Discovery and optimization of aminopyrimidinones as potent and state-dependent Nav1.7 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.11.111

Clinical genetic data have shown that the product of the SCN9A gene, voltage-gated sodium ion channel Nav1.7, is a key control point for pain perception and a possible target for a next generation of analgesics. Sodium channels, however, historically have been difficult drug targets, and many of the existing structure-activity relationships (SAR) have been defined on pharmacologically modified channels with indirect reporter assays. Herein we describe the discovery, optimization, and SAR of potent aminopyrimidinone Nav1.7 antagonists using electrophysiology-based assays that measure the ligand-receptor interaction directly. Within this series, rapid functionalization at the polysubstituted aminopyrimidinone head group enabled exploration of SAR and of pharmacokinetic properties. Lead optimized N-Me-aminopyrimidinone 9 exhibited improved Nav1.7 potency, minimal off-target hERG liability, and improved rat PK properties.

Full text of DOI:10.1016/j.bmcl.2011.11.111

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1055–1060
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of aminopyrimidinones as potent
and state-dependent Nav1.7 antagonists
Hanh Nho Nguyen a, , Howie Bregman a, John L. Buchanan a, Bingfan Du a, Elma Feric b, Liyue Huang c,
⇑
Xingwen Li c, Joseph Ligutti d, Dong Liu d, Annika B. Malmberg b, David J. Matson b, Jeff S. McDermott b,
Vinod F. Patel a, , Ben Wilenkin b, Anruo Zou c, Stefan I. McDonough b, Erin F. DiMauro a
a Department of Chemistry Research and Discovery, Amgen Inc., 360 Binney St., Cambridge, MA 02142, USA
b Department of Neuroscience, Amgen Inc., 360 Binney St., Cambridge, MA 02142, USA
c Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 360 Binney St., Cambridge, MA 02142, USA
d Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Clinical genetic data have shown that the product of the SCN9A gene, voltage-gated sodium ion channel
Nav1.7, is a key control point for pain perception and a possible target for a next generation of analgesics.
Sodium channels, however, historically have been difficult drug targets, and many of the existing structure–
activity relationships (SAR) have been defined on pharmacologically modified channels with indirect repor-
ter assays. Herein we describe the discovery, optimization, and SAR of potent aminopyrimidinone Nav1.7
antagonists using electrophysiology-based assays that measure the ligand–receptor interaction directly.
Within this series, rapid functionalization at the polysubstituted aminopyrimidinone head group enabled
exploration of SAR and of pharmacokinetic properties. Lead optimized N-Me-aminopyrimidinone 9
exhibited improved Nav1.7 potency, minimal off-target hERG liability, and improved rat PK properties.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 28 October 2011
Revised 23 November 2011
Accepted 28 November 2011
Available online 6 December 2011
Keywords:
Nav1.7
Voltage-gated sodium channels
Nav channels
Sodium channel blockers
Sodium channel inhibitors
Sodium channel antagonists
Congenital indifference to pain
Paroxysmal extreme pain disorder
Primary erythromelalgia
Aminopyrimidinone
Voltage-gated sodium (Nav) channels govern directly the excit-
ability of pain-sensing neurons and are key to pain perception.1 In
addition, sodium channel antagonists including carbamazepine,
oxcarbazepine, lidocaine, and mexiletine in some circumstances
are effective analgesics.2 These inhibitors, however, have little selec-
tivity within the family of nine sodium channel isoforms and have
limited clinical utility due to dose-limiting side effects on the cardiac
and central nervous systems. Accordingly, inhibition of the sodium
channel isoform(s) that govern pain without inhibition of the
sodium channels governing cardiac, CNS, or motor function is a
major goal of the field.
