Discovery of selective, orally bioavailable, N-linked arylsulfonamide Nav1.7 inhibitors with pain efficacy in mice

Article abstract of DOI:10.1016/j.bmcl.2017.03.085

The voltage-gated sodium channel Na_v1.7 is a genetically validated target for the treatment of pain with gain-of-function mutations in man eliciting a variety of painful disorders and loss-of-function mutations affording insensitivity to pain.

Full text of DOI:10.1016/j.bmcl.2017.03.085

Accepted Manuscript
Discovery of Selective, Orally Bioavailable, N-linked Arylsulfonamide Na 1.7
v
Inhibitors with Pain Efficacy in Mice
Anthony J. Roecker, Melissa Egbertson, Kristen L.G. Jones, Robert Gomez,
Richard L. Kraus, Yuxing Li, Amy Jo Koser, Mark O. Urban, Rebecca Klein,
Michelle Clements, Jacqueline Panigel, Christopher Daley, Jixin Wang,
Eleftheria N. Finger, John Majercak, Vincent Santarelli, Irene Gregan, Matthew
Cato, Tracey Filzen, Aneta Jovanovska, Ying-Hong Wang, Deping Wang, Leo
A. Joyce, Edward C. Sherer, Xuanjia Peng, Xiu Wang, Haiyan Sun, Paul J.
Coleman, Andrea K. Houghton, Mark E. Layton
PII:
S0960-894X(17)30335-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.085
BMCL 24837
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
3 March 2017
24 March 2017
28 March 2017
Please cite this article as: Roecker, A.J., Egbertson, M., Jones, K.L.G., Gomez, R., Kraus, R.L., Li, Y., Jo Koser,
A., Urban, M.O., Klein, R., Clements, M., Panigel, J., Daley, C., Wang, J., Finger, E.N., Majercak, J., Santarelli,
V., Gregan, I., Cato, M., Filzen, T., Jovanovska, A., Wang, Y-H., Wang, D., Joyce, L.A., Sherer, E.C., Peng, X.,
Wang, X., Sun, H., Coleman, P.J., Houghton, A.K., Layton, M.E., Discovery of Selective, Orally Bioavailable, N-
linked Arylsulfonamide Na 1.7 Inhibitors with Pain Efficacy in Mice, Bioorganic & Medicinal Chemistry Letters
v
(
2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.085
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Discovery of Selective, Orally Bioavailable, N-linked Arylsulfonamide
Na 1.7 Inhibitors with Pain Efficacy in Mice
v
a
a
a
a
Anthony J. Roecker, Melissa Egbertson, Kristen L. G. Jones, Robert Gomez, Richard
b
b
b
b
c
L. Kraus, Yuxing Li, Amy Jo Koser, Mark O. Urban, Rebecca Klein, Michelle
c
c
b
c
b
Clements, Jacqueline Panigel, Christopher Daley, Jixin Wang, Eleftheria N. Finger,
b
c
b
b
b
John Majercak, Vincent Santarelli, Irene Gregan, Matthew Cato, Tracey Filzen,
b
d
e
f
Aneta Jovanovska, Ying-Hong Wang, Deping Wang, Leo A. Joyce, Edward C.
f
g
g
g
a
Sherer, Xuanjia Peng, Xiu Wang, Haiyan Sun, Paul J. Coleman, Andrea K.
b
a
Houghton, Mark E. Layton,
a
b
c
d
Department of Discovery Chemistry, Pharmacology, Neuroscience, Drug Metabolism,
e
f
Chemistry Modeling & Informatics, Analytical Chemistry Merck & Co., Inc., West
g
Point, PA 19486 USA; WuXi AppTec Co., Ltd, Shanghai, China
*
Corresponding author. Tel.: + 1-215-652-8257; fax: + 1-215-652-3971; e-mail:
anthony_roecker@merck.com
1

