Highly potent and selective NaV1.7 inhibitors for use as intravenous agents and chemical probes

Article abstract of DOI:10.1016/j.bmcl.2017.09.056

The discovery and selection of a highly potent and selective Na_V1.7 inhibitor PF-06456384, designed specifically for intravenous infusion, is disclosed. Extensive in vitro pharmacology and ADME profiling followed by in vivo preclinical PK and efficacy model data are discussed. A proposed protein–ligand binding mode for this compound is also provided to rationalise the high levels of potency and selectivity over inhibition of related sodium channels. To further support the proposed binding mode, potent conjugates are described which illustrate the potential for development of chemical probes to enable further target evaluation.

Full text of DOI:10.1016/j.bmcl.2017.09.056

Accepted Manuscript
Highly potent and selective NaV1.7 inhibitors for use as intravenous agents and
chemical probes
R. Ian Storer, Andy Pike, Nigel A. Swain, Aristos J Alexandrou, Bruce M.
Bechle, David C. Blakemore, Alan D. Brown, Neil A. Castle, Matthew S.
Corbett, Neil J. Flanagan, David Fengas, M. Scott Johnson, Lyn H. Jones, Brian
E. Marron, C. Elizabeth Payne, David Printzenhoff, David J. Rawson, Colin R.
Rose, Thomas Ryckmans, Jianmin Sun, Jonathan W Theile, Rubben Torella,
Elaine Tseng, Joseph S. Warmus
PII:
S0960-894X(17)30965-4
https://doi.org/10.1016/j.bmcl.2017.09.056
BMCL 25320
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 July 2017
17 September 2017
27 September 2017
Please cite this article as: Storer, R.I., Pike, A., Swain, N.A., Alexandrou, A.J., Bechle, B.M., Blakemore, D.C.,
Brown, A.D., Castle, N.A., Corbett, M.S., Flanagan, N.J., Fengas, D., Scott Johnson, M., Jones, L.H., Marron, B.E.,
Elizabeth Payne, C., Printzenhoff, D., Rawson, D.J., Rose, C.R., Ryckmans, T., Sun, J., Theile, J.W., Torella, R.,
Tseng, E., Warmus, J.S., Highly potent and selective NaV1.7 inhibitors for use as intravenous agents and chemical
probes, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.09.056
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Highly potent and selective NaV1.7 inhibitors for use as intravenous agents and
chemical probes
R. Ian Storer a,#,∗, Andy Pikeb,#, Nigel A. Swaina, Aristos J Alexandrouc, Bruce M. Bechlee, David C.
Blakemorea, Alan D. Browna, Neil A. Castlef, Matthew S. Corbette, Neil J. Flanagand, David Fengasg , M.
Scott Johnsonf, Lyn H. Jonesh, Brian E. Marronf, C. Elizabeth Paynec, David Printzenhofff, David J.
Rawsoni, Colin R. Rosee, Thomas Ryckmansi, Jianmin Sune, Jonathan W Theilef, Rubben Torellaa, Elaine
Tsengj and Joseph S. Warmuse.
aWorldwide Medicinal Chemistry, bPharmacokinetics, Dynamics and Metabolism, cElectrophysiology, dPharmaceutical Sciences, Pfizer Ltd, The Portway,
Granta Park, Cambridge, CB21 6GS, UK.
eWorldwide Medicinal Chemistry, Pfizer Inc. Eastern Point Road, Groton, Connecticut 06340, USA.
fIcagen Inc, 4222 Emperor Blvd #350, Durham, NC 27703, USA.
gPeakdale Molecular, Ltd, Discovery Park House, Ramsgate Road, Sandwich, Kent, CT13 9ND, UK.
hWorldwide Medicinal Chemistry, Pfizer Inc, 610 Main Street, Cambridge, MA 02139, USA.
iWorldwide Medicinal Chemistry, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK.
jPharmacokinetics, Dynamics and Metabolism, Pfizer Inc, Eastern Point Road, Groton, Connecticut 06340, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The discovery and selection of a highly potent and selective NaV1.7 inhibitor PF-06456384,
designed specifically for intravenous infusion, is disclosed. Extensive in vitro pharmacology and
ADME profiling followed by in vivo preclinical PK and efficacy model data are discussed. A
proposed protein-ligand binding mode for this compound is also provided to rationalise the high
levels of potency and selectivity over inhibition of related sodium channels. To further support
the proposed binding mode, potent conjugates are described which illustrate the potential for
development of chemical probes to enable further target evaluation.
