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N-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase

Identifieur interne : 001C99 ( Istex/Corpus ); précédent : 001C98; suivant : 001D00

N-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase

Auteurs : Igor A. Schepetkin ; Andrei I. Khlebnikov ; Mark T. Quinn

Source :

Journal of Medicinal Chemistry [ 0022-2623 ] ; 2007.

RBID : ISTEX:7D573D68DFABAD5CFFF14F38AE7CB3CB92016E6B

Abstract

Human neutrophil elastase (NE) plays an important role in the pathogenesis of pulmonary disease. Using high-throughput chemolibrary screening, we identified 10 N-benzoylpyrazole derivatives that were potent NE inhibitors. Nine additional NE inhibitors were identified through further screening of N-benzoylpyrazole analogues. Evaluation of inhibitory activity against a range of proteases showed high specificity for NE, although several derivatives were also potent inhibitors of chymotrypsin. Analysis of reaction kinetics and inhibitor stability revealed that N-benzoylpyrazoles were pseudoirreversible competitive inhibitors of NE. Structure−activity relationship (SAR) analysis demonstrated that modification of N-benzoylpyrazole ring substituents modulated enzyme selectivity and potency. Furthermore, molecular modeling of the binding of selected active and inactive compounds to the NE active site revealed that active compounds fit well into the catalytic site, whereas inactive derivatives contained substituents or conformations that hindered binding or accessibility to the catalytic residues. Thus, N-benzoylpyrazole derivatives represent novel structural templates that can be utilized for further development of efficacious NE inhibitors.

Url:

https://api.istex.fr/ark:/67375/TPS-4V3LMDFN-6/fulltext.pdf

DOI: 10.1021/jm070600+

Links to Exploration step

ISTEX:7D573D68DFABAD5CFFF14F38AE7CB3CB92016E6B

Le document en format XML

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-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase</title>
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<front><div type="abstract">Human neutrophil elastase (NE) plays an important role in the pathogenesis of pulmonary disease. Using high-throughput chemolibrary screening, we identified 10 N-benzoylpyrazole derivatives that were potent NE inhibitors. Nine additional NE inhibitors were identified through further screening of N-benzoylpyrazole analogues. Evaluation of inhibitory activity against a range of proteases showed high specificity for NE, although several derivatives were also potent inhibitors of chymotrypsin. Analysis of reaction kinetics and inhibitor stability revealed that N-benzoylpyrazoles were pseudoirreversible competitive inhibitors of NE. Structure−activity relationship (SAR) analysis demonstrated that modification of N-benzoylpyrazole ring substituents modulated enzyme selectivity and potency. Furthermore, molecular modeling of the binding of selected active and inactive compounds to the NE active site revealed that active compounds fit well into the catalytic site, whereas inactive derivatives contained substituents or conformations that hindered binding or accessibility to the catalytic residues. Thus, N-benzoylpyrazole derivatives represent novel structural templates that can be utilized for further development of efficacious NE inhibitors.</div>
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 Montana State University.</p>
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<p>Human neutrophil elastase (NE) plays an important role in the pathogenesis of pulmonary disease. Using
high-throughput chemolibrary screening, we identified 10 <hi rend="italic">N</hi>-benzoylpyrazole derivatives that were potent
NE inhibitors. Nine additional NE inhibitors were identified through further screening of <hi rend="italic">N</hi>-benzoylpyrazole
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although several derivatives were also potent inhibitors of chymotrypsin. Analysis of reaction kinetics and
inhibitor stability revealed that <hi rend="italic">N</hi>-benzoylpyrazoles were pseudoirreversible competitive inhibitors of NE.
Structure−activity relationship (SAR) analysis demonstrated that modification of <hi rend="italic">N</hi>-benzoylpyrazole ring
substituents modulated enzyme selectivity and potency. Furthermore, molecular modeling of the binding of
selected active and inactive compounds to the NE active site revealed that active compounds fit well into
the catalytic site, whereas inactive derivatives contained substituents or conformations that hindered binding
or accessibility to the catalytic residues. Thus, <hi rend="italic">N</hi>-benzoylpyrazole derivatives represent novel structural
templates that can be utilized for further development of efficacious NE inhibitors.
</p>
</abstract>
<textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term>
</keywords>
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<istex:document><article article-type="research-article" specific-use="acs2jats-2.0.3"><front><journal-meta><journal-id journal-id-type="acspubs">jm</journal-id>
<journal-id journal-id-type="coden">jmcmar</journal-id>
<journal-title-group><journal-title>Journal of Medicinal Chemistry</journal-title>
<abbrev-journal-title>J. Med. Chem.</abbrev-journal-title>
</journal-title-group>
<issn pub-type="ppub">0022-2623</issn>
<issn pub-type="epub">1520-4804</issn>
<publisher><publisher-name>American Chemical Society</publisher-name>
</publisher>
<self-uri>pubs.acs.org/jmc</self-uri>
</journal-meta>
<article-meta><article-id pub-id-type="doi">10.1021/jm070600+</article-id>
<article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title><italic>N</italic>
-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Schepetkin</surname>
<given-names>Igor A.</given-names>
</name>
<xref rid="jm0706001AF2"><sup>†</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Khlebnikov</surname>
<given-names>Andrei I.</given-names>
</name>
<xref rid="jm0706001AF3"><sup>‡</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Quinn</surname>
<given-names>Mark T.</given-names>
</name>
<xref rid="jm0706001AF1">*</xref>
<xref rid="jm0706001AF2"><sup>†</sup>
</xref>
</contrib>
<aff>Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, and Department of Chemistry,
Altai State Technical University, Barnaul 656038, Russia
</aff>
</contrib-group>
<author-notes><fn id="jm0706001AF2"><label>†</label>
<p>
 Montana State University.</p>
</fn>
<fn id="jm0706001AF3"><label>‡</label>
<p>
 Altai State Technical University.</p>
</fn>
<corresp id="jm0706001AF1">
 To whom correspondence should be addressed. Phone:  406-994-5721.
Fax:  406-994-4303. E-mail:  mquinn@montana.edu.</corresp>
</author-notes>
<pub-date pub-type="epub"><day>12</day>
<month>09</month>
<year>2007</year>
</pub-date>
<pub-date pub-type="ppub"><day>04</day>
<month>10</month>
<year>2007</year>
</pub-date>
<volume>50</volume>
<issue>20</issue>
<fpage>4928</fpage>
<lpage>4938</lpage>
<supplementary-material xlink:href="jm070600+si20070715_051030.pdf"></supplementary-material>
<history><date date-type="received"><day>22</day>
<month>05</month>
<year>2007</year>
</date>
<date date-type="asap"><day>12</day>
<month>09</month>
<year>2007</year>
</date>
<date date-type="issue-pub"><day>04</day>
<month>10</month>
<year>2007</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2007 American Chemical Society</copyright-statement>
<copyright-year>2007</copyright-year>
<copyright-holder>American Chemical Society</copyright-holder>
</permissions>
<abstract><graphic content-type="abstract-graphic" xlink:href="jm-2007-00600+_0001.tif"></graphic>
<p>Human neutrophil elastase (NE) plays an important role in the pathogenesis of pulmonary disease. Using
high-throughput chemolibrary screening, we identified 10 <italic>N</italic>-benzoylpyrazole derivatives that were potent
NE inhibitors. Nine additional NE inhibitors were identified through further screening of <italic>N</italic>-benzoylpyrazole
analogues. Evaluation of inhibitory activity against a range of proteases showed high specificity for NE,
although several derivatives were also potent inhibitors of chymotrypsin. Analysis of reaction kinetics and
inhibitor stability revealed that <italic>N</italic>-benzoylpyrazoles were pseudoirreversible competitive inhibitors of NE.
Structure−activity relationship (SAR) analysis demonstrated that modification of <italic>N</italic>-benzoylpyrazole ring
substituents modulated enzyme selectivity and potency. Furthermore, molecular modeling of the binding of
selected active and inactive compounds to the NE active site revealed that active compounds fit well into
the catalytic site, whereas inactive derivatives contained substituents or conformations that hindered binding
or accessibility to the catalytic residues. Thus, <italic>N</italic>-benzoylpyrazole derivatives represent novel structural
templates that can be utilized for further development of efficacious NE inhibitors.
</p>
</abstract>
<custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name>
<meta-value>jm070600+</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body><sec id="d7e142"><title>Introduction</title>
<p>Acute respiratory distress syndrome (ARDS<italic><sup>a</sup>
</italic>
<sup></sup>
<xref rid="jm0706001n00001"></xref>), chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF)
are progressive diseases that are frequently fatal.<named-content content-type="bibref-group"><xref rid="jm0706001b00001" ref-type="bibr"></xref>
−<xref rid="jm0706001b00002" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="jm0706001b00003" ref-type="bibr"></xref>
</named-content> Unfortunately, there are currently few effective therapeutic treatments
for these syndromes. Inflammation associated with these
pulmonary diseases is predominantly due to neutrophils and is
associated with excessive release of neutrophil granule proteases,
such as neutrophil elastase (NE, EC 3.4.21.37).<named-content content-type="bibref-group"><xref rid="jm0706001b00004" ref-type="bibr"></xref>
,<xref rid="jm0706001b00005" ref-type="bibr"></xref>
</named-content> NE is a serine
protease that is synthesized in neutrophils and stored in
azurophilic granules.<xref rid="jm0706001b00006" ref-type="bibr"></xref> While the primary role of NE appears to
be in microbial killing in the phagosome, excessive NE release
into extracellular fluids can cause major tissue damage.<xref rid="jm0706001b00007" ref-type="bibr"></xref> For
example, NE is released in large amounts during pulmonary
inflammation, resulting in a protease/antiprotease imbalance, and
this imbalance appears to be a major pathogenic determinant
in COPD and ARDS.<named-content content-type="bibref-group"><xref rid="jm0706001b00005" ref-type="bibr"></xref>
,<xref rid="jm0706001b00008" ref-type="bibr"></xref>
−<xref rid="jm0706001b00009" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="jm0706001b00010" ref-type="bibr"></xref>
</named-content>
</p>
<p>NE is a member of the chymotrypsin family of serine
proteases and is expressed primarily in neutrophils but is also
present in monocytes and mast cells. It can degrade a variety
of extracellular matrix proteins, including elastin, fibronectin,
laminin, collagen, and proteoglycans (reviewed in refs <xref rid="jm0706001b00011" specific-use="ref-style=base-text" ref-type="bibr"></xref> and
<xref rid="jm0706001b00012" specific-use="ref-style=base-text" ref-type="bibr"></xref>). NE also can activate several matrix metalloproteinases
(MMP-2, -3, and -9)<sup>13</sup> and seems to play an important
physiologic role in tissue repair through its ability to regulate
growth factors and modulate cytokine expression at epithelial
and endothelial surfaces.<named-content content-type="bibref-group"><xref rid="jm0706001b00014" ref-type="bibr"></xref>
,<xref rid="jm0706001b00015" ref-type="bibr"></xref>
</named-content> However, excessive NE activity
can lead to severe pathology through the degradation of elastin
and collagen in the airways, resulting in microvascular injury
and interstitial edema.<xref rid="jm0706001b00016" ref-type="bibr"></xref>
</p>
<p>Given the destructive potential of unregulated NE, it is not
surprising that inhibition of NE activity in pulmonary tissues
has been considered a promising strategy to improve the
outcome of pulmonary diseases.<named-content content-type="bibref-group"><xref rid="jm0706001b00017" ref-type="bibr"></xref>
,<xref rid="jm0706001b00018" ref-type="bibr"></xref>
</named-content> A number of therapeutic
strategies have focused on the use of recombinant or purified
preparations of two endogenous NE inhibitors described above,
α<sub>1</sub>
-antitrypsin and secretory leukocyte protease inhibitor; however, use of these inhibitors has been problematic.<xref rid="jm0706001b00019" ref-type="bibr"></xref> Many types
of peptide and nonpeptide inhibitors, employing both reversible
and irreversible mechanisms of action, have also been reported
(reviewed in refs <xref rid="jm0706001b00015" specific-use="ref-style=base-text" ref-type="bibr"></xref>
, <xref rid="jm0706001b00019" specific-use="ref-style=base-text" ref-type="bibr"></xref>, and 20). Among the most potent NE
inhibitors are β-lactams,<xref rid="jm0706001b00021" ref-type="bibr"></xref>
 <italic>tert</italic>
-butyloxadiazoles,<xref rid="jm0706001b00022" ref-type="bibr"></xref> and peptidyl
trifluoromethyl ketones.<xref rid="jm0706001b00023" ref-type="bibr"></xref> Nevertheless, the primary chemical
scaffolds of the most potent NE inhibitors were discovered 15−20 years ago,<named-content content-type="bibref-group"><xref rid="jm0706001b00024" ref-type="bibr"></xref>
,<xref rid="jm0706001b00025" ref-type="bibr"></xref>
</named-content> and modification of these scaffolds is still
the current focus of most NE inhibitor development because
moving away from these core scaffolds would be difficult and
time-consuming.<xref rid="jm0706001b00026" ref-type="bibr"></xref> Conversely, we propose that new NE inhibitors with different structural and/or physicochemical properties
from those described so far could lead to novel and useful leads
for the development of anti-inflammatory drugs. Although some
novel approaches have been developed for high-throughput
screening (HTS) to identify NE inhibitors on a large scale, there
are still only a few reports on HTS of elastase inhibitors.<named-content content-type="bibref-group"><xref rid="jm0706001b00027" ref-type="bibr"></xref>
−<xref rid="jm0706001b00028" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="jm0706001b00029" ref-type="bibr"></xref>
</named-content>
Thus, we utilized HTS of a chemical diversity library containing
10 000 druglike molecules to identify novel inhibitors of NE
that have core structures distinct from currently known leads.
Notably, the hits obtained from our screen included 10 <italic>N</italic>-benzoylpyrazole derivatives, which were potent NE inhibitors.
Furthermore, analysis of 43 additional <italic>N</italic>-benzoylpyrazole
derivatives resulted in the identification of nine more potent
NE inhibitors with <italic>K</italic>
<sub>i</sub> ≤ 1 μM. Evaluation of target specificity
showed that most of NE inhibitors were selective for NE and
chymotrypsin but not other proteases tested. Finally, molecular
modeling approaches demonstrated that active <italic>N</italic>-benzoylpyrazole derivatives were able to effectively dock within the NE
catalytic site so that Michaelis complex formation and synchronous proton transfer were favored, while binding of inactive
derivatives into the pocket was sterically hindered or catalytically unfavorable.
</p>
</sec>
<sec id="d7e237"><title>Results and Discussion</title>
<p><bold>Primary High-Throughput Screening.</bold> To identify novel
compounds that inhibit NE protease activity, we screened a
chemical diversity library of 10 000 druglike compounds with
molecular weights from 200 to 550 Da. This library of
commonly accepted pharmaceutical hit structures was randomly
assembled to maximize chemical diversity and includes 6118
compounds containing an amide linker. However, the library
does not contain β-lactam derivatives, which have been reported
previously as NE inhibitors.<named-content content-type="bibref-group"><xref rid="jm0706001b00021" ref-type="bibr"></xref>
,<xref rid="jm0706001b00030" ref-type="bibr"></xref>
</named-content> Further details on the composition of the parent library are described in our previous
studies.<named-content content-type="bibref-group"><xref rid="jm0706001b00031" ref-type="bibr"></xref>
,<xref rid="jm0706001b00032" ref-type="bibr"></xref>
</named-content>
</p>
<p>A compound was defined as a hit if it exhibited >90%
inhibition of NE activity at a final compound concentration of
20 μg/mL in fluorescence-based microplate assays. From the
primary enzymatic screening, 432 inhibitory compounds were
selected (4.3% hit rate). The size of the hit set was further
reduced by applying a series of experimental filters. For
example, compounds that formed crystals or aggregates in
aqueous buffer were eliminated, as they could inhibit enzymes
nonspecifically by absorption of enzyme molecules to/into the
aggregates.<xref rid="jm0706001b00033" ref-type="bibr"></xref> We also determined the dose−response relationship for enzyme inhibition by hits filtered from the primary
screen.<named-content content-type="bibref-group"><xref rid="jm0706001b00031" ref-type="bibr"></xref>
,<xref rid="jm0706001b00034" ref-type="bibr"></xref>
</named-content> As an example, a representative curve for compound
<bold>7</bold>
 is shown in Figure <xref rid="jm0706001f00001"></xref>. Sixteen compounds with the highest
inhibitory activity for NE were selected as a set of prospective
NE inhibitors, and the inhibition constants (<italic>K</italic>
<sub>i</sub>) were determined
for these compounds using Dixon plots.<xref rid="jm0706001b00035" ref-type="bibr"></xref> The structures of these
compounds and their activity are presented in Table <xref rid="jm0706001t00001"></xref>
. Compounds <bold>1</bold>
−<bold>10</bold>
 were quite potent, all with <italic>K</italic>
<sub>i</sub> ≤ 110 nM. This
inhibition is likely not due to quenching of product fluorescence
by the test compounds, as nanomolar concentrations of compounds do not appear to cause this problem.<xref rid="jm0706001b00036" ref-type="bibr"></xref> In addition, we
mixed selected lead compounds with cleaved substrate and
found no effect on the fluorescence signal intensity at excitation
and emission wavelengths of 355 and 460 nm, respectively (data
not shown).
<fig id="jm0706001f00001" position="float"><label>1</label>
<caption><p>Inhibition of NE activity by a representative compound identified with HTS. NE was incubated with the indicated concentrations of<bold>7</bold>, and cleavage of the fluorogenic NE substrate (25 μM) was
monitored, as described. The percent inhibition of NE activity is plotted
vs the logarithm of inhibitor concentration. The data are presented as
the mean ± SEM of triplicate samples from a representative experiment
of three independent experiments.</p>
</caption>
<graphic xlink:href="jm-2007-00600+_0002.tif"></graphic>
</fig>
<table-wrap id="jm0706001t00001" position="float"><label>1</label>
<caption><p>Chemical Structures and Inhibitory Activity of the Most Potent NE Inhibitors Identified by High-Throughput Screening</p>
</caption>
<oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1"><graphic xlink:href="jm-2007-00600+_0003.tif"></graphic>
</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
</oasis:table>
</table-wrap>
</p>
<p>An examination of the 16 most potent NE inhibitors showed
that 10 of these compounds (62%) had <italic>N</italic>-benzoylpyrazole
scaffolds (Table <xref rid="jm0706001t00001"></xref>). This scaffold has been reported among
compounds with diuretic,<xref rid="jm0706001b00037" ref-type="bibr"></xref>
 antidiabetic,<xref rid="jm0706001b00038" ref-type="bibr"></xref>
 antivirus,<xref rid="jm0706001b00039" ref-type="bibr"></xref>
 and anti-inflammatory<xref rid="jm0706001b00040" ref-type="bibr"></xref> properties. However, to our knowledge, no other
potent NE inhibitors have been reported to be <italic>N</italic>-benzoylpyrazole
derivatives. Indeed, our findings suggest the possibility that the
anti-inflammatory effects reported for <italic>N</italic>
-benzoylpyrazoles<sup>40</sup> may
be due, in part, to inhibition of NE. On the basis of this
discovery, we selected 43 additional <italic>N</italic>-benzoylpyrazole derivatives from the TimTec stock library, which contains 224 700
compounds, for evaluation of NE inhibitory activity. All of these
derivatives were soluble in aqueous buffer at the highest tested
concentrations (50−55 μM), and dose−response analysis showed
that eight of these compounds (compounds <bold>17</bold>
−<bold>24</bold>) were
relatively potent (<italic>K</italic>
<sub>i</sub>
 ≤ 300 nM) NE inhibitors (Table <xref rid="jm0706001t00002"></xref> and
Supporting Information Table S1). Thus, we selected the 17
most potent <italic>N</italic>
-benzoylpyrazole derivatives (compounds <bold>1</bold>
−<bold>9</bold> and
<bold>17</bold>
−<bold>24</bold>) for further characterization and SAR analysis. Dixon
plots and double-reciprocal Lineweaver−Burk plots of substrate

