8-Aryl- and alkyloxycaffeine analogues as inhibitors of monoamine oxidase

Article abstract of DOI:10.1016/j.ejmech.2011.05.014

Recently it was reported that a series of 8-benzyloxycaffeine analogues are potent reversible inhibitors of human monoamine oxidase (MAO) A and B. In an attempt to discover additional C8 oxy substituents of caffeine that lead to potent MAO inhibition, a series of related 8-aryl- and alkyloxycaffeine analogues were synthesized and their MAO-A and -B inhibition potencies were compared to those of the 8-benzyloxycaffeines. The results document that while the 8-substituted-oxycaffeine analogues inhibited both human MAO isoforms, they displayed a high degree of selectivity for MAO-B. 8-(3-Phenylpropoxy)caffeine, 8-(2-phenoxyethoxy)caffeine and 8-[(5-methylhexyl)oxy]caffeine were found to be the especially potent MAO-B inhibitors with IC₅₀ values ranging from 0.38 to 0.62 μM. These inhibitors are therefore 2.5-4.6 fold more potent MAO-B inhibitors than is 8-benzyloxycaffeine (IC₅₀ = 1.77 μM). It is also demonstrated that, analogous to 8-benzyloxycaffeine, halogen substitution on the phenyl ring of the C8 substituent significantly enhances MAO binding affinity. For example, the most potent MAO-B inhibitor of the present series is 8-[2-(4-bromophenoxy)ethoxy]caffeine with an IC₅₀ value of 0.166 μM. This study also reports possible binding orientations of selected oxy caffeines within the active site cavities of MAO-A and MAO-B.

Full text of DOI:10.1016/j.ejmech.2011.05.014

European Journal of Medicinal Chemistry 46 (2011) 3474e3485
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
8-Aryl- and alkyloxycaffeine analogues as inhibitors of monoamine oxidase
*
Belinda Strydom, Jacobus J. Bergh, Jacobus P. Petzer
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently it was reported that a series of 8-benzyloxycaffeine analogues are potent reversible inhibitors of
human monoamine oxidase (MAO) A and B. In an attempt to discover additional C8 oxy substituents of
caffeine that lead to potent MAO inhibition, a series of related 8-aryl- and alkyloxycaffeine analogues
were synthesized and their MAO-A and -B inhibition potencies were compared to those of the 8-ben-
zyloxycaffeines. The results document that while the 8-substituted-oxycaffeine analogues inhibited both
human MAO isoforms, they displayed a high degree of selectivity for MAO-B. 8-(3-Phenylpropoxy)
caffeine, 8-(2-phenoxyethoxy)caffeine and 8-[(5-methylhexyl)oxy]caffeine were found to be the espe-
Received 21 April 2011
Received in revised form
5 May 2011
Accepted 5 May 2011
Available online 12 May 2011
Keywords:
cially potent MAO-B inhibitors with IC50 values ranging from 0.38 to 0.62
m
M. These inhibitors are
M). It is
Monoamine oxidase
Reversible inhibition
8-Aryloxycaffeine
8-Alkyloxycaffeine
Caffeine
therefore 2.5e4.6 fold more potent MAO-B inhibitors than is 8-benzyloxycaffeine (IC50 ¼ 1.77
m
also demonstrated that, analogous to 8-benzyloxycaffeine, halogen substitution on the phenyl ring of the
C8 substituent significantly enhances MAO binding affinity. For example, the most potent MAO-B
inhibitor of the present series is 8-[2-(4-bromophenoxy)ethoxy]caffeine with an IC50 value of
Molecular docking
0.166
mM. This study also reports possible binding orientations of selected oxy caffeines within the active
site cavities of MAO-A and MAO-B.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
disease (PD) [6,7]. The inhibition of the MAO-B catalyzed metabo-
lism of dopamine in the PD striatum may preserve the depleted
dopamine supply [8] and elevate the levels of dopamine produced
for exogenously administered levodopa [9,10]. For example, litera-
ture reports that MAO-B inhibitors enhance the elevation of dopa-
mine levels in the striatum of primates treated with levodopa [9,10].
For these reasons, MAO-B inhibitors are frequently used as adju-
vants to levodopa in the therapy of PD [6,11]. MAO-B inhibitors may
also block the central production and accumulation of potentially
neurotoxic species such as dopaldehyde and H2O2 which are formed
during the MAO-B catalyzed metabolism of dopamine [12e14].
Based on this effect, MAO-B inhibitors are thought to protect against
the neurodegenerative processes associated with PD [15]. This
property of MAO-B inhibitors may be of particular relevance since
the density and activity of MAO-B increases with age in most brain
regions [16e18]. Examples of MAO-B inhibitors which are clinically
used in PD therapy are (R)-deprenyl and rasagiline [19]. Both these
drugs are selective for the MAO-B isoform and are propargylamine-
containing mechanism-based inhibitors of the enzyme. Of interest
is the observation that, even though MAO-A activity is much lower
than MAO-B activity in the striatum, both MAO-A and eB may
contribute to catabolism of dopamine in the primate brain [18,20].
Literature reports that the extent to which the MAO-A selective
inhibitor, clorgyline, enhances the elevation of dopamine levels in
the striatum of primates treated with levodopa is similar to that
The monoamine oxidases (MAO) A and B are mitochondrial
bound flavin adenine dinucleotide (FAD) containing enzymes
which catalyze the a-carbon oxidation of a variety of endogenous
and dietary aminyl substrates in the brain and peripheral tissues
[1,2]. The two MAO isoforms display different specificities towards
substrates and inhibitors [3]. MAO-A catalyses the oxidation of
serotonin and norepinephrine and is irreversibly inhibited by the
mechanism-based inhibitor, clorgyline. MAO-B, on the other hand,
catalyses the oxidation of benzylamine and is irreversibly inhibited
by (R)-deprenyl. Both isoforms employ dopamine as substrate [3].
Because of their roles in the metabolism of neurotransmitter
amines, MAO-A and -B are considered to be drug targets for the
treatment of neuropsychiatric and neurodegenerative disorders.
Inhibitors of MAO-A may elevate central serotonin levels and are
therefore used in the treatment of depressive illness [4]. Moclobe-
mide is an example of a selective MAO-A inhibitor that is currently
being used as an antidepressant [5]. Since MAO-B is a major dopa-
mine metabolizing enzyme in the basal ganglia of the primate brain,
inhibitors of MAO-B are employed in the treatment of Parkinson’s
* Corresponding author. Tel.: þ27 18 299 2206; fax: þ27 18 299 4243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.014

B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
3475
obtained with (R)-deprenyl [9]. It has therefore been suggested that
non-selective inhibitors of MAO may be more effective in the inhi-
bition of dopamine catabolism and the treatment of PD [1].
analogues act as potent reversible competitive inhibitors of both
human MAO-A and -B [27]. In this series, 8-benzyloxycaffeine (2)
was found to inhibit MAO-B with an Ki value of 0.59
mately 6000 fold more potent than caffeine. 8-Benzyloxycaffeine
also displayed affinity for MAO-A with an Ki value of 0.43 M.
mM, approxi-
While inactivators of MAO have been used extensively as clinical
drugs, irreversible inhibition has a number of disadvantages [21].
These include the loss of isoform selectivity as a result of repeated
drug administration and a slow and variable rate of enzyme
recovery following withdrawal of the drug [22]. For example, the
turnover rate for the biosynthesis of MAO-B in the human brain
may require as much as 40 days [23]. In contrast, for reversible
inhibition, enzyme activity is recovered when the inhibitor is
eliminated from the tissues and the risk of loss of selectivity is
reduced because of a shorter duration of action. Furthermore,
where the mode of inhibition is competitive, the inhibition may be
relieved when the substrate concentration is increased. Irreversible
MAO-A inhibitors have the added disadvantage that they may
potentiate the cardiovascular effects of indirectly acting sympa-
thomimetic amines such as tyramine [1,4]. Inactivation of MAO-A in
the gut wall greatly increases the quantity of tyramine entering the
systemic circulation while reversible inhibition allows for the
inhibitor to be displaced by tyramine which is subsequently nor-
mally metabolized by the enzyme. For these reasons several
research groups are interested in the discovery of novel MAO
inhibitors which interact reversibly with the enzymes.
m
Substitution on the benzyloxy ring with halogens enhances the
MAO-A and -B inhibition potencies by an even greater extent. The
benzyloxy side chain appears to be particularly effective in
enhancing the binding affinities of reversible inhibitors for
the active site of MAO-B. For example, both safinamide (3) and
7-(3-chlorobenzyloxy)-4-formylcoumarin (4) are potent MAO-B
inhibitors and also contain halogen substituted-benzyloxy side
chains (Fig. 1) [28]. The biological properties of the benzyloxy side
chain with respect to its ability to bind to MAO-B may be best
explained by considering the binding orientations of these inhibi-
tors within the active site of MAO-B. The crystal structure of MAO-B
in complex with safinamide show that the 3-fluorobenzyloxy side
chain of safinamide extends into the entrance cavity of the MAO-B
active site while the propanamidyl moiety binds within the
substrate cavity [28]. In the crystal structure of MAO-B in complex
with 7-(3-chlorobenzyloxy)-4-formylcoumarin, a similar binding
orientation is observed with the 3-chlorobenzyloxy side chain
located within the entrance cavity of the enzyme while the
coumarin ring occupies the substrate cavity [28]. Modelling studies
suggest that 8-benzyloxycaffeine may also bind to MAO-B with the
caffeine moiety situated within the substrate cavity and the ben-
zyloxy side chain extending towards the entrance cavity [27]. The
interesting observation that 8-benzyloxycaffeines are also MAO-A
inhibitors may possibly be attributed to the relatively large
degree of rotational freedom of the benzyloxy side chain at the
carboneoxygen ether bond [27]. Previous studies have suggested
that structures with a relatively larger degree of conformational
freedom may be better suited for binding to MAO-A than relatively
rigid structures [25].
