Inhibition of myeloperoxidase: Evaluation of 2H-indazoles and 1H-indazolones

Article abstract of DOI:10.1016/j.bmc.2014.09.044

Myeloperoxidase (MPO) produces hypohalous acids as a key component of the innate immune response; however, release of these acids extracellularly results in inflammatory cell and tissue damage. The two-step, one-pot Davis-Beirut reaction was used to synth

Full text of DOI:10.1016/j.bmc.2014.09.044

Accepted Manuscript
Inhibition of Myeloperoxidase: Evaluation of 2H-Indazoles and 1H-Indazolones
Aaron Roth, Sean Ott, Kelli M. Farber, Teresa A. Palazzo, Wayne E. Conrad,
Makhluf J. Haddadin, Dean J. Tantillo, Carroll E. Cross, Jason P. Eiserich, Mark
J. Kurth
PII:
S0968-0896(14)00698-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.09.044
BMC 11831
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 July 2014
13 September 2014
20 September 2014
Please cite this article as: Roth, A., Ott, S., Farber, K.M., Palazzo, T.A., Conrad, W.E., Haddadin, M.J., Tantillo,
D.J., Cross, C.E., Eiserich, J.P., Kurth, M.J., Inhibition of Myeloperoxidase: Evaluation of 2H-Indazoles and 1H-
Indazolones, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.09.044
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Leave this area blank for abstract info.
Inhibition of Myeloperoxidase: Evaluation of
2H-Indazoles and 1H-Indazolones
Aaron Rotha, Sean Ottb, Kelli M. Farbera, Teresa A. Palazzoa, Wayne E. Conrada, Makhluf J. Haddadinc, Dean
J. Tantilloa, Carroll E. Crossb, Jason P. Eiserichb, and Mark J. Kurtha
Department of Chemistrya & Department of Internal Medicineb, University of California, Davis
Department of Chemistryc, American University of Beirut, Beirut, Lebanon
N
N
S
O
O

Bioorganic & Medicinal Chemistry
jo u rn al h ome p ag e : w ww .e lse vie r .c om
Inhibition of Myeloperoxidase: Evaluation of 2H-Indazoles and 1H-Indazolones
Aaron Rotha, Sean Ottb, Kelli M. Farbera, Teresa A. Palazzoa, Wayne E. Conrada, Makhluf J. Haddadinc,
Dean J. Tantilloa, Carroll E. Crossb, Jason P. Eiserichb, and Mark J. Kurtha,*
aDepartment of Chemistry, University of California, One Shields Avenue, Davis, California 95616
bDepartment of Internal Medicine, Division of Pulmonary/Critical Care Medicine, 4150 V Street, Suite 3100, Sacramento, CA 95817
cDepartment of Chemistry, American University of Beirut, Beirut, Lebanon
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Myeloperoxidase (MPO) produces hypohalous acids as a key component of the innate immune
response; however, release of these acids extracellularly results in inflammatory cell and tissue
damage. The two-step, one-pot Davis-Beirut reaction was used to synthesize a library of 2H-
indazoles and 1H-indazolones as putative inhibitors of MPO. A structure-activity relationship
study was undertaken wherein compounds were evaluated utilizing taurine-chloramine and
MPO-mediated H2O2 consumption assays. Docking studies as well as toxicophore and Lipinski
analyses were performed. Fourteen compounds were found to be potent inhibitors with IC50
values <1 µM, suggesting these compounds could be considered as potential modulators of pro-
oxidative tissue injury pertubated by the inflammatory MPO:H2O2:HOCl/ HOBr system.
Available online
Keywords:
Myeloperoxidase
Davis-Beirut reaction
2H-indazole
Structure-activity relationship
Computational docking
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Neutrophil-derived MPO, known to represent the strongest
and most abundant oxidative effector system in neutrophils, is
Myeloperoxidase (MPO), primarily found in azurophilic
granules of polymorphonuclear neutrophils (PMNs), is a key
component of the innate immune response.1-5 Specifically, MPO
acts as an important antimicrobial effector through its enzymatic
production of hypohalous acids (e.g. HOCl, HOBr). These acids
promote phagosomal oxidative antimicrobicidal activities, but if
present in CF RT fluids in early stages of the disease8 and in
increasingly large quantities in later stages of the disease.9
Indeed, both circulating and RT MPO levels have been found to
correlate with disease severity in CF.7,10,11 MPO’s extracellular
releases are suspected to play an important role in the
pathophysiology of numerous other inflammatory diseases
including both RT and cardiovascular diseases through its (i)
direct oxidation and (ii) production of hypohalous acids.5,12-16
secreted or released extracellularly, can present
a potent
instigator of cell and tissue damage in the inflammatory milieu.2
Active MPO has been found in or associated with a variety of
disorders including atherosclerosis, Alzheimer’s disease,
neutrophil mediated glomerular injury, and idiopathic pulmonary
fibrosis.2
In CF patients with advanced RT disease, opportunities exist
for the development of small molecules to target the major
molecular components of the neutrophil proteolytic and oxidative
related mediators of RT pathobiology.12,13 One such pathway is
represented by MPO. Despite its high expression in CF RT
disease,9,14 implications that MPO polymorphisms leading to
greater levels of MPO expression are associated with more
severe RT CF diseases,13,17 and recognition as a viable drug
target18 with animal experimental evidence,19 we know of no
studies to date that have directly addressed the effects of
inhibition or absence of MPO in CF RT pathobiology. This is the
case even though several promising recent experimental
approaches have been designed to therapeutically target pro-
oxidative MPO activities.20,21
Mediation of the effects of MPO related oxidative stress in
patients with cystic fibrosis is a particularly pressing challenge.
Cystic fibrosis (CF) is an autosomal recessive disease caused by
mutations in a single gene encoding the glycoprotein CF
transmembrane conductance regulator (CFTR). Loss of CFTR
function in respiratory tract (RT) tissues results in a thickened,
under-hydrated RT mucus secretion. This in turn leads to reduced
mucociliary airway clearance, airway bacterial colonization,
infection, and an overly exuberant dysregulated RT inflammatory
response characterized by an intense airway neutrophil influx.6
Overactive recruited neutrophil effector proteolytic and oxidative
processes are believed to represent the most debilitating feature
of CF leading to progressively more severe RT disease and
eventual RT failure, which characterizes end stage CF.6,7
The structural similarity of 2H-indazoles to previously
reported inhibitors, such as indoles and hydroxamic acids,
inspired our exploration of their inhibitory activity. Furthermore,
we hypothesized that 2H-indazoles and 1H-indazolones would
have greater bioavailability due to the second polar nitrogen in
*Corresponding author. Tel.: 530-554-2145; fax: 530-752-8995.
E-mail address: mjkurth@ucdavis.edu (M. J. Kurth)

