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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Functional antagonists of the Apelin (APJ) receptor - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]">
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<meta name="citation_title" content="Functional antagonists of the Apelin (APJ) receptor">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/03/07">
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<meta name="citation_author" content="Patrick R. Maloney">
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<meta name="citation_author" content="Pasha Khan">
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<meta name="citation_author" content="Michael Hedrick">
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<meta name="citation_author" content="Palak Gosalia">
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<meta name="citation_author" content="Monika Milewski">
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<meta name="citation_author" content="Linda Li">
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<meta name="citation_author" content="Gregory P. Roth">
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<meta name="citation_author" content="Eduard Sergienko">
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<meta name="citation_author" content="Eigo Suyama">
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<meta name="citation_author" content="Eliot Sugarman">
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<meta name="citation_author" content="Kevin Nguyen">
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<meta name="citation_author" content="Alka Mehta">
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<meta name="citation_author" content="Stefan Vasile">
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<meta name="citation_author" content="Ying Su">
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<meta name="citation_author" content="Derek Stonich">
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<meta name="citation_author" content="Hung Nguyen">
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<meta name="citation_author" content="Fu-Yue Zeng">
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<meta name="citation_author" content="Arianna Mangravita Novo">
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<meta name="citation_author" content="Michael Vicchiarelli">
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<meta name="citation_author" content="Jena Diwan">
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<meta name="citation_author" content="Thomas D.Y. Chung">
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<meta name="citation_author" content="Anthony B. Pinkerton">
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<meta name="citation_author" content="Layton H. Smith">
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<meta name="citation_pmid" content="23658961">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK133430/">
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<meta name="DC.Title" content="Functional antagonists of the Apelin (APJ) receptor">
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<meta name="DC.Type" content="Text">
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<meta name="DC.Publisher" content="National Center for Biotechnology Information (US)">
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<meta name="DC.Contributor" content="Patrick R. Maloney">
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<meta name="DC.Contributor" content="Pasha Khan">
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<meta name="DC.Contributor" content="Michael Hedrick">
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<meta name="DC.Contributor" content="Palak Gosalia">
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<meta name="DC.Contributor" content="Monika Milewski">
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<meta name="DC.Contributor" content="Linda Li">
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<meta name="DC.Contributor" content="Gregory P. Roth">
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<meta name="DC.Contributor" content="Eduard Sergienko">
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<meta name="DC.Contributor" content="Eigo Suyama">
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<meta name="DC.Contributor" content="Eliot Sugarman">
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<meta name="DC.Contributor" content="Kevin Nguyen">
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<meta name="DC.Contributor" content="Alka Mehta">
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<meta name="DC.Contributor" content="Stefan Vasile">
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<meta name="DC.Contributor" content="Ying Su">
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<meta name="DC.Contributor" content="Derek Stonich">
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<meta name="DC.Contributor" content="Hung Nguyen">
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<meta name="DC.Contributor" content="Fu-Yue Zeng">
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<meta name="DC.Contributor" content="Arianna Mangravita Novo">
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<meta name="DC.Contributor" content="Michael Vicchiarelli">
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<meta name="DC.Contributor" content="Jena Diwan">
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<meta name="DC.Contributor" content="Thomas D.Y. Chung">
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<meta name="DC.Contributor" content="Anthony B. Pinkerton">
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<meta name="DC.Contributor" content="Layton H. Smith">
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<meta name="DC.Date" content="2013/03/07">
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<meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK133430/">
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<meta name="description" content="The recently discovered apelin receptor (APJ, AGTRL-1, APLNR) system has emerged as a critical mediator of cardiovascular homeostasis and is involved in the pathogenesis of hypertension, heart failure, atherosclerosis, and other cardiovascular diseases. We disclose the first discovery and characterization of a potent (1.7 – 2.2 μM), kojic acid based small molecule APJ functional antagonist in cell-based assays that is >37 fold selective over the closely related angiotensin II type 1 (AT1) receptor, derived from an high throughput screening (HTS) of the ~330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid receptor (<50%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented. This probe molecule provides a useful tool compound for investigators interested in understanding apelin receptor pharmacology and function.">
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<meta name="og:description" content="The recently discovered apelin receptor (APJ, AGTRL-1, APLNR) system has emerged as a critical mediator of cardiovascular homeostasis and is involved in the pathogenesis of hypertension, heart failure, atherosclerosis, and other cardiovascular diseases. We disclose the first discovery and characterization of a potent (1.7 – 2.2 μM), kojic acid based small molecule APJ functional antagonist in cell-based assays that is >37 fold selective over the closely related angiotensin II type 1 (AT1) receptor, derived from an high throughput screening (HTS) of the ~330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid receptor (<50%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented. This probe molecule provides a useful tool compound for investigators interested in understanding apelin receptor pharmacology and function.">
