Butyl 2-(4-[1.1′-biphenyl]-4-yl-1H-imidazol-2-yl)ethylcarbamate, a potent sodium channel blocker for the treatment of neuropathic pain

Article abstract of DOI:10.1016/j.bmcl.2006.12.064

A series of 4-arylimidazole carbamates was synthesized and their binding affinities to the site-2 sodium (Na⁺) channel were determined. SAR studies led to the identification of compound 10, a potent Na⁺ channel blocker which was efficacious in pain models in vivo.

Full text of DOI:10.1016/j.bmcl.2006.12.064

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1746–1749
Butyl 2-(4-[1.10-biphenyl]-4-yl-1H-imidazol-2-yl)ethylcarbamate, a
potent sodium channel blocker for the treatment of neuropathic pain
Anne-Marie Liberatore,* Jocelyne Schulz, Christine Favre-Guilmard, Jacques Pommier,
Jacques Lannoy, Emilia Pawlowski, Marie-Anne Barthelemy, Marion Huchet,
Michel Auguet, Pierre-Etienne Chabrier and Dennis Bigg
Ipsen Research Laboratories, Institut Henri Beaufour, 5 avenue du Canada, 91966 Les Ulis Cedex, France
Received 7 November 2006; revised 14 December 2006; accepted 16 December 2006
Available online 22 December 2006
Abstract—A series of 4-arylimidazole carbamates was synthesized and their binding affinities to the site-2 sodium (Na+) channel
were determined. SAR studies led to the identification of compound 10, a potent Na+ channel blocker which was efficacious in pain
models in vivo.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Neuropathic pain is initiated or caused by a primary le-
sion or dysfunction in the nervous system (e.g., diabetic
neuropathy, post-herpetic neuralgia). This development
of abnormal primary sensory neuron excitability may be
explained by an upregulation of voltage-gated sodium
channels (VGSCs) and a modification of sodium cur-
rents.1–3 Anticonvulsant drugs such as carbamazepine,
lamotrigine or lidocaine block VGSCs and show some
efficacy in the treatment of neuropathic pain.4 They
have, however, low affinity for the Na channel5 (IC50
in the 30–200 lM range) and poor side-effect profiles.6
The compounds were prepared according to the reaction
sequence11 shown in Scheme 1.
The amino acid 3 was converted to the corresponding
N-carbamate 4 using chloroformate derivatives under
basic conditions.12 When the halo-derivatives were not
commercially available, chloroformylation was carried
out using the appropriate alcohol and triphosgene under
mild conditions.13
The imidazoles 5–24 were prepared by reacting the caesi-
um salt of the acids 4 with an aryl bromoketone 2 to afford
an intermediate keto-ester which on heating with an ex-
cess of ammonium acetate gave the required imidazoles.
TheneedformorepotentandselectiveVGSCblockershas
led to the identification of second generation compounds
such as ralfinamide,7 which is currently in phase II clinical
trials and the preclinical candidate PPPA8 (Fig. 1).
Most of the required aryl bromoketones 2 are commer-
cially available except those needed for 17, 22, 23 and 24
which were prepared using either poly(vinyl-
pyridiniumhydrobromide perbromide) resin14 or bro-
mine15 in ethanol as the brominating agent.
We report here the synthesis and structure–activity rela-
tionship (SAR) of a structurally novel series of VGSC
blockers.
In a course of a pain research programme,9 the imidaz-
ole 1 (Fig. 2) was identified as a hit by screening of our
in-house sample collection (IC50 = 150 nM for the site 2
Na+ channel10), and we present here the synthesis and
structure–activity relationships (SARs) for a series of
compounds. For the most potent analogue, in vivo
activity is also reported.
Initial SAR studies began with the modulation of the
central linker.
F
NH2
O
N
O
H
O
N
O
Cl
NH2
N
F
Keywords: Sodium channel modulators; Treatment; Neuropathic pain.
*
ralfinamide
PPPA
Corresponding author. Fax: +33 169 07 3802; e-mail: anne-marie.
liberatore@ipsen.com
Figure 1. Structures of new drugs with Na+ channel activity.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.064

