The adenosine A2A antagonistic properties of selected C8-substituted xanthines

Article abstract of DOI:10.1016/j.bioorg.2013.06.006

The adenosine A₂A receptor is considered to be an important target for the development of new therapies for Parkinson's disease. Several antagonists of the A₂A receptor have entered clinical trials for this purpose and many research

Full text of DOI:10.1016/j.bioorg.2013.06.006

Bioorganic Chemistry 49 (2013) 49–58
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The adenosine A2A antagonistic properties of selected C8-substituted
xanthines
Mietha M. Van der Walt a, Gisella Terre’Blanche a, Anél Petzer b, Anna C.U. Lourens a, Jacobus P. Petzer a,
⇑
a Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Centre of Excellence for Pharmaceutical Sciences, 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 18 December 2012
Available online 4 July 2013
The adenosine A2A receptor is considered to be an important target for the development of new therapies
for Parkinson’s disease. Several antagonists of the A2A receptor have entered clinical trials for this purpose
and many research groups have initiated programs to develop A2A receptor antagonists. Most A2A recep-
tor antagonists belong to two different chemical classes, the xanthine derivatives and the amino-substi-
tuted heterocyclic compounds. In an attempt to discover high affinity A2A receptor antagonists and to
further explore the structure–activity relationships (SARs) of A2A antagonism by the xanthine class of
compounds, this study examines the A2A antagonistic properties of series of (E)-8-styrylxanthines, 8-
(phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines. The results document that among these
series, the (E)-8-styrylxanthines have the highest binding affinities with the most potent homologue,
(E)-1,3-diethyl-7-methyl-8-[(3-trifluoromethyl)styryl]xanthine, exhibiting a Ki value of 11.9 nM. This
compound was also effective in reversing haloperidol-induced catalepsy in rats, providing evidence that
it is in fact an A2A receptor antagonist. The importance of substitution at C8 with the styryl moiety was
demonstrated by the finding that none of the 8-(phenoxymethyl)xanthines and 8-(3-phenylpro-
pyl)xanthines exhibited high binding affinities for the A2A receptor.
Keywords:
Adenosine A2A receptors
Antagonism
Xanthine
Haloperidol-induced catalepsy
Parkinson’s disease
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
antagonistic effect on D2 receptors [6,7]. This antagonistic relation-
ship provides the basis for a role of A2A receptors in the treatment
The purine nucleoside, adenosine, is involved in numerous
functions in the central nervous system (CNS) and mediates most
of its effects through the activation of four guanine nucleotide-
binding protein (G protein)-coupled receptor (GPCR) subtypes,
A1, A2A, A2B and A3 [1]. Through its actions at these receptors, aden-
osine regulates a variety of physiological responses and adenosine
receptors have become targets for the treatment of a variety of
disease states [2]. Of particular interest is a potential role for the
A2A receptor subtype in the therapy of Parkinson’s disease [3].
A2A receptors are highly enriched in striatopallidal neurons in the
striatum where they are colocalized with the dopamine D2
receptors [4,5]. A2A and D2 receptors are thought to interact with
each other, with A2A receptor stimulation exerting a functional
of Parkinson’s disease. Blockade of the A2A receptor potentiates D2
receptor-mediated neurotransmission and therefore reduces the
effects of striatal dopamine depletion in Parkinson’s disease [8,9].
These observations suggest that A2A antagonism also may possibly
potentiate the motor actions of levodopa and dopamine agonists
[10–12]. An example of this behaviour is found with (E)-
1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KW-6002,
istradefylline, 1), an A2A antagonist which has entered clinical trials
for the treatment of Parkinson’s disease (Fig. 1) [11,12]. It was
demonstrated that, in Parkinson’s disease patients, KW-6002
potentiates the symptomatic benefits of a reduced dose of levo-
dopa and produces motor enhancement that is comparable to that
of an optimal levodopa dose [10]. In addition, KW-6002 prolongs
the therapeutic action of a full dose of levodopa [13]. Pharmacolog-
ical and epidemiological evidence suggest that A2A antagonists
may possess neuroprotective properties. For example, selective
A2A antagonists, and not A1 antagonists, exert a protective effect
against the neurotoxic actions of 1-methyl-4-phenyl-1,2,3,6-tetra-
hydropyridine (MPTP) [14,15] and 6-hydroxydopamine [15,16] in
animals. Furthermore, the consumption of caffeine (2), a nonselec-
tive A1/A2A antagonist, has been correlated with a reduced risk of
developing Parkinson’s disease [17–20]. This protective effect is
Abbreviations: CNS, central nervous system; CPA, cyclopentyladenosine; CSC,
(E)-8-(3-chlorostyryl)caffeine; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; EDAC, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide; GPCR, guanine
nucleotide-binding protein (G protein)-coupled receptor; MAO-B, monoamine
oxidase B; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; [3H]NECA, N-
[3H]ethyladenosin-50-uronamide; [3H]DPCPX, 1,3-[3H]-dipropyl-8-cyclopentylxan-
thine; SAR, structure–activity relationship; SI, selectivity index.
⇑
Corresponding author. Fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.06.006

50
M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
the xanthine ring are appropriate for A2A antagonism. These
O
N
substituents include the methyl, ethyl, propyl and propargyl
functional groups [23,24,29,30]. Based on these observations, the
present study aims to discover high affinity xanthine derived A2A
antagonists and to further explore the SARs of A2A antagonism by
the xanthine class of compounds. For this purpose, the A2A antag-
onistic properties of series of novel (E)-8-styrylxanthines (4–7) will
be investigated and compared to those of 8-(phenoxymethyl)
xanthines (8) and 8-(3-phenylpropyl)xanthines (9), chemical clas-
ses which have not previously been examined for potential A2A
antagonistic properties. The selection of compounds from series 8
and 9 is based on a recent report that these compounds also are
highly potent MAO-B inhibitors [31]. Should they also possess
A2A antagonistic properties, these derivatives may be viewed as
promising dual-target-directed antiparkinsonian agents. To ex-
plore chemical space, these series (4–9) will be comprised of
homologues with different CH3/C2H5 substitution patterns on N1,
N3 and N7 of the xanthine ring. For the (E)-8-styrylxanthines (4–
7), the effects on A2A antagonism by different halogen (Cl, Br)
and halogen containing (CF3) substituents on the styryl phenyl ring
will also be examined. This study also reports, for selected xanth-
ines, the binding affinities to the adenosine A1 receptor as well as
the human MAO-B inhibitory potencies.
OCH3
N
N
N
(E)
OCH3
O
KW-6002 (1)
O
N
N
N
O
N
Caffeine (2)
O
N
Cl
N
N
N
(E)
O
CSC (3)
2. Results and discussion
Fig. 1. The structures of KW-6002 (1), caffeine (2) and CSC (3).
2.1. Chemistry
The target xanthine derivatives, series 4–9, were synthesized
according to the literature procedure (Fig. 2) [32]. 1,3-Dimethyl-
(10a) and 1,3-diethyl-5,6-diaminouracil (10b), synthesized accord-
ing to literature, served as key starting materials [33]. The uracils
were reacted with the appropriate carboxylic acids, the (E)-cin-
namic acids (11), the phenoxyacetic acids (12) and the 4-phenylb-
utanoic acids (13), in the presence of N-(3-dimethylaminopropyl)-
N0-ethylcarbodiimide (EDAC) as dehydrating agent to yield the cor-
responding intermediary amides. Treatment of the amides with
NaOH yielded the corresponding 1,3-dialkyl-7H-xanthine ana-
logues (14). In order to obtain the desired 7-alkylated xanthine
analogues (4–9), the xanthines 14 were reacted with an excess of
iodomethane or iodoethane in the presence of K2CO3. The target
compounds (yields of 7–93%) were purified by recrystallization
from a suitable solvent, and in each instance, the structures and
purities were verified by 1H NMR, 13C NMR, mass spectrometry
and HPLC analysis as cited in the Supplementary material.
thought to be related to blockade of A2A receptors by caffeine [14].
The symptomatic and protective benefits of A2A antagonists there-
fore make them attractive agents for the therapy of Parkinson’s
disease.
Based on these considerations, several research groups have ini-
tiated programs to develop A2A receptor antagonists and at least
five compounds have entered clinical trials for the treatment of
Parkinson’s disease [21]. Most A2A receptor antagonists may be
classified into two different chemical classes, the xanthine deriva-
tives and the amino-substituted heterocyclic compounds [22]. KW-
6002 and (E)-8-(3-chlorostyryl)caffeine (CSC, 3; IC50 = 36–54 nM),
a frequently used reference A2A antagonist, are examples of xan-
thine derived A2A antagonists [23,24]. It is noteworthy that certain
xanthine derived A2A antagonists also are highly potent mono-
amine oxidase B (MAO-B) inhibitors. For example, CSC is reported
to inhibit MAO-B with a Ki value of 80.6 nM, a value that is compa-
rable to that reported for its antagonism of A2A receptors [25,26]. In
Parkinson’s disease, MAO-B inhibitors potentiate the motor restor-
ative effects of levodopa by reducing the metabolic breakdown of
dopamine in the central nervous system [27]. In addition, MAO-B
inhibitors may also protect against the underlying neurodegenera-
tive processes in Parkinson’s disease. Since MAO-B inhibitors are
considered useful in the therapy of Parkinson’s disease, dual-tar-
get-directed compounds such as CSC may possess enhanced thera-
peutic value. The combination of A2A antagonism and MAO-B
inhibition in a single drug may be particularly advantageous as
an adjunct to levodopa therapy [28].
