1,3,7-Triethyl-substituted xanthines - Possess nanomolar affinity for the adenosine A1 receptor

Article abstract of DOI:10.1016/j.bmc.2015.09.012

Adenosine A₁ receptors are attracting great interest as drug targets for their role in cognitive deficits. Antagonism of the adenosine A₁ receptor may offer therapeutic benefits in complex neurological diseases, such as Alzheimer's and Parkinson's disease. The aim of this study was to discover potential selective adenosine A₁ receptor antagonists. Several analogs of 8-(3-phenylpropyl)xanthines (3), 8-(2-phenylethyl)xanthines (4) and 8-(phenoxymethyl)xanthines (5) were synthesized and assessed as antagonists of the adenosine A₁ and A_2A receptors via radioligand binding assays. The results indicated that the 1,3,7-triethyl-substituted analogs (3d, 4d, and 5d), among each series, displayed the highest affinity for the adenosine A₁ receptor with K_i values in the nanomolar range. This ethyl-substitution pattern was previously unknown to enhance adenosine A₁ receptor binding affinity. The 1,3,7-triethyl-substituted analogs (3d, 4d, and 5d) behaved as adenosine A₁ receptor antagonists in GTP shift assays performed with either rat cortical or whole brain membranes expressing adenosine A₁ receptors. Further, in vivo evaluation of 3d showed reversal of adenosine A₁ receptor agonist-induced hypolocomotion. In conclusion, the most potent evaluated compound, 8-(3-phenylpropyl)-1,3,7-triethylxanthine (3d), showed both in vitro and in vivo activity, and therefore represent a novel adenosine A₁ receptor antagonist that may have potential as a drug candidate for dementia disorders.

Full text of DOI:10.1016/j.bmc.2015.09.012

Bioorganic & Medicinal Chemistry 23 (2015) 6641–6649
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,3,7-Triethyl-substituted xanthines—possess nanomolar affinity for
the adenosine A1 receptor
b
Mietha M. Van der Walt a, , Gisella Terre’Blanche
⇑
a Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2015
Revised 28 August 2015
Accepted 6 September 2015
Available online 7 September 2015
Adenosine A1 receptors are attracting great interest as drug targets for their role in cognitive deficits.
Antagonism of the adenosine A1 receptor may offer therapeutic benefits in complex neurological diseases,
such as Alzheimer’s and Parkinson’s disease. The aim of this study was to discover potential selective ade-
nosine A1 receptor antagonists. Several analogs of 8-(3-phenylpropyl)xanthines (3), 8-(2-phenylethyl)
xanthines (4) and 8-(phenoxymethyl)xanthines (5) were synthesized and assessed as antagonists of
the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that the 1,3,7-
triethyl-substituted analogs (3d, 4d, and 5d), among each series, displayed the highest affinity for the
adenosine A1 receptor with Ki values in the nanomolar range. This ethyl-substitution pattern was previ-
ously unknown to enhance adenosine A1 receptor binding affinity. The 1,3,7-triethyl-substituted analogs
(3d, 4d, and 5d) behaved as adenosine A1 receptor antagonists in GTP shift assays performed with either
rat cortical or whole brain membranes expressing adenosine A1 receptors. Further, in vivo evaluation of
3d showed reversal of adenosine A1 receptor agonist-induced hypolocomotion. In conclusion, the most
potent evaluated compound, 8-(3-phenylpropyl)-1,3,7-triethylxanthine (3d), showed both in vitro and
in vivo activity, and therefore represent a novel adenosine A1 receptor antagonist that may have potential
as a drug candidate for dementia disorders.
Keywords:
Adenosine A1 receptors
8-(3-Phenylpropyl)xanthines
8-(2-Phenylethyl)xanthines
8-(Phenoxymethyl)xanthines
Alzheimer’s disease
Parkinson’s disease
GTP shift assay
Hypolocomotion
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Adenosine A1 and A3 receptors mediate inhibition of adenyl
cyclase, whereas adenosine A2A and A2B mediate stimulation of
Numerous research groups have attempted to discover selective
adenosine receptor agonists and antagonists for different thera-
peutic applications.1,2 Adenosine receptors are involved in several
nervous system disorders, for example, Parkinson’s disease, cogni-
tive disorders ischemia, epilepsy and stroke.2 The stimulation of
adenosine receptors is responsible for a broad variety of effects
produced by adenosine throughout several organ systems, making
the regulation of these receptors a beneficial therapeutic target.3 In
the central nervous system (CNS), adenosine is a neuromodulator
that acts via G-protein-coupled receptors.4 Four human adenosine
receptor subtypes have been identified, namely A1, A2A, A2B and A3.
adenyl cyclase.5,6
In complex neurological conditions, such as Alzheimer’s disease
(AD) and Parkinson’s disease (PD), it is suggested that antagonism
of the adenosine A1 and/or A2A receptor subtypes may offer thera-
peutic benefits. The adenosine A1 receptors are mainly involved as
drug targets in renal conditions and cognitive deficits.7,8 Adenosine
A1 receptors are widely expressed throughout the human brain,9,10
including the hippocampus and pre-frontal cortex, that are
considered important areas for cognitive function.11 Post-mortem
samples of AD patients showed a reduced density of adenosine
A1 receptors12,13 and in animal studies the blockade of these recep-
tors have been reported previously to enhance cognition in
rodents.14 Furthermore, the blockade of adenosine A1 receptors
on cholinergic terminals has been shown to increase extracellular
levels of acetylcholine that is particularly important for cognitive
processing and are dramatically decreased in the brains of patients
with AD.15 Therefore, based on the latter studies, the antagonism of
adenosine A1 receptors has been targeted to enhance cognitive
impairment associated with AD. On the other hand, antagonism
Abbreviations: PD, Parkinson’s disease; AD, Alzheimer’s disease; CNS, central
nervous system; [3H]DPCPX, [3H]-dipropyl-8-cyclopentylxanthine; [3H]NECA,
N-[3H]-ethyladenosin-50-uronamide; CPA, N6-cyclopentyladenosine; CSC, 1,3,7-
trimethyl-8-(3-chlorostyryl)caffeine;
4-dimethoxystyryl)xanthine; EDAC, N-(3-dimethylaminopropyl)-N0-ethylcarbodi-
KW-6002,
1,3-diethyl-7-methyl-8-(3,
imide; SEM, standard error of the mean.
⇑
Corresponding author. Tel.: +27 18 2992262; fax: +27 18 2994243.
