Fluorinated phenylcyclopropylamines. Part 4: Effects of aryl substituents and stereochemistry on the inhibition of monoamine oxidases by 1-aryl-2-fluoro-cyclopropylamines

Article abstract of DOI:10.1016/j.bmc.2005.01.043

A series of para-ring-substituted (E)- and (Z)-1-aryl-2- fluorocyclopropylamines were examined as inhibitors of recombinant human liver monoamine oxidase A (MAO A) and B (MAO B). Unlike the parent 1-phenylcyclopropylamine, which is a selective inhibitor of MAO B, both (E)- and (Z)-diastereomers of derivatives having fluorine at the 2-position of the cyclopropane ring were potent and selective irreversible inhibitors of MAO A. Both electron releasing groups (Me, OMe) and electron attracting groups (Cl, F) substituted in the para-position caused a modest increase in activity. Geminal difluoro-substitution caused a loss of potency of 100-fold compared to either (E)- or (Z)-monofluorinated analogue. Surprisingly, (1S,2R)-2-fluoro-1- phenylcyclopropylamine and the (1R,2S)-enantiomer were essential equally potent as inhibitors of MAO A and MAO B. None of the tested 1-aryl-2- fluorocyclopropylamines exhibited significant inhibition of tyramine oxidase.

Full text of DOI:10.1016/j.bmc.2005.01.043

Bioorganic & Medicinal Chemistry 13 (2005) 2489–2499
Fluorinated phenylcyclopropylamines. Part 4: Effects of
aryl substituents and stereochemistry on the inhibition
of monoamine oxidases by 1-aryl-2-fluoro-cyclopropylamines
Song Ye,a Shinichi Yoshida,b Roland Frohlich,c Gunter Haufec and Kenneth L. Kirka,*
¨
¨
aLaboratory of Bioorganic Chemistry, National Institute of Diabetes, and Digestive and Kidney Diseases,
National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA
bIndustrial Research Institute of Tottori Prefecture, Tottori 689-1112, Japan
cOrganisch-Chemisches Institut, Universita¨t Mu¨nster, Corrensstr. 40, D-48149 Mu¨nster, Germany
Received 19November 2004; accepted 21 January 2005
Abstract—A series of para-ring-substituted (E)- and (Z)-1-aryl-2-fluorocyclopropylamines were examined as inhibitors of recom-
binant human liver monoamine oxidase A (MAO A) and B (MAO B). Unlike the parent 1-phenylcyclopropylamine, which is a selec-
tive inhibitor of MAO B, both (E)- and (Z)-diastereomers of derivatives having fluorine at the 2-position of the cyclopropane ring
were potent and selective irreversible inhibitors of MAO A. Both electron releasing groups (Me, OMe) and electron attracting
groups (Cl, F) substituted in the para-position caused a modest increase in activity. Geminal difluoro-substitution caused a loss
of potency of 100-fold compared to either (E)- or (Z)-monofluorinated analogue. Surprisingly, (1S,2R)-2-fluoro-1-phenylcycloprop-
ylamine and the (1R,2S)-enantiomer were essential equally potent as inhibitors of MAO A and MAO B. None of the tested 1-aryl-
2-fluorocyclopropylamines exhibited significant inhibition of tyramine oxidase.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Recognition of the importance of monoamine oxidases
as targets for drug intervention for treatment of a vari-
ety of conditions has produced an enormous interest in
development of inhibitors of these enzymes. Of particu-
lar importance in this approach is the development of
potent inhibitors selective for specific classes (e.g.,
CAO vs MAO) and/or subclasses (e.g., MAO A vs
MAO B) of amine oxidase. Examples of inhibitors
investigated in this research include 1- and 2-phenylcy-
clopropylamines, among the most extensively studied
MAO inhibitors4 and fluorinated allylamines.5 In the
latter example, the presence of fluorine was critical to
activity. In our own research in this area, we have
prepared two series of fluorinated inhibitors, 1-aryl-2-
fluorocyclopropylamines (1) and 2-aryl-2-fluorocyclo-
propylamines (2), compounds that incorporate two
structural units present separately in previously synthe-
sized active MAO inhibitors.6 We initially examined
these as inhibitors of CAO and have reported that cer-
tain of the 2-aryl-2-fluorocyclopropylamines, but not
1-phenyl-2-fluorocyclopropylamine, are potent and
selective inhibitors of CAO.6,7 Whereas the 2-aryl-2-flu-
orocyclopropylamines were less active as inhibitors
of MAO than CAO, preliminary data revealed that
The enzymatic oxidation of amines to aldehydes is a
critical biochemical process in all organisms, including
mammals, plants, and both prokaryotic and eukaryotic
microorganisms. They have been classified into two
groups, copper- (EC: 1.4.3.6) and flavin- (EC: 1.4.3.4)
containing amine oxidases.1 Copper-containing amine
oxidases (CAO) require copper and an organic co-fac-
tor, for example, 2,4,5-trihydroxyphenylalanine qui-
none, for activity and are strongly inhibited by
semicarbazide.2 The flavin-containing monoamine oxi-
dases exist in two forms, MAO A and MAO B that
are characterized by different substrate and inhibitor
selectivities. MAO A and B are composed of 527 and
520 amino acids, respectively, and have a 70% amino
acid identity.3 Each isozyme has a flavin co-factor cova-
lently linked to a cysteine residue in the active center.
Keywords: Monoamine oxidase; Tyramine oxidase; Fluorinated phen-
ylcyclopropylamine; Irreversible inhibition; Stereochemistry.
*
Corresponding author. Tel.: +1 301 496 2619; fax: +1 301 402
4182; e-mail: kennethk@bdg8.niddk.nih.gov
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.043

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S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
(E)-2-fluoro-1-phenylcyclopropylamine ((E)-1a) dis-
played a dramatic reversal of MAO A versus MAO B
selectivity.8 Thus, the parent 1-phenylcyclopropylamine
(3) is selective for MAO B. In marked contrast, (E)-1a
had lower activity at this isozyme, but had dramatically
increased activity at MAO A. We have now extended
our studies to include an examination of the diastereo-
selectivity, enantioselectivity and aromatic ring substi-
tuent effects on potency and selectivity of inhibition of
MAO A and MAO B. In this report we describe the
syntheses of (E)- and (Z)-para-aromatic ring-sub-
stituted 1-aryl-2-fluorocyclopropylamines ((E)- and
(Z)-1a–e).
The (E/Z) configurations of cyclopropylamines ((E)-
and (Z)-1a) were assigned by analysis of their NOESY
spectra in DMSO-d6 (Fig. 1). Thus, NOE is observed be-
tween the phenyl proton and the proton of CHF in the
NOESY spectra of amine (Z)-1a, while no NOE is ob-
served between phenyl proton and the proton of CHF
in the corresponding spectra of amine (E)-1a. Subse-
quent X-ray analysis of one of the diastereomeric enan-
tiopure amides confirmed its stereochemical assignment
as (1R,2S)-1a (see below).
The synthetic route to 2,2-difluoro-1-phenylcyclopropyl-
amine (4) was based on similar chemistry from ethyl 3,3-
difluoro-2-phenylacrylate, prepared as reported by
McDonald et al.5
In addition, we report the preparation and purification
of the individual enantiomers of (E)-2-fluoro-1-phenyl-
cyclopropylamine ((1R,2S)-1a and (1S,2R)-1a) and
assignment of absolute configuration by X-ray analysis.
The effects of stereochemistry and ring substitution on
inhibition of recombinant MAO A and MAO B were
measured and these results are discussed. In addition,
inhibition of tyramine oxidase by 1-aryl-2-fluoro-cyclo-
propylamines was investigated.
