N-benzoylpyrazoles are novel small-molecule inhibitors of human neutrophil elastase

Article abstract of DOI:10.1021/jm070600+

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.

Full text of DOI:10.1021/jm070600+

4
928
J. Med. Chem. 2007, 50, 4928-4938
N-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase
†
‡
,†
Igor A. Schepetkin, Andrei I. Khlebnikov, and Mark T. Quinn*
Department of Veterinary Molecular Biology, Montana State UniVersity, Bozeman, Montana 59717, and Department of Chemistry,
Altai State Technical UniVersity, Barnaul 656038, Russia
ReceiVed May 22, 2007
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.
Introduction
has been considered a promising strategy to improve the
1
7,18
a
outcome of pulmonary diseases.
A number of therapeutic
Acute respiratory distress syndrome (ARDS ), chronic ob-
structive pulmonary disease (COPD), and cystic fibrosis (CF)
strategies have focused on the use of recombinant or purified
preparations of two endogenous NE inhibitors described above,
R1-antitrypsin and secretory leukocyte protease inhibitor; how-
are progressive diseases that are frequently fatal.1-3 Unfortu-
nately, 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).4 NE is a serine
protease that is synthesized in neutrophils and stored in
azurophilic granules. 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. For
19
ever, use of these inhibitors has been problematic. Many types
of peptide and nonpeptide inhibitors, employing both reversible
and irreversible mechanisms of action, have also been reported
(reviewed in refs 15, 19, and 20). Among the most potent NE
,5
2
1
22
inhibitors are â-lactams, tert-butyloxadiazoles, and peptidyl
6
23
trifluoromethyl ketones. Nevertheless, the primary chemical
scaffolds of the most potent NE inhibitors were discovered 15-
7
24,25
2
0 years ago,
and modification of these scaffolds is still
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.5
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 11 and
the current focus of most NE inhibitor development because
moving away from these core scaffolds would be difficult and
time-consuming. Conversely, we propose that new NE inhibi-
26
,8-10
tors 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.2
Thus, we utilized HTS of a chemical diversity library containing
7-29
1
(
2). NE also can activate several matrix metalloproteinases
MMP-2, -3, and -9)13 and seems to play an important
1
0 000 druglike molecules to identify novel inhibitors of NE
physiologic role in tissue repair through its ability to regulate
growth factors and modulate cytokine expression at epithelial
that have core structures distinct from currently known leads.
Notably, the hits obtained from our screen included 10 N-
benzoylpyrazole derivatives, which were potent NE inhibitors.
Furthermore, analysis of 43 additional N-benzoylpyrazole
derivatives resulted in the identification of nine more potent
NE inhibitors with Ki e 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 N-benzoylpyra-
zole derivatives were able to effectively dock within the NE
catalytic site so that Michaelis complex formation and synchro-
nous proton transfer were favored, while binding of inactive
derivatives into the pocket was sterically hindered or catalyti-
cally unfavorable.
and endothelial surfaces.1
4,15
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.16
Given the destructive potential of unregulated NE, it is not
surprising that inhibition of NE activity in pulmonary tissues
*
To whom correspondence should be addressed. Phone: 406-994-5721.
Fax: 406-994-4303. E-mail: mquinn@montana.edu.
†
Montana State University.
Altai State Technical University.
Abbreviations: ARDS, adult respiratory distress syndrome; COPD,
‡
a
chronic obstructive pulmonary disease; HTS, high-throughput screening;
NE, neutrophil elastase; RP-HPLC, reverse-phase high-performance liquid
chromatography.
1
0.1021/jm070600+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/12/2007

