Human and rat monoamine oxidase-A are differentially inhibited by (S)-4-alkylthioamphetamine derivatives: Insights from molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2007.05.021

Four enantiomerically pure (S)-4-alkylthioamphetamine derivatives were evaluated as monoamine oxidase (MAO) inhibitors using the human and rat isoforms of the enzyme. Molecular dockings were performed in order to gain insights regarding the binding mode o

Full text of DOI:10.1016/j.bmc.2007.05.021

Bioorganic & Medicinal Chemistry 15 (2007) 5198–5206
Human and rat monoamine oxidase-A are differentially inhibited
by (S)-4-alkylthioamphetamine derivatives: Insights from
molecular modeling studies
a
a
b,c
Angelica Fierro, Mauricio Osorio-Olivares, Bruce K. Cassels, Dale E. Edmondson,d
´
Silvia Sepu´lveda-Bozae and Miguel Reyes-Paradac,e,
*
aFaculty of Chemistry and Biology, University of Santiago de Chile, Alameda 3363, Santiago, Chile
bDepartment of Chemistry, Faculty of Sciences, University of Chile, Casilla 653, Santiago, Chile
cMillennium Institute for Cell Dynamics and Biotechnology, Beauchef 861, Santiago, Chile
dDepartments of Biochemistry and Chemistry, Emory University, Atlanta, GA 30322, USA
eSchool of Medicine, Faculty of Medical Sciences, University of Santiago de Chile, Alameda 3363, Santiago, Chile
Received 13 April 2007; accepted 8 May 2007
Available online 22 May 2007
Abstract—Four enantiomerically pure (S)-4-alkylthioamphetamine derivatives were evaluated as monoamine oxidase (MAO) inhib-
itors using the human and rat isoforms of the enzyme. Molecular dockings were performed in order to gain insights regarding the
binding mode of these inhibitors. All compounds were potent and selective MAO-A inhibitors although different rank orders of
potencies were observed against the enzymes from different species. This behavior can be rationalized on the basis of different bind-
ing modes to each enzyme, as determined in silico. These findings further support the concept that MAO inhibitory activity of novel
compounds, determined with enzymes from diverse mammalian species, should be considered with caution if human MAO is the
final target to be addressed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
tallizes and behaves hydrodynamically in solution as a
monomer,5 whereas structural data suggest that
rMAO-A forms a dimeric structure in vivo.6 In addition,
the volume of the active site cavity of rMAO-A
Compounds that inhibit monoamine oxidase (MAO,
E.C. 1.4.3.4) have shown therapeutic value in a variety
of conditions including affective disorders and neurode-
generative diseases.1 In mammals, two kinds of MAOs
(MAO-A and MAO-B) are present in most tissues.
Crystal structures of both isoforms of human,2–5 and
of rat MAO-A (rMAO-A),6 complexed with pharmaco-
logically relevant inhibitors, have been recently de-
scribed. The similarities and differences between
human MAO-A (hMAO-A) and that from the rat have
provided new insights that may be important in drug
development.1,7 Although hMAO-A and rMAO-A exhi-
bit ꢀ90% sequence identity, both enzymes show impor-
tant differences in their quaternary structures, as
revealed by X-ray diffraction data. Thus, hMAO-A crys-
3
˚
(ꢀ450 A ) is smaller than that found in hMAO-A
3
˚
(ꢀ550 A ), which is due to a different conformation of
the cavity-shaping loop (residues 210–216) present in
both enzymes.1,5 Even though it has been consistently
pointed out,8 the aforementioned data lend further sup-
port to the notion that results obtained with non-human
forms of MAO (e.g., the evaluation of the inhibitory
properties of a given compound) cannot be unambigu-
ously extrapolated to the situation in humans.
Several phenylisopropylamine derivatives (often referred
to in the literature as ‘substituted amphetamines’) have
been shown by us and others to be potent MAO inhib-
itors.9–11 Structure–activity relationship (SAR) studies,
including quantitative analyses (QSAR),10–12 have
shown that the presence of electron-donating, un-
branched, alkylthio substituents at the para position of
the aromatic ring of the amphetamine scaffold generates
potent and selective MAO-A inhibitors. In addition, a
Keywords: Monoamine oxidase; MAO inhibitors; Amphetamine deri-
vatives; Molecular dynamics simulation.
*
Corresponding author. Tel./fax: +56 2 681 7602; e-mail: mreyes@
usach.cl
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.021

A. Fierro et al. / Bioorg. Med. Chem. 15 (2007) 5198–5206
5199
few studies have shown that the (S)-isomers of substi-
tuted amphetamines (which are always dextrorotatory)
are the eutomers.13–15 However, little is known about
the binding mode of this class of compounds, in spite
of their close structural similarity to endogenous mono-
aminergic neurotransmitters which are the main physio-
logical substrates of MAOs. In this sense, molecular
docking is a useful approach since protein–ligand con-
figurations produced by the docking programs allow a
visual analysis of protein–ligand interactions and facili-
tate an intuitive interpretation and understanding of the
binding process at the protein-binding site.16 Further-
more, recent studies have shown that molecular model-
ing yields good correlations between the calculated and
experimental Ki values for a series of known and new
MAO inhibitors.17,18
ones with triethylsilane, and deprotection of the amino
group by acid hydrolysis, according to a previously
reported procedure.19,20 Hydrochloride salts were
obtained by adding gaseous HCl (Scheme 1).
