The stereoselectivity of inhibition of rat liver mitochondrial MAO-A and MAO-B by the enantiomers of 2-phenylpropylamine and their derivatives

Article abstract of DOI:10.1016/S0223-5234(99)80080-4

As part of a study of the stereoselectivity of inhibition of the different forms of monoamine oxidase (MAO-A and MAO-B), the enantiomers of 2- phenylpropylamine, N-methyl-2-phenylpropylamine and N-methyl-N-propargyl-2- phenylpropylamine have been prepared. The K(i) values for each enantiomer when competitively inhibiting both MAO-A and MAO-B are reported. The enantiomers of N-methyl-N-propargyl-2-phenylpropylamine were also evaluated as irreversible inhibitors (first order rate constant [k₂] for formation of the covalent adduct). These compounds represent a series of enantiomers in which asymmetry is due to the presence of a hydrophobic (-CH₃) substituent at the carbon atom β to the amino function. The results are discussed in comparison to previous studies of similar enantiomeric compounds in which the asymmetry was present at the carbon atom α to the amino function.

Full text of DOI:10.1016/S0223-5234(99)80080-4

Eur. J. Med. Chem. 34 (1999) 293−300
© Elsevier, Paris
293
Original article
The stereoselectivity of inhibition of rat liver mitochondrial MAO-A and MAO-B
by the enantiomers of 2-phenylpropylamine and their derivatives
Ronald Bocchinfuso, J. Barry Robinson*
Faculty of Pharmacy, University of Toronto 19 Russell Street, Toronto, Ontario M5S 1A1, Canada
(Received 4 June 1998; revised 15 December 1998; accepted 16 December 1998)
Abstract – As part of a study of the stereoselectivity of inhibition of the different forms of monoamine oxidase (MAO-A and MAO-B), the
enantiomers of 2-phenylpropylamine, N-methyl-2-phenylpropylamine and N-methyl-N-propargyl-2-phenylpropylamine have been prepared.
The Ki values for each enantiomer when competitively inhibiting both MAO-A and MAO-B are reported. The enantiomers of
N-methyl-N-propargyl-2-phenylpropylamine were also evaluated as irreversible inhibitors (first order rate constant [k2] for formation of the
covalent adduct). These compounds represent a series of enantiomers in which asymmetry is due to the presence of a hydrophobic (-CH3)
substituent at the carbon atom â to the amino function. The results are discussed in comparison to previous studies of similar enantiomeric
compounds in which the asymmetry was present at the carbon atom a to the amino function. © Elsevier, Paris
enantiomeric 2-phenylpropylamines and derivatives / MAO-A and MAO-B / stereoselectivity / competitive inhibition / irreversible
inhibition
1. Introduction
from a chemical standpoint, are identical. Both com-
pounds N-alkylate the reduced flavin prosthetic group of
the enzyme [4] through formation of an intermediate
radical cation [5]; for recent review see [6]. Such a
suicide inhibitory mechanism shows time-dependent ir-
reversible kinetics (formation of EI*) preceded by a
competitive reversible phase (formation of the initial EI
complex) and represented as:
The two forms of the enzyme monoamine oxidase
(MAO) (monoamine: O2 oxido-reductase (deaminating)
(flavin containing) (E.C. 1.4.3.4.)) within tissue or crude
enzyme preparations are detected and differentiated by
their response to selective substrates and inhibitors. Thus,
MAO-A
catalysed
oxidative
deamination
of
k1
k2
5-hydroxytryptamine (5-HT; serotonin) (MAO-A selec-
tive substrate) is inhibited by clorgyline (figure 1, com-
pound 1; approximately 10–8 M) [1], whereas MAO-B
catalysed oxidative deamination of â-phenylethylamine
or benzylamine (MAO-B selective substrates) is inhibited
by [R]-(-)-deprenyl (Selegiline) (figure 1, compound 2;
approximately 10–8 M) [2]. Other endogenous amine
substrates e.g. tryptamine, tyramine etc. are metabolised
by both forms of the enzyme [3].
