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<title>Small Molecule Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1) - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="Small Molecule Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2013/02/28" /><meta name="citation_author" content="Ganesha Rai" /><meta name="citation_author" content="Vaddadi N. Vyjayanti" /><meta name="citation_author" content="Dorjbal Dorjsuren" /><meta name="citation_author" content="Anton Simeonov" /><meta name="citation_author" content="Ajit Jadhav" /><meta name="citation_author" content="David M. Wilson" /><meta name="citation_author" content="David J. Maloney" /><meta name="citation_pmid" content="23658942" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK133448/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Small Molecule Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Ganesha Rai" /><meta name="DC.Contributor" content="Vaddadi N. Vyjayanti" /><meta name="DC.Contributor" content="Dorjbal Dorjsuren" /><meta name="DC.Contributor" content="Anton Simeonov" /><meta name="DC.Contributor" content="Ajit Jadhav" /><meta name="DC.Contributor" content="David M. Wilson" /><meta name="DC.Contributor" content="David J. Maloney" /><meta name="DC.Date" content="2013/02/28" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK133448/" /><meta name="description" content="Probe compound, ML199, and related inhibitors of Apurinic/apyrimidinic (AP) endonuclease (APE1) are reported herein. APE1 is a key component of the base excision repair (BER) pathway that is responsible for repair of DNA damage caused by many anti-cancer agents such as bleomycin and temozolomide. As a result, inhibition of APE1 has been postulated as a viable strategy for sensitizing tumor cells to chemotherapy. ML199 and its related analogs belong to a drug-like series that was identified and optimized through a focused medicinal chemistry effort to afford compounds which display competitive inhibition of APE1 activity in the low micromolar potency range. On target effect of the ML199 was demonstrated through a concentration depended inhibition of AP site incision activity in whole cell HeLa extracts. Moreover, ML199 potentiated the cytotoxicity of the DNA alkylating agent methylmethane sulfonate (MMS) at non-cytotoxic doses of the probe compound. The probe and its general class of compounds have shown to have good kinetic solubility, Caco-2 permeability, metabolic stability and other favorable physicochemical properties, thus making ML199 an ideal starting point for further pre-clinical development of anti-cancer agents." /><meta name="og:title" content="Small Molecule Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)" /><meta name="og:type" content="book" /><meta name="og:description" content="Probe compound, ML199, and related inhibitors of Apurinic/apyrimidinic (AP) endonuclease (APE1) are reported herein. APE1 is a key component of the base excision repair (BER) pathway that is responsible for repair of DNA damage caused by many anti-cancer agents such as bleomycin and temozolomide. As a result, inhibition of APE1 has been postulated as a viable strategy for sensitizing tumor cells to chemotherapy. ML199 and its related analogs belong to a drug-like series that was identified and optimized through a focused medicinal chemistry effort to afford compounds which display competitive inhibition of APE1 activity in the low micromolar potency range. On target effect of the ML199 was demonstrated through a concentration depended inhibition of AP site incision activity in whole cell HeLa extracts. Moreover, ML199 potentiated the cytotoxicity of the DNA alkylating agent methylmethane sulfonate (MMS) at non-cytotoxic doses of the probe compound. The probe and its general class of compounds have shown to have good kinetic solubility, Caco-2 permeability, metabolic stability and other favorable physicochemical properties, thus making ML199 an ideal starting point for further pre-clinical development of anti-cancer agents." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK133448/" /><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/ml199/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK133448/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK133448_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK133448_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/ml201/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml194/" 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="_NBK133448_"><span class="title" itemprop="name">Small Molecule Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)</span></h1><p class="contrib-group"><span itemprop="author">Ganesha Rai</span>, <span itemprop="author">Vaddadi N. Vyjayanti</span>, <span itemprop="author">Dorjbal Dorjsuren</span>, <span itemprop="author">Anton Simeonov</span>, <span itemprop="author">Ajit Jadhav</span>, <span itemprop="author">David M. Wilson</span>, and <span itemprop="author">David J. Maloney</span>.</p><a data-jig="ncbitoggler" href="#__NBK133448_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK133448_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Ganesha Rai</span>,<sup>a</sup> <span itemprop="author">Vaddadi N. Vyjayanti</span>,<sup>b</sup> <span itemprop="author">Dorjbal Dorjsuren</span>,<sup>a</sup> <span itemprop="author">Anton Simeonov</span>,<sup>a</sup> <span itemprop="author">Ajit Jadhav</span>,<sup>a</sup> <span itemprop="author">David M. Wilson</span>,<sup>b</sup> and <span itemprop="author">David J. Maloney</span><sup>a</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>a</sup>
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NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Rockville, Maryland.</div><div class="affiliation"><sup>b</sup>
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National Institute on Aging, National Institutes of Health, Bethesda, Maryland.</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed: NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Building B, Bethesda, MD 20892-3370. Phone: 301-217-4381. Fax: 301-217-5736. Email:
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<a href="mailto:dev@null" data-email="vog.hin.liam@dyenolam" class="oemail">vog.hin.liam@dyenolam</a></div></div><p class="small">Received: <span itemprop="datePublished">October 29, 2010</span>; Last Update: <span itemprop="dateModified">February 28, 2013</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Probe compound, <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a>, and related inhibitors of Apurinic/apyrimidinic (AP) endonuclease (APE1) are reported herein. APE1 is a key component of the base excision repair (BER) pathway that is responsible for repair of DNA damage caused by many anti-cancer agents such as bleomycin and temozolomide. As a result, inhibition of APE1 has been postulated as a viable strategy for sensitizing tumor cells to chemotherapy. <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> and its related analogs belong to a drug-like series that was identified and optimized through a focused medicinal chemistry effort to afford compounds which display competitive inhibition of APE1 activity in the low micromolar potency range. On target effect of the <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> was demonstrated through a concentration depended inhibition of AP site incision activity in whole cell HeLa extracts. Moreover, <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> potentiated the cytotoxicity of the DNA alkylating agent methylmethane sulfonate (MMS) at non-cytotoxic doses of the probe compound. The probe and its general class of compounds have shown to have good kinetic solubility, Caco-2 permeability, metabolic stability and other favorable physicochemical properties, thus making <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> an ideal starting point for further pre-clinical development of anti-cancer agents.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> <a href="/nuccore/1543677917" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">MH086444</a></p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Assay Submitter & Institution:</b> David M. Wilson III, NIH, National Institute on Aging</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2324" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2324</a></p><div id="ml199.s1"><h2 id="_ml199_s1_">Probe Structure & Characteristics</h2><div id="ml199.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133448/bin/ml199fu1.jpg" alt="ML199." /></div><h3><span class="title">ML199</span></h3></div><div id="ml199.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133448/table/ml199.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml199.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml199.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID/ML#</th><th id="hd_h_ml199.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml199.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml199.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml199.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml199.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml199.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml199.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 46925884/<a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a></td><td headers="hd_h_ml199.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">APE1</td><td headers="hd_h_ml199.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">6000 nM IC<sub>50</sub> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430950</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2572" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2572</a>]</td><td headers="hd_h_ml199.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Thiazole orange counterscreen</td><td headers="hd_h_ml199.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 57 μM IC<sub>50</sub> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430950</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2741" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2741</a>]</td><td headers="hd_h_ml199.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 10 fold</td><td headers="hd_h_ml199.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">RIA gel assay: 1000 nM IC<sub>50</sub> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430950</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488940" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488940</a>]</td></tr></tbody></table></div></div></div><div id="ml199.s2"><h2 id="_ml199_s2_">Recommendations for Scientific Use of the Probe</h2><p>The novel optimized probe described in this report (<a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a>) can be used for target validation of APE1 in enzymatic and cell-based systems. It can also be used in combination with known DNA cancer chemotherapeutics, such as temozolomide (TMZ), to potentiate the activity of these agents. It can be utilized in studies involving the BER pathway and the potentiation of known cancer chemotherapeutics. Lastly, the general class of this probe and its analogs has good <i>in vitro</i> ADME properties, making the probe series useful in further optimization for potential <i>in vivo</i> exposure.</p></div><div id="ml199.s3"><h2 id="_ml199_s3_">1. Introduction</h2><p>The goal of this project is to develop chemical probes directed at the human BER enzyme, APE1.
