2-Arylthiomorpholine derivatives as potent and selective monoamine oxidase B inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.01.029

2-Arylthiomorpholine and 2-arylthiomorpholin-5-one derivatives, designed as rigid and/or non-basic phenylethylamine analogues, were evaluated as rat and human monoamine oxidase inhibitors. Molecular docking provided insight into the binding mode of these inhibitors and rationalized their different potencies. Making the phenylethylamine scaffold rigid by fixing the amine chain in an extended six-membered ring conformation increased MAO-B (but not MAO-A) inhibitory activity relative to the more flexible α-methylated derivative. The presence of a basic nitrogen atom is not a prerequisite in either MAO-A or MAO-B. The best K_i values were in the 10^-8 M range, with selectivities towards human MAO-B exceeding 2000-fold.

Full text of DOI:10.1016/j.bmc.2010.01.029

Bioorganic & Medicinal Chemistry 18 (2010) 1388–1395
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Arylthiomorpholine derivatives as potent and selective monoamine oxidase
B inhibitors
Susan Lühr a, Marcelo Vilches-Herrera a, Angélica Fierro b,c, Rona R. Ramsay d, Dale E. Edmondson e,
Miguel Reyes-Parada c,f, Bruce K. Cassels a,c, Patricio Iturriaga-Vásquez a,c,
*
a Department of Chemistry, Faculty of Sciences, University of Chile, Casilla 653, Santiago, Chile
b Faculty of Chemistry and Biology, University of Santiago de Chile, Alameda 3363, Santiago, Chile
c Millennium Institute for Cell Dynamics and Biotechnology, Beauchef 861, Santiago, Chile
d School of Biology, Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews KY16 9ST, UK
e Departments of Biochemistry and Chemistry, Emory University, Atlanta, GA 30322, USA
f School of Medicine, Faculty of Medical Sciences, University of Santiago de Chile, Alameda 3363, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Arylthiomorpholine and 2-arylthiomorpholin-5-one derivatives, designed as rigid and/or non-basic
phenylethylamine analogues, were evaluated as rat and human monoamine oxidase inhibitors. Molecular
docking provided insight into the binding mode of these inhibitors and rationalized their different poten-
cies. Making the phenylethylamine scaffold rigid by fixing the amine chain in an extended six-membered
Received 18 November 2009
Revised 11 January 2010
Accepted 12 January 2010
Available online 15 January 2010
ring conformation increased MAO-B (but not MAO-A) inhibitory activity relative to the more flexible a-
methylated derivative. The presence of a basic nitrogen atom is not a prerequisite in either MAO-A or
MAO-B. The best Ki values were in the 10ꢀ8 M range, with selectivities towards human MAO-B exceeding
2000-fold.
Keywords:
Monoamine oxidase
MAO inhibitors
Arylthiomorpholine derivatives
Amphetamine
Ó 2010 Elsevier Ltd. All rights reserved.
Human and rat MAO
Docking
1. Introduction
sion of Parkinson’s disease and of symptoms associated with Alz-
heimer’s disease.3–6
Functional impairment of the dopamine, serotonin, and nor-
adrenaline neurotransmitter systems has been advocated as an
important feature of neurodegenerative diseases, particularly
Parkinson’s disease, and neuropsychiatric disorders such as
depression and anxiety.1 As the neuronal levels of these neuro-
transmitters are altered under these conditions, the enzyme mono-
amine oxidase (MAO, E.C. 1.4.3.4) is an attractive drug target due to
its central role in monoaminergic neurotransmitter metabolism.
This FAD dependent enzyme is bound to the mitochondrial out-
er membrane and catalyzes the oxidative deamination of a range of
endogenous and exogenous monoamines. The two mammalian
isoforms named MAO-A and MAO-B differ in tissue localization,
substrate preference, and inhibitor selectivity.2 Thus, MAO-A is
selectively inhibited by clorgyline and metabolizes serotonin pref-
erentially, whereas MAO-B is inhibited by l-deprenyl and prefers
benzylamine and phenylethylamine as substrates. Selective
MAO-A inhibitors (MAOAI) are used clinically as antidepressants
and anxiolytics, while MAOBI are used for reduction of the progres-
Our group has studied a large number of phenylethylamine
derivatives as MAOIs. Modifications include substitutions on the
amine nitrogen, alkylation of the
tions with respect to the amine function, and different substitu-
tions on the aromatic ring including extension of the system
(Fig. 1). Several of these compounds, particularly -methylphenyl-
a and oxygenation of the b-posi-
p
a
ethylamines with a single alkoxy or alkylthio substituent at the
β
H
N
R2
α
R3
R1
Figure 1. Different modifications (dashed line) to a phenylethylamine scaffold
(bold and solid line).
* Corresponding author. Tel.: +56 2 978 7253; fax: +56 2 271 3888.
E-mail address: iturriag@uchile.cl (P. Iturriaga-Vásquez).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.029

S. Lühr et al. / Bioorg. Med. Chem. 18 (2010) 1388–1395
1389
para position, have proved to be potent and selective MAOAI with
2.2. Biochemistry and molecular docking
negligible MAOBI activity.7–12
These compounds have in common a basic amino group linked
to a flexible ethyl chain. A number of structurally unrelated selec-
tive MAOBI that contain a basic nitrogen atom held in a relatively
rigid environment, for example, forming part of a heterocycle, are
known.13,14 Diverse MAOBI have also been described in which
either the basicity is reduced or there is no basic group at all.15 If
flexibility around the amino group and its basic character are not
prerequisites for potent MAO-B inhibition, it seems reasonable to
explore modifications around the ethylamine side chain leading
to greater rigidity and including non-basic analogues as possible
means of generating a novel series of phenylethylamine-derived
MAOBI.
In the present study we tested these hypotheses by evaluating
the MAO inhibitory properties of a series of phenylethylamine
derivatives in which the nitrogen atom is incorporated into a six-
membered sulfur containing heterocycle. The influence of the basic
character of the molecule was evaluated by comparing compounds
containing a secondary amino group with others in which the
nitrogen atom was part of an amide functionality. In addition, as
species differences in the pharmacology of MAOIs have been re-
ported,10,16,17 we studied our compounds as inhibitors of both
the human and the rat enzymes. Finally, in order to rationalize
our biochemical results, docking experiments were performed
using models built on the basis of the crystal structures of human
MAO-A and -B.
Table 1 summarizes the effects and selectivity ratios of the 2-
arythiomorpholin-5-one (4a–f) and 2-arylthiomorpholine (5a–f)
derivatives (in all cases as the racemic mixtures), upon human
(h) and rat (r) MAO-A and MAO-B. Also included are the reported
values for d-amphetamine (6).12,21,22
In contrast to what had been seen in previous studies with
phenylethylamine derivatives, most of the compounds exhibited
some selectivity for MAO-B with Ki values in the low micromolar
and submicromolar range, but only 4f and 5f are highly selective
for this isoform.
Reversible and competitive inhibition of hMAO-A and hMAO-B
was demonstrated for all compounds tested. Figure 2 depicts the
results of kinetic measurements (initial velocities) of hMAO-B in
the presence of different concentrations of 5f, the most potent drug
described in this paper.
