Synthesis of 4-methyl-thio-phenyl-propylamine and the evaluation of its interaction with different amine oxidases

Article abstract of DOI:10.1016/j.bmc.2003.10.007

A new molecule, the 4-methyl-thio-phenyl-propylamine (PrNH₂) was synthesized and its biological interaction with different amine oxidases such as semicarbazide sensitive amine oxidase (SSAO) [E.C.1.4.3.6], and monoamine oxidase [E.C.1.4.3.4] un

Full text of DOI:10.1016/j.bmc.2003.10.007

Bioorganic & Medicinal Chemistry 12 (2004) 273–279
Synthesis of 4-methyl-thio-phenyl-propylamine and the
evaluation of its interaction with different amine oxidases
Alejandra Gallardo-Godoy, Mar Hernandez, Elisenda Sanz and Mercedes Unzeta*
´
Institut de Neurociencies-Departament de Bioquımica i Biologia Molecular, Facultat de Medicina,
`
Universitat Autonoma de Barcelona, Campus Universitari de Bellaterra, E-08193 Bellaterra, Barcelona, Spain
´
Received 16 April 2003; accepted 1 October 2003
Abstract—A new molecule, the 4-methyl-thio-phenyl-propylamine (PrNH2) was synthesized and its biological interaction with dif-
ferent amine oxidases such as semicarbazide sensitive amine oxidase (SSAO) [E.C.1.4.3.6], and monoamine oxidase [E.C.1.4.3.4]
under its two isoforms, MAO A and MAO B, has been assessed. The substrate specifities of MAO and SSAO overlap to some
extent. In this context, the search of new molecules, able to discriminate between these different amine oxidases is very important as
it will allow greater elucidation of the SSAO’s role in physiological and pathological conditions. We report for the first time, the
synthesis and evaluation of a new molecule which has a high affinity towards the SSAO family of enzymes, more so than previously
described and furthermore an ability to discriminate between the different amine oxidases.
# 2003Elsevier Ltd. All rights reserved.
1. Introduction
and does not interact with MAO. Both enzymes catalyse
the oxidative deamination of amines to ammonia,
hydrogen peroxide and the corresponding aldehyde,
according to the overall reaction:
The term semicarbazide sensitive amine oxidase (SSAO)
is used to describe a group of enzymes that catalyse the
oxidative deamination of aliphatic and aromatic primary
amines. They are classificated as E.C.1.4.3.6 (amine:
oxygen oxidoreductase (deaminating) (copper-contain-
ing), and all of them have 2,4,5-trihydroxyphenylalanine
(TPQ) as cofactor. Semicarbazide is frequently used to
distinguish the SSAOs from the monoamine oxidase,
[amine: oxygen oxidoreductase (deaminating) (flavin-
containing), E.C.1.4.3.4; MAO], that contains flavin
adenine dinucleotide (FAD) as cofactor and are sensitive
to acetylenic inhibitors such as clorgyline and l-deprenyl,
but are not affected by semicarbazide. The substrate spe-
cificities of MAO-A and SSAO overlap to some extent
but, whereas MAO catalyses the oxidative deamination
of primary, secondary and tertiary amines, SSAO
activity appears to be restricted to primary amines. At
present methylamine, which arises from the metabolism
of adrenaline, lecithin, sarcosine, and creatinine, is the
only physiological substrate known that can dis-
criminate between both amine oxidases. Methylamine is
exclusively metabolised by SSAO from many sources1,2
RCH2NH2 þ O2 þ H2O ) þRCHO þ NH3 þ H2O2
SSAO is tightly associated with membranes in several
mammalian tissues and also occurs as a soluble form in
blood plasma.3,4 The membrane-bound SSAO shows
high activity in vascular tissue and it appears to be
associated with smooth muscle cells.5,6 SSAO activity
has been also found in other non-vascular cell types
such as chondrocytes,7 bovine eye,8 in dental pulp,9 and
in adipocytes from rat white and brown fat.10 The
physiological role of SSAO is still far from clear, despite
its wide distribution in mammalian tissues.11 SSAO
located in the plasma membrane may act as a scavenger
of circulating toxic amines and in this context, the
enzyme present in the microsomal fraction of human
and bovine lung12,13 may be important in inhaled vola-
tile amines metabolism.14 This enzyme could also be
involved in the control of cellular activities through the
generation of H2O2.15 It has been recently reported that
SSAO co-localizes with GLUT4 glucose transporter in
endosomal compartment in rat adipocytes and that
SSAO substrates cause a marked stimulation of glucose
transport, mimicking the effects of insulin.16
Keywords: 4-Methyl-thio-phenyl-propylamine; Synthesis; Biological
evaluation; Amine oxidases inhibitor; SSAO; MAO.
