Dual inhibition of monoamine oxidase B and antagonism of the adenosine A2A receptor by (E,E)-8-(4-phenylbutadien-1-yl)caffeine analogues

Article abstract of DOI:10.1016/j.bmc.2008.07.088

The adenosine A_2A receptor has emerged as an attractive target for the treatment of Parkinson's disease (PD). Evidence suggests that antagonists of the A_2A receptor (A_2A antagonists) may be neuroprotective and may help to alleviate the symptoms of PD. We have reported recently that several members of the (E)-8-styrylcaffeine class of A_2A antagonists also are potent inhibitors of monoamine oxidase B (MAO-B). Since MAO-B inhibitors are known to possess anti-parkinsonian properties, dual-target-directed drugs that block both MAO-B and A_2A receptors may have enhanced value in the management of PD. In an attempt to explore this concept further we have prepared three additional classes of C-8 substituted caffeinyl analogues. The 8-phenyl- and 8-benzylcaffeinyl analogues exhibited relatively weak MAO-B inhibition potencies while selected (E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues were found to be exceptionally potent reversible MAO-B inhibitors with enzyme-inhibitor dissociation constants (K_i values) ranging from 17 to 149 nM. Furthermore, these (E,E)-8-(4-phenylbutadien-1-yl)caffeines acted as potent A_2A antagonists with K_i values ranging from 59 to 153 nM. We conclude that the (E,E)-8-(4-phenylbutadien-1-yl)caffeines are a promising candidate class of dual-acting compounds.

Full text of DOI:10.1016/j.bmc.2008.07.088

Bioorganic & Medicinal Chemistry 16 (2008) 8676–8684
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dual inhibition of monoamine oxidase B and antagonism of the adenosine
A2A receptor by (E,E)-8-(4-phenylbutadien-1-yl)caffeine analogues
Judey Pretorius a, Sarel F. Malan a, Neal Castagnoli Jr. b, Jacobus J. Bergh a, Jacobus P. Petzer a,
*
a Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Department of Chemistry, Virginia Tech and Edward Via College of Osteopathic Medicine, Blacksburg, VA 24061, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The adenosine A2A receptor has emerged as an attractive target for the treatment of Parkinson’s disease
(PD). Evidence suggests that antagonists of the A2A receptor (A2A antagonists) may be neuroprotective
and may help to alleviate the symptoms of PD. We have reported recently that several members of the
(E)-8-styrylcaffeine class of A2A antagonists also are potent inhibitors of monoamine oxidase B (MAO-
B). Since MAO-B inhibitors are known to possess anti-parkinsonian properties, dual-target-directed drugs
that block both MAO-B and A2A receptors may have enhanced value in the management of PD. In an
attempt to explore this concept further we have prepared three additional classes of C-8 substituted caf-
feinyl analogues. The 8-phenyl- and 8-benzylcaffeinyl analogues exhibited relatively weak MAO-B inhi-
bition potencies while selected (E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues were found to be
exceptionally potent reversible MAO-B inhibitors with enzyme–inhibitor dissociation constants (Ki val-
ues) ranging from 17 to 149 nM. Furthermore, these (E,E)-8-(4-phenylbutadien-1-yl)caffeines acted as
potent A2A antagonists with Ki values ranging from 59 to 153 nM. We conclude that the (E,E)-8-(4-phe-
nylbutadien-1-yl)caffeines are a promising candidate class of dual-acting compounds.
Received 20 June 2008
Revised 29 July 2008
Accepted 30 July 2008
Available online 5 August 2008
Keywords:
Monoamine oxidase B
Adenosine A2A receptor
Reversible inhibition
Antagonism
Dual-target-directed drug
Caffeine
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
A particularly well-characterized A2A antagonist, (E)-1,3-diethyl-
8-(3,4-dimethoxystyryl)-7-methylxanthine (KW-6002; 1) (Scheme
Currently, the therapy of Parkinson’s disease (PD) is largely fo-
cused on dopamine replacement strategies with the dopamine pre-
cursor levodopa and dopamine agonist drugs.1 Although these
strategies are highly effective in controlling the early stages of
the disease, long-term treatment is associated with drug-related
complications such as a loss of drug efficacy, the onset of dyskine-
sias and the occurrence of psychosis and depression.2,3 The inade-
quacies of dopamine replacement therapy have prompted the
search for alternative drug targets. The adenosine A2A receptor
has emerged as one such target and antagonists of this receptor
(A2A antagonists) are considered promising agents for the symp-
tomatic treatment of PD.4 Additionally, evidence suggests that
A2A antagonists may slow the course of the disease by protecting
against the underlying neurodegenerative processes5,6 and may
prevent the development of dyskinesias that are normally associ-
ated with levodopa treatment.7 Furthermore, since the symptom-
atic relief conferred by A2A antagonists are additive to the effect
produced by dopamine replacement therapy, it may be possible
to reduce the dose of the dopaminergic drugs and therefore the
occurrence of side effects.2,8 A2A antagonists are therefore a
promising adjunctive to dopamine replacement therapy.9
1), is currently undergoing clinical trials for the treatment of the mo-
tor symptoms associated with PD.10 Another A2A antagonist and
structural analogue of KW-6002, (E)-8-(3-chlorostyryl)caffeine
(CSC; 2b) (Scheme 1), is frequently used when examining the in vivo
pharmacological effects of A2A antagonists.11,12 We have previously
reported that CSC is also a potent reversible inhibitor of monoamine
oxidase B (MAO-B) with an enzyme–inhibitor dissociation constant
(Ki value)of 128 nM.5,13,14 Inhibitors ofMAO-B alsoare consideredto
be useful for the treatment of age-related neurodegenerative dis-
eases such as Alzheimer’s disease and PD.15,16 Since MAO-B appears
to be predominantly responsible for dopamine metabolism in the
basal ganglia,17,18 inhibition of this enzyme in the brain may con-
serve the depleted supply of dopamine. MAO-B inhibitors are used
in combination with levodopa as dopamine replacement therapy
in patients diagnosed with early PD.19 MAO-B inhibitors have been
shownto elevatedopaminelevelsinthestriatumofprimatestreated
with levodopa.20 Furthermore, for each mole of dopamine oxidized
by MAO-B, one mole of hydrogen peroxide (H2O2) is produced.
H2O2 may interact with free iron to form highly reactive hydroxyl
radicals that may contribute to neurodegenerative processes.21 Inhi-
bition of MAO-B, therefore, may also exert a protective effect by
reducing H2O2 production in the brain.22 These effects of MAO-B
inhibitors are especially relevant when considering that the brain
shows an age-related increase in MAO-B activity.23,24 This increase
* Corresponding author. Tel.: +27 18 2992206; fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.088

J. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8676–8684
8677
OCH3
OCH3
O
R
O
N
N
N
N
N
N
N
(E)
O
N
O
O
N
O
4a: R = H
4b: R = Cl
4c: R = CF3
KW-6002 (1)
Cl
O
N
N
R
N
N
(E)
N
N
CSC (2b)
O
N
O
5a: R = H
5b: R = Cl
5c: R = CF3
O
R1
N
N
R3
N
N
O
R2
(E)
N
N
(E)
Caffeine (3)
N
O
N
Scheme 1. The structures of the adenosine A2A receptor antagonists KW-6002 (1),
CSC (2b), and caffeine (3).