Keywords:
Acid isostere
Ion channel
Pain
2009 Elsevier Ltd. All rights reserved
.
Voltage-gated
———
∗ Corresponding author. e-mail: ian.storer@astrazeneca.com

Voltage-gated sodium (NaV) channels comprise a family of
nine members NaV1.1-NaV1.9. They are members of the 6-TM
ion channel sub-family that are structurally composed from a
main transmembrane α-subunit of approximately 260 kDa with
multiple lower molecular weight associated β-subunits.1 NaV
channels have been established to play important roles in the
control of neuronal excitability by regulating the threshold of
firing and duration of inter-spike interval underlying the
formation and propagation of action potentials.2 Unselective
sodium channel blockers are known and include established
clinical agents (eg mexiletine 1, lidocaine 2 and lamotrigine 3)
that have been used successfully to modulate neuronal firing
patterns in a range of conditions including epilepsy and chronic
pain (Figure 1).3,4 However, due to a lack of selectivity across the
NaV family, these drugs suffer narrow therapeutic indices over
adverse effects due to inhibition of sodium channels in the brain
(eg NaV1.1, NaV1.2) and heart (eg NaV1.5).5
infusion. This required a compound with high potency, to at least
partially offset the high clearance and minimise dose
requirements, as well as good solubility in order to be compatible
with IV formulations and minimise infusion volumes.
To design a suitable intravenous (IV) infusion compound,
SAR knowledge gained from our oral NaV1.7 program was
incorporated to deliver optimal NaV1.7 inhibition potency and
solubility, ultimately resulting in PF-06456384 (5) (Figure 2).30
Figure 2. Oral candidate22 and IV lead compounds
Some key SAR which guided the design that led to compound
5 is illustrated in Table 1. A strategy that comprised combining
the most lipophilic efficient (lipE)31 fragments was adopted in
order to deliver optimal potency and selectivity. Accordingly, it
had previously been observed that compounds containing a polar
1,2,4-thiadiazole sulfonamide acidic headgroup in combination
with a nitrile substituted core ring (compounds 5-8, 13) provided
the most lipophilic efficient and intra-NaV selective compounds.32
However, despite the potency and selectivity advantages, for the
oral program this combination had often led to poor in vivo
intestinal absorption due to low permeability, likely driven by
high topological polar surface area (TPSA >150 Å2) and acidity
of the heterocyclic sulfonamide. However, in the case of IV
administration intestinal permeability was no longer of relevance.
Also it had previously been established that scope for further
variation of either the amino heterocycle or core substituents was
relatively limited.32 However, several other key combinations
were also known to be reasonably efficient such as 4-thiazole (4,
11) or 1,3,4-thiadiazole (9-10, 12) in combination with a F,Cl di-
substituted core ring. In contrast, broader variation of substitution
around the left hand ring of the diaryl ether group (as drawn) and
of the pendant ortho-ring could be readily tolerated, providing
opportunity for gains in potency and lipE plus the introduction of
groups to specifically modulate the bulk physical properties of
the molecules such as polar and ionisable groups.
Figure 1. pan-NaV inhibitor drugs
It is only in the last 10-15 years that a better understanding of
which specific NaV channels are likely to provide greatest
therapeutic value has emerged. Human genetic data has pointed
to a number of specific NaV subtypes as playing a critical role in
pain signal generation and transduction. Of these, NaV1.7 has
emerged as a particularly compelling target based on evidence of
both gain-of and loss-of function mutations in the encoding
SCN9A gene showing a strong association with pain and itch.6-13
In particular, in 2006 it was reported that a loss-of-function
mutation in this gene was responsible for a rare human genetic
condition that manifests as a congenital insensitivity to all types
of pain.6 On this basis over the last decade there has been a
significant interest in the discovery of subtype selective inhibitors
of NaV1.7.14-19
The design of subtype selective NaV inhibitors remains a
highly challenging prospect that is compounded by a lack of
robust structural biology understanding of ion channel function
and binding sites for functional modulation by small molecules.
