Structure- and Ligand-Based Discovery of Chromane Arylsulfonamide Nav1.7 Inhibitors for the Treatment of Chronic Pain

Article abstract of DOI:10.1021/acs.jmedchem.9b00141

Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Na_v1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Na_v1.7. Compound 24 showed a robust PK/PD response in a Na_v1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.

Full text of DOI:10.1021/acs.jmedchem.9b00141

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Article
Structure- and ligand-based discovery of chromane
v
arylsulfonamide Na1.7 inhibitors for the treatment of chronic pain
Steven John McKerrall, Teresa Nguyen, Kwong Wah Lai, Philippe Bergeron, Lunbin Deng,
Antonio DiPasquale, Jae H. Chang, Jun Chen, Tania Chernov-Rogan, David H. Hackos,
Jonathan Maher, Daniel Fred Ortwine, Jodie Pang, Jian Payandeh, William R. Proctor, Shannon
D. Shields, Jennifer Vogt, Pengfei Ji, Wenfeng Liu, Elisa Ballini, Lilia Schumann, Glauco
Tarozzo, Girish Bankar, Sultan Chowdhury, Abid Hasan, James Philip Johnson, Kuldip Khakh,
Sophia Lin, Charles J. Cohen, Christoph M Dehnhardt, Brian S. Safina, and Daniel P. Sutherlin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00141 • Publication Date (Web): 03 Apr 2019
Downloaded from http://pubs.acs.org on April 3, 2019
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Structure- and ligand-based discovery of chromane
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arylsulfonamide Na 1.7 inhibitors for the treatment
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of chronic pain
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Steven J. McKerrall* , Teresa Nguyen , Kwong Wah Lai , Philippe Bergeron , Lunbin Deng ,
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Antonio DiPasquale , Jae H. Chang , Jun Chen , Tania Chernov-Rogan , David H. Hackos ,
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Jonathan Maher , Daniel F. Ortwine , Jodie Pang , Jian Payandeh , William R. Proctor ,
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Shannon D. Shields , Jennifer Vogt , Pengfei Ji , Wenfeng Liu , Elisa Ballini , Lilia Schumann ,
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Glauco Tarozzo , Girish Bankar , Sultan Chowdhury , Abid Hasan , James P. Johnson, Jr. ,
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Kuldip Khakh , Sophia Lin , Charles J. Cohen , Christoph M. Dehnhardt , Brian S. Safina ,
Daniel P. Sutherlin1
1Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080
2WuXi AppTec Co., Ltd. 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131,
People’s Republic of China.
3Evotec AG, Essener Bogen 7, 22419 Hamburg, Germany
4Xenon Pharmaceuticals, Inc., 200-3650 Gilmore Way, Burnaby, BC V5G 4W8, Canada
ABSTRACT
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Using structure- and ligand-based design principles, a novel series of piperidyl chromane
arylsulfonamide Na 1.7 inhibitors was discovered. Early optimization focused on improvement of
v
potency through refinement of the low energy ligand conformation and mitigation of high in vivo
clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of
lipophilicity and LLE to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype
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selective inhibitor of Na 1.7. Compound 24 showed a robust PK/PD response in a Nav1.7
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dependent mouse model and site-directed mutagenesis was used to identify residues critical for the
isoform selectivity profile of 24.
Introduction
Chronic pain is a significant unmet medical need, with an estimated 11% of people in the
developed world—25.3 million people in the US alone—reporting pain every day for longer than
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3
months. The voltage gated sodium channel Na 1.7 is critical for the transmission of pain signals
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,3
in humans and has been extensively investigated as a potential target for pain therapeutics. The
most compelling data that supports the role of Na 1.7 in pain signaling is the impact of SCN9A
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loss of function mutations in some forms of congenital insensitivity to pain (CIP), an inherited
condition characterized by a complete absence of pain sensation without additional cognitive or
sensory deficits.4,5 The dramatic loss of function phenotype underlies Na 1.7’s necessary role in
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the initiation of action potentials that transmit pain signals in the peripheral nervous system.
Furthermore, CIP patients show only anosmia as an additional effect, suggesting that selective
blockade of Na 1.7 may result in few side effects. Insensitivity to pain due to Na 1.7 knock-out
v
v
has also been recapitulated in both germ-line and conditional knock-out mice, both validating the
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target and showing the translational potential of mouse pharmacodynamic (PK/PD) and efficacy
studies.6,7
The key challenge in identifying a safe Na 1.7 inhibitor is achieving subtype selectivity
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over the other voltage gated sodium channel isoforms that have diverse functions in bodily systems
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including the CNS (Na 1.1, Na 1.2, Na 1.6), skeletal muscle (Na 1.4), and heart (Na 1.5).
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Unintended systemic administration of non-selective local anesthetic sodium channel blockers is
associated with severe cardiovascular and CNS side effects, so the safety consequences of
compounds with poor subtype selectivity are potentially serious.10
Figure 1. Selected previous arylsulfonamide Na 1.7 inhibitors.
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The strong human and rodent genetic validation of Na 1.7 as a pain target has motivated
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significant efforts to develop subtype selective inhibitors for clinical use. Early breakthroughs in
this area led to the discovery of two series of inhibitors (acylsulfonamides and arylsulfonamides)
that bind to the fourth voltage sensing domain (VSD4) and block the channel by locking it into the
inactivated state.3,11 We have previously disclosed our efforts in the discovery and optimization of
1
2,13
novel acylsulfonamide Na 1.7 inhibitors.