Regarding our first hypothesis, the incorporation of the phenyl-
ethylamine amino group into a ring containing a sulfur atom in the
b-position (4a and 5a), produced an increase of the rMAO-B inhib-
itory potency (4a Ki = 9.58
amphetamine (the -methylated phenylethylamine analogue 6, Ki
>100 M), whereas the inhibitory activity of these compounds to-
wards rMAO-A (4a Ki = 9.19 M, 5a Ki = 10.6 M) is practically the
same as that of amphetamine (Ki = 12.2 M). Interestingly, neither
lM, 5a Ki = 2.23 lM) as compared with
a
l
l
l
l
the benzene ring-unsubstituted thiomorpholin-5-one (4a) nor its
reduced derivative (5a) exhibited any significant inhibition of
either human isoform (Ki >100 lM in both cases). These results
are in agreement with the observation that striking differences in
inhibitory properties can be observed when MAOs from different
species are compared.10,16,17
2. Results and discussion
2.1. Chemistry
Although 4a and 5a do not bind with high affinity to the human
enzyme isoforms, reducing the mobility of the ethylamine chain by
including it in a thiomorpholine ring elicits an increase of the
inhibitory potency towards rMAO-B. These results prompted us
to further explore the effect of some simple structural modifica-
tions of this scaffold. Thus, the introduction of n-alkoxyl groups
containing 2–4 carbon atoms, at the para position of the aromatic
ring (4c–e, 5c–e) produces a progressive increase of the inhibitory
activity against both human and rat MAO-B, reaching the lowest
submicromolar Ki values for the butoxy derivatives (4e and 5e).
In the case of hMAO-A, all these compounds showed poor inhibi-
tory potencies and low- to medium selectivity ratios.
The ( )-2-arylthiomorpholin-5-one (4a–f) and ( )-2-arylthi-
omorpholine (5a–f) derivatives were synthesized following pub-
lished methods (Scheme 1), which involved a Henry–Knoevenagel
condensation of the appropriately substituted aromatic aldehydes
1a–f with nitromethane and the subsequent Michael addition of
methyl thioglycolate to the nitrostyrenes 2a–f to generate the
corresponding nitroester 3a–f.18 Sulfur was selected over other
heteroatoms, for example, oxygen, because its greater nucleophi-
licity ensures better yields of the intermediates. Finally, a one-
pot reaction involving a nucleophilic attack on the ester group of
the amine, obtained by reduction of the nitro group with Zn0, affor-
ded the heterocycles 4a–f.19 Reduction of the lactams using DIBAL-
H, gave the desired thiomorpholine derivatives 5a–f.20
Docking experiments suggest that the (S)-enantiomers of both
thiomorpholine and thiomorpholin-5-one derivatives and their
O
S
OCH3
NO2
O
NO2
a
b
H
X
X
X
c
2a-f
3a-f
1a-f
X, a = H-
X, b = CH3O-
O
S
S
X, c = CH3CH2O-
X, d = CH3CH2CH2O-
X, e = CH3CH2CH2CH2O-
X, f = PhCH2O-
NH
d
NH
X
X
5a-f
4a-f
Scheme 1. Reagents and conditions: (a) CH3NO2/cyclohexylamine/CH3COOH, 40 °C, 2 h; (b) HSCH2COOMe/N(Et)3/THF, rt, 2 h; (c) Zn/CH3COOH, 80 °C, 48 h; (d) DIBAL-n-
hexane/THF, reflux, 2 h.

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S. Lühr et al. / Bioorg. Med. Chem. 18 (2010) 1388–1395
Table 1
Inhibition constants of 2-arylthiomorpholin-5-one (4a–f), 2-arylthiomorpholine (5a–5f) derivatives (racemic mixtures), and d-amphetamine (6)
S
X
NH2
NH
6
4a-f
5a-f
R
Compd
R
X
Ki (
l
M) humana
MAO-B
Ki (
l
M) ratb
MAO-B
Selectivity A/Bc
Human Rat
dMAO-A
MAO-A
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
H–
CH3O–
–C@O
–C@O
–C@O
–C@O
–C@O
–C@O
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
>100
>100
60 8.57
40 1.85
10 0.34
>100
>100
>100
9.19 1.43
9.28 2.09
12.3 1.14
8.68 1.12
50.9 6.19
27.5 4.62
10.6 0.41
6.39 0.14
2.17 0.13
3.69 0.39
14.1 1.22
19.0 0.36
9.58 1.75
13.6 2.16
3.40 1.01
1.49 0.03
0.16 0.01
0.074 0.003
2.23 0.03
3.85 0.01
2.17 0.31
0.98 0.11
0.27 0.02
0.13 0.004
>100g
—
—
1
0.68
3.6
6
318
373
5
1.7
1
4
CH3CH2O–
CH3(CH2)2O–
CH3(CH2)3O–
C6H5CH2O–
H–
16.9 1.73
2.4 0.31
0.46 0.18
0.048 0.03
>100
3.5
16.7
22
>2000
—
—
5
51
37
>100
CH3O–
16.2 2.29
9.60 1.63
6.62 0.34
2.50 0.25
>100
>100
CH3CH2O–
CH3(CH2)2O–
CH3(CH2)3O–
C6H5CH2O–
2.1 0.28
0.13 0.011
0.068 0.016
0.038 0.003
280 10f
52
146
>0.12
>2600
0.05
6
14.4 0.5e
12.2 2.72g
a
b
c
Calculated directly from the respective full kinetic determinations.
Calculated from IC50 values using the Cheng–Prusoff equation: Km (5-HT, MAO-A) = 100
Selectivity was expressed as the ratio of the Ki of MAO-A to the Ki of MAO-B.
lM, Km (DMAPEA, MAO-B) = 5 lM.
d
Standard errors for hMAO-A calculated for a single kinetic analysis based on Km and Vmax values at 6–8 concentrations of inhibitor, not comparable with the standard
deviation calculated on the average of different assays for hMAO-B, rMAO-A and rMAO-B.
e
Ref. 21.
Ref. 22.
Ref. 12.
f
g
Figure 3. Superimposed structures of compounds 4b (gray), 4c (light rose), 4d
(blue) and 4e (red) docked into the active site of hMAO-B. Main active site amino
acid residues (green) and FAD (yellow) are rendered as stick models.
