Discovery of 2-(Imidazo[1,2- b]pyridazin-2-yl)acetic Acid as a New Class of Ligands Selective for the γ-Hydroxybutyric Acid (GHB) High-Affinity Binding Sites

Article abstract of DOI:10.1021/acs.jmedchem.9b00131

Gabazine, a γ-aminobutyric acid type A (GABA_A) receptor antagonist, has previously been reported to inhibit the binding of [³H]NCS-382, a representative ligand of the high-affinity binding site for the neuroactive substance γ-hydroxy

Full text of DOI:10.1021/acs.jmedchem.9b00131

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Article
Discovery of 2-(Imidazo[1,2-b]pyridazin-2-yl)acetic
Acid as a New Class of Ligands Selective for the #-
Hydroxybutyric Acid (GHB) High-Affinity Binding Sites
Jacob Krall, Francesco Bavo, Christina B. Falk-Petersen, Claus Hatt Jensen, Julie Olsson Nielsen,
Yongsong Tian, Valeria Anglani, Kenneth Thermann Kongstad, Louise Piilgaard, Birgitte Nielsen, David
E. Gloriam, Jan Kehler, Anders Jensen, Kasper Harpsøe, Petrine Wellendorph, and Bente Frølund
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00131 • Publication Date (Web): 14 Feb 2019
Downloaded from http://pubs.acs.org on February 14, 2019
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Discovery of 2-(Imidazo[1,2-b]pyridazin-2-yl)acetic
Acid as a New Class of Ligands Selective for the γ-
Hydroxybutyric Acid (GHB) High-Affinity Binding
Sites
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†
†,ƒ,‡
† ,‡
†
Jacob Krall, Francesco Bavo, Christina B. Falk-Petersen, Claus H. Jensen, Julie O.
†
†
†
†
†
Nielsen, Yongsong Tian, Valeria Anglani, Kenneth T. Kongstad, Louise Piilgaard, Birgitte
†
†
§
†
†
Nielsen, David E. Gloriam, Jan Kehler, Anders A. Jensen, Kasper Harpsøe, Petrine
†
,
†,
Wellendorph, *Bente Frølund *
†
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,
ƒ
University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Department of
§
Pharmaceutical Sciences, University of Milan, via Mangiagalli 25, 20133 Milano, Italy. Discovery
Chemistry and DMPK, H. Lundbeck A/S, Ottiliavej, DK-2500 Valby, Denmark
KEYWORDS γ-Hydroxybutyric acid (GHB), gabazine (SR-95531), high-affinity GHB binding site,
NCS-382, structure-affinity studies, pharmacophore model, prodrug.
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ABSTRACT. Gabazine, a GABA
A
receptor antagonist, has previously been reported to inhibit the
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binding of [ H]NCS-382, a representative ligand of the high-affinity binding site for the neuroactive
substance GHB. We herein report a study on the structural determinants of gabazine for binding to i)
A
the orthosteric binding site of the GABA receptor and ii) the high-affinity GHB binding site.
Expanding the structural diversity of available ligands for the high-affinity GHB binding sites, this
study identified 2-(imidazo[1,2-b]pyridazin-2-yl)acetic acid as a novel ligand-scaffold leading to
3
analogs with relatively high affinity (K
i
0.19–2.19 µM) and >50 times selectivity for the [ H]NCS-
3
3
82 over [ H]muscimol binding sites. These results highlight that gabazine interacts with the high-
A
affinity GHB and orthosteric GABA receptor binding sites differently, and that distinct analogs can
be generated to select between them. To facilitate further in vivo studies a promising prodrug
candidate for brain delivery was identified.
Introduction
γ-Hydroxybutyric acid (GHB), a metabolite of γ-aminobutyric acid (GABA), is present in
1
micromolar concentration in the mammalian brain. The pharmacology of GHB is complex and it has
been shown to bind to both high- and low affinity targets. The observed clinical and behavioral effects
of GHB (e.g. sedation, hypothermia, sleep regulation, and rewarding effects) are predominantly
2
-6
mediated via the GABA receptors where GHB acts as a weak partial agonist (EC 5 mM).
B
50
However, a group of subtypes of the GABA
A
A 4
receptors (GABA Rs) consisting of α β1–3δ has shown
7
apparent implication in GHB high-affinity binding.
The GABA
central nervous system. A high degree of structural heterogeneity of the GABA
A
Rs are ligand-gated ion channels that mediate the overall inhibitory signaling in the
Rshas been revealed
A
and is reflected in multiple receptor subtypes built up as pentameric assemblies comprised of 19
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A
different GABA R subunits; α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3. A rich and complex pharmacology
has been observed based on multiple subtypes, allosteric binding sites and diverse subcellular and
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regional localization. GABA
A
Rs containing the γ subunit are the major synaptic receptors whereas
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δ-containing GABA
to the low extrasynaptic GABA concentration.
More detailed structural insight is emerging into the GABA
structures of related receptors and recently the release of a β
A
Rs are thought to be mainly extrasynaptic, mediating a tonic current in response
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0
A
Rs in terms of full-length crystal
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1-14
3
A
homopentameric GABA R.
Furthermore, extensive structure-activity relationship (SAR) studies have been performed over the
15, 16
years.
A
Consequently, a large number of potent and selective ligands for the orthosteric GABA R
1
7
binding site have been reported, e.g. the classical antagonist gabazine (SR-95531) and a series of
1
8-20
highly potent competitive antagonists
including compounds 1a,b (Figure 1).
O
O
O
HO
HO
H N
2
OH
OH
OH
GHB
HO
GABA
MeO
HOCPCA
COOH
N
N
COOH
NH
Gabazine
NCS-382
R1
OH
N
O
N
HN
COOH
R2
OH
BnOPh-GHB (2)
1
1
a: R = 3-biphenyl, R = H
1
2
b: R = H, R2 = 3-biphenyl
1
Figure 1. Chemical structures of GHB, GABA, HOCPCA, NCS-382, gabazine, GABA R
A
antagonists 1a,b, and BnOPh-GHB (2).
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In contrast, the molecular identity and structural composition of the high-affinity GHB binding sites
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, 21, 22
have for long been elusive.
In order to achieve more knowledge and understanding of these
binding sites, we have previously published two series of selective ligands with mid to high
nanomolar affinity: i) The conformationally restricted GHB analog 3-hydroxycyclopenten-1-
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enecarboxylic acid (HOCPCA, Figure 1), displaying 27 times higher affinity than GHB, and ii) the
4-substituted biaromatic GHB analogs, represented by 4-(4-benzyloxy)phenyl)-4-hydroxybutanoic
acid (BnOPh-GHB, 2, Figure 1), displaying 39 times improved affinity compared to GHB. The latter
2
4
series indicates the presence of a spacious cavity in the vicinity of the GHB scaffold binding area.
However, our recent study, aimed at expanding the SAR for HOCPCA, and reports on GHB elude to
2
1, 25, 26
a very tight binding of the core scaffold.
affinity GHB binding site is low and, in essence, only represented by the core structures of GHB,
HOCPCA, and NCS-382 ((E)-2-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-
Overall, the chemical diversity of ligands for the high-
ylidene)acetic acid). To advance the field, exploring chemical space for novel chemical entities for
the high-affinity GHB binding sites is needed.
Numerous GABA
A
R ligands, orthosteric agonists and antagonists as well as modulators, have been
3
tested for their ability to inhibit binding of [ H]NCS-382, which is selective for the high-affinity GHB
7
3
binding site. Only gabazine was found to inhibit [ H]NCS-382 binding in the micromolar range (K
i
7
16 µM), whereas GABA was in the millimolar range (K
i
2.1 mM). Additionally, gabazine was found
125
3
27
to inhibit the binding of the GHB specific radioligands [ I]BnOPh-GHB and [ H]HOCPCA in a
7
, 28
regionally specific manner similar to GHB as shown by autoradiography in rodent brain slices.
Inspired by the above-mentioned dual activities of gabazine, the objective of the present study was
to investigate the structural determinants of gabazine for recognizing either the high-affinity GHB
binding site or GABA Rs in rat cortical synaptic membranes. Toward this aim, systematic SAR
A
studies, including fragmentation, substitution pattern at the aryl ring, and conformational restriction
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of the gabazine structure were performed. To obtain knowledge on the three-dimensional (3D)
structure of the high-affinity GHB ligand binding site in comparison to the orthosteric GABA binding
2
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site, we incorporated the obtained SAR data into a recently developed 3D-pharmacophore model.
