Discovery of imidazo[1,2-b][1,2,4]triazines as GABAA α2/3 subtype selective agonists for the treatment of anxiety

Article abstract of DOI:10.1021/jm051200u

The identification of a series of imidazo[1,2-b][1,2,4]triazines with high affinity and functional selectivity for the GABA_A α3-containing receptor subtype is described, leading to the identification of a clinical candidate, 11. Compound 11 shows good bioavailability and half-life in preclinical species, and it is a nonsedating anxiolytic in both rat and squirrel monkey behavioral models.

Full text of DOI:10.1021/jm051200u

J. Med. Chem. 2006, 49, 1235-1238
1235
Chart 1
Discovery of Imidazo[1,2-b][1,2,4]triazines as
GABAA r2/3 Subtype Selective Agonists for the
Treatment of Anxiety
Michael G. N. Russell,* Robert W. Carling, Leslie J. Street,
David J. Hallett, Simon Goodacre, Elena Mezzogori,
Michael Reader, Susan M. Cook, Frances A. Bromidge,
Robert Newman, Alison J. Smith, Keith A. Wafford,
George R. Marshall, David S. Reynolds, Rebecca Dias,
Pushpindar Ferris, Jo Stanley, Rachael Lincoln,
Spencer J. Tye, Wayne F. A. Sheppard, Bindi Sohal,
Andrew Pike, Maria Dominguez, John R. Atack, and
Jose´ L. Castro
rivative (4) were identified as being worthy of further
development. Both compounds exhibit functional selectivity for
the R2 and R3 subtype-containing receptors over the R1 and
R5 subtypes and are orally bioavailable. However, although 3
has low clearance and a long half-life in rat and dog, its efficacy
is low at the R3 subtype. On the other hand, 4 has higher
efficacy at the R3 subtype but with a short half-life in animals.
Therefore, a compound with higher efficacy at the R3 subtype
and a long half-life was sought. In this manuscript, we describe
the identification of a series of imidazo[1,2-b][1,2,4]triazines
leading to the selection of a clinical candidate that meets the
above criteria.
The binding affinities of compounds were measured by the
inhibition of [3H]Ro15-1788 binding to human recombinant
GABAA receptor subtypes stably expressed in L(tk-) cells
containing R1, R2, R3, or R5 subunits in combination with â3
and γ2.13,14 The efficacies of most compounds were determined
using whole-cell patch clamp recordings on the same receptor
subtype combinations. A concentration of GABA was used that
elicited a response that was 20% of the maximum GABA current
(EC20). Modulation of this current by increasing concentrations
of the test compounds was measured.15 Alternatively, efficacies
were determined in the same cell lines by measuring the
modulation of 36Cl ion flux produced by the EC20 concentration
of GABA in the presence of a concentration of 1000 × Ki of
the test compound.16
When exploring initial changes to the heterocyclic core, it
was found that the imidazo[1,2-b]pyridazine (5, Scheme 1)
retained the functional selectivity for the R3 subtype (Table 1),
even though affinity and efficacy were slightly reduced relative
to the corresponding imidazo[1,2-a]pyridine (6).12 The previ-
ously reported introduction of another nitrogen atom to the top
of the six-membered ring, to give imidazo[1,2-a]pyrimidine
(7),12 showed a 10-fold increase in affinity, and it was gratifying
to find that this effect translated into the imidazo[1,2-b][1,2,4]-
triazine (8). Like that of 5 compared to 6, compound 8 had
lower efficacy than 7 at both R1 and R3 subtypes but retained
the selectivity window. The general synthetic route outlined in
Scheme 2 was used to explore the SAR of this new scaffold.
The 3-substituted imidazotriazine core (16) was synthesized by
condensation of the corresponding 5-substituted-3-amino-1,2,4-
triazine (15) with bromoacetaldehyde (formed immediately
beforehand by treatment of bromoacetaldehyde diethyl acetal
with concentrated hydrobromic acid). The 3-aminotriazines 15
were in turn prepared by the condensation of aminoguanidine
and the dibromoketone (14) (prepared by bromination of the
corresponding methyl ketone). This condensation was nonselec-
tive, producing a mixture of aminotriazines, but a regioselective
synthesis of aminotriazines using bis-morpholine ketoaminals
has since been reported.17 The imidazotriazine was then bro-
minated selectively in the 3-position to give 17, which was then
coupled with the required biaryl boronate (18) in a Suzuki
reaction to give the final compounds.
The Neuroscience Research Centre, Merck Sharp & Dohme
Research Laboratories, Terlings Park, Eastwick Road,
Harlow, Essex, CM20 2QR, UK
ReceiVed NoVember 29, 2005
Abstract: The identification of a series of imidazo[1,2-b][1,2,4]triazines
with high affinity and functional selectivity for the GABAA R3-
containing receptor subtype is described, leading to the identification
of a clinical candidate, 11. Compound 11 shows good bioavailability
and half-life in preclinical species, and it is a nonsedating anxiolytic
in both rat and squirrel monkey behavioral models.
The GABAA receptor is a ligand-gated chloride ion channel
that has a pentameric structure composed from a family of at
least 16 subunits (R1-6, â1-3, γ1-3, δ, ꢀ, π, and θ), but
comprising generally of R, â, and γ subunits in a 2:2:1
stoichiometry. The benzodiazepine (BZ) binding site is an
allosteric site located between the R and γ2 subunits.1-3 Hence,
benzodiazepines, exemplified by diazepam (1, Chart 1) interact
with GABAA receptors containing â and γ2 subunits in
conjunction with an R1, R2, R3, or R5 (but not R4 or R6) subunit
and are full agonists at the BZ recognition site of these four
subtypes, leading to a positive modulation of the actions of
GABA.4 Diazepam displays anxiolytic, anticonvulsant, sedative-
hypnotic, anaesthetic, and muscle-relaxant activities, but it is
also associated with side effects such as amnesia, tolerance,
dependence, and alcohol potentiation. Although sedation and
myorelaxation are of clinical utility for treating sleep disorders
or premedication prior to procedures such as endoscopy, in terms
of treating general anxiety disorder (GAD) they are liabilities.
Consequently, there is a need for a nonsedating anxiolytic.5,6
It has been shown using R1H101R mutant mice, in which
the R1 subunit is rendered insensitive to diazepam, that diazepam
retains its anxiolytic but not its sedative effect.7,8 There is also
evidence in mice lacking the R5 subunit that this subtype is
associated with memory and learning and is not involved in
anxiety.9 This suggests that GABAA receptors containing the
R1 subtype are involved in the sedative effects of BZs whereas
the R2 and/or R3 subtypes mediate the anxiolytic effects.10,11
Therefore, BZ site agonists with selectivity for the R2/3 subtypes
may be anxiolytics with a potential for reduced side effects.6
We have previously reported on the generation of a series of
imidazopyrimidines that are structurally related to 2 (NS-2710,
Chart 1).12 In particular, the 7-trifluoromethyl-3-(2′-cyano-2,4′-
difluorobiphenyl-5-yl) analogue (3, Table 1) and the 7-(1-
hydroxy-1-methylethyl)-3-[4-fluoro-3-(pyridine-3-yl)phenyl de-
* Correspondence to Dr. M. G. N. Russell. Tel: 44 1279 440419. Fax:
44 1279 440390. E-mail: michael_russell@merck.com.
10.1021/jm051200u CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/01/2006