Figure 2. Lineweaver–Burk plot for the inhibition by 5f of human MAO-B assayed
with benzylamine as the substrate.
of the enzyme.23 Additionally, the sulfur atom docks close to
Tyr435, suggesting that it might interact with the amino acid’s
phenol group. Similar interactions between the aromatic cage tyro-
sines and H bond acceptor heteroatoms, have been described as
important for binding of different MAO-B and MAO-A
inhibitors.10,24,25
The MAO-B active site consists of two cavities, the relatively
polar substrate binding site facing the flavin cofactor, and the
so-called entrance cavity, which displays a highly hydrophobic
environment.26 Visual inspection of the best poses for compounds
belonging to both the amide (4) and amine (5) series indicated that
these drugs can bind in an extended conformation occupying both
antipodes bind to the hMAO-B active site in very similar orienta-
tions (see below), with the heterocycle facing the isoalloxazine ring
of the enzyme cofactor, and the aromatic ring located in such a way
as to establish interactions with the Tyr326 hydroxyl group, which
is at a distance of approximately 3 Å (Fig. 3 illustrates the binding
modes obtained for compounds (S)-4b–e; henceforth all figures
show the S-isomer unless stated otherwise). These types of attrac-
tive hydrogen–p interactions, which involve hydroxyl groups and
aromatic rings, have been previously reported and although they
are weaker than classical hydrogen bonds they can be decisive in
determining the preferred pose of the ligand in the binding site

S. Lühr et al. / Bioorg. Med. Chem. 18 (2010) 1388–1395
1391
cavities and that differences in potencies of alkoxylated derivatives
might be attributed to differential hydrophobic interactions of the
n-alkoxyl chain and several aromatic and aliphatic residues includ-
ing Phe99, Phe103, Trp119, Leu164, Leu167, Phe168, Ile199 and
Ile316, where the longer substituents interact more strongly with
the entrance cavity.
To increase our understanding of the main binding interactions
at the hMAO active site and explain the high selectivity of 4f and 5f
toward MAO-B, we superimposed the structures of MAO-B docked
with 5f on the hMAO-A model. Figure 5 shows that the fit of 5f into
the active site of hMAO-A might be hindered by steric interference
with the side chain of Phe208, which corresponds to Ile199 of
hMAO-B.32
The role of the sulfur atom deserves some further comment. As
indicated above, this atom seems to interact with the tyrosine res-
idues forming the aromatic cage with the isoalloxazine ring. These
tyrosine residues are conserved in the catalytic site of both iso-
forms, and it may be significant that the hMAO-A-selective 4-alkyl-
thiophenylisopropylamines dock with their sulfur atom between
the OH groups of Tyr407 and Tyr444. Although the latter com-
pounds are highly selective for MAO-A, the propyl-, butyl- and
higher alkylthio homologues are also weak inhibitors of both rat
and human MAO-B.10,12
The existence of these aromatic residues in the entrance cavity
suggests that replacement of the aliphatic substituents in 4a–e,
5a–e by an aromatic entity might lead to stronger interactions.
In previous CoMFA studies of indolyl derivatives in rMAO-B it
was noted that a benzyloxy group can be placed at a location con-
gruent with the para position of our phenylethylamine deriva-
tives.27 Moreover, the literature records
a large number of
compounds where the inclusion of a benzyloxy group affords po-
tent and selective MAOBI,13,25,28,29 thus justifying the synthesis of
derivatives with a benzyloxy group at the para position of the aro-
matic ring (4f and 5f). In fact, this substituent resulted in strongly
increased potency and selectivity of thiomorpholinone 4f and thi-
In agreement with our hypothesis that the basic character of the
nitrogen is not a prerequisite for potent MAO-B inhibition, Table 1
shows that thiomorpholin-5-one derivatives 4e–f, lacking a basic
group, are potent inhibitors of MAO-B from both species.2
omorpholine 5f towards MAO-B of both species (4f Ki = 0.048
lM,
selectivity >2000; 5f Ki = 0.038
lM, selectivity >2600), with little or
no binding to hMAO-A.
As shown in Figure 4A, docking experiments in human MAO-B
revealed that the benzylated compounds 4f and 5f exhibit similar
binding modes, where their heterocycle points toward the isoallox-
azine ring of FAD. In addition, the orientation of both R- and S-iso-
mers of 5f showed very similar poses into the active site of MAO-B
(Fig. 4B). In these cases the sulfur atom in the heterocycle appears
to interact with Tyr398 rather than Tyr435 as seems to happen
with 4b–e. These tyrosine residues may show some degree of flex-
ibility and it is reasonable to assume that binding interactions of
the inhibitor in the catalytic site may arise from interactions with
either amino acid. The position of the central aromatic ring is
highly conserved and is the same as in the n-alkoxy derivatives,
underlining the likely importance of a phenol–benzene ring inter-
action with the Tyr326 residue. As expected, 4f and 5f dock in ex-
tended conformations occupying both cavities. The benzyloxy
group is embedded in a hydrophobic environment delimited by
Phe99, Phe103, Pro104, Trp119, Leu164, Leu167, Phe168, Ile199
and Ile316. This location is similar to that occupied by a phenyl
ring of safinamide, 7-(3-chlorobenzyloxy)-4-(methylamino)-
methylcoumarin, 7-(3-chlorobenzyloxy)-4-formylcoumarin, and
1,4-diphenyl-2-butene cocrystallized with hMAO-B,25,30 and in
the docking poses of safinamide and 5- and 6-styrylisatin
analogues.29,31
Figure 5. Superimposition of hMAO-B (green) binding 5f (rose) onto hMAO-A
(blue). Main active site amino acid residues are rendered as stick models.
Figure 4. A.- Docking poses of 4f (light blue) and 5f (rose) into hMAO-B. Main active site aminoacid residues (green) and FAD (yellow) are rendered as stick models. B.-
Superimposition of R- (rose) and S- (blue) 5f docked into the hMAO-B catalytic site.

1392
S. Lühr et al. / Bioorg. Med. Chem. 18 (2010) 1388–1395
All reagents and solvents were commercially available and were
3. Conclusions
used without further purification.
In this study, two series of rigid phenylethylamine analogues
have been synthesized to test the hypotheses that fixing the amine
chain in an extended six-membered ring conformation could lead
to increased potency as MAO-B inhibitors, and that the basic nitro-
gen atom is not a prerequisite for this activity. Indeed, the thi-
omorpholine (5a) and thiomorpholinone (4a) analogues of
4.1.1. General procedure for the preparation of 4-alkoxybenz-
aldehydes (1c–f)
To
a stirred solution of 4-hydroxybenzaldehyde (7.00 g,
51 mmol) in CH3CN (100 mL), K2CO3 (14.2 g, 103 mmol) and the
appropriate alkyl halide (51 mmol) were added. The reaction mix-
ture was refluxed and stirred for 24 h and then the solvent was re-
moved by rotary evaporation. The product (a white solid) was
extracted with EtOAc and 25% NaOH (2 ꢁ 100 mL) and then with
H2O (2 ꢁ 100 mL). The organic layer was dried over Na2SO4 and
concentrated.
phenylethylamine compare favorably with amphetamine (a-meth-
ylphenylethylamine) in this regard, retaining very similar activities
toward rat MAO-A and showing an increase in MAOBI activity by
more than an order of magnitude. Unexpectedly, we found that
the sulfur atom in the heterocycle docked into the hMAO-B cata-
lytic site interacts with Tyr398 and/or Tyr435. Furthermore, the
docking poses of a series of 4-alkylthiophenylisopropylamines in
hMAO-A10 showed similar interactions with the corresponding
Tyr407 and/or 444. This similar binding mode could explain the
residual MAO-A inhibitory activity for 4a–e and 5a–e. The 40-n-alk-
oxy (methoxy to butoxy) derivatives 4b–e and 5b–e exhibit a trend
toward progressively higher potencies, and the benzyloxy ana-
logues 4f and 5f were the most potent and selective for both
rMAO-B and hMAO-B. In the case of the human enzyme, Ki values
were in the 10ꢀ8 M range, and selectivities for MAO-B rather than
MAO-A exceed 2000-fold. Docking experiments indicate that the
alkoxy groups extend into the entrance cavity of the enzyme, and
that the benzyloxy group occupies a similar position to the benzyl-
oxy group of safinamide25 and appropriately placed aromatic sub-
stituents of other MAOBI. The successful design of this series
provides information about three specific interactions that could
increase the potency and selectivity of MAO inhibitors: an aro-
4.1.1.1. 4-Ethoxybenzaldehyde (1c). Yield 92% (yellow oil), 1H
NMR (CDCl3) d 1.45 (t, J = 7.4 Hz, 3H, CH3), 4.10 (m, 2H, OCH2),
6.99 (d, J = 8.6 Hz, 2H, ArH), 7.80 (d, J = 8.6 Hz, 2H, ArH), 9.85 (s,
1H, CHO).
