Synthesis, structure-activity relationships at the GABAA receptor in rat brain, and differential electrophysiological profile at the recombinant human GABAA receptor of a series of substituted 1,2-diphenylimidazoles

Article abstract of DOI:10.1021/jm049120y

A series of new 1,2-diphenylimidazole derivatives (1a-x) were synthesized and evaluated for their ability to potentiate γ-aminobutyric acid (GABA)-evoked currents in Xenopus laevis oocytes expressing recombinant human GABA_A receptors. Many of t

Full text of DOI:10.1021/jm049120y

2638
J. Med. Chem. 2005, 48, 2638-2645
Synthesis, Structure-Activity Relationships at the GABAA Receptor in Rat
Brain, and Differential Electrophysiological Profile at the Recombinant Human
GABAA Receptor of a Series of Substituted 1,2-Diphenylimidazoles
Battistina Asproni,*,† Giuseppe Talani,‡ Fabio Busonero,‡ Amedeo Pau,† Sebastiano Sanna,† Riccardo Cerri,†
Maria Paola Mascia,§ Enrico Sanna,‡ and Giovanni Biggio‡,§
Dipartimento Farmaco Chimico Tossicologico, Universita` di Sassari, Sassari, Dipartimento di Biologia Sperimentale “B.
Loddo”, Sezione di Neuroscienze and Centro di Eccellenza per le Dipendenze, Universita` di Cagliari, Cagliari, and
Istituto di Neuroscienze, Sezione di Cagliari, Consiglio Nazionale delle Ricerche, Cagliari, Italy
Received November 3, 2004
A series of new 1,2-diphenylimidazole derivatives (1a-x) were synthesized and evaluated for
their ability to potentiate γ-aminobutyric acid (GABA)-evoked currents in Xenopus laevis oocytes
expressing recombinant human GABAA receptors. Many of these compounds enhanced GABA
action with potencies (EC50 ) 0.19-19 µM) and efficacies (maximal efficacies of up to 640%)
similar to or greater than those of anesthetics such as etomidate, propofol, and alphaxalone.
Structure-activity relationship analysis revealed that the presence of an ester moiety in the
imidazole ring was required for full agonist properties, while modifications made in the phenyl
rings affected potency and efficacy, with ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-
4-imidazolecarboxylate showing the highest potency. These compounds potentiated the [3H]-
GABA binding to rat brain membranes, suggesting a site of interaction different from that of
GABA. As for etomidate, mutation of asparagine-265 in the â2 subunit of the GABAA receptor
into serine reduced the ability of derivative 1i to modulate the GABA function.
Introduction
The GABAA receptor is also a major target of several
chemically unrelated volatile or injectable general an-
esthetics such as halothane, isoflurane, pentobarbital,
etomidate, propofol, and alphaxalone. These compounds
enhance GABA-evoked Cl- currents at both neuronal
and recombinant GABAA receptors10-12 and exhibit
specific structure-activity relationships (SARs) and
stereoselectivity in this regard.10,13-15 In addition, en-
hancement of the GABAA receptor function by many of
these compounds may depend on the receptor subunit
composition. Moreover, site-directed mutagenesis has
revealed that specific amino acids are important deter-
minants of the action of several GABA-ergic drugs and
in particular of volatile or intravenous anesthetics.16
A variety of other agents, such as cocaine and cloza-
pine, known for their effects at other receptors, also
interact with and affect the function of GABAA receptors
at pharmacologically relevant concentrations. These
compounds may provide structural leads for the devel-
opment of more specific GABAA receptor ligands.7
In the course of a research program aimed at obtain-
ing antipsychotic agents with clozapine-like activities,
we recently developed a series of (1,2-diphenylimida-
zolyl)piperazine derivatives that are endowed with
substantial affinities for both D2 dopamine receptors as
well as 5-HT1A and 5-HT2A serotonin receptors, com-
pound I of which is representative (Chart 1). Compound
I was also found to inhibit in a concentration-dependent
manner (0.1-300 µM) GABA-evoked Cl- currents in
Xenopus laevis oocytes expressing recombinant human
GABAA receptors composed of R1, â2, and γ2L sub-
units.17 This finding prompted us to determine whether
the 1,2-diphenylimidazole framework of I might serve
γ-Aminobutyric acid (GABA) is the major inhibitory
neurotransmitter in the vertebrate central nervous
system and interacts physiologically with two types of
receptors, designated GABAA and GABAB. GABAA
receptors are ligand-activated Cl- channels, whereas
GABAB receptors are coupled to G proteins.1,2 Activation
of the GABAA receptor by the binding of GABA results
in an increase in the Cl- conductance of the neuronal
membrane, consequent membrane hyperpolarization,
and a reduction in neuronal excitability.3 The GABAA
receptor is a pentamer assembled from a large number
of potential subunits (R1-6, â1-3, γ1-3, δ, ꢀ, F1-3, π,
and θ).2,4 Most GABAA receptors are heterooligomers
that contain R, â, and γ subunits with a stoichiometry
of 2:1:2 or 2:2:1.2
In addition to the GABA-binding site, GABAA recep-
tors contain a large number of allosteric modulatory
sites for structurally unrelated compounds, including
benzodiazepines, barbiturates, steroids, avermectins,
picrotoxin, and loreclezole, that regulate receptor affin-
ity for GABA.5-8 Benzodiazepines exhibit a continuum
of intrinsic activity, ranging from full agonists (positive
allosteric modulators of GABA-evoked Cl- currents with
anxiolytic, hypnotic, and anticonvulsant activities)
through antagonists devoid of intrinsic activity to
inverse agonists (negative allosteric modulators of
GABA-evoked Cl- currents with proconvulsant, con-
vulsant, and anxiogenic activities).9
* To whom correspondence should be addressed. Phone: +39-079-
228-746. Fax: +39-079-228-727. E-mail: asproni@uniss.it.