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Such probes will enhance our understanding of the role of APE1 in normal and cancer cells and may
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provide opportunities for developing novel anticancer agents that potentiate genotoxic treatments. BER is the major pathway for dealing with spontaneous hydrolytic, oxidative and alkylative base and sugar damage to DNA<sup><a class="bk_pop" href="#ml199.r1">1</a></sup>. Central to this process is incision at an apurinic/apyrimidinic (AP) site that is generated either spontaneously or via the enzymatic activity of a DNA repair glycosylase. This strand cleavage step is performed by the main, if not sole, mammalian AP endonuclease, APE1<sup><a class="bk_pop" href="#ml199.r2">2</a>,<a class="bk_pop" href="#ml199.r3">3</a></sup>. APE1 has been found to be essential for not only animal viability, as deletion of both alleles of the APE1 gene in mice leads to embryonic lethality<sup><a class="bk_pop" href="#ml199.r4">4</a></sup>, but also for cell viability in culture<sup><a class="bk_pop" href="#ml199.r5">5</a>,<a class="bk_pop" href="#ml199.r6">6</a></sup>. Moreover, past studies incorporating either antisense or RNAi strategies have revealed that APE1-deficient cells exhibit hypersensitivity to a number of “DNA-damaging” agents, including the laboratory chemicals methyl methanesulfonate (MMS), hydrogen peroxide, menadione and paraquat, and the anticancer agents ionizing radiation, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea (a.k.a. Carmustine), TMZ, gemcitabine, and the nucleoside analog ß-L-dioxolane-cytidine (a.k.a. Troxacitabine)<sup><a class="bk_pop" href="#ml199.r7">7</a></sup>. Recent work from the Wilson group employing a dominant-negative APE1 protein (termed ED), which binds with high affinity to substrate DNA and blocks subsequent repair steps<sup><a class="bk_pop" href="#ml199.r8">8</a></sup>, has shown that ED augments the cell killing of 5-fluorouracil (~fivefold) and 5-fluorodeoxyuridine (~thirtyfold), implicating BER in the cellular response to such antimetabolites as well. These data underscore the potential of APE1 as an inhibitor target in the effort to improve therapeutic efficacy of clinical DNA-interactive drugs through inactivation of DNA repair responses<sup><a class="bk_pop" href="#ml199.r9">9</a></sup>.</p><p>Most drugs employed to eradicate neoplastic disease (e.g. alkylators, cross-linking agents, intercalating agents, topoisomerase inhibitors, DNA cleaving agents and certain antimetabolites) operate by inducing the formation of complex DNA lesions/intermediates that ultimately block replication of rapidly dividing cells, such as cancer cells, and activate cell death responses. A primary goal of current studies is to devise combinatorial methods that (a) protect normal cells from the toxic effects of anti-cancer agents and (b) enhance the sensitivity of tumor cells. As alluded to above, DNA repair systems represent a major protective mechanism against the cytotoxic effects of clinical DNA-interactive compounds. Thus, recent efforts have focused on the design of novel small molecule inhibitors of specific DNA repair proteins, for instance, the DNA strand break response protein poly(ADP)ribose polymerase PARP1<sup><a class="bk_pop" href="#ml199.r10">10</a></sup>.</p><p>Whereas extensive studies have been performed using various genetic and molecular techniques to
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delineate the cellular effects of a BER deficiency, experiments employing inhibitor molecules are comparatively lacking and would complement existing knowledge and potentially provide novel biological insights. Moreover, recent evidence has implicated BER capacity as an important factor in determining individual responsiveness to clinical DNA-interactive agents, most notably alkylators (e.g. TMZ) and antimetabolites (e.g. 5-fluorouracil and certain nucleoside analogs) used in the treatment of cancer, and in some instances, viral infections<sup><a class="bk_pop" href="#ml199.r11">11</a></sup>. Alkylators induce cell killing through the formation of alkylated bases, many of which are either lost spontaneously to form abasic sites or are substrates for DNA glycosylases<sup><a class="bk_pop" href="#ml199.r12">12</a></sup>; TMZ is a promising drug recently added to the arsenal of alkylating agents for the adjuvant chemotherapy of brain cancers due to its ability to readily cross the blood-brain barrier<sup><a class="bk_pop" href="#ml199.r13">13</a></sup>. The antimetabolite 5-fluorouracil, which has been used clinically for ~40 years and is commonly employed in the eradication of colorectal and pancreatic cancers, induces cell death by promoting the introduction of uracil and fluorouracil bases into DNA, which are ultimately processed by BER glycosylases<sup><a class="bk_pop" href="#ml199.r14">14</a>,<a class="bk_pop" href="#ml199.r15">15</a></sup>. All told, BER inhibitors or activators would provide novel resources that may improve efficacy of current treatment paradigms by promoting selective sensitization or protection of diseased and normal cells, respectively. <i>In vitro</i> inhibitors of APE1 identified within are prospective reagents for the design of high affinity compounds with potential therapeutic value in combinational treatments.</p><div id="ml199.s4"><h3>Prior Art</h3><p>Compound <b>1</b><sup><a class="bk_pop" href="#ml199.r16">16</a></sup> (see <a class="figpopup" href="/books/NBK133448/table/ml199.t1/?report=objectonly" target="object" rid-figpopup="figml199t1" rid-ob="figobml199t1">Table 1</a>) was the first reported direct APE1 AP endonuclease inhibitor and is currently sold by Sigma-Aldrich chemical company; however, several groups have failed to reproduce the APE1 inhibitory activity of this compound. In agreement with these findings, our group was also unable to reproduce the activity in both the HTS and radiotracer incision assays. Arylstilbonic acid <b>2</b> displays potent activity against APE1 with a submicromolar IC<sub>50</sub> (low micromolar in our assay); however, this class of compounds has limited efficacy in cellular assays due to poor cellular uptake<sup><a class="bk_pop" href="#ml199.r17">17</a></sup>. Zawahir <i>et al</i>. described the utilization of a pharmacophore model to identify compounds such as <b>3</b>, which display low μM inhibition against APE1 incision activity. However, given the presence of three carboxylic acid moieties in many of their described inhibitors, it is unlikely that these compounds will have acceptable cell permeability<sup><a class="bk_pop" href="#ml199.r18">18</a></sup>. Additionally, this report contained limited biochemical data using a purified enzyme assay; thus, the compounds’ utility as APE1 probes is unknown.</p><p>Most recently, Kelley and co-workers<sup><a class="bk_pop" href="#ml199.r19">19</a></sup> described the HTS of 60,000 small molecules and found several low micromolar hits against APE1. The most promising was compound <b>4</b>, which had a reported IC<sub>50</sub> of 2.1 μM in whole cell extract AP endonuclease assays and inhibited the repair of AP sites in SF767 glioblastoma cells. Moreover, AR03 was reported to potentiate the cytotoxicity of the alkylators methyl methansulfonate (MMS) and TMZ in SF767 cells. This compound is actually a part of the MLSMR library (MLS000552981) and had weak activity in our assay, ~31 μM. While the reason for this discrepancy is not fully understood, it may likely be due to the differences in AP endonuclease conditions used in the respective studies. Nevertheless, these studies provide us a direct comparison of our probe compound with previously reported APE1 inhibitors.</p><p>During the course of our assay development and LOPAC screening, we identified aurintricarboxylic acid (ATA, a stable radical polymer), 6-hydroxy-DL DOPA, Tyrphostin AG 538, myricetin, thiolactomycin, and mitoxantrone as inhibitors of APE1 AP site incision activity<sup><a class="bk_pop" href="#ml199.r20">20</a></sup> (<i>see</i><a class="figpopup" href="/books/NBK133448/figure/ml199.f1/?report=objectonly" target="object" rid-figpopup="figml199f1" rid-ob="figobml199f1">Figure 1</a>). These compounds have been noted to affect multiple targets, including numerous DNA-processing enzymes. In addition to their known promiscuous nature, none of the compounds represented a good starting point for extensive optimization.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml199f1" co-legend-rid="figlgndml199f1"><a href="/books/NBK133448/figure/ml199.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml199f1" rid-ob="figobml199f1"><img class="small-thumb" src="/books/NBK133448/bin/ml199f1.gif" src-large="/books/NBK133448/bin/ml199f1.jpg" alt="Figure 1. Hit compounds identified from the LOPAC screen." /></a><div class="icnblk_cntnt" id="figlgndml199f1"><h4 id="ml199.f1"><a href="/books/NBK133448/figure/ml199.f1/?report=objectonly" target="object" rid-ob="figobml199f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Hit compounds identified from the LOPAC screen. </p></div></div></div></div><div id="ml199.s5"><h2 id="_ml199_s5_">2. Materials and Methods</h2><p><b>General Methods for Chemistry.</b> Unless otherwise stated, all reactions were carried out under an atmosphere of dry argon or nitrogen in dried glassware. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (rt) is noted as 25 °C. All solvents were of anhydrous quality, purchased from Aldrich Chemical Co. and used as received. Commercially available starting materials and reagents were purchased from Aldrich and were used as received. Analytical thin layer chromatography (TLC) was performed with Sigma Aldrich TLC plates (5 × 20 cm, 60 Å, 250 μm). Visualization was accomplished by irradiation under a 254 nm UV lamp. Chromatography on silica gel was performed using forced flow (liquid) of the indicated solvent system on Biotage KP-Sil pre-packed cartridges and the Biotage SP-1 automated chromatography system. <sup>1</sup>H- and <sup>13</sup>C NMR spectra were recorded on a Varian Inova 400 MHz spectrometer. Chemical shifts are reported in ppm with the solvent resonance as the internal standard (CDCl<sub>3</sub> 7.26 ppm, 77.00 ppm, DMSO-<i>d</i><sub>6</sub> 2.49 ppm, 39.51 ppm for <sup>1</sup>H, <sup>13</sup>C respectively). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants, and number of protons. Low resolution mass spectra (electrospray ionization) were acquired on an Agilent Technologies 6130 quadrupole spectrometer coupled to the HPLC system. High resolution mass spectral data were collected in-house using an Agilent 6210 time-of-flight mass spectrometer, also coupled to an Agilent Technologies 1200 series HPLC system. If needed, products were purified via a Waters semi-preparative HPLC equipped with a Phenomenex Luna<sup>®</sup> C18 reverse phase (5 micron, 30 × 75 mm) column having a flow rate of 45 mL/min. The mobile phase was a mixture of acetonitrile (0.025% TFA) and H<sub>2</sub>O (0.05% TFA), and the temperature was maintained at 50 °C.</p><p>Samples were analyzed for purity on an Agilent 1200 series LC/MS equipped with a Luna® C18 reverse phase (3 micron, 3 ×75 mm) column having a flow rate of 0.8–1.0 mL/min over a 7-minute gradient and an 8.5 minute run time. Purity of final compounds was determined to be >95%, using a 3 μL injection with quantitation by AUC at 220 and 254 nm (Agilent Diode Array Detector).