Inhibition of monoamine oxidase by 8-phenoxymethylcaffeine derivatives

Article abstract of DOI:10.1016/j.bmc.2012.05.048

A recent study has reported that a series of 8-benzyloxycaffeines are potent and reversible inhibitors of both human monoamine oxidase (MAO) isoforms, MAO-A and -B. In an attempt to discover additional caffeine derivatives with potent MAO inhibitory activities, and to contribute to the known structure-activity relationships of MAO inhibition by caffeine derived compounds, the present study investigates the MAO inhibitory potencies of series of 8-phenoxymethylcaffeine and 8-[(phenylsulfanyl)methyl]caffeine derivatives. The results document that the 8-phenoxymethylcaffeine derivatives act as potent reversible inhibitors of MAO-B, with IC₅₀ values ranging from 0.148 to 5.78 μM. In contrast, the 8-[(phenylsulfanyl)methyl]caffeine derivatives were found to be weak inhibitors of MAO-B, with IC₅₀ values ranging from 4.05 to 124 μM. Neither the 8-phenoxymethylcaffeine nor the 8-[(phenylsulfanyl)methyl]caffeine derivatives exhibited high binding affinities for MAO-A. While less potent than the 8-benzyloxycaffeines as MAO-B inhibitors, this study concludes that 8-phenoxymethylcaffeines may act as useful leads for the design of MAO-B selective inhibitors. Such compounds may find application in the therapy of neurodegenerative disorders such as Parkinson's disease. Using molecular docking experiments, this study also proposes possible binding orientations of selected caffeine derivatives in the active sites of MAO-A and -B.

Full text of DOI:10.1016/j.bmc.2012.05.048

Bioorganic & Medicinal Chemistry 20 (2012) 4336–4347
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
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Inhibition of monoamine oxidase by 8-phenoxymethylcaffeine derivatives
Thokozile Okaecwe a, Abraham J. Swanepoel a, Anél Petzer 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 Unit for Drug Research and Development, 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:
A recent study has reported that a series of 8-benzyloxycaffeines are potent and reversible inhibitors of
both human monoamine oxidase (MAO) isoforms, MAO-A and -B. In an attempt to discover additional
caffeine derivatives with potent MAO inhibitory activities, and to contribute to the known structure–
activity relationships of MAO inhibition by caffeine derived compounds, the present study investigates
the MAO inhibitory potencies of series of 8-phenoxymethylcaffeine and 8-[(phenylsulfanyl)methyl]caf-
feine derivatives. The results document that the 8-phenoxymethylcaffeine derivatives act as potent
Received 28 March 2012
Revised 11 May 2012
Accepted 17 May 2012
Available online 24 May 2012
Keywords:
reversible inhibitors of MAO-B, with IC50 values ranging from 0.148 to 5.78
ylsulfanyl)methyl]caffeine derivatives were found to be weak inhibitors of MAO-B, with IC50 values
ranging from 4.05 to 124 M. Neither the 8-phenoxymethylcaffeine nor the 8-[(phenylsulfa-
lM. In contrast, the 8-[(phen-
Monoamine oxidase
Reversible inhibition
Competitive
l
nyl)methyl]caffeine derivatives exhibited high binding affinities for MAO-A. While less potent than the
8-benzyloxycaffeines as MAO-B inhibitors, this study concludes that 8-phenoxymethylcaffeines may
act as useful leads for the design of MAO-B selective inhibitors. Such compounds may find application
in the therapy of neurodegenerative disorders such as Parkinson’s disease. Using molecular docking
experiments, this study also proposes possible binding orientations of selected caffeine derivatives in
the active sites of MAO-A and -B.
Selectivity
Caffeine
Phenoxymethylcaffeine
8-[(Phenylsulfanyl)methyl]caffeine
Molecular docking
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
selectively oxidizes serotonin while MAO-B selectively catalyzes
the oxidation of the arylalkylamines, benzylamine and b-phenyl-
The monoamine oxidases (MAO) A and B are apoenzymes,
which require flavin adenine dinucleotide (FAD) as cofactor.1 The
MAO enzymes are bound to the outer surface of mitochondria
and are expressed in most human tissues, including the brain.2,3
An important function of the MAOs is to catalyze the oxidative
catabolism of neurotransmitters, including serotonin, dopamine
and epinephrine, thereby terminating their actions.4 The MAOs
also function as metabolic barriers by restricting the access of
extraneous amines to the systemic circulation and the central ner-
vous system. By metabolizing false neurotransmitters such as ben-
zylamine and b-phenylethylamine,4,5 brain microvessel MAO-B
limits their passage into the brain tissue and thus protects neurons
from their stimulatory effects.6 In the gut wall, intestinal MAO-A
metabolizes tyramine, an indirectly-acting sympathomimetic
amine which is present in certain foods. MAO-A thereby restricts
the amount of tyramine that enters the systemic circulation and
prevents the tyramine-induced release of catecholamines from
peripheral neurons.7
ethylamine. Both isoforms utilize dopamine, epinephrine and nor-
epinephrine as substrates.4 The interest in the MAOs as drug
targets stems from their roles in the catabolism of these neuro-
transmitters. MAO-A inhibitors have been employed as antide-
pressant agents while MAO-B inhibitors have been developed as
therapy for Parkinson’s disease.4,9 These clinical applications of
MAO-A and -B inhibitors are thought to be, for the most part,
dependent on the blocking of the central catabolism of serotonin
and dopamine, respectively. In Parkinson’s disease therapy,
MAO-B inhibitors are frequently combined with levodopa, the
metabolic precursor of dopamine.10 The inhibition of the MAO-B
catalyzed oxidation of dopamine in the brain may elevate the lev-
els of dopamine derived from levodopa and may allow for a
reduction of the levodopa dose required for a therapeutic effect.11
MAO-B inhibitors are of particular relevance in Parkinson’s dis-
ease since MAO-B is the major isoform in the basal ganglia, the
affected brain region in Parkinson’s disease.12,13 Furthermore,
MAO-B activity and density increases in most brain regions with
age while MAO-A activity remains constant.14,15 This presents a
further rationale for targeting MAO-B in age-related neurodegen-
erative disorders such as Parkinson’s disease.16 Another point of
interest is that the MAOs may be major sources of hydrogen per-
oxide in cells. Following the oxidation of a substrate molecule, the
reoxidation of the flavin requires the reduction of molecular
Although the amino acid sequences of MAO-A and -B are
approximately 73% identical, they exhibit different substrate
and inhibitor specificities.8 Most notably, the MAO-A isoform
⇑
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 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.048

T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
4337
oxygen to yield hydrogen peroxide. Hydrogen peroxide may lead
O
to pathological oxidative stress and promote the neurodegenera-
tive processes associated with Parkinson’s disease.17,18 Also, the
aldehydic products, formed as a result of the MAO catalyzed oxi-
dative deamination of amines, may act as neurotoxic metabolites
by reacting with exocyclic amino groups of nucleosides, and N-
N
N
R
R
N
O
S
O
N
O
3
terminal and lysine e-amino groups of proteins. MAO-B inhibitors
may therefore also exert a neuroprotective effect by reducing the
formation of these potentially toxic by-products.1
Considering the pharmacological importance of MAO inhibitors,
the present study aims to discover new inhibitors of the MAOs with
particular emphasis on the MAO-B isoform. For this purpose, caf-
feine derived structures will serve as lead compounds (Fig. 1). Re-
cent studies have reported that 8-benzyloxycaffeines (1) act as
potent reversible inhibitors of both MAO-A and -B.19 A homologous
series of and 8-sulfanylcaffeines (2) has similarly been shown to in-
N
N
N
N
O
O
N
O
4
hibit MAO-B, and to
a
lesser degree MAO-A.20 The 8-ben-
N
zyloxycaffeines are of particular interest since, unlike most
caffeine derived MAO inhibitors, they also possess high binding
affinities for MAO-A. The ability of these compounds to also bind
to MAO-A may be attributed to the relatively large degree of rota-
tional freedom of the C8 side chain at the carbon–oxygen ether
bond. It has been suggested that structures with a relatively larger
degree of conformational freedom may be better suited for binding
to MAO-A than relatively rigid structures.19 MAO-A inhibition may
also have a role in Parkinson’s disease therapy since, in the primate
brain, MAO-A also contributes to the catabolism of dopamine.21
Nonselective MAO-A/B inhibitors may therefore be more effica-
cious in blocking the oxidation of dopamine and thereby prolonging
its effect in the basal ganglia.1 Based on the promising activities of
the 8-benzyloxycaffeines and 8-sulfanylcaffeines, the present study
investigates the human MAO inhibitory potencies of series of 8-
phenoxymethylcaffeine (3) and 8-[(phenylsulfanyl)methyl]caffeine
(4 and 5) derivatives (Fig. 2). The 8-phenoxymethylcaffeines (3) are
isomers of 1 while the 8-[(phenylsulfanyl)methyl]caffeines (4 and
5) are close structural analogues of 2. The major difference between
the structures of compounds 3–5 and that of 8-benzyloxycaffeine is
in the placement of the hetero atom in the C8 side chain. In com-
pounds 3–5 the position of the hetero atom is distal from the caf-
N
S
N
R
5
Figure 2. The structures of the 8-phenoxymethylcaffeine (3), 8-[(phenylsulfa-
nyl)methyl]caffeine (4) and 8-[(phenylsulfanyl)ethyl]caffeine (5) derivatives that
were investigated in this study.
