Search for α3β2/3γ2 subtype selective ligands that are stable on human liver microsomes

Article abstract of DOI:10.1016/j.bmc.2012.10.057

Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABA_A) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β_(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100 mg/kg and was devoid of sedative properties.

Full text of DOI:10.1016/j.bmc.2012.10.057

Bioorganic & Medicinal Chemistry 21 (2013) 93–101
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Search for
a3b2/3
c2 subtype selective ligands that are stable on human
liver microsomes
Ojas A. Namjoshi a, Zhi-jian Wang a, Sundari K. Rallapalli a, Edward Merle Johnson Jr. a, Yun-Teng Johnson a,
Hanna Ng b, Joachim Ramerstorfer c, Zdravko Varagic c, Werner Sieghart c, Samarpan Majumder d,
Bryan L. Roth d, James K. Rowlett e, James M. Cook a,
⇑
a Department of Chemistry, University of Wisconsin-Milwaukee, PO Box 413, Milwaukee, WI 53211, USA
b Biosciences Division, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA
c Department of Biochemistry & Molecular Biology, Center for Brain Research, Medical University Vienna, A-1090 Vienna, Austria
d Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
e Harvard Medical School, New England Primate Research Center, One Pine Hill Drive, PO Box 9102, Southborough, MA 01772, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABAA)
receptor ion channels has been identified as an important method for separating out the variety of phar-
Received 31 August 2012
Revised 22 October 2012
Accepted 31 October 2012
Available online 15 November 2012
macological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both
a2b(2/3)
c
2 ( 2BzR) and 3BzR (and/or 2/ 3) BzR subtype selective ligands exhibit anxiolytic effects with little
a
a
a a
or no sedation. Previously we have identified several such ligands; however, three of our parent ligands
exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs
are reported which were designed to circumvent this liability by utilizing a rational replacement of the
ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver
microsomes, four compounds were found to undergo slower metabolic transformation, as compared to
their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Addi-
tionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at
doses of 10 and 100 mg/kg and was devoid of sedative properties.
Keywords:
GABA
Benzodiazepines
Bioisostere
Anxiolytic
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The majority of GABAA receptors is composed of 1
subunits. The classical benzodiazepines, such as diazepam or fluni-
trazepam, bind at the extracellular domain of the 2-interface of
these receptors8 and exhibit a high affinity for receptors composed
of 1b(2/3) 2, 2b(2/3) 2, 3b(2/3) 2 or 5b(2/3) 2 subunits (diaze-
pam sensitive receptors). Different receptor subtypes reside within
anatomically distinct regions of the brain and are responsible for
different physiological and pathological processes.9,10 Data from
c, 2a, and 2b
Gamma amino butyric acid-A (GABAA) receptors are the major
inhibitory neurotransmitter receptors of the central nervous sys-
tem (CNS) and the site of action of a variety of pharmacologically
and clinically important drugs. Thus, benzodiazepines, barbitu-
rates, neuroactive steroids, anesthetics and convulsants exert their
action by modulating the function of these receptors.1 From the ac-
tion of these drugs it is clear that GABAA receptors regulate numer-
ous neurological functions including convulsions, anxiety,
sedation, ataxia and sleep activity, as well as memory and learning
processes.2–5 GABAA receptors are composed of 5 subunits that
form a central chloride channel and can belong to different subunit
a+c
a
c
a
c
a
c
a
c
knock in mice indicate that the
the sedative, anticonvulsant, ataxic effects, anterograde amnesia
and abuse liability,11,12 while the 313,14 receptor subtypes
2 and
a1 containing receptors mediate
a
a
mediate anxiolytic activity.15–17 The
a5 containing GABAA receptors
have been implicated in memory and learning processes,18,19 but
seem neither to influence anxiolysis nor motor effects,9,19 although
classes. A total of 19 subunits (6a, 3b, 3c, 1d, 1e, 1p, 1h, 3q) of the
GABAA receptor have been cloned and sequenced from the mam-
malian nervous system.6,7 The homology within each subunit class
is about 60–80%, while the homology between the subunit classes
is about 30–40%.
some reports of a decrease in locomoter effects due to
type specific ligands have also been reported.20 It has been shown
that a substantial amount of agonist activity at the 1 BzR is re-
quired for effects on spatial learning and memory impairments,
a5 BzR sub-
a
while much weaker, but simultaneous activity at
a1 and a5 to-
gether is sufficient for eliciting sedation.21 Tolerance to some of
the effects of benzodiazepines, most notably the anticonvulsant
⇑
Corresponding author.
