Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential treatment of cognition in neurological disorders

Article abstract of DOI:10.1016/j.bmcl.2017.07.071

Previous research has shown that bicyclic 6:5-fused heteroaromatic compounds with two N-atoms have variable degrees of adenosine A₁ receptor antagonistic activity. Prompted by this imidazo[1,2-α]pyridine analogues were synthesized and evaluated

Full text of DOI:10.1016/j.bmcl.2017.07.071

Accepted Manuscript
Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential
treatment of cognition in neurological disorders.
Roslyn Lefin, Mietha M van der Walt, Pieter J Milne, Gisella Terre'Blanche
PII:
S0960-894X(17)30778-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.071
BMCL 25184
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
15 May 2017
26 July 2017
27 July 2017
Please cite this article as: Lefin, R., van der Walt, M.M., Milne, P.J., Terre'Blanche, G., Imidazo[1,2-α]pyridines
possess adenosine A1 receptor affinity for the potential treatment of cognition in neurological disorders., Bioorganic
& Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.071
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Imidazo[1,2-α]pyridines possess adenosine
A1 receptor affinity for the potential
treatment of cognition in neurological
disorders.
Roslyn Lefina, Mietha M van der Walt b Pieter J Milne a,b and Gisella Terre’Blanchea,b*
aPharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
bCentre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520,
South Africa
Abstract
Previous research has shown that bicyclic 6:5-fused heteroaromatic compounds with two N-atoms
have variable degrees of adenosine A1 receptor antagonistic activity. Prompted by this imidazo[1,2-
α]pyridine analogues were synthesized and evaluated for their adenosine A1 and A2A receptor affinity
via radioligand binding studies and subjected to a GTP shift assay to determine its adenosine A1
receptor agonistic or antagonistic functionality. Imidazo[1,2-α]pyridine, the parent scaffold, was
found devoid of affinity for the adenosine A1 and A2A receptors. The influence of substitution on
position C2 showed no improvement for either adenosine A1 or A2A receptor affinity. The addition of
an amino or a cyclohexylamino group to position C3 also showed no improvement of adenosine A1 or
A2A receptor affinity. Surprisingly para-substitution on the phenyl ring at position C2 in combination
with a cyclohexylamino group at position C3 led to adenosine A1 receptor affinity in the low
micromolar range with compound 4d showing: (1) the highest affinity for the adenosine A1 receptor
with a Ki value of 2.06 µM and (2) adenosine A1 receptor antagonistic properties. This pilot study
concludes that para-substituted 3-cyclohexylamino-2-phenyl-imidazo[1,2-α]pyridine analogues
represent an interesting scaffold to investigate further structure-activity relationships in the design of
novel imidazo[1,2-α]pyridine-based adenosine A1 receptor antagonists for the treatment of
neurodegenerative disorders.
Keywords: Alzheimer’s disease, Parkinson’s disease, imidazo[1,2-α]pyridine analogues, Adenosine A1 and A2A receptor antagonists
*Corresponding author. Tel: +27 18 2992264; fax: +27 18 2994243; E-mail address: Gisella.Terreblanche@nwu.ac.za (G. Terre’Blanche).

