Synthesis and evaluation of 2-(2′-((dimethylamino)methyl)-4′-(2-fluoroethoxy-substituted)phenylthio)benzenamine derivatives as potential positron emission tomography imaging agents for serotonin transporters

Article abstract of DOI:10.1016/j.bioorg.2020.103654

A series of diphenylsulfide derivatives with various substitutions at the 4-position on phenyl ring A and different lengths of the 2-fluoroethoxy-substituted side-chain at the 4′-position on ring B were synthesized and evaluated as potential positron emission tomography (PET) imaging agents for serotonin transporters (SERT). These ligands exhibited high SERT binding affinities (K_i = 0.11–1.3 nM) and the 4-methyl-substituted (4-Me) compounds 7a and 8a displayed excellent selectivity for SERT versus norepinephrine transporters (NET) (392- and 700-fold, respectively). In the parallel artificial membrane permeability assay (PAMPA), these ligands demonstrated moderate to high brain penetration, and the 4-Me analogs showed higher BBB permeability than the corresponding 4-F analogs. The 2-fluoroethoxy-substituted ligands showed higher metabolic stability and lower lipophilicity than 4-F-ADAM. [¹⁸F]7a-c were readily prepared using an automatic synthesizer and exhibited significant uptake and slow washout in rat brains. At 120 min after iv injection, [¹⁸F]7a exhibited the highest uptake in the midbrain, whereas [¹⁸F]7b exhibited the highest uptake in the hypothalamus and midbrain. After treatment with citalopram, a SERT-selective ligand, the uptake of [¹⁸F]7a in the hypothalamus and striatum was significantly decreased. The potent and highly selective SERT binding and the selective and reversible accumulation in SERT-rich brain regions suggested that [¹⁸F]7a is a promising lead for the further development of novel [¹⁸F]-labeled PET imaging agents for SERT binding sites in the brain.

Full text of DOI:10.1016/j.bioorg.2020.103654

Journal Pre-proofs
Synthesis and Evaluation of 2-(2’-((Dimethylamino)methyl)-4’-(2-fluoroe-
thoxy-substituted)phenylthio)benzenamine Derivatives as Potential Positron
Emission Tomography Imaging Agents for Serotonin Transporters
Ya-Yao Huang, Li-Te Chang, Hsin-Yi Shen, Ying-Heng Chen, Kai-Yuan
Tzen, Chyng-Yann Shiue, Ling-Wei Hsin
PII:
S0045-2068(19)31863-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103654
YBIOO 103654
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
4 November 2019
30 January 2020
5 February 2020
Please cite this article as: Y-Y. Huang, L-T. Chang, H-Y. Shen, Y-H. Chen, K-Y. Tzen, C-Y. Shiue, L-W. Hsin,
Synthesis
and
Evaluation
of
2-(2’-((Dimethylamino)methyl)-4’-(2-fluoroethoxy-
substituted)phenylthio)benzenamine Derivatives as Potential Positron Emission Tomography Imaging Agents for
Serotonin Transporters, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103654
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Synthesis and Evaluation of 2-(2’-((Dimethylamino)methyl)-4’-(2-fluoroethoxy-
substituted)phenylthio)benzenamine Derivatives as Potential Positron Emission
Tomography Imaging Agents for Serotonin Transporters
a,§
b,§
b
b
a
Ya-Yao Huang, Li-Te Chang, Hsin-Yi Shen, Ying-Heng Chen, Kai-Yuan Tzen, Chyng-
a
b,c*
*
Yann Shiue, Ling-Wei Hsin
aPET Center, Department of Nuclear Medicine, National Taiwan University Hospital, 7,
b
c
Chung-Shan S. Road, Taipei, Taiwan. School of Pharmacy, College of Medicine, and Center
for Innovative Therapeutics Discovery, National Taiwan University, 17, Xuzhou Road, Room
9
36, Taipei 10055, Taiwan.
*
To whom correspondence should be addressed. Ling-Wei Hsin, Tel.: +886-2-3366-8696. Fax:
+
886-2-2351-2086. E-mail: lwhsin@ntu.edu.tw. Chyng-Yann Shiue, Tel.: +886-2-2312-3456,
ext.65938. Fax: +886-2-2391-1506. E-mail: shiue@ntuh.gov.tw.
These authors contributed equally to this article.
§
Abbreviations:
DAST: (diethylamino)sulfur trifluoride
DAT: dopamine transporters
NET: norepinephrine transporters
PET: potential positron emission tomography
SERT: serotonin transporters
PAMPA: parallel artificial membrane permeability assay
SUV: standardized uptake value
SUR: standardized uptake value ratio (expressed as SUVtarget region / SUVcerebellum)
1

Abstract
A series of diphenylsulfide derivatives with various substitutions at the 4-position on phenyl
ring A and different lengths of the 2-fluoroethoxy-substituted side-chain at the 4′-position on
ring B were synthesized and evaluated as potential positron emission tomography (PET)
imaging agents for serotonin transporters (SERT). These ligands exhibited high SERT binding
affinities (K = 0.11~1.3 nM) and the 4-methyl-substituted (4-Me) compounds 7a and 8a
i
displayed excellent selectivity for SERT versus norepinephrine transporters (NET) (392- and
7
00-fold, respectively). In the parallel artificial membrane permeability assay (PAMPA), these
ligands demonstrated moderate to high brain penetration, and the 4-Me analogs showed higher
BBB permeability than the corresponding 4-F analogs. The 2-fluoroethoxy-substituted ligands
1
8
showed higher metabolic stability and lower lipophilicity than 4-F-ADAM. [ F]7a-c were
readily prepared using an automatic synthesizer and exhibited significant uptake and slow
1
8
washout in rat brains. At 120 min after iv injection, [ F]7a exhibited the highest uptake in the
1
8
midbrain, whereas [ F]7b exhibited the highest uptake in the hypothalamus and midbrain.
1
8
After treatment with citalopram, a SERT-selective ligand, the uptake of [ F]7a in the
hypothalamus and striatum was significantly decreased. The potent and highly selective SERT
binding and the selective and reversible accumulation in SERT-rich brain regions suggested
1
8
18
that [ F]7a is a promising lead for the further development of novel [ F]-labeled PET imaging
agents for SERT binding sites in the brain.
Keywords: serotonin transporter, positron emission tomography, imaging agent, structure-
activity relationship, synthesis
2

1
.
Introduction
Serotonin (5-HT) is involved in a wide variety of physiological functions through its
interaction with 14 serotonin receptor subtypes. Dysregulation of serotonin transmission is
known to be involved in various neuropsychiatric and neurodegenerative diseases/disorders,
such as depression, anxiety, obsessive-compulsive disorder, eating disorder, addiction,
1
-8
Parkinson’s disease, and Alzheimer’s disease. In the central nervous system (CNS), serotonin
transporters (SERT) are responsible for the reuptake of extracellular 5-HT from the synapse
into the presynaptic nerves to terminate its downstream action, and high densities of SERT have
been observed in the raphe nuclei, caudate, putamen, thalamus, hypothalamus, and amygdala,
9
with lower densities in the cortex and cerebellum. SERT is known to play critical roles in many
psychiatric and neurodegenerative disorders and has been an attractive target for the diagnosis
and treatment of serotonin-related brain disorders.
SERT is the target of selective serotonin reuptake inhibitors (SSRIs), which are among the
most commonly prescribed class of antidepressants in recent decades.10-11 In addition, SERT
inhibitors have been proven to have potential for treating obsessive compulsive disorder,
schizophrenia, bipolar disorder, anxiety, and eating disorders. However, there is a lag time
before the onset of the antidepressant effect of SSRIs, more than 30% of patients are
5
nonresponders, and the mechanisms of SSRIs are not yet known. From the precision and
personalized medicine point of view, SERT brain imaging would be valuable for more accurate
categorization of patients with distinct psychiatric and neurodegenerative disorders and
determination of an appropriate dose for individual patients to provide more effective treatment
with minimal side effects.12
Positron emission tomography (PET) is a noninvasive molecular imaging technique,
which has been used frequently to study monoaminergic systems in the brains of live animals
and humans. Direct assessment of the density and ligand occupancy of SERT binding sites in
3

