Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Nav1.8 sodium channel with efficacy in a model of neuropathic pain

Article abstract of DOI:10.1016/j.bmc.2010.09.057

Na_v1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, a

Full text of DOI:10.1016/j.bmc.2010.09.057

Bioorganic & Medicinal Chemistry 18 (2010) 7816–7825
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and biological evaluation of potent, selective, orally bioavailable,
pyrazine-based blockers of the Nav1.8 sodium channel with efficacy
in a model of neuropathic pain
Marc J. C. Scanio a, , Lei Shi a, Irene Drizin a, Robert J. Gregg a, Robert N. Atkinson b, James B. Thomas b,
⇑
Matthew S. Johnson b, Mark L. Chapman b, Dong Liu b, Michael J. Krambis b, Yi Liu b, Char-Chang Shieh a,
XuFeng Zhang a, Gricelda H. Simler a, Shailen Joshi a, Prisca Honore a, Kennan C. Marsh a, Alison Knox a,
Stephen Werness b, Brett Antonio b, Douglas S. Krafte b, Michael F. Jarvis a, Connie R. Faltynek a,
Brian E. Marron b, Michael E. Kort a
a Global Pharmaceutical Research and Development, Abbott Laboratories, Dept R4PM, Bldg. AP9A, 100 Abbott Park Road, Abbott Park, IL 60064-6117, United States
b Icagen, Inc., 4222 Emperor Blvd., Durham, NC 27703, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2010
Revised 16 September 2010
Accepted 22 September 2010
Available online 29 September 2010
Nav1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that
is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of
inflammatory and neuropathic pain, and we envisioned that selective blockade of Nav1.8 would be
analgesic, while reducing adverse events typically associated with non-selective VGSC blocking
therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyr-
azinecarboxamides, which are potent blockers of the human Nav1.8 channel and also block TTx-r sodium
currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human
Nav1.2. We further demonstrate that an example from this series is orally bioavailable and produces
antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Sodium channels
Nav1.8
PN3
Neuropathic pain
1. Introduction
pain.7,8 Consistent with this hypothesis, non-selective blockade of
sodium channels contributes to the analgesic activity of a number
Voltage-gated sodium channels (VGSCs) are transmembrane
proteins that open in response to changes in membrane potential
to allow selective permeability of sodium ions.1–4 These channels
belong to a multi-gene family consisting of nine members that
are distinguished pharmacologically on the basis of their
sensitivity to blockade by natural toxins, particularly tetrodotoxin
(TTx).2,3 VGSCs contribute to the initiation and propagation of
action potentials in excitable tissues such as nerve and muscle,
and participate in many physiological processes including locomo-
tion, cognition, and nociception.4–6
of clinically used agents, such as mexiletine, lamotrigine, and
carbamazepine, all of which were developed for other indications.
Unfortunately, the adverse events observed with these drugs limits
their clinical utility for the treatment of chronic pain.9
Nav1.8 (also known as PN3) is a TTx-resistant (TTx-r) VGSC that is
preferentially expressed on sensory neurons10 and carries a major
portion of the TTX-r current in peripheral nerves.11 The highly local-
ized distribution of Nav1.8 in nociceptive small diameter neurons
suggests this channel may be involved in the generation of action
potentials in response to painful stimuli.12 Increased expression of
Nav1.8 in chronic pain states,13 gene knockout data,14–16 and analge-
sic effects of Nav1.8-specific antisense oligodeoxynucleotides17–19
support the premise that Nav1.8 is an attractive target for analgesic
drug discovery.20–23 Herein we describe a series of subtype-selective
small molecule Nav1.8 antagonists and their characterization as
novel antihyperalgesic agents.
Considerable data indicate that hyperexcitability and spontane-
ous action potential firing mediated by VGSCs in peripheral
sensory neurons play a role in the pathophysiology of chronic
Abbreviations: TTx, tetrodotoxin; TTx-r, tetrodotoxin-resistant; VGSC, voltage
gated sodium channels; DRG, dorsal root ganglia; ip, intraperitoneal; iv, intrave-
nous; po, per os; F, bioavailability; T1/2, half-life; Clp, plasma clearance; CEREP,
cell-surface receptors, ion channels and enzymes; Vss, volume of distribution at
steady state; Cmax, maximum concentration.
Our combined groups have previously disclosed the 5-aryl-2-
furfuramides (Fig. 1) as potent and selective inhibitors of
Nav1.8.24 A-803467, the most thoroughly characterized compound
from the 5-aryl-2-furfuramides series, has excellent potency at
⇑
Corresponding author. Tel.: +1 847 935 1064.
E-mail addresses: marc.scanio@abbott.com, mscanio@stanfordalumni.org (M.J.C.
recombinant human Nav1.8 (IC50 = 0.008 lM), inhibits TTx-r
Scanio).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.057

M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
7817
Scheme 4. Conditions: (a) H2NR, MgCl2, THF.
2. Chemistry
Methyl 6-chloro-2-pyrazinecarboxylate (3)28,29 served as an
intermediate for our exploration of 6-substituted 2-pyrazinecarb-
oxamides and could be readily prepared in two steps on multigram
scale (Scheme 1). 4-Oxo-2-pyrazinecarboxylate (2) was formed by
oxidation of methyl pyrazinecarboxylate (1) with mCPBA.30
N-Oxide 2 was then refluxed in thionyl chloride to afford the key
intermediate 3.31 The regiochemistry of 3 is consistent with litera-
ture precedence,31 and was confirmed by an HMBC NMR experi-
ment, which showed a long-range correlation between H5 and
C3, indicating that the ester and chloro substitutions are at the
2- and 6-positions.
Figure 1. Expanding SAR by switching central cores.
The majority of analogs for this study were synthesized by the
route illustrated in Scheme 2. Suzuki coupling of aryl boronic acids
and 3 afforded methyl 6-aryl-2-pyrazinecarboxylates 4–6. The
methyl esters were saponified to carboxylic acids 7–9 and subse-
quently converted to acid chlorides 11–13. A variety of amide ana-
logs (15–25, 29, 30, 32, 33, 35 and 36) were prepared by reacting
11–13 with amines in the presence of triethylamine.
Under the aqueous conditions used in the Suzuki coupling of 4-
ethoxyphenylbornic acid to 3 (Scheme 3), the ester functionality
underwent hydrolysis to provide carboxylic acid 10. Compound
10 was subsequently converted to amide analogs 26–28 via acid
chloride 14. An alternative method for amide formation involved
direct conversion of esters 5 and 6 to amides 31 and 34, respec-
tively, mediated by MgCl2 (Scheme 4).32
Scheme 1. Conditions: (a) mCPBA, 1,2-dichloroethane; (b) SOCl2.
currents (IC50 = 0.14 lM) in rat DRG neurons and is selective versus
other sodium channels (Nav1.2, Nav1.3, Nav1.5, Nav1.7) and the
hERG channel.25,26,34 It is active in Chung (EC50 = 47 mg/kg), Ben-
nett (EC50 = 85 mg/kg), and CFA (EC50 = 41 mg/kg) rodent models
of pain following intraperitoneal (ip) administration, and is now
commercially available as a tool compound. A-803467 has low oral
bioavailability (F = 13% at 10 mg/kg) in rat, moderate in vitro
intrinsic clearance (140 l
L minꢀ1 mgꢀ1, rat microsomes) and lacks
linear increases in plasma exposure upon escalating oral dose, pre-
sumably due to solubility limited absorption (aqueous solubility
<0.1 l
g/mL).27
3. Biological evaluation
We were interested in evaluating other central core series in an
attempt to identify new compounds with improved aqueous solu-
bility and oral bioavailability relative to the furan series, while
maintaining or improving potency (Fig. 1). Pyridine, oxazole, thia-
zole, oxadiazole, thiazole and thiadiazole central core replace-
ments have recently been reported.27 As a continuing part of this
strategy, pyrazine derivatives were synthesized and found to pos-
sess encouraging levels of Nav1.8 potency and isoform selectivity.
These preliminary findings set the stage for a more detailed SAR
investigation of 6-aryl-2-pyrazinecarboxamide derivatives, the
syntheses of which are detailed in Schemes 1–4.
All compounds were first evaluated for the ability to block the
recombinant mouse Nav1.8 (mNav1.8) sodium channel stably
expressed in HEK293 cells using a modified version of an isotopic
efflux assay.33 The activity of selected compounds was confirmed
by demonstrating inhibition of sodium currents in human Nav1.8
(hNav1.8) expressing HEK293 cells using a conventional voltage-
clamp electrophysiology procedure.25 Electrophysiological evalua-
tion of inhibition of native TTX-r sodium current in acutely
dissociated DRG neurons was carried out for a subset of these
Scheme 2. Conditions: (a) ArB(OH)2, PdCl2(PPh3)2, Cs2CO3, DMF; (b) NaOH, H2O/EtOH; (c) (COCl)2, cat. DMF, CH2Cl2; (d) H2NR, TEA, CH2Cl2.
