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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="Development of the First Potent, Selective and CNS penetrant M5 Negative Allosteric Modulator (NAM)" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2014/09/18" /><meta name="citation_author" content="Patrick R. Gentry" /><meta name="citation_author" content="Masaya Kokubo" /><meta name="citation_author" content="Thomas M. Bridges" /><meta name="citation_author" content="J. Scott Daniels" /><meta name="citation_author" content="Colleen M. Niswender" /><meta name="citation_author" content="Emery Smith" /><meta name="citation_author" content="Peter Chase" /><meta name="citation_author" content="Peter S. Hodder" /><meta name="citation_author" content="Hugh Rosen" /><meta name="citation_author" content="P. Jeffrey Conn" /><meta name="citation_author" content="Julie Engers" /><meta name="citation_author" content="Katrina A. Brewer" /><meta name="citation_author" content="Michael R. Wood" /><meta name="citation_author" content="Craig W. Lindsley" /><meta name="citation_pmid" content="25506971" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK259190/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Development of the First Potent, Selective and CNS penetrant M5 Negative Allosteric Modulator (NAM)" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Patrick R. Gentry" /><meta name="DC.Contributor" content="Masaya Kokubo" /><meta name="DC.Contributor" content="Thomas M. Bridges" /><meta name="DC.Contributor" content="J. Scott Daniels" /><meta name="DC.Contributor" content="Colleen M. Niswender" /><meta name="DC.Contributor" content="Emery Smith" /><meta name="DC.Contributor" content="Peter Chase" /><meta name="DC.Contributor" content="Peter S. Hodder" /><meta name="DC.Contributor" content="Hugh Rosen" /><meta name="DC.Contributor" content="P. Jeffrey Conn" /><meta name="DC.Contributor" content="Julie Engers" /><meta name="DC.Contributor" content="Katrina A. Brewer" /><meta name="DC.Contributor" content="Michael R. Wood" /><meta name="DC.Contributor" content="Craig W. Lindsley" /><meta name="DC.Date" content="2014/09/18" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK259190/" /><meta name="description" content="A recently completed functional, high throughput screen of the Molecular Libraries Probe Production Center's Network (MLPCN) screening deck of ∼360,000 compounds conducted by Scripps Research Institute Molecular Screening Center (SRIMSC) against three of the five muscarinic receptor subtypes (M1, M4 and M5) provided a number of interesting hits. As this was the first time a directed effort had been undertaken to identify M5 selective leads, we were very pleased by the identification of nine M5 PAMs and nine M5 antagonists (or negative allosteric modulators, NAMs), despite the complete absence of M5 agonist hits. The most promising M5 inhibitor hit (CID 5189681), was quickly developed into a potent, selective and CNS penetrant probe molecule ML375 (hM5 NAM IC50 = 300 nM, hM1-4 IC50s >30 μM) displaying enantioselective inhibition. Detailed molecular pharmacology studies demonstrated that ML375 is the first M5 NAM ever described, and ML375 show good rat and cyno pharmacokinetics to support in vivo studies in rodents and non-human primates." /><meta name="og:title" content="Development of the First Potent, Selective and CNS penetrant M5 Negative Allosteric Modulator (NAM)" /><meta name="og:type" content="book" /><meta name="og:description" content="A recently completed functional, high throughput screen of the Molecular Libraries Probe Production Center's Network (MLPCN) screening deck of ∼360,000 compounds conducted by Scripps Research Institute Molecular Screening Center (SRIMSC) against three of the five muscarinic receptor subtypes (M1, M4 and M5) provided a number of interesting hits. As this was the first time a directed effort had been undertaken to identify M5 selective leads, we were very pleased by the identification of nine M5 PAMs and nine M5 antagonists (or negative allosteric modulators, NAMs), despite the complete absence of M5 agonist hits. The most promising M5 inhibitor hit (CID 5189681), was quickly developed into a potent, selective and CNS penetrant probe molecule ML375 (hM5 NAM IC50 = 300 nM, hM1-4 IC50s >30 μM) displaying enantioselective inhibition. Detailed molecular pharmacology studies demonstrated that ML375 is the first M5 NAM ever described, and ML375 show good rat and cyno pharmacokinetics to support in vivo studies in rodents and non-human primates." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK259190/" /><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/ml375/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK259190/" /><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="#__NBK259190_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK259190_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/ml372/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml377/" 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="_NBK259190_"><span class="title" itemprop="name">Development of the First Potent, Selective and CNS penetrant M5 Negative Allosteric Modulator (NAM)</span></h1><p class="contrib-group"><span itemprop="author">Patrick R. Gentry</span>, <span itemprop="author">Masaya Kokubo</span>, <span itemprop="author">Thomas M. Bridges</span>, <span itemprop="author">J. Scott Daniels</span>, <span itemprop="author">Colleen M. Niswender</span>, <span itemprop="author">Emery Smith</span>, <span itemprop="author">Peter Chase</span>, <span itemprop="author">Peter S. Hodder</span>, <span itemprop="author">Hugh Rosen</span>, <span itemprop="author">P. Jeffrey Conn</span>, <span itemprop="author">Julie Engers</span>, <span itemprop="author">Katrina A. Brewer</span>, <span itemprop="author">Michael R. Wood</span>, and <span itemprop="author">Craig W. Lindsley</span>.</p><a data-jig="ncbitoggler" href="#__NBK259190_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK259190_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Patrick R. Gentry</span>,<sup>#</sup> <span itemprop="author">Masaya Kokubo</span>,<sup>#</sup> <span itemprop="author">Thomas M. Bridges</span>,<sup>#</sup> <span itemprop="author">J. Scott Daniels</span>,<sup>#</sup> <span itemprop="author">Colleen M. Niswender</span>,<sup>#</sup> <span itemprop="author">Emery Smith</span>,<sup>%</sup> <span itemprop="author">Peter Chase</span>,<sup>%</sup> <span itemprop="author">Peter S. Hodder</span>,<sup>%</sup> <span itemprop="author">Hugh Rosen</span>,<sup>%</sup> <span itemprop="author">P. Jeffrey Conn</span>,<sup>#</sup> <span itemprop="author">Julie Engers</span>,<sup>#</sup> <span itemprop="author">Katrina A. Brewer</span>,<sup>#</sup> <span itemprop="author">Michael R. Wood</span>,<sup>#</sup> and <span itemprop="author">Craig W. Lindsley</span><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup><sup>#</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>#</sup>
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Vanderbilt University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.tlibrednav@yelsdnil.giarc" class="oemail">ude.tlibrednav@yelsdnil.giarc</a></div></div><div class="affiliation"><sup>%</sup>
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Scripps Research Institute</div><div><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author.</div></div><p class="small">Received: <span itemprop="datePublished">December 9, 2013</span>; Last Update: <span itemprop="dateModified">September 18, 2014</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>A recently completed functional, high throughput screen of the Molecular Libraries Probe Production Center's Network (MLPCN) screening deck of ∼360,000 compounds conducted by Scripps Research Institute Molecular Screening Center (SRIMSC) against three of the five muscarinic receptor subtypes (M<sub>1</sub>, M<sub>4</sub> and M<sub>5</sub>) provided a number of interesting hits. As this was the first time a directed effort had been undertaken to identify M<sub>5</sub> selective leads, we were very pleased by the identification of nine M<sub>5</sub> PAMs and nine M<sub>5</sub> antagonists (or negative allosteric modulators, NAMs), despite the complete absence of M<sub>5</sub> agonist hits. The most promising M<sub>5</sub> inhibitor hit (CID 5189681), was quickly developed into a potent, selective and CNS penetrant probe molecule <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> (hM<sub>5</sub> NAM IC<sub>50</sub> = 300 nM, hM<sub>1-4</sub> IC<sub>50</sub>s >30 μM) displaying enantioselective inhibition. Detailed molecular pharmacology studies demonstrated that <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> is the first M<sub>5</sub> NAM ever described, and <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> show good rat and cyno pharmacokinetics to support <i>in vivo</i> studies in rodents and non-human primates.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> X01 MH077607-1 and 1X01 <a href="/nuccore/1540627243" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">MH077606</a></p><p><b>Screening Center Name & PI</b>: Scripps Research Institute Molecular Screening Center (SRIMSC), Hugh Rosen</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 M. 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/624103" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">624103</a></p><div id="ml375.s1"><h2 id="_ml375_s1_">Probe Structure & Characteristics</h2><div id="ml375.f1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK259190/bin/ml375f1.jpg" alt="(S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one." /></div><h3><span class="title">(<i>S</i>)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1<i>H</i>-imidazo[2,1-<i>a</i>]isoindol-5(9b<i>H</i>)-one</span></h3></div><div id="ml375.t1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259190/table/ml375.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml375.t1_lrgtbl__"><table><thead><tr><th id="hd_h_ml375.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml375.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml375.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml375.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_ml375.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_ml375.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml375.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml375.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 71598521/<a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a></td><td headers="hd_h_ml375.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">M<sub>5</sub> (NAM)</td><td headers="hd_h_ml375.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">300 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/71598521" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 71598521</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743026" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743026</a>]</td><td headers="hd_h_ml375.