This synthetic route proceeded with complete retention
of chirality, supporting the versatility of using chiral ala-
nine derivatives,20 previously exploited in the synthesis
of amphetamine21 and cathinone analogs.22
Enantiomeric excesses were determined to be >98% in
all cases, as evaluated by the comparison of the 1H
NMR signals of the enantiomer with those of the
corresponding racemic mixture, both complexed with
europium tris-[3-(heptafluoropropylhydroxymethylene)-
(+)-camphorate] (Eu(hfc)3) used as chiral shift reagent.
In the present study, four enantiomerically pure (S)-4-
alkylthiophenylisopropylamine derivatives were pre-
pared and evaluated as MAO inhibitors using the
human and rat enzyme isoforms. In addition, using
the crystal structures of the proteins, molecular docking
was performed in order to gain insights regarding the
binding modes of these inhibitors and also to explain
observed differences in their inhibitory properties with
MAO from different species.
2.2. Biochemistry
Table 2 summarizes the effects of compounds 1–4 upon
MAO-A and -B from human and rat. As previously
shown for 1,15 and racemic 2,10 all (S)-4-alkylthioam-
phetamines selectively inhibited MAO-A from both spe-
cies with Ki values in the nanomolar range. This
inhibition was competitive in all cases, as shown for
(S)-4-ethylthioamphetamine 2 in Figure 1. Inhibition
of both hMAO-B and rMAO-B was observed only in
the case of the derivatives bearing a propylthio (3) or
butylthio (4) substituent, but the potency was at least
20-fold lower than that observed for the corresponding
MAO-A.
2. Results and discussion
2.1. Chemistry
The synthesis of the (S)-4-alkylthiophenylisopropyl-
amine derivatives 1–4 (Table 1) was carried out by a
reaction sequence involving initial Friedel–Crafts acyla-
tion of the corresponding alkyl-phenyl thioethers with
(S)-N-trifluoroacetylalanyl chloride, followed by reduc-
tion of the (S)-2-trifluoroacetamido-1-arylpropan-1-
Regarding the MAO-A inhibitory potency, it was simi-
lar and followed the same trend for both species in the
case of 1 and 2, that is, lengthening of the substituent
from a methylthio to an ethylthio group produced a
decrease of the Ki, which agrees with results obtained
with racemic mixtures10 and other alkylthio-substituted
Table 1. Yields and physical properties of (S)-2-amino-1-arylpropane derivatives 1–4
NH
2
H
CH
3
RS
20
½aꢁD
a
Compound
R
Mp (ꢁC)
Yield (%)
m/z (free base)
1
2
3
4
CH3
CH3CH2
224–225
211–213
135–137
141–143
47
58
50
38
+7.9
+6.4
+5.9
+6.8
181.09
195.11
209.12
223.13
CH3CH2CH2
CH3CH2CH2CH2
a (c 1, MeOH).
O
NH2.HCl
iv)
v)
NHCOCF3
HOOC
H3C
NH2
i), ii)
H3C
H
H3C
H
R4
iii) R4-Ar-H
H
R4
Scheme 1. Reagents and conditions: (i) 1,1,3,3-tetramethylguanidine/CF3CO2Et/MeOH; (ii) (CO)2Cl2/Py/CH2Cl2; (iii) AlCl3; (iv) (Et3)3SiH/
CF3COOH, reflux; (v) 12 M HCl/2-propanol, 70 ꢁC.

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A. Fierro et al. / Bioorg. Med. Chem. 15 (2007) 5198–5206
Table 2. MAO inhibitory activity of phenylisopropylamine derivatives 1–4
Compound
Rat
Human
Kia (lM)
Ki (lM)
MAO-A
MAO-B
MAO-A
MAO-B
1
2
3
4
0.245 0.030
0.054 0.009
0.115 0.016
0.392 0.033
>100
>100
0.133 0.020
0.075 0.015
0.030 0.001
0.022 0.001
NE
>100
15.000 1.430
8.925 1.575
14.03 1.082
4.58 0.850
NE, no effect at the highest concentration tested (100 lM).
a Ki values for MAOs from rat brain were determined from the IC50 values using the Cheng–Prusoff equation (see Section 4).
