Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain

Article abstract of DOI:10.1016/j.bmcl.2010.10.017

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.

Full text of DOI:10.1016/j.bmcl.2010.10.017

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7479–7482
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel class of biphenyl pyrazole sodium channel blockers
for treatment of neuropathic pain
Sriram Tyagarajan a, , Prasun K. Chakravarty a, Bishan Zhou a, Brett Taylor c, Ronsar Eid a, Michael H. Fisher a,
⇑
William H. Parsons a, Mathew J. Wyvratt a, Kathryn A. Lyons b, Tracy Klatt b, Xiaohua Li b, Sanjeev Kumar b,
Brande Williams c, John Felix c, Birgit T. Priest c, Richard M. Brochu c, Vivien Warren c, McHardy Smith c,
Maria Garcia c, Gregory J. Kaczorowski c, William J. Martin d, Catherine Abbadie d, Erin McGowan d,
Nina Jochnowitz d, Ann Weber a, Joseph L. Duffy a
a Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
b Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, Rahway, NJ 07065, USA
c Department of Ion Channels, Merck Research Laboratories, Rahway, NJ 07065, USA
d Department of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers
for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve
ligation (SNL) model of neuropathic pain.
Received 23 July 2010
Revised 1 October 2010
Accepted 5 October 2010
Available online 14 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Sodium channel blockers
Nav1.7 blocker
Neuropathic pain
Biaryl pyrazole
Chung model
In the pathogenesis of neuropathic pain, voltage gated sodium
channel blockers have been shown to play a key role. Indeed, hu-
man specific genetic studies have shown that a loss of function
mutation in Nav1.7 is associated with congenital insensitivity to
painful stimuli.1 Neuropathic pain is caused by injury to the
peripheral or central nervous system, and is often treated using
weak sodium channel antagonists.2 Certain subtypes of sodium
channels (Nav1.7 and Nav1.8) are over expressed at the site of inju-
ries.3,4 Sodium channel blockers currently used in the clinic pro-
vide only partial relief and have adverse effects that limit their
use. We reasoned that development of more potent state-depen-
dent Nav1.7 blockers may afford minimal CNS and cardiac side ef-
fects and improved efficacy for treating neuropathic pain.5
underwent extensive hydrolysis of the amide to the acid in dog
hepatocytes. The amide to acid hydrolysis was reduced by the
introduction of the pyrazole ring as shown in 2.7 The pyrazole
O
N
S
N
N
NH2
OCF3
F3C
CF3
NH2
O
1
2
Merck biphenyl thiazole
Biphenyl pyrazole
State-dependent sodium
channel blocker
State-dependent sodium
channel blocker
We have previously disclosed a novel class of biphenyl thiazole
carboxamides, represented by 1 (Fig. 1), as state dependent sodium
channel blockers.6 These compounds displayed good hNav1.7 func-
tional block (Nav1.7 EP Ki = 0.015 lM) and good in vivo efficacy in
various animal models of neuropathic pain. However, pharmacoki-
Nav1.7: Ki = 0.23 µM
Nav1.7: Ki = 0.34 µM
O
N
N
3
R4
NH2
5 5
R
R
netic metabolite analysis revealed that this class of compounds
3
N-Biphenyl pyrazole
⇑
Corresponding author. Tel.: +1 732 594 6226; fax: +1 732 594 5350.
E-mail address: sriram_tyagarajan@merck.com (S. Tyagarajan).
Figure 1. Design concept of N-biphenyl pyrazole Nav1.7 blockers.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.017

7480
S. Tyagarajan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7479–7482
analog 2 displayed good in vitro Nav1.7 block and excellent in vivo
efficacy but exhibited poor solubility. The compound was very
stable during incubation with human, dog, and monkey liver micro-
somes, but 2 was extensively metabolized in rat liver microsomes.
We set out to modify pyrazole 3 while retaining Nav1.7 potency
and in vivo efficacy with the hypothesis that introduction of polarity
in the molecule could address some of these issues.