</p><div id="ml199.s6"><h3>2.1. Assays</h3><p><b>APE1 qHTS Assay:</b> Inhibition of APE1 activity was screened (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2517" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2517</a>) by utilizing double-stranded short substrate containing a tetrahydrofuran (THF) abasic site labeled with rhodamine-type fluorophore (TAMRA) at the 5′-end and with non-fluorescent Black Hole Quencher-2 (BHQ-2) at the opposing 3′-end. An increase in the fluorescence intensity due to incision of the abasic site by APE1 was used to measure the enzyme activity. Three μL of enzyme were dispensed to 1536-well Greiner black solid bottom plates. Compounds (23 nL) were transferred via Kalypsys pintool. The plates were incubated for 15 min at room temperature, and then 1 μL of substrate solution was added to start the reaction. The plates were immediately transferred into a ViewLux High-throughput CCD imager (Perkin-Elmer) in order to measure the reaction progress in kinetic mode (three reads every 60 seconds) using a 525 nm excitation and 598 nm emission fluorescence protocol. The fluorescence intensity difference between the third and the first time points was used to compute reaction progress. Reagents and Controls: Substrate: 50 nM final concentration of TAMRA/BHQ-2 substrate dispensed throughout the plate. Enzyme: 0.75 nM APE1 final concentration in columns 1, 2, 5–48. Column 1 is neutral (100% activity). Control: Pintool transfer of control inhibitor NSC-13755 to column 2 of all assay plates. Two-fold, 16-pt dilution in duplicate to produce final concentrations in the 5.75 μM – 0.175 nM range. Buffer columns in columns 3 and 4 were used as negative control (no enzyme).</p><p><b>Thiazole Orange Assay for Promiscuous DNA Binding:</b> In order to distinguish true inhibitors of DNA-processing enzymes from promiscuous DNA-binding compounds among screening assay hits, we developed a homogeneous and miniaturized assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2741" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2741</a>) based on fluorescent dye displacement test as originally described by Tse and Boger<sup><a class="bk_pop" href="#ml199.r21">21</a></sup>. The assay is based on the strong enhancement of fluorescence when Thiazole Orange (ThO) binds to double-stranded DNA; conversely, in the presence of a DNA-binding compound, Thiazole Orange is displaced from the DNA and its fluorescence is reduced. Three μL of reaction buffer (50 mM Tris-HCl pH7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT and 0.01% Tween-20) containing 250 nM ThO and 50 nM dsDNA (upper strand 5′ TC ACC FTC GTA CGA CTC, lower strand 3′ AG TGG GAG CAT GCT GAG, F denotes tetrahydrofuran) were dispensed into a 1536-well Greiner black assay plate via solenoid-valve based nanoliter dispensers. Compounds (23 nL each in columns 5–48) and control (23 nL each in column 2 as dose-response) were transferred via a Kalypsys pintool equipped with a 1536-pin array. The plates were incubated for 15 min at room temperature and were then transferred into a ViewLux high-throughput CCD imager, where the endpoint fluorescence was measured using a 480 nm excitation and 540 nm emission protocol.</p><p><b>Radiotracer Incision Assay (RIA)</b>. Recombinant wild type APE1 protein was purified as previously described<sup><a class="bk_pop" href="#ml199.r22">22</a></sup>. Fifty pg of APE1 (~140 pM) was incubated without (positive control containing 1% DMSO) or with 100 μM of the indicated inhibitor at room temperature in RIA buffer (50 mM Tris pH 7.5, 25 mM NaCl, 1 mM MgCl<sub>2</sub>, 1 mM DTT, 0.01% Tween -20) for 15 min. One-half pmol of <sup>32</sup>P 5′-radiolabeled AP-DNA substrate (18 mer) was added to a 10 μL final volume<sup><a class="bk_pop" href="#ml199.r23">23</a></sup>, and the reactions were incubated at 37 °C for 5 min and stopped by adding stop buffer (0.05% Bromophenol blue/ Xylene cyanol dissolved in 95% formamide, 20 mM EDTA) and heating at 95 °C for 10 min. Intact substrate was separated from incised product on a 15% polyacrylamide denaturing gel in tris boric acid EDTA buffer. Following electrophoresis, the gel was subjected to standard phosphoimager analysis using the ImageQuant 5.2 software, and the percent incision activity (amount of substrate converted to product) was calculated. For IC<sub>50</sub> determinations (i.e. the concentration of inhibitor at which 50% inactivation was observed), 50 pg of APE1 (~140 pM) was incubated without or with increasing concentrations (1 nM to 100 μM) of the indicated inhibitor as above, and the percent incision activity was determined. IC<sub>50</sub>s were extrapolated from duplicate experimental sets after plotting the results using GraphPad Prism 4.0. PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488940" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488940</a>.</p><p><b>Electrophoretic Mobility Shift Assay (EMSA).</b> Three hundred ng of APE1 (~0.8 μM) was incubated without inhibitor (positive control, 1% DMSO) or with increasing concentrations of inhibitor (1, 3, 10, 30 pr 100 μM) in binding buffer (50 mM Tris pH 7.5, 25 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.01% Tween 20) for 10 min on ice, and then radiolabeled <sup>32</sup>P AP-DNA substrate (100 fmol) was added to a 10 μL final volume. Following incubation on ice for 5 min, samples were subjected to non-denaturing polyacrylamide gel electrophoresis (20 mM Tris pH 7.5, 10 mM sodium acetate, 0.5 mM EDTA, 8% polyacrylamide, 2.5% glycerol) for 2 hr at 120 V in electrophoresis buffer (20 mM Tris pH 7.5, 10 mM sodium acetate, 0.5 mM EDTA) to separate the APE1-DNA complex from unbound radiolabeled DNA<sup><a class="bk_pop" href="#ml199.r24">24</a></sup>. After electrophoresis, the gel was subjected to standard phosphoimager analysis as above, and the percentage of substrate DNA in complex with APE1 was determined.</p><p><b>HeLa Whole Cell Extract Incision Assay.</b> HeLa cells maintained in DMEM with 10% fetal bovine serum and 1% Penicillin-Streptomycin were harvested, washed with 1X PBS, and re-suspended in hypotonic ice cold lysis buffer (50 mM Tris pH 7.4, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.5 mM PMSF). The suspension was frozen at −80 °C for at least 30 min and then slowly thawed at 4 ºC for ~1 hr. KCl was then added to the cell suspension to a final concentration of 222 mM, followed by incubation on ice for 30 min and clarification by centrifugation at 12,000 xg for 15 min at 4 ºC. The supernatant (whole cell extract) was retained, the protein concentration determined using the Bio-Rad Bradford reagent, and aliquots stored until needed at −80 ºC. Three hundred ng of HeLa whole cell extract was incubated with 0, 50 or 100 μM of the indicated inhibitor at room temperature for 15 min prior to the addition of 0.5 pmol of <sup>32</sup>P radiolabeled AP-DNA substrate (final volume of 10 μL). The reaction mix was then transferred to 37 ºC for 5 min to allow for incision. Following addition of stop buffer and heat denaturation, the reaction products were analyzed as above.</p><p><b>MMS Potentiation Cell-based Assay.</b> HeLa cells were plated by multichannel pipette or Multidrop Combi dispenser (Thermo) at 6K/25 μL/well in DMEM culture medium, plus 10% FBS, into white solid bottom 384-well cell culture plates. The plates were incubated at 37 ºC overnight for cell attachment. The following day, the cell medium in each well was replaced with fresh medium containing MMS (0.4 mM final concentration) and/or serial dilutions of compounds of interest, except for the vehicle controls. The plates were incubated for 24 hr at 37 ºC. Cell viability was evaluated by adding 15 μL of CellTiter Glo reagent (Promega, Madison, WI) and incubating the plate at room temperature for 30 min, and subsequently measuring the luminescence in ViewLux reader. Percent viability was calculated for each concentration of tested compounds in duplicates, relating the luminescence to the negative DMSO control. MMS potentiation trends were defined as follows: (−), if cell viability was the same as that of the DMSO control; (+), if the cell viability decrease was between 20–40%; and (++), if the cell viability decrease was more than 40% in presence of test compounds.</p></div><div id="ml199.s7"><h3>2.2. Probe Chemical Characterization</h3><div id="ml199.fu2" class="figure"><div class="graphic"><img src="/books/NBK133448/bin/ml199fu2.jpg" alt="Image ml199fu2" /></div></div><p>*Purity >95% as judged by LC/MS and <sup>1</sup>H NMR</p><p><b><i>N</i></b><b>-(3-(benzo[d]thiazol-2-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-2-yl)acetamide</b>: LC-MS: rt (min) = 3.98; <sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ 2.36 (s, 3 H), 4.54 (s, 2 H), 4.68 (s, 2 H), 7.49 (t, <i>J</i> = 7.6 Hz, 1 H), 7.61 (t, <i>J</i> = 7.7 Hz, 1 H), 8.17 (t, <i>J</i> = 7.5 Hz, 2 H), 9.94 (brs, 1 H), and 12.03 (brs, 1 H); HRMS (ESI) <i>m</i>/<i>z</i> (M+Na)<sup>+</sup> calcd. for C<sub>15</sub>H<sub>13</sub>N<sub>3</sub>OS<sub>2</sub>Na, 338.0403; found 338.0403.</p><div id="ml199.s8"><h4>LC/MS conditions</h4><ul><li class="half_rhythm"><div>LC/MS (Agilent system) Retention time t<sub>1</sub> (short) = 2.88 min and t<sub>2</sub> (long) = 3.88.</div></li><li class="half_rhythm"><div>Column: 3 × 75 mm Luna C18, 3 micron</div></li><li class="half_rhythm"><div>Run time: 4.5 min (short); 8.5 min (long)</div></li><li class="half_rhythm"><div>Gradient: 4 % to 100 %</div></li><li class="half_rhythm"><div>Mobile phase: Acetonitrile (0.025 % TFA), water (0.05 % TFA)</div></li><li class="half_rhythm"><div>Flow rate: 0.8 to 1.0 mL</div></li><li class="half_rhythm"><div>Temperature: 50°C</div></li><li class="half_rhythm"><div>UV wavelength: 220 nm, 254 nm</div></li></ul></div><div id="ml199.s9"><h4>MLSMR Numbers for Probe and Analogs</h4><div id="ml199.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133448/table/ml199.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml199.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml199.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS ID</th><th id="hd_h_ml199.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC ID</th><th id="hd_h_ml199.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th id="hd_h_ml199.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th id="hd_h_ml199.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">ML</th><th id="hd_h_ml199.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Type</th></tr></thead><tbody><tr><td headers="hd_h_ml199.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003178551</td><td headers="hd_h_ml199.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185090</td><td headers="hd_h_ml199.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430950</a></td><td headers="hd_h_ml199.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 46925884</td><td headers="hd_h_ml199.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a></td><td headers="hd_h_ml199.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe</td></tr><tr><td headers="hd_h_ml199.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003178552</td><td headers="hd_h_ml199.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184830</td><td headers="hd_h_ml199.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430935" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430935</a></td><td headers="hd_h_ml199.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 4055306</td><td headers="hd_h_ml199.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml199.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td></tr><tr><td headers="hd_h_ml199.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003178553</td><td headers="hd_h_ml199.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184832</td><td headers="hd_h_ml199.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430937" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430937</a></td><td headers="hd_h_ml199.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 46925888</td><td headers="hd_h_ml199.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml199.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td></tr><tr><td headers="hd_h_ml199.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003178554</td><td headers="hd_h_ml199.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184834</td><td headers="hd_h_ml199.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430938" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430938</a></td><td headers="hd_h_ml199.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 46925885</td><td headers="hd_h_ml199.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml199.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td></tr><tr><td headers="hd_h_ml199.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003178555</td><td headers="hd_h_ml199.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185094</td><td headers="hd_h_ml199.