For the design of reversible MAO inhibitors, a variety of scaffolds
have been employed. Among these is caffeine (1) (Fig. 1). While
caffeine (Ki ¼ 3.6 mM) is a weak MAO-B inhibitor, literature reports
that substitution of the caffeine ring at C8 with a variety of groups
yields compounds endowed with greatly enhanced MAO-B inhi-
bition activities compared to caffeine [24e26]. For example,
a recent report documents that a series of 8-benzyloxycaffeine
O
O
N
N
In the present study, caffeine was substituted with a variety of
C8 oxy substituents and the resulting analogues (5aen) were
evaluated as inhibitors of human MAO-A and -B (Table 1). The
inhibition potencies of these analogues were compared to those of
the previously reported 8-benzyloxycaffeines in an attempt to
discover additional C8 oxy substituents of caffeine that lead to
potent MAO inhibition [27]. For selected homologues, the effect of
halogen substitution on the phenyl ring of the C8 substituent on
MAO binding affinity was also investigated. As mentioned above,
halogen substitution on the phenyl ring of 8-benzyloxycaffeine
markedly enhances MAO inhibition potency [27]. In addition, in
order to establish whether the test compounds bind reversibly with
the MAO isozymes, the time-dependency of inhibition by selected
analogues was investigated. As illustrated in Table 1, the oxy
substituents that were selected for this study include phenyl,
cycloalkane and cyclohexene rings attached via a variety of linkers
at C8 of caffeine. In addition, two alkyloxy substituents (5k, 5l)
were also included. The most potent 8-aryloxycaffeine (5i) identi-
fied in this study (see Results section) was further investigated by
substitution with chlorine and bromine on the phenyl ring (5m, 5n)
and the measured MAO-A and -B inhibition potencies were
compared to the corresponding halogen substituted 8-benzylox-
ycaffeines (5d, 5e).
N
N
N
N
O
O
N
O
N
8-Benzyloxycaffeine (2)
Caffeine (1)
H
CH3
O
NH2
N
F
H
O
Safinamide (3)
CHO
Cl
O
O
O
7-(3-Chlorobenzyloxy)-4-formylcoumarin (4)
2. Results
OH
2.1. Chemistry
The 8-aryl- and alkyloxycaffeine analogues (5a, b, fen) that
were investigated as potential MAO inhibitors were synthesized
according to the general method previously described for the
trans,trans-Farnesol (7)
Fig. 1. The structures of known MAO inhibitors reported in this study.

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B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
Table 1
2.2. Enzymology
The structures of the C8 aryl- and alkyloxy substituted caffeine analogues (5aen)
that were synthesized and investigated in this study.
For the purpose of determining the MAO inhibition potencies of
the study compounds, recombinant human MAO-A and -B were
employed. The activities of both MAO isoforms were measured by
using kynuramine as substrate [32]. Kynuramine, a mixed MAO-A/B
substrate, is non-fluorescent and is oxidized by MAO to yield
4-hydroxyquinoline. The rates of MAO catalysis can be conveniently
determined by measuring the formation of 4-hydroxyquinoline via
fluorescence spectrophotometry at excitation and emission wave-
lengths of 310 nm and 400 nm, respectively. None of the inhibitors
investigated in this study fluoresced at these excitation/emission
wavelengths or quenched the fluorescence of 4-hydroxyquinoline
at the inhibitor concentrations used. The experimentally deter-
mined IC50 values (Fig. 2) for the inhibition of the MAO isoforms
were converted to the corresponding enzymeeinhibitor dissocia-
tion constants (Ki values) using the ChengePrusoff equation
[26,33]. For this purpose Km values for the oxidation of kynuramine
by MAO-A and -B were used from previous studies. These are
O
N
N
O
N
R
O
N
-R
-R
5a
5b
5c
5d
5e
5h
5i
O
16.1 mM for human MAO-A and 22.7 mM for human MAO-B [27].
These Ki values enabled the calculation of the MAO-A/B selectivity
ratios [SI ¼ Ki(MAO-B)/Ki(MAO-A)].
As shown in Table 2, all of the 8-aryl- and alkyloxycaffeine
analogues (5aen) were found to be inhibitors of both recombinant
human MAO-A and -B. In general, the analogues exhibited rela-
tively weak inhibition potencies towards MAO-A compared to
MAO-B. In fact, with the exception of 5h (SI ¼ 1.46) which displayed
a slight selectivity for MAO-A, all of the analogues evaluated were
selective inhibitors of MAO-B (Table 2). Compound 5l was essen-
tially non-selective (SI ¼ 1.08). Interestingly, the most potent MAO-
A inhibitors were those containing halogen substitution on the
phenyl ring of the C8 substituent. For example, the most potent
MAO-A inhibitors of the present series are the halogen (chlorine
and bromine) substituted homologues, 5d and 5e, with IC50 values
5j
O
Cl
Br
5k
5l
of 1.34 mM and 1.30 mM, respectively. It can therefore be concluded
that halogen substitution on the phenyl ring of the C8 substituent is
a general strategy to enhance MAO-A binding affinity of caffeine
derived inhibitors.
In contrast to their inhibition potencies towards MAO-A, the
caffeine analogues 5aen were relatively potent MAO-B inhibitors.
Among the most potent MAO-B inhibitors was the phenylpropoxy
substituted caffeine analogue 5a with an IC50 value of 0.615 mM.
O
O
Cl
Br
5f
5m
Interestingly, a reduction of the length of the substituent at C8 of
caffeine is associated with a reduction of affinity for MAO-B. For
example, both the benzyloxy (2) and phenylethoxy (5b) substituted
caffeines are weaker MAO-B inhibitors than 5a with IC50 values of
1.77
analogue containing the shortest C8 substituent, the phenoxy
substituent, was particularly weak as MAO-B inhibitor
M). In agreement with these findings, the results also
mM [27] and 2.94 mM, respectively. The 8-aryloxycaffeine
5g
5n
a
(IC50 ¼ 10.7
m
document that compound 5i, with a C8 substituent (phenox-
yethoxy) that is approximately equal in length to that of 5a, is
synthesis of C8 oxy substituted caffeine analogues [27,29]. As
shown in Scheme 1 the target caffeine analogues were obtained by
reacting 8-chlorocaffeine (6) with the appropriately substituted
alcohol at high temperatures (150 ꢀC) in the presence of metallic
sodium. After recrystallization from ethanol, the desired products
(5a, b, fen) were obtained in low to moderate yields of 7e40%. For
the synthesis of 8-aryloxycaffeine analogues (5cee) the reaction
between the alcohol and 8-chlorocaffeine were carried out in the
presence of potassium hydroxide [30]. 8-Chlorocaffeine was
conveniently obtained in high yield by reacting chlorine with
caffeine in chloroform [31]. The structures of all the compounds
were verified by mass spectrometry, 1H NMR and 13C NMR while
the purities were estimated by HPLC.
another potent MAO-B inhibitor with an IC50 value of 0.383 mM. A
further increase in the length of the C8 substituent yields
compounds with relatively lower MAO-B inhibition potencies. For
example, 5j with a benzyloxyethoxy side chain exhibits a reduction
in MAO-B inhibition potency with an IC50 value of 3.77
mM,
approximately 9 fold less potent than 5i.
Replacement of the benzyloxy phenyl ring of 2 with cyclo-
hexenyl (5f), cyclohexyl (5g) and cyclopentyl (5h) rings also yielded
interesting results. Compared to 2 (IC50 ¼ 1.77
cyclohexylmethoxy substituent (5g) slightly improved MAO-B
inhibition potency (IC50 1.37 M) while the cyclo-
hexenemethoxy substituent (5f) slightly reduced affinity for MAO-
B (IC50 ¼ 2.99 M). The cyclopentylmethoxy substituent of caffeine
mM) [27], the
¼
m
m

B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
3477
endowed with potent MAO-B inhibitory properties. Also of note is
the observation that compound 2 has a markedly lower IC50 value
O
O
N
N
N
N
(IC50 ¼ 1.24
mM) [27] for the inhibition of MAO-A than do
N
N
i
compounds 5feh. This suggests that the phenyl ring is more
optimal for MAO-A inhibition than the cyclohexenyl (5f), cyclo-
hexyl (5g) and cyclopentyl (5h) rings.
Cl
O
N
O
O
N
Interestingly, the results also document that the branched-chain
8-alkyloxycaffeine analogues 5k and 5l are also MAO inhibitors. In
fact, 5k containing a 5-methylhexyloxy substituent at C8 of the
caffeine ring proved to be avery potent and selective MAO-B inhibitor
1
6
O
with an IC50 value of 0.381 mM. Since the caffeine ring is thought to
N
N
bind within the substrate cavity of MAO-B and the C8 side chain
within the entrance cavity [27], this observation suggests that a ring
system is not a requirement for interactionwith the entrance cavityof
the enzyme. This finding is in agreement with literature reports of the
aliphatic alcohol, trans, trans-farnesol (7), acting as a selective MAO-B
inhibitor (Fig.1) [34]. Trans, trans-farnesol also exhibits a dual binding
mode to the active site of MAO-B with the alcohol binding in close
proximity to the FAD cofactor in the substrate cavity while the
aliphatic chain extends towards the entrance cavity [34].