We sought to expand our approach into the related
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class of arylsulfonamide inhibitors, broadly represented by 1–4 (Figure 1). Illustrative compounds,
such as PF-05089771 (1) and AM-0466 (2) from this series are high MW (>500 g/mol) lipophilic
acids with a large number of aromatic rings, which generally leads to high levels of plasma protein
binding, challenging pharmacokinetic (PK) properties, and high plasma concentrations required to
see activity in vivo.14–16 Furthermore, clinical development of 1 was recently discontinued due to
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lack of activity in a Phase 2a diabetic neuropathy trial. While minimal preclinical efficacy data
for 1 has been published, a related IV-dosed lead molecule showed no activity in a mouse model
of inflammatory pain despite achieving unbound plasma concentrations well above the in vitro
potency.18
The lack of in vivo efficacy of 1 and related compounds highlights the importance of
demonstrating PK/PD relationships in vivo, and the continued need for additional high-quality tool
molecules to profile the effects of Na 1.7 inhibition in vivo. More recently, zwitterionic inhibitors
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exemplified by 3 and 4 have been disclosed that occupy a distinct physicochemical property space
with lower molecular weight and lipophilicity; however, their zwitterionic nature can introduce
PK challenges such as poor permeability, high clearance, and modest oral bioavailability.19,20 In
support of further exploration and validation of Na 1.7 as a target for pain, we sought to develop
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structurally novel arylsulfonamide Na 1.7 inhibitors and to optimize them to a novel lead series
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with improved PK properties and in vivo activity.
Results and Discussion
Figure 2. Nav1.7 X-ray structure and initial piperidine design.
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a) X-ray co-crystal structure of 5 bound to VSD4 with a Na 1.7 construct. (PDB: 5EK0) . b) Design of piperidine scaffold 6.
To further advance the arylsulfonamide class of Na 1.7 inhibitors, we sought to utilize our
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landmark co-crystal structure of GX-936 (5) bound to a VSD4 within a Na 1.7 construct to design
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a novel scaffold. (Figure 2) In this co-crystal structure, the anionic arylsulfonamide A-ring makes
essential interactions with R1608 and R1602, important residues for the function of VSD4 and
channel gating. The pyrazole D-ring occupies a pocket between Y1537 and W1538 (‘YW pocket’)
and the CF group occupies a highly lipophilic channel formed by W1538, V1541, and F1583
3
(
‘WF channel’). The YW pocket interaction is critical for subtype selectivity, as this YW pair is
varied in all other isoforms except Na 1.2 and Na 1.6. We sought to maintain the key interactions
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between the anionic head-group and R1608, while redesigning the B/C/D/E rings and reducing the
number of aromatic rings by saturating the linking C-ring. To this end we envisioned that saturating
the C-ring via replacement with a piperidine, as in 6, would improve physicochemical properties
3
22
with its higher fraction of sp carbons and better occupy the available space in the pocket.
Molecular modeling suggested that vectors of appropriate geometry to occupy the YW pocket and
WF channel could be afforded by a 2,4-cis-substituted piperidine in a low energy, all equatorial
conformation. This transformation would require replacing the ether linker with a carbon atom to
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provide maximum chemical stability. While we were mindful about the conformational impact of
this change (vide infra) we felt these compounds would provide a simple proof of concept.
Table 1. Discovery of (2R,4S)-4-trifluoromethyl-2-aryl piperidine
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aHuman Na
1.7 K
from a radioligand binding assay (mean ± SEM). LLE was calculated using LogD7.4 (LLE = pK
b
v
i
i
– LogD7.4). Stereochemistry
of 9 and 10 is assigned as described in the text.
Compounds 7 and 8 were prepared as initial design probes, but showed weak binding to
human Na 1.7 in a VSD4 radioligand binding (RLB) assay with 7 showing a K of ~500 nM (Table
v
i
1
). We suspected the low potency was due to the compounds only filling the YW pocket and
leaving the WF channel unoccupied, so we evaluated cis-4-CF substituted analogs 9 and 10. The
3
two enantiomers showed a >10-fold difference in Na 1.7 potency with the more potent enantiomer
v
showing moderate binding in the RLB assay. While we were unable to crystalize 9 or synthetic
intermediates to determine the absolute configuration, 9 was tentatively assigned as the (2R,4S)
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enantiomer on the basis of the relative potency of 7 and 8 and computational docking (see
supporting information). Testing in functional electrophysiology (EP) assays—which are designed
favor the inactivated state of the channel—showed 9 inhibited Na 1.7 with an IC of 15.3 nM and
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possessed >600-fold selectivity over Na 1.5. The basic amine pK of 9 was measured as 5.50,
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indicating the piperidine is unlikely to be protonated. We were encouraged by the functional
potency and high selectivity over Na 1.5, both of which indicated that 9 achieved our main design
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goals of occupying the YW pocket and WF channel.
Figure 3. Torsion analysis of 9 and 11 and docking model of 11 in Na 1.7.
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a) Quantum-mechanical torsion scan of C-N-C-C torsion indicated in red in model of 9. The vertical line indicates the angle observed in the
docked model. b) Computational scan of C-N-C-C torsion indicated in red in model of 11. Vertical line indicates angle observed in docking
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model. c) Docked model of 11 in Na 1.7 X-ray structure (PDB: 5EK0).