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">M<sub>1</sub>-M<sub>4</sub> (> 10x hM<sub>5</sub>)</td><td headers="hd_h_ml375.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">M<sub>1</sub>-M<sub>4</sub> > 30 µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/71598521" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 71598521</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743098" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743098</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743099" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743099</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743103" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743103</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743105" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743105</a>]</td><td headers="hd_h_ml375.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 100</td><td headers="hd_h_ml375.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">M<sub>5</sub> rat IC<sub>50</sub>: 790 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/71598521" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 71598521</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743104" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743104</a>]</td></tr></tbody></table></div></div></div><div id="ml375.s2"><h2 id="_ml375_s2_">1. Recommendations for scientific use of the probe</h2><ul><li class="half_rhythm"><div>What limitations in the current state of the art is the probe addressing?</div></li></ul><p>Prior to the discovery of <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a>, there was a complete paucity of selective M<sub>5</sub> inhibitors (few antagonists and no NAMs). <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> is the most potent and selective M<sub>5</sub> NAM, and the only M<sub>5</sub> NAM, currently known and its demonstrated CNS exposure will allow it to find utility in a wide range of in vivo and ex vivo studies. <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> will find utility in electrophysiology experiments and animal models exploring the function of M<sub>5</sub> receptors (cognition, migraine, ischemia, and addiction/withdrawal), where dose-exposure relationships can now be obtained. Importantly, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> is first in class as a selective M<sub>5</sub> NAM.</p><ul><li class="half_rhythm"><div>How will the probe be used?</div></li></ul><p>Given <a href="/pcsubstance/?term=ML75[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML75</a>'s sub-micromolar potency (hM<sub>5</sub> NAM IC<sub>50</sub> = 300 nM, rM<sub>5</sub> NAM IC<sub>50</sub> = 790 nM) and demonstrated CNS penetrance (vide infra), this probe will find utility in various animal models designed to elucidate the specific functions of the M<sub>5</sub> receptor. Importantly, being <i>in vivo</i> quantifiable, exposure-response relationships will be obtainable rather than just dose-response relationships. Additionally, <i>ex vivo</i> electrophysiology experiments on slice preparations from various regions of the brain could help define the neurological functioning of the M<sub>5</sub> receptor. These future experiments will be greatly aided by the very high M<sub>5</sub> receptor selectivity displayed by <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> over the M<sub>1</sub> – M<sub>4</sub> receptors.</p><ul><li class="half_rhythm"><div>Who in the research community will use the probe?</div></li></ul><p>Through the use of knock-out (KO) mutant mice, the research community has learned of numerous potential indications for selective M<sub>5</sub> ligands, for example, in models of cognition, addiction and cerebral blood flow. Those researchers interested in confirming and extending those early behavioral studies with the KO mice will find our M<sub>5</sub> NAM probe invaluable in exploring numerous disease states, just as addiction, and identifying new therapeutic possibilities implicated in the genetic models.</p><ul><li class="half_rhythm"><div>What is the relevant biology to which the probe can be applied?</div></li></ul><p>In particular, M<sub>5</sub> receptor activation has been implicated in modifying cognitive impairment (such as that associated with Alzheimer's disease) and cerebrovascular blood flow (potential use with migraines, ischemia and cognitive enhancement), while M<sub>5</sub> receptor inhibition has been implicated in addiction/withdrawal mechanisms (substance abuse). Validating any of these potential modes of action could help spur increased medical research around selective M<sub>5</sub> ligand development, with the potential to significantly impact human health. It is essential to validate genetic M<sub>5</sub> knock-out data with pharmacological M<sub>5</sub> inhibition.</p><p><b>Introduction.</b> Acetylcholine (ACh) serves as the endogenous agonist for both the nicotinic (nAChR) and muscarinic acetylcholine receptors. There are five mAChR subtypes (M<sub>1</sub> – M<sub>5</sub>) widely expressed in both the central nervous system (CNS) and periphery of mammals. These receptors undertake critical roles in regulating a variety of diverse physiological processes. Within the CNS, the M<sub>1</sub>, M<sub>4</sub> and M<sub>5</sub> subtypes are believed to be the most important with respect to normal neuronal functioning. Of these three, M<sub>5</sub> is the least studied as a combined result of its lower expression levels(<a class="bk_pop" href="#ml375.r1">1</a>) (< 2% of total muscarinic receptor population within the brain) and, until recently, a near absence of highly selective M<sub>5</sub> receptor ligands. The majority of our current beliefs surrounding the function of M<sub>5</sub> has come from M<sub>5</sub> receptor localization, phenotypic observations of M<sub>5</sub> knock-out mice(<a class="bk_pop" href="#ml375.r2">2</a>) and studies conducted with non-selective muscarinic ligands.(<a class="bk_pop" href="#ml375.r3">3</a>) It is intriguing to note that the M<sub>5</sub> receptor is the only muscarinic receptor observed the substantia nigra pars compacta (based on mRNA detection),<a class="bk_pop" href="#ml375.r4">4</a>,<a class="bk_pop" href="#ml375.r5">5</a> leading to the prediction that M<sub>5</sub> functions in addiction/reward mechanisms. This hypothesis was supported in man through the clinically observed correlation between a specific M<sub>5</sub> gene mutation and an increase in cigarette consumption (+ 27%), as well as, an increased risk for cannabis dependence (+ 290%).(<a class="bk_pop" href="#ml375.r6">6</a>) M<sub>5</sub> receptors have also been localized on the cerebrovascular system and shown to be critical in the ACh-induced dilation necessary for normal CNS blood perfusion, without an effect extending into the periphery.(<a class="bk_pop" href="#ml375.r7">7</a>) In a broader sense, M<sub>5</sub> KO mice show decreased prepulse inhibition,(<a class="bk_pop" href="#ml375.r8">8</a>) CNS neuronal abnormalities and cognitive deficits.(<a class="bk_pop" href="#ml375.r9">9</a>) In summary, these studies support the potential for M<sub>5</sub> ligands in the treatment of numerous CNS indications including schizophrenia, Alzheimer's disease, addiction, ischemia and migraine. However, in the absence of highly selective M<sub>5</sub> tool compounds, and ultimately pharmaceuticals, this potential will remain unrealized. There is a critical need for subtype selective antagonists, negative allosteric modulators (NAMs) and positive allosteric modulators (PAMs) to truly understand the physiological, and potential therapeutic role, of the M<sub>5</sub> receptor.</p><p><b>Prior Art.</b> To date, only three publications have described the development of M<sub>5</sub> selective ligands. These manuscripts disclosed the successive improvements of a structurally related class of positive allosteric modulators (PAMs) and the development of three MLPCN probe molecules: <a href="/pcsubstance/?term=ML129[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML129</a>(<a class="bk_pop" href="#ml375.r10">10</a>) <a href="/pcsubstance/?term=ML172[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML172</a>(<a class="bk_pop" href="#ml375.r11">11</a>) and <a href="/pcsubstance/?term=ML326[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML326</a>(<a class="bk_pop" href="#ml375.r12">12</a>) (<a class="figpopup" href="/books/NBK259190/figure/ml375.f2/?report=objectonly" target="object" rid-figpopup="figml375f2" rid-ob="figobml375f2">Figure 1</a>). There is little in the way of prior art for M<sub>5</sub> selective inhibitors (either orhtosteric antagonists of NAMs). To date, the majority of mAChR antagonists (<a class="figpopup" href="/books/NBK259190/figure/ml375.f2/?report=objectonly" target="object" rid-figpopup="figml375f2" rid-ob="figobml375f2">Figure 1</a>) are unselective ligands, such as scopolamine (<b>1</b>), atropine (<b>2</b>) or moderately selective ligands such as the ∼10-fold M<sub>1</sub>-preferring pirenzepine (<b>3</b>). Recently in 2013, the first M<sub>5</sub>-preferring antagonist (based on binding data – not functional) was reported.(<a class="bk_pop" href="#ml375.r13">13</a>) Compound <b>4</b> bound to M<sub>5</sub> with a K<sub>i</sub> of 2.2 μM with 11-fold selectivity versus M<sub>1</sub>-M<sub>4</sub>, but no DMPK or CNS penetration data was disclosed. Moreover, there are no known M<sub>5</sub> NAMs, and no highly selective and CNS penetrant M<sub>5</sub> antagonists/NAMs. <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> marks a major advancement in the field that will enable detailed <i>in vitro</i> and <i>in vivo</i> studies to be performed.(<a class="bk_pop" href="#ml375.r14">14</a>)</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml375f2" co-legend-rid="figlgndml375f2"><a href="/books/NBK259190/figure/ml375.f2/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml375f2" rid-ob="figobml375f2"><img class="small-thumb" src="/books/NBK259190/bin/ml375f2.gif" src-large="/books/NBK259190/bin/ml375f2.jpg" alt="Figure 1. Prior Art: M5 PAM Probes ML129, ML172 and ML326, and examples of known mAChR antagonists 1-4, which are lacking in terms of mAChR selectivity and/or DMPK profiles." /></a><div class="icnblk_cntnt" id="figlgndml375f2"><h4 id="ml375.f2"><a href="/books/NBK259190/figure/ml375.f2/?report=objectonly" target="object" rid-ob="figobml375f2">Figure 1</a></h4><p class="float-caption no_bottom_margin">Prior Art: M5 PAM Probes ML129, ML172 and ML326, and examples of known mAChR antagonists 1-4, which are lacking in terms of mAChR selectivity and/or DMPK profiles. </p></div></div></div><div id="ml375.s3"><h2 id="_ml375_s3_">2. Materials and Methods</h2><p><b>Cell culture and transfections.