E-mail address: 13035134@nwu.ac.za (M.M. Van der Walt).
http://dx.doi.org/10.1016/j.bmc.2015.09.012
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

6642
M. M. Van der Walt, G. Terre’Blanche / Bioorg. Med. Chem. 23 (2015) 6641–6649
of the adenosine A2A receptors affords neuroprotection in AD.16
Thus, selective adenosine A1 receptor antagonists may be consid-
ered for non-cholinergic symptomatic treatment, whereas adeno-
sine A2A receptor antagonism may offer neuroprotection in the
treatment of AD.
with a dissociation constant value (Ki value) of 55 lM and 50 lM
documented for the adenosine A1 and A2A receptors, respectively.26
Substitution at the C8 position was found to enhance adenosine
receptor affinity, where 8-phenylcaffeine (2b) exhibited a Ki value
of 17 lM for the adenosine A1 receptor and a Ki value of 27 lM for
In the literature, several of the novel approaches to treat PD
involve non-dopaminergic strategies. Adenosine receptor antago-
nists are recognized as such a strategy. Adenosine A1 receptors
are expressed presynaptically in the striatum on both glutamater-
gic and dopaminergic neurons,9,14,17 whereas adenosine A2A recep-
tors are concentrated mainly in the striatum and co-localized with
dopamine D2 receptors.17 Adenosine A2A receptor antagonists may
improve PD-related motor impairment18 via the blockade of ade-
nosine A2A receptors by facilitating dopamine receptor signaling
and thereby restoring motor function in animal models of PD.19
Additionally, blockade of the adenosine A2A receptors also exerts
neuroprotective effects.20 However, the role of adenosine A1 recep-
tors should not be ignored in PD.21 Antagonism of both adenosine
A1 and A2A receptors results in a synergistic motor activating effect,
where inhibition of the adenosine A1 receptor facilitates presynap-
tic dopamine release, whereas inhibition of the adenosine A2A
receptor enhances postsynaptic responses to dopamine.17
Compared to the motor symptoms, the PD-related non-motor
symptoms are under-recognized clinically and as a result under-
treated.22 This may be partly due to evidence suggesting that
non-motor symptoms do not respond to dopaminergic replace-
ment therapy.22,23 PD-associated cognitive impairment has been
identified as one of the non-motor symptoms that is unresponsive
to dopaminergic treatment.22 Antagonism of the adenosine A1
receptor has been implicated in enhancing PD-associated cognitive
dysfunction.24 Therefore selective adenosine A1 receptor antago-
nists may demonstrate therapeutic value in treating cognitive
impairment in PD and dual-acting adenosine A1/A2A receptor
antagonists may be beneficial as a synergistic motor symptom
treatment for PD. In addition, antagonism of the adenosine A2A
receptors may exhibit neuroprotective properties as part of a dis-
ease modifying approach PD means to address.
the adenosine A2A receptor,26 thus showing a slight degree of ade-
nosine A1 receptor selectivity (approximately one fold). During the
1990s the 8-styrylxanthines were developed and are currently one
of the most recognized xanthine classes for adenosine receptor
activity. Two compounds worth mentioning are 1,3,7-trimethyl-
8-(3-chlorostyryl)caffeine (CSC, 2c) and 1,3-diethyl-7-methyl-8-
(3,4-dimethoxystyryl)xanthine (KW-6002, 2d). CSC is often used
as a reference adenosine A2A receptor antagonist in in vivo phar-
macological studies,27 and exhibits Ki values of 28
0.054
M for adenosine A1 and A2A receptors, respectively.21 KW-
lM and
l
6002 was one of the first orally active xanthine drugs developed
and reported with high adenosine receptor affinity (A1 Ki
value = 0.230 lM; A2A Ki value = 0.0022 lM), resulting in an
approximate 104-fold selectivity for rat adenosine A2A recep-
tors.1,28,29 Even though the styryl double bond endows this class
with high adenosine A2A receptor affinity and selectivity, a reduc-
tion of adenosine receptor affinity is observed and may be
explained by E/Z-isomerization of these compounds due to light-
sensitivity.28 Furthermore, affinity and subtype selectivity are
influenced by the substituent chosen at positions N1, N3 and N7
of the xanthine core.25 Some of the alkyl-substituents that have
been explored include the methyl, ethyl and propyl functional
groups.21,25 Caffeine (2a), 8-phenylcaffeine (2b) and CSC (2c) con-
tain methyl substitution at position N1/N3, whereas KW-6002 (2d)
has a 1,3-diethyl-substitution (Fig. 1). Based on the latter, 1,3-di-
ethyl-substitution at the xanthine core may be considered as an
ideal structural requirement for improving adenosine A1 and A2A
receptor affinity.
Recently, we reported on the synthesis and adenosine A2A
receptor affinity (at a maximum tested concentration of 1 lM) of
two xanthine classes, namely 8-(3-phenylpropyl)xanthines and
8-(phenoxymethyl)xanthines.30 The xanthine scaffold may also
be used to design compounds for adenosine A1 receptor affinity.8
Based on the above and the need to explore the adenosine A1
receptor affinity, prompted us to explore further the adenosine
A1 and A2A affinity of the latter reported xanthines. Furthermore,
we introduced another aryl-substituent at position C8, namely
8-(2-phenylethyl). The 8-(2-phenylehtyl)xanthines possess one
methylene group less at C8 than the 8-(3-phenylpropyl)xanthines
Over the past decades, various compounds have been synthe-
sized via the xanthine scaffold (1) to possess adenosine receptor
affinity (Fig. 1). To date, structurally modified xanthines represent
some of the most effective and selective adenosine receptor
antagonists.25 The first reported xanthines displayed poor selectiv-
ity between the adenosine A1 and A2A receptors, for example,
caffeine (2a) is a well-known non-selective xanthine derivative
O
O
O
R7
R1
N
N
N
N
N
N
N
R8
N
N
O
N
O
N
O
N
R3
1
2b
2a
A
A
1 Ki = 17 µM
2A Ki = 27 µM
A
A
1 Ki = 55 µM
2A Ki = 50 µM
O
N
O
N
OCH
N
3
Cl
N
N
N
N
(E)
N
OCH
(E)
O
3
O
2d
1 Ki = 0.230 µM
2A Ki = 0.0022 µM
2c
A
A
A
A
1 Ki = 28 µM
2A Ki = 0.054 µM
Figure 1. The general structure of the xanthine scaffold (1) indicating positions identified in the literature for structural modifications (R1, R3, R7, R8) and the structures of
caffeine (2a), 8-phenylcaffeine (2b), CSC (2c) and KW-6002 (2d) are also shown.

M. M. Van der Walt, G. Terre’Blanche / Bioorg. Med. Chem. 23 (2015) 6641–6649
6643
O
and do not contain an oxygen in the carbon chain at C8 like the
8-(phenoxymethyl)xanthines (Fig. 2). Therefore, in a continuing
effort to identify potential compounds with antagonism for the
adenosine receptor, three xanthine classes (3–5) were investigated
for both adenosine A1 and A2A receptor affinity, at a maximum
tested concentration of 0.1 mM. Structure–activity relationship
studies have shown that a variety of alkyl-substituents at N1, N3
and N7 are appropriate to enhance adenosine A1 and A2A receptor
affinity. Based on these findings, variations at positions N1, N3 and
N7 were included. By analogy with caffeine (2a), 8-phenylcaffeine
(2b) and CSC (2c) the 1,3-dimethyl substitution pattern and by
analogy with KW-6002 (2d) the 1,3-diethyl substitution pattern,
were chosen. Furthermore, the selected 1,3-dialkyl substitution
patterns were combined with either methyl or ethyl substitution
at position N7 (Fig. 2).
