3-(4-Phenoxyphenyl)pyrazoles: A Novel Class of Sodium Channel Blockers

Article abstract of DOI:10.1021/jm030498q

A series of 3-(4-phenoxyphenyl)-1H-pyrazoles were synthesized and characterized as potent state-dependent sodium channel blockers. A limited SAR study was carried out to delineate the chemical requirements for potency. The results indicate that the distal

Full text of DOI:10.1021/jm030498q

J . Med. Chem. 2004, 47, 1547-1552
1547
3-(4-P h en oxyp h en yl)p yr a zoles: A Novel Cla ss of Sod iu m Ch a n n el Block er s
J i Yang,*,† Parviz Gharagozloo,† J iangchao Yao,† Victor I. Ilyin,† Richard B. Carter,‡ Phong Nguyen,§
Silvia Robledo,§ Richard M. Woodward,† and Derk J . Hogenkamp#
Discovery Research, Purdue Pharma L.P., 6 Cedar Brook Drive, Cranbury, New J ersey 08512, Novartis Pharmaceuticals Corp.,
One Health Plaza, East Hanover, New J ersey 07936, Allergan, Inc., 2525 Dupont Drive, Irvine, California 92612, and
Department of Pharmacology, College of Medicine, University of California, Irvine, California 92697
Received October 3, 2003
A series of 3-(4-phenoxyphenyl)-1H-pyrazoles were synthesized and characterized as potent
state-dependent sodium channel blockers. A limited SAR study was carried out to delineate
the chemical requirements for potency. The results indicate that the distal phenyl group is
critical for activity but will tolerate lipophilic (+π) electronegative (+σ) substituents at the
ortho and/or para position. Substitution at the pyrazole nitrogen with a H-bond donor improves
potency. Compound 18 showed robust activity in the rat Chung neuropathy paradigm.
Ch a r t 1
In tr od u ction
Neuropathic pain syndromes are caused by injury to
the nervous system. The injury can result from meta-
bolic disorders, infection, or physical trauma. Examples
include painful diabetic neuropathy, postherpetic neu-
ralgia (shingles), complex regional pain syndrome, and
chronic postoperative pain.1,2 A significant proportion
(∼10%) of lower back pain is also thought to have a
neuropathic component.3 Neuropathic pain is chronic
in nature and is characterized by allodynia, hyperalge-
sia, and hyperpathia.4 The molecular mechanisms un-
derlying these pain states remain unclear, and treat-
ment options are limited.5
A common feature of peripheral neuropathic pain is
pathological firing patterns in primary afferent neurons,
including ectopic discharge.6 There is now a growing
body of evidence that voltage-gated Na+ channels play
a role in establishing and maintaining these firing
patterns.7,8 In particular, the expression of various Na+
channel subunits in primary afferent neurons changes
in animal models of nerve injury and neuropathic
pain.9-12 Furthermore, nociception is modified in animal
studies where specific Na+ channel subunits have been
knocked out or where expression has been decreased by
use of intrathecal antisense.13,14 It is not surprising,
therefore, that drugs such as carbamazepine (1), lam-
otrigine (2), and phenytoin (3), which block Na+ channel
function, have shown clinical efficacy in the treatment
of neuropathic pain (Chart 1).15,16 Still, these compounds
were developed as anticonvulsants and are only weakly
active at Na+ channels. Subsequent efforts to optimize
the Na+ channel potencies have led to more agents (e.g.,
4 and 5) with improved efficacies in models of inflam-
matory and neuropathic pain.17,18
in hippocampal neurons, and its MED in the Chung
model is ∼2.5 mg/kg.19-22 An in vivo metabolism study
in rats suggested that the semicarbazone moiety of 6 is
a major metabolic site leading to the formation of
several metabolites.23 In addition, the compound suffers
from poor aqueous solubility. It was envisaged that
heterocyclic bioisosteres of the semicarbazone moiety
would give analogues with improved metabolic stability
and solubility. Our efforts led to compound 10, where a
pyrazole ring was found to be a suitable replacement.
In this paper, we report the synthesis of several
analogues of 10 with high sodium channel blocking
potency.
