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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Identification of phenanthroindolizines and phenanthroquinolizidines as novel potent anti-coronaviral agents for porcine enteropathogenic coronavirus transmissible gastroenteritis virus and human severe acute respiratory syndrome coronavirus</title><author><name sortKey="Yang, Cheng Wei" sort="Yang, Cheng Wei" uniqKey="Yang C" first="Cheng-Wei" last="Yang">Cheng-Wei Yang</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation><affiliation><nlm:aff id="aff0010">Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Lee, Yue Zhi" sort="Lee, Yue Zhi" uniqKey="Lee Y" first="Yue-Zhi" last="Lee">Yue-Zhi Lee</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Kang, Iou Jiun" sort="Kang, Iou Jiun" uniqKey="Kang I" first="Iou-Jiun" last="Kang">Iou-Jiun Kang</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Barnard, Dale L" sort="Barnard, Dale L" uniqKey="Barnard D" first="Dale L." last="Barnard">Dale L. Barnard</name><affiliation><nlm:aff id="aff0015">Institute for Antiviral Research, Utah State University, Logan, UT, USA</nlm:aff></affiliation></author><author><name sortKey="Jan, Jia Tsrong" sort="Jan, Jia Tsrong" uniqKey="Jan J" first="Jia-Tsrong" last="Jan">Jia-Tsrong Jan</name><affiliation><nlm:aff id="aff0020">Genomics Research Center, Academia Sinica, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Lin, Du" sort="Lin, Du" uniqKey="Lin D" first="Du" last="Lin">Du Lin</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Huang, Chun Wei" sort="Huang, Chun Wei" uniqKey="Huang C" first="Chun-Wei" last="Huang">Chun-Wei Huang</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Yeh, Teng Kuang" sort="Yeh, Teng Kuang" uniqKey="Yeh T" first="Teng-Kuang" last="Yeh">Teng-Kuang Yeh</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Chao, Yu Sheng" sort="Chao, Yu Sheng" uniqKey="Chao Y" first="Yu-Sheng" last="Chao">Yu-Sheng Chao</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Lee, Shiow Ju" sort="Lee, Shiow Ju" uniqKey="Lee S" first="Shiow-Ju" last="Lee">Shiow-Ju Lee</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">20727913</idno><idno type="pmc">7114283</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114283</idno><idno type="RBID">PMC:7114283</idno><idno type="doi">10.1016/j.antiviral.2010.08.009</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">000D90</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D90</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Identification of phenanthroindolizines and phenanthroquinolizidines as novel potent anti-coronaviral agents for porcine enteropathogenic coronavirus transmissible gastroenteritis virus and human severe acute respiratory syndrome coronavirus</title><author><name sortKey="Yang, Cheng Wei" sort="Yang, Cheng Wei" uniqKey="Yang C" first="Cheng-Wei" last="Yang">Cheng-Wei Yang</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation><affiliation><nlm:aff id="aff0010">Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Lee, Yue Zhi" sort="Lee, Yue Zhi" uniqKey="Lee Y" first="Yue-Zhi" last="Lee">Yue-Zhi Lee</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Kang, Iou Jiun" sort="Kang, Iou Jiun" uniqKey="Kang I" first="Iou-Jiun" last="Kang">Iou-Jiun Kang</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Barnard, Dale L" sort="Barnard, Dale L" uniqKey="Barnard D" first="Dale L." last="Barnard">Dale L. Barnard</name><affiliation><nlm:aff id="aff0015">Institute for Antiviral Research, Utah State University, Logan, UT, USA</nlm:aff></affiliation></author><author><name sortKey="Jan, Jia Tsrong" sort="Jan, Jia Tsrong" uniqKey="Jan J" first="Jia-Tsrong" last="Jan">Jia-Tsrong Jan</name><affiliation><nlm:aff id="aff0020">Genomics Research Center, Academia Sinica, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Lin, Du" sort="Lin, Du" uniqKey="Lin D" first="Du" last="Lin">Du Lin</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Huang, Chun Wei" sort="Huang, Chun Wei" uniqKey="Huang C" first="Chun-Wei" last="Huang">Chun-Wei Huang</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Yeh, Teng Kuang" sort="Yeh, Teng Kuang" uniqKey="Yeh T" first="Teng-Kuang" last="Yeh">Teng-Kuang Yeh</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Chao, Yu Sheng" sort="Chao, Yu Sheng" uniqKey="Chao Y" first="Yu-Sheng" last="Chao">Yu-Sheng Chao</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author><author><name sortKey="Lee, Shiow Ju" sort="Lee, Shiow Ju" uniqKey="Lee S" first="Shiow-Ju" last="Lee">Shiow-Ju Lee</name><affiliation><nlm:aff id="aff0005">Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</nlm:aff></affiliation></author></analytic><series><title level="j">Antiviral Research</title><idno type="ISSN">0166-3542</idno><idno type="eISSN">1872-9096</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The discovery and development of new, highly potent anti-coronavirus agents and effective approaches for controlling the potential emergence of epidemic coronaviruses still remains an important mission. Here, we identified tylophorine compounds, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines, as potent <italic>in vitro</italic> inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV). The potent compounds showed 50% maximal effective concentration (EC<sub>50</sub>) values ranging from 8 to 1468 nM as determined by immunofluorescent assay of the expression of TGEV N and S proteins and by real time-quantitative PCR analysis of viral yields. Furthermore, the potent tylophorine compounds exerted profound anti-TGEV replication activity and thereby blocked the TGEV-induced apoptosis and subsequent cytopathic effect in ST cells. Analysis of the structure–activity relations indicated that the most active tylophorine analogues were compounds with a hydroxyl group at the C14 position of the indolizidine moiety or at the C3 position of the phenanthrene moiety and that the quinolizidine counterparts were more potent than indolizidines. In addition, tylophorine compounds strongly reduced cytopathic effect in Vero 76 cells induced by human severe acute respiratory syndrome coronavirus (SARS CoV), with EC<sub>50</sub> values ranging from less than 5 to 340 nM. Moreover, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of 7-methoxycryptopleurine (52.7%) and the naturally occurring tylophorine (65.7%) in rats. Thus, our results suggest that tylophorine compounds are novel and potent anti-coronavirus agents that may be developed into therapeutic agents for treating TGEV or SARS CoV infection.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Abe, I" uniqKey="Abe I">I. Abe</name></author><author><name sortKey="Yukiko" uniqKey="Yukiko">Yukiko</name></author><author><name sortKey="Yamauchi, T" uniqKey="Yamauchi T">T. Yamauchi</name></author><author><name sortKey="Honda, K" uniqKey="Honda K">K. Honda</name></author><author><name sortKey="Hayashi, N" uniqKey="Hayashi N">N. Hayashi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Barnard, D L" uniqKey="Barnard D">D.L. Barnard</name></author><author><name sortKey="Day, C W" uniqKey="Day C">C.W. Day</name></author><author><name sortKey="Bailey, K" uniqKey="Bailey K">K. Bailey</name></author><author><name sortKey="Heiner, M" uniqKey="Heiner M">M. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Antiviral Res</journal-id><journal-id journal-id-type="iso-abbrev">Antiviral Res</journal-id><journal-title-group><journal-title>Antiviral Research</journal-title></journal-title-group><issn pub-type="ppub">0166-3542</issn><issn pub-type="epub">1872-9096</issn><publisher><publisher-name>Elsevier B.V.