Inhibition of monoamine oxidase by selected phenylalkylcaffeine analogues

Article abstract of DOI:10.1111/jphp.12193

Objectives Caffeine represents a useful scaffold for the design of monoamine oxidase (MAO) type B inhibitors. Specifically, substitution on the C8 position yields structures which are high-potency MAO-B inhibitors. To explore the structure-activity relati

Full text of DOI:10.1111/jphp.12193

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Research Paper
Journal of Pharmacy
And Pharmacology
Inhibition of monoamine oxidase by selected
phenylalkylcaffeine analogues
Anél Petzera, Paul Groblerb, Jacobus J. Berghb and Jacobus P. Petzerb
aCentre of Excellence for Pharmaceutical Sciences and bDepartment of Pharmaceutical Chemistry, School of Pharmacy, North-West University,
Potchefstroom, South Africa
Keywords
caffeine; competitive; monoamine oxidase;
Abstract
Objectives Caffeine represents a useful scaffold for the design of monoamine
oxidase (MAO) type B inhibitors. Specifically, substitution on the C8 position
yields structures which are high-potency MAO-B inhibitors. To explore the
structure–activity relationships of MAO-B inhibition by caffeine-derived com-
pounds, this study examines the MAO inhibitory properties of a series of
phenylalkylcaffeine analogues.
Methods Employing the recombinant human enzymes, the potencies (IC50
values) by which the caffeine analogues inhibit MAO-A and MAO-B were meas-
ured. The reversibility of inhibition of a selected inhibitor was determined by
measuring the recovery of enzyme activity after dilution and dialysis of enzyme-
inhibitor mixtures.
reversible inhibition; structure–activity
relationship
Correspondence
Jacobus P. Petzer, Department of
Pharmaceutical Chemistry, North-West
University, Private Bag X6001, Potchefstroom,
2520, South Africa.
E-mail: jacques.petzer@nwu.ac.za
Received June 26, 2013
Accepted October 30, 2013
Key findings The results document that the phenylalkylcaffeine analogues are
reversible and selective MAO-B inhibitors with a competitive mode of inhibition.
The most potent analogue, 8-(7-phenylheptyl)caffeine, exhibits IC50 values for
the inhibition of MAO-A and MAO-B of 3.01 μm and 0.086 μm, respectively.
Increasing the length of the alkyl side chain leads to enhanced MAO-A and
MAO-B inhibitory potency while introduction of a carbonyl group reduces
MAO-B inhibitory potency.
doi: 10.1111/jphp.12193
Conclusions Phenylalkylcaffeines represent a new class of high-potency MAO-B
inhibitors with the longer alkyl side chains yielding enhanced inhibitory activity.
Such compounds may represent useful leads for the development of anti-
parkinsonian therapies.
Introduction
The monoamine oxidases (MAOs) are mitochondrial
bound enzymes, which are present in most mammalian
tissues.[1] The two known MAO isoforms, MAO-A and
MAO-B, are products of different genes and are expressed
in a tissue-selective manner.[2] While MAO-A and MAO-B
are both found in human liver tissue,[3] MAO-A is the main
isoform in human intestinal[4] and placental tissues.[5] Both
isoforms are present in the human brain, with MAO-B
present in higher amounts.[6,7] The primary function of
MAO is the catabolism of monoamine neurotransmitters in
central and peripheral tissues. The oxidative deamination
by MAO-B is considered to be one of the major catabolic
pathways of dopamine in the human brain, and this process
is therefore a target in the treatment of Parkinson’s
disease.[8] Inhibitors of MAO-B may safeguard the depleted
supply of dopamine in the parkinsonian brain and prolong
the activity of dopamine derived from its metabolic precur-
sor, levodopa.[9,10] MAO-B inhibitors are thus recommended
as adjunctive therapy in patients treated with levodopa. It is
noteworthy that MAO-B activity, as well as density, increase
with age in most brain regions while MAO-A activity
remains unchanged.[7,11,12] The inhibition of the MAO-B-
catalysed oxidation of dopamine is therefore of enhanced
relevance in the aged parkinsonian brain. It should be noted
that both MAO-A and MAO-B oxidize dopamine in the
brain.[1] MAO-A inhibition, however, is in general not a
desired property of drugs used in Parkinson’s disease
therapy since inhibitors of MAO-A potentiates the
sympathomimetic effects of dietary amines such as
tyramine, which may lead to serious adverse effects.[13,14]
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Phenylalkylcaffeines as MAO inhibitors
Anél Petzer et al.
O
N
N
N
N
O
Caffeine (1)
1,4-Diphenyl-2-butene
O
Cl
N
N
N
OH
O
N
(E)-8-(3-Chlorostyryl)caffeine (2)
O
Trans,trans-farnesol
Br
N
N
N
O
O
N
8-(3-Bromobenzyloxy)caffeine (3)
Figure 1 The structures of caffeine, (E)-8-(3-chlorostyryl)caffeine, 8-(3-bromobenzyloxy)caffeine, 1,4-diphenyl-2-butene and trans,trans-farnesol.
MAO inhibitors used for Parkinson’s disease therapy should
therefore be selective for the B isoform.
Among these, caffeine (1) has emerged as a useful scaffold
for the design of potent and reversible MAO-B inhibitors
(Figure 1).[18,19] In this regard, substitution on the C8
position of caffeine yields structures which are high potency
reversible MAO-B inhibitors. C8 substituents, which
have been shown to be particularly effective, are the
styryl and benzyloxy moieties. Examples of such struc-
tures are (E)-8-(3-chlorostyryl)caffeine (2) and 8-(3-
bromobenzyloxy)caffeine (3).[18,19] These compounds are
potent inhibitors of MAO-B with IC50 values 0.128 μm
and 0.068 μm, respectively. To further explore the structure–
activity relationships (SARs) of MAO-B inhibition by
caffeine-derived compounds, the present study examines
the MAO inhibitory properties of a series of caffeine ana-
logues substituted on C8 with a variety of phenylalkyl
groups. For this purpose, a homologous series (4a–f) con-
taining the −(CH2)n-C6H5 (n = 2–7) moieties at C8 were
synthesized and examined as inhibitors of human MAO-A
and MAO-B. These homologues were designed to examine
the effect of differing chain lengths of the C8 substituent
on MAO-B inhibition activity and selectivity. In addi-
tion the −(CH2)2-C6H11 containing homologue (4g) was
included to determine the requirement of a terminal phenyl
ring for MAO-B inhibition. Two carbonyl containing
homologues, compounds 4h and 4i, served to determine if
hydrogen bonding potential of the C8 side chain may
further improve MAO-B inhibitory activity. Literature
reports that several amino acid residues in the MAO-B
active site may be involved in such hydrogen bonding.