The selective inhibitors clorgyline and Deprenyl (Sel-
egiline) are both derivatives of N-methylpropargylamine
and, although selective towards different forms of the
enzyme, exert their inhibition by mechanisms which,
[E] + [I]
[EI]
[EI*]
k
1
where Ki = k–1/k1
Since the chemical mechanism of inhibition of the two
forms of MAO are identical for the substituted propargy-
lamine inhibitors, selectivity towards a particular form
will be largely dependent upon affinity differences (as
reflected in Ki values) towards the two forms arising from
differences in lipophilicity and/or differences in the steric
and stereochemical requirements for inhibition. Quanti-
tative structure activity relationship studies have sug-
gested that, in addition to differences in the steric
requirements for optimal inhibition, the two forms of the
enzyme also differ in their lipophilicity requirements [7,
8].
*Correspondence and reprints

294
(1)
(2)
Figure 1. Structures of compounds 1 and 2.
Aspects of the interaction of monoamine oxidase with
substrates and inhibitors, particularly stereochemical as-
pects of such interactions, have been reviewed [9]. Stud-
ies employing the enantiomers of Selegiline and of
MAO-B. The enantiomers of the N-methyl-N-propargyl
derivative (figure 2, compounds 5a and 5b) have also
been prepared and studied under conditions leading to
both reversible (competitive) and irreversible inhibition.
α-methylpargyline
(N-methyl-N-propargyl-1-phenyl-
ethylamine) have shown that, at the competitive phase of
the inhibition, although each form of the enzyme is
sensitive to configurational differences and show a pref-
erence towards the [R]-configuration enantiomer, such
differences are more pronounced in the MAO-B form of
the enzyme [10–12]. At the irreversible phase of the
inhibition, although N-alkylation appears to proceed at a
slightly faster rate with MAO-A than with MAO-B, the
influence of configuration is negligible. Such is consistent
with the generally accepted inhibitory mechanism [6]
wherein formation of a radical cation intermediate is
followed by proton loss from the â-carbon atom leading
to a planar (racemic) radical as the alkylating species.
All of the above compounds are MAO inhibitors in
which the chiral centre is at the carbon atom α to the
basic nitrogen atom. Compounds in which the chiral
centre is at the â-carbon have not been extensively
studied. The present work therefore reports on the syn-
thesis and testing of the enantiomers of 2-phenyl-
propylamine (figure 2, compounds 3a and 3b) and
N-methyl-2-phenylpropylamine (figure 2, compounds 4a
and 4b) as competitive inhibitors of both MAO-A and
2. Results and discussion.
2.1. Chemistry
(±)-2-Phenylpropylamine was synthesised by reaction
of formamide with (±)-2-phenylpropionaldehyde (Leuck-
art reaction) and the product resolved into its individual
enantiomers using both (+)- and (-)-tartaric acid [13]. The
(+)-2-phenylpropylamine enantiomer has been previously
established as having the [R] configuration by synthesis
from [R](L)-(-)-2-phenylpropionic acid [14] and conver-
sion of [R](D)-(-)-atrolactic acid to [S](D)-(+)-2-
phenylpropionic acid [15]. The N-methylation reactions
were performed by reaction of dimethylsulphate on the
benzilidine derivative of the primary amine and the
products further alkylated using propargyl bromide.
2.2. Biological studies
A mitochondrial enzyme preparation of MAO derived
from rat liver, and known to contain both MAO-A and
MAO-B [16], was employed in the inhibition studies.
[R]
[S]
Figure 2. 3a (R1 = R2 = H)
4a (R1 = CH3; R2 = H)
3b (R1 = R2 = H)
4b (R1 = CH3; R2 = H)
5b (R1 = CH3; R2 = Ch2C§CH)
5a(R1 = CH3; R2 = CH2C§CH)

295
Figure 3. a. Double-reciprocal plot of inhibition of oxidative deamination of PEA by [R]-(-)-N-methyl-N-propargyl-2-
phenylpropylamine hydrochloride (5a). All points are the mean of duplicate determinations. Inhibitor concentrations are 0 µM (■);
37.66 µM (M); 75.32 µM (▲);150.6 µM (•). b. Replot of gradient vs. inhibitor concentration, data from figure 3a.