Scheme 3. Conditions: (a) 4-etoxyphenylboronic acid, PdCl2(PPh3)2, Na2CO3, iPrOH/H2O; (b) (COCl)2, cat. DMF, CH2Cl2; (c) H2NR, TEA, CH2Cl2.

7818
M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
compounds.24,25 Screening against human Nav1.2 provided an
assessment of VGSC subtype selectivity. Inhibition of the hERG
channel was also evaluated as a potential liability.33 All of the final
responsible for the loss of potency, while incorporating a water
solubilizing morpholino group. Compound 22 (aqueous solubil-
ity = 2.7 lg/mL) was potent at Nav1.8 and TTx-r, while maintaining
compounds, 15–36, demonstrated hERG IC50 >10
l
M, which was
selectivity versus Nav1.2. Perturbing the position of the pyridine
nitrogen, as in isomers 23 and 24 led to a dramatic loss of Nav1.8
potency. Replacing the morpholino group of 22 with a pyrrolidino
in compound 25 resulted in a compound with comparable potency
at hNav1.8 and TTx-r, but less selectivity versus Nav1.2 and lower
aqueous solubility than either 21 or 22 (Table 1). The in vitro
not considered to be a major liability. Selected compounds were
evaluated for aqueous solubility. Key compounds were also evalu-
ated for in vitro microsomal stability in rat microsomes.
Electrophysiological protocols were designed to set the mem-
brane potential to the midpoint of voltage-dependent steady-state
inactivation (i.e., the voltage at which 50% of channels are inacti-
vated or V1/2): V1/2 = ꢀ40 mV for hNav1.8, TTx-r; V1/2 = ꢀ60 mV
for hNav1.2. For each channel, compounds were evaluated at 1–3
concentrations, with 2–6 measurements at each concentration.
For compounds with multiple single-point data, the average of
the various estimated values is reported.
turnover of 22 was high (410 l
L minꢀ1 mgꢀ1, rat microsomes)
prompting us to look for compounds with both improved solubility
and stability.
We next examined various aromatic substitutions with anilides,
benzyl amides and their heterocyclic analogs (Table 2). The 4-eth-
oxy, 4-cyano and 4-trifluoromethoxy aromatic substitutions were
selected in an attempt to improve the overall solubility and stabil-
ity of these compounds. The 4-ethoxyphenyl derivatives 26–28
were very potent at both Nav1.8 and TTx-r. Collectively, however,
Following an assessment of the pharmacokinetic profile, candi-
date compounds were further evaluated in the L5/L6 spinal nerve
tight ligation (Chung)18,35,40 model of neuropathic pain.
they possessed greater activity against Nav1.2 (IC50 61 lM), and
the ethoxy group presented a potential metabolic liability. For
4. Results and discussion
example, 28 proved particularly susceptible to microsomal turn-
over (630 l
L minꢀ1 mgꢀ1, rat microsomes). The 4-cyanophenyl
Initially, we chose to examine 6-(4-chlorophenyl)pyrazine-2-
carboxamides (Table 1) based on our SAR experience with the re-
lated furan series,24 wherein 4-chlorophenyl substitution generally
led to potent Nav1.8 inhibitors. Compounds with an IC50 value
3,5-dimethylanilde analog 29, maintained the high Nav1.8 and
TTx-r potency of other 3,5-dimethylanilide derivatives, such as
17, 26 and 32. This activity did not, however, extend to the other
4-cyanophenyl derivatives, 30 and 31, which showed weaker
TTx-r potency (IC50 >100 nM). Compound 29 had modest in vitro
<3
more rigorous electrophysiological evaluation; compounds with
an IC50 value >3 M in the mNav1.8 flux assay generally showed
lM in the mNav1.8 flux assay were advanced for additional,
intrinsic clearance (140
poor aqueous solubility (0.03
l
L minꢀ1 mgꢀ1, rat microsomes), but had
g/mL) and no oral bioavailability.
l
l
poor activity in electrophysiological evaluation of hNav1.8 or
TTx-r. We elected to use the recombinant mouse Nav1.8 cell line
for compound triage because of concerns with intellectual prop-
erty. In general, compounds displaying the most potent activity
in the recombinant mouse were among the most potent blockers
in the recombinant human assay. It is unclear to us whether the
observed left-shift in potency observed in the hNav1.8 assay de-
rives from a species difference (mouse vs human) or a screening
platform difference (flux vs electrophysiological evaluation).
The anilides (15–17) were the first compounds to be evaluated.
These analogs were inhibitors in the mNav1.8 flux assay, with IC50
The 4-trifluoromethoxy derivatives 32–36 tended toward good po-
tency at both Nav1.8 and TTx-r. Morpholine 36 was designed to mi-
mic the solubilizing strategy used in 22, and was also more potent
at Nav1.8 and TTx-r than 22. In fact, 36 was markedly more water
soluble (>50
addition, 36 had modest in vitro intrinsic clearance (98
mgꢀ1, rat microsomes). Compound 36 was more active at Nav1.2
l
g/mL) than the other compounds in this series. In
l
L minꢀ1
than 22, but its IC50 was greater than 1 lM and was still 79-fold
selective relative to hNav1.8.
Given its improved aqueous solubility and favorable clearance
values, 36 was selected for pharmacokinetic evaluation (Table 3),
where it demonstrated modest oral bioavailability and exposure.
Oral administration of 36 resulted in dose-dependent attenuation
of mechanical allodynia in the L5/L6 spinal nerve injury (Chung)
model of neuropathic pain (ED50 = 78 mg/kg) (Fig. 2). A comparison
of 36 and 29 demonstrates the importance of physiochemical
properties in this series, as the two compounds had similar
potency at Nav1.8, TTx-r and Nav1.2. Compound 36 was much more
values <2 lM in all cases. In electrophysiological assays, the
anilides were very potent inhibitors (IC50 <10 nM) of hNav1.8.
The anilides also demonstrated good TTx-r inhibition (IC50
<300 nM) and generally had favorable selectivity (>1000-fold) for
hNav1.8 versus Nav1.2. For example, the most potent anilide, 17,
was a 3 nM hNaV1.8 blocker (TTx-r IC50 52 nM) and >1600-fold
selectivity against Nav1.2. The benzyl amine 18 maintained compa-
rable potency relative to the anilides in the mNav1.8 flux, hNav1.8
and TTx-r assays and showed excellent selectivity against Nav1.2
water soluble than 29 (>50 lg/mL vs 0.03 lg/mL), supporting the
hypothesis that increasing solubility would increase exposure
and oral activity. Compounds 36 and 29 were also tested in a broad
screening panel (N = 70) of cell-surface receptors, ion channels and
enzymes (CEREP, Poitiers, France) and showed no or weak (IC50
(<5% inhibition at 3 lM). Further extension of the linker between
the amide and aromatic group to phenethylamine derivative 19
resulted in conserved in vitro potency in the hNav1.8 and TTx-r
assays, but with a substantial loss of selectivity against Nav1.2
(53-fold selective). Therefore we elected to focus the majority of
our effort on exploring the SAR of anilides, benzyl amides and their
heterocyclic analogs.
>2
10
l
l
M) activity, 36 showed modest activity at three receptors at
M (Table 3).
As the simple anilides tended to be very water insoluble (e.g., the
5. Conclusion
aqueous solubility of 17 <0.1 lg/mL), steps were taken to increase
water solubility by incorporating heteroatoms, such as in pyridyl
analog 20 and in 2-picolinyl derivative 21. Although the potency
of 21 was weak, its increased aqueous solubility (1 lg/mL) was a
10-fold improvement relative to 17 making it an attractive starting
point for the design of compounds with enhanced physiochemical
properties. In analog 22, for example, the objective was to attenuate
the basicity of the pyridine nitrogen, which we thought might be
We have identified a novel series of pyrazine-based derivatives
that are potent, selective blockers of the Nav1.8 sodium channel.