In Parkinson’s disease, MAO-B inhibitors may also pre-
serve dopaminergic nigrostriatal neurons by protecting
against underlying neurodegenerative processes. MAO-B
inhibitors may reduce the formation of potentially neuro-
toxic metabolic by-products of the MAO catalytic cycle.[1]
The catalytic cycle of MAO-B stoichiometrically yields one
mole each of an iminium intermediate that is hydrolysed to
the aldehyde product (which may react with exocyclic
amino groups of nucleosides and N-terminal and lysine
ε-amino groups of proteins), and H2O2 (which may lead to
oxidative damage) for each mole of monoamine substrate
oxidized.[1]
Based on the above considerations, several research
groups pursue the design of MAO-B selective inhibitors for
Parkinson’s disease therapy. While irreversible inhibitors of
MAO-B have been used clinically, reversible inhibitors may
have a number of potential advantages. For example, in
contrast to reversible inhibition, at high drug concentra-
tions or following repeated drug administration, irreversible
inhibitors may lead to the loss of isoform selectivity.[15] Also,
following withdrawal of an irreversible inhibitor, the rate by
which the activity of the MAO enzyme is recovered may be
variable and full recovery may require several weeks.[16,17]
Following the administration of reversible inhibitors,
enzyme activity is recovered when the inhibitor is cleared
from the tissues. A variety of oxygen and nitrogen contain-
ing heterocycles has been employed as MAO-B inhibitors.
2
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Anél Petzer et al.
Phenylalkylcaffeines as MAO inhibitors
Examples of these are Gln206 and Tyr326.[20,21] Finally, the
phenylpropenyl moiety (4j) was included to compare the
MAO-B inhibition potency of a compound containing an
isolated double bond to that of (E)-8-styrylcaffeine in
which the vinyl group is conjugated with the caffeine
ring.[18] These novel caffeine analogues are therefore a con-
tinuation of our efforts to discover highly potent and selec-
tive MAO-B inhibitors for PD therapy. As the results will
show, longer alkyl side chains on C8 of the caffeine moiety
yield highly potent and selective MAO-B inhibitors.
and are expressed as mean SD. The Kruskal–Wallis test
with Dunn’s post-hoc test was used to determine if statisti-
cal differences exist between the means of the residual
enzyme rates recorded before and after dilution and dialy-
sis. A P value of <0.05 is judged as being statistical signifi-
cantly different. These analyses were carried out with the
Prism 5 software package (GraphPad, San Diego, CA, USA).
The synthesis of caffeine analogues (4a–j)
1,3-Dimethyl-5,6-diaminouracil[23] (5, 10 mmol) and N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlo-
ride (EDAC; 13.4 mmol) were dissolved in 100 ml dioxane/
H2O (1 : 1), and the appropriate carboxylic acid (6,
10 mmol) was added. The pH of the resulting suspension
was adjusted to 5 with HCl (2 M), and stirring was con-
tinued for an additional 2 h. The reaction mixture was
neutralized with NaOH (1 M), cooled to 0°C and the
precipitate was collected by filtration. The crude product
was dissolved in 100 ml NaOH (1 M)/dioxane (1 : 1)
and heated for 2 h under reflux. The reaction mixture
was cooled to 0°C, acidified to a pH of 4 with 4 M aqueous
hydrochloric acid and the precipitate was collected
by filtration. The resulting 1,3-dimethyl-8-substituted-
7H-xanthinyl analogues (7) were used in the subse-
quent reaction without further purification. Compound
Materials and Methods
Unless otherwise noted, all starting materials were obtained
from Sigma-Aldrich (St. Louis, MO, USA) and were used
without purification. Proton (1H) and carbon (13C) nuclear
magnetic resonance (NMR) spectra were recorded on a
Bruker Avance III 600 spectrometer at frequencies of
600 MHz and 150 MHz, respectively (Bruker, Karlsruhe,
Germany). All NMR measurements were conducted in
CDCl3, and the chemical shifts are reported in parts per
million (δ) downfield from the signal of tetramethylsilane
added to the deuterated solvent. Spin multiplicities are
given as s (singlet), d (doublet), t (triplet), q (quartet), qn
(quintet) or m (multiplet). High-resolution mass spectra
(HRMS) were obtained on a Double focusing magnetic
sector high resolution magnetic sector mass spectrometer
(Thermo Electron Corporation, Waltham, MA, USA) in
electrospray ionization (ESI) mode or with a Bruker
micrOTOF-Q II mass spectrometer in atmospheric-
pressure chemical ionization (APCI) mode. Melting points
(mp) were determined on a Stuart SMP10 melting point
apparatus (Stuart, Staffordshire, UK) and are uncorrected.
To determine the purities of the synthesized compounds,
high-performance liquid chromatography (HPLC) analyses
were conducted as described previously.[15] HPLC grade
acetonitrile (Merck, Darmstadt, Germany) and Milli-Q
water (Millipore, Billerica, MA, USA) were used for the
chromatography. For fluorescence spectrophotometry, a
Varian Cary Eclipse fluorescence spectrophotometer
(Agilent Technologies, Santa Clara, CA, USA) was
employed. The following were obtained from Sigma-
Aldrich: microsomes from insect cells containing recombi-
nant human MAO-A and MAO-B (5 mg/ml), kynuramine
dihydrobromide, (R)-deprenyl hydrochloride and toloxa-
tone. Lazabemide hydrochloride was synthesized according
to the patented method.[22]
7
(0.20 mmol) was dissolved in 5 ml N,N-
dimethylformamide (DMF) and potassium carbonate
(0.50 mmol) was added. To the resulting suspension,
iodomethane (0.40 mmol) was added. Stirring was con-
tinued at 60°C for 60 min, the insoluble materials were
removed by filtration and sufficient water (∼100 ml) was
added to the filtrate to precipitate the product (4), which
was collected by filtration. Following crystallization from
a
methanol analytically pure samples of 4a–j were
obtained.[24]
8-(2-Phenylethyl)caffeine (4a)
The title compound was prepared from 3-phenylpropanoic
acid in a yield of 88%: mp 154–157°C (methanol). 1H NMR
(CDCl3) δ 2.98 (t, 2H, J = 7.2 Hz), 3.05 (t, 2H, J = 7.2 Hz),
3.34 (s, 3H), 3.55 (s, 3H), 3.57 (s, 3H), 7.08 (d, 2H,
J = 7.2 Hz), 7.18 (t, 1H, J = 7.2 Hz), 7.24 (m, 2H); 13C NMR
(CDCl3) δ 27.8, 28.9, 29.7, 31.3, 34.0, 107.1, 126.6, 128.3,
128.7, 139.9, 147.9, 151.6, 153.3, 155.2; ESI-HRMS m/z:
calcd for C16H19N4O2 (MH+), 299.1508, found 299.1502;
Purity (HPLC): 99.7%.
