Discovery and optimisation of potent and highly subtype selective Nav1.8 inhibitors with reduced cardiovascular liabilities

Article abstract of DOI:10.1039/c6md00281a

Voltage-gated sodium channels, in particular Na_v1.8, can be targeted for the treatment of neuropathic and inflammatory pain. Herein, we describe the discovery and optimisation of a Na_v1.8 inhibiting phenyl imidazole series that delivers chemical equity that possesses high potency and selectivity and is capable of demonstrating good oral pharmacokinetics.

Full text of DOI:10.1039/c6md00281a

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K. Omoto and J. S. Warmus, Med. Chem. Commun., 2016, DOI: 10.1039/C6MD00281A.
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DOI: 10.1039/C6MD00281A
Discovery and optimisation of potent and highly
subtype selective Nav1.8 inhibitors with reduced
cardiovascular liabilities
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Sharan K. Bagal,*,a Mark I. Kemp,b Peter J. Bungay,c Tanya L. Hay, d Yoshihisa Murata,b C.
Elizabeth Payne, e Edward B. Stevens,e Alan Brown,a David C. Blakemore, a Matthew S.
Corbett,f Duncan C. Miller,b Kiyoyuki Omoto,a Joseph S. Warmus.f
www.rsc.org/
Voltage-gated sodium channels, in particular Nav1.8, can be targeted for the treatment of neuropathic and inflammatory
pain. Herein is described the discovery and optimisation of a Nav1.8 inhibiting phenyl imidazole series that delivers
chemical equity that possesses high potency and selectivity and is capable of demonstrating good oral pharmacokinetics.
nociceptors,5,6 and its expression and function is modulated by
agents that cause pain.7,8 Genetic ablation of NaV1.8 in
rodents results in deficits in nociception following
Introduction
Voltage-gated sodium channels (Nav) are members of the ion
channel family and are composed of a transmembrane
subunit of approximately 260kDa with associated
transmembrane -subunits of lower molecular weight. The
inflammation, but not neuropathic pain,9,10,11,12 while recent
human genetic evidence suggest that gain of function
mutations in NaV1.8 contribute to painful peripheral
neuropathy.13 A-803467 is a selective Nav1.8 inhibitor that has
been used to demonstrate a link between Nav1.8 knockdown
and inflammatory and neuropathic pain (Figure 1). However,
A-803467 exhibits poor oral pharmacokinetics in preclinical
species.14 We recently described the optimisation of Nav1.8
modulator series to deliver subtype selective, state and use
dependent chemical matter. This work culminated in the
identification of PF-04531083 and PF-01247324 whereby both
compounds were shown to be efficacious in preclinical models
of neuropathic and inflammatory pain (Figure 1).15 The present
article discusses the discovery of a novel subtype selective
Nav1.8 series identified via pharmacophore based screening.
This series has an improved potency, selectivity and solubility
profile when compared with PF-04531083 and PF-01247324.
α
-
β
family is comprised of nine members Nav1.1-Nav1.9 which can
be subdivided into tetrodotoxin-sensitive (TTX-S) and
tetrodotoxin-resistant (TTX-R) subtypes. Navs play a key role in
controlling excitability of neurons by regulating the threshold
of firing, underlying the upstroke of the action potential and
controlling the duration of interspike interval.1 Non-selective
Nav blockers (e.g. lamotrigine, lacosamide and mexilitine) have
been successfully used in the clinic to treat pathological firing
patterns of neurons that occur in a range of conditions such as
chronic pain and epilepsy. However, such drugs have a narrow
therapeutic window due to inhibition of sodium channels in
the heart and throughout the central nervous system.
