Discovery and Optimization of Selective Nav1.8 Modulator Series That Demonstrate Efficacy in Preclinical Models of Pain

Article abstract of DOI:10.1021/acsmedchemlett.5b00059

Voltage-gated sodium channels, in particular Na_v1.8, can be targeted for the treatment of neuropathic and inflammatory pain. Herein, we described the optimization of Na_v1.8 modulator series to deliver subtype selective, state, and use-dependent chemical matter that is efficacious in preclinical models of neuropathic and inflammatory pain. (Chemical Presented).

Full text of DOI:10.1021/acsmedchemlett.5b00059

Letter
pubs.acs.org/acsmedchemlett
Discovery and Optimization of Selective Nav1.8 Modulator Series
That Demonstrate Efficacy in Preclinical Models of Pain
Sharan K. Bagal,*,† Peter J. Bungay,§ Stephen M. Denton,‡ Karl R. Gibson,‡ Melanie S. Glossop,‡
Tanya L. Hay,‡ Mark I. Kemp,‡ Charlotte A. L. Lane,‡ Mark L. Lewis,‡ Graham N. Maw,‡
William A. Million,‡ C. Elizabeth Payne,§ Cedric Poinsard,‡ David J. Rawson,‡ Blanda L. Stammen,‡
Edward B. Stevens,§ and Lisa R. Thompson‡
†Worldwide Medicinal Chemistry, Pfizer Neusentis, The Portway Building, Granta Park, Great Abington, Cambridge CB21 6GS, U.K.
‡Worldwide Medicinal Chemistry, Pfizer Global R&D, Sandwich CT13 9NJ, U.K.
§Neusentis U.K., Pfizer Global R&D, The Portway Building, Granta Park, Cambridge CB21 6GS, U.K.
S
* Supporting Information
ABSTRACT: Voltage-gated sodium channels, in particular
Nav1.8, can be targeted for the treatment of neuropathic and
inflammatory pain. Herein, we described the optimization of
Nav1.8 modulator series to deliver subtype selective, state, and
use-dependent chemical matter that is efficacious in preclinical
models of neuropathic and inflammatory pain.
KEYWORDS: Voltage-gated sodium channels, sodium channel drugs, Nav1.8, SCN10A, TTX-R
oltage-gated sodium channels (Nav) are a family of
transmembrane (TM) ion channel proteins. Structurally,
V
they are members of the 6-TM ion channel family and are
composed of a TM α-subunit of approximately 260 kDa with
associated transmembrane β-subunits of lower molecular
weight. The family comprises 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
Nonselective 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 (CNS).
Figure 1. Discovery of candidate compound 3. IC50 data generated in
VSP-FRET at hNav1.8 in HEK293 cells (in house cell line) with at
least three tests on three different assay runs. TTX-S data generated in
VSP-FRET in SHSY5Y neuroblastoma cell line expressing hNav1.2,
hNav1.3, and hNav1.7.16
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 preclinical studies have implicated
Nav1.3, 1.7, 1.8, and 1.9, which are expressed in DRG (dorsal
root ganglion neurons) and trigeminal neurones, in nociceptive
processing.2 Nav1.8 is highly (but not exclusively) expressed in
nociceptors,3,4 and its expression and function is modulated by
agents that cause pain5,6 Genetic ablation of NaV1.8 in rodents
results in deficits in nociception following inflammation, but
not neuropathic pain,7−10 while recent human genetic evidence
suggest that gain of function mutations in NaV1.8 contributes to
painful peripheral neuropathy.11 A-803467 is one of the first
compounds in the public domain that demonstrated selectivity
across human Nav subtypes and attenuated pain sensitivity in
models of both nerve injury and inflammation induced pain
Received: February 4, 2015
Accepted: April 29, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00059
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Table 1. Compounds 2, 3, 13, and 18: Pharmacokinetic
Studies in Rat (R) and Dog (D)
Dose (mg/kg)
route
T1/2
(h)
Tmax
(h)
Plasma
a
Vd
(L/kg)
Oral F
(%)
Cmpd
CL
2 (R)
3 (R)
3, p.o.
2, i.v.
4.1
0.75
1.0
204
11.7
13.3
6.7
N.D.
91
4.0
3.0
5, p.o.
1, i.v.