the core scaffold. Specifically, we sought to examine chain
length tolerance at the synthetically accessible N1, N2, and C3
positions as wells as the effect of polar, non-polar and aromatic
substituents. Since terminal polar substituents on long alkyl
chains have been shown to be a beneficial structural motif in
previous MPO inhibitors,22 a subset of this initial library
containing this feature was synthesized in hopes of replicating
these previous successes. Herein, we describe the synthesis,
computational analysis, structure-activity relationship (SAR)
insights, and evaluation of 2H-indazoles and 1H-indazolones as
inhibitors of MPO.
Finally, the R1-constrained 2H-indazole analogs
– i.e.,
containing N2C3 fused ring systems – employed in this study
(see 9 ⇒ 7 and 12 ⇒ 11; Scheme 2) were obtained by
intramolecular nucleophilic attack onto the corresponding DBR
nitroso-imine intermediate (cf., 8 → 9). Conventionally, hydroxyl
has been the internal nucleophile utilized in R1-constrained
DBRs, which provides access to oxazolino- (9; n = 1) and
oxazino-2H-indazoles (9; n = 2; Scheme 2). However, recent
advances have expanded the scope of the R1-constrained DBR to
include thiol as the internal nucleophile.30 In this modification, N-
(2-nitrobenzyl)-2-(trityl-thio)alkylamine 10, formed from 2 via
Method B, is trityl deprotected with TFA to give thiol 11, which
is then subjected to R1-constrained DBR conditions to deliver the
thiazolino- (12a, b; n = 1) and thiazino-2H-indazoles (12b, c; n =
2) in 66 – 83% yield.
2. Results and Discussion
2.1 Chemistry
The 3-alkoxy-2H-indazoles employed in this study were
prepared by the versatile Davis-Beirut reaction (DBR)23-29 on 2-
nitrobenzylamine 3, in turn prepared by one of two complimen-
tary methods: (i) commercially available 2-nitrobenzyl bromide
(1) was used to N-alkylate the requisite amine to form
intermediate 3 (Scheme 1/Method A) or (ii) 3 was prepared by
reductive amination of the requisite 2-nitrobenzaldehyde
(Scheme 1/Method B). Regardless of the route to 3, this 2-
nitrobenzylamine intermediate need not be isolated. Rather, the
DBR is initiated by direct addition of alcoholic potassium
hydroxide to the Method A or Method B reaction mixture
containing 3, which leads to the targeted 3-alkoxy-2H-indazole 4
in good to excellent overall yield (49–87% for this two-step, one-
pot process).
NO2
Method A
(a)
NO2
O
Br
1
H
N
O
n
OH
R1 R2
7
NO2
O
(b)
O
O
(c)
Method B
O
N
H
2
O
O
(b)
Method B
N
n
OH
R1 R2
8
Likewise, the 1H-indazolones (5 and 6; Scheme 1) employed
in this study were prepared through one of two complimentary
methods from the DBR-derived 3-alkoxy-2H-indazole 4. In the
first method (C), 3-alkoxy-2H-indazole 4 was heated to 100 ºC in
0.3 M methanolic H2SO4 to affect a proto-dealkylation (e.g., loss
of R2) reaction to deliver the targeted N1-unsubstituted 1H-
indazolone 5 (52 – 94% yield). In the second method (D), N1-
substituted 1H-indazolone 6 was obtained by an electrophile-
mediated domino N1-alkylation/O-dealkylation process, which
provides the targeted heterocycle in excellent yield (70-95%).
NO2
O
O
O
N
R1
R2
Ph
Ph
H
N
N
O
O
S
Ph
n
n
10
9a-e
(d)
NO2
O
O
N
O
O
N
S
H
N
(c)
SH
n
n
12a-d
11
Scheme 2. Reagents and conditions: (a) H2N(CH2)nOH, DIPEA, iPrOH; (b)
H2N(CH2)nOH (→ 9) or H2N(CH2)nSTr (→ 10), DIPEA, iPrOH; then
NaCNBH3; (c) KOH in 10% H2O (v/v), 65oC; (d) TFA, TES, DCM, 1 h, r.t.
NO2
Method A
(a)
1
NO2
Br
O
O
2.2 Molecular Docking
H
N
R1
Computational docking, performed using FRED (OpenEye
software suite),31-33 was utilized to help characterize interactions
within the active site. Docking study validation was accomplish-
3
NO2
O
O
O
Method B
(b)
(c)
N1
N2
2
H
O
O
N
N
R1
C3
R2
Method C
O
4a-q
H
N
(d)
R1
Method D
(e)
N
R3
N
O
5a-f
N
R1
O
6a-f
Scheme 1. Reagents and conditions: (a) H2NR1, DIPEA, R2OH (solvent); (b)
H2NR1, AcOH, R2OH (solvent) then NaCNBH3 ; (c) KOH in 10% H2O (v/v),
65oC; (d) 0.3 M methanolic H2SO4/sealed vial, 100 °C; (e) R3X, CH3CN,
reflux.
Figure 1. Structural layover of naturally bound HX1 (green) and
docked HX1 (grey) indicating an effective docking model
(RMSD = 1.2 Å).