2. Results
2.1. Chemistry
The syntheses of the 8-phenoxymethylcaffeine derivatives 3a–j
were accomplished using the literature procedure (Scheme 1).22
1,3-Dimethyl-5,6-diaminouracil (6)23 was allowed to react with
the appropriate phenoxyacetic acid (7) in the presence of the
carbodiimide activating reagent, N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride (EDAC). The resulting amide
feine ring and these compounds are expected to exhibit
a
differing degree of flexibility than 8-benzyloxycaffeine. Since the
degree of flexibility of the 8-benzyloxycaffeine C8 side chain plays
an important role in its binding to particularly MAO-A, compounds
3–5 may differ in their MAO inhibitory properties compared to the
8-benzyloxycaffeines. The possibility therefore exists that com-
pounds 3–5 may display limited inhibition of MAO-A, making them
MAO-B selective inhibitors. This study aims to explore this possibil-
ity. This study may also contribute to the known structure–activity
relationships of MAO inhibition by caffeine derived compounds.
O
NH2
N
R
HO2C
+
O
O
N
NH2
7
6
O
H
O
N
a, b
N
N
R
R
R
N
N
N
N
O
O
O
O
N
O
O
S
8
N
O
O
N
O
1
N
c
N
N
N
N
3
N
R
2
Scheme 1. Synthetic pathway to the 8-phenoxymethylcaffeine derivatives 3.
Reagents and conditions: (a) EDAC dioxane/H2O, rt; (b) dioxane/NaOH (aq), reflux;
(c) CH3I, K2CO3, DMF, 90 °C.
Figure 1. The structures of 8-benzyloxycaffeines (1) and 8-sulfanylcaffeines (2).

4338
T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
intermediate was treated under reflux with aqueous sodium
hydroxide to yield the corresponding 8-phenoxymethyltheophyl-
line intermediate (8). Without further purification, the crude the-
ophylline was 7N-methylated with an excess of iodomethane to
yield the target compounds, the 8-phenoxymethylcaffeine deriva-
tives 3a–j. The 8-[(phenylsulfanyl)methyl]caffeine derivatives,
4a–i and 5a–e, were prepared in a similar manner by reacting
1,3-dimethyl-5,6-diaminouracil with an appropriate 2-(phen-
ylsulfanyl)acetic acid (9) or 3-(phenylsulfanyl)propanoic acid (10),
respectively (Scheme 2). After ring closure of the resulting amide,
the theophylline intermediate (11) was obtained. Methylation of
11 in the presence of iodomethane yielded the target 8-[(phen-
ylsulfanyl)methyl]caffeine derivatives 4a–i and 5a–e. Following
crystallization, the structures of all compounds were verified by
mass spectrometry, 1H NMR and 13C NMR. In certain instances
the phenoxyacetic acids (7), 2-(phenylsulfanyl)acetic acids (9)
and 3-(phenylsulfanyl)propanoic acids (10), which were required
for the synthesis of 3–5, were not commercially available (Table
1). These reagents were thus synthesized according to literature
procedures by reacting the appropriate phenol or thiophenol with
chloroacetic acid or 3-chloropropionic acid in the presence of base
(Scheme 3).24,25
measurements of 4-hydroxyquinoline concentrations in the pres-
ence of inhibitors 3a–j and 5a–e were not possible since these
inhibitors fluoresce under the conditions described above, and
interfere with the fluorometric determination of 4-hydroxyquino-
line at higher inhibitor concentrations. The determination of MAO
catalytic rates in the presence of compounds 3a–j and 5a–e was
accomplished by measuring the amount of hydrogen peroxide that
is produced in the MAO oxidation cycle. In a horseradish peroxi-
dase-coupled reaction, hydrogen peroxide reacts with Ampliflu
Red to yield resorufin, a fluorescent compound.27 Concentration
measurement of resorufin may be readily made via fluorescence
spectrophotometry at excitation and emission wavelengths of
560 nm and 590 nm, respectively. Under these conditions, inhibi-
tors 3a–j and 5a–e do not fluoresce, or quench the fluorescence of
resorufin. The MAO inhibition potencies of the inhibitors are ex-
pressed as the IC50 values which were estimated from sigmoidal
curves of the rate of MAO catalysis versus the logarithm of the
inhibitor concentration (Fig. 3).
2.2.1. IC50 values for the inhibition of MAO-B
The MAO inhibition potencies of the caffeine derivatives, com-
pounds 3–5, are presented in Tables 2–4. The results show that,
among the compounds evaluated, the 8-phenoxymethylcaffeine
derivatives, 3a–j, are the most potent inhibitors of MAO-B with
2.2. Inhibition studies
IC50 values ranging from 0.148 to 5.78
inhibitor among the 8-phenoxymethylcaffeine derivatives is the
unsubstituted derivative 3a (IC50 = 5.78 M). Substitution on the
meta and para positions of the phenyl ring with both halogens (Cl,
Br and F) and alkyl groups (CF3, CH3, OCH3) leads to enhanced inhi-
bition. With the exception of 3d, the meta fluorine substituted
lM. The weakest MAO-B
The inhibitory potencies of the caffeine derivatives, compounds
3–5, towards MAO were examined by employing recombinant hu-
man MAO-A and -B as enzyme sources. Kynuramine, a MAO-A/B
mixed substrate, served as substrate for the inhibition studies with
both enzymes. The determination of MAO catalytic rates in the
presence of compounds 4a–i was accomplished by measuring the
concentration of 4-hydroxyquinoline, the MAO catalyzed oxidation
product of kynuramine, via fluorescence spectrophotometry.26
4-Hydroxyquinoline is a fluorescent compound which is readily
measured in the presence of the nonfluorescent kynuramine at
excitation and emission wavelengths of 310 nm and 400 nm,
respectively. Under these conditions, inhibitors 4a–i do not fluo-
resce, or quench the fluorescence of 4-hydroxyquinoline. Analogous
l
homologue (IC50 = 1.61 lM), halogen containing substituents on
the phenyl ring yields compounds with IC50 values in the submi-
cromolar range. Bromine substitution on the meta (3c) and para
(3i) positions was found to be particularly appropriate for MAO-B
inhibition and yielded the most potent inhibitors of this study with
IC50 values of 0.148 lM and 0.189 lM, respectively. These com-
pounds are 30- to 39-fold more potent as MAO-B inhibitors than
the unsubstituted homologue 3a. Compared to halogen substitu-
tion, substitution on the meta position with the methyl (3f,
IC50 = 1.23 lM) and methoxy (3g, IC50 = 1.96 lM) groups led to a
modest enhancement of the MAO-B inhibition, with these com-
pound being only 3- to 4-fold more potent than 3a.
The results of the MAO inhibition studies with the 8-[(phen-
ylsulfanyl)methyl]caffeine derivatives, 4a–i, are presented in Ta-
ble 3. As shown, compounds 4a–i were found to be relatively
weak inhibitors of MAO-B with IC50 values ranging from 4.05 to
O
NH2
N
HO2C (CH2)n
S
+
R
O
N
NH2
9: n = 1
10: n = 2
23.4
l
M. As observed for the 8-phenoxymethylcaffeine derivatives,
M) was
6
the unsubstituted homologue, compound 4a (IC50 = 23.4
l
the weakest MAO-B inhibitor of this series, and substitution on
the meta and para positions of the phenyl ring leads to enhanced
inhibition. Also similar to the results obtained with the 8-phen-
oxymethylcaffeine derivatives, meta (4c) and para (4i) substitution
with bromine yielded the most potent MAO-B inhibitors of this
O
H
N
a, b
N
N
(CH2)n
S
S
N
11
R
R
O
O
N
O
series with IC50 values of 4.90 lM and 4.05 lM, respectively. Com-
pared to the bromine substituted 8-phenoxymethylcaffeines (3c
and 3i), compounds 4c and 4i are, however, 21- to 33-fold weaker
as MAO-B inhibitors. This observation leads to the conclusion that
phenoxymethylcaffeines are better suited as lead compounds for
the design of MAO-B inhibitors than 8-[(phenylsulfa-
nyl)methyl]caffeines. To explore the possibility of improving the
MAO inhibition properties of 4a–i, a series of 8-[(phenylsulfa-
nyl)ethyl]caffeine derivatives, 5a–e, were examined as MAO inhib-
itors. As shown in Table 4, compounds 5a–e proved to be weak
MAO-B inhibitors, in most instances weaker than the correspond-
ing 8-[(phenylsulfanyl)methyl]caffeine derivatives (4). It may thus
be concluded that extending the C8 side chain of 8-[(phenylsulfa-
N
N
c
(CH2)n
N
4: n = 1
5: n = 2
Scheme 2. Synthetic pathway to 8-[(phenylsulfanyl)methyl]caffeines (4) and 8-
[(phenylsulfanyl)ethyl]caffeines (5). Reagents and conditions: (a) EDAC dioxane/
H2O, rt; (b) dioxane/NaOH (aq), reflux; (c) CH3I, K2CO3, DMF, 90 °C.

