Discovery of Arylsulfonamide Nav1.7 Inhibitors: IVIVC, MPO Methods, and Optimization of Selectivity Profile

Article abstract of DOI:10.1021/acsmedchemlett.1c00218

The voltage-gated sodium channel Nav1.7 continues to be a high-profile target for the treatment of various pain afflictions due to its strong human genetic validation. While isoform selective molecules have been discovered and advanced into the clinic, to date, this target has yet to bear fruit in the form of marketed therapeutics for the treatment of pain. Lead optimization efforts over the past decade have focused on selectivity over Nav1.5 due to its link to cardiac side effects as well as the translation of preclinical efficacy to man. Inhibition of Nav1.6 was recently reported to yield potential respiratory side effects preclinically, and this finding necessitated a modified target selectivity profile. Herein, we report the continued optimization of a novel series of arylsulfonamide Nav1.7 inhibitors to afford improved selectivity over Nav1.6 while maintaining rodent oral bioavailability through the use of a novel multiparameter optimization (MPO) paradigm. We also report in vitro-in vivo correlations from Nav1.7 electrophysiology protocols to preclinical models of efficacy to assist in projecting clinical doses. These efforts produced inhibitors such as compound 19 with potency against Nav1.7, selectivity over Nav1.5 and Nav1.6, and efficacy in behavioral models of pain in rodents as well as inhibition of rhesus olfactory response indicative of target modulation.

Full text of DOI:10.1021/acsmedchemlett.1c00218

pubs.acs.org/acsmedchemlett
Letter
Discovery of Arylsulfonamide Nav1.7 Inhibitors: IVIVC, MPO
Methods, and Optimization of Selectivity Profile
Anthony J. Roecker,* Mark E. Layton, Joseph E. Pero, Michael J. Kelly, III, Thomas J. Greshock,
Richard L. Kraus, Yuxing Li, Rebecca Klein, Michelle Clements, Christopher Daley, Aneta Jovanovska,
Jeanine E. Ballard, Deping Wang, Fuqiang Zhao, Andrew P. J. Brunskill, Xuanjia Peng, Xiu Wang,
Haiyan Sun, Andrea K. Houghton, and Christopher S. Burgey
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ABSTRACT: The voltage-gated sodium channel Nav1.7 continues to
be a high-profile target for the treatment of various pain afflictions due
to its strong human genetic validation. While isoform selective
molecules have been discovered and advanced into the clinic, to date,
this target has yet to bear fruit in the form of marketed therapeutics for
the treatment of pain. Lead optimization efforts over the past decade
have focused on selectivity over Nav1.5 due to its link to cardiac side
effects as well as the translation of preclinical efficacy to man. Inhibition
of Nav1.6 was recently reported to yield potential respiratory side effects
preclinically, and this finding necessitated a modified target selectivity
profile. Herein, we report the continued optimization of a novel series of
arylsulfonamide Nav1.7 inhibitors to afford improved selectivity over
Nav1.6 while maintaining rodent oral bioavailability through the use of a
novel multiparameter optimization (MPO) paradigm. We also report in vitro−in vivo correlations from Nav1.7 electrophysiology
protocols to preclinical models of efficacy to assist in projecting clinical doses. These efforts produced inhibitors such as compound
19 with potency against Nav1.7, selectivity over Nav1.5 and Nav1.6, and efficacy in behavioral models of pain in rodents as well as
inhibition of rhesus olfactory response indicative of target modulation.
KEYWORDS: Nav1.7, Nav1.6, ion channel, pain, arylsulfonamide, selectivity, olfaction
The difficulty with increasing degree of channel blockade
relates to the requisite increase in selectivity profile over
homologous Nav1.x channels necessary to safely interrogate
this hypothesis. High levels of selectivity over Nav1.4 and
Nav1.5 blockade, inhibition of which affords known skeletal
muscle and cardiac toxicity, respectively, have been achieved
within the arylsulfonamide class of Nav1.7 inhibitors
represented by compounds 1, 2, and 3 as reported previously
(Table 1).6 Lack of central inhibition of Nav1.1 and Nav1.2 has
been achieved via limiting CNS penetration with this class of
zwitterionic Nav1.7 blocker, affording some safety margin to
established CNS-related toxicities.7 However, recent studies
from our laboratory revealed significant respiratory inhibition
findings potentially related to Nav1.6 blockade in the phrenic
he discovery and development of novel nonopioid pain
Tmedications has been highlighted as a critical area of
unmet medical need in recent years with a number of high-
profile reviews and popular articles covering the topic.1 The
strong level of genetic validation for the voltage-gated sodium
channel Nav1.7 as a novel, nonopioid target for pain relief has
afforded an enormous amount of effort in the scientific
community focused on the discovery of potent and selective
inhibitors of this channel.2 Multimodality efforts across
industry and academic groups have produced a number of
clinical candidates advancing toward or into human studies.3
However, no selective Nav1.7 inhibitors have demonstrated
clinical efficacy in chronic or acute human pain conditions to
date.4 This clinical experience has left the scientific field to
ponder whether the ligands studied owned the necessary
profiles to adequately test the mechanism regarding target
engagement or if the translation of the human genetic
condition into therapeutics was possible via the current
approaches.5 Increasing target inhibition relative to what has
been currently achieved in clinical study would add value
toward validating or invalidating this important target.