† Universita` di Sassari.
‡ Universita` di Cagliari.
§ Consiglio Nazionale delle Ricerche.
10.1021/jm049120y CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/25/2005

Synthesis of Substituted 1,2-Diphenylimidazoles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2639
Chart 1
greater than those of anesthetics such as etomidate,
propofol, and alphaxalone.
Chemistry
Title compounds 1 (Table 1) were synthesized accord-
ing to the reaction pathways shown in Schemes 1-4.
The procedure for preparation of compounds 1b-f and
1h-n (Scheme 1) is the same as that previously
described17 for compounds 1a,g (Table 1). In brief, the
carbinol intermediates 3 were synthesized from ap-
propriate N-phenylbenzamidines 2 with ethylbromopy-
ruvate in the presence of NaHCO3 and were then
dehydrated in the presence of p-toluenesulfonic acid to
give the corresponding esters 1 in almost quantitative
yields. The alkylation-cyclization reaction yields either
only carbinol (3b,e,f) or a mixture of carbinol intermedi-
ate and target imidazole (3h-n and 1h-n, respec-
tively). In the case of 1c and 1d, no trace of carbinol
was detected and the reaction proceeded directly to the
desired imidazole. The amidines 2 were synthesized in
satisfactory yields from appropriate benzonitrile and
aniline compounds in toluene via trimethylaluminum
amide generated in situ.
The nitro-substituted compound 1m (Scheme 2) was
reduced by SnCl2-HCl to aniline derivative 1o, which
was converted either to iodide 1p by reaction of its
diazonium salt with KI or to amide 1q by treatment
with acetic anhydride in tetrahydrofuran (THF).
The 1,2-diphenylimidazoles containing H (1r) or CH3
(1s) at position 4 of the imidazole ring (Table 1) were
synthesized in a three-step process (Scheme 3) from
commercially available 2-phenylimidazole or 2-phenyl-
4-methylimidazole. Aromatic nucleophilic substitution
of 4-fluoronitrobenzene with 2-phenylimidazoles in
as the basis for development of a new series of more
specific allosteric modulators of the GABAA receptor.
Ethyl 1,2-diphenyl-1H-4-imidazolecarboxylate (1a),
an intermediate in our synthesis of I, was first selected
as a potential template for this investigation. We now
show that 1a and several analogues are effective posi-
tive allosteric modulators of human recombinant GABAA
receptors with potencies and efficacies similar to or
Table 1. Potency (EC50) and Maximal Efficacy of 1a-x, 8, Propofol, Etomidate, and Alphaxalone for Modulation of GABA-Evoked Cl-
Currents in Xenopus Oocytes Expressing Recombinant Human GABAA Receptorsa
EC50
(µM)
maximal
efficacy (%)
compd
R
X
X′
1a
1b
1c
1d
1e
1f
1g
1h
1i
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
H
H
H
H
H
H
6.4 ( 1.9
4.2 ( 1.1
4.1 ( 1.1
9.1 ( 1.1
3.0 ( 1.2
1.5 ( 1.1
3.1 ( 1.2
2.3 ( 1.7
0.6 ( 1.3
1.8 ( 1.7
1.0 ( 1.6
1.9 ( 1.5
2.4 ( 1.0
0.19 ( 2.1
4.3 ( 1.1
0.45 ( 1.8
19 ( 1.5
ND
638 ( 7
Cl
458 ( 20
625 ( 12
369 ( 25
350 ( 27
302 ( 9
CH3
OCH3
H
3-Cl
Cl
4-Cl
4-F
F
569 ( 38
521 ( 33
390 ( 22
508 ( 32
568 ( 42
362 ( 27
413 ( 32
388 ( 57
406 ( 19
180 ( 37
209 ( 41
-5.3 ( 2
-25 ( 20
258 ( 33
537 ( 82
-14 ( 4
32.4 ( 2
58.2 ( 12.6
256 ( 25
461 ( 39
501 ( 60
322 ( 38
214.5 ( 25
Cl
3,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
H
Cl
1j
1k
1l
H
CH3
OCH3
1m
1n
1o
1p
1q
1r
1s
1t
1u
1v
1w
1x
8
NO2
Br
NH2
I
NHCOCH3
H
H
CH3
H
ND
COOCH3
COOC3H7
COOCH2CH2N(CH2)5
CON(C2H5)2
CON(CH2CH2)2NCH3
COOH
Br
Br
Br
Cl
Cl
Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
3.4 (1.6
1.7 ( 2
ND
4.4 ( 2.8
1.3 ( 2.2
8.7 ( 2.5
11.2 ( 1.2
3.5 ( 1.1
2.0 ( 1.5
50.0 ( 1.1
propofol
etomidate
alphaxalone
pentobarbital
a See Chart 1 for the structure of compounds 1. ND ) not determinable.

2640 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Asproni et al.
Scheme 1a
a Reagents: (i) Al(CH3)3, toluene; (ii) BrCH2COCOOC2H5, NaHCO3, iPrOH; (iii) p-TsOH, toluene.
Scheme 2a
Scheme 4a
a Reagents: (i) SnCl2‚2H2O, concentrated HCl, CH3OH; (ii)
NaNO2, HCl, KI; (iii) acetic anhydride, THF.