E-mail address: capncook@uwm.edu (J.M. Cook).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.057

94
O. A. Namjoshi et al. / Bioorg. Med. Chem. 21 (2013) 93–101
and analgesic actions are mediated by the simultaneous action of
1 and
5 BzR.22,23
Agents selective for specific BzR subtypes should permit one to
a
a
separate out the pharmacological activities of these different iso-
forms and reduce the chances of tolerance and abuse poten-
tial.12,23–27 The GABAA/
a1-selective positive allosteric modulators
alpidem and zolpidem are clinically prescribed as hypnotic agents
suggesting that at least much of the sedation associated with
known anxiolytic drugs which act at the BzR binding site is medi-
ated through GABAA receptors containing the
tioned. Consequently, novel benzodiazepine-like drugs that have
pharmacological selectivity for 2GABAA and/or 3GABAA recep-
tors and low receptor efficacy at 1GABAA and 5GABAA receptors
a1 subunit, as men-
a
a
a
a
may be particularly useful as anxiolytics lacking sedative, ataxic,
and amnestic side effects as well as little or no abuse potential.11,12
Recently, with the help of GABAA receptor point mutated mice,
Figure 2. Pharmacokinetically-based bioisosteric analogs of the lead compounds
1–3 to increase stability.
it was also demonstrated that
largely responsible for the spinal antihyperalgesic actions of classi-
cal benzodiazepines, while
1 receptors do not contribute.28 Inter-
estingly, in 1-GABAA receptor point-mutated mice, which are
protected from the sedative effects of diazepam, pronounced anal-
gesia against formalin-induced pain has also been reported after
systemic treatment with diazepam.29 These results suggest that
a2 and/or a3 receptor subtypes are
In another study in mouse models of neuropathic and inflam-
a
matory pain,
a2/a3 ligand 2 exhibited a dose-dependent antihy-
a
peralgesic effect and was as efficacious as gabapentin.31 This
antihyperalgesic activity was antagonized by flumazenil and hence
mediated via the benzodiazepine-binding site of GABAA receptors.
At doses producing maximal antihyperalgesia, ligand 2 was devoid
of sedation and motor impairment, and showed no loss of analgesic
activity during a 9-day chronic treatment period (i.e., no tolerance
development) versus acute treatment.23
a
1 sparing (non-sedative) benzodiazepine-site agonists should ex-
ert a genuine analgesic effect after systemic treatment.29
Through the iterative process of computational modeling and
synthesis, benzodiazepine derivatives, XHe-II-053 (1), HZ-166 (2),
and JY-XHe-053 (3) have been synthesized in Milwaukee (Fig. 1).
All three compounds 1–3 were exceptionally selective (hence non-
In preclinical studies 1 was shown to be safe and effective (anx-
iolytic but not sedating, not ataxic, and had reduced abuse poten-
tial in rodents and was anxiolytic in non-human primate
models).30 Because of its encouraging results in pre-clinical testing,
1 had been taken into Phase I studies in humans for anxiety disor-
ders (Bristol-Myers Squibb, unpublished results of Phase I trials).
Ligand 1 was found to be safe in humans. It was found to be stable
in human blood, plasma, brain and kidney (Mithridion, unpub-
lished results). However, in human liver, ligand 1 was largely
transformed into the inactive metabolite XHe-II-053-acid (4) via
hepatic enzymes, resulting in sub-optimal pharmacokinetics
(unpublished results). The related analog mining was an effort to
circumvent the metabolic liability of these compounds in human
liver microsomes versus the control anxiolytic 1, in an effort to de-
velop nonsedating anxiolytics with longer half lives in humans.
sedating) GABAA
a2/a3 agonists in in vitro electrophysiological
experiments (Fig. 2).21,30 In a rhesus monkey conflict procedure,
over the dose range tested (0.1–10 mg/kg), 1 and 2 produced an
anti-conflict effect without producing diazepam-like alterations
in the absence of response-contingent electric shock (non-
suppressed responding). Both the compounds were anxiolytic,
but lacked sedative, ataxic, muscle relaxant and amnestic side ef-
fects.30 The 20-F ligand 3 also produced a robust anti-conflict effect;
however, it also produced some reduction in response rates at the
highest dose tested. This was, presumably, due to its greater effi-
cacy at GABAA
clearly illustrating the subtle differences that structures have on
efficacy of ligands at GABAA 1 BzR.
a1 receptors as compared to a2 or a3 BzR ligands,
a
Figure 1. Structures of imidazobenzodiazepine-related ligands 1–5.

O. A. Namjoshi et al. / Bioorg. Med. Chem. 21 (2013) 93–101
95
Table 1
project funded by the National Institute of Mental Health (NIMH).
In this study the test compounds were incubated with pooled hu-
man liver microsomes and the aliquots were analyzed at various
time points using LC–MS/MS. Illustrated in Table 1 are the results
from this study. Significant metabolic lability was observed at both
In vitro metabolic stability of 1–5 using human liver microsomes by SRI International
Test article
Time (min)
Mean % remaining versus T = 0 mina
Mb Mb
10
1
l
l
15
30
60
41.4
11.2
1.5
47.6
13.9
1.7
1 and 10 lM for 1 (less than 14% remaining at 30 min) and 3 (less
1
than 20% remaining at 15 min), while the key ligand 2 underwent
minimal metabolism (80% remaining at 60 min). The correspond-
ing carboxylic acid controls of 1 and 2, that is, acids 4 and SR-II-
54 (5), respectively did not exhibit any metabolism during the test
period. Additionally, all the compounds when incubated with heat-
inactivated human liver microsomes underwent no significant
change in the % remaining of any of the compounds, suggesting
that the compounds were stable under the control incubation con-
ditions. These results indicated that the main site of metabolism in
human liver was due to the pendant ethyl ester moiety, which was
not unexpected but designed as a method of clearance (metabolic
switching).
