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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK280048" /><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="/NBK280048/" /><meta name="ncbi_pagename" content="The discovery and characterization of a centrally penetrant (ML396) and a peripherally restricted (ML397) pan-Group III mGlu positive allosteric modulators - 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>The discovery and characterization of a centrally penetrant (ML396) and a peripherally restricted (ML397) pan-Group III mGlu positive allosteric modulators - 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="The discovery and characterization of a centrally penetrant (ML396) and a peripherally restricted (ML397) pan-Group III mGlu positive allosteric modulators" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2015/01/16" /><meta name="citation_author" content="Nidhi Jalan-Sakrikar" /><meta name="citation_author" content="Julie Roper-Field" /><meta name="citation_author" content="Rebecca Klar" /><meta name="citation_author" content="Margrith Mattman" /><meta name="citation_author" content="Adam G. Walker" /><meta name="citation_author" content="Rocio Zamorano" /><meta name="citation_author" content="Zixiu Xiang" /><meta name="citation_author" content="C. Frank Byers" /><meta name="citation_author" content="Anna L. Blobaum" /><meta name="citation_author" content="Darren Engers" /><meta name="citation_author" content="C. David Weaver" /><meta name="citation_author" content="Emily Days" /><meta name="citation_author" content="Thomas J. Utley" /><meta name="citation_author" content="Bruce Melancon" /><meta name="citation_author" content="J. Scott Daniels" /><meta name="citation_author" content="Michael R. Wood" /><meta name="citation_author" content="Craig W. Lindsley" /><meta name="citation_author" content="P. Jeffrey Conn" /><meta name="citation_author" content="Corey R. Hopkins" /><meta name="citation_author" content="Colleen M. Niswender" /><meta name="citation_pmid" content="25834902" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK280048/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="The discovery and characterization of a centrally penetrant (ML396) and a peripherally restricted (ML397) pan-Group III mGlu positive allosteric modulators" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Nidhi Jalan-Sakrikar" /><meta name="DC.Contributor" content="Julie Roper-Field" /><meta name="DC.Contributor" content="Rebecca Klar" /><meta name="DC.Contributor" content="Margrith Mattman" /><meta name="DC.Contributor" content="Adam G. Walker" /><meta name="DC.Contributor" content="Rocio Zamorano" /><meta name="DC.Contributor" content="Zixiu Xiang" /><meta name="DC.Contributor" content="C. Frank Byers" /><meta name="DC.Contributor" content="Anna L. Blobaum" /><meta name="DC.Contributor" content="Darren Engers" /><meta name="DC.Contributor" content="C. David Weaver" /><meta name="DC.Contributor" content="Emily Days" /><meta name="DC.Contributor" content="Thomas J. Utley" /><meta name="DC.Contributor" content="Bruce Melancon" /><meta name="DC.Contributor" content="J. Scott Daniels" /><meta name="DC.Contributor" content="Michael R. Wood" /><meta name="DC.Contributor" content="Craig W. Lindsley" /><meta name="DC.Contributor" content="P. Jeffrey Conn" /><meta name="DC.Contributor" content="Corey R. Hopkins" /><meta name="DC.Contributor" content="Colleen M. Niswender" /><meta name="DC.Date" content="2015/01/16" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK280048/" /><meta name="description" content="Herein we report the discovery and characterization of two, structurally distinct, positive allosteric modulators (PAM) of the Group III metabotropic glutamate receptors (mGlus). The first probe molecule (ML397) was discovered after a high-throughput screening campaign of the original Molecular Libraries Small Molecule Repository (MLSMR) library. ML397 is equipotent as a PAM against mGlu7, mGlu8 and mGlu4 and is selective against Group I and Group II mGlu receptors as well as against the EuroFins Lead Profiling Screen. This probe is unique in that it is peripherally restricted, which allows for distinction from other PAMs or agonists of the Group III mGlus. The second probe molecule (ML396) was discovered after a screen of Vanderbilt's Center for Neuroscience Drug Discovery mGlu4-biased library and is related to a previous ML probe for mGlu4, ML128. ML396 is more potent (∼100 nM against mGlu7, mGlu8 and mGlu4) and displays good brain penetration. By virtue of possessing activity at all group III receptors but not groups I and II, we will be able to use these tools to identify common allosteric binding sites among these receptors which are important targets for drug discovery. Additionally, by capitalizing on a synapse in the hippocampus that exclusively expresses mGlu7, we have validated that each of these probes potentiate agonist responses in a native tissue preparation." /><meta name="og:title" content="The discovery and characterization of a centrally penetrant (ML396) and a peripherally restricted (ML397) pan-Group III mGlu positive allosteric modulators" /><meta name="og:type" content="book" /><meta name="og:description" content="Herein we report the discovery and characterization of two, structurally distinct, positive allosteric modulators (PAM) of the Group III metabotropic glutamate receptors (mGlus). The first probe molecule (ML397) was discovered after a high-throughput screening campaign of the original Molecular Libraries Small Molecule Repository (MLSMR) library. ML397 is equipotent as a PAM against mGlu7, mGlu8 and mGlu4 and is selective against Group I and Group II mGlu receptors as well as against the EuroFins Lead Profiling Screen. This probe is unique in that it is peripherally restricted, which allows for distinction from other PAMs or agonists of the Group III mGlus. The second probe molecule (ML396) was discovered after a screen of Vanderbilt's Center for Neuroscience Drug Discovery mGlu4-biased library and is related to a previous ML probe for mGlu4, ML128. ML396 is more potent (∼100 nM against mGlu7, mGlu8 and mGlu4) and displays good brain penetration. By virtue of possessing activity at all group III receptors but not groups I and II, we will be able to use these tools to identify common allosteric binding sites among these receptors which are important targets for drug discovery. Additionally, by capitalizing on a synapse in the hippocampus that exclusively expresses mGlu7, we have validated that each of these probes potentiate agonist responses in a native tissue preparation." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK280048/" /><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/ml397/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK280048/" /><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="/core/mathjax/2.7.9/MathJax.js?config=/corehtml/pmc/js/mathjax-config-classic.3.4.js"></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="#__NBK280048_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK280048_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/ml395/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml398/" 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="_NBK280048_"><span class="title" itemprop="name">The discovery and characterization of a centrally penetrant (ML396) and a peripherally restricted (ML397) pan-Group III mGlu positive allosteric modulators</span></h1><p class="contrib-group"><span itemprop="author">Nidhi Jalan-Sakrikar</span>, <span itemprop="author">Julie Roper-Field</span>, <span itemprop="author">Rebecca Klar</span>, <span itemprop="author">Margrith Mattman</span>, <span itemprop="author">Adam G. Walker</span>, <span itemprop="author">Rocio Zamorano</span>, <span itemprop="author">Zixiu Xiang</span>, <span itemprop="author">C. Frank Byers</span>, <span itemprop="author">Anna L. Blobaum</span>, <span itemprop="author">Darren Engers</span>, <span itemprop="author">C. David Weaver</span>, <span itemprop="author">Emily Days</span>, <span itemprop="author">Thomas J. Utley</span>, <span itemprop="author">Bruce Melancon</span>, <span itemprop="author">J. Scott Daniels</span>, <span itemprop="author">Michael R. Wood</span>, <span itemprop="author">Craig W. Lindsley</span>, <span itemprop="author">P. Jeffrey Conn</span>, <span itemprop="author">Corey R. Hopkins</span>, and <span itemprop="author">Colleen M. Niswender</span>.