The inhibition of monoamine oxidase by 8-(2-phenoxyethoxy)caffeine analogues

Article abstract of DOI:10.1055/s-0032-1323662

Previous studies have documented that substituted 8-oxycaffeines act as inhibitors of human monoamine oxidase (MAO) B. A particularly potent inhibitor among the reported compounds was 8-(2-phenoxyethoxy)caffeine with an IC ⁵⁰ value of 0.383M towards MAO-B. In an attempt to improve on the inhibition potency of this compound and to discover highly potent reversible MAO-B inhibitors, in the present study, a series of 8-(2-phenoxyethoxy)caffeine analogues containing various substituents on C4 of the phenoxy ring, were synthesized and evaluated as inhibitors of human MAO-A and -B. The results show that the 8-(2-phenoxyethoxy)caffeine analogues are selective and reversible MAO-B inhibitors with the most potent homologue, 8-{2-[4-(trifluoromethyl) phenoxy]ethoxy}caffeine, exhibiting an IC⁵⁰ value of 0.061μM. These highly potent inhibitors are useful leads in the design of therapies for neurodegenerative disorders such as Parkinsons disease. Georg Thieme Verlag KG Stuttgart New York.

Full text of DOI:10.1055/s-0032-1323662

Original Article 513
The Inhibition of Monoamine Oxidase by
8-(2-Phenoxyethoxy)Caffeine Analogues
Authors
B. Strydom, J. J. Bergh, J. P. Petzer
Affiliation
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Potchefstroom, South Africa
Key words
Abstract
▼
taining various substituents on C4 of the phe-
noxy ring, were synthesized and evaluated as
▶
monoamine oxidase
caffeine
●
▶
●
Previous studies have documented that substi-
tuted 8-oxycaffeines act as inhibitors of human
monoamine oxidase (MAO) B. A particularly
potent inhibitor among the reported compounds
was 8-(2-phenoxyethoxy)caffeine with an IC50
value of 0.383μM towards MAO-B. In an attempt
to improve on the inhibition potency of this com-
pound and to discover highly potent reversible
MAO-B inhibitors, in the present study, a series
of 8-(2-phenoxyethoxy)caffeine analogues con-
inhibitors of human MAO-A and -B. The results
show that the 8-(2-phenoxyethoxy)caffeine
analogues are selective and reversible MAO-B
inhibitors with the most potent homologue,
8-{2-[4-(trifluoromethyl)phenoxy]ethoxy}caf-
feine, exhibiting an IC50 value of 0.061μM. These
highly potent inhibitors are useful leads in the
design of therapies for neurodegenerative disor-
ders such as Parkinson’s disease.
▶
8-(2-phenoxyethoxy)caffeine
reversible inhibition
selective inhibition
structure-activity relation-
ship
●
▶
●
▶
●
▶
●
Introduction
reversible MAO-A inhibitor, moclobemide [5].
Since MAO-B is considered to be the major
dopamine metabolizing enzyme in the basal gan-
glia of the brain, MAO-B inhibitors are used in the
treatment of Parkinson’s disease [6,7]. In the basal
ganglia, inhibitors of MAO-B are thought to reduce
the depletion of dopamine, and to enhance
dopamine levels after treatment with levodopa
[8,9]. MAO-B inhibitors may also protect against
the neurodegenerative events associated with
Parkinson’s disease by reducing the levels of
potentially neurotoxic aldehydes and H2O2, which
are generated as by-products in the catalytic cycle
of MAO-B [1]. (R)-Deprenyl and rasagiline are
examples of MAO-B inhibitors currently being
used as adjuncts to levodopa therapy in the treat-
ment of Parkinson’s disease [10]. While these
inhibitors have been widely used, their irreversi-
ble mode of inhibition may be associated with
certain disadvantages. These include a slow rate of
enzyme recovery after drug withdrawal and a loss
of selectivity after repeated administration
[11,12]. For these reasons, the discovery of new
MAO-B inhibitors with a reversible mode of action
is being pursued by various research groups.
▼
Monoamine oxidase (MAO) is a flavin adenine
dinucleotide (FAD) containing enzyme which is
bound to the outer mitochondrial membrane.
