Synthesis and evaluation of (4-chlorobenzhydryl) piperazine amides as sodium channel Nav1.7 inhibitors

Article abstract of DOI:10.1002/bkcs.10446

Blockage of voltage-gated sodium channels is used to treat neuropathic pain which is chronic and can become debilitating. Sodium channels Nav1.7-1.9 are especially attractive targets for drug discovery because of the broad therapeutic potential of their m

Full text of DOI:10.1002/bkcs.10446

Article
DOI: 10.1002/bkcs.10446
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S. K. Back et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Evaluation of (4-Chlorobenzhydryl) Piperazine Amides as
Sodium Channel Nav1.7 Inhibitors
Seung Keun Back,† Yoo Lim Kam,‡ Jung Ae Oh,‡ Heung Sik Na,§,
*
‡,
Uhtaek Ih,¶, and Hea-Young Park Choo
*
*
†Pharmaceutics & Biotechnology, College of Medical Engineering, Konyang University,
Chungnam 320-711, Republic of Korea
‡College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University,
Seoul 120-750, Republic of Korea. *E-mail: hypark@ewha.ac.kr
§Department of Physiology, Korea University College of Medicine, Seoul 136-705, Republic of Korea.
*E-mail: hsna@korea.ac.kr
¶Sensory Research Center, CRI, College of Pharmacy, Seoul National University, Seoul 151-742,
Republic of Korea. *E-mail: utoh@snu.ac.kr
Received February 27, 2015, Accepted May 21, 2015, Published online August 25, 2015
Blockage of voltage-gated sodium channels is used to treat neuropathic pain which is chronic and can become
debilitating. Sodium channels Nav1.7–1.9 are especially attractive targets for drug discovery because of the
broad therapeutic potential of their modulation. For a neuropathic pain therapy, anticonvulsant like lamotri-
gine, carbamazepine and a topical anesthetic such as Lidocaine are used. A growing number of clinical reports
suggest that selective inhibitors of Nav1.7 are likely to be the powerful analgesics for treating a broad range of
pain conditions. Therefore we evaluated 108 amide derivatives synthesized on human Nav1.7 (hNav1.7) by
VIPR (voltage/ion probe reader), a fluorescence image plate reader (FLIPR) assay that used voltage-sensor
fluorescence dye and stable HEK-293 cell lines expressing hNaV1.7. Ten compounds demonstrated inhibitory
activity, and the two most active compounds (5 and 6) had IC50 values of 8–10 μM.
Keywords: Nav1.7, Voltage Ion Probe Reader assay, Formalin test, Neuropathic pain
Introduction
Of the nine voltage-gated sodium channel isoforms that
have been cloned and functionally expressed to date, three
(Nav1.3, Nav1.7, and Nav1.8) appear to play roles in pain sig-
naling. These subtypes are expressed in peripheral nerves and
seen as potential targets in the development of novel analge-
sics. However, blocking the activity of subtypes Nav1.2,
Nav1.4, Nav1.5, and Nav1.6 must be avoided due to the risk
of CNS, cardiac, and neuromuscular side effects. Recent data
from human genetic studies have implicated Nav1.7, a sub-
type located primarily in the PNS, as a key constituent in pain
signaling.7 Therefore, selective inhibitors of Nav1.7 are pow-
erful analgesics for treating a broad range of painful condi-
tions. As shown in Figure 1, ralfinamide and lacosamide
were developed as selective sodium channel blockers. Ralfi-
namide, a (halobenzyloxy) benzyl-amino-propanamide deriv-
ative, inhibits Nav1.3, Nav1.7, and Nav1.8 in rat DRG
neurons.8 Lacosamide acts by enhancing the slow inactivation
ofVGSCs. PPPAis astate-dependent Nachannelblocker hav-
ing an effect on nociception-induced animals.9 The imidazo-
pyridine-derived compound, Merck-1, is hNav1.7 blocker
(hNav1.7 IC50 = 80 nM), and a study by Merck found it
reversed allodynia by 52 and 41% at 2 and 4 h postdose,
respectively, in the Chung rat spinal nerve ligation model of
neuropathic pain when dosed orally at 10 mg/kg.10
There are more than 160 million people all over the world suf-
fering from chronic pain, and they spend over $20 billion a
year on treatment. Neuropathic pain results from primary
lesions or dysfunctions in the peripheral nervous system
(PNS) or central nervous system (CNS) due to diseases like
diabetes, and infections such as shingles and AIDS.1 It is char-
acterized byspontaneous burning pain, hyperalgesia, and allo-
dynia.2 Enhanced pain sensitivities under neuropathic or
inflammatory conditions are known to be driven by spontane-
ous ectopic discharges, or hyperexcitability of primary affer-
ent neurons,3 which are involved in the dynamic changes of
the voltage-gated sodium channels (VGSCs), such as altera-
tions in channel activity and subtype profile.4,5 Therefore,
VGSCs are important pharmaceutical targets for the treatment
of chronic pain, and their blockers as novel analgesics have
been extensively studied during the last decades. Although
several VGSC blockers have been used to treat chronic pain,
there are a few clinical trials in which new oral VGSC modu-
lators have successfully treated patients with chronic pain. For
neuropathic pain therapy, conventionally anticonvulsants
such as lamotrigine, carbamazepine, and topical anesthetic
lidocaine are used.6 These drugs are sodium channel blockers
combining the site located in the inner pore region of the mam-
malian sodium channel.
