Discovery of a potent, selective and orally active canine COX-2 inhibitor, 2-(3-difluoromethyl-5-phenyl-pyrazol-1-yl)-5-methanesulfonyl-pyridine

Article abstract of DOI:10.1016/j.bmcl.2003.10.004

Structure-activity relationship (SAR) studies of 2-[3-di(and tri)fluoromethyl-5-arylpyrazol-1-yl]-5-methanesulfonylpyridine derivatives for canine COX enzymes are described. This led to the identification of 12a as a lead candidate for further progression. The in vitro and in vivo activity of 12a for the canine COX-2 enzyme as well as its in vivo efficacy and pharmacokinetic properties in dog are highlighted.

Full text of DOI:10.1016/j.bmcl.2003.10.004

Bioorganic & Medicinal Chemistry Letters 14 (2004) 95–98
Discovery of a potent, selective and orally active canine
COX-2 inhibitor, 2-(3-difluoromethyl-5-phenyl-pyrazol-1-yl)-5-
methanesulfonyl-pyridine
Jin Li,* Kristin M. Lundy DeMello, Henry Cheng, Subas M. Sakya, Brian S. Bronk,
Robert J. Rafka, Burton H. Jaynes, Carl B. Ziegler, Carolyn Kilroy, Donald W. Mann,
Eric L. Nimz, Michael P. Lynch, Michelle L. Haven, Nicole L. Kolosko,
Martha L. Minich, Chao Li, Jason K. Dutra, Bryson Rast, Rhonda M. Crosson,
Barry J. Morton, Glen W. Kirk, Kathleen M. Callaghan, David A. Koss,
Andrei Shavnya, Lisa A. Lund, Scott B. Seibel, Carol F. Petras and Annette Silvia
Veterinary Medicine Research and Development, Pfizer Inc., Groton, CT 06340, USA
Received 7 July 2003; revised 30 September 2003; accepted 5 October 2003
Abstract—Structure–activity relationship (SAR) studies of 2-[3-di(and tri)fluoromethyl-5-arylpyrazol-1-yl]-5-methanesulfonylpyr-
idine derivatives for canine COX enzymes are described. This led to the identification of 12a as a lead candidate for further pro-
gression. The in vitro and in vivo activity of 12a for the canine COX-2 enzyme as well as its in vivo efficacy and pharmacokinetic
properties in dog are highlighted.
# 2003 Elsevier Ltd. All rights reserved.
Nonsteroidal anti-inflammatory drugs (NSAIDs) used
for the treatment of inflammatory conditions act by
inhibition of cyclooxygenase (COX)1À3 which catalyzes
the first step in arachidonic acid metabolism.4 The iso-
zyme COX-1 is constitutive and responsible for the
physiological production of prostaglandins while COX-
2 is inducible and responsible for the elevated produc-
tion of prostaglandins during inflammation.5 COX-3, a
new isozyme of COX, was reported recently and its
biological significance is under evaluation.6 It is believed
that a selective COX-2 inhibitor will greatly reduce the
side effect profile, including gastric ulceration, that is
commonly associated with the chronic use of non-selec-
tive NSAIDs.7À10 Several marketed COX-2 selective
inhibitors,11À14 including celecoxib (Celebrex1), valde-
coxib (Bextra1), rofecoxib (Vioxx1), and etoricoxib
(Arcoxia1) have shown excellent efficacy in humans
with few side effects (Fig. 1).
Progressive degenerative joint disease, or osteoarthritis,
is the most common cause of chronic pain in dogs.15 It
is estimated that one out of every five adult dogs, or
* Corresponding author. Tel.:+1-860-715-3552; fax:+1-860-715-9259;
e-mail: jin_li@groton.pfizer.com
Figure 1. Structures of selected COX-2 inhibitors.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.004