* Corresponding author. Tel.: +34-93-581-1523; fax: +34-93-581-
1573; e-mail: mercedes.unzeta@uab.es
0968-0896/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.007

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A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 12 (2004) 273–279
The vascular-adhesion protein VAP-1, present in endo-
thelial cells, has been shown to have an identical amino-
acid sequence to SSAO.17 Because VAP-1 is induced in
inflammation and it is involved in lymphocyte migration
to lymphoid organs, a new physiological role in cellular
trafficking has been proposed for SSAO.18 In this con-
text, design and synthesis of new compounds that
behave as specific SSAO substrates or inhibitors could
be useful for discriminating between SSAO and MAO.
Furthermore the search of new substrates with high
affinity towards SSAO may have therapeutic value in
diabetes, due to its importance in glucose transport
mimicking insulin effects.
Elemental analyses (C, H, N) were carried out using
Perkin-Elmer 240 B or 240 C instruments and the
results were within ꢀ0.4% of the calculated values.
Thin-layer chromatography (TLC) was performed
under standard conditions using silica gel 60-F254 plates,
0.2 mm thickness (Merck). Abbreviations for the fol-
lowing solvents were used: MeCN, acetonitrile; CH2Cl2,
dichloromethane; Et2O, diethyl ether; EtOH, ethanol;
IPA, propan-2-ol; MeOH, methanol; py, pyridine;
THF, tetrahydrofuran.
2.2.1. 3-(4-Methylthiophenyl)-acrylonitrile (1). To a sus-
pension of KOH (1.5 g, 26.7 mmol; fine powder) in
20 mL of acetonitrile was added 4-methylthiobenzalde-
hyde (4.0 g, 26.0 mmol). The reaction mixture was heated
at reflux for 15 min with magnetic stirring. The mixture
was poured onto 50 g of crushed ice and the organic layer
separated. After washing with water, the organic layer
was removed under reduced pressure resulting in a thick
yellow oil, which was purified using bulb to bulb dis-
tillation (145–150 ꢁC/0.02 Torr) to give 3.4 g (75% yield)
Starting from the 4-methyl-thio-amphetamine (4-MTA),
a high selective and reversible MAO-A inhibitor and
poor MAO-B inhibitor,19 we have studied the effect of
side-chain enlargement on its amine oxidase inhibitory
potency. Here, we report by the first time, the design and
synthesis of a new molecule, the 4-methyl-thio-phenyl-
propylamine (PrNH2), and its biological evaluation as
inhibitor or substrate of different amine oxidases.
1
of an E/Z mixture in a 4:1 ratio (by H NMR analysis).
1H NMR (CDCl3): d 7.40 (d, J=8.4 Hz, 2H, Ph-3-H, Ph-
5-H), 7.30 (d, J=12.6 Hz, 1H, PhCHCHCN), 7.20 (d,
J=8.4 Hz, 2H, Ph-2-H, Ph-6-H), 5.80 (d, J=12.6 Hz,
1H, PhCHCHCN), 2.50 (s, 3H, CH3S).
2. Results
2.1. Synthesis
2.2.2. 3-(4-Methylthiophenyl)-propionitrile (2). To a stir-
red solution of 1 (1.0 g, 5.7 mmol) in 10 mL of pyridine
and 3mL of MeOH was added in portions, NaBH 4
(0.22 g, 5.7 mmol). The reaction mixture was heated at
reflux for 3h. After cooling it was poured into 100 mL
of 10% v/v HCl in ice water. The solution was extracted
with 2 ꢂ 50mL CH2Cl2, the organic layer separated and
the solvent removed under reduced pressure affording a
colorless oil which was purified using bulb to bulb dis-
tillation (123–125ꢁC/0.02Torr) to give 700 mg (70%
yield) of the title compound. 1H NMR (CDCl3): d 7.24 (d,
J=8.4Hz, 2H, Ph-3-H, Ph-5-H), 7.15 (d, J=8.4 Hz, 2H,
Ph-2-H, Ph-6-H), 2.91 (t, J=7.6 Hz, 2H, PhCH2CH2CN),
2.60 (t, J=7.6 Hz, 2H, PhCH2CH2CN), 2.47 (s, 3H,
CH3S). Anal. calcd for C10H11NS: C, 67.76; H, 6.25; N,
7.90; S, 18.09. Found: C 68.05; H 6.11; N, 7.84; S, 18.16.