The established unselective pan-NaV inhibitors are believed to
bind to an intracellular site within the channel pore. However,
design of subfamily selectivity at this binding site has proved
challenging, likely owing to high sequence homology within this
region across the NaV subfamily.20,21
Beginning with zwitterionic compound 6 as a reasonably
potent (NaV1.7 IC50 8.5 nM) and lipophilic efficient (lipE 4.5)
lead, various modifications were combined to maximise ligand
potency. Firstly, it was observed that the primary benzylic amine
in compound 6 could be replaced with either secondary (7) or
tertiary (8) amines whilst maintaining good levels of lipE (4.2-
4.5) and therefore modulating potency in line with lipophilicity.
Secondly, by comparing compounds 9 (lipE 2.6) and 10 (lipE
4.3), a notable increase in ∆lipE >1.0 could be obtained by
introduction of a nitrogen into the ortho ring, rendering this a 2-
substituted pyridine (10). By cross-comparing a selection of
favoured core/amino heterocycle combinations (compounds 8-
11) it appeared that the 1,2,4-thiadiazole in combination with a
nitrile core remained the most lipophilic efficient as observed in
Despite these challenges we were able to pioneer a series of
acidic heterocyclic sulfonamide NaV1.7 inhibitors that exhibited
good levels of potency and intra-family selectivity, culminating
in the delivery of an oral clinical candidate PF-05089771 (4)
(Figure 2).22,23 Also, via application of site directed mutagenesis,
the NaV subtype selectivity inherent to this series was rationalised
to result from binding to a novel site in the domain IV voltage
sensor of the alpha subunit.24 Further supporting evidence was
subsequently provided by an X-ray co-crystal structure of a
Pfizer patent compound by Genentech and Xenon25 who, along
with several other companies, had also opted to pursue acidic
sulfonamide inhibitors.26-29
the oral drug discovery campaign. Thirdly,
a meta-CF3
substituted phenyl ring was introduced para to the diaryl ether
linkage. Previous experience had shown this group to be
tolerated, essentially driving increased potency through
additional lipophilicity. As a result, the ortho 2-pyridine lipE
enhancing group was combined with the meta-CF3 system to
maximise potency in compounds 12 (NaV1.7 IC50 1.5 nM, lipE
As an alternative therapeutic modality a program was initiated
to design a compound for intravenous (IV) infusion to treat acute
traumatic, perioperative pain or postoperative pruritus in a
clinical setting where oral dosing is not convenient. The product
concept included targeting rapid clearance to minimise any
residual on or off target drug effects following the end of

3.7) and 13 (NaV1.7 PX IC50 0.5 nM, lipE 5.1). However, despite
the good levels of potency, these compounds were poorly soluble
in the desired IV formulation pH3-8 range (compound 12
solubility in aqueous buffer: pH 3.0 = 6 µg/mL, pHs 4.2 / 5.1 /
7.2 = <0.5 µg/mL).
whilst simultaneously maintaining excellent potency. Initial
automated electrophysiology profiling of compound 5 on a
PatchXpress® (PX) platform gave NaV1.7 PX IC50= 0.58 nM
(lipE 3.8). Although very potent, this was weaker than
anticipated based on previous SAR, which had suggested that this
compound should have a lipE >5 which would in turn result in a
NaV1.7 IC50 <100 pM. Further investigation via conventional
single cell patch clamp electrophysiology (EP) showed that both
compounds 13 and 5 were indeed more potent (EP IC50s 100 pM
and 10 pM respectively) in line with the anticipated lipE of
around 5.5. Further investigation suggested that the
PatchXpress® platform struggled to accurately determine potent
(<1 nM) IC50s due to slow equilibration kinetics, therefore
requiring longer duration recordings that were only possible by
conventional EP. (see SI section for details of the
electrophysiology protocols used and for a more full discussion
of protocols see ref. 22).
Table 1. Selected SAR used in the design of compound 5.
NaV1.7 PX
IC50 (nM)a
No.
Structure
clogP lipEc
6
8.5
3.6 4.5
7
0.70
4.8 4.4
Recently Genentech and Xenon reported a NaV1.7 X-ray co-
crystal structure,25 in the presence of Pfizer compound PF-
05196233 (GX-936) from the same chemotype as compound 5
(Figure 3).23 To rationalise the extreme potency compound 5, was
docked using this published structure as a reference protein.