Scheme 3a
a Reagents: (i) NaOH, H2O/CH3OH; (ii) (COCl)2/DMF, CH2Cl2,
TEA; secondary amine, nPrOH, or (CH2)4NCH2CH2OH.
gave aniline derivatives 6 and 7, which were allowed
to react with NaNO2 and H3PO2 to give compounds 1r
and 1s.
The esters 1i and 1n were hydrolyzed, and the
resulting acids 8 and 9 (Scheme 4) were converted to
the corresponding acid chlorides, which were then
reacted with appropriate alcohols or secondary amines
to give, respectively, esters 1u and 1v and carboxamides
1w and 1x. Analogue 1t (Table 1), which was considered
useful for SAR analysis, was obtained by treatment of
1v with CHCl3-CH3OH. This result can be easily
explained assuming a transesterification reaction of
1v.18
a Reagents: (i) K2CO3, DMF; (ii) SnCl2‚2H2O, concentrated HCl,
CH3OH; (iii) NaNO2, HCl, H3PO2.
Results and Discussion
Electrophysiology with Xenopus Oocytes Ex-
pressing Human GABAA Receptors. We first used
electrophysiology to screen all compounds for the ability
K2CO3 afforded the nitro intermediates 4 and 5 in high
yields. Reduction of the nitro group with SnCl2-HCl

Synthesis of Substituted 1,2-Diphenylimidazoles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2641
to modulate GABA-evoked Cl- currents in X. laevis
oocytes expressing recombinant human GABAA recep-
tors (R2â2γ2L). Most of the 1,2-diphenylimidazole de-
rivatives enhanced the action of GABA at the EC10 (the
concentration of GABA that elicits a response that is
10% of the maximal effect observed with 10 mM GABA)
in a concentration-dependent manner, with EC50 values
ranging from 0.19 to 19 µM and maximal efficacies of
up to 638% (Table 1). In many instances, these values
were similar to or greater than those of the anesthetics
etomidate, propofol, and alphaxalone, which were in-
cluded in the screening for comparison purposes. The
parent compound (1a) thus manifested an efficacy
(638%) higher than those of all three of these anesthetics
and an EC50 (6.4 µM) most similar to that of etomidate.
For SAR studies, we introduced several substituents
at the phenyl groups attached to positions N-1 and C-2
of the imidazole ring of 1a, exploring the effects of
substituents such as halogens (F, Cl, Br, I), CH3, OCH3,
NO2, NH2, and NHCOCH3. Congeners 1b-d are para-
substituted derivatives at the C-2 phenyl ring, whereas
1e is an example of a meta substitution at the N-1
phenyl ring. Among these analogues, compounds 1b, 1c,
and 1e possessed a higher potency (EC50 of 3.0-4.2 µM)
than did 1a, whereas that of the p-methoxy congener
1d was reduced (EC50 of 9.1 µM). This finding is
indicative of a positive influence of substituent lipo-
philicity. The para-dichlorinated derivative 1f exhibited
a greater potency (EC50 of 1.5 µM) than did 1b, whereas
the difluorinated analogue 1g possessed a potency (EC50
of 3.1 µM) similar to those of 1b, 1c, and 1e. The
introduction into 1f of an additional chlorine atom in
the ortho position of the N-1 phenyl ring to give
analogue 1i yielded a further improvement in potency
(EC50 of 0.6 µM), whereas moving the same substituent
from the ortho to the meta position, to give positional
isomer 1h, resulted in a decrease in potency (EC50 of
2.3 µM). This result is indicative of a negative influence
of the substituent steric effect (influence of the sub-
stituent size).
Maintaining the 2,4-dichloro substitution pattern in
1i, we next investigated the para position of the C-2
phenyl ring, either by reintroduction of CH3 or OCH3
groups or by exploring new substituents such as NO2,
Br, NH2, I, and NHCOCH3 (1k-q). The potency of the
para-unsubstituted congener 1j (EC50 of 1.8 µM) was
reduced compared with that of 1i but was similar to that
of its positional isomer 1f. The presence of a methyl
group in 1k (EC50 of 1.0 µM) resulted in a slight
decrease in potency compared with that of 1i, whereas
the presence of a methoxy group in 1l (EC50 of 1.9 µM)
was greater tolerated than was that in 1d. The p-nitro
derivative 1m exhibited a reduced potency (EC50 of 2.4
µM), whereas the para-substituted bromine (1n) and
iodine (1p) compounds showed the highest potencies
among all derivatives tested in this study, with EC50
values of 0.19 and 0.45 µM, respectively. Introduction
of the hydrophilic and electron-donating amino group
(1o) resulted in a marked decrease in potency (EC50 of
4.3 µM), whereas the acetylamino derivative 1q pos-
sessed a lower potency (EC50 of 19 µM) than did all of
the derivatives 1a-p. Despite its hydrophilic character,
both the potency and efficacy of compound 1o were
similar to those of etomidate.
Table 2. Potency (EC50) of Compounds 1a, 1b, 1f, 1g, 1i, and
8 for Enhancement of [3H]GABA Binding to Rat Cerebrocortical
Membranesa
compd
EC50 (nM)
compd
EC50 (nM)
1a
1b
1f
12390 ( 87.3
1591 ( 62.8
684 ( 43.4
1g
1i
8
2087 ( 102.5
87.79 ( 8.9
ND
a ND ) not determinable.
To evaluate the importance of the ester function of
the parent compound (1a) for modulatory activity, we
synthesized the analogue 1r, which lacks the 4-COOC2H5
group, and the 4-CH3 derivative 1s. Neither of these
analogues enhanced GABA-evoked Cl- currents, al-
though a small inhibitory effect was observed. These
results suggest that the carbonyl oxygen might partici-
pate in a hydrogen-bond-mediated interaction that is
essential for positive modulatory activity. The carboxylic
acid derivative 8 retained the ability to enhance GABAA
receptor function. Although this enhancement by 8
occurred with a lower potency (EC50 of 8.7 µM) and
efficacy (256%) compared with those of other 1,2-
diphenylimidazole derivatives, these values were similar
to those of the intravenous anesthetics propofol and
alphaxalone.