NoVel Inhibitors of Neutrophil Elastase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4929
Table 1. Chemical Structures and Inhibitory Activity of the Most
Potent NE Inhibitors Identified by High-Throughput Screening
Figure 1. Inhibition of NE activity by a representative compound
identified with HTS. NE was incubated with the indicated concentra-
tions of 7, 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.
Results and Discussion
Primary High-Throughput Screening. 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 â-lactam derivatives, which have been reported
previously as NE inhibitors.2 Further details on the composi-
tion of the parent library are described in our previous
studies.3
Table 2. Effect of N-Benzoylpyrazoles 17-33 on NE Activitya
1,30
K
i
compd
R
1
R
2
R
3
R
4
R
5
R
6
R
7
R
8
(nM)
1,32
1
18
7
CH
3
H
H
H
NO
Cl
Br
Br
H
Cl
H
Br
Cl
Cl
Cl
H
H
H
H
CH
CH
H
CH
H
Cl
CH
H
H
F
OCH
H
H
H
H
CH3
H
H
H
H
F
H
H
H
H
H
3
OCH
3
OCH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH
3
3
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
21
28
46
A compound was defined as a hit if it exhibited >90%
inhibition of NE activity at a final compound concentration of
NO
NO
H
H
H
2
H
H
H
H
H
H
H
H
H
H
H
CH
H
3
19
20
21
2
2
3
H
H
H
H
65
2
0 µg/mL in fluorescence-based microplate assays. From the
3
230
250
300
300
1000
3400
7200
9000
9000
10700
24500
29900
50900
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
22
23
24
25
26
H
3
CH
H
3
NHCOCH
H
H
F
tert-butyl
H
Cl
OCH3
H
3
H
H
H
3
27
28
29
30
CH
CH
3
CH
CH
CH3
H
3
3
3
aggregates. We also determined the dose-response relation-
ship for enzyme inhibition by hits filtered from the primary
screen.3 As an example, a representative curve for compound
3
3
H
H
Br
H
31
32
CH3 Br
H
1,34
H
CH
33
CH
3
3
CH
3
OCH
3
OCH
3
7
is shown in Figure 1. Sixteen compounds with the highest
a
inhibitory activity for NE were selected as a set of prospective
NE inhibitors, and the inhibition constants (Ki) were determined
for these compounds using Dixon plots.35 The structures of these
compounds and their activity are presented in Table 1. Com-
pounds 1-10 were quite potent, all with Ki e 110 nM. This
inhibition is likely not due to quenching of product fluorescence
by the test compounds, as nanomolar concentrations of com-
pounds do not appear to cause this problem.36 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).
See Supporting Information Table S1 for analysis results of compounds
4-59.
3
potent NE inhibitors have been reported to be N-benzoylpyrazole
derivatives. Indeed, our findings suggest the possibility that the
40
anti-inflammatory effects reported for N-benzoylpyrazoles may
be due, in part, to inhibition of NE. On the basis of this
discovery, we selected 43 additional N-benzoylpyrazole deriva-
tives 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 17-24) were
relatively potent (Ki e 300 nM) NE inhibitors (Table 2 and
Supporting Information Table S1). Thus, we selected the 17
most potent N-benzoylpyrazole derivatives (compounds 1-9 and
17-24) for further characterization and SAR analysis. Dixon
plots and double-reciprocal Lineweaver-Burk plots of substrate
An examination of the 16 most potent NE inhibitors showed
that 10 of these compounds (62%) had N-benzoylpyrazole
scaffolds (Table 1). This scaffold has been reported among
3
7
38
39
compounds with diuretic, antidiabetic, antivirus, and anti-
inflammatory4 properties. However, to our knowledge, no other
0

4
930 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Schepetkin et al.
Figure 2. Kinetics of NE inhibition by selected N-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.
hydrolysis by NE in the absence and presence of the selected
compounds showed competitive inhibition (Figure 2 shows
representative plots).
Stability and Kinetic Features of N-Benzoylpyrazole
Derivatives. The set of 17 most potent NE inhibitors, as well
as selected low activity and inactive N-benzoylpyrazoles, were
evaluated for their chemical stability in aqueous buffer. Spon-
taneous 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 3 shows that the absorbance maxima at 266 and 262 nm
decreased over time for compounds 1 and 2, respectively, while
a new peak appeared at 226 nm, which is indicative of benzoic
acid formation by N-benzoylpyrazole hydrolysis. Compound
hydrolysis was verified by following hydrolysis of compound
1
(
1
using reverse-phase high-performance liquid chromatography
RP-HPLC). RP-HPLC traces of the progression of compound
hydrolysis revealed the formation of two major product peaks,
one at 3.7 min and the other at 4.0 min (Figure 4). The peak
eluting at 4.0 min is very close to that of pure benzoic acid (tR
)
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 1 was dissolved
in 85% DMSO-d6/15% D2O, incubated for 0 h (control) or 36
1
h at 37 °C, and analyzed by 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 (p-fluorobenzoic acid and 4-chloropy-
razole), as well as the signals of nonhydrolyzed compound 1
(see Supporting Information Table S4). Thus, these data verify
that we are detecting compound hydrolysis using our spectro-
photometric assay.
Using specific absorbance maxima for the other N-ben-
zoylpyrazoles under investigation (Table 3), we monitored
hydrolysis of the remaining set of selected compounds and used
these data to create semilogarithmic plots for determining rate
constants (k′) of spontaneous hydrolysis (see Supporting Infor-
mation Figure S1 for example plots). As shown in Table 3,
Figure 3. Analysis of changes in compound absorbance resulting from
spontaneous hydrolysis of N-benzoylpyrazole derivatives. The changes
in absorbance spectra of compounds 1 and 2 (20 µM in 0.05 M
phosphate buffer, pH 7.3, 25 °C) were monitored over time in solution.
Representative scans are from three independent experiments.

