Inhibition of monoamine oxidase by 8-[(phenylethyl)sulfanyl]caffeine analogues

Article abstract of DOI:10.1016/j.bmc.2012.10.005

In a previous study we have investigated the monoamine oxidase (MAO) inhibitory properties of a series of 8-sulfanylcaffeine analogues. Among the compounds studied, 8-[(phenylethyl)sulfanyl]caffeine (IC₅₀ = 0.223 μM) was found to be a particularly potent inhibitor of the type B MAO isoform. In an attempt to discover potent MAO inhibitors and to further examine the structure-activity relationships (SAR) of MAO inhibition by 8-sulfanylcaffeine analogues, in the present study a series of 8-[(phenylethyl)sulfanyl]caffeine analogues were synthesized and evaluated as inhibitors of human MAO-A and -B. The results document that substitution on C3 and C4 of the phenyl ring with alkyl groups and halogens yields 8-[(phenylethyl)sulfanyl]caffeine analogues which are potent and selective MAO-B inhibitors with IC₅₀ values ranging from 0.017 to 0.125 μM. The MAO inhibitory properties of a series of 8-sulfinylcaffeine analogues were also examined. The results show that, compared to the corresponding 8-sulfanylcaffeine analogues, the 8-sulfinylcaffeins are weaker MAO-B inhibitors. Both the 8-sulfanylcaffeine and 8-sulfinylcaffeine analogues were found to be weak MAO-A inhibitors. This study also reports the MAO inhibition properties of selected 8-[(phenylpropyl)sulfanyl]caffeine analogues.

Full text of DOI:10.1016/j.bmc.2012.10.005

Bioorganic & Medicinal Chemistry 20 (2012) 7040–7050
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of monoamine oxidase by 8-[(phenylethyl)sulfanyl]caffeine
analogues
Samantha Mostert a, , Wayne Mentz 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:
In a previous study we have investigated the monoamine oxidase (MAO) inhibitory properties of a series
of 8-sulfanylcaffeine analogues. Among the compounds studied, 8-[(phenylethyl)sulfanyl]caffeine
(IC50 = 0.223 lM) was found to be a particularly potent inhibitor of the type B MAO isoform. In an attempt
to discover potent MAO inhibitors and to further examine the structure–activity relationships (SAR) of
MAO inhibition by 8-sulfanylcaffeine analogues, in the present study a series of 8-[(phenylethyl)sulfa-
nyl]caffeine analogues were synthesized and evaluated as inhibitors of human MAO-A and -B. The results
document that substitution on C3 and C4 of the phenyl ring with alkyl groups and halogens yields 8-
[(phenylethyl)sulfanyl]caffeine analogues which are potent and selective MAO-B inhibitors with IC50 val-
Received 27 July 2012
Revised 1 October 2012
Accepted 8 October 2012
Available online 16 October 2012
Keywords:
Monoamine oxidase
Sulfanylcaffeine
ues ranging from 0.017 to 0.125 lM. The MAO inhibitory properties of a series of 8-sulfinylcaffeine ana-
Sulfinylcaffeine
logues were also examined. The results show that, compared to the corresponding 8-sulfanylcaffeine
analogues, the 8-sulfinylcaffeins are weaker MAO-B inhibitors. Both the 8-sulfanylcaffeine and 8-sulfinyl-
caffeine analogues were found to be weak MAO-A inhibitors. This study also reports the MAO inhibition
properties of selected 8-[(phenylpropyl)sulfanyl]caffeine analogues.
Reversible inhibition
Selective inhibition
Caffeine
Structure–activity relationship
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
employed as antiparkinsonian drugs. Examples of MAO inhibitors
that are currently in clinical use are the reversible MAO-A inhibi-
The monoamine oxidases (MAO) A and B are flavin adenine
dinucleotide (FAD) containing enzymes which are bound to the
outer membranes of mitochondria.1 Although, the two MAO iso-
forms are distinctive enzymes and products of separate genes,2
they exhibit several similarities. MAO-A and -B have approxi-
mately 70% identity at the amino acid level and their crystallo-
graphic structures show that the amino acid residues comprising
their active sites and their relative geometries are highly similar.2
Only six of the 16 active site amino acids differ between the two
enzymes.3–5 In spite of these similarities, MAO-A and -B exhibit
different substrate and inhibitor specificities. For example, seroto-
nin acts as a MAO-A selective substrate while the arylalkylamines,
benzylamine and b-phenethylamine, are MAO-B selective sub-
strates. Dopamine, epinephrine and norepinephrine are employed
by both MAO isoforms as substrates.6
tor, moclobemide, and the irreversible MAO-B inhibitors, (R)-
deprenyl and rasagiline.7,8 This study is particularly interested in
the discovery of new MAO-B inhibitors. In Parkinson’s disease ther-
apy, MAO-B inhibitors are frequently combined with the dopamine
precursor, levodopa.8 Inhibition of the MAO-catalyzed catabolism
of dopamine is thought to lead to a dopamine sparing effect in
the brain and subsequently a symptomatic benefit for Parkinson’s
disease patients.9 Furthermore, inhibitors of MAO-B may enhance
an elevation of the concentration of dopamine derived from levo-
dopa and thus allow for a reduction of the dosage of levodopa that
is necessary for a therapeutic response.10 A reduction of the dosage
of levodopa is also expected to diminish levodopa associated side
effects.11 By inhibiting the degradation of b-phenethylamine,
MAO-B inhibitors may also indirectly induce an increase of extra-
cellular dopamine concentrations.12 This effect may possibly be
attributed to the release of dopamine and inhibition of active dopa-
mine uptake by b-phenethylamine.13 Although, MAO-A may also
metabolize dopamine in the primate and possibly human brain,10
in the aged brain MAO-B is thought to be the major dopamine
metabolizing enzyme.14,15 One reason for this is that the activity
and density of MAO-B increase in most brain regions with age
while MAO-A activity remains unchanged.16,17 In the aged parkin-
sonian brain, MAO-B is therefore considered to be the principal
drug target for reducing the oxidative metabolism of dopamine.
Based on their roles in the degradation of neurotransmitter
amines, MAO-A and -B have attracted attention as pharmacological
targets for the treatment of neurological and neuropsychiatric dis-
orders.6 Inhibitors of MAO-A are used in the management of anxi-
ety disorder and depression while MAO-B inhibitors have been
⇑
Corresponding author. Tel.: +27 18 2992206; fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.005

S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
7041
Furthermore, the clinical use of MAO-A inhibitors have declined in
recent years because of concerns of severe side effects that may
arise from the indirectly-acting sympathomimetic amine, tyra-
mine. Intestinal MAO-A metabolizes tyramine, which is present
in certain foods, and thus reduces the amount of tyramine that en-
ters the systemic circulation. MAO-A inhibitors may enhance tyra-
mine blood levels and lead to a tyramine-induced release of
norepinephrine from peripheral neurons.18 It should, however, be
noted that, in contrast to irreversible inhibitors, reversible MAO-
A inhibitors do not, in general, elevate tyramine levels to such an
extent as to result in sympathomimetic side effects.19
substituents at these positions.28–30 Furthermore, a series of 8-sul-
finylcaffeine analogues (2a–d) was synthesized and their MAO
inhibitory potencies were measured (Fig. 2). The purpose with
these compounds was to compare the MAO inhibitory properties
of the 8-sulfinylcaffeine analogues (2) with those of the 8-sulfanyl-
caffeine analogues (1). This study also reports the MAO inhibition
properties of selected 8-[(phenylpropyl)sulfanyl]caffeine (3a–c)
and 8-(benzylsulfanyl)caffeine analogues (4a–b).
2. Results
MAO-B inhibitors may also have significance in the treatment of
neurodegenerative disorders due to a putative neuroprotective ef-
fect. In the central nervous system, MAO-B inhibitors are thought
to reduce the formation of aldehydes and hydrogen peroxide,
which are produced by the MAO-B-catalyzed oxidation of
amines.20–23 These harmful metabolic by-products may lead to
neurotoxicity and accelerate the neurodegenerative processes
associated with Parkinson’s disease. The aldehydic product derived
from the MAO-catalyzed oxidation of dopamine has been impli-
2.1. Chemistry
The target 8-sulfanylcaffeine analogues, compounds 1a–l, 3a–c
and 4a–b were synthesized according to the literature procedure
as shown in Scheme 1.31 8-Chlorocaffeine (5) was reacted with
an appropriate mercaptan (6) in the presence of NaOH, with 50%
aqueous ethanol serving as solvent. This gave the target 8-sulfanyl-
caffeine analogues in yields of 6.4–83%. 8-Chlorocaffeine, in turn,
was conveniently synthesized in high yield by reacting chlorine
with caffeine in chloroform.32 In certain instances, the mercaptan
starting materials were not commercially available and were thus
synthesized according the literature procedure.33 For this purpose
an appropriate alkylbromide was reacted with thiourea (7) in eth-
anol (Scheme 2). The resulting thiouronium salt (8) was hydro-
lyzed in the presence of NaOH to yield the target mercaptan (6).
