Synthesis and evaluation of biaryl derivatives for structural characterization of selective monoamine oxidase B inhibitors toward Parkinson's disease therapy

Article abstract of DOI:10.1016/j.bmc.2017.11.036

Benzyloxyphenyl moiety is a common structure of highly potent, selective and reversible inhibitors of monoamine oxidase B (MAO-B), safinamide and sembragiline. We synthesized 4-(benzyloxy)phenyl and biphenyl-4-yl derivatives including halogen substituents on the terminal aryl unit. In addition, we modified the carbon linker between amine group and the biaryl linked unit. Among synthesized compounds, 12c exhibited the most potent and selective MAO-B inhibitory effect (hMAO-B IC₅₀: 8.9 nM; >10,000-fold selectivity over MAO-A) as a competitive inhibitor. In addition, 12c showed greater MAO-B inhibitory activity and selectivity compared to well-known MAO-B inhibitors such as selegiline, safinamide and sembragiline. In the MPTP-induced mouse model of Parkinson's disease (PD), 12c significantly protected the tyrosine hydroxylase (TH)-immunopositive DAergic neurons and attenuated the PD-associated behavioral deficits. This study suggests characteristic structures as a MAO-B inhibitor that may provide a good insight for the development of therapeutic agents for PD.

Full text of DOI:10.1016/j.bmc.2017.11.036

Accepted Manuscript
Synthesis and Evaluation of Biaryl Derivatives for Structural Characterization
of Selective Monoamine Oxidase B Inhibitors toward Parkinson’s Disease
Therapy
Seul Ki Yeon, Ji Won Choi, Jong-Hyun Park, Ye Rim Lee, Hyeon Jeong Kim,
Su Jeong Shin, Bo Ko Jang, Siwon Kim, Yong Sun Bahn, Gyoonhee Han, Yong
Sup Lee, Ae Nim Pae, Ki Duk Park
PII:
S0968-0896(17)32116-8
https://doi.org/10.1016/j.bmc.2017.11.036
BMC 14088
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
30 October 2017
22 November 2017
23 November 2017
Please cite this article as: Yeon, S.K., Choi, J.W., Park, J-H., Lee, Y.R., Kim, H.J., Shin, S.J., Jang, B.K., Kim, S.,
Bahn, Y.S., Han, G., Lee, Y.S., Pae, A.N., Park, K.D., Synthesis and Evaluation of Biaryl Derivatives for Structural
Characterization of Selective Monoamine Oxidase B Inhibitors toward Parkinson’s Disease Therapy, Bioorganic
&
Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.11.036
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Synthesis and Evaluation of Biaryl Derivatives for Structural
Characterization of Selective Monoamine Oxidase B Inhibitors toward
Parkinson’s Disease Therapy
a,b,1
a,b,1
a,1
a,c,1
a,b
Seul Ki Yeon , Ji Won Choi , Jong-Hyun Park , Ye Rim Lee , Hyeon Jeong Kim , Su
a,b
a
a,d
b
b
c
Jeong Shin , Bo Ko Jang , Siwon Kim , Yong Sun Bahn , Gyoonhee Han , Yong Sup Lee , Ae
a,c,d
a,c,d,
Nim Pae , Ki Duk Park
*
a
From the Convergence Research Center for Diagnosis, Treatment and Care system of Dementia,
b
Korea Institute of Science and Technology, Seoul, Department of Biotechnology, Yonsei University,
c
Seoul, KHU-KIST Department of Converging Science and Technology, Kyung Hee University,
d
Seoul, Division of Bio-Medical Science &Technology, KIST School, Korea University of Science
and Technology, Seoul, Republic of Korea
1
These authors contributed equally to the work.
a
Address correspondence to: Ki Duk Park, Ph.D., Convergence Research Center for Diagnosis,
Treatment and Care system of Dementia, Korea Institute of Science and Technology, 5 Hwarang-ro,
1
4-gil, Seongbuk-gu, Seoul 136-791, South Korea; Email: kdpark@kist.re.kr; TEL: 822-958-5132;
FAX: 822-958-5189

Abstract
Benzyloxyphenyl moiety is a common structure of highly potent, selective and reversible inhibitors
of monoamine oxidase B (MAO-B), safinamide and sembragiline. We synthesized 4-
(
benzyloxy)phenyl and biphenyl-4-yl derivatives including halogen substituents on the terminal aryl
unit. In addition, we modified the carbon linker between amine group and the biaryl linked unit.
Among synthesized compounds, 12c exhibited the most potent and selective MAO-B inhibitory
effect (hMAO-B IC50: 8.9 nM; >10,000-fold selectivity over MAO-A) as a competitive inhibitor. In
addition, 12c showed greater MAO-B inhibitory activity and selectivity compared to well-known
MAO-B inhibitors such as selegiline, safinamide and sembragiline. In the MPTP-induced mouse
model of Parkinson’s disease (PD), 12c significantly protected the tyrosine hydroxylase (TH)-
immunopositive DAergic neurons and attenuated the PD-associated behavioral deficits. This study
suggests characteristic structures as a MAO-B inhibitor that may provide a good insight for the
development of therapeutic agents for PD.
Keywords
Benzyloxyphenyl derivatives, MAO-B inhibitor, MPTP mouse model, Parkinson’s disease

Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized by
1
marked motor deficits such as resting tremor, bradykinesia, rigidity and postural instability. These
motor symptoms are mostly associated with dopamine (DA) depletion caused by a profound loss of
dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). However, the exact
2
-4
mechanisms causing neurodegeneration are not yet known.
Recently, several studies have reported that the expression level of monoamine oxidase B (MAO-B)
in human brain increases with age, and activity is highly increased in the substantia nigra of PD
5
,6
patients. MAOs are localized on the outer membrane of mitochondria, particularly in the liver and
7
brain, and catalyze the oxidative deamination of monoamine neurotransmitters such as dopamine. In
addition, the reaction catalyzed by MAOs result in the production of hydrogen peroxide (H
2
2
O )
8
,9
which causes oxidative stress and neuronal cell death. There are two isoforms, MAO-A and MAO-
B. MAO-A is localized in catecholaminergic neurons and selectively inhibited by low concentrations
of clorgyline (1), whereas MAO-B is abundant in serotonergic neurons and astrocytes, and
1
0-12
selectively inhibited by potent inhibitors selegiline (2) and rasagiline (3) (Fig. 1).
Fig. 1. Chemical structures of irreversible MAO-B inhibitors
Selegiline and rasagiline, selective and potent MAO-B inhibitors, are the most widely used drugs for
the therapy of PD by either mono-therapy or in combination with the levodopa, the dopamine