1 with HIc microsomes 60
107.4
106.0
95.6
80.2
93.8
101.8
103.4
90.4
76.1
95.4
20.0
3.6
15
30
60
2
2 with HIc microsomes 60
15
30
60
13.5
2.2
3
0.3
0.6
3 with HIc microsomes 60
101.9
107.6
110.4
110.7
105.8
101.9
104.6
109.6
97.0
110.5
95.3
94.8
94.8
97.2
91.4
86.1
85.9
88.3
15
30
60
4
4 with HIc microsomes 60
15
30
60
5
2. Design of novel ligands
5 with HIc microsomes 60
a
b
c
Based on these findings it was decided to design novel analogs
with bioisosteric replacement of the labile ethyl ester moiety of the
lead analogs (Fig. 1). Amides are well known bioisosteric replace-
ments for substituted esters. Hence compounds 6–8 were synthe-
sized (Fig. 2). The N,N-dimethylamide ligand 9 was included to
determine the importance of a H-bond donor at that position as
% Remaining at T = 0 is 100%.
Samples were assayed in duplicates.
HI = Heat Inactivated, for control purposes.
A metabolic stability study of these compounds was undertaken
at SRI International by Dr. Ng and co-workers as a collaborative
Scheme 1. Synthesis of 6–13.

96
O. A. Namjoshi et al. / Bioorg. Med. Chem. 21 (2013) 93–101
compared to amide 7. Previously, in the case of GABAA modulators
in the benzodiazepine series of compounds, it has been demon-
strated that replacement of the ethyl ester at the 3-position by a
1,2,4-oxadiazole moiety resulted in higher intrinsic efficacy at ben-
zodiazepine receptors, as compared to the corresponding ethyl es-
ters.32 Substituted 1,2,4-oxadiazoles are also metabolically stable
and slightly less lipophilic than the corresponding ester deriva-
tives.33 Hence compounds 10–13 were synthesized based on
molecular modeling to limit the number of compounds.34,35
analog 2 (HZ-166). Amide 8 (HJ-I-37, approximately 92% remaining
at the end of 60 min) was significantly more stable than the corre-
sponding ethyl ester analog 3, which had underwent biotransfor-
mation almost quantitatively. Substituted 1,2,4-oxadiazole
analogs 11 (EMJ-I-026) and 12 (ZJW-II-40) were significantly more
stable than 1 and 2 respectively, as expected, based on medicinal
chemistry precedents.
Further assessment of these compounds in regard to agonist
efficacy at BzR was carried out by in vitro electrophysiological
studies on recombinant GABAA receptor subtypes expressed in
Xenopus oocytes.
3. Synthesis
4.1.1. In vitro electrophysiological studies on 6–8 and 11–13 for
efficacy at Bz/GABAergic receptor subtypes
The dose–response curves for the stimulation of GABA-induced
currents by BDZs 6–8 and 11–13 in oocytes, which expressed GA-
Ligands 6–8 were prepared by heating 1–3 with methylamine at
50 °C in ethanol in a sealed vessel (Scheme 1). Ligand 9 was syn-
thesized by heating acyl chloride of 5 (obtained by treatment of
5 with thionyl chloride at room temperature) with dimethyl amine
at reflux in the presence of triethylamine. Ligands 10 and 11 were
prepared by treatment of 1 with sodium hydride and N-hydroxy-
propanimidamide or N-hydroxy-2-methyl-propanimidamide,
respectively, in anhydrous THF at reflux in the presence of 4 Å
molecular sieves. Ligands 12 and 13 were prepared from 2 and 3,
respectively, by treatment with sodium hydride followed by
N-hydroxy-2-methyl-propanimidamide in anhydrous THF at reflux
in the presence of molecular sieves (4 Å).
BAA receptors of the subtypes
5b3 2, are illustrated in Figures 3–8. Amide 6 exhibited the most
selective efficacy at 3b3 2 receptor subtypes. At 100 nM concen-
tration it exhibited very weak agonistic effects at
and 5b3 2; while acting as an antagonist at 1b3
a1b3c2, a2b3c2, a3b3c2, and
a
c
a
c
a
1b3
c2 and
c
2,
a
2b3
c
2
2
a
c
a
a5b3
c
4. Results and discussion
4.1. In vitro metabolic stability of ligands 6–12
BzR ligands 6–8, 11 and 12 were evaluated for metabolic stabil-
ity (Table 2). Bioisosteric replacement of the ester moiety with an
amide improved the metabolic stability of amides 6–8 on human
microsomes. Monomethyl amide 6 (YT-III-31) exhibited a de-
creased rate of metabolism, as compared to the corresponding
ethyl ester analog 1 in liver microsomes; however, over the period
of 60 min almost 95% of the compound was metabolized. However
the N-methyl amide analog 7 (HJ-I-40) of anxiolytic/analgesic 2
was not metabolized during the 60 min of incubation and exhib-
ited improved metabolic stability as compared to the ethyl ester
Figure 3. Concentration–effect curves for 6 (YT-III-31) on
a1b3c2 (j), a2b3c2 (N),
a
3b3 5b3 2 (.) GABAA receptors, using an EC3 GABA concentration.
c
2 (ꢀ), and
a
c
Data points represent the mean SEM from four oocytes (for each receptor
subtype) from 2 batches. Stimulation of GABA EC3 by compound 6 at 100 nM or
1 lM concentration was 132 11 or 192 30, 211 23 or 287 31, 412 41 or
831 106, and 257 28 or 307 36, for
a1b3c2, a2b3c2, a3b3c2, and a5b3c2
Table 2
receptors, respectively.