2.2. In vitro binding to A2A and A1 receptors
The affinities of compounds 4–9 for A2A receptors were exam-
ined by the radiolig and binding protocol described in literature
[34]. Binding of compounds from series 4–9 to the A2A receptors
were investigated by measuring the displacement of N-[3H]ethyla-
denosin-50-uronamide ([3H]NECA) from rat striatal membranes.
These studies were carried out in the presence of N6-cyclopentyl-
adenosine (CPA) to minimize the binding of [3H]NECA to adenosine
A1 receptors. As positive controls the known A2A antagonists, KW-
6002 (1), CSC (3) and ZM 241385, were included in this study. As
shown in Table 1, KW-6002, CSC and ZM 241385 displayed Ki val-
ues for the antagonism of A2A receptors of 7.94 nM, 26.2 nM and
2.31 nM, respectively. These values correspond well with the liter-
ature values of 2.2 nM [29], 36–54 nM [23,24] and 2 nM [22] for
these antagonists, respectively.
An important structural feature of KW-6002 and CSC is the sty-
ryl moiety of the (E) configuration at C8 of the xanthine ring. While
a wide variety of substituents on the styryl phenyl ring are toler-
ated, modification of the styryl double bond is usually associated
with a loss of A2A antagonistic activity. For example, (E)-8-styryl-
caffeine displays a Ki value for the antagonism of A2A receptors of
94 nM, while the corresponding phenyl substituted homologue is
a weak A2A receptor antagonist with a Ki value of 19
Structure–activity relationship (SAR) studies have further shown
that a variety of substituents on the N1, N3 and N7 positions of
l
M [24].
The affinities of the (E)-8-styrylxanthine (4–7), 8-(phenoxy-
methyl)xanthine (8) and 8-(3-phenylpropyl)xanthine (9) deriva-
tives for A2A receptors are given in Tables 2–4. From the results,

M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
51
O
O
H
N
R1
R1
NH2
N
N
R8
a, b
R8
HO2C
11−13
+
N
NH2
O
O
N
N
R3
R3
10a: R1 = R3 =CH3
10b: R1 = R3 = C2H5
14
O
R7
R1
N
N
c
R8
N
O
N
R3
4−9
Fig. 2. Synthetic pathway to xanthine derivatives 4–9. Reagents and conditions: (a) EDAC, dioxane:H2O (1:1); (b) NaOH (aq), reflux; (c) CH3I or C2H5I, K2CO3, DMF, ethanol or
acetone.
Table 1
(7e) possessing a Ki value <100 nM. This analysis suggests that
among the four series of (E)-8-styrylxanthines (4–7) examined,
the 1,3-diethyl-7-methylxanthine substitution pattern, as found
with compounds 6a–f, is most appropriate for high affinity binding
to A2A receptors.
The Ki values for the competitive inhibition of [3H]NECA binding to rat striatal
adenosine A2A receptors by KW-6002, CSC and ZM 241385.
Ki values (nM)a
KW-6002
CSC
ZM 241385
7.94 2.05 (2.2)b
26.2 3.49 (36)c (54)d
2.31 1.96 (2)e
To examine the selectivities of the xanthines for A2A receptors,
the affinities of four homologues for adenosine A1 receptors were
examined. The xanthines selected for this purpose were com-
pounds 4c, 5c, 6f and 7e. These compounds are representative of
the four classes of xanthines (compounds 4–7) which exhibited
affinity for A2A receptors and include the most potent compounds
identified in this study. The binding of the test compounds to A1
receptors were investigated by measuring the displacement of
1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) from rat
whole brain membranes [34]. As positive controls, the affinities
of the antagonists, caffeine and ZM 241385, for A1 receptors were
examined. As shown in Table 5, caffeine and ZM 241385 displayed
Ki values for the binding to A1 receptors of 43,907 nM and 273 nM,
respectively. These values correspond well with the literature val-
ues of 41,000 nM [24] and 225 nM [22] for these compounds,
respectively. In addition, the recorded affinity of the agonist, CPA,
for A1 receptors corresponded to that reported in literature (Ta-
ble 5). Compared to their affinities for binding to A2A receptors,
compounds 4c, 5c and 6f displayed relatively weaker binding to
a
b
c
All Ki values were determined in duplicate and are expressed as mean SD.
Value obtained from Ref. [29].
Value obtained from Ref. [24].
Value obtained from Ref. [23].
Value obtained from Ref. [22].
d
e
it is evident that the 8-(phenoxymethyl)xanthine (8) and (E)-8-(3-
phenylpropyl)xanthine (9) derivatives do not possess affinities for
A2A receptors with only one homologue, compound 8a (Ki = 137
nM), exhibiting moderate affinity. Since different CH3/C2H5 substi-
tution patterns on N1, N3 and N7 do not yield compounds with
high binding affinities, it may be concluded that, in contrast to sty-
ryl substitution, phenoxymethyl and phenylpropyl substitution on
C8 of the xanthine ring are in general not suitable for binding to
A
2A receptors. In accordance with literature, the (E)-8-styrylxanth-
ines were found to possess affinities for the A2A receptor
[23,24,32]. Compounds with good binding affinities for the A2A
receptor (Ki < 100 nM) were identified among all four series 4–7
of (E)-8-styrylxanthines examined. The compound with the high-
est binding affinity among the compounds examined was 6f, the
CF3 substituted 1,3-diethyl-7-methylxanthine homologue. This
compound exhibited a Ki value of 11.9 nM (Fig. 3), a value similar
to that of KW-6002 (Ki = 7.94 nM). It is noteworthy that all of the
1,3-diethyl-7-methylxanthine homologues, 6a–f, exhibited high
affinities for A2A receptors. This result suggests that the CH3/C2H5
substitution pattern of 6a–f is particularly suitable for binding to
A2A receptors by xanthine derived compounds. High affinity com-
pounds were, however, also found among the 1,3,7-trimethylx-
anthines (4a–e) and the 1,3-dimethyl-7-ethylxanthines (5a–f)
with Ki values as low as 28.4 nM (compound 4c). The 1,3,7-trieth-
ylxanthines (7a–f) on the other hand displayed comparatively
weaker affinities for A2A receptors with three homologues (7a, 7b
A1 receptors. Compound 4c exhibited a Ki value >1 lM for binding
to A1 receptors while 5c and 6f were 16-and 17-fold, respectively,
more selective for A2A receptors. Interestingly, 7e was found to
bind to A1 receptors (Ki = 56.9 nM) with higher affinity than to
A2A receptors (Ki = 83.0 nM). From these results it may be con-
cluded that compound 4c is a suitable lead were high potency
and selective A2A binding is desired, while 7e is an example of a
compound which exhibits high potency binding to both A1 and
A2A receptors. Although not as selective as ZM 241385 (SI = 118),
compounds 5c and 6f may still be considered to possess high
potency and selectivity for A2A receptors. These interesting SARs
merit further investigation.
2.3. In vivo reversal of haloperidol-induced catalepsy
To obtain evidence of an antagonistic mechanism of action and
of an in vivo efficacy, the abilities of two compounds with high A2A
and 7f) exhibiting Ki values of >1 lM and only one homologue

52
M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
Table 2
The Ki values for the competitive inhibition of [3H]NECA binding to rat striatal adenosine A2A receptors by (E)-8-styrylxanthines 4–7.
X
Ki values (nM)a
X
Ki values (nM)a
4a
4b
4c
4d
4e
4-Cl
3,4-Cl
3-Br
4-Br
3-CF3
169 61.4b
44.8 4.11
28.4 2.40
5a
5b
5c
5d
5e
5f
3-Cl
4-Cl
3,4-Cl
3-Br
4-Br
3-CF3
421 105b
95.3 11.3
32.9 6.27
58.2 2.91
546 223b
93.8 2.01
127 0.01
125 24.2 (134)c
6a
6b
6c
6d
6e
6f
3-Cl
4-Cl
3,4-Cl
3-Br
4-Br
3-CF3
24.3 1.57
18.9 1.88
47.9 8.55
17.2 4.80
28.9 4.93
11.9 1.34
7a
7b
7c
7d
7e
7f
3-Cl
4-Cl
3,4-Cl
3-Br
4-Br
3-CF3
>1
>1
l
l
M
M
428 491b
161 223b
83.0 50.9b
>1 lM
a
b
c
All Ki values were determined in duplicate and are expressed as mean SD.