T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
4339
Table 1
The structures of carboxylic acid derivatives 7, 9 and 10
O
S
S
CO2H
R
CO2H
R
CO2H
R
7
9
10
R
Mp (°C)
Lit. mp (°C)
R
mp (°C)
Lit. mp (°C)
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
Commercially available (98–100)
9c
9d
9e
9f
9g
9h
9i
10a
10b
10c
10d
10e
3-Br
3-F
3-CH3
3-OCH3
3-OCH2CH3
4-Cl
4-Br
H
3-Cl
3-Br
90
74
70
72
106
107
117
63
83
91
86.8–87.6c
73.5–74.2c
66.8–67.4c
62.3–63.5c
New compd
107–108c
118–118.5c
58.5–59.5d
77–78e
3-Cl
3-Br
3-F
3-CF3
3-CH3
3-OCH3
4-Cl
4-Br
4-F
108–109
107–108
114–115
92–93
101–105
117–118
108–110a
107–108.8a
111–112a
93.5–94.5b
102–103.5a
111–118a
Commercially available (157)
Commercially available (149–153)
Commercially available (104)
Commercially available (60–63)
7j
9a
9b
90–91e
H
3-Cl
4-Cl
4-Br
88
116
90–91e
86
81.5–82.2c
114–115e
a
b
c
Value obtained from Ref. 37.
Value obtained from Ref. 38.
Value obtained from Ref. 39.
Value obtained from Ref. 40.
Value obtained from Ref. 41.
d
e
R
O
O
R
HO2C
HO
HS
O
Cl
Cl
+
+
HO
HO
NaOH (aq)
NaOH (aq)
7
R
HO2C (CH2)n
S
R
( )n
9: n = 1
10: n = 2
Scheme 3. Synthetic pathway to carboxylic acid derivatives 7, 9 and 10.
tent inhibitor, compound 3e, exhibiting an IC50 value of 4.59 lM.
100
All of the 8-phenoxymethylcaffeine derivatives were, however,
selective for the MAO-B isoform with selectivity indices ranging
from 4 to 230. The most selective inhibitor, compound 3c, also is
the most potent MAO-B inhibitor of the compounds investigated.
It may thus be concluded that 3c represents a potent MAO-B selec-
tive inhibitor. The 8-[(phenylsulfanyl)methyl]caffeine (4a–i) and
8-[(phenylsulfanyl)ethyl]caffeine (5a–e) derivatives were similarly
found to be weak MAO-A inhibitors with the most potent homo-
75
50
25
logue, compound 4b, exhibiting an IC50 value of 19.4 lM. Interest-
ingly, 5b exhibited no MAO-A inhibition, thus making it a highly
selective MAO-B inhibitor. This structure may therefore serve as
a lead in design studies where absolute selectivity is required.
-2
-1
0
1
2
Log[3d]
2.2.3. Reversibility of MAO inhibition
Caffeine derived MAO inhibitors have been shown to interact
reversibly with the MAO enzymes.28 This is a desirable property
since de novo synthesis is not required for the recovery of enzyme
activity. Enzyme activity is recovered when the inhibitor is cleared
fromthe tissues. Since the 8-phenoxymethylcaffeines may represent
a promising new class of MAO inhibitors, thisstudyverified that they
interact reversibly with MAO-A and -B. For this purpose the time-
dependence of MAO inhibition by 2 selected inhibitors was exam-
ined. Compounds 3e and 3c were selected as representative inhibi-
tors since they were found to be the most potent inhibitors of
MAO-A and -B, respectively, among the 8-phenoxymethylcaffeines.
Figure 3. An example sigmoidal dose–response curve of the initial rate of oxidation
of kynuramine by recombinant human recombinant human MAO-B versus the
logarithm of concentration of an inhibitor (expressed in lM). These determinations
were carried out in duplicate and the values are expressed as the mean SD.
nyl)methyl]caffeine derivatives does not improve their MAO-B
inhibitory potencies.
2.2.2. IC50 values for the inhibition of MAO-A
As shown in Table 2, the 8-phenoxymethylcaffeine derivatives,
3a–j, were relatively weak inhibitors of MAO-A, with the most po-

4340
T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
Table 2
Table 4
The IC50 values for the inhibition of recombinant human MAO-A and -B by
The IC50 values for the inhibition of recombinant human MAO-A and -B by
compounds 3a–j
compounds 5a–e
O
O
N
N
N
N
R
N
O
N
S
O
N
O
N
R
3
5
SIc
a,b
R
IC50
(lM)
MAO-A
MAO-B
a,b
R
IC50
(lM)
SIc
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
3-Cl
21.1 3.23
5.78 0.935
0.334 0.010
0.148 0.002
1.61 0.723
0.641 0.010
1.23 0.088
1.96 0.065
0.250 0.040
0.189 0.018
0.825 0.044
4
—
230
8
7
15
—
82
57
10
MAO-A
MAO-B
No inhibition
34.0 31.5d
13.2 7.74
4.59 1.06
18.8 1.52
No inhibition
20.4 7.27
10.7 2.89
8.22 0.336
3-Br
3-F
3-CF3
3-CH3
3-OCH3
4-Cl
5a
5b
5c
5d
5e
H
139 8.82
No inhibition
372 121
142 40.6
117 13.7
108 32.7
5.67 3.55
64.1 9.73
7.79 0.66
124 14.2
1.3
—
6
18
0.9
3-Cl
3-Br
4-Cl
4-Br
a
All values are expressed as the mean SD of triplicate determinations.
The horseradish peroxidase-coupled reaction with Ampliflu Red was used to
4-Br
4-F
b
3j
measure MAO activities.
a
c
All values are expressed as the mean SD of duplicate determinations.
The selectivity index is the selectivity for the MAO-B isoform and is given as the
b
The horseradish peroxidase-coupled reaction with Ampliflu Red was used to
ratio of IC50(MAO-A)/IC50(MAO-B).
measure MAO activities.
c
The selectivity index is the selectivity for the MAO-B isoform and is given as the
ratio of IC50(MAO-A)/IC50(MAO-B).
d
Limited solubility restricts the determination of an accurate IC50 value.
preincubated with MAO-A and -B, respectively, no time-dependent
reductions of the catalytic activities occurred, even following a per-
iod of 60 min preincubation with the enzyme. These data are consis-
tent with a reversible interaction of 3e and 3c with MAO-A and -B,
respectively.
Table 3
To provide further evidence for the reversible interaction of 8-
phenoxymethylcaffeines with MAO, Lineweaver–Burk plots were
constructed for the inhibition of MAO-B by compound 3c. The
MAO-B catalytic rates were measured in the absence of inhibitor,
and presence of three different concentrations of 3c. These mea-
surements were carried out using four different concentrations of
The IC50 values for the inhibition of recombinant human MAO-A and -B by
compounds 4a–i
O
N
N
R
the substrate, kynuramine (15–90 lM). A set of 4 Lineweaver–Burk
N
S
O
N
plots were constructed from these experiments and is given in Fig-
ure 5. The results show that the Lineweaver–Burk plots are linear
and intersect at the y-axis. This indicates that the inhibition of
MAO-B by 3c is competitive, and is further support that 3c is a
reversible MAO-B inhibitor.
4
a,b
R
IC50
(lM)
SIc
MAO-A
MAO-B
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
3-Cl
3-Br
3-F
3-CH3
3-OCH3
3-OCH2CH3
4-Cl
66.2 5.32
19.4 0.35
23.8 0.95
50.1 1.84
51.8 4.20
41.3 2.56
44.7 2.29
31.6 0.54
20.8 1.62
23.4 9.83
6.12 0.45
4.90 1.04
22.3 3.82
17.9 0.99
8.42 0.10
18.4 0.70
6.91 0.65
4.05 0.77
3
3
5
2
3
5
2
5
5
2.2.4. Modelling studies
The results of the MAO inhibition studies have shown that 3c
acts as a potent MAO-B inhibitor while its sulfanyl homologue,
compound 4c, is approximately 33-fold less potent as an MAO-B
inhibitor. Also, 3c is a highly selective inhibitor of MAO-B, with a
230-fold selectivity over the MAO-A isoform. To provide additional
insight, possible binding orientations of 3c and 4c within the MAO
active sites were explored. For this purpose, molecular docking
with the CDOCKER module of Discovery Studio 3.1 (Accelrys)29
was carried out. The crystallographic structures of human MAO-A
cocrystallized with harmine (PDB entry: 2Z5X)30 and human
MAO-B cocrystallized with 7-(3-chlorobenzyloxy)-4-formylcoum-
arin (PDB entry: 2V60)31 served as protein models. These protein
models and docking protocol have previously been shown to be
appropriate for predicting binding orientations of ligands in the ac-
tive sites of MAO-A and -B.32
4-Br
a
All values are expressed as the mean SD of triplicate determinations.