In order to prepare the individual enantiomers of (E)-1a,
the racemic compound was converted to the diastereo-
meric
N-(2-fluoro-1-phenylcyclopropyl)-(2R)-2-hydr-
oxy-2-phenyl-acetamides (10a,b) (Scheme 2). The
diastereomers were separated chromatographically and
hydrolyzed to give (1R,2S)-1a and (1S,2R)-1a as hydro-
chloride salts (Scheme 2). The lower Rf diastereomer 10b
was crystallized from ethyl acetate/n-hexane to give a
crystal suitable for X-ray analysis. This was shown to
be the (1R,2S)-isomer (Fig. 2).
H
F
F
H
NH2-HCl
2.2. Enzyme inhibition results
NH2-HCl
R
R
Stock solutions of the human liver mitochondrial outer
membrane MAO A and B, expressed in the methylo-
trophic yeast Pichia pastoris, were used for enzyme
assays. The activities of MAO A and MAO B in the
(E)-1a-e
(Z)-1a-e
(a: R = H; b R = F; c R = Cl; d R = Me; e R = OMe)
F
F
F
NH2-HCl
NH2-HCl
NH2-HCl
NOE
NOE
NOE
Ph
Hb
Ha
F
Ph
Hb
H
3
2
4
+H3N
+H3N
H
F
Ha
2. Results
NOE
(E)-1a
(Z)-1a
2.1. Chemistry
Figure 1. The NOEs observed in the NOESY spectrum of (E)-1a and
(Z)-1a (DMSO-d6, 300 MHz).
(E)-2-Fluoro-1-phenylcyclopropylamine (1a) was previ-
ously prepared6 from ethyl (E)-3-fluoro-2-phenylacryl-
ate, synthesized by the procedure reported by
McDonald et al.5 A key step in the synthesis is cycload-
dition of diazomethane followed by photochemical
extrusion of nitrogen. The latter step occurs with a mod-
est loss of stereo integrity to produce mainly (E)-2-flu-
orocyclopropyl ester ((E)-5) along with lesser amounts
of (Z)-5 (Scheme 1). The formation of both isomers al-
lows both (E)- and (Z)-1-aryl-2-fluorocylopropylamines
to be synthesized from the common starting (E)-2-aryl-
3-fluoro esters. The mixture of diastereomeric cyclopro-
pyl esters 5 was converted to the hydrazides 6. Curtius
rearrangement of the derived carbonyl azides 7, as re-
ported for the synthesis of (E)-1a,6 gave the t-butylcar-
bamates 8 (Scheme 1). The (E)- and (Z)-carbamates
were separated by chromatography and hydrolyzed to
give (E)- and (Z)-1a–e as single diastereomers.
F1
C2
C54
C55
O1
C53
C56
C1
C3
N1
C11
C12
C52
C51
C4
C16
C5
C13
C15
C14
O2
Figure 2. X-ray structure of N-[(1R,2S)-2-fluoro-1-phenylcycloprop-
yl]-(2R)-2-hydroxy-2-phenyl-acetamide (10b).

S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
2491
Series
E: R1 = F, R2 = H
Z: R1 = H, R2 = F
a: R3 = H
b: R3 = F
c; R3 = Cl
F
d: R3 = Me
e: R3 = OMe
f. R1 = R2 = F. R3 = H
CO2Et
a
R3
R2
R2
R1
R1
b
CO2Et
CONHNH2
R3
a
F
F
R3
6
5
CO2Et
R2
R2
NHCO2 Bu
R1
R1
R2
CON3
R1
d
e
c
t
NH2-HCl
R3
R3
R3
8
7
(E)-1a-e, R1 = F, R2 = H
(Z)-1a-e, R1 = H, R2 = F
3, R1 = R2 = R3 = H
4,
R1 = R2 = F, R3 = H
Scheme 1. Reagents and conditions: (a) (i) CH2N2; (ii) hm, ether; (b) hydrazine, rt; (c) HCl, NaNO2, ether, rt; (d) t-BuOH, reflux; (e) 2 N HCl, ether.
presence of inhibitors were monitored spectrometrically
at 25 ꢁC using 1 mM kynuramine hydrobromide and
1 mM benzylamine, respectively, as substrates. The inhi-
bition assay was carried out in the presence of 6% of
dimethylsulfoxide (DMSO) to improve the solubility
of inhibitors used in this study. MAO A and B retained
87% and 86% of the original activities, respectively, after
the addition of DMSO to the assay mixture. The relative
activity was calculated based on the standard assay sys-
tem containing 6% DMSO but not containing inhibitor.
were mathematically calculated by using Figure 3B data
and are summarized in Table 1.
Figure 4 shows the inhibition curve of (Z)-isomers and
difluoro-compound 4 for MAO B. In the control exper-
iment, for (Z)-1c, (Z)-1e and the difluoro-compound 4,
no increase of absorbance at 250 nm was observed in
control experiments as above. However, an increase of
the absorbance was seen with (Z)-1a, (Z)-1b, and (Z)-
1d, so inhibition curves were corrected by subtraction
of the control data as shown in Figure 4B, and the
IC50 values were calculated from these results.
2.2.1. Inhibition of MAO B. Figure 3 shows the inhibi-
tion curve of (E)-1a–e for MAO B. As shown in Figure
3A, an increase of the relative activity was observed at
higher inhibitor concentration. For this reason, IC50 val-
ues could not be estimated from these data. To investi-
gate this complication, control experiments were
carried out. The absorbance increase at 250 nm was also
observed in the reaction mixture when both benzylamine
and MAO B were omitted. Therefore, the control data
were subtracted from the data of complete system to
give the curves shown in Figure 3B. The IC50 values
We have no clear explanation for this increase in
absorption at 250 nm in the absence of benzylamine
and MAO B, a phenomenon more pronounced for the
(E)-isomers. This may reflect relative stabilities of the
(E)- and (Z)-isomers under the assay conditions.
As can be seen from the data in Table 1, all cyclopro-
pane ring-fluorinated derivatives 1 were relatively weak
inhibitors of MAO B. The (Z)-isomers were from 2-fold
Ph
Ph
O
O
F
F
Ph
F
a,b
HO
+
HO
N
H
H
N
H
H
H3N
H
H
H
Ph
Ph
10b
(+)-(1R,2S)-1a
10a
(+)-(1S,2R)-1a
c
c
Scheme 2. Reagents and conditions: (a) (R)-O-acetylmandelic acid, DCC, DMAP, DCM, 0 ꢁC to rt, 74%; (b) 1% triethylamine (1.1 equiv) in
methanol, rt, 10a (38%, dr = 94:6), 10b (36%; dr = 93:7); (c) 2 N HCl (aq), reflux, (+)-1a (80%), (ꢀ)-1a 68%.

2492
S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
1-phenylcyclopropylamine 3 had an IC50 of 730 lM.8
The difluoro analogue 4 was a much weaker inhibitor
(IC50 = 110 lM) than the mono-fluoro compounds
(Fig. 5).
-1a
-1b
-1c
-1d
-1e
(E)
(E)
(E)
(E)
(E)
120
A
100
80
60
40
20
0
2.2.3. Enantioselectivity in the inhibition of MAO A and
B. To investigate the effect of absolute stereochemistry
in the inhibition of MAO A and B, the two enantiomers
of 2-fluoro-1-phenylcyclopropylamine ((1R,2S)-1a and
(1S,2R)-1a) were examined as inhibitors. No significant
enantioselectivity was observed for both isoforms of
MAO (Fig. 6).
