The discovery of a potent Nav1.3 inhibitor with good oral pharmacokinetics

Article abstract of DOI:10.1039/c7md00131b

In this article, we describe the discovery of an aryl ether series of potent and selective Na_v1.3 inhibitors. Based on structural analogy to a similar series of compounds we have previously shown bind to the domain IV voltage sensor region of Na_v channels, we propose this series binds in the same location. We describe the development of this series from a published starting point, highlighting key selectivity and potency data, and several studies designed to validate Na_v1.3 as a target for pain.

Full text of DOI:10.1039/c7md00131b

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RESEARCH ARTICLE
The discovery of a potent Nav1.3 inhibitor with
good oral pharmacokinetics†
Cite this: DOI: 10.1039/c7md00131b
D. C. Pryde,
a N. A. Swain,a P. A. Stupple,a C. W. West,c B. Marron,c
*
‡
C. J. Markworth,c D. Printzenhoff,c Z. Lin,c P. J. Cox,b R. Suzuki,b S. McMurray,b
G. J. Waldron,b C. E. Payne,b J. S. Warmusd and M. L. Chapmanc
Received 14th March 2017,
Accepted 26th April 2017
In this article, we describe the discovery of an aryl ether series of potent and selective Nav1.3 inhibitors.
Based on structural analogy to a similar series of compounds we have previously shown bind to the do-
main IV voltage sensor region of Nav channels, we propose this series binds in the same location. We de-
scribe the development of this series from a published starting point, highlighting key selectivity and po-
tency data, and several studies designed to validate Nav1.3 as a target for pain.
DOI: 10.1039/c7md00131b
rsc.li/medchemcomm
Voltage-gated sodium channels (Nav) comprise a family of
nine transmembrane proteins (Nav1.1–Nav1.9) that span
cellular membranes.1 The main pore-forming α-subunit has a
6-transmembrane topology of approximately 260 kDa size
which assembles as a pseudo-tetramer into four distinct do-
mains each of which contribute pore-forming helices and a
spatially separated voltage sensor region to allow the channel
to respond to voltage changes across the cell membrane.2 The
sequence homology of mammalian Nav subtypes is very high,
particularly in the pore region, making subtype selectivity of
ligand interactions challenging. Nav channels selectively con-
duct sodium ions across cell membranes to create action po-
tentials and nerve impulses in electrically excitable cells.
These action potentials in turn elicit a plethora of physiologi-
cal effects to, for example, control muscle contraction, cardiac
function and neurological processing. Nav channel modula-
tors have therefore been targeted as potential treatments for
diseases as diverse as chronic pain, epilepsy and cardiac ar-
rhythmias leading to a number of successful drug launches.3
Fig. 1 shows some selected Nav channel drugs 1–7. All of
these drugs show weak and non-selective activity across the
Nav family which has limited their utility due to central and/
or cardiovascular adverse events.4 Significant research has
been dedicated to the identification of subtype selective inhib-
itors of Nav channels, as potentially safer alternatives to these
older, non-selective examples.
There has been considerable work carried out to character-
ise and modify various animal toxins and other natural prod-
ucts which have been shown to engage Nav channels,5 but de-
spite some recent advances, no advanced clinical candidates
from this approach have as yet been described. There have
been significant efforts to obtain protein crystal structures of
bacterial sodium channels6 and carry out modelling studies7
to elucidate ligand binding sites and modes of modulation. In
a notable recent publication,8 a bacterial Nav channel was
engineered to contain features of the human Nav1.7 voltage-
sensor region and a crystal structure obtained of this chimera
a Worldwide Medicinal Chemistry, Pfizer Neuroscience and Pain Research Unit,
Portway Building, Granta Park, Cambridge, CB21 6GS, UK.
E-mail: david@curadev.co.uk
b Pfizer Neuroscience and Pain Research Unit, Portway Building, Granta Park,
Cambridge, CB21 6GS, UK
c Pfizer Neuroscience and Pain Research Unit, 4222 Emperor Boulevard, Suite 350,
Durham, North Carolina, NC27703, USA
d Worldwide Medicinal Chemistry, Pfizer Neuroscience and Pain Research Unit,
Groton, CT, USA
† All authors are, or were at the time of the work being undertaken, employees
of Pfizer.
‡ Current address: Curadev Pharma Ltd., Innovation House, Discovery Park,
Sandwich, Kent, CT13 9ND, UK.
Fig. 1 Selected Nav channel ligands.
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bound to an inhibitor to elucidate the drivers of subtype selec-
tivity within this region of the protein. Homology models
using this structure to explore subtype selectivity of other Nav
channels, such as Nav1.3, are anticipated.
For several years now, we have pursued medicinal chemis-
try approaches to identify potent and selective inhibitors of
several Nav channels to aid in the elucidation of the role of in-
dividual channels in pain transmission.9 As part of this larger
effort, we have identified a series of aryl sulphonamides that
show potent and for the most part selective inhibition of the
Nav1.3 channel. This paper describes our efforts to success-
fully improve the physicochemical and pharmacokinetic prop-
erties of this series while retaining excellent Nav1.3 potency
and broader Nav subtype selectivity.
There are very few reports of potent Nav1.3 inhibitors, and
those reports that have emerged tend to describe poorly se-
lective Nav blockers that also carry Nav1.3 activity such as
lacosamide 810 (Fig. 2). There have been some more recent
reports of aryl sulphonamides from Vertex 911 and Icagen
1012 that have greater Nav1.3 potency and it has been the lat-
ter series that we have focussed our efforts on.