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430953" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430953</a></td><td headers="hd_h_ml199.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 46925861</td><td headers="hd_h_ml199.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml199.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td></tr><tr><td headers="hd_h_ml199.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003178556</td><td headers="hd_h_ml199.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00186101</td><td headers="hd_h_ml199.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430962" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99430962</a></td><td headers="hd_h_ml199.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 46925887</td><td headers="hd_h_ml199.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml199.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td></tr></tbody></table></div></div></div><div id="ml199.s10"><h4>Probe <i>in vitro</i> ADME properties</h4><ul><li class="half_rhythm"><div>Aqueous (kinetic) solubility in PBS buffer (pH 7.4 @ 25 °C): 51.6 μM</div></li><li class="half_rhythm"><div>Caco-2 permeability (P<sub>app</sub> × 10<sup>6</sup>)/cm-s<sup>−1</sup>: 6.8</div></li><li class="half_rhythm"><div>Efflux ratio (B→A)/(A→B): 0.81</div></li><li class="half_rhythm"><div>hERG inhibition-Qpatch-whole-cell patch clamp (IC<sub>50</sub>): 8.04 μM (note original “hit” was 0.66 μM)</div></li><li class="half_rhythm"><div>mouse plasma stability (T ½) = >120 min</div></li></ul><div id="ml199.f2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133448/bin/ml199f2.jpg" alt="Figure 2. Buffer Stability (48 hrs @ 25 °C) of ML199." /></div><h3><span class="label">Figure 2</span><span class="title">Buffer Stability (48 hrs @ 25 °C) of ML199</span></h3></div></div></div><div id="ml199.s11"><h3>2.3. Probe Preparation</h3><p>The synthesis commenced using the Gewald’s 2-aminothiophene reaction<sup><a class="bk_pop" href="#ml199.r25">25</a></sup> of commercially available <i>t</i>-butyl-3-oxopyrrolidine-1-carboxylate in the presence of sulfur and morpholine to afford the fused product<sup><a class="bk_pop" href="#ml199.r26">26</a></sup> (see <a class="figpopup" href="/books/NBK133448/figure/ml199.f9/?report=objectonly" target="object" rid-figpopup="figml199f9" rid-ob="figobml199f9">Scheme 1</a>). The regiochemistry of the formed product was determined by NMR spectroscopy. Acetylation of the 2-amino group was achieved with acetyl choride in the presence of triethyl amine, and subsequent Boc-deprotection with TFA gave the probe molecule in good yield.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml199f9" co-legend-rid="figlgndml199f9"><a href="/books/NBK133448/figure/ml199.f9/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml199f9" rid-ob="figobml199f9"><img class="small-thumb" src="/books/NBK133448/bin/ml199f9.gif" src-large="/books/NBK133448/bin/ml199f9.jpg" alt="Scheme 1. Synthetic route to ML199." /></a><div class="icnblk_cntnt" id="figlgndml199f9"><h4 id="ml199.f9"><a href="/books/NBK133448/figure/ml199.f9/?report=objectonly" target="object" rid-ob="figobml199f9">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthetic route to ML199. </p></div></div><p><b>Preparation of</b><b><i>t</i></b><b>-butyl 2-amino-3-(benzo[d]thiazol-2-yl)-4H-thieno[3,2-c]pyrrole-5(6H)-carboxylate (Step 1):</b> A mixture of <i>t</i>-butyl 3-oxopyrrolidine-1-carboxylate (1.0 g, 5.4 mmol, 1 eq.), 2-(benzo[d]thiazol-2-yl)acetonitrile (0.94 g, 5.4 mmol, 1 eq.), sulfur (0.17 g, 5.4 mmol, 1 eq.) and morpholine (0.47 mL, 5.4 mmol, 1 eq.) in ethanol (25 mL) was refluxed for 1 hr. Excess solvent was removed under diminished pressure and the crude product was recrystallized from ethanol. Yield: 1.6 g (79%).</p><p><b>Preparation of</b><b><i>t</i></b><b>-butyl 2-acetamido-3-(benzo[d]thiazol-2-yl)-4H-thieno[3,2-c]pyrrole-5(6H)-carboxylate (Step 2)</b>: To a mixture of <i>t</i>-butyl 2-amino-3-(benzo[d]thiazol-2-yl)-4H-thieno[3,2-c]pyrrole-5(6H)-carboxylate (1.0 g, 2.7 mmol, 1 eq.) and triethylamine (1.1 mL, 8.0 mmol, 3 eq.) in dichloromethane (25 mL) was added acetyl chloride (0.29 mL, 4.0 mmol, 1.5 eq.) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 hrs, then diluted with dichloromethane, and the organic layer was successively with water, saturated bicarbonate solution and brine. The organic layer was dried on sodium sulfate and evaporated to afford the crude solid which was further purified on a Biotage® silica gel column. Gradient elution with 2% methanol in dichloromethane gave the product. Yield: 0.99 g (89%).</p><p><b>Preparation of</b><b><i>N</i></b><b>-(3-(benzo[d]thiazol-2-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-2-yl)acetamide (step 3):</b><i>t</i>-Butyl 2-acetamido-3-(benzo[d]thiazol-2-yl)-4H-thieno[3,2-c]pyrrole-5(6H)-carboxylate (0.5 g, 1.59 mmol) in dichloromethane (10 mL) was added with TFA (5 mL) and was stirred at room temperature for 1 h. The volatiles were removed under reduced pressure and the crude product was purified in a preparative HPLC (see general methods for details) to afford the pure product.</p><p><b>General Procedure for reductive amination (for analogs containing substitution on piperidine nitrogen):</b> Sodium cyanoborohydride (3 mmol, 3 eq.) and few drops of acetic acid were added to a mixture of appropriate amine (1 mmol, 1 eq.) and acetone/appropriate carbonyl compound (10 mmol, 10 eq.) in a mixture of MeOH/THF (0.2 molar reaction concentration). The reaction mixture was stirred at room temperature for 6–10 h. Volatiles were removed and the crude solid was purified on a preparative HPLC.</p></div></div><div id="ml199.s12"><h2 id="_ml199_s12_">3. Results</h2><div id="ml199.s13"><h3>3.1. Summary of Screening Results</h3><p>A robust modified donor/quencher-based assay was further miniaturized into a 1536-well format and used to identify small molecule inhibitors of APE1. Briefly, a deoxyoligonucleotide containing a tetrahydrofuran (THF) abasic-site analog labeled on the 5′-end with 6-TAMRA was annealed to its complementary strand, where the 3′-end was labeled with a Black Hole Quencher-2 (BHQ2), to create an artificial DNA substrate for the APE1 enzyme. Upon cleavage of the abasic site by APE1, a short deoxyoligonucleotide product labeled with TAMRA was spontaneously released from the remaining DNA, causing the fluorophore emission to increase by up to 10-fold under the assay conditions. Prior to the full-collection screen, the assay was tested and found to perform reproducibly by screening the LOPAC<sup>1280</sup> (library of pharmacologically active compounds) in triplicate using a fully-integrated robotic system. The assay was then applied to screen a 239,513-compound library arrayed as seven-point titrations ranging from 57 μM to 2.9 nM. In total, 1,339 assay plates, containing 2,056,704 wells, were screened in one continuous robotic run lasting approximately 84 hrs. Overall, the assay performed well during the entire course of the screen: intra-plate control titration (NSC 13755) was stable, with an average IC<sub>50</sub> of 0.035 μM and a minimum significant ratio of 1.9; the Z’ factor remained consistent without fluctuations, at an average of 0.79.</p><p>In contrast to traditional HTS, qHTS provides a concentration response curve (CRC) for each compound and allows for calculation of an AC<sub>50</sub> value (defined as the half-maximal activity concentration) for each compound in the primary screen. In this screen, the inhibition associated with each well was computed from the alteration in fluorescence intensity over the time-course measurement period, normalized against control wells. Analysis of the qHTS results revealed that the top filtered samples (based on curve quality and potency) led to 121 clusters and 154 singletons, consisting of approximately 1,100 compounds with full concentration-response curves and IC<sub>50</sub> values of less than 30 μM. Results can be seen in <a class="figpopup" href="/books/NBK133448/figure/ml199.f3/?report=objectonly" target="object" rid-figpopup="figml199f3" rid-ob="figobml199f3">Figure 3</a>. To define the structure activity relationship (SAR) between the various compounds that elicited inhibition of APE1 activity, we clustered the structures showing high and low quality CRC by structural similarity. This enabled us to find a series of inhibitors with a common core and to query the entire qHTS dataset to identify those related structures associated with a weaker CRC or inactivity. This approach allows determination of a complete SAR from the qHTS to be developed for novel lead compounds. After exclusion of strong auto-fluorescent compounds, compounds active in the thiazole orange dye displacement DNA binding assay, antimony-containing compounds, and various mercury and other heavy-metal-containing molecules, 471 compounds remained. These were tested in a radiotracer assay for orthogonal confirmation, resulting in the identification of the present chemotype (<i>see</i><a class="figpopup" href="/books/NBK133448/figure/ml199.f4/?report=objectonly" target="object" rid-figpopup="figml199f4" rid-ob="figobml199f4">Figure 4</a>). The initial active for probe chemotype is CID 3581333 (MLS000419194, resynthesized as NCGC00183664-01).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml199f3" co-legend-rid="figlgndml199f3"><a href="/books/NBK133448/figure/ml199.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml199f3" rid-ob="figobml199f3"><img class="small-thumb" src="/books/NBK133448/bin/ml199f3.gif" src-large="/books/NBK133448/bin/ml199f3.jpg" alt="Figure 3. Activity from the primary qHTS screen (A) and distribution of the assay response against the chemical library (B)." /></a><div class="icnblk_cntnt" id="figlgndml199f3"><h4 id="ml199.f3"><a href="/books/NBK133448/figure/ml199.f3/?report=objectonly" target="object" rid-ob="figobml199f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Activity from the primary qHTS screen (A) and distribution of the assay response against the chemical library (B). </p></div></div></div><div id="ml199.s14"><h3>3.2. Dose Response Curves for Probe</h3><div id="ml199.f4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133448/bin/ml199f4.jpg" alt="Figure 4. Dose response from ML199 in Radiotracer Incision Assay and structure of the probe." /></div><h3><span class="label">Figure 4</span><span class="title">Dose response from ML199 in Radiotracer Incision Assay and structure of the probe</span></h3></div></div><div id="ml199.s15"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>While many of the series found in the qHTS contained reactive functional groups, the series chosen for probe development did not contain any serious liabilities.</p></div><div id="ml199.s16"><h3>3.4. SAR Tables</h3><div id="ml199.t1" class="table"><h3><span class="label">Table 1</span><span class="title">SAR Table - APE1 inhibition: representative analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133448/table/ml199.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml199.t1_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml199.t1_1_1_1_1" colspan="8" rowspan="1" style="text-align:center;vertical-align:bottom;">
