Synthesis and evaluation of highly potent GABAA receptor antagonists based on gabazine (SR-95531)

Article abstract of DOI:10.1016/j.bmcl.2011.05.067

A selection of highly potent analogues based on the gabazine structure is described. Their syntheses are carried out in just four steps, and their potencies for antagonism at the GABA_A receptor were measured. All antagonists showed significantly higher potencies compared to the parent competitive antagonist, gabazine.

Full text of DOI:10.1016/j.bmcl.2011.05.067

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4252–4254
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of highly potent GABAA receptor antagonists
based on gabazine (SR-95531)
Favaad Iqbal a, Ryan Ellwood a, Martin Mortensen b, Trevor G. Smart b, James R. Baker a,
⇑
a Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, United Kingdom
b Department of Neuroscience, Physiology & Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
A selection of highly potent analogues based on the gabazine structure is described. Their syntheses are
carried out in just four steps, and their potencies for antagonism at the GABAA receptor were measured.
All antagonists showed significantly higher potencies compared to the parent competitive antagonist,
gabazine.
Received 4 May 2011
Revised 16 May 2011
Accepted 18 May 2011
Available online 25 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Gabazine
GABAA receptor
Pyridazines
GABAA antagonists
Antagonist
GABAA receptors are ligand-gated ClÀ channels belonging to the
Cys-loop superfamily of ionotropic receptors.1 These receptors are
expressed in a variety of isoforms. They play an important role in
inhibiting cell excitation in the central nervous system.2 In addi-
inception, gabazine has become a widespread tool in scientific re-
search of the GABAA receptor.12–15
In our ongoing studies into the development of labelled ana-
logues of gabazine as probes for the GABAA receptor, we discovered
that a relatively minor addition to the gabazine skeleton signifi-
cantly enhances its antagonist potency. Herein, we report on highly
potent gabazine analogues that inhibit GABAA receptor function,
describing their synthesis and pharmacological evaluation.
Our initial target was the benzyl analogue of gabazine (7a,
Scheme 1), the testing of which would indicate whether substitu-
ents in this position are tolerated.
The preparation of novel arylpyridazine 4 outlined in Scheme
1 adopts a microwave Suzuki–Miyaura protocol for the coupling
of 3-amino-6-chloropyridazine 3 and 4-hydroxybenzeneboronic
acid affording 4 in 72% yield.16–18 We installed the benzyl motif
in 5a via a Williamson ether synthesis with benzyl bromide,
achieving the synthesis of 5a in a reasonable 41% yield. The abil-
ity to achieve selective N(2)-alkylation of arylpyridazines stems
from a combination of steric effects from the adjacent phenyl
tion to mediating the effects of endogenous c-aminobutyric acid
(GABA), GABAA receptors are also modulated by an array of com-
pounds including neurosteroids and benzodiazepines.3
A number of small molecule antagonists are known for the
GABAA receptor.4–9 Heaulme et al. showed SR 95103 (1, Fig. 1) to
be a selective antagonist at the GABA binding sites on GABAA
receptors.10 Subsequent structure–activity relationship studies
demonstrated that a series of aminopyridazine derivatives acted
as selective, competitive antagonists at GABAA receptors.10,11 These
reports showed that the potency of these antagonists was clearly
linked to the presence of an aromatic ring at the 6-position of
the pyridazine ring and that the carboxylic acid side-chain was
essential for antagonism at the GABAA receptor agonist binding
sites. Furthermore, they also noted that substituents attached to
the aromatic ring influenced antagonist potency. The most potent
GABAA antagonist reported was the para-methoxy analogue, gab-
azine, also known as SR-95531 (2, Fig. 1).11 Ueno et al. demon-
strated that gabazine partially inhibited direct activation of the
receptor by the barbiturate pentobarbital and by the steroid alpha-
xolone, not by blocking their binding, but possibly by acting as an
allosteric inhibitor of GABAA receptor channel opening.7 Since its
5
4
5
4
3
3
6
6
NH.HBr
O
O
NH.HBr
O
N
N
N
N
2
1
1
2
3
3
Gabazine
SR 95531 (2)
SR 95103 (1)
OH
OH
⇑
Corresponding author. Tel.: +44 (0)20 7679 2653; fax: +44 (0)20 7679 7463.
E-mail address: j.r.baker@ucl.ac.uk (J.R. Baker).
Figure 1. Structure of SR 95103 (1) and gabazine (2).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.067