O
H
N
R1
NH2
NH2
R1
R8
N
N
a
HO2C R8
O
+
O
N
O
N
NH2
7-9
R3
R3
6a: R1=R3=CH3
6b: R1=R3=C2H5
10
b
O
N
O
R7
H
N
R1
R1
N
N
N
c
R8
R8
N
N
O
O
N
R3
11
R3
3-5
2. Results and discussion
2.1. Synthetic preparation
Scheme 1. Synthetic pathway to xanthine derivatives 3–5. Reagents and condi-
tions: (a) EDAC, dioxane/H2O (1:1), room temperature; (b) NaOH (aq), reflux; (c)
CH3I or C2H5I, K2CO3, acetone.
The key starting materials, 1,3-dimethyl- (6a) and 1,3-diethyl-
5,6-diaminouracil (6b), was synthesized and prepared in good
yields via a previously described protocol.31 Subsequently, the tar-
get xanthines (3–5) were synthesized as reported in the litera-
ture.30 The 1,3-dialkyl-5,6-diaminouracils (6a, b) were reacted
with the appropriate commercially available carboxylic acid, such
as 4-phenylbutanoic acid (7), phenylpropanoic acid (8) and
phenoxyacetic acid (9), in the presence of the coupling reagent
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide (EDAC) at room
temperature. From this acylation reaction the 1,3-dialkyl-6-amino-
5-carboxamidouracil (10) intermediates were obtained. Cycliza-
tion of the intermediates is achieved under strong basic conditions
(aqueous sodium hydroxide solution) to yield the corresponding
1,3-dialkyl-7H-xanthine derivatives (11). Without purification,
the 1,3-dialkyl-7H-xanthine derivatives (11) was directly treated
with excess iodomethane or iodoethane in the presence of potas-
sium carbonate to give the desired range of 7-alkylated xanthine
derivatives (3–5) (Scheme 1). The target compounds (yields of
27–93%) were purified by recrystallization from a suitable solvent,
and in each instance, the structures and purities were verified by
1H NMR, 13C NMR, and mass spectrometry analysis as cited in
Supplementary material.
adenosine A2A receptors in rat striatal membranes are documented
in Tables 1 and 2. These experiments were carried out in the pres-
ence of 0.1 nM [3H]-8-cylcopentyl-1,3-dipropylxanthine ([3H]
DPCPX)33 and 4 nM 50-N-[3H]-ethylcarboxamideadenosine ([3H]
NECA)34 as radioligands for the adenosine A1 and A2A receptors,
respectively. In addition, the adenosine A2A receptor binding stud-
ies were determined in the presence of N6-cyclopentyladenosine
(CPA) to minimize the binding of [3H]NECA to adenosine A1 recep-
tors. All incubations were carried out in triplicate and the Ki values
(dissociation constants) are expressed as the mean standard
error of mean (SEM). Three reference compounds (CPA, DPCPX
and ZM 241385) were included in the study and the results are
in accordance with literature values (Table 1).
2.2.1. Affinity for the adenosine A1 receptor
Compound 3d was recorded with the highest affinity for the
adenosine A1 receptor among the compounds evaluated (series 3,
4 and 5) with a selectivity index (SI) of 17 towards the adenosine
A1 receptor. Decreasing the chain length of the N1/N3 position of
3d (Ki value = 0.164 lM) by one methylene group, compound 3b
2.2. Radioligand binding assays
Table 1
The affinities of the three investigated xanthine classes (3–5) at
rat adenosine A1 and A2A receptor subtypes were determined with
radioligand competition experiments as described previously.32
The experimental results of the radioligand binding experiments
to adenosine A1 receptors in rat whole brain membranes and
The dissociation constant values (Ki values) for the binding of reference compounds
(CPA, DPCPX, ZM 241385) to rat adenosine A1 and A2A receptors
Compound
Ki SEMa (nM)
SIc
b
b
A1 versus
[3H]DPCPX
A2A versus
[3H]NECA
(A2A/A1)
CPA (A1 agonist)
15.30 2.49
(7.9)d
0.553 0.101
(0.56)d (0.3)f
n.d.e
331 84
(460)d
22
n.d.e
958
O
R7
DPCPX (A1 antagonist)
ZM 241385 (A2A antagonist)
530 136
(340)d,f
(1130)f
n.d.e
R1
N
1.359 0.072
(2.2)g
N
(CH )
2 n
X
N
a
O
N
All Ki values determined in triplicate and expressed as mean SEM.
Rat receptors were used (A1: rat whole brain membranes; A2A: rat striatal
b
R3
membranes).
c
Selectivity index (SI) for the adenosine A1 receptor isoform calculated as the
3: R1=R3 = CH3 or C2H5; R7 = CH3 or C2H5; n = 2; X = C
4: R1=R3 = CH3 or C2H5; R7 = CH3 or C2H5; n = 1; X = C
5: R1=R3 = CH3 or C2H5; R7 = CH3 or C2H5; n = 1; X = O
ratio of Ki (A2A)/Ki (A1).
d
Literature values: [3H]DPCPX and [3H]NECA used for adenosine A1 and A2A
receptors, respectively.33
e
Not determined.
f
Literature values: [3H]PIA and [3H]NECA used for adenosine A1 and A2A recep-
Figure 2. The general structures of the 8-(3-phenylpropyl)xanthines (3), 8-(2-
phenylethyl)xanthines (4) and 8-(phenoxymethyl)xanthines (5) that were exam-
ined in this study.
tors, respectively.6
Literature values: [3H]NECA used for adenosine A2A receptors.30,46
g

6644
M. M. Van der Walt, G. Terre’Blanche / Bioorg. Med. Chem. 23 (2015) 6641–6649
Table 2
The dissociation constant values (Ki values) for the binding of the test compounds (3–5) to rat adenosine A1 and A2A receptors
R7
O
R1
N
N
R8
N
O
N
R3
Compound
R1 = R3
R7
R8
Ki SEMa
(lM)
SIc
A1 versus [3H]DPCPX
b
A2A versus [3H]NECA
(A2A/A1)
b
8-(3-Phenylpropyl)xanthines (3)
3a
3b
3c
3d
–CH3
–CH3
–C2H5
–C2H5
–CH3
–C2H5
–CH3
–C2H5
–(CH2)3–C6H5
–(CH2)3–C6H5
–(CH2)3–C6H5
–(CH2)3–C6H5
1.55 0.06
0.259 0.019
1.24 0.09
6.80 0.49
4.93 0.69
3.93 0.48
2.71 0.08
4
19
3
0.164 0.014
17
8-(2-Phenylethyl)xanthines (4)
4a
4b
4c
4d
–CH3
–CH3
–C2H5
–C2H5
–CH3
–C2H5
–CH3
–C2H5
–(CH2)2–C6H5
–(CH2)2–C6H5
–(CH2)2–C6H5
–(CH2)2–C6H5
5.74 1.19
1.09 0.04
0.816 0.053
0.636 0.084
4.95 0.19
3.11 0.24
2.36 0.52
4.97 0.39
0.86
3
3
8
8-(Phenoxymethyl)xanthines (5)
5a
5b
5c
5d
–CH3
–CH3
–C2H5
–C2H5
–CH3
–C2H5
–CH3
–C2H5
–CH2–O–C6H5
–CH2–O–C6H5
–CH2–O–C6H5
–CH2–O–C6H5
3.09 0.40
1.10 0.04
0.874 0.024
0.416 0.042
2.37 0.42
2.25 0.36
1.91 0.29
2.64 0.38
0.77
2
2
6
a
b
c
All Ki values determined in triplicate and expressed as mean SEM.