NoVel Inhibitors of Neutrophil Elastase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4931
Figure 4. Analysis of hydrolysis of N-benzoylpyrazole derivatives by RP-HPLC. Changes in the chromatographic profile of compound 1 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.
a
Table 3. Kinetic Constants for the Spontaneous Hydrolysis of Selected
N-Benzoylpyrazoles
the selected nitropyrazoles were also potent NE inhibitors
Tables 1 and 2). 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
(
compd
λ (nm)b
k′ (min-1 × 103)
t1/2 (h)
1
2
3
4
5
6
7
8
9
266
262
262
265
270
262
270
255
254
265
260
272
270
266
262
264
266
290
262
245
266
265
260
265
266
270
250
258
258
258
260
270
268
256
7.26
40.5
56.6
56.2
291.9
22.4
7.75
10.59
70.4
3.92
2.76
117.0
348.9
21.5
2.76
2.3
1.99
1.15
3.92
9.44
16.12
5.65
2.30
4.38
10.4
4.6
1.6
0.29
0.22
0.21
0.04
0.51
2.1
1.1
0.16
3.1
(spontaneous hydrolysis) or by the Ser195 hydroxyl group in
42-44
the NE catalytic site.
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 7 (a potent NE
inhibitor), compound 28 (a weak NE inhibitor), and inactive
compound 56 all had similar rates of hydrolysis (t1/2 ) 2.1-
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
4
4
5
5
5
5
5
5
3
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
2
3
3
5
6
7
8
9
2
.2 h). In general, the most potent NE inhibitors, excluding the
4.2
0.10
0.033
0.55
4.2
5.1
6.2
10.0
3.0
1.2
0.73
2.2
5.3
2.6
1.1
2.5
5.6
25.1
7.2
12.5
2.2
3.1
2.1
nitropyrazoles (compounds 5, 18, and 19), were more stable
than 6-acylamino-2-[1-(ethylsulfonyl)oxy]-1H-isoindole-1,3-di-
45
30
ones but less stable than â-lactam, 1,2,5-thiadiazolidin-3-
4
6
47
one 1,1-dioxide, and 2-azetidinone inhibitors of NE.
The relatively rapid rate of spontaneous hydrolysis of
N-benzoylpyrazole inhibitors allowed us to evaluate reversibility
of NE inhibition over time. As shown in Figure 5, NE was
rapidly inhibited, with no lag period after addition of compound,
and inhibition was maximal during the first 30 min with com-
pounds 1, 7, and 18 at nanomolar concentrations (panels A, C,
and E). However, inhibition by the most rapidly hydrolyzable
inhibitor (compound 18) 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 5B). 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 5B). In comparison, reversal of inhibition by
compounds 1 and 7 was much slower, and NE was still inhibited
by g0.5 µM compounds 1 and 7 at 2 and 5 h after treatment,
respectively (parts D and F of Figure 5). Thus, these results
suggest that N-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.48 To
support this conclusion, we treated NE with a range of stable
(t1/2 > 2 h) and unstable inhibitors (t1/2 < 1 h) at a relatively
high concentration (25 µM), removed free inhibitor by ultra-
filtration of the enzyme/inhibitor mixture, and then moni-
2.1
0.46
1.6
0.92
6.1
3.7
5.5
1.15
10.0
a
The kinetic constants were obtained at pH 7.3 and 25 °C. b Absorption
maxima that were used for monitoring spontaneous hydrolysis.
compounds 7, 13, 17, 21-25, and 28-30 were the most stable
with t1/2 > 2 h. The presence of a nitro group (compounds 3, 5,
, and 18-20) appears to increase the rate of spontaneous
hydrolysis, with an average t1/2 of ∼11 min for the nitropyra-
zoles. Nitro groups are strong electron-withdrawing moieties
and have previously been reported to increase the activity of
pyrazolooxadiazinone inhibitors of serine proteases.41 Likewise,
9