Enzyme preparations containing a single active form of
the enzyme were prepared from the above by incubation
with either Selegiline (MAO-B selective inhibitor) or
Clorgyline (MAO-A selective inhibitor) followed by
washing and centrifugation to remove the excess inhibi-
tor. Enzyme activity in such preparations was determined
at 37 °C and pH 7.4 by a radiochemical method [17, 18]
as previously reported [7, 12] using the appropriate spe-
cific substrate (14C-labelled 5-HT or PEA). In studies of
the competitive phase of the inhibition and in order to
prevent any time dependent irreversible inhibition by
covalent adduct (EI*) formation, the enzyme catalysed
reaction was initiated by addition of enzyme to a solution
of substrate and inhibitor i.e. preincubation of the enzyme
with the inhibitor was omitted and the enzyme catalysed
reaction was allowed to proceed for only 3 min. Employ-
ing such conditions, all plots of resulting data were
indicative of competitive inhibition kinetics (see figures
3a and 3b for a typical set of data). The computed Ki
values are given in table I.
In studies of the irreversible phase of the inhibition,
aliquots of an enzyme/inhibitor mixture were removed at
intervals, diluted with substrate solution and residual
enzyme activity determined (amount of product formed
during 3 min incubation). Kinetic data was interpreted as
previously reported [12] using the method developed by
Kitz and Wilson [19]. A typical set of results are pre-
sented graphically in figures 4a and 4b and the computed
values of k2, the rate constant for formation of the
covalent adduct (EI*), are shown in table I.
Table I. Competitive Ki values and kinetic constants (k2) for the time-dependent inhibition of MAO-A and MAO-B by the enantiomers of
2-Phenylpropylamine and their derivatives.
Compound
MAO-A
MAO-B
Km(5-HT) 28.6 (± 6.2) µM
Ki (µM)
Km(PEA) 3.46 (± 0.48) µM
Ki (µM)
k2 (min-1)
k2 (min-1)
3a
3b
[R]-(+)-
[S]-(-)-
133.5 (± 8)
584 (± 18)
na
na
14.1 (± 1.3)
156 (± 5.5)
na
na
4a
4b
[R]-(+)-
[S]-(-)-
169 (± 10)
614 (± 72)
na
na
47.8 (± 8.4)
149 (± 33)
na
na
5a
5b
[R]-(-)-
[S]-(+)-
11.3 (± 3.2)
181.6 (± 40)
0.14 (± 0.03)
0.18 (± 0.04)
24.9 (± 2.9)
44.9 (± 7.8)
0.42 (± 0.03)
0.38 (± 0.005)

296
Figure 4. a. Time-dependent inhibition of MAO-B by [R]-(-)-N-methyl-N-propargyl-2-phenylpropylamine hydrochloride (5a)
(typical data from one experiment). Plot of log (% residual activity) against incubation time. All points are the mean of duplicate
determinations. Concentrations of 5a are 0 µM (M); 1.66 µM (•); 3.32 µM (_); 6.64 µM (m); 13.29 µM (●); 33.22 µM (▲). b. Plot
of 1/kobs (data from figure 4a plus additional experiments) against 1/[I]. The line is that of best fit by linear regression.
Data regarding the influence of a chiral centre at the
carbon atom â to the amino function with respect to either
stereoselectivity of inhibition, substrate selectivity or
selectivity towards a particular form of the enzyme is
sparse. Thus, while the compounds [R]-(-)-noradrenaline
and [R]-(-)-adrenaline are selective substrates for MAO-
A [20, 21] the opposite enantiomers of the above com-
pounds do not appear to have been studied in detail. The
results of studies of the enantiomers of â-phenylethano-
lamine suggest that the [D]-(-)- enantiomer is a substrate
for rat liver mitochondrial MAO-A, whereas both enan-
tiomers are substrates for MAO-B [22]. The racemic
form of compounds having a chiral centre at the â-carbon
atom and carrying an N-methyl-N-propargyl function
have been tested as irreversible inhibitors of MAO-A and
MAO-B, but showed only limited ability to differentiate
between the two forms of the enzyme [23, 24].
In all the examples quoted above, the substituent at the
â-carbon atom has been a polar hydroxyl group. The
present work therefore differs from previous studies in
that a hydrophobic methyl substituent has been intro-
duced at the â-carbon atom. Further, the results are
readily compared with similar studies upon the enanti-
omers of amphetamine, methamphetamine and selegiline
(Deprenyl series of compounds) [10], and of
α-methylbenzylamine, N-methyl-a-methylbenzylamine
and N-propargyl-N-methyl-α-methylbenzylamine (α-
methyl-Pargyline series of compounds) [11].