patients using PET and a suitable SERT probe would allow identification of the underlying
mechanisms of SERT-related disorders, diagnosis and monitoring of the progression of diseases
in patients, as well as the discovery and development of novel therapeutic strategies for these
brain diseases and disorders. 13-14
[11
C] (+)McN 5652 was the first useful PET imaging agent for studying SERT in
1
5
humans. However, this agent has high nonspecific binding and has only moderate signal
1
6
contrast in human studies. Among the numerous structural classes of SERT ligands that have
1
1
18
9, 14
been radiolabeled with C or F and studied in humans as potential PET radiotracers,
diphenylsulfides possessed high SERT binding affinity and selectivity over the dopamine
transporters (DAT), are easier to prepare, and have been extensively evaluated as SERT
imaging agents (Chart 1).17-23
[11
C]DASB ([ C]1) demonstrated high signal-to-noise ratio and suitable kinetics, and is
11
the current “gold standard tracer” for SERT imaging using PET in humans.24-25 [11C]MADAM
1
1
(
[ C]2) is another promising SERT probe, in which the 4-methyl substituent seems to be
2
6
11
11
responsible for its high SERT selectivity. Although [ C]1 and [ C]2 have been translated to
1
1
human PET imaging use, the short half-life of C tracers limits their widespread use in the
1
8
clinic. Because of the lower positron energy and longer physical half-life of the F isotope (t
1
/2
1
1
18
=
109.8 min vs 20.3 min for C), F-labeled PET radiotracers can provide imaging with higher
spatial resolution, longer times for radiosynthesis and a more sufficient time for data acquisition
as well as tracer kinetic studies, and therefore could serve many more patients and clinical sites.
1
8
18
A number of F-labeled biphenylsulfides have been reported including [ F]4-F-ADAM
[ F]3), [ F]AFM, [18F]ACF,28 and [ F]FPBM ([ F]4).
1
8
18
27
18
18
9, 13, 29-30
[18F]3 demonstrated
(
promising tracer kinetic properties, and specific uptake in SERT-rich brain areas in preliminary
human studies.31-32 However, a nucleophilic substitution with [ F]fluoride on the less activated
18
1
8
aromatic ring is required in the radiosynthesis of [ F]3, which resulted in relatively low
4

3
3
radiochemical yield (<5%) and therefore hampered its routine application in the clinic. 4’-
1
8
(
Fluoroalkoxy)-substituted biphenylsulfides, such as [ F]4, were studied, in which the
[18
F]fluoride was introduced via a more feasible nucleophilic reaction at an aliphatic position
with high radiochemical yields and short reaction times.13, 29 [18F]4 showed a high
hypothalamus to cerebellum ratio and promising pharmacokinetic profile.34 The 4’-
fluoroalkoxy group on ring B seems to be responsible for high brain uptake and significant
1
8
brain washout. However, [ F]4 exhibited high norepinephrine transporter (NET) affinity, and
thus lower SERT selectivity versus NET.29
Currently, the fluorinated diphenylsulfides that have been developed have not
1
1
11
demonstrated superior pharmacokinetic profiles in human studies relative to [ C]1 or [ C]2,
1
8
and therefore a suitable F-labeled PET radiotracer with high SERT affinity and specificity as
well as optimal pharmacokinetic properties and a metabolic profile for SERT imaging in the
1
8
living brain is still an unmet need. Thus, research to identify new F-labeled candidates for
SERT imaging continues.
R2
N
[11
C]1: R = CN, R = CH , R = H; [11C]DASB
11
NH2
1 2 3 3
[11C]2: R = CH , R = CH , R = H; [11C]MADAM
11
2
2'
1
S
1'
3'
4'
1 3 2 3 3
3
4
[18F]3: R = F, R = CH , R = H; [18F]4-F-ADAM
18
A
B
1 2 3 3
[18
F]4: R = H, R = CH , R = O(CH ) 18F; [18F]4'-FPBM
1 2 3 3 2 3
R1
R3
5
1
1
18
Chart 1. Chemical structures of C- and F-labeled diphenylsulfides.
Based on the previous structure-activity relationship (SAR) of diphenylsulfides on SERT,
the 4-substituents on ring A provide high SERT selectivity relative to NET, e.g., compounds 1
and 2, whereas no substituent on the 4-position (4-H) significantly increases NET affinity and
reduces SERT selectivity, e.g., 4. 4’-Fluoroalkoxy-substitution maintained high SERT affinity
and provided more desirable kinetic properties for PET imaging. In addition, the USFDA
approved AmyvidTM (Florbetapir F-18 injection, [ F]AV-45) and Neuraceq (Florbetaben F-
18
TM
5

1
8
1
8 injection, [ F]BAY94-9172), which contain end-capped fluoro-triethyleneglycolyl (FEEE)
3
5
side chains, as brain imaging drugs for Alzheimer’s disease (AD). The FEEE substituent could
modulate the lipophilicity of the core structure and maintain a relatively small total polar surface
area (tPSA), resulting in sufficient brain penetration ability.
1
1
In this study, 4-methyl-substituted (4-Me) [ C]2 was chosen as the lead compound. The
2
-fluoroethoxy (FE), 2-(2-fluoroethoxy)ethoxy (FEE), and 2-(2-(2-
fluoroethoxy)ethoxy)ethoxy (FEEE) groups were incorporated in the 4’-position on ring B to
improve the feasibility of 18F-labeling and provide desirable physicochemical and
pharmacokinetic properties. The nucleophilic aliphatic fluorination on ring B allows more
1
8
feasible introduction of [ F]fluoride to diphenylsulfides. Novel biphenylsulfides 7a-g with
various 4-substituents and 4’-fluoroethoxy-substitution were synthesized. The SERT binding
affinity, monoamine transporter selectivity, lipophilicity, metabolic stability, and BBB
1
8
permeability of compounds 7 were evaluated. In addition, the 4-Me-substituted [ F]7a-c were
1
8
prepared for PET imaging studies for the discovery and development of new F-labeled PET
tracers for SERT brain imaging. Part of this work has been presented in abstract form.36
6

2
.
Results and discussion
.1. Chemistry
The 4’-fluoroethoxy-substituted biphenylsulfides 7a-g were synthesized as shown in
2
Schemes 1 and 2. 2-Amino-5-methoxybenzoic acid was readily converted to the corresponding
diphenylsulfide 11 via the literature procedure with modification using 1-bromo-4-methyl-2-
nitrobenzene (Scheme 1).29, 37 Treatment of benzoic acid 11 with oxalyl chloride and coupling
with dimethylamine yielded the amide 12. Selective O-demethylation of 12 using BBr -Me S
3
2
in 1,2-dichloroethane produced the phenol 13. Compounds 14a and 14c were synthesized by
alkylation of phenol 13 with 2-fluoroethyl tosylate and 1-(2-(2-chloroethoxy)ethoxy)-2-
fluoroethane, respectively.
Selective reduction of amides 14a and 14c using five equivalents of BH -THF provided
3
amines 15a and 15c in 73% and 72% yields, respectively. Further reduction of the nitro group
in compounds 15a and 15c by using SnCl in conc. HCl and MeOH yielded the desired anilines
2
7
a and 7c in 36% and 33% yields, and unexpected byproducts 8a and 8c in 18% and 16% yields,
respectively. The structures of 8a and 8c were elucidated by using NMR and mass spectrometry,
3
5
37
in which the two singlet signals on ring A and the characteristic Cl/ Cl isotope peaks in a
ratio of 3/1 confirmed that the chlorination occurred at the 5-position of ring A. To the best of
our knowledge, this is the first case demonstrating that p-chloroaniline was produced via a one-
pot reduction of ortho-sulfur-substituted nitrobenzene. The most similar simultaneous
reduction and chlorination reported previously was the reduction of an ortho-oxygenated
3
8
nitrobenzene, in which 10% of p-chloroaniline product was formed. Therefore, using SnCl
2
for the reduction of nitro groups in radiosynthesis may result in a similar side reaction.
Alternatively, reduction of compound 15a using ten equivalents of BH -THF gave 7a in 65%
3
yield (Table 1, method II). Therefore, to increase the yields of compounds 7 and prevent the
formation of the undesired chlorinated byproducts 8, simultaneous reduction of both amide and
7