</b><a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> Chinese hamster ovary (CHO-K1) cells stably expressing rat (r)M<sub>1</sub> were purchased from the American Type Culture Collection and cultured according to their indicated protocol. CHO cells stably expressing human (h)M<sub>2</sub>, hM<sub>3</sub>, and hM<sub>5</sub> were generously provided by A. Levey (Emory University, Atlanta, GA); hM<sub>1</sub> and hM<sub>4</sub> cDNAs were purchased from Missouri S&T cDNA Resource; rM<sub>4</sub> cDNA was provided by T. I. Bonner (National Institutes of Health, Bethesda, MD). hM<sub>1</sub>, hM<sub>4</sub>, and rM<sub>4</sub> cDNAs were used to stably transfect CHO-K1 cells purchased from the American Type Culture Collection using Lipofectamine2000. To make stable hM<sub>2</sub>, rM<sub>2</sub>, hM<sub>4</sub> and rM<sub>4</sub> cell lines for use in calcium mobilization assays, these cells also were stably transfected with a chimeric G-protein (G<sub>qi5</sub>) (provided by B.R. Conklin, University of California, San Francisco) using Lipofectamine 2000. rM<sub>1</sub>, hM<sub>3</sub>, and hM<sub>5</sub> cells were grown in Ham's F-12 medium containing 10% heat-inactivated fetal bovine serum (FBS), 20mM HEPES, and 50µg/mL G418 sulfate. hM<sub>2</sub>–G<sub>qi5</sub> and hM<sub>4</sub>–G<sub>qi5</sub> cells were grown in the same medium also containing 500 µg/mL Hygromycin B. Stable rM<sub>4</sub>–G<sub>qi5</sub> cells were grown in DMEM containing 10% heat-inactivated FBS, 20 mM HEPES, 400 µg/mL G418 sulfate, and 500 µg/mL Hygromycin B. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.</p><p><b>Calcium Mobilization Assays - Potency determinations.</b><a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> Assays were performed within the Vanderbilt Center for Neuroscience Drug Discovery's Screening Center. CHO cell lines expressing muscarinic acetylcholine receptors were plated (15,000 cells/20 μL/well) in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in Ham's F-12, 10% FBS, 20 mM HEPES. The cells were grown overnight at 37 °C in the presence of 5% CO<sub>2</sub>. The following day, plated cells had their medium exchanged to Assay Buffer (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) using an ELX405 microplate washer (BioTek), leaving 20 μL/well, followed by addition of with 20 µL of 4.5 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 for 45 minutes at 37 °C. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 μL/well and the plates were incubated at room temperature for 10 min prior to assay. Test compounds were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA) and then diluted into Assay Buffer to generate a 2x stock in 0.6% DMSO (0.3% final). Acetylcholine (ACh) EC<sub>20</sub> and EC<sub>80</sub> were prepared at a 5X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 37 °C using a Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan) kinetic plate reader according to the following protocol. Cells were preincubated with test compound (or vehicle) for 144 seconds prior to the addition of an EC<sub>20</sub> concentration of the agonist, ACh. 86 seconds after this addition, an EC<sub>80</sub> concentration of ACh was added. Control wells also received a maximal ACh concentration (1 mM) for eventual response normalization. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to ACh. Microsoft XLfit (IDBS, Bridgewater, NJ) was utilized for curve fitting and EC<sub>50</sub> value determination using a four point logistical equation. Compounds showing dose-dependency were assigned ‘Outcome’ = ‘Active’, EC<sub>50</sub>=’Value’, and % ACh min=’Value’.</p><p><b><i>[</i></b><i><sup>3</sup></i><b><i>H]-NMS Competition Binding Assay with CRCs.</i></b><a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> Membranes were prepared from stable CHO cell lines constitutively expressing human M<sub>5</sub> receptors according to a previously described protocol (Marlo <i>et al.</i>, 2009; Brady <i>et al.</i>, 2008). Binding reactions were carried out in 2 mL, clear, 96-well, deep well plates (Axygen Scientific) and contained 0.3 nM [<sup>3</sup>H]-<i>N</i>-methylscopolamine ([<sup>3</sup>H]-NMS, obtained commercially from PerkinElmer), 10 μg of membrane protein, and an 11 point concentration range of test compound or atropine in a total volume of 500 μL assay buffer (100 mM NaCl, 10 mM MgCl<sub>2</sub>, 20 mM HEPES, pH 7.4). Nonspecific binding was determined in the presence of 10 μM atropine. The <i>K<sub>D</sub></i> of [<sup>3</sup>H]-NMS was determined empirically to be 0.264 nM. Binding reactions were performed at ambient temperature and allowed to incubate for 3 hours on a Lab-Line Titer plate shaker at setting 7 (∼750 rpm). Reactions were terminated by rapid filtration through GF/B filter plates (1 μm pore size) using a 96-well Brandel harvester and washed 3X with ice-cold harvesting buffer (50 mM Tris-HCl, 0.9% NaCl, pH 7.4). Filter plates were dried overnight and counted in a PerkinElmer TopCount scintillation counter (PerkinElmer Life and Analytical Sciences). Actual [<sup>3</sup>H]-NMS concentration was back-calculated after counting aliquots of 10X [<sup>3</sup>H]-NMS used in the reaction. Atropine <i>K<sub>i</sub></i> was determined to be ∼2.45 nM. For all assays, radioligand depletion was kept to approximately 15% or less. Data was plotted and atropine <i>K<sub>i</sub></i> was calculated using the curve-fitting software of GraphPad Prism (version 5.01). Data shown represent mean values obtained from three independent determinations performed using three or more replicates (error bars represent +/- SEM).</p><p><b><i>[</i></b><i><sup>3</sup></i><b><i>H]-NMS Dissociation Kinetics Assays</i>.</b><a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> Membranes were prepared from stable CHO cell lines constitutively expressing human M<sub>5</sub> receptors cells according to a previously described protocol (Marlo <i>et al</i>., 2009; Brady <i>et al</i>., 2008). Binding reactions were carried out in 2 mL, clear, 96-well, deep well plates (Axygen Scientific) and initially contained M<sub>5</sub> CHO cell membranes (10 μg ) with 0.3 nM [<sup>3</sup>H]-NMS. Binding was allowed to equilibrate at ambient temperature for 3 hours on a Lab-Line Titer plate shaker at setting 7 (∼750 rpm). Atropine (10 μM) and either vehicle or test compound (10 μM) were then added at various time points over 3.5 hours to prevent radioligand reassociation in a total volume of 500 μL. Plate agitation was continued between additions. Nonspecific binding was determined in the presence of 10 μM atropine. Reactions were terminated by rapid filtration through GF/B filter plates (1 μm pore size) using a 96-well Brandel harvester and washed 3X with ice-cold harvesting buffer (50 mM Tris-HCl, 0.9% NaCl, pH 7.4). Filter plates were dried overnight and counted in a PerkinElmer TopCount scintillation counter (PerkinElmer Life and Analytical Sciences). Actual [<sup>3</sup>H]-NMS concentration was back-calculated after counting aliquots of 10X [<sup>3</sup>H]-NMS used in the reaction. For all assays, radioligand depletion was kept to approximately 15% or less. Data was plotted and half-life was calculated using the curve-fitting software of GraphPad Prism (version 5.01). Data shown represent mean values obtained from three independent determinations performed using three or more replicates (error bars represent +/- SEM).</p><div id="ml375.s4"><h3>DMPK Methods</h3><p><b>Plasma protein binding</b>.<a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> The protein binding of each compound was determined in plasma via equilibrium dialysis employing RED Plates (ThermoFisher Scientific, Rochester, NY). Plasma was added to the 96 well plate containing test compound and mixed thoroughly for a final concentration of 5 µM. Subsequently, an aliquot of the plasma-compound mixture was transferred to the <i>cis</i> chamber (red) of the RED plate, with phosphate buffer (25 mM, pH 7.4) in the <i>trans</i> chamber. The RED plate was sealed and incubated for 4 hours at 37°C with shaking. At completion, aliquots from each chamber were diluted 1:1 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 containing internal standard (50 ng/mL carbamazepine) (2 volumes) was added to extract the matrices. The plate was centrifuged (3000 rcf, 10 min) and supernatants transferred and diluted 1:1 (supernatant: water) into a new 96 well plate, which was then sealed in preparation for LC/MS/MS analysis. Each compound was assayed in triplicate within the same 96-well plate. Fraction unbound was determined using the following equation</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml375.eq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">
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<math id="ml375.m1" display="block"><mrow><msub><mi>F</mi><mi>u</mi></msub><mo>=</mo><mfrac><mrow><msub><mtext mathvariant="italic">Conc</mtext><mtext mathvariant="italic">buffer</mtext></msub></mrow><mrow><msub><mtext mathvariant="italic">Conc</mtext><mtext mathvariant="italic">plasma</mtext></msub></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"></span></div></div></div><p><b>Intrinsic clearance</b>.<a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> Human or rat hepatic microsomes (0.5 mg/mL) and 1 µM test compound were incubated in 100 mM potassium phosphate pH 7.4 buffer with 3 mM MgCl<sub>2</sub> at 37°C with constant shaking. After a 5 min preincubation, the reaction was initiated by addition of NADPH (1 mM). At selected time intervals (0, 3, 7, 15, 25, and 45 min), aliquots were taken and subsequently placed into a 96-well plate containing cold acetonitrile with internal standard (50 ng/mL carbamazepine). Plates were then centrifuged at 3000 rcf (4° C) for 10 min, and the supernatant was transferred to a separate 96-well plate and diluted 1:1 with water for LC/MS/MS analysis. The <i>in vitro</i> half-life (T<sub>1/2</sub>, min, <a href="#ml375.eq2">Eq. 1</a>), intrinsic clearance (CL<sub>int</sub>, mL/min/kg, <a href="#ml375.eq3">Eq. 2</a>) and subsequent predicted hepatic clearance (CL<sub>hep</sub>, mL/min/kg, <a href="#ml375.eq4">Eq. 3</a>) was determined employing the following equations:</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml375.eq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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<math id="ml375.m2" display="block"><mrow><msub><mi>T</mi><mrow><mn>1</mn><mo stretchy="false">/</mo><mn>2</mn></mrow></msub><mo>=</mo><mfrac><mrow><mi>L</mi><mi>n</mi><mo stretchy="false">(</mo><mn>2</mn><mo stretchy="false">)</mo></mrow><mi>k</mi></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">1</span></div></div></div><p>where k represents the slope from linear regression analysis of the natural log percent remaining of test compound as a function of incubation time <sup>a</sup>scale-up factors: of 20 (human) or 45 (rat).