Standard errors probably due to low aqueous solubility at concentrations approaching the Ki value.
Value obtained from Ref. [23].
Table 3
binding affinities, compounds 4c and 6f, to attenuate haloperidol-
induced catalepsy in rats were examined. For this purpose rats
(n = 6/group) were treated intraperitoneally (i.p.) with haloperidol
(5 mg/kg) followed 30 min later with vehicle or test compound (4c
or 6f) at 0.1, 0.4, 1 and 2 mg/kg. At 90 min post haloperidol treat-
ment, the degree of catalepsy of the rats were evaluated using the
standard bar test according to the literature protocol [35,36]. Rats
which were similarly treated with KW-6002 served as positive
controls. The results presented in Fig. 4 show that both 4c and 6f
reverse haloperidol-induced catalepsy in a concentration depen-
dent manner. At doses of 0.1–2 mg/kg, 4c significantly (p < 0.05)
reduces mean catalepsy time by 20–42% compared to the vehicle.
At doses of 0.4, 1 and 2 mg/kg, 6f significantly (p < 0.05) reduces
mean catalepsy time by 17–39% compared to the vehicle. For com-
parison, KW-6002 at doses of 0.1–2 mg/kg reduces mean catalepsy
time by 7–44% compared to the vehicle. These data demonstrates
that both 4c and 6f are in vivo active A2A receptor antagonists. It
should be noted that haloperidol-induced catalepsy may also be
reversed by pharmacological agents acting at other biochemical
targets such as dopamine and serotonin receptors. The current
study cannot rule out that 6f does not reverse haloperidol-induced
catalepsy by acting at such alternative targets. It may, however, be
expected that, if 6f is an A2A agonist (rather than an antagonist), its
effect on the reversal of catalepsy would not be so pronounced
since A2A agonism would enhance haloperidol-induced catalepsy.
The Ki values for the competitive inhibition of [3H]NECA binding to rat striatal
adenosine A2A receptors by 8-(phenoxymethyl)xanthines 8.
Ki values (nM)a
8a
8b
8c
8d
R1 = R3 = R7 = CH3
137 50.8
R1 = R3 = CH3; R7 = C2H5
R1 = R3 = C2H5; R7 = CH3
R1 = R3 = R7 = C2H5
>1
>1
>1
l
l
l
M
M
M
a
All Ki values were determined in duplicate and are expressed as mean SD.
Table 4
The Ki values for the competitive inhibition of [3H]NECA binding to rat striatal
adenosine A2A receptors by (E)-8-(3-phenylpropyl)xanthines 9.
2.4. In vitro inhibition of MAO-B
Ki values (nM)a
The MAO-B inhibitory potencies of selected potent A2A antago-
nists (compounds 4c, 5c, 6f, 7e) were examined using recombinant
human MAO-B as enzyme source and kynuramine as enzyme sub-
strate. For this purpose the amount of hydrogen peroxide that is
produced in the MAO oxidation cycle was measured. In a horserad-
ish peroxidase-coupled reaction, hydrogen peroxide reacts with
9a
9b
9c
9d
R1 = R3 = R7 = CH3
>1
>1
>1
>1
l
l
l
l
M
M
M
M
R1 = R3 = CH3; R7 = C2H5
R1 = R3 = C2H5; R7 = CH3
R1 = R3 = R7 = C2H5
a
Determined in duplicate.

M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
53
100
75
50
25
0
A2A Receptor
100
75
50
25
0
A1 Receptor
-2
-1
0
1
2
3
4
-2
0
2
4
6
Log [6f]
Log [6f]
Fig. 3. The sigmoidal dose–response curve for the antagonism of [3H]NECA binding to rat striatal A2A receptors and [3H]DPCPX binding to rat whole brain A1 receptors by
compound 6f (expressed in nM). The bound cpm values were adjusted for nonspecific binding and are expressed as percentage of the cpm values recorded in the absence of
6f.
Table 5
3. Conclusion
The Ki values for the competitive inhibition of [3H]DPCPX binding to rat whole brain
adenosine A1 receptors by selected xanthines and reference compounds. The IC50
values for the inhibition of MAO-B are also listed.
In conclusion, this study shows that 8-(phenoxymethyl)xan-
thine and 8-(3-phenylpropyl)xanthine derivatives, in general, do
A1 Antagonism
MAO-B inhibition
IC50
M)c
not exhibit potent binding affinities to A2A receptors. In accordance
to literature, (E)-8-styrylxanthines, on the other hand, have high
affinities for A2A receptors, with particularly the 1,3-diethyl-7-
methylxanthine substitution being most appropriate for high affin-
ity binding. This behaviour is exemplified by compound 6f, which
binds to rat striatal A2A receptors with a Ki value of 11.9 nM. An
investigation of the affinities of selected compounds for A1 recep-
tors documents that 6f, as well as 4c and 5c, display selective bind-
ing to A2A receptors compared to A1 receptors. Among these
compounds 4c is particularly selective for A2A receptors. Interest-
ingly 7e was found to be bind relatively non-selectively to both
A1 and A2A receptors while still displaying good affinities for both
receptor subtypes. An examination of the MAO-B inhibitory poten-
cies of selected potent A2A antagonists reveals that 4c also is a
highly potent MAO-B inhibitor. This compound therefore repre-
sents a potential dual-acting antiparkinsonian agent with affinity
for both A2A receptors and MAO-B.
Ki (nM)a
SIb
(l
4c
5c
6f
7e
>1
lM
–
0.112 0.020
2.05 1.03
No inhibition
No inhibition
NDg
562 4.85
194 17.3
56.9 2.45
17
16
0.7
–
118
–
Caffeine
ZM 241385
CPA
43,907 3151 (41,000)d
273 4.73 (225)e
10.4 1.57 (7.9)f
NDg
NDg
a
All Ki values were determined in duplicate and are expressed as mean SD.
The selectivity index is the selectivity for the A2A receptor isoform and is given
as the ratio of Ki (A1)/Ki (A2A).
b
c
All IC50 values were determined in triplicate and are expressed as mean SD.
Value obtained from Ref. [24].
Value obtained from Ref. [22].
Value obtained from Ref. [41].
d
e
f
g
Not determined.
Ampliflu Red to yield resorufin, a fluorescent compound, which is
readily quantifiable via fluorescence spectrophotometry. The re-
sults, presented in Table 5, show that only 4c and 5c exhibit mean-
4. Experimental section
ingful MAO-B inhibition with IC50 values of 0.112 lM and 2.05 lM,
respectively. In fact, 4c may be considered to be a highly potent
MAO-B inhibitor since its IC50 value is in the nanomolar range. It
is interesting to note that the active MAO-B inhibitors, compounds
4c and 5c, contain the di-CH3 substitution pattern on N1 and N3 of
the xanthine ring, while introduction of the C2H5 substituent on
these positions (compounds 6f and 7e) eliminate MAO-B inhibi-
tion. In addition, a CH3 on N7 is more suitable for MAO-B inhibition
than the C2H5 substituent on this position since 4c is approxi-
mately 18-fold more potent as a MAO-B inhibitor than 5c. It
should, however, be noted that different substitution on the phenyl
rings of these compounds may also contribute to their different
MAO-B inhibitory activities. These interesting relationships may
be of value in future design studies and should be examined
further.
Considering these results, compound 4c is a particularly prom-
ising therapeutic compound for Parkinson’s disease since it acts as
a high affinity A2A antagonist as well as a potent MAO-B inhibitor.
In addition, 4c displays a high degree of selectivity for A2A recep-
tors over A1 receptors. A further point of interest is that, in addition
to A2A antagonism, MAO-B inhibition may also contribute to the
in vivo anticataleptic activity of 4c. Since compound 6f is a selec-
tive A2A antagonist with no affinity for MAO-B, its in vivo anticat-
aleptic activity is, however, most likely not mediated by MAO-B
inhibition.
4.1. Chemicals and instrumentation
All reagents and solvents were obtained from Sigma–Aldrich
and were used without further purification. Proton (1H) and carbon
(
13C) NMR spectra were recorded on a Bruker Avance III 600 spec-
trometer in CDCl3. The chemical shifts are reported in parts per
million (d) relative to the signal of tetramethylsilane. Spin multi-
plicities are abbreviated as follows: s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quartet), qn (quintet) or m
(multiplet). High resolution mass spectra (HRMS) were recorded
with a Bruker micrOTOF-Q II mass spectrometer in atmospheric-
pressure chemical ionization (APCI) mode or on a DFS high resolu-
tion magnetic sector mass spectrometer (Thermo Electron Corpo-
ration) in electrospray ionization (ESI) mode. Melting points (mp)
were measured with a Buchi M-545 melting point apparatus and
are uncorrected. Counting of radio activities were performed using
a Packard Tri-Carb 2100 TR liquid scintillation counter. 1,3-Di-
methyl-(10a) and 1,3-diethyl-5,6-diaminouracil (10b) were pre-
pared according to
a previously reported procedure [33].