In considering the competitive inhibition studies, the
results reported show very similar trends to those dem-
onstrated in the α-methyl-Pargyline series of compounds.
The major difference shown is in affinity, where a higher
affinity (greater potency) is shown among the present
compounds. However, using the present data as an
example, it will be noted that, for any enantiomeric pair
of compounds, whether they be primary, secondary or
tertiary amines, it is the [R]-configuration enantiomer
which is the more potent inhibitor. Further, when com-
paring the activity of any enantiomeric pair of com-
pounds as inhibitors of MAO-A and MAO-B, the
MAO-B form of the enzyme is more sensitive to inhibi-
tion (only in the case of compound Va is this observation
reversed).
However, within the present series of compounds (and
similarly within the Deprenyl and α-methyl-Pargyline
series) it should be noted that there are considerable
differences in the pKa values of the tertiary amines
relative to the primary and secondary amines. Thus,
whereas the pKa of â-phenylpropylamine is reported as
9.80 at 25 °C [25], and N-methylation will slightly in-
crease the basicity, the introduction of an N-propargyl
substituent is known to produce significant base weaken-
ing effects of about two pK units [26] (pKa of Deprenyl
is reported to be 7.4 at 25 °C [27]. Thus, within the
present series, compounds 3 and 4 would be 99% ionised
at pH 7.4 whereas compound 5 would be less than 50%

297
ionised at the same pH value. The active species, either as
a substrate or as an inhibitor, of the enzyme MAO is the
base form [28–30] and correction of the above Ki values
for the concentration of unionised species show that the
enantiomers of N-methyl-N-propargyl-â-phenylpropy-
lamine (compounds 5a and 5b) have considerably less
affinity towards both MAO-A and MAO-B than do the
enantiomers of â-phenylpropylamine (compounds 3a and
3b) and N-methyl-â-phenylpropylamine (compounds 4a
and 4b). This is in direct contrast to similar calculations
performed on the compounds within the Deprenyl [10]
and α-methyl-Pargyline series, in which both [R]-(-)-N-
tra (in CCl4 solution) from a Varian T60 spectrometer
using tetramethylsilane as external standard. Determina-
tion of Cl– was by non-aqueous titration in glacial acetic
acid in the presence of mercuric acetate and using
perchloric acid in glacial acetic acid as titrant. Analyses
of all synthesised compounds were within 0.4% of the
theoretical value unless otherwise stated.
3.1.1. (±)-2-phenylpropylamine
(±)-2-Phenylpropionaldehyde (35 g; 0.23 mol) and for-
mamide (75 mL; 1.88 mol) were mixed and the partially
immiscible liquids refluxed for 10 h in a flask equipped
with a wide necked air condenser topped with a Leibig
condenser (ammonium carbonate tends to sublime and to
clog the condenser if the air space provided by the air
condenser is not available). At the elevated temperature
of the reaction the two materials become miscible and the
reaction mixture darkens rapidly.
Sodium hydroxide solution (150 mL; 30%) was added
and the mixture further refluxed for 15 h after which the
solution was steam distilled, the distillate being trapped
in HCl solution. The 1 000 mL of distillate was evapo-
rated to dryness under reduced pressure, the residue
dissolved in water and the solution made alkaline with
ammonia. The mixture was extracted with ether
(3 × 50 mL), the extracts dried (Na2SO4), filtered and the
ether distilled. The residue was fractionally distilled, the
fraction b.p. 106–108 °C at 20 mm (7.2 g) (literature
b.p. = 97–98 °C at 19 mm; [31]) being collected as a
colourless liquid. I.R. (liquid film) 3 380 and 3 300 cm–1
(-NH2; H-bonding); 1 600, 1 560, 770, 705 cm–1
(monosubstituted aromatic) n.m.r. (CCl4 solution) δ 7.1
(s, 5H, aromatic); 2.66–3.0 (m, 3H, benzylic -CH- and
-CH2-N), 1.86 (broad, 2H, -NH2, 1.3 (d, J = 6 cycles s–1,
C-CH3).
methyl-N-propargyl-1-phenylisopropylamine
([R]-(-)-
Deprenyl) and [R]-(+)-N-methyl-N-propargyl-α-
methylbenzylamine display greater affinity towards
MAO-B than any of the enantiomers of the corresponding
primary and secondary amines. Thus, the introduction of
a nonpolar asymmetric centre on the carbon atom â to the
amino function causes, with respect to competitive inhi-
bition of MAO-B, a considerable loss of affinity and
stereoselectivity.