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match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK133430_"><span class="title" itemprop="name">Functional antagonists of the Apelin (APJ) receptor</span></h1><p class="contribs">Maloney PR, Khan P, Hedrick M, et al.</p><p class="fm-aai"><a href="#_NBK133430_pubdet_">Publication Details</a></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>The recently discovered apelin receptor (APJ, AGTRL-1, APLNR) system has emerged as a critical mediator of cardiovascular homeostasis and is involved in the pathogenesis of hypertension, heart failure, atherosclerosis, and other cardiovascular diseases. We disclose the first discovery and characterization of a potent (1.7 – 2.2 μM), kojic acid based small molecule APJ functional antagonist in cell-based assays that is >37 fold selective over the closely related angiotensin II type 1 (AT1) receptor, derived from an high throughput screening (HTS) of the ~330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid receptor (<50%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial <i>in vitro</i> pharmacologic characterization are also presented. This probe molecule provides a useful tool compound for investigators interested in understanding apelin receptor pharmacology and function.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1R21NS059422-01</p><p><b>Screening Center Name & PI:</b> Sanford-Burnham Center for Chemical Genomics (BCCG) & John C. Reed (PI)</p><p><b>Chemistry Center Name & PI:</b> Sanford-Burnham Center for Chemical Genomics (BCCG) & John C. Reed (PI)</p><p><b>Assay Submitter & Institution:</b> Layton H. Smith, Sanford-Burnham Medical Research Institute</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2569" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2569</a></p><div id="ml221.s1"><h2 id="_ml221_s1_">Probe Structure & Characteristics</h2><p>This Center Probe Report describes the first reported small molecule APJ antagonist, <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> that is also selective against the AT1 receptor and cell active</p><div id="ml221.fu1" class="figure"><div class="graphic"><img src="/books/NBK133430/bin/ml221fu1.jpg" alt="Image ml221fu1" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml221t1"><a href="/books/NBK133430/table/ml221.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml221t1" rid-ob="figobml221t1"><img class="small-thumb" src="/books/NBK133430/table/ml221.t1/?report=thumb" src-large="/books/NBK133430/table/ml221.t1/?report=previmg" alt="Table 1. Biological activity summary & AIDs for Probe." /></a><div class="icnblk_cntnt"><h4 id="ml221.t1"><a href="/books/NBK133430/table/ml221.t1/?report=objectonly" target="object" rid-ob="figobml221t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Biological activity summary & AIDs for Probe. </p></div></div></div><div id="ml221.s2"><h2 id="_ml221_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>The recently discovered apelin system is emerging as a critical mediator of cardiovascular homeostasis, whose importance in the pathogenesis of hypertension, heart failure, atherosclerosis, and other cardiovascular diseases, is the subject of intense investigation<sup><a class="bibr" href="#ml221.r1" rid="ml221.r1">1</a>–<a class="bibr" href="#ml221.r7" rid="ml221.r7">7</a></sup>. These investigations are limited by the paucity of research tools and reagents needed to fully understand the role of apelin in physiology and pathology. One particular challenge is the lack of a robust apelin receptor deficient (knock-out) mouse. Although this mouse has been created and reported in the literature, it is difficult to breed homozygous, apelin receptor null mice. Therefore a small molecule antagonist of the apelin receptor (a.k.a APJ, AGTRL-1, APLNR) would advance apelin research significantly. While not a replacement of a transgenic apelin receptor deficient mouse, a small molecule antagonist would be a useful tool for understanding the pharmacology of APJ and ultimately to validate the importance of this system in animal models of cardiovascular disease. To date, there are no small antagonists for the apelin receptor. In this report we disclose the discovery and characterization of a potent selective apelin receptor antagonist. Synthetic methodology, SAR, and activity of our most efficacious and selective compound in a cell-based assay of apelin receptor inhibition are presented. The probe molecule can be used as a tool compound by investigators interested in understanding apelin receptor pharmacology and function.</p></div><div id="ml221.s3"><h2 id="_ml221_s3_">2. Materials and Methods</h2><div id="ml221.s4"><h3>2.1. Assays</h3><p><a class="figpopup" href="/books/NBK133430/table/ml221.t2/?report=objectonly" target="object" rid-figpopup="figml221t2" rid-ob="figobml221t2">Table 2</a> summarizes the details for the assays that drove this probe project and can be found in the “Assay Description” section under the listed AIDs in <a class="figpopup" href="/books/NBK133430/table/ml221.t2/?report=objectonly" target="object" rid-figpopup="figml221t2" rid-ob="figobml221t2">Table 2</a> as viewable in PubChem Bioassays.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml221t2"><a href="/books/NBK133430/table/ml221.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml221t2" rid-ob="figobml221t2"><img class="small-thumb" src="/books/NBK133430/table/ml221.t2/?report=thumb" src-large="/books/NBK133430/table/ml221.t2/?report=previmg" alt="Table 2. Summary of Assays and AIDs." /></a><div class="icnblk_cntnt"><h4 id="ml221.t2"><a href="/books/NBK133430/table/ml221.t2/?report=objectonly" target="object" rid-ob="figobml221t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Summary of Assays and AIDs. </p></div></div><p>All assays in <a class="figpopup" href="/books/NBK133430/table/ml221.t2/?report=objectonly" target="object" rid-figpopup="figml221t2" rid-ob="figobml221t2">Table 2</a>, with the exception of the β-galactosidase counterscreen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/485352" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 485352</a>), utilize the DiscoveRx PathHunter<sup>®</sup> β-arrestin assay technology. Unlike imaging or other second messenger assays, the DiscoveRx β-arrestin assay allows for a direct measure of GPCR activation by detection of β-arrestin binding to the APJ, and in the case of the counterscreen, the AT1 receptor. In this system, β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor of EA) and the GPCR of interest is fused to a smaller (42 amino acids), weakly complementing fragment termed ProLink™. In cells that stably express these fusion proteins, ligand stimulation results in the interaction of β-arrestin and the ProLink-tagged GPCR, forcing the complementation of the two β-galactosidase fragments and resulting in the formation of a functional enzyme that converts substrate to detectable luminescent signal.</p></div><div id="ml221.s5"><h3>2.2. Probe Chemical Characterization</h3><div id="ml221.s6"><h4>Chemical name of probe compound & structure including stereochemistry</h4><p>The IUPAC name of the probe is 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4<i>H</i>-pyran-3-yl 4-nitrobenzoate. The actual batch prepared, tested and submitted to the MLSMR is archived as <a href="https://pubchem.ncbi.nlm.nih.gov/substance/103061721" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103061721</a> corresponding to CID 7217941. The probe <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> has no chiral centers (<a class="figpopup" href="/books/NBK133430/figure/ml221.f1/?report=objectonly" target="object" rid-figpopup="figml221f1" rid-ob="figobml221f1">Figure 1</a>). This probe is not commercially available. A 25 mg sample of <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> synthesized at SBCCG has been deposited in the MLSMR (Bio-Focus DPI).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml221f1" co-legend-rid="figlgndml221f1"><a href="/books/NBK133430/figure/ml221.