ed through comparative docking of the 5-fluoroindole scaffold
described by Soubhye,22 and HX120 bound in the active site of
MPO [Figure 1; (PDB ID 4C1M), RMSD docked vs. natural is
1.2 Å]. Also included in the docking studies was 4-aminobenzoic
acid hydrazide, the internal control for the assays discussed
herein, which consistently displayed an IC50 of 81 +/- 19 nM.
acid production. Select IC50 values were confirmed utilizing a
H2O2-specific electrode allowing for real time hydrogen peroxide
concentration measurements (Supporting Information). Upon
generation of an initial library of 2H-indazoles (Table 1), a
consistent trend was found wherein, as the length of the chain
increased, there was a dramatic decrease in inhibitory potency.
For example, increasing the length of the chain from four (4f) to
five (4g) carbons resulted in a three-fold decrease in potency.
Compound 4l was included to ensure that this decrease in
efficacy was not a result of reduced solubility. The compounds
synthesized containing terminal polar substituents (4j – 4l, Table
1) failed to elicit a significant increase in inhibitory activity. It is
possible that these modifications were not effective due to a
change in orientation of the inhibitor with respect to the indole
scaffold or because the alcohol moiety failed to carry a positive
charge necessary to lower binding energy. A series of N2-aryl
analogues (4m – 4q) was included to explore the effects of more
rigid, hydrophobic substituents that likely extend into the pocket
of the MPO active site, but these compounds failed to produce an
appreciable increase in inhibitor activity.
Lastly, by lengthening the alkoxy substituent at C3, we also
sought to probe the relationship between substituents on the 3-
position of both scaffolds [e.g.; 4a -OMe → 4b -OEt → 4c –O-
(CH2)2OMe]. With this SAR study, we noted decreased inhibition
of MPO as the length of the alkoxy chain was extended past two
carbons. This trend is likely the result of increased inhibitor size
within the relatively small distal portion of the active site.
Figure 2. Compound 4a docked into the active site of MPO (crystal structure
obtained from PDB ID 4C1M).
These docking studies indicated that the 2H-indazoles, like
previously reported inhibitors, likely sit in a stacked fashion over
the heme lending credence to our inhibitor design approach. For
example, compound 4a (Figure 2) is positioned in such a way
that the alkyl chain on the N2-position extends out toward the
hydrophobic pocket, while the aromatic core scaffold stacks over
the pyrrole rings of the heme porphyrin.
As before, docking studies of the synthesized inhibitors
indicate that the aromatic portion of the inhibitors is positioned in
a π-stacking fashion over pyrrole ring D of the heme, while the
hydrophobic portion extends into the cavernous hydrophobic
pocket. If hydrogen bond donors are present, they are generally
oriented toward the carboxylate groups of the heme. Because the
same orientation was maintained, it followed that increasing
steric bulk at the C3-position would decrease inhibitor activity
due to the constraints of the active site. Interestingly, docking
2.3 Structure-Activity relationship
4-Aminobenzoic acid hydrazide was used as an internal
control while MPO inhibitory activity was assessed using a
taurine-chloramine assay to measure enzymatic hypochlorous
IC50 (nM)
IC50 (nM)
IC50 (nM)
Compound
Compound
4g
Compound
4m
N
N
N
N
N
N
4a
704 +/- 243
3876 +/- 2550
2029 +/- 1542
3
OMe
OMe
OMe
N
Cl
N
N
N
N
N
4b
4c
722 +/- 123
4h
4i
>10,000
>10,000
4n
4o
4771 +/- 2811
2000 +/- 615
6
OMe
N
OMe
OEt
N
N
N
N
N
I
1404 +/- 105
6
OEt
O(CH2)2OMe
OMe
I
N
N
O
O
N
N
N
N
711 +/- 300
910 +/- 530
1787 +/- 1221
1539 +/- 476
1660 +/- 1391
1102 +/- 318
4d
4e
4j
4p
4q
OH
3
OMe
OMe
OMe
N
N
N
N
N
N
4k
OH
4
2
OMe
OMe
OMe
N
N
N
N
807 +/- 402
1078 +/- 227
4f
4l
O
2
OEt
OMe
Table 1. IC50 values of 3-alkoxy-2H-indazoles assessed with a Taurine chlorination assay (154 mM NaCl, 30 µM H2O2, and 2 nM MPO). All IC50 values are
expressed in nM as a mean of ≥ 3 trials +/- standard deviation.

IC50 (nM)
IC50 (nM)
>10,000
Compound
Compound
Compound
IC50 (nM)
H
H
O
N
N
660 +/- 222
N
N
5a
5b
5f
2121 +/- 1474
N
O
N
6d
O
O
O
O
H
N
O
O
S
O
O
792 +/- 380
>10,000
O
N
O
N
S
O
N
6a
>10,000
>10,000
>10,000
Cl
N
O
>10,000
6e
N
H
N
O
5c
5d
5e
O
N
O
N
6
O
N
6b
6c
N
O
H
N
N
O
N
N
2469 +/- 1251
1948 +/- 915
N
6f
>10,000
O
OH
OH
Br
N
O
O
O
H
N
N
N
2
O
O
Table 2: IC50 values of 1H-indazolones assessed with a Taurine chlorination assay (154 mM NaCl, 30 µM H2O2, and 2 nM MPO). All IC50 values are expressed
in nM as a mean of ≥ 3 trials +/- standard deviation.
N1-unsubstituted 1H-indazolones. We hypothesize that
studies indicated that incorporation of the benzodioxole ring (4a
substituents at the N1-position results in the loss of a crucial
→ 4d) changes the inhibitor orientation in the active site. Here,
interaction with either the carboxylate groups of the heme or the
glutamate anion within the active site.35 These observations are
consistent with the findings of Sliskovic35 wherein N-alkylation
of the indole scaffold resulted in reduced inhibitor potency.
Based on these SAR results, it is clear that either the accessible
lone pair or nucleophilic nitrogen is essential for inhibitory
activity.35
rather than the aromatic core of the inhibitor occupying the space
directly above the iron, the inhibitor is shifted such that the
benzodioxole ring is positioned above the heme iron center.
When studying the 5-fluoroindole scaffold, Soubhye22 found
that compounds with longer alkyl chains in the 3-position
displayed better inhibitory efficacy than their “less greasy”
counterparts. In the case of these indazolo analogues, it is
interesting to note that increasing the length of the alkyl chain of
N2 or C3 was only beneficial to a certain point. For example, a
branched alkyl chain (isopropyl; 4a) was well tolerated, while an
n-pentyl (4g) or n-octyl chain (4h) decreased inhibitory activity.
It is important to note that increasing the length of an alkyl chain
can cause issues with solubility or with the molecule’s ability to
enter the active site, even if it may be beneficial within the active
site.
Figure 3. Compound 4d in the MPO active site. The benzodioxole moitey
now resides over the heme center while the tethered ring extends into the
hydrophobic pocket.
We next set out to explore the use of constrained analogues of
the 2H-indazole scaffold – i.e., containing N2C3 fused ring
systems – in hopes that this would facilitate passage through the
narrow pore to the exposed heme. A series of both substituted
and unsubstituted oxazolino- and oxazinoindazoles were
synthesized. Also, taking advantage of newly developed
methodology to access substituted and unsubstituted thiazolino-
and thiazino-2H-indazoles,30 we were able to explore variation of
the heteroatom substituent at the C3-position. Indeed, it was
found that both ring size and the nature of the heteroatom are
determining factors in inhibitory potency. Constraining the N2-
position resulted in significantly increased inhibitory activity by
possibly facilitating entrance into the active site (Table 3).
Furthermore, by changing from an oxygen substituent (9c) to a
sulfur substituent (12c) in the tethering ring, a 40% improvement
in potency was observed. Additional improvement (56%) was
observed when the tethering ring size was reduced from six-
(12c) to five-membered [12a; note that the O-analog of 12a (i.e.,
“S” replaced with “O”), unlike substituted versions 9a and 9b, is
Owing to the presence of a basic nitrogen in the core scaffold
and mildly acidic assay conditions, a portion of the inhibitor is
expected to exist in the protonated form. Therefore, a protonated
species of each molecule was docked for comparison. It was
found that, in some cases, protonation created the opportunity for
additional hydrogen bond and charge-charge interactions with the
carboxylate groups of the heme. It should be noted that, in this
instance, a direct correlation between IC50 values and docking
scores could not generally be obtained through the use of
conventional docking programs.34
Interestingly, conversion of 3-alkoxy-2H-indazoles to the
corresponding N1-substituted 1H-indazolones resulted in
significantly decreased inhibitory activity while the unsubstituted
1H-indazolones retained inhibitory potency (Table 2). Docking
studies indicate that if N1-substituted 1H-indazolones were to
enter the active site, they would likely adopt a pose similar to the