Rat receptors were used (A1: rat whole brain membranes; A2A: rat striatal membranes).
Selectivity index (SI) for the A1 receptor isoform calculated as the ratio of Ki (A2A)/Ki (A1).
(Ki value = 0.259
lM) was obtained with the second highest
seems to favor ethyl over methyl substitution for enhanced adeno-
affinity and a SI of approximately 19 for the adenosine A1 receptor.
Compounds 3b and 3d were compared to their homologs 3a and
3c, respectively. Noteworthy, 3a and 3c was substituted with a
methyl group at N7 oppose to an ethyl group at that same position
for 3b and 3d. It was found that 3b and 3d possess 6 and 8 fold
higher affinities for the adenosine A1 receptor than 3a and 3c,
respectively. It may be concluded, among the compounds of series
3, that ethyl substitution at N7 is important for increased affinity
for the adenosine A1 receptor.
sine A1 receptor affinity. For example, 5d possess an improved Ki
value of 0.416
Similarly, compound 5b (Ki value = 1.10
fold more potent than 5a (Ki value = 3.09
l
M over the Ki value of 0.874
M) is approximately 3
M). The 1,3,7-triethyl-
lM reported for 5c.
l
l
substituted derivative, 5d, was documented for series 5 with the
best affinity for the adenosine A1 receptor. It seems that substitu-
tion of the C8 chain with a phenoxymethyl side chain (series 5)
compared to a phenylethyl side chain (series 4) moderately
affected the adenosine A1 receptor affinity. Compound 5d (Ki
Furthermore, series 4 was obtained by decreasing the C8 chain
length of series 3 with one methylene group. Comparing the homo-
logs of series 3 and 4, the importance of the C8 chain length of
series 3 is illustrated by the decreased adenosine A1 receptor
affinity observed for series 4. None the less, the two most potent
value = 0.416
sine A1 receptor than 4d (Ki value = 0.639
l
M) possess a 1.5 fold higher affinity for the adeno-
M).
l
2.2.2. Affinity for the adenosine A2A receptor
Evidence suggests that ethyl substitution at N1/N3 in combina-
tion with methyl substitution at N7 may result in an enhancement
of binding affinity towards the adenosine A2A receptor. The litera-
ture supports the findings that enhancement of the adenosine A2A
receptor binding activity may be achieved with ethyl and methyl
substitution on the xanthine base at position N1/N328 and
N7,25,28 respectively. For example, among series 4 and 5, the 1,3-di-
ethyl-7-methyl-substituted xanthines, 4c and 5c, was documented
with the highest affinity towards the adenosine A2A receptor. Com-
8-(2-phenylethyl)xanthines
value = 0.816 M) and 4d (Ki value = 0.636
below 1 M. On examination of the three xanthine classes (3, 4 and
5), compound 4a was documented with the weakest affinity for the
adenosine A1 receptor. In fact, 4a (Ki value = 5.74 M) is 9 and 35
fold weaker than 4d (Ki value = 0.636 M) and 3d (Ki
value = 0.164 M), respectively. This may be attributed to the
(series
4),
namely
4c
(Ki
l
lM), exhibited Ki values
l
l
l
l
observation that compounds 3d and 4d possess a 1,3,7-triethyl
substitution on the xanthine ring, in contrast to the 1,3,7-trimethyl
substitution of 4a. Not only does ethyl substitution at N7 enhance
the adenosine A1 receptor affinity within series 4, but simultane-
ous ethyl substitution of N1/N3 may also increase the affinity.
For example, the affinity for the adenosine A1 receptor of com-
pound 1,3,7-trimethyl-8-(2-phenylethyl)xanthine (4a) diminished
by 5 and 7 fold when compared to the homologs 1,3-dimethyl-7-
ethyl-8-(2-phenylethyl)xanthine (4b) and 1,3-diethyl-7-methyl-
8-(2-phenylethyl)xanthine (4c), respectively.
pound 5c was found to possess a Ki value of 1.91 lM. Compound 5c
also possess the highest affinity for the adenosine A2A receptor
among the investigated compounds (series 3, 4 and 5) in this study.
Contrary to the expected for series 3, compound 3d, and not 3c,
was found with a greater affinity for the adenosine A2A receptor.
Noteworthy, 3c was only found to be one fold less potent than 3d.