The 8-sulfinylcaffeine analogues, 2a–d, were synthesized by react-
ing the 8-sulfanylcaffeines with H2O2 in the presence of glacial ace-
tic acid and acetic anhydride (Scheme 3).31 Both MS and NMR
indicated that the structures of 2a–d were those of the 8-sul-
finylcaffeines and not the corresponding 8-sulfonylcaffeines. This
was apparent from the 1H NMR spectra which yielded two distinc-
tive signals, multiplets integrating for 1 proton each, for the pro-
tons of the –S–CH2– moiety of 2a–c. For the 8-sulfonylcaffeines,
these protons are expected to be equivalent and would lead to a
single 1H NMR signal, a triplet integrating for 2 protons. The 1H
NMR signal of the benzylic CH2 of 2d is a multiplet, indicating non-
equivalence of these protons. With the 8-sulfonylcaffeine homo-
logue, the benzylic protons are expected to be equivalent and
would lead to a singlet. The purities of the target compounds were
estimated via HPLC analysis.
cated in the aggregation of a-synuclein, a process which is associ-
ated with the pathogenesis of Parkinson’s disease.24 Hydrogen
peroxide, in turn, causes oxidative damage and promotes apoptotic
signaling events.25 Considering that MAO-B activity increases in
the brain with age,16,17 the generation of these metabolic by-prod-
ucts by the MAO-B isoform may be especially relevant in Parkin-
son’s disease. Although, irreversible MAO-B inhibitors have been
used in the therapy of Parkinson’s disease, these compounds may
have certain disadvantages. Among these are slow and variable
rates of enzyme recovery following withdrawal of the irreversible
inhibitor.26 For example, the turnover rate for the biosynthesis of
MAO-B in the human brain may be as much as 40 days.27 In con-
trast, following withdrawal of a reversible inhibitor, enzyme activ-
ity is recovered when the inhibitor is eliminated from the tissues.
For these reasons the discovery of new reversible inhibitors may be
of value.
As mentioned above, the present study aims to discover new
reversible inhibitors of the MAO enzymes, particularly the MAO-B
isoform. This study is a continuation of an investigation of the
MAO inhibitory properties of caffeine derived compounds.28,29 We
have recently reported that a series of 8-sulfanylcaffeine analogues
acts as selective inhibitors of human MAO-B.30 Among the com-
pounds examined, 8-[(phenylethyl)sulfanyl]caffeine (1a) was
found to be a particularly potent MAO-B inhibitor with an IC50 value
of 0.223 lM (Fig. 1). In an attempt to further enhance the MAO-B
O
inhibition potency of 1a, and possibly to discover highly potent
MAO-B inhibitors, a series of 8-[(phenylethyl)sulfanyl]caffeine ana-
logues (1a–l) was synthesized and evaluated as inhibitors of human
MAO-A and -B. For the purpose of this study 8-[(phenylethyl)sulfa-
nyl]caffeine homologues containing C3 and C4 alkyl (CF3, CH3,
OCH3) and halogen (Cl, Br, F) substituents on the phenyl ring were
considered. Similar substitution of a series of 8-benzyloxycaffeine
analogues has previously been shown to be beneficial for MAO-B
inhibition.28,29 For the purpose of this study, substitution on C3
and C4 of the phenyl ring was considered since literature reports
that the MAO inhibitory activities of 8-benzyloxycaffeine and
8-sulfanylcaffeine analogues may be significantly attenuated with
O
N
N
N
N
S
S
S
N
O
O
O
N
O
R
2
N
4
N
R
N
O
3
3
O
R
N
N
N
N
N
S
4
N
1a
O
N
Figure 2. The general structures of the 8-sulfinylcaffeine analogues (2), 8-[(phen-
ylpropyl)sulfanyl]caffeine analogues (3) and 8-(benzylsulfanyl)caffeine analogues
(4) which were examined in this study.
Figure 1. The structure of 8-[(phenylethyl)sulfanyl]caffeine (1a).

7042
S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
Table 1
O
N
The IC50 values for the inhibition of recombinant human MAO-A and -B by
compounds 1a–l
N
N
N
Cl
O
+
HS
R
R
O
4
O
5
6
N
3
N
S
N
O
N
N
N
a
N
1a−l
S
R
O
N
R
IC50
(
lM)
SIb
a
MAO-A
MAO-B
1, 3,and 4
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
18.7 0.31
8.46 0.98
108 18.2
14.7 0.94
0.271 0.006
0.020 0.005
0.019 0.003
0.071 0.012
0.019 0.003
0.072 0.012
0.023 0.003
0.043 0.0085
0.040 0.0017
0.017 0.007
0.051 0.005
0.125 0.021
69
4-Cl
4-Br
4-F
4-CF3
4-CH3
4-OCH3
3-Cl
3-Br
423
5684
207
–
1736
7304
132
143
8294
235
385
Scheme 1. Synthetic pathway to 8-sulfanylcaffeine analogues 1, 3 and 4. Reagents
and conditions: (a) NaOH, H2O/ethanol, reflux.
No inhc
125 9.12
168 9.12
R
S
S
Br −
NH2
5.66 0.187
5.70 1.87
141 26.8
12.0 2.39
48.1 5.93
a
Br
R
+
N
NH2
H2
H2N
1j
1k
1l
3-CF3
3-CH3
3-OCH3
7
8
a
b,c
All values are expressed as the mean SD of triplicate determinations.
The selectivity index is the selectivity for the MAO-B isoform and is given as the
HS
R
b
ratio of IC50(MAO-A)/IC50(MAO-B).
6
c
No inhibition observed at a maximal tested concentration of 100 lM.
Scheme 2. Synthetic pathway to the mercaptans (6) which were required for the
synthesis of 8-sulfanylcaffeine analogues. Reagents and conditions: (a) ethanol,
reflux; (b) NaOH, reflux; (c) H2SO4.
0.091 lM under the same conditions (unpublished data from our
laboratory). Interestingly, both alkyl (CF3, CH3, OCH3) and halogen
(Cl, Br, F) substitution lead to highly potent MAO-B inhibition. It
may therefore be concluded that substitution on C3 and C4 is a
general strategy to enhance the MAO-B inhibition potency of 8-
[(phenylethyl)sulfanyl]caffeine (1a). This result is in agreement
with a previous observation that the human MAO-B inhibition po-
O
N
O
N
N
N
O
N
N
N
a
S
S
R
N
O
R
tency of 8-(benzylsulfanyl)caffeine (4c; IC50 = 1.86 lM) may be im-
O
proved with halogen (Cl, Br and F) substitution on the para position
2
of the benzyl ring, yielding compounds with IC50 values ranging
Scheme 3. Synthetic pathway to 8-sulfinylcaffeine analogues 2. Reagents and
conditions: (a) H2O2, glacial acetic acid, acetic anhydride, rt.
from 0.167 to 0.348
substitution with chlorine (4a; IC50 = 0.227
IC50 = 0.199 M) enhances the MAO-B inhibition potency of 8-
l
M.30 The present study also shows that meta
lM) and bromine (4b;
l
(benzylsulfanyl)caffeine (4c). The 8-[(phenylethyl)sulfanyl]caf-
feine analogues are, however, significantly more potent MAO-B
inhibitors than the corresponding 8-(benzylsulfanyl)caffeines. For
example, the 8-[(phenylethyl)sulfanyl]caffeine analogues substi-
2.2. MAO inhibition studies
The MAO inhibitory properties of the test compounds were
examined using the recombinant human enzymes. The mixed
MAO-A/B substrate, kynuramine, was employed as substrate for
both enzymes. Kynuramine exhibits similar Km values for the
tuted with chlorine on the meta (1h; IC50 = 0.043
(1b; IC50 = 0.020 M) positions of the phenyl ring are fivefold and
ninefold, respectively, more potent than the corresponding meta
(4a; IC50 = 0.227 M) and para (IC50 = 0.192
M)30 chlorine substi-
lM) and para
l
two isozymes of 16.1 and 22.7 l
M, respectively.28 Kynuramine is
l
l
oxidized by the MAOs to yield 4-hydroxyquinoline, a fluorescent
compound, which is readily measurable in the presence of the
non-fluorescent kynuramine and the test inhibitors investigated
in the current study.