Selective block of Nav channels as pain targets gained traction
with the recognition that some Nav subtypes showed
preferential or exclusive expression in peripheral sensory
neurons. A number of studies have implicated Nav1.3, 1.7, 1.8
and 1.9, which are expressed in dorsal root ganglion (DRG)
neurons and trigeminal neurons, in nociceptive processing.2,3,4
Nav1.8 is highly (but not exclusively) expressed in
Results
Discovery and optimisation of lead matter
Structural analysis of existing subtype selective NaV1.8
inhibitors suggested 5,6- and 6,6- biaryl motifs with at least
one hydrogen bond donor (HBD) may have a selective NaV1.8
inhibition profile (Figure 1). Based on this hypothesis, file
screening of 5,6- and 6,6- biaryl motifs residing in rule of five
physicochemical space and having ≥ 1 HBD was conducted.
Data generated in recombinantly expressed hNav1.8/β1
(Merck Millipore) led to the identification of oxadiazoles 1a
and 1b (Scheme 1).
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ARTICLE
Journal Name
At this stage analogue
4
was used as a scaffold to identify the
optimal amine fragment expression, the data for which are
shown in Table 1. Amines substituted with lipophilic groups
e.g. 4-7 and polar substituents e.g.
increased LipE relative to . There was no significant difference
in hNav1.8 potency between enantiomers when small lipophilic
groups were appended e.g. 2a/b 6-7. Phenyl imidazoles such
as , can be synthesised from β-amino acids leading to a 2
9-10 were tolerated and
4
,
4
carbon spacer between the imidazole and amine (Scheme 2).
Synthesis using α-amino acids led to a 1 carbon spacer
(Scheme 2). The latter compounds were very potent and LipE
Figure 1. Subtype selective Nav1.8 inhibitors.14,15
efficient e.g. as shown by comparison of
lower cLogP (2.7) and a higher potency (IC50 = 0.053 µM)
resulting in > 1 unit LipE enhancement when compared with
5 and 13. 13 has a
5
.
For a single carbon spacer, if larger lipophilic moieties were
appended a 10-fold difference between eutomer and distomer
emerged e.g. 11-12, suggesting that whilst there is a large
binding pocket in this region it has size and directionality
limitations.
Gem dimethyl derivative 13 was profiled against other sodium
channel subtypes in order to assess subtype selectivity (Table
2). Pleasingly, 13 was 80-500-fold selective over the other
sodium channel subtypes. However 13 was also active at the
human Ether-à-go-go-Related Gene (hERG) ion channel in the
patch express (PX) assay (hERG PX IC50 = 2.6 µM) leading to a
hNav1.8 selectivity of ca. 50-fold over hERG. 13 was incubated
in human liver microsomes (HLM) and hepatocytes (hHep) in
order to understand metabolic liability. Low intrinsic clearance
(CLint) was observed (HLM CLint <8 uL/min/mg, n = 4; hHep CLint
<6 uL/min/million cells, n = 2) which predicted low metabolic
systemic blood clearance (CL) in human (<5ml/min/kg) and
corresponding oral bioavailability of >75%. When combined
with in silico prediction of volume of distribution at steady
state (Vss) effective half-life in human was predicted to be ~24
hours.
Scheme 1. Phenyl oxadiazole and imidazole Nav1.8 inhibitors. IC50 values were
determined at recombinantly expressed hNav1.8/β1 (Merck Millipore) using
manual patch clamp electrophysiology with a conditioning pulse at the V0.5 of
inactivation. IC50 data were generated with at least 2 tests on 2 different assay
runs.
Oxadiazoles 1a and 1b were low µM inhibitors of hNav1.8 but
were non-selective over the cardiac ion channel hNav1.5.