MAO activities were determined by measuring 4-hydroxyquinoline formation
b
via fluorescence spectrophotometry.
c
The selectivity index is the selectivity for the MAO-B isoform and is given as the
ratio of IC50(MAO-A)/IC50(MAO-B).
These inhibitors were preincubated with MAO-A or -B for time peri-
ods of 0, 15, 30 and 60 min. These preincubations were conducted at
inhibitor concentrations of approximately twofold their measured
IC50 values for the inhibition of the respective MAO enzymes. Follow-
ing these preincubations the reactions were diluted twofold, to yield
inhibitor concentrations equal to their IC50 values, and the residual
MAO catalytic activities were measured. The results of these
reversibility studies are shown in Figure 4. When 3e and 3c were
The highest ranked solution of 3c within the MAO-B active site
is illustrated in Figure 6. The orientations of the ten best solutions
differed by less than 0.08 Å from the highest ranked solution. As
shown, the caffeine ring of the inhibitor binds within the substrate
cavity of the enzyme, in proximity to the FAD cofactor. In the space
directly in front of the FAD cofactor, which is considered to be the

T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
4341
entrance cavity constitutes a highly hydrophobic environment,
the C8 substituents are expected to be stabilized principally by
Van der Waals interactions within this space.33 An analysis of the
interaction energies suggests particular favorable Van der Waals
interactions with Ile-199 and Leu-171. Figure 7 illustrates the
highest ranked solution of 4c within the MAO-B active site. In this
instance, the orientations of the ten best solutions differed by less
than 0.07 Å from the highest ranked solution. Compound 4c exhib-
its a similar orientation with respect to the placement of the caf-
feine ring and C8 side chain to that observed for 3c. The most
notable difference, however, is that the polar functional groups of
the caffeine ring of 4c are not placed within hydrogen bond inter-
action distance to residues and waters in the substrate cavity. Cal-
culation of the interaction energies show that an unfavorable
interaction may exist between the thioether sulfur of 4c and Tyr-
326. This interaction is most likely due to close contact between
these moieties. This unfavorable interaction with Tyr-326 and
the loss of potential hydrogen bonding may explain the reduced
binding affinity of 4c for the MAO-B active site compared 3c.
The highest ranked docking orientation of 3c within the MAO-A
active site is presented in Figure 8. Similar to its orientation in
MAO-B, compound 3c binds with the caffeine ring in the proximity
of the FAD cofactor while the C8 phenoxymethyl side chain extends
towards the entrance of the MAO-A active site. The inhibitor may
A
100
75
50
25
0
0
15
30
60
Preincubation time (min)
B
100
75
50
25
0
undergo
p–p and p–r interactions with Tyr-407 and Phe-208,
respectively, as well as hydrogen bonding with an active site water
molecule. The most notable difference between the binding orien-
tations of 3c in MAO-A and -B is that, in MAO-A, 3c binds in a folded
conformation, while exhibiting an extended conformation in the
MAO-B active site. The folded conformation observed in the MAO-
A active site is, to a large degree, imposed by the bulk of the phenyl
side chain of Phe-208. This residue may impede the binding of rel-
atively large inhibitors in the MAO-A active site, and, in order to
avoid structural overlap with Phe-208, larger inhibitors adopt a
folded conformation. In the MAO-B active site, the residue occupy-
ing the analogous position to Phe-208 is Ile-199. The side chain of
Ile-199, which is comparatively smaller compared to the phenyl
ring of Phe-208, is able to rotate from the MAO-B active site cavity,
which enables larger inhibitors to bind in an extended
0
15
30
60
Preincubation time (min)
Figure 4. Time-dependant inhibition of recombinant human MAO-A (Panel A) and
recombinant human MAO-B (Panel B) by 3e and 3c, respectively. The enzymes were
preincubated for various periods of time (0–60 min) with 3e (MAO-A) and 3c
(MAO-B) at inhibitor concentrations of 9.7
l
M and 0.3
l
M, respectively.
100
75
50
25
0
-0.02
0.00
0.02
1/[S]
0.04
0.06
Figure 5. Lineweaver–Burk plots of the recombinant human MAO-B catalyzed
oxidation of kynuramine in the absence (filled squares) and presence of various
concentrations of 3c. The concentrations employed for 3c were 0.0375
squares), 0.075 M (filled circles), 0.15 M (open circles).
lM (open
l
l
polar part of the active site, the C6 carbonyl oxygen of the caffeine
ring undergoes potential hydrogen bonding with the phenolic
hydrogen of Tyr-435 and a water molecule. Furthermore, the caf-
feine ring is orientated approximately parallel to the phenyl rings
of Tyr-435 and Tyr-398, an orientation that is restricted by the flat
shape of the substrate cavity. This places the caffeine ring within
p–p
interaction distance to Tyr-398. The C8 phenoxymethyl side
chain of 3c extends into the entrance cavity of MAO-B. Since the
Figure 6. The predicted binding orientation of 3c within the MAO-B active site.

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T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
3. Discussion and conclusion
The present study examines the possibility that series of 8-
phenoxymethylcaffeine (3) and 8-[(phenylsulfanyl)methyl]caf-
feine (4 and 5) derivatives may act as inhibitors of human MAO.
The results demonstrate that the 8-phenoxymethylcaffeines are
the most active MAO inhibitors, with 6 compounds exhibiting
IC50 values in the submicromolar range. The most potent inhibitor,
compound 3c, was also shown to interact reversible and competi-
tively with MAO-B. In contrast, the phenylsulfanyl derivatives 4
and 5 proved to be comparatively weak MAO-B inhibitors. Model-
ling studies suggest that, while homologues 3c and 4c exhibit sim-
ilar binding orientations in the MAO-B active site, an unfavorable
Van der Waals interaction between the thioether sulfur of 4c and
Tyr-326 may explain, in part, the low MAO-B inhibition potency
of this inhibitor compared to 3c. It may therefore be concluded that
the 8-phenoxymethylcaffeines, particularly compound 3c, repre-
sent promising lead compounds for the development of reversible
MAO-B selective inhibitors. The results obtained with compounds
4 and 5 show that differing substitution on the phenyl ring and
modification of the linker between the caffeine and phenyl ring
do not improve the MAO inhibitory properties of the 8-[(phen-
ylsulfanyl)methyl]caffeines. Considering these results, it is appar-
ent that 8-[(phenylsulfanyl)methyl]caffeines are not suitable
leads for the design of MAO-B inhibitors, and minor structural
modifications would not lead to improved inhibitory properties.
Compared with the previously reported 8-benzyloxycaffeines
Figure 7. The predicted binding orientation of 4c within the MAO-B active site.
(1)19 the 8-phenoxymethylcaffeines
3 are, however, weaker
MAO-B inhibitors. For example, 8-(3-bromobenzyloxy)caffeine,
the benzyloxycaffeine homologue of 3c, inhibits human MAO-B
with an IC50 value of 0.068 lM, which is approximately twofold
more potent than the inhibition potency of 3c. This result suggests
that, the placement of the ether oxygen in the C8 side chain of caf-
feine derived inhibitors is an important consideration in the design
of MAO-B inhibitors. When comparing the MAO-B inhibition
potencies of previously reported 8-(benzylsulfanyl)caffeines (2)20
with the potencies of the 8-[(phenylsulfanyl)methyl]caffeine
derivatives 4, a similar conclusion may be drawn. For example,
8-(benzylsulfanyl)caffeines inhibit human MAO-B with IC50 values
as low as 0.167
nyl)methyl]caffeine derivative examined here, compound 4i,
inhibits MAO-B with an IC50 value of 4.05 M. Therefore, the posi-
lM, while the most potent 8-[(phenylsulfa-
l
tion of the thioether sulfur in the C8 side chain of caffeine derived
inhibitors also has large effects on the MAO-B inhibition potencies.
These results demonstrate that, when a heteroatom is introduced
into the C8 side chain of caffeine derived structures, substitution
directly on C8 of the caffeine ring is likely to lead to the most po-
tent MAO-B inhibition.
Interestingly, compared to the previously reported 8-ben-
zyloxycaffeines (1) the 8-phenoxymethylcaffeine (3) exhibited
weak inhibition potencies towards MAO-A. As mentioned in Sec-
tion 1, the ability of 8-benzyloxycaffeines to bind within the
MAO-A active site may be attributed to the relatively large degree
of rotational freedom of the C8 side chain at the carbon–oxygen
ether bond.19 This rotational freedom presumably enables the
inhibitor to adopt the folded conformation required for binding
to MAO-A. Although, the 8-phenoxymethylcaffeines also contain
a carbon–oxygen ether bond in the C8 side chain, they exhibit
weak binding to the MAO-A active site. The position of the ether
oxygen in the C8 side chain of caffeine derived inhibitors therefore,
in part, determines the affinities of these inhibitor for MAO-A. One
potential reason for this observation is that the placement of the
ether oxygen distal from the caffeine ring does not allow for the
correct measure of flexibility of the C8 side chain for the inhibitor
to be accommodated well in the MAO-A binding site. This reduced/
modified degree of flexibility may, in part, be accounted for by
Figure 8. The predicted binding orientation of 3c within the MAO-A active site.
conformation in MAO-B.16 An analysis of the interaction energies
suggests that the binding orientation of 3c in MAO-A imposes an
unfavorable interaction between the N7 methyl group of the inhib-
itor and the side chain of Asn-181. Since the extended conformation
results in no unfavorable interactions with the MAO-B active site,
the ability of 3c to bind in an extended conformation to MAO-B
may explain its higher binding affinity for MAO-B. Also, the ex-
tended conformation may allow for more and improved productive
interactions between the inhibitor and enzyme than the folded
inhibitor conformation.