hydrolysis by NE in the absence and presence of the selected
compounds showed competitive inhibition (Figure <xref rid="jm0706001f00002"></xref> shows
representative plots).
<fig id="jm0706001f00002" position="float"><label>2</label>
<caption><p>Kinetics of NE inhibition by selected<italic>N</italic>-benzoylpyrazole derivatives. Representative Dixon plots (A and C) and double-reciprocal
Lineweaver−Burk plots (B and D) are shown. Representative plots are from three independent experiments.</p>
</caption>
<graphic xlink:href="jm-2007-00600+_0004.tif"></graphic>
</fig>
<table-wrap id="jm0706001t00002" position="float"><label>2</label>
<caption><p>Effect of<italic>N</italic>
-Benzoylpyrazoles <bold>17</bold>
−<bold>33</bold>
 on NE Activity<italic><sup>a</sup>
</italic>
<sup></sup>
<graphic xlink:href="jm-2007-00600+_0005.tif"></graphic>
</p>
</caption>
<oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="10"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:colspec colnum="5" colname="5"></oasis:colspec>
<oasis:colspec colnum="6" colname="6"></oasis:colspec>
<oasis:colspec colnum="7" colname="7"></oasis:colspec>
<oasis:colspec colnum="8" colname="8"></oasis:colspec>
<oasis:colspec colnum="9" colname="9"></oasis:colspec>
<oasis:colspec colnum="10" colname="10"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">compd</oasis:entry>
<oasis:entry namest="2" nameend="2">R<sub>1</sub>
</oasis:entry>
<oasis:entry namest="3" nameend="3">R<sub>2</sub>
</oasis:entry>
<oasis:entry namest="4" nameend="4">R<sub>3</sub>
</oasis:entry>
<oasis:entry namest="5" nameend="5">R<sub>4</sub>
</oasis:entry>
<oasis:entry namest="6" nameend="6">R<sub>5</sub>
</oasis:entry>
<oasis:entry namest="7" nameend="7">R<sub>6</sub>
</oasis:entry>
<oasis:entry namest="8" nameend="8">R<sub>7</sub>
</oasis:entry>
<oasis:entry namest="9" nameend="9">R<sub>8</sub>
</oasis:entry>
<oasis:entry namest="10" nameend="10"><italic>K</italic>
<sub>i</sub>
(nM)
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>17</bold>
</oasis:entry>
<oasis:entry colname="2">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="3">H
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">OCH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="7">OCH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="8">OCH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">21
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>18</bold>
</oasis:entry>
<oasis:entry colname="2">NO<sub>2</sub>
</oasis:entry>
<oasis:entry colname="3">H
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">28
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>19</bold>
</oasis:entry>
<oasis:entry colname="2">NO<sub>2</sub>
</oasis:entry>
<oasis:entry colname="3">H
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">46
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>20</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">NO<sub>2</sub>
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">65
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>21</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">Cl
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">230
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>22</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">Br
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">250
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>23</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">Br
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">300
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>24</bold>
</oasis:entry>
<oasis:entry colname="2">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="3">H
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">NHCOCH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">300
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>25</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">Cl
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">Cl
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">1000
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>26</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">H
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">3400
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>27</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">Br
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">F
</oasis:entry>
<oasis:entry colname="7">F
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">Cl
</oasis:entry>
<oasis:entry colname="10">7200
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>28</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">Cl
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7"><italic>tert</italic>-butyl
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">9000
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>29</bold>
</oasis:entry>
<oasis:entry colname="2">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="3">Cl
</oasis:entry>
<oasis:entry colname="4">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="5">F
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">9000
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>30</bold>
</oasis:entry>
<oasis:entry colname="2">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="3">Cl
</oasis:entry>
<oasis:entry colname="4">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">Cl
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">10700
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>31</bold>
</oasis:entry>
<oasis:entry colname="2">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="3">Br
</oasis:entry>
<oasis:entry colname="4">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">OCH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">24500
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>32</bold>
</oasis:entry>
<oasis:entry colname="2">H
</oasis:entry>
<oasis:entry colname="3">H
</oasis:entry>
<oasis:entry colname="4">H
</oasis:entry>
<oasis:entry colname="5">Br
</oasis:entry>
<oasis:entry colname="6">H
</oasis:entry>
<oasis:entry colname="7">H
</oasis:entry>
<oasis:entry colname="8">H
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">29900
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>33</bold>
</oasis:entry>
<oasis:entry colname="2">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="3">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="4">CH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="5">H
</oasis:entry>
<oasis:entry colname="6">OCH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="7">OCH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="8">OCH<sub>3</sub>
</oasis:entry>
<oasis:entry colname="9">H
</oasis:entry>
<oasis:entry colname="10">50900</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
</oasis:table>
<table-wrap-foot><p><italic><sup>a</sup>
</italic>
<sup></sup> See Supporting Information Table S1 for analysis results of compounds
<bold>34</bold>
−<bold>59</bold>
.</p>
</table-wrap-foot>
</table-wrap>
</p>
<p><bold>Stability and Kinetic Features of </bold>
<bold><italic>N</italic>
</bold>
<bold>-Benzoylpyrazole
Derivatives.</bold> The set of 17 most potent NE inhibitors, as well
as selected low activity and inactive <italic>N</italic>-benzoylpyrazoles, were
evaluated for their chemical stability in aqueous buffer. Spontaneous hydrolysis rates and reaction orders of the inhibitors
were measured in phosphate buffer at pH 7.3 and 25 °C using
spectrophotometry to detect compound hydrolysis. As examples,
Figure <xref rid="jm0706001f00003"></xref> shows that the absorbance maxima at 266 and 262 nm
decreased over time for compounds <bold>1</bold>
 and <bold>2</bold>, respectively, while
a new peak appeared at 226 nm, which is indicative of benzoic
acid formation by <italic>N</italic>-benzoylpyrazole hydrolysis. Compound
hydrolysis was verified by following hydrolysis of compound
<bold>1</bold> using reverse-phase high-performance liquid chromatography
(RP-HPLC). RP-HPLC traces of the progression of compound
<bold>1</bold> hydrolysis revealed the formation of two major product peaks,
one at 3.7 min and the other at 4.0 min (Figure <xref rid="jm0706001f00004"></xref>). The peak
eluting at 4.0 min is very close to that of pure benzoic acid (<italic>t</italic>
<sub>R</sub>
= 3.9 min), suggesting it is a benzoate derivative, whereas the
peak at 7.7 min represents nonhydrolyzed starting material and
the peak at 3.7 min likely represents the unsubstituted pyrazole,
as it elutes with a retention time close to that of pure pyrazole
(3.3 min). To confirm hydrolysis, compound <bold>1</bold> was dissolved
in 85% DMSO-<italic>d</italic>
<sub>6</sub>
/15% D<sub>2</sub>O, incubated for 0 h (control) or 36
h at 37 °C, and analyzed by <sup>1</sup>H NMR. Compared to the 0 h
sample, two new signals at δ = 7.28 ppm and δ = 7.98 ppm
were present in the 36 h sample, corresponding to protons of
the hydrolytic products (<italic>p</italic>
-fluorobenzoic acid and 4-chloropyrazole), as well as the signals of nonhydrolyzed compound <bold>1</bold>
(see Supporting Information Table S4). Thus, these data verify
that we are detecting compound hydrolysis using our spectrophotometric assay.
<fig id="jm0706001f00003" position="float"><label>3</label>
<graphic xlink:href="jm-2007-00600+_0006.tif"></graphic>
</fig>
<fig id="jm0706001f00004" position="float"><label>4</label>
<caption><p>Analysis of hydrolysis of<italic>N</italic>
-benzoylpyrazole derivatives by RP-HPLC. Changes in the chromatographic profile of compound <bold>1</bold> over
time in solution are shown. Relevant peaks discussed in the text (a and b) are indicated. Representative chromatogram profiles are from three
independent experiments.</p>
</caption>
<graphic xlink:href="jm-2007-00600+_0007.tif"></graphic>
</fig>
</p>
<p>Using specific absorbance maxima for the other <italic>N</italic>
-benzoylpyrazoles under investigation (Table <xref rid="jm0706001t00003"></xref>), we monitored
hydrolysis of the remaining set of selected compounds and used
these data to create semilogarithmic plots for determining rate
constants (<italic>k</italic>
‘) of spontaneous hydrolysis (see Supporting Information Figure S1 for example plots). As shown in Table <xref rid="jm0706001t00003"></xref>,

