Discovery of Acyl-sulfonamide Nav1.7 Inhibitors GDC-0276 and GDC-0310

Article abstract of DOI:10.1021/acs.jmedchem.1c00049

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profil

Full text of DOI:10.1021/acs.jmedchem.1c00049

pubs.acs.org/jmc
Drug Annotation
Discovery of Acyl-sulfonamide Nav1.7 Inhibitors GDC-0276 and
GDC-0310
Brian S. Safina, Steven J. McKerrall,* Shaoyi Sun, Chien-An Chen, Sultan Chowdhury, Qi Jia, Jun Li,
Alla Y. Zenova, Jean-Christophe Andrez, Girish Bankar, Philippe Bergeron, Jae H. Chang, Elaine Chang,
Jun Chen, Richard Dean, Shannon M. Decker, Antonio DiPasquale, Thilo Focken, Ivan Hemeon,
Kuldip Khakh, Amy Kim, Rainbow Kwan, Andrea Lindgren, Sophia Lin, Jonathan Maher,
Janette Mezeyova, Dinah Misner, Karen Nelkenbrecher, Jodie Pang, Rebecca Reese, Shannon D. Shields,
Luis Sojo, Tao Sheng, Henry Verschoof, Matthew Waldbrook, Michael S. Wilson, Zhiwei Xie,
Clint Young, Tanja S. Zabka, David H. Hackos, Daniel F. Ortwine, Andrew D. White, J.P. Johnson, Jr.,
C. Lee Robinette, Christoph M. Dehnhardt, Charles J. Cohen, and Daniel P. Sutherlin*
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ABSTRACT: Nav1.7 is an extensively investigated target for pain
with a strong genetic link in humans, yet in spite of this effort, it
remains challenging to identify efficacious, selective, and safe
inhibitors. Here, we disclose the discovery and preclinical profile of
GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors
that have completed Phase 1 trials. Our initial search focused on
close-in analogues to early compound 3. This resulted in the
discovery of GDC-0276 (1), which possessed improved metabolic
stability and an acceptable overall pharmacokinetics profile. To
further derisk the predicted human pharmacokinetics and enable
QD dosing, additional optimization of the scaffold was conducted,
resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by
blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and
pharmacokinetic profile over 1.
focused on acylsulfonamide and arylsulfonamide inhibitor
scaffolds that bind to voltage-sensing domain IV (VSD4) and
stabilize an inactivated state of the channel.9−13 In particular,
this effort has been motivated by the recognition that
compounds in these series are capable of exhibiting selectivity
over other members of the voltage gated sodium channel
family. This is a critical challenge in the discovery of safe
Nav1.7 inhibitors as other Nav isoforms are responsible for an
array of essential functions in, among many other tissues, the
CNS (Nav1.1, Nav1.2, Nav1.6), skeletal muscle (Nav1.4), and
heart (Nav1.5).14,15 Despite progress, balancing potency,
selectivity, in vivo activity, and DMPK properties remains
challenging in these series of compounds.8
INTRODUCTION
■
Chronic pain is a highly prevalent unmet medical need, with
the CDC estimating that in the United States (US) alone, 50.0
million people experience chronic painpain on most days or
every day for the past 6 monthsand 19.6 million people
experience chronic pain that limits their life or work activities.1
The voltage-gated sodium channel Nav1.7 is an essential
mediator of painful stimuli.2 In particular, Nav1.7 has been
linked to pain sensation in humans through the identification
of loss-of-function mutations in SCN9A, the gene that encodes
Nav1.7, which causes profound insensitivity to pain.3,4 These
results have been further recapitulated and extensively studied
through genetic experiments in both germ-line and adult-
inducible knockout mice.5,6 Together, these data provide
strong validation that inhibition of Nav1.7 could produce
substantial pain relief and support the use of preclinical rodent
models for evaluation of pharmacodynamic and analgesic
effects.
Received: January 11, 2021
Published: March 8, 2021
The wealth of information linking Nav1.7 to pain has
motivated substantial efforts to discover potent and subtype-
selective Nav1.7 inhibitors.7,8 Recently, significant efforts have
https://dx.doi.org/10.1021/acs.jmedchem.1c00049
© 2021 American Chemical Society
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Journal of Medicinal Chemistry
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Drug Annotation
We have previously disclosed our efforts to optimize a series
of acylsulfonamide Nav1.7 inhibitors resulting in the discovery
of 3 and 4 (Figure 1).16 These compounds contain a common
and 4, respectively, conferred excellent Nav1.7 potency and
provided reasonable selectivity over Nav1.5, which we viewed
as the most critical off-target isoform due to the relationship
between Nav1.5 function and human cardiotoxicity.15 How-
ever, these saturated groups also led to significant metabolic
liabilities for these compounds that resulted in poor predicted
pharmacokinetics in humans. Here, we describe our efforts to
improve on the pharmacokinetic (PK) properties of 3,
resulting in 1 (GDC-0276) and 2 (GDC-0310), two
development candidates which have progressed to Phase 1
studies.
RESULTS AND DISCUSSION
■
Our optimization campaign began with sulfonamide 3 (Table
1). Compound 3 showed excellent Nav1.7 potency in both
radioligand binding and sodium influx assays and moderate
selectivity against Nav1.5 as measured in a sodium influx assay.
This assay format tended to underpredict the selectivity seen in
electrophysiology (EP) assays; however, it was useful for rank
ordering compounds and identifying significant changes in
selectivity. Due to the extremely high plasma protein binding
of this series of inhibitors, compounds were also assayed in the
influx assay in the presence of 3 mg/mL of bovine serum
albumin (Flux PS). Compounds showing shifts >2-fold in this
assay tended to perform poorly in our in vivo PK/PD model
despite high levels of potency and were thus avoided. The
major liability of 3 was its high clearance in vitro, with scaled
hepatic clearances in human liver microsomes and hepatocytes
nearly at the rate of human liver blood flow (20.7 mL/min/
kg). To improve the predicted half-life and allow for
Figure 1. GDC-0276 (1), GDC-0310 (2), and previously described
acylsulfonamide Nav1.7 inhibitors.
anionic sulfonamide moiety that makes critical interactions
with a conserved arginine residue in the binding site.16 Key to
the identification of these compounds was the recognition that
saturated groups, such as the adamantane or cyclohexane in 3
Table 1. Structure Activity Relationships Leading to GDC-0276 (1)
a
b
c
hNav1.7 Ki from a radioligand binding assay. IC50 of sodium influx in cells transfected with hNav1.7 or hNav1.5. IC50 of sodium influx in the
d
e
presence of 3 mg/mL BSA. Scaled hepatic clearance from human liver microsomes and hepatocytes. LLE was calculated using Nav1.7 Ki and
LogD7.4 (LLE = pKi − LogD7.4).
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Drug Annotation
Table 2. Discovery of the N-Benzyl Piperidine Scaffold
a
b
c
hNav1.7 Ki from a radioligand binding assay. IC50 of sodium influx in cells transfected with hNav1.7 or hNav1.5. IC50 of sodium influx in the
d
e
presence of 3 mg/mL BSA. Scaled hepatic clearance from human liver microsomes and hepatocytes. LLE was calculated using Nav1.7 Ki and
LogD7.4 (LLE = pKi − LogD7.4).
reasonable dosing, achieving a decrease in clearance was
essential.
acceptable Nav1.7 potency and an improvement in HLM and
HH stability, poor Nav1.5 selectivity was a concern, as at least
10-fold was desired in this assay. Pyrrolidine 8 also showed an
increase in metabolic stability but with only modest Nav1.5
selectivity and a poor LLE. Azetidine 1 had increased potency,
acceptable Nav1.5 selectivity, improved HLM and HH stability,
and the highest LLE of all sulfamoyl-containing compounds
surveyed. After additional characterization (vide infra), 1 was
predicted to have acceptable human pharmacokinetics and was
selected as our first development candidate, GDC-0276.
While we believed 1 had improved predicted pharmacoki-
netics over 3, high plasma protein binding and low predicted
volume of distribution led to significant uncertainty in the
predicted half-life. Additionally, 1 was predicted to require BID
dosing to achieve sufficient target coverage, and QD dosing
was viewed as a significant advantage. Therefore, we continued
our optimization campaign to find compounds with further
improved metabolic stability to improve the PK profile and
derisk uncertainty in the predicted PK of 1. Furthermore, there
was room for improvement in the selectivity profile of 1, in
particular over Nav1.5, which we viewed as the key peripheral
off-target.
While metabolite identification experiments consistently
indicated the adamantane group was a major site of
metabolism, substitution of the adamantane with polar or
blocking groups, including fluorine, universally reduced
potency to unacceptable levels.16 Additionally, while the
adamantane group could be replaced with other saturated
groups, as in 4, the overall profile still did not result in
acceptable predicted human pharmacokinetics.16 We hypothe-
sized that compounds of this class could be metabolized by
P450 isoforms that recognize anionic substrates such as 2C8,
2C9, and 2C19 and that modifying the anionic sulfonamide
could result in improved stability (see Supporting Informa-
tion).17 Subsequent CYP reaction phenotyping on 1 confirmed
that it was primarily metabolized by CYP2C8 and provided
validation of our hypothesis. To this end, we prepared a
number of analogues modifying the methylsulfonamide, a
selection of which are shown in Table 1. Cyclopropane 5 was
representative of the general profile of nonpolar alkyl
sulfonamides, which had good potency and Nav1.5 selectivity
but showed no improvement in metabolic stability. More polar
sulfonamides such as oxetane 6 tended to slightly reduce the
potency and provide improvements in metabolic stability
which were too inconsistent to be leveraged.