Scheme 1 shows the route used to prepare the novel
compound (3), starting from 4-methylthiobenzaldehyde,
following the method published for the synthesis of
p-methoxy-cinnamonitrile.20 The condensation of ace-
tonitrile with the aldehyde, catalyzed by powdered
KOH, afforded a 4:1 mixture of (E)- and (Z)-4-methyl-
thio-cinnamonitrile, which was used without the
separation of isomers. Although there are published
routes21,22 for the synthesis of intermediate 1, this
method was chosen for the simplicity and low cost of
the route. Direct reduction of compound 1 by LiAlH4
gave none of the desired product, and therefore a two-
step sequence using NaBH4 and LiAlH4 successively
was used, giving 3 (via 2) in very good overall yield.
2.2. Chemistry, general procedures
2.2.3. 3-(4-Methylthiophenyl)-propylamine (PrNH2). A
solution of 2 (1.0g, 5.6 mmol) in 5 mL of THF was added
dropwise to a suspension of LiAlH4 (0.5g, 13.0 mmol) in
10mL of freshly distilled THF. The reaction mixture was
heated at reflux for 6 h. The excess LiAlH4 was decom-
posed by successive addition of 0.5 mL of distilled water,
0.5 mL 15% w/v NaOH and 1.5 mL of distilled water. The
cake was filtered and washed with 3 ꢂ 10mL of THF. The
solvent was removed under reduced pressure and the resi-
due was purified by bulb to bulb distillation (110–115 ꢁC/
0.02Torr). The resulting oil was crystallized as the hydro-
chloride in IPA/Et2O to give 300 mg of white microcrystals
1H NMR spectra were recorded on a Bruker ARX 300
spectrometer. Chemical shifts are expressed in parts per
million downfield from internal Me4Si as a reference. 1H
NMR data are reported in the order: multiplicity (s,
singlet; d, doublet; t, triplet; m, multiplet; number of
protons, and approximate coupling constant in Hz).
1
(61% yield). H NMR (D2O): d 7.29 (d, J=8.4 Hz, 2H,
Ph-3-H, Ph-5-H), 7.21 (d, J=8.4 Hz, 2H, Ph-2-H, Ph-6-
H), 2.95 (t, J=7.6 Hz, 2H, PhCH2CH2CH2NH2), 2.65 (t,
J=7.6Hz, 2H, PhCH2CH2CH2NH2), 2.45 (s, 3H, CH3S),
1.88–1.90 (m, 4H, PhCH2CH2CH2NH2). Anal. calcd for
C10H15NS.HCl: C, 55.16; H, 7.41; N, 6.43; S, 14.73.
Found: C 55.15; H 7.39; N, 6.48; S, 14.81.
Scheme 1. (a) KOH, MeCN, reflux; (b) NaBH4, MeOH, py, reflux;
(c) (i) LiAlH4, THF, reflux; (ii) HCl, IPA, Et2O.

A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 12 (2004) 273–279
275
2.3. Biology
B inhibitor, IC50=3ꢂ10ꢃ8 M) than by PrNH2, whereas
in case of SSAO, it showed similar behaviour towards
semicarbazide (specific SSAO inhibitor, IC50=10ꢃ4 M)
and PrNH2. When the assays were repeated with a pre-
vious incubation of 30 min, MAO A and MAO B resul-
ted to be no time-dependent inhibited, whereas in case of
SSAO a time-dependent inhibition was observed.