4
932 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Schepetkin et al.
Figure 5. Evaluation of NE inhibition by representative N-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.
tored recovery of NE activity over time. As shown in Figure 6,
inhibition of NE by the selected inhibitors was fully reversible,
although with differing kinetics between compounds. For
example, inhibition by compound 20 was rapidly reversed after
compound removal (<2 h for full recovery of enzymatic
activity), reversal of inhibition by compound 21 was slower
(
∼16 h), and reversal of compound 24 inhibition was very slow
(>24 h). These data suggest varying degrees of stability of the
enzyme-inhibitor complexes formed with individual com-
pounds.
Specificity of N-Benzoylpyrazoles. To evaluate inhibitor
specificity, we analyzed the effects of the 53 N-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
Figure 6. N-Benzoylpyrazole derivatives are reversible inhibitors of
NE. NE was incubated with 25 µM of the indicated inhibitors or without
inhibitor (control) in buffer A. After 15 min, excess inhibitor was
removed by ultrafiltration. Control samples were treated under the same
conditions. The samples were then resuspended in buffer A, and aliquots
were withdrawn at the indicated times and assayed for enzyme activity.
Representative curves are from two independent experiments.
(
D (EC 3.4.23.5)]. As shown in Table 4 and Supporting
Information Table S2, none of the N-benzoylpyrazole derivatives
inhibited aminopeptidase M and cathepsin D, and most deriva-
tives either did not inhibit kallikrein or inhibited this enzyme

NoVel Inhibitors of Neutrophil Elastase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4933
Table 4. Analysis of N-Benzoylpyrazole Inhibitory Specificitya
IC50 (nM)
amino-
peptidase cathepsin
compd chymotrypsin kallikrein thrombin urokinase
M
D
1
2
3
4
5
6
7
8
9
3
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
40
15
340
120
2700
57
15
125
1200
1800
50
190
7000
240
4700
2700
2300
NI
1400
NI
7300
1500
3500
4700
7600
NI
6600
11900
11300
NI
6900
51
2500
120
170
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
680
120
11900
390
4700
2800
2800
NI
4800
4300
11000
12100
NI
1100
18200
1500
1700
2600
1500
32300
24900
3100
47900
1300
NI
NI
12300
6200
5700
4400
NI
42200
6100
18800
NI
Figure 7. Geometric parameters important for formation of a Michaelis
complex in the NE active site. The important distances (d) and relevant
angle (R), as specified in the text, are indicated. The information is
based on the model of synchronous proton transfer from the oxyanion
hole in NE.5
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
1,58
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
two potent inhibitors (compounds 2 and 9) with R-substituents
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
(R3) in the pyrazole ring. Note, however, that compound 2 also
51200
33600
18400
NI
28300
4700
NI
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 R3 and/or R4/R8. Thus, it appears that an ortho
substituent in the benzene ring and/or R-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.
NI
NI
NI
NI
5600
NI
To investigate binding of N-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
a
NI: no inhibition seen at the highest concentration of compound tested
(
55 µM). See Supporting Information Table S2 for analysis results of
compounds 34-59.
at high micromolar concentrations. A few of the N-benzoylpyra-
zole derivatives inhibited thrombin (compounds 2-4 and 6) or
urokinase (compounds 2 and 3) at nanomolar concentrations,
whereas most of the N-benzoylpyrazole derivatives that were
potent NE inhibitors also inhibited chymotrypsin (Table 4 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 chymot-
rypsin inhibitors.4 While enzyme selectivity between NE and
chymotrypsin was low for our most potent NE inhibitors, the
differential selectivity observed among this set of N-ben-
zoylpyrazole derivatives suggests that differences in ring
substituents (R1-R8) 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 1 and 2), and N-benzoyl-4-
nitropyrazole (compound 5), which has only a nitro group as
substituent R2, was a relatively selective NE inhibitor versus
all other proteases tested, including chymotrypsin. Compound
4
2
ketone inhibitor (see Experimental Section). As reported by
5
1
Vergely et al., 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-CdO
fragment involves interaction between the inhibitor’s carbonyl
9,50
4
6,51,52
group and Ser195
site.
and/or other targets in the active
2
0,50,53,54
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 d1 in Figure 7), while
the angle of Ser195 Oγ‚‚‚CdO, denoted as angle R in Figure
5
1,55
2
4, which is a more stable derivative (t1/2 ) 10 h), was also
7, is limited to 80-120°.
quite selective for NE. On the other hand, compound 54 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 N-benzoylpyrazole-based inhibitors that possess both high
potency and chemical stability.
The most favorable binding modes for five active (compounds
1, 4, 6, 7, and 17) and four inactive (compounds 35, 39, 53,
and 54) 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 8, Supporting Information Figure S2, and
Table 5. The d1 and R values show that in highly active
inhibitors (compounds 1, 4, 6, 7, and 17) the carbonyl carbon
atom is slightly more accessible to the Ser195 hydroxyl oxygen,
compared to inactive benzoylpyrazoles (d1 ) 2.98 ( 0.29 versus
3.41 ( 0.74 Å, respectively) (Table 5). Two of the inactive
compounds (53 and 54) also had relatively short distances d1;
however, the Ser195 Oγ‚‚‚CdO angle was too acute (R ) 51°),
making them unfavorable for nucleophilic attack by Ser195 in
the oxyanion hole (Figure 8D and F). For example, Figure 8D
SAR Analysis and Molecular Modeling. Analysis of the
series of N-benzoylpyrazoles indicated that the presence or
absence of ring substituents significantly affected inhibitor
activity. For example, the presence of methyl groups at both
the R1 and R3 positions decreased inhibitory activity. There were
only two potent (Ki < 200 nM) inhibitors (compounds 9 and
2
0) 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

