Synthesis of thiocarbohydrazide and carbohydrazide derivatives as possible biologically active agents
Identifieur interne : 000297 ( Pmc/Corpus ); précédent : 000296; suivant : 000298Synthesis of thiocarbohydrazide and carbohydrazide derivatives as possible biologically active agents
Auteurs : Kiran Gangarapu ; Sarangapani Manda ; Anvesh Jallapally ; Sreekanth Thota ; Subhas S. Karki ; Jan Balzarini ; Erik De Clercq ; Harukuni TokudaSource :
- Medicinal Chemistry Research [ 1054-2523 ] ; 2013.
Abstract
A series of new β-isatin aldehyde-
Url:
DOI: 10.1007/s00044-013-0684-3
PubMed: NONE
PubMed Central: 7080202
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PMC:7080202Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Synthesis of thiocarbohydrazide and carbohydrazide derivatives as possible biologically active agents</title><author><name sortKey="Gangarapu, Kiran" sort="Gangarapu, Kiran" uniqKey="Gangarapu K" first="Kiran" last="Gangarapu">Kiran Gangarapu</name><affiliation><nlm:aff id="Aff1">Department of Pharmaceutical Chemistry, Kakatiya Institute of Pharmaceutical Sciences, Pembarthy (V), Hasanparthy (M), Warangal, 506 371 AP India</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.411828.6</institution-id><institution-id institution-id-type="ISNI">0000 0001 0683 7715</institution-id><institution>Center for Pharmaceutical Sciences, IST, JNTU,</institution></institution-wrap>Kukatpally, Hyderabad, 500085 India</nlm:aff></affiliation></author><author><name sortKey="Manda, Sarangapani" sort="Manda, Sarangapani" uniqKey="Manda S" first="Sarangapani" last="Manda">Sarangapani Manda</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.411990.4</institution-id><institution-id institution-id-type="ISNI">0000000123346125</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>University College of Pharmaceutical Sciences, Kakatiya University,</institution></institution-wrap>Warangal, 506 009 AP India</nlm:aff></affiliation></author><author><name sortKey="Jallapally, Anvesh" sort="Jallapally, Anvesh" uniqKey="Jallapally A" first="Anvesh" last="Jallapally">Anvesh Jallapally</name><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="GRID">grid.418280.7</institution-id><institution-id institution-id-type="ISNI">0000000417943160</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>Acharya and BM Reddy College of Pharmacy,</institution></institution-wrap>Soldevanahalli, Bangalore, KN India</nlm:aff></affiliation></author><author><name sortKey="Thota, Sreekanth" sort="Thota, Sreekanth" uniqKey="Thota S" first="Sreekanth" last="Thota">Sreekanth Thota</name><affiliation><nlm:aff id="Aff5"><institution-wrap><institution-id institution-id-type="GRID">grid.47894.36</institution-id><institution-id institution-id-type="ISNI">0000000419368083</institution-id><institution>Department of Chemistry,</institution><institution>Colorado State University,</institution></institution-wrap>Fort Collins, 80523 CO USA</nlm:aff></affiliation></author><author><name sortKey="Karki, Subhas S" sort="Karki, Subhas S" uniqKey="Karki S" first="Subhas S." last="Karki">Subhas S. Karki</name><affiliation><nlm:aff id="Aff6"><institution-wrap><institution-id institution-id-type="GRID">grid.411053.2</institution-id><institution-id institution-id-type="ISNI">0000 0001 1889 7360</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>KLE University’s College of Pharmacy,</institution></institution-wrap>Rajajinagar, Bangalore, KN India</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="Aff7"><institution-wrap><institution-id institution-id-type="GRID">grid.415751.3</institution-id><institution>Rega Institute for Medical Research, KU Leuven,</institution></institution-wrap>Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="De Clercq, Erik" sort="De Clercq, Erik" uniqKey="De Clercq E" first="Erik" last="De Clercq">Erik De Clercq</name><affiliation><nlm:aff id="Aff7"><institution-wrap><institution-id institution-id-type="GRID">grid.415751.3</institution-id><institution>Rega Institute for Medical Research, KU Leuven,</institution></institution-wrap>Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="Tokuda, Harukuni" sort="Tokuda, Harukuni" uniqKey="Tokuda H" first="Harukuni" last="Tokuda">Harukuni Tokuda</name><affiliation><nlm:aff id="Aff8"><institution-wrap><institution-id institution-id-type="GRID">grid.9707.9</institution-id><institution-id institution-id-type="ISNI">0000000123083329</institution-id><institution>Department of Complementary and Alternative Medicine,</institution><institution>Clinical R&D, Graduate School of Medical Science Kanazawa University,</institution></institution-wrap>Kanazawa, Japan</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmc">7080202</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080202</idno><idno type="RBID">PMC:7080202</idno><idno type="doi">10.1007/s00044-013-0684-3</idno><idno type="pmid">NONE</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000297</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000297</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Synthesis of thiocarbohydrazide and carbohydrazide derivatives as possible biologically active agents</title><author><name sortKey="Gangarapu, Kiran" sort="Gangarapu, Kiran" uniqKey="Gangarapu K" first="Kiran" last="Gangarapu">Kiran Gangarapu</name><affiliation><nlm:aff id="Aff1">Department of Pharmaceutical Chemistry, Kakatiya Institute of Pharmaceutical Sciences, Pembarthy (V), Hasanparthy (M), Warangal, 506 371 AP India</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.411828.6</institution-id><institution-id institution-id-type="ISNI">0000 0001 0683 7715</institution-id><institution>Center for Pharmaceutical Sciences, IST, JNTU,</institution></institution-wrap>Kukatpally, Hyderabad, 500085 India</nlm:aff></affiliation></author><author><name sortKey="Manda, Sarangapani" sort="Manda, Sarangapani" uniqKey="Manda S" first="Sarangapani" last="Manda">Sarangapani Manda</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.411990.4</institution-id><institution-id institution-id-type="ISNI">0000000123346125</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>University College of Pharmaceutical Sciences, Kakatiya University,</institution></institution-wrap>Warangal, 506 009 AP India</nlm:aff></affiliation></author><author><name sortKey="Jallapally, Anvesh" sort="Jallapally, Anvesh" uniqKey="Jallapally A" first="Anvesh" last="Jallapally">Anvesh Jallapally</name><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="GRID">grid.418280.7</institution-id><institution-id institution-id-type="ISNI">0000000417943160</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>Acharya and BM Reddy College of Pharmacy,</institution></institution-wrap>Soldevanahalli, Bangalore, KN India</nlm:aff></affiliation></author><author><name sortKey="Thota, Sreekanth" sort="Thota, Sreekanth" uniqKey="Thota S" first="Sreekanth" last="Thota">Sreekanth Thota</name><affiliation><nlm:aff id="Aff5"><institution-wrap><institution-id institution-id-type="GRID">grid.47894.36</institution-id><institution-id institution-id-type="ISNI">0000000419368083</institution-id><institution>Department of Chemistry,</institution><institution>Colorado State University,</institution></institution-wrap>Fort Collins, 80523 CO USA</nlm:aff></affiliation></author><author><name sortKey="Karki, Subhas S" sort="Karki, Subhas S" uniqKey="Karki S" first="Subhas S." last="Karki">Subhas S. Karki</name><affiliation><nlm:aff id="Aff6"><institution-wrap><institution-id institution-id-type="GRID">grid.411053.2</institution-id><institution-id institution-id-type="ISNI">0000 0001 1889 7360</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>KLE University’s College of Pharmacy,</institution></institution-wrap>Rajajinagar, Bangalore, KN India</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="Aff7"><institution-wrap><institution-id institution-id-type="GRID">grid.415751.3</institution-id><institution>Rega Institute for Medical Research, KU Leuven,</institution></institution-wrap>Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="De Clercq, Erik" sort="De Clercq, Erik" uniqKey="De Clercq E" first="Erik" last="De Clercq">Erik De Clercq</name><affiliation><nlm:aff id="Aff7"><institution-wrap><institution-id institution-id-type="GRID">grid.415751.3</institution-id><institution>Rega Institute for