Discovery of a novel indole pharmacophore for the irreversible inhibition of myeloperoxidase (MPO)

Article abstract of DOI:10.1016/j.bmc.2020.115548

Myeloperoxidase (MPO) activity and subsequent generation of hypochlorous acid has been associated with the killing of host-invading microorganisms (e.g. bacteria, viruses, and fungi). However, during oxidative stress, high MPO activity can damage host tissue and is linked to several chronic inflammatory conditions. Herein, we describe the development of a novel biaryl, indole-pyrazole series of irreversible mechanism-based inhibitors of MPO. Derived from an indole-containing high-throughput screen hit, optimization efforts resulted in potent and selective 6-substituted indoles with good oral bioavailability and in vivo activity.

Full text of DOI:10.1016/j.bmc.2020.115548

Journal Pre-proofs
Discovery of a Novel Indole Pharmacophore for the Irreversible Inhibition of
Myeloperoxidase (MPO)
Anup Patnaik, Laura Axford, Lin Deng, Evan Cohick, Xianglin Ren, Sally
Loi, Sam Kecman, Micah Hollis-Symynkywicz, Tyler J. Harrison, Julien P.N.
Papillo, Natalie Dales, Lawrence G. Hamann, Lac Lee, Jean B. Regard, Jovita
Marcinkeviciene, Martin L. Marro, Andrew W. Patterso
PII:
S0968-0896(20)30378-3
https://doi.org/10.1016/j.bmc.2020.115548
BMC 115548
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 February 2020
1 May 2020
5 May 2020
Please cite this article as: A. Patnaik, L. Axford, L. Deng, E. Cohick, X. Ren, S. Loi, S. Kecman, M. Hollis-
Symynkywicz, T.J. Harrison, J.P.N. Papillo, N. Dales, L.G. Hamann, L. Lee, J.B. Regard, J. Marcinkeviciene,
M.L. Marro, A.W. Patterso, Discovery of a Novel Indole Pharmacophore for the Irreversible Inhibition of
Myeloperoxidase (MPO), Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.
2020.115548
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Discovery of a Novel Indole Pharmacophore for the
Irreversible Inhibition of Myeloperoxidase (MPO)
Anup Patnaik*1, Laura Axford2, Lin Deng3, Evan Cohick2, Xianglin Ren2, Sally Loi2, Sam
Kecman2, Micah Hollis-Symynkywicz2, Tyler J. Harrison,1 Julien P.N. Papillon,1 Natalie Dales1,
Lawrence G. Hamann1, Lac Lee,2 Jean B. Regard2, Jovita Marcinkeviciene2, Martin L. Marro2,
Andrew W. Patterson*1
Global Discovery Chemistry1, Cardiovascular and Metabolic Diseases2, PK Sciences3, Novartis
Institutes for BioMedical Research, Cambridge MA, 02139.
Corresponding authors
*For A.P., 617-871-3605; E-mail, anup.patnaik@novartis.com
*or A.W.P., 617-871-4024; E-mail, andrew.patterson@novartis.com
1

ABSTRACT
Myeloperoxidase (MPO) activity and subsequent generation of hypochlorous acid has been
associated with the killing of host-invading microorganisms (e.g. bacteria, viruses, and fungi).
However, during oxidative stress, high MPO activity can damage host tissue and is linked to
several chronic inflammatory conditions. Herein, we describe the development of a novel biaryl,
indole-pyrazole series of irreversible mechanism-based inhibitors of MPO. Derived from an
indole-containing high-throughput screen hit, optimization efforts resulted in potent and selective
pyrazole-indoles with good oral bioavailability and in vivo activity.
NH
NH
CF3
N
NH2
N
N
N
N
Linker
NH2
OH
N
O
H
HTS indole hit
Lead pyrazole-indoles
MPO kinact/Ki: 23,000 M-1s-1
MPO kinact/Ki: 56,000-230,000 M-1s-1
hERG binding IC50: 2.8 µM
hERG binding IC50: >30 µM
2