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml221f1" rid-ob="figobml221f1"><img class="small-thumb" src="/books/NBK133430/bin/ml221f1.gif" src-large="/books/NBK133430/bin/ml221f1.jpg" alt="Figure 1. Chemical structure of ML221." /></a><div class="icnblk_cntnt" id="figlgndml221f1"><h4 id="ml221.f1"><a href="/books/NBK133430/figure/ml221.f1/?report=objectonly" target="object" rid-ob="figobml221f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Chemical structure of ML221. </p></div></div><p><b>Synthetic Route</b> is in <a class="figpopup" href="/books/NBK133430/figure/ml221.f8/?report=objectonly" target="object" rid-figpopup="figml221f8" rid-ob="figobml221f8">Scheme 1</a> and structure proof by 1HNMR & LC-MS is in <a class="figpopup" href="/books/NBK133430/figure/ml221.f2/?report=objectonly" target="object" rid-figpopup="figml221f2" rid-ob="figobml221f2">Figures 2</a> and <a class="figpopup" href="/books/NBK133430/figure/ml221.f3/?report=objectonly" target="object" rid-figpopup="figml221f3" rid-ob="figobml221f3">3A</a> and <a class="figpopup" href="/books/NBK133430/figure/ml221.f4/?report=objectonly" target="object" rid-figpopup="figml221f4" rid-ob="figobml221f4">3B</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml221f8" co-legend-rid="figlgndml221f8"><a href="/books/NBK133430/figure/ml221.f8/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml221f8" rid-ob="figobml221f8"><img class="small-thumb" src="/books/NBK133430/bin/ml221f8.gif" src-large="/books/NBK133430/bin/ml221f8.jpg" alt="Scheme 1. Synthesis of ML221, conditions." /></a><div class="icnblk_cntnt" id="figlgndml221f8"><h4 id="ml221.f8"><a href="/books/NBK133430/figure/ml221.f8/?report=objectonly" target="object" rid-ob="figobml221f8">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of ML221, conditions. a. neat SOCl<sub>2</sub> (17 eq); b. pyrimidine-2-thiol (1 eq), NaOMe (1 eq), MeCN; c. 4-nitrobenzoyl chloride (1.4 eq), Cs<sub>2</sub>CO<sub>3</sub> (1 eq), MeCN. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml221f2" co-legend-rid="figlgndml221f2"><a href="/books/NBK133430/figure/ml221.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml221f2" rid-ob="figobml221f2"><img class="small-thumb" src="/books/NBK133430/bin/ml221f2.gif" src-large="/books/NBK133430/bin/ml221f2.jpg" alt="Figure 2. 1H NMR Spectra for ML221." /></a><div class="icnblk_cntnt" id="figlgndml221f2"><h4 id="ml221.f2"><a href="/books/NBK133430/figure/ml221.f2/?report=objectonly" target="object" rid-ob="figobml221f2">Figure 2</a></h4><p class="float-caption no_bottom_margin"><sup>1</sup>H NMR Spectra for ML221. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml221f3" co-legend-rid="figlgndml221f3"><a href="/books/NBK133430/figure/ml221.f3/?report=objectonly" target="object" title="Figure 3A" class="img_link icnblk_img figpopup" rid-figpopup="figml221f3" rid-ob="figobml221f3"><img class="small-thumb" src="/books/NBK133430/bin/ml221f3.gif" src-large="/books/NBK133430/bin/ml221f3.jpg" alt="Figure 3A. LC of LC-MS Data for ML221." /></a><div class="icnblk_cntnt" id="figlgndml221f3"><h4 id="ml221.f3"><a href="/books/NBK133430/figure/ml221.f3/?report=objectonly" target="object" rid-ob="figobml221f3">Figure 3A</a></h4><p class="float-caption no_bottom_margin">LC of LC-MS Data for ML221. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml221f4" co-legend-rid="figlgndml221f4"><a href="/books/NBK133430/figure/ml221.f4/?report=objectonly" target="object" title="Figure 3B" class="img_link icnblk_img figpopup" rid-figpopup="figml221f4" rid-ob="figobml221f4"><img class="small-thumb" src="/books/NBK133430/bin/ml221f4.gif" src-large="/books/NBK133430/bin/ml221f4.jpg" alt="Figure 3B. MS of LC-MS Data for ML221." /></a><div class="icnblk_cntnt" id="figlgndml221f4"><h4 id="ml221.f4"><a href="/books/NBK133430/figure/ml221.f4/?report=objectonly" target="object" rid-ob="figobml221f4">Figure 3B</a></h4><p class="float-caption no_bottom_margin">MS of LC-MS Data for ML221. </p></div></div></div><div id="ml221.s7"><h4>Solubility and Stability of probe in PBS at room temperature</h4><p>The stability and solubility of <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> was investigated in PBS buffer at room temperature (<a class="figpopup" href="/books/NBK133430/figure/ml221.f5/?report=objectonly" target="object" rid-figpopup="figml221f5" rid-ob="figobml221f5">Figure 4</a>). Initial experiments examining the stability of <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> at its solubility limit suggested that the probe degrades relatively rapidly in PBS buffer. Thus, we subsequently examined the stability of <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> with 50% acetonitrile/PBS. In 50% acetonitrile/PBS buffer the compound is soluble, and appears to be stable out to 48 hr (92% remaining).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml221f5" co-legend-rid="figlgndml221f5"><a href="/books/NBK133430/figure/ml221.f5/?report=objectonly" target="object" title="Fig. 4" class="img_link icnblk_img figpopup" rid-figpopup="figml221f5" rid-ob="figobml221f5"><img class="small-thumb" src="/books/NBK133430/bin/ml221f5.gif" src-large="/books/NBK133430/bin/ml221f5.jpg" alt="Fig. 4. Stability of ML221 in 1:1 PBS:ACN at ambient temp." /></a><div class="icnblk_cntnt" id="figlgndml221f5"><h4 id="ml221.f5"><a href="/books/NBK133430/figure/ml221.f5/?report=objectonly" target="object" rid-ob="figobml221f5">Fig. 4</a></h4><p class="float-caption no_bottom_margin">Stability of ML221 in 1:1 PBS:ACN at ambient temp. </p></div></div></div><div id="ml221.s8"><h4>MLS# of probe molecule and five related samples that were submitted to the SMR collection</h4><p>Samples of the probe (25 mg) and each of five analogs (20 mg) synthesized at SBCCG were submitted to MLSMR (<a class="figpopup" href="/books/NBK133430/table/ml221.t3/?report=objectonly" target="object" rid-figpopup="figml221t3" rid-ob="figobml221t3">Table 3</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml221t3"><a href="/books/NBK133430/table/ml221.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml221t3" rid-ob="figobml221t3"><img class="small-thumb" src="/books/NBK133430/table/ml221.t3/?report=thumb" src-large="/books/NBK133430/table/ml221.t3/?report=previmg" alt="Table 3. Probe and Analog Submissions to MLSMR (BioFocus DPI) for APJ Antagonists." /></a><div class="icnblk_cntnt"><h4 id="ml221.t3"><a href="/books/NBK133430/table/ml221.t3/?report=objectonly" target="object" rid-ob="figobml221t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Probe and Analog Submissions to MLSMR (BioFocus DPI) for APJ Antagonists. </p></div></div></div></div><div id="ml221.s9"><h3>2.3. Probe Preparation</h3><div id="ml221.s10"><h4>Details of Synthesis and Structural Verification Information of probe SID 103061721 corresponding to CID 7217941</h4><p>Step 1: A mixture of 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one (Kojic acid) (0.55 g, 3.87 mmol) was dissolved in thionyl chloride (5 ml, 68.5 mmol) and was stirred at ambient temperature for 3 hours. Excess reagent was removed <i>in vacuo</i> to provide 0.61 g. (98%) of 2-(chloromethyl)-5-hydroxy-4H-pyran-4-one as an off-white solid. <sup>1</sup>H NMR. (500 MHz, DMSO-<i>d</i>6): δ (ppm) 8.13 (s, 1H), 6.57 (s, 1H), 4.66 (s, 2H).</p><p>Step 2: A mixture of pyrimidine-2-thiol (161 mg, 1.433 mmol) in 2 ml methanol was treated with sodium methoxide solution (310 mg, 1.433 mmol) and stirred until dissolved. Acetonitrile (10 ml) was added followed by 2-(chloromethyl)-5-hydroxy-4H-pyran-4-one (230 mg, 1.433 mmol) and the mixture was stirred at ambient temperature for 3 hours at which time analysis by LC/MS indicated the reaction to be complete. The solvent was removed <i>in vacuo</i> to provide 406 mg (96%) of a yellow solid containing crude 5-hydroxy-2-((pyrimidin-2-ylthio)methyl)-4H-pyran-4-one and an equimolar amount of sodium chloride which was used without further purification. <sup>1</sup>H NMR. (500 MHz, CDCl<sub>3</sub>): δ (ppm) 8.52 (d, 2H, <i>J</i>= 4.9 Hz), 7.80 (s, 1H), 7.02 (t, 1H, <i>J</i>= 4.8 Hz), 6.63 (s, 1H), 4.23 (s, 2H).