T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
4343
differences in the resonance of the phenoxy and the oxycaffeinyl
systems.
EIMS 300; EI-HRMS m/z: calcd for C15H16O3N4, 300.1222, found
300.1218.
4.2.2. 8-(3-Chlorophenoxymethyl)caffeine (3b)
4. Experimental section
The title compound was prepared from 3-chlorophenoxyacetic
acid in a yield of 83%: mp 201–203 °C (methanol/ethyl acetate
7:5). 1H NMR (CDCl3) d 3.38 (s, 3H), 3.56 (s, 3H), 4.02 (s, 3H),
5.15 (s, 2H), 6.87 (dd, 1H, J = 2.6, 8.3 Hz), 6.97 (m, 1H), 7.01 (t,
1H, J = 2.3 Hz), 7.20 (t, 1H, J = 8.3 Hz); 13C NMR (CDCl3) d 27.9,
29.7, 32.4, 62.1, 108.8, 113.0, 115.4, 122.3, 130.5, 135.1, 147.2,
147.4, 151.5, 155.4, 158.2; EI-HRMS m/z: calcd for C15H15O3N4Cl,
334.0833, found 334.0843.
4.1. Chemicals and instrumentation
Unless otherwise noted, all starting materials were obtained
from Sigma–Aldrich and were used without further purification.
1,3-Dimethyl-5,6-diaminouracil (6) was prepared according to
the literature procedure.23 Proton (1H) and carbon (13C) NMR spec-
tra were recorded on a Bruker Avance III 600 spectrometer at fre-
quencies of 600 MHz and 150 MHz, respectively. All NMR
measurements were conducted in CDCl3 and the chemical shifts
are reported in parts per million (d) downfield from the signal of
tetramethylsilane. Spin multiplicities are given as s (singlet), br s
(broad singlet), d (doublet), dd (doublet of doublets), t (triplet), q
(quartet) or m (multiplet). High resolution mass spectra (HRMS)
were obtained on a DFS high resolution magnetic sector mass spec-
trometer (Thermo Electron Corporation) in electron ionization (EI)
mode. Melting points (mp) were determined on a Buchi M-545
melting point apparatus and are uncorrected. For fluorescence
spectrophotometry, a Varian Cary Eclipse fluorescence spectropho-
tometer was employed. The following chemical and biological
agents were obtained from Sigma–Aldrich: microsomes from in-
sect cells containing recombinant human MAO-A or MAO-B
(5 mg/mL), kynuramineꢀ2HBr, Ampliflu Red (10-acetyl-3,7-dihydr-
oxyphenoxazine), horseradish peroxidase, (R)-deprenylꢀHCl, clo-
rgylineꢀHCl and H2O2 (3%).
4.2.3. 8-(3-Bromophenoxymethyl)caffeine (3c)
The title compound was prepared from 3-bromophenoxyacetic
acid in a yield of 78%: mp 200–204 °C (methanol/ethyl acetate 7:5).
1H NMR (CDCl3) d 3.36 (s, 3H), 3.55 (s, 3H), 4.01 (s, 3H), 5.14 (s, 2H),
6.91 (d, 1H, J = 7.5 Hz), 7.12 (m, 3H); 13C NMR (CDCl3) d 28.0, 29.7,
32.4, 62.1, 108.8, 113.5, 118.2, 123.0, 125.2, 130.8, 147.2, 147.4,
151.5, 155.4, 158.2; EI-HRMS m/z: calcd for
C15H15O3N4Br,
378.0328, found 378.0301.
4.2.4. 8-(3-Fluorophenoxymethyl)caffeine (3d)
The title compound was prepared from 3-fluorophenoxyacetic
acid in a yield of 67%: mp 198–201 °C (methanol/ethyl acetate
7:5). 1H NMR (CDCl3) d 3.36 (s, 3H), 3.55 (s, 3H), 4.02 (s, 3H),
5.15 (s, 2H), 6.71 (m, 2H), 6.76 (dd, 1H, J = 2.3, 8.3 Hz), 7.22 (m,
1H); 13C NMR (CDCl3) d 28.0, 29.7, 32.4, 62.2, 102.7 (d), 108.8,
108.9 (d), 110.3 (d), 130.5 (d), 147.3 (d), 151.6, 155.4, 158.7 (d),
162.7, 164.3; EI-HRMS m/z: calcd for C15H15O3N4F, 318.1128, found
318.1131.
4.2. Synthesis of 8-phenoxymethylcaffeine derivatives (3a–j)
4.2.5. 8-[3-(Trifluoromethyl)phenoxymethyl]caffeine (3e)
The title compound was prepared from 2-[3-(trifluoro-
methyl)phenoxy]acetic acid in a yield of 52%: mp 172–174 °C
(methanol/ethyl acetate 7:5). 1H NMR (CDCl3) d 3.38 (s, 3H), 3.56
(s, 3H), 4.04 (s, 3H), 5.22 (s, 2H), 7.17 (m, 1H), 7.25 (d, 1H,
J = 7.5 Hz), 7.29 (s, 1H), 7.40 (t, 1H, J = 8.3 Hz); 13C NMR (CDCl3) d
28.0, 29.7, 32.4, 62.1, 108.8, 111.7 (q), 118.2, 118.7 (q), 122.8,
124.6, 130.3, 132.2 (q), 147.0, 147.4, 151.5, 155.4, 157.6; EI-HRMS
m/z: calcd for C16H15O3N4F3, 368.1096, found 368.1081.
The syntheses of 3a–j were accomplished using the literature
procedure.22 1,3-Dimethyl-5,6-diaminouracil23 (4 mmol) and N-
(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride
(EDAC; 5.36 mmol) were dissolved in 42 mL dioxane/H2O (1:1)
and the appropriate phenoxyacetic acid (4 mmol) was added. A
thick suspension was obtained and the pH was adjusted to 5–6
with 4 N aqueous HCl. The reaction was stirred for 2 h at room
temperature and neutralized with aqueous NaOH (1 N). The pre-
cipitate was collected by filtration and subsequently dissolved in
40 mL dioxane/H2O (1:1). The reaction was heated under reflux
for 2 h, cooled to 0 °C and acidified to a pH of 4 with 4 N aqueous
HCl. The resulting precipitate, the corresponding 1,3-dimethyl-8-
substituted-7H-xanthinyl analogue, was collected by filtration,
washed with 50 mL H2O and dried at 50 °C. The 7H-xanthinyl ana-
logue was used in the subsequent reaction without further purifi-
4.2.6. 8-(3-Methylphenoxymethyl)caffeine (3f)
The title compound was prepared from 3-methylphenoxyacetic
acid in a yield of 72%: mp 195–196 °C (methanol/ethyl acetate 7:5).
1H NMR (CDCl3) 1H NMR (Bruker Avance III 600, CDCl3) d 2.30 (s,
3H), 3.70 (s, 3H), 3.55 (s, 3H), 4.02 (s, 3H), 5.14 (s, 2H), 6.80 (m,
3H), 7.16 (t, 1H, J = 8.3 Hz); 13C NMR (CDCl3) d 21.4, 27.9, 29.7,
32.4, 61.9, 108.7, 111.3, 115.5, 122.8, 129.4, 139.8, 147.3, 148.0,
cation.
The
corresponding
1,3-dimethyl-8-substituted-7H-
xanthinyl analogue (2 mmol) was dissolved in a minimum amount
of DMF (approximately 20 mL) at 90 °C. K2CO3 (5 mmol), followed
by iodomethane (4 mmol), and the reaction mixture was stirred at
90 °C for 1 h. The reaction progress was followed with TLC employ-
ing neutral alumina sheets and ethyl acetate/dichloromethane
(1:1) as mobile phase. The insoluble materials were removed by fil-
tration. H2O (350 ml) was added to the filtrate and the mixture was
cooled on ice for 3 h. The precipitate that formed was collected by
filtration and dried overnight at room temperature. The products
were recrystallized from methanol/ethyl acetate (7:5).
151.5, 155.4, 157.5; EI-HRMS m/z: calcd for
314.1379, found 314.1386.