4.1.1.2. 4-Propoxybenzaldehyde (1d). Yield 97% (orange oil), 1H
NMR (CDCl3) d 1.05 (t, J = 7.5 Hz, 3H, CH3), 1.75–1.90 (m, 2H, CH2),
4.00 (t, J = 6.6 Hz, 2H, OCH2), 6.99 (d, J = 8.8 Hz, 2H, ArH), 7.80 (d,
J = 8.8 Hz, 2H, ArH), 9.85 (s, 1H, CHO).
4.1.1.3. 4-Butoxybenzaldehyde (1e). Yield 94% (orange oil), 1H
NMR (CDCl3) d 0.95 (t, J = 7.4 Hz, 3H, CH3), 1.40–1.55 (m, 2H,
CH2), 1.70–1.85 (m, 2H, CH2), 4.05 (t, J = 6.6 Hz, 2H, OCH2), 7.00
(d, J = 8.6 Hz, 2H, ArH), 7.80 (d, J = 8.6 Hz, 2H, ArH), 9.85 (s, 1H,
CHO).
4.1.1.4. 4-Benzyloxybenzaldehyde (1f). Yield 94%, mp 68.5–
69.3 °C (white crystals from MeOH), 1H NMR (CDCl3) d 5.15 (s,
2H, CH2), 7.10 (d, J = 8.6 Hz, 2H, ArH), 7.25–7.48 (m, 5H, ArH),
7.85 (d, J = 8.7 Hz, 2H, ArH), 9.90 (s, 1H, CHO).
matic ring that can participate in a probably hydrogen–p interac-
tion with Tyr326; a heterocycle with a heteroatom that can
interact with Tyr398 and/or Tyr435 (in our case, a sulfur atom);
and a benzyl substituent at the opposite end of the molecule,
establishing interactions with the primarily aromatic residues of
the entrance cavity and thus determining selectivity for the B iso-
form. These structural features are found in previously described
MAO-B inhibitors, namely a central phenyl ring flanked by a benzyl
substituent and a heterocycle incorporating an atom that might
interact with the tyrosine residues in the aromatic cage. The ratio-
nalization of these common aspects should contribute to the devel-
opment of new selective and reversible MAO-B inhibitors with a
potential for use in the therapy of neurodegenerative diseases. As
mentioned above, all the compounds were evaluated as racemic
mixtures. Further experiments are necessary to elucidate which
enantiomer is the eutomer. Finally, our data confirm the impor-
tance of measuring drug inhibitory properties using the human
isoforms of the enzyme, but at the same time support the use of
rat MAO as a useful model, since in our case a whole series of po-
tent derivatives might have been disregarded due to the lack of
activity of the parent compounds towards the human enzymes,
while they showed an interesting activity against both rat MAOs.
4.1.2. General procedure for the preparation of arylnitroethe-
nes (2a–f)
The appropriate aldehyde (82 mmol), nitromethane (50 g,
82 mmol) and cyclohexylamine (32.5 g, 328 mmol) were dissolved
in acetic acid (100 mL). This reaction mixture was kept at 50 °C for
24 h, after which the solvent was evaporated. The products were
recrystallized from MeOH.
4.1.2.1. 2-Phenyl-1-nitroethene (2a). Yield 46%, mp 49.8–
50.7 °C (yellow needles), 1H NMR (CDCl3) d 7.35–7.57 (m, 5H,
ArH), 7.60 (d, J = 13.9 Hz, 1H, CH), 8.00 (d, J = 13.7 Hz, 1H, CH).
4.1.2.2. 2-(40-Methoxyphenyl)-1-nitroethene (2b). Yield 83%,
mp 85–87 °C (yellow needles), 1H NMR (CDCl3) d 3.87 (s, 3H,
OCH3), 6.97 (d, J = 8.8 Hz, 2H, ArH), 7.50 (d, J = 8.8 Hz, 2H, ArH),
7.55 (d, J = 13.6 Hz, 1H, ArCH), 7.98 (d, J = 13.7 Hz, 1H, CHNO2).
4.1.2.3. 2-(40-Ethoxyphenyl)-1-nitroethene (2c). Yield 92%, mp
4. Experimental section
4.1. Chemistry
111–112.8 °C (yellow needles), 1H NMR (CDCl3)
d 1.45 (t,
J = 7.4 Hz, 3H, CH3), 4.08 (m, 2H, OCH2), 6.93 (d, J = 8.6 Hz, 2H,
ArH), 7.50 (d, J = 8.4 Hz, 2H, ArH), 7.52 (d, J = 12.6 Hz, 1H, ArCH),
7.98 (d, J = 13.7 Hz, 1H, CHNO2).
Melting points are uncorrected and were determined with a
Reichert Galen III hot plate microscope. 1H NMR spectra were re-
corded using Bruker AMX 400 and AMX 300 spectrometers at
400 or 300 MHz, respectively. Chemical shifts are reported relative
to TMS (d = 0.00) or HDO (d = 4.79) and coupling constants (J) are
given in hertz. The elemental analyses for C, H, N, O and S were per-
formed on a CE Instruments (model EA 1108) analyzer. Reactions
and product mixtures were routinely monitored by thin-layer
chromatography (TLC) on silica gel-precoated F254 Merck plates.
4.1.2.4. 2-(40-Propoxyphenyl)-1-nitroethene (2d). Yield 60%,
mp 93.8–94.2 °C (yellow needles), 1H NMR (CDCl3) d 1.05 (t,
J = 7.3 Hz, 3H, CH3), 1.75–1.90 (m, 2H, CH2), 3.98 (t, J = 6,6 Hz, 2H,
OCH2), 6.92 (d, J = 8.7 Hz, 2H, ArH), 7,44 (d, J = 8.2 Hz, 2H, ArH),
7.51 (d, J = 13.3 Hz, 1H, ArCH), 7.94 (d, J = 13.7 Hz, 1H, CHNO2).