The enantiomeric propargylamine derivatives (com-
pounds 5a and 5b) are also capable of irreversible
inhibition of both MAO-A and MAO-B and the deter-
mined values of k2 (the first order rate constant for
formation of the covalent adduct) are reported in table I.
As with previous studies, stereoselectivity is negligible or
absent while the rate constants are slower than those
previously found for the enantiomers of Deprenyl and
α-methyl-Pargyline [12].
Thus, while treatment of Parkinson’s disease by means
of irreversible inhibition of MAO requires the use of
inhibitors highly stereoselective towards MAO-B, such
stereoselectivity is not attained when employing a non-
polar asymmetric substituent at the carbon atom â to the
amino function. This loss of stereoselectivity is accom-
panied by both a loss of affinity and a reduction in the rate
of development of irreversible inhibition. In the absence
of significant knowledge of the active site and the
surrounding area of MAO-A and MAO-B it is not
currently possible to present any logical reason for these
findings.
3.1.2. [R]-(+)-2-Phenylpropylamine (3a)
(±)-2-Phenylpropylamine (18 g) was resolved using
(+)-tartaric acid using a literature method [13]. After
three recrystallisations from methanol, the salt (8.7 g) had
20
m.p. 137–139 °C and [α]D = +30.13° (c,4.3, H2O)
20
(literature [α]D = +31.7° (c,4, H2O) after 23 crystalli-
sations [32]). From the crystalline tartrate salt m.p.
137–139 °C, the base was isolated by dissolving in water,
making the solution alkaline with ammonia and extract-
ing with ether. The extracts were dried (Na2SO4), filtered
and the ether evaporated. The residual oil was distilled,
the fraction b.p. 82–84 °C at 8 mm being collected.
3. Experimental protocols
3.1. Chemistry
22
Melting points were determined using a Thomas
Hoover Unimelt capillary melting point apparatus and are
uncorrected. IR spectra were obtained from a Perkin
Elmer 1330 Infra-red spectrophotometer and NMR spec-
[α]D20 = +31.9° (c,0.985, EtOH) (literature reports [α]D
= +35.4° (c,2, EtOH) [32], suggesting a 90% enantio-
meric excess in the above product). The hydrochloride
salt was prepared by passing dry HCl gas through an

298
ether solution of the base. The solid product was filtered
and recrystallised from ethyl propyl ketone and had m.p.
138.5–139.5 °C. Anal. C9H14NCl (Cl–).
(3.5 g) added, followed by propargyl bromide solution
(2.2 mL of 80% solution in toluene; 2.26 g; 0.02 mol of
bromo compound). The solution was refluxed on a steam
bath for 20 h.
3.1.3. [S]-(-)-2-Phenylpropylamine (3b)
The mixture was carefully acidified with dilute acetic
acid, extracted with ether and the extracts discarded. The
solution was made alkaline with ammonia, extracted with
ether (3 × 30 mL), the extracts dried (Na2SO4), filtered
and the solvent evaporated under reduced pressure. The
residue was distilled, the fraction b.p. 112–114 °C at
2-Phenylpropylamine enriched in the [S]-(-)-
enantiomer was isolated from the mother liquors from the
above reaction. The material was further resolved using
(-)-tartaric acid as the resolving acid [13]. Three recrys-
tallisations of the salt from methanol yielded product,
m.p. 138–139 °C (8 g) from which the base was isolated,
3.5 mm being collected; [α]D = –20.32° (c, 3.11,
20
20
EtOH). Yield 1.5 g. I.R. (liquid film) 3 300 cm–1 (C§C
stretch); n.m.r. (CCl4) δ 6.7 (s, 5H, aromatic); 3.07 (d, J
= 2 cycles s–1, 2H, -CH2-C(C); 2.9–2.2 (m, 3H, benzylic
-CH- and -CH2-N); 2.1 (s, 3H, N-CH3); 1.9 (t, J = 2
cycles s–1, 1H, C(CH); 1.15 (d, J = 6 cycles s–1, 3H,
C-CH3). The hydrochloride salt after recrystallisation
from methyl propyl ketone had m.p. 128–129 °C. Anal.