4.6
13 (R)
18 (D)
3.9
2.25
5.3
2, p.o.
0.1, i.v.
0.25, p.o.
N.D.
9.7
1.3
11.4
6.2
59
N.D.
3.5
10.0
63
a
Plasma CL (i.v.) or CL/F (p.o.) in mL/min/kg.
a
Table 2. Aryl Ring SAR in Picolinamide Series
Figure 2. Antiallodynic effects of 13 in the TNT model of neuropathic
pain. Error bars represent the SEM. A single dose of 40 mg/kg of 13
equivalent to a free plasma exposure of 0.2 μM, significantly shifted the
50% paw withdrawal threshold in the ipsilateral paw from a baseline of
1.7 0.3 to 7.3 1.8 g, 1.5 h after dosing (*P < 0.05). These effects
were comparable to 10 mg/kg of Pregabalin, which shifted the 50%
paw withdrawal threshold in the ipsilateral hindpaw from 1.7 0.2 to
5.8 1.7 g, 1.5 h after dosing (P = 0.07).
Cmpd
Aryl
Nav1.8 IC50 (μM)
cLogP
LipE
3
4
5
6
7
8
2,3,5-trichlorophenyl
2-chlorophenyl
2.6
3.8
2.5
2.8
2.8
3.3
3.5
1.8
selective Nav1.8 modulators with good oral pharmacokinetics in
preclinical studies.
>32
>32
>32
12
NA
NA
NA
1.6
3-chlorophenyl
Lamotrigine is a first generation sodium channel modulator and
an anticonvulsant used in the treatment of epilepsy and bipolar
disorder (Figure 1). Lamotrigine is a weak Nav1.8 inhibitor and
shows no selectivity for Nav1.8 over TTX-S channels (measured
in fluorescence based assays). Compound 1 was identified
through file screening and suggested that trichloroaryl and
aminopyridine units offered a potent Nav1.8 and TTX-S channel
inhibition profile. Modification of the core to a diaminopyridine
unit coupled with the introduction of 6-acetamide led to
compound 2, which displayed a significant improvement in
Nav1.8 potency together with approximately 20-fold selectivity
over TTX-S channels.
4-chlorophenyl
2,5-dichlorophenyl
3,5-dichlorophenyl
>32
NA
a
IC50 data generated in VSP-FRET at hNav1.8 in HEK293 cells (in house
cell line) with at least three tests on three different assay runs.16 cLogP
was calculated using BioByte program version 4.3.
(the latter with the exception of the formalin challenge paw
withdrawal assay).12 This provided the first pharmacological
evidence supporting a role for Nav1.8 in both inflammatory and
neuropathic pain.
Existing subtype selective Nav1.8 inhibitors, for example,
A-803467, exhibit poor oral pharmacokinetics in preclinical
species.12 In this article, we discuss the discovery of subtype
Compound 2 was assessed in an oral pharmacokinetic (PK)
study in rat where it demonstrated high in vivo clearance (CL)
(Table 1). The high CL was likely to be mediated by amide
Table 3. hNav1.8 Potency (IC50), Selectivity (IC50), and Antiallodynic Effects in Rodent Models of Neuropathic Pain for 3, 13,
a
and 18
hNav subtype
selectivity
TTX-r
Rat DRG
TTX-R
human DRG
hNav1.8
hERG
Unbound
exposure
(μM)
IC50
IC50
IC50
Cmpd
3
(μM)
n
IC50 (μM)
n
(μM)
n
IC50 (μM)
n
(μM)
Model
Effect significance
0.19
5
Nav1.1 = 13
Nav1.2 = 12.8
Nav1.5 = 9.0
Nav1.7 = 19
Nav1.1 = 37
Nav1.5 = 37
Nav1.7 = 36
5
5
5
5
2
2
2
5
4
0.44
4
0.31
4
30
SNL hypersensitivity P < 0.05 (equal to
0.25
100 mg/kg gabapentin)
13
18
0.19
0.26
2
4
0.54
0.33
4
6
0.20
ND
3
>30
>30
TNT mechanical
allodynia
SP < 0.05 (comparable to
10 mg/kg pregabalin)
0.20
0.19
b
SHSY5Y = 10
TNT mechanical
allodynia
P < 0.05 (comparable to
10 mg/kg pregabalin)
Nav1.5 = 12
a
IC50 values for 3, 13, and 18 at recombinantly expressed hNav1.8/β1 (Merck Millipore) and at TTX-R in rat and human DRG were determined
using manual patch clamp electrophysiology. hNav subtype selectivity for 3 was also measured using manual patch clamp electrophysiology. IC50
values determined using manual patch clamp electrophysiology were determined at the respective V0.5 of inactivation for TTX-R and each channel
b
isoform. For 13 and 18, human sodium channel subtype selectivity was measured using IonWorks Quattro and FRET, respectively. SHSY5Y cells
expressing hNav1.2, 1.3, and 1.7 were also used. The voltage protocol for the IonWorks Quattro and assay methodology for the FRET assay is
detailed in the Supporting Information.