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">20727913</article-id><article-id pub-id-type="pmc">7114283</article-id><article-id pub-id-type="publisher-id">S0166-3542(10)00696-0</article-id><article-id pub-id-type="doi">10.1016/j.antiviral.2010.08.009</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Identification of phenanthroindolizines and phenanthroquinolizidines as novel potent anti-coronaviral agents for porcine enteropathogenic coronavirus transmissible gastroenteritis virus and human severe acute respiratory syndrome coronavirus</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Cheng-Wei</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="aff0010" ref-type="aff">b</xref><xref rid="fn0005" ref-type="fn">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Yue-Zhi</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="fn0005" ref-type="fn">1</xref></contrib><contrib contrib-type="author"><name><surname>Kang</surname><given-names>Iou-Jiun</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Barnard</surname><given-names>Dale L.</given-names></name><xref rid="aff0015" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Jan</surname><given-names>Jia-Tsrong</given-names></name><xref rid="aff0020" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Du</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Chun-Wei</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Yeh</surname><given-names>Teng-Kuang</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Chao</surname><given-names>Yu-Sheng</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Shiow-Ju</given-names></name><email>slee@nhri.org.tw</email><xref rid="aff0005" ref-type="aff">a</xref><xref rid="cor0005" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff0005"><label>a</label>Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, ROC</aff><aff id="aff0010"><label>b</label>Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, ROC</aff><aff id="aff0015"><label>c</label>Institute for Antiviral Research, Utah State University, Logan, UT, USA</aff><aff id="aff0020"><label>d</label>Genomics Research Center, Academia Sinica, Taiwan, ROC</aff><author-notes><corresp id="cor0005"><label>⁎</label>Corresponding author at: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC. Tel.: +886 37 246166x35715, fax: +886 37 586456. <email>slee@nhri.org.tw</email></corresp><fn id="fn0005"><label>1</label><p>Authors with equal contribution.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>19</day><month>8</month><year>2010</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>11</month><year>2010</year></pub-date><pub-date pub-type="epub"><day>19</day><month>8</month><year>2010</year></pub-date><volume>88</volume><issue>2</issue><fpage>160</fpage><lpage>168</lpage><history><date date-type="received"><day>11</day><month>6</month><year>2010</year></date><date date-type="rev-recd"><day>7</day><month>8</month><year>2010</year></date><date date-type="accepted"><day>12</day><month>8</month><year>2010</year></date></history><permissions><copyright-statement>Copyright © 2010 Elsevier B.V. All rights reserved.</copyright-statement><copyright-year>2010</copyright-year><copyright-holder>Elsevier B.V.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract><p>The discovery and development of new, highly potent anti-coronavirus agents and effective approaches for controlling the potential emergence of epidemic coronaviruses still remains an important mission. Here, we identified tylophorine compounds, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines, as potent <italic>in vitro</italic> inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV). The potent compounds showed 50% maximal effective concentration (EC<sub>50</sub>) values ranging from 8 to 1468 nM as determined by immunofluorescent assay of the expression of TGEV N and S proteins and by real time-quantitative PCR analysis of viral yields. Furthermore, the potent tylophorine compounds exerted profound anti-TGEV replication activity and thereby blocked the TGEV-induced apoptosis and subsequent cytopathic effect in ST cells. Analysis of the structure–activity relations indicated that the most active tylophorine analogues were compounds with a hydroxyl group at the C14 position of the indolizidine moiety or at the C3 position of the phenanthrene moiety and that the quinolizidine counterparts were more potent than indolizidines. In addition, tylophorine compounds strongly reduced cytopathic effect in Vero 76 cells induced by human severe acute respiratory syndrome coronavirus (SARS CoV), with EC<sub>50</sub> values ranging from less than 5 to 340 nM. Moreover, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of 7-methoxycryptopleurine (52.7%) and the naturally occurring tylophorine (65.7%) in rats. Thus, our results suggest that tylophorine compounds are novel and potent anti-coronavirus agents that may be developed into therapeutic agents for treating TGEV or SARS CoV infection.</p></abstract><kwd-group><title>Keywords</title><kwd>Coronavirus</kwd><kwd>Human severe acute respiratory syndrome coronavirus</kwd><kwd>Porcine transmissible gastroenteritis virus</kwd><kwd>Phenanthroindolizine</kwd><kwd>Phenanthroquinolizidine</kwd><kwd>Tylophorine</kwd></kwd-group></article-meta></front><body><sec id="sec0005"><label>1</label><title>Introduction</title><p id="par0005">Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical symmetry. As animal viruses, they belong to the family <italic>Coronaviridae</italic> and are divided into three groups based on serological cross-reactivity and confirmed by genome sequence analysis. Group I coronaviruses include animal pathogens, such as porcine transmissible gastroenteritis coronavirus (TGEV) and human coronaviruses HCoV-229E. Group II also includes pathogens of veterinary relevance, such as murine coronavirus mouse hepatitis virus (MHV)-JHM strain and human severe acute respiratory syndrome coronavirus (SARS CoV) as well as Group III includes only avian coronaviruses (<xref rid="bib0125" ref-type="bibr">Weiss and Navas-Martin, 2005</xref>). They primarily infect the upper respiratory and gastrointestinal tract of birds and mammals (<xref rid="bib0020" ref-type="bibr">Brian and Baric, 2005</xref>).</p><p id="par0010">Vaccines are currently available for some animal coronaviruses. Some vaccines are efficacious, and others cause adverse side effects. For instance, some vaccines against feline coronaviruses exacerbated disease in vaccinated animals exposed to the wild-type virus (<xref rid="bib0095" ref-type="bibr">Olsen, 1993</xref>). Moreover, antibody enhancement of disease is a potential risk of SARS CoV vaccines in humans (<xref rid="bib0125" ref-type="bibr">Weiss and Navas-Martin, 2005</xref>). Although no cases of SARS have been reported since April 2004, the nature of the unpredictable outbreak of SARS CoV is still a potential threat to the global economy and public health (<xref rid="bib0125" ref-type="bibr">Weiss and Navas-Martin, 2005</xref>). No effective therapy exists for SARS CoV infection, despite tremendous effort invested in finding anti-SARS CoV drugs, including targeting SARS CoV-specific main protease or viral attachment, entry, and fusion for intervention. Thus, SARS CoV or some variants thereof could easily remerge to cause disease. The discovery and development of new, highly potent anti-coronavirus agents and effective approaches for controlling the potential emergence of epidemic coronaviruses remains an important mission.