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Results
A series of gabazine analogs was synthesized in order to discern the structural determinants for the
binding of gabazine to GHB high-affinity binding sites and GABA Rs. The structural design of
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analogs was based on a systematic investigation of different parts of the 6-aryl-3-
aminopyridazinebutanoic acid scaffold constituting the core scaffold of gabazine (Figure 2): the
aromatic ring system and substituents (compound series 3), the flexibility of the butyric acid side
chain (compound series 4 and 5), and the pyridazine moiety (compounds 6 and 7).
R
R
N
COOH
N
COOH
N
N
NH
3
4
4
R
X
COOH
N
COOH
N
N
N
N
5
6: X = N
7: X = C
Figure 2. General structures of target compounds 3–7.
The synthesized gabazine analogs were characterized pharmacologically in binding studies using
rat cortical synaptic membranes, where the binding affinities of compounds 3a–g,k–m, 4a–c,h,i, 5b–
e,h–j, 6, and 7 at the high-affinity GHB binding sites and at native GABA Rs were measured by
A
3
3
displacement of [ H]NCS-382 and [ H]muscimol, respectively (Table 1 and 2). Functional
characterization of selected compounds was further performed at the human α β γ GABA R
1
2
2S
A
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transiently expressed in tsA201 cells using the FLIPR Membrane Potential (FMP) Blue assay as
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described previously.
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The new gabazine analogs 3a–g,k–m, 4a–c,h,i, 5b–e,h–j, and 6 were synthesized as depicted in
3
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31
31
Scheme 1 to 3. Compounds 3a, 6, and 15 were synthesized according to procedures reported in
3
2
the literature, while compounds 8a–i were synthesized according to a modified procedure previously
described in the literature (please refer to Experimental Section for further information). Compound
7
was obtained from a commercial vendor.
Structure-activity studies on the phenyl moiety of gabazine (3a–g,k–m)
We first explored the influence of the substitution pattern of the 6-phenyl group of gabazine on the
affinity for the GHB high-affinity binding sites and the orthosteric GABA R binding site. A series of
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compounds in which the 3- or 4-position of the 6-phenyl ring in gabazine was substituted by a variety
of electron-donating and electron-withdrawing groups were synthesized as depicted in Scheme 1 and
2
. The 6-aryl-pyridazin-3-amines (8a–j) for the three series of gabazine analogs (3a–g,k–m, 4a–c,h,i,
and 5b–e,h–j) were synthesized starting from the commercially available 6-chloropyridazin-3-amine
(
9) by a Suzuki-Myiaura cross coupling with suitable arylboronic acids following a modified
3
2
literature procedure using conventional heating compared to microwave-assisted synthesis.
a
Scheme 1
a Ph
b 3-Cl-Ph
f 3-NO -Ph
2
Cl
N
R
N
g 4-NO -Ph
2
N
i
N
c 4-Cl-Ph
h 3-OMe-Ph
NH2
NH2 d 3-BnO-Ph
e 4-BnO-Ph
i
j
4-OMe-Ph
3,4-Cl -Ph
9
8a-j
2
a
Reagents and conditions: (i) 6-chloropyridazine-3-amine (9), R-B(OH) , Pd(PPh ) , Na CO or
2
3 4
2
3
K CO , EtOH/H O (4:1), reflux.
2
3
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The selectivity of N - over N -alkylation of 6-aryl-3-aminopyridazines has previously been
2
1
1
7, 32, 33
described in the literature
and following these findings, 10a–g were obtained by N -alkylation
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of 8a–g using ethyl 4-bromobutyrate in the presence of potassium carbonate and conventional
heating. The 3-aniline substituted analog 10k was obtained by a catalytic reduction of the
corresponding 3-nitrophenyl substituted analog 10f using palladium on carbon and hydrogen in
ethanol. Deprotection of compounds 10a–g,k was performed by using either sodium hydroxide or
hydrochloric acid to obtain compounds 3a–g,k–m. Using hydrochloric acid in the deprotection of the
O-benzyl substituted analogs 10d,e provided the corresponding hydroxyl substituted analogs 3l,m.
a
Scheme 2
R
N
N
NH2
8
a-g
10a Ph
0b 3-Cl-Ph
10c 4-Cl-Ph
10e 4-BnO-Ph
1
10f 3-NO
10g 4-NO
2
2
-Ph
-Ph
i
ii
10d 3-BnO-Ph 10k 3-NH -Ph
2
R
N
N
COOEt
NH
0a–g,k
3a Ph
3f 3-NO -Ph
2
1
3
3
3
b 3-Cl-Ph
c 4-Cl-Ph
3g 4-NO -Ph
2
3k 3-NH -Ph
2
3
3
a,b,f,g,k-m: iii
c-e: iv
d 3-BnO-Ph 3l 3-OH-Ph
3e 4-BnO-Ph 3m4-OH-Ph
R
N
N
COOH
NH
3a–g,k–m
a
Reagents and conditions: (i) Ethyl 4-bromobutyrate, K CO , DMF, 80 °C, (ii) Pd/C, H , EtOH, rt.,
2
3
2
(iii) AcOH, 35% HCl, 100 °C or reflux, (iv) NaOH, H O, THF, rt.
2
Pharmacological evaluation of compounds 3a–g,k–m revealed only minor differences in the SAR
pattern at the two target proteins. Hence, the following description of the SAR of these compounds
will mainly focus on observed trends rather than on pronounced differences.
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Like gabazine, all of the synthesized compounds 3a–g,k–m displayed mid to high nanomolar
affinity for the orthosteric GABA R binding site. Electron-donating as well as electron-withdrawing
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groups are tolerated showing affinities for the GABA R binding site comparable to, or slightly higher
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or lower than, that of gabazine. Relative to the unsubstituted 3a, a 10 and 20 times increase in affinity
was observed when going from a 3-nitro (3f) to a 3-chloro (3b) substituent, respectively. In contrast,
no change in affinity was observed changing the chloro to a nitro group in the 4-position (3c to 3g).
The latter also applies to having a hydroxyl group in either the 3- or 4-position (3l and 3m,
respectively). Thus, for the small substituents, there seems to be no clear preference for the 3- or 4-
position. However, introduction of a benzyloxy group showed a ~40-fold preference for the 4-
position with a 43-fold increase in affinity of 3e relative to the unsubstituted 3a, while a similar
structural change in the 3-position (3d) had no effect.
3
As observed for the GABA R, compounds 3a–g,k–m were found to inhibit [ H]NCS-382 binding
A
with K values in the mid to low micromolar range, being around five times higher or lower than that
i
of gabazine despite electronic properties and position of the substituents. For the GHB high-affinity
binding sites, the introduction of a benzyloxy substituent, in the 3- or 4-position, resulted in the
highest affinity compounds 3d and 3e with 25 and 33 times increase in affinity, respectively,
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compared to the unsubstituted 3a. However, in contrast to the orthosteric GABA R binding site, there
was no marked difference between the 3- and 4-positions. This observation also applies to the chloro,
nitro, and hydroxyl substituents, which in all cases result in an increase in affinity below 10 times
relative to 3a.
The functional properties of selected analogs (3f,g,k–m) were characterized at the human α β γ
2 2S
1
GABA R transiently expressed in tsA201 cells in the FMP Blue assay. Here, the analogs were found
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to be moderately potent antagonists, exhibiting low-to-mid-micromolar IC values at the receptor,
5
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and the rank order of these were in good agreement with their respective binding affinities at the
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native GABA Rs in the [ H]muscimol binding assay (Table 1).
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Pharmacological data for GABA, GHB, 1a,b, 2, gabazine, and synthesized compounds 3a–
g,k–m: GABA R and GHB binding affinities at rat cortical synaptic membranes and functional
A
properties at the human α β γ GABA R transiently expressed in tsA201 cells in the FMP Blue
1
2
2S
A
assay.