B
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a
Table 4. Aryl Ring and Amide Moiety SAR Observed in the Acetamide Series
a
IC50 data generated in VSP-FRET at hNav1.8 in HEK293 cells (in house cell line) with at least three tests on three different assay runs.16 cLogP was
calculated using BioByte program version 4.3.
deacetylation as evidenced by rapid formation of the cor-
responding diaminopyridine metabolite in vivo. Reversal of
the amide produced 3, which was more stable toward amide
hydrolysis based on in vitro ADME data. Compound 2 readily
underwent metabolism in rat and human liver microsomes
(RLM and HLM), with intrinsic clearance (CLint) values of
29 and 41 μL/min/mg protein in RLM and HLM, res-
pectively, while 3 displayed very little turnover (CLint < 9 and
7.1 μL/min/mg protein in RLM and HLM, respectively).
Moreover, this analogue retained Nav1.8 activity (albeit weaker
activity when compared with 2) and was Nav1.8 selective.
Oral and i.v. rat PK studies with 3 exhibited low CL with high
oral bioavailability of 91% (Table 1). Allometric scaling from rat
data predicted low CL in human, oral bioavailability >90%, and
half-life 8−28 h.13 Compound 3 was profiled through manual
electrophysiology in order to assess potency and selectivity
across multiple ion channels. The IC50 value for 3 at hNav1.8,
was 0.19 μM with ≥50-fold selectivity for Nav1.8 over the other
ion channels studied, including hERG. Furthermore, 3 inhibited
native TTX-R currents in both human and rat DRG neurons
(Table 3). The block of both human recombinant Nav1.8 and
TTX-R currents from rat DRG neurons was found to be frequency
and state dependent.14 State dependence of NaV modulators
results from different affinities for each channel state, whereby
binding to the inactivated state is often preferred. Many NaV
modulators also demonstrate use dependence, which occurs when
potency increases with NaV channel firing frequency. As 3 was
both state and frequency dependent, it may be possible to achieve
functional selectivity by preferentially targeting high frequency
firing rates associated with neuroma ectopic activity and sparing
the low frequency firing rates of the normal somatosensory system
leading to an improved therapeutic index.15
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In order to improve the lipophilic efficiency (LipE) of 3
above 1.8, the trichloroaryl ring was varied (Table 2).17 In this
picolinamide series, Nav1.8 potency was found to be highly
dependent on aryl polychlorination with 3 being one of the
most potent and LipE efficient compounds synthesized.18
In a parallel effort, the instability of the acetamide in 2
was also addressed. Through variation of the amide moiety
(Table 4) pyrazole 9 and isoxazole 10 were found to be two of
the most potent amides identified. Both of these heterocyclic
amides retained potency and Nav1.8 subtype selectivity over
TTX-S while demonstrating improved amide stability in in vitro
systems.18−20 These amides may be chemically stable due to
increased conjugation between the carbonyl and heterocycle in
comparison to the acetamide group. However, 9 and 10 were
poorly soluble (<0.1 μg/mL at pH 7.2), which was attributable
to their combination of high cLogP and planar shape.21 In
order to reduce lipophilicity, the pyridyl core was replaced by
a pyrazine core. Although cLogP was reduced by the pyridine
to pyrazine switch (data not shown), the simultaneous potency
loss of greater than 10-fold led to Nav1.8 inhibitors that were
too weak to permit progression.
