In vitro and in vivo profile of 2-(3-di-fluoromethyl-5-phenylpyrazol-1-yl)- 5-methanesulfonylpyridine, a potent, selective, and orally active canine COX-2 inhibitor

Article abstract of DOI:10.1016/j.bmc.2004.11.048

The synthesis of a novel canine COX-2 selective inhibitor, 2-(3-difluoromethyl-5-phenylpyrazol-1-yl)-5-methanesulfonylpyridine, and its in vitro and in vivo profile are described. Pyrazole 8 demonstrated excellent potency and selectivity for canine COX-2

Full text of DOI:10.1016/j.bmc.2004.11.048

Bioorganic & Medicinal Chemistry 13 (2005) 1805–1809
In vitro and in vivo profile of 2-(3-di-fluoromethyl-5-
phenylpyrazol-1-yl)-5-methanesulfonylpyridine, a potent,
selective, and orally active canine COX-2 inhibitor
Jin Li,* Michael P. Lynch, Kristin Lundy DeMello, Subas M. Sakya, Hengmiao Cheng,
Robert J. Rafka, Brian S. Bronk, Burton H. Jaynes, Carolyn Kilroy, Donald W. Mann,
Michelle L. Haven, Nicole L. Kolosko, Carol Petras, Scott B. Seibel and Lisa A. Lund
Pfizer Inc, Groton, CT 06340, USA
Received 12 October 2004; revised 24 November 2004; accepted 24 November 2004
Available online 19 December 2004
Abstract—The synthesis ofa novel canine COX-2 selective inhibitor, 2-(3-difluoromethyl-5-phenylpyrazol-1-yl)-5-methanesulfonyl-
pyridine, and its in vitro and in vivo profile are described. Pyrazole 8 demonstrated excellent potency and selectivity for canine COX-
2 in both in vitro and ex vivo whole blood assays. This novel COX-2 inhibitor also showed a good pharmacokinetic profile (pk)
following oral (po), intravenous (iv), and subcutaneous (sc) dosing and demonstrated excellent in vivo efficacy in a canine synovitis
model.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
leads to excess prostaglandin production during injury
resulting in pain and inflammation.7,8 Thus, the inhibi-
tion ofCOX-2 alone is thought to be enough to provide
the anti-inflammatory activity.9–12 A moderately COX-2
selective agent, carprofen13 (1), is the most prescribed
drug currently used for the treatment of inflammation
and pain in dogs and has been shown to have reduced
or no side effects associated with gastric ulceration.14,15
Deracoxib16 (2), another COX-2 selective NSAID, has
been recently marketed by Novartis for dogs. Here we
disclose the synthesis and the in vitro and in vivo profile
ofa highly potent and selective novel canine COX-2
inhibitor, 2-(3-difluoromethyl-5-phenylpyrazol-1-yl)-5-
methanesulfonylpyridine17 (Fig. 1).
A major trend in veterinary medicine is the increasing
recognition ofthe need ofr pain management and the
associated benefits to pet health and quality oflife. Pro-
gressive degenerative joint disease (osteoarthritis) is the
most common cause ofchronic pain in dogs. It is esti-
mated that one out ofevery five dogs, or approximately
eight million dogs, have osteoarthritis, yet nearly half
1
2
(48%) ofthe patients go untreated. Nonsteroidal anti-
inflammatory drugs (NSAIDs), whose mechanism ofac-
tion is the inhibition ofcyclooxygenase enzymes (both
COX-1 and COX-2) involved in the conversion ofara-
chidonic acid to the prostaglandins,3–5 have been used
most commonly to treat pain and inflammation in
dogs.6 The chronic use ofsuch non-selective NSAIDs
has been associated with side effects such as gastric
ulceration, bleeding and renal function suppression
and is attributed to the inhibition ofthe constitutively
expressed COX-1 enzyme necessary for normal platelet
activity and homeostatic maintenance ofthe gastric mu-
cosa and renal function. The inducible COX-2 enzyme
2. Chemistry
2-(3-Difluoromethyl-5-phenylpyrazol-1-yl)-5-methane-
sulfonylpyridine (8) was synthesized as outlined in
Scheme 1. The commercially available 2,5-dibromopyr-
idine (3) was converted to 2-bromo-5-methylsulfanyl-
pyridine (4) by a lithium–halogen exchange reaction
followed by the addition of methyldisulfanylmethane.
