 Hz, C<italic>C</italic>OP), 65.0 (s, PC<italic>C</italic>O), 69.1 (s, O<italic>C</italic>CN), 129.9 and 137.5 (2 s, C=C), 156.5 and 161.3 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 28.5 ppm.</p></sec><sec id="d29e4228"><title><italic>Diethyl 3</italic>-<italic>[4,5</italic>-<italic>bis(hydrazinocarbonyl)</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl]</italic>-<italic>2</italic>-<italic>hydroxypropylphosphonate</italic> (<bold>10e</bold>, C<sub>11</sub>H<sub>22</sub>N<sub>7</sub>O<sub>6</sub>P)</title><p>From 0.290 g diester <bold>8e</bold> (0.764 mmol) dihydrazide <bold>10e</bold> was obtained as a white amorphous solid (0.140 g, 48 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, v/v) and crystallisation from ethanol. M.p.: 140–142 °C; IR (KBr): <inline-formula id="IEq18"><alternatives><tex-math id="M35">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M36"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq18.gif"></inline-graphic></alternatives></inline-formula> = 3,336, 3,275, 2,980, 2,930, 1,668, 1,578, 1,549, 1,443,1,260, 1,217, 1,067, 1,027, 957 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 600 MHz): <italic>δ</italic> = 1.34 and 1.35 (2 t, <italic>J</italic> = 7.1 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.12 (ddd, <italic>J</italic> = 23.6, 15.4, 8.2 Hz, 1H, PC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 2.18 (ddd, <italic>J</italic> = 20.1, 15.4, 4.7 Hz, 1H, PCH<sub>a</sub><italic>H</italic><sub>b</sub>), 4.11–4.19 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.44–4.47 (m, 1H, PCC<italic>H</italic>), 4.96 (dd, <italic>J</italic> = 13.4, 8.4 Hz, 1H, PCCC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 5.05 (dd, <italic>J</italic> = 13.4, 4.1 Hz, 1H, PCCCH<sub>a</sub><italic>H</italic><sub>b</sub>) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 150 MHz): <italic>δ</italic> = 15.3 (d, <italic>J</italic> = 5.8 Hz, <italic>C</italic>COP), 30.8 (d, <italic>J</italic> = 141.6 Hz, PC), 56.4 (d, <italic>J</italic> = 17.3 Hz, PCC<italic>C</italic>), 61.9 and 62.2 (2 d, <italic>J</italic> = 6.5 Hz, C<italic>C</italic>OP), 65.6 (d, <italic>J</italic> = 3.6 Hz, PC<italic>C</italic>), 130.1 and 137.9 (2 s, C=C), 156.7 and 160.6 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 243 MHz): <italic>δ</italic> = 28.9 ppm.</p></sec><sec id="d29e4436"><title><italic>Diethyl 3</italic>-<italic>[4,5</italic>-<italic>bis(hydrazinocarbonyl)</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl]</italic>-<italic>1</italic>-<italic>hydroxypropylphosphonate</italic> (<bold>10f</bold>, C<sub>11</sub>H<sub>22</sub>N<sub>7</sub>O<sub>6</sub>P)</title><p>From 0.262 g diester <bold>8f</bold> (0.691 mmol) dihydrazide <bold>10f</bold> was obtained as a white amorphous solid (0.155 g, 60 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 30:1, v/v). M.p.: 119–120 °C; IR (KBr): <inline-formula id="IEq19"><alternatives><tex-math id="M37">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M38"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq19.gif"></inline-graphic></alternatives></inline-formula> = 3,316, 3,266, 3,160, 2,976, 2,930, 1,659, 1,572, 1,532, 1,249, 1,210, 1,072, 1,022, 955 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 300 MHz): <italic>δ</italic> = 1.31 (t, <italic>J</italic> = 7.5 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.13-2.29 (m, 1H, PCC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 2.31–2.44 (m, 1H, PCCH<sub>a</sub><italic>H</italic><sub>b</sub>), 3.91 (ddd, <italic>J</italic> = 10.8, 7.8, 3.3 Hz, 1H, PCH), 4.09–4.21 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.96–5.15 (m, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 75 MHz): <italic>δ</italic> = 17.0 (d, <italic>J</italic> = 5.4 Hz, <italic>C</italic>COP), 33.4 (s, PC<italic>C</italic>), 48.4 (d, <italic>J</italic> = 18.9 Hz, PCC<italic>C</italic>), 64.2 and 64.5 (d, <italic>J</italic> = 7.1 Hz, C<italic>C</italic>OP), 65.6 (d, <italic>J</italic> = 166.7 Hz, PC), 130.9 and 139.2 (2 s, C=C), 157.7 and 161.8 (2 s, C=O × 2) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 121 MHz): <italic>δ</italic> = 25.5 ppm.