INTRODUCTION
Neutrophils contain a rich supply of the green heme enzyme myeloperoxidase (MPO), a lysosomal
enzyme that plays an important role in the human innate immunity system.1 During phagocytosis
of a microorganism, MPO is secreted into the phagolysosome where it catalyzes the conversion of
hydrogen peroxide (H2O2) to hypochlorous acid (HOCl), a powerful oxidant that plays a key role
in host defense against invading microbes.
However, not all MPO-dependent oxidations are desirable. In addition to HOCl, primary oxidation
products of MPO include hypobromous acid, hypothiocyanite, tyrosyl radial, and oxidized amine
species such as nitrogen dioxide.2 MPO-dependent reactive species can then damage host tissues,
proteins, and lipids contributing to the initiation and progression of many inflammatory diseases.
For example, it has been reported that MPO and its oxidation products can oxidize high density
lipoprotein (HDL),3 apolipoprotein B100,4 and nitric oxide.5 The oxidation of endogenous
tyrosine to 3-chlorotyrosine by HOCl has been identified as a specific product of MPO-dependent
oxidation and thus serves as a useful biomarker for MPO activity.6
Elevated levels of MPO and DNA polymorphism associations implicate MPO in cardiac
dysfunction and major adverse cardiac events,7,8 atherosclerosis,9 respiratory diseases10 and central
nervous system diseases.1 Increased neuronal MPO has been described in patients with
Alzheimer’s disease,11 and has been linked to reduced cognitive function in recurrent depressive
disorder.12 Further, in brain tissue, elevated MPO levels have been reported in several
neurodegenerative disorders, including Parkinson’s and Huntington’s disease.13,14
While there has been widespread interest in the development of MPO inhibitors, the identification
of potent and selective in vivo tools and progression to clinical candidates has proven challenging.
Reversible MPO inhibitor classes containing hydroxamic acids,15 hydrazines,16 anilines,17
3

phenols,18 and tryptamines19 have not demonstrated sufficient in vivo activity. However, recent
reports describe reversible triazolopyrimidine and triazolopyridine class that demonstrated
inhibition of neutrophil production of HOCl in an acute mouse model.20
In contrast to reversible MPO inhibitors, irreversible mechanism-based inhibitors of MPO with in
vivo activity have been identified. These inhibitors utilize the oxidative reactivity of MPO to
generate covalent MPO-inhibitor adducts which arrest the enzyme in an unreactive state, blocking
further oxidative activity or re-activation by H2O2.21-23 To-date, no approved MPO inhibitors have
made it to market, but a thiouracil-based pharmacophore class of mechanism-based irreversible
MPO inhibitors has progressed into clinical trials. The most advanced examples are AZD3241
(now BHV-3241/verdiperstat) for the treatment of neurodegenerative diseases24 and PF-06282999
for cardiovascular diseases (reportedly terminated in 2017)25-26 (Figure 1). With verdiperstat
advancing to Phase 3 studies for multiple system atrophy24e and amyotrophic lateral sclerosis,24f
there is evidence that irreversible inhibition of MPO is a viable mechanism for pharmacological
targeting and is a sufficiently safe mechanism for advanced human evaluation.
O
O
H
N
HN
HN
O
S
N
S
N
O
O
NH2
Cl
AZD3241 / BHV-3241
verdiperstat
MPO IC50: 630 nM
PF-06282999
MPO kinact/Ki: 11,600 M-1s-1
Figure 1. Representative thiouracil-based MPO inhibitors verdiperstat24 and PF-0628299925 that
have advanced into clinical trials. MPO activity values as reported.24d, 25a
4

In this article, we report the development of a 6-substituted indole series as a novel class of
irreversible, mechanism-based MPO inhibitors. Derived from an indole-containing high-
throughput screen hit, in vivo activity for the class was confirmed in a mouse peritonitis model.
Optimization efforts resulted in potent pyrazole-indoles with high selectivity over thyroid
peroxidase (TPO), improved in vitro hERG safety profiles relative to the starting screening hit,
and good oral bioavailability.
RESULTS AND DISCUSSION
Hit identification and in vitro assays for compound evaluation
To identify irreversible mechanism-based MPO inhibitors, a high-throughput screen of a >1.4-
million compound library was initiated using a fluorescent MPO peroxidation assay. A dilution
experiment was then conducted on the 4,700 top-scoring hits to identify compounds that inhibited
MPO at 20 µM compound concentration and retained full inhibition after 1000-fold dilution (a
behavior characteristic of irreversible inhibitors). The resulting hit series were confirmed as
irreversible inhibitors via dialysis experiments. Selectivity against TPO, a peroxidase enzyme
prevalent in the thyroid and a near homologue of MPO, is a key concern for MPO inhibitors,21,24-
25 therefore a counterscreen against TPO was conducted.
Two hit series emerged with MPO IC50 <0.5 µM and >100-fold selectivity against TPO. Of the
two classes, we previously described our work on the 2-aminopyridine series,27 and herein we
describe our work on the 6-indole series of irreversible MPO inhibitors. This is represented by
compound 1 (Table 1), featuring a novel 6-substituted indole pharmacophore. In a single point
dilution experiment, compound 1 (Figure 2A) maintained a high level of residual MPO inhibition
after being diluted 1000-fold. While this alone is not confirmatory of an irreversible mechanism,
this was a rapid method for triaging screening hits to find compounds most likely to be irreversible
5