1236 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Letters
Table 1. Binding Affinities and Efficacies at Cloned Human GABAA Receptor Subtypes
a Inhibition of [3H]Ro15-1788 binding to recombinant human GABAA receptor subtypes stably expressed in L(tk-) cells. Values are the geometric mean
( SEM for n ) 2-12. Six to eight concentrations of each test compound incremented in half-log units were used in the determinations. b Efficacy measured
at GABAA receptors stably expressed in L(tk-) 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. Values are quoted relative to the maximum response seen with the full agonist chlordiazepoxide (CDZ, efficacy
) 1.00) except for those figures quoted as percentages. c Modulation of chloride ion flux in cells expressing â3γ2 plus either R1 or R3 produced by an EC20
equivalent concentration of GABA in the presence of an approximate 1000 × Ki concentration of test compound. Values are the mean of at least seven
independent experiments.
Table 2. Pharmacokinetic Data for Selected Compoundsa
Scheme 1a
compd
species
Clp (mL/min/kg)
t1/2 (h)
Vd (L/kg)
F (%)
10
11
11
11
12
rat
rat
dog
rhesus
rat
1.1
2.4
4.6
5.8
1.0
38
11
6.5
4.9
69
3.4
2.1
1.9
2.1
5.8
NDb
77
22
29
64
a Reagents and conditions: (i) Br2, NaOAc, AcOH, rt, 20 min; (ii) 18a,
Pd(PPh3)4, K3PO4, DMA, 80 °C, 21 h.
a Dose: 1 mg/kg iv; 1 mg/kg po. b Oral dose not administered.
On carrying out optimization studies, we prepared the
imidazotriazine analogue (9) of 3 and found similar decreases
on the functional efficacy to that observed with the methyl
derivatives (8 and 7). The lower intrinsic efficacy of this core
compared to the imidazopyrimidines proved useful when
optimizing substitution in this series and allowed us to have
different substituents to the imidazopyrimidines while still
retaining antagonism at the R1 subtype. Thus, imidazotriazine