Subsequent optimization efforts focused on replacing the
adamantane group with polar saturated groups that might
provide a significant change in the overall metabolism. Further
exploration of replacements for the adamantane led to the
discovery that suitably substituted azetidine and piperidine
rings could provide a tractable scaffold (Table 2). Benzhydryl
substituted azetidine 9 and piperidine 10 showed reasonable
Modifying the acylsulfonamide to an acylsulfamoyl group
proved more fruitful. In particular, sulfamoyl groups sub-
stituted with a cyclic amine provided the best balance of
potency and metabolic stability. While morpholine 7 had
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Drug Annotation
Table 3. Discovery of GDC-0310 (2)
a
b
c
hNav1.7 Ki from a radioligand binding assay. IC50 of sodium influx in cells transfected with hNav1.7 or hNav1.5. IC50 of sodium influx in the
d
e
presence of 3 mg/mL BSA. Scaled hepatic clearance from human liver microsomes and hepatocytes. LLE was calculated using Nav1.7 Ki and
LogD7.4 (LLE = pKi − LogD7.4).
potency and selectivity over Nav1.5 and an overall reduction in
lipophilicity relative to 3. Additionally, piperidine 10 showed a
promising increase in metabolic stability in both human liver
microsomes and hepatocytes. Paring back the large and
lipophilic benzhydryl group to a benzyl group in azetidine 11
or piperidine 12 resulted in a significant reduction in potency.
However, this potency reduction was accompanied by a
substantial reduction in LogD7.4, which resulted in 11 and 12
having an LLE comparable to 3. In spite of its low lipophilicity,
12 showed mixed results in metabolic stability assays, with high
clearance observed in human hepatocytes. Taken together,
these results led us to continue our investigation of N-benzyl
substituted piperidines with the aim of improving the potency
and metabolic stability of this novel scaffold.
Further optimization focused on improving potency through
incorporation of lipophilic substituents on the benzyl group
(Table 3). Simple substitutions such as para-chloro 13 and
para-trifluoromethoxy 14 led to improved potency, a
maintenance of high LLE, and acceptable Nav1.5 selectivity.
After extensive exploration of phenyl substituents, we arrived at
meta-dichloro 15 as possessing the best balance of potency,
selectivity, and LLE. While this compound represented a
promising lead, it continued to suffer from poor hepatocyte
stability. In contrast to our experience with 1, replacing the
methanesulfonamide with sulfamoyl groups did not mean-
ingfully improve the metabolic stability in the scaffold. In vitro
metabolism studies on this series of compounds indicated that
they were consistently metabolized via N-dealkylation, leading
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Drug Annotation
to the unsubstituted piperidine. Therefore, we sought to
improve the metabolic stability through substitution of the
metabolically labile benzylic position.
further evaluation of 1 and 2 as selective Nav1.7 inhibitors and
reinforced the improved profile of 2 over 1.
The DMPK profiles of 1 and 2 are shown in Table 5. In
addition to the improvement in human hepatocyte stability
Gratifyingly, we found that piperidine amide 16 provided a
significant improvement in the hepatocyte stability over 15.
While this validated our hypothesis, 16 showed decreased
potency and a high shift in the presence of plasma protein
(vide supra). After additional SAR investigations, gem-
dimethyl compound 17 was found to possess an excellent
balance of potency, Nav1.5 selectivity, and hepatocyte stability.
While the overall profile of 17 looked promising, we were
concerned about the high lipophilicity and low LLE. To
determine if we could maintain the potency/selectivity profile
while reducing the lipophilicity, we prepared the monomethyl
analogues 18 and 2. Both 18 and 2 showed good potency,
Nav1.5 selectivity, and lipophilicity; however, they surprisingly
differed dramatically in their hepatocyte stability. While the R
enantiomer 18 had a scaled hepatic clearance of 16 mL/min/
kg in hepatocytes, comparable to parent 15, the S enantiomer
2 showed clearance of <6.2 mL/min/kg. This is a particularly
stark example of the impact of chirality on P450 mediated
metabolism.18 Based on the overall profile described below, 2
was progressed as our second development candidate, GDC-
0310.
a
Table 5. DMPK Properties of 1 and 2
kinetic
HH CIHep
(a) (mL/min/kg)
MDCK P
hFu
v
solubility
CYP3A4
a
apbp
d
(106 cm/s)
(%)
(μM)
IC50 (μM)
1
2
11
<6.2
4.7
11
1
0.025
0.196
5.3
52
>10
>10
2
(b)
Hep CIHep
rat
dog
cyno
18
rat
dog
cyno
13
23
<7.8
11
<7.8
a
e
(mL/min/kg)
b
Fu (%)
CIp
(mL/min/kg)
0.013
5.1
0.005
0.40
0.025
2.3
0.278
1.4
0.157
0.091
0.129
0.87
e
Vss (L/kg)
0.38
0.95
34
0.17
5.5
50
0.30
1.6
30
0.60
5.0
68
0.33
46
∼100
0.29
4.4
82
e
T1/2 (hr)
F%
e
a
(a) In vitro human pharmacokinetic profile of 1 and 2. (b) In vitro
and in vivo pharmacokinetic profile in Sprague−Dawley rats, Beagle
dogs, and cynomolgus monkeys. Scaled hepatic clearance from
hepatocytes. Passive A−B permeability in a WT-MDCKI transwell
assay. Unbound fraction reported as a percentage (e.g., 0.025% =
0.00025). Kinetic solubility of a DMSO solution in pH 7.4 PBS. In
vivo pharmacokinetic parameters determined from 1 mg/kg (rat,
cyno) or 0.5 mg/kg (dog) IV dose. Determined using 5 mg/kg (rat),
b
v
The Nav selectivities of 1 and 2 were characterized by
voltage-clamp studies using the PatchExpress platform (Table
4). Due to the compound’s preferential binding to the
d
e
f
g
a
Table 4. Nav Selectivity of 1 and 2
1.0 mg/kg (dog), or 2.0 mg/kg (cyno) oral dose in a suspension of
0.5% methylcellulose, 0.2% Tween 80 in water.
hNav
1.7
IC50
hNav 1.1
IC50
(fold)
hNav 1.2
IC50
(fold)
hNav 1.4
IC50
(fold)
hNau 1.5
IC50
(fold)
hNav 1.6
IC50
(fold)
1
2
0.4
nM
10.6 nM
(26×)
202 nM
(337×)
18.9 nM
(47×)
38 nM
(63×)
8.5 nM
(21×)
3.4 nM
(6×)
51 nM
(127×)
551 nM
(918×)
480 nM
(1200×)
198 nM
(330×)
described above, 2 also showed improved permeability in
MDCK cells and increased kinetic solubility. The improvement
in permeability was surprising in light of the strongly
zwitterionic nature of 2, which has an acidic pKa of 3.9 and
a basic pKa of 8.6. The improvement in metabolic stability was
also reflected in the improved in vivo PK of 2 compared to 1.
Compound 1 showed low clearance across all species, however,
the high plasma protein binding resulted in a low volume of
distribution and a short to moderate half-life of 0.95−5.5 h
across species. Additionally, 1 had low to moderate oral
bioavailability, likely due to its relatively poor solubility. Both
of these effects were observed in the human PK of 1, which
exhibited a less than dose proportional exposure and evidence
of delayed absorption using a powder in capsule formulation.19
When dosed with a cyclodextrin solution formulation, 1
showed a half-life of 3.8−5.3 h, consistent with the range of
half-lives observed in preclinical species. This further validated
the need for a backup compound. In contrast, 2 showed
extremely low clearance in vivo, resulting in a half-life of 4.4−
46 h in preclinical species in spite of the low volume of
distribution. The high permeability and improved solubility of
2 also resulted in moderate to high oral bioavailability across
species. Overall, 2 provided a superior in vitro and in vivo
pharmacokinetic profile over 1.
0.6
nM
a
Nav1.X potency measured by voltage clamp on the PatchExpress
platform.
inactivated state of the channel, inhibition of each isoform
was measured while maintaining a holding potential at
depolarizing voltages that promote steady-state inactivation
of the channel.13 Slow equilibration of potent compounds
requires a protocol that maintains channels in the high affinity
state (inactivated) for a prolonged period of time to facilitate
equilibration of block. This approach allowed for accurate
determination of the potency and molecular selectivities of 1
and 2 across Navs. Compound 1 showed a high level of
selectivity over Nav1.6, >100-fold selectivity over Nav1.5, and
moderate selectivity over Nav1.1, Nav1.2, and Nav1.4. In
contrast, 2 had significantly improved selectivity over a number
of isoforms, with >300-fold selectivity over Nav1.1, Nav1.5, and
Nav1.6. The selectivity over Nav1.2 was 63-fold, while the
selectivity over Nav1.4 was a relatively low 6-fold. In spite of
the low selectivity versus Nav1.4, no obvious in vivo effects due
to blocking Nav1.4 were observed. This is potentially due to
both the marked state-dependence of this series of Nav1.7
inhibitors such that inhibition is much weaker at voltages in
which inactivation is removed and the fact that muscle fibers
that express Nav1.4 maintain a significantly more negative
resting membrane potential relative to the sensory neurons
that express Nav1.7. Overall, the selectivity data supported
To confirm the activity of 1 and 2 in vivo we tested both
compounds in a previously described inherited erythromelalgia
(IEM) mouse model (Figure 2).20 Briefly, this model tests the
ability of compounds to block a response to aconitine, a plant
alkaloid that causes abnormal opening of sodium channels, in a
transgenic mouse containing a human Nav1.7 gain-of function
variant. Aconitine causes a specific effect in these transgenic
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Drug Annotation
Chemistry. The synthesis of GDC-0276 (1) and its
analogues began with benzoic acid 19 (Scheme 1).16 tert-
Butyl ester formation with Boc2O and DMAP provided tert-
butyl ester 20. Next, the adamantane group was incorporated
through an SNAr reaction with 1-adamantane methanol, giving
21 as a single regioisomer. Introduction of the aryl cyclo-
propane was affected via an alkyl-Suzuki reaction with
cyclopropylboronic acid catalyzed by Pd(OAc)2 and PCy3 to
give 22. Next, deprotection of the tert-butyl ester was
facilitated by treatment with TFA to give penultimate benzoic
acid 23. Finally, formation of the acylsulfamoyl group was
completed by activation of the carboxylic acid with CDI and
reaction with sulfonamide 24 to form GDC-0276 (1). The
overall sequence was highly efficient, producing GDC-0276 in
5 linear steps from commercial starting materials. The route
was also highly scalable and could be further improved to
support 100 kg synthesis.21 Compounds 5−8 were prepared
using an analogous sequence by substituting in the requisite
sulfonamide.
