Discovery and hit-to-lead evaluation of piperazine amides as selective, state-dependent NaV1.7 inhibitors

Article abstract of DOI:10.1039/c6md00578k

Na_V1.7 is a particularly compelling target for the treatment of pain. Herein, we report the discovery and evaluation of a series of piperazine amides that exhibit state-dependent inhibition of Na_V1.7. After demonstrating significant pharmacodynamic activity with early lead compound 14 in a Na_V1.7-dependent behavioural mouse model, we systematically established SAR trends throughout each sector of the scaffold. The information gleaned from this modular analysis was then applied additively to quickly access analogues that encompass an optimal balance of properties, including Na_V1.7 potency, selectivity over Na_V1.5, aqueous solubility, and microsomal stability.

Full text of DOI:10.1039/c6md00578k

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RESEARCH ARTICLE
Discovery and hit-to-lead evaluation of piperazine
amides as selective, state-dependent NaV1.7
inhibitors†‡
Cite this: DOI: 10.1039/c6md00578k
Brian A. Sparling, a S. Yi,a J. Able,c H. Bregman,a Erin F. DiMauro,§a R. S. Foti,d
*
H. Gao,e A. Guzman-Perez,a H. Huang,¶a M. Jarosh,b T. Kornecook,c J. Ligutti,c
B. C. Milgram,a B. D. Moyer,c B. Youngblood,c V. L. Yub and M. M. Weissa
NaV1.7 is a particularly compelling target for the treatment of pain. Herein, we report the discovery and
evaluation of a series of piperazine amides that exhibit state-dependent inhibition of NaV1.7. After demon-
strating significant pharmacodynamic activity with early lead compound 14 in a NaV1.7-dependent behav-
ioural mouse model, we systematically established SAR trends throughout each sector of the scaffold. The
information gleaned from this modular analysis was then applied additively to quickly access analogues that
encompass an optimal balance of properties, including NaV1.7 potency, selectivity over NaV1.5, aqueous
solubility, and microsomal stability.
Received 14th October 2016,
Accepted 30th November 2016
DOI: 10.1039/c6md00578k
www.rsc.org/medchemcomm
expressed in atrial and ventricular myocytes, has been shown
to prolong the cardiac QRS wave in humans, causing a de-
Introduction
The voltage-gated sodium channel NaV1.7 plays a key role in
the propagation of pain signalling in humans.1,2 NaV1.7 is
expressed in high levels in nociceptive sensory neurons within
the dorsal root ganglia,3 and mutations of the gene encoding
for NaV1.7, SCN9A, are linked to a variety of pain disorders.4
Given that current pain treatments suffer from either lack of
efficacy or dose-limiting toxicity,5 NaV1.7 is a particularly com-
pelling target for the treatment of pain. However, despite ex-
tensive studies by various organizations,6 a NaV1.7-selective
inhibitor has yet to reach patients.7 One challenge associated
with targeting NaV1.7 for pain is garnering selectivity over
other members of the NaV family of channels (NaV1.1–
NaV1.9). In particular, inhibition of NaV1.5, which is
crease in conduction velocity which may ultimately lead to ad-
verse cardiovascular events.8
Recently, we have reported the lead optimization of
heteroaryl sulfonamides and biaryl acyl sulfonamides as po-
tent NaV1.7 inhibitors possessing a high degree of selectivity
over other NaV isoforms.9 Despite excellent potency and
NaV1.7 selectivity, the development of these sulfonamides
has been hampered by several factors. In addition to PXR ac-
tivation and inhibition of multiple CYP isoforms, lipophilic
acids such as these are frequently subject to transporter-
mediated clearance pathways, and wide variance of such
clearance routes amongst preclinical species leads to chal-
lenges in accurately predicting human dose.7c Accordingly,
we pursued the advancement of alternative chemotypes with
diverse physicochemical properties, particularly those outside
of lipophilic acid chemical space.
Furthermore, we were interested in developing chemical
matter that displayed use- or state-dependent inhibition of
NaV1.7.10 An abnormally hyperactive nociceptive neuron with
frequent action potential firing or a depolarized membrane
potential significantly increases the proportion of channels in
certain inactivated states including fast- and slow-inactivated
states.11 A compound that has selective affinity towards
NaV1.7 in these inactive conformations should not only in-
hibit nociceptive hyperactivity but also possess functional se-
lectivity over other NaV channels in cells firing at lower rates
or with less depolarized membrane potentials. This functional
selectivity would potentially garner a larger margin of safety
over a comparable inhibitor that lacks state dependence.
a Department of Medicinal Chemistry, Amgen Inc., 360 Binney Street, Cambridge,
MA, 02142, USA. E-mail: brian.sparling@amgen.com
b Department of Neuroscience, Amgen Inc., 360 Binney Street, Cambridge, MA,
02142, USA
c Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand
Oaks, CA, 91320, USA
d Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 360 Binney
Street, Cambridge, MA, 02142, USA
e Department of Molecular Engineering, Amgen Inc., 360 Binney Street,
Cambridge, MA, 02142, USA
† The authors declare no competing financial interests.
‡ Electronic supplementary information (ESI) available: Assay protocols, experi-
mental procedures, spectra of key compounds. See DOI: 10.1039/c6md00578k
§ Current address: Merck & Co., 33 Avenue Louis Pasteur, Boston, MA, 02115,
USA.
¶ Current address: Assistant Professor, Baylor College of Medicine, One Baylor
Plaza, BCM315, Houston, TX, 77030, USA.
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Our interest in a state- or use-dependent, nonacidic Results and discussion
NaV1.7 inhibitor led to the discovery of a novel series of pi-
Owing to our interest in developing state-dependent inhibi-
tors of NaV1.7, we evaluated compounds using two distinct
electrophysiological assays run on IonWorks Barracuda (IWB)
and PatchXpress (PX) automated electrophysiology platforms.