</p><p id="par0015">The phenanthroindolizidine alkaloids, such as tylophorine, tylocrebrine, and tylophorinine, are characteristic constituents of <italic>Cynanchum</italic>, <italic>Pergularia</italic>, <italic>Tylophora</italic>, and other genera of the <italic>Asclepiadaceae</italic>. These phenanthroindolizidines have activities against leukemia, asthma, anaphylaxis, other inflammations and bacterial infection (<xref rid="bib0015" ref-type="bibr">Bhutani et al., 1987</xref>, <xref rid="bib0025" ref-type="bibr">Chemler, 2009</xref>, <xref rid="bib0045" ref-type="bibr">Gopalakrishnan et al., 1980</xref>), as well as against cancer (<xref rid="bib0035" ref-type="bibr">Gao et al., 2004</xref>, <xref rid="bib0075" ref-type="bibr">Li et al., 2001b</xref>, <xref rid="bib0120" ref-type="bibr">Staerk et al., 2002</xref>, <xref rid="bib0140" ref-type="bibr">Yang et al., 2006</xref>, <xref rid="bib0145" ref-type="bibr">Yang et al., 2007a</xref>). The herbal plant <italic>Tylophora indica</italic> has been traditionally used in India as a folk remedy to treat bronchial asthma, bronchitis, rheumatism, and dermatitis (<xref rid="bib0070" ref-type="bibr">Li et al., 2001a</xref>). In a clinical trial, six <italic>Tylophora</italic> leaves (one per day) were efficacious as an herbal medicine for asthma (<xref rid="bib0110" ref-type="bibr">Shivpuri et al., 1968</xref>). <italic>Tylophora ovata</italic> has been used as traditional herbal medicine for rheumatism, asthma, and even traumatic injury even though it possesses mildly toxic properties (<xref rid="bib0080" ref-type="bibr">Lin, 2003</xref>). Phenanthroquinolizidines, typified by cryptopleurine, isolated from <italic>Boehmeria siamensis</italic> or through chemical synthesis, have anti-cancer and anti-inflammatory activities (<xref rid="bib0030" ref-type="bibr">Chuang et al., 2006</xref>, <xref rid="bib0075" ref-type="bibr">Li et al., 2001b</xref>, <xref rid="bib0085" ref-type="bibr">Luo et al., 2003</xref>, <xref rid="bib0140" ref-type="bibr">Yang et al., 2006</xref>). <italic>Tylophora</italic> plants used as alternative medicine and their derived alkaloids have been under extensive investigation for drug development (<xref rid="bib0025" ref-type="bibr">Chemler, 2009</xref>).</p><p id="par0020">Here, we report on the discovery, synthesis, isolation and identification of tylophorine compounds as novel, potent inhibitors of TGEV and SARS CoV. Structure–activity relations were also analyzed. This inhibition of TGEV and SARS CoV by tylophorine compounds with high potency <italic>in vitro</italic> at low nanomolar range and high oral availability in rats suggests that they may be potential therapeutic agents for coronavirus infections such as TGEV and SARS CoV.</p></sec><sec id="sec0010"><label>2</label><title>Materials and methods</title><sec id="sec0015"><label>2.1.1</label><title>Cells, viruses, immunofluorescent assay (IFA), and cytopathic effect (CPE)</title><p id="par0025">Swine testicular (ST) epithelial cells and the Taiwan field-isolated virulent strain of TGEV were grown and propagated as described (<xref rid="bib0150" ref-type="bibr">Yang et al., 2007b</xref>). IFA and CPE and cytotoxicity assays were also as described (<xref rid="bib0150" ref-type="bibr">Yang et al., 2007b</xref>). Briefly, ST cells in 96-well plates, with or without a 2 h pretreatment with test compounds, were infected with TGEV at a multiplicity of infection (MOI) of 10 for IFA. The IFA was performed at 6 h post-infection (hpi) with antibodies against the spike (S) and nucleocapsid (N) proteins of TGEV. The cells were treated with 10 different concentrations of test compounds. The results of these assays were used to obtain the dose–response curves from which 50% maximal effective concentration (EC<sub>50</sub>) values were determined. For the CPE assay, TGEV was inoculated into a monolayer of ST cells in 12-well plates at 5 MOI, and the CPE was determined at 24 hpi for detection of anti-TGEV activity. TGEV-induced CPE is characterized by a rounding and enlargement of cells, formation of syncytia, and detachment of cells into the medium. Results presented are representative of at least 3 independent experiments. For cytotoxicity assay, ST cells cultured in minimal essential medium (Invitrogen, Inc.) and 10% fetal bovine serum (HyClone Co.) in 96-well plates were treated with 10 different concentrations of test compounds for 24 h. The resulting dose–response curves were used to determine 50% maximal cytotoxic concentration (CC<sub>50</sub>) values.</p><p id="par0030">Vero 76 cells and SARS CoV Urbani strain were grown and propagated as described (<xref rid="bib0010" ref-type="bibr">Barnard et al., 2006</xref>). SARS CoV-related CPE inhibition and neutral red uptake assays for determination of antiviral efficacy (EC<sub>50</sub>) and compound cytotoxicity (CC<sub>50</sub>) were performed as described (<xref rid="bib0010" ref-type="bibr">Barnard et al., 2006</xref>).</p></sec><sec id="sec0020"><label>2.2</label><title>Chemicals, western blot analysis, viral RNA isolation and relative quantification by real-time RT-PCR</title><p id="par0035">These assays were performed as described (<xref rid="bib0150" ref-type="bibr">Yang et al., 2007b</xref>); antibody against human GAPDH was purchased from Cell Signaling Technology Inc. (MA, USA). DMSO, PEG400 and DMA were purchased from Sigma Aldrich Inc. (MO, USA). Infergen™ was interferon alfacon-1 lot 002586 kindly provided by Intermune, Inc. (Brisbane, CA).</p></sec><sec id="sec0025"><label>2.3</label><title>Pharmacokinetic analysis</title><p id="par0040">The Sprague–Dawley rats for the pharmacokinetic study were obtained from BioLASCO Taiwan Co. (Ilan, Taiwan) and housed in the animal facility at the National Health Research Institutes, Taiwan. The animal studies were performed according to committee-approved procedures. Male rats (330–380 g, 9–10 weeks old) were quarantined for 1 week before use. The animals were surgically implanted with a jugular-vein cannula 1 day before treatment and were fasted before treatment. Compounds <bold>1a</bold> and <bold>1c</bold> were given to rats (<italic>n</italic> = 3) at 3 mg/kg by intravenous or oral administration. <bold>1a</bold> was prepared in a mixture of DMSO/PEG400 (90/10, v/v) for intravenous and oral administration and <bold>1c</bold> in 100% DMA for oral administration and in a mixture of DMSO/PEG400 (80/20, v/v) for intravenous administration. The volume of the dosing solution given was adjusted according to the body weight recorded before the drug was administered. At 0 (immediately before dosing), 2, 5 (intravenous only), 15 and 30 min and 1, 2, 4, 6, 8, 12 and 24 h after compound administration, a blood sample (150 μl) was taken from each animal via the jugular-vein cannula and stored in ice (0–4 °C). The processing of the plasma and subsequent analysis by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS) were as described (<xref rid="bib0155" ref-type="bibr">Yao et al., 2007</xref>). The plasma concentration data were analyzed by a standard non-compartmental method with the Kinetica software (InnaPhase, Philadelphia, PA, USA).</p></sec><sec id="sec0030"><label>2.4</label><title>Chemical synthesis</title><p id="par0045">Tylophorine (<bold>1a</bold>) and analogues <bold>1c</bold>, <bold>2a</bold>, <bold>2c</bold>, <bold>3a</bold>, <bold>3b</bold> and <bold>3c</bold> were prepared as described (<xref rid="bib0030" ref-type="bibr">Chuang et al., 2006</xref>, <xref rid="bib0140" ref-type="bibr">Yang et al., 2006</xref>). NMR data for compounds <bold>2a</bold>, <bold>2c</bold>, <bold>3a</bold>, and <bold>3c</bold> were previously reported (<xref rid="bib0030" ref-type="bibr">Chuang et al., 2006</xref>) and identification for <bold>3b</bold> were previously reported (<xref rid="bib0135" ref-type="bibr">Wu et al., 2002</xref>, <xref rid="bib0140" ref-type="bibr">Yang et al., 2006</xref>). A novel and concise route with high yield was developed to synthesize a novel intermediate, 2,3,5,6,7-pentamethoxy-phenanthrene-9-carbaldehyde. An intermediate, 2,3,6-trimethoxy-phenanthrene-9-carbaldehyde, was synthesized by the same scheme. This high yield-synthesis scheme will be published elsewhere. These two intermediates were further used for the synthesis of <bold>1b</bold>, <bold>1d</bold>, <bold>1i</bold>, <bold>1j</bold>, <bold>2b</bold>, and <bold>2d</bold>. Consequently, two naturally