Fig. 3 Screening lead compound.
Compound 11 was shown to bind in the voltage-sensor re-
gion of voltage-gated sodium channels, based on chimeric
channels of Nav1.3 domains combined with varying homolo-
gous domains of either Nav1.7 or Nav1.5. These studies
suggested domain 4 to be the interaction site for this com-
pound, a region of the protein that we believed would support
subtype-selective Nav1.3 channel blockers with the appropri-
ate optimisation, as we had observed large reductions in po-
tency when the S2–S4 regions within domain 4 were mutated
away from Nav1.3 sequences to those of Nav1.5 or Nav1.7.13
This region also showed the greatest similarity in amino acid
sequence between Nav1.3 and Nav1.1 channels,13 explaining
the equivalent potency of 11 at these two channel subtypes.
In developing this lead further, analogues of 11 were pre-
pared to explore peripheral and core template functionality
and their effect on Nav1.3 channel potency (Table 1) and
subtype selectivity. Compounds were tested for Nav1.3 inhibi-
Identifying Nav1.3 pharmacological
tools
In a previous disclosure, we described the identification of a
diphenylmethyl amide 11 (Fig. 3) of this aryl sulphonamide
series13 which showed good potency at Nav1.3 and some se-
lectivity for this channel over other Nav subtypes. Compound
11 showed excellent potency at human and rat Nav1.3 with
good selectivity against all other human subtypes tested with
the exception of Nav1.1 which mirrored Nav1.3 activity. Inter-
estingly, while 11 was very weak at human Nav1.7, it showed
significantly greater potency at the rat orthologue. This com-
pound showed poor passive permeability in RRCK and mod-
erate efflux in an MDR1 cell line, and was of modest aqueous
solubility, features that we sought to address in subsequent
analogues.
tory activity in
a
HEK cell-based electrophysiology
PatchXpress platform.
Simply excising one of the phenyl rings (18) led to a sharp
drop in potency. Similarly, constraining the aniline function
into a ring system such as 13 concomitant with excising one
of the phenyl rings also produced a significant loss in activ-
ity. Inverting the amide group in 19 was essentially
equipotent with 18, which was an important observation as
we were concerned that amidolysis of the amide linkage in
18 would produce sulfathiazole which is a known toxin.14 It
was the addition of lipophilic substituents, for example the
trifluoromethyl group in 12, which gave significant boosts in
potency and 12 was now only some 10-fold less active than
the starting 11. Other amide replacements such as the sul-
fone 14 and the sulphonamide 15 were of similar potency to
12, as were the amide isosteric triazoles 16 and 17. It was
clear at this stage that several truncated amides or their iso-
steres could afford potent Nav1.3 activity provided they
retained a lipophilic aryl substituent. We chose the syntheti-
cally most accessible amide linkage, in the non-anilinic re-
versed configuration, to further optimise. A trifluoromethoxy
group in 20 was similarly potent to the trifluoromethyl conge-
ner 12 and further optimisation of the functionality on both
phenyl rings led to the chloro derivative 21 and the fluoro-
chloro 22 which had excellent potency at Nav1.3, equivalent
to the starting point 11. Further profiling (Fig. 4) confirmed
its potency at both human and rat Nav1.3 and that 22
retained good selectivity over all subtypes except Nav1.1, at
which it was equipotent.
Fig. 2 Selected literature Nav1.3 ligands.
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Table 1 SAR exploration around compound 11
Compound
R
clog P (log D)
Nav1.3 IC50 (nM)
17
11
3.8 (3.3)
12
13
3.5 (2.5)
2.6 (1.8)
147
2506
14
15
16
3.7 (ND)
3.2 (ND)
4.1 (ND)
155
160
45
17
3.6 (ND)
124
18
19
2.9 (ND)
2.6 (ND)
3790
4450
20
21
4.0 (2.6)
4.3 (3.1)
197
20
22
4.5 (3.3)
11
Evaluating Nav1.3 as a pain target
macology and in vivo efficacy, but as 22 was profiled more ex-
tensively, it became apparent that it had some liabilities as a
lead due to low aqueous solubility, low passive membrane
permeability and inhibition of both CYP3A4 and CYP2C9
Despite the equivalent levels of Nav1.1 activity, 22 was an ex-
cellent early tool compound to further explore Nav1.3 phar-
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Cynomolgus monkeys were then dosed with 30 mg kg−1 23
which resulted in a plasma exposure of 22 in excess of some
62 fold the in vitro IC50. This was accompanied by no clinical
signs, no changes in heart rate and mean, diastolic or systolic
blood pressure, no effects on any ECG parameter and no
changes in left ventricular pressure parameters. Our conclu-
sion from this study was that the original observations were
likely rat-specific and allowed us to proceed with more confi-
dence in the safety of Nav1.3 as a target.
Based on previous reports of an upregulation of Nav1.3
channels in the DRG and their potential involvement in pain
pathogenesis,17 we investigated the ability of 22 to alter neu-
ronal excitability using ex vivo skin nerve recording and fur-
thermore evaluated the effects of 23 in vivo, using spinal cord
recording in models of peripheral nerve injury. Exogenous
administration to the skin-tibial nerve with 22 at 0.01, 0.03,
0.1, 0.3 and 1 μM had no effect on heat evoked afferent firing
or ongoing nerve activity. Intravenous administration of 23
produced significant reductions of the noxious pinch evoked
activity (3 mg kg−1: difference −759, 95% CI: −1393 to −126, p
= 0.02; 10 mg kg−1: difference −788, 95% CI: −1421 to −154, p
= 0.02), where exposures up to 15× IC50 of 22 were achieved.