In the IWB assay protocol, a series of high frequency
depolarizing pulses at 5 Hz are applied to cells following a
four-minute period where cells are unclamped, with a resting
membrane potential around −20 mV, to induce a slow-
inactivated state. Within this series of pulses, a potency shift
between the first tonic pulse and after the entire sequence is
indicative of use dependence. In the PX assay protocol, cells
are voltage-clamped at a membrane potential resulting in 20–
50% channel inactivation. We postulated that compounds
exhibiting enhanced potency against NaV1.7 in the IWB assay
protocol when compared to the PX assay protocol would dem-
onstrate increased affinity to a slow-inactivated channel state
and/or exhibit use-dependent block.15
As previously mentioned, selectivity over NaV1.5 is critical
for establishing a safety window. NaV1.5 is expressed in the
heart, where it controls the upstroke of the cardiac action po-
tential.8 We determined NaV1.5 potency using a PX assay,
where channels are partially inactivated. The IWB assay volt-
age parameters are not relevant to normal NaV1.5 function,
as human cardiomyocytes are not exposed to high frequency
pulses or prolonged periods of depolarization under physio-
logical conditions. Our decision to use the PX assay to deter-
mine selectivity over NaV1.5 was substantiated through
Langendorff isolated rabbit heart studies16 using several of
our piperazine amides; QRS prolongation was more corre-
lated with NaV1.5 PX potency rather than NaV1.5 IWB
potency.17
perazine amides. Herein we report our efforts to understand
the structure–activity relationship of the series, which led to
the development of potent inhibitors of NaV1.7 possessing
favourable physicochemical and pharmacokinetic properties
as well as selectivity over NaV1.5. We also provide preliminary
results of a representative early lead piperazine amide in a
histamine-induced mouse pruritus model of NaV1.7
engagement.9
Chemistry
In order to quickly access derivatives of key positions around
the scaffold, we employed two distinct synthetic routes
(Scheme 1). An Ugi three-component coupling using benzyl
isocyanate (1), a benzaldehyde 2, and N-Boc-piperazine (3)
allowed us to access key intermediate 4 in short order
(Scheme 1a).12 Subsequent acid-mediated Boc removal and
amide coupling with a benzoic acid derivative 5 afforded ra-
cemic final compounds 6. Analogues containing variations to
the piperazine core were also made using this methodology.
While the Ugi three-component protocol worked well to
rapidly deliver a variety of analogues, limitations to the isocy-
anate component scope as well as scalability concerns
prompted us to develop alternative methodology
(Scheme 1b). AIBN-promoted bromination of an α-toluic acid
ester 7 provided bromides 8.13 Bromide displacement using
either N-Boc-piperazine (3) or a suitably pre-functionalized pi-
perazine amide derivative 9 afforded esters 10. Subsequent
ester saponification and amide coupling with a primary
amine 11 facilitated the synthesis of analogues 12 directly
when 9 was employed and an intermediate similar to 4 when
3 was employed. Chiral separation of late-stage intermediates
(e.g., 4) or final compounds was used to furnish enantio-
enriched products.14 Compounds were generally resistant to
racemization/epimerization at the carbonyl α-methine unless
exposed to strongly basic conditions for prolonged periods.
Screening Amgen's compound collection identified com-
pound 13 (Fig. 1) as a potent inhibitor of NaV1.7.19 Subse-
quent analysis revealed that this compound was 35-fold less
potent in the NaV1.7 PX assay compared to the IWB assay,
highlighting preferential block of a slow-inactivated state or
use-dependent block.20 In addition to its potency, this
Scheme 1 (a) Ugi three-component coupling and (b) α-toluic acid ester routes to piperazine amide analogues. Reagents and conditions: (a) HOAc,
TFE; (b) TFA, CH2Cl2; (c) T3P, NEt3, CH2Cl2; (d) NBS, AIBN, CCl4, 85 °C; (e) K2CO3, DMF; (f) LiOH, THF, H2O, MeOH; (g) T3P, NEt3, CH2Cl2.
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dermal injection of histamine to the nape of the neck. The
number of scratching bouts was then recorded by an experi-
menter blinded to compound treatment over a 30 minute pe-
riod. At this dose, 14 produced a statistically significant
(76%) reduction in scratching bouts compared to vehicle.
This reduction was similar in magnitude to that produced by
a 30 mg kg−1 oral dose of diphenhydramine (DPH), an anti-
histamine that served as a positive control. The plasma con-
centration of 14 measured immediately after the study
(plasma Cu = 0.82 μM, 1.5 h) indicated a 1.8-fold coverage
over the mouse IWB IC50. Also noteworthy is the lack of sig-
nificant effects associated with a 300 mg kg−1 oral dose of
compound 14 in a mouse open-field assay, which afforded
similar exposure as the histamine-induced itch study.25 This
indicates that the reduction in scratching behaviour in the
histamine pruritus model is unlikely to be the result of
nonspecific alterations in locomotor activity.26
Having shown in vivo target engagement with a member
of this series, we sought to make further improvements to
microsomal stability and selectivity over NaV1.5 via judicious
SAR analysis. While we could leverage in silico tools to guide
our subsequent SAR studies concerning microsomal stability,
the lack of structural information concerning the differential
binding of 14 to both NaV1.7 and NaV1.5 motivated us to si-
multaneously evaluate a variety of SAR hypotheses in a rapid,
efficient manner. With this in mind, we divided the scaffold
of 14 into four distinct domains (see Fig. 2a) and leveraged
our synthetic methodology to simultaneously explore SAR
across all sectors. Moreover, we aspired to establish if SAR,
particularly concerning selectivity over NaV1.5, would be addi-
tive across these domains and would allow us to quickly ac-
cess high quality lead compounds for subsequent in vivo
analysis.
Fig. 1 Structure and properties of screening hit 13.
compound exhibited good permeability and was not a sub-
strate for Pgp-mediated efflux.21 Despite these advantageous
properties, 13 exhibited high intrinsic clearance in human
and mouse liver microsomes and lacked selectivity over
NaV1.5.