C16H18O3N4,
4.2.7. 8-(3-Methoxyphenoxymethyl)caffeine (3g)
The title compound was prepared from 3-methoxyphenoxyace-
tic acid in a yield of 36%: mp 165–167 °C (methanol/ethyl acetate
7:5). 1H NMR (CDCl3) d 3.36 (s, 3H), 3.54 (s, 3H), 3.75 (s, 3H),
4.01 (s, 3H), 5.14 (s, 2H), 6.53 (m, 2H), 6.56 (m, 1H), 7.17 (t, 1H,
J = 8.7 Hz); 13C NMR (CDCl3) d 27.9, 29.7, 32.4, 55.3, 62.0, 101.2,
106.6, 107.5, 108.7, 130.1, 147.3, 147.8, 151.5, 155.4, 158.7,
160.9; EI-HRMS m/z: calcd for
C16H18O4N4, 330.1328, found
4.2.1. 8-Phenoxymethylcaffeine (3a)
330.1320.
The title compound was prepared from phenoxyacetic acid in a
yield of 73%: mp 197–199 °C (methanol/ethyl acetate 7:5). 1H NMR
(CDCl3) d 3.37 (s, 3H), 3.56 (s, 3H), 4.03 (s, 3H), 5.17 (s, 2H), 6.99 (m,
3H), 7.29 (t, 2H, J = 7.53 Hz); 13C NMR (CDCl3) d 27.9, 29.7, 32.4,
62.0, 108.7, 114.6, 122.0, 129.7, 147.4, 147.9, 151.6, 155.4, 157.5;
4.2.8. 8-(4-Chlorophenoxymethyl)caffeine (3h)
The title compound was prepared from 4-chlorophenoxyacetic
acid in a yield of 52%: mp 181–183 °C (methanol/ethyl acetate
7:5). 1H NMR (CDCl3) d 3.36 (s, 3H), 3.54 (s, 3H), 4.01 (s, 3H),

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T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
5.13 (s, 2H), 6.91 (d, 2H, J = 8.7 Hz), 7.22 (d, 2H, J = 8.7 Hz); 13C
NMR (CDCl3) d 27.9, 29.7, 32.4, 62.2, 108.7, 116.0, 127.0, 129.6,
147.3, 147.4, 151.5, 155.4, 156.0; EI-HRMS m/z: calcd for
4.3.5. 8-{[(3-Methylphenyl)sulfanyl]methyl}caffeine (4e)
The title compound was prepared from 2-[(3-methyl-
phenyl)sulfanyl]acetic acid in a yield of 37%: mp 124–126 °C
(methanol/ethylacetate 7:2). 1H NMR (CDCl3) d, 2.32 (s, 3H), 3.39
(s, 3H), 3.53 (s, 3H) 3.85 (s, 3H), 4.18 (s, 2H), 7.09 (br s, 1H), 7.19
(m, 2H), 7.24 (m, 1H); 13C NMR (CDCl3) d; 21.2, 27.8, 29.6, 30.7,
32.0, 107.9, 128.6, 128.7, 128.9, 132.3, 133.1, 139.0, 147.5, 149.5,
151.5, 155.2; EI-HRMS m/z: calcd for C16H18N4O2S, 330.1150, found
330.1157.
C15H15O3N4Cl, 334.0833, found 334.0838.
4.2.9. 8-(4-Bromophenoxymethyl)caffeine (3i)
The title compound was prepared from 4-bromophenoxyacetic
acid in a yield of 80%: mp 195–197 °C (methanol/ethyl acetate 7:5).
1H NMR (CDCl3) d 3.35 (s, 3H), 3.53 (s, 3H), 4.00 (s, 3H), 5.13 (s, 2H),
6.86 (d, 2H, J = 9.0 Hz), 7.35 (d, 2H, J = 9.0 Hz); 13C NMR (CDCl3) d
27.9, 29.7, 32.4, 62.1, 108.7, 114.3, 116.4, 132.5, 147.3, 151.5,
155.3, 156.5; EI-HRMS m/z: calcd for C15H15O3N4Br, 378.0328,
found 378.0317.
4.3.6. 8-{[(3-Methoxyphenyl)sulfanyl]methyl}caffeine (4f)
The title compound was prepared from 2-[(3-methoxy-
phenyl)sulfanyl]acetic acid in a yield of 41%: mp 161–165 °C
(methanol/ethylacetate 7:2). 1H NMR (CDCl3) d; 3.36 (s, 3H), 3.50
(s, 3H), 3.75 (s, 3H), 3.84 (s, 3H), 4.16 (s, 2H), 6.78 (dd, 1H,
J = 1.9, 7.9 Hz), 6.93 (m, 2H), 7.17 (t, 1H, J = 7.9 Hz); 13C NMR
(CDCl3) d; 27.9, 29.7, 30.5, 32.1, 55.3, 108.0, 113.6, 116.5, 123.3,
130.0, 134.8, 147.5, 149.4, 151.5, 155.2, 159.9; EI-HRMS m/z: calcd
for C16H18N4O3S, 346.1100, found 346.1091.
4.2.10. 8-(4-Fluorophenoxymethyl)caffeine (3j)
The title compound was prepared from 4-fluorophenoxyacetic
acid in a yield of 79%: mp 212–213 °C (methanol/ethyl acetate
7:5). 1H NMR (CDCl3) d 3.37 (s, 3H), 3.55 (s, 3H), 4.02 (s, 3H),
5.13 (s, 2H), 6.94 (m, 4H); 13C NMR (CDCl3) d 27.9, 29.7, 32.4,
62.6, 108.7, 115.8, 115.9, 116.0, 116.2, 147.3, 147.6, 151.5, 153.6,
155.4, 157.1, 158.7; EI-HRMS m/z: calcd for
318.1128, found 318.1114.
C
15H15O3N4F,
4.3.7. 8-{[(3-Ethoxyphenyl)sulfanyl]methyl}caffeine (4g)
The title compound was prepared from 2-[(3-ethoxyphenyl)sul-
fanyl]acetic acid in a yield of 55%: mp 147–149 °C (methanol/eth-
ylacetate 7:2). 1H NMR (CDCl3) d 1.35 (t, 3H, J = 6.8 Hz), 3.34 (s, 3H),
3.48 (s, 3H), 3.82 (s, 3H), 3.95 (q, 2H, J = 6.8 Hz), 4.15 (s, 2H), 6.75
(d, 1H, J = 8.3 Hz), 6.91 (m, 2H), 7.14 (t, 1H, J = 7.5 Hz); 13C NMR
(CDCl3) d 14.7, 27.8, 29.6, 30.5, 32.0, 63.5, 108.0, 114.1, 117.1,
123.2, 129.9, 134.7, 147.5, 149.5, 151.5, 155.2, 159.2; EI-HRMS
m/z: calcd for C17H20N4O3S, 360.1256, found 360.1261.
4.3. Synthesis of 8-[(phenylsulfanyl)methyl]caffeine (4a–i)
Compounds 4a–i were synthesized from 1,3-dimethyl-5,6-dia-
minouracil23 and an appropriate 2-(phenylsulfanyl)acetic acid
according to the procedure described above for the syntheses of
3a–j.22
4.3.1. 8-[(Phenylsulfanyl)methyl]caffeine (4a)
4.3.8. 8-{[(4-Chlorophenyl)sulfanyl]methyl}caffeine (4h)
The title compound was prepared from 2-[(4-chlorophenyl)sul-
fanyl]acetic acid in a yield of 59%: mp 149–150 °C (methanol/eth-
ylacetate 7:2). 1H NMR (CDCl3) d, 3.34 (s, 3H), 3.46 (s, 3H), 3.86 (s,
The title compound was prepared from 2-(phenylsulfanyl)acetic
acid in a yield of 74%: mp 154–156 °C (methanol/ethylacetate 7:2).
1H NMR (CDCl3) d 3.34 (s, 3H), 3.47 (s, 3H), 3.78 (s, 3H), 4.13 (s, 2H),
7.24 (m, 3H), 7.35 (m, 2H); 13C NMR (CDCl3) d 27.8, 29.6, 30.8, 32.0,
107.9, 128.0, 129.1, 131.9, 133.3, 147.5, 149.4, 151.5, 155.2; EI-
HRMS m/z: calcd for C15H16N4O2S, 316.0994, found 316.0988.
3H), 4.12 (s, 2H), 7.23 (d, 2H, J = 7.5 Hz), 7.29 (d, 2H, J = 7.5 Hz); 13
C
NMR (CDCl3) d; 27.9, 29.6, 30.8, 32.1, 108.0, 129.3, 131.8, 133.0,
134.1, 147.4, 149.0, 151.4, 155.2; EI-HRMS m/z: calcd for
C15H15ClN4O2S, 350.0604, found 350.0597.
