Synthesis and serotonin transporter activity of sulphur-substituted α-alkyl phenethylamines as a new class of anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2009.07.027

The discovery that some serotonin reuptake transporter (SERT) ligands have the potential to act as pro-apoptotic agents in the treatment of cancer adds greatly to their diverse pharmacological application. 4-Methylthioamphetamine (MTA) is a selective ligand for SERT over other monoamine transporters. In this study, a novel library of structurally diverse 4-MTA analogues were synthesised with or without N-alkyl and/or C-α methyl or ethyl groups so that their potential SERT-dependent antiproliferative activity could be assessed. Many of the compounds displayed SERT-binding activity as well as cytotoxic activity. While there was no direct correlation between these two effects, a number of derivatives displayed anti-tumour effects in lymphoma, leukaemia and breast cancer cell lines, showing further potential to be developed as possible chemotherapeutic agents.

Full text of DOI:10.1016/j.ejmech.2009.07.027

European Journal of Medicinal Chemistry 44 (2009) 4862–4888
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and serotonin transporter activity of sulphur-substituted
phenethylamines as a new class of anticancer agents
a-alkyl
,
Suzanne M. Cloonan a, John J. Keating b 1, Stephen G. Butler b, Andrew J.S. Knox a,
Anne M. Jørgensen c, Gu¨nther H. Peters c, Dilip Rai d, Desmond Corrigan b,
,
David G. Lloyd a, D. Clive Williams a, Mary J. Meegan b
*
a School of Biochemistry and Immunology, Trinity College Dublin, Ireland
b School of Pharmacy and Pharmaceutical Sciences, Centre for Synthesis and Chemical Biology, Trinity College Dublin, Dublin 2, Ireland
c EMPHYS – Center for Biomembrane Physics, Department of Chemistry, Technical University of Denmark, Building 206, 2800 Kgs. Lyngby, Denmark
d Centre for Synthesis and Chemical Biology, School of Chemistry & Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery that some serotonin reuptake transporter (SERT) ligands have the potential to act as
pro-apoptotic agents in the treatment of cancer adds greatly to their diverse pharmacological application.
4-Methylthioamphetamine (MTA) is a selective ligand for SERT over other monoamine transporters. In
this study, a novel library of structurally diverse 4-MTA analogues were synthesised with or without
Received 28 March 2009
Received in revised form
8 July 2009
Accepted 30 July 2009
Available online 6 August 2009
N-alkyl and/or C-
a methyl or ethyl groups so that their potential SERT-dependent antiproliferative
activity could be assessed.
Many of the compounds displayed SERT-binding activity as well as cytotoxic activity. While there was
no direct correlation between these two effects, a number of derivatives displayed anti-tumour effects in
lymphoma, leukaemia and breast cancer cell lines, showing further potential to be developed as possible
chemotherapeutic agents.
Keywords:
SERT
4-MTA
Antiproliferative activity
Apoptosis
Ó 2009 Elsevier Masson SAS. All rights reserved.
a
-Alkylphenethylamine
1. Introduction
disorders such as depression, anxiety, substance abuse, suicide,
autism, Alzheimer’s and Parkinson’s disease [1].
The serotonin transporter (SERT) transports 5-hydroxytrypta-
mine (5-HT) from central and enteric nervous system synapses
back into pre-synaptic neurons determining the duration and
magnitude of 5-HT responses. Alterations in SERT activity, binding
site density and polymorphisms of the SERT gene have implicated
the transporter in many psychiatric and neurodegenerative
The serotonin transporter is an important pharmacological
target that has high affinity in vivo for tricyclic antidepressants
(TCAs) such as imipramine, serotonin-selective reuptake inhibitors
(SSRIs), like fluoxetine and nonselective stimulants including
cocaine and amphetamines [2]. Apart from its high expression in
the nervous system, SERT is expressed in a wide range of speci-
alised non-neuronal cells [3–5] and more interestingly, it has been
found in a number of B cell malignancies including diffuse large B
cell lymphoma, multiple myeloma and Burkitt’s lymphoma [6].
Recently, a new pharmacological application for SERT has been
reported, linking it with drug-induced apoptosis in lymphoma
[6–8], where it is thought to act as a pro-apoptotic target for SERT
ligands. It has been implicated in the mechanism of cytotoxicity
associated with the amphetamine analogues, fenfluramine and
3,4-methylenedioxymethamphetamine (MDMA) and SERT-target-
ing antidepressants [6,9,10]. The SSRI class of antidepressants
including, fluoxetine, paroxetine and citalopram have been repor-
ted to induce apoptosis in a range of malignant cell lines [8,11–14]
and the tricyclic antidepressants have been implicated in apoptotic
activities over a range of cell lines both neuronal and malignant
Abbreviations: 4-MTA, 4-methylthioamphetamine; 5-HT, 5-hydroxytryptamine;
ALEPH-2, 1-(2,5-dimethoxy-4-ethylthiophenyl)-2-aminopropane; AMPH, amphet-
amine; BBB, blood–brain barrier; BCB, blood–cerebrospinal fluid barrier; BL, Bur-
kitt’s lymphoma; DAT, dopamine transporter; EBV, Epstein barr virus; FACS,
fluorescent-activated cell sorting; HEK, human embryonic kidney cells; MDMA, 3,4-
methylenedioxymethamphetamine; NAT, noradrenaline transporter; NR, Neutral
Red; PBS, phosphate buffered saline; PCD, programmed cell death; PI, propidium
iodide; PMA, para-methoxyamphetamine (4-methoxyamphetamine); SERT, sero-
tonin transporter; SSRI, selective serotonin reuptake inhibitors; TCA, tricyclic
antidepressant.
* Corresponding author. Tel.: þ353 1 8962798; fax: þ353 1 8962793.
E-mail address: mmeegan@tcd.ie (M.J. Meegan).
Present address: School of Pharmacy, Cavanagh Pharmacy Building, University
1
College Cork (UCC), Ireland.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.027

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4863
[12,15–17]. Some reports have questioned the role of SERT in such
apoptosis [18]. High concentrations of MDMA have been shown to
induce SERT-independent cytotoxic effects in a number of cell lines
[19] and non-serotonergic effects of fluoxetine have been reported
[20]. This suggests that (a) molecular target(s) other than SERT may
exist on the lymphoma cell with the possibility that some
antidepressants preferentially target the proliferating B cell [8].
Because of its growing association with HIV infection, Burkitt’s
lymphoma (BL) is becoming a more common malignancy in
humans [21]. It is a tumour of B cells that accounts for 30–50% of
lymphomas in children [22] and remains a serious health problem
in those areas where it is endemic: namely the malarial belts of
equatorial Africa, north-eastern Brazil, and Papua New Guinea [23].
With combination and CNS chemotherapy, the survival rate of
patients with BL is reported to be at least 60%. Patients with bone
marrow and CNS involvement have a poor prognosis and adults
with the disease, especially those in the advanced stage, do more
poorly than affected children with reoccurrence common. The
development of novel selective apoptotic drugs and drug targets is
therefore imperative to the future of effective anticancer drug
design.
requirements and to determine if there are any structural correla-
tions relating to SERT binding and any observed toxicity.
Based on existing data linking SERT as a pro-apoptotic target
for SERT-targeting ligands [6–8] in B cell malignancies, the ulti-
mate aim of the present study is to use the 4-MTA scaffold as
a starting point to synthesise a number of possible SERT-targeting
agents. We wish to evaluate these agents for activity as novel
antiproliferative agents. This will not only allow for the develop-
ment and synthesis of more potent, selective analogues that offer
the potential to be used as future anticancer agents but will also
help confirm or contradict reports of SERT’s potential role as a
pro-apoptotic target.
2. Results and discussion
2.1. Chemistry
The structures of the sulphur-substituted
a-alkyl phenethyl-
amines and related compounds investigated in the present study
are arranged in six distinct groups, Types I–VI (see Tables 1–4;
Schemes 1–5):
Amphetamine analogues substituted at the 4-position with an
alkylthio group are
a
pharmacologically important class of
Type I. Structures based on 4-MTA scaffold with N-alkyl, N,
N-dialkyl, N-hydroxyl or N-alkoxy substituents, compounds 4a–4r.
Type II. Structures based on thiomethyl substituted a-methyl-
phenethylamine scaffold with alkyl, aryl or benzyl type sulphur
compounds which demonstrate potent biochemical activity in the
central nervous system. The parent member of the series, 4-MTA
(4-methylthioamphetamine) [24] (Fig. 1) is a ligand for SERT. It is
a potent inhibitor of serotonin reuptake in the rat brain [25],
a serotonin releasing agent in rat brain synaptosomes and has been
shown to have inhibitory effects on 5-HT-mediated vascular
contraction in isolated rat aortas [26]. It is also a selective reversible
monoamine oxidase-A (MAO-A) inhibitor [27].
substituent, compounds 9a–9e.
Type III. Structures based on ethylthio-substituted
phenethylamines, compounds 15a–15d.
a-methyl-
Type IV. Structures based on ethylthio-substituted
phenethylamines, compounds 15e–15i.
a
-ethyl-
-ethyl-
In the present study we report the synthesis of a series of novel
Type V. Structures based on thiomethyl substituted
a
4-MTA analogues, with or without N-alkyl and/or C-
a
methyl or
phenethylamine scaffold with N-alkyl, N,N-dialkyl, N-hydroxyl or
N-alkoxy substituents, compounds 19a–19q.
Type VI. Structures based on 1-phenylpropylamine, compounds
25a, 25b.
ethyl groups as well as the synthesis of the structurally related
4-thioethyl series. The aim of this study is to investigate the SERT-
binding activity and the SERT-selective cytotoxic activity of these
derivatives. We wish to determine if any observed toxicity is related
to the ability of each compound to bind to SERT. SERT binding will
be tested using an [3H] 5-HT uptake inhibition assay with the
specific structural requirements of these sulphur-substituted
2.1.1. Type I compounds
The synthetic routes which were utilised to obtain the sulphur-
substituted a-alkyl phenethylamines are illustrated in Schemes 1–4
a
-alkyl phenethylamines at the serotonin transporter examined
and are based on modifications of previously described processes
[27,29–33]. 4-MTA, (4a) together with the N-substituted analogues
4b–r are obtained in good yield by a two step synthesis via the
nitropropene 2 which was obtained from a modified Henry
(Knovenegel) reaction of 4-methylthiobenzaldehyde (1) with
nitroethane and N,N-dimethylamine and KF (Scheme 1). The
nitropropene 2 was then reduced with Fe iron powder to afford the
propanone 3 which was then treated with sodium cyanoborohy-
dride and the appropriate amine to afford the Type I products
4a–4n. 4-MTA (4a) was also obtained in 80% yield by direct
reduction of the nitrostyrene 2 with LiAlH4. N-Hydroxy and
N-alkoxy compounds 4o–r were prepared by sodium cyanobor-
ohydride reduction of the corresponding oximes 5a–d (isolated as
syn/anti mixture from the ketone 3 by reaction with the appro-
priated amines). Oxidation of the 4-MTA with mCPBA afforded the
sulfone 4s [34].
using a recently developed homology model for human SERT [28].
The results obtained from such experiments can then be used in
molecular modelling studies to rationalise SERT-binding
CH3
NH2
O
O
Methylenedioxyamphetamine (MDA)
CH3
NH2
S
4-Methylthioamphetamine (4-MTA)
2.1.2. Type II compounds
CH3
The required benzaldehydes 7a–e for Type II compounds 9a–e
were prepared by reaction of 4-fluorobenzaldehyde (6) with the
appropriate thiol in a sealed tube at 120 ꢀC in high yield in
a modification of the method of Tanaka [35] (Scheme 2). Conden-
sation of the aldehydes 7a–e with nitroethane afforded the nitro-
styrenes 8a–e which were then reduced with lithium aluminium
O
HN
O
CH3
Methylenedioxymethamphetamine (MDMA)
Fig. 1. The structures of methylenedioxyamphetamine, (MDA), methylenediox-
ymethamphetamine (MDMA) and 4-methylthioamphetamine (4-MTA).
hydride
to
afford
the
desired
amines
9a–e.
4-