[3H]NECA (specific activity 25 Ci/mmol) and [3H]DPCPX (specific
activity 120 Ci/mmol) were obtained from Amersham Biosciences
and PerkinElmer, respectively. Adenosine deaminase (type X from
calf spleen; 250 units) and CPA were from Sigma–Aldrich. What-
manÒ GF/B 25 mm diameter filters and dimethyl sulfoxide (DMSO)

54
M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
125
6f
125
100
75
50
25
0
4c
100
75
*
*
*
*
50
*
*
*
25
0
100
80
60
40
20
0
KW-6002
*
*
*
*
Fig. 4. Attenuation of haloperidol-induced catalepsy by 4c, 6f and KW-6002. Rats (n = 6/group) were treated with haloperidol (5 mg/kg) followed 30 min later with vehicle,
compounds 4c, 6f or KW-6002 at 0.1, 0.4, 1 and 2 mg/kg. At 90 min post haloperidol treatment, catalepsy time were evaluated using the standard bar test. ꢂOne way ANOVA
followed by Dunnet’s multiple comparison test p < 0.05.
were obtained from Merck. Filter-count was purchased from Per-
kin Elmer. The reference compounds, KW-6002 (1) and CSC (3),
were synthesized as previously reported [29,37], while ZM -
241385 was obtained from Ascent Scientific. The syntheses and
characterizations of 4a [38], 4b [39] and 8a [31] have been previ-
ously reported. For fluorescence spectrophotometry, a Varian Cary
Eclipse fluorescence spectrophotometer was employed. The
following chemical and biological agents were obtained from Sig-
ma–Aldrich: microsomes from insect cells containing recombinant
human MAO-B (5 mg/mL), kynuramineꢀ2HBr, Ampliflu Red
(10-acetyl-3,7-dihydroxyphenoxazine), horseradish peroxidase,
(R)-deprenylꢀHCl and H2O2 (3%).
HCl and the resulting precipitate was collected by filtration and
washed with 50 mL water. For the synthesis of 9b–d, 1 g of sodium
hydroxide was dissolved in H2O (1 mL) and added to the reaction.
The reaction was heated under reflux for 2 h, cooled to 0 °C and
acidified with 4 N HCl. The resulting precipitate, the 7H-xanthine
analogues (yields 29–75%), were collected via filtration and
washed with 50 mL H2O.
Synthesis of 4–7: Without further purification, the crude 7H-
xanthine analogue (14, 1.6 mmol) was dissolved in a minimum
amount of N,N-dimethylformamide (DMF). K2CO3 (4 mmol) was
added followed by iodomethane or iodoethane (3.2 mmol) and
the reaction was stirred for 2 h at 90 °C. The progress of the reac-
tion was monitored with neutral alumina thin layer chromatogra-
phy (TLC). This was followed by adding 50 mL H2O to the reaction,
yielding a precipitate that was collected via filtration and dried
overnight in a fume cupboard. Analytical pure samples were ob-
tained after recrystallization from the appropriate solvent as cited
below.
Synthesis of 8b–d and 9a–d: Without further purification, the
crude 7H-xanthine analogue (14, 1.27 mmol) was dissolved in a
minimum amount of either dry acetone (for the synthesis of 8b–
d and 9a–b) or anhydrous ethanol (for the synthesis of 9c and
9d). K2CO3 (0.55 mmol) was added followed by iodomethane or
iodoethane (1.5 mmol). The reaction was heated under reflux
(90 °C) for 1–2 h. The progress of the reaction was monitored with
neutral alumina TLC. The solvent was removed in vacuo and H2O
(30 mL) was added to the residue. The product was extracted to
CHCl3 (3 ꢁ 30 mL), the organic phase was dried over anhydrous
MgSO4 and concentrated in vacuo. Analytical pure samples were
obtained after recrystallization from the appropriate solvent as ci-
ted below.
4.2. General procedure for the synthesis of xanthine analogues (4–9)
Synthesis of intermediary 1,3-dialkyl-7H-xanthine analogues (14):
1,3-Dimethyl- (10a) or 1,3-diethyl-5,6-diaminouracil (10b)
(10 mmol) was dissolved in a minimum amount of dioxane/H2O
(1:1). EDAC (13.4 mmol) and the appropriate carboxylic acid
(10 mmol) was added and the pH was adjusted to 5 with 4 N
HCl. The reaction was stirred for 2 h at room temperature. Except
for the synthesis of compounds 8c, 8d and 9b–d, the reaction
was subsequently neutralized (pH 7) with the addition of 1 N
NaOH. The precipitate was collected via filtration and suspended
in 100 mL of dioxane/H2O (1:1). The reaction was then heated at
reflux for 2 h at room temperature. After cooling to 0 °C, the precip-
itate that formed was collected by filtration and washed with
50 mL H2O. In the case of compounds 8c and 8d, H2O (30 mL)
was added to the reaction and the reaction was subsequently ex-
tracted to CHCl3 (9 ꢁ 25 mL). The combined organic phases were
washed with H2O (4 ꢁ 80 mL) followed with brine (4 ꢁ 80 mL),
dried over anhydrous MgSO4 and finally concentrated under re-
duced pressure. The crude residue was dissolved in a mixture of
30 mL aqueous NaOH (1 M)/dioxane (1:1) and heated under reflux
for 45 min. After cooling to 0 °C, the reaction was acidified with 4 N
4.2.1. (E)-8-(3-Bromostyryl)-1,3,7-trimethylxanthine (4c)
The title compound (yellow crystals) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-3-bromocinnamic acid and iodo-

M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
55
methane in a yield of 61%: mp 229.9 °C (lit. 229–231 °C) [39] (eth-
anol); 1H NMR (CDCl3) d 3.39 (s, 3H), 3.59 (s, 3H), 4.05 (s, 3H), 6.88
(d, 1H, J = 15.4 Hz), 7.25 (d, 1H, J = 6.4 Hz), 7.45 (dd, 2H, J = 1.5,
7.9 Hz), 7.70 (d, 1H, J = 15.4 Hz), 7.71 (s, 1H); 13C NMR (CDCl3) d
27.9, 29.7, 31.6, 108.1, 112.5, 123.1, 126.2, 129.8, 130.4, 132.2,
136.4, 137.6, 148.5, 149.3, 151.6, 155.2; APCI-HRMS m/z: Calcd
4.2.7. (E)-8-(3-Bromostyryl)-1,3-dimethyl-7-ethylxanthine (5d)
The title compound (yellow needles) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-3-bromocinnamic acid and iodoeth-
ane in a yield of 76%: mp 227.5 °C (ethanol); 1H NMR (CDCl3) d 1.45
(t, 3H, J = 7.2 Hz), 3.40 (s, 3H), 3.60 (s, 3H), 4.48 (q, 2H, J = 7.2 Hz),
6.87 (d, 1H, J = 15.8 Hz), 7.26 (d, 1H, J = 7.9 Hz), 7.45 (dd, 2H, J = 1.9,
7.9 Hz), 7.71 (s, 1H), 7.72 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3) d
16.7, 28.0, 29.7, 40.1, 107.3, 112.5, 123.1, 126.2, 129.7, 130.4,
132.2, 136.5, 137.6, 148.4, 148.7, 151.7, 154.8; APCI-HRMS m/z:
Calcd for C17H18BrN4O2 (MH+), 389.0614. Found: 389.0599; Purity
(HPLC): 99%.
for
C
16H16BrN4O2 (MH+), 375.0457. Found: 375.0440; Purity
(HPLC): 100%.
4.2.2. (E)-8-(4-Bromostyryl)-1,3,7-trimethylxanthine (4d)
The title compound (yellow needles) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-4-bromocinnamic acid and iodo-
methane in a yield of 88%: mp 255.7 °C (ethanol); 1H NMR
(CDCl3) d 3.38 (s, 3H), 3.59 (s, 3H), 4.03 (s, 3H), 6.87 (d, 1H,
J = 15.4 Hz), 7.41 (d, 2H, J = 8.7 Hz), 7.50 (d, 2H, J = 8.7 Hz), 7.70
(d, 1H, J = 15.8 Hz); 13C NMR (CDCl3) d 27.9, 29.7, 31.5, 108.0,
111.7, 123.5, 128.7, 132.1, 134.3, 136.8, 148.5, 149.5, 151.6,
155.2; APCI-HRMS m/z: Calcd for C16H16BrN4O2 (MH+), 375.0457.
Found: 375.0442; Purity (HPLC): 100%.