R
N
N
COOH
NH
3a–g,k–m
ꢀ
3
3
[
H]muscimolꢀbindingꢀ [ H]NCS-382ꢀbindingꢀ
1 2 2S
α β γ ꢀtsA201ꢀcellꢀlineꢀ
Compoundꢀ
Rꢀ
a
a
b
K
i
ꢀ(µM) ꢀ[pK
i
ꢀ±ꢀSEM]ꢀ
K
i
ꢀ(µM) ꢀ[pK
i
ꢀ±ꢀSEM]ꢀ
50 50
IC ꢀ(µM) ꢀ[pIC ꢀ±ꢀSEM]ꢀ
c
d
GABAꢀ
GHBꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0.049 ꢀ
2100 ꢀ
ꢀ
ꢀ
e
d
>1000 ꢀ
4.3 ꢀ
f
f
1
aꢀ
0.0028 ꢀ
>100ꢀ
0.024 ꢀ
f
f
1bꢀ
0.030 ꢀ
>100ꢀ
0.21 ꢀ
g
g
2
ꢀ
>100 ꢀ
0.12 ꢀ
ꢀ
h
d
h
Gabazineꢀ 4-OMe-Phꢀ
0.074 ꢀ
16 ꢀ
0.24 ꢀ
3
aꢀ
3bꢀ
cꢀ
3dꢀ
eꢀ
fꢀ
Phꢀ
0.81ꢀ[6.10ꢀ±ꢀ0.04]ꢀ
0.039ꢀ[7.41ꢀ±ꢀ0.01]ꢀ
0.61ꢀ[6.23ꢀ±ꢀ0.06]ꢀ
0.87ꢀ[6.07ꢀ±ꢀ0.05]ꢀ
0.016ꢀ[7.81ꢀ±ꢀ0.07]ꢀ
0.078ꢀ[7.11ꢀ±ꢀ0.03]ꢀ
0.75ꢀ[6.13ꢀ±ꢀ0.01]ꢀ
1.1ꢀ[5.95ꢀ±ꢀ0.02]ꢀ
0.13ꢀ[6.89ꢀ±ꢀ0.01]ꢀ
0.22ꢀ[6.66ꢀ±ꢀ0.02]ꢀ
100ꢀ[4.01ꢀ±ꢀ0.08]ꢀ
21ꢀ[4.66ꢀ±ꢀ0.09]ꢀ
11ꢀ[4.99ꢀ±ꢀ0.08]ꢀ
4.2ꢀ[5.39ꢀ±ꢀ0.09]ꢀ
3.1ꢀ[5.52ꢀ±ꢀ0.06]ꢀ
13ꢀ[4.90ꢀ±ꢀ0.05]ꢀ
14ꢀ[4.84ꢀ±ꢀ0.02]ꢀ
30ꢀ[4.53ꢀ±ꢀ0.04]ꢀ
16ꢀ[4.81ꢀ±ꢀ0.04]ꢀ
15ꢀ[4.83ꢀ±ꢀ0.02]ꢀ
n.d.ꢀ
n.d.ꢀ
n.d.ꢀ
n.d.ꢀ
3-Cl-Phꢀ
4-Cl-Phꢀ
3-BnO-Phꢀ
4-BnO-Phꢀ
3
i
3
0.011 ꢀ
3
3-NO
4-NO
3-NH
2
2
-Phꢀ
-Phꢀ
4.4ꢀ[5.36ꢀ±ꢀ0.06]ꢀ
~30ꢀ[4.5]ꢀ
3
3
gꢀ
kꢀ
2
-Phꢀ
~30ꢀ[4.5]ꢀ
3
lꢀ
3-OH-Phꢀ
4-OH-Phꢀ
3.9ꢀ[5.40ꢀ±ꢀ0.03]ꢀ
11ꢀ[4.94ꢀ±ꢀ0.01]ꢀ
3
mꢀ
a
IC values were extracted from the concentration–inhibition curves and converted into Ki values
50
using the Cheng–Prusoff equation. The mean K values are given, along with pK ± SEM values, and
i
i
b
are based on 3–4 independent experiments. The mean IC values are given, along with pIC ± SEM
5
0
50
values, and are based on at least three independent experiments using GABA EC –EC as agonist
7
0
90
c
34 d
7 e
concentration. From Lolli et al., 2006. From Absalom et al., 2012. From Wellendorph et al.,
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
23 f
18 g
24 h
20 i
2
005. From Møller et al., 2010. From Høg et al., 2008. From Frølund et al., 2005. From
3
5
Iqbal et al., 2011. n.d.; not determined.
Exploration of conformationally restricted gabazine analogs (4a–c,h,i, 5b–e,h–j, and 6)
The next step was to study the importance of the conformational flexibility of the butyric acid side
chain of gabazine. In these analogs, the butyric acid side chain was partly locked via the exo-amino
group into a dihydroimidazopyridazine or imidazopyridazine scaffold.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3
R
N
i
ii
N
COOEt
NH
11
R
N
iii
R
N
COOEt iv
R
N
COOH
N
N
N
NH2
N
N
8
a-e,h-j,14
12b 3-Cl-Ph
12c 4-Cl-Ph
4a Ph
4b 3-Cl-Ph
4c 4-Cl-Ph
1
1
2h 3-MeO-Ph
2i 4-MeO-Ph
4h 3-MeO-Ph
4-MeO-Ph
4
i
R
N
COOEt
R
ii or iv
N
COOH
v
N
N
N
N
13b 3-Cl-Ph
5b 3-Cl-Ph
5c 4-Cl-Ph
5d 3-BnO-Ph
5e 4-BnO-Ph
5h 3-MeO-Ph
5i 4-MeO-Ph
1
1
3c 4-Cl-Ph
3d 3-BnO-Ph
13e 4-BnO-Ph
1
1
3h 3-MeO-Ph
3i 4-MeO-Ph
13j 3,4-Cl
-Ph
5j 3,4-Cl -Ph
6
H
2
2
1
5
H
a
Reagents and conditions: (i) 8a, ethyl 4-bromocrotonate, DMF, 80 °C, (ii) NaOH, H O, THF, rt.,
iii) a) 8b,c,h,i, ethyl 4-bromocrotonate, DMF, 80 °C, b) K CO , DMF, rt., (iv) 4M HCl, 45 °C, (v)
b–e,h–j and 14, ethyl 4-chloroacetoacetate, EtOH, 80 °C.
2
(
8
2 3
The synthesis of the dihydroimidazopyridazine containing gabazine analogs 4a–c,h,i (Scheme 3)
was inspired by the reported intramolecular cyclisation of a α,β-unsaturated ethyl ester substituted
3
6
compounds either spontaneously in water or in the presence of base. Alkylation of 8a using ethyl 4-
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2
3
4
bromocrotonate provided the ethyl-protected α,β-unsaturated analog 11 (Scheme 3). The isolation of
11 proved troublesome and spontaneous cyclization of compound 11 was observed using standard
column chromatography. Instead, 11 was isolated by pouring the crude reaction mixture into cold
ethyl acetate followed by filtration. Cyclization followed by basic hydrolysis afforded compound 4a.
The remaining dihydroimidazopyridazine analogs 4b,c,h,i were synthesized by applying a one-pot
synthesis strategy. Compounds 8b,c,h,i were alkylated using ethyl 4-bromocrotonate and upon
completion of the alkylation (followed by HPLC analysis), excess of solid potassium carbonate was
added to the crude reaction mixture and stirring was continued until completion of the cyclization
(followed by HPLC analysis). Work-up and purification afforded compounds 12b,c,h,i, which were
converted into 4b,c,h,i using hydrochloric acid.
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The imidazopyridazine analogs (5b–e,h–j, Scheme 3) were synthesized by inspiration from a
procedure previously published by Luraschi et al., starting from 8b–e,h–j and ethyl 4-
31
ethylchloroacetoacetate in ethanol. The ethyl protected imidazopyridazine analogs (13b–e,h–j)
were deprotected to afford gabazine analogs 5b–e,h–j. To further investigate the importance of the
distal aryl-group in this series of gabazine analogs at the GABA R and the GHB binding sites, the
A
unsubstituted imidazopyridazine analog 6 was synthesized from compound 14 as described by
31
Luraschi et al. (Scheme 3).
Table 2. Pharmacological data for GABA, GHB, 1a,b, 2, gabazine, and compounds 4a–c,h,i, 5b–
e,h–j, 6, and 7: GABA R and GHB binding affinities at rat cortical synaptic membranes.