Abstract
The voltage-gated sodium channel Na 1.7 is a genetically validated target for the
v
treatment of pain with gain-of-function mutations in man eliciting a variety of painful
disorders and loss-of-function mutations affording insensitivity to pain. Unfortunately,
drugs thought to garner efficacy via Na 1 inhibition have undesirable side effect profiles
v
due to their lack of selectivity over channel isoforms. Herein we report the discovery of a
novel series of orally bioavailable arylsulfonamide Na 1.7 inhibitors with high levels of
v
selectivity over Na 1.5, the Na isoform responsible for cardiovascular side effects,
v
v
through judicious use of parallel medicinal chemistry and physicochemical property
optimization. This effort produced inhibitors such as compound 5 with excellent potency,
selectivity, behavioral efficacy in a rodent pain model, and efficacy in a mouse itch
model suggestive of target modulation.
Keywords: Na 1.7, ion channel, pain, arylsulfonamide, selectivity
v
2

It is estimated that 15% of the US population currently suffers from chronic pain
conditions. In addition to the debilitating nature of these ailments, this spectrum of
diseases confers a significant economic burden of approximately $600 billion per year in
1
the US due to increased medical costs and lost productivity in the workforce. A range of
pharmaceutical agents are utilized to treat chronic pain. First line treatments include
2
anticonvulsants (pregabalin and gabapentin), antidepressants (i.e. duloxetine and
3
desipramine), and non-steroidal anti-inflammatory drugs (NSAIDs, i.e. ibuprofen and
4
naproxen). All current first-line treatment options have significant limitations including
incomplete efficacy and undesirable side effect profiles. Opioids (i.e. morphine,
oxycodone, and hydrocodone) are effective agents for chronic pain management;
however, these are not considered first line options due to the potential for tolerance,
potential for addiction and abuse, and narrow therapeutic indices with adverse effects
5
, 6
consisting of constipation, decreased respiration, sedation, and nausea.
For these
reasons, novel, safe, and effective chronic pain treatments remain an area of significant
7
unmet medical need and a focus of pharmaceutical research.
A more recent option for chronic pain management is the topical application of
®
lidocaine in the form of a patch (Lidoderm ) approved for the treatment of postherpetic
8
neuralgia, a painful skin condition resulting from shingles. The mechanism of action of
lidocaine is thought to arise from non-selective blockade of Na 1 voltage-gated sodium
v
channels (VGSCs); key regulators of electrical signaling in a variety of tissues through
the generation and propagation of action potentials via sodium ion flux in response to
9
changes in membrane voltage potential. There are nine VGSCs currently known, Na 1.1
v
1
0
–
1.9, that possess differential tissue distribution patterns and associated pharmacology.
3

Na 1.1, 1.2, and 1.3 are expressed in the central nervous system (CNS) while Na 1.4 and
v
v
1
.5 are expressed in skeletal tissue and cardiac muscle, respectively. Na 1.6 is expressed
v
widely throughout the CNS and peripheral nervous system (PNS), and Na 1.7, 1.8, and
v
1
.9 are restricted mainly to the PNS. Recent human genetic studies have revealed that
loss-of-function (LOF) mutations in Na 1.7 afford channelopathy-associated insensitivity
v
to pain suggesting that blockade of this VGSC might be a contributing factor to efficacy
1
1
for agents such as lidocaine. Additional investigations revealed that gain-of-function
GOF) mutations in Na 1.7 elicit painful conditions such as erythromelalgia and
(
v
1
2
paroxysmal extreme pain disorder further linking Na 1.7 and pain. Unfortunately,
v
systemic exposure to local anesthetics such as lidocaine can engender severe
cardiovascular and CNS side effects through blockade of Na 1.5 in the heart and Na
v
v
channels in the CNS (Na 1.1 / Na 1.2), respectively, thus limiting their therapeutic
v
v
1
3
utility. The combination of human genetic validation for Na 1.7 and marketed
v
therapeutics which target Na 1 channels has drawn extremely high levels of interest and
v
investment from the scientific community with the goal of developing a safe and
1
4
effective therapeutic for patients suffering from chronic pain.
Historically, identifying selective inhibitors of Na 1.7 has been extremely
v
challenging due to high levels of homology among the Na 1 channels. Indeed, the
v
binding site for lidocaine resides in the pore region of the channels which is almost
1
5
completely conserved; consistent with the non-selective nature of the drug. Pioneering
work by Pfizer and Icagen described the discovery of arylsulfonamides such as PF-
0
4856264 (1a, Figure 1) that potently inhibited Na 1.7 through a binding site distinct
v
from the pore region (DIV = domain four: S2-S3 = between segments 2 and 3) with
4

