Imidazo[1,2-b][1,2,4]triazines as α2/α3 subtype selective GABAA agonists for the treatment of anxiety

Article abstract of DOI:10.1016/j.bmcl.2005.12.044

Imidazo[1,2-a]pyrimidines and imidazo[1,2-b][1,2,4]triazines are ligands for the benzodiazepine binding site of GABA_A receptors that are functionally selective for the α2/α3 subtypes over the α1 subtype. SAR studies to optimise this functional

Full text of DOI:10.1016/j.bmcl.2005.12.044

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1477–1480
Imidazo[1,2-b][1,2,4]triazines as a2/a3 subtype selective
GABAA agonists for the treatment of anxiety
Andrew S. R. Jennings,* Richard T. Lewis, Michael G. N. Russell, David J. Hallett,
´
Leslie J. Street, Jose L. Castro, John R. Atack, Susan M. Cook, Rachael Lincoln,
Joanna Stanley, Alison J. Smith, David S. Reynolds, Bindi Sohal, Andrew Pike,
George R. Marshall, Keith A. Wafford, Wayne F. A. Sheppard and Spencer J. Tye
Neuroscience Research Centre, Merck Sharp and Dohme Research Laboratories, Terlings Park,
Eastwick Road, Harlow, Essex CM20 2QR, UK
Received 22 November 2005; revised 12 December 2005; accepted 12 December 2005
Available online 4 January 2006
Abstract—Imidazo[1,2-a]pyrimidines and imidazo[1,2-b][1,2,4]triazines are ligands for the benzodiazepine binding site of GABAA
receptors that are functionally selective for the a2/a3 subtypes over the a1 subtype. SAR studies to optimise this functional selec-
tivity, pharmacokinetic and behavioural data are described.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Inhibition of neurotransmission in the central nervous
system is mediated predominantly by chloride ion flux
into nerve cells through GABAA receptors. These recep-
tors are ligand-gated ion channels which open in
response to the binding of the neurotransmitter
c-aminobutyric acid (GABA). They are composed of
five transmembrane subunits that come from a family
of 19 (a1-6, b1–3, c1-3, d, e, p, h and q1-3), most frequently
consisting of a, b and c subunits in a 2:2:1 ratio.1,2 The
GABAA receptors are the site of action of the benzodi-
azepine (BZ) class of molecules which allosterically
modulate the GABA-mediated chloride ion flux.3 The
major BZ-sensitive receptors contain b, c2 and either
a1, a2, a3 or a5 subunits. Studies with transgenic mice
and subtype selective compounds indicate that receptors
containing different a subtypes mediate the various
pharmacological effects of the non-subtype selective
BZ agonists, such as diazepam, i.e., the a1-containing
receptors are responsible for the sedative/muscle relax-
ant properties, while the a2 and/or the a3-containing
receptors mediate the anxiolytic properties.4,5 The goal
of our research has been to identify ligands for the
GABAA receptor that are selective a2/a3 agonists which
could be potential anxiolytics without the concurrent
sedation/ataxia observed with unselective BZs.
The imidazo[1,2-a]pyrimidine 1a (Table 1) was identified
as a high affinity ligand for the GABAA receptor BZ
binding site, which also had the desired functional selec-
tivity between a subtypes, i.e., antagonism at the a1 sub-
type and partial agonism at the a2 and a3 subtypes. The
compound, as had been hypothesised for such a profile,
proved to function as an anxiolytic in several animal
behavioural models without showing the pronounced
sedation and ataxia observed with unselective BZs.6
However, compound 1a has modest pharmacokinetics
(PK) in rat and dog (half-lives of 1.7 and 1.0 h, respec-
tively—Table 3) and the desire was to find a compound
with a longer half-life whilst maintaining the beneficial
efficacy profile.
The pyridyl N-oxide of 1a was observed as a major
metabolite in vivo, therefore initial work focused on
modification of the 3-pyridyl moiety. This was achieved
using the route shown in Scheme 17 by forming the
biaryl ring system 5, which was then coupled to
the imidazopyrimidine core 3. Chemistry to introduce
the diversity as the final step by Suzuki coupling to a late
stage intermediate (1 where R = Br or B(OH)2) was
explored, but proved to be problematic due to compet-
ing Dimroth rearrangement.8
Keywords: Imidazo[1,2-a]pyrimidines; Imidazo[1,2-b][1,2,4]triazines;
GABAA receptors; a2/a3 subtype selective; Anxiolytic.
*
Corresponding author. Tel.: +44 1279 440810; fax: +44 1279
440187; e-mail: andrew_jennings@merck.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.044