The synthesis of GDC-0310 (2) began with tert-butyl ester
20 (Scheme 2). Nucleophilic aromatic substitution with
alcohol 25 provided Boc-piperidine intermediate 26 as a single
regioisomer. Introduction of the aryl cyclopropane was again
achieved via an alkyl-Suzuki reaction with cyclopropylboronic
acid catalyzed by Pd(OAc)2 and PCy3 to give 27. Treatment
with SOCl2 in MeOH affected deprotection of both the
piperidine Boc and tert-butyl ester followed by subsequent
methyl ester formation to provide the unprotected piperidine
28. Synthesis of the chiral electrophile 31 began with
acetophenone 29. Reduction of the carbonyl with BH3·DMS
and (S)-CBS catalyst produced alcohol 30 in 98.3% ee.
Subsequent mesylation with Ms2O produced mesylate 31.
Combining amine 28 and mesylate 31 in the presence of
Cs2CO3 effected smooth SN2 substitution to give N-benzyl
piperidine 32 with the requisite (S)-stereochemistry. Hydrol-
ysis of the methyl ester with NaOH provided benzoic acid 33,
which could be activated with CDI and reacted with
methansulfonamide to produce GDC-0310 (2). The absolute
configuration of 2 was unambiguously confirmed through a
small molecule X-ray crystal structure. The overall synthesis
provided 2 in 7 linear steps from commercially available
Figure 2. IEM aconitine assay PK/PD of 1 and 2. (a) PK/PD of 1 in
the IEM model. (b) PK/PD of 2 in the IEM model. Each dot
represents an individual animal measurement.
mice while having no impact on wild type mice, and the
resulting PK/PD relationship is dependent on human Nav1.7
potency. Nav1.7 inhibitors show comparable activity in this
model to traditional inflammatory and neuropathic pain
models.20 In this model, 1 and 2 both showed a concentration
dependent reduction in nociceptive events, with a EC50s of 1.7
μM and 1.1 μM, respectively. Correcting for mouse PPB
indicated an EC50,u of 0.42 nM for 1 and an EC50,u of 3.5 nM
for 2. Both values are comparable to the IC50 for inhibition of
hNav1.7 reported above. Notably, both 1 and 2 compared very
favorably with the previously disclosed development candidate
PF-05089771, which had an EC50 > 18 μM in this assay.9,20
a
Scheme 1. Synthesis of GDC-0276 (1)
a
Reagents and conditions: (a) Boc2O, DMAP, t-BuOH, 60 °C, 18 h, 90%. (b) Cs2CO3, 1-adamantane methanol, DMSO, 80 °C, 4 h, 52%. (c)
Pd(OAc)2, cyclopropyl boronic acid, PCy3·HBF4, K3PO4, PhMe, H2O, 100 °C, 18 h, 86%. (d) TFA, CH2Cl2, rt, 2 h, 71%. (e) CDI, azetidine-1-
sulfonamide, DBU, THF, rt, 16 h, 91%.
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Drug Annotation
Scheme 2. Synthesis of GDC-0310 (2)
a
Reagents and conditions: (a) Cs2CO3, 25, DMSO, rt, 1 h, 60%. (b) Pd(OAc)2, PCy3·HBF4, cyclopropylboronic acid, K3PO4, PhMe, H2O, 100 °C,
16 h, 74%. (c) SOCl2, MeOH, reflux, 16 h, 70%. (d) (S)-CBS catalyst, BH3·DMS, THF, rt, 4 h, 79%, 98.3% ee. (e) Ms2O, Et3N, CH2Cl2, 0 °C to rt,
2 h, 94%. (f) 31, K2CO3, 4-methyl-2-pentanone, H2O, 85 °C, 16 h, 99%. (g) NaOH, THF, H2O, 70 °C, 16 h, 97%. (h) CDI, MsNH2, K2CO3,
EtOAc, 70 °C, 4 h, 79%. Absolute stereochemistry of 2 was unambiguously confirmed by single-crystal X-ray diffraction.
homogeneous materials unless otherwise stated. NMR experiments
were performed with Bruker Avance III 300, 400, or 500 MHz
starting materials. Again, the synthesis was highly scalable and
an improved early development synthesis allowed the
preparation of 6.5 kg of 2.22 Analogues 9−18 were prepared
in an analogous fashion as described in the Experimental
Section.
1
spectrometers. H, 19F, and 13C NMR data are reported as chemical
shifts (δ) in parts per million (ppm) and are calibrated using residual
undeuterated solvent as an internal reference. Proton spectra are
reported as chemical shift, multiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, and br = broad), coupling constant
in Hz, and number of protons. Carbon and fluorine NMR spectra are
reported as chemical shift alone. High resolution mass spectrometry
was performed on a Dionex Ultimate 3000 coupled with Thermo
Scientific Q Exactive HRMS using ESI as ionization source. The LC
separation was done on a Phenomenex XB-C18, 1.7 mm, 50 × 2.1
mm column at a flow rate of 0.4 mL/min. MPA (mobile phase A) was
water with 0.1% FA and MPB (mobile phase B) was acetonitrile with
0.1% FA. The gradient started at 2% MPB and ended at 98% MPB
over 7 min and held at 98% MPB for 1.5 min following an
equilibration for 1.5 min. LC column temperature was 40 °C. UV
absorbance was collected by a DAD detector and mass spec full scan
was applied to all experiments. Optical rotation was measured on an
Autopol VI Automatic Polarimeter from Rudolph Research Analytical.
Reactions were monitored by walkup Shimadzu HPLC/UV system
with LC-10AD solvent pump, FVC-10AL, DGU-14A degasser, SPD-
M10A UV detector, CTO-10AC column oven, using the elution
gradient 10−80% (2.5 ML/4LTFA in acetonitrile) over 4 min and
holding at 80% for 2 min at a flow rate of 1.2 mL/min. Routine
purification was performed using Teledyne Isco Combiflash Rf
utilizing Silicycle HP columns. Reverse-phase purifications were
carried out using a Gilson GX-281 system with a 322 solvent pump,
159 UV−vis detector and a Xtimate C18 150 × 25 mm × 5 μm
column using MeCN/H2O gradients described below. Preparative
SFC separations were carried out as described below. Analytical purity
of all final compounds was greater than 95% as determined by LCMS
using 254 nM UV detection unless otherwise stated.
CONCLUSION
■
Acylsulfonamide Nav1.7 inhibitors 3 and 4 showed promising
biological activity and selectivity but suffered from inadequate
ADME properties. Initial optimization focused on the
hypothesis that modifying the anionic sulfonamide could
improve the metabolic stability, and resulted in the discovery
of acylsulfamoyl groups which exhibited superior profiles in
liver microsomes and hepatocytes. This ultimately led to the
discovery of GDC-0276 (1) which possessed improved
metabolic stability sufficient for selection as a development
candidate.19 To further derisk the pharmacokinetic properties
of 1 and to allow for QD dosing, we conducted a search for a
novel scaffold, ultimately replacing the lipophilic adamantane
group with more polar saturated amines. The resulting N-
benzyl piperidine scaffold still suffered from poor in vitro
metabolic stability, which could be improved by substituting
the metabolically labile benzylic position, resulting in GDC-
0310 (2). GDC-0310 (2) showed substantially improved Nav
selectivity and ADME properties, which supported its selection
as our second development candidate. GDC-0276 (1) and
GDC-0310 (2) have both completed Phase 1 studies, but their
subsequent development has been discontinued.19 Full results
of our clinical and nonclinical experience with 1 and 2 will be
reported in due course.
4-((Adamantan-1-yl)methoxy)-N-(azetidin-1-ylsulfonyl)-5-
cyclopropyl-2-fluorobenzamide (1, GDC-0276). A mixture of 23
(12.4 g, 36.1 mmol) in anhydrous tetrahydrofuran (250 mL) was
treated with carbonyldiimidazole (7.3 g, 45.1 mmol). The resulting
mixture was refluxed under nitrogen for 1 h. The reaction mixture was
cooled to room temperature and then treated with azetidine-1-
EXPERIMENTAL SECTION
■
General Methods. Unless otherwise specified, commercially
available reagents and solvents were used without further purification.
Yields refer to chromatographically and spectroscopically (1H NMR)
2959
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pubs.acs.org/jmc
Drug Annotation
sulfonamide (6.4 g, 46.9 mmol) and 1,8-diazabicycloundec-7-ene
(13.5 mL, 90.3 mmol). The resulting solution was stirred for 16 h at
ambient temperature under nitrogen. The reaction solution was
quenched with anhydrous methanol (12 mL) and stirred for 2 h. The
mixture was diluted with ethyl acetate, washed with a 1:1 mixture of 3
M hydrochloric acid:brine, a 1:1 mixture of 1 M hydrochloric
acid:brine, and brine. The organic layer was dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The solid was crystallized
3.26 (ddd, J = 5.4, 3.0, 1.3 Hz, 3H), 2.02 (dt, J = 17.4, 3.4 Hz, 4H),
1.78−1.63 (m, 12H), 0.96−0.83 (m, 2H), 0.74−0.62 (m, 2H).
HRMS (ESI-FTMS) m/z [M + H]+ Calcd for C25H34O5N2FS
493.2167; Found 493.2169.
4-((Adamantan-1-yl)methoxy)-5-cyclopropyl-2-fluoro-N-
(pyrrolidin-1-ylsulfonyl)benzamide (8). Compound 8 was
synthesized using the general procedure for the synthesis of 1,
substituting azetidine-1-sulfonamide for pyrrolidine-1-sulfonamide.
1H NMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H), 7.10 (d, J = 8.1 Hz,
1
with methanol to yield the title compound (15.13 g, 91%). H NMR
1H), 6.90 (d, J = 12.9 Hz, 1H), 3.64 (s, 2H), 3.44−3.35 (m, 4H),
2.09−1.96 (m, 4H), 1.88−1.77 (m, 4H), 1.77−1.63 (m, 12H), 0.95−
0.83 (m, 2H), 0.74−0.59 (m, 2H). HRMS (ESI-FTMS) m/z [M +
H]+ Calcd for C25H34O4N2FS 477.2218; Found 477.2217.