In order to quickly drive a decision whether or not to pur-
sue this series, we sought to evaluate either 13 or a close de-
rivative in vivo. In order to address the high microsomal in-
trinsic clearance of this compound, we initially hypothesized
that eliminating the metabolically labile piperazinyl benzylic
amine functionality would impart a degree of stability while
lowering clog P.22 Indeed, replacement of the benzylic amine
with an aryl amide in compound 14 not only lowered micro-
somal turnover but also increased selectivity over NaV1.5
while maintaining potency in our NaV1.7 IWB assay (Fig. 2a).
Oral administration at a 100 mg kg−1 dose in mice provided
sufficient exposure to warrant further study.
Piperazine amide 14 was subsequently evaluated in a
mouse histamine-induced pruritus model of NaV1.7 engage-
ment (Fig. 2b). In humans, a gain-of-function NaV1.7 muta-
tion has been reported to cause episodes of spontaneous
itch.23 Furthermore, NaV1.7 knockout mice fail to exhibit
scratching behaviour following an intradermal histamine
challenge.4a The robustness and reliability of this behaviour,
the ease of study execution, and this assay's translational po-
tential to human studies make this model particularly useful
for the in vivo evaluation of NaV1.7 inhibition,24 as we have
previously reported.9a In the current experiment, 14 (or a ve-
hicle control solution) was administered orally at a dose of
300 mg kg−1 to C57BL/6 male mice 60 minutes prior to intra-
First, we further explored the SAR of the amide A-ring ex-
emplified by compounds 15–18 (Table 1).27 In line with 14,
these amides generally possessed levels of selectivity over
NaV1.5 greater than benzyl piperazine 13 and were signifi-
cantly more metabolically stable. 3,5- and 2,5-disubstitution
provided the optimal balance of NaV1.7 potency and selectiv-
ity over NaV1.5. This balance is exemplified with analogue 16;
accordingly, a 3,5-difluorophenyl A-ring was selected for
Fig. 2 (a) In vitro and in vivo parameters of 14. Sectors of SAR analysis are labelled in the structure of 14. (b) Reduction of scratching behaviour
observed with 14 (300 mg kg−1, p.o.) and diphenhydramine (DPH, 30 mg kg−1, p.o.) in a mouse histamine-induced pruritus model compared to ve-
hicle, with exposure levels of 14. ***, p < 0.001; ****, p < 0.0001 versus vehicle group (one-way ANOVA followed by Dunnet’s tests).
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Table 1 SAR of A-ring amide sector
NaV1.7 IWB
IC50 (μM)
NaV1.5 PX
IC50 (μM)
NaV1.5/1.7
selectivity
HLM Clint
Solubility at
pH 7.4 (μM)
No.
Structure
LE/LipEa
0.26/1.4
(μL min−1 mg−1
)
clog P
14
0.37
0.90
2.4×
74
96
5.0
15
16
1.48
0.26
0.24/2.0
0.27/2.1
9.1
6.2×
27
454
137
3.9
4.4
1.3
5.0×
3.8×
1.1×
95
29
50
17
18
0.62
0.35
0.25/2.4
0.24/1.5
3.1
248
44
3.8
5.0
0.40
a
LipE = −log(NaV1.7 IWB IC50) − clog P.
Table 2 SAR of the B-ring aryl sector
NaV1.7 IWB
IC50 (μM)
NaV1.5 PX
IC50 (μM)
NaV1.5/1.7
selectivity
HLM Clint
Solubility at
pH 7.4 (μM)
No.
Structure
LE/LipEa
(μL min−1 mg−1
<14
)
clog P
19
0.80
0.24/1.7
6.5
8.1×
74
4.4
20
0.20
0.25/2.0
0.50
2.4×
33
30
4.7
a
LipE = −log(NaV1.7 IWB IC50) − clog P.
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inclusion in subsequent SAR analysis of other regions along
the scaffold. A meta-nitrile substituent (17) was effective for
lowering clog P and decreasing microsomal turnover. Inser-
tion of a nitrogen atom to form pyridine 15 resulted in a loss
of NaV1.7 potency but also an increase in selectivity over
NaV1.5 and microsomal stability. While large, lipophilic
groups tended to increase NaV1.7 potency, these groups did
not necessarily lead to gains in lipophilic efficiency and fre-
quently led to decreases in both selectivity over NaV1.5 and
aqueous solubility, as illustrated by analogue 18. We sur-
mised that this region of our scaffold may be residing in a li-
pophilic pocket of both NaV1.5 and NaV1.7; however, it
seemed probable that a key polar or π-stacking interaction
driven by electron-withdrawing substituents along this ring
engendered differential binding in favour of NaV1.7.
We next turned our attention to modifications of the re-
mainder of the scaffold. After exploring various mono-
substitution patterns along the B-ring, we found that 4-cyano
substitution (19) provided a desirable combination of NaV1.7
potency and selectivity over NaV1.5 (Table 2). In addition, this
group also blocked a putative site of oxidative metabolism
which in combination with its greater polarity led to a lower
observed intrinsic clearance in human liver microsomes. Fur-
ther modifications based upon 19 in attempts to improve
NaV1.7 potency, such as derivative 20, led to erosions in selec-
tivity over NaV1.5 with concomitant increases in microsomal
turnover and decreases in aqueous solubility.
The SAR around the benzylic amide C-ring position was
extensively explored (Table 3). Methylation of the secondary
amide to afford 21 resulted in a significant loss of NaV1.7 po-
tency, illustrating the importance of this hydrogen-bond do-
nor. We hypothesized that several positions along this region
would be particularly prone to oxidative metabolism. Both
blocking the para position of the aryl ring with
a
trifluoromethyl group and increasing polarity via the use of a
pyridine in 22 resulted in an increase in microsomal stability
and selectivity over NaV1.5.