4864
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
Table 1
The effects of the 4-methylthioamphetamine derivatives 4a–i, 4l–p (Type I) on SERT activity (IC50), on SERT expressing HEK cells (EC50) and on the malignant cell lines, DG-75
and SHSY-5Y.
O
NH2
S
O
N
S
R1
R2
Compound
R1
R2
Yield (%)
c log P
SERT reuptake inhibition,
IC50 M)
HEK293,
pEC50 ꢁ SE
4.73 ꢁ 0.14
4.71 ꢁ 0.13
4.79 ꢁ 0.10
4.51 ꢁ 0.16
HEK293 hSERT,
pEC50 ꢁ SE
4.65 ꢁ 0.15
4.77 ꢁ 0.18
4.90 ꢁ 0.10
4.60 ꢁ 0.11
DG-75,
pEC50 ꢁ SE
3.466 ꢁ 0.73
3.89 ꢁ 0.28
3.00 ꢁ 0.17
3.47 ꢁ 0.24
SH-SY5Y,
pEC50 ꢁ SE
3.216 ꢁ 0.52
2.81 ꢁ 0.23
2.73 ꢁ 0.21
2.71 ꢁ 0.29
a
a
a
a
(
m
4a (4-MTAb)
4b
4c
4d
H
CH3
CH2CH3
CH(CH3)2
H
H
H
H
85
49
35
36
2.30
2.45
2.98
3.29
0.207 ꢁ 0.029
0.417 ꢁ 0.031
0.963 ꢁ 0.076
1.854 ꢁ 0.618
4e
H
21
2.80
0.702 ꢁ 0.234
4.73 ꢁ 0.10
4.71 ꢁ 0.14
3.50 ꢁ 0.17
3.43 ꢁ 0.26
4f
4g
4h
4i
4l
4m
4n
4o
4p
CH2C]CH2
–C^CH
CH2CH2OH
CH2CH2OCH3
CH3
OH
OCH3
OH
OCH3
–
H
H
H
H
CH3
CH3
CH3
H
H
–
23
31
68
28
48
20
73
68
81
67
–
3.22
3.05
1.97
2.60
2.99
1.82
2.96
1.86
2.67
0.59
2.15
1.64
1.77
0.524 ꢁ 0.121
0.688 ꢁ 0.110
0.868 ꢁ 0.117
3.583 ꢁ 0.816
0.520 ꢁ 0.088
1.875c
25.06 ꢁ 3.41
0.436 ꢁ 0.040
0.664 ꢁ 0.057
100% reuptake at 100
1.06
4.81 ꢁ 0.12
4.61 ꢁ 0.15
5.00 ꢁ 0.09
4.64 ꢁ 0.13
4.70 ꢁ 0.06
4.62 ꢁ 0.12
3.28 ꢁ 0.24
4.42 ꢁ 0.13
4.96 ꢁ 0.10
<2
4.76 ꢁ 0.15
4.62 ꢁ 0.17
4.89 ꢁ 0.089
4.68 ꢁ 0.18
4.54 ꢁ 0.14
4.53 ꢁ 0.11
2.61 ꢁ 0.24
4.40 ꢁ 0.13
4.55 ꢁ 0.11
2.67 ꢁ 0.58
3.31 ꢁ 0.21
3.16 ꢁ 0.34
2.97 ꢁ 0.24
3.72 ꢁ 0.29
4.42 ꢁ 0.20
2.75 ꢁ 0.21
4.48 ꢁ 0.21
3.31 ꢁ 0.18
3.13 ꢁ 0.17
<2
3.4 ꢁ 0.16
2.74 ꢁ 0.20
<2
3.91 ꢁ 0.32
2.63 ꢁ 0.18
2.64 ꢁ 0.18
2.87 ꢁ 0.24
2.73 ꢁ 0.29
3.64 ꢁ 0.14
<2
3.24 ꢁ 0.29
2.61 ꢁ 0.21
<2
4s
mM
MDMA
MDA
PMA
–
–
–
–
3.61 ꢁ 0.19
3.49 ꢁ 0.18
3.34 ꢁ 0.21
4.49 ꢁ 0.21
2.72 ꢁ 0.25
4.49 ꢁ 0.17
<2
<2
<2
–
0.996
0.173
The selective serotonin reuptake inhibitor, citalopram was used as a positive control for SERT uptake inhibition (IC50 of 3.17 nM), whereas sodium azide (30 mM) and Triton-X
(2%) acted as positive controls for cytotoxicity resulting in 80–90% cytotoxicity to all cells.
TREx SERT-FLAG cells (1 ꢂ 105) were incubated with drug and [3H] 5-HT. After 2 min, uptake was stopped by removing the 5-HT solution and immediately adding cold buffer
containing 1 mM paroxetine (to inhibit 5-HT transport). Cells were washed, lysed and radioactivity determined by liquid scintillation counting. Non-specific uptake was
defined as the uptake in the presence of 10
m
M paroxetine subtracted from the total uptake to obtain specific uptake. For the Neutral Red assay, 5 ꢂ 104 cells/well were seeded
treated for 48 h. Supernatant was removed and the cells incubated for 3 ꢁ 1 h with Neutral Red dye solution. NR solution was removed, washed before the addition NR assay
solubilisation solution. Absorbance was read at 540 nm (690 nm, background) within 1 h. Cell viability was expressed as percent of vehicle treated cells.
a
pEC50 (ꢃ(ꢃlog EC50 is the log[Dose] when response ¼ 50%)) values were calculated from % Cell Viability versus ꢃlog concentration curves, using 4 concentrations in
triplicate on two independent days. Data was subjected to non-linear regression analysis using a sigmoidal dose–response (Hill slope ¼ 1) using GRAPHPAD Prism4 software
(Graphpad software Inc., San Diego, CA).
b
At 1 mM, 4-MTA is selectively more toxic to the hSERT cell line compared to the control HEK293 cell line P < 0.05 based on two-way ANOVA test (GRAPHPad Prism4) with
no matching followed by a Bonferroni Post Test to compare replicate means by row to the control cell line HEK293 for each concentration.
c
Single determination.
Ethylthioamphetamine (9a) [27] was also obtained by reductive
amination of the ketone 14a (Scheme 2).
reduction of the nitrostyrene 17. Reductive amination of the ketone
18 afforded the required products 19a–l and 19n. The N-hydroxy
derivative 19m, together with the N-alkoxy compounds 19o–q
were obtained via reduction of the oximes 20a–d which were
obtained from the ketone 18 by condensation reaction with the
appropriate amines (Scheme 4).
2.1.3. Type III and IV compounds
The synthetic route to the Type III compounds 15a–d and Type IV
compounds 15e–i is illustrated in Scheme 3 and required the
common precursor ketones 12a, b. Henry condensation of nitro-
ethane and nitropropane with 4-bromobenzaldehyde (10) afforded
the nitrostyrenes 11a, b which were then reduced to the corre-
sponding ketones 12a, b. Quantitative conversion to the 1,3-dioxo-
lanes 13a, b was followed by lithiation and reaction with diethyl
sulphide. Reductive amination of the ketones 14a, b with the
appropriate amines afforded the requiredproducts 15a, b, d–g, i. The
N-hydroxy compounds 15c, h were obtained via sodium cyanobor-
ohydride reduction (at pH 3) of the oximes 16a, b respectively
(Scheme 3). In the 1H NMR spectrum of 15h, the OH and NH protons
2.1.5. Type VI compounds
Synthesis of the Type VI compounds 25a and 25b was achieved
in moderate yield by reductive amination of the ketones 22 and 24
respectively (Scheme 5). Treatment of the thioether 23 (obtained
by alkylation of methythiobenzene with n-butyl iodide and
sec-butyl lithium [36,37]) with propionyl chloride afforded the
ketone 24, while Grignard alkylation of 4-methylthiobenzaldehyde
(1) provided the alcohol 21 which was oxidised to ketone 22
using PCC.
are initially displayed as two broad singlets at
integrating for one proton. In subsequent 1H NMR spectra acquired
over a period of 480 s, a broad signal at 5.80, integrating for two
d 5.20 and d 6.25; each
2.2. Inhibition of SERT activity
d
protons had replaced these two signals. This exchange may be due to
trace of acid present and is also observed for compound 15c.
Amphetamines (AMPHs) are thought to release stores of
catecholamines from nerve endings by converting the respective
molecular transporters into open channels [38]. They are thought
to compete with substrate for the transporters, reversing the
transport of monoamines by either binding to the transporter as
a substrate or binding without being transported. It has been
difficult to determine if amphetamines are genuine substrates of
2.1.4. Type V compounds
Synthesis of the Type V compounds 19a–q is illustrated in
Scheme 4. Ketone 18 was obtained by Henry condensation of
4-methylthiobenzaldehyde (1) with nitropropane and subsequent