N
ii
ROH
R
O
N
5a-n
Scheme 1. Synthetic pathway to the C8 oxy substituted caffeine analogues 5aen.
Reagents and conditions: (i) Cl2, CHCl3, (ii) Na or KOH, 150 ꢀC.
5h was found to be the least favourable for MAO-B inhibition with
5h being the weakest MAO-B inhibitor of the present series. These
results indicate that replacement of the benzyloxy phenyl ring at C8
of caffeine with a cyclohexyl ring, may also lead to compounds
In the present study, compounds 5m and 5n, the caffeine
analogues containing halogens on the phenyl ring of the C8
substituent, were also found to be exceptionally potent inhibitors of
MAO-B. These inhibitors, substituted with chlorine and bromine,
Panel A
30
20
10
-3
-2
-1
0
1
2
3
Log[5m]
6
Panel B
4
2
-3
-2
-1
0
1
2
3
Log[5m]
Fig. 2. The recombinant human MAO-A (Panel A) and MAO-B (Panel B) catalyzed oxidation of kynuramine in the presence of various concentrations of inhibitor 5m. The
sigmoidal doseeresponse curves are constructed from the initial rates of oxidation of kynuramine (expressed as nmoles 4-hydroxyquinoline formed/min/mg protein) versus the
logarithm of concentration of inhibitor 5m (expressed in nM). The concentrations of kynuramine used were 45 and 30 mM for the studies with MAO-A and MAO-B, respectively.

3478
B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
Table 2
The IC50 and calculated Ki values for the inhibition of human MAO-A and -B by the C8 aryl- and alkyloxy substituted caffeine analogues 5aen.
O
N
N
O
N
R
O
N
Compd.
R
IC50 MAO-A (human)
m
M
IC50 MAO-B (human)
m
M
Ki MAO-A (human)
m
Ma
Ki MAO-B (human)
m
Ma
SIb
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
C6H5(CH2)3e
C6H5CH2CH2e
C6H5e
4-ClC6H4CH2e
4-BrC6H4CH2e
C6H9CH2e
69.335 ꢂ 1.69
15.925 ꢂ 2.906
75.19 ꢂ 7.849
1.337 ꢂ 0.052
1.304 ꢂ 0.034
13.755 ꢂ 1.605
18.415 ꢂ 3.175
22.81 ꢂ 1.103
20.35 ꢂ 16.476
167.75 ꢂ 13.223
15.17 ꢂ 0.325
27.34 ꢂ 2.079
1.83 ꢂ 0.013
0.615 ꢂ 0.082
2.943 ꢂ 0.281
10.705 ꢂ 0.983
0.065 ꢂ 0.001
0.062 ꢂ 0.005
2.99 ꢂ 0.17
24.27
5.57
26.32
0.47
0.46
4.81
6.45
7.98
77.12
58.71
5.31
9.57
0.64
0.58
0.45
2.16
7.84
0.048
0.045
2.19
1.00
11.65
0.28
2.76
0.28
0.019
0.39
0.30
0.10
0.099
0.45
C6H11CH2e
C5H9CH2e
1.365 ꢂ 0.057
15.915 ꢂ 3.401
0.383 ꢂ 0.021
3.772 ꢂ 0.215
0.381 ꢂ 0.082
14.13 ꢂ 2.079
0.183 ꢂ 0.005
0.166 ꢂ 0.003
0.16
1.46
C6H5OCH2CH2e
C6H5CH2OCH2CH2e
(CH3)2CH(CH2)4e
(CH3)2CH(CH2)2e
4-ClC6H4OCH2CH2e
4-BrC6H4OCH2CH2e
0.0036
0.047
0.053
1.08
0.203
0.207
10.35
0.13
0.12
1.65 ꢂ 0.087
All values are expressed as the mean ꢂ SD of duplicate determinations.
a
The Ki values were calculated from the experimental IC50 values according to the equation by Cheng and Prusoff: Ki ¼ IC50/(1 þ [S]/Km) with [S] ¼ 30
(kynuramine) ¼ 22.7 M for human MAO-B while [S] ¼ 45 M and Km (kynuramine) ¼ 16.1 M for human MAO-A [33].
The selectivity index is the selectivity for the A isoform and is given as the ratio of Ki(MAO-B)/Ki(MAO-A).
m
M and Km
m
m
m
b
exhibited IC50 values towards MAO-B of 0.183
m
M and 0.166
m
M,
To provide further evidence for the reversibility of inhibition of
MAO-A and -B by the oxycaffeine analogues examined in this study,
sets of LineweavereBurk plots were constructed for the inhibition
of both enzymes by 5n, the selected representative inhibitor
(Fig. 4A and B). Since for both the inhibition of both MAO-A and -B,
the LineweavereBurk plots are linear and intersect at the y-axis, it
may be concluded that 5n interacts competitively and therefore
reversibly with both enzymes. From the replot of the slopes of the
LineweavereBurk plots versus the inhibitor concentrations, Ki
respectively. This result demonstrates that, analogous to the
8-benzyloxycaffeines studied previously [27], halogen substitution
on the phenyl ring significantly enhances MAO-B binding affinity.
The same result was obtained in the present study with 8-benzy-
loxycaffeines, 5d and 5e, exhibiting IC50 values of 0.065
mM
and 0.062 M, respectively. These compounds are approximately
m
27 fold more potent as MAO-B inhibitors than the parent
unsubstituted 8-benzyloxycaffeine (2).
values of 1.18 mM and 0.088 mM were calculated for the inhibition of
MAO-A and -B, respectively, by 5n.
2.3. Reversibility studies
2.4. Molecular modelling
As discussed in the Introduction, reversible inhibition of MAO has
certain advantages over irreversible inactivation of the MAO
isozymes. One goal of this study is therefore to identify new oxy-
caffeine analogues that interact reversibly and with high affinity with
MAO-A and/or -B. To investigate the reversibility of the interaction
between the oxycaffeine analogues and MAO-A and -B, the time-
dependence of inhibition by one representative inhibitor,
compound 5n, was evaluated. The selection of compound 5n as
representative inhibitor is based on the observation that it acts as
a potent inhibitor of both MAO-A and -B. For this purpose, 5n was
preincubated with recombinant human MAO-A or-B for periods of 0,
15, 30 and 60 min and the residual rates by which MAO-A and -B
catalyze the oxidation of kynuramine were measured. The concen-
trations of 5n that were selected for evaluating the time-dependence
The results of this study demonstrate that 8-aryl- and alkylox-
ycaffeine analogues (5aen) are reversible inhibitors of both
recombinant human MAO-A and -B and the analogues are in
general selective for the MAO-B isoform. Interestingly, different oxy
substituents at C8 of caffeine lead to relatively large differences in
MAO inhibition potencies. For example the IC50 values for the
inhibition of MAO-A ranged from 1.3 to 167
mM while the IC50
values for the inhibition of MAO-B ranged from 0.062 to 15.9
mM.
The MAO inhibition potencies appear to be dependent upon the
length of the substituent at C8 of caffeine and the presence of
halogens on the phenyl ring of the C8 substituent. To provide
additional insight, the binding modes of selected 8-aryloxycaffeine
analogues (5a and 5i) in the active site cavities of MAO-A and -B
were examined using molecular docking. These analogues were
selected since they were found to be potent inhibitors of MAO-B.
For the purpose of the docking experiments, the crystallo-
graphic structures of human MAO-A in complex with harmine (PDB
entry: 2Z5X) [37] and human MAO-B in complex with safinamide
(PDB entry: 2V5Z) [28] were selected. These protein models were
selected based on the high resolution of the crystallographic
structures and the observation that, in the model of MAO-B, the
side chain of Ile-199 occupies an alternate conformation which
permits for the fusion of the substrate and entrance cavities. This is
a requirement for the binding of relatively large inhibitors which
extend from the substrate cavity into the entrance cavity [34]. The
preparation of the protein models and the docking experiments
of inhibitionof MAO-Aand-Bwere3.29mM and0.33mM,respectively.
These concentrations are approximately 2 fold the measured IC50
values for the inhibition of the respective enzymes by 5n.
The results of the reversibility studies are shown in Fig. 3A and B.
The graphs indicate that the rates of MAO-A and -B catalyzed
oxidation of kynuramine do not decrease in a time-dependent
manner when 5n and the respective enzyme preparations are pre-
incubated for various periods of time. These results demonstrate
that 5n interacts reversibly with both MAO-A and -B, at least at the
inhibitor concentrations (2 ꢁ IC50) and time period (60 min) used for
these studies. This finding is in agreement with literature reports
that the interactions between the MAO isozymes and C8 substituted
caffeines are in general reversible [24,27,35,36].