We next examined the conformation of the carbon linkage connecting the piperidine ring
to the central phenyl ring. Quantum-mechanical (QM) torsional scans of the C-C and C-N bonds
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were carried out to assess the energetic energy landscape and the impact on the associated bond
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vectors leading to the YW pocket and WF channel. The proposed bound structure of 9 requires
a nearly coplanar C-C dihedral angle. While this conformation is within 1 kcal/mol of the global
minimum, there is significant rotational flexibility which could contribute to an entropic penalty
on binding (see supporting information, Figure S5). More significantly, a torsional scan of the C-
N bond indicated the desired conformation was a high energy rotamer ~3 kcal/mol above the global
minimum (Figure 3a). We hypothesized that restricting the rotational flexibility around both bonds
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to favor the desired conformation could lead to an increase in binding affinity. To achieve this,
1
1 was designed to restrict the conformational freedom of the ligand. We envisioned that the
cyclization into a chromane ring would both remove free rotation around the C-C bond and
significantly bias the C-N bond into the desired torsion angle. This was supported by a quantum
torsional scan of the C-N bond in a model of 11, which revealed the desired conformation was
effectively locked in (Figure 3b). Gratifyingly, 11 showed a 0.97 nM K in the RLB assay, a 75-
i
fold increase in binding affinity over 9 (Table 2). This increase appears to be due to an increase in
specific binding since the lipophilic ligand efficiency (LLE) also increased by ~1 unit. We believe
this result also validates the tentative structural assignment of 9, as 11 can be unambiguously
assigned through X-ray crystallographic analysis of related analog 16 (vide infra). Finally, 11
maintained excellent functional potency and selectivity, with a Na 1.7 IC of 0.57 nM and 509-
v
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fold selectivity over Na 1.5 in an EP assay.
v
Table 2. Chromane core and arylsulfonamide heterocycle SAR.
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aHuman Na
1.7 K
from a radioligand binding assay (mean ± SEM). LLE was calculated using LogD7.4 (LLE = pK
b
– LogD7.4). Scaled hepatic
c
v
i
i
d
clearance from human (HH) and rat (RH) hepatocytes. Total plasma clearance from a 1 mg/kg rat IV pharmacokinetic study.
Further profiling of 11 revealed moderate LLE and moderate stability in human and rat
2
5
hepatocytes (Table 2). Computational metabolic soft-spot prediction using MetaSite suggested
that the phenyl group was a potential site of metabolism, so para-fluoro compound 12 was
prepared. While this change did not substantially improve the hepatocyte stability, metabolite
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identification in human hepatocytes on 12 indicated no oxidation of the aromatic ring, so the para-
fluoro group was preserved in subsequent analogs to reduce the risk of arene metabolism. Profiling
of 12 in vivo revealed that the compound was subject to high clearance in rat after IV
administration, approximately at the rate of liver blood flow. This was unexpected given the
moderate in vitro stability and high plasma protein binding (>99.9%) of 12. Examination of the in
vivo fate of the compound revealed that a large proportion was excreted in the bile as parent (data
not shown). We hypothesized that the anionic arylsulfonamide group may interact with
transporters in the liver and contribute to clearance, so we altered the sulfonamide heterocycle to
reduce the acidity.26,27 Subsequent analogs also incorporated the core fluorine (Table 2, R ) in 9
which was left out of the earlier chromane analogs for simplicity. Notably, while the thiadiazole
showed high clearance, several other heterocycles commonly employed in reported
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arylsulfonamide series dramatically reduced the clearance in rat. While 5-fluoropyridine 13 and
pyridazine 14 showed significantly improved hepatocyte stability and decreased clearance, both
suffered from reduced potency and LLE. Isoxazole 15 and pyrimidine 16 were more promising,
showing excellent potency, LLE, and clearance in rat. Consistent with known relationships
between acidic pK and OATP-mediated liver uptake, less acidic sulfonamides showed universally
a
improved in vivo clearance, with 16 best balancing the pK to maintain the critical interaction with
a
2
7
R1608. Compound 16 was chosen for subsequent profiling due to its increased potency and
overall physicochemical properties.
Table 3. Comparison of in vitro and in vivo PK properties of 12 and 16.
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aPassive A–B permeability in a WT-MDCKI transwell assay, 10-6 cm/s. Scaled hepatic clearance from mouse (MH) and rat (RH) hepatocytes.
b
c
d
Total plasma clearance and volume of distribution from a 1 mg/kg IV pharmacokinetic study estimated non-compartmental analysis. Total
liver/plasma ratios in rat after oral administration.
Further comparison of 12 and 16 using in vitro and in vivo PK experiments revealed that
active transport in the liver may indeed underlie the stark differences in their PK properties (Table
). Compound 12 showed significantly higher clearance in both mouse and rat than expected based
3
on its intrinsic stability and plasma protein binding. In contrast, 16 showed 5-fold lower mouse
Clblood and 77-fold lower rat Cl despite having no discernible differences in hepatocyte stability
p
or plasma protein binding (PPB). Interestingly, 12 also showed significantly higher apparent
volume of distribution in both mouse and rat which is not explained by tissue binding differences
(
data not shown). Compound 16 showed higher passive permeability than 12 which was
hypothesized to decrease its susceptibility to transport-mediated clearance.27,28 Most compellingly,
when liver and plasma concentrations were examined in rats, 12 showed a large excess (>200-
fold) of total drug in the liver while 16 showed only a modest 4-fold liver/plasma (L/P) ratio. While
unbound L/P ratios are challenging to determine due to the high binding of the compounds, these
data suggest significant active uptake into the liver for 12 but not 16. Taken together, the likely
explanation is that the high clearance of 12 is mediated by transporter activity, which was reduced
in 16.