2.3.1. Inhibitory behavior of PrNH2 versus different
amine oxidases. MAO-A and MAO-B activities from
rat liver mitochondria were assayed at the 0-min pre-
incubationin at 37 ꢁC, in the presence of different con-
centration of 4-methyl-thio-phenyl propylamine
(PrNH2) [10ꢃ8–10ꢃ3 M], and the activity remaining was
measured against [14C]5-HT, (100 mM), serotonin, a
specific MAO-A substrate, and against [14C] PEA
(22 mM), a specific MAO-B substrate, respectively (see
Table 1). In the case of SSAO, bovine lung microsomes
were used, and the activity remaining was determined
towards (50 mM) [14C] benzylamine as substrate. Figure
1 shows the inhibition curve of MAO A by PrNH2 as
representative of the inhibition curves of the rest of the
oxidases. The IC50 values were determined from the
corresponding inhibition curves and this new molecule
showed the highest inhibitory potency towards MAO-A
(IC50=5.8 mM), followed by SSAO (IC50=38.7 mM)
and the lowest inhibition was observed with MAO-B
(IC50=175 mM). PrNH2, showed a high inhibitory
selectivity towards MAO-A, expressed in terms of IC50
MAO-A/IC50 MAO-B (0.033) and a high selectivity
towards SSAO compared with MAO-B (IC50 SSAO/
IC50 MAO-B=0.22). However, these enzymes were
more potently inhibited by clorgyline (specific MAO A
inhibitor, IC50=10ꢃ8 M) and l-deprenyl, (specific MAO
2.3.2. Kinetic behavior of PrNH2 as substrate of MAO-
A, MAO-B and SSAO. Since PrNH2 was an amine, it
might potentially behave as a substrate of any amine
oxidases. Mitochondria from rat liver that contains
both MAO-A and MAO-B, were pretreated by deprenyl
10ꢃ6 M or clorgyline 10ꢃ6 M in order to inhibit MAO B
and MAO A, respectively, and avoid interferences
between them. When PrNH2 (15–500 mM) was assayed
as MAO A substrate, identical progress curves were
obtained at any PrNH2 concentrations used (data not
shown), suggesting that this compound was not a
MAO-A substrate. When PrNH2 was assayed as possi-
ble MAO-B substrate, the results showed that PrNH2
was oxidized by the enzyme. The kinetic parameters
were calculated by non-lineal regression analysis, and
they resulted to be Km=91.5 mM and Vmax=1742 pmol/
min (see Fig. 2). The catalytic efficacy of MAO-B
towards PrNH2, expressed as Vmax/Km was 19.04. When
SSAO bovine lung microsomes12,13 were assayed, a con-
centration range of PrNH2 between 1 and 30 mM was
used. The kinetic parameters were Km=8.35 mM
Vmax=2669 pmol/min (see Fig. 3) and the catalytic effi-
cacy was (Vmax/Km) 319.6 pmol/min mM. Table 2 shows
all the kinetic parameters for PrNH2 in comparison with
the common MAO A and MAO B substrates (5HT for
MAO A and PEA for MAO B, respectively23). Thus,
SSAO had the highest activity towards PrNH2 as a
substrate, which was oxidised more slowly by MAO B
and not at all by MAO A. In this regard, it is worth to
remark that PrNH2 resulted to be the best exogenous
substrate for SSAO yet discovered, with a Km=8.35 mM
versus benzylamine, the commonly used SSAO sub-
strate (Km=50 mM). Furthermore, the catalytic efficacy
Vmax/Km of SSAO versus PrNH2 as substrate, was also
higher than for benzylamine as substrate.
Table 1. Chemical structures of b-phenylethylamine (PEA), 5-OH
tryptamine (5HT), benzylamine (Bz), and PrNH2
Figure 1. Amine oxidases inhibition by different concentration of
PrNH2 by measuring the remaining activity towards 100 mM 14C-5HT
(MAO A), 20 mM 14C PEA (MAO B) and 50 mM 14C Benzylamine
(SSAO) as substrates. The data are expressed in percentage of control
activities determined in absence of PrNH2 as mean ꢀSD of three
independent experiments performed in duplicate with error bar repre-
senting the standard deviation.
Figure 2. Double reciprocal plot of MAO B activity towards PrNH2
as substrate. The concentration range used was (0–500 mM). MAO B
activity was measured as described in the Experimental. Each value is
the mean ꢀSD of two independent experiments performed in tripli-
cate, with error bars representing the standard deviation.