B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
3479
Panel A
Panel A
0.45
A
6
5
4
3
2
1
0
0.30
0.15
0.00
-0.02
0.00
0.02
1/[S]
0.04
0.06
0
15
30
60
Incubation time (min)
0.8
0.4
0.0
B 2.5
Panel B
Panel B
2.0
1.5
1.0
0.5
0.0
-0.02
0.00
0.02
1/[S]
0.04
0.06
0
15
30
60
Incubation time (min)
Fig. 4. LineweavereBurk plots of the oxidation of kynuramine by recombinant human
MAO-A (Panel A) and -B (Panel B) in the absence (filled squares) and presence of
various concentrations of 5n. For the studies with MAO-A (Panel A) the concentrations
Fig. 3. Time-dependence of the inhibition of the oxidation of kynuramine by
recombinant human MAO-A (Panel A) and -B (Panel B). MAO-A and -B were pre-
incubated for various periods of time (0e60 min) with 5n at concentrations of 3.29
and 0.33 M, respectively, and the residual catalytic rates were determined. The rates
are expressed as nmoles 4-hydroxyquinoline formed/min/mg protein and the catalytic
rates recorded in the absence of the inhibitors are 9.19 ꢂ 0.31 and 6.38 ꢂ 0.37 nmol/
min/mg for MAO-A and -B, respectively.
of 5n were: 0.4125
circles). For the studies with MAO-B (Panel B) the concentrations of 5n were:
0.0425 M (open squares), 0.085 M (filled circles) and 0.17 M (open circles). The
mM (open squares), 0.825 mM (filled circles) and 1.65 mM (open
mM
m
m
m
m
rates (V) are expressed as nmol product formed/min/mg protein.
The predicted binding orientations of compounds 5a and 5i in
the active site of MAO-B are shown in Fig. 5AeB. In all instances, the
caffeine rings of the inhibitors bind within the substrate cavity of
the enzyme, just in front of the flavin ring and approximately
parallel the phenolic side chains of the Tyr-398 and Tyr-435. This
orientation is restricted by the flat shape of the substrate cavity of
MAO-B [40]. Interestingly, the N1 methyl carbons of the caffeine
rings of the inhibitors are positioned relatively closely to the FAD
cofactor, approximately 3.6e4.1 Å from the flavin N5. This binding
orientation brings the carbonyl oxygen at C6 of the caffeine ring
within hydrogen bond interaction distance to an active site water
molecule (HOH 1155) and the phenolic hydrogen of Tyr-435. A
possible hydrogen bond interaction may also exist between the
carbonyl oxygen at C2 of the caffeine ring and HOH 1346. Another
productive interaction between the caffeine ring and the MAO-B
were carried out according to a previously reported protocol with
the Discovery Studio 1.7 modelling software [27,38]. After the
valences of the FAD cofactor and the co-crystallised ligands were
corrected, hydrogen atoms were added to the MAO-A and -B
models and a three-step energy minimization procedure of the
protein models was carried out (see Experimental). For this
purpose, the backbones of the proteins were kept constrained. The
co-crystallized ligands were subsequently extracted from the
models and the binding cavities of the proteins were determined by
the flood-filling algorithm. All crystal water molecules were
removed except for those in the MAO-A and -B active sites that are
considered conserved and non-displaceable (see Experimental).
The structures of the ligands to be docked (5a and 5i) were con-
structed and their geometries optimized within Discovery Studio
and the ligands were docked into the active sites of the models with
the LigandFit application within Discovery Studio. This protocol has
been shown to be suitable for examining the binding of inhibitors
to MAO-A and -B since redocking of harmine (RMSD ¼ 0.64 Å) and
safinamide (RMSD ¼ 1.54 Å) into the active sites of the two
enzymes, respectively, yielded binding orientations that exhibited
relatively small RMSD values from the position of the co-
crystallized ligands [39].
active site is a possible
pep interactions with the amide func-
tional group of the Gln-206 side chain with an interplane distance
of approximately 3.5 Å [37]. These binding orientations and inter-
actions are similar to those predicted by docking experiments
previously for the binding of 8-benzyloxycaffeine (2) to MAO-B
[27]. The results of the docking study also show that the C8 oxy
substituents of caffeine extend towards the entrance cavity of the
active site. The orientations of the C8 substituents of 5a and 5i

3480
B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
most part, account for the potent MAO-B inhibition properties of
compounds 5aen compared to caffeine.
The predicted binding orientations of compound 5a in the active
site of MAO-A (Fig. 6) show that the caffeine rings adopt similar
binding orientations to that observed in MAO-B, with the carbonyl
oxygens at C2 of the caffeine rings located approximately 5.0e5.5 Å
from the flavin N5. Only one potential hydrogen bond interaction
between the inhibitors and the MAO-A active site is observed e
between the carbonyl oxygen at C2 of the caffeine ring and the
phenolic hydrogen of Tyr-407. A possible pep interaction between
the amide functional group in the side chain of Gln-215 and the
caffeine ring may represent another stabilizing factor (interplane
distance, 3.5 Å) [37]. Similar to MAO-B, the C8 oxy substituent
extends towards the entrance of the active site cavity. Also note-
worthy is the observation that, in the MAO-A active site, the C8 oxy
side chain of 5a is bent by a larger extent from the plane of the
caffeine ring compared the corresponding orientations that are
adopted in the MAO-B active site (Fig. 7). A possible reason for this
is that the caffeine rings bind comparatively distant from the FAD
cofactor in the MAO-A active site than in the MAO-B active site. This
reduces the space available for relatively long C8 side chains to bind
within MAO-A and as a result, in order for the inhibitor to be
accommodated within the active site, these side chains exist in
a bent conformation relative to the plane of the caffeine ring.
3. Discussion
In the present study, a series of C8 oxy substituted caffeine
analogues 5aen were synthesized and evaluated as inhibitors of
Fig. 5. Predicted binding modes of 5a (Panel A) and 5i (Panel B) within the active site
of MAO-B (2V5Z.pdb). The inhibitors are shown in yellow, the FAD cofactor in magenta
and the active site amino acid residues in grey. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
within the entrance cavity are very similar. Since the entrance
cavity is lined by the side chains of hydrophobic amino acid resi-
dues (Phe-103, Trp-119, Leu-164, Leu-167, Phe-168 and Ile-316), the
C8 substituents are stabilized principally by Van der Waals inter-
actions within the entrance cavity [32]. As discussed in the Intro-
duction, the interaction of C8 substituted caffeine derivatives with
both the substrate and entrance cavities of MAO-B may, for the
Fig. 6. Predicted binding mode of 5a within the active site of MAO-A (2Z5X.pdb). The
inhibitor is shown in cyan, the FAD cofactor in magenta and the active site amino acid
residues in grey. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
3481
both the MAO-A and -B binding affinities of 8-benzyloxycaffeins as
shown with compounds 5d and 5e. Literature reports that halogen
substitution also enhances the MAO-B inhibition potencies of (E)-
8-styrylcaffeiens [36] and (E)-2-styryl-1-methylbenzimidazoles
[35]. A possible explanation may be that halogens may facilitate
charge transfer or dipole interactions in the entrance cavity in
addition to enhancing lipophilic interactions between the side
chain and the hydrophobic environment of the entrance cavity [27].
In agreement with this view, literature reports that the MAO-B
inhibition potencies of caffeinyl derived inhibitors frequently
correlate with electronic properties as well as the lipophilicities
of the substituents attached the phenyl ring of the C8 side chain
[27,36].
Interestingly, the caffeine analogue containing
a cyclo-
hexylmethoxy substituent (5g) was found to be slightly more
potent as an MAO-B inhibitor than 8-benzyloxycaffeine (2) [27].
This indicates that the cyclohexyl and phenyl rings both form
productive interactions with the entrance cavity of the enzyme that
are approximately equal in strength. Substitution at C8 with side
chains containing these two rings therefore enhances the MAO-B
inhibition potency of caffeine to a similar extent. Another note-
worthy observation is the finding that the branched-chain 8-alky-
loxycaffeine analogue 5k acts as a potent MAO-B inhibitor. Since
the (methylhexyl)oxy side chain of 5k would interact via hydro-
phobic burial with the residues of the entrance cavity, this finding
demonstrates the importance of hydrophobic interactions between
an inhibitor and the entrance cavity of MAO-B for the stabilization
of the enzymeeinhibitor complex.
In conclusion, compounds 5a and 5i, which contain phenyl-
propoxy and phenoxyethoxy substituents at C8 of the caffeine ring
may be viewed as promising lead compounds for the development
of reversible MAO-B inhibitors. Both these compound exhibit
improved MAO-B inhibition potencies compared to 8-benzylox-
ycaffeine which may be further improved via appropriate halogen
substitution at the C8 substituent phenyl ring. This study demon-
strates the importance of the length of the C8 side chain for the
inhibition of MAO-B by caffeine analogues.
Fig. 7. The predicted binding orientations of 5a within the active sites of MAO-A (cyan)
and MAO-B (yellow). This illustration was generated by superimposing the caffeine
rings of the respective orientations. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
recombinant human MAO-A and -B. The results demonstrate that,
although the analogues inhibit both isozymes, they exhibit greater
binding affinities for MAO-B. Molecular docking studies suggest
that, in the MAO-B active site, the caffeine rings bind in closer
proximity to the flavin cofactor compared to the binding orienta-
tion adopted in the MAO-A active site. This enables the caffeine ring
to interact with MAO-B via 3 possible hydrogen bonds (2 to integral
water molecules and 1 to Tyr-435) while only 1 hydrogen bond
(with Tyr-407) is formed between the caffeine ring and MAO-A.
These observations may explain, in part, the MAO-B selective
action of most of the oxy caffeines examined here. The C8
substituents may also play a role in determining the selectivity of
caffeine analogues. For example, the previously studied 8-benzy-
loxycaffeine [27] exhibit little MAO-A/B selectivity. The molecular
basis for this behaviour, however, remains unclear. One possible
reason may be that the relatively shorter C8 benzyloxy side chain
may be better suited for binding to MAO-A than MAO-B. Also, by
examining the interactions of one representative inhibitor (5e)
with the enzymes it was concluded that the inhibition is reversible
and competitive. This finding is in agreement with a previous
report that 8-benzyloxyacffeine analogues also are competitive
reversible inhibitors of human MAO-A and -B [27].