A
R
N
COOH
R
N
COOH
X
COOH
N
N
N
N
N
N
4a–c,h,i
5b–e,h–j
6: X=N
: X=C
7
ꢀ
3
3
Compoundꢀ
Rꢀ
[ H]muscimolꢀ
[ H]NCS-382ꢀ
bindingꢀ
bindingꢀ
a
a
K
i
ꢀ(µM) ꢀꢀ
K
i
ꢀ(µM) ꢀꢀ
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[
pK
i
ꢀ±ꢀSEM]ꢀ
[pK
i
ꢀ±ꢀSEM]ꢀ
b
c
GABAꢀ
GHBꢀ
1aꢀ
ꢀ
0.049 ꢀ
2100 ꢀ
d
c
ꢀ
>1000 ꢀ
4.3 ꢀ
e
ꢀ
0.0028 ꢀ
>100ꢀ
>100ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
e
1
bꢀ
ꢀ
0.030 ꢀ
f
f
2ꢀ
ꢀ
>100 ꢀ
0.12 ꢀ
g
c
Gabazineꢀ
ꢀ
0.074 ꢀ
15.6 ꢀ
4aꢀ
Phꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
>100ꢀ
427ꢀ[3.41ꢀ±ꢀ0.14]ꢀ
>100ꢀ
4
bꢀ
4cꢀ
hꢀ
4iꢀ
3-Cl-Phꢀ
4-Cl-Phꢀ
3-OMe-Phꢀ
4-MeO-Phꢀ
3-Cl-Phꢀ
4-Cl-Phꢀ
3-BnO-Phꢀ
4-BnO-Phꢀ
3-MeO-Phꢀ
4-MeO-Phꢀ
146ꢀ[3.84ꢀ±ꢀ0.06]ꢀ
>100ꢀ
4
45ꢀ[4.35ꢀ±ꢀ0.06]ꢀ
0.23ꢀ[6.65ꢀ±ꢀ0.05]ꢀ
0.22ꢀ[6.67ꢀ±ꢀ0.06]ꢀ
0.51ꢀ[6.33ꢀ±ꢀ0.12]ꢀ
2.2ꢀ[5.67ꢀ±ꢀ0.07]ꢀ
0.44ꢀ[6.36ꢀ±ꢀ0.01]ꢀ
0.83ꢀ[6.09ꢀ±ꢀ0.05]ꢀ
0.19ꢀ[6.77ꢀ±ꢀ0.10]ꢀ
9.9ꢀ[5.01ꢀ±ꢀ0.04]ꢀ
72ꢀ[4.14ꢀ±ꢀ0.05]ꢀ
5
bꢀ
5cꢀ
5dꢀ
5eꢀ
5hꢀ
5
iꢀ
jꢀ
5
2
3,4-Cl -Phꢀ
6
ꢀ
ꢀ
ꢀ
ꢀ
7
a
IC50 values were extracted from the concentration–inhibition curves and converted into K
using the Cheng–Prusoff equation. The mean K values are given, along with pK ± SEM values, and
are based on 3–4 independent experiments. From Lolli et al., 2006. From Absalom et al., 2012.
From Wellendorph et al., 2005.
i
values
i
i
b
34 c
7
d
23 e
18 f
24 g
From Møller et al., 2010. From Høg et al., 2008.
From
2
0
Frølund et al., 2005.
The pharmacological evaluation of 4a–c,h,i, 5b–e,h–j, 6, and 7 at the GABA Rs and GHB high-
A
affinity binding sites revealed a number of interesting observations (Table 2). Linking the exo-amino
group to the butyric acid side chain afforded two series of analogs containing
dihydroimidazopyridazine (4a–c,h,i) or imidazopyridazine (5b–e,h–j) scaffolds with complete loss
3
of GABA R affinity. Dihydroimidazopyridazine analogs 4a–c,h were poor at displacing [ H]NCS-
A
3
82, yielding K
i
values higher than 100 µM. In contrast, the imidazopyridazine analogs 5b–e,h–j
3
were found to inhibit [ H]NCS-382 binding with K values in the high nanomolar range. An
i
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3
illustrative example of binding curves obtained in the [ H]NCS-382 binding assay for 3c, 4c, and 5c
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
is given in Figure 3.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
Figure 3. Concentration-dependent inhibition of [ H]NCS-382 binding to rat cortical synaptic
membranes by the 4-chloro substituted gabazine analogs 3c, 4c, and 5c. The affinities were
determined as described in the Experimental section. Results are given as mean ± SD of a single
representative experiment performed in triplicate. Two additional experiments gave similar results
(the data is summarized in Table 1 and Table 2).
The decreased affinity of the unsubstituted imidazopyridazine analog 6 for the high-affinity GHB
binding site reflects that a 3- and/or 4-substituted phenyl is generally favorable for affinity. As for the
corresponding 3-amino-pyridazine analogs, no clear preference for substituents in either the 3- or 4-
position was observed. Additionally, there was apparently no marked preference for the nature of the
substituent as a chloro, a benzyloxy or methoxy (5b, 5d, or 5h) in the 3-position, practically resulting
in similar affinity as when the substituent is in the 4-position (maximum two times difference in the
K values). Only a ten times difference in affinity between a chloro and a benzyloxy (5c vs 5e) in the
i
4
-position with a methoxy group (5i) in-between was observed. Also the 3,4-dichloro analog (5j)
showed similar affinity as the two monosubstituted analogs 5b,c.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To test the impact of the nitrogen atoms in the imidazopyridazine scaffold, the imidazopyridine
compound 7 was included in the study. The absence of the N -nitrogen led to a seven times reduction
5
in affinity relative to that of the corresponding imidazopyridazine analog 6.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Using 5c as representative for the novel class of compounds discovered in the present study an
assessment of brain exposure was performed. We investigated the brain-to-plasma distribution of 5c
in mice after i.p. administration at a dose of 10 mg/kg. The measured brain and plasma concentrations
were 59±5.8 ng/g and 7604±2287 ng/mL, respectively, resulting in a brain-to-plasma distribution
ratio of 0.008±0.002. Despite remarkable high plasma concentration which indicates high
bioavailability no brain penetrance was observed.
Optimization of brain exposure of 5c using the prodrug approach
To facilitate efficient brain penetration for 5c we decided to follow a prodrug approach masking
the polar carboxylic acid as biological convertible esters. The ethyl, tert-butyl, acetoxymethyl and
tert-butoxymethyl esters were pursued. The synthesis of the target ester compounds 16a–c was
carried out as illustrated in Scheme 4. Esterification of the carboxylic acid group of 5c with
trichloroacetimidate in the presence of a Lewis acid or with the appropriate alkyl halide gave the
corresponding tert-butyl, acetoxymethyl and tert-butoxymethyl esters 16a, b, c, respectively.
a
Scheme 4
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3
4
Cl
Cl
5
6
7
8
9
N
COOH
N
COOR
N
N
i or ii
N
N
5
c
16a tert-Bu
6b Acetoxymethyl
6c tert-Butoxymethyl
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (i) For 16a; tert-butyl 2,2,2-trichloroacetimidate, BF O(C H ) , DMF,
0 °C, (ii) for 16b,c; chloromethyl acetate or chloromethyl pivalate, K CO , DMF, 70 °C.
2 3
3
2
5 2
5
The metabolic stabilities of 5c and the ester prodrugs 13c and 16a–c were determined in vitro in
mouse plasma and mouse liver microsomes. The remaining percentages of the compounds after
incubation are summarized in Table 3. The tert-butyl ester 16a showed high stability in mouse liver
microsomes and fair stability in mouse plasma (t ≈ 60 min), however, prodrugs 13c and 16b,c were
1
/2
highly chemical/metabolic unstable and converted to the desirable parent compound 5c instantly.
Chemical or enzymatic degradation of 5c was not observed during metabolic stability study
correlating well with the high bioavailability observed in the brain exposure study. The corresponding
loss of prodrug compounds and formation of parent drug 5c was determined by UHPLC-MS/MS.
TABLE 3 Metabolic stability of 5c and prodrugs 13c, 16a–c in mouse liver microsomes (MLM) and
mouse plasma (MP)
Metabolicꢀstabilityꢀinꢀmouseꢀliverꢀmicrosomesꢀ
ꢀ
ꢀ
ꢀ
Metabolicꢀstabilityꢀinꢀmouseꢀplasmaꢀ
bꢀ
a
ꢀ
%ꢀremainingꢀinꢀMLM
ꢀ
%ꢀremainingꢀinꢀMP ꢀ
c
Compdꢀ
cꢀ
0ꢀmin ꢀ
30ꢀminꢀ
60ꢀminꢀ
Compdꢀ
0ꢀminꢀ
100ꢀ
-ꢀ
60ꢀminꢀ
120ꢀminꢀ
5
1
1
1
100ꢀ
-ꢀ
105ꢀ
-ꢀ
100ꢀ
-ꢀ
ꢀ
5cꢀ
97ꢀ
-ꢀ
99ꢀ
-ꢀ
3cꢀ
6aꢀ
6bꢀ
ꢀ
ꢀ
ꢀ
ꢀ
13cꢀ
16aꢀ
16bꢀ
16cꢀ
100ꢀ
-ꢀ
90ꢀ
-ꢀ
98ꢀ
-ꢀ
100ꢀ
-ꢀ
51ꢀ
-ꢀ
36ꢀ
-ꢀ
16cꢀ
-ꢀ
-ꢀ
-ꢀ
-ꢀ
-ꢀ
-ꢀ
a
b
Mean value of three biological replicates Percentage of prodrug remaining after metabolism was
calculated by the ratio of the internal standard-corrected peak area at respective time (min) to peak
area found at 0 min multiplied by 100: %remaining = [(peak area at respective time (min))/(peak area
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c
at 0 min)] × 100; The peak area at 0 min was compared to the peak area of freshly prepared prodrug
in neat buffer and MeCN; '-' at 0 min means that the prodrug has been substantially degraded during
sample preparation and pre-incubation.