tuted 8-(benzylsulfanyl)caffeines. Similarly, the 8-[(phenyl-
ethyl)sulfanyl]caffeines containing bromine on the meta (1i;
IC50 = 0.040
phenyl ring are fivefold and eightfold, respectively, more potent
than the corresponding meta (4b; IC50 = 0.199 M) and para
(IC50 = 0.167
M)30 bromine substituted 8-(benzylsulfanyl)caffe-
lM) and para (1c; IC50 = 0.019 lM) positions of the
l
2.2.1. Inhibition of MAO-B
l
The IC50 values for the inhibition of human MAO by the 8-sul-
fanylcaffeines 1a–l are given in Table 1. As shown, the lead com-
pound for this study, compound 1a, inhibits MAO-B with an IC50
ines. Based on these analyses, it may be concluded that 8-[(phen-
ylethyl)sulfanyl]caffeines with substituents on the phenyl ring
are exceptionally potent MAO-B inhibitors and suitable lead com-
pounds for the design of novel inhibitors of this enzyme.
value of 0.271
by us (IC50 = 0.223
l
M. This value is similar to that previously reported
l
M).30 The results further show that substitu-
The IC50 values for the inhibition of human MAO by the 8-sul-
finylcaffeines 2a–d are given in Table 2. The results document
that the 8-sulfinylcaffeines also are inhibitors of MAO-B with IC50
tion on the phenyl ring of 1a leads to a considerable enhancement
of its MAO-B inhibition potency, with all of the substituted homo-
logues exhibiting more potent MAO-B inhibition than 1a. The IC50
values recorded for these homologues (1b–l) ranged from 0.017–
values of 0.471–131 lM. Compared to the 8-sulfanylcaffeines,
these homologues are, however, weaker inhibitors. For example,
0.125 lM, making them twofold to 16-fold more potent MAO-B
the most potent 8-sulfinylcaffeine inhibitor, compound 2b
inhibitors than the lead compound. For comparison, the reversible
(IC50 = 0.471 lM), is approximately 25-fold weaker as a MAO-B
MAO-B selective inhibitor, lazabemide, exhibits an IC50 value of

S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
7043
inhibitor than its corresponding 8-sulfanylcaffeine homologue,
compound 1c (IC50 = 0.019 M). Similarly, the 8-sulfinylcaffeine
2a (IC50 = 0.781 M) is 39-fold weaker as a MAO-B inhibitor than
its corresponding 8-sulfanylcaffeine homologue, compound 1b
(IC50 = 0.02 M). It may, therefore, be concluded that 8-sul-
finylcaffeines are comparatively weak MAO-B inhibitors and less
suited for the design of high potency MAO-B inhibitors.
This study also examined the MAO inhibitory properties of a
limited series of 8-[(phenylpropyl)sulfanyl]caffeine analogues
(3a–c). The results are given in Table 3, and shows that these com-
pounds are also inhibitors of MAO-B with IC50 values of 0.061–
are also MAO-A inhibitors with IC50 values of 0.708–6.48
noteworthy that 3b–c are the most potent MAO-A inhibitors among
the compounds evaluated in this study. In fact, 3b (IC50 = 0.708 M)
l
M. It is
l
l
l
is the only compound with an IC50 value for the inhibition of MAO-A
in the submicromolar range. Although, more potent as MAO-A
inhibitors, compounds 3b–c are still MAO-B selective inhibitors
with SI values of 12 and 57, respectively. The 8-[(phenylpropyl)sul-
fanyl]caffeines 3a–c display lower degrees of selectivity for MAO-B
than the corresponding 8-[(phenylethyl)sulfanyl]caffeines. For
example, compounds 3a–c exhibit SI values of 12–57, while the
equivalently substituted 8-[(phenylethyl)sulfanyl]caffeine homo-
logues, compounds 1a, 1b and 1h, display SI values of 69–423.
l
0.500 lM. Those homologues substituted with chlorine on the para
and meta positions of the phenyl ring, compounds 3b–c, were
found to be exceptionally potent inhibitors with IC50 values of
2.3. Reversibility of inhibition
0.061 and 0.062
less active than the corresponding 8-[(phenylethyl)sulfanyl]caf-
feine homologues, compounds 1h (IC50 = 0.043 M) and 1b
(IC50 = 0.020 M). It may therefore be concluded that, although,
less active than the 8-[(phenylethyl)sulfanyl]caffeines, 8-[(phenyl-
propyl)sulfanyl]caffeine substituted on the phenyl ring may repre-
sent suitable lead compounds for the design of MAO-B inhibitors.
lM, respectively. These compounds are slightly
8-(Benzylsulfanyl)caffeine analogues have previously been
shown to interact reversibly with human MAO-A and -B.30 This is
advantageous since the activity of the MAO enzymes are recovered
when treatment is terminated and the inhibitor is cleared from the
tissues. For irreversible inhibitors, de novo synthesis is required for
enzyme activity to recover. Although, it is likely that 8-[(phenyl-
ethyl)sulfanyl]caffeines also interact reversibly with MAO-A and -
B, this study verified this expectation with a representative inhib-
itor, compound 1b. In order to evaluate reversibility, the time-
dependence of MAO inhibition was examined. Compound 1b, at
concentrations approximately twofold its measured IC50 values,
was preincubated with human MAO-A or -B for 0, 15, 30 and
60 min. Following these preincubations the reactions were diluted
twofold, to yield concentrations of the inhibitor that are equal to its
IC50 values for the inhibition of the respective enzymes. The resid-
ual MAO catalytic activities were subsequently measured. The re-
sults of these reversibility studies are shown in Figure 3. As
shown, when 1b is preincubated with MAO-A no time-dependent
reductions of the catalytic activities are observed, even following
a period of 60 min preincubation with the enzymes. After preincu-
bation with MAO-B, only a slight time-dependent reduction of
catalysis is observed. These data are consistent with a reversible
interaction of 1b with both MAO enzymes.
The reversible interaction of 1b with MAO-B was further inves-
tigated by evaluating the recovery of the enzymatic activity after
dilution of the enzyme-inhibitor complex. For this purpose,
MAO-B was preincubated with compound 1b at concentrations
of 10 Â IC50 and 100 Â IC50 for 30 min. The reactions were subse-
quently diluted 100-fold to 0.1 Â IC50 and 1 Â IC50, respectively.
The results are given in Figure 4 and show that, after dilution to
0.1 Â IC50 and 1 Â IC50, the MAO-B catalytic activities are recovered
to 58% and 26%, respectively, of the control value. For reversible
enzyme inhibition, the enzyme activities are expected to recover
to levels of approximately 90% and 50%, respectively, after 100-fold
dilution of the preincubations containing inhibitor concentrations
of 10 Â IC50 and 100 Â IC50. After preincubation of MAO-B with
the irreversible inhibitor (R)-deprenyl (at 10 Â IC50), and dilution
of the resulting complex to 0.1 Â IC50, MAO-B activity is not recov-
ered (3.0% of control). These data indicate that 1b interacts revers-
ibly with MAO-B. Interestingly, after dilution, the enzyme activities
are not recovered to 90% and 50%, respectively, as expected. This
result suggests that the binding of 1b may possess a quasi-revers-
ible or tight-binding component. This behavior was also observed
with other 8-[(phenylethyl)sulfanyl]caffeines. For example, after
similar preincubation of compound 1j with MAO-B and dilution
of the resulting enzyme-inhibitor complexes, the MAO-B catalytic
activities are recovered to only 35% and 22%, respectively, of the
control value (Fig. 4). This also supports a tight-binding mecha-
nism, and further investigation is necessary to clarify this point.
The interaction of inhibitors via a potential tight-binding mecha-
nism has been reported previously. For example, chromone car-
l
l
2.2.2. Inhibition of MAO-A
The results of the human MAO inhibition studies show that the
8-sulfanylcaffeines 1a–l are relatively weak MAO-A inhibitors with
IC50 values of 5.66–168 lM (Table 1). Compound 1e did not exhibit
inhibition towards MAO-A. Interestingly, those compounds substi-
tuted on the meta position (1h–1l) of the phenyl ring were more
potent MAO-A inhibitors than the corresponding homologues
substituted on the para (1b–1g) position. For example, the meta
substituted chlorine and bromine homologues, compounds 1h
(IC50 = 5.66
respectively, more potent than the corresponding para substituted
homologues, compounds 1b (IC50 = 8.46 M) and 1c
(IC50 = 108 M). As evident from the selectivity indices (SI values),
lM) and 1i (IC50 = 5.70 lM), were 1.5-fold and 19-fold,
l
l
compounds 1a–l were all selective inhibitors of the MAO-B iso-
form. Five compounds (1c, 1e, 1f, 1g, and 1j) exhibited SI values
in excess of 1000. Since these compounds are also highly potent
MAO-B inhibitors, they represent suitable leads for the design of
potent and selective MAO-B inhibitors.