</p><a data-jig="ncbitoggler" href="#__NBK280048_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK280048_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Nidhi Jalan-Sakrikar</span>, <span itemprop="author">Julie Roper-Field</span>, <span itemprop="author">Rebecca Klar</span>, <span itemprop="author">Margrith Mattman</span>, <span itemprop="author">Adam G. Walker</span>, <span itemprop="author">Rocio Zamorano</span>, <span itemprop="author">Zixiu Xiang</span>, <span itemprop="author">C. Frank Byers</span>, <span itemprop="author">Anna L. Blobaum</span>, <span itemprop="author">Darren Engers</span>, <span itemprop="author">C. David Weaver</span>, <span itemprop="author">Emily Days</span>, <span itemprop="author">Thomas J. Utley</span>, <span itemprop="author">Bruce Melancon</span>, <span itemprop="author">J. Scott Daniels</span>, <span itemprop="author">Michael R. Wood</span>, <span itemprop="author">Craig W. Lindsley</span>, <span itemprop="author">P. Jeffrey Conn</span>, <span itemprop="author">Corey R. Hopkins</span>, and <span itemprop="author">Colleen M. Niswender</span>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup> Vanderbilt Specialized Chemistry Center for Accelerated Probe Development</div></div><p class="small">Received: <span itemprop="datePublished">April 15, 2014</span>; Last Update: <span itemprop="dateModified">January 16, 2015</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>Herein we report the discovery and characterization of two, structurally distinct, positive allosteric modulators (PAM) of the Group III metabotropic glutamate receptors (mGlus). The first probe molecule (<a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a>) was discovered after a high-throughput screening campaign of the original Molecular Libraries Small Molecule Repository (MLSMR) library. <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> is equipotent as a PAM against mGlu<sub>7</sub>, mGlu<sub>8</sub> and mGlu<sub>4</sub> and is selective against Group I and Group II mGlu receptors as well as against the EuroFins Lead Profiling Screen. This probe is unique in that it is peripherally restricted, which allows for distinction from other PAMs or agonists of the Group III mGlus. The second probe molecule (<a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a>) was discovered after a screen of Vanderbilt's Center for Neuroscience Drug Discovery mGlu<sub>4</sub>-biased library and is related to a previous ML probe for mGlu<sub>4</sub>, <a href="/pcsubstance/?term=ML128[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML128</a>. <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> is more potent (∼100 nM against mGlu<sub>7</sub>, mGlu<sub>8</sub> and mGlu<sub>4</sub>) and displays good brain penetration. By virtue of possessing activity at all group III receptors but not groups I and II, we will be able to use these tools to identify common allosteric binding sites among these receptors which are important targets for drug discovery. Additionally, by capitalizing on a synapse in the hippocampus that exclusively expresses mGlu<sub>7</sub>, we have validated that each of these probes potentiate agonist responses in a native tissue preparation.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R03 NS053536-01</p><p><b>Screening Center Name & PI:</b> Vanderbilt Screening Center for GPCRs, Ion Channels, and Transporters/Vanderbilt Center for Neuroscience Drug Discovery, David Weaver</p><p><b>Chemistry Center Name & PI:</b> Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley</p><p><b>Assay Submitter & Institution:</b> Colleen Niswender, Vanderbilt University</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488969" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">488969</a></p><div id="ml397.s1"><h2 id="_ml397_s1_">Probe Structures & Characteristics</h2><div id="ml397.f1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml397f1&p=BOOKS&id=280048_ml397f1.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/NBK280048/bin/ml397f1.jpg" alt="Image ml397f1" class="tileshop" title="Click on image to zoom" /></a></div></div><div id="ml397.t1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280048/table/ml397.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml397.t1_lrgtbl__"><table><thead><tr><th id="hd_h_ml397.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml397.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml397.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EC<sub>50</sub>/(nM) [SID, AID]</th><th id="hd_h_ml397.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml397.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml397.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml397.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml397.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 73058451/<a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a></td><td headers="hd_h_ml397.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">mGlu<sub>8</sub>, mGlu<sub>7</sub>, mGlu<sub>4</sub></td><td headers="hd_h_ml397.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">110 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/173028078" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 173028078</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743462" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743462</a>], 146 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743384" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743384</a>], 130 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743423" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743423</a>]</td><td headers="hd_h_ml397.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">mGlu<sub>1</sub>, mGlu<sub>2</sub>, mGlu<sub>3</sub>, mGlu<sub>5</sub>, mGlu<sub>6</sub></td><td headers="hd_h_ml397.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/1730280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 1730280</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743429" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743429</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743433" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743433</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743431" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743431</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743419" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743419</a>]; 3.5 FS [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/734421" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 734421</a>]</td><td headers="hd_h_ml397.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>30</td><td headers="hd_h_ml397.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2.3 ± 0.4 mGlu<sub>8</sub> Fold-Shift [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/1730280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 1730280</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743447" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743447</a>]; 2.2 ± 0.2 mGlu<sub>7</sub> Fold-Shift [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/1730280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 1730280</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743386" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743386</a>]; 3.1 ± 0.4 mGlu<sub>4</sub> Fold-Shift [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/1730280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 1730280</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743466" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743466</a>]</td></tr><tr><td headers="hd_h_ml397.