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Encouraged by the promising metabolic profile of 16a an assessment of brain exposure was
performed as described for 5c. The measured brain and plasma concentrations were 798±296 ng/g
and 212±92 ng/mL, respectively, resulting in a brain-to-plasma distribution ratio of 3.68±0.92,
disclosing 16a as a very promising tool for facilitated brain exposure of 5c.
Ligand-based approach to define a binding mode at the GHB high-affinity binding site
Since the protein and location of the high-affinity GHB binding site is still unknown, we are limited
to ligand-based approaches to gain structural insight of the binding pocket. Recently, we described
2
5
the first pharmacophore model of ligands for the high-affinity GHB binding site, constructed on
derivatives of the endogenous ligand GHB, its constrained analogs trans-4-hydroxycrotonic acid and
the cyclic analog HOCPCA. To obtain binding conformations for discussing SAR, this model,
consisting of a negatively charged carboxylic acid, a hydrogen bond donor, a hydrogen bond acceptor,
an aromatic ring, and exclusion volumes, was applied to all the new ligands synthesized in this paper
upon quantum mechanical determination of protonation states (see below). Requiring a match of three
out of the four pharmacophore elements, all the high- (pK ≥ 6) and intermediate-affinity (4 < pK <
i
i
6) imidazopyridazine derivatives (5b–j), all the intermediate-affinity derivatives of gabazine (5a–
g,k–m), and gabazine itself fitted the pharmacophore model as binding compounds. Neither of the
enantiomers of the inactive compounds (pK ≤ 4; 1a,b and 4a–c,h–i) fitted the pharmacophore model,
i
while compounds 6 and 7 were discarded because they could fit only two out of four pharmacophore
elements.
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Physicochemical properties; Determination of pK values
a
Since the protonation state of the compounds under study is important for the interpretation of the
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binding modes suggested, the effect on protonation states by incorporating the amino group of
gabazine into a dihydroimidazopyridazine or imidazopyridazine scaffold was estimated by
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calculating the pK of all the new compounds reported in this work (Please refer to Table S2 in
a
Supporting Information). At physiological pH, the imidazopyridazine ring of analogs 5b–e,h–j were
predicted to be non-protonated (pK 4.8–5.3), while the 3-aminopyridazine ring of 3a–g,k–m and
a
dihydroimidazopyridazine ring of 4a–c,h,i were predicted to be in their zwitterionic form (pK 9.7–
a
10.3). Additionally, we experimentally determined the pK of compounds 4c (pK 9.7) and 5c (pK
a
a
a
4
.5), with good agreement between the calculated and experimental values.
Discussion
Gabazine, in which the γ-aminobutyric acid moiety is incorporated by its N-terminal group into a
3
-amino-4-methyl-6-phenylpyridazine system, was initially reported in 1987 by Wermuth et al. as a
1
7
potent competitive GABA R antagonist. Since its introduction, gabazine has become a widely used
A
tool compound in the research field of GABA Rs, and SAR studies have been carried out on gabazine,
A
35
identifying more potent analogs. Recently, Mortensen et al. used the scaffold of gabazine to develop
3
7
photoactive ligands for tracking GABA R mobility. Besides the effect on GABA Rs, gabazine has
A
A
3
been identified as an inhibitor of [ H]NCS-382 binding to the elusive high-affinity GHB binding
7
3
sites. Whereas GABA inhibits [ H]NCS-382 binding with a K value of 2.1 mM, gabazine was shown
i
3
7
to inhibit [ H]NCS-382 binding to a greater extent with a K value of 15.6 µM. The referred study,
i
also included a series of GABA R ligands with various pharmacological profiles and structural
A
3
8
classes e.g. the orthosteric GABA R antagonist bicuculline and the positive modulator diazepam.
A
3
Interestingly, only gabazine affected the [ H]NCS-382 binding. To test if this is a general trend for
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orthosteric GABA R antagonists we included the high-affinity GABA R antagonists 1a,b (Figure 1),
A
A
3
structurally diverse from gabazine, in the present study and observed no effect on [ H]NCS-382
binding (data not shown).
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In the present study, we sought to discern the structural determinants of gabazine for respective
recognition of the high-affinity binding site of GHB and the orthosteric GABA R binding site. Three
A
classes of gabazine analogs, based upon the 3-aminopyridazine group in gabazine and the bicyclic
dihydroimidazopyridazine and imidazopyridazine ring systems, were synthesized and
pharmacologically characterized. In general, the 3-aminopyridazine analogs showed binding affinity
for the GABA R binding site in the mid nano- to micromolar range (K 0.016–1.8 µM), in line with
A
i
1
7, 35
previous reported data,
and in the micromolar range for the GHB site (K 3.1–100 µM). Both the
i
imidazopyridazine and dihydroimidazopyridazine analogs were completely devoid of affinity for the
GABA R binding site. At the high-affinity GHB binding site, our previously developed
A
2
5
pharmacophore model divided the three series in accordance with the affinities into binders, 3-
aminopyridazines (K 3.1–100 µM) and imidazopyridazines (K 0.19–2.9 µM), and non-binders,
i
i
dihydroimidazopyridazines (K > 100 for 4 out of 5 compounds, K 45 µM for 4i). The non-planarity
i
i
of the dihydroimidazopyridazines (exemplified by 4c) changes the relative orientation/direction of
the carboxylic acid and nitrogen acceptor and they were discarded by dis-allowed regions, confirming
the previously reported poor steric tolerance of the GHB binding site in close proximity to the
2
5
carboxylic acid functionality (Figure 4).
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Figure 4. Pharmacophore model for the high-affinity GHB binding site showing the proposed binding
modes of compounds 3c (light blue stick representation, K 10.9 µM), 4c (purple stick representation,
i
K 146 µM), and 5c (green stick representation, K 0.22 µM). The yellow spheres indicate unfavorable
i
i
positions of substituents (exclusion volumes) in the high affinity GHB binding site, while the white
sphere represents the volume (VdW radii) of the "out-of-the-plane" hydrogen atom of 4c that clashes
with the exclusion volume behind it. The remaining spheres indicate positions favorable for: aromatic
ring systems (purple), hydrogen bond acceptor (orange), hydrogen bond donor (blue), and negatively
charged groups (red).
The fact that gabazine and some analogs thereof are able to bind to both classes of binding sites,
shows that they do share some common structural features, e.g. a large hydrophobic pocket
encompassing the 4-methoxy-phenyl substituent (Table 1). However, the fact that the
imidazopyridazines do not bind to the GABA R binding site and the differences in the SAR for the
A
GABA R and the GHB high-affinity binding site, e.g. similar affinity of 3d and 3e in the GHB site
A
and >40 times affinity difference in the GABA R binding site, highlights fundamental differences in
A
the binding pockets (Table 1 and 2).
The orthosteric GABA R binding site was previously explored by, among others, the rigid
A
compounds 1a and 1b. In these studies, the butanoic acid fragment of gabazine was suggested to bind
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15, 39
in a completely extended conformation.
This results in a distance of 6.8 Å between the protonated
nitrogen and the carboxylic acid carbon of gabazine (Figure 5). In the dihydroimidazopyridazine and
imidazopyridazine analogs, the two functional groups are, however, constrained at a shorter distance
of ~3.8 Å comparable to the inter-site distance of the shorter gabazine analog described by Wermuth
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et al. (1987), with a 2-carboxyethyl and a 30-fold reduction in affinity towards the GABA Rs. The
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fit of gabazine to our GHB binding site pharmacophore model shows a non-extended conformation
with an inter-site distance of 5.0 Å (Figure 5). The fact that 1a does not match the phamacophore
elements further supports that the two binding sites have different optimal requirements for the
relative distance between the carboxylic acid and the other functional pharmacophore elements, i.e.
the donor and/or acceptor and the aromatic ring. The non-linear binding conformation of the gabazine
butanoic acid fragment in the GHB site additionally results in a different angle of the carboxylic acid
relative to the other functional groups compared to the practically linear and planar relationship in
1
5
the GABA R binding site (Figure 5). This provides an explanation for why the so-called arginine
A
39
flip is not enough to accommodate the dihydroimidazopyridazines and imidazopyridazines in the
GABA R binding site.
A
Figure 5. Superposition of the extended GABA R binding site conformation of gabazine (orange
A
stick representation) and the gabazine conformation matching the high-affinity GHB binding site
pharmacophore model (cyan stick representation). Distances between the protonated nitrogen and
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acidic carbon are shown as dotted lines and values, given in Ångstrøm (Å), in the same color as the
conformations.