MAO is classified into 2 distinct isoforms, MAO-A
and -B, which share a 70% sequence identity and
are products of separate genes [1,2]. The princi-
pal function of the MAO isozymes is the oxidative
deamination of primary amines of an endog-
enous and dietary nature. In the central and
peripheral tissues, MAO is responsible for the
termination of the action of neurotransmitter
amines such as serotonin, dopamine, epine-
phrine and norepinephrine [1,3]. The 2 MAO iso-
forms may be distinguished by their different
substrate specificities. MAO-A preferentially cat-
alyzes the oxidation of serotonin while MAO-B
favours benzylamine and 2-phenethylamine as
substrates [3]. Dopamine, epinephrine, nore-
pinephrine and tyramine are considered to be
substrates for both MAO isoforms [3].
received 11.05.2012
accepted 24.07.2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1323662
Published online:
August 31, 2012
Arzneimittelforschung 2012;
62: 513–518
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
Correspondence
Prof. J. P. Petzer, PhD
Pharmaceutical Chemistry
School of Pharmacy
North-West University
Private Bag X6001
2520, Potchefstroom
South Africa
Tel.: +27/18/2992206
Fax: +27/18/2994243
jacques.petzer@nwu.ac.za
MAO has acted as a target for the treatment of
central nervous system diseases [3]. Selective
MAO-A inhibitors are reported to be effective in
the treatment of depression by increasing the lev-
els of serotonin, norepinephrine and dopamine in
the brain [4]. An example of such a drug is the
For the design of reversible MAO inhibitors,
C8-substituted caffeine analogues have been pre-
viously employed [13–16]. While caffeine is a
Strydom B et al. Phenoxyethoxycaffeines as MAO Inhibitors… Arzneimittelforschung 2012; 62: 513–518

514 Original Article
with a Buchi® M-545 melting point apparatus and are uncor-
rected. Fluorescence spectrophotometry was conducted with a
Varian® Cary Eclipse fluorescence spectrophotometer. Micro-
somes from insect cells containing recombinant MAO-A or -B
(5mg/mL) and kynuramine·2HBr were obtained from Sigma-
Aldrich®. 8-Chlorocaffeine (4) was prepared according to a pre-
viously reported procedure [19,20] by the reaction of caffeine
with chlorine in chloroform. The 2-phenoxyethanol analogues
(5), which were required as reactants for the synthesis of 3, were
prepared by reacting the appropriately substituted phenol (6)
with bromoethanol in the presence of acetone and potassium
carbonate [21,22]. The purities of previously unreported com-
pounds were determined by HPLC analysis as described previ-
ously [15].
O
O
N
Cl
N
N
N
N
N
O
N
N
O
O
8-Benzyloxycaffeine (1)
CSC (2)
O
N
N
O
O
N
O
N
3a
Fig. 1 The structures of 8-benzyloxycaffeine (1), CSC (2) and 8-(2-phe-
noxyethoxy)caffeine 3a.
Procedures for the synthesis of the
weak MAO-B inhibitor, substitution with a variety of moieties at
C8 often yields compounds that are highly potent and selective
MAO-B inhibitors [17]. For example, a series of 8-benzyloxycaf-
8-(2-phenoxyethoxy)caffeine analogues (3)
For the synthesis of compounds 3a–d a method described in lit-
erature was followed with minor modifications [23]. Metallic
sodium (1.5mmol) and the appropriately substituted phenox-
yethanol analogue (5, 21mmol) were allowed to react at room
temperature. Following consumption of the sodium, 8-chloro-
caffeine (4, 1.5mmol) was added and the resulting mixture was
stirred for 6h at 150 °C. The reaction was cooled to room tem-
perature and the 8-(2-phenoxyethoxy)caffeine analogue was
recrystallized (at 4 °C) after the addition of ethanol (10–20mL)
to the reaction mixture. The melting points and structure char-
acterizations of compounds 3a–c have been previously reported
[14]. The physical data of 3d are cited below. The 8-(2-phenox-
yethoxy)caffeine analogues 3e–j were synthesized according to
the literature procedure by Baumann et al. [24]. Potassium
hydroxide (2mmol) was dissolved in 1mL distilled water and
the appropriately substituted 2-phenoxyethanol analogue
(1.85mmol) was added to yield a solution. 8-Chlorocaffeine (4,
1.5mmol) was added and the resulting reaction mixture was
stirred at 150 °C for 6h. The reaction was cooled to room tem-
perature and ethanol (10mL) was added. The resulting solution
was allowed to recrystallize (at 4 °C).