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 4043841/<a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a></td><td headers="hd_h_ml397.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">mGlu<sub>8</sub>, mGlu<sub>7</sub>, mGlu<sub>4</sub></td><td headers="hd_h_ml397.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3.43 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99361191" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99361191</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743423" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743423</a>], 1.5 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743384" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743384</a>], 0.93 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743423" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743423</a>]</td><td headers="hd_h_ml397.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">mGlu<sub>1</sub>, mGlu<sub>2</sub>, mGlu<sub>3</sub>, mGlu<sub>5</sub>, mGlu<sub>6</sub></td><td headers="hd_h_ml397.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/9936119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 9936119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743429" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743429</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743433" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743433</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743431" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743431</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743419" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743419</a>]; 2.6 FS [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/734421" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 734421</a>]</td><td headers="hd_h_ml397.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>30</td><td headers="hd_h_ml397.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">7.8 ± 1.1 mGlu<sub>8</sub> Fold-Shift [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/9936119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 9936119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743447" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743447</a>]; 4.9 ± 0.3 mGlu<sub>7</sub> Fold-Shift [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/9936119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 9936119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743386" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743386</a>]; 1.8 ± 0.1 mGlu<sub>4</sub> Fold-Shift [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/9936119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 9936119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743466" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743466</a>]</td></tr></tbody></table></div></div></div><div id="ml397.s2"><h2 id="_ml397_s2_">Recommendations for scientific use of the probe</h2><p><a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> (CID 73058451) and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> (CID 4043841) are both positive allosteric modulators of the group III mGlus. It is important to note that no PAMs of mGlu<sub>7</sub> have yet been reported and, for <i>in vitro</i> based structure function studies, both probes represent invaluable tools as well as important positive controls for screens of new modulators of mGlu<sub>7</sub> and mGlu<sub>8</sub> that are now ongoing at Vanderbilt. <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> is particularly interesting, as it possesses approximately 100 nanomolar activity at each of the group III mGlus but also exerts limited cooperativity with orthosteric agonist; compounds with this profile are predicted to have high affinity for their targets, suggesting a path forward for eventual radioligand development. Additionally, we have performed extensive studies showing that mGlu<sub>7</sub> is the only presynaptic metabotropic glutamate receptor expressed at the Shaffer Collateral-CA1 synapse in the hippocampus. Coupled with a more selective mGlu<sub>7</sub> orthosteric agonist, we have shown that both of these PAMs significant potentiate agonist-induced inhibition of synaptic transmission, further validating a unique role for mGlu<sub>7</sub> at this synapse. As hippocampal synaptic plasticity underlies certain forms of learning and memory, these new tools are allowing us to explore the potential of mGlu<sub>7</sub> potentiation in affecting synaptic plasticity, providing validation for mGlu<sub>7</sub> PAMs in improving learning and memory. Portions of this work has been published in Jalan-Sakrikar et al., ACS Chem Neurosci, 2014 [<a class="bk_pop" href="#ml397.r6">6</a>].</p></div><div id="ml397.s3"><h2 id="_ml397_s3_">2. Materials and Methods</h2><div id="ml397.s4"><h3>In vitro pharmacology</h3><p><b>Cell culture.</b> Human mGlu<sub>4</sub>/G<sub>qi5</sub>/CHO cells were grown in 90% Dulbecco's Modified Eagle Media (DMEM), 10% dialyzed fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 20 μg/ml proline, 2 mM glutamine, 400 μg/ml G418 sufate (Mediatech, Inc., Herndon, VA) and 5 nM methotrexate (Calbiochem, EMD Chemicals, Gibbstown, NJ). Rat mGlu<sub>7</sub>/Gα15/HEK and rat mGlu<sub>8</sub>/Gα15/HEK cells were grown in in 90% Dulbecco's Modified Eagle Media (DMEM), 10% FBS, 100 units/ml penicillin/streptomycin, 20 mM HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine, 1× nonessential amino acids, 700 μg/mL G418, and 0.6 μg/mL puromycin. Rat mGlu<sub>4</sub>/HEK/GIRK cells, as well as HEK/GIRK lines expressing rat mGlu<sub>2</sub>, mGlu<sub>3</sub>, mGlu<sub>7</sub>, mGlu<sub>8</sub>, and human mGlu<sub>6</sub> were cultured in 45% DMEM, 45% F-12, 10% FBS, 20 mM HEPES, 2 mM l-glutamine, 100 units/ml penicillin/streptomycin, 1× nonessential amino acids, 1 mM sodium pyruvate, 700 μg/mL G418, and 0.6 μg/mL puromycin. Rat mGlu<sub>1</sub> and mGlu<sub>5</sub> HEK cell lines were grown in DMEM, 10% FBS, 20 mM HEPES, 2 mM l-glutamine, 1× nonessential amino acids, 1 mM sodium pyruvate, 500 μg/mL G418. All cell culture reagents were from Invitrogen (Carlsbad, CA) unless otherwise noted.</p></div><div id="ml397.s5"><h3>GIRK-mediated thallium flux assays</h3><p>Human embryonic kidney cell lines co-expressing rat mGlu<sub>4</sub>, rat mGlu<sub>7</sub>, or rat mGlu<sub>8</sub> with GIRK 1/2 potassium channel subunits were used for these experiments, and potency studies were performed as described in Niswender et al., 2008, Molecular Pharmacology and Jones et al., 2012, JPET. Cells were plated into 384 well, black-walled, clear-bottom poly-D-lysine coated plates (Greiner) at a density of 15,000 cells/20 μl/well in DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 100 units/ml penicillin/streptomycin (Assay Media). Plated cells were incubated overnight at 37°C in the presence of 5% CO2. The following day, the medium was removed from the cells and 20 μl/well of 330 nM Fluo Zn2 (Invitrogen; prepared as a stock in DMSO and mixed in a 1:1 ratio with pluronic acid F-127) in Assay Buffer (Hanks Balanced Salt Solution (Invitrogen) containing 20 mM HEPES pH 7.3) was added to the plated cells. Cells were incubated for one hour at room temperature and the dye was replaced with the 20 μl of Assay Buffer. Glutamate was diluted in Thallium Buffer (125 mM sodium bicarbonate (added fresh the morning of the experiment), 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM thallium sulfate, 10 mM HEPES, pH 7.3) at 5× the final concentration to be assayed. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10 point concentration response curves in DMSO, transferred to daughter plates using the Echo, and diluted in Assay Buffer to a 2× final concentration. Cell plates and compound plates were loaded onto a Hamamatsu FDSS 6000 or 7000 kinetic imaging plate reader. Baseline readings were taken (10 images at 1 Hz, excitation, 470±20 nm emission, 540±30 nm) and test compounds were added in a 20 μl volume and incubated for 2.5 minutes prior to the addition of 10 μl of Thallium Buffer ± agonist. After the addition of agonist, data were collected for an additional 2 min. Data were analyzed using Microsoft Excel. Raw data were opened in Excel and each data point in a given trace was divided by the first data point from that trace (static ratio). For experiments in which PAMs were added, data were again normalized by dividing each point by the fluorescence value immediately before the agonist addition to correct for any subtle differences in the baseline traces after the compound incubation period. The slope of the fluorescence increase beginning five seconds after thallium/agonist addition and ending fifteen seconds after thallium/agonist addition was calculated. Curves were fitted using a four point logistical equation using GraphPad Prism (La Jolla, CA). For fold shift experiments, a 10 μM concentration of compound was applied two minutes prior to a full agonist concentration-response (either glutamate for mGlu<sub>4</sub> and mGlu<sub>8</sub> or L-AP4 for mGlu<sub>7</sub>). The leftward “fold shift” of the concentration-response was calculated as the potency in the absence of compound divided by the potency in the presence of a 10 μM concentration of PAM.</p></div><div id="ml397.s6"><h3>Calcium Assays</h3><p>Human mGlu<sub>4</sub>/G<sub>qi5</sub>/CHO cells (30,000 cells/20 μl/well), rat mGlu<sub>7</sub>/G<sub>a15</sub>/HEK cells (15,000 cells/20 μl/well), and rat mGlu<sub>8</sub>/G<sub>a15</sub>/HEK cells (15,000 cells/20 μl/well) were plated in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in DMEM containing 10% dialyzed FBS, 20 mM HEPES, 100 units/ml penicillin/streptomycin, and 1 mM sodium pyruvate (Plating Medium). The cells were grown overnight at 37°C in the presence of 5% CO<sub>2</sub>. The next day, the medium was removed and replaced with 20 μL of 1 μM Fluo-4, AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) for 45 minutes at 37°C. Dye was removed and replaced with 20 μL of Assay Buffer. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10 point concentration response curves in DMSO, transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA) Echo, and diluted in Assay Buffer to a 2× final concentration. Ca<sup>2+</sup> flux was measured using the Functional Drug Screening System 6000 (FDSS6000, Hamamatsu, Japan). After establishment of a fluorescence baseline for 4 seconds (4 images at 1 Hz; excitation, 470 ± 20 nm; emission, 540 ± 30 nm), 20 μl of test compounds were added to the cells, and the response was measured. 142 seconds later, 10 μl (5×) of an EC<sub>20</sub> concentration of glutamate was added to the cells, and the response of the cells was measured; after an additional 120 seconds, 12 μl (5×) of an EC<sub>80</sub> concentration of agonist was added and readings taken for an additional 40 seconds. Calcium fluorescence was recorded as fold over basal fluorescence and raw data were normalized to the maximal response to glutamate. Potency (EC<sub>50</sub>) and maximum response (% Glu Max) for compounds were determined using a four parameter logistical equation in GraphPad Prism (La Jolla, CA). For efficacy experiments, a constant amount of compound was applied prior to the addition of a full glutamate concentration-response curve and the left shift of the EC<sub>50</sub> of the curves was calculated as “fold shift”.</p></div><div id="ml397.s7"><h3>Selectivity testing</h3><p>Selectivity testing across the mGlus was performed as described in Niswender et al. 2008 and Jones et al., 2012 [<a class="bk_pop" href="#ml397.r1">1</a>, <a class="bk_pop" href="#ml397.r2">2</a>].</p></div><div id="ml397.s8"><h3>DMPK Methods</h3><p><u>In vitro:</u> The metabolism of <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> was investigated in human, rat and mouse hepatic microsomes (BD Biosciences, Billerica, MA) using substrate depletion methodology (% test article remaining). A potassium phosphate-buffered reaction mixture (0.1 M, pH 7.4) of test article (1 μM) and microsomes (0.5 mg/mL) was pre-incubated (5 min) at 37°C prior to the addition of NADPH (1 mM). The incubations, performed in 96-well plates, were continued at 37 °C under ambient oxygenation and aliquots (80 μL) were removed at selected time intervals (0, 3, 7, 15, 25 and 45 min). Protein was precipitated by the addition of chilled acetonitrile (160 μL), containing glyburide as an internal standard (50 ng/mL), and centrifuged at 3000 rpm (4°C) for 10 min. Resulting supernatants were transferred to new 96-well plates in preparation for LC/MS/MS analysis. The in vitro half-life (<i>t</i><sub>1/2</sub>, min, <a href="#ml397.eq1">Eq. 1</a>), intrinsic clearance (CL<sub>int</sub>, mL/min/kg, <a href="#ml397.eq2">Eq. 2</a>) and subsequent predicted hepatic clearance (CL<sub>hep</sub>, mL/min/kg, <a href="#ml397.eq3">Eq. 3</a>) were determined employing the following equations:
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<div class="pmc_disp_formula whole_rhythm clearfix" id="ml397.eq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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<i>t</i><sub>1/2</sub> = Ln(2) / <i>k</i> ; where <i>k</i> represents the slope from linear regression analysis (% test article remaining)</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap">1</span></div></div></div>
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<div class="pmc_disp_formula whole_rhythm clearfix" id="ml397.eq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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CL<sub>int</sub> = (0.693 / <i>t</i><sub>1/2</sub>) (rxn volume / mg of microsomes) (45 mg microsomes / gram of liver) (20<i><sup>a</sup></i> gm of liver / kg body weight); <i><sup>a</sup></i>scale-up factors of 20 (human) and 45 (rat)</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap">2</span></div></div></div>
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<div class="pmc_disp_formula whole_rhythm clearfix" id="ml397.eq3"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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<math id="ml397.m1" display="block"><mrow><mtext mathvariant="italic">CLhep</mtext><mo>=</mo><mfrac><mrow><mi>Q</mi><mo>·</mo><mtext mathvariant="italic">CL</mtext><mtext>int</mtext></mrow><mrow><mi>Q</mi><mo>+</mo><mtext mathvariant="italic">CL</mtext><mtext>int</mtext></mrow></mfrac></mrow></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap">3</span></div></div></div></p><p><b>Plasma Protein Binding.</b> Protein binding of <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> was determined in human, rat and mouse plasma via equilibrium dialysis employing Single-Use RED Plates with inserts (ThermoFisher Scientific, Rochester, NY). Briefly plasma (220 μL) was added to the 96 well plate containing test article (5 μL) and mixed thoroughly. Subsequently, 200 μL of the plasma-test article mixture was transferred to the <i>cis</i> chamber (red) of the RED plate, with an accompanying 350 μL of phosphate buffer (25 mM, pH 7.4) in the <i>trans</i> chamber. The RED plate was sealed and incubated 4 h at 37 °C with shaking. At completion, 50 μL aliquots from each chamber were diluted 1:1 (50 μL) with either plasma (<i>cis</i>) or buffer (<i>trans</i>) and transferred to a new 96 well plate, at which time ice-cold acetonitrile (2 volumes) was added to extract the matrices. The plate was centrifuged (3000 rpm, 10 min) and supernatants transferred to a new 96 well plate. The sealed plate was stored at -20 °C until LC/MS/MS analysis.</p><p><b>Liquid Chromatography/Mass Spectrometry Analysis</b>. <i>In vitro experiments.</i>