These compounds further demonstrate that the furan core of
A-803467 can be replaced with heteroaromatics, while maintain-
ing potency at blocking Nav1.8 channels and DRG neurons.27 In
general, good activity was achieved with a variety of substitutions
on the general pyrazine scaffold but with different SAR from the

M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
7819
Table 1
In vitro characterization of 6-(4-chlorophenyl)pyrazine-2-carboxamide Nav1.8 channel blockers 15–25 and A-803467 for comparison
Cl
O
N
N
R
N
H
Compound
A-803467
R
mNav1.8
IC50 M)
hNav1.8
Est. IC50
TTx-r
Est. IC50
hNav1.2
Est. IC50
Solubility (
l
g/mL)
Rat microsomal
a
a
a
(
l
(
lM)
(lM)
(
lM)
clearance (
lL/min/mL)
0.85 0.3b
0.008b
0.14b
7.4b
<0.1d
140d
OMe
15
1.07 0.34
0.006
0.19
6.2
<0.1
OMe
16
17
0.96 0.20
0.49 0.30
0.006
0.003
0.28
8.2
<0.1
<0.1
0.052
5.0
83
2% @
18
19
1.34 0.12
2.68 0.46
0.027
0.019
0.11
0.36
<0.1
<0.1
3 l
Mc
Cl
1.0
20
21
18 2.0
N
N
N
11 1.48
1.0
2.7
O
N
11% @
Mc
22
23
2.50 0.25
0.037
0.32
0.28
410
3
l
O
N
4.14 2.29
N
O
N
24
25
29.1 3.0
N
N
0.21 0.09
0.030
0.55
1.0
0.65
N
a
b
c
Estimated IC50 values from electrophysiology data generated at multiple testing concentrations (see Section 6.7).
Data from Ref. 25.
Percent inhibition of hNaV1.2 at 3
Data from Ref. 27.
lM.
d
furan core. The physicochemical and pharmacokinetic properties of
many of these compounds limited the in vivo evaluation of their
analgesic profiles. However, consistent with its in vitro profile
and improved solubility, the oral administration of 36 produced
dose-dependent anti-nociceptive effects in a rodent model of neu-
ropathic pain.
6. Experimental section
6.1. General procedures
Nuclear magnetic resonance spectra were obtained on a 300,
400, or 500 MHz instruments with chemical shifts (d) reported rel-

7820
M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
Table 2
In vitro characterization of pyrazine Nav1.8 channel blockers 26–36
R1
O
N
R2
N
H
N
Compound
R1
R2
mNav1.8
IC50 M)
hNav1.8
Est. IC50
TTx-r
Est. IC50
hNav1.2
Est. IC50
Solubility (lg/mL)
Rat microsomal
clearance ( L/min/mL)
a
a
a
(
l
(l
M)
(lM)
(lM)
l
26
–OEt
0.056 0.1
0.002
0.012
0.36
<0.1
27
28
–OEt
–OEt
0.65 0.13
0.11 0.05
0.001
0.002
0.068
0.019
1.0
<0.1
0.14
0.47
630
29
–CN
0.47 0.07
0.73 0.28
0.008
0.084
1.6
0.03
140
120
30
31
–CN
–CN
0.049
0.15
0.13
0.45
9.1
0.08
0.35
O
9.4
8
1.0
93% @
32
–OCF3
0.058 0.05
0.021
0.034
<0.1
<0.1
3 l
Mb
33
34
–OCF3
–OCF3
0.066 0.05
0.24 0.06
0.005
0.018
0.016
0.17
2.2
1.2
Cl
35
36
–OCF3
–OCF3
1.03 0.4
0.099
0.019
0.090
0.085
3.9
1.5
143
98
N
N
O
N
0.48 0.09
>50
a
Estimated IC50 values from electrophysiology data generated at multiple testing concentrations (see Section 6.7).
Percent inhibition of hNaV1.2 at 3 lM
b
ative to tetramethylsilane as internal standard. Mass spectra deter-
minations were obtained using an electrospray (ESI) technique or
by direct chemical ionization (DCI) methods employing ammonia.
Melting points were determined with capillary apparatus and are
uncorrected. Elemental analyses were performed by Robertson
Microlit Laboratories, Inc., Madison, NJ. Analytical thin layer
chromatography was done on 2 ꢁ 6 cm Kieselgel 60 F-254 plates
pre-coated with 0.25 mm thick silica gel distributed by E. Merck.
LC–MS analyses were performed on ThermoQuest Navigator sys-
tems using 10–100% acetonitrile:10 mM ammonium acetate gradi-
ent with MS data obtained using atmospheric pressure chemical
ionization (APCI) positive ionization over the range of m/z from
170 to 1200. Unless otherwise specified, column chromatography
was performed on silica gel (230–400 mesh). The term in vacuo re-
fers to solvent removal using a rotary evaporator. Unless otherwise
specified, solvents and reagents were purchased from Aldrich
Chemical Co. and were used without further purification unless
otherwise specified.
6.2. High-throughput mouse Nav1.8 and hERG isotopic flux
assays
HEK293 cells stably expressing mouse Nav1.8 sodium channels
or CHO cells stably expressing hERG potassium channels were
loaded overnight with an appropriate radiotracer, followed by
stimulation using variations on protocols previously described.33,36

M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
7821
Table 3
becco’s media, 10% Fetal Bovine Serum, 2 mM sodium pyruvate,
G418. For whole-cell voltage-clamp recordings, patch pipettes
were pulled from borosilicate glass on a Flaming-Brown micropi-
pette puller (Sutter Instruments, Inc). Pipettes had a tip resistance
In vivo activitya and pharmacokinetic profileb of Nav1.8 blockers 36 and 29
36
29
Neuropathic pain, Chung
po (ED50, mg/kg)
F, po (%)
Clp (L/(h kg))
T1/2 (h)
78
<20% @ 100 l
Mc
of 0.8–2.5 MX using the internal solutions (mM): 135 CsF, 10 CsCl,
18.2 2.5
2.04 0.10
1.9 (iv)
0%
2.0
1.6 (iv)
5 EGTA, 5 NaCl, 10 HEPES-free acid, pH to 7.3 with 5 M CsOH and
voltage offset was zeroed prior to seal formation. The external buf-
fer consisted of (mM): 132 NaCl, 5.4 KCl, 0.8 MgCl2, 1.8 CaCl2, 5
Glucose, 10 HEPES-free acid, pH to 7.3 with 6 N NaOH. After estab-
lishment of a whole-cell recording, cellular capacitance was mini-
mized using the analog compensation available on the recording
1.6 (po)
Cmax, po (
iv Vss (L/kg)
lg/mL)
0.15 0.03
3.3 0.3
0
5.1
Plasma protein binding, rat (%)
93.2 0.4
b2 (h), BZD
peripheral, NK1 (h)
55.2 1.6
None
Cerep (@ 10 l
M)d
amplifier (Axopatch 200B). Series resistance was less than 5 M
and was compensated >85% in all experiments, resulting in a final
series resistance no greater than 0.75 M . Signals were low-pass
X
a
Values shown represent the mean for experiments in rats, n = 6 per dose group.
Pharmacokinetic parameters determined in rats (n = 3) following administra-
X
b
filtered at 5–10 kHz, digitized at 20–50 kHz, and stored on a com-
puter for later analysis. Voltage protocols were generated and data
acquisition and analysis were performed using pCLAMP software
(Version 8.1, Axon Instruments, Inc.). All experiments were per-
formed at room temperature. Liquid junction potentials were
<10 mV and were not corrected.
tion of a 3 mg/kg iv or po dose.
c
Percent inhibition at 100
Displayed >60% inhibition of control specific binding at indicated receptor.
l
M.
d
The radiotracer efflux was measured at a single time point which
had been previously established to be on the linear portion of
the efflux curve. Percentage inhibition of efflux was calculated as:
6.4. Electrophysiological recordings. Rat dorsal root ganglion
neurons37,38
½Effluxðtest compoundÞ ꢀ Effluxðref blockerÞꢂ
% inhibition ¼ 1 ꢀ
½EffluxðControlÞ ꢀ Effluxðref blockerÞꢂ
Whole-cell patch clamp recordings were performed on dissoci-
ated rat small diameter DRG neurons (18–25 lm) from the L4 and
ꢁ 100
L5 lumbar region at room temperature. Coverslips were mounted
in a small flow-through chamber on the stage of an inverted micro-
scope and were continuously perfused with bath external solution
(see below). Cells were voltage clamped via the whole-cell config-
uration of the patch clamp with an Axopatch-200B amplifier
(Molecular Devices/Axon Instruments, Foster City, CA) using stan-
dard techniques. Micropipettes were pulled from thin-walled
borosilicate glass capillaries (TW-150F, World Precision Instru-
ments, Sarasota, FL) with a Flaming-Brown micropipette puller
(P97, Sutter Instrument, Novato, CA) and polished on a microforge
Reference blockers for Nav1.8 and hERG assays were tetracaine
(30 lM) and terfenadine (30 lM), respectively. Concentration
dependent activity was established via eight-point concentra-
tion–response curves and assays were performed in duplicate.
