The Synthesis and Evaluation of C7-Substituted α-Tetralone Derivatives as Inhibitors of Monoamine Oxidase

Article abstract of DOI:10.1111/cbdd.12508

Based on a previous report that α-tetralone (3,4-dihydro-2H-naphthalen-1-one) is a promising scaffold for the design of highly potent inhibitors of the enzyme, monoamine oxidase, the present study investigates the monoamine oxidase inhibitory properties of a synthetic series of fifteen C7-substituted α-tetralone derivatives. Arylalkyloxy substitution on C7 of the α-tetralone moiety yielded compounds with high inhibition potencies toward the human monoamine oxidase-B isoform with all compounds possessing IC₅₀ values in the submicromolar range (0.00089-0.047 μm). The C7-substituted α-tetralones also were highly potent monoamine oxidase-A inhibitors with thirteen (of fifteen) compounds possessing IC₅₀ values in the submicromolar range (0.010-0.741 μm). The α-tetralones were, however, in each instance selective for monoamine oxidase-B over the monoamine oxidase-A isoform. Dialyses of enzyme-inhibitor mixtures show that, while a representative inhibitor acts as a reversible monoamine oxidase-A inhibitor, inhibition of monoamine oxidase-B is not readily reversed by dialysis. Using a molecular modeling approach, possible binding orientations and interactions of selected α-tetralones with the active sites of the monoamine oxidases are also proposed. This study suggests that C7-substituted α-tetralones are promising monoamine oxidase inhibitors and may represent lead compounds for the development of therapies for Parkinson's disease and depression. C7-Substituted α-tetralones act as high potency reversible inhibitors of human MAO-A and MAO-B. This class of compounds represent promising leads for the development of therapies for Parkinson's disease and depression.

Full text of DOI:10.1111/cbdd.12508

Received Date : 29-Oct-2014
Revised Date : 09-Dec-2014
Accepted Date : 15-Dec-2014
Article type : Research Letter
The synthesis and evaluation of C7-substituted α-tetralone derivatives as
inhibitors of monoamine oxidase
Lesetja J. Legoabe 1,*, Anél Petzer 1 and Jacobus P. Petzer 1,2
1.
Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom,
2520, South Africa
2.
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom,
2520, South Africa
3.
*Corresponding author: Lesetja J. Legoabe, Tel.: +27 18 2992182, fax: +27 18 2994243,
lesetja.legoabe@nwu.ac.za
Running title: α-Tetralones as MAO inhibitors
Keywords: monoamine oxidase, MAO, inhibition, reversible, competitive, dialysis, α-tetralone.
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'Accepted Article', doi: 10.1111/cbdd.12508
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Abstract: Based on a previous report that α-tetralone (3,4-dihydro-2H-naphthalen-1-one) is a promising
scaffold for the design of highly potent inhibitors of the enzyme, monoamine oxidase (MAO), the
present study investigates the MAO inhibitory properties of a synthetic series of fifteen C7-substituted
α-tetralone derivatives. Arylalkyloxy substitution on C7 of the α-tetralone moiety yielded compounds
with high inhibition potencies towards the human MAO-B isoform with all compounds possessing IC50
values in the submicromolar range (0.00089–0.047 µM). The C7-substituted α-tetralones also were
highly potent MAO-A inhibitors with thirteen (of fifteen) compounds possessing IC50 values in the
submicromolar range (0.010–0.741 µM). The α-tetralones were, however, in each instance selective for
MAO-B over the MAO-A isoform. Dialyses of enzyme–inhibitor mixtures show that, while a
representative inhibitor acts as a reversible MAO-A inhibitor, inhibition of MAO-B is not readily reversed
by dialysis. Using a molecular modelling approach, possible binding orientations and interactions of
selected α-tetralones with the active sites of the MAOs are also proposed. This study suggests that C7-
substituted α-tetralones are promising MAO inhibitors and may represent lead compounds for the
development of therapies for Parkinson’s disease and depression.
Parkinson’s disease is a progressive neurodegenerative disorder and is the result of the death of the
dopamine-containing neurons which project from the pars compacta component of the substantia nigra
to the corpus striatum (1). The loss of these nigrostriatal neurons leads to a deficiency of dopamine at
the nerve terminals in the corpus striatum, and it is this deficiency that is responsible for the major
motor symptoms observed in Parkinson’s disease. The mainstay of Parkinson’s disease therapy is based
on replacing the lost dopamine by administering its immediate metabolic precursor,
L-3,4-
dihydroxyphenylalanine ( -dopa) (2). Because -dopa undergoes extensive metabolism in the periphery,
L
L
it is often combined with peripheral dopa decarboxylase inhibitors or catechol-O-methyl transferase
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inhibitors (3,4). These drugs allow for the reduction of the
L
-dopa dose and thus the occurrence of the
-Dopa is frequently also combined with
debilitating adverse effects associated with -dopa therapy (5).