1
3,14
prodrug, resulting in the increase of dopamine.
However, selegiline and rasagiline are irreversible
MAO-B inhibitors and showed undesirable adverse effects in long term treatment of PD such as
hallucinations and headaches. Recent studies have been shown that the irreversibility of MAO-B
1
5
inhibition may contribute short-lived action. Accordingly, the development of selective and
reversible MAO-B inhibitors could reduce undesirable adverse effects and maintain the effectiveness
TM
in long-term use for the treatment of neurodegenerative diseases. Recently, safinamide (4, Xadago ,
Fig. 2), a highly selective and reversible MAO-B inhibitor, was approved for the treatment of mild-
to-late PD patients as an add-on therapy to stable dose of levodopa alone or in combination with
1
6
other PD medications. However, safinamide may cause disadvantageous effects due to other
biological properties such as selective sodium channel blockade, calcium channel modulation, and
1
7-20
inhibition of stimulated release of glutamate.
Sembragiline (5, RG-1577, Fig. 2) is another potent
reversible MAO-B inhibitor for the treatment of Alzheimer’s disease (AD). MAO-B activity is
linked to the production of reactive oxygen species (ROS) that can cause neuronal damage.
Sembragiline was expected to slow progression of neurodegeneration by reducing oxidative stress.
2
1,22
However, it failed to demonstrate benefit of the primary endpoint in clinical trial phase 2.
Interestingly, two recent potent and selective MAO-B inhibitors have common moieties, ((3-
fluorobenzyl)oxy)phenyl group (Fig. 2, red box).

Fig. 2. Chemical structures of reversible MAO-B inhibitors.
In this study, we asked if compounds conforming to the biaryl linked unit exhibited MAO-B
inhibitory activity. First, we synthesized substituted (4-(benzyloxy)phenyl)-methylammonium
chlorides. Next, we modified the carbon linker between amine group and the biaryl linked unit
(
Fig.3.). In addition, we prepared biphenyl-4-yl derivatives wherein the aryl linker (X) is a single
bond (Fig.3.). We reported that the substituted (4-(benzyloxy)phenyl)-ethylammonium chlorides
exhibited potent and selective MAO-B inhibitory activities. One of the compounds with the highest
potency was further evaluated for in vivo efficacy in the MPTP-induced mouse model of PD.
Fig. 3. Representative chemical structures of biaryl derivatives.
Recently, some biaryl derivatives in our study have been reported their biological activities such as
2
3
24
anticonvulsant activity and trace amine-associated receptor agonist effects . However, this is the

first report to propose structural features for the excellent activity of MAO-B inhibitors by variously
modifying the biaryl linker and the carbon linker. Thus, this study can provide insight into the
development of new potential MAO-B inhibitors for PD therapy.
Results and Discussion
2
.1. Selection of compounds
According to recent literature, safinamide (4) has emerged as a new therapeutic agent for the
treatment of PD through MAO-B inhibition. We divided safinamide into two parts (the blue and the
red boxes in Fig. 2) to determine the part that affected the MAO-B inhibitory effects. We found that
(
4-((3-fluorobenzyl)oxy)phenyl)methanaminium chloride (7f) corresponding to the red box showed
much potent than compound 15 corresponding to blue box (IC50 hMAO-B = 0.926 M vs >10 M).
Sembragiline (5, RG-1577), a reversible MAO-B inhibitor, has a ((3-fluorobenzyl)oxy)phenyl group
in common with safinamide. We observed that 4-((3-fluorobenzyl)oxy)benzenaminium chloride (10e)
corresponding to the red box in 5 inhibited MAO-B activity. Accordingly, we prepared 4-
(
benzyloxy)phenyl and biphenyl-4-yl derivatives which included various substituents on the
terminal aryl unit. We also modified the carbon linker between amine group and the biaryl linked
unit.
2
.2. Synthesis
Compounds 7 and 8 were prepared in two steps (Scheme 1). Using a slightly modified method from
the previous method, we prepared the Williamson ether coupled products 6 by treating 4-
cyanophenol with the appropriate substituted benzyl bromide and potassium carbonate (K
2
CO ).
3
Lithium aluminum hydride (LiAlH ) reduction of the nitrile group in 6a-6k afforded the amines,
4

which were immediately converted to the corresponding aminium chlorides 7a-7k. The conversion
of nitriles (6a-6k) to amides by partial hydrolysis with KOH afforded benzamide derivatives 8a-8f.
To prepare 4-nitrophenoxy benzene derivatives 9a-9f, we synthesized 4-nitrophenol and the
appropriate substituted benzyl bromide using the similar method above except reaction solvent
(
DMF) (Scheme 2). 4-nitrophenoxy benzene derivatives 9a-9f were reduced to benzenaminium
chlorides 10a-10f by platinum (II) oxide under hydrogen gas (Scheme 2). Compounds 11 were
prepared by Williamson ether coupling using tert-butyl (4-hydroxyphenethyl)carbamate and the
substituted benzyl bromide. Deprotection of boc-group by 4.0M hydrogen chloride in dioxane gave
ethanaminium chlorides 12a-12j (Scheme 3). The (biphenyl-4-yl)ethanaminum chlorides were
prepared using Suzuki coupling of N-boc-2-(4-bromophenyl)ethylamine (13) with the appropriate,
commercially available substituted aryl boronic acids to give the boc-protected amines, which then
were immediately deprotected and converted to their hydrochloride salts 14a-14j using HCl (Scheme
4
).

Scheme 1. General Procedure for Substituted (Benzyloxyphenyl)methanaminium Chlorides and Substituted (Benzyloxy)benzamides.

Scheme 2. General Procedure for Substituted (Benzyloxy)benzenaminium Chlorides.
Scheme 3. General Procedure for Substituted (Benzyloxyphenyl)ethanaminium Chlorides.

Scheme 4.
General Procedure for Substituted (Biphenyl)ethanaminium Chlorides.