1
6
unprecedented selectivity over Na 1.5,
potentially ameliorating the risk of
v
1
7
cardiotoxicity. This important finding has inspired further chemistry efforts from Pfizer
1
8
affording the clinical candidate PF-05089771 (1b, Figure 1), an X-ray crystal structure
of GX-936 (1c, Figure 1) bound to DIV of human Na 1.7 from scientists at Genentech
v
1
9
and Xenon, and several arylsulfonamide subseries efforts reported by Amgen (1d,
2
0
21
22
Figure 1), Genentech / Xenon (1e, Figure1), and MSD (1f, Figure 1). One hallmark
feature of the arylsulfonamide series referenced above is the use of aromatic motifs in
Western SAR. This observation suggested that Western aromatic motifs might be
necessary to achieve adequate target potency. Herein we describe initial parallel
chemistry efforts to identify novel Western SAR devoid of aromatic motifs and further
medicinal chemistry efforts to optimize potency, selectivity, physicochemical properties,
oral bioavailability, and behavioral pain efficacy in mice.
5

Figure 1. Known arylsulfonamide (1a – 1f) Na 1.7 inhibitors from Pfizer / Icagen,
v
Genentech, Amgen, Xenon, and MSD.
The generic aryl sulfonamide scaffold (2) shown in Figure 2 was utilized as a
template for design of nucleophilic aromatic substitution (S Ar) libraries taking
N
advantage of the MSD collection of alcohol- and amine-containing building blocks
lacking aromatic motifs. Initial library exploration afforded several interesting leads
including compound
3
containing
a
novel (tetrahydro-1H-pyrrolizin-7a(5H)-
2
3
yl)methanamine (3.3.0 amine hereafter) Western substituent. This compound possessed
2
4
moderate potency against Na 1.7 (inactivated state potency) of 112 nM, no activity
v
against Na 1.5 (> 30 M), and good binding affinity to the same binding site as PF-
v
2
5
0
4856264. Encouraged by this finding, compound 3 was profiled further and was found
to be very polar (LogD = 0.0) with low permeability (Papp = 2; Table 1). Arylsulfonamide
had limited oral bioavailability (%F = 4), consistent with its permeability and
3
lipophilicity profile, and low intrinsic clearance (CL ) in rat. Hence, additional
int
optimization focused on increasing lipophilicity without compromising pharmacokinetics.
Figure 2. Generic arylsulfonamide scaffold (2) utlized in library design via S Ar
N
chemistry yielding novel Na 1.7 inhibitor leads such as 3.
v
Modification of the 5-position fluorine to chlorine on the central aromatic ring (4,
Table 1) increased lipophilicity (LogD = 0.4), improved Na 1.7 potency by an order of
v
6

magnitude, and maintained selectivity over Na 1.5. Unfortunately, rat CL increased
v
int
significantly and permeability was not improved. Exploration of substituted thiazole
sulfonamides based upon compound 4 afforded compounds 5 – 11 which all possessed
moderate to excellent Na 1.7 potency, high levels of selectivity over Na 1.5, and
v
v
increased LogD (0.7 – 1.3; Table 1). Surprisingly, compounds 5 -7 and 9 still displayed
low permeability (Papp = 2 - 6) but were orally bioavailable in rat, indicating that high
2
6
levels of permeability were not a prerequisite for oral bioavailability. A selection of 6-
membered ring sulfonamides was also synthesized to probe SAR, and representative
examples 12 and 13 showed decreased Na 1.7 potency compared to 5 suggestive of tight
v
sulfonamide SAR. Although 12 significantly increased permeability, the increase in rat
CLint deterred further analog design using this core template. Arylsulfonamide 5
possessed the best balance of potency, selectivity, rat CL , and oral bioavailability and
int
was utilized for further compound design.
Table 1. Arylsulfonamide core SAR.
b
d
Na 1.7 IC
Na 1.5 IC
LogD /
Papp
Rat CLint
/ %F
v
50
v
50
Compound
3
Ar
X
F
-6
c
a
a
(
nM)
(nM)
PSA (Å) (10 cm/s)
112
>30000
7200
0.0 / 96
0.4 / 95
2
2
62 / 4
4
Cl
10
660 / ND
7