R1
R2
R2
R3
6a:
6b: Cl
6c: Br
6d:
6e:
6f:
H
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
CH3
CH3
CH3
CH3
C2H5
CH3
C2H5
may be attributed to glial cell proliferation, since central MAO-B is
predominantly located in glial cells.25 In the aged parkinsonian
brain, inhibition of MAO-B may, therefore, counter the effects of in-
creased MAO-B activity and protect against further
neurodegeneration.26
F
H
H
H
6g:
Based on these observations, dual-target-directed drugs, com-
pounds that inhibit MAO-B and antagonize A2A receptors, may have
enhanced value in the management of PD. Using CSC as a lead com-
pound wehave, in the present study, attempted toidentifyadditional
dual-acting compounds, possibly with enhanced MAO-B inhibition
and A2A antagonism potencies. Such compounds may deepen our
understanding of the structural requirements of C-8 substituted caf-
feinyl analogues to act as dual inhibitors of MAO-B and antagonists of
the A2A receptor. The (E)-styryl group at C-8 appears to be critical for
the dual-action of CSC since caffeine (3) is only a moderate A2A antag-
Scheme 2. The structures of the C-8 substituted caffeinyl analogues that
were investigated in the present study: 8-phenylcaffeinyl analogues 4a–c,
8-benzylcaffeinyl analogues 5a–c, and (E,E)-8-(4-phenylbutadien-1-yl)caffeinyl
analogues 6a–g.
previously reported for the preparation of (E)-8-styrylcaffeinyl
analogues.29,14 The key starting materials for the procedure, 1,3-di-
methyl- (7a) and 1,3-diethyl-5,6-diaminouracil (7b),30 were
allowed to react with the appropriate carboxylic acid in the pres-
ence of the carbodiimide activating reagent N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide hydrochloride (EDAC) (Scheme 3).
The commercially available benzoic acids 8a–c, phenylacetic acids
9a–c, and newly synthesized (E,E)-5-phenyl-2,4-pentadienoic
acids 10a–d (see below) were used for the preparation of 4a–c,
5a–c, and 6a–g, respectively. The resulting amidyl intermediates
underwent ring closure when heated under reflux in aqueous so-
dium hydroxide to yield the corresponding 1,3-dialkyl-8-sustitut-
ed-7H-xanthinyl analogues (11). Without further purification, the
crude xanthinyl intermediates were selectively 7N-alkylated with
an excess of iodomethane (4a–c, 5a–c, 6a–d, and 6f) or iodoethane
(6e and 6g) and potassium carbonate to yield the target com-
pounds 4–6. Following crystallization from a suitable solvent, the
structures and purity of all compounds were verified by mass spec-
trometry, 1H NMR and 13C NMR. The trans–trans geometry about
onist (Ki = 22 l
M)12 and a weak MAO-B inhibitor.5 In this study, we
further investigate the importance of the (E)-styryl functional group
for dual-action by preparing and evaluating three additional classes
of C-8 substituted caffeinyl analogues. These are the 8-phenylcaffei-
nyl analogues 4a–c, the 8-benzylcaffeinyl analogues 5a–c and the
(E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues 6a–d (Scheme
2). The particularly potent series of (E,E)-8-(4-phenylbutadien-1-
yl)caffeinylanalogues6a–dwasexpandedtoincludetheethylhomo-
logs, 6e–g. The MAO-B inhibition properties of the test compounds
were first investigated, and the most potent inhibitors were then fur-
ther evaluated for binding at the A2A receptor.
As part of this effort we have measured both the Ki and IC50 val-
ues (concentration of inhibitor producing 50% inhibition) for the
inhibition of MAO-B of a subset of the test compounds. The results
have provided an opportunity to examine the validity of the rela-
tionship between Ki and IC50 of a competitive inhibitor of a mono-
substrate reaction that is described by the Cheng-Prusoff equation,
Ki = IC50/(1 + [S]/Km),27 by comparing experimentally determined
Ki values to those calculated from the IC50 values.28
the conjugated ethenyl
p-bonds of 6a–g was confirmed by pro-
ton–proton coupling constants in the range of 14.6–15.5 Hz for
the olefinic proton signals.
The (E,E)-5-phenyl-2,4-pentadienoic acids (10a–d) required for
the preparation of 6a–g were conveniently prepared by allowing
the appropriately substituted cinnamylidenemalonic acid (12a–d)
to react with refluxing acetic anhydride and acetic acid (Scheme
4).31 A solution of the resulting crude 5-phenyl-2,4-pentadienoic
acids in chloroform containing a crystal of iodine was exposed to
ambient light for 5 h. This photochemical reaction converts the
allo-styryl-acrylic acid into the desired trans–trans geometry.31
2. Results
2.1. Chemistry
The C-8 substituted caffeinyl analogues 4a–c, 5a–c, and 6a–g
(Scheme 2) were prepared in high yield according to the procedure

8678
J. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8676–8684
absorb light at this wavelength, it is possible to measure the rates
O
of substrate oxidation spectrophotometrically. We have employed
baboon liver mitochondrial preparations as the enzyme source.
Even though MMTP is a MAO-A/B mixed substrate, its oxidation
by baboon liver mitochondria is exclusively attributed to the action
of MAO-B since baboon liver tissue exhibits a high degree of MAO-
B catalytic activity while MAO-A activity is negligible.35 Also, the
interaction of reversible inhibitors with MAO-B obtained from ba-
boon liver tissue appears to be similar to the interaction with the
human form of the enzyme, since inhibitors such as CSC are
approximately equipotent with both enzyme sources.14 The incu-
bation time of the enzyme catalyzed reaction was 10 min since
the rate of MMTP oxidation was found to be linear (Fig. 1) for at
R2
NH2
NH2
N
+
HO2C Ar
8-10
O
N
R2
7a: R2 = CH3
7b: R2 = C2H5
O
H
R2
N
N
i, ii
Ar
N
O
N
least 10 min at all substrate concentrations (30–120 lM) used in
the inhibition studies. From Lineweaver–Burke plots generated
from data obtained in the absence of inhibitor, we have estimated
the Km value for the oxidation of MMTP by baboon liver MAO-B to
R2
11
O
R3
be 68.3 1.60
lM, a value consistent with the reported value of
R2
M.35 This Km was used in the studies where Ki values for
N
60.8
l
N
iii
Ar
N
the inhibition of MAO-B were calculated from the corresponding
IC50 values (see below).