inhibitors. In this same dilution experiment, compound 1 did not have residual inhibition of
peroxidases TPO and horseradish peroxidase (HRP). To confirm an irreversible mechanism, we
next conducted a dialysis experiment (Figure 2B). Compound 1 was incubated with human MPO
and H2O2 followed by 72-hour dialysis and dilution, with >95% of residual MPO inhibition
maintained. While reversible indole-containing inhibitors of MPO have been well-studied,
typically with 3-substitution and containing a pendant amino group, (i.e. tryptophan, melatonin,
and related structures),19,28 this is to our knowledge the first example of an irreversible series of
indole inhibitors.
Figure 2. Compounds 1 and 2 are irreversible MPO inhibitors. A) Residual MPO, TPO, and HRP
inhibition activity of compound 1 measured after 1000-fold dilution. B) Residual MPO activity of
compounds 1 and 2 after 72-hour dialysis and subsequent 200-fold dilution.
6

The enzyme inactivation rate (kinact/Ki) was measured on a stopped-flow instrument, as described
in the Materials and Methods section, via fluorescence monitoring of product formation over time.
Compound 1 displayed rapid kinetics of inhibition (23,000 M-1s-1, Table 1), comparing favorably
to that reported for clinical compound PF-06282999 (11,600 M-1s-1, Figure 1).25a Partition ratio
(k3/k4) is another important parameter for a mechanism-based inhibitor, particularly in the case of
MPO inhibitors where there is potential for escape of active radical species from the enzyme active
site generated by an initial reaction with oxidatively active MPO. A low k3/k4 value indicates
efficient mechanism-based inhibition upon binding to the enzyme. The partition ratio was
determined using an amplex red-based fluorescent assay as described in Materials and Methods.
Although compound 1 demonstrated an improved kinact/Ki over PF-06282999, it was a less efficient
mechanism-based inhibitor (k3/k4: 141, Table 1), requiring further optimization.
Structure Activity Relationship (SAR) Studies
As shown in Table 1, while compound 1 demonstrated good starting kinetics and high selectivity
against TPO and cytochromes P450 (CYP) inhibition, off-target liabilities include human ether-a-
go-go-related gene (hERG) potassium channel binding and inhibition of multiple kinases.
Optimization of compound 1 focused on lowering the partition ratio, reducing hERG binding, and
improving kinase selectivity while simultaneously progressing towards a pharmacological tool for
in vivo validation.
Replacement of, or substitutions on, the indole ring were either not tolerated or reversed the
mechanism of inhibition to reversible inhibition, implying that the indole group was important for
the irreversible mechanism of inhibition. Thus, we hypothesized that MPO might be rapidly
oxidizing the indole C2‒C3 bond and generating a radical-species capable of covalently binding
to the heme group or at residues within the enzyme active site. Unfortunately, mass spectrometry
7

and X-ray crystallography techniques utilized successfully for the mechanism of action
determination of the thiouracil class of inhibitors21-22 were unsuccessful when applied to this indole
series. Having established that a 6-substituted indole was required for effective inhibition, we then
turned our attention to optimizing the core 4-amino-pyrrolopyrimidine and N-aryl moieties.
Core optimization of 1 focused on replacing the aminopyrimidine portion of the 4-amino-
pyrrolopyrimidine core, which we suspected may serve as a hinge binder to kinases and may be
responsible for the observed kinase inhibition.29 Replacement of the 4-amino-pyrrolopyrimidine
with a 4-chloro-pyrrolopyridine yielded compound 2, which displayed an improved kinase
selectivity profile. In general, for analogues where this amino group was not present, no kinase
activity was observed. Compound 2 also demonstrated improved MPO inhibition kinetics with a
higher kinact/Ki and lower k3/k4 than 1, and confirmed as an irreversible inhibitor via dialysis
experiments (Figure 2B).
We next turned our attention to improving TPO selectivity, as well as reducing CYP inhibition
and hERG binding. Truncating and replacing the 4-chloro-pyrrolopyridine core of 2 with a 3-
trifluoromethyl-1H-pyrazole core yielded compound 3, which displayed the highest kinact/Ki
activity (430,000 M-1s-1) yet reported. Compound 3 also demonstrated low k3/k4 (0.4) and was
highly selective against TPO (IC50 > 30 µM). Through our medicinal chemistry efforts, we learned
that an electron-withdrawing group at the 3-position of the pyrazole, as in 3, proved important for
MPO activity. Furthermore, the hERG binding IC50 of 3 was 2-fold higher than that of 2,
presumably arising from reduced lipophilicity (ΔcLogP = 1.8). Further optimization focused on
modification of the alkyl amine tail to improve hERG and CYP selectivity while maintaining MPO
activity and TPO selectivity.
8