compounds <bold>7</bold>
, <bold>13</bold>
, <bold>17</bold>
, <bold>21</bold>
−<bold>25</bold>
, and <bold>28</bold>
−<bold>30</bold> were the most stable
with <italic>t</italic>
<sub>1/2</sub>
 > 2 h. The presence of a nitro group (compounds <bold>3</bold>
, <bold>5</bold>,
<bold>9</bold>
, and <bold>18</bold>
−<bold>20</bold>) appears to increase the rate of spontaneous
hydrolysis, with an average <italic>t</italic>
<sub>1/2</sub> of ∼11 min for the nitropyrazoles. Nitro groups are strong electron-withdrawing moieties
and have previously been reported to increase the activity of
pyrazolooxadiazinone inhibitors of serine proteases.<xref rid="jm0706001b00041" ref-type="bibr"></xref> Likewise,
the selected nitropyrazoles were also potent NE inhibitors
(Tables <xref rid="jm0706001t00001"></xref>
 and <xref rid="jm0706001t00002"></xref>). In these compounds, the nitro substituent
enhances positive charge on the carbonyl carbon atom, making
it more susceptible to nucleophilic attack by a water molecule
(spontaneous hydrolysis) or by the Ser195 hydroxyl group in
the NE catalytic site.<named-content content-type="bibref-group"><xref rid="jm0706001b00042" ref-type="bibr"></xref>
−<xref rid="jm0706001b00043" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="jm0706001b00044" ref-type="bibr"></xref>
</named-content> Aside from the nitropyrazoles,
however, there was no direct correlation between rates of
spontaneous hydrolysis and anti-NE activity for the other
inhibitors tested. For example, compound <bold>7</bold> (a potent NE
inhibitor), compound <bold>28</bold> (a weak NE inhibitor), and inactive
compound <bold>56</bold>
 all had similar rates of hydrolysis (<italic>t</italic>
<sub>1/2</sub> = 2.1−2.2 h). In general, the most potent NE inhibitors, excluding the
nitropyrazoles (compounds <bold>5</bold>
, <bold>18</bold>
, and <bold>19)</bold>, were more stable
than 6-acylamino-2-[1-(ethylsulfonyl)oxy]-1<italic>H</italic>
-isoindole-1,3-diones<xref rid="jm0706001b00045" ref-type="bibr"></xref>
 but less stable than β-lactam,<xref rid="jm0706001b00030" ref-type="bibr"></xref>
 1,2,5-thiadiazolidin-3-one 1,1-dioxide,<xref rid="jm0706001b00046" ref-type="bibr"></xref>
 and 2-azetidinone<xref rid="jm0706001b00047" ref-type="bibr"></xref> inhibitors of NE.
<table-wrap id="jm0706001t00003" position="float"><label>3</label>
<caption><p>Kinetic Constants<italic><sup>a</sup>
</italic>
<sup></sup> for the Spontaneous Hydrolysis of Selected
<italic>N</italic>
-Benzoylpyrazoles</p>
</caption>
<oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">compd</oasis:entry>
<oasis:entry namest="2" nameend="2">λ (nm)<italic><sup>b</sup>
</italic>
<sup></sup>
</oasis:entry>
<oasis:entry namest="3" nameend="3"><italic>k</italic>
‘ (min<sup>-1</sup>
 × 10<sup>3</sup>
)</oasis:entry>
<oasis:entry namest="4" nameend="4"><italic>t</italic>
<sub>1/2</sub> (h)
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>1</bold>
</oasis:entry>
<oasis:entry colname="2">266
</oasis:entry>
<oasis:entry colname="3">7.26
</oasis:entry>
<oasis:entry colname="4">1.6
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>2</bold>
</oasis:entry>
<oasis:entry colname="2">262
</oasis:entry>
<oasis:entry colname="3">40.5
</oasis:entry>
<oasis:entry colname="4">0.29
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>3</bold>
</oasis:entry>
<oasis:entry colname="2">262
</oasis:entry>
<oasis:entry colname="3">56.6
</oasis:entry>
<oasis:entry colname="4">0.22
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>4</bold>
</oasis:entry>
<oasis:entry colname="2">265
</oasis:entry>
<oasis:entry colname="3">56.2
</oasis:entry>
<oasis:entry colname="4">0.21
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>5</bold>
</oasis:entry>
<oasis:entry colname="2">270
</oasis:entry>
<oasis:entry colname="3">291.9
</oasis:entry>
<oasis:entry colname="4">0.04
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>6</bold>
</oasis:entry>
<oasis:entry colname="2">262
</oasis:entry>
<oasis:entry colname="3">22.4
</oasis:entry>
<oasis:entry colname="4">0.51
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>7</bold>
</oasis:entry>
<oasis:entry colname="2">270
</oasis:entry>
<oasis:entry colname="3">7.75
</oasis:entry>
<oasis:entry colname="4">2.1
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>8</bold>
</oasis:entry>
<oasis:entry colname="2">255
</oasis:entry>
<oasis:entry colname="3">10.59
</oasis:entry>
<oasis:entry colname="4">1.1
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>9</bold>
</oasis:entry>
<oasis:entry colname="2">254
</oasis:entry>
<oasis:entry colname="3">70.4
</oasis:entry>
<oasis:entry colname="4">0.16
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>13</bold>
</oasis:entry>
<oasis:entry colname="2">265
</oasis:entry>
<oasis:entry colname="3">3.92
</oasis:entry>
<oasis:entry colname="4">3.1
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>17</bold>
</oasis:entry>
<oasis:entry colname="2">260
</oasis:entry>
<oasis:entry colname="3">2.76
</oasis:entry>
<oasis:entry colname="4">4.2
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>18</bold>
</oasis:entry>
<oasis:entry colname="2">272
</oasis:entry>
<oasis:entry colname="3">117.0
</oasis:entry>
<oasis:entry colname="4">0.10
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>19</bold>
</oasis:entry>
<oasis:entry colname="2">270
</oasis:entry>
<oasis:entry colname="3">348.9
</oasis:entry>
<oasis:entry colname="4">0.033
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>20</bold>
</oasis:entry>
<oasis:entry colname="2">266
</oasis:entry>
<oasis:entry colname="3">21.5
</oasis:entry>
<oasis:entry colname="4">0.55
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>21</bold>
</oasis:entry>
<oasis:entry colname="2">262
</oasis:entry>
<oasis:entry colname="3">2.76
</oasis:entry>
<oasis:entry colname="4">4.2
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>22</bold>
</oasis:entry>
<oasis:entry colname="2">264
</oasis:entry>
<oasis:entry colname="3">2.3
</oasis:entry>
<oasis:entry colname="4">5.1
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>23</bold>
</oasis:entry>
<oasis:entry colname="2">266
</oasis:entry>
<oasis:entry colname="3">1.99
</oasis:entry>
<oasis:entry colname="4">6.2
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>24</bold>
</oasis:entry>
<oasis:entry colname="2">290
</oasis:entry>
<oasis:entry colname="3">1.15
</oasis:entry>
<oasis:entry colname="4">10.0
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>25</bold>
</oasis:entry>
<oasis:entry colname="2">262
</oasis:entry>
<oasis:entry colname="3">3.92
</oasis:entry>
<oasis:entry colname="4">3.0
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>26</bold>
</oasis:entry>
<oasis:entry colname="2">245
</oasis:entry>
<oasis:entry colname="3">9.44
</oasis:entry>
<oasis:entry colname="4">1.2
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>27</bold>
</oasis:entry>
<oasis:entry colname="2">266
</oasis:entry>
<oasis:entry colname="3">16.12
</oasis:entry>
<oasis:entry colname="4">0.73
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>28</bold>
</oasis:entry>
<oasis:entry colname="2">265
</oasis:entry>
<oasis:entry colname="3">5.65
</oasis:entry>
<oasis:entry colname="4">2.2
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>29</bold>
</oasis:entry>
<oasis:entry colname="2">260
</oasis:entry>
<oasis:entry colname="3">2.30
</oasis:entry>
<oasis:entry colname="4">5.3
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>30</bold>
</oasis:entry>
<oasis:entry colname="2">265
</oasis:entry>
<oasis:entry colname="3">4.38
</oasis:entry>
<oasis:entry colname="4">2.6
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>34</bold>
</oasis:entry>
<oasis:entry colname="2">266
</oasis:entry>
<oasis:entry colname="3">10.4
</oasis:entry>
<oasis:entry colname="4">1.1
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>35</bold>
</oasis:entry>
<oasis:entry colname="2">270
</oasis:entry>
<oasis:entry colname="3">4.6
</oasis:entry>
<oasis:entry colname="4">2.5
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>42</bold>
</oasis:entry>
<oasis:entry colname="2">250
</oasis:entry>
<oasis:entry colname="3">2.1
</oasis:entry>
<oasis:entry colname="4">5.6
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>43</bold>
</oasis:entry>
<oasis:entry colname="2">258
</oasis:entry>
<oasis:entry colname="3">0.46
</oasis:entry>
<oasis:entry colname="4">25.1
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>53</bold>
</oasis:entry>
<oasis:entry colname="2">258
</oasis:entry>
<oasis:entry colname="3">1.6
</oasis:entry>
<oasis:entry colname="4">7.2
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>55</bold>
</oasis:entry>
<oasis:entry colname="2">258
</oasis:entry>
<oasis:entry colname="3">0.92
</oasis:entry>
<oasis:entry colname="4">12.5
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>56</bold>
</oasis:entry>
<oasis:entry colname="2">260
</oasis:entry>
<oasis:entry colname="3">6.1
</oasis:entry>
<oasis:entry colname="4">2.2
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>57</bold>
</oasis:entry>
<oasis:entry colname="2">270
</oasis:entry>
<oasis:entry colname="3">3.7
</oasis:entry>
<oasis:entry colname="4">3.1
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>58</bold>
</oasis:entry>
<oasis:entry colname="2">268
</oasis:entry>
<oasis:entry colname="3">5.5
</oasis:entry>
<oasis:entry colname="4">2.1
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>59</bold>
</oasis:entry>
<oasis:entry colname="2">256
</oasis:entry>
<oasis:entry colname="3">1.15
</oasis:entry>
<oasis:entry colname="4">10.0</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
</oasis:table>
<table-wrap-foot><p><italic><sup>a</sup>
</italic>
<sup></sup>
 The kinetic constants were obtained at pH 7.3 and 25 °C.<italic><sup>b</sup>
</italic>
<sup></sup> Absorption
maxima that were used for monitoring spontaneous hydrolysis.
</p>
</table-wrap-foot>
</table-wrap>
</p>
<p>The relatively rapid rate of spontaneous hydrolysis of
<italic>N</italic>-benzoylpyrazole inhibitors allowed us to evaluate reversibility
of NE inhibition over time. As shown in Figure <xref rid="jm0706001f00005"></xref>, NE was
rapidly inhibited, with no lag period after addition of compound,
and inhibition was maximal during the first 30 min with compounds <bold>1</bold>
, <bold>7</bold>
, and <bold>18</bold> at nanomolar concentrations (panels A, C,
and E). However, inhibition by the most rapidly hydrolyzable
inhibitor (compound <bold>18</bold>) was soon reversed, and full recovery
of NE enzyme was observed after ∼7 h after treatment with up
to 2 μM of the compound (Figure <xref rid="jm0706001f00005"></xref>B). Indeed, subsequent
addition of a more fluorogenic substrate showed that the enzyme
was still active after the 7 h incubation period (indicated by
arrow in Figure <xref rid="jm0706001f00005"></xref>B). In comparison, reversal of inhibition by
compounds <bold>1</bold>
 and <bold>7</bold> was much slower, and NE was still inhibited
by ≥0.5 μM compounds <bold>1</bold>
 and <bold>7</bold> at 2 and 5 h after treatment,
respectively (parts D and F of Figure <xref rid="jm0706001f00005"></xref>). Thus, these results
suggest that <italic>N</italic>-benzoylpyrazoles may be pseudoirreversible
inhibitors of NE that covalently attack the enzyme active site
but can be reversed by hydrolysis of the acyl−enzyme complex.
Indeed, other known inhibitors of NE, such as Sivelestat (ONO-5046), utilize a similar pseudoirreversible mechanism.<xref rid="jm0706001b00048" ref-type="bibr"></xref> To
support this conclusion, we treated NE with a range of stable
(<italic>t</italic>
<sub>1/2</sub>
 > 2 h) and unstable inhibitors (<italic>t</italic>
<sub>1/2</sub> < 1 h) at a relatively
high concentration (25 μM), removed free inhibitor by ultrafiltration of the enzyme/inhibitor mixture, and then 
monitored recovery of NE activity over time. As shown in Figure <xref rid="jm0706001f00006"></xref>,
inhibition of NE by the selected inhibitors was fully reversible,
although with differing kinetics between compounds. For
example, inhibition by compound <bold>20</bold> was rapidly reversed after
compound removal (<2 h for full recovery of enzymatic
activity), reversal of inhibition by compound <bold>21</bold> was slower
(∼16 h), and reversal of compound <bold>24</bold> inhibition was very slow
(>24 h). These data suggest varying degrees of stability of the
enzyme−inhibitor complexes formed with individual compounds.
<fig id="jm0706001f00005" position="float"><label>5</label>
<caption><p>Evaluation of NE inhibition by representative<italic>N</italic>-benzoylpyrazole derivatives over extended periods of time. NE was incubated with the
indicated compounds, and kinetic curves monitoring substrate cleavage catalyzed by NE during the first 30 min (A, C, and E) and from 0 to 7 h
(B, D, and F) are shown. Representative curves are from three independent experiments.</p>
</caption>
<graphic xlink:href="jm-2007-00600+_0008.tif"></graphic>
</fig>
<fig id="jm0706001f00006" position="float"><label>6</label>
<graphic xlink:href="jm-2007-00600+_0009.tif"></graphic>
</fig>
</p>
<p><bold>Specificity of </bold>
<bold><italic>N</italic>
</bold>
<bold>-Benzoylpyrazoles.</bold> To evaluate inhibitor
specificity, we analyzed the effects of the 53 <italic>N</italic>-benzoylpyrazole
derivatives on six different proteases other than NE. These
proteases included four serine proteases [human pancreatic
chymotrypsin (EC 3.4.21.1), human plasma kallikrein (EC
3.4.21.34), human thrombin (EC 3.4.21.5), and human urokinase
(urokinase-type plasminogen activator precursor, EC 3.4.21.73)],
a zinc-dependent protease [porcine kidney aminopeptidase M
(EC 3.4.11.2)], and an aspartic protease [human spleen cathepsin
D (EC 3.4.23.5)]. As shown in Table <xref rid="jm0706001t00004"></xref> and Supporting
Information Table S2, none of the <italic>N</italic>-benzoylpyrazole derivatives
inhibited aminopeptidase M and cathepsin D, and most derivatives either did not inhibit kallikrein or inhibited this enzyme