The methylsulfanyl functional group was then trans-
formed into methanesulfonyl moiety in 5 by MCPBA-
mediated oxidation. Displacement ofthe bromine
Keywords: COX-2; Inflammation; Pyrazole; Osteoarthritis.
*
Corresponding author. Tel.: +1 860 715 3552; fax: +1 860 715
9259; e-mail: jin_li@groton.pfizer.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.048

1806
J. Li et al. / Bioorg. Med. Chem. 13 (2005) 1805–1809
O
H2N
O
O
S
S
H3C
Cl
N
O
N
N
CO2H
N
N
N
H
CHF2
CHF2
F
MeO
Deracoxib
1
Carprofen
2
8
Figure 1. Structures ofselected canine COX-2 inhibitors.
Table 1. In vitro activity ofcarprofen ( 1), deracoxib (2), and pyrazole
(8)
CH3
CH3
S O
SMe
Br
O
S
O
O
Compound
Ratio, COX-1/2
IC50 (lg/mL)
a
b
c
COX-1
13.5
9.13
16.8
COX-2
N
N
N
N
1
2
8
5
36.5
153
2.70
0.25
0.11
Br
Br
Br
NHNH2
HCl
5
6
3
4
8 would be expected to exhibit an excellent therapeutic
index and safety profile based on its selectivity.
O
CH3
O S
H3C
O
CHF2
S
O
N
O
The pharmacokinetics ofpyrazole 8 were assessed in
female beagle dogs.19 Table 2 summarizes the pk data
for a 2 mg/kg dose in female beagle dogs following single
po (0.5% methylcellulose suspension), sc (80% PEG400/
20% ethanol), and iv administration (80% PEG400/20%
water). The pyrazole 8 exhibited multi-exponential plas-
ma kinetics following iv dosing with slow plasma clear-
ance. Following po and sc administration, pyrazole 8
was detected in the plasma within 30 min. Peak plasma
concentrations were achieved more rapidly following
po dosing (Tmax = 1.75 h) compared to sc dosing
(Tmax = 13.25 h) and maximal plasma concentrations
were approximately twofold greater following po dosing
than sc dosing. Due to the prolonged absorption phase,
elimination ofthe compound ofllowing sc dosing
seemed to be absorption rate dependent (flip-flop kinet-
ics). The prolonged absorption phase following sc dos-
ing helped maintain plasma concentrations of 8 above
163 69 ng/mL for at least 24 h. This may be advanta-
geous in maintaining efficacy for 24 h or longer once de-
sired plasma concentrations (2–3X COX-2 IC50) are
achieved with an appropriate dose. Unlike sc dosing,
po administration provided mean plasma concentra-
tions at 24 h (83 106 ng/mL) that were lower than
the COX-2 IC50. The bioavailability following sc dosing
was approximately twofold greater than with po dosing
(Fig. 2).
O
d
N
N
+
CHF2
N
NHNH2
HCl
8
6
7
Scheme 1. Reagents and conditions: (a) n-BuLi, Et2O, À78 ꢁC,
(MeS)2; (b) MCPBA, DCM, rt; (C) i. NH2NH2, EtOH, reflux, ii.