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<a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> were analyzed via electrospray ionization (ESI) on an AB Sciex API-4000 (Foster City, CA) triple-quadrupole instrument that was coupled with Shimadzu LC-10AD pumps (Columbia, MD) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Analytes were separated by gradient elution using a Fortis C18 2.1 × 50 mm, 3.5 μm column (Fortis Technologies Ltd, Cheshire, UK) thermostated at 40 °C. HPLC mobile phase A was 0.1% NH<sub>4</sub>OH (pH unadjusted), mobile phase B was acetonitrile. The gradient started at 30% B after a 0.2 min hold and was linearly increased to 90% B over 0.8 min; held at 90% B for 0.5 min and returned to 30% B in 0.1 min followed by a re-equilibration (0.9 min). The total run time was 2.5 min and the HPLC flow rate was 0.5 mL/min. The source temperature was set at 500°C and mass spectral analyses were performed using multiple reaction monitoring (MRM) utilizing a Turbo-Ionspray® source in positive ionization mode (5.0 kV spray voltage). LC/MS/MS analysis was performed employing a TSQ Quantum<sup>ULTRA</sup> that was coupled to a ThermoSurveyor LC system (Thermoelectron Corp., San Jose, CA) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Chromatographic separation of analytes was achieved with an Acquity BEH C18 2.1 × 50 mm, 1.7 μm column (Waters, Taunton, MA).</p></div><div id="ml397.s9"><h3>2.1. Assays</h3><dl class="temp-labeled-list"><dt>2.1.1.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504480" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504480</a>: Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Summary (mGlu8_Summary)-Already deposited in PubChem as part of <a href="/pcsubstance/?term=ML297[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML297</a> report</p></dd><dt>2.1.2.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488969" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488969</a>: Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Primary Screening (mGlu8_Primary)-Already deposited in PubChem as part of <a href="/pcsubstance/?term=ML297[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML297</a> report</p></dd><dt>2.1.3.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623911" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623911</a>: hGIRK1/2_mGlu8_WaveformData (GIRK1/2_Confirmatory)-Already deposited in PubChem as part of <a href="/pcsubstance/?term=ML297[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML297</a> report</p></dd><dt>2.1.4.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623869" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623869</a>: rmGlu8_Gqi9_Counterscreen-Already deposited in PubChem as part of <a href="/pcsubstance/?term=ML297[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML297</a> report</p></dd><dt>2.1.5.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623868" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623868</a>: mGlu8_nonGIRK_Counterscreen- Already deposited in PubChem as part of <a href="/pcsubstance/?term=ML297[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML297</a> report</p></dd><dt>2.1.6.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743462" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743462</a>: Potency at rat mGlu<sub>8/</sub>GIRK</p></dd><dt>2.1.7.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743425" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743425</a>: Potency at rat mGlu<sub>8/</sub>calcium</p></dd><dt>2.1.8.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743384" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743384</a>: Potency at rat mGlu<sub>7/</sub>calcium</p></dd><dt>2.1.9.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743423" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743423</a>: Potency at human mGlu<sub>4/</sub>calcium</p></dd><dt>2.1.10.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743429" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743429</a>: Selectivity at rat mGlu<sub>1</sub>/calcium</p></dd><dt>2.1.11.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743433" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743433</a>: Selectivity at rat mGlu<sub>2</sub>/GIRK</p></dd><dt>2.1.12.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743431" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743431</a>: Selectivity at rat mGlu<sub>3</sub>/GIRK</p></dd><dt>2.1.13.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743419" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743419</a>: Selectivity at rat mGlu<sub>5</sub>/Calcium</p></dd><dt>2.1.13.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743421" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743421</a>: Selectivity at human mGlu<sub>6</sub>/GIRK</p></dd><dt>2.1.14.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743427" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743427</a>: Fold shift at rat mGlu<sub>8</sub>/calcium</p></dd><dt>2.1.15.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743385" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743385</a>: Fold shift at rat mGlu<sub>7</sub>/calcium</p></dd><dt>2.1.16.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743393" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743393</a>: Fold shift at human mGlu<sub>4</sub>/calcium</p></dd><dt>2.1.17.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743447" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743447</a>: Fold shift using selectivity assay protocol at rat mGlu<sub>8</sub>/GIRK</p></dd><dt>2.1.18.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743386" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743386</a>: Fold shift using selectivity assay protocol at rat mGlu<sub>7</sub>/GIRK</p></dd><dt>2.1.19.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743466" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743466</a>: Fold shift using selectivity assay protocol at rat mGlu<sub>4</sub>/GIRK</p></dd></dl></div><div id="ml397.s10"><h3>2.2. Probe Chemical Characterization</h3><div id="ml397.f2" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml397f2&p=BOOKS&id=280048_ml397f2.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/NBK280048/bin/ml397f2.jpg" alt="Image ml397f2" class="tileshop" title="Click on image to zoom" /></a></div></div><p>Probe compound <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> (CID 4043841, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/99361191" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99361191</a>, VU0155094-5) is a commercially available compound and had the following characterization. <b>Methyl 4-(3-(2-((4-acetamidophenyl)thio)acetyl)-2,5-dimethyl-1H-pyrrol-1-yl)benzoate,</b>
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<a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a>. LCMS: R<sub>T</sub> = 2.481 min, >99% @ 254 nm, >99% @ 215 nm; <i>m/z</i> [M + H]<sup>+</sup> = 437. <sup>1</sup>H NMR (400 MHz, CDCl3, δ ppm): 8.18 (dd; <i>J</i> = 6.8, 2 Hz, 2 H), 7.42 (d; <i>J</i> = 8.8 Hz, 2 H), 7.38 (d; <i>J</i> = 8.8 Hz; 2 H), 7.34-7.29 (m; 6 H), 6.33 (s; 1 H), 4.03 (s; 2H), 3.97 (s, 3 H), 2.30 (s; 3 H), 2.16 (s; 3 H), 1.99 (s; 3 H). HRMS calculated for C<sub>24</sub>H<sub>24</sub>N<sub>2</sub>O<sub>4</sub>S [M + H]<sup>+</sup>
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<i>m/z</i>: 437.1535, measured: 437.1539.</p><p>Probe compound <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> (CID 73058451, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/173028078" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 173028078</a>) was prepared according to the above scheme and had the following characterization. <b>N-(3-chloro-4-((5-chloropyridin-2-yl) methyl)phenyl)picolinamide,</b>