2
9
nitro groups of 14a using ten equivalents of BH -THF was conducted, and only compound 7a
3
was afforded in 55% yield without any detectable byproduct 8a (Table 1, method III).
Scheme 1a
O
OH
O
OH
O
N
NO2
NO2
H N
2
S
a
b
S
OMe
OMe
OMe
11
12
O
N
N
NO2
NO2
O
N
NO2
S
S
c
d
e
S
F
F
n
O
n
O
OH
1
4a: n = 1
15a: n = 1
5b: n = 2
15c: n = 3
13
14b: n = 2
1
14c: n = 3
h
g
f or g
i
O
N
N
N
NO2
NH2
S
NH2
S
S
+
OH
2
F
F
n
O
O
n
O
Cl
7a: n = 1
7b: n = 2
7c: n = 3
8a: n = 1
8c: n = 3
16b
a
Reagents and conditions: (a) (i) 50% NaOH , NaNO , H O, rt, (ii) HCl(concd), -10°C, (iii)
(aq)
2
2
KS COEt, H O, 80°C, (iv) 1-bromo-4-methyl-2-nitrobenzene, EtONa, EtOH, reflux; (b) (i)
2
2
(
COCl) , DMF, CH Cl , rt, (ii) Et N, Me NH, CH Cl , rt; (c) BBr -Me S, 1,2-dichloroethane,
2 2 2 3 2 2 2 3 2
7
0°C; (d) 2-fluoroethyl-4-toluenesulfonate, Cs CO , DMF, 75°C, for 14a and 1-(2-(2-
2 3
chloroethoxy)ethoxy)-2-fluoroethane, NaI, Cs CO , DMF, 75°C, for 14c; (e) (i) BH -THF (5
2
3
3
eqiv), THF, reflux, (ii) HCl(concd), (iii) H O, reflux; (f) SnCl , HCl(conc), MeOH, rt; (g) (i) BH3-
2
2
THF (10 eqiv), THF, reflux, (ii) HCl(concd), (iii) H O, reflux; (h) 2-(2-chloroethoxy)ethanol,
2
NaI, Cs CO , DMF, 75°C; (i) Et NSF , CH Cl , -78°C~rt.
2
3
2
3
2
2
Alkylation of the phenol group of compound 13 with 2-(2-chloroethoxy)ethanol produced
alcohol 16b. Treatment of 16b with DAST obtained fluoride 14b in moderate yield. Reduction
of 14b by using different amounts of BH -THF (5 and 10 equiv) gave 15b and 7b in 71% and
3
8

6
3% yields, respectively.
Table 1. Reagents and yields for the synthesis of compound 7a.
Starting
material
Reagent &
conditions
Total yield
Method
Product
Yield
(From 14a)
I
15a
15a
14a
7a
7a
7a
SnCl , HCl
36%
65%
55%
26%
47%
55%
2
II
10 equiv BH -THF
3
III
10 equiv BH -THF
3
Synthesis of the 4-H and 4-F-substituted diphenylsulfides 7d-g is illustrated in Scheme 2.
The key intermediates phenolic compounds 17 and 18 were prepared using the same method
for the preparation of compound 13.29 Treatment of compounds 17 and 18 with 2-(2-
chloroethoxy)ethanol and 2-(2-(2-chloroethoxy)ethoxy)ethanol afforded compounds 16d-g.
Alcohols 16d-g were directly converted to the fluorides 14d-g by treatment with DAST in
moderate yields. Compounds 7d-g were synthesized by simultaneous reduction of nitro and
amide groups of 14d-g using ten equivalents of BH -THF in 48~63% yields. The purity of
3
target compounds 7 and 8 used for the bioassay was confirmed by using HPLC analysis and
showed greater than 95% purity.
1
8
The O-mesylate precursors 20a-c for radiosynthesis of [ F]7a-c were synthesized as
shown in Scheme 3. Phenol 13 was alkylated with corresponding haloalcohols to give
compounds 16a-c in 73-80% yields. Selective reduction of the amide group of 16a-c by using
five equivalents of BH -THF provided compounds 19a-c in 65-76% yields. Treatment of
3
alcohols 19a-c with methanesulfonyl chloride furnished the desired precursors 20a-c in 70-80%
1
8
yields. [ F]7a-c were synthesized via a one-pot two-step labeling reaction following previous
1
8
31
procedures for the preparation of [ F]3 and the reaction conditions were not optimized.
9

Scheme 2a
O
N
O
N
NO2
NO2
S
S
a
OH
n
R
O
R
OH
1
1
7: R = H
8: R = F
16d: R = H, n = 2
16e: R = H, n = 3
1
1
6f: R = F, n = 2
6g: R = F, n = 3
O
N
N
NO2
NH2
S
S
b
c
F
n
F
n
R
O
R
O
14d: R = H, n = 2
14e: R = H, n = 3
14f: R = F, n = 2
14g: R = F, n = 3
7d: R = H, n = 2
7e: R = H, n = 3
7f: R = F, n = 2
7g: R = F, n = 3
aReagents and conditions: (a) 2-(2-chloroethoxy)ethanol or 2-(2-(2-
chloroethoxy)ethoxy)ethanol, NaI, Cs CO , DMF, 75°C; (b) Et NSF , CH Cl , -78°C~rt; (c)
2
3
2
3
2
2
(
i) BH -THF (10 eqiv), THF, reflux, (ii) HCl
, (iii) H O, reflux.
3
(concd)
2
All radiosynthesis in this study was conducted in a modified automatic synthesizer, in
which a radio-HPLC system was connected to a General Electric TracerLab FxFDG module, and
1
8
provided sufficient amounts of [ F]7a-c for in vivo PET imaging experiments. Treatment of
1
8
mesylate precursors 20a-c with [ F]fluoride in the presence of Kryptofix 222 and potassium
carbonate in DMSO at 120°C for 10 min, followed by in situ reduction of the intermediates
1
8
with NaBH /Cu(OAc) in EtOH at 80°C for 20 min provided [ F]7a-c (Scheme 3). The crude
4
2
1
8
product was directly purified by semiprep HPLC and formulated via SPE to obtain [ F]7a-c in
.0, 2.3, and 2.6% radiochemical yields (RCY, n＞3), respectively, with >98% radiochemical
purity. The synthesis time, including HPLC purification and formulation, was ~120 min from
2
1
8
EOB. The specific activities of [ F]7a-c were 9.6, 4.2, and 3.4 Ci/μmol (EOS), respectively.
1
8
The identities of each F-labeled ligand were verified by comparing its HPLC chromatogram
1
0

with the authentic samples.
Scheme 3a
O
N
N
NO2
NO2
S
S
a
b
13
OH
n
OH
n
O
O
16a: n = 1
16b: n = 2
16c: n = 3
19a: n = 1
19b: n = 2
19c: n = 3
N
N
NO2
NH2
S
S
c
d
OMs
n
18F
n
O
O
1
8F]7a: n = 1
F] : n = 2
F]7c: n = 3
2
2
2
0a: n = 1
0b: n = 2
0c: n = 3
[
[
[
18 7b
18
aReagents and conditions: (a) 2-bromoethanol (for 16a), 2-(2-chloroethoxy)ethanol (for 16b),
and 2-(2-(2-chloroethoxy)ethoxy)ethanol (for 16c), NaI, Cs CO , DMF, 75°C; (b) BH -THF
2
3
3
(
5 eqiv), THF, reflux; (ii) HCl(conc), (iii) H O, reflux; (c) MsCl, Et N, CH Cl , rt; (d) (i)
2 3 2 2
1
8
K[ F], K , DMSO, 120°C; (ii) NaBH , Cu(OAc) , EtOH, 80°C.
2
22
4
2
2
2
.2. Evaluation
.2.1. SERT binding affinity and selectivity
The binding affinities of this series of diphenylsulfides for SERT, NET, and DAT were
determined through in vitro competition assays using SERT-, NET-, and DAT-overexpressing
human CHO cells,39-43 and are shown in Table 2. All 4’-(2-fluoroethoxy)-substituted
diphenylsulfide derivatives demonstrated high SERT binding affinity (K = 0.11-1.32 nM),
i
moderate NET binding affinity (K = 4.1-98 nM), and weak DAT binding affinity (6~23%
i
inhibition at 0.1 M), which is consistent with the binding profiles reported in the literature.17,
2
0-21, 23, 28, 44-45
When the length of the 4’-side chain increased, both the SERT binding affinity
and SERT selectivity vs. NET decreased. For example, in the 4-Me series, the rank order of the
SERT binding affinity and selectivity is 7a > 7b > 7c. In addition, compounds 7a and 8a showed
1
1