IC50 (nM)
IC50 (nM)
IC50 (nM)
87 +/- 7
Compound
Compound
Compound
O
O
N
N
N
N
900 +/- 466
9a
9d
12b
4148 +/- 128
N
S
N
N
O
N
O
O
O
O
N
N
12c
12d
9e
403 +/- 128
94 +/- 18
213 +/- 22
140 +/- 33
N
N
1028 +/- 427
359 +/- 89
9b
9c
O
S
N
N
O
N
N
N
S
N
12a
O
O
S
Table 3. IC50 values of constrained 2H-indazole analogues assessed with a Taurine chlorination assay (154 mM NaCl, 30 µM H2O2, and 2 nM MPO). All IC50
vales are expressed in nM as a mean of ≥ 3 trials +/- standard deviation.
unstable23]. Seeking to build upon these successes, we set out to
µM, those containing less than two HBD and HBA’s scored
incorporate the benzodioxole moiety into the S-constrained
scaffold (i.e., 12a → 12b), which docking studies suggest causes
a change in the orientation of the inhibitor in the active site (see
Supporting Information/12b/pg S25). To our delight, incorpora-
tion of the 1,3-benzodioxole ring provided markedly improved
inhibitory activity (see 12b and 12d; Table 3) and we speculate
that addition of the benzodioxole ring reduces the likelihood of
better with our docking methods. It is interesting to note that
even with relatively few HBD and HBA’s, active 2H-indazoles
and 1H-indazolones still maintain satisfactory calculated LogP
vales. For example, compound 12b was calculated to have a
LogP of 1.72, while the protonated form of 4e, containing a
comparatively greasy butyl substituent, has a calculated LogP of
3.94. In addition, Veber and Kopple found an increased
probability of satisfactory oral bioavalibility through passive
diffusion existed if compounds contained a PSA of less than 140
Å2 and fewer than ten rotatable bonds.37 Additionally, Hou and
Xu found that reduced polar surface area and molecular weight
below 350 Daltons facilitates passage through the blood brain
barrier.38 Efficient passage through this barrier could prove
essential in the treatment of diseases such as multiple sclerosis,
Alzheimer’s disease, and Parkinson’s disease through inhibition
of MPO.2,39 Furthermore, it was found that all 2H-indazoles and
1H-indazolones were unlikely to act as aggregators based on a
structure based screen. This analysis underwrites the further
consideration of the potential efficacy of these compounds as
drugs. A complete tabulation of this analysis is available in the
Supporting Information.
electrophilic attack and/or MPO compound
I mediated
oxidation.36 To confirm that the observed active inhibitors 12a-d
are the unoxidized thiazino- or thiazolino-systems, rather than the
corresponding sulfoxides and/or sulfones, inhibitors 12a-d were
treated with excess NaOCl and examined by liquid
chromatography/mass spectrometry (LC/MS) at 1 h and 24 h.
Only trace sulfoxide formation was detected at 24 h (detectable
by MS, but not diode array).
12b
12a
12d
12c
9c
0
0
0
0
0
0
1
0
0
0
1
0
0
0
3
1
3
1
2
4
2
2
4
2
2
2
2
2
1.72
1.99
2.21
2.48
1.75
1.48
2.03
2.76
2.49
3.16
2.48
3.61
1.79
3.21
36.28
17.82
36.28
17.82
27.05
45.51
37.79
27.05
45.51
27.05
37.79
27.05
27.05
27.05
220.3
176.2
234.3
190.3
174.2
218.2
176.2
190.2
234.3
204.3
190.2
218.3
174.2
204.3
-44.74
-46.65
-41.23
-43.61
-38.09
-35.45
-39.94
-47.20
-63.55
-50.03
-48.13
-53.95
-39.72
-50.64
87
94
3. Conclusion
140
213
359
403
660
704
711
722
792
807
900
910
A
series of 2H-indazoles and 1H-indazolones were
synthesized utilizing the Davis-Beirut reaction, and evaluated as
potential MPO inhibitors. Fourteen of these compounds
displayed potent IC50 values (nanomolar range) as assessed
utilizing taurine-chloramine inhibition and H2O2 consumption
assays. Compounds containing N1-substituents were not effective
inhibitors, while compounds with relatively small substituents at
N2 and C3 were most active. The most potent compounds in the
series were constrained 2H-indazole analogues – i.e., containing
N2C3 fused ring systems – with the most potent also
incorporating a 1,3-benzodioxole ring. The present data suggests
that, like the 2-thioxanthines,21 the recently described modified
9e
5a
4a
4d
4b
5b
4f
9a
40
hydroxamates,20 the tetramethyl and tetraethyl nitroxides and
4e
targeted MPO-modulating oligopeptide,41 2H-indazoles and 1H-
indazolones should be considered for possible therapeutic
inhibition of unwarranted MPO activity in inflammatory diseases
characterized by episodes of heightened neutrophil activation and
convincing evidences of MPO-related pro-oxidative tissue injury.
Such investigations will need to take into consideration
toxicology and pharmacokinetic activities in complex biological
fluids such as plasma, joint fluids, respiratory tract mucus
secretions, and in complex inflammatory milieu.
Table 4. Drug like properties of select compounds (mean IC50 values are
shown); see Supporting Information for a complete tabulation of results.
Further computational analysis was undertaken to examine the
drug-like properties for both classes of inhibitors using OpenEye
software. As detailed in Table 4, the parameters examined
included the presence of hydrogen bond donors (HBD) and
hydrogen bond acceptors (HBA), LogP, 2D Polar Surface Area
(PSA), and the potential for aggregation. It was found that, of the
fourteen compounds which demonstrate potencies less than 1