</p><p>Step 3: A mixture of 5-hydroxy-2-((pyrimidin-2-ylthio)methyl)-4H-pyran-4-one (200 mg, 0.847 mmol), cesium carbonate (276 mg, 0.847 mmol), and 4-nitrobenzoyl chloride (220 mg, 1.185 mmol) in acetonitrile (8 ml) was stirred at ambient temperature overnight. The solvent was removed <i>in vacuo</i> to provide a pale yellow solid, which was partitioned with approximately 20 ml of 1:1 ethyl acetate and water. The desired product remained insoluble in the biphase and was collected by filtration. The solid was dried <i>in vacuo</i> to yield 202 mg (62%) as a tan solid. <sup>1</sup>H NMR. (500 MHz, DMSO-<i>d6</i>): δ (ppm) 8.69 (d, 2H, <i>J</i>=4.8 Hz), 8.68 (s, 1H), 8.40 (d, 2H, <i>J</i>=8.8 Hz), 8.29 (d, 2H, <i>J</i>=8.8 Hz), 7.29 (t, 1H, <i>J</i>= 4.9 Hz), 6.65 (s, 1H), 4.45 (s, 2H). <sup>13</sup>C NMR. (125 MHz, DMSO-<i>d6</i>): δ (ppm) 171.2, 168.8, 165.9, 161.7, 158.1, 150.8, 149.9, 140.3, 133.0, 131.4, 124.2, 118.0, 114.6, 31.2.</p></div></div></div><div id="ml221.s11"><h2 id="_ml221_s11_">3. Results</h2><div id="ml221.s12"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml221.f6" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%205.%20Representative%20dose%20response%20curve%20for%20ML221%20in%20the%20DiscoveRx%20assay%20for%20apelin%20receptor%20(APJ)%20antagonist%20activity.&p=BOOKS&id=133430_ml221f6.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/NBK133430/bin/ml221f6.jpg" alt="Figure 5. Representative dose response curve for ML221 in the DiscoveRx assay for apelin receptor (APJ) antagonist activity." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 5</span><span class="title">Representative dose response curve for ML221 in the DiscoveRx assay for apelin receptor (APJ) antagonist activity</span></h3></div></div><div id="ml221.s13"><h3>3.2. Cellular Activity</h3><p>All of the primary and selectivity assays are cell based.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml221t4"><a href="/books/NBK133430/table/ml221.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml221t4" rid-ob="figobml221t4"><img class="small-thumb" src="/books/NBK133430/table/ml221.t4/?report=thumb" src-large="/books/NBK133430/table/ml221.t4/?report=previmg" alt="Table 4. Cellular Activity of Probe ML221." /></a><div class="icnblk_cntnt"><h4 id="ml221.t4"><a href="/books/NBK133430/table/ml221.t4/?report=objectonly" target="object" rid-ob="figobml221t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Cellular Activity of Probe ML221. </p></div></div></div><div id="ml221.s14"><h3>3.3. Profiling Assays</h3><p>The probe was evaluated in a detailed <i>in vitro</i> pharmacology panel as shown in <a class="figpopup" href="/books/NBK133430/table/ml221.t5/?report=objectonly" target="object" rid-figpopup="figml221t5" rid-ob="figobml221t5">Table 5</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml221t5"><a href="/books/NBK133430/table/ml221.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml221t5" rid-ob="figobml221t5"><img class="small-thumb" src="/books/NBK133430/table/ml221.t5/?report=thumb" src-large="/books/NBK133430/table/ml221.t5/?report=previmg" alt="Table 5. Summary of in vitro ADME Properties of APJ Antagonist probe ML221." /></a><div class="icnblk_cntnt"><h4 id="ml221.t5"><a href="/books/NBK133430/table/ml221.t5/?report=objectonly" target="object" rid-ob="figobml221t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Summary of <i>in vitro</i> ADME Properties of APJ Antagonist probe ML221. </p></div></div><p><a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> is poorly soluble in aqueous media at pH 5.0/6.2/7.4, although the solubility appears pH-dependent as it is almost three-fold higher in pH 7.4 than in either pH6.2 or pH5.0. We note that the aqueous solubilities obtained at physiological pH are 5 – 14 fold higher than the obtained potency of the probe.</p><p>The PAMPA (<b>P</b>arallel <b>A</b>rtificial <b>M</b>embrane <b>P</b>ermeability <b>A</b>ssay) assay is used as an <i>in vitro</i> model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. Consistent with its solubility data, <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> exhibits moderate permeability that increased with pH.</p><p>Plasma protein binding is a measure of a drug’s efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> was undetectable in the plasma protein binding assay indicating that the compound is likely rapidly metabolized in plasma.</p><p>Plasma stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which strongly can influence the <i>in vivo</i> efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> shows very poor stability in human plasma (<1% remaining) after 3 hr. This data explains the lack of compound detected in the plasma protein binding assay, as that assay involves an 18h incubation.</p><p>The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> shows poor stability (4.2 % & 4.9% remaining at 60 min) in both human and mouse liver homogenates. Neither the plasma nor the microsomal stability assay results are surprising given the ester linkage in this probe. Attempts to replace the ester with a more stable functional group resulted in inactive compounds, although replacements were not widely investigated. Ultimately this limits the utility of this probe to <i>in vitro</i> studies or apelin receptor or <i>in vivo</i> studies using acute intravenous doses to avoid metabolism.</p><p><a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> shows no toxicity (>50 μM) toward human hepatocytes.</p><p><b>Profiling against other GPCRs.</b> The probe, <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> (CID7217941), was submitted to the Psychoactive Drug Screening Program (PDSP) at the University of North Carolina (PDSP, Bryan Roth, PI) and the data against a GPCR binding assay panel is shown in <a class="figpopup" href="/books/NBK133430/figure/ml221.f7/?report=objectonly" target="object" rid-figpopup="figml221f7" rid-ob="figobml221f7">Figure 6</a>. Overall, the compound shows a relatively clean binding profile with the only significant activity at the kappa opioid receptor.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml221f7" co-legend-rid="figlgndml221f7"><a href="/books/NBK133430/figure/ml221.f7/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml221f7" rid-ob="figobml221f7"><img class="small-thumb" src="/books/NBK133430/bin/ml221f7.gif" src-large="/books/NBK133430/bin/ml221f7.jpg" alt="Figure 6. GPCR profiling panel for ML221." /></a><div class="icnblk_cntnt" id="figlgndml221f7"><h4 id="ml221.f7"><a href="/books/NBK133430/figure/ml221.f7/?report=objectonly" target="object" rid-ob="figobml221f7">Figure 6</a></h4><p class="float-caption no_bottom_margin">GPCR profiling panel for ML221. </p></div></div></div></div><div id="ml221.s15"><h2 id="_ml221_s15_">4. Discussion</h2><p>Currently there are no small molecule tools to investigate the biological functions of apelin and its receptor. Apelin is the endogenous peptide ligand<sup><a class="bibr" href="#ml221.r8" rid="ml221.r8">8</a></sup> for the G-protein coupled receptor (GPCR) APJ (angiotensin II receptor-like 1, AGTRL-1 and APLNR). Until the discovery of apelin, APJ was an orphan GPCR. APJ is coupled to Gαi, and has been shown in cell culture to inhibit adenylate cyclase. The APJ gene encodes a receptor that most closely resembles the angiotensin receptor AT1. However, the APJ receptor does not bind angiotensin II<sup><a class="bibr" href="#ml221.r9" rid="ml221.r9">9</a></sup>. Underscoring the emerging importance of the apelin/APJ system, recent studies have shown that apelin reduces the extent of atherosclerotic lesions in ApoE−/− mice (7), and opposes the development of abdominal aortic aneurysms<sup><a class="bibr" href="#ml221.r10" rid="ml221.r10">10</a></sup>. Additionally, work in Dr. Smith’s lab has revealed that APJ forms a heterodimer with the Ang II receptor AT1<sup><a class="bibr" href="#ml221.r11" rid="ml221.r11">11</a></sup>, and that this complex facilitates antagonism of Ang II signaling by apelin. Despite these exciting results, there remains a multitude of unanswered questions regarding the role of apelin and APJ in the physiology and pathology, so chemical biological tools would be an advance.