Another interesting observation of the present study was the
finding that among the aryloxy substituted caffeine analogues,
compounds 5a and 5i, were particularly potent MAO-B inhibitors.
The compounds contain phenylpropoxy and phenoxyethoxy C8
substituents, respectively, which are approximately equal in length.
Analogues containing the shorter benzyloxy (2) [27] and phenyl-
ethoxy (5b) substituents as well as longer substituents such as the
benzyloxyethoxy side chain (5j) proved to be weaker MAO-B
inhibitors. These observations suggest that for the aryloxy
substituted caffeiens, a linker consisting of 4 atoms separating the
caffeine and the phenyl ring may be optimal for MAO-B inhibition.
This linker length would allow for the best placement of the phenyl
ring in the entrance cavity of the enzyme and hence the most
productive interactions with the hydrophobic residues lining this
space.
4. Experimental
4.1. Chemicals and instrumentation
All starting materials, unless noted elsewhere, were obtained
from SigmaeAldrich and were used without purification. Proton
(1H) and carbon (13C) NMR spectra were recorded in CDCl3 on
a Bruker Avance III 600 spectrometer at frequencies of 600 MHz
and 150 MHz, respectively. Chemical shifts are reported in parts per
million (d) downfield from the signal of tertamethylsilane added to
the CDCl3 (Merck). Spin multiplicities are given as s (singlet),
d (doublet), t (triplet), q (quartet), quin (quintet), sept (septet) or m
(multiplet). Direct insertion electron impact ionization (EIMS) and
high resolution mass spectra (HRMS) were obtained on a DFS high
resolution magnetic sector mass spectrometer (Thermo Electron
Corporation). Melting points (mp) were determined on a Stuart
SMP10 melting point apparatus and are uncorrected. The purities of
the synthesized compounds were estimated by HPLC analyses
which were carried out with an Agilent 1100 HPLC system equip-
ped with a quaternary pump and an Agilent 1100 series diode array
detector (see Supplementary material). For this purpose, HPLC
grade acetonitrile (Merck) and Milli-Q water (Millipore) was used
for the chromatography. Fluorescence spectrophotometry was
conducted with a Varian Cary Eclipse fluorescence spectropho-
tometer. Microsomes from insect cells containing recombinant
MAO-A and -B (5 mg/mL) and kynuramine$2HBr that were
required for the enzymatic reactions were obtained from
Caffeine analogues 5m and 5n, containing halogens on the
phenyl ring of the C8 substituent, were also found to be remarkably
potent inhibitors of MAO-B. Halogen substitution appears to be
a general strategy to improve the inhibition potencies of candidate
MAO-A and -B inhibitors since substitution with halogens enhances

3482
B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
SigmaeAldrich. 8-Chlorocaffeine (6) was prepared according to
a previously reported procedure [31] by reaction of caffeine with
chlorine in chloroform. The melting point (188 ꢀC) as well as the
NMR data correlated to the corresponding published values [41,42].
4.2.5. 8-(4-Bromobenzyloxy)caffeine (5e)
The title compound was prepared from 8-chlorocaffeine (6) and
4-bromobenzyl alcohol in a yield of 17%: mp 179 ꢀC (ethanol). 1H
NMR (Bruker Avance III 600, CDCl3)
(s, 3H), 5.45 (s, 2H), 7.36 (d, 2H, J ¼ 7.9 Hz), 7.56 (d, 2H, J ¼ 7.9 Hz);
13C NMR (Bruker Avance III 600, CDCl3)
; 27.8, 29.8, 29.9, 71.7,
d 3.40 (s, 3H), 3.55 (s, 3H), 3.72
4.2. Procedures for the synthesis of the 8-aryl- and alkyloxycaffeine
d
analogues (5aen)
103.6, 123.1, 130.1, 131.9, 134.0, 146.1, 151.7, 154.9, 155.3; EIMS 378;
HRMS m/z: calcd for C15H15BrN4O3, 378.0328, found 378.0339;
Purity (HPLC): 96.3%.
For the synthesis of the target 8-aryl- and alkyloxycaffeine
analogues 5a, b, fen, a method described in literature was followed
with minor modifications [29]. Metallic sodium (1.5 mmol) and an
appropriately substituted alcohol (21 mmol) were allowed to react
at room temperature. Following consumption of the sodium,
8-chlorocaffeine (6, 1.5 mmol) was added to the mixture and the
resulting reaction was stirred for 6 h at a temperature of 150 ꢀC. The
reaction was cooled to room temperature and the desired C8
substituted oxycaffeine analogues were recrystallized (at 4 ꢀC) after
the addition of ethanol (10e20 mL) to the reaction mixture.
For the synthesis of 8-aryloxycaffeine analogues (5cee) the
following literature method was followed [30]: Potassium
hydroxide (2 mmol) was dissolved in 1 mL water and the appro-
priately substituted alcohol (1.85 mmol) was added to yield
a solution. 8-Chlorocaffeine (6, 1.5 mmol) was added and the
resulting reaction mixture was stirred at 150 ꢀC for 6 h. The reac-
tion was cooled to room temperature and ethanol (10 mL) was
added. The resulting precipitate was recrystallized (at 4 ꢀC) from
ethanol.
4.2.6. 8-(Cyclohex-3-en-1-ylmethoxy)caffeine (5f)
The title compound was prepared from 8-chlorocaffeine (6) and
3-cyclohexen-1-ylmethanol in a yield of 18%: mp 170 ꢀC (ethanol).
1H NMR (Bruker Avance III 600, CDCl3)
d 1.39 (m, 1H), 1.84 (m, 2H),
2.09 (m, 4H), 3.35 (s, 3H), 3.48 (s, 3H), 3.67 (s, 3H), 4.33 (d, 2H,
J ¼ 6.4 Hz), 5.68 (m, 2H); 13C NMR (Bruker Avance III 600, CDCl3)
d;
24.3, 25.0, 27.7, 27.8, 29.7, 33.3, 75.2, 103.3, 125.2, 127.1, 146.3, 151.7,
154.8, 155.9; EIMS 304; HRMS m/z: calcd for C15H20N4O3, 304.1535,
found 304.1539; Purity (HPLC): 96.9%.
4.2.7. 8-Cyclohexylmethoxycaffeine (5g)
The title compound was prepared from 8-chlorocaffeine (6) and
cyclohexylmethanol in a yield of 24%: mp 120 ꢀC (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3)
d 1.03 (m, 2H), 1.17 (m, 1H), 1.25 (m,
2H), 1.79 (m, 6H), 3.33 (s, 3H), 3.46 (s, 3H), 3.65 (s, 3H), 4.22 (d, 2H,
J ¼ 6.0 Hz); 13C NMR (Bruker Avance III 600, CDCl3)
d; 25.5, 26.3,
27.6, 29.4, 29.6, 29.7, 37.3, 76.1, 103.3, 146.3, 151.7, 154.7, 156.0; EIMS
306; HRMS m/z: calcd for C15H22N4O3, 306.1692, found 306.1690;
Purity (HPLC):100%.
4.2.1. 8-(3-Phenylpropoxy)caffeine (5a)
The title compound was prepared from 8-chlorocaffeine (6) and
3-phenylpropanol in a yield of 26%: mp 109 ꢀC (ethanol). 1H NMR
4.2.8. 8-Cyclopentylmethoxycaffeine (5h)
(Bruker Avance III 600, CDCl3)
3.40 (s, 3H), 3.51 (s, 3H), 3.68 (s, 3H), 4.50 (t, 2H, J ¼ 6.4 Hz), 7.23 (m,
3H), 7.31 (m, 2H); 13C NMR (Bruker Avance III 600, CDCl3)
; 27.7,
d
2.18 (m, 2H), 2.81 (t, 2H, J ¼ 7.5 Hz),
The title compound was prepared from 8-chlorocaffeine (6) and
cyclopentylmethanol in a yield of 33%: mp 112 ꢀC (ethanol). 1H NMR
d
(Bruker Avance III 600, CDCl3) d 1.33 (m, 2H), 1.61 (m, 4H), 1.79 (m,
29.7, 30.3, 32.0, 70.4, 103.4, 126.2, 128.4, 128.5, 140.8, 146.3, 151.7,
154.8, 155.7; EIMS 328; HRMS m/z: calcd for C17H20N4O3, 328.1535,
found 328.1528; Purity (HPLC): 96.1%.
2H), 2.36 (sept, 1H, J ¼ 7.5 Hz), 3.36 (s, 3H), 3.48 (s, 3H), 3.66 (s, 3H),
4.31 (d, 2H, J ¼ 7.2 Hz); 13C NMR (Bruker Avance III 600, CDCl3)
d;
25.3, 27.7, 29.1, 29.6, 29.7, 38.7, 74.9, 103.3, 146.3, 151.7, 154.8, 155.9;
EIMS 292; HRMS m/z: calcd for C14H20N4O3, 292.1535, found
292.1538; Purity (HPLC): 100%.