Figure 4. In vivo activity of 16.
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Antinociceptive activity of 16 in a) IEM aconitine PK/PD model (significance is versus Vehicle group, by one-way ANOVA then Dunnett’s test)
and b) Complete Freund's Adjuvant (CFA) hyperalgesia model. Significance is versus Pre-CFA measurement, except where indicated for the 50
mg/kg dose (by two-way ANOVA followed by Sidak’s multiple comparisons test). *: p<0.05, **: p<0.005, n.s.: not significant.
To evaluate the activity of 16 in vivo we first turned to a mouse model that is dependent
29
on a transgenic hNa 1.7 variant (inherited erythromelalgia (IEM) model). IEM is a disorder
v
caused by gain of function mutations in Na 1.7 that result in heightened sensitivity to painful
v
stimuli. Animals are dosed with Aconitine, a plant alkaloid that causes abnormal opening of
sodium channels, and the dose is chosen so that there is little effect on wild type mice, but a
profound effect on transgenic IEM mice. Thus, the assay is designed to measure in vivo blockade
of hNa 1.7. Compound 16 showed a dose dependent reduction in the number of nociceptive events,
v
with 75% and 85% reduction at 15 and 50 mg/kg PO. Compound 16 was further evaluated in a
mouse model of Complete Freund's Adjuvant (CFA) induced inflammatory pain. Administration
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of CFA causes a significant reduction in paw withdrawal latency to heat stimulus, which was
reversed by ~50% at 15 mg/kg and completely reversed at 50 mg/kg (both were not significant
versus the pre-CFA latency). The 50mg/kg dose also provided a significant reversal in
hypersensitivity versus the post-CFA, pre-dose measurement. Mean total plasma exposures for
doses of 15 and 50 mg/kg were 33 M and 58 M, respectively (see supporting information). The
high PPB makes assessment of the unbound PK/PD relationship challenging; however, we viewed
the in vivo activity as promising and warranting further optimization to increase potency and
reduce the high total plasma concentration required to see activity.
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Table 4. YW pocket heterocycle SAR and identification of 24.
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aHuman Na
1.7 K
from a radioligand binding assay (mean ± SEM). LLE was calculated using LogD7.4 (LLE = pK
b
– LogD7.4). Cytotoxicity
c
v
i
i
d
-6
e
(
total ATP) in human hepatocyte derived liver microtissues. Passive A–B permeability in a WT-MDCKI transwell assay, 10 cm/s. Scaled
d
hepatic clearance from human (HH) and rat (RH) hepatocytes. Total plasma clearance from a 1 mg/kg rat IV pharmacokinetic study.
In the course of a routine assessment of the in vitro safety profile, we identified that 16
0
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showed significant cytotoxicity in 3D human liver microtissues (hLiMT), with an IC <10 M.
5
0
Cytotoxicity in hLiMT has been shown to be predictive of clinical hepatotoxicity in a retrospective
3
0
analysis. Given the high plasma concentrations required for activity and the chronic nature of the
intended pain therapy, we viewed this as a significant liability for 16 and sought to mitigate this
risk through further optimization. We reasoned that the relatively high lipophilicity of 16 (LogD7.4
of 3.2) could contribute to the cytotoxicity observed. The fluorophenyl group was identified as a
possible group to modify to incorporate additional polarity and a range of heterocyclic
replacements were synthesized (Table 4). Pyrazole and N-methylpyrazole replacements 17–20
showed excellent potency, a substantial reduction in lipophilicity, and importantly, a marked
decrease in the observed in vitro cytotoxicity in hLiMT. Furthermore, the LLE was increased by
1
.4–2 units, suggesting that heterocyclic rings in the YW pocket provide a specific favorable
binding interaction, potentially through increased edge-to-face and -stacking interactions with
the key Y1537 and W1538 residues. Unfortunately, the in vivo clearances of 17–20 were greatly
increased, ranging from 75–>100% of liver blood flow (LBF) in rats. Oxazole 21 showed a similar
profile as the pyrazoles. The increase in clearance may be due in part to the substantial increase in
unbound fraction, but nevertheless, the observed clearance was much higher than expected based
on the hepatocyte stability and PPB, a profile reminiscent of 12. In addition to a relationship with
pK discussed above, OATP-mediated liver uptake is also correlated with low LogD and reduced
a
7.4
permeability, and we hypothesized the increased polarity of 17-21 resulted in a return to
transporter-mediated clearance.27,28 Thus, we targeted less polar 6-membered pyridines to slightly
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increase the lipophilicity and permeability. While 4- and 3-substituted isomers 22 and 23 showed
similarly high clearance, the 2-pyridyl isomer 24 showed comparable potency, low cytotoxicity in
hLiMT, and improved rat clearance. More polar diazine isomers 25–27 showed substantially
lower passive permeability and, where profiled, inferior rat clearance. The human hepatocyte
stability of 24 was excellent and it showed the highest passive permeability of the analogs profiled.
While we have not examined the in vivo metabolic fate of 24 in as much detail as 12 and 16, we
suspect that an optimal balance of lipophilicity and permeability reduces transporter activity and
contributes to the reduced in vivo clearance.