Medical Research, KU Leuven,</institution></institution-wrap>Leuven, Belgium</nlm:aff></affiliation></author><author><name sortKey="Tokuda, Harukuni" sort="Tokuda, Harukuni" uniqKey="Tokuda H" first="Harukuni" last="Tokuda">Harukuni Tokuda</name><affiliation><nlm:aff id="Aff8"><institution-wrap><institution-id institution-id-type="GRID">grid.9707.9</institution-id><institution-id institution-id-type="ISNI">0000000123083329</institution-id><institution>Department of Complementary and Alternative Medicine,</institution><institution>Clinical R&D, Graduate School of Medical Science Kanazawa University,</institution></institution-wrap>Kanazawa, Japan</nlm:aff></affiliation></author></analytic><series><title level="j">Medicinal Chemistry Research</title><idno type="ISSN">1054-2523</idno><idno type="eISSN">1554-8120</idno><imprint><date when="2013">2013</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 new β-isatin aldehyde-<italic>N</italic>,<italic>N</italic>′-thiocarbohydrazone, bis-β-isatin thiocarbohydrazones, bis-β-isatin carbohydrazones was synthesized by condensation of 5-substituted isatin with thiocarbohydrazide or carbohydrazide. The chemical structures of the newly synthesized compounds were confirmed by FT-IR, <sup>1</sup>H NMR, and mass spectral analysis. The synthesized compounds were evaluated for in vitro antiviral activity against various strains of DNA and RNA viruses, but exhibited moderate antiviral activity compared with the reference compounds. Among all the compounds <bold>6c</bold> exhibited the highest chemoprevention activity in a two-stage mouse-skin carcinogenesis test.</p></sec><sec><title>Graphical abstract</title><p><graphic position="anchor" xlink:href="44_2013_684_Figa_HTML" id="MO1"></graphic></p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Akhaja, Tn" uniqKey="Akhaja T">TN Akhaja</name></author><author><name sortKey="Raval, Jp" uniqKey="Raval J">JP Raval</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Alcaide, B" uniqKey="Alcaide B">B Alcaide</name></author><author><name sortKey="Almendros, P" uniqKey="Almendros P">P Almendros</name></author><author><name sortKey="Aragoncillo, C" uniqKey="Aragoncillo C">C Aragoncillo</name></author><author><name sortKey="Campillos, Gg" uniqKey="Campillos G">GG Campillos</name></author><author><name sortKey="Arn, M" uniqKey="Arn M">M Arnó</name></author><author><name sortKey="Domingo, 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Med Chem Res</journal-id><journal-id journal-id-type="iso-abbrev">Med Chem Res</journal-id><journal-title-group><journal-title>Medicinal Chemistry Research</journal-title></journal-title-group><issn pub-type="ppub">1054-2523</issn><issn pub-type="epub">1554-8120</issn><publisher><publisher-name>Springer US</publisher-name><publisher-loc>New York</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmc">7080202</article-id><article-id pub-id-type="publisher-id">684</article-id><article-id pub-id-type="doi">10.1007/s00044-013-0684-3</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Research</subject></subj-group></article-categories><title-group><article-title>Synthesis of thiocarbohydrazide and carbohydrazide derivatives as possible biologically active agents</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><name><surname>Gangarapu</surname><given-names>Kiran</given-names></name><address><phone>+91-99-12345280</phone><email>gangakiran1905@gmail.com</email></address><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Manda</surname><given-names>Sarangapani</given-names></name><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Jallapally</surname><given-names>Anvesh</given-names></name><xref ref-type="aff" rid="Aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Thota</surname><given-names>Sreekanth</given-names></name><xref ref-type="aff" rid="Aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Karki</surname><given-names>Subhas S.</given-names></name><xref ref-type="aff" rid="Aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Balzarini</surname><given-names>Jan</given-names></name><xref ref-type="aff" rid="Aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>De Clercq</surname><given-names>Erik</given-names></name><xref ref-type="aff" rid="Aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>Tokuda</surname><given-names>Harukuni</given-names></name><xref ref-type="aff" rid="Aff8">8</xref></contrib><aff id="Aff1"><label>1</label>Department of Pharmaceutical Chemistry, Kakatiya Institute of Pharmaceutical Sciences, Pembarthy (V), Hasanparthy (M), Warangal, 506 371 AP India</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="GRID">grid.411828.6</institution-id><institution-id institution-id-type="ISNI">0000 0001 0683 7715</institution-id><institution>Center for Pharmaceutical Sciences, IST, JNTU,</institution></institution-wrap>Kukatpally, Hyderabad, 500085 India</aff><aff id="Aff3"><label>3</label><institution-wrap><institution-id institution-id-type="GRID">grid.411990.4</institution-id><institution-id institution-id-type="ISNI">0000000123346125</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>University College of Pharmaceutical Sciences, Kakatiya University,</institution></institution-wrap>Warangal, 506 009 AP India</aff><aff id="Aff4"><label>4</label><institution-wrap><institution-id institution-id-type="GRID">grid.418280.7</institution-id><institution-id institution-id-type="ISNI">0000000417943160</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>Acharya and BM Reddy College of Pharmacy,</institution></institution-wrap>Soldevanahalli, Bangalore, KN India</aff><aff id="Aff5"><label>5</label><institution-wrap><institution-id institution-id-type="GRID">grid.47894.36</institution-id><institution-id institution-id-type="ISNI">0000000419368083</institution-id><institution>Department of Chemistry,</institution><institution>Colorado State University,</institution></institution-wrap>Fort Collins, 80523 CO USA</aff><aff id="Aff6"><label>6</label><institution-wrap><institution-id institution-id-type="GRID">grid.411053.2</institution-id><institution-id institution-id-type="ISNI">0000 0001 1889 7360</institution-id><institution>Department of Pharmaceutical Chemistry,</institution><institution>KLE University’s College of Pharmacy,</institution></institution-wrap>Rajajinagar, Bangalore, KN India</aff><aff id="Aff7"><label>7</label><institution-wrap><institution-id institution-id-type="GRID">grid.415751.3</institution-id><institution>Rega Institute for Medical Research, KU Leuven,</institution></institution-wrap>Leuven, Belgium</aff><aff id="Aff8"><label>8</label><institution-wrap><institution-id institution-id-type="GRID">grid.9707.9</institution-id><institution-id institution-id-type="ISNI">0000000123083329</institution-id><institution>Department of Complementary and Alternative Medicine,</institution><institution>Clinical R&D, Graduate School of Medical Science Kanazawa University,</institution></institution-wrap>Kanazawa, Japan</aff></contrib-group><pub-date pub-type="epub"><day>23</day><month>8</month><year>2013</year></pub-date><pub-date pub-type="ppub"><year>2014</year></pub-date><volume>23</volume><issue>2</issue><fpage>1046</fpage><lpage>1056</lpage><history><date date-type="received"><day>23</day><month>3</month><year>2013</year></date><date date-type="accepted"><day>25</day><month>7</month><year>2013</year></date></history><permissions><copyright-statement>© Springer Science+Business Media New York 2013</copyright-statement><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract id="Abs1"><sec><title>Abstract</title><p>A series of new β-isatin aldehyde-<italic>N</italic>,<italic>N</italic>′-thiocarbohydrazone, bis-β-isatin thiocarbohydrazones, bis-β-isatin carbohydrazones was synthesized by condensation of 5-substituted isatin with thiocarbohydrazide or carbohydrazide. The chemical structures of the newly synthesized compounds were confirmed by FT-IR, <sup>1</sup>H NMR, and mass spectral analysis. The synthesized compounds were evaluated for in vitro antiviral activity against various strains of DNA and RNA viruses, but exhibited moderate antiviral activity compared with the reference compounds. Among all the compounds <bold>6c</bold> exhibited the highest chemoprevention activity in a two-stage mouse-skin carcinogenesis test.