2.3. Functional characterization of selected test compounds
Upon evaluation of compounds 5a–d the Ki values ranged
between 0.416 and 3.09 lM. Among the 8-(phenoxymethyl)xan-
thines (series 5), it was found that 5c and 5d displayed the highest
affinity for the adenosine A1 receptor. In general, the N7 position
The regulation of the G-protein-coupled receptors’ affinity via
the effects of guanosine triphosphate (GTP) is well documented.35
The influence of GTP on binding of adenosine A1 and A2A receptors
have been studied previously with both rat and human species.35,36

M. M. Van der Walt, G. Terre’Blanche / Bioorg. Med. Chem. 23 (2015) 6641–6649
6645
It is thought that GTP act by uncoupling the receptors from G-pro-
teins and this leads to a decrease in affinity for agonists.4,35,36 The
regulation of GTP and interactions with endogenous adenosine in
binding studies were found to be similar for both rat and human
expressing adenosine A1 and A2A receptors.35
8-Cylcopentyl-1,3-dipropylxanthine (DPCPX) is documented as
an antagonist with a high affinity and selectivity for the adenosine
A1 receptor.6 In order to study adenosine A1 receptors, [3H]DPCPX
is frequently used as radioligand in both rat and human
species.6,33,35 The affinity of [3H]DPCPX to the adenosine A1 recep-
tors are affected in the presence of GTP. However, GTP up to
0.1 mM concentration did not affect the binding of [3H]DPCPX in
rat brain membranes and agonists still competed for the binding
of [3H]DPCPX.6
Therefore, by investigating competition curves of a selected
compound, in the presence and absence of GTP, one may be able
to determine if that particular compound may function as an ago-
nist or as an antagonist. The GTP shifts are calculated by dividing
the Ki value obtained in the presence of GTP by the Ki value obtained
in the absence of GTP.37 GTP do not affect competition curves of an
antagonist5 and a compound with a calculated GTP shift of approx-
imately 1 is considered a full antagonist.37 On the other hand, the
presence of GTP affects the competition curves of an agonist and
shifts the curve to the right. Full agonists possess a greater calcu-
lated GTP shift than partial agonists.5 Literature documents the
GTP shifts for unlabelled DPCPX and CPA with [3H]DPCPX as radioli-
gand to rat brain membranes. It was found that unlabelled DPCPX
act as a full antagonist with a GTP shift of 138 and the CPA as a full
agonist with GTP shifts reported as 6 and 32.5,37
shift assay using rat whole brain membrane with the addition of
0.1 mM GTP and the assay conditions were kept the same. Simi-
larly to the above described GTP shift assay in rat cortical mem-
branes (see Section 2.3.1), the two reference compounds (DPCPX
and CPA) and three test compounds (3d, 4d and 5d) were evalu-
ated. On examination, the GTP shift assay carried out with either
membranes prepared from rat brain cortical or whole brains lead
to similar Ki values both in the absence and presence of GTP. This
demonstrated that whole brain membranes—expressing adenosine
A1 receptors—may be used similarly to cortical membranes in
order to determine functionality of a compound via a GTP shift
assay. Consequently, the calculated GTP shifts for 3d, 4d and 5d
was approximately 1, thus confirming that these compounds func-
tion as adenosine A1 receptor antagonists (Table 4). As mentioned
above, the three xanthine homologs 3d, 4d and 5d are representa-
tive of the three classes of xanthines currently evaluated, suggest-
ing that the compounds of the three xanthine classes function as
antagonists (Fig. 3). An advantage of using the rat whole brain
membranes, with the GTP shift assay, is that these membranes
may not only be easily obtained in large quantities, but may also
be prepared with ease. Taking the above results into account, the
1,3,7-triethyl-substituted analogs (3d, 4d, and 5d) behave as ade-
nosine A1 receptor antagonists in GTP shift assays performed with
either rat cortical or whole brain membranes using [3H]DPCPX as
radioligand. Nonspecific binding was defined by the addition of
100 lM CPA or 10 lM DPCPX (unlabelled). The two competitors
resulted in the same nonspecific binding determination with the
radioligand [3H]DPCPX to either rat cortical or whole brain
membranes.
2.3.1. GTP shift assay performed with rat cortical membranes
The full antagonist DPCPX and full agonist CPA was chosen to
validate the GTP shift assay of the current study. The binding of
0.4 nM [3H]DPCPX to rat brain cortical membranes for the selected
test compounds (3d, 4d and 5d) and two reference compounds
(DPCPX and CPA) were determined in triplicate. The affinities were
determined in the absence and presence of 0.1 mM GTP and are
summarized in Tables 3 and 4 with the calculated GTP shifts. The
results document a GTP shift of approximately 1 for the unlabelled
DPCPX (full antagonist) and approximately 14 for CPA (full ago-
nist), thus, corresponding with literature values (Table 3). Three
xanthine classes (compounds 3–5) were evaluated in this study.
Among each class the 1,3,7-triethyl-substituted xanthines (3d, 4d
and 5d) was identified with the highest degree of binding to the
adenosine A1 receptor. Therefore, these three xanthine homologs
(3d, 4d and 5d) are representative of the three classes of xanthines
(compounds 3–5) currently investigated and were selected to
assess whether these compounds function as agonists or antago-
nists (as expected). Compounds 3d, 4d and 5d were found to pos-
sess a calculated GTP shift of approximately 1 (Table 4). Therefore,
it may be concluded that these compounds function as adenosine
A1 receptor antagonists.
2.4. Evaluation of adenosine A1 receptor agonist-induced
hypolocomotion in rats
Further evidence for adenosine A1 antagonism of compound 3d
was explored using the CPA-induced locomotor depression model
in vivo, where simple agonist/antagonist competition for occu-
pancy of adenosine A1 receptors would result in the attenuation
of the hypolocomotion.14
Locomotor activity was significantly reduced in the CPA
(0.06 mg/kg, ip) treated control group compared to the normal
group (P <0.05). DPCPX, a known selective adenosine A1 antagonist,
significantly attenuated the hypolocomotion induced by CPA with
a dosage of 0.03 and 0.1 mg/kg as shown in Figure 4. As expected
from the GTP shift assay results, intraperitoneal administration of
compound 3d antagonized selective adenosine A1 receptor agonist
CPA-induced hypolocomotion (0.06 and 0.15 mg/kg), indicating
that compound 3d is able to block central adenosine A1 receptors
in vivo and that it has blood–brain barrier permeability (Fig. 4).
3. Conclusion
Selective adenosine A1 receptor antagonists are recognized as a
potential treatment for cognitive impairments, found in AD and
PD.39 In general, modifications of the xanthine scaffold at position
C8 with aryl or cycloalkyl groups has shown promise for identify-
ing novel adenosine A1 and A2A receptor antagonists.40 The effects
of receptor subtype selectivity of methyl and propyl substitution at
the N1, N3 and N7 positions and various substituents at C8 of the
xanthine core have been explored in detail.41 However, the influ-
ence of a 1,3,7-triethyl-substitution pattern on adenosine A1 recep-
tor affinity has not been explored, yet. Accordingly, the aim of this
study was to discover potential selective adenosine A1 receptor
antagonists by comparing various methyl and ethyl substitution
patterns at positions N1, N3 and N7 on three selected C8-sub-
stitued xanthine scaffolds.
2.3.2. GTP shift assay performed with rat whole brain
membranes
Lohse and co-workers6 stated that [3H]DPCPX bind swiftly and
selectively to adenosine A1 receptors of various tissues. This is
strengthened by the observation that the binding of [3H]DPCPX
to adenosine A1 receptors expressed in whole brain or cortical
membranes (in the absence of GTP) results in similar Ki values
for reference compounds CPA and DPCPX (Table 3). For this reason,
the binding of 0.1 nM [3H]DPCPX to whole brain membranes was
explored additionally in the presence of GTP to assess if whole
brain membranes would result in similar calculated GTP shifts as
obtained with cortical membranes. The adenosine A1 radioligand
binding assay described in Section 4.2.1 was modified for a GTP

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M. M. Van der Walt, G. Terre’Blanche / Bioorg. Med. Chem. 23 (2015) 6641–6649
Table 3
The adenosine A1 affinities (in the absence and presence of GTP) and GTP shifts of CPA (A1 agonist) and DPCPX (A1 antagonist) as reference compounds
Compound
Ki SEMa (nM)
Rat brain cortical membranesb
+GTP (0.1 mM)
Rat whole brain membranesc
+GTP (0.1 mM)
ꢀ GTP
4.43 1.05
(5.9)e
GTPd shift
ꢀ GTP
15.30 2.48
GTPd shift
6.48
CPA
(A1 agonist)
63.08 3.2
(35.2)e
14.24
(6)e
99.21 14.83
(32)f
0.92
(1)g
DPCPX
(A1 antagonist)
0.229 0.079
0.211 0.020
0.553 0.101
0.477 0.048
0.86
a
b
c
d
e
f
All Ki values determined in triplicate and expressed as mean SEM.