We reported the design and synthesis of compounds with
1–3 amide bonds and a lipophilic ring system in each
Correction added on 01 October 2015, after first online publication: ISSN (Print) has been corrected.
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molecule, and some of the prepared compounds showed anal-
gesic effects on neuropathic pain in rats.11–15 As our continu-
ous effort to find analgesics for neuropathic pain, we evaluated
the 108compounds to determine theirNav1.7 inhibitory activ-
ity by Voltage Ion Probe Reader (VIPR) assay, and 10 active
compounds are reported in this paper.
As outlined in Scheme 1, a mixture of N-((tert-butyloxy)
carbonyl)iminodiacetic acid (2.13 mmol) and EDCI (2.13
mmol) in DMF was stirred at room temperature (RT) for 1
h. Various amines (2.13 mmol) were added to the resulting
solution and the solutions were stirred further for 20 h. The
reaction mixture was diluted with EtOAc, and washed sequen-
tially with 10% aqueous HCl and saturated aqueous NaCl. The
organic layer was dried over Na2SO4, and the solvent was
removed using rotary evaporator.
Experimental
The monoamides thus obtained was converted to diamides
following the procedures shown below. N-Boc-iminodiacetic
acid monoamide (1.31 mmol), 1-(4-chlorobenzhydryl) piper-
azine (1.441 mmol), and PyBOP (1.441 mmol) were dis-
solved in DMF, and i-Pr2NEt (2.62 mmol) was added. After
stirring for 16 h at rt, the reaction mixture was diluted with
EtOAc. The organic layer collected after sequential washings
with 10% aqueous HCl, saturated aqueous NaHCO3, and sat-
urated aqueous NaCl was dried over Na2SO4. The solvent was
then removed in vacuo to afford the N-Boc-iminodiacetic acid
diamide. To purify the crude diamides, CHCl3 and 4 M HCl–
dioxane were added to the diamide and the mixture was
allowed to stand for 3 h. The solvent was removed in vacuo
to afford the desired diamides (5, 6, 11–17).
Materials and Methods. All melting points of the synthe-
sized compounds were taken in Pyrex capillaries using an
electrothermal digital melting point apparatus (Buchi, Flawil,
1
Switzerland) and were not corrected. H NMR spectra were
recorded on a 400 MHz Varian FT-NMR using tetramethylsi-
lane as an internal standard. Chemical shifts were expressed in
ppm (δ), and peaks were listed as singlet (s), doublet (d), triplet
(t), quintet (q), and multiplet (m), with coupling constants (J)
expressed in Hertz. Mass spectra data were obtained on an
Agilent 6220 Accurate-Mass TOF LC/MS (Palo Alto, CA,
USA). Most of the reagents were purchased from Aldrich
Chemical Company (St. Louis, MO, USA) and Merck
(Darmstadt, Germany). TLC was run on the silica gel-coated
plastic sheets (Silica Gel 60, 230–400 mesh; Merck) and
visualized in UV light (254 or 365 nm).
Synthetic Procedure for Synthesis of Amide Derivatives
Synthesis ofDiamides 1–4. Thesynthesis ofdiamides, includ-
ing compounds 1–4, was previously reported.13
Synthesis of Diamides 5–6. The synthesis of diamide 6 was
reported elsewhere,15 and the typical synthetic procedure for
the other eight diamides that were not previously reported is
presented here.