Homologation of the carboxyethyl function in 1n (EC50
of 0.19 µM), to give compounds 1t (EC50 of 3.4 µM) and
1u (EC50 of 1.7 µM), revealed that the carboxyethyl
function is optimal in terms of potency. The introduction
into the ester function of 1n of a basic nitrogen to give
the amino ester 1v abolished positive modulatory activ-
ity at the GABAA receptor, likely because of an unfavor-
able effect of the protonated site (at physiological pH).
Isosteric replacement of the ester function in 1i by a
carboxamide group to give 1w was detrimental for
modulatory activity (efficacy of 32.4%), and a similar
effect was observed for the amino amide derivative 1x.
The lack of activity of the amide 1w when compared to
the ester 1i could be ascribed to conformational factors;
the amide might adopt an unfavorable binding confor-
mation due to the reduced freedom of OC-N bond
rotation. Negative steric effects due to the second ethyl
group could also be invoked to account for the difference
in activity.
Receptor-Binding Assays. Given that many of the
1,2-diphenylimidazole derivatives tested in our study
were potent positive modulators of GABA-evoked Cl-
currents mediated by recombinant human GABAA
receptors, we also assessed the ability of some of these
compounds to affect the binding of [3H]GABA to rat
cerebral cortical membranes (Table 2). All compounds
tested, with the exception of the carboxylic acid deriva-
tive 8, enhanced [3H]GABA binding, with EC50 values
ranging from 88 to 12390 nM. The potency of these
compounds for enhancement of [3H]GABA binding ap-
peared to correlate with that determined for potentia-
tion of GABA-evoked Cl- currents, suggesting that these
derivatives affect the function of GABAA receptors in
an allosteric manner through interaction with a site
distinct from the GABA-binding site.
In the present study, we have thus investigated a
series of 1,2-diphenylimidazole derivatives for the ability
to modulate GABA-evoked Cl- currents in Xenopus
oocytes expressing recombinant human GABAA recep-
tors, and we found that many of these compounds act

2642 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Asproni et al.
Figure 1. Concentration-response curves for the potentiation
by compound 1i, etomidate, and lorazepam of GABA-induced
Cl- currents in Xenopus oocytes expressing recombinant
human GABAA receptors. Data are expressed as percentage
potentiation of the response induced by GABA at the EC10
value and are means ( SEM of values from 8-12 oocytes from
two or more batches.
as positive allosteric modulators at these receptors. The
potency and efficacy for enhancement of GABAA recep-
tor function by several of the tested compounds were
higher than those of benzodiazepines such as lorazepam
as well as those of the general anesthetic etomidate
(Figure 1), which, among previously identified GABAA
receptor modulators, is most related structurally to our
1,2-diphenylimidazoles (Chart 1). The 1,2-diphenylimi-
dazole scaffold thus appears to be appropriate for the
development of potent and efficacious allosteric modula-
tors of GABAA receptor function. Our SAR analysis for
this class of compounds revealed that the presence of
the ester moiety in the imidazole core is required for
full agonist properties. Moreover, receptor modulatory
activity is greatly affected by the pattern of chemical
substitution on the phenyl rings at positions N-1 and
C-2 of the imidazole ring. Combination of the 2,4-
dichloro substitution pattern on the N-1 phenyl ring
with a p-bromophenyl group at C-2 (1n) gave the
highest potency among all derivatives tested.
In vitro radioligand displacement assays with rat
brain membranes revealed that the 1,2-diphenylimida-
zole derivatives do not bind to the GABA recognition
site but rather enhance GABAA receptor function in an
allosteric manner through interaction with a distinct
binding site. In addition, these compounds did not act
at the benzodiazepine-binding site. In fact, flumazenil
was not able to block the enhancement of the GABAA
receptor function induced by 1i.19 Electrophysiological
studies performed in X. laevis oocytes expressing
R1â1γ2L and R1â2γ2L have demonstrated that 1i, like
the general anesthetic etomidate, is selective for GABAA
receptors containing â2 subunits.19 In addition, muta-
tion of asparagine-265 of the â3 subunit of the GABAA
receptor to serine was previously shown to result in a
marked reduction in the extent of potentiation of the
GABA response by the general anesthetic etomidate.16
Given that the â subunit selectivity of etomidate is
determined by a single amino acid, asparagine 265 in
â2 and â3 and serine in â1, we mutated asparagine 265,
located in the transmembrane domain 2 (TM2),
Figure 2. Concentration dependence of the potentiation of
GABA-induced Cl- current by etomidate (A) or 1i (B) in
Xenopus oocytes expressing R1â1γ2L, R1â2γ2L, or R1â2-
(N265S)γ2L combinations of GABAA receptor subunits. Data
are expressed as percentage potentiation of the response to
GABA at the EC10 and are means ( SEM of values from 3-8
oocytes. A single asterisk indicates p < 0.05 and double
asterisks indicate p < 0.001 versus the corresponding values
for R1â1γ2L or R1â2(N265S)γ2L receptors.
nant R1â1γ2L, R1â2γ2L, or R1â2(N265S)γ2L GABAA
receptors in X. laevis oocytes. Mutation of asparagine-
265 of the GABAA receptor â2 subunits to serine greatly
reduced the potentiation of the GABAA receptor function
induced by 1i. This reduction was similar to the one
induced in mutated R1â2(N265S)γ2L GABAA receptors
by the general anesthetic etomidate (Figure 2) and
suggests that derivative 1i may bind to the same
binding site of etomidate.