Displacement of [3H]DPCPX (final concentration 0.4 nM; Kd = 0.28 nM).
Displacement of [3H]DPCPX (final concentration 0.1 nM; Kd = 0.36 nM).
GTP shifts calculated by dividing the Ki in the presence of GTP by the Ki in the absence of GTP.
Literature Ki values and GTP shift determined with [3H]DPCPX.37
A literature GTP shift of 32 was obtained with [3H]DPCPX.5
A literature GTP shift of 1 was obtained with [3H]DPCPX.38
g
Table 4
The adenosine A1 affinities (in the absence and presence of GTP) and GTP shifts of selected test compounds (3d, 4d and 5d)
Compound
Ki SEMa
(lM)
Rat brain cortical membranesb
+GTP (0.1 mM)
Rat whole brain membranesc
+GTP (0.1 mM)
ꢀ GTP
GTPd shift
ꢀ GTP
GTPd shift
3d
4d
5d
0.139 0.01
0.543 0.14
0.356 0.04
0.258 0.03
0.681 0.07
0.368 0.02
1.85
1.25
1.03
0.164 0.01
0.636 0.08
0.416 0.04
0.209 0.05
0.763 0.01
0.397 0.08
1.27
1.20
0.95
a
b
c
All Ki values determined in triplicate and expressed mean SEM.
Displacement of [3H]DPCPX (final concentration 0.4 nM; Kd = 0.28 nM).
Displacement of [3H]DPCPX (final concentration 0.1 nM; Kd = 0.36 nM).
d
GTP shifts calculated by dividing the Ki in the presence of GTP by the Ki in the absence of GTP.
B. Adenosine A1 binding
A. Adenosine A1 binding
(rat whole brain membranes)
(rat whole brain membranes)
100
100
+ GTP (0.1 mM)
- GTP
+ GTP (0.1 mM)
- GTP
50
0
50
0
-1
0
1
2
3
4
-1
0
1
2
3
4
Log [DPCPX]
Log [CPA]
D. Adenosine A1 binding
C. Adenosine A1 binding
(rat whole brain membranes)
(rat corꢀcal membranes)
100
100
+ GTP (0.1 mM)
- GTP
+ GTP (0.1 mM)
- GTP
50
0
50
0
-3
-2
-1
0
1
2
-2
-1
0
1
2
Log [3d]
Log [3d]
Figure 3. GTP shift assays at adenosine A1 receptors determined with [3H]DPCPX as radioligand in various rat membranes (cortical and whole brains). (A) Binding curve for
the adenosine A1 agonist CPA performed with rat whole brain membranes; GTP shift is 6.48. (B) Binding curve of the adenosine A1 antagonist DPCPX performed with rat
whole brain membranes; GTP shift is 0.86. (C) Binding curve of the adenosine A1 antagonist 3d performed with rat cortical membranes; GTP shift is 1.85. (D) Binding curve of
the adenosine A1 antagonist 3d performed with rat whole brain membranes; GTP shift is 1.27.
This study describes three xanthine classes, namely 8-(3-
phenylpropyl)xanthines (3a–d), 8-(2-phenylethyl)xanthines (4a–
d) and 8-(phenoxymethyl)xanthines (5a–d), as novel adenosine
A1 receptor antagonists. In general, the compounds evaluated were
found to be selective for the adenosine A1 receptor, especially
compounds 3b and 3d. Overall, ethyl substitution compared to

M. M. Van der Walt, G. Terre’Blanche / Bioorg. Med. Chem. 23 (2015) 6641–6649
6647
may be obtained at either rat cortical or whole brain membranes
with [3H]DPCPX as radioligand. The most potent test compound,
8-(3-phenylpropyl)-1,3,7-triethylxanthine (3d), showed both
in vitro and in vivo activity, confirming that xanthine adenosine
A1 receptor antagonists is an attractive therapeutic candidate for
dementia disorders.
5000
4000
3000
2000
1000
0
*
*
*
*
*
4. Experimental section
4.1. Synthetic preparation
General information: Unless otherwise noted, all the commer-
cially available reagents were obtained from Sigma–Aldrich and
were used without further purification. Melting points (mp): The
melting points (mp) were measured with a Buchi M-545 melting
point apparatus and are uncorrected. Nuclear Magnetic Resonance
(NMR) spectra: The proton (1H) and carbon (13C) NMR spectra were
acquired on a Bruker Avance III 600 spectrometer in CDCl3. The
chemical shifts was reported in parts per million (d) and
tetramethylsilane (TMS) was used as internal standard. Coupling
constants (J value) are given in hertz (Hz). Spin multiplicities are
abbreviated as follow: s (singlet), d (doublet), t (triplet), q (quartet)
or qn (quintet). Mass spectra (MS): High resolution mass spectra
(HRMS) were recorded with a Bruker micrOTOF-Q II mass spec-
trometer in atmospheric pressure chemical ionization (APCI)
mode. The 1,3-dialkyl-5,6-diaminouracils (6a, b) were synthesized
following the protocol developed by Blicke and Godt.31 The test
compounds 3a, 3b, 3c, 3d, 5a, 5b, 5c, 5d30 and 4a42 were prepared
as described in literature. The analytical results of the compounds
4b, 4c and 4d are documented in Supplementary material.
The synthetic procedure for the preparation of the xanthines
derivatives 4b, 4c and 4d: 1,3-Dialkyl-6-amino-5-carboxamidouracil
(10): The 1,3-dialkyl-6-amino-5-carboxamidouracil (10) interme-
diates were acquired by dissolving 10 mmol of the appropriate
1,3-dialkyl-5,6-diaminouracils (6a or 6b) in a minimum amount
of dioxane/H2O (1:1). Subsequently 13.4 mmol EDAC and 10 mmol
phenylpropanoic acid (8) was added, the pH was adjusted to 5 with
4 N HCl and stirred for 2 h at room temperature. 1,3-dialkyl-7H-
xanthine derivatives (11): To the 1,3-dialkyl-6-amino-5-carboxam-
idouracil (10) reaction mixture, 1 g of sodium hydroxide (dissolved
in 1 mL H2O) was added. The reaction was heated under reflux for
1 h, cooled to 0 °C and acidified with 4 N HCl. The resulting precip-
itate yielded the corresponding 1,3-dialkyl-7H-xanthine derivative
(11). Desired xanthine derivatives (4b–4d): Without further purifica-
tion, the crude 7H-xanthine analog (11, 1.42 mmol) was dissolved
in a minimum amount of dry acetone and treated with either iodo-
methane or iodoethane (3.3 mmol) in the presence of K2CO3
(1.1 mmol). The reaction was heated under reflux for 1–2 h. The
progress of the reaction was monitored with neutral alumina
TLC. The solvent was removed in vacuo, H2O (30 mL) was added
to the residue and the product extracted to ethylacetate
(3 ꢁ 30 mL). The organic phase was combined, dried over anhy-
drous MgSO4 and concentrated in vacuo. Analytical pure samples
were obtained after recrystallization from ethanol.