A.-M. Liberatore et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1746–1749
1747
The carbamate arylimidazole derivatives were tested for
their ability to displace radiolabelled batrachotoxin
([3H]BTX) from the sodium channel binding in synapto-
somal preparations of rat cerebral cortex.16 The
functional activity of the more potent compounds was
assessed by their capacity to protect SH-SY5Y cells
N
HN
N
O
H
O
1
à
against veratridine-induced cytotoxicity.17
Figure 2. Structure of compound 1.
As shown in Table 1, the two-carbon linker 5 appears to
be somewhat more active than 1 or 6.
R1
R1
Preparation of cortex membranes for Na+ binding. Cortex membranes
were prepared as follows: rats were decapitated, the brains rapidly
removed and the cortices dissected and weighed. The isolated cortices
were homogenised by means of a Teflon-glass homogenizer in 10
volumes of ice-cold 0.32 M sucrose 5 mM potassium hydrogen
phosphate (pH 7.4, 4 ꢁC.) solution. The resulting homogenate was
centrifuged at 1000g (4 ꢁC) for 10 min and the supernatant was
further centrifuged at 20,000g (4 ꢁC) for 15 min. The pellet was
suspended and washed in 0.32 M sucrose buffer and centrifuged again
at 20,000g (4 ꢁC) for 15 min. The residue was recovered. The
membrane sample thus obtained was suspended in Na-free assay
buffer (50 mM Hepes, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM
glucose, 130 mM choline chloride, pH 7.4) to give a final concentra-
tion of about 4 mg protein/ml and stored at ꢀ80 ꢁC until use. Protein
concentration was determined by the Bradford method using bovine
serum albumin as a standard.
a
Br
O
O
2
O
O
H
N
b
O
H2N
n
OH
n
OH
R2
O
3
4
R1
c
Na+ binding protocol. Binding studies were carried out as follow:
100ll of the above membrane sample preparation (75 lg/ml) was
added to buffer containing 1 lM tetrodotoxin, 50 lg/ml scorpion
venom, 5 nM [3H]BTX (34.0 ci/mmol) and the compound at different
concentrations to make a final volume of 0.5 ml. The reaction was
carried out at 25 ꢁC and was terminated after 90 min by the addition
of 2 ml of ice-cold washing buffer (5 mM Hepes, 1.8 mM CaCl2,
0.8 mM MgSO4, 130 mM choline chloride, pH 7.4). The mixture was
immediately vacuum-filtered on a unifilter GF/C (Packard) presoaked
with 0.1% poly-ethylene-imine. The filter was washed once with 2 mL
of ice-cold washing buffer. Bound [3H]BTX was determined by liquid
scintillation spectrometry in Microscint scintillation cocktail (Pack-
ard). Non-specific binding (NSB) was determined in the presence of
100 lM flunarizine, and the total binding was obtained without
inhibitor.For each concentration of compound, percent of control is
calculated as follows:
N
H
N
O
R2
N
n
H
O
5-24
Scheme 1. Formation of arylimidazole carbamates 5–24. Reagents and
condition: (a) Br2, AcOH; (b) NaOH, H2O, R2OCOCl; (c) i—Cs2CO3,
MeOH; ii—bromoketone 2, DMF; iii—NH4OAc, xylene, reflux.
Table 1. In vitro binding potencies
ððcpmcompound ꢀ cpmNSBÞ ꢁ 100Þ=ðcpmcontrol ꢀ cpmNSBÞ:
Points are performed in duplicate.
For each compound the values included in the linear part of each
experiment’s sigmoid were retained in a linear regression analysis
and were used to estimate the 50% inhibitory concentration (IC50).
N
H
N
O
à
N
n
Veratridine-induced cytotoxicity on SH-SY5Y cell line.SH-SY5Y
H
neuroblastoma cells were seeded on 96-well plates 24 h prior to
treatment. Cells were then pre-incubated with compounds for 20 min
before addition of 100 lM veratridine and 1 mM ovabain mixture.
After 3 h exposure, the medium was replaced by a culture medium
containing 10% alamar blue and incubated for 18–24 h. Cytotoxicity
was evaluated after plate reading on a spectrophotometer at double
wavelength (570 and 620 nM). For each concentration of compound
the percentage of cell injury was calculated as follows:
O
Na+ binding IC50 (nM)
a
Compound
n
1
5
6
1
2
3
144
64
184
a Data represent means of two experiments.
ðODvalues of veratridine-treated cells
ꢀ ODvalues of control cellsÞ=ðODvalues of veratridine-untreated cells
ꢀ ODvalues of control cellsÞ ꢂ 100
The two-carbon linker was conserved for studying the
impact of the carbamate group on biological activity.
Each OD was initial OD ꢀ blank OD.
The IC50 value was the concentration which decreased the % of cell
injury by 50% and was derived by extrapolation from graphs of
dose–response curves. Values used for calculations are the mean val-
ues of triplicates.
As shown in Table 2, most R2 alkyl group analogues
show potent binding affinity with the exception of meth-
yl 7 and tert-butyl -CH2 12.