</p></sec><sec id="d29e4613"><title><italic>Diethyl 2</italic>-<italic>[4,5</italic>-<italic>bis(hydrazinocarbonyl)</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazol</italic>-<italic>1</italic>-<italic>yl]</italic>-<italic>1</italic>-<italic>hydroxyethylphosphonate</italic> (<bold>10</bold><bold>g</bold>, C<sub>10</sub>H<sub>20</sub>N<sub>7</sub>O<sub>6</sub>P)</title><p>From 0.450 g diester <bold>8g</bold> (1.23 mmol) dihydrazide <bold>10g</bold> was obtained as a white amorphous solid (0.128 g, 28 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 30:1, v/v). M.p.: 149–152 °C; IR (KBr): <inline-formula id="IEq20"><alternatives><tex-math id="M39">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M40"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq20.gif"></inline-graphic></alternatives></inline-formula> = 3,334, 3,207, 2,980, 2,930, 2,870, 1,668, 1,578, 1,449, 1,217, 1,067, 1,028, 953 cm<sup>−1</sup>; <sup>1</sup>H NMR (D<sub>2</sub>O, 600 MHz): <italic>δ</italic> = 1.31 (t, <italic>J</italic> = 7.0 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 4.18–4.24 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.53–4.58 (m, 1H, PCH), 4.94–5.01 (m, 1H, PCC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 5.11–5.16 (m, 1H, PCCH<sub>a</sub><italic>H</italic><sub>b</sub>) ppm; <sup>13</sup>C NMR (D<sub>2</sub>O, 150 MHz): <italic>δ</italic> = 15.7 (d, <italic>J</italic> = 4.6 Hz, <italic>C</italic>COP), 51.9 (d, <italic>J</italic> = 11.2 Hz, PC<italic>C</italic>N), 64.7 and 64.8 (2 d, <italic>J</italic> = 7.4 Hz, C<italic>C</italic>OP), 65.5 (d, <italic>J</italic> = 165.8 Hz, PC), 130.4 and 137.5 (2 s, C=C), 156.5 and 160.2 (2 s, C=O) ppm; <sup>31</sup>P NMR (D<sub>2</sub>O, 243 MHz): <italic>δ</italic> = 21.8 ppm.</p></sec></sec><sec id="Sec11"><title>Synthesis of 1,2,3-triazolopyridazinediones <bold>11a</bold>–<bold>11g</bold> (general procedure)</title><p>A mixture of the dihydrazide <bold>10a</bold>–<bold>10g</bold> (0.50 mmol) and 5 cm<sup>3</sup> 10 % hydrochloric acid was heated at 90 °C for 2.5 h. After concentration in vacuo, crude products were purified on a silica gel column with chloroform/methanol or crystallised from the appropriate solvent.</p><sec id="d29e4802"><title><italic>Diethyl 2</italic>-<italic>(4,7</italic>-<italic>dioxo</italic>-<italic>5,6</italic>-<italic>dihydro</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazolo[4,5</italic>-<italic>d]pyridazin</italic>-<italic>1</italic>-<italic>yl)ethylphosphonate</italic> (<bold>11a</bold>, C<sub>10</sub>H<sub>16</sub>N<sub>5</sub>O<sub>5</sub>P)</title><p>From 0.167 g dihydrazide <bold>10a</bold> (0.478 mmol) compound <bold>11a</bold> was obtained as a white amorphous solid (0.045 g, 30 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, 10:1, v/v) followed by crystallisation from ethanol. M.p.: 198–200 °C; IR (KBr): <inline-formula id="IEq21"><alternatives><tex-math id="M41">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M42"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq21.gif"></inline-graphic></alternatives></inline-formula> = 3,385, 2,922, 2,672, 1,687, 1,582, 1,460, 1,262, 1,214, 1,014, 1,019, 980 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 300 MHz): <italic>δ</italic> = 1.28 (t, <italic>J</italic> = 6.6 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.58 (dt, <italic>J</italic> = 14.7 Hz, <italic>J</italic> = 7.5 Hz, PCH<sub>2</sub>), 4.03–4.13 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 5.12 (dt, <italic>J</italic> = 12.6 Hz, <italic>J</italic> = 7.5 Hz, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 150 MHz): <italic>δ</italic> = 17.5 (d, <italic>J</italic> = 5.7 Hz, <italic>C</italic>COP), 28.2 (d, <italic>J</italic> = 141.0 Hz, PC), 46.5 (d, <italic>J</italic> = 3.0 Hz, PC<italic>C</italic>N), 64.7 (d, <italic>J</italic> = 6.6 Hz, C<italic>C</italic>OP), 131.3 and 141.8 (2 s, C=C), 152.0 and 154.6 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 121 MHz): <italic>δ</italic> = 27.7 ppm.