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4865
Table 2
The effects of the 4-methylthioamphetamine derivatives 9a–e (Type II) on SERT activity (IC50), on SERT expressing HEK cells (EC50) and on the malignant cell lines, DG-75 and
SHSY-5Y.
R
NH2
S
a
a
a
a
Compound
R
Yield (%) c log P SERT reuptake inhibition, IC50
(m
M) HEK293, pEC50 ꢁ SE hSERT, pEC50 ꢁ SE DG-75, pEC50 ꢁ SE SH-SY5Y, pEC50 ꢁ SE
9a
9b
9c
9d
CH2CH3
C(CH3)3
CH2C6H5
CH2CH2N(CH3)2 52
C6H5
75
58
71
2.83
3.69
3.87
2.23
4.09
2.15
1.64
1.77
1.403 ꢁ 0.250
100% 100
0.679 ꢁ 0.025
43.29 ꢁ 9.154
14.33 ꢁ 0.355
1.06
4.82 ꢁ 0.10
5.14 ꢁ 0.13
5.53 ꢁ 0.10
5.06 ꢁ 0.11
5.56 ꢁ 0.13
3.61 ꢁ 0.19
3.49 ꢁ 0.18
3.34 ꢁ 0.21
4.85 ꢁ 0.12
5.4 ꢁ 0.1563
5.43 ꢁ 0.15
5.09 ꢁ 0.13
5.60 ꢁ 0.09
3.31 ꢁ 0.21
3.16 ꢁ 0.34
2.97 ꢁ 0.24
4.64 ꢁ 0.19
5.11 ꢁ 0.19
5.31 ꢁ 0.14
4.76 ꢁ 0.18
5.53 ꢁ 0.12
4.49 ꢁ 0.21
2.72 ꢁ 0.25
4.49 ꢁ 0.17
4.19 ꢁ 0.16
4.69 ꢁ 0.16
4.71 ꢁ 0.14
3.62 ꢁ 0.16
4.88 ꢁ 0.14
<2
mM
9e
47
–
–
MDMA
MDA
PMA
–
–
0.996
0.173
<2
<2
The selective serotonin reuptake inhibitor, citalopram was used as a positive control for SERT uptake inhibition (IC50 of 3.17 nM), whereas sodium azide (30 mM) and Triton-X
(2%) acted as positive controls for cytotoxicity resulting in 80–90% cytotoxicity to all cells.
TREx SERT-FLAG cells (1 ꢂ 105) were incubated with drug and [3H] 5-HT. After 2 min, uptake was stopped by removing the 5-HT solution and immediately adding cold buffer
containing 1 mM paroxetine (to inhibit 5-HT transport). Cells were washed, lysed and radioactivity determined by liquid scintillation counting. Non-specific uptake was
defined as the uptake in the presence of 10
m
M paroxetine subtracted from the total uptake to obtain specific uptake. For the Neutral Red assay, 5 ꢂ 104 cells/well were seeded
treated with for 48 h. Supernatant was removed and the cells incubated for 3 ꢁ 1 h with Neutral Red dye solution. NR solution was removed, washed before the addition NR
assay solubilisation solution. Absorbance was read at 540 nm (690 nm, background) within 1 h. Cell viability was expressed as percent of vehicle treated cells.
a
pEC50 (ꢃ(ꢃlog EC50 is the log[Dose] when response ¼ 50%)) values were calculated from % Cell Viability versus ꢃlog concentration curves, using 4 concentrations in
triplicate on two independent days. Data was subjected to non-linear regression analysis using a sigmoidal dose–response (Hill slope ¼ 1) using GRAPHPAD Prism4 software
(Graphpad software Inc., San Diego, CA).
SERT, due to their lipophilic nature and to the lack of a crystal
structure of human SERT [39]. The amphetamine analogues,
4-MTA, MDMA, 3,4-methylenedioxyamphetamine (MDA) and
para-chloroamphetamine (PCA) have all been previously shown to
inhibit SERT activity with IC50 values of 74 nM (4-MTA) [29],
425 nM (MDMA) [40], 478 nM (MDA) [25] and 182 nM (PCA) [25]
respectively. These amphetamines also inhibit the activity of the
noradrenaline transporter (NAT) (2375 nM (4-MTA), 405 nM
(MDMA), 266 nM (MDA) and 207 nM (PCA)) and inhibit the
activity of the dopamine transporter (DAT) (3073 nM (4-MTA),
1442 nM (MDMA), 890 nM (MDA) and 424 nM (PCA)) [25,29,41].
para-Methoxyamphetamine
(PMA)
is
another
common
substituted amphetamine that is thought to act in a similar way to
MDMA, with evidence of PMA decreasing SERT-binding sites in rat
forebrains [42] and synpatosomal 5-HT uptake and content
[43,44].
To determine the potential SERT-binding activity of these
derivatives, the 4-MTA library of compounds synthesised was
Table 3
The effects of the 4-methylthioamphetamine derivatives 15a–d (Type III), and 15e–i (Type IV) on SERT activity (IC50), on SERT expressing HEK cells (EC50) and on the malignant
cell lines, DG-75 and SHSY-5Y.
N
N
S
R1
R2
S
R1
15a-d
R2
15e-i
a
a
a
a
Compound R1
R2 Yield (%) c log P SERT reuptake inhibition, IC50
(m
M) HEK293, pEC50 ꢁ SE HEK293 SERT, pEC50 ꢁ SE DG-75, pEC50 ꢁ SE SH-SY5Y, pEC50 ꢁ SE
15a
15b
15c
15d
15e
15f
15g
15h
15i
MDMA
MDA
PMA
CH3
CH2CH3
OH
CH3
H
CH2CH2
CH3
OH
H
H
H
34
17
86
2.99
3.50
2.39
2.35
3.36
4.03
3.50
2.92
2.89
2.15
1.64
1.77
0.724 ꢁ 0.104
0.478 ꢁ 0.036
0.625 ꢁ 0.165
1.575 ꢁ 0.030
1.290 ꢁ 0.247
1.804 ꢁ 0.035
5.12 ꢁ 0.11
5.20 ꢁ 0.07
4.49 ꢁ 0.13
4.91 ꢁ 0.09
5.29 ꢁ 0.06
3.60 ꢁ 0.17
4.71 ꢁ 0.13
4.78 ꢁ 0.13
4.59 ꢁ 0.18
4.62 ꢁ 0.15
5.23 ꢁ 0.10
4.213 ꢁ 0.14
3.88 ꢁ 0.12
3.85 ꢁ 0.19
3.91 ꢁ 0.32
4.73 ꢁ 0.21
4.16 ꢁ 0.20
3.45 ꢁ 0.16
3.55 ꢁ 0.19
3.52 ꢁ 0.20
3.15 ꢁ 0.12
4.36 ꢁ 0.15
4.27 ꢁ 0.18
3.61 ꢁ 0.17
OH 35
H
H
H
H
73
27
28
73
c
c
c
c
>100
m
Mb
–
–
–
–
1.446 ꢁ 0.482
2.003 ꢁ 0.170
1.06
0.996
0.173
4.94 ꢁ 0.08
4.29 ꢁ 0.15
3.61 ꢁ 0.19
3.49 ꢁ 0.18
3.34 ꢁ 0.21
4.82 ꢁ 0.13
4.35 ꢁ 0.14
3.31 ꢁ 0.21
3.16 ꢁ 0.34
2.97 ꢁ 0.24
4.11 ꢁ 0.27
3.40 ꢁ 0.15
4.49 ꢁ 0.21
2.72 ꢁ 0.25
4.49 ꢁ 0.17
2.74 ꢁ 0.13
3.68 ꢁ 0.13
<2
<2
<2
CH3
–
–
OH 62
–
–
–
–
The selective serotonin reuptake inhibitor, citalopram was used as a positive control for SERT uptake inhibition (IC50 of 3.17 nM), whereas sodium azide (30 mM) and Triton-X
(2%) acted as positive controls for cytotoxicity resulting in 80–90% cytotoxicity to all cells.
TREx SERT-FLAG cells (1 ꢂ 105) were incubated with drug and [3H] 5-HT. After 2 min, uptake was stopped by removing the 5-HT solution and immediately adding cold buffer
containing 1 mM paroxetine (to inhibit 5-HT transport). Cells were washed, lysed and radioactivity determined by liquid scintillation counting. Non-specific uptake was
defined as the uptake in the presence of 10
m
M paroxetine subtracted from the total uptake to obtain specific uptake. For the Neutral Red assay, 5 ꢂ 104 cells/well were seeded
treated with for 48 h. Supernatant was removed and the cells incubated for 3 ꢁ 1 h with Neutral Red dye solution. NR solution was removed, washed before the addition NR
assay solubilisation solution. Absorbance was read at 540 nm (690 nm, background) within 1 h. Cell viability was expressed as percent of vehicle treated cells. The cytotoxic
potency of each compound was quantified by a pEC50 value determined by non-linear regression analysis of sigmoidal log concentration dependence curves whereby pEC50 is
ꢃ[ꢃlog EC50] ꢁ SE (log EC50 is the log[Dose] when response is equal to 50% cell viability).
a
pEC50 (ꢃ(ꢃlog EC50 is the log[Dose] when response ¼ 50%)) values were calculated from % Cell viability versus ꢃlog concentration curves, using 4 concentrations in
triplicate on two independent days. Data was subjected to non-linear regression analysis using a sigmoidal dose–response (Hill slope ¼ 1) using GRAPHPAD Prism4 software
(Graphpad software Inc., San Diego, CA).
b
26% reuptake inhibition at 1
m
M.
c
Compound not included in Neutral Red cytotoxicity screen.