higher SERT selectivity vs. NET than the 4-Me-substituted lead compound 2.
The SERT binding affinity of these ligands is also highly dependent on the 4-substituents
on the ring A, and the rank order of their SERT binding affinity is 4-Me-5-Cl ≈ 4-F > 4-Me ≈
4
2
5
-H. Previously, incorporation of a fluoro-substituent on the 5-position of ring A in compound
2
3
significantly reduced its SERT binding affinity (i.e., > 100-fold), whereas in this study the
-chloro-substituted 8a and 8c showed higher SERT binding affinities (0.11 and 0.21 nM,
respectively) than their corresponding 5-nonsubstituted analogues 7a and 7c (0.25 and 1.32 nM,
respectively).
The size of the 4-substituent on ring A of the diphenylsulfide played a significant role in
the binding affinity for NET, in which a larger 4-substituent resulted in weaker binding affinity
toward NET.29, 46 Consistent with the previous SAR, the 4-H-substituted analogues (7d and 7e)
displayed the highest NET binding affinity (K = 4~6 nM), and the 4-F-substituted analogues
i
(
7f and 7g) demonstrated high NET affinity (K = 9~12 nM), whereas the 4-Me-substituted
i
derivatives (7a-c, 8a, and 8c) exhibited the lowest binding affinity for NET (K = 54~98 nM).
i
In this series, the rank order of NET binding affinity is 4-H > 4-F >> 4-Me ≈ 4-Me-5-Cl.
As a result, the SERT binding selectivity vs. NET is highly dependent on the substitution
on C-4 of ring A in the rank order of 4-Me-5-Cl > 4-Me ≈ 4-F > 4-H. In contrast to the influence
of 4-substitution on SERT binding affinity, the length of the 4’-side chain did not show a
significant influence on NET binding affinity of these 4’-fluoroethyl-substituted
diphenylsulfides. The SERT selectivity vs. NET was decreased when the length of the 4’-side
chain increased, which is mainly due to the decreased SERT binding affinity, not the differences
in their NET binding affinity.
The 4-Me analogues 7a-c and 4-Me-5-Cl compounds 8a and 8c displayed high SERT
binding affinities, low NET affinity, and therefore much higher SERT selectivity vs. NET than
their corresponding 4-H and 4-F analogues 7d-g. On the other hand, the 4-H derivatives 7d-e
1
2

possessed the lowest SERT selectivity in the series. The 4-Me-5-Cl compound 8a is the most
potent and selective SERT ligand in this study.
Table 2. Binding affinities of 4’-fluoroethoxy-substituted diphenylsulfides.
a
K (nM)
i
K ratio
i
compd R1 R2
R3
SER
T
NET/SER
T
NET
DATb
7
a
CH3
H
O(CH2)2F
0.25
98
60
55
77
54
4.1
5.6
9.2
12
13%
392
120
41
7
b
CH3 H [O(CH2)2]2F 0.50
CH3 H [O(CH2)2]3F 1.32
11%
11%
18%
6%
7
c
8
a
CH3 Cl O(CH2)2F
0.11
700
257
14
8
c
CH3 Cl [O(CH2)2]3F 0.21
7
d
H
H
H [O(CH ) ] F 0.29
23%
17%
9%
2
2 2
7
e
H [O(CH ) ] F 0.43
13
2
2 3
7
f
F
H [O(CH ) ] F 0.12
77
2
2 2
7
g
F
H [O(CH ) ] F 0.20
14%
60
2
2 3
2
CH3
H
H
1.19c
320c >10,000c
269
a
Inhibition constants (K ) were determined in duplicate with semiquantitative data. SERT:
i
3
using human recombinant HEK-293 cells, 0.40 nM [ H] paroxetine, 10.0 M imipramine;
1
25
NET: using human recombinant MDCK cells, 0.20 nM [ I] RTI-55, 10.0 M nomifensine;
1
25
DAT: using human recombinant CHO-K1 cells, 0.15 nM [ I] RTI-55, 10.0 M desipramine.
b
c
Inhibition percentage determined at 0.1 M of test compound. Data were determined by
using human transporter expressed HEK-293 cells.23
2
.2.2.
Lipophilicity is an important physicochemical parameter for correlating structure with the
blood-brain barrier (BBB) permeability, nonspecific binding in the brain, and washout kinetics
Lipophilicity and BBB permeability
1
3

in vivo. The experimental LogP7.4 values of compounds 7 and 8 were determined using the
4
7
HPLC method as shown in Table 3. The LogP of 7a-g were in the range of 3.30-3.70, and
7
.4
were slightly decreased when the length of the 4’-side chain increased (n=1 > n=2 > n=3). The
-substituents on ring A had a more prominent effect on the lipophilicity of diphenylsulfides,
4
in which the rank order of lipophilicity is 4-Me-5-Cl > 4-F > 4-Me > 4-H. The 5-Cl-substitution
significantly increased the lipophilicity of compounds 8. The calculated total polar surface areas
(
TPSA) of compounds 7 and 8 were between 38.5~57.0 and are in the range known for BBB-
permeable ligands.
The BBB permeability of this series of 4’-fluoroethoxy-substituted diphenylsulfides was
4
8
investigated using an in vitro parallel artificial membrane permeability assay (PAMPA), and
all the new compounds possessed moderate to high BBB permeability (Table 3). The 4-Me
compounds 7a-c and 4-H compounds 7d-e exhibited high BBB permeability (Pe = 38.1~46.6
-6
×
10 cm/s), whereas the more lipophilic 4-F-substituted compounds 7f-g showed moderate
-6
BBB permeability (Pe = 32.5~35.6 × 10 cm/s), and the most lipophilic 4-Me-5-Cl compounds
-6
8
demonstrated the lowest brain penetration ability (Pe = 17.0~25.5 × 10 cm/s). The increased
lipophilicity was detrimental to BBB permeability for this series of 4’-fluoroethoxy-substituted
diphenylsulfides.
1
4

2
.2.3.
Metabolic stability
Table 3. Physicochemical properties and metabolic stability of 4’-fluoroethoxy-substituted
diphenylsulfide derivatives.
N(CH3)2
NH2
S
F
R4
O
n
R5
TPSA,
Ǻ2)b
PAMPA, Pe
Stability, Metabolite
compd
n
1
2
3
R4
R5
H
H
H
LogP7.4a
3.64
-6
c
(%)d
(m/z)e
(
(10 cm/s)
M+16
M–14
7
a
Me
Me
Me
38.5
47.7
57.0
42.0±1.4
42.8±8.1
38.1±3.3
41
M+16
M–14
7
b
3.53
60
62
M+16
M–14
7
c
3.43
M+16
M–14
7
d
2
3
2
3
H
H
F
H
H
H
H
3.37
3.30
3.70
3.63
47.7
57.0
47.7
57.0
42.8±3.2
46.6±4.1
35.6±1.9
32.5±1.5
54
---
64
---
7
e
---
M+16
M–14
7
f
7
8
g
a
F
---
M–28
M+16
M–14
1
Me
Cl
4.55
4.38
38.5
17.0±2.7
18
8
c
3
-
Me
F
Cl
H
57.0
29.3
25.5±9.1
---
0
---
M+16
M+32
3
3.87
---
a
b
c
Determined by HPLC method. Calculated by ChemDraw 17.1.0.105. Determined by in
-6
48 d
vitro PAMPA assay (n ≥ 3) and calculated as Pe (10 cm/s). The parent compound
concentrations (%) remaining in the incubation solution at 30 min were determined by HPLC
e
and calculated according to the concentration of parent compound at 0 min. The m/z of
1
5