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Figure 5. Relationship between LogD and in vitro cytotoxicity in hepatic spheroids
7.4
1Correlation between measured LogD7.4 and hLiMT cytotoxicity IC50. Points indicated with > or < had IC50 values greater or less than the
indicated value. NPV and PPV values are calculated using hLiMT cutoffs of >50 M (negative) and <50 M (positive)
Examination of the correlation between cytotoxicity in spheroid hLiMT and lipophilicity
revealed that measured LogD alone was a very strong predictor of toxicity (Figure 5). A LogD
7.4
7
.4
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cutoff of 2.0 was able to separate compounds with 81% (>50 M, low risk) and 92% (<50 M,
high risk) negative and positive predictive values. This relationship provided clear design criteria
during optimization and reinforced the value of reducing lipophilicity to resolve the in vitro
cytotoxicity risk and prioritizing compounds on the basis of LLE.
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Figure 6. Pharmacokinetic properties and IEM PK/PD of 24.
a) Pharmacokinetic profile of 24. b) PK/PD of 24 in IEM model, each dot represents and individual animal measurement (Black = vehicle, light
a
blue = 3 mg/kg, blue = 10 mg/kg, dark blue = 30 mg/kg. Square = male, circle = female). Scaled hepatic clearance from human (HH) and rat
b
c
(
RH) hepatocytes. In vivo pharmacokinetic parameters determined from 1 mg/kg (rat) or 0.5 mg/kg (dog, cyno) IV dose. Determined using 5
mg/kg (rat) or 0.5 mg/kg (dog, cyno) oral dose in MCT vehicle.
The in vivo pharmacokinetic profile of 24 was evaluated in rats, beagle dogs, and
cynomolgus monkeys (Figure 6a). Compound 24 showed low or moderate clearance (8–44% of
LBF) across all species and low volume of distribution, consistent with the high degree of PPB.
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This resulted in modest 2–4 hour half-lives across species. The oral bioavailability was variable,
with high exposure in rat (73%) and relatively low exposure in monkey (18%). Additional
formulation options beyond MCT suspension were not evaluated to address this potential liability.
In addition, the in vivo PK/PD relationship of 24 was evaluated in the IEM mouse model (Figure
1
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b). Compound 24 showed robust, concentration dependent reduction in nociceptive events with
an EC of 740 nM and an EC of 9.6 nM. While the comparison of EC50,u with 16 is difficult
50
50,u
due to its high PPB, 24 has >10-fold lower total plasma EC than 16, fulfilling the goal of reduced
5
0
total plasma exposure.
a
Table 5. Potency and selectivity of 24 on Na 1.X isoforms.
v
aNa
1.X K determined by Dynaflow Manual Patch Clamp (mean ± SEM). Fold selectivity is relative to hNa 1.7 K . h = human, r = rat, d = beagle dog.
v d v d
Figure 7. Mutagenesis and structural rationale for the observed Na 1.X isoform selectivity.
v
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a) Sequence alignment of a section of VSD4 S2 helix of hNa
the surface of the binding pocket in the X-ray structure, while the residues in red were selected for mutagenesis studies. b) Mutagenesis identified
residues essential for selectivity for Na 1.7 over other Na isoforms (mean ± SEM). c) Structural docking model and rationale for the importance
v
1.X isoforms showing residues important for selectivity. Highlighted residues form
v
v
of Y1537, W1538, and V1541 in conferring selectivity (derived from PDB: 5EK0).
The selectivity of 24 over other Na isoforms was assessed using manual patch clamp
v
electrophysiology (Table 5). Compound 24 is a highly potent inhibitor of Na 1.7 in this assay,
v
showing a K of 0.38 nM. The observed selectivity over the majority of other isoforms tested was
d
very high, showing >1000 nM K and >2500-fold selectivity over Na 1.1, Na 1.3, Na 1.4, and
d
v
v
v
Na 1.5. Selectivity over Nav1.2 and Nav1.6 was more modest at 31- and 73-fold, respectively.
v
Consistent with previous reports, the potency of 24 on the rat Na 1.7 channel was substantially
v
reduced compared with human Nav1.7, while the potency was roughly equivalent for dog.31 No
inhibition of the tetrodotoxin resistant (TTX-R) current in mouse dorsal root ganglion (DRG)
neurons was seen up to >3000 nM, indicating a high level of selectivity over mNa 1.8 and
v
7
mNa 1.9.
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To better understand the origin of selectivity, we examined sequence differences across
isoforms in the VSD4 binding pocket (Figure 7). In particular, we focused on residues in the S2
helix where there is high sequence variability across isoforms. Consistent with our proposed
binding mode, we suspected that Y1537 and W1538 (YW) play a crucial role in dictating isoform
selectivity.21 This pair of residues is varied in all of the isoforms where 24 has >2500-fold
selectivity. We examined the effect of mutating the YW pair to SR—residues that are found in
Na 1.1 and Na 1.3—and found the potency of 24 was reduced by 448-fold. We believe these
1
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residue differences disrupt -stacking and edge-to-face interactions between the pyridine in 24 and
the adjacent residues, which are responsible for much for the activity. While this variation
explained the selectivity over most other Na isoforms, the origin of modest selectivity over Na 1.2
v
v
and Na 1.6 was less clear since both isoforms maintain the YW pair. V1541 forms significant
v
contacts with the CF and piperidine and is unique to Na 1.7, so we suspected it may play a role
3
v
in selectivity. The V1541L mutation, found in most of the other isoforms including Na 1.2 and
v
Na 1.6, reduced the potency of 24 by 10-fold, consistent with the observed selectivity against both
v
isoforms. The larger leucine may clash with the CF substitution and modify the surface the
3
piperidine ring packs against, either of which could be responsible for the observed selectivity.