C13H18NCl (N, Cl–).
b.p. 75–77 °C at 3 mm, [α]D = –33.75° (c,3.5, EtOH)
(95% enantiomeric excess based on data of Brode &
Raasch [32]). The hydrochloride salt, prepared as re-
ported above for the (+)-enantiomer had m.p.
145–146 °C. Anal. C9H14NCl (N, Cl–).
3.1.4. [R]-(+)-N-Methyl-2-Phenylpropylamine (4a)
(+)-2-Phenylpropylamine (4.4 g; 0.033 mol) was dis-
solved in benzene (10 mL) and benzaldehyde (10 mL;
10.4 g; 0.1 mol) was added and the mixture allowed to
stand over a Molecular Sieve, type 4A overnight. To the
filtered solution was added dimethyl sulphate (10 mL)
and the mixture warmed on a steam bath overnight. Water
(20 mL) was then added and the mixture refluxed for 4 h.
The aqueous phase was separated, the organic phase
washed with dilute HCl and the extracts added to the
separated aqueous phase. The aqueous solution was made
alkaline with ammonia, extracted with ether (3 × 40 mL)
and the combined ether extracts dried (Na2SO4), filtered
and the ether evaporated. The residue was fractionally
distilled, the fraction b.p. 80–83 °C at 5 mm (3.3 g) being
3.1.7.
[S]-(+)-N-Methyl-N-propargyl-2-phenylpropyl-
amine (5b)
Similarly prepared, but using [S]-(-)-N-methyl-2-
phenylpropylamine (1.5 g). The product amine had b.p.
20
107–112 °C at 3 mm (0.7g) [α]D = +15.7° (c, 2.73,
EtOH). I.R. (liquid film) identical to that of the
R-configuration enantiomer. The hydrochloride salt after
recrystallisation from methyl propyl ketone had m.p.
108–113 °C. Anal.: Found N, 5.96; Cl– 16.51%, 16.49%.
C13H18NCl requires N, 6.25; Cl– 15.84%. [N.B. The
above analytical data (N, Cl– and specific rotation are
consistent with the presence of approximately 10% of the
starting secondary amine within this product].
20
collected. [α]D = +26.5° (c, 4.02, EtOH). Hydrochlo-
ride salt m.p. = 143–144 °C after recrystallisation from
methyl propyl ketone. I.R. (liquid film) 3 300 cm–1
(broad; bonded N-H); 1 600, 1 560, 770, 705 cm–1 (aro-
matic stretching and bending) n.m.r. (CCl4 solution) δ 6.9
(s, 5H, aromatic); 2.4–3.0, (m, 3H, benzylic -CH- and
-CH2-N); 2.1 (s, 3H, N-CH3); 1.1 (d, J = 6 cycles s-1, 3H,
C-CH3).
3.2. Enzyme studies
Livers of male Sprague-Dawley rats were used as the
enzyme source, and preparations of MAO-A and MAO-B
were prepared by incubation of the crude mitochondrial
enzyme preparation with (-)-Deprenyl and clorgyline
respectively [7, 33]. The washed suspension of each form
of the enzyme was stored at –20 °C until required.
Enzyme activity was determined by radiochemical meth-
ods [17, 18]. [14C]-labelled 2-phenylethylamine hydro-
chloride (50 mCi mmol–1) and 5-hydroxytryptamine
binoxalate (50 mCi mmol–1) were purchased from New
England Nuclear, Boston, MA.
3.1.5. [S]-(-)-N-Methyl-2-Phenylpropylamine (4b)
Similarly prepared but using [S]-(-)-2-Phenyl-
propylamine (2.5 g). The product amine (1.9 g) had b.p.
81–84 °C at 3.5 mm; [α]D20 = –26.6° (c, 2.4, EtOH). I.R.
spectrum identical with that of the [R]-(+)-enantiomer.