Compounds 3, 13, and 18 were efficacious in preclinical in
vivo models of neuropathic pain: 3 was efficacious in the rat
model of spinal nerve injury, while 13 and 18 were efficacious
in the tibial nerve transection (TNT) induced mechanical
allodynia model in rat (see Table 3 and Figure 2). Analysis of
compound concentrations in plasma and cerebrospinal fluid
samples suggested that 13 readily crossed the blood−brain
barrier in rats (cerebrospinal fluid: unbound plasma concen-
tration ratio 0.75:1). Moreover, an oral dose of 13 (250 mg/kg)
achieving unbound exposures up to 0.33 μM in rats had no
effect on either horizontal or vertical movements when compared
to vehicle control animals indicating little or no effect of 13 on
either the peripheral or central nervous system.
Based on the favorable Nav1.8 potency, LipE, selectivity and
in vivo PK profiles of 3, 13, and 18 these compounds were
selected as candidates for further progression.
Schemes 1 and 2 demonstrate a typical synthetic route for both
the picolinamide and acetamide series, in this case illustrated by
Scheme 1. Preparation of Compound 3
The aryl unit was also varied (Table 4). Substitution at the
2- and 5-aryl positions with lipophilic groups tended to offer
profiles with potency in the submicromolar range, e.g., 11, 12,
and 13. In the case of 18, the larger 2-trifluoromethoxy moiety
is sufficient to achieve submicromolar potency without the
requirement of a 5-aryl substituent. Moreover, the observed SAR
exhibited relatively steep activity cliffs, whereby, for instance,
addition of a 4-F atom to compound 15 to give 21 decreased
Nav1.8 potency from 4.9 to >31 μM.22
The amide group was optimized further (Table 4). Aliphatic
amides were less stable to amide hydrolysis in vitro than
aromatic amides as exemplified by 16, which readily underwent
amide hydrolysis in buffer solutions at pH 7.4 such that in vitro
ADME measurements were precluded. As before, this may
be attributed to increased conjugation between the carbonyl
and aromatic amides in comparison to the aliphatic amides.
Furthermore, it appeared that the optimal heterocyclic amide
was heavily dependent on the aryl unit it was combined with.
For instance, a comparison of compounds 17−20 suggested
that the ideal heterocyclic amide to combine with the
2-trifluoromethoxyaryl moiety was 3-methylisoxazole. However,
analysis of the 2-chloro, 5-methoxy, and 2,3,5-trichlorophenyl
aryl groups present in 9, 10, 12, 13, and 16 indicated very little
difference in Nav1.8 potency between the 1-methyl-1H-pyrazole
amides and 3-methylisoxazole amides.
Scheme 2. Preparation of Compound 13
SAR development led to the selection of 13 and 18 as
compounds of interest to progress based on highest LipE
(13 and 18 have a LipE two units greater than 3), Nav1.8 potency
and acceptable solubility of ∼10 μg/mL. PK studies in rat with 13
indicated a low CL compound with good bioavailability ∼60%
(Table 1). Allometric scaling predicted low CL of 13 in human
(CL < 5 mL/min/kg).13 Preclinical oral PK studies of 18 also
yielded low human CL estimates. Compounds 13 and 18 were
profiled through manual electrophysiology. In a consistent
manner to 3, 13 and 18 were selective for hNav1.8 over the
other human sodium channel subtypes studied and the hERG
channel. Compounds 13 and 18 also inhibited native TTX-R
currents in rodent DRG neurons and the IC50 for the inhibition
of TTX-R currents in human DRG neurons was determined for
13 (Table 3). The block of both human recombinant Nav1.8 and
TTX-R currents from rat DRG neurons by both 13 and 18 were
found to be frequency and state dependent.14,15
the synthesis of analogues 3 and 13. Further synthesis details are
available in the Supporting Information.23
In conclusion, optimization of a biaryl lead has led to highly
selective Nav1.8 series. Three key compounds 3, 13, and 18
have also demonstrated good pharmacokinetics in preclinical
species leading to low human CL projections. Moreover, these
compounds are efficacious in preclinical studies of neuropathic
and inflammatory pain. Further data on the progression of 3,
13, and 18 will be reported in due course.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and analytical data for the preparation
of compounds 2−21, additional biological data, PK and efficacy
study information, and experimental details for the in vitro
assays. The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/
acsmedchemlett.5b00059.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sharan.bagal@pfizer.com.
Notes
■
The authors declare no competing financial interest.