4. Experimental
Da, 20 V cone voltage, and Xterra MS C18 column (2.1 mm x 50
mm x 3.5 µm).
4.1 Docking protocols
4.4.1 General procedure for synthesis of 3-
alkoxy-2H-indazoles – Method A (4a-4l)
All docking was performed using the OpenEye Suite of
programs.31-33 A receptor to model the MPO active site was set
up using the Protein Data Bank (PDB) file 4C1M20 utilizing the
FRED receptor program. The series of molecules studied herein
were built using Gaussview 5.0.9 associated with Gaussian0942
and saved as a library (.oeb.gz). The 5-fluoroindole with
inhibitory activity previously published by Soubhye22 was
included in this library to ensure that our docking methods were
sound. All small molecules in the library were subject to a
conformational search using Omega2. The conformers of each
small molecule were then docked into the active site using
FRED. Chemgauss3 scoring was employed.
2-Nitrobenzyl bromide (15.31 mmol) was dissolved in
alcoholic solvent (typically MeOH or EtOH, 115 mL) and added
dropwise to a stirring solution of requisite primary amine (61.2
mmol), DIPEA (61.2 mmol), and 38 mL of alcoholic solvent
over 6 h and then left stirring overnight. Water (15 mL) and
KOH (76.6 mmol) were then added and this mixture was heated
o
at 60 C for 24 h and monitored by thin layer chromatography.
When the starting material had been consumed, 50 mL of water
was added and the alcoholic solvent was removed under reduced
pressure. This aqueous mixture was then extracted twice with
dichloromethane. The combined organic extracts were washed
with water and brine, dried over sodium sulfate, and
concentrated. The crude mixture was purified by flash
chromatography to afford the title compound.23
4.2 Taurine chlorination assay
Taurine chloramine was measured using a modified method
from Dypbukt et al.43 In a volume of 125 µL, the final reaction
mixture contained 6.8 mM taurine, 2nM MPO, 105 mM NaCl,
and various inhibitor concentrations in 20 mM phosphate buffer,
pH 6.5. The reaction was initiated with 40 µM H2O2, and after 5
min. was terminated with the addition of 4 ug catalase. To
determine the amount of taurine chloramine produced, an equal
volume of color developing solution containing 500 uM 3,3′,5,5′-
Tetramethylbenzidine and 100 µM sodium iodide in 300 mM
4.4.2 General procedure for preparation of 3-
alkoxy-2H-Indazoles – Method B (4m-4p)
2-Nitrobenzaldehyde (5.22 mmol) and the requisite amine
(6.26 mmol) were dissolved in alcoholic solvent (typically
MeOH or EtOH, 13 mL) in a 250 mL round bottom flask. Glacial
acetic acid (26.1 mmol) and sodium cyanoborohydride (6.26
mmol) were added and this solution was stirred at room
temperature for 2.5 h. Additional alcoholic solvent (37 mL) and a
solution of KOH (78.3 mmol) in water (6 mL) were added, a
water-cooled reflux condenser was attached and this solution was
heated at reflux for 2 h. The reaction mixture was then cooled to
room temperature, water (20 mL) was added, and the alcoholic
solvent was removed by rotary evaporation. This aqueous
mixture was then neutralized with 1N HCl and extracted with
ethyl acetate (3 X 30 mL). The combined organic extracts were
then washed with 1N HCl (40 mL), saturated sodium carbonate
(40 mL), water (40 mL), and brine (40 mL), dried over
anhydrous sodium sulfate, and concentrated and purified by flash
chromatography to afford the title compound.24
acetate buffer, pH 5.4. Absorbance was measured in
a
spectrophotometric microplate reader (PowerWave; Biotek
Instruments Inc.) at a wavelength of 650 nm.
4.3 H2O2 mediated MPO inhibition assay
H2O2 was measured using a H2O2-specific electrode (World
Precision Instruments, Sarasota, FL). The electrode was
submerged in a continuously stirred reaction vessel containing 30
µM H2O2, 154 mM NaCl, and various inhibitor concentrations in
10 mM phosphate buffer, pH 7.4. The reaction was initiated with
the addition of 10 nM MPO. Data was collected using a free
radical analyzer (World Precision Instruments, Sarasota, FL) and
a PowerLab data acquisition system (ADInstruments, Colorado
Springs, CO).
4.4.3 Synthesis of 2-(4-ethynylphenyl)-3-meth-
oxy-2H-indazole (4q)
4.4 Chemistry
Compound 4o (10.05 mmol) was added to a flame dried 250
mL round bottomed flask and dissolved in dry Tetrahydrofuran
(16 mL) under Nitrogen gas. PdCl2(PPh3)2 (0.50 mmol), CuI
(1.00 mmol), and triethylamine (16 mL) were added and the
All solvents and reagents were purchased from commercial
suppliers and used without further purification. For reactions run
in sealed microwave vials, oven-dried Biotage® 5-10 mL or 10-
20 mL vials containing a Teflon-coated stirrer bar and sealed
with a Teflon-lined septum were used. Analytical thin layer
chromatography was carried out on pre-coated plates (Silica gel
60 F254, 250 µm thickness) and visualized with UV light. Flash
chromatography was performed with 60 Å, 35-70 µM silica gel
(Acros Organics). Concentration refers to rotary evaporation
under reduced pressure.
1H NMR spectra were recorded on Varian spectrometers
operating at 600 MHz at ambient temperature with CDCl3 or
DMSO-d6 as solvent. 13C NMR spectra were recorded on Varian
spectrometers operating at 150 MHz at ambient temperature with
CDCl3 or DMSO- d6 as solvent. Data for 1H NMR are recorded as
follows: chemical shift (δ, ppm), multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; br,
broad; app, apparent), integration, coupling constant (Hz).
Chemical shifts are reported in parts per million relative to
CDCl3 (1H, δ 7.26, 13C, δ 77.16), DMSO (1H, δ 2.50, 13C, δ 39.5)
or TMS (1H, δ 0.00, 13C, δ 0.00). Infrared spectra were recorded
on an ATI-FTIR spectrometer. The specifications of the LC/MS
are as follows: electrospray (+) ionization, mass range 150-1500
o
reaction mixture cooled to 0 C. Trimethylsilylacetylene (15.10
mmol) was added dropwise, then the reaction allowed to stir at
room temperature. Formation of the protected silyl intermediate
was complete after 1 h and 20 min. as shown by TLC.
Approximately 5 g of sodium carbonate was added to the
reaction and allowed to stir for 12 h to effect removal of silyl
protecting group. Water (10 mL) was added to the reaction and
the reaction mixture was extracted with ethyl acetate (3 x 30
mL). The organic extracts were combined, washed with brine,
dried over anhydrous sodium sulfate, and purified by flash
chromatography (10% ethyl acetate/hexanes) to afford 4q (1.952
g, 78% yield) as an off-white solid.
4.4.4 General procedures for the synthesis of
substituted [1,3]oxazino[3,2-b]indazoles and 2, 3-
dihydrooxazolo[3,2-b]indazoles (9a-9d)
2-Nitrobenzyl bromide (15.31 mmol) was dissolved in THF
(115 mL) and added dropwise to a stirring solution of requisite
amino alcohol (61.2 mmol), DIPEA (61.2 mmol), and 38 mL of