0
20
40
60
80
100
120
140
160
2.2.4. Inhibition of tyramine oxidase. Commercially
available tyramine oxidase was used as a model for cop-
per-containing amine oxidase in this study.6 None of the
compounds in this series was a potent inhibitor of tyr-
amine oxidase (Fig. 7, Table 2). The (Z-isomers of para-
fluoro- and para-methyl-substituted compounds ((Z)-1b
and (Z)-1d) showed weak irreversible inhibition
(IC50 = 110 and 220 lM, respectively). The low or ab-
sent inhibition by 1-aryl-2-fluorocyclopropylamines
stands in contrast to our results with the isomeric 2-
aryl-2-fluorocyclopropylamines.6,7
Inhibitor (µM)
120
100
80
60
40
20
0
B
(E)
(E)
(E)
(E)
(E)
-1a
-1b
-1c
-1d
-1e
0
20
40
60
80
100
120
140
160
3. Discussion
Inhibitor (µM)
Substitution of fluorine on the cyclopropyl ring of MAO
B-selective 1-phenylcyclopropylamine (3) (IC50
Figure 3. Inhibition curves of MAO B by p-aromatic substituted (E)-2-
fluoro-1-phenylcyclopropylamines. (A) Without correction by the
control data. (B) Subtracted control data from complete experiments
data. Exponential fitting curves for (E)-1b,d,e were made by using the
relative activity values at inhibitor concentrations below 60 lM,
because good correlations were not obtained if data at higher
concentrations were used. For 1c data at inhibitor concentration
below 50 lM were used, and for 1a data obtained at all inhibitor
concentrations were used for fitting. IC50 values were calculated by
using these fitting curves (see Table 1).
=
190 lM) produced a potent and selective inhibitor of
MAO A. Both (E)- and (Z)-1a were comparably potent
and selective inhibitors of MAO A (IC50 = 1.1 and
0.9 lM, respectively) and both showed only weak inhibi-
tion of MAO B (IC50 = 290 and 72 lM, respectively).
1-Phenylcyclopropylamine (3) had an IC50 of 730 lM
for MAO A. The presence of electron donating (Me,
OMe) or electron withdrawing (Cl, F) groups substi-
tuted on the para-position caused a modest increase on
the inhibition of MAO A. The most potent compounds,
(E)- and (Z)-1d (p-Me), had IC50 values of 0.2 lM,
about 3600 times lower than the parent 1-phenylcyclo-
propylamine (3).
((Z)-1c vs (E)-1c) to 4-fold ((Z)-1a vs (E)-1a) more po-
tent inhibitors of MAO B than were the (E)-isomers.
For both (E)- and (Z)-isomers, para-aromatic substitu-
tion increased the potency of inhibition of MAO B,
and the most effective inhibitor, (Z)-1b (para-fluorine)
had an IC50 of 13 lM. The difluoro analogue 4 showed
no inhibition of MAO B. Under our conditions, 1-phen-
ylcyclopropylamine 3 had an IC50 of 190 lM for inhi-
bition of MAO B.8
The most striking feature of these results is the dramatic
increase in inhibitory activity for MAO A that is caused
by substitution of fluorine on the cyclopropane of
1-arylcyclopropylamines. Together with the accompany-
ing decrease in potency as an MAO B inhibitor, this
represents a very substantial reversal of MAO A versus
MAO B selectivity (about 1000-fold). That this effect is
independent of relative or absolute stereochemistry is
noteworthy. Assuming the aryl ring and amino substitu-
ent occupy the same position when interacting with the
enzyme, this suggests that the fluorine substituent is
accommodated with equal facility when in either of four
positions (Fig. 8). The lowered activity of the difluoro
analogue 4 is difficult to reconcile with this, although
pKa considerations may be appropriate. In this regard,
there is ample evidence that the unprotonated amine is
the active species, and this should favor binding of 4
at physiological pH.3
2.2.2. Inhibition of MAO A. Figure 5 shows the inhibi-
tion curve of (E)- and (Z)-1a–e and 4 for MAO A. In
preliminary control experiments for all compounds
examined, no increase of absorbance at 316 nm in the
reaction system without enzyme and substrate was ob-
served. Therefore, the data obtained were not corrected.
All compounds were more potent inhibitors of MAO A
than of MAO B. There was very little effect of relative
configuration of the fluorine. The (Z)-diastereomers
were equal or only slightly more potent than the (E)-dia-
stereomers (Table 1). The para-methyl derivatives, (E)-
and (Z)-1d, were the most potent inhibitors
(IC50 = 0.2 lM). In contrast, the nonfluorinated parent

S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
2493
Table 1. IC50 values and inhibition type for p-aromatic substituted 2-fluoro-1-phenylcyclopropylamines (MAO A and B)
Compound
Ra
Isomer typeb
MAO A
Inhibition typeh
MAO B
Inhibition typeh
IC50 (lM)e
IC50 (lM)e
(E)-1a
(Z)-1a
(E)-1b
(Z)-1b
(E)-1c
(Z)-1c
(E)-1d
(Z)-1d
(E)-1e
(Z)-1e
3g
H
trans
cis
1.1 0.1
0.9 0.1
1.2 0.0
0.6 0.0
0.7 0.1
0.5 0.0
0.2 0.0
0.2 0.0
0.7 0.0
0.6 0.1
730 150
110 10
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
NDd
290 50
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
NDd
H
72
38
13
39
24
1
F
trans
cis
0
F
Cl
1
trans
cis
1
Cl
1
CH3
CH3
OMe
OMe
—
trans
cis
110 0f
40
81
23
2
5
0
trans
cis
—
—
190 20
NIc
4
—
Irreversible
a p-Aromatic substituent.
b Relative configuration of fluorine and amine-containing side chain.
c No inhibition was observed.
d Could not be determined.
e Refer to the legend of Figures 3–5.
f In Figure 3B, IC50 values for 1d could not be correctly estimated, because the relative activity values at high inhibitor concentrations is still high even
after the data were corrected by subtraction of the control data. Therefore, the IC50 value for this compound was mathematically predicted from the
inhibition curve fitted by the data at below 60 lM inhibitor concentrations (Fig. 3B).
g From Ref. 8.
h The term ÔirreversibleÕ was used when both time- and concentration-dependent inhibitions were observed under conditions described in
Experimental.
-1a
-1b
-1c
-1d
120
100
80
60
40
20
0
(Z)
(Z)
(Z)
(Z)
(Z)-1e
4
-1a
(E)
A
120
100
80
60
40
20
0
A
(E)-1b
(E)
-1c
(E)
-1d
(E)
-1e
0
20
40
60
80
100
120
140
160
0
2
4
6
8
Inhibitor (µM)
Inhibitor (µM)
120
100
80
60
40
20
0
B
(Z)
(Z)
(Z)
(Z)
(Z)
120
100
80
60
40
20
0
-1a
B
-1b
-1c
-1d
-1e
4
-1a
(Z)
(Z)-1b
(Z)
(Z)
(Z)
-1c
-1d
-1e
4
0
20
40
60
80
100
120
140
160
Inhibitor (µM)
0
2
4
6
8
Figure 4. Inhibition curves of MAO B by p-aromatic substituted (Z)-2-
fluoro-1-phenylcyclopropylamines and 2,2-difluoro-1-phenylcycloprop-
ylamine. (A) Without correction by the control data. (B) Subtracted
control data from complete experiments data. For (Z)-1b,c,e the
relative activity values at inhibitor concentrations below 60 lM were
used. For (Z)-1a, data at inhibitor concentrations below 100 lM and
for (Z)-1d, data at concentration below 70 lM were used to obtain
exponential fitting curves, because good correlations were not obtained
including the data at higher concentrations. For 4, values at all
inhibitor concentrations were used for fitting. IC50 values were
calculated by using these fitting curves (see Table 1).
Inhibitor (µM)
Figure 5. Inhibition curves of MAO A by p-aromatic substituted (E)-
and (Z)-2-fluoro-1-phenylcyclopropylamines, and 2,2-difluoro-1-phen-
ylcyclopropylamine. (A) (E)-1a–e. (B) (Z)-1a–e and difluoroanalogue
4. For all compounds, except for (E)-1a, the relative activity values at
all inhibitor concentrations were used to obtain exponential fitting
curves. For (E)-1a, the values at inhibitor concentrations below 4 lM
of were used for fitting, because good correlations were not obtained
including the data above this concentration. IC50 values were
calculated by using these fitting curves (see Table 1).