Received: April 19, 2021
Accepted: May 26, 2021
Published: June 1, 2021
https://doi.org/10.1021/acsmedchemlett.1c00218
© 2021 American Chemical Society
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Compounds 1, 2, and 3 in Table 1 were previously reported
examples of arylsulfonamide Nav1.7 blockers that display state-
dependent channel inhibition. Previous potency values have
been reported using a depolarized assay protocol, which placed
the channel in a population of states that favor the inactivated
state.9 All three compounds have very similar mouse potency
in this assay protocol with IC50 values of 13, 18, and 15 nM,
respectively. These compounds were studied in a number of
assay protocols including a hyperpolarized assay protocol,
which placed the channel in a population of states favoring the
resting state. Under this protocol, the mouse potency shifted
significantly from 17- and 26-fold for 1 and 3 to more than
270-fold in the case of compound 2. All three compounds were
evaluated for efficacy in the mouse formalin paw test (mFPT)
model, and the unbound concentration in plasma necessary for
90% reversal of the phase 2 effect was related to in vitro
potency measures. While shifts were high (17−200-fold)
relating phase 2 mFPT efficacy concentrations to depolarized
potency, the ratios of mFPT efficacy to hyperpolarized potency
were almost unity in all three cases. Notably, this ratio was able
to distinguish potency differences between enantiomers 1 and
2 with similar depolarized potency values but disparate
hyperpolarized potency values.10 Figure 1 demonstrates the
relationship between hyperpolarized Nav1.7 mouse potency
and unbound concentration for 90% mFPT efficacy across a
wide range of potency values within the arylsulfonamide series
with an R2 of 0.68. The hyperpolarized assay protocol was also
evaluated for Nav1.6, and this value was found to approximate
the unbound concentrations necessary for measurable
respiratory findings in rodents.8 With this data in hand, the
Nav1.7 and Nav1.6 hyperpolarized assay protocols were utilized
for further optimization of potency and selectivity.
Table 1. Mouse Formalin Paw Efficacy and Mouse Nav1.7
Inhibition Relationships under Depolarized and
Hyperpolarized Assay Protocols
a
Estimated depolarized or hyperpolarized state potency as measured
by PatchXpress in HEK293 cells stably expressing mouse Nav1.7, IC50
values are estimated from ≥3 cellular measurements at varying
compound concentrations and standard deviation <50% of average
b
value shown. Mouse formalin paw test performed as described in ref
c
22. Estimated hyperpolarized state potency as measured by Qube in
HEK293 cells stably expressing human Nav1.7 or Nav1.6; IC50 values
are estimated from ≥3 cellular measurements at varying compound
concentrations; * = estimated value.
nerve, thus necessitating optimization of selectivity over this
related channel.8 Notably, published compounds 1, 2, and 3
have limited to no selectivity over Nav1.6 (Table 1). To
address the potential increase in channel blockade necessary
for efficacy, our efforts focused on establishing in vitro−in vivo
correlations (IVIVC) between Nav1.7 potency in different
assay protocols to effects in a behavioral model of pain efficacy
and a rhesus model of target modulation. The result of these
efforts would give us a method to effectively assess increases in
on-target potency in vitro related to pharmacodynamic end
points. Our efforts also targeted establishing SAR in the
arylsulfonamide series that significantly increased selectivity
over Nav1.6 to widen our therapeutic index.
The hyperpolarized potency values for human Nav1.6 and
Nav1.7 for compounds 1, 2, and 3 are displayed at the bottom
of Table 1. All compounds have limited to inverted selectivity
over Nav1.6, necessitating additional SAR to provide analogues
with improved profiles for program advancement. While
rodent bioavailability was not an issue for analogues 1−3,
limited rat oral bioavailability in the arylsulfonamide class was
an issue identified across a variety of analogues in zwitterionic
space, potentially due to low permeability.11 An optimization
approach was taken to establish SAR to improve selectivity
Figure 1. In vitro−in vivo correlation between mouse formalin paw test and mouse Nav1.7 potency under hyperpolarized assay protocol.