4866
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
Table 4
The effects of the 4-methylthioamphetamine derivatives 19a–h, 19m–n (Type V) and related analogues 25a–b (Type VI) on SERT activity (IC50), on SERT expressing HEK cells
(EC50) and on the malignant cell lines, DG-75 and SHSY-5Y.
NH2
N
R1
S
R1
R2
19m-n
S
19a-h,
25a-b
Compound
R1
R2
Yield (%)
c log P
SERT reuptake inhibition,
IC50 M)
HEK293,
pEC50 ꢁ SE
HEK293 hSERT,
pEC50 ꢁ SE
DG-75,
pEC50 ꢁ SE
SH-SY5Y,
pEC50 ꢁ SE
a
a
a
a
(m
19a
19b
19c
19d
19e
H
CH3
CH2CH3
CH2CH2CH3
CH(CH3)2
H
H
H
H
H
53
77
28
26
15
2.83
2.99
3.50
4.03
3.81
0.611 ꢁ 0.016
0.437 ꢁ 0.056
0.823 ꢁ 0.274
0.621 ꢁ 0.124
1.939 ꢁ 0.628
4.83 ꢁ 0.11
4.30 ꢁ 0.15
5.00 ꢁ 0.10
3.519 ꢁ 0.17
4.76 ꢁ 0.08
4.755 ꢁ 0.13
4.26 ꢁ 0.10
4.679 ꢁ 0.11
3.57 ꢁ 0.27
4.67 ꢁ 0.13
3.87 ꢁ 0.27
4.33 ꢁ 0.23
3.55 ꢁ 0.19
3.59 ꢁ 0.36
3.57 ꢁ 0.19
3.36 ꢁ 0.20
3.08 ꢁ 0.23
3.82 ꢁ 0.13
2.52 ꢁ 0.36
3.47 ꢁ 0.19
19f
H
30
3.33
0.872 ꢁ 0.214
4.97 ꢁ 0.11
4.68 ꢁ 0.16
4.22 ꢁ 0.25
<2
19g
19h
19m
19n
25a
–C^CH
CH2CH2OCH3
OH
OCH3
–(CH2)4CH3
CH3
–
H
H
H
CH3
–
–
–
–
26
39
30
51
48
46
–
3.58
3.13
2.39
2.35
3.98
2.32
2.15
1.64
1.77
0.399 ꢁ 0.030
2.821 ꢁ 0.081
0.598 ꢁ 0.199
0.695 ꢁ 0.232
4.69 ꢁ 0.12
4.79 ꢁ 0.09
5.07 ꢁ 0.07
4.92 ꢁ 0.12
5.50 ꢁ 0.12
2.57 ꢁ 0.20
3.61 ꢁ 0.19
3.49 ꢁ 0.18
3.34 ꢁ 0.21
4.724 ꢁ 0.16
4.718 ꢁ 0.14
4.98 ꢁ 0.09
4.84 ꢁ 0.13
5.59 ꢁ 0.1
3.80 ꢁ 0.18
2.72 ꢁ 0.15
4.11 ꢁ 0.20
3.51 ꢁ 0.23
5.32 ꢁ 0.13
<2
4.49 ꢁ 0.21
2.72 ꢁ 0.25
4.49 ꢁ 0.17
3.405 ꢁ 0.16
2.41 ꢁ 0.21
4.50 ꢁ 0.25
4.02 ꢁ 0.28
4.71 ꢁ 0.14
<2
<2
<2
<2
>25 mM
25b
65% reuptake at 10
1.06
0.996
0.173
m
M
2.71 ꢁ 0.15
3.31 ꢁ 0.21
3.16 ꢁ 0.34
2.97 ꢁ 0.24
MDMA
MDA
PMA
–
–
The selective serotonin reuptake inhibitor, citalopram was used as a positive control for SERT uptake inhibition (IC50 of 3.17 nM), whereas sodium azide (30 mM) and Triton-X
(2%) acted as positive controls for cytotoxicity resulting in 80–90% cytotoxicity to all cells.
TREx SERT-FLAG cells (1 ꢂ 105) were incubated with drug and [3H] 5-HT. After 2 min, uptake was stopped by removing the 5-HT solution and immediately adding cold buffer
containing 1 mM paroxetine (to inhibit 5-HT transport). Cells were washed, lysed and radioactivity determined by liquid scintillation counting. Non-specific uptake was
defined as the uptake in the presence of 10
m
M paroxetine subtracted from the total uptake to obtain specific uptake. For the Neutral Red assay, 5 ꢂ 104 cells/well were seeded
treated with for 48 h. Supernatant was removed and the cells incubated for 3 ꢁ 1 h with Neutral Red dye solution. NR solution was removed, washed before the addition NR
assay solubilisation solution. Absorbance was read at 540 nm (690 nm, background) within 1 h. Cell viability was expressed as percent of vehicle treated cells.
a
pEC50 (ꢃ(ꢃlog EC50 is the log[Dose] when response ¼ 50%)) values were calculated from % Cell Viability versus ꢃlog concentration curves, using 4 concentrations in
triplicate on two independent days. Data was subjected to non-linear regression analysis using a sigmoidal dose–response (Hill slope ¼ 1) using GRAPHPAD Prism4 software
(Graphpad software Inc., San Diego, CA).
screened using an [3H] 5-HT uptake inhibition assay with HEK293
(Human Embryonic Kidney) TREx cells stably expressing rSERT. The
percentage reuptake of [3H] 5-HT for the compounds was initially
decrease in the binding capabilities of the N-hydroxy, N-methyl
analogue 4n (IC50 25.06 M) was observed compared with 4a.
m
Oxidation of 4-MTA to the sulfone product 4s resulted in very
low activity for SERT reuptake inhibition (100% reuptake at
100 mM), possibly due to poor solubility of the product. No clear
correlation was observed between the calculated lipophilicity
(c log P) of the compounds 4a–s and the SERT reuptake activity
(Table 1).
The effect of the nature and size of the sulphur substituent was
investigated in the Type II compounds (Table 2). Introduction of the
ethyl or benzyl substituent on the sulphur resulted in small
determined at concentrations of 1
for reuptake inhibition of the more potent compounds (>90%
uptake inhibition at 100 M) was then determined from the
mM and 100 mM. The IC50 value
m
appropriate sigmoidal dose-dependent curves. The SSRI citalopram
was used as a positive control and was found to have an IC50 of
3.17 nM, while the amphetamines MDMA, MDA and PMA gave
inhibition of serotonin reuptake with IC50 values of 1.06
mM,
0.996 and 173 nM respectively (Tables 1–4). The results
mM
obtained for the SERT reuptake experiments for the series of
compounds synthesised together with the c log P values are given
in Tables 1–4. Compounds 4-MTA and PMA which are para
substituted with electron donating groups –SCH3 and –OCH3
respectively, show considerably more potent SERT activity than
3,4-methylenedioxy substituted MDA and MDMA.
decrease in SERT activity (IC50 values of 1.403 mM and 0.679 mM
respectively for compounds 9a and 9c) compared with 4-MTA (4a).
Introduction of the t-butyl and aryl sulphur substituents in
compounds 9b and 9e and basic substituent in compound 9d
resulted in decrease in activity.
The effect of N-substitution in the S-ethyl compounds was next
examined (Type III compounds, Table 3). Small substituents such as
Me, (15a), Et (15b) and OH (15c) resulted in improved activity over
the unsubstituted compound 9a. In the series of 4-ethylthio-
Examination of the data from the Type I compounds (Table 1)
showed that 4-MTA (4a) was the most active compound in the
series as a serotonin reuptake inhibitor with IC50 0.207
(previously reported SERT reuptake inhibition potency for 4-MTA
was hSERT Ki 0.45 þ 0.10 M [45] and whole rat brain synaptosome
determinations IC50 0.074 ꢁ 0.010 M [25]). It was found to be more
potent than MDMA (1.06 M) and MDA (0.996 M), had a similar
mM
substituted
primary amine compound 15e was the most potent, with
M. Introduction of single N-substituents (Et or OH) or
a-ethylphenethylamines (Type IV compounds), the
m
m
IC50 ¼ 1.290
m
m
m
disubstituted (OH and Me) resulted in a small decrease in activity as
expected (Table 3).
activity to PMA (173 nM) but was not as potent as the SSRI,
citalopram at SERT (3.17 nM).
Substantial SERT activity is preserved for small substituents
on the primary amine group e.g. compounds 4b, 4l, 4o, 4p which
The effect on SERT reuptake inhibition of the elongation of
amphetamine carbon scaffold of 4-MTA to a 4 carbon chain was also
examined in Type V compounds (Table 4). Most compounds in the
series displayed good SERT activity (IC50 values in the range
have IC50 values in the range 0.417–0.664 mM. A dramatic