2494
S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
120
120
100
80
60
40
20
0
-1a
-1b
-1c
-1d
-1e
(E)
(E)
(E)
(E)
(E)
A
A
(1R,2S)-1a
100
(1S,2R)-1a
80
60
40
20
0
0
20
40
60
80
100
Inhibitor (µM)
0
2
4
6
8
-1a
-1b
-1c
-1d
-1e
(Z)
(Z)
(Z)
(Z)
(Z)
4
Inhibitor (µM)
120
100
80
60
40
20
0
B
(1R,2S)-1a
(1S,2R)-1a
120
100
80
60
40
20
0
B
0
20
40
60
Inhibitor (µM)
80
100
Figure 7. Effect of concentration of 2-fluoro-1-phenylcyclopropylam-
ines on inhibition of tyramine oxidase (A: (E)-isomers; B: (Z)-isomers).
cause of the greater activity as an inhibitor of MAO B
shown by the (Z)-diastereomers, this effect on selectivity
is more pronounced with the (E)-diastereomers. There
was no obvious correlation with electronic effects on
the effect of para-substituents on activity with either en-
zyme. Both electron donating (Me, OMe) and electron
withdrawing (Cl, F) substituents increased activity, par-
ticularly toward MAO B.
0
50
100
150
Inhibitor (µM)
Figure 6. Effect of enantiomers of 2-fluoro-1-phenylcyclopropylamine
((1S,2R)-1a, (1R,2S)-1a) on monoamine oxidase activity (A: for MAO
A; B: for MAO B).
The significant loss of potency of inhibition of MAO B
that results from fluorine substitution is also notewor-
thy. This result makes more striking the effect of fluorine
substitution on MAO A versus MAO B selectivity. Be-
Enzyme-mediated opening of the cyclopropane ring has
been implicated in the inhibition mechanism of 1- and 2-
phenylcyclopropylamines. This is supported by the iso-
Table 2. IC50 values and inhibition type for p-aromatic substituted 2-fluoro-1-phenylcyclopropylamines (tyramine oxidase)
Compound
Ra
Isomer typeb
IC50 (lM)
Inhibition typee
(E)-1a
(Z)-1a
(E)-1b
(Z)-1b
(E)-1c
(Z)-1c
(E)-1d
(Z)-1d
(E)-1e
(Z)-1e
4
H
trans
cis
NIc
NIc
NIc
NDd
H
NDd
F
trans
cis
NDd
F
Cl
110
NIc
NIc
NIc
0
Irreversible
NDd
NDd
NDd
trans
cis
Cl
CH3
CH3
OMe
OMe
—
trans
cis
220 10
NIc
NIc
NDd
Irreversible
NDd
NDd
NDd
trans
cis
—
a p-Aromatic substituent.
b Relative configuration of fluorine and amine-containing side chain.
c No inhibition was observed.
d Could not be determined.
e The term ÔirreversibleÕ was used when both time- and concentration-dependent inhibitions were observed under conditions described in
Experimental.

S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
2495
The clear solution was filtered and the solvent was re-
moved at reduced pressure to give an oil. The oil was
dissolved in acetone (150 mL) and irradiated (Rayonette
F
H
F
H
H
F
H
F
NH2
NH2
NH2
NH2
˚
apparatus) at 3500 A for 4 days. The solvent was
removed at reduced pressure and the residue was puri-
fied by chromatography on silica gel (n-hexane/ethyl
acetate, 20:1) to give cyclopropane 5a as a mixture of
(E)- and (Z)-isomers (13.1 g, 86%, E:Z = 4:1).
(1S,2R)-1a
(1S,2S)-1a
(1R,2S)-1a
(1R,2R)-1a
Figure 8. Position of the fluorine substituent in the four isomers of
2-fluoro-1-phenylcyclopropylamine.
4.2.1. Compound 5a (Ar = Ph) mixture of (E)- and (Z)-
isomers. H NMR (CDCl3/TMS): 7.4–7.2 (m, 5H), 5.08
lation of ring-opened products covalently attached to
the enzyme.3,4,9 A fluorine substituent should facilitate
this ring opening due to the increased ring strain.10
However, this does not explain the unusual MAO A
selectivity exhibited by these compounds. In addition,
the expectation that the presence of two fluorine substit-
uents would increase activity was not realized. It is
apparent that more detailed examination of the behav-
ior of these compounds will be required to determine
the mechanism of increased MAO A inhibition, and
such experiments will be pursued.
1
(ddd, J = 65.4, 6.3, 3.6 Hz, 0.8H), 4.86 (ddd, J = 64.2,
6.0, 3.9Hz, 0.2H), 4.2–4.0 (m, 2H), 2.37 (ddd,
J = 22.5, 6.9, 3.6 Hz, 0.2H), 1.89 (dt, J = 13.2, 6.6 Hz,
0.8H), 1.72 (ddd, J = 21.9, 6.6, 3.6 Hz, 0.8H), 1.46 (dt,
J = 13.5, 6.6 Hz, 0.2H), 1.4–1.1 (m, 3H).
4.2.2. Compound 5b (Ar = p-FC6H4) mixture of (E)- and
(Z)-isomers. H NMR (CDCl3/TMS): 7.4–7.0 (m, 4H),
1
5.07 (ddd, J = 65.1, 6.0, 3.3 Hz, 0.8H), 4.82 (ddd,
J = 64.8, 6.3, 3.6 Hz, 0.2H), 4.4–4.0 (m, 2H), 2.37
(ddd, J = 22.5, 7.2, 3.9Hz, 0.2H), 1.88 (dt, J = 13.5,
6.6 Hz, 0.8H), 1.68 (ddd, J = 21.9, 6.9, 3.6 Hz, 0.8H),
1.5–1.4 (m, 0.2H), 1.3–1.1 (m, 3H).
4. Experimental
4.1. General
4.2.3. Compound 5c (Ar = p-ClC6H4) mixture of (E)- and
(Z)-isomers. H NMR (CDCl3/TMS): 7.4–7.2 (m, 4H),
All reactions were performed under an atmosphere of
N2 in oven (90 ꢁC) dried glassware unless otherwise sta-
ted. Anhydrous solvents were purchased from Aldrich
and used as received. Chromatography was performed
1
5.07 (ddd, J = 65.1, 6.3, 3.6 Hz, 0.8H), 4.81 (ddd,
J = 64.5, 6.0, 3.6 Hz, 0.2H), 4.3–4.0 (m, 2H), 2.38
(ddd, J = 22.5, 7.2, 3.9Hz, 0.2H), 1.89 (dt, J = 13.2,
6.3 Hz, 0.8H), 1.68 (ddd, J = 21.9, 6.9, 3.6 Hz, 0.8H),
1.5–1.4 (m, 0.2H), 1.3–1.1 (m, 3H).
˚
on ICN SiliTech 60 A silica gel purchased from Bod-
1
man. H, 13C, and 19F NMR spectra were recorded on
a Varian Gemini-300 FT spectrometer. 19F chemical
shifts are expressed in d ppm upfield (minus sign) from
CCl3F. Melting points were determined on a Thomas–
Hoover capillary melting point apparatus and are
uncorrected. Optical rotations were determined on a
Perkin–Elmer Polarimeter 241. Chiral HPLC was per-
formed on an HPLC system from GBC using an analyt-
ical CHIRACEL OD column.
4.2.4. Compound 5d (Ar = p-CH3C6H4) mixture of (E)-
and (Z)-isomers. H NMR (CDCl3/TMS): 7.3–7.0 (m,
1
4H), 5.06 (ddd, J = 65.4, 6.3, 3.6 Hz, 0.8H), 4.83 (ddd,
J = 64.8, 6.6, 3.9Hz, 0.2H), 4.3–4.0 (m, 2H), 2.35 (s,
0.8 · 3H), 2.33 (s, 0.2 · 3H), 2.34 (ddd, J = 22.5, 7.2,
3.6 Hz, 0.2H), 1.85 (dt, J = 13.2, 6.6 Hz, 0.8H), 1.70
(ddd, J = 21.6, 6.6, 3.3 Hz, 0.8H), 1.48–1.38 (m, 0.2H),
1.3–1.1 (m, 3H).