O
N
All of the C-8 substituted caffeinyl analogues tested were found
to be inhibitors of MAO-B. As demonstrated with (E,E)-8-(4-phe-
nylbutadien-1-yl)caffeine (6a) (Fig. 2), the lines of the Linewe-
aver–Burke plots intersected at the y-axis, indicating the mode of
inhibition to be competitive. Competitive inhibition has also been
observed with (E)-8-styrylcaffeinyl analogues.13,14,36 The en-
zyme–inhibitor dissociation constants (Ki values) for the inhibition
of MAO-B by the test compounds are presented in Tables 1–3. The
8-phenylcaffeinyl analogues 4a–c (Table 1) and 8-benzylcaffeinyl
analogues 5a–c (Table 2) were found to be relatively weak inhibi-
4a-c
5a-c
6a-g
R2
Scheme 3. Synthetic pathway to the C-8 substituted caffeinyl analogues 4a–c, 5a–
c, and 6a–g. Reagents and conditions: (i) EDAC, dioxane/H2O; (ii) NaOH (aq), reflux;
(iii) CH3I or C2H5I, K2CO3, DMF.
i, ii
CHO
CHO
tors with Ki values ranging from 36.0 to 97.6
and 5a were especially weak inhibitors with only 24.0% and
18.0% inhibition at a concentration of 1000 M. In contrast, the
lM. Compounds 4c
R1
13a-d
R1
iii
14b-d
l
(E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues 6a–d (Table 3)
were exceptionally potent MAO-B inhibitors. The most potent
inhibitor was (E,E)-8-[4-(3-bromophenyl)butadien-1-yl]caffeine
(6c) with a Ki value of 17.2 nM, approximately 3.6 times more po-
tent than that of the corresponding (E)-8-(3-bromostyryl)caffeinyl
analogue 2c (Table 4).36 The second most potent inhibitor was
(E,E)-8-[4-(3-chlorophenyl)butadien-1-yl]caffeine (6b) with a Ki
value of 42.1 nM. This is approximately 2.5 times more potent than
that of the corresponding (E)-8-(3-chlorostyryl)caffeinyl analogue
a: R1 = H
b: R1 = Cl
c: R1 = Br
d: R1 = F
R1
C(CO2H)2
12a-d
iv, v
(E)
(E)
R1
CO2H
10a-d
Scheme 4. Synthetic pathway to the (E,E)-5-phenyl-2,4-pentadienoic acids (10a–
d). Reagents and conditions: (i) NaOH, CH3CHO; (ii) Ac2O, 120 °C; (iii) CH2(CO2H)2,
pyridine, 100 °C; (iv) Ac2O, CH3CO2H, reflux; (v) I2, light.
16
12
8
Following recrystallization from benzene, 10a–d were obtained in
good yield and with a high degree of purity. The required cinnam-
ylidenemalonic acids (12a–d) were in turn prepared in high yield
from the corresponding cinnamaldehydes (13a–d) and malonic
acid in pyridine.32 Except for cinnamaldehyde (13a), which is com-
mercially available, the other substituted cinnamaldehydes (13b–
d) were prepared by reacting the corresponding benzaldehydes
(14b–d) with acetaldehyde under basic conditions.33,34 The result-
ing cinnamaldehydes were purified by neutral aluminum oxide
column chromatography.
4
0
0
2
4
6
8
10
12
2.2. MAO-B inhibition studies
Time (min)
Enzyme activity measurements were based on the MAO-B cat-
alyzed oxidation of 1-methyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-
tetrahydropyridine (MMTP) to the corresponding dihydropyridini-
um metabolite (MMDP+).35 MMDP+ absorbs light maximally at a
wavelength of 420 nm. Since neither MMTP nor the test inhibitors
Figure 1. Linearity in the oxidation of MMTP by baboon liver MAO-B (0.15 mg
protein/mL of the mitochondrial preparation). The concentration of MMDP+
produced was measured spectrophotometrically following termination of the
enzyme catalyzed reaction at time points of 2.5, 5, and 10 min. The concentrations
of MMTP used in this study were 30 lM (filled circles) and 120 lM (open triangles).

J. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8676–8684
8679
Table 3
30
20
10
0
The Ki and IC50 values for the inhibition of MAO-B by (E,E)-8-(4-phenylbutadien-1-
yl)caffeinyl analogues 6a–g
R1
1.2
1.0
0.8
0.6
0.4
0.2
0.0
R3
O
R2
(E)
N
-200
0
200 400 600
N
(E)
[I], nM
N
O
N
R2
R2
R3
Exp. Kia (nM)
Exp. IC50 (nM)
Calcd Kic (nM)
a,b
R1
6a
6b
6c
6d
6e
6f
H
Cl
Br
F
H
H
H
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
CH3
CH3
CH3
CH3
C2H5
CH3
C2H5
148.6
42.1
17.2
46.4
1712
—
383 5.65
96.3 22.2
37.9 7.42
89.0 6.20
2371 42.5
32.7%d
221.1
55.6
21.9
51.4
1369
—
-0.01
0.00
0.01
1/[S], μM-1
0.02
0.03
0.04
6g
—
14.6%d
—
Figure 2. Lineweaver–Burke plots of the oxidation of MMTP by baboon liver MAO-
B in the absence (filled circles) and presence of various concentrations of 6a (open
circles, 0.1 lM; filled triangles, 0.2 lM; open triangles, 0.4 lM). The concentration
of the baboon liver mitochondrial preparation was 0.15 mg protein/mL, and the
rates are expressed as nmol of MMDP+ formed/mg protein/min. The inset is the
replot of the slopes versus the inhibitor concentrations.
a
The enzyme source used was baboon liver mitochondrial MAO-B.
The IC50 values were experimentally determined by fitting the rate data to the
b
one site competition model incorporated into the Prism software package.
c
The Ki values were calculated from the experimental IC50 values according to
the equation by Cheng and Prusoff: Ki = IC50/(1 + [S]/Km) with [S] = 50
(MMTP) = 68.3 1.60
M.27
Percentage inhibition at an inhibitor concentration of 30
lM and Km
l
d
lM. Due to limited
solubility in the aqueous incubation solvent, higher concentrations were not tested,
and Ki values were not determined.
Table 1
The Ki values for the inhibition of MAO-B by 8-phenylcaffeinyl analogues 4a–c
Table 4
O
R
The Ki and IC50 values for the inhibition of MAO-B by (E)-8-styrylcaffeinyl analogues
2a–d
N
N
O
N
O
N
N
N
(E)
R
Ki valuea
(lM)
N
R
O
N
4a
4b
4c
H
Cl
CF3
86.2
36.0
24.0%b
a,b
R
Exp. Kia (nM)
Exp. IC50 (nM)
Calcd Kic (nM)
2864d
Not determinede
146 1.42
107 4.59
—
a
2a
2b
2c
2d
H
The enzyme source used was baboon liver mitochondrial MAO-B.
Percentage inhibition at an inhibitor concentration of 1000 lM. Due to limited
3-Cl
3-Br
3,4-Cl2
80.6 1.96; 128d
62.7 2.73; 83f
18.9 0.89; 36f
84.3
61.8
16.4
b
solubility in the aqueous incubation solvent, higher concentrations were not tested.
28.4 1.07
a
The enzyme source used was baboon liver mitochondrial MAO-B.