4.2.8. (E)-8-(4-Bromostyryl)-1,3-dimethyl-7-ethylxanthine (5e)
The title compound (yellow needles) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-4-bromocinnamic acid and iodoeth-
ane in a yield of 63%: mp 233.7 °C (ethanol/toluene); 1H NMR
(CDCl3) d 1.44 (t, 3H, J = 7.2 Hz), 3.38 (s, 3H), 3.59 (s, 3H), 4.46 (q,
2H, J = 7.2 Hz), 6.86 (d, 1H, J = 15.8 Hz), 7.41 (d, 2H, J = 8.3 Hz),
7.50 (d, 2H, J = 8.7 Hz), 7.73 (d, 1H, J = 15.8 Hz); 13C NMR (CDCl3)
d 16.7, 27.9, 29.7, 40.0, 107.2, 111.7, 123.5, 128.7, 132.1, 134.4,
136.8, 148.6, 148.7, 151.7, 154.8; APCI-HRMS m/z: Calcd for C17H18-
BrN4O2 (MH+), 389.0614. Found: 389.0600; Purity (HPLC): 98%.
4.2.3. (E)-8-[3-(Trifluoromethyl)styryl]-1,3,7-trimethylxanthine (4e)
The title compound (yellow needles) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-3-(trifluoromethyl)cinnamic acid
and iodomethane in a yield of 58%: mp 243.0 °C (lit. 243.7 °C)
[38] (ethanol); 1H NMR (CDCl3) d 3.40 (s, 3H), 3.60 (s, 3H), 4.08
(s, 3H), 6.96 (d, 1H, J = 15.8 Hz), 7.51 (t, 1H, J = 7.5 Hz), 7.59 (d,
1H, J = 7.9 Hz), 7.72 (d, 1H, J = 7.9 Hz), 7.80 (s, 1H), 7.81 (d, 1H,
J = 15.4 Hz); 13C NMR (CDCl3) d 27.9, 29.7, 31.6, 108.2, 112.9,
123.0, 123.7, 123.7, 124.8, 125.8, 125.8, 129.5, 130.5 131.4, 131.6,
136.2, 136.4, 148.5, 149.2, 151.6, 155.3; APCI-HRMS m/z: Calcd
4.2.9. (E)-8-[3-(Trifluoromethyl)styryl]-1,3-dimethyl-7-ethylxanthine
(5f)
The title compound (yellow needles) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-3-(trifluoromethyl)cinnamic acid
and iodoethane in a yield of 60%: mp 256.7 °C (ethanol/toluene);
1H NMR (CDCl3) d 1.47 (t, 3H, J = 7.2 Hz), 3.40 (s, 3H), 3.61 (s,
3H), 4.50 (q, 2H, J = 7.2 Hz), 6.94 (d, 1H, J = 15.8 Hz), 7.52 (t, 1H,
J = 7.5 Hz), 7.59 (d, 1H, J = 7.5 Hz), 7.72 (d, 1H, J = 7.5 Hz), 7.80 (s,
1H), 7.84 (d, 1H, J = 15.8 Hz); 13C NMR (CDCl3) d 16.8, 28.0, 29.7,
40.1, 107.4, 112.9, 123.0, 123.7, 123.7, 124.8, 125.8, 129.5, 129.5,
130.5, 131.1, 131.3, 131.6, 136.3, 136.5, 148.3, 148.7, 151.7,
154.8; APCI-HRMS m/z: Calcd for C18H18F3N4O2 (MH+), 379.1383.
Found: 379.1374; Purity (HPLC): 99%.
for
C
17H16F3N4O2 (MH+), 365.1226. Found: 365.1218; Purity
(HPLC): 100%.
4.2.4. (E)-8-(3-Chlorostyryl)-1,3-dimethyl-7-ethylxanthine (5a)
The title compound (yellow needles) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-3-chlorocinnamic acid and iodoeth-
ane in a yield of 44%: mp 213.1 °C (ethanol); 1H NMR (CDCl3) d 1.46
(t, 3H, J = 7.2 Hz), 3.40 (s, 3H), 3.61 (s, 3H), 4.48 (q, 2H, J = 7.2 Hz),
6.88 (d, 1H, J = 15.8 Hz), 7.30–7.34 (m, 2H), 7.42 (d, 1H, J = 6.4 Hz),
7.55 (s, 1H), 7.74 (d, 1H, J = 15.4 Hz); 13C NMR (CDCl3) d 16.7, 28.0,
29.8, 40.1, 107.3, 112.5, 125.8, 126.8, 129.3, 130.2, 134.9, 136.6,
137.3, 148.4, 148.7, 151.7, 154.8; APCI-HRMS m/z: Calcd for C17H18-
ClN4O2 (MH+), 345.1119. Found: 345.1114; Purity (HPLC): 99%.
4.2.10. (E)-8-(3-Chlorostyryl)-1,3-diethyl-7-methylxanthine (6a)
The title compound (yellow powder) was prepared from 1,3-
diethyl-5,6-diaminouracil, (E)-3-chlorocinnamic acid and iodo-
methane in a yield of 55%: mp 178.6 °C (ethanol); 1H NMR (CDCl3)
d 1.23 (t, 3H, J = 7.2 Hz), 1.35 (t, 3H, J = 7.2 Hz), 4.04 (s, 3H), 4.05 (q,
2H, J = 7.2 Hz), 4.18 (q, 2H, J = 7.2 Hz), 6.89 (d, 1H, J = 15.4 Hz),
7.28–7.32 (m, 2H), 7.41 (d, 1H, J = 6.8 Hz), 7.54 (s, 1H), 7.70 (d,
1H, J = 15.8 Hz); 13C NMR (CDCl3) d 13.3, 13.4, 31.5, 36.3, 38.4,
108.3, 112.6, 125.6, 126.9, 129.2, 130.1, 134.9, 136.4, 137.4,
148.0, 149.2, 150.7, 155.0; APCI-HRMS m/z: Calcd for C18H20ClN4O2
(MH+), 359.1276. Found: 359.1262; Purity (HPLC): 100%.
4.2.5. (E)-8-(4-Chlorostyryl)-1,3-dimethyl-7-ethylxanthine (5b)
The title compound (yellow needles) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-4-chlorocinnamic acid and iodoeth-
ane in a yield of 47%: mp 210.6 °C (ethanol); 1H NMR (CDCl3) d 1.45
(t, 3H, J = 7.2 Hz), 3.39 (s, 3H), 3.60 (s, 3H), 4.46 (q, 2H, J = 7.2 Hz),
6.84 (d, 1H, J = 15.8 Hz), 7.35 (d, 2H, J = 8.7 Hz), 7.48 (d, 2H,
J = 8.3 Hz), 7.75 (d, 1H, J = 15.4 Hz); 13C NMR (CDCl3) d 16.7, 27.9,
29.7, 40.0, 107.2, 111.6, 128.5, 129.2, 134.0, 135.2, 136.8, 148.6,
148.7, 151.7, 154.8; APCI-HRMS m/z: Calcd for C17H18ClN4O2
(MH+), 345.1119. Found: 345.1105; Purity (HPLC): 100%.
4.2.11. (E)-8-(4-Chlorostyryl)-1,3-diethyl-7-methylxanthine (6b)
The title compound (yellow needles) was prepared from 1,3-
diethyl-5,6-diaminouracil, (E)-4-chlorocinnamic acid and iodo-
methane in a yield of 48%: mp 214.6 °C (ethanol); 1H NMR (CDCl3)
d 1.23 (t, 3H, J = 7.2 Hz), 1.35 (t, 3H, J = 7.2 Hz), 4.03 (s, 3H), 4.05 (q,
2H, J = 7.2 Hz), 4.18 (q, 2H, J = 7.2 Hz), 6.86 (d, 1H, J = 15.8 Hz), 7.35
(d, 2H, J = 8.3 Hz), 7.49 (d, 2H, J = 8.3 Hz), 7.71 (d, 1H, J = 15.8 Hz);
13C NMR (CDCl3) d 13.3, 13.4, 31.5, 36.3, 38.4, 108.2, 111.8, 128.5,
129.1, 134.0, 135.2, 136.6, 148.1, 149.5, 150.7, 155.0; APCI-HRMS
m/z: Calcd for C18H20ClN4O2 (MH+), 359.1276. Found: 359.1265;
Purity (HPLC): 100%.
4.2.6. (E)-8-(3,4-Dichlorostyryl)-1,3-dimethyl-7-ethylxanthine (5c)
The title compound (yellow needles) was prepared from 1,3-di-
methyl-5,6-diaminouracil, (E)-3,4-dichlorocinnamic acid and iodo-
ethane in a yield of 46%: mp 216.6 °C (ethanol); 1H NMR (CDCl3) d
1.46 (t, 3H, J = 7.2 Hz), 3.40 (s, 3H), 3.60 (s, 3H), 4.48 (q, 2H,
J = 7.2 Hz), 6.86 (d, 1H, J = 15.8 Hz), 7.37 (dd, 1H, J = 1.9, 8.3 Hz),
7.45 (d, 1H, J = 8.3 Hz), 7.64 (d, 1H, J = 1.9 Hz), 7.70 (d, 1H,
J = 15.8 Hz); 13C NMR (CDCl3) d 16.8, 28.0, 29.8, 40.1, 107.4,
112.8, 126.5, 128.6, 130.9, 133.2, 133.2, 135.5, 135.6, 148.2,
148.7, 151.7, 154.8; APCI-HRMS m/z: Calcd for C17H17Cl2N4O2
(MH+), 379.0729. Found: 379.0715; Purity (HPLC): 98%.