potential metabolites.
The in vitro metabolic stability of selected 4’-fluoroethoxy-substituted diphenylsulfides
was investigated using rat liver S9 fraction49-50 and the results are shown in Table 2. Extension
of the length of the 4’-side chain increased the metabolic stability: n=3 (7c) > n=2 (7b) > n=1
(
7a). This is consistent with the idea that polyethyleneglycol modification can enhance the
stability of the parent compound in blood circulation. When possessing the same 4’-side chain
n=2), the 4-substituents exhibited minor influence on the metabolic stability, in which the rank
(
order of stability is 4-F (7f) > 4-Me (7b) > 4-H (7d). With the same 4-fluoro-substitution, 64%
of compound 7f remained after 30 min, whereas compound 3 was completely metabolized in
1
8
3
0 min, which is consistent with the previous data, in which [ F]3 was metabolized rapidly in
3
3
rats. This fast metabolism may play an important role in the fast washout of 3 from the brain.
In this series, 8a was the most metabolically unstable compound, as only 18% of 8a remained
after 30 min (Table 3).
The major metabolites were analyzed by using LC/MS. Most metabolites were formed due
to oxidation (i.e., hydroxylation or sulfoxide formation, in which mono-oxidation (M+16) and
di-oxidation (M+32) were found) and demethylation (i.e., mono-demethylation (M–14) and bis-
demethylation (M–28)) reactions. All major metabolites were found to be more hydrophilic
than the parent compounds based on their retention times in RP-HPLC. Compound 3 was highly
unstable metabolically, and oxidation reactions (i.e., M+16 and M+32) were the major
metabolic pathway. The 4’-fluoroethoxy side-chain significantly increased the metabolic
stability of compounds 7, in which 41-64% of parent compounds remained after 30 min. Both
oxidation and demethylation reactions were observed in compounds 7 and 8, and a longer 4’-
side chain length resulted in higher metabolic stability. Incorporation of the 5-Cl-substituent
significantly enhanced the metabolism of 8a, in which significantly increased demethylation
reactions including the bis-demethylation metabolites were found (i.e., M–14 and M–28). It
seems that high lipophilicity may enhance their accessibility to metabolic enzymes, and
1
6

therefore increased hydrophilicity would reduce their ability to access the metabolic enzymes
and provide higher metabolic stability.
2
.2.4.
In vivo PET studies
To investigate the in vivo SERT binding profiles of compounds 7a-c, the brain uptake and
1
8
18
regional brain distribution of [ F]3 and [ F]7a-c in rats were measured by using micro-PET.
[18
F]3 demonstrated a kinetics profile similar to that previously reported, in which the uptake
1
8
33
of [ F]3 in the midbrain peaked at ≈20 min after tail vein injection and then declined quickly.
[18
F]-7a and [ F]-7b readily penetrated the BBB and exhibited high uptake in the SERT-rich
18
brain regions, such as the midbrain, hypothalamus, striatum, and thalamus. (Figure 1). The
1
8
18
18
18
amounts of initial brain uptake were in the rank order of [ F]3 > [ F]7a ≈ [ F]7b > [ F]7c,
and therefore increasing the number of ethoxy units at the 4’-position on ring B resulted in
1
8
18
18
lower brain uptake. The maximum brain uptake of [ F]3, [ F]7a, and [ F]7b in SERT-rich
regions peaked at 20 min, 60 min, and 180 min, respectively. The notably different brain
uptakes between 20 and 180 min for these ligands provided information on the relative kinetics
1
8
18
18
of brain washout. [ F]7a and [ F]7b had much slower brain washout than [ F]3 from the
SERT-rich brain regions and therefore demonstrated sustained high brain uptake.
The time-activity curves (TACs) in various brain regions were expressed as normalized
standard uptake values (SUVs). The specific uptake ratios (SURs) in various brain regions were
expressed as SUVtarget region / SUVcerebellum. The maximal SURs in SERT-rich regions
1
8
18
18
peaked at 60 min for [ F]3 (2.0-2.5), 180 min for [ F]7a (1.6-2.3), and 180 min for [ F]7b
(2.0-2.5), respectively (Supporting information, Table 4). The SURs in the midbrain were in
1
8
18
18
18
18
18
the rank order of [ F]3 ≈ [ F]7b > [ F]7a >> [ F]7c at 60 min and [ F]7a ≈ [ F]7b >>
[18
F]7c >> [ F]3 at 180 min. As a result, [ F]-7a and [ F]-7b possessed comparable SURs at
18
18
18
1
8
6
0 min and higher SURs at 120 min than [ F]3.
1
7

1
8
18
Figure 1. SUVs and SURs of [ F]3 and [ F]7a-c in rat brains.
1
8

1
8
18
To evaluate the specificity and reversibility of the brain uptake of [ F]3 and [ F]7a-c in
rats, blocking studies were conducted by using SERT-selective citalopram at 80 min (Figure
1
8
2
). In the citalopram-treated group at 150 min: the SUVs of [ F]3 in the hypothalamus and
1
8
striatum were decreased by 31 and 27%, respectively, and the SUVs of [ F]7a in the
hypothalamus and striatum were decreased by 37 and 33%, respectively (Supporting
1
8
information, Table 5). Under the same experimental conditions, the SUVs of [ F]7b in both
the hypothalamus and striatum were only decreased by 9%.
1
8
18
Figure 2. Dynamic PET images of [ F]3 and [ F]7a-c in rat brain. (A) The tracers were iv-
injected and then scanned for 180 min. (B) For the blocking study: the tracers were iv-injected
at 0 min, and citalopram-HBr (1.5 mg/kg in 0.5 mL) was iv-injected at 80 min. The data
between 0-180 min were collected.
1
9

2
.2.5.
Discussion
The 4-Me-substituted diphenylsulfides 7a-c possessed high binding affinity and
selectivity for SERT versus NET, moderate lipophilicity, and high BBB permeability, while
the 4-Me-5-Cl analogs 8a and 8c had high affinity and selectivity for SERT vs. NET, high
lipophilicity, and relatively low BBB permeability (Table 4). The 4-H diphenylsulfides 7d
and 7e exhibited low binding selectivity for SERT vs. NET, low lipophilicity, and high BBB
permeability, whereas the corresponding 4-F derivatives 7f and 7g showed moderate
selectivity for SERT vs. NET, high lipophilicity, and moderate BBB permeability. As a result,
the 4-Me derivatives 7a-c maintained the high BBB permeability of their 4-H analogs and
demonstrated much higher SERT binding selectivity than their corresponding 4-H derivatives.
In contrast to the previous SAR, the additional 5-Cl-substitution on ring A maintained high
SERT binding affinity and selectivity. However, the 5-Cl-substitution was detrimental to the
BBB permeability of 8a and 8c.
Table 4. SERT binding affinity, selectivity, lipophilicity, and BBB permeability of 4-
substituted diphenylsulfides.
4
-Me
a-c
4-H
4-F
4-Me-5-Cl
Compound
7
7d-e
7f-g
8a, 8c
SERT binding
affinity
high
high
high
low
high
high
high
SERT vs NET
selectivity
moderate
Lipophilicity
LogP
++
+
+++
++++
3.4~3.6
3.3~3.4
3.6~3.7
4.4~4.6
BBB permeability
PAMPA assay
high
high
moderate
low
2
0

These 4’-(2-fluoroethoxy)-substituted diphenylsulfides are relatively easy to prepare, and
1
8
the radiosynthesis of [ F]7a-c via an aliphatic nucleophilic substitution reaction is much more
1
8
feasible than using aromatic nucleophilic substitution reaction in the preparation of [ F]3.
Incorporation of the 4’-(2-fluoroethoxy)-substituents to diphenylsulfides appeared to reduce the
rate of clearance from the brain, which is consistent with a previous observation that similar
substitution at the 4’-position on the B ring resulted in slow washout kinetics in the brain.51
1
8
The initial brain uptake of [ F]7a-c was proportional to their lipophilicity (i.e., LogP ),
7
.4
1
8
18
and lower lipophilicity resulted in lower initial brain uptake. [ F]-7a and [ F]-7b exhibited
substantial brain uptake and moderate target to nontarget ratios in rat brains, but these results
were not satisfactory as expected due to the slow in vivo kinetics. Fast kinetics may need fast
clearance, including fast metabolism and lack of lipophilic BBB-permeable metabolites. The
1
8
18
slow brain washout of [ F]-7a and [ F]-7b from the brain tissues may be caused by their high
1
8
SERT binding affinity and higher metabolic stability in comparison to [ F]3. The fast
1
8
metabolism of [ F]3 facilitated its clearance from the blood circulation, which should be
beneficial to faster brain washout and equilibrium, and therefore higher specific binding. The
ethoxy moiety increased the metabolic stability of compounds 7, and therefore the clearance of
7
was slowed down and resulted in slow equilibrium and brain washout. The brain washout of
[18
F]3 and [ F]-7a were significantly increased after treatment with SERT-selective citalopram.
18
1
8
18
The blockade data suggested that [ F]3 and [ F]-7a demonstrated similar specificity and
1
8
reversibility of SERT binding in rat brains, whereas most brain uptake of [ F]-7b was due to
1
8
nonspecific and/or irreversible binding in brains. Although the slow brain washout of [ F]-7a
may limit its application in a quantitative imaging study, the selective and reversible uptake in
SERT-rich regions in a longer period of time (60-180 min) may be an advantage for a qualitative
PET imaging study or diagnosis.
2
1