6.3. Electrophysiology. Recombinant human sodium channels25
Human embryonic kidney (HEK-293) cells expressing recombi-
nant sodium channels were grown in DMEM/High Glucose Dul-
Figure 2. Effects of 36 on mechanical allodynia observed in the Chung model of neuropathic pain (n = 6). Compound was administered (10, 30, 100 mg/kg po) 60 min before
von Frey testing. Squares represent paw withdraw threshold ipsilateral to the injury (left). The circle represents paw withdrawal threshold contralateral to the injury. Data
*
**
represent mean SEM. , significantly different (P <0.05) from vehicle-treated animals (n = 6); , significantly different (P <0.01) from vehicle-treated animals (n = 6).

7822
M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
(Narishige, Tokyo) to obtain electrode resistances ranging from
1.0–2.5 M . The pipette solution contained (mM): 135 CsF, 5 NaCl,
channels are inactivated. V1/2 = ꢀ40 mV for hNav1.8, TTx-r; V1/2
ꢀ60 mV for hNav1.2.
=
X
10 HEPES, 10 CsCl, and 5 EGTA, pH 7.4 with CsOH (295 mOSM).
Capacity transients were cancelled and series resistance was com-
pensated (>80%) using the facilities of the amplifier. The bath solu-
tion contained (mM): 110 choline-Cl, 22 NaCl, 5.4 KCl, 1.8 CaCl2,
0.8 MgCl2, 10 HEPES, and 5 glucose, plus 500 nM tetrodotoxin
6.7.1. Methyl 4-oxy-2-pyrazinecarboxylate (2)
Methyl 2-pyrazinecarboxylate (1) (Pyrazine Specialists, 10.04 g,
72.2 mmol) was suspended in 1,2-dichloroethane (100 mL). To the
reaction mixture was added mCPBA (32.35 g, 77%, 144 mmol). The
reaction mixture was stirred at 60 °C for 16 h, then allowed to cool
to ambient temperature and diluted with CH2Cl2 (300 mL). The
precipitate was filtered off and washed with additional CH2Cl2
(3 ꢁ 35 mL). The filtrates were combined, dried over K2CO3, filtered
and concentrated. The residue was suspended in hexane (50 mL).
The title compound was isolated by filtration, washed with addi-
tional hexane (2 ꢁ 50 mL) to afford a slightly yellow solid (7.22 g,
64%): 1H NMR (DMSO-d6, 300 MHz) d 3.91 (s, 3H), 8.54 (dd,
J = 4.07, 1.69 Hz, 1H), 8.64–8.67 (m, 2H) ppm. 13C NMR (DMSO-
d6, 100 MHz) d 53.0, 135.2, 136.2, 147.1, 148.8, 162.68 ppm; MS
(DCI/NH3) m/z 155 (M+H)+; Anal. (C6H6N2O3) C, H, N.
(TTx) and 50 lM CdCl2, pH 7.4 with NaOH (300 mOSM). The pipette
potential was zeroed before seal formation. Calculated liquid junc-
tion potentials were 65 mV and were not corrected. Whole-cell cur-
rents were filtered at 5 kHz and acquired at 20 kHz using Clampex
8.2 software (Molecular Devices/Axon Instruments) and analyzed
using Clampfit 8.2 (Molecular Devices/Axon Instruments). All
experiments were performed at room temperature (21–25 °C).
Compounds were dissolved in DMSO (10 mM) and added to extra-
cellular solution (final DMSO concentration <0.1%) immediately
prior to use. Compounds were applied directly to the voltage-
clamped cells at a flow rate of 1–2 mL/min via a custom-made
perfusion manifold connected to an array of gravity-fed reservoirs.
Sodium currents were evoked from
a
holding potential of
6.7.2. Methyl 6-chloro-2-pyrazinecarboxylate (3)
ꢀ100 mV by a test pulse of 0 mV for 20 ms and pulsed every 15 s
until stabilized. To measure drug effects, sodium currents were
evoked with 20 ms steps to 0 mV following 8 s prepulses to either
ꢀ100 mV or ꢀ40 mV. The prepulse was followed by a brief
(20 ms) repolarization to ꢀ100 mV to relieve fast inactivation.
Methyl 4-oxy-2-pyrazinecarboxylate (2) (7.18 g, 45.9 mmol)
was dissolved in SOCl2 (50 mL, 687 mmol). The reaction mixture
was heated to reflux for 8 h and then allowed to cool to ambient
temperature. The SOCl2 was removed under reduced pressure,
and the residue was quenched with water (50 mL) at 0 °C. The mix-
ture was neutralized by the addition of 1 M K2CO3 (aq) and ex-
tracted with CH2Cl2 (5 ꢁ 100 mL). The organic extracts were
combined and washed with brine (100 mL), dried over Na2SO4,
filtered and concentrated. The residue was purified by silica gel
chromatography (5% EtOAc in CH2Cl2, Rf = 0.35) to afford the title
compound as a thick oil that slowly solidified (7.16 g, 67%): 1H
NMR (DMSO-d6, 500 MHz) d 3.95 (s, 3H), 9.07 (s, 1H), 9.18 (s,
1H) ppm; 13C NMR (DMSO-d6, 125 MHz) d 52.9, 142.1, 143.6,
144.9, 148.1, 162.8 ppm; MS (DCI/NH3) m/z 190 (M+NH4)+; Anal.
(C6H5ClN2O2) C, H, N.
6.5. In vivo evaluation
Male Sprague–Dawley rats (Charles River, Wilmington, MA)
weighing 200–300 g were utilized. All animals were group housed
in AAALAC approved facilities at Abbott Laboratories in a temper-
ature-regulated environment with lights on between 0700 and
2000 h. Food and water were available ad libitum except during
testing. All animal handling and experimental protocols were ap-
proved by an institutional animal care and use committee (IACUC).
All experiments were performed during the light cycle. Unless
otherwise noted, all experimental and control groups contained
6.7.3. Representative procedure for Suzuki coupling (Scheme 2,
step a). Methyl 6-(4-chlorophenyl)-2-pyrazinecarboxylate (4)
Methyl 6-chloro-2-pyrazinecarboxylate (3) (1.01 g, 5.85 mmol)
was dissolved in DMF (23 mL). 4-Chlorophenylboronic acid
(1.34 g, 8.57 mmol), PdCl2(PPh3)2 (204.5 mg, 0.29 mmol) and ce-
sium carbonate (4.44 g, 13.63 mmol) were added, and the reaction
mixture was stirred at 65 °C for 16 h. The reaction mixture was
then diluted with water (200 mL) and extracted with EtOAc
(3 ꢁ 100 mL). The organic extracts were combined, washed with
water (2 ꢁ 100 mL) and brine (200 mL), dried over Na2SO4, filtered
and concentrated. The residue was purified by silica gel chroma-
tography (5% EtOAc in CH2Cl2, Rf = 0.39) to afford the title com-
pound as a white solid (789.7 mg, 54%): 1H NMR (DMSO-d6,
300 MHz) d 3.97 (s, 3H), 7.65–7.68 (m, 2H), 8.23–8.25 (m, 2H),
9.17 (s, 1H), 9.52 (s, 1H) ppm. MS (DCI/NH3) m/z 249 (M+H)+.
at least six animals per group and data are expressed as mean
-
SEM. Data analysis was conducted using analysis of variance and
appropriate post-hoc comparisons (P <0.05) as previously de-
scribed.39 ED50 values were estimated using least squares linear
regression.
6.6. Spinal nerve (L5/L6) ligation model of neuropathic pain
As previously described in detail by Kim and Chung,40 a 1.5 cm
incision was made dorsal to the lumbosacral plexus in anesthe-
tized rats. The paraspinal muscles (left side) were separated from
the spinous processes, the L5 and L6 spinal nerves were isolated
and tightly ligated with 3–0 silk threads. Following hemostasis,
the wound was sutured and coated with antibiotic ointment. The
rats were allowed to recover and then placed in a cage with soft
bedding for 14 days before behavioral testing for mechanical allo-
dynia. The vehicle for compound administration and vehicle con-
trol animals was 10% DMSO in PEG400. Paw withdraw threshold
was determined with von Frey hairs and reported as mean SEM.
6.7.3.1. Methyl 6-(4-cyanophenyl)-2-pyrazinecarboxylate (5).
Yield 51%; 1H NMR (DMSO-d6, 300 MHz) d 3.98 (s, 3H), 8.05–
8.09 (m, 2H), 8.37–8.41 (m, 2H), 9.24 (s, 1H), 9.61 (s, 1H) ppm;
MS (DCI/NH3) m/z 240 (M+H)+.