We additionally pursued several strategies for masking the
potentially metabolically labile benzylic methylene group at-
tached to the C-ring. Excision of this methylene entirely,
resulting in aniline 23, also led to an increase in microsomal
stability and enhanced selectivity over NaV1.5. Further optimi-
zation led to discovery of the 6-(2,2,2-trifluoroethoxy)pyridin-3-
amine group, found in 24, which conferred a marked im-
provement in potency and microsomal stability. Despite these
improvements, the anilines and pyridinamines generally suf-
fered from poor aqueous solubility and significantly higher
molecular weight. We therefore targeted small, alkyl
Table 3 SAR of C-ring benzyl amide sector
NaV1.7 IWB
IC50 (μM)
NaV1.5 PX
IC50 (μM)
NaV1.5/1.7
selectivity
HLM Clint
Solubility at
pH 7.4 (μM)
No.
Structure
LE/LipEa
0.24/1.3
(μL min−1 mg−1
)
clog P
21
1.22
10
8.4×
306
220
4.6
22
0.21
0.25/2.7
7.9
22×
14
225
4.0
23
24
0.42
0.11
0.24/0.8
0.25/1.7
9.5
3.0
23×
27×
24
0
5.6
5.2
<14
19
25
26
0.95
0.31
0.27/2.5
0.27/2.4
7.7
6.7
8.1×
22×
<14
<14
472
346
3.5
4.1
a
LipE = −log(NaV1.7 IWB IC50) − clog P.
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replacements to this benzylic moiety to not only address
these shortcomings but also to achieve our general goal of in-
creasing metabolic stability. Cyclopropyl 25 and homologated
derivative 26 provided competent replacements to the aryl
ring, resulting in compounds with lower molecular weight
and microsomal turnover, increased LipE and increased solu-
bility, and adequate NaV1.7 potency while maintaining
existing levels of selectivity over NaV1.5.
To culminate our SAR explorations around the scaffold,
we investigated replacements for the piperazine D-ring
(Table 4). We hypothesized that rigidifying this ring would re-
sult in increased selectivity over NaV1.5, presumably by
disfavouring conformations that preferentially engage
NaV1.5. In this regard, a particularly effective strategy was the
deployment of bridging methylene and ethylene units along
the piperazine ring. Greater selectivity over NaV1.5 was ob-
served with analogues containing bridging elements placed
toward the A-ring-connected portion of the piperazine. Both
diazabicyclo[3.1.1]heptane 27 and diazabicyclo[3.2.1]octane
28 were significantly more selective over NaV1.5 than parent
piperazine 14 with comparable NaV1.7 potency. In general,
these bridged piperazines suffered from increased micro-
somal clearance and lower aqueous solubility.
perhaps targeting analogues that contain particularly solubi-
lizing moieties in other portions of the scaffold would im-
prove solubility while maintaining or enhancing the benefits
imparted by this group. Accordingly, analogue 29 was pre-
pared. Through the strategic use of the pyridyl A-ring, this
analogue featured increased solubility and selectivity over
NaV1.5 while maintaining high levels of microsomal stability
and NaV1.7 potency. The attachment of a para-nitrile to the
toluic acid ring in 30 also engendered improved selectivity
over NaV1.5.
We also focused efforts on improving the NaV1.7 potency
of the alkyl-substituted C-ring amides and found that cyclo-
propylmethyl amides 31 and 32, both bearing diaza-
bicyclo[3.2.1]octane and para-nitrile toluic acid rings, not only
possessed increased potency, low microsomal clearance, and
high aqueous solubility but also significantly improved levels
of selectivity over NaV1.5 (Table 5). These compounds had par-
ticularly high levels of NaV1.7 lipophilic efficiency and an excel-
lent overall balance of properties. Similar explorations were
undertaken with derivatives of cyclopropylethyl amide 26.
3-Cyano-5-fluorophenyl A-ring amide 33 maintained a similar
level of NaV1.7 potency with a substantial increase in selectiv-
ity over NaV1.5. A significant loss of potency was observed for
With explorations of each individual sector completed, we
were now in a position to test our hypothesis that SAR would
be additive once these individual components were aggregated
in new compounds (Table 5). In addition to primarily address-
ing selectivity over NaV1.5, we also sought to ameliorate any is-
sues associated with specific functionality through prudent
combinations of groups across the scaffold. For instance,
while the 6-(2,2,2-trifluoroethoxy)pyridin-3-amine C-ring in 24
imparted stability and potency, aqueous solubility worsened;
most derivatives bearing a para-nitrile B-ring; however,
through the use of the lipophilic 2-chloro-5-fluorophenyl am-
ide, 34 bore only a twofold loss of potency compared to 26
while maintaining >40-fold selectivity over NaV1.5, good
aqueous solubility, and microsomal stability. Owing to their
favourable properties, analogues 33 and 34 were subjected to
further analysis. Both compounds exhibited low intrinsic
clearance in mouse liver microsomes, particularly when com-
pared to 14. Oral administration of 33 and 34 in mice at 100
Table 4 SAR of piperazine sector
NaV1.7 IWB
IC50 (μM)
NaV1.5 PX
IC50 (μM)
NaV1.5/1.7
selectivity
HLM Clint
Solubility at
pH 7.4 (μM)
No.
Structure
LE/LipEa
0.26/1.7
(μL min−1 mg−1
)
clog P
27
0.35
2.4
7.0×
>399
83
4.8
28
0.46
0.25/1.0
4.2
9.0×
270
35
5.3
a
LipE = −log(NaV1.7 IWB IC50) − clog P.
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Table 5 Additive SAR reveals piperazine analogues with good overall balance of propertiesa
NaV1.7 IWB
IC50 (μM)
NaV1.5 PX
IC50 (μM)
NaV1.5/1.7
selectivity
HLM Clint
Solubility at
pH 7.4 (μM)
No.