Combining the mutations in a triple YWV to SRL mutant—again present in Na 1.1 and Na 1.3—
v
v
reduced the potency of 24 by >2000-fold, reproducing virtually all of the observed selectivity over
these isoforms. While all of the other variations at these three residues across isoforms have not
been examined, we believe that variations at Y1537, W1538, and V1541 account for the majority
of selectivity seen for 24 and other compounds in this class.
Chemistry
Scheme 1. Synthesis of 7-10
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Reagents and conditions: a) Pd(OAc)
0ºC. e) (R)- or (S)-2-phenylpiperdine, NaI, K
2, NaI, K CO , MeCN, 80ºC. DMB = 2,4-dimethoxybenzyl. 36, 37, 9, and 10 are assigned as described in the text.
2
, DPE-phos, Cs
2
CO
3
, CO, MeOH/dioxane, 60ºC. b) TFA, DCM, r.t. c) NaBH
4
, MeOH, r.t. d) SOCl
2
,
9
2
CO , MeCN, 80ºC. f) Pd(OAc)
3
2
, Phenylboronic acid, K CO , EtOH/H O. g) Pd/C, H
2
3
2
2
, AcOH. h)
3
2
3
The synthesis of 7-10 began with previously reported 28, which was subjected to
32
palladium-catalyzed carboxymethylation to produce ester 29. Removal of the DMB protecting
group with TFA produced 30, which was reduced to the primary alcohol 31 then converted into
the penultimate alkyl chloride 32 with thionyl chloride. Alkylation of commercially available (R)-
2
-phenylpiperidine gave 7, while reaction with (S)-2-phenylpiperidine gave 8. The synthesis of 9
and 10 began with the cross coupling of 33 with phenylboronic acid to produce 34. Compound 34
was reduced with H and Pd/C to give rac-35, which was produced without any detectable trans-
2
stereoisomers. Racemic 35 was resolved into by chiral SFC to produce single enantiomers 36 and
3
7, which were alkylated with 32 to produce 9 and 10 respectively.
Scheme 2. Synthesis of chromane cores 45 and 47.
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1
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
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4
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4
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5
5
5
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0
1
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9
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9
0
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7
8
9
0
Reagents and conditions: a) 3-chloropropanoyl chloride, AlCl
3
, DCM, r.t. b) TfOH, 85ºC. c) (S)-2-methyl-2-propanesulfinamide, Ti(OEt)
4
, THF,
80ºC. d) NaBH
4
, THF, –15ºC. e) HCl, dioxane, r.t. f) TFAA, Et
3
N, DCM, r.t. g) BnSH, Pd
2
(dba)
3
, Xantphos, iPr
2
NEt, dioxane, 120ºC. h) 1,3-
dichloro-5,5-dimethylhydantoin, THF/AcOH/H
2
O, 0ºC.
Synthesis of chromane intermediate 45 began with reaction of 38 with 3-chloropropanoyl
chloride followed by cyclization to form 39. Condensation with (S)-tert-butylsulfinamide gave 40,
which was smoothly reduced with sodium borohydride to produce S amine 41. No diastereomeric
R amine could be detected. Removal of the tert-butylsulfinyl group with HCl gave 42 and
protection of the free amine as the trifluoroacetamide gave 43. The aryl bromide was cross coupled
with benzyl thiol with palladium catalysis to give 44. Oxidation with 1,3-dichloro-5,5-
dimethylhydantoin then gave the sulfonyl chloride 45. The identical sequence of steps could be
followed beginning with commercially available chromanone 46 to produce core intermediate 47.
Scheme 3. Synthesis of 11–27
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
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0
1
2
3
4
5
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7
8
9
0
1
2
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5
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: a) (R)-2-methylpropane-2-sulfinamide, Ti(OEt)
4
, DCM, r.t. b) LDA, ethyl 4,4,4-trifluorobut-2-enoate, THF, –78ºC to
0
ºC. c) HCl (aq.), EtOH, r.t. d) LiAlH
4
, THF, 0ºC. e) Dess-Martin periodinane, DMF, 0ºC. f) DABCO, DCM, r.t. g) NaOH (aq.), THF/MeOH,
4 3 2
40ºC. h) Ti(OiPr) , NaBH(OAc) , PhMe, r.t. g) HCO H, r.t. DMB = 2,4-dimethoxybenzyl. Non-stereogenic hydrogens omitted for clarity in the
X-ray structure of 16. Thermal ellipsoids drawn at 50% probability. X-ray image generated using ORTEP-3.33
The preparation of final compounds 11–27 began with methyl ketones 48a–m.