</p><div id="ml221.s16"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>There are no reports of APJ antagonists in the literature, including in the patent literature. Thus, <a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a> represents the first tool compound to probe APJ receptor biology. A recent SciFinder search (March 14, 2011) around the scaffold (holding the benzoate ester and thiopyrimidine constant) shows that it is not widely cited and thus is unlikely to be a promiscuous compound.</p></div></div><div id="ml221.s17"><h2 id="_ml221_s17_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml221.r1">De Falco M, De Luca L, Onori N, Cavallotti I, Artigiano F, Esposito V, De Luca B, Laforgia V, Groeger AM, De Luca A. Apelin expression in normal human tissues. <span><span class="ref-journal">In Vivo. </span>2002;<span class="ref-vol">16</span>:333–336.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12494873" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12494873</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml221.r2">Kleinz MJ, Davenport AP. Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells. <span><span class="ref-journal">Regul Pept. </span>2004;<span class="ref-vol">118</span>:119–125.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15003827" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15003827</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml221.r3">Kleinz MJ, Skepper JN, Davenport AP. Immunocytochemical localization of the apelin receptor, APJ, to human cardiomyocytes, vascular smooth muscle and endothelial cells. <span><span class="ref-journal">Regul Pept. </span>2005;<span class="ref-vol">126</span>:233–240.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15664671" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15664671</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml221.r4">Quazi R, Palaniswamy C, Frishman WH. The emerging role of apelin in cardiovascular disease and health. <span><span class="ref-journal">Cardiol Rev. </span>2009;<span class="ref-vol">17</span>:283–286.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19829178" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19829178</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml221.r5">Sorli SC, van den Berghe L, Masri B, Knibiehler B, Audigier Y. Therapeutic potential of interfering with apelin signaling. <span><span class="ref-journal">Drug Discov Today. </span>2006;<span class="ref-vol">11</span>:1100–1106.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17129829" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17129829</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml221.r6">Ashley EA, Powers J, Chen M, Kundu R, Finsterbach T, Caffarelli A, Deng A, Eichhorn J, Mahajan R, Agrawal R, et al. The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading <em>in vivo</em>. <span><span class="ref-journal">Cardiovasc Res. </span>2005;<span class="ref-vol">65</span>:73–82.</span> [<a href="/pmc/articles/PMC2517138/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2517138</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15621035" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15621035</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml221.r7">Chun HJ, Ali ZA, Kojima Y, Kundu RK, Sheikh AY, Agrawal R, Zheng L, Leeper NJ, Pearl NE, Patterson AJ, et al. Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis. <span><span class="ref-journal">J Clin Invest. </span>2008;<span class="ref-vol">118</span>:3343–3354.</span> [<a href="/pmc/articles/PMC2525695/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2525695</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18769630" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18769630</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml221.r8">Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, Osmond DH, George SR, O’Dowd BF. Characterization of apelin, the ligand for the APJ receptor. <span><span class="ref-journal">J Neurochem. </span>2000;<span class="ref-vol">74</span>:34–41.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10617103" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10617103</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml221.r9">O’Dowd BF, Heiber MM, Chan AA, Heng HHH, Tsui LLC, Kennedy JJL, Shi XX, Petronis AA, George SSR, Nguyen TT. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. <span><span class="ref-journal">Gene. </span>1993;<span class="ref-vol">136</span>:355.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8294032" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8294032</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml221.r10">Leeper NJ, Tedesco MM, Kojima Y, Schultz GM, Kundu RK, Ashley EA, Tsao PS, Dalman RL, Quertermous T. Apelin prevents aortic aneurysm formation by inhibiting macrophage inflammation. <span><span class="ref-journal">Am J Physiol Heart Circ Physiol. </span>2009;<span class="ref-vol">296</span>:H1329–1335.</span> [<a href="/pmc/articles/PMC2685356/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2685356</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19304942" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19304942</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml221.r11">Lee DK, Lanca AJ, Cheng R, Nguyen T, Ji XD, Gobeil F Jr, Chemtob S, George SR, O’Dowd BF. Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>:7901–7908.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14645236" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14645236</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK133430_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Patrick R. Maloney</span>, <span itemprop="author">Pasha Khan</span>, <span itemprop="author">Michael Hedrick</span>, <span itemprop="author">Palak Gosalia</span>, <span itemprop="author">Monika Milewski</span>, <span itemprop="author">Linda Li</span>, <span itemprop="author">Gregory P. Roth</span>, <span itemprop="author">Eduard Sergienko</span>, <span itemprop="author">Eigo Suyama</span>, <span itemprop="author">Eliot Sugarman</span>, <span itemprop="author">Kevin Nguyen</span>, <span itemprop="author">Alka Mehta</span>, <span itemprop="author">Stefan Vasile</span>, <span itemprop="author">Ying Su</span>, <span itemprop="author">Derek Stonich</span>, <span itemprop="author">Hung Nguyen</span>, <span itemprop="author">Fu-Yue Zeng</span>, <span itemprop="author">Arianna Mangravita Novo</span>, <span itemprop="author">Michael Vicchiarelli</span>, <span itemprop="author">Jena Diwan</span>, <span itemprop="author">Thomas D.Y. Chung</span>, <span itemprop="author">Anthony B. Pinkerton</span>, and <span itemprop="author">Layton H. Smith</span>.</p><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">March 22, 2011</span>; Last Update: <span itemprop="dateModified">March 7, 2013</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p>National Center for Biotechnology Information (US), Bethesda (MD)</p><h3>NLM Citation</h3><p>Maloney PR, Khan P, Hedrick M, et al. Functional antagonists of the Apelin (APJ) receptor. 2011 Mar 22 [Updated 2013 Mar 7]. 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></p></div><div class="small-screen-prev"><a href="/books/n/mlprobe/ml225/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/mlprobe/ml220/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobml221fu1"><div id="ml221.fu1" class="figure"><div class="graphic"><img data-src="/books/NBK133430/bin/ml221fu1.jpg" alt="Image ml221fu1" /></div></div></article><article data-type="table-wrap" id="figobml221t1"><div id="ml221.