occurring compounds, 4-methoxytylophorine (<xref rid="bib0100" ref-type="bibr">Rao, 1970</xref>) and Boehmeriasin A (<xref rid="bib0085" ref-type="bibr">Luo et al., 2003</xref>), <bold>1b</bold> and <bold>1j</bold>, were chemically synthesized for the first time. Compounds 4, 7-dimethoxycryptoplerine (<bold>1i</bold>) (a novel compound) and deoxypergularinine (<bold>1d</bold>) were similarly synthesized. Collectively, compounds <bold>1b</bold>, <bold>1d</bold>, <bold>1i</bold>, <bold>1j</bold>, <bold>2b</bold>, and <bold>2d</bold> were synthesized from phenanthrene-9-carbaldehyde intermediates and further completely synthesized with slight modifications of a reported method (<xref rid="bib0030" ref-type="bibr">Chuang et al., 2006</xref>). Synthesized compounds were verified on HPLC–MS (data not shown), <sup>1</sup>H NMR, and <sup>13</sup>C NMR. Compounds <bold>4a</bold> (tylophorine N-oxide, a naturally occurring compound) and <bold>4b</bold> (7-methoxycryptopleurine N-oxide, a novel compound) were synthesized by use of <bold>1a</bold> and <bold>1c</bold>, respectively, and reacted with H<sub>2</sub>O<sub>2</sub>, as follows. Compounds <bold>1a</bold> or <bold>1c</bold> (10 mg, 0.025 mmol) were dissolved in acetone (15 ml) before the drop-wise addition of 30% H<sub>2</sub>O<sub>2</sub> (16.8 ml, 0.15 mmol). The resulting solutions were stirred at room temperature for 24 h and then extracted with CHCl<sub>3</sub> (4 × 30 ml). The organic phases were combined, dried, and evaporated. Finally, the residue was purified by column chromatography on silica gel (CH<sub>2</sub>Cl<sub>2</sub>:MeOH = 15:1) to give the compound <bold>4a</bold> (yield 80.1%) and the novel compound <bold>4b</bold> (yield 60.5%). All compounds derived from chemical synthesis are racemic except <bold>3a</bold>–<bold>3c</bold> compounds. See <xref rid="fig0005" ref-type="fig">Fig. 1</xref>for chemical structures and the <xref rid="sec0080" ref-type="sec">supplementary data</xref> for the physical properties of the synthesized tylophorine compounds.<fig id="fig0005"><label>Fig. 1</label><caption><p>Chemical structures of phenanthroindolizidine and phenanthroquinolizidine compounds. Phenanthroindolizidines, <bold>1a</bold> (<bold>1a′</bold>), <bold>1b</bold>, <bold>1d</bold>–<bold>1h</bold>, <bold>2a</bold>, <bold>2b</bold>, <bold>2d</bold>, <bold>3a</bold>, <bold>3b</bold>, and <bold>4a</bold>, consist of the moieties of phenanthrene and indolizidine and phenanthroquinolizidines, <bold>1c</bold>, <bold>1i, 1j</bold>, <bold>2c</bold>, <bold>3c</bold>, and <bold>4b</bold> of phenanthrene and quinolizidine. These phenanthroindolizidines and phenanthroquinolizidines are tri-, tetra-, or penta-methoxylated at C2, C3, C4, C6, or C7 in the moiety of phenanthrene and with or without hydroxylated or OAc- at C14, formation of C9-one, or N-oxide in the indolizidine/quinolizidine moiety as shown.</p></caption><graphic xlink:href="gr1"></graphic></fig></p></sec><sec id="sec0035"><label>2.5</label><title>Isolation and purification of tylophorine compounds from <italic>T. indica</italic> and <italic>T. ovata</italic></title><p id="par0050">The tylophorine compounds <bold>1a′</bold>, <bold>1e</bold>, <bold>1g</bold>, and <bold>1h</bold> were obtained from the methanol extracts of <italic>T. indica</italic> and <italic>T. ovata</italic>, which were further purified by a chromatography series including silica-gel open-column chromatography and HPLC. The details of isolation and purification will be published elsewhere. LC–MS, HREIMS, <sup>1</sup>H NMR, <sup>13</sup>C NMR, DEPT, COSY, NOESY, HSQC, and HMBC were used to identify and verify the structure of the obtained tylophorine analogues (data not shown except <sup>1</sup>H NMR and <sup>13</sup>C NMR).</p><p id="par0055">Compounds <bold>1a′</bold> and <bold>1e</bold> were validated as tylophorine and tylophorinine with <sup>1</sup>H NMR and <sup>13</sup>C NMR data previously reported (<xref rid="bib0005" ref-type="bibr">Abe et al., 1995</xref>, <xref rid="bib0030" ref-type="bibr">Chuang et al., 2006</xref>, <xref rid="bib0090" ref-type="bibr">Nordlander and Njoroge, 1987</xref>, <xref rid="bib0160" ref-type="bibr">Zeng and Chemler, 2008</xref>, <xref rid="bib0165" ref-type="bibr">Zhen et al., 2002</xref>). Tylophorine analogue <bold>1f</bold> was semi-synthesized by mixing acetic anhydride (Ac<sub>2</sub>O), pyridine, and <bold>1e</bold> with overnight stirring at room temperature (<xref rid="bib0050" ref-type="bibr">Govindachari et al., 1973</xref>) and validated with <sup>1</sup>H NMR and <sup>13</sup>C NMR data previously reported (<xref rid="bib0050" ref-type="bibr">Govindachari et al., 1973</xref>, <xref rid="bib0165" ref-type="bibr">Zhen et al., 2002</xref>). Detailed information about compounds <bold>1g</bold> and <bold>1h</bold> will be published elsewhere; the compounds were identified as 3,14a-dihydroxy-4,6,7-trimethoxyphenanthroindolizidine and 3,14a-dihydroxy-6,7-dimethoxyphenanthroindolizidine (<xref rid="bib0060" ref-type="bibr">Komatsu et al., 2001</xref>). See <xref rid="fig0005" ref-type="fig">Fig. 1</xref> for chemical structures and the <xref rid="sec0080" ref-type="sec">supplementary data</xref> for the physical properties of the isolated tylophorine compounds.</p></sec></sec><sec id="sec0040"><label>3</label><title>Results</title><sec id="sec0045"><label>3.1</label><title>Discovery of tylophorine and 7-methoxycryptopleurine as potent anti-TGEV agents</title><p id="par0060">Because of the laboratory constraints of biosafety levels 3 and 4, ST cells infected with TGEV and MRC5 or Vero E6 cells infected with human CoV 229E were used as surrogate systems for screening agents that inhibit the activity of SARS CoV. Our laboratory has established cell-based and 3CL<sup>pro</sup> enzymatic assays for TGEV to search for anti-viral agents for TGEV and SARS CoV. Some benzothiazolium compounds, e.g. A38120, were found to inhibit 3CL<sup>pro</sup> enzymatic activities of TGEV and SARS CoV through hit compounds obtained from IFA screening of agents against TGEV, which may reflect the highly conserved substrate specificity and structure of CoV 3CL<sup>pro</sup>s. These active benzothiazolium compounds inhibit TGEV with EC<sub>50</sub> values or 50% maximal inhibition concentration (IC<sub>50</sub>) values ranging from ∼2 to ∼36 μM (<xref rid="bib0150" ref-type="bibr">Yang et al., 2007b</xref>).</p><p id="par0065">Tylophorine compounds were not included in the compound library for our primary IFA screening for agents against TGEV or SARS CoV because of a shortage of compound resources at that time, although we carried out studies of the anti-inflammatory and anti-cancerous mechanisms of tylophorine (<xref rid="bib0130" ref-type="bibr">Wu et al., 2009</xref>, <xref rid="bib0140" ref-type="bibr">Yang et al., 2006</xref>). Accidentally, our laboratory found that tylophorine (<bold>1a</bold>) and 7-methoxycryptopleurine (<bold>1c</bold>) potently inhibited TGEV replication, as assessed by IFA with antibodies against the N and S proteins of TGEV. EC<sub>50</sub> values for the compounds were 58 ± 4 and 20 ± 1 nM, respectively (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>A and <xref rid="tbl0005" ref-type="table">Table 1</xref>). However, the compounds at up to 20 μM did not significantly inhibit TGEV or SARS CoV 3CL<sup>pro</sup> enzymatic activity (data not shown). Moreover, the two compounds exerted long-lasting, for at least 64 hpi, inhibition of virus CPE in TGEV-infected ST cells, in contrast to TGEV-infected ST cells without compound treatment which exhibited significant CPE for about 20–24 hpi (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>B). We examined the other apoptosis indicator for TGEV-infected ST cells, activation of caspase 3, and found good agreement with the anti-CPE effects of tylophorine (<bold>1a</bold>) and 7-methoxycryptopleurine (<bold>1c</bold>) (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>C).