An orally bioavailable, functionally selective inverse agonist at the benzodiazepine site of GABAA α5 receptors with cognition enhancing properties

Article abstract of DOI:10.1021/jm040863t

(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2, 4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (13) has been identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA_A α5 receptors. 13 is orall

Full text of DOI:10.1021/jm040863t

J. Med. Chem. 2004, 47, 5829-5832
5829
Chart 1. GABAA Benzodiazepine Site Ligands
An Orally Bioavailable, Functionally
Selective Inverse Agonist at the
Benzodiazepine Site of GABAA r5
Receptors with Cognition Enhancing
Properties
Mark S. Chambers,* John R. Atack,
Robert W. Carling, Neil Collinson, Susan M. Cook,
Gerard R. Dawson, Pushpindar Ferris,
Sarah C. Hobbs, Desmond O’Connor,
George Marshall, W. Rycroft, and Angus M. MacLeod
Merck Sharp & Dohme Research Laboratories,
The Neuroscience Research Centre, Terlings Park,
Eastwick Road, Harlow, Essex, CM20 2QR, U.K.
efficacy), and such compounds have found therapeutic
use as anxiolytics, hypnotics, and anticonvulsants.5
However, they also impair learning and memory pro-
cesses.6,7 Currently, all clinically effective BZ ligands
are full agonists at the BZ binding site on the GABAA
receptor. In contrast, methyl 6,7-dimethoxy-4-ethyl-â-
carboline-3-carboxylate (DMCM; 2) is a nonselective full
inverse agonist,8,9 and it has been shown that nonselec-
tive BZ inverse agonists enhance cognitive performance
in animal models10 but are anxiogenic,11 convulsant,12
and proconvulsant13 and may alter attentional process-
ing.14 Using genetically modified, point-mutated mice,
it has been demonstrated that GABAA receptors con-
taining an R1-subunit mediate the sedative/muscle
relaxant effects of benzodiazepines, whereas R2- or R3-
subunit-containing receptors mediate the anxiolytic and
anticonvulsant effects.15-18 Recently, Collinson et al.19
and Crestani et al.20 have used R5 ‘knock-out’ and point-
mutated mice, respectively, to demonstrate a role for
the GABAA R5-subtype in cognitive processing. In adult
brain, R5-subunit-containing GABAA receptors have a
relatively restricted distribution, being primarily ex-
pressed in the hippocampus, a region of the brain
associated with learning and memory, and, although R5
receptors account for less than 5% of the total GABAA
receptor population in the brain, in the hippocampus
they represent around 20% of all GABAA receptors.4,21,22
Given the molecular genetic evidence and the relative
abundance of GABAA R5 receptors in the hippocampus,
we proposed that an inverse agonist acting at the BZ
site of R5-containing GABAA receptors (hereafter re-
ferred to as the R5-subtype) may have therapeutic utility
as a cognition-enhancing agent and, furthermore, that
an R5-selective inverse agonist may also lack the
unwanted side effects associated with inverse agonist
activity at other GABAA receptor subtypes (i.e., anxio-
genesis or convulsant or proconvulsant activity).
Received July 20, 2004
Abstract: (3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-
1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (13)
has been identified as a functionally selective, inverse agonist
at the benzodiazepine site of GABAA R5 receptors. 13 is orally
bioavailable, readily penetrates the CNS, and enhances per-
formance in animal models of cognition. It does not exhibit
the convulsant, proconvulsant, or anxiogenic activity associ-
ated with nonselective GABAA inverse agonists.
γ-Aminobutyric acid (GABA) is the major inhibitory
neurotransmitter in the central nervous system. GABAA
receptors are GABA-gated chloride ion channels, com-
posed of pentameric assemblies of members of the
GABAA receptor gene family (R1-6, â1-3, γ1-3, δ, ꢀ,
θ, and π)1,2 with the majority of GABAA receptors
comprising of R-, â-, and γ-subunits arranged in a 2:2:1
stoichiometry.3 The binding of GABA to its receptor can
be modulated by simultaneous binding of chemical
entities to allosteric sites on the ion channel complex,
the most studied of which is the benzodiazepine (BZ)
binding site. Upon the basis of their modulatory effects
on GABA-induced GABAA receptor activation, BZ site
ligands are categorized as either agonists (positive
allosteric modulators), inverse agonists (negative allos-
teric modulators) or antagonists. BZ agonists exert their
effect by increasing the frequency of channel opening
in the presence of GABA resulting in an increased
chloride flux through the ion channel to give a net
hyperpolarization of the neuron and a decreased excit-
ability. Conversely, BZ inverse agonists decrease the
frequency of channel opening in the presence of GABA
and thereby increase neuronal excitability. Spanning
these efficacy extremes are partial agonists, antagonists
(which bind to the BZ site on the GABAA receptor but
have no intrinsic efficacy), and partial inverse agonists.
GABAA receptor subtypes which possess a BZ binding
site (and which comprise around 75% of all GABAA
receptor subtypes present in the brain)4 contain â- and
γ2-subunits in conjunction with either an R1-, R2-, R3-,
or R5-subunit.4 Diazepam (1; Valium) is classified as a
nonselective full BZ agonist (i.e. binds to the different
GABAA receptor subtypes with similar affinity and
Although there are many structural classes of com-
pound which bind to the BZ site on the GABAA ion
channel complex, relatively few have been reported as
being selective ligands for the R5-subtype. The imida-
zobenzodiazepines23 such as L-655,708 (3)24 and some
diazepam analogues25 exhibit binding selectivity for the
R5-subtype compared to the other receptor subtypes.
From our laboratories, a series of 6,7-dihydro-2-ben-
zothiophen-4(5H)-ones26,27 has been described which has
binding selectivity for the R5-subtype and from which
* To whom correspondence should be addressed. Tel: (01144)-1279-
440417. Fax: (01144)-1279-440390. E-mail: mark_chambers@
merck.com.
10.1021/jm040863t CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/16/2004