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The sub-micromolar affinity of the 3-aminopyridazines and imidazopyridazines for the GABA R
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and GHB binding sites, respectively, confirmed the presence of at least one large lipophilic cavity in
both binding sites, which was explored through the introduction of different substituents on the
aromatic ring. In the GABA R binding site, the 3-position seemed to be most favorable for the smaller
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substituents with 20 times increase in affinity for the 3-chloro substituent (3b vs. 3a) while the larger
benzyloxy substituent was 44 times more favorable in the 4-position (3e vs. 3d). However, as
substitution in both positions displayed high affinity, it indicates that this lipophilic pocket is
relatively large and/or is comprised by flexible amino acid side chains, which is in agreement with
15, 39
previous reports.
A similar pattern, but with even less specificity, was observed for the high-
affinity GHB binding site. As clearly illustrated by the ~50 times gain in affinity (5j vs. 6), an
aromatic substituent is highly favored for the imidazopyridazines, but regardless of whether it is
substituted in the 3- or 4- position, or both (5b, 5c, 5j), similar affinities are obtained. In fact, even
the large benzyloxy substituent, which is tolerated in the GHB binding site without an affinity
contribution compared to the smaller substituents, only displays approximately four times affinity
difference between the 3- and 4-position (Table 2).
Unexpectedly, and even though the pharmacophore model performed well in identifying all the
high and intermediate affinity ligands, the 3-aminopyridazine analogs generally ranked better than
imidazopyridazine analogs, despite their lower affinities. This discrepancy is likely explained by
differences in pK , which contributes to the binding affinity that the pharmacophore cannot account
a
for, except for the qualitative ionization state of the input structures. As predicted by quantum
chemistry calculations and experimentally confirmed for 5c, the imidazopyridazine analogs are
without protonation of the imidazopyridazine ring while the 3-aminopyridazine analogs are in their
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zwitterionic form at physiological pH (Please refer to Table S2 in Supporting Information). This
suggests that the pK value is important for binding affinity. The protolytical properties of the 3-
a
aminopyridazine and imidazopyridazines qualitatively correspond to those of GABA and GHB,
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respectively, and while the zwitterionic nature is obviously an advantage in the GABA R site, the
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presence of a positive charge is disfavored in the high-affinity GHB binding site. Furthermore, as
confirmed for 4c, the dihydroimidazopyridazines were also found to be zwitterionic at physiological
pH, which likely also contribute to their poor affinity. Also, the unsubstituted imidazopyridazine, 6,
shows seven times higher affinity compared to the benzimidazole analog, 7, which could be related
to the lower pK of the basic group (Please refer to Table S2 in Supporting Information).
a
One additional observation concerning the employed pharmacophore model of ligands for the high-
affinity GHB binding site arises from the compound series in this work. In addition to the negative
and aromatic pharmacophore elements, the original model contained both a hydrogen bond donor and
2
5
acceptor, but as illustrated by the imidazopyridazines, a hydrogen bond acceptor is apparently
sufficient to retain the high affinity towards the GHB binding site.
In conclusion, the identification of dihydroimidazopyridazine or imidazopyridazine scaffolds as
preferred GHB binding site ligands confirms that GHB high-affinity binding sites and GABA R
A
orthosteric binding sites are distinct binding sites that, coincidentally, can both bind gabazine. This
3
is supported by the inability of GHB, HOCPCA and NCS-382 to displace [ H]muscimol binding and,
conversely, that GABA orthosteric ligands like THIP, muscimol, bicuculline and compounds 1a,b
A
3
cannot displace [ H]NCS-382 binding.
Both binding sites contain areas favorable for a negative and an aromatic moiety in the distal parts
of the molecules, but with a longer and more linear inter-site relationship in the GABA R orthosteric
A
binding site relative to that of the high-affinity GHB binding site. Additionally, both binding sites can
encompass aromatic moieties of similar size and substitution patterns, indicating a cavity of a
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considerable size or comprised of flexible side chains. In contrast, the two binding sites show
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significantly different requirements for the central part, where the GABA R orthosteric binding site
A
favors positively charged hydrogen bond donors and the high-affinity GHB binding site favors a
neutral hydrogen bonding group, which can be devoid of the donor functionality. The latter finding
additionally serves as a refinement of the employed pharmacophore model, which can be used for
rational ligand design towards the high-affinity GHB binding site.
0
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The physicochemical properties of the imidazopyridazine moiety makes it suitable for bioisosteric
replacement of the hydroxyl group in GHB. Hence, the present work has provided a new lead
compound synthesized by an efficient minimal-step synthesis, suitable for further development. As a
further step towards future in vivo studies a tert-butyl ester of 5c (16a) was identified, based on
metabolic stability and brain exposure studies, as a promising tool to facilitate brain delivery of 5c.
Experimental section
Chemistry
General procedures. Compound 7 obtained from Combi-Blocks Inc. (San Diego, USA),
3
0
31
31
compounds 3a, 6, and 15 were synthesized as described in the literature, and compounds 8a–i
32
40
were synthesized after a modified literature procedure. The spectroscopic data for compounds 8a,
4
1
40
32
35
40
17
41
40
8
b, 8c, 8d, 8e, 8f, 8g, 8h, and 8i, is in agreement with data previously reported in the
literature. All reagents and solvents (reagent or chromatography grade) were obtained from
commercial suppliers and used without further purification. All air- and/or moisture-sensitive
reactions were performed under a nitrogen atmosphere using syringe-septum cap techniques and
flame or oven dried glassware. Anhydrous solvents were obtained by using a solvent purification
system (THF, DMF, and CH Cl ) or by storage over 3 Å molecular sieves. Thin-layer
2
2
chromatography (TLC) was performed using Merck silica gel 60 F254 sheets and compounds were
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visualized by UV (254 or 365 nm), KMnO4 or ninhydrin spray reagent. Flash column
chromatography (FC) and dry column vacuum chromatography (DCVC) were performed using
1
13
18
Merck silica gel (0.040‒0.063 mm or 0.015–0.040 mm, respectively). H, C, and F NMR were
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19
recorded on a Bruker Avance 400 MHz spectrometer equipped with a 5 mm PABBO BB( H, F) Z-
GRD probe or a Bruker Avance 600 MHz spectrometer equipped with a cryogenically cooled 5 mm
1
3
1
CPDCH C( H) Z-GRD probe, at 300 K. Data are tabulated in the following order: Chemical shift
(δ) [multiplicity (b, broad ; s, singlet ; d, doublet ; t, triplet ; q, quartet; m, multiplet), coupling
4
2
constant(s) J (Hz), number of protons]. The solvent residual peak was used as reference. Melting
points were determined on a Stanford Research Systems OptiMelt instrument in an open capillary
tubes and are uncorrected. Analytical High performance liquid chromatography (anal. HPLC) was
performed on a Merck-Hitachi HPLC system consisting of an L-7100 pump, an L-7200 auto sampler,
and an L-7400 UV detector (210 or 254 nm), using a Chromolith SpeedROD RP-18 column (4.6 ×
5
0 mm). A linear gradient elution was applied with eluent A (H O/TFA 100:0.01) containing 0% of
2
solvent B (MeCN/H O/TFA, 90:10:0.01) rising to 100% of B over 3.5 min with a flow rate of 4.0
2
mL/min. The purity of the analyzed compounds are ≥95% unless otherwise stated. Data were acquired
and processed using the EZChrom Elite Software version 3.1.7 by Hitachi. Elemental analyses were
performed by Mr. J. Theiner, Department of Physical Chemistry, University of Vienna, Austria and
results are within ±0.4% unless otherwise stated.
4
-(3-(3-Chlorophenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid (3b). Compound 10b (71 mg,
0
.4 mmol) was dissolved in AcOH (3.8 mL) and 35% HCl (1.1 mL) was added. The resulting solution
was heated at 100 °C for 24 h. where upon it was cooled to rt., filtered, and evaporated in vacuo to
1
afford compound 3b (54 mg, 30%) as brown solid: mp 225–228 °C. H NMR (400 MHz, CD OD):
3
δ 8.34 (d, J = 9.4 Hz, 1H), 8.05–8.02 (m, 1H), 7.95–7.91 (m, 1H), 7.68 (d, J = 9.4 Hz, 1H), 7.60–
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.51 (m, 2H), 4.48 (t, J = 7.1 Hz, 2H), 2.57 (t, J = 6.7 Hz, 2H), 2.24 (quin, J = 7.1 Hz, 2H). C NMR
(
100 MHz, CD OD): δ 175.0, 153.1, 149.6, 135.0, 134.8, 131.4, 130.6, 130.5, 126.3, 125.6, 124.8,
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55.7, 29.6, 21.2.