The results document that the 8-sulfinylcaffeines 2a–d are weak
MAO-A inhibitors with IC50 values of 57.3–250 lM (Table 2). The SI
values demonstrate that these compounds are MAO-B selective
inhibitors, although, to a lesser degree than the 8-sulfanylcaffeines
1a–l. Table 3 shows that 8-[(phenylpropyl)sulfanyl]caffeines 3a–c
Table 2
The IC50 values for the inhibition of recombinant human MAO-A and -B by
compounds 2a–d
R
O
O
N
O
O
N
N
N
N
S
S
N
N
O
N
O
2d
2a−c
R
IC50
(
l
M)a
MAO-B
SIb
MAO-A
2a
Cl
Br
F
203 14.4
57.3 12.8
239 17.2
250 18.1
0.781 0.048
0.471 0.057
3.50 0.083
131 33.4
260
122
68
2b
2c
2d
H
2
a
All values are expressed as the mean SD of triplicate determinations.
b
The selectivity index is the selectivity for the MAO-B isoform and is given as the
ratio of IC50(MAO-A)/IC50(MAO-B).

7044
S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
Table 3
The IC50 values for the inhibition of recombinant human MAO-A and -B by compounds 3a–c and 4a–c
O
O
R
N
N
N
N
S
4
S
N
N
R
O
N
O
N
3
3a−c
4a−c
R
IC50
(l
M)a
SIb
MAO-A
MAO-B
3a
3b
3c
4a
4b
4c
H
6.48 0.802
0.708 0.124
3.53 0.381
6.43 1.55
37.9 1.39
8.22c
0.500 0.041
0.061 0.004
0.062 0.008
0.227 0.024
0.199 0.039
1.86c
13
12
57
28
190
4-Cl
3-Cl
Cl
Br
H
4.4
a
b
c
All values are expressed as the mean SD of triplicate determinations.
The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of IC50(MAO-A)/IC50(MAO-B).
Taken form reference.30
boxamides display a quasi-reversible binding to human MAO-
B,34,35 while oxadiazolones are reported to bind reversibly and
time-dependently to rat brain MAO-B.36 Mazouz and colleagues
has also described a series of reversible and extremely tight-bind-
ing MAO-B specific inhibitors.37
The reversibility of MAO-B inhibition by compound 1b was fur-
ther demonstrated by constructing a set of Lineweaver–Burk plots
for the inhibition of MAO-B. For this purpose, the initial catalytic
rates were measured in the presence of three different concentra-
tions of 1b, and in the absence of inhibitor. These measurements
were carried out using four different concentrations of the sub-
2.4. Lipophilicity
To further examine the potential of 8-[(phenylethyl)sulfa-
nyl]caffeines to serve as lead compounds for the treatment of cen-
tral nervous system disorders, the octanol/water partition
coefficients (P) of selected homologues were measured. The parti-
tion coefficient is a measure of the lipophilicity of a compound, a
property which has a major effect on the permeability of a com-
pound through biological membranes. Lipophilicity estimates
may thus be used to predict the likelihood of a compound to pen-
etrate the blood–brain barrier. As shown in Table 4, all of the eval-
uated compounds may be viewed as lipophilic since they display
LogP values in excess of 2.55. As a general guide, LogP values rang-
ing from 0–3 are optimal for passive diffusion permeability and
such compounds are expected to display good oral bioavailability
and blood–brain barrier permeation.39 While the measurement of
additional properties such as solubility, permeability, metabolism
and protein binding will provide a clearer picture, these prelimin-
ary results suggest that particularly compounds 1c and 1g may
possess suitable properties to gain access to the central nervous
system. These compounds are of further interest since they also
display potent MAO-B inhibition.
strate, kynuramine (15–90 lM). Fig. 5 illustrates the set of Linewe-
aver–Burk plots that were obtained from these studies. The results
show that the Lineweaver–Burk plots are linear and intersect at
y = 0. This indicates that the inhibition of MAO-B by 1b is compet-
itive, and is further evidence that 1b is a reversible inhibitor of
MAO-B. From a replot of the slopes of the Lineweaver–Burk plots
versus inhibitor concentration, a Ki value of 0.013 lM was obtained
for the inhibition of MAO-B by 1b. Since this inhibitor may exhibit
tight-binding to MAO-B, the inhibition data were analyzed accord-
ing to the equation by Morrison.38 This analysis gave a Ki value of
0.0098 0.002 nM, a value that is similar to that obtained from the
Lineweaver–Burk plots.
100
100
MAO-A
MAO-B
75
75
50
*
*
*
50
25
0
*
*
*
25
0
No Inhibitor
0
15
30
60
No Inhibitor
0
15
30
60
Incubation time (min)
Incubation time (min)
Figure 3. The time-dependency of inhibition of recombinant human MAO-A and -B by inhibitor 1b. The MAO enzymes were preincubated for various periods of time (0–
60 min) in the presence of 1b at concentrations of 16.92 and 0.04 lM, respectively. These concentrations are equal to twofold the recorded IC50 values for the inhibition of
MAO-A and -B, respectively. The preincubation mixtures were diluted twofold and the residual MAO catalytic rates were measured. ⁄Statistically significantly different from
the mean at t = 0.

S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
7045
A
B
100
75
50
25
0
100
75
50
25
0
l
r
r
o
y
yl
n
C50
C50
C50
=
C50
o
t
t
i
n
i
I
e
I
e
r
b
b
i
r
i
x
h
p
h
n
I
p
n
I
e
1 x I
1 x
1
.
1 x I
.
D
De
-
0
=
0
-
)
)
]
]
No
=
I
[
No
=
]
I
R
[
R
(
]
(
I
I
[
[
Figure 4. Reversibility of inhibition of MAO-B by 1b (panel A) and 1j (panel B). The enzyme was preincubated with 1b or 1j at 10 Â IC50 and 100 Â IC50 for 30 min and then
diluted to 0.1 Â IC50 and 1 Â IC50, respectively. As control, MAO-B was also preincubated with (R)-deprenyl at 10 Â IC50 and subsequently diluted to 0.1 Â IC50. The residual
enzyme activities were subsequently measured.
100
A
1500
B
75
1000
50
500
25
0
-0.02
0.00
0.02
0.04
0.06
-0.01
0.00
0.01
0.02
1/[S]
[I], µM
Figure 5. Lineweaver–Burk plots of the oxidation of kynuramine by recombinant human MAO-B (Panel A). The plots were constructed in the absence (filled squares) and
presence of various concentrations of 1b. The concentrations of 1b employed were 0.005 M (open squares), 0.01 M (filled circles) and 0.02 M (open circles), respectively. A
l
l
l
replot of the slopes of the Lineweaver–Burk plots versus inhibitor concentration (Panel B).
however, yields homologues with potent MAO-B inhibition activi-
ties. In this respect para substitution generally leads to slightly
more potent MAO-B inhibitors than meta substitution. Although,
the MAO-B inhibition potencies of the 8-[(phenylethyl)sulfa-
nyl]caffeine analogues are very similar, the following additional
structure–activity relationships (SARs) are apparent: (1) substitu-
tion on both C3 and C4 of the phenyl ring yields improved inhibi-
tory potency; (2) methoxy substitution on C3 yields the weakest
MAO-B inhibitor of the series, compound 1l with an IC50 value of
Table 4
The LogP values of selected compounds as estimated by the shake flask method.
LogP valuea
LogP valuea
1a
1c
1e
1f
3.22 0.008
2.98 0.029
3.21 0.024
3.15 0.008
2.55 0.026
1j
3.14 0.019
3.30 0.030
3.59 0.059
3.76 0.051
3a
3b
3c
1g
a
All values are expressed as the mean SD of triplicate determinations.