Our prior exploration in the area of biphenyl thiazolidine-2,4-
diones, thiazoles, and pyrazoles as Nav1.7 blockers established that
meta arrangement of the biphenyl (1,3) is more potent than the
para arrangement (1,4). We observed that lipophilic substituents
like the trifluoromethoxy or trifluoromethyl group at C-2 position
of the biphenyl were the preferred substituents.
at the C-4 position of the pyrazole afforded compound 5, which
gave excellent Nav1.7 functional block, but was weakly active in
the in vivo SNL assay. Introduction of a methyl substituent at C-5
afforded 6, which did not yield significant improvement over pyr-
azole 4. The tricarboxamide pyrazole 7 had a profile similar to pyr-
azole 4 but with increased affinity toward MK-0499 binding. The
pyrazole 8 afforded similar potency to 6 and 7, but had an im-
proved in vivo efficacy in the Chung model of neuropathic pain
and reduced affinity toward Ikr. Pyrazoles 10 and 11 illustrate
the importance of a primary carboxamide at the C-3 position of
the pyrazole in this design for Nav1.7 potency. Based on the above
data, we decided to further examine a series of dicarboxamide pyr-
azole similar in design to pyrazole 8.
The synthesized compounds were evaluated for their ability to
block voltage gated hNav1.7 using a voltage-fluorescence (VIPR) as-
say.8 Selected compounds were screened for activity against ancil-
lary targets including IKr (MK-0499 binding assay), CYP inhibition,
and Cav1.2 (diltiazem (DLZ) binding).9 Compounds selective in
these ancillary assays were examined in the rat spinal nerve liga-
tion (SNL, Chung) model of neuropathic pain and their pharmaco-
kinetic profiles were evaluated.10
The biphenyl-3,5-carboxamide-1H-pyrazoles were synthesized
by the route outlined in Scheme 1. Copper TMEDA complex medi-
ated coupling of the commercially available 3,5-pyrazole carbox-
ylic acid ethyl ester 12 with the 3-bromophenyl boronic acid, 13
afforded the biphenyl acetophenone 14.11 Aminolysis of the diester
afforded the dicarboxamide pyrazole 15. A palladium catalyzed
cross coupling of the pyrazole 15 with aryl boronic acids afforded
the desired biphenyl pyrazole dicarboxamide 16.
Keeping the core design of the pyrazole 3 intact, we introduced
polar groups at the various positions of the pyrazole. Table 1 shows
the various polar changes which were incorporated in the pyrazole
and the associated activity. Introduction of a second carboxamide
Table 2 shows a series of novel N-aryl pyrazole-3,5-dicarboxam-
ide sodium channel blockers that were prepared from this diamide
configuration. Consistent with our prior work, we found that lipo-
philic substituents like the trifluoromethyl or trifluoromethoxy
were preferred at the R2 position. Most of these pyrazoles dis-
played good hNav1.7 block. As a measure of the subtype selectivity
of these compounds, several were also tested for block of the re-
lated analgesia target hNav1.8. As illustrated in Table 2, the com-
pounds exhibited only modest selectivity (2–8Â) in favor of
potency for block of Nav1.7 over Nav1.8.
Table 1
The effect of substitution on the pyrazole ring of N-pyrazole biphenyl Nav1.7 blockers
R1
Pyrazole 8, with a trifluoromethoxy group at the R2 position of
the biphenyl, had a very good sodium channel block but moderate
efficacy in the in vivo Chung model. Pyrazole 17, a dichloro analog,
and pyrazole 18, with a trifluoromethyl substituent, had similar
Nav1.7 functional block but poor in vivo efficacy compared to pyr-
azole 8. Incorporation of a second trifluoromethyl group brought
interesting results. A 2,4-CF3 analog (19) had poor Nav1.7 block,
but the adjacent 2,5-CF3 pyrazole 20 exhibited improved func-
tional block of Nav1.7 and modest block of Nav1.8, and interest-
ingly provided robust efficacy in vivo efficacy in the Chung
model. A trifluoromethoxy substituted pyrazole incorporating a
second substituent either a fluoro group at R6 position (23) or a tri-
fluoromethyl group at R5 position (24) led to good improved
OCF3
Entry R1
Nav1.7 VIPR MK-0499 Rat SNL
(IC50 (% inh at % reversal
or % inh 10 M)
@ 10 M)
Rat SNL
,
l
M
% reversal
l
of allodyniaa of allodyniaa
l
(2 h)
(4 h)
O
N
N
N
4
5
0.224
0.049
69%
1%
33%
11%
NH2
O
N
N
NH2
18%
8%
8%
NH2
O
O
N
NH2
NH2
6
7
0.185
0.115
15%
49%
16%,
O
O
O
OEt
a
N
NH2
NH2
N
EtO
+
ND
ND
N
N
H
Br
B(OH)2
O
O
O
NH2
12
13
O
N
N
O
8
9
0.132
0.010
21%
72%
35%,
ND
19%
ND
NH2
b
NH2
Br
Br
N
N
O
N
N
N
N
O
H2N
O
EtO
O
O
NH2
NH2
O
OEt
N
OEt
O
c
14
15
N
N
O
10
11
0.029
20%
27%
ND
ND
ND
ND
NH2
NH
N
Ar
N
N
O
O
H2N
O
NH2
1%
HN
O
NH
16
a
Reversal of rat spinal nerve ligation induced allodynia (Chung model—Von Frey)
vs pre-surgery sensitivity.