6.7. Estimated IC50 values
6.7.3.2. Methyl 6-(4-trifluoromethoxyphenyl)-2-pyrazinecarb-
oxylate (6). Yield 61%; 1H NMR (DMSO-d6, 300 MHz) d 3.97 (s,
3H), 7.57–7.60 (m, 2H), 8.31–8.36 (m, 2H), 9.19 (s, 1H), 9.53 (s,
1H) ppm; MS (DCI/NH3) m/z 299 (M+H)+.
Estimated IC50 values were calculated from single-point electro-
physiology data using the following equation: [(100% ꢀ % inhibi-
tion)/% inhibition] ꢁ (test concentration in
lM). Inhibition values
<20% and >80% are excluded from the calculation. For compounds
with multiple single-point data, the average of the various esti-
mated values was reported. Data were collected using an inacti-
vated state protocol (the prepulse voltage at which 50% of
6.7.4. Representative procedure for ester saponification (Scheme
2, step b). 6-(4-Chlorophenyl)-pyrazine-2-carboxylic acid (7)
Methyl 6-(4-chlorophenyl)-2-pyrazinecarboxylate (4) (764.5 mg,
3.07 mmol) was suspended in EtOH (15 mL). 1 M NaOH (15 mL)

M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
7823
was added, and the reaction mixture stirred at ambient tempera-
ture for 1 h. The reaction mixture was acidified to pH ꢃ 3 with
1 M HCl (aq). The mixture was then diluted with water (100 mL)
and extracted with EtOAc (5 ꢁ 50 mL). The organic extracts were
combined, washed with brine (100 mL), dried over Na2SO4, filtered
and concentrated to provide the title compound as a white solid
(715.1 mg, 99%): 1H NMR (DMSO-d6, 300 MHz) d 7.63–7.67 (m,
2H), 8.24–8.29 (m, 2H), 9.15 (s, 1H), 9.49 (s, 1H), 13.82 (br s, 1H)
ppm; MS (DCI/NH3) m/z 235 (M+H)+.
0.1 M HCl (100 mL) and extracted with CH2Cl2 (3 ꢁ 50 mL). The or-
ganic extracts were combined, washed with 0.1 M HCl (50 mL) and
brine (50 mL), dried over Na2SO4, filtered and concentrated. The
residue was purified by silica gel chromatography (10% EtOAc in
CH2Cl2, Rf = 0.26) to afford the title compound as a white solid
(391.0 mg, 67%): mp 161–162 °C; 1H NMR (DMSO-d6, 300 MHz) d
1.30 (d, J = 5.76 Hz, 6H), 4.57–4.65 (m, 1H), 6.75 (dd, J = 7.80,
2.03 Hz, 1H), 7.29 (t, J = 8.14 Hz, 1H), 7.43–7.46 (m, 1H), 7.53 (t,
J = 2.20 Hz, 1H), 8.07–8.11 (m, 2H), 8.66–8.69 (m, 2H), 9.29 (s,
1H), 9.63 (s, 1H), 10.59 (s, 1H) ppm; MS (DCI/NH3) m/z 359
(M+H)+; Anal. (C21H18N4O2) C, H, N.
6.7.4.1. 6-(4-Cyanophenyl)-pyrazine-2-carboxylic acid (8). Yield
98%; 1H NMR (DMSO-d6, 300 MHz) d 8.06 (d, J = 8.48 Hz, 2H), 8.43
(d, J = 8.48 Hz, 2H), 9.21 (s, 1H), 9.58 (s, 1H), 13.89 (br s, 1H) ppm;
MS (DCI/NH3) m/z 226 (M+H)+.
6.7.7.1. N-(3,5-Dimethoxyphenyl)-6-(4-chlorophenyl)-pyrazine-
2-carboxamide acid (15). Yield 75%; mp 156–157 °C; 1H NMR
(DMSO-d6, 300 MHz) d 3.77 (s, 6H), 6.35 (t, 1H, J = 2.1 Hz), 7.20
(d, 2H, J = 2.0 Hz), 7.67 (d, 2H, J = 8.8 Hz), 8.49 (d, 2H, J = 8.8 Hz),
9.23 (s, 1H), 9.54 (s, 1H), 10.53 (s, 1H). MS (ESI) m/z 370 (M+H)+;
Anal. (C19H16ClN3O3ꢄ0.3 H2O) C, H, N.
6.7.4.2.
6-(4-Trifluoromethoxyphenyl)-pyrazine-2-carboxylic
acid (9). Yield 99%; 1H NMR (DMSO-d6, 300 MHz) d 7.57–7.59
(m, 2H), 8.34–8.37 (m, 2H), 9.16 (s, 1H), 9.50 (s, 1H), 13.82 (br s,
1H) ppm; MS (DCI/NH3) m/z 285 (M+H)+.
6.7.7.2. N-(m-Tolyl)-6-(4-chlorophenyl)-pyrazine-2-carboxam-
ide (16). Purified by preparative HPLC on a Waters Nova-PakÒ
HR C18 Prep-PakÒ cartridge column (40 mm ꢁ 100 mm) using a
gradient of 10–100% acetonitrile in 0.1% aqueous TFA over
12 min at a flow rate of 70 mL/min to provide the title compound:
mp 136–137 °C; 1H NMR (DMSO-d6, 300 MHz) d 3.35 (s, 3H), 7.01
(d, 1H, J = 7.5 Hz), 7.30 (t, 1H, J = 8.50 Hz), 7.67 (d, 1H, J = 8.8 Hz),
7.66–7.77 (m, 3H), 8.51 (d, 2H, J = 8.5 Hz), 9.23 (s, 1H), 9.54 (s,
1H), 10.55 (s, 1H). MS (ESI) m/z 324 (M+H)+; Anal. (C18H14ClN3Oꢄ0.4
TFA) C, H, N.
6.7.5. 6-(4-Ethoxyphenyl)-pyrazine-2-carboxylic acid (10)
Methyl 6-chloro-2-pyrazinecarboxylate (3) (440 mg, 2.55 mmol)
was dissolved in 75% aqueous isopropanol solution (50 mL). 4-Eth-
oxyphenylboronic acid (487 mg, 2.93 mmol), PdCl2(PPh3)2 (72 mg,
0.10 mmol) and sodium carbonate (676 mg, 6.37 mmol) were
added, and the reaction mixture was stirred at refluxing tempera-
ture for 4 h. The reaction mixture was then diluted with ethyl ace-
tate (100 mL) and was partitioned between ethyl acetate (100 mL)
and water (250 mL). The organic layer was washed with 2 M NaOH
(100 mL). The aqueous layers were combined and acidified to pH
2–3. The acidified aqueous layer was extracted with ethyl acetate
(2 ꢁ 150 mL). The organic extracts were combined, dried over
Na2SO4, filtered and concentrated in vacuo to afford the desired
product as an off-white solid (600 mg, 96%): 1H NMR (MeOH-d4,
400 MHz) d 1.42 (t, J = 7.1 Hz, 3H), 4.13 (q, J = 7.0 Hz, 2H), 7.00–
7.15 (m, 2H), 8.11–8.17 (m, 2H), 9.07 (s, 1H), 9.22 (s, 1H) ppm;
MS (APCI) m/z 245 (M+H)+.
6.7.7.3. N-(3,5-Dimethylphenyl)-6-(4-chlorophenyl)-pyrazine-2-
carboxamide (17). Purified by preparative HPLC on a Waters
Nova-PakÒ HR C18 Prep-PakÒ cartridge column (40 mm ꢁ
100 mm) using a gradient of 10–100% acetonitrile in 0.1% aqueous
TFA over 12 min at a flow rate of 70 mL/min to provide the title
compound: yield 78%; mp 164 °C; 1H NMR (DMSO-d6, 300 MHz)
d 2.31 (s, 6H), 6.85–6.82 (m, 1H), 7.52 (s, 2H), 7.67 (d, 2H,
J = 8.8 Hz), 8.52 (d, 2H, J = 8.8 Hz), 9.22 (s, 1H), 9.54 (s, 1H), 10.47
(s, 1H). MS (ESI) m/z 338 (M+H)+; Anal. (C19H16ClN3Oꢄ0.1 TFA) C,
H, N.
6.7.6. Representative procedure for acid chloride formation
(Scheme 2, step c). 6-(4-Cyanophenyl)-pyrazine-2-carbonyl
chloride (12)
6-(4-Cyanophenyl)-pyrazine-2-carboxylic acid (8) (1.36 g,
6.04 mmol.) was suspended in dichloromethane (60 mL). Oxalyl
chloride (0.95 mL, 10.89 mmol) and DMF (30 lL, 0.31 mmol) were
added, and the reaction mixture was stirred at ambient tempera-
ture for 1 h. The mixture was concentrated and placed under high
vacuum to provide the crude product (1.45 g, 99%), which was used
without additional purification.