Thus, while potency against hMAO-A increased for
the compounds with longer alkyl chains attached to
the sulfur atom at the para position (with the four-
carbon derivative, i.e., 4, being the most potent), pro-
pyl- and butylthio derivatives 3 and 4 were less potent
rMAO-A inhibitors than the ethylthio analog 2. These
results are in the same line as a recent study by
Novaroli et al.,24 which showed that some coumarin
and 5H-indeno[1,2-c]pyridazin-5-one derivatives exhib-
ited striking divergences in their inhibitory potency
when evaluated against human or rat MAO-B. As
stressed by these authors, species-related differences
on inhibitor activity such as that of derivative 4 (more
than one order of magnitude between rMAO-A and
hMAO-A) not only support the idea that results
might greatly vary depending on the enzyme source
used, but also have important implications for the
selection of hit compounds for lead generation, which
is an interesting finding considering that both enzymes
are ꢀ90% identical in sequence. Accordingly, the dif-
ferent rank orders of potency shown by the com-
pounds tested here strongly suggest that, even
though both hMAO-A and rMAO-A are able to
accommodate alkylthio-substituted amphetamines in
their active sites, the interactions that account for
the binding properties in each enzyme, at the least
for compounds 3 and 4, are probably different.
Figure 1. Lineweaver–Burk plot for the inhibition of hMAO-A by 2.
amphetamine derivatives,11 using MAO from rat brain.
However, a clear difference between hMAO-A and
rMAO-A was observed when the inhibitory properties
of compounds 3 and 4 were compared. This different
behavior is demonstrated in the plots in Figure 2 where
log Ki is plotted versus the van der Waals volume of the
alkyl substituent. A linear correlation is observed with
hMAO-A, while a strong deviation from linearity is
observed for the binding properties of compounds 3
and 4 with rMAO-A.
2.3. Molecular docking
In order to test the aforementioned hypothesis, com-
pounds 1–4 were docked into the active sites of
rMAO-A and hMAO-A, using the Autodock 3.05
suite,25 and the available crystal structures of both en-
zymes (PDB: 1O5W or 2BXS, respectively). Results
are summarized in Figures 3 (rat) and 4 (human), which
show the most stable conformations for each drug–en-
zyme complex.
In rMAO-A, compounds 1 and 2 exhibit binding modes
where the amino group points away from the flavin ring
forming a hydrogen bond with the carbonyl group of
Phe208, whereas the alkylthio chain is located between
Tyr407 and Tyr444, a position that would favor interac-
tions between the highly polarizable sulfur atom and
either or both aromatic moieties or the hydroxyl groups
of the tyrosines (Figs. 3A and B).
Figure 2. Plot of logKi versus van der Waals volume of para-alkyl
substituents for the inhibition of human and rat MAO-As by (S)-4-
alkylthioamphetamine analogs. Vw values for the various alkyl
substituents were calculated according to Bondi.23
In these conformations, the aromatic rings of 1 and 2
seem to be almost identically positioned between the

A. Fierro et al. / Bioorg. Med. Chem. 15 (2007) 5198–5206
5201
Figure 3. Binding modes of phenylisopropylamine derivatives 1–4 to rMAO-A (PDB: 1O5W).
side chains of Gln215 and Ile180, and perpendicular to
the flavin ring. A similar binding mode has been
recently described for the naturally occurring eugenol
(4-allyl-2-methoxyphenol), which has clear structural
similarities with phenylisopropylamines, although in
that case the aromatic ring of the ligand was located
somewhat closer to the isoalloxazine ring.26 As previ-
ously discussed for similar results obtained with hetero-
arylisopropylamine derivatives,27 this binding mode
provides a rationale for the observed inhibitory activ-
ity, since while blocking the access of any substrate
to the active site, these phenylisopropylamines could
avoid deamination by adopting a conformation where
the amino group is remote from the influence of the
flavin ring. In contrast, docking of compounds 3 and
4 yielded binding modes where the inhibitor molecules
adopted an almost opposite orientation to derivatives 1
and 2. Thus, the most energetically favorable confor-
mations of 3 and 4 were those where the aminopropyl
chain was positioned between Tyr407 and Tyr444, and
the alkylthio chain appeared folded into a space sur-
rounded by Ile335, Phe208, Val210 and Thr336, in-
stead of being extended into the hydrophobic channel
pointing to the binding site entrance (Figs. 3C and
D). The aromatic rings of 3 and 4 were almost in the
same position and orientation as those of derivatives
1 and 2. Although different from the foregoing, this
binding mode is also consistent with the rMAO-A
inhibitory activity of compounds 3 and 4. In spite of
an intense debate about the mechanism underlying
MAO-catalyzed amineoxidation,28 the two most ac-
cepted schemes, that is, the single electron transfer29
and the polar nucleophilic mechanisms,30 involve the
abstraction of the pro-R a-proton of the amine by
the N5 atom of the flavin ring, which would act as a
base. As can be seen in Figures 3C and D, a possible
interaction between the sole a-proton of phenylisopro-
pylamine derivatives 3 and 4 and the FAD N5 is made

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A. Fierro et al. / Bioorg. Med. Chem. 15 (2007) 5198–5206
less likely by the presence of the a-methyl group, which
prefers to face the re side of the isoalloxazine ring,
leaving the pro-R a-proton far from the isoalloxazine
N5. Beyond these considerations, the striking differ-
ences in the binding modes of derivatives 1–2 and
3–4 to rMAO-A generated by computational simula-
tions can explain the trend of their inhibitory poten-
cies. Thus, no hydrogen bonds between the inhibitor
and active site residues were apparent for the best
binding conformations of 3 and 4, which agree with
a less stable interaction and higher Ki values.