Statistical methods
8-(3-Phenylpropyl)caffeine (4b)
IC50 values were determined in triplicate and are expressed
as mean standard deviation (SD). For the recovery of
enzyme activity after the dilution and dialysis experiments,
residual enzyme catalytic rates were determined in triplicate
The title compound was prepared from 4-phenylbutanoic
acid in a yield of 66%: mp 115–117°C (methanol). 1H NMR
(CDCl3) δ 2.09 (qn, 2H, J = 7.5 Hz), 2.71 (q, 4H,
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
3

Phenylalkylcaffeines as MAO inhibitors
Anél Petzer et al.
J = 7.5 Hz), 3.37 (s, 3H), 3.55 (s, 3H), 3.80 (s, 3H), 7.17 (m,
3H), 7.28 (t, 2H, J = 7.5 Hz); 13C NMR (CDCl3) δ 26.0, 27.8,
28.7, 29.7, 31.6, 35.1, 107.3, 126.2, 128.4, 128.5, 140.9, 148.0,
151.7, 153.9, 155.3; ESI-HRMS m/z: calcd for C17H21N4O2
(MH+), 313.1665, found 313.1659; Purity (HPLC): 99.4%.
128.3, 142.6, 147.9, 151.6, 154.4, 155.2; APCI-HRMS m/z:
calcd for C21H29N4O2 (MH+), 369.2291, found 369.2289;
Purity (HPLC): 99.2%.
8-(2-Cyclohexylethyl)caffeine (4g)
The title compound was prepared from 3-
cyclohexylpropanoic acid in a yield of 78%: mp 133–134°C
(methanol). H NMR (CDCl3) δ 0.95 (m, 2H), 1.20 (m,
8-(4-Phenylbutyl)caffeine (4c)
1
The title compound was prepared from 5-phenylpentanoic
acid in a yield of 68%: mp 131–133°C (methanol). 1H NMR
(CDCl3) δ 1.69–1.79 (m, 4H), 1.79 (t, 2H, J = 7.2 Hz), 1.77
(t, 2H, J = 7.2 Hz), 3.37 (s, 3H), 3.54 (s, 3H), 3.85 (s, 3H),
7.15 (m, 3H), 7.25 (m, 2H); 13C NMR (CDCl3) δ 26.7, 27.1,
27.8, 29.7, 30.9, 31.7, 35.5, 107.3, 125.9, 128.3, 128.4, 141.8,
148.0, 151.7, 154.1, 155.3; ESI-HRMS m/z: calcd for
C18H23N4O2 (MH+), 327.1821, found 327.1812; Purity
(HPLC): 99.2%.
4H), 1.57–1.75 (m, 7H), 2.70 (t, 2H, J = 7.9 Hz), 3.37 (s,
3H), 3.54 (s, 3H), 3.88 (s, 3H); 13C NMR (CDCl3) δ 24.4,
26.2, 26.5, 27.8, 29.7, 31.6, 33.0, 34.9, 37.5, 107.3, 148.0,
151.7, 154.8, 155.3; ESI-HRMS m/z: calcd for C16H25N4O2
(MH+), 305.1978, found 305.1977; Purity (HPLC): 99.8%.
8-(3-Oxo-3-phenylpropyl)caffeine (4h)
The title compound was prepared from 4-oxo-4-
phenylbutanoic acid in a yield of 59%: mp 191–194°C
1
(methanol). H NMR (CDCl3) δ 3.11 (t, 2H, J = 7.4 Hz),
8-(5-Phenylpentyl)caffeine (4d)
3.36 (s, 3H), 3.46 (s, 3H), 3.58 (t, 2H, J = 7.4 Hz), 4.01 (s,
3H), 7.46 (t, 2H, J = 7.5 Hz), 7.57 (t, 1H, J = 7.5 Hz), 7.98
(d, 2H, J = 7.5 Hz); 13C NMR (CDCl3) δ 20.7, 27.8, 29.6,
31.7, 35.6, 107.4, 128.1, 128.7, 133.4, 136.4, 147.9, 151.7,
153.2, 155.3, 198.1; ESI-HRMS m/z: calcd for C17H19N4O3
(MH+), 327.1457, found 327.1469; Purity (HPLC): 92.2%.
The title compound was prepared from 6-phenylhexanoic
acid in a yield of 44%: mp 114–116°C (methanol). 1H NMR
(CDCl3) δ 1.41 (qn, 2H, J = 7.5 Hz), 1.66 (qn, 2H,
J = 7.9 Hz), 1.75 (qn, 2H, J = 7.5 Hz), 2.61 (t, 2H,
J = 7.5 Hz), 2.69 (t, 2H, J = 7.9 Hz), 3.38 (s, 3H), 3.54 (s,
3H), 3.87 (s, 3H), 7.15 (m, 3H), 7.25 (m, 2H); 13C NMR
(CDCl3) δ 26.8, 27.5, 27.8, 28.3, 29.7, 31.0, 31.7, 35.7, 107.3,
125.8, 128.3, 128.4, 142.3, 148.0, 151.7, 154.3, 155.3; ESI-
HRMS m/z: calcd for C19H25N4O2 (MH+), 341.1978, found
341.1974; Purity (HPLC): 99.0%.