Table 1. Core optimization of high throughput screen hit compound 1 to lead pyrazole-indole 3.
NH
NH
NH
Core
Optimization
Cl
N
NH2
N
F3C
N
N
N
N
N
OH
N
O
N
NH2
H
1
3
2
MPO Peroxidation IC50 (µM)
Plasma MPO IC50 (µM)
MPO kinact/Ki (M-1s-1)
MPO k3/k4
0.22
--
0.12
2.6
0.03
0.36
23,000
141
210,000
3.7
430,000
0.4
TPO IC50 (µM)
30
5.3
>30
pH 6.8 solubility (µM)
hERG binding IC50 (µM)
CYP3A4/2D6 (µM)
cLogP/cLogD7.4/calc. pKa
Kinase Activity
46
21
62
2.8
1.7
4.0
>50/>50
17/21
5.8/4.6/8.6
>10 uM
3.8/1.4
3.3/1.9/8.8
<1 uM for
4.1/2.8/9.2
--
6/16 kinases tested
50 kinases tested
9

Table 2. Lead N-aryl pyrazole-indole analogs and entry into N-alkyl derivatives.
NH
NH
NH
N-aryl
to
F3C
F3C
F3C
N-alkyl
N
N
N
N
N
N
N
NH2
NH2
NH
F3C
N
6
4
5
MPO Peroxidation IC50 (µM)
0.25
23
0.42
18
0.33
0.9
Plasma MPO IC50 (µM)
MPO kinact/Ki (M-1s-1)
MPO k3/k4
106,000
12
122,000
17
61,000
42
TPO IC50 (µM)
5.9
>30
>30
pH 6.8 solubility (µM)
hERG binding IC50 (µM)
CYP3A4/2D6 (µM)
cLogP/cLogD7.4/calc. pKa
11
21
73
>30
>30
1.9
6.9/0.5
4.8/4.8/--
17/21
4.8/4.8 /4.5
>50/43
2.9/0.9/9.4
In order to reduce hERG binding, our initial strategy focused on the modification of the amino
group (Table 2). Introduction of a triazole (4) in order to replace the basic amine while maintaining
hydrogen bonding potential resulted in a hERG IC50 >30 µM with maintained MPO peroxidation
IC50. However, this modification resulted in reduced TPO selectivity and increased CYP
inhibition. Another strategy employed to mitigate the hERG liability was to reduce the pKa of the
amino group. Introduction of a CF3 group at the benzylic position in 3 furnished compound 5
10

(ΔpKa = 4.7), which had a hERG IC50 > 30 µM and ablated TPO activity. The electron-
withdrawing CF3 group also had a significant effect on reducing CYP inhibition.
Next, the ability to inhibit MPO in the presence of human plasma was tested.27 We hypothesized
that targeting the inhibition of extracellular MPO (as opposed to intracellular MPO in neutrophils)
would be beneficial for pharmacologic effect without causing deleterious effects on the innate
immune system. Both 4 and 5 displayed a significant IC50 shift between the buffer and plasma
conditions, with 5 demonstrating a plasma MPO IC50 = 18 µM. However, as we explored replacing
the N-aryl linker with N-alkyl groups to reduce lipophilicity, we discovered that compound 6 was
20-fold more active in the plasma MPO assay (IC50 = 0.9 µM) with only a 3-fold shift between
buffer and plasma conditions. In general, a reduced shift between plasma and buffered
peroxidation IC50 values was observed for compounds with lower clogD values. By lowering the
lipophilicity of compound 6 relative to 5 (ΔclogD7.4 = 3.9), solubility and CYP inhibition were
also both improved. Based on these results, we focused on optimization of the N-alkyl series to
further improve MPO kinetic potency while continuing to reduce hERG binding.
To mitigate the hERG risk of compound 6, which contains a basic amine (calc. pKa: 9.4), various
amide and carboxylic acid group replacements were explored. Unfortunately, these modifications
resulted in a loss of MPO potency (compounds 7‒9, Table 3). The effect of increasing the steric
bulk and rigidifying the amine tail on hERG binding (compounds 10 and 11, Table 3) was also
explored. These changes led to the discovery of compound 11, which was potent, selective over
TPO, and demonstrated improved hERG and CYP inhibition.
11