Gain-of-function and loss-of-function mutations in human
point to Nav1.7 as the most promising drug target among the nine
sodium channel isoforms (Nav1.1–Nav1.9).3 Fueling interest in
Nav1.7, recent data show that small molecules can be made selec-
tive for Nav1.7, as previously shown for Nav1.3 and Nav1.8.4,5 In
general, however, sodium channels have proven difficult to screen,
and no subtype-selective sodium channel inhibitors have been
made into drugs. The channels are voltage-activated and normally
open and inactivate within milliseconds, making standard multi-
well fluorescence-based live cell assays difficult. Few high-affinity
ligands are available for binding assays; and sodium channels exist
in many voltage-sensitive states with different pharmacology.6
In an effort to developpotent and selectiveNav1.7 antagonists for
the treatment of chronic pain, we carried out an electrophysiology-
based screening and hit-to-lead campaign using the IonWorksÒ
Quattro (IWQ) and PatchXpressÒ (PX) automated electrophysiology
platforms, respectively.7 Using automated electrophysiology assays
allowed us to quantitate directly receptor–ligand interactions with a
readout of functional inhibition. Moreover, the precise voltage con-
trol of electrophysiology-based screening and driver assays enabled
compound potency to be assessed against known gating states of the
channel. Sodium channels inactivate after opening, and the nonse-
lective sodium channel inhibitors used clinically likely gain their
small therapeutic window via preferential inhibition or stabilization
of inactivated states. Accordingly, compound potency was assessed
⇑
Corresponding author. Tel.: +1 617 444 5254; fax: +1 617 577 9822.
E-mail address: hanhn@amgen.com (H.N. Nguyen).
Present address: Sanofi-Aventis, Cambridge, MA 02139, USA.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.111

1056
H. N. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1055–1060
that compounds indeed were preferential inhibitors/stabilizers of
inactivated states.
O
N
NH
Aminopyrimidinone 1 was identified as a potent and state-depen-
dent inhibitor of hNav1.7 from a screening campaign (Fig. 1).8,9 This
hit was unique in structure compared to reported Nav blockers.5,10
Although 1 lacked selectivity over the cardiac hNav1.5 (also
measured with the PX system on channels with average 20% inactiva-
tion) and exhibited high clearance in rats, its favorable selectivity
over hERG channels compared to other screening hits was promising.
In addition, we anticipated that selectivity against hNav1.5 would be
achieved if hNav1.7 potency could be further improved.
Flexible chemistry was developed in order to access analogs of 1
bearing different side-chains and head groups. Side-chain analogs
8 and 14–24 were prepared by selective S-alkylation of aminothio-
pyrimidinone 2 with alkyl bromides (Scheme 1, Eq. 1). Similarly,
compounds 912–13 with different head groups could be prepared
by selective S-benzylation of the corresponding aminothiopyrimid-
inones (313–7) and 1-(bromomethyl)-4-(tert-butyl)benzene (Eq. 2).
Further derivatization of compound 8 was readily achieved to
explore modifications of the aminopyrimidinone head group
(Scheme 2). For example, the amino-group of 8 was transformed
to amides (25–26), carbamate (27), and sulfonamide (28) via acyl-
ation, peptide coupling, or reaction with either (Boc)2O or mesyl
chloride. Taking advantage of the enaminone reactivity of 8, regio-
selective chlorination afforded 29. Finally, 9 and 30 were prepared
via a Mitsunobu reaction of 8 and MeOH.
H2N
S
0.94a
0.40 [1.4]a
1.2a
hNav1.7 PX IC50 (µM)
hNav1.7 Man IC50 (µM)
hNav1.5 PX IC50 (µM)
>15
hERG dofetilide binding, derived Ki
(µM)
257 / 224
HLM / RLM CLint (µL/min/mg)
Solubility11 - SIF, PBS, 0.01 N HCl (µg/mL)
14, 2, 3
21 / 1.1b
Permeability (10-6 cm/s) / Efflux ratio
CL (L/h/kg)
Vss (L/kg)
t1/2 (h)
6.5c
1.3
0.25
Figure 1. Data for aminopyrimidinone hit 1. aElectrophysiology IC50 of partially
inactivated channels is shown. Where applicable, bracket indicates IC50 against fully
noninactivated channels determined with Man electrophysiology. Inhibitory activ-
ity represents an average of at least two determinations. bData obtained from
MDR1-LLC-PK1 cells, pig kidney cells expressing human MDR1, at 5 lM in the
presence of 0.1% BSA and permeability estimated by averaging apparent perme-
ability values in the apical to basolateral and basolateral to apical directions. cMale
Sprague–Dawley rats were dosed intravenously at 0.5 mg/kg in 0.5 mL/kg of 100%
DMSO. (See above-mentioned reference for further information.)
on hNav1.7 with an average 20% contribution from inactivated
channels using the PX system. For key compounds, potency was ver-
ified with manual (Man) electrophysiology, both on average 20%
inactivated channels and on fully noninactivated channels to verify
The synthesis of triazinone 34 was achieved with three consec-
utive SNAr reactions of trichloro triazine 31, with 4-tert-butylbenzyl
O
O
a
NH
NH
Eq 1
R
R-Br
50-70%
H2N
N
S
H2N
N
H
2
S
8, 14-24
O
O
R3
R2
R4
R3
R1
S
N
Br
a
N
Eq 2
N
S
N
11-83%
R1
R2
9-13
3-7
Scheme 1. Preparation of 8–24 via chemoselective S-alkylation of 2–7 with alkyl bromides. Reagents and conditions: (a) NaOH in EtOH/H2O, 50 °C.