8-(4-Oxo-4-phenylbutyl)caffeine (4i)
The title compound was prepared from 5-oxo-5-
phenylpentanoic acid in a yield of 62%: mp 145–146°C
(methanol). 1H NMR (CDCl3) δ 2.20 (qn, 2H, J = 7.15 Hz),
2.83 (t, 2H, J = 7.5 Hz), 3.11 (t, 2H, J = 6.4 Hz), 3.37 (s, 3H),
3.48 (s, 3H), 3.93 (s, 3H), 7.44 (t, 2H, J = 7.5 Hz), 7.55 (t,
1H, J = 7.2 Hz), 7.92 (d, 2H, J = 7.2 Hz); 13C NMR (CDCl3)
δ 21.6, 26.0, 27.9, 29.6, 31.8, 37.0, 107.5, 127.9, 128.7, 133.3,
136.7, 147.9, 151.7, 153.5, 155.3, 199.2; ESI-HRMS m/z:
calcd for C18H21N4O3 (MH+), 341.1614, found 341.1606;
Purity (HPLC): 98.9%.
8-(6-Phenylhexyl)caffeine (4e)
The title compound was prepared from 6-phenylheptanoic
acid in a yield of 44%: mp 114–116°C (methanol). 1H NMR
(CDCl3) δ 1.41 (m, 4H), 1.65 (qn, 2H, J = 7.5 Hz), 1.75 (qn,
2H, J = 7.5 Hz), 2.62 (t, 2H, J = 7.9 Hz), .2.71 (t, 2H,
J = 7.9 Hz), 3.40 (s, 3H), 3.57 (s, 3H), 3.89 (s, 3H), 7.17 (d,
2H, J = 7.9 Hz), 7.27 (t, 3H, J = 7.5 Hz); 13C NMR (CDCl3) δ
26.7, 27.4, 27.7, 28.7, 29.0, 29.6, 31.1, 31.6, 35.7, 107.1,
125.6, 128.2, 128.3, 142.4, 147.9, 151.6, 154.3, 155.2; APCI-
HRMS m/z: calcd for C20H27N4O2 (MH+), 355.2134, found
355.2134; Purity (HPLC): 99.4%.
8-((2E)-3-Phenylprop-2-en-1-yl)caffeine (4j)
The title compound was prepared from trans-styrylacetic
acid in a yield of 76%: mp 169–171°C (methanol). 1H NMR
(CDCl3) δ 3.38 (s, 3H), 3.57 (s, 3H), 3.93 (s, 3H), 3.69 (d,
2H, J = 6.4 Hz), 6.28 (m, 1H), 6.44 (d, 1H, J = 15.8 Hz), 7.22
(m, 1H), 7.29 (t, 2H, J = 7.5 Hz), 7.32 (d, 2H, J = 7.5 Hz);
13C NMR (CDCl3) δ 27.9, 29.8, 30.9, 31.9, 107.7, 122.5,
126.3, 127.9, 128.6, 133.3, 136.3, 148.0, 151.7, 155.3; ESI-
HRMS m/z: calcd for C17H19N4O2 (MH+), 311.1508, found
311.1493; Purity (HPLC): 99.2%.
8-(7-Phenylheptyl)caffeine (4f)
The title compound was prepared from 6-phenyloctanoic
acid in a yield of 44%: mp 114–116°C (methanol). 1H NMR
(CDCl3) δ 1.30 (m, 6H), 1.54 (qn, 2H, J = 7.2 Hz), 1.65 (qn,
2H, J = 7.2 Hz), 2.52 (t, 2H, J = 7.9 Hz), .2.63 (t, 2H,
J = 7.9 Hz), 3.31 (s, 3H), 3.48 (s, 3H), 3.81 (s, 3H), 7.09 (m,
3H), 7.18 (m, 2H); 13C NMR (CDCl3) δ 26.7, 27.5, 27.7,
28.9, 29.0, 29.1, 29.6, 31.3, 31.6, 35.8, 107.1, 125.5, 128.1,
IC50 value determinations
The IC50 values for the inhibition of the recombinant
human MAO-A and MAO-B were determined according to
4
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Anél Petzer et al.
Phenylalkylcaffeines as MAO inhibitors
a previously reported protocol.[25] Potassium phosphate
buffer (100 mM, pH 7.4, made isotonic with KCl) served
as solvent for the enzyme reactions (500 μl final volume).
The reactions contained the MAO-A/B mixed substrate
kynuramine (45 μm for MAO-A and 30 μm for MAO-B)
and the test inhibitors (0.003–100 μm). The test inhibitors
were dissolved in dimethyl sulfoxide (DMSO) and were
added to the reactions to yield a final concentration of 4%
DMSO. Similar reactions were also carried out in the
absence of inhibitor and also using the reference MAO
inhibitors, toloxatone and lazabemide, as test inhibitors.
MAO-A or MAO-B (0.0075 mg protein/ml) were added,
and the reactions were incubated for 20 min at 37°C. The
reactions were terminated with the addition of 400 μl
NaOH (2 N) and 1000 μl water, and the concentrations of
4-hydroxyquinoline, the MAO-catalysed oxidation product
of kynuramine, were measured by fluorescence spec-
trophotometry (λex = 310; λem = 400 nm).[26] For this
purpose, linear calibration curves (4-hydroxyquinoline:
0.047–1.5 μm) were constructed. The resulting MAO cata-
lytic rates were fitted to the one site competition model
incorporated into the Prism 5 software package. The IC50
values were determined in triplicate from the resulting
sigmoidal concentration–inhibition curves and are
expressed as mean SD.
capacity of 0.5–3 ml. MAO-B (0.03 mg/ml) and 4f, at a con-
centration equal to 4 × IC50, were preincubated for 15 min
at 37°C. These reactions were conducted in potassium
phosphate buffer (100 mM, pH 7.4) containing 5% sucrose
to final volume of 0.8 ml, and DMSO (4%) was added as
cosolvent. As negative and positive controls, MAO-B was
also preincubated in the absence of inhibitor and presence
of a concentration equal to 4 × IC50 of the irreversible
inhibitor, (R)-deprenyl (IC50(MAO-B) = 0.079 μm).[25] The
reactions were subsequently dialysed at 4°C in 80 ml of
outer buffer (100 mM potassium phosphate, pH 7.4, 5%
sucrose). The outer buffer was replaced with fresh buffer at
3 h and 7 h after the start of dialysis. At 24 h after dialysis
was started, the reactions were diluted twofold with the
addition of kynuramine (dissolved in potassium phosphate
buffer, 100 mM, pH 7.4, made isotonic with KCl), and the
residual MAO-B activities were measured as described
above. The final concentration of kynuramine in these reac-
tions was 50 μm while the final inhibitor concentrations
were equal to twofold their IC50 values for the inhibition of
MAO-B. For comparison, undialysed mixtures of MAO-B
with 4f were maintained at 4°C over the same time period.