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4867
CH3
NO2
CHO
CH3
(b)
(a)
H3C
H3C
H3C
O
S
S
S
1
2
3
(c)
(f)
(d)
(e)
CH3
CH3
H3C
N
H3C
N
S
OR
S
R1
R2
5a R = H
4a R1 = R2 = H
4b
5b R = CH3
5c
R1 =CH3 ,R2 = H
R = CH2CH3
5d R = CH2CH=CH2
4c R1 =CH2CH3 ,R2 = H
4d
R1 =CH(CH3)2 ,R2 = H
4e R1
=
,R2 = H
CH3
NH2
(g)
O
S
O
H3C
4f R1 =CH2CH=CH2, R2 = H
4g
R1 =
,R = H
C CH
4h R1 =CH2CH2OH 2,R2 = H
4s
4i
R
1 = H, R2 = CH2CH2OCH3
R1 =CH2CH2SH, R2 = H
1 =CH2CH2CH3 ,R2 = H
4j
4k
4l
R
R1 = R2 = CH3
4m R1 =CH3 ,R2 = OH
4n R1 =CH3 ,R2 = OCH3
4o
R1 =OH ,R2 = H
R1 =OCH3 ,R2 = H
4p
4q
R1 =OCH2CH3 ,R2 = H
4r R1 =OCH2CH=CH2, R2 = H
aReagents and conditions: (a) CH3CH2NO2, (CH3)2NH.HCl, KF, reflux ; (b) Fe, CH3COOH, 100o, 2h, (c)
NaCNBH3, NH4OCOCH3 or R1R2NH.HCl, CH3OH,rt, 72h; (d) RONH2HCl, Pyridine, EtOH, reflux, 2h; (e)
NaCNBH3, MeOH, rt, 72h; (f ) LiAlH4, THF, reflux, 12h; (g) mCPBA, CH2Cl2.
Scheme 1. Synthesis of 4-methylthioamphetamine analogues Type 1. Reagents and conditions: (a) CH3CH2NO2, (CH3)2NH$HCl, KF, reflux; (b) Fe, CH3COOH, 100 ꢀC, 2 h; (c)
NaCNBH3, NH4OCOCH3 or R1R2NH$HCl, CH3OH, rt, 72 h; (d) RONH2HCl, pyridine, EtOH, reflux, 2 h; (e) NaCNBH3, MeOH, rt, 72 h; (f) LiAlH4, THF, reflux, 12 h; (g) mCPBA, CH2Cl2.
0.399–2.821
by compounds having a methyl or propargryl nitrogen substituent,
e.g. compounds 19b (IC50 ¼ 0.437 M) and 19g (IC50 ¼ 0.399 M).
The related 1-aminophenethylamines 25a and 25b were found to
be inactive at SERT with IC50 > 25 M, indicating that the position
of the primary amine in the phenethylamine structure is critical for
SERT activity.
The majority of derivatives inhibited the uptake of 5-HT with
IC50 values in the high nanomolar range, similar to the IC50 values
m
M). The optimum activity for the series was displayed
of other known amphetamines at SERT. These IC50 values were
found to be significantly higher than the IC50 value of a known
inhibitor of SERT (citalopram, 3.17 nM). We can therefore
hypothesise that these derivatives are most likely acting in the
same way as other amphetamines at SERT, by reversing the
transport of 5-HT, by either binding as a substrate or binding
without being transported. Further experiments, such as 5-HT-
release experiments or in vivo studies would be needed to verify
such speculations.
m
m
m
CH3
NO2
CH3
NH2
CHO
(b)
(c)
CHO
(a)
R
R
S
R
S
F
S
6
8a R = CH2CH3
8b R = C(CH3)3
8c R = CH2C6H5
8d R = CH2CH2N(CH3)2
8e R = C6H5
9a R = CH2CH3
9b R = C(CH3)3
9c R = CH2C6H5
9d R = CH2CH2N(CH3)2
9e R = C6H5
7a R = CH2CH3
7b R = C(CH3)3
7c R = CH2C6H5
7d R = CH2CH2N(CH3)2
7e R = C6H5
aReagents and conditions: (a) R-SH, K2CO3, DMF, 120o, 4h; (b) CH3CH2NO2, (CH3)2NH.HCl, KF, reflux ; (b)
LiAlH4, THF, reflux, 12h.
Scheme 2. Synthesis of 4-methylthioamphetamine analogues Type II. Reagents and conditions: (a) R–SH, K2CO3, DMF, 120 ꢀC, 4 h; (b) CH3CH2NO2, (CH3)2NH$HCl, KF, reflux; (c)
LiAlH4, THF, reflux, 12 h.

4868
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
R
CHO
R
(b)
(c)
(a)
NO2
O
Br
Br
Br
10
11a R = CH3
12a R = CH3
12b
11b R = CH2CH3
R = CH2CH3
R
O
R
R
O
(d)
O
(f)
N
H3C
S
H3C
S
OH
Br
16a R = CH3
16b
13a R = CH3
13b
14a R = CH3
R = CH2CH3
14b
R = CH2CH3
R = CH2CH3
(e)
(g)
R
N
H3C
S
R1
R2
15a R = R1 = CH3, R2 = H
15b
R = CH3, R1 = CH2CH3, R2 = H
15c R = CH3, R1 = OH, R2 = H
15d R = R2 = CH3, R1 = OH
15e
R = C2H5, R1 = R2 = H
15f R = R1 = C2H5, R2 = H
15g
R = C2H5, R1 = CH3, R2 = H
15h R = C2H5, R1 = OH, R2 = H
15i R = C2H5, R1 = OH, R2 =CH3
a
Reagents and conditions: (a) CH3CH2NO2or CH3CH2CH2NO2, (CH3)2NH.HCl, KF, reflux ; (b) Fe,
CH3COOH, 100o, 2h; (c) HOCH2CH2OH, p-TSA, toluene, reflux, 18h; (d) (i) n-BuLi, (CH3CH2S)2, THF, -78oC
(ii) 20%aq HCl, EtOH, reflux, 2h; (e) NaCNBH3, NH4OCOCH3 or R1R2NH.HCl, CH3OH, rt, 72h; (f )
NH2OH.HCl, Pyridine, EtOH, reflux, 2h; (g) NaCNBH3, MeOH, pH 3, rt, 72h.
Scheme 3. Synthesis of 4-methylthioamphetamine analogues Type III and Type IV. Reagents and conditions: (a) CH3CH2NO2 or CH3CH2CH2NO2, (CH3)2NH$HCl, KF, reflux; (b) Fe,
CH3COOH, 100 ꢀC, 2 h; (c) HOCH2CH2OH, p-TSA, toluene, reflux, 18 h; (d) (i) n-BuLi, (CH3CH2S)2, THF, ꢃ78 ꢀC, (ii) 20% aq. HCl, EtOH, reflux, 2 h; (e) NaCNBH3, NH4OCOCH3 or
R1R2NH$HCl, CH3OH, rt, 72 h; (f) NH2OH$HCl, pyridine, EtOH, reflux, 2 h; (g) NaCNBH3, MeOH, pH 3, rt, 72 h.
2.3. Molecular modelling study
unsubstituted, it will interact (H-bond) with all three residues,
Ala96, Asp98, Phe335 whereas, if there is a substituent on the
terminal nitrogen, as in MDMA, only interactions with Asp98 and
Phe335 occur. Interactions with Ala96, Asp98, Phe335 were also
found to be important for the representative 4-MTA derivative, 9a
binding in the 5-HT binding site (Fig. 2D). Recent findings by
Walline et al. from hSERT mutant studies of transmembrane helix
III (TMHIII) indicate that key residue Ile172 is involved in the
binding interaction of amphetamines, while Tyr95 and Ile172 have
been previously reported as key residues [49–51]. These interac-
tions are identified in our studies of 5-HT, MDMA, 4-MTA, and its
related analogue 9a. 5-HT also makes additional interactions with
Thr439 to ensure efficacious binding of the endogenous ligand. The
interaction of Thr439 is also present in, MDMA, 9a and escitalo-
pram binding to SERT but is not present in the 4-MTA model.
Reuptake inhibitors such as escitalopram are thought to inhibit
SERT function by binding to the substrate-binding site, where they
are thought to interact via the protonated ligand amine of escita-
lopram and the Asp98 residue on SERT. Several hydrophobic
contacts between aromatic parts of the ligand and residues Ile172,
Phe341, Gly442 and Tyr176 are also thought to be important for
escitalopram binding to SERT [28]. It has also been recently
demonstrated by Anderson et al., the importance of a direct contact
between Ser438 and aminopropyl groups of SSRIs and tricyclic
2.3.1. Homology model of hSERT
To date no crystal structure of human SERT (hSERT) exists to
avail of in the ligand binding site docking process. A recent
homology model of hSERT was constructed by Jorgensen et al. [28]
using LeuT as a template (which belongs to the same transporter
family as SERT) and containing escitalopram as a bound ligand.
Human SERT is 92% homologous to the rat protein [46] (rSERT used
in SERT inhibition study) and has similar residues involved in
ligand and sodium binding. Subsequent investigation of the
flexibility of the binding site in complex with the natural substrate
(5-HT) was undertaken to determine key protein–ligand interac-
tions through MD simulation in a membrane environment [47].
In the present study FlexE [48] was selected as a docking plat-
form to allow these binding site variations to be taken into account.
To establish the key interactions that the series of 4-MTA analogues
make, it was necessary to initially determine the binding modes for
5-HT and the related methylenedioxymethamphetamine (MDMA)
within the SERT homology model. Fig. 2 illustrates the key inter-
actions that are observed for 5-HT, 4-MTA and MDMA within the
active site of SERT where the cationic head is paramount to these
interactions and residues Ala96, Asp98 and Phe335 are common to
the binding mode. If the terminal nitrogen of the ligand is