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A. S. R. Jennings et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1477–1480
Table 1. Affinity and efficacy at a1 and a3 subtype GABAA receptors for imidazo[1,2-a]pyrimidines varying the terminal pyridyl ring
Compound
R
Ki (nM)a
Flux efficacy
(vs CDZ)b
Patch–clamp
efficacy (vs CDZ)c
a1
a2
a3
a5
a1
a3
a1
a3
1a
1b
1c
0.85
3.70
4.00
0.53
0.06
0.18
0.38
0.50
0.05
0.53
0.68
1.60
2.25
1.94
0.31
0.42
0.47
0.57
0.42
0.40
1d
1e
1f
0.80
1.79
1.43
0.60
0.42
3.76
3.64
0.28
1.24
1.13
0.67
0.14
0.44
0.18
0.26
0.30
0.68
0.21
0.52
0.59
1g
a Affinity was determined by the inhibition of [3H]Ro 15-1788 (flumazenil) binding to human recombinant GABAA receptors containing b3c2 plus
either a1 or a3 stably expressed in L(tkÀ) cells. Values are means of 3–10 separate determinations.9
b Modulation of chloride ion flux in cells expressing b3c2 plus either a1 or a3 produced by an EC20 equivalent concentration of GABA in the presence
of an approximate 1000 · Ki concentration of test compound. Efficacy is expressed relative to the full agonist chlordiazepoxide (CDZ) (relative
efficacy = 1.0), from at least seven independent experiments.10
c Measured with GABAA receptors stably expressed in L(tkÀ) cells using whole cell patch-clamp recording and represents the effect of the test
compound on the current produced by an EC20-equivalent of GABA relative to the full agonist chlordiazepoxide (CDZ) (relative efficacy = 1.0).
Data show mean maximal efficacy from at least four individual cells.4
cyano substituents could also be introduced to give
promising compounds (1c–g) with high affinity. In our
high throughput chloride ion flux efficacy assay,10 all
the compounds showed a window between the agonism
produced at the a3 and a1 subtypes, however none acted
as an antagonist at a1 (confirmed for compounds 1b and
1c by using a whole cell patch–clamp efficacy assay).4
It was noted, during concurrent work modifying the
core heterocyclic-ring system, that replacing the imi-
dazo[1,2-a]pyrimidine by an imidazo[1,2-b][1,2,4]triazine
conferred a lowered agonism profile at both a3 and a1
subtypes whilst maintaining an equivalent level of bind-
ing affinity.11 For example, the direct analogue of 1a,
compound 2a (Table 2), has equivalent affinity to 1a
and exhibits lower efficacy in the patch–clamp assay:
0.24 relative to the standard full agonist chlordiazepox-
ide (CDZ) at the a3 subtype compared to 0.53 for 1a.
Scheme 1. Reagents and conditions: (i) Bis(pinacolato)diboron,
Pd(dppf)Cl2, KOAc, 1,4-dioxane, 90 ꢁC; (ii) R-Cl/Br, Pd2(dba)3, PtBu3,
KF, THF, rt to 50 ꢁC; (iii) SnCl2Æ2H2O, EtOH; (iv) NaNO2, 48% HBr
(aq), CuBr, 0–50 ꢁC; (v) Pd(PPh3)4, Na2CO3 (aq), DME, 80 ꢁC.
With this in mind several of the pyridine compounds
Some key compounds to come from this work are
shown in Table 1. In terms of affinity, the pyridyl nitro-
gen was tolerated at all three positions (1a–c). Fluoro or
were made in the imidazotriazine series (conveniently
by coupling the previously synthesised biaryls 5 to
bromide 4 as shown in Scheme 1)12 with the aim of