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A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 12 (2004) 273–279
of SSAO and a relatively weak MAO-B inhibitor. The
IC50 determined at 0 and 30 min pre-incubation time,
suggested a reversible inhibitory behaviour in case of
both MAO isoforms and a time-dependent inhibition in
case of SSAO. PrNH2 had 4 times higher selectivity
towards SSAO compared to MAO-B (IC50 MAO-B/
IC50 SSAO). PrNH2 had a 30 times lower inhibitory
potency towards MAO-A than 4 MTA (IC50=0.2 mM),
whereas the inhibitory potency towards MAO-B19 was
higher. These results suggest that the rearrangement and
elongation of the side chain, results in a loss of affinity
towards MAO-A, and an increasing affinity towards
MAO-B. When PrNH2 was assayed as a possible sub-
strate, it proved to be a very good SSAO substrate fol-
lowed by MAO-B, however this molecule was not
metabolised by MAO-A at all. Considering the kinetic
behaviour of PrNH2 as a substrate, SSAO showed 11
times higher activity than MAO-B in terms of Km
values, and 1.5 times higher Vmax values. Consequently
the catalytic efficacy of SSAO towards PrNH2, expres-
sed in terms of Vmax/Km was 319.6 versus 19 in the case
of MAO-B. When the kinetic behaviour of PrNH2 was
assayed as a MAO-A inhibitor, it behaved as a potent
and reversible mixed-type inhibitor, indicating that
PrNH2 is able to bind whether to the enzyme–substrate
complex or to the free enzyme at a different binding site
of the active site.
Figure 3. Double reciprocal plot of SSAO activity towards PrNH2 as
substrate. The concentration range used was (0–40 mM). SSAO activity
was measured as described in the Experimental. Each value is the
mean ꢀSD of two independent experiments performed in triplicate,
with error bars representing the standard deviation.
2.3.3. Kinetic behavior of PrNH2 as inhibitor of MAO-
A. PrNH2 was a mixed-type inhibitor of rat liver mito-
chondrial MAO-A, as shown in Figure 4. Plots of 1/v
versus PrNH2 concentration (Dixon, 1953)24 (see Fig. 4
insert), confirmed a mixed-type inhibition. The Kic and
the Kiu values were calculated from the equation Vapp
¼
V=1 þ i=Kiu and Kmapp ¼ Kmð1 þ i=KicÞ=1 þ i=Kiu. The
Kiu corresponds to the inhibition constant when the
PrNH2 binds to the complex ES, and its value resulted
to be 13.35 mM, whereas Kic expresses the inhibition
constant of PrNH2 when binds to the free enzyme and
showed to be 2.76 mM.
The active sites of different known Cu-amine oxidases
are structurally very similar. The TPQ cofactor exhibits
different degrees of mobility and it is clearly flexible in
all cases. Asp 383 in the active site performs multiple
roles in stabilizing the TPQ in an off-copper conforma-
tion and assisting substrate binding to TPQ and
abstracting the C-H proton from the substrate.29
Recently, some authors30 have reported the structure of
human MAO-B. The structure of the enzyme shows a
flat cavity lined with aromatic and aliphatic aminoacids
that constitutes the substrate binding site providing a
highly hydrophobic environment. Located between the
active site and the protein surface, there is another
smaller, hydrophobic cavity. Some residues constitute a
loop that separates both cavities and this loop allows
the substrate to access to the active site through the
small entrance cavity.
3. Discussion
Several phenylisopropylamines, including ampheta-
mine, have been evaluated as monoamine oxidase inhi-
bitors.25 Substituents on the aromatic ring of the
phenylisoproprylamine molecule (in particular at the
para position), such as amino,26 halogens27 and alkyl-
thio groups,19 lead to an increase in the potency and
selectivity towards MAO-A compared with the parent
compound, amphetamine.