Scheme 1. Reagents and conditions: (a) [Cu(OH)TMEDA]2Cl2, DCM, 3 Å mol. sieves;
(b) 7 N Ammonia, MeOH; (c) ArB(OH)2, PdCl2(dppf), TEA, EtOH.

S. Tyagarajan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7479–7482
7481
Table 2
The effect of SAR around the biphenyl region
R7
R8
N
R2
R5
R3
R4
NH2
O
N
6 O
R
NH2
Entry
R2
R3
R4
R5
R6
R7
R8
Nav1.7 VIPR (IC50
,
lM)
Nav1.8 VIPR (IC50
,
lM)
SNL % reversala (t = 2 h)
SNL % reversala (t = 4 h)
8
17
18
19
20
21
22
23
24
25
26
27
28
29
OCF3
Cl
CF3
CF3
CF3
CF3
F
OCF3
OCF3
OCF3
OCH2CF2CF3
OCH2CF2CF3
OCH2CF2CF3
OCH2CF2CF3
H
Cl
H
H
H
H
OCF3
H
H
H
H
H
OCF3
CF3
H
H
H
CF3
H
H
H
H
H
H
F
H
H
H
H
CF3
H
H
H
CF3
H
H
H
H
H
H
CF3
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.132
0.195
0.155
9%
0.554
ND
0.915
ND
2000
ND
1513
0.802
1181
ND
35
15
9
19
7
2
ND
34
ND
8
9
6
ND
8
9
ND
44
ND
28
15
29
ND
28
25
6
0.810
0.715
0.282
0.106
0.325
0.477
0.106
0.046
0.064
0.147
H
F
H
H
H
H
H
H
ND
H
H
H
0.149
0.203
0.299
3
8
22
a
Reversal of rat spinal nerve ligation induced allodynia (Chung model—Von Frey) vs pre-surgery sensitivity.
in vitro sodium channel functional block but only modest in vivo
efficacy. A fluoro group at R7 position (25) resulted in decreased
Nav1.7 functional block. We found that increasing the lipophilicity
of the molecule with a pentafluoroethoxy group at the R2 position
and substituents at R3 or R4 or R5 position (26–29) provided com-
pounds with distinctly improved Nav1.7 and Nav1.8 functional
block but only modest in vivo efficacy.
Table 5 shows the pharmacokinetic profile in rat and dog for
compound 20. In rat, the compound had low clearance (5 mL/
min/kg), high oral exposure (AUC = 5 lM h), moderate plasma half
life (3 h), and desirable bioavailability (60%). In dog, the PK profile
followed a similar trend with low clearance, high oral exposure,
and longer half life of 12 h. The oral exposure increased in a
dose-dependent manner (AUC = 372 lM h) following a single
From these results, pyrazole 20 was identified for additional
characterization based on the remarkable efficacy in the SNL
(Chung) model. Counter screening compound 20 against a panel
of receptors, enzymes, and ion channels (MDS Pharma Services)
established pyrazole 20 to be remarkably selective for sodium
channels. Binding at IKr, Cav1.2, and Nav1.5 was measured as a gen-
eral indicator of ion channel activities.12 As illustrated in Table 3,
pyrazole 20 has a favorable profile against calcium and potassium
ion channel targets but little subtype selectivity for sodium
channels.
20 mpk po dose. Compound 20 afforded remarkable exposure at
even higher doses (single 60 mg/kg po dose).
This pyrazole dicarboxamide was efficacious in various preclin-
ical pain models. In the SNL model of neuropathic pain, 20 reversed
mechanical allodynia at 2 and 4 h in a dose-dependent manner
(Table 6) achieving maximally 59% reversal of allodynia following
a 30 mg/kg dose. In the rat CFA (complete Freund’s adjuvant-in-
duced inflammatory pain) model, when dosed orally at 30 mg/kg,
compound 20 produced 46% and 53% reversal of mechanical hyper-
algesia at 2 h and 4 h, respectively.