</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml375.eq3"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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<math id="ml375.m3" display="block"><mrow><mi>C</mi><msub><mi>L</mi><mo>int</mo></msub><mo>=</mo><mfrac><mn>0.693</mn><mrow><mi>i</mi><mi>n</mi><mspace width="0.2em"></mspace><mtext mathvariant="italic">vitro</mtext><msub><mi>T</mi><mrow><mn>1</mn><mo stretchy="false">/</mo><mn>2</mn></mrow></msub></mrow></mfrac><mo>×</mo><mfrac><mtext mathvariant="italic">mL incubation</mtext><mtext mathvariant="italic">mg microsomes</mtext></mfrac><mo>×</mo><mfrac><mrow><mn>45</mn><mtext mathvariant="italic">mg microsomes</mtext></mrow><mtext mathvariant="italic">gram liver</mtext></mfrac><mo>×</mo><mfrac><mrow><msup><mn>20</mn><mi>a</mi></msup><mtext mathvariant="italic">gram liver</mtext></mrow><mtext mathvariant="italic">kg body wt</mtext></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">2</span></div></div></div><div class="pmc_disp_formula whole_rhythm clearfix" id="ml375.eq4"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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<math id="ml375.m4" display="block"><mrow><mi>C</mi><msub><mi>L</mi><mtext mathvariant="italic">hep</mtext></msub><mo>=</mo><mfrac><mrow><msub><mi>Q</mi><mi>h</mi></msub><mo>·</mo><mi>C</mi><mi>L</mi><mo>int</mo></mrow><mrow><msub><mi>Q</mi><mi>h</mi></msub><mo>+</mo><mi>C</mi><mi>L</mi><mo>int</mo></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>where Q<sub>h</sub> (hepatic blood flow, mL/min/kg) is 21 (human) or 70 (rat).</p><p><b>LC/MS/MS Bioanalysis of Samples from Plasma Protein Binding and Intrinsic Clearance Assays</b>.<a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> Samples were analyzed on a Thermo Electron TSQ Quantum Ultra triple quad mass spectrometer (San Jose, CA) via electrospray ionization (ESI) with two Themo Electron Accella pumps (San Jose, CA), and a Leap Technologies CTC PAL autosampler (Carrboro, NC). Analytes were separated by gradient elution on a dual column system with two Thermo Hypersil Gold (2.1 × 30 mm, 1.9 µm) columns (San Jose, CA) thermostated at 40°C. HPLC mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The gradient started at 10% B after a 0.2 min hold and was linearly increased to 95% B over 0.8 min; hold at 95% B for 0.2 min; returned to 10% B in 0.1 min. The total run time was 1.3 min and the HPLC flow rate was 0.8 mL/min. While pump 1 ran the gradient method, pump 2 equilibrated the alternate column isocratically at 10% B. Compound optimization, data collection and processing was performed using Thermo Electron's QuickQuan software (v2.3) and Xcalibur (v2.0.7 SP1).</p><p><b>Inhibition of Cytochrome P450 Enzymes.</b><a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> A cocktail of substrates for cytochrome P450 enzymes (1A2: Phenacetin, 10 µM; 2C9: Diclofenac, 5 µM; 2D6: Dextromethorphan, 5 µM; 3A4: Midazolam, 2 µM) were mixed for cocktail analysis. For P450 2C19, the substrate stock (Mephenytoin, 40 µM )and substrate mix were prepared separately for discrete analysis. The positive control for pan-P450 inhibition (miconazole) was included alongside each test compound in analysis. A reaction mixture of 100 mM Kpi, pH 7.4, 0.1 mg/mL human liver microsomes (HLM) and Substrate Mix is prepared and aliquoted into a 96-deepwell block. Test compound and positive control (in duplicate) were then added such that the final concentration of test compound ranged from 0.1 – 30 µM. The plate was vortexed briefly and then pre-incubated at 37°C while shaking for 15 minutes. The reaction was initiated with the addition of NADPH (1 mM final concentration). The incubation continued for 8 min and the reaction quenched by 2x volume of cold acetonitrile containing internal standard (50 nM carbamazepine). The plate was centrifuged for 10 minutes (4000 rcf, 4°C) and the resulting supernatant diluted 1:1 with water for LC/MS/MS analysis. A 12 point standard curve of substrate metabolites over the range of 0.98 nM to 2000 nM. Samples 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 3.0 × 50 mm, 3 µm column (Fortis Technologies Ltd, Cheshire, UK) thermostated at 40°C. HPLC mobile phase A was 0.1% formic acid in water (pH unadjusted), mobile phase B was 0.1% formic acid in acetonitrile (pH unadjusted). The gradient started at 10% B after a 0.2 min hold and was linearly increased to 90% B over 1.2 min; held at 90% B for 0.1 min and returned to 10% 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), with transitions specific for each compound utilizing a Turbo-Ionspray® source in positive ionization mode (5.0 kV spray voltage).</p><p>The IC<sub>50</sub> values for each compound were obtained for the individual CYP enzymes by quantitating the inhibition of metabolite formation for each probe substrate. A 0 µM compound condition (or control) was set to 100% enzymatic activity and the effect of increasing test compound concentrations on enzymatic activity could then be calculated from the % of control activity. Curves were fitted using XLfit 5.2.2 (four-parameter logistic model, equation 201) to determine the concentration that produces half-maximal inhibition (IC<sub>50</sub>).</p><p><b>In vivo DMPK experimental.</b><a class="bk_pop" href="#ml375.r10">10</a>,<a class="bk_pop" href="#ml375.r14">14</a> Compounds were formulated as 10% tween 80 micro suspensions in sterile water at the concentration of 1 mg/ml and administered intraperitoneally to male Sprague- Dawley rats weighing 225 to 250 g (Harlan, Inc., Indianapolis, IN) at the dose of 10 mg/kg. The rat blood and brain were collected at 0.25 hr. Animals were euthanized and decapitated, and the brains were removed, thoroughly washed in cold phosphate buffered saline and immediately frozen on dry ice. Trunk blood was collected in EDTA Vacutainer tubes, and plasma was separated by centrifugation and stored at -80°C until analysis. Plasma was separated by centrifugation (4000 rcf, 4°C) and stored at 80°C until analysis. On the day of analysis, frozen whole-rat brains were weighed and diluted with 1:3 (w/w) parts of 70:30 isopropanol:water. The mixture was then subjected to mechanical homogenation employing a Mini-Beadbeater™ and 1.0 mm Zirconia/Silica Beads (BioSpec Products) followed by centrifugation. The sample extraction of plasma (20 μL) or brain homogenate (20 μL) was performed by a method based on protein precipitation using three volumes of ice-cold acetonitrile containing an internal standard (50 ng/mL carbamazepine). The samples were centrifuged (3000 rcf, 5 min) and supernatants transferred and diluted 1:1 (supernatant: water) into a new 96 well plate, which was then sealed in preparation for LC/MS/MS analysis.</p><p><i>In vivo</i> samples were analyzed via electrospray ionization (ESI) on an AB Sciex API-5500 QTrap (Foster City, CA) instrument that was coupled with Shimadzu LC-20AD pumps (Columbia, MD) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Analytes were separated by gradient elution using a Fortis C18 3.0 × 50 mm, 3 µm column (Fortis Technologies Ltd, Cheshire, UK) thermostated at 40°C. HPLC mobile phase A was 0.1% formic acid in water (pH unadjusted), mobile phase B was 0.1% formic acid in acetonitrile (pH unadjusted). 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), with transitions specific for each compound utilizing a Turbo-Ionspray® source in positive ionization mode (5.0 kV spray voltage). The calibration curves were constructed in blank plasma. All data were analyzed using AB Sciex Analyst software v1.5.1.</p><p><b><i>In vivo pharmacokinetic study in non-human primate.</i></b>(<a class="bk_pop" href="#ml375.r14">14</a>) An <i>in vivo</i> PK study in non-human primates (NHP) was performed by contract with Frontage Laboratories (Exton, PA). VU0483253 was administered IV as a single dose (1 mg/kg) in 10% ethanol 70% PEG400 20% saline vehicle (1 mg/mL) to male cynomolgus monkeys (<i>n</i> = 3), and plasma was collected serially at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours. NCA was performed to obtain PK parameters.</p></div><div id="ml375.s5"><h3>Crystallographic Data for Compound ML375(<a class="bk_pop" href="#ml375.r14">14</a>)</h3><div id="ml375.t2" class="table"><h3><span class="title">Crystal Data</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259190/table/ml375.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml375.t2_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">C<sub>23</sub>H<sub>14</sub>ClF<sub>2</sub>N<sub>2</sub>O<sub>2</sub></td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>F</i>(000) = 868</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>M<sub>r</sub></i> = 423.81</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>D</i><sub>x</sub> = 1.473 Mg m<sup>−3</sup></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Monoclinic, <i>C</i>2</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cu <i>K</i>α radiation, λ = 1.54178 Å</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Hall symbol: C 2y</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cell parameters from 14922 reflections</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>a</i> = 23.2072 (4) Å</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">θ = 3.7–1.0°</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>b</i> = 6.83657 (6) Å</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">μ = 2.15 mm<sup>−1</sup></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>c</i> = 14.4411 (3) Å</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>T</i> = 293 K</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">β = 123.469 (3)°</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rectangular block</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>V</i> = 1911.26 (11) Å<sup>3</sup></td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">× × mm</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>Z</i> = 4</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td></tr></tbody></table></div></div><div id="ml375.t3" class="table"><h3><span class="title">Data Collection</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259190/table/ml375.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml375.t3_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Agilent Xcalibur PX II diffractometer</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>R</i><sub>int</sub> = 0.024</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Radiation source: Enhance (Cu) X-ray Source</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">θ<sub>max</sub> = 72.0°, θ<sub>min</sub> = 3.7°</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">confocal, multilayer</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>h</i> = −28→27</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">17619 measured reflections</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>k</i> = −8→8</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3641 independent reflections</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>l</i> = −17→17</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3544 reflections with <i>I</i> > 2σ(<i>I</i>)</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td></tr></tbody></table></div></div><div id="ml375.t4" class="table"><h3><span class="title">Refinement</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259190/table/ml375.