(400 MHz, DMSO-d6) δ 11.57 (s, 1H), 7.15 (d, J = 8.2 Hz, 1H), 6.93
(d, J = 12.9 Hz, 1H), 4.04 (t, J = 7.7 Hz, 4H), 3.65 (s, 2H), 2.22−2.11
(m, 2H), 2.09−1.96 (m, 4H), 1.78−1.63 (m, 12H), 0.95−0.87 (m,
2H), 0.71−0.65 (m, 2H). 13C NMR (101 MHz, DMSO) δ 162.32 (d,
J = 238.8 Hz), 162.21 (d, J = 10.7 Hz), 158.62, 127.96, 127.00, 100.42
(d, J = 26.4 Hz), 78.79, 51.97 (2C), 39.31 (3C), 37.04 (3C), 34.00,
27.97 (3C), 15.10, 9.64, 7.56 (2C). 19F NMR (376 MHz, DMSO-d6)
δ −112.31. HRMS (ESI-FTMS) m/z [M + H]+ Calcd for
C24H32O4N2FS 463.2061; Found 463.2061.
(S)-5-Cyclopropyl-4-((1-(1-(3,5-dichlorophenyl)ethyl)-
piperidin-4-yl)methoxy)-2-fluoro-N-(methylsulfonyl)-
benzamide (2, GDC-0310). To a solution of 33 (40 g, 85.7 mmol)
in EtOAc (400 mL) was added CDI (21.6 g, 120 mmol). The mixture
was heated to 35 °C and stirred for 2 h. Methanesulfonamide (12.6 g,
128 mmol) and K2CO3 (23.7 g, 171 mmol) were added, and the
mixture was heated to 70 °C and stirred for 4 h. Water was added, and
the mixture was extracted with EtOAc. The organic fractions were
concentrated and suspended in EtOAc. The suspension was heated to
70 °C and cooled to 10 °C over 10 h. The resulting solids were
collected by filtration to yield 2 (37.0 g, 79% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ 7.41 (t, J = 1.9 Hz, 1H), 7.36 (d, J
= 1.9 Hz, 2H), 7.18 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 13.0 Hz, 1H),
3.94 (d, J = 5.9 Hz, 2H), 3.66 (q, J = 6.8 Hz, 1H), 3.23 (s, 3H), 2.99−
2.93 (m, 1H), 2.83 (d, J = 11.6 Hz, 1H), 2.16−2.05 (m, 2H), 2.00 (tt,
J = 8.4, 5.3 Hz, 1H), 1.86−1.72 (m, 3H), 1.48−1.33 (m, 2H), 1.32 (d,
J = 6.8 Hz, 3H), 0.92−0.83 (m, 2H), 0.68−0.60 (m, 2H).
Exchangeable NH not observed. 13C NMR (101 MHz, DMSO-d6)
δ 164.57, 160.90 (d, J = 18.1 Hz), 159.59 (d, J = 259.6 Hz), 147.98,
133.92 (2C), 127.20, 126.94, 126.41 (2C), 126.15, 114.55 (d, J = 9.4
Hz), 100.25 (d, J = 26.4 Hz), 72.99, 62.57, 49.89, 48.76, 41.30, 35.37,
28.56 (2C), 17.71, 9.14, 6.88 (2C). 19F NMR (376 MHz, DMSO-d6)
δ −112.66. HRMS (ESI-FTMS) m/z [M + H]+ Calcd for
C25H30O4N2Cl2FS 543.1282; Found 543.1281. [α]D +0.008 (c 2.0,
CHCl3).
4-((Adamantan-1-yl)methoxy)-5-cyclopropyl-N-(cyclopro-
pylsulfonyl)-2-fluorobenzamide (5). Compound 5 was synthe-
sized using the general procedure for the synthesis of 1, substituting
azetidine-1-sulfonamide for cyclopropylanesulfonamide. 1H NMR
(400 MHz, DMSO-d6) δ 11.78 (s, 1H), 7.14 (d, J = 8.2 Hz, 1H), 6.93
(d, J = 13.1 Hz, 1H), 3.65 (s, 2H), 3.07 (tt, J = 7.7, 5.1 Hz, 1H),
2.10−1.96 (m, 4H), 1.78−1.63 (m, 12H), 1.19−1.04 (m, 4H), 0.96−
0.84 (m, 2H), 0.74−0.62 (m, 2H). HRMS (ESI-FTMS) m/z [M +
H]+ Calcd for C24H31O4NFS 448.1952; Found 448.1953.
4-((Adamantan-1-yl)methoxy)-5-cyclopropyl-2-fluoro-N-
(oxetan-3-ylsulfonyl)benzamide (6). Compound 6 was synthe-
sized using the general procedure for the synthesis of 1, substituting
azetidine-1-sulfonamide for oxetane-3-sulfonamide. 1H NMR (400
MHz, DMSO-d6) δ 7.33 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 13.6 Hz,
1H), 5.34 (t, J = 5.7 Hz, 1H), 4.96 (dd, J = 12.7, 3.6 Hz, 1H), 4.80
(ddd, J = 13.1, 9.3, 7.1 Hz, 2H), 3.98−3.77 (m, 1H), 3.65 (d, J = 18.7
Hz, 2H), 2.02 (ddt, J = 14.0, 5.8, 2.9 Hz, 4H), 1.73 (d, J = 12.2 Hz,
3H), 1.69−1.63 (m, 9H), 0.98−0.90 (m, 1H), 0.94−0.83 (m, 1H),
0.68−0.55 (m, 2H). Exchangeable NH not observed. HRMS (ESI-
FTMS) m/z [M + H]+ Calcd for C24H31O5NFS 464.1901; Found
464.1902.
4-((Adamantan-1-yl)methoxy)-5-cyclopropyl-2-fluoro-N-
(morpholinosulfonyl)benzamide (7). Compound 7 was synthe-
sized using the general procedure for the synthesis of 1, substituting
azetidine-1-sulfonamide for morpholine-4-sulfonamide. 1H NMR
(400 MHz, DMSO-d6) δ 11.69 (s, 1H), 7.12 (d, J = 8.2 Hz, 1H),
6.91 (d, J = 12.9 Hz, 1H), 3.68−3.60 (m, 6H), 3.45−3.34 (m, 1H),
4-((1-Benzhydrylazetidin-3-yl)methoxy)-5-cyclopropyl-2-
fluoro-N-(methylsulfonyl)benzamide (9). Compound 9 was
prepared from S2 and methansulfonamide following the general
procedure for the synthesis of 2. See Supporting Information for the
1
synthesis of S2. H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H),
7.45−7.39 (m, 4H), 7.28 (t, J = 7.6 Hz, 4H), 7.22−7.13 (m, 3H),
6.94 (d, J = 12.8 Hz, 1H), 4.50 (s, 1H), 4.21 (d, J = 6.2 Hz, 2H), 3.27
(s, 2H), 3.24 (s, 3H), 3.03 (s, 2H), 2.89 (d, J = 14.5 Hz, 1H), 2.05 (tt,
J = 8.4, 5.3 Hz, 1H), 0.94−0.85 (m, 2H), 0.70−0.61 (m, 2H). HRMS
(ESI-FTMS) m/z [M + H]+ Calcd for C28H30O4N2FS 509.1905;
Found 509.1906.
4-((1-Benzhydrylpiperidin-4-yl)methoxy)-5-cyclopropyl-2-
fluoro-N-(methylsulfonyl)benzamide (10). Compound 10 was
prepared using the general procedure for the synthesis of 2,
substituting 31 for benzhydryl chloride in the reaction preparing
32. 1H NMR (400 MHz, DMSO-d6) δ 11.88 (s, 1H), 7.77−7.58 (m,
5H), 7.56−7.33 (m, 5H), 7.13 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 13.0
Hz, 1H), 5.55 (d, J = 9.1 Hz, 1H), 3.99 (d, J = 5.9 Hz, 2H), 3.28 (s,
3H), 3.15−2.97 (m, 2H), 2.15−1.95 (m, 4H), 1.95−1.79 (m, 2H),
1.73−1.54 (m, 2H), 0.93−0.81 (m, 2H), 0.72−0.62 (m, 2H). HRMS
(ESI-FTMS) m/z [M + H]+ Calcd for C30H34O4N2FS 537.2218;
Found 537.2217.
4-((1-Benzylazetidin-3-yl)methoxy)-5-cyclopropyl-2-fluoro-
N-(methylsulfonyl)benzamide (11). Compound 11 was prepared
using the general procedure for the synthesis of 9, substituting S1 for
(1-benzylazetidin-3-yl)methanol in the reaction preparing S2 (see
1
Supporting Information). H NMR (400 MHz, DMSO-d6) δ 7.49−
7.31 (m, 5H), 7.23 (d, J = 8.5 Hz, 1H), 6.80 (d, J = 12.7 Hz, 1H),
4.18 (d, J = 6.1 Hz, 2H), 4.08 (s, 2H), 3.92−3.78 (m, 2H), 3.68 (d, J
= 8.1 Hz, 2H), 3.18−3.05 (m, 1H), 2.99 (s, 3H), 2.11−1.94 (m, 1H),
0.94−0.81 (m, 2H), 0.65−0.53 (m, 2H). Exchangeable NH not
observed. HRMS (ESI-FTMS) m/z [M + H]+ Calcd for
C22H26O4N2FS 433.1592; Found 433.1583.
4-((1-Benzylpiperidin-4-yl)methoxy)-5-cyclopropyl-2-fluo-
ro-N-(methylsulfonyl)benzamide (12). Compound 12 was
prepared using the general procedure for the synthesis of 2,
1
substituting 31 for benzyl chloride in the reaction preparing 32. H
NMR (300 MHz, CDCl3) δ 7.55 (d, J = 9.04 Hz, 1H), 7.44−7.36 (m,
5H), 6.52 (d, J = 14.25 Hz, 1H), 4.16−4.02 (m, 2H), 3.94−3.86 (m,
2H), 3.64−3.43 (m, 2H), 3.39 (s, 3H), 2.68−2.43 (m, 2H), 2.09−
1.87 (m, 6H), 0.97−0.85 (m, 2H), 0.65−0.55 (m, 2H). Exchangable
NH not observed. HRMS (ESI-FTMS) m/z [M + H]+ Calcd for
C24H30O4N2FS 461.1905; Found 461.1896.