3
.
Conclusion
In summary, a novel series of 4’-fluoroethoxy-substituted diphenylsulfide derivatives was
synthesized and demonstrated high binding affinity and selectivity for SERT versus NET and
DAT. These ligands exhibited desirable lipophilicity and BBB permeability in the PAMPA
assay. The 4-Me derivatives possessed both the high SERT binding affinity and selectivity of
the corresponding 4-F analogs and the high BBB permeability of the corresponding 4-H
derivatives. In the series, compounds 7a and 7b demonstrated very high and selective SERT
binding affinity with excellent BBB permeability, and compound 8a demonstrated the highest
1
8
SERT binding affinity and selectivity. [ F]7a-c were automatically synthesized by a one-pot
1
8
two-step method using a modified GE TraceLab FxFDG module and compared with [ F]3 as
1
8
18
SERT imaging agents in animal PET studies. [ F]-7a and [ F]-7b showed moderate brain
1
8
uptake and sustained retention after iv injection in rats. [ F]-7a selectively and reversibly
accumulated in SERT-rich regions of rat brains. Thus, the 4-Me-substituted compounds 7a, 7b,
and 8a, which could provide more precise SERT imaging than those with lower SERT
1
8
selectivity, are promising leads for the further development of novel [ F]-labeled PET
molecular imaging probes for SERT-related diseases and disorders..
2
2

4
.
Experimental
4
.1. Chemistry
NMR spectra were recorded on Bruker DPX-200, AV-400 and AV-600 FT-NMR
spectrometers. Chemical shifts are expressed in parts per million (ppm) on the  scale relative
to a tetramethylsilane (TMS) internal standard. Electron spray ionization (ESI) mass spectra
and high-resolution mass measurements (HRMS) were obtained using an Esquire 2000 and
Bruker Daltonik micro TOF mass spectrometer, respectively. Thin-layer chromatography (TLC)
was performed on Merck (art. 5715) silica gel plates and visualized under UV light (254 nm),
upon treatment with iodine vapor, or upon heating after treatment with 5% phosphomolybdic
acid in ethanol. Flash column chromatography was performed with Merck (art. 9385) 40–63
m silical gel 60. Anhydrous tetrahydrofuran (THF) was distilled from sodium-benzophenone
prior to use. 1,2-Dichloroethane, triethylamine (Et N) and dichloromethane (DCM) were
3
distilled from calcium hydride prior to use. Methanesulfonyl chloride (MsCl) was distilled from
phosphorus pentoxide under reduced pressure prior to use. No attempt was made to optimize
yields.
4
.1.1.
2-(2-((Dimethylamino)methyl)-4-(2-fluoroethoxy)phenylthio)-5-methyl-
benzenamine
(7a)
and
4-chloro-2-(2-((dimethylamino)methyl)-4-(2-fluoro-
ethoxy)phenylthio)-5-methyl-benzenamine (8a). Method I: To a solution of compound 15a
(
82 mg, 0.22 mmol) in MeOH (3 mL)and HCl (concd, 1 mL) was added SnCl (410 mg, 2.2
2
o
mmol) at 0 C. The reaction mixture was stirred at rt under N overnight. The mixture was
2
quenched with water (25 mL), basified with 1 M NaOH to pH = 11, extracted with CH Cl (25
2
2
mL × 3), washed with brine, dried over MgSO and evaporated. The crude product was
4
chromatographed (silica gel, NH OH/MeOH/CH Cl = 0.1/1/49) to afford 7a (27 mg, 36%) and
4
2
2
8
a (15 mg, 18%) as oils. Method II: To a solution of compound 15a (40 mg, 0.11 mmol) in
2
3

o
THF (3 mL) was added BH -THF (1 M in THF, 1.65 mL, 1.65 mmol) at 0 C. The mixture was
3
refluxed under N for 36 h and then cooled to rt. HCl (concd, 0.3 mL) was added to the mixture,
2
stirred at rt under N for 10 min and evaporated to afford an oily residue. Water (10 mL) was
2
added to the residue and the solution was refluxed under N for 30 min. The solution was
2
adjusted to pH = 10-11 with Na CO
, extracted with CH Cl (10 mL × 3), washed with
3(sat.) 2 2
2
brine, dried over MgSO and evaporated. The crude residue was chromatographed (silica gel,
4
NH OH/MeOH/CH Cl = 0.1/1/49) to afford 7a (24 mg, 65%) as an oil. Method III: To a
4
2
2
solution of compound 14a (10 mg, 0.027 mmol) in THF (3 mL) was added BH -THF (1 M in
3
o
THF, 0.35 mL, 0.35 mmol) at 0 C. The mixture was refluxed under N for 5 h and then cooled
2
to rt. The HCl(concd) was added to the mixture, stirred at rt under N for 10 min and evaporated
2
to afford a yellow oil. Water was added to the oil and the mixture was refluxed under N for 30
2
min. The solution was adjusted to pH = 10-11 by saturated Na CO , extracted with CH Cl ,
2
3
2
2
washed with brine, dried over MgSO and evaporated. The crude product was chromatographed
4
1
(
silica gel, 2% MeOH in CH Cl ) to afford 7a (55%) as an oil. 7a: H NMR (400 MHz, CDCl )
2
2
3

4
2
2.27 (s, 3H), 2.30 (s, 6H), 3.54 (s, 2H), 4.16 (dt, J = 28.0, 4.1 Hz, 2H), 4.34 (bs, 2H, -NH2),
.71 (dt, J = 47.4, 4.1 Hz, 2H), 6.53-6.55 (m, 2H), 6.67 (dd, J = 8.6, 3.0 Hz, 1H), 6.87-6.90 (m,
1
3
H), 7.30 (d, J = 8.0 Hz, 1H); C NMR (100 MHz, CDCl )  21.5, 45.5, 45.5, 62.4, 67.3 (d, J
3
=
20.0 Hz), 82.0 (d, J = 170.0 Hz), 113.3, 114.3, 116.0, 116.6, 119.6, 128.7, 129.9, 136.9, 138.8,
+
1
8
4
40.9, 148.6, 156.8; ESIHRMS calcd for C H FN OS [M+H] , 335.1593; found, 335.1593.
1
8
24
2
1
a: H NMR (600 MHz, CDCl )  2.28 (s, 3H), 2.29 (s, 6H), 3.52 (s, 2H), 4.17 (dt, J = 27.7,
3
.2 Hz, 2H), 4.51 (bs, 2H), 4.72 (dt, J = 47.4, 4.2 Hz, 2H), 6.57 (s, 1H), 6.70 (dd, J = 8.5, 2.8
1
3
Hz, 1H), 6.89 (d, J = 2.8 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H), 7.37 (s, 1H), C NMR (150 MHz,
CDCl )  20.1, 45.4, 45.4, 62.5, 67.4 (d, J = 21.0 Hz), 82.0 (d, J = 169.5 Hz), 114.4, 115.7,
3
1
16.9, 117.3, 122.7, 127.7, 131.0, 136.0, 138.2, 139.4, 147.2, 157.2; ESIHRMS calcd for
+
C H ClFN OS [M+H] ,369.1204; found, 369.1218.
1
8
23
2
2
4