isopropanol over 6 h and then left stirring overnight. Water (15
mL) and KOH (76.6 mmol) were then added and this mixture
was heated at 60 oC for 24 h and monitored by thin layer
chromatography. When the starting material had been consumed,
50 mL of water was added and the alcoholic solvent was
removed under reduced pressure. This aqueous mixture was then
extracted twice with dichloromethane. The combined organic
extracts were washed with water and brine, dried over sodium
sulfate, and concentrated. The crude mixture was purified by
flash chromatography to afford the title compound. 25-27
Institutes of Health (Grants HL092506, DK72517 and
GM089153) for generous financial support of this work and
gratefully acknowledge OpenEye for providing software. A.R.
and S.O. contributed equally to this work.
Supplementary data
Supplementary data, including docking images, 1H and 13C
NMR spectra, and full experimental protocols can be found, in
the online version, at …
References
4.4.5 General Procedure for conversion of 2H-
indazoles into 1H-indazalones (5a-5f)
1. Arnhold, J.; Flemmig, J. Arch. Biochem. Biophys. 2010, 500, 92.
2. Klebanoff, S. J. J. Leukoc. Biol. 2005, 77, 598.
3. Metzler, K. D.; Fuchs, T. A.; Nauseef, W. M.; Reumaux, D.; Roesler, J.;
Schulze, I.; Wahn, V.; Papayannopoulos, V.; Zychlinsky, A. Blood 2011,
117, 953.
4. Prokopowicz, Z.; Marcinkiewicz, J.; Katz, D. R.; Chain, B. M. Arch.
Immunol. Ther. Exp. (Warsz) 2012, 60, 43.
5. van der Veen, B. S.; de Winther, M. P. J.; Heeringa, P. Antioxid. Redox.
Sign. 2009, 11, 2899.
6. Rowe, S. M.; Miller, S.; Sorscher, E. J. New. Engl. J. Med. 2005, 352,
1992.
7. Garner, H. P.; Phillips, J. R.; Herron, J. G.; Severson, S. J.; Milla, C. E.;
Regelmann, W. E. J. Lab. Clin. Med. 2004, 144, 127.
8. Thompson, E.; Brennan, S.; Senthilmohan, R.; Gangell, C. L.; Chapman,
A. L.; Sly, P. D.; Kettle, A. J. Free Radical Bio. Med. 2010, 49, 1354.
9. Van Der Vliet, A.; Nguyen, M. N.; Shigenaga, M. K.; Eiserich, J. P.;
Marelich, G. P.; Cross, C. E. Am. J. Physiol. – Lung C. 2000, 279, 537.
10. Regelmann, W. E.; Siefferman, C. M.; Herron, J. G.; Elliott, G. R.;
Clawson, C. C.; Gray, B. H. Pediatr. Pulm. 1996, 19, 1.
11. Van Der Vliet, A.; Eiserich, J. P.; Marelich, G. P.; Halliwell, B.; Cross,
C. E. Advances in Pharmacology 1997, 38, 491.
12. Lucas, S.; Costa, E.; Guedes, R. C.; Moreira, R. Med. Res. Rev. 2013, 33,
E73.
13. Cantin, A. M.; White, T. B.; Cross, C. E.; Forman, H. J.; Sokol, R. J.;
Borowitz, D. Free Radical Biol. Med. 2007, 42, 15.
14. Kettle, A. J.; Chan, T.; Osberg, I.; Senthilmohan, R.; Chapman, A. L.;
Mocatta, T. J.; Wagener, J. S. Am. J. Respir. Crit. Care Med. 2004, 170,
1317.
15. Gorudko, I. V.; Grigorieva, D. V.; Shamova, E.V.; Kostevich, V. A.;
Sokolov, A. V.; Mikhalchik, E. V.; Cherenkevich, S. N.; Arnhold, J.;
Panasenko, O. M. Free Radical Biol. Med. 2014, 68, 326.
16. Huang, Y.; DiDonato, J. A.; Levison, B. S.; Schmitt, D.; Li, L.; Wu, Y.;
Buffa, J,; Kim, T.; Gerstenecker, G. S.; Gu, X.; Kadiyala, C. S.; Wang,
Z.; Culley, M. K.; Hazen, J. E.; DiDonato, A. J,; Fu, X.; Berisha, S. Z.;
Peng, D.; Nguyen, T. T.; Liang, S.; Chuang, C.-C.; Cho, L.; Plow, E. F.;
Fox, P. L.; Gogonea, V.; Tang, W. H. W.; Parks, J. S.; Fisher, E. A.;
Smith, J. D.; Hazen, S. L. Nat. Med. 2013, 20, 193.
The precursor 2H-indazole (0.813 mmol) was dissolved in
MeOH (4 mL) in a sealed microwave vial. H2SO4 (4.07 mmol)
was added and heated at 100 oC overnight. This solution was then
allowed to room temperature and neutralized with a saturated
solution of NaHCO3. This aqueous solution was extracted with
EtOAc (3 X 25 mL) and the combined organic extracts were
dried over anhydrous sodium sulfate and concentrated. This
crude material was then purified by flash chromatography to
afford the title compound.28
4.4.6 General procedure for the synthesis of
N1, N2-disubstituted-1H-indazolones (6a-6f)
The requisite indazole (205 mg, 1.08 mmol) was added to a
two-neck round bottom flask fitted with a water-cooled reflux
condenser and an N2 balloon, then dissolved in dry acetonitrile
(10 mL). R2X (334 µL, 4.32 mmol) was added and the solution
was heated at reflux until TLC analysis showed consumption of
the indazole. The acetonitrile was then removed under reduced
pressure and the crude material was dissolved in ethyl acetate (30
mL). This solution was then poured into saturated sodium
carbonate (20 mL) and the layers were separated. The organic
extracts were then washed with water, brine, dried over sodium
sulfate, and concentrated. This crude material was purified by
flash chromatography to afford the title compound.29
4.4.7 General procedure for the synthesis of
thiazolo- and thiazolino-2H-indazoles (12a-12d)
2-Nitrobenzaldehyde or 6-nitropiperonal (1.1 eq) and requisite
S-trityl protected 1o-aminothioalkane (1 eq) were dissolved in
MeOH (0.4 M) in a 50 mL round-bottomed flask. The solution
was stirred until TLC showed consumption of the starting 1o-
aminothioalkane. Sodium cyanoborohydride (2 eq) was added
portionwise and this solution was stirred until TLC showed
consumption of imine. The solution was concentrated and the
resulting residue was redissolved in methylene chloride. The
organic layer was washed twice with 1M HCl, dried over sodium
sulfate and concentrated to ~15 mL. To this solution was added
an equal volume of TFA, followed by triethylsilane (3 eq). The
solution was stirred for 30 min. then concentrated. KOH (15 eq),
MeOH (0.1 M) and 10% H2O were added to the flask and the
17. Reynolds, W. F.; Rhees, J.; Maciejewski, D.; Paladino, T.; Sieburg, H.;
Maki, R. A.; Masliah, E. Exp. Neurol. 1999, 155, 31.
18. Malle, E.; Furtmuller, P. G.; Sattler, W.; Obinger, C. Br. J. Pharmacol.
2007, 152, 838.
19. Churg, A.; Marshall, C. V.; Sin, D. D.; Bolton, S.; Zhou, S.; Thain, K.;
Cadogan, E. B.; Maltby, J.; Soars, M. G.; Mallinder, P. R.; Wright, J. L.
Am. J. Respir. Crit. Care Med. 2012, 185, 34.
20. Forbes, L. V.; Sjogren, T.; Auchere, F.; Jenkins, D. W.; Thong, B.;
Laughton, D.; Hemsley, P.; Pairaudeau. G.; Turner, R.; Eriksson, H.;
Unitt, J. F.; Kettle, A. J. J. Biol. Chem. 2013, 51, 36636.
21. Tidén, A. K.; Sjögren T.; Svensson, M.; Bernlind, A.; Senthilmohan, R.;
Auchère, F.; Norman, H.; Markgren, P. O.; Gustavsson, S.; Schmidt, S.;
Lundquist, S.; Forbes, L. V.; Magon, N. J.; Paton, L. N.; Jameson, G. N.;
Eriksson, H.; Kettle, A. J. J. Biol. Chem., 2011, 43, 37578.
22. Soubhye, J.; Prevost, M.; Van Antwerpen, P.; Zouaoui Boudjeltia, K.;
Rousseau, A.; Furtmuller, P. G.; Obinger, C.; Vanhaeverbeek, M.;
Ducobu, J.; Neve, J.; Gelbcke, M.; Dufrasne, F. O. J. Med. Chem. 2010,
53, 8747.
23. Mills, A. D.; Nazer, M. Z.; Haddadin, M. J.; Kurth, M. J., N,N-Bond-
Forming Heterocyclization:ꢀ J. Org. Chem. 2006, 71, 2687.
24. Oakdale, J. S.; Solano, D. M.; Fettinger, J. C.; Haddadin, M. J.; Kurth, M.
J. Org. Lett. 2009, 11, 2760.
o
solution was heated in a 90 C oil bath for 3-6 h. Upon reaction
completion, the MeOH was evaporated and the remaining
aqueous solution was partitioned with EtOAc. The aqueous layer
was extracted twice with EtOAc and then the organic extracts
were combined, washed with brine, dried over Na2SO4, and
concentrated. Purification by silica gel chromatography provided
products 12a-12d.30
25. Butler, J. D.; Solano, D. M.; Robins, L. I.; Haddadin, M. J.; Kurth, M. J.
J. Org. Chem. 2008, 73, 234.
Acknowledgment
26. Donald, M. B.; Conrad, W. E.; Oakdale, J. S.; Butler, J. D.; Haddadin, M.
J.; Kurth, M. J. Org. Lett. 2010, 12, 2524.
We thank the Tara K. Telford Fund for Cystic Fibrosis
Research at the University of California-Davis and the National