The IC50 values were experimentally determined by fitting the rate data to the
Table 2
b
The Ki values for the inhibition of MAO-B by 8-benzylcaffeinyl analogues 5a–c
one site competition model incorporated into the Prism software package.
c
O
The Ki values were calculated from the experimental IC50 values according to
the equation by Cheng and Prusoff: Ki = IC50/(1 + [S]/Km) with [S] = 50
(MMTP) = 68.3 1.60
M.27
Value obtained from Ref. 14.
lM and Km
R
N
N
l
N
d
e
For weak inhibitors limits of aqueous solubility prevents accurate IC50
O
N
determinations.
f
Value obtained from Ref. 36.
R
Ki valuea
(lM)
18.0%b
54.6
feine (2a) with a reported Ki value of 2864 nM.14 In view of the
exceptional MAO-B inhibition potencies of 6a–d, we have ex-
panded the series to include the ethyl homologs 6e–g. These com-
pounds provided an opportunity to examine the effects that
homologation at positions 1, 3, and 7 of the caffeinyl ring would
have on MAO-B inhibition potency and A2A antagonism activity.
As shown in Table 3, 6e–g proved to be much weaker MAO-B
inhibitors than the corresponding methyl analogues 6a–d. For
example, the Ki value for the inhibition of MAO-B by 6e
(1712 nM) was over 10 times higher than the corresponding value
5a
5b
5c
H
Cl
CF3
97.6
a
The enzyme source used was baboon liver mitochondrial MAO-B.
Percentage inhibition at an inhibitor concentration of 1000 lM. Due to limited
solubility in the aqueous incubation solvent, higher concentrations were not tested.
b
CSC (2b) that has a reported Ki value for the inhibition of baboon
liver MAO-B of 128 nM.14 The Ki value for CSC determined in this
study is 80.6 nM (Table 4). This trend also exists for the other
(E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues 6a and 6d with
the most dramatic difference in activities found with 6a
(Ki = 148.6 nM) that is 19 times more potent than (E)-8-styrylcaf-
for 6a. Similarly, at a relatively high concentration of 30 lM, 6f and
6g exhibited only 32.7% and 14.6% MAO-B inhibition, respectively.
For the (E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues 6a–
e we have also measured the IC50 values for the inhibition of

8680
J. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8676–8684
MAO-B. An example of the data routinely obtained for the IC50
determinations is illustrated by (E,E)-8-[4-(3-chlorophenyl)buta-
dien-1-yl]caffeine (6b) (Fig. 3). Considering that the substrate
known A2A antagonists CSC (2b) and KW-6002 (1). As reported in
Table 5, a Ki value of 30.2 5.40 nM was observed for CSC. This va-
lue corresponds well with the lit. values of 36 6 nM12 and
54 19 nM.11 The Ki value obtained for KW-6002 (1) was
4.46 1.13 nM that compares favorably with the lit. value of
2.2 0.34 nM.10
concentration used for the IC50 determinations was 50
the Km value of MMTP oxidation by baboon liver mitochondrial
MAO-B is 68.3 1.60 M, we have also calculated the Ki values
lM and
l
from the measured IC50 values. As mentioned in the introduction,
the relationship between Ki and IC50 of a competitive inhibitor of
a monosubstrate reaction is described by the Cheng–Prusoff
equation: Ki = IC50/(1 + [S]/Km).27 As shown in Table 3, the calcu-
lated Ki values closely approximate those that were experimen-
tally determined, and the differences are within the range
expected for experimental error. For example, the Ki value for
the reversible interaction of 6a with MAO-B was measured as
148.6 nM, while the value calculated from the IC50 was found to
be 221.1 nM. The same trend is observed for (E)-8-styrylcaffeinyl
analogues 2b–d (Table 4) where the experimentally determined
Ki value of CSC (2b), for example, was 80.6 nM while the value
calculated from the corresponding IC50 was 84.3 nM. It should
be noted that the accuracy of an IC50 determination is dependent
upon adequately defining the sigmoid curve (obtained from plot-
ting the MAO-B catalyzed MMTP oxidation rate versus the loga-
rithm of the inhibitor concentration) at both low and high
inhibitor concentrations. For relatively weak inhibitors such as
2a, the limit of solubility in the aqueous incubation solvent pre-
vents the definition of the curve at higher inhibitor concentra-
tions (maximal inhibition) and an accurate IC50 determination is
therefore not possible.
As shown in Table 5 all of the (E,E)-8-(4-phenylbutadien-1-
yl)caffeinyl analogues (6a–g) tested were found to be potent antag-
onists of the A2A receptor. The dose–inhibition curves for the test
compounds versus [3H]NECA that were routinely observed are
illustrated by example with 6f (Fig. 4). This analogue was found
to be the most potent antagonist with
a
Ki value of
2.74 0.35 nM. Since 6f is approximately 55 times more potent
than the corresponding caffeinyl analogue 6a, 1,3-diethyl substitu-
tion of the xanthinyl ring leads to enhanced A2A antagonism po-
tency compared to 1,3-dimethyl substitution.
3. Discussion
We have recently reported that several (E)-8-styrylcaffeines act
as potent reversible inhibitors of MAO-B.13,14,36 (E)-8-Styrylcaffe-
ines have also been shown to be antagonists of adenosine A2A
receptors.11,12,29 In the present study, using (E)-8-styrylcaffeine
as the lead compound, we have attempted to identify additional
dual-target-directed compounds, possibly with enhanced MAO-B
inhibition and A2A antagonism potencies. This study also served
to elucidate the structural requirements of C-8 substituted caffei-
nyl analogues to act as dual inhibitors of MAO-B and antagonists
of the adenosine A2A receptor. Since caffeine (3) is a weak inhibitor
of MAO-B5 and only a moderately potent adenosine A2A antago-
nist,12 it can be concluded that the (E)-styryl group at C-8 plays
an important role in the dual-action of (E)-8-styrylcaffeines. In this
study we have prepared three additional classes of C-8 substituted
caffeinyl analogues 4, 5, and 6. The series of (E,E)-8-(4-phenylbuta-
dien-1-yl)caffeinyl analogues 6a–d was expanded to include the
ethyl homologs 6e–g that provided additional insight into the ste-
2.3. Adenosine A2A receptor antagonism studies
The (E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues 6a–g
were selected for further evaluation as potential antagonists of
the adenosine A2A receptor. The potencies by which the test com-
pounds antagonize A2A receptors were determined by the radioli-
gand binding procedure described in lit.37 Binding to the A2A
receptors was evaluated with N-[3H]ethyladenosin-50-uronamide
([3H]NECA) in rat striatal membranes in the presence of N6-cyclo-
pentyladenosine (CPA) to minimize the adenosine A1 receptor
binding component of [3H]NECA. This procedure is frequently used
to identify compounds that exhibit high binding affinity and selec-
tivity to A2A receptors.11,12,29 As positive controls we included the
Table 5
The Ki values for the competitive inhibition of [3H]NECA binding to rat striatal
adenosine A2A receptors by selected caffeinyl analogues
R1
R3
O
6
5
4
3
2
1
0
R2
(E)
N
N
(E)
N
O
N
R2
R1
R2
R3
Ki valuea (nM)
6a
6b
6c
6d
6e
6f
6g
1
2b
H
Cl
Br
F
H
H
H
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
CH3
CH3
CH3
CH3
C2H5
CH3
C2H5
153 5.40
104 1.50
59.1 15.8
114 14.2
13.5 4.87
2.74 0.35
7.73 2.80
KW-6002
CSC
4.46 1.13; (2.2)b
30.2 5.40; (36)c; (54)d
0
1
2
3
a
The Ki values for the competitive inhibition of [3H]NECA (Kd = 15.3 nM) binding
Log[6b]
was calculated from the corresponding IC50 values according to the Cheng–Prusoff
equation.27 The IC50 values were in turn determined by fitting the data, using
nonlinear least-squares regression analysis, to the one site competition model
incorporated into the Prism software package.