4
.1.2.
2-(2-((Dimethylamino)methyl)-4-(2-(2-fluoroethoxy)ethoxy)phenylthio)-5-
methylbenzenamine (7b). Compound 7b was synthesized from 15b according to the Method
1
II for the preparation of 7a and afforded as a white oil (63%). H NMR (400 MHz, CDCl ) 
3
2
.26 (s, 3H), 2.29 (s, 6H), 3.53 (s, 2H), 3.74-3.77 (m, 1H), 3.82-3.86 (m, 3H), 4.09 (d, J = 2.4
Hz, 2H), 4.58 (dt, J = 47.6 ,4.2 Hz, 2H), 6.52-6.54 (m, 2H), 6.67 (dd, J = 8.7, 2.5 Hz, 1H), 6.87-
1
3
6
6
1
3
.89 (m, 2H), 7.29 (d, J = 8.3 Hz, 1H); C NMR (100 MHz, CDCl )  21.5, 45.4, 45.4, 62.4,
3
7.7, 70.0, 70.7 (d, J = 19.5 Hz), 83.3 (d, J = 168.0 Hz), 113.5, 114.3, 116.0, 116.7, 119.6,
+
28.2, 129.9, 136.8, 138.7, 140.7, 148.4, 157.1; ESIHRMS calcd for C H FN O S [M+H] ,
2
0
28
2
2
79.1856; found, 379.1853.
4
.1.3.
2-(2-((Dimethylamino)methyl)-4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-
phenylthio)-5-methylbenzenamine (7c) and 4-chloro-2-(2-((dimethylamino)- methyl)-4-
2-(2-(2-fluoroethoxy)ethoxy)-ethoxy)phenylthio)-5-methyl- benzenamine (8c). Compound
(
7
c and 8c were synthesized from 15c according to the Method I for the preparation of 7a and
1
afforded as white oils 7c (33%) and 8c (16%). 7c: H NMR (400 MHz, CDCl )  2.26 (s, 3H),
3
2
.29 (s, 6H), 3.53 (s, 2H), 3.64-3.73 (m, 5H), 3.80-3.85 (m, 3H), 4.06-4.10 (m, 2H), 4.55 (dt, J
47.7, 4.2 Hz, 2H), 6.52-6.54 (m, 2H), 6.67 (dd, J = 8.7, 2.8 Hz, 1H), 6.87-6.89 (m, 2H), 7.30
=
1
3
(
d, J = 7.5 Hz, 1H); C NMR (100 MHz, CDCl )  21.5, 45.4, 45.4, 62.4, 67.6, 69.9, 70.6 (d,
3
J = 19.0 Hz), 71.0, 71.0, 83.3 (d, J = 168.0 Hz), 113.5, 114.4, 116.0, 116.7, 119.6, 128.1, 130.0,
+
1
4
36.8, 138.7 140.8, 148.4, 157.2; ESIHRMS calcd for C H FN O S [M+H] , 423.2118; found,
2
2
32
2
3
1
23.2116. 8c: H NMR (600 MHz, CDCl )  2.27 (s, 3H), 2.28 (s, 6H), 3.52 (s, 2H), 3.69-3.73
3
(m, 5H), 3.77 (m, 1H), 3.83 (t, J = 4.8 Hz, 2H), 4.09 (t, J = 4.8 Hz, 2H), 4.56 (dt, J = 47.7, 4.2
Hz, 2H), 6.56 (s, 1H), 6.70 (dd, J = 8.6, 2.8 Hz, 1H), 6.87 (d, J = 2.8Hz, 1H), 6.95 (d, J = 8.6
1
3
Hz, 1H), 7.36 (s, 1H) ; C NMR (150 MHz, CDCl ) 20.1, 45.4, 45.4, 62.6, 67.7, 69.9, 70.6 (d,
3
J = 19.5 Hz), 71.0, 71.0, 83.3 (d, J = 168.0 Hz), 114.5, 115.9, 116.9, 117.3, 122.6, 127.1, 131.1,
2
5

+
1
35.9, 138.1, 139.3, 147.1, 157.6; ESIHRMS calcd for C H ClFN NaO S [M+Na] , 479.1547;
22 30 2 3
found 479.1547.
4
.1.4.
2-(2-((Dimethylamino)methyl)-4-(2-(2-fluoroethoxy)-ethoxy)phenylthio)-
benzenamine (7d). Compound 7d was synthesized from 14d according to the Method III for
1
the preparation of 7a and afforded as a white oil (54%). H NMR (400 MHz, CDCl )  2.29 (s,
3
6
4
7
6
1
H), 3.54 (s, 2H), 3.75-3.77 (m, 1H), 3.82-3.86 (m, 3H), 4.08-4.11 (m, 2H), 4.57 (dt, J = 47.7,
.2 Hz, 2H), 6.67-6.70 (m, 3H), 6.90 (dd, J = 8.5, 2.9 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 7.12-
1
3
.17 (m, 1H), 7.38 (dd, J = 8.0, 1.6 Hz, 1H); C NMR (100 MHz, CDCl )  45.4, 45.4, 62.4,
3
7.6, 70.0, 70.7 (d, J = 20.0 Hz), 83.3 (d, J = 168.0 Hz), 114.3, 115.3, 116.8, 117.1, 118.4,
27.5, 130.2, 130.7, 136.5, 139.2, 148.3, 157.3; ESIHRMS calcd for C H FN NaO S
1
9
25
2
2
+
[
M+Na] , 387.1518; found, 387.1503.
4
.1.5.
2-(2-((Dimethylamino)methyl)-4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-
phenylthio)benzenamine (7e). Compound 7e was synthesized from 14e according to the
1
Method III for the preparation of 7a and afforded as a white oil (63%). H NMR (400 MHz,
CDCl )  2.29 (s, 6H), 3.54 (s, 2H), 3.69-3.74 (m, 5H), 3.78 (t, J = 4.2 Hz, 1H), 3.83 (t, J = 4.8
3
Hz, 2H), 4.07-4.10 (m, 2H), 4.55 (dd, J = 47.7, 4.2 Hz, 2H), 6.67-6.71 (m, 3H), 6.89 (d, J = 2.8
1
3
Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 7.13-7.17 (m, 1H), 7.38 (dd, J = 8.0, 1.5 Hz, 1H) ; C NMR
100 MHz, CDCl )  45.4, 45.4, 62.5, 67.6, 69.9, 70.6 (d, J = 19.0 Hz), 71.0, 71.0, 83.3 (d, J =
(
3
1
68.0 Hz), 114.4, 115.4, 116.8, 117.2, 118.4, 127.5, 130.2, 130.8, 136.5, 139.3, 148.4, 157.4;
+
ESIHRMS calcd for C H FN NaO S [M+Na] , 431.1781; found, 431.1777.
2
1
29
2
3
4
5
.1.6.
2-(2-((Dimethylamino)methyl)-4-(2-(2-fluoroethoxy)ethoxy)phenylthio)-
-fluorobenzenamine (7f). Compound 7f was synthesized from 14f according to the Method
1
III for the preparation of 7a and afforded as a white oil (55%). H NMR (400 MHz, CDCl ) 
3
2
6