</p></sec><sec><title>Graphical abstract</title><p><graphic position="anchor" xlink:href="44_2013_684_Figa_HTML" id="MO1"></graphic></p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>Isatin</kwd><kwd>Thiocarbohydrazide</kwd><kwd>Carbohydrazide</kwd><kwd>Antiviral</kwd><kwd>Anti-influenza</kwd><kwd>Carcinogenesis</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© Springer Science+Business Media New York 2014</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Introduction</title><p>Isatin is a versatile lead molecule for designing potential antiviral agents. Isatin derivatives display broad spectrum pharmacological properties, including cytotoxic (Reddy <italic>et al.</italic>, <xref ref-type="bibr" rid="CR24">2012</xref>; Alcaide <italic>et al.</italic>, <xref ref-type="bibr" rid="CR2">2012</xref>), antibacterial (Akhaja and Raval, <xref ref-type="bibr" rid="CR1">2011</xref>), antifungal (Reddy <italic>et al.</italic>, <xref ref-type="bibr" rid="CR23">2011</xref>), antiviral (Kang <italic>et al.</italic>, <xref ref-type="bibr" rid="CR15">2011</xref>; Bacani <italic>et al.</italic>, <xref ref-type="bibr" rid="CR3">2011</xref>; Sin <italic>et al.</italic>, <xref ref-type="bibr" rid="CR27">2009</xref>), anti-HIV (Bal <italic>et al.</italic>, <xref ref-type="bibr" rid="CR4">2005</xref>), and antimalarial (Hans <italic>et al.</italic>, <xref ref-type="bibr" rid="CR13">2011</xref>) activities. The first clinically approved antiviral agent, <italic>N</italic>-methylisatin-β-thiosemicarbazone (methisazone) was active against smallpox and vaccinia viruses (Bauer, <xref ref-type="bibr" rid="CR5">1985</xref>). <italic>N</italic>,<italic>N</italic>′-disubstituted thiosemicarbazone derivatives of isatin were inhibitory to HIV-1 replication (Teitz and Ronen, <xref ref-type="bibr" rid="CR28">1984</xref>). <italic>N</italic>-methylisatin-β-thiosemicarbazone is an inhibitor of Japanese encephalitis virus infection (Sebastain <italic>et al.</italic>, <xref ref-type="bibr" rid="CR25">2008</xref>). The general configuration of the isatin molecule resembles that of the purines and also tryptophan and its derivatives, but there is no evidence available as to whether isatin-β-thiosemicarbazone acts as an antimetabolite to these compounds (Bauer and Sadler, <xref ref-type="bibr" rid="CR6">1960</xref>). Recent studies have shown that isatin could inhibit the protease, caspase, MAO and COX-2 enzymes (Medvedev <italic>et al.</italic>, <xref ref-type="bibr" rid="CR18">2007</xref>).</p><p>Viral pathogens are responsible for many human diseases and examples of diseases caused by viruses include the common cold, smallpox, AIDS, cold sores, human herpes virus, papilloma virus, etc. (Prado-Prado <italic>et al.</italic>, <xref ref-type="bibr" rid="CR21">2009</xref>). The worldwide market for antiviral drugs is estimated to be approximately 20 billion US dollars and is expected to grow rapidly as new therapies become available. The need for effective antiviral drugs is further emphasized by the lack of vaccines for most respiratory tract virus infections [adenovirus, rhinovirus, parainfluenza virus and respiratory syncytial virus (RSV), the widely occurring human papilloma viruses, and herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein–Barr virus, cytomegalovirus (CMV), and the vast array of hemorrhagic fever viruses (De Clercq, <xref ref-type="bibr" rid="CR9">2002</xref>)]. Millions of people worldwide are affected by infectious diseases caused by viruses. Further, widespread viral resistance has renewed the interest in the quest for new antiviral agents. Earlier work could clearly define the structural requirements necessary for the retention of antiviral activity by heterocyclic thiosemicarbazone in neurovaccinia infections in mice. Viral infections primarily affect the respiratory and gastrointestinal system; epithelium, mucous membranes and endothelium of skin, mouth and genitals; lymphoid tissue, liver and other organs; and the central nervous system (Bruce <italic>et al.</italic>, <xref ref-type="bibr" rid="CR7">2007</xref>).</p><p>Recently, we have reported that β-isatin-<italic>N</italic>,<italic>N</italic>′-thiocarbohydrazone derivatives could have antimicrobial and antioxidant activity (Kiran <italic>et al.</italic>, <xref ref-type="bibr" rid="CR16">2012a</xref>). Thiosemicarbazones were the first antiviral compounds recognized to possess broad-spectrum antiviral activity against a range of DNA and RNA viruses [Castro <italic>et al.</italic>, <xref ref-type="bibr" rid="CR8">2011</xref>]. Earlier reports have described antiviral activity of isatin-β-thiosemicarbazone and isatin-β-semicarbohydrazone against different viral strains [Quenelle <italic>et al.</italic>, <xref ref-type="bibr" rid="CR22">2006</xref>; Pandeya <italic>et al.</italic>, <xref ref-type="bibr" rid="CR20">2005</xref>]. However, the biological activities of isatin-β-thiocarbohydrazone and isatin-β-carbohydrazone have not been evaluated against DNA and RNA viruses. In connection with our ongoing work on isatin (Kiran <italic>et al.</italic>, <xref ref-type="bibr" rid="CR17">2012b</xref>), we are now reporting a simple and efficient method for the synthesis of some β-isatin-<italic>N</italic>,<italic>N</italic>′-thiocarbohydrazone (<bold>4a</bold>–<bold>g</bold>), bis-β-isatin thiocarbohydrazone, and bis-β-isatin thiocarbohydrazone derivatives (<bold>6a</bold>–<bold>e</bold>) as depicted in Schemes <xref rid="Sch1" ref-type="fig">1</xref> and <xref rid="Sch2" ref-type="fig">2</xref>. These compounds were screened for in vitro antiviral activity and also in a two stage mouse-skin carcinogenesis test.<fig id="Sch1"><label>Scheme 1</label><caption><p>Schematic steps of β-isatin aldehyde-<italic>N</italic>,<italic>N</italic>′-thiocarbohydrazones derivatives synthesis</p></caption><graphic xlink:href="44_2013_684_Sch1_HTML" id="MO2"></graphic></fig><fig id="Sch2"><label>Scheme 2</label><caption><p>Schematic steps of Bis-isatin derivatives synthesis</p></caption><graphic xlink:href="44_2013_684_Sch2_HTML" id="MO3"></graphic></fig></p></sec><sec id="Sec2"><title>Experimental</title><sec id="Sec3"><title>Materials and methods</title><p>Melting points (m.p.) were determined in open capillaries, using the Toshniwal melting point apparatus, expressed in °C and were uncorrected. FT-IR spectra (KBr) were recorded on a thermo Nicolet Nexus 670S series. NMR spectra were recorded on an Avance-300 MHz in DMSO-<italic>d</italic><sub>6</sub> solvent using TMS as an internal standard (chemical shifts in δ, ppm). Mass spectra were obtained on a LC-MSD-Trap-SL. Reactions were monitored using thin layer chromatography (TLC) on aluminum-backed precoated silica gel 60 F<sub>254</sub> plates (E Merck). The purity of the compounds was checked on silica gel-coated aluminum sheets by TLC. Column chromatography was performed by using Qualigen’s silica gel (60–120 mesh). All the solvents were AR grade and distilled before use. 5-Substituted isatins were purchased from Himedia (Mumbai, India). Indole-3-carboxaldehyde was obtained from E. Merck (India). All substituted aldehydes were purchased from Sd. Fine Chemicals.</p></sec><sec id="Sec4"><title>General procedure</title><sec id="Sec5"><title>Synthesis of isatin derivatives</title><p>The synthesis of isatin derivatives was carried out using a modified Sandmeyer methodology (Henry and Blatt, <xref ref-type="bibr" rid="CR14">1964</xref>). <italic>N</italic>-benzyl isatin is prepared by a microwave method as reported in the literature (Shmidt <italic>et al.</italic>, <xref ref-type="bibr" rid="CR26">2008</xref>). All the isatin derivatives exhibit same physical properties as reported in the literature.</p></sec><sec id="Sec6"><title>Synthesis of thiocarbohydrazide (<bold>1</bold>)</title><p>The synthesis of thiocarbohydrazide was carried out by the Taguchi method. The physical data of the synthesized compound are comparable and show m.p 170–172 °C similar to that reported in the literature (Zhou <italic>et al.</italic>, <xref ref-type="bibr" rid="CR30">2010</xref>).</p></sec><sec id="Sec7"><title>Synthesis of monothiocarbohydrazones (<bold>2a</bold>–<bold>g</bold>)</title><p>An equimolar mixture of thiocarbohydrazide (0.01 mol), substituted aldehyde (0.01 mol), and a few drops of glacial acetic acid were taken in 30 ml of ethanol in 250 ml conical flask. The resulting mixture was heated under reflux for 1 h. Then the reaction mixture was cooled overnight and the precipitate was collected by filtration. Further it was purified by recrystallization with ethanol. The synthesized compounds were comparable with the reported literature (Mohamed <italic>et al</italic>., <xref ref-type="bibr" rid="CR19">1991</xref>).