4-((1-(4-Chlorobenzyl)piperidin-4-yl)methoxy)-5-cycloprop-
yl-2-fluoro-N-(methylsulfonyl)benzamide (13). Compound 13
was prepared using the general procedure for the synthesis of 2,
substituting 31 for p-chlorobenzyl chloride in the reaction preparing
32. 1H NMR (400 MHz, DMSO-d6) δ 7.42 (d, J = 8.5 Hz, 2H), 7.37
(d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 12.8 Hz,
1H), 3.91 (d, J = 5.9 Hz, 2H), 3.67 (s, 2H), 3.08−2.87 (m, 6H),
2.31−2.14 (m, 2H), 2.00 (tt, J = 8.5, 5.4 Hz, 1H), 1.89−1.73 (m,
3H), 1.41 (d, J = 12.5 Hz, 2H), 0.93−0.81 (m, 2H), 0.65−0.52 (m,
2H). HRMS (ESI-FTMS) m/z [M + H]+ Calcd for C24H29O4N2ClFS
495.1515; Found 495.1513.
5-Cyclopropyl-2-fluoro-N-(methylsulfonyl)-4-((1-(4-
(trifluoromethoxy)benzyl)piperidin-4-yl)methoxy)benzamide
(14). Compound 14 was prepared using the general procedure for the
synthesis of 2, substituting 31 for 1-(chloromethyl)-4-
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Drug Annotation
1
(trifluoromethoxy)benzene in the reaction preparing 32. H NMR
(400 MHz, DMSO-d6) δ 10.91 (s, 1H), 7.55−7.46 (m, 2H), 7.38 (d,
J = 8.1 Hz, 2H), 7.17 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 12.8 Hz, 1H),
3.94 (d, J = 6.0 Hz, 2H), 3.81 (s, 2H), 3.12 (s, 3H), 3.04 (d, J = 11.5
Hz, 2H), 2.37 (s, 2H), 2.01 (tt, J = 8.5, 5.3 Hz, 1H), 1.94−1.81 (m,
3H), 1.45 (q, J = 11.9 Hz, 2H), 0.94−0.81 (m, 2H), 0.69−0.57 (m,
2H). HRMS (ESI-FTMS) m/z [M + H]+ Calcd for C25H29O5N2F4S
545.1728; Found 545.1729.
tert-Butyl-4-((adamantan-1-yl)methoxy)-5-chloro-2-fluoro-
benzoate (21). To a solution of 20 (230 g, 0.927 mol) in anhydrous
dimethyl sulfoxide (1 L) was added 1-adamantane methanol (154 g,
0.927 mol) and Cs2CO3 (393g, 1.20 mol), and the suspension was
stirred at 80 °C for 4 h. Water and EtOAc were added to the mixture,
and the aqueous layer was extracted with EtOAc. The combined
organic layers were dried and concentrated to give a crude residue,
which was triturated with methanol. The resulting white solid was
filtered and dried to give 21 (193 g, 52% yield). 1H NMR (300 MHz,
CDCl3) δ 7.84 (d, J = 7.8 Hz, 1H), 6.59 (d, J = 12.3 Hz, 1H), 3.53 (s,
2H), 2.01 (s, 3H), 1.78−1.61 (m, 12H), 1.55 (s, 9H).
tert-Butyl-4-((adamantan-1-yl)methoxy)-5-cyclopropyl-2-
fluorobenzoate (22). To a solution of 21 (15.80 g, 40 mmol),
cyclopropylboronic acid (5.16 g, 60 mmol), potassium phosphate
(38.2 g, 180 mmol) and tricyclohexylphosphine tetrafluoroborate
(1.47 g, 3.99 mmol) in toluene (160 mL) and water (8 mL) under a
nitrogen atmosphere was added palladium acetate (0.45 g, 2.00
mmol). The reaction mixture was heated to 100 °C for 18 h and then
cooled to ambient temperature. Water was added, and the mixture
extracted with ethyl acetate, the combined organics were washed with
brine; dried over anhydrous Na2SO4 and concentrated. Purification of
the residue by column chromatography (5% ethyl acetate in hexanes)
afforded 22 (13.8 g, 86% yield). 1H NMR (300 MHz, CDCl3) δ 7.37
(d, J = 8.4 Hz, 1H), 6.47 (d, J = 12.9 Hz, 1H), 3.49 (s, 2H), 2.05−
1.95 (m, 4H), 1.78−1.61 (m, 12H), 1.55 (s, 9H), 0.91−0.84 (m, 2H),
0.64−0.58 (m, 2H).
4-((Adamantan-1-yl)methoxy)-5-cyclopropyl-2-fluoroben-
zoic acid (23). To a solution of 22 (257g, 642 mmol) in
dichloromethane (500 mL), was added trifluoroacetic acid (250
mL). The reaction mixture was stirred at ambient temperature for 2 h
and then concentrated in vacuo. The residue was triturated in
methanol (500 mL), and the solid was filtered and dried to give 23
(158 g, 71% yield). 1H NMR (300 MHz, DMSO-d6) δ 12.77 (s, 1H),
7.29 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 13.2 Hz, 1H), 3.59 (s, 2H),
2.04−1.92 (m, 4H), 1.71−1.58 (m, 12H), 0.91−0.83 (m, 2H), 0.59−
0.52 (m, 2H). LCMS m/z 345.1 [M + H]+.
tert-Butyl-4-((4-(tert-butoxycarbonyl)-2-chloro-5-
fluorophenoxy)methyl)piperidine-1-carboxylate (26). To a
mixture of 20 (10.0 g, 46.3 mmol) and 25 (12.6 g, 50.9 mmol) in
DMSO (200 mL) was added Cs2CO3 (18.1 g, 55.6 mmol). The
reaction mixture was stirred at rt for 1 h, poured into ice-water, and
extracted with EtOAc. The combined organics were washed with
brine, dried over anhydrous Na2SO4, and concentrated. The crude
residue was purified by silica gel chromatography (eluting with
petroleum ether/EtOAc, from 20/1 to 5/1) to give 26 (12.3 g, 60%)
as yellow oil. 1H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 7.6 Hz, 1H),
6.59 (d, J = 11.9 Hz, 1H), 4.23−4.05 (m, 2H), 3.84 (d, J = 6.2 Hz,
2H), 2.81−2.64 (m, 2H), 2.10−1.94 (m, 1H), 1.87−1.77 (m, 2H),
1.55 (s, 9H), 1.44 (s, 9H), 1.36−1.20 (m, 2H). LCMS m/z 466.1 [M
+ Na]+.
5-Cyclopropyl-4-((1-(3,5-dichlorobenzyl)piperidin-4-yl)-
methoxy)-2-fluoro-N-(methylsulfonyl)benzamide (15). Com-
pound 15 was prepared using the general procedure for the synthesis
of 2, substituting 31 for 1,3-dichloro-5-(chloromethyl)benzene in the
1
reaction preparing 32. H NMR (400 MHz, DMSO-d6) δ 11.43 (s,
1H), 7.53 (t, J = 2.0 Hz, 1H), 7.40 (d, J = 2.0 Hz, 2H), 7.17 (d, J = 8.4
Hz, 1H), 6.86 (d, J = 12.9 Hz, 1H), 3.94 (d, J = 5.9 Hz, 2H), 3.64 (s,
2H), 3.15 (s, 3H), 2.93 (d, J = 11.2 Hz, 2H), 2.19 (s, 2H), 2.01 (tt, J
= 8.4, 5.3 Hz, 1H), 1.81 (d, J = 12.6 Hz, 3H), 1.41 (q, J = 11.8 Hz,
2H), 0.93−0.79 (m, 2H), 0.70−0.58 (m, 2H). HRMS (ESI-FTMS)
m/z [M + H]+ Calcd for C24H28O4N2Cl2FS 529.1125; Found
529.1127.
5-Cyclopropyl-4-((1-(3,5-dichlorobenzoyl)piperidin-4-yl)-
methoxy)-2-fluoro-N-(methylsulfonyl)benzamide (16). Com-
pound 16 was prepared from S6 and following the general procedure
for the synthesis of 2, analogous to the sequence beginning with 32.
1
See Supporting Information for the synthesis of S6. H NMR (400
MHz, DMSO-d6) δ 11.87 (s, 1H), 7.71 (t, J = 1.9 Hz, 1H), 7.45 (d, J
= 1.9 Hz, 2H), 7.15 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 12.9 Hz, 1H),
4.48 (d, J = 12.8 Hz, 1H), 3.98 (d, J = 6.3 Hz, 2H), 3.52 (d, J = 13.2
Hz, 1H), 3.28 (s, 3H), 3.12 (s, 1H), 2.84 (s, 1H), 2.15−1.96 (m, 2H),
1.95−1.83 (m, 1H), 1.76 (d, J = 12.3 Hz, 1H), 1.35 (s, 2H), 0.94−
0.84 (m, 2H), 0.74−0.61 (m, 2H). HRMS (ESI-FTMS) m/z [M +
H]+ Calcd for C24H26O5N2Cl2FS 543.0918; Found 543.0920.
5-Cyclopropyl-4-((1-(2-(3,5-dichlorophenyl)propan-2-yl)-
piperidin-4-yl)methoxy)-2-fluoro-N-(methylsulfonyl)-
benzamide (17). Compound 17 was prepared from S7 and
following the general procedure for the synthesis of 2, analogous to
the sequence beginning with 32. See Supporting Information for the
1
synthesis of S7. H NMR (400 MHz, DMSO-d6) δ 11.85 (s, 1H),
7.51 (s, 2H), 7.45 (s, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 12.9
Hz, 1H), 3.94 (d, J = 5.7 Hz, 2H), 3.24 (s, 3H), 2.75 (s, 2H), 2.19−
2.06 (m, 1H), 2.01 (tt, J = 8.5, 5.4 Hz, 1H), 1.82−1.74 (m, 4H),
1.46−1.16 (m, 8H), 0.97−0.79 (m, 2H), 0.74−0.57 (m, 2H). HRMS
(ESI-FTMS) m/z [M + H]+ Calcd for C26H32O4N2Cl2FS 557.1438;
Found 557.1443.