N-(1-Methyl-3-phenyl)propyl-N0-1-(4-chlorobenzhydryl)
piperazineiminodiacetic acid diamide (5). Yellow solid,
30 mg(100%), mp 83–85 ꢀC, 1H-NMR (DMSO-d6, 400 MHz)
δ 12.953 (s, 1H), 9.096 (s, 2H), 8.471 (d, J = 8.0 Hz, 1H),
7.957 (s, 3H), 7.440–7.520 (m, 4H), 7.144–7.286 (m, 4H),
5.755 (s, 1H), 4.358 (s, 1H), 4.084 (s, 2H), 3.746–3.850 (m,
3H), 3.692 (d, J = 4.4 Hz, 2H), 3.353 (dd, J = 6.8 and 11.6 Hz,
1H), 3.250 (s, 2H), 3.003–3.108 (m, 4H), 2.523-–2.646 (m,
2H), 1.657–1.737 (m, 2H), 1.096 (d, J = 6.4 Hz, 3H). HR-
FABMS Calcd for C31H38ClN4O2 (M+ + H): 533.2678. Found:
533.2686.
N-Benzyl-N-1-(4-chlorobenzhydryl) piperazineimino-
diacetic acid diamide (11). Yellow solid, 21 mg (99%), mp
75–77 ꢀC, 1H-NMR (DMSO-d6, 400 MHz) δ 12.949 (s,
1H), 9.147 (s, 1H), 9.031 (t, J = 5.6 Hz, 1H), 7.960 (s, 3H),
7.237–7.520 (m, 9H), 5.755 (s, 1H), 4.345 (d, J = 6.0 Hz,
2H), 4.100 (s, 2H), 3.819–3.955 (m, 1H), 3.765 (s, 3H),
3.329–3.370 (m, 1H), 3.249 (s, 1H), 3.035–3.107 (m, 3H).
HR-FABMS Calcd for C28H32ClN4O2 (M+ + H):491.2208.
Found: 491.2216.
Cl
OCF3
S
O
N
H
N
C
F
3
N
N
H
N
O
N
N
O
NH
O
O
O
N
O
e
OM
e
M
Merck1 4)
Merck2
Merck3
SO2NH2
Figure 1. Developed selective sodium channel Nav1.7 blockers.
N-(4-Methylbenzyl)-N0-1-(4-chlorobenzhydryl) pipera-
zineiminodiacetic acid diamide (12). Yellow oil, 6 mg
1
(96%), H-NMR (DMSO-d6, 400 MHz) δ 8.268 (s, 1H),
7.309–7.439 (m, 7H), 7.094–7.209 (m, 5H), 4.359 (s, 1H),
4.228 (s, 2H), 4.208 (d, J = 7.2 Hz, 1H), 3.459 (s, 4H),
3.140 (s, 2H), 3.104 (s, 2H), 2.245 (s, 7H), 1.987 (s, 2H).
HR-FABMS Calcd for C29H34ClN4O2 (M+ + H): 505.2365.
Found: 505.2375.
N-(4-Methoxybenzyl)-N0-1-(4-chlorobenzhydryl)
piperazineiminodiacetic acid diamide (13). Yellow solid,
18.9 mg (67%) obtained: mp 110–120 ꢀC, 1H-NMR (DMSO-
d6, 400 MHz) δ 12.816 (s, 1H), 9.069 (s, 2H), 8.848 (s, 1H),
7.930 (s, 1H), 7.421 (d, J = 8.4 Hz, 5H), 7.199–7.220
Scheme 1. Synthesis of diamides and triamides.11 Reagents and con-
ditions: (i) EDCI; (ii) R1NH2, DMAP, DMF; (iii) R2, PyBOP, DMF,
rt, 16 h; (iv) 4 M HCl–dioxane, CHCl3, 3 h; (v) R3COOH, PyBOP.
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Amide Derivatives for Sodium Channel Nav1.7
KOREAN CHEMICAL SOCIETY
(m, 2H), 6.881–6.903 (m, 2H), 5.675 (s, 1H), 4.432 (s, 1H),
4.267 (d, J = 6.0 Hz, 2H), 4.054 (s, 2H), 3.943 (s, 1H), 3.731
(d, J = 2.0 Hz, 5H), 3.331 (s, 4H). HR-FABMS Calcd for
C29H32ClN4O3 (M++ H): 519.2168. Found: 519.2171.