Finally, compound 1i was chosen to assess the be-
havioral effects of the 1,2-diphenylimidazole derivatives.
This compound induced a reversible loss of the righting
reflex (an effect often used to evaluate anesthesia) in
Xenopus tadpoles with an EC50 of 1.2 µM (Figure 3).This
value of EC50 is comparable to the one previously found
for propofol (EC50 of 1.9 ( 0.2 µM)20 and for etomidate
(EC50 of 2.3 ( 0.13 µM).15 Again this result suggests
that 1i as well as other 1,2-diphenylimidazole deriva-
tives may constitute a new class of positive modulators
of GABAA receptors with a molecular mechanism com-
parable to that of etomidate.
Etomidate is one of the most potent general anesthet-
ics employed in clinical practice. The R-(+) enantiomer,
which is the one used in the clinical formulation,15 is
an order of magnitude more potent than the S-(-)
enantiomer. This enantioselectivity affords a valuable
tool to assess the fundamental mechanisms underlying
of the â2 subunit to serine, and we expressed recombi-

Synthesis of Substituted 1,2-Diphenylimidazoles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2643
7.67 (s, 1H, CH-5). API-ES: m/z 376.0 (MH+). Anal. (C18H15-
Cl2N3O2) C, H, N.
Ethyl 1-(2,4-Dichlorophenyl)-2-(4-iodophenyl)-1H-4-
imidazolecarboxylate (1p). To a cold solution (0 °C) of amine
1o (0.3 g, 0.79 mmol) in concentrated HCl (4 mL) and ice (8 g)
was added dropwise a 1.0 M solution of NaNO2 (797 µL, 0,79
mmol). The resulting yellow solution was further stirred at
the same temperature for 30 min and then transferred into a
solution of KI (0.44 g, 7.95 mmol) in water (4 mL). The mixture
was stirred at room temperature for 5 h and then extracted
with CH2Cl2. The combined organic extracts were dried and
evaporated in vacuo to leave a residue which was purified by
flash silica gel chromatography eluting with AcOEt/petroleum
ether (30:70 and 50:50) to give 1p (0.29 g, 74%) as a white
solid. Mp: 197 °C (CHCl3/CH3OH). TLC (AcOEt/petroleum
Figure 3. Anesthetic action of compound 1i in Xenopus
tadpoles. The anesthetic effect of compound 1i (0.01-30 µM)
was assessed from the percentage of tadpoles (n ) 10) that
manifested loss of the righting reflex (LORR) at each drug
concentration.
1
ether, 4:6): Rf ) 0.73. IR (KBr): 1710, 1586 cm-1. H NMR
(CDCl3): δ 1.41 (t, J ) 8.0 Hz, 3H, CH3), 4.43 (q, J ) 8.0 Hz,
2H, OCH2), 7.12 (d, J ) 8.8 Hz, 2H, ArH), 7.24 (d, J ) 9.4 Hz,
1H, ArH), 7.38 (d, J ) 8.6 Hz, 1H, ArH), 7.56 (s, 1H, ArH),
7.60 (d, J ) 8.6 Hz, 2H, ArH), 7.72 (s, 1H, CH-5). API-ES:
m/z 486.9 (MH+). Anal. (C18H13Cl2IN2O2) C, H, N.
anesthesia. However, from a chemical and pharmaco-
logical point of view, the development of a single
enantiomer is more difficult since it is necessary to
prevent possible racemization or it requires extensive
pharmacological research.21 A distinct advantage of the
new 1,2-diphenylimidazoles described in this paper is
that they do not possess any chiral center, and this
feature could facilitate their development.
Ethyl
2-[4-(Acetylamino)phenyl]-1-(2,4-dichloro-
phenyl)-1H-4-imidazolecarboxylate (1q). To a solution of
amine 1o (0.08 g, 0.21 mmol) in anhydrous THF (2 mL) was
added at room temperature acetic anhydride (60 µL, 0.63
mmol). The resulting solution was stirred at the same tem-
perature for 30 min and then evaporated in vacuo to give 1q
(65 mg, 75%) as a white solid. Mp: 233-234 °C (CHCl3/CH3-
OH). TLC (AcOEt/petroleum ether, 8:2): Rf ) 0.27. IR (KBr):
3289, 1736, 1684, 1591 cm-1. 1H NMR (CDCl3): δ 1.39 (t, J )
7.2 Hz, 3H, CH3), 2.12 (s, 3H, CH3), 4.39 (q, J ) 7.2 Hz, 2H,
OCH2), 7.25-7.56 (m, 7H, ArH), 7.72 (s, 1H, CH-5), 9.41 (s,
1H, NH, D2O exchanged). API-ES: m/z 418.1 (MH+). Anal.
(C20H17Cl2N3O3) C, H, N.
Experimental Section
Chemistry. Reactions were monitored by TLC with Poly-
gram SIL and ALOX N/UV254 precoated plastic sheets (0.2
mm), with detection by exposure to iodine vapor or UV light.
Pure compounds yielded a single spot on TLC. Flash chroma-
tography was performed with Merck silica gel type 60 (size
230-240 mesh ASTM). Melting points were determined with
a Kofler hot-stage microscope and are uncorrected. Infrared
spectra were recorded with a Perkin-Elmer Paragon 500 FT
IR spectrophotometer (KBr pellets for solid samples). 1H NMR
spectra were recorded with the use of a Varian XL 200 FT
NMR spectrometer, with CDCl3 as solvent and tetramethyl-
silane as internal standard. Chemical shifts are expressed in
δ (ppm) downfield from tetramethylsilane, and coupling
constants (J) are expressed in hertz. Mass spectroscopy was
performed with an Agilent 1100 series LC/MSD spectrometer.