▶
feine analogues (1) ( Fig. 1) has been shown to act as potent
●
inhibitors of human MAO-B with IC50 values ranging from 0.068
to 1.77μM [13]. Interestingly, the 8-benzyloxycaffeine analogues
were also found to be potent reversible MAO-A inhibitors with
IC50 values ranging from 0.397 to 3.72μM [13]. Modelling stud-
ies have indicated that the ability of the 8-benzyloxycaffeine
analogues to also bind to the MAO-A active site may depend on
the large degree of rotational freedom of the benzyloxy side
chain at the carbon-oxygen ether bond [13]. More rigid C8-sub-
stituted caffeine analogues such as (E)-8-(3-chlorostyryl)caf-
feine (CSC, 2) typically do not inhibit MAO-A [13,17,18]. Another
C8 oxy substituent that leads to potent MAO-B inhibition is the
2-phenoxyethoxy moiety. In fact, 8-(2-phenoxyethoxy)caffeine
(3a) (IC50 =0.383μM) is a more potent inhibitor of human MAO-B
than is 8-benzyloxycaffeine (IC50 =1.77μM) [14]. A structure-
activity relationship (SAR) study has indicated that for C8-sub-
stituted oxycaffeine analogues, a linker consisting of 4 atoms
separating the caffeine and the terminal phenyl ring may be par-
ticularly suited for MAO-B inhibition [14]. Since this require-
ment is satisfied by the 8-(2-phenoxyethoxy)caffeine structure,
this compound may be considered to be a promising lead com-
pound for the design of highly potent MAO-B inhibitors.
8-[2-(4-Fluorophenoxy)ethoxy]caffeine (3d): The title com-
pound was prepared from 8-chlorocaffeine (4) and 2-(4-fluor-
ophenoxy)ethanol. (63%) cream coloured solid: mp 177 ºC (from
ethanol). 1H NMR (600MHz, CDCl3, Me4Si) 3.37 (3H, s, CH3), 3.49
(3H, s, CH3), 3.68 (3H, s, CH3), 4.29 (2H, t, CH2, J=4.52Hz), 4.77
(2H, t, CH2, J=4.52Hz), 6.85 (2H, m, phenyl), 6.97 (2H, m, phe-
nyl); 13C NMR (150MHz, CDCl3, Me4Si) 27.76, 29.75, 29.90, 66.48,
69.20, 103.62, 115.69 (d), 115.90, 116.06, 146.07, 151.67, 154.45,
154.83, 155.27; HRMS m/z: calcd for C16H17FN4O4, 348.1234,
found 348.1214; Purity (HPLC): 98%.
Based on these observations, in the present study the MAO-A
and -B inhibition properties of a series of ten 8-(2-phenox-
yethoxy)caffeine analogues (3a–j) were examined. The ana-
logues considered, differed by substitution on the para position
of the phenoxy ring. Previous studies reported that halogen sub-
stitution at this position leads to a superior enhancement of the
MAO-B inhibition potency of 8-benzyloxycaffeine [13,14] with
improved selectivity compared to meta substitution.
8-{2-[4-(Trifluoromethyl)phenoxy]ethoxy}caffeine (3e): T h e
title compound was prepared from 8-chlorocaffeine (4) and
2-[4-(trifluoromethyl)phenoxy]ethanol. (3%) white solid: mp
130°C (from ethanol). 1H NMR (600MHz, CDCl3, Me4Si) 3.36
(3H, s, CH3), 3.49 (3H, s, CH3), 3.67 (3H, s, CH3), 4.37 (2H, t, CH2,
J=3.39Hz), 4.81 (2H, t, CH2, J=3.01Hz), 6.97 (2H, d, phenyl,
J=7.91Hz), 7.54 (2H, d, phenyl, J=7.91Hz); 13C NMR (150MHz,
CDCl3, M4Si) 27.75, 29.74, 29.90, 65.89, 68.86, 103.65, 114.52,
125.14 (q), 127.01, 127.03, 146.03, 151.65, 154.83, 155.16,
160.71; HRMS m/z: calcd for C17H17F3N4O4, 398.1202, found
398.1198; Purity (HPLC): 93%.