2
.3. Inhibitory Activities of the Synthesized Compounds against Monoamine Oxidase B
The synthesized compounds were evaluated for inhibitory activities of human MAO-A and MAO-B
using recombinant enzymes. The enzyme inhibition assay on test compounds was performed using
Amplex Red reagents and the inhibitory activity was determined by spectrophotometrically
measuring the resorufin formation at 570 nm. The inhibitory potencies (IC50 values) for all
synthesized compounds are summarized in Table 1, with comparative values of the positive controls,
selegiline, safinamide and sembragiline.
First, we prepared 11 (benzyloxyphenyl)methanaminium chlorides with various functional groups on
the terminal aryl ring (Table 1, compounds 7). The first set of compounds 7 contained methylamine
hydrochloride groups at one end of the biaryl linked unit. Compound 7f, which is the biaryl linked
unit of safinamide (the red box on the left in Fig. 2), exhibited moderate MAO-B inhibitory activity
(
IC50 = 0.926 M). We introduced a variety of electron-withdrawing groups and electron-donating
group into the terminal aryl ring (7a-7k). We observed that the two groups did not exhibit significant
activity differences, but most of them showed moderate inhibitory activities (IC50: 0.136 M ~ 0.926

M).
Next, we changed the number of carbon atoms between the amine group and the biaryl linked unit of
compound 7 from one to zero (10) and two (12). Compound 10e, which is the biaryl linked unit of
sembragiline (the red box on the right in Fig. 2), has lower MAO-B inhibitory activity (IC50 = 2.483

M) than compound 7f. All compound 10, in which no carbon was present between the amine
groups and the biaryl linked units, exhibited lower inhibitory activity (IC50: 0.402 M ~ >10 M)
than compound 7 having one carbon at the corresponding position. We investigated whether adding
one carbon to this position of compound 7 improved MAO-B inhibitory activities. Interestingly, this
modification resulted in dramatic improvement of inhibitory activity in most compounds (IC50: 0.009

M ~ 0.396 M). For the CF
3
-substituted compounds 12a-12c, we found that the 4’-CF derivative
3

1
2c was more potent than the corresponding 2’- and 3’-CF
3
derivatives 12a and 12b (12c, IC50 =
0
.009 M; 12a, IC50 =1.803 M; 12b, IC50 = 0.110 M). A similar trend on the inhibitory activities
of 2’, 3’, and 4’ regioisomers was observed in the F- or Cl-substituted compounds 12d-12i. In
addition, we modified the methanaminum chloride group of compound 7 to an amide group, and
found that the substitution with the amide group did not significantly affect MAO-B inhibitory
activity.
Finally, we prepared (biphenyl-4-yl)ethanaminium chlorides 14a-14j wherein the aryl linker is a
single bond. In this case, the compounds substituted with bulky electron-withdrawing groups (CF
and OCF ) exhibit far superior inhibitory activity than the F- or Cl-substituted compounds 14c-14h.
In particular, 4’-CF derivatives 14b showed much better MAO-B inhibitory activities than 4’-F and
’-Cl derivatives 14e and 14h (14b, IC50 = 0.041 M; 14e, IC50 = 6.806 M; 14h, IC50 = 0.625 M).
3
3
3
4

Table 1. Inhibitory effects of the synthesized compounds against hMAOs.
1
2
3
MAO-B a
MAO-A
(IC50, μM)
b
1
2
3
MAO-B a
MAO-A
(IC50, μM)
b
Compd
n
R
R
R
SI
Compd
n
R
R
R
SI
(
IC50, μM)
(IC50, μM)
e
7
7
a
1
1
1
1
1
1
1
1
1
1
1
0
0
0
H
CF
H
H
H
H
H
3.225±0.06
3.146±0.40
0.387±0.08
0.136±0.01
1.462±0.19
0.926±0.03
0.452±0.07
0.257±0.02
0.440±0.02
0.175±0.01
0.283±0.02
>10
>100
>100
>100
>100
>100
>100
>100
>100
>30
>31
>32
10d
10e
10f
12a
12b
12c
12d
12e
12f
12g
12h
12i
0
0
0
2
2
2
2
2
2
2
2
2
2
F
H
H
F
H
H
F
>10
>30
>100
>100
>100
>100
>100
>10
nd
>40
b
3
2.483±0.07
3.324±0.11
1.803±0.12
0.110±0.02
0.009±0.001
0.396±0.02
0.193±0.01
0.109±0.01
2.849±0.07
0.062±0.01
0.059±0.01
0.305±0.03
7
c
CF
H
3
H
>250
>730
>68
H
H
H
CF
H
H
F
>30
7
d
H
CF
3
CF
3
H
H
CF
H
H
F
>55
7
e
F
H
H
H
3
>900
>11000
>25
7
f
H
F
H
>100
>220
>380
>68
H
3
7
7
g
H
H
F
F
h
H
Cl
H
H
H
>100
>100
>10
>510
>910
>3.5
>1600
>1600
>33
7
i
H
Cl
H
H
H
H
Cl
H
7
j
H
OMe
H
>30
>170
>350
Cl
H
H
H
H
H
Cl
H
7
k
H
OMe
H
>100
>100
>100
>100
>100
>100
>10
e
1
0a
0b
0c
CF
3
H
nd
1
H
CF
3
H
1.507±0.03
0.402±0.02
>66
12j
OMe
1
H
H
CF
3
>240

1
2
3
MAO-B a
MAO-A
(IC50, μM)
b
1
2
3
MAO-B a
MAO-A
(IC50, μM)
b
Compd
R
R
R
SI
Compd
R
R
R
SI
(
IC50, μM)
(IC50, μM)
8
8
a
CF
H
H
F
3
H
CF
H
H
H
2.023±0.20
1.108±0.17
0.642±0.09
1.055±0.07
1.550±0.05
0.271±0.01
>1
>100
>100
>49
>90
14a
14b
14c
14d
14e
14f
14g
14h
14i
H
H
F
CF
H
H
F
3
H
CF
H
1.568±0.04
0.041±0.02
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>6.4
b
3
3
>240
e
8
c
CF
3
>100
>150
>95
nd
e
8
d
H
H
>100
H
H
Cl
H
H
H
H
H
>10
nd
8
e
H
H
F
H
>100
>65
H
H
Cl
H
F
6.806±0.09
8.340±0.12
2.110±0.07
0.625±0.05
5.003±0.23
5.757±0.19
>1.5
>1.2
>4.7
>16
8
f
H
F
>100
>360
H
c
e
Clorgyline (1)
0.008±0.001
1.5±0.02
>100
nd
H
d
Selegiline (2)
0.011±0.001
0.017±0.002
0.016±0.001
>130
Cl
H
d
Safinamide (4)
>5800
OMe
H
>1.9
>1.7
d
e
Sembragiline (5)
nd
14j
OMe
e
1
5
>10
>10
nd
a
b
c
Inhibition data are reported as IC50 (μM) ± SEM.
SI = selectivity index, the selectivity for the MAO-B isoform and is given as the ratio of IC50(MAO-A)/IC50(MAO-B).
Positive controls (MAO-A inhibitor); clorgyline: irreversible inhibitor
d
Positive controls (MAO-B inhibitor); selegiline: irreversible inhibitor, (S)-safinamide & sembragiline: reversible inhibitor.
e
nd: not determined
.