0.125 lM; (3) in general, both halogen and alkyl substitution yields
highly potent MAO-B inhibitors. For the inhibition of MAO-A, no
clear SAR trends is apparent. It is however noteworthy that the
most potent MAO-A inhibitors among the 8-[(phenylethyl)sulfa-
nyl]caffeine analogues are the C3 chlorine and bromine substituted
homologues. The relatively large degree of tolerance for different
substituents and substitution patterns makes 8-[(phenylethyl)sul-
fanyl]caffeine analogues good candidates for the design of MAO-B
inhibitors since structural modifications that may lead to better
drug properties are less likely to be associated with a loss of activ-
ity. This study also shows that 8-[(phenylethyl)sulfanyl]caffeine
analogues are significantly more potent as MAO-B inhibitors than
3. Discussion and conclusion
The present study examines the possibility of improving the
MAO inhibitory properties of 1a, a compound previously shown
to be a potent and selective MAO-B inhibitor.30 The results show
that substitution on the phenyl ring of 1a leads to an enhancement
of MAO-B inhibition potency. The most potent inhibitor of this
study, the 3-CF3 substituted homologue (1j), is approximately
16-fold more potent as a MAO-B inhibitor than 1a, and exhibits a
higher degree of selectivity for MAO-B. A variety of substituents,

7046
S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
the corresponding 8-(benzylsulfanyl)caffeines investigated
previously.30
reflux for 60 min and then cooled to room temperature. The white
precipitate present in the reaction was collected by filtration and
washed with 30 mL ethanol. The product was recrystallized from
30 mL ethanol at room temperature and the crystals were washed
with 30 mL ethanol.31 TLC was conducted using silica gel sheets
(Merck) and ethylacetate/n-hexane (60:30) as mobile phase. (Rf
ꢀ0.45).
Interestingly 8-[(phenylpropyl)sulfanyl]caffeine analogues (3a–
c) are also highly potent MAO-B inhibitors with comparable poten-
cies to those of the 8-[(phenylethyl)sulfanyl]caffeines. Compounds
3a–c are, however, less selective for MAO-B. In fact, 3b is the most
potent MAO-A inhibitor among the investigated compounds, and
the only inhibitor with an IC50 value in the submicromolar range.
Although, dopamine is metabolized by both MAO-A and -B in the
human brain, the inhibition of MAO-A is associated with poten-
tially dangerous side effects, and highly selective MAO-B inhibitors
may therefore be more desirable for the treatment of Parkinson’s
disease. Furthermore, the combination of MAO-A inhibitors and
levodopa in PD therapy should be avoided since this may lead to
a hypertensive response.40 Based on this analysis, 8-[(phenyl-
ethyl)sulfanyl]caffeines may be better suited as antiparkinsonian
drugs than 8-[(phenylpropyl)sulfanyl]caffeines, since several 8-
4.2.1. 8-[(Phenylethyl)sulfanyl]caffeine (1a)
The title compound was prepared from 2-phenylethane-1-thiol
in a yield of 83%: mp 95 °C (ethanol). 1H NMR (Bruker Avance III
600, CDCl3) d 3.04 (t, 2H, J = 7.5 Hz), 3.36 (s, 3H), 3.48 (t, 2H,
J = 7.5 Hz); 3.55 (s, 3H), 3.78 (s, 3H), 7.21 (m, 3H), 7.28 (m, 2H);
13C NMR (Bruker Avance III 600, CDCl3) d 27.8, 29.6, 32.1, 33.8,
36.0, 108.5, 126.7, 128.5, 139.3, 148.4, 150.8, 151.4, 154.5; EI-
HRMS m/z: Calcd for C16H18N4O2S, 330.1151. Found 330.1147; Pur-
ity (HPLC): 98%.
[(phenylethyl)sulfanyl]caffeines are highly potent (IC50 < 0.05 lM)
MAO-B inhibitors with SI values in excess of 1000. The 8-sulfinyl-
caffeine analogues on the other hand exhibited comparatively
weak MAO-B inhibition and are thus not suited for the design of
MAO inhibitors.
4.2.2. 8-{[2-(4-Chlorophenyl)ethyl]sulfanyl}caffeine (1b)
The title compound was prepared from 2-(4-chlorophenyl)eth-
ane-1-thiol in a yield of 69%: mp 164 °C (ethanol). 1H NMR (Bruker
Avance III 600, CDCl3) d 3.01 (t, 2H, J = 7.5 Hz), 3.35 (s, 3H), 3.45 (t,
2H, J = 7.5 Hz); 3.54 (s, 3H), 3.78 (s, 3H), 7.12 (d, 2H, J = 8.3 Hz),
7.23 (d, 2H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) d
27.8, 29.6, 32.1, 33.6, 35.4, 108.5, 128.6, 129.9, 132.5, 137.7,
148.4, 150.5, 151.4, 154.5; EI-HRMS m/z: Calcd for C16H17ClN4O2S,
364.0761. Found 364.0756; Purity (HPLC): 99%.
4. Experimental section
4.1. Chemicals and instrumentation
Unless otherwise noted, all starting materials and reagents
were obtained from Sigma–Aldrich and were used without further
purification. Proton (1H) and carbon (13C) NMR spectra were re-
corded on a Bruker Avance III 600 spectrometer at frequencies of
600 MHz and 150 MHz, respectively, with CDCl3 serving as NMR
solvent. All chemical shifts are reported in parts per million (d)
downfield from the signal of Si(CH3)4 added to the CDCl3. Spin mul-
tiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet),
qn (quintet) or m (multiplet). High resolution mass spectra (HRMS)
were recorded on a DFS high resolution magnetic sector mass spec-
trometer (Thermo Electron Corporation) in electron ionization (EI)
mode or on a Bruker micrOTOF-Q II mass spectrometer in atmo-
spheric-pressure chemical ionization (APCI) mode. Melting points
(mp) were determined with a Buchi M-545 melting point appara-
tus and are uncorrected. Thin layer chromatography (TLC) was car-
ried out using silica gel 60 (Merck) with UV254 fluorescent
indicator. The purities of the synthesized compounds were esti-
mated via HPLC analyses. For this purpose an Agilent 1100 HPLC
system equipped with a quaternary pump and an Agilent 1100 ser-
ies diode array detector were employed (see Supplementary data).
HPLC grade acetonitrile (Merck) and Milli-Q water (Millipore) were
used for the chromatography. The mercaptan starting materials
(6), which were not commercially available, were synthesized
according to the literature procedure.33 Fluorescence spectropho-
tometry was carried out with a Varian Cary Eclipse fluorescence
spectrophotometer while UV–vis spectrophotometry was carried
out with a Shimadzu MultiSpec-1501 UV–vis photodiode array
spectrophotometer. Microsomes from insect cells containing re-
combinant human MAO-A and -B (5 mg/mL), and kynur-
amineÁ2HBr were obtained from Sigma–Aldrich.
4.2.3. 8-{[2-(4-Bromophenyl)ethyl]sulfanyl}caffeine (1c)
The title compound was prepared from 2-(4-bromophenyl)eth-
ane-1-thiol in a yield of 38%: mp 165 °C (ethanol). 1H NMR (Bruker
Avance III 600, CDCl3) d 3.01 (t, 2H, J = 7.5 Hz), 3.37 (s, 3H), 3.45 (t,
2H, J = 7.5 Hz); 3.54 (s, 3H), 3.79 (s, 3H), 7.07 (d, 2H, J = 8.7 Hz),
7.39 (d, 2H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) d
27.8, 29.7, 32.1, 33.6, 35.5, 108.6, 120.6, 130.3, 131.6, 138.2,
148.4, 150.5, 151.5, 154.5; EI-HRMS m/z: Calcd for C16H17BrN4O2S,
408.0256. Found 408.0258; Purity (HPLC): 97%.
4.2.4. 8-{[2-(4-Fluorophenyl)ethyl]sulfanyl}caffeine (1d)
The title compound was prepared from 2-(4-fluorophenyl)eth-
ane-1-thiol in a yield of 53%: mp 145 °C (ethanol). 1H NMR (Bruker
Avance III 600, CDCl3) d 3.01 (t, 2H, J = 7.5 Hz), 3.36 (s, 3H), 3.45 (t,
2H, J = 7.5 Hz); 3.55 (s, 3H), 3.78 (s, 3H), 6.97 (t, 2H, J = 8.7 Hz), 7.16
(m, 2H); 13C NMR (Bruker Avance III 600, CDCl3) d 27.8, 29.7, 32.1,
33.8, 35.2, 108.6, 115.4, 130.0, 135.0, 135.1, 148.4, 150.7, 151.4,
154.5, 160.9, 162.5; EI-HRMS m/z: Calcd for
C16H17FN4O2S,
348.1056. Found 348.1045; Purity (HPLC): 99%.
4.2.5. 8-{[2-(4-(Trifluoromethyl)phenyl)ethyl]sulfanyl}caffeine
(1e)
The title compound was prepared from 2-[4-(trifluoro-
methyl)phenyl]ethane-1-thiol in a yield of 37%: mp 178 °C (etha-
nol). 1H NMR (Bruker Avance III 600, CDCl3) d 3.12 (t, 2H,
J = 7.5 Hz), 3.36 (s, 3H), 3.49 (t, 2H, J = 7.5 Hz); 3.54 (s, 3H), 3.79
(s, 3H), 7.33 (d, 2H, J = 7.9 Hz), 7.54 (d, 2H, J = 8.2 Hz); 13C NMR
(Bruker Avance III 600, CDCl3) d 27.8, 29.6, 32.1, 33.3, 35.8, 108.6,
121.4, 123.2, 125.0, 125.5 (q), 126.8, 128.7, 128.9, 129.2, 129.4,
143.3, 148.4, 150.4, 151.4, 154.5; EI-HRMS m/z: Calcd for
C17H17F3N4O2S, 398.1024. Found 398.1007; Purity (HPLC): 99%.