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<a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a>. LCMS:</p><p><b>Solubility.</b> Solubility for <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> in PBS was determined to be <5 μM, which is >35-fold higher than the cellular EC<sub>50</sub> for PAM activity.</p><p>Solubility for <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> in PBS was determined to be <6.4 μM, which is >5-fold higher than the cellular EC<sub>50</sub> for PAM activity.</p><p><b>Stability.</b> Stability was determined for <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%). After 48 hours, ∼39% of the initial concentration of <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> remained in solution. The loss is most likely due to limited solubility in buffer. However, as evidenced by the in vivo PK study (vide infra), the compound is present in the plasma and brain, again suggesting solubility issues with the solution for this study.</p><div id="ml397.t2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280048/table/ml397.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml397.t2_lrgtbl__"><table><thead><tr><th id="hd_h_ml397.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_h_ml397.t2_1_1_1_2" colspan="6" rowspan="1" style="text-align:center;vertical-align:middle;">Percent Remaining (%)</th></tr><tr><th headers="hd_h_ml397.t2_1_1_1_1" id="hd_h_ml397.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th headers="hd_h_ml397.t2_1_1_1_2" id="hd_h_ml397.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0 min</th><th headers="hd_h_ml397.t2_1_1_1_2" id="hd_h_ml397.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">24 min</th><th headers="hd_h_ml397.t2_1_1_1_2" id="hd_h_ml397.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44 min</th><th headers="hd_h_ml397.t2_1_1_1_2" id="hd_h_ml397.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">102 min</th><th headers="hd_h_ml397.t2_1_1_1_2" id="hd_h_ml397.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">24 Hour</th><th headers="hd_h_ml397.t2_1_1_1_2" id="hd_h_ml397.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">48 Hour</th></tr></thead><tbody><tr><td headers="hd_h_ml397.t2_1_1_1_1 hd_h_ml397.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a>, CID 73058507</td><td headers="hd_h_ml397.t2_1_1_1_2 hd_h_ml397.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100 ± 0.0</td><td headers="hd_h_ml397.t2_1_1_1_2 hd_h_ml397.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">95.0 ± 1.3</td><td headers="hd_h_ml397.t2_1_1_1_2 hd_h_ml397.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">90.6 ± 1.6</td><td headers="hd_h_ml397.t2_1_1_1_2 hd_h_ml397.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">84.1 ± 1.1</td><td headers="hd_h_ml397.t2_1_1_1_2 hd_h_ml397.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">48.1 ± 1.8</td><td headers="hd_h_ml397.t2_1_1_1_2 hd_h_ml397.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">38.7 ± 0.4</td></tr></tbody></table></div></div><p>Stability was determined for <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%). After 48 hours, ∼94% of the initial concentration of <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> remained in solution.</p><div id="ml397.t3" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280048/table/ml397.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml397.t3_lrgtbl__"><table><thead><tr><th id="hd_h_ml397.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_h_ml397.t3_1_1_1_2" colspan="6" rowspan="1" style="text-align:center;vertical-align:middle;">Percent Remaining (%)</th></tr><tr><th headers="hd_h_ml397.t3_1_1_1_1" id="hd_h_ml397.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th headers="hd_h_ml397.t3_1_1_1_2" id="hd_h_ml397.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0 min</th><th headers="hd_h_ml397.t3_1_1_1_2" id="hd_h_ml397.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">22 min</th><th headers="hd_h_ml397.t3_1_1_1_2" id="hd_h_ml397.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">42 min</th><th headers="hd_h_ml397.t3_1_1_1_2" id="hd_h_ml397.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">104 min</th><th headers="hd_h_ml397.t3_1_1_1_2" id="hd_h_ml397.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">24 Hour</th><th headers="hd_h_ml397.t3_1_1_1_2" id="hd_h_ml397.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">48 Hour</th></tr></thead><tbody><tr><td headers="hd_h_ml397.t3_1_1_1_1 hd_h_ml397.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a>, CID 73058451</td><td headers="hd_h_ml397.t3_1_1_1_2 hd_h_ml397.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100 ± 0.0</td><td headers="hd_h_ml397.t3_1_1_1_2 hd_h_ml397.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">102.1 ± 0.6</td><td headers="hd_h_ml397.t3_1_1_1_2 hd_h_ml397.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100.3 ± 0.6</td><td headers="hd_h_ml397.t3_1_1_1_2 hd_h_ml397.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">94.5 ± 0.8</td><td headers="hd_h_ml397.t3_1_1_1_2 hd_h_ml397.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">94.1 ± 0.7</td><td headers="hd_h_ml397.t3_1_1_1_2 hd_h_ml397.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">93.8 ± 0.2</td></tr></tbody></table></div></div><p><b>Compounds added to the SMR collection (MLS#s):</b> MLS005886349 (<a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a>, CID 73058507, 26.6 mg); MLS005886350 (CID 73058493, 6.2 mg); MLS005886351 (CID 73058438, 5.8 mg); MLS005886352 (CID 73058449, 6.9 mg); MLS005886353 (CID 73058466, 5.6 mg); MLS005886354 (CID 73058446, 6.2 mg)</p><p>MLS005886355 (<a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a>, CID 73058451, 25.0 mg); MLS005886356 (CID 73058454, 30.0 mg); MLS005886357 (CID 73058498, 27.7 mg); MLS005886358 (CID 73058456, 28.6 mg; MLS005886359 (CID 73058443, 29.7 mg); MLS005886360 (CID 73058492, 32.1 mg)</p></div><div id="ml397.s11"><h3>2.3. Probe Preparation</h3><p>Probe compound <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> was purchased from a commercial vendor for this report.</p><div id="ml397.f3" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml397f3&p=BOOKS&id=280048_ml397f3.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/NBK280048/bin/ml397f3.jpg" alt="Image ml397f3" class="tileshop" title="Click on image to zoom" /></a></div></div><p>Probe compound <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> (CID 73058451, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/173028078" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 173028078</a>) was prepared according to the above scheme and had the following characterization. <b>N-(3-chloro-4-((5-chloropyridin-2-yl) methyl)phenyl)picolinamide,</b>
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<a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a></p><p><b>3-chloro-4-((5-chloropyridin-2-yl)oxy)aniline (3).</b> To a microwave vial (20 mL) was added 4-amino-2-chlorophenol (1) (1.0 g, 6.97 mmol, 1.0 eq), 5-chloro-2-fluoropyridine (2) (0.7 mL, 6.97 mmol, 1.0 eq), K<sub>2</sub>CO<sub>3</sub> (1.44 g, 10.45 mmol, 1.5 eq) and DMF (10 mL). The rxn mixture was subjected to microwave irradiation at 150°C for 45 min. The rxn was added to EtOAc:water (1:1, 1000 mL) and the organic layer was separated. The water layer was re-extracted with EtOAc (3 × 50 ml) and the combined organic layers were washed with water (2 × 50 mL), brine (50 mL), dried (MgSO4), filtered and concentrated. The residue was purified on a Biotage Isolera One (Zip 80 column, 50-100% EtOAc:hexanes) to afford 3-chloro-4-((5-chloropyridin-2-yl)oxy)aniline (3)(1.36g, 76% yield). LCMS: RT = 0.796 min, >98% @ 215 and 254 nM, m/z = 254.8 [M + H]+.</p><p><b>N-(3-chloro-4-((5-chloropyridin-2-yl)oxy)phenyl)picolinamide (5).</b> To a solution of 3-chloro-4-((5-chloropyridin-2-yl)oxy)aniline (1.36g, 5.35 mmol, 1.0 eq) and Hunig's Base (2.05 mL, 11.78 mmol, 2.2 eq) in DMF (30 mL) at 0°C was added picolinoyl chloride hydrochloride (1.05g, 5.89 mmol, 1.1 eq). After 15 min, the ice bath was removed. After 12 h at rt, the rxn was added to EtOAc:H<sub>2</sub>O (1:1, 500 mL). The separated organic layer was washed with NaHCO<sub>3</sub> (aq) (50 mL), H<sub>2</sub>O (3 × 50 mL), Brine (50 mL) and dried (MgSO<sub>4</sub>). The mixture was filtered and the solvent removed under vacuo. The residue was purified by recrystalization (EtOH) to afford N-(3-chloro-4-((5-chloropyridin-2-yl)oxy)phenyl)picolinamide (5) (1.17 g, 59% yield). LCMS: R<sub>T</sub> = 1.214 min, >98% @ 215 and 254 nM, <i>m/z</i> = 359.6 [M + H]<sup>+</sup>; <sup>1</sup>H NMR (400 MHz, , d-DMSO): δ 10.93 (br s, 1H), 8.77 (d, <i>J</i> = 4.0 Hz, 1H), 8.23 (d, <i>J</i> = 2.4 Hz, 1H), 8.20-8.18 (m, 2H), 8.09 (ddd, <i>J</i> = 7.6, 7.6, 1.6 Hz, 1H), 7.99 (dd, <i>J</i> = 8.8, 2.7 Hz, 1H), 7.92 (dd, <i>J</i> = 8.8, 2.5 Hz, 1H), 7.71 (ddd, <i>J</i> = 7.6, 4.8, 1.2 Hz, 1H), 7.35 (d, <i>J</i> = 8.8 Hz, 1H), 7.20 (d, <i>J</i> = 8.8 Hz, 1H); HRMS, calc'd for C<sub>17</sub>H<sub>12</sub>N<sub>3</sub>O<sub>2</sub>Cl<sub>2</sub> [M + H]<sup>+</sup>, 360.0307; found 360.0304.