8
9
1.6
8.6
4.6 4.2
N
N
O
O
O
O
N
N
F
C
S
S
3
S
S
N
F
C
N
3
H
H
O
O
5.5 2.6
N
CN
CN
N
H
N
N
N
H N
PF-05196233 (GX-936)
5
Figure 3. Structures of compound GX-936 & IV lead compound 5.
10
11
12
13
4.8
13.2
1.5
4.0 4.3
4.0 3.9
5.1 3.7
Compound 5 (orange) is predicted by docking to bind in a
similar manner and orientation to GX-936 (yellow), making
consistent interactions between the acidic sulfonamide and
arginine residues in the binding site (Figure 4).
0.50
5.1
4.2
(EP 0.1)b
(5.8)
0.58
3.8
5.4
Figure 4. Docking pose of compound 5 (orange), overlaid with X-ray
pose of GX936 (transparent yellow). The described new set of interactions
with Phe1583, Trp1537, Asp1586 and Glu1589 is shown by yellow dashes
5
(EP 0.01)b
(5.6)
aPX=PatchXpress® electrophysiology; bEP=Conventional
Patch Clamp electrophysiology; clipE=-logIC50-clogP. IC50
values are based on the average of >2 determinations (see SI for
details).
However, there are some interesting differences that can be
highlighted from this analysis which likely account for the
excellent potency of compound 5: i) the additional meta-
trifluoromethyl substituted aromatic ring in compound 5 is
predicted to embed into the membrane, flanked by two aromatic
residues (Phe1583 and Trp1537) which can both make π−π
stacking interactions; ii) the pyridine ring in compound 5 can π-
stack with Trp1537 in an analogous manner to the pyrazole in
GX-936; iii) the additional flexibility of the benzylamine in
compound 5 relative to the rigid azetidine in GX-936 allows for a
more optimal salt bridge interaction with Asp1586 and; iv) the
Therefore, based on the earlier SAR observed for compound
7, a dibasic amino-substituted piperidine group was introduced to
provide compound 5. The rationale for this was primarily to
provide an overall weakly basic compound with the other two
ionisable centres existing as a zwitterion at neutral pH, thereby
maximising solubility and compatibility with IV formulations,

basic nitrogen of the piperidine in compound 5 can form an
additional interaction with Glu1589 at the top of the voltage-
sensor domain (VSD). Taken together, these additional
interactions likely rationalise why compound 5 confers such high
levels of potency.
tetrakis(triphenylphosphine)palladium(0), proceeded smoothly,
but the subsequent Suzuki-Miyaura coupling reactions with
boronic acid 23 proved to be low yielding in both cases using
standard catalysts. However, application of Buchwald’s water
soluble SPhos proved superior, providing high yields of either the
methoxy acid 24 or phenol acid 26.40 Furthermore, use of this
catalyst also enabled the two couplings to be completed in one-
pot by simply adding the second boronic acid to the reaction after
the first coupling was deemed complete by HPLC.
Interestingly, the proposed positioning of the piperidine at the
mouth of the pocket pointing into solvent suggests that this might
be a suitable handle from which further groups could be
appended without significantly impacting the binding energy due
to exiting the extracellular binding site. It was decided to explore
this hypothesis further with a view to being able to potentially
exploit this feature for the introduction of pharmacokinetic (PK)
modulating conjugations or provision of functional probes. To
initially investigate whether amide attachment and conjugate
linkers could be tolerated acyl capped (14) and PEG-12 amide
(15) compounds were synthesized (Table 2). Pleasingly, both of
these modifications maintained good potency, giving complete
channel block at low nM levels (no accurate IC50 was determined
in these cases). Furthermore PEG-12 amide (15) was also tested
for activity at NaV1.5 for which it showed no significant activity,
thereby suggesting that the high levels of selectivity inherent to
the headgroup had been maintained. To further build on this
initial result, a BODIPY®FL (16) fluorescent conjugate,37 and
benzophenone photoprobe (17) compound (potentially useful for
binding or target engagement studies,35,36 or for determination of
the NaV1.7 interactome) were synthesised.38,39 Interestingly, these
conjugates all maintained excellent sub-nanomolar levels of
NaV1.7 potency (Table 2). The apparently flat NaV1.7 IC50 SAR
for the conjugate groups supports the initial proposal that the
piperidine moiety on the headgroup offers a trajectory out of the
binding site, thereby positioning the conjugate groups into
solvent leading to them not significantly impacting binding
affinity.