6.7.7.4. N-(2-Methylbenzyl)-6-(4-chlorophenyl)-pyrazine-2-car-
boxamide (18). Yield 86%; mp 178 °C; 1H NMR (DMSO-d6,
300 MHz) d 4.56 (d, 2H, J = 6.1 Hz), 7.12–7.21 (m, 3H), 7.23–7.29
(m, 1H), 7.63 (d, 2H, J = 8.6 Hz), 8.46 (d, 2H, J = 8.5 Hz), 9.51 (s,
1H), 9.54 (t, 1H, J = 6.4 Hz). MS (ESI) m/z 338 (M+H)+; Anal.
(C19H16ClN3O) C, H, N.
6.7.7.5. N-(4-Chlorophenethyl)-6-(4-chlorophenyl)pyrazine-2-
carboxamide (19). Yield 89%; mp 157 °C; 1H NMR (DMSO-d6,
300 MHz) d ppm 2.91 (t, J = 7.5 Hz, 2H), 3.54–3.63 (m, 2H), 7.28–
7.39 (m, 4H), 7.65 (d, J = 8.5 Hz, 2H), 8.42 (d, J = 8.5 Hz, 2H),
9.08–9.15 (m, 2H), 9.49 (s, 1H); MS (DCI/NH3) m/z 372 (M+H)+;
Anal. (C19H15Cl2N3O) C, H, N.
6.7.6.1. 6-(4-Chlorophenyl)-pyrazine-2-carbonyl chloride (11).
Yield 95%.
6.7.6.2.
6-(4-Trifluoromethoxyphenyl)-pyrazine-2-carbonyl
chloride (13). Yield 99%.
6.7.6.3. 6-(4-Ethoxyphenyl)-pyrazine-2-carbonyl chloride (14).
6.7.7.6. N-(Pyridin-3-yl)-6-(4-chlorophenyl)-pyrazine-2-carbox-
amide (20). Yield 49%; mp 213–215 °C; 1H NMR (DMSO-d6,
300 MHz) d ppm 7.47 (dd, J = 8.1, 4.7 Hz, 1H), 7.61–7.78 (m, 2H),
8.28 (ddd, J = 8.3, 2.4, 1.5 Hz, 1H), 8.40 (dd, J = 4.7, 1.4 Hz, 1H),
8.48–8.57 (m, 2H), 9.04 (d, J = 2.4 Hz, 1H), 9.25 (s, 1H), 9.57 (s,
1H), 10.85 (s, 1H); MS (DCI/NH3) m/z 311 (M+H)+; Anal.
(C16H11ClN4Oꢄ0.75 H2O) C, H, N.
Yield 95%.
6.7.7. Representative procedure for amide formation from acid
chlorides (Scheme 2, step d). N-(3-Isopropoxyphenyl)-6-(4-
cyanophenyl)-pyrazine-2-carboxamide (29)
6-(4-Cyanophenyl)-pyrazine-2-carbonyl chloride (12) (394.4 mg,
1.62 mmol) was dissolved in dichloromethane (16 mL). Triethyl-
amine (0.33 mL, 2.37 mmol) and 3-isopropoxyaniline (0.36 mL,
2.44 mmol) were added, and the reaction mixture was stirred at
ambient temperature for 3 h. The mixture was then poured into
6.7.7.7. N -(Pyridin-3-ylmethyl)-6-(4-chlorophenyl)-pyrazine-2-
carboxamide (21). Yield 56%; mp 167–168 °C; 1H NMR (DMSO-
d6, 300 MHz) d ppm 4.60 (d, J = 6.4 Hz, 2H), 7.36 (ddd, J = 7.8, 4.7,

7824
M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
0.7 Hz, 1H), 7.59–7.71 (m, 2H), 7.77 (ddd, J = 7.8, 2.4, 1.7 Hz, 1H),
8.42–8.49 (m, 3H), 8.60 (d, J = 1.7 Hz, 1H), 9.15 (s, 1H), 9.51 (s,
1H), 9.71 (t, J = 6.3 Hz, 1H); MS (DCI/NH3) m/z 325 (M+H)+; Anal.
(C17H13ClN4Oꢄ0.7 H2O) C, H, N.
8.07–8.10 (m, 2H), 8.67–8.70 (m, 2H), 9.28 (s, 1H), 9.62 (s, 1H),
10.51 (s, 1H) ppm. MS (DCI/NH3) m/z 329 (M+H)+; Anal.
(C20H16N4O) C, H, N.
6.7.7.16. N-(3,5-Dimethylphenyl)-6-(4-trifluoromethoxyphenyl)-
pyrazine-2-carboxamide (32). Yield 65%; mp 135–136 °C; 1H
NMR (DMSO-d6, 300 MHz) d 2.30 (s, 6H), 6.83 (s, 1H), 7.52 (s, 2H),
7.58–7.60 (m, 2H), 8.57–8.62 (m, 2H), 9.24 (s, 1H), 9.55 (s, 1H),
10.47 (s, 1H) ppm. MS (DCI/NH3) m/z 388 (M+H)+; Anal.
(C20H16F3N3O2) C, H, N.
6.7.7.8. N-((2-Morpholin-4-yl-pyridin-3-yl)methyl)-6-(4-chloro-
phenyl)-pyrazine-2-carboxamide (22). Yield 40%; mp 140 °C; 1H
NMR (DMSO-d6, 300 MHz) d ppm 3.06–3.12 (m, 4H), 3.75–3.81 (m,
4H), 4.62 (d, J = 6.1 Hz, 2H), 7.03 (dd, J = 7.5, 4.7 Hz, 1H), 7.58–7.66
(m, 3H), 8.20 (dd, J = 4.9, 1.9 Hz, 1H), 8.47 (d, J = 8.8 Hz, 2H), 9.17 (s,
1H), 9.53 (s, 1H), 9.67 (t, J = 6.1 Hz, 1H). MS (ESI) m/z 410 (M+H)+;
Anal. (C21H20ClN5O2ꢄ0.2 H2O) C, H, N.
6.7.7.17. N-(m-Tolyl)-6-(4-trifluoromethoxyphenyl)-pyrazine-2-
carboxamide (33). Purified by preparative HPLC; yield 79%; mp
124–125 °C; 1H NMR (DMSO-d6, 300 MHz) d 2.35 (s, 3H), 7.00–
7.02 (m, 1H), 7.27–7.33 (m, 1H), 7.58–7.61 (m, 2H), 7.69–7.72
(m, 2H), 8.57–8.62 (m, 2H), 9.25 (s, 1H), 9.55 (s, 1H), 10.56 (s,
1H) ppm. MS (DCI/NH3) m/z 374 (M+H)+; Anal. (C19H14F3N3O2ꢄ0.08
TFA) C, H, N.
6.7.7.9. N-((3-Morpholinopyridin-4-yl)methyl)-6-(4-chlorophe-
nyl)-pyrazine-2-carboxamide (23). Yield 60%; mp 93–95 °C; 1H
NMR (DMSO-d6, 300 MHz) d ppm 2.98–3.07 (m, 4H), 3.72–3.87
(m, 4H), 4.68 (d, J = 6.4 Hz, 2H), 7.26 (d, J = 5.1 Hz, 1H), 7.55–7.74
(m, 2H), 8.26 (d, J = 5.1 Hz, 1H), 8.39 (s, 1H), 8.44–8.53 (m, 2H),
9.16 (s, 1H), 9.54 (s, 1H), 9.70 (t, J = 6.3 Hz, 1H); MS (DCI/NH3) m/
z 410 (M+H)+; Anal. (C21H20ClN5O2ꢄ0.25 H2O) C, H, N.
6.7.7.18. N-((2-Chloropyridin-3-yl)methyl)-6-(4-(trifluorometh-
oxy)phenyl)pyrazine-2-carboxamide (35). Yield 27%; mp 137–
138 °C; 1H NMR (DMSO-d6, 300 MHz) d 4.64 (d, J = 6.10 Hz, 2H),
7.42 (dd, J = 7.80, 4.75, Hz, 1H), 7.58, (d, J = 7.80 Hz, 2H), 7.80 (dd,
J = 7.63, 1.86, Hz, 1H), 8.33 (dd, J = 4.75, 1.70, Hz, 1H), 8.53–8.58
(m, 2H), 9.17 (s, 1H), 9.55 (s, 1H), 9.72 (t, J = 6.10 Hz, 1H) ppm.