</p></div></div><div id="ml397.s12"><h2 id="_ml397_s12_">3. Results</h2><div id="ml397.s13"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml397.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20VU0155094%20potentiates%20mGlu4%2C%20mGlu7%2C%20and%20mGlu8%20with%20similar%20potencies.&p=BOOKS&id=280048_ml397f4.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/NBK280048/bin/ml397f4.jpg" alt="Figure 1. VU0155094 potentiates mGlu4, mGlu7, and mGlu8 with similar potencies." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">VU0155094 potentiates mGlu<sub>4</sub>, mGlu<sub>7</sub>, and mGlu<sub>8</sub> with similar potencies</span></h3><div class="caption"><p>When applied prior at an EC<sub>20</sub> concentration of glutamate or L-AP4 (mGlu<sub>7</sub>), VU0155094 induced a concentration-dependent potentiation of agonist responses. Potencies: mGlu<sub>4</sub> pEC<sub>50</sub>=5.48±0.03 μM, 3.4 μM, mGlu<sub>7</sub>=pEC<sub>50</sub>=5.86±0.06, EC<sub>50</sub>=1.5 μM, mGlu<sub>8</sub>=pEC<sub>50</sub>=6.07±0.07, 929 nM. The variations in the maximal responses result from apparent differential coupling efficiencies of various receptors with chimeric or promiscuous G proteins. Data represent three independent experiments performed in triplicate.</p></div></div><div id="ml397.f5" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20VU0422288%20(ML396)%20potentiates%20mGlu4%2C%20mGlu7%2C%20and%20mGlu8%20with%20similar%20potencies.&p=BOOKS&id=280048_ml397f5.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/NBK280048/bin/ml397f5.jpg" alt="Figure 2. VU0422288 (ML396) potentiates mGlu4, mGlu7, and mGlu8 with similar potencies." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">VU0422288 (ML396) potentiates mGlu<sub>4</sub>, mGlu<sub>7</sub>, and mGlu<sub>8</sub> with similar potencies</span></h3><div class="caption"><p>When applied prior at an EC<sub>20</sub> concentration of glutamate or L-AP4 (mGlu<sub>7</sub>), VU0422288 induced a concentration-dependent potentiation of agonist responses. Potencies were: mGlu<sub>4</sub> pEC<sub>50</sub>=6.98±0.04; mGlu<sub>7</sub>; pEC<sub>50</sub>=6.85±0.04, EC<sub>50</sub>=146 nM, mGlu<sub>8</sub>=pEC<sub>50</sub>= 6.93±0.05, EC<sub>50</sub>=125 nM. The variations in the maximal responses result from apparent differential coupling efficiencies of various receptors with chimeric or promiscuous G proteins. N=3 independent experiments performed in triplicate.</p></div></div></div><div id="ml397.s14"><h3>3.2. Cellular Activity</h3><p>The primary screening assay for mGlu<sub>7</sub>, mGlu<sub>8</sub>, and mGlu<sub>4</sub> are cell-based assay, indicating that <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> can gain access to its molecular target when applied to cells. The compound did not exhibit acute toxicity in cell based assays at concentrations up to 30 μM.</p></div><div id="ml397.s15"><h3>3.3. Profiling Assays</h3><p>To more fully characterize these potent, selective pan Group III mGlu positive allosteric modulators, <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> were tested using EuroFins (formerly MDS Pharma's) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM). Included in the EuroFin screening panel are a number of ion channels (Calcium Channel, L-Type and N-Type; Potassium channel [K<sub>ATP</sub>]; Potassium channel [hERG]) and class A GPCRs (D<sub>1-5</sub>, H<sub>1-3</sub>, etc…). <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> did not inhibit any of the 68 targets conducted (inhibition of radio ligand binding > 50% at 10 μM), highlighting a clean ancillary pharmacology profile. <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> did not show any inhibition for 67 of the 68 targets conducted, also highlighting a clean ancillary pharmacology profile. The only target that showed any inhibition was the serotonin norepinephrine transporter (NET) which showed 51% at 10 μM. Table 10 highlights calculated properties for <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a>, which compare favorably with the MDDR.</p></div></div><div id="ml397.s16"><h2 id="_ml397_s16_">4. Discussion</h2><div id="ml397.s17"><h3>4.1. Comparison to existing art and how the new probes are an improvement</h3><p>There are no known positive allosteric modulators of mGlu<sub>7</sub>, nor are there any pan-Group III PAMs. As such, <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> represent critically important advances for the field. The fact that both of these compounds potentiate multiple receptors within the mGlu family, but not the group I and group II receptors, suggest a common and unappreciated binding site on the group III receptor proteins that is not shared with other members of the receptor family. The two compounds do exhibit different in vitro profiles, PK properties and are of different structures. The lack of selectivity among the group III receptors is actually of value for certain <i>in vitro</i> pharmacology experiments, as there are no existing mGlu<sub>7</sub> PAMs and only one mGlu<sub>8</sub> PAM that has been reported in the literature. The ability of a ligand to interact with multiple receptors will now allow us to perform structure-function studies across the group III receptors to identify and characterize a common allosteric binding site. Additionally, a new and exciting area that we and others are pursuing are studies to characterize heterodimerization between various subunits of the mGlus [<a class="bk_pop" href="#ml397.r3">3</a>-<a class="bk_pop" href="#ml397.r5">5</a>]; tools that bind <i>in vitro</i> with one or multiple receptors will now be critically important for advancing our understanding of how mGlus interact in heterodimeric format and how these interactions impact pharmacology.</p></div><div id="ml397.s18"><h3>4.2. Mechanism of Action Studies</h3><p>These classes of Group III mGlu PAMs increase the efficacy of orthosteric agonists (glutamate and L-AP4) for mGlu<sub>4,7,8</sub>. These compounds do not interact with the group I and group II mGlus and possess very clean profiles in the Ricerca lead profiling screen. Additionally, these compounds potentiate mGlu<sub>7</sub> responses in a native tissue preparation, resulting in clearly measurable electrophysiological changes.</p></div><div id="ml397.s19"><h3>4.3. Efficacy in Cell-Based Assays</h3><p>The primary screening assays for mGlu<sub>7</sub>, mGlu<sub>8</sub>, and mGlu<sub>4</sub> are cell-based assay, indicating that <a href="/pcsubstance/?term=ML396[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML396</a> and <a href="/pcsubstance/?term=ML397[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML397</a> can gain access to its molecular target when applied to cells. Additionally, the compounds are active in potentiating mGlu<sub>7</sub> responses at SC-CA1 synapses, indicating that they are functional in a native tissue preparation and can modulate synaptic transmission. The compounds did not exhibit acute toxicity in cell based assays at concentrations up to 30 μM.</p></div></div><div id="ml397.s20"><h2 id="_ml397_s20_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml397.r1">Jones CK, et al. The mGlu4 positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine A2A antagonist in preclinical rodent models of Parkinson's disease. <span><span class="ref-journal">J. Pharmacol. Exp. Ther. </span>2012;<span class="ref-vol">340</span>:404–421.