The aromatic rings of the inhibitors occupy a position
close to that found in rMAO-A, but they are located
in a clearly less perpendicular orientation to the flavin
as compared with that observed in the rat enzyme. This
positioning might favor stacking interactions with the
aromatic rings of Phe208 and Tyr69 (both about 5 A
from the inhibitors’ aromatic moieties), as shown in
Figure 5.
˚
In addition, stabilizing hydrogen bonds between the
amino group and the side chain of Ser209, the backbone
carbonyl of Val210, or both, were detected for all com-
pounds. The similarities in the binding modes of 1–4 to
hMAO-A suggest that the different potencies of these
compounds determined in biochemical assays could be
correlated with differential interactions at the active site,
mainly associated with the different properties of the
alkylthio chain attached to the para position. It has been
shown that the affinities of 4-substituted benzylamine
In the case of hMAO-A, the binding modes obtained for
compounds 1–4 from docking simulations were similar
in all cases and resemble those observed in rMAO-A
for 1 and 2, that is, the amino group was oriented away
from the flavin ring and the alkylthio chain was posi-
tioned in the so-called aromatic cage, between Tyr407
and Tyr444 (Fig. 4).
Figure 4. Binding modes of phenylisopropylamine derivatives 1–4 to hMAO-A (PDB: 2BXS).

A. Fierro et al. / Bioorg. Med. Chem. 15 (2007) 5198–5206
5203
Finally, our results further support the concept that
MAO inhibitory activity of novel compounds, deter-
mined with enzymes from diverse mammalian species,
should be considered with caution if human MAO is
the final target to be addressed.
4. Experimental
4.1. Chemistry
All reagents and solvents were commercially available
and were used without further purification. Melting
points are uncorrected and were determined with an
Electrothermal apparatus. NMR spectra were recorded
using either a Bruker AMX 300 or a Bruker Advance
400 spectrometer at 300 or 400 (1H) and 75 (13C)
MHz, employing tetramethylsilane as an internal stan-
dard. Chemical shifts are reported relative to TMS
(d = 0.00) and coupling constants (J) are given in Hz.
Europium tris[3-(heptafluoropropylhydroxymethylene)-
(+)-camphorate] was purchased from Aldrich. Optical
rotation values were obtained with a Perkin-Elmer 341
polarimeter. The elemental analyses for C, H, N, and
S were performed on a CE Instruments (model EA
1108) analyzer.
Figure 5. Superimposed structures of compounds 1 (gray), 2 (green), 3
(yellow), and 4 (blue), docked into the active site of hMAO-A.
and phenethylamine analogs for hMAO-A are more
favorable when the size of the para substituent increases,
and QSAR analyses have shown that this effect depends
exclusively on steric parameters of the para substituent
(van der Waals volume and/or Taft steric parameter
Es).31,32 The results presented here completely agree with
this notion, and an excellent correlation (r2 = 0.97) was
found between logKi and the van der Waals volume23
of the para substituent of compounds 1–4. In the case
of rMAO-A, this SAR does not hold for the compounds
tested here, since (as shown by docking experiments) the
binding mode is not the same for the whole series. Other
examples of arylalkylamine derivatives, in which differ-
ential binding modes to MAO from different species
have been advocated to explain different inhibitory
potencies, include phentermine33 and benzylamine
analogs.34
4.1.1. Preparation of (S)-N-trifluoroacetylalanine. (S)-N-
Trifluoroacetylalanine was prepared following a proce-
dure described previously from L-alanine (Aldrich),
ethyl trifluoroacetate, and 1,1,3,3-tetramethylguani-
dine.22 The product (86% yield) had a melting point of
62–64 ꢁC (lit.19 70–71 ꢁC) and was sufficiently pure for
all subsequent uses.
4.1.2. General procedure for the preparation of (S)-2-
trifluoroacetamido-1-aryl-1-propanones. (S)-Trifluoro-
acetamido-1-aryl-1-propanones were prepared by Fri-
edel–Crafts acylation of the corresponding arenes with
(S)-N-trifluoroacetylalanyl chloride, obtained in situ by
reaction of (S)-N-trifluoroacetylalanine and oxalyl
chloride, following previously reported procedures.19,22
Final compounds were purified by column chromatog-
raphy on silica gel 60, eluting with CH2Cl2. (S)-Methyl-
thio and (S)-ethylthio derivatives were recognized by
melting points and NMR spectra from the literature,22
and (S)-propylthio and (S)-butylthio derivatives were
identified by comparison with the previously reported
NMR signals for the same aromatic substitutions in
the racemic compounds.35
3. Conclusions
The results presented here show that some 4-alkylthi-
ophenylisopropylamine derivatives are able to differen-
tially inhibit MAO-A from rat and human, a behavior
that can be rationalized on the basis of differing binding
modes to each enzyme exhibited by the compounds in
docking studies. These results might be related to the
differences in the volume and shape of the active sites
of rMAO-A and hMAO-A demonstrated by structural
studies, but further experiments are necessary to fully
elucidate how these differences influence the binding of
this class of compounds to MAO-A. In addition, we
have described two binding modes, consistent with
inhibitory properties, for the interaction of substituted
amphetamine derivatives with MAO-A. It should there-
fore be kept in mind that drugs with similar activities
may not necessarily bind to their receptor(s) in the same
orientation, and that differences of this sort must be con-
sidered when analyzing SAR.