In the same study, pregabalin, an α2δ ligand licensed for the
treatment of neuropathic pain robustly inhibited pinch
evoked activity (9 mg kg−1: difference −711, 95% CI: −1345 to
−76, p = 0.03; 18 mg kg−1 difference −1354, 95% CI: −1989 to
−719, p = 0.0003). Notably, the magnitude of pregabalin ef-
fects were greater compared to that seen with 23. Responses
to innocuous brush, mechanical punctate stimuli and heat
evoked activity were not significantly modulated by 23. This
was in contrast to pregabalin, where clear inhibitions were
seen against these parameters. These data indicated that in
this study, Nav1.3 blockade did not drive a strong inhibitory
response against the majority of the endpoints measured.
However, Nav1.3 gene expression is known to be significantly
upregulated in a number of nerve injury models of pain in-
cluding SNT.18–20 Furthermore, data from the Waxman group
has shown profound effects of Nav1.3 gene knockdown on
several other evoked endpoints in different pain models.18
The increased availability of Nav1.3 could contribute to the
pain hypersensitivity in these models.
Fig. 4 Early Nav1.3 tool compound.
isoforms in high throughput inhibition assays. In common
with similar chemotypes developed as selective Nav1.7
blockers,15 22 was highly bound to plasma proteins in all spe-
cies tested (rat and human ppb 99.8% as measured by equi-
librium dialysis, dog ppb >99.8%). Nonetheless, 22 did allow
us to probe Nav1.3 as a drug target more fully at this stage.
An oral 100 mg kg−1 dose of 22 produced exposure levels
in the rat of approximately 12 fold the Nav1.3 IC50, at which
concentrations a drop in heart rate, an increase in blood
pressure and a decrease in core body temperature were ob-
served. A similar finding was seen at a 500 mg kg−1 dose,
which resulted in similar exposure levels,
a result of
solubility-limited absorption. These findings raised some
concern that blockade of Nav1.3 could produce a cardiac
safety liability. It was not believed that the findings were con-
founded by the inherent activity of 22 at the primarily brain-
expressed Nav1.1 channel and it became important to check
if this observation was purely a rat-specific effect. A follow-up
study was conducted in cynomolgus monkey, in which we
wished to target in excess of the 12 fold IC50 levels achieved
in the rat study. In order to do this with 22, a phosphate
prodrug was designed with significantly enhanced solubility
(Fig. 5).16 We demonstrated first that 23 was rapidly and
completely converted to the parent 22 upon oral dosing to
the rat and an equivalent dose of 23 resulted in a signifi-
cantly higher exposure of parent 22 than dosing 22 directly.
Gene expression levels of Nav1.3 in rat DRGs following spi-
nal nerve transection (SNT) were measured in-house. Ipsilat-
eral versus contralateral Ct values were compared using a
paired T-test. The house keeping gene beta actin was not sta-
tistically different between ipsi- and contralateral samples.
However both Nav1.3 and ATF3 genes were significantly dif-
ferent at the 5% level when ipsi- and contralateral levels were
compared (see Table 2).
Ipsilateral Ct values were lower for both Nav1.3 and ATF3
indicative of higher gene expression. In line with previous
studies,18 Nav1.3 and ATF3 genes were upregulated in ipsilat-
eral DRG following SNT.
The fact that Nav1.3 blockade did not drive a strong inhib-
itory response for the majority of the endpoints measured in
either the ex vivo skin nerve or in vivo spinal nerve
Fig. 5 Phosphate prodrug of 22.
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Table 2 Changes in gene expression levels of Nav1.3 in rat DRG
Ct value change
Lower 95% CI
Upper 95% CI
Std error
P value
Nav1.3
ATF3
β-Actin
3.325
4.629
−0.348
1.906
2.076
−0.876
4.744
7.181
0.180
0.552
0.993
0.205
0.0018
0.0055
0.1508
All values compared contralateral and ipsilateral measurements.
recordings, despite showing an increase in Nav1.3 gene ex-
pression in the neuronal cell bodies, is in contrast to reports
in the literature, which have shown effects of Nav1.3 knock-
down on behavioural and electrophysiological endpoints in
different pain models. One could speculate that this could be
due to differences in the experimental approach i.e. genetic
modification to reduce expression versus a pharmacological
inhibition, or that the increase in Nav1.3 gene expression was
not recapitulated at the protein level in our model. Finally, it
could be that a higher multiple of the primary pharmacology
is required to drive efficacy against the endpoints measured
(maximum of 15× IC50 plasma free drug levels achieved in
our in vivo studies, approximately 200 nM; the free drug
levels in DRG or spinal cord were not measured and could be
markedly less). Interestingly it is worth noting that Nassar
et al. showed using Nav1.3 null mutant mice, that Nav1.3 is
neither necessary nor sufficient for the development of nerve-
injury related pain.21 Further study is required to elucidate
these points.
acidic head group for activity at Nav1.3. The aryl sulfonyl
amino-thiazole head group in 22 had an acidic pKa of 6.4. We
explored a number of other head groups (Table 3) that cov-
ered a range of disparate pKa's from more to less acidic than
the thiazole in 22. There did not appear to be a strong corre-
lation between the acidic pKa of the head group and Nav1.3
potency, with the substantially more acidic thiadiazole group
24 being equipotent with the thiazole congener, while the
similarly acidic pyrimidines 25 and 26 were substantially
weaker. Head groups of intermediate acidity such as the iso-
meric pyrimidine 27 and the pyridazine 28 were very weak in-
hibitors of Nav1.3.