96
J. Li et al. / Bioorg. Med. Chem. Lett. 14 (2004) 95–98
approximately 8 million animals, have osteoarthritis,
yet nearly half (48%) of these patients are untreated.16
As in people, chronic use of NSAIDs in dogs is often
associated with GI side effects.17 Carprofen (4) (Rima-
dyl1)18 and deracoxib (3) (DeramaxxTM),19 two of the
marketed agents for the treatment of inflammation and
pain for dogs, have demonstrated moderate COX-2
selectivity.17 Firocoxib (2), a new class of COX-2 inhi-
bitor for animal health, is under further clinical eval-
uation.20 In this paper, we disclose the synthesis and
SAR of a novel class of canine selective COX-2 inhibi-
tors, pyridylsulfones. This research effort culminated in
the identification of 12a. A general synthetic route for
the synthesis of the pyrazole analogues21 is described in
Scheme 1.
tional group was transformed to the methanesulfonyl
moiety in 7 by MCPBA mediated oxidation. Displace-
ment of bromine with hydrazine furnished 5-methane-
sulfonyl-pyridin-2-yl-hydrazine which was converted to
its HCl salt 8 in situ. The 1,3-diketones 10a and 10b
were synthesized11 using Claisen condensation of the
aryl-substituted ketones
9 with either ethyl tri-
fluoroacetate or ethyl difluoroacetate in DME. The
condensation of hydrazine 8 with the 1,3-dicarbonyl
10a/10b in ethanol then provided the 1,5-diarylpyrazole
(11 or 12) as the major product,11 which was readily
separated from its 1,4-diarylpyrazole isomer 13 by flash
chromatography. It was found that the ratio of the two
regioisomers (1,5- vs 1,4-diarylpyrazole) was affected by
different acids and solvents. Generally, a better product
ratio (1,5- vs 1,4-diarylpyrazole) was obtained by
refluxing the mixture of 8 and 10 in ethanol containing
concentrated sulfuric acid.22 The 4-chloro/bromo pyra-
zole analogues (14/15) were obtained in high yield by
stirring the pyrazole (11/12) with NCS or NBS in DMF
at room temperature.
The pyrazoles were prepared via condensation of the
hydrazine 8 with the respective diketone 10. The com-
mercially available 2,5-dibromopyridine (5) was con-
verted to 2-bromo-5-methylsulfanylpyridine (6) by a
lithium–halogen exchange reaction followed by addition
of methyldisulfanylmethane. The methylsulfanyl func-
Numerous 11/12 analogues were prepared and tested in
the in vitro canine whole blood COX inhibition
assays.23 Data for a select group of compounds are lis-
ted in Tables 1–3. Generally, analogues with halogen
substitution on the 4-position of the phenyl ring had
good potency against canine COX-2 enzyme in both
the-CF3 and-CHF2 analogue series. The selectivity
(COX-2 vs COX-1), on the other hand, tended to
decrease from F to Br (e.g., 11a–d, 12a–d). Analogues
which contained an electron donating group at the 4
position of the phenyl ring tended to have increased
COX-1 and COX-2 potency, with decreased COX-1/
COX-2 selectivity (e.g., 11e–g, 12e–f, with the exception
of 11h). The potency against COX-1 could be modu-
lated without affecting COX-2 potency by introducing a
halogen or a methyl substituent at the 3-position of the
phenyl ring in both-CF3 and -CHF2 analogue series
(11i–k, 12g–i). The selectivity and potency tended to
drop with the introduction of a halogen at the 2-posi-
tion (11m, 12j). Limited improvement in activity was
achieved in analogues with substitution at 3-position or
2-position only or di-substitution in both the 2- and 4-
positions of the aryl ring. Increasing the steric bulk at
the 4-position of the phenyl ring reduced the potency
towards the COX-2 enzyme (12k–n). Celecoxib (1) and
deracoxib (3) were tested in the in vitro canine whole
blood COX inhibition assays as well (Table 2). In gen-
eral, the pyridyl sulfone series (11 and 12) demonstrated
improved potency and selectivity toward COX-2 com-
pared to the benzylsulfonamide series (celecoxib and
deracoxib). As illustrated in Table 3, chloro or bromo
substitution at the 4-position of the pyrazole ring gen-
erally increased the potency towards both COX-1 and
COX-2 enzymes and resulted in a slight decline in
selectivity.
With many potent compounds in hand, we next
focused on identifying the best pharmacokinetic (PK)
profile. In vivo pharmacokinetic evaluations of the
analogues were carried out in beagle dogs by oral
gavage at 5 mg/kg.
Scheme 1. Reagents and conditions: (a) n-BuLi, Et2O, À78 ꢀC;
(MeS)2, 98%; (b) MCPBA, DCM, 95%; (c) NH2NH2, EtOH, reflux;
HCl/MeOH, 100%; (d) NaOMe, DME, rt, 60–95%; (e) EtOH, reflux,
50–95%; (f) NCS or NBS, DMF, rt, >95%.