Hydrochloride salt (recrystallised from methyl propyl
ketone) m.p. 146–147 °C. Anal. C10H16NCl (N, Cl–).
3.3. Competitive inhibition studies
3.1.6.
[R]-(-)-N-Methyl-N-propargyl-2-phenylpropyl-
amine (5a)
Immediately before use, the MAO-B enzyme prepara-
tion (5–7 mg protein per mL) were diluted 1 to 10 with
phosphate buffer (10 mM; pH 7.4). The MAO-A enzyme
[R]-(+)-N-Methyl-2-phenylpropylamine
0.019 mol) was dissolved in ethanol (15 mL), K2CO3
(2.9 g;

299
preparation was used undiluted. Within these studies, the
inhibitor (50 µL) was incubated at 35 °C for 3 min in the
presence of substrate in phosphate buffer (10 mM; pH
7.4) and the enzyme catalysed reaction then initiated by
the addition of the enzyme preparation (50 µL). The final
volume of the incubation mixture was 300 µL. The
reaction was allowed to proceed for 3 min and was
stopped by the addition of HCl (200 µL; 2 M). A ten-fold
range of substrate concentration was employed and en-
zyme activity determined in the presence and absence of
at least a three fold concentration range of inhibitor. All
determinations were carried out in duplicate.
For studies of MAO-B activity and inhibition (PEA as
substrate), deaminated products were extracted by addi-
tion of toluene (6 mL), the mixture extracted by shaking
in a vortex mixer and then centrifuged. A portion (4 mL)
of the toluene layer was removed and added to scintilla-
tion fluid (5 mL) (prepared from Liquifluor concentrate,
New England Nuclear), and containing 0.4% w/v of PPO
(2,5-diphenyloxazole) and 0.005% w/v of POPOP (1,4-
bis-[2-(4-methyl-5-phenyl-oxazolyl]-benzene in the final
toluene solution) and counted in a Beckman LS7500
liquid scintillation counter. All counts were corrected for
quenching (counting efficiency 96%).
The enzyme catalysed reaction was stopped by addition
of HCl (200 µL, 2 M) and the metabolites extracted and
counted as reported above.
Similar studies employing the MAO-A enzyme prepa-
ration used inhibitor solution (1 mL) and enzyme prepa-
ration (1 mL) and the residual enzyme activity was
determined using 14C-labelled 5-hydroxytryptamine as
substrate (350 µL; approximately 100 µM).
All determinations were performed in duplicate and at
least two independent experiments were performed, each
employing 4–5 different concentrations of inhibitor.
Kinetic data derived from the studies of the irreversible
phase of the inhibition were interpreted using the method
developed by Kitz and Wilson [19]. The apparent first
order rate constant for loss of enzyme activity (kobs) was
obtained by linear regression of a plot of log (% residual
activity) against incubation time (gradient = –kobs/2.303)
and linear regression of a plot of 1/kobs against 1/[I]
yielded Ki/k2 (gradient) [12]. A typical set of experimen-
tal data is shown in figures 4a and 4b and the computed
results for all inhibition studies are presented in table I.
References
For studies of MAO-A activity and inhibition (5-HT as
substrate), deaminated products were extracted into
benzene/ethyl acetate (1:1) (6 mL) and after vortex mix-
ing and centrifuging, the mixture was refrigerated for 4 h
at –10 °C. A sample (4 mL) of the organic layer was
added to scintillation cocktail (5 mL) and counted as
described above.
[1] Johnston J.P., Biochem. Pharmacol. 17 (1968) 1285–1297.
[2] Yang H.Y.T., Neff N.H., J. Pharmacol. Exp. Ther. 187 (1973)
365–371.
[3] Fowler C.J., Tipton K.F., J. Pharm. Pharmacol. 36 (1984) 111–115.
[4] Singer T.P., Salach J.I., in: Youdim M.B.H., Paykel E.S. (Eds.),
Monoamine Oxidase Inhibitors- The State of the Art, John Wiley &
Sons, London, 1981, pp. 17–29.