In vitro metabolic stability of 6–12 on human liver microsomes
Test article
Time (min) Mean % remaining versus T = 0 mina
Mb Mb
10
1
l
l
15
30
60
60
15
30
60
60
15
30
60
60
15
30
60
87.3
37.5
5.2
114.2
108.7
126.0
136.4
109.6
97.3
107.4
104.3
82.8
113.6
104.4
106.3
110.0
109.5
96.8
102.8
103.3
108.5
109.2
108.0
96.6
118.9
105.6
99.3
6
6 with HIc microsomes
7
7 with HIc microsomes
8
95.9
92.3
8 with HIc microsomes
116.2
100.0
109.7
90.8
137.5
97.8
98.7
89.5
112.7
11
11 with HIc microsomes 60
15
30
60
Figure 4. Concentration–effect curves for 7 (HJ-I-40) on
a1b3c2 (j), a2b3c2 (N),
12
a
3b3 5b3 2 (.) GABAA receptors, using an EC3 GABA concentration.
c
2 (ꢀ), and
a
c
95.1
91.7
12 with HIc microsomes 60
Data points represent the mean SEM from at least three oocytes (for each receptor
subtype) from P2 batches. Stimulation of GABA EC3 by compound 7 at 100 nM or
a
b
c
% Remaining at T = 0 is 100%.
Samples were assayed in duplicates.
HI = Heat Inactivated, for control purposes.
1
l
M
concentration was 117
376 27, and 122 or 182 9, for
receptors, respectively.
3
or 171 12, 140 15 or 226 35, 163
5
or
5
a
1b3 2, 2b3 2, 3b3 2, and
c
a
c
a
c
a
5b3c2

O. A. Namjoshi et al. / Bioorg. Med. Chem. 21 (2013) 93–101
97
Figure 5. Concentration–effect curves for 8 (HJ-I-37) on
a1b3c2 (j), a2b3c2 (N),
a
3b3 5b3 2 (.) GABAA receptors, using an EC3 GABA concentration.
c
2 (ꢀ), and
a
c
Figure 8. Concentration–effect curves for 13 (YT-III-40) on
a1b3c2 (j), a2b3c2
Data points represent the mean SEM from at least three oocytes (for each receptor
(N),
a
3b3 5b3 2 (.) GABAA receptors, using an EC3 GABA concentra-
c
2 (ꢀ), and
a
c
subtype) from 2 batches. Stimulation of GABA EC3 by compound 8 at 100 nM or
tion. Data points represent the mean SEM from four oocytes (for each receptor
1
l
M concentration was 152 6 or 201 15, 241 12 or 287 16, 349 60 or
485 37, and 326 or 361 4, for 1b3 2, 2b3 2, 3b3 2, and 5b3 2
receptors, respectively.
subtype) from P2 batches. Stimulation of GABA EC3 by compound 13 at 100 nM or
9
a
c
a
c
a
c
a
c
1
lM
concentration was 140
8
or 234 37, 154
2
or 225 9, 226 53 or
2, 3b3 2, and 5b3c2
460 53, and 239 23 or 331 21, for
receptors, respectively.
a1b3
c
2,
a2b3
c
a
c
a
Figure 6. Concentration–effect curves for 11 (EMJ-I-026) on
a1b3c2 (j), a2b3c2
(N),
a
3b3 5b3 2 (.) GABAA receptors, using an EC3 GABA concentra-
c
2 (ꢀ), and
a
c
tion. Data points represent the mean SEM from at least three oocytes (for each
receptor subtype) from 2 batches. Stimulation of GABA EC3 by compound 11 at
100 nM or 1
or 381 45, and 175 66 or 291 13, for
receptors, respectively.
l
M concentration was 107 4 or 150 10, 160 3 or 281 13, 195
5
2
a1b3c2, a2b3c2, a3b3c2, and a5b3c
Figure 9. The light/dark cycle test and locomotor activity test on oxadiazole agonist
11 (EMJ-I-026).
Figure 7. Concentration–effect curves for 12 (ZJW-II-40) on
3b3 2 (.), and 5b3
tion. Data points represent the mean SEM from four oocytes (for each receptor
a
1b3
c
2 (j),
a
2b3
c
2
(N),
a
c
a
c
2 (ꢀ) GABAA receptors, using an EC3 GABA concentra-
subtype) from P2 batches. Stimulation of GABA EC3 by compound 12 at 100 nM or
at higher concentration, which is extremely desirable. This indi-
cates that 6 is a potential nonsedating anxiolytic. Amides 7 and 8
exhibited selective efficacy at a3b3c2 BzR at higher concentration.
1
l
M
concentration was 117
375 52, and 130 or 252 11, for
receptors, respectively.
2
or 178 19, 139
6
or 236 37, 143
2, 3b3 2, and
5
or
1
a
1b3 2, 2b3
c
a
c
a
c
a5b3c2

98
O. A. Namjoshi et al. / Bioorg. Med. Chem. 21 (2013) 93–101
Ligand 7, however was relatively less potent than 8. Dose–response
curves for 11 indicated a maximum separation between efficacies
6. Experimental
at
vant concentrations (100 nM). Oxadiazole 12 exhibited selective
efficacy at 3b3 2, however it was far less potent than 6 and 11.