Compound 20 did not inhibit any of the major CYP isoform en-
zymes as shown in Table 4. The pyrazole also exhibited a low po-
tential for CYP3A4 induction as measured by an hPXR induction
assay. Compound 20 was stable in liver microsome preparations
from human, rat, and dog, with >99% parent remaining after
Table 5
Pharmacokinetic profile of pyrazole 20
Dose (iv/po)
(mg/kg)
t1/2
(h)
Cmax
M)
Clp (mL/
mg/kg)
PO AUC
M h)
F
(%)
(
l
(
l
60 min incubation (10 lM substrate).
Rat
1/3
3.1
12
2.64
2.92
20.33
24.44
5
0.5
5.6
50
372
650
60
33
Dog 0.5/2
Dog 20 po
Dog 60 po
Table 3
Ion channel profile for compound 20
Entry MK-0499
bindinga
hERG
EPb
DLZ
Nav1.5 VIPR (IC50
lM)
bindinga
Table 6
In vivo activity and exposure of compound 20
20
17%
15%
0%
1.32
a
CF3
%Inh at 10
%Inh at 30
l
l
M.
M.
b
O
N
N
NH2
H2N
CF3
O
Entry
% Reversal of allodynia (2 h)
% Reversal of allodynia (4 h)
Table 4
CYP profile for compound 20 (IC50
,
l
M)
CYP2C9
20.3
SNL (3 mpk)
SNL (10 mpk)
SNL (30 mpk)
CFA (30 mpk)
18%
44%
56%
46%
30%
34%
59%
53%
Entry
CYP3A4
>100
CYP2D6
>100
PXR
>25
20

7482
S. Tyagarajan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7479–7482
In summary, pyrazole 20 exhibited greater in vitro metabolic
Baseline
2 hour post-dose
stability than pyrazole carboxamide 2, in that hydrolysis to the cor-
responding acid was reduced in rat liver preparations. A new class
of sodium channel blockers based on a biphenyl pyrazole core with
a primary carboxamide at C-3 and C-5 position has been identified
and optimized. Compound 20 has excellent efficacy in the Chung
model of neuropathic pain and outstanding exposure in the rat
and dog pharmacokinetic profile. The compound exhibited no
CNS ancillary pharmacology and limited preclinical CV effects at
exposures well above those required for preclinical efficacy.
3:21
2:52
2:24
1:55
1:26
0:57
0:28
0:00
-2%
-11%
18%
Vehicle
Vehicle = Imwitor /Tween 80
30 mg/kg p.o. 100 mg/kg p.o.
Figure 2. Effect of compound 20 on motor coordination in the rat rota-rod model.
References and notes
In the rat rotarod model of motor coordination, no significant
effect was observed at both 30 mg/kg and 100 mg/kg doses for pyr-
azole 20 which resulted in 3.5 h plasma exposure of 31.6 lM
1. Cox, J. J.; Reimann, F.; Nicholas, A. K.; Thornton, G.; Roberts, E.; Springell, K.;
Karbani, G.; Jafri, H.; Mannan, J.; Raashid, Y.; Al Gazali, L.; Hamamy, H.; Valente,
E. M.; Gorman, S.; Williams, R.; Mchale, D. P.; Wood, J. N.; Gribble, F. M.;
Woods, C. G. Nature 2006, 444, 894.
(Fig. 2). In contrast, marketed drugs mexiletine and Gabapentin
at subefficacious oral dose of 100 mg/kg significantly reduced la-
tency in rats on the rotarod model.13,14
2. Taylor, R. S. Eur. J. Neurol. 2006, 13, 315.
3. Catterall, W. A. Neuron 2000, 26, 13.
4. Djouhri, L.; Newton, R.; Levinson, S. R.; Berry, C. M.; Carruthers, B.; Lawson, S. N.
J. Physiol. 2003, 546, 565.
The cardiovascular effects of pyrazole 20 were examined in bar-
biturate-anesthetized and ventilated dogs at rising doses of 1, 3,
and 10 mg/kg IV. Compound 20 had no treatment-related effect
on mean arterial pressure, heart rate, or QRS and QTc intervals over
this dose range. A small but significant increase was observed in
the PR interval, reaching maximally 6% at 10 mg/kg. Average plas-
ma concentrations measured after infusion of 1, 3, and 10 mg/kg
5. Priest, B. T.; Garcia, M. L.; Middleton, R. E.; Brochu, R. M.; Clark, S.; Dai, G.; Dick,
I. E.; Felix, J. P.; Liu, C. J.; Reiseter, B. S.; Schmalhofer, W. A.; Shao, P. C. P.; Tang,
Y. S.; Chou, M. Z.; Kohler, M. G.; Smith, M. M.; Warren, V. A.; Williams, B. S.;
Cohen, C. J.; Martin, W. J.; Meinke, P. T.; Parsons, W. H.; Wafford, K. A.;
Kaczorowski, G. J. Biochemistry 2004, 43, 9866.