at high micromolar concentrations. A few of the <italic>N</italic>
-benzoylpyrazole derivatives inhibited thrombin (compounds <bold>2</bold>
−<bold>4</bold>
 and <bold>6</bold>) or
urokinase (compounds <bold>2</bold>
 and <bold>3</bold>) at nanomolar concentrations,
whereas most of the <italic>N</italic>-benzoylpyrazole derivatives that were
potent NE inhibitors also inhibited chymotrypsin (Table <xref rid="jm0706001t00004"></xref> and
Supporting Information Table S2). This is not surprising, as
NE and chymotrypsin belong to same enzyme family, and
several known NE inhibitors are also relatively potent chymotrypsin inhibitors.<named-content content-type="bibref-group"><xref rid="jm0706001b00049" ref-type="bibr"></xref>
,<xref rid="jm0706001b00050" ref-type="bibr"></xref>
</named-content> While enzyme selectivity between NE and
chymotrypsin was low for our most potent NE inhibitors, the
differential selectivity observed among this set of <italic>N</italic>-benzoylpyrazole derivatives suggests that differences in ring
substituents (R<sub>1</sub>
−R<sub>8</sub>) could be exploited to optimize selectivity.
For example, derivatives bearing nitro groups were weaker
chymotrypsin inhibitors compared to compounds with halogen
or methyl substituents (Tables <xref rid="jm0706001t00001"></xref>
 and <xref rid="jm0706001t00002"></xref>
), and <italic>N</italic>
-benzoyl-4-nitropyrazole (compound <bold>5</bold>), which has only a nitro group as
substituent R<sub>2</sub>, was a relatively selective NE inhibitor versus
all other proteases tested, including chymotrypsin. Compound
<bold>24</bold>
, which is a more stable derivative (<italic>t</italic>
<sub>1/2</sub> = 10 h), was also
quite selective for NE. On the other hand, compound <bold>54</bold> was a
potent chymotrypsin inhibitor with high selectivity versus the
other proteases tested, including NE. These observations suggest
that it may be possible to exploit such differences to design
novel <italic>N</italic>-benzoylpyrazole-based inhibitors that possess both high
potency and chemical stability.
<table-wrap id="jm0706001t00004" position="float"><label>4</label>
<caption><p>Analysis of<italic>N</italic>
-Benzoylpyrazole Inhibitory Specificity<italic><sup>a</sup>
</italic>
<sup></sup>
</p>
</caption>
<oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="7"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:colspec colnum="5" colname="5"></oasis:colspec>
<oasis:colspec colnum="6" colname="6"></oasis:colspec>
<oasis:colspec colnum="7" colname="7"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry>
<oasis:entry namest="2" nameend="7">IC<sub>50</sub>
 (nM)</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry namest="1" nameend="1">compd</oasis:entry>
<oasis:entry namest="2" nameend="2">chymotrypsin</oasis:entry>
<oasis:entry namest="3" nameend="3">kallikrein</oasis:entry>
<oasis:entry namest="4" nameend="4">thrombin</oasis:entry>
<oasis:entry namest="5" nameend="5">urokinase</oasis:entry>
<oasis:entry namest="6" nameend="6">aminopeptidase
M</oasis:entry>
<oasis:entry namest="7" nameend="7">cathepsin
D
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>1</bold>
</oasis:entry>
<oasis:entry colname="2">40
</oasis:entry>
<oasis:entry colname="3">6600
</oasis:entry>
<oasis:entry colname="4">6900
</oasis:entry>
<oasis:entry colname="5">2500
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>2</bold>
</oasis:entry>
<oasis:entry colname="2">15
</oasis:entry>
<oasis:entry colname="3">11900
</oasis:entry>
<oasis:entry colname="4">51
</oasis:entry>
<oasis:entry colname="5">120
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>3</bold>
</oasis:entry>
<oasis:entry colname="2">340
</oasis:entry>
<oasis:entry colname="3">11300
</oasis:entry>
<oasis:entry colname="4">680
</oasis:entry>
<oasis:entry colname="5">170
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>4</bold>
</oasis:entry>
<oasis:entry colname="2">120
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">120
</oasis:entry>
<oasis:entry colname="5">1100
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>5</bold>
</oasis:entry>
<oasis:entry colname="2">2700
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">11900
</oasis:entry>
<oasis:entry colname="5">18200
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>6</bold>
</oasis:entry>
<oasis:entry colname="2">57
</oasis:entry>
<oasis:entry colname="3">12300
</oasis:entry>
<oasis:entry colname="4">390
</oasis:entry>
<oasis:entry colname="5">1500
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>7</bold>
</oasis:entry>
<oasis:entry colname="2">15
</oasis:entry>
<oasis:entry colname="3">6200
</oasis:entry>
<oasis:entry colname="4">4700
</oasis:entry>
<oasis:entry colname="5">1700
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>8</bold>
</oasis:entry>
<oasis:entry colname="2">125
</oasis:entry>
<oasis:entry colname="3">5700
</oasis:entry>
<oasis:entry colname="4">2800
</oasis:entry>
<oasis:entry colname="5">2600
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>9</bold>
</oasis:entry>
<oasis:entry colname="2">1200
</oasis:entry>
<oasis:entry colname="3">4400
</oasis:entry>
<oasis:entry colname="4">2800
</oasis:entry>
<oasis:entry colname="5">1500
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>13</bold>
</oasis:entry>
<oasis:entry colname="2">1800
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">32300
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>17</bold>
</oasis:entry>
<oasis:entry colname="2">50
</oasis:entry>
<oasis:entry colname="3">42200
</oasis:entry>
<oasis:entry colname="4">4800
</oasis:entry>
<oasis:entry colname="5">24900
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>18</bold>
</oasis:entry>
<oasis:entry colname="2">190
</oasis:entry>
<oasis:entry colname="3">6100
</oasis:entry>
<oasis:entry colname="4">4300
</oasis:entry>
<oasis:entry colname="5">3100
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>19</bold>
</oasis:entry>
<oasis:entry colname="2">7000
</oasis:entry>
<oasis:entry colname="3">18800
</oasis:entry>
<oasis:entry colname="4">11000
</oasis:entry>
<oasis:entry colname="5">47900
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>20</bold>
</oasis:entry>
<oasis:entry colname="2">240
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">12100
</oasis:entry>
<oasis:entry colname="5">1300
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>21</bold>
</oasis:entry>
<oasis:entry colname="2">4700
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">NI
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>22</bold>
</oasis:entry>
<oasis:entry colname="2">2700
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">NI
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>23</bold>
</oasis:entry>
<oasis:entry colname="2">2300
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">51200
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>24</bold>
</oasis:entry>
<oasis:entry colname="2">NI
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">33600
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>25</bold>
</oasis:entry>
<oasis:entry colname="2">1400
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">18400
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>26</bold>
</oasis:entry>
<oasis:entry colname="2">NI
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">NI
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>27</bold>
</oasis:entry>
<oasis:entry colname="2">7300
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">28300
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>28</bold>
</oasis:entry>
<oasis:entry colname="2">1500
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">4700
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>29</bold>
</oasis:entry>
<oasis:entry colname="2">3500
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">NI
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>30</bold>
</oasis:entry>
<oasis:entry colname="2">4700
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">NI
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>31</bold>
</oasis:entry>
<oasis:entry colname="2">7600
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">NI
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>32</bold>
</oasis:entry>
<oasis:entry colname="2">NI
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">NI
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>33</bold>
</oasis:entry>
<oasis:entry colname="2">5600
</oasis:entry>
<oasis:entry colname="3">NI
</oasis:entry>
<oasis:entry colname="4">NI
</oasis:entry>
<oasis:entry colname="5">NI
</oasis:entry>
<oasis:entry colname="6">NI
</oasis:entry>
<oasis:entry colname="7">NI</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
</oasis:table>
<table-wrap-foot><p><italic><sup>a</sup>
</italic>
<sup></sup> NI:  no inhibition seen at the highest concentration of compound tested
(55 μM). See Supporting Information Table S2 for analysis results of
compounds <bold>34</bold>
−<bold>59</bold>
.</p>
</table-wrap-foot>
</table-wrap>
</p>
<p><bold>SAR Analysis and Molecular Modeling.</bold> Analysis of the
series of <italic>N</italic>-benzoylpyrazoles indicated that the presence or
absence of ring substituents significantly affected inhibitor
activity. For example, the presence of methyl groups at both
the R<sub>1</sub>
 and R<sub>3</sub> positions decreased inhibitory activity. There were
only two potent (<italic>K</italic>
<sub>i</sub>
 < 200 nM) inhibitors (compounds <bold>9</bold> and
<bold>20</bold>) among the benzoylpyrazoles with ortho substituents in the
benzene ring, whereas the other 18 ortho-substituted derivatives
were inactive or only weak inhibitors. Likewise, there are only
two potent inhibitors (compounds <bold>2</bold>
 and <bold>9</bold>) with α-substituents
(R<sub>3</sub>
) in the pyrazole ring. Note, however, that compound <bold>2</bold> also
has a methylated carboxyl group and thus may differ from the
other inhibitors in its coordination with the enzyme. Overall,
most low-activity and inactive compounds had substituents in
positions R<sub>3</sub>
 and/or R<sub>4</sub>
/R<sub>8</sub>. Thus, it appears that an ortho
substituent in the benzene ring and/or α-substituent in the
pyrazole rings may hinder rotation of the rings that is needed
to achieve optimal conformation for interaction with the active
site of the enzyme.
</p>
<p>To investigate binding of <italic>N</italic>-benzoylpyrazole derivatives to
the NE active site and obtain insight into the differences in
inhibitory activity of the various derivatives, we performed
molecular modeling studies using a 23-residue model of the
NE binding site that was derived from the 1.84 Å resolution
crystal structure of NE complexed with a peptide chloromethyl
ketone inhibitor<sup>42</sup> (see Experimental Section). As reported by
Vergely et al.,<xref rid="jm0706001b00051" ref-type="bibr"></xref> optimization of this geometry as a constituent
part of whole enzyme does not lead to noticeable changes in
atom positions. We imported this structure of the NE binding
site into ArgusLab and performed docking studies to identify
binding modes of the flexible ligands that were favorable for
the interaction between the hydroxyl group of Ser195 and the
carbonyl group of the benzoylpyrazole molecule. This approach
is based on a number of reports showing that the mechanism
of action of serine protease inhibitors containing an N−CO
fragment involves interaction between the inhibitor's carbonyl
group and Ser195<named-content content-type="bibref-group"><xref rid="jm0706001b00046" ref-type="bibr"></xref>
,<xref rid="jm0706001b00051" ref-type="bibr"></xref>
,<xref rid="jm0706001b00052" ref-type="bibr"></xref>
</named-content> and/or other targets in the active
site.<named-content content-type="bibref-group"><xref rid="jm0706001b00020" ref-type="bibr"></xref>
,<xref rid="jm0706001b00050" ref-type="bibr"></xref>
,<xref rid="jm0706001b00053" ref-type="bibr"></xref>
,<xref rid="jm0706001b00054" ref-type="bibr"></xref>
</named-content> This mechanism of interaction has been shown to
involve formation of an intermediate Michaelis complex where
the inhibitor carbonyl carbon atom is located 1.8−2.6 Å from
the Ser195 hydroxyl oxygen (distance <italic>d</italic>
<sub>1</sub>
 in Figure <xref rid="jm0706001f00007"></xref>), while
the angle of Ser195 Oγ···CO, denoted as angle α in Figure
<xref rid="jm0706001f00007"></xref>
, is limited to 80−120°.<named-content content-type="bibref-group"><xref rid="jm0706001b00051" ref-type="bibr"></xref>
,<xref rid="jm0706001b00055" ref-type="bibr"></xref>
</named-content>
<fig id="jm0706001f00007" position="float"><label>7</label>
<caption><p>Geometric parameters important for formation of a Michaelis complex in the NE active site. The important distances (<italic>d</italic>) and relevant
angle (α), as specified in the text, are indicated. The information is
based on the model of synchronous proton transfer from the oxyanion
hole in NE.<named-content content-type="bibref-group"><xref rid="jm0706001b00051" ref-type="bibr"></xref>
,<xref rid="jm0706001b00058" ref-type="bibr"></xref>
</named-content>
</p>
</caption>
<graphic xlink:href="jm-2007-00600+_0010.tif"></graphic>
</fig>
</p>
<p>The most favorable binding modes for five active (compounds
<bold>1</bold>
, <bold>4</bold>
, <bold>6</bold>
, <bold>7</bold>
, and <bold>17</bold>
) and four inactive (compounds <bold>35</bold>
, <bold>39</bold>
, <bold>53</bold>,
and <bold>54</bold>) benzoylpyrazole derivatives were chosen from ligand
docking poses lying within a 2 kcal/mol energy gap above the
lowest-energy binding mode for each compound and are
presented in Figure <xref rid="jm0706001f00008"></xref>, Supporting Information Figure S2, and
Table <xref rid="jm0706001t00005"></xref>
. The <italic>d</italic>
<sub>1</sub> and α values show that in highly active
inhibitors (compounds <bold>1</bold>
, <bold>4</bold>
, <bold>6</bold>
, <bold>7</bold>
, and <bold>17</bold>) the carbonyl carbon
atom is slightly more accessible to the Ser195 hydroxyl oxygen,
compared to inactive benzoylpyrazoles (<italic>d</italic>
<sub>1</sub> = 2.98 ± 0.29 versus
3.41 ± 0.74 Å, respectively) (Table <xref rid="jm0706001t00005"></xref>). Two of the inactive
compounds (<bold>53</bold>
 and <bold>54</bold>
) also had relatively short distances <italic>d</italic>
<sub>1</sub>;
however, the Ser195 Oγ···CO angle was too acute (α = 51°),
making them unfavorable for nucleophilic attack by Ser195 in
the oxyanion hole (Figure <xref rid="jm0706001f00008"></xref>
D and F). For example, Figure <xref rid="jm0706001f00008"></xref>D