(R)-5-Cyclopropyl-4-((1-(1-(3,5-dichlorophenyl)ethyl)-
piperidin-4-yl)methoxy)-2-fluoro-N-(methylsulfonyl)-
benzamide (18). Compound 18 was prepared using the general
procedure for the synthesis of 2, substituting 31 for its enantiomer
which could be prepared from an identical sequence as 31 using the
(R)-CBS catalystin the reaction preparing 32. 1H NMR (400 MHz,
DMSO-d6) δ 7.54 (s, 1H), 7.43 (s, 2H), 7.15 (d, J = 8.4 Hz, 1H), 6.87
(d, J = 12.9 Hz, 1H), 3.92 (d, J = 5.8 Hz, 2H), 3.78 (s, 1H), 3.20 (s,
3H), 3.11−3.06 (m, 1H), 3.00−2.81 (m, 1H), 2.18−2.12 (m, 2H),
2.00 (tt, J = 8.5, 5.3 Hz, 1H), 1.90−1.72 (m, 3H), 1.37 (d, J = 6.3 Hz,
5H), 0.92−0.81 (m, 2H), 0.71−0.59 (m, 2H). Exchangable NH not
observed. HRMS (ESI-FTMS) m/z [M + H]+ Calcd for
C25H30O4N2Cl2FS 543.1282; Found 543.1281.
tert-Butyl-4-((1-benzylpiperidin-4-yl)methoxy)-5-cycloprop-
yl-2-fluorobenzoate (27). To a mixture of 26 (5 g, 11.3 mmol),
cyclopropylboronic acid (2.91 g, 33.9 mmol), K3PO4 (7.18 g, 339
mmol), and P(Cy)3·HBF4 (1.81g, 4.5 mmol) in toluene (200 mL)
and water (10 mL) under N2 was added Pd(OAc)2 (230 mg, 1.13
mmol). The reaction mixture was heated to 100 °C for 16 h and
cooled to rt. Water was added, and the mixture was extracted with
ethyl acetate. The combined organics were washed with brine, dried
over anhydrous Na2SO4, and concentrated. The residue was purified
by silica gel chromatography (eluting with petroleum ether/EtOAc,
from 10/1 to 2/1) to give 27 (3.7 g, 74%) as yellow oil. LCMS m/z
472.2 [M + Na]+.
Methyl-5-cyclopropyl-2-fluoro-4-(piperidin-4-ylmethoxy)-
benzoate (28). To a solution of 27 (9.0 g, 20.0 mmol) in MeOH
(110 mL) was added SOCl2 (10 mL) dropwise. The reaction mixture
was stirred at reflux for 16 h, cooled to rt, and concentrated. The
residue was slurried in petroleum ether/ethyl acetate (20/1), filtered,
and dried to give 28 (4.3 g, 70% yield) as a HCl salt and gray solid.
LCMS m/z 308.2 [M + H]+.
tert-Butyl-5-chloro-2,4-difluorobenzoate (20). To a stirred
solution of 5-chloro-2,4-difluorobenzoic acid (19, 200 g, 1.04 mol) in
t-BuOH (1 L) was added (Boc)2O (453 g, 2.08 mol), and then
DMAP (26 g, 0.208 mmol) was added slowly to the mixture. The
reaction was heated to 60 °C and stirred overnight. The mixture was
concentrated to give a crude product; water and EtOAc were added,
and the aqueous layer was extracted with EtOAc. The organic layer
was combined and dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
column chromatography eluting with petroleum ether/EtOAc = 1:1
1
to give compound 20 (232 g, 90% yield) as a yellow oil. H NMR
(300 MHz, CDCl3) δ 7.94 (t, J = 7.8 Hz, 1H), 6.95 (t, J = 9.4 Hz,
1H), 1.59 (s, 9H).
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(R)-1-(3,5-Dichlorophenyl)ethan-1-ol (30). A 1 M solution of
(S)-2-methyl-CBS-oxazaborolidine (84.6 mL, 84.6 mmol) in toluene
and BH3·DMS (47.3 mL, 508 mmol) were dissolved in THF (1.4 L).
The mixture was stirred at 20 °C for 30 min before the addition of a
solution of 29 (80.0 g, 423 mmol) in THF (500 mL) dropwise over
1.5 h. The mixture was stirred at 20 °C for 2 h. The mixture was
quenched with MeOH dropwise at 0 °C, followed by the addition of 2
M HCl. The mixture was extracted with DCM, washed with water and
brine, dried over anhydrous Na2SO4, filtered, and concentrated. The
crude product was heated in MeOH followed by the addition of water
After overnight incubation (37◦C, 5% CO2), culture media was
removed and cells were loaded with 5 μM ANG2 dye for 1 h in Buffer
1 (155 mM NMDG (N-methyl-D-glucamine), 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 10 mM HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid buffer), 10 mM glucose, adjusted with
Tris to pH 7.4). Excess dye was removed, and cells were incubated
with and without test compound for 1 h in Buffer 1 containing 50 μM
veratridine or 500 nM veratridine plus 100 nM anthropleurin C for
hNaV1.7 and hNaV1.5 cells, respectively, at room temperature. A
Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K
challenge buffer (145 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15
mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH
7.4) and simultaneously read plates at excitation wavelength of 530
nm and emission wavelength of 558 nm. Nonsodium channel
mediated sodium influx was determined in the presence of 1 μM
tetrodotoxin (TTX) for NaV1.7 and 25 μM TTX for NaV1.5. This
background signal was subtracted from the total sodium influx signal
in the absence of compound, and all data in the presence of
compound was normalized to this control. Percent inhibition of
sodium ion influx was calculated for each test compound at each test
concentration to determine the IC50 values. The use of veratridine and
anthropleurin C in the influx assay but not in the voltage clamp
studies and the longer time course of the influx assay presumably
account for the difference in selectivity between NaV1.7 and NaV1.5 in
the two assays. For Protein Shift (FluxPS) experiments, 3 mg/mL
human serum albumin was added to Buffer 1.
The percent inhibition in the sodium influx assays was determined
and IC50 values were calculated using a 4-parameter logistic model
using IDBS XLfit integrated with Microsoft Excel 2013: % inhibition
= (A + ((B − A)/(1 + (x/C)D)), where A and B are the maximum
and minimum inhibition, respectively; C is the IC50 concentration; D
is the (Hill) slope; and x is the concentration of test compound.
Voltage Clamp Whole Cell Recordings. Cell Lines. Electro-
physiology experiments were performed with human embryonic
kidney cells (HEK), permanently transfected with an expression
vector containing the full-length cDNA coding for the human sodium
channel α-subunit, grown in culture media containing 10% FBS, 1%
PSG, and 0.5 mg/mL G418 at 37 °C with 5% CO2. The β1 subunit
was coexpressed in all cell lines. The hNaV1.6 cell line (NM_014191)
was purchased as a CHO cell line from Genionics.
Whole Cell Recordings. Sodium currents were measured using the
patch clamp technique in the whole-cell configuration using the
PatchXpress (PX) automated voltage clamp amplifier. The pipet was
filled with a solution comprised of: 5 mM NaCl, 10 mM CsCl, 120
mM CsF, 0.1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM
EGTA; and adjusted to pH 7.2 with CsOH. The external solution had
the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2,
1 mM MgCl2, 10 mM HEPES; and adjusted to pH 7.4 with NaOH.
Generally, in studies by PX, the external sodium was reduced by
equimolar replacement with choline. Osmolarity in the internal and
external solutions was adjusted to 300 and 310 mOsm/kg with
glucose, respectively.
Currents were recorded at 40 kHz sampling frequency, filtered at 5
Hz, and stored using a Digidata-1322A analogue/digital interface with
the pClamp software (Axon Instruments). Series resistance
compensation was applied at 55%. For inactivated state block, general
holding voltages were −60 mV (NaV1.7 and NaV1.5), −35 mV
(NaV1.1, NaV1.2, and NaV1.6), and −50 mV (NaV1.4). Test pulses
were preceded by a 20 ms hyperpolarization to −150 mV to remove
fast inactivation.
1
to precipitate 30 (63.1 g, 79% yield) as a white solid. H NMR (400
MHz, CDCl3) δ 7.26 (s, 3H), 4.85 (q, J = 6.4 Hz, 1H), 2.19−1.97 (m,
1H), 1.48 (d, J = 6.4 Hz, 3H). SFC analysis showed the product was
98.3% ee.
(R)-1-(3,5-Dichlorophenyl)ethylmethanesulfonate (31). To a
solution of 30 (6.7 g, 35 mmol) and Ms2O (6.7 g, 38.5 mmol) in
DCM (175 mL) at 0 °C was added Et3N (5.4 mL, 38.5 mmol)
dropwise. The reaction was allowed to warm to rt and stirred for 2 h.
Saturated NH4Cl solution (20 mL) was added, and the solution was
extracted with DCM. The organic layer was collected, dried with
MgSO4, and concentrated to produce 31 as a clear, colorless oil (8.8
1
g, 94% yield). H NMR (400 MHz, CDCl3) δ 7.36 (t, J = 1.9 Hz,
1H), 7.28 (d, J = 1.9 Hz, 2H), 5.66 (q, J = 6.6 Hz, 1H), 2.90 (s, 3H),
1.69 (d, J = 6.6 Hz, 3H), 1.58−1.45 (m, 1H).
Methyl-(S)-5-cyclopropyl-4-((1-(1-(3,5-dichlorophenyl)-
ethyl)piperidin-4-yl)methoxy)-2-fluorobenzoate (32). To a
solution of 28 (7.7 g, 25 mmol) in 4-methyl-2-pentanone (125
mL) were added 31 (8.8 g, 33 mmol), water (12.5 mL), and K2CO3
(5.2 g, 37.5 mmol). The mixture was heated to 85 °C and stirred for
16 h. Reaction was diluted with water and extracted with EtOAc. The
organic layer was collected, dried with MgSO4, and concentrated. The
crude product was purified by column chromatography to produce 32
1
as a clear colorless oil (11.8 g, 99% yield). H NMR (400 MHz,
CDCl3) δ 7.44 (d, J = 8.4 Hz, 1H), 7.31−7.19 (m, 3H), 6.53 (d, J =
12.7 Hz, 1H), 3.90−3.80 (m, 5H), 3.40 (q, J = 6.7 Hz, 1H), 3.03 (d, J
= 11.0 Hz, 1H), 2.82 (d, J = 11.3 Hz, 1H), 2.09−1.92 (m, 3H), 1.91−
1.75 (m, 4H), 1.51−1.24 (m, 4H), 0.95−0.84 (m, 2H), 0.68−0.59
(m, 2H). LCMS m/z 480.2 [M + H]+.