It seemed more likely that the binding region of the head
group required a combination of an acidic grouping of suffi-
cient compactness to be tolerated, with the activity observa-
tions for those head groups investigated being quite subtle.
These SAR had identified the thiadiazole as an equivalent
head group to the thiazole in terms of potency at Nav1.3.
Sulfonamide linkers
Identifying a non-amino thiazole tool
compound
In parallel to the above head group alterations, we also
returned to explore a number of sulfonamide linkers which
in the early analogues were of similar potency to the amides.
These were made as both secondary and tertiary sulfon-
amides; a selection of the tertiary sulphonamides made lack-
ing an H-bond donor are shown in Table 4 with different
linked saturated heterocycles (29, 30, 31 and 32) and vectors
We were concerned that the amino thiazole group could rep-
resent a toxicity liability and were very keen at this early stage
to identify alternate groups that would avoid a potential
structural alert in the amino thiazole group.22 The analogous
1,2,4-thiadiazole 24 demonstrated very similar pharmacology
and physicochemical properties but interestingly showed very
low oral bioavailability, perhaps due to this more acidic
headgroup being more susceptible to transporter-mediated
clearance and having a higher overall topological polar sur-
face area (TPSA) and H-bond count (Fig. 6).
We began to further explore structure activity relation-
ships in this chemotype to specifically improve the physico-
chemical and pharmacokinetic properties of the series in a
non-thiazole containing headgroup and identify an improved
compound that retained the favourable potency and selectiv-
ity characteristics of 22. In particular, the areas we wished to
explore in more depth were variations to the linker region to
reduce H-bonding character and PSA, and alterations to the
head group structure to improve permeability.
Head group SAR
Consistent with the arginine-rich putative binding site for
this series, it was highly likely that we would require an
Fig. 6 Non-amino thiazole version of 22.
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Table 3 Head group variations on the aryl amide core of 22
Compound
R
clog P (log D)
pKa (cpKa)
Nav1.3 IC50 (nM)
13
22
4.5 (3.3)
6.4 (6.6)
24
25
26
27
3.7 (2.1)
3.9 (2.1)
3.8 (1.8)
3.8 (2.0)
3.3 (4.3)
(3.8)
13
993
(3.9)
374
4.6 (4.7)
>3000
28
3.6 (2.4)
(4.1)
1793
Table 4 Sulfonamide linker analogues of 22
Compound
R–N–R
clog P (log D)
MWt.
451.0
Nav1.3 IC50 (nM)
959
29
3.0 (1.4)
30
3.3 (ND)
534.0
851
31
32
4.1 (2.6)
3.1 (ND)
534.4
532.6
945
1640
33
34
4.1 (ND)
2.8 (ND)
529.6
519.6
1830
>3000
of attachment (33 and 34) but all were significantly weaker
than 22. These data were replicated in the case of secondary
sulphonamides (data not shown) and all subsequent ana-
logues did not feature this linker.
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Table 5 Linker variations of the thiadiazole head group derivative 13
Compound
Structure
clog P (log D)
MWt.
510.9
Nav1.3 IC50 (nM)
13
24
3.7 (2.1)
35
3.6 (1.3)
524.9
522
36
37
4.65 (2.5)
0.9
451.8
516.5
296
6675
38
2.20 (0.4)
434.5
1273
39
40
4.40 (2.6)
2.68
465.9
582.6
22
1955
41
3.71 (1.30)
548.9
237
Other linker variations
good metabolic stability and much improved aqueous solubil-
ity compared to 22.
We then explored a number of alternative linker variations in
the thiadiazole series, designed to reduce the H-bonding
character of the linking group and improve membrane per-
meability (Table 5). For example the amide linker of 24 was
capped off with a methyl group (35) or replaced with a phenyl
ether linker (36), changes which resulted in more than 20
fold losses in potency. A number of tertiary amides were
made (37 and 38 and 40), which continued the trend of large
potency losses seen for the tertiary sulfonamides. However,
we found that benzyl ether linkers were equipotent with the
amide linker, while being of similar lipophilicity. The ether
39 had an IC50 at Nav1.3 of 22 nM. Other expressions of an
ether linker were less successful, for example 41 in which we
had incorporated the ether into an azetidine-linked amide
which was some 10 fold weaker than the benzyl ether.
Its broader Nav selectivity profile was also improved with the
window over Nav1.1 now increased to 20 fold with good win-
dows retained over the other Nav channels Nav1.2, Nav1.7 and
Nav1.8. 39 showed no appreciable activity at the hERG channel.