4.2. Synthesis of 8-sulfanylcaffeine analogues (1, 3 and 4)
4.2.6. 8-{[2-(4-Methylphenyl)ethyl]sulfanyl}caffeine (1f)
To a solution of NaOH (4 mmol) in 3.5 mL water was added a
volume of 3.5 mL ethanol. The mixture was cooled in an ice bath
and an appropriate thiol (4 mmol) was added. While the reaction
mixture was stirred, 8-chlorocaffeine (4 mmol) was added in a sin-
gle portion to yield a suspension. The reaction was heated under
The title compound was prepared from 2-(4-methylphenyl)eth-
ane-1-thiol in a yield of 36%: mp 122 °C (ethanol). 1H NMR (Bruker
Avance III 600, CDCl3) d 2.29 (s, 3H), 2.99 (t, 2H, J = 7.5 Hz), 3.36 (s,
3H), 3.46 (t, 2H, J = 7.5 Hz), 3.55 (s, 3H), 3.78 (s, 3H), 7.09 (m, 4H);
13C NMR (Bruker Avance III 600, CDCl3) d 21.0, 27.8, 29.6, 32.1,

S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
7047
34.0, 35.6, 108.4, 128.4, 129.2, 136.3, 148.4, 150.9, 151.5, 154.5; EI-
HRMS m/z: Calcd for C17H20N4O2S, 344.1307. Found 344.1307; Pur-
ity (HPLC): 97%.
29.7, 32.1, 33.8, 36.1, 55.1, 108.5, 111.6, 114.6, 120.9, 129.5,
140.9, 148.5, 150.8, 151.5, 154.5, 159.7; EI-HRMS m/z: Calcd for
C17H20N4O3S, 360.1256. Found 360.1255; Purity (HPLC): 97%.
4.2.7. 8-{[2-(4-Methoxyphenyl)ethyl]sulfanyl}caffeine (1g)
The title compound was prepared from 2-(4-methoxy-
phenyl)ethane-1-thiol in a yield of 45%: mp 125 °C (ethanol). 1H
NMR (Bruker Avance III 600, CDCl3) d 2.98 (t, 2H, J = 7.5 Hz), 3.36
(s, 3H), 3.44 (t, 2H, J = 7.5 Hz); 3.55 (s, 3H), 3.76 (s, 3H), 3.79(s,
3H), 6.82 (d, 2H, J = 8.7 Hz), 7.11 (d, 2H, J = 8.7 Hz); 13C NMR (Bru-
ker Avance III 600, CDCl3) d 27.8, 29.7, 32.1, 34.1, 35.2, 55.2, 108.5,
113.9, 129.5, 131.4, 148.5, 151.0, 151.5, 154.5, 158.4; EI-HRMS m/z:
Calcd for C17H20N4O3S, 360.1256. Found 360.1248; Purity (HPLC):
99%.
4.2.13. 8-[(Phenylpropyl)sulfanyl]caffeine (3a)
The title compound was prepared from 3-phenylpropane-1-
thiol in a yield of 21%: mp 76.4–78.3 °C (ethanol). 1H NMR (Bruker
Avance III 600, CDCl3) d 2.07 (qn, 2H, J = 7.5 Hz), 2.75 (t, 2H,
J = 7.5 Hz), 3.24 (t, 2H, J = 7.5 Hz), 3.36 (s, 3H), 3.50 (s, 3H), 3.81
(s, 3H), 7.16–7.19 (m, 3H), 7.24–7.28 (m, 2H); 13C NMR (Bruker
Avance III 600, CDCl3) d 27.8, 29.6, 31.1, 32.0, 32.1, 34.5, 108.5,
126.1, 128.4, 128.4, 140.7, 148.4, 151.0, 151.5, 154.5; EI-HRMS
m/z: Calcd for C17H20N4O2S, 344.1307. Found 344.1308; Purity
(HPLC): 99%.
4.2.8. 8-{[2-(3-Chlorophenyl)ethyl]sulfanyl}caffeine (1h)
The title compound was prepared from 2-(3-chlorophenyl)eth-
ane-1-thiol in a yield of 6.4%: mp 125.5–126.1 °C (ethanol). 1H
NMR (Bruker Avance III 600, CDCl3) d 3.02 (t, 2H, J = 7.2 Hz), 3.36
(s, 3H), 3.47 (t, 2H, J = 7.2 Hz); 3.55 (s, 3H), 3.78 (s, 3H), 7.08 (d,
1H, J = 7.5 Hz), 7.16–7.22 (m, 3H); 13C NMR (Bruker Avance III
600, CDCl3) d 27.8, 29.7, 32.1, 33.5, 35.8, 108.6, 126.8, 126.9,
128.7, 129.7, 134.3, 141.2, 148.4, 150.5, 151.5, 154.5; EI-HRMS
m/z: Calcd for C16H17ClN4O2S, 364.0761. Found 364.0757; Purity
(HPLC): 97%.
4.2.14. 8-{[3-(4-Chlorophenyl)propyl]sulfanyl}caffeine (3b)
The title compound was prepared from 3-(4-chlorophenyl)pro-
pane-1-thiol in a yield of 27%: mp 95.5–98.0 °C (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3) d 2.04 (qn, 2H, J = 7.5 Hz), 2.72 (t, 2H,
J = 7.5 Hz), 3.22 (t, 2H, J = 7.5 Hz), 3.36 (s, 3H), 3.50 (s, 3H), 3.81 (s,
3H), 7.09 (d, 2H, J = 8.7 Hz), 7.22 (d, 2H, J = 8.3 Hz); 13C NMR (Bru-
ker Avance III 600, CDCl3) d 27.8, 29.6, 31.0, 31.7, 32.1, 33.8, 108.5,
128.5, 129.7, 131.9, 139.1, 148.4, 150.8, 151.5, 154.5; EI-HRMS m/z:
Calcd for
C17H19ClN4O2S, 378.0917. Found 378.0903; Purity
(HPLC): 94%.
4.2.9. 8-{[2-(3-Bromophenyl)ethyl]sulfanyl}caffeine (1i)
4.2.15. 8-{[3-(3-Chlorophenyl)propyl]sulfanyl}caffeine (3c)
The title compound was prepared from 3-(3-chlorophenyl)pro-
pane-1-thiol in a yield of 24%: mp 87.7–89.3 °C (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3) d 2.07 (qn, 2H, J = 7.5 Hz), 2.73 (t, 2H,
J = 7.5 Hz), 3.23 (t, 2H, J = 7.5 Hz), 3.36 (s, 3H), 3.50 (s, 3H), 3.81 (s,
3H), 7.04 (d, 1H, J = 7.5 Hz), 7.15 (m, 2H), 7.19 (m, 1H); 13C NMR
(Bruker Avance III 600, CDCl3) d 27.8, 29.7, 30.8, 31.7, 32.1, 34.2,
108.5, 126.3, 126.6, 128.5, 129.7, 134.2, 142.7, 148.4, 150.8,
151.5, 154.5; EI-HRMS m/z: Calcd for C17H19ClN4O2S, 378.0917.
Found 378.0902; Purity (HPLC): 98%.
The title compound was prepared from 2-(3-bromophenyl)eth-
ane-1-thiol in a yield of 32%: mp 122.6–124.3 °C (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3) d 3.01 (t, 2H, J = 7.2 Hz), 3.36 (s, 3H),
3.47 (t, 2H, J = 7.2 Hz); 3.55 (s, 3H), 3.77 (s, 3H), 7.11–7.15 (m, 2H),
7.31–7.32 (m, 1H), 7.35 (m, 1H); 13C NMR (Bruker Avance III 600,
CDCl3) d 27.8, 29.7, 32.1, 33.5, 35.8, 108.6, 122.5, 127.3, 129.8,
130.0, 131.7, 141.5, 148.4, 150.5, 151.5, 154.5; EI-HRMS m/z: Calcd
for C16H17BrN4O2S, 408.0256. Found 408.0251; Purity (HPLC): 99%.
