Synthesis and SAR of heteroaryl-phenyl-substituted pyrazole derivatives as highly selective and potent canine COX-2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.01.059

The discovery of heteroaryl-phenyl-substituted pyrazole derivatives as canine selective COX-2 inhibitors is described. Structure-activity relationship (SAR) studies of this class of compounds led to the identification of compound 1 which demonstrated a ca

Full text of DOI:10.1016/j.bmcl.2006.01.059

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2076–2080
Synthesis and SAR of heteroaryl-phenyl-substituted
pyrazole derivatives as highly selective and
potent canine COX-2 inhibitors
Hengmiao Cheng,a,* Kristin M. Lundy DeMello,a Jin Li,a Subas M. Sakya,a Kazuo Ando,b
K. Kawamura,b Tomoki Kato,b Robert J. Rafka,a Burton H. Jaynes,a Carl B. Ziegler,a
Rod Stevens,b Lisa A. Lund,a Donald W. Mann,a Carolyn Kilroy,a Michelle L. Haven,a
Erik L. Nimz,a Jason K. Dutra,a Chao Li,a Martha L. Minich,a Nicole L. Kolosko,a
Carol Petras,a Annette M. Silviaa and Scott B. Seibela
aVeterinary Medicine Research and Development Pfizer Inc., Groton, CT 06340, USA
bGlobal Research and Development, Nagoya Laboratories, Pfizer Inc., 5-2 Taketoyo, Aichi 470-2393, Japan
Received 19 December 2005; revised 12 January 2006; accepted 17 January 2006
Available online 7 February2006
Abstract—The discovery of heteroaryl-phenyl-substituted pyrazole derivatives as canine selective COX-2 inhibitors is described.
Structure–activity relationship (SAR) studies of this class of compounds led to the identification of compound 1 which demonstrated
a canine whole blood COX-2 inhibitory IC50 of 12 nM and selectivity ratio of COX-1/COX-2 greater than 4000-fold.
Ó 2006 Elsevier Ltd. All rights reserved.
Nonsteroidal anti-inflammatory drugs (NSAIDs) such
as aspirin exhibit their anti-inflammatory effect by
inhibiting cyclooxygenase (COX)1–3 which catalyzes
the first step in arachidonic acid metabolism.4 It was
realized in the late 1980s that the isozyme COX-1 is
constitutive and responsible for the physiological pro-
duction of prostaglandins, while COX-2 is inducible
and responsible for the elevated production of prosta-
glandins during inflammation.5 COX-3, another isozy-
me of COX, was also reported recently.6 The chronic
use of NSAIDs to treat pain and inflammation is often
accompanied by side effects such as gastric ulceration,
bleeding, and renal function suppression. It is believed
that a selective COX-2 inhibitor will greatly reduce
these side effects.7–10 Several COX-2Òselective inhibi-
tors, including celecoxib (ÒCelebrex ),11 valdecoxib
(BextraÒ),12 rofecoxib (Vioxx ),13 and etoricoxib (Arr-
oxinÒ), have shown excellent efficacy in humans with
few side effects.
Progressive degenerative joint disease, or osteoarthritis,
is the most common cause of chronic pain in dogs.14 It is
estimated that one out of every five adult dogs, or
approximately 8 million animals, has osteoarthritis, yet
nearly half (48%) of these patients are untreated.15 As
with humans, chronic use of NSAIDs in dogs is often
associated with GI side effects.16 Carprofen (RimadylÒ)
and deracoxib (DeramaxxÒ), with moderate COX-2
selectivity, are marketed agents for the treatment of
pain and inflammation for dogs.17a,17b Another canine
COX-2 selective inhibitor under clinical evaluation is
firocoxib.18 In the search for the next generation canine
COX-2 selective inhibitor, in addition to the pyrazole
derivatives reported recently,19 we discovered that
heteroaryl-phenyl-substituted pyrazole derivatives such
as compound 1 are highly selective and potent canine
COX-2 inhibitors. The synthesis and SAR of this class
of compounds will be discussed.
The synthesis of 20-pyridine-4-methylsulfone pyrazole
derivatives20 is outlined in Scheme 1. The pyrazoles were
prepared via condensation of the hydrazine with the
respective diketone. The commercially available 2,5-
dibromopyridine 2 was converted to 2-bromo-5-methyl-
sulfanylpyridine 3 by a lithium–halogen exchange
Keywords: Cox-2 inhibitor; Anti-inflammation; Pain; Dog.
*
Corresponding author. Tel.: + 1 858 622 3208; fax.: +1 858 526
4151; e-mail: henry.cheng@pfizer.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.059

H. Cheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2076–2080
2077
H2NO2S
MCPBA-mediated oxidation. Displacement of bromine
with hydrazine furnished 5-methanesulfonylpyridin-2-
yl-hydrazine, which was converted to its HCl salt 5
in situ. The 1,3-diketones 7a and 7b were synthesized
using Claisen condensation of the heteroaryl-substituted
acetophenones 6 with either ethyl trifluoroacetate or
ethyl difluoroacetate in DME. The condensation of
hydrazine 5 with the 1,3-dicarbonyl 7 in ethanol then
provided the 1,5-diarylpyrazole 8 as the major product,
which was readily separated from its 1,4-diarylpyrazole
isomer 9 by flash chromatography.
H3CO2S
N
N
CF3
O
O
O
Celecoxib
Firocoxib
H2NO2S
Cl
N
N
CO2H
CHF2
N
H
The preparation of the heteroaryl-substituted acetophe-
nones 6 is illustrated in Scheme 2. The hydroxy group in
acetophenone 10 was activated by treatment with triflic
anhydride. The resultant triflate 11 was reacted with
2-furyl-tributyltin by palladium-mediated coupling to
afford acetophenone derivative 6a. When compound 11
was reacted with 2-thiazolyl-tributyltin, acetophenone
derivative 6b was obtained. To synthesize acetophenone
derivative 6c, the a-bromoketone 12 was condensed with
thioformamide to give 13, which was then converted to
acetophenone 6c by palladium mediated coupling with
tributyl-(1-ethoxy-vinyl)-stannane.
F
O
Carprofen
Deracoxib
MeO2S
N
N
N
CF3
Cl
O
1
The chemistry that was employed to prepare 6a was fol-
lowed to synthesize the acetophenone precursors for
pyrazole analogs 18, 19, 20, 21, and 29.
reaction followed by addition of methyldisulfanylme-
thane. The methylsulfanyl functional group was
transformed to a methanesulfonyl moiety in 4 by
O
O
Me
Me
OH
OTf
R2
R2
O S O
R2
Br
SMe
N
O S O
SnBu3
b
a
a
c
b
N
N
N
O
O
O
NHNH2 HCl
Br
Br
Br
11
10
6a
4
2
3
5
R2: H, F, Cl, or Me
S
b
N
R2
R2
O
SnBu3
O
d
R1
R1
O
S
R
N
6
7 a R = CF3
7 b R = CF2H
R1
R1 = Meheteroaryl
R2 = H, F, Cl
5
7
+
O
6b
e
MeO2S
O
Br
Br
N
EtO
SnBu3
MeO2S
c
N
N
N
R
d
N
N
R2
R1
R1
+
O
N
R
N
R2
S
Br
S
9
8 a R = CF3
8 b R = CF2H
12
13
6c
Scheme 1. Reagents and conditions: (a) n-BuLi, Et2O, ꢀ78 °C;
(MeS)2, 98%; (b) MCPBA, DCM, 95%; (c) NH2NH2, EtOH, reflux,
then conversion to HCl salt, 100%; (d) RCOOEt, NaOMe, DME, rt,
60–95%. (e) EtOH, reflux, 50–95%.
Scheme 2. Reagents and conditions: (a) (CF3SO2)2O, Et3N, ꢀ40 °C,
15 min, 100%; (b) Pd(PPh3)4, LiCl, dioxane, 110 °C, 1 h, 70%; (c)
HCSNH2, dioxane, reflux, 95%; (d) (1) Pd(PPh3)4, LiCl, dioxane,
reflux; (2) 2 M HCl, THF, reflux, 78%.

2078
H. Cheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2076–2080
Table 1. In vitro whole blood COX-1 and COX-2 inhibition data for CF3 substituted pyrazole derivatives
MeO2S
N
N
N
CF3
R3
R1
R2
Compound
R1
R2
R3
WB IC50 (lM)
Ratio
COX-1
COX-2
COX 1/2
1
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Cl
H
H
H
H
H
H
H
H
H
H
H
F
>50
0.012
>4167
759
154
558
340
4.9
O
O
O
O
O
O
O
H
18.2
2
0.020
0.013
0.050
0.01
CN
F
27.9
3.4
2
Me
C(O)NH2
CO2Me
CN
H
0.41
—
>0.500
0.11
—
N
23.4
>50
15.5
16
212
>313
172
>320
78
S
N
N
N
N
N
0.16
S
S
S
S
O
Me
Cl
0.09
<0.05
0.29
H
22.7
12.7
11.1
—
Me
Me
H
H
H
H
H
H
H
0.03
487
171
—
N
0.065
>0.5
0.21
O
N
S
N
H
50
238
15
S
N
H
2.7
—
0.18
S
N
S
H
>0.5
—

H. Cheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2076–2080
2079
Compounds were tested in canine whole blood in vitro
10
9
8
7
6
5
4
3
2
1
assays for inhibition against COX-1 and COX-2 en-
zymes.21 The biological activities of the trifluoromethyl
substituted pyrazoles are summarized in Table 1. Com-
pounds with furyl, oxazole or thiazole at the para posi-
tion of the phenyl ring demonstrated good COX-2
inhibitory activity and selectivity. Interestingly, com-
pound 29 only exhibited moderate selectivity when the
thiazole is attached to the phenyl ring at position 50
compared to position 20 in the case of 28 or position
40 in the case of 21. When the heteroaryl ring is attached
to the phenyl ring at meta position as shown in 30, or
there is a methyl group on the heteroaryl ring as shown
in 27, no COX-2 inhibitory activity was observed.
1
*
*
*
Inject
knee
*
\ \
16 hr
19 hr
20 hr
21 hr
22 hr
Next, we examined the effects of other substituents (R2,
R3) on the biological activity of these compounds. When
the substituents are F, Cl, Me or CN, the resultant pyr-
azoles demonstrated very good potency and high selec-
tivity. However, when the substituents are formamide,
or methoxcarbonyl, the resultant pyrazole derivatives
showed little activity. These results indicate the SAR is
very sensitive for this class of pyrazoles in the canine
whole blood in vitro assay.
Figure 1. Comparison of lameness scores (bars show mean + standard
error of the mean) for dogs given vehicle or 1 prior to induction of
acute knee joint synovitis in one hind leg.22 The asterisks over the error
bars indicate statistical differences in the means at each time point
(p 6 0.001) as determined by an unpaired t-test.
The analgesic effect of 1 was evaluated in a beagle
acute inflammatory model in which lameness develops
following induction of synovitis in one knee joint.
Dogs were dosed orally once a day with either vehicle
or 1 at a dosage of 6.6 mg/kg for 3 consecutive days to
achieve steady-state plasma concentrations and synovi-
tis induced 16 h after the last drug dose. Compared to
placebo dogs, lameness was improved at all time points
from 19 to 22 h following the last dose as illustrated in
Figure 1.
Finally, we investigated the effects of changing CF3 to
CF2H on the pyrazole ring; the latter analogs being
more polar than the CF3 analogs. As illustrated in
Table 2, when the CF3 is replaced by CF2H, the
potency of the resultant compounds remained about
the same, however, the selectivity was slightly nega-
tively impacted.
In summary, the heteroaryl-phenyl-substituted pyrazole
derivatives demonstrated potent in vitro activity inhibit-
ing canine COX-2 with very high selectivity in a canine
whole blood assay. This resulted in the discovery of
compound 1 which demonstrated canine whole blood
COX-2 IC50 of 12 nM, COX-1/COX-2 selectivity greater
than 4000, and an excellent efficacy profile for the treat-
ment of pain and inflammation in dogs.
Pharmacokinetic studies with compound 1 demonstrat-
ed 34% oral bioavailability and 16.4 h half-life. It was
then progressed to a canine in vivo synovitis model.
Table 2. In vitro whole blood COX-1 and COX-2 inhibition data for
CF2H-substituted pyrazole derivatives
MeO2S
N
N
N
References and notes
CF2H
R2
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21. Canine whole blood was collected by venal puncture into
two tubes with and without heparin. All analogs were
dissolved in DMSO. Microtubes containing 2 ll of various
drugs at concentrations ranging from 500 to 0.005 lM
were prepared ahead of time. Following collection, 500 ll
of whole blood without heparin was immediately added to
the drug tubes for COX-1 measurement. Samples were
incubated for 1 h at 37 °C. Samples without drug were
included as controls for maximum production of TXB2.
COX-2 activity was measured by 500 ll of heparinized
blood to tubes containing drug and 10 lg/ml of LPS to
stimulate production of prostaglandin E2 (PGE2). LPS
and vehicle-only controls were included for maximum
PGE2 production and background values, respectively.
Samples were incubated overnight at 37 °C. Following the
respective COX-1 and COX-2 incubations, EDTA, 0.3%
final concentration, was added to the samples to alleviate
clotting upon sample thaw. Samples were centrifuged, and
serum/plasma was collected into 96-well microtiter plates
and stored at ꢀ20 °C until ready for analysis. Cayman
EIA kits were used according to the manufacturer’s
instructions for measuring production of TBX2 and
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the kit standards (1/10,000 for TXB2 and 1/1000 for
PEG2).
22. Synovitis was induced by injection of media containing
pro-inflammatory cytokines generated from a lipopoly-
saccharide-stimulated culture of canine histiocytes.23 Ani-
mals were anesthetized and knee joints were injected 16
hours following the last administration of drug. Lameness
was scored hourly from 3 to 6 h post-injection (19–22 h
post-final dose) using a numerical rating scale scoring
system where 1 represents no lameness and 10 the most
severe lameness. Aqueous 80% PEG-400 vehicle was used
to prepare a solution of 1, which was administered by oral
gavage to provide a dose of 6.6 mg/kg. Placebo animals
received vehicle-only by oral gavage. An unpaired t-test
was used to compare the mean lameness scores at each
time point with significant differences (p 6 0.001) indicated
by an asterisk over the error bars (Fig. 1).
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