Condensation of (R)-tert-butylsulfinamide proceeded smoothly to give 49a–m. Deprotonation
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with LDA and addition of ethyl (E)-4,4,4-trifluorobut-2-enoate provided 50a–m in >20:1 d.r. in
all cases, setting the requisite R-CF configuration. The chiral auxiliary could be removed with
3
hydrochloric acid to produce 51a–m. Global reduction with lithium aluminum hydride provided
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
2a–m as a mixture of inconsequential diastereomers, which were smoothly oxidized with Dess-
Martin periodinane to provide the key keto-aldehydes 53a–m. Formation of the sulfonamide could
be accomplished by combining 45 or 47 with DMB protected anilines 54a–e to give sulfonamides
5
5a–f. Deprotection of the trifluoroacetamide with sodium hydroxide proceeded smoothly to give
key amine intermediates 56a–f. The union of 53a–m and 56a–f could be affected through
treatment with titanium isopropoxide followed by sodium triacetoxy borohydride to facilitate a
double reductive amination, set the final stereocenter without any observed trans-diastereomer,
and form 57a–q. Removal of the DMB protecting group with formic acid provided final
compounds 11–27. The stereochemical outcome of this sequence was established by an X-ray
crystal structure of 16, which confirmed the desired stereochemistry and the stereochemical
identity of the intermediates described above. Additionally, the small molecule X-ray structure of
1
6 agreed well with the predicted bound conformation and supports the conformational arguments
presented herein.
Conclusion
Examination of the arylsulfonamide Na 1.7 literature revealed that existing chemical
v
matter suffered from poor physiochemical properties which resulted in poor PK properties and
high plasma exposures required to see effects in vivo. An X-ray co-crystal structure of an
arylsulfonamide bound to VSD4 of Na 1.7 afforded the opportunity to pursue structure-based drug
v
design in tandem with physiochemical property optimization. This effort led initially to the design
of inhibitor scaffold 9. Further refinement of the ligand conformation resulted in the discovery of
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9
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a novel chromane core, which produced a dramatic improvement of potency. Optimization of the
in vivo PK profile in rats led to 16, which showed a promising PK profile and substantial activity
in PK/PD and efficacy models. Further optimization of potency, LLE, and in vitro cytotoxicity led
to the discovery of 24 which possesses excellent potency, metabolic stability, and good selectivity
over other Na isoforms. Compound 24 shows robust activity in a Na 1.7-dependent PK/PD model
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
v
v
with an EC of 740 nM and EC
of 9.6 nM, a significant improvement over the majority of
50,u
50
3
published Na 1.7 inhibitors. Finally, examination of the proposed binding mode of 24 in Na 1.7
v
v
revealed residues important for isoform selectivity that were confirmed by mutagenesis,
suggesting opportunities for further improvement of isoform selectivity. This work was supported
by a highly efficient asymmetric synthesis of this complex scaffold. GNE-616 (24) represents a
valuable tool molecule for evaluation of the utility of Na 1.7 inhibition in the treatment of chronic
v
pain.
Experimental Section
General Methods. Unless otherwise specified, commercially available reagents and solvents were
used without further purification. Compounds 33, 38, and 46 were obtained commercially and the
starting materials used in Scheme 3 are described in the supporting information Figure S1. Yields
1
refer to chromatographically and spectroscopically ( H NMR) homogeneous materials, unless
otherwise stated. NMR experiments were performed with Bruker Avance III 300, 400, or 500 MHz
1
19
13
spectrometers. H, F, and C 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
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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, 1.7mm, 50 × 2.1
mm column at a flow rate of 0.4 ml / minute. 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
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
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5
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9
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1
2
3
4
5
6
7
8
9
0
ended at 98% MPB over 7 min and held at 98% MPB for 1.5 min following an equilibration for
o
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%
(2.5ML/4LTFA in acetonitrile) over 4 minutes and holding at 80% for 2 minutes 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*25mm*5um column using
MeCN/H O gradients described below. Preparative SFC separations were carried out as described
2
below. Analytical purity of all final compounds was greater than 95% as determined by LCMS
using 254 nM UV detection unless otherwise stated. A detailed listing of starting materials used
in Scheme 3 and their source is provided in the supporting information.
(R)-2,5-difluoro-4-((2-phenylpiperidin-1-yl)methyl)-N-(1,2,4-thiadiazol-5-
yl)benzenesulfonamide (7). The title compound was prepared from benzyl chloride 32 and (R)-
+
2
-phenylpiperidine in a manner analogous to 9. HRMS (ESI-FTMS) m/z [M+H] Calcd for
1
C H F N O S 451.1069; Found 451.1062. H NMR (400 MHz, DMSO-d ) δ 7.95 (s, 1H), 7.48
2
0
20
2
4
2
2
6
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
-
7.24 (m, 7H), 3.51 - 3.47 (m, 1H), 3.23 - 3.15 (m, 2H), 2.95 - 2.86 (m, 1H), 2.33 - 2.31 (m, 1H),
.75 - 1.56 (m, 5H), 1.44 - 1.35 (m, 1H). Exchangeable NH not observed.
1
(S)-2,5-difluoro-4-((2-phenylpiperidin-1-yl)methyl)-N-(1,2,4-thiadiazol-5-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
yl)benzenesulfonamide (8). The title compound was prepared from benzyl chloride 32 and (S)-2-
+
phenylpiperidine in a manner analogous to 9. HRMS (ESI-FTMS) m/z [M+H] Calcd for
1
C H F N O S 451.1069; Found 451.1063. H NMR (400 MHz, DMSO-d ) δ 7.90 (s, 1H), 7.40
20 20
2
4
2
2
6
-
7.19 (m, 7H), 3.47 - 3.43 (m, 1H), 3.25 - 3.20 (m, 2H), 3.08 - 3.04 (m, 1H), 2.84 - 2.81 (m, 1H),
.74 - 1.39 (m, 5H), 1.36 - 1.33 (m, 1H). Exchangeable NH not observed.