Materials and Methods
▼
Materials
All starting materials, unless otherwise noted, were acquired
from Sigma-Aldrich®. Proton (1H) and carbon (13C) NMR spectra
were recorded in CDCl3 and DMSO-d6 with a Bruker® Avance III
600 spectrometer at frequencies of 600MHz and 150MHz,
respectively. Direct insertion high resolution mass spectra
(HRMS) were obtained on a DFS high resolution magnetic sector
mass spectrometer (Thermo Electron® Corporation) in electron
impact ionization mode. Melting points (mp) were measured
Strydom B et al. Phenoxyethoxycaffeines as MAO Inhibitors… Arzneimittelforschung 2012; 62: 513–518

Original Article 515
8-[2-(4-Methylphenoxy)ethoxy]caffeine (3f): The title com-
pound was prepared from 8-chlorocaffeine (4) and 2-(4-methyl-
phenoxy)ethanol. (83%) white solid: mp 144°C (from ethanol).
1H NMR (600MHz, CDCl3, Me4Si) 2.27 (3H, s, CH3-phenyl), 3.49
(3H, s, CH3), 3.64 (3H, s, CH3), 3.67 (3H, s, CH3), 4.3 (2H, t, CH2,
J=4.52Hz), 4.77 (2H, t, CH2, J=4.52Hz), 6.81 (2H, d, phenyl,
J=8.69Hz), 7.07 (2H, d, phenyl, J=8.28Hz); 13C NMR (150MHz,
CDCl3, Me4Si) 20.46, 27.75, 29.75, 29.89, 65.92, 69.37, 103.58,
114.51, 130.01, 130.74, 146.11, 151.68, 154.83, 155.37, 156.21;
HRMS m/z: calcd for C17H20N4O4, 344.1485, found 344.1480;
Purity (HPLC): 98%.
500μL. The reactions contained the MAO-A/B mixed substrate
kynuramine (45μM for MAO-A and 30μM for MAO-B), different
inhibitor concentrations (0.003–100μM) and DMSO as co-sol-
vent (4%). The reactions were initiated with the addition of
MAO-A or MAO-B (0.0075mg protein/mL), incubated for 20min
at 37 ºC and terminated by the addition of 400μL NaOH (2N)
and 1000μL water. The concentrations of the MAO generated
4-hydroxyquinoline were subsequently measured by fluores-
cence spectrophotometry (λem =310; λex =400nm) [26]. Employ-
ing a linear calibration curve (4-hydroxyquinoline: 0.047–1.56μM),
the enzyme catalytic rates were calculated and fitted to the one
site competition model incorporated into the Prism software
package (GraphPad). The IC50 values were determined in tripli-
cate and are expressed as mean±standard deviation (SD).
8-[2-(4-Methoxyphenoxy)ethoxy]caffeine (3g): The title com-
pound was prepared from 8-chlorocaffeine (4) and 2-(4-methox-
yphenoxy)ethanol. (5%) sand coloured solid: mp 138°C (from
ethanol). 1H NMR (600MHz, CDCl3, Me4Si) 3.36 (3H, s, CH3), 3.49
(3H, s, CH3), 3.67 (3H, s, CH3), 3.75 (3H, s, OCH3), 4.27 (2H, t, CH2,
J=4.52Hz), 4.76 (2H, t, CH2, J=4.52Hz), 6.83 (2H, m, phenyl), 6.84
(2H, m, phenyl); 13C NMR (150MHz, CDCl3, Me4Si) 27.73, 29.73,
29.88, 55.70, 66.59, 69.40, 103.56, 114.69, 115.74, 146.09, 151.66,
152.43, 154.31, 154.81, 155.35; HRMS m/z: calcd for C17H20N4O5,
360.1434, found 360.1434; Purity (HPLC): 98%.
Recovery of enzyme activity after dilution
Compound 3h or pargyline (IC50 =12.97μM) at concentrations
equal to 100×IC50 (92.4μM and 1.30mM for the 2 inhibitors,
respectively) for the inhibition of MAO-A were preincubated
with recombinant human MAO-A (0.75mg/ml) for 30min at
37°C in potassium phosphate buffer (100mM, pH 7.4, made iso-
tonic with KCl). Compound 3e or (R)-deprenyl (IC50 =0.079μM)
[27] were similarly preincubated with recombinant human
MAO-B (0.75mg/ml) at concentrations equal to 100×IC50
(6.1μM and 7.9μM for the 2 inhibitors, respectively). Control
incubations were conducted in the absence of inhibitor, and
DMSO (4%) was added as co-solvent to all preincubations. The
reactions were diluted 100-fold with the addition of kynuramine
8-[2-(4-Iodophenoxy)ethoxy]caffeine (3h): The title com-
pound was prepared from 8-chlorocaffeine (4) and 2-(4-iodo-
phenoxy)ethanol. (4%) white solid: mp 160°C (from ethanol). 1H
NMR (600MHz, CDCl3, Me4Si) 3.36 (3H, s, CH3), 3.49 (3H, s, CH3),
3.67 (3H, s, CH3), 4.29 (2H, m, CH2), 4.77 (2H, m, CH2), 6.69 (2H,
d, phenyl, J=7.91Hz), 7.55 (2H, d, phenyl, J=7.53Hz); 13C NMR
(150MHz, CDCl3, Me4Si) 27.74, 29.74, 29.90, 65.85, 68.98, 83.54,
103.61, 116.95, 138.35, 146.03, 151.64, 154.81, 155.19, 158.20;
HRMS m/z: calcd for C16H17IN4O4, 456.0294, found 456.0268;
Purity (HPLC): 98%.