shows that the docked compound <bold>53</bold> is strongly anchored in a
narrow groove of the binding site; however, rotation of the
molecule to a more appropriate α angle is impossible without
significantly increasing <italic>d</italic>
<sub>1</sub>. In support of our observations that
the presence of methyl groups at R<sub>1</sub>
 and R<sub>3</sub> decreases inhibitor
activity, the docked structure for inactive benzoylpyrazole <bold>53</bold>
shows that the pyrazole methyl groups collide with the borders
of the binding pocket and hinder penetration of molecule <bold>53</bold>
into the oxyanion hole.
<fig id="jm0706001f00008" position="float"><label>8</label>
<caption><p>Molecular docking of active and inactive<italic>N</italic>-benzoylpyrazole derivatives into the binding site of NE. The NE Ser195 hydroxyl oxygen
is orange. For all ligand structures, carbon is gray, hydrogen is white, nitrogen is violet, oxygen is the small red sphere, chlorine is green, fluorine
is blue, and bromine is the large red sphere.</p>
</caption>
<graphic xlink:href="jm-2007-00600+_0011.tif"></graphic>
</fig>
<table-wrap id="jm0706001t00005" position="float"><label>5</label>
<caption><p>Results of Docking and Subsequent Molecular Optimization for Favorable Poses<italic><sup>a</sup>
</italic>
<sup></sup> in the Binding Site of NE for Selected Active and Inactive
<italic>N</italic>
-Benzoylpyrazoles</p>
</caption>
<oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="8"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:colspec colnum="5" colname="5"></oasis:colspec>
<oasis:colspec colnum="6" colname="6"></oasis:colspec>
<oasis:colspec colnum="7" colname="7"></oasis:colspec>
<oasis:colspec colnum="8" colname="8"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry>
<oasis:entry colname="2"></oasis:entry>
<oasis:entry colname="3"></oasis:entry>
<oasis:entry namest="4" nameend="8">distances (Å)<italic><sup>b</sup>
</italic>
<sup></sup>
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"></oasis:entry>
<oasis:entry namest="2" nameend="3">α (deg)<italic><sup>b</sup>
</italic>
<sup></sup>
</oasis:entry>
<oasis:entry colname="4"></oasis:entry>
<oasis:entry namest="5" nameend="8">optimization</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
<oasis:tgroup cols="9"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:colspec colnum="5" colname="5"></oasis:colspec>
<oasis:colspec colnum="6" colname="6"></oasis:colspec>
<oasis:colspec colnum="7" colname="7"></oasis:colspec>
<oasis:colspec colnum="8" colname="8"></oasis:colspec>
<oasis:colspec colnum="9" colname="9"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">compd</oasis:entry>
<oasis:entry namest="2" nameend="2">docking</oasis:entry>
<oasis:entry namest="3" nameend="3">optimization</oasis:entry>
<oasis:entry namest="4" nameend="4">docking
<italic>d</italic>
<sub>1</sub>
</oasis:entry>
<oasis:entry namest="5" nameend="5"><italic>d</italic>
<sub>1</sub>
</oasis:entry>
<oasis:entry namest="6" nameend="6"><italic>d</italic>
<sub>2</sub>
</oasis:entry>
<oasis:entry namest="7" nameend="7"><italic>d</italic>
<sub>3</sub>
</oasis:entry>
<oasis:entry namest="8" nameend="8"><italic>L</italic>
 = <italic>d</italic>
<sub>2,min</sub>
 + <italic>d</italic>
<sub>3</sub>
</oasis:entry>
<oasis:entry namest="9" nameend="9">molecular deformation
Δ<italic>E</italic>
 (kcal/mol)<italic><sup>c</sup>
</italic>
<sup></sup>
</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
<oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1">Active
</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
<oasis:tgroup cols="9"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:colspec colnum="5" colname="5"></oasis:colspec>
<oasis:colspec colnum="6" colname="6"></oasis:colspec>
<oasis:colspec colnum="7" colname="7"></oasis:colspec>
<oasis:colspec colnum="8" colname="8"></oasis:colspec>
<oasis:colspec colnum="9" colname="9"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1"><bold>1</bold>
</oasis:entry>
<oasis:entry colname="2">102
</oasis:entry>
<oasis:entry colname="3">114
</oasis:entry>
<oasis:entry colname="4">2.94
</oasis:entry>
<oasis:entry colname="5">2.45
</oasis:entry>
<oasis:entry colname="6">2.03/2.08
</oasis:entry>
<oasis:entry colname="7">2.99
</oasis:entry>
<oasis:entry colname="8">5.02
</oasis:entry>
<oasis:entry colname="9">5.13
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>4</bold>
</oasis:entry>
<oasis:entry colname="2">69
</oasis:entry>
<oasis:entry colname="3">84
</oasis:entry>
<oasis:entry colname="4">3.39
</oasis:entry>
<oasis:entry colname="5">2.27
</oasis:entry>
<oasis:entry colname="6">1.98/2.08
</oasis:entry>
<oasis:entry colname="7">2.83
</oasis:entry>
<oasis:entry colname="8">4.81
</oasis:entry>
<oasis:entry colname="9">1.49
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>6</bold>
</oasis:entry>
<oasis:entry colname="2">74
</oasis:entry>
<oasis:entry colname="3">86
</oasis:entry>
<oasis:entry colname="4">3.05
</oasis:entry>
<oasis:entry colname="5">2.28
</oasis:entry>
<oasis:entry colname="6">2.27/2.30
</oasis:entry>
<oasis:entry colname="7">2.82
</oasis:entry>
<oasis:entry colname="8">5.09
</oasis:entry>
<oasis:entry colname="9">2.32
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>7</bold>
</oasis:entry>
<oasis:entry colname="2">96
</oasis:entry>
<oasis:entry colname="3">96
</oasis:entry>
<oasis:entry colname="4">2.58
</oasis:entry>
<oasis:entry colname="5">2.58
</oasis:entry>
<oasis:entry colname="6">1.64/2.62
</oasis:entry>
<oasis:entry colname="7">2.99
</oasis:entry>
<oasis:entry colname="8">4.63
</oasis:entry>
<oasis:entry colname="9">0
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>17</bold>
</oasis:entry>
<oasis:entry colname="2">95
</oasis:entry>
<oasis:entry colname="3">86
</oasis:entry>
<oasis:entry colname="4">2.92
</oasis:entry>
<oasis:entry colname="5">2.39
</oasis:entry>
<oasis:entry colname="6">2.13/2.38
</oasis:entry>
<oasis:entry colname="7">2.90
</oasis:entry>
<oasis:entry colname="8">5.03
</oasis:entry>
<oasis:entry colname="9">2.95</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
<oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1">Inactive
</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
<oasis:tgroup cols="9"><oasis:colspec colnum="1" colname="1"></oasis:colspec>
<oasis:colspec colnum="2" colname="2"></oasis:colspec>
<oasis:colspec colnum="3" colname="3"></oasis:colspec>
<oasis:colspec colnum="4" colname="4"></oasis:colspec>
<oasis:colspec colnum="5" colname="5"></oasis:colspec>
<oasis:colspec colnum="6" colname="6"></oasis:colspec>
<oasis:colspec colnum="7" colname="7"></oasis:colspec>
<oasis:colspec colnum="8" colname="8"></oasis:colspec>
<oasis:colspec colnum="9" colname="9"></oasis:colspec>
<oasis:tbody><oasis:row><oasis:entry colname="1"><bold>35</bold>
</oasis:entry>
<oasis:entry colname="2">121
</oasis:entry>
<oasis:entry colname="3">99
</oasis:entry>
<oasis:entry colname="4">4.14
</oasis:entry>
<oasis:entry colname="5">2.32
</oasis:entry>
<oasis:entry colname="6">2.71/3.97
</oasis:entry>
<oasis:entry colname="7">3.54
</oasis:entry>
<oasis:entry colname="8">6.25
</oasis:entry>
<oasis:entry colname="9">2.94
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>39</bold>
</oasis:entry>
<oasis:entry colname="2">92
</oasis:entry>
<oasis:entry colname="3">103
</oasis:entry>
<oasis:entry colname="4">3.94
</oasis:entry>
<oasis:entry colname="5">2.67
</oasis:entry>
<oasis:entry colname="6">3.42/3.66
</oasis:entry>
<oasis:entry colname="7">3.57
</oasis:entry>
<oasis:entry colname="8">6.99
</oasis:entry>
<oasis:entry colname="9">6.60
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>53</bold>
</oasis:entry>
<oasis:entry colname="2">51
</oasis:entry>
<oasis:entry colname="3">82
</oasis:entry>
<oasis:entry colname="4">2.64
</oasis:entry>
<oasis:entry colname="5">2.52
</oasis:entry>
<oasis:entry colname="6">4.23/4.96
</oasis:entry>
<oasis:entry colname="7">4.95
</oasis:entry>
<oasis:entry colname="8">9.18
</oasis:entry>
<oasis:entry colname="9">11.40
</oasis:entry>
</oasis:row>
<oasis:row><oasis:entry colname="1"><bold>54</bold>
</oasis:entry>
<oasis:entry colname="2">51
</oasis:entry>
<oasis:entry colname="3">80
</oasis:entry>
<oasis:entry colname="4">2.91
</oasis:entry>
<oasis:entry colname="5">2.40
</oasis:entry>
<oasis:entry colname="6">2.01/2.60
</oasis:entry>
<oasis:entry colname="7">3.28
</oasis:entry>
<oasis:entry colname="8">5.29
</oasis:entry>
<oasis:entry colname="9">37.36</oasis:entry>
</oasis:row>
</oasis:tbody>
</oasis:tgroup>
</oasis:table>
<table-wrap-foot><p><italic><sup>a</sup>
</italic>
<sup></sup>
 A docking pose was regarded as favorable if it had α and <italic>d</italic>
<sub>1</sub>
 values closest to the optimal ranges and within 2 kcal/mol above the lowest-energy pose.<italic><sup>b</sup>
</italic>
<sup></sup>
 The angle and distances are as indicated in Figure <xref rid="jm0706001f00007"></xref>
.<italic><sup>c</sup>
</italic>
<sup></sup>
 Energy of molecular deformation of a selected molecule during optimization.</p>
</table-wrap-foot>
</table-wrap>
</p>
<p>While docking studies revealed that active compounds <bold>6</bold> and
<bold>7</bold> were almost planar in their favorable binding modes, with
dihedral angles of ∼3° between heterocycle and benzoyl ring,