The X-ray data set was collected with an Enraf-Nonius
CAD4 diffractometer. Programs used: data collection
EXPRESS (Nonius, B. V., 1994), data reduction
MOLEN (Fair, K.; Enraf-Nonius B.V., 1990), structure
solution SHELXS-97 (Ref. 15), structure refinement
SHELXL-97 (Sheldrick, G. M. Universita¨t Go¨ttingen,
1997), graphics SCHAKAL (Keller, E. Universita¨t Frei-
burg, 1997).
4.2.5. Compound 5e (Ar = p-CH3OC6H4) mixture of (E)-
and (Z)-isomers. 1H NMR (CDCl3/TMS): 7.27 (d,
J = 8.4 Hz, 2H), 6.89(d, J = 8.7 Hz, 2H), 5.06 (ddd,
J = 65.4, 6.3, 3.3 Hz, 0.8H), 4.82 (ddd, J = 66.0, 6.6,
3.9Hz, 0.2H), 4.4–4.0 (m, 2H), 3.81 (s, 0.8 · 3H), 3.80
(s, 0.2 · 3H), 2.33 (ddd, J = 23.1, 7.2, 3.9Hz, 0.2H),
1.85 (dt, J = 13.2, 6.3 Hz, 0.8H), 1.68 (ddd, J =
21.6, 6.6, 3.6 Hz, 0.8H), 1.5–1.3 (m, 0.2H), 1.3–1.1 (m,
3H).
4.2. 1-Aryl-2-fluoro-1-ethoxycarbonylcyclopropane (5).
Typical procedure
Our published procedure6 with modifications was used.
An ethereal solution of diazomethane, prepared in situ
from diazald (17.1 g, 79.6 mmol), was added dropwise
to a solution of ethyl 2-phenyl-3-fluoroacrylate (15 g,
70.4 mmol), prepared according to the literature proce-
dure,5 in anhydrous ether while stirring at 0 ꢁC. After
addition, the reaction mixture was allowed to warm to
room temperature and was stirred overnight. The excess
diazomethane was removed by treatment with MgSO4.
4.2.6. Compound 5f (R1 = R2 = F, R3 = H). 1H NMR
(CDCl3/TMS): 7.4–7.2 (m, 5H), 4.17 (q, J = 7.22 Hz,
2H), 2.62 (ddd, J = 12.6, 8.1, 6.0 Hz, 1H), 1.92 (ddd,
J = 12.9, 7.8, 4.2 Hz, 1H), 1.21 (t, J = 7.2 Hz, 3H); 13C
NMR (CDCl3/TMS, 75 MHz): 167.05, 130.46, 130.43,
1
128.49, 128.42, 111.23 (t, JCF = 289.1 Hz), 62.27,
2
39.72 (t, JCF = 10.9Hz), 21.93 (t, 1JCF = 10.0 Hz),
14.01.

2496
S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
4.3. 2-Fluoro-1-phenylcyclopropane carboxyhydrazide
(6a). Typical procedure
2H), 2.80 (dt, J = 14.4, 7.2 Hz, 1H), 1.84 (ddd,
J = 12.6, 7.8, 4.8 Hz, 1H).
Hydrazine monohydrate (30 equiv, 54 mL) was added to
a solution of an (E/Z)-mixture of ethyl 2-fluoro-1-phen-
ylcyclopropanecarboxylate (5a) (7.7 g, 37 mmol) in
EtOH (50 mL). After the reaction mixture was stirred
overnight, the solvent was removed at reduced pressure.
The residue was purified by chromatography on silica
gel (DCM/MeOH, 20:1) to give 6.9g (96%, ( E/
Z) = 4:1) of carboxyhydrazide 6a as a colorless oil.
Small amount of pure (E)- and (Z)-isomers were ob-
tained by careful chromatography for characterization.
4.4. t-Butyl (E)- and (Z)-2-fluoro-1-arylcyclopropane
carbamate (8a). Typical procedure
A
suspension of carboxyhydrazide 6a (1.53 g,
7.88 mmol) in water (30 mL) was topped with 30 mL
of ether. Then 6 N HCl (19.5 mL) was added dropwise
at 0 ꢁC. After several minutes, an aqueous solution of
sodium nitrite (1.0 M, 12 mL) was added dropwise.
The reaction mixture was stirred for additional 40 min
at 0 ꢁC. The mixture was extracted with ether, washed
with brine, dried over MgSO4, and evaporated at re-
duced pressure. The residue was dissolved in anhydrous
tert-butanol, and the solution was refluxed overnight.
The solvent was evaporated, and the residue was puri-
fied by column chromatography on silica gel (n-hex-
ane/ethyl acetate, 9:1) to give (Z)-8a (173 mg, 9%,
white solid) and (E)-8a (360 mg, 18%, white solid).
4.3.1. Compound 6a (Ar = C6H5) mixture of (E)- and (Z)-
isomers. H NMR (CDCl3/TMS): 7.5–7.3 (m, 5H), 6.68
1
(br, 0.2H), 6.54 (br, 0.8H), 5.13 (ddd, J = 65.4, 6.3,
3.6 Hz, 0.8H), 4.91 (ddd, J = 63.6, 6.3, 3.9Hz, 0.2H),
3.75 (br, 2H), 2.49(ddd, J = 22.8, 6.9, 3.9 Hz, 0.2H),
1.92 (dt, J = 13.8, 6.6 Hz, 0.8H), 1.60 (ddd, J = 22.2,
6.3, 3.3 Hz, 0.8H), 1.39(dt, J = 13.2, 6.3 Hz, 0.2H).
1
4.4.1. Compound (E)-8a (Ar = Ph). H NMR (CDCl3/
TMS): 7.5–7.2 (m, 5H), 5.1 (br, 1H), 4.87 (br d,
J = 66.0 Hz, 1H), 1.7–1.2 (m, 2H), 1.43 (s, 9H).
4.3.2. Compound 6b (Ar = p-FC6H4) mixture of (E)- and
(Z)-isomers (E/Z = 4:1). H NMR (CDCl3/TMS), 7.4–
1
7.3 (m, 2H), 7.2–7.0 (m, 2H), 6.6 (br, 0.2H), 6.5 (br,
0.8H), 5.11 (ddd, J = 65.1, 6.3, 3.6 Hz, 0.8H), 4.88
(ddd, J = 64.8, 6.6, 3.9Hz, 0.2H), 3.78 (br, 2H), 2.45
(ddd, J = 22.8, 7.2, 4.2 Hz, 0.2H), 1.93 (dt, J = 13.8,
6.3 Hz, 0.8H), 1.56 (ddd, J = 22.5, 6.3, 3.3 Hz, 0.8H),
1.37 (dt, J = 13.2, 6.6 Hz, 0.2H).
1
4.4.2. Compound (Z)-8a. H NMR (CDCl3/TMS): 7.4–
7.2 (m, 5H), 5.37 (br, 1H), 4.67 (br d, J = 62.4 Hz,
1H), 1.8–1.2 (m, 2H), 1.45 (s, 9H).
4.4.3. Compound (E)-8b (Ar = p-FC6H4). 1H NMR
(CDCl3/TMS): 7.6–7.4 (m, 2H), 7.1–6.9(m, 2H), 5.03
(br, 1H), 4.85 (br d, J = 64.5 Hz, 1H), 1.64 (ddd,
J = 22.5, 8.1, 3.6 Hz, 2H), 1.6–1.4 (m, 1H), 1.41 (s, 9H).
4.3.3. Compound 6c (Ar = p-ClC6H4) (E)-isomer. 1H
NMR (CDCl3/TMS): 7.41 (d, J = 8.4 Hz, 2H), 7.34 (d,
J = 8.4 Hz, 2H), 6.51 (br, 1H), 5.12 (ddd, J = 65.1, 6.3,
3.3 Hz, 1H), 3.78 (br, 2H), 1.93 (dt, J = 13.5, 6.3 Hz,
1H), 1.57 (ddd, J = 22.5, 6.6, 3.6 Hz, 1H). (Z)-Isomer:
1H NMR (CDCl3/TMS): 7.4–7.2 (m, 4H), 5.23 (br,
1H), 4.88 (ddd, J = 64.8, 6.6, 3.9Hz, 1H), 3. 31 (br,
2H), 2.41 (ddd, J = 22.8, 7.2, 3.9Hz, 1H), 1.4–1.2 (m,
1H).