<fig id="fig0010"><label>Fig. 2</label><caption><p>Anti-transmissible gastroenteritis virus (anti-TGEV) activities of tylophorine (<bold>1a</bold>) and 7-methoxycryptopleurine (<bold>1c</bold>). (A) Immunofluorescent assay against S and N protein of TGEV in ST cells infected with TGEV (10 multiplicities of infection [MOI]) at 6 hpi treated with vehicle (1% DMSO) and 100 nM <bold>1a</bold> and <bold>1c</bold>. The compounds were co-administered or administered 2 h before or 1 h after viral infection for prophylactic and therapeutic effects on anti-viral replication. (B) Anti-cytopathic effect (CPE) in ST cells infected with TGEV at 5 MOI. Shown are the CPE of ST cells with TGEV infection and mock infection at 24 hpi, as well as compound treatments (500 nM) at 48 and 64 hpi in TGEV-infected ST cells. (C) Western blot analysis for effect of compounds <bold>1a</bold> and <bold>1c</bold> at the indicated concentrations on caspase 3 activation, N protein production, and β-actin (as an internal loading control) in TGEV (5 MOI)-infected ST cells at 14 and 18 hpi. Shown for A, B, and C are representative of 3 independent experiments. (D) Effect of the compounds <bold>1a</bold> and <bold>1c</bold> on TGEV yield. Real time quantification of RT-PCR involved viral RNA extracted from TGEV (10 MOI)-infected ST cell lysates and was performed at 6 hpi with or without compound treatment as described (<xref rid="bib0150" ref-type="bibr">Yang et al., 2007b</xref>). Shown are means ± S.D. from 3 independent experiments.</p></caption><graphic xlink:href="gr2"></graphic></fig><table-wrap position="float" id="tbl0005"><label>Table 1</label><caption><p><italic>In vitro</italic> anti-transmissible gastroenteritis virus (anti-TGEV) activity of phenanthroindolizidines and phenanthroquinolizidines in ST cells.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Compound</th><th align="left">Source</th><th align="left">EC<sub>50</sub> (nM)</th><th align="left">CC<sub>50</sub> (nM)</th><th align="left">SI</th></tr></thead><tbody><tr><td align="left"><bold>1a</bold></td><td align="left">Synthesis</td><td align="char">58 ± 4</td><td align="char">>100,000</td><td align="char">>1715</td></tr><tr><td align="left"><bold>1a′</bold></td><td align="left"><italic>T. indica</italic></td><td align="char">95 ± 17</td><td align="char">>100,000</td><td align="char">>1053</td></tr><tr><td align="left"><bold>1b</bold></td><td align="left">Synthesis</td><td align="char">207 ± 25</td><td align="char">83,826 ± 3288</td><td align="char">406</td></tr><tr><td align="left"><bold>1c</bold></td><td align="left">Synthesis</td><td align="char">20 ± 1</td><td align="char">43,522 ± 7404</td><td align="char">2232</td></tr><tr><td align="left"><bold>1d</bold></td><td align="left">Synthesis</td><td align="char">83 ± 9</td><td align="char">71,541 ± 2148</td><td align="char">859</td></tr><tr><td align="left"><bold>1e</bold></td><td align="left"><italic>T. indica</italic></td><td align="char">82 ± 8</td><td align="char">>100,000</td><td align="char">>1220</td></tr><tr><td align="left"><bold>1f</bold></td><td align="left"><italic>T. indica</italic>/semi-synthesis</td><td align="char">403 ± 22</td><td align="char">>100,000</td><td align="char">>248</td></tr><tr><td align="left"><bold>1g</bold></td><td align="left"><italic>T. ovata</italic></td><td align="char">8 ± 2</td><td align="char">59,943 ± 2786</td><td align="char">7685</td></tr><tr><td align="left"><bold>1h</bold></td><td align="left"><italic>T. ovata</italic></td><td align="char">18 ± 1</td><td align="char">31,632 ± 1192</td><td align="char">1719</td></tr><tr><td align="left"><bold>1i</bold></td><td align="left">Synthesis</td><td align="char">170 ± 21</td><td align="char">48,306 ± 3071</td><td align="char">284</td></tr><tr><td align="left"><bold>1j</bold></td><td align="left">Synthesis</td><td align="char">313 ± 46</td><td align="char">41,876 ± 6917</td><td align="char">134</td></tr><tr><td align="left"><bold>2a</bold></td><td align="left">Synthesis</td><td align="char">>100,000</td><td align="char">>100,000</td><td align="char">>1</td></tr><tr><td align="left"><bold>2b</bold></td><td align="left">Synthesis</td><td align="char">12,798 ± 1567</td><td align="char">>100,000</td><td align="char">>8</td></tr><tr><td align="left"><bold>2c</bold></td><td align="left">Synthesis</td><td align="char">74,743 ± 5377</td><td align="char">>100,000</td><td align="char">>1</td></tr><tr><td align="left"><bold>2d</bold></td><td align="left">Synthesis</td><td align="char">19,949 ± 1501</td><td align="char">71,541 ± 2148</td><td align="char">4</td></tr><tr><td align="left"><bold>3a</bold></td><td align="left">Synthesis</td><td align="char">>100,000</td><td align="char">>100,000</td><td align="char">>1</td></tr><tr><td align="left"><bold>3b</bold></td><td align="left"><italic>Ficus septica</italic><xref rid="tblfn0005" ref-type="table-fn">a</xref></td><td align="char">14,906 ± 2468</td><td align="char">>100,000</td><td align="char">>7</td></tr><tr><td align="left"><bold>3c</bold></td><td align="left">Synthesis</td><td align="char">22,595 ± 2825</td><td align="char">>100,000</td><td align="char">>4</td></tr><tr><td align="left"><bold>4a</bold></td><td align="left">Synthesis</td><td align="char">1468 ± 110</td><td align="char">>50,000</td><td align="char">>34</td></tr><tr><td align="left"><bold>4b</bold></td><td align="left">Synthesis</td><td align="char">363 ± 45</td><td align="char">>50,000</td><td align="char">>137</td></tr><tr><td align="left">A38120<xref rid="tblfn0010" ref-type="table-fn">b</xref></td><td align="left">ChemDiv<xref rid="tblfn0010" ref-type="table-fn">b</xref></td><td align="char">6.2 ± 6 (μM)<xref rid="tblfn0010" ref-type="table-fn">b</xref></td><td align="char">>50 (μM)<xref rid="tblfn0010" ref-type="table-fn">b</xref></td><td align="char">>8<xref rid="tblfn0010" ref-type="table-fn">b</xref></td></tr></tbody></table><table-wrap-foot><fn><p>The compounds and their sources from chemical synthesis or isolation from <italic>Tylophora indica</italic> and <italic>T. ovata</italic> plants are shown. The 50% maximal effective concentration (EC<sub>50</sub>) (nM) values for TGEV replication were determined by immunofluorescent assay at 6 h post-infection (hpi). The 50% maximal cytotoxic concentration (CC<sub>50</sub>) (nM) values for cytotoxicity of each compound in ST cells were obtained at 24 hpi, and the selectivity index (SI) values, CC<sub>50</sub>/EC<sub>50</sub>, were calculated. Shown are means ± S.D. from 3 to 5 independent experiments, each performed in duplicate.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tblfn0005"><label>a</label><p><xref rid="bib0140" ref-type="bibr">Yang et al. (2006)</xref>.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tblfn0010"><label>b</label><p><xref rid="bib0150" ref-type="bibr">Yang et al. (2007b)</xref>.</p></fn></table-wrap-foot></table-wrap></p><p id="par0070">The effect of these compounds on inhibiting virus yields was also examined by real-time quantitative PCR with two primer pairs corresponding to 3CL<sup>pro</sup> (ORF1a/ORF1ab) and the N protein regions at the 5′-end and 3′-end of the TGEV genome, respectively (<xref rid="bib0020" ref-type="bibr">Brian and Baric, 2005</xref>, <xref rid="bib0105" ref-type="bibr">Sawicki and Sawicki, 2005</xref>). TGEV virus yields were profoundly reduced by compounds <bold>1a</bold> and <bold>1c</bold>, with EC<sub>50</sub> values comparable to those measured from IFA (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>D to A). Moreover, compounds <bold>1a</bold> and <bold>1c</bold> prophylactically and therapeutically inhibited TGEV replication because pre-, co-, or post-treatments of <bold>1a</bold> and <bold>1c</bold> in TGEV-infected ST cells potently reduced fluorescent staining (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>A). Thus, these compounds strongly protected ST cells against TGEV infection.