5
Cl
8
>30000
0.7 / 77
2
210 / 40
6
7
Cl
Cl
5
1900
1.0 / 77
1.3 / 77
2
3
230 / 55
780 / 45
12
17600
8
9
Cl
Cl
142
44
24200
22500
1.1 / 78
1.1 / 78
9
6
560 / ND
71 / 36
1
0
Cl
30
>30000
1.3 / 78
5
630 / ND
e
1
1
1
2
Cl
Cl
91
45
>30000
19000
0.7 / 78
1.0 / 77
20
ND / ND
610 / 26
23
2
1
3
Cl
215
>30000
-0.2 / 92
61 / ND
®
(
a) Estimated inactivated state potency as measured by PatchXpress in HEK293 cells stably expressing
human Nav1.7 or Nav1.5; IC50 values are estimated from > 3 cellular measurements at varying compound
concentrations; (b) MSD HPLC LogD assay; (c) Monolayer assay in MDCK cells; (d) Rat CLint = (84*rat
CL) / [rat f * (84-rat CL)]; units mL/min/kg; IV: 0.05 mpk cassette dosing or 2 mpk single dosing in
u
DMSO/PEG400/water (20/60/20); PO: 10 mpk in PEG400/Tween90/water (40/10/50); (e) permeability
determined in LLCPK cell line.
Western amine SAR utilizing the core template of arylsulfonamide 5 is displayed
in Table 2. Addition of one carbon atom to the linker of 5 afforded 14 which decreased
Na 1.7 potency by over an order of magnitude, increased rat CL substantially, and had
v
int
no impact on permeability. Somewhat surprisingly, the (octahydro-9aH-quinolizin-9a-
2
7
yl)methanamine (4.4.0) substituent in 15 dramatically increased permeability (P
=
app
8

3
2) compared to 5, albeit at the expense of potency and pharmacokinetics. Modifying the
basicity of the 3.3.0 amine via the beta-fluorine effect improved permeability for
2
8
compounds 16 and 18, however, potency was diminished on Na 1.7. A substitution
v
pattern which was tolerated with regard to Na 1.7 potency was dimethyl-3.3.0 ring
v
system in 19 and 20. This pair of enantiomers had excellent selectivity over Na 1.5,
v
moderate permeability (9 – 11), lipophilicity (LogD = 1.7), rat CLint and oral
bioavailability. Compound 19 possessed a very comparable profile to 5 (Table 1) with
slight differentiation in permeability (Papp = 9), hence both compounds were moved
forward for additional characterization.
a
Table 2. Western Amine SAR.
Na 1.7 IC
Na 1.5 IC
LogD /
PSA (Å)
P
app
(10 cm/s)
v
50
v
50
Compound
R
-6
Rat CLint / %F
1600 / 30
(
nM)
(nM)
1
4
129
136
24200
0.6 / 80
2.2 / 76
2
1
1
5
6
>30000
>30000
>30000
32
23
3000 / ND
NA / NA
ND / ND
3
1
75
60
1.7 / 77
1.5 / 77
(
racemic)
1
7
ND
(
enant. A)
9

1
8
2
10
5700
1.5 / 77
11
810 / 57
(
enant. B)
1
9
1
2
7
>30000
>30000
1.7 / 78
1.7 / 78
9
460 / 61
850 / 45
(
R)
2
0
2
13
(
S)
(
a) See Table 1 legend for definition of terms.
Representative synthetic routes for the compounds in Tables 1 and 2 are
exemplified in Schemes 1 and 2. Compound 5 was synthesized from commercially
2
9
available (tetrahydro-1H-pyrrolizin-7a(5H)-yl)methanamine 21
and known 2,4-
3
0
dimethyoxybenzyl-protected sulfonamide scaffold 22 under nucleophilic substitution
reaction conditions, deprotected under acidic conditions, and transformed to the HCl salt
to afford 5 in moderate overall yield. This synthetic approach was utilized for all
compounds in the preceding tables.
Scheme 1. Synthesis of compound 5. Reagents and reaction conditions: (a) Et N, DMF, 40 °C, 18 h, 62%;
3
(
b) TFA, DCM, 25 °C, 1 h, then 2.0 M HCl / MeOH, 80%: DMF = N,N-dimethylformamide, DCM =
dichloromethane.
Substituted or homologated 3.3.0 amines were assembled using several methods
3
1
(
Scheme 2). Known intermediate 23 was ozonized and subsequently difluorinated to
afford 24 which was globally reduced to afford 25 in moderate yield (Scheme 2A).
1
0