2
.4. Mode of MAO-B inhibition of 12c
To examine the interaction mode of MAO-B inhibition, the most potent compound 12c was
evaluated in substrate-dependent kinetic experiments, and both the corresponding progression curves
and the Linewever-Burk plots were generated. The initial rates of the MAO-B-catalyzed oxidation of
benzylamine at seven different substrate concentrations in the absence and in the presence of three
different concentrations of 12c were measured. The results are depicted in Fig. 4. The Lineweaver-
Burk plots for different concentration of 12c were linear and intersected at the y-axis. Using
GraphPad Prism, the maximal velocity (Vmax), the Michaelis constant (K
m
) and the inhibition
= 4.093 and Ki = 6.88e ). These results indicate
that 12c is a competitive MAO-B inhibitor. (Fig. 4)
+
7
-4
-9
constant (K
i
) were calculated (Vmax = 5.864e , K
m
Fig. 4. Mode of 12c with the binding site of MAO-B. The catalytic rates were measured at different
concentrations of benzylamine (0.065, 0.125, 0.25, 0.5, 1, 2 and 4 mM) in the absence and in the
presence of different concentrations (0.3, 1 and 3 nM) of 12c. In the Lineweaver-Burk plot, the
reciprocal MAO-B inhibitory activity was plotted against the reciprocal substrate concentration. The
maximal velocity (Vmax), the Michaelis constant (K
m
) and the inhibition constant (K ) were calculated
i

+
7
-4
-9
®
(
V
max = 5.864e , K = 4.093 and Ki = 6.88e ) using Sigma plot .
m
2
.5. Alleviative effect of compound 12c in MPTP-induced motor impairment
To verify in vivo efficacy of the compound 12c as a MAO-B inhibitor, we conducted an animal
model for Parkinson’s disease generated by MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,
+
administration. The dyskinesia in Parkinson’s disease occurs when MPP , 1-methyl-4-
phenylpyridinium, is converted by MAO-B in glia cells, cause the death of dopamine(DA)ergic
neurons in substantia nigra pars compacta (SNpc). We conducted treatment of compound 12c (30
mg/kg, p.o. injection) for 3 consecutive days including before and after MPTP administration (4
injections of MPTP, 2 h intervals; 20 mg/kg, i.p. injection), and assessed whether treatment can
restore motor ability (Fig. 5A). Commonly, transient weight loss is observed in the acute injection
2
5
method of MPTP. As shown in Fig. 5B, MPTP-treated mice showed weight loss compared to
saline-treated control mice. In the case of treatment with compound 12c, the weight loss was restored
more rapidly. In coat-hanger experiment, MPTP-induced mouse showed a falling or slipping to the
first hanging position on the base of coat hanger (score 2.0 ± 0.58). In contrast to the vehicle-treated
control mouse climbing to the top of the central vertical handle (score 4.75 ± 0.25). In comparison,
the compound 12c oral administrated mice recovered ascending ability to reach a stable position
(
score 3.83 ± 0.40) (Fig. 5C). Similarly, we found that mice treated with compound 12c though a
vertical grid test showed significant improvement in motor impairment by measuring the total time
spent on the test, the time of rotation at the top of the grid and time to go down (Fig. 5D). These
results indicate that oral administration of compound 12c can inhibit MAO-B activity in the brain
and alleviates the MPTP-induced motor dysfunction.

Fig. 5. Alleviation in MPTP-induced motor impairment by 12c oral treatment A) The experimental
protocol of MPTP treatment (20 mg/kg, i.p.) with or without 12c compound (30 mg/kg, p.o.). Score
diagram in coat hanger test and schematic diagram of vertical grid test. B) Weight change curve over
time by MPTP treatment. C) Coat hanger score (F = 9.094, p = 0.005), D) Total time taken in
vertical grid test (left, F = 10.87, p < 0.001), time to turn at the top of the grid (middle, F = 7.384, p
=
0.003) and time to climb down (right, F = 15.55, P < 0.001). ***p < 0.001, **p < 0.01, *p < 0.05,
and n.s., not significant (One-way ANOVA with Tukey’s test), n refers to the number of mice. Data
are means±s.e.m.
2
.6. Protection against reduction of MPTP-induced tyrosine hydroxylase (TH)-positive
dopamine neurons by oral treatment of compound 12c
To determine whether the recovery of motor ability by compound 12c was due to the protection of

DAergic neurons, we measured the amount of tyrosine hydroxylase (TH) by immunohistochemistry
Fig. 6A). TH is an enzyme involved in the synthesis of dopamine in DAergic neurons of SNpc.
(
Thus, TH-positive neurons are considered markers of DAergic neurons. The number of TH-positive
DAergic neurons in SNpc was significantly reduced by MPTP, whereas the number of TH-positive
DAergic neurons in compound 12c-treated group was reduced by only 38%. Moreover, although a
sharp decrease in TH-immunoreactive fiber density was observed in striatum of MPTP treated mice
(
7% compared to vehicle-treated mice by density measurement analysis), the decrease was also
prevented in mice administered with compound 12c (Fig. 6B).

Fig. 6. Protection in MPTP-induced Tyrosine hydroxylase (TH) positive cell reduction by 12c oral
treatment A) Representative images of immunostaining was conducted with TH on SNpc (substantia
nigra pars compacta) and striatum sections. B) Left, TH-immuno-positive cells in SNpc were
counted (F = 63.31, p < 0.001), right, The TH-immuno-positive fibers in the striatum were
quantitated by optical density (F = 196, p < 0.001). ***p < 0.001 (One-way ANOVA with Tukey’s
test). Data are means±s.e.m.

Conclusion
In this study, we synthesized a series of 4-(benzyloxy)phenyl and biphenyl-4-yl derivatives by
modifying the benzyloxyphenyl moiety, a common structure of potent, selective, and reversible
MAO-B inhibitors such as safinamide and sembragiline. Significant activity was observed for many
of these compounds. Comparison of CF
3
substituted aryl regioisomers (2’, 3’, and 4’) on the terminal
aryl ring demonstrated that 4’-CF derivatives exhibited the most potent inhibitory activity against
3
MAO-B. The compound with the highest potency (12c) significantly protected the tyrosine
hydroxylase (TH)-immunopositive DAergic neurons and attenuated the PD-associated behavioral
deficits in the MPTP-induced mouse model of PD. In conclusion, we present a potent and selective
MAO-B inhibitor with structural features that can be a good starting point for development of
therapeutic agents for PD.