HCl/EtOH; (d) CF3CH2OH, reflux.
moiety with hydrazine furnished 5-methanesulfonylpyri-
din-2-ylhydrazine, which was converted to its hydro-
chloride salt 6 in situ. The condensation ofhydrazine
6 with the 1,3-dicarbonyl 7 in trifluoroethanol then pro-
vided the 1,5-diarylpyrazole 8 in 70–80% yield.18 The
structure ofpyrazole 8 was verified by NMR, MS,
CHN elemental analysis, and X-ray crystallography.
3. Results and discussion
Carprofen (1), deracoxib (2), and pyrazole (8) were all
tested in the in vitro canine whole blood COX inhibition
assays17 as shown in Table 1. The pyrazole 8 is about 20-
fold more potent toward the COX-2 enzyme compared
to carprofen (1), slightly more potent than deracoxib
(2). Both pyrazole 8 and deracoxib (2) demonstrated
excellent selectivity compared to carprofen (1). Pyrazole
The excellent selectivity ofpyrazole 8 for the COX-2
enzyme was also demonstrated by ex vivo studies.20
Table 2. Mean ( SD) pharmacokinetic parameters of 8 in female beagles following a single intravenous (iv), oral (po), or subcutaneous (sc) dose at
2 mg/kg
Tmax (h)
Cmax (ng/mL) C24 (ng/mL) AUC (ng h/mL) T1/2 (h) CL (mL/kg h) Vss (mL/kg) Kel
F (%)
iv
sc
NA
1929 (369)
181 (62)
163 (69)
83 (106)
13993 (4467)
9483 (4529)
5788 (5682)
13
21
8
155 (49)
NA
NA
2500 (693)
NA
NA
0.0544 (0.0226) NA
0.0331 (0.0176) 66 (13)
0.0864 (0.0504) 38 (29)
13.25 (12.5) 183 (54)
339 (114)
po 1.75 (1.5)

J. Li et al. / Bioorg. Med. Chem. 13 (2005) 1805–1809
1807
10000
1000
100
10
10
8
(4.0 mg/kg)
Carprofen
Score Range
Non-treated
8
6
4
2
0
(4.4 mg/kg)
Deracoxib
(2.0 mg/kg)
c
c
b
b
a
IV-2 mg/Kg
PO-2 mg/Kg
SC-2 mg/kg
a
Inject knee
↓
1
0
20
40
60
80
12 hr
15 hr
17 hr
Time (hr)
Time Post Dose
Figure 2. Pharmacokinetics ofpyrazole 8 in female beagles following a
single intravenous (iv), oral (po), or subcutaneous (sc) dose at 2 mg/kg.
Figure 5. Comparison oflameness scores (bars show mean + standard
error ofthe mean) of r dogs given 8, carprofen (1), and deracoxib (2)
prior to induction ofacute knee joint synovitis in one hind leg.
Four dogs per group were dosed at 10 and 15 mg/kg via
oral administration for 5 days. The whole blood ex vivo
assessment was conducted at 0, 2, 8, and 24 h on days 1
and 5. On day 1, COX-2 inhibition reached a high of
76% at 10 mg/kg and 93% at 15 mg/kg at the 2 h time-
point (Fig. 3). Inhibition levels remained at 55% and
85% for 10 and 15 mg/kg through 24 h, respec-
tively. On day 5, inhibition was at 88% and 97% at the
Ô0Õ h time-point and remained at 92% and 98% for 10
and 15 mg/kg 24 h after the last dose (Fig. 4). No
COX-1 inhibitory activity was detected throughout the
study.
The analgesic effect ofpyrazole 8 was evaluated in an
acute inflammatory model in beagles in which lameness
occurs following induced synovitis of the stifle (knee)
joint.17 Dogs were dosed orally for five consecutive days
with either 8, carprofen (1) or deracoxib (2) and then
synovitis was induced 12 h after the last dose. Compared
to non-treated dogs, lameness was improved in all three
groups ofdrug-treated animals. A once-a-day 4.0 mg/kg
dose of 8 demonstrated efficacy superior to carprofen (1)
dosed once daily at 4.4 mg/kg and deracoxib (2) at
2.0 mg/kg (Fig. 5).