</p></sec><sec id="d29e4972"><title><italic>Diethyl 3</italic>-<italic>(4,7</italic>-<italic>dioxo</italic>-<italic>5,6</italic>-<italic>dihydro</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazolo[4,5</italic>-<italic>d]pyridazin</italic>-<italic>1</italic>-<italic>yl)propylphosphonate</italic> (<bold>11b</bold>, C<sub>11</sub>H<sub>18</sub>N<sub>5</sub>O<sub>5</sub>P)</title><p>From 0.055 g dihydrazide <bold>10b</bold> (0.151 mmol) compound <bold>11b</bold> was obtained as a white amorphous solid (0.033 g, 66 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, 10:1, v/v). M.p.: 148–150 °C; IR (KBr): <inline-formula id="IEq22"><alternatives><tex-math id="M43">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M44"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq22.gif"></inline-graphic></alternatives></inline-formula> = 3,446, 3,074, 2,991, 2,960, 1,662, 1,555, 1,462, 1,294, 1,218, 1,032, 972, 805 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 300 MHz): <italic>δ</italic> = 1.31 (t, <italic>J</italic> = 6.9 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 1.84–1.95 (m, 2H, PCH<sub>2</sub>C<italic>H</italic><sub>2</sub>), 2.24–2.38 (m, 2H, PCH<sub>2</sub>), 4.02–4.15 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.93 (t, <italic>J</italic> = 6.9 Hz, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 75 MHz): <italic>δ</italic> = 16.7 (d, <italic>J</italic> = 5.7 Hz, <italic>C</italic>COP), 22.7 (d, <italic>J</italic> = 141.9 Hz, PC), 23.9 (s, PC<italic>C</italic>), 50.5 (d, <italic>J</italic> = 18.5 Hz, PCC<italic>C</italic>), 62.6 (d, <italic>J</italic> = 6.5 Hz, C<italic>C</italic>OP), 129.1 and 139.7 (2 s, C=C), 150.5 and 152.7 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 121 MHz): <italic>δ</italic> = 32.6 ppm.</p></sec><sec id="d29e5145"><title><italic>Diethyl [2</italic>-<italic>(4,7</italic>-<italic>dioxo</italic>-<italic>5,6</italic>-<italic>dihydro</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazolo[4,5</italic>-<italic>d]pyridazin</italic>-<italic>1</italic>-<italic>yl)ethoxy]methylphosphonate</italic> (<bold>11c</bold>, C<sub>11</sub>H<sub>18</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.090 g dihydrazide <bold>10c</bold> (0.237 mmol) compound <bold>11c</bold> was obtained as a white amorphous solid (0.043 g, 52 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, v/v). M.p.: 170–172 °C; IR (KBr): <inline-formula id="IEq23"><alternatives><tex-math id="M45">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M46"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq23.gif"></inline-graphic></alternatives></inline-formula> = 3,348, 2,985, 2,930, 2,918, 1,658, 1,552, 1,420, 1,260, 1,070, 1,028, 975 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 600 MHz): <italic>δ</italic> = 1.25 (t, <italic>J</italic> = 7.0 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 3.89 (d, <italic>J</italic> = 8.5 Hz, 2H, PCH<sub>2</sub>), 4.01–4.04 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.16 (t, <italic>J</italic> = 5.1 Hz, 2H, OC<italic>H</italic><sub>2</sub>CH<sub>2</sub>N), 5.09 (t, <italic>J</italic> = 5.1 Hz, 2H, OCH<sub>2</sub>C<italic>H</italic><sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 150 MHz): <italic>δ</italic> = 15.2 (d, <italic>J</italic> = 5.7 Hz, <italic>C</italic>COP), 49.2 (s, CN), 62.7 (d, <italic>J</italic> = 6.6 Hz, C<italic>C</italic>OP), 63.9 (d, <italic>J</italic> = 164.8 Hz, PC), 70.9 (d, <italic>J</italic> = 12.1 Hz, PCO<italic>C</italic>), 129.2 and 139.3 (2 s, C=C), 149.6 and 152.3 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 243 MHz): <italic>δ</italic> = 21.2 ppm.