Experimental Section
1
13
General Methods. NMR spectra were obtained at either 400 MHz ( H) and 100 MHz ( C) or 300
1
13
MHz( H) and 75 MHz ( C) using TMS as an internal standard. Chemical shifts (δ) are reported in
parts per million (ppm) from tetramethylsilane (TMS). Reactions were monitored by analytical thin-
layer chromatography (TLC) plates (Merck, Cat # 1.05715) and analyzed with 254 nm light. The
reactions were purified by column chromatography using silica gel (Merck, Cat # 1.07734&
1
.09385). All chemicals and solvents were reagent grade and used as obtained from commercial
sources without further purification. Yields reported are for purified products and were not optimized.
1
13
Compounds were checked by H and C NMR.
General procedure for the preparation of (4-(benzyloxy)phenyl)methanaminium chloride
derivatives (Method A). To a THF (400 mL) solution of LiAlH (3.0 equiv) was added a THF (30
4
mL) solution of the 4-(benzyloxy)benzonitrile derivative dropwise at 0 °C. The reaction mixture was
stirred at room temperature (16 h). Distilled water (4 mL) was added dropwise at 0 °C followed by
an aqueous NaOH solution (2 mL, 15% w/w), then distilled water (4 mL) again. The product mixture
was stirred at room temperature (1•2 h) for quenching the reaction, and was filtered through celite.
The filtered solution was concentrated in vacuo. The product residue was dissolved in CH
.0M HCl in dioxane was added dropwise for salt formation. The product mixture was evaporated in
vacuo and washed with EtOAc and n-hexane.
2
Cl , and
2
4
General procedure for the preparation of 4-(benzyloxy)benzamide derivatives (Method B). To
a 4-(benzyloxy)benzonitrile derivative dissolved in tert-butanol was added potassium hydroxide and
reflux for 2 h. After the reaction is completed, product mixture is extracted with EtOAc and brine

(
200 mLｘ3). Organic layer is dried with sodium sulfate. The residue is recrystallized.
General procedure for the preparation of 4-(benzyloxy)benzenaminium chloride (Method C).
To a 1-(benzyloxy)-4-nitrobenzene derivative dissolved in MeOH was added Platinum (II) oxide.
The reaction mixture was stirred for 1-2 h under H gas at room temperature. The product mixture
2
was filtered through celite and evaporated in vacuo. To a product residue dissolved in EtOAc was
added 4.0M HCl in dioxane for salt formation. The product is washed with MeOH and ether.
General procedure for the preparation of 2-(4-(benzyloxy)phenyl)ethan-1-aminium chloride
(
Method D). To a tert-butyl (4-(benzyloxy)phenethyl)carbamate derivative dissolved in CH
2
Cl was
2
added 4.0M HCl in dioxane and stirred for overnight at room temperature. After the reaction, the
product mixture was evaporated in vacuo and washed with EtOAc and n-hexane.
General Procedure for the preparation of 2-([1,1'-biphenyl]-4-yl)ethan-1-amine hydrochloride
derivatives (Method E). A mixture of N-boc-2-(4-bromophenyl)ethylamine, the desired arylboronic
acid (a-m) (1.2 equiv), tetrakis(triphenylphosphine)palladium(0) (0.04 equiv), Na
degassed toluene/H O (5/2) was refluxed for 18 h. The reaction mixture was filtered through celite
and concentrated in vacuo. The resulting residue was dissolved in in EtOAc (200 mL), washed with
O (200 mL 2) and brine (200 mL). The organic layer was dried with anhydrous Na SO and
concentrated in vacuo. The residue was purified by column chromatography on SiO2.
2
CO
3
(5 equiv) in
2
H
2
2
4
Preparation of (4-(benzyloxy)phenyl)methanaminium chloride (7a). Using Method A, 6a (0.50 g,

2
.4 mmol), LiAlH
4
(0.27 g, 7.2 mmol), and NaOH (0.2 mL) gave 7a as a white solid (0.60 g, 100%):
1
R
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 400 MHz)  3.91 (s, CH
2
NH ), 5.13 (s,
3
OCH
ArH), 8.40 (s, NH
27.7, 128.4, 130.5, 130.7, 137.0 (11 ArC), 158.3 (1 ArC).
2
), 7.03 (d, J = 8.6 Hz, 2 ArH), 7.29-7.35 (m, 1 ArH), 7.36-7.41 (m, 3 ArH), 7.41-7.46 (m, 3
1
3
3
); C NMR (DMSO-d
6
, 100 MHz)  41.5 (CH
2
NH
2
), 69.2 (OCH ), 114.8, 126.3,
2
1
Preparation of (4-((2-(trifluoromethyl)benzyl)oxy)phenyl)methanaminium chloride (7b). Using
Method A, 6b (1.50 g, 5.4 mmol), LiAlH
white solid (0.52 g, 34%): R
CH NH ), 5.25 (s, OCH
ArH), 7.69-7.78 (m, 2 ArH), 7.78-7.83 (m, 1 ArH), 8.24 (s, NH
1.6 (CH NH ), 66.3 (OCH ), 114.7, 124.3 (CF , q, JC-F = 275.5 Hz), 126.2, 126.9 (q, JC-F = 31.7
4
(0.62 g, 16.2 mmol), and NaOH (0.6 mL) gave 7b as a
1
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 400 MHz)  3.95 (s,
2
3
2
), 7.05 (d, J = 8.6 Hz, 2 ArH), 7.42 (d, J = 8.6 Hz, 2 ArH), 7.57-7.62 (m, 1
1
3
3
); C NMR (DMSO-d , 75 MHz) 
6
4
2
2
2
3
Hz), 128.8, 130.5, 130.7, 132.8, 134.8 (11 ArC), 158.1 (1 ArC).
Preparation of (4-((3-(trifluoromethyl)benzyl)oxy)phenyl)methanaminium chloride (7c). Using
Method A, 6c (0.50 g, 1.8 mmol), LiAlH
white solid (0.20 g, 40%): R
CH NH ), 5.24 (s, OCH
ArH), 7.68-7.73 (m, 1 ArH), 7.74-7.78 (m, 1 ArH), 7.80-7.82 (m, 1 ArH), 8.13 (s, NH
DMSO-d , 75 MHz)  41.6 (CH NH ), 68.3 (OCH ), 114.8, 124.0, 124.2 (CF , q, JC-F = 270.5 Hz),
24.6, 126.5, 129.2 (q, JC-F = 31.3 Hz), 129.6, 130.6, 131.6, 138.6 (11 ArC), 158.1 (1 ArC).
4
(0.20 g, 5.4 mmol), and NaOH (0.2 mL) gave 7c as a
1
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 400 MHz)  3.94 (s,
2
3
2
), 7.07 (d, J = 8.7 Hz, 2 ArH), 7.40 (d, J = 8.6 Hz, 2 ArH), 7.61-7.67 (m, 1
1
3
3
); C NMR
(
6
2
2
2
3
1
Preparation of (4-((4-(trifluoromethyl)benzyl)oxy)phenyl)methanaminium chloride (7d). Using