In summary, a novel class ofpotent and selective canine
COX-2 inhibitors were synthesized and evaluated. These
efforts resulted in the discovery ofpyrazole 8 with good
canine COX-2 potency, selectivity and an excellent effi-
cacy profile for the treatment of pain and inflammation
in dogs.
100
80
60
40
20
0
4. Experimental
Proton and carbon magnetic spectra were recorded on
Bruker DMX500, Varian XL-300, or Varian Unity
400 spectrometers. Chemical shifts are expressed in parts
per million (d) downfield from TMS. Low-resolution
mass spectra were obtained on a Hewlett-Packard
5889A spectrometer using ammonia as the source of
chemical ionization. All reagents, solvents, and drying
agents were obtained from commercial sources and were
used without further purification. Analytical thin-layer
chromatography was carried out using silica plates (E.
Merck Kieselgel 60 F254). Melting points were recorded
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (hours)
Figure 3. COX-2 inhibition (time-point control) of 8 at 10 mg/kg
(dotted line) and 15 mg/kg (solid line) po day 1.
100
80
60
40
20
0
on a Buchi 510 apparatus and are uncorrected. Combus-
¨
tion analyses were performed on Perkin–Elmer 2400 ele-
mental analyzer.
4.1. Preparation of 5-methanesulfonylpyridin-2-ylhydr-
azine hydrochloride (6)
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (hours)
4.1.1. 5-Methylthio-2-bromopyridine (4). To a solution of
2,5-dibromopyridine (3) (23.4 g, 99 mmol) in ether
Figure 4. COX-2 inhibition (time-point control) of 8 at 10 mg/kg
(dotted line) and 15 mg/kg (solid line) po day 5.

1808
J. Li et al. / Bioorg. Med. Chem. 13 (2005) 1805–1809
(500 mL), n-BuLi (1.52 M in n-hexane, 68 mL,
103 mmol) was added dropwise at À78 ꢁC and the mix-
ture was stirred for 1 h at that temperature. Dimethyldi-
sulfide (9.8 mL, 110 mmol) was added slowly at À78 ꢁC
and the mixture was stirred for 1 h at that temperature
and further 1 h at 0 ꢁC. The mixture was quenched with
aqueous 1 N HCl (200 mL) and extracted with ether,
dried over MgSO4, and concentrated gave compound
4 (18.9 g, 94%). An analytical sample can be obtained
product was purified by flash chromatography on silica
gel. The product obtained was then crystallized from
isopropyl alcohol to furnish 5.29 g of 2-(3-difluoro-
methyl-5-phenylpyrazol-1-yl)-5-methanesulfonylpyridine
8 (71%), mp 124–125 ꢁC; 1H NMR (500, CDCl3): d
3.01 (3H, s), 6.73 (1H, s), 6.77 (1H, t, J = 54.8 Hz),
7.29 (2H, m), 7.38 (3H, m), 7.82 (1H, d, J =
8.7 Hz), 8.26 (1H, dd, J = 8.7, 2.1 Hz), 8.76 (1H, d,
J = 2.1 Hz); 13C NMR (CDCl3): d 45.31, 107.99,
109.39, 111.26, 113.12, 118.14, 128.86, 129.10, 129.42,
130.55, 135.77, 138.35, 146.82, 148.20, 155.61; MS m/z
350 (M++1). Anal. Calcd for C16H13F2N3O2S: C,
55.01; H, 3.75; N, 12.03. Found: C, 55.11; H, 3.67; N,
11.98.
by crystallization ofthe product rfom hexane.
1H
NMR (400, CDCl3): d 2.48 (3H, s), 7.37 (1H, dd,
J = 8.3, 0.83 Hz), 7.43 (1H, dd, J = 8.3, 2.49 Hz), 8.22
(1H, dd, J = 2.49, 0.83 Hz); 13C NMR (CDCl3): d
16.08, 128.07, 135.44, 137.05, 138.56, 147.98; MS spec-
trum m/z 204 (M++1).