(S)-5-Cyclopropyl-4-((1-(1-(3,5-dichlorophenyl)ethyl)-
piperidin-4-yl)methoxy)-2-fluorobenzoic acid (33). To a
solution of 32 (11.8 g, 25 mmol) in THF (100 mL) and water (50
mL) was added NaOH (15 g, 375 mmol). The reaction was then
heated to reflux (70 °C) and stirred for 16 h. The crude mixture was
allowed to cool to 0 °C, and conc HCl was added to precipitate 33
(11.3 g, 97% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ 7.45 (dt, J = 10.9, 2.0 Hz, 1H), 7.37 (dd, J = 7.2, 1.9 Hz, 2H), 7.22
(d, J = 8.6 Hz, 1H), 6.73 (d, J = 12.5 Hz, 1H), 3.87 (d, J = 5.8 Hz,
2H), 3.55 (q, J = 6.7 Hz, 1H), 2.93 (d, J = 11.1 Hz, 1H), 2.78 (d, J =
11.2 Hz, 1H), 2.04−1.85 (m, 3H), 1.83−1.66 (m, 3H), 1.42−1.22
(m, 6H), 0.90−0.81 (m, 2H), 0.59−0.50 (m, 2H). LCMS m/z 466.1
[M + H]+.
Nav1.X Cell Lines. HEK cell lines stably expressing full-length
cDNAs coding each Nav α subunit were generated as previously
described,13 except in the cases of hNav1.1 and hNav1.6, which were
purchased as Chinese Hamster Ovary (CHO) cell lines from
Chantest (now part of Charles River Laboratories) and Genionics
AG, respectively. GenBank accession numbers for α-subunits were:
hNav1.2 (NM_021007), hNav1.4 (NM_000334), hNav1.5
(AC137587; SCN5A), and hNav1.7 (NM_002977).
Radioligand Binding Assays. Radioligand binding assays were
conducted as previously described.12,13,16
Data Analysis. For voltage clamp studies, percent inhibition was
determined at the holding potential for near complete inactivation
and fast inactivation of compound-free channels was removed by a
brief, strong hyperpolarization. When analyzing grouped data, IC50
values were determined at different concentrations for multiple cells
and a single dose−response curve was generated in Prism software
Sodium Influx Assays. TREX HEK 293 cells were stably
transfected with an inducible expression vector containing the full-
length cDNA coding for the desired human sodium channel α-
subunit, full-length cDNA coding for inward rectifying potassium
channel, Kir1.1, and with an expression vector containing full length
cDNA coding for the β1-subunit. Sodium channel expressing cell lines
were induced with tetracycline (1 μg/mL) and plated on 384-well
poly-D-lysine (PDL)- coated plates at a density of 25K cells/well in
culture media (DMEM, containing 10% FBS and 1% L-glutamine).
̂
using equation (I/I0 = 1/(1 + 10((LogIC50 − X) × Hill slope)); I/Io
is the fraction of sodium current remaining in the presence of drug,
[Drug] is the concentration of drug, LogIC50 is the log of the IC50
concentration, and n is the Hill coefficient describing the
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stoichiometry of compound binding to the sodium channel. IC50
values on the PatchXpress automated voltage-clamp platform were
generated from Hill equation fits to pooled data, for which SD is not
applicable.
LogD and pKa. LogD measurements were determined as
previously described.23 pKa values were determined using published
UV-metric procedures.24
per route and dose, and were not fasted before dosing. Mice were
administered compounds intravenously (IV) at 1 mg/kg (formulated
in 5% DMSO, 35% PEG400, and 60% phosphate buffered saline) or
post oral (PO) at 5 mg/kg (formulated in 0.5% (w/v)
methylcellulose, 0.2% (w/v) Tween 80 in water (MCT)) at a dose
volume of 5 mL/kg. Blood samples (15 μL) were collected via tail
nick from each animal at 0.25, 0.5, 1, 2, 4, and 6 h postdose and added
to 60 μL of EDTA (1.7 mg/mL) water. Test article concentration in
each blood sample was determined by a nonvalidated LCMS/MS
assay at Genentech.
Rat PK. Male Sprague−Dawley (SD) rats (9−11 weeks old)
ranging from 250 to 300 g were obtained from Charles River
Laboratories (Hollister, CA). Animals were fasted overnight before
oral dose administration of compounds. Three rats were administered
compounds intravenously (IV) at 1 mg/kg (formulated in 10%
DMSO, 50% PEG400, and 40% phosphate buffered saline) or post
oral (PO) at 5 mg/kg (formulated in MCT). Blood samples
(approximately 0.2 mL per sample) were collected from each animal
into tubes containing K2EDTA at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6,
and 8 h after dose administration. Blood was centrifuged for 10 min to
harvest plasma. The concentration of test compound in each plasma
sample was determined by a nonvalidated LCMS/MS assay at
Genentech. Pharmacokinetic analysis was performed at Genentech
using noncompartmental methods.
Dog PK. Pharmacokinetics of test compound was determined in
male Beagle dogs following a single IV (formulated in 10% DMSO,
50% PEG400, and 40% PBS) or oral (formulated in 0.5% (w/v)
methylcellulose, 1.0% (w/v) Tween 80 in Water) administration.
Non-naive male Beagle dogs ages ranging from 6 months to 3 years
old and weighing between 6 and 12 kg were obtained from Marshall
Bioresources (Beijing, China). Animals were not fasted before IV
dosing. They were fasted overnight prior to PO dosing through
approximately 4 h postdose.
Blood samples were collected in tubes containing K2EDA at
predose and at 0.033 (IV only), 0.083, 0.25, 0.5, 1, 3, 6, 9, and 24 h
post IV or oral administration. Blood was centrifuged for 10 min to
harvest plasma. The concentration of test compound in each plasma
sample was determined by a nonvalidated LCMS/MS assay at Wuxi
AppTech, Inc. Pharmacokinetic analysis was performed at Genentech
using noncompartmental methods.
Cyno PK. Pharmacokinetics of test compound was determined in
male cynomolgus monkey following a single 0.5 mg/kg IV
(formulated in 10% DMSO, 50% PEG400 and 40% PBS) or 0.5
mg/kg oral (formulated in 0.5% (w/v) methylcellulose, 1.0% (w/v)
Tween 80 in water) administration. Non-naive male cynomolgus
monkeys ages greater than 2 years old and weighing between 2.5 and
6 kg were obtained from Hainan Jingang Laboratory Animal Co. Ltd.
(Hainan, China). Animals were not fasted before IV dosing, but were
fasted overnight prior to PO dosing through approximately 4 h
postdose. Blood samples were collected in tubes containing K2EDTA
at predose and at 0.033 (IV only), 0.083, 0.25, 0.5, 1, 3, 6, 9, and 24 h
post IV or oral administration. Blood was centrifuged for 10 min to
harvest plasma. The concentration of test compound in each plasma
sample was determined by a nonvalidated LCMS/MS assay at Wuxi
AppTech, Inc. Pharmacokinetic analysis was performed at Genentech
using noncompartmental methods.
PK/PD Studies in Human NaV1.7 I848T (IEM) Transgenic
Mice. Transgenic mice were prepared as described previously.20 Male
and female FVB hSCN9a transgenic mice used for the experiments
were seven to 10 weeks old and weighed 20−30 g. The test
compound was administered post oral (PO) in 0.5% w/w methyl
cellulose and 0.2% v/v Tween 80 in deionized water (MCT) at a dose
volume of 10 mL/kg. An injection of 20 μL of 39 μM aconitine
(Sigma-Aldrich) was made subcutaneously into the dorsum of the
hind paw at the predicted Tmax after oral test compound
administration (as determined previously in separate PK experi-
ments). Video data acquisition begins immediately after injection of
aconitine and continues for up to 70 min. The first 30 min of each
video (or the 5 min period spanning 10−15 min after aconitine
injection) was analyzed and scored for the number of instances of
Metabolic Stability Study in Liver Microsomes. In vitro
metabolism was evaluated in pooled liver microsomes (Celsis,
Chicago, IL) from humans (n = 150; BioIVT, Hicksville, NY). The
incubation mixture was prepared in 0.1 M-potassium phosphate buffer
(pH 7.4) containing 0.5 mg/mL microsomal protein, 1 mM NADPH,
and 1 μM of compound. Reactions were initiated with the addition of
NADPH. Samples were incubated at 37 °C and aliquots were sampled
at 0, 20, 40, and 60 min. Reactions were quenched with 95/5
acetonitrile/water (v/v) containing propranolol as the internal
standard at each time point. Samples were centrifuged at 3000g for
10 min. Supernatant was diluted with water (1:2 ratio) and the
percentage of G# remaining was determined by LC−MS/MS using
the t = 0 peak area ratio values as 100%. The in vitro Clint and scaled
hepatic Cl were determined as previously described;25,26 standard
deviation is not applicable.
Metabolic Stability Study in Cryopreserved Hepatocytes.
Metabolic stability was evaluated in cryopreserved hepatocytes from
Sprague−Dawley rats, cynomolgus monkey, Beagle dog (Invitrogen
Corporation, Carlsbad, CA), and humans (Celsis, Baltimore, MD).
The cells were seeded at a density of 0.5 × 106 cells/mL; reactions
were initiated with the addition of test compound at a final substrate
concentration of 1 μM. Samples were incubated at 37 °C in 5%
carbon dioxide with saturating humidity, and aliquots were sampled at
0, 1, 2, and 3 h. Reactions were quenched with acetonitrile containing
propranolol as the internal standard at each time point. Samples were
centrifuged at 3000g for 10 min. Supernatant was diluted with water
(1:2 ratio), and the percentage of test compound remaining was
determined by LC/MS/MS. Using the t = 0 peak area ratio values as
100%, the in vitro Clint and scaled hepatic Clhep were determined as
previously described;25,26 standard deviation is not applicable.