1748
A.-M. Liberatore et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1746–1749
Table 2. In vitro binding potencies and cytoprotection against
veratridine-induced cell cytotoxicity
its cytoprotective activity since it proved to be rapidly
metabolized by human microsomes.19
Compound 10 was selected for in vivo evaluation in two
rat models of pain: in the carrageenan hyperalgesia
model,20 compound 10 elicits an anti-hyperalgesic effect
at 10 and 30 mg/kg, intra-peritoneally (ip) (Fig. 3A). In
the chronic constriction injury (CCI)-induced neuro-
pathic pain model,21 compound 10 affords significant
protection at 30 mg/kg, ip (Fig. 3B). Little, or no-effect
was seen on the contralateral paw in either model, sug-
gesting an anti-hyperalgesic rather than an analgesic ef-
fect. Furthermore, no significant drug-related effect was
observed on motor performance in the rotarod test at
30 mg/kg (ip) (data not shown), suggesting a lack of
O
N
R2
N
O
N
H
H
Compound R2
Na+ binding Cytoprotection
a
IC50 (nM)
a
IC50 (nM)
5
7
tert-Butyl
Methyl
Ethyl
64
282
71
—
—
8
—
—
9
Propyl
n-Butyl
iso-Butyl
86
32
10
11
12
13
14
15
16
230
610
—
26
tert-Butyl–CH2
Hexyl
Cyclohexyl
372
15
670
650
870
700
26
52
Cyclopentyl
Cyclohexylmethyl
21
a Data represent means of two experiments.
In the functional (cell-based) assay, the compounds
tested show little difference in activity, with the excep-
tion of compound 10.
The left-hand-side compound of 1018 was retained as a
template for optimization studies on the R1 group.
As shown in Table 3, a selection of aryl substituents
failed to improve the cytoprotective activity, although
certain compounds (17 and 21) showed good affinity
for the Na+ channel. Compound 17 was not tested for
Table 3. In vitro binding potencies and cytoprotection against
veratridine-induced cell cytotoxicity
R1
N
O
N
N
O
H
H
Compound
R1
Na+ binding
a
IC50 (nM)
Cytoprotection
a
IC50 (nM)
10
17
18
19
20
21
22
23
24
Phenyl
32
7
230
—
Cyclohexyl
F
Br
Figure 3. In vivo activity of compound 10 in two rat models:
carrageenan-induced hyperalgesia (A) and chronic constriction injury
(B). Compound or vehicle was administered intraperitoneally 30 min
prior to measurement of paw withdrawal thresholds after mechanical
stimulus (Randall and Selitto method)22 on carrageenan-inflamed paw
and on contralateral (non-inflamed paw) (A) or on injured paw and on
non-injured paw (B) comparison between vehicle and treated groups
was made using a two-sided Student/Dunnet test means SEM
(n = 4–6).23 *p < 0.05; **p < 0.01.
1000
210
453
14
—
900
—
CF3
Pyrolidine
iso-Butyl
Propyl
Ethyl
1120
750
3990
3220
41
62
96
a Data represent means of two experiments.