2
2
.29 (s, 6H), 3.52 (s, 2H), 3.75-3.77 (m, 1H), 3.82-3.87 (m, 3H), 4.08-4.10 (m, 2H), 4.58 (m,
H), 6.36-6.42 (m, 2H), 6.69 (d, J = 8.6 ,2.8 Hz, 1H), 6.85 (d, J = 2.8 Hz, 1H), 6.90 (d, J = 8.6
1
3
Hz, 1H), 7.39-7.42 (m, 1H); C NMR (100 MHz, CDCl )  45.4, 45.4, 62.6, 67.7, 70.0, 70.7
3
(d, J = 19.0 Hz), 83.3 (d, J = 168.0 Hz), 101.7 (d, J = 25.0 Hz), 105.1 (d, J = 22.0 Hz), 112.0
(d, J = 2.0 Hz), 114.4, 117.0, 127.9, 130.5, 138.7 (d, J= 10.0 Hz), 138.9, 150.5 (d, J= 12.0 Hz),
+
1
57.3, 164.7 (d, J = 244.0 Hz); ESIHRMS calcd for C H F N NaO S [M+Na] , 405.1424;
1
9
24
2
2
2
found, 405.1412.
4
.1.7.
2-(2-((Dimethylamino)methyl)-4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-
phenylthio)-5-fluorobenzenamine (7g). Compound 7g was synthesized from 14g according
1
to the Method III for the preparation of 7a and afforded a white oil (48%). H NMR (400 MHz,
CDCl )  2.29 (s, 6H), 3.52 (s, 2H), 3.68-3.74 (m, 5H), 3.77-3.79 (m, 1H), 3.82-3.84 (m, 2H),
3
4
.07-4.09 (m, 2H), 4.55 (dt, J = 47.7, 4.2 Hz, 2H), 4.85 (bs, 2H, -NH ), 6.36-6.41 (m, 2H), 6.69
2
(d, J = 8.6 ,2.8 Hz, 1H), 6.84 (d, J = 2.8 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 7.41 (dd, J = 8.1, 6.7
1
3
Hz, 1H); C NMR (100 MHz, CDCl )  45.4, 45.4, 62.6, 67.6, 69.9, 70.6 (d, J = 20.0 Hz),
3
7
1
2
1.0, 71.0, 83.3 (d, J = 168.0 Hz), 101.7 (d, J = 25.0 Hz), 105.1 (d, J = 22.0 Hz), 112.1, 114.4,
17.0, 127.8, 130.5, 138.7 (d, J = 10.0 Hz), 138.9, 150.4 (d, J = 11.0 Hz), 157.3, 164.7 (d, J =
+
44.0 Hz); ESIHRMS calcd for C H F N NaO S [M+Na] ,449.1686; found, 449.1686.
2
1
28
2
2
3
4
.1.8.
5-Methoxy-2-(4-methyl-2-nitrophenylthio)benzoic acid (11). To a solution of 2-
amino-5-methoxybenzoic acid (4.0 g, 24 mmol) in 50% aqueous sodium hydroxide (2.6 mL,
4
7 mmol) was added a solution of NaNO (1.6 g, 24 mmol) in water (1.5 mL). The resulting
2
o
mixture was poured into a solution of HCl (concd, 8.2 mL, 94 mmol) at -10 C. The reaction
was stirred under N for 1 h and then neutralized by addition of potassium acetate to pH = 5-6.
2
The mixture was carefully added to a solution of potassium O-ethyl dithiocarbonate (KS COEt,
2
o
1
1.5 g, 72.0 mmol,) in water (50 mL) and stirred at 80 C under N for 30 min. The solution was
2
2
7

acidified by concd HCl to pH = 2-3, extracted with IPA/EtOAc (1/7, 400 mL × 3) and the
organic layer was evaporated. Toluene was added to the crude mixture for azeotropic removal
of residual water and a brown solid was obtained. To the solution of the brown solid in EtOH
(115 mL), a solution of EtONa (52.2 mmol) in ethanol (55 mL) and 1-bromo-4-methyl-2-
nitrobenzene (6.22 g, 28.8 mmol) were added in sequence. The mixture was refluxed under N2
for 16 h. After cooled to rt, the solvent was evaporated and then water (400 mL) was added to
the crude residue. The resulting mixture was acidified by concd HCl in a ice bath, and then
extracted with IPA/EtOAc (1/7, 400 mL × 3). The organic layer was washed with brine, dried
over MgSO , filtered, and evaporated. The residue was chromatographed (silica gel,
4
1
HOAc/EtOAc/n-hexane = 1/50/50) to afford 11 (4.1 g, 54%) as a yellow solid. H NMR (200
MHz, DMSO-d )  2.35 (s, 3H), 3.80 (s, 3H), 6.84 (d, J = 8.3 Hz, 1H), 7.17 (dd, J = 8.6, 2.9
6
1
3
Hz, 1H), 7.36-7.52 (m, 3H), 8.01-8.02 (m, 1H), 13.33 (bs, 1H, -COOH); C NMR (50 MHz,
DMSO-d )  19.9, 55.7, 115.3, 118.1, 121.3, 125.2, 130.0, 133.5, 135.0, 136.7, 137.6, 138.2,
6
+
1
3
46.0, 160.0, 167.5; ESIHRMS calcd for C H NNa O S [M-H+2Na] , 364.0232; found,
1
5
12
2
5
64.0231.
4
.1.9.
5-Methoxy-2-(4-methyl-2-nitrophenylthio)-N,N-dimethylbenzamide (12). To a
o
solution of compound 11 (1.8 g, 5.6 mmol) in CH Cl (50 mL) and DMF (0.2 mL) at 0 C,
2
2
oxalyl chloride (2 M in CH Cl , 5.6 mL, 11.2 mmol) was added dropwise and the mixture was
2
2
stirred at rt under N for 2 h. The reaction mixture was evaporated and treated with CH Cl (50
2
2
2
mL). To the solution were added Et N (4 mL, 30 mmol) and Me NH (2.9 M in CH Cl , 9.6 mL,
3
2
2
2
2
7.8 mmol). The mixture was stirred at rt under N for 30 min and evaporated. The residue was
2
treated with water (70 mL), extracted with CH Cl (70 mL × 3), washed with brine, dried over
2
2
MgSO , filtered and evaporated. The residue was chromatographed (silica gel, 10-20% EtOAc
4
1
in n-hexane) to afford 12 (1.4 g, 75%) as a yellow oil. H NMR (200 MHz, CDCl )  2.33 (s,
3
2
8

3
6
7
1
H), 2.86 (s, 3H), 3.03 (s, 3H), 3.88 (s, 3H), 6.83 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 2.7 Hz, 1H),
.99 (dd, J = 8.5, 2.8 Hz, 1H), 7.18 (ddd, J = 8.4, 2.0, 0.5 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H),
1
3
.98-7.99 (m, 1H); C NMR (50 MHz, CDCl )  20.5, 34.6, 38.5, 55.6, 112.5, 116.4, 118.1,
3
25.6, 129.1, 134.7, 135.4, 135.6, 139.0, 144.8, 145.1, 161.5, 169.0; ESIHRMS calcd for
+
C H N NaO S [M+Na] , 369.0885; found, 369.0886.
1
7
18
2
4
4
.1.10. 5-Hydroxy-2-(4-methyl-2-nitrophenylthio)-N,N-dimethylbenzamide (13). To a
solution of compound 12 (1.3 g, 3.8 mmol) in 1,2-dichloroethane (38 mL) was added BBr3-
o
(
CH ) S (1 M in CH Cl , 7.6 mL, 7.6 mmol). The mixture was heated at 70 C under N for 15
3
2
2
2
2
h, and then poured into a mixture of water (25 mL) and NaHCO3(sat.) (25 mL) in an ice bath.
The resulting mixture was extracted with CH Cl (50 mL × 3), washed with brine, dried over
2
2
MgSO and evaporated. The crude was chromatographed (silica gel, 15-50% EtOAc in n-
4
1
hexane) to afford 13 (1.00 g, 78%) as a yellow solid. H NMR (200 MHz, CDCl )  2.36 (s,
3
3
8
1
H), 2.91 (s, 3H), 3.08 (s, 3H), 6.81-6.90 (m, 3H), 7.21-7.27 (m, 1H), 7.37 (d, J = 8.4 Hz, 1H),
1
3
.00-8.01 (m, 1H), 9.32 (bs, 1H, -OH); C NMR (50 MHz, CDCl )  20.6, 35.1, 39.0, 115.3,
3
16.4, 118.8, 125.8, 129.2, 135.1, 135.4, 135.8, 139.3, 143.1, 144.8, 159.5, 170.7; ESIHRMS
+
calcd for C H N NaO S [M+Na] , 355.0728; found, 355.0728.
1
6
16
2
4
4
.1.11. 5-(2-Fluoroethoxy)-2-(4-methyl-2-nitrophenylthio)-N,N-dimethylbenzamide
(
14a). To a solution of compound 13 (210 mg, 0.63 mmol) in DMF (10 mL) were added Cs CO
2
3
(0.49 g, 1.5 mmol) and 2-fluoroethyl-4-toluenesulfonate (150 l, 0.91 mmol) in sequence. The
o
mixture was stirred at 75 C under N for 1 h. The reaction was treated with water (100 mL) and
2
extracted with CH Cl (100 mL × 3). The organic layer was washed with brine, dried over
2
2
MgSO and evaporated. The residue was chromatographed (silica gel, 30-60% EtOAc in n-
4
1
hexane) to afford 14a (210 mg, 87%) as a yellow oil. H NMR (200 MHz, CDCl )  2.33 (s,
3
3
H), 2.85 (s, 3H), 3.02 (s, 3H), 4.28 (dt, J = 27.7, 4.1 Hz, 2H), 4.79 (dt, J = 47.3, 4.2 Hz, 2H),
2
9