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Biological activity summary & AIDs for Probe</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133430/table/ml221.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml221.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml221.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml221.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml221.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_ml221.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_ml221.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_ml221.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th></tr></thead><tbody><tr><td headers="hd_h_ml221.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 7217941<br /><a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a></td><td headers="hd_h_ml221.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">APJ<br />Receptor</td><td headers="hd_h_ml221.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1750 nM<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/103061721" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103061721</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/492986" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 492986</a></td><td headers="hd_h_ml221.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">AT1 Receptor</td><td headers="hd_h_ml221.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>79 μM<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/103061721" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103061721</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/492984" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 492984</a></td><td headers="hd_h_ml221.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>45</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml221t2"><div id="ml221.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Summary of Assays and AIDs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133430/table/ml221.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml221.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml221.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PubChemBioAssay Name</th><th id="hd_h_ml221.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">AIDs</th><th id="hd_h_ml221.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe Type</th><th id="hd_h_ml221.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Type</th><th id="hd_h_ml221.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Format</th><th id="hd_h_ml221.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Detection & well format</th></tr></thead><tbody><tr><td headers="hd_h_ml221.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Summary assay for small molecule antagonists of the APJ receptor [Summary]</td><td headers="hd_h_ml221.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2569" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2569</a></td><td headers="hd_h_ml221.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Antagonist</td><td headers="hd_h_ml221.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Summary</td><td headers="hd_h_ml221.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N/A</td><td headers="hd_h_ml221.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N/A</td></tr><tr><td headers="hd_h_ml221.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">uHTS identification of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay [Primary Screening]</td><td headers="hd_h_ml221.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2521" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2521</a></td><td headers="hd_h_ml221.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Antagonist</td><td headers="hd_h_ml221.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml221.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cell-based</td><td headers="hd_h_ml221.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence-DiscoveRx β-arrestin & 1536</td></tr><tr><td headers="hd_h_ml221.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Single concentration confirmation of uHTS hits from a small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay [Primary Screening]</td><td headers="hd_h_ml221.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2766" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2766</a></td><td headers="hd_h_ml221.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Antagonist</td><td headers="hd_h_ml221.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Confirmatory</td><td headers="hd_h_ml221.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cell-based</td><td headers="hd_h_ml221.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence-DiscoveRx β-arrestin & 1536</td></tr><tr><td headers="hd_h_ml221.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Dose Response confirmation of uHTS hits from a small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay<br />[Confirmatory]</td><td headers="hd_h_ml221.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2784" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2784</a></td><td headers="hd_h_ml221.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Antagonist</td><td headers="hd_h_ml221.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Confirmatory</td><td headers="hd_h_ml221.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cell-based</td><td headers="hd_h_ml221.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence-DiscoveRx β-arrestin & 1536</td></tr><tr><td headers="hd_h_ml221.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SAR analysis of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay [Confirmatory]</td><td headers="hd_h_ml221.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463109" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463109</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488803" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488803</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488992" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488992</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/492986" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 492986</a></td><td headers="hd_h_ml221.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Antagonist</td><td headers="hd_h_ml221.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity<br />SAR</td><td headers="hd_h_ml221.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cell-based</td><td headers="hd_h_ml221.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence-DiscoveRx β-arrestin & 1536</td></tr><tr><td headers="hd_h_ml221.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">HTS Dose response counterscreen for assays utilizing the enzyme, beta-galactosidase [Confirmatory]</td><td headers="hd_h_ml221.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/485352" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 485352</a></td><td headers="hd_h_ml221.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Antagonist</td><td headers="hd_h_ml221.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Counterscreen</td><td headers="hd_h_ml221.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cell-based</td><td headers="hd_h_ml221.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence & 1536</td></tr><tr><td headers="hd_h_ml221.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SAR analysis of antagonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule antagonists hits of the APJ receptor [Confirmatory]</td><td headers="hd_h_ml221.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488994" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488994</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488810" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488810</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/492984" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 492984</a></td><td headers="hd_h_ml221.