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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK169449" /><meta name="ncbi_domain" content="mlprobe" /><meta name="ncbi_report" content="record" /><meta name="ncbi_type" content="fulltext" /><meta name="ncbi_objectid" content="" /><meta name="ncbi_pcid" content="/NBK169449/" /><meta name="ncbi_pagename" content="Discovery, optimization, and characterization of a novel series of dopamine D2 versus D3 receptor selective antagonists - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf" /><meta name="ncbi_bookparttype" content="chapter" /><meta name="ncbi_app" content="bookshelf" />
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<title>Discovery, optimization, and characterization of a novel series of dopamine D2 versus D3 receptor selective antagonists - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="Discovery, optimization, and characterization of a novel series of dopamine D2 versus D3 receptor selective antagonists" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2013/09/03" /><meta name="citation_author" content="Jingbo Xiao" /><meta name="citation_author" content="R. Benjamin Free" /><meta name="citation_author" content="Elena Barnaeva" /><meta name="citation_author" content="Jennie Conroy" /><meta name="citation_author" content="Trevor Doyle" /><meta name="citation_author" content="Marthe Bryant-Genevier" /><meta name="citation_author" content="Mercedes K. Taylor" /><meta name="citation_author" content="Noel Southall" /><meta name="citation_author" content="Xin Hu" /><meta name="citation_author" content="Marc Ferrer" /><meta name="citation_author" content="Steve Titus" /><meta name="citation_author" content="Wei Zheng" /><meta name="citation_author" content="David R. Sibley" /><meta name="citation_author" content="Juan J. Marugan" /><meta name="citation_pmid" content="24260782" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK169449/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Discovery, optimization, and characterization of a novel series of dopamine D2 versus D3 receptor selective antagonists" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Jingbo Xiao" /><meta name="DC.Contributor" content="R. Benjamin Free" /><meta name="DC.Contributor" content="Elena Barnaeva" /><meta name="DC.Contributor" content="Jennie Conroy" /><meta name="DC.Contributor" content="Trevor Doyle" /><meta name="DC.Contributor" content="Marthe Bryant-Genevier" /><meta name="DC.Contributor" content="Mercedes K. Taylor" /><meta name="DC.Contributor" content="Noel Southall" /><meta name="DC.Contributor" content="Xin Hu" /><meta name="DC.Contributor" content="Marc Ferrer" /><meta name="DC.Contributor" content="Steve Titus" /><meta name="DC.Contributor" content="Wei Zheng" /><meta name="DC.Contributor" content="David R. Sibley" /><meta name="DC.Contributor" content="Juan J. Marugan" /><meta name="DC.Date" content="2013/09/03" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK169449/" /><meta name="description" content="Dopamine receptors (DARs) are members of the G protein-coupled receptors (GPCRs) superfamily which play a critical role in cell signaling processes, especially modulating the transfer of information within the nervous system [1]. Amongst DARs, the D2 receptor is arguably one of the most validated drug targets in neurology and psychiatry [2]. There is a strong correlation between the clinical doses of neuroleptics and their affinity for brain D2 receptors [3]. Despite numerous attempts of producing D2 selective modulators, current drugs display poor selectivity between D2 and D3 DARs. This is because D2 is closest to D3 in terms of sequence homology and signaling transduction pathways hence it pose a challenge to selectively regulate the two receptors [4, 5]. However given the success, a potent and selective D2 DAR antagonist would be of particular interest for the treatment of a variety of related CNS diseases [6, 7]. Here we present the discovery of a novel series of selective small molecule D2 DAR antagonists from a quantitative high-throughput screen (qHTS) campaign. Optimized lead compound in this series exhibits an excellent D2versus D1, D3, D4 and D5 receptor selectivity. In a panel of GPCR binding assays, ML321 shows a cleaner profile compared to the best previously reported selective D2 DAR antagonist. Furthermore, ML321 showed good in vitro ADME data and in vivo pharmacokinetic (PK) properties. We therefore believe that this probe can be a very useful pharmacological tool to perform proof-of-concept studies in animal models and may be an ideal starting point for further development into drug-like molecules for the treatment of a variety of CNS diseases including Tourette’s syndrome, tardive dyskinesia, dystonia, Huntington’s chorea, and especially schizophrenia." /><meta name="og:title" content="Discovery, optimization, and characterization of a novel series of dopamine D2 versus D3 receptor selective antagonists" /><meta name="og:type" content="book" /><meta name="og:description" content="Dopamine receptors (DARs) are members of the G protein-coupled receptors (GPCRs) superfamily which play a critical role in cell signaling processes, especially modulating the transfer of information within the nervous system [1]. Amongst DARs, the D2 receptor is arguably one of the most validated drug targets in neurology and psychiatry [2]. There is a strong correlation between the clinical doses of neuroleptics and their affinity for brain D2 receptors [3]. Despite numerous attempts of producing D2 selective modulators, current drugs display poor selectivity between D2 and D3 DARs. This is because D2 is closest to D3 in terms of sequence homology and signaling transduction pathways hence it pose a challenge to selectively regulate the two receptors [4, 5]. However given the success, a potent and selective D2 DAR antagonist would be of particular interest for the treatment of a variety of related CNS diseases [6, 7]. Here we present the discovery of a novel series of selective small molecule D2 DAR antagonists from a quantitative high-throughput screen (qHTS) campaign. Optimized lead compound in this series exhibits an excellent D2versus D1, D3, D4 and D5 receptor selectivity. In a panel of GPCR binding assays, ML321 shows a cleaner profile compared to the best previously reported selective D2 DAR antagonist. Furthermore, ML321 showed good in vitro ADME data and in vivo pharmacokinetic (PK) properties. We therefore believe that this probe can be a very useful pharmacological tool to perform proof-of-concept studies in animal models and may be an ideal starting point for further development into drug-like molecules for the treatment of a variety of CNS diseases including Tourette’s syndrome, tardive dyskinesia, dystonia, Huntington’s chorea, and especially schizophrenia." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK169449/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/mlprobe/ml321/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK169449/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK169449_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK169449_dtls__"><div>Bethesda (MD): National Center for Biotechnology Information (US); 2010-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/mlprobe/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/mlprobe/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/mlprobe/ml322/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/y4/" title="Next page in this title">Next ></a></div></div></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK169449_"><span class="title" itemprop="name">Discovery, optimization, and characterization of a novel series of dopamine D<sub>2</sub> versus D<sub>3</sub> receptor selective antagonists</span></h1><p class="contrib-group"><span itemprop="author">Jingbo Xiao</span>, <span itemprop="author">R. Benjamin Free</span>, <span itemprop="author">Elena Barnaeva</span>, <span itemprop="author">Jennie Conroy</span>, <span itemprop="author">Trevor Doyle</span>, <span itemprop="author">Marthe Bryant-Genevier</span>, <span itemprop="author">Mercedes K. Taylor</span>, <span itemprop="author">Noel Southall</span>, <span itemprop="author">Xin Hu</span>, <span itemprop="author">Marc Ferrer</span>, <span itemprop="author">Steve Titus</span>, <span itemprop="author">Wei Zheng</span>, <span itemprop="author">David R. Sibley</span>, and <span itemprop="author">Juan J. Marugan</span>.</p><a data-jig="ncbitoggler" href="#__NBK169449_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK169449_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Jingbo Xiao</span>,<sup>a</sup> <span itemprop="author">R. Benjamin Free</span>,<sup>b</sup> <span itemprop="author">Elena Barnaeva</span>,<sup>a</sup> <span itemprop="author">Jennie Conroy</span>,<sup>b</sup> <span itemprop="author">Trevor Doyle</span>,<sup>b</sup> <span itemprop="author">Marthe Bryant-Genevier</span>,<sup>a</sup> <span itemprop="author">Mercedes K. Taylor</span>,<sup>a</sup> <span itemprop="author">Noel Southall</span>,<sup>a</sup> <span itemprop="author">Xin Hu</span>,<sup>a</sup> <span itemprop="author">Marc Ferrer</span>,<sup>a</sup> <span itemprop="author">Steve Titus</span>,<sup>a</sup> <span itemprop="author">Wei Zheng</span>,<sup>a</sup> <span itemprop="author">David R. Sibley</span>,<sup>b</sup> and <span itemprop="author">Juan J. Marugan</span><sup>a</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>a</sup>
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NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850.</div><div class="affiliation"><sup>b</sup>
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Molecular Neuropharmacology Section, National Institute of Neurological Disorders & Stroke, National Institutes of Health, 5625 Fishers Lane, Room 4S-04, MSC-9405, Bethesda, MD 20892.</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed: Email:
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<a href="mailto:dev@null" data-email="vog.hin.liam@jnaguram" class="oemail">vog.hin.liam@jnaguram</a> or 301-217-9198.</div></div><p class="small">Received: <span itemprop="datePublished">December 25, 2012</span>; Last Update: <span itemprop="dateModified">September 3, 2013</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Dopamine receptors (DARs) are members of the G protein-coupled receptors (GPCRs) superfamily which play a critical role in cell signaling processes, especially modulating the transfer of information within the nervous system [<a class="bk_pop" href="#ml321.r1">1</a>]. Amongst DARs, the D<sub>2</sub> receptor is arguably one of the most validated drug targets in neurology and psychiatry [<a class="bk_pop" href="#ml321.r2">2</a>]. There is a strong correlation between the clinical doses of neuroleptics and their affinity for brain D<sub>2</sub> receptors [<a class="bk_pop" href="#ml321.r3">3</a>]. Despite numerous attempts of producing D<sub>2</sub> selective modulators, current drugs display poor selectivity between D<sub>2</sub> and D<sub>3</sub> DARs. This is because D<sub>2</sub> is closest to D<sub>3</sub> in terms of sequence homology and signaling transduction pathways hence it pose a challenge to selectively regulate the two receptors [<a class="bk_pop" href="#ml321.r4">4</a>, <a class="bk_pop" href="#ml321.r5">5</a>]. However given the success, a potent and selective D<sub>2</sub> DAR antagonist would be of particular interest for the treatment of a variety of related CNS diseases [<a class="bk_pop" href="#ml321.r6">6</a>, <a class="bk_pop" href="#ml321.r7">7</a>]. Here we present the discovery of a novel series of selective small molecule D<sub>2</sub> DAR antagonists from a quantitative high-throughput screen (qHTS) campaign. Optimized lead compound in this series exhibits an excellent D<sub>2</sub><i>versus</i> D<sub>1</sub>, D<sub>3</sub>, D<sub>4</sub> and D<sub>5</sub> receptor selectivity. In a panel of GPCR binding assays, <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> shows a cleaner profile compared to the best previously reported selective D<sub>2</sub> DAR antagonist. Furthermore, <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> showed good <i>in vitro</i> ADME data and <i>in vivo</i> pharmacokinetic (PK) properties. We therefore believe that this probe can be a very useful pharmacological tool to perform proof-of-concept studies in animal models and may be an ideal starting point for further development into drug-like molecules for the treatment of a variety of CNS diseases including Tourette’s syndrome, tardive dyskinesia, dystonia, Huntington’s chorea, and especially schizophrenia.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R21-NS064831</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Assay Submitter & Institution:</b> David Sibley, Institute of Neurological Disorders & Stroke, National Institute of Health</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/485359" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">485359</a></p><div id="ml321.s1"><h2 id="_ml321_s1_">Probe Structure & Characteristics</h2><div id="ml321.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169449/bin/ml321fu1.jpg" alt="ML321." /></div><h3><span class="title">ML321</span></h3></div><div id="ml321.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169449/table/ml321.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml321.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml321.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML#</th><th id="hd_h_ml321.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml321.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (nM) [SID, AID]</th><th id="hd_h_ml321.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml321.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (μM) [SID, AID]</th><th id="hd_h_ml321.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fold Selective</th><th id="hd_h_ml321.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml321.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 57377246/<a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a></td><td headers="hd_h_ml321.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">D<sub>2</sub> DAR receptor</td><td headers="hd_h_ml321.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">D<sub>2</sub> Ca<sup>2+</sup> AC<sub>50:</sub> 70 nM, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882616" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 136882616</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624496" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624496</a>]</td><td headers="hd_h_ml321.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">D<sub>3</sub> DAR receptor</td><td headers="hd_h_ml321.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">D<sub>3</sub> β-arrestin: AC<sub>50:</sub> 12,893 nM, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882616" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 136882616</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624500" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624500</a>]</td><td headers="hd_h_ml321.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">17–22 fold</td><td headers="hd_h_ml321.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">D<sub>2</sub> β-arrestin: AC<sub>50:</sub> 725 nM, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882616" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 136882616</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624495" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624495</a>]</td></tr><tr><td headers="hd_h_ml321.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml321.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml321.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">D<sub>2</sub> binding <i>K</i>i: 120 nM, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882616" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 136882616</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624494" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624494</a>]</td><td headers="hd_h_ml321.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml321.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">D<sub>3</sub> binding <i>K</i>i: 2,650 nM, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882616" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 136882616</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624502" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624502</a>]</td><td headers="hd_h_ml321.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml321.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td></tr></tbody></table></div></div></div><div id="ml321.s2"><h2 id="_ml321_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>Preferential inhibition remains to be a limitation for D<sub>2</sub> prior art antagonists. The probe <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> demonstrates excellent D<sub>2</sub><i>versus</i> D<sub>3</sub> DAR selectivity with a very clean selectivity profile in a panel of GPCR binding assays. The selective D<sub>2</sub> (<i>versus</i> D<sub>3</sub>) <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> probe will be of interest to biologist to further elucidate the role of the D<sub>2</sub> receptors on CNS diseases. <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> will be used by researchers as a pharmacological tool for the dissection of D<sub>2</sub> DAR signaling pathways <i>in vitro</i> and <i>in vivo</i> in response to a variety of biological conditions. Given its reasonable ADME and pharmacokinetic properties, <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> will also provide scientists a tool to perform proof-of-concept studies in animal model. Moreover, <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> can be further development into drug-like molecules subsequently giving clinicians a starting point for the development of novel or improved treatments (with less side effects) for Tourette’s syndrome, tardive dyskinesia, dystonia, Huntington’s chorea, and especially schizophrenia where D<sub>2</sub> receptor is observed to play a crucial role.</p></div><div id="ml321.s3"><h2 id="_ml321_s3_">2. Materials and Methods</h2><p><b>General Methods for Chemistry.</b> All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, <i>N</i>,<i>N</i>-dimethylformamide (DMF), acetonitrile, methanol and triethylamine were purchased from Sigma-Aldrich (St. Louis, MO). Preparative purification was performed on a Waters semi-preparative HPLC system (Waters Corp., Milford, MA). The column used was a Phenomenex Luna C<sub>18</sub> (5 micron, 30 × 75 mm; Phenomenex, Inc., Torrance, CA) at a flow rate of 45.0 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection at 220 nM. Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7-minute gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8-minute run time at a flow rate of 1.0 mL/min. Method 2: A 3-minute gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5-minute run time at a flow rate of 1.0 mL/min. A Phenomenex Luna C<sub>18</sub> column (3 micron, 3 × 75 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent diode array detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. <sup>1</sup>H NMR spectra were recorded on Varian 400 MHz spectrometers (Agilent Technologies, Santa Clara, CA). Chemical shifts are reported in ppm with undeuterated solvent (DMSO at 2.49 ppm) as internal standard for DMSO-<i>d</i><sub>6</sub> solutions. All of the analogs tested in the biological assays have a purity of greater than 95% based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight (TOF) LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (Version B.02).</p><div id="ml321.s4"><h3>2.1. Assays</h3><p><b>D<sub>2</sub> Ca</b><sup><b>2+</b></sup>
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<b>Primary qHTS of Sytravon Library and Confirmatory Screen.</b> Stable cell line that expresses the D<sub>2</sub> DAR regulated by Tetracycline-Regulated Expression (HEK293 T-REx™) and chimeric G-protein (G<sub>qi5</sub>) to allow coupling of the D<sub>2</sub> DAR to calcium release was developed for the D<sub>2</sub> Ca<sup>2+</sup> Assay described in detail in <a class="figpopup" href="/books/NBK169449/table/ml321.t1/?report=objectonly" target="object" rid-figpopup="figml321t1" rid-ob="figobml321t1">Table 1</a>. In this system, D<sub>2</sub> DAR gene expression is induced by addition of Tet to the cells prior to the assay. Dopamine stimulation of the D<sub>2</sub> DAR activates the chimeric G<sub>qi5</sub> G-protein, which in turn acts on PLC which hydrolyses the membrane phospholipid PIP2 to form Inositol trisphosphate (IP3) and diacylglycerol (DAG). IP<sub>3</sub> diffuses to the ER, binds to its receptor (IP3 receptor), which is a Ca<sup>2+</sup> channel and, releases Ca<sup>2+</sup> from the ER to the cytosol. The Screen Quest™ Fluo-8 Calcium Assay Kit (AAT Bioquest, Sunnyvale, CA) was used to measure the cytosolic Ca<sup>2+</sup> accumulation. This kit contains a non-polar moleculue, acetoxymethyl (AM) esters bound to Fluo-8™ dye that easily penetrates live cell membranes transporting the dye inside the cell where they are rapidly hydrolyzed by cellular esterases. As Fluo-8™ is freed from AM esters; it binds to Ca<sup>2+</sup> and emits a fluorescent signal at 514 nm that escalates with increasing cytosolic Ca<sup>2+</sup>. A high throughput screening device which allows optical detection of signal transmissions within living cells in a time-resolving fashion, the Functional Drug Screening System (FDSS) (Hamamatsu, Japan) was then used to measure the calcium flux signal.