The data obtained were plotted as Lineweaver-Burk
double reciprocal plots, the best straight line being
determined by a least squares regression analysis
(figure 3a). A re-plot of the gradient of the Lineweaver-
Burk plot against inhibitor concentration allowed deter-
mination of the Ki from the values of the slope and
intercept (y-axis). All such plots were linear, indicating
the absence of detectable non-competitive kinetics under
the employed conditions (figures 3a and 3b).
[5] Silverman R.B., Hoffman S.J., Catus W.B., J. Amer. Chem. Soc. 102
(1980) 7126–7128.
[6] Silverman R.B., Acc. Chem. Res. 28 (1995) 335–342.
[7] Ali A., Robinson J.B., J. Pharm. Pharmacol. 43 (1991) 750–757.
[8] Wang Y.X., Castagnoli N., J. Med. Chem. 38 (1995) 1904–1911.
[9] Dostert P.L., Strolin Benedetti M., Tipton K.F., Medicinal Res. Rev.
9 (1989) 45–89.
[10] Robinson J.B., Biochem. Pharmacol. 34 (1985) 4105–4108.
[11] Robinson J.B., J. Pharm. Pharmacol. 37 (1985) 72P.
[12] Robinson J.B., Bocchinfuso R., Khalil A., J. Pharm. Pharmacol. 47
(1995) 324–328.
3.4. Irreversible inhibition studies
[13] Biere H., Rufer C., Ahrens H., Loge O., Schroder E., J. Med. Chem.
17 (1974) 716–721.
[14] Levene P.A., Mikeska L.A., Passoth K., J. Biol. Chem. 88 (1930)
27–59.
To solutions of the inhibitor prepared in phosphate
buffer (10 mM, pH 7.4) (2 mL) was added MAO-B
enzyme preparation (500 µL) and the mixture incubated
at 35 °C. Aliquots (50 µL) were removed at fixed time
intervals up to 30 min (up to 15 min in the case of the
more concentrated inhibitor solutions) and immediately
added to 14C-labelled phenylethylamine hydrochloride
solution (350 µL) (approximately 20 µM in phosphate
buffer, 10 mM, pH 7.4) and incubated at 35 °C for 3 min.
[15] Eliel E.L., Freeman J.P., J. Amer. Chem. Soc. 74 (1952) 923–928.
[16] Fowler C.J., Callingham B.A., Mantle T.J., Tipton K.F., Biochem.
Pharmacol. 27 (1978) 97–101.
[17] Wurtman F.J., Axelrod J., Biochem. Pharmacol. 12 (1963)
1439–1440.
[18] Squires R.F., Adv. Biochem. Psychopharmacol. 5 (1972) 355–370.
[19] Kitz R., Wilson I.R., J. Biol. Chem. 237 (1962) 3245–3249.
[20] Goridis C., Neff N.H., Brit. J. Pharmacol. 43 (1971) 814–818.

300
[21] Houslay M.D., Tipton K.F., Biochem. J. 139 (1974) 645–652.
[22] Williams C.H., Biochem. Soc. Trans. 5 (1977) 1770–1771.
[23] Williams C.H., Biochem. Pharmacol. 33 (1984) 334–337.
[24] Williams C.H., J. Pharm. Pharmacol. 34 (1982) 386–387.
[25] Girault-Vexlearschi G., Bull. Soc. Chim. Fr. (1956) 589–606.
[28] McEwen C.M., Sasaki G., Lenz W.R., J. Biol. Chem. 243 (1968)
5217–5225.
[29] McEwen C.M., Sasaki G., Jones D.C., Biochemistry 8 (1969)
3952–3962.
[30] Williams C.H., Biochem. Pharmacol. 23 (1974) 615–628.
[31] Weston A.W., Ruddy A.W., Suter S.M., J. Am. Chem. Soc. 65 (1943)
674–677.
[26] Perrin D.D., in: Yalkowsky S.H., Sinkula A.A., Valvani S.C. (Eds.),
Physical Chemical Properties of Drugs, Marcel Dekker, New York,
1980, pp. 1–48.
[32] Brode W.R., Raasch M.S., J. Am. Chem. Soc. 64 (1942) 1449–1450.
[33] Fowler C.J., Tipton K.F., Biochem. Pharmacol. 30 (1981)
3329–3332.
[27] Chafetz L., Desai M.P., Sukonic L., J. Pharm. Sci. 83 (1994)
1250–1252.