</p></sec><sec id="Sec8"><title>Synthesis of β-isatin aldehyde-<italic>N</italic>,<italic>N</italic>′-thiocarbohydrazones (<bold>4a</bold>–<bold>g</bold>)</title><p>To a solution of monothiocarbohydrazone (<bold>2a</bold>–<bold>g</bold>, 0.01 mol) and substituted isatin (<bold>3</bold>, 0.01 mol) in ethanol, a few drops of glacial acetic acid were added to initiate the reaction. The progress of the reaction was monitored by TLC. The residue was cooled, kept overnight and the precipitate was collected by filtration. The solid was purified by column chromatography, and subsequently by recrystallization with ethanol.</p></sec><sec id="Sec9"><title>Synthesis of bis-isatin derivatives (<bold>6a</bold>–<bold>e</bold>)</title><p>A mixture of thiocarbohydrazide or carbohydrazide (0.01 mol) was mixed with appropriate substituted isatin derivatives (0.02 mol) in ethanol–acetic acid (1:1). The reaction mixture was refluxed for 2 h. Then the reaction mixture was cooled, kept overnight and the precipitate was collected by filtration. The resulting solid was purified by column chromatography and subsequently by recrystallization with ethanol.</p></sec><sec id="Sec10"><title>Spectral data of the synthesized compounds</title><sec id="Sec11"><title>3-[(1H-indol-3-ylmethylidene)amino]-1-{[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}thiourea (<bold>4a</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3226 (br, N–H, lactam), 3138, 3070 (w, C–H, Ar), 1705 (s, C=O, lactam), 1337 (s, C=S), 1538 (s, C=N), 1463 (s, C=C). <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) δ ppm: 14.1 (s, 1H, NH), 12.2 (s, 1H, NH), 11.7 (s, 1H, NH), 11.3 (s, 1H, NH), 8.67 (1H, =CH–), 8.47 (1H, N=CH), 7.87 (dd, <italic>J</italic> = 7.4, 1.6 Hz, 1H), 7.64–7.52 (m, 3H), 7.49–7.40 (m, 2H), 7.34 (td, <italic>J</italic> = 7.4, 1.5 Hz, 1H), 6.59 (d, <italic>J</italic> = 7.5 Hz, 1H). <sup>13</sup>C NMR (DMSO-<italic>d</italic><sub>6</sub>) δ ppm: 173.9 (C=S), 162 (C=O), 143 (C=N), 110–137 (Ar–C); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 363 (M+1, 100 %), 385 (M+Na, 80 %). Elemental analysis for C<sub>18</sub>H<sub>14</sub>N<sub>6</sub>OS: calculated (%) C: 59.65; H: 3.89; N: 23.19; found: C: 59.29; H: 3.24; N: 23.45.</p></sec><sec id="Sec12"><title>3-{[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}-1-[thiophen-2-ylmethylidene)amino]thiourea (<bold>4b</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3316 (N–H), 3026 (w, C–H, Ar), 1708 (s, C=O, lactam), 1368 (s, C=S), 1395 (s, C=N), 1528 (s, CH=N). <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) δ ppm: 14.7 (s, 1H, NH), 13.3 (s, 1H, –NH), 11.9 (s, 1H, –NH), 8.90 (s, 1H), 7.65–7.59 (m, 2H), 7.57–7.45 (m, 2H), 7.31 (dd, <italic>J</italic> = 7.5, 2.8 Hz, 1H), 7.27–7.20 (m, 1H), 6.96 (dd, <italic>J</italic> = 7.5, 1.5 Hz, 1H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 330 (M+1, 100 %). Elemental analysis for C<sub>14</sub>H<sub>11</sub>N<sub>5</sub>OS<sub>2</sub>: calculated (%) C: 51.05; H: 3.37; N: 21.26; found: C: 51.02; H: 3.34; N: 21.18.</p></sec><sec id="Sec13"><title>1-({[4-(dimethylamino)phenyl]methylidene}amino)-3-{[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}thiourea (<bold>4c</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3218 (–NH), 3016 (w, C–H, Ar), 2700 (C–H, <italic>str</italic>), 1702 (s, C=O, lactam), 1358 (s, C=S), 1528 (s, C=N), 1528 (s, CH=N). <sup>1</sup>H-NMR (DMSO- <italic>d</italic><sub>6</sub>) δ ppm: 14.9 (s, 1H, –NH), 14.5 (s, 1H, –NH), 13.0 (s, 1H, –NH), 8.06 (s, 1H), 7.76 (dd, <italic>J</italic> = 7.5, 1.5 Hz, 1H), 7.61 (dd, <italic>J</italic> = 7.5, 1.7 Hz, 1H), 7.53–7.46 (m, 3H), 7.28–7.20 (m, 1H), 6.85–6.79 (m, 2H), 3.02 (s, 6H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 367 (M+1, 100 %), 389 (M+Na, 40 %). Elemental analysis for C<sub>18</sub>H<sub>18</sub>N<sub>6</sub>OS: calculated (%) C: 59.00; H: 4.95; N: 22.93; found: C: 59.02; H: 4.86; N: 22.78.</p></sec><sec id="Sec14"><title>1-{[(4-Nitrophenyl)methylidene]amino}-3-{[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}thiourea (<bold>4d</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3120 (–NH), 3006 (w, C–H, Ar), 1690 (s, C=O, lactam), 1358 (s, C=S), 1528 (s, C=N), 1528 (s, CH=N). <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) δ ppm: 14.2 (s, 1H,–NH), 13.6 (s, 1H, –NH), 12.0 (s, 1H, –NH), 8.06 (s, 1H), 7.64 (dd, <italic>J</italic> = 7.5, 1.5 Hz, 1H), 7.64 (dd, <italic>J</italic> = 7.5, 1.7 Hz, 1H), 7.42–7.26 (m, 3H), 7.28–7.20 (m, 1H), 6.85–6.79 (m, 2H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 369 (M+1, 100 %), 391 (M+Na, 30 %). Elemental analysis for C<sub>16</sub>H<sub>12</sub>N<sub>6</sub>O<sub>3</sub>S: calculated (%) C: 52.17; H: 3.28; N: 22.81 found: C: 52.12; H: 3.24; N: 22.76.</p></sec><sec id="Sec15"><title>1-{[(4-Chlorophenyl)methylidene]amino}-3-{[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}thiourea (<bold>4e</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3345 (N–H), 3050 (C–H, Ar), 1630 (C=O, lactam), 900–690 (Ar–H), 785 (C–Cl); <sup>1</sup>H-NMR (DMSO- <italic>d</italic><sub>6</sub>) δ ppm; 14.7 (s, 1H), 13.6 (s, 1H), δ 11.2 (s, 1H), 7.93 (s, 1H), 7.85–7.79 (m, 2H), 7.68 (dd, <italic>J</italic> = 7.5, 1.6 Hz, 1H), 7.60 (dd, <italic>J</italic> = 7.4, 1.6 Hz, 1H), 7.48 (td, <italic>J</italic> = 7.4, 1.5 Hz, 3H), 7.27–7.20 (m, 1H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 357 (M+, 100 %), 359 (M+2, 38 %). Elemental analysis for C<sub>16</sub>H<sub>12</sub>N<sub>6</sub>O<sub>3</sub>S: calculated (%) C: 53.71; H: 3.38; N: 19.57; found: C: 53.68; H: 3.42; N: 19.76.</p></sec><sec id="Sec16"><title>1-{[(3,4-Dimethoxyphenyl)methylidene]amino}-3-{[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}thiourea (<bold>4f</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3348 (N–H), 3020 (C–H, Ar), 1648 (C=O), 1313 (C=S); <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) δ ppm; 14.7 (s, 1H), 13.1 (s, 1H), δ 10.9 (s, 1H), 7.93 (s, 1H), 7.71–7.57 (m, 4H), 7.48 (td, <italic>J</italic> = 7.5, 1.5 Hz, 1H), 7.14–7.08 (m, 2H), 3.79 (s, 6H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 384 (M+1, 100 %). Elemental analysis for C<sub>24</sub>H<sub>21</sub>N<sub>5</sub>OS: calculated (%) C: 56.38; H, 4.47; N, 18.27; found: C: 56.34; H: 4.46; N: 18.23.</p></sec><sec id="Sec17"><title>3-{[(3Z)-1-benzyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}-1-[(phenylmethylidene)amino]thiourea (<bold>4g</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3360 (N–H), 3050 (C–H, Ar), 1630 (C=O, lactam), 900–690 (Ar, oop); <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) δ ppm; 14.7 (s, 1H), 13.6 (s, 1H), δ 11.2 (s, 1H), 7.93 (s, 1H), 6.8–7.5 (Ar–H), 3.2 (s, –CH<sub>2</sub>, 2H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 428 (M+, 100 %), 450 (M+Na, 38 %). Elemental analysis for C<sub>18</sub>H<sub>17</sub>N<sub>5</sub>O<sub>3</sub>S: calculated (%) C: 66.81; H: 4.63; N: 16.94; found: C: 66.78; H: 4.58; N: 16.78.</p></sec><sec id="Sec18"><title>1,3-Bis({[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino})urea (<bold>6a</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3350–3050 (NH, NH), 3070 (C–H, Ar), 1742 (s, C=O, lactam), 1608, 1472 (C=C, Ar), 1650 (C=O), 1610 (C=N). <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) ppm; δ 13.2 (s, 2H), 7.93 (s, 2H), 7.15–7.79 (m, 6H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 349 (M+1, 100 %), 371 (M+Na, 52 %). Elemental analysis for C<sub>17</sub>H<sub>12</sub>N<sub>6</sub>O<sub>3</sub>: calculated (%) C:58.62; H:3.47; N:24.13; found: C: 58.58; H: 3.46; N: 24.20.</p></sec><sec id="Sec19"><title>1,3-Bis({[(3Z)-5-chloro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino})urea (<bold>6b</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3250–3050 (NH, NH), 3010 (C–H, Ar), 1642 (s, C=O, lactam), 1608, 1472 (C=C, Ar), 1650 (C=O), 1610 (C=N). <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) ppm; δ 13.2 (s, 2H), 7.93 (s, 2H), 7.15–7.79 (m, 6H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 417 (M+1, 100 %), 439 (M+Na, 46 %).Elemental analysis for C<sub>17</sub>H<sub>10</sub>Cl<sub>2</sub>N<sub>6</sub>O<sub>3</sub>: calculated (%) C:48.94; H:2.42;N: 20.14; found: C: 48.89; H: 2.46; N: 20.20.