-(3-(4-Chlorophenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid (3c). Compound 10c (89 mg,
.3 mmol) was dissolved THF (1 mL) and water (1 mL) and aq. NaOH (0.4935 M, 0.565 mL, 0.3
0
1
2
3
4
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7
8
9
0
4
0
mmol) was added. The resulting solution was stirred at rt. 48 h where upon it was evaporated in vacuo
1
to afford compound 3c (85 mg, 96%) as brown solid: mp > 230 °C. H NMR (400 MHz, CD OD): δ
3
7
.87–7.82 (m, 2H), 7.50–7.42 (m, 3H), 7.03 (d, J = 9.7 Hz, 1H), 4.32 (t, J = 7.0 Hz, 0.3H), 4.17 (t, J
1
3
= 7.2 Hz, 1.7H), 2.34–2.28 (m, 2H), 2.10–2.09 (m, 2H). C NMR (150 MHz, CD OD): δ 180.1,
3
1
41.8, 134.6, 133.6, 129.9, 128.7, 128.5, 126.7, 125.1, 52.8, 34.5, 23.6.
-(3-(3-(Benzyloxy)phenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid (3d). Compound 10d
100 mg, 0.3 mmol) was dissolved in THF (3 mL) and water (3 ml) and aq. NaOH (0.5 M, 0.55 mL,
4
(
0.3 mmol) was added. The resulting solution was stirred at rt. for 22 h. where upon it was filtered and
1
evaporated in vacuo to afford compound 3d (71 mg, 72%) as pink solid: mp 162–163 °C. H NMR
(600 MHz, DMSO-d ): δ 11.57 (s, 1H), 8.21 (d, J = 9.6 Hz, 1H), 7.59 (d, J = 9.6 Hz, 1H), 7.52–7.50
6
(m, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.41–7.35 (m, 3H), 7.31 (t, J = 7.6 Hz, 2H), 7.26–7.22 (m, 1H),
7
.12 (dd, J = 1.9, 7.8 Hz, 1H), 5.10 (s, 2H), 4.32 (t, J = 7.1 Hz, 2H), 2.39 (t, J = 6.9 Hz, 2H), 2.06
1
3
(
quin, J = 7.0 Hz, 2H). C NMR (150 MHz, DMSO-d ): δ 174.4, 159.3, 152.7, 149.9, 137.0, 134.5,
6
1
31.6, 130.6, 128.7, 128.2, 128.0, 126.0, 119.2, 117.4, 113.2, 69.8, 55.6, 30.3, 21.8.
-(3-(4-(Benzyloxy)phenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid (3e). Compound 10e (92
4
mg, 0.2 mmol) was dissolved in THF (3 mL) and water (3 mL) and aq. NaOH (0.5 M, 0.47 mL, 0.2
mmol) was added. The resulting solution was stirred at rt. for 24 h where upon it was filtered and
1
evaporated in vacuo to afford compound 3e (59 mg, 65%) as white solid: mp 182–183 °C. H NMR
(
600 MHz, DMSO-d ): δ 11.75 (s, 1H), 8.26 (d, J = 9.5 Hz, 1H), 7.89 (d, J = 9.2 Hz, 2H), 7.57 (d, J
6
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=
9.5 Hz, 1H), 7.43 (d, J = 6.9 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.29 (t, J = 7.2 Hz, 1H), 7.14 (d, J =
8
.8 Hz, 2H), 5.16 (s, 2H), 4.31 (t, J = 7.0 Hz, 2H), 2.39 (t, J = 7.0 Hz, 2H), 2.06 (quin, J = 7.1 Hz,
1
3
2H). C NMR (150 MHz, DMSO-d ): δ 174.2, 160.9, 152.4, 149.8, 137.0, 131.4, 128.8, 128.4, 128.3,
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28.0, 126.0, 125.7, 115.8, 69.8, 55.6, 30.5, 22.0.
-(6-Imino-3-(3-nitrophenyl)pyridazin-1(6H)-yl)butanoic acid (3f). Compound 10f (150 mg,
4
0.5 mmol) was dissolved in AcOH (9 mL) and 35% HCl (5 mL) was added. The resulting mixture
was heated at reflux for 21 h where upon it was cooled to rt., filtered, and evaporated in vacuo to
1
afford compound 3f (139 mg, 91%) as white solid: mp 221–222 °C. H NMR (600 MHz, DMSO-d ):
6
δ 12.18 (bs, 1H), 10.03 (bs, 1H), 8.75–8.73 (m, 1H), 8.55 (d, J = 9.4 Hz, 1H), 8.44–8.39 (m, 2H),
13
7
.90–7.86 (m, 2H), 4.46 (t, J = 7.3 Hz, 2H), 2.46 (t, J = 7.3 Hz, 2H), 2.08 (quin, J = 7.3 Hz, 2H). C
NMR (150 MHz, DMSO-d ): δ 174.2, 153.2, 148.9, 135.1, 133.3, 131.6, 131.4, 126.6, 125.7, 121.5,
6
55.9, 30.7, 22.2.
4
-(6-Imino-3-(4-nitrophenyl)pyridazin-1(6H)-yl)butanoic acid (3g). Compound 10g (150 mg,
0
.5 mmol) was dissolved in AcOH (9 mL) and 35% HCl (5 mL) was added. The resulting mixture
was heated at reflux for 21 h where upon it was cooled to rt., filtered, and evaporated in vacuo to
1
afford compound 3g (145 mg, 95%) as brown solid: mp 226–227 °C. H NMR (600 MHz, DMSO-
d6): δ 10.12 (bs, 1H), 9.39 (bs, 1H), 8.52 (d, J = 9.5 Hz, 1H), 8.39 (d, J = 8.9 Hz, 2H), 8.25 (d, J =
8.9 Hz, 2H), 7.91 (d, J = 9.5 Hz, 1H), 4.45 (t, J = 7.0 Hz, 2H), 2.46 (t, J = 7.0 Hz, 2H), 2.08 (quin, J
13
=
7.0 Hz, 2H). C NMR (150 MHz, DMSO-d ): δ 173.7, 152.7, 148.5, 147.4, 138.8, 131.1, 127.8,
6
1
26.1, 124.2, 55.4, 30.2, 21.8.
4-(3-(3-Aminophenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid (3k). Compound 10k (0.28 g,
.9 mmol) was dissolved in AcOH (20 mL) and 35% HCl (7 mL) was added. The resulting mixture
0
was heated at reflux for 21 h where upon it was cooled to rt., filtered, and evaporated in vacuo afford
1
compound 3k (0.26 g, 79%) as light yellow solid: mp 156–157 °C. H NMR (600 MHz, DMSO-d ):
6
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Journal of Medicinal Chemistry
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δ 9.92 (bs, 2H), 9.22 (bs, 1H), 8.34 (d, J = 9.7 Hz, 1H), 7.87–7.80 (m, 3H), 7.60 (t, J = 8.4 Hz, 1H),
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8
9
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1
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7
.47–7.43 (m, 1H), 4.41 (t, J = 7.4 Hz, 2H), 2.46 (t, J = 7.4 Hz, 2H), 2.07 (quin, J = 7.4 Hz, 2H). C
NMR (150 MHz, DMSO-d ): δ 173.5, 152.5, 148.6, 141.8, 136.2, 134.2, 131.0, 130.4, 126.1, 123.8,
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0
1
2
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19.1, 55.3, 30.2, 21.9.
-(3-(3-Hydroxyphenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid (3l). Compound 10d (100
4
mg, 0.3 mmol) was dissolved in AcOH (5 mL) and 35% HCl (3 mL) was added. The resulting mixture
was heated at reflux for 21 h where upon it was cooled to rt. and evaporated in vacuo to afford
1
compound 3l (79 mg, 95%) as white solid: mp 99–100 °C. H NMR (600 MHz, CD OD): δ 8.27 (d,
3
J = 9.5 Hz, 1H), 7.65 (d, J = 9.5 Hz, 1H), 7.45–7.41 (m, 2H), 7.37 (t, J = 7.9 Hz, 1H), 6.99 (dd, J =
13
0
.8, 7.5 Hz, 1H), 4.47 (t, J = 7.2 Hz, 2H), 2.59 (t, J = 6.7 Hz, 2H), 2.26 (quin, J = 7.0 Hz, 2H). C
NMR (150 MHz, CD OD): δ 175.0, 158.1, 152.8, 150.8, 134.1, 131.5, 130.0, 125.3, 117.8, 117.47,
3
112.9, 55.6, 29.6, 21.3.