</p></sec><sec id="d29e5328"><title><italic>Diethyl 2</italic>-<italic>[2</italic>-<italic>(4,7</italic>-<italic>dioxo</italic>-<italic>5,6</italic>-<italic>dihydro</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazolo[4,5</italic>-<italic>d]pyridazin</italic>-<italic>1</italic>-<italic>yl)ethoxy]ethylphosphonate</italic> (<bold>11d</bold>, C<sub>12</sub>H<sub>20</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.165 g dihydrazide <bold>10d</bold> (0.419 mmol) compound <bold>11d</bold> was obtained as a white amorphous solid (0.087 g, 58 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, v/v) and crystallisation from ethyl acetate/chloroform. M.p.: 134–135 °C; IR (KBr): <inline-formula id="IEq24"><alternatives><tex-math id="M47">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M48"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq24.gif"></inline-graphic></alternatives></inline-formula> = 3,433, 2,974, 2,928, 2,872, 1,715, 1,645, 1,206, 1,113, 1,065, 1,026, 982, 952 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>, 300 MHz): <italic>δ</italic> = 1.26 (t, <italic>J</italic> = 7.2 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.07 (dt, <italic>J</italic> = 15.0, 7.2 Hz, 2H, PCH<sub>2</sub>), 3.73 (dt, <italic>J</italic> = 13.5, 7.5 Hz, 2H, PCH<sub>2</sub>C<italic>H</italic><sub>2</sub>O), 3.96–4.10 (m, 6H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP and OC<italic>H</italic><sub>2</sub>CH<sub>2</sub>N), 5.01 (t, <italic>J</italic> = 5.1 Hz, 2H, OCH<sub>2</sub>C<italic>H</italic><sub>2</sub>N), 10.49 (brs, 2H, NH) ppm; <sup>13</sup>C NMR (CDCl<sub>3</sub>, 75 MHz): <italic>δ</italic> = 16.6 (d, <italic>J</italic> = 6.3 Hz, <italic>C</italic>COP), 26.8 (d, <italic>J</italic> = 138.9 Hz, PC), 49.9 (s, OC<italic>C</italic>N), 62.2 (d, <italic>J</italic> = 6.5 Hz, C<italic>C</italic>OP), 65.0 (s, PC<italic>C</italic>O), 68.8 (s, O<italic>C</italic>CN), 129.4 and 139.4 (2 s, C=C), 151.1 and 152.8 (2 s, C=O) ppm; <sup>31</sup>P NMR (CDCl<sub>3</sub>, 121 MHz): <italic>δ</italic> = 29.3 ppm.</p></sec><sec id="d29e5526"><title><italic>Diethyl 3</italic>-<italic>(4,7</italic>-<italic>dioxo</italic>-<italic>5,6</italic>-<italic>dihydro</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazolo[4,5</italic>-<italic>d]pyridazin</italic>-<italic>1</italic>-<italic>yl)</italic>-<italic>2</italic>-<italic>hydroxypropylphosphonate</italic> (<bold>11e</bold>, C<sub>11</sub>H<sub>18</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.270 g dihydrazide <bold>10e</bold> (0.719 mmol) compound <bold>11e</bold> was obtained as a white amorphous solid (0.087 g, 39 %) after chromatography on a silica gel column with chloroform/methanol (20:1, 10:1, v/v) and crystallisation from water. M.p.: 168–170 °C; IR (KBr): <inline-formula id="IEq25"><alternatives><tex-math id="M49">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M50"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq25.gif"></inline-graphic></alternatives></inline-formula> = 3,476, 2,982, 2,932, 2,867, 1,655, 1,550, 1,427, 1,405, 1,256, 1,222, 1,108, 1,054, 957 cm<sup>−1</sup>; <sup>1</sup>H NMR (D<sub>2</sub>O, 600 MHz): <italic>δ</italic> = 1.30 and 1.31 (2 t, <italic>J</italic> = 6.1 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.25 (td, <italic>J</italic> = 16.1, 8.8 Hz, 1H, PC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 2.35 (td, <italic>J</italic> = 15.8, 3.2 Hz, 1H, PCH<sub>a</sub><italic>H</italic><sub>b</sub>), 4.10–4.17 