0.03
0.1
0.06
normal control
0.15
_______
_______
DPCPX (mg/kg)
3d (mg/kg)
______________________
CPA (0.06 mg/kg)
Figure 4. Adenosine A1 receptor antagonistic activity of DPCPX and 3d determined
in vivo. DPCPX (0.03 or 0.1 mg/kg) and 3d (0.06 or 0.15 mg/kg) attenuated
hypolocomotion induced by the adenosine A1 agonist CPA (0.06 mg/kg) in rats.
Data are represented as the mean SEM (rats n = 7–14). *P <0.05 statistically
significant compared with control group (One way ANOVA with Tukey’s post hoc
test).
methyl substitution seemed to enhance adenosine A1 receptor
affinity. Comparing homologs with either methyl or ethyl substitu-
tion at position N7 (N1@N3@CH3), showed that ethyl substitution,
in each case, improved adenosine A1 receptor affinity (3a vs 3b; 4a
vs 4b; 5a vs 5b). Furthermore, ethyl substitution on both N1 and
N3 (N1@N3@N7@CH2CH3) showed the same pattern, resulting in
higher affinity for the adenosine A1 receptors (3a vs 3d; 4a vs
4d; 5a vs 5d). C8 substitution included the 8-(3-phenylpropyl)
(3), the 8-(2-phenylethyl) (4) and the 8-(phenoxymethyl) (5) sub-
stituents. The 8-(3-phenylpropyl) substituent (series 3) was identi-
fied as showing the most promising adenosine A1 receptor affinity
with compounds 3b and 3d exhibiting the highest affinity and
selectivity among all three investigated series and bearing ethyl
substitution on N7. These compounds may be considered as selec-
tive adenosine A1 receptor antagonists that can potentially be used
in the treatment of AD- and PD-associated cognitive impairments.
Compounds 4a and 5a (1,3,7-trimethyl-substituted) may be con-
sidered as good non-selective adenosine receptor antagonists,
compared to caffeine (2a), and may therefore find therapeutic
value in treating PD-related motor symptoms via a synergistic
mechanism. Jacobson and co-workers21 suggested that the motor
effects exerted by caffeine can not only be attributed to the block-
ade of the adenosine A2A receptors and that the role of adenosine
A1 receptors should not be rejected.
The 1,3,7-triethyl-substituted compounds (representative of
each xanthine class) were found to possess a calculated GTP shift
of approximately 1, thereby emphasizing that 3d, 4d and 5d may
act as antagonists of the adenosine A1 receptor and that 1,3,7-tri-
ethyl-substitution may govern adenosine A1 receptor affinity.
Furthermore, the results indicate that among these 1,3,7-triethyl-
substituted xanthines (3d, 4d and 5d), compound 3d exhibited
the highest binding affinity and was subsequently further evalu-
ated in vivo with an adenosine A1 receptor agonist-induced
hypolocomotor animal model. The results provided evidence that
compound 3d not only possesses in vitro activity, but also in vivo
activity as an adenosine A1 receptor antagonist.
4.1.1. 8-(2-Phenylethyl)-1,3-dimethyl-7-ethylxanthine (4b)
The title compound (white crystals) was prepared from 1,3-
dimethyl-5,6-diaminouracil (6a), phenylpropanoic acid (8) and
iodoethane in a yield of 72%: mp 255 °C (ethanol); 1H NMR (CDCl3)
d 1.23 (t, 3H, J = 7.2 Hz), 2.98 (t, 2H, J = 7.2 Hz), 3.09 (t, 2H,
J = 7.5 Hz), 3.37 (s, 3H), 3.57 (s, 3H), 4.09 (q, 2H, J = 7.2 Hz), 7.14
(d, 2H, J = 7.5 Hz), 7.20 (t, 1H, J = 7.2 Hz), 7.26 (t, 2H, J = 7.2 Hz);
13C NMR (CDCl3) d 16.1, 27.9, 28.8, 29.7, 34.1, 40.1, 106.3, 126.6,
128.3, 128.7, 140.1, 148.2, 151.7, 152.5, 154.8; APCI-HRMS m/z:
Calcd for C17H21N4O2 (MH+), 313.1664. Found: 313.1635.
In conclusion it was found that ethyl substitution at the N1, N3
and N7 positions of the three xanthine classes explored, enhances
adenosine A1 receptor affinity, thus emphasizing that, the 1,3,7-tri-
ethyl-substituted xanthines (3d, 4d and 5d) represent novel ade-
nosine A1 receptor antagonists that are suitable for the design of
compounds with nanomolar affinity for the adenosine A1 receptor.
This study is the first to report that similar calculated GTP shifts

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M. M. Van der Walt, G. Terre’Blanche / Bioorg. Med. Chem. 23 (2015) 6641–6649
4.1.2. 8-(2-Phenylethyl)-1,3-diethyl-7-methylxanthine (4c)
The title compound (light yellow needles) was prepared from
1,3-diethyl-5,6-diaminouracil (6b), phenylpropanoic acid (8) and
iodomethane in a yield of 27%: mp 92.3 °C (ethanol); 1H NMR
(CDCl3) d 1.21 (t, 3H, J = 7.2 Hz), 1.32 (t, 3H, J = 7.2 Hz), 2.99 (t,
2H, J = 7.5 Hz), 3.05 (t, 2H, J = 7.2 Hz), 3.61 (s, 3H), 4.03 (q, 2H,
J = 7.2 Hz), 4.15 (q, 2H, J = 7.2 Hz), 7.11 (d, 2H, J = 6.8 Hz), 7.20 (t,
1H, J = 7.2 Hz), 7.25 (t, 2H, J = 7.2 Hz); 13C NMR (CDCl3) d 13.3,
13.4, 28.9, 31.3, 34.0, 36.3, 37.4, 107.4, 126.6, 128.3, 128.7, 140.0,
147.5, 150.7, 153.2, 155.0; APCI-HRMS m/z: Calcd for C18H23N4O2
(MH+), 327.1821. Found: 327.1802.
the adenosine A1 receptor radioligand binding assays and for the
adenosine A2A receptor radioligand binding assays. The competi-
tion experiments were performed in triplicate.