Compound 39 had a measured pKa of 4.1, similar to that of
previous thiadiazoles investigated in this series. The com-
pound was of low clearance in the rat (2 mL min−1 kg−1, Vd 1 L
kg−1), of high bioavailability (F 73%) and had an elimination
half-life of some 7 h, a pharmacokinetic profile that was not
typical of other thiadiazoles in the series. It is interesting to
compare 39 to the previous thiadiazole 24. The latter com-
pound is larger and of higher TPSA and has very low oral bio-
availability compared to 39. It is likely that controlling molecu-
lar weight, H-bond count and TPSA in this series of acidic
compounds helps to modulate transporter affinity, particularly
within the more acidic thiadiazole series. Therefore, whilst the
thiadiazole head group typically brings poor oral
Further work on 39 investigated its physicochemical and
pharmacokinetic properties (Fig. 7). The compound showed
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4-(Benzylthio)-3-fluorobenzoic acid
3,4-Difluorobenzoic acid (50 g, 320 mmol), caesium carbonate
(206 g, 632 mmol) and benzyl mercaptan (37.4 mL, 320
mmol) were combined in DMSO (500 mL) and the reaction
mixture heated at 70 °C for 3 hours. The reaction mixture
was cooled to room temperature and then poured into water
(1.5 L) before extraction with EtOAc (2 × 750 mL). The aque-
ous was acidified to pH = 1 with 4N HCI (aq) and the precipi-
tate filtered and dried in vacuo to provide the title compound
as a pale pink coloured solid (84.7 g, 320 mmol). 1HNMR
(CDCI3): 4.2 (s, 2H), 7.3 (m, 6H), 7.75 (m, 2H). LCMS (0.05%
formic acid in MeCN) Rt = 1.55 min. MS m/z 261 [M − H]−.
Fig. 7 Profile of the optimised non-amino thiazole tool compound 39.
bioavailability, by careful control of physical and structural
properties, a well absorbed compound can be identified.
Being a moderately lipophilic acid, the free fraction in rat
of 39 was 0.00024. It was very selective in a broad panel of en-
zymes, channels and receptors (<35% inhibition across the
panel at 10 μM), although it retained inhibition of CYP2C9
(85% inhibition @ 3 μM) and CYP3A4 (75% inhibition @ 3
μM) in line with both 22 and 24.
Methyl 4-(benzylthio)-3-fluorobenzoate
Conclusions
Through a focussed SAR exercise starting from a previous Nav1.3
active compound 11, we were rapidly able to identify a potent
and selective and more aqueous soluble compound 39 devoid of
the toxicity-suspect amino-thiazole headgroup, which was well-
absorbed upon oral dosing. These properties gave a more attrac-
tive overall profile than our initial tool compound 22 in a non-
thiazole series with similar levels of Nav1.3 potency. Further
studies with compound 39 will be disclosed in due course.
A suspension of 4-(benzylthio)-3-fluorobenzoic acid (81.4 g,
0.310 mol) in MeOH (800 mL) and concentrated H2SO4 (2
mL) was heated at reflux for 36 hours. The mixture was
cooled to room temperature and the resulting precipitate fil-
tered and washed with hexane. The filtrate was concentrated
in vacuo and the resulting solid filtered and washed with hex-
ane. The combined solids were dried in vacuo to provide the
1
title compound as a white solid (60.9 g, 0.217 mol). HNMR
(CDCI3): 3.90 (s, 3H), 4.18 (s, 2H), 7.22–7.35 (m, 6H), 7.65–
7.72 (m, 2H).
Experimental
An example of the synthetic routes followed to prepare the
compounds contained herein is shown below for compounds
22 and 39.
Methyl 4-(chlorosulfonyl)-3-fluorobenzoate
Synthesis of compound 22
To a vigorously stirred solution of methyl 4-(benzylthio)-3-
fluorobenzoate (30.4 g, 0. 110 mol) in DCM (860 mL) and
4N HCI (aq) (860 mL) at 5 °C was added sodium hypochlo-
rite (445 mL) dropwise, keeping the temperature below 10
°C throughout. Upon complete addition the reaction mix-
ture was allowed to warm to room temperature over 1
hour. Two reactions were carried out on identical scale in
parallel (2 × 0.11 mol scale) and combined at this point for
work-up and purification. The layers were separated and
the aqueous extracted with DCM (500 mL). The combined
organics were washed with a 10% aqueous solution of so-
dium metabisulfite (2 × 200 mL), brine (200 mL), dried
(MgSO4) and concentrated in vacuo. The residue was puri-
fied via column chromatography on silica gel eluting with
hexane/EtOAc (95 : 5 to 4 : 1) to provide the title compound
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as a white solid (53.1 g, 0.210 mol). 1HNMR (CDCI3): 3.99
(s, 3H), 7.95–8.08 (m, 3H).
continued for 8 hours. The reaction was then cooled to
room temperature and diluted with brine (300 mL) and
TBME (300 mL). The organic layer was separated, dried
(Na2SO4) then concentrated in vacuo to provide the title
compound as a yellow oil (9.0 g, crude quant.). This mate-
rial was used crude for subsequent reactions. 1H NMR (d6-
DMSO): 3.71 (s, 2H), 7.37–7.41 (m, 1H), 7.44–7.48 (m, 1H),
7.63 (d, 1H).
Methyl 3-fluoro-4-[(1,3-thiazol-2-ylamino)sulfonyl]benzoate
To a solution of 2-aminothiazole (105.2 g, 1.05 mol) in pyri-
dine (250 mL) was added methyl 4-(chlorosulfonyl)-3-
fluorobenzoate (53.1 g, 0.21 mol) and the mixture stirred at
50 °C for 18 hours. The reaction was then allowed to cool to
room temperature and added portionwise into 2N HCI (aq)
(300 mL) and the mixture acidified to pH 1 with 4N HCI (aq).