27. Avila, B.; Solano, D. M.; Haddadin, M. J.; Kurth, M. J. Org. Lett. 2011,
13, 1060.
28. Conrad, W. E.; Fukazawa, R.; Haddadin, M. J.; Kurth, M. J. Org. Lett.
2011, 13, 3138.
29. Conrad, W. E.; Rodriguez, K. X.; Nguyen, H. H.; Fettinger, J. C.;
Haddadin, M. J.; Kurth, M. J. Org. Lett. 2012, 14, 3870.
30. Farber, K. M.; Haddadin, M. J.; Kurth, M. J. J. Org. Chem. 2014, 79,
6939.
31. Hawkins, P. C. D.; Skillman, A. G.; Warren, G. L.; Ellingson, B. A.;
Stahl, M. T. J. Chem. Inf. Model. 2010, 50, 572.
32. OpenEye Scientific Software, I, version 1.7.4 ed.; 2010, OpenEye: Santa
Fe, NM, http://www.eyesopen.com. 2010.
33. Hawkins, P. C. D.; Nicholls, A. J. Chem. Inf. Model. 2012, 52, 2919.
34. Soubhye, J.; Aldib, I.; Elfving, B.; Gelbcke, M.; Furtmüller, P. G.;
Podrecca, M.; Conotte, R.; Colet, J.-M.; Rousseau, A.; Reye, F.; Sarakbi,
A.; Vanhaeverbeek, M.; Kauffmann, J.-M.; Obinger, C.; Nève, J.;
Prévost, M.; Zouaoui Boudjeltia, K.; Dufrasne, F.; Van Antwerpen, P. J.
Med. Chem. 2013, 56, 3943.
35. Sliskovic, I.; Abdulhamid, I.; Sharma, M.; Abu-Soud, H. M. Free
Radical Biol. Med. 2009, 47, 1005.
36. Van Antwerpen, P.; Prévost, M.; Zouaoui-Boudjeltia, K.; Babar, S.;
Legssyer, I.; Moreau, P.; Moguilevsky, N.; Vanhaeverbeek, M.; Ducobu,
J.; Nève, J.; Dufrasne, F. Bioorgan. Med. Chem. 2008, 16, 1702.
37. Veber, D. F.; Johnson, S. R.; Cheng, H.; Smith, B. R.; Ward, K. W.;
Kopple, K. D. J. Med. Chem. 2002, 45, 2615
38. Hou, T. J.; Xu, X. J. J. Chem. Inf. Comput. Sci. 2003, 43, 2137.
39. Choi, D. K.; Pennathur, S.; Perier, C.; Tieu, K.; Teismann, P.; Wu, D. C.;
Jackson-Lewis, V.; Vila, M.; Vonsattel, J.; Heinecke, J. W.;
Prezedborski, S. J. Neurosci. 2005, 25, 6594.
40. Kajer, T. B.; Fairfull-Smith, K. E.; Yamasaki, T.; Yamada, K.; Fu, S.;
Bottle, S. E.; Hawkins, C. L.; Davies, M. J. Free Radical Biol. Med.
2014, 70, 96.
41. van der Does, A. M.; Hensbergen, P. J.; Bogaards, S. J.; Cansoy, M.;
Deelder, A. M.; van Leeuwen, H. C.; Drijfhout, J. W.; van Dissel, J. T.;
Nibbering, P. H. J. Immunol. 2012, 10, 5012.
42. Frisch, M.J. Gaussian 09, Revision B.01 Gaussian, Inc. Wallingford, CT,
2010 (for full reference, see Supporting Information).
43. Dypbukt, J. M.; Bishop, C.; Brooks, W. M.; Thong, B.; Eriksson, H.;
Kettle, A. J. Free Radical Biol. Med. 2005, 39, 1468.