V102862 (6) is a potent, state-dependent Na+ channel
blocker that is efficacious in animal models of neuro-
pathic pain. It has a Ki value of ∼0.6 µM on Na currents
Ch em istr y
The procedure for synthesizing the pyrazole series is
shown in Scheme 1.20,24 Reaction of phenols with 4-fluo-
roacetophenone gave the desired products in high yields.
Subsequent reaction of the phenoxyphenyl ketones with
N,N-dimethylformamide dimethyl acetal afforded the
* To whom correspondence should be addressed. Telephone: 609-
409-5164. Fax: 609-409-6930. E-mail: ji.yang@pharma.com.
† Purdue Pharma L. P.
‡ Novartis Pharmaceuticals Corp.
§ Allergan, Inc.
#
University of California, Irvine.
10.1021/jm030498q CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/10/2004

1548 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Yang et al.
Sch em e 1a
a
Reagents and conditions: (a) K2CO3, DMA, 150 °C; (b) HC(OMe)2NMe2, DMF, reflux; (c) hydrazine, EtOH, reflux; (d) MeI, NaOH,
b
n-Bu4NBr; (e) NaOCN, HOAc; (f) methyl isoyanate, CH2Cl2; (g) DBU, dimethylcarbamoyl chloride, THF. Available commercially.
corresponding â-(dimethylamino)-R,â-unsaturated ke-
tones, which upon treatment with hydrazine-hydrate
in ethanol furnished unsubstituted pyrazoles (e.g., 10).
Methylation of 10 with MeI gave 13 in 80% yield.
Conversion of the pyrazole intermediates (9-12) to the
respective primary amides 14, 15, 18, and 19 was
affected with sodium cyanate in 90% aqueous acetic acid
in 50-60% yield. The reaction of 10 with methyl
isocyanate or dimethylcarbamoyl chloride gave the
secondary and tertiary amides 16 and 17, respectively,
in >80% yields.
position of the phenyl group with the more lipophilic
electron-withdrawing NO2 group (i.e., 18) has little
effect. Inclusion of a second fluorine atom in the 2-posi-
tion of the phenyl ring (i.e., 19) does not change potency.
As mentioned earlier, V102862 (6) is a state-depend-
ent Na+ channel blocker with 150-fold selectivity be-
tween the inactive (Ki) and resting (Kr) states. The
selectivity is important to reduce the liability of produc-
ing anesthetic-like “nerve blocks” and disruption of
normal sensation. The structural modifications incor-
porated in compounds 9, 10, and 13-19 appear to have
little effect on the resting state potency and in general
follow a similar (albeit less pronounced) SAR trend to
that observed at the inactivated state. Thus, the major-
ity of the analogues exhibit good to excellent selectivity
between the inactivated and resting states of the
channels. Compound 19 is the most potent and state-
dependent sodium channel blocker reported to date.
Compound 18 was tested in an experimental model
of peripheral neuropathy produced by segmental spinal
nerve ligation in the rat (the Chung model, Figure 1)
and compared with carbamazepine (1).26 The data
indicate that 18, at a dose of 10 mg/kg po, shows robust
antiallodynic effects comparable to carbamazepine at
100 mg/kg po across all time points.
Resu lts a n d Discu ssion
The compounds listed in Table 1 were evaluated in
electrophysiological assays for their sodium channel
blocking activity. Inhibition of voltage-gated sodium
currents was recorded in HEK-293 cells stably express-
ing hSkM1 sodium channels. The blocking effects of
these compounds are generally highly sensitive to the
holding voltage, indicating they bind to voltage-sensitive
sodium channels in their inactivated states (measured
as Ki) and have weaker potency toward resting states
(measured as Kr).25
At the inactivated state Ki, a comparison of the
structures 7-9 reveals that the presence of the distal
phenyl group is critical to the potency of the molecules.