N-(3-(5-tert-Butylisoxazole))-N0-1-(4-chlorobenzhy-
dryl) piperazineiminodiacetic acid diamide (14). Yellow
solid, 13 mg (98%), mp 85–88 ꢀC, 1H-NMR (DMSO-d6,
400 MHz) δ 12.868 (s, 1H), 11.538 (s, 1H), 9.271 (s, 3H),
7.944 (s, 2H), 7.422 (d, J = 7.2 Hz, 7H), 6.568 (d, J = 4.0
Hz, 1H), 5.698 (s, 1H), 4.362 (s, 1H), 4.132 (s, 3H),
3.011–3.166 (m, 4H), 1.987 (s, 1H), 1.283 (d, J = 10.4 Hz,
9H). HR-FABMS Calcd for C28H35ClN5O3 (M+ + H):
524.2423. Found: 524.2428.
incubated in Dulbecco's Modified Eagle F-12 until they
reached confluency. Cells were plated in 96-well plates and
incubated with a voltage-sensor dye, Membrane Potential
Assay Kit (R8034; Molecular Devices, Sunnyvale, CA,
USA). Cells were incubated with veratridine (50 μM) and test
compounds (1–10 μM). Fluorescence intensity was recorded
every 5 s.
Formalin Test. The analgesic effect of compound 7 on noci-
ceptive inflammatory pain was evaluated by the formalin test.
A mixture of methyl pyrrolidone, Tween 80, and sterile saline
(1:1:8)wasusedassolventtodissolveCompound7. Rats were
habituated to an observation chamber (20 × 20 × 20 cm)
equipped with a mirror placed behind the chamber to
allow an unobstructed view of the paw for 30 min. Then,
a mixture of formalin (5%, 50 μL) and compound 7 (6 or
60 μg/10 μL) was injected into the plantar surface of the right
hind paw of rats using a 30-gauge syringe. Animals were
returned tothechamber immediatelyandthetime spentlifting,
licking, shaking, or biting the injected paw was recorded for
every 5 min up to a 60-min period. The response time of each
rat was recorded by researchers who were not aware of the
treatment status of the rats. The early and late phases were
defined as 10 min after formalin injection and the following
50 min, respectively.
N-Cyclohexyl-N0-1-(4-chlorobenzhydryl) piperazinei-
minodiacetic acid diamide (15). Yellow solid, 160 mg
1
(66%), mp 135–145 ꢀC. H-NMR (DMSO-d6, 400 MHz) δ
12.902 (s, 1H), 9.024 (s, 2H), 8.381 (d, J = 7.6 Hz, 1H),
7.903 (s, 2H), 7.245–7.487 (m, 6H), 5.753 (s, 1H), 3.920 (s,
2H), 3.324–3.367 (m, 5H), 2.993–3.111 (m, 6H), 1.986 (s,
4H), 1.732–1.925 (m, 2H), 1.674 (d, J = 12.8 Hz, 1H),
1.545 (d, J = 12.4 Hz, 1H), 1.244 (d, J = 12.0 Hz, 1H). HR-
FABMS Calcd for C27H36ClN4O2 (M+ + H): 483.2521.
Found: 483.2527.
N-(5-(3-Methylisoxazole)-N0-1-(4-chlorobenzhydryl)
piperazineiminodiacetic acid diamide (16): Yellow solid,
16 mg (99%), mp 93–95ꢀC, 1H-NMR (DMSO-d6, 400
MHz) δ 12.870 (s, 1H), 11.495 (s, 1H), 9.259 (s, 2H), 8.845
(s, 1H), 7.929 (s, 2H), 7.393–7.434 (m, 7H), 6.629 (d, J =
4.4 Hz, 1H), 5.754 (s, 1H), 4.369 (s, 1H), 4.120 (s, 2H),
3.951 (s, 4H), 3.084 (t, J = 5.6 Hz, 2H), 2.397 (s, 3H). HR-
FABMS Calcd for C25H29ClN5O3 (M+ + H): 482.1953.
Found: 482.1955.
Neuropathic Pain Test
Rat Tail Nerve Injury. Under enflurane anesthesia, animals
were subjected to unilateral transection of the superior and
inferior caudal trunks at the level between the S1 and S2 spinal
nerves. To prevent possible rejoining of the proximal and dis-
tal nerve ending, 2 mm of the nerve was removed from the dis-
tal nerve ending. This surgery injured the S1 spinal nerve
innervating the tail.
Behavioral Testing. Mechanical allodynia of rat tails was
evaluated by measuring the withdrawal threshold in response
to a series of calibrated von Frey filaments (3.92, 5.88, 9.80,
19.60, 39.20, 58.80, 78.40, and 147. mN; Stoeling, Wood
Dale, IL, USA; equivalent to 0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0,
and 15.0 g, respectively) in an up–down fashion. The 50%
withdrawal threshold was determined by the Dixon method.16
A brisk tail withdrawal to the von Frey filament application
was regarded as a positive response.