Method A, 6d (0.3 g, 1.1 mmol), LiAlH
white solid (0.18 g, 59%): R
CH NH ), 5.26 (s, OCH
ArH); C NMR (DMSO-d
4
(0.12 g, 3.2 mmol), and NaOH (0.12 mL) gave 7d as a
1
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 400 MHz)  3.93 (s,
2
3
2
), 7.06 (d, J = 8.5 Hz, 2 ArH), 7.41 (d, J = 8.5 Hz, 2 ArH), 7.63-7.79 (m, 4
, 75 MHz)  41.5 (CH NH ), 68.2 (OCH ), 114.8, 124.2 (CF
71.6 Hz), 125.3 (q, JC-F = 3.8 Hz), 126.5, 127.9, 128.3 (q, JC-F = 31.5 Hz), 130.6, 141.9 (11 ArC),
58.0 (1 ArC).
1
3
6
2
2
2
3
, q, JC-F =
2
1
Preparation of (4-((2-fluorobenzyl)oxy)phenyl)methanaminium chloride (7e). Using Method A,
6
e (0.50 g, 2.2 mmol), LiAlH
4
(0.25 g, 6.6 mmol), and NaOH (0.2 mL) gave 7e as a white crystalline
1
(
0.08 g, 16%): R
f
= 0.00 (EtOAc 1 : n-hexane 5): H NMR (DMSO-d
6
, 400 MHz)  3.92 (s,
CH
2
NH
3
), 5.15 (s, OCH ), 7.05 (d, J = 8.5 Hz, 2 ArH), 7.20-7.30 (m, 2 ArH), 7.38-7.46 (m, 3 ArH),
2
1
3
7
.55 (t, J = 7.6 Hz, 1 ArH), 8.40 (s, NH
OCH ), 114.7, 115.4 (d, JC-F = 20.7 Hz), 123.7 (d, JC-F = 14.3 Hz), 124.5, 126.7, 130.4, 130.5, 130.6,
30.7 (10 ArC), 158.2 (1 ArC), 160.4 (1 ArC, d, JC-F = 244.7 Hz).
3
); C NMR (DMSO-d
6
, 100 MHz)  41.6 (CH
2
NH
2
), 63.6
(
2
1
Preparation of (4-((3-fluorobenzyl)oxy)phenyl)methanaminium chloride (7f). Using Method A,
6
f (3.00 g, 13.2 mmol), LiAlH
4
(1.50 g, 39.6 mmol), and NaOH (2.4 mL) gave 7f as a white solid
1
(
2.70 g, 89%): R
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 400 MHz)  3.92 (s, CH
2
NH ),
3
5
.16 (s, OCH ), 7.01-7.07 (m, 2 ArH), 7.11-7.19 (m, 1 ArH), 7.24-7.30 (m, 2 ArH), 7.37-7.47 (m, 3
2
1
3
ArH), 8.13 (s, NH
21.7 Hz), 114.3, 114.5, 114.6, 123.3, 123.4, 128.3, 130.4 (d, JC-F = 8.2 Hz), 140.2 (10 ArC), 156.8
1 ArC), 162.2 (1 ArC, d, JC-F = 244.7 Hz).
3
); C NMR (DMSO-d
6
, 100 MHz)  44.6 (CH
2
NH
2
), 68.3 (OCH
2
), 114.1 (d, JC-F
=
(

Preparation of (4-((4-fluorobenzyl)oxy)phenyl)methanaminium chloride (7g). Using Method A,
6
g (1.00 g, 4.4 mmol), LiAlH
4
(0.50 g, 13.2 mmol), and NaOH (0.40 mL) gave 7g as a white solid
1
(
1.0 g, 97%): R
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 300 MHz)  3.92 (s, CH
2
NH
3
),
);
), 114.5, 115.2 (d, JC-F = 21.2 Hz),
28.2, 129.8 (d, JC-F = 8.2 Hz), 133.5, 136.5, 156.8 (11 ArC), 161.7 (1 ArC, d, JC-F = 242.0 Hz).
5
.11 (s, OCH
2
), 7.00-7.09 (m, 2 ArH), 7.16-7.29 (m, 2 ArH), 7.50-7.65 (m, 4 ArH), 8.45 (s, NH
3
1
3
C NMR (DMSO-d , 100 MHz)  45.0 (CH NH ), 68.4 (OCH
6
2
2
2
1
Preparation of (4-((3-chlorobenzyl)oxy)phenyl)methanaminium chloride (7h). Using Method A,
6
h (0.40 g, 1.6 mmol), LiAlH
4
(0.19 g, 4.9 mmol), and NaOH (0.16 mL) gave 7h as a white solid
1
(
0.20 g, 50%): R
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 300 MHz)  3.94 (s, CH
2
NH
3
),
);
), 115.3, 125.8, 125.8, 126.5, 127.0,
5
.26 (s, OCH
2
), 7.03-7.10 (m, 2 ArH), 7.36-7.43 (m, 2 ArH), 7.62-7.80 (m, 4 ArH), 8.15 (s, NH
C NMR (DMSO-d , 75 MHz)  42.1 (CH NH ), 68.8 (OCH
28.2, 128.4, 128.6, 129.0, 129.4, 131.1, 142.4, 158.5 (12 ArC).
3
1
3
6
2
2
2
1
Preparation of (4-((4-chlorobenzyl)oxy)phenyl)methanaminium chloride (7i). Using Method A,
6
i (0.40 g, 1.6 mmol), LiAlH
4
(0.19 g, 4.9 mmol), and NaOH (0.16 mL) gave 7i as a white solid
1
(
0.16 g, 39%): R
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 300 MHz)  3.93 (s, CH
2
NH
3
),
);
), 115.3, 126.9, 128.9, 129.9, 131.0,
5
.14 (s, OCH
2
), 7.01-7.07 (m, 2 ArH), 7.36-7.42 (m, 2 ArH), 7.44-7.49 (m, 4 ArH), 8.20 (s, NH
C NMR (DMSO-d , 75 MHz)  42.1 (CH NH ), 68.8 (OCH
32.8, 136.5, 158.6 (12 ArC).
3
1
3
6
2
2
2
1
Preparation of (4-((3-methoxybenzyl)oxy)phenyl)methanaminium chloride (7j). Using Method
A, 6j (1.00 g, 4.2 mmol), LiAlH (0.48 g, 12.5 mmol), and NaOH (0.4 mL) gave 7j as a white solid
4