Oxadiazole 13, at lower concentration, selectively modulated
5b3 2 and 3b3 2 receptor subtypes. At higher concentration
this compound modulated 3b3 2 receptors with higher efficacy
than 5b3 2 receptors. In general, all of the ligands exhibited the
desired selective efficacy at the 3b3 2 Bz receptor subtype. Bio-
a1b3c2 and those at the other subtypes at physiologically rele-
6.1. General methods for organic synthesis
a
c
Proton and carbon nuclear magnetic resonance spectra were
obtained on a Bruker 300-MHz spectrometer. Chemical shifts are
reported as parts per million (d) using an internal standard of
residual CHCl3 (7.26 ppm). The low resolution mass spectra (LRMS)
were obtained on an electron impact (EI, 70 eV) mass spectrome-
ter, which were recorded on a Hewlett-Packard 5985B gas chroma-
tography–mass spectrometer, while high resolution mass spectra
(HRMS) were recorded on a VG Autospec (Manchester England)
mass spectrometer. Infra-red spectra were recorded on a Thermo
Nicolet Nexus 870 FT-IR or a Perkin Elmer 1600 series FT-IR spec-
trometer. Elemental analyses were performed on a Carlo Erba
model EA-1110 carbon, hydrogen, and nitrogen analyzer. All sam-
ples submitted for CHN analyses were first dried under high vac-
uum for a minimum of 6 h using a drying pistol with isopropyl
alcohol as the solvent with potassium hydroxide pellets in the dry-
ing bulb. Melting points were taken on an Electrothermal model
IA8100 digital melting point apparatus and are uncorrected. Ana-
lytical thin layer chromatography plates employed were Dynamic
Adsorbents Inc UV active silica gel on plastic, while silica gel
60A, grade 60 for flash and gravity chromatography, were pur-
chased from E. M. Science. Tetrahydrofuran (THF) was dried by dis-
tillation from sodium-benzophenone ketyl. Dichloromethane was
distilled from calcium hydride. Acetonitrile was used directly as
received.
a
c
a
c
a
c
a
c
a
c
isostere 11 (EMJ-I-026) was selected for in vivo assessment for
three reasons—it exhibited relatively more selective efficacy at
a3b3c2 receptor subtypes at physiologically relevant concentra-
tions, it was metabolically stable as indicated in the liver micro-
somal assays, and it is a novel 1,2,4-oxadiazole analog.
4.1.2. Behavioral studies
4.1.2.1. Anxiolytic effect of 11 (EMJ-I-026) in the mouse model—
the light/dark cycle test and locomotor activity test.
‘Pre-
liminary anxiolytic-like behavior’ of subtype selective and meta-
bolically stable oxadiazole 11 (EMJ-I-026) was tested in rodents
using the light/dark cycle test (Fig. 9). Increased time spent in
the lighted area compared to vehicle indicates anti-anxiety effects.
Diazepam was used as a positive control. Diazepam was signifi-
cantly anxiolytic as compared to vehicle, as expected. Oxadiazole
11 exhibited anxiolytic activity at 10 and 100 mg/kg. Amphet-
amine-induced locomotor activity was a measure of the animal’s
activity related to sedation. Although this is not a direct measure
of sedation, it gives some idea about the sedative nature of a com-
pound. Diazepam was used as a positive control. Diazepam signif-
icantly reduced locomotor activity over vehicle that is, an indicator
6.1.1. 8-Ethynyl-N-methyl-6-phenyl-4H-benzo-[f]-imidazo-[1,5-
a]-[1,4]-diazepine-3-carboxamide (6)36
of sedation, while
and 100 mg/kg increased locomotor activity. From this preliminary
data, it was clear that 3 ligand 11 did not decrease locomotor
activity. In these mice, there appeared to be no effects from either
ataxia or sedation. This indicated in this paradigm that 11 (EMJ-I-
026) was a potential nonsedating anxiolytic.
a3 subtype selective oxadiazole 11 at doses of 10
Ester 1 (355 mg, 1 mmol) was treated with methylamine (33%
wt solution in ethanol, 10 mL). The suspension which resulted
was stirred in a sealed vessel at 45–50 °C for 24 h during which
time it became a clear solution. After removal of the ethanol and
methylamine under reduced pressure, the residue was purified
by a wash column (silica gel, gradient elution CH2Cl2–0.5% MeOH
in CH2Cl2) to afford amide 6 as a white powder (238 mg, 0.7 mmol,
70%): mp 237–238 °C; IR (KBr) 3284, 1651, 1607, 1567, 1525, 1409,
1303, 1256, 1208, 1010, 942, 826, 782, 765, 743, 698 cmꢀ1. 1H NMR
(CDCl3) d 2.99–2.97 (d, 3H, J = 5.04 Hz), 3.17(s, 1H), 4.09–4.05(d,
1H, J = 12.1 Hz), 6.29–6.26 (d, 1H, J = 10.7 Hz), 7.09–7.08 (t, 1H),
7.59–7.36 (m, 7H), 7.78–7.75 (dd, 1H, J = 1.8 Hz and 1.6 Hz), 7.85
(s, 1H); MS (EI) m/e (relative intensity) 340 (M+, 100). HRMS(TOF)
Calcd for C21H16N4ONa (M+Na)+ 363.1222, found: 363.1202. CHN
Analysis: Anal. Calcd for C21H16N4Oꢁ0.95 H2O: C, 70.56; H, 5.05;
N, 15.67. Found: C, 70.58; H, 4.82; N, 15.20.