Adenosine has many physiological functions throughout the body as well as a role in numerous
nervous system disorders including cognitive disorders, epilepsy, ischemia, stroke, Alzheimer’s
disease (AD) and Parkinson’s disease (PD) [1]. The effects of adenosine in neurodegenerative
disorders are predominately mediated by the A1 adenosine receptors (ARs) expressed in the
hippocampus and the A2A ARs expressed in the striatum [2–4]. The A2A ARs are important for motor
function [5] and display neuroprotective [6] and antidepressant effects [7], whereas the A1 ARs play
an important role in cognitive function [8]. Antagonism of the A1 AR represents a beneficial strategy
in the treatment of AD and PD since they have cognitive enhancing abilities that are still inadequately
treated with available treatment in both disorders. Furthermore, the A1 and A2A ARs exert a
synergistic effect on motor function as the A1 ARs enhance the presynaptic release of dopamine (DA)
[9], while the A2A ARs improve the postsynaptic DA activity [10,11]. For the abovementioned reasons
A1 and A2A AR antagonists have valuable potential as treatment agents as it will synergistically
improve motor function via A1 and A2A AR antagonism (PD), enhance cognitive function through A1
AR antagonism (AD and PD), provide neuroprotective properties to sufferers by means of A2A AR
antagonism (AD and PD) and alleviate depressive symptoms via A2A AR antagonism.
Compounds with affinity for the A1 and A2A ARs are generally divided into xanthine derivatives and
the amino-substituted heterocyclic compounds (non-xanthine) [12]. The xanthine derivatives are one
of the most researched AR classes and consist of a bicyclic fused 6:5-membered N-containing ring
system. Various non-xanthine heterocyclic compounds have previously been documented to possess
affinity for the A1 AR with the majority of these compounds considered as fused bi-or-tri-heterocyclic
systems that are extensions of the xanthine scaffold [13]. The pyrazolo[1,5-α]pyridines, imidazo[1,2-
α]pyridines and benzimidazoles (Figure 1) currently constitutes the largest group of non-xanthine 6:5-
fused heteroaromatic A1 AR antagonists [13]
N
N
N
N
N
N
H
benzimidazole
pyrazolo[1,5-α]pyridine
imidazo[1,2-α]pyridine
Figure 1. The general scaffolds of bicyclic 6:5 fused N-containing heteraromatic compounds.
Compounds containing the imidazo[1,2-α]pyridine ring system have been shown to possess a broad
range of useful pharmacological properties, including antibacterial, antifungal, anthelmintic, antiviral,
antiprotozoal, anti-inflammatory, anticonvulsant, anxiolytic, hypnotic (e.g., zolpidem),
gastrointestinal, antiulcer and immunomodulatory activities [14–17]. Until recently imidazo[1,2-
α]pyridines have only been described in patent literature for their selective A1 AR antagonistic
properties [18]. However, a study conducted by Reutlinger and co-workers [19] identified N-benzyl-
2-phenylimidazo[1,2-α]pyridin-3-amine as a potential A1 AR ligand with 89% binding at 100 µM
(Figure 2). Based on the latter finding, the imidazo[1,2-α]pyridines scaffold represent an interesting
class of 6:5-fused bicyclic compounds and prompted the current pilot study to investigate the potential
of several structurally related 3-cyclohexylamino-2-phenylimidazo[1,2-α]pyridine analogues
possessing affinity for the A1 and A2A ARs. Furthermore, in order to find new insights into the
structural requirements for AR binding, substitution at position C2 and C3 was explored (Figure 2).