1
(
0.37 g 37%): R
.92 (s, CH NH
ArH), 7.38-7.44 (m, 2 ArH), 8.35 (s, NH
OCH ), 69.0 (OCH ), 113.1, 113.2, 114.6, 114.8, 119.7, 127.3, 128.3, 129.6, 130.4, 138.6, 158.2,
59.4 (12 ArC).
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 400 MHz)  3.75 (s, OCH ),
3
3
2
3
), 5.10 (s, OCH ), 6.85-6.91 (m, 1 ArH), 6.97-7.06 (m, 4 ArH), 7.26-7.33 (m, 1
2
1
3
3
); C NMR (DMSO-d
6
, 100 MHz)  42.0 (CH
2
NH ), 55.1
2
(
3
2
1
Preparation of (4-((4-methoxybenzyl)oxy)phenyl)methanaminium chloride (7k). Using Method
A, 6k (0.30 g, 1.3 mmol), LiAlH
0.25 g, 71%): R
.93 (s, CH NH ), 5.09 (s, OCH
NMR (DMSO-d
29.9, 131.0, 159.5, 162.3 (12 ArC).
4
(0.14 g, 3.8 mmol), and NaOH (0.12 mL) gave 7k as a white solid
1
(
f
= 0.00 (EtOAc 9 : acetone 1): H NMR (DMSO-d
6
, 300 MHz)  3.75 (s, OCH ),
3
1
3
3
2
3
2
), 6.90-7.06 (m, 4 ArH), 7.32-7.43 (m, 4 ArH), 8.26 ((s, NH ); C
3
6
, 75 MHz)  42.1 (CH
2
NH
2
), 55.6 (OCH
3
), 69.4 (OCH ), 114.3, 115.3, 126.5, 129.3,
2
1
Preparation of 4-((2-(trifluoromethyl)benzyl)oxy)benzamide (8a). Using Method B, 6b (0.50 g,
1
0
7
.8 mmol) and potassium hydroxide (0.76 g, 13.5 mmol) gave 8a as a baige solid (0.47 g, 89%): R =
f
1
.00 (EtOAc 1 : n-hexane 5): H NMR (CDCl
3
, 400 MHz)  5.33 (s, OCH ), 6.98-7.05 (m, 2 ArH),
2
1
3
.41-7.48 (m, 1 ArH), 7.55-7.61 (m, 1 ArH), 7.69-7.75 (m, 2 ArH), 7.77-7.83 (m, 2 ArH); C NMR
, 100 MHz)  66.8 (OCH ), 114.6, 120.7, 124.8 (CF , q, JC-F = 272.2 Hz), 126.7 (q, JC-F
.7 Hz), 127.0, 127.5 (q, JC-F = 30.3 Hz), 127.6, 127.9, 128.9, 129.3, 129.9, 131.1, 133.3, 135.0 (11
(
DMSO-d
6
2
3
=
5
ArC), 160.8 (1 ArC), 167.8 (C(O)).
Preparation of 4-((3-(trifluoromethyl)benzyl)oxy)benzamide (8b). Using Method B, 6c (0.20 g,
0
.7 mmol) and potassium hydroxide (0.30 g, 5.4 mmol) gave 8b as a white solid (0.19 g, 91%): R =
f

1
0
.00 (EtOAc 1 : n-hexane 5): H NMR (DMSO-d
6
, 400 MHz)  5.28 (s, OCH ), 7.06-7.14 (m, 2
2
ArH), 7.16-7.27 (m, 1 ArH), 7.62-7.70 (m, 1 ArH), 7.70-7.76 (m, 1 ArH), 7.77-7.82 (m, 1 ArH),
1
3
7
1
1
.82-7.93 (m, 2 ArH, C(O)NH
24.2 (CF , q, JC-F = 270.7 Hz), 124.6, 127.0, 129.2 (q, JC-F = 29.2 Hz), 129.6, 131.7, 138.3 (11 ArC),
60.4 (1 ArC), 167.4 (C(O)).
2
); C NMR (DMSO-d
6
, 75 MHz)  68.4 (OCH ), 114.3, 124.1, 124.1,
2
3
Preparation of 4-((4-(trifluoromethyl)benzyl)oxy)benzamide (8c). Using Method B, 6d (0.30 g,
1
0
.1 mmol) and potassium hydroxide (0.45 g, 8.1 mmol) gave 8c as a brown solid (0.29 g, 92%): R =
f
1
.00 (EtOAc 1 : n-hexane 5): H NMR (DMSO-d
6
, 400 MHz)  5.30 (s, OCH ), 7.05-7.14 (m, 2
2
ArH), 7.16-7.27 (m, 1 ArH), 7.66-7.74 (m, 2 ArH), 7.76-7.82 (m, 2 ArH), 7.82-7.90 (m, 1 ArH,
1
3
C(O)NH
25.8, 125.8, 127.4, 128.5, 128.9 (q, JC-F = 31.6 Hz), 129.9, 142.1 (11 ArC), 160.8 (1 ArC), 167.8
C(O)).
2
); C NMR (DMSO-d
6
, 75 MHz)  68.9 (OCH
2
), 114.7, 124.7 (CF , q, JC-F = 272.0 Hz),
3
1
(
Preparation of 4-((2-fluorobenzyl)oxy)benzamide (8d). Using Method B, 6e (0.50 g, 2.2 mmol)
and potassium hydroxide (0.93 g, 16.5 mmol) gave 8d as a brown powder (0.50 g, 93%): R = 0.00
f
1
(
EtOAc 1 : n-hexane 5): H NMR (DMSO-d
6
, 400 MHz)  5.20 (s, OCH ), 7.06-7.11 (m, 2 ArH),
2
7
.18-7.30 (m, 3 ArH), 7.40-7.48 (m, 1 ArH), 7.55-7.61 (m, C(O)NHH), 7.83-7.89 (m, 2ArH,
1
3
C(O)NHH); C NMR (DMSO-d
6
, 100 MHz)  63.6 (OCH
23.6, 124.5, 124.6, 126.9, 129.4, 130.5 (d, JC-F = 8.3 Hz), 130.8, 130.8, 160.4 (d, JC-F = 244.9 Hz),
60.5 (12 ArC), 167.3 (C(O)).
2
), 114.1, 115.4 (d, JC-F = 20.8 Hz), 123.4,
1
1
Preparation of 4-((3-fluorobenzyl)oxy)benzamide (8e). Using Method B, 6f (2.0 g, 8.8 mmol) and