6. Tyagarajan, S.; Chakravarty, P. K.; Zhou, B.; Fisher, M. H.; Wyvratt, M. J.; Lyons,
K. A.; Klatt, T.; Li, X.; Kumar, S.; Williams, B.; Felix, J. P.; Priest, B. T.; Brochu, R.
M.; Warren, V. A.; Smith, M. M.; Garcia, M. L.; Kaczorowski, G. J.; Martin, W. J.;
Abbadie, C.; McGowan, E.; Jochnowitz, N.; Parsons, W. H. Bioorg. Med. Chem.
Lett. 2010, 20, 5536.
7. Tyagarajan, S.; Chakravarty, P. K.; Zhou, B.; Taylor, B.; Fisher, M. H.; Wyvratt, M.
J.; Lyons, K. A.; Klatt, T.; Li, X.; Kumar, S.; Williams, B.; Felix, J. P.; Priest, B. T.;
Brochu, R. M.; Warren, V. A.; Smith, J. M.; Garcia, M. L.; Kaczorowski, G. J.;
Martin, W. J.; Abbadie, C.; McGowan, E.; Jochnowitz, N.; Parsons, W. H. Bioorg.
Med. Chem. Lett. 2010, 20, 5480.
8. Felix, J. P.; Williams, B. S.; Priest, B. T.; Brochu, R. M.; Dick, I. E.; Warren, V. A.;
Yan, L. Z.; Slaughter, R. S.; Kaczorowski, G. J.; Smith, M. M.; Garcia, M. L. Assay
Drug Dev. Technol. 2004, 2, 260.
doses were 7.3 3.6, 16.2 8.0, and 78.2 33.6
respectively.
lM (mean SD),
Pyrazole 20 was also given to conscious mice to determine the
potential effects on central nervous system (CNS) function, behav-
ior, motor activity, and body temperature. No treatment-related
changes were observed at 2, 5, and 24 h after a 100 mg/kg oral
dose.
9. Schoemaker, H.; Hicks, P. E.; Langer, S. Z. J. Cardiovasc. Pharmacol. 1987, 9, 173.
10. Chaplan, S. R.; Bach, F. W.; Pogrel, J. W.; Chung, J. M.; Yaksh, T. L. J. Neurosci.
Methods 1994, 53, 55.
While compound 20 was chosen for further characterization
based on the high level of pharmacological efficacy in the SNL as-
say, we note that efficacy in this series was not accurately pre-
dicted by potency in the Nav1.7 or Nav1.8 VIPR assay. Indeed,
several structurally related compounds in this series afforded
superior potency in these assays but exhibited diminished efficacy
in vivo. There is a possibility that the sodium channel VIPR assays
do not fully reproduce the physiologic state of the ion channel
in vivo. Additionally, we cannot rule out the possibility that 20
could be active on additional targets for analgesia.
11. Collman, J. P.; Zhong, M. Org. Lett. 2000, 2, 1233.
12. Joshi, S. K.; Mikusa, J. P.; Hernandez, G.; Baker, S.; Shieh, C. C.; Neelands, T.;
Zhang, X. F.; Niforatos, W.; Kage, K.; Han, P.; Krafte, D.; Faltynek, C.; Sullivan, J.
P.; Jarvis, M. F.; Honore, P. Pain 2006, 123, 75.
13. Brochu, R. M.; Dick, I. E.; Tarpley, J. W.; McGowan, E.; Gunner, D.; Herrington, J.;
Shao, P. P.; Ok, D.; Li, C. S.; Parsons, W. H.; Stump, G. L.; Regan, C. P.; Lynch, J. J.;
Lyons, K. A.; McManus, O. B.; Clark, S.; Ali, Z.; Kaczorowski, G. J.; Martin, W. J.;
Priest, B. T. Mol. Pharmacol. 2006, 69, 823.
14. McGowan, E.; Hoyt, S. B.; Li, X. H.; Lyons, K. A.; Abbadie, C. Anesth. Analg. 2009,
109, 951.