H
H
N
N
3
3
4'
N
N
2
2
X
N
N
N-benzyl-2-phenylimidazo[1,2-α]pyridin-3-amine
Imidazo[1,2-α]pyridine derivatives
Figure 2. N-benzyl-2-phenylimidazo[1,2-α]pyridin-3-amine used as lead to investigate 3-
cyclohexylamino-2-phenylimidazo[1,2-α]pyridine analogues.
The selected imidazo[1,2-α]pyridine analogues were either obtained commercially (1, 2a–d and 3) or
synthesized (4a–i) according to literature procedures. Compound 1, imidazo[1,2-α]pyridine, is seen as
the parent compound of the study (Figure 1). The structure-activity relationships of the imidazo[1,2-
α]pyridine scaffold was further explored via substitution at the C2 position alone (2-
phenylimidazo[1,2-α]pyridines, 2a–d) and in combination with position C3 (2-phenylimidazo[1,2-
α]pyridin-3-amine, 3 and 3-cyclohexylamino-2-phenylimidazo[1,2-α]pyridines, 4a–i). The C2
substitution explored the unsubstituted phenyl ring (2a) as well as para-substitution which included
OH- (2b), OCH3- (2c) and Br- (2d). The simultaneous substitution of C2 and C3 included C2 phenyl
substitution combined with a C3 amino- (3) and C3 cyclohexyl amino-group (4a). The effect of para-
substitution at the C2 phenyl ring was further explored with the C3 cyclohexyl amino series and
included OH- (4b) OCH3- (4c), CH3- (4d), Br- (4e), Cl- (4f), F- (4g) CF3- (4h) and NO2- (4i) (Figure
3; Table 1)
C3 cyclohexyl
amino substitution
C2 phenyl substitution,
with C4' para-substitution
H
N
3
4'
N
2
X
N
1
X= H, OH, OCH3, CH3, Br, Cl, F, CF3, NO2
Figure 3. The imidazo[1,2-α]pyridine scaffold and suggested structural modifications.
Compounds 1, 2a–d and 3 were commercially available from Sigma-Aldrich® and subsequently used
without any further purification. The synthetic route of the known synthesized imidazo[1,2-
α]pyridine-based derivatives (4a–i) is outlined in Scheme 1. Compounds 4a–i was successfully
synthesized via a modified catalyst- and solvent-free three-compound procedure described in the
literature [20]. Briefly,
a
mixture of 2-aminopyridine, the appropriate aldehyde and
cyclohexylisocyanide was refluxed with no solvent at 120°C (4a–g and 4i) or 60°C (4h) until
completion of the reaction, as indicated by TLC. The crude compounds were purified by
recrystallization from either hexane:ethyl acetate (4a, 4d, 4e, 4f, 4g, 4h) or ethyl acetate:ethanol (4b,
4c, 4i) (Scheme 1). Compounds (4a–i) were successfully synthesized and the structures confirmed by
NMR spectrometry and supported by MS results. Both the 1H and 13C NMR of each test compound
corresponded with the proposed structures and the spectra reported in the literature [21].

H
N
3
O
H
4'
NC
N
2
a, b
X
+
+
X
NH2
N
1
X= H, OH, OCH3, CH3, Br, Cl, F, CF3, NO2
Scheme 1: The catalyst- and solvent-free synthetic procedure that was utilized to obtain the
corresponding imidazo[1,2-α]pyridines. Reagents and conditions: (a) heated at 120°C (4a, 4b, 4c, 4d,
4e, 4f, 4g, 4i) or 60°C (4h), (b) reflux for the appropriate time.
The A1 and A2A AR affinity of all the test compounds (1, 2a–d, 3 and 4a–i) was determined via
radioligand binding assays via a previously described procedure [22]. In short, the A1 AR affinity was
determined in the presence of the radioligand [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX)
with rat whole brain membranes expressing the A1 AR, while the A2A AR affinity was performed in
the presence of 5’-N-[3H]-ethylcarboxamideadenosine ([3H]NECA) as radioligand with rat striatal
membranes expressing the A2A ARs. In order to minimize [3H]NECA’s binding to the A1 AR the A1
AR agonist N6-cyclopentyladenosine (CPA) was also included in the A2A AR radioligand binding
experiment. Nonspecific binding was defined by the addition of 100 µM CPA. CPA was used as
reference compound and its assay results confirmed validity of the radioligand binding assays. The Ki
values were obtained by determining the IC50 values from sigmoidal-dose response curves by means
of the Graphpad Software Inc. package. The corresponding Ki value for the competitive inhibition by
the test compounds of [3H]DPCPX (Kd = 0.36 nM) or [3H]NECA (Kd = 15.3 nM) were subsequently
calculated from the IC50 values [22]. The sigmoidal-dose response curves were obtained by plotting
the specific binding (i.e. the eight concentrations of each test compound ranging between 0 µM and
100 µM) versus the logarithm of the test compound’s concentrations [22]. The in vitro A1 and A2A AR
affinity results of the test compounds and CPA are summarized in Table 1.
Table 1
The dissociation constant values (Ki values) for the binding of the imidazo[1,2-α]pyridine analogues
to rat adenosine A1 and A2A receptors
H
NH2
N
N
N
N
N
X
X
N
N
N
N
1
2a-d
4a-i
3
Ki SEM (µM)a / % displacementb
A1 vs [3H]DPCPX A2A vs [3H]NECA
A1 + GTPc vs
[3H]DPCPX
GTP
Shiftd
Compd
X
-
> 100 (100%)b
> 100 (49%)b
> 100 (47%)b
> 100 (76%)b
> 100 (86%)b
> 100 (52%)b
> 100 (75%)b
> 100 (52%)b
> 100 (85%)b
> 100 (61%)b
> 100 (87%)b
> 100 (86%)b
1
-
-
-
-
-
-
-
-
-
-
-
-
H
2a
2b
2c
2d
3
OH
OCH3
Br
-