Measurements were performed in the positive or negative ion
mode [(MH+) or (M - H)-], with an atmospheric pressure
ionization electrospray (API-ES). Electron ionization mass
spectra (70 eV) were recorded with a Hewlett-Packard 5790-
5970 MSD gas chromatograph-mass spectrometer. Elemental
analyses were performed with a Perkin-Elmer 2400 analyzer,
and results were within (0.40% of the theoretical values. All
moisture-sensitive reactions were performed under a nitrogen
atmosphere, with the use of oven-dried glassware and syringes
to transfer solutions. Organic extracts were dried over anhy-
drous MgSO4 prior to solvent evaporation. All starting materi-
als and reagents were obtained from Aldrich.
1-(4-Nitrophenyl)-2-phenyl-1H-imidazole (4). To a solu-
tion of 2-phenylimidazole (0.5 g, 3.46 mmol) in anhydrous DMF
(28 mL) was added K2CO3 (0.96 g). After 15 min of stirring at
room temperature, 4-fluoronitrobenzene (0.61 g, 4.32 mmol)
was added. The resulting mixture was heated at 130 °C for 5
h, cooled to room temperature, and poured onto ice. The
mixture was extracted with AcOEt, and the combined extracts
were dried and evaporated in vacuo. The residue was subjected
to flash silica gel chromatography eluting with AcOEt/hexane
(60:40) to give 4 (0.76 g, 84%) as a yellow solid. Mp: 127 °C
(triturated with Et2O). TLC (AcOEt/petroleum ether, 1:1): Rf
) 0.37. IR (KBr): 1525, 1345 cm-1. 1H NMR (CDCl3): δ 7.23-
7.40 (m, 9H, ArH), 8.26 (d, J ) 9.0 Hz, 2H, ArH). MS: m/z
265.0 (M+, base). Anal. (C15H11N3O2) C, H, N.
4-(2-Phenyl-1H-1-imidazolyl)aniline (6). Compound 6
was prepared following the procedure reported for compound
1o. Yield: 91%. Mp: 178-200 °C (triturated with Et2O). TLC
(AcOEt/petroleum ether, 1:1): Rf ) 0.27. IR (KBr): 3481, 3443,
1
1643 cm-1. H NMR (CDCl3): δ 4.90 (br s, 2H, ArNH2, D2O
exchanged), 6.62-6.66 (m, 2H, ArH), 6.89-6.93 (m, 2H, ArH),
7.12-7.41 (m, 7H, ArH). MS: m/z 235.0 (M+, base). Anal.
(C15H13N3) C, H, N.
1,2-Diphenyl-1H-imidazole (1r).22 To a cold solution (-10
°C) of amine 6 (0.35 g, 1.49 mmol) in concentrated HCl (10
mL) was added NaNO2 (0.14 g, 2.0 mmol). The resulting yellow
solution was further stirred at the same temperature for 1 h
and then added dropwise to 50% H3PO2 (10 mL); stirring was
continued for 5 h. The solution was basified with 20% NaOH
and then extracted with AcOEt. The combined organic extracts
were dried and evaporated in vacuo to leave an oily residue
which was purified by flash silica gel chromatography eluting
with AcOEt/petroleum ether (70:30) to give 1r (0.21 g, 65%)
as a white solid. Mp: 85-86 °C (iPr2O). TLC (AcOEt/petroleum
Synthesis of Ethyl 1,2-Diphenyl-1H-4-imidazolecar-
boxylates 1. Ethyl 1,2-diphenyl-1H-4-imidazolecarboxylates
1b-f,h-n (Scheme 1) were synthesized according to the
procedure applied to compounds 1a,g.17
Ethyl 2-(4-Aminophenyl)-1-(2,4-dichlorophenyl)-1H-4-
imidazolecarboxylate (1o). To a solution of 1m (0.8 g, 1.97
mmol) in CH3OH (12 mL) and concentrated HCl (12 mL) was
added at 0 °C SnCl2‚2H2O (2.22 g, 9.75 mmol), and the reaction
mixture was stirred for 2 h. The reaction mixture was basified
(to pH 14) with a 3 N aqueous solution of NaOH and extracted
with AcOEt. The combined extracts were dried and evaporated
to give 1o (0.73 g, 98%) as a white powder. Mp: 217-219 °C
(CHCl3/CH3OH). TLC (AcOEt/petroleum ether, 8:2): Rf ) 0.55.
1
ether, 1:1): Rf ) 0.54. IR (KBr): 3095, 1596, 1498 cm-1. H
NMR (CDCl3): δ 7.16-7.41 (m, 12H, ArH). MS m/z 220.0 (M+,
88), 219.0 (base). Anal. (C15H12N2) C, H, N.
IR (KBr): 3424, 3300, 3206, 1679, 1585 cm-1 1H NMR
.
(CDCl3): δ 1.40 (t, J ) 7.2 Hz, 3H, CH3), 3.81 (br s, 2H, ArNH2,
D2O exchanged), 4.42 (q, J ) 7.2 Hz, 2H, OCH2), 6.52 (d, J )
8.4 Hz, 2H, ArH), 7.15-7.34 (m, 4H, ArH), 7.54 (s, 1H, ArH),
Compounds 5, 7, and 1s were prepared following the
procedures reported for compounds 4, 6, and 1r, respectively.