(12) Jarvis, M. F.; Honore, P.; Shieh, C.-C.; Chapman, M.; Joshi, S.;
Zhang, X.-F.; Kort, M.; Carroll, W.; Marron, B.; Atkinson, R.; Thomas,
J.; Liu, D.; Krambis, M.; Liu, Y.; McGaraughty, S.; Chu, K.; Roeloffs,
R.; Zhong, C.; Mikusa, J. P.; Hernandez, G.; Gauvin, D.; Wade, C.;
Zhu, C.; Pai, M.; Scanio, M.; Shi, L.; Drizin, I.; Gregg, R.; Matulenko,
M.; Hakeem, A.; Gross, M.; Johnson, M.; Marsh, K.; Wagoner, P. K.;
Sullivan, J. P.; Faltynek, C. R.; Krafte, D. S. A-803467, a potent and
selective Nav1.8 sodium channel blocker, attenuates neuropathic and
inflammatory pain in the rat. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
8520−8525.
(13) Caldwell, G. W.; Masucci, J. A.; Yan, Z.; Hageman, W.
Allometric scaling of pharmacokinetic parameters in drug discovery:
Can human CL, Vss and t1/2 be predicted from in-vivo rat data? Eur.
J. Drug Metab. Pharmacokinet. 2004, 29, 133−143.
(14) Payne, C. E. Brown, A. R.; Theile, J. W.; Mahoney, J. H.; Loucif,
A. J. C.; Alexandrou, A. J.; Fuller, M. D.; Antonio, B. M.;. Gerlach, A.
C.; Prime, R. L.; Stockbridge, G.; Kirkup, A. J.; Bagal, S. K.; Bannon, A.
W.; England, S.; Chapman, M. L.; Roeloffs, R.; Bungay, P.; Anand, U.;
Anand, P.; Kemp, M.; Butt, R. P.; Stevens, E. B. A novel oral NaV 1.8
blocker PF-01247324 attenuates nociception and neuronal excitability. Br.
J. Pharmacol. 2015, 172 (10), 2654−2670; DOI: 10.1111/bph.13092.
(15) Bagal, S. K.; Brown, A. D.; Cox, P. J.; Omoto, K.; Owen, R. M.;
Pryde, D. C.; Sidders, B.; Skerratt, S. E.; Stevens, E. B.; Storer, R. I.;
Swain, N. A. Ion channels as therapeutic targets: a drug discovery
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ACKNOWLEDGMENTS
■
We thank Chan W. Huh and Wendy B. Wang for the
generation of compound characterization data. Compound 13
(PF-04531083) is commercially available via Sigma-Aldrich
(catalog # PZ0273). Compound 3 (PF-01247324) is
commercially available via Sigma-Aldrich (catalog # PZ0274).
DEDICATION
■
This publication is dedicated to the memory of Bill Million.
ABBREVIATIONS
■
Cmpd, compound; PK, pharmacokinetics; ADME, absorption
distribution, metabolism, excretion; CL, clearance; PK, pharma-
cokinetics; DRG, dorsal root ganglion neuron; TNT, tibial nerve
transection; TM, transmembrane; TTX-S, tetrodotoxin-sensitive;
TTX-R, tetrodotoxin-resistant; LipE, lipophilic efficiency; SNL,
spinal nerve injury; CNS, central nervous system; L/kg, liters per
kilogram; μg/mL, microgram per milliliter; h, hour; i.v.,
intravenous; p.o., pharmacokinetic study with oral administration;
T
1/2, pharmacokinetic half-life; Tmax, time of maximum concen-
tration in vivo; Vd, volume of distribution; oral F, oral bioavaibility
(16) hNav1.8 data generated in VSP-FRET hNav1.8 in HEK293 cells
with at least three tests on three different assay runs. TTX-S data
generated in VSP-FRET in SHSY5Y neuroblastoma cell line
expressing hNav1.2, hNav1.3, and hNav1.7.
(17) Freeman-Cook, K. D.; Hoffman, R. L.; Johnson, T. W.
Lipophilic efficiency: the most important efficiency metric in medicinal
chemistry. Future Med. Chem. 2013, 5 (2), 113−115.
(18) Lane, C. A. L.; Maw, G. N.; Rawson, D. J.; Thompson, L. R.
Pyridine derivatives. WO2006011050, 2006.
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