Testing for cold or warm allodynia was carried out by mea-
suring tail withdrawal latency after immersing the tail into a
cold (4 ꢀC) or warm (40 ꢀC) water bath. An abrupt tail move-
ment within the cut-off time of 15 s was considered as a pos-
itive withdrawal response. The testing was repeated five times
in 5-min intervals, and the average latency of the tail response
was calculated.
Onthe14thdayfollowingtailnerveinjury, ratsweregivena
baseline test and then subjected to intraperitoneal injection of
compound 7 (6 and 60 mg/kg) dissolved in a vehicle (Methyl
pyrrolidone:Tween80:Saline = 1:1:8). Behavioral tests for
mechanical, cold, and warm allodynia were conducted at
1, 3, and 5 h, respectively, after the compound 7 treatment
in a blinded fashion. The percent of maximum possible effect
(%MPE), where %MPE = (Postinjection value − Baseline)/
N-(3,4-Dibenzyloxyphenethyl)-N0-1-(4-chlorobenzhy-
dryl) piperazineiminodiacetic acid diamide (17). Yellow
oil, 7 mg (99%), 1H-NMR (DMSO-d6, 400 MHz)
δ
7.803–7.957 (m, 1H), 7.277–7.454 (m, 17H), 7.200 (t, J =
7.2 Hz, 1H), 6.931–6.968 (m, 2H), 6.702 (d, J = 8.0 Hz,
1H), 5.070 (t, J = 5.2 Hz, 5H), 3.448 (s, 2H), 3.236 (s, 7H),
2.991–3.055 (m, 4H), 2.626 (t, J = 7.2 Hz, 2H), 2.230 (s,
2H), 1.730 (t, J = 3.2 Hz, 1H). HR-FABMS Calcd for
C43H46ClN4O4 (M+ + H): 717.3202. Found: 717.3202.
Synthesis of Triamide 7. The synthesis of triamides including
compound 7 was previously reported.11
Synthesis of Monoamides 8–10. The synthesis of monoa-
mides including compound 8–10 was previously reported.14
VIPR Assay. Stable HEK-293 cell lines expressing hNaV1.7
were obtained from Millipore (Darmstadt, Germany). All tis-
sue culture media were from obtained Invitrogen Corp.
(Carlsbad, CA, USA), CC2-DMPE and DiSBAC2 were pur-
chased from Invitrogen. Tetracaine hydrochloride was
obtained from Sigma-Aldrich (St. Louis, MO, USA). Veratri-
dine was from BIOMOL Research Laboratories Inc.
(Plymouth Meeting, PA, USA).
The HEK cells stably expressing hNaV1.7 were plated at
approximately 100 000 cells/well in poly-L-lysine-coated
96-well plates and incubated overnight at 37 ꢀC. Cells were
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Amide Derivatives for Sodium Channel Nav1.7
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(Presurgical value − Baseline) × 100, of each dose was
calculated.
Compound 7 Attenuates Inflammatory Pain in the Forma-
lin Test In Vivo. As illustrated in Figure 2, the control rats
treated with the mixture of formalin and vehicle spent a signif-
icantly longer time lifting, licking, shaking, or biting the
affected paw during both the early and late phases, indicating
formalin-induced nociceptive and inflammatory pain. How-
ever, an injection of compound 7 clearly reduced the forma-
lin-induced response duration in both phases (**p < 0.01 and
*p < 0.05 vs. the vehicle group [one-way ANOVA followed
by Bonferroni t-test]). The response durations in the early
phase of the vehicle, low (6 μg) and high (60 μg) dose com-
pound 7 groups were 47.05 ꢁ 8.53, 0.36 ꢁ 0.25**, and
4.71 ꢁ 2.16 s**, respectively (Figure 2(b)). In the late phase,
the time spent lifting, licking, shaking, or biting the paw in
each group was 334.21 ꢁ 51.10, 48.68 ꢁ 19.44**, and
60.40 ꢁ 20.38 s**, respectively (Figure 2(b)). However, there
was no statistical difference between the two compound
7 treatment groups. This result indicates that local compound
7 has an analgesic effect on both nociceptive and
inflammatory pain.
Statistical Analysis. All data were presented as mean ꢁ SEM
and statistically analyzed using one-way ANOVA and one-
way repeated measured ANOVA tests. A difference of p <
0.05 was considered statistically significant. Sigma Stat
(ver. 3.5; Systat Software Inc., Chicago, IL, USA) was utilized
for data analyses.