Figure 3. The sigmoidal dose–response curve of the initial rates of oxidation of
MMTP versus the logarithm of concentration of inhibitor 6b (expressed in nM). The
concentration of the baboon liver mitochondrial preparation was 0.15 mg protein/
mL; the rates are expressed as nmol MPDP+ formed/mg protein/min; the concen-
lM. The determinations were carried out in duplicate
and the values are expressed as mean SEM.
b
Value obtained from Ref. 10.
Value obtained from Ref. 12.
Value obtained from Ref. 11.
c
tration of MMTP used was 50
d

J. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8676–8684
8681
corresponding 1,3-dimethyl analogue 6a (Ki = 153 nM). Although
functional antagonism has not been demonstrated, it is reasonable
to assume caffeine derived structures would act in an antagonistic
manner since caffeine analogues are well-known antagonists of the
adenosine A2A receptor.
We conclude that the (E,E)-8-(4-phenylbutadien-1-yl)caffeines,
6a–d, are promising lead compounds for the development of dual-
target-directed compounds that inhibit MAO-B and antagonize A2A
receptors. Although 1,3-diethyl substitution of the caffeinyl ring
leads to enhanced potency of A2A antagonism, the opposite effect
was observed on MAO-B inhibition potency.
In the second part of this study it was shown that for potent
inhibitors, the Ki values for competitive interaction with MAO-B
could be accurately calculated from the corresponding IC50 values
using the Cheng–Prusoff equation. This would facilitate the com-
parison of IC50 and Ki values from different lit. sources and the
employment of the inhibitors in QSAR studies.
1800
1500
1200
900
-1
0
1
2
3
Log[6f]
4. Experimental
Figure 4. The sigmoidal dose–response curve for the inhibition of [3H]NECA
binding to rat striatal A2A receptors by antagonist 6f (expressed in nM). The IC50
value was determined by fitting the data, using nonlinear least-squares regression
analysis, to the one site competition model incorporated into the Prism software
package (GraphPad Software Inc.). The Ki value (2.74 nM) for the competitive
Caution: MMTP is a structural analogue of the nigrostriatal neu-
rotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),
and should be handled using disposable gloves and protective eye-
wear. Procedures for the safe handling of MPTP have been de-
scribed previously.38
inhibition of [3H]NECA (Kd = 15.3 nM) binding was calculated with the Cheng–
27
Prusoff equation.
In this experiment the non-saturable, nonspecific binding was
623 cpm. The determinations were carried out in duplicate and the values are
expressed as mean SEM.
4.1. Chemicals and instrumentation
All starting materials, unless mentioned elsewhere, were ob-
tained from Sigma–Aldrich and were used without purification.
The oxalate salt of MMTP,39 KW-6002 (1),13,29 compounds 2b–
2d,14,36 1,3-dimethyl- (7a) and 1,3-diethyl-5,6-diaminouracil
(7b)30 were prepared according to previously reported procedures.
Because of chemical instability, compounds 7a–b were used within
24 h of preparation. Proton and carbon NMR spectra were recorded
on a Varian Gemini 300 spectrometer. Proton (1H) spectra were re-
corded in CDCl3 or DMSO-d6 at a frequency of 300 MHz and carbon
ric features associated with the potencies of MAO-B inhibition and
A2A antagonism.
The results of MAO-B inhibition studies have shown that the 8-
phenylcaffeinyl (4a–c) and 8-benzylcaffeinyl (5a–c) analogues
were weak inhibitors of MAO-B while the (E,E)-8-(4-phenylbutadi-
en-1-yl)caffeinyl analogues 6a–d were found to be exceptionally
potent inhibitors. For example, the (E,E)-8-(4-phenylbutadien-1-
yl)caffeinyl analogue 6b substituted with chlorine at C-3 of the
phenyl ring (Ki = 42.1 nM) was approximately 855 and 1269 times
more potent as an MAO-B inhibitor than was the corresponding
C-3 chlorine substituted 8-phenylcaffeinyl analogue 4b (Ki =
(
13C) spectra at 75 MHz. Chemical shifts are reported in parts per
million (d) downfield from the signal of tetramethylsilane added
to the deuterated solvent. Spin multiplicities are given as s (sin-
glet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets),
or m (multiplet) and the coupling constants (J) are given in hertz
(Hz). Direct insertion electron impact ionization (EIMS) and high
resolution mass spectra (HRMS) were obtained on a VG 7070E
mass spectrometer. Melting points (mp) were determined on a
Stuart SMP10 melting point apparatus and are uncorrected. UV–
vis spectra were recorded on a Shimadzu UV-2100 double-beam
spectrophotometer. Thin layer chromatography (TLC) was carried
out with neutral aluminum oxide 60 (Merck) containing UV254
fluorescent indicator. [3H]NECA was obtained from Amersham
(specific activity 25 Ci/mmol), while adenosine deaminase (type
X from calf spleen) and CPA were from Sigma–Aldrich. Counting
of radio activities was performed using a Packard Tri-Carb 2100
TR liquid scintillation counter.