4.1.3. General procedure for the preparation of (S)-2-
trifluoroacetamido-1-arylpropanes. The (S)-trifluoroace-
tamido-1-arylpropanes were prepared following
a
previously reported procedure,20 with some modifica-
tions. To a stirred solution of (S)-2-trifluoroacetamido-
1-aryl-1-propanone (7 mmol) in trifluoroacetic acid
(3 ml) was added triethylsilane (0.55 ml, 35 mmol). The
reaction mixture was refluxed and stirred for 6 h. Final-
ly, the mixture was cooled to room temperature and
made basic with a saturated solution of NaHCO3. The
mixture was extracted with CH2Cl2 (2· 15 ml) and
washed with HCl (1 N). The organic layer was dried

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A. Fierro et al. / Bioorg. Med. Chem. 15 (2007) 5198–5206
over anhydrous Na2SO4 and concentrated by rotatory
evaporation at 40 ꢁC to afford the crude (S)-trifluoro-
acetamido-1-arylpropane. The product was purified by
column chromatography on silica gel 60, eluting with
CH2Cl2.
fluoroacetamido-1-aryl-1-propane derivatives (0.7 mmol)
were dissolved in 2-propanol (16 ml) and concentrated
HCl (12 ml). The resulting solutions were then stirred
at 70 ꢁC for 10 h. The mixtures were allowed to cool
to room temperature and the solvent was eliminated
by rotary evaporation. The solid mixtures were treated
with 1 N NaOH (15 ml) and extracted with CH2Cl2
(2· 15 ml). The organic phases were combined and
extracted with 1 N HCl and the aqueous layers were
separated and made basic with 2 N NaOH. These solu-
tions were extracted with CH2Cl2 (2· 15 ml) and the
organic phases were dried over anhydrous Na2SO4
and concentrated. The residual amines were dissolved
in dry ether (10 ml) and the hydrochlorides precipitated
with gaseous HCl.
4.1.3.1. (S)-2-Trifluoroacetamido-1-(4-methylthiophe-
nyl)-propane. Yield 90%. 1H NMR (CDCl3) 1.14 (d, 3H,
J = Hz, CHCH3), 2.40 (s, 3H, CH3S), 2.66 (dd, 1H,
Ja,b = 13.3 Hz, Ja,c = 9.1 Hz, CHaHbCHc), 2.78 (dd,
1H, Jb,a = 13.4 Hz, Jb,c = 5.1 Hz, CHaHbCHc), 4.18 (m,
1H, CHCH3), 6.04 (s, 1H, NH), 7.30 (d, 2H,
J = 8.7 Hz, ArH-3,5), 7.16 (d, 2H, J = 8.7 Hz, ArH-
2,6). 13C NMR (CDCl3) 15.9 (CHCH3), 19.4 (CH3S),
41.2 (CH2CH), 47.2 (CHCH3), 117.0 (q, COCF3),
127.0 (ArC-3,5), 129.8 (ArC-2,6), 133.5 (ArC-4), 137.1
(ArC-1), 157.0 (q, COCF3).
4.1.4.1. (S)-2-Amino-1-(4-methylthiophenyl)-propane
hydrochloride (1). 1H NMR (DMSO-d6) 1.11 (d,
3H, J = 6.6 Hz, CHCH3), 2.44 (s, 3H, CH3S), 2.64
(dd, 1H, Ja,b = 13.3 Hz, Ja,c = 9.1 Hz, CHaHbCHc),
3.02 (dd, 1H, Jb,a = 13.4 Hz, Jb,c = 5.1 Hz, CHa
HbCHc), 3.31 (br, 1H, CHCH3), 7.18 (d, 2H,
J = 8.7 Hz, ArH-3,5), 7.22 (d, 2H, J = 8.7 Hz,
ArH-2,6), 8.23 (s, 3H, NH3). 13C NMR (DMSO-
d6) 15.2 (CHCH3), 17.9 (CH3S), 39.8 (CH2CH),
48.4 (CHCH3), 126.7 (ArC-3,5), 130.3 (ArC-2,6),
4.1.3.2. (S)-2-Trifluoroacetamido-1-(4-ethylthiophe-
nyl)-propane. Yield 93%. 1H NMR (CDCl3) 1.14 (d,
3H, J = 6.6 Hz, CHCH3), 1.23 (t, 3H, J = 7.3 Hz,
CH3CH2S), 2.71 (dd, 1H, Ja,b = 13.9 Hz, Ja,c = 7.9 Hz,
CHaHbCHc), 2.79 (dd, 1H, Jb,a = 13.8 Hz, Jb,c = 7.0 Hz,
CHa HbCHc), 2.85 (q, 2H, J = 7.3 Hz, CH3CH2S), 4.18
(m, 1H, CHCH3), 6.00 (s, 1H, NH), 7.10 (d, 2H,
J = 8.1 Hz, ArH-3,5), 7.21 (d, 2H, J = 8.6 Hz, ArH-
2,6). 13C NMR (CDCl3) 14.4 (CHCH3), 19.4
(CH3CH2S), 27.8 (CH3CH2S), 41.3 (CH2CH), 47.2
(CHCH3), 115.7 (q, COCF3) 129.4 (ArC-3,5), 129.8
(ArC-2,6), 134.3 (ArC-4), 135.3 (ArC-1), 156.3 (q,
COCF3).