A. S. R. Jennings et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1477–1480
1479
Table 2. Affinity and efficacy at a1 and a3 subtype GABAA receptors for imidazo[1,2-b][1,2,4]triazines varying the terminal pyridyl ring
Compound
R
Ki (nM)a
Patch–clamp efficacy (vs CDZ)b
a1
a2
a3
a5
a1
a2
a3
a5
2a
2b
2c
2d
2e
0.81
1.63
0.50
0.00
0.24
0.55
1.45
2.01
0.67
0.92
2.3
0.52
2.77
3.42
4.50
0.50
1.24
1.57
2.71
0.06
0.15
0.24
0.00
0.50
0.52
0.50
3.44
8.43
1.54
0.39
0.37
0.23
0.09
0.20
0.41
2f
1.28
9.53
9.34
0.32
0.01
0.69
2g
2h
0.76
0.67
1.06
3.30
0.00
0.01
0.37
0.34
a Affinity was determined by the inhibition of [3H]Ro 15-1788 (flumazenil) binding to human recombinant GABAA receptors containing b3c2 plus
either a1 or a3 stably expressed in L(tkÀ) cells. Values are the mean of 3–10 separate determinations.9
b Measured with GABAA receptors stably expressed in L(tkÀ) cells using whole cell patch–clamp recording and represents the effect of the test
compound on the current produced by an EC20-equivalent of GABA relative to the full agonist chlordiazepoxide (CDZ) (relative efficacy = 1.0).
Data shows mean maximal efficacy from at least four individual cells.4
reducing the efficacy at the a1 subtype to antagonism
levels. The resulting compounds are shown in Table 2.
Gratifyingly, the anticipated change in efficacy profile
did indeed manifest itself; 4-pyridyl and 2-pyridyl-4-flu-
oro compounds 2b and 2c when compared with ana-
logues 1b and 1c showed a reduction in their a1
efficacy of approximately 0.3 units in the patch–clamp
assay. Utilising this discovery, several compounds with
very interesting selective efficacy profiles (2e–h) were
identified.
Having measured the affinity and efficacy for these com-
pounds at the a2 and a5 subtypes (Table 2), all were
shown to be partial agonists, but compounds 2e and
2f stood out: 2f due to its high efficacy at the a3 subtype
and 2e due to its low efficacy at the a5 subtype (a5 effi-
cacy has been implicated in memory/learning
impairment).13
The pharmacokinetic profiles of 2e and 2f were exam-
ined in rat and dog (Table 3). Both were found to have
Table 3. Pharmacokinetic parameters in rat and dog
Compound
Rata
Dogb
F (%)
Cl (ml/min/kg)
T1/2 (h)
Vdis (l/kg)
F (%)
Cl (ml/min/kg)
T1/2 (h)
Vdis (l/kg)
1a
2e
2f
77
69
9.1
1.2 0.1
2.4 0.3
1.7
22
1.0
2.2 0.1
1.7 0.4
46
64 28
99 32
8.1
0.5 0.1
2.2 0.7
1.0
9/65c
9.7
0.6
2.3 0.3
1.5 0.1
8
102 20
7.9
a Determined in six male Sprague–Dawley rats. Three dosed 1 mg/kg iv and three dosed 1 mg/kg po.
b Determined in six female beagle dogs. Three dosed 1 mg/kg iv and three dosed 1 mg/kg po.
c Two-phase elimination (2–10 h/10–48 h).