In the present work, we have studied the effect of the
side-chain rearrangement and elongation of 4-methyl-
thioamphetamine (4-MTA),19 a high potent MAO-A
inhibitor, and highly selective non-neurotoxic serotonin
releasing and uptake blocking agent.28 The IC50 values
showed that this new molecule, had high potency as an
inhibitor of MAO-A. It was less potent as an inhibitor
Despite these structural differences in the active site of
both types of amine oxidases, the two MAO isoforms
and SSAO, all of them have in common an ability to
recognize hydrophobic structures in their substrates.31
The present data reported herein on the synthesis and
biological evaluation of PrNH2, shows that this mole-
Table 2. Kinetic parameters of MAO A, MAO B and SSAO towards Pr NH2, PEA, Bz and 5Htas substratesa
>
Pr NH2
PEA
5-HT
Bz
Km
Vmax
Vmax/Km
Km
Vmax
Vmax/Km
Km
Vmax
Vmax/Km
Km
Vmax
Vmax/Km
MAO A
MAO B 91.5ꢀ91 1742ꢀ180 19
SSAO 320
8.35ꢀ76 2669ꢀ38
84.2ꢀ5.9 5467ꢀ249 164
9.7ꢀ0.5 5057ꢀ12 522
312ꢀ13980 ꢀ99 3.14
40ꢀ32010 ꢀ165 50.25
Km: mM; Vmax: pmols/min/mg prot.
a Each value is the meanꢀSD of three independent experiments performed in duplicate.

A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 12 (2004) 273–279
277
Figure 4. Double reciprocal plot of MAO A inhibition by PrNH2. The PrNH2 concentration range used was (1–10mM). MAO A activity was measured
towards Kynuramine as substrate as stated in the Experimental. Each value is the mean ꢀSD of two independent experiments performed in triplicate,
with error bars representing the standard deviation. Insert: the Dixon plot (1/v versus inhibitor concentration) of MAO A by PrNH2 as inhibitor.
cule is able to interact with different types of amine
oxidases. The presence of a para methyl-thio group, is
responsible for its MAO-A inhibitory behaviour, how-
ever the rearrangement of the alpha-methyl group in the
linear side chain, results in some loss of its inhibitory
potency towards this MAO isoform. On the other hand,
its hydrophobicity and the presence of the primary
amine group on the side chain, confirm that PrNH2 is a
good MAO-B and SSAO substrate. However, this mol-
ecule shows better affinity towards SSAO than MAO-B,
so it allows discriminating both enzymatic activities,
although the amino propynyl side chain probably fits
much better in the active site of SSAO than in the MAO-
B’s. The existence in the human MAO B structure30 of an
entrance cavity, which connects the surface of the protein
to the hydrophobic substrate cavity, suggests that
PrNH2, could find some structural restrictions in the
entry to the first small entrance cavity that could not
allow the long side chains to pass to the real active site.
the side-chain 2-[14C]-5-hydroxy tryptamine sulphate
(serotonin) (55 mCi/mmol) (0.2 mCi/mL) and ethyl-2-
[14C phenylethylamine HCl (57 mCi/mmol) (0.2 mCi/
mL) were supplied by Amersham International.
l-Deprenyl was from Research Biochemical Interna-
tional (R.B.I., USA) and kynuramine dihydrobromide
and other common reagents, were from Sigma-
Aldrich.
4.2. Preparation of bovine lung microsomes
Bovine lung was obtained from the abattoir after
slaughter, packed in ice and transported immediately to
the laboratory. After removal of the connective tissue,
the lung was weighed, chopped into small pieces with
scissors and washed extensively with saline (0.9%, w/v,
NaCl) to eliminate blood as a potential source of con-
taminating plasma amine oxidase. The tissue was then
homogenised in 1:10 (w/v) of 20 mM Tris/HCl buffer,
pH 7.2, containing 0.25 M sucrose, in a Waring blender,
and filtered through two layers of cheesecloth. The
homogenate was subjected to differential centrifugation
and the microsomal fraction was obtained by adding
10 mM CaCl2 to the post-mitochondrial supernatant
and centrifugation as previously described.32 The final
microsomal pellet was resuspended in 50 mM potassium
phosphate buffer, pH 7.2, the protein concentration was
adjusted to 10 mg/mL and samples were stored in ali-
quots at ꢃ20 ꢁC until required. The resulting prepara-
tion, referred to as crude microsomes, contained the
SSAO enzyme.
The design and synthesis of new substrates or inhibitors
capable to discriminate between different amine oxi-
dases, is very important to elucidate the SSAO role in
physiological conditions and to develop new therapies
for SSAO related disorders.