Structure
LE/LipE
0.23/1.8
(μL min−1 mg−1
)
clog P
29
0.34
11
32×
<14
49
4.6
30
31
32
0.25
0.22/2.0
0.25/3.0
0.25/2.9
9.6
39×
<14
15
322
326
4.7
3.3
3.3
0.58
0.72
>42
>73×
19
42
59×
<14
33
34
0.35
0.64
0.26/3.0
0.24/2.1
14
27
41×
42×
<14
<14
405
154
3.5
4.1
a
LipE = −log(NaV1.7 IWB IC50) − clog P.
mg kg−1 revealed improved coverage of the mouse IWQ28
IC50: 1.8-fold and 5.4-fold, respectively (Table 6).
Table 6 In vitro and in vivo parameters of 33 and 34
Conclusions
33
34
Human NaV1.7 IWB IC50 (μM)
Mouse NaV1.7 IWQ28 IC50 (μM)
HLM/MLM Clint (μL min−1 mg−1
Mouse plasma fu
0.35
0.91
<14/134
0.13
470/3.5/76
0.64
0.60
<14/59
0.11
505/4.1/76
Through high-throughput screening efforts, we identified 13,
a novel chemotype that displayed state-dependent inhibition
of NaV1.7. Metabolic liability was mitigated through
piperazinyl amide analogue 14, which also possessed im-
proved selectivity over NaV1.5. In a histamine-induced pruri-
tus assay in mice, oral dosing of piperazine amide 14 at 300
mg kg−1 resulted in a significant decrease in scratching
)
MW/clog P/tPSA
Mouse PK (100 mg kg−1 P. O. dose)
Cmax (μM)
Cmax,unbound (μM)
12.8
1.66
29.8
3.28
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bouts, indicative of the effectiveness of this series toward en-
gaging NaV1.7 in a therapeutically relevant manner. Subse-
quent SAR explorations of each sector of the scaffold allowed
us to quickly access analogues of this compound with an em-
phasis on balancing NaV1.7 potency, selectivity over NaV1.5,
and microsomal stability. Enabled by these modular SAR
studies, we circumvented the need for a time- and resource-
intensive matrix SAR effort and were able to rapidly access
highly desirable analogues with enhanced selectivity over
NaV1.5 and a good overall balance of properties, exemplified
by compounds 31–34, with 33 and 34 displaying favourable
mouse PK. It is noteworthy that we were able to significantly
increase selectivity over NaV1.5 in this series while concur-
rently maintaining consistent NaV1.7 LE and improving
NaV1.7 LipE. Our hope is that the unique properties of this
series may be leveraged to deliver tools for use by the broader
scientific community to further elucidate NaV1.7 biology and
function.
Na2SO4, and concentrated under reduced pressure to provide
2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic acid (20
g, 84% yield) as a white amorphous solid. 1H NMR (400
MHz, DMSO-d6) δ 7.48–7.33 (m, 5H), 4.22 (s, 1H), 2.56–2.51
(m, 4H), 2.47–2.40 (m, 4H), 1.37 (s, 9H). LCMS (ESI) m/z:
321.2 (M + H)+.
To
a
CH2Cl2 (105 mL) solution of 2-(4-(tert-
butoxycarbonyl)piperazin-1-yl)-2-phenylacetic acid (21 g, 66
mmol) and phenylmethanamine (9.13 g, 85.0 mmol) was
added triethylamine (27.4 mL, 197 mmol) and propyl-
phosphonic anhydride (31.3 g, 98 mmol). The reaction mix-
ture was stirred at rt for 12 h. The reaction was diluted with
CH2Cl2 (250 mL) and H2O (300 mL). The layers were sepa-
rated, and the organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The material was pu-
rified by column chromatography using 60–120 mesh silica
gel, eluting with 20% EtOAc in hexane, to provide tert-butyl
4-(2-(benzylamino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate
(18 g, 67% yield) as a white amorphous solid. 1H NMR (400
MHz, DMSO-d6) δ 7.47–7.40 (m, 2H), 7.41–7.26 (m, 5H), 7.24–
7.20 (m, 1H), 7.18–7.12 (m, 2H), 4.32–4.20 (m, 2H), 3.90 (d, J
= 2.3 Hz, 1H), 3.32 (d, J = 8.6 Hz, 4H), 2.28 (q, J = 4.0, 3.2 Hz,
4H), 1.38 (d, J = 2.1 Hz, 9H). LCMS (ESI) m/z: 410.2 (M + H)+.
Experimental section
In vitro assays
Protocols for IWB and PX assays are provided in the ESI.‡
To
a
1,4-dioxane (74 mL) solution of tert-butyl
4-(2-(benzylamino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate
(14.7 g, 35.9 mmol) was added hydrogen chloride (4.0 M in
1,4-dioxane, 40. mL, 180 mmol), and the reaction mixture
was stirred at rt for 12 h. The reaction mixture was then con-
centrated in vacuo. The material was then stirred with Et2O
(50 mL) and filtered to afford racemic N-benzyl-2-phenyl-2-
(piperazin-1-yl)acetamide hydrochloride as white amorphous
solid. This racemate was subjected to preparatory SFC purifi-
cation using a (S,S) Whelk-O column (5 μm, 5 × 15 cm), elut-
ing with 30% MeOH containing 0.2% HNEt2 at a flowrate of
350 mL min−1 with diode array detection at 225 nm to
provide (R)-N-benzyl-2-phenyl-2-(piperazin-1-yl)acetamide (5.08
g, 16.4 mmol, 46% yield, >99% ee) as a white amorphous
In vivo studies
Descriptions of mouse P. O. PK studies and the histamine-
induced pruritus experiment are provided in the ESI.‡
General synthetic methodology
Representative experimental details for the syntheses of 14
and 34 are found below. Further experimental details and
spectral data are found in the ESI.‡
(R)-N-Benzyl-2-(4-(2-chloro-5-fluorobenzoyl)piperazin-1-yl)-
2-phenylacetamide (14). To
a DMF (1.0 L) solution of
tert-butyl piperazine-1-carboxylate (3, 100 g, 538 mmol) was
added potassium carbonate (80 g, 58 mmol) and methyl
2-bromo-2-phenylacetate (73.5 g, 320 mmol). The reaction
contents were stirred at rt for 12 h. The reaction mixture was
then filtered through a bed of celite, and the filtrate was con-
centrated in vacuo. The material was purified by column
chromatography using 60–120 mesh silica gel, eluting with
20% EtOAc in heptane, to obtain tert-butyl 4-(2-methoxy-2-
oxo-1- phenylethyl)piperazine-1-carboxylate (100 g, 67% yield)
as a colourless oil. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J =
6.0 Hz, 2H), 7.42–7.33 (m, 3H), 4.06 (s, 1H), 3.71 (d, J = 1.1
Hz, 3H), 3.48 (s, 4H), 2.58–2.30 (m, 4H), 1.46 (d, J = 1.1 Hz,
9H). LCMS (ESI) m/z: 335.2 (M + H)+.