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml375.t4_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Refinement on <i>F</i><sup>2</sup></td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary atom site location: difference Fourier map</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Least-squares matrix: full</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Hydrogen site location: inferred from neighboring sites</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>R</i>[<i>F</i><sup>2</sup> > 2σ(<i>F</i><sup>2</sup>)] = 0.027</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H atoms treated by a mixture of independent and constrained refinement</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>wR</i>(<i>F</i><sup>2</sup>) = 0.073</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>w</i> = 1/[σ<sup>2</sup>(<i>F</i><sub>o</sub><sup>2</sup>) + (0.050<i>P</i>)<sup>2</sup> + 0.6517<i>P</i>] where <i>P</i> = (<i>F</i><sub>o</sub><sup>2</sup> + 2<i>F</i><sub>c</sub><sup>2</sup>)/3</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>S</i> = 1.02</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">(Δ/σ)<sub>max</sub> = 0.001</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3641 reflections</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Δρ<sub>max</sub> = 0.15 e Å<sup>−3</sup></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">281 parameters</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Δρ<sub>min</sub> = −0.27 e Å<sup>−3</sup></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 restraint</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Absolute structure: Flack H D (1983), Acta Cryst. A39, 876-881</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary atom site location: structure-invariant direct methods</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Flack parameter: 0.002 (10)</td></tr></tbody></table></div></div><div id="ml375.s6"><h4>Special Details</h4><p><b>Geometry</b>. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.</p><p><b>Refinement</b>. Refinement of F<sup>2</sup> against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F<sup>2</sup>, conventional R-factors R are based on F, with F set to zero for negative F<sup>2</sup>. The threshold expression of F<sup>2</sup> > 2sigma(F<sup>2</sup>) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F<sup>2</sup> are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger.</p><div id="ml375.f3" class="figure"><div class="graphic"><img src="/books/NBK259190/bin/ml375f3.jpg" alt="Image ml375f3" /></div></div></div></div><div id="ml375.s7"><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/624103" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624103</a>: Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5, summary</p></dd><dt>2.1.2.1.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743025" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743025</a>: Discovery of Novel Negative Allosteric Modulators (NAM) of Muscarinic Receptor M5: Single Point Assay</p></dd><dt>2.1.2.2.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743026" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743026</a>: Discovery of Novel Negative Allosteric Modulators (NAM) of Muscarinic Receptor M5: CRC Assay</p></dd><dt>2.1.2.3.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743098" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743098</a>: Discovery of Novel Negative Allosteric Modulators of Muscarinic Receptor M5: Counterscreen against human M1</p></dd><dt>2.1.2.4.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743099" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743099</a>: Discovery of Novel Negative Allosteric Modulators of Muscarinic Receptor M5: Counterscreen against human M2</p></dd><dt>2.1.2.5.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743103" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743103</a>: Discovery of Negative Allosteric Modulators of Muscarinic Receptor M5: Counterscreen against human M3</p></dd><dt>2.1.2.6.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743105" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743105</a>: Discovery of Novel Negative Allosteric Modulators of Muscarinic Receptor M5: Counterscreen against human M4</p></dd><dt>2.1.2.7.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743104" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743104</a>: Discovery of Novel Negative Allosteric Modulators of Muscarinic Receptor M5: Counterscreen against RAT M5</p></dd><dt>2.1.2.8.</dt><dd><p class="no_top_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743249</a>: <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> M5 NAM Competition in Radioligand Binding assays (Eurofins PanLabs)</p></dd></dl></div><div id="ml375.s8"><h3>2.2. Probe Chemical Characterization</h3><p>Probe compound <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> (CID 71598521, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/163678873" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 163678873</a>, VU0483253) was prepared according to <a class="figpopup" href="/books/NBK259190/figure/ml375.f7/?report=objectonly" target="object" rid-figpopup="figml375f7" rid-ob="figobml375f7">scheme 1</a> and provided the following characterization. (<b><i>S</i>)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1<i>H</i>-imidazo[2,1-<i>a</i>]isoindol-5(9b<i>H</i>)-one</b>: <sup>1</sup>H NMR (400.1 MHz, CDCl<sub>3</sub>) δ (ppm): 8.04-7.99 (m, 1H); 7.90-7.85 (m, 1H); 7.65-7.56 (m, 2H); 7.38-7.30 (m, 3H); 7.25-7.19 (m, 2H); 7.18-7.14 (m, 2H); 4.38-4.30 (m, 1H); 4.01-3.93 (m, 1H); 3.82-3.75 (m, 1H); 3.34-3.25 (m, 1H). <sup>13</sup>C NMR (100.6 MHz, CDCl<sub>3</sub>) δ (ppm): 172.07, 166.84, 151.81 (dd, <i>J</i><i><sub>CF</sub></i> = 254 Hz, 12.7 Hz), 150.33 (dd, <i>J<sub>C-F</sub></i> = 252 Hz, 13 Hz) 145.77, 136.65, 134.94, 133.55, 132.91 (t, <i>J</i> = 4.8 Hz), 131.88, 130.61, 129.06, 128.97, 127.53, 124.03, 123.62 (dd, <i>J</i> = 6.8 Hz, 4 Hz), 117.94 (d, <i>J</i> = 17 Hz), 116.83 (d, <i>J</i> = 18 Hz), 87.37, 52.24, 39.70. SFC (214 nM) <i>R<sub>T</sub></i> = 3.591 min (>98%). HRMS (TOF, ES+) C<sub>23</sub>H<sub>16</sub>N<sub>2</sub>O<sub>2</sub>F<sub>2</sub>Cl [M+H]<sup>+</sup> calc. mass 425.0868, found 425.0872. Specific rotation [α]
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<math id="ml375.m5" display="inline"><mrow><mfrac><mn>23</mn><mi>D</mi></mfrac></mrow></math></span> = -168.6° (<i>c</i> = 0.75, CHCl<sub>3</sub>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml375f7" co-legend-rid="figlgndml375f7"><a href="/books/NBK259190/figure/ml375.f7/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml375f7" rid-ob="figobml375f7"><img class="small-thumb" src="/books/NBK259190/bin/ml375f7.gif" src-large="/books/NBK259190/bin/ml375f7.jpg" alt="Scheme 1. Preparation of ML375." /></a><div class="icnblk_cntnt" id="figlgndml375f7"><h4 id="ml375.f7"><a href="/books/NBK259190/figure/ml375.f7/?report=objectonly" target="object" rid-ob="figobml375f7">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Preparation of ML375. </p></div></div><p><b>Solubility:</b> Solubility for <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> in PBS (@ pH = 7.4, 23 °C, final DMSO concentration 1%) was determined to be < 4 μM, which is not optimal, but not unexpected in light of its structure. Future efforts will be directed towards improving the solubility for this novel series of M<sub>5</sub> NAMs through the introduction of weakly basic nitrogens or other solubility enhancing groups, and the truncation of lipophilic regions, as allowed by SAR.</p><p><b>Stability:</b> Stability was determined for <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> at 23 °C in PBS (no antioxidants or other protectorants, initial <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> concentration = 1.0 µM and final DMSO concentration 10%). After 48 hours, 89% of the initial concentration of <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> remained, indicating that <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> was fairly stable to these buffer conditions, or that a small percentage of <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> precipitated from solution over the course of the experiment despite the presence of additional DMSO relative to the solubility experiment.</p><div id="ml375.t5" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259190/table/ml375.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml375.t5_lrgtbl__"><table><thead><tr><th id="hd_h_ml375.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_h_ml375.t5_1_1_1_2" colspan="6" rowspan="1" style="text-align:center;vertical-align:middle;">Percent Remaining (%)</th></tr><tr><th headers="hd_h_ml375.t5_1_1_1_1" id="hd_h_ml375.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th headers="hd_h_ml375.t5_1_1_1_2" id="hd_h_ml375.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0 Min</th><th headers="hd_h_ml375.t5_1_1_1_2" id="hd_h_ml375.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15 Min</th><th headers="hd_h_ml375.t5_1_1_1_2" id="hd_h_ml375.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">30 Min</th><th headers="hd_h_ml375.t5_1_1_1_2" id="hd_h_ml375.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">90 min.</th><th headers="hd_h_ml375.t5_1_1_1_2" id="hd_h_ml375.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">24 Hour</th><th headers="hd_h_ml375.t5_1_1_1_2" id="hd_h_ml375.t5_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_ml375.t5_1_1_1_1 hd_h_ml375.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a>, CID 71598521</td><td headers="hd_h_ml375.t5_1_1_1_2 hd_h_ml375.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100</td><td headers="hd_h_ml375.t5_1_1_1_2 hd_h_ml375.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100</td><td headers="hd_h_ml375.t5_1_1_1_2 hd_h_ml375.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100</td><td headers="hd_h_ml375.t5_1_1_1_2 hd_h_ml375.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">97</td><td headers="hd_h_ml375.t5_1_1_1_2 hd_h_ml375.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">92</td><td headers="hd_h_ml375.t5_1_1_1_2 hd_h_ml375.t5_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">89</td></tr></tbody></table></div></div><p><b>Compounds added to the SMR collection (MLS#s):</b> MLS005000729 (<a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a>, CID 71598521, 26.2 mg); MLS005000730 (CID 71598550, 8 mg); MLS005000731 (CID 71598549, 6.6 mg); MLS005000732 (CID 12204567, 7.1 mg); MLS005000733 (CID 2999410, 5.7 mg); MLS005000734 (CID 71598548, 6.0 mg).</p></div><div id="ml375.s9"><h3>2.3. Probe Preparation</h3><p>(<b><i>S</i>)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1<i>H</i>-imidazo[2,1-<i>a</i>]isoindol-5(9b<i>H</i>)-one, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a></b>: To a mixture of 2-(4-chlorobenzoyl)benzoic acid (5.21 g, 20.0 mmol, 1 eq.) and ethylenediamine (2.67 mL, 40.0 mmol, 2 eq.) in toluene (30 mL, 0.67 M) was added <i>p</i>-toluenesulfonic acid monohydrate (∼0.1 g, 3 mol%). A Dean-Stark trap was used to remove water while the mixture was allowed to stir at reflux for 4 hours. After cooling to ambient temperature, the reaction mixture was dissolved in dichloromethane. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and then with brine. Solvent was removed under reduced pressure and the crude product was recrystallized from ethanol to give 2.86 g of pure 9b-(4-chlorophenyl)-2,3-dihydro-1<i>H</i>-imidazo[2,1-<i>a</i>]isoindol-5(9b<i>H</i>)-one (50 % yield).</p><p>To a solution of 9b-(4-chlorophenyl)-2,3-dihydro-<i>1H</i>-imidazo[2,1-<i>a</i>]isoindol-5(9b<i>H</i>)-one (15 mg, 0.053 mmol, 1.0 eq.) and DIPEA (18 μL, 0.105 mmol, 2.0 eq.) in DCM (0.53 mL, 0.1 M) was added 3,4-difluorobenzoyl chloride (9.9 μL, 0.079 mmol, 1.5 eq.). The reaction was allowed to stir at ambient temperature for 2 hours. The reaction was quenched with methanol and the organics were concentrated on a heated air-drying block. Crude product was purified via Gilson preparative LC to obtain 12.0 mg of 9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1<i>H</i>-imidazo[2,1-<i>a</i>]isoindol-5(9b<i>H</i>)-one (53 % yield).</p><p>The second eluting pure enantiomer of 9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1<i>H</i>-imidazo[2,1-<i>a</i>]isoindol-5(9b<i>H</i>)-one (<a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a>) was separated via CO<sub>2</sub> supercritical fluid chromatography (Lux cellulose-3 10 × 250 mm column at 40 °C, backpressure regulated at 100 bar, MeOH co-solvent, 10% isocratic prep over 7 minutes at 15 mL/min) and was determined to have an ee of >98% by chiral HPLC analysis (Lux cellulose-3 4.6 × 250 mm column at 40 °C, backpressure regulated at 100 bar, MeOH co-solvent, 5-50% over 7 minutes at 3.5 mL/min.</p></div></div><div id="ml375.s10"><h2 id="_ml375_s10_">3. Results</h2><p>Functional, triple-add assay protocols were provided to the screening center detailing a heterogeneous assay conducted in a 384-well format for M<sub>1</sub>, M<sub>4</sub> and M<sub>5</sub> human receptor transfected CHO cells and CHO-K1 untransfected cells (assay provider at Vanderbilt). The screening center (SRIMSC) then adapted and modified these procedures into a homogeneous, 1536-well format to facilitate a more rapid and economical screening of the MLSMR (∼360, 000 compounds). From this effort, we identified CID 5189681 (<b>5</b>) as a weak (M<sub>5</sub> IC<sub>50</sub> >10 μM, 41% ACh min), but selective (versus M<sub>1</sub> and M<sub>4</sub>) antagonist. <a class="figpopup" href="/books/NBK259190/figure/ml375.f4/?report=objectonly" target="object" rid-figpopup="figml375f4" rid-ob="figobml375f4">Figure 2</a> shows the structure of <b>5</b>, the CRC window from the confirmation screen at SCRIPPS as well as a CRC generated at Vanderbilt from the HTS DMSO stock. To confirm the structure of the compound, we resynthesized 5 in a two-step sequence (<a class="figpopup" href="/books/NBK259190/figure/ml375.f5/?report=objectonly" target="object" rid-figpopup="figml375f5" rid-ob="figobml375f5">Scheme 2</a>). Commercial acid 6 was condensed with ethylene diamine to produce tricycle 7, followed by acylation with 4-fluorobenzoyl chloride to afford the HTS hit 5 in 74% overall yield. We then assayed fresh powder of 5, and were very pleased to find that new material was significantly more potent (hM<sub>5</sub> IC<sub>50</sub> = 3.49 μM, rM<sub>5</sub> IC<sub>50</sub> = 5.67 μM, M<sub>1</sub>-M<sub>4</sub> IC<sub>50</sub>s >30 μM), yet retained M<sub>5</sub> selectivity (<a class="figpopup" href="/books/NBK259190/figure/ml375.f6/?report=objectonly" target="object" rid-figpopup="figml375f6" rid-ob="figobml375f6">Figure 3</a>). Thus, we prepared libraries of over 120 analogs, following slight modifications to <a class="figpopup" href="/books/NBK259190/figure/ml375.f7/?report=objectonly" target="object" rid-figpopup="figml375f7" rid-ob="figobml375f7">Scheme 1</a>, surveying SAR in five regions of the HTS hit 5 (<a class="figpopup" href="/books/NBK259190/figure/ml375.f6/?report=objectonly" target="object" rid-figpopup="figml375f6" rid-ob="figobml375f6">Figure 3</a>), and screened in full CRC mode against human M<sub>5</sub>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml375f4" co-legend-rid="figlgndml375f4"><a href="/books/NBK259190/figure/ml375.f4/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml375f4" rid-ob="figobml375f4"><img class="small-thumb" src="/books/NBK259190/bin/ml375f4.gif" src-large="/books/NBK259190/bin/ml375f4.jpg" alt="Figure 2. A) HTS confirmatory data from SCRIPPS for CID 5189681 (5) showing the antagonist window CRCs on M1, M4, M5 and non-transferred parental CHO cell line, highlighting the selectivity of 5 for M5." /></a><div class="icnblk_cntnt" id="figlgndml375f4"><h4 id="ml375.f4"><a href="/books/NBK259190/figure/ml375.f4/?report=objectonly" target="object" rid-ob="figobml375f4">Figure 2</a></h4><p class="float-caption no_bottom_margin">A) HTS confirmatory data from SCRIPPS for CID 5189681 (5) showing the antagonist window CRCs on M1, M4, M5 and non-transferred parental CHO cell line, highlighting the selectivity of <i>5</i> for M5. B) Confirmation CRC at VUMC for on human M5 (a weak M5 inhibitor: <a href="/books/NBK259190/figure/ml375.f4/?report=objectonly" target="object" rid-ob="figobml375f4">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml375f5" co-legend-rid="figlgndml375f5"><a href="/books/NBK259190/figure/ml375.f5/?report=objectonly" target="object" title="Scheme 2" class="img_link icnblk_img figpopup" rid-figpopup="figml375f5" rid-ob="figobml375f5"><img class="small-thumb" src="/books/NBK259190/bin/ml375f5.gif" src-large="/books/NBK259190/bin/ml375f5.jpg" alt="Scheme 2. Synthetic route to prepare the HTS hit, CID 5189681 (5)." /></a><div class="icnblk_cntnt" id="figlgndml375f5"><h4 id="ml375.f5"><a href="/books/NBK259190/figure/ml375.f5/?report=objectonly" target="object" rid-ob="figobml375f5">Scheme 2</a></h4><p class="float-caption no_bottom_margin">Synthetic route to prepare the HTS hit, CID 5189681 (5). </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml375f6" co-legend-rid="figlgndml375f6"><a href="/books/NBK259190/figure/ml375.f6/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml375f6" rid-ob="figobml375f6"><img class="small-thumb" src="/books/NBK259190/bin/ml375f6.gif" src-large="/books/NBK259190/bin/ml375f6.jpg" alt="Figure 3. Rat and human M5 potency and mAChR selectivity of fresh powder of CID 5189681 (5), and the chemical optimization plan to establish SAR for the series en route to an MLPCN probe." /></a><div class="icnblk_cntnt" id="figlgndml375f6"><h4 id="ml375.f6"><a href="/books/NBK259190/figure/ml375.f6/?report=objectonly" target="object" rid-ob="figobml375f6">Figure 3</a></h4><p class="float-caption no_bottom_margin">Rat and human M5 potency and mAChR selectivity of fresh powder of CID 5189681 (5), and the chemical optimization plan to establish SAR for the series en route to an MLPCN probe. </p></div></div><p>SAR was very shallow/flat, and reminiscent of allosteric SAR. Very quickly, one compound stood out form all those prepared, CID 71598549 (8) a potent, sub-micromolar human M<sub>5</sub> antagonist/NAM (hM<sub>5</sub> IC<sub>50</sub> = 480 nM, rM<sub>5</sub> IC<sub>50</sub> = 1,100 nM), with no activity at hM<sub>1</sub>-M<sub>4</sub> (IC<sub>50</sub>s >30 mM) (<a class="figpopup" href="/books/NBK259190/figure/ml375.f8/?report=objectonly" target="object" rid-figpopup="figml375f8" rid-ob="figobml375f8">Figure 4</a>). As CID 71598549 (8) was a racemic molecule, we then separated the enantiomers by chiral super critical fluid chromatography to >99% ee. Opticcal rotations were obtained, and all of the M5 inhibitory activity was found to reside in the (-)-enantiomer (a ∼-160°). Thus, whereas the racemic <b>8</b> had a hM<sub>5</sub> IC<sub>50</sub> of 480 nM, the (-)-enantiomer CID71598521 (<b>9</b>) had a hM<sub>5</sub> IC<sub>50</sub> of 300 nM, while the (+)-enantiomer's (CID 71598550, <b>10</b>) hM<sub>5</sub> IC<sub>50</sub> was >30 mM (<a class="figpopup" href="/books/NBK259190/figure/ml375.f9/?report=objectonly" target="object" rid-figpopup="figml375f9" rid-ob="figobml375f9">Figure 5</a>). This was an exciting discovery, but we still did not know the absolute stereochemistry at the 9b stereocenter. Thus, we initiated an effort to grow X-ray quality crystals in an attempt to utilize X-ray crystallography to elucidate the absolute stereochemistry at 9b. In short order, crystals were grown that displayed excellent diffraction and enabled our small molecule X-ray facility to definitively assign the absolute stereochemistry of the 9b stereocenter in CID 71598521 (<b>9</b>) as the (<i>S</i>)-enantiomer. Thus, this series displayed enantiospecific inhibition of the M<sub>5</sub> receptor. With the single enantiomer in hand, we assessed mAChR selectivity at both rat and human M<sub>1</sub>-M<sub>4</sub> (<a class="figpopup" href="/books/NBK259190/figure/ml375.f10/?report=objectonly" target="object" rid-figpopup="figml375f10" rid-ob="figobml375f10">Figure 7</a>, <a href="#ml375.s11">section 3.1</a>), where CID 71598521 (<b>9</b>) was devoid of activity at M<sub>1</sub>-M<sub>4</sub> (IC<sub>50</sub>s >30 mM) for both species. Subsequent detailed molecular pharmacology studies were performed to establish its mechanism of action. To determine the mechanism of action of (<i>S</i>)-CID 71598521 (<b>9</b>), whether orthosteric or allosteric, we first performed competition binding experiments with the orthosteric mAChR antagonist [<sup>3</sup>H]-NMS and compared this to atropine. (<i>S</i>)-CID 71598521 (<b>9</b>) displayed no competition [<sup>3</sup>H]-NMS for binding to hM<sub>5</sub>, suggesting an allosteric mode of receptor inhibition. To further investigate an allosteric mechanism, we also performed [<sup>3</sup>H]-NMS dissociation kinetic experiments, with hM<sub>5</sub> cell membranes, which revealed that (<i>S</i>)-CID 71598521 (<b>9</b>) decreased the dissociation rate of [<sup>3</sup>H-NMS], further confirming an allosteric effect of (<i>S</i>)-CID 71598521 (<b>9</b>) on the orthosteric site. Thus, (<i>S</i>)-CID 71598521 (<b>9</b>) is the first M<sub>5</sub>-selective negative allosteric modulator (NAM), and declared an MLPCN probe, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml375f8" co-legend-rid="figlgndml375f8"><a href="/books/NBK259190/figure/ml375.f8/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml375f8" rid-ob="figobml375f8"><img class="small-thumb" src="/books/NBK259190/bin/ml375f8.gif" src-large="/books/NBK259190/bin/ml375f8.jpg" alt="Figure 4. Structure and mAChR selectivity of CID 71598549 (8), the first submicrobolar hM5 NAM." /></a><div class="icnblk_cntnt" id="figlgndml375f8"><h4 id="ml375.f8"><a href="/books/NBK259190/figure/ml375.f8/?report=objectonly" target="object" rid-ob="figobml375f8">Figure 4</a></h4><p class="float-caption no_bottom_margin">Structure and mAChR selectivity of CID 71598549 (8), the first submicrobolar hM5 NAM. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml375f9" co-legend-rid="figlgndml375f9"><a href="/books/NBK259190/figure/ml375.f9/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml375f9" rid-ob="figobml375f9"><img class="small-thumb" src="/books/NBK259190/bin/ml375f9.gif" src-large="/books/NBK259190/bin/ml375f9.jpg" alt="Figure 5. Structures and M5 activity of racemic 8, the (-)-enantiomer 9 and the (+) –enantiomer 10." /></a><div class="icnblk_cntnt" id="figlgndml375f9"><h4 id="ml375.f9"><a href="/books/NBK259190/figure/ml375.f9/?report=objectonly" target="object" rid-ob="figobml375f9">Figure 5</a></h4><p class="float-caption no_bottom_margin">Structures and M5 activity of racemic 8, the (-)-enantiomer 9 and the (+) –enantiomer 10. All of the M5 activity resides in the (-)-enantiomer . </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml375f10" co-legend-rid="figlgndml375f10"><a href="/books/NBK259190/figure/ml375.f10/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml375f10" rid-ob="figobml375f10"><img class="small-thumb" src="/books/NBK259190/bin/ml375f10.gif" src-large="/books/NBK259190/bin/ml375f10.jpg" alt="Figure 7. Dose Response Curves ML375." /></a><div class="icnblk_cntnt" id="figlgndml375f10"><h4 id="ml375.f10"><a href="/books/NBK259190/figure/ml375.f10/?report=objectonly" target="object" rid-ob="figobml375f10">Figure 7</a></h4><p class="float-caption no_bottom_margin">Dose Response Curves ML375. A) Human mAChR selectivity. ML375 is selective for hM<sub>5</sub> (hM5 IC<sub>50</sub> = 300 nM, hM<sub>1</sub>-M<sub>4</sub> IC<sub>5o</sub>s >30 μM); B) rat mAChR selectivity ML375 is selective for rM<sub>5</sub> (rM5 IC<sub>50</sub> = 790 nM rM<sub>1</sub>-M<sub>4</sub> IC<sub>5o</sub>s >30 μM)</p></div></div><p>We next assessed the DMPK profile and ancillary pharmacology profile of <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> (<a class="figpopup" href="/books/NBK259190/table/ml375.t6/?report=objectonly" target="object" rid-figpopup="figml375t6" rid-ob="figobml375t6">Tables 1</a>-<a class="figpopup" href="/books/NBK259190/table/ml375.t8/?report=objectonly" target="object" rid-figpopup="figml375t8" rid-ob="figobml375t8">3</a>, <a href="#ml375.s13">section 3.3</a>). <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> was stable and possessed acceptable solubility. Evaluation of the <i>in vitro</i> and <i>in vivo</i> DMPK profile of <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> revealed the compound to possess high metabolic stability with low hepatic microsomal intrinsic clearance (CL<sub>int;</sub>; human 2.6 mL/min/kg, cynomolgus monkey (cyno), 20 mL/min/kg, rat, 24 mL/min/kg) and a corresponding low predicted hepatic clearance in multiple species (CL<sub>hep</sub>; human, 2.3 mL/min/kg, cyno, 14 mL/min/kg rat, 18 mL/min/kg). Correspondingly, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> exhibited low clearance (CL<sub>p</sub>, 2.5 mL/min/kg) and a long elimination half-life (t<sub>1/2</sub>, 80hr) in rodents (male, Sprague-Dawley rat, 1 mg/kg IV, <i>n</i> = 2) and nonhuman primates (male, cynomolgus monkey, 1 mg/kg, CL<sub>p</sub>, 3.0 mL/min/kg, t<sub>1/2</sub>, 10 hr, <i>n</i> = 3). Consistent with a low clearance compound, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> also demonstrated high oral bioavailability (%F, 80) following administration of a suspension-dose to male SD rats (<i>n</i>=2) with a maximal plasma concentration (C<sub>max</sub>) of 1.4 µM and a corresponding time to reach C<sub>max</sub> (T<sub>max</sub>) of 7 hours. The distribution of <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> was characterized by a low fraction unbound in plasma (f<sub>u,p</sub>; human: 0.013, cyno: 0.001, rat: 0.029) and a high nonspecific binding in brain homogenate (f<sub>u,br</sub>; rat: 0.003). Following an oral CNS distribution study in rat (male, Sprague-Dawley, <i>n</i> = 2; 10 mg/kg) we observed total and unbound brain-plasma partition coefficients of 1.8 and 0.2 (K<sub>p</sub>, K<sub>p,uu</sub>, respectively) one hour post-administration. <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> displayed an acceptable human cytochrome P450 inhibition profile producing acceptable IC<sub>50</sub> values for 3A4 (16 µM), 1A2 (25 µM, 2C9 (7.4 µM) and 2D6 (26 µM). Moreover, in a Eurofins radioligand binding panel of 68 GPCRs, ion channels and transporter, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> displayed significant binding (>50% inhibition @10 μM) at only 1 target (CB<sub>1</sub>, 66%), but no functional activity at this target in a subsequent assay.(<a class="bk_pop" href="#ml375.r14">14</a>)</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml375t6"><a href="/books/NBK259190/table/ml375.t6/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml375t6" rid-ob="figobml375t6"><img class="small-thumb" src="/books/NBK259190/table/ml375.t6/?report=thumb" src-large="/books/NBK259190/table/ml375.t6/?report=previmg" alt="Table 1. Pan Labs profiling of ML375." /></a><div class="icnblk_cntnt"><h4 id="ml375.t6"><a href="/books/NBK259190/table/ml375.t6/?report=objectonly" target="object" rid-ob="figobml375t6">Table 1</a></h4><p class="float-caption no_bottom_margin">Pan Labs profiling of ML375. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml375t8"><a href="/books/NBK259190/table/ml375.t8/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml375t8" rid-ob="figobml375t8"><img class="small-thumb" src="/books/NBK259190/table/ml375.t8/?report=thumb" src-large="/books/NBK259190/table/ml375.t8/?report=previmg" alt="Table 3. In vitro and in vivo DMPK profile of ML375." /></a><div class="icnblk_cntnt"><h4 id="ml375.t8"><a href="/books/NBK259190/table/ml375.t8/?report=objectonly" target="object" rid-ob="figobml375t8">Table 3</a></h4><p class="float-caption no_bottom_margin"><i>In vitro</i> and <i>in vivo</i> DMPK profile of ML375. </p></div></div><div id="ml375.f11" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%206.%20X-ray%20structure%20of%20the%20active%20(-)-enantiomer%2C%20CID%2071598521%20(9)%2C%20which%20proved%20to%20have%20the%20(S)-configuration.&p=BOOKS&id=259190_ml375f11.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/NBK259190/bin/ml375f11.jpg" alt="Figure 6. X-ray structure of the active (-)-enantiomer, CID 71598521 (9), which proved to have the (S)-configuration." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 6</span><span class="title">X-ray structure of the active (-)-enantiomer, CID 71598521 (9), which proved to have the (S)-configuration</span></h3></div><div id="ml375.s11"><h3>3.1. Dose Response Curves for Probe</h3><p><a class="figpopup" href="/books/NBK259190/figure/ml375.f10/?report=objectonly" target="object" rid-figpopup="figml375f10" rid-ob="figobml375f10">Figure 7</a> shows dose response curves for <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> for both human and rat M<sub>5</sub>, as well as mAChR selectivity across M<sub>1</sub>-M<sub>5</sub>.</p></div><div id="ml375.s12"><h3>3.2. Cellular Activity</h3><p>The primary screening assays and the muscarinic selectivity (M<sub>1</sub> – M<sub>4</sub>) assays are cell-based assays, indicating that <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> can interact with its molecular target (the M<sub>5</sub> receptor) when applied to cells (at a final DMSO concentration of < 0.5 %). The probe compound did not exhibit acute toxicity in cell-based assays at concentrations up to 30 µM.</p></div><div id="ml375.s13"><h3>3.3. Profiling Assays</h3><p>To more fully characterize <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a>, and to better inform the scientific community about its potential off target activities, this structurally novel M<sub>5</sub> NAM was tested using Eurofins' Lead Profiling Screen. This battery of radioligand binding assays consists of 68 common GPCRs, ion channels and transporters where the test compound (<a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a>) was present at 10 µM. Responses were considered significant if > 50% inhibition was observed. However it should be pointed out that these are only single-point values and that functional selectivity may be significantly better than suggested by these “% inhibitions.” <a class="figpopup" href="/books/NBK259190/table/ml375.t6/?report=objectonly" target="object" rid-figpopup="figml375t6" rid-ob="figobml375t6">Table 1</a> presents the Pan Labs results for <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a>, which showed only a single significant response in just the cannabinoid CB<sub>1</sub> assay. However, in a subsequent functional assay, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> exhibited no activity at CB<sub>1</sub>.</p><p>Additionally, a set of calculated physical properties were determined for <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> and appear in <a class="figpopup" href="/books/NBK259190/table/ml375.t7/?report=objectonly" target="object" rid-figpopup="figml375t7" rid-ob="figobml375t7">Table 2</a>. For comparison, this table also shows the averaged values for compounds appearing in the MDDR database (MDL Drug Data Report database, 2010) at two stages of clinical development (Phase I and Launched). <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> compares reasonably well with the average values for both Phase I and Launched compounds, but obviously has room for improvement with respect to cLogP and solubility. <a class="figpopup" href="/books/NBK259190/table/ml375.t8/?report=objectonly" target="object" rid-figpopup="figml375t8" rid-ob="figobml375t8">Table 3</a> summarizes key DMPK parameters for <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> in both rat and cyno monkey. In rat, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> displayed low clearance (2.5 mL/min/kg) with a t<sub>1/2</sub> of 80 hr and with 80% oral bioavailability. In cyno monkey, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> possessed similarly low clearance (3.5 mL/min/kg) with a t<sub>1/2</sub> of 10 hours. In a 10 mg/kg oral plasma-brain level (PBL) study, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> possessed a brain:plasma ratio of 1.8, indicating excellent CNS exposure.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml375t7"><a href="/books/NBK259190/table/ml375.t7/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml375t7" rid-ob="figobml375t7"><img class="small-thumb" src="/books/NBK259190/table/ml375.t7/?report=thumb" src-large="/books/NBK259190/table/ml375.t7/?report=previmg" alt="Table 2. Calculated property comparison between ML375 and MDDR compounds." /></a><div class="icnblk_cntnt"><h4 id="ml375.t7"><a href="/books/NBK259190/table/ml375.t7/?report=objectonly" target="object" rid-ob="figobml375t7">Table 2</a></h4><p class="float-caption no_bottom_margin">Calculated property comparison between ML375 and MDDR compounds. </p></div></div></div></div><div id="ml375.s14"><h2 id="_ml375_s14_">4. Discussion</h2><div id="ml375.s15"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p>To date, the majority of mAChR antagonists are unselective ligands, such as scopolamine, atropine or moderately selective ligands such as the moderately M<sub>1</sub>-preferring pirenzepine Recently in 2013, the first M<sub>5</sub>-preferring antagonist (based on binding data – not functional) was reported.