a
5. Summary
In this report, eight bioisosteric analogs of XHe-II-053 were de-
signed in order to circumvent any potential metabolic liability in
humans of the previously described ligand. In fact, the 20N-analog
nonsedating anxiolytic HZ-166 (2), was nearly 80-fold more stable
than XHe-II-053 (1) after 1 h (see Table 1). Interestingly the 20-F
analog JY-XHe-053 (3) was not very stable. Importantly, the 5 bio-
isosteric ligands tested comprised of amides (6–9) and oxadiazoles
(10–13) were much more stable on human liver microsomes than
1 again indicating these bioisosteres (6–13) are potential nonse-
dating anxiolytics as well as HZ-166 (2) useful for treatment of
anxiety disorders in human populations. Gratifyingly, ligands 6,
6.1.2. 8-Ethynyl-N-methyl-6-(pyridine-2-yl)-4H-benzo-[f]-
imidazo-[1,5-a]-[1,4]-diazepine-3-carboxamide (7)
Target 7 was prepared in 80% yield from 230 using the procedure
as described above. Compound 7: mp 215–216 °C; 1H NMR (CDCl3)
d 2.97–2.99 (d, 3H, J = 4.9 Hz), 3.16 (s, 1H), 4.12 (br s, 1H), 6.29 (br
s, 1H), 7.14–7.15 (br s, 1H), 7.33–7.37 (t, 1H), 7.51–7.57 (m, 2H),
7.71–7.84 (m, 3H), 8.12–8.15 (d, 1H), 8.55–8.57 (d, 1H).
7, and 11 were clearly a3 Bz/GABAergic receptor subtype selective
ligands at pharmacologically relevant doses (approximately
100 nM) and, presumably, provide agents to study physiological
processes mediated by
tion were much more stable on human liver microsomes than 1. In
this regard 3 subtype selective ligand oxadiazole 11 (EMJ-I-026)
a3 subtypes including anxiety and in addi-
C
20H15N5O; MS (EI) m/e (relative intensity) 341 (M+, 100), 342
(22). HRMS(TOF) Calcd for C20H16N5O(M+H)+ 342.1355, found:
342.1365.
a
has been evaluated in the light dark paradigm and clearly is an
anxiolytic, wherein this ligand was anxiolytic with less sedative
properties, in vivo, as compared to diazepam in this paradigm. This
study indicated that the ester function in these molecules can be
replaced with a metabolically more stable ester bioisostere and
still retain anxiolytic activity. The in depth study of these ligands
in animal models and other receptor systems is underway and will
be reported in due course.
6.1.3. 8-Ethynyl-N-methyl-6-(2-fluorophenyl)-4H-benzo-[f]-
imidazo-[1,5-a]-[1,4]-diazepine-3-carboxamide (8)
Amide 8 was prepared in 75% yield from 331 using the proce-
dure analogous to the one used for the synthesis of 6. Compound
8: mp 235–236 °C; 1H NMR (CDCl3)
d
2.96–2.98 (d, 3H,
J = 4.57 Hz), 3.16 (s, 1H), 4.10 (br s, 1H), 6.29 (br s, 1H),

O. A. Namjoshi et al. / Bioorg. Med. Chem. 21 (2013) 93–101
99
6.96–7.02 (t, 1H), 7.14 (br s, 1H), 7.21–7.26 (t, 1H), 7.39–7.46 (m,
2H), 7.52–7.55 (d, 1H), 7.65–7.73 (m, 2H), 7.86 (s, 1H). C21H15FN4O;
MS (EI) m/e (relative intensity) 358 (M+, 100), 359 (23). HRMS(TOF)
Calcd for C20H15FN4ONa (M+Na)+ 381.1128, found: 381.1140. CHN
Analysis: Anal. Calcd for C21H15FN4Oꢁ0.17 CH2Cl2: C, 68.21; H, 4.15;
N, 15.03. Found: C, 68.29; H, 4.28; N, 14.71.
cooled to rt, after which acetic acid (56 mg, 0.931 mmol) was
added. After the solution was stirred for 10 min, the mixture was
filtered through celite. The filtrate was diluted with CH2Cl2
(75 mL) and washed with water, brine and dried (K2CO3). Evapora-
tion of the solvent under reduced pressure afforded a pale yellow
solid, which was purified by flash column chromatography (silica
gel, EtOAc/hexane, 2:3) to furnish 11 as a white solid (82 mg,
6.1.4. 8-Ethynyl-N,N-dimethyl-6-(pyridine-2-yl)-4H-benzo-[f]-
imidazo-[1,5-a]-[1,4]-diazepine-3-carboxamide (9)36
0.209 mmol, 45%): mp 190 °C; IR (KBr)
m 3291, 3057, 2972, 1613,
1574, 1494, 1466, 1303, 1264, 939, 832, 781, 734, 699, 666 cmꢀ1
.