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Figure 2. Distribution of properties (MW, HBD, cLogD, and PSA; Y axis = compound count) of analogues evaluated in rat oral PK studies (%F ≥
20%, green; %F < 20%, red) and property functions comprising custom oral bioavailability MPO paradigm.
over Nav1.6 while concomitantly exploring space enriched for
significant rodent oral bioavailability. A range of physicochem-
ical properties were evaluated to determine potential values
that could increase enrichment for rat oral bioavailability
within this zwitterionic space for utility in prospective design.
The results of four properties with the best enrichment data
are shown in Figure 2.
Oral bioavailability (defined as rat %F ≥ 20%: green bars
≥20% and red bars <20%) was generally enriched with
reductions in molecular weight (MW), limiting H-bond donor
(HBD) count, balancing cLogD in a desirable range, and
lowering polar surface area (PSA). These findings were
aggregated into a simple four-point scoring system, with each
property given a score of 1. The scoring functions are depicted
in Figure 2, with a monotonic declining function for MW and
PSA, a step function for HBD, and a hump function for
cLogD.12 Enrichment in rat bioavailability utilizing this
multiparameter optimization (MPO) paradigm is shown in
Figure 3. Increased enrichment was observed upon increasing
MPO score, with molecules scoring below 2.5, all exhibiting
<20% rat oral bioavailability. Increasing bioavailability enrich-
ment was observed between 2.5 and 3.8, and significant
enrichment was observed with scores >3.8, with almost 70% of
compounds owning significant bioavailability. Compounds 1
and 3 were utilized as leads for optimization with efficacy in
mFPT upon oral dosing, low to moderate permeability, and
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Figure 3. Categorical enrichment in rat oral bioavailability as a function of MPO score (%F ≥ 20%, green; %F < 20%, red).
limited selectivity over Nav1.6. Analogue designs were
prioritized for synthesis through MPO evaluation and
breakthroughs in selectivity over Nav1.6, which could be
rationalized via homology modeling (vide infra).
this lead, isosteric replacements for the phenyl ring in 14 were
explored.
Replacing the phenyl substituent with cycloalkyl motifs
afforded analogues 16 and 17, which both reduced Nav1.7
potency 4- and 14-fold, respectively, as well as reduced MPO
score (via reduced lipophilicity; cLogD < 1.5 for each) and
permeability as shown in Table 3. Notably, cyclopropane 17
significantly lowered clearance compared to 14 or 16 and
maintained excellent oral bioavailability, suggesting that
increasing sp3 character could improve PK profiles in the
series. Incorporation of a quaternary methyl group in 18
restored potency to within 2-fold of lead 14 and increased
permeability. Opening the cyclopropane ring to afford the tert-
butyl motif in 19 resulted in slightly improved potency
compared to 14, high MPO score, moderate permeability and
selectivity, and excellent rat bioavailability. Utilizing 19 as
design inspiration, trimethylsilyl analogue 20 provided the
most potent Nav1.7 blocker to date in the series with moderate
selectivity over Nav1.6, reduced clearance, and good bioavail-
ability.17 As a known phenyl isostere, the bicyclo[1.1.1]-
pentane was targeted, providing 21 with desired potency and
selectivity, but increased clearance was observed; for this
reason, 21 was not characterized further. Compound 19
provided improved selectivity over Nav1.6, permeability, and
bioavailability compared to initial lead compound 3 and was
selected as an exemplar in the series for further character-
ization.
