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Novel sulfanyl- and sulfinylcaffeine analogues as inhibitors of monoamine oxidase / Wayne Mentz

Parkinson’s disease (PD) is a neurodegenerative disorder, which is progressive in nature and
usually associated with the elderly. It is the second most common age-related
neurodegenerative disorder after Alzheimer’s disease (AD). PD occurs when there is a dramatic
loss of dopamine (DA) in the striatum, a substructure of the basal ganglia, of the brain due to
the degeneration of the nigrostriatal pathway that contains the dopaminergic neurons. Motor
symptoms of PD include bradykinesia, muscular rigidity and resting tremors. Non-motor
symptoms include speech and sleep problems, hallucinations and depression. Diverse
treatment options are available to treat the symptoms of PD, including levodopa (L-Dopa), DA
agonists and monoamine oxidase B (MAO-B) inhibitors.
The MAOs are flavoproteins that are bound to the outer membrane of the mitochondria and
catalyze the oxidative deamination of neurotransmitters such as serotonin (5-HT), noradrenaline
(NA) and DA. Two isoforms occur, namely MAO-A and –B, which share a 70% sequence
identity. MAO-A catalyzes the oxidation of 5-HT and MAO-B has a substrate specificity towards
benzylamine and 2-phenylethylamine. DA, NA, adrenaline and tryptamine are oxidized by both
forms. MAO-A plays an important role in depression while MAO-B plays an important role in PD.
The two isoforms are not evenly distributed in the brain. Of particular relevance to PD is the
observation that, in the basal ganglia, MAO-B is the predominant form and the oxidation of DA
in this region is largely due to MAO-B activity. Also, with an increase in age, there is an up to
fourfold increase in MAO-B activity in the brain. In the aged parkinsonian brain, MAO-B is
therefore a major DA metabolizing enzyme and MAO-B inhibitors have an important role in the
therapy of PD. MAO-B inhibitors may potentially reduce the metabolic destruction of DA and
thereby provide relief from the symptoms of PD. MAO-B inhibitors may also exert a
neuroprotective effect in PD. In the catalytic cycle of MAO-B, one mole each of an aldehyde,
hydrogen peroxide and ammonia are formed for each mole of primary amine substrate oxidized.
Ferrous iron, which is abundant in the basal ganglia, may react with the hydrogen peroxide to
form hydroxyl radicals in the Fenton reaction. The hydroxyl radical damages virtually all types of
biomolecules including proteins, DNA, lipids, carbohydrates and amino acids. The aldehyde, in turn, may react with amino groups of proteins, and thus lead to cell injury. Inhibitors of MAO-B
may reduce the MAO-catalyzed formation of hydrogen peroxide and aldehydes in the basal
ganglia, and thus act as neuroprotective agents.
MAO-B inhibitors that are currently being used in the treatment of PD are selegiline and
rasagiline. Both are irreversible inhibitors of MAO-B. While irreversible inhibitors of MAO have
been used extensively as drugs, irreversible inhibition has a number of disadvantages. These
include the loss of selectivity as a result of repeated drug administration and a slow and variable
rate of enzyme recovery following termination of drug treatment. The turnover rate for the
biosynthesis of MAO-B in the human brain may require as much as 40 days while with
reversible inhibition, enzyme activity is recovered when the inhibitor is eliminated from the
tissues. For these reasons the discovery of novel MAO-B inhibitors, which interact reversibly
with the enzymes are of value in the therapy of PD.