These reactions were carried out in triplicate and the resid-
ual enzyme catalytic rates were expressed as mean SD.
Construction of Lineweaver–Burk plots
Recovery of enzyme activity after dilution
Lineweaver–Burk double reciprocal plots (1/V vs. 1/(S))
were constructed to examine the mode of MAO-B inhibi-
tion by compound 4f. For this purpose, a plot was con-
structed in the absence of the test inhibitor while five plots
were each constructed in the presence of different concen-
trations of the test inhibitor. The concentrations of 4f for
these studies were one-fourth × IC50, one-half × IC50,
three-fourth × IC50, 1 × IC50 and 1 and one-fourth × IC50.
For each Lineweaver–Burk plot, six different concentrations
(15–250 μm) of kynuramine were employed. All enzymatic
reactions were carried out as described for the determina-
tion of IC50 values above with the exception that the con-
centration of recombinant human MAO-B employed was
0.015 mg/ml. The rates of the MAO-B-catalysed formation
of 4-hydroxyquinoline were measured by fluorescence
spectrophotometry as described above. Linear regression
analysis was performed using Prism 5, and the Ki value was
estimated from the x-axis intercept (–Ki) of a replot of
the slopes of the Lineweaver–Burk plots versus inhibitor
concentration.
Potassium phosphate buffer (100 mM, pH 7.4, made iso-
tonic with KCl) served as solvent for these studies. Com-
pound 4f (IC50(MAO-B) = 0.086 μm) at concentrations
equal to 10 × IC50 and 100 × IC50 was preincubated with
recombinant human MAO-B (0.75 mg/ml) for 30 min at
37°C. (R)-Deprenyl (IC50(MAO-B) = 0.079 μm) were simi-
larly preincubated with MAO-B at a concentration equal to
10 × IC50.[25] Control incubations were conducted in the
absence of inhibitor, and DMSO (4%) was added as
cosolvent to all preincubations. The reactions were diluted
100-fold with the addition of kynuramine to yield final con-
centrations of 4f equal to 0.1 × IC50 and 1 × IC50, and
of (R)-deprenyl equal to 0.1 × IC50. The final concentration
of kynuramine was 30 μm, and the final concentration of
MAO-B was 0.0075 mg/ml. The reactions were incubated
for a further 20 min at 37°C, terminated and the residual
rates of 4-hydroxyquinoline formation were measured as
described above. The residual enzyme catalytic rates were
expressed as mean SD of triplicate determinations.
Dialysis
Results
The reversibility of the MAO-B inhibition by 4f was investi-
gated by dialysis, employing Slide-A-Lyzer® dialysis cas-
settes (Thermo Scientific, Waltham, MA, USA) with a
molecular weight cut-off of 10 000 and a sample volume
Synthesis of caffeine analogues
The syntheses of the caffeine derivatives 4a–j were accom-
plished using the literature procedure (Figure 2).[24] 1,3-
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
5

Phenylalkylcaffeines as MAO inhibitors
Anél Petzer et al.
inhibitor the MAO-B isoform.[19] Three homologues, 4d–f,
display IC50 values for the inhibition of MAO-B in the
submicromolar range. In fact, 4f (IC50 = 0.086 μm), the
most potent MAO-B inhibitor among the compounds
evaluated, may be considered as a highly potent MAO-B
inhibitor and equipotent to the reference inhibitor
lazabemide (IC50 = 0.091 μm) (Figure 3). Compound 4f
also inhibited MAO-A (IC50 = 3.01 μm), although 35-fold
weaker compared with its inhibitory potency towards
MAO-B. In spite of this, 4f may still be considered as
a moderately potent MAO-A inhibitor since it is slightly
more potent than the reference inhibitor toloxatone
(IC50 = 3.92 μm). It is interesting to note that 4f also was
the most potent MAO-A inhibitor among the compounds
evaluated. This suggests that structural modifications made
to the caffeine analogues, which result in enhanced MAO-B
inhibition potency, in general, also enhances MAO-A
inhibition potency. As shown below, increasing the alkyl
chain length of the C8 substituent is an example of such a
structural modification. Among homologues 4a–f, which
contain the –(CH2)n-C6H5 (n = 2–7) moieties at C8, inter-
esting SARs are apparent. The general order of MAO-B
inhibition potency of these homologues are 4f (n = 7) > 4e
(n = 6) > 4d (n = 5) > 4c (n = 4) > 4a (n = 2) > 4b (n = 3).
This result demonstrates that increasing the alkyl chain
length of the C8 substituent is associated with enhanced
MAO-B inhibition potency. The only exception is the
increase of the chain length by one methylene unit from 4a
to 4b, which leads to a twofold reduction in MAO-B inhibi-
tion potency. The general order of MAO-A inhibition
potency of these homologues are 4f (n = 7) > 4e
(n = 6) > 4d (n = 5) > 4b (n = 3) > 4c (n = 4) > 4a (n = 2).
For the inhibition of MAO-A, the same trend exists as
observed for the inhibition of MAO-B, where increasing the
alkyl chain length of the C8 substituent enhances MAO-A
inhibition potency. The only exception is found with 4b
(IC50 = 117 μm) and 4c (IC50 = 125 μm), which exhibit
equipotent inhibitory potencies towards MAO-A. As men-
tioned above, increasing the alkyl chain length of the C8
substituent is thus a structural modification that leads to
enhanced inhibition of both MAO-A and MAO-B.
O
NH2
N
HO2C
6
R
+
NH2
O
O
N
5
O
N
H
N
N
N
(a), (b)
(c)
N
N
R
R
N
O
N
O
7
4a−j
Figure 2 Synthetic pathway to caffeine derivatives 4a–j. Reagents
and conditions: (a) EDAC dioxane/H2O, rt; (b) dioxane/NaOH (aq),
reflux; (c) CH3I, K2CO3, DMF, 60°C.
Dimethyl-5,6-diaminouracil[23] (5) was reacted with a
carboxylic acid derivative (6) in the presence of the
carbodiimide dehydrating reagent, EDAC. The amide inter-
mediate thus obtained was treated with aqueous sodium
hydroxide to yield the 7H-xanthinyl analogues (7). Without
further purification, the crude xanthine was 7N-methylated
with an excess of iodomethane to yield the target caffeine
derivatives 4a–j. Following crystallization, the structures of
1
all compounds were verified by mass spectrometry, H
NMR and 13C NMR, and the purities were estimated by
HPLC analyses.