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<div class="graphic"><img src="/books/NBK133448/bin/ml199fu3.jpg" alt="Image ml199fu3.jpg" /></div></th></tr><tr><th headers="hd_h_ml199.t1_1_1_1_1" id="hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Entry</th><th headers="hd_h_ml199.t1_1_1_1_1" id="hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th headers="hd_h_ml199.t1_1_1_1_1" id="hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th headers="hd_h_ml199.t1_1_1_1_1" id="hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC IDs</th><th headers="hd_h_ml199.t1_1_1_1_1" id="hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>1</sub></th><th headers="hd_h_ml199.t1_1_1_1_1" id="hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>2</sub></th><th headers="hd_h_ml199.t1_1_1_1_1" id="hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>3</sub></th><th headers="hd_h_ml199.t1_1_1_1_1" id="hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">HTS potency (μM) [RIA IC50 (μM)], n= replicates</th></tr></thead><tbody><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3581333</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430932" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430932</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00183664-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">10 [19], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925879</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430934" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430934</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184829-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">6 [5], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925863</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430933" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430933</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184828-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Boc</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">31 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4055306</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430935" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430935</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184830-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">6 [5], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4532325</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430936" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430936</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184831-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Bn</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">18 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4348310</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430945" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430945</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185083-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Ac</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">10 [5], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925878</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430951" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430951</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185091-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">>56 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925860</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430960" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430960</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00186096-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2-furan</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">18 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>9</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925883</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430961" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430961</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00186097-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2-thiophene</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">36 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>10</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925887</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430962" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430962</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00186101-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2-benzofuran</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">18 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>11</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925876</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430963" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430963</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00187565-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2-indole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">>56 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>12</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925880</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430964" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430964</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00187566-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2-thiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">23 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>13</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925872</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430959" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430959</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00186075-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Ph</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">>56 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>14</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925888</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430937" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430937</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184832-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzoxazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">23 [13], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>15</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925885</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430938" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430938</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184834-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2-(4-phenylthiazole)</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">6 [3], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>16</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4138521</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430940" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430940</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184838-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C(CH<sub>3</sub>)<sub>3</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">25 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>17</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4053204</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430941" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430941</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184839-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">cyclopropane</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">18 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>18</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">43950654</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430942" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430942</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184840-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">cyclopropane</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">11 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>19</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4107484</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430939" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430939</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184837-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Ph</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">16 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>20</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925867</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430944" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430944</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185082-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CF<sub>3</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">31 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>21</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925862</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430967" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430967</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00189087-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OMe</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">6 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>22</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925861</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430953" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430953</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185094-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CHF<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">3 [4], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>23</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925869</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430946" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430946</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185085-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">(CH<sub>2</sub>)<sub>2</sub>C≡CH</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">14 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>24</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925865</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430947" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430947</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185086-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">(CH<sub>2</sub>)<sub>2</sub>C≡CH</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">11 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>25</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925871</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430966" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430966</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00189084-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK133448/bin/ml199fu4.jpg" alt="Image ml199fu4.jpg" /></div></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">>56 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>26</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925873</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430965" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430965</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00189083-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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||
<div class="graphic"><img src="/books/NBK133448/bin/ml199fu5.jpg" alt="Image ml199fu5.jpg" /></div></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">23 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>27</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925877</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430969" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430969</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00189234-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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||
<div class="graphic"><img src="/books/NBK133448/bin/ml199fu6.jpg" alt="Image ml199fu6.jpg" /></div></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">16 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>28</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925868</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430968" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430968</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00189233-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK133448/bin/ml199fu7.jpg" alt="Image ml199fu7.jpg" /></div></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">16 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>29</b></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925866</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/46925866" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">46925866</a></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00184843-01</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NH<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">benzothiazole</td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t1_1_1_1_1 hd_h_ml199.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">41 [ND], n = 2</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt></dt><dd><div id="ml199.tfn5"><p class="no_margin">Note: all compounds in table were synthesized at NCGC. ND = not determined.</p></div></dd></dl></div></div></div><div id="ml199.t2" class="table"><h3><span class="label">Table 2</span><span class="title">SAR Table - APE1 inhibition: representative analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133448/table/ml199.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml199.t2_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml199.t2_1_1_1_1" colspan="6" rowspan="1" style="text-align:center;vertical-align:bottom;">