the coplanarity of the two rings does not seem to correlate with
high inhibitory activity. For example, the docked geometries
of active inhibitors <bold>1</bold>
 and <bold>4</bold> are nonplanar, with dihedral angles
of 48° and 51°, respectively. It is interesting to note that the
user-specified, constrained planarity of molecule <bold>35</bold>, which
contains a bulky chlorine atom in the ortho position of the
phenyl ring, leads to the appearance of a “good” docking pose
with <italic>d</italic>
<sub>1</sub> = 2.57 Å and α = 105°. In reality, this molecule cannot
become planar, and its docking without constraints produces
no binding modes suitable for the interaction with Ser195
(Figure <xref rid="jm0706001f00008"></xref>
B and Table <xref rid="jm0706001t00005"></xref>). Thus, the presence of a large ortho
substituent on the phenyl ring blocks rotation of the molecule
into a conformation that can inhibit NE.
</p>
<p>As indicated above, distances <italic>d</italic>
<sub>1</sub> for NE inhibitors were
generally shorter than those of their inactive analogues (Table
<xref rid="jm0706001t00005"></xref>
). However, <italic>d</italic>
<sub>1</sub> was >2.6 Å for some active compounds, which
exceeds the maximum distance specified for Michaelis complex
formation.<xref rid="jm0706001b00051" ref-type="bibr"></xref> Most likely, this difference is due to rigidity of
the binding site used in our docking study, which does not allow
any degrees of freedom for Ser195 and His57, which is located
in proximity to Ser195. These two residues, along with Asp102,
form a conserved, active site catalytic triad that catalyzes
synchronous proton transfer from Ser195 to Asp102, with His57
acting as a general base to enhance nucleophilicity of the Ser195
hydroxyl oxygen and activate the hydrolytic H<sub>2</sub>
O<sup>42-44</sup> (illustrated
in Figure <xref rid="jm0706001f00007"></xref>). While rotational lability of the catalytic triad
residues could result in a more favorable environment for
substrate binding and cleavage, certain rotational freedom could
also destroy the conformationally sensitive “channel” of proton
migration. To explore this issue, five torsion angles (<italic>r</italic>
<sub>1</sub>
−<italic>r</italic>
<sub>5</sub>) in
Ser195, His57, and Asp102 (Figure <xref rid="jm0706001f00007"></xref>) were varied during
optimization with the MM+ force field, while concurrently
applying harmonic restraints to <italic>d</italic>
<sub>1</sub> and α. This approach allowed
us to evaluate if rotational flexibility in Ser195, His57, and
Asp102 could lead to a favorable orientation for Michaelis
complex formation with an optimized ligand. Differences in the
energies (Δ<italic>E</italic>) of a benzoylpyrazole molecule before and after
MM+ optimization within the partially flexible binding site
showed that eight benzoylpyrazole derivatives (compounds <bold>1</bold>,
<bold>4</bold>
, <bold>6</bold>
, <bold>7</bold>
, <bold>17</bold>
, <bold>35</bold>
, <bold>39</bold>
, and <bold>53</bold>
) bound with suitable <italic>d</italic>
<sub>1</sub> and α and
without strong distortion of their optimal molecular geometry,
as indicated by relatively low Δ<italic>E</italic> values. In addition, optimization of binding modes for these compounds did not lead to steric
collisions between nonbonded atoms. Alternatively, a favorable
binding mode for molecule <bold>54</bold> was not attainable because its
penetration to Ser195 under the forces of harmonic restraints
led to dramatic molecular distortion characterized by Δ<italic>E</italic> =
37.36 kcal/mol. Consequently, the lack of inhibitory activity of
compound <bold>54</bold> can be explained by steric hindrances to formation
of a Michaelis complex. The other inactive benzoylpyrazoles
investigated by molecular modeling (compounds <bold>35</bold>
, <bold>39</bold>, and
<bold>53</bold>
) required significant rotations <italic>r</italic>
<sub>3</sub>
 and/or <italic>r</italic>
<sub>4</sub> of His57 in order
to interact with Ser195. As a result of these rotations, the
distances <italic>d</italic>
<sub>2</sub>
 and <italic>d</italic>
<sub>3</sub> generally became higher than in the initial
enzyme structure. The sum of the distances <italic>L</italic>
 = <italic>d</italic>
<sub>2</sub>
(min) + <italic>d</italic>
<sub>3</sub>
can be regarded as the length of synchronous proton transfer
between Ser195 and Asp102 (Table <xref rid="jm0706001t00005"></xref>
). For compound <bold>7</bold>,
suitable <italic>d</italic>
<sub>1</sub> and α characteristics were achieved after docking,
and no further optimization was necessary. Thus, the <italic>L</italic> value
of 4.63 Å for compound <bold>7</bold> corresponds to the initial enzyme
architecture. For the inactive benzoylpyrazoles (compounds <bold>35</bold>,
<bold>39</bold>
, and <bold>53</bold>), the length of proton transfer increased by 1.6−4.5
Å (<italic>L</italic> = 7.06 ± 1.68), which was sufficient to interfere with
general acid−base catalysis and led to loss of inhibitory activity
despite the ligand being in a suitable position for Michaelis
complex formation. In contrast, the active inhibitors (compounds
<bold>1</bold>
, <bold>4</bold>
, <bold>6</bold>
, <bold>7</bold>
, and <bold>17</bold>
) had shorter proton-transfer distances (<italic>L</italic> =
4.92 ± 0.2), as well as <italic>d</italic>
<sub>1</sub>
 and α values (Table <xref rid="jm0706001t00005"></xref>) that favored
interaction of the carbonyl group with Ser195 and enzyme
inhibition.<named-content content-type="bibref-group"><xref rid="jm0706001b00046" ref-type="bibr"></xref>
,<xref rid="jm0706001b00051" ref-type="bibr"></xref>
,<xref rid="jm0706001b00052" ref-type="bibr"></xref>
</named-content>
</p>
</sec>
<sec id="d7e3011"><title>Conclusions</title>
<p>We utilized HTS of a large chemical diversity library to select
unique small-molecule inhibitors of NE and identified a novel
class of NE inhibitors that is based on an <italic>N</italic>-benzoylpyrazole
scaffold. A number of these compounds were highly active, with
<italic>K</italic>
<sub>i</sub> values of 6−300 nM, making them promising candidates
for further evaluation and development. These <italic>N</italic>-benzoylpyrazole derivatives were competitive, pseudoirreversible inhibitors
of NE activity and were relatively specific for NE and
chymotrypsin, compared to a range of other proteases tested.
Evaluation of compound stability in physiological buffer showed
that some of the selected compounds were unstable while others
were quite stable, and these differences in stability were
correlated with variation in ring substituents. Clearly, the
understanding of these features will be essential in future
development of this class of inhibitors.
</p>
<p>Molecular docking of the active <italic>N</italic>-benzoylpyrazole inhibitors
to the NE catalytic site revealed favorable binding for Michaelis
complex formation. Furthermore, the carbonyl group of the
bound inhibitor was positioned in the oxyanion hole of the NE
binding site with a favorable orientation for the interaction with
the catalytic triad. In contrast, inactive benzoylpyrazoles either
had unfavorable orientation for nucleophilic attack by Ser195
or were sterically hindered from optimal binding. Overall, our
molecular docking and SAR analyses provide key details that
could be exploited in the development of <italic>N</italic>-benzoylpyrazole
analogues, and we suggest that the <italic>N</italic>-benzoylpyrazole scaffold
represents a novel direction for developing NE inhibitors that
exhibit high enzyme selectivity and potency.
</p>
</sec>
<sec id="d7e3038"><title>Experimental Section</title>
<p><bold>Compounds and Reagents.</bold> The ActiProbe-10K chemical
diversity set was obtained from TimTec, Inc. (Newark, DE). This
commercial library is comprised of a random selection of 10 000
druglike compounds. Additional <italic>N</italic>-benzoylpyrazole derivatives
were selected from the Actimol database and purchased from
TimTec. The purity of the most active target compounds was
confirmed by dissolving compounds directly in acetonitrile/H<sub>2</sub>O
(60/40 or 65/35, v/v) and analyzing immediately by RP-HPLC, as
described below. All target compounds eluted as a single peak and
were found to be >98% pure (Supporting Information Table S3).
Compound identity was verified by <sup>1</sup>H NMR analysis at 200, 300,
or 500 MHz using Bruker AC200, AC300, or Avance NMR
spectrometers (Bruker BioSpin, Billerica, MA), respectively, at 23
or 30 °C and with the samples dissolved in deuterated dimethyl
sulfoxide (DMSO-<italic>d</italic>
<sub>6</sub>) (Supporting Information Table S4; NMR
analyses were preformed by TimTec, Inc.). NE, human chymotrypsin, human kallikrein, human thrombin, human urokinase,
porcine aminopeptidase, and human cathepsin D as well as their
substrates were purchased from Calbiochem (San Diego, CA).
DMSO, DMSO-<italic>d</italic>
<sub>6</sub>
, deuterated water (D<sub>2</sub>O), pyrazole, and benzoic
acid were purchased from Sigma Chemical Co. (St. Louis, MO).
HPLC grade acetonitrile was from EMD Chemicals (Gibbstown,
NJ), and HPLC grade H<sub>2</sub>O and trifluoroacetic acid (TFA) were
from Mallinckrodt Baker Inc. (Phillipsburg, NJ).
</p>
<p><bold>Library Screening and Kinetic Measurements.</bold> We used HTS
to screen the compound library for hits with NE inhibitory activity.
Stock compounds were dissolved in 100% DMSO at a concentration
of 2 mg/mL. The final concentration of DMSO in the reactions
was 1%, and this level of DMSO had no effect on enzyme activity.