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml321t1"><a href="/books/NBK169449/table/ml321.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml321t1" rid-ob="figobml321t1"><img class="small-thumb" src="/books/NBK169449/table/ml321.t1/?report=thumb" src-large="/books/NBK169449/table/ml321.t1/?report=previmg" alt="Table 1. Screen Quest™ Fluor-8 of D2 Ca2+ qHTS assay protocol." /></a><div class="icnblk_cntnt"><h4 id="ml321.t1"><a href="/books/NBK169449/table/ml321.t1/?report=objectonly" target="object" rid-ob="figobml321t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Screen Quest™ Fluor-8 of D<sub>2</sub> Ca<sup>2+</sup> qHTS assay protocol. </p></div></div><p><b>DiscoveRx D<sub>2</sub> β-arrestin Secondary Assay.</b> For a secondary-screen and selectivity assays, DAR PathHunter® β-arrestin GPCR cell lines from DiscoveRx (Fremont, CA) were used as described in the protocol below (<a class="figpopup" href="/books/NBK169449/table/ml321.t2/?report=objectonly" target="object" rid-figpopup="figml321t2" rid-ob="figobml321t2">Table 2</a>). In the D<sub>2</sub> Receptor PathHunter® β-arrestin GPCR cell line, the D<sub>2</sub> GPCR receptor (DAR) is overexpressed and fused with a small 42-amino acid fragment of β-galactosidase called ProLink™ on a CHO cellular background expressing a fusion protein of β-arrestin and a larger <i>N</i>-terminal deletion mutant of β-galactosidase enzyme acceptor. When DAR is activated by dopamine, it stimulates binding of β-arrestin to the ProLink-tagged DAR and the two complementary parts of β-galactosidase form a functional enzyme. When PathHunter® Detection reagent is added, β-galactosidase hydrolyzes it and generates a chemiluminescent signal.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml321t2"><a href="/books/NBK169449/table/ml321.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml321t2" rid-ob="figobml321t2"><img class="small-thumb" src="/books/NBK169449/table/ml321.t2/?report=thumb" src-large="/books/NBK169449/table/ml321.t2/?report=previmg" alt="Table 2. Detailed protocol of DiscoveRx D2 β-arrestin secondary assay." /></a><div class="icnblk_cntnt"><h4 id="ml321.t2"><a href="/books/NBK169449/table/ml321.t2/?report=objectonly" target="object" rid-ob="figobml321t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Detailed protocol of DiscoveRx D<sub>2</sub> β-arrestin secondary assay. </p></div></div><p><b>D<sub>2</sub> Binding Secondary Assay.</b> Compounds were tested for differences in affinity between three dopamine receptor subtypes using radio-labeled ligand binding assays (<a class="figpopup" href="/books/NBK169449/table/ml321.t3/?report=objectonly" target="object" rid-figpopup="figml321t3" rid-ob="figobml321t3">Table 3</a>). The first assay was to determine the <i>K</i>i value of the compounds using the D<sub>2</sub> DAR subtype using stable HEK cell lines expressing the D<sub>2L</sub> human dopamine receptors (Codex Biosciences, Gaithersburg, MD). Cells were cultured in Dulbecco’s modified Eagle’s Medium containing 10% FBS, 1,000 units/mL Penicillin, 1,000 mg/mL Streptomycin, 100 mM Sodium Pyruvate, 1 μg/mL Gentamicin, and 250 mg/mL G418. All cells were maintained at 37 °C in 5% CO<sub>2</sub> and 90% humidity. For radioligand binding assays, cells were removed mechanically using calcium and magnesium-free Earle’s Balanced Salt Solution (EBSS(−)). Intact cells were collected by centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl<sub>2</sub> at pH 7.4 in a glass homogenizer. Homogenates were centrifuged at 20,000 × g for 30 minutes. The membranes were re-suspended in EBSS (pH 7.4) and protein concentration was determined using a Bradford assay according to the manufacturer’s recommendations (Bio-Rad). Membranes were diluted to 24 mg/mL. It was determined in preliminary experiments that this protein concentration gave optimal binding with minimal ligand depletion. Membrane preparations were incubated for 90 min at room temperature with various concentrations of radioligand in a reaction volume of 250 μL EBSS containing 200 mM sodium metabisulfite. Non-specific binding was determined in the presence of 4 μM (+)-butaclamol. Bound ligand was separated from unbound by filtration through GF/C filters using a PerkinElmer cell harvester with ice cold EBSS (4 washes) and quantified on a Top-count (PerkinElmer) after addition of scintillation solution. Saturation experiments generated a <i>K</i><sub>d</sub> value of 0.2 nM and a B<sub>max</sub> of ~4,200 fmol/mg for [<sup>3</sup>H]-methylspiperone binding to D<sub>2</sub> receptors. In order to determine the affinity of a given compound for a receptor type, competition-binding assays were performed. For these assays the reaction mixture was incubated with a single concentration of radiolabeled ligand (0.2 nM [<sup>3</sup>H]methylspiperone) and various concentrations of competing compound. Reactions were incubated, terminated, and quantified as indicated above. <i>K</i><sub>i</sub> values of compounds were determined from observed IC<sub>50</sub> values using the Cheng-Prussoff equation.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml321t3"><a href="/books/NBK169449/table/ml321.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml321t3" rid-ob="figobml321t3"><img class="small-thumb" src="/books/NBK169449/table/ml321.t3/?report=thumb" src-large="/books/NBK169449/table/ml321.t3/?report=previmg" alt="Table 3. Detailed protocol for the D2 binding secondary assay." /></a><div class="icnblk_cntnt"><h4 id="ml321.t3"><a href="/books/NBK169449/table/ml321.t3/?report=objectonly" target="object" rid-ob="figobml321t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Detailed protocol for the D<sub>2</sub> binding secondary assay. </p></div></div><p><b>DiscoveRx D<sub>3</sub> β-arrestin Selectivity Assay.</b> To determine the functional selectivity of the compounds for D<sub>2</sub><i>versus</i> D<sub>3</sub> receptor antagonism, we used a D<sub>3</sub> PathHunter® β-arrestin cell line from DiscoveRx (Fremont, CA) using the detailed protocol below (<a class="figpopup" href="/books/NBK169449/table/ml321.t4/?report=objectonly" target="object" rid-figpopup="figml321t4" rid-ob="figobml321t4">Table 4</a>). A CHO cell line was engineered to overexpress D<sub>3</sub> dopamine receptor (DAR) and fused with a small 42-amino acid fragment of β-gal called ProLink™. In addition, these cells stably express a fusion protein of β-arrestin and a larger <i>N</i>-terminal deletion mutant of β-galactosidase (“enzyme acceptor”). When DAR is activated by dopamine, it stimulates binding of β-arrestin to ProLink-tagged DARs, and the two complementary parts of β-galactosidase form a functional enzyme. When substrate (PathHunter® Detection reagent) is added, β-galactosidase hydrolyzes it and generates a chemiluminescent signal.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml321t4"><a href="/books/NBK169449/table/ml321.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml321t4" rid-ob="figobml321t4"><img class="small-thumb" src="/books/NBK169449/table/ml321.t4/?report=thumb" src-large="/books/NBK169449/table/ml321.t4/?report=previmg" alt="Table 4. Detailed protocol for the DiscoveRx D3 PathHunter® β-arrestin selectivity assay." /></a><div class="icnblk_cntnt"><h4 id="ml321.t4"><a href="/books/NBK169449/table/ml321.t4/?report=objectonly" target="object" rid-ob="figobml321t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Detailed protocol for the DiscoveRx D<sub>3</sub> PathHunter® β-arrestin selectivity assay. </p></div></div><p><b>D<sub>3</sub> Binding Selectivity Assay.</b> Compounds were counter screened for affinity for the D<sub>3</sub> dopamine receptor using a radioligand assay (<a class="figpopup" href="/books/NBK169449/table/ml321.t5/?report=objectonly" target="object" rid-figpopup="figml321t5" rid-ob="figobml321t5">Table 5</a>). This was accomplished by determining the <i>K</i><sub>i</sub> values for the compounds using stable (HEK293 based) cell lines expressing the D<sub>3</sub> human dopamine receptors (Codex Biosciences, Gaithersburg, MD). Cells were cultured in Dulbecco’s modified Eagle’s Medium containing 10 % FBS, 1,000 units/mL Penicillin, 1,000 mg/mL Streptomycin, 100 mM Sodium Pyruvate, 1 μg/mL Gentamicin, and 250 mg/mL G418. All cells were maintained at 37 °C in 5% CO<sub>2</sub> and 90% humidity. For radioligand binding assays, cells were removed mechanically using calcium and magnesium-free Earle’s Balanced Salt Solution (EBSS(−)). Intact cells were collected by centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl<sub>2</sub> at pH 7.4 in a glass homogenizer. Homogenates were centrifuged at 20,000 × g for 30 min. The membranes were re-suspended in EBSS (pH 7.4), and protein concentration was determined using a Bradford assay according to the manufacturer’s recommendations (Bio-Rad). Membranes were diluted to 80 mg/mL, the predetermined optimal protein concentration for binding but minimal ligand depletion. Membrane preparations were incubated for 90 min at room temperature with various concentrations of radioligand in a reaction volume of 250 μL EBSS containing 200 mM sodium metabisulfite. Non-specific binding was determined in the presence of 4 μM (+)-Butaclamol. Bound ligand was separated from unbound by filtration through GF/C filters using a PerkinElmer cell harvester with ice cold EBSS (4 washes) and quantified on a Top-count (PerkinElmer) after addition of scintillation solution. Saturation experiments generated a <i>K</i><sub>d</sub> value of 0.125 nM and a Bmax of ~600 fmol/mg for [<sup>3</sup>H]-methylspiperone binding to D<sub>3</sub> receptors. In order to determine the affinity of a given compound for a receptor type, competition-binding assays were performed. For these assays the reaction mixture was incubated with a single concentration of radiolabeled ligand (0.5 nM [<sup>3</sup>H]methylspiperone) and various concentrations of competing compound. Reactions were incubated, terminated, and quantified as indicated above. <i>K</i><sub>i</sub> values of compounds were determined from observed IC<sub>50</sub> values using the Cheng-Prussoff equation.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml321t5"><a href="/books/NBK169449/table/ml321.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml321t5" rid-ob="figobml321t5"><img class="small-thumb" src="/books/NBK169449/table/ml321.t5/?report=thumb" src-large="/books/NBK169449/table/ml321.t5/?report=previmg" alt="Table 5. Detailed protocol for the D3 binding selectivity assay." /></a><div class="icnblk_cntnt"><h4 id="ml321.t5"><a href="/books/NBK169449/table/ml321.t5/?report=objectonly" target="object" rid-ob="figobml321t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Detailed protocol for the D<sub>3</sub> binding selectivity assay. </p></div></div></div><div id="ml321.s5"><h3>2.2. Probe Chemical Characterization</h3><div id="ml321.