</p></sec><sec id="Sec20"><title>1,3-Bis({[(3Z)-5-methyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino})urea (<bold>6c</bold>)</title><p>IR (ν cm<sup>−1</sup>) 3198 (NH), 3010 (C–H, Ar), 2980 (-CH str), 1730 (C=O), 1680 (s, C=O, lactam), 1608, 1472 (C=C, Ar), 1620 (C=N). <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) ppm; δ 11.2 (s, 2H), 10.2 (s, 2H), 7.15–7.79 (m, 6H) 2.2 (s, –CH<sub>3</sub>); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 377 (M+1, 100 %), 386 (M+Na, 51 %). Elemental analysis for C<sub>19</sub>H<sub>16</sub> N<sub>6</sub>O<sub>3</sub>: calculated (%) C: 60.63; H: 4.28; N: 22.33; found: C: 60.69; H: 4.26; N: 22.32.</p></sec><sec id="Sec21"><title>1,3-Bis({[(3Z)-5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino})urea (<bold>6d</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3097 (NH), 3050 (C–H, Ar), 1702 (C=O), 1618 (s, C=O, lactam), 1608, 1472 (C=C, Ar), 1626 (C=N) 1355 (–NO<sub>2</sub>). <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) ppm; δ 13.5 (s, 2H), 11.5 (s, 2H), 7.15–7.79 (m, 6H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 439 (M+1, 100 %). Elemental analysis for C<sub>17</sub>H<sub>10</sub> N<sub>8</sub>O<sub>7</sub>: calculated (%) C: 46.58; H: 2.30; N: 25.56; found: C: 46.53; H: 2.26; N: 25.54.</p></sec><sec id="Sec22"><title>1,3-Bis({[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino})thiourea (<bold>6e</bold>)</title><p>IR (ν cm<sup>−1</sup>): 3150, 3010 (NH, NH) 1650 (C=O), 1610 (C=N), 1350 (NCSN). <sup>1</sup>H-NMR (DMSO-<italic>d</italic><sub>6</sub>) δ ppm; δ 13.5 (s, 2H), 7.93 (s, 2H), 7.15–7.79 (m, 6H); mass (ESI–MS): <italic>m</italic>/<italic>z</italic> 365 (M+1, 100 %). Elemental analysis for C<sub>17</sub>H<sub>12</sub> N<sub>6</sub>O<sub>2</sub>S: calculated (%) C: 56.04; H: 3.32; N: 23.06; found: C: 56.03; H: 3.36; N: 23.04.</p></sec></sec></sec><sec id="Sec23"><title>Biological activities</title><sec id="Sec24"><title>Antiviral activity assays</title><p>The synthesized compounds were evaluated for antiviral activity according to well-established procedures by De Clercq (<xref ref-type="bibr" rid="CR10">1981</xref>, <xref ref-type="bibr" rid="CR11">1986</xref>). The compounds were evaluated for antiviral activity by using the following viruses: human cytomegalovirus (HCMV) strains AD-169 and Davis, 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, vaccinia virus, vesicular stomatitis virus (VSV), VZV strain Oka, TK<sup>-</sup> VZV strain 07-1, RSV strain Long, VSV, coxsackie B4, parainfluenza 3, Reovirus-1, Sindbis, Punta Toro, feline coronavirus (FIPV: feline infectious peritonitis virus), influenza A virus subtypes H1N1 and H3N2, and influenza B virus. Antiviral assays were carried out in Crandell–Rees feline kidney cells (feline corona virus and feline herpes virus), HeLa cell cultures (vesicular stomatitis virus, coxsackie virus B4, and RSV), human embryonic lung (HEL) cell cultures [HSV-1 (KOS), HSV-2 (G), vaccinia virus, vesicular stomatitis virus and HSV-1 TK<sup>-</sup> KOS ACV<sup>r</sup>], Madin Darby canine kidney (MDCK) cells (influenza A H1N1 subtype, influenza A H3N2 subtype, and influenza B) and Vero cell cultures (parainfluenza-3 virus, reovirus-1, sindbis virus, coxsackie virus B4, and Punta Toro virus). The HEL, Vero, and HeLa cell lines used in this study were monitored for mycoplasma contamination and were found to be mycoplasma-free.</p></sec><sec id="Sec25"><title>Screening for inhibition of virus-induced cytopathic effect in vitro</title><p>Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID<sub>50</sub> of virus (1 CCID<sub>50</sub> being the virus dose to infect 50 % of the cell cultures) or with 20 plaque forming units (PFU) (for VZV) and the cell cultures were incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation (VZV) was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC<sub>50</sub> or compound concentration required to reduce virus-induced cytopathicity or viral plaque formation by 50 %.</p></sec><sec id="Sec26"><title>Cytotoxicity assays</title><p>Cytostatic activity were based on the inhibition of HEL cell growth. HEL cells were seeded at 5 × 10<sup>3 </sup>cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at 37 °C, the cell number was determined with a Coulter counter. The cytostatic concentration was calculated as CC<sub>50</sub> (the compound concentration required to reduce cell growth by 50 % relative to the number of cells in the untreated controls). CC<sub>50</sub> values were estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds. Cytotoxicity was expressed as minimum cytotoxic concentration (MCC) or the compound concentration that causes a microscopically detectable alteration of cell morphology.</p></sec><sec id="Sec27"><title>Anti-influenza virus activity</title><p>The synthesized compounds were evaluated for their antiviral activity against three influenza virus subtypes [A/Puerto Rico/8/34 (H1N1); A/Hong Kong/7/87 (H3N2), and B/Hong Kong/5/72]. Antiviral activity was estimated from the inhibitory effect on virus-induced cytopathic effect, as determined by microscopical examination and/or the formazan-based MTS cell viability test. The EC<sub>50</sub> (50 % Effective concentration), or concentration producing 50 % inhibition of virus-induced cytopathic effect, was determined by visual scoring of the CPE, or by measuring the cell viability with the colorimetric formazan-based MTS assay. Cytotoxicity of the test compounds was expressed as the compound concentration causing minimal changes in cell morphology (MCC), or the concentration causing 50 % cytotoxicity (CC<sub>50</sub>), as determined by the MTS assay.</p></sec><sec id="Sec28"><title>Two-stage mouse-skin carcinogenesis test</title><p>Specific pathogen-free female ICR mice (6 weeks old, body weight approx. 30 g) were obtained from Japan SLC Inc., Shizuoka, Japan, and the animals were housed, five per polycarbonate cage, in a temperature-controlled room at 24 ± 2 °C and given food and water ad libitum throughout the experiment. Animals were divided into three experimental groups containing 15 mice each. The back (2 × 8 cm<sup>2</sup>) of each mouse was shaved with surgical clippers, and the mice were topically treated with 7,12-dimethyl benz[a]anthracene (DMBA) (100 μg, 390 nmol) in acetone (0.1 ml) an initiating treatment. One week after the initiation, papilloma formation was promoted twice weekly by the application of 12-<italic>O</italic>-tetradecanoylphorbol-13-acetate (TPA) (1 μg, 1.7 nmol) in acetone (0.1 ml) to the skin (Fig. <xref rid="Fig1" ref-type="fig">1</xref>). One hour before each treatment with TPA, the mice were treated with the samples (85 nmol) in acetone (0.1 ml). The incidence of papillomas was examined weekly over a period of 20 weeks (Tanaka <italic>et al</italic>., <xref ref-type="bibr" rid="CR29">2001</xref>), the percentages of mice bearing papillomas (Fig. <xref rid="Fig2" ref-type="fig">2</xref>a) and the average number of papillomas per mouse (Fig. <xref rid="Fig2" ref-type="fig">2</xref>b) were recorded.<fig id="Fig1"><label>Fig. 1</label><caption><p>Inhibitory effects of compounds <bold>6b</bold> and <bold>6c</bold> on mouse-skin carcinogenesis induced by DMBA and TPA. <bold>a</bold> Positive control [DMBA (390 nmol) + TPA (1.7 nmol)]; <bold>b</bold> TPA + 85 nmol of <bold>6b</bold>; <bold>c</bold> TPA + 85 nmol of <bold>6c</bold></p></caption><graphic xlink:href="44_2013_684_Fig1_HTML" id="MO4"></graphic></fig><fig id="Fig2"><label>Fig. 2</label><caption><p>Inhibition effects of compounds <bold>6b</bold> and <bold>6c</bold> on DMBA–TPA-induced carcinogenesis. Tumor formation in all mice was initiated with DMBA (390 nmol) and promoted with TPA (1.7 nmol) twice weekly, 1 week after initiation. <bold>a</bold> Percentage of mice with papillomas. <bold>b</bold> Average number of papillomas/mouse. <italic>Circle</italic> control [DMBA (390 nmol) + TPA (1.7 nmol)]; <italic>filled square</italic> TPA + 85 nmol of <bold>6b</bold>; <italic>triangle</italic> TPA + 85 nmol of <bold>6c</bold></p></caption><graphic xlink:href="44_2013_684_Fig2_HTML" id="MO5"></graphic></fig></p></sec></sec></sec><sec id="Sec29"><title>Results and discussion</title><p>A series of new β-isatin aldehyde-<italic>N</italic>,<italic>N</italic>′-thiocarbohydrazone, bis-β-isatin thiocarbohydrazones, and bis-β-isatin carbohydrazones derivatives was synthesized by microwave-oriented reaction by two-step procedure as depicted in Schemes <xref rid="Sch1" ref-type="fig">1</xref> and <xref rid="Sch2" ref-type="fig">2</xref>. The synthesized compounds were purified by column chromatography. All of the derivatives were characterized by FT-IR, <sup>1</sup>H NMR, and mass spectral data. The FT-IR spectra of the compounds revealed absorption bands in the region 1,509–1,610 cm<sup>−1</sup> corresponding to C=N stretching bands. Absence of carbonyl (C=O) peak around 1,715 cm<sup>−1</sup> revealed the formation of Schiff bases. The <sup>1</sup>H NMR spectrum of the synthesized compounds showed only one set of signals for the aromatic protons around 6.73–7.80 ppm, while the –NH of the isatin and –NH of hydrazones gave rise to two different signals in the 11–14 ppm range.