(<a class="bk_pop" href="#ml375.r13">13</a>) Compound <b>4</b> bound to M<sub>5</sub> with a K<sub>i</sub> of 2.2 μM M<sub>5</sub> antagonist with 11-fold selectivity versus M<sub>1</sub>-M<sub>4</sub>, but no DMPK or CNS penetration data was disclosed. Moreover, there are no known M<sub>5</sub> NAMs, and no highly selective and CNS penetrant M<sub>5</sub> antagonists/NAMs. <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> marks a major advancement in the field. With an M<sub>5</sub> IC<sub>50</sub> of 300 nM, no activity at M<sub>1</sub>-M<sub>4</sub> IC<sub>50</sub>s >30 μM), clean ancillary pharmacology, good DMPK profile in both rat and cyno (moderate clearance, 80 %F) and high brain exposure (B:P ratio of 1.8, when dosed orally) make <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> a unique tool to selectively probe M5 inhibition in cells, via electrophysiology and <i>in vivo</i> – a true first in class probe. Finally, <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> rests firmly in the public domain, which will allow the MLPCN to freely provide this probe to the biomedical research community. We just published this probe in <i>J. Med. Chem</i>.,(<a class="bk_pop" href="#ml375.r14">14</a>) and have already filled request for over 10 g of <a href="/pcsubstance/?term=ML375[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML375</a> to outside labs.</p></div></div><div id="ml375.s16"><h2 id="_ml375_s16_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml375.r1">Yasuda RP, Ciesla W, Flores LR, Wall SJ, Li M, Satkus SA, Weisstein JS, Spagnola BV, Wolfe BB. <span><span class="ref-journal">Mol. Pharmacol. </span>1993;<span class="ref-vol">43</span>:149–157.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8429821" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8429821</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml375.r2">Wess J, Eglen RM, Gautam D. <span><span class="ref-journal">Nat. Rev. Drug Discov. </span>2007;<span class="ref-vol">6</span>:721–733.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17762886" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17762886</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml375.r3">Steidl S, Miller AD, Blaha CD. Yeomans. <span><span class="ref-journal">PLoS ONE. </span>2011;<span class="ref-vol">6</span>(11):e27538.</span> [<a href="/pmc/articles/PMC3216953/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3216953</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22102904" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22102904</span></a>] [<a href="http://dx.crossref.org/10.1371/journal.pone.0027538" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml375.r4">Vilaro MT, Palacios JM, Mengod G. <span><span class="ref-journal">Neurosci. Lett. </span>1990;<span class="ref-vol">114</span>:154–159.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/2395528" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2395528</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml375.r5">Weiner DM, Levey AI, Brann MR. <span><span class="ref-journal">Proc. Natl. Acad. Sci. USA. </span>1990;<span class="ref-vol">87</span>:7050–7054.</span> [<a href="/pmc/articles/PMC54680/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC54680</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/2402490" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2402490</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml375.r6">Anney RJL, Lotfi-Miri M, Olsson CA, Reid SC, Hemphill SA, Patton GC. <span><span class="ref-journal">BMC Genetics. </span>2007;<span class="ref-vol">8</span>(46)</span> [<a href="/pmc/articles/PMC1978498/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1978498</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17608938" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17608938</span></a>] [<a href="http://dx.crossref.org/10.1186/1471-2156-8-46" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml375.r7">Yamada M, Lamping KG, Duttaroy A, Zhang W, Cui Y, Bymaster FP, McKinzie DL, Felder CC, Deng CX, Faraci FM, Wess J. <span><span class="ref-journal">Proc. Natl. Acad. Sci. USA. </span>2001;<span class="ref-vol">98</span>:14096–14101.</span> [<a href="/pmc/articles/PMC61174/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC61174</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11707605" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11707605</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml375.r8">Thomsen M, Wörtwein G, Fink-Jessen A, Woldbye DPD, Wess J, Caine SB. <span><span class="ref-journal">Psychopharmacology. </span>2007;<span class="ref-vol">192</span>:97–110.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17310388" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17310388</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml375.r9">Araya R, Noguchi T, Yuhki M, Kitamura N, Higuchi M, Saido TC, Seki K, Itohara S, Kawano M, Tanemura K, Takashima A, Yamada K, Kondoh Y, Kanno I, Wess J, Yamada M. <span><span class="ref-journal">Neurobiol. Dis. </span>2006;<span class="ref-vol">24</span>:334–344.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16956767" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16956767</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml375.r10">Bridges TM, Marlo JE, Niswender CM, Jones CK, Jadhav SB, Gentry PR, Plumley HC, Weaver CD, Conn PJ, Lindsley CW. <span><span class="ref-journal">J. Med. Chem. </span>2009;<span class="ref-vol">52</span>:3445–3448.</span> [<a href="/pmc/articles/PMC3875304/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3875304</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19438238" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19438238</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml375.r11">Bridges TM, Kennedy JP, Cho HP, Breininger ML, Gentry PR, Hopkins CR, Conn PJ, Lindsley CW. <span><span class="ref-journal">Bioorg. Med. Chem. Lett. </span>2010;<span class="ref-vol">20</span>:558–562.</span> [<a href="/pmc/articles/PMC3177601/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3177601</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20004578" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20004578</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml375.r12">Gentry PR, Bridges TM, Lamsal A, Vinson PN, Smith E, Chase P, Hodder PS, Engers JL, Niswender CM, Daniels JS, Conn PJ, Wood MR, Lindsley CW. <span><span class="ref-journal">Bioorg. Med. Chem. Lett. </span>2013;<span class="ref-vol">23</span>:2996–3000.</span> [<a href="/pmc/articles/PMC3634896/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3634896</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23562060" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23562060</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml375.r13">Zheng G, Smith AM, Huang X, Subramanian KL, Siripurapu KB, Deaciuc A, Zhan CG, Dwoskin LP. <span><span class="ref-journal">J. Med. Chem. </span>2013;<span class="ref-vol">56</span>:1693–1703.</span> [<a href="/pmc/articles/PMC3676450/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3676450</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23379472" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23379472</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml375.r14">Gentry PR, Kokubo M, Bridges TM, Kett NR, Harp JM, Cho HP, Smith E, Chase P, Hodder PS, Niswender CM, Daniels JS, Conn PJ, Wood MR, Lindsley CW. <span><span class="ref-journal">J. Med. Chem. </span>2013;<span class="ref-vol">56</span>:9351–9355.</span> [<a href="/pmc/articles/PMC3876027/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3876027</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/24164599" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24164599</span></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/NBK259190/?report=reader">PubReader</a></li><li><a href="/books/NBK259190/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK259190" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK259190" style="display:none" title="Cite this Page"><div class="bk_tt">Gentry PR, Kokubo M, Bridges TM, et al. Development of the First Potent, Selective and CNS penetrant M5 Negative Allosteric Modulator (NAM) 2013 Dec 9 [Updated 2014 Sep 18]. 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="#ml375.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml375.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml375.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml375.s10" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml375.s14" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml375.s16" ref="log$=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=3318648" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3318648" ref="log$=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=3318648" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/25834908" ref="ordinalpos=1&linkpos=1&log$=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> Development of the First CNS penetrant M5 Positive Allosteric Modulator (PAM) Based on a Novel, non-Isatin Core.</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> Development of the First CNS penetrant M5 Positive Allosteric Modulator (PAM) Based on a Novel, non-Isatin Core.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Gentry PR, Kokubo M, Bridges TM, Daniels JS, Niswender CM, Smith E, Chase P, Hodder PS, Rosen H, Conn PJ, 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/25834910" ref="ordinalpos=1&linkpos=2&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Development of a Novel, Orthosteric M5 Antagonist Possessing a High Degree of Muscarinic Subtype Selectivity.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Development of a Novel, Orthosteric M5 Antagonist Possessing a High Degree of Muscarinic Subtype Selectivity.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Gentry PR, Kokubo M, Bridges TM, Daniels JS, Niswender CM, Smith E, Chase P, Hodder PS, Rosen H, Conn PJ, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" 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Epub 2021 Aug 5.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/25542588" ref="ordinalpos=1&linkpos=5&log$=relatedarticles&logdbfrom=pubmed">Further optimization of the M5 NAM MLPCN probe ML375: tactics and challenges.</a><span class="source">[Bioorg Med Chem Lett. 2015]</span><div class="brieflinkpop offscreen_noflow">Further optimization of the M5 NAM MLPCN probe ML375: tactics and challenges.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Kurata H, Gentry PR, Kokubo M, Cho HP, Bridges TM, Niswender CM, Byers FW, Wood MR, Daniels JS, Conn PJ, 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. 2015 Feb 1; 25(3):690-4. 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