O
NH
R2
R1
N
H
N
S
O
O
Cl
a
25, R2 = COMe
NH
NH
d
R1
R1
2
26
27
, R = CO(cyclopropyl)
H2N
N
S
H2N
N
S
b
c
, R2 = CO2t-Bu
29
8
, R2 = SO2Me
28
R
1 = CH2-4-t-Bu-C6H4
e
Me
O
O
Me
N
N
+
R1
R1
H2N
N
S
H2N
N
S
9
30
Scheme 2. Synthesis of analogs by derivatization of lead 8. Reagents and conditions: (a) AcCl or cyclopropanecarbonyl chloride, Et3N, DCM, rt (25, 22%; 26, 71%); (b) (Boc)2,
DMAP, Et3N, DCM, rt (47%); (c) MsCl, Et3N, DCM, rt (69%); (d) NCS, CHCl3, rt (60%); (e) MeOH, DEAD, PPh3, THF, rt (9, 30%; 30, 21%).

H. N. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1055–1060
1057
Cl
O
N
Cl
N
Cl
N
N
N
N
NH
N
N
a
c
b
N
N
Cl
N
S
H2N
S
H2N
S
Cl
Cl
32
34
31
33
Scheme 3. Preparation of triazinone 34. Reagents and conditions: (a) 4-tert-butylbenzyl mercaptan, (i-Pr)2NEt, THF, 0 °C (96%); (b) NH3 in MeOH, rt (50%); (c) TFA, water, rt
(100%).
Table 1
Alkyl thio modifications
O
NH
O
H2N
N
S
NH
R
H2N
N
S
0.68a
0.34 [2.2]a
0.30a
hNav1.7 PX IC50 (µM)
hNav1.7 Man IC50 (µM)
Compound
R
hNav1.7 PX IC50
M)
HLM/RLM
CLint L/min/mg)
(l
(l
hNav1.5 PX IC50 (µM)
1
0.94
1.3
257/224
49/124
>15
hERG dofetilide binding, derived Ki (µM)
HLM / RLM CLint (µL/min/mg)
<14 / 28
14
Solubility11 - SIF, PBS, 0.01 N HCl (µg/mL)
Permeability (10-6 cm/s) / Efflux ratio
CL (L/h/kg)
67, 5, 4
27 / 0.50b
15
16
>30
20
ND/ND
5.1c
7.9
2.7
<14/<14
Vss (L/kg)
t1/2 (h)
Figure 2. Data for aminopyrimidinone lead 8. aElectrophysiology IC50 of partially
inactivated channels is shown. Where applicable, bracket indicates IC50 against fully
noninactivated channels determined with Man electrophysiology. Inhibitory activ-
ity represents an average of at least two determinations. bData obtained from
Table 2
Structure–activity relationship of benzyl analogs
MDR1-LLC-PK1 cells, pig kidney cells expressing human MDR1, at 5 lM in the
O
presence of 0.1% BSA and permeability estimated by averaging apparent perme-
ability values in the apical to basolateral and basolateral to apical directions. cMale
Sprague–Dawley rats were dosed intravenously at 0.5 mg/kg in 0.5 mL/kg of 100%
DMSO.
NH
H2N
N
S
R1
Compound R1
hNav1.7 PX IC50
(l
M) HLM/RLM CLint (lL/min/mg)
at the para-position of the phenyl ring (Table 2). The IC50 was
improved by fourfold with a chlorine substituent (18). As the
4-substituent increased in size, the potency further improved
(17–21, 8). However, the para-phenyl variant 22 was not active
16
17
18
19
20
21
8
22
23
24
H
4-F
4-Cl
4-Me
4-CF3
4-OCF3
4-t-Bu
4-Ph
20
<14/<14
ND/ND
22/<14
ND/ND
<14/<14
<14/25
<14/28
ND/ND
ND/ND
ND/ND
>30
4.7
5.4
3.2
2.2
0.68
>30
at concentrations up to 30 lM. Incorporation of polar groups such
as SO2Me (23), and CN (24) was detrimental to inhibitory activity.