The goal of this study was to design novel and reversible inhibitors of MAO-B, which may find
application in the therapy of PD. In the current study, caffeine was used as scaffold for the
design of new MAO inhibitors. Caffeine is reported to be a weak inhibitor of MAO-B, with an IC50
value of 5084 μM. Substitution at C-8 of the caffeine moiety, however, yields compounds with
potent MAO-B inhibitory properties. Of particular importance to this study is a recent report that
a series of 8-sulfanylcaffeine analogues acts as selective inhibitors of human MAO-B. Among
the compounds examined, 8-[(phenylethyl)sulfanyl]caffeine was found to be a particularly potent
MAO-B inhibitor with an IC50 value of 0.223 μM. In an attempt to further enhance the MAO-B
inhibition potency of 8-[(phenylethyl)sulfanyl]caffeine, and possibly to discover highly potent
MAO-B inhibitors, a series of five 8-[(phenylethyl)sulfanyl]caffeine analogues was synthesized
and evaluated as inhibitors of human MAO-A and –B. For the purpose of this study 8-
[(phenylethyl)sulfanyl]caffeine homologues containing C-3 alkyl (CF3, CH3 and OCH3) and
halogen (Cl and Br) substituents on the phenyl ring were considered. Furthermore, a series of
two 8-sulfinylcaffeine analogues and one 8-sulfonylcaffeine were synthesized and their MAO
inhibitory potencies were measured. The purpose with these compounds was to compare the
MAO inhibitory properties of the 8-sulfinylcaffeine analogues and 8-sulfonylcaffeine with those
of the 8-sulfanylcaffeine analogues. This study also investigates the MAO inhibition properties of
three selected 8-[(phenylpropyl)sulfanyl]caffeine and two 8-(benzylsulfanyl)caffeine analogues.
Chemistry: The target 8-sulfanylcaffeine analogues were synthesized according to the literature
procedure. 8-Chlorocaffeine was reacted with an appropriate mercaptan in the presence of NaOH, to yield the target 8-sulfanylcaffeine analogues in yields of 6.4–50.7%. 8-Chlorocaffeine,
in turn, was conveniently synthesized in high yield by reacting chlorine with caffeine in
chloroform. In certain instances, the mercaptan starting materials were not commercially
available and were thus synthesized according to the literature procedure by reacting an
appropriate alkylbromide with thiourea. The resulting thiouronium salt was hydrolyzed in the
presence of NaOH to yield the target mercaptan. The 8-sulfinylcaffeine analogues and 8-
sulfonylcaffeine were synthesized by reacting the 8-sulfanylcaffeines with H2O2 in the presence
of glacial acetic acid and acetic anhydride. The structures and the purities of the inhibitors were
verified by NMR, MS and HPLC analyses.
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 and the inhibition potencies were expressed as the
IC50 values.
The 8-[(phenylethyl)sulfanyl]caffeine analogues were found to be highly potent inhibitors of
MAO-B. The IC50 values recorded for these homologues ranged from 0.017–0.125 μM, making
them twofold to 13-fold more potent MAO-B inhibitors than the lead compound, 8-
[(phenylethyl)sulfanyl]caffeine (IC50 = 0.223 μM). For comparison, the reversible MAO-B
selective inhibitor, lazabemide, exhibits an IC50 value of 0.091 μM under the same conditions
(unpublished data from our laboratory). Interestingly, both alkyl (CF3, CH3 and OCH3) and
halogen (Cl and Br) substitution lead to highly potent MAO-B inhibition. It may therefore be
concluded that substitution on C-3 is a general strategy to enhance the MAO-B inhibition
potency of 8-[(phenylethyl)sulfanyl]caffeine. The results of the MAO inhibitory studies with the 8-
[(phenylpropyl)sulfanyl]caffeine analogues showed that these compounds are also inhibitors of
MAO-B with IC50 values of 0.061–0.500 μM. Those homologues substituted with chlorine on the
para and meta positions of the phenyl ring were found to be exceptionally potent inhibitors with
IC50 values of 0.061 μM and 0.062 μM, respectively. For the series of 8-
(benzylsulfanyl)caffeines, meta substitution with chlorine (IC50 = 0.227 μM) and bromine
(IC50 = 0.199 μM) was also found to enhance the MAO-B inhibition potency of 8-
(benzylsulfanyl)caffeine (IC50 = 1.86 μM). The results document that the 8-sulfinylcaffeines are
also inhibitors of MAO-B with IC50 values of 11.8–131 μM. The 8-sulfonylcaffeine was also found
to be a MAO-B inhibitor. Compared to the 8-sulfanylcaffeines, these homologues are, however,
weaker inhibitors. It may, therefore, be concluded that 8-sulfinylcaffeines and 8-sulfonylcaffeines are comparatively weak MAO-B inhibitors and less suited for the design of high potency MAO-B
inhibitors.