4
934 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Schepetkin et al.
Figure 8. Molecular docking of active and inactive N-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.
a
Table 5. Results of Docking and Subsequent Molecular Optimization for Favorable Poses in the Binding Site of NE for Selected Active and Inactive
N-Benzoylpyrazoles
distances (Å)b
R (deg)b
optimization
optimization
docking
d1
molecular deformation
c
compd
docking
d1
d2
d3
L ) d2,min + d3
∆E (kcal/mol)
Active
1
4
6
7
102
69
74
96
95
114
84
86
96
86
2.94
3.39
3.05
2.58
2.92
2.45
2.27
2.28
2.58
2.39
2.03/2.08
1.98/2.08
2.27/2.30
1.64/2.62
2.13/2.38
2.99
2.83
2.82
2.99
2.90
5.02
4.81
5.09
4.63
5.03
5.13
1.49
2.32
0
1
7
2.95
Inactive
3
3
5
5
5
9
3
4
121
92
51
99
103
82
4.14
3.94
2.64
2.91
2.32
2.67
2.52
2.40
2.71/3.97
3.42/3.66
4.23/4.96
2.01/2.60
3.54
3.57
4.95
3.28
6.25
6.99
9.18
5.29
2.94
6.60
11.40
37.36
51
80
a
A docking pose was regarded as favorable if it had R and d1 values closest to the optimal ranges and within 2 kcal/mol above the lowest-energy pose.
The angle and distances are as indicated in Figure 7. c Energy of molecular deformation of a selected molecule during optimization.
b
shows that the docked compound 53 is strongly anchored in a
narrow groove of the binding site; however, rotation of the
molecule to a more appropriate R angle is impossible without
significantly increasing d1. In support of our observations that
the presence of methyl groups at R1 and R3 decreases inhibitor
activity, the docked structure for inactive benzoylpyrazole 53
shows that the pyrazole methyl groups collide with the borders
of the binding pocket and hinder penetration of molecule 53
into the oxyanion hole.
While docking studies revealed that active compounds 6 and
7 were almost planar in their favorable binding modes, with
dihedral angles of ∼3° between heterocycle and benzoyl ring,