4.1.2. 5-Methylsulfonyl-2-bromopyridine (5). To a solu-
tion of5-methylthio-2-bromopyridine ( 4) from step 1
(18.9 g, 93 mmol) in DCM (600 mL), MCPBA (48 g,
190 mmol) was added portion wise at 0 ꢁC and the mix-
ture was stirred for 2 h at rt. Aqueous saturated Na2SO3
(200 mL) was added and stirred for 15 min and the or-
ganic phase was separated and washed with aqueous sat-
urated NaHCO3 (200 mL), dried (MgSO4), and
concentrated. The crude product was purified through
flash chromatography on silica gel to give the title com-
pound (20.9 g, 95%). 1H NMR (400, CDCl3): d 3.11
(3H, s), 7.71 (1H, d, J = 8.3 Hz), 8.05 (1H, dd, J = 8.3,
2.49 Hz), 8.89 (1H, d, J = 2.49 Hz); 13C NMR (CDCl3):
d 45.14, 129.07, 136.55, 137.47, 148.20, 149.46; MS m/z
236 (M++1).
References and notes
1. Fox, S. M.; Johnston, S. A. J. Am. Vet. Med. Assoc. 1997,
210, 1493.
2. Pfizer Animal Health data file.
3. Carter, J. S. Exp. Opin. Ther. Patents 1998, 8, 21.
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Funk, C. D.; Smith, W. L. J. Pharmacol. Exp. Ther. 1994,
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5. OÕNeill, G. P.; Mancini, J. A.; Kargman, S.; Yergey, J.;
Kwan, M. Y.; Falgueyret, J. P.; Abramovitz, M.; Ken-
nedy, B. P.; Ouellet, M.; Cromlish, W. Mol. Pharmacol.
1994, 45, 245.
6. Ricketts, A. P.; Lundy, K. M.; Seibel, S. B. Am. J. Vet.
Res. 1998, 59, 1441.
7. Otta, J. C.; Smith, W. L. J. Lipid Mediators Cell Signal
1995, 12, 139.
4.1.3. 5-Methanesulfonylpyridin-2-ylhydrazine hydrochlo-
ride (6). A mixture of5-methylsulfonyl-2-bromopyridine
(5) (20.9 g, 89 mmol) and anhydrous hydrazine (5.6 mL,
180 mmol) in ethanol (200 mL) was refluxed for 4 h.
After cooling to room temperature the mixture was con-
centrated. The residual solid was washed with aqueous
saturated NaHCO3 (100 mL) and H2O and collected
by filtration. The solid was then treated with 10%
HCl/MeOH and the precipitate was collected by filtra-
tion to give the title compound 6 (9.8 g, 50%). 1H
NMR (400, DMSO-d6): d 3.16 (3H, s), 7.0 (1H, d,
J = 9.1 Hz), 8.03 (1H, dd, J = 9.1, 2.49 Hz), 8.58 (1H,
d, J = 2.49 Hz); 13C NMR (DMSO-d6): d 44.82,
109.24, 129.22, 137.27, 147.79, 159.57; MS m/z 188
(M++1).
8. Prasit, P.; Riendeau, D. Ann. Rep. Med. Chem. 1997, 32,
211.
9. Clive, D. M.; Stoff, J. S. New Engl. J. Med. 1984, 310,
563.
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D.; Russell, R. I. New Engl. J. Med. 1992, 327, 749.
11. Griswold, D. E.; Adams, J. L. Med. Res. Rev. 1996, 16,
181.
12. Cryer, B.; Dubois, A. Prostag. Other Lipid Mediators
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A.; Polanski, M.; Kopp, B.; Sito, E.; Oleksy, J. Hepato-
gastroenterol. 1983, 30, 261.
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Vet. Ther. 2002, 3, 453.