1
4.4.4. Compound (Z)-8b. H NMR (CDCl3/TMS): 7.3–
7.1 (m, 2H), 7.1–6.9(m, 2H), 5.36 (br, 1H), 4.64 (br d,
J = 63.9Hz, 1H), 1.8–1.2 (m, 2H).
4.4.5. Compound (E)-8c (Ar = p-ClC6H4). 1H NMR
(CDCl3/TMS): 7.4–7.2 (m, 4H), 5.1 (br, 1H), 4.86 (br,
d, J = 65.1 Hz, 1H), 1.7–1.2 (m, 2H), 1.41 (s, 9H). MS-
FAB 286.2 (MH+).
4.3.4. Compound 6d (Ar = p-CH3C6H4) mixture of (E)-
and (Z)- isomers (E/Z = 4:1). H NMR (CDCl3/TMS):
1
1
4.4.6. Compound (Z)-8c. H NMR (CDCl3/TMS): 7.28
(d, J = 7.3 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 5.38 (br,
1H), 4.63 (br d, J = 63.6 Hz, 1H), 1.8–1.2 (m, 2H).
8.05 (br, 0.2H), 7.95 (br, 0.8H), 7.4–7.2 (m, 4H), 5.19
(ddd, J = 65.4, 6.0, 3.6 Hz, 0.8H), 4.92 (ddd, J = 64.8,
6.3, 3.9Hz, 0.2H), 2.57 (ddd, J = 22.8, 6.9, 3.9 Hz,
0.2H), 2.39(s, 0.8 · 3H), 2.36 (s, 0.2 · 3H), 2.1–1.9(m,
0.8H), 1.60 (ddd, J = 22.5, 6.6, 3.6 Hz, 0.8H), 1.5–1.3
(m, 0.2H).
1
4.4.7. Compound (E)-8d (Ar = p-CH3C6H4). H NMR
(CDCl3/TMS): 7.5–7.2 (m, 4H), 5.0 (br, 1H), 4.86 (br
d, J = 65.1 Hz, 1H), 2.34 (s, 3H), 1.7–1.2 (m, 2H), 1.41
(s, 9H).
1
4.3.5. Compound 6e (Ar = p-CH3OC6H4) (E)-isomer. H
1
4.4.8. Compound (Z)-8d. H NMR (CDCl3/TMS): 7.2–
7.0 (m, 4H), 5.35 (br, 1H), 4.63 (br d, J = 62.1 Hz,
1H), 2.31 (s, 3H), 1.8–1.2 (m, 2H), 1.44 (s, 9H).
NMR (CDCl3/TMS): 7.31 (d, J = 8.7 Hz, 2H), 6.94 (d,
J = 8.7 Hz, 2H), 6.56 (br, 1H), 5.10 (ddd, J = 65.4, 6.3,
3.6 Hz, 1H), 3.83 (s, 3H), 1.88 (dt, J = 13.5, 6.3 Hz,
1H), 1.56 (ddd, J = 22.2, 6.3, 3.6 Hz, 1H). (Z)-Isomer:
1H NMR (CDCl3/TMS): 7.3–7.2 (m, 2H), 7.0–6.9(m,
2H), 6.78 (br, 1H), 4.87 (ddd, J = 65.1, 6.3, 3.9Hz,
1H), 3.83 (s, 3H), 2.44 (ddd, J = 22.8, 6.9, 3.6 Hz, 1H),
1.35 (dt, J = 13.2, 6.6 Hz, 1H).
1
4.4.9. Compound (E)-8e (Ar = p-CH3OC6H4). H NMR
(CDCl3/TMS): 7.40 (d, J = 8.1 Hz 2H), 6.88 (d,
J = 8.7 Hz, 2H), 5.0 (br, 1H), 4.82 (br d, J = 66 Hz,
1H), 3.80 (s, 3H), 1.7–1.2 (m, 2H), 1.41 (s, 9H).
4.3.6. Compound 6f (R1 = R2 = F, R3 = H). 1H NMR
(CDCl3/TMS): 7.5–7.3 (m, 5H), 6.71 (br, 1H), 4.82 (br,
1
4.4.10. Compound (Z)-8e. H NMR (CDCl3/TMS): 7.17
(d, J = 8.4 Hz, 2H), 6.85 (d, J = 9.6 Hz, 2H), 5.35 (br,

S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
2497
1H), 4.63 (br d, J = 64.2 Hz, 1H), 3.79(s, 3H), 1.7–1.2
(m, 2H), 1.43 (s, 9H).
2H), 5.21 (ddd, J = 61.8, 7.2, 3.6 Hz, 1H), 3.87 (s, 3H),
2.0–1.7 (m, 2H).
1
4.4.11. Compound 8f (R1 = R2 = F, R3 = H). H NMR
4.5.10. Compound (Z)-1e. 1H NMR (D2O): 7.49(d,
J = 15.3 Hz, 2H), 7.07 (d, J = 15.3 Hz, 2H), 5.08 (ddd,
J = 63.0, 6.6, 3.3 Hz, 1H), 3.86 (s, 3H), 1.9–1.7 (m, 2H).
(CDCl3/TMS): 7.5–7.2 (m, 5H), 5.46 (br, 1H), 2.17 (br,
1H), 1.94 (br, 1H), 1.40 (s, 9H); 13C NMR (CDCl3/
TMS, 75 MHz): 154.81, 135.17, 128.61, 128.19, 128.14,
1
112.42 (t, JCF = 289.2 Hz), 80.70, 42.48, 28.41, 24.36;
4.5.11. Compound 4 (R1 = R2 = F, R3 = H). 1H NMR
(D2O): 7.4–7.2 (m, 5H), 5.42 (br), 2.17 (br, 1H), 1.93
(br, 1H).
19F NMR (CDCl3/TMS, 282 MHz): ꢀ137.7 (d,
J = 152.4 Hz), ꢀ140.1 (d, J = 155.8 Hz).
4.5. (E)-2-Fluoro-1-phenylcyclopropylamine hydrochlo-
ride (1a). Typical procedure
4.6. The resolution of (E)-2-fluoro-1-phenylcyclopropyl-
amine ((E)-1a)
Using a procedure modified from our previous report,6
the carbamate (E)-8a (1.43 mmol, 360 mg) was dissolved
in 7.2 mL of 2 N HCl in ether. The reaction mixture was
stirred overnight at room temperature. The mixture was
centrifuged and the precipitate was collected and washed
with ether to give a white solid (178 mg, 66%).
(E)-2-Fluoro-1-phenylcyclopropylamine hydrochloride
((E)-1a) (439mg, 2.34 mmol), ( R)-O-acetylmandelic acid
(908 mg, 4.68 mmol) and DMAP (144 mg, 0.94 mmol)
were dissolved in anhydrous dichloromethane (40 mL)
and cooled to 0 ꢁC in an ice bath. A solution of dicy-
clohexylcarbodiimide (1.93 g, 9.36 mmol) in dichloro-
methane (15 mL) was added dropwise. The reaction
mixture was gradually warmed to rt and stirred for
24 h. The white precipitate that formed was removed
by filtration and the filtrate was concentrated in vacuo.
The residue was purified by column chromatography
on silica gel (n-hexane/ethyl acetate, 4:1) to give
568 mg (74%, dr = 1:1, determined by 1H NMR) of
(R)-O-acetylmandelic cyclopropylamides (9a,b) as a
white solid.