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.52–4.59 (m, 1H, PCCH), 4.91 (dd, <italic>J</italic> = 12.8, 9.2 Hz, 1H, PCCC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 4.98 (dd, <italic>J</italic> = 12.8, 2.6 Hz, 1H, PCCCH<sub>a</sub><italic>H</italic><sub>b</sub>) ppm; <sup>13</sup>C NMR (D<sub>2</sub>O, 150 MHz): <italic>δ</italic> = 15.6 (d, <italic>J</italic> = 5.7 Hz, <italic>C</italic>COP), 30.0 (d, <italic>J</italic> = 139.9 Hz, PC), 55.7 (d, <italic>J</italic> = 17.8 Hz, PCC<italic>C</italic>), 63.5 and 63.6 (2 d, <italic>J</italic> = 6.4 Hz, C<italic>C</italic>OP), 65.4 (d, <italic>J</italic> = 3.8 Hz, PC<italic>C</italic>), 130.1 and 140.1 (2 s, C=C), 149.8 and 153.4 (2 s, C=O) ppm; <sup>31</sup>P NMR (D<sub>2</sub>O, 243 MHz): <italic>δ</italic> = 30.3 ppm.</p></sec><sec id="d29e5740"><title><italic>Diethyl 3</italic>-<italic>(4,7</italic>-<italic>dioxo</italic>-<italic>5,6</italic>-<italic>dihydro</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazolo[4,5</italic>-<italic>d]pyridazin</italic>-<italic>1</italic>-<italic>yl)</italic>-<italic>1</italic>-<italic>hydroxypropylphosphonate</italic> (<bold>11f</bold>, C<sub>11</sub>H<sub>18</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.163 g dihydrazide <bold>10f</bold> (0.430 mmol) compound <bold>11f</bold> was obtained as a white amorphous solid (0.086 g, 58 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 20:1, 10:1, v/v). M.p.: 66–68 °C; IR (KBr): <inline-formula id="IEq26"><alternatives><tex-math id="M51">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M52"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq26.gif"></inline-graphic></alternatives></inline-formula> = 3,362, 2,987, 2,920, 1,666, 1,580, 1,449, 1,212, 1,050, 1,021, 970 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 300 MHz): <italic>δ</italic> = 1.32 (t, <italic>J</italic> = 7.2 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 2.19–2.28 (m, 1H, PCC<italic>H</italic><sub>a</sub>H<sub>b</sub>), 2.31–2.44 (m, 1H, PCCH<sub>a</sub><italic>H</italic><sub>b</sub>), 3.89 (ddd, <italic>J</italic> = 10.5, 7.2, 3.0 Hz, 1H, PCH), 4.10–4.21 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.95–5.12 (m, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 75 MHz): <italic>δ</italic> = 17.0 (d, <italic>J</italic> = 5.4 Hz, <italic>C</italic>COP), 33.3 (d, <italic>J</italic> = 4.0 Hz, PC<italic>C</italic>), 48.1 (d, <italic>J</italic> = 16.2 Hz, PCC<italic>C</italic>), 64.2 and 64.5 (d, <italic>J</italic> = 6.9 Hz, C<italic>C</italic>OP), 65.4 (d, <italic>J</italic> = 167.3 Hz, PC), 130.2 and 140.8 (2 s, C=C), 148.9 and 152.2 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 121 MHz): <italic>δ</italic> = 25.3 ppm.</p></sec><sec id="d29e5929"><title><italic>Diethyl 2</italic>-<italic>(4,7</italic>-<italic>dioxo</italic>-<italic>5,6</italic>-<italic>dihydro</italic>-<italic>1H</italic>-<italic>1,2,3</italic>-<italic>triazolo[4,5</italic>-<italic>d]pyridazin</italic>-<italic>1</italic>-<italic>yl)</italic>-<italic>1</italic>-<italic>hydroxyethylphosphonate</italic> (<bold>11g</bold>, C<sub>10</sub>H<sub>16</sub>N<sub>5</sub>O<sub>6</sub>P)</title><p>From 0.100 g dihydrazide <bold>10g</bold> (0.274 mmol) compound <bold>11g</bold> was obtained as a colourless oil (0.036 g, 40 %) after chromatography on a silica gel column with chloroform/methanol (50:1, 10:1, v/v). IR (film): <inline-formula id="IEq27"><alternatives><tex-math id="M53">\documentclass[12pt]{minimal}