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1237
Scheme 2a
Figure 2. The anxiolytic-like effects of 11 in the rat elevated plus
maze test. The mean time (( SEM, n ) 18/group) spent on the open
arms (expressed as a percentage of the total time, 5 min, on the maze)
after 30 min pretreatment with vehicle (0.5% methyl cellulose, po), 11
(0.3, 1 or 3 mg/kg po) or chlordiazepoxide (CDZ, 5 mg/kg ip). The
basis of this assay is that the less anxious an animal is, then the more
time it will spend on the open arms compared to the safer, closed arms.
The % figures denote the % occupancy at each dose. * ) significantly
different from control (vehicle), p < 0.05.
a Reagents and conditions: (i) (a) NaOAc‚3H2O, H2O, reflux, 30 min;
(b) aminoguanidine bicarbonate, 4 N NaOH(aq) or KHCO3, rt, 1.5-39 h;
(ii) BrCH2CHO, EtOH, 50-80 °C, 1-2 d; (iii) Br2, NaOAc, AcOH, rt, 0.25-6
h; (iv) 18, Pd(PPh3)4, 2 M Na2CO3(aq) or K3PO4, THF or DME or DMA,
70-80 °C, 16-24 h.
Table 3. Binding Affinity and Efficacy of Compound 11
subtype
Kia (nM)
efficacy/CDZb
R1â3γ2
R2â3γ2
R3â3γ2
R5â3γ2
0.73 ( 0.21
2.0 ( 0.4
1.8 ( 0.4
1.1 ( 0.2
0.03 ( 0.01
0.38 ( 0.04
0.50 ( 0.02
0.37 ( 0.04
a,b See corresponding footnotes in Table 1.
Figure 3. The lack of sedative effect of 11 in the rat chain-pulling
test even at a dose, 10 mg/kg, corresponding to 99% occupancy. The
mean rate of responding (( SEM; n ) 11-12/group) over a 60 min
trial period immediately following treatment with either vehicle, 11 or
diazepam (diaz.). The % figures denote the % occupancy at each dose.
** ) significantly different from control (vehicle), p < 0.01.
Figure 1. Efficacy of 11 at human recombinant GABAA receptors
containing â3, γ2, and various R subunits. The ability of 11 to potentiate
currents produced by a GABA concentration that produced a response
20% of the maximum (EC20) was measured using whole-cell patch
clamp electrophysiology. In comparison, a nonselective full agonist
such as diazepam or chlordiazepoxide would produce a potentiation
of 100-150% at each subtype. Values shown are mean ( SEM (n )
4-6).
Figure 4. Nonsedating anxiolytic properties of 11 in nonhuman
primates. In the squirrel monkey conditioned emotional response (CER)
assay 11 has anxiolytic-like activity. In CER assay, all doses of 11
(0.3, 1, and 3 mg/kg po) reversed the inhibition of lever-pressing
response produce by the presentation of the stimulus (i.e. the monkey
was less anxious about the possibility of receiving an electric shock
when presented with the light cue). In the sedation assay, the rate of
lever pressing to obtain a fruit juice reward in squirrel monkeys was
measured following treatment with either vehicle (0.5% methyl
cellulose) or 11 (1, 3, or 10 mg/kg po). Values shown are mean (
SEM (n ) 10-11/group). * ) significantly different from control
(vehicle), p < 0.05.
10, for which the imidazopyrimidine analogue had too high
efficacy at the R1 subtype,12 was found to have a good binding
and efficacy profile and a very long half-life in rat (38 h, Table
2). Additional fluorination on the terminal aryl ring led to the
4,6-difluoro analogue (12) with a similar profile to 10, but the
optimum efficacy and half-life across three species was given
by the 6-fluoro derivative (11).
The full binding and efficacy profile of 11 is shown in Figure
1 and Table 3, which shows that it also has somewhat lower
efficacy at both the R2 and R5 subtypes compared to its efficacy
at R3. 11 had no significant activity at 133 other receptors and
in 36 enzyme assays. In a measurement of central BZ receptor
occupancy by displacement of [3H]Ro15-1788 binding in rat,18
it was found to achieve high occupancy (Occ50 0.3 mg/kg po)
at relatively low plasma levels (56 nM). In animal models of
anxiety, 11 was active in the rat elevated plus maze19 at 1 mg/
kg po (at 88% occupancy, Figure 2) and showed no sedation
up to 10 mg/kg po in the rat chain-pulling assay20 (where 99%
occupancy was achieved, Figure 3). In the squirrel monkey
conditioned emotional response model21 a significant anxiolytic
effect was seen at 0.3 mg/kg po, but no significant sedation
was observed up to 10 mg/kg po as measured by the response
rates during the RPI30 schedule (Figure 4). 11 also showed no
impairment of performance in the mouse rotarod assay11 up to
a dose of 10 mg/kg po corresponding to 100% occupancy
(Figure 5).
Thus, on the basis of its excellent efficacy and pharmacoki-
netic profile, compound 11 was chosen as a clinical candidate.
The multi-kilogram scale synthesis of this compound has already
been described.22 In Phase I clinical trials, it demonstrated a
23
PK/PD profile suitable for once daily dosing in man.

1238 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
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Figure 5. Lack of effect of 11 in the mouse rotarod assay of sedation
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of impaired performance whereas at the same dose, diazepam (Diaz.)
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8/group). The % figures denote the % occupancy at each dose. * )
significantly different from control (vehicle), p < 0.05.
Supporting Information Available: Full experimental details
and data for all final compounds. Squirrel monkey RPI lever
pressing assay. Elemental analyses and HPLC data. This information
is available free of charge via the Internet at http://pubs.acs.org.
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JM051200U