F. Iqbal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4252–4254
4253
(a)
(b)
O
NH2
Cl
NH2
HO
NH2
N
N
N
N
N
N
5a
(c)
4
3
O
NH.HBr
O
O
NH.HBr
O
N
N
N
N
(d)
6a
7a
3
3
O
OH
Scheme 1. Reagents and conditions: (a) 4-hydroxybenzeneboronic acid, bis(triphenylphosphine)palladium(II) dichloride, potassium carbonate, MeCN/H2O, 120 °C, 72%;
(b) sodium hydride, benzyl bromide, DMF, 0 °C, 41%; (c) allyl-4-bromobutyrate, DMF, 80 °C, 59%; (d) palladium(II) acetate, triethyl phosphite, dimedone, THF/H2O, 56%.
Table 1
120
Structure–activity relationships of gabazine (2) and its analogues (7a–c)
100
80
60
40
20
0
RO
NH.HBr
O
N
N
3
OH
Entry
Compound
IC50 (nM)
Relative potency
i
2 (R = CH3)
7a (R = C6H5CH2)
7b (R = m-MeOC6H4CH2)
7c (R = m-NO2C6H4CH2)
349
11
7
1
32
50
ii
iii
iv
3
116
[Antagonist], nM
IC50 values are mean (n = 5–8). Relative potency is determined as an IC50 ratio with
Figure 2. Concentration inhibition curves for gabazine 2 (open squares), 7a (filled
squares), 7b (open circles) and 7c (filled triangles) on recombinant 1b2 2S GABAA
receptors activated by EC50 GABA (10 M).
respect to gabazine (= 1).
a
c
l
group and resonance stabilisation.11 N(2)-alkylation of arylpyrid-
azines can be achieved through reaction with an appropriate
bromoester; ethyl-4-bromobutyrate is commonly used. However,
the harsh deprotection conditions that would be required were of
concern.11 We chose to react 5a with the corresponding allyl es-
ter, affording the protected antagonist 6a in 59% yield. Allyl ester
6a was easily deprotected using a relatively benign Pd-mediated
method in 56% yield, thus affording the desired analogue 7a in
just four steps.
O
NH.HBr
O
a, b, c
4
N
N
7d
3
OH
Scheme 2. Reagents and conditions: (a) propargyl bromide, sodium hydride, DMF,
0 °C, 71%; (b) allyl-4-bromobutyrate, DMF, 80 °C, 88%; (c) tetrakis(triphenylphos-
phine)palladium(0), morpholine, 86%.
We examined the potency of this compound, using patch clamp
In summary, we have developed a novel series of GABAA recep-
tor antagonists. We have demonstrated that by the simple addition
of a benzyl group, the antagonist potency of arylpyridazine ana-
logues of GABA can be greatly increased. This is exemplified by
7a, where we observed a 30-fold increase in potency compared
to gabazine. We also found the inclusion of an electron-donating
methoxy-group or an electron-withdrawing nitro-group on the
meta-position of the benzyl ring (7b and 7c, respectively) were
not only tolerated but also enabled further increases in potency. Fi-
nally, we demonstrated that the presence of an additional benzyl
group is not the only means of eliciting increments in antagonist
potency. In the case of 7d, a simple propargyl group will clearly
suffice. The versatility of the alkyne and benzyl groups provides
useful tools for further structural exploration of the core antagonist
structure.
electrophysiology with recombinant
a1b2c2S GABAA receptors
transiently expressed in HEK293 cells. By constructing concentra-
tion inhibition curves for the response to EC50 GABA, the lower
IC50 for 7a (IC50 = 11 nM, Table 1, entry ii) indicated a 32-fold in-
crease in antagonist potency compared to gabazine (IC50 = 349 nM,
entry i). We also synthesised the meta-methoxy analogue 7b in or-
der to assess the feasibility of attaching tags to this position. The
potency of this compound was increased further (IC50 = 7 nM, en-
try iii). Interestingly, installing an electron-withdrawing nitro-
group in 7c even further enhanced antagonist potency (IC50 = 3 nM,
entry iv).
Our findings indicate that there is significant scope to modify
the gabazine skeleton on the alkoxy substituent and considerably
enhance antagonist potency at the GABAA receptor (Fig. 2).
In our final analogue 7d (Scheme 2), we installed a propargyl-
oxy group in place of the methoxy-group on 2, thereby creating
an alternative diverse point of attachment for prospective labelling
groups. By employing a similar synthetic strategy as previous, we
were able to isolate 7d, which again showed an increased potency
(IC50 = 40 nM; n = 6) compared to gabazine.
Acknowledgements
This work has been supported by an MRC programme grant and
an EPSRC studentship.

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have all been previously described.19 Supplementary data (experi-
mental procedures and characterisation of compounds) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.05.067.
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