4.2.2. 8-(3-Phenylethoxy)caffeine (5b)
The title compound was prepared from 8-chlorocaffeine (6) and
2-phenylethanol in a yield of 40%: mp 144 ꢀC (ethanol). 1H NMR
4.2.9. 8-(2-Phenoxyethoxy)caffeine (5i)
(Bruker Avance III 600, CDCl3)
3.52 (s, 3H), 3.66 (s, 3H), 4.68 (t, 2H, J ¼ 7.2 Hz), 7.28 (m, 3H), 7.34
(m, 2H); 13C NMR (Bruker Avance III 600, CDCl3)
; 27.7, 29.7, 35.4,
d
3.14 (t, 2H, J ¼ 7.2 Hz), 3.39 (s, 3H),
The title compound was prepared from 8-chlorocaffeine (6) and
2-phenoxyethanol in a yield of 37%: mp 175 ꢀC (ethanol). 1H NMR
d
(Bruker Avance III 600, CDCl3) d 3.36 (s, 3H), 3.48 (s, 3H), 3.66 (s,
71.3, 103.4, 126.8, 128.61, 128.9, 137.1, 146.3, 151.7, 154.8, 155.3; EIMS
314; HRMS m/z: calcd for C16H18N4O3, 314.1379, found 314.1374;
Purity (HPLC): 100%.
3H), 4.32 (t, 2H, 4.9 Hz), 4.78 (t, 2H, 4.9 Hz), 6.91 (d, 2H, J ¼ 7.5 Hz),
6.96 (t, 1H, J ¼ 7.5 Hz), 7.28 (t, 2H, J ¼ 7.5 Hz); 13C NMR (Bruker
Avance III 600, CDCl3) d; 27.7, 29.7, 29.8, 65.6, 69.3, 103.5, 114.6,
121.4, 129.5, 146.040, 151.6, 154.8, 155.3, 158.3; EIMS 330; HRMS m/
z: calcd for C16H18N4O4, 330.1328, found 330.1323; Purity (HPLC):
94.9%.
4.2.3. 8-Phenoxycaffeine (5c)
The title compound was prepared from 8-chlorocaffeine (6) and
phenol in a yield of 24%: mp 140 ꢀC (ethanol). 1H NMR (Bruker
Avance III 600, CDCl3)
d
3.41 (s, 3H), 3.46 (s, 3H), 3.89 (s, 3H), 7.32
4.2.10. 8-[2-(Benzyloxy)ethoxy]caffeine (5j)
(m, 3H), 7.44 (m, 2H); 13C NMR (Bruker Avance III 600, CDCl3)
d;
The title compound was prepared from 8-chlorocaffeine (6) and
27.8, 29.9, 30.4, 103.8, 119.4, 125.7, 129.8, 145.9, 151.6, 153.4, 153.5,
154.9; EIMS 286; HRMS m/z: calcd for C14H14N4O3, 286.1066, found
286.1067; Purity (HPLC): 92.9%.
2-(benzyloxy)ethanol in a yield of 7%: mp 90 ꢀC (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3)
3H), 3.81 (t, 2H, J ¼ 4.5 Hz), 4.58 (s, 2H), 4.61 (t, 2H, J ¼ 4.5 Hz), 7.27
(m, 1H), 7.32 (m, 4H); 13C NMR (Bruker Avance III 600, CDCl3)
d 3.36 (s, 3H), 3.47 (s, 3H), 3.68 (s,
d
;
4.2.4. 8-(4-Chlorobenzyloxy)caffeine (5d)
27.7, 29.7, 29.9, 67.7, 70.1, 73.2, 103.487, 127.7, 127.9, 128.5, 137.6,
146.1, 151.7, 154.8, 155.5; EIMS 344; HRMS m/z: calcd for
C17H20N4O4, 344.1485, found 344.1482; Purity (HPLC): 94.7%.
The title compound was prepared from 8-chlorocaffeine (6) and
4-chlorobenzyl alcohol in a yield of 32%: mp 163 ꢀC (ethanol). 1H
NMR (Bruker Avance III 600, CDCl3)
(s, 3H), 5.46 (s, 2H), 7.40 (m, 4H); 13C NMR (Bruker Avance III 600,
CDCl3) ; 27.7, 29.7, 29.8, 71.7, 103.6, 129.0, 129.9, 133.5, 134.9, 146.1,
d 3.39 (s, 3H), 3.54 (s, 3H), 3.71
4.2.11. 8-[(5-Methylhexyl)oxy]caffeine (5k)
d
The title compound was prepared from 8-chlorocaffeine (6) and
151.7, 154.8, 155.3; EIMS 334; HRMS m/z: calcd for C15H15ClN4O3,
5-methyl-1-hexanol in a yield of 10%: mp 79 ꢀC (ethanol). 1H NMR
334.0833, found 334.0833; Purity (HPLC): 96.9%.
(Bruker Avance III 600, CDCl3)
d
0.87 (d, 6H, J ¼ 6.8 Hz),1.22 (m, 2H),

B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
3483
1.41 (m, 2H), 1.54 (sept, 1H, J ¼ 6.8 Hz), 1.77 (quin, 2H, J ¼ 7.5 Hz),
3.36 (s, 3H), 3.49 (s, 3H), 3.66 (s, 3H), 4.43 (t, 2H, J ¼ 6.8 Hz); 13C
different inhibitor concentrations spanning at least 3 orders of
a magnitude were used. To calculate the IC50 values, these data
were fitted to the one site competition model incorporated into the
Prism software package (GraphPad). All experiments were carried
out in duplicate and the IC50 values are expressed as
mean ꢂ standard deviation (SD) [27]. The IC50 values were con-
verted to the corresponding Ki values according to the equation by
Cheng and Prusoff: Ki ¼ IC50/(1 þ [S]/Km) [33].
NMR (Bruker Avance III 600, CDCl3) d; 22.5, 23.4, 27.7, 27.8, 29.1,
29.7, 38.4, 71.2, 103.3, 146.4, 151.7, 154.8, 155.9; EIMS 308; HRMS m/
z: calcd for C15H24N4O3, 308.1848, found 308.1855; Purity (HPLC):
100%.
4.2.12. 8-(3-Methylbutoxy)caffeine (5l)
The title compound was prepared from 8-chlorocaffeine (6) and
3-methyl-1-butanol in a yield of 37%: mp 130 ꢀC (ethanol). 1H NMR
4.4. Time-dependence of inhibition
(Bruker Avance III 600, CDCl3)
d
0.96 (d, 6H, J ¼ 6.4 Hz), 1.68 (q, 2H,
J ¼ 6.8 Hz), 1.77 (sept, 1H, J ¼ 6.8 Hz), 3.36 (s, 3H), 3.49 (s, 3H), 3.66
To determine whether the 8-aryl- and alkyloxycaffeine
analogues act as reversible inhibitors or inactivators of MAO-A and
-B, the time-dependence of inhibition was investigated over
a period of 60 min. For this purpose, compound 5n was selected as
a representative inhibitor of the series. Compound 5n was pre-
incubated for periods of 0, 15, 30, 60 min at 37 ꢀC with recombinant
human MAO-A or human MAO-B (0.03 mg/mL) in potassium
phosphate buffer (100 mM, pH 7.4, made isotonic with KCl). The
(s, 3H), 4.46 (t, 2H, J ¼ 6.8 Hz); 13C NMR (Bruker Avance III 600,
CDCl3) d; 22.5, 24.9, 27.7, 29.7, 29.7, 37.6, 69.8, 103.3, 146.3, 151.7,
154.8, 155.9; EIMS 280; HRMS m/z: calcd for C13H20N4O3, 280.1535,
found 280.1537; Purity (HPLC): 100%.
4.2.13. 8-[2-(4-Chlorophenoxy)ethoxy]caffeine (5m)
The title compound was prepared from 8-chlorocaffeine (6) and
2-(4-chlorophenoxy)ethanol in a yield of 7.5%: mp 157 ꢀC (ethanol).
concentrations of 5n employed for these reactions were 3.29
mM
1H NMR (Bruker Avance III 600, CDCl3)
d
3.35 (s, 3H), 3.48 (s, 3H),
and 0.33 M for the incubations with MAO-A and -B, respectively,
m
3.66 (s, 3H), 4.29 (t, 2H, J ¼ 4.52 Hz), 4.77 (t, 2H, J ¼ 4.52 Hz), 6.83 (d,
and are approximately 2 fold the measured IC50 values for the
inhibition of the respective MAO preparations. These preincubated
enzyme preparations were then diluted 2 fold with the addition of
2H, J ¼ 9.04 Hz), 7.22 (d, 2H, J ¼ 9.04 Hz); 13C NMR (Bruker Avance
III 600, CDCl3) d; 27.71, 29.70, 29.86, 66.05, 69.03, 103.57, 115.85,
126.28, 129.41, 146.00, 151.6, 154.76, 155.18, 156.89; Purity (HPLC):
92.1%.
kynuramine, at final concentrations of 45 mM and 30 mM for the
reactions containing MAO-A and -B, respectively, and the resulting
reactions were incubated at 37 ꢀC for 15 min. The final volumes of
4.2.14. 8-[2-(4-Bromophenoxy)ethoxy]caffeine (5n)
these incubations were 500
preparations were 0.015 mg/mL and the final concentrations of 5n
were 1.65 M and 0.165 M for MAO-A and MAO-B, respectively.