The results also document that the 8-[(phenylethyl)sulfanyl]caffeines are relatively weak MAO-A
inhibitors with IC50 values of 5.66–141 μM, with one homologue exhibiting no inhibition under
the experimental conditions. As evident from the selectivity indices (SI values), the 8-
[(phenylethyl)sulfanyl]caffeines were all selective inhibitors of the MAO-B isoform. Two
compounds 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 8-sulfinylcaffeines and 8-sulfonylcaffeine were found to be weak MAO-A
inhibitors with IC50 values of 166–250 μM. The SI values demonstrate that these compounds are
MAO-B selective inhibitors, although to a lesser degree than the 8-
[(phenylethyl)sulfanyl]caffeines. The 8-[(phenylpropyl)sulfanyl]caffeines are also MAO-A
inhibitors with IC50 values of 0.708–6.48 μM. It is noteworthy that these homologues are the
most potent MAO-A inhibitors among the compounds evaluated in this study. In fact, one of the
8-[(phenylpropyl)sulfanyl]caffeines, 8-{[3-(4-chlorophenyl)propyl]sulfanyl}caffeine (IC50 = 0.708
μM), is the only compound with an IC50 value for the inhibition of MAO-A in the submicromolar
range. The 8-[(phenylpropyl)sulfanyl]caffeines display, in general, lower degrees of selectivity
for MAO-B than the corresponding 8-[(phenylethyl)sulfanyl]caffeines.
Reversibility studies: The reversibility of the interaction of a representative inhibitor, 8-{[2-(3-
(trifluoromethyl)phenyl)ethyl]sulfanyl}caffeine, with MAO-B was investigated by evaluating the
recovery of the enzymatic activity after dilution of the enzyme-inhibitor complex. For this
purpose, MAO-B was preincubated with the test compound at concentrations of 10 × IC50 and
100 × IC50 for 30 min. The reactions were subsequently diluted 100-fold to 0.1 × IC50 and 1 ×
IC50, respectively. The results show that, after dilution to 0.1 × IC50 and 1 × IC50, the MAO-B
catalytic activities are recovered to 35% and 22%, 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 × IC50 and 100 × IC50. After preincubation of MAO-B
with the irreversible inhibitor (R)-deprenyl (at 10 × IC50), and dilution of the resulting complex to
0.1 × IC50, MAO-B activity is not recovered (3.0% of control). These data indicate that the test
compound does indeed react reversibly with MAO-B but because enzyme activities are not
recovered to the expected 90% and 50% respectively, it may suggest that the test compound
possess a quasi-reversible or tight-binding component. Hansch-type structure activity relationship studies: A limited Hansch-type QSAR study was
performed for the inhibition of MAO by the 8-[(phenylethyl)sulfanyl]caffeines. For this purpose,
five parameters were used to describe the physicochemical properties of the C-3 substituents
on the phenyl rings of the inhibitors. The Van der Waals volume (Vw) and Taft steric parameter
(Es) served as descriptors of the bulkiness of the substituents, while the lipophilicities were
described by the Hansch constant (π). The electronic properties were described by the classical
Hammett constant (σm) and the Swain-Lupton constant (F). A one-parameter fit with the Taft
steric parameter versus the inhibition potency (logIC50) yielded the best correlation with a
correlation coefficient (R2) of 0.912 and a statistical F value of 41.27 (Fmax = 35). The positive
sign of the Es (+0.47) parameter coefficient indicated that the inhibition potencies of the 8-
[(phenylethyl)sulfanyl]caffeines towards MAO-B may be enhanced by substitution with sterically
large groups at C-3 of the phenyl rings of the inhibitors. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Identiferoai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/9521
Date January 2013
CreatorsMentz, Wayne
PublisherNorth-West University
Source SetsSouth African National ETD Portal
LanguageEnglish
Detected LanguageEnglish
TypeThesis

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