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169449/bin/ml321fu2.jpg" alt="Probe ML321 (CID 57377246)." /></div><h3><span class="title">Probe ML321 (CID 57377246)</span></h3><div class="caption"><p></p></div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml321.fn2"><p class="no_top_margin">Purity > 95% as determined by LC/MS and <sup>1</sup>H NMR analyses.</p></div></dd></dl></div></div><p><b>10-Methyl-11-oxo-</b><b><i>N</i></b><b>-(2-(thiophen-2-yl)ethyl)-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide 5-(</b><b><i>S</i></b><b>)-oxide (<a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a>).</b> LC-MS Retention Time: t<sub>1</sub> (Method 1) = 5.348 min; t<sub>2</sub> (Method 2) = 3.188 min; <sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ ppm 8.76 (t, <i>J</i>=5.7 Hz, 1 H), 7.94 (d, <i>J</i>=2.0 Hz, 1 H), 7.86 (dd, <i>J</i>=8.2, 2.0 Hz, 1 H), 7.69 – 7.77 (m, 2 H), 7.67 (d, <i>J</i>=8.2 Hz, 2 H), 7.51 – 7.60 (m, 1 H), 7.31 (dd, <i>J</i>=5.1, 1.2 Hz, 1 H), 6.92 (dd, <i>J</i>=5.1, 3.1 Hz, 1 H), 6.83 – 6.90 (m, 1 H), 3.55 (s, 3 H), 3.43 – 3.52 (m, 2 H), 3.02 (t, <i>J</i>=7.0 Hz, 2 H); <sup>13</sup>C NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ ppm 13C NMR (101 MHz, DMSO-<i>d</i><sub>6</sub>) δ ppm 165.16, 165.10, 147.66, 145.98, 141.75, 137.87, 137.02, 132.92, 131.69, 131.23, 128.20, 127.39, 126.51, 125.70, 124.57, 124.43, 121.05, 119.40, 41.55, 38.03, 29.49; HRMS (ESI) <i>m/z</i> (M+H)<sup>+</sup> calcd. for C<sub>21</sub>H<sub>19</sub>N<sub>2</sub>O<sub>3</sub>S<sub>2</sub> [M+H<sup>+</sup>] 411.0832, found 411.0831.</p><div id="ml321.f1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Chemical%20stability%20evaluation%20for%20ML321%20for%2048%20hours%20at%20room%20temperature%20in%20(A)%20D2%20Ca2%2B%20assay%20buffer%20(Glucose%2C%2010%25%20FBS%2C%201%D7%20NEAA%2C%20and%20Pen%2FStrep)%2C%20(B)%20PathHunter%AE%20%003B2-arrestin%20GPCR%20assay%20buffer%20(DiscoveRx%2C%20Catalog%20%23%20056R2A)%20and%20(C)%20PBS%2B%20buffer%20(DPBS%2C%201%20mM%20CaCl2%2C%200.&p=BOOKS&id=169449_ml321f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK169449/bin/ml321f1.jpg" alt="Figure 1. Chemical stability evaluation for ML321 for 48 hours at room temperature in (A) D2 Ca2+ assay buffer (Glucose, 10% FBS, 1× NEAA, and Pen/Strep), (B) PathHunter® β-arrestin GPCR assay buffer (DiscoveRx, Catalog # 056R2A) and (C) PBS+ buffer (DPBS, 1 mM CaCl2, 0." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span></h3><div class="caption"><p>Chemical stability evaluation for <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> for 48 hours at room temperature in <b>(A)</b> D<sub>2</sub> Ca<sup>2+</sup> assay buffer (Glucose, 10% FBS, 1× NEAA, and Pen/Strep), <b>(B)</b> PathHunter® β-arrestin GPCR assay buffer (DiscoveRx, Catalog # 056R2A) and <b>(C)</b> PBS+ buffer (DPBS, 1 mM CaCl<sub>2</sub>, 0.5 mM MgCl<sub>2</sub>, 0.05% BSA, 0.005% Tween 20).</p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml321f2" co-legend-rid="figlgndml321f2"><a href="/books/NBK169449/figure/ml321.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml321f2" rid-ob="figobml321f2"><img class="small-thumb" src="/books/NBK169449/bin/ml321f2.gif" src-large="/books/NBK169449/bin/ml321f2.jpg" alt="Figure 2. Structures of the ML321 and five analogs (with corresponding SID) that have been submitted to the MLSMR compound repository." /></a><div class="icnblk_cntnt" id="figlgndml321f2"><h4 id="ml321.f2"><a href="/books/NBK169449/figure/ml321.f2/?report=objectonly" target="object" rid-ob="figobml321f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Structures of the ML321 and five analogs (with corresponding SID) that have been submitted to the MLSMR compound repository. </p></div></div><div id="ml321.t6" class="table"><h3><span class="label">Table 6</span><span class="title">List of the D<sub>2</sub> DAR inhibitor probe ML321 and related analogs and their corresponding identification numbers</span></h3><div class="caption"><p>Their corresponding molecular structures are shown above in <a class="figpopup" href="/books/NBK169449/figure/ml321.f2/?report=objectonly" target="object" rid-figpopup="figml321f2" rid-ob="figobml321f2">Figure 2</a>.</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169449/table/ml321.t6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml321.t6_lrgtbl__"><table><thead><tr><th id="hd_h_ml321.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Internal ID</th><th id="hd_h_ml321.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS ID</th><th id="hd_h_ml321.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th id="hd_h_ml321.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th id="hd_h_ml321.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">ML #</th><th id="hd_h_ml321.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Type</th><th id="hd_h_ml321.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml321.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00250206</td><td headers="hd_h_ml321.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004497249</td><td headers="hd_h_ml321.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882616" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">136882616</a></td><td headers="hd_h_ml321.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">57377246</td><td headers="hd_h_ml321.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b><a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a></b></td><td headers="hd_h_ml321.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe</td><td headers="hd_h_ml321.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml321.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241677</td><td headers="hd_h_ml321.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004497250</td><td headers="hd_h_ml321.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882617" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">136882617</a></td><td headers="hd_h_ml321.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">57377245</td><td headers="hd_h_ml321.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml321.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml321.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241689</td><td headers="hd_h_ml321.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004497251</td><td headers="hd_h_ml321.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882618" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">136882618</a></td><td headers="hd_h_ml321.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">57377247</td><td headers="hd_h_ml321.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml321.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml321.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00109352</td><td headers="hd_h_ml321.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004497252</td><td headers="hd_h_ml321.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882619" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">136882619</a></td><td headers="hd_h_ml321.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16007752</td><td headers="hd_h_ml321.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml321.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml321.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00248394</td><td headers="hd_h_ml321.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004497253</td><td headers="hd_h_ml321.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882620" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">136882620</a></td><td headers="hd_h_ml321.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">57377248</td><td headers="hd_h_ml321.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml321.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml321.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241680</td><td headers="hd_h_ml321.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004497254</td><td headers="hd_h_ml321.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136882621" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">136882621</a></td><td headers="hd_h_ml321.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">57377249</td><td headers="hd_h_ml321.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml321.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr></tbody></table></div></div></div><div id="ml321.s6"><h3>2.3. Probe Preparation</h3><p>Preparation of 10-methyl-11-oxo-<i>N</i>-(2-(thiophen-2-yl)ethyl)-10,11-dihydrodibenzo[b,f][1,4] thiazepine-8-carboxamide 5-(<i>S</i>)-oxide (<b><a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a></b>) is a multi-step process with 8 compound intermediates illustrated above (<a class="figpopup" href="/books/NBK169449/figure/ml321.f6/?report=objectonly" target="object" rid-figpopup="figml321f6" rid-ob="figobml321f6">Scheme 1</a>) and summarized below.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml321f6" co-legend-rid="figlgndml321f6"><a href="/books/NBK169449/figure/ml321.f6/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml321f6" rid-ob="figobml321f6"><img class="small-thumb" src="/books/NBK169449/bin/ml321f6.gif" src-large="/books/NBK169449/bin/ml321f6.jpg" alt="Scheme 1. The synthetic route to ML321 is a multi-step process with 8 compound intermediates (A – H)." /></a><div class="icnblk_cntnt" id="figlgndml321f6"><h4 id="ml321.f6"><a href="/books/NBK169449/figure/ml321.f6/?report=objectonly" target="object" rid-ob="figobml321f6">Scheme 1</a></h4><p class="float-caption no_bottom_margin">The synthetic route to ML321 is a multi-step process with 8 compound intermediates (A – H). </p></div></div><ol><li class="half_rhythm"><div>A solution of methyl 4-fluoro-3-nitrobenzoate and methyl 2-mercaptobenzoate in dimethylformamide (DMF) was treated with caesium carbonate (Cs<sub>2</sub>CO<sub>3</sub>) at room temperature. The reaction mixture was stirred at 40 °C for 4 hour and then cooled to room temperature. Ice water was added to induce the precipitation. The precipitate was filtered, washed with water, and dried to give 21.0 g (99%) of methyl 4-(2-(methoxycarbonyl)phenylthio)-3-nitrobenzoate (<b>intermediate A</b>). A yellow solid which was used directly in the next reaction without further purification.</div></li><li class="half_rhythm"><div>A solution of <b>intermediate A</b> and water was treated at room temperature with lithium hydroxide (LiOH). The reaction mixture was stirred at 60 °C for 2 hr. The organic solvent was removed and the aqueous solution was washed with ethyl acetate (EtOAc) and acidified with 2 N hydrogen chloride (HCl) until pH = ~2. The yellow precipitate was filtered, washed with water, and dried to give 19.1 g (99%) of 4-(2-Carboxyphenylthio)-3-nitrobenzoic acid (<b>intermediate B</b>); a yellow solid which was used directly in the next reaction without further purification.