4a
4b
4c
H
> 100 (57%)b
5.53 0.86a
7.61 1.25a
2.06 0.08a
3.90 0.65a
> 100 (61%)b
> 100 (63%)b
> 100 (76%)b
> 100 (99%)b
> 100 (77%)b
> 100 (27%)b
> 100 (55%)b
> 100 (83%)b
> 100 (54%)b
> 100 (64%)b
> 100 (90%)b
> 100 (74%)b
> 100 (69%)b
-
-
OH
-
-
OCH3
-
-
4d
4e
CH3
Br
2.00 0.24a
0.97
-
-
-
-
-
-
-
-
-
-
Cl
F
4f
4g
4h
CF3
NO2
4i
0.015e
0.99e
CPA
0.331e
6.48e
a All Ki values determined in triplicate and expressed as mean SEM.
b Percentage displacement of the radioligand at the indicated concentration.
c GTP shift assay, where the 100 µM GTP was added to the A1 AR radioligand binding assay.
d GTP shifts calculated by dividing the Ki in the presence of GTP by the Ki in the absence of GTP.
e Literature values obtained from references [22].
Compound 4d was further evaluated via a GTP shift assay to determine the functionality of the test
compounds towards the A1 AR. The GTP shift assay was carried out as described previously with rat
whole brain membranes and [3H]DPCPX in the absence and presence of 100 µM GTP (Table 1;
Figure 4). Nonspecific binding was defined by the addition of 10 µM DPCPX (unlabeled). A
compound with a calculated GTP shift of approximately 1 is considered an antagonist, in turn the
presence of GTP affects the competition curve of an agonist and shifts the curve to the right [22].
Figure 4. The sigmoidal-dose response curves of compound 4d (Panel A) and CPA (Panel B,
reference agonist) displaying the binding affinity to A1 ARs in the absence (-) and presence (+) of
GTP. (A) GTP shift of 0.97 calculated for the A1 AR antagonist compound 4d, and (B) GTP shift of
6.48 calculated for the A1 AR agonist CPA.
Previous research [19] identified N-benzyl-2-phenylimidazo[1,2-α]pyridin-3-amine as a lead for
designing compounds with A1 AR affinity (Figure 2). Imidazo[1,2-α]pyridine (1), the parent scaffold
of this pilot study, is unsubstituted at positions C2 and C3 and was devoid of A1 and A2A AR affinity.
The influence of C2 substitution on A1 and A2A AR affinity was investigated by comparing compound