However, the inclusion of a fluorine atom (present in
6) in the 4-position of the phenyl group (10) has little
or no effect on the potency (e.g., compare 9 and 10, and
14 and 15). N-methylation of the pyrazole ring (13)
lowers the potency by about 3-fold, while introduction
of a primary amide increases it by about 8-fold. This
observation is supported by two comparisons (i.e., 9 and
14, and 10 and 15). The results may suggest the
requirement of a hydrogen-bond donor in this region of
the molecule. This is further supported by the secondary
and/or tertiary amide analogues (16 and 17) where up
to a 10-fold drop in potency results over the primary
amide (15). Replacement of the fluorine atom in the para
Con clu sion s
3-(4-Phenoxyphenyl)pyrazoles are a novel class of
state-dependent sodium channel blockers that have high
in vitro potencies. Initially we identified compound 10
to be equipotent to V102862 (6). Our SAR results
indicate the requirement of a H-bond donor at the
pyrazole nitrogen. Finally, at one-tenth of the dose,
compound 18 is as efficacious as carbamazapine (1) in
an in vivo model of neuropathic pain.
Exp er im en ta l Section
Ch em istr y. All purchased reagents were used without
further purification. Flash chromatography was performed on
E. Merck silica gel 60, 230-440 mesh. TLC analysis was

3-(4-Phenoxyphenyl)pyrazoles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1549
Ta ble 1. In Vitro Potencies at the Inactivated (Ki) and Resting (Kr) States of Sodium Channelsa
a
The potencies were measured on the human skeletal muscle hSkM1 Na+ cell line (hosted in HEK-293 cells). The Ki and Kr values are
the measured potencies at the inactivated and resting states of sodium channels, respectively. The numbers in parentheses are run
b
times. Available commercially.
Agilent 1100 series LC/MSD. HRMS experiments were per-
formed on a Bruker Apex II 4.7T FTMS.
3-[4-(4-Flu or oph en oxy)ph en yl]-1H-pyr azole (10). A mix-
ture of 4-fluoroacetophenone (20 g, 0.14 mol, 1.0 equiv),
4-fluorophenol (17.8 g, 0.16 mmol, 1.1 equiv), and potassium
carbonate (18 g, 0.13 mol, 0.9 equiv) in N,N-dimethylacetamide
(80 mL) was heated at 150 °C for 16 h. The mixture was cooled
to room temperature, diluted with CHCl3 (200 mL), and
washed with 2 N aqueous sodium hydroxide solution (2 × 100
mL). The organic layer was dried over sodium sulfate, filtered,
and evaporated under reduced pressure to afford the crude
product (30 g). This was dried in vacuo (40 °C/1 mmHg) for 2
h to give 4-(4-fluorophenoxy)acetophenone as a viscous brown
1
oil (27.5 g, 83%). H NMR (CDCl3) δ 7.93 (d, J ) 8.7 Hz, 2H),
F igu r e 1. Rat Chung neuropathy test for carbamazepine and
7.09-7.04 (m, 4H), 6.96 (d, J ) 8.4 Hz, 2H), 2.57 (s, 3H).
18: dose- and time-response relation for compound-induced
changes in the mechanical force required to produce paw
4-(4-Fluorophenoxy)acetophenone (23 g, 0.1 mol) and N,N-
dimethylformamide dimethylacetal (13 g, 0.11 mol) in DMF
(70 mL) were heated at 150 °C for 16 h. The solution was cooled
to room temperature, and water (250 mL) was added over a
period of 20 min. The yellow precipitate was collected by
filtration, washed with water (3 × 200 mL), and dried in vacuo
at 60 °C for 24 h (27 g). The product (5 g, 17.5 mmol) was
dissolved in ethanol (70 mL), followed by the addition of 35%
aqueous hydrazine (10.0 g, 0.11 mol). The solution was heated
at 80 °C for 1.5 h and then taken to dryness. The crude product
was redissolved in ethyl acetate and washed with water (2 ×
50 mL) and brine (50 mL). The residue was further purified
on a silica gel column, eluting with 1:1 EtOAc/hexane to give
withdrawal in rat (mechanical threshold). The threshold was
determined manually on the left plantar surface with von Frey
filaments using the up-down method. Neuropathic injury
involved tightly ligating the left L5 and L6 spinal nerves. Each
point is the mean ( SEM for a group of eight animals. Pre-op
represents the preoperative control. The zero time values show
the hypersensitivity (allodynia) that developed during the 1
week following surgery.
carried out on a glass plate precoated with silica gel 60 F254
.