Acid 5 (328 mg, 1 mmol) was dissolved in CH2Cl2 (5 mL). To the
suspension which resulted was added SOCl2 (0.5 mL) and the mix-
ture was stirred at rt for 1 h during which time it became a clear
solution. After removing the solvent and SOCl2 under reduced pres-
sure, the residue was redissolved in CH2Cl2 (5 mL) and was treated
with Et3N (0.2 mL) and Me2NH (0.2 mL). After stirring at reflux for
8 h, the reaction was quenched with water (5 mL). The organic
layer was washed with brine (5 mL) and dried (Na2SO4). After re-
moval of the solvent under reduced pressure, the residue was puri-
fied by a wash column (silica gel, gradient elution CH2Cl2–0.5%
MeOH in CH2Cl2) to afford amide 9 (178 mg, 0.5 mmol, 50%) as a
light yellow powder: mp 193–194 °C. 1H NMR (CDCl3) d 3.10(s,
3H), 3.16(s, 1H), 3.35(s, 1H), 4.17–4.21(d, 1H, J = 12.09 Hz), 5.89–
5.93(d, 1H, J = 12 Hz), 7.35–7.39(m, 1H), 7.52–7.55(m, 2H), 7.73–
7.86(m, 3H), 8.10–8.07(d, 1H, J = 7.8 Hz), 8.58–8.59(d, 1H,
J = 4.8 Hz); 13C NMR (CDCl3) d 35.9, 39.1, 45.2, 79.3, 81.8, 120.9,
122.7, 124, 124.7, 127, 132.6, 133.3, 135.2, 135.8, 136.2, 136.8,
136.9, 148.7, 156.7, 164.5, 167.4. HRMS (ESI) m/z calcd for
1H NMR (CDCl3) d 8.07(s, 1H), 7.81–7.79(dd, 1H), 7.64–7.61(m,
2H), 7.53–7.37(m, 5H), 6.14(d, 1H, J = 13.1 Hz), 4.19(d, 1H,
J = 12.8 Hz), 3.20(s, 1H), 3.24–3.15(m, 1H), 1.44–1.41(d, 6H,
J = 6.93 Hz); MS(EI) m/e (relative intensity) 393(M+, 100). HRMS
(TOF) Calcd for C24H19N5ONa(M+Na)+ 416.1487, found: 416.1501.
CHN Analysis: Anal. Calcd for C24H19N5Oꢁ0.37 CH2Cl2: C, 68.89; H,
4.68; N, 16.48. Found: C, 68.94; H, 4.59; N, 16.32. (CHN sample
was transferred to a vial for drying with CH2Cl2 which may explain
the contaminant.)
6.1.7. 5-(8-Ethynyl-6-(pyridin-2-yl)-4H-benzo-[f]-imidazo-[1,5-
a]-[1,4]-diazepine-3-yl)-3-isopropyl-1,2,4-oxadiazole (12)
Oxadiazole 12 was prepared in 40% yield from 2 using the same
procedure employed for the synthesis of 11. Compound 12: mp
200 °C; 1H NMR (CDCl3) d 1.42–1.44 (d, 6H, J = 6.93 Hz), 3.13–
3.27 (m, 2H), 4.27–4.31 (d, 1H, J = 10.8 Hz), 6.14–6.18 (d, 1H,
J = 10.8 Hz), 7.36–7.40 (m, 1H), 7.59–7.62 (m, 2H), 7.77–7.86 (m,
2H), 8.04–8.09 (m, 2H), 8.58–8.60 (m, 1H); 13C NMR (300 MHz,
CDCl3) d 20.5, 26.7, 44.8, 79.5, 81.5, 121.3, 122.7, 123.9, 124.8,
127, 135.2, 135.3, 135.7, 136, 136.2, 136.8, 148.7, 156.3, 167.8,
170.6, 175.2, 190.2; HRMS (ESI) Calcd for C23H19N6O (M+H)+
395.1620, found: 395.1635.
C
21H18N5O (M+H)+ 356.1511, found: 356.1528.
6.1.5. 5-(8-Ethynyl-6-phenyl-4H-benzo-[f]-imidazo-[1,5-a]-
[1,4]-diazepine-3-ethyl-1,2,4-oxadiazole (10)23
Ethyl amido oxime (59.5 mg, 0.676 mmol) was added to a stir-
red suspension of powdered 4 Å molecular sieves (75 mg) in anhy-
drous THF (15 mL) under argon. After the mixture was allowed to
stir at rt for 10 min, NaH (60% dispersion in mineral oil;
0.676 mmol) was added to the mixture. After the mixture had stir-
red for another 30 min, a solution of the forgoing ester 1 (120 mg,
0.338 mmol) in THF (20 mL) was added. The mixture, which re-
sulted, was heated at reflux for 8 h. The reaction mixture was then
cooled to rt, after which acetic acid (40.6 mg, 0.676 mmol) was
added. After the solution was stirred for 10 min, the mixture was
filtered through celite. The filtrate was diluted with CH2Cl2
(50 mL) and washed sequentially with water, brine, and then dried
(K2CO3). Evaporation of the solvent under reduced pressure affor-
ded a pale yellow solid, which was purified by flash column chro-
matography (silica gel, EtOAc/hexane, 2:3) to furnish 10 as a white
6.1.8. 5-(8-Ethynyl-6-(2-fluorophenyl)-4H-benzo-[f]-imidazo-
[1,5-a]-[1,4]-diazepine-3-yl)-3-isopropyl-1,2,4-oxadiazole (13)
Oxadiazole 13 was prepared in 45% yield from 3 using the pro-
cedure for the synthesis of 11. Compound 13: mp 160–165 °C; IR
(neat)
1394, 1367, 1342, 1312, 1259, 1221, 1071, 1011, 940, 903, 862,
793, 767, 754, 697, 671 cmꢀ1 1H NMR (CDCl3) d 8.09(s, 1H),
m 3194, 2961,2924, 2854, 1631, 1610, 1495, 1450, 1414,
;
7.80(dd, 1H, J = 1.78, 1.78 Hz), 7.69(m, 3H), 7.51(m, 2H), 7.07(m,
1H), 6.26(br s, 1H), 4.40(br s, 1H), 3.24 (m, 2H), 1.43(d, 6H,
J = 6.93 Hz); MS (EI) m/e (relative intensity) 411(43), 383(M+, 98),
325(100), 299(74), 178(74), 57(57); HRMS(ESI) Calcd for
C
24H18FN5O (M+H)+ 412.1644, found: 412.1628.