to yield final concentrations of the inhibitors equal to 1×IC50
.
The final concentration of MAO-A and -B were 0.0075mg/mL
and the concentrations of kynuramine were 45μM and 30μM
for MAO-A and -B, respectively. The reactions were incubated for
a further 20min at 37°C, terminated and the residual rates of
4-hydroxyquinoline formation were measured as described
above. These reactions were carried out in triplicate and the
residual enzyme catalytic rates were expressed as mean±SD.
8-[2-(4-Cyanophenoxy)ethoxy]caffeine (3i): The title com-
pound was prepared from 8-chlorocaffeine (4) and 2-(4-cyano-
phenoxy)ethanol. (8%) white solid: mp >300°C (from ethanol).
1H NMR (600MHz, DMSO-d6, Me4Si) 3.18 (3H, s, CH3), 3.35 (3H,
s, CH3), 3.56 (3H, s, CH3), 4.43 (2H, m, CH2), 4.77 (2H, m, CH2),
7.02 (2H, d, phenyl, J=9.04Hz), 7.83 (2H, d, phenyl, J=8.66Hz);
13C NMR (150MHz, DMSO-d6, Me4Si) 27.38, 29.50, 29.64, 65.89,
69.43, 102.69, 114.03, 126.89, 129.39, 145.53, 150.87, 153.85,
154.98, 160.47, 167.31; HRMS m/z: calcd for C17H17N5O4,
355.1281, found 355.1275; Purity (HPLC): 98%.
Results
▼
Chemistry
The 8-(2-phenoxyethoxy)caffeine analogues (3a–j) were syn-
thesized according to the protocol previously described for the
synthesis of C8 substituted caffeines [14,24]. The target com-
pounds were obtained by reacting 8-chlorocaffeine (4) with an
appropriately substituted 2-phenoxyethanol (5) at high temper-
atures (150°C) in the presence of sodium or potassium hydrox-
▶
8-[2-(4-Nitrophenoxy)ethoxy]caffeine (3j): The title com-
pound was prepared from 8-chlorocaffeine (4) and 2-(4-nitro-
phenoxy)ethanol. (63%) white solid: mp 178°C (from ethanol).
1H NMR (600MHz, CDCl3, Me4Si) 3.35 (3H, s, CH3), 3.48 (3H, s,
CH3), 3.68 (3H, s, CH3), 4.43 (2H, t, CH2, J=4.52Hz), 4.83 (2H, t,
CH2, J=4.52Hz), 6.98 (2H, d, phenyl, J=9.41Hz), 8.19 (2H, d,
phenyl, J=9.04Hz); 13C NMR (150MHz, CDCl3, Me4Si) 27.72,
29.72, 29.91, 66.35, 68.55, 103.64, 114.48, 125.93, 141.92,
145.93, 151.58, 154.76, 154.98, 163.17; HRMS m/z: calcd for
C16H17N5O6, 375.1179, found 375.1172; Purity (HPLC): 98%.