4
-(3-(4-Hydroxyphenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid (3m). Compound 10e (86
mg, 0.2 mmol) was dissolved in AcOH (5 mL) and 35% HCl (3 mL) was added. The resulting mixture
was heated reflux for 23 h where upon it was cooled to rt., filtered, and evaporated in vacuo to afford
1
compound 3m (68 mg, 89%) as yellow solid: mp 109–110 °C. H NMR (600 MHz, CD OD): δ 8.27
3
(
d, J = 9.4 Hz, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.60 (d, J = 9.3 Hz, 1H), 6.94 (d, J = 8.4 Hz, 2H), 4.44
1
3
(t, J = 7.0 Hz, 2H), 2.57 (t, J = 6.6 Hz, 2H), 2.24 (quin, J = 6.9 Hz, 2H). C NMR (150 MHz, CD OD):
3
δ 175.0, 160.4, 152.3, 150.9, 131.2, 128.1, 125.1, 123.8, 115.7, 55.4, 29.6, 21.3.
2
-(6-Phenyl-2,3-dihydroimidazo[1,2-b]pyridazin-2-yl)acetic acid (4a). Compound 11 (100 mg,
0.3 mmol) was dissolved in THF (0.5 mL) and water (1 mL) and aq. NaOH (0.4935 M, 1.13 mL, 0.6
mmol) was added. The resulting solution was stirred at rt. for 24 h where upon it was filtered and
1
evaporated in vacuo to afford compound 4a (99 mg, 68%) as light brown solid: mp >230 °C. H NMR
(
400 MHz, CD OD): δ 7.77–7.73 (m, 2H), 7.45 (d, J = 10.0 Hz, 1H), 7.43–7.34 (m, 3H), 6.85 (d, J
3
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=
10.0 Hz, 1H), 4.49–4.35 (m, 2H), 4.05–3.98 (m, 1H), 2.74–2.67 (m, 1H), 2.39–2.31 (m, 1H). C
NMR (100 MHz, CD OD): δ 179.4, 155.1, 145.4, 136.5, 130.1, 130.0, 129.6, 126.4, 124.2, 60.7,
3
58.5, 46.4.
0
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2
-(6-(3-Chlorophenyl)2,3-dihydroimidazo[1,2-b]pyridazin-2-yl)acetic acid (4b). Compound
1
2b (48 mg, 0.2 mmol) was dissolved in aq. HCl (4 M, 5 mL) and heated at 45 °C for 24 h. Upon
cooling to rt., the reaction mixture was filtered and evaporated in vacuo to afford compound 4b (41
1
mg, 84%) as brown solid: mp 236–237 °C. H NMR (600 MHz, CD OD): δ 8.42–8.38 (m, 1H), 8.02
3
(t, J = 1.8 Hz, 1H), 7.91 (dt, J = 1.7, 7.3 Hz, 1H), 7.64 (d, J = 9.5 Hz, 1H), 7.58–7.52 (m, 2H), 5.04
(
dd, J = 11.6, 12.7 Hz, 1H), 4.82–4.77 (m, 1H), 4.67 (dd, J = 8.1, 12.8 Hz, 1H), 2.96 (d, J = 6.2 Hz,
13
2
1
H). C NMR (150 MHz, CD OD): δ 173.3, 155.4, 152.1, 136.6, 136.5, 135.3, 135.2, 132.1, 127.7,
3
26.2, 121.2, 59.1, 53.3, 39.7.
2
-(6-(4-Chlorophenyl)-2,3-dihydroimidazo[1,2-b]pyridazin-2-yl)acetic acid (4c). Compound
1
2c (48 mg, 0.2 mmol) was dissolved in aq. HCl (4 M, 5 mL) and heated at 45 °C for 24 h. Upon
cooling to rt., the reaction mixture was filtered and evaporated in vacuo to afford compound 4c (40
1
mg, 74%) as white solid: mp 237–240 °C. H NMR (400 MHz, CD OD): δ 8.39 (d, J = 9.5 Hz, 1H),
3
8
.00–7.95 (m, 2H), 7.64 (d, J = 9.5 Hz, 1H), 7.59–7.54 (m, 2H), 5.03 (dd, J = 11.4, 12.6 Hz, 1H),
1
3
4.84–4.75 (m, 1H), 4.66 (dd, J = 6.7, 13.0 Hz, 1H), 2.96 (d, J = 6.1 Hz, 2H). C NMR (100 MHz,
CD OD): δ 173.3, 152.4, 138.4, 135.2, 133.2, 130.7, 129.4, 121.1, 59.1, 53.2, 39.7.
3
2
-(6-(3-Methoxyphenyl)-2,3-dihydroimidazo[1,2-b]pyridazin-2-yl)acetic acid (4h). Compound
12h (0.12 g, 0.4 mmol) was dissolved in aq. HCl (4M, 12 mL) and stirred at 45 °C for 24 h. Upon
cooling to rt., the reaction mixture filtered and evaporated in vacuo to afford compound 4h (94 mg,
1
77%) as off-white solid: mp 195–197 °C. H NMR (400 MHz, CD OD): δ 8.38 (d, J = 9.8 Hz, 1H),
3
7
1
.62 (d, J = 9.8 Hz, 1H), 7.55–7.50 (m, 2H), 7.46 (t, J = 8.1 Hz, 1H), 7.12 (ddd, J = 1.0, 2.5, 8.3 Hz,
H), 5.03 (dd, J = 11.3, 12.5 Hz, 1H), 4.84–4.74 (m, 1H), 4.70–4.61 (m, 1H), 3.88 (s, 3H), 2.96 (d, J
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13
=
6.1 Hz, 2H). C NMR (150 MHz, CD OD): δ 173.3, 162.1, 155.3, 153.4, 135.8, 135.5, 131.7,
3
1
20.9, 120.1, 117.8, 113.1, 59.1, 56.1, 53.2, 39.8.
7
8
9
2-(6-(4-Methoxyphenyl)-2,3-dihydroimidazo[1,2-b]pyridazin-2-yl)acetic acid (4i). Compound
2i (60 mg 0.2 mmol) was dissolved in aq. HCl (4 M, 6 mL) and heated at 45 °C for 24 h. Upon
1
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0
1
cooling to rt., the reaction mixture was filtered and evaporated in vacuo to afford compound 4i (53
1
mg, 83%) as white solid: mp 253–254 °C. H NMR (400 MHz, CD OD): δ 8.35 (d, J = 9.5 Hz, 1H),
3
7
.96–7.91 (m, 2H), 7.58 (d, J = 9.5 Hz, 1H), 7.11–7.06 (m, 2H), 5.00 (dd, J = 11.5, 12.5 Hz, 1H),
1
3
4.82–4.71 (m, 1H), 4.63 (dd, J = 7.9, 12.6 Hz, 1H), 3.88 (s, 3H), 2.95 (d, J = 6.4 Hz, 2H). C NMR
(
150 MHz, CD OD): δ 173.4, 163.8, 154.9, 153.4, 135.0, 129.4, 126.7, 120.8, 115.9, 59.1, 56.2, 53.1,
3
3
9.8.
2
-(6-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-2-yl)acetic acid (5b). Compound 13b (40 mg,
0
.1 mmol) was dissolved in aq. HCl (4 M, 5 mL) and heated at 45 °C for 24 h. Upon cooling to rt.,
the reaction mixture was filtered and evaporated in vacuo to afford compound 5b (42 mg, quant.) as
1
brown solid: mp 205–207 °C. H NMR (600 MHz, CD OD): δ 8.51 (s, 1H), 8.47–8.44 (m, 1H), 8.41–
3
8
.38 (m, 1H), 8.20 (t, J = 1.8 Hz, 1H), 8.08 (dt, J = 1.5, 7.7 Hz, 1H), 7.66–7.63 (m, 1H), 7.63–7.59
1
3
(
m, 1H), 4.13 (s, 2H). C NMR (150 MHz, CD OD): δ 171.1, 156.3, 136.9, 136.7, 133.8, 132.7,
3
132.2, 128.7, 127.3, 125.3, 122.8, 119.2, 31.83.
-(6-(4-Chlorophenyl)imidazo[1,2-b]pyridazin-2-yl)acetic acid (5c). Compound 13c (140 mg,
.4 mmol) was dissolved in aq. HCl (4 M, 15 mL) and heated at 45 °C for 48 h. Upon cooling to rt.,
2
0
the reaction mixture was filtered and evaporated in vacuo to afford compound 5c (61 mg, 42%) as
1
light brown solid: mp 195–197 °C. H NMR (400 MHz, CD OD): δ 8.48 (d, J = 0.7 Hz, 1H), 8.42 (d,
3
J = 9.8 Hz, 1H), 8.36 (d, J = 9.8 Hz, 1H), 8.18–8.13 (m, 2H), 7.66–7.61 (m, 2H), 4.12 (d, J = 0.7 Hz,
1
3
2
1
H). C NMR (150 MHz, CD OD): δ 170.3, 156.4, 139.1, 137.1, 133.7, 130.8, 130.4, 130.4, 124.8,
3
22.9, 119.1, 32.0.
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