</span> [<a href="/pmc/articles/PMC3263969/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3263969</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22088953" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22088953</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml397.r2">Niswender CM, et al. Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4): Part I. Discovery of pyrazolo[3,4-d]pyrimidines as novel mGluR4 positive allosteric modulators. <span><span class="ref-journal">Bioorg. Med. Chem. Lett. </span>2008;<span class="ref-vol">18</span>(20):5626–5630.</span> [<a href="/pmc/articles/PMC3182458/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3182458</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18793851" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18793851</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml397.r3">Yin S, et al. Selective actions of novel allosteric modulators reveal functional heteromers of metabotropic glutamate receptors in the CNS. <span><span class="ref-journal">J Neurosci. </span>2014;<span class="ref-vol">34</span>(1):79–94.</span> [<a href="/pmc/articles/PMC3866496/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3866496</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/24381270" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24381270</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml397.r4">Doumazane E, et al. A new approach to analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate receptors. <span><span class="ref-journal">FASEB J. </span>2011;<span class="ref-vol">25</span>(1):66–77.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20826542" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20826542</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml397.r5">Kammermeier PJ. Functional and pharmacological characteristics of metabotropic glutamate receptors 2/4 heterodimers. <span><span class="ref-journal">Mol Pharmacol. </span>2012;<span class="ref-vol">82</span>(3):438–47.</span> [<a href="/pmc/articles/PMC3422699/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3422699</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22653971" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22653971</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml397.r6">Jalan-Sakrikar N., et al. Identification of positive allosteric modulators VU0155094 (ML397) and VU0422288 (ML396) reveals new insights into the biology of metabotropic glutamate receptor 7. <span><span class="ref-journal">ACS Chem. Neurosci. </span>2014;<span class="ref-vol">5</span>:1221–1237.</span> DOIPMID. [<a href="/pmc/articles/PMC4306484/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4306484</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/25225882" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25225882</span></a>] [<a href="http://dx.crossref.org/10.1021/cn5000153z" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</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/NBK280048/?report=reader">PubReader</a></li><li><a href="/books/NBK280048/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK280048" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK280048" style="display:none" title="Cite this Page"><div class="bk_tt">Jalan-Sakrikar N, Roper-Field J, Klar R, et al. The discovery and characterization of a centrally penetrant (ML396) and a peripherally restricted (ML397) pan-Group III mGlu positive allosteric modulators. 2014 Apr 15 [Updated 2015 Jan 16]. 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="#ml397.s1" ref="log$=inpage&link_id=inpage">Probe Structures & Characteristics</a></li><li><a href="#ml397.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml397.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml397.s12" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml397.s16" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml397.s20" ref="log$=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=3413730" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3413730" ref="log$=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=3413730" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/25225882" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Identification of positive allosteric modulators VU0155094 (ML397) and VU0422288 (ML396) reveals new insights into the biology of metabotropic glutamate receptor 7.</a><span class="source">[ACS Chem Neurosci. 2014]</span><div class="brieflinkpop offscreen_noflow">Identification of positive allosteric modulators VU0155094 (ML397) and VU0422288 (ML396) reveals new insights into the biology of metabotropic glutamate receptor 7.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Jalan-Sakrikar N, Field JR, Klar R, Mattmann ME, Gregory KJ, Zamorano R, Engers DW, Bollinger SR, Weaver CD, Days EL, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">ACS Chem Neurosci. 2014 Dec 17; 5(12):1221-37. Epub 2014 Oct 9.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/25518990" ref="ordinalpos=1&linkpos=2&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Novel metabotropic glutamate receptor 4 and glutamate receptor 8 therapeutics for the treatment of anxiety.</a><span class="source">[Expert Opin Investig Drugs. 2015]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Novel metabotropic glutamate receptor 4 and glutamate receptor 8 therapeutics for the treatment of anxiety.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Raber J, Duvoisin RM. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Expert Opin Investig Drugs. 2015 Apr; 24(4):519-28. Epub 2014 Dec 17.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23658969" ref="ordinalpos=1&linkpos=3&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Discovery of a novel metabotropic glutamate receptor 4 (mGlu(4)) positive allosteric modulator (PAM) extended probe: Characterization of ML292, a potent and selective mGlu(4) PAM which produces efficacy alone or in combination with L-DOPA in preclinical rodent models of Parkinson's disease.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Discovery of a novel metabotropic glutamate receptor 4 (mGlu(4)) positive allosteric modulator (PAM) extended probe: Characterization of ML292, a potent and selective mGlu(4) PAM which produces efficacy alone or in combination with L-DOPA in preclinical rodent models of Parkinson's disease.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Engers DW, Jones CK, Bubser M, Thompson AD, Blobaum AL, Sheffler DJ, Zamorano R, Carrington SJS, Bridges TM, Morrison RD, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/29057060" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">Discovery of VU6005649, a CNS Penetrant mGlu(7/8) Receptor PAM Derived from a Series of Pyrazolo[1,5-a]pyrimidines.</a><span class="source">[ACS Med Chem Lett. 2017]</span><div class="brieflinkpop offscreen_noflow">Discovery of VU6005649, a CNS Penetrant mGlu(7/8) Receptor PAM Derived from a Series of Pyrazolo[1,5-a]pyrimidines.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Abe M, Seto M, Gogliotti RG, Loch MT, Bollinger KA, Chang S, Engelberg EM, Luscombe VB, Harp JM, Bubser M, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">ACS Med Chem Lett. 2017 Oct 12; 8(10):1110-1115. Epub 2017 Sep 1.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/32890686" ref="ordinalpos=1&linkpos=5&log$=relatedarticles&logdbfrom=pubmed">Synthesis and SAR of a series of mGlu(7) NAMs based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinoline carboxylate core.</a><span class="source">[Bioorg Med Chem Lett. 2020]</span><div class="brieflinkpop offscreen_noflow">Synthesis and SAR of a series of mGlu(7) NAMs based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinoline carboxylate core.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Kalbfleisch JJ, Reed CW, Park C, Spearing PK, Quitalig MC, Jenkins MT, Rodriguez AL, Blobaum AL, Conn PJ, Niswender CM, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Bioorg Med Chem Lett. 2020 Nov 15; 30(22):127529. 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