36.0 lM) and 8-benzylcaffeinyl analogue 5b (Ki = 54.6 lM), respec-
tively. The (E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues
6a–d were also more potent inhibitors than the corresponding
(E)-8-styrylcaffeinyl analogues 2a–c. For example, chloro substi-
tuted analogue 6b (Ki = 42.1 nM) was approximately 1.9 times
more potent than CSC (2b) (Ki = 80.6 nM) while the unsubstituted
(E,E)-8-(4-phenylbutadien-1-yl)caffeinylanalogue6a(Ki = 148.6 nM)
was almost 20 times more potent than the corresponding unsubsti-
tuted (E)-8-styrylcaffeinyl analogue 2a (Ki = 2864 nM). Compounds
6e–g were found to be relatively weak inhibitors of MAO-B indicating
that ethylsubstitutionat positions1, 3, and 7ofthecaffeinylring hasa
negative effect on the potency of MAO-B inhibition. This result sug-
gests that 1, 3, and 7 methyl substitution is probably optimal for the
design of xanthine-based reversible MAO-B inhibitors. In the case of
(E)-8-styrylcaffeines, 1,3-diethyl substitution of the caffeine ring has
also been shown to reduce the MAO-B inhibition potency compared
to 1,3-dimethyl substitution.13
4.2. General procedure for the synthesis of (E,E)-5-phenyl-2,4-
pentadienoic acids (10a–d)
Since the series of (E,E)-8-(4-phenylbutadien-1-yl)caffeinyl ana-
logues was found to include exceptionally potent reversible MAO-
B inhibitors, they were selected for further evaluation as potential
antagonists of the adenosine A2A receptor. All the (E,E)-8-(4-phe-
nylbutadien-1-yl)caffeinyl analogues 6a–g were found to be rela-
tively potent A2A antagonists. In contrast to its effect on MAO-B
inhibition potency, ethyl substitution at positions 1, 3, and 7
of the caffeinyl ring had a positive effect on the potency of A2A
antagonism. For example, the 1,3-diethyl analogue 6f
(Ki = 2.74 nM) was approximately 55 times more potent than the
Compounds 10a–d were prepared from the corresponding cinn-
amylidenemalonic acids (12a–d)32 according to the previously de-
scribed procedure.31 Cinnamylidenemalonic acids (12a–d) (1 g)
were allowed to reflux for 60 min with acetic anhydride (5 mL)
and acetic acid (3 mL). The reaction was cooled to room tempera-
ture and poured into 100 mL water. After 3 h, the resulting
precipitate was collected by filtration. The crude product and a
crystal of iodine were dissolved in 20 mL CHCl3 and incubated in

8682
J. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8676–8684
ambient light for 5 h. The CHCl3 was removed under reduced pres-
sure and the residue was recrystallized from benzene. For previ-
ously described 10a–c, the melting points were recorded as
in a yield of 33.6%: mp 192 °C; 1H NMR (CDCl3) d 3.39 (s, 3H), 3.59
(s, 3H), 4.05 (s, 3H), 7.61–7.67 (m, 1H), 7.73–7.77 (m, 1H), 7.84–
7.88 (m, 1H), 7.95–7.96; 13C NMR (CDCl3) d 27.97, 29.74, 33.87,
108.84, 121.74, 126.14 (q), 126.93 (q), 129.32, 129.46, 131.62 (q),
132.26, 148.20, 150.23, 151.59, 155.52; EIMS m/z 338 (M.+); HRMS
calcd 338.09906; found: 338.09735.
follows: 10a 194 °C, lit. 178 °C31; 10b 190 °C, lit. 173–174 °C40
10c 187 °C, lit. 179–180.41
;
4.2.1. (E,E)-5-(3-Fluorophenyl)-2,4-pentadienoic acid (10d)
The title compound was prepared from 12d in a yield of 42%.
Mp 164 °C; 1H NMR (DMSO-d6) d 6.03 (d, 1H, J = 15.0 Hz), 7.10–
7.45 (m, 7H) 13C NMR (DMSO-d6) d 112.90, 113.16, 115.33,
123.18, 123.51, 128.09, 130.63, 130.74, 138.19, 143.66, 167.31;
EIMS m/z 192 (MÅ+); HRMS calcd 192.05866; found: 192.05936.
4.3.3. 8-(3-Chlorobenzyl)caffeine (5b)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), 3-chlorophenylacetic acid (9b) and iodomethane in a
yield of 42%: mp 132 °C; 1H NMR (CDCl3) d 3.36 (s, 3H), 3.56 (s, 3H),
3.79 (s, 3H), 4.10 (s, 2H), 7.02–7.05 (m, 1H), 7.14–7.16 (m, 1H),
7.21–7.24 (m, 2H); 13C NMR (CDCl3) d 27.83, 29.74, 31.00, 32.96,
107.85, 126.38, 127.54, 128.35, 130.16, 134.84, 137.00, 147.87,
151.20, 151.59, 155.30; EIMS m/z 319 (MÅ+); HRMS calcd
318.08835; found: 318.08702.
4.3. General procedure for the synthesis of caffeinyl analogues
(4a–c, 5a–c, and 6a–g)
The C-8 substituted caffeinyl analogues examined in this study
were prepared according to the procedure described in lit.29 1,3-
Dimethyl- (7a) or 1,3-diethyl-5,6-diaminouracil (7b) (3.50 mmol)
and N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochlo-
ride (EDAC; 5.11 mmol) were dissolved in 40 mL dioxane/H2O
(1:1) and the appropriate carboxylic acid [benzoic acids (8a–c),
phenylacetic acids (9a–c), or (E,E)-5-phenyl-2,4-pentadienoic acids
(10a–d), 3.81 mmol] was added. A suspension was obtained and
the pH was adjusted to 5 with 2 M aqueous hydrochloric acid.
The reaction mixture was stirred for an additional 2 h and then
neutralized with 1 M aqueous sodium hydroxide. After cooling to
0 °C, the precipitate that formed was collected by filtration. A solu-
tion of this crude amide in 40 mL aqueous sodium hydroxide
(1 M)/dioxane (1:1) was heated under reflux for 2 h, cooled to
0 °C and then acidified to a pH of 4 with 4 M aqueous hydrochloric
acid. For the preparation of 4a–c, 5a–c, and 6a–e, the resulting pre-
cipitate, the corresponding 1,3-dimethyl-8-substituted-7H-xanthi-
nyl analogue (11), was collected by filtration and used in the
subsequent reaction without further purification. For the prepara-
tion of 6f–g, the resulting precipitate was removed via filtration
and the filtrate was extracted to CHCl3 (2ꢀ 100 mL). The organic
phase was dried over anhydrous MgSO4 and removed under re-
duced pressure to yield a yellow oily residue, the 1,3-diethyl-8-
substituted-7H-xanthinyl analogues (11). To a stirred suspension
of 11 (0.20 mmol) and potassium carbonate (0.50 mmol) in 5 mL
DMF was added iodomethane (4a–c, 5a–c, 6a–d, and 6f) or iodo-
ethane (6e and 6g) (0.40 mmol). Stirring was continued at 60 °C
for 60 min, and the insoluble materials were removed by filtration.
Sufficient water was added to the filtrate to precipitate the product
(4–6) which was collected by filtration. Following crystallization
from a mixture of methanol/ethyl acetate (9:1) (4a–c, 5a–c, and
6a–e) or ethanol (6f–g), analytically pure samples of the target
compounds were obtained. For previously described 4a and 5a,
we found the melting points to be 180 and 165 °C [from metha-
nol/ethyl acetate (9:1)] while the reported melting points are
178 °C12 and 161–163 °C,42 respectively.
4.3.4. 8-(3-Trifluoromethylbenzyl)caffeine (5c)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), 3-(trifluoromethyl)phenylacetic acid (9c), and iodo-
methane in a yield of 49%: mp 163 °C; 1H NMR (CDCl3) d 3.36 (s,
3H), 3.55 (s, 3H), 3.81 (s, 3H), 4.18 (s, 2H), 7.34–7.52 (m, 4H); 13C
NMR (CDCl3) d 27.83, 29.72, 31.00, 33.14, 107.86, 122.00, 124.23
(q), 125.08 (q), 125.61, 129.45, 131.37 (q), 131.62, 136.09,
147.90, 151.00, 151.60, 155.31; EIMS m/z 352 (M.+); HRMS calcd
352.11471; found: 352.11570.