L
L
monoamine oxidase (MAO) inhibitors in Parkinson’s disease (6). Since MAO represents a major catabolic
pathway of dopamine in the central nervous system, inhibitors of this enzyme limit the central
breakdown of dopamine (6). In combination with
levels derived from -dopa and also allow for a reduction in the dose of
effect (7–9). As monotherapy, MAO inhibitors may delay the emergence of the motor symptoms that
require the initiation of -dopa therapy (10).
L
-dopa, MAO inhibitors enhance central dopamine
L
L
-dopa required for a therapeutic
L
The MAO enzyme consists of two isoforms, MAO-A and MAO-B. These isoforms are encoded by
separate genes and share approximately 70% sequence identity at the amino acid level (11). While both
isoforms metabolize dopamine in the central nervous system, MAO-B is expressed in higher
concentrations in the brain than MAO-A (12–14), and MAO-B activity is higher than that of MAO-A in the
human basal ganglia, the affected region in Parkinson’s disease (13,15). Furthermore, as the human
brain ages, MAO-B activity as well as density increases in most brain areas, including the basal ganglia
(16). MAO-A activity, on the other hand, remains unchanged with age (12). This suggests that, with
ageing, the central metabolism of dopamine is progressively more dependent on MAO-B than MAO-A.
For these reasons, MAO-B inhibitors, and not MAO-A inhibitors, are established therapy in Parkinson’s
disease. Since MAO-A also catalyzes the central breakdown of dopamine, it has been suggested that, by
acting synergistically, dual-target-directed MAO-A/B inhibitors may be more effective in the therapy of
Parkinson’s disease compared to MAO-B selective inhibitors (9,17). MAO-A inhibitors may also have a
role as antidepressants in Parkinson’s disease. MAO-A is predominantly responsible for the breakdown
of serotonin in the human brain (6) and inhibitors of MAO-A have been employed as antidepressant
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agents (18,19). A significant fraction of patients exhibit signs of depression, and the antidepressant
action of MAO-A inhibitors may thus be of value in Parkinson’s disease (20). It should be noted that the
use of MAO-A inhibitors is restricted by the their ability to provoke a severe hypertensive response
when combined with tyramine-containing food (21). Dietary tyramine is normally metabolized by MAO-
A in the intestinal endothelial cells, which reduces the entry of tyramine into the systemic circulation.
Inhibition of MAO-A thus leads to high systemic levels of tyramine, and since tyramine induces the
release of norepinephrine from peripheral neurons, this may result in severe hypertension (21). While
this adverse effect is regularly observed with irreversible MAO-A inhibitors, reversible inhibitors, such as
moclobemide and toloxatone, are safer and do not provoke the tyramine-induced hypertensive
response (22,23). From this analysis it is evident that, for the discovery and development of MAO-B and
mixed MAO-A/B inhibitors for Parkinson’s disease therapy, the mode of inhibition should be reversible,
especially towards MAO-A. Such compounds should possess an acceptable safety profile and act as
treatment of both motor and depressive symptoms of Parkinson’s disease.
For the design of new MAO inhibitors a variety of structures have been shown to be suitable scaffolds,
and numerous highly potent inhibitors have been reported. Recently it has been shown that α-tetralone
(1; 3,4-dihydro-2H-naphthalen-1-one) is an example of a useful scaffold for the design of MAO
inhibitors, and a series of C6-substituted α-tetralone derivatives was found to be highly potent MAO-B
inhibitors with most compounds exhibiting selective inhibition of MAO-B over the MAO-A isoform
(Figure 1) (24). Many of the C6-substituted α-tetralone derivatives also are potent MAO-A inhibitors. The
potent MAO inhibition properties of the α-tetralones are exemplified by 6-(3-iodobenzyloxy)-3,4-
dihydro-2H-naphthalen-1-one [2; IC50(MAO-B) = 0.0045 µM] and 6-(3-cyanobenzyloxy)-3,4-dihydro-2H-
naphthalen-1-one [3; IC50(MAO-A) = 0.024 µM], respectively the most potent MAO-B and MAO-A
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inhibitors of the reported series. Compound 3, in particular, represents a dual-target-directed MAO-A/B
inhibitor since it also inhibits MAO-B with an IC50 value of 0.078 µM. It is noteworthy that compound 3
was found to be a reversible MAO-A inhibitor. As discussed above, from a drug safety point of view
reversibility of inhibition, particularly towards MAO-A, is a desirable property. The present study aims to
expand on the structure-activity relationships (SARs) of MAO inhibition by α-tetralone derivatives by
synthesizing a series of fifteen α-tetralone derivatives (4a–o) with substitution on the C7 position. For
this purpose, the benzyloxy, phenylethoxy and phenylpropoxy substituents were selected to yield 4a–c.