These concentrations of the inhibitor are approximately equal to
the IC50 values for the inhibition of the respective enzyme prepa-
mL, the final concentrations of the MAO
The title compound was prepared from 8-chlorocaffeine (6) and
2-(4-Bromophenoxy)ethanol in
a
yield of 9.9%: mp 165 ꢀC
m
m
(ethanol). 1H NMR (Bruker Avance III 600, CDCl3)
d
3.35 (s, 3H), 3.48
(s, 3H), 3.67 (s, 3H), 4.29 (t, 2H, J ¼ 4.52 Hz), 4.77 (t, 2H, J ¼ 4.52 Hz),
6.79 (d, 2H, J ¼ 9.04 Hz), 7.36 (d, 2H, J ¼ 9.04 Hz); 13C NMR (Bruker
rations by 5n. The reactions were terminated with 400
mL NaOH
Avance III 600, CDCl3)
d; 27.74, 29.73, 29.89, 65.99, 69.02, 103.61,
(2 N) and 1000 L distilled water was added to each reaction. The
m
113.60, 116.38, 132.38, 146.03, 151.64, 154.80, 155.21, 157.42; EIMS
408; HRMS m/z: calcd for C16H17BrN4O4, 408.0433, found 408.0440;
Purity (HPLC): 100%.
rates of the MAO-catalyzed production of 4-hydroxyquinoline were
measured as described above. All measurements were carried out
in triplicate and are expressed as mean ꢂ SD [27,43,44].
4.3. IC50 determination for the inhibition of human MAO-A and -B
4.5. Mode of inhibition
Microsomes from baculovirus infected insect cells expressing
recombinant human MAO-A or -B (5 mg/mL) were obtained from
SigmaeAldrich and were pre-aliquoted and stored at ꢃ70 ꢀC. All
enzymatic reactions were carried out in 2 mL microcentrifuge tubes
in potassium phosphate buffer (100 mM, pH 7.4) which were made
isotonic with KCl (20.2 mM). The final volumes of the reactions
To evaluate the mode of inhibition by the 8-aryl- and alkylox-
ycaffeine analogues, sets of LineweavereBurk plots for the inhibi-
tion of MAO-A and -B by compound 5n were constructed. The initial
rates of the MAO-catalyzed oxidation of kynuramine at four
different substrate concentrations (15e90 mM) in the absence and
presence of three different concentrations of 5n were measured.
The concentrations of 5n that were selected ranged from 0.4125 to
were 500
mL and contained MAO-A or MAO-B (0.0075 mg/mL) and
various concentrations of the test inhibitor (0e100
m
M). Stock
1.65 mM and 0.0425e0.17 mM for the studies with MAO-A and -B,
solutions of the test inhibitors were prepared in DMSO and added
to the reactions to yield a final concentration of 4% (v/v) DMSO.
Kynuramine served as enzyme substrate and were added to the
respectively. The enzymatic reactions and measurements were
carried out as described above for the determination of the IC50
values. The only exception was that the concentrations of MAO-A
and -B in the reactions were 0.015 mg/mL. Linear regression anal-
ysis was performed using the Prism software package [27].
reactions to yield final concentrations of 45 mM and 30 mM where
MAO-A and -B, respectively, were used as enzymes. The reactions
were incubated in a water bath at 37 ꢀC for 20 min and terminated
with the addition of 400
mL NaOH (2 N). Distilled water (1000
mL)
4.6. Molecular modelling studies
was subsequently added to each reaction, and the concentrations of
the MAO generated 4-hydroxyquinoline in the reactions were
measured by fluorescence spectrophotometry (lexcitation ¼ 310 nm,
lemission ¼ 400 nm) [32]. For this purpose, a linear calibration curve
was constructed from solutions of authentic 4-hydroxyquinoline
Molecular docking of selected inhibitors (5a and 5i) into the
active sites of MAO-A and -B were carried out in the Windows based
Discovery Studio 1.7 molecular modelling software [38]. The ligands
to be docked were constructed in Discovery Studio and the hydrogen
atoms were added according tothe appropriateprotonationstates at
pH 7.4. Since flexible ligand docking were employed, their geome-
tries required only brief optimization using a fast Dreiding-like
forcefield (1000 iterations) in Discovery Studio. The atom potential
types and partial charges were then automatically assigned with the
(0.047e1.5
mM) dissolved in potassium phosphate buffer (100 mM,
pH 7.4). To each standard 400
m
L NaOH (2 N) and 1000 L distilled
m
water was added. Sigmoidal doseeresponse curves were con-
structed by plotting the initial rates of kynuramine oxidation versus
the logarithm of the inhibitor concentration. For each curve, 9

3484
B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
Momany and Rone CHARMm forcefield. The X-ray crystallographic
structures of MAO-A in complex with harmine (PDB code: 2Z5X)
[37] and MAO-B in complex with safinamide (PDB code: 2V5Z) [28]
were obtained from the Brookhaven Protein Data Bank (www.rcsb.
org/pdb). Hydrogen atoms were added to the receptor models
according to the appropriate protonation states of the ionizable
amino acids at pH 7.4 and the valences of the FAD cofactors (oxidized
state) and co-crystallized ligands were corrected and hydrogen
atoms were added, also according to the appropriate protonation
states at pH 7.4. The resulting models were automatically typed with
the Momany and Rone CHARMm forcefield, the protein backbone
was constrained and the models were subjected to a three-step
energy minimization cascade. The first step was a steepest descent
minimization which was followed by conjugate gradient minimi-
zation. For both protocols the termination criterion was set to
a maximum of 2500 steps or a minimum value of 0.1 for the root
mean square of the energy gradient. The third step was an adopted
basis NewtoneRapheson minimization with the termination
criteria set to a maximum of 5000 steps or a minimum value for the
root mean square of the energy gradient of 0.01. For this minimi-
zation cascade the implicit generalized Born solvation model with
simple switching was used with the dielectric constant set to 4. The
co-crystallized ligands were subsequently deleted from the active
sites of the protein models, the backbone constraints were deleted
and the binding site was identified by a flood-filling algorithm. The
crystallized water molecules were removed with the exception of 4
active site water molecules. X-ray crystal structures of MAO-B have
shown that only three active site water molecules (HOH 1155, 1170
and 1351; A-chain) are conserved, all in the vicinity of the FAD
cofactor [28]. Preliminary docking studies in our laboratory suggest
that HOH 1346 (MAO-B, A-chain) may also be involved in stabilizing
caffeine analogues (by hydrogen bond interaction with the carbonyl
oxygen at C-6 of the caffeine ring) and were thus also retained. For
the docking studies with MAO-A, the crystal waters were also
deleted with the exception of HOH 710, 739 and 725 which occupies
the analogous positions in the MAO-A active site to those positions
that are occupied in the MAO-B active site by the waters cited above.
HOH 726 which is located between the aromatic residues Tyr-407
and Tyr-444 was also retained since previous docking studies sug-
gested that the caffeine do not bind in close proximity to this polar
space [27]. Automated docking was subsequently carried out with
theLigandFit applicationof DiscoveryStudio. This protocoluses total
ligand flexibility whereby the final ligand conformations are deter-
mined by the Monte Carlo conformation search method set to
a variable number of trial runs. The docked ligands were further
refined using in situ ligand minimization with the Smart Minimizer
algorithm. All parameters for the docking runs were set to their
default values and tenpossible binding solutions were computed for
each docked ligand. The best-ranked binding conformation of each
ligand was determined according to the DockScore values. The
illustrations were prepared in PyMOL [45].
Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2011.05.014.
References
[1] M.B.H. Youdim, Y.S. Bakhle, Monoamine oxidase: isoforms and inhibitors in
Parkinson’s disease and depressive illness, Br. J. Pharmacol. 147 (2006)
S287eS296.
[2] C. Binda, P. Newton-Vinson, F. Hubalek, D.E. Edmondson, A. Mattevi, Structure
of human monoamine oxidase B, a drug target for the treatment of neuro-
logical disorders, Nat. Struct. Biol. 9 (2002) 22e26.
[3] M.B.H. Youdim, D.E. Edmondson, K.F. Tipton, The therapeutic potential of
monoamine oxidase inhibitors, Nat. Rev. Neurosci. 7 (2006) 295e309.
[4] M.B.H. Youdim, M. Weinstock, Therapeutic applications of selective and non-
selective inhibitors of monoamine oxidase A and B that do not cause signifi-
cant tyramine potentiation, Neurotoxicology 25 (2004) 243e250.
[5] U. Bonnet, Moclobemide: therapeutic use and clinical studies, CNS. Drug Rev.
9 (2003) 97e140.
[6] H.H. Fernandez, J.J. Chen, Monoamine oxidase-B inhibition in the treatment of
Parkinson’s disease, Pharmacotherapy 27 (2007) 174Se185S.
[7] G.G.S. Collins, M. Sandler, E.D. Williams, M.B.H. Youdim, Multiple forms of
human brain monoamine oxidase, Nature 225 (1970) 817e820.
[8] P. Riederer, M.B.H. Youdim, Monoamine oxidase activity and monoamine
metabolism in brains of parkinsonian patients treated with l-deprenyl,
J. Neurochem. 46 (1986) 1359e1365.
[9] D.A. Di Monte, L.E. DeLanney, I. Irwin, J.E. Royland, P. Chan, M.W. Jacowec,
J.W. Langston, Monoamine oxidase-dependent metabolism of dopamine in
the striatum and substantia nigra of L-DOPA-treated monkeys, Brain Res. 738
(1996) 53e59.
[10] J.P. Finberg, J. Wang, K. Bankiewich, J. Harvey-White, I.J. Kopin, D.S. Goldstein,
Increased striatal dopamine production from L-DOPA following selective
inhibition of monoamine oxidase
B
by R(þ)-N-propargyl-1-aminoindan
(rasagiline) in the monkey, J. Neural Transm. Suppl. 52 (1998) 279e285.
[11] W. Birkmayer, P. Riederer, M.B.H. Youdim, W. Linaur, The potentiation of the
anti-akinetic effect after L-dopa treatment by an inhibitor of MAO-B, L-dep-
renyl, J. Neural Transm. 36 (1975) 303e326.