In Vitro Plasma Protein Binding. Equilibrium dialysis was
conducted using a Single-Use Plate Rapid Equilibrium Dialysis (RED)
device (Thermo Scientific, Rockford, IL). In brief, 300 μL of plasma
(set at pH 7.4) spiked with drug (final concentration of 5 μM) was in
the donor chamber, and 500 μL phosphate buffered saline containing
133 mM potassium phosphate and 150 mM NaCl was in the receiver
chamber. The RED device was placed on a shaking incubator at 37 °C
with 5% CO2 at 450 rpm (VWR SymphonyTM). Following 6-h
incubation, plasma and buffer samples from the RED device were
equalized with equal amount of either blank buffer and plasma,
respectively. Samples were then quenched in 1:3 (sample:
acetonitrile) ratio with ice cold HPLC grade acetonitrile (EMD
Millipore, Billerica, MA) with propranolol as the internal standard.
The resulting mixture was shaken for 15 min at 500 rpm (Thermo
Scientific Compact Digital MicroPlate Shaker), and the supernatant
was collected by centrifuging for 15 min at 3750 rpm (Beckman
Coulter Allegra X 12R). Subsequently, the supernatant was diluted
with water (1:2 ratio), for LC-MS/MS analysis. Fraction unbound
was calculated by dividing the peak area from the receiver chamber by
the peak area from the donor chamber. Experiments were conducted
in triplicate.
Use of Animal Subjects. All studies involving animals were either
approved by the Animal Care Committee of Xenon Pharmaceuticals
(Burnaby, BC, Canada) and performed in compliance with the
Canadian Council on Animal Care guidelines or approved by the
Institutional Animal Care and Use Committee of Genentech (South
San Francisco, CA, United States) and performed in accordance with
the recommendations of the International Association for the Study of
Pain.
Pharmacokinetic Studies in Mouse, Rat, Dog, and Cyno.
Mouse PK. Male FVB mice between 6 and 7 weeks of age with body
weight ranging from 23 to 30 g were obtained from Charles River
Laboratories (Hollister, CA). Animals were divided into groups of 3
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Drug Annotation
flicking or lifting the injected paw, or paw licking that lasts for 3 s. At
the end of the observation period, mice were euthanized by CO2
inhalation. Blood samples were collected via cardiac puncture into
K2EDTA tubes and processed into plasma by centrifugation at
17 000g for 2 min and collection of supernatant. Plasma samples were
stored at −80 °C until bioanalysis by LC MS/MS to determine test
compound concentration.
Antonio DiPasquale − Genentech, Inc., South San Francisco,
California 94080, United States
Thilo Focken − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada; orcid.org/0000-0003-
1993-2476
Ivan Hemeon − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada
Kuldip Khakh − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Amy Kim − Genentech, Inc., South San Francisco, California
94080, United States
Rainbow Kwan − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Andrea Lindgren − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Sophia Lin − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada
Jonathan Maher − Genentech, Inc., South San Francisco,
California 94080, United States
Janette Mezeyova − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00049.
Additional synthetic methods, plasma exposure of in
vivo PK/PD studies, X-ray structure determination of 2,
HPLC purity of tested compounds, representative
HPLC chromatograms, NMR spectra of 1 and 2, and
modeling of compounds in the active site of CYP2C8
(PDF)
SMILES molecular formula strings (CSV)
Dinah Misner − Genentech, Inc., South San Francisco,
AUTHOR INFORMATION
■
California 94080, United States
Corresponding Authors
Karen Nelkenbrecher − Xenon Pharmaceuticals, Inc.,
Burnaby, British Columbia V5G 4W8, Canada
Jodie Pang − Genentech, Inc., South San Francisco, California
94080, United States
Rebecca Reese − Genentech, Inc., South San Francisco,
California 94080, United States
Steven J. McKerrall − Genentech, Inc., South San Francisco,
California 94080, United States; orcid.org/0000-0002-
2966-5810; Phone: (650) 467-4347;
Email: mckerrall.steven@gene.com
Daniel P. Sutherlin − Genentech, Inc., South San Francisco,
California 94080, United States; Phone: (650) 225-3171;
Email: sutherlin.dan@gene.com
Shannon D. Shields − Genentech, Inc., South San Francisco,
California 94080, United States
Luis Sojo − Xenon Pharmaceuticals, Inc., Burnaby, British
Authors
Columbia V5G 4W8, Canada
Brian S. Safina − Genentech, Inc., South San Francisco,
California 94080, United States
Shaoyi Sun − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada
Chien-An Chen − Chempartner, Shanghai 201203, P.R.
China
Sultan Chowdhury − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Qi Jia − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada
Jun Li − Genentech, Inc., South San Francisco, California
94080, United States
Alla Y. Zenova − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Jean-Christophe Andrez − Xenon Pharmaceuticals, Inc.,
Burnaby, British Columbia V5G 4W8, Canada
Girish Bankar − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Philippe Bergeron − Genentech, Inc., South San Francisco,
California 94080, United States
Jae H. Chang − Genentech, Inc., South San Francisco,
California 94080, United States; orcid.org/0000-0003-
3457-7695
Elaine Chang − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada
Jun Chen − Genentech, Inc., South San Francisco, California
94080, United States
Richard Dean − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Shannon M. Decker − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Tao Sheng − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada
Henry Verschoof − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Matthew Waldbrook − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Michael S. Wilson − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Zhiwei Xie − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada
Clint Young − Xenon Pharmaceuticals, Inc., Burnaby, British
Columbia V5G 4W8, Canada
Tanja S. Zabka − Genentech, Inc., South San Francisco,
California 94080, United States
David H. Hackos − Genentech, Inc., South San Francisco,
California 94080, United States
Daniel F. Ortwine − Genentech, Inc., South San Francisco,
California 94080, United States
Andrew D. White − Chempartner, Shanghai 201203, P.R.
China; orcid.org/0000-0001-7869-0118
J.P. Johnson, Jr. − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
C. Lee Robinette − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
Christoph M. Dehnhardt − Xenon Pharmaceuticals, Inc.,
Burnaby, British Columbia V5G 4W8, Canada;
orcid.org/0000-0001-5213-6354
Charles J. Cohen − Xenon Pharmaceuticals, Inc., Burnaby,
British Columbia V5G 4W8, Canada
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(9) Swain, N. A.; Batchelor, D.; Beaudoin, S.; Bechle, B. M.; Bradley,
P. A.; Brown, A. D.; Brown, B.; Butcher, K. J.; Butt, R. P.; Chapman,
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S.; de Groot, M. J.; Glossop, M. S.; Gurrell, I. K.; Hannam, J.;
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Discovery of clinical candidate 4-[2-(5-Amino-1H-pyrazol-4-yl)-4-
chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfona-
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Foti, R. S.; Gao, H.; Graceffa, R.; Gunaydin, H.; Guzman-Perez, A.;
Huang, H.; Huang, L.; Jarosh, M.; Kornecook, T.; Kreiman, C. R.;
Ligutti, J.; La, D. S.; Lin, M.-H. J.; Liu, D.; Moyer, B. D.; Nguyen, H.
N.; Peterson, E. A.; Rose, P. E.; Taborn, K.; Youngblood, B. D.; Yu,
V.; Fremeau, R. T. Sulfonamides as selective NaV1.7 inhibitors:
optimizing potency and pharmacokinetics while mitigating metabolic
liabilities. J. Med. Chem. 2017, 60, 5969−5989.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00049
Funding
Thanks to Genentech, a member of the Roche Group, for
research funding.
Notes
The authors declare the following competing financial
interest(s): The authors are employees of Xenon Pharmaceut-
icals or Genentech, part of the Roche Group, and may hold
stock in those companies.
ACKNOWLEDGMENTS
■
We would like to thank the members of the synthetic
chemistry teams at Shanghai ChemPartner and Xenon
Pharmaceuticals. We thank Genentech Analytical, Purification,
DMPK, Safety Assessment, and in vivo Studies Group
colleagues for their contributions.
(11) La, D. S.; Peterson, E. A.; Bode, C.; Boezio, A. A.; Bregman, H.;
Chu-Moyer, M. Y.; Coats, J.; DiMauro, E. F.; Dineen, T. A.; Du, B.;
Gao, H.; Graceffa, R.; Gunaydin, H.; Guzman-Perez, A.; Fremeau, R.,
Jr.; Huang, X.; Ilch, C.; Kornecook, T. J.; Kreiman, C.; Ligutti, J.;
Jasmine Lin, M. H.; McDermott, J. S.; Marx, I.; Matson, D. J.;
McDonough, S. I.; Moyer, B. D.; Nguyen, H. N.; Taborn, K.; Yu, V.;
Weiss, M. M. The discovery of benzoxazine sulfonamide inhibitors of
NaV1.7: tools that bridge efficacy and target engagement. Bioorg. Med.
Chem. Lett. 2017, 27, 3477−3485.
(12) McKerrall, S. J.; Nguyen, T.; Lai, K. W.; Bergeron, P.; Deng, L.;
DiPasquale, A.; Chang, J. H.; Chen, J.; Chernov-Rogan, T.; Hackos,
D. H.; Maher, J.; Ortwine, D. F.; Pang, J.; Payandeh, J.; Proctor, W.
R.; Shields, S. D.; Vogt, J.; Ji, P.; Liu, W.; Ballini, E.; Schumann, L.;
Tarozzo, G.; Bankar, G.; Chowdhury, S.; Hasan, A.; Johnson, J. P.;
Khakh, K.; Lin, S.; Cohen, C. J.; Dehnhardt, C. M.; Safina, B. S.;
Sutherlin, D. P. Structure- and ligand-based discovery of chromane
arylsulfonamide Nav1.7 inhibitors for the treatment of chronic pain. J.
Med. Chem. 2019, 62, 4091−4109.
ABBREVIATIONS
■
CIP, congenital insensitivity to pain; EP, electrophysiology;
IEM, inherited erythromelalgia; LLE, lipophilic ligand
efficiency; PK, pharmacokinetic; PPB, plasma protein binding;
RLB, radioligand binding; VSD4, Nav1.7 fourth voltage sensing
domain
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