28 R1 = CHO, R = Me
iv)
v)
NH2
Cl
Br
F3C
N
Boc
i)
ii)
29
OR
OR
vi)
Cl
F3C
HO2C
N
OR
OR
B(OH)2
18
B(OH)2
23
HO2
C
N
R1
N
30
vii) R1 = CH2Cl, R = H
19
R = Me
20 R = H
21
22
24
25
26
27
R = Me
R = H
R1 = CO2H, R = Me
R1 = CH2OH, R = Me
R1 = CO2H, R = H
R1 = CH2OH, R = H
iii)
iii)
H
viii)
N
Nos
N
31
Boc
vi)
N
O
O
ix)
N
N
O
O
S
S
N
H
N
S
S
F3C
F3C
N
F
H
33
CN
OH
O
CN
H
N
H
N
N
N
N
N
34 R2 = Boc
x)
Boc
R2
32
5
R2 = H
Scheme 1. Reagents and conditions: i) 19 or 20, Pd(PPh3)4,
Na2CO3, dioxane/H2O. ii) 23, Pd(OAc)2, s-SPhos, K2CO3,
H2O. iii) BH3-THF, THF. iv) TEMPO, NaClO, DCM (95%).
v) 29, NaBH4, MgSO4, DCM (87%). vi) dodecane thiolate,
NaOH, DMSO. vii) POCl3, DMF, DCM. viii) 30, NaH,
DMAc (65%). ix) 33, K2CO3, DMSO (80%). x) HCl, MeOH
(76%).
To incorporate the requisite amino-piperidine to access
compound 32, a reductive amination with primary amine 29
could be carried out. However, it proved necessary to have the
phenol masked as the methyl ether 25 in order to successfully
prosecute this reductive amination. Alternatively a Fukuyama
amine synthesis using nosyl protected amine 31 could be applied
as the key bond forming reaction.41 In this case, the free phenol
27 could be successfully reacted to provide phenol 32 which was
subsequently coupled via SNAr to give compound 34 followed by
deprotection to lead compound 5 (Scheme 1).
Table 2. NaV1.7 activities for amide conjugated derivatives.
NaV1.7
NaV1.5
No.
14
R Group
IC50 (nM)
IC50 (nM)
<4a
nt
Having identified compound 5 as a highly potent lead
compound, extensive selectivity profiling was carried out across
NaV channel human subtypes and relevant orthologues using
15
16
<10a
>3000a
conventional EP. Pleasingly, compound
5 proved to be
exquisitely selective over sodium channels that are associated
with cardiovascular and CNS activities (>300x selective over all
other human NaVs) as shown in Table 3. Further wide ligand
secondary pharmacology assessment across a broad panel of
target classes was also carried out (see SI section 6 for table of
this data). All pharmacologies tested in this panel also showed
>1000-fold weaker activity than the primary NaV1.7 IC50.
0.10b
0.12b
nt
nt
17
b
aPatchXpress® electrophysiology; Conventional Patch Clamp
electrophysiology.
Solubility assessment showed compound 5 to have very good
solubility in aqueous buffer of >900 µg/mL across the relevant
pH 3-7 range which enabled formulations to solutions of 10
mg/mL at pH 5-6.
In order to prepare compound 5 and derivatives, a concise
convergent synthetic route via phenol intermediate 32 was
developed allowing late stage SNAr coupling with fluorophenyl
33 (Scheme 1). The biphenyl pyridyl in compound 32 was
installed using sequential Suzuki-Miyaura reactions. The
couplings of the 4-bromonicotinic acid 18 with either methoxy-
phenylboronic acid 19 or phenolboronic acid 20, catalysed by
In vitro ADME profiling of compound 5 was carried out
across a panel of assays. This included metabolic stability assays
and showed low Clint in both microsomes and hepatocytes from
mouse, rat, dog and human. This finding is consistent with low
metabolic clearance of Compound 5, albeit the low microsomal fu
may be confounding this measurement. However, the rate

limiting clearance step of the oral clinical compound PF-5089771
(4) and the related series was usually active hepatic uptake via
organic anion transporting polypeptides (OATPs).33
uptake of compound 5 in OATP expressing cell lines vs wild
type was assessed. Despite the altered physicochemistry, these
suggested that compound 5 was indeed an OATP substrate
(Table 3).