4.3.5. (E,E)-8-(4-Phenylbutadien-1-yl)caffeine (6a)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), (E,E)-5-phenyl-2,4-pentadienoic acid (10a), and
iodomethane in a yield of 27%: mp 253 °C; 1H NMR (CDCl3) d
3.37 (s, 3H), 3.58 (s, 3H), 3.96 (s, 3H), 6.44 (d, 1H, J = 15.0 Hz),
6.84–6.95 (m, 2H), 7.24–7.36 (m, 3H), 7.42–7.46 (m, 2H), 7.56
(dd, 1H, J = 15.0, 10.0 Hz); 13C NMR (CDCl3) d 27.86, 29.66, 31.35,
107.82, 114.46, 126.90, 127.30, 128.65, 128.77, 136.40, 138.14,
138.50, 148.61, 149.98, 151.64, 155.13; EIMS m/z 322 (MÅ+); HRMS
calcd 322.14298; found: 322.14186.
4.3.6. (E,E)-8-[4-(3-Chlorophenyl)butadien-1-yl]caffeine (6b)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), (E,E)-5-(3-chlorophenyl)-2,4-pentadienoic acid
(10b), and iodomethane in a yield of 10%: mp 249 °C; 1H NMR
(CDCl3) d 3.39 (s, 3H), 3.59 (s, 3H), 3.99 (s, 3H), 6.49 (d, 1H,
J = 15.0 Hz), 6.80 (d, 1H, J = 15.5 Hz), 6.92–7.01 (m, 1H), 7.24–
7.31 (m, 3H), 7.44 (d, 1H, J = 0.41 Hz), 7.55 (dd, 1H, J = 14.7, 10.9
Hz); 13C NMR (CDCl3) d 27.92, 29.71, 31.42, 108.01, 115.47,
125.15, 126.61, 128.48, 128.67, 130.01, 134.85, 136.34, 137.87,
138.32, 148.65, 149.72, 151.68, 155.21; EIMS m/z 357 (MÅ+); HRMS
calcd 356.10400; found: 356.10571.
4.3.7. (E,E)-8-[4-(3-Bromophenyl)butadien-1-yl]caffeine (6c)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), (E,E)-5-(3-bromophenyl)-2,4-pentadienoic acid
(10c), and iodomethane in a yield of 40%: mp 246 °C; 1H NMR
(CDCl3) d 3.38 (s, 3H), 3.58 (s, 3H), 3.98 (s, 3H), 6.49 (d, 1H,
J = 15.0 Hz), 6.78 (d, 1H, J = 15.5 Hz), 6.95 (dd, 1H, J = 15.5,
10.9 Hz), 7.17–7.22 (m, 1H), 7.33–7.40 (m, 2H), 7.55 (dd, 1H,
J = 14.6, 10.9 Hz), 7.59 (d, 1H, J = 1.8 Hz); 13C NMR (CDCl3) d
27.91, 29.70, 31.40, 108.00, 115.50, 123.01, 125.58, 128.70,
129.53, 130.27, 131.38, 136.20, 137.82, 138.60, 148.63, 149.70,
151.67, 155.20; EIMS m/z 400, 402 (MÅ+); HRMS calcd 400.05349;
found: 400.05193.
4.3.1. 8-(3-Chlorophenyl)caffeine (4b)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), 3-chlorobenzoic acid (8b), and iodomethane in a
yield of 91%: mp 202 °C; 1H NMR (CDCl3) d 3.39 (s, 3H), 3.58 (s,
3H), 4.04 (s, 3H), 7.43–7.48 (m, 2H), 7.53–7.56 (m, 1H), 7.67–
7.69 (m, 1H); 13C NMR (CDCl3) d 27.97, 29.72, 33.90, 108.73,
127.07, 129.28, 130.10, 130.42, 135.05, 148.16, 150.40, 151.60,
155.51; EIMS m/z 304 (MÅ+); HRMS calcd 304.07270; found:
304.07108.
4.3.2. 8-(3-Trifluoromethylphenyl)caffeine (4c)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), 3-trifluoromethylbenzoic acid (8c), and iodomethane
4.3.8. (E,E)-8-[4-(3-Fluorophenyl)butadien-1-yl]caffeine (6d)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), (E,E)-5-(3-fluorophenyl)-2,4-pentadienoic acid

J. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8676–8684
8683
(10d), and iodomethane in a yield of 22.1%: mp 223 °C; 1H NMR
(CDCl3) d 3.22 (s, 3H), 3.44 (s, 3H), 3.95 (s, 3H), 6.87 (d, 1H,
J = 15.0 Hz), 7.01 (d, 1H, J = 15.4 Hz), 7.03–7.15 (m, 1H), 7.26 (dd,
1H, J = 15.4, 11.0 Hz), 7.35–7.44 (m, 3H), 7.47 (dd, 1H, J = 14.6,
11.0 Hz); EIMS m/z 340 (MÅ+); HRMS calcd 340.13355; found:
340.13367.
orders of a magnitude (3–1000
l
M). The stock solutions of the
inhibitors were prepared in DMSO and were added to the incuba-
tion mixtures to yield a final DMSO concentration of 4% (v/v).
DMSO concentrations higher than 4% are reported to inhibit
MAO-B.45 The reactions were incubated at 37 °C for 10 min and
then terminated by the addition of 10 lL perchloric acid (70%).
The MAO-B catalyzed production of MMDP+ was found to be lin-
ear for the first 10 min of incubation under these conditions. The
samples were centrifuged at 16,000g for 10 min, and the concen-
trations of the MAO-B generated product, MMDP+, were measured
4.3.9. (E,E)-1,3-Dimethyl-8-(4-phenylbutadien-1-yl)-7-
ethylxanthine (6e)
The title compound was prepared from 1,3-dimethyl-5,6-diami-
nouracil (7a), (E,E)-5-phenyl-2,4-pentadienoic acid (10a), and
iodoethane in a yield of 6.9%: mp 227 °C; 1H NMR (CDCl3) d 1.46
(t, 3H, J = 7.2 Hz), 3.43 (s, 3H), 3.63 (s, 3H), 4.45 (q, 1H,
J = 7.2 Hz), 6.50 (d, 1H, J = 14.9 Hz), 6.92 (d, 1H, J = 15.5 Hz), 7.01
(dd, 1H, J = 15.5, 11.0 Hz), 7.31 (t, 1H, J = 7.3 Hz), 7.38 (t, 2H,
J = 7.4 Hz), 7.49 (d, 2H, J = 7.4 Hz), 7.63 (dd, 1H, J = 14.9, 10.9 Hz);
13C NMR (CDCl3) d 16.65, 28.12, 29.93, 40.18, 107.76, 115.16,
127.73, 128.16, 129.48, 129.63, 137.30, 139.00, 139.40, 149.78,
150.10, 152.69, 155.77; EIMS m/z 336 (MÅ+); HRMS calcd
336.15863;found:336.15872.
spectrophotometrically at 420 nm (
natant fractions.35 For the Ki determinations, the initial rates of
oxidation at four different substrate concentrations (30–120 M)
e
= 25,000 Mꢁ1) in the super-
l
in the absence and presence of three different concentrations of
the inhibitors were used to construct Lineweaver–Burke plots.