|
||
<div class="graphic"><img src="/books/NBK133448/bin/ml199fu8.jpg" alt="Image ml199fu8.jpg" /></div></th></tr><tr><th headers="hd_h_ml199.t2_1_1_1_1" id="hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Entry</th><th headers="hd_h_ml199.t2_1_1_1_1" id="hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">CID</th><th headers="hd_h_ml199.t2_1_1_1_1" id="hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">SID</th><th headers="hd_h_ml199.t2_1_1_1_1" id="hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC IDs</th><th headers="hd_h_ml199.t2_1_1_1_1" id="hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R</th><th headers="hd_h_ml199.t2_1_1_1_1" id="hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">HTS potency (μM) [RIA IC50 (μM)], n= replicates</th></tr></thead><tbody><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>30</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925881</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430948" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430948</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185087-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">O</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">18 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>31</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925864</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430949" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430949</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185089-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">S</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>32</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925882</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430955" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430955</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185096-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">S(O)<sub>2</sub></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">11<sup><a class="bk_pop" href="#ml199.tfn1">*</a></sup> [77], n = 2</td></tr><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>33</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925874</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430954" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430954</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185095-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NH</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">45 [ND], n = 2</td></tr><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>34</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925884</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430950</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185090-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NH</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6 [1], n = 2</td></tr><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>35</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925875</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430952</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185093-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N(CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3 [4], n = 2</td></tr><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>36</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925870</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430958" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430958</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185100-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N(CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">18 [13], n = 2</td></tr><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>37</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925889</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430956" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430956</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185097-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N(CH(CH<sub>3</sub>)<sub>2</sub></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16 [14], n = 2</td></tr><tr><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>38</b></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46925886</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99430957" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99430957</a></td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00185099-01</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NH</td><td headers="hd_h_ml199.t2_1_1_1_1 hd_h_ml199.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">18 [7], n = 2</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt></dt><dd><div id="ml199.tfn6"><p class="no_margin">Note: all compounds in table were synthesized at NCGC. ND = not determined.</p></div></dd><dt>*</dt><dd><div id="ml199.tfn1"><p class="no_margin">low maximum response in HTS.</p></div></dd></dl></div></div></div></div><div id="ml199.s17"><h3>3.5. Cellular Activity</h3><div id="ml199.s18"><h4>Cytotoxicity Studies (MTT Assay) of ML199</h4><ul><li class="half_rhythm"><div>HeLa cells: % viability- (98% @ 5 μM), (79% @ 20 μM), (~5% @ 50 μM)</div></li><li class="half_rhythm"><div>T98G cells: % viability- (99% @ 5 μM), (95% @ 20 μM), (~80% @ 50 μM)</div></li><li class="half_rhythm"><div>U-87 MG cells: % viability- (35% @ 5 μM), (20% @ 20 μM), (~20% @ 50 μM)</div></li><li class="half_rhythm"><div>U-138 MG cells: (92% @ 5 μM), (80% @ 20 μM), (~50% @ 50 μM)</div></li></ul></div><div id="ml199.s19"><h4>MMS Potentiation</h4><div id="ml199.f5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133448/bin/ml199f5.jpg" alt="Figure 5. Potentiation of probe ML199 in the presence or absence of MMS." /></div><h3><span class="label">Figure 5</span><span class="title">Potentiation of probe ML199 in the presence or absence of MMS</span></h3></div></div></div><div id="ml199.s20"><h3>3.6. Profiling Assays</h3><div id="ml199.s21"><h4>ADME profiling</h4><p>As shown in the <a class="figpopup" href="/books/NBK133448/table/ml199.t3/?report=objectonly" target="object" rid-figpopup="figml199t3" rid-ob="figobml199t3">Table 3</a>, both compound <b>1</b> (CID 3581333) and the probe molecule <b>34</b> (<a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a>, <i>see</i><a class="figpopup" href="/books/NBK133448/table/ml199.t2/?report=objectonly" target="object" rid-figpopup="figml199t2" rid-ob="figobml199t2">Table 2</a>) have many favorable attributes, yet a few liabilities exist. Compound <b>34</b> was chosen as the probe molecule because of its greatly improved potency in the secondary radiotracer assay (1 μM for <b>34</b> vs. 19 μM for <b>1</b>). The probe molecule also exhibits improved kinetic solubility, Caco-2 permeability and 10-fold less inhibition of the hERG channel or the HTS “hit” compound. Though the probe molecule is susceptible to mouse liver microsomes, compound <b>1</b> shows favorable stability. This seems to indicate that the general core scaffold is metabolically stable, and through additional structural modifications, this potential liability of the probe molecule could be addressed while maintaining potency.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml199t3"><a href="/books/NBK133448/table/ml199.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml199t3" rid-ob="figobml199t3"><img class="small-thumb" src="/books/NBK133448/table/ml199.t3/?report=thumb" src-large="/books/NBK133448/table/ml199.t3/?report=previmg" alt="Table 3. In vitro ADME to date." /></a><div class="icnblk_cntnt"><h4 id="ml199.t3"><a href="/books/NBK133448/table/ml199.t3/?report=objectonly" target="object" rid-ob="figobml199t3">Table 3</a></h4><p class="float-caption no_bottom_margin"><i>In vitro</i> ADME to date. </p></div></div><p>No additional profiling studies have been conducted on the probe compound. This novel probe does not have any additional data in literature or in PubChem.</p></div></div></div><div id="ml199.s22"><h2 id="_ml199_s22_">4. Discussion</h2><p>Our initial SAR investigations involved modification of the piperidine nitrogen moiety (see entries <b>1–6</b> in <a class="figpopup" href="/books/NBK133448/table/ml199.t1/?report=objectonly" target="object" rid-figpopup="figml199t1" rid-ob="figobml199t1">Table 1</a>). Changes to this position were well tolerated except for the introduction of the Boc-group (entry <b>3</b>), which resulted in a 3-fold loss of activity. Removal of the isopropyl group (R<sub>3</sub> = H, entry <b>2</b>) led to an increase in potency [IC<sub>50</sub> = 6 μM (HTS assay) and 5 μM (RIA)]. Consequently, many of the subsequent analogs were synthesized without the isopropyl group, which saved one synthetic step.</p><p>The next area of SAR exploration involved modification of the benzothiazole moiety at the 3-position of the thiophene. As shown in the SAR Table above, many of the changes were not well tolerated; however, select modifications maintained activity of the parent compound. Removal of the benzothiazole (R<sub>2</sub> = H, entry <b>7</b>) resulted in essentially complete loss in activity. Other heterocycles were synthesized (entries <b>8</b>–<b>12</b>, <b>14–15</b>); however, only the 2-(4-phenylthiazole) analog (<b>15</b>) displayed potent inhibition. Interestingly, several simple phenyl substituted analogs (entry <b>13</b>) and other differentially substituted phenyl ring analogs (data not shown), failed to show even moderate inhibition of APE1. These results suggest that the thiazole moiety (or analogous heterocycyle) is involved in an essential interaction in the APE1 binding site and should be maintained in some capacity in future analogs. Given that entry <b>15</b> had comparable potency to the requisite benzothiazole moiety, a phenyl ring scan of the pendant aryl ring may prove fruitful.</p><p>The goal of entries <b>16–29</b> was to gain a better understanding of the SAR associated with the N-acetyl moiety of the lead compound <b>1</b>. As such, a variety of different analogs were prepared, including numerous amides, carbamates, des-acetyl, carboxylic acids, esters, and amide bioisosteres (1,2,4-oxadiazoles and 1,3,4-oxadiazoles). Our first interest was to look at the des-acetyl analog (entry <b>29</b>); as shown in the above SAR Table, this modification was clearly unfavorable, with a 4-fold loss in activity. Many of the amide analogs resulted in a decrease in potency (e.g. <i>t</i>-Butyl (<b>16</b>) = 25 μM, cyclopropane (<b>17</b>) = 18 μM, CF<sub>3</sub> (<b>20</b>) = 31 μM). However, other amide analogs exhibited comparable if not improved potency (e.g. homo-propargyl (<b>24</b>) = 11 μM, CHF<sub>2</sub> (<b>22</b>) = 3 μM, and cyclopentane (<b>18</b>) = 11 μM). Replacing the amide with other functional groups, such as a carboxylic acid or ester, resulted in a dramatic loss of potency (data not shown); however, methylcarbamate (<b>21</b>) showed a 2–3-fold improvement in activity over compound <b>1</b>. We were also eager to explore the tolerance of amide bioisosteres, such as 1,2,4-oxadiazoles and 1,3,4-oxadiazoles. Toward this end, both analogs that lacked the 2-amino group (<b>25</b> and <b>26</b>) and those that maintained this moiety (<b>27</b> and <b>28</b>) were investigated. Interestingly, both the 1,3,4-oxadiazole (<b>27</b>) and 1,2,4-oxadiazole analog (<b>28</b>) showed comparable activity to the lead compound <b>1</b> (16 μM vs. 10 μM), providing the possibility of utilizing such modifications if necessary, based on future pharmacokinetic studies on this class of compounds.