The resulting precipitate was filtered, washed with water and
dried in vacuo to provide the title compound as a tan
coloured solid (31.97 g, 0.10 mol). This material was used
crude for subsequent reactions. 1HNMR (d6-DMSO): 3.88 (s,
3H), 6.88 (d, 1H), 7.29 (d, 1H), 7.77–7.99 (m, 3H).
N-[3-Chloro-4-(trifluoromethoxy)benzyl]-3-fluoro-4-[(1,3-
thiazol-2-ylamino)sulfonyl]benzamide, compound 22
To
a
suspension of 3-fluoro-4-[(1,3-thiazol-2-ylamino)-
sulfonyl]benzoic acid (2.88 g, 9.54 mmol) and 1-[3-chloro-4-
(trifluoromethoxy)phenyl]methanamine (3.00 g, 11.4 mmol)
and Et3N (4.81 ml, 34.5 mmol) in DMSO (30 mL) was
added HBTU (4.34 g, 34.5 mmol) and the mixture stirred
at room temperature for 18 hours. The reaction was then
diluted with EtOAc (100 ml) before washing with water
(100 mL) and brine (3 × 100 mL). The EtOAc layer was
dried (Na2SO4), filtered and concentrated in vacuo leaving
a brown solid (6.38 g). The crude product was triturated
with MTBE, dried in vacuo and stirred in EtOAc for 20
minutes and the resulting off-white solid filtered off and
dried in vacuo overnight to provide the title compound
(3.97 g, 7.8 mmol). 1H NMR (d6-DMSO): 4.50 (d, 2H),
6.90 (d, 1H), 7.30 (d, 1H), 7.40 (d, 1H), 7.50 (d, 1H), 7.60
(s, 1H), 7.75 (s, 1H), 7.80 (d, 1H), 7.90 (t, 1H), 9.25 (t,
1H). LCMS (0.05% formic acid in MeCN) Rt = 1.49 min.
MS m/z 510 [M + H]+.
3-Fluoro-4-[(1,3-thiazol-2-ylamino)sulfonyl]benzoic acid
To
a
suspension of methyl 3-fluoro-4-[(1,3-thiazol-2-
ylamino)sulfonyl] benzoate (32.0 g, 0.101 mol) in dioxane : wa-
ter (1 : 1, 150 mL) at 5 °C was added lithium hydroxide (12.1 g,
0.505 mol). The mixture was allowed to warm to room temper-
ature and stirred for 4.5 hours. The dioxane was then removed
in vacuo and the aqueous washed with EtOAc (2 × 100 mL).
The aqueous was acidified by addition to 2N HCI (aq) (1000
mL) and the resulting precipitate filtered and washed with wa-
ter. Purification was accomplished via recrystallisation from
DCM : MeOH (10 : 1) to provide the title compound as a tan
coloured solid (12.9 g, 0.043 mol). 1HNMR (d6-DMSO): 6.88
(d, 1H), 7.29 (d, 1H), 7.72–7.98 (m, 3H). LCMS (0.05% formic
acid in MeCN) Rt = 1.44 min. MS m/z 303 [M + H]+.
Synthesis of phosphate prodrug 23
1-[3-Chloro-4-(trifluoromethoxy)phenyl]methanamine
To a solution of 3-chloro-4-(trifluoromethoxy)benzyl bromide
(10.0 g, 34.5 mmol) in DMF (100 mL) was added
phthalimide (5.6 g, 38.0 mmol) and potassium carbonate
(7.15 g, 51.8 mmol) and the mixture stirred at 80 °C for 5
hours. The mixture was cooled and treated with water (100
mL) and the resulting white precipitate filtered, washed
with water and dried in vacuo. This crude solid (16 g) was
suspended in methylamine (100 mL of a 40% aqueous so-
lution) and water (100 mL) and heated in a steel bomb at
100 °C for 3 hours. Additional methylamine (100 mL of a
40% aqueous solution) was added and heating at 100 °C
Compound 12 (294 mg, 0.58 mmol) was dissolved in 6 mL
DMF and cesium carbonate (595 mg, 1.7 mmol) and di-tert-
butyl chloromethyl-phosphate (242 mg, 0.94 mmol) added to
the mixture. The whole was stirred for 18 h at 60 °C, allowed
to cool to room temperature, diluted with EtOAc and then
washed with saturated NaHCO3. The mixture was extracted
with DCM and the combined organic layers were dried over
MgSO4, filtered and evaporated to give a yellow oil. Purifica-
tion using automated flash column chromatography with
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0–10% MeOH/DCM as eluant gave the product as a pale
yellow oil. This was dissolved in 5 mL DCM and 1 mL
TFA, and then stirred for 2 h. The mixture was then puri-
fied directly by preparative HPLC using MeCN and H2O as
the mobile phase to give a white solid of the title com-
pound (102 mg, 59%). 1HNMR (d4-MeOH): 4.61 (s, 2H),
5.78 (d, 2H), 6.87 (m, 1H), 7.42 (m, 3H), 7.80 (m, 1H),
7.78 (m, 2H), 8.09 (m, 1H). 19FNMR (d4-MeOH): 60.0.
LCMS (0.05% formic acid in MeCN) Rt = 1.16 min. MS m/
z 618 [M − H].