ide ( Fig. 2). After recrystallization from ethanol, compounds
●
3a–j were obtained in low to high yields (3–83%). The low yields
obtained with some of the reactions are most likely the result of
incomplete recrystallization after adding ethanol to the reaction
mixture. Although inefficient in some instances, this procedure
effectively separated the desired product from unreacted 8-chlo-
rocaffeine. 8-Chlorocaffeine was obtained in high yield from a
reaction between chlorine and a solution of caffeine in chloro-
form [19]. In certain instances, the 2-phenoxyethanol analogues
that were required for the synthesis of 3a–j were not commer-
cially available and were thus synthesized according to literature
methods [21,22]. For this purpose an appropriately substituted
phenol (6) was reacted with 2-bromoethanol in the presence of
potassium carbonate in acetone. The structures of the 8-(2-phe-
IC50 determinations
IC50 values for the inhibition of MAO-A and -B were determined
using the recombinant human enzymes as described previously
[25]. Incubations were carried out at pH 7.4 (potassium phos-
phate 100mM, made isotonic with KCl) to a final volume of
Strydom B et al. Phenoxyethoxycaffeines as MAO Inhibitors… Arzneimittelforschung 2012; 62: 513–518

516 Original Article
enzyme substrate. Kynuramine, a non-fluorescent compound,
undergoes MAO catalyzed oxidation to yield the fluorescent
compound, 4-hydroxyquinoline, as end product. The extent to
which kynuramine is oxidized to 4-hydroxyquinoline by MAO
was subsequently measured via fluorescence spectrophotome-
try [26]. None of the inhibitors investigated in this study fluo-
resced at these excitation/emission wavelengths or quenched
the fluorescence of 4-hydroxyquinoline at the inhibitor concen-
trations used. Sigmoidal dose-response curves were constructed
for the inhibition of the MAO isozymes and the inhibition poten-
cies of the test compounds were expressed as the corresponding
a
HO
R
HO
+
Br
6
HO
O
R
5
b
O
N
IC50 values.
N
N
▶
N
The results of the MAO inhibition studies are given in Table 1.
●
HO
O
R
Cl
+
The 8-(2-phenoxyethoxy)caffeine analogues (3a–j) were found
to act as inhibitors of MAO-B. The most potent inhibitor of the
series was the CF3 substituted homologue, compound 3e, which
exhibited an IC50 value of 0.061μM for the inhibition of MAO-B.
Compound 3e is therefore approximately 6-fold more potent than
the corresponding unsubstituted homologue 3a (IC50 =0.383μM).
The results show that a variety of substituents on C4 of the phe-
noxy ring lead to enhanced MAO-B inhibition compared to 3a.
These substituents are notably halogens. For example, the Cl
(IC50 =0.183μM), Br (IC50 =0.166μM), F (IC50 =0.115μM) and I
(IC50 =0.128μM) substituted homologues inhibited MAO-B with
potencies approximately 2–3-fold higher than that of the unsub-
stituted homologue 3a. Non-halogen substituents on C4 of the
phenoxy ring of the 8-(2-phenoxyethoxy)caffeine analogues
resulted in a reduction of MAO-B inhibition potency compared
to the unsubstituted homologue 3a. For example, the CH3
(IC50 =1.41μM) and OCH3 (IC50 =1.53μM) substituted homo-
logues were 3.5–4-fold weaker inhibitors of MAO-B than the
unsubstituted homologue 3a. Similarly, those homologues con-
taining the CN (IC50 =6.98μM) and NO2 (IC50 =0.852μM) substit-
uents were found to be 18-fold and 2-fold weaker inhibitors
than 3a, respectively.
O
5
4
O
N
N
N
O
O
R
N
O
3
Fig. 2 Synthetic pathway to the 8-(2-phenoxyethoxy)caffeine analogues
3. Reagents and conditions: a Acetone, K2CO3 b Na or KOH, 150°C.
Table 1 The IC50 values for the inhibition of recombinant human MAO-A
and –B by the 8-(2-phenoxyethoxy)caffeine analogues 3a–ja.
O
N
N
O
O
R
N
O
N
3
The 8-(2-phenoxyethoxy)caffeine analogues (3a–j) were also
found to inhibit MAO-A. As shown by the SI values, which ranged
from 5–79, all of the compounds were however selective inhibi-
tors of the MAO-B isoform. The only homologue which exhibited
an IC50 value in the sub-micromolar range was compound 3h,
which inhibited MAO-A with an IC50 value of 0.924μM. With the
exception of the CN substituted homologue 3i (IC50 =35.5μM),
substitution on C4 of the phenoxy ring led to an enhancement
of MAO-A inhibition potency compared to the unsubstituted
homologue 3a (IC50 =20.4μM). With the exception of the F sub-
stituted homologue (IC50 =9.08μM), those homologues with
halogen containing substituents (3b, 3c, 3e and 3h) were the
most potent MAO-A inhibitors of the series with IC50 values
ranging from 0.924–2.22μM.
IC50 values (μM)
Compd.