To further explore chemical space, the benzyloxy-substituted homologue 4a, was substituted on the
benzyloxy phenyl ring with halogens (F, Cl, Br, I) and alkyl groups (CH3, CN, CF3). This yielded a
homologous series of C7-substituted α-tetralone derivatives (4a–o) that are similar to the series of C6-
substituted α-tetralone derivatives reported previously. Thus, besides deriving additional SARs for the
inhibition of the MAOs and potentially discovering highly potent MAO inhibitors, this study will also
facilitate the comparison of the MAO inhibition properties of C7-substituted α-tetralone derivatives to
those of the C6-substituted homologues reported earlier (24). An important consideration is that C7-
substituted α-tetralone derivatives should interact reversibly with MAO-A. As will be shown below, a
representative C7-substituted α-tetralone derivative interacts reversibly with MAO-A. It is in this respect
that our study contributes much, as a number of C7-substituted α-tetralone derivatives are highly
potent MAO-A inhibitors. As mentioned above, a major limitation of the MAO-A inhibitors on the
market are that they act irreversibly and their use is thus restricted because of serious adverse effects
(21). Based on recent reviews that emphasize the untapped potential of MAO-A inhibitors as
antidepressants, C7-substituted α-tetralones represent promising compounds (19). This study further
shows that C7-substituted tetralones are potent MAO-B inhibitors. While selective MAO-B inhibition
would be appropriate for Parkinson’s disease therapy, dual-target-directed MAO-A/B inhibitors may
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have enhanced therapeutic value, making such compounds ideal adjuvants to L-Dopa treatment in
Parkinson’s disease, particularly were Parkinson’s disease is associated with depression.
For the synthesis of the C7-substituted α-tetralone derivatives 4a–o, a similar approach was followed to
that reported for the synthesis of the C6-substituted α-tetralones (Scheme 1) (24). A suspension of 7-
hydroxy-1-tetralone (5) in acetone was treated with the required alkyl bromide in the presence of K2CO3.
The reaction mixture was heated at reflux for 18 h, cooled and filtered through a pad of celite. The crude
product, present in the filtrate, was subsequently purified by recrystallization and obtained in fair yields
(43–88%). The structures of the C7-substituted α-tetralones were solved by 1H NMR, 13C NMR and mass
spectrometry as cited in the supplementary material. 7-Hydroxy-1-tetralone, in turn, was prepared by
treating commercially available 7-methoxy-1-tetralone (6) with aluminum trichloride (25).
To measure the inhibition potencies of the C7-substituted α-tetralones, the recombinant human MAO-A
and MAO-B enzymes were used (26). The non-selective MAO-A/B substrate, kynuramine, served as
enzyme substrate for these studies. Kynuramine is oxidized by MAO-A and MAO-B to ultimately yield 4-
hydroxyquinoline. This metabolite can be readily measured by fluorescence spectrophotometry since it
fluoresces in alkaline media (λex = 310 nm; λem = 400 nm) (27). Neither kynuramine nor the α-tetralone
derivatives investigated in this study fluoresce under the specific assay conditions and concentrations
used, and thus did not interfere with the fluorescence measurements. The MAO catalytic activities that
were recorded in the presence of various concentrations (0.001–100 µM) of the C7-substituted α-
tetralones were graphed versus the logarithm of inhibitor concentration. This yielded sigmoidal rate-
concentration curves from which the corresponding IC50 values were calculated as a measure of the
inhibition potencies.
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The IC50 values for the inhibition of human MAO-A and MAO-B by the C7-substituted α-tetralone
derivatives 4a–o, are given in table 1. The IC50 values show that the α-tetralones are highly potent
inhibitors of MAO-B since all derivatives display IC50 values in the submicromolar range (IC50 values <
0.047 µM). It is also evident that the C7-substituted α-tetralone derivatives are selective for MAO-B over
the MAO-A isoform with selectivity index (SI) values ranging from 8–195. The most potent MAO-B
inhibitor of the series is 7-(4-fluorobenzyloxy)-3,4-dihydro-2H-naphthalen-1-one (4e) with an IC50 value
of 0.00089 µM. This compound exhibited modest selectivity for MAO-B with a SI value of 13. Compared
to the reference MAO-B-selective inhibitor, lazabemide (IC50 = 0.091 µM), compound 4e is
approximately 100-fold more potent as a MAO-B inhibitor under identical experimental conditions (28).