Melting points were determined on a MEL-TEMP II capillary
melting point apparatus and are uncorrected. 1H NMR spectra
were recorded on a Bruker Avance 400 spectrometer and were
referenced to TMS, and all chemical shifts are reported as δ
(ppm) values. LC/MS experiments were performed on an
1
10 as an off-white solid (5.3 g, 95%). H NMR (CDCl3) δ 10.6
(bs, 1H), 7.71 (d, J ) 8.4, 2H), 7.60 (d, J ) 2.1 Hz, 1H), 7.04-

1550 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
6.99 (m, 6H), 6.57 (d, J ) 2.4 Hz, 1H). HRMS Calcd for C15H11
Yang et al.
-
Biology. Electr op h ysiology. Cell Cu ltu r e. The human
skeletal muscle Na+ (hSKM1) cell line (hosted in HEK-293
cells) was a generous gift from Dr. A. L. George, J r. (Vanderbilt
University Medical Center, Nashville,TN). For electrophysi-
ology, cells were plated onto 35 mm Petri dishes (precoated
with poly-D-lysine) at a density of ∼104 cells/dish on the day
of reseeding from confluent cultures. HEK-293 cells were
suitable for recordings for 3-4 days after plating.
P a tch -Cla m p Recor d in gs. Whole-cell voltage-clamp re-
cordings were made using conventional patch-clamp tech-
niques at room temperature (ca. 22 °C).27a The patch-clamp
pipettes were pulled from thick-walled borosilicate glass (WPI,
Sarasota, FL). Currents were recorded using an Axopatch
200A amplifier (Axon Instruments, Union City, CA) and were
leak-subtracted (P/4 or by analog built-in circuitry), low-pass-
filtered (3 kHz, 8-pole Bessel), digitized (20-50 µs intervals),
and stored using Digidata-1200 B interface and Pclamp8.1/
Clampex software (Axon Instruments, Union City, CA). When
necessary, residual series access resistance was largely (75-
80%) canceled using built-in amplifier circuitry. External
solution contained the following (in mM): 150 NaCl, 5.4 KCl,
1.8 CaCl2, 1 MgCl2, 10 D-glucose, 5 HEPES; pH 7.4 (NaOH).
The internal solution contained the following (in mM): 130
CsF, 20 NaCl, 1 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES; pH 7.3
(CsOH). Osmolality was set at ∼10 mmol/kg lower than that
for external solution.
Dr u g Ap p lica tion . Drug solutions and intervening inter-
vals of wash were applied through a linear array of flow pipes
(Drummond Microcaps, 2 µL, 64 mm length). Drugs were
dissolved in dimethyl sulfoxide (DMSO) to make 1-30 mM
stock solutions. The stock solutions were diluted into external
solution to generate final concentrations of 0.003-10 µM. At
the highest concentration, the concentration of DMSO was
0.1%. At this level, DMSO itself had only marginal inhibitory
effects on the peak Na+ current.
Stim u la tion P r otocol a n d Da ta An a lysis. To assess tonic
(resting state) block membrane voltage was held at -120 mV
at which all hSkM1 Na+ channels are in the resting state.
From this holding voltage, a depolarizing testing pulse to 0
mV was applied to cause maximal sodium current. The size
of the current was measured in the control and in the presence
of high (3 µM) concentration of an antagonist. The ratio of
these amplitudes defined FR (fractional response). The con-
stant, Kr, for inhibition was determined using the following
equation (a modified form of the logistic equation with the
slope factor n ) 1):27b
FN2O: 254.0855. Found: 254.0856. Anal. (C15H11FN2O) C, H,
N.
3-[4-(4-F lu or op h e n oxy)p h e n yl]-1-m e t h yl-1H -p yr a -
zole (13). A mixture of 10 (0.4 g, 1.6 mmol), CH3I (0.3 g, 2.1
mmol), NaOH (6N, 1 mL), and n-Bu4NBr (20 mg) in MeOH (4
mL) was stirred at room temperature for 24 h. The mixture
was partitioned between EtOAc and water. The organic layer
was separated, dried over MgSO4, and concentrated in vacuo.