MS (DCI/NH3) m/z 409 (M+H)+; Anal. (C18H12ClF3N4O2ꢄ0.18 H2O)
C, H, N.
6.7.7.10. N-((4-Morpholinopyridin-3-yl)methyl)-6-(4-chlorophe-
nyl)-pyrazine-2-carboxamide (24). Yield 45%; mp 165–167 °C;
1H NMR (DMSO-d6, 300 MHz) d ppm 3.36–3.42 (m, 4H), 3.64–
3.70 (m, 4H), 4.44 (d, J = 6.1 Hz, 2H), 6.81 (d, J = 8.8 Hz, 1H), 7.59
(dd, J = 8.8, 2.4 Hz, 1H), 7.61–7.67 (m, 2H), 8.16 (d, J = 2.0 Hz, 1H),
8.39–8.48 (m, 2H), 9.13 (s, 1H), 9.49 (s, 1H), 9.55 (t, J = 6.3 Hz,
1H); MS (DCI/NH3) m/z 410 (M+H)+; Anal. (C21H20ClN5O2ꢄ0.1 H2O)
C, H, N.
6.7.7.19. N-((2-Morpholin-4-yl-pyridin-3-yl)methyl)-6-(4-cya-
nophenyl)-pyrazine-2-carboxamide (36). Yield 94%; mp 98–
99 °C; 1H NMR (DMSO-d6, 400 MHz) d 3.07–3.09 (m, 4H), 3.75–
3.77 (m, 4H), 4.62 (d, J = 5.83 Hz, 2H), 7.01 (dd, J = 7.52, 4.60 Hz,
1H), 7.50–7.52 (m, 2H), 7.61 (d, J = 7.36 Hz, 1H), 8.81 (d,
J = 4.60 Hz, 1H), 8.51–8.53 (m, 2H), 9.17 (s, 1H), 9.50 (s, 1H), 9.64
(t, J = 6.14 Hz, 1H) ppm. MS (DCI/NH3) m/z 460 (M+H)+; Anal.
(C22H20F3N5O3) C, H, N.
6.7.7.11. N -((2-Pyrrolidin-1-yl-pyridin-3-yl)methyl)-6-(4-chlo-
rophenyl)-pyrazine-2-carboxamide (25). mp 160–161 °C; 1H
NMR (DMSO-d6, 300 MHz) d ppm 1.86–1.93 (m, 4H), 3.46–3.52
(m, 4H), 4.60 (d, J = 6.1 Hz, 2H), 6.72 (dd, J = 7.5, 4.7 Hz, 1H), 7.45
(dd, J = 7.5, 1.7 Hz, 1H), 7.63 (d, J = 8.8 Hz, 2H), 8.01 (dd, J = 4.7,
2.0 Hz, 1H), 8.46 (d, J = 8.8 Hz, 2H), 9.15 (s, 1H), 9.52 (s, 1H), 9.55
(t, J = 6.4 Hz, 1H). MS (ESI) m/z 394 (M+H)+; Anal. (C21H20ClN5Oꢄ0.4
H2O) C, H, N.
6.7.8. Representative procedure for amide formation from
methyl esters (Scheme 4, step a). N-(2-Methylbenzyl)-6-(4-
cyanophenyl)-pyrazine-2-carboxamide (31)
Methyl 6-(4-cyanophenyl)-2-pyrazinecarboxylate (5) (71.6 mg,
0.30 mmol) and MgCl2 (62.4 mg, 0.66 mmol) were suspended in
THF (3 mL) and stirred at ambient temperature for 5 min. 2-Meth-
6.7.7.12. N-(3,5-Dimethylphenyl)-6-(4-ethoxyphenyl)-pyrazine-
2-carboxamide (26). Yield 44%; mp 162–163 °C; 1H NMR (DMSO-
d6, 300 MHz) d ppm 1.38 (t, J = 7.0 Hz, 3H),, 2.30 (s, 6H), 4.15 (q,
J = 7.1 Hz, 2H), 6.83 (s, 1H), 7.12 (d, J = 8.8 Hz, 2H), 7.53 (s, 2H),
8.42 (d, J = 8.8 Hz, 2H), 9.12 (s, 1H), 9.45 (s, 1H), 10.42 (s, 1H). MS
(DCI/NH3) m/z 348 (M+H)+; Anal. (C21H21N3O2ꢄ0.8 H2O) C, H, N.
ylbenzylamine (100 lL, 0.81 mmol) was added and the reaction
mixture was stirred at 40 °C for 4 h. The solid material was re-
moved by filtration and washed with CH2Cl2 (3 ꢁ 2 mL). The com-
bined filtrates were concentrated and purified by preparative
HPLC; yield 64%; mp 161 °C; 1H NMR (DMSO-d6, 300 MHz) d 2.36
(s, 3H), 4.57 (d, J = 6.44 Hz, 2H), 7.13–7.28 (m, 4H), 8.05–8.08 (m,
2H), 8.63–8.65 (m, 2H), 9.22 (s, 1H), 9.57–9.61 (m, 2H) ppm; MS
(DCI/NH3) m/z 329 (M+H)+; Anal. (C20H16N4Oꢄ0.04 TFA) C, H, N.
6.7.7.13. N-(m-Tolyl)-6-(4-ethoxyphenyl)-pyrazine-2-carboxam-
ide (27). Yield 46%; mp 152 °C; 1H NMR (DMSO-d6, 300 MHz) d
ppm 1.38 (t, J = 7.0 Hz,3H), 2.35 (s, 3H), 4.15 (q, J = 6.9 Hz, 2H),
7.00 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 7.25–7.33 (m, 1H),
7.67–7.74 (m, 2H), 8.42 (d, J = 8.8 Hz, 2H), 9.12 (s, 1H), 9.46 (s,
1H), 10.50 (s, 1H). MS (DCI/NH3) m/z 334 (M+H)+; Anal.
(C20H19N3O2ꢄ0.4 H2O) C, H, N.
6.7.8.1. N-(2-Methylbenzyl)-6-(4-trifluoromethoxyphenyl)-pyr-
azine-2-carboxamide (32). Purified by preparative HPLC; yield
79%; mp 130 °C; 1H NMR (DMSO-d6, 300 MHz) d 2.36 (s, 3H),
4.56 (d, J = 6.44 Hz, 2H), 7.12–7.28 (m, 4H), 7.55–7.58 (m, 2H),
8.52–8.57 (m, 2H), 9.17 (s, 1H), 9.52–9.56 (m, 2H) ppm; MS (DCI/
NH3) m/z 388 (M+H)+; Anal. (C20H16F3N3O2) C, H, N, F.
6.7.7.14.
N-(2-Methylbenzyl)-6-(4-ethoxyphenyl)-pyrazine-2-
carboxamide (28). Purified by preparative HPLC; yield 51%; mp
128 °C; 1H NMR (DMSO-d6, 300 MHz) d ppm 1.37 (t, J = 7.0 Hz,
3H), 2.36 (s, 3H), 4.13 (q, J = 6.9 Hz, 2H), 4.56 (d, J = 6.4 Hz, 2H),
7.09 (d, J = 9.2 Hz, 2H), 7.12–7.21 (m, 3H), 7.23–7.29 (m, 1H),
8.36 (d, J = 8.8 Hz, 2H), 9.05 (s, 1H), 9.42 (d, J = 0.7 Hz, 1H), 9.46
(t, J = 6.3 Hz, 1H); MS (ESI) m/z 348 (M+H)+; Anal. (C21H21N3O2ꢄ0.16
TFA) C, H, N.
References and notes
1. Anger, T.; Madge, D. J.; Mulla, M.; Riddall, D. J. Med. Chem. 2001, 44, 115.
2. Goldin, A. L.; Barchi, R. L.; Caldwell, J. H.; Hofmann, F.; Howe, J. R.; Hunter, J. C.;
Kallen, R. G.; Mandel, G.; Meisler, M. H.; Netter, Y. B.; Noda, M.; Tamkun, M. M.;
Waxman, S. G.; Wood, J. N.; Catterall, W. A. Neuron 2000, 28, 365.
3. Catterall, W. A.; Goldin, A. L.; Waxman, S. G. Pharmacol. Rev. 2005, 57, 397.
4. Catterall, W. A. Neuron 2000, 26, 13.
6.7.7.15. N-(3,5-Dimethylphenyl)-6-(4-cyanophenyl)-pyrazine-
2-carboxamide (29). Yield 86%; mp 167–168 °C; 1H NMR
(DMSO-d6, 300 MHz) d 2.31 (s, 6H), 6.84 (s, 1H), 7.52 (s, 2H),

M. J. C. Scanio et al. / Bioorg. Med. Chem. 18 (2010) 7816–7825
7825
5. Lai, J.; Porreca, F.; Hunter, J. C.; Gold, M. S. Annu. Rev. Pharmacol. Toxicol. 2004,
44, 371.