NoVel Inhibitors of Neutrophil Elastase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4935
the coplanarity of the two rings does not seem to correlate with
high inhibitory activity. For example, the docked geometries
of active inhibitors 1 and 4 are nonplanar, with dihedral angles
of 48° and 51°, respectively. It is interesting to note that the
user-specified, constrained planarity of molecule 35, which
contains a bulky chlorine atom in the ortho position of the
phenyl ring, leads to the appearance of a “good” docking pose
with d1 ) 2.57 Å and R ) 105°. In reality, this molecule cannot
become planar, and its docking without constraints produces
no binding modes suitable for the interaction with Ser195
despite the ligand being in a suitable position for Michaelis
complex formation. In contrast, the active inhibitors (compounds
1, 4, 6, 7, and 17) had shorter proton-transfer distances (L )
4.92 ( 0.2), as well as d1 and R values (Table 5) that favored
interaction of the carbonyl group with Ser195 and enzyme
4
6,51,52
inhibition.
Conclusions
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 N-benzoylpyrazole
scaffold. A number of these compounds were highly active, with
(Figure 8B and Table 5). Thus, the presence of a large ortho
substituent on the phenyl ring blocks rotation of the molecule
into a conformation that can inhibit NE.
K values of 6-300 nM, making them promising candidates
i
for further evaluation and development. These N-benzoylpyra-
As indicated above, distances d1 for NE inhibitors were
zole 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.
generally shorter than those of their inactive analogues (Table
5
). However, d1 was >2.6 Å for some active compounds, which
exceeds the maximum distance specified for Michaelis complex
5
1
formation. 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
Molecular docking of the active N-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 N-benzoylpyrazole
analogues, and we suggest that the N-benzoylpyrazole scaffold
represents a novel direction for developing NE inhibitors that
exhibit high enzyme selectivity and potency.
acting as a general base to enhance nucleophilicity of the Ser195
hydroxyl oxygen and activate the hydrolytic H2O42-44 (illustrated
in Figure 7). 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 (r1-r5) in
Ser195, His57, and Asp102 (Figure 7) were varied during
optimization with the MM+ force field, while concurrently
applying harmonic restraints to d1 and R. 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 (∆E) of a benzoylpyrazole molecule before and after
MM+ optimization within the partially flexible binding site
showed that eight benzoylpyrazole derivatives (compounds 1,
Experimental Section
Compounds and Reagents. 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 N-benzoylpyrazole derivatives
were selected from the Actimol database and purchased from
TimTec. The purity of the most active target compounds was
4
, 6, 7, 17, 35, 39, and 53) bound with suitable d1 and R and
without strong distortion of their optimal molecular geometry,
as indicated by relatively low ∆E values. In addition, optimiza-
tion of binding modes for these compounds did not lead to steric
collisions between nonbonded atoms. Alternatively, a favorable
binding mode for molecule 54 was not attainable because its
penetration to Ser195 under the forces of harmonic restraints
led to dramatic molecular distortion characterized by ∆E )
2
confirmed by dissolving compounds directly in acetonitrile/H 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).
1
Compound identity was verified by H NMR analysis at 200, 300,
3
7.36 kcal/mol. Consequently, the lack of inhibitory activity of
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
compound 54 can be explained by steric hindrances to formation
of a Michaelis complex. The other inactive benzoylpyrazoles
investigated by molecular modeling (compounds 35, 39, and
6
sulfoxide (DMSO-d ) (Supporting Information Table S4; NMR
5
3) required significant rotations r3 and/or r4 of His57 in order
analyses were preformed by TimTec, Inc.). NE, human chymot-
rypsin, 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-d , deuterated water (D O), pyrazole, and benzoic
6 2
acid were purchased from Sigma Chemical Co. (St. Louis, MO).
HPLC grade acetonitrile was from EMD Chemicals (Gibbstown,
to interact with Ser195. As a result of these rotations, the
distances d2 and d3 generally became higher than in the initial
enzyme structure. The sum of the distances L ) d2(min) + d3
can be regarded as the length of synchronous proton transfer
between Ser195 and Asp102 (Table 5). For compound 7,
suitable d1 and R characteristics were achieved after docking,
and no further optimization was necessary. Thus, the L value
of 4.63 Å for compound 7 corresponds to the initial enzyme
architecture. For the inactive benzoylpyrazoles (compounds 35,
2
NJ), and HPLC grade H O and trifluoroacetic acid (TFA) were
from Mallinckrodt Baker Inc. (Phillipsburg, NJ).
Library Screening and Kinetic Measurements. 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.
3
9, and 53), the length of proton transfer increased by 1.6-4.5
Å (L ) 7.06 ( 1.68), which was sufficient to interfere with
general acid-base catalysis and led to loss of inhibitory activity