HTS was performed in black flat-bottom 96-well microtiter plates.
Briefly, a solution containing buffer A (200 mM Tris-HCl, pH 7.5,
0.01% bovine serum albumin, and 0.05% Tween-20) and up to 20
mU/mL of NE (Calbiochem) was added to wells containing 20 μg/mL of each compound. The reaction was initiated by addition of
25 μM elastase substrate (<italic>N</italic>-methylsuccinyl-Ala−Ala-Pro-Val-7-amino-4-methylcoumarin, Calbiochem) in a final reaction volume
of 100 μL/well. Kinetic measurements were obtained every 30 s
for 10 min at 25 °C using a Fluoroskan Ascent FL fluorescence
microplate reader (Thermo Electron, MA) with excitation and
emission wavelengths at 355 and 460 nm, respectively.
</p>
<p>For selected lead compounds, the inhibition constant (<italic>K</italic>
<sub>i</sub>) values
were determined using Dixon plots of three to four different
concentrations of the substrate.<xref rid="jm0706001b00035" ref-type="bibr"></xref> At each substrate concentration,
rates were determined with four to five different inhibitor concentrations, and the inverse of the velocities was plotted against the
final inhibitor concentration. <italic>K</italic>
<sub>i</sub>
 was determined from the intersection of the plotted lines (<italic>R</italic>
<sup>2</sup> > 0.98 for each line).
</p>
<p>Reactivation of the NE−inhibitor complex was investigated as
reported previously.<xref rid="jm0706001b00046" ref-type="bibr"></xref> Briefly, NE (20 mU/mL) was incubated with
excess inhibitor at final concentrations of 25 μM in 2 mL of buffer
A. After 20 min, a 50 μL aliquot was removed and assayed to verify
complete inhibition of NE enzymatic activity. Excess inhibitor was
removed via Centricon-10 filtration by centrifuging at 5000<italic>g</italic> for 1
h at 5 °C, adding 2 mL of buffer A to the retentate, and centrifuging
again under the same conditions. This process was repeated again,
and the final retentate was suspended in 1.5 mL of buffer A at
25 °C. At the indicated times, 50 μL aliquots were removed and
added to microplate wells containing 25 μM of the fluorogenic NE
substrate dissolved in 50 μL of buffer A. A control containing NE
(20 mU/mL) and 0.25% DMSO was run under the same conditions.
</p>
<p><bold>Analysis of Compound Stability.</bold> Spontaneous hydrolysis of
selected <italic>N</italic>-benzoylpyrazoles was evaluated at 25 °C in 0.05 M
phosphate buffer, pH 7.3. Kinetics of <italic>N</italic>-benzoylpyrazole hydrolysis
was monitored by measuring changes in absorbance spectra over
incubation time using a SpectraMax Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Absorbance (<italic>A</italic>
<italic><sub>t</sub>
</italic>) at
the characteristic absorption maxima of each <italic>N</italic>-benzoylpyrazole
was measured at the indicated times until no further absorbance
decreases occurred (<italic>A</italic>
<sub>∞</sub>
).<xref rid="jm0706001b00056" ref-type="bibr"></xref> Using these measurements, we created
semilogarithmic plots of log(<italic>A</italic>
<italic><sub>t</sub>
</italic>
 − <italic>A</italic>
<sub>∞</sub>) vs time (see Supporting
Information Figure S1), and <italic>k</italic>‘ values were determined from the
slopes of these plots. Half-conversion times were calculated using
<italic>t</italic>
<sub>1/2</sub>
 = 0.693/<italic>k</italic>‘.
</p>
<p>For selected compounds, the kinetics of hydrolysis was also
confirmed by RP-HPLC. Reactions were initiated by addition of 2
μL from a 10 mM stock solution (in DMSO) of the respective
compound to 200 μL of 0.05 M phosphate buffer (pH 7.3) at
25 °C. At the indicated times, aliquots (5 μL) were separated by
RP-HPLC on an automated HPLC system (Shimadzu, Torrance,
CA) with a Phenomenex Jupiter C18 300A column (5 μm, 25 cm
× 0.46 cm) eluted with acetonitrile/water (60%/40%, v/v) containing 0.1% (v/v) TFA at a flow rate of 1 mL/min at 25 °C. The elution
was monitored using a diode array detector (Shimadzu SPD-M10A
VP) set to spectrum max plot (wavelength at which the highest
absorbance occurs) in the region of 200−300 nm.
</p>
<p>To verify compound hydrolysis, compound <bold>1</bold> was dissolved in
85% DMSO-<italic>d</italic>
<sub>6</sub>
/15% D<sub>2</sub>O (3 mg/mL). The mixture was incubated
for 36 h at 37 °C, and the samples were analyzed by <sup>1</sup>H NMR on
a Bruker DRX-500 spectrometer (Bruker BioSpin, Billerica, MA).
Control samples were dissolved in mixture of 85% DMSO-<italic>d</italic>
<sub>6</sub>/15%
D<sub>2</sub>
O and analyzed immediately. <sup>1</sup>H NMR spectra were recorded at
20 °C using 3-(trimethylsilyl)propionic-2,2,3,3-<italic>d</italic>
<sub>4</sub> acid sodium salt
as an internal reference.
</p>
<p><bold>Analysis of Inhibitor Specificity.</bold> Selected compounds were
evaluated for their ability to inhibit a range of proteases in 100 μL
reaction volumes at 25 °C. Analysis of chymotrypsin inhibition
was performed in reaction mixtures containing 0.05 M Tris-HCl,
pH 8.0, 30 nM human pancreas chymotrypsin, test compounds, and
100 μM substrate (Suc-Ala−Ala-Pro-Phe-7-amino-4-methylcoumarin). Thrombin inhibition was evaluated in reaction mixtures
containing 0.25 M sodium phosphate, pH 7.0, 0.2 M NaCl, 0.1%
PEG 8000, 1.7 U human plasma thrombin, test compounds, and
20 μM substrate (benzoyl-Phe-Val-Arg-7-amino-4-methylcoumarin). Analysis of kallikrein inhibition was performed in reaction
mixtures containing 0.05 M Tris-HCl, pH 8.0, 0.1 M NaCl, 0.05%
Tween-20, 2 nM human plasma kallikrein, test compounds, and
50 μM substrate (benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin). Analysis of urokinase inhibition assay was performed
in reaction mixtures containing 0.1 M Tris-HCl, pH 8.0, 30 U/mL
human urine urokinase, test compounds, and 30 μM substrate
(benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin). Aminopeptidase M inhibition was determined in reaction mixtures
contained 0.1 M Tris-HCl, pH 7.0, 0.05% Tween-20, 2 mU porcine
kidney aminopeptidase M, test compounds, and 0.4 mM substrate
(<sc>l</sc>
-alanyl-<italic>p</italic>-nitroanilide). Analysis of cathepsin D inhibition was
performed in reaction mixtures containing 0.1 M sodium acetate,
pH 5.0, 0.1 U/mL human spleen cathepsin D, test compounds, and
5 μM substrate (MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-<sc>d</sc>
-Arg-NH<sub>2</sub>). Reactions for aminopeptidase M activity
were monitored at 405 nm using a SpectraMax Plus microplate
reader. Cathepsin D assays were monitored with a Fluoroskan
Ascent FL microtiter plate reader at excitation and emission
wavelengths of 340 and 390 nm, respectively. For all serine
proteases (chymotrypsin, thrombin, kallikrein, and urokinase)
activity was monitored at excitation and emission wavelengths of
355 and 460 nm, respectively. For all compounds tested, the
concentration of inhibitor that causes 50% inhibition of the
enzymatic reaction (IC<sub>50</sub>) was calculated by plotting % inhibition
vs logarithm of inhibitor concentration (at least six points), and
the data are the mean values of at least three experiments with
relative standard deviations of <15%.
</p>
<p><bold>Molecular Modeling.</bold> For computer-assisted molecular modeling
we employed HyperChem, version 7.0 (Hypercube, Inc., Waterloo,
ON, Canada) and ArgusLab 4.0.1 (Planaria Software LLC, Seattle,
WA). Molecular structures of <italic>N</italic>
-benzoylpyrazole derivatives <bold>1</bold>
, <bold>4</bold>,
<bold>6</bold>
, <bold>7</bold>
, <bold>17</bold>
, <bold>35</bold>
, <bold>39</bold>
, <bold>53</bold>
, and <bold>54</bold> were first created and optimized by
HyperChem with the use of the molecular mechanics MM+ force
field. These structures were then subjected to docking computations
by ArgusLab with AScore scoring functions<xref rid="jm0706001b00057" ref-type="bibr"></xref> to find the lowest-energy binding modes. Flexibility of an inhibitor was accounted
for around all rotatable bonds automatically identified by ArgusLab.
The binding site of NE was considered to be rigid in the docking
procedure, and its geometry was obtained by downloading a crystal
structure of NE complexed with a peptide chloromethyl ketone
inhibitor<sup>42</sup>
 from the Protein Data Bank (code <ext-link ext-link-type="pdb" xlink:href="1HNE">1HNE</ext-link>). Amino acid
residues within 7 Å of any non-hydrogen atom of the inhibitor were
regarded as belonging to the binding site, and 22 residues satisfied
this condition (Phe41, Cys42, Ala55, His57, Cys58, Leu99B,
Asp102, Val190, Cys191, Phe192, Gly193, Asp194, Ser195,
Gly196, Ser197, Ala213, Ser214, Phe215, Val216, Arg217A,
Gly218, and Tyr224). Although Ala56 is 8.3 Å away from the
nearest inhibitor atom, we included Ala55 in the binding site to
maintain continuous sequence from Ala55 to Cys58. We then
removed the chloromethyl ketone inhibitor and cocrystallized water
molecules to obtain the 23-residue model of the NE binding site
that was used for docking.
</p>
<p>Binding modes found for each inhibitor within a 2 kcal/mol gap
above the lowest-energy mode were examined for the potential to
form a Michaelis complex between the hydroxyl group of Ser195
and the inhibitor's carbonyl group, and values of <italic>d</italic>
<sub>1</sub> and α (Figure
<xref rid="jm0706001f00007"></xref>) were determined for each docked compound. These modes were
further optimized by molecular mechanics with the MM+ force
field using HyperChem. During this optimization, we varied torsion
angles <italic>r</italic>
<sub>1</sub>
−<italic>r</italic>
<sub>5</sub> in Ser195, His57, and Asp102 and also applied
harmonic restraints to <italic>d</italic>
<sub>1</sub> and α. Energy terms for the restraints
were calculated as <italic>E</italic>
<sub>d</sub>
 = <italic>K</italic>
<sub>d</sub>
(<italic>d</italic>
<sub>1</sub>
 − 1.5)<sup>2</sup>
 and <italic>E</italic>
<sub>α</sub>
 = <italic>K</italic>
<sub>α</sub>
(α − 100°),<xref rid="jm0706001b00002" ref-type="bibr"></xref>
where <italic>K</italic>
<sub>d</sub>
 = 15 kcal·mol<sup>-1</sup>
·Å<sup>-1</sup>
 and <italic>K</italic>
<sub>α</sub>
 = 3 kcal·mol<sup>-1</sup>
·deg<sup>-1</sup>. These
restraints served as driving forces to attain the conditions of
Michaelis complex formation through simultaneous cooperative
movements of key residues within the binding site. Again, values
of <italic>d</italic>
<sub>1</sub>
 and α were determined, as well as distances <italic>d</italic>
<sub>2</sub>
 and <italic>d</italic>
<sub>3</sub> (Figure