4.2.2. GTP shift assays
Membrane preparations: The collection of tissue samples for the
adenosine A1 receptor GTP shift assay was approved by the
Research Ethics Committee of the North-West University (applica-
tion number NWU-0035-10-A5). GTP shift assay (rat cortical mem-
branes): The membrane preparation entailed that the tissues
collected after dissection were immediately placed in 0.32 mol/L
sucrose (4 °C) and prepared according to the protocol described
by Lohse and co-workers36 except that the membranes were finally
resuspended in 50 mM Tris–HCl buffer (pH 7.4 at 25 °C) to a vol-
ume of 5 mL/g original tissue weight, then aliquoted into micro-
centrifuge tubes and stored at ꢀ70 °C until needed. GTP shift
assay (rat whole brain membranes): The membrane preparation
was performed under the same conditions as described above for
the adenosine A1 receptor radioligand binding assay (Sec-
tion 4.2.1).32,33 The protein content of all membrane preparations
were determined with Bradford reagent according to the method
described by Bradford.43
4.1.3. 8-(2-Phenylethyl)-1,3,7-triethylxanthine (4d)
The title compound (yellow solid) was prepared from 1,3-di-
ethyl-5,6-diaminouracil (6b), phenylpropanoic acid (8) and
iodoethane in a yield of 32%: mp 105.7 °C (ethanol); 1H NMR
(CDCl3) d 1.24 (qn, 6H, J = 7.2 Hz), 1.34 (t, 3H, J = 7.2 Hz), 2.99 (t,
2H, J = 7.9 Hz), 3.08 (t, 2H, J = 7.9 Hz), 4.05 (q, 2H, J = 7.2 Hz), 4.11
(q, 2H, J = 7.2 Hz), 4.16 (q, 2H, J = 7.2 Hz), 7.15 (d, 2H, J = 6.8 Hz),
7.20 (t, 1H, J = 7.2 Hz), 7.27 (t, 2H, J = 7.2 Hz); 13C NMR (CDCl3) d
13.3, 13.5, 16.2, 28.8, 34.8, 36.3, 38.4, 40.0, 106.6, 126.6, 128.4,
128.7, 140.2, 147.8, 150.8, 152.4, 154.6; APCI-HRMS m/z: Calcd
for C19H25N4O2 (MH+), 341.1977. Found: 341.1941.
GTP shift assay protocol: The GTP shift assays performed with
either rat cortical or whole brain membranes were carried out in
a final assay volume of 1 mL of the appropriate 50 mM Tris–HCl
buffer. The GTP shift assay performed with rat cortical membranes
was conducted as previously described.36,37 The 50 mM Tris–HCl
buffer used had a pH of 7.4 at 25 °C.36 The radioligand [3H]DPCPX
(Kd = 0.28 nM) was used at a concentration of 0.4 nM as docu-
mented by Van der Wenden and co-workers.37 The incubations
4.2. In vitro evaluation
General information: All commercially available reagents were
obtained from various manufacturers (Sigma–Aldrich, Ascent Sci-
entific and Merck). The radioligands [3H]NECA (specific activity
25 Ci/mmol) and [3H]DPCPX (specific activity 120 Ci/mmol) were
obtained from Amersham Biosciences and PerkinElmer, respec-
tively. Filter-count was purchased from PerkinElmer, while What-
man GF/B 25 mm diameter filters was obtained from Merck.
Counting of radio activities were performed using a Packard Tri-
CARB 2810 TR liquid scintillation counter.
contained 100 l
g membrane protein suspension, 0.4 nM [3H]
DPCPX, 0.2 units/mL adenosine deaminase, with or without
0.1 mM GTP, the test compound and 1% DMSO. The GTP shift assay
performed with rat whole brain membranes followed the protocol
mentioned in Section 4.2.1,32,33 except with the addition of 0.1 mM
GTP. The 50 mM Tris–HCl buffer used had a pH of 7.7 at 25 °C.33
The radioligand [3H]DPCPX (Kd = 0.36 nM) was used at a concentra-
tion of 0.1 nM as documented in the literature.32,33 The incubations
4.2.1. Radioligand binding assays
Membrane preparations: The collection of tissue samples for the
adenosine A1 and A2A receptor binding studies was approved by
the Research Ethics Committee of the North-West University
(application number NWU-0035-10-A5). Male Sprague–Dawley
rats were dissected in order to obtain rat whole brains (excluding
cerebellum and brain stem) for the adenosine A1 receptor compe-
tition experiments, while rat striata were collected for the adeno-
sine A2A receptor binding assays. After dissection, the tissues
were immediately snap frozen with liquid nitrogen and stored at
ꢀ70 °C. On the day of the membrane preparation, the rat whole
brains and rat striata were prepared according to the protocol
described in literature.32 The protein content of all membrane
preparations were determined with Bradford reagent according
to the method described by Bradford.43
contained 120 l
g membrane protein suspension, 0.1 nM [3H]
DPCPX, 0.1 units/mL adenosine deaminase, with or without
0.1 mM GTP, the test compound and 1% DMSO. The addition of
GTP to a final concentration of 0.1 mM was done according to
literature specifications.4,6 Nonspecific binding was defined by
the addition of either 100 lM CPA or 10 lM DPCPX (unlabelled).
The two competitors resulted in the same nonspecific binding
determination with the radioligand [3H]DPCPX to either rat cortical
or whole brain membranes.
4.3. In vivo evaluation
4.3.1. Adenosine A1 receptor agonist-induced hypolocomotion
in rats
Radioligand binding assay protocol: For the radioligand binding
assays at adenosine A1 receptors, the incubations contained
Animals: Male Sprague–Dawley rats weighing between 260 and
300 g were used to test the agonist/antagonist behavior. Animals
were housed in groups of three animals per cage at 22 °C 1 and
relative humidity 50% 10 with a light cycle from 06:00 to
18:00, and were given free access to standard laboratory food
and water throughout the study. All efforts were made to minimize
animal suffering, and all experiments were carried out in accor-
dance with the guidelines of the Experimental Laboratory Animal
Committee of the North West University (NWU-0035-10-A5).
Methods: Locomotor activity was measured with a Digiscan Ani-
mal Activity Monitor system (Omnitech Electronics Inc., Columbus,
OH, USA). Rats were placed individually into the locomotor cages
(L = 20; W = 20; H = 30 cm) and ambulatory counts were registered
when consecutive light beams were interrupted, and stereotypical
120 lg of membrane protein suspension (rat whole brain mem-
branes), 0.1 nM [3H]DPCPX, 0.1 units/mL adenosine deaminase,
the test compound and 1% DMSO.32,33 Whereas the radioligand
binding studies at adenosine A2A receptors were incubated with
120 lg of membrane protein suspension (rat striatal membranes),
4 nM [3H]NECA, 50 nM CPA, 10 mM MgCl2, 0.2 units/mL adenosine
deaminase, the test compound and 1% DMSO.32,34 DMSO was used
to prepare all stock solutions of the test compounds. The incuba-
tion tubes containing a final volume of 1 mL 50 mM Tris–HCl buffer
(pH 7.7 at 25 °C). The assays were performed as described in
literaure.32 Nonspecific binding was defined by the addition of
either 100
petitors resulted in the same nonspecific binding determination for
l l
M CPA32,34 or unlabelled 10 M DPCPX.5 The two com-

M. M. Van der Walt, G. Terre’Blanche / Bioorg. Med. Chem. 23 (2015) 6641–6649
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The NMR and MS spectra data were recorded by Andre Joubert
and Johan Jordaan of the SASOL Centre for Chemistry, North-West
University. This work is based on the research supported in part by
the National Research Foundation (NRF) and Medical Research
Council (MRC) of South Africa. The Grant holders acknowledge that
opinions, findings and conclusions or recommendations expressed
in any publication generated by the NRF and MRC supported
research are that of the authors and that the NRF and MRC accepts
no liability whatsoever in this regard.
Supplementary data
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