These modifications revealed that 4-t-butyl analog 8 was compara-
bly potent to hit 1 and highlighted the overall potency trend that
lipophilic substitution at the para-position was preferred. Notably,
these benzyl analogs were in general more stable in liver micro-
somes compared to their alkyl counterparts (Table 2 vs Table 1).
Manual patch-clamp on 20% inactivated channels confirmed the
potency of lead 8 determined by the PX studies (Fig. 2).9 Potency
was approximately sixfold weaker on fully noninactivated chan-
nels, confirming the property of state dependence. Although 8 still
lacked selectivity over hNav1.5, the maintenance of hERG selectiv-
ity and the improvement in microsomal clearance over hit 1 were
promising. Unfortunately, the low CLint did not translate in vivo, as
rat clearance of 8 exceeded hepatic blood flow.
Although the CLint in liver microsomes was low, preliminary
data showed that phase II metabolism involving O-glucuronidation
was the predominant clearance mechanism.14 Since the glucuron-
idation was likely to occur on the aminopyrimidinone head group
of 8, we decided to probe the site of glucuronidation and to inves-
tigate the SAR around this aminopyrimidinone ring (Table 3). First,
the des-amino 3515 and hydroxyl 11 analogs were inactive up to
4-SO2Me >30
4-CN >30
mercaptan, followed by NH3, then water in the presence of TFA
(Scheme 3).
We initiated medicinal chemistry efforts around aminopyrimid-
inone hit 1 with the goals of improving potency, selectivity, and PK
properties while maintaining state-dependence and hERG selectiv-
ity. All potent hNav1.7 blockers within this series were tested
against the cardiac channel hNav1.5 assay. At the onset, our strat-
egy was to improve PK properties by removing the potential sites
of oxidative metabolism. Thus, truncating the methyl chain by
one carbon (14) reduced the intrinsic clearance (CLint) in liver
microsomes (Table 1). However, further truncation eventually
abolished potency (15). The potency could be rescued somewhat
by capping the aminopyrimidinone head group with a benzyl
(16) that imparted significantly improved microsomal clearance
because most of the metabolic soft spots from the linear carbon
chain of hit 1 had been removed.
30 lM, suggesting that the 6-amino group was critical. The acetyl
25 or the cyclopropylcarbonyl 26 analog was tolerated but these
compounds were unstable in human and rat liver microsomes.16
Carbamate 27 was moderately active and sulfonamide 28 was
From a structural overlay of 1 and 16, we hypothesized that the
potency of microsomally stable compound 16 could be substan-
tially improved by strategic incorporation of hydrophobic groups

1058
H. N. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1055–1060
Table 3
Next, we sought to cap the pyrimidinone with a small methyl
Amino group at R6 position is important for potency
group at either the oxygen or the ring nitrogens. This exercise could
also allow us to determine the active tautomer of the aminopyri-
midinone 8 and provide insightful information on how aminopyri-
midinone antagonists interact with the sodium channel to guide
further SAR exploration (Table 4). Based on ground state energies,17
we suspected that the pyrimidin-4(3H)-one tautomer (8) rather
than either the pyrimidin-4-ol (36) or pyrimidin-4(1H)-one tauto-
mer (37) was predominant. In the proton NMR of 8, we observed a
broad singlet at 11.5 ppm corresponding to an NH-amide; however,
we were not able to conclusively determine the predominant tauto-
mer.18 In order to probe the active species, we selectively installed a
methyl group at each reactive position (8?9, 36?30, and
37?10).19 As expected, pyrimidin-4(3H)-one containing compound
9 was the active methylated compound.
O
N
O
N
R5
R6
NH
N
NH
or
S
H2N
S
34
8, 11-13, 25-29, 35
Compound
R6
R5
hNav1.7 PX IC50 (lM)
8
35
11
0.68
>30
>30
H2N
H
H
H
H
H
Methylation served to increase the potency of our lead 8 by
fourfold as measured by manual electrophysiology (Fig. 3).9,20 Lead
9 showed state dependence when tested with manual electrophys-
HO
O
25
26
1.2
2.5
N
iology (IC50 = 0.50 lM on fully noninactivated channels). In addi-
H
O
tion to exceptional potency and state dependence, hERG activity
was also minimal. Importantly, installation of this methyl group
led to a substantially improved pharmacokinetic profile as clear-
ance was reduced significantly (CL = 1.1 L/h/kg), possibly due to
suppressed glucuronidation. Unfortunately, compound 9 showed
no selectivity over hNav1.5, a concern for cardiac toxicity. Due to
its excellent PK properties, compound 9 was studied in vivo via
H
H
H
N
H
O
27
28
7.8
O
O
N
H
O
S
intraperitoneal injection ([unbound plasma] = 0.024 lM; [brain]/
>30
N
H
[plasma] = 3.3). Unfortunately, in open field studies, compound 9
caused severe reductions in total basic movement (81%) and an al-
most complete reduction in rearing behavior (98%). This obviated
drawing conclusions from other in vivo models, as the rats were
no longer moving. Therefore, due to poor tolerability, apparent
off-target behavioral effects and a risk for cardiotoxicity, this series
was not further pursued.