</p><sec id="Sec30"><title>Antiviral activity</title><p>The novel β-isatin aldehyde-<italic>N</italic>,<italic>N</italic>′-thiocarbohydrazones (<bold>4a</bold>–<bold>g</bold>) and bis-isatin (<bold>6a</bold>–<bold>e</bold>) were evaluated against various strains of DNA viruses (i.e., human cytomegalovirus (HCMV), vaccinia virus (VACV), HSV type 1 (HSV-1) and type 2 (HSV-2), acyclovir-resistant, ACV<sup>r</sup>), VZV (Table <xref rid="Tab1" ref-type="table">1</xref>). The test compounds were also evaluated against several RNA viruses (i.e., VSV, coxsackie B4 virus, RSV, parainfluenza virus type 3, reovirus-1, sindbis virus and Punta Toro virus (Table <xref rid="Tab2" ref-type="table">2</xref>). The EC<sub>50</sub> was calculated as the effective concentration of compound that decreased the percentage of formazan production in compound treated, virus-infected cells to 50 % of that produced by compound-free uninfected cells. The 50 % cytostatic concentration (CC<sub>50</sub>) was calculated as the concentration required to reduce cell growth by 50 %. MCC is the minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. The HEL, Vero, and HeLa cell lines used in this study were regularly monitored for mycoplasma contamination and found to be mycoplasma-free. The antiviral activity was measured for twelve isatin derivatives using a cell-protection assay. The data are recorded in Tables <xref rid="Tab1" ref-type="table">1</xref>, <xref rid="Tab2" ref-type="table">2</xref>, <xref rid="Tab3" ref-type="table">3</xref>, and <xref rid="Tab4" ref-type="table">4</xref>.<table-wrap id="Tab1"><label>Table 1</label><caption><p>Inhibitory activity of synthesized isatin derivatives against DNA viruses</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="3">S. no.</th><th align="left" rowspan="3">Compounds</th><th align="left" colspan="7">Human embryonic cell culture (HEL) EC<sub arrange="stack">50</sub><sup arrange="stack">a</sup> (μM)</th><th align="left" colspan="2">Crandell–Rees feline kidney cell culture (CRFK)</th><th align="left" colspan="2">Human embryonic cell culture (HEL)</th></tr><tr><th align="left" colspan="2">Cytomegalovirus</th><th align="left" rowspan="2">Herpes simplex virus-1 (KOS)</th><th align="left" rowspan="2">Herpes simplex virus-2 (G)</th><th align="left" rowspan="2">Vaccinia virus</th><th align="left" rowspan="2">Vesicular stomatitis virus</th><th align="left" rowspan="2">Herpes simplex virus-1TK-KOS ACV<sup>r</sup></th><th align="left" rowspan="2">Feline corona virus</th><th align="left" rowspan="2">Feline herpes virus</th><th align="left" colspan="2">Varicella-zoster virus (VZV)</th></tr><tr><th align="left">AD-169 strain</th><th align="left">Davis strain</th><th align="left">TK + VZV strain</th><th align="left">TK-VZV strain</th></tr></thead><tbody><tr><td align="left">1</td><td align="left"><bold>4a</bold></td><td align="left">12 ± 3.5</td><td align="left">15 ± 0.4</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>4</td><td align="left">7.4</td></tr><tr><td align="left">2</td><td align="left"><bold>4b</bold></td><td align="left">16 ± 3.7</td><td align="left">11.32 ± 0.08</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td></tr><tr><td align="left">3</td><td align="left"><bold>4c</bold></td><td align="left">>20</td><td align="left">>20</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left">4</td><td align="left"><bold>4d</bold></td><td align="left">>20</td><td align="left">>20</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>20</td><td align="left">>20</td></tr><tr><td align="left">5</td><td align="left"><bold>4e</bold></td><td align="left">>20</td><td align="left">>20</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>20</td><td align="left">>20</td></tr><tr><td align="left">6</td><td align="left"><bold>4f</bold></td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>100</td><td align="left">>100</td><td align="left">>20</td><td align="left">>20</td></tr><tr><td align="left">7</td><td align="left"><bold>4g</bold></td><td align="left">>20</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>20</td></tr><tr><td align="left">8</td><td align="left"><bold>6a</bold></td><td align="left">>20</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>20</td><td align="left">>100</td></tr><tr><td align="left">9</td><td align="left"><bold>6b</bold></td><td align="left">>20</td><td align="left">>20</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">19.7</td><td align="left">>20</td><td align="left">>20</td></tr><tr><td align="left">10</td><td align="left"><bold>6c</bold></td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">55.1</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left">11</td><td align="left"><bold>6d</bold></td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>20</td><td align="left">>20</td></tr><tr><td align="left">12</td><td align="left"><bold>6e</bold></td><td align="left">>20</td><td align="left">>20</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>20</td><td align="left">>20</td></tr><tr><td align="left">13</td><td align="left">Ganciclovir</td><td align="left">8.1 ± 0.2</td><td align="left">5.5 ± 2.36</td><td align="left">0.02</td><td align="left">0.01</td><td align="left">>100</td><td align="left">>100</td><td align="left">4</td><td align="left">>100</td><td align="left">1.4</td><td align="left">–</td><td align="left">–</td></tr><tr><td align="left">14</td><td align="left">Cidofovir</td><td align="left">1.0 ± 0.05</td><td align="left">0.78 ± 0.49</td><td align="left">2</td><td align="left">1</td><td align="left">22</td><td align="left">>250</td><td align="left">0.9</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td></tr><tr><td align="left">15</td><td align="left">Brivudin</td><td align="left">–</td><td align="left">–</td><td align="left">0.04</td><td align="left">112</td><td align="left">4</td><td align="left">>250</td><td align="left">50</td><td align="left">–</td><td align="left">–</td><td align="left">2.1</td><td align="left">143</td></tr><tr><td align="left">16</td><td align="left">Acyclovir</td><td align="left">–</td><td align="left">–</td><td align="left">0.4</td><td align="left">0.2</td><td align="left">>250</td><td align="left">>250</td><td align="left">22</td><td align="left">–</td><td align="left">–</td><td align="left">0.019</td><td align="left">104.5</td></tr><tr><td align="left">17</td><td align="left">HHA (μg ml<sup>−1</sup>)</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">4.2</td><td align="left">11.0</td><td align="left">–</td><td align="left">–</td></tr><tr><td align="left">18</td><td align="left">UDA (μg ml<sup>−1</sup>)</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">–</td><td align="left">1.6</td><td align="left">1.2</td><td align="left">–</td><td align="left">–</td></tr></tbody></table><table-wrap-foot><p>“–” not performed</p><p><sup>a</sup>Required to reduce virus-induced cytopathogenicity by 50 %</p></table-wrap-foot></table-wrap><table-wrap id="Tab2"><label>Table 2</label><caption><p>Inhibitory activity of synthesized isatin derivatives against RNA Viruses</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="3">S. no.