R
MAO-A
MAO-B
SIb
5 3
1 0
9.9
3a
3b
3c
3d
3e
3f
-H
20.4±16.5c
1.83±0.013c
1.65±0.087c
9.08±1.38
0.383±0.021c
0.183±0.005c
0.166±0.003c
0.115±0.004
0.061±0.003
1.41±0.068
1.53±0.185
0.128±0.013
6.98±0.433
0.852±0.007
-Cl
-Br
-F
79
36
- C F 3
- C H 3
2.22±0.068
13.4±0.549
7.57±0.187
0.924±0.031
35.5±3.04
9.5
3g
3h
3i
- O C H 3
-I
5.0
7.2
5.1
5.8
-CN
3j
- N O 2
4.92±0.286
a Values are expressed as the mean±SD of triplicate determinations
b The selectivity index (SI) is the selectivity for the MAO-B isoform and is given as the
ratio of IC50(MAO-A)/IC50(MAO-B)
c Values obtained from literature [14]
Reversibility of inhibition
As mentioned in the introduction, literature reports that 8-oxy-
caffeines interact reversibly with both MAO-A and -B [14]. To
verify that the most potent MAO-A inhibitor of the present
series, compound 3h, and the most potent MAO-B inhibitor,
compound 3e, are reversible inhibitors, the recoveries of the
enzymatic activities after dilution of the enzyme-inhibitor com-
plexes were evaluated. The MAO enzymes were preincubated
with compounds 3h and 3e at concentrations of 100×IC50 for
noxyethoxy)caffeine analogues (3a–j) were verified by mass
spectrometry, 1H NMR and 13C NMR while the purities were
estimated by chromatographic analysis.
Enzymology
To examine the MAO-A and -B inhibition properties of the
8-(2-phenoxyethoxy)caffeine analogues (3a–j), the commer-
cially available recombinant human enzymes were employed
and kynuramine, a MAO-A/B nonselective substrate, served as
30min and then diluted to 1×IC50. The results are presented
▶
in Fig. 3 and show that after diluting 3h and 3e to concentra-
●
tions equal to 1×IC50, the MAO-A and -B activities were recov-
Strydom B et al. Phenoxyethoxycaffeines as MAO Inhibitors… Arzneimittelforschung 2012; 62: 513–518

Original Article 517
Fig. 3 Reversibility of inhibition of MAO-A and –B
MAO-A
MAO-B
by compounds 3h and 3e. MAO-A was preincubat-
ed with 3h and pargyline (left panel), and MAO-B
was preincubated with 3e and (R)-deprenyl (right
panel), at 100×IC50 for 30min and then diluted
to 1×IC50. The residual enzyme activities were
subsequently measured.
100
75
50
25
0
100
75
50
25
0
No Inhibitor
[I] = 1 x IC50
Pargyline
No Inhibitor
[I] = 1 x IC50
(R)-Deprenyl
ered to levels of 47% and 44% of the control values, respectively.
This behavior is consistent with a reversible interaction of 3h
with MAO-A and 3e with MAO-B. In contrast, after similar treat-
ment of MAO-A and -B with the irreversible inhibitors pargyline
and (R)-deprenyl, respectively, the MAO-A and -B activities were
not recovered.
The 8-(2-phenoxyethoxy)caffeine analogues were also found to
be inhibitors of human MAO-A. With the exception of the CN
substituted homologue 3i, substitution on C4 of the phenoxy
ring resulted in an enhancement of MAO-A inhibition potency
compared to the unsubstituted homologue 3a. Since the proper-
ties of the substituents considered are relatively diverse, this
enhancement may, similar to MAO-B, be dependent on a combi-
nation of properties of the C4 substituent. In this regard, both
electron withdrawing functional groups on C4 of the phenoxy
ring, such as the halogen containing groups, and electron releas-
ing substituents (CH3 and OCH3) enhance the MAO-A inhibition
potency of 8-(2-phenoxyethoxy)caffeine. Similarly, substitution
which results in both enhanced (halogens, CF3 and CH3) and
reduced lipophilicity (OCH3 and NO2) leads to higher MAO-A
inhibition potency. The 8-(2-phenoxyethoxy)caffeine analogues
are, however, MAO-B selective inhibitors. A possible explanation
for this observation may be that the active site of MAO-B is more
suitable to accommodate relatively larger inhibitors than the
active site of MAO-A [28]. In the MAO-B active site, residue Ile-
199 may rotate from the active site cavity to allow larger inhibi-
tors to span both the substrate and entrance cavities. In MAO-A,
the residue that corresponds to Ile-199 in MAO-B, is Phe-208
[29]. The relatively larger size of the phenyl side chain of this
residue may, in certain instances, restrict the binding of larger
inhibitors to MAO-A. Although the MAO-B inhibition potencies
of the 8-(2-phenoxyethoxy)caffeine analogues are similar to
those of the 8-benzyloxycaffeine analogues, they exhibit
improved selectivity for the MAO-B isoform [13]. For example,
the most potent MAO-B inhibitor among the 8-(2-phenox-
yethoxy)caffeines, compound 3e (IC50 =0.061μM), exhibits an SI
value of 36 while the most potent 8-benzyloxycaffeine, 8-(3-bro-
mobenzyloxy)caffeine (IC50 =0.068μM) exhibits an SI value of
only 1.7 [13]. Considering that MAO-A inhibition may lead to
serious side effects such as potentiating the sympathomimetic
effects of dietary amines [1], selective MAO-B inhibition is a
desirable property.