Alcohol 25 was converted to primary amine 26 via standard mesylate formation, azide
displacement, and hydrogenation sequence in modest yield. Alternatively, the ozonolysis
product of intermediate 23 could be stereoselectively reduced with NaBH to afford
4
alcohol 27 which can be inverted through a DAST fluorination to afford 28 (Scheme 2B).
This intermediate underwent the same alcohol to primary amine interconversion sequence
as before to afford 29. Finally, methyl benzylprolinate 30 was alkylated with ethyl-3-
bromopropionate, de-benzylated and cyclized to afford 31 which was selectively reduced
(
NaBH ) and TBS protected (32). Dimethylation of the lactam was achieved under LDA /
4
MeI conditions to afford 33 in modest yield after TBS deprotection. The resulting alcohol
was taken through a phthalimide Mitsunobu / hydrazine deprotection sequence to afford
amines 36 and 37, respectively, with a chiral resolution occurring at the phthalimide stage
3
2
(Scheme 2C).
Scheme 2. Representative synthesis of amine monomers. Reagents and reaction conditions: A) (a) ozone,
DCM, 25 °C, then Me S, 67%; (b) DAST, THF, 0 °C, 18 h, 57%; (c) LiAlH , THF, 0 °C to reflux, 4 h,
2
4
8
0%; (d) MsCl Et N, DCM, 0 °C, 1 h, aqueous work up, then NaN , DMF, 80 °C, 18 h, 83%; (e) 10% Pd/C,
3
3
MeOH, H (1 atm), 0 °C, 2 h, ~ 100%; B) (a) see A-a; 67%; (f) NaBH , MeOH, 0 °C, 5 min, 86%; (b)
2
4
DAST, DCM, 0 °C to 25 °C, 18 h; 74%; (c); see A-c; 99%; (d) see A-d; 71%; (e); see A-e; ~ 100%; C) (g)
1
1

LDA, HMPA, 3-bromopropionate, THF, -78 °C to 25 °C, 16 h, 22%; (h) 10% Pd/C, cat. formic acid,
MeOH, H (50 psi), 25 °C, 10 h, 92%; (i) toluene, reflux, 16 h, 81%; (j) NaBH , MeOH, 0 °C, 30 min,
2
4
7
5%; (k) TBSCl, imidazole, DCM, 0 °C to 25 °C, 5 h, 100%; (l) LDA, MeI, THF, -78 °C, (reaction
performed twice for bis-addition), 3 h, 36% overall; (m) 2.0 M HCl, MeOH, 1 h, 76%; (n) phthalimide,
PPh , DEAD, THF, 0 °C to 25 °C, 18 h, 71%; (o) SFC separation; IC column, 5 to 40% EtOH in CO ; (p)
3
2
hydrazine hydrate, EtOH, reflux, 4 h, 54%; (q) BH •DMS, THF, 25 °C ~ quant. DAST =
3
diethylaminosulfurtrifluoride; MsCl = methanesulfonyl chloride; LDA = lithium diisopropylamide; HMPA
=
hexamethylphosphoramide; TBSCl = tert-butyldimethylsilyl chloride; DEAD = diethylazodicarboxylate;
LiHMDS = lithium bis(trimethylsilyl)amide.
Table 3. Additional profiling of compounds 5 and 19.
Compound
Compound 5
Compound 19
459 g/mol
7.0, 9.3
Molecular Weight
431 g/mol
6.7, 9.8
159, 43
0.7 / 2
pK (sulfonamide / amine)
a
a
Solubility (M, pH 2 and 7)
143, 28
1.7 / 9
b
c
app
HPLC LogD / P
d
Mouse Na 1.7 IC (nM)
15 nM
13 nM
v
50
Mouse PPB
59%
82%
CYP IC (3A4/2C9/2D6, M)
> 50, > 50, 32
16
> 50, > 50, 3.1
5.6
50
PXR EC (M)
5
0
(
(
a) MSD HPLC kinetic solubility assay; (b) MSD HPLC logD assay; (c) Monolayer assay in MDCK cells;
d) Estimated inactivated state inhibition 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.
Compounds 5 and 19 possessed moderate molecular weight, zwitterionic
character, and moderate kinetic solubility at neutral and acidic pH (Table 3). As stated
above, the two compounds represented a 1 log unit range of lipophilicity (0.7 versus 1.7)
-6
and apparent differentiation in permeability (2 versus 9 x 10 cm/s), and the impact of
these properties on in vivo performance was of high interest to our team. Na 1.7 potency
v
3
3
shifts in preclinical species, especially rodents, were recently identified in the literature.
Hence, we were delighted to observe that compounds 5 and 19 possessed a mouse Na 1.7
v
IC of 15 and 13 nM, respectively; sufficient to support in vivo efficacy evaluation. Both
5
0
compounds had adequate unbound fraction in mouse plasma to interpret our in vivo
1
2