In conclusion, we have described the discovery and the hit-to-
lead optimization of aminopyrimidinones as a new class of potent
and state-dependent hNav1.7 antagonists. Even though the po-
tency of lead 9 was improved by fivefold compared to hit 1, the
selectivity ratio against the cardiac hNav1.5 channel was very sim-
ilar. Our strategy to remove the oxidative metabolic sites resulted
in low CLint in liver microsomes. However further examination re-
vealed phase II mediated clearance. Gratifyingly, a chemoselective
N-methylation of the lead 8 improved the inhibitory activity and
29
12
4.3
H2N
Cl
>30
H2N
N
H2N
13
>30
HN
inactive. Second, modulating the steric and electronic properties of
the aminopyrimidinone ring at the five position with a chlorine 29
reduced potency. 5-Amino compound 12 was inactive. Finally,
bicyclic analog 13 and triazinone 34 (Scheme 3) were also inactive
up to 30 lM.
Table 4
Determination of the preferred binding tautomer
O
N
OH
O
N
NH
N
H2N
H2N
N
S
S
H2N
N
H
S
H-Analog
pyrimidin-4(3H)-one
pyrimidin-4-ol
pyrimidin-4(1 )-one
H
Compound
8
0.0
36
37
Enthalpy (kcal/mol)a
4.9
4.3
O
N
Me
O
O
N
Me
S
N
N
N
Me-Analog
Compound
H2N
H2N
S
H2N
N
S
Me
9
30
10
hNav1.7 PX IC50
(lM)
0.42
>30
>30
a
All of the calculations were done at the HF/6-31+G(d) level of theory by using Gaussian03. Solvation corrections were computed by using CPCM method with UAKS cavity
model.

H. N. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1055–1060
1059
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O
Me
N
H2N
N
S
hNav1.7 PX IC50 (µM)
0.42a
8. PX protocol: hNav1.7 or hNav1.5-expressing HEK 293 cells were voltage
hNav1.7 Man IC50 (µM) 0.080 [0.50]a
clamped at
a membrane potential that produced average 20% fractional
hNav1.5 PX IC50 (µM)
0.20a
>15
inactivation. Dose–response curves were built from an ensemble of single-
concentration tests on many cells. For details of electrophysiology and in vivo
assays, see Bregman, H. et al.
hERG dofetilide binding, derived Ki (µM)
HLM / RLM CLint (µL/min/mg)
9. Manual electrophysiology was used to determine compound potency on
noninactivated channels (holding voltage set to À140 mV) and on channels
with holding voltage set and adjusted as necessary during the experiment to
produce steady 20% inactivation. Values are average of n = 2 cells. Compounds
1 and 8 blocked and reversed with fast kinetics. Compound 9 had a slower off-
rate but still blocked with rapid onset.
164 / 57
14, 10, 1
16 / 1.0b
Solubility11 - SIF, PBS, 0.01 N HCl (µg/mL)
Permeability (10-6 cm/s) / Efflux ratio
CL (L/h/kg)
Vss (L/kg)
t1/2 (h)
1.1c
2.6
3.9
10. Marron, B. Annu. Rep. Med. Chem. 2006, 41, 59.
11. Tan, H.; Semin, D.; Wacker, M.; Cheetham, J. JALA 2005, 10, 364.
12. An alternative synthesis of compound 9 via a Mitsunobu reaction is shown in
Scheme 2.
13. This compound was purchased from HDH Pharma Inc. LC–MS (ESI) Calcd for
C5H7N3OS: [M]+ = 157. Found [M+H]+ = 158. 1H NMR (Bruker, 400 MHz, DMSO-
d6) d ppm 11.73 (br s, 1H), 6.38 (br s, 2H), 4.86 (s, 1H), 3.43 (s 3H).