4.2.10. 8-{[2-(3-(Trifluoromethyl)phenyl)ethyl]sulfanyl}caffeine
(1j)
4.2.16. 8-[(3-Chlorobenzyl)sulfanyl]caffeine (4a)
The title compound was prepared from 2-[3-(trifluoro-
The title compound was prepared from (3-chlorophenyl)meth-
anethiol in a yield of 51%: mp 156.5–158.1 °C (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3) d 3.35 (s, 3H), 3.56 (s, 3H), 3.73 (s,
3H), 4.39 (s, 2H), 7.21 (m, 3H), 7.38 (s, 1H); 13C NMR (Bruker
Avance III 600, CDCl3) d 27.8, 29.7, 32.2, 36.3, 108.8, 127.1, 128.0,
129.3, 129.9, 134.3, 138.7, 148.3, 149.6, 151.4, 154.5; EI-HRMS
m/z: Calcd for C15H15ClN4O2S, 350.0604. Found 350.0600; Purity
(HPLC): 99%.
methyl)phenyl]ethane-1-thiol in
a yield of 18%: mp 130.4–
132.7 °C (ethanol). 1H NMR (Bruker Avance III 600, CDCl3) d 3.12
(t, 2H, J = 7.5 Hz), 3.37 (s, 3H), 3.50 (t, 2H, J = 7.5 Hz), 3.55 (s, 3H),
3.78 (s, 3H), 3.40–3.42 (m, 2H), 3.46–3.48 (m, 2H); 13C NMR (Bru-
ker Avance III 600, CDCl3) d27.8, 29.7, 32.12, 33.4, 35.9, 108.6,
123.1 (d), 125.3 (d), 129.0, 130.9 (q), 132.0, 140.2, 148.4, 150.4,
151.5, 154.5; EI-HRMS m/z: Calcd for C17H17F3N4O2S, 398.1024.
Found 398.1031; Purity (HPLC): 99%.
4.2.17. 8-[(3-Bromobenzyl)sulfanyl]caffeine (4b)
4.2.11. 8-{[2-(3-Methylphenyl)ethyl]sulfanyl}caffeine (1k)
The title compound was prepared from 2-(3-methylphenyl)eth-
ane-1-thiol in a yield of 28%: mp 110.5–112.0 °C (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3) d 2.31 (s, 3H), 3.00 (t, 2H, J = 7.5 Hz),
3.37 (s, 3H), 3.48 (t, 2H, J = 7.5 Hz), 3.56 (s, 3H), 3.79 (s, 3H), 7.00–
7.03 (m, 3H), 7.17 (t, 1H, J = 7.5 Hz); 13C NMR (Bruker Avance III
600, CDCl3) d 21.3, 27.8, 29.7, 32.1, 33.9, 36.0, 108.5, 125.5,
127.4, 128.4, 129.3, 138.1, 139.3, 148.5, 150.9, 151.5, 154.5; EI-
HRMS m/z: Calcd for C17H20N4O2S, 344.1307. Found 344.1259; Pur-
ity (HPLC): 99%.
The title compound was prepared from (3-bromophenyl)meth-
anethiol in a yield of 46%: mp 143.9–145.7 °C (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3) d 3.35 (s, 3H), 3.56 (s, 3H), 3.73 (s,
3H), 4.38 (s, 2H), 7.14 (t, 1H, J = 7.9 Hz), 7.26 (d, 1H, J = 7.5 Hz),
7.37 (d, 1H, J = 8.3 Hz), 7.54 (s, 1H); 13C NMR (Bruker Avance III
600, CDCl3) d 27.8, 29.7, 32.2, 36.3, 108.8, 122.4, 127.5, 130.1,
130.9, 132.2, 139.0, 148.3, 149.5, 151.4, 154.5; EI-HRMS m/z: Calcd
for C15H15BrN4O2S, 394.0099. Found 394.0113; Purity (HPLC): 99%.
4.3. Synthesis of 8-sulfinylcaffeine analogues (2a–d)
4.2.12. 8-{[2-(3-Methoxyphenyl)ethyl]sulfanyl}caffeine (1l)
The title compound was prepared from 2-(3-methoxy-
phenyl)ethane-1-thiol in a yield of 41%: mp 126.6–127.5 °C (etha-
nol). 1H NMR (Bruker Avance III 600, CDCl3) d 3.01 (t, 2H,
J = 7.5 Hz), 3.36 (s, 3H), 3.48 (t, 2H, J = 7.5 Hz), 3.55 (s, 3H), 3.76
(s, 3H), 3.78 (s, 3H), 6.74 (m, 2H), 6.79 (d, 1H, J = 7.5 Hz), 7.20 (t,
1H, J = 7.5 Hz); 13C NMR (Bruker Avance III 600, CDCl3) d 27.8,
The appropriate 8-sulfanylcaffeine analogue (4 mmol) was dis-
solved in a mixture of glacial acetic acid (8 mL) and acetic anhy-
dride (2 mL). The reaction was stirred at room temperature and
2 mL of a 30% hydrogen peroxide solution was added dropwise.
The reaction was stirred for 4 h and 25 mL of distilled water was
added. The product, which precipitated from the reaction mixture,
was collected by filtration and purified by recrystallization from

7048
S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
ethyl acetate.31 TLC of the product was conducted using silica gel
sheets (Merck) and ethylacetate/n-hexane (60:30) as mobile phase
(Rf ꢀ0.23).
4.5. Inhibition of human MAO
Microsomes prepared from insect cells, which contain recombi-
nant human MAO-A and -B, were obtained from Sigma–Aldrich.
Potassium phosphate buffer (100 mM, pH 7.4, made isotonic with
KCl 20.2 mM) was used for all the enzyme reactions and dilutions.
The final volume of the enzyme reactions was 500
reaction contained kynuramine (45 M for MAO-A and 30
MAO-B), various concentrations of the test inhibitors (0–100
and 4% DMSO as co-solvent. After the reactions were started with
the addition of MAO-A or -B (0.0075 mg protein/mL), they were
incubated in a water bath at 37 °C for 20 min. The reactions were
4.3.1. 8-{[2-(4-Chlorophenyl)ethyl]sulfinyl}caffeine (2a)
The title compound was prepared from 8-{[2-(4-chloro-
phenyl)ethyl]sulfanyl}caffeine in a yield of 72%: mp 155 °C (etha-
nol). 1H NMR (Bruker Avance III 600, CDCl3) d 3.12 (m, 2H, CH2),
3.37 (s, 3H, NCH3), 3.50 (m, 1H, O@SCH2), 3.51 (s, 3H, NCH3);
3.73 (m, 1H, O@SCH2), 4.20 (s, 3H, NCH3), 7.15 (d, 2H, J = 8.3 Hz,
ArH); 7.23 (d, 2H, J = 8.3 Hz, ArH); 13C NMR (Bruker Avance III
600, CDCl3) d 27.5, 28.1, 29.8, 33.3, 53.7, 110.0, 128.9, 129.9,
132.8, 136.2, 147.1, 149.1, 151.2, 155.0; APCI-HRMS m/z:
Calcd for C16H18ClN4O3S 381.0788. Found 381.0789; Purity (HPLC):
92%.
l
L, and each
M for
M)
l
l
l
subsequently terminated by the addition of 400
lL NaOH (2 N). A
volume of 1000 L water was added to each reaction, the reactions
l
were centrifuged at 16,000g (10 min) and the concentrations of the
MAO generated 4-hydroxyquinoline in the supernatants were
measured by fluorescence spectrophotometry (kex = 310 nm;
kem = 400 nm).29,30,41 For the concentration calculations, linear cal-
ibration curves were constructed by measuring the fluorescence of
samples containing known amounts of 4-hydroxyquinoline
4.3.2. 8-{[2-(4-Bromophenyl)ethyl]sulfinyl}caffeine (2b)
The title compound was prepared from 8-{[2-(4-bromophe-
nyl)ethyl]sulfanyl}caffeine in a yield of 45%: mp 166 °C (ethanol).
1H NMR (Bruker Avance III 600, CDCl3) d 3.11 (t, 2H, J = 7.5 Hz,
CH2), 3.37 (s, 3H, NCH3), 3.50 (m, 1H, O@SCH2), 3.51 (s, 3H,
NCH3); 3.74 (m, 1H, O@SCH2), 4.20 (s, 3H, NCH3), 7.09 (d, 2H,
J = 7.9 Hz, ArH); 7.38 (d, 2H, J = 7.5 Hz, ArH); 13C NMR (Bruker
Avance III 600, CDCl3) d 27.5, 28.1, 29.8, 33.3, 53.6, 110.0, 120.8,
130.3, 131.8, 136.7, 147.1, 149.1, 151.2, 155.0; EI-HRMS m/z: Calcd
for C16H17BrN4O3S 424.0205. Found 424.0200; Purity (HPLC):
96%.