</div></li><li class="half_rhythm"><div><b>Intermediate B</b> was treated at room temperature with platinum (IV) oxide (PtO<sub>2</sub>) and palladium on carbon (Pd/C). A balloon containing hydrogen (H<sub>2</sub>) was connected to the flask and the reaction flask was repeatedly evacuated and refilled with H<sub>2</sub>. After 16 hr, additional Pd/C (10%, 600 mg, 5.64 mmol) was added and the reaction mixture was stirred under H<sub>2</sub> balloon for an additional 32 hr. The reaction mixture was filtered through a pad of celite and concentrated to give 7.80 g (99%) of 3-amino-4-(2-carboxyphenylthio)benzoic acid (<b>intermediate C</b>); a grey-yellow solid which was used directly in the next reaction without further purification.</div></li><li class="half_rhythm"><div>A solution of <b>intermediate C</b> in THF was treated at 0 °C with 1,1′-carbonyldiimidazole (CDI) via several portions. The reaction mixture was warmed and stirred at room temperature overnight. The reaction mixture was poured into 140 mL of ice water containing concentrated HCl and stirred for 1 hr. The white precipitate was filtered, washed with water, and dried to give 3.89 g (87%) of 11-Oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid (<b>intermediate D</b>), a white solid which was used directly in the next reaction without further purification.</div></li><li class="half_rhythm"><div>A solution of <b>intermediate D</b> in DMF was treated at 0 °C with sodium hydride (NaH). The reaction mixture was warmed and stirred at room temperature for 1 hr. Then, a solution of methyl iodide (MeI) in DMF was added dropwise to the mixture. The reaction mixture was stirred at room temperature for 1.5 hr. Water was carefully added and the aqueous layer was washed with EtOAc. The aqueous layer was acidified with HCl to induce the precipitation which was filtered, washed, and dried to give 200 mg (91%) of methyl 10-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylate (<b>intermediate E</b>); a yellow solid which was used directly in the next reaction without further purification.</div></li><li class="half_rhythm"><div>A solution of <b>intermediate E</b> in tetrahydrofuran (THF), methanol (MeOH) and water was treated at room temperature with LiOH. The reaction mixture was stirred at room temperature for 1 hr, diluted with water, and acidified with HCl. The aqueous mixture was extracted with 20% of MeOH in dichloromethane. The organic layer was separated, dried and concentrated to give 140 mg (98%) of 10-Methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid (<b>intermediate F</b>); a grey solid which was used directly in the next reaction without further purification.</div></li><li class="half_rhythm"><div>A suspension of <b>intermediate F</b> in acetic acid was treated at room temperature with hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) for 8 hr. Upon completion, the reaction mixture was poured into a cold saturated solution of sodium thiosulphate (Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>) in water and stirred at room temperature for 3 hr. The mixture was then extracted with 20% of MeOH in dichloromethane (DCM). The organic layer was separated, dried, and concentrated to give 595 mg (85%) of 10-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid 5-oxide (<b>intermediate G</b>); a white solid containing ~ 5% of dioxide as a by-product.</div></li><li class="half_rhythm"><div><b>Intermediate G</b> was enantiomerically purified to > 98% purity to yield 10-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid 5-(<i>S</i>)-oxide (<b>intermediate H</b>) using supercritical fluid chromatography (SFC) preparative systems at Lotus Separations, LLC (Princeton, NJ, USA). For preparative separation, an IC (2 × 15 cm) column was used with an eluent of 40% MeOH (0.1% DEA)/CO<sub>2</sub>, 100 bar. Flow rate was 60 mL/min and detection wavelength was 220 nm. For analytical separation, an IC (15 × 0.46 cm) column was used with an eluent of 40% MeOH/CO<sub>2</sub>, 100 bar. Flow rate was 3 mL/min and detection wavelengths were 220 and 280 nm. Retention time was 3.42 min. Retention time for <i>R</i>-configuration enantiomer was 2.40 min. The material was used directly in the next coupling reaction.</div></li><li class="half_rhythm"><div>A solution <b>intermediate H</b> in DMF (5.00 mL) was treated at room temperature with the peptide coupling reagent HATU and diisopropylethylamine followed by 2-(thiophen-2-yl)ethanamine. The reaction mixture was stirred at room temperature for 3 h and poured into ice water. The white precipitate was filtered and dried to give a white solid, which was purified via silica gel chromatography using a gradient of 10–100% of EtOAc in hexanes to give 118 mg (87%) of 10-methyl-11-oxo-<i>N</i>-(2-(thiophen-2-yl)ethyl)-10,11-dihydrodibenzo[b,f][1,4] thiazepine-8-carboxamide 5-(<i>S</i>)-oxide (<a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a>).</div></li></ol></div></div><div id="ml321.s7"><h2 id="_ml321_s7_">3. Results</h2><div id="ml321.s8"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml321.f3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169449/bin/ml321f3.jpg" alt="Figure 3. Dose response activity of ML321 in (A) D2 Ca2+ assay (green, AC50 = 0." /></div><h3><span class="label">Figure 3</span></h3><div class="caption"><p>Dose response activity of <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> in <b>(A)</b> D<sub>2</sub> Ca<sup>2+</sup> assay (green, AC<sub>50</sub> = 0.070 μM), D<sub>2</sub> β-arrestin assay (blue, AC<sub>50</sub> = 0.725 μM), and D<sub>3</sub> β-arrestin assay (red, AC<sub>50</sub> = 12.9 μM). <b>(B)</b> Graphical representation of the dose response curves of <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> in binding assays for D<sub>1</sub> (black, <i>K</i><sub>i</sub> = 67.1 μM), D<sub>2</sub> (blue, <i>K</i><sub>i</sub> = 0.1 μM), D<sub>3</sub> (red, <i>K</i><sub>i</sub> = 2.9 μM), D<sub>4</sub> (green, <i>K</i><sub>i</sub> = 8.48 μM) and D<sub>5</sub> (brown) showing preferential inhibition for the D<sub>2</sub> receptor.</p></div></div></div><div id="ml321.s9"><h3>3.2. Cellular Activity</h3><div id="ml321.t7" class="table"><h3><span class="label">Table 7</span><span class="title">Activity profile of the probe ML321 and relative analogs against D<sub>2</sub> Ca<sup>2+</sup> assay in D<sub>2</sub> expressing HK293 T-Rex™ cell line, D<sub>2</sub> β-arrestin assay in D<sub>2</sub> receptor PathHunter® β-arrestin cells, D<sub>3</sub> β-arrestin assay in D<sub>3</sub> PathHunter® β-arrestin cells, and D<sub>2</sub> or D<sub>3</sub> radio-ligand binding assays in HEK cell line expressing either D<sub>2L</sub> or D<sub>3</sub> human dopamine receptor</span></h3><div class="caption"><p>Results showed probe <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> is a potent (<1 μM AC<sub>50</sub>) and selective (>17 fold preference) D<sub>2</sub> antagonist.</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169449/table/ml321.t7/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml321.t7_lrgtbl__"><table><thead><tr><th id="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Internal ID</th><th id="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Entry</th><th id="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">D<sub>2</sub> Ca<sup>2+</sup> AC<sub>50</sub> (μM)</th><th id="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">D<sub>2</sub> β-arrestin AC<sub>50</sub> (μM)</th><th id="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">D<sub>3</sub> β-arrestin AC<sub>50</sub> (μM)</th><th id="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">D<sub>2</sub> Ki AC<sub>50</sub> (μM)</th><th id="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">D<sub>3</sub> Ki AC<sub>50</sub> (μM)</th><th id="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">D<sub>3/</sub>D<sub>2</sub> β-arrestin</th><th id="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">D<sub>3/</sub>D<sub>2</sub> Ki</th></tr></thead><tbody><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250206</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">55 <b>(<a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a>)</b></td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.070</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.725</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12.9</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.1</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.9</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">17.8</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">29.0</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250207</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>R</i>-55</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00248415</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">32 <i>Racemic</i>-55</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.089</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.913</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20.4</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.23</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.6</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">22.3</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">28.7</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00109414</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.281</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.89</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.76</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.08</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.48</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.0</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.0</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00238612</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">38</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.141</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.15</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.89</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.5</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250200</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">47</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.056</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.576</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.24</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.09</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.23</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.6</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.6</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250203</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">48</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