4-((3-Chloro-4-(trifluoromethoxy)benzyl)oxy)benzene-1-sulfo-
nyl chloride
To a solution of 4-((3-chloro-4-(trifluoromethoxy)benzyl)oxy)-
benzenesulfonic acid (1.80 g, 4.70 mmol) in dichloromethane/
dimethylformamide (50 mL/5 mL) was added thionyl chloride
(2.88 mL, 47 mmol) dropwise at room temperature. The reac-
tion mixture was stirred at room temperature for 16 hours then
concentrated in vacuo and the solid remaining was dried with
a high vacuum pump for 3 hours to afford the title compound
as a green solid (4.1 g) which was taken on crude into the next
step. LCMS (0.05% formic acid in MeCN) Rt = 4.03 min.
Synthesis of compound 39
4-((3-Chloro-4-(trifluoromethoxy)benzyl)oxy)-N-(2,4-dimethoxy-
benzyl)-N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide
To a solution of N-(2,4-dimethoxybenzyl)-1,2,4-thiadiazol-5-
amine (714 mg, 3.0 mmol) in anhydrous dry tetrahydrofuran
(15 mL) at −60 °C was added a solution of sodium hexa-
methyldisilazane (1 M in tetrahydrofuran, 3.0 mL, 3.0 mmol)
and the reaction mixture was allowed to warm up to 0 °C and
stirred for 1 hour. The reaction mixture was then cooled to
−60 °C and a solution of 4-((3-chloro-4-(trifluoromethoxy)-
benzyl)oxy)benzenesulfonic acid (1.0 g, 2.5 mmol) in
tetrahydrofuran/dimethyformamide (5 mL/1 mL) was added
dropwise and the reaction mixture was allowed to warm up
and stir at room temperature for 16 hours. An aqueous solu-
tion of ammonium chloride (20 mL) was added to the reac-
tion mixture and the organics were extracted into ethylacetate
(3 × 20 mL). The combined organic phases were washed with
brine (3 × 30 mL), dried over MgSO4 filtered and evaporated
to give the title compound as a yellow oil (860 mg). This ma-
terial was taken to the final deprotection step without any
further purification.
4-((3-Chloro-4-(trifluoromethoxy)benzyl)oxy)benzenesulfonic
acid
To a solution of sodium 4-hydroxybenzenesulfonate (836 mg,
3.60 mmol) in aqueous sodium hydroxide solution (1 M, 5.0
mL) at room temperature was added (dropwise)
4-(bromomethyl)-2-chloro-1-(trifluoromethoxy)benzene (1.0 g,
3.60 mmol) in ethanol (5 mL). The reaction mixture was
heated up to 100 °C and allowed to stir for 16 hours. It was
then cooled to room temperature and an aqueous solution of
hydrogen chloride (2 M, 10 mL) was added (pH = 1). The or-
ganic phase was extracted into ethyl acetate (3 × 10 mL),
these were combined and washed with brine (2 × 20 mL),
dried over MgSO4, filtered and evaporated to afford the title
compound as a white solid (1.80 g, 0.0047 mol). 1HNMR
(CDCl3): 5.10 (s, 1H), 6.55 (d, 2H), 7.20 (br s, 1H), 7.45 (d,
2H), 7.50–7.60 (m, 2H), 7.85 (d, 1H). 19FNMR (CDCl3) −57 (s).
LCMS (0.05% formic acid in MeCN) Rt = 2.63 min. MS m/z
381 [MH]+.
4-((3-Chloro-4-(trifluoromethoxy)benzyl)oxy)-N-(1,2,4-
thiadiazol-5-yl)benzenesulfonamide, compound 39
To a solution of 4-((3-chloro-4-(trifluoromethoxy)benzyl)oxy)-N-
(2, 4-dim ethoxybenzyl)-N -(1, 2, 4-thiadiazol- 5-yl)-
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benzenesulfonamide (860 mg, 1.40 mmol) in dioxan (3 mL) at
room temperature was added dropwise, hydrogen chloride in
dioxan (4 M, 3 mL). The reaction mixture was stirred at room
temperature for 2 hours. The solvent was then evaporated in
vacuo and the crude oil was purified by reverse phase chro-
matography (eluting with acetonitrile/water + 0.1% formic
acid, 5% to 95% gradient) followed by HTAP using basic con-
ditions to afford the title compound as the diethylamine salt;
white solid (18.9 mg, 0.04 mmol). 1HNMR (d4-MeOH): 1.21
(t, 2H), 3.0 (t, 1H), 5.15 (s, 2H), 7.05 (m, 2H), 7.40 (d, 1H),
7.45 (d, 1H), 7.65 (s, 1H), 7.82 (d, 2H), 7.95 (s, 1H). 19FNMR
(d4-MeOH): −60.0. LCMS (0.05% formic acid in MeCN) Rt =
2.90 min. MS m/z 464 [MH]+.