experiments, and compound 5 displayed excellent functional selectivity in a general off-
3
4
target screening panel with no hits of less than 400-fold selectivity. Both compounds
3
5
were not potent CYP inhibitors with the exception of moderate activity at CYP2D6, and
did elicit moderate PXR activation. Compound 5 demonstrated minimal CNS penetration
when dosed orally to mice with less than 1% and 6% of the plasma exposure existing in
the CSF and brain compartments (30 mpk oral dose), respectively. While this same
experiment was not performed on 19, this compound was a Pgp substrate in the rat
3
6
(
LLCPK cell line) with a BA/AB ratio of 17. These two experiments partially alleviated
concerns around CNS penetration of these molecules which could elicit known toxicity
from blockade of Na 1 isoforms (Na 1.1 and 1.2) expressed in the CNS (vide supra). On
v
v
the basis of the data above, both compounds 5 and 19 were advanced into behavioral pain
models in mice.
A common assay to evaluate antinociceptive effects of potential pain therapeutic
3
7
agents is the mouse formalin paw test assay. When a dilute solution of formalin is
injected into the hind paw of a mouse, characteristic biting and licking of the affected
area is elicited. The efficacy assessment is composed of two phases; an acute phase (or
Phase I) generally reflective of direct activation of nociceptors and a tonic phase (or
Phase II) related to inflammatory responses. Compounds 5 and 19 were administered
orally two hours prior to formalin challenge, and both compounds demonstrated
statistically significant, dose-dependent reversal of these effects in the acute phase of the
experiment (0 – 5 minute period post formalin injection) and the tonic phase of the
experiment (20 – 35 minute period post formalin injection) with full reversal of formalin
effects in the tonic phase (Figure 3). Plasma concentrations were evaluated at the end of
1
3

the experiment with an unbound plasma concentration IC of 170 nM (C = 420 nM) and
5
0
p
4
5 nM (C = 250 nM) for 5 and 19, respectfully, during the tonic phase. These exposures
p
were 11-fold (5) and 3.5-fold (19) over the mouse Na 1.7 IC value, respectively.
v
50
Recently, GOF mutations in Na 1.7 have been linked to paroxysmal itch in
v
3
8
humans. Hence, a model of histamine-induced itch was developed to compare the pain
efficacy observed for the compounds characterized above to effects that our team viewed
as suggestive of Na 1.7 target modulation. Briefly, intradermal treatment of histamine to
v
mice engendered a significant increase in the number of scratching events over the course
of 15 minutes (Figure 4). Oral administration of compound 5 two hours prior to histamine
challenge demonstrated dose-dependent blockade of these scratching events with
statistically significant, complete blockade occurring at the same oral dose as that used in
the mouse formalin paw test (30 mpk). This suggested that the efficacy observed in the
mouse formalin paw test was mediated through the inhibition of Na 1.7.
v
In conclusion, parallel synthetic efforts utilizing an arylsulfonamide core template
led to the discovery of the novel Western 3.3.0-amine class of arylsulfonamide Na 1.7
v
inhibitors represented by compound 3. Medicinal chemistry targeting the balance of
potency, selectivity over Na 1.5, PSA, and lipophilicity afforded advanced compounds 5
v
and 19: potent, selective, and orally bioavailable inhibitors of Na 1.7 with limited CNS
v
penetration. Arylsulfonamides 5 and 19 were effective at reversal of formalin-induced
nociceptive events in mice, and the efficacy of 5 was consistent with Na 1.7 target
v
modulation as evidenced by efficacy in the mouse itch assay. Additional research in the
Na 1.7 inhibitor arena will be published in due course.
v
1
4

