First in class, potent, and orally bioavailable NADPH oxidase isoform 4 (Nox4) inhibitors for the treatment of idiopathic pulmonary fibrosis

Article abstract of DOI:10.1021/jm100773e

We describe the design, synthesis, and optimization of first-in-class series of inhibitors of NADPH oxidase isoform 4 (Nox4), an enzyme implicated in several pathologies, in particular idiopathic pulmonary fibrosis, a life-threatening and orphan disease. Initially, several moderately potent pyrazolopyridine dione derivatives were found during a high-throughput screening campaign. SAR investigation around the pyrazolopyridine dione core led to the discovery of several double-digit nanomolar inhibitors in cell free assays of reactive oxygen species (ROS) production, showing high potency on Nox4 and Nox1. The compounds have little affinity for Nox2 isoform and are selective for Nox4/1 isoforms. The specificity of these compounds was confirmed in an extensive in vitro pharmacological profile, as well as in a counterscreening assay for potential ROS scavenging. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo, allowing further clinical trials for the potential treatment of fibrotic diseases, cancers, and cardiovascular and metabolic diseases.

Full text of DOI:10.1021/jm100773e

J. Med. Chem. 2010, 53, 7715–7730 7715
DOI: 10.1021/jm100773e
First in Class, Potent, and Orally Bioavailable NADPH Oxidase Isoform 4 (Nox4) Inhibitors for the
Treatment of Idiopathic Pulmonary Fibrosis
ꢀ
Beno^ıt Laleu, Francesca Gaggini, Mike Orchard, Laetitia Fioraso-Cartier, Laurene Cagnon, Sophie Houngninou-Molango,
ꢁ
Angelo Gradia, Guillaume Duboux, Cedric Merlot, Freddy Heitz, Cedric Szyndralewiez, and Patrick Page*
ꢁ
Genkyotex, S.A., 14 Chemin des Aulx, CH-1228 Plan-Les-Ouates, Switzerland
Received June 23, 2010
We describe the design, synthesis, and optimization of first-in-class series of inhibitors of NADPH
oxidase isoform 4 (Nox4), an enzyme implicated in several pathologies, in particular idiopathic pulmo-
nary fibrosis, a life-threatening and orphan disease. Initially, several moderately potent pyrazolopyridine
dione derivatives were found during a high-throughput screening campaign. SAR investigation around
the pyrazolopyridine dione core led to the discovery of several double-digit nanomolar inhibitors in
cell free assays of reactive oxygen species (ROS) production, showing high potency on Nox4 and Nox1.
The compounds have little affinity for Nox2 isoform and are selective for Nox4/1 isoforms. The speci-
ficity of these compounds was confirmed in an extensive in vitro pharmacological profile, as well as in a
counterscreening assay forpotential ROSscavenging. Concomitant benefitsare goodoralbioavailability
and high plasma concentrations in vivo, allowing further clinical trials for the potential treatment of
fibrotic diseases, cancers, and cardiovascular and metabolic diseases.
Introduction
(O2) to superoxide (O2•-), the primary product of Nox acti-
vation.14 Nox2 (also known as gp91 phox) was the historically
first identified NADPH oxidase (Nox) family member. Ini-
tially found in phagocytes, it plays a role in host defense by
giving an outward burst of ROS.15 In the late 1990s, Nox2 and
its homologues were defined as a protein family of seven
different isoforms including the phagocyte Nox2 isoform and
Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2, which were
identified in non-phagocytes cells.14,16 Although all seven Nox
isoforms catalyze the reduction of molecular oxygen, they
differ in their tissue distribution, subcellular localization, sub-
unit requirements, domain structure, and the mechanism by
which they are activated.14 Today, it is becoming more recog-
nized that the Nox enzyme protein family participates as an
important, and in many cases predominant, source of ROS in a
large variety of diseases.17,18
There is compelling evidence indicating the production of
low molecular weight second messengers, so-called reactive
oxygen species (ROSa), as cell-signaling molecules and their
implications in signal transduction.1,2 The molecules involved
in signaling include the three successive reduction products of
molecular oxygen such as superoxide (O2•-), hydrogen per-
oxide (H2O2), and hydroxyl radical (HO•), as well as reactive
nitrogen species (RNS) such as NO, peroxynitrite, and peroxy-
chloride.3-5 Each of these species possesses chemical properties
that potentially affect their signaling function. ROS and RNS
act as signaling molecules and key regulators of cell function
and are produced in specific compartments and tissues where
they fine-tune the fragile intracellular redox cell-signaling
balance.6-10 Superoxide (O2•-) production is the first chemi-
cal step of initiation of the oxidative stress cascade.11
The NADPH oxidase isoform 4 (Nox4) has gained consider-
able and growing attention during the past years because of its
potential key involvement in a variety of diseases, some of which
have high unmet medical needs, such as idiopathic pulmonary
fibrosis (IPF),19 pulmonary arterial hypertension (PAH),20,21
diabetic nephropathy (DN),22 and complications such as dia-
betic cardiomyopathy23 and neuropathy and retinopathy24 or
cancers25 like metastatic renal cell carcinoma (RCC).26,27
Nox4 is constitutively active and requires a two-transmem-
brane (2-TM) protein counterpart called p22phox in its active
form. In humans, Nox4 is most abundantly expressed in the
kidney,28,29 hence originally termedrenal oxidase (renox),30 as
well as in the lung.31,32 Nox4 is distributed in the renal vascu-
lature, mesangial cells, podocytes, tubular cells as well as myo-
fibroblasts and epithelial cells, the last two types of cells being
known to play a key role in the pathogenesis of idiopathic
pulmonary fibrosis (IPF).33 In the lung, Nox4 regulates myo-
fibroblast synthesis of fibronectin and contractile responses to
Although ROS are produced by a variety of intracellular
mechanisms, it is now well established that the predominant
cellular sources of these molecules area family of multisubunit
enzymes known as the NADPH oxidases.12,13 NADPH oxi-
dase enzymes (Nox) are a six-transmembrane (6-TM) domain
proteinfamily thatcatalyze the reduction of molecular oxygen
*To whom correspondence should be addressed. Phone: þ41228801025.
Fax: þ41228801013. E-mail: patrick.page@genkyotex.com.
a Abbreviations: ADME, absorption, distribution, metabolism, excre-
tion; ADMET, absorption, distribution, metabolism, excretion, toxicity;
Cl, clearance; DIPEA, N,N-diisopropylethylamine; DPI, diphenyliodo-
nium; Fu, fraction unbound; Fz, bioavailability; hERG, the human ether-
a-go-go-related gene; HPLC, high performance liquid chromatography;
hFLMC, human fetal lung mesenchymal cells; HTS, high throughput
screening; IPF, idiopathic pulmonary fibrosis; NADPH, nicotinamide
adenine dinucleotide phosphate; Nox, nicotinamide adenine dinucleotide
phosphate oxidase; QH, liver blood flow; RNS, reactive nitrogen species;
ROS, reactive oxygen species; rt, room temperature; SAR, structure-
activity-relationship; TGF, transforming growth factor.
r
2010 American Chemical Society
Published on Web 10/13/2010
pubs.acs.org/jmc

7716 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Laleu et al.
Figure 1. Structures of some known or claimed NADPH oxidases inhibitors: DPI (1), apocynin (2), VAS2870 (3), Shionigi compound (4),
S17834 (5), and fulvene-5 (6).
the multifunctional cytokine, transforming growth factor-β1
(TGF-β1).19 Nox4 is specifically induced by TGF-β1 in a time-
dependent manner in hFLMCs and mediates H2O2 secretion
by myofibroblasts.19 The severity of idiopathic pulmonary
fibrosis has been correlated with increased levels of H2O2 in
antioxidant structural features.38 Despite a recent publication
that shows apocynin being not an inhibitor of Nox enzymes
but an antioxidant,39 it is still used as a reference compound
although the resulting data may have to be carefully interpreted.
Furthermore, legions of molecules described in the litera-
ture wrongly named “Nox inhibitors” or “indirect Nox inhib-
itors” add to the general confusion.40,41 These molecules
such as 3 (VAS2870) are interfering with protein targets and
signaling pathways that may affect Nox enzymes.42 For in-
stance, ARBs, ACEs, phosphodiesterase, eicosanoids, corti-
costeroids, MAP kinase, and protein kinase C inhibitors42
such as Shionigi compound (4) and many others including
potential antioxidant, catechol-like molecule 5 (S17834) or
fluorescent dyes and/or unstable molecules like fulvene-5 (6)
have all been claimed to be Nox inhibitors43-46 without any
data demonstrating inhibition of Nox enzymes in cell free
assays or counterscreening assays to eliminate potential ROS-
scavenging properties.42-47 Today, there is urgency toidentify
truly specific Nox inhibitors in order to unravel the promises
of this protein family. These molecules should meet key
requirements such as lack of unspecific ROS scavenging or
antioxidant ability, demonstrating off-targets selectivity, low
adverse effects profile, and strong in vivo pharmacological
activity in relevant diseases models.
the expired breath condensate (EBC) of IPF patients,34
a
biomarker directly linked to the NADPH oxidase 4 isoform.
Nox4 mediates myofibroblasts differentiation and contractil-
ity and is induced in fibrotic lung injury in vivo.19 Further
data in animal models likewise point to a potential role for
Nox4 modulation in idiopathic pulmonary fibrosis. RNAi-
mediated inhibition of Nox4 protected against fibrotic lung
injury. Furthermore, pharmacological unspecific inhibition
of Nox flavoenzyme(s) activity by the irreversible inhibitor
diphenyliodonium (1, Figure 1) dramatically diminished the
deposition of collagen in a curative murine model of bleomycine-
induced pulmonary fibrosis.19
Despite compelling evidence for the involvement of Nox
enzymes in many signaling pathways and the great potential
for the treatment of several pathologies, there is as of today no
specific inhibitor of NADPH oxidases.35 The above statement
is very important and needs to be highlighted because a large
number of studies investigating the role of Nox enzymes in
pathological conditions rely on the use of molecules that are
claimed to be specific. For instance, 1, a toxic and nondruglike
molecule, remains today the gold standard of Nox inhibitors
although this molecule was demonstrated to be an irreversible
and unselective inhibitor of several flavin-dependent enzymes
such as nitric oxide synthase, xanthine oxidase, and others.35
Also, 1 is an inhibitor of acetylcholinesterase and butyryl cho-
linesterase as well as of the internal Ca2þ pump,36 which in
addition to its intrinsic toxicity raises major concerns on the
interpretation of studies performed with this molecule. Apoc-
ynin (2) is the claimed by some to be a Nox inhibitor.37
However, this potentially reactive molecule is inactive in cell
and membranes overexpressing Nox enzymes and decom-
poses in vivo in several entities which all contain catechol-like
Particularly, the validation of in vivo efficacy of druglike
and selective Nox4 inhibitors is needed and will provide new
insights on the role of Nox4 in idiopathic pulmonary fibrosis
and chronic kidney diseases (CKD).
We set out to discover and develop first-in-class, potent,
selective, and orally active Nox4 inhibitors as potential new
therapeutic agents for the pharmacological management of
IPF and CKD.
Results and Discussion
Primary Screening Results. A high-throughput screening
(HTS) campaign using a cell free assay of ROS production
on human Nox4 membranes was performed on 136 000

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7717
compounds. Several pyrazolopyridine diones (structure 7,
Figure 2) were moderately potent as confirmed in a concen-
tration-response IC50 determination experiment (Table 1).
The most potent compound was 12 with Ki(hNox4) = 373 nM.
Incorporation of an acetyl group at R4 position furnished deriv-
ative 18 almost equipotent to 12, whereas the introduction of
a benzyl group gave poorly active or inactive compounds. The
very preliminary SAR obtained from this limited set of com-
pounds suggested that the picolyl group at position R3 could
bring a beneficial effect although various groups gave some
activity. Besides, phenyl substituents appeared to be preferred
over benzothiazole groups at position R1 and a triazolopyr-
idazine resulted in a loss of activity.
oxidase assay). Its in vitro early ADME profile showed a high
intrinsic clearance in rat liver microsomes but a good perme-
ability in the Caco-2 cell assay. The in vivo pharmacokinetic
profile of 12 was therefore assessed in rat (Table 3). A high
clearance was observed after intravenous dosing (Cl > liver
blood flow, QH = 3.3 (L/kg)/h), which was in line with the in
vitro data obtained for the microsomal metabolism. The short
half-life of 12 might at least in part be related to high first-pass
metabolism and translated in an apparent low oral bioavail-
ability and poor exposure. The suspected strong first-pass
metabolism was somehow corroborated by the really good
bioavailability (FZ = 87%) obtained after subcutaneous ad-
ministration. Nevertheless, this compound had an acceptable
volume of distribution (Vss = 2.36 L/kg after iv injection).
Compound 12 was further evaluated in in vitro enzymatic
assays and for early ADME properties (Table 2). This com-
pound displayed a good Nox4/Nox2 selectivity (>12-fold)
and was also deprived of antioxidant properties based on a
counterscreening assay for potential ROS scavenging (xanthine
Table 2. Pharmacological and Early in Vitro ADME Profile for Hit
Compound 12
parameter
Ki (hNox4)a
373 ( 37 nM
4720 ( 1280 nM
>30000 nM
Ki (hNox2)b
Ki (xanthine oxidase)c
Cl (r-LM)d
Caco-2 (Papp
a Cell free assay of ROS production on Nox4 membranes. b Cell free
assay ofROS production on Nox2membranes(see Experimental Section).
c In vitro enzymatic assay. d Microsome stability experiment. r-LM, rat
liver microsomes (see Experimental Section). e Caco-2 cell permeability
assay (see Experimental Section). Papp, permeability coefficient.
97.1 (μL/min)/mg protein
79.2 ꢀ 10-6 cm/s
e
)
Figure 2. Structure of pyrazolopyridine dione core structure.
Table 1. Pyrazolopyridine Dione Derivatives from HTS Screening: Preliminary Exploration
a Cell free assay of ROS production on human Nox4 membranes (see Experimental Section).

7718 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Laleu et al.
Table 3. In Vivo Pharmacokinetic Profile in Rat for Hit Compound 12
route
dose (mg/kg)
AUC¥ (h ng/mL)
FZ (%)
Cmax (ng/mL)
tmax (h)
t1/2 (h)
Cl ((L/kg)/h)
5.11
Vss (L/kg)
3
iv
sc
os
1
5
196
815
417
271
678
575
0.08
0.50
0.25
0.40
0.70
1.20
2.36
87
22
10
Scheme 1. Synthesis of Hit Compound 12 (General Procedure A)a
a Reagents and conditions: (a) phenylhydrazine, benzene, reflux, 80%; (b) Ac2O, MeC(OEt)3, reflux, 50%; (c) 3-aminomethylpyridine, toluene,
reflux, 60%; (d) i-PrONa/i-PrOH, reflux, 58%.
On the basis of these data, compound 12 was considered
as a suitable starting point for lead optimization. In the
following chapters, we describe the SAR and the synthesis of
pyrazolopyridine dione core structures.
Chemistry. The synthesis of pyrazolopyridine dione scaf-
fold 7 was versatile and allowed the development of a quite
extensive lead optimization program involving substitutions
of the four diversity points of the scaffold (R1, R2, R3, R4, as
illustrated in Figure 2).
The synthetic route of hit compound 12 consisted of a four-
step procedure described in Scheme 1. In the first approach,
the pyrazolone moiety 21 was built by condensation of com-
mercially available dimethyl 1,3-acetonedicarboxylate 20 and
phenylhydrazine48 in benzene warmed to reflux. In presence
of an excess of triethyl orthoacetate, pyrazolone 21 formed the
enol ether 2249 which was easily converted into the corre-
sponding enamine 23 in presence of 3-picolylamine in toluene
heating at reflux.48,50 The driving force of the reaction was the
formation of final enamine which precipitated from the reac-
tion mixture in good yield and high purity. Treatment of 23
with freshly prepared sodium isopropanolate51,52 afforded the
final product 12 in 14% overall yield. This procedure was
applied to the synthesis of compounds 8-17 (Table 1).
The synthesis of final acetylated or alkylated analogues 18
and 19 (Table 1) and 41-48 (Table 4) was performed using
different procedures. Acylation of 12 was achieved in 1 h with
sodium acetate in acetic anhydride at 40 ꢀC. Instead, DIPEA
in DMF was used when a more hindered acyl chloride was
selected (Scheme 2). Compounds 19 and 41-45 were obtained
using standard protocol with several alkylating agents in the
presence of triethylamine in THF (Scheme 3).
performed at variable temperature (25-60 ꢀC) depending on
the intrinsic reactivity of the hydrazine. The final pyrazolone
derivatives 28 were usually collected by precipitation in
moderate to good yields. A key improvement concerned the
second step: the use of a catalytic amount of glacial acetic
acid rather than an excess of acetic anhydride resulted in
much cleaner formation of the enol ether intermediates 29.
This modified route is outlined in Scheme 4. Following these
procedures several libraries of this series of compounds
(R1 =Me) were synthesized (over 300 molecules), and the
data for a representative set are shown in Table 4.
In order to investigate further the diversity point R2, a
modification of the synthetic strategy already illustrated was
pursued and two different methods were developed: acyl
chloride and 2-chloro-1,1,1-triethoxyethane pathways (Schemes
5 and 6, respectively). In the first case, condensation of commer-
cially available acyl chlorides and pyrazolone 28 in 1,4-dioxane
followed by Fries rearrangement was performed in the presence
of calcium hydroxide under refluxing conditions (Scheme 5).53,54
The obtained acylpyrazolone 33 was functionalized following
the procedure already described: the enamine 34, formed in acid
conditions, was cyclized in milder conditions. The final product
35 was obtained after treatment with freshly prepared sodium
methanolate solution at room temperature.
In the second approach, the reaction of pyrazolone 28
with an excess of 2-chloro-1,1,1-triethoxyethane under acid
catalysis gave the possibility of exploring another route for
lead optimization. Interestingly, and in contrast with the
method described in Scheme 4, the expected enol ether 36
was never observed under UPLC conditions. Most probably
in situ hydrolysis of enol ether 36 generated the R-chloroke-
tone 37, the only species detected by UPLC. Its intrinsic
instability precluded any purification and characterization.
The R-chloroketone 37 afforded the enamine 38 by reaction
with a primary amine in acid conditions. In a subsequent
step, the chlorine was displaced with a secondary amine in
the presence of DIPEA under refluxing conditions. Final
derivatives of type 40 were generated by cyclization of the
Alternative routes for the synthesis of pyrazolone deriva-
tive were also designed to overcome issues due to solvent
toxicity. In the first instance, toluene replaced benzene and
addition of DIPEA accelerated the condensation between
the suitable hydrazine and the dimethyl 1,3-acetonedicar-
boxylate 20. The second later improvement consisted of
the use of methanol in the condensation step which was

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7719
Table 4. SAR: R4 Modification
a Cell free assay of ROS production on human Nox4 membranes.
intermediate compounds 39 with freshly prepared sodium
methanolate solution.
halogens. A loss of potency was observed in the case of a
p-benzyloxyphenyl substituted compound 55, whereas ortho
substitutions afforded the most potent compounds, for
instance, compounds 54 and 49 (Ki ≈ 215 nM) harboring a
p-fluoro-o-chlorophenyl and an o-chlorophenyl group, re-
spectively. Interestingly, the incorporation of an ortho sub-
stituent also allowed a dramatic decrease of the microsomal
metabolism compared to meta-substituted derivatives (e.g.,
49.3 ( μL/min)/mg protein for 49 and 163.0 ( μL/min)/mg pro-
tein for 57).
After the o-chlorophenyl group was determined as the
potential optimal substituent for R1, the SAR at R3 position
was explored, keeping R2 as a methyl group (Table 6). A wide
variety of substituents were tolerated (benzylic, acetylenic,
aliphatic groups with H-bond donor or acceptor moieties,
etc.) as well as various functionalities (acyl hydrazine, alco-
hol, piperazine, morpholine, sulfonamide, and pyridine).
Compounds 64 and 66 were found to present solubility
issues. Among the tested compounds, 63 and 67 were the
Biological Activity. At first, the SAR around the R4 part of
the molecule was explored (Table 4). All tested N-alkylated
derivatives were found inactive on Nox4, which confirmed the
primary screening results. In contrast, acylated products were
in the same range of potency as the hit 12. Unfortunately,
these compounds proved to be unstable (readily hydrolyzed at
pH 6.5-8). These results discouraged us from further inves-
tigations on R4-acylated compounds; therefore, lead optimi-
zation was focused on compounds bearing R4 = H.
The influence of the R1 substitution was investigated
(R2 = R3 = Me). Table 5 shows the data for a representative
set of compounds. 53 can be seen as a benchmark compound
for the pyrazolopyridine dione core structure (Ki = 2175 nM
on hNox4). Heterocyclic substitution at R1 position gave
disappointing results. On the other hand, meta-substituted
phenyl substituent compounds were more active than the hit
12 with a large tolerance in functionalities: amide, nitrile,

7720 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Laleu et al.
most potent compounds with Ki ≈ 160 nM. The potency of
61, 67, and 68 advocated for a beneficial effect of having a
basic center at a three-atom distance from the core structure.
Interestingly, R-substitution of the pyridine nitrogen atom of
67 with a morpholine resulted only in a slight decrease in
activity.
The high potency demonstrated by 67 led us to investigate
further the SAR and to look at the preferential combinations
at R1 with 2-picolyl as R3 group (Table 7). Once again, sever-
al substituents were tolerated. Chloroquinoline was found
as a possible heterocyclic substitution although the affinity
for hNox4 remained unsatisfying (Ki = 458 nM for com-
pound 80). Benzylic and aliphatic groups were revealed to
be potential alternatives to the phenyl susbtituents pre-
viously identified. Furthermore, incorporation of halogens
into benzylic substituents brought beneficial effects: up to a
2-fold increase in potency compared to the unsusbtituted
benzyl group (compounds 74, 76, and 77 vs 75). Regarding
phenyl derivative analogues of 67, the o-tolyl group in com-
pound 79 gave a product almost equipotent with 67 whereas
the p-fluoro-o-chlorophenyl moiety engendered a weaker
affinity (Ki = 235 nM for 78). The length of the spacer con-
necting the pyrazolopyridine dione core to the aryl moiety
was also studied, and no loss of activity was observed up to a
three-atom chain (compounds 72 and 73).
Finally, the SAR at R2 position was explored while per-
forming limited variations at R1 and R3 positions to fine-
tune the best possible combinations (Table 8). The choice of
R1 group was restricted to o-chlorophenyl or p-fluoro-o-
chlorophenyl with the exception of 82. R3 was chosen among
a simple methyl group, alkyl ether chains, or heteroaryl deriv-
atives such as picolyl groups. At first, the rigid structure of
compound 91 showed an interesting affinity (Ki = 128 nM
with R2 = o-F-Ph). The spacer between the core structure
and the aryl moiety was then investigated for the R2 position.
All compounds displayed substantial potencies (Ki < 172 nM)
with a good tolerance of functionalities within the flexible
chain introduced: aniline, amine, halogens, phenoxy, piper-
azine, and pyridine. Phenoxymethyl derivatives 82 and 90
were less potent (Ki ≈ 114-153 nM) than the benzyloxy-
methyl analogues 88 and 89 (Ki ≈ 82-95 nM). Replacement
of the oxygen by a nitrogen atom in the spacer consolidated
the SAR around R2: more flexibility and/or additional basic
centers (Ki ≈ 47 nM for 87) seemed to bring beneficial effects
over the anilinomethyl spacer present in 84 and 86 (Ki ≈
151-172 nM). Incorporation of nitrogen heterocycles at R2,
such as pyrrolidine and piperazine, gave as well excellent
results (compounds 83 and 85). In summary, this optimiza-
tion allowed the discovery of several double-digit nanomolar
inhibitors (Ki down to 47 nM for 87) with several possible
combinations for R1, R2, and R3 groups.
Scheme 2. Acylation Methods A and Ba
a Reagents and conditions: (a) Ac2O/AcONa, 40 ꢀC; (b) ArCH2C-
(O)Cl, DIPEA, DMF, 0 ꢀC to rt.
In order to select the best compounds for pharmacokinetic
studies, the most active compounds were subjected to in vitro
ADME profiling, in particular with respect to oxidative
metabolism in human and rat liver microsomes and Caco-2
cellular permeation models. These results are combined in
Table 9.
Scheme 3. Alkylation methoda
Most compounds showed moderate to low stability to
microsomal metabolism in both species studied. Compounds
63, 81, 89, and 90 exhibited a particularly high intrinsic
a Reagents and conditions: (a) R4X (X = Cl, Br), Et3N, THF, reflux.
Scheme 4. Synthesis of Compounds of Type 31 (R2 = Me) (General Procedure B)a
a Reagents and conditions: (a) R1NHNH2, DIPEA, toluene, reflux; (b) glacial acetic acid (0.1 equiv), excess of MeC(OEt)3, 60-70 ꢀC; (c) R3NH2,
toluene, reflux; (d) i-PrONa/i-PrOH, reflux.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7721
Scheme 5. Synthesis of Final Compounds of Type 35 (General Procedure C)a
a Reagents and conditions: (a) R1NHNH2, MeOH, 25-60 ꢀC; (b) Ca(OH)2 (2 equiv), R2C(O)Cl, 1,4-dioxane, reflux; (c) glacial acetic acid (0.1 equiv),
R3NH2, toluene, 70 ꢀC; (d) MeONa/MeOH, rt.
Scheme 6. Synthesis of Final Compounds of Type 40 (General Procedure D)a
a Reagents and conditions: (a) R1NHNH2, MeOH, 25-60 ꢀC; (b) glacial acetic acid (0.1 equiv), ClCH2C(OEt)3 (3 equiv), CH3CN, 60-70 ꢀC; (c)
R3NH2, CH3CN, rt; (d) R0R00NH, DIPEA, CH3CN, reflux; (e) MeONa/MeOH, rt.
clearance in the rat liver microsomes (>125 ( μL/min)/mg
protein). In the specific case of 90, the metabolism was however
moderate for human liver microsomes (49.3 ( μL/min)/mg
protein). On the other hand, 82, 83, and 88 had acceptable
clearance values. The huge difference noted between the two
structural analogues 88 and 89 could be explained by the
higher lipophilicity of the latter compound making it more
prone to phase I oxidative metabolism. Compound 67 dis-
played a slightly high clearance in the rat liver microsomes
but a good stability to the human microsomes.
Concerning in vitro permeability, the Papp observed in
Caco-2 experiments varied between 21.4 and 78.6 ꢀ 10-6 cm/s
for most compounds. The very low values obtained for 82
and 87 can be explained in terms of log D properties (-0.35
and 1.15, respectively). These products were also character-
ized by a conspicuous efflux ratio that could be due to active
transport (data not shown). Besides, compounds 83, 84,
and 87 have at least two basic centers that can be readily
protonated, which might preclude them from easily crossing
membranes.
Also, most molecules tested show no potential problem
associated with CYP 450 inhibition (tested on five human
isoforms CYP 1A2, 2C19, 2C9, 2D6, 3A4; data not shown).
There were only a couple of exceptions: 84 showed multiple
moderate to high inhibitions probably due to the presence of
the easily accessible pyridine nitrogen in the para position
(IC50 ≈ 4.1 μM on CYP 2C9, IC50 ≈ 6.5 μM on CYP 2C19,
IC50 ≈ 1.1 μM on CYP 3A4, and IC50 ≈ 3.6 μM on CYP
2C8), and 81 inhibited primarily only one isoform (IC50
0.9 μM on CYP 2C9, IC50 ≈ 13 μM on CYP 2C19).
≈
On the basis of the data gathered, five compounds were
selected for in vivo pharmacokinetics studies in rat. The
results are summarized in Table 10. As expected from in vitro
data, 82 gave poor results with a very low oral bioavailability

7722 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Table 5. SAR: R1 Modification
Laleu et al.
a Cell free assay of ROS production on human Nox4 membranes.
and high in vivo clearance presumably because of its low
permeability along with its physical properties (Papp = 1.4 ꢀ
10-6 cm/s with efflux and log D = -0.35). Despite its low
stability to rat liver microsomes but its extremely high
plasma protein binding (Fu = 0.0% in 50% plasma protein),
compound 90 showed suitable in vivo clearance and plasma
concentrations and moderate oral bioavailability but low
volume of distribution. Interestingly, the pharmacokinetic
profile for both iv and per os routes strongly suggested the
presence of enterohepatic circulation. This phenomenon is
known to be potentially problematic because of its possible
hepatotoxicity. 88 was a promising candidate with its high
permeability ranking and reasonable stability to microsomal
metabolism. A good exposure was indeed observed, but this
compound displayed moderate bioavailability (29%) and a
low volume of distribution (Vss = 0.35 L/kg after iv dosing).
Compound 83 proved to have an acceptable oral bioavail-
ability (39%) with moderate plasma concentrations and
volume of distribution. Finally, compound 67 displayed a
good oral bioavailability associated with high plasma con-
centrations. After intravenous dosing, a half-life of 3.6 h
was observed as well as acceptable distribution into tissues
(Vss =1.08 L/kg). These in vivo data shed light on the bene-
ficial effect of the o-chloro subtituent over the phenyl group
at R1 position with a dramatic improvement compared to the
in vivo pharmacokinetic profile of the hit 12. Compound 67
constitutes a good compromise between plasma protein
binding, microsomal metabolism, permeability, and physical
properties (log D, solubility, etc.).
The Nox4 specificity of 67 was confirmed vs Nox2 isoform
(9-fold selectivity ratio) as well as in a counterscreening assay
for potential ROS scavenging (Table 11). It is worth men-
tioning that this compound was proven to be a dual Nox4/
Nox1 inhibitor (Ki = 165 and 160 nM, respectively). The
most potent pyrazolopyridine diones on Nox4 also exhibited
these key features (data not shown). The specificity of 67 was
further confirmed in an extensive in vitro pharmacological
profile (Table 12). Compound 67 had an excellent early in
vitro ADMET profile (Table 13), and no potential problem
was found regarding CYP 450 inhibition. This molecule was
not mutagenic or genotoxic based on mini Ames and in vitro
micronucleus tests. It also exhibited a low probability of
generating QT prolongation based on lack of inhibition of
hERG. Finally, the pyrazolopyridine dione 67 was demon-
strated to have no toxicity up to 1000 mg/kg (per os) over a
2 week dose range finding in mice (data not shown), which
confers a very large safety window to this compound.
Conclusion
We have identified a novel family of highly potent Nox4
inhibitors with dual activity on Nox1 and selectivity over the
isoform Nox2. Exploration of the pyrazolopyridine dione
class58 focusing on R1, R2, R3, and R4 modifications afforded
several compounds that showed good to excellent potency in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7723
Table 6. SAR: R3 Modification
a Cell free assay of ROS production on human Nox4 membranes.
our cell free assay of ROS production on human Nox4 mem-
branes. Starting from our hit compound 12, further improve-
ment of the physical properties and pharmacological profiles
led to the identification of the highly potent, orally bioavail-
able lead compound 67 (GKT136901). This compound dem-
onstrated not only a straightforward synthetic procedure
but also an excellent in vitro Nox isoforms specificity and
pharmacological and safety profiles. Also, several molecules
with Ki in the two digit nanomolar range on Nox4 and Nox1
were discovered. Those molecules proved to be highly potent
in in vitro assays on human fibroblasts differentiation and
epithelial cells apoptosis and epithelial-mesenchymal transi-
tion (EMT), as well as in preventive and curative murine
models of bleomycin induced pulmonary fibrosis. The best
pyrazolopyridine dione derivatives deserve further clinical
trials for the treatment of idiopathic pulmonary fibrosis.
electrospray. Analytical HPLC data were obtained using a Waters
2690 separation module (Alliance) equipped with a Zorbax Ex-
tend C-18 column (50 mm ꢀ 4.6 mm, 1.8 μm). Detection was con-
ducted at 254 nm and full scan at 210-400 nm with a photodiode
array detector. The mobile phase was a linear gradient with a flow
rate of 0.8 mL/min using a 10:90 A/B to 100:0 A/B mixture over
4 min. Solvent A was 0.1% formicacidinwater, and solvent B was
0.1% formic acid in acetonitrile. Silica flash and reverse phase
chromatography were performed using an Armen apparatus Spot
Flash equipped with silica SI60 15-40 μm and RP18 25-40 μm
cartridges respectively. For TLC (thin layer chromatography)
alumina sheets from Merck coated with silica gel 60 F254 were
used. All tested compounds possessed a purity of at least 95%
estabilished by HPLC. When the purity of a particular compound
was less than 95%, the percent of purity was specified at the end of
its synthesis in the Experimental Section. Reported yields were not
optimized, the emphasis being on purity of product rather than
quantity.
General Procedure A. Synthesis of Hit Compound 12. Methyl
(5-Hydroxy-1-phenyl-1H-pyrazol-3-yl)acetate (21). A stirred
mixture of dimethyl 1,3-acetonedicarboxylate (10 mmol, 1.74 g)
and phenylhydrazine (10 mmol, 1.08 g) in dry benzene (50 mL)
was heated under reflux for 8 h. The solvent was removed in vacuo
and the product was separated by recrystallization from i-PrOH
(20mL), yielding a white solid(80%yield) which was pureenough
for the following step. Mp 140-141 ꢀC (parent). 1H NMR (400
MHz, DMSO-d6) δ 11.10 (1H, bs), 7.78 (2H, d, J = 7.9 Hz), 7.35
(2H, t, J=7.6 Hz), 7.12 (1H, t, J = 7.4 Hz), 5.43 (1H, s), 3.68
(3H, s), 3.49 (2H, s). LC-MS 233.4 (M þ H)þ.
Experimental Section
Chemistry. Solvents and chemicals were reagent grade or
better and were obtained from commercial sources. All experi-
ments dealing with moisture sensitive compounds were conducted
€
under nitrogen. Melting points were measured with a Buchi B-540
melting point apparatus and were uncorrected. NMR spectra
were recorded on Bruker AMX 500, 400, and 300 MHz spectro-
meters. Chemical shifts were referenced with respect to the resid-
ual solvent signals (DMSO, 2.50 ppm; CHCl3, 7.26 ppm). Data
were reported as follows: chemical shift (δ), integration, multi-
plicity (s = singlet, bs = broad singlet, d = doublet, t = triplet,
dd = doublet of doublets, ddd = doublet of doublet of doublets,
dt=doublet of triplets, td=triplet of doublets, q=quadruplet,
m=multiplet). Coupling constants (J ) are in hertz. UPLC (ultra-
performance liquid chromatography) data were obtained using a
Waters Acquity SQ detector operating with positive or negative
Methyl [(4Z)-4-(1-Ethoxyethylidene)-5-oxo-1-phenyl-4,5-di-
hydro-1H-pyrazol-3-yl]acetate (22). A mixture of the obtained
pyrazolone 21 (1.85 g, 7.97 mmol), acetic anhydride (1.00 mL),
and triethyl orthoacetate (4.50 mL) was heated under reflux for
1 h and left overnight at room temperature. The resulting pre-
cipitate was collected by filtration and washed with diethyl ether

7724 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Table 7. SAR: R1 Modification
Laleu et al.
a Cell free assay of ROS production on human Nox4 membranes.
1
product was obtained as a beige solid (0.46 g, 58% yield). H
(10 mL) in order to obtain the crude product (1.20 g, 80% HPLC
purity, yellow solid) in 50% yield. The product was used for the
following step without further manipulation. 1H NMR (400
MHz, CDCl3) δ 7.97 (2H, d, J = 8.0 Hz), 7.37 (2H, t, J =
7.6 Hz), 7.16 (1H, t, J = 7.2 Hz), 4.31 (2H, q, J = 6.9 Hz), 3.76
(2H, s), 3.73 (3H, s), 2.78 (3H, s), 1.42 (3H, t, J = 7.1 Hz). LC-
MS 303.4 (M þ H)þ.
NMR (400 MHz, DMSO-d6) δ 10.80 (1H, bs), 8.49 (2H, bs), 7.73
(2H, d, J = 8.0 Hz), 7.56 (1H, d, J = 7.7 Hz), 7.45 (2H, t, J = 7.6
Hz), 7.36(1H, m), 7.18(1H, t, J=7.5Hz), 5.82(1H, s), 5.38(2H, s),
2.77 (3H, s). LC-MS 333.4 (M þ H)þ.
General Procedure B for Compounds of Type 31. Step 1:
Synthesis of Pyrazolone Intermediate of Type 28. Pyrazolone
Method A. To a suspension of the appropriate hydrazine (10.16
mmol) in anhydrous toluene (50 mL) were added DIPEA
(3.55 mL, 20.32 mmol) and dimethyl 1,3-acetonedicarboxylate
(20) (1.77 g, 10.16 mmol). The resulting mixture was heated at
reflux for 18 h before being concentrated in vacuo. The resulting
brown oil was purified by flash chromatography over SiO2
(CH2Cl2/MeOH, gradient).
Methyl [(4Z)-5-Oxo-1-phenyl-4-{1-[(pyridin-3-ylmethyl)amino]-
ethylidene}-4,5-dihydro-1H-pyrazol-3-yl]acetate (23). A stirred mix-
ture of compound 22 (1.20 g, 3.97 mmol) and 3-aminomethylpyr-
idine (0.45 g, 4 mmol) was heated under reflux in toluene (20 mL)
for 0.5 h and left overnight at room temperature. The resulting
enamine 23 (0.87 g) was collected by filtration and washed with
ethyl ether (20 mL), yielding a white solid (60% yield). 1H NMR
(400 MHz, CDCl3) δ 12.17 (1H, bs), 8.63 (2H, bs), 7.97 (2H, d,
J = 8.0 Hz), 7.70 (1H, d, J = 7.7 Hz), 7.38 (3H, m), 7.18 (1H, t,
J = 7.2 Hz), 4.71 (2H, d, J = 5.9 Hz), 3.82 (2H, s), 3.71 (3H, s),
2.38 (3H, s). LC-MS 365.6 (M þ H)þ.
Pyrazolone Method B. To the solution of the appropriate
hydrazine (10.16 mmol) in MeOH (25 mL) was added dimethyl
1,3-acetonedicarboxylate (20) (1.77 g, 10.16 mmol). The result-
ing mixture was heated at 40-65 ꢀC for 1 h. The solvent was
removed by evaporation, and the residue was purified by flash
chromatography over SiO2 (CH2Cl2/MeOH, gradient).
Step 2: Synthesis of Enol Ether Derivatives of Type 29. A
stirred mixture of compound from step 1 (3.75 mmol), glacial
acetic acid (21 μL, 0.375 mmol), and an excess of triethyl
orthoacetate (2.0 mL) was heated at 75 ꢀC for 1 h. The resulting
solution was concentrated in vacuo to afford a syrup that was
washed with diethyl ether and then dried in vacuo. Because of its
relative instability, no further purification was attempted.
4-Methyl-2-phenyl-5-(pyridin-3-ylmethyl)-1H-pyrazolo[4,3-c]-
pyridine-3,6(2H,5H)-dione (12). A stirred solution of compound
23 (0.87 g, 2.39 mmol) in anhydrous i-PrOH (10 mL) was treated
with a freshly prepared solution of i-PrONa-i-PrOH, obtained
by dissolving sodium (0.055 g, 2.39 mmol) in i-PrOH (50 mL)
under nitrogen. The reaction mixture was heated under reflux
for 9 h, then cooled and neutralized to pH 7 with 20% HCl aque-
ous solution. The precipitate formed was filtered off, washed
with water (20 mL), and air-dried. Chromatographically pure

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7725
Table 8. SAR: R1, R2, and R3 Optimization
a Cell free assay of ROS production on human Nox4 membranes.
Step 3: Synthesis of Enamine Derivative of Type 30. A stirred
mixture of compound from step 2 (3.75 mmol) and appropriate
amine (3.75 mmol) in toluene (20 mL) was stirred at room
temperature for 15 min. The solvent was removed in vacuo. The
resulting residue was dissolved in a minimum of CH2Cl2 and
precipitated with diethyl ether. The desired product was usually
obtained as a precipitate in good to moderate yields.
Step 4: Cyclization. Compound from step 3 (3.57 mmol) was
treated with an isopropanolic solution of i-PrONa, obtained by
dissolving sodium (0.082 g, 3.57 mmol) in i-PrOH (35 mL). The
reaction mixture was heated under reflux for 1 h, then cooled
and neutralized to pH 7 by addition of 20% HCl aqueous solu-
tion. The organic solvent was removed in vacuo, and water
was added before placing the flask in the fridge overnight. The

7726 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Laleu et al.
precipitate formed was filtered off, washed with diethyl ether,
and dried in vacuo to afford the desired pure product.
Following the above-described general procedure B, using the
appropriate hydrazines and primary amines, compounds 49, 53,
54, 63, 67, 68, 78, 79 were obtained with 20-52% overall yields.
2-(2-Chlorophenyl)-4,5-dimethyl-1H-pyrazolo[4,3-c]pyridine-
3,6(2H,5H)-dione (49). Isolated as a white solid. 1H NMR (400
MHz, DMSO-d6) δ 10.56 (1H, bs), 7.61-7.59 (1H, m), 7.52-
7.49 (1H, m), 7.45-7.42 (1H, m), 5.62 (1H, s), 3.43 (3H, s), 2.74
(3H, s). LC-MS 290.5 (M þ H)þ.
lated as a yellowish solid (92% purity by HPLC). 1H NMR (400
MHz, DMSO-d6) δ 7.63-7.60 (1H, m), 7.57-7.52 (2H, m),
7.48-7.36 (2H, m), 6.77 (1H, d, J = 8.8 Hz), 6.48 (1H, d, J = 7.2
Hz), 5.70 (1H, s), 5.27 (2H, s), 3.62 (2H, t, J = 5.2 Hz), 3.36 (2H, t,
J = 5.2 Hz), 2.77 (3H, s). LC-MS 452.3 (M þ H)þ.
2-(2-Chloro-4-fluorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-
1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (78). Isolated as a
yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (1H, d, J =
4.4 Hz), 7.94 (1H, td, J = 8.0 Hz, J = 2.0 Hz), 7.67-7.59 (2H,
m), 7.45-7.40 (2H, m), 7.35 (1H, td, J = 8.0 Hz, J = 2.0 Hz),
5.67 (1H, s), 5.43 (2H, s), 2.77 (3H, s). LC-MS 385.0 (M þ H)þ.
4-Methyl-2-(2-methylphenyl)-5-(pyridine-2-ylmethyl)-1H-pyra-
zolo[4,3-c]pyridine-3,6(2H,5H)-dione (79). Isolated as a yellow
2,4,5-Trimethyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione
1
(53). Isolated as a yellow solid (94% HPLC purity). H NMR
(400 MHz, DMSO-d6) δ 5.48 (1H, s), 3.40 (3H, s), 3.16 (3H, s),
2.72 (3H, s). LC-MS 194.3 (M þ H)þ.
solid. H NMR (400 MHz, DMSO-d6) δ 10.71 (1H, bs), 8.47
1
2-(2-Chloro-4-fluorophenyl)-4,5-dimethyl-1H-pyrazolo[4,3-c]-
pyridine-3,6(2H,5H)-dione (54). Isolated as a yellowish solid. 1H
NMR (400 MHz, DMSO-d6) δ 10.56 (1H, bs), 7.64 (1H, dd, J =
8.8 Hz, J = 3.2 Hz), 7.58 (1H, dd, J = 8.8 Hz, J = 5.6 Hz), 7.34
(1H, td, J = 8.8 Hz, J = 3.2 Hz), 7.25-7.20 (2H, m), 5.63 (1H, s),
3.43 (3H, s), 2.74 (3H, s). LC-MS 308.7 (M þ H)þ.
(2H, d, J = 3.2 Hz), 7.78 (1H, t, J = 6.0 Hz), 7.38-7.28 (6H, m),
5.66 (1H, s), 5.41 (2H, s), 2.79 (3H, s), 2.25 (3H, s). LC-MS 347.5
(M þ H)þ.
General Procedure C for the Synthesis of Compounds of Type
35. Starting from the appropriate pyrazolone of type 28 whose
synthesis has been already described (pyrazolone method B), the
following molecules were generated.
2-(2-Chlorophenyl)-4-methyl-5-(3-phenylprop-2-yn-1-yl)-1H-
pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (63). Isolated as a yel-
lowish solid. 1H NMR (400 MHz, DMSO-d6) δ 10.81 (1H, bs),
7.63-7.61 (1H, m), 7.55-7.52 (1H, m), 7.37-7.33 (3H, m), 4.35
(3H, m), 5.68 (2H, s), 5.17 (2H, s), 2.95 (3H, s). LC-MS 390.8
(M þ H)þ.
Step 1: Synthesis of C-Acylated Pyrazolone 33. A stirred
solution of the appropriate pyrazolone 28 (1.9 mmol) in anhy-
drous 1,4-dioxane (10 mL) was treated with Ca(OH)2 (0.282 g,
3.8 mmol) and the suitable acyl chloride (1.9 mmol) under N2.
The mixture was heated at 70 ꢀC, and the rearrangement was
easily monitored by HPLC or TLC system because the acylated
oxygen product was less polar than the desired product (1-3 h).
The solvent was concentrated in vacuo, and the residue was
partitioned between dichloromethane (50 mL) and a mixture of
2-(2-Chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo-
[4,3-c]pyridine-3,6(2H,5H)-dione (67). Isolated as a pale yellow
1
solid. Mp 210-212 ꢀC (HCl salt, dec). H NMR (500 MHz,
DMSO-d6) δ 10.74 (1H, bs), 8.48-8.46 (1H, m), 7.79 (1H, td, J =
7.6 Hz, J = 1.9 Hz), 7.67-7.64 (1H, m), 7.60-7.56 (1H, m), 7.50-
7.47 (2H, m), 7.33-7.28 (2H, m), 5.66 (1H, s), 5.41 (1H, s), 2.78
(13H, s). LC-MS 367.1 (M þ H)þ.
Table 11. Activities of Lead Compound 67 on Flavoenzymes
parameter
2-(2-Chlorophenyl)-4-methyl-5-[(6-morpholin-4-ylpyridin-2-yl)-
methyl]-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (68). Iso-
Ki (hNox4)a
165 ( 5 nM
160 ( 10 nM
1530 ( 90 nM
>30000 nM
Ki (hNox1)b
Ki (hNox2)c
Ki (xanthine oxidase)d
Table 9. In Vitro ADME Characterization for Selected Compounds
Cl (h-LM),a (μL/ Cl (r-LM),b (μL/ Caco-2 (Papp),c
a Cell free assay of ROS production on Nox4 membranes. b Cell free
assay of ROS production on Nox1 membranes. c Cell free assay of ROS
production on Nox2 membranes. d In vitro enzymatic assay.
compd min)/mg protein
min)/mg protein
(10-6 cm/s) log D d
63
178.0
70.2
71.6
3.18
67
68
78
81
82
83
84
85
87
88
89
90
24.8
21.4
1.59
2.21
1.73
2.03
-0.35
1.61
3.23
2.22
1.15
1.55
2.77
2.39
Table 12. In Vitro Pharmacological Profile of Lead 67
37.6
96.4
45.1
45.4
76.3
57.9
24.8
32.4
212.0
49.3
84.2
13.4
39.9
1.4
inhibition
at 10 μM
(%)
inhibition
at 10 μM
(%)
248.0
45.3
62.3
off target assay
off target assay
7.3
4.6
12-lipoxygenase (h)
15-lipoxygenase
AMPK (r)
2.0
0
p38 MAP kinase (h)
MEK1 (h)
10.0
0
78.0
121.0
74.1
49.5
0
myeloperoxidase (h)
PKCR,β,δ (h)
PI 3KR,β,γ,δ
PTP1B (h)
0
0.5
78.6
62.6
35.5
ASK1 (h)
Ca2þ channel
Cl- channel
0
0
0
0
210.0
125.0
0
0
Naþ channel
4.0
0
iNOS
eNOS (h)
ETA (h)
0
a Microsome stability experiment. h-LM, human liver microsomes.
b Microsome stability experiment. r-LM, rat liver microsomes. c Caco-2 cell
permeability assay. Papp, permeability coefficient. d Calculated at pH 7.4.
K
þ channel
MAO-B
6.0
0
0
Table 10. In Vivo Pharmacokinetic Profile in Rats for Selected Compounds
compd
route
dose (mpk)
AUC¥ (h ng/mL)
FZ (%)
Cmax (ng/mL)
Tmax (h)
t1/2 (h)
Cl ((L/kg)/h)
0.47
Vss (L/kg)
3
67
iv
5
10
5
10979
10937
822
12126
3147
4637
132
3.63
1.08
os
iv
54
6
1.50
0.25
0.33
0.25
1.17
82
83
88
90
6.52
96.50
1.61
4.39
0.33
1.20
0.77
2.92
2.95
1.49
0.35
0.80
os
iv
10
5
109
2.35
3.30
1.13
3.14
2.38
3.47
3.03
3130
2427
15381
8933
6528
3633
6191
2834
36754
6083
30383
558
os
iv
10
5
39
29
28
os
iv
10
5
os
10

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7727
1
Table 13. Early in Vitro ADMET Profile of 67
yellow solid. Mp 86-87 ꢀC (HCl salt). H NMR (400 MHz,
DMSO-d6) δ 8.57(1H, s), 8.44(2H, m), 7.65-7.58 (2H, m), 7.48-
7.46 (2H, m), 7.21-7.20 (3H, m), 7.12-7.10 (2H, m), 5.80 (1H, s),
5.48 (2H, s), 5.18 (2H, s), 4.49 (2H, s). LC-MS 474.1 (M þ H)þ.
4-{[(2-Chlorobenzyl)oxy]methyl}-2-(2-chlorophenyl)-5-methyl-
1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (89). Isolated as a
parameter
IC50 (1A)a
>25 μM
>25 μM
>25 μM
>25 μM
IC50 (2C19)a
IC50 (2C9)a
IC50 (2D6)a
IC50 (3A4)a
Cl (h-LM)b
Cl (r-LM)c
Caco-2 (Papp
1
yellow solid (15% yield). H NMR (400 MHz, DMSO-d6) δ
20.9 μM
10.76 (1H, bs), 7.68-7.64 (1H, m), 7.60-7.57 (1H, m), 7.52-7.49
(3H, m), 7.45-7.43.39 (1H, m), 7.35-7.32 (2H, m), 5.82 (1H, s),
5.23 (2H, s), 4.67 (2H, s), 3.56 (3H, s). LC-MS 431.4 (M þ H)þ.
2-(2-Chlorophenyl)-4-[(4-fluorophenoxy)methyl]-5-methyl-1H-
pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (90). Isolated as a yel-
low solid. Mp 129-130 ꢀC (parent). 1H NMR (400 MHz, DMSO-
d6) δ 10.85 (1H, bs), 7.64-7.55 (2H, m), 7.48-7.45 (2H, m), 7.12-
7.09 (4H, m), 5.84 (1H, s), 5.68 (1H, s), 3.55 (3H, s). LC-MS 400.0
(M þ H)þ.
24.8 (μL/min)/mg protein
70.2 (μL/min)/mg protein
21.4 ꢀ 10-6 cm/s
20.5%
d
)
e
Fu
hERG f
0% at 1 μM
negative
negative
mini AMES testg
in vitro micronucleush
a Inhibition of cytochrome P450 isoforms (see Experimental Section).
b Microsome stability experiment. h-LM, human liver microsomes.
c Microsome stability experiment. r-LM, rat liver microsomes. d Caco-
2 cell permeability assay. Papp, permeability coefficient. e Plasma protein
binding assay (in 50% plasma protein). f Conventional patch-clamp.
g Performed at 100 μM. h Performed at 500 μM.
General Procedure D for the Synthesis of Compounds of Type
40. Starting from the appropriate pyrazolone 28 whose synthesis
has been already described (pyrazolone method B), the follow-
ing molecules were generated.
Step 1: Synthesis of r-Chloroketone Derivative of Type 37. A
stirred mixture of the appropriate pyrazolone 28 (2.35 mmol) in
acetonitrile (10 mL) was treated with 2-chloro-1,1,1-triethoxy-
ethane (1.34 mL, 7.05 mmol) and glacial acetic acid (27 μL, 0.47
mmol) under N2. The reaction mixture was heated to 70 ꢀC and
monitored by LC-MS until disappearance of starting material.
The evaporation of the solvent until dryness by rotoevaporation
at 45 ꢀC allowed the reaction to go to completion. Thecrude dark
oil was used in the following step without further purification.
Step 2: Synthesis of Enamine of Type 38. Compound from step
1 (2.25 mmol) was dissolved in acetonitrile (10 mL), and the
appropriate amine (2.03 mmol) was added at room temperature
under N2. The reaction was monitored by UPLC-MS showing
the disappearance of the starting material after only 10 min. The
solvent was evaporated, and the crude product was used in the
following step without further purification.
Step 3: Chloride Displacement. Compound from step 2 (2.25
mmol) was dissolved in acetonitrile (10 mL), and the appro-
priate amine (2.03 mmol) followed by DIPEA (0.392 mL, 2.25
mmol) was added at room temperature under N2. The mixture
was warmed to 50 ꢀC until the disappearance of starting mate-
rial. The solvent was evaporated, and the crude mixture was
partitioned between ethyl acetate and an aqueous solution of
saturated sodium bicarbonate. The combined organic layers
were dried over Na2SO4 and concentrated in vacuo. The crude
product was used in the following step without further manip-
ulation or purified by silica plug.
Step 4: Cyclization. The enamine from step 3 (1.4 mmol) was
treated with a freshly prepared solution of MeONa in MeOH
(2 M, 20 mL). The solution was stirred at room temperature until
disappearance of the starting enamine (0.5-2 h). The reaction
mixture was concentrated in vacuo to evaporate MeOH, and the
crude was dissolved in ethyl acetate (80 mL) and extracted with
water (3 ꢀ 30 mL). The combined aqueous layers were acidified
to pH 6 and extracted with ethyl acetate (3 ꢀ 30 mL). Then the
combined organic layers were dried over Na2SO4. The crude mix-
ture was purified by silica chromatography or reverse phase chro-
matography to afford the desired product.
brine (10 mL) and 2 M HCl (1 mL, 2.00 mmol). The organic
phase was separated, and the aqueous one was extracted with
dichloromethane (50 mL) and then ethyl acetate (50 mL). The
combined organic phases were dried over Na2SO4 and the
solvent was evaporated to give the final product pure enough
to be used for the following step without further purification.
Step 2: Synthesis of Enamine of Type 34. Compound from step
1 (1.9 mmol) was dissolved in toluene (20 mL), and the appro-
priate amine (1.9 mmol) and glacial acetic acid (0.12 mL, 2.09
mmol) were added at room temperature under N2. The reaction
mixture was stirred at 70 ꢀC and monitored by LC-MS showing
the disappearance of the starting material. The reaction mixture
was evaporated until dryness. The residue was dissolved with a
minimum amount of ethyl acetate (containing 0.1% triethylamine)
and passed through a silica gel plug, eluting with ethyl acetate/
0.1% triethylamine. Evaporation of the solvent gave the desired
enamine which was used in the following step without further
purification.
Step 3: Cyclization. The enamine from step 2 (1.4 mmol) was
treated with a freshly prepared solution of MeONa in MeOH
(2 M, 20 mL). The solution was stirred at room temperature
until disappearance of the starting enamine (0.5-2 h). The
reaction mixture was concentrated in vacuo to eliminate MeOH,
and the crude was dissolved in ethyl acetate (80 mL) and extracted
with water (3 ꢀ 30 mL). Then combined inorganic layers were
acidified to pH 6, extracted with ethyl acetate (3 ꢀ 30 mL)
and the combined organic layers dried over Na2SO4. The crude
was purified by silica chromatography or reverse phase chro-
matography to afford the desired product.
Following the above-described general procedure C, using
the appropriate hydrazines, acyl chloride, and primary amines,
compounds 82, 84, and 88-90 were obtained with 15-30%
overall yield.
4-[(4-Fluorophenoxy)methyl]-5-(2-methoxyethyl)-2-(2-morpholin-
4-ylethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (82).
1
Isolated as red solid. Mp 48-49 ꢀC (HCl salt). H NMR (400
MHz, DMSO-d6) δ 10.78 (1H, bs), 7.18-7.09 (4H, m), 5.91 (1H,
s), 5.68 (2H, s), 4.35-4.25 (2H, m), 4.10-4.00 (2H, m), 3.98-3.90
(2H, m), 3.80-3.65 (2H, m), 3.60-3.40 (6H, m), 3.20 (3H, s),
3.15-3.05 (2H, m). LC-MS 447.3 (M þ H)þ.
Following the described general procedure D, using the
appropriate hydrazines and primary and secondary amines,
compounds 81, 83, 85, and 87 were obtained with 15-30%
overall yield.
2-(2-Chlorophenyl)-4-{[methyl(phenyl)amino]methyl}-5-(pyridin-
4-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (84). Iso-
4-{[Benzyl(methyl)amino]methyl}-2-(2-chloro-4-fluorophenyl)-
5-(3-methoxypropyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione
(81). Isolated as a yellow solid (93% HPLC purity). H NMR
1
lated as a yellow solid. H NMR (400 MHz, DMSO-d6) δ 11.26
1
(1H, bs), 8.55 (2H, d, J = 6.4 Hz), 7.70-7.63 (2H, m), 7.54-7.52
(2H, m), 7.26 (2H, d, J = 6.4 Hz), 7.04 (2H, t, J = 4.4 Hz), 6.70
(2H, t, J = 7.2 Hz), 6.50 (2H, d, J = 7.2 Hz), 5.92 (1H, s), 5.35 (2H,
s), 5.11 (2H, s), 2.48 (3H, s). LC-MS 472.2 (M þ H)þ.
(400 MHz, DMSO-d6) δ 10.62 (1H, bs), 7.68 (1H, dd, J = 7.0 Hz,
J = 2.2 Hz), 7.64 (1H, dd, J = 7.2 Hz, J = 4.8 Hz), 7.33-7.24
(6H, m), 5.72 (1H, s), 4.29 (2H, t, J = 6.2 Hz), 4.13 (2H, m), 3.60
(2H, s), 3.23 (3H, s), 2.14 (3H, s), 1.86-1.81 (2H, m). LC-MS
486.5 (M þ H)þ.
4-[(Benzyloxy)methyl]-2-(2-chlorophenyl)-5-(pyrazin-2-ylmethyl)-
1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (88). Isolated as a

7728 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Laleu et al.
2-(2-Chloro-4-fluorophenyl)-5-(2-pyridin-2-ylethyl)-4-(pyrrolidin-
1-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (83). Iso-
lated as a beige solid. Mp 163-164 ꢀC (2HCl salt). 1H NMR (400
MHz, DMSO-d6) δ 8.8 (1H, d, J = 6.0 Hz), 8.56 (1H, t, J = 8.0
Hz), 8.07 (1H, d, J=8.0Hz), 7.99(1H, t, J=6.8Hz),7.70(1H, dd,
J=8.8Hz,J=5.6Hz),7.53(1H,dd, J=8.4Hz, J= 2.8 Hz), 7.32
(1H, td, J = 8.8 Hz, J = 2.8 Hz), 6.10 (1H, s), 5.35 (2H, s), 4.78
(2H, t, J = 7.2 Hz), 3.76-3.56 (4H, m), 3.65 (2H, t, J = 7.2 Hz),
2.25-2.10 (4H, m). LC-MS 468.1 (M þ H)þ.
the absence of agonist. Antagonist activity of compounds was
measured in the presence of increasing concentrations ranging
from 1 nM to 100 μM. After 20 min of incubation at 37 ꢀC, ROS
levels were measured using a BMG Labtech microplate reader.
Data Analysis. Data were analyzed using Prism (GraphPad
Software Inc., San Diego, CA). Ki values were calculated using
the Cheng-Prusoff equation and represent the average of at
least three individual experiments performed in triplicate.
Oxidative Metabolism. Pooled human liver microsomes
(pooled male and female) and pooled rat liver microsomes
(male Sprague-Dawley rats) were used to screen the metabolic
instability resulting from phase I oxidation. Microsomes (final
concentration of 0.5 mg/mL), 0.1 M phosphate buffer, pH 7.4,
and test compound (final substrate concentration of 0.5 μM;
final DMSO concentration of 0.25%) were added to the assay
plate and preincubated at 37 ꢀC.
NADPH solution (final incubation concentration of 1 mM)
was added to initiate the reaction. Each compound is incubated
for 0, 5, 15, 30, and 45 min. The control (minus NADPH) was
incubated for 45 min only. The reactions were stopped by the
addition of 50 μL of methanol containing internal standard at
the appropriate time points. The samples were centrifuged at
2500 rpm for 20 min at 4 ꢀC to precipitate the protein. Samples
were analyzed by LC/MS-MS. The percentage of compound
disappearance and the in vitro intrinsic clearance were calcu-
lated according to the literature.57
Cytochrome P450 Inhibition. Six human recombinant cyto-
chrome P450 isoenzymes (BD Gentest, Woburn, MA) were tested
(CYP 1A2, CYP 2C8, CYP 2C9, CYP 2C19, CYP 2D6, and
CYP 3A4). Test compound (0.1-25 μM) was incubated (final
DMSO concentration of 0.3%) with specific species liver micro-
somes and NADPH in the presence of a cytochrome P450 isoform-
specific probe substrate. For the CYP 2C8, CYP 2C9, CYP 2C19,
CYP 2D6, and CYP 3A4 specific reactions, the metabolites were
monitored by mass spectrometry. CYP 1A2 activity was mon-
itored by measuring the formation of a fluorescent metabolite. A
decrease in the formation of the metabolite compared to the vehicle
control was used to calculate an IC50 value.
2-(2-Chlorophenyl)-4-{[4-(3-methoxyphenyl)piperazin-1-yl]-
methyl}-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione
(85). Isolated as a yellow solid. 1H NMR (400 MHz, DMSO-d6)
δ 7.67-7.61 (2H, m), 7.51-7.48 (2H, m), 7.15 (1H, t, J = 8.2
Hz), 6.65-6.50 (2H, m), 6.50-7.40 (1H, m), 5.92 (1H, s), 3.70-
3.3 (14H, m). LC-MS 480.1 (M þ H)þ.
2-(2-Chlorophenyl)-5-[(1-methyl-1H-pyrazol-3-yl)methyl]-4-
{[methyl(pyridin-3-ylmethyl)amino]methyl}-1H-pyrazolo[4,3-c]-
pyridine-3,6(2H,5H)-dione (87). Isolated as a yellow solid. H
1
NMR(400 MHz, DMSO-d6) δ10.67(1H, bs),8.44(1H, d, J= 1.6
Hz), 8.42 (1H, dd, J = 3.6 Hz, J = 1.2 Hz), 7.68-7.62 (2H, m),
7.60-7.55 (2H, m), 7.48 (2H, dd, J = 4.8 Hz, J = 2.8 Hz), 7.29
(1H, dd, J = 6.0 Hz, J = 3.6 Hz), 6.05 (1H, d, J = 1.6 Hz), 5.68
(1H, s), 5.55 (2H, s), 4.31-4.24 (2H, m), 3.73 (3H, s), 2.22 (3H, s).
LC-MS 491.2 (M þ H)þ.
Biological Assays. Plasmid Construction. Human Nox1
(NM_007052.4) cDNA was cloned into the pcDNA5/TO
(Invitrogen). Human p22 (NM_000101.2) was cloned into
pcDNA3.1/Zeo (þ) (Invitrogen), and human NOXA1
(AY255769.1) and human NOXO1 (AB097667) were cloned
into the bicistronic pVITRO1-neo-mcs plasmid (Invivogen).
Cell Culture and Stable Transfected Cell Line Generation.
T-REx-CHO cells (Invitrogen) were cultured in Ham’s F12
containing 4.5 g/L glucose supplemented with 10% fetal calf
serum, 2 mM glutamine, penicillin (100 U/mL), streptomycin
(100 μg/mL), and blasticidin (5 μg/mL). Cells stably expressing
functional human Nox1 (hNOX1 T-REx-CHO) were obtained
by co-transfecting T-REx-CHO with human NOX1, human
p22, human NOXA1, and human NOXO1 using the FuGENE 6
method (Roche, 11988387).
Caco-2 Permeability. Caco-2 cells were obtained from Amer-
ican Type Culture Collection (Rockville, MD). The cells were
seeded onto Millipore multiscreen Caco-2 plates at 1 ꢀ 105 cell/cm2
and grown in Dulbecco’s modified Eagle’s medium, and media
were changed every 2 or 3 days. Permeability studies were
performed with the monolayers cultured for 20 days. On day
20, the monolayers were prepared by rinsing both basolateral
and apical surfaces twice with HBSS at 37 ꢀC. Prior to all
experiments, the cell monolayer integrity was evaluated by trans
T-REx-293 cell line expressing hNOX4 (hNOX4 T-REx-293)
was a gift from the Department of Immunology and Pathology,
University of Geneva, Switzerland.55 hNOX4 T-REx-293 cells
were cultured in DMEM containing 4.5 g/L glucose supplemented
with 10% fetal calf serum, penicillin (100 U/mL), and streptomy-
cin (100 μg/mL) at 37 ꢀC in air with 5% CO2. Human Nox1 and
human Nox4 expression were induced with tetracycline (1 μg/mL)
for 24 h prior to the membrane preparation.
Membrane Preparation. Membranes from human polymor-
phonuclear (PMN) cells (expressing high levels of Nox2) or from
cells overexpressing Nox1 or Nox4 were prepared as previously
described.56 After resuspension in sonication buffer (11% su-
crose, 120 mM NaCl, 1 mM EGTA in PBS, pH 7.4, for Nox4-
expressing cells) or in relax buffer (10 mM Pipes, 3 mM NaCl,
3.5 mM MgCl2, 0.1 M KCl, pH 7.4), cells were broken by sonica-
tion and centrifuged (200g, 10 min). The supernatant was layered
onto a 17/40% (w/v) discontinuous sucrose gradient and centri-
fuged (150000g for 30 min). Membrane fractions were collected
from the 17/40% interface and were stored at -80 ꢀC. Protein
concentration was determined with Bradford reagent.
ROS Production Measurement. Reactive oxygen species (ROS)
production by membranes expressing human Nox1, human Nox2,
or human Nox4 or by xanthine oxidase were measured using the
Amplex Red (AR) method following a slightly modified version
of the manufacturer’s instruction manual (Invitrogen). Briefly,
membranes expressing different Nox subunits or xanthine
oxidase were incubated in PBS with Amplex Red, horseradish
peroxidase (HRP), and appropriate cofactors. ROS production
was induced by addition of NADPH to Nox expressing mem-
branes or by addition of xanthine to xanthine oxidase. Non-
specific signal was measured in the absence of membranes or in
epithelial electrical resistance; values greater than 800 Ω cm2
3
were used. Hank’s balanced salt solution buffer was then
removed from either the apical compartment (for apical to
basolateral transport, A to B) or basolateral compartment (for
basolateral to apical transport, B to A) side of the monolayer
and replaced with test compound dosing solutions. The perme-
ability studies were initiated by adding an appropriate volume of
solution made by diluting 10 mM test compound in DMSO with
HBSS pH 6.5 buffer to give a final test compound concentration
of 10 μM (final DMSO concentration of 1%). The monolayers
were then placed in an incubator at 37 ꢀC. At the end of the
incubation time (2 h), samples were taken from both the apical
and basolateral compartments. Test and control compounds
were quantified by LC-MS/MS cassette analysis using a five-
point calibration with appropriate dilution of the samples. The
permeability coefficient (Papp) was calculated according to the
following equation: Papp = (dQ/dt)/(C0A), where dQ/dt is the
rate of permeation of the drug across the cell, C0 is the donor
compartment concentration at time zero, and A is the area of the
cell monolayer. C0 is obtained from analysis of donor and
receiver compartments at the end of the incubation period.
In Vivo Pharmacokinetic Studies in Rat. These studies were
conducted to estimate the plasma pharmacokinetic parameters

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7729
and the oral bioavailability of compounds in the male Sprague-
Dawley rat. These studies were carried out using a parallel
design using six male rats. Three animals in group 1 were given
5 mg/kg iv dose. Three animals in group 2 were given 10 mg/kg
oral dose. Blood samples were collected from the carotid arterial
cannula at various times up to 24 h following administration of
compound. Plasma concentrations of compound were quanti-
fied by LC/MS/MS (LLOQ = 1.00 ng/mL). Noncompartmen-
tal methods were used for pharmacokinetic data analysis.
The vehicle used for per os administration was an aqueous
solution of 0.5% CMC (carboxymethylcellulose) and 0.25%
Tween 20. The vehicle used for iv injection was an aqueous
solution of EDPW (10% EtOH, 10% DMA, 30% PG, and 50%
water).
(18) Lambeth, J. D.; Krause, K. H.; Clark, R. A. NOX enzymes as novel
targets for drug development. Semin. Immunopathol. 2008, 30, 339–
363.
(19) Hecker, L.; Vittal, R.; Jones, T.; Jagirdar, R.; Luckhardt, T. R.;
Horowitz, J. C.; Pennathur, S.; Martinez, F. J.; Thannickal, V. J.
NADPH oxidase-4 mediates myofibroblast activation and fibro-
genic responses to lung injury. Nat. Med. 2009, 15, 1077–1081.
(20) Li, S.; Tabar, S. S.; Malec, V.; Eul, B. G.; Klepetko, W.; Weissmann,
€
N.;Grimminger, F.; Seeger, W.;Rose, F.; Hanze, J. NOX4regulates
ROS levels under normoxic and hypoxic conditions, triggers pro-
liferation, and inhibits apoptosis in pulmonary artery adventitial
fibroblasts. Antioxid. Redox Signaling 2008, 10, 1687–1697.
(21) Sanders, K. A.; Hoidal, J. R. The NOX on pulmonary hyperten-
sion. Circ. Res. 2007, 101, 224–226.
(22) Tojo, A.; Asaba, K.; Onozato, M. L. Suppressing renal NADPH
oxidase to treat diabetic nephropathy. Expert Opin. Ther. Targets
2007, 11, 1011–1018.
(23) Matsushima, S.; Kinugawa, S.; Yokota, T.; Inoue, N.; Ohta,
Y.; Hamaguchi, S; Tsutsui, H. Increased myocardial NAD(P)H
oxidase-derived superoxide causes the exacerbation of postinfarct
heart failure in type 2 diabetes. Am. J. Physiol.: Heart Circ. Physiol.
2009, 297, 409–416.
Acknowledgment. We thank He Haiying, Cao Yafeng, Lei
Jianguang (WuXi AppTec, Shanghai), and the Wuxi AppTec
chemistry team for their participation in this work.
(24) Gao, L.; Mann, G. E. Vascular NADPH oxidase activation in
diabetes: a double-edged sword in redox signalling. Cardiovasc.
Res. 2009, 82, 9–20.
(25) Juhasz, A.; Ge, Y.; Markel, S.; Chiu, A.; Matsumoto, L.; Van
Balgooy, J.; Roy, K.; Doroshow, J. H. Expression of NADPH
oxidase homologues and accessorygenes in human cancer cell lines,
tumours and adjacent normal tissues. Free Radical Res. 2009, 43,
523–532.
Supporting Information Available: Synthetic methods for
compounds 18, 19, 41-45, 46-48; characterization data for
compounds 8-11, 13-17, 50-52, 55-62, 64-66, 69-77, 80, 86,
91; spectroscopic and analytical data for compounds 67, 82, 83,
88, 90. This material is available free of charge via the Internet at
http://pubs.acs.org.
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factor 2-A transcriptional activity in von Hippel-Lindau-deficient
renal cell carcinoma. Cancer Res. 2005, 65, 9190–9193.
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Clark, R. A.; Yoneda, T.; Abboud, H. E. NAD(P)H oxidases
regulate HIF-2alpha protein expression. J. Biol. Chem. 2007, 282,
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