5830 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Letters
Table 1. Binding Affinity and Efficacy of 13, 5, 2, and 14 at Human Recombinant GABAA Rxâ3γ2 Receptor Subtypes
Ki (nM) human GABAA Rxâ3γ2 receptorsa
efficacy at human GABAA Rxâ3γ2 receptors (%)b
compound
R1
R2
R3
R5
R1
R2
R3
R5
13
5
0.83 ( 0.08
0.88 ( 0.19
10 ( 1
0.85 ( 0.16
0.58 ( 0.17
13 ( 5
0.77 ( 0.10
0.61 ( 0.17
7.5 ( 1.2
1.4 ( 0.4
0.66 ( 0.14
2.2 ( 1.0
-16 ( 2
-15 ( 3
-71 ( 2
+100 ( 3
+6 ( 5
+16 ( 4
-53 ( 3
+111 ( 4
-9 ( 2
-7 ( 4
-62 ( 2
+113 ( 4
-55 ( 2
-40 ( 1
-57 ( 1
+99 ( 2
2
14c
605 ( 136
392 ( 73
471 ( 164
368 ( 66
a Displacement of [3H]Ro 15-1788 binding from recombinant human GABAA receptor subtypes with a Rxâ3γ2 composition (where x )
1, 2, 3, or 5) stably expressed in mouse fibroblast L(tk-) cells. Ki values are the geometric mean ( SEM of four independent determinations.
b Maximum modulation of the current produced by 13, 5, 2, or 14 relative to a submaximal (EC20) GABA response. Values are the mean
maximum modulation ( SEM from at least five individually fitted concentration-response curves for each receptor subtype. c Efficacy
values are the mean ( SEM from 31 to 37 cells produced using [14] ) 3 µM.
Scheme 1a
ing 6 with hydrazine hydrate afforded the pyrazole 7
in quantitative yield. The C3-hydroxyl was selectively
protected in 67% yield as the tosylate 8. Protection of
this hydroxyl functionality was necessary in order to
ensure efficient oxidation of the primary alcohol, to give
aldehyde 9. Condensing 9 with 5-methyl-3-isoxazole-
carbohydrazide (10)30 produced, after concomitant cy-
clization, the pyrazolotriazine 11. Removal of the tosyl
group, under basic conditions, gave the hydroxypyra-
zolotriazine 12 in 85% yield, and subsequent alkylation
with 5-chloromethyl-1-methyl-1H-1,2,4-triazole31 pro-
duced 13.
a Reagents: (i) NH2NH2‚H2O, EtOH, reflux; (ii) p-toluenesulfo-
nyl chloride, Et3N, DCM; (iii) MnO2, CHCl3, reflux; (iv) xylene,
reflux; (v) 4 N NaOH, MeOH, 65 °C; (vi) K2CO3, DMF, 5-chloro-
methyl-1-methyl-1H-1,2,4-triazole.
As shown in Table 1, 13 has high in vitro binding
affinity32 at all four BZ-sensitive GABAA receptor
subtypes, ranging from 0.8 to 1.4 nM. It has very weak
affinity at the GABAA R4â3γ2-subtype (Ki 395 ( 173
nM) and is essentially inactive at the GABAA R6â3γ2
receptor (Ki > 4000 nM). Furthermore, when examined
in 147 radioligand binding and enzyme assays,33 13
showed no significant off-target activity (IC50 values >
10 µM). The efficacy values shown in Table 1 were
determined using whole cell patch clamp recordings34
from mouse fibroblast cells stably expressing the human
GABAA receptor subtypes.32 The in vitro efficacy is
measured as the percentage maximum modulation of
the GABA-evoked current using a submaximal (EC20)
GABA concentration. Positive values represent a po-
tentiation of the GABA-induced current (agonist) whereas
negative values represent an attenuation (inverse ago-
nist). The values for 13 are compared to the clinical
compound 5, the nonselective full inverse agonist 2,9 and
the nonselective full agonist chlordiazepoxide (CDZ; 14).
13 is a full inverse agonist at the R5-subtype and is
functionally selective over the R1-, R2-, and R3-subtypes.
In mouse fibroblast L(tk-) cells expressing the R5â3γ2-
subtype, 13 attenuates the GABA-evoked current by
55% (efficacy ) -55%), which is essentially identical to
that of the full inverse agonist 2 (efficacy ) -57%) and
greater than that produced by 5 (efficacy ) -40%). In
contrast, efficacy at the R1-, R2-, and R3-subtypes is
much lower, with respective efficacy values of -16%,
+6%, and -9% being in the range of weak partial
inverse agonists or antagonists. The efficacy of 13 at
the R1- and R3-subtypes is comparable with the record-
ings obtained for 5; whereas at the R2-subtype 13 is
essentially an antagonist (efficacy ) +6%), 5 exhibits
partial agonism (efficacy ) +16%). Indeed, in our quest
for a compound with full inverse agonism at the R5-
subtype and little functional activity at the other
GABAA receptor subtypes, pyrazolotriazine 13 has a
more impressive efficacy profile compared to the clinical
thiophene 4 was identified as a functionally selective
R5-subtype inverse agonist.27 Recently, we have also
described work leading to the identification of the
triazolophthalazine 5, which was chosen for clinical
evaluation as a potential treatment for cognitive disor-
ders.28
In this manuscript, we describe the synthesis and
biological data of a structurally novel, orally bioavail-
able, and functionally selective R5-subtype inverse
agonist which has also been selected for clinical evalu-
ation as a potential cognition-enhancing agent.
In the search for such a compound, our strategy was
to modify the triazolophthalazine core of 5. Various
benzofused and nonbenzofused heterocyclic alternatives
were investigated. In the course of these studies, a series
of pyrazolo[1,5-d][1,2,4]triazines was identified as pro-
viding high affinity R5-subtype inverse agonists. Within
this class of compounds, a tert-butyl group and a
5′-methyl-3′-isoxazolyl substituent at the C3 and C7
positions, respectively, of the pyrazolotriazine core were
the optimum groups for providing high R5 inverse
agonism. As was the case in the triazolophthalazine
series,28 small changes in the heteroarylmethyloxy
substituent at C3 could have a profound effect on
efficacy at one or more of the GABAA receptor subtypes,
and, while the pyrazolotriazines did not exhibit signifi-
cant binding selectivity for any particular subtype,
varying degrees of functional (i.e., efficacy) selectivity
for the GABAA R5-subtype could be attained. Following
an extensive study of the series, the optimum compound
was identified as (3-tert-butyl-7-(5-methylisoxazol-3-yl)-
2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-
d][1,2,4]triazine (13).
As shown in Scheme 1, 13 was synthesized in six
steps from 3-tert-butyl-4-hydroxy-2-furanone (6).29 Heat-

Letters
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5831
Figure 1. Occupancy of GABAA receptor benzodiazepine
binding site by 13 as a function of dose 0.75 h after oral
administration in a 0.5% Methocel vehicle as measured using
an in vivo [3H]Ro 15-1788 binding assay. Values shown are
mean ( SEM (n ) 4-8/group).
Figure 2. Enhanced performance of rats dosed with pyrazo-
lotriazine 13 in the delayed ‘matching-to-place’ water maze
test.39 13 was dosed po at 0.3, 1, and 3 mg/kg in 0.5% Methocel.
Vehicle-treated animals were dosed po with 0.5% Methocel.
The difference in time taken between trial 1 and trial 2
(savings) to find the hidden platform over a five-day test period
is shown. Rats dosed orally at 1 and 3 mg/kg with 13 made
significantly higher savings (*) compared with vehicle-treated
animals. Values shown are mean ( SEM (n ) 9-10/group).
compound 5. The EC50 value of 13 at the R5-subtype is
3.0 nM, which complements its in vitro binding affinity
of 1.4 nM.
The inhibition of in vivo binding of [3H]Ro 15-1788
by 13 was used to determine the occupancy of this
compound at the BZ site of rat brain GABAA receptors.
[3H]Ro 15-1788 binds with equal affinity to the BZ site
of GABAA receptors containing R1-, R2-, R3-, and R5-
subunits, and, therefore, in vivo binding of [3H]Ro 15-
1788 reflects binding to this combined receptor popu-
lation. However, since 13 also binds with equal affinity
to GABAA receptors containing R1-, R2-, R3-, and R5-
subtypes, the dose of 13 required to occupy the R5-
subtype is the same as that required to occupy the
combined R1-, R2-, R3-, and R5-subtypes. As shown in
Figure 1, the occupancy of 13 in rats was dose-depend-
ent after oral dosing, with the mean dose of 13 required
to occupy 50% of GABAA receptor subtypes (0.75 h
postdose) being 0.35 mg/kg.
varies day-to-day but is fixed on any given day) on the
first and second trial is used as an index of how well
the rat has remembered the position of the platform.
Spatial cues around the pool assist the rat in navigating
its way to the platform.39 As can be seen in Figure 2,
13 showed a dose-dependent (0.3-3 mg/kg po) improve-
ment in performance between trial 1 and trial 2
compared with vehicle-treated animals (p < 0.05). The
occupancy of BZ sites on GABAA receptors in a satellite
group of rats dosed at 0.3, 1, and 3 mg/kg po was 61%,
72%, and 85%, respectively. Since 13 was without effect
on swim speed, these data indicates that the function-
ally selective GABAA R5 inverse agonist 13 significantly
enhanced performance in a dose-dependent manner in
this hippocampal-dependent memory task. These data
complements the findings obtained with the structurally
diverse thiophene 427 and triazolophthalazine 528 and
supports the hypothesis that a selective R5-subtype
inverse agonist may have therapeutic utility for the
treatment of cognitive disorders.
In conclusion, the structurally novel, functionally
selective GABAA R5-subtype inverse agonist 13 en-
hances cognitive performance in a hippocampal-depend-
ent memory task, without the anxiogenic, convulsant,
or proconvulsant side effects associated with nonselec-
tive BZ GABAA inverse agonists. 13 has greater inverse
agonism at the R5-subtype and reduced R2 agonism
compared with the clinical compound 5, and, on the
basis of its overall in vitro and in vivo profile, 13 was
selected for clinical evaluation as a potential cognition-
enhancing agent.
The oral bioavailability of 13 in rats and dogs is 52%
and 8%, respectively.
The proconvulsant potential of 13 was determined in
mice.36 At doses up to 10 mg/kg ip, where 99% of GABAA
receptor subtypes are occupied, 13 did not potentiate
pentylenetetrazole-induced convulsions in mice. This
contrasts with the significant convulsant effects reported
in mice for the nonselective BZ full inverse agonist 212
and the proconvulsant effects reported for the nonselec-
tive BZ partial inverse agonist 15 (FG 7142).13 While
nonselective BZ partial inverse agonists are without
direct convulsant effects in mice when given acutely,
they can induce seizures if given daily over a number
of days, a process known as ‘kindling’.13 Repeated dosing
of 13 (30 mg/kg po for 20 days) did not induce seizures,
although seizures were induced with 15 (40 mg/kg ip
for 20 days) which occupied fewer receptors than 13 and
had a shorter duration. The rat elevated plus maze
test37 is sensitive to the anxiogenic effects of nonselec-
tive BZ inverse agonists such as 15.11 In this test, 13
was without significant anxiogenic-like effects in rats
at doses that occupy >95% of BZ binding sites. In
contrast, 15 produced a robust anxiogenic effect.
To investigate the effect that 13 has on learning and
memory we assessed the performance of rats dosed
orally with 13 in the delayed ‘matching-to-place’ variant
of the Morris water maze, a hippocampal-dependent
cognitive test.38,39 In this assay, the difference in time
taken to find a hidden platform (the position of which
Acknowledgment. The authors would like to thank
Elizabeth Norris, Jonathan Rose, Paul Scott-Stevens,
and Nicola Szeto for performing and analyzing the
pharmacokinetic studies, Dave Tattersall for technical
assistance with the PTZ assay, and Luis Castro for
helpful discussions.
Supporting Information Available: Details of the syn-
thesis and characterization of 13 and intermediates (chemistry,
NMR, MS, microanalytical data and melting points), as well
as a detailed description of the biological test methods used.
Data showing the lack of proconvulsant effect produced by 13
on pentylenetetrazole-induced seizures in mice. Data showing

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