MAO inhibitory potencies of
the caffeine analogues
For the purpose of this study, recombinant human MAO-A
and MAO-B were used to examine the MAO inhibitory
properties of the phenylalkylcaffeines, 4a–j. The MAO-A/B
mixed substrate, kynuramine, served as enzyme substrate.
Kynuramine is oxidized by the MAOs to ultimately
yield 4-hydroxyquinoline, which can be readily monitored
by fluorescence spectrophotometry (λex = 310 nm; λem
=
400 nm). This protocol is free from interference of the test
inhibitors and kynuramine since these species are non-
fluorescent under the measurement conditions.[26]
The results of the MAO inhibitory studies are given in
Table 1. As shown, the caffeine analogues 4a–j are inhibitors
of both MAO-A and MAO-B. With the exception of com-
pound 4h, all of the homologues are selective for the
MAO-B isoform with selectivity index (SI) values ranging
from 2 to 79. Among the compounds examined, 4d
is the most selective MAO-B inhibitor with a SI value of
79. Compared with the MAO-B selective inhibitor,
lazabemide (SI = 2219), these selectivities for the MAO-B
isoform are moderate. The previously studied 8-(3-
bromobenzyloxy)caffeine (3) is reported to possess a SI
value of 13.8, which also makes it a moderately selective
The −(CH2)2-C6H11 containing homologue (4g) was also
found to be a MAO inhibitor with IC50 values of 6.59 μm
and 73.3 μm for the inhibition of MAO-B and MAO-A,
respectively. A comparison of the MAO inhibitory potencies
of 4g with its phenyl-substituted homologue 4a shows that
4g is a superior inhibitor of both isoforms than 4a. These
data suggests that, as a terminal moiety on the C8 substitu-
ent of caffeines, the cyclohexyl moiety is more suitable than
the phenyl ring for MAO inhibition. A possible explanation
for this observation is that the higher degree of lipophilicity
of the cyclohexyl ring compared with the phenyl ring facili-
tates more productive Van der Waals interactions with the
6
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Anél Petzer et al.
Phenylalkylcaffeines as MAO inhibitors
Table 1 The IC50 values for the inhibition of recombinant human MAO-A and MAO-B by compounds 4a–j, and the reference inhibitors
lazabemide and toloxatone. The selectivity index (SI) values are also provided
O
N
N
R
N
O
N
IC50 (μM)a
MAO-B
R
MAO-A
SIb
4a
4b
4c
4d
4e
4f
−(CH2)2-C6H5
−(CH2)3-C6H5
−(CH2)4-C6H5
−(CH2)5-C6H5
−(CH2)6-C6H5
−(CH2)7-C6H5
−(CH2)2-C6H11
−(CH2)2-CO-C6H5
−(CH2)3-CO-C6H5
−(CH2)-CH = CH-C6H5
Lazabemide
172 26.5
117 0.071
125 22.1
51.6 2.37
19.2 0.74
3.01 0.24
73.3 10.0
10.4 0.767
216 5.73
68.4 1.94
202 26
26.0 5.57
43.3 6.20
3.25 0.176
0.656 0.0048
0.442 0.015
0.086 0.003
6.59 1.54
187 7.21
46.9 7.65
33.1 4.50
0.091 0.015
–
7
3
0.41
0.39
38 8.9
79 4.2
43 0.33
35 3.7
11 4.2
4g
4h
4i
0.06 0.002
5
2
0.64
0.22
4j
2219 703
–
Toloxatone
3.92 0.015
a
MAO, monoamine oxidase. All values are expressed as the mean SD of triplicate determinations. bThe SI is the selectivity for the MAO-B isoform
and is given as the ratio of IC50(MAO-A)/IC50(MAO-B). The values are reported as mean SD.
MAO-A
100
MAO-B
100
Laz
4f
75
50
25
0
75
50
25
0
4f
Tol
−2
−1
0
1
2
−2
−1
0
1
2
Log[I]
Log[I]
Figure 3 The sigmoidal dose–response curves (filled circles) of the monoamine oxidase (MAO)-A and MAO-B catalytic rates in the presence
of compound 4f (concentration expressed in μM). For comparison, the dose-response curves (open circles) for the inhibition of MAO-A and MAO-B
by toloxatone (Tol) and lazabemide (Laz), respectively, are also provided. The values are given as mean standard deviation (SD) of triplicate
determinations.
MAO active sites. Since the active sites of both MAO-A and
MAO-B are lined mostly by hydrophobic residues,
lipophilic moieties of ligands are expected to interact pro-
ductively with hydrophobic patches in the MAO active
sites.[27,28] This proposal is supported by the observation that
increasing the alkyl chain length of the C8 substituent
enhances MAO inhibition potency, since an increasing alkyl
chain length would also enhance the lipophilicities of the
caffeine-derived inhibitors.
The two carbonyl containing homologues, compounds
4h and 4i, were found to be relatively weak MAO-B inhibi-
tors with IC50 values of 187 μm and 46.9 μm, respectively.
These MAO-B inhibition potencies are the weakest rec-
orded for the series, which suggests that the introduction of
a carbonyl group in the C8 side chain does not enhance
binding to MAO-B, and potential hydrogen bonding with
these carbonyl groups does not contribute to productive
interactions with the MAO-B active site. While 4i was a
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
7

Phenylalkylcaffeines as MAO inhibitors
Anél Petzer et al.
weak MAO-A inhibitor (IC50 = 216 μm), 4h proved to be
the second most potent MAO-A inhibitor of the series with
an IC50 value of 10.4 μm. These data suggest that for 4h, the
carbonyl group in the C8 side chain interacts productively
with the MAO-A active site. It is, however, unclear why 4i
acts as a weak MAO-A inhibitor. Further investigation of
the molecular interactions of 4i with MAO-A would be of
interest.
100
75
50
25
0
The phenylpropenyl-substituted homologue 4j was found
to be a moderately potent MAO-A and MAO-B inhibitor
with IC50 values of 68.4 μm and 33.1 μm, respectively.