Table 3. Physical properties, in vitro pharmacology and
ADME data for compound 5.
Compound 5 was subsequently profiled in preclinical in vivo
pharmacokinetic studies (Table 4). It exhibited high total blood
clearance, close to or exceeding liver blood flow, and very high
unbound clearance in all species. In bile duct cannulated rats,
74% of the dose was recovered unchanged in bile, supporting
active uptake and excretion as a major clearance mechanism of
this compound in this species. Renal clearance was not
significant in either rat or dog. The volume of distribution
approximated body water in all species. However, in dog an
extended β phase of elimination was observed which may
indicate more extensive distribution of a fraction of the dose.
Taken together this extensive profiling suggested suitability of
compound 5 for progression in vivo efficacy studies as a
potential candidate drug.
Property
Compound 5
Physicochemical properties
Molecular weight (Da)
clogP
719.8
5.37
logD (pH7.4
pKa (acid)
pKa (base)
)
2.37
2.6
9.3, 6.6
178
TPSA (Å2)
Solubility at pH3.2 (µg/mL)
Solubility at pH4.2 (µg/mL)
Solubility at pH5.5 (µg/mL)
Solubility at pH7.1 (µg/mL)
NaV Pharmacologya
>906
>1172
>1835
>2573
Table 4. Preclinical in vivo IV PK.
Species/Property
hNaV1.1 IC50 (nM)
314
3
Compound 5
hNaV1.2 IC50 (nM)
hNaV1.3 IC50 (nM)
6438
1451
2592
5.8
Mouse
Clp (mL/min/kg)
Clu (mL/min/kg)
Clb (mL/min/kg) b
T1/2 (h)
hNaV1.4 IC50 (nM)
36.5
12989
67.2
hNaV1.5 IC50 (nM)
hNaV1.6 IC50 (nM)
hNaV1.7 IC50 (nM)
0.01
26000
<0.1
75
0.66
hNaV1.8 IC50 (nM)
0.54
Vss (L/kg)
mNaV1.7 IC50 (nM)
Rat
83.1 (59.8a)
30218
178
rNaV1.7 IC50 (nM)
Clp (mL/min/kg)
Clu (mL/min/kg)
Clb (mL/min/kg) b
T1/2 (h)
ADME in vitro profile
RRCK (x10-6 cm/sec)
HLM, Clint (µL/min/mg)
RLM, Clint (µL/min/mg)
DLM, Clint (µL/min/mg)
MLM, Clint (µL/min/mg)
HHEP, Clint (µL/min/106 cells)
RHEP, Clint (µL/min/106 cells)
DHEP, Clint (µL/min/106 cells)
MHEP, Clint (µL/min/106 cells)
Human microsomal binding
hppb (fraction unbound)
rppb (fraction unbound)
dppb (fraction unbound)
mppb (fraction unbound)
Human blood:plasma
Rat blood:plasma
0.62
<8.0
0.21
<14.1
<18.1
ND
0.59
Vss (L/kg)
44.0
Biliary (mL/min/kg)
Renal (mL/min/kg)
Dog
0.0322
6.5
<6.0
Clp (mL/min/kg)
Clu (mL/min/kg)
Clb (mL/min/kg)b
T1/2 (h) α/β
17.8
4759
<6.0
<3.0
39.8
0.0012
0.00497
0.00275
0.00374
0.00281
0.573
0.467
0.447
0.543
0.057/5.9
0.46
Vss (L/kg)
0.0012
Renal (mL/min/kg)
aClp value from bile duct cannulated rat study. bClb calculated
from plasma clearance and measured blood:plasma.
Lead compound 5 was therefore tested for analgesic efficacy
in a mouse formalin model following continuous IV infusion.
Published data in NaV1.7 knockout mice suggests these animals
recapitulate the human phenotype, showing congenital
insensitivity to all pain and minimal response in the formalin test,
indicating this model to be relevant to NaV1.7 pharmacology.11
Compound 5 was tested for analgesic efficacy in a wild type
mouse formalin model with continuous infusion. However,
despite achieving unbound plasma concentration of ≥60x the
mouse NaV1.7 IC50, there was no significant effect of the
compound in this model relative to Gabapentin as the positive
control (Figure 3).