1 to compound 2a–d containing a/an (un)substituted C2 phenyl ring. Although the specific binding
percentages of these compounds improved compared to compound 1, they did not display improved
A1 and A2A AR affinity. The addition of an amino group at position C3 (3 vs. 2a) also showed no
improvement for the A1 and A2A AR affinity. Alternatively the combination of a para-substituted
phenyl ring at position C2 together with a cyclohexylamino group at position C3 displayed A1 AR
affinity in the low micromolar range (4a vs. 4b, 4c, 4d, and 4e). However, no significant A2A AR
affinity was obtained. It seems that the more bulky electron donating groups (-OH, -OCH3, -CH3)
displayed good A1 AR affinity, while the electron withdrawing groups (-Cl, -F, -CF3, -NO2) showed
no A1 AR affinity with the exception of the bromo group (4e) that possessed good A1 AR affinity
(Table 1).
The compound documented with the best A1 AR affinity, was compound 4d with a para-methyl
substituent (Ki = 2.06 µM). Compound 4e (para-bromo substituent) displayed the second highest A1
AR affinity (Ki = 3.90 µM), followed by the para-hydroxy (4b) and para-methoxy (4c) analogues,
exhibiting Ki values of 5.53 µM and 7.61 µM respectively for the A1 AR. Unfortunately, none of these
structural modifications favored A2A AR affinity. Furthermore, selective A1 AR affinity was only
obtained with para-substitution of the phenyl ring at position C2 in combination with a
cyclohexylamino substitution on position C3, thus requiring substitution of both position C2 and C3
to obtain A1 AR affinity
In order to demonstrate if the compound possessing the highest A1 AR binding affinity, compound 4d,
acted as an antagonist or agonist, a GTP shift experiment was performed. Generally, a rightward shift
of the binding curve in the presence of GTP (due to an uncoupling of the A1 AR from its Gi protein) is
expected for an A1 AR agonist. In the case of an A1 AR antagonist, no significant shift is anticipated
in the presence of GTP [22,23]. As expected compound 4d showed no significant shift of the binding
curve in the presence of GTP, thus compound 4d may be considered an antagonist of the A1 AR
(Table 1; Figure 4). Based on the structural similarity of compound 4d to compounds 4b, 4c and 4e,
the latter compounds are also expected to act as A1 AR antagonists
For future optimization of the 3-cyclohexylamino-imidazo[1,2-α]pyridine scaffold the results of a
study by Novellino and co-workers [24] gave valuable insight for future scaffold modifications. They
explored the A1 AR affinity of novel N-alkyl- and N-acyl-(7-substituted-2-phenylimidazo[1,2-
α][1,3,5]triazin-4-yl)amines (ITAs). Their findings (Figure 5) highlighted the importance of a CH2
spacer and CO linker. The CO linker enhanced A1 AR affinity; however the affinity of the A2A AR
was diminished. When an acetyl group was introduced at the N4 position, it reduced the binding
affinity of the A1 AR, while the binding affinity of the A2A AR remained unchanged. The A1 AR
affinity was improved when a CO-cyclohexyl and CO-phenyl group was introduced although the
binding affinity of the A2A AR showed no improvement. The addition of a methylene spacer between
the N4 position and the acetyl group enhanced A1 AR affinity. When a CO-cyclopentyl was
introduced, it enhanced the affinity for the A1 AR and affinity for the A2A AR was gained.
Substitution of the C7 position with a phenyl moiety rather than a methyl group combined with the
CO-cyclopentyl maintained affinity for the A1 AR, while A2A AR affinity was further increased [24].