Modification of the oxadiazole core to imidazole improved
hNav1.8 selectivity over hNav1.5 to ~ 20-fold as exemplified by
2a. Based on hNav1.8 potency and selectivity the phenyl
imidazole series was prioritised over the phenyl oxadiazole
series. Variation of the chlorophenyl ring of 2a identified p-CN
and p-OCF3 phenyl as suitable replacements (Figure 2). When
Optimisation of hERG liability
The profile of compound 13 suggested that the phenyl
imidazole series could deliver an acceptable combination of
potency, selectivity and pharmacokinetics. However, the hERG
selectivity needed improvement with an ideal therapeutic
window ≥ 300-fold.16 13 is a basic compound with a measured
pKa of 7.9. In order to improve hERG liability a fluorescence
polarisation (FP) competition assay was used because it had a
higher throughput when compared with the hERG PX assay.17
The hERG FP assay has been shown to be predictive of
compound QT prolongation effects via hERG blockade and can
be run in a high throughput 384-well plate manner.17 In this
assay 13 had a hERG FP IC50 of 1.8 µM giving an apparent
therapeutic index of ~34-fold. There are a number of known
methods to reduce hERG liability,18 one of which is removal of
the basic centre. A number of structurally related neutral
compounds were prepared 14-17 which reduced the hERG FP
potency to an IC50 >16 µM. However there was a concomitant
> 20-fold loss in hNav1.8 potency (IC50 >1 µM). Another known
method to reduce hERG liability is via discrete structural
modifications to aryl units which are thought to interact with a
compared with 2a p-CN analogue
3 was weaker at hNav1.8 but
had a higher LipE whereas the p-OCF3 derivative
potent with a lower LipE.
4 was more
Figure 2. Variation of the phenyl imidazole aryl ring. IC50 values were determined
at recombinantly expressed hNav1.8/β1 (Merck Millipore) using manual patch
clamp electrophysiology with a conditioning pulse at the V0.5 of inactivation. IC50
data were generated with at least 2 tests on 2 different assay runs.
2 | J. Name., 2012, 00, 1-3
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key phenylalanine and/or tyrosine at the hERG ion channel.18 unique binding mode was identified (data not shown). This
Modification of the p-OCF3 phenyl group in 13 to a range of suggested that the phenyl imidazole compounds described in
aryl units did indeed reduce the hERG FP potency although this work are bound to the fenestration site. Interestingly, the
there was always an associated loss in hNav1.8 potency such fenestration site has been suggested to be a hNav1.8 inhibitor
that the hERG selectivity did not improve sufficiently. Some binding site in the literature. Browne et al. reported that site-
representative examples of aryl variations are given in Table 3. directed mutagenesis of F1710, which constitutes the
If the imidazole core was varied to N-aryl linked imidazole or a fenestration site, to an alanine residue decreased the
triazole there was a substantial reduction in potency in both dissociation constants of Tetracaine and A-803467 with
the hERG FP assay (IC50 >16 µM), and hNav1.8 assay (IC50 >1 hNav1.8 by 3- and 6- fold, respectively.22 The F1710 residue is
µM). Based on the SAR generated thus far it appeared that the conserved across all mammalian sodium channel subtypes and
hNav1.8 pharmacophore required the p-OCF3 aryl unit, a significant drop in affinity or activity of sodium channel
imidazole core and basic centre for adequate activity at inhibitors, such as Etidocaine,23 Lidocaine,24 and Tetracaine,25
hNav1.8. One region remaining that could be exploited to by mutation of this residue has been reported in several
reduce hERG liability was the gem dimethyl groups. These publications. Wallace et al. have recently published the X-ray
methyls could be modified to reduce the pKa of the amine in co-crystal structure of a hNav1.8 inhibitor PI1 with the NavMS
13 (pKa 7.9), thereby reducing hERG activity. Appending one bacterial sodium channel which suggested that PI1 is bound to
or two F atoms as in 18 and 19 respectively, reduced the pKa the fenestration site.21 PI1 has the phenyl imidazole scaffold
significantly from 7.9 in 13 to 6.4 and 5.9 respectively (Table with significant structural similarity to the ligands described in
1). Gratifyingly, both these fluorinated derivatives were 10- this manuscript, supporting the binding site hypothesis made
fold weaker at the hERG channel (hERG FP 18 IC50 14 µM and from the docking simulations.