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A. S. R. Jennings et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1477–1480
4. (a) McKernan, R. M.; Rosahl, T. W.; Reynolds, D. S.;
Sur, C.; Wafford, K. A.; Atack, J. R.; Farrar, S.; Myers, J.;
Cook, G.; Ferris, P.; Garrett, L.; Bristow, L.; Marshall,
G.; Macaulay, A.; Brown, N.; Howell, O.; Moore, K. W.;
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excellent parameters in the two species, with significant-
ly increased half-lives when compared to 1e.
Compound 2e was an anxiolytic in the rat elevated
plus maze assay14 giving a statistically significant in-
crease in time spent on the open arms at an oral dose
of 1 mg/kg (which corresponded with an occupancy of
84% as measure by in vivo displacement of [3H]Ro 15-
1788)15, and showed no ataxia in the mouse rotarod
assay4 either at oral doses up to 30 mg/kg or in the
presence of a sub-threshold level of ethanol (1.5 g/kg
ip) at an oral dose of 3 mg/kg (92% occupancy). Com-
pound 2e was also effective in the squirrel monkey
conditioned emotional response (CER) paradigm, a
more stringent test of anxiolytic potential, at an oral
dose of 0.3 mg/kg.
In conclusion, we have shown that certain changes to
the pyridyl moiety of imidazo[1,2-a]pyrimidine, 1a,
are tolerated in terms of affinity but lose the desired
efficacy profile. This can be regained by modulating
the efficacy profile with an imidazo[1,2-b][1,2,4]tri-
azine ring system, which leads to compounds that
are selective agonists at the a2/a3 subtypes of the
GABAA receptor over a1. These compounds also
exhibit improved pharmacokinetics in rat and dog
over 1a and 2e has shown anxiolysis in two species
at levels which show no effect in an animal model
of ataxia.
8. Guerret, P.; Jacquier, R.; Maury, G. Bull. Soc. Chim. Fr.
1972, 9, 3503.
9. Hadingham, K. L.; Wingrove, P.; Le Bourdelles, B.;
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col. 1993, 43, 970.
10. Smith, A. J.; Alder, L.; Silk, J.; Adkins, C.; Fletcher, A. E.;
Scales, T.; Kerby, J.; Marshall, G.; Wafford, K. A.;
McKernan, R. M.; Atack, J. R. Mol. Pharmacol. 2001, 59,
1108.
11. Russell, M. G. N.; Carling, R. W.; Street, L. J.; Hallett, D.
J.; Mezzogori, E.; Atack, J. R.; Cook, S. M.; Bromidge, F.
A.; Wafford, K. A.; Marshall, G. R.; Reynolds, D. S.;
Stanley, J.; Lincoln, R.; Tye, S. J.; Sheppard, W. F. A.;
Sohal, B.; Pike, A.; Dominguez, M.; Castro, J. L. J. Med.
Chem. in press.
12. Bettati, M.; Blurton, P.; Carling, W. R.; Chambers, M. S.;
Hallett, D. J.; Jennings, A.; Lewis, R. T.; Russell, M. G. N.;
Street, L. J.; Szekeres, H. J.; Van Niel, M. B. WO
2002038568.
Acknowledgments
The authors thank Sarah Kelly, Dr. Robert Carling and
Alec Guiblin for the synthesis of compounds 1a, 1b, 2c
and 2g. We also thank Alison Macaulay and Nicola
Brown for their contributions to the electrophysiology
data.
13. Collinson, N.; Kuenzi, F. M.; Jarolimek, W.; Maubach,
K. A.; Cothliff, R.; Sur, C.; Smith, A.; Otu, F. M.; Howell,
O.; Atack, J. R.; McKernan, R. M.; Seabrook, G. R.;
Dawson, G. R.; Whiting, P. J.; Rosahl, T. W. J. Neurosci.
2002, 22, 5572.
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