2-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-1H-4-imida-
zolecarboxylic Acid (8). A mixture of ester 1i (1.15 g, 2.90

2644 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Asproni et al.
mmol) in CH3OH (20 mL) and NaOH (1 N) (20 mL) was
refluxed for 1.5 h, cooled, and acidified with concentrated HCl.
The precipitate was filtered, washed with water, and dried to
give 8 as a white solid (0.9 g, 85%). Mp: 238-239 °C (CH3-
OH). TLC (CHCl3/CH3OH, 8:2): Rf ) 0.48. IR (KBr): 1714,
R1, â2, and γ2L; or R1, â2(N265S), and γ2L receptor subunits
(total of 1.5 ng of cDNA in 30 nL in a 1:1:1 ratio) was injected
into the animal pole of oocytes with the use of a microdispenser
(Drummond Scientific, Broomwall, PA). The injected oocytes
were maintained at 15 °C in sterile modified Barth’s solution
[MBS; 88 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.82 mM
MgSO4, 2.4 mM NaHCO3, 0.91 mM CaCl2, and 0.33 mM Ca-
(NO3)2, adjusted to pH 7.5] supplemented with streptomycin
(10 mg/L), penicillin (10000 U/L), gentamicin (50 mg/L),
theophylline (90 mg/L), and pyruvate (220 mg/L).
1562 cm-1 1H NMR (CDCl3): δ 4.83 (br s, 1H, OH, D2O
.
exchanged), 7.27-7.57 (m, 7H, ArH), 7.74 (s, 1H, CH-5). API-
ES: m/z 367.0 (MH+). Anal. (C16H9Cl3N2O2) C, H, N.
Compound 9 was obtained following the procedure reported
for compound 8.
N4,N4-diethyl-2-(4-chlorophenyl)-1-(2,4-dichloro-
phenyl)-1H-4-imidazolecarboxamide (1w). To a solution
of acid 8 (0.15 g, 0.41 mmol) in CH2Cl2 (15 mL) was added
two drops of DMF. Oxalyl chloride (0.11 mL, 1.23 mmol) was
then added and the solution stirred at room temperature for
30 min and then evaporated in vacuo. The acid chloride was
solubilized in CH2Cl2 (15 mL) and cooled at 0 °C (ice bath). At
this temperature triethylamine (0.060 mL, 0.43 mmol) and
diethylamine (0.037 mL, 0.36 mmol) were added. The reaction
mixture was stirred at room temperature for 30 min and then
washed with 5% aqueous NaHCO3. The dried organic layer,
evaporated in vacuo, gave a crude residue which was purified
by flash silica gel chromatography using AcOEt/petroleum
ether (8:2) as eluent to give 1w (0.087 g, 51%) as a white solid.
Mp: 289-290 °C (CHCl3/CH3OH). TLC (AcOEt/petroleum
Electrophysiology. Electrophysiological measurements
were performed 1-4 days after injection of oocytes with
expression plasmids. The oocytes were placed individually in
a rectangular chamber (volume ∼100 mL) and perifused (1.7
mL/min) with MBS through 18-gauge polyethylene tubing
(Clay Adams, Parsippany, NJ) with the use of a roller pump
(Cole-Parmer, Chicago, IL). Oocytes were impaled at the
animal pole with two glass electrodes (0.5-10 MΩ) filled with
3 M KCl and were clamped at -70 mV with an Axo-Clamp
2A amplifier (Axon Instruments, Union City, CA). Currents
were continuously plotted with a chart recorder (Cole-Parmer,
Vernon Hills, IL). GABA (Sigma, St. Louis, MO) was dissolved
in MBS and applied for 30 s. Test compounds were dissolved
in dimethyl sulfoxide (DMSO) at 100 mM and then diluted in
MBS to a final DMSO concentration not exceeding 0.1%
(DMSO at this concentration had no effect on Cl- current).
Drugs were applied for 60 s either in the absence of GABA or
in its presence at the EC10. For each experiment, control
responses were determined before and 10 min after application
of test agents.
Assay of [3H]GABA Binding. Male Sprague-Dawley rats
with body masses of 200-225 g were killed by decapitation,
and their brains were rapidly removed. The fresh cortical
tissue was homogenized with a Polytron PT 10 disrupter for
30 s in 10 volumes of ice-cold water, and the homogenate was
centrifuged at 48000g for 10 min at 0 °C. The resulting pellet
was washed once by resuspension and centrifugation in the
original volume of a solution containing 20 mM potassium
phosphate buffer (pH 7.4) and 50 mM KCl. The membrane
pellet was then stored at -20 °C until binding analysis (1-5
days later). On the day of the assay, the membranes were
thawed and washed a total of four times by resuspension and
centrifugation in the same ice-cold solution.
For the [3H]GABA-binding assay, the membranes were
resuspended in 50 volumes of the same solution, and 300 µL
of membrane suspension (∼300 µg of protein) was added to
plastic minivials containing the drugs to be tested. The assay
was performed in a final volume of 500 µL, started by the
addition of [3H]GABA (specific activity 80-100 Ci/mmol,
Perkin-Elmer, Boston, MA) to a final concentration of 10 nM,
and stopped after incubation for 10 min at 0 °C by centrifuga-
tion at 48000g for 10 min at 0 °C. The pellet was washed with
4 mL of ice-cold water and then dissolved in 3 mL of
scintillation fluid. The membrane-associated radioactivity was
determined with a scintillation counter (TRI-CARB 2100TR,
Packard). Drug stock solutions (20 mM) were prepared in
DMSO; the concentration of DMSO in the binding reaction
mixture did not exceed 0.2% and did not affect [3H]GABA
binding. Binding data were corrected for nonspecific binding,
which was determined in the presence of 1 mM GABA.