</p></sec><sec id="sec0050"><label>3.2</label><title>Anti-TGEV activities of synthesized and natural tylophorine analogues</title><p id="par0075">In addition to tylophorine (<bold>1a</bold>) and 7-methoxycryptopleurine (<bold>1c)</bold>, another 18 compounds were obtained and analyzed for anti-TGEV activity, EC<sub>50</sub> values, and cytotoxicity to ST cells with CC<sub>50</sub> values (<xref rid="tbl0005" ref-type="table">Table 1</xref>). Selective index (SI) values were calculated (CC<sub>50</sub>/EC<sub>50</sub>). The dehydro-tylophorine analogues, <bold>3a</bold>–<bold>3c</bold>, were the least inhibitory of the compounds tested, with EC<sub>50</sub> values ranging from 14906 ± 2468 to >100,000 nM (<xref rid="tbl0005" ref-type="table">Table 1</xref>). The 9-one-tylophorine analogues <bold>2a</bold>–<bold>2d</bold> were moderate inhibitors of TGEV replication, with EC<sub>50</sub> values ranging from 12798 ± 1567 to >100,000 nM (<xref rid="tbl0005" ref-type="table">Table 1</xref>). The tylophorine analogues <bold>1b</bold>, <bold>1d</bold>–<bold>1j</bold>, <bold>4a</bold>, and <bold>4b</bold> were the most potent and selective inhibitors of virus replication, with EC<sub>50</sub> values from 8 to 1468 nM and high SI values from >34 to 7685 (<xref rid="tbl0005" ref-type="table">Table 1</xref>). Moreover, the anti-TGEV activities of these compounds derived from the CPE assay were consistent with the EC<sub>50</sub> values determined by IFA (<xref rid="fig0015" ref-type="fig">Fig. 3</xref>A and B and <xref rid="tbl0005" ref-type="table">Table 1</xref>). As with <bold>1a</bold> and <bold>1c</bold>, these potent analogues (<bold>1b</bold>, <bold>1d</bold>–<bold>1j</bold>) exerted prolonged inhibition of CPE, inhibiting the appearance of CPE at least up to 48 h (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>, <xref rid="fig0015" ref-type="fig">Fig. 3</xref>). Thus, they blocked TGEV replication and induced apoptosis, as indicated by the reduction of TGEV N protein expression and caspase 3 activation in the infected ST cells (<xref rid="fig0015" ref-type="fig">Fig. 3</xref>C).<fig id="fig0015"><label>Fig. 3</label><caption><p>Anti-CPE and anti-apoptosis activities of phenanthroindolizidines and phenanthroquinolizidines. Shown are the CPE of ST cells with mock infection and TGEV infection (5 MOI) in the absence and in the presence of 500 nM compound treatments at 24 (A) and 48 (B) hpi and western blot analysis for protein expression of caspase 3, TGEV N protein and GAPDH (as an internal loading control) at 18 hpi (C) in the presence of 500 nM compound treatments. Shown are representative of 3 independent experiments.</p></caption><graphic xlink:href="gr3"></graphic></fig></p></sec><sec id="sec0055"><label>3.3</label><title>Tylophorine compounds show potent anti-SARS CoV activity <italic>in vitro</italic></title><p id="par0080">Efforts have been initiated to identify small-molecule agents for anti-SARS CoV infection. Thus far, inhibitors targeting the SARS CoV protease have been reported to have IC<sub>50</sub> values ranging from 0.5 to 7 μM and the SARS CoV papain-like protease in the 100 nM range. However, the anti-enzymatic activity of these inhibitors may not exhibit corresponding anti-SARS CoV activity in cells (<xref rid="bib0040" ref-type="bibr">Ghosh et al., 2008</xref>). Because we have used porcine TGEV as a surrogate system to search for potential anti-SARS CoV agents (<xref rid="bib0145" ref-type="bibr">Yang et al., 2007a</xref>), we tested the tylophorine compounds <bold>1a</bold>, <bold>1c</bold>, <bold>1e</bold>, <bold>4a</bold>, and <bold>4b</bold> for anti-CPE activity induced by SARS CoV (Urbani strain) in Vero 76 cells. These compounds were found to have profound activity with EC<sub>50</sub> values ranging from 0.34 μM to <0.005 μM and selective index (SI) values from 10 to >100 determined by CPE assay under microscopic observation whereas their EC<sub>50</sub> values ranging from 0.62 μM to <0.005 μM and SI values from 6.8 to >15 determined by Neutral red uptake assay, respectively (<xref rid="tbl0010" ref-type="table">Table 2</xref>). The CPE assay in Vero 76 cells induced by SARS CoV takes 3–4 days (<xref rid="bib0010" ref-type="bibr">Barnard et al., 2006</xref>) and tylophorine compounds have been reported to inhibit cell growth (<xref rid="bib0035" ref-type="bibr">Gao et al., 2004</xref>, <xref rid="bib0065" ref-type="bibr">Lee et al., 2003</xref>, <xref rid="bib0130" ref-type="bibr">Wu et al., 2009</xref>), so that on the day for assay the final cell numbers would have 2- to 16-fold difference and the cell volume usually larger for those treated with tylophorine compounds (our unpublished data). This also was manifested by comparing the cell numbers and cell sizes of SARS CoV infected Vero 76 cells treated by compound <bold>1a</bold> to those without SARS infected control in the equal area under microscope (<xref rid="fig0020" ref-type="fig">Fig. 4</xref>). Therefore, it is conceivable that lower SI values were obtained from Neutral red uptake assay determining relative cell numbers than those from CPE assay by observation under microscope. These compounds also decreased the formation of large syncytia and/or multinucleated giant cells resulting from virus-induced fusion of cell membranes in a dose-dependent manner (<xref rid="fig0020" ref-type="fig">Fig. 4</xref>). Thus, these tylophorine compounds are potent small molecules for anti-SARS CoV protection in cell systems.<table-wrap position="float" id="tbl0010"><label>Table 2</label><caption><p>Anti-SARS CoV activity of phenanthroindolizidines and phenanthroquinolizidines in Vero 76 cells.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Compound</th><th colspan="3" align="left">Visual assay<hr></hr></th><th colspan="3" align="left">Neutral red uptake assay<hr></hr></th></tr><tr><th></th><th align="left">EC<sub>50</sub> (μM)<xref rid="tblfn0015" ref-type="table-fn">a</xref></th><th align="left">CC<sub>50</sub> (μM)<xref rid="tblfn0020" ref-type="table-fn">b</xref></th><th align="left">SI<xref rid="tblfn0025" ref-type="table-fn">c</xref></th><th align="left">EC<sub>50</sub> (μM)</th><th align="left">CC<sub>50</sub> (μM)</th><th align="left">SI</th></tr></thead><tbody><tr><td align="left"><bold>1a</bold></td><td align="char">0.018</td><td align="char">1.6</td><td align="char">88</td><td align="char">0.066</td><td align="char">1.1</td><td align="char">17</td></tr><tr><td align="left"><bold>1c</bold></td><td align="char"><0.005</td><td align="char">0.5</td><td align="char">>100</td><td align="char"><0.005</td><td align="char">0.0084</td><td align="char">>1.7</td></tr><tr><td align="left"><bold>1e</bold></td><td align="char"><0.005</td><td align="char">0.39</td><td align="char">>78</td><td align="char"><0.005</td><td align="char">>0.077</td><td align="char">>15</td></tr><tr><td align="left"><bold>4a</bold></td><td align="char">0.34</td><td align="char">3.4</td><td align="char">10</td><td align="char">0.62</td><td align="char">4.2</td><td align="char">6.8</td></tr><tr><td align="left"><bold>4b</bold></td><td align="char">0.039</td><td align="char">0.73</td><td align="char">19</td><td align="char">0.056</td><td align="char">0.56</td><td align="char">10</td></tr><tr><td align="left">Infergen™ (μg/ml)</td><td align="char"><0.32</td><td align="char">>100</td><td align="char">>320</td><td align="char">2.4</td><td align="char">>130</td><td align="char">>320</td></tr></tbody></table><table-wrap-foot><fn><p>The EC<sub>50</sub> and CC<sub>50</sub> (μM) values were determined visually by microscopy and neutral red uptake assay on the same test plates for anti-SARS CoV-induced cytopathic effect and cytotoxicity, respectively, for the indicated compounds in Vero 76 cells as described (<xref rid="bib0010" ref-type="bibr">Barnard et al., 2006</xref>). The SI values and CC<sub>50</sub>/EC<sub>50</sub> were also calculated.