4.2.12. (E)-8-(3,4-Dichlorostyryl)-1,3-diethyl-7-methylxanthine (6c)
The title compound (yellow needles) was prepared from 1,3-
diethyl-5,6-diaminouracil, (E)-3,4-dichlorocinnamic acid and iodo-
methane in a yield of 65%: mp 215.5 °C (ethanol); 1H NMR (CDCl3)
d 1.23 (t, 3H, J = 7.2 Hz), 1.35 (t, 3H, J = 7.2 Hz), 4.05 (s, 3H), 4.06 (q,

56
M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
2H, J = 6.8 Hz), 4.17 (q, 2H, J = 7.2 Hz), 6.87 (d, 1H, J = 15.8 Hz), 7.37
(d, 1H, J = 6.8 Hz), 7.44 (d, 1H, J = 8.3 Hz), 7.64 (s, 1H), 7.66 (d, 1H,
J = 15.6 Hz); 13C NMR (CDCl3) d 13.3, 13.4, 31.5, 36.4, 38.4, 108.4,
113.0, 126.4, 128.7, 130.9, 133.1, 133.2, 135.2, 135.6, 148.0,
149.0, 150.6, 155.0; APCI-HRMS m/z: Calcd for C18H19Cl2N4O2
(MH+), 393.0886. Found: 393.0873; Purity (HPLC): 100%.
4.07 (q, 2H, J = 7.2 Hz), 4.19 (q, 2H, J = 7.2 Hz), 4.47 (q, 2H,
J = 7.2 Hz), 6.85 (d, 1H, J = 15.4 Hz), 7.36 (d, 2H, J = 8.3 Hz), 7.49
(d, 2H, J = 8.7 Hz), 7.75 (d, 1H, J = 15.8 Hz); 13C NMR (CDCl3) d
13.4, 13.4, 16.7, 36.4, 38.4, 40.0, 107.5, 111.8, 128.5, 129.2, 134.1,
135.1, 136.6, 148.3, 148.6, 150.8, 154.6; APCI-HRMS m/z: Calcd
for
C
19H22ClN4O2 (MH+), 373.1432. Found: 373.1417; Purity
(HPLC): 99%.
4.2.13. (E)-8-(3-Bromostyryl)-1,3-diethyl-7-methylxanthine (6d)
The title compound (yellow powder) was prepared from 1,3-
diethyl-5,6-diaminouracil, (E)-3-bromocinnamic acid and iodo-
methane in a yield of 30%: mp 189.9 °C, (lit. mp 187.3–188.2 °C)
[40] (ethanol); 1H NMR (CDCl3) d 1.24 (t, 3H, J = 7.2 Hz), 1.35 (t,
3H, J = 7.2 Hz), 4.05 (s, 3H), 4.06 (q, 2H, J = 7.2 Hz), 4.18 (q, 2H,
J = 7.2 Hz), 6.89 (d, 1H, J = 15.8 Hz), 7.26 (d, 1H, J = 7.9 Hz), 7.46
(t, 2H, J = 7.2 Hz), 7.69 (d, 1H, J = 15.8 Hz), 7.71 (s, 1H); 13C NMR
(CDCl3) d 13.3, 13.4, 31.5, 36.4, 38.4, 108.3, 112.7, 123.1, 126.1,
129.8, 130.4, 132.1, 136.3, 137.7, 148.1, 149.2, 150.7, 155.1;
APCI-HRMS m/z: Calcd for C18H20BrN4O2 (MH+), 403.0770. Found:
403.0757; Purity (HPLC): 100%.
4.2.18. (E)-8-(3,4-Dichlorostyryl)-1,3,7-triethylxanthine (7c)
The title compound (light yellow powder) was prepared from
1,3-diethyl-5,6-diaminouracil, (E)-3,4-dichlorocinnamic acid and
iodoethane in a yield of 7%: mp 177.9 °C (ethanol); 1H NMR (CDCl3)
d 1.20 (t, 3H, J = 7.2 Hz), 1.31 (t, 3H, J = 7.2 Hz), 1.41 (t, 3H,
J = 7.2 Hz), 4.02 (q, 2H, J = 7.2 Hz), 4.13 (q, 2H, J = 7.2 Hz), 4.43 (q,
2H, J = 7.2 Hz), 6.81 (d, 1H, J = 15.4 Hz), 7.33 (dd, 1H, J = 1.8,
8.3 Hz), 7.40 (d, 1H, J = 8.3 Hz), 7.60 (d, 1H, J = 1.9 Hz), 7.65 (d,
1H, J = 15.8 Hz); 13C NMR (CDCl3) d 13.3, 13.4, 16.8, 36.4, 38.4,
40.0, 107.6, 113.0, 126.5, 128.7, 130.8, 133.0, 133.2, 135.2, 135.7,
148.1, 148.2, 150.7, 154.6; APCI-HRMS m/z: Calcd for C19H21Cl2N4-
O2 (MH+), 407.1042. Found: 407.1026; Purity (HPLC): 98%.
4.2.14. (E)-8-(4-Bromostyryl)-1,3-diethyl-7-methylxanthine (6e)
The title compound (yellow needles) was prepared from 1,3-
diethyl-5,6-diaminouracil, (E)-4-bromocinnamic acid and iodo-
methane in a yield of 26%: mp 210.5 °C, (lit. mp 198.5–198.9 °C)
[40] (ethanol); 1H NMR (CDCl3) d 1.23 (t, 3H, J = 7.2 Hz), 1.35 (t,
3H, J = 7.2 Hz), 4.03 (s, 3H), 4.06 (q, 2H, J = 7.2 Hz), 4.18 (q, 2H,
J = 7.2 Hz), 6.88 (d, 1H, J = 15.8 Hz), 7.42 (d, 2H, J = 8.3 Hz), 7.50
(d, 2H, J = 8.7 Hz), 7.70 (d, 1H, J = 15.8 Hz); 13C NMR (CDCl3) d
13.3, 13.4, 31.5, 36.4, 38.4, 108.3, 111.9, 123.4, 128.7, 132.1,
134.5, 136.7, 148.1, 149.5, 150.7, 155.1; APCI-HRMS m/z: Calcd
4.2.19. (E)-8-(3-Bromostyryl)-1,3,7-triethylxanthine (7d)
The title compound (white needles) was prepared from 1,3-
diethyl-5,6-diaminouracil, (E)-3-bromocinnamic acid and iodoeth-
ane in a yield of 14%: mp 166.2 °C (ethanol); 1H NMR (CDCl3) d 1.24
(t, 3H, J = 7.2 Hz), 1.36 (t, 3H, J = 7.2 Hz), 1.46 (t, 3H, J = 7.2 Hz), 4.07
(q, 2H, J = 7.2 Hz), 4.19 (q, 2H, J = 7.2 Hz), 4.48 (q, 2H, J = 7.2 Hz),
6.87 (d, 1H, J = 15.4 Hz), 7.26–7.27 (m, 1H), 7.46 (t, 2H,
J = 7.2 Hz), 7.71–7.73 (m, 2H); 13C NMR (CDCl3) d 13.4, 13.4, 16.8,
36.4, 38.4, 40.0, 107.6, 112.6, 123.1, 126.2, 129.8, 130.4, 132.1,
136.3, 137.7, 148.3, 148.3, 150.7, 154.6; APCI-HRMS m/z: Calcd
for
C
18H20BrN4O2 (MH+), 403.0770. Found: 403.0758; Purity
(HPLC): 100%.
for C
19H22BrN4O2 (MH+), 417.0927. Found: 417.0914; Purity
(HPLC): 99%.
4.2.15. (E)-8-[3-(Trifluoromethyl)styryl]-1,3-diethyl-7-
methylxanthine (6f)
4.2.20. (E)-8-(4-Bromostyryl)-1,3,7-triethylxanthine (7e)
The title compound (white powder) was prepared from 1,3-
diethyl-5,6-diaminouracil, (E)-3,4-(trifluoromethyl)cinnamic acid
and iodomethane in a yield of 58%: mp 212.8 °C, (lit. mp 214.8–
215.3 °C) [40] (ethanol); 1H NMR (CDCl3) d 1.24 (t, 3H, J = 7.2 Hz),
1.36 (t, 3H, J = 7.2 Hz), 4.06 (q, 2H, J = 7.2 Hz), 4.07 (s, 3H), 4.19
(q, 2H, J = 7.2 Hz), 6.96 (d, 1H, J = 15.8 Hz), 7.51 (t, 1H, J = 7.9 Hz),
7.58 (d, 1H, J = 7.9 Hz), 7.72 (d, 1H, J = 7.9 Hz), 7.80 (s, 1H), 7.80
(d, 1H, J = 15.8 Hz); 13C NMR (CDCl3) d 13.3, 13.4, 31.5, 36.4, 38.4,
108.4, 113.1, 122.9, 123.7, 123.8, 124.8, 125.7, 125.7, 129.4,
130.4, 131.3, 131.5, 136.2, 136.3, 148.0, 149.1, 150.7, 155.1;
APCI-HRMS m/z: Calcd for C19H20F3N4O2 (MH+), 393.1539. Found:
393.1525; Purity (HPLC): 100%.