6.2. Electrophysiological experiments
solid (52 mg, 40%): mp 221–222 °C; IR (KBr)
m 3297, 3105, 1603,
1570, 1495, 1310, 938 cmꢀ1 1H NMR (CDCl3) d 8.07 (s, 1H), 7.80
.
Xenopus oocytes were injected with rat cDNA’s of GABAA recep-
(dd, 1H, J1 = 8.4 Hz, J2 = 1.8 Hz), 7.64–7.6 (m, 2H), 7.53–7.37 (m,
5H), 6.12 (d, 1H, J = 12.9 Hz), 4.21 (d, 1H, J = 12.9 Hz), 3.20 (s,
1H), 2.88 and 2.83 (Abq, 2H, J = 7.6 Hz), 1.41 (t, 3H, J = 7.6 Hz);
13C NMR (300 MHz, CDCl3) d 171.8, 170.6, 168.8, 139.1, 136.6,
135.8, 135.4 (2C), 135.1, 130.7, 129.3 (2C), 128.3 (2C), 128.1,
124.7, 122.7, 121.6, 81.2, 80, 44.7, 19.7, 11.5. MS(EI) m/e (relative
intensity) 379 (M+, 100).
tor b3 and c2 subunits as well as a1, a2, a3, or a
5 subunits.21 36 h
after injection, the enveloping follicle cell layers of the oocytes
were removed and oocytes were placed on a nylon-grid in a bath
of Xenopus Ringer solution (XR, containing 90 mM NaCl, 5 mM
HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM KCl and 1 mM CaCl2).
For current measurements the oocytes were impaled with two
microelectrodes (2–3 mX), which were filled with 2 mM KCl. The
oocytes were constantly washed by a flow of 6 mL/min XR, which
could be switched to XR containing GABA and/or drugs. Drugs were
diluted into XR from DMSO-solutions resulting in a final concen-
tration of 0.1% DMSO perfusing the oocytes. Drugs were preapplied
for 30 s before the addition of GABA, which was coapplied with the
drugs until a peak response was observed. Between two applica-
tions, oocytes were washed in XR for up to 15 min to ensure full
recovery from desensitization. All recordings were performed at
room temperature at a holding potential of ꢀ60 mV using a War-
ner OC-725C two-electrode voltage clamp. Data were digitized, re-
corded and measured using a Digidata 1322A data acquisition
6.1.6. 5-(8-Ethynyl-6-phenyl-4H-benzo-[f]-imidazo-[1,5-a]-
[1,4]-diazepine-3-isopropyl-1,2,4-oxadiazole (11)36
Isopropyl amido oxime (95 mg, 0.931 mmol) was added to a
stirred suspension of powdered 4 Å molecular sieves (100 mg) in
anhydrous THF (30 mL) under argon. After the mixture was al-
lowed to stir at rt for 10 min, NaH (60% dispersion in mineral oil;
0.931 mmol) was added to the mixture. After the mixture was stir-
red for a further 30 min, a solution of the forgoing ester 1 (165 mg,
0.465 mmol) in THF (30 mL) was added. The mixture which re-
sulted was heated to reflux for 8 h. The reaction mixture was then

100
O. A. Namjoshi et al. / Bioorg. Med. Chem. 21 (2013) 93–101
system. Results of concentration response experiments were fitted
using GraphPad Prism 3.00 (GraphPad Software, San Diego, CA).
Acknowledgements
The equation used for fitting concentration response curves was
We wish to acknowledge the NIH (MH46851), the Lynde and
Harry Bradley Foundation and the Research Growth Initiative of
the University of Wisconsin-Milwaukee for generous financial sup-
port. We thank Dr. Jamie Driscoll and Dr. Linda Brady (NIMH) for
their interest in this work and for support of the metabolic studies.
J.K.R. was supported, in part, by ORIP grant OD011103, and NIDA
grants DA011792 and DA033795.
ꢀX) ꢂ HillSlope))
Y = Bottom + (Top-Bottom)/(1 + 10((LogEC
; X repre-
50
sents the logarithm of concentration, Y represents the response;
Y starts at Bottom and goes to Top with a sigmoid shape. This is
identical to the ‘four parameter logistic equation’.
6.3. Metabolic stability for GABAA receptor ligands using human
liver microsomes (SRI study No. 400–10)
References and notes
The test articles were incubated at two concentrations (1 and
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100
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l
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