To a THF (125 mL)/H2O (62.5 mL) solution of tert-butyl
4-(2-methoxy-2-oxo-1-phenylethyl)piperazine-1-carboxylate (25
g, 74.8 mmol, 1.0 equiv.) was added lithium hydroxide (8.9 g,
370 mmol). The reaction mixture was stirred at rt for 12 h.
The reaction mixture was then concentrated in vacuo. The
material was acidified to pH 5 using 0.5 N aq. HCl. The layers
were separated, and the aqueous layer was extracted thrice
with CH2Cl2. The organic extracts were combined, dried over
1
solid. H NMR (400 MHz, DMSO-d6) δ 7.69–7.54 (m, 2H), 7.47
(dd, J = 5.3, 2.9 Hz, 3H), 7.31–7.18 (m, 3H), 7.16–7.03 (m,
2H), 5.03 (s, 1H), 4.31 (q, J = 9.1, 7.2 Hz, 2H), 3.35 (q, J =
14.6, 12.9 Hz, 4H), 3.18 (d, J = 31.3 Hz, 4H). LCMS (ESI) m/z:
310.2 (M + H)+.
To a CH2Cl2 (5.4 mL) solution of 2-chloro-5-fluorobenzoic
acid (0.283 g, 1.62 mmol) and (R)-N-benzyl-2-phenyl-2-
(piperazin-1-yl)acetamide (0.502 g, 1.62 mmol) was added tri-
ethylamine (0.34 mL, 2.4 mmol) and propylphosphonic anhy-
dride (50 wt% in EtOAc, 1.5 mL, 2.4 mmol). The solution was
stirred at rt for 16 h. Sat. aq. NaHCO3 was added. The mix-
ture was stirred at rt for 30 min and partitioned. The aqueous
layer was twice with CH2Cl2. The combined organic extracts
were combined, washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The material was absorbed
onto a plug of silica gel and purified by chromatography
through a Biotage Snap Ultra pre-packed silica gel column
(25 g), eluting with a gradient of 10% to 70% EtOAc in hep-
tane, to provide 14 (0.69 g, 1.5 mmol, 91% yield) as a white
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1
amorphous solid. H NMR (500 MHz, DMSO-d6) δ 8.68 (t, J =
6.1 Hz, 1H), 7.58–7.53 (m, 1H), 7.42 (d, J = 7.0 Hz, 2H), 7.36–
7.28 (m, 5H), 7.27–7.23 (m, 2H), 7.22–7.17 (m, 1H), 7.14 (d, J
= 7.8 Hz, 2H), 4.33–4.21 (m, 2H), 3.95 (s, 1H), 3.73–3.56 (m,
2H), 3.17 (t, J = 4.9 Hz, 2H), 2.48–2.38 (m, 2H), 2.38–2.28 (m,
2H). 13C NMR (101 MHz, DMSO-d6) δ 169.98, 164.04, 161.94,
159.50, 139.38, 137.40, 137.32, 136.94, 131.42, 131.34, 128.61,
128.18, 127.76, 127.01, 126.68, 124.49, 124.46, 117.64, 117.41,
115.17, 114.93, 79.15, 73.89, 73.87, 50.43, 46.09, 41.95, 41.09,
19F NMR (376 MHz, DMSO-d6) δ −115.02 (s, 1F). [α]2D2 = −65.7°
(c 1.35, CHCl3). HPLC (System A) Rt 1.00 min, >95% purity.
HRMS (ESI) m/z: [M + H]+ calculated for C26H25ClFN3O2:
466.1692, found 466.1697.
thrice with CH2Cl2. The organic extracts were combined,
washed with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo to afford racemic 2-(4-(2-chloro-5-
fluorobenzoyl)piperazin-1-yl)-2-(4-cyanophenyl)acetic acid (15.0
g, 37.3 mmol, 78% yield) as a white amorphous solid. This
racemate was subjected to preparatory SFC purification using
a Chiralpak AD-H column (5 μm, 3 × 25 cm), eluting with
30% MeOH containing 0.5% HNEt2 at a flowrate of 120 mL
min−1 with diode array detection at 230 nm to provide (R)-2-
(4-(2-chloro-5-fluorobenzoyl)piperazin-1-yl)-2-(4-cyanophenyl)-
1
acetic acid (4.11 g, 96.7% ee) as a white amorphous solid. H
NMR (400 MHz, DMSO-d6): δ = 7.70 (d, J = 7.9 Hz, 2H), 7.65–
7.49 (m, 3H), 7.31 (dd, J = 8.4, 6.6 Hz, 2H), 3.84–3.56 (m, 4H),
3.22–3.11 (m, 2H), 2.75 (q, J = 7.1 Hz, 1H), 2.43–2.31 (m, 1H),
2.24 (dt, J = 10.6, 5.1 Hz, 1H). LCMS (ESI) m/z: 402.1 (M +
H)+.