<xref rid="jm0706001f00007"></xref>
). The distance <italic>d</italic>
<sub>2</sub> is measured between the NH hydrogen in the
His57 imidazole ring and either one of the carboxylic oxygens in
Asp102. It defines the synchronous proton transfer from the
oxyanion hole.<xref rid="jm0706001b00058" ref-type="bibr"></xref> The distance between the hydroxyl proton in
Ser195 and the basic pyridine-type nitrogen in His57 is also
important for this transfer. However, because of easy rotation of
the hydroxyl about the C−O bond in Ser195, we measured <italic>d</italic>
<sub>3</sub>
between the oxygen in Ser195 and the basic nitrogen in His57 (see
Figure <xref rid="jm0706001f00007"></xref>).
</p>
<p>Molecular mechanics optimization of a binding mode also caused
deformation of the benzoylpyrazole molecule, and the change in
energy (Δ<italic>E</italic>
) due to deformation was calculated as Δ<italic>E</italic>
 = <italic>E</italic>
<sub>2</sub>
 − <italic>E</italic>
<sub>1</sub>,
where <italic>E</italic>
<sub>1</sub> is the MM+ conformational energy of the inhibitor
molecule in the docked structure chosen as the favorable binding
mode and where <italic>E</italic>
<sub>2</sub> is the conformational energy of the inhibitor
in its geometry attained in the optimized docked structure.
</p>
</sec>
</body>
<back><ack><title>Acknowledgments</title>
<p>We thank Dr. Scott Busse from the
Department of Chemistry and Biochemistry at Montana State
University for providing expert NMR analysis. This work was
supported in part by Department of Defense Grant W9113M-04-1-0001, National Institutes of Health Grant RR020185, and
the Montana State University Agricultural Experimental Station.
The U.S. Army Space and Missile Defense Command, 64
Thomas Drive, Frederick, MD 21702, is the awarding and
administering acquisition office. The content of this report does
not necessarily reflect the position or policy of the U.S.
Government.
</p>
</ack>
<notes notes-type="si"><sec id="d7e3404"><title><ext-link xlink:href="/doi/suppl/10.1021%2Fjm070600%2B">Supporting Information Available</ext-link>
</title>
<p>Table S1 on the effect of
<italic>N</italic>
-benzoylpyrazoles <bold>34</bold>
−<bold>59</bold> on NE activity, Table S2 on the analysis
of inhibitory specificity for compounds <bold>34</bold>
−<bold>59</bold>; Table S3 on the
HPLC analysis of selected compounds, Table S4 on the <sup>1</sup>H NMR
analysis of selected compounds, Figure S1 showing examples of
semilogarithmic plots used to determine rate constants for <italic>N</italic>-benzoylpyrazole spontaneous hydrolysis, and Figure S2 showing
further examples of molecular docking of <italic>N</italic>-benzoylpyrazoles into
the active site of NE. This material is available free of charge via
the Internet at <uri xlink:href="http://pubs.acs.org">http://pubs.acs.org</uri>.
</p>
</sec>
</notes>
<fn-group><fn id="jm0706001n00001"><p>Abbreviations:  ARDS, adult respiratory distress syndrome; COPD,
chronic obstructive pulmonary disease; HTS, high-throughput screening;
NE, neutrophil elastase; RP-HPLC, reverse-phase high-performance liquid
chromatography.</p>
</fn>
</fn-group>
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<mods version="3.6"><titleInfo><title>N-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase</title>
</titleInfo>
<titleInfo contentType="CDATA"><title>N-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase</title>
</titleInfo>
<name type="personal"><namePart type="family">SCHEPETKIN</namePart>
<namePart type="given">Igor A.</namePart>
<affiliation>Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, and Department of Chemistry,Altai State Technical University, Barnaul 656038, Russia</affiliation>
<affiliation> Montana State University.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal"><namePart type="family">KHLEBNIKOV</namePart>
<namePart type="given">Andrei I.</namePart>
<affiliation>Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, and Department of Chemistry,Altai State Technical University, Barnaul 656038, Russia</affiliation>
<affiliation> Altai State Technical University.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal" displayLabel="corresp"><namePart type="family">QUINN</namePart>
<namePart type="given">Mark T.</namePart>
<affiliation>Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, and Department of Chemistry,Altai State Technical University, Barnaul 656038, Russia</affiliation>
<affiliation> Montana State University.</affiliation>
<affiliation> To whom correspondence should be addressed. Phone:  406-994-5721.Fax:  406-994-4303. E-mail:  mquinn@montana.edu.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<typeOfResource>text</typeOfResource>
<genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre>
<originInfo><publisher>American Chemical Society</publisher>
<dateCreated encoding="w3cdtf">2007-09-12</dateCreated>
<dateIssued encoding="w3cdtf">2007-10-04</dateIssued>
<copyrightDate encoding="w3cdtf">2007</copyrightDate>
</originInfo>
<language><languageTerm type="code" authority="iso639-2b">eng</languageTerm>
<languageTerm type="code" authority="rfc3066">en</languageTerm>
</language>
<abstract>Human neutrophil elastase (NE) plays an important role in the pathogenesis of pulmonary disease. Using high-throughput chemolibrary screening, we identified 10 N-benzoylpyrazole derivatives that were potent NE inhibitors. Nine additional NE inhibitors were identified through further screening of N-benzoylpyrazole analogues. Evaluation of inhibitory activity against a range of proteases showed high specificity for NE, although several derivatives were also potent inhibitors of chymotrypsin. Analysis of reaction kinetics and inhibitor stability revealed that N-benzoylpyrazoles were pseudoirreversible competitive inhibitors of NE. Structure−activity relationship (SAR) analysis demonstrated that modification of N-benzoylpyrazole ring substituents modulated enzyme selectivity and potency. Furthermore, molecular modeling of the binding of selected active and inactive compounds to the NE active site revealed that active compounds fit well into the catalytic site, whereas inactive derivatives contained substituents or conformations that hindered binding or accessibility to the catalytic residues. Thus, N-benzoylpyrazole derivatives represent novel structural templates that can be utilized for further development of efficacious NE inhibitors.</abstract>
<relatedItem type="host"><titleInfo><title>Journal of Medicinal Chemistry</title>
</titleInfo>
<titleInfo type="abbreviated"><title>J. Med. Chem.</title>
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<identifier type="ISSN">0022-2623</identifier>
<identifier type="eISSN">1520-4804</identifier>
<identifier type="acspubs">jm</identifier>
<identifier type="coden">JMCMAR</identifier>
<identifier type="uri">pubs.acs.org/jmc</identifier>
<part><date>2007</date>
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N-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase

N-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase

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