4.3.2. 8-{[(3-Chlorophenyl)sulfanyl]methyl}caffeine (4b)
The title compound was prepared from 2-[(3-chlorophenyl)sul-
fanyl]acetic acid in a yield of 56%: mp 174–177 °C (methanol/eth-
ylacetate 7:2). 1H NMR (CDCl3) d 3.35 (s, 3H), 3.49 (s, 3H), 3.90 (s,
3H), 4.17 (s, 2H), 7.19 (m, 2H), 7.23 (m, 1H), 7.41 (m, 1H); 13C NMR
(CDCl3) d 27.9, 29.6, 30.1, 32.1, 108.1, 127.7, 128.9, 130.1, 130.6,
134.7, 135.5, 147.4, 148.8, 151.5, 155.2; EI-HRMS m/z: calcd for
4.3.9. 8-{[(4-Bromophenyl)sulfanyl]methyl}caffeine (4i)
The title compound was prepared from 2-[(4-bromophenyl)sul-
fanyl]acetic acid in a yield of 73%: mp 160–164 °C (methanol/eth-
ylacetate 7:2). 1H NMR (CDCl3) d, 3.39 (s, 3H), 3.51 (s, 3H), 3.92 (s,
3H), 4.17 (s, 2H), 7.27 (d, 2H, J = 7.5 Hz), 7.42 (d, 2H, J = 7.5 Hz); 13
C
NMR (CDCl3) d; 27.9, 29.6, 30.6, 32.1, 108.0, 122.1, 132.2, 132.5,
133.0, 147.4, 149.0, 151.4, 155.2; EI-HRMS m/z: calcd for
C15H15ClN4O2S, 350.0604, found 350.0615.
C15H15BrN4O2S, 394.0099, found 394.0092.
4.3.3. 8-{[(3-Bromophenyl)sulfanyl]methyl}caffeine (4c)
The title compound was prepared from 2-[(3-bromophenyl)sul-
fanyl]acetic acid in a yield of 75%: mp 151–152 °C (methanol/eth-
ylacetate 7:2). 1H NMR (CDCl3) d, 3.35 (s, 3H), 3.49 (s, 3H), 3.89 (s,
3H), 4.16 (s, 2H), 7.12 (t, 1H, J = 7.9 Hz), 7.27 (m, 1H), 7.34 (m, 1H),
7.56 (m, 1H); 13C NMR (CDCl3) d 27.9, 29.6, 30.1, 32.1, 108.1, 122.8,
129.4, 130.3, 130.6, 133.4, 135.8, 147.4, 148.8, 151.5, 155.2; EI-
HRMS m/z: calcd for C15H15BrN4O2S, 394.0099, found 394.0100.
4.4. Synthesis of 8-[(phenylsulfanyl)ethyl]caffeine derivatives
(5a–e)
Compounds 5a–e were synthesized from 1,3-dimethyl-5,6-dia-
minouracil23 and an appropriate 3-(phenylsulfanyl)propanoic acid
according to the procedure described above for the syntheses of
3a–j.22
4.4.1. 8-[(Phenylsulfanyl)ethyl]caffeine (5a)
4.3.4. 8-{[(3-Flourophenyl)sulfanyl]methyl}caffeine (4d)
The title compound was prepared from 2-[(3-flourophenyl)sul-
fanyl]acetic acid in a yield of 30%: mp 155–156 °C (methanol/eth-
ylacetate 7:2). 1H NMR (CDCl3) d, 3.45 (s, 3H), 3.48 (s, 3H), 3.89 (s,
3H), 4.18 (s, 2H), 6.92 (m, 1H), 7.13 (m, 2H), 7.22 (m, 1H); 13C NMR
(CDCl3) d; 27.9, 29.6, 30.1, 32.1, 108.1, 114.5. 114.6, 117.5, 117.6,
126.3, 130.3, 130.4, 135.8, 135.9, 147.4, 148.8, 151.5, 155.2,
161.8, 163.4; EI-HRMS m/z: calcd for C15H15FN4O2S, 334.0900,
found 334.0893.
The title compound was prepared from 3-(phenylsulfanyl)prop-
anoic acid in a yield of 45%: mp 143–145 °C (methanol/ethylace-
tate 7:2). 1H NMR (CDCl3) d, 3.00 (t, 2H, J = 7.2 Hz), 3.35 (s, 3H),
3.35 (t, 2H, J = 7.2 Hz), 3.50 (s, 3H), 3.79 (s, 3H), 7.15 (m, 1H),
7.24 (m, 2H), 7.31 (m, 2H); 13C NMR (CDCl3) d; 26.9, 27.8, 29.6,
31.7, 31.8, 107.3, 126.6, 129.0, 129.7, 134.7, 147.9, 151.6, 151.9,
155.2; EI-HRMS m/z: calcd for C16H18N4O2S, 330.1150, found
330.1148.

T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
4345
4.4.2. 8-{[(3-Chlorophenyl)sulfanyl]ethyl}caffeine (5b)
4.5.2. 2-(Phenylsulfanyl)acetic acids (9) and 3-
(phenylsulfanyl)propanoic acids (10)
The title compound was prepared from 3-[(3-chlorophenyl)sul-
fanyl]propanoic acid in a yield of 3%: mp 181–183 °C (methanol/
ethylacetate 7:2). 1H NMR (CDCl3) d, 3.04 (t, 2H, J = 6.8 Hz), 3.37
(s, 3H), 3.39 (t, 2H, J = 6.8 Hz); 3.52 (s, 3H), 3.84 (s, 3H), 7.12 (m,
1H), 7.18 (m, 2H); 7.24 (m, 1H); 13C NMR (CDCl3) d; 26.9, 27.8,
29.7, 31.6, 31.8, 107.3, 126.5, 127.4, 128.7, 130.0, 134.7, 137.0,
An appropriate thiophenol (10 mmol) was dissolved in ethanol
(5 mL) and a solution of NaOH (10 mmol) in H2O (3 mL) was added.
In a separate flask, chloroacetic acid or 3-chloropropionic acid
(11 mmol) was dissolved in H2O (3 mL) and Na2CO3 (5.5 mmol)
was added. The resulting solution was added to the thiophenol
containing reaction and the reaction was allowed to stir at room
temperature for 3 h. The reaction was subsequently heated under
reflux for 1 h, cooled to room temperature and acidified with 2 N
HCl to pH 2. H2O (25 mL) was added and the brown oil fraction
was extracted to diethylether (50 mL). The ether fraction was ex-
tracted with an aqueous solution of 5% Na2CO3 (25 ml). The Na2CO3
fraction was acidified to pH 2 with concentrated HCl and the
resulting precipitate was collected by filtration and oven dried to
obtain the required acids.25 2-[(3-Ethoxyphenyl)sulfanyl]acetic
acid (9g): Yield 85%: mp 106 °C. 1H NMR (CDCl3) d 1.38 (t, 3H,
J = 7.2 Hz), 3.37 (s, 2H), 3.99 (q, 2H, J = 7.2 Hz), 6.75 (dd, 1H,
J = 2.3, 8.3 Hz), 6.92–6.95 (m, 2H), 7.17 (t, 1H, J = 8.3 Hz); 13C
NMR (CDCl3) d 14.7, 36.2, 63.5, 113.6, 115.5, 121.6, 130.0, 135.6,
159.3, 174.3.
147.8, 151.5, 155.2; EI-HRMS m/z: calcd for
C16H17ClN4O2S,
364.0761, found 364.0743.
4.4.3. 8-{[(3-Bromophenyl)sulfanyl]ethyl}caffeine (5c)
The title compound was prepared from 3-[(3-bromophe-
nyl)sulfanyl]propanoic acid in a yield of 61%: mp 175–177 °C
(methanol/ethylacetate 7:2). 1H NMR (CDCl3) d, 2.90 (t, 2H,
J = 6.4); 3.23 (s, 3H); 3.25 (t, 2H, J = 6.4 Hz); 3.38 (s, 3H), 3.70 (s,
3H), 6.97 (t, 1H, J = 7.5 Hz); 7.09 (d, 1H, J = 7.5 Hz); 7.13 (d, 1H,
J = 6.8 Hz), 7.24 (s, 1H); 13C NMR (CDCl3) d; 27.0, 27.8, 29.7,
31.7, 31.7, 107.3, 122.8, 127.9, 129.4, 130.2, 131.6, 137.3, 147.8,
151.5, 151.5, 155.1; EI-HRMS m/z: calcd for
C16H17BrN4O2S,
408.0256, found 408.0255.
4.4.4. 8-{[(4-Chlorophenyl)sulfanyl]ethyl}caffeine (5d)
The title compound was prepared from 3-[(4-chlorophenyl)sul-
fanyl]propanoic acid in a yield of 23%: mp 148–150 °C (methanol/
ethylacetate 7:2). 1H NMR (CDCl3) d, 2.99 (t, 2H, J = 6.8 Hz); 3.34 (t,
2H, J = 6.8 Hz), 3.36 (s, 3H), 3.50 (s, 3H), 3.82 (s, 3H), 7.20 (d, 2H,
J = 8.3 Hz); 7.23 (d, 2H, J = 8.3 Hz); 13C NMR (CDCl3) d 26.9, 27.9,
29.6, 31.8, 32.1, 107.3, 129.1, 131.1, 132.7, 133.3, 147.9, 151.6,
151.6, 155.2; EI-HRMS m/z: calcd for C16H17ClN4O2S, 364.0761,
found 364.0755.
4.6. Determination of IC50 values
Microsomes from baculovirus infected insect cells expressing
either recombinant human MAO-A or MAO-B (5 mg/mL) were ob-
tained from Sigma–Aldrich, pre-aliquoted and stored at ꢂ70 °C. All
enzymatic reactions were carried out to a final volume of 500 lL in
2 mL microcentrifuge tubes. Potassium phosphate buffer (100 mM,
pH 7.4, made isotonic with KCl) served as reaction solvent.