Starting with compound 3, opening the [3.3.0] ring system
to a pyrrolidine scaffold afforded analogues 4 through 7, which
significantly reduced Nav1.7 potency including inactive
compounds 6 and 7 (Table 2). Compound 5 was screened
for rat pharmacokinetics (PK) and had bioavailability of 14%,
consistent with the enrichment for a compound with an MPO
of 3.2. Addition of a phenyl ring to compound 5 yielded 8,
with a 5-fold improvement in Nav1.7 potency, and importantly,
moderate selectivity over Nav1.6. While this analogue had an
MPO score of 4.0, bioavailability was only 7%, likely due to
high rat clearance (CLint = 5830) potentially due to
metabolism.13 The pyrrolidine ring was opened to afford 9
and 10, with reduced Nav1.7 potency but maintained moderate
selectivity over Nav1.6 for compound 10. Surprisingly,
removing the quaternary center affording 11 increased potency
compared to 10 and increased selectivity over Nav1.6 to more
than 100-fold. The MPO score of 4.0 for 11 translated to 25%
oral bioavailability despite high clearance. Removal of the
methyl group from the terminal amine to afford 12 lost all
Nav1.7 potency, and addition of a methyl group (13) also
reduced on-target potency by 4-fold. Despite the promising
selectivity PK profile for compound 11, the 2-aminothiazole
sulfonamide was still a risk for potential toxicity via known
metabolic activation pathways, which were observed in rat and
human microsomal incubations.14 A survey of thiazole
replacements that would reduce the potential for metabolic
activation revealed very limited options without compromising
on target potency.15 Fluorothiazole 14 maintained the overall
profile of 11 regarding potency, selectivity, and oral
bioavailability while also having the potential to diminish
oxidative metabolism on the thiazole ring.16 Thiadiazole 15
also had the potential to reduce oxidative metabolism as on the
sulfonamide aryl ring, however, potency and MPO score were
reduced through this design element. While 14 was considered
a promising new lead, clearance was significantly higher
compared with initial lead 3, potentially due to incorporation
of the phenyl ring, which increased lipophilicity and offered
additional opportunity for metabolism. To further optimize
An interesting observation was made that compound 19 is
essentially an open form of lead compound 1 that increased
selectivity over Nav1.6. To explore this selectivity phenomen-
on, compounds 1 and 19 were docked to the binding site with
18
̈
Glide v7.7 of Schrodinger software using a known X-ray
crystal structure (PDB 5EK0) containing the human Nav1.7
binding site for the arylsulfonamide class of inhibitors (top of
S1−S4 in domain 4 of the channel).19 Also, a homology model
of Nav1.6 VSD4 was built using Prime in Maestro v11.4.20
Then, a 100 ns molecular dynamics (MD) simulation was
performed for both compounds 1 and 19 in Nav1.7 and Nav1.6
in the presence of explicit water and membrane using
Desmond v5.2. Last, 400 snapshots of each simulation were
used to estimate binding free energy using prime_mmgbsa in
Maestro v11.4. The results were shown in Figure 4.
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Table 2. Western SAR and Selectivity over Nav1.6
a
Estimated hyperpolarized state potency as measured by Qube in HEK293 cells stably expressing human Nav1.7 or Nav1.6; IC50 values are
b
estimated from ≥3 cellular measurements at varying compound concentrations and standard deviation <50% of average value shown. Monolayer
c
assay in MDCK cells. Rat CLint = (84 × rat CL)/[rat fu × (84-rat CL)]; units mL/min/kg; IV, 0.05 mpk cassette dosing or 2 mpk single dosing in
DMSO/PEG400/water (20/60/20); PO, 10 mpk in PEG400/Tween90/water (40/10/50).
Not surprisingly, several key interactions were maintained
between the two compounds: interaction of the acidic
sulfonamide with R1608, terminal basic amine interaction
with D1586, and key π−π stacking interaction with Y1537 (not
labeled, but central in Figure 4) with the aromatic core of the
molecule. The MD simulation also provided slight differences
in docked versions of 19 and 1, namely the tert-butyl motif in
19 reached higher into the binding pocket toward the top or
loop region of S2, where several amino acid residue differences
were observed between human Nav1.7 and Nav1.6 (Q1530K
and T1590K shown; others removed for clarity). Steric clashes
between 19 and the larger amino acid residues in Nav1.6 could
underly the difference in selectivity. Relative binding free
energy calculations were also performed for both 1 and 19
using the MM_GBSA method. Compound 19 was calculated
to bind approximately 2 kcal/mol more strongly to Nav1.7 than
Nav1.6, while compound 1 was predicted to have similar
binding energy between the two channels, consistent with the
selectivity profiles of both compounds. It should be noted that
the standard deviations were overlapping these differences,
however, this methodology might represent a prospective
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Table 3. Phenyl Replacements and Selectivity over Nav1.6
a
Estimated hyperpolarized state potency as measured by Qube in HEK293 cells stably expressing human Nav1.7 or Nav1.6; IC50 values are
b
estimated from ≥3 cellular measurements at varying compound concentrations and standard deviation <50% of average value shown. Monolayer
c
assay in MDCK cells. Rat CLint = (84 × rat CL)/[rat fu × (84-rat CL)]; units mL/min/kg; IV, 0.05 mpk cassette dosing or 2 mpk single dosing in
DMSO/PEG400/water (20/60/20); PO, 10 mpk in PEG400/Tween90/water (40/10/50).