A.-M. Liberatore et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1746–1749
1749
16. Biological method described by McKinnon, A. C.; Wyatt,
K. M.; McGivern, J. G.; Sheridan, R. D.; Brown, C. M.
Br. J. Pharmacol. 1995, 115, 1103.
17. Biological method described by Pauwels, P. J.; Van
Assouw, H. P.; Leysen, J. E.; Janssen, P. A. Mol.
Pharmacol. 1989, 36, 525.
myorelaxant or sedative effect; an observation that
could be of relevance in terms of the side-effect profile
of the compound.
In conclusion, in the SAR studies of the 4-arylimidazole
carbamate series derived from 1, a lead compound 10
has been identified which shows high affinity for the
sodium channel with functional and in vivo activity in
two animal models of pain.
18. Selected data for compound 10 Typical experimental
procedure:Synthesis of 10: butyl 2-(4-[1.10-biphenyl]-4-yl-
1H-imidazol-2-yl)ethylcarbamate (10). N-(butoxycarbon-
yl)-b-alanine. A solution containing b-alanine (8.9 g,
0.1 mol) and 100 ml of a 1-N solution of sodium hydrox-
ide were cooled to 10 ꢁC. n-Butyl chloroformate (13.66 g,
0.1 mol) and 50 ml of a 2-N solution of sodium hydroxide
was added simultaneously. After stirring for 16 h at 23 ꢁC,
approximately 10 ml of a solution of concentrated hydro-
chloric acid (approximately 11 N) was added in order to
adjust the pH to 4–5. The oil obtained was extracted with
ethyl acetate (2 · 50 ml), washed with water then dried
over magnesium sulfate. The product crystallized from
isopentane in the form of a white powder (68% yield). Mp:
50.5 ꢁC. Butyl 2-(4-[1.10-biphenyl]-4-yl-1H-imidazol-2-
yl)ethylcarbamate. A mixture of N-(butoxycarbonyl)-b-
alanine (5.67 g, 0.03 mol) and caesium carbonate (4.89 g,
0.015 mol) in 100 ml of ethanol was stirred at 23 ꢁC for
1 h. The ethanol was eliminated by evaporation under
reduced pressure. The mixture obtained was dissolved in
100 ml of dimethylformamide and 4-phenyl-bromoace-
tophenone (8.26 g, 0.03 mol) was added. After stirring for
16 h, the solvent was evaporated under reduced pressure.
The mixture obtained was taken up in ethyl acetate and
the caesium bromide removed by filtration. The filtrate
was evaporated and the reaction oil was taken up in a
mixture of xylenes (100 ml) and ammonium acetate
(46.2 g, 0.6 mol). The reaction medium was heated at
reflux and, after cooling, a mixture of ice-cold water and
ethyl acetate was poured into the reaction medium. After
decantation, the organic phase was washed with a satu-
rated solution of sodium bicarbonate, dried over magne-
sium sulfate, and evaporated under reduced pressure; The
solid obtained was filtered and washed with ether to afford
a pale beige-coloured powder (50% yield). Mp: 136–
137 ꢁC. 1H NMR (400 MHz, DMSO-d6, d): 12.01 (br s,
1H), 7.83–7.19 (m, 11H), 3.94 (m, 2H); 3.33 (m, 2H), 2.8
(m, 2H), 1.51 (m, 2H), 1.32 (m, 2H); 0.8–0.83 (m, 3H).
Elemental analysis for C26H24N2: Theoretical: C 72.70%,
H 6.93%, N 11.56%. Found: C 72.72%, H 6.85%, N
11.54%.
Acknowledgment
´
We thank Jose Camara and his team for mass spectral
and NMR H analysis.
1
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