These activities are more potent than those recorded for
the corresponding n = 3 homologue 4b. This suggests that
the introduction of a C–C double bond into the C8 side
chain is beneficial for MAO inhibition. Compared with
the MAO-B inhibition potency of (E)-8-styrylcaffeine
(IC50 = 2.7 μm), 4j is, however, a 12-fold weaker inhibi-
tor.[18] This result demonstrates that substitution on C8 of
caffeine with the styryl moiety is more favourable for
MAO-B inhibition than the phenylpropenyl moiety. A pos-
sible explanation for this behaviour is that the coplanar
characteristic of the caffeine and styryl phenyl ring pro-
motes binding of (E)-8-styrylcaffeine to the MAO-B active
site, which is reported to be a relatively narrow and flat
cavity.[28] In contrast, structures such as 4j, where the caf-
feine and side chain phenyl ring are not coplanar, may be
less suitable for fitting into the narrow MAO-B active site
cavity.
*
No Inhibitor
[4f] = 0.1 x IC50
(R)-Deprenyl
[4f] = 1 x IC50
Figure 4 The reversibility of monoamine oxidase (MAO)-B inhibition
of by compound 4f. MAO-B was preincubated with 4f at 10 × IC50
and 100 × IC50 for 30 min and then diluted to 0.1 × IC50 and
1 × IC50. The residual enzyme activities were subsequently measured.
For comparison, MAO-B was similarly treated with (R)-deprenyl at
10 × IC50 and subsequently diluted to 0.1 × IC50. *Statistical signifi-
cantly different from the mean of No inhibitor. The values are given as
mean standard deviation (SD) of triplicate determinations.
respectively, of the control value. For comparison, after
similar treatment of MAO-B with the irreversible inhibitor
(R)-deprenyl (at 10 × IC50) and dilution of the resulting
complexes to 0.1 × IC50, MAO-B activity is not recovered
(1.5% of control). It may thus be concluded that 4f interacts
reversibly with MAO-B. For reversible enzyme inhibition,
the enzyme activities are expected to recover to levels of
approximately 90 and 50%, respectively, after dilution of the
inhibitor to 0.1 × IC50 and 1 × IC50. It is interesting to note
that, after dilution, MAO-B catalytic activity is not recov-
ered to 90 and 50%, respectively, as expected. This result
suggests that the binding of 4f may possess a quasi-
reversible or tight-binding component. Although further
investigation is necessary to clarify this point, it is note-
worthy that the side chains of several residues lining the
MAO-B active site are flexible. The side chain of Ile199, in
particular, may adopt at least two alternate conforma-
tions.[28] It is thus conceivable that upon binding of certain
inhibitors (such as 4f), a conformational change of the
MAO-B active site may occur, which results in slow disso-
ciation of the inhibitor from the active site. Such a scenario
may result in tight-binding of the inhibitor.
The reversibility of MAO-B inhibition
Caffeine analogues have previously been shown to act as
reversible inhibitors of human MAO-B.[29] As mentioned in
the introduction, reversible inhibition has the advantage
that MAO activity is recovered when treatment is termi-
nated and the inhibitor is cleared from the tissues. In con-
trast, after the termination of treatment with an irreversible
inhibitor, de novo protein synthesis is required for enzyme
activity to recover. To examine the possibility that the most
potent MAO-B inhibitor of the present series, compound
4f, is a reversible inhibitor, the reversibility of the interac-
tion of 4f with MAO-B was investigated. Since none of the
compounds examined were potent MAO-A inhibitors, the
reversibility of MAO-A inhibition was not further studied.
To investigate the reversibility of inhibition, the recoveries
of the MAO-B catalytic activity after dilution of enzyme-
inhibitor complexes were examined. Compound 4f was
combined and preincubated with MAO-B at concentrations
of 10 × IC50 and 100 × IC50 for 30 min. The reactions were
subsequently diluted 100-fold to yield inhibitor concentra-
tions of 0.1 × IC50 and 1 × IC50. As shown in Figure 4, after
dilution of the inhibitor to 0.1 × IC50 and 1 × IC50, the
MAO-B catalytic activities are recovered to 69 and 39%,
The reversibility of MAO-B inhibition by 4f was also
investigated by measuring the recoveries of MAO-B activity
8
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Anél Petzer et al.
Phenylalkylcaffeines as MAO inhibitors
1200
800
100
75
50
25
0
100
75
50
25
0
400
−0.08 −0.04 0.00 0.04 0.08 0.12
[I], µM
*
−0.02
0.00
0.02
1/(S)
0.04
0.06
Figure 6 Lineweaver–Burk plots for the inhibition of monoamine
oxidase (MAO)-B in the absence (filled squares) and presence of various
concentrations of 4f. The inset is a plot of the slopes of the double
reciprocal plots versus inhibitor concentration.
4f - dialysed
Depr - dialysed
4f - undialysed
No Inhibitor - dialysed
Figure 5 Reversibility of the inhibition of monoamine oxidase
(MAO)-B by 4f. MAO-B and 4f (at 4 × IC50) were preincubated for
15 min, dialysed for 24 h and the residual enzyme activity was meas-
ured (4f–dialysed). MAO-B was similarly preincubated in the absence
(No inhibitor–dialysed) and presence of the irreversible inhibitor, (R)-
deprenyl (Depr–dialysed), and dialysed. For comparison, the residual
MAO activity of undialysed mixtures of MAO-B with 4f is also shown
(4f–undialysed). *Statistical significantly different from the mean of No
inhibitor–dialysed. The values are given as mean standard deviation
(SD) of triplicate determinations.
concentrations of 4f employing six substrate concentrations
(15–250 μm). The resulting Lineweaver–Burk plots are
shown in Figure 6. For the inhibition of MAO-B, the
Lineweaver–Burk plots are linear and intersect at a single
point on the y-axis. This suggests that 4f most likely inter-
acts competitively with MAO-B and is further support for a
reversible mode of inhibition. From the replot of the slopes
of the Lineweaver–Burk plots versus the inhibitor concen-
trations, a Ki value of 0.064 μm is estimated for the inhibi-
tion of MAO-B by 4f.
after dialysis of enzyme-inhibitor mixtures. MAO-B and 4f,
at a concentration of 4 × IC50, were preincubated for a
period of 15 min and subsequently dialysed for 24 h. The
results show that MAO-B inhibition by 4f is almost com-
pletely reversed after 24 h of dialysis (Figure 5). After dialy-
sis, MAO-B activity is recovered to a level of 95% of the
control value (recorded in the absence of inhibitor). In con-
trast, the MAO-B activity in undialysed mixtures are 29%
of the control value. This result is consistent with a revers-
ible interaction between MAO-B and 4f. After similar
preincubation and dialysis of samples containing MAO-B
and the irreversible inhibitor, (R)-deprenyl, the enzyme
activities are not recovered with MAO-B activity at a level of
only 1.9% of the control value.