Figure 4. Docked models and binding free energy calculations for 1 and 19 in hNav1.7 and hNav1.6.
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a
Scheme 1. Synthesis of Compounds 11 (A) and 19 (B)
a
Reagents and reaction conditions: (A) (a) BH3·THF, THF, 0−25 °C, 0.5 h, 97%; (b) phthalimide, DTAD, PS−PPh3, THF, 25 °C, 0.5 h, 78%;
(c) hydrazine hydrate, MeOH, reflux, 2 h, 97%; (d) core 1, Hunig’s base, NMP, 100 °C, microwave, 20 min, 77%; (e) TFA, DCM, 25 °C, 3 h, 79%.
(B) (f) N-bromosuccinimide, CCl4, 80 °C, 2 h 96%; (g) triethylphosphite, THF, 75 °C, 16 h, 98%; (h) pivaldehyde, tetramethylguanidine, THF,
−78 to 25 °C, 9 h, 57%; (i) NaBH4, MeOH, 25 °C, 2 h, 64%; (j) Raney nickel, H2, EtOH, 25 °C, 2 h, 63%; (k) core 2, TEA, DMF, 25 °C, 16 h,
83%; (l) SFC separation, Chiralpak AD-3 150 mm × 4.6 mm I.D.; mobile phase, 2-propanol (0.05% DEA) in CO2 from 5% to 40%; flow rate, 2.4
mL/min; wavelength, 220 nM; enantiomer 1 = 30 (Rt = 5.0 min), eantiomer 2 = 29 (Rt = 5.4 min); (m) TFA, DCM, 25 °C, 1 h; (n) reverse phase
HPLC (acetonitrile/0.05% HCl in water), 61% over 2 steps.
method to prioritize analogue designs based on potential for
increased selectivity over Nav1.6.
(Table 4). The potency of 19 was right-shifted on mouse
Nav1.7 (IC = 8.8 μM) and less right-shifted in rhesus
experiments (IC50 = 310 nM) compared to human potency
consistent with previously evaluated compounds in the series.
The physicochemical properties of 19 are within targeted
MPO ranges that resulted in high rat bioavailability. This
analogue was also a substrate for human and rat P-glycoprotein
(Pgp) efflux transporters and, consistent with this finding,
limited central penetration was observed upon oral dosing in
mice, with a CSF-to-unbound plasma ratio of 0.02. This
zwitterionic analogue had good kinetic solubility under low
and neutral pH conditions, had limited CYP inhibition potency
against three major isoforms, moderate PXR activation
potential, and no significant activity in broad off-target
profiling. Pharmacokinetics in dog revealed low clearance, a
6 h half-life, and moderate oral bioavailability. Rhesus
bioavailability was negligible; however, pharmacokinetics
were sufficient to evaluate this compound in our target
modulation assay via IV administration. Finally, metabolic
profiling in human, rat, dog, and rhesus hepatocytes revealed
N-demethylation (metabolite potency; hNav1.7 IC50 = 3.5
μM) as the major route of metabolism with no metabolism of
the thiazole motif. This finding suggested that fluorination of
Representative synthetic routes to the compounds above are
described in Scheme 1. The synthesis of compound 11 began
with a borane reduction of N-Boc-N-methyl-L-phenylalanine
(22) followed by Mitsunobu with phthalimide to afford 23.
Phthalimide removal afforded 24, which underwent an SNAr
with core 1 and acidic deprotection to afford 11. The synthesis
of 19 began with the bromination/phosphonate sequence from
25 to afford 26. Horner−Wadsworth−Emmons olefination
with pivaldehyde followed by conjugate reduction afforded 27
in modest yield. Reduction with Raney nickel and hydrogen
afforded 28, which was engaged in an SNAr reaction with core
2, which afforded 29 and 30 after chiral separation.
Deprotection of 30 afforded the hydrochloride salt of 19.21
The identity and absolute stereochemistry of 22 was
determined by X-ray crystallography.22
Compound 19 underwent additional profiling prior to in
vivo evaluation for pain efficacy and target modulation. In
preparation for these studies, manual electrophysiology (EP)
experiments were performed using the hyperpolarized assay
protocol, and potency values were found to be within 3-fold of
screening potency values with recapitulation of selectivity
profiles over Nav1.6 (60-fold) and limited activity on Nav1.5
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d
and unbound concentration for full mFPT efficacy. To
investigate further, the team decided to assess target
modulation orthogonally in a model of rhesus olfaction to
further assist with the determination of target concentration for
human efficacy.