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\begin{document}$$ \bar{V} $$\end{document}</tex-math><mml:math id="M54"><mml:mover accent="true"><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:math><inline-graphic xlink:href="706_2013_1137_Article_IEq27.gif"></inline-graphic></alternatives></inline-formula> = 3,362, 2,988, 2,971, 1,667, 1,580, 1,212, 1,021 cm<sup>−1</sup>; <sup>1</sup>H NMR (CD<sub>3</sub>OD, 600 MHz): <italic>δ</italic> = 1.38 and 1.39 (2 t, <italic>J</italic> = 7.0 Hz, 6H, C<italic>H</italic><sub>3</sub>CH<sub>2</sub>OP), 4.23–4.29 (m, 4H, CH<sub>3</sub>C<italic>H</italic><sub>2</sub>OP), 4.64–4.69 (m, 1H, PCH), 5.09–5.15 (m, 2H, CH<sub>2</sub>N) ppm; <sup>13</sup>C NMR (CD<sub>3</sub>OD, 150 MHz): <italic>δ</italic> = 15.3 and 15.4 (d, <italic>J</italic> = 5.2 Hz, <italic>C</italic>COP), 51.3 (d, <italic>J</italic> = 11.6 Hz, PC<italic>C</italic>N), 63.3 and 63.5 (2 d, <italic>J</italic> = 6.8 Hz, COP), 66.4 (d, <italic>J</italic> = 166.2 Hz, PC), 129.2 and 139.4 (2 s, C=C), 150.1 and 152.4 (2 s, C=O) ppm; <sup>31</sup>P NMR (CD<sub>3</sub>OD, 243 MHz): <italic>δ</italic> = 20.5 ppm.</p></sec></sec><sec id="Sec12"><title>Assays for antiviral activity other than HIV</title><p>The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK<sup>−</sup>) HSV-1 KOS strain resistant to ACV (ACV<sup>r</sup>), herpes simplex virus type 2 (HSV-2) strain G, cytomegalovirus (strains AD-169 and Davis), varicella-zoster virus (VZV) (strains OKA and YS), vaccinia virus Lederle strain, respiratory syncytial virus (RSV) strain Long, vesicular stomatitis virus (VSV), coxsackie b4 virus, parainfluenza 3, influenza virus A (subtypes H1N1, H3N2), influenza virus B, reovirus-1, Sindbis virus and Punta Toro virus. The antiviral assays were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cervix carcinoma cells (HeLa) or 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 20 or 100 plaque forming units (PFU) (for VZV and CMV, respectively) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation 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 to reduce virus-induced cytopathogenicity or viral plaque formation by 50 %.</p></sec><sec id="Sec13"><title>Anti-HIV activity assays</title><p>Inhibition of HIV-1(III<sub>B</sub>)- and HIV-2(ROD)-induced cytopathicity in CEM cell cultures was measured in microtiter 96-well plates containing 3 × 10<sup>5</sup> CEM cells/cm<sup>3</sup> infected with 100 CCID50 of HIV per cm<sup>3</sup> and containing appropriate dilutions of the test compounds. After 4–5 days of incubation at 37 °C in a CO<sub>2</sub>-controlled humidified atmosphere, CEM giant cell (syncytium) formation was examined microscopically. The EC<sub>50</sub> (50 % effective concentration) was defined as the compound concentration required to inhibit HIV-induced giant cell formation by 50 %.</p></sec><sec id="Sec14"><title>Cytostatic activity assays</title><p>All assays were performed in 96-well microtiter plates. To each well were added (5–7.5) × 10<sup>4</sup> tumour cells and a given amount of the test compound. The cells were allowed to proliferate for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37 °C in a humidified CO<sub>2</sub>-controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The IC<sub>50</sub> (50 % inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50 %.</p></sec></sec><sec sec-type="supplementary-material"><title>Electronic supplementary material</title><sec id="Sec15"><p>Below is the link to the electronic supplementary material.
<supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="706_2013_1137_MOESM1_ESM.pdf"><caption><p>Supplementary material 1 (PDF 1043 kb)</p></caption></media></supplementary-material></p></sec></sec></body><back><ack><p>The authors are grateful to Leen Ingels, Leentje Persoons, Frieda De Meyer, Lizette van Berckelaer, Anita Camps, Steven Carmans and Lies Van den Heurck for excellent technical assistance. The work was supported by the KU Leuven (GOA 10/14) and by the Medical University of Lodz (502-34-009 and 503/3-014-01/503-1).</p></ack><ref-list id="Bib1"><title>References
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