Behavioral Effects in Tadpoles. X. laevis tadpoles (43-
50 days old) were maintained in an aquarium at 20-22 °C.
For determination of loss of the righting reflex, each of 10
tadpoles was placed in separate beakers containing 300 mL
of tap water with or without 1i (0.01-30 µM). With the
exception of the tap water control, all beakers contained DMSO
at 0.1%, a concentration that did not affect animal behavior.
Anesthesia was defined as the absence of a purposeful and
sustained swimming response after inversion of the tadpole
with a smooth glass rod. The number of anesthetized tadpoles
was recorded every 10 min for up to 120 min, after which the
tadpoles were returned to fresh tap water. Normal swimming
activity was restored within 30 min.
1
ether, 1:1): Rf ) 0.60. IR (KBr): 1592, 1570 cm-1. H NMR
(CDCl3): δ 1.25-1.32 (m, 6H, 2 × CH3), 3.45-3.60 (m, 2H,
CH2), 4.02-4.12 (m, 2H, CH2), 7.22-7.39 (m, 6H, ArH), 7.55
(s, 1H, ArH), 7.62 (s, 1H, CH-5). API-ES: m/z 422.1 (MH+).
Anal. (C20H18Cl3N3O) C, H, N.
[2-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-1H-4-imi-
dazolyl](4-methylpiperazino]methanone (1x). Compound
1x was obtained following the above procedure in the absence
of triethylamine. The crude product was purified by recrys-
tallization. Yield: 86%. Mp: 269-270 (CHCl3/CH3OH). TLC
(CHCl3/CH3OH, 9:1): Rf ) 0.57. IR (KBr): 1593, 1534 cm-1
.
1H NMR (CDCl3): δ 2.35 (s, 3H, CH3), 2.49-2.59 (m, 4H, 2 ×
CH2), 3.70-3.90 (m, 2H, CH2), 4.31-4.50 (m, 2H, CH2), 7.24-
7.39 (m, 6H, ArH), 7.56 (s, 1H, ArH), 7.65 (s, 1H, CH-5). API-
ES: m/z 449.0 (MH+). Anal. (C21H19Cl3N4O) C, H, N.
Propyl 2-(4-Bromophenyl)-1-(2,4-dichlorophenyl)-1H-
4-imidazolecarboxylate (1u). Compound 1u was synthe-
sized using the procedure applied to compound 1w except that
1-propanol was used instead of diethylamine. The reaction
mixture was stirred at room temperature for 6 h. The crude
product was purified by flash silica gel chromatography using
AcOEt/petroleum ether (7:3) as eluent to give 1u as a white
solid. Yield: 40%. Mp: 142-143 °C (iPr2O/CH2Cl2). TLC
(AcOEt/petroleum ether, 3:7): Rf ) 0.55. IR (KBr): 1685, 1588
1
cm-1. H NMR (CDCl3): δ 1.01 (t, J ) 7.6 Hz, 3H, CH3), 1.80
(m, 2H, CH2), 4.32 (t, J ) 6.8 Hz, 2H, CH2O), 7.22-7.43 (m,
6H, ArH), 7.55 (s, 1H, ArH), 7.71 (s, 1H, CH-5). MS: m/z 454
(M+, 57), 368 (base). Anal. (C19H15BrCl2N2O2) C, H, N.
2-Piperidinoethyl 2-(4-Bromophenyl)-1-(2,4-dichlo-
rophenyl)-1H-4-imidazolecarboxylate (1v). Compound 1v
was synthesized using the procedure applied to compound 1w
except that 1-piperidine ethanol was used instead of dieth-
ylamine. The crude product was purified by recrystallization.
Yield: 77%. Mp: 142-145 °C (iPr2O/CH2Cl2). TLC (CHCl3/
CH3OH, 9:1): Rf ) 0.46. IR (KBr): 1709, 1550 cm-1. 1H NMR
(CDCl3): δ 1.44-1.47 (m, 2H, CH2), 1.61-1.65 (m, 4H, 2 ×
CH2), 2.51-2.54 (m, 4H, 2 × CH2), 2.78 (t, J ) 6.0 Hz, 2H,
CH2N), 4.49 (t, J ) 6.0 Hz, 2H, CH2O), 7.22-7.43 (m, 6H,
ArH), 7.56 (s, 1H, ArH), 7.74 (s, 1H, CH-5). API-ES: m/z 523.8
(MH+). Anal. (C23H22BrCl2N3O2) C, H, N.
Mutagenesis and Expression of GABAA Receptor Sub-
units. Site-directed mutagenesis (asparagine-265 to serine,
N265S) of the â2 subunit of the human GABAA receptor was
performed with the corresponding cDNA subcloned into pCDM8
and a Quik-Change site-directed mutagenesis kit (Stratagene,
La Jolla, CA). The mutation was verified by partial sequencing
of the sense and antisense DNA strands.
Expression of Human GABAA Receptors in Xenopus
oocytes. A mixture of plasmids encoding the R1, â1, and γ2L;

Synthesis of Substituted 1,2-Diphenylimidazoles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2645
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and Volatile Anaesthetic Action on GABAA and Glycine Recep-
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Acknowledgment. This work was supported by a
grant from the Ministero dell’Universita` e della Ricerca
Scientifica e Tecnologica. We thank Mr. Domenico
Serra, Mrs. Paola Manconi, and Maria Orecchioni for
their technical assistance in recording mass and 1H
NMR spectra.
Supporting Information Available: Complete physical
and spectral data for new compounds 1b-f,h-n,s,t and
intermediates 2b-f,h-n, 3b,e,f,h-n, 5, 7, and 9 and elemen-
tal analysis (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
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