The title compound (light yellow needles) was prepared from
1,3-diethyl-5,6-diaminouracil, (E)-4-bromocinnamic acid and
iodoethane in a yield of 20%: mp 189.2 °C (ethanol); 1H NMR
(CDCl3) d 1.24 (t, 3H, J = 7.2 Hz), 1.36 (t, 3H, J = 7.2 Hz), 1.45 (t,
3H, J = 7.2 Hz), 4.07 (q, 2H, J = 7.2 Hz), 4.19 (q, 2H, J = 7.2 Hz),
4.46 (q, 2H, J = 7.2 Hz), 6.85 (d, 1H, J = 15.8 Hz), 7.42 (d, 2H,
J = 8.3 Hz), 7.51 (d, 2H, J = 8.3 Hz), 7.73 (d, 1H, J = 15.8 Hz); 13C
NMR (CDCl3) d 13.3, 13.4, 16.7, 36.4, 38.4, 40.0, 107.5, 111.9,
123.4, 128.7, 132.1, 134.5, 136.7, 148.3, 148.6, 150.7, 154.6;
APCI-HRMS m/z: Calcd for C19H22BrN4O2 (MH+), 417.0927. Found:
417.0919; Purity (HPLC): 98%.
4.2.21. (E)-8-[3-(Trifluoromethyl)styryl]-1,3,7-triethylxanthine (7f)
4.2.16. (E)-8-(3-Chlorostyryl)-1,3,7-triethylxanthine (7a)
The title compound (light yellow powder) was prepared from
The title compound (light yellow crystals) was prepared from
1,3-diethyl-5,6-diaminouracil, (E)-3-chlorocinnamic acid and iodo-
ethane in a yield of 93%: mp 165.0 °C (ethanol); 1H NMR (CDCl3) d
1.20 (t, 3H, J = 7.2 Hz), 1.32 (t, 3H, J = 7.2 Hz), 1.41 (t, 3H, J = 7.2 Hz),
4.02 (q, 2H, J = 7.2 Hz), 4.14 (q, 2H, J = 7.2 Hz), 4.43 (q, 2H,
J = 7.2 Hz), 6.83 (d, 1H, J = 15.8 Hz), 7.24–7.28 (m, 2H), 7.37 (d,
1H, J = 6.8 Hz), 7.50 (s, 1H), 7.68 (d, 1H, J = 15.4 Hz); 13C NMR
(CDCl3) d 13.3, 13.4, 16.7, 36.4, 38.4, 40.0, 107.6, 112.6, 125.7,
126.8, 129.2, 130.1, 134.9, 136.4, 137.4, 148.2, 148.3, 150.7,
154.6; APCI-HRMS m/z: Calcd for C19H22ClN4O2 (MH+), 373.1432.
Found: 373.1433; Purity (HPLC): 99%.
1,3-diethyl-5,6-diaminouracil,
(E)-3-(trifluoromethyl)cinnamic
acid and iodoethane in a yield of 11%: mp 163.2 °C (ethanol); 1H
NMR (CDCl3) d 1.25 (t, 3H, J = 7.2 Hz), 1.37 (t, 3H, J = 7.2 Hz), 1.48
(t, 3H, J = 7.2 Hz), 4.07 (q, 2H, J = 7.2 Hz), 4.19 (q, 2H, J = 7.2 Hz),
4.50 (q, 2H, J = 7.2 Hz), 6.94 (d, 1H, J = 15.8 Hz), 7.52 (t, 1H,
J = 7.5 Hz), 7.59 (d, 1H, J = 7.5 Hz), 7.73 (d, 1H, J = 7.5 Hz), 7.80 (s,
1H), 7.83 (d, 1H, J = 15.4 Hz); 13C NMR (CDCl3) d 13.3, 13.4, 16.8,
36.4, 38.4, 40.0, 107.7, 113.0, 123.0, 123.8, 123.8, 124.8, 125.7,
125.7, 129.4, 130.4, 131.1, 131.3, 131.5, 136.3, 136.4, 148.2,
148.3, 150.8, 154.7; APCI-HRMS m/z: Calcd for C20H22F3N4O2
(MH+), 407.1695. Found: 407.1698; Purity (HPLC): 98%.
4.2.17. (E)-8-(4-Chlorostyryl)-1,3,7-triethylxanthine (7b)
4.2.22. 8-Phenoxymethyl-1,3-dimethyl-7-ethylxantine (8b)
The title compound (light yellow crystals) was prepared from
1,3-diethyl-5,6-diaminouracil, (E)-4-chlorocinnamic acid and iodo-
ethane in a yield of 46%: mp 171.7 °C (ethanol); 1H NMR (CDCl3) d
1.24 (t, 3H, J = 7.2 Hz), 1.36 (t, 3H, J = 7.2 Hz), 1.45 (t, 3H, J = 7.2 Hz),
The title compound (white crystals) was prepared from 1,3-di-
methyl-5,6-diaminouracil, phenoxyacetic acid and iodoethane in a
yield of 93% mp 141.9 °C (ethanol); 1H NMR (CDCl3) d 1.46 (t, 3H,
J = 7.2 Hz), 3.39 (s, 3H), 3.57 (s, 3H), 4.41 (q, 2H, J = 7.2 Hz), 5.16

M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
57
(s, 2H), 6.98–7.00 (m, 3H), 7.28–7.31 (m, 2H); 13C NMR (CDCl3) d
16.5, 28.0, 29.7, 41.5, 61.9, 108.0, 114.6, 122.0, 129.7, 147.2,
147.7, 151.6, 154.9, 157.5; APCI-HRMS m/z: Calcd for C16H19N4O3
(MH+), 315.1458. Found: 315.1461; Purity (HPLC): 93%.
(t, 3H, J = 7.2 Hz), 1.31 (t, 3H, J = 7.2 Hz), 1.33 (t, 3H, J = 7.2 Hz),
2.08 (qn, 2H, J = 7.5 Hz), 2.69–2.73 (m, 4H), 4.04 (q, 2H,
J = 7.2 Hz), 4.12 (q, 2H, J = 7.2 Hz), 4.19 (q, 2H, J = 7.2 Hz), 7.16–
7.19 (m, 3H), 7.24–7.28 (m, 2H); 13C NMR (CDCl3) d 13.3, 13.4,
16.3, 25.9, 29.4, 35.2, 36.2, 38.3, 40.1, 106.6, 126.1, 128.4, 128.4,
141.0, 147.7, 150.7, 153.0, 154.6; APCI-HRMS m/z: Calcd for
4.2.23. 8-Phenoxymethyl-1,3-diethyl-7-methylxanthine (8c)
The title compound (white powder) was prepared from 1,3-
diethyl-5,6-diaminouracil, phenoxyacetic acid and iodomethane
in a yield of 91% mp 135.2 °C (ethanol); 1H NMR (CDCl3) d 1.17
(t, 3H, J = 7.2 Hz), 1.27 (t, 3H, J = 7.2 Hz), 3.98 (s, 3H), 3.99 (q, 2H,
J = 7.2 Hz), 4.09 (q, 2H, J = 7.2 Hz), 5.12 (s, 2H), 6.92–6.94 (m, 3H),
7.19–7.24 (m, 2H); 13C NMR (CDCl3) d 12.3, 12.4, 31.4, 35.4, 37.5,
61.0, 107.9, 113.7, 121.0, 128.7, 145.9, 146.8, 149.6, 154.2, 156.5;
APCI-HRMS m/z: Calcd for C17H21N4O3 (MH+), 329.1614. Found:
329.1616; Purity (HPLC): 100%.
C
87%.