</th><th align="left" rowspan="3">Compound</th><th align="left" colspan="8">Antiviral activity EC<sub arrange="stack">50</sub><sup arrange="stack">a</sup> (μM)</th></tr><tr><th align="left" colspan="3">HeLa cell cultures</th><th align="left" colspan="5">Vero cell cultures</th></tr><tr><th align="left">Vesicular stomatitis virus</th><th align="left">Coxsackie virus B4</th><th align="left">Respiratory syncytial virus</th><th align="left">Para-influenza-3 virus</th><th align="left">Reovirus-1</th><th align="left">Sindbis virus</th><th align="left">Coxsackie virus B4</th><th align="left">Punta Toro virus</th></tr></thead><tbody><tr><td align="left">1</td><td align="left"><bold>4a</bold></td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td></tr><tr><td align="left">2</td><td align="left"><bold>4b</bold></td><td char=">" align="char">>4</td><td char=">" align="char">>4</td><td char=">" align="char">>4</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td></tr><tr><td align="left">3</td><td align="left"><bold>4c</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td></tr><tr><td align="left">4</td><td align="left"><bold>4d</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td></tr><tr><td align="left">5</td><td align="left"><bold>4e</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td></tr><tr><td align="left">6</td><td align="left"><bold>4f</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td></tr><tr><td align="left">7</td><td align="left"><bold>4g</bold></td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td></tr><tr><td align="left">8</td><td align="left"><bold>6a</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td></tr><tr><td align="left">9</td><td align="left"><bold>6b</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td></tr><tr><td align="left">10</td><td align="left"><bold>6c</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td></tr><tr><td align="left">11</td><td align="left"><bold>6d</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td></tr><tr><td align="left">12</td><td align="left"><bold>6e</bold></td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td><td char=">" align="char">>20</td></tr><tr><td align="left">13</td><td align="left">DS—5,000 (μg ml<sup>−1</sup>)</td><td char=">" align="char">1</td><td char=">" align="char">>100</td><td char=">" align="char">0.8</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td><td char=">" align="char">100</td><td char=">" align="char">>100</td><td char=">" align="char">>100</td></tr><tr><td align="left">14</td><td align="left">(S)-DHPA</td><td char=">" align="char">>250</td><td char=">" align="char">>250</td><td char=">" align="char">>250</td><td char=">" align="char">>250</td><td char=">" align="char">>250</td><td char=">" align="char">>250</td><td char=">" align="char">>250</td><td char=">" align="char">>250</td></tr><tr><td align="left">15</td><td align="left">Ribavirin</td><td char=">" align="char">4</td><td char=">" align="char">112</td><td char=">" align="char">4</td><td char=">" align="char">85</td><td char=">" align="char">146</td><td char=">" align="char">>250</td><td char=">" align="char">250</td><td char=">" align="char">25</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>Required to reduce virus-induced cytopathogenicity by 50 %</p></table-wrap-foot></table-wrap><table-wrap id="Tab3"><label>Table 3</label><caption><p>Anti-influenza virus activity and cytotoxicity of compounds in MDCK cell cultures</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="3">Compound</th><th align="left" colspan="2">Cytotoxicity</th><th align="left" colspan="6">Antiviral EC<sub arrange="stack">50</sub><sup arrange="stack">c</sup> (μM)</th></tr><tr><th align="left" rowspan="2">CC<sub arrange="stack">50</sub><sup arrange="stack">a</sup>(μM)</th><th align="left" rowspan="2">Minimum cytotoxic concentration<sup>b</sup></th><th align="left" colspan="2">Influenza A H1N1 subtype</th><th align="left" colspan="2">Influenza A H3N2 subtype</th><th align="left" colspan="2">Influenza B</th></tr><tr><th align="left">Visual CPE score</th><th align="left">MTS</th><th align="left">Visual CPE score</th><th align="left">MTS</th><th align="left">Visual CPE score</th><th align="left">MTS</th></tr></thead><tbody><tr><td align="left"><bold>4a</bold></td><td align="left">51</td><td char="." align="char">20</td><td align="left">>4</td><td align="left">>4</td><td align="left">>4</td><td align="left">>4</td><td align="left">>4</td><td align="left">>4</td></tr><tr><td align="left"><bold>4b</bold></td><td align="left">46</td><td char="." align="char">20</td><td align="left">>4</td><td align="left">>4</td><td align="left">>4</td><td align="left">>4</td><td align="left">>4</td><td align="left">>4</td></tr><tr><td align="left"><bold>4c</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left"><bold>4d</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left"><bold>4e</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left"><bold>4f</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">39 ± 5.5</td><td align="left">37 ± 25</td><td align="left">27 ± 7.0</td><td align="left">27 ± 7.3</td><td align="left">45 ± 0.0</td><td align="left">37 ± 8.75</td></tr><tr><td align="left"><bold>4g</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left"><bold>6a</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left"><bold>6b</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left"><bold>6c</bold></td><td align="left">46</td><td char="." align="char">≥20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td><td align="left">>20</td></tr><tr><td align="left"><bold>6d</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">47 ± 2.5</td><td align="left">55 ± 26</td><td align="left">72 ± 27</td><td align="left">33 ± 1.7</td><td align="left">47 ± 2.5</td><td align="left">38 ± 1.3</td></tr><tr><td align="left"><bold>6e</bold></td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">72 ± 27</td><td align="left">61 ± 32</td><td align="left">72 ± 27</td><td align="left">34 ± 9.5</td><td align="left">39 ± 5.5</td><td align="left">48 ± 31</td></tr><tr><td align="left">Oseltamivir carboxylate</td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">0.4 ± 0.0</td><td align="left">0.35 ± 0.2</td><td align="left">10.25 ± 9.7</td><td align="left">5.45 ± 4.05</td><td align="left">6.7 ± 5.3</td><td align="left">2.5 ± 0.7</td></tr><tr><td align="left">Ribavirin</td><td align="left">>100</td><td char="." align="char">>100</td><td align="left">2.5 ± 0.5</td><td align="left">2.8 ± 0.25</td><td align="left">3 ± 1.0</td><td align="left">2 ± 0.2</td><td align="left">2.4 ± 1.6</td><td align="left">2.45 ± 0.25</td></tr><tr><td align="left">Amantadine</td><td align="left">>200</td><td char="." align="char">>200</td><td align="left">5</td><td align="left">3.2</td><td align="left">0.9</td><td align="left">0.8</td><td align="left">>200</td><td align="left">>200</td></tr><tr><td align="left">Rimantadine</td><td align="left">>200</td><td char="." align="char">>200</td><td align="left">>200</td><td align="left">>200</td><td align="left">0.4</td><td align="left">0.6</td><td align="left">>200</td><td align="left">>200</td></tr></tbody></table><table-wrap-foot><p><italic>MDCK cells</italic> Madin Darby canine kidney cells</p><p><sup>a</sup>50 % Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay</p><p><sup>b</sup>Minimum compound concentration that causes a microscopically detectable alteration of normal cell morphology</p><p><sup>c</sup>50 % Effective concentration, or concentration producing 50 % inhibition of virus-induced cytopathic effect, as determined by visual scoring of the CPE, or by measuring the cell viability with the colorimetric formazan-based MTS assay</p></table-wrap-foot></table-wrap><table-wrap id="Tab4"><label>Table 4</label><caption><p>Cytotoxicity for synthesized isatin derivatives in different cell cultures</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2">S.no</th><th align="left" rowspan="2">Compound</th><th align="left">Human embryonic lung cell cultures</th><th align="left">Crandell-Rees feline kidney cells</th><th align="left">HeLa cell cultures</th><th align="left">Vero cell cultures</th></tr><tr><th align="left">Minimum cytotoxic concentration<sup>a</sup> (μM)</th><th align="left">Minimum cytotoxic concentration<sup>a</sup> (μM)</th><th align="left">Minimum cytotoxic concentration<sup>a</sup> (μM)</th><th align="left">Minimum cytotoxic concentration<sup>a</sup> (μM)</th></tr></thead><tbody><tr><td