Results
Chemistry. For the syntheses of most of the target com-
pounds, well-documented solution phase combinatorial syn-
thetic routes reported were employed.17 Most of the
compounds synthesized were reported in our previous papers
as well. Amides studied in this paper include 49 triamides,11
33 diamides,13 and 11 monoamides.14 On top of these
93 amides, diamides, which were not previously reported,
are characterized in this paper.
The combinatorial synthesis of the eight diamides was
accomplished by coupling reactions (Scheme 1). Condensa-
tion was carried between the anhydride and various amines
to obtain monoamides. Each product was treated with 1-(4-
chlorobenzhydryl) piperazine and PyBOP resulting in Boc-
protected diamides. Crude Boc-protected diamides were
effectively purified by acid and base extractions. Removal
of Boc group was accomplished by acid hydrolysis with 4
M HCl–dioxane. Each individual compound synthesized
was characterized by 1H NMR and high-resolution MS.
Biological Evaluation: VIPR Assay. All synthesized com-
pounds were evaluated in vitro against the hNav1.7 using
the VIPR assay. VIPR is a FLIPR assay that uses a voltage-
sensitive dye for detecting the depolarization of cell mem-
branes due to the opening of hNav1.7. Veratridine (50 μM)
was used for the activation of hNav1.7. A potent Nav channel
blocker tetracaine (50 μM) was used as a positive control.
Three different doses (1, 3, and 10 μM) of synthesized com-
pounds were applied to the VIPR assay.
The structures and inhibitory activities of 10 active com-
pounds are illustrated in Table 1. The compound 6 showed
the highest activity with 51.2% inhibition at 10 μM concentra-
tion, which was observed as sodium channel inhibition in an
electrophysiograph and displayed analgesic activity in vivo.15
The structurally related compound 5 showed similar activity
with 48.9% inhibition. Compounds 1–4 showed weak activity
with 8–35% inhibition. The triamide compound 7 showed
24.9% inhibition while the other 50 structurally related tria-
mides that were previously reported did not show any activity.
Monoamides 8–10 showed fairly good activity with 25–33%
inhibition.
Compound 7 Relieves Neuropathic Pain In Vivo. Before
tail nerve injury, Animals exhibited little tail withdrawal in
response to any given stimuli, such as von Frey filaments, cold
(4 ꢀC), and warm (40 ꢀC) water stimuli (Figure 3). Fourteen
days (BL) after TNI, however, the tail withdrawal threshold
to von Frey filaments and latencies in response to cold and
warm stimuli were significantly decreased compared to the
presurgical values (Pre)(p < 0.05 vs. Pre, Paired t-test), indi-
cating the presence of nerve injury-induced pain called
mechanical and cold and warm allodynia, respectively. These
neuropathic animals were then subjected to compound 7 or
vehicle treatment.
Asshownin**Figure3(c)and(e), compound7significantly
attenuated the signs of cold and warm allodynia, in a dose-
dependent manner (*p < 0.05 vs. baseline [BL], one-way
repeated measured ANOVA followed by Bonferroni's t-
test]). The maximum possible effects (%MPE) of the high
dose (60 mg/kg) on cold and warm allodynia were 70.41 ꢁ
13.04%##ψ and 78.97 ꢁ 14.95%##ψ, respectively (##p < 0.01
vs. the vehicle group; ψp < 0.05 vs. the low dose group,
one-way ANOVA followed by Bonferroni's t-test). Analgesic
effects on both cold and warm allodynia were sustained for at
least 3 h after the injection of high dose compound 7 without
any adverse effects that interfered with behavioral testing. In
the present study, mechanical allodynia was barely affected by
compound 7 at the two doses (Figures 3(a) and (b)). Our result
indicatesthatcompound7ishighlyeffective inrelievingnerve
injury-induced pain, at least cold and warm allodynia.
Discussion
Blocking of the Sodium Channel Site 2 by Compound 7.
Compound 7 was found to be a strong blocker against the rat
sodium channel site2, knownas the batrachotoxin-binding site,
whentestedbyMDSPharmaServices(MDSPS PT#1064545).
It exhibited 108% inhibition at a concentration of 10 μM.
Even though sodium channel blockers have drawn research-
ers’ attention as a viable target for the development of novel
analgesics, the clinical use of sodium channel blockers is cur-
rently very limited because of their nonselectivity, relatively
narrow therapeutic window, and adverse side effects. As
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Amide Derivatives for Sodium Channel Nav1.7
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Table 1. The structures and activity of active compounds having
Table 1 (continued)
Nav1.7 inhibition.
No.
Structure
% inhibition at 10 μM
No.