potassium hydroxide (3.70 g, 66.0 mmol) gave 8e as a beige crystalline (1.89 g, 88%): R = 0.00
f
1
(
EtOAc 1 : n-hexane 5): H NMR (DMSO-d
6
, 400 MHz)  5.19 (s, OCH
2
), 7.04-7.10 (m, 2 ArH),
);
), 114.3, 114.4, 114.7 (d, JC-F = 20.8 Hz), 123.6, 126.9,
29.4, 130.5 (d, JC-F = 8.2 Hz), 139.7, 139.7, 160.4, 162.2 (d, JC-F = 242.2 Hz) (12 ArC), 167.38
7
.13-7.26 (m, 2 ArH), 7.27-7.34 (m, 2 ArH), 7.40-7.48 (m, 1 ArH), 7.83-7.90 (m, 1 ArH, C(O)NH
2
1
3
C NMR (DMSO-d
6
, 100 MHz)  68.5 (OCH
2
1
(
C(O)).
Preparation of 4-((4-fluorobenzyl)oxy)benzamide (8f). Using Method B, 6g (0.3 g, 1.3 mmol) and
potassium hydroxide (0.56 g, 9.9 mmol) gave 8f as a white crystalline (0.32 g, 98%): R = 0.00
f
1
(
EtOAc 1 : n-hexane 5): H NMR (DMSO-d
6
, 400 MHz)  5.14 (s, OCH ), 7.02-7.08 (m, 2 ArH),
2
1
3
7
1
1
.15-7.26 (m, 3 ArH), 7.47-7.55 (m, 2 ArH), 7.80-7.87 (m, 1 ArH, C(O)NH
00 MHz)  69.0 (OCH ), 114.7, 115.7 (d, JC-F = 21.2 Hz), 127.2, 129.8, 130.5 (d, JC-F = 8.2 Hz),
33.4, 161.0, 162.3 (d, JC-F = 242.3 Hz) (12 ArC), 167.8 (C(O)).
2
); C NMR (DMSO-d ,
6
2
Preparation of 4-((2-(trifluoromethyl)benzyl)oxy)benzenaminium chloride (10a). Using Method
C, 9a (0.20 g, 0.7 mmol), platinum oxide, (0.016 g, 0.7 mmol), and 4.0M hydrogen chloride gave
1
1
0a as a white powder (0.11 g, 53%): R
MHz)  5.25 (s, OCH
.79 (m, 2 ArH), 7.79-7.83 (m, 1 ArH), 10.02 (s, NH
OCH ), 116.1, 124.7 (CF , q, JC-F = 272.1 Hz), 125.0, 125.4, 126.6, 126.7, 127.4 (q, JC-F = 30.3 Hz),
29.3, 131.1, 133.3, 135.0, 157.9 (12 ArC).
f
= 0.00 (EtOAc 1 : n-hexane 5): H NMR (DMSO-d
6
, 400
2
), 7.09-7.14 (m, 2 ArH), 7.29-7.34 (m, 2 ArH), 7.57-7.63 (m, 1 ArH), 7.70-
13
7
3
); C NMR (DMSO-d , 100 MHz)  67.0
6
(
2
3
1
Preparation of 4-((3-(trifluoromethyl)benzyl)oxy)benzenaminium chloride (10b). Using Method

C, 9b (0.20 g, 0.7 mmol), platinum oxide, (0.016 g, 0.7 mmol), and 4.0M hydrogen chloride gave
1
1
0b as a white powder (0.13 g, 63%): R
f
= 0.00 (EtOAc 1 : n-hexane 5): H NMR (DMSO-d
6
, 400
MHz)  5.24 (s, OCH
2
), 7.12-7.17 (m, 2 ArH), 7.30-7.36 (m, 2 ArH), 7.62-7.84 (m, 4 ArH), 10.13 (s,
); C NMR (DMSO-d , 100 MHz)  68.6 (OCH ), 115.8, 124.2 (CF , q, JC-F = 270.9 Hz), 124.1,
24.5, 124.6, 124.7, 129.2 (q, JC-F = 31.4 Hz), 129.6, 131.7, 138.3, 157.5 (12 ArC).
1
3
NH
3
6
2
3
1
Preparation of 4-((4-(trifluoromethyl)benzyl)oxy)benzenaminium chloride (10c). Using Method
C, 9c (0.20 g, 0.7 mmol), platinum oxide, (0.016 g, 0.7 mmol), and 4.0M hydrogen chloride gave 10c
1
as a white powder (0.15 g, 72%): R
.26 (s, OCH
ArH), 10.15 (s, NH
70.5 Hz), 125.0, 125.3, 125.8 (q, JC-F = 3.8 Hz), 128.5, 128.8 (q, JC-F = 31.4 Hz), 142.1, 157.9 (12
ArC).
f
= 0.00 (EtOAc 1 : n-hexane 5): H NMR (CDCl
3
, 400 MHz) 
5
2
), 7.11-7.16 (m, 2 ArH), 7.30-7.35 (m, 2 ArH), 7.65-7.70 (m, 2 ArH), 7.75-7.80 (m, 2
1
3
3
); C NMR (DMSO-d6, 75 MHz)  69.1 (OCH
2
), 116.2, 124,7 (CF , q, JC-F =
3
2
Preparation of 4-((2-fluorobenzyl)oxy)benzenaminium chloride (10d). Using Method C, 9d (0.15
g, 0.6 mmol), platinum oxide, (0.012 g, 0.06 mmol), and 4.0M hydrogen chloride gave 10d as a
1
white solid (0.04 g, 30%): R
f
= 0.59 (5% CH
2
Cl
2
/MeOH): H NMR (DMSO-d
6
, 400 MHz)  5.10 (s,
OCH
2
), 7.08-7.14 (m, 2 ArH), 7.19-7.26 (m, 2 ArH), 7.29-7.35 (m, 2 ArH), 7.47-7.54 (m, 2 ArH)
1
3
1
1
1
0.19 (s, NH
3
); C NMR (DMSO-d6, 100 MHz)  68.8 (OCH ), 115.3 (d, JC-F = 21.3 Hz), 115.3,
2
15.5, 115.7, 115.7, 123.4, 123.6, 124.5, 124.6, 124.7, 130.0 (d, JC-F = 8.2 Hz), 130.5, 130.6, 130.7,
30.8, 133.0, 157.6, 157.6, 160.4 (d, JC-F = 245.0 Hz) (12 ArC).
Preparation of 4-((3-fluorobenzyl)oxy)benzenaminium chloride (10e). Using Method C, 9e (0.10