Compound
IC50 (nM)
Compound
IC50 (nM)
Compound
IC50 (nM)
N
N
N
N
N
N
( )
3
OMe
4g
OMe
4m
OMe
4a
704 243
3876 2550
2029 1542
Cl
N
N
N
N
N
N
( )
6
OMe
4h
OMe
4n
OEt
4b
722 123
>10,000
>10,000
4771 2811
N
N
N
N
N
N
I
( )
6
O(CH2)2OMe
OEt
4i
OMe
4o
4c
1404 105
2000 615
I
N
N
N
O
O
N
N
N
( )OH
3
OMe
4j
OMe
4p
OMe
4d
N
711 300
1787 1221
1660 1391
N
N
N
N
N
( )
( )
OH
2
4
OMe
4e
OMe
4q
OMe
4k
910 530
1539 476
1102 318

N
N
N
N
O
( )
2
OMe
4l
OEt
4f
807 402
1078 227
Table 1. IC50 values of 3-alkoxy-2H-indazoles assessed with a Taurine chlorination assay (154 mM NaCl, 30 µM H2O2, and 2 nM
MPO). All IC50 values are expressed in nM as a mean of ≥ 3 trials +/- standard deviation.
Compound
IC50 (nM)
Compound
IC50 (nM)
Compound
IC50 (nM)
O
N
O
H
N
H
N
N
N
N
N
( )
OH
2
O
5e
O
O
6c
5a
660 222
1948 915
>10,000
O
H
N
H
N
N
N
N
N
O
O
O
O
O
O
5b
5f
6d
O
792 380
2121 1474
>10,000

O
O
S
S
O
O
H
N
Cl
N
N
N
N
N
( )
6
O
O
O
5c
6a
6e
>10,000
>10,000
>10,000
O
N
N
O
O
H
N
N
N
N
N
Br
OH
O
O
O
5d
6b
6f
2469 1251
>10,000
>10,000
Table 2: IC50 values of 1H-indazolones assessed with a Taurine chlorination assay (154 mM NaCl, 30 µM H2O2, and 2 nM MPO). All
IC50 values are expressed in nM as a mean of ≥ 3 trials +/- standard deviation.
Compound
IC50 (nM)
Compound
IC50 (nM)
Compound
IC50 (nM)
N
N
N
N
N
O
O
N
O
S
O
9a
9d
12b
900 466
4148 128
87
7

N
N
N
O
O
N
O
N
N
S
O
9b
9e
12c
1028 427
403 128
213 22
N
N
N
O
O
N
S
N
N
O
S
9c
12a
12d
359 89
94 18
140 33
Table 3. IC50 values of constrained 2H-indazole analogues assessed with a Taurine chlorination assay (154 mM NaCl, 30 µM H2O2,
and 2 nM MPO). All IC50 vales are expressed in nM as a mean of ≥ 3 trials +/- standard deviation.

Molecular
XLogP 2D PSA Weight
Cmpd
HBD
HBA
Score IC50 (nM)
12b
12a
12d
12c
9c
9e
5a
4a
0
0
0
0
0
0
1
0
0
0
1
0
0
0
3
1
3
1
2
4
2
2
4
2
2
2
2
2
1.72
1.99
2.21
2.48
1.75
1.48
2.03
2.76
2.49
3.16
2.48
3.61
1.79
3.21
36.28
17.82
36.28
17.82
27.05
45.51
37.79
27.05
45.51
27.05
37.79
27.05
27.05
27.05
220.3
176.2
234.3
190.3
174.2
218.2
176.2
190.2
234.3
204.3
190.2
218.3
174.2
204.3
-44.74
-46.65
-41.23
-43.61
-38.09
-35.45
-39.94
-47.20
-63.55
-50.03
-48.13
-53.95
-39.72
-50.64
87
94
140
213
359
403
660
704
711
722
792
807
900
910
4d
4b
5b
4f
9a
4e
Table 4. Drug like properties of select compounds (mean IC50 values are shown); see Supporting Information for a complete
tabulation of results.