1
Structure
% inhibition at 10 μM
O
O
H
N
O
O
N
N
N
O
O
16.3
H
O
O
N
N
H
O
N
O
Cl
7
24.9
Cl
O
2
3
35.0
14.3
O
C
O
O
O
N
N
N
H
N
N
H
N
O
N
Cl
O
8
27.1
32.9
25.7
Cl
NH-Boc
N
O
O
N
N
N
H
N
CH3
Cl
O
Cl
9
Cl
N
N
O
O
H
4
5
8.0
N
Cl
N
N
H
N
O
Cl
Cl
10
O
H
N
O
O
CH3
48.9
N
H
N
N
N
N
N
Cl
Cl
11
Tetracaine
81.5
6
51.2
Figure2. Theeffectofcompound7onspontaneouspainbehaviorsinducedbyformalin. Compound7(6and60 μg/10 μL)orvehicle(10 μL)was
co-injected with formalin (For, 5%, 50 μL) under the plantar surface of the right hind paw. (a) Time spent lifting, licking, shaking, or biting the
injected paw was plotted against every 5 min after the injection over a 60-min testing period. (b) Analgesic effect of compound 7 on pain beha-
viors during both the early (0–10 min) and late (10–60) phases. *p < 0.05 and **p < 0.001 vs. For + vehicle (one-way ANOVA followed by Bon-
ferroni's t-test).
Bull. Korean Chem. Soc. 2015, Vol. 36, 2290–2297
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Amide Derivatives for Sodium Channel Nav1.7
KOREAN CHEMICAL SOCIETY
Table 2. The structure of diamide compounds 11–17.
No.
11
R
No.
15
Structure
12
13
16
17
14
Compounds 5 and 6 have the identical linker and are struc-
turally related. Compound 5 showed good inhibitory activity
with 48.9% inhibition. However, seven compounds (com-
pounds 11–17; Table 2) structurally related to compound
6 showed no activity. In these compounds, one portion of
the diamide functionality is fixed as a 4-chlorobenzhydryl
piperazine moiety, and the other part is varied by introducing
a benzyl, methylbenzyl, 3-(5-tert-butylisoxazol)yl, cyclo-
hexyl, 5-(3-methylisoxazol)yl, and 3,4-dibenzyloxyphe-
nethyl, respectively.
When the linker was changed to ethyl or 2-methylpropyl,
compounds 1–4 showed weak activity with only 8–35% inhi-
bition. Only compound 2 with cyclohexylamide showed 35%
inhibition activity. When compound 6 and 1 were compared,
the only one nitrogen structural difference being the linker,
activity of 1 was decreased to 10%. When the linker was chan-
ged to dimethyl propyl or ethenyl, none of the derivatives
showed activity. Compound 7 was the only active triamide,
which showed 24.9% inhibition while 50 other structurally
related triamides we reported on previously did not show
any activity. Monoamide compounds 9–10 showed fairly
good activity with 25–33% inhibition. However, the other
eight structurally related compounds we previously reported
on did not show any activity.
Figure 3. Effect of compound 7 on (a, b) mechanical, (c, d) cold, and
(e, f ) warm allodynia induced by rat tail nerve injury (TNI). (a, c, e)
Time courses of TNI-induced pain behaviors and antiallodynic effect
of compound 7. ‘Pre’ represents the day prior to nerve injury. At 2
weeks after nerve injury, the rats underwent a baseline (BL) test
and were then injected with compound 7 (6 and 60 mg, i.p.). Behav-
ioral tests were performed 1, 3, and 5 h after the injection. *p < 0.05
vs. BL (one-way repeated measured ANOVA followed by Bonferro-
ni's t-test). (b, d, f ) The percentage of maximum possible effect
(%MPE) of compound
7 on mechanical, cold, and warm
allodynia, respectively. #p < 0.05 and ##p < 0.01 vs. the vehicle
group; ψp < 0.05 vs. the low-dose group, one-way ANOVA followed
by Bonferroni's t-test).
subtype Nav1.7 is overexpressed at the site of a peripheral
nerve injury and genetic evidence implicated Nav1.7 in noc-
ciceptive signaling, the selective inhibitors of Nav1.7 are
likely to be the powerful analgesics for treating a broad range
of painful conditions.18–20
With the goal of finding potent peripherally active Nav1.7
blockers, we evaluated 108 compounds we prepared, which
were reported previously as Bradykinin antagonists, sodium
channel or calcium channel blockers.11–15 The synthesized
compounds were tested for their Nav1.7 blocking activity
by VIPR assay at concentration of 10 μM.