4.5.1. Compound (E)-1a (Ar = Ph). Mp 159 ꢁC (de-
comp.). H NMR (D2O): 7.5–7.2 (m, 5H), 5.26 (ddd,
1
J = 62.1, 7.5, 3.6 Hz, 1H), 1.97 (ddd, J = 23.4, 9.6,
3.6 Hz, 1H), 1.83–1.75 (m, 1H). 19F NMR (D2O,
282 MHz): ꢀ214.6 (ddd, J = 62.6, 24.7, 13.7 Hz).
4.5.2. Compound (Z)-1a. Mp 154 ꢁC (decomp.). 1H
NMR (D2O): 7.51 (br, 4H), 5.12 (ddd, J = 63.0, 6.6,
3.6 Hz, 1H), 2.0–1.7 (m, 2H).
To a solution of O-acetylmandelic amides 9a,b (470 mg,
1.44 mmol) in methanol (22 mL) was added triethyl-
amine (220 lL, 1.58 mmol). The resulting mixture was
stirred at rt for 3 d. The solvent was removed under re-
duced pressure and the residue was carefully purified by
column chromatography on silica gel (n-hexane/ethyl
acetate, 4:1 then 7:3) to give 155 mg (38%) mandelic
amide 10a (Rf = 5.0 (n-hexane/ethyl acetate, 1:1), dr
(10a/10b) = 94:6 (HPLC: Chiralcel OD, n-hexane/2-pro-
panol = 80/20, 0.6 mL/min; tR = 16.56 min (minor),
26.12 min (major)) followed by 231 mg mandelic amide
10b, dr (10b/10a) = 5:1 (1H NMR)), which was further
recrystallized to give amide 10b (148 mg, 36%, dr (10b/
10a) = 93:7 (HPLC: Chiralcel OD, n-hexane/2-propa-
nol = 80/20, 0.6 mL/min; tR = 16.76 min (major),
27.02 min (minor)) as colorless crystals.
4.5.3. Compound (E)-1b (Ar = p-FC6H4). 1H NMR
(D2O): 7.7–7.5 (m, 2H), 7.4–7.2 (m, 2H), 5.26 (ddd,
J = 62.7, 7.5, 3.9Hz, 1H), 1.94 (ddd, J = 21.9, 9.3,
2.4 Hz, 1H), 1.86–1.76 (m, 1H).
1
4.5.4. Compound (Z)-1b. H NMR (D2O): 7.6–7.5 (m,
2H), 7.3–7.1 (m, 2H), 5.13 (ddd, J = 63.0, 6.6, 3.3 Hz,
1H), 2.0–1.6 (m, 2H).
4.5.5. Compound (E)-1c (Ar = p-ClC6H4). Mp 164 ꢁC
(decomp.). H NMR (D2O): 7.62 (d, J = 8.7 Hz, 2H),
1
7.56 (d, J = 8.4 Hz, 2H), 5.33 (ddd, J = 61.8, 7.5,
3.9Hz, 1H), 1.7–1.2 (m, 2H). FAB-MS 186.0 (M +).
HRMS (FAB): calcd for (M+, C9H10ClFN) 186.0486.
Found: 186.0484.
4.5.6. Compound (Z)-1c. Mp 146 ꢁC (decomp.). 1H
NMR (D2O): 7.6–7.4 (m, 4H), 5.11 (ddd, J = 63.0, 6.6,
3.6 Hz, 1H), 2.0–1.7 (m, 2H). HRMS (FAB): calcd for
(M+, C9H10ClFN) 186.0486. Found: 186.0486.
4.6.1. Amide 10a. White solid, mp 121–123 ꢁC,
20
1
½aꢁ ꢀ57.6 (c 0.217, CH2Cl2). H NMR (CDCl3/TMS):
D
7.4–7.2 (m, 9H), 6.59 (s, 1H), 4.97 (d, J = 3.3 Hz, 1H),
4.86 (dd, J = 63.3, 6.9, 3.3 Hz, 1H), 3.36 (d,
J = 3.6 Hz, 1H), 1.80 (ddd, J = 22.8, 8.4, 3.6 Hz, 1H),
1.41 (ddd, J = 13.2, 8.4, 6.9Hz, 1H). 13C NMR
(CD3OD): 175.79, 141.57, 138.08, 129.52, 129.25,
129.12, 128.35, 127.96, 77.43 (d, J = 226.5 Hz), 75.66,
39.25 (d, J = 12.5 Hz), 21.13 (d, J = 10.3 Hz).
1
4.5.7. Compound (E)-1d (Ar = p-CH3C6H4). H NMR
(D2O): 7.56 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.1 Hz,
2H), 5.26 (ddd, J = 62.1, 7.2, 3.6 Hz, 1H), 2.41 (s, 3H),
1.95 (ddd, J = 23.1, 9.6, 3.3 Hz, 1H), 1.86–1.75 (m, 1H).
4.5.8. Compound (Z)-1d. 1H NMR (D2O): 7.43 (d,
J = 8.1 Hz, 2H), 7.36 (d, J = 7.8 Hz, 2H), 5.1 (br d,
J = 63 Hz, 1H), 2.38 (s, 3H), 2.0–1.7 (m, 2H).
4.6.2. Amide 10b. Colorless crystals, mp 121–123 ꢁC,
20
1
½aꢁ ꢀ35.7 (c 0.213, CH2Cl2). H NMR (CDCl3/TMS):
D
7.4–7.2 (m, 9H), 6.67 (s, 1H), 4.94 (d, J = 3.6 Hz, 1H),
4.94 (dd, J = 63.0, 6.6, 3.3 Hz, 1H), 3.40 (d,
J = 3.3 Hz, 1H), 1.74 (ddd, J = 22.5, 8.4, 3.6 Hz, 1H),
1.44 (ddd, J = 12.9, 8.1, 6.6 Hz, 1H). 13C NMR
1
4.5.9. Compound (E)-1e (Ar = p-CH3OC6H4). H NMR
(D2O): 7.59(d, J = 9.0 Hz, 2H), 7.11 (d, J = 8.4 Hz,

2498
S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
(CD3OD): 175.76, 141.55, 138.10, 129.57, 129.50,
129.23, 129.12, 128.35, 127.93, 77.50 (d, J = 227.0 Hz),
75.68, 39.22 (d, J = 12.5 Hz), 21.22 (d, J = 10.2 Hz).
umn, Amersham Biosciences) pre-equilibrated with
50 mM potassium phosphate (pH 7.2) containing 0.8%
octyl-glucoside. The activity of MAO A was measured
spectrometrically at 25 ꢁC by the modified method of Li
et al.11 using 0.7 mL of standard reaction mixture
containing 1 mM kynuramine hydrobromide, 50 mM
potassium phosphate buffer (pH 7.2), 0.5% Triton · 100
(reduced), 6% DMSO and MAO A. The reaction was
monitored at 316 nm, which is the maximum absorption
wavelength of 4-hydroxyquinoline. The enzyme activity
was calculated by using 12,300 Mꢀ1 cmꢀ1 as the extinc-
tion coefficient of 4-hydroxyquinoline at 316 nm. One
unit of the enzyme oxidizes 1 lmol of kynuramine to
4-hydroxyquinoline per 1 min.
4.6.3.
(+)-(1S,2R)-2-Fluoro-1-phenylcyclopropylamine
hydrochloride ((+)-(1S,2R)-1a). A suspension of amide
10a (66 mg, 0.23 mmol) in 2.3 mL 2 N HCl (aq) was
heated to reflux until it becomes a clear solution (about
1 day). The solvent was removed under reduced pressure
and the residue was washed with ether and collected by
centrifugation to give a white solid (34.7 mg, 80.4%).
1
The H NMR spectrum is identical to racemic (E)-1a.
20
D
Mp 169 ꢁC (decomp.), ½aꢁ +28.3 (c 0.13, H2O).