19 IC50 15 µM). However, both these fluorinated derivatives A potential binding mode for 13 in hNav1.8 is illustrated in
also had > 10-fold reduced activity on hNav1.8 leading to Figure 3. The binding site extends from the fenestration site to
weaker compounds with no improvement in hERG therapeutic the p-loop and the selectivity filter. Backbone carbonyl oxygen
index. The methyl ether derivative 20 appeared more atoms in the selectivity filter from Glu355, Thr1659 and
promising in that this structural change was tolerated by Thr1365 form hydrogen bonds with the protonated amine in
hNav1.8 (IC50 = 0.084 µM). 20 was weakly basic at physiological the R1 fragment and the imidazole NH and this might
pH having measured pKa 7.1 although the reduction in hERG contribute to the significant activity of 13 (hNav1.8 IC50 0.053
liability was modest at best (hERG FP IC50 3.7 µM). Cyclisation µM). Similar hydrogen bonds with the p-loop or selectivity
of the methyl ether to give an oxetane derivative led to the filter have been reported for interactions of inhibitors with
identification of 21. This compound retained hNav1.8 activity other voltage-gated ion channels (Kv, Nav, and Cav).26-34 This
(IC50 0.047 µM), and had a measured pKa 6.4 which led to a docking model was also consistent with the lower activity of 14
hERG FP IC50 14 µM and an apparent therapeutic index of (hNav1.8 IC50 > 1 µM) that lacks the hydrogen bond donor in
~300-fold. Moreover, 21 was moderately lipophilic (cLogP 2.3) R1. There are very few hydrophobic residues around the
resulting in a respectable LipE of 5. The selectivity and LipE selectivity filter and the t-Bu group in R1 is unable to make an
parameters of 21 were noteworthy improvements when effective interaction with the protein. The lower activity of
compared with 13 (apparent hERG FP therapeutic index 34- alcohol 15 (hNav1.8 IC50 > 1 µM) can probably be explained by
fold, cLogP 2.7, LipE 4.6). Oxetane derivative 21 was profiled the alcohol being a weaker hydrogen bond donor when
against the other sodium channel subtypes along with hERG PX compared with protonated amines.
There are other
in order to assess selectivity (Table 2). Pleasingly, 21 was ca. unsatisfied hydrogen bond acceptors in the selectivity filter
350-fold selective over the other sodium channel subtypes and (for example, backbone carbonyls of Cys847 and Gly848) and
ca. 200-fold selective over the hERG channel (hERG PX IC50 10 this may have allowed the moderate structural changes in R1
µM). 21 was screened against additional ion channels including such as carbon insertion or cyclization without a significant
KCNQ1, GABA, CaV1.2 and CaV3.2 where it exhibited minimal change in hNav1.8 IC50. Such modifications would alter the
activity (% inhibition ˂ 40% at 10 µM). Pharmacokineꢀc (PK) orientations and positions of the R1 amines and weaken the
studies in rat with 21 demonstrated low blood CL original hydrogen bond interactions of 13, but can create new
(~5ml/min/kg) with oral bioavailability of 49-52% (n = 2).
interactions with other hydrogen bond acceptors in the
selectivity filter. The p-OCF3 aryl unit of 13 was positioned in
the centre of the fenestration site which was formed by four
residues (L1361, T1365, I1706, and F1710), with a significant
π−π stacking interaction with F1710 and van der Waals
interactions with L1361, T1365 and I1706. Similar models have
been proposed by Tikhonov et al. for the interaction between
Docking simulation analysis
Docking simulation analysis was applied in order to understand
the hNav1.8 protein-ligand interaction mode. As far as we are
aware, there are three binding sites for small molecule sodium
channel inhibitors, the pore cavity,19 the voltage-sensor
domain,20 and the fenestration site.21 Docking analysis was
applied to all of the three binding sites, however only docking
poses of the fenestration site showed consistency. Docking
poses of the other two binding sites were diverse and no
Tetracaine and hNav
1.4.35 In their docking model, the phenyl
core of Tetracaine made hydrophobic interactions with F1586,
(equivalent to F1710 of Nav1.8).
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Figure 3. Docking pose of 13 in a human Nav1.8 homology model. The four
domains, D1, D2, D3, and D4 are coloured blue, purple, pink, and yellow,
respectively.