Compound 6 showed the highest activity with IC50 value of
8.5 μM. We reported that this compound was capable of bind-
ing to VGSCs and has also potent inhibitory activity based on
the results of the electrophysiograph against the TTX-R
sodium currents of the rat DRG sensory neuron. In addition,
the effectiveness of compound 6 against nociceptive, inflam-
matory, and neuropathic pain in vivo was reported.15
As the in vivo activity of compound 6 was already reported,
compound 7 was subjected to the experiments to determine
in vivo analgesic activity. In the present study, we provide evi-
dence that compound 7, a novel triamide compound, has an
analgesic effect on formalin-induced and nerve injury-
induced pain in rat models. The formalin test is a useful model
of acute and tonic pain for evaluating the analgesic efficacies
of chemical agents. In this test, local treatment of compound
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Amide Derivatives for Sodium Channel Nav1.7
KOREAN CHEMICAL SOCIETY
7 significantly reduced the time rats spent lifting, licking,
shaking, or biting the formalin-injected paw during both the
early and late phases, which correlate to nociceptive and
inflammatory pain behaviors, respectively (Figure 2). When
exposed to inflammatory irritants, increased VGSC activities
of primary afferent neurons appear to play a crucial role in the
generation of pain behaviors. Recent studies have revealed
that Nav1.7, a subtype of VGSC, is a major contributor to pain
signaling.21 It has been reported that Nav1.7 in DRG neurons
was upregulated in response to peripheral inflammation,22 and
knockout or knockdown of Nav1.7 expression impaired the
manifestation of inflammatory pain.23,24 Thus, the strong inhi-
bition of INav1.7 by compound 7 in DRG neurons accounts for
how the local injection of compound 7 could relieve formalin-
induced pain behaviors in the present study.
analgesic efficacy, no adverse effects were detectable during
behavioral testing, making compound 7 a valuable candidate
for the treatment of chronic pain.
Conclusion
We synthesized 108 amide derivatives and evaluated
their activities on Nav1.7 by VIPR assay using stable HEK-
293 cell lines expressing hNaV1.7. Ten compounds showed
inhibitory activity, and the two most active compounds
(5 and 6) had IC50 values of 8–10 μM. Our novel compound 7,
which showed 24.9% inhibition in VIPR assay, ameliorated
not only nociceptive pain but also chronic inflammatory
and neuropathic pain. Thus, it may be useful for the
treatment of inflammatory pain and for certain types of
neuropathic pain.
Compound 7 has also proven to be effective in
relieving nerve injury-induced pain when treated systemati-
cally. Central sensitization in neuropathic conditions has been
known to be mediated by ongoing discharges that arise from
ectopic loci of injured nerves and their surrounding area.
Peripheral nerve injury leads to the changes in expression of
VGSC subtypes; while Nav1.1, Nav1.6, Nav1.7, Nav1.8,
and Nav1.9 in DRG are downregulated, expression of
Nav1.3, a potent candidate generator of ectopic firing in
neuroma,25 is highly upregulated in DRG after peripheral
nerve injuries. In addition to Nav1.3, TTX-sensitive Nav1.7
also appears to have an important role in the generation of
neuropathic pain. It has been reported that Nav1.7 accumu-
lates within neuromas that are often formed at axotomized
nerve endings, suggesting its predictable role in ectopic
firing.26,27 In the present study using a rat tail model of neuro-
pathic pain, compound 7 significantly ameliorated cold and
warm allodynia in a dose-dependent manner. Therefore, these
analgesic effects might be closely associated with the inhibi-
tory activity of compound 7 on Nav1.7. However, it is note-
worthy that compound 7 failed to relieve mechanical
allodynia. Consistent with our results, previous studies have
reported that knocking out or knocking down Nav1.7 expres-
sion in mouse DRG prevented the manifestation of heat hyper-
algesia, but not mechanical allodynia following nerve
injury.28,29 It has been well known that Nav1.7 is expressed
in the majority of small unmyelinated DRG neurons,21 while
mechanical allodynia is mediated by large myelinated Aβ
fibers,30 suggesting that Nav1.7 specific blockers may be use-
ful for the treatment of pain which are mediated by small
unmyelinated afferents.
Further study on Nav1.5 is needed to avoid the cardiac side
effect.
Acknowledgments.
This research was supported by
Basic Science Research Program through the National
Research Foundation of Korea (NRF), and funded by the
Ministry of Education, Science and Technology (NRF-
2013R1A1A2011145).
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Bull. Korean Chem. Soc. 2015, Vol. 36, 2290–2297
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