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4869
CHO
(b)
(a)
O
NO2
S
S
S
18
1
17
(e)
(c)
(d)
N
N
S
R1
R2
S
OR
20a R = H
19a
R1 = R2 = H
20b
R = CH3
19b
R1 = CH3, R2 = H
20c
R = CH2CH3
19c R1 = CH2CH3, R2 = H
20d R = CH2CH=CH2
19d
R1 = CH2CH2CH3, R2 = H
19e
R1 = C(CH3)3, R2 = H
19f R1
=
R2 = H
19g
R =
, R = H
2
H2C C CH
19h R11 = CH2CH2OCH3, R2 = H
19i R1 = CH2CH=CH2, R2 = H
19j
R1 = CH2CH2OH, R2 = H
19k R1 = R2 = CH3
19l R1 = OCH3, R2 = CH3
19m
R1 = OH, R2 = H
19n
R1 = OH, R2 = CH3
19o R1 = OCH3, R2 = H
19p R1 = OCH2CH3, R2 = H
19q
R1 = OCH2CH=CH2, R2 = H
aReagents and conditions: (a) CH3CH2CH2NO2, (CH3)2NH.HCl, KF, reflux ; (b) Fe, CH3COOH, 100o, 2h, (c)
H2NOR.HCl, Pyridine, EtOH, reflux, 2h; (d) NaCNBH3, MeOH, rt, 72h, (e) NaCNBH3, NH4OCOCH3 or
R1R2NH.HCl, CH3OH, rt, 72h;
Scheme 4. Synthesis of 4-methylthioamphetamine analogues Type V. Reagents and conditions: (a) CH3CH2CH2NO2, (CH3)2NH$HCl, KF, reflux; (b) Fe, CH3COOH, 100 ꢀC, 2 h; (c)
H2NOR$HCl, pyridine, EtOH, reflux, 2 h; (d) NaCNBH3, MeOH, rt, 72 h; (e) NaCNBH3, NH4OCOCH3 or R1R2NH$HCl, CH3OH, rt, 72 h.
antidepressants as a critical determinant for their potency at SERT
[52] and it is suggested that an overlap of substrate and inhibitor
sites in hSERT exists. This implies that, antidepressants may act by
a mechanism that involves direct occlusion of the 5-HT site. In the
present study, a similar binding pattern was observed for escita-
lopram (Fig. 2E) as with 5-HT, 4-MTA, MDMA and 9a (Fig. 2A–D),
identifying key interactions of each ligand with residues Tyr95,
Ile172, Asp98 and Phe335 on SERT, conclusive with previous
findings. The main difference observed is the presence of an arene–
cation bond between escitalopram and Tyr95, an interaction that is
absent in the MDMA, 5-HT, 4-MTA and 9a models (Fig. 2). In all
cases, it is important to note that the interactions illustrated may
vary slightly depending upon the docking routine and subsequent
minimisation protocols chosen.
The importance of a direct contact between Ser438 and ami-
nopropyl groups of SSRIs and tricyclic antidepressants as a critical
determinant for the potency observed [52] was found in our
modelling studies, where the positioning of Ser438 w4.5 Å from
the escitalopram aminopropyl group, effectively shapes the 5-HT
binding site. This interaction is also present in the 4-MTA, MDMA,
9a and 5-HT models supporting the theory that antidepressants
may act by a mechanism that involves direct occlusion of the 5-HT
site. These results therefore appear to be consistent with the ability
of 9a and the other novel 4-MTA derivatives binding to SERT in the
5-HT binding site in a similar way to MDMA, 5-HT and 4-MTA.
2.3.2. Structure–activity relationship – SERT-binding and inhibition
A detailed molecular modelling study was undertaken to
rationalise the observed SERT-binding activity for the 4-MTA type
compounds synthesised. An initial examination of the docked
structure of compound 4a (4-MTA) with the internal vDW surface
of the cavity mapped shows that there is ample space in the SERT
ligand binding site to accommodate the N-alkyl substituent of these
ligands (Fig. 2). Chemical analogues in the Type I series with
increasing chain length substitutions close to the cationic head of
4-MTA generate steric hindrance and in turn prevent interaction
other than salt bridge interaction with additional residues such as
Phe335 and Ala96. This translates to a sequential decrease in SERT
activity observed as the size of the alkyl or alkoxy nitrogen
substituent increases. The introduction of both hydroxyl and
methyl substituents rather than the cationic primary amine in 4a
neutralises any positive charge at pH 7.4 and consequently may
interfere with the ability of 4m to form a salt bridge with Asp98
(Fig. 3A). As a result a weaker H-bond is formed to Asp98 through
the oxygen of the hydroxylamine 4m when compared with inter-
actions proposed for primary amine 4a (Fig. 3A) and SERT reuptake
inhibition is reduced. This result underlines the fact that a positive
charge at this position is a crucial determinant of SERT reuptake.
The effect could also be explained by steric effects of the nitrogen
substituents which prevent the cationic nitrogen from interacting
with Asp98.

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S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
OH
O
CHO
(a)
(b)
S
S
S
(c)
S
1
22
21
NH2
R
(c)
25a R = CH3
25b R = (CH2)4CH3
O
(e)
(d)
S
S
23
S
24
aScheme reagents and conditions: (a) CH3CH2MgBr, Et2O, 34o, 3h (b) PCC, CH2Cl2, 40o, 3h (c) (i)
HMDS, TiCl4, CH2Cl2, 20h, rt (ii) MeOH, NaCNBH3, rt (d) secBuLi, CH3CH2CH2CH2I, THF, -78o,
3.5h, (e) CH3CH2COCl, AlCl3, CH2Cl2, 0o, 3h
Scheme 5. Synthesis of 4-methylthioamphetamine analogues Type VI. Reagents and conditions: (a) CH3CH2MgBr, Et2O, 34 ꢀC, 3 h; (b) PCC, CH2Cl2, 40 ꢀC, 3 h; (c) (i) HMDS, TiCl4,
CH2Cl2, 20 h, rt, (ii) MeOH, NaCNBH3, rt, (d) sec-BuLi, CH3CH2CH2CH2I, THF, ꢃ78 ꢀC, 3.5 h; (e) CH3CH2COCl, AlCl3, CH2Cl2, 0 ꢀC, 3 h.
The 2D depiction of the docked complex 9a (4-ethyl-
thioamphetamine, Type II) in the SERT-binding site is illustrated in
Fig. 2, where the contact with Phe335 and Asp98 is clearly evident.
The decrease in activity observed on introduction of larger S
substituents, as in compounds 9b, 9d and 9e is possibly due to
increased solvent exposure correlating with substituent size. The
docked structure of the S-ethylsubstituted compound 15c (Type III)
in the SERT-binding site is illustrated in Fig. 3B showing the key
interactions of the hydroxyl with Asn101 and Asp98 and the
nitrogen with Asp98 and Tyr176. Many of the compounds of Type V
with any change in the amount of the dye incorporated by the cells
indicating the degree of cytotoxicity caused by the test material.
The cytotoxic potentials of a library of 4-MTA derivatives on
HEK293 and HEK293 hSERT cells are presented in Tables 1–4 and
Fig. 4. It was found that MDMA, MDA and PMA did not show any
selective cytotoxic effects toward SERT expressing cells.
2.4.1.1. Selective toxicity of 4-MTA derivatives 4a–r (Type I) and
derivatives 19a–q (Type V) on hSERT overexpressing HEK cell lines in
vitro. All of the Type I and Type V derivatives displayed similar low
micromolar cytotoxic potencies implying that neither show any
obvious selective cytotoxic activity toward SERT (based on an
unpaired T-test with P < 0.05 representing a significant difference)
(Tables 1 and 5, Fig. 4). Compounds 4n, 4s, 19b and 19d displayed
the least cytotoxic effect on both cell lines with EC50 values in the
high micromolar range. The N-hydroxy, N-methyl analogue 4n (IC50
with a-ethyl substitution retain SERT-binding activity. The docked
structure of compound 19b in the SERT-binding site is illustrated in
Fig. 3C, which illustrating that the contacts of the nitrogen with
Phe335 and Asp98 are preserved for this scaffold structure.
2.4. Biological activity
25.06 mM) showed decreased SERT-binding activity compared with
2.4.1. Cytotoxicity in HEK293 and hSERT overexpressing cell lines
4-MTA has previously been shown to be toxic to rat hypotha-
lamic cultures [53] and to CYP2D6 expressing cells [54]. MDMA and
its related metabolites, including MDA have been found to be toxic
to numerous cell lines [19,55–59] while there is little information
on the in vitro cellular toxicity of PMA or PCA. Metabolic pathways
for 4-MTA have been demonstrated in vivo in mice [60] and in vitro
in primary hepatocytes [61]. In humans, metabolism of 4-MTA
could involve the following: oxidative deamination to a ketone
metabolite, that can be further reduced to the corresponding
alcohol or suffer degradation of the side chain into the 4-methyl-
thiobenzoic acid metabolite, ring hydroxylation to a phenolic
4a and oxidation of 4-MTA to the sulphone product 4s resulted in
very low activity for SERT reuptake inhibition. However, derivative
19b showed optimum SERT-binding activity (IC50 ¼ 0.437
the Type V series and 19d also had good SERT inhibitory activity
M). These
mM) for
(IC50 ¼ 0.621
m
M) compared to 4-MTA (IC50 ¼ 0.207
m
results imply that there is no correlation between the SERT-binding
and inhibitory activities of these derivatives with their cytotoxic
activities.
Statistical analysis of mean values for each concentration group
revealed that at 1 mM, 4-MTA (4a) and compound 19h showed
a selective cytotoxic effect to SERT expressing cells (P < 0.05) (data
not shown). Although 4-MTA is highly selective for serotonin
uptake inhibition compared to dopamine (ratio of IC50 value for
serotonin versus dopamine: 3.07 ꢂ 103) [25] and noradrenaline
(ratio IC50 value for serotonin versus noradrenaline: 2.37 ꢂ 103)
[25], the cytotoxic effect of 4-MTA on HEK cells overexpressing the
dopamine transporter (DAT) and noradrenaline transporter (NAT)
proteins was also determined and found to be very similar indi-
cating that the cytotoxicity observed for the compounds cannot be
directly related to SERT activity. (For 4a pEC50 values for HEK cells,
4.73 ꢁ 0.14; SERT expressing HEK cells, 4.65 ꢁ 0.15; DAT expressing
HEK cells, 4.64 ꢁ 0.14; NAT expressing HEK cells, 4.46 ꢁ 0.14). It
structure or
b-hydroxylation of the side chain to 4-methyl-
thioephedrine, oxidation of thioether [54]. The metabolism of the
new derivatives reported in the present study is most likely
the same.
To assess the potential SERT-dependent toxic effects of 4-MTA
and related analogues, the cytotoxicity of the series was deter-
mined in HEK cells previously shown to stably express the human
monoamine transporter, hSERT (Supplemental Fig. 1) using an in
vitro cytotoxicity assay. The Neutral Red (NR) assay is a simple,
accurate reproducible system where viable cells take up the NR dye