Compounds 13 and 21 exhibited species based differential
activity at Nav1.8. 13 and 21 did not inhibit native TTX-R
currents in rodent DRG neurons at 10 µM (mouse and rat), but
did inhibit native TTX-R currents in cynomolgus monkey DRG
neurons at 10 µM (Table 2). The lack of Nav1.8 activity in
rodent precluded preclinical in vivo efficacy studies in these
species. Based on the favourable Nav1.8 potency, LipE,
selectivity, in vivo PK, and in vitro cardiovascular risk profile
oxetane derivative 21 was selected as a pre-candidate for
further progression.
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hNav1.8 IC50
(µM)
hNav1.8 IC50
(µM)
Cmpd
R1
cLogP
4.0
LipE
2.4
3.5
3.9
4.0
Cmpd
13
R1
cLogP
2.7
LipE
4.6
NA
NA
NA
4
5
6
7
0.39
0.23
0.32
0.26
0.053
>1
3.1
14
4.8
2.6
15
>1
2.9
H
N
O
2.6
16
>1
2.7
H
N
O
8
9
0.62
1.6
3.3
0.7
2.9
5.1
17
18
>1
2.5
2.6
NA
3.5
NH2
0.79
10
3.2
1.6
3.9
19
>1
2.3
NA
11
12
0.036
0.34
3.8
3.8
3.6
2.7
20
21
0.084
0.047
2.4
2.3
4.7
5.0
Table 1. Amine fragment SAR observed in the phenyl imidazole series. IC50 values were determined at recombinantly expressed hNav1.8/β1 (Merck Millipore) using
manual patch clamp electrophysiology with a conditioning pulse at the V0.5 of inactivation. IC50 data were generated with at least 2 tests on 2 different assay runs.
cLogP was calculated using BioByte program version 4.3.
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hNav1.8
IC50
hNav subtype
selectivity
TTX-R Rat
DRG IC50
TTX-R Cyno
DRG IC50
hERG PX
IC50
Cmpd
Nav1.1 11 µM
Nav1.2 16 µM
Nav1.5 27 µM
Nav1.6 4.2 µM
Nav1.7 7.0 µM
Nav1.1 43 µM
Nav1.2 17 µM
Nav1.5 36 µM
Nav1.6 46 µM
Nav1.7 24 µM
13
0.053 µM
0.047 µM
>10 µM
>10 µM
0.132 µM
0.070 µM
2.6 µM
10 µM
21
Table 2. hNav1.8 potency (IC50) and selectivity (IC50) for 13 and 21. IC50 values for 13 and 21 at recombinantly expressed hNav1.8
/
β1 (Merck Millipore) and at TTX-R
in rat and cynomologous monkey (Cyno) DRG were determined using manual patch clamp electrophysiology. hNav subtype selectivity for 13 and 21 was measured
using manual patch clamp (Nav1.1, Nav1.2, Nav1.6 and Nav1.8) or PatchXpress (Nav1.5 and Nav1.7) electrophysiology. IC50 values determined using patch clamp
electrophysiology were determined at the respective V0.5 of inactivation for TTX-R and each channel isoform. IC50 data were generated with at least 2 tests on 2
different assay runs.
Cmpd
13
Ar
hNav1.8 IC50 (µM)
cLogP
2.9
hERG FP IC50 (µM)
hERG FP / hNav1.8
4-(trifluoromethoxy)phenyl
Phenyl
0.053
> 1.0
0.71
> 1.0
2.2
1.8
> 16
> 16
> 16
9.0
34
-
22
1.7
23
4-chlorophenyl
2.5
> 23
-
24
4-(methylsulfonyl)phenyl
3-(trifluoromethoxy)phenyl
4-methoxyphenyl
0.1
25
2.9
4
26
1.0
1.8
> 16
> 16
Table 3. Aryl unit SAR observed in the phenyl imidazole series. IC50 values were determined at recombinantly expressed hNav1.8/β1 (Merck Millipore) using manual
patch clamp electrophysiology with a conditioning pulse at the V0.5 of inactivation. IC50 data were generated with at least 2 tests on 2 different assay runs. cLogP was
calculated using BioByte program version 4.3. hERG measured using a fluorescence polarisation assay using a Cy3B tagged ligand that binds to the hERG product
expressed in HEK-293S cells.36
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Synthesis
good potency, excellent subtype selectivity and in vivo PK but
suffered from an insufficient therapeutic index over the hERG
ion channel. Through a combination of reducing amine pKa,
sterically hindering the amine and introducing cyclic
conformational restraints, an acceptable potency and
Scheme 2 demonstrates a general conversion of α- and β-
amino acids to give phenyl imidazoles with a 1 or 2 carbon
spacer between the imidazole and amine respectively. In this
sequence, a bromoketone is reacted with the carboxylic acid of
the amino acid to form a ketoester that is cyclised with
selectivity profile was achieved with oxetane derivative 21
.