133.9
(ArC-4),
136.8
(ArC-1).
HRMS
m/z
181.0926, calculated for C10H15NS (MꢂHCl)+,
181.0925. Elemental analysis of this compound was
reported previously.15
4.1.4.2. (S)-2-Amino-1-(4-ethylthiophenyl)-propane
hydrochloride (2). 1H NMR (D2O) 1.26 (t, 3H,
J = 7.3 Hz, CH3CH2S), 1.28 (d, 3H, J = 6.6 Hz,
CHCH3), 2.88 (dd, 1H, Ja,b = 13.9 Hz, Ja,c = 7.3 Hz,
CHaHbCHc), 2.93 (dd, 1H, Jb,a = 13.8 Hz, Jb,c = 7.0 Hz,
CHa HbCHc) 2.98 (q, 2H, J = 7.3 Hz, CH3CH2S), 3.60
(m, 1H, CHCH3), 7.25 (d, 2H, J = 8.1 Hz, ArH-3,5),
7.38 (d, 2H, J = 8.6 Hz, ArH-2,6). 13C NMR (D2O)
16.2 (CH CH3), 20.0 (CH3CH2S), 29.5 (CH3CH2S),
42.2 (CH2CH), 51.6 (CHCH3), 131.8 (ArC-3,5), 132.8
(ArC-2,6), 136.7 (ArC-4), 137.0 (ArC-1). HRMS m/z
195.1078, calculated for C11H17NS (MꢂHCl)+,
195.1082. (C11H18ClNS): C, 57.07%; H, 8.57%; N,
6.05%; S, 15.07% (calcd: C, 57.00%; H, 7.83%; N,
6.04%; S, 13.83%).
4.1.3.3. (S)-2-Trifluoroacetamido-1-(4-propylthiophe-
nyl)-propane. Yield 90%. 1H NMR (CDCl3) 0.95 (t, 3H,
J = Hz, CH3CH2CH2S), 1.14 (d, 3H, J = Hz, CHCH3),
1.60 (m, 2H, CH3CH2CH2S), 2.75 (m, 4H, CH2CH,
CH3CH2CH2S), 4.17 (m, 1H, CHCH3), 5.98 (s, 1H,
NH), 7.02 (d, 2H, J = 8.6 Hz, ArH-3,5), 7.23 (d, 2H,
J = 8.6 Hz, ArH-2,6). 13C NMR (CDCl3) 13.4
(CHCH3), 19.4 (CH3CH2CH2S), 22.5 (CH3CH2CH2S),
35.8 (CH3CH2CH2S), 41.3 (CH2CH), 47.2 (CHCH3),
116.0 (q, COCF3) 129.3 (ArC-3,5), 129.8 (ArC-2,6),
134.2 (ArC-4), 135.7 (ArC-1), 156.6 (q, COCF3).
4.1.3.4. (S)-2-Trifluoroacetamido-1-(4-butylthiophe-
nyl)-propane. Yield 95%. 1H NMR (CDCl3) 0.84 (t,
3H, J = 7.4 Hz, CH3CH2CH2CH2S), 1.13 (d, 3H,
J = 6.6 Hz, CHCH3), 1.37 (m, 2H, CH3CH2CH2CH2S),
1.53 (m, 2H, CH3CH2CH2CH2S), 2.68 (dd, 1H,
Ja,b = 13.9 Hz, Ja,c = 7.4 Hz, CHaHbCHc), 2.77 (dd,
1H, Jb,a = 13.4 Hz, Jb,c = 7.0 Hz, CHaHbCHc), 2.84 (t,
2H, J = 7.2 Hz, CH3CH2CH2CH2S), 4.18 (m, 1H,
CHCH3), 6.00 (s, 1H, NH), 6.99 (d, 2H, J = 8.5 Hz,
ArH-3,5), 7.20 (d, 2H, J = 8.5 Hz, ArH-2,6). 13C
4.1.4.3. (S)-2-Amino-1-(4-propylthiophenyl)-propane
hydrochloride (3). 1H NMR (D2O) 0.96 (t, 3H,
J = 7.3 Hz, CH3CH2CH2S), 1.28 (d, 3H, J = 6.6 Hz,
CHCH3), 1.62 (m, 2H, CH3CH2CH2S), 2.94 (t, 2H,
J = 7.3 Hz, CH3CH2CH2S), (m, 4H, CH2CH,
CH3CH2CH2S) 3.60 (m, 1H, CHCH3), 7.24 (d, 2H,
J = 8.6 Hz, ArH-3,5), 7.37 (d, 2H, J = 8.6 Hz, ArH-
2,6). 13C NMR (D2O) 15.2 (CHCH3) 20.0
NMR
(CDCl3)
13.6
(CHCH3),
19.4
(CH3CH2CH2CH2S), 22.0 (CH3CH2CH2CH2S), 31.2
(CH3CH2CH2CH2S), 33.4 (CH3CH2CH2CH2S), 41.3
(CH2CH), 47.2 (CHCH3), 115.7 (q, COCF3) 129.2
(ArC-3,5), 129.8 (ArC-2,6), 134.1 (ArC-4), 135.8 (ArC-
1), 156.0 (q, COCF3).