.112</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.257</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.62</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.023</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.13</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.3</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.7</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250211</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">49</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.223</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.29</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9.13</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.68</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.88</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.1</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.3</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250215</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">50</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.353</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.89</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">32.4</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.21</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.4</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11.2</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">16.2</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00248384</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">51</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.280</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.89</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10.2</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.5</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250205</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">52</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.089</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.02</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.57</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.24</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.37</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.5</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.5</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250213</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">53</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.445</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.89</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">14.5</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.31</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">21.0</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.0</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">70.0</td></tr><tr><td headers="hd_h_ml321.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00250209</td><td headers="hd_h_ml321.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">54</td><td headers="hd_h_ml321.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.070</td><td headers="hd_h_ml321.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.636</td><td headers="hd_h_ml321.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.57</td><td headers="hd_h_ml321.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.21</td><td headers="hd_h_ml321.t7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.35</td><td headers="hd_h_ml321.t7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.2</td><td headers="hd_h_ml321.t7_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.7</td></tr></tbody></table></div></div></div><div id="ml321.s10"><h3>3.3. Profiling Assays</h3><div id="ml321.t8" class="table"><h3><span class="label">Table 8</span><span class="title"><i>In vitro</i> ADME profile for probe ML321 and a relative analog NCGC00109414 (CID16007814)</span></h3><div class="caption"><p>Results showed compound <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> has good stability and permeability.</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169449/table/ml321.t8/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml321.t8_lrgtbl__"><table><thead><tr><th id="hd_h_ml321.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th id="hd_h_ml321.t8_1_1_1_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Aqueous Kinetic Solubility</th><th id="hd_h_ml321.t8_1_1_1_3" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Liver Microsomal Stability (T<sub>1/2</sub> in min)</th><th id="hd_h_ml321.t8_1_1_1_4" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Plasma Stability (% remaining after 2 h)</th><th id="hd_h_ml321.t8_1_1_1_5" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Caco-2 Permeability (10<sup>−6</sup>, cm/s)</th></tr><tr><th headers="hd_h_ml321.t8_1_1_1_1" id="hd_h_ml321.t8_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th><th headers="hd_h_ml321.t8_1_1_1_2" id="hd_h_ml321.t8_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">μg/mL</th><th headers="hd_h_ml321.t8_1_1_1_2" id="hd_h_ml321.t8_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">μM</th><th headers="hd_h_ml321.t8_1_1_1_3" id="hd_h_ml321.t8_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Mouse</th><th headers="hd_h_ml321.t8_1_1_1_3" id="hd_h_ml321.t8_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Human</th><th headers="hd_h_ml321.t8_1_1_1_4" id="hd_h_ml321.t8_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Mouse</th><th headers="hd_h_ml321.t8_1_1_1_4" id="hd_h_ml321.t8_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Human</th><th headers="hd_h_ml321.t8_1_1_1_5" id="hd_h_ml321.t8_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P<sub>app (A-B)</sub></th><th headers="hd_h_ml321.t8_1_1_1_5" id="hd_h_ml321.t8_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P<sub>app (B-A)</sub></th></tr></thead><tbody><tr><td headers="hd_h_ml321.t8_1_1_1_1 hd_h_ml321.t8_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b><a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a></b></td><td headers="hd_h_ml321.t8_1_1_1_2 hd_h_ml321.t8_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">46.5</td><td headers="hd_h_ml321.t8_1_1_1_2 hd_h_ml321.t8_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">113.3</td><td headers="hd_h_ml321.t8_1_1_1_3 hd_h_ml321.t8_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">24.7</td><td headers="hd_h_ml321.t8_1_1_1_3 hd_h_ml321.t8_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">26.4</td><td headers="hd_h_ml321.t8_1_1_1_4 hd_h_ml321.t8_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">95.2</td><td headers="hd_h_ml321.t8_1_1_1_4 hd_h_ml321.t8_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">98.4</td><td headers="hd_h_ml321.t8_1_1_1_5 hd_h_ml321.t8_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">18.8</td><td headers="hd_h_ml321.t8_1_1_1_5 hd_h_ml321.t8_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23.8</td></tr><tr><td headers="hd_h_ml321.t8_1_1_1_1 hd_h_ml321.t8_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID16007814</td><td headers="hd_h_ml321.t8_1_1_1_2 hd_h_ml321.t8_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">11.5</td><td headers="hd_h_ml321.t8_1_1_1_2 hd_h_ml321.t8_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">26.4</td><td headers="hd_h_ml321.t8_1_1_1_3 hd_h_ml321.t8_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">35.9</td><td headers="hd_h_ml321.t8_1_1_1_3 hd_h_ml321.t8_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N/A</td><td headers="hd_h_ml321.t8_1_1_1_4 hd_h_ml321.t8_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N/A</td><td headers="hd_h_ml321.t8_1_1_1_4 hd_h_ml321.t8_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N/A</td><td headers="hd_h_ml321.t8_1_1_1_5 hd_h_ml321.t8_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">21.6</td><td headers="hd_h_ml321.t8_1_1_1_5 hd_h_ml321.t8_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16.2</td></tr></tbody></table></div></div><div id="ml321.t9" class="table"><h3><span class="label">Table 9</span><span class="title">Summary of in vivo pharmacokinetic (PK) data for ML321 in C57BL/6 mice (BQL = Below quantifiable limit of 1 ng/mL for ML321 in male C57BL/6 plasma. NA = Not available)</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169449/table/ml321.t9/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml321.t9_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml321.t9_1_1_1_1" colspan="10" rowspan="1" style="text-align:center;vertical-align:top;">Individual and mean plasma concentration-time data of <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> after an IP dose of 30 mg/kg in male C57BL/6 mice</th></tr><tr><th headers="hd_h_ml321.t9_1_1_1_1" id="hd_h_ml321.t9_1_1_2_1" colspan="10" rowspan="1" style="text-align:left;vertical-align:top;">
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<span class="hr"></span></th></tr><tr><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" id="hd_h_ml321.t9_1_1_3_1" rowspan="3" colspan="1" style="text-align:center;vertical-align:top;">Dose (mg/kg)</th><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" id="hd_h_ml321.t9_1_1_3_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:top;">Dose route</th><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" id="hd_h_ml321.t9_1_1_3_3" rowspan="3" colspan="1" style="text-align:center;vertical-align:top;">Sampling Time (hr)</th><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1" id="hd_h_ml321.t9_1_1_3_4" colspan="3" rowspan="1" style="text-align:center;vertical-align:top;">Concentration (ng/mL)</th><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" id="hd_h_ml321.t9_1_1_3_5" rowspan="3" colspan="1" style="text-align:center;vertical-align:top;">Mean (ng/mL)</th><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" id="hd_h_ml321.t9_1_1_3_6" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">μM</th><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" id="hd_h_ml321.t9_1_1_3_7" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">SD</th><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" id="hd_h_ml321.t9_1_1_3_8" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">CV (%)</th></tr><tr><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4" id="hd_h_ml321.t9_1_1_4_1" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">