4
936 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Schepetkin et al.
HTS was performed in black flat-bottom 96-well microtiter plates.
Briefly, a solution containing buffer A (200 mM Tris-HCl, pH 7.5,
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-methylcou-
marin). 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-methyl-
coumarin). 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). Ami-
nopeptidase 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
(L-alanyl-p-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-
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 (N-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.
i
For selected lead compounds, the inhibition constant (K ) values
were determined using Dixon plots of three to four different
concentrations of the substrate.35 At each substrate concentration,
rates were determined with four to five different inhibitor concen-
trations, and the inverse of the velocities was plotted against the
i
final inhibitor concentration. K was determined from the intersec-
2
tion of the plotted lines (R > 0.98 for each line).
Reactivation of the NE-inhibitor complex was investigated as
2
Lys(Dnp)-D-Arg-NH ). Reactions for aminopeptidase M activity
46
reported previously. 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 5000g 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
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 (IC50) 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%.
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.
Analysis of Compound Stability. Spontaneous hydrolysis of
Molecular Modeling. 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 N-benzoylpyrazole derivatives 1, 4,
6, 7, 17, 35, 39, 53, and 54 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 functions57 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
inhibitor42 from the Protein Data Bank (code 1HNE). 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.
selected N-benzoylpyrazoles was evaluated at 25 °C in 0.05 M
phosphate buffer, pH 7.3. Kinetics of N-benzoylpyrazole hydrolysis
was monitored by measuring changes in absorbance spectra over
incubation time using a SpectraMax Plus microplate spectropho-
tometer (Molecular Devices, Sunnyvale, CA). Absorbance (A ) at
t
the characteristic absorption maxima of each N-benzoylpyrazole
was measured at the indicated times until no further absorbance
56
decreases occurred (A
∞
). Using these measurements, we created
) vs time (see Supporting
semilogarithmic plots of log(A
Information Figure S1), and k′ values were determined from the
t
- A
∞
slopes of these plots. Half-conversion times were calculated using
t1/2 ) 0.693/k′.
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
2
5 °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) contain-
ing 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.
To verify compound hydrolysis, compound 1 was dissolved in
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
8
6 2
5% DMSO-d /15% D O (3 mg/mL). The mixture was incubated
1
for 36 h at 37 °C, and the samples were analyzed by H NMR on
a Bruker DRX-500 spectrometer (Bruker BioSpin, Billerica, MA).
and the inhibitor’s carbonyl group, and values of d and R (Figure
1
Control samples were dissolved in mixture of 85% DMSO-d
6
/15%
O and analyzed immediately. H NMR spectra were recorded at
0 °C using 3-(trimethylsilyl)propionic-2,2,3,3-d acid sodium salt
as an internal reference.
7) were determined for each docked compound. These modes were
1
D
2
further optimized by molecular mechanics with the MM+ force
field using HyperChem. During this optimization, we varied torsion
angles r -r in Ser195, His57, and Asp102 and also applied
2
4
1
5
Analysis of Inhibitor Specificity. 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
harmonic restraints to d
were calculated as E ) K
R
where K ) 15 kcal‚mol ‚Å and K ) 3 kcal‚mol ‚deg . These
1
and R. Energy terms for the restraints
2
2
d
d 1 R R
(d - 1.5) and E ) K (R - 100°),
-1
-1
-1
-1
d
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
1
00 µM substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcou-
marin). Thrombin inhibition was evaluated in reaction mixtures
of d
1
2 3
and R were determined, as well as distances d and d (Figure

NoVel Inhibitors of Neutrophil Elastase
). The distance d 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.58 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
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4937
7
2
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275, H385-H392.
Molecular mechanics optimization of a binding mode also caused
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(
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2
- E
1
,
1
(
(
(
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Acknowledgment. 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-
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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.
(
Supporting Information Available: Table S1 on the effect of
N-benzoylpyrazoles 34-59 on NE activity, Table S2 on the analysis
of inhibitory specificity for compounds 34-59; Table S3 on the
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HPLC analysis of selected compounds, Table S4 on the H NMR
analysis of selected compounds, Figure S1 showing examples of
semilogarithmic plots used to determine rate constants for N-
benzoylpyrazole spontaneous hydrolysis, and Figure S2 showing
further examples of molecular docking of N-benzoylpyrazoles into
the active site of NE. This material is available free of charge via
the Internet at http://pubs.acs.org.
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