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Antagonist</td><td headers="hd_h_ml221.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity<br />SAR</td><td headers="hd_h_ml221.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cell-based</td><td headers="hd_h_ml221.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence-DiscoveRx β-arrestin & 1536</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml221f1"><div id="ml221.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133430/bin/ml221f1.jpg" alt="Figure 1. Chemical structure of ML221." /></div><h3><span class="label">Figure 1</span><span class="title">Chemical structure of ML221</span></h3></div></article><article data-type="fig" id="figobml221f8"><div id="ml221.f8" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133430/bin/ml221f8.jpg" alt="Scheme 1. Synthesis of ML221, conditions." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of ML221, conditions</span></h3><div class="caption"><p>a. neat SOCl<sub>2</sub> (17 eq); b. pyrimidine-2-thiol (1 eq), NaOMe (1 eq), MeCN; c. 4-nitrobenzoyl chloride (1.4 eq), Cs<sub>2</sub>CO<sub>3</sub> (1 eq), MeCN.</p></div></div></article><article data-type="fig" id="figobml221f2"><div id="ml221.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%201H%20NMR%20Spectra%20for%20ML221.&p=BOOKS&id=133430_ml221f2.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 data-src="/books/NBK133430/bin/ml221f2.jpg" alt="Figure 2. 1H NMR Spectra for ML221." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title"><sup>1</sup>H NMR Spectra for ML221</span></h3></div></article><article data-type="fig" id="figobml221f3"><div id="ml221.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203A.%20LC%20of%20LC-MS%20Data%20for%20ML221.&p=BOOKS&id=133430_ml221f3.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 data-src="/books/NBK133430/bin/ml221f3.jpg" alt="Figure 3A. LC of LC-MS Data for ML221." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3A</span><span class="title">LC of LC-MS Data for ML221</span></h3></div></article><article data-type="fig" id="figobml221f4"><div id="ml221.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133430/bin/ml221f4.jpg" alt="Figure 3B. MS of LC-MS Data for ML221." /></div><h3><span class="label">Figure 3B</span><span class="title">MS of LC-MS Data for ML221</span></h3></div></article><article data-type="fig" id="figobml221f5"><div id="ml221.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133430/bin/ml221f5.jpg" alt="Fig. 4. Stability of ML221 in 1:1 PBS:ACN at ambient temp." /></div><h3><span class="label">Fig. 4</span><span class="title">Stability of ML221 in 1:1 PBS:ACN at ambient temp</span></h3></div></article><article data-type="table-wrap" id="figobml221t3"><div id="ml221.t3" class="table"><h3><span class="label">Table 3</span><span class="title">Probe and Analog Submissions to MLSMR (BioFocus DPI) for APJ Antagonists</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133430/table/ml221.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml221.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml221.t3_1_1_1_1" colspan="8" rowspan="1" style="text-align:center;vertical-align:middle;">Probe - CID7217941</th></tr><tr><th headers="hd_h_ml221.t3_1_1_1_1" id="hd_h_ml221.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe/Analog</th><th headers="hd_h_ml221.t3_1_1_1_1" id="hd_h_ml221.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS_ID (SBCCG)</th><th headers="hd_h_ml221.t3_1_1_1_1" id="hd_h_ml221.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS_ID (MLSMR)</th><th headers="hd_h_ml221.t3_1_1_1_1" id="hd_h_ml221.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml221.t3_1_1_1_1" id="hd_h_ml221.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml221.t3_1_1_1_1" id="hd_h_ml221.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Source (vendor or SBCCG syn)</th><th headers="hd_h_ml221.t3_1_1_1_1" id="hd_h_ml221.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Amt (mg)</th><th headers="hd_h_ml221.t3_1_1_1_1" id="hd_h_ml221.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Date ordered/Submitted</th></tr></thead><tbody><tr><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe<br /><a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a></td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0437359</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003389520</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID 7217941</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/103061721" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103061721</a></td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">03/20/11</td></tr><tr><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 1</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0437362</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003389521</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID 7217946</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167731" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 110167731</a></td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">03/20/11</td></tr><tr><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 2</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0437497</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003389522</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID 46905029</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/104222780" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 104222780</a></td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">03/20/11</td></tr><tr><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 3</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0437386</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003389524</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID 7217947</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99213141" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99213141</a></td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">03/20/11</td></tr><tr><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 4</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0437385</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003389523</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID 7217942</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/104222782" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 104222782</a></td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">03/20/11</td></tr><tr><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 5</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0437462</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003389525</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID 46905018</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/104222781" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 104222781</a></td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml221.t3_1_1_1_1 hd_h_ml221.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">03/20/11</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml221f6"><div id="ml221.f6" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%205.%20Representative%20dose%20response%20curve%20for%20ML221%20in%20the%20DiscoveRx%20assay%20for%20apelin%20receptor%20(APJ)%20antagonist%20activity.&p=BOOKS&id=133430_ml221f6.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 data-src="/books/NBK133430/bin/ml221f6.jpg" alt="Figure 5. Representative dose response curve for ML221 in the DiscoveRx assay for apelin receptor (APJ) antagonist activity." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 5</span><span class="title">Representative dose response curve for ML221 in the DiscoveRx assay for apelin receptor (APJ) antagonist activity</span></h3></div></article><article data-type="table-wrap" id="figobml221t4"><div id="ml221.t4" class="table"><h3><span class="label">Table 4</span><span class="title">Cellular Activity of Probe ML221</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133430/table/ml221.