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<span class="hr"></span></th></tr><tr><th headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1" id="hd_h_ml321.t9_1_1_5_1" colspan="3" rowspan="1" style="text-align:center;vertical-align:top;">Individual</th></tr></thead><tbody><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">30</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IP</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BQL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BQL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BQL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BQL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.083</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6639</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6779</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6897</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6771</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16.495</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">129</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.91</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.25</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8016</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7749</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">9061</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8275</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20.159</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">694</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8.38</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.5</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5893</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7473</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6778</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6715</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16.357</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">792</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">11.8</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2257</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2539</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2986</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2594</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6.319</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">368</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">14.2</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">256</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">236</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">257</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">250</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.608</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">11.6</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.66</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">13.0</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">9.37</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8.06</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">10.2</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.025</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.58</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">25.4</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">67.1</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">75.7</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">94.3</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">79.0</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.193</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">13.9</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">17.6</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">12</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">34.9</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">10.7</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">13.5</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">19.7</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.048</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">13.2</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">67.2</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">24</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BQL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BQL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BQL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BQL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1 hd_h_ml321.t9_1_1_3_2 hd_h_ml321.t9_1_1_3_3 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1 hd_h_ml321.t9_1_1_3_5 hd_h_ml321.t9_1_1_3_6 hd_h_ml321.t9_1_1_3_7 hd_h_ml321.t9_1_1_3_8" colspan="10" rowspan="1" style="text-align:left;vertical-align:top;">
|
||
<span class="hr"></span></td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1 hd_h_ml321.t9_1_1_3_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;">PK parameters</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Unit</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1 hd_h_ml321.t9_1_1_3_5 hd_h_ml321.t9_1_1_3_6 hd_h_ml321.t9_1_1_3_7 hd_h_ml321.t9_1_1_3_8" colspan="7" rowspan="1" style="text-align:center;vertical-align:top;">Estimate</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1 hd_h_ml321.t9_1_1_3_2 hd_h_ml321.t9_1_1_3_3 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1 hd_h_ml321.t9_1_1_3_5 hd_h_ml321.t9_1_1_3_6 hd_h_ml321.t9_1_1_3_7 hd_h_ml321.t9_1_1_3_8" colspan="10" rowspan="1" style="text-align:left;vertical-align:top;">
|
||
<span class="hr"></span></td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1 hd_h_ml321.t9_1_1_3_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;">T<sub>max</sub></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Hr</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1 hd_h_ml321.t9_1_1_3_5 hd_h_ml321.t9_1_1_3_6 hd_h_ml321.t9_1_1_3_7 hd_h_ml321.t9_1_1_3_8" colspan="7" rowspan="1" style="text-align:center;vertical-align:top;">0.25</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1 hd_h_ml321.t9_1_1_3_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;">C<sub>max</sub></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ng/mL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1 hd_h_ml321.t9_1_1_3_5 hd_h_ml321.t9_1_1_3_6 hd_h_ml321.t9_1_1_3_7 hd_h_ml321.t9_1_1_3_8" colspan="7" rowspan="1" style="text-align:center;vertical-align:top;">8275</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1 hd_h_ml321.t9_1_1_3_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;">Terminal t<sub>1/2</sub></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Hr</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1 hd_h_ml321.t9_1_1_3_5 hd_h_ml321.t9_1_1_3_6 hd_h_ml321.t9_1_1_3_7 hd_h_ml321.t9_1_1_3_8" colspan="7" rowspan="1" style="text-align:center;vertical-align:top;">1.67</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1 hd_h_ml321.t9_1_1_3_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;">AUC<sub>last</sub></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">h*ng/mL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1 hd_h_ml321.t9_1_1_3_5 hd_h_ml321.t9_1_1_3_6 hd_h_ml321.t9_1_1_3_7 hd_h_ml321.t9_1_1_3_8" colspan="7" rowspan="1" style="text-align:center;vertical-align:top;">7796</td></tr><tr><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_1 hd_h_ml321.t9_1_1_3_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;">AUC<sub>INF</sub></td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">h*ng/mL</td><td headers="hd_h_ml321.t9_1_1_1_1 hd_h_ml321.t9_1_1_2_1 hd_h_ml321.t9_1_1_3_4 hd_h_ml321.t9_1_1_4_1 hd_h_ml321.t9_1_1_5_1 hd_h_ml321.t9_1_1_3_5 hd_h_ml321.t9_1_1_3_6 hd_h_ml321.t9_1_1_3_7 hd_h_ml321.t9_1_1_3_8" colspan="7" rowspan="1" style="text-align:center;vertical-align:top;">7844</td></tr></tbody></table></div></div><div id="ml321.f4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169449/bin/ml321f4.jpg" alt="Figure 3. Mean plasma and brain concentration-time profiles of ML321 after an IP dose of 30 mg/kg in male C57BL/6 mice (N = 3)." /></div><h3><span class="label">Figure 3</span><span class="title">Mean plasma and brain concentration-time profiles of ML321 after an IP dose of 30 mg/kg in male C57BL/6 mice (N = 3)</span></h3><div class="caption"><p>The mice appeared less activity at 5 min post dosing, and it lasted for about 2 hr. The IP dosing solution was prepared in 10% NMP + 20% PEG400 + 70% of 25% HP-β-CD in water.</p></div></div></div></div><div id="ml321.s11"><h2 id="_ml321_s11_">4. Discussion</h2><div id="ml321.s12"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>A recent group of publications appeared on a series of <i>N</i>-(1-benzylpiperidin-4-yl)pyridazin-3-amines exemplified by the clinical candidate JNJ-37822681 showing D<sub>2</sub> inhibition [<a class="bk_pop" href="#ml321.r8">8</a>]. However, JNJ-37822681 (<a class="figpopup" href="/books/NBK169449/figure/ml321.f5/?report=objectonly" target="object" rid-figpopup="figml321f5" rid-ob="figobml321f5">Figure 4</a>) is not very selective between D<sub>2</sub> (<i>K</i><sub>i</sub> = 158 nM) and D<sub>3</sub> (<i>K</i><sub>i</sub> = 1,159 nM) in displacement assays. Another compound L741,626 (<a class="figpopup" href="/books/NBK169449/figure/ml321.f5/?report=objectonly" target="object" rid-figpopup="figml321f5" rid-ob="figobml321f5">Figure 4</a>), a close analog of Haloperidol, had claimed selectivity of ~10–15 fold in D<sub>2</sub> and D<sub>3</sub> displacement assays [<a class="bk_pop" href="#ml321.r9">9</a>]. This compound showed a 3-fold selectivity in functional D<sub>2</sub> and D<sub>3</sub> β-arrestin assays. The binding selectivity was significantly improved through several rounds of medicinal chemistry efforts to obtain analogs <b>57</b> and <b>58</b> (<a class="figpopup" href="/books/NBK169449/figure/ml321.f5/?report=objectonly" target="object" rid-figpopup="figml321f5" rid-ob="figobml321f5">Figure 4</a>) which was observed to have D<sub>2</sub> vs. D<sub>3</sub> selectivity greater than 100 fold [<a class="bk_pop" href="#ml321.r10">10</a>]. However, no further reports related to ADME profiling and <i>in vivo</i> animal data was reported. It is well known that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its derivatives are potential neurotoxins [<a class="bk_pop" href="#ml321.r11">11</a>] and could be a reason for lack of <i>in vivo</i> data for this chemical series. Hence, the reference compounds <b>57</b> and <b>58</b> were resynthesized in house and assayed side-by-side with the probe <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a>. A stability and GPCR panel inhibition profile for compounds <b>58</b> and <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> were also assessed. Compound <b>58</b> was observed to have a 60-fold D<sub>2</sub> vs. D<sub>3</sub> selectivity in the β-arrestin functional assay and 48.6-fold D<sub>2</sub> vs. D<sub>3</sub> selectivity in the binding assay (<a class="figpopup" href="/books/NBK169449/table/ml321.t10/?report=objectonly" target="object" rid-figpopup="figml321t10" rid-ob="figobml321t10">Table 10</a>). However it showed a very short half-life time of 2.4 min in rat liver microsomal test. Results of the GPCR panel inhibition profile also showed that compound <b>58</b> at 10 μM is very promiscuous; targeting