6. Wood, J. N.; Boorman, J. Curr. Top. Med. Chem. 2005, 5, 529.
7. Lai, J.; Hunter, J. C.; Porreca, F. Curr. Opin. Neurobiol. 2003, 13, 291.
8. Waxman, S. G.; Cummins, T. R.; Dib-Hajj, S.; Fjell, J.; Black, J. A. Muscle Nerve
1999, 22, 1177.
Mikusa, J. P.; Hernandez, G.; Gauvin, D.; Wade, C.; Zhu, C.; Pai, M.; Scanio, M. J.
C.; Shi, L.; Drizin, I.; Gregg, R. J.; Matulenko, M. A.; Ahmed Hakeem, A. H.; Gross,
M. F.; Johnson, M. S.; Marsh, K. C.; Wagoner, K.; Sullivan, J. P.; Faltynek, C. R.;
Krafte, D. S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8520.
26. McGaraughty, S.; Chu, K. L.; Scanio, M. J. C.; Kort, M. E.; Faltynet, C. R.; Jarvis, M.
F. J. Pharm. Exp. Ther. 2008, 324, 1204.
9. Kinloch, R. A.; Cox, P. J. Expert Opin. Ther. Targets 2005, 9, 685.
10. Renganathan, M.; Cummins, T. R.; Waxman, S. G. J. Neurophysiol. 2001, 86, 629.
11. Lai, J.; Gold, M. S.; Kim, C.-S.; Bian, D.; Ossipov, M. H.; Hunter, J. C.; Porreca, F.
Pain 2002, 95, 143.
12. Amir, R.; Argoff, C. E.; Bennett, G. J.; Cummins, T. R.; Durieux, M. E.; Gerner, P.;
Gold, M. S.; Porreca, F.; Strichartz, G. R. J. Pain 2006, 7, S1.
27. Kort, M. E.; Atkinson, R. N.; Thomas, J. B.; Drizin, I.; Johnson, M. S.; Secrest, M.
A.; Gregg, R. J.; Scanio, M. J. C.; Shi, L.; Hakeem, A. H.; Matulenko, M. A.;
Chapman, M. L.; Krambis, M. J.; Liu, D.; Shieh, C.-C.; Zhang, X.; Hernandez, G.;
Mikusa, J. P.; Zhong, C.; Joshi, S.; Honore, P.; Roeloffs, R.; Werness, S.; Antonio,
B.; Marsh, K. C.; Faltynek, C. R.; Krafte, D. S.; Jarvis, M. F.; Marron, B. E. Bioorg.
Med. Chem. Lett. 2010. doi:10.1016/j.bmcl.2010.08.121.
13. Coggeshall, R. E.; Tate, S.; Carlton, S. M. Neurosci. Lett. 2004, 355, 45.
14. Laird, J. M. A.; Souslova, V.; Wood, J. N.; Cervero, F. J. Neurosci. 2002, 22, 8352.
15. Nassar, M. A.; Levato, A.; Stirling, L. C.; Wood, J. N. Mol. Pain 2005, 1, 24.
16. Akopian, A. N.; Souslova, V.; England, S.; Okuse, K.; Ogata, N.; Ure, J.; Smith, A.;
Kerr, B. J.; McMahon, S. B.; Boyce, S.; Hill, R.; Stanfa, L. C.; Dickenson, A. H.;
Wood, J. N. Nat. Neurosci. 1999, 2, 541.
28. Uchimaru, F.; Okada, S.; Kosasayama, A.; Konno, T. Chem. Pharm. Bull. 1971, 19,
1337.
29. Okada, S.; Kosasayama, A.; Konno, T.; Uchimaru, F. Chem. Pharm. Bull. 1971, 19,
1344.
30. Imai, K.-I.; Mano, M.; Seo, T.; Matsuno, T. Chem. Pharm. Bull. 1981, 29, 88.
31. Sato, N.; Fuji, M. J. Heterocycl. Chem. 1994, 31, 1177.
17. Gold, M. S.; Weinreich, D.; Kim, C.-S.; Wang, R.; Treanor, J.; Porreca, F.; Lai, J. J.
Neurosci. 2003, 23, 158.
32. Guo, Z.; Dowdy, E. D.; Li, W.-S.; Polniaszek, R.; Delaney, E. Tetrahedron Lett.
2001, 42, 1843.
18. Joshi, S. K.; Mikusa, J. P.; Hernandez, G.; Baker, S.; Shieh, C. C.; Neelands, T.;
Zhang, X.; Niforatos, W.; Kage, K.; Han, P.; Krafte, D.; Faltynek, C.; Sullivan, J. P.;
Jarvis, M. F.; Honore, P. Pain 2006, 123, 75.
19. Porreca, F.; Lai, J.; Bian, D.; Wegert, S.; Ossipov, M. H.; Eglen, R. M.; Kassotakis,
L.; Novakovic, S.; Rabert, D. K.; Sangameswaran, L.; Hunter, J. C. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 7640.
20. Kalso, E. Curr. Pharm. Des. 2005, 11, 3005.
21. Priestley, T. Curr. Drug Targets: CNS Neurol. Disord. 2004, 3, 441.
22. Weiser, T. Drugs Future 2006, 31, 597.
23. Matelenko, M. A.; Scanio, M. J. C.; Kort, M. E. Curr. Top. Med. Chem. 2009, 9, 362.
24. Kort, M. E.; Drizin, I.; Gregg, R. J.; Scanio, M. J. C.; Shi, L.; Gross, M. F.; Atkinson,
R. N.; Johnson, M. S.; Pacofsky, G. J.; Thomas, J. B.; Carroll, W. A.; Krambis, M. J.;
Liu, D.; Shieh, C.-C.; Zhang, X.; Hernandez, G.; Mikusa, J. P.; Zhong, C.; Joshi, S.;
Honore, P.; Roeloffs, R.; Marsh, K. C.; Murray, B. P.; Liu, J.; Werness, S.; Faltynek,
C. R.; Krafte, D. S.; Jarvis, M. F.; Chapman, M. L.; Marron, B. E. J. Med. Chem.
2008, 51, 407.
25. Jarvis, M. F.; Honore, P.; Shieh, C. C.; Chapman, M.; Joshi, S. K.; Zhang, X. F.; Kort,
M. E.; Carroll, W. A.; Marron, B. E.; Atkinson, R. N.; Thomas, J. B.; Liu, D.;
Krambis, M. J.; Liu, Y.; McGaraughty, S.; Chu, K.; Roeloffs, C. R.; Zhong, C.;
33. Daniel, S.; Malkowitz, L.; Wang, H.-C.; Beer, B.; Blume, A. J.; Ziai, M. R. J.
Pharmacol. Meth. 1991, 25, 185.
34. Tang, W.; Kang, J.; Wu, X.; Rampe, D.; Wang, L.; Shen, H.; Li, Z.; Dunnington, D.;
Garyantes, T. J. Biomol. Screen. 2001, 6, 325.
35. Joshi, S. K.; Honore, P. Expert Opin. Drug Disc. 2006, 1, 323.
36. Worley, J. F.; Main, M. J. Receptors Channels 2002, 8, 269.
37. Jarvis, M. F.; Burgard, E. C.; McGaraughty, S.; Honore, P.; Lynch, K.; Brennan, T.
J.; Subieta, A.; Van Biesen, T.; Cartmell, J.; Bianchi, B.; Niforatos, W.; Kage, K.;
Yu, H.; Mikusa, J.; Wismer, C. T.; Zhu, C. Z.; Chu, K.; Lee, C. H.; Stewart, A. O.;
Polakowski, J.; Cox, B. F.; Kowaluk, E.; Williams, M.; Sullivan, J.; Faltynek, C.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 17179.
38. Zhang, X. F.; Zhu, C. Z.; Thimmapaya, R.; Choi, W. S.; Honore, P.; Scott, V. E.;
Kroeger, P. E.; Sullivan, J. P.; Faltynek, C. R.; Gopalakrishnan, M.; Shieh, C. C.
Brain Res. 2004, 1009, 147.
39. Joshi, S. K.; Hernandez, G.; Mikusa, J. P.; Zhu, C. Z.; Zhong, C.; Salyers, A.;
Wismer, C. T.; Chandran, P.; Decker, M. W.; Honore, P. Neuroscience 2006, 143,
587.
40. Kim, S. H.; Chung, J. M. Pain 1992, 50, 355.