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4871
Fig. 2. Derivatives 4a (4-MTA) and 9a display similar key interactions in the 5-HT SERT-binding site to 5-HT, MDMA and escitalopram. 2-D rendering of ligand–protein interactions
using LigX module of MOE was used to create docked structures of 5-HT, 4-MTA, MDMA, 9a, and escitalopram in the 5-HT binding site using a recent homology model of hSERT,
constructed by Jorgensen et al. [28] containing escitalopram as a bound ligand. Clearly similar contacts are observed in all cases with the exception of an arene–cation bond formed
with Tyr95 in the escitalopram docked structure.

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S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
further proving the lack of correlation between cytotoxic effect and
SERT inhibitory activity.
2.4.1.3. Selective cytotoxicity toward SERT expressing Burkitt’s lym-
phoma. The DG-75 cell line is a B-lymphocyte, chemoresistant
Burkitt’s lymphoma cell line derived from a metastatic pleural
effusion (lung) of a sporadic case of EBV (Epstein Barr Virus)
negative Burkitt’s lymphoma [62] that has been shown to express
SERT (Supplemental Fig. 1). The SHSY-5Y cell line is a neuroblas-
toma cell line established from a metastatic bone tumour [63] that
was chosen to use in the screen as it has been shown not to express
SERT [64]. In this study using the Neutral Red assay it was found
that a number of 4-MTA derivatives in particular the Type IIa
derivatives showed potent cytotoxic activity toward the chemo-
resistant SERT expressing BL cell line DG-75 (Tables 1–5) and to the
SHSY-5Y cell line with approximate pEC50 values of 5 correlating to
an EC50 range of between 1
showed a small decrease in SERT activity (IC50 values of 1.403
and 0.679 M respectively for compounds 9a and 9c) compared
mM and 5 mM. These Type II derivatives
m
M
m
with 4-MTA (4a). The introduction of the t-butyl and aryl sulphur
substituents in compounds 9b and 9e and basic substituent in
compound 9d resulted in decrease in activity. The lack of potent
SERT binding by 9e and the result that compound 9e was found to
have the most potent cytotoxic effect on both the DG-75 and SHSY-
5Y as well as on the other cell lines (with no evidence of selective
activity toward the HEK293 hSERT) (Table 2) cell line implies that
SERT is most likely not involved in such cytotoxicity. MDMA and
PMA were also found to more cytotoxic to the DG-75 cell line,
compared to MDA and 4-MTA, whereas 4-MTA was the only one of
these controls that showed cytotoxicity to the SHSY-5Y neuro-
blastoma cell line.
2.4.2. Antiproliferative activity
To assess if the observed cytotoxicity of these compounds was
due to an antiproliferative effect, compound 9e was chosen as
a representative example using the Alamar Blue assay for anti-
proliferative activity in a range of malignant cell lines. 9e was
found to have potent antiproliferative activity in two BL cell lines
(MUTU-I and DG-75) (Fig. 5A). After 24 h (MUTU-I) and 72 h (DG-
75), only 10% of cells were found to be viable upon treatment with
Fig. 3. The interaction of 4-MTA analogues with Asp98 and Phe335 is important for
SERT inhibition activity. A positive charge at Asp98 is a crucial determinant of SERT
reuptake, where a weaker H-bond with Asp98 through the oxygen of the hydroxyl-
amine 4m when compared with interactions proposed for primary amine 4a is
important for SERT inhibition (A). Interactions of a hydroxyl group with Asn101 and
Asp98 and the nitrogen with Asp98 and Tyr176 are important for SERT-binding
activity, illustrated by the docked structure of the S-ethylsubstituted compound 15c
(Type III) in the SERT-binding site (B). The contacts of the nitrogen with Phe335 and
Asp98 are preserved for the 4-MTA analogues, contributing to their SERT-binding
abilities illustrated by the docked structure of compound 19b in the SERT-binding site
(C). 2-D rendering of ligand–protein interactions using LigX module of MOE was used
using a recent homology model of hSERT, constructed by Jorgensen et al. [28] con-
taining escitalopram as a bound ligand.
50 mM of 9e, implying 90% of the cells ceased to grow (Fig. 5A).
Derivative 9e was also found to have a potent antiproliferative
effect in three breast carcinoma cell lines (MCF-7, MDA-MB231,
4TI) (Fig. 5A and B) having a potent effect (w90% antiproliferative
activity) in the 4TIs and MDA-MB321 cells at 50 mM and leaving
only 40% of MCF-7 cells viable after 24 h treatments (Fig. 5B). The
microtubule targeting agent, Paclitaxel (Taxol) was used as posi-
tive control for antiproliferative activity as it has been shown to
have effects in BL cell lines with IC50s of between 10 and 40 nM
[65,66]. In other cell lines IC50 values for Taxol have ranged from 4
to 35 ng/mL with cytotoxic effects greater on proliferating cells
over quiescent cells [67].
therefore appears that SERT expression alone may not be sufficient
to confer 4-MTA toxicity to HEK cell lines. To the best of our
knowledge this study is the first report comparing the cytotoxic-
ities of a range of different 4-MTA derivatives on a human serotonin
transporter overexpressing cell line hSERT HEK293.
2.4.3. Apoptotic activity
Designing drugs that can induce programmed cell death (PCD),
namely apoptosis, of a cancer cell, whilst ignoring the ‘normal cells’
of the body is imperative to the future development of safe effective
anticancer agents. A drug that can induce PCD in vitro in malignant
cell lines has the potential to become an anticancer agent. In order
to determine if derivative 9e induces apoptosis in Burkitt’s
lymphoma propidium iodide (PI) FACS (fluorescent-activated cell
sorting) analysis was carried out. PI was chosen as the preferred
method to detect apoptosis to allow rapid FACS analysis to occur
allowing any pro-apoptotic and cell cycle effects to be easily iden-
tified. Annexin V staining was avoided, as phosphatidyl serine
2.4.1.2. Lack of selective toxicity of 4-MTA derivatives 9a–d (Type II),
15a–d (Type III) and 15e–i (Type IV) on hSERT overexpressing HEK
cell lines in vitro. All of the Type II and Type IV derivatives displayed
similar (P > 0.05) low micromolar cytotoxic potencies implying that
there is no obvious selective cytotoxic activity toward SERT.
Compounds 15f and 15i had less of a general cytotoxic effect on
both cell lines with EC50 values in the high micromolar range
(Tables 2 and 3) despite their moderate SERT inhibitory activity
with IC50 values of 1.804 mM and 2.003 mM respectively, again