4.6.1. IC50. measurements of 4a–i
4.4.5. 8-{[(4-Bromophenyl)sulfanyl]ethyl}caffeine (5e)
The title compound was prepared from 3-[(4-bromophenyl)sul-
fanyl]propanoic acid in a yield of 26%: mp 159–161 °C (methanol/
ethylacetate 7:2). 1H NMR (CDCl3) d, 3.00 (t, 2H, J = 6.8 Hz), 3.34 (t,
2H, J = 6.8 Hz), 3.35 (s, 3H), 3.49 (s, 3H), 3.81 (s, 3H), 7.15 (d, 2H,
J = 8.3 Hz), 7.34 (d, 2H, J = 8.3 Hz); 13C NMR (CDCl3) d 26.9, 27.8,
29.6, 31.7, 31.9, 107.2, 120.4, 131.2, 131.9, 133.9, 147.8, 151.5,
151.5, 155.2; EI-HRMS m/z: calcd for C16H17BrN4O2S, 408.0256,
found 408.0255.
The reactions contained the MAO-A/B mixed substrate, kynur-
amine (45
l
M and 30
lM for MAO-A and -B, respectively), and var-
ious concentrations of the test inhibitor (0.003–100
lM). Stock
solutions of the test inhibitors were prepared in DMSO and added
to the reactions to yield a final concentration equal to 4% (v/v)
DMSO. The reactions were initiated with the addition of MAO-A
or -B (0.0075 mg/mL) and incubated at 37 °C for 20 min in a water
bath. The reactions were subsequently terminated with the addi-
tion of 400
lL NaOH (2 N). After the addition of distilled water
(1000 L), the concentrations of the MAO generated 4-hydroxy-
l
4.5. Synthesis of carboxylic acid derivatives (7, 9 and 10)
quinoline were measured by fluorescence spectrophotometry
(kex = 310 nm, kem = 400 nm).26 Quantitative estimations of 4-
hydroxyquinoline concentrations were made via a linear calibra-
tion curve, which was constructed from solutions of 4-hydroxy-
When not commercially available, the phenoxyacetic acids (7),
2-(phenylsulfanyl)acetic acids (9) and 3-(phenylsulfanyl)propa-
noic acids (10) required for the synthesis of 3–5 were synthesized
according to literature procedures.24,25 The experimentally deter-
mined as well as literature melting points of these acids are given
in Table 1. The physical data of compound 9g, previously unknown,
is given below.
quinoline (0.047–1.5
standard solution (500
(2 N) and 1000 L distilled water were added. The initial rate of
l
M) in potassium phosphate buffer. To each
l
L), 4% DMSO as co-solvent, 400 L NaOH
l
l
kynuramine oxidation was graphed versus the logarithm of the
inhibitor concentration to yield sigmoidal dose–response curves.
For each curve, at least six different inhibitor concentrations span-
ning at least 3 orders of magnitude were used. The IC50 values were
determined by fitting the sigmoidal curves to the one site compe-
tition model incorporated into the Prism 5 software package
(GraphPad). All experiments were carried out in triplicate and
the IC50 values are expressed as mean standard deviation (SD).19
4.5.1. Phenoxyacetic acids (7)
An appropriately substituted phenol (50 mmol) was added to a
solution of NaOH (187.5 mmol) in 15 mL of H2O. Chloroacetic acid
(85 mmol) was added slowly at to the resulting solution at 40 °C
and the reaction mixture was heated to 85 °C. Stirring was contin-
ued for 2 h, the reaction was cooled to room temperature and
100 mL H2O was added. The reaction was filtered and the filtrate
was acidified with concentrated HCl to pH 1–2. The brown oil frac-
tion which formed was extracted to diethylether (2 ꢁ 50 ml). The
ether fraction, in turn, was extracted with an aqueous solution of
5% Na2CO3 (2 ꢁ 37.5 ml). The combined Na2CO3 fractions were
acidified to pH 1–2 with concentrated HCl and the resulting precip-
itate was collected by filtration and oven dried to obtain the re-
quired acids.24
4.6.2. IC50. measurements of 3a–j and 5a–e
The reactions contained kynuramine (45
MAO-A and -B, respectively), various concentrations of the test
inhibitor (0.003–100 M), horseradish peroxidase (1 unit/mL) and
Ampliflu Red (200 M). Stock solutions of the test inhibitors were
lM and 30 lM for
l
l
prepared in DMSO and added to the reactions to yield a final con-
centration of 4% (v/v) DMSO. The reactions were initiated with the
addition of MAO-A or -B (0.0075 mg/mL), incubated at 37 °C for

4346
T. Okaecwe et al. / Bioorg. Med. Chem. 20 (2012) 4336–4347
20 min in a water bath and terminated with the addition of 10
(R)-deprenyl (5 mM) for MAO-B and 10 L clorgyline (5 mM) for
MAO-A. Distilled water (1400 L) was added to each reaction and
the concentrations of resorufin in the reactions were determined
by fluorescence spectrophotometry (kex = 560 nm, kem = 590 nm).27
Quantitative estimations of resorufin were made by means of a lin-
ear calibration curve, which was constructed from solutions of
l
L
fixed atom constraint was applied to the backbone of the enzyme
models and the energies of the models were minimized using the
Smart Minimizer protocol. For this purpose the maximum steps
were set to 50000 and the implicit generalized Born solvation
model with molecular volume was used. The cocrystallized ligands
present in the active sites and the crystal water molecules were
subsequently removed from the models and the binding sites were
identified from the existing enzyme cavities. For the purpose of the
docking procedure, the backbone constraint was also removed
from the models. The following active site waters are considered
conserved and were retained for the docking calculations: In the
MAO-B active site, HOH 1159, 1166 and 1309 in the A-chain of
2V60.31 In the MAO-A active site, HOH 710, 718 and 739 of
2Z5X. The structures of the selected inhibitors were constructed
within Discovery Studio. Hydrogen atoms were added to the inhib-
itors and starting geometries of the structures were computed
using a Dreiding-like forcefield (5000 iterations). Atom potential
types and partial charges were assigned with the MMFF forcefield
and the inhibitors were docked into the MAO models with the
CDOCKER protocol. Allowance was made for the generation of 10
random ligand conformations, a heating target temperature of
700 K. The full potential was used for CDOCKER and in situ ligand
minimization of the docking solutions was finally carried out using
the Smart Minimizer algorithm. Unless otherwise specified, all the
application modules within Discovery Studio were set to their de-
fault values. The illustrations were prepared with PyMOL.36
l
l
H2O2 (0.05–1.6
final volume of 500
tained horseradish peroxidase (1 unit/mL), Ampliflu Red (200
4% DMSO as co-solvent, 10 L (R)-deprenyl (5 mM) for MAO-B and
10 L clorgyline (5 mM) for MAO-A. Distilled water (1400 L) was
l
M). Each calibration standard was prepared to a
L in potassium phosphate buffer and also con-
M),
l
l
l
l
l
added to each standard. The IC50 values were determined as de-
scribed above.
4.7. Time-dependence of inhibition
The time-dependencies of the inhibition of MAO-A and -B by se-
lected inhibitors, compounds 3e and 3c, were examined. Recombi-
nant human MAO-A and -B (0.03 mg/mL) were preincubated with
compounds 3e and 3c, respectively for various periods of time (0,
15, 30, 60 min) at 37 °C. Potassium phosphate buffer (100 mM,
pH 7.4, made isotonic with KCl) served as reaction solvent and
the concentrations of inhibitors 3e (9.7 lM) and 3c (0.3 lM) were
approximately twofold the measured IC50 value for the inhibition
of MAO-A and -B, respectively. The preincubated reactions were
subsequently diluted twofold by the addition of kynuramine to
yield final kynuramine concentrations of 45 lM and 30 lM for
Acknowledgements
MAO-A and -B, respectively. After dilution, the final concentrations
of the 3e and 3c were approximately equal to the IC50 values and
the final MAO concentration was 0.015 mg/mL. The reactions,
which were carried out in 2 mL microcentrifuge tubes to a volume
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 Ser-
vice, University of the Witwatersrand. This work was supported by
grants from the National Research Foundation and the Medical Re-
search Council, South Africa.
of 500
nated with the addition of 400
1000 L distilled water, the rates of the MAO generated of 4-
lL, were incubated for a further 15 min at 37 °C and termi-
l
L NaOH (2 N). After the addition of
l
hydroxyquinoline were measured spectrophotometrically as de-
scribed above. All measurements were carried out in triplicate
and are expressed as mean SD.34,35
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.048.
4.8. Lineweaver–Burk plots
Since the caffeine derivatives examined here exhibit selective
MAO-B inhibition, the mode of MAO-B inhibition by a selected
inhibitor, compound 3c, was investigated. For this inhibitor, a set
consisting of 4 Lineweaver–Burk plots were constructed. One plot
was constructed in the absence of inhibitor. To construct the
remaining 3 plots, the concentrations selected for inhibitor 3c were
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