Table 3. Optimization of MPO activity of the N-alkyl pyrazole-indole series
NH
CF3
N
N
R
Compound
R
MPO Peroxidation IC50 (uM)
MPO k3/k4
TPO IC50 (uM)
4.2
>200
>30
7
O
NH2
8
9
O
NH2
2.0
>200
>30
5.2
>200
6.7
---
O
OH
0.17
>30
10
NH2
11
0.07
3.1
>30
NH2
12

Table 4. In vitro profile of compound 11
NH
F3C
N
N
NH2
11
MPO Peroxidation IC50 (µM)
Plasma MPO IC50 (µM)
MPO kinact/Ki (M-1s-1)
MPO k3/k4
0.07
0.17
230,000
3.1
TPO IC50 (µM)
>30
pH 6.8 Solubility (µM)
hERG binding IC50 (µM)
CYP3A4/2D6 (uM)
cLogP/cLogD7.4/calc. pKa
% PPB Human
587
17
34/>50
2.7/0.7/9.4
96.6
Kinase Activity
>10 µM
26 kinases tested
13

In vivo studies
Pharmacokinetics of compounds 2 and 11 in mice
Both compounds 2 and 11 had promising MPO activity as well as in vitro ADME properties. We
next sought to determine if either could be used for in vivo pharmacodynamic studies. Exposure
profiles for the two compounds were measured in mice, dosed by both intravenous (i.v.) and oral
(p.o.) routes of administration, with the results described in Table 5.
For Compound 2, plasma concentrations in mice decreased in a biphasic manner after the 1 mg/kg
intravenous administration. The mean plasma elimination half-life (T1/2) was 2.6 h. The mean
systemic plasma clearance (CL) was 64.7 mL/min/kg. The mean volume of distribution at steady
state (Vss = 9.8 L/kg) was 14.7 times higher than that of total body water of 0.67 L/kg, indicating
that 2 was highly distributed systemically. Following oral administration of 10 mg/kg, Cmax
reached 500 nM, with unbound Cmax (Cmax,un) of 2 nM based on 99.6% mouse plasma protein
binding. Compound 2 showed prolonged exposure, likely due to slow dissolution from the
suspension formulation based upon modest solubility (21 µM). Despite the slow absorption profile,
2 had good oral bioavailability (F = 61%).
Compound 11 displayed lower CL relative to 2 (16.1 vs. 64.7 mL/min•kg), and higher AUC, Cmax
,
and T1/2 values. While Compound 11 would serve as a more promising lead for future drug
development, the greater peak-to-trough exposure ratio provided by Compound 2 made it more
desirable for initial in vivo evaluation.
14

Table 5. Mouse pharmacokinetic parameter comparison of compounds 2 and 11
Parameter
Dose
Compound 2
Compound 11
1/10 mg/kg i.v./p.o.
0.5/10 mg/kg i.v./p.o.
Vss (L/kg)
T1/2 (h)
9.8
2.6
2.1
4.5
AUCinf (nM•h) p.o.
Cmax (nM) p.o.
PPB (%)
2,486
502
99.6
61
22,862
1986
97.3
40
F (%)
Pharmacodynamics of compound 2
The in vivo pharmacodynamic activity of compound 2 was evaluated in a mouse peritonitis model
of acute inflammation (Figure 3), a model which leads to an upregulation of MPO activity.21 Not
knowing how in vitro MPO activity would translate into an in vivo effect, a low oral dose (5 mg/kg)
and a high oral dose (90 mg/kg) were chosen for this exploratory study. Mice were administered
successive intraperitoneal doses of thioglycolate and zymosan A to promote neutrophil infiltration
into the peritoneal cavity and degranulation, respectively, resulting in high local levels of MPO in
the peritoneum as well as elevated plasma MPO levels. 3-Chlorotyrosine, a specific biomarker of
MPO activity, and tyrosine concentrations from the peritoneal exudate were quantified by mass
spectrometry using a previously described free amino acid (FAA) detection method.27 Control
experiments comparing the FAA detection method to a total protein hydrolysis method30 are
described in the Materials and Methods section. MPO protein levels were measured in peritoneal
exudate by ELISA. Normalized MPO activity was measured by expressing the ratios of 3-
15