4.1.2.5. 2-(40-Butoxyphenyl)-1-nitroethene (2e). Yield 78%, mp
63.2–63.8 °C (yellow needles), 1H NMR (CDCl3)
d 0.96 (t,

S. Lühr et al. / Bioorg. Med. Chem. 18 (2010) 1388–1395
1393
J = 7.3 Hz, 3H, CH3), 1.43–1.58 (m, 2H, CH2), 1.73–1.82 (m, 2H,
CH2), 4.00 (t, J = 6,6 Hz, 2H, OCH2), 6.92 (d, J = 8.8 Hz, 2H, ArH),
7.47 (d, J = 8.5 Hz, 2H, ArH), 7.52 (d, J = 13.4 Hz, 1H, ArCH), 7.95
(d, J = 13.6 Hz, 1H, CHNO2).
(500 mL) at room temperature. Stirring was continued at 70 °C
for 72 h. After cooling, the mixture was filtered through Celite.
The solid was washed with CH2Cl2 (5 ꢁ 50 mL) and the filtrate
was evaporated. The residue was dissolved in 100 mL of CH2Cl2
and washed with H2O made alkaline with NH3 (2 ꢁ 100 mL). The
organic phase was dried, filtered and evaporated. The products
were recrystallized from absolute EtOH.
4.1.2.6. 2-(40-Benzyloxyphenyl)-1-nitroethene (2f). Yield 65%,
mp 117.4–118.2 °C (yellow needles), 1H NMR (CDCl3) d 5.13 (s,
2H, OCH2), 7.04 (d, J = 8.6 Hz, 2H, ArH), 7.35–7.45 (m, 5H, ArH),
7.50 (d, J = 8.6 Hz, 2H, ArH), 7.55 (d, J = 13.7 Hz, 1H, ArCH), 7.97
(d, J = 13.7 Hz, 1H, CHNO2).
4.1.4.1. 2-Phenylthiomorpholin-5-one (4a). Yield 59%, mp
144.3–145 °C (white needles), 1H NMR (CDCl3) d 3.45 (d, J = 16.2 Hz,
1H, CH2), 3.55 (d, J = 16.2 Hz, 1H, CH2), 3.78–3.85 (m, 2H, CH2), 4.35
(dd, J1 = 4.6 Hz, J2 = 8.6 Hz, 1H, SCH), 6.15–6.25 (br s, 1H, NHCO),
7.33–7.50 (m, 5H, ArH). Anal. Calcd for C10H11NOSꢂ0.5H2O: C, 59.38;
H, 5.98; N, 6.92; S, 15.85. Found: C, 58.98; H, 5.96; N, 6.86; S, 16.88.
4.1.3. General procedure for the preparation of 1-methylthio-
glycolyl-1-aryl-2-nitroethanes (3a–f)
To a stirred solution of the appropriate nitrostyrene (17 mmol)
in THF (50 mL) were added methyl thioglycolate (2.02 g, 19 mmol)
and Et3N (2.02 g, 20 mmol). The mixture was stirred at room tem-
perature for 2 h. The reaction was quenched with concentrated HCl
(5–10 drops), and the volatiles were removed in vacuo. The residue
was partitioned between 2 M HCl (2 mL) and CH2Cl2 (3 ꢁ 100 mL).
The combined organic extracts were dried over Na2SO4, filtered
and evaporated.
4.1.4.2. 2-(40-Methoxyphenyl)thiomorpholin-5-one (4b). Yield
48%, mp 158.3–159.1 °C (white needles), 1H NMR (CDCl3) d 3.43
(d, J = 16.2 Hz, 1H, CH2), 3.55 (d, J = 16.2 Hz, 1H, CH2), 3.70–3.80
(m, 2H, CH2), 3.85 (s, 3H, CH3), 4.,25–4.33 (dd, J1 = 4.6 Hz,
J2 = 9.1 Hz, 1H, SCH), 6.25–6.35 (br s, 1H, NHCO), 6.91 (d,
J = 8.6 Hz, 2H, ArH), 7.36 (d, J = 8.6 Hz, 2H, ArH). Anal. Calcd for
C11H13NO2S: C, 59.17; H, 5.87; N, 6.27; S, 14.36. Found: C, 58.91;
H, 6.24; N, 6.30; S, 15.53.
4.1.3.1. 1-Methylthioglycolyl-1-phenyl-2-nitroethane (3a).
Yield 97% (brown oil), 1H NMR (CDCl3) d 3.05 (d, J = 15.1 Hz, 1H,
SCH2), 3.15 (d, J = 15.1 Hz, 1H, SCH2), 3.70 (s, 3H, OCH3), 4.76–4.84
(m, 3H, CHCH2NO2), 7.33–7.48 (m, 5H, ArH).
4.1.4.3. 2-(40-Ethoxyphenyl)thiomorpholin-5-one (4c). Yield
62%, mp 161.5–162.5 °C (white needles), 1H NMR (CDCl3) d 1.45
(t, J = 6.9 Hz, 3H, CH3), 3.45 (d, J = 15.9 Hz, 1H, CH2), 3.50 (d,
J = 15.9 Hz, 1H, CH2), 3.60–3.70 (m, 2H, CH2), 4.00–4.10 (m, 2H,
OCH2), 4.25–4.33 (dd, J1 = 4.5 Hz, J2 = 8.8 Hz, 1H, SCH), 6.40–6.50
(br s, 1H, NHCO), 6.90 (d, J = 8.6 Hz, 2H, ArH), 7.35 (d, J = 8.6 Hz,
2H, ArH). Anal. Calcd for C12H15NO2S: C, 60.73; H, 6.37; N, 5.90;
S, 13.51. Found: C, 60.66; H, 7.13; N, 5.88; S, 14.85.
4.1.3.2. 1-Methylthioglycolyl-1-(40-methoxyphenyl)-2-nitroeth-
ane (3b). Yield 68% (green oil), 1H NMR (CDCl3) d 3.05 (d,
J = 15.1 Hz, 1H, SCH2), 3.15 (d, J = 15.1 Hz, 1H, SCH2), 3.70 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 4.75–4.85 (m, 3H, CHCH2NO2), 6.85 (d,
J = 8.6 Hz, 2H, ArH), 7.25 (d, J = 8.4 Hz, 2H, ArH).
4.1.3.3. 1-Methylthioglycolyl-1-(40-ethoxyphenyl)-2-nitroetha-
ne (3c). Yield 81%, mp 47.8–48.5 °C (white crystals from absolute
EtOH), 1H NMR (CDCl3) d 1.39 (t, J = 6.9 Hz, 3H, CH3), 3.05 (d,
J = 15.1 Hz, 1H, SCH2), 3.15 (d, J = 15.1 Hz, 1H, SCH2), 3.70 (s, 3H,
OCH3), 4.02 (m, 2H, OCH2), 4.75–4.87 (m, 3H, CHCH2NO2), 6.85
(d, J = 8.6 Hz, 2H, ArH), 7.25 (d, J = 8.8 Hz, 2H, ArH).
4.1.4.4. 2-(40-Propoxyphenyl)thiomorpholin-5-one (4d). Yield
50%, mp 163.7–164.3 °C (white needles), 1H NMR (CDCl3) d 1.05
(t, J = 7.6 Hz, 3H, CH3), 1.75–1.90 (m, 2H, CH2), 3.35 (d,
J = 16.2 Hz, 1H, CH2), 3.53 (d, J = 16.2 Hz, 1H, CH2), 3.68–3.78 (m,
2H, CH2), 3.95 (t, J = 6.6 Hz, 2H, OCH2), 4.25–4.30 (dd, J1 = 4.5 Hz,
J2 = 8.8 Hz, 1H, SCH), 6.20–6.30 (br s, 1H, NHCO), 6.90 (d,
J = 8.8 Hz, 2H, ArH), 7.35 (d, J = 8.5 Hz, 2H, ArH). Anal. Calcd for
C13H17NO2S: C, 62.12; H, 6.82; N, 5.57; S, 12.76. Found: C, 62.05;
H, 7.29; N, 5.87; S, 15.12.