a large number of receptors with > 50% inhibition (<a class="figpopup" href="/books/NBK169449/table/ml321.t11/?report=objectonly" target="object" rid-figpopup="figml321t11" rid-ob="figobml321t11">Table 11</a>). Not only did it inhibit dopamine receptors 2–4, but also targeted Sigma 1–2, SERT, MOR and Alpha2 A-B with > 90% inhibition. In contrast, <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> at 10 μM gave a more selective profile; with > 90% inhibition only against the D<sub>2</sub> receptor while the rest (5-HT2C, 5-HT7, D<sub>3</sub>) were only ~ 60% inhibition. Until this probe, it was very difficult to interpret data using existing compounds in <i>ex vivo</i> preparation because so many of them are “contaminated” by influence from the D<sub>3</sub> receptor or the D<sub>4</sub> receptor; both of which is antagonized by all existing molecules. Although <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> (and members of its chemical series) is less potent than the compound <b>58</b>, <b><a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a></b> showed a good D<sub>2</sub> vs. D<sub>3</sub> receptor selectivity and good ADME and PK properties with fewer chemical scaffold liabilities. Therefore, probe <a href="/pcsubstance/?term=ML321[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML321</a> provides sufficient selectivity towards D<sub>2</sub> giving scientist a tool to better understand it contributions to dopamine signaling pathways both <i>in vitro</i> and <i>in vivo</i>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml321f5" co-legend-rid="figlgndml321f5"><a href="/books/NBK169449/figure/ml321.f5/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml321f5" rid-ob="figobml321f5"><img class="small-thumb" src="/books/NBK169449/bin/ml321f5.gif" src-large="/books/NBK169449/bin/ml321f5.jpg" alt="Figure 4. Known chemical series of selective D2versus D3 DAR antagonist." /></a><div class="icnblk_cntnt" id="figlgndml321f5"><h4 id="ml321.f5"><a href="/books/NBK169449/figure/ml321.f5/?report=objectonly" target="object" rid-ob="figobml321f5">Figure 4</a></h4><p class="float-caption no_bottom_margin">Known chemical series of selective D<sub>2</sub><i>versus</i> D<sub>3</sub> DAR antagonist. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml321t10"><a href="/books/NBK169449/table/ml321.t10/?report=objectonly" target="object" title="Table 10" class="img_link icnblk_img figpopup" rid-figpopup="figml321t10" rid-ob="figobml321t10"><img class="small-thumb" src="/books/NBK169449/table/ml321.t10/?report=thumb" src-large="/books/NBK169449/table/ml321.t10/?report=previmg" alt="Table 10. Comparison of dopamine receptor inhibition assay for ML321 and current D2 antagonists." /></a><div class="icnblk_cntnt"><h4 id="ml321.t10"><a href="/books/NBK169449/table/ml321.t10/?report=objectonly" target="object" rid-ob="figobml321t10">Table 10</a></h4><p class="float-caption no_bottom_margin">Comparison of dopamine receptor inhibition assay for ML321 and current D<sub>2</sub> antagonists. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml321t11"><a href="/books/NBK169449/table/ml321.t11/?report=objectonly" target="object" title="Table 11" class="img_link icnblk_img figpopup" rid-figpopup="figml321t11" rid-ob="figobml321t11"><img class="small-thumb" src="/books/NBK169449/table/ml321.t11/?report=thumb" src-large="/books/NBK169449/table/ml321.t11/?report=previmg" alt="Table 11. GPCR panel screening for ML321 and compound 58." /></a><div class="icnblk_cntnt"><h4 id="ml321.t11"><a href="/books/NBK169449/table/ml321.t11/?report=objectonly" target="object" rid-ob="figobml321t11">Table 11</a></h4><p class="float-caption no_bottom_margin">GPCR panel screening for ML321 and compound <i>58</i>. Data represent mean % inhibition (N = 4 determinations) for compound tested at receptor subtypes. > 50% of inhibition (marked red) is considered as significant inhibition. In cases where negative <a href="/books/NBK169449/table/ml321.t11/?report=objectonly" target="object" rid-ob="figobml321t11">(more...)</a></p></div></div></div></div><div id="ml321.s13"><h2 id="_ml321_s13_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml321.r1">Kuhar MJ, Couceyro PR, Lambert P. Dopamine Receptors. In: Siegel G, Agranoff B, Albers R, et al., editors. <span class="ref-journal">Basic Neurochemistry: Molecular Cellular and Medical Aspects.</span> 6th edition. Philadelphia: Lippincott-Raven; 1999. NCBI Bookshelf ID: NBK27980.</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml321.r2">Beaulieu JM, Gainetdinov RR. The Physiology, Signaling, and Pharmacology of Dopamine Receptors. <span><span class="ref-journal">Pharmacol. Rev. </span>2011;<span class="ref-vol">63</span>(1):182–217.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21303898" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21303898</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml321.r3">Seeman P. Dopamine Receptors: Clinical Correlates. In: Sibley D, editor. <span class="ref-journal">Psychopharmacology – 4th Generation of Progress.</span> New York: Nature Publishing Group; 2000. </div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml321.r4">Cho DI, Zheng M, Kim KM. Current Perspectives on the Selective Regulation of Dopamine D<sub>2</sub> and D<sub>3</sub> Receptors. <span><span class="ref-journal">Arch Pharm Res. </span>2010;<span class="ref-vol">33</span>(10):1521–1538.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21052931" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21052931</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml321.r5">Miyake N, Miyamoto S, Jarskog LF. New seratonin/dopamine antagonists for the treatment of schizophrenia: are we making real progress? <span><span class="ref-journal">Clin Schizophr Relat Psychoses. </span>2012;<span class="ref-vol">6</span>(3):122–133.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/23006237" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23006237</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml321.r6">Ginovart N, Kapur S. Role of Dopamine D<sub>2</sub> receptors for antipsychotic activity. <span><span class="ref-journal">Handb Exp Pharmacol. </span>2012;<span class="ref-vol">212</span>:27–52.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/23129327" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23129327</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml321.r7">Le Foll B, Gallo A, Le Strat Y, Gorwood P. Genetics of dopamine receptors and drug addiction: a comprehensive review. <span><span class="ref-journal">Behav Pharmacol. </span>2009;<span class="ref-vol">20</span>(1):1–17.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19179847" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19179847</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml321.r8">Langlois X, Megens A, Lavreysen H, Atack J, Cik M, te Riele P, Peeters L, Wouters R, Vermeire J, Hendrickx H, Macdonald G, De Bruyn M. Pharmacology of JNJ-37822681, a specific and fast-dissociating D2 antagonist for the treatment of schizophrenia. <span><span class="ref-journal">J Pharmacol Exp Ther. </span>2012;<span class="ref-vol">342</span>(1):91–105.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/22490380" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22490380</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml321.r9">Grundt P, Husband SL, Luedtke RR, Taylor M, Newman AH. Analogues of the dopamine D2 receptor antagonist L741,626: Binding, function, and SAR. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2007;<span class="ref-vol">17</span>(3):745–749.</span> [<a href="/pmc/articles/PMC1851912/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1851912</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17095222" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17095222</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml321.r10">Vangveravong S, Taylor M, Xu J, Cui J, Calvin W, Babic S, Luedtke RR, Mach RH. Synthesis and characterization of selective dopamine D2 receptor antagonists. 2. Azaindole, benzofuran, and benzothiophene analogs of L-741,626. <span><span class="ref-journal">Bioorg Med Chem. </span>2010;<span class="ref-vol">18</span>(14):5291–5300.</span> [<a href="/pmc/articles/PMC2946321/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2946321</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20542439" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20542439</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml321.r11">Pitts SMM, SP, Murphy DL, Weisz A, editors. <span class="ref-journal">Recommended practices for the safe handling of MPTP MPTP - A Neurotoxin Producing a Parkinsonian Syndrome.</span> Academic Press; New-York: 1995. </div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml321.r12">Receptor binding profiles was generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract # HHSN-271-2008-025C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA.</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK169449/?report=reader">PubReader</a></li><li><a href="/books/NBK169449/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK169449" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK169449" style="display:none" title="Cite this Page"><div class="bk_tt">Xiao J, Free RB, Barnaeva E, et al. Discovery, optimization, and characterization of a novel series of dopamine D2 versus D3 receptor selective antagonists. 2012 Dec 25 [Updated 2013 Sep 3]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></div></div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ml321.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml321.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml321.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml321.s7" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml321.s11" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml321.s13" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3060638" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=3060638" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3060638" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3060638" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24666157" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Discovery, optimization, and characterization of novel D2 dopamine receptor selective antagonists.</a><span class="source">[J Med Chem. 2014]</span><div class="brieflinkpop offscreen_noflow">Discovery, optimization, and characterization of novel D2 dopamine receptor selective antagonists.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Xiao J, Free RB, Barnaeva E, Conroy JL, Doyle T, Miller B, Bryant-Genevier M, Taylor MK, Hu X, Dulcey AE, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Med Chem. 2014 Apr 24; 57(8):3450-63. 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