a
Figure 3. Oral efficacy of compounds 5 and 19 in mouse formalin paw test.
(
a) Measurement of formalin-induced nociceptive behaviors in mice (C57BL/6 mice) following
administration of vehicle (40% PEG400 / 10% Tween 80 / 50% water) or rising doses of compounds 5
Panel A) and 19 (Panel B) PO. Compounds were administered 2 hours prior to formalin injection to
(
coincide with Tmax from separate pharmacokinetic studies (3 hours). Pharmacokinetics were evaluated 3
hours post administration. Data were analyzed using within-subject ANOVA to determine main effects and
one sample t-test to compare to vehicle (N = 8 / group); * P > 0.01, ** > 0.001.
a
Figure 4. Oral efficacy of compound 5 in mouse itch assay.
1
5

(
a) Measurement of histamine-induced nociceptive behaviors in mice (C57BL/6 mice) following
administration of vehicle (40% PEG400 / 10% Tween 80 / 50% water), 3, 10, and 30 mg/kg of compound
PO. Compound 5 was administered 2 hours prior to histamine injection to coincide with Tmax from
5
separate pharmacokinetic studies (3 hours). Data were analyzed using within-subject ANOVA to determine
main effects and one sample t-test to compare to vehicle (N = 8 or 9 / group); * P > 0.01.
Acknowledgments
The authors would like to thank Dr. Christopher J. Bungard and Dr. Christopher S.
Burgey for review of this manuscript. The authors would also like to thank Mr. Andrew
Danzinger for data analysis for the mouse itch assay.
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(
a) Baidya, D. K.; Agarwal, A.; Khanna, P.; Kumar, M. J. Anesth. Clin. Pharmacol. 2011, 27, 307-314;
(
b)Thomas, B.; Farquhar-Smith, P. Expert Opin. Pharmacother. 2011, 12, 2565-2571.
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2
3
All oxygen-linked variants of the 3.3.0 amine template were greater than 20-fold less potent than the
analogous nitrogen-linked compounds.
24
Please see the Supporting Information section for experimental procedures regarding Na 1.7 and Na 1.5
v
v
potency determinations via PatchXpress electrophysiology assays.
1
7

25
The “arylsulfonamide” binding site potency for 3 was measured by a radioligand displacement assay
with a K = 42 nM using a proprietary Merck radioligand residing in the structural class from reference 22.
i
No binding was detected for compound 3 in a standard local anesthetic binding site displacement assay
with K > 30 M.
i
26
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27
(
octahydro-9aH-quinolizin-9a-yl)methanamine was utilized to synthesize compound 15 and is
commercially available from Advance Building Blocks Corporation.
28
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4
252.
(
2
9
Tetrahydro-1H-pyrrolizin-7a(5H)-yl)methanamine 21 is available for purchase from Fisher Scientific,
and can be synthesized according to: Sparatore, A.; Basilico, N.; Casagrande, M.; Parapini, S.; Taramelli,
D.; Brun, R.; Wittlin, S.; Sparatore, F. Bioorg. Med. Chem. Lett.2008, 18, 3737-3740.
3
3
3
0
1
2
Babich, O.; Luo, R. Z.; Wang-Fisher, Y.; Pallin, D. J.; Venkatachalan, S. P. WO 2104151472.
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33
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D.; Waxman, S.G.; Patel, M.K.; Butt, R.P.; Stevens, E.B. PLoS ONE 2016, 11(4);
DOI:10.1371/journal.pone.0152405
34
General off-target screening (Ricerca panel, 145 assays) of compound 5 revealed several initial binding
hits including adrenergic activity (1D, 2A, 2B), serotonin (5HT ), and muscarinic (M1) activites.
2B
Second messenger assays for adrenergic activities resulted in functional antagonist activity with an IC50
~
8
M (all 3 isoforms listed), M1 IC50 and EC50 > 30 M, and 5HT2B IC50 = 3.8M. Compound 19 was
only tested in selected off-target assays and demonstrated a similar profile to 5.
35
CYP inhibition has been identified as an issue for the arylsulfonamide series of Na 1.7 inhibitors (see
v
reference 21.
36
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3
7
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38
(
a) Devigili, G.; Eleopra, R.; Pierro, T.; Lombardi, R.; Rinaldo, S.; Lettieri, C.; Faber, C. G.; Merkies, I.
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analogous fashion to that reported in reference 20.
1
8