chlorotyrosine to total tyrosine (Figure 3A) and the ratio of 3-chlorotyrosine to total MPO (Figure
3B). Plasma and exudate concentrations of 2 were measured at the same terminal time point (5
hours post-compound administration, 4 hours post-zymosan A administration) by mass
spectrometry and are presented in Figure 3C. Administration of 90 mg/kg of 2, which corresponds
to low-micromolar terminal total drug concentrations and low-nanomolar terminal unbound drug
concentrations in both plasma and exudate, resulted in a ~50% reduction in the MPO activity. With
~1 µM four-hour total compound concentration corresponding to ~50% inhibition of in vivo MPO
activity, this does compare favorably to an in vitro MPO plasma activity IC50 of 2.6 µM. However,
a more detailed kinetic evaluation across a greater number of examples would be needed to draw
a more conclusive correlation between the two measurements.
A
B
C
10000
100
50
0
100
50
0
1000
100
10
**
**
1
0.1
0.01
Total
Free
g
g
g
g
k
k
k
/
k
/
/
/
g
a
g
m
g
g
m
m
m
5
-
5
0
0
9
9
-
-
-
a
e
t
a
e
t
sm
d
a
a
sm
d
l
xu
a
l
P
xu
E
P
E
Figure 3. Compound 2 inhibits MPO in vivo in a mouse peritonitis model. A) MPO activity as
measured by 3-chlorotyrosine (Cl-Tyr) levels normalized to total tyrosine from peritoneal exudate
four hours post-Zymosan A administration. B) MPO activity as measured by 3-chlorotyrosine (Cl-
Tyr) levels normalized to total MPO from peritoneal exudate four hours post-Zymosan A
16

administration. C) Plasma and peritoneal exudate concentrations of compound 2 were measured
(total) and calculated (free) four hours post-Zymosan A administration.
Chemistry
Compound 2 was synthesized according to Scheme 1. 4-Chloro-7-azaindole 12 was iodinated at
the C3-position with NIS (100%). Chan-Lam coupling of 13 with an appropriate boronic acid
(23%), followed by Suzuki coupling with 6-indolyl boronic acid, provided the desired product 2
(23%).
Scheme 1.
NH
Cl
N
I
Cl
N
Cl
N
Cl
N
I
c
a
b
N
N
N
N
H
H
N
12
14
13
2
N
Reagents and conditions: (a) N-Iodosuccinimide, rt, 16 h; 100%; (b) 4-((dimethylamino)methyl
phenyl) boronic acid, Cu(OAc)2, Et3N, rt,; 23%; (c) 6-indolylboronic acid, Pd(PPh3)4, Na2CO3, 95
°C, 16 h; 23%.
The synthesis of compound 11 is shown in Scheme 2. Mesylation of tert-butyl ((1R,3S)-3-
hydroxycyclopentyl)carbamate 15 (93%), followed by nucleophilic displacement with 4-iodo-3-
(trifluoromethyl)-1H-pyrazole, afforded the desired pyrazole regioisomer 17 (65%) from a 5:1
mixture of regioisomers. Iodide 17 then underwent a Suzuki coupling with 6-indolylboronic acid
to provide pyrazole-indole 18 (59%). Cleavage of the N-Boc group of was then performed under
17

neutral conditions through microwave irradiation in trifluoroethanol/water (3:1) to avoid
decomposition that was noticed under acidic deprotection conditions, yielding compound 11
(23%).
Scheme 2.
O
I
CF3
N
S
O
O
OH
a
b
N
NHBoc
NHBoc
15
NHBoc
17
16
NH
NH
CF3
N
CF3
N
c
d
N
N
NHBoc
NH2
18
11
Reagents and conditions: (a) Methanesulfonyl chloride, Et3N, 4-DMAP, 0 °C to rt, 16 h; 93%; (b)
4-iodo-3-(trifluoromethyl)-1H-pyrazole, Cs2CO3, 100 °C, 16 h; 65%; (c) 6-indolylboronic acid, X-
Phos palladacycle, Na2CO3, 100 °C, 16 h; 59%; (d) trifluoroethanol/water (3:1), microwave, 150
°C, 4 h; 23%.
18

SUMMARY
We have described the discovery of a novel mechanism-based irreversible indole pharmacophore
for the inhibition of MPO. Pyrrolopyridine 2 demonstrated in vivo validation for this indole
pharmacophore, demonstrating inhibition of MPO activity in an acute mouse model of
inflammation. Subsequent core optimization led to the identification of a pyrazole-indole series,
notably compound 3, which has a low k3/k4 (0.4), high selectivity against TPO (>1000-fold based
on IC50), and the highest MPO kinact/Ki (430,000 M-1s-1) reported to-date. Optimization to reduce
hERG binding and CYP inhibition led to compound 11, which also had a good oral exposure and
will serve as a lead scaffold for future drug discovery efforts.
19

REFERENCES
1. Klebanoff, S. J. Myeloperoxidase: friend and foe. J. Leukocyte Biol. 2005, 77, 598.
2. Malle, E.; Marsche, G.; Arnhold, J.; Davies, M.J. Modification of low-density
lipoprotein by myeloperoxidase-derived oxidants and reagent hypochlorous acid. Biochim
Biophys Acta 2006, 1761, 392.
3. Shao, B.; Oda, M.N.; Oram, J.F.; Heinecke, J.W. Myeloperoxidase: An oxidative pathway
for generating dysfunctional high-density lipoprotein. Chem. Res. Toxicol. 2010, 23, 447.
4. Marsche, G.; Zimmermann, R.; Horiuchi, S.; Tandon, N.N.; Sattler, W.; Malle, E. Class B
scavenger receptors CD36 and SR-BI are receptors for hypochlorite-modified low density
lipoprotein. J. Biol. Chem. 2003, 278, 47562.
5. Eiserich, J.P.; Baldus, S. et al. Myeloperoxidase, a leukocyte-derived vascular NO oxidase.
Science 2002, 296, 2391.
6. Hazen, S.; Heinecke, J. W. 3-Chlorotyrosine, a specific marker of myeloperoxidase-
catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human
atherosclerotic intima. J. Clin. Invest. 1997, 99, 2075.
7. Mayyas, F. A.; Al-Jarrah, M. I.; Ibrahim, K. S.; Alzoubi, K. H. Level and significance of
plasma myeloperoxidase and the neutrophil to lymphocyte ratio in patients with coronary
artery disease. Exp. Ther. Med. 2014, 8, 1951.
8. Nussbaum, K.; Klinke, A. et al. Myeloperoxidase: A leukocyte-derived protagonist of
inflammation and cardiovascular disease. Antioxid. Redox Signal 2013, 18, 692.
9. Weber, C.; Noels, H. Atherosclerosis: Current pathogenesis and therapeutic options.
Nature Med. 2011, 17, 1410.
20

10. 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. Late intervention with a
myeloperoxidase inhibitor stops progression of experimental chronic obstructive
pulmonary disease. Am. J. Respir. Crit. Care Med. 2012, 185, 34.
11. Green, P.S.; Mendez, A.J. et al. Neuronal expression of myeloperoxidase is increased in
Alzheimer's disease. J. Neurochem. 2004, 90, 724.
12. Talarowska, M.; Szemraj J.; Galecki, P. Myeloperoxidase gene expression and cognitive
functions in depression. Adv. Med. Sci. 2015, 60, 1.
13. Choi, D. K.; Pennathur, S. et al. Ablation of the inflammatory enzyme myeloperoxidase
mitigates features of Parkinson’s disease in mice. J. Neurosci. 2005, 25, 6594.
14. Gellhaar, S.; Sunnemark, D. et al. Myeloperoxidase-immunoreactive cells are significantly
increased in brain areas affected by neurodegeneration in Parkinson’s and Alzheimer’s
disease. Cell Tissue Res. 2017, 369, 445.
15. Forbes L. V.; Tove Sjögren, T. et al. Potent reversible inhibition of myeloperoxidase by
aromatic hydroxamates. J. Biol. Chem. 2013, 288, 36636.
16. Kettle, A. J.; Gedye, C. A. et al. Inhibition of myeloperoxidase by benzoic acid hydrazides.
Biochem. J. 1995, 308, 559.
17. Kettle A. J. et al. Winterbourn C. C. Mechanism of inhibition of myeloperoxidase by anti-
inflammatory drugs. Biochemical Pharmacology, 1991, 41, 1485.
18. Aldib, I.; Soubhye, J. et al. Evaluation of new scaffolds of myeloperoxidase inhibitors by
rational design combined with high-throughput virtual screening. J. Med. Chem. 2012, 55,
7208.
19. a) Soubhye, J.; Prevost, M. et al. Structure-based design, synthesis, and pharmacological
21

evaluation of 3-(aminoalkyl)-5-fluoroindoles as myeloperoxidase inhibitors. J. Med.
Chem. 2010, 53, 8747. b) Soubhye, J.; Aldib, I. et al. Design, synthesis, and structure
activity relationship studies of novel 3-alkylindole derivatives as selective and highly
potent myeloperoxidase inhibitors. J. Med. Chem. 2013, 56, 3943. c) Aldib, I.; Gelbcke,
M. et al. Novel bis-arylalkylamines as myeloperoxidase inhibitors: Design, synthesis, and
structure-activity relationship study. Eur. J. Med. Chem. 2016, 123, 746.
20. a) Duclos, F.; Abell, L.M. et al. Triazolopyrimidines identified as reversible
myeloperoxidase inhibitors. Med. Chem. Commun., 2017, 8, 2093. b) Wurtz, N.R.; Viet,
A. et al. Potent Triazolopyridine Myeloperoxidase Inhibitors. ACS Med Chem Lett. 2018,
12, 1175-1180.
21. Tidén, A.-K.; Sjögren, T. et al. 2-Thioxanthines are mechanism-based inactivators of
myeloperoxidase that block oxidative stress during inflammation. J. Biol. Chem. 2011, 286,
37578.
22. Geoghegan, K.F.; Varghese, A.H. et al. Deconstruction of activity-dependent covalent
modification of heme in human neutrophil myeloperoxidase by multistage mass
spectrometry (MS4). Biochemistry 2012, 51, 2065.
23. Soubhye, J.; Alard, I.A. et al. Discovery of novel potent reversible and irreversible
myeloperoxidase inhibitors using virtual screening procedure. J. Med. Chem. 2017, 60,
6563.
24. a) Johnström, P.; Bergman, L. et al. Development of rapid multistep carbon-11
radiosynthesis of the myeloperoxidase inhibitor AZD3241 to assess brain exposure by PET
microdosing. Nucl. Med. Biol. 2015, 42, 555. b) Jucaite, A.; Svenningsson, P. et al. Effect
of the myeloperoxidase inhibitor AZD3241 on microglia: a PET study in Parkinson's
22

disease. Brain. 2015, 138, 2687. c) Kaindlstorfer, C.; Sommer, P. et al. Failure of
Neuroprotection despite microglial suppression by delayed-start myeloperoxidase
inhibition in a model of advanced multiple system atrophy: clinical implications. Neurotox.
Res. 2015, 28, 185. d) https://ncats.nih.gov/files/AZD3241-2016.pdf. e)
https://clinicaltrials.gov/ct2/show/NCT03952806.
https://clinicaltrials.gov/ct2/show/NCT04297683.
f)
25. a) Ruggeri, R. B.; Buckbinder, L. et al. Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-
oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999): highly
A
selective mechanism-based myeloperoxidase inhibitor for the treatment of cardiovascular
diseases. J. Med. Chem. 2015, 58, 8513. b) Dong, J.Q.; Varma, M.V. et al.
Pharmacokinetics and disposition of the thiouracil derivative PF-06282999, an orally
bioavailable, irreversible inactivator of myeloperoxidase enzyme, across animals and
humans. Drug Metab. Dispos. 2016, 44, 209.
26. Li, T. Jiang, S. et al. Discontinued drugs for the treatment of cardiovascular disease from
2016 to 2018. Int. J. Mol. Sci. 2019, 20, 4513.
27. Marro, M.L.; Patterson, A.W. et al. Discovery of 1-((6-aminopyridin-3-yl)methyl)-3-(4-
bromophenyl) urea as a potent, irreversible myeloperoxidase inhibitor. J. Pharmacol. Exp.
Ther. 2018, 367, 147.
28. a) Galijasevic, S.; Proteasa, G.; Abdulhamid, I.; Abu-Soud, H. M. Melatonin is a potent
inhibitor for myeloperoxidase. Biochemistry 2008, 47, 2668. b) Galijasevic, S.; Proteasa,
G.; Abdulhamid, I.; Abu-Soud, H. M. Potential role of tryptophan and chloride in the
inhibition of human myeloperoxidase. Free Radic. Biol. Med. 2008, 44, 1570. c) Sliskovic,
I.; Abdulhamid, I.; Sharma, M.; Abu-Soud, H.M. Free Radic. Biol. Med. 2009, 47, 1005.
23

29. Xing, L.; Klug-Mcleod, J. et al. Kinase hinge binding scaffolds and their hydrogen bond
patterns. Bioorg. Med. Chem. 2015, 23, 6520.
30. a) Hazen, S.L.; Crowley, J.R.; Mueller, D.M.; Heinecke, J.W. Mass spectrometric
quantification of 3-chlorotyrosine in human tissues with attomole sensitivity: A sensitive
and specific marker for myeloperoxidase-catalyzed chlorination at sites of inflammation.
Free. Radic. Biol. Med. 1997, 23, 909. b) Brennan, M.-L.; Wu, W. et al. Defining both the
role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and
myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen
species. J. Biol. Chem. 2002, 277, 17145.
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NH
NH
CF3
N
NH2
N
N
N
N
Linker
NH2
OH
N
O
H
HTS indole hit
Lead pyrazole-indoles
MPO kinact/Ki: 23,000 M-1s-1
MPO kinact/Ki: 56,000-230,000 M-1s-1
hERG binding IC50: 2.8 µM
hERG binding IC50: >30 µM
25

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
26

Conflict of Interest.
27