(0.047–1.56
lM) dissolved in 500
l
L potassium phosphate buffer.
L NaOH (2 N) and 1000
To each calibration sample, 400
l
lL water
were added. The MAO catalytic rates were calculated from the end-
point concentration of 4-hydroxyquinoline (nM) in the superna-
tants, the incubation time (20 min) and the enzyme
concentration (0.0075 mg protein/mL), and were expressed as
nmol 4-hydroxyquinoline formed/min mg protein. For each inhib-
itor evaluated, the appropriate control samples were analyzed in
order to confirm that the test inhibitors do not fluoresce or quench
the fluorescence of 4-hydroxyquinoline under the assay conditions.
From the enzyme rate data, sigmoidal dose–response curves (plots
of the initial rate of kynuramine oxidation versus the logarithm of
inhibitor concentration) were constructed. For the construction of
each sigmoidal curve, at least six different inhibitor concentrations
were employed, and the inhibitor concentrations covered at least
three orders of magnitude. IC50 values were estimated from the
sigmoidal curves with the aid of the Prism 5 software package
(GraphPad), employing the one site competition model. IC50 values
were determined in triplicate and are expressed as mean stan-
dard deviation (SD).
4.3.3. 8-{[2-(4-Fluorophenyl)ethyl]sulfinyl}caffeine (2c)
The title compound was prepared from 8-{[2-(4-fluoro-
phenyl)ethyl]sulfanyl}caffeine in a yield of 38.8%: mp 126 °C (eth-
anol). 1H NMR (Bruker Avance III 600, CDCl3) d 3.13 (m, 2H, CH2),
3.37(s, 3H, NCH3), 3.50 (m, 1H, O@SCH2), 3.53 (m, 3H, NCH3),
3.74 (m, 1H, O@SCH2), 4.21 (s, 3H, NCH3), 6.98 (t, 2H, J = 6.4 Hz,
ArH); 7.18 (m, 2H, ArH); 13C NMR (Bruker Avance III 600, CDCl3)
d 27.4, 28.1, 29.8, 33.3, 54.0, 110.1, 115.6, 130.1, 133.5, 147.2,
149.3, 151.2, 155.0, 161.0, 162.6; EI-HRMS m/z: Calcd for
C16H17FN4O3S 364.1006. Found 364.0995; Purity (HPLC): 96%.
4.3.4. 8-(Benzylsulfinyl)caffeine (2d)
The title compound was prepared from 8-(benzylsulfanyl)caf-
feine30 in a yield of 42%: mp 165.6–169.7 °C (ethanol). 1H NMR
(Bruker Avance III 600, CDCl3) d 3.20 (s, 3H, NCH3), 3.44 (s, 3H,
NCH3), 3.58 (s, 3H, NCH3), 4.60 (m, 2H, O@SCH2), 7.15–7.17 (m,
2H, ArH), 7.30–7.35 (m, 3H, ArH); 13C NMR (Bruker Avance III
600, CDCl3) d 27.7, 29.6, 32.2, 59.3, 108.8, 128.5, 128.6, 129.2,
130.5, 146.9, 149.4, 150.8, 154.4; EI-HRMS m/z: Calcd for
4.6. Time-dependency of MAO-A and -B inhibition
The time-dependency of the inhibition of MAO-A and -B by a se-
lected inhibitor, compound 1b, was investigated. Compound 1b
was firstly incubated with recombinant human MAO-A and -B
(0.03 mg/mL) for different periods of time (0, 15, 30, 60 min) at
37 °C. The buffer used for this purpose was potassium phosphate
buffer (100 mM, pH 7.4, made isotonic with KCl), the reaction vol-
C15H16N4O3S, 332.0943. Found 332.0932; Purity (HPLC): 99%.
ume was 250
the measured IC50 value for the inhibition of MAO-A (16.92
and MAO-B (0.04 M), respectively. Kynuramine, dissolved in
potassium phosphate buffer, was subsequently added to these
reactions at a volume of 250 L. The final volume of the reactions
was 500 L, the MAO enzyme concentrations were 0.015 mg/mL
l
L and the concentration of 1b was equal to twofold
4.4. Synthesis of mercaptan reagents 6
lM)
l
Thiourea (7 mmol) was dissolved in 13.5 mL ethanol and the
appropriate alkylbromide (7 mmol) was added. The mixture was
stirred and heated under reflux for 2 h after which the reaction be-
came homogenous. The reaction was allowed to cool to room tem-
perature and the ethanol solvent was removed under reduced
pressure. A solution of NaOH (10.5 mmol) in 8.75 mL water was
added to the residue, and the reaction mixture was heated under
reflux (120 °C) for 2 h. The reaction was subsequently cooled to
room temperature and aqueous H2SO4 (15%) was added. The mer-
captan separated as oil droplets and was extracted to diethylether
(30 mL). The organic phase was washed twice with water, dried
over anhydrous Na2SO4 and removed under reduced pressure. This
yielded the desired mercaptans which were used without further
purification for the synthesis of 1, 3 and 4.
l
l
and concentrations of 1b were equal to the IC50 values for the inhi-
bition of MAO-A and -B, respectively. The concentrations of kynur-
amine were 45 and 30
reactions were subsequently incubated at 37 °C for a further
15 min and terminated with the addition of 400 L NaOH (2 N).
A volume of 1000 L distilled water was added to each reaction
lM for MAO-A and -B, respectively. The
l
l
and the rates of formation the MAO generated of 4-hydroxyquino-
line were measured by fluorescence spectrophotometry as de-
scribed above. All measurements were carried out in triplicate
and are expressed as mean SD.42 One-way analyses of variances
(ANOVA) with Tukey’s post hoc test was used to determine if

S. Mostert et al. / Bioorg. Med. Chem. 20 (2012) 7040–7050
7049
statistical differences exist between the means of the MAO cata-
lytic rates recorded at each time point.
analysis. For this purpose,
(4.60 Â 150 mm, 5
of 75% acetonitrile and 25% Milli-Q water at a flow rate of 1 mL/
min. A volume of 20 L of the water phases were injected into
a
Venusil XBP C18 column
lm) was used and the mobile phase consisted
4.7. Recovery of enzyme activity after dilution
l
the HPLC system and the eluent was monitored at a wavelength
of 292 nm. To quantify the analytes, linear calibration curves were
constructed by similarly analyzing known amounts of the analytes
(0.05–10 lM) in 50% acetonitrile. These studies were carried out in
triplicate for each analyte and the LogP values are expressed as
Compound 1b [IC50(MAO-B) = 0.02
l
M] or 1j [IC50(MAO-
B) = 0.017
l
M] at 10 Â IC50 and 100 Â IC50 were preincubated with
recombinant human MAO-B (0.75 mg/mL) for 30 min at 37 °C. The
reaction solvent was potassium phosphate buffer (pH 7.4, 100 mM,
made isotonic with KCl). DMSO (4%) served as co-solvent in all
preincubations. A control reaction was also carried out in the ab-
sence of inhibitor. The reactions were subsequently diluted 100-
fold with the addition of kynuramine to yield final concentrations
mean SD.
Acknowledgments
of kynuramine equal to 30
l
M, and of MAO-B equal to 0.0075 mg/
The NMR and MS spectra were recorded by André Joubert of the
SASOL Centre for Chemistry, North-West University and by the
Mass Spectrometry Service, University of the Witwatersrand. This
work was supported by Grants from the National Research Founda-
tion and the Medical Research Council, South Africa, and the Unit
for Drug Research and Development, North-West University.
mL. The final concentrations of 1a and 1j were 0.1 Â IC50 and
1 Â IC50. The reactions were incubated for a further 20 min at
37 °C, terminated and the residual rates of 4-hydroxyquinoline for-
mation were measured as described above.43 For comparison, (R)-
deprenyl (IC50 = 0.079 lM) was similarly preincubated with MAO-
B at 10 Â IC50 and diluted 100-fold with the addition of kynur-
amine to yield a final concentration of (R)-deprenyl equal to
Supplementary data
43
0.1 Â IC50
.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.005.
4.8. The construction of Lineweaver–Burk plots
The mode of MAO-B inhibition was investigated with the aid of
a set consisting of four Lineweaver–Burk plots. The first plot was
constructed in the absence of inhibitor while the remaining three
plots were constructed in the presence of different concentrations
of an inhibitor. For this study, compound 1b was selected as repre-
sentative inhibitor at the following concentrations: 0.005, 0.01 and
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