4.6.4.
hydrochloride ((ꢀ)-(1R,2S)-1a). This enantiomer was
(ꢀ)-(1R,2S)-2-Fluoro-1-phenylcyclopropylamine
The activity of MAO B was measured spectrophotomet-
rically at 25 ꢁC by using 0.7 mL of standard reaction
mixture containing 1 mM benzylamine, 0.1 M potas-
sium phosphate buffer (pH 7.2), 6% DMSO, and MAO
B. The reaction was monitored at 250 nm, which is the
maximum absorption wavelength of benzaldehyde.
The enzyme activity was calculated by using
13,800 Mꢀ1 cmꢀ1 as extinction coefficient of benzalde-
hyde at 250 nm. One unit of the enzyme oxidizes 1 lmol
of benzylamine to benzaldehyde per 1 min.
prepared from amide 10b by the same procedure as
above. The H NMR spectrum is identical to racemic
1
20
D
1a. Mp 169 ꢁC (decomp.), ½aꢁ ꢀ35.7 (c 0.20, H2O).
4.7. X-ray crystallographic study
4.7.1. N-[(1R,2S)-2-Fluoro-1-phenylcyclopropyl]-(2R)-2-
hydroxy-2-phenyl-acetamide (10b). Formula C17H16-
FNO2, M = 285.31, colorless crystal 0.25 · 0.20 ·
˚
0.15 mm, a = 8.965(1), b = 9.835(1), c = 15.674(1) A,
Each inhibitor was dissolved in DMSO and diluted with
the same solvent to give the appropriate concentration.
The solution was immediately divided into several vials
and wrapped with aluminum foil. These vials were
stocked in an ice bath until used for inhibition experi-
ments. Inhibition experiments were carried out as fol-
lows: varying concentrations of inhibitor were added
to the reaction mixture described above (without sub-
strate), and allowed to stand for 10 min at 10 ꢁC. The
reaction was started by the addition of substrate stock,
and the time course of the absorption increase of the
reaction product was monitored as described above.
Control experiments were carried out by using the reac-
tion mixture omitting both benzylamine and MAO B
from the standard assay solution.
V = 1382.0(2) A , qcalcd = 1.371 g cmꢀ3, l = 8.16 cmꢀ1
,
3
˚
empirical absorption correction via
w scan data
(0.822 6 T 6 0.887), Z = 4, orthorhombic, space group
˚
P212121 (No. 19), k = 1.54178 A, T = 223 K, x/2h scans,
1630 reflections collected (ꢀh, ꢀk, ꢀl), [(sinh)/
k]=0.62 Aꢀ1, 1630 independent and 1446 observed
˚
reflections [I P 2r(I)], 196 refined parameters, R =
0.037, wR2 = 0.113, Flack parameter 0.2(3), max. resid-
ual electron density 0.18 (ꢀ0.19) e Aꢀ3, hydrogens cal-
˚
culated and refined as riding atoms.
Crystallographic data (excluding structure factors) for
this structure have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publica-
tion CCDC-254590. Copies of the data can be obtained
free of charge on application to The Director, CCDC,
12 Union Road, Cambridge CB2 1EZ, UK [fax: int.
code +44 (1223) 336 033, e-mail: deposit@
ccdc.cam.ac.uk].
4.8.2. Tyramine oxidase. Tyramine oxidase was pur-
chased from Sigma and dissolved in 25 mM potassium
phosphate buffer (pH 7.2). The enzyme activity was
measured spectrophotometrically at 30 ꢁC as described
in the MAO B assay method, using 0.7 mL of standard
reaction mixture containing 0.6 mM benzylamine, 0.1 M
potassium phosphate buffer (pH 7.2), 6% DMSO, and
tyramine oxidase. Inhibition experiments were carried
out using the same method as for MAO B after letting
the mixture stand for 10 min at 25 ꢁC without substrate.
Control experiments were also carried out by using the
reaction mixture omitting both benzylamine and tyra-
mine oxidase from the standard assay solution. One unit
of the enzyme oxidizes 1 lmol of benzylamine to benzal-
dehyde per 1 min.
4.8. Enzyme assay
4.8.1. MAO A and MAO B. Stock solutions of human
liver mitochondrial outer membrane MAO A and
MAO B, expressed in the methylotrophic yeast, Pichia
pastoris, were kindly provided by Professor Dale E.
Edmondson, Departments of Biochemistry and Chemis-
try, Emory University, Atlanta, GA, USA. Enzyme
assays for MAO A and MAO B were performed by
modified procedures of Li et al.11 and Houslay and Tip-
ton,12 respectively, as described in our previous publica-
tion.8 Protein concentration was determined by the
method of Bradford.13
4.8.3. Time- and concentration-dependent inhibition
experiments. Time- and concentration-dependent inhibi-
tion experiments were carried out by the previously de-
scribed method of Kitz and Wilson.14 The incubation
of MAO A with inhibitor was carried out at 4 ꢁC in
Prior to use, the stock solution of MAO A was passed
through a gel-filtration column (PD 10 desalting col-

S. Ye et al. / Bioorg. Med. Chem. 13 (2005) 2489–2499
2499
0.1 mL of 50 mM potassium phosphate (pH 7.2) con-
References and notes
taining 0.5% of Triton X-100 (reduced), 15 lg of enzyme,
6% of DMSO and different concentration of inhibitor.
The incubation of MAO B with inhibitor was carried
out at 4 ꢁC in 0.1 mL of 100 mM potassium phosphate
(pH 7.2), 10 lg of enzyme, 6% of DMSO and different
concentrations of inhibitor. A lower incubation temper-
ature (4 ꢁC) was used in this study for two reasons. First,
this reduced spontaneous inactivation of the MAO dur-
ing the incubation. In addition, for some compounds it
was necessary to work at a lower temperature in order
to reduce the rate of inactivation to a readily measurable
rate. Aliquots (20 lL) were taken out periodically from
the mixture, and diluted with 0.68 mL of each enzyme as-
say solution. The incubation of tyramine oxidase with
inhibitor was carried out at 25 ꢁC in the 0.05 mL of
100 mM potassium phosphate (pH 7.2), 12 lg of enzyme,
6% of dimethylsulfoxide and different concentrations of
inhibitor. Aliquots (10 lL) were taken out periodically
from the mixture, and diluted with 0.24 mL of each en-
zyme assay solution. The increase of absorbance at
316 nm for MAO A, and at 250 nm for MAO B and tyra-
mine oxidase was monitored as described above.
1. Singer, T. P.; von Korff, R. W.; Murphy, D. L.
Monoamine Oxidase: Structure, Function and Altered
Functions; Academic: New York, 1979.
2. Mure, M.; Mills, S. A.; Klinman, J. P. Biochemistry 2002,
41, 9269.
3. Edmondson, D. E.; Mattevi, E.; Binda, C.; Li, M.;
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Hubalek, F. Curr. Med. Chem. 2004, 11, 1983.
4. Mitchell, D. J.; Nikolic, D.; Rivera, E.; Sablin, S. O.;
Choi, S.; van Breemen, R. B.; Singer, T. P.; Silverman, R.
B. Biochemistry 2001, 40, 5447, and references cited
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5. McDonald, I. A.; Lacoste, J. M.; Bey, P.; Palfreyman, M.
G.; Zreika, M. J. Med. Chem. 1985, 28, 186.
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Haufe, G. J. Med. Chem. 2004, 47, 5860.
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Ye, S.; Haufe, G.; Kirk, K. L. Bioorg. Med. Chem. 2004,
12, 2645.
9. Mitchell, D. J.; Nikolic, D.; van Breemen, R. B.; Silver-
man, R. B. Bioorg. Med. Chem. Lett. 2001, 11, 1757.
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´
11. Li, M.; Hubalek, F.; Newton-Vinson, P.; Edmondson,
Acknowledgements
D. E. Protein Exp. Purif. 2002, 24, 152.
12. Houslay, M. D.; Tipton, K. F. Biochem. J. 1973, 135,
735.
13. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
14. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
15. Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467.
We thank Professor Dale Edmondson, Departments of
Chemistry and Biochemistry, Emory University, Atlan-
ta, GA, for the generous gift of membrane preparations
of MAO and MAO B.