Figure 3. Potent block of Nav1.7 by N-methyl aminopyrimidinone lead 9. A. Percent
Nav1.7 current remaining as a function of concentration of compound 9, at holding
potentials where Nav1.7 was fully noninactivated and where Nav1.7 was 20%
inactivated (average of n = 2 cells for each condition). Curves were fitted to the
equation I/I0 = 1/{1+(concentration/IC50)n}. B. aElectrophysiology IC50 of partially
inactivated channels is shown. Where applicable, bracket indicates IC50 of fully
noninactivated channels. Inhibitory activity represents an average of at least two
determinations. bData obtained from MDR1-LLC-PK1 cells, pig kidney cells
14. Metabolite identification in fresh rat hepatocytes confirmed that lead
8
underwent glucuronidation. Full scan positive ion mode of the incubated
material showed parent 8 at 20.1 min, m/z = 290 and glucuronide adduct at
18.7 min, m/z = 466.
15. This analog was purchased from Otava.
16. The parent compound was not detected on the LC–MS after 15 min of
incubation with rat liver microsomes.
expressing human MDR1, at 5 lM in the presence of 0.1% BSA and permeability
estimated by averaging apparent permeability values in the apical to basolateral
and basolateral to apical directions. cMale Sprague–Dawley rats were dosed
intravenously at 0.5 mg/kg in 0.5 mL/kg of 100% DMSO.
17. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Montgomery Jr., J. A.; Vreven, T.; Kudin, K. N.; Burant, J.
C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi,
M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara,
M.; Toyota, K.; Fukuda, R. H., J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.;
Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;
Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P.
Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski,
V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.;
Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko,
A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham,
M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A., Gaussian
03, Revision C.02; Gaussian, Inc., Wallingford CT, 2004.
reduced rat in vivo clearance possibly by suppressing glucuronida-
tion, while maintaining state-dependence and excellent selectivity
over hERG.
Acknowledgements
We would like to thank Dr. Steve Hollis and Paul Krolikowski for
structural determination, Dr. Hakan Gunaydin for modeling/com-
putation, Dan Waldon for glucuronidation determination, Grace
Bi and Larry Miller for purification support, and Dr. Margaret Y.
Chu-Moyer for proofreading the manuscript.
18. The carbon shift data supported the structure of pyrimidin-4(1H)-one 37.
However, line-broadening in proton and carbon spectra suggested the double-
bond was likely moving between the two ring-nitrogens suggesting that
pyrimidin-4(1H)-one 37 and aminopyrimidin-4(3H)-one 8 were in equilibrium
in DMSO-d6.
Supplementary data
19. Each structure was confirmed by 1D and 2D NMR analyses. 1H NMR chemical
shifts in DMSO-d6, LCMS and HPLC retention times distinguished and
confirmed each methylated isomer. Chemical shifts of methyl group were
3.21, 3.33, and 3.78 ppm for 9, 10, and 30 respectively. The 1H NMR chemical
shift of the O-methylated analog (30) was shifted down field as expected. 1H
NMR (Bruker, 400 MHz, DMSO-d6) d ppm for 9: 7.31–7.41 (dd, J = 26, 8.5 Hz,
4H), 6.51 (br s, 1H), 4.93 (s, 1H), 4.37 (s, 2H), 3.21 (s, 3H), 1.26 (s, 9 H); for 10:
7.34 (m, 4H), 6.55 (s, 2H), 4.94 (s, 1H), 4.30 (s, 2H), 3.33 (s, 3H), 1.26 (s, 9H); for
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.111.
References and notes
1. Cummins, T. R.; Sheets, P. L.; Waxman, S. G. Pain 2007, 131, 243.

1060
H. N. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1055–1060
30: 7.33 (m, 4H), 6.72 (s, 2H), 5.45 (s, 1H), 4.28 (s, 2H), 3.78 (s, 3H), 1.25 (s, 9H).
20. Eighty nanomolar is approaching the most potent sodium channel inhibitors
reported. Most nonselective sodium channel inhibitors have potency well over
a micromolar. In our hands, mexiletine IC50 on 20% inactivated channels was
56 mM (Bregman, H. et al.).
LC–MS retention times were 2.33, 2.65, and 1.62 min for 9, 30, and 10,
respectively. HPLC retention times were 7.51, 7.25, and 6.37 min for 9, 30, and
10, respectively.