(CH3CH2CH2S)
24.6
(CH3CH2CH2S),
37.4
(CH3CH2CH2S) 42.2 (CH2CH), 51.6 (CHCH3), 131.8
(ArC-3,5) 132.8 (ArC-2,6) 136.6 (ArC-4) 137.2 (ArC-
1). HRMS m/z 209.1240, calculated for C12H19NS
(MꢂHCl)+, 209.1238. (C12H20ClNS): C, 57.77%; H,
9.01%; N, 5.56%; S, 14.44% (calcd: C, 58.63%; H,
8.20%; N, 5.70%; S, 13.04%).
4.1.4. General procedure for the preparation of (S)-2-
amino-1-arylpropane hydrochlorides (1–4). The (S)-2-tri-

A. Fierro et al. / Bioorg. Med. Chem. 15 (2007) 5198–5206
5205
4.1.4.4.
(S)-2-Amino-1-(4-butylthiophenyl)-propane
without inhibitors) versus ꢂlog inhibitor concentration.
Ki were determined from the IC50 values using the
Cheng–Prusoff equation: Ki = IC50/(1+[S]/Km).36
hydrochloride (4). 1H NMR (D2O) 0.85 (t, 3H,
J = 7.4 Hz, CH3CH2CH2CH2S), 1.26 (d, 3H,
J = 6.6 Hz, CHCH3), 1.38 (m, 2H, CH3CH2CH2CH2S),
1.56 (m, 2H, CH3CH2CH2CH2S), 2.86 (dd, 1H,
Ja,b = 13.9 Hz, Ja,c = 7.4 Hz, CHaHbCHc), 2.91 (dd,
1H, Jb,a = 13.4 Hz, Jb,c = 7.0 Hz, CHaHbCHc), 2.95 (t,
2H, J = 7.2 Hz, CH3CH2CH2CH2S), 3.58 (m, 1H,
CHCH3), 7.22 (d, 2H, J = 8.5 Hz, ArH-3,5), 7.36 (d,
2H, J = 8.5 Hz, ArH-2,6). 13C NMR (D2O) 15.5
4.2.2. Human MAO. Human recombinant MAO-A and
MAO-B were expressed in Pichia pastoris and purified
as described previously.37,38 Both enzymes (1–2 mg)
were desalted from glycerol stock solutions using a
G-25 (fine) Sephadex column (1 · 20 cm, Sigma) in
50 mM potassium phosphate buffer, pH 7.5, containing
0.8% (w/v) b-octyl-glucoside before use. Enzymatic
activity measurements were carried out as previously de-
scribed.39 Briefly, hMAO-A and hMAO-B activities
were determined spectrophotometrically, in the presence
or absence of inhibitors, using kynuramine and benzyl-
amine (purchased from Sigma) as substrates, respec-
tively, in 50 mM Hepes buffer, pH 7.5, containing
0.5% (w/v) reduced Triton X-100, at 25 ꢁC. The oxida-
tion of kynuramine catalyzed by MAO-A was moni-
tored at 316 nm (absorption maximum for aldehyde
product, extinction coefficient = 11,800 Mꢂ1 cmꢂ1), and
that of benzylamine by MAO-B was monitored at
250 nm (maximum absorption by benzaldehyde, extinc-
tion coefficient = 12,800 Mꢂ1 cmꢂ1). All spectral data
were obtained using a Perkin-Elmer Lambda 2 UV–vis
double-beam spectrophotometer with thermostatted
cuvette holders. Kinetic data were evaluated and plotted
using Microcal Origin or Prism Graph Pad software.
(CHCH3),
20.0
(CH3CH2CH2CH2S),
23.9
(CH3CH2CH2CH2S), 33.2 (CH3CH2CH2CH2S), 35.0
(CH3CH2CH2CH2S), 42.2 (CH2CH), 51.6 (CHCH3),
131.8 (ArC-3,5), 132.7 (ArC-2,6), 136.6 (ArC-4), 137.3
(ArC-1). HRMS m/z 223.1392, calculated for
C13H21NS (MꢂHCl)+, 223.1395. (C13H22ClNS): C,
59.10%; H, 9.39%; N, 5.26%; S, 14.03% (calcd: C,
60.09%; H, 8.53%; N, 5.39%; S, 12.34%).
1
4.1.5. Determination of the enantiomeric excess by H
NMR Using (Eu(hfc)3) as chiral shift reagent. Trifluoro-
acetamides, suitable for the 1H NMR analysis in CDCl3,
were prepared from the (S)-2-amino-1-(4-alkylthiophe-
nyl)-propane hydrochlorides. Trifluoroacetic anhydride
(0.1 ml) was added separately to suspensions of 5 mg
(0.022 mmol) of (R,S) and (S)-2-amino-1-(4-alkylthi-
ophenyl)-propane hydrochlorides in CH2Cl2. The reac-
tion mixtures were stirred for 1 hour and rotary
evaporated to give solid (R,S) and (S)-2-trifluoroacetam-
ido-1-(4-alkylthiophenyl)-propane, respectively. To each
solid, 0.5 equiv of Eu(hfc)3 was added and the complexes
were dissolved in CDCl3 for analysis. Inspection of the 1H
NMR signals indicated that in the case of the (S)-enanti-
omers, the methyl group linked to the asymmetric center
gave a doublet in the spectrum with a chemical shift rela-
tive to TMS close to 1.44 ppm, whereas the racemic mix-
ture gave rise to two partially overlapping doublets.
4.3. Molecular simulation
The crystallographic data of rMAO-A or hMAO-A
(PDB: 1O5W or 2BXS, respectively) were used for all
calculations. The Autodock 3.05 suite25 was then used
to perform the docking simulations and conditions were
as previously reported,27,35 with some modifications.
Briefly, the hydrogen atoms of the protein and the
FAD molecule were built using Insight II,40 and the
structures were relaxed following a minimization proto-
col using Discover_3 and the ESFF force field. The grid
maps were calculated using the autogrid3 option and
were centered on the putative ligand-binding site. The
volumes chosen for the grid maps were made up of
40 · 40 · 40 points, with a grid-point spacing of
4.2. Biochemistry
4.2.1. Rat MAO. All the chemicals used were of the
highest grade commercially available. Tetrahydrofuran
and acetonitrile were of Merck HPLC grade. 5-HT,
5-hydroxyindoleacetic acid, yeast aldehyde dehydroge-
nase, and b-nicotinamide dinucleotide were from Sigma
(St. Louis, MO, USA). The effects of the enantiomers on
MAO-A or MAO-B activities were studied using a crude
rat brain mitochondrial suspension (male Sprague–
Dawley rats weighing 180–220 g, sacrificed by decapita-
tion), using 5-HT (100 lM) and 4-dimethylaminophen-
ethylamine (DMAPEA, 5 lM) as selective substrates
for MAO-A and -B, respectively, and detecting these
compounds and their metabolites by HPLC with elec-
trochemical detection (HPLC-ED) as described previ-
ously.10 A C18 reverse phase column (Lichrospher
250 mm · 4.6 mm, 5 lm) and an amperometric detector
(Merck-Recipe L3500A) were used to analyze the reac-
tion mixtures. All other conditions were as previously
described.10,15 IC50 values (mean SD from at least
two-independent experiments, each in triplicate) were
determined using Prism Graph Pad software, from plots
of inhibition percentages (calculated in relation to a
sample of the enzyme treated under the same conditions
˚
0.375 A. The partial charges of 4-alkylthiophenylisopro-
pylamine derivatives 1–4 were corrected using RESP
methodology.41 The dielectric constant was adjusted
either to 10 (hMAO-A) or to 2 (rMAO-A) in the grid
parameter file (gpf) of the Autodock suite. This param-
eter adjustment gave the best correlations between the
calculated docking energies and biochemical results.
The docked compound complexes were built using the
lowest free-energy binding positions.
Acknowledgments
This work was partially funded by FONDECYT Grant
1060199 and DICYT Grant 020591RPRP. D.E.E.
acknowledges research support from the National Insti-
tutes of Health (GM29433). The compounds described
were synthesized by MOO during a stay in Prof. E. Bre-
itmaier’s laboratory at the University of Bonn as the re-

5206
A. Fierro et al. / Bioorg. Med. Chem. 15 (2007) 5198–5206
18. Chimenti, F.; Bolasco, A.; Manna, F.; Secci, D.; Chimenti,
cipient of a DAAD grant. We also thank Dr. Juan
Guerrero for his assistance in the NMR spectroscopy
with the chiral shift reagent, Ms. Milagros Aldeco for
the preparation of purified recombinant human MAO
A and MAO B, and Drs. G. Zapata-Torres and C. Mas-
cayano and Prof. M. C. Rezende for their contribution
to the computational work. A.F. was the recipient of
CONICYT and MECESUP scholarships.
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