[12] M. Gesi, A. Santinami, R. Ruffoli, G. Conti, F. Fornai, Novel aspects of dopamine
oxidative metabolism. Confounding outcomes take place of certainties,
Pharmacol. Toxicol. 89 (2001) 217e224.
[13] F. Fornai, G. Battaglia, M. Gesi, F.S. Giorgi, F. Orzi, F. Nicoletti, Time-course and
doseeresponse study on the effects of chronic L-DOPA administration on
striatal dopamine levels and dopamine transporter following MPTP toxicity,
Brain Res. 887 (2000) 110e117.
[14] S.A. Marchitti, R.A. Deitrich, V. Vasiliou, Neurotoxicity and metabolism of the
catecholamine-derived 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydrox-
yphenylglycolaldehyde: the role of aldehyde dehydrogenase, Pharmacol. Rev.
59 (2007) 125e150.
[15] J.P. Petzer, N. Castagnoli Jr., M.A. Schwarzschild, J. Chen, C.J. Van der Schyf,
Dual-target-directed drugs that block monoamine oxidase B and adenosine
A2A receptors for Parkinson’s disease, Neurotherapeutics 6 (2009) 141e151.
[16] A. Nicotra, F. Pierucci, H. Parvez, O. Santori, Monoamine oxidase expression
during development and aging, Neurotoxicology 25 (2004) 155e165.
[17] J.S. Fowler, N.D. Volkow, G.J. Wang, J. Logan, N. Pappas, C. Shea, R. MacGregor,
Age-related increases in brain monoamine oxidase B in living healthy human
subjects, Neurobiol. Aging 18 (1997) 431e435.
[18] R.N. Kalaria, M.J. Mitchell, S.I. Harik, Monoamine oxidases of the human brain
and liver, Brain 111 (1988) 1441e1451.
[19] J.M. Rabey, I. Sagi, M. Huberman, E. Melamed, A. Korczyn, N. Giladi,
R. Inzelberg, R. Djaldetti, C. Klein, G. Berecz, Rasagiline mesylate, a new MAO-
B inhibitor for the treatment of Parkinson’s disease: a double-blind study as
adjunctive therapy to levodopa, Clin. Neuropharmacol. 23 (2000) 324e330.
[20] C.J. Fowler, A. Wiberg, L. Oreland, B. Winblad, Titration of human brain type-B
monoamine oxidase, Neurochem. Res. 5 (1980) 697e708.
[21] L.H.A. Prins, J.P. Petzer, S.F. Malan, Inhibition of monoamine oxidase by indole
and benzofuran derivatives, Eur. J. Med. Chem. 45 (2010) 4458e4466.
[22] K.F. Tipton, S. Boyce, J. O’Sullivan, G.P. Davey, J. Healy, Monoamine oxidases:
certainties and uncertainties, Curr. Med. Chem. 11 (2004) 1965e1982.
[23] J.S. Fowler, N.D. Volkow, J. Logan, G. Wang, R.R. MacGregor, D. Schlyer,
A.P. Wolf, N. Pappas, D. Alexoff, C. Shea, E. Dorflinger, L. Kruchowy, K. Yoo,
E. Fazzini, C. Patlak, Slow recovery of human brain MAO B after L-deprenyl
(selegeline) withdrawal, Synaps 18 (1994) 86e93.
[24] J.P. Petzer, S. Steyn, K.P. Castagnoli, J. Chen, M.A. Schwarzchild, C.J. Van Der
Schyf, N. Castagnoli, Inhibition of monoamine oxidase B by selective adeno-
sine A2A receptor antagonists, Bioorg. Med. Chem. 11 (2003) 1299e1310.
[25] E.M. Van der Walt, E.M. Milczek, S.F. Malan, D.E. Edmondson, N. Castagnoli,
J.J. Bergh, J.P. Petzer, Inhibition of monoamine oxidase by (E)-styrylisatin
analogues, Bioorg. Med. Chem. Lett. 19 (2009) 2509e2513.
Acknowledgements
The NMR spectra were recorded by André Joubert of the SASOL
Centre for Chemistry, North-West University while the MS spectra
were recorded by Marelize Ferreira of the Mass Spectrometry
Service, University of the Witwatersrand. This work was supported
by grants from the National Research Foundation and the Medical
Research Council, South Africa. The financial assistance of the
National Research Foundation (DAAD-NRF) towards this research is
hereby acknowledged. Opinions expressed and conclusions arrived
at, are those of the authors and are not necessarily to be attributed
to the DAAD-NRF.
[26] J. Pretorius, S.F. Malan, N. Castagnoli, J.J. Bergh, J.P. Petzer, Dual inhibition of
monoamine oxidase B and antagonism of the adenosine A2A receptor by (E,
E)-8-(4-phenylbutadien-1-yl)caffeine analogues, Bioorg. Med. Chem. 16
(2008) 8676e8684.

B. Strydom et al. / European Journal of Medicinal Chemistry 46 (2011) 3474e3485
3485
[27] B. Strydom, S.F. Malan, N. Castagnoli, J.J. Bergh, J.P. Petzer, Inhibition of
monoamine oxidase by 8-benzyloxycaffeine analogues, Bioorg. Med. Chem.
18 (2010) 1018e1028.
[36] N. Vlok, S.F. Malan, N. Castagnoli, J.J. Bergh, J.P. Petzer, Inhibition of mono-
amine oxidase B by analogues of the adenosine A2A receptor antagonist (E)-8-
(3-chlorostyryl)caffeine (CSC), Bioorg. Med. Chem. 14 (2006) 3512e3521.
[37] S.-Y. Son, J. Ma, Y. Kondou, M. Yoshimura, E. Yamashita, T. Tsukihara, Structure of
human monoamine oxidase A at 2.2-A resolution: the control of opening the
entry for substrates/inhibitors, Proc. Natl. Acad. Sci. U.S.A 105 (2008) 5739e5744.
[38] Accelrys Discovery Studio 1.7. Accelrys Software Inc., San Diego, CA, USA,
2006.http://www.accelrys.com.
[28] C. Binda, J. Wang, L. Pisani, C. Caccia, A. Carotti, P. Salvati, D.E. Edmondson,
A. Mattevi, Structures of human monoamine oxidase
B complexes with
selective noncovalent inhibitors: safinamide and coumarin analogs, J. Med.
Chem. 50 (2007) 5848e5852.
[29] R.C. Huston, W.F. Allen, Caffeine derivatives. I. The 8-ethers of caffeine, J. Am.
Chem. Soc. 56 (1934) 1356e1358.
[30] W. Baumann, Die nervenkrankenfürsorge der stadt essen, Z. Gesamte Neurol.
Psychiatr. 15 (1913) 114e121.
[39] C.I. Manley-King, J.J. Bergh, J.P. Petzer, Inhibition of monoamine oxidase by
selected C5- and C6-substituted isatin analogues, Bioorg. Med. Chem. 19
(2011) 261e274.
[31] E. Fischer, L. Reese, Ueber caffeïn, xanthin und guanin, Justus Liebigs Ann.
Chem. 221 (1883) 336e344.
[40] C. Binda, R. Angelini, R. Federico, P. Ascenzi, A. Mattevi, Structural bases for
inhibitor binding and catalysis in polyamine oxidase, Biochemistry 40 (2001)
2766e2776.
[41] E. Fischer, Ueber caffeïn, Theobromin, xanthin und guanin, Justus Liebigs Ann.
Chem. 215 (1882) 253e320.
[42] M. Sono, N. Toyoda, K. Shimizu, E. Noda, Y. Shizuri, M. Tori, Functionalization
including fluorination of nitrogen-containing compounds using electro-
chemical oxidation, Chem. Pharm. Bull. 44 (1996) 1141e1145.
[43] M.O. Ogunrombi, S.F. Malan, G. Terre’Blanche, N. Castagnoli, J.J. Bergh, J.P. Petzer,
Structure-activity relationships in the inhibition of monoamine oxidase B by 1-
methyl-3-phenylpyrroles, Bioorg. Med. Chem. 16 (2008) 2463e2472.
[44] C.I. Manley-King, G. Terre’Blanche, N. Castagnoli, J.J. Bergh, J.P. Petzer, Inhi-
bition of monoamine oxidase B by N-methyl-2-phenylmaleimides, Bioorg.
Med. Chem. 17 (2009) 3104e3110.
[32] L. Novaroli, M. Reist, E. Favre, A. Carotti, M. Catto, P.A. Carrupt, Human
recombinant monoamine oxidase B as reliable and efficient enzyme source for
inhibitor screening, Bioorg. Med. Chem. 13 (2005) 6212e6217.
[33] Y.C. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1)
and the concentration of inhibitor which causes 50 per cent inhibition (I50) of
an enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108.
[34] F. Hubálek, C. Binda, A. Khalil, M. Li, A. Mattevi, N. Castagnoli, D.E. Edmondson,
Demonstration of isoleucine 199 as a structural determinant for the selective
inhibition of human monoamine oxidase B by specific reversible inhibitors,
J. Biol. Chem. 280 (2005) 15761e15766.
[35] D. Van den Berg, K.R. Zoellner, M.O. Ogunrombi, S.F. Malan, G. Terre’Blanche,
N. Castagnoli, J.J. Bergh, J.P. Petzer, Inhibition of monoamine oxidase B by
selected benzimidazole and caffeine analogues, Bioorg. Med. Chem. 15 (2007)
3692e3702.
[45] W.L. DeLano, The PyMOL Molecular Graphics System. DeLano Scientific, San
Carlos, USA, 2002.