ammonium acetate in toluene (or xylene) at reflux.37, The
38
Oxetane 21 was progressed to preclinical toxicology studies
and further development data will be reported in due course.
amino group of the amino acid is protected with a tert-
butyloxycarbonyl (Boc) or carboxybenzyl (CBZ) group in this
reaction. This cyclisation approach proved both broad in
scope and straightforward to carry out and its use is illustrated
in the synthesis of analogue 21 as shown in Scheme 3.
Starting from oxetan-3-one, Horner-Wadsorth-Emmons
Notes
All studies with animals were conducted in compliance with
appropriate national regulations and subject to review by the
Pfizer Animal Ethics Committee which includes external
members.
reaction gave the enoate 28
.
Subsequent reaction with
CBZ
ammonia in methanol gave the beta-amino acid 29
.
protection of the amine was followed by ester hydrolysis to
give aminoacid 30. This was reacted with bromoketone 31 to
give the cyclisation precursor, 32. Ketoester 32 was refluxed
with ammonium acetate in toluene forming the desired
imidazole 33. Deprotection of the CBZ protected amine gave
Abbreviations
Boc, tert-butyloxycarbonyl; Cmpd, compound; CBZ,
Carboxybenzyl; hERG, human Ether-à-go-go-Related Gene
product; hERG PX, hERG patch express assay; hERG FP, hERG
fluorescence polarisation assay; CL, Clearance; DRG, dorsal
root ganglion neuron; HBD, hydrogen bond donor; i.v.,
intravenous; L/Kg, litres per kilogram; μg/mL, microgram per
millilitre; PK, Pharmacokinetics; TTX-S, tetrodotoxin-sensitive;
TTX-R, tetrodotoxin-resistant. LipE, lipophilic efficiency; p.o.
pharmacokinetic study with oral administration.
the desired product 21
.
R
R
N
OH
PHN
N
H
NH2
Ar
O
O
-amino acid
α
+
Br
Ar
N
PHN
OH
N
H
NH2
Ar
R
O
R
-amino acid
β
References
P = Boc, CBZ
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O
O
O
NH3, MeOH
100oC, Dioxane
P
i) CBZCl,
OEt Hunigs, MeCN CBZN
O
EtO
OEt
OH
OEt
OEt
H2
N
O
nBuLi
THF
100%
O
O
ii) NaOH, EtOH
O
O
O
73%
96%
27
30
28
29
O
O
OH
O
O
CBZN
Cs2CO3
MeCN
Br
CBZN
OCF3
+
O
O
CF3
O
Quant
O
31
30
32
7. England S, Bevan S, Docherty RJ. J Physiol 1996, 495, 429.
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N
N
NCBZ
NH2
NH4OAc
130oC, toluene
H2, Pd(OH)2, 40oC, MeOH
N
N
O
O
63%
46%
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al. Nat Neurosci. 1999, 2, 541.
CF3
O
CF3O
33
21
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2001, 12, 3077.
Scheme 3. Preparation of compound 21.
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Proc Natl Acad Sci U S A. 2012, 109, 19444.
In conclusion, optimisation of an oxadiazole lead has delivered
a highly selective Nav1.8 series. Compound 13 demonstrated
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