Table 4. Additional Profiling of Compound 19
property
compd 19
human Nav1.7/1.6/1.5 IC50 [nM]
87/5230 (60×)/ >34000
(>390×)
a
(fold-selectivity)
a
mouse/rhesus Nav1.7 IC50 (nM)
8800/310
Loss-of-function mutations in Nav1.7 not only cause
insensitivity to painful stimuli but also cause anosmia (lack
of sense of smell) through inactivation of Nav1.7 in the
olfactory bulb.24 A target modulation assay was developed
using functional magnetic resonance imaging (fMRI) to
measure odorant-induced olfaction in the olfactory bulb
(OB) of anesthetized rhesus monkeys (nonhuman primates,
or NHPs), previously reported from our laboratories as a
potential translative clinical biomarker.25,26 Briefly, the study
was performed with with NHPs each for vehicle control and
for compound 19. The fMRI measurement paradigm for
odorant-induced olfaction was 1 min for baseline, 1 min for
odor stimulation, and 2 min for recovery (4 min total for each
fMRI measurement). Thirty fMRI measurements were made
for each NHP during a 2 h period. Compound 19 or vehicle
delivery was started 1 h after acquisition of data and was
continuously infused for the following hour. The strength of
fMRI response was quantified by averaging the amplitudes of
fMRI signals during the stimulation period. The inhibition on
the olfaction was expressed as percentage inhibition of the
strength of fMRI response in the control period. Time courses
of fMRI signals in olfactory bulb upon a 40 s odor
administration (isoamyl acetate: red bars) before/after the
vehicle, and compound 19 delivery are shown in parts A and B
of Figure 6, respectively. With the vehicle administration, the
odorant-induced fMRI activations in the OB are similar before
and after the administration, indicating that the vehicle has no
significant effect on the olfaction. The odorant-induced fMRI
activations in the OB after the administration of 19 were
weaker than the fMRI responses before the administration,
suggesting that the compound 19 inhibits the odorant-induced
olfaction in the NHPs with statistical significance (Figure 6C).
Compound 19 reduced olfactory response by approximately
35%, with an unbound plasma concentration of 1.5 μM and a
ratio compared to rhesus potency of 4.8 (1500/310 nM).
These results were more consistent with previous compounds
studied that demonstrated that full efficacy in mFPT correlated
to approximately a 40% reduction in rhesus olfactory response
at unbound concentrations approximately equivalent to Nav1.7
mouse/rhesus/human PPB
MW/HBD/log D /PSA
Pgp (BA/AB ratio; h/r)
mouse brain/plasma/CSF (μM)
94%/98%/98%
453 g/mol/3/1.7/89 Å2
4.9/>14
0.1/4.9/0.005
5.4/9.0
170, 148 μM
>50, 28, 20
10/80%
no hits <10 μM
29, 1670, 2.1, 2.5, 100%
4.5, 200, 0.8, 6.0, 25%
11, 610, 0.3, 1.3, 2%
c
e
pKa (sulfonamide/amine)
b
solubility (pH 2, 7)
CYP IC50 (3A4/2C9/2D6, μM)
PXR EC50 (μM)/% max
Panlabs (97 assays)
rat PK: CL, CLint, Vdss, T1/2, F
dog PK: CL, CLint, Vdss, T1/2, F
f
f
f
rhesus PK: CL, CLint, Vdss, T1/2, F
a
Estimated hyperpolarized protocol inhibition potency as measured
by manual electrophysiology in HEK293 cells stably expressing
human, rhesus, or mouse Nav1.7; IC50 values are estimated from ≥3
cellular measurements at varying compound concentrations and
b
standard deviation <50% of average value shown. MSD HPLC
c
d
kinetic solubility assay. MSD HPLC log D assay. Monolayer assay in
e
f
MDCK cells. 100 mpk PO, 1.25 h sampling. CL(int) units = mL/
min/kg, Vdss = L/kg, T1/2 = hours.
the thiazole reduced the potential metabolic liability of the
aminothiazole sulfonamide.
To estimate pain efficacy in a rodent behavioral pain assay,
compound 19 was evaluated in the mFPT assay and
administered orally to mice at three dose levels 15 min prior
to formalin injection (Figure 5).23 Plasma sampling was
performed 1.5 h after compound administration to coincide
with Tmax values provided by satellite PK experiments. Limited
reversal of the phase 1 (acute phase) effects of formalin
administration were observed at all three dose levels.
Significant and full reversal of these effects were observed in
phase 2 (tonic phase) of the experiment, with unbound
concentrations at EC90 of 360 nM. This represents a ratio of
0.04 (360/8800 nM) when normalizing for mouse potency.