mega ohms and >85% series resistance compensation (50%
for PatchXpress) was routinely achieved. As a result, voltage
errors were negligible and no correction was applied. Current
records were acquired at 20 to 50 kHz and filtered at 5 to 10
kHz. HEK cells stably transfected with hSCN3A were viewed
under Hoffman contrast optics and placed in front of an ar-
ray of flow pipes emitting either control or compound-
containing extracellular solutions. All compounds were
dissolved in dimethyl sulfoxide to make 10 mM stock solu-
tions, which were then diluted into extracellular solution to
attain the final concentrations desired. The final concentra-
tion of dimethyl sulfoxide (<0.3% dimethyl sulfoxide) was
found to have no significant effect on hSCN3A sodium cur-
rents. The voltage-dependence of inactivation was deter-
mined by applying a series of depolarizing prepulses (8 s long
in 10 mV increments) from a negative holding potential. The
voltage was then immediately stepped to 0 mV to assess the
magnitude of the sodium current. Currents elicited at 0 mV
were plotted as a function of prepulse potential to allow esti-
mation of the voltage midpoint of inactivation (V1/2). Cells
were then voltage clamped at the empirically determined
compounds were tested for their ability to inhibit hSCN3A so-
dium channels by activating the channel with a 20 ms voltage
step to 0 mV following an 8 second conditioning prepulse to
the empirically determined V1/2. Compound effect (% inhibi-
tion) was determined by difference in current amplitude be-
fore and after application of test compounds. For ease of
comparison, “estimated IC50” (EIC50) values were calculated
from single point electrophysiology data by the following
equation, (tested concentration, μM) × (100% inhibition/%
inhibition). Inhibition values <20% and >80% were excluded
from the calculation. In some cases electrophysiological as-
says were conducted with PatchXpress 7000 hardware and as-
sociated software (Molecular Devices Corp). All assay buffers
and solutions were identical to those used in conventional
whole-cell voltage clamp experiments described above.
hSCN3A cells were grown as above to 50–80% confluency and
harvested by trypsinization. Trypsinized cells were washed
and resuspended in extracellular buffer at a concentration of
1 × 106 cells per ml. The onboard liquid handling facility of
the PatchXpress was used for dispensing cells and applica-
tion of test compounds. Determination of the voltage mid-
point of inactivation was as described for conventional
whole-cell recordings. Cells were then voltage-clamped to the
empirically determined V1/2 and current was activated by a 20
ms voltage step to 0 mV. Electrophysiological assays were also
conducted using the lonworks Quattro automated electro-
physiological platform (Molecular Devices Corp). Intracellular
and extracellular solutions were as described above with the
following changes, 100 mg ml−1 amphotericin was added to
the intracellular solution to perforate the membrane and al-
low electrical access to the cells. hSCN3A cells were grown
and harvested as for PatchXpress and cells were resuspended
in extracellular solution at a concentration of 3–4 × 106 cells
per ml. The onboard liquid handling facility of the lonworks
Quattro was used for dispensing cells and application of test
Cell line construction and maintenance
Human embryonic kidney (HEK) cells were transfected with
an hSCN3A construct using lipofectamine reagent
(Invitrogen), using standard techniques. Cells stably express-
ing the hSCN3A constructs were identified by their resistance
to G-418 (400 μg ml−1). Clones were screened for expression
using the whole-cell voltage-clamp technique.
Cell culture
HEK cells stably transfected with hSCN3A were maintained
in DMEM medium supplemented with 10% heat-inactivated
fetal bovine serum and 400 μg ml−1 G418 sulfate in an incu-
bator at 37 °C with a humidified atmosphere of 10% CO2.
For HTS, cells were harvested from flasks by trypsinization
and replated in an appropriate multi-well plate (typically 96
or 384 wells per plate) such that confluence would be
achieved within 24 hours of plating. For electrophysiological
studies, cells were removed from the culture flask by brief
trypsinization and replated at low density onto glass cover
slips. Cells were typically used for electrophysiological experi-
ments within 24 to 72 h after plating.
Electrophysiological recording
Cover slips containing HEK cells expressing hSCN3A were
placed in a bath on the stage of an inverted microscope and
perfused (approximately 1 ml min−1) with extracellular solu-
tion of the following composition: 138 mM NaCI, 2 mM
CaCI2, 5.4 mM KCI, 1 mM MgCI2, 10 mM glucose, and 10
mM HEPES, pH 7.4, with NaOH. Pipettes were filled with an
intracellular solution of the following composition: 135 mM
CsF, 5 mM CsCI2, 2 mM MgCI2, 10 mM EGTA, 10 mM
HEPES, pH 7.3 to 7.4, and had a resistance of 1 to 2 mega
ohms. The osmolarity of the extracellular and intracellular
solutions was 300 mmol kg−1 and 295 mmol kg−1, respec-
tively. All recordings were made at room temperature (22–24
°C) using AXOPATCH 200B amplifiers and PCLAMP software
(Axon Instruments, Burlingame, CA) or PatchXpress 7000
hardware and associated software (Axon Instruments, Burlin-
game, CA). hSCN3A currents in HEK cells were measured
using the whole-cell configuration of the patch-clamp tech-
nique. Uncompensated series resistance was typically 2 to 5
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compounds. A voltage protocol was then applied that com- Acknowledgements
prised of a voltage step to fully inactivate the sodium chan-
The authors would like to thank Kiyoyuki Omoto and Andy
Pike for helpful discussions during the course of these
studies.
nels, followed by a brief hyperpolarized recovery period to al-
low partial recovery from inactivation for unblocked sodium
channels, followed by a test depolarized voltage step to assess
magnitude of inhibition by test compound. Compound effect
was determined based on current amplitude difference be-
tween the pre-compound addition and post-compound addi-
tion scans.
Notes and references
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All experimental procedures were conducted in accordance
with UK Animals (Scientific Procedures) Act 1986 and
followed the guidelines of the International Association for
the Study of Pain. All studies were reviewed and approved
by the internal Pfizer Institutional Animal Care and Use
committee prior to any in vivo experiments commencing.
Animals received surgery for tibial nerve transection (TNT) 2
to 3 weeks prior to recordings. All surgical procedures were
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parylene-coated tungsten electrodes (A-M Systems, Sequim,
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Gene expression data
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