Dog blood:plasma
Mouse blood:plasma
CYP inhibition IC50
CYP1A2 (µM)
>30
6.17
3.75
1.32
8.83
1.15
CYP2C8 (µM)
CYP2C9 (µM)
CYP2C19 (µM)
CYP2D6 (µM)
CYP3A4 (µM)
OATP cell uptake
OATP1B1 (transfected/wild type)
OATP1B3 (transfected/wild type)
OATP2B1 (transfected/wild type)
3.9
3.4
3.6
This lack of preclinical efficacy in the mouse formalin model
was unexpected given the published NaV1.7 mouse knock out
data.11 Ultimately this data contributed to compound 5 being
discontinued as a potential intravenous drug candidate for pain.
However, the exquisite potency and selectivity coupled with the
opportunity to exploit the free piperidine for the synthesis of
probe derivatives, suggest that these compounds could potentially
be used as tools to enable further work to better understand target
aSee SI for details of electrophysiology assay protocols.
Given the significant changes in physicochemical properties
between compound 5 and the oral series, including increased
molecular weight and the addition of the pendent dibasic
sidechain, it was unclear if this would still be the case. As a result

engagement and how selective chemical modulation of NaV1.7
might translate into pain efficacy.34,35,36
glycol; PK, pharmacokinetics; ppb, plasma protein binding; PX,
PatchXpress® electrophysiology; RHEP, rat hepatocytes; RLM,
rat liver microsomes; RRCK, Ralph Russ canine kidney cell line;
SAR, structure activity relationship; SNAr, nucleophilic aromatic
substitution; T1/2, half-life; TEA, trimethylamine; TEMPO,
(2,2,6,6-Tetramethylpiperidin-1-yl)oxyl; THF, tetrahydrofuran;
TPSA, topological polar surface area; Vss, apparent volume of
distribution at steady state.
Supplementary Material
Electronic Supplementary Information (ESI) available:
Chemistry experimental procedures and characterisation for
1
compounds, HPLC, MS, H NMR, and 13C NMR spectra for key
compound 5, electrophysiology assay protocols and in vivo
pharmacology.
Author Information
#Current address: Astrazeneca, Darwin Building, 310
Cambridge Science Park, Milton Road, Cambridge, CB4 0FZ,
UK.
Figure 3. Mouse formalin efficacy model. To achieve steady state plasma
concentrations, IV infusion of compound commenced 90 min prior to
formalin injection (0 min on graph) and continued throughout the testing
phase. Dashed lines indicate unbound plasma concentration measured at the
end of the experiment. However, these are expected to be representative of
the entire testing period.
References and notes
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In summary, a highly potent and subtype selective NaV1.7
inhibitor PF-06456384 (5) was designed and synthesised for
application as an IV infusion agent. The compound was
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Abbreviations
ADME, absorption, distribution, metabolism and excretion;
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DMAc,
dimethylacetamide;
DMF,
dimethylformamide; DMSO, dimethyl sulfoxide EP, manual
electrophysiology; F, bioavailability; fu, fraction unbound;
HHEP, human hepatocytes; HLM, human liver microsomes, IC50,
half-maximum inhibitory concentration; IV, intravenous; lipE,
lipophilic efficiency; NHS, N-hydroxysuccinamide; OATP,
organic anion-transporting polypeptide; PEG, polyethylene
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Graphical Abstract
Highly potent and selective NaV1.7 inhibitors for
use as intravenous agents and chemical probes
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R. Ian Storer∗, Andy Pike, Nigel A. Swain, Aristos J Alexandrou, Bruce M. Bechle, David C. Blakemore, Alan D. Brown, Neil A. Castle, Matthew S.
Corbett, Neil J. Flanagan, David Fengas, M. Scott Johnson, Lyn H. Jones, Brian E. Marron, C. Elizabeth Payne, David Printzenhoff, David J. Rawson,
Colin R. Rose, Thomas Ryckmans, Jianmin Sun, Jonathan W Theile, Rubben Torella, Elaine Tseng and Joseph S. Warmus