20H27N4O2 (MH+), 355.2135. Found: 355.2138; Purity (HPLC):
4.3. Radioligand binding studies
The collection of tissue samples for the adenosine A2A and A1
receptor binding studies was approved by the Research Ethics
Committee of the North-West University (application number
NWU-0035-10-A5). The adenosine A2A and A1 receptor binding
studies were carried out according to the procedure described in
literature [34]. For studies with the A2A receptor, the dissected
striata of male Sprague–Dawley rats were used while for the stud-
ies with the A1 receptor whole rat brain (excluding cerebellum
and brain stem) were employed. After dissection, the tissues were
snap frozen with liquid nitrogen and stored at ꢃ70 °C. The tissue
were thawed on ice, weighed and disrupted for 30 s with the aid
of a Polytron homogenizer (model: Polytron PT 10–35 GT) in 10
volumes of ice-cold 50 mM Tris buffer (pH 7.7 at 25 °C). The
resulting homogenate was centrifuged at 20,000g for 10 min at
4 °C and the pellet was resuspended in 10 volumes of ice-cold Tris
buffer, again with the aid of a Polytron homogenizer as above. The
resulting suspension was recentrifuged and the pellet obtained
was suspended in Tris buffer (pH 7.7 at 25 °C) to a volume of
5 mL/g original striatal weight. The striatal and whole brain mem-
branes were aliquoted into microcentrifuge tubes and stored at
ꢃ70 °C until needed. The incubations were carried out in 4 mL
polypropylene tubes that were precoated with SigmacoteÒ (Sig-
ma–Aldrich). All incubations were prepared with Tris buffer (pH
7.7 at 25 °C) to a volume of 1 mL. For the A2A receptor binding
study, the incubations contained 10 mg of the original tissue
weight of the striatal membranes, 4 nM [3H]NECA, 50 nM CPA,
10 mM MgCl2, 0.2 units/mL adenosine deaminase, the test com-
pound and 1% DMSO. DMSO was used to prepare all stock solu-
tions of the compounds to be tested. For the A1 receptor binding
study, the incubations contained 5 mg of the original tissue
weight of the whole brain membranes, 0.1nM [3H]DPCPX,
0.2 units/mL adenosine deaminase, the test compound and 1%
DMSO. The incubations were vortexed and incubated for 60 min
at 25 °C in a shaking water bath. Half an hour after incubation
was started, the incubations were vortexed again. The incubations
were terminated via filtration through a prewetted 2.5 cm What-
man glass microfiber filter (grade GF/B) under reduced pressure
using a Hoffeler vacuum system. The tubes were washed twice
with 4 mL ice-cold Tris buffer and the filters were washed once
more with 4 mL ice-cold Tris buffer. The damp filters were placed
in scintillation vials and 4 mL of scintillation fluid (Filter-Count)
was added. The vials were shaken and incubated for 2 h before
being counted (Packard Tri-CARB 2100 TR). An estimate of the
nonspecific binding was obtained from binding studies in the
4.2.24. 8-Phenoxymethyl-1,3,7-triethylxanthine (8d)
The title compound (white crystals) was prepared from 1,3-
diethyl-5,6-diaminouracil, phenoxyacetic acid and iodoethane in
a yield of 58%: mp 95.0 °C (ethanol); 1H NMR (CDCl3) d 1.23 (t,
3H, J = 7.2 Hz), 1.33 (t, 3H, J = 7.2 Hz), 1.46 (t, 3H, J = 7.2 Hz), 4.05
(q, 2H, J = 7.2 Hz), 4.15 (q, 2H, J = 7.2 Hz), 4.41 (q, 2H, J = 7.2 Hz),
5.16 (s, 2H), 7.99–7.00 (m, 3H), 7.27–7.30 (m, 2H); 13C NMR
(CDCl3) d 13.3, 13.4, 16.5, 36.5, 38.5, 41.4, 61.9, 108.2, 114.6,
121.9, 129.7, 147.1, 147.2, 150.7, 154.7, 157.6; APCI-HRMS m/z:
Calcd for C18H23N4O3(MH+), 343.1770. Found: 343.1722; Purity
(HPLC): 89%.
4.2.25. 8-(3-Phenylpropyl)-1,3,7-trimethylxanthine (9a)
The title compound (white crystals) was prepared from 1,3-di-
methyl-5,6-diaminouracil, 4-phenylbutanoic acid and iodometh-
ane in a yield of 66%: mp 115–117 °C (methanol). 1H NMR
(CDCl3) d 2.09 (qn, 2H, J = 7.5 Hz), 2.71 (q, 4H, J = 7.5 Hz), 3.37 (s,
3H), 3.55 (s, 3H), 3.80 (s, 3H), 7.17 (m, 3H), 7.28 (t, 2H,
J = 7.5 Hz); 13C NMR (CDCl3) d 26.0, 27.8, 28.7, 29.7, 31.6, 35.1,
107.3, 126.2, 128.4, 128.5, 140.9, 148.0, 151.7, 153.9, 155.3; ESI-
HRMS m/z: Calcd for
C
17H21N4O2 (M+), 313.1665. Found:
313.1659; Purity (HPLC): 99%.
4.2.26. 8-(3-Phenylpropyl)-1,3-dimethyl-7-ethylxanthine (9b)
The title compound (white crystals) was prepared from 1,3-di-
methyl-5,6-diaminouracil, 4-phenylbutanoic acid and iodoethane
in a yield of 55%: mp 129.0 °C (ethanol); 1H NMR (CDCl3) d 1.28
(t, 3H, J = 7.2 Hz), 2.05 (qn, 2H, J = 7.5 Hz), 2.64–2.69 (m, 4H),
3.32 (s, 3H), 3.50 (s, 3H), 4.41 (q, 2H, J = 7.2 Hz), 7.11–7.14 (m,
3H), 7.20–7.24 (m, 2H); 13C NMR (CDCl3) d 16.3, 25.9, 27.8, 29.2,
29.7, 35.2, 40.2, 106.4, 126.1, 128.4, 128.5, 140.9, 148.2, 151.7,
153.1, 154.8; APCI-HRMS m/z: Calcd for
327.1822. Found: 327.1823; Purity (HPLC): 94%.
C
18H23N4O2 (MH+),
4.2.27. 8-(3-Phenylpropyl)-1,3-diethyl-7-methylxanthine (9c)
The title compound (light yellow crystals) was prepared from
1,3-diethyl-5,6-diaminouracil, 4-phenylbutanoic acid and iodo-
methane in a yield of 80%: mp 61.7 °C (ethanol); 1H NMR (CDCl3)
d 1.17 (t, 3H, J = 7.2 Hz), 1.27 (t, 3H, J = 7.2 Hz), 2.02 (qn, 2H,
J = 7.5 Hz), 2.64–2.67 (m, 4H), 3.75 (s, 3H), 3.99 (q, 2H, J = 7.2 Hz),
4.08 (q, 2H, J = 7.2 Hz), 7.11–7.14 (m, 3H), 7.20–7.24 (m, 2H); 13C
NMR (CDCl3) d 12.3, 12.5, 25.0, 27.9, 30.5, 34.1, 35.2, 37.3, 106.5,
125.1, 127.4, 127.5, 140.0, 146.5, 149.7, 152.8, 154.1; APCI-HRMS
m/z: Calcd for C19H25N4O2 (MH+), 341.1978. Found: 341.1980; Pur-
ity (HPLC): 100%.
presence of 100 lM CPA. The IC50 values were determined by
plotting the count values (adjusted for nonspecific binding) vs.
the logarithm of the inhibitor concentrations to obtain a sigmoidal
dose–response curve. This kinetic data were fitted to the one site
competition model incorporated into the Prism software package
(GraphPad Software Inc.). The Ki values for the competitive inhibi-
tion of [3H]NECA (Kd = 15.3 nM) [34] or [3H]DPCPX (Kd = 0.36 nM)
[41] binding by the test compounds were calculated from the IC50
values according to the Cheng and Prusoff equation [42]. All incu-
bations were carried out in duplicate and the Ki values are ex-
pressed as mean standard deviation (SD).
4.2.28. 8-(3-Phenylpropyl)-1,3,7-triethylxanthine (9d)
The title compound (light red crystals) was prepared from 1,3-
diethyl-5,6-diaminouracil, 4-phenylbutanoic acid and iodoethane
in a yield of 46%: mp 73.6 °C (ethanol); 1H NMR (CDCl3) d 1.22

58
M.M. Van der Walt et al. / Bioorganic Chemistry 49 (2013) 49–58
4.4. Evaluation of the haloperidol-induced catalepsy
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(500
buffer (100 mM, pH 7.4, made isotonic with KCl) and contained
kynuramine (30 M) as substrate, various concentrations of the
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and Ampliflu Red (200 M). Stock solutions of the test inhibitors
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l
l
l
reactions were terminated with the addition of 10
lL (R)-deprenyl
(5 mM) and distilled water (1400 L) was added. The concentra-
l
tions of resorufin in the reactions were subsequently determined
by fluorescence spectrophotometry (kex = 560 nm, kem = 590 nm)
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mined by fitting the sigmoidal curves to the one site competition
model incorporated into the Prism 5 software package (GraphPad).
All experiments were carried out in triplicate and the IC50 values
are expressed as mean standard deviation (SD).
Acknowledgments
The NMR and MS spectra were recorded by André Joubert and
Johan Jordaan of the SASOL Centre for Chemistry, North-West Uni-
versity. This work was supported by grants from the National Re-
search Foundation and the Medical Research Council, South
Africa. Any opinion, findings and conclusions or recommendations
expressed in this material are those of the authors and therefore
the NRF do not accept any liability in regard thereto.
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2013.06.
006.