4.1.3.4. 1-Methylthioglycolyl-1-(40-propoxyphenyl)-2-nitroeth-
ane (3d). Yield 86%, mp 68.6–69.3 °C (white crystals from abso-
lute EtOH), 1H NMR (CDCl3) d 1.00 (t, J = 7.4 Hz, 3H, CH3), 1.72–
1.85 (m, 2H, CH2), 3.05 (d, J = 15.3 Hz, 1H, CH2), 3.15 (d,
J = 15.3 Hz, 1H, CH2), 3.70 (s, 3H, OCH3), 3.90 (t, J = 6.5 Hz, 2H,
OCH2), 4.70–4.85 (m, 3H, CHCH2NO2), 6.85 (d, J = 8.6 Hz, 2H,
ArH), 7.25 (d, J = 8.8 Hz, 2H, ArH).
4.1.4.5. 2-(40-Butoxyphenyl)thiomorpholin-5-one (4e). Yield
30%, mp 166.1–167.6 °C (white needles), 1H NMR (CDCl3) d 0.95
(t, J = 7.3 Hz, 3H, CH3), 1.40–1.53 (m, 2H, CH2), 1.68–1.80 (m, 2H,
CH2), 3.35 (d, J = 16.2 Hz, 1H, CH2), 3.45 (d, J = 16.2 Hz, 1H, CH2),
3.60–3.75 (m, 2H, CH2), 3.90 (t, J = 6.3 Hz, 2H, OCH2), 4.20–4.25
(dd, J1 = 4.6 Hz, J2 = 8.8 Hz, 1H, SCH), 6.40–6.50 (br s, 1H, NHCO),
6.85 (d, J = 8.8 Hz, 2H, ArH), 7.25 (d, J = 8.6 Hz, 2H, ArH). Anal. Calcd
for C14H19NO2S: C, 60.36; H, 7.22; N, 5.28; S, 12.08. Found: C,
60.52; H, 8.49; N, 5.33; S, 14.22.
4.1.3.5. 1-Methylthioglycolyl-1-(40-butoxyphenyl)-2-nitroetha-
ne (3e). Yield 91%, mp 48.5–49.2 °C (white crystals from absolute
EtOH), 1H NMR (CDCl3) d 0.95 (t, J = 7.4 Hz, 3H, CH3), 1.40–1.50 (m,
2H, CH2), 1.70–1.80 (m, 2H, CH2), 3.05 (d, J = 15.3 Hz, 1H, CH2), 3.15
(d, J = 15.3 Hz, 1H, CH2), 3.68 (s, 3H, OCH3), 3.90 (t, J = 7.4 Hz, 2H,
OCH2), 4.73–4.85 (m, 3H, CHCH2NO2), 6.84 (d, J = 8.8 Hz, 2H,
ArH), 7.23 (d, J = 8.8 Hz, 2H, ArH).
4.1.4.6. 2-(40-Benzyloxyphenyl)thiomorpholin-5-one (4f). Yield
62%, mp 193.9–194.6 °C (white needles), 1H NMR (CDCl3) d 3.35 (d,
J = 16.2 Hz, 1H, CH2), 3.45 (d, J = 16.2 Hz, 1H, CH2), 3.62–3.70 (m,
2H, CH2), 4.25–4.30 (dd, J1 = 4.8 Hz, J2 = 8.9, Hz 1H, SCH), 5.02 (s,
2H, CH2), 6.20–6.30 (br s, 1H, NHCO), 6.92 (d, J = 8.6 Hz, 2H, ArH),
7.30 (d, J = 8.8 Hz, 2H, ArH), 7.30–7.43 (m, 5H, ArH). Anal. Calcd
for C17H17NO2S: C, 68.20; H, 5.72; N, 4.68; S, 10.71. Found: C,
67.92; H, 6.10; N, 4.73; S, 12.84.
4.1.3.6. 1-Methylthioglycolyl-1-(40-benzyloxyphenyl)-2-nitroe-
thane (3f). Yield 93%, mp 90.0–96.8 °C (golden crystals from
absolute EtOH), 1H NMR (CDCl3) d 3.05 (d, J = 15.3 Hz, 1H, CH2),
3.15 (d, J = 15.3 Hz, 1H, CH2), 3.65 (s, 3H, OCH3), 4.70–4.85 (m,
3H, CHCH2NO2), 5.03 (s, 2H, OCH2), 6.85 (d, J = 8.6 Hz, 2H, ArH),
7.25 (d, J = 8.8 Hz, 2H, ArH), 7.28–7.45 (m, 5H, ArH).
4.1.4. General procedure for the preparation of 2-arylthiomor-
pholin-5-ones (4a–f)
Zinc powder (24.4 g, 400 mmol) was added in portions to a stir-
red solution of the nitro ester (20 mmol) in glacial acetic acid
4.1.5. 2-Arylthiomorpholine oxalates (5a–f)
To a refluxing solution of the appropriate lactam (3 mmol) in
10 mL of THF was added 12 mL of 1 M DIBAL-H solution in n-hex-
ane (12 mmol), and the mixture was refluxed for 2 h with stirring.

1394
S. Lühr et al. / Bioorg. Med. Chem. 18 (2010) 1388–1395
Then 50 mL of H2O was added to the reaction mixture and ex-
tracted with CH2Cl2 (2 ꢁ 30 mL). The organic layer was dried over
Na2SO4, filtered and evaporated to give a yellow oil. The oil was
dissolved in EtOH and treated with oxalic acid in Et2O. The precip-
itated oxalate salt was collected by filtration and recrystallized
from EtOH.
metabolites were detected by HPLC with electrochemical detection
as described previously.7,8 IC50 values (mean SD from at least two
independent experiments, each in triplicate) were determined
using Graph Pad Prism software, from plots of inhibition percent-
ages (calculated in relation to a sample of the enzyme treated un-
der the same conditions without inhibitors) versus ꢀlog inhibitor
concentration. Ki were determined from the IC50 values using the
Cheng–Prusoff equation: Ki = IC50/(1 + [S]/Km),33 Km (5-HT, MAO-
4.1.5.1. 2-Phenylthiomorpholine oxalate (5a). Yield 40%, mp
226.9–227.5 °C (pale yellow crystals), 1H NMR (DMSO-d6) d 2.90
(m, 1H, CH2), 3.05 (m, 1H, CH2), 3.15 (m, 1H, CH2), 3.35 (m, 1H,
CH2), 3.60 (m, 2H, CH2), 4.30 (m, 1H, SCH), 7.25–7.48 (m, 5H,
ArH). Anal. Calcd for C20H26NSꢂC2H2O4ꢂH2O: C, 56.63; H, 6.48; N,
6.00; S, 13.74. Found: C, 56.67; H, 6.56; N, 5.82; S, 15.83.
A) = 100
lM, Km (DMAPEA, MAO-B) = 5 lM.
4.2.2. Human MAO
4.2.2.1. Mao-A (purified human recombinant).34 Initial rates of
oxidation were measured spectrophotometrically at 30 °C in
50 mM potassium phosphate, pH 7.4, containing 0.05% Triton X-
100. Ki values were determined using a substrate range of 0.1–
0.9 mM kynuramine (purchased from Sigma)35 at six different
inhibitor concentrations over a 10-fold range. The formation of
the product was followed spectrophotometrically at 314 nm
4.1.5.2. 2-(40-Methoxyphenyl)thiomorpholine oxalate (5b). Yield
14%, mp 195.6–196.1 °C (pale yellow crystals), 1H NMR (DMSO-d6) d
2.75 (m, 1H, CH2), 2.95 (m, 1H, CH2), 3.10 (m, 1H, CH2), 3.25 (m, 1H,
CH2), 3.48 (m, 2H, CH2), 3.68 (s, 3H, OCH3), 4.24 (m, 1H, SCH), 6.86
(d, J = 8.6 Hz, 2H, ArH), 7.22 (d, J = 8.6 Hz, 2H, ArH). Anal. Calcd for
C11H15NOSꢂC2H2O4ꢂ0.25H2O: C, 51.39; H, 5.81; N, 4.61; S, 10.55.
Found: C, 51.35; H, 6.02; N, 4.77; S, 12.56.
(e
= 12,300 Mꢀ1 cmꢀ1), and the kinetic constants determined using
Shimadzu software.
4.2.2.2. Mao-b (purified human recombinant).36 The activities
were determined spectrophotometrically, using benzylamine (pur-
chased from Sigma) as substrate, in 50 mM Hepes buffer, pH 7.5,
containing 0.5% (w/v) reduced Triton X-100, at 25 °C. The oxidation
of benzylamine by MAO-B was monitored at 250 nm (maximum
absorption by benzaldehyde, extinction coefficient = 12,800 Mꢀ1
cmꢀ1).37 All spectral data were obtained using a Unicam Helios-
beta spectrophotometer. Kinetic data were evaluated and plotted
using Prism Graph Pad software.
4.1.5.3. 2-(40-Ethoxyphenyl)thiomorpholine oxalate (5c). Yield
29%, mp 204.3–204.9 °C (pale yellow crystals), 1H NMR (DMSO-
d6) d 1.30 (m, 3H, CH3), 2.65 (m, 1H, CH2), 2.90 (m, 1H, CH2), 3.05
(m, 1H, CH2), 3.15 (m, 1H, CH2), 3.40 (m, 2H, CH2), 3.90 (m, 2H,
OCH2), 4.00 (m, 1H, SCH), 6.90 (d, J = 8.7 Hz, 2H, ArH), 7.25 (d,
J = 8.6 Hz, 2H, ArH). Anal. Calcd for C24H34NOSꢂC2H2O4: C, 58.18;
H, 6.76; N, 5.22; S, 11.95. Found: C, 57.75; H, 6.91; N, 5.19; S, 13.59.
4.1.5.4. 2-(40-Propoxyphenyl)thiomorpholine oxalate (5d). Yield
39%, mp 197.3–197.8 °C (pale yellow crystals), 1H NMR (DMSO-d6) d
0.93 (m, 3H, CH3), 1.60–1.75 (m, 2H, CH2), 2.80 (m, 1H, CH2), 3.05
(m, 1H, CH2), 3.15 (m, 1H, CH2), 3.25 (m, 1H, CH2), 3.55 (m, 2H, CH2),
3.90 (m, 2H, OCH2), 4.28 (m, 1H, SCH), 6.90 (d, J = 8.7 Hz, 2H, ArH),
7.15 (d, J = 8.7 Hz, 2H, ArH). Anal. Calcd for C13H19NOSꢂ-
C2H2O4ꢂ0.5H2O: C, 53.55; H, 6.59; N, 4.16; S, 9.53. Found: C, 53.40;
H, 6.85; N, 4.18; S, 11.84.
4.3. Molecular simulation
The crystal structures of human MAO-B (PDB: 1S3E) and MAO-A
(PDB: 2BXS) were used for all simulations with the Autodock 4.0
suite.38 All other docking conditions were as previously pub-
lished.10,12 Grid maps were calculated using the autogrid4 option
and were centered on the putative ligand-binding site. The vol-
umes chosen for the grid maps were made up of 60 ꢁ 60 ꢁ 60
points, with a grid-point spacing of 0.375 Å. Partial charges of the
different compounds were corrected using ESP methodology.39
The docked compound complexes were built using the lowest
docked-energy binding positions. Both enantiomers of the com-
pounds were investigated and the energies and orientations found
are very similar with a good correlation with biological results.
Therefore in all cases the results obtained for the (S)-isomers were
used for the analysis.
4.1.5.5. 2-(40-Butoxyphenyl)thiomorpholine oxalate (5e). Yield
33%, mp 202.1–202.9 °C (pale yellow crystals), 1H NMR (DMSO-
d6) d 0.96 (m, 3H, CH3), 1.30–1.50 (m, 2H, CH2), 1.60–1.75 (m,
2H, CH2), 2.85 (m, 1H, CH2), 3.05 (m, 1H, CH2), 3.15 (m, 1H, CH2),
3.30 (m, 1H, CH2), 3.55 (m, 2H, CH2), 3.95 (m, 2H, OCH2), 4.30
(m, 1H, SCH), 6.90 (d, J = 8.6 Hz, 2H, ArH), 7.25 (d, J = 8.7 Hz, 2H,
ArH). Anal. Calcd for C14H21NOSꢂC2H2O4ꢂH2O: C, 53.46; H, 7.01; N,
3.90; S, 8.92. Found: C, 53.13; H, 7.05; N, 3.83; S, 10.57.
4.1.5.6. 2-(40-Benzyloxyphenyl)thiomorpholine oxalate (5f). Yield
14%, mp 201.3–201.6 °C (pale yellow crystals), H NMR (DMSO-d6) d
Acknowledgments
1
2.83 (m, 1H, CH2), 3.05 (m, 1H, CH2), 3.15 (m, 1H, CH2), 3.30 (m, 1H,
CH2), 3.55 (m, 2H, CH2), 4.25 (m, 1H, SCH), 5.10 (s, 2H, OCH2), 6.95 (d,
J = 8.4 Hz, 2H, ArH), 7.30 (d, J = 8.5 Hz, 2H, ArH), 7.37–7.48 (m, 5H,
ArH). Anal. Calcd for C17H19NOSꢂC2H2O4ꢂ0.5H2O: C, 59.36; H, 5.77; N,
3.64; S, 8.34. Found: C, 59.72; H, 5.91; N, 3.70; S, 10.88.
This work was partially funded by FONDECYT Grants 1060199,
1090037, 11085002, PBCT PDA-23 and ICM Grant P05-001-F. S.L.
was the recipient of a CONICYT scholarship and a MeceSup travel
grant. The authors thank Mr. Marco Rebolledo-Fuentes for expert
experimental support. D.E.E. acknowledges support from NIH
Grant GM-29433 and technical assistance from Ms. Milagros
Aldeco in purifying the recombinant human MAOB used in this
study.
4.2. Biochemistry
4.2.1. Rat MAO
The effects of the compounds (the lactams dissolved with the
aid of DMSO and the amines, as salts, in buffer) on MAO-A or
MAO-B activities were studied following previously reported
methodologies, using a crude rat brain mitochondrial suspension
References and notes
1. Filip, V.; Kolibás, E. J. Psychiat. Neurosci. 1999, 24, 234.
2. Reyes-Parada, M.; Fierro, A.; Iturriaga-Vásquez, P.; Cassels, B. K. Curr. Enzyme
Inhib. 2005, 1, 85.
3. Cesura, A. M.; Pletscher, A. Prog. Drug Res. 1992, 38, 171.
4. Pacher, P.; Kecskeméti, V. Curr. Med. Chem. 2004, 11, 925.
5. Youdim, M. B. H.; Edmondson, D.; Tipton, K. F. Nat. Rev. Neurosci. 2006, 7, 295.
as a source of enzyme.7,8 Serotonin (100
ophenylethylamine (5
MAO-A and -B, respectively, and these compounds and their
lM) and 4-dimethylamin-
l
M) were used as selective substrates for

S. Lühr et al. / Bioorg. Med. Chem. 18 (2010) 1388–1395
22. Li, M. Ph.D, Dissertation, Emory University: Atlanta, 2006.
1395
6. Sterling, J.; Herzig, Y.; Goren, T.; Finkelstein, N.; Lerner, D.; Goldenberg, W.;
Miskolczi, I.; Molnár, S.; Rantal, F.; Tamás, T.; Tóth, G.; Zagyva, A.; Zekany, A.;
Lavian, G.; Gross, A.; Friedman, R.; Razin, M.; Huang, W.; Krais, B.; Chorev, M.;
Youdim, M. B. H.; Weinstock, M. J. Med. Chem. 2002, 45, 5260.
7. Scorza, M. C.; Carrau, C.; Silveira, R.; Zapata-Torres, G.; Cassels, B. K.; Reyes-
Parada, M. Biochem. Pharmacol. 1997, 54, 1361.
23. Meyer, E. A.; Castellano, R. K.; Diederich, F. Angew. Chem., Int. Ed. 2003, 42,
1210.
24. Yelekçi, K.; Karahan, Ö.; Toprakçi, M. J. Neural Transm. 2007, 114, 725.
25. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.
E.; Mattevi, A. J. Med. Chem. 2007, 50, 5848.
8. Hurtado-Guzmán, C.; Fierro, A.; Iturriaga-Vásquez, P.; Sepúlveda-Boza, S.;
Cassels, B. K.; Reyes-Parada, M. J. Enzyme Inhib. Med. Chem. 2003, 18, 339.
9. Gallardo-Godoy, A.; Fierro, A.; McLean, T.; Castillo, M.; Cassels, B. K.; Reyes-
Parada, M.; Nichols, D. E. J. Med. Chem. 2005, 48, 2407.
26. Binda, C.; Newton-Vinson, P.; Hubálek, F.; Edmondson, D. E.; Mattevi, A. Nat.
Struct. Biol. 2002, 9, 22.
27. Moron, J. A.; Campillo, M.; Pérez, V.; Unzeta, M.; Pardo, L. J. Med. Chem. 2000,
43, 1684.
10. Fierro, A.; Osorio-Olivares, M.; Cassels, B. K.; Edmondson, D. E.; Sepúlveda-
Boza, S.; Reyes-Parada, M. Bioorg. Med. Chem. 2007, 15, 5198.
11. Osorio-Olivares, M.; Rezende, M. C.; Sepúlveda-Boza, S.; Cassels, B. K.; Fierro, A.
Bioorg. Med. Chem. 2004, 12, 4055.
12. Vilches-Herrera, M.; Miranda-Sepúlveda, J.; Rebolledo-Fuentes, M.; Fierro, A.;
Lühr, S.; Iturriaga-Vásquez, P.; Cassels, B. K.; Reyes-Parada, M. Bioorg. Med.
Chem. 2009, 17, 2452.
13. Mazouz, F.; Gueddari, S.; Burstein, C.; Mansuy, D.; Milcent, R. J. Med. Chem.
1993, 36, 1157.
14. Mazouz, F.; Lebreton, L.; Milcent, R.; Burstein, C. Eur. J. Med. Chem. 1990, 25,
659.
28. Pérez, V.; Marco, J. L.; Fernández-Álvarez, E.; Unzeta, M. Br. J. Pharm. 1999, 127,
869.
29. Leonetti, F.; Capaldi, C.; Pisani, L.; Nicolotti, O.; Muncipinto, G.; Stefanachi, A.;
Cellamare, S.; Caccia, C.; Carotti, A. J. Med. Chem. 2007, 50, 4909.
30. Binda, C.; Li, M.; Hubálek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 9750.
31. Van der Walt, E. M.; Milczek, E. M.; Malan, S. F.; Edmondson, D. E.; Castagnoli,
N., Jr.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem. Lett. 2009, 19, 2509.
32. De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684.
33. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
34. Li, M.; Hubálek, F.; Newton-Vinson, P.; Edmondson, D. E. Protein Exp. Purif.
2002, 24, 152.
15. Wouters, J.; Moureau, F.; Evrard, G.; Koenig, J.; Jegham, S.; George, P.; Durant, F.
Bioorg. Med. Chem. 1999, 7, 1683.
16. Krueger, M. J.; Mazouz, F.; Ramsay, R. R.; Milcent, R.; Singer, T. P. Biochem.
Biophys. Res. Commun. 1995, 206, 556.
17. Novaroli, L.; Daina, A.; Favre, E.; Bravo, J.; Carotti, A.; Leonetti, F.; Catto, M.;
Carrupt, P.; Reist, M. J. Med. Chem. 2006, 49, 6264.
18. Beck, J. J.; Stermitz, F. R. J. Nat. Prod. 1995, 58, 1047.
19. Battersby, A.; Baker, M.; Broadbent, H.; Fookes, C.; Leeper, F. J. Chem. Soc., Perkin
Trans. 1 1987, 2027.
35. Hynson, R. M. G.; Wouters, J.; Ramsay, R. R. Biochem. Pharmacol. 2003, 65, 1867.
36. Newton-Vinson, P.; Hubálek, F.; Edmondson, D. E. Protein Exp. Purif. 2000, 20,
334.
37. Hubálek, F.; Binda, C.; Li, M.; Herzig, Y.; Sterling, J.; Youdim, M. B. H.; Mattevi,
A.; Edmondson, D. E. J. Med. Chem. 2004, 47, 1760.
38. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comp. Chem. 1998, 19, 1639.
20. Yamashita, M.; Ojima, I. J. Am. Chem. Soc. 1983, 105, 6339.
21. Vintém, A. P.; Price, N. T.; Silverman, R. B.; Ramsay, R. R. Bioorg. Med. Chem.
2005, 13, 3487.
39. Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. J. Phys. Chem. 1993, 97,
10269.