It is interesting to note that all of the C7-substituted α-tetralone derivatives evaluated are more potent
inhibitors than lazabemide. Among the compounds evaluated, compound 4k is the most selective MAO-
B inhibitor with a 195-fold selectivity for MAO-B over the MAO-A isoform. Similar to the other C7-
substituted α-tetralone derivatives, 4k also is a highly potent MAO-B inhibitor [IC50(MAO-B) = 0.0038
µM]. As discussed above, compounds that display selective inhibition of MAO-B are suitable for the
therapy of Parkinson’s disease. Interesting SARs may be derived from the MAO-B inhibition data. The
results show that benzyloxy substitution on the C7 position of the α-tetralone moiety is more favourable
for MAO-B inhibition than phenylethoxy and phenylpropoxy substitution. In this regard, the benzyloxy-
substituted homologue 4a (IC50 = 0.0072 µM) is a modestly more potent MAO-B inhibitor than the
phenylethoxy- [4b; IC50 = 0.047 µM] and phenylpropoxy-substituted [4c; IC50 = 0.022 µM] homologues.
The results further show that alkyl and halogen substituents on the meta and para positions of the
benzyloxy ring of 4a enhance MAO-B inhibition potency. With the exception of 4j and 4o, all compounds
with substituents on the benzyloxy ring (4d–o) are more potent MAO-B inhibitors than 4a, the
benzyloxy-substituted homologue. Compound 4o (IC50 = 0.0077 µM) and 4a may be considered
equipotent as MAO-B inhibitors. High potency inhibition was found with compounds substituted on
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both the meta and para positions of the benzyloxy ring, and thus no clear correlation between the
position (meta or para) of the substituent and MAO-B inhibition potency exists. Clear correlations
between the nature (alkyl and halogen) of the substituent on the benzyloxy ring and MAO-B inhibition
potency also are not apparent, and high potency inhibitors are found with substitution with halogens (F,
Cl, Br, I) and alkyl groups (CH3, CN, CF3). The results further document that 7-hydroxy-1-tetralone (5) is a
weak MAO-B inhibitor with an IC50 value of 187 µM. Compared to the benzyloxy-substituted derivative
4a, compound 5 is therefore 25000-fold weaker as a MAO-B inhibitor. This result shows that a C7
substituent is a prerequisite for MAO-B inhibition by C7-substituted α-tetralone derivatives.
The C7-substituted α-tetralone derivatives 4a–o also are potent inhibitors of human MAO-A. Among the
α-tetralones evaluated, thirteen (of fifteen) compounds display IC50 values in the submicromolar range
(< 0.741 µM). As mentioned above, the α-tetralones are, however, in each instance selective for the
MAO-B isoform. The most potent MAO-A inhibitor of the series is 7-(4-cyanobenzyloxy)-3,4-dihydro-2H-
naphthalen-1-one [4m; IC50 = 0.010 µM]. For comparison, the reversible MAO-A inhibitor, toloxatone, is
reported to inhibit human MAO-A with an IC50 value of 3.92 µM under identical experimental conditions
(28). All of the C7-substituted α-tetralone derivatives are thus more potent MAO-A inhibitors than
toloxatone. Compound 4m is an example of a dual-target-directed MAO-A/B inhibitor since it also is a
potent MAO-B inhibitor with an IC50 value of 0.0012 µM. It is noteworthy that 4e [IC50(MAO-A) = 0.012
µM], the most potent MAO-B inhibitor of the series, also is a highly potent MAO-A inhibitor, and thus
also represents a promising dual-target-directed compound. As discussed above, such dual-target-
directed inhibitors may have additional value in Parkinson’s disease by potentially treating both motor
en depression symptoms. Most C7-substituted α-tetralones examined here, however, represent dual-
target-directed MAO-A/B inhibitors with high inhibition potencies for both MAO isoforms. In fact 13 of
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the 15 derivatives examined possess IC50 values for the inhibition of both MAO-A and MAO-B in the
submicromolar range. In this respect, those derivatives (4e, 4g, 4i, 4l and 4m) with IC50 values < 0.05 µM
for the inhibition of both isoforms may be emphasized. Interesting SARs may be derived from the MAO-
A inhibition data. Among the benzyloxy- [4a; IC50 = 0.443 µM], phenylethoxy- [4b; IC50 = 0.366 µM] and
phenylpropoxy-substituted [4c; IC50 = 1.17 µM] homologues, 4b is the most potent MAO-A inhibitor. This
suggests that, in order to design highly potent MAO-A inhibitors, 4b is the most suitable lead among
these. In spite of this, substitution on the benzyloxy ring of 4a results in highly potent MAO-A inhibition
and 4a thus also represents a useful lead. It is noteworthy that 4c is at least 2.6-fold less potent as a
MAO-A inhibitor than 4a and 4b. A possible explanation may be that, because of its larger size, the
MAO-A active site accommodates 4c less well than 4a and 4b. Because of the architecture of its active
site, MAO-A binds larger inhibitors less well than smaller inhibitors, foremost because structural overlap
with Phe-208 may occur with larger inhibitors (27). In this respect, both alkyl and halogen substituents
on the meta and para positions of the benzyloxy ring enhance the MAO-A inhibition potency of 4a. With
the exception of 4k and 4o, all compounds with substituents on the benzyloxy ring (4d–o) are more
potent MAO-A inhibitors than 4a, the benzyloxy-substituted homologue. The results suggest that
substitution on the para position may by more favourable for MAO-A inhibition than the meta position
since, with the exception of 4n/o, the para substituted homologues are more potent MAO-A inhibitors
than the corresponding meta substituted homologues. Correlations between MAO-A inhibition potency
and the nature (alkyl and halogen) of the substituent on the benzyloxy ring, however, do not exist, and
among the five most potent MAO-A inhibitors of the series (4e, 4g, 4i, 4l and 4m) halogen (F, Cl, Br) and
alkyl groups (CN) are represented as substituents. Similar to MAO-B, the C7 substituent is a requirement
for the MAO-A inhibition since 7-hydroxy-1-tetralone [5; IC50(MAO-A) = 39.4 µM] was found to be
relatively weak MAO-A inhibitor. Compound 5 is approximately 88-fold weaker as a MAO-A inhibitor
than the benzyloxy-substituted derivative 4a.