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml221.t4_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml221.t4_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml221.t4_1_1_1_1" style="text-align:center;vertical-align:top;">Probe</th><th id="hd_h_ml221.t4_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml221.t4_1_1_1_2" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml221.t4_1_1_1_3" rowspan="2" colspan="1" headers="hd_h_ml221.t4_1_1_1_3" style="text-align:center;vertical-align:top;">SID</th><th id="hd_h_ml221.t4_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml221.t4_1_1_1_4" style="text-align:center;vertical-align:top;">MLS-#</th><th id="hd_h_ml221.t4_1_1_1_5" rowspan="2" colspan="1" headers="hd_h_ml221.t4_1_1_1_5" style="text-align:center;vertical-align:top;">S/P<sup>*</sup></th><th id="hd_h_ml221.t4_1_1_1_6" rowspan="2" colspan="1" headers="hd_h_ml221.t4_1_1_1_6" style="text-align:center;vertical-align:top;">Chemical Structure</th><th id="hd_h_ml221.t4_1_1_1_7" colspan="3" rowspan="1" style="text-align:center;vertical-align:top;">Potency (μM)<sup>a</sup></th></tr><tr><th headers="hd_h_ml221.t4_1_1_1_7" id="hd_h_ml221.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">APJ</th><th headers="hd_h_ml221.t4_1_1_1_7" id="hd_h_ml221.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">AT1</th><th headers="hd_h_ml221.t4_1_1_1_7" id="hd_h_ml221.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SI</th></tr></thead><tbody><tr><td headers="hd_h_ml221.t4_1_1_1_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a></td><td headers="hd_h_ml221.t4_1_1_1_2" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">CID 7217941</td><td headers="hd_h_ml221.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99213078" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99213078</a></td><td headers="hd_h_ml221.t4_1_1_1_4" rowspan="2" colspan="1" style="text-align:center;vertical-align:top;">0437359</td><td headers="hd_h_ml221.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">P</td><td headers="hd_h_ml221.t4_1_1_1_6" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK133430/bin/ml221fu2.jpg" alt="Image ml221fu2.jpg" /></div></td><td headers="hd_h_ml221.t4_1_1_1_7 hd_h_ml221.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.16 ± 0.96</td><td headers="hd_h_ml221.t4_1_1_1_7 hd_h_ml221.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>79</td><td headers="hd_h_ml221.t4_1_1_1_7 hd_h_ml221.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>37</td></tr><tr><td headers="hd_h_ml221.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/103061721" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103061721</a></td><td headers="hd_h_ml221.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">S</td><td headers="hd_h_ml221.t4_1_1_1_7 hd_h_ml221.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.75 ± 0.19</td><td headers="hd_h_ml221.t4_1_1_1_7 hd_h_ml221.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>79</td><td headers="hd_h_ml221.t4_1_1_1_7 hd_h_ml221.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>45</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ml221.tfn1"><p class="no_margin">S = Synthesized P = purchased</p></div></dd></dl><dl class="bkr_refwrap"><dt>a</dt><dd><div id="ml221.tfn4a"><p class="no_margin">Ave. ± <i>S.E.M.</i> (n = 4)<i>, if replicates is different than n=4 it is noted in parentheses (AIDs in</i>
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<a class="figpopup" href="/books/NBK133430/table/ml221.t1/?report=objectonly" target="object" rid-figpopup="figml221t1" rid-ob="figobml221t1">Table 1</a>). SI = Selectivity Index: (IC<sub><i>50</i></sub>
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<i>AT1)/(IC</i><sub><i>50</i></sub>
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<i>APJ)</i></p></div></dd></dl></dl></div></div></div></article><article data-type="table-wrap" id="figobml221t5"><div id="ml221.t5" class="table"><h3><span class="label">Table 5</span><span class="title">Summary of <i>in vitro</i> ADME Properties of APJ Antagonist probe ML221</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133430/table/ml221.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml221.t5_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml221.t5_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml221.t5_1_1_1_1" style="text-align:center;vertical-align:top;">Probe<br />Probe ML#<br />SBCCG MLS-#</th><th id="hd_h_ml221.t5_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml221.t5_1_1_1_2" style="text-align:center;vertical-align:top;">Aqueous Solubility<br />(μg/mL)<br />(pH5.0/6.2/7.4)<br /><br /><i>[μM]</i><sup>a</sup></th><th id="hd_h_ml221.t5_1_1_1_3" rowspan="2" colspan="1" headers="hd_h_ml221.t5_1_1_1_3" style="text-align:center;vertical-align:top;">Aqueous Solubility<br />in 1× PBS (μg/mL)<br />pH 7.4<br /><i>[μM]</i></th><th id="hd_h_ml221.t5_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml221.t5_1_1_1_4" style="text-align:center;vertical-align:top;">PAMPAPe<br />(x10<sup>−6</sup> cm/s)<br />Donor pH: 5.0/6.2/7.4<br />Acceptor pH: 7.4</th><th id="hd_h_ml221.t5_1_1_1_5" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;">Plasma Protein Binding (% Bound)</th><th id="hd_h_ml221.t5_1_1_1_6" rowspan="2" colspan="1" headers="hd_h_ml221.t5_1_1_1_6" style="text-align:center;vertical-align:top;">Plasma Stability<br />(%Remaining @3hrs)<br />Human/Mouse<br />Plasma:1× PBS, pH 7.4, 1:1<br />1× PBS, pH 7.4</th><th id="hd_h_ml221.t5_1_1_1_7" rowspan="2" colspan="1" headers="hd_h_ml221.t5_1_1_1_7" style="text-align:center;vertical-align:top;">Hepatic Microsome Stability<br />Human/ Mouse</th><th id="hd_h_ml221.t5_1_1_1_8" rowspan="2" colspan="1" headers="hd_h_ml221.t5_1_1_1_8" style="text-align:center;vertical-align:top;">Hepatic Toxicity<br />LC50 (μM)</th></tr><tr><th headers="hd_h_ml221.t5_1_1_1_5" id="hd_h_ml221.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Human<br />1μM</th><th headers="hd_h_ml221.t5_1_1_1_5" id="hd_h_ml221.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Mouse<br />1μM</th></tr></thead><tbody><tr><td headers="hd_h_ml221.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID7217941<br /><a href="/pcsubstance/?term=ML221[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML221</a><br />MLS-0437359</td><td headers="hd_h_ml221.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.0/3.6/9.8<i>[7.8/9.3/25]</i></td><td headers="hd_h_ml221.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.6 <i>[9.3]</i></td><td headers="hd_h_ml221.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">195/240/271</td><td headers="hd_h_ml221.t5_1_1_1_5 hd_h_ml221.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td><td headers="hd_h_ml221.t5_1_1_1_5 hd_h_ml221.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td><td headers="hd_h_ml221.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.26/0.88<br />46.97/42.18</td><td headers="hd_h_ml221.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.19/4.89</td><td headers="hd_h_ml221.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>50</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>a</dt><dd><div id="ml221.tfn2"><p class="no_margin">Solubility also expressed in molar units (μM) as indicated in <i>italicized [bracketed values],</i> in addition to more traditional μg/mL units.</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml221f7"><div id="ml221.f7" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%206.%20GPCR%20profiling%20panel%20for%20ML221.&p=BOOKS&id=133430_ml221f7.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 data-src="/books/NBK133430/bin/ml221f7.jpg" alt="Figure 6. GPCR profiling panel for ML221." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 6</span><span class="title">GPCR profiling panel for ML221</span></h3></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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