The residue was purified by column chromatography (EtOAc/
hexanes 3:7) to afford 13 as a colorless oil (0.32 g, 80%). 1H
NMR (CDCl3) δ 7.78 (d, 2H, J ) 8.7 Hz), 7.39 (d, J ) 2.2 Hz,
1H), 6.09-7.09 (m, 6H), 6.51 (d, J ) 2.4 Hz, 1H), 3.96 (s, 3H).
HRMS Calcd for C16H13FN2O: 268.1012. Found: 268.1012.
Anal. (C16H13FN2O) C, H, N.
3-[4-(4-F lu or op h en oxy)p h en yl]-1H-p yr a zole-1-ca r box-
a m id e (15). A solution of sodium cyanate (1.4 g, 20.7 mmol)
in water (5 mL) was added to a solution of 10 (4.4 g, 17.3 mmol)
in 90% aqueous acetic acid (65 mL). The reaction mixture was
stirred at room temperature for 16 h. Water was added, and
the precipitate was collected by filtration. The residue was
chromatographed on silica gel (EtOAc/hexanes 1:1) to give 15
1
as a white solid (2.79 g, 52%): mp ) 141-143 °C; H NMR
(DMSO-d6) δ 8.28 (d, J ) 3.0 Hz, 1H), 7.94 (d, J ) 8.7 Hz,
2H), 7.84 (bs, 2H), 7.24 (t, J ) 8.4 Hz, 2H), 7.13-7.08 (m, 2H),
7.04 (d, J ) 8.4 Hz, 2H), 6.94 (d, J ) 2.7 Hz, 1H). HRMS Calcd
for C16H12FN3O2: 297.0914. Found: 297.0913. Anal. (C16H12
FN3O2) C, H, N.
-
3-[4-(4-F lu or op h en oxy)p h en yl]-1H-p yr a zole-1-ca r box-
ylic Acid Meth yla m id e (16). A solution of 10 (0.4 g, 1.6
mmol) and methyl isocyanate (0.1 g, 1.8 mmol) in CH2Cl2 (4
mL) was stirred at room temperature for 24 h. The solvent
was removed, and the residue was chromatographed on silica
gel (EtOAc/hexanes 3:7) to afford 16 as a colorless oil (0.4 g,
1
80%). H NMR (CDCl3) δ 8.28 (d, J ) 2.6 Hz, 1H), 7.81 (d, J
) 8.7 Hz, 2H), 7.15-7.25 (br, 1H), 7.05-7.10 (m, 6H), 6.67 (d,
J ) 2.8 Hz, 1H), 3.09 (d, J ) 5.0 Hz, 3H). HRMS Calcd for
C
17H14FN3O2: 311.1070. Found: 311.1070. Anal. (C17H14-
FN3O2) C, H, N.
3-[4-(4-F lu or op h en oxy)p h en yl]-1H-p yr a zole-1-ca r box-
ylic Acid Dim eth yla m id e (17). A solution of 10 (0.4 g, 1.6
mmol), DBU (0.4 g), and dimethylcarbamoyl chloride (0.25 g,
1.5 mmol) in THF (10 mL) was stirred at room temperature
for 24 h. The reaction mixture was partitioned between EtOAc
and water. The organic layer was separated, washed with
brine, dried over MgSO4, and concentrated. The crude product
was chromatographed on silica gel (EtOAc/hexanes 3:7) to
afford 15 as a colorless oil (0.42 g, 84%). 1H NMR (CDCl3) δ
8.18 (d, J ) 2.6 Hz, 1H), 7.82 (d, J ) 8.8 Hz, 2H), 7.02-7.09
(m, 6H), 6.66 (d, J ) 2.6 Hz, 1H), 3.24-3.36 (br, 6H). HRMS
Calcd for C18H16FN3O2: 325.1227. Found: 325.1223. Anal.
(C18H16FN3O2) C, H, N.
FR
1 - FR
Kr )
[antag]
where [antag] is the concentration of the antagonist used. To
measure the affinity toward the inactivated state of the
channel, two distinct experimental paradigms were used. For
most of the compounds tested, we used depolarizing prepulse
protocol. Here, cells again were held at a holding potential of
-120 mV to keep nearly all the channels in the closed state.