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tblfn0015"><label>a</label><p>EC<sub>50</sub>: the 50% maximal effective concentration.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tblfn0020"><label>b</label><p>CC<sub>50</sub>: the 50% maximal cytotoxic concentration.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tblfn0025"><label>c</label><p>SI: selectivity index, CC<sub>50</sub>/EC<sub>50</sub>.</p></fn></table-wrap-foot></table-wrap><fig id="fig0020"><label>Fig. 4</label><caption><p>Anti-SARS CoV activity of tylophorine. SARS CoV and the indicated concentrations of tylophorine (<bold>1a</bold>) were added in equal volumes to near-confluent cell monolayers of Vero 76 cells in culture plates. The MOI used was ranged from 0.01 to 0.025 to produce virus cytopathic effects in 100% of the cells in the virus control wells within 3–4 days along with a mock infection control. The plates were incubated at 37 °C until the cells in the virus control wells showed complete viral CPE as observed by light microscopy and stained with neutral red as described (<xref rid="bib0010" ref-type="bibr">Barnard et al., 2006</xref>). Equal field areas of each treatment were shown from microscopic observation at 100× magnification.</p></caption><graphic xlink:href="gr4"></graphic></fig></p></sec><sec id="sec0060"><label>3.4</label><title>Analysis of structure–activity relations</title><p id="par0085">The increased planarity and rigidity in structure and more dehydrogenated in indolizidine/quinolizidine moiety of compounds <bold>2a</bold>–<bold>2d</bold> and <bold>3a</bold>–<bold>3c</bold> are suggested to be responsible for their moderate and least activities of anti-TGEV compared to the other potent compounds, <bold>1a</bold>–<bold>1j</bold> and <bold>4a</bold>–<bold>4b</bold> and these structure–activity relations were also found stand for their anti-inflammatory and anti-cancerous activities (<xref rid="bib0140" ref-type="bibr">Yang et al., 2006</xref>, <xref rid="bib0145" ref-type="bibr">Yang et al., 2007a</xref>). Among the most potent tylophorine analogues inhibiting TGEV were compounds <bold>1e</bold>, <bold>1g</bold>, and <bold>1h</bold> with a hydroxyl group at C14 or at the phenanthrene moiety C3. EC<sub>50</sub> values for these compounds were 82 ± 8, 8 ± 2 and 18 ± 1 nM, respectively. Thus, these hydroxyl groups contributed significantly to the inhibitory activity exhibited by the tylophorine analogues. However, introduction of an OAc-group into the C14 position decreased the activity by ∼5-fold as compared with compounds with a hydrogen or a hydroxyl group-replacement (<bold>1f</bold> to <bold>1d</bold> and <bold>1e</bold>, respectively). Although <bold>1d</bold> and <bold>1e</bold> had similar potency, they differed in cytotoxicity and thus in selectivity (<xref rid="tbl0005" ref-type="table">Table 1</xref>), which is an important concern for future drug development. Moreover, introduction of a methoxyl group to position C4 of tylophorine decreased the inhibitory activity of the parent compound ∼4- to 8-fold for both the indolizidine and the quinolizidine counterparts (<bold>1a</bold> to <bold>1b</bold> and <bold>1c</bold> to <bold>1i</bold>). Similarly, replacing the methoxyl with a hydrogen group at position C2 of tylophorine decreased the inhibitory activity of both the indolizidine and the quinolizidine counterparts but to a lesser extent (<bold>1a</bold> to <bold>1d</bold> and <bold>1c</bold> to <bold>1j</bold>). In addition, compounds <bold>4a</bold> and <bold>4b</bold> with an oxygen atom introduced to the reactive nitrogen to form an N-oxide as compared with <bold>1a</bold> and <bold>1c</bold>, respectively, exerted less potency, by ∼8- to 25-fold. Introduction of the N-oxide to the indolizidine/quinolizidine moiety changed the nitrogen reactivity and might affect the position projection of hydrogen atoms for potency because dehydrogenation in this moiety profoundly decreased activity, e.g. <bold>1a</bold> to <bold>2a</bold>/<bold>3a</bold> and <bold>1c</bold> to <bold>2c</bold>/<bold>3c</bold>. Similarly, the phenanthroquinolizidines <bold>1c</bold> and <bold>4b</bold> were more potent than their phenanthroindolizidine counterparts <bold>1a</bold> and <bold>4a</bold> for anti-SARS CoV activity, and the hydroxyl group at C14 contributed significantly to the inhibitory activity exhibited by compound <bold>1e</bold>.</p></sec><sec id="sec0065"><label>3.5</label><title>Pharmacokinetic studies of the tylophorine compounds</title><p id="par0090">Because tylophorine is the main active constituent of <italic>T. indica</italic>, which has been used as traditional herbal medicine in India taken as dry leaves for anti-asthma, for example, we performed a pharmacokinetic study to determine the oral bioavailability and other parameters of tylophorine (<bold>1a</bold>) and its quinolizidine counterpart 7-methoxycryptopleurine (<bold>1c</bold>) (<xref rid="tbl0015" ref-type="table">Table 3</xref>). 7-Methoxycryptopleurine is a synthetic compound that is not naturally occurring (<xref rid="bib0145" ref-type="bibr">Yang et al., 2007a</xref>). After the intravenous administration of <bold>1a</bold> and <bold>1c</bold> at 3 mg/kg, the total body clearances were 66.8 ± 15.3 and 101.0 ± 8.8 ml/min/kg for <bold>1a</bold> and <bold>1c</bold>, respectively. The volumes of distribution at steady state (<italic>V</italic><sub>ss</sub>) were 16.6 ± 2.3 and 31.6 ± 4.1 L/kg for <bold>1a</bold> and <bold>1c</bold>, respectively, which suggests a wide distribution of these compounds to the extravascular tissues. The apparent elimination half-life (<italic>t</italic><sub>1/2</sub>) with an intravenous dose was long, at ∼4 h for both compounds. After oral administration, the compounds were rapidly absorbed; the <italic>C</italic><sub>max</sub> values of 31.9 ± 17.5 and 16.5 ± 10.4 ng/ml were reached at 1.8 and 1.0 h for <bold>1a</bold> and <bold>1c</bold>, respectively, after administration. The oral bioavailability of <bold>1c</bold> was estimated to be 52.7%, which was comparable to that of <bold>1a</bold>, 65.7%, and suggests conserved oral bioavailability for these compounds.<table-wrap position="float" id="tbl0015"><label>Table 3</label><caption><p>Pharmacokinetic properties of tylophorine (<bold>1a</bold>) and 7-methoxycryptopleurine (<bold>1c</bold>) in rats.</p></caption><table frame="hsides" rules="groups"><thead><tr><th></th><th></th><th colspan="2" align="left"><bold>1a</bold><hr></hr></th><th colspan="2" align="left"><bold>1c</bold><hr></hr></th></tr><tr><th align="left">Parameter<xref rid="tblfn0030" ref-type="table-fn">a</xref></th><th align="left">Unit</th><th align="left">Intravenous dose</th><th align="left">Oral dose</th><th align="left">Intravenous dose</th><th align="left">Oral dose</th></tr></thead><tbody><tr><td align="left">N</td><td></td><td align="char">3</td><td align="char">3</td><td align="char">3</td><td align="char">3</td></tr><tr><td align="left">Dose</td><td align="left">mg/kg</td><td align="char">3.0</td><td align="char">3.0</td><td align="char">3.0</td><td align="char">3.0</td></tr><tr><td align="left"><italic>t</italic><sub>1/2</sub></td><td align="left">h</td><td align="char">3.9<xref rid="tblfn0035" ref-type="table-fn">b</xref></td><td align="char">30.7</td><td align="char">4.2</td><td align="char">15.1</td></tr><tr><td align="left">Clearance</td><td align="left">ml/min/kg</td><td align="char">66.8</td><td></td><td align="char">101.0</td><td></td></tr><tr><td align="left"><italic>V</italic><sub>ss</sub></td><td align="left">L/kg</td><td align="char">16.6</td><td></td><td align="char">31.6</td><td></td></tr><tr><td align="left"><italic>C</italic><sub>max</sub></td><td align="left">ng/ml</td><td></td><td align="char">31.9</td><td></td><td align="char">16.5</td></tr><tr><td align="left"><italic>C</italic><sub>trough</sub></td><td align="left">ng/ml</td><td align="char">1.1</td><td align="char">3.0</td><td align="char">1.4</td><td align="char">5.3</td></tr><tr><td align="left"><italic>T</italic><sub>max</sub></td><td align="left">h</td><td></td><td align="char">1.8</td><td></td><td align="char">1.0</td></tr><tr><td align="left">AUC<sub>(0–inf.)