Discussion
Caffeine acts as a useful scaffold for the design of MAO
inhibitors. Previous studies have shown that substitution of
caffeine on C8 with a variety of moieties yields highly
potent MAO inhibitors.[18,19] In this regard, the selection of
the C8 moiety has a significant effect on the potency of
MAO-B inhibition as well as the selectivity over the MAO-A
isoform. For example, styryl substitution on C8 yields
highly selective MAO-B inhibitors while benzyloxy-
substituted caffeines possess affinities for both MAO-A and
MAO-B. Phenoxy substitution, in contrast, results in weak
MAO-B inhibitory activity.[30] The present study shows that,
while shorter phenylalkyl substituents (−(CH2)n-C6H5,
n = 2–4) on C8 of caffeine results in moderate MAO-B inhi-
bition, substitution with longer phenylalkyl substituents
yields highly potent MAO-B inhibitors. In this regard,
substitution of caffeine with the −(CH2)n-C6H5 (n = 5–7)
moieties yields inhibitors 4d–4f with IC50 values in
the submicromolar range. These inhibitors also display
Competitive mode of MAO-B inhibition
As shown above, compound 4f is a reversible MAO-B
inhibitor. To examine whether the mode of inhibition of
MAO-B is competitive, sets of Lineweaver–Burk plots were
constructed. For this purpose, the MAO-B catalytic activity
was recorded in the absence and presence of five different
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
9

Phenylalkylcaffeines as MAO inhibitors
Anél Petzer et al.
relatively good selectivities (SI = 35–79) for the MAO-B
isoform. The observation that substitution with longer
phenylalkyl substituents results in potent MAO-B inhibition
may be explained, at least in part, by considering the archi-
tecture of the MAO-B active site. The MAO-B active site is
reported to consist of two cavities, an entrance cavity and a
substrate cavity.[28] Substrate and inhibitor molecules must
pass through the entrance cavity to reach the substrate
cavity where amine oxidation takes place. While small mol-
ecules bind within the substrate cavity of MAO-B, larger
structures such as 1,4-diphenyl-2-butene and trans,trans-
farnesol (Figure 1) span both entrance and substrate cav-
ities. In order for larger molecules to be able to bind to
MAO-B, the side chain of residue Ile199 rotates out of its
normal conformation to allow for the fusion of the two cav-
ities.[31] Larger inhibitors thus interact with residues and
water molecules in both the substrate and entrance cavities
while smaller molecules interact with only the substrate-
binding cavity. Modelling studies have shown that
substituents on C8 of caffeine most likely bind within the
entrance cavity, which is reported to be a hydrophobic
space.[19] The caffeine moiety is located in the substrate
cavity where the carbonyl oxygens may interact with polar
amino acid residues in proximity to the flavin adenine
dinucleotide cofactor. The possibility therefore exists that
extended phenylalkyl substituents may protrude deeper into
the entrance cavity thereby maximizing the formation of
productive Van der Waals interactions with the hydrophobic
entrance cavity. This is expected to lead to enhanced
binding affinity and MAO-B inhibition. An explanation for
the observation that substitution with longer alkylphenyl
substituents also enhances MAO-A inhibition is not appar-
ent. In certain instances, compounds with extended struc-
tures may not bind well to MAO-A.[27] The reason for this is
that, in the MAO-A active site, the residue corresponding to
Ile199 in MAO-B is Phe208. The increased size of the Phe
side chain compared with the side chain of Ile prevents it
from rotating from the MAO-A active site cavity into an
alternative conformation.[27] The binding of certain inhibi-
tors may thus by hindered by Phe208 in the MAO-A active
site. The possibility that the caffeine analogues examined
here may undergo potential unfavourable interactions with
Phe208 may, at least partly, explain why this series are
MAO-B selective inhibitors. Possible reasons why the two
carbonyl containing homologues, compounds 4h and 4i, act
as weak MAO-B inhibitors are also not apparent. One pos-
sibility is that the introduction of a sp2 carbon may restrict
the flexibility of the C8 side chain, thus hindering it from
adopting the conformation required for maximal interac-
tion with the MAO-B entrance cavity.
Conclusion
The present study shows that substitution of caffeine on C8
with extended side chains yields structures with potent
MAO-B inhibitory activities. This behaviour is exemplified
by 4f (Ki = 0.064 μm), which possesses the 7-phenylheptyl
moiety on the C8 position. Compound 4f is not only a high
potency MAO-B inhibitor, but displays relatively good selec-
tivity for MAO-B over the MAO-A isoform. A high degree
of selectivity is a desirable attribute for anti-parkinsonian
drugs since MAO-A inhibition is associated with potentially
serious side effects. Compound 4d is also a notable inhibitor
since it displayed the highest degree of selectivity for
MAO-B while possessing relatively potent MAO-B inhibi-
tory activity. The results also document that 4f is a revers-
ible MAO-B inhibitor, which from a safety point of view is a
desirable property. In contrast to irreversible inhibitors, de
novo protein synthesis is not required for enzyme activity to
recover after termination of treatment with a reversible
inhibitor. It may thus be concluded that 4f is a promising
lead for the development of reversible and selective MAO-B
inhibitors, which may be used in the treatment of Parkin-
son’s disease.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Acknowledgements
The NMR and MS spectra were recorded by André Joubert
and Johan Jordaan of the SASOL Centre for Chemistry,
North-West University, and Marelize Ferreira of the Mass
Spectrometry Service, University of the Witwatersrand. This
work is based on the research supported in part by the
Medical Research Council and National Research Founda-
tion (NRF) of South Africa (Grant specific unique reference
numbers (UID) 85642 and 80647). The Grant holders
acknowledge that opinions, findings and conclusions or rec-
ommendations expressed in any publication generated by
the NRF-supported research are that of the authors, and
that the NRF accepts no liability whatsoever in this regard.
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(Suppl. 1): S287–S296.
3. Inoue H et al. Species-dependent dif-
ferences in monoamine oxidase A and
B-catalyzed oxidation of various C4
substituted 1-methyl-4-phenyl-1,2,3,
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