J. Li et al. / Bioorg. Med. Chem. Lett. 14 (2004) 95–98
97
Table 1. In vitro COX-1 and COX-2 inhibition data of 11a–m (CF3
analogues, R1=H)
Table 4. Pharmacokinetic data
Compd
Cmax
(ng/mL)
Tmax
(h)
AUC
(ng h/mL)
Oral T1/2
(h)
C24
(ng/mL)
Compd
R2
Ratio
COX 1/2
IC50 (mM)
COX-1
COX-2
12a
12b
11b
12c
11a
14a
11n
11c
12g
12o
11j
12l
12i
12h
11k
15b
1
1089
509
540
412
611
473
338
95
89
208
69
45
36
27
42
1.5
1.3
0.8
1
0.8
0.75
1.5
1
1
0.5
1
2.5
1
1
0.8
16541
7652
4817
4285
4277
2654
2098
913
594
516
396
249
26
17
17.5
14
13.1
14.7
13.3
16.2
6.3
18.5
10.4
7
12.4
17.7
2.6
0.8
3.3
443
329
134
138
77
34
28
26
4.6
3.6
5
11a
11b
11c
11d
11e
11f
11g
11h
11i
H
4-F
4-Cl
4-Br
4-OMe
4-SMe
4-OEt
NMe2
>122
116
77
>50
27.9
19.4
28.6
0.83
1.9
0.05
24.2
40.7
47.8
0.41
0.24
0.25
0.45
0.044
0.069
0.13
0.12
0.27
0.31
0.30
1.50
0.06
64
19
28
0.38
202
151
154
>167
>33
47
3-Me, 4-OMe
3-F, 4-OMe
3-Cl, 4-OMe
3-Br, 4-OMe
2-F, 4-OMe
11j
11k
11l
0
2
2.1
4
>50
>50
2.8
185
145
115
26
11m
13
540
N/A
N/A
0
N/A
3100
Table 2. In vitro COX-1 and COX-2 inhibition data of 12a–n (CHF2
analogues, R1=H), celecoxib (1) and deracoxib (3)
Compd
R2
Ratio
COX 1/2
IC50 (mM)
COX-1
COX-2
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
Celecoxib (1)
Deracoxib (3)
H
4-F
4-Cl
4-Br
4-OMe
4-SMe
155
47
173
3.4
3.1
48.0
14.0
20.7
1.0
0.2
0.9
>50
9.9
>50
36.4
10
0.31
0.30
0.12
0.29
0.064
0.12
0.17
0.05
0.33
0.39
0.18
0.4
7.5
3-Me, 4-OMe
3-F, 4-OMe
3-Cl, 4-OMe
2-F, 4-OMe
4-Me
>294
198
>152
93
56
30
>25
>6.2
6.2
4-Et
4-n-pr
4-i-pr
12
>50
>50
5.57
23
2
8.1
0.90
0.63
36.5
Figure 2. Comparison of lameness scores (bars show mean+standard
error of the mean) for dogs given 12a, carprofen or deracoxib prior to
induction of acute knee joint synovitis in one hind leg.24 Different let-
ters over the error bars indicate statistical differences in the means
(p<0.10). The range of scores for non-treated dogs falls between the
parallel dashed lines above each time point.
Table 3. In vitro COX-1 and COX-2 inhibition data
Compd
R1
R2
Ratio
COX 1/2
IC50 (mM)
COX-1
COX-2
14a
15a
15b
15c
11n
12o
Cl
Cl
Cl
Br
Me
Me
H
H
132
7.5
24
25
>294
48
7.9
0.33
1.5
0.06
0.044
0.062
0.08
0.17
0.19
12a was identified for further in vivo profiling based on
its potency and PK properties. The analgesic effect of
12a was evaluated in a beagle acute pain model in which
lameness develops following induced synovitis of the
knee joint. Dogs were dosed orally for 5 consecutive
days to achieve steady-state plasma concentration and
synovitis induced 12 h after the last drug dose. Com-
pared to nontreated dogs, lameness was improved in all
groups of drug-treated animals. A once-a-day 4.0 mg/kg
dose of 12a demonstrated efficacy superior to carprofen
dosed once daily at 4.4 mg/kg and deracoxib dosed once
daily at 2.0 mg/kg (Fig. 2).
3-Cl, 4-OMe
H
H
H
2
>50
9.1
PK data for select analogues and celecoxib (1) are sum-
marized in Table 4. The –CHF2 analogues showed
higher AUCs values compared to the –CF3 analogues
(e.g., 12a and b vs 11a and b), presumably due to
increased polarity (clogP of the –CHF2 analogues were
1 unit lower compared to the –CF3 analogues). Either
unsubstituted phenyl or mono-substituted 4-phenyl had
better PK profiles overall than di-substituted analogues
(e.g., 11a–c vs 11j–k, 12a–c vs 12g–i). Halogen or methyl
substitution at the 4-pyrazole position did not improve
the PK of the analogues (e.g., 14a, 15b, 11n, 12o). Thus,
In summary, a novel class of potent and selective canine
COX-2 inhibitors were synthesized and evaluated. Ana-
logue 12a demonstrated good COX-2 selectivity as well as
desirable potency and PK properties, and was selected
for in vivo efficacy testing. Excellent control of pain was
shown in a canine synovitis model.

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J. Li et al. / Bioorg. Med. Chem. Lett. 14 (2004) 95–98
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23. Whole blood was collected by venal puncture into two
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at concentrations ranging from 500 to 0.005 mM were
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