1
2
,5-difluoro-4-(((2R,4S)-2-phenyl-4-(trifluoromethyl)piperidin-1-yl)methyl)-N-(1,2,4-
thiadiazol-5-yl)benzenesulfonamide (9). A mixture of 4-(chloromethyl)-2,5-difluoro-N-(1,2,4-
thiadiazol-5-yl)benzenesulfonamide (150 mg, 0.46 mmol), (2R,4S)-2-phenyl-4-
trifluoromethyl)piperidine (106 mg, 0.46 mmol), sodium iodide (104 mg, 0.69 mmol) and
(
o
potassium carbonate (191 mg, 1.38 mmol) in acetonitrile (7.5 mL) was stirred at 80 C for 2 h
under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was filtered
and the filtrate was concentrated in vacuo. The residue was purified by reverse phase
chromatography (acetonitrile 45-75% / 0.225% formic acid in water) to give the title compound
(42 mg, 18%) as a white solid. The structure was assigned tentatively as described in the text (vide
+
supra). HRMS (ESI-FTMS) m/z [M+H] Calcd for C H F N O S 519.0942; Found 519.0932.
2
1
20
5
4
2 2
1
H NMR (400 MHz, DMSO-d ) δ 8.47 (s, 1H), 7.52 (dd, J = 8.7, 5.5 Hz, 1H), 7.49 – 7.40 (m, 3H),
6
7.35 (t, J = 7.4 Hz, 2H), 7.28 (t, J = 7.3 Hz, 1H), 3.56 (s, 2H), 3.03 (s, 2H), 2.60 (s, 1H), 2.41 (s,
0
H), 2.39 – 2.21 (m, 1H), 1.93 – 1.82 (m, 2H), 1.68 – 1.60 (m, 3H). Exchangeable NH not observed.
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,5-difluoro-4-(((2S,4R)-2-phenyl-4-(trifluoromethyl)piperidin-1-yl)methyl)-N-(1,2,4-
thiadiazol-5-yl)benzenesulfonamide (10). The title compound was prepared from benzyl
chloride 32 and (2S,4R)-2-phenyl-4-(trifluoromethyl)piperidine in a manner analogous to 9. The
structure was assigned tentatively as described in the text (vide supra). HRMS (ESI-FTMS) m/z
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
[M+H] Calcd for C H F N O S 519.0942; Found 519.0937. H NMR (400 MHz, DMSO-d ) δ
21 20 5 4 2 2 6
8
.47 (s, 1H), 7.52 (dd, J = 8.7, 5.5 Hz, 1H), 7.49 – 7.40 (m, 3H), 7.35 (t, J = 7.4 Hz, 2H), 7.28 (t,
J = 7.2 Hz, 1H), 3.58 – 3.53 (m, 2H), 3.05 – 3.00 (m, 2H), 2.62 – 2.57 (m, 1H), 2.39 – 2.26 (m,
H), 1.93 – 1.82 (m, 2H), 1.69 – 1.61 (m, 3H). Exchangeable NH not observed.
1
(S)-4-((2R,4S)-2-phenyl-4-(trifluoromethyl)piperidin-1-yl)-N-(1,2,4-thiadiazol-5-
yl)chromane-7-sulfonamide (11). The title compound was prepared from 45, 48a, and 54a in a
+
manner analogous to 24. HRMS (ESI-FTMS) m/z [M+H] Calcd for C H F N O S 525.1236;
2
3
23
3
4
3
1
Found 525.1232. H NMR (400 MHz, DMSO-d ) δ: 8.47 (s, 1H), 7.84-7.82 (m, 1H), 7.49-7.47
6
(m, 2H), 7.38-7.28 (m, 4H), 7.03 (s, 1H), 4.27-4.25 (m, 1H), 3.82-3.73 (m, 4H), 2.58-2.50 (m, 1H),
2
.41-2.31 (m, 1H), 2.03-1.80 (m, 4H), 1.67-1.46 (m, 2H). Exchangeable NH not observed.
(S)-4-((2R,4S)-2-(4-fluorophenyl)-4-(trifluoromethyl)piperidin-1-yl)-N-(1,2,4-thiadiazol-5-
yl)chromane-7-sulfonamide (12). The title compound was prepared from 45, 48b, and 54a in a
+
manner analogous to 24. HRMS (ESI-FTMS) m/z [M+H] Calcd for C H F N O S 543.1142;
2
3
22
4
4
3 2
1
Found 543.1135. H NMR (400 MHz, DMSO-d ) δ 8.23 (s, 1H), 7.78 (d, J = 8 Hz, 1H), 7.55-7.52
6
(m, 2H), 7.33-7.16 (m, 3H), 7.03 (s, 1H), 4.22-4.20 (m, 1H), 3.78-3.76 (m, 2H), 3.68-3.65 (m, 2H),
2.54-2.50 (m, 2H), 2.01-1.73 (m, 4H), 1.76-1.744 (m, 2H). Exchangeable NH not observed.
(S)-6-fluoro-4-((2R,4S)-2-(4-fluorophenyl)-4-(trifluoromethyl)piperidin-1-yl)-N-(5-
fluoropyridin-2-yl)chromane-7-sulfonamide (13). The title compound was prepared from 47,
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