(R)-2-(4-(2-chloro-5-fluorobenzoyl)piperazin-1-yl)-2-(4-
cyanophenyl)-N-(2-cyclopropyl-2,2-difluoroethyl)acetamide
(34). To a CH2Cl2 (800 mL) solution of tert-butyl 4-(1-(4-
cyanophenyl)-2-methoxy-2-oxoethyl)piperazine-1-carboxylate
(80.0 g, 223 mmol) was added trifluoroacetic acid (86.0 mL,
1.12 mol). The reaction mixture was stirred at rt for 2 h. The
reaction mixture was then concentrated in vacuo. The mate-
rial was dissolved in H2O and washed twice with MTBE. The
aqueous layer was basified with 20% NaHCO3 (aq.) to adjust
to about pH 9 and then extracted twice with 10% MeOH in
CH2Cl2. The organic extracts were combined, washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo
to afford methyl 2-(4-cyanophenyl)-2-(piperazin-1-yl)acetate
To
a CH2Cl2 (0.6 mL) mixture of 2-cyclopropyl-2,2-
difluoroethanamine (0.035 g, 0.25 mmol) and (R)-2-(4-(2-
chloro-5-fluorobenzoyl)piperazin-1-yl)-2-(4-cyanophenyl)acetic
acid (0.050 g, 0.12 mmol) was added triethylamine (0.035
mL, 0.25 mmol) and propylphosphonic anhydride (50 wt% in
EtOAc, 0.16 ml, 0.25 mmol). The mixture was shaken for 16 h
at rt. The mixture was filtered and purified via high through-
put parallel purification to provide 34 (36.1 mg, 0.0715 mmol,
60% yield) as a white amorphous solid. 1H NMR (500 MHz,
DMSO-d6) δ 8.64 (br s, 1H), 7.82 (br d, J = 8.30 Hz, 2H), 7.61
(br d, J = 7.92 Hz, 2H), 7.56 (br s, 1H), 7.25–7.37 (m, 2H),
4.18 (br s, 1H), 3.53–3.74 (m, 4H), 3.14–3.20 (m, 2H), 2.26–
2.47 (m, 4H), 1.27 (br s, 1H), 0.39–0.54 (m, 4H). 13C NMR
(101 MHz, DMSO-d6) δ 169.35, 164.05, 161.95, 159.50, 142.39,
137.33, 137.26, 132.16, 131.44, 131.36, 129.56, 124.48, 124.45,
122.00, 118.67, 117.68, 117.45, 115.19, 114.94, 110.60, 72.72,
50.53, 50.38, 50.01, 49.80, 46.07, 43.38, 43.08, 41.05, 13.92,
13.64, 13.37, 0.82. 19F NMR (376 MHz, DMSO-d6) δ = −105.61–
107.76 (m, 2F), −115.01 (s, 1F). [α]2D2 = −47.3° (c 0.63, CHCl3).
HPLC (System A) Rt 1.07 min, >95% purity. HRMS (ESI) m/z:
[M + H]+ calculated for C25H24ClF3N4O2: 505.1613, found
505.1621.
1
(50.0 g, 193 mmol, 87% yield) as a colorless syrup. H NMR
(400 MHz, DMSO-d6) δ = 7.98–7.78 (m, 2H), 7.66–7.56 (m,
2H), 4.45 (s, 1H), 3.64 (s, 3H), 3.56 (s, 1H), 2.90 (t, J = 4.9 Hz,
4H), 2.48–2.41 (m, 4H). LCMS (ESI) m/z: 260.1 (M + H)+.
To
a
CH2Cl2 (300 mL) solution of methyl
2-(4-cyanophenyl)-2-(piperazin-1-yl)acetate (20.0 g, 77.0 mmol)
was added 2-chloro-5-fluorobenzoic acid (8.0 g, 77 mmol). Tri-
ethylamine (21.5 mL, 154 mmol) and propylphosphonic an-
hydride (50 wt% in EtOAc, 49.1 g, 154 mmol) was added to
the reaction mixture after cooling to 0 °C. The reaction mix-
ture was then stirred for 16 h while slowly warming to rt. The
reaction mixture was then quenched with sat. aq. NaHCO3
solution and was extracted twice with CH2Cl2. The organic ex-
tracts were combined, dried over Na2SO4, filtered, and con- Abbreviations
centrated in vacuo. The material was purified by column
chromatography through a Redi-Sep pre-packed silica gel col-
umn (120 g), eluting 0% to 50% EtOAc in hexane, to provide
BCRP Breast cancer resistance protein
clog P Calculated logarithm of the octanol/water partition
coefficient
Clint Intrinsic clearance
Cmax Maximum plasma concentration
methyl
2-(4-(2-chloro-5-fluorobenzoyl)piperazin-1-yl)-2-(4-
cyanophenyl)acetate (20.0 g, 48.1 mmol, 62% yield) as a white
amorphous solid. 1H NMR (400 MHz, DMSO-d6): δ = 7.96–
7.78 (m, 2H), 7.68–7.50 (m, 3H), 7.36–7.22 (m, 2H), 4.46 (d, J
= 2.9 Hz, 1H), 3.64 (d, J = 2.1 Hz, 3H), 3.14 (t, J = 4.8 Hz, 4H),
2.47–2.30 (m, 4H). LCMS (ESI) m/z: 416.1 (M + H)+.
To a THF (100 mL)/MeOH (100 mL)/H2O (200 mL) solu-
tion of methyl 2-(4-(2-chloro-5-fluorobenzoyl)piperazin-1-yl)-2-
(4-cyanophenyl)acetate (20.0 g, 48.1 mmol) was added lithium
hydroxide (5.76 g, 240 mmol), and the reaction mixture was
stirred at rt for 2 h. Volatiles were then removed in vacuo.