Although outliers of this magnitude were observed in Figure 1,
this result was surprising given the correlation previously
acquired, with most compounds demonstrating approximately
a ratio unity comparing mouse hyperpolarized Nav1.7 potency
Figure 5. Oral efficacy of compound 19 in mouse formalin paw test. Measurement of formalin-induced nociceptive behaviors in mice (C57BL/6
mice) following administration of vehicle (10% Tween 80; dose volume = 5 mL/kg) or rising oral doses of compound 19. Compound 19 was
administered 15 min prior to formalin injection and plasma concentrations were evaluated 1.5 h postadministration. Data were analyzed using
within-subject ANOVA to determine main effects and 1 sample t test to compare to vehicle (N = 8/group); **P > 0.001.
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Figure 6. Target modulation efficacy of compound 19 in rhesus olfaction fMRI. Measurement of odorant-induced olfaction in the olfactory bulb of
NHPs following administration of vehicle (30% Captisol, dose volume = ∼8 mL/animal) or compound 19 (19.2 mg/kg). (A) Time courses of
fMRI signals which represent the odorant-induced olfaction in the OB of NHPs before and after vehicle administration (mean STD, n = 4). (B)
Time courses of fMRI signals before and after compound 19 (C′22) administration (mean STD, n = 4). (C) Compare the inhibition by
compound 22 and the effect from the vehicle (“*” statistical student t test, P < 0.012). Red bars in (A,B): odorant stimulations.
potency.27 These disparate behavioral efficacy, mFPT overall
correlations and target modulation data made it difficult to
project an appropriate dose estimate for efficacy in clinical
evaluation. The team chose to bracket human dose predictions
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
■
sı
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00218.
using the three efficacy or target modulation data sets. Table 5
Synthetic procedures; assay protocols; MPO calculation
methods; X-ray crystallographic data; compound char-
acterization data (PDF)
Roecker Nav1.7 ACS MCL spectra (PDF)
Table 5. Estimated Human Dose Projections Based on
Changing Ctrough Targets of Compound 19
Ctrough target
regimen
dose (mg)
0.04× Nav1.7 IC50
1× Nav1.7 IC50
4.8× Nav1.7 IC50
BID
BID
BID
40
960
4600
AUTHOR INFORMATION
Corresponding Author
■
Anthony J. Roecker − Discovery Chemistry, Merck & Co.,
Inc., West Point, Pennsylvania 19486, United States;
orcid.org/0000-0002-7688-7651;
displays the estimated BID (“bis en die” or dosing twice per
day) human dose projections via allometric scaling for 19 using
the three end points to set trough concentration efficacy
targets in man.28 While dose projections from behavioral
efficacy and IVIVC studies were considered clinically feasible
(40−960 mg BID), accessing the top end of the efficacy curve
set by rhesus olfaction (4.6 g BID) would likely not be
possible. This data suggested the need for significant additional
optimization toward clinical candidate identification to
confidently interrogate Nav1.7 as a therapeutic target.
Email: anthony_roecker@merck.com
Authors
Mark E. Layton − Discovery Chemistry, Merck & Co., Inc.,
West Point, Pennsylvania 19486, United States
Joseph E. Pero − Discovery Chemistry, Merck & Co., Inc.,
West Point, Pennsylvania 19486, United States; Present
Address: (J.E.P.) GlaxoSmithKline, 1250 South
Collegeville Road, Collegeville, Pennsylvania, 19426;
orcid.org/0000-0003-2674-4713
In conclusion, a correlation was established between
hyperpolarized Nav1.7 and Nav1.6 assay protocols to efficacy
and off-target end points: behavioral pain efficacy and target
modulation for Nav1.7 and respiratory side effects for Nav1.6.
A novel MPO paradigm was developed to enrich design cycles
for analogues with adequate rodent bioavailability concomitant
with selectivity optimization. This strategy afforded analogues
with a more than 100-fold selectivity over Nav1.6 with high
rodent oral bioavailability. Molecular dynamics simulations and
free energy binding calculations were performed, which
provided rationale for selectivity differences between structur-
ally related compounds. These tools could potentially be
utilized for prospective design of analogues with increased
potency, selectivity, and oral bioavailability. An exemplar
compound (19) has demonstrated behavioral pain efficacy in
mFPT and target modulation in a rhesus olfaction fMRI assay,
however, dose predictions for leading analogues suggest the
need for significant optimization toward candidate selection to
adequately test the mechanism. Future efforts will focus on the
identification of analogues with more aligned ratios in pain
efficacy and target modulation compared to potency and
reduction of predicted dose estimates via potency and PK
optimization. These efforts will be reported in due course.