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Since α-tetralones are reported to act as reversible MAO inhibitors, the present study investigated the
reversibility of MAO-A and MAO-B inhibition by the C7-substituted α-tetralone derivative, 4e. This
compound represents a potent inhibitor of MAO-A and MAO-B of the present series, and is in fact the
most potent MAO-B inhibitor discovered in this study. The reversibility of MAO inhibition was
investigated by measuring the recoveries of the MAO activities after dialysis of mixtures containing the
enzymes and test inhibitor (29). For this purpose, MAO-A and MAO-B were incubated in the presence of
4e, at concentrations equal to 4 × IC50, for 15 min and subsequently dialyzed for 24 h. The results given
in figure 2 show that the inhibition of MAO-A by 4e is completely reversed after 24 h of dialysis, with
MAO-A catalytic activity recovering to 110% of the control value (activity after dialysis in the absence of
inhibitor). In undialyzed mixtures of MAO-A and 4e, inhibition is still observed with the MAO-A activity
at 68% of the control value. After dialysis of mixtures containing MAO-A and the irreversible inhibitor,
pargyline, the enzyme activity is not recovered, with the residual enzyme activity at a level of only 1.6%
of the control value. These data suggest that 4e is a reversible MAO-A inhibitor. Interestingly, the results
given in figure 3 show that, after 24 h of dialysis of mixtures of MAO-B and 4e, MAO-B activity is not
recovered, with the MAO-B activity at 34% of the control value. This value is similar to that observed in
undialyzed mixtures of MAO-B and 4e, with MAO-B activity at 29% of the control value. After dialysis of
mixtures containing MAO-B and the irreversible inhibitor, (R)-deprenyl, the enzyme activity is not
recovered, with only 1.6% activity remaining. The finding that MAO-B activity is not recovered by dialysis
suggests that 4e may act as a tight-binding inhibitor. This finding is similar to that observed with the C6-
substituted α-tetralone derivative, compound 2 (24). Dialysis of mixtures containing MAO-B and 2 also
failed to completely result in recovery of enzyme activity. Further investigation is necessary to
determine the molecular basis of this observation.
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Based on the observation that the C7-substituted α-tetralone derivative, 4e, is a reversible MAO-A
inhibitor, its mode of inhibition was further investigated. For this purpose, Lineweaver-Burk graphs were
constructed. For each graph, the catalytic rates of MAO-A and MAO-B at 8 different kynuramine
concentrations (15–250 µM) were measured. For each MAO isozyme, Lineweaver-Burk graphs were
constructed in the absence of inhibitor, and presence of five different concentrations of the test
inhibitor, 4e. The sets of Lineweaver-Burk graphs for the inhibition of MAO-A and MAO-B thus obtained
are given in figure 4 and figure 5, respectively. For both sets, the lines of Lineweaver-Burk graphs are
linear and intersect on the y-axis. This suggests that 4e is a competitive inhibitor of human MAO-A and
human MAO-B, and serves as additional support for the observation above that that this inhibitor is a
reversible MAO inhibitor, at least of the MAO-A isoform. From graphs of the slopes of the Lineweaver-
Burk graphs versus the concentrations of the test inhibitors, a Ki value of 0.019 µM for the inhibition of
MAO-A by 4e is estimated, while a Ki value of 0.0018 µM for the inhibition of MAO-B by 4e is estimated.
The availability of the X-ray crystal structures of human MAO-A and MAO-B greatly assists in the design
of inhibitors of these enzymes and may provide a structural explanation for the observed inhibitory
properties of inhibitors. To determine potential binding orientations and interactions of the α-tetralone
derivatives with the MAO enzymes, compound 4l, a highly potent inhibitor of both isoforms was
selected for molecular docking studies. Compound 4l was selected because it may be compared to the
corresponding C6-substituted α-tetralone derivative, compound 3, which was also included in the study.