The concentrate was cooled to 0 °C and acidified with 1.5 N
HCl to about pH 6. The aqueous layer was then extracted
fu
HLM Human liver microsomes
IC50 Half-maximum inhibitory concentration
IWB IonWorks Barracuda assay
LE Ligand efficiency
Fraction unbound
LipE Lipophilic efficiency
MLM Mouse liver microsomes
MW Molecular weight
P-gp P-glycoprotein
Papp
Apparent permeability coefficient
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PK
PX
SAR
t1/2
Pharmacokinetics
Patel, M. Fantini and M. Rivara, Curr. Top. Med. Chem.,
2009, 9, 396.
PatchXpress assay
Structure–activity relationship
Half life
7 (a) For examples of NaV1.7-selective inhibitors in the clinic,
see: N. Price, R. Namdari, J. Neville, K. J. W. Proctor, S.
Kaber, J. Vest, M. Fetell, R. Malamut, R. Sherrington, S. N.
Pimstone and Y. P. Goldberg, Clin. J. Pain, 2016, DOI:
10.1097/AJP.0000000000000408; (b) L. Cao, A. McDonnell, A.
Nitzsche, A. Alexandrou, P.-P. Saintot, A. J. C. Loucif, A. R.
Brown, G. Young, M. Mis, A. Randall, S. G. Waxman, P.
Stanley, S. Kirby, S. Tarabar, A. Gutteridge, R. Butt, R. M.
McKernan, P. Whiting, Z. Ali, J. Bilsland and E. B. Stevens,
Sci. Transl. Med., 2016, 8, 335ra356; (c) H. M. Jones, R. P.
Butt, R. W. Webster, I. Gurrell, P. Dzygiel, N. Flanagan, D.
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Phipps, J. Segelbacher, B. Speed and K. Beaumont, Clin.
Pharmacokinet., 2016, 55, 875.
tPSA Topological polar surface area
Acknowledgements
We thank Grace Bi, Jonathan Carlson, Larry Miller, Laszlo
Varady, and Yinong Zhang for purification support; Paul
Krolikowski and Steve Hollis for analytical support; Dan Gillie
for electrophysiology support; and Margaret Y. Chu-Moyer for
proofreading this manuscript.
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Waxman, Mol. Pain, 2012, 8, 82; (b) N. A. Singh, C. Pappas,
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14 In general, one enantiomer was more potent than the other.
15 For an example of state dependence with other chemical
matter, we obtained the following human NaV1.7 IC50 values
for a heteroaryl sulfonamide from ref. 9a (compound 16):
0.010 μM (IWB) and 0.16 μM (PX).
16 J.-P. Valentin, P. Hoffman, F. De Clerck, T. G. Hammond
and L. Hondeghem, J. Pharmacol. Toxicol. Methods, 2004, 49,
171.
17 For example, significant QRS increases were not observed in
the isolated rabbit heart experiment at concentrations up to
30 μM compound 25. We obtained the following human
NaV1.5 IC50 values for this compound: 7.7 μM (PX) and 0.71
μM (IWB). It should be noted that the comparison of data
across different assay platforms could be potentially
misleading; however, in this instance, relevance of the
NaV1.7 IWB platform was substantiated through the
pharmacodynamic effect of 14 in the histamine-induced pru-
ritus model, and the relevance of the NaV1.5 PX platform
was substantiated through the isolated rabbit heart experi-
ment of 25.
22 Benzylic amines such as that found in 13 are found to be
metabolic liabilities and are accordingly flagged as
structural alerts. For more information, see: P. J. Edwards
and C. Sturino, Curr. Med. Chem., 2011, 18, 3116.
23 G. Devigili, R. Eleopra, T. Pierro, R. Lombardi, S. Rinaldo, C.
Lettieri, C. G. Faber, I. S. Merkies, S. G. Waxman and G.
Lauria, Pain, 2014, 155, 1702.
24 Histamine does not interact directly with NaV1.7; it acts
upstream via histamine receptors on C-fibre nerves that ex-
press NaV1.7, causing Ca2+ entry into the cell and subse-
quent depolarization that eventually leads to action potential
firing or NaV1.7-dependent signal transduction. However,
NaV1.7-knockout mice display an almost complete lack of re-
sponse to intradermal histamine application, and potent
and NaV1.7-selective sulfonamide inhibitors reduce itching
behaviour in an exposure- and dose-dependent manner. See
ref. 4a and 9a for further details.
18 All solubility data reported herein are thermodynamic (i.e.,
equilibrium) solubilities.
19 High-throughput screening of Amgen's compound collection
was conducted using a NaV1.7 IonWorks Quattro (IWQ)
electrophysiology platform, which is very similar to the IWB
platform. For more information, see: D. J. Gillie, S. J. Novick,
B. T. Donovan, L. A. Payne and C. Townsend, J. Pharmacol.
Toxicol. Methods, 2013, 67, 33.
20 Compound 13 was threefold less potent in the IWB assay
when comparing the first pulse to the last pulse, indicative
of minor use dependence. This trend was consistently
observed for other members of this series and was also
observed across all NaV1.7 species isoforms (e.g., human,
mouse, and rat) in the IWB assay. We observed a high
degree of state-dependent block of NaV1.7 (i.e., the differen-
tial potency of the IWB and PX assays).
25 See ESI‡ for details.
26 It should be noted that inhibition of multiple NaV
isoforms in addition to NaV1.7 cannot be ruled out as an
explanation for efficacy of 14 in the histamine-induced
pruritus model.
27 All data reported herein are the average of multiple
replicates (n ≥ 2). Also it should be noted that while ligand
efficiency (LE) is formally a function of binding energy, we
utilized it herein as a function of NaV1.7 IWB IC50 in order
to gauge improvements as the series progressed.
21 Both 13 and 14 have high permeability and low transporter-
mediated efflux, which may be indicative of blood–brain bar-
28 These data originate from an IonWorks Quattro (IWQ)
electrophysiology platform. See ref. 19 for more information.
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