Then a 3 s depolarizing step (“prepulse”) was made to the
voltage (usually 0 mV) where the maximum current was
elicited. At the end of this depolarization, all the channels
would be in the inactivated state. A 3 ms hyperpolarizing step
back to -120 mV was then made in order to remove some
channels from the inactivated state. These channels comprised
the fraction that had no antagonist bound and thus recovered
from inactivation caused by the prepulse very rapidly (>95%
recovery within 2 ms).27c A final depolarizing step (“test pulse”)
was used to assay the fraction of sodium channels available
for activation. Sodium currents were measured at this test
pulse before and after application; FR values were calculated
for different concentrations of an antagonist. The latter were
used to plot partial concentration inhibition curves. Each
individual curve was fitted by the logistic expression
The syntheses of 14, 18, and 19 were carried out following
the same experimental procedures that were described for 15.
3-(4-P h en oxyp h en yl)-1H-p yr a zole-1-ca r boxa m id e (14):
mp ) 132-133 °C; 1H NMR (CDCl3) δ 8.24 (d, J ) 2.7 Hz,
1H), 7.80 (d, J ) 8.7 Hz, 2H), 7.37 (t, J ) 8.4 Hz, 2H), 7.14 (t,
J ) 7.5 Hz, 1H), 7.08-7.05 (m, 2H), 7.06 (d, J ) 9.0 Hz, 2H),
6.90 (d, J ) 3.0 Hz, 1H), 5.30 (bs, 2H). HRMS Calcd for
C
16H13N3O2: 279.1008. Found: 279.0998. Anal. (C16H13N3O2)
C, H, N.
3-[4-(4-Nitr op h en oxy)p h en yl]-1H-p yr a zole-1-ca r boxa -
1
m id e (18): mp ) 145-147 °C; H NMR (CDCl3) δ 8.28 (d, J
) 2.4 Hz, 1H), 8.23 (d, J ) 9.0 Hz, 2H), 7.91 (d, J ) 8.4 Hz,
2H), 7.16 (d, J ) 8.7 Hz, 2H), 7.07 (d, J ) 9.3 Hz, 2H), 6.73 (d,
J ) 3.0 Hz, 1H), 5.3 (bs, 2H). HRMS Calcd for C16H12N4O4:
324.0859. Found: 324.0861. Anal. (C16H12N4O4) C, H, N.
3-[4-(2,4-Diflu or op h en oxy)p h en yl]-1H-p yr a zole-1-ca r -
1
boxa m id e (19): mp ) 132-134 °C; H NMR (CDCl3) δ 8.24
(d, J ) 2.7 Hz, 1H), 7.79 (d, J ) 8.7 Hz, 2H), 7.15-7.08 (m,
2H), 6.99 (d, J ) 8.4 Hz, 2H), 6.94-6.85 (m, 1H), 6.67 (d, J )
3.0 Hz, 1H), 5.30 (bs, 2H). HRMS Calcd for C16H11F2N3O2:
315.0819. Found: 315.0821. Anal. (C16H11F2N3O2) C, H, N.
1
1 + ([antag]/Ki)n

3-(4-Phenoxyphenyl)pyrazoles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1551
using approximation programs within the Origin 5.0 software
to get the dissociation constant Ki for inactivated channels.
This protocol was applicable only for antagonists that, upon
binding to the channel, cause a significant retardation of
recovery from inactivation.27d Compounds 7 and 8 did not cause
any retardation. To assess their inhibitory effect on inactivated
channels, we measured the shift in the midpoint of steady-
state inactivation.27e For this aim, double-pulse protocol was
used to collect the steady-state inactivation curve. Cells were
held at a holding voltage of -120 mV to remove residual
steady-state inactivation. A series of 10-12 depolarizing
conditioning prepulses (each 3 s in duration) incrementing in
10 mV steps immediately followed by a 5 ms testing pulse to
preparation, Zhengrong Xu and Xun Chen for animal
surgeries, Leon Zhou for HRMS analysis, and Dr. Ravi
Upasani for helpful discussions.
Su p p or tin g In for m a tion Ava ila ble: Additional experi-
mental procedures for compound syntheses, electrophysiology,
and in vivo pharmacology. This material is available free of
charge via the Internet at http://pubs.acs.org.
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exp(-∆V1/2/k)
- 1
[
]
1 - a
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