Compound 3 was shown in a previous study to be a highly potent inhibitor of both MAO isoforms (24).
The structures of human MAO-A cocrystallized with harmine (PDB: 2Z5X) (30) and human MAO-B
cocrystallized with safinamide (PDB: 2V5Z) (31) served as protein models. All calculations were carried
out with the Windows based Discovery Studio 3.1 modeling software (32). The preparation of the
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models and the docking calculations were carried out according to the literature procedure (33). For this
purpose, the CDOCKER protocol was used for the docking calculations and the structural integrities of
the highest ranked docking orientations were further explored via short (500 ps) molecular dynamics
simulations of the enzyme–inhibitor complexes. A snapshot of the production run was taken at each 1
ps, thus yielding 500 frames. Based on its ability to correctly predict orientations of cocrystallized
ligands, the CDOCKER protocol has previously been deemed suitable for predicting the binding
orientations of ligands in the active sites of MAO-A and MAO-B (33).
The highest ranked solutions for the binding of 4l and 3 to the active site of MAO-A, show that the α-
tetralone moieties of these inhibitors bind in close proximity to the FAD cofactor of the enzyme, while
the 3-cyanobenzyloxy substituents extend towards the entrance to the active site cavity (Figure 6). The
trajectory analysis for 4l shows that the binding mode to MAO-A is relatively stable with an average root
mean square deviation (RMSD) of 0.58 ± 0.10 Å. For 4l, hydrogen bonding is observed between the
nitrile group of the inhibitor and the amide nitrogen of Val-210. This interaction is, however, only
present in 2 of 500 frames. The most prominent Van der Waals interactions occur with Gln-215 and Phe-
208. These interactions contribute to 25.2 ± 3.15% of the total interaction energy between 4l and the
MAO-A active site. The integrities of these interactions are confirmed by the finding that the Van der
Waals interactions remain highly significant (14.1–37.9% of the total interaction energy) throughout the
production run. The principal difference between the binding modes of 4l and 3 is that the α-tetralone
moiety of 3 is rotated by approximately 180 ° in the MAO-A active site compared to that of 4l. The
cyanobenzyloxy side chains of the inhibitors, however, occupy the same space in the active site. The
consequence of this is that 3 may undergo more extensive hydrogen bonding in the MAO-A active site
with the carbonyl oxygen involved in interactions with Tyr-444 (496 of 500 frames) and HOH739 (490 of
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500 frames). Interactions of the carbonyl oxygen of 3 with HOH718 (131 of 500 frames) and the nitrile
group of the inhibitor and the amide nitrogen of Val-218 (160 of 500 frames) are also present in certain
orientations of 3. As for 4l, the most prominent Van der Waals interactions occur with Gln-215 and Phe-
208 (23.5 ± 3.19% of the total interaction energy). Although 4l does not undergo extensive hydrogen
bonding to MAO-A (in contrast to 3), 4l and 3 display similar MAO-A inhibition potencies. It may thus be
concluded that hydrogen bonding plays a relatively smaller role in inhibitor stabilization compared to
Van der Waals interactions, which seems to be similar for the two inhibitors. In spite of the differing
orientations of the two inhibitors, similar Van der Waals interactions remain possible.
The highest ranked solutions for the binding of 4l and 3 to the active site of MAO-B, show that these
inhibitors bind in a similar orientation to that observed in MAO-A, with the α-tetralone moieties in close
proximity to the FAD cofactor (Figure 7). The trajectory analysis for 4l shows that its binding mode to
MAO-B is stable with a root mean square deviation (RMSD) of 0.56 ± 0.17 Å. For 4l, hydrogen bonding is
observed between the nitrile group of the inhibitor and the amide nitrogen of Tyr-326. This interaction
is, however, only present in 53 of 500 frames. The most important Van der Waals interactions occur
with Leu-171, Gln-206 and Tyr-326, which together contribute to 30.0 ± 20.6% of the total interaction
energy, and remain significant throughout the production run (21.4–36.2% of the total interaction
energy). As in MAO-A, the α-tetralone moiety of 4l is rotated by approximately 180 ° in the MAO-B
active site compared to that of 3. For 3, hydrogen bonding occurs between the carbonyl oxygen and
HOH1155, with a frequency of only 12 of 500 frames. Hydrogen bonding thus appears to also play a
relatively small role in inhibitor stabilization in MAO-B. As for 4l, the principal Van der Waals interactions
between 3 and MAO-B occur with Leu-171, Gln-206 and Tyr-326, with Ile-199 also contributing
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significantly to inhibitor stabilization. Interactions with these residues contribute to 40.4 ± 2.22% of the
total interaction energy.
The binding modes of 4l and 3 to MAO-B are, in certain respects, similar to that of safinamide, the co-
crystallized ligand. As for 4l and 3 the polar moiety of safinamide, the propanamidyl group, binds in the
substrate cavity in close proximity to the FAD, while the 3-fluorobenzyloxy side chain of safinamide is
located in the entrance cavity where it forms Van der Waals interactions similar to those observed for 4l
and 3 (31). Similar to 4l and 3, for safinamide the most important Van der Waals interactions occur with
Leu-171, Gln-206, Tyr-326 and Ile-199, which together contribute 36.1% of the total interaction energy.
Similar to compound 3, safinamide also undergoes a hydrogen bond interaction with a water molecule,
but an additional hydrogen bond interaction with Gln-206 is observed for safinamide, which is absent
from the interactions of 4l and 3 with MAO-B.
To determine the molecular basis for the lower degree of isoform selectivity of compounds 4k (SI = 195)
and 4o (SI = 164), these compounds were also docked into the active sites of MAO-A and MAO-B. The
results are given in Figures 8 and 9, and show that the binding modes of these two inhibitors are almost
identical, and similar to those observed for 4l. For binding of 4k and 4o to MAO-A, the most prominent
Van der Waals interactions occur with Phe-208 and Ile-180. Interestingly non-productive interactions
occur with Gln-215 and Ile-335, which may, at least in part, explain the lower MAO-A inhibitory
potencies of these compounds. For MAO-B, the principal difference between the binding modes of 4k
and 4o and that of 4l, is that the C7 side chains of 4k and 4o protrude deeper into the entrance cavity
where they are stabilized by Van der Waals interactions, most notably with residues Leu-171, Gln-206
and Ile-199. This stabilization in the entrance cavity may explain why these inhibitors have comparable
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MAO-B inhibition potencies to 4l, even though their binding modes differ with respect to the placement
of the C7 side chain.
In conclusion, this study finds that C7-substituted α-tetralone derivatives act as high potency inhibitors
of human MAO-A and MAO-B. All homologues of the series exhibited higher inhibition potencies for
MAO-B compared to the MAO-A isoform. The IC50 values recorded for the inhibition of MAO-B by 4a–o
ranged from 0.00089–0.047 µM. It may thus be concluded that substitution with a wide variety moieties
on C7 of the α-tetralone moiety yields compounds that are highly active MAO-B inhibitors. Compared to
the previously studied C6-substituted α-tetralone derivatives (24), the leads for the current study, the
C7-substituted α-tetralones have similar MAO-B inhibition potencies. This may be best illustrated by the
observation that the homologous series of C6-substituted α-tetralones is reported to inhibit MAO-B with
IC50 values ranging from 0.0045–0.078 µM.
The C7-substituted α-tetralones also were highly potent MAO-A inhibitors with IC50 values ranging from
0.010–1.26 µM. Among the fifteen α-tetralones evaluated, thirteen compounds possess IC50 values in
the submicromolar range. Compared to the previously studied C6-substituted α-tetralone derivatives
(24), the C7-substituted α-tetralones are in general more potent MAO-A inhibitors. For example, among
a homologous series of C6-substituted α-tetralone, only one compound, compound 3 (IC50 = 0.024 µM),
displays an IC50 value < 50 µM, while five C7-substituted α-tetralones display IC50 values <50 µM.
Although most C7-substituted α-tetralones inhibited both MAO-A and MAO-B, derivatives 4e, 4g, 4i, 4l
and 4m, with IC50 values < 0.05 µM for the inhibition of both MAO isoforms, may be highlighted. Such
compounds may thus be considered dual-target-directed MAO-A/B inhibitors with potential application
in the therapy of both Parkinson’s disease and depressive illness. In this regard it is important to note
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that these α-tetralones are reversible MAO-A inhibitors (as exemplified by 4e), and thus unlikely to
cause tyramine-induced hypertension. Modelling has shown that, in MAO-A and MAO-B, hydrogen
bonding plays a relatively smaller role in inhibitor stabilization compared to Van der Waals interactions.
Although C6- and C7-substituted α-tetralones adopt differing orientations in the MAO active sites, they
remain capable of forming similar Van der Waals interactions. This may explain, at least in part, their
similar MAO-B inhibition potencies. An explanation for the enhanced MAO-A inhibition potencies for C7-
substituted α-tetralones compared to the C6-substituted homologues are, however, not apparent.
Acknowledgements
The NMR and MS spectra were recorded by André Joubert and Johan Jordaan of the SASOL Centre for
Chemistry, North-West University. This work is based on the research supported in part by the Medical
Research Council and National Research Foundation of South Africa (Grant specific unique reference
numbers (UID) 85642, 80647 and 80637). The Grantholders acknowledge that opinions, findings and
conclusions or recommendations 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.
Conflict of Interest
The authors have no conflicts of interest to declare
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Table 1: The IC50 values for the inhibition of recombinant human MAO-A and MAO-B by C7-
substituted α-tetralone derivatives 4a–o and 7-hydroxy-1-tetralone (5).
O
R
4
R
IC50 (µM)a
SIb
MAO-A
MAO-B
4a
4b
4c
4d
4e
4f
C6H5CH2O–
0.443 ± 0.044
0.366 ± 0.063
1.17 ± 0.105
0.129 ± 0.031
0.012 ± 0.002
0.161 ± 0.018
0.033 ± 0.006
0.328 ± 0.076
0.034 ± 0.003
0.283 ± 0.043
0.741 ± 0.061
0.026 ± 0.006
0.010 ± 0.001
0.375 ± 0.035
1.26 ± 0.141
39.4 ± 3.91
–
0.0072 ± 0.002
0.047 ± 0.002
0.022 ± 0.004
0.0013 ± 0.00006
0.00089 ± 0.0003
0.0040 ± 0.0006
0.0041 ± 0.0007
0.0033 ± 0.0007
0.0035 ± 0.001
0.020 ± 0.001
0.0038 ± 0.0008
0.0031 ± 0.0004
0.0012 ± 0.0004
0.0038 ± 0.0004
0.0077 ± 0.001
187 ± 26.7
62
8
C6H5(CH2)2O–
C6H5(CH2)3O–
53
99
13
40
8
(3-FC6H4)CH2O–
(4-FC6H4)CH2O–
(3-ClC6H4)CH2O–
(4-ClC6H4)CH2O–
(3-BrC6H4)CH2O–
(4-BrC6H4)CH2O–
(4-IC6H4)CH2O–
(4-CH3C6H4)CH2O–
(3-CNC6H4)CH2O–
(4-CNC6H4)CH2O–
(3-CF3C6H4)CH2O–
(4-CF3C6H4)CH2O–
HO–
4g
4h
4i
99
10
14
195
8
4j
4k
4l
4m
4n
4o
5
8
99
164
0.2
–
Lazabemide
Toloxatone
0.091c
3.92c
–
–
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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).
c Value obtained from ref (28).
Figures and schemes:
Figure 1: The structures of compounds discussed in the text.
Figure 2: Dialysis of mixtures containing MAO-A and 4e. MAO-A was preincubated with 4e (4 × IC50) for
15 min and dialyzed for 24 h. The residual enzyme activity was subsequently measured (4e–dialyzed).
MAO-A was similarly preincubated in the presence of the irreversible inhibitor, pargyline (Parg–
dialyzed), and in the absence of inhibitor (No inhibitor–dialyzed), and dialyzed. The residual MAO-A
activity of undialyzed mixtures of MAO-A with 4e was also measured (4e–undialyzed).
Figure 3: Dialysis of mixtures containing MAO-B and 4e. MAO-B was preincubated with 4e (4 × IC50) for
15 min and dialyzed for 24 h. The residual enzyme activity was subsequently measured (4e–dialyzed).
MAO-B was similarly preincubated in the presence of the irreversible inhibitor, (R)-deprenyl (Depr–
dialyzed), and in the absence of inhibitor (No inhibitor–dialyzed), and dialyzed. The residual MAO-B
activity of undialyzed mixtures of MAO-B with 4e was also measured (4e–undialyzed).
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Figure 4: Lineweaver-Burk graphs of the inhibition of human MAO-A by 4e (IC50 = 0.012 µM). The
concentrations of 4e used for the construction of these graphs ranged from ¼ × IC50 to 1¼ × IC50. The
inset is a graph of the slopes of the Lineweaver-Burk graphs versus inhibitor concentration (Ki = 0.019
µM).
Figure 5: Lineweaver-Burk graphs of the inhibition of human MAO-B by 4e (IC50 = 0.00089 µM). The
concentrations of 4e used for the construction of these graphs ranged from ¼ × IC50 to 1¼ × IC50. The
inset is a graph of the slopes of the Lineweaver-Burk graphs versus inhibitor concentration (Ki = 0.0018
µM).
Figure 6: The highest ranked docking solutions of 4l (magenta) and 3 (cyan) for binding to the active site
of MAO-A.
Figure 7: The highest ranked docking solutions of 4l (magenta) and 3 (cyan) for binding to the active site
of MAO-B.
Figure 8: The highest ranked docking solutions of 4k (yellow) and 4o (green) for binding to the active site
of MAO-A.
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Figure 9: The highest ranked docking solutions of 4k (yellow) and 4o (green) for binding to the active site
of MAO-B.
Scheme 1: Synthetic route to the C7-substituted α-tetralone derivatives 4a–o. Reagents and conditions:
(a) AlCl3, toluene; reflux; (b) acetone, K2CO3, reflux.
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