Michael J. Kelly, III − Discovery Chemistry, Merck & Co.,
Inc., West Point, Pennsylvania 19486, United States
Thomas J. Greshock − Discovery Chemistry, Merck & Co.,
Inc., West Point, Pennsylvania 19486, United States;
orcid.org/0000-0002-3313-9733
Richard L. Kraus − Pharmacology, Merck & Co., Inc., West
Point, Pennsylvania 19486, United States
Yuxing Li − Pharmacology, Merck & Co., Inc., West Point,
Pennsylvania 19486, United States
Rebecca Klein − Pharmacology, Merck & Co., Inc., West
Point, Pennsylvania 19486, United States
Michelle Clements − Pharmacology, Merck & Co., Inc., West
Point, Pennsylvania 19486, United States
Christopher Daley − Pharmacology, Merck & Co., Inc., West
Point, Pennsylvania 19486, United States
Aneta Jovanovska − Pharmacology, Merck & Co., Inc., West
Point, Pennsylvania 19486, United States
Jeanine E. Ballard − Pharmacokinetic, Pharmacodynamics,
and Drug Metabolism, Merck & Co., Inc., West Point,
Pennsylvania 19486, United States
Deping Wang − Computational and Structural Chemistry,
Merck & Co., Inc., West Point, Pennsylvania 19486, United
States
1046
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pubs.acs.org/acsmedchemlett
Letter
Opin. Anaesthesiol. 2015, 28, 379−397. (d) Busserolles, J.; Lolignier,
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Pharmacol. Ther. 2020, 210, 107519. (e) Time Staff Writers. The
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Fuqiang Zhao − Translational Imaging and Biomarkers,
Merck & Co., Inc., West Point, Pennsylvania 19486, United
States
Andrew P. J. Brunskill − Molecular and Materials
Characterization, Merck & Co., Inc., Rahway, New Jersey
07065, United States
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Xuanjia Peng − HitS Unite, WuXi AppTec Co., Ltd.
(Shanghai), Shanghai 200131, China; orcid.org/0000-
0001-6067-0311
Xiu Wang − IDSU, WuXi AppTec Co., Ltd. (Shanghai),
Shanghai 200131, China
Haiyan Sun − IDSU, WuXi AppTec Co., Ltd. (Shanghai),
Shanghai 200131, China
Andrea K. Houghton − Pharmacology, Merck & Co., Inc.,
West Point, Pennsylvania 19486, United States
Christopher S. Burgey − Discovery Chemistry, Merck & Co.,
Inc., West Point, Pennsylvania 19486, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00218
Author Contributions
All authors have given approval to the final version of the
manuscript. Conceived and designed experiments: A.J.R.,
M.E.L., J.E.P., D.W., F.Z., R.K., R.L.K., A.K.H., and C.S.B.
Designed compounds and/or contributed to synthetic route
design: A.J.R., M.E.L., J.E.P., M.J.K., III, T.G., X.P., X.W., and
H.S. Analyzed data: R.L.K., Y.L., M.C., C.D., A.J., J.B., and
D.W. Wrote the paper: A.J.R., D.W., and F.Z. Edited the paper:
C.S.B. and M.E.L.
Notes
The authors declare the following competing financial
interest(s): The authors are or were employees of Merck
and Co., Inc. during the period of this work, and may hold
stock in those companies.
ACKNOWLEDGMENTS
■
We thank Justin Newman for small-molecule X-ray support.
We also thank Wilfredo Pinto for high-resolution mass
spectrometry support.
ABBREVIATIONS
■
CNS, central nervous system; SAR, structure−activity relation-
ship; HBD, hydrogen bond donor; cLogD, calculated water/
octanol partition coefficient at pH 7.4; PK, pharmacokinetics;
PSA, polar surface area; SNAr, nucleophilic aromatic
substitution; THF, tetrahydrofuran; MeOH, methanol;
DTAD, di-tert-butylazodicarboxylate; NMP, N-methylpyrroli-
dinone; TFA, trifluoroacetic acid; DCM, dichloromethane;
EtOH, ethanol; TEA, triethylamine; DMF, dimethylforma-
mide; PS, polymer-supported; CLint, intrinsic clearance;
mFPT, mouse formalin paw test; NHP, nonhuman primate;
MPO, multiparameter optimization; fMRI, functional magnetic
resonance imaging; IVIVC, in vitro−in vivo correlation; BID,
“bis in die” or dosing twice per day; PPB, plasma protein
binding
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