</sub></td><td align="left">ng/ml*h</td><td align="char">772</td><td align="char">507</td><td align="char">503</td><td align="char">265</td></tr><tr><td align="left">Oral bioavailability</td><td align="left">%</td><td></td><td align="char">65.7</td><td></td><td align="char">52.7</td></tr></tbody></table><table-wrap-foot><fn id="tblfn0030"><label>a</label><p><italic>t</italic><sub>1/2</sub>: apparent elimination half-life; <italic>V</italic><sub>ss</sub>: volume of distribution at steady state; <italic>C</italic><sub>max</sub>: maximal concentration; AUC<sub>(0–inf.)</sub>: area under concentration curve from time 0 to infinity.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tblfn0035"><label>b</label><p>Data are mean values.</p></fn></table-wrap-foot></table-wrap></p></sec></sec><sec id="sec0070"><label>4</label><title>Discussion</title><p id="par0095">Plant materials are great resources for human therapeutic medicine, for use as alternative medicines and as great reservoirs for the discovery of naturally occurring compounds for developing therapeutic agents. The content of phenanthroindolizidines differs in each <italic>Tylophora</italic> plant and could slightly vary by harvest season and location. For instance, tylophorine is the main active constituent of <italic>T. indica</italic> (<xref rid="bib0045" ref-type="bibr">Gopalakrishnan et al., 1980</xref>), tylophorinine in <italic>T. atrofolliculata</italic> (<xref rid="bib0055" ref-type="bibr">Huang et al., 2004</xref>), and 14-hydroxytylophorine in <italic>T. ovata</italic> (our unpublished data). In addition, the potency and toxicity of each phenanthroindolizidine may vary depending on the plant source (<xref rid="tbl0005" ref-type="table">Table 1</xref> and our unpublished data). Thus, the doses to achieve efficacy and selectivity must be investigated as part of the drug development process, regardless of the formulation, of pure compounds or herbal medicine. Our study is the first to report and compare the pharmacokinetics of naturally occurring tylophorine and synthesized 7-methoxycryptopleurine. We demonstrated their good oral bioavailability. Because of the efficacy, selectivity and oral bioavailability of the tylophorine compounds we report, efforts to develop and optimize more drug-like derivatives of these compounds are warranted.</p><p id="par0100">The SI values for <bold>1a</bold>, <bold>1c</bold>, and <bold>1e</bold> are 88, >100, and >78 from microscopic observation results (<xref rid="tbl0010" ref-type="table">Table 2</xref>) are different when comparing the values obtained for the TGEV assays and to the values obtained for the SARS CoV assay with neutral red uptake due entirely different cells from different species origins were used for each assays, the length of time the compound was exposed to cells was very different for each assay, and the type of cytotoxicity tests used were different. The compounds were exposed to the Vero 76 cells in the SARS CoV tests for 72 h, which gives the compounds longer to become toxic to cells, the neutral red uptake assays used are more sensitive evaluation of cytotoxicity or growth inhibition (<xref rid="bib0115" ref-type="bibr">Smee et al., 2002</xref>), and there always tissue to tissue differences when assessing cytotoxicity (<xref rid="bib0115" ref-type="bibr">Smee et al., 2002</xref>). Cells derived from swine testicular tissue apparently are less sensitive to toxicity than cells derived from monkey kidneys.</p><p id="par0105">The main effect of the tylophorine compounds is to inhibit TGEV replication in ST cells thereby blocking TGEV-induced apoptosis signaling in ST cells (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>, <xref rid="fig0015" ref-type="fig">Fig. 3</xref>). The tylophorine compounds may be administered prophylactically and therapeutically because both administration regimens greatly inhibited TGEV replication, as measured by IFA (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>A). Therefore, tylophorine compounds are likely to inhibit TGEV replication event(s) after the viruses enter the cells. Moreover, the ability of these compounds to inhibit other coronaviruses such as SARS CoV in Vero 76 cells (<xref rid="tbl0010" ref-type="table">Table 2</xref> and <xref rid="fig0020" ref-type="fig">Fig. 4</xref>) and increase the survival rate of mice infected with MHV-JHM (data not shown) suggests that these compounds inhibit a common or highly conserved target among coronaviruses involved in coronaviral transcriptional complex, e.g. RdRp, helicase, N protein etc., or common cellular factors those are crucial for coronaviral replication. Further detailed mechanism studies of the effect of tylophorine compounds at molecular levels on TGEV replication in infected ST cells are under investigation.</p><p id="par0110">For development and optimization of phenanthroindolizidines into therapeutic drugs, the variety of tylophorine derivatives must be expanded to provide fundamental data and to better understand the relationship between structure and activity. Here we have synthesized and isolated from natural sources to obtain a variety of tylophorine and cryptopleurine compounds and described the previously unreported anti-coronavirus activity of tylophorine compounds and provided more insight into structure–activity relations. Moreover, because of the compounds’ clinical relevance, with potent activity in the low nanomolar range (<xref rid="tbl0005" ref-type="table">Table 1</xref>, <xref rid="tbl0010" ref-type="table">Table 2</xref>) and high oral bioavailability (<xref rid="tbl0015" ref-type="table">Table 3</xref>), the most active tylophorine compounds should be able to safely achieve clinically acceptable doses. 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Sin.</source><volume>44</volume><year>2002</year><fpage>349</fpage><lpage>353</lpage></element-citation></ref></ref-list><sec id="sec0080" sec-type="supplementary-material"><label>Appendix A</label><title>Supplementary data</title><p id="par0125"><supplementary-material content-type="local-data" id="upi0005"><media xlink:href="mmc1.pdf"></media></supplementary-material></p></sec><ack><title>Acknowledgements</title><p>This work was supported by the National Science Council of Taiwan, ROC [Grants 99-2320-B-400-009-MY3 and 95-2320-B-400-009-MY3] and the National Health Research Institutes, Taiwan, ROC [Grants BP-098-PP-05 and BP-099-PP-04]. Cheng-Wei Yang is a Ph.D. student in the Graduate Program of Biotechnology in Medicine sponsored by the National Tsing Hua University and the National Health Research Institutes. The authors also acknowledge Dr. Yu-Cheng Chou for some of the <sup>1</sup>H NMR and LC/MS measurements and Dr. Chi-Min Chen for providing antibodies for TGEV. The work contributed by Dr. Barnard was supported in part by Contract N01-AI-30048 from the US National Institutes of Health, NIAID, Division of Virology.</p></ack><fn-group><fn id="sec0075" fn-type="supplementary-material"><label>Appendix A</label><p id="par0120">Supplementary data associated with this article can be found, in the online version, at <ext-link ext-link-type="doi" xlink:href="10.1016/j.antiviral.2010.08.009">doi:10.1016/j.antiviral.2010.08.009</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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