4.2. Preparation of 2-(3-difluoromethyl-5-phenylpyrazol-
1-yl)-5-methanesulfonylpyridine (8)
17. Li, J.; DeMello, K. M. L.; Cheng, H.; Sakya, S. M.;
Bronk, B. S.; Rafka, R. J.; Jaynes, B. H.; Ziegler, C. B.;
Kilroy, C.; Mann, D. W.; Nimz, E. L.; Lynch, M. P.;
Haven, M. L.; Kolosko, N. L.; Minich, M. L.; Li, C.;
Dutra, J. K.; Rast, B.; Crossan, R.; Morton, B. J.; Kirk,
G. W.; Callaghan, K. M.; Koss, D. A.; Shavnya, A.;
Lund, L. A.; Seibel, S. B.; Petras, C. F.; Silvia, A. Bioorg.
Med. Chem. Lett. 2004, 14, 95.
18. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J.
S.; Collins, P. W.; Doctor, S.; Graneto, M. J.; Lee, L. F.;
Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn,
J. N.; Gregory, S. A.; Koboldt, C. M.; Perkins, W. E.;
Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.; Isakson, P.
C. J. Med. Chem. 1997, 40, 1347.
5-Methanesulfonylpyridin-2-ylhydrazine hydrochloride
(6) (5.55 g, 21.2 mmol) and 4,4-difluoro-1-phenylbu-
tane-1,3-dione (7) (4.21 g, 21.2 mmol) were dissolved in
trifluoroethanol (250 mL). Concentrated H2SO4 (2 mL)
was added to the reaction mixture. The mixture was re-
fluxed overnight. The reaction mixture was cooled to rt
and the solvent was removed under reduced pressure.
The residue was then partitioned between EtOAc
(200 mL) and NaHCO3 (satd 200 mL). The aqueous
layer was separated and extracted with EtOAc
(3 · 50 mL). The organic extracts were combined and
dried (Na2SO4). After removing the solvent the crude

J. Li et al. / Bioorg. Med. Chem. 13 (2005) 1805–1809
1809
19. No gender effects on the pk profile ofpyrazole 8 would
be expected based on several earlier pk studies ofpyrazole
8.
added (500 lL) and incubated at 37 ꢁC overnight. EDTA
(10 lL, 0.3% final concn) was added after incubation
(prevents coagulation ofplasma that sometimes occurs
after thawing samples), samples were centrifuged at 4 ꢁC,
and plasma (ꢀ200 lL) was collected and stored at À20 ꢁC
in polypropylene 96-well plates. For the clotted blood
COX-1 assay, vacutainer clot tubes were directly incu-
bated at 37 ꢁC for 1 h and centrifuged at approximately
4000 rpm for 15 min. Serum was removed and stored at
À20 ꢁC. To run the endpoints, Cayman EIA plates were
used according to kit instructions to measure production
20. The time-points evaluated in the whole blood ex vivo were
0, 2, 8 and 24 h on days 1 and 5. Four dogs per group were
dosed at 10 and 15 mg/kg po, and blood from four
untreated dogs was used as controls for each time-point.
Drug was prepared in a 0.5% methylcellulose vehicle and
dosed by oral gavage for 5 days. The best method for
solubilizing the compound fully in a homogenous suspen-
sion was by the use ofa tissue homogenizer. Drug
solutions were made fresh each day. Dogs were not fed
prior to dosing. Heparin tubes were used to collect blood
for the LPS COX-2 assay. Microassay tubes were
prepared with 2 lL ofLPS (10 lg/mL final concn) as well
as vehicle controls for background values. Blood was
ofTXB and PGE2 for COX-1 and COX-2, respectively,
2
utilizing competitive binding ofa tracer to antibody and a
colorimetric endpoint. Samples were diluted to approxi-
mate the range ofthe kit standards (1/10,000 for TXB 2, 1/
1000 for PGE2).