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4873
4-MTA derivatives
4-MTA derivatives
4a-r (Type 1)
4-MTA derivatives
9a-e, (Type IIa)
4-MTA derivatives,
19a-q (Type V)
15a-d (Type IIb)
15e-i (Type IV)
HEK293
hSERT
1000
500
60
50
40
30
20
10
0
Compund Number
Fig. 4. The majority of 4-MTA derivatives are cytotoxic to hSERT and wildtype HEK cells with EC50 values in the low micromolar range. 5 ꢂ104 cells were seeded and treated for
48 h. Cells were incubated for 3 ꢁ 1 h with Neutral Red dye solution. Absorbance was read at 540 nM (690 nm background). Relative cell viability was expressed as percent of vehicle
treated cells. The cytotoxic potency of each compound was quantified by an EC50 value determined by non-linear regression analysis of sigmoidal log concentration dependence
curves where EC50 is the dose at 50% cell viability. Derivatives 4s, 15f, 15g and 25b were not included as EC50 values and were greater than 100 mM.
exposure can result from multiple activation pathways in the cell
and is not the best indicator of apoptosis to use in B cell-derived cell
lines from its cross reactivity with the B cell receptor [68]. PI FACS
these PBMCs to approximately 50% (compared to <10% in the DG-
75, MUTU-I, and breast cancer derived cell lines, Fig. 5). Derivative
25a had less of an effect in the PBMCs at 50 mM (Fig. 6B) compared
analysis was carried out initially using 50
m
M concentration of
to its pro-apoptotic effect in the MUTU-I, HL-60 and DG-75 cell
lines. Such results are consistent with these agents selectively tar-
geting cells of malignant origin over ‘normal cells’ of the body.
To assess the anticancer therapeutic potential of these deriva-
tives, a number of issues require discussion. The use of 4-MTA
derivatives as anticancer agents may pose the question of these
agents having CNS activity or toxicity. Both derivatives 9e and 25a
compounds 9e and 25a (as this also had a potent effect in the DG-75
cell line from the NR assay) in two BL cell lines and in the human
promoeolytic leukaemia cell line (HL-60). It was found that both 9e
and 25a showed strong pro-apoptotic activity (40–65% cell death)
in all of the cell lines analysed (Fig. 5C). 10 mM Taxol was used as
a positive control for apoptosis as it has been previously shown to
induce apoptosis in HL-60 cells [69]. Further characterisation of
these effects using sigmoidal dose–concentration curves (data not
shown) revealed EC50 values for the pro-apoptotic effects of both
derivative 9e and 25a in the low micromolar range (e.g. for
have very little SERT inhibitory activity (IC50s of 14.33
mM and
>25 M respectively compared to 4-MTA (IC50 0.207 M)) implying
m
m
possible differential pharmacological activity to 4-MTA and hence
a lack of CNS activity. Further safety-toxicology studies on these
derivatives will assess such concerns. Alternatively, most anti-
cancer agents have limited brain penetration resulting in a greater
need for new pharmacological approaches to enhance delivery into
the brain. Despite aggressive therapy, the majority of primary and
metastatic brain tumour patients have a poor prognosis with brief
survival periods as before systemically administered drugs can
distribute into the CNS, they must cross two membrane barriers,
the blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier
(BCB) [70]. Assessing the effects of these agents against primary and
metastatic brain tumour derived cell lines is also worth further
future exploration.
MUTU-c179 cell line, EC50 values of 14.21
respectively) (Table 5).
mM and 21.02 mM
2.4.4. Selective anticancer activity
In order to assess the selectivity of these two derivatives toward
proliferating malignant cells over the ‘normal’ cells of the body, the
effects of 9e and 25a were assessed in peripheral blood mono-
nuclear cells (PBMCs). After 24 h, it was found that at 10
derivatives 9e and 25a had little effect on the viability of PBMCs
compared to the effect of 10 M 9e on a range of malignant cells
M, derivative 9e reduced the viability of
mM,
m
lines (Figs. 5 and 6). At 50
m
A comparison of the potency of these agents to induce apoptosis
to other effective anticancer agents would place them in middle
range between anti-metabolites and receptor targeted agents. The
EC50 values of the lead 4-MTA derivatives places them agents
behind the efficacy of other known chemotherapeutic agents (14–
Table 5
Compounds 9e and 25a induce dose-dependent apoptosis in a range of haemato-
poietic malignancies.
Compound Burkitt’s lymphoma
Human-promyelocytic leukaemia
20 mM for the 4-MTA derivatives compared to 10–40 nM range of
DG-75 (72 h) MUTU-c179 (24 h) HL-60 (24 h)
the other agents). As these derivatives are of amphetamine origin,
they may be having an indirect SERT-mediated effect, whereby
these agents may release 5-HT from intracellular stores, initiating
a variety of secondary effects, which could in turn induce PCD in the
malignant cell lines. As these derivatives were found from an initial
screen, with little information regarding their suspected target-
based mechanism of action, extensive further investigations are
required to develop more potent analogues and to identify their
mechanism of action.
9e
25a
23.68
33.76
14.21
21.02
19.61
29.50
EC50 values (mM).
Cells were seeded at a density of 7 ꢂ 105 cells/5 mL treated with 9e and 25a for the
indicated time, harvested by centrifugation and fixed overnight in 70% ethanol. FACS
analysis was carried out upon incubation with propidium iodide and RNase A.
10,000 cells were counted using appropriate gates. EC50 values were determined by
non-linear regression analysis of sigmoidal log concentration dependence curves
where EC50 is the dose at 50% apoptotic effect.

4874
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
120
110
100
90
A
B
C
80
70
60
50
40
30
20
10
0
l
l
l
o
n
n
n
o
o
M
M
M
M
M
M
µ
o
o
o
x
x
x
a
µ
µ
µ
µ
µ
C
C
C
a
a
0
0
0
0
0
0
5
T
T
T
1
5
1
5
1
DG-75 24h
DG-75 72h
MUTU-I 24h
120
110
100
90
80
70
60
50
40
30
20
10
0
4TI 24h
MDA-MB231 24h
MCF-7 24h
I
II
100
100
90
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
l
e
e
l
c
l
o
l
l
o
e
e
e
l
c
l
l
c
l
c
e
9
o
x
e
9
a
5
a
a
o
c
i
i
9e
5
2
5
2
i
x
i
x
i
x
a
T
h
e
h
e
2
a
a
a
h
h
h
Taxol
Taxol
e
V
T
e
T
e
V
T
V
V
Vehicle
V
MUTU-I
HL-60
DG-75
MUTU-I
HL-60
DG-75
Fig. 5. Derivative 9e is a potent antiproliferative pro-apoptotic agent. 1–5 ꢂ104 cells/200
m
l (A and B) and 7 ꢂ 105 cells/5 mL (C) were seeded and treated with the 10
mM or 50 mM
(CI) 9e and 25a (50 mM) (CII) for the indicated times. 10 ml of Alamar Blue reagent was added to each well (A and B), fluorescence was read as emission 590 nm/excitation 544 nm
and values represent the mean value ꢁ S.E.M. of six data points (recording in triplicate on two independent days) (A and B). Cells were harvested by centrifugation and fixed
overnight in 70% ethanol (C). FACS analysis was carried out upon incubation with propidium iodide and RNase A. 10,000 cells were counted using appropriate gates (C). Values
represent the mean ꢁ SEM of three independent experiments.
3. Conclusion
concentrations below 1 mM, implying that they behave in a similar
way to 4-MTA and other amphetamines. The structure–activity
relationship molecular modelling study of the SERT inhibition for
these compounds revealed similar specific molecular binding
requirements for 4-MTA and related analogues at the 5-HT binding
site, again implying these derivatives behave in similar way to
4-MTA and MDMA.
The original rationale behind this study was to design and
synthesise a series of potentially potent novel SERT ligands based
on the structure of 4-MTA, working on the hypothesis that SERT
could act as a pro-apoptotic target in Burkitt’s lymphoma. In the
present work, a series of 4-MTA analogues, with or without N-alkyl
and/or C-
a
methyl or ethyl groups were synthesised and tested for
No SERT-dependent cytotoxic effect was found for any of the
compounds despite the ability of these 4-MTA derivatives to reduce
the viability of both HEK293 cells and HEK cells overexpressing
SERT binding. It was found that the majority of these novel
compounds were found to inhibit the reuptake of serotonin at

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4875
the in vivo toxic concentrations (25–480
m
M) [74,75] mentioned in
A
100
75
50
25
0
the reported fatalities. However as MTA is a relatively new
amphetamine derivative and the number of reported poisonings is
limited, the pathology findings and the mechanism of death due to
this product have not yet been fully evaluated. Further safety-
toxicology studies on 4-MTA derivatives will assess such concerns.
In addition, both derivatives 9e and 25a have very little SERT
inhibitory activity (IC50s of 14.33
mM and >25 mM respectively
compared to 4-MTA (IC50 0.207 M)) implying possible differential
m
pharmacological activity to 4-MTA.
Burkitt’s lymphoma remains a serious health problem in areas
where it is endemic [23]. In certain regions of equatorial Africa and
other tropical locations between latitudes 10ꢀ South and 10ꢀ North,
incidence is 100 per million children [79]. Despite its incidence in
developing countries it is also rapidly increasing in developed
countries. Burkitt’s and Burkitt’s-like/atypical Burkitt’s lymphomas
make up the largest group of HIV-associated non-Hodgkin
lymphomas, comprising up to 35–50% of these neoplasms [80] with
the relative risk of non-Hodgkin lymphoma increased 60- to
200-fold in HIV-infected patients [81]. The need for the develop-
ment of selective, potent economical alternatives in the treatment
of Burkitt’s lymphoma is worthy of further exploration and interest.
The pro-apoptotic abilities of derivatives 9e and 25a provide
sufficient justification for the further development of more potent
selective 4-MTA related analogues as anticancer agents and to
search for their possible target-based mechanisms of action.
10 µM
50 µM
9e
100
75
50
25
0
B
10 µM
50 µM
25a
4. Experimental
Fig. 6. The effects of derivatives 9e and 25a on peripheral blood mononuclear cells. 1–
mM 35 (A), 34 (B) for 24 h.
10 ml of Alamar Blue reagent was added to each well, fluorescence was read as emis-
5 ꢂ104 cells/200 mL were seeded and treated with 0.1–100
4.1. Materials and methods
sion 590 nm/excitation 544 nm and values represent the mean value þ S.E.M. of six
data points (recording in triplicate on two independent days).
4.1.1. Chemistry
Uncorrected melting points were measured on a Gallenkamp
apparatus. Infra-red (IR) spectra were recorded on a Perkin Elmer
FT-IR Paragon 1000 spectrometer. 1H, 13C and 19F nuclear magnetic
resonance (NMR) spectra were recorded at 27 ꢀC on a Brucker DPX
400 spectrometer (400.13 MHz, 1H; 100.61 MHz, 13C; 376.47 MHz,
19F) in either CDCl3 (internal standard tetramethylsilane (TMS)) or
CD3OD. