N-Substituted pyrazole-3-carboxamides as inhibitors of human 15-lipoxygenase

Article abstract of DOI:10.1016/j.bmcl.2015.05.007

High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LOX-1). One hit, a 1-benzoyl substituted pyrazole-3-carboxanilide (1a), was used as a starting point in a program to develop potent and selective 15-LOX-1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2015.05.007

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3017–3023
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Substituted pyrazole-3-carboxamides as inhibitors of human
15-lipoxygenase
b
Benjamin Pelcman a, , Andrei Sanin , Peter Nilsson a,b, Wesley Schaal a,b, Kristofer Olofsson b,
⇑
Christian Krog-Jensen b, Pontus Forsell b, Anders Hallberg a, Mats Larhed a, Thomas Boesen d,
Hasse Kromann d, Hans-Erik Claesson b,c
a Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden
b Biolipox AB, Berzelius väg 3, SE-171 65 Solna, Sweden
c Department of Medicine, Building A3:02, Karolinska University Hospital Solna and Karolinska Institutet, SE-171 76 Stockholm, Sweden
d MedChem ApS, Fruebjergvej 3, DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LOX-1).
One hit, a 1-benzoyl substituted pyrazole-3-carboxanilide (1a), was used as a starting point in a program
to develop potent and selective 15-LOX-1 inhibitors.
Received 3 March 2015
Revised 5 May 2015
Accepted 6 May 2015
Available online 15 May 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
15-Lipoxygenase
Inhibitors
Arachidonic acid
Pyrazole
The mammalian lipoxygenases belong to a family of struc-
turally related lipid peroxidizing enzymes that are implicated in
the pathogenesis of asthma and other inflammatory diseases.
Human cells express three main types of lipoxygenases, which
catalyse the insertion of molecular oxygen into arachidonic acid
(AA) at carbon 5, 12 and 15, and hence are usually named 5-, 12-
and 15-lipoxygenase, respectively. 5-Lipoxygenase is the key
enzyme in leukotriene (LT) synthesis, and together with the 5-
lipoxygenase-activating protein (FLAP), it catalyses the first step
in the conversion of arachidonic acid to leukotrienes (Scheme 1).1
Human 15-lipoxygenase exists in two forms, named 15-LOX-1
(also named 12/15-LOX, 15-LO-1) and 15-LOX-2.2 Several reports
indicate that 15-LOX-1 has a pathophysiological role in respiratory
inflammatory diseases, in particular asthma.3 Increased activity of
15-LOX-1 is displayed in the respiratory tract in patients with
asthma. 15-LOX-1 is expressed in distinct sets of human cells, for
example, airway epithelial cells, eosinophils, mast cells, dendritic
cells and macrophages, and an increased expression of the enzyme
is often observed in inflammatory diseases, especially those driven
by IL-4/IL-13.3 Studies of both human airway epithelial cells and in
rodents indicate that 15-LOX-1 might be involved in mucus
production in the respiratory tract.4,5 Increased expression and/or
activity of 15-LOX-1 have also been demonstrated in Alzheimer’s
disease, indicating a possible pathophysiological function in this
condition.6,7 There are also reports indicating a role of 15-LOX-1
in cell differentiation and maturation of erythrocytes.2 Some stud-
ies also suggest a role of 15-LOX-1 in neoplasia such as prostate
cancer and colon carcinoma cells.3 Finally, inhibition of 15-LOX-1
has been investigated for the treatment of stroke.8
The predominant arachidonic acid metabolite formed via the
15-LOX-1 pathway is 15(S)-hydroperoxyeicosatetraenoic acid
(15(S)-HPETE) which in turn can be reduced to 15(S)-hydroxye-
icosatetraenoic acid (15(S)-HETE) (Scheme 1). Increased expression
of 15-LOX-1 in the respiratory tract and elevated levels of 15-HETE
in broncoalveolar lavage fluid have been found in asthmatic
patients compared to control subjects.3,4 In addition, 15(S)-HPETE
can also be converted to the epoxide eoxin A4 (EXA4).9 This
metabolite can be conjugated with glutathione yielding EXC4,
which in turn can be converted to EXD4 and EXE4 (Scheme 1).
The biosynthesis of eoxins has been demonstrated in eosinophils,
mast cells, nasal polyps, and in the Hodgkin lymphoma cell line
L1236.3 Eoxins have also been found in exhaled breath condensates
from children with asthma.10 Although our knowledge of the bio-
logical role of the eoxins is still limited, these mediators have been
shown to increase the permeability of an endothelial cell
⇑
Corresponding author. Tel.: +46 70 4411 261.
E-mail address: benjamin.pelcman@orgfarm.uu.se (B. Pelcman).
http://dx.doi.org/10.1016/j.bmcl.2015.05.007
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

3018
B. Pelcman et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3017–3023
COOH
arachidonic acid (AA)
5-LOX
15-LOX-1
Leukotrienes
Eoxins
OOH
COOH
COOH
COOH
OOH
OH
5-HPETE
15-HPETE
15-HETE
O
COOH
COOH
COOH
O
LTA4
EXA4
OH
COOH
HO
S
H
N
COOH
LTC4
S
HN
H
N
HOOC
O
COOH
EXC4
O
HN
NH2
HOOC
O
O
NH2
OH
COOH
COOH
HO
S
H
N
COOH
LTD4
S
H2N
H
N
O
COOH
EXD4
H2N
O
OH
COOH
COOH
HO
S
S
H2N
COOH
H2N
COOH
LTE4
EXE4
Scheme 1. The formation of leukotrienes and eoxins from arachidonic acid.
monolayer in vitro, indicating
a
pro-inflammatory role.9
inflammation and/or atherosclerosis. More recently, Maloney and
Holman used 15-LOX-1 in their primary screen to develop molec-
Overexpression of 15-LOX-1 in cultured human lung epithelial
cells has been shown to stimulate the expression and release of
chemokines.11 Recently, it was reported that 15-LOX-1 activity
was greater in eosinophils derived from patients with severe
asthma or aspirin-intolerant asthma (AIA) compared to eosinophils
isolated from patients with mild asthma or healthy controls.12
There are numerous publications regarding 15-LOX-1 inhibi-
tors, but many studies are conducted using 15-LOX-1 from non-
human species, most notably soybean (see, e.g., Refs. 13–15).
Such studies are of limited relevance for developing compounds
for treatment of human disorders. The groups at Parke–
Davis/Warner–Lambert (now Pfizer)16–22 and Bristol-Myers
Squibb23–26 used the rabbit reticulocyte 15-LOX-1 for their primary
screen, followed by a cell-based assay with recombinant 15-LOX-1
or a rabbit in vivo model to design compounds for the treatment of
ular tools27 and compounds aimed for anti-stroke therapy.8
A
selection of 15-LOX-1 inhibitors reported by the groups mentioned
above are depicted in Figure 1.
Herein we describe some of our efforts to develop 15-LOX-1
inhibitors for the potential use in the treatment of asthma and
chronic obstructive pulmonary disease (COPD). Parts of this work,
including experimental procedures, have been disclosed in patent
applications.28,29
To find
a suitable starting point we attempted several
approaches: HTS, virtual screening and synthesis of analogues of
known lipoxygenase inhibitors. The HTS approach was found to
be the most fruitful so the others were soon abandoned. A chemical
library comprising a diverse set of 50000 compounds were assayed
as inhibitors of 15-LOX-1 at 10 lM using linoleic acid (LA) as the

B. Pelcman et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3017–3023
3019
Cl
Cl
O
O
S
N
H
N
H
N
H
MeO
MeO
H
N
Cl
NH2
HN
S
Parke-Davis / Warner-Lambert 15LO inhibitors
Cl
H
H
N
N
S
O
N
S
O
S
O
O
N
N
N
F
N
H
N
H
N
Bristol-Myers Squibb 15LO inhibitors
F
CN
Me
N
N
N
O
N
O
S
H
O
Cl
O
Maloney / Holman inhibitors
Figure 1. Selected 15-LOX-1 inhibitors from patents and literature.
substrate. Although there is a difference in the absolute value
when AA or LA is used as a substrate, the cost and chemical stabil-
ity favoured LA in the hit finding phase. However, we would like to
emphasize the importance of using human 15-LOX-1 in the pri-
mary screen, either in an enzyme- or cell-based assay, and then
(or concomitantly8) ascertain if the compounds also inhibit the
proper lipoxygenase from the species that will be used in the
in vivo model. For example, compounds reported to be highly
active as inhibitors of the rabbit enzyme in our hands did not
always show activity when tested in our human enzyme- and
cell-based assays. Thus, using, for example, rabbit reticulocyte
15-LO in the primary screen may lead to erroneous conclusions
and to the rejection of good inhibitors of human 15-LOX-1.
Our 250 best hits were retested to confirm the results. Out of
these, 125 were selected for a dose-response study and in the next
step 40 selected compounds were tested in a cell-based assay and
for selectivity.
patentability point of view. The compound was however not active
on the mouse ortholog of 15-LOX-1. The differences of activity for
different species will be discussed in an accompanying Letter.30
The syntheses of the target compounds are straightforward
(Scheme 2).28 Heating pyrazole 3-carboxylic acid 2a with SOCl2
in THF gives the corresponding acid chloride 3 that after concentra-
tion is used as such without further purification. When treated
with an amine it readily gives the carboxamides 5. For relatively
non-nucleophilic amines, for example, aminopyridines, the anion
of the amine (formed by deprotonation with NaH) is used. A more
practical route is to dimerize pyrazole-3-carboxylic acid to the
stable diketopiperazine 4 by heating in neat SOCl2, preferably in
the presence of a catalytic amount of DMF or DMAP (4-dimethy-
laminopyridine). Due to the extremely low solubility, 4 is difficult
to purify and we were never able to fully characterize it.
Carboxamides 5 are then obtained by heating 4 with an excess of
the amine neat (e.g., in a microwave reactor)31 or in the presence
of DMAP, or at rt with equimolar amounts of the amine using,
for example, DMAP in CH2Cl2.28 The sequential dimerization of
pyrazole-3-carboxylic acids followed by addition of nucleophiles
is well known32–38 and has also been used to develop anti-inflam-
matory 15-LOX-1 inhibitors, albeit using soybean and potato
lipoxygenase assays in combination with a rat carrageenan edema
model.34 Acylation of 5 with acid chlorides affords the final product
1. Typically, the reaction is performed by first deprotonating 5 with
NaH in THF at rt and then adding the acid chloride, or by heating a
mixture of 5 and the acid chloride in CH2Cl2 in the presence of
DMAP for 2 d. The latter method is also used for the synthesis of
A set of compounds that we found particularly interesting were
1,3-disubstituted pyrazoles exemplified by compound 1a (Fig. 2).
Its structure is simple; it has good activity, both with AA and LA
as the substrate, good selectivity over some other relevant targets
(5-LOX, 12-LOX, COX-1 and COX-2) and seemed promising from a
15-LOX-1 = 62 %
O
15-LOX-1 = 66 % (LA as substrate)
5-LOX
= 8 % (enzyme)
N
H
Cl
5-LOX = 16 % (cell)
12-LOX = 18 %
carbamates
6 using chloroformates. Heating 5 with aryliso-
N
cyanates0 in toluene, either at 100 °C for 24 h or at 180 °C for
10 min in a microwave reactor gives ureas 7. Thioureas (8) are
obtained from 5 and isothiocyanates, the sulfonyl substituted pyra-
zoles 9 from the sulfonyl chlorides and N-alkylation to 10 is per-
formed using, for example, alkyl halides. An alternative strategy
where pyrazole-3-carboxylic acid are N-acylated before the assem-
bly of the amide bond was also investigated, but this route was less
COX1 = 11 %
N
COX2 = no inhibition
Cl
O
1a
Figure 2. Inhibitory activities for the HTS hit 1a on some relevant targets. Unless
otherwise stated the %-inhibitory data are obtained with 10
substrate.
lM of 1a and AA as the

3020
B. Pelcman et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3017–3023
O
Cl
R1
R1
O
O
O
N
N
a
b
c
N
N
H
N
H
OH
3
H
d, e, f,
g or h
1 L= C(O)
6 L= C(O)O
7 L= C(O)NH
8 L= C(S )NH
9 L= S O
N
N
N
H
N
L
N
H
5
O
R2
N
c
N
2a
10 L= CH22
N
N
O
4
Scheme 2. Reagents and conditions: (a) SOCl2/THF, rx, 1 h; (b) SOCl2/DMAP, rx, 3 d; (c) R0NH2, see text; (d) NaH/THF, rt, 1 h then R2C(O)Cl, rt, 1 h; (e) R2COCl, R2OC(O)Cl or
R2SO2Cl/DMAP/CH2Cl2, 40 °C, 16–48 h; (f) R2NCO/PhMe, 100 °C, 18–24 h; (g) R2NCO/PhMe, 180 °C, 10 min (microwave); (h) R2NCS or R2CH2Br/K2CO3/acetone, rt.
F
F
O
O
O
N
OH
2b
N
H
N
H
Cl
Cl
S iMe3
a, b
c, d
e
N
N
Me3S i
Me3S i
N
H
N
H
N
H
N2
COOE t
F
5a
5b
O
O
N
OE t
11
O
Cl
H
f
b, g, d
OE t
N
N
N
N
H
O
O
5c
H
Scheme 3. Reagents and conditions: (a) 100 °C, 3 d; (b) NaOH/EtOH/H2O, 80 °C, 15 min; (c) EDCI/DMAP/CH2Cl2, 50 °C, 2 d, 75%; (d) ArNH2, DMAP, 100 °C, 18 h; (e) TBAF/THF,
rt, 18 h; (f) N2H4xH2O/EtOH, rx, 2 h, 57%; (g) SOCl2/toluene, rx, 1 h.
pyrazole-3-carboxylic acid. The 4- and 5-methyl substituted pyra-
F
zole-3-carboxanilides are prepared according to Scheme 3.29 The
O
[2,3]-dipolar cycloaddition between 1-trimethylsilylpropyne and
O
N
N
diazoacetic acid ethyl ester followed by hydrolysis furnish 4-
methyl-5-trimethylsilylpyrazole-3-carboxylic acid (2b) in good
yield. The reaction also works with 1-triisopropylsilylpropyne,
but the yield is substantially lower (15%). To keep the TMS group
intact we used 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDCI)/DMAP instead of SOCl2 to convert the acid into a dike-
topiperazine, as we wanted to use the TMS group as a handle to
introduce other substituents in the 5-position of the pyrazole.
The reaction with 2-chloro-4-fluoroaniline afforded the anilide 5a
that on treatment with tributylammonium fluoride (TBAF) in THF
gives the 4-methylpyrazole-3-carboxanilide 5b. 4-Butyl substi-
tuted pyrazoles were similarly prepared from 1-trimethylsilyl-1-
hexyne. Even though 5-methylpyrazole-3-carboxylic acid ethyl
ester (11) is commercially available, we choose to synthesize it
N
H
N
Cl
N
O
N
H
5d
12
Scheme 4. Reagents and conditions: hexylNCO/PhMe, 180 °C, 10 min (microwave).
efficient. This strategy is however practical for the synthesis of 1N-
alkylated pyrazoles (not shown).
We wanted to investigate the influence of substituents on the
pyrazole ring on the activity, but only a few substituted pyra-
zole-3-carboxylic acids/esters were commercially available in use-
ful amounts at a reasonable price, for example, 4- and 5-nitro-
F
F
F
O
O
N
O
O
N
N
H
N
H
N
H
N
H
Cl
Cl
Cl
Cl
N
N
OCF
3
N
N
N
N
S
O
O
N
H
O
N
H
O
N
H
O
50 % hydrolyzed
3 h, 50 °C
3.5 % hydrolyzed
24 h, 50 °C
10 % hydrolyzed
24 h, 20 °C
5 % methanolyzed
7 d, 20 °C
Figure 3. Chemical stability for selected compounds as measured by 1H NMR either as hydrolysis in DMSO-d6 or by methanolysis in MeOD.

B. Pelcman et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3017–3023
3021
Table 1
15-LOX-1 inhibitory activity of 1-substituted pyrazolyl-3-carboxanilides
O
RN
N
H
R2
Inhibition @10
l
(LA)
Inhibition @10
l
(AA)
IC50/(Imax) enzyme (AA)
IC50/(Imax) cell (AA)
N
Compd
N
R1
R1
RN
R2
H
%
%
nM (%)
NT
nM (%)
NT
O
1a
66
62
Cl
Cl
Cl
F
1b
1c
6
H
H
H
H
H
H
H
H
H
NT
73
15
NT
NT
NT
50
NT
NT
NT
91
75
96
96
94
60
NT
NT
NT
NI
O
O
Cl
428
NT
292
Cl
O
O
Cl
NT
Cl
Cl
Cl
Cl
H
F
F
F
N
O
7a
7b
7c
7d
8
109
130
250
NT
47
O
O
H
N
408
O
H
N
141
OEt
O
O
O
NMe2
NT
O
Cl
Cl
H
N
F
F
F
NT
17 (57)
1062 (59)
O
S
CF3
O
S
O
9a
1944 (70)
Cl
Cl
NMe2
S
O
9b
H
NT
NI
NT
NI
NT
NT
NT
NI
NT
NI
O
10a
10b
H
Cl
Cl
F
Me
4-Cl
NT
NT
H
O
F
F
N
O
7e
7f
4-Me
4-Bu
5-Me
NT
NT
NT
NT
NT
NT
NT
NI
222 (48)
O
H
F
F
N
NI
Cl
Cl
O
H
N
7g
395
85
O
O
N
N
Cl
Me
7h
NI
NI
NT
NT
N
O
N
H
NI = no inhibition; NT = not tested; when no Imax is given it is >90%.

3022
B. Pelcman et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3017–3023
using the convenient reaction between 2,4-dioxovaleric acid ethyl
ester and hydrazine hydrate.39 Dimerization with SOCl2, followed
by the reaction with 2-chloro-4-fluoroaniline as before, gives the
the IC50 of thioureas, for example, 8, is low, the compounds could
not completely inhibit the enzyme (the Imax did not approach
100%). Phenylsulfonyl substituted compounds, for example, 9a,
have substantially lower activity and also do not inhibit the
enzyme completely. The sulfamide 9b as well as the 1N-benzyl
(10a) and 1N-methyl (10b) substituted pyrazoles lacks activity
completely. Finally, methylation of the nitrogen of the anilide in
the pyrazole 3-position (e.g., 7h) gave compounds devoid of inhibi-
tory activity.
In conclusion we have prepared several 1N-substituted pyra-
zole-3-carboxanilides that are good inhibitors of the human 15-
LOX-1. The best compounds are found among the ureas, in par-
ticular 7a and 7g, but they slowly decompose on standing in
solutions containing traces of water or other nucleophiles, which
makes them unsuitable for further drug development. However,
the 1N-unsubstituted pyrazole-3-carboxanilides 5 are active 15-
LOX-1 inhibitors and these were pushed forward and developed
as clinical candidates, which is discussed in detail in a following
Letter.30
desired
5-methylpyrazole-3-carboxanilide
5c.
5-tert-
Butylpyrazoles are obtained in a similar fashion.40
Both the 4- and 5-alkyl substituted pyrazole-3-carboxylic acids
are conveniently transformed to the anilides employing the dike-
topiperazine approach depicted above in Scheme 2. Again, treat-
ment with arylisocyanates gives the corresponding ureas. One
exception is the 5-tert-butylpyrazolecarboxanilide 5d, which on
treatment with hexylisocyanate expels the aniline portion of the
molecule to give the imidazopyrazoledione 12 (Scheme 4). Here
the bulky tert-butyl substituent forces the acylation to take place
on the 2-nitrogen of the pyrazole. Only minor amounts of 2-substi-
tuted pyrazole-3-carboxanilides could sometimes be isolated on
acylation of 5 with benzoyl chlorides and on alkylations.
Early on in the program we realized that chemical instability of
the pyrazole N-acyl bond might be an issue. Actually, some tar-
geted compounds could not be isolated, presumably due to hydrol-
ysis during aqueous workup. Judged by recording NMR spectra of
compounds upon standing in DMSO-d6, the order of stability
seemed to be alkyl ꢀ phenylsulfonyl > phenylurea P benzoyl. It
was also seen that alkylureas are more stable than arylureas and
that 5-substitution of the pyrazole core decreased stability. In con-
trast, N-sulfonyl substituted pyrazoles were stable DMSO-d6 but
were slowly methanolysed upon prolonged standing in MeOD
(Fig. 3). It must be emphasized that the isolated compounds were
stable enough to be evaluated as 15-LOX-1 inhibitors in our assay
systems. This is particularly important as also intermediates 5 pos-
sess considerably 15-LOX inhibitory activity.30
Before analyzing the results and the structure–activity relation-
ships (SAR) it is important to discuss the assays used and the inter-
pretation of the data in Table 1. In the beginning of the project
various assay conditions were evaluated in our primary assay:
enzyme or cell based assays, LA or AA as a substrate. IC50’s were
only determined for the most interesting compounds. As the work
progressed and we became more confident in how to run assays
and interpret data, we normally recorded the IC50’s for all com-
pounds both in an enzyme and a cellular assay using AA as a sub-
strate for both. It has also been pointed out that Imax, that is, the
extent of how much of the activity of 15-LOX-1 that can be inhib-
ited (where an Imax of 100% means complete inhibition of the enzy-
matic activity) varied for different compounds. Usually,
compounds with low IC50’s also had an Imax approaching 100%,
but for weaker inhibitors the Imax could be substantially lower.
Eventually, robust enzyme and cellular assays were developed,
which were also amenable to HTS.41 Thus, the data presented in
Table 1 must be interpreted with great care.
Acknowledgment
This work was financially supported by Biolipox AB.
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Table 1 shows a selection of compounds that illustrate the SAR
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discussed in a following Letter30 but 2-chloro-4-fluoroanilides is
one of the preferred. Using 1a as a benchmark one can deduce that
insertion of a methylene between the carbonyl group and the phe-
nyl group—benzoyl 1a versus phenacetyl 1b—completely abolishes
activity, but that the simple N-acetyl compound 1c show consider-
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activity comparable to the N-benzoyl substituted compounds, but
here there is a large difference depending on the substrate used
in the assay; when LA was used it showed only minute activity
whereas it showed good activity with AA. The discrepancy is hard
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