align="left">1</td><td align="left"><bold>4a</bold></td><td align="left">60</td><td align="left">86</td><td align="left">≥20</td><td align="left">100</td></tr><tr><td align="left">2</td><td align="left"><bold>4b</bold></td><td align="left">100</td><td align="left">63</td><td align="left">20</td><td align="left">100</td></tr><tr><td align="left">3</td><td align="left"><bold>4c</bold></td><td align="left">≥100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left">4</td><td align="left"><bold>4d</bold></td><td align="left">100</td><td align="left">>100</td><td align="left">>100</td><td align="left">100</td></tr><tr><td align="left">5</td><td align="left"><bold>4e</bold></td><td align="left">100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left">6</td><td align="left"><bold>4f</bold></td><td align="left">100</td><td align="left">100</td><td align="left">>100</td><td align="left">≥20</td></tr><tr><td align="left">7</td><td align="left"><bold>4g</bold></td><td align="left">≥100</td><td align="left">≥100</td><td align="left">100</td><td align="left">>100</td></tr><tr><td align="left">8</td><td align="left"><bold>6a</bold></td><td align="left">≥100</td><td align="left">>100</td><td align="left">>100</td><td align="left">100</td></tr><tr><td align="left">9</td><td align="left"><bold>6b</bold></td><td align="left">100</td><td align="left">>100</td><td align="left">>100</td><td align="left">100</td></tr><tr><td align="left">10</td><td align="left"><bold>6c</bold></td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td><td align="left">100</td></tr><tr><td align="left">11</td><td align="left"><bold>6d</bold></td><td align="left">≥100</td><td align="left">>100</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left">12</td><td align="left"><bold>6e</bold></td><td align="left">100</td><td align="left">>100</td><td align="left">>100</td><td align="left">100</td></tr><tr><td align="left">13</td><td align="left">Acyclovir</td><td align="left">>400</td><td align="left">–</td><td align="left">–</td><td align="left">–</td></tr><tr><td align="left">14</td><td align="left">Brivudin</td><td align="left">>300</td><td align="left">–</td><td align="left">–</td><td align="left">–</td></tr><tr><td align="left">15</td><td align="left">Ganciclovir</td><td align="left">>350</td><td align="left">>100</td><td align="left">–</td><td align="left">–</td></tr><tr><td align="left">16</td><td align="left">Cidofovir</td><td align="left">>300</td><td align="left">–</td><td align="left">–</td><td align="left">–</td></tr><tr><td align="left">17</td><td align="left">DS-5000 (μg ml)</td><td align="left">–</td><td align="left">–</td><td align="left">>100</td><td align="left">>100</td></tr><tr><td align="left">18</td><td align="left">(S)-DHPA</td><td align="left">–</td><td align="left">–</td><td align="left">>250</td><td align="left">>250</td></tr><tr><td align="left">19</td><td align="left">Ribavirin</td><td align="left">–</td><td align="left">–</td><td align="left">>250</td><td align="left">>250</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>Minimum cytotoxic concentration that causes a microscopically detectable alteration of normal cell morphology</p></table-wrap-foot></table-wrap></p><p>There was only moderate activity of the <bold>4a</bold> and <bold>4b</bold> compounds against both strains of HCMV. Although at concentrations close to their toxicity levels, no specific antiviral effects were noted with the evaluated compounds against HSV-1, (HSV-1 strain KOS), HSV-2 (HSV-2 strain G), HSV-1 (ACV resistant KOS strain), vaccinia virus, vesicular stomatitis virus, coxsackie virus B4, respiratory syncytial virus, parainfluenza-3 virus, reovirus-1, sindbis virus, and Punta Toro virus. None of the compounds exhibited specific antiviral effects (i.e., minimal antiviral effective concentration ≥ fivefold lower than minimal cytotoxic concentration) against any of the viruses evaluated.</p></sec><sec id="Sec31"><title>Anti-influenza virus activity</title><p>The anti-influenza virus activities of a series of isatin derivatives were determined by measuring their inhibitory effects on virus replication in MDCK cells using three strains of the influenza subtype. The antiviral data obtained by microscopic evaluation of the virus-induced CPE were confirmed by the MTS cell viability assay and were expressed as the EC<sub>50</sub>. The cytotoxicities of the compounds were expressed as the MCC (the compound concentration causing minimal alterations in cell morphology as estimated by microscopy) or the CC<sub>50</sub> (the compound concentration causing 50 % reduction in cell viability based on the MTS assay).</p><p>None of the twelve analogs were able to inhibit the cytopathic effects of three influenza virus subtypes i.e., H1N1, H3N2, and influenza B (Table <xref rid="Tab3" ref-type="table">3</xref>), except <bold>4f</bold>, <bold>6d</bold>, and <bold>6e</bold> that showed some activity below the non-toxic concentration of 100 µM.</p></sec><sec id="Sec32"><title>Two-stage mouse-skin carcinogenesis test</title><p>In the present study, we selected two compounds <bold>6b</bold> and <bold>6c</bold> among the ten compounds to examine their effects on in vivo two-stage carcinogenesis using mouse-skin papillomas induced by DMBA as an initiator and TPA as a promoter. During the test, no significant toxic effects, such as inflammation and lesional damage (edema, erosion, and ulcer), were observed on the areas of mouse skin topically treated with test compounds. In our experiment, the body weight of the animals was not affected by the treatment with test compounds. As shown in Fig. <xref rid="Fig2" ref-type="fig">2</xref>a, the positive control (DMBA and TPA) group in papilloma-bearing mice appears rapidly from week 6, and reached 100 % within 10 weeks of promotion. On the other hand, treatment with compound <bold>6c</bold> (85 nmol) along with DMBA/TPA reduced the percentage of papilloma-bearing mice to 40–70 % during weeks 10–15, and thereafter 90 % during week 18 and reached 100 % at week 20. As shown in Fig. <xref rid="Fig2" ref-type="fig">2</xref>b, in the positive control group, the number of papillomas per mouse formed increased rapidly after week 6 and reached 7.9 papillomas/mouse at week 20. On the other hand, mice treated with compound <bold>6c</bold> bore only 5.1 papillomas/mouse over the period of week 20. A moderate protection is observed for the compound <bold>6b</bold>. The percentage of papillomas increased rapidly from week 7 and reached 100 % after week 17, and in this group only 1.2, 3.9, and 6.1 papillomas were formed per mouse at 10, 15, and 20 weeks of promotion. From these results, the inhibitory effects of compound <bold>6c</bold> on mouse two-stage carcinogenesis induced by DMBA and TPA were apparently more potent than those of compound <bold>6b</bold>. Thus, as demonstrated above, the in vivo anti-tumor promoting activity of compound <bold>6c</bold> showed stronger chemoprevention activity than that of compound <bold>6b</bold>. These results suggest that isatin lead molecules may be useful as cancer chemopreventive agents.</p></sec></sec><sec id="Sec33"><title>Conclusion</title><p>We have synthesized twelve compounds by reacting substituted/unsubstituted 2,3-dihydro-2,3-dioxindole and thiocarbohydrazide/carbohydrazide in alcohol in the presence of glacial acetic acid. All synthesized compounds were characterized by FT-IR, NMR, and mass spectra. All the compounds were screened for antiviral activity against a various strains of DNA and RNA viruses and some of them, exhibited moderate antiviral activity compared with the reference compounds. Further, the anti-carcinogenic activity of compounds <bold>6b</bold> and <bold>6c</bold> was examined by a two stage-carcinogenesis test using DMBA as initiator and TPA as a promoter. The data suggested that compound <bold>6c</bold> exhibited stronger chemoprevention activity than compound <bold>6b</bold>. Thus these results suggest that isatin lead molecules could also be useful as cancer chemopreventive agents.</p></sec></body><back><ack><p>The authors are very much thankful to M. Sadalaxmi, President, Kakatiya Institute of Pharmaceutical Sciences, for providing the laboratory facilities. The antiviral research was performed by grants of the KU Leuven (GOA 10/14). 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