The slopes of the Lineweaver–Burke plots were plotted versus
the inhibitor concentration and the Ki value were determined
from the abscissa intercept (intercept = ꢁKi). Linear regression
analysis was performed using the SigmaPlot software package
(Systat Software Inc.). Each Ki value reported here is representa-
tive of a single determination where the correlation coefficient
(R2 value) of the replot of the slopes versus the inhibitor concen-
trations was at least 0.98. The IC50 values were determined by
plotting the initial rates of oxidation versus 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 4 software package (GraphPad
Software Inc.). The IC50 values were determined in duplicate and
are expressed as mean standard error of the mean (SEM).
4.3.10. (E,E)-1,3-Diethyl-8-(4-phenylbutadien-1-yl)-7-
methylxanthine (6f)
The title compound was prepared from 1,3-diethyl-5,6-diami-
nouracil (7b), (E,E)-5-phenyl-2,4-pentadienoic acid (10a),and iodo-
methane in a yield of 24%: mp 156–157 °C; 1H NMR (CDCl3) d 1.19
(t, 3H, J = 7.1 Hz), 1.30 (t, 3H, J = 7.0 Hz), 3.93 (s, 3H), 4.01 (q, 2H,
J = 6.9 Hz), 4.13 (q, 2H, J = 7.0 Hz), 6.41(d, 1H, J = 15.0 Hz), 6.84 (d,
1H, J = 15.5 Hz), 6.92 (dd, 1H, J = 15.5, 11.2 Hz), 7.23 (m, 1H), 7.30
(t, 2H, J = 7.4 Hz), 7.40 (d, 2H, J = 7.8 Hz), 7.53 (dd, 1H, J = 14.9,
10.9 Hz); 13C NMR (CDCl3) d 13.49, 29.90, 31.57, 36.60, 38.68,
108.76, 115.30, 127.73, 128.20, 129.48, 129.64, 137.33, 138.93,
139.38, 149.10, 150.90, 151.70, 156.00; EIMS m/z 350 (MÅ+); HRMS
calcd 350.17428; found: 350.17277.
4.5. Adenosine A2A receptor antagonism studies
The adenosine A2A receptor binding studies were carried out
according the procedure described in lit.37 The striata of male Spra-
gue–Dawley rats (n = 40) were dissected, immediately frozen in li-
quid nitrogen and stored at ꢁ70 °C. The striata were thawed on ice,
weighed and disrupted for 30 s with the aid of a Polytron homog-
enizer in 10 volumes of ice-cold 50 mM TrisꢂHCl (pH 7.7 at 25 °C).
The resulting homogenate was centrifuged at 50,000g for 10 min
and the pellet was resuspended in 10 volumes of ice-cold TrisꢂHCl,
again with the aid of a Polytron homogenizer as above. The result-
ing suspension was recentrifuged and the pellet obtained was sus-
pended in TrisꢂHCl to a volume of 5 mL/g original striatal weight.
The striatal membranes were aliquoted into microcentrifuge tubes
and stored at ꢁ70 °C. The incubations were carried out in 4 mL
polypropylene tubes, previously coated with Sigmacote (Sigma–Al-
drich) and were prepared in 1 mL TrisꢂHCl containing 5 mg of the
original tissue weight striatal membranes, 4 nM [3H]NECA, 50 nM
CPA, 10 mM MgCl2, 0.1 U/mL adenosine deaminase, and various
concentrations of the test compounds. The stock solutions of the
compounds to be tested as well as that of CPA were prepared in
DMSO. On the day of the study, CPA and [3H]NECA were diluted
to concentrations of 500 nM and 40 nM, respectively. A membrane
suspension was prepared that contained 5 mg/0.79 mL striatal
membranes and sufficient amounts of MgCl2 and adenosine deam-
inase to produce concentrations of 10 mM and 0.1 U/mL, respec-
tively, in the final 1 mL incubation mixtures. The order of
4.3.11. (E,E)-1,3-Diethyl-8-(4-phenylbutadien-1-yl)-7-
ethylxanthine (6g)
The title compound was prepared from 1,3-diethyl-5,6-diami-
nouracil (7b), (E,E)-5-phenyl-2,4-pentadienoic acid (10a), and
iodoethane in a yield of 21%: mp 177–178 °C; 1H NMR (CDCl3) d
1.19 (t, 3H, J = 7.1 Hz), 1.31 (t, 3H, J = ꢁ7.0 Hz), 1.38 (t, 3H,
J = 7.2 Hz), 4.01 (q, 2H, J = 7.0 Hz), 4.13 (q, 2H, J = 7.0 Hz), 4.36 (q,
2H, J = 7.2 Hz), 6.41 (d, 1H, J = 14.9 Hz), 6.84 (d, 1H, J = 15.5 Hz),
6.93 (dd, 1H, J = 15.5, 11.0 Hz), 7.22 (m, 1H), 7.29 (t, 2H,
J = 7.6 Hz), 7.41 (d, 2H, J = 8.0 Hz), 7.55 (dd, 1H, J = 14.9, 11.0 Hz);
13C NMR (CDCl3) d 13.47, 16.68, 29.89, 36.62, 38.64, 40.10,
108.00, 115.38, 127.70, 128.26, 129.43, 129.63, 137.37, 138.73,
139.23, 149.33, 149.98, 151.74, 155.57; EIMS m/z 364 (MÅ+); HRMS
calcd 364.18993; found: 364.18904.
4.4. MAO-B inhibition studies
The mitochondrial fraction of baboon liver tissue was isolated
as described previously43 and stored at –70 °C. Following addition
of an equal volume of sodium phosphate buffer (100 mM, pH 7.4)
containing glycerol (50%, w/v) to the mitochondrial isolate, the
protein concentration was determined by the method of Bradford
using bovine serum albumin as reference standard.44 MMTP
additions was as follows: test compound (10
lL), CPA (100 lL),
(Km = 68.3 1.60
l
M for baboon liver MAO-B),35 served as sub-
[3H]NECA (100
lL), and the membrane suspension (0.79 mL). The
strate for the inhibition studies. The enzymatic reactions were
prepared in sodium phosphate buffer (100 mM, pH 7.4) and con-
incubations were vortexed and incubated for 60 min at 25 °C in a
shaking water bath. Halfway through the experiment, the incuba-
tions were vortexed again. The incubations were filtered through a
prewetted 2.4 cm Whatman glass microfiber filter (grade GF/B) un-
der reduced pressure. The tubes were washed with 4 mL ice-cold
TrisꢂHCl and the filters were washed twice more with 4 mL ice-cold
TrisꢂHCl. The damp filters were place in scintillation vials, 4 mL of
tained MMTP (30–120
protein/mL) and various concentrations of the test inhibitors.
The final volume of the incubations was 500 L. For the IC50
determinations, a fixed substrate concentration of 50 M was
used and the inhibitor concentrations spanned at least three
lM), the mitochondrial isolate (0.15 mg
l
l

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Acknowledgments
We are grateful to Cor Bester and Antoinette Fick of the Animal
Research Centre as well as the staff of the Laboratory for Applied
Molecular Biology, North-West University for their support. The
NMR and MS spectra were recorded by André Joubert, Johan Jorda-
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West University. This work was supported by grants from the Na-
tional Research Foundation and the Medical Research Council,
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