Discussion
▼
Based on a previous report that 8-(2-phenoxyethoxy)caffeine
(3a) is a potent MAO-B inhibitor and potentially suitable lead for
the design of reversible MAO-B inhibitors, the present study
investigates the SAR for the inhibition of human MAO-A and -B
by a series of 8-(2-phenoxyethoxy)caffeine analogues contain-
ing different substituents on the para position of the phenoxy
ring [14]. The results document that, compared to 3a, substitu-
tion with halogen containing groups leads to an enhancement in
MAO-B inhibition potency while non-halogen substituents
reduce the MAO-B inhibition potencies of the 8-(2-phenox-
yethoxy)caffeine analogues. The enhancement in MAO-B inhibi-
tion potency may be attributed to the electron withdrawing
properties of the halogen containing substituents, since those
substituents that may be considered electron releasing (CH3 and
OCH3), reduce the MAO-B inhibition potencies of 8-(2-phenox-
yethoxy)caffeine. This finding is in accordance with similar stud-
ies reported in literature. For example, for the inhibition of
MAO-B by a series of 8-benzyloxycaffeine analogues, the inhibi-
tion potencies may be improved with electron withdrawing sub-
stituents on the benzyloxy ring [13]. Interestingly, although the
CN and NO2 groups are electron withdrawing, substitution with
these groups do not result in enhanced MAO-B inhibition. This
suggests that additional properties of the C4 substituents also
determine binding to MAO-B. The lipophilicity of the para sub-
stituents may also play a role since halogen substitution is
expected to also result in analogues that are more lipophilic. The
active site cavity of the MAO-B is considered to be a mostly
hydrophobic space, and it may therefore be expected that C4
substituents with enhanced lipophilicity may interact more
favourably via Van der Waals interactions and thus should lead
to improved MAO-B inhibition. The finding that OCH3 CN and
NO2 substitution reduces binding affinity to MAO-B is in agree-
ment with this analysis since these functional groups are
expected to reduce the lipophilicity of the caffeine analogues.
The observation that the CH3 group, which is considered to be a
lipophilic substituent, does not enhance MAO-B inhibition
potency further supports the proposal that a combination of
properties of the C4 substituent play a role in the binding to
MAO-B.
In conclusion, the present study has shown that, with the appro-
priate substitution, 8-(2-phenoxyethoxy)caffeine analogues act
as highly potent and selective MAO-B inhibitors. In addition, the
compounds bind reversibly to the enzyme, which may be con-
sidered a desired property for further development as antipar-
kinsonian therapy.
Acknowledgments
▼
The NMR spectra were recorded by André Joubert of the SASOL
Centre for Chemistry, North-West University while the MS spec-
tra were recorded by Marelize Ferreira of the Mass Spectrometry
Strydom B et al. Phenoxyethoxycaffeines as MAO Inhibitors… Arzneimittelforschung 2012; 62: 513–518

518 Original Article
13 Strydom B, Malan SF, Castagnoli N et al. Inhibition of monoamine
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Service, University of the Witwatersrand. This work was sup-
ported by grants from the National Research Foundation and the
Medical Research Council, South Africa. The financial assistance
of the National Research Foundation (DAAD-NRF) towards this
research is hereby acknowledged. Opinions expressed and con-
clusions arrived at, are those of the authors and are not neces-
sarily to be attributed to the DAAD-NRF.
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Conflict of Interest
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The authors declare that they have no conflicts of interest to
disclose.
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