</p><p>Having explored the various functional groups around the core of the molecule, we then aimed to investigate modifications to the piperidine ring. As shown in <a class="figpopup" href="/books/NBK133448/table/ml199.t1/?report=objectonly" target="object" rid-figpopup="figml199t1" rid-ob="figobml199t1">Tables 1</a> and <a class="figpopup" href="/books/NBK133448/table/ml199.t2/?report=objectonly" target="object" rid-figpopup="figml199t2" rid-ob="figobml199t2">2</a>, most of these changes we met with limited success; however, a few interesting lessons were learned. Replacing the nitrogen with oxygen (entry <b>30</b>) or sulfur (entry <b>31</b>) had a moderate effect on potency, with IC<sub>50</sub> values of 18 and 16 μM, respectively. Sulfone derivative <b>32</b> (low max response in HTS) and pyridine analog (data not shown) both resulted in a loss of activity. Next, we explored various ring sizes and found that pyrrolidine analogs <b>34</b> and <b>35</b> displayed 19-fold and 5-fold improvement in potency, respectively, over compound <b>1</b> in the radiotracer assay (RIA). In the HTS assay, both compounds had comparable potency; however, the radiotracer incision assay results are considered the “golden standard” for final IC<sub>50</sub> determination. It should be noted that this assay is more laborious and very low throughput, so only select compounds are profiled in this system. Differentially substituted piperidine analog <b>33</b> displayed lower potency (45 μM); however, the seven-membered ring analogs <b>36–38</b> maintained comparable activity to <b>1</b>.</p><p>Compound <b>34</b> (<a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a>) exhibited good potency in the secondary radiotracer assay and gave a favorable ADME profile (see <a href="#ml199.s20">Section 3.6</a>). Furthermore, <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> was shown to potentiate the toxicity of DNA-damaging agent MMS in a subsequent cell-based assay (<a class="figpopup" href="/books/NBK133448/figure/ml199.f5/?report=objectonly" target="object" rid-figpopup="figml199f5" rid-ob="figobml199f5">Figure 5</a>) while exhibiting only moderate cytotoxicity with the probe compound alone. Mechanism of action of the probe and the original hit were explored, and both compounds were shown to act as competitive inhibitors of APE1 (<a class="figpopup" href="/books/NBK133448/figure/ml199.f6/?report=objectonly" target="object" rid-figpopup="figml199f6" rid-ob="figobml199f6">Figure 6</a>). An electrophoretic mobility shift assay (EMSA), was employed to examine the stability of the APE1-DNA substrate complex in the presence of <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> (<a class="figpopup" href="/books/NBK133448/figure/ml199.f7/?report=objectonly" target="object" rid-figpopup="figml199f7" rid-ob="figobml199f7">Figure 7</a>). The percent APE1-DNA complex decreased with the increasing concentration of the probe compound, further supporting the notion that <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> acts by competitive inhibition and binds to Ape1 at the same site as the DNA substrate. On target effect of the probe was explored using HeLa whole cell extract incision assay in which <a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> was shown to inhibit AP Site incision activity which comparable activity as the purified enzyme assay (<a class="figpopup" href="/books/NBK133448/figure/ml199.f8/?report=objectonly" target="object" rid-figpopup="figml199f8" rid-ob="figobml199f8">Figure 8</a>).</p><div id="ml199.s23"><h3>SAR Summary</h3><div id="ml199.fu3" class="figure"><div class="graphic"><img src="/books/NBK133448/bin/ml199fu9.jpg" alt="Image ml199fu9" /></div></div><p><b>R<sub>1</sub></b> = various groups are tolerated here. (e.g. Me, CHF<sub>2</sub>, OMe, homopropargyl, amide bioisosteres)</p><p><b>R<sub>2</sub></b> = tight SAR, benzothiazole (and thiazole analogs) preferred. Simple replacement with phenyl group results in complete loss in activity. Removal of benzothiazole also kills activity.</p><p><b>R<sub>3</sub> =</b> (ordered in decreasing potency) H ~ Me > Ac ~ isopropyl > Bn > Boc</p><p><b>Piperidine ring changes:</b> (ordered in decreasing potency) pyrrolidine > piperidine ~ azepane > tetrahydropyran ~ tetrahydrothiopyran > differentially substituted piperidine > pyridine</p></div><div id="ml199.s24"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p><a href="/pcsubstance/?term=ML199[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML199</a> is more selective inhibitor of APE1 when compared to Compounds <b>1–4</b>. It is an ideal starting point for further development of the probe as a pre-clinical lead for drug discovery.</p></div><div id="ml199.s25"><h3>4.2. Mechanism of Action Studies</h3><div id="ml199.s26"><h4>Enzyme Kinetic Studies</h4><p>Mode of Inhibition: <b>Competitive</b></p><div id="ml199.f6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133448/bin/ml199f6.jpg" alt="Figure 6. Kinetic parameters without and with ML199 or Compound 1." /></div><h3><span class="label">Figure 6</span><span class="title">Kinetic parameters without and with ML199 or Compound <b>1</b></span></h3><div class="caption"><p>Ten pg of APE1 (~28 pM) was incubated without or with 5 μM of the indicated inhibitor at room temperature for 15 min. Increasing concentrations of radiolabeled abasic DNA substrate (i.e. 5, 10, 25, 50, or 100 nM) were then added, and the reactions were incubated at 37 ºC for 5 min before the addition of stop buffer. Intact substrate was separated from incised product on a 15% polyacrylamide denaturing gel, and the percent conversion was determined by standard phosphorimager analysis. Lineweaver – Burk plots of 1/V versus 1/[S] were used to determine K<sub>M</sub> and k<sub>cat</sub> (shown). The plotted data points (averages and standard deviations) were derived from 11 independent values for the no inhibitor reactions, and 5 values for each of the inhibitor points.</p></div></div></div><div id="ml199.s27"><h4>Interference of APE1-DNA Complex Formation</h4><div id="ml199.f7" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133448/bin/ml199f7.jpg" alt="Figure 7. Stability of the APE1-DNA substrate complex in the presence of ML199." /></div><h3><span class="label">Figure 7</span><span class="title">Stability of the APE1-DNA substrate complex in the presence of ML199</span></h3><div class="caption"><p>(<b>A</b>) Representative EMSA. Three hundred ng of APE1 (~0.8 μM) was incubated without inhibitor (final concentration 1% DMSO) or with the indicated inhibitor (1, 3, 10, 30 or 100 μM) for 10 min on ice. One hundred fmol of abasic DNA substrate (10 nM) was then added, and the binding reaction was incubated on ice for an additional 5 min. At that time, samples were subjected to non-denaturing polyacrylamide gel electrophoresis to separate the APE1-DNA complex (C) from unbound radiolabeled DNA (DNA). Inh = inhibitor. (<b>B</b>) Relative complex formation without (“0”) or with the indicated inhibitor (in μM). Shown is the average and standard deviation of three independent experimental data points, all relative to the APE1 control, without inhibitor.</p></div></div></div><div id="ml199.s30"><h4>Inhibition of AP Site Incision Activity of Whole Cell HeLa Extracts</h4><div id="ml199.f8" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133448/bin/ml199f8.jpg" alt="Figure 8. Inhibition of HeLa whole cell extract AP site incision activity with ML199." /></div><h3><span class="label">Figure 8</span><span class="title">Inhibition of HeLa whole cell extract AP site incision activity with ML199</span></h3><div class="caption"><p>Three hundred ng of HeLa whole cell extract was incubated with 0, 50 or 100 μM of the indicated inhibitor at room temperature for 15 min, prior to the addition of 0.5 pmol radiolabeled AP-DNA substrate and subsequent transfer of the reaction mix (final volume of 10 μL) to 37 ºC for 5 min to allow for incision. Following addition of stop buffer and heat denaturation, the reaction products were subjected to 15% polyacrylamide denaturing gel electrophoresis. Shown is a bar graph reporting the relative percent incision activity in comparison to the no inhibitor control, arbitrarily set at 100. The values reported represent the averages and standard deviation of three independent experimental data points.</p></div></div></div></div><div id="ml199.s28"><h3>4.3. Planned Future Studies</h3><p>Future studies of this probe molecule include submitting the probe and related analogs to the NCI-60 panel, analysis of the pharmacokinetics (mouse species), and additional pre-clinical experiments, such as Xenograft studies (primarily as a combination therapy with known DNA damaging cancer chemotherapeutics such as TMZ). Additional planned experiments include TMZ potentiation with U-87 glioma line, AP site accumulation studies and isothermal titration calorimetry (ITC) characterization. We plan to submit an extended probe characterization proposal to provide the funds to carry out such studies.</p></div></div><div id="ml199.s29"><h2 id="_ml199_s29_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml199.r1">Wilson DM III, Bohr VA. The mechanics of base excision repair, and its relationship to aging and disease. <span><span class="ref-journal">DNA Repair (Amst). </span>2007;<span class="ref-vol">6</span>:544–559.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17112792" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17112792</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml199.r2">Wilson DM III, Barsky D. The major human abasic endonuclease: formation, consequences and repair of abasic lesions in DNA. <span><span class="ref-journal">Mutat Res. </span>2001;<span class="ref-vol">485</span>:283–307.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11585362" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11585362</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml199.r3">Demple B, Sung JS. Molecular and biological roles of APE1 protein in mammalian base excision repair. <span><span class="ref-journal">DNA Repair (Amst). </span>2005;<span class="ref-vol">4</span>:1442–1449.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16199212" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16199212</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml199.r4">Xanthoudakis S, Smeyne RJ, Wallace JD, Curran T. The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice. <span><span class="ref-journal">Proc Natl Acad Sci U S A, </span>1996;<span class="ref-vol">93</span>:8919–8923.</span> [<a href="/pmc/articles/PMC38569/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC38569</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/8799128" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8799128</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml199.r5">Fung H, Demple B. A vital role for ape1/ref1 protein in repairing spontaneous DNA damage in human cells. <span><span class="ref-journal">Mol Cell. </span>2005;<span class="ref-vol">17</span>:463–470.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15694346" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15694346</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml199.r6">Izumi T, Brown DB, Naidu CV, Bhakat KK, MacInnes MA, Saito H, Chen DJ, Mitra S. Two essential but distinct functions of the mammalian abasic endonuclease. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2005;<span class="ref-vol">102</span>:5739–5743.</span> [<a href="/pmc/articles/PMC556297/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC556297</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15824325" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15824325</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml199.r7">Fishel ML, Kelley MR. The DNA base excision repair protein APE1/Ref-1 as a therapeutic and chemopreventive target. <span><span class="ref-journal">Mol Aspects Med. </span>2007;<span class="ref-vol">28</span>:375–395.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17560642" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17560642</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml199.r8">McNeill DR, Wilson DM III. A dominant-negative form of the major human abasic endonuclease enhances cellular sensitivity to laboratory and clinical DNA-damaging agents. <span><span class="ref-journal">Mol Cancer Res. </span>2007;<span class="ref-vol">5</span>:61–70.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17259346" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17259346</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml199.r9">Lieberman HB. DNA damage repair and response proteins as targets for cancer therapy. <span><span class="ref-journal">Curr Med Chem. </span>2008;<span class="ref-vol">15</span>:360–367.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18288990" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18288990</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml199.r10">Zaremba T, Curtin NJ. PARP inhibitor development for systemic cancer targeting. <span><span class="ref-journal">Anticancer Agents Med Chem. </span>2007;<span class="ref-vol">7</span>:515–523.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17896912" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17896912</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml199.r11">Horton JK, Wilson SH. Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency. <span><span class="ref-journal">DNA Repair (Amst). </span>2007;<span class="ref-vol">6</span>:530–543.</span> [<a href="/pmc/articles/PMC1911606/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1911606</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17113833" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17113833</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml199.r12">Wyatt MD, Pittman DL. Methylating agents and DNA repair responses: Methylated bases and sources of strand breaks. <span><span class="ref-journal">Chem Res Toxicol. </span>2006;<span class="ref-vol">19</span>:1580–1594.</span> [<a href="/pmc/articles/PMC2542901/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2542901</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17173371" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17173371</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml199.r13">Mutter N, Stupp R. Temozolomide: a milestone in neuro-oncology and beyond? <span><span class="ref-journal">Expert. Rev Anticancer Ther. </span>2006;<span class="ref-vol">6</span>:1187–1204.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16925485" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16925485</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml199.r14">Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. <span><span class="ref-journal">Nat Rev Cancer. </span>2003;<span class="ref-vol">3</span>:330–338.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12724731" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12724731</span></a>]</div></dd><dt>15.</dt><dd><div class="bk_ref" id="ml199.r15">An Q, Robins P, Lindahl T, Barnes DE. 5-Fluorouracil incorporated into DNA is excised by the Smug1 DNA glycosylase to reduce drug cytotoxicity. <span><span class="ref-journal">Cancer Res. </span>2007;<span class="ref-vol">67</span>:940–945.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17283124" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17283124</span></a>]</div></dd><dt>16.</dt><dd><div class="bk_ref" id="ml199.r16">Madhusudan S, Smart F, Shrimpton P, Parsons JL, Gardiner L, Houlbrook S, Talbot D, Hammonds T, Freemont PA, Sternberg MJE, Dianov GL, Hickson ID. Isolation of a small molecule inhibitor of DNA base excision repair. <span><span class="ref-journal">Nucleic Acids Res. </span>2005;<span class="ref-vol">33</span>:4711–4724.</span> [<a href="/pmc/articles/PMC1188083/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1188083</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16113242" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16113242</span></a>]</div></dd><dt>17.</dt><dd><div class="bk_ref" id="ml199.r17">Seiple LA, Cardellina JH 2nd, Akee R, Stivers JT. Potent inhibition of human apurinic/apyrimidinic endonuclease 1 by arylstilbonic acids. <span><span class="ref-journal">Mol. Pharmacol. </span>2008;<span class="ref-vol">73</span>:669–677.</span> [<a href="/pmc/articles/PMC2720577/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2720577</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18042731" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18042731</span></a>]</div></dd><dt>18.</dt><dd><div class="bk_ref" id="ml199.r18">Zawahir Z, Dayam R, Deng J, Pereira C, Neamati N. Pharmacophore guided discovery of small-molecule human apurinic/apyrimidinic endonuclease APE1. <span><span class="ref-journal">J. Med. Chem. </span>2009;<span class="ref-vol">52</span>:20–32.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19072053" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19072053</span></a>]</div></dd><dt>19.</dt><dd><div class="bk_ref" id="ml199.r19">Bapat A, Glass LS, Luo M, Fishel ML, Long EC, Georgiadis MM, Kelley MR. Novel small molecule inhibitor of APE1 endonuclease blocks proliferation and reduces viability of glioblastoma cells. <span><span class="ref-journal">J. Pharmacol. Exp. Ther. </span>2010;<span class="ref-vol">334</span>:988–998.</span> [<a href="/pmc/articles/PMC2939666/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2939666</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20504914" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20504914</span></a>]</div></dd><dt>20.</dt><dd><div class="bk_ref" id="ml199.r20">Simeonov A, Kulkarni A, Dorjsuren D, Jadhav A, Shen M, McNeill DR, Austin CP, Wilson DM III. Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1. <span><span class="ref-journal">PLos ONE. </span>2009;<span class="ref-vol">4</span>:e5740.</span> [<a href="/pmc/articles/PMC2685009/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2685009</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19484131" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19484131</span></a>]</div></dd><dt>21.</dt><dd><div class="bk_ref" id="ml199.r21">Tse W, Boger DL. A Fluorescent Intercalator Displacement Assay for Establishing DNA Binding Selectivity and Affinity. <span><span class="ref-journal">Acc Chem Res. </span>2004;<span class="ref-vol">37</span>(1):61–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14730995" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14730995</span></a>]</div></dd><dt>22.</dt><dd><div class="bk_ref" id="ml199.r22">Erzberger JP, Barsky D, Scharer OD, Colvin ME, Wilson DM. III Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases. <span><span class="ref-journal">Nucleic Acids Res. </span>1998;<span class="ref-vol">26</span>:2771–2778.</span> [<a href="/pmc/articles/PMC147600/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC147600</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/9592167" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9592167</span></a>]</div></dd><dt>23.</dt><dd><div class="bk_ref" id="ml199.r23">Wilson DM III, Takeshita M, Grollman AP, Demple Bs. Incision activity of human apurinic endonuclease (Ape) at abasic site analogs in DNA. <span><span class="ref-journal">J. Biol. Chem. </span>1995;<span class="ref-vol">270</span>:16002–16007.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7608159" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7608159</span></a>]</div></dd><dt>24.</dt><dd><div class="bk_ref" id="ml199.r24">Wilson DM III, Takeshita M, Demple B. Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites. <span><span class="ref-journal">Nucleic Acids Res. </span>1997;<span class="ref-vol">25</span>:933–939.</span> [<a href="/pmc/articles/PMC146540/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC146540</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/9023101" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9023101</span></a>]</div></dd><dt>25.</dt><dd><div class="bk_ref" id="ml199.r25">Gewald K, Schinke E, Böttcher H. 2-Amino-thiophene aus methylenaktiven Nitrile carbonylverbindiungen und Schwefel. <span><span class="ref-journal">Chem. Ber. </span>1966;<span class="ref-vol">99</span>:94–100.</span></div></dd><dt>26.</dt><dd><div class="bk_ref" id="ml199.r26">Andersen HS, Olsen OH, Iversen LF, Sorensen ALP, Mortensen SB, Christensen MS, Branner S, Hansen T, Lau JF, Jeppesen L, Moran EJ, Su J, Bakir F, Judge L, Shahbaz M, Collins T, Vo T, newman MJ, Ripka WC, Moller NPH. Discovery and SAR of a Novel Selective and Orally Bioavailable Nonpeptide Classical Competitive Inhibitor Class of Protein-Tyrosine Phosphatase 1B. <span><span class="ref-journal">J. Med. Chem. </span>2002;<span class="ref-vol">45</span>:4443–4459.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12238924" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12238924</span></a>]</div></dd></dl></div><div style="display:none"><div style="display:none" id="figml199f4"><img alt="Image ml199f4" src-large="/books/NBK133448/bin/ml199f4.jpg" /></div><div style="display:none" id="figml199f5"><img alt="Image ml199f5" src-large="/books/NBK133448/bin/ml199f5.jpg" /></div><div style="display:none" id="figml199f6"><img alt="Image ml199f6" src-large="/books/NBK133448/bin/ml199f6.jpg" /></div><div style="display:none" id="figml199f7"><img alt="Image ml199f7" src-large="/books/NBK133448/bin/ml199f7.jpg" /></div><div style="display:none" id="figml199f8"><img alt="Image ml199f8" src-large="/books/NBK133448/bin/ml199f8.jpg" /></div></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/NBK133448/?report=reader">PubReader</a></li><li><a href="/books/NBK133448/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK133448" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK133448" style="display:none" title="Cite this Page"><div class="bk_tt">Rai G, Vyjayanti VN, Dorjsuren D, et al. Small Molecule Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1) 2010 Oct 29 [Updated 2013 Feb 28]. 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><li><a href="/books/NBK133448/pdf/Bookshelf_NBK133448.pdf">PDF version of this page</a> (992K)</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="#ml199.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml199.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml199.s3" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml199.s5" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml199.s12" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml199.s22" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml199.s29" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3025402" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=3025402" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3025402" ref="log$=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=3025402" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/22455312" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Synthesis, biological evaluation, and structure-activity relationships of a novel class of apurinic/apyrimidinic endonuclease 1 inhibitors.</a><span class="source">[J Med Chem. 2012]</span><div class="brieflinkpop offscreen_noflow">Synthesis, biological evaluation, and structure-activity relationships of a novel class of apurinic/apyrimidinic endonuclease 1 inhibitors.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Rai G, Vyjayanti VN, Dorjsuren D, Simeonov A, Jadhav A, Wilson DM 3rd, Maloney DJ. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Med Chem. 2012 Apr 12; 55(7):3101-12. Epub 2012 Mar 28.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/15330152" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Inhibition of the human apurinic/apyrimidinic endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone.</a><span class="source">[Anticancer Res. 2004]</span><div class="brieflinkpop offscreen_noflow">Inhibition of the human apurinic/apyrimidinic endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Luo M, Kelley MR. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Anticancer Res. 2004 Jul-Aug; 24(4):2127-34. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23110144" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.</a><span class="source">[PLoS One. 2012]</span><div class="brieflinkpop offscreen_noflow">Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Dorjsuren D, Kim D, Vyjayanti VN, Maloney DJ, Jadhav A, Wilson DM 3rd, Simeonov A. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">PLoS One. 2012; 7(10):e47974. 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