O
CH2 spacer
O
H
N
H
N
NH2
N
N
N
N
N
N
N
N
N
N
N
N
KiA1 = 12 nM
KiA1 = 332 nM
KiA2A = 682 nM
KiA1 = 855 nM
KiA2A > 10000 nM
KiA2A > 10000 nM
C7 phenyl
CO linker
cyclopentyl
cyclohexyl
O
O
O
H
N
H
H
N
H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
KiA1 = 231 nM
KiA1 = 116 nM
KiA1 = 4.4 nM
KiA2A = 4300 nM
KiA1 = 17 nM
KiA2A > 10000 nM
KiA2A > 10000 nM
KiA2A = 2500 nM
Figure 5. Structural modification to ITA analogues to gain A1 and A2A AR affinity [24].
A possible explanation for our low AR affinity can be ascribed, in part, to the research done by
Gillespie and co-workers [25], where the increased number of nitrogens in the heterocyclic ring (from
a pyridine to a pyrimidine) enhanced both A1 and A2A AR affinities. They compared the affinities of
triazine, pyrimidine and pyridine scaffolds and observed that the pyridine scaffold was sevenfold less
potent than the triazine and 45-fold less potent than the corresponding aminopyrimidine, thereby
concluding that two nitrogens in the ring are optimum for both A1 and A2A AR affinity [25,26]. This
trend was also observed in the ITA analogues synthesized by Novellino and co-workers [24], where
the 6:5 fused bicyclic rings containing four nitrogen atoms, displayed better AR affinity in
comparison to the current investigated imidazo[1,2-α]pyridine analogues containing only two
nitrogens in the fused heterocyclic rings
Therefore we hypothesize that the introduction of additional nitrogens in the heterocyclic ring may
increase both A1 and A2A AR affinity (Figure 6). Moreover the findings of Novellino and co-workers
[24] can be used for further structural modifications (Figure 6) to 3-cyclohexylamino-2-(4’-
methylphenyl)imidazo[1,2-α]pyridine (4d) to optimize the scaffold for improved A1 and A2A AR
affinity (Figure 6). These modifications include the replacement of the cyclohexyl ring with a
cyclopentyl ring to increase both A1 and A2A AR affinity and the insertion of a CO group and a
methylene spacer between the NH and the CO group to increase A1 AR affinity.
CO linker
cyclopentyl
C7 phenyl
CH2 spacer
O
H
Additional N-atoms (X=N)
N
X
X
X
X
N
N
Figure 6. Proposed future structural modifications to compound 4d.

In conclusion, the newly proposed structural optimization of the investigated imidazo[1,2-α]pyridine
analogues may be implemented in future studies to potentially improve the A1 AR affinity and
possibly gain A2A AR binding. Among the test compounds, 4d possessing a para-substituted methyl
functional group, was identified as the best selective A1 AR antagonist (Ki = 2.06 µM) and may find
therapeutic relevance to enhance PD-associated cognitive dysfunction. Since the study provided proof
of concept that the imidazo[1,2-α]pyridine derivatives possess A1 AR affinity, this scaffold can thus
be used for further structure-activity relationship studies to design novel imidazo[1,2-α]pyridine-
based A1 AR antagonists for the potential treatment of cognition in neurological disorders.
Acknowledgements
We are grateful to Dr. J. Jordaan and Mr. A. Joubert of the SASOL Centre for Chemistry, North-West
University, for recording the NMR and MS spectra, respectively. The financial assistance of the
North-West University, the South African Medical Research Council and the National Research
Foundation towards this research is hereby acknowledged. Opinions expressed and conclusions
arrived at are those of the authors and are not necessarily to be attributed to the NRF.
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Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the
potential treatment of cognition in neurological disorders.
R Lefin, MM van der Walt PJ Milne and G Terre’Blanche
Highlights:
•
•
•
•
A1 receptors are considered drug targets for neurological disorders.
Known 3-cyclohexylamino-2-phenyl-imidazo[1,2-α]pyridines were synthesized.
Para-substituted analogs favor A1 affinity with diminished A2A affinity.
Para-methyl-substituted compound 4d possess highest Ki value of 2.06 µM.

Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential
treatment of cognition in neurological disorders.
R Lefin, MM van der Walt PJ Milne and G Terre’Blanche
Graphical abstract
H
H
N
N
N
N
N
VS
VS
CH3
N
N
N
KiA1 = 2.06 µM
KiA2A > 100 µM
KiA1 > 100 µM
KiA2A > 100 µM
KiA1 > 100 µM
KiA2A > 100 µM