4. Experimental
4.1. Materials
The radioactive substrates [7-14C]-benzylamine hydro-
chloride (specific activity 57 mCi/mmol), (0.2 mCi/mL),

278
A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 12 (2004) 273–279
4.3. Preparation of rat liver mitochondria
was proportional to the amount of hydrogen peroxide
released during the amine oxidase reaction. The extinc-
tion coefficient is 4654 Mꢃ1 cmꢃ1. Kinetic parameters
were calculated by linear-regression analysis using the
Rat livers were recovered from male Sprague–Dawley
rats (weighing 200–250 g) which had been fasted for
12 h. The liver homogenates were prepared in 10 vol
(w/v) of a 50 mM potassium phosphate buffer (pH 7.2)
by use of a Dounce homogenizer. The mitochondrial
fraction was prepared by a standard differential cen-
trifugation method.33 The pellets were resuspended in
the same buffer and frozen as small aliquots at ꢃ20 ꢁC
until required.
Graph-Pad Prism-3program. When PrNH
was
2
assayed as a MAO-B substrate, rat liver mitochondria
was previously inhibited by clorgyline (10ꢃ6 M) in order
to leave only MAO-B activity in the assay mixture. In
order to assay PrNH2 as a SSAO substrate, MAOB was
previously inhibited by preincubation with deprenyl
(10ꢃ6 M).
4.4. Radiochemical assays
4.6. Kinetic behavior of PrNH2 as a MAO-A inhibitor
SSAO activity in bovine lung microsomes, was deter-
mined radiochemically, by the method of Fowler and
Tipton,34 using 25 mL of [14C]-benzylamine (3mCi/
mmol) (50 mM) in the assay mixture. The reaction was
carried out at 37 ꢁC in a final volume of 200 mL of
50 mM potassium phosphate buffer, pH 7.2, and the
incubation was stopped by the addition of 100 mL of 2 M
citric acid. Radioactive-labeled products were extracted
into toluene/ethyl acetate (1:1, v/v) containing 0.6%
(w/v) 2,5-diphenyloxazole (PPO) before liquid scintilla-
tion counting. In the case of MAO-A and MAO-B, [14C]-
serotonin (100 mM) and [14C phenylethylamine (22 mM)
were used as specific substrates, respectively.
PrNH2 was assayed as a MAO-A inhibitor and the
kinetic parameters determined using the spectro-
photometric method with kynuramine as substrate in
the presence of different PrNH2 concentration
(1–10 mM). Spectrophotometric assays for MAO-A
activities were performed at 37 ꢁC using kynuramine
(40 mM) as substrate, in 50 mM potassium phosphate
buffer (pH 7.2) containing 500 mM of mitochondrial
preparation in a total volume of 3mL. The appearance
of the product was measured at 324 nm35 and the
absorbance coefficient at this wavelength was
20,000 Mꢃ1 cmꢃ1. Since kynuramine is a common sub-
strate of both MAO forms, it was necessary to inhibit
MAO-B with l-deprenyl (10ꢃ6 M) to ensure that only
MAO-A activity was present.
Selective inhibition of SSAO without affecting MAO-B
activity was achieved pre-incubating the enzyme with
1 mM semicarbazide for 30 min at 37 ꢁC before the
addition of the substrate. MAO-B activity was selectively
inhibited, by preincubating the bovine lung microsomes
with 10ꢃ6 M of l-deprenyl for 30 min at 37 ꢁC, before
adding the substrate. Time course assays were used to
ensure that initial rates of the reaction were determined
and proportionality to enzyme concentration was also
established in each case. The IC50 values of PrNH2
towards different amine oxidases, were determined after
incubating the compound with the enzyme preparation
for either 0 or 30 min at 37 ꢁC in the concentration range
of 10ꢃ10–10ꢃ3 M. The activities remaining, were then
measured against the specific substrates and expressed
as percentages of the controls, which have been incu-
bated in the same way but in the absence of PrNH2.
4.7. Protein quantification
Protein was measured by the method of Bradford,37
using bovine-serum albumin (BSA) as standard.
Acknowledgements
This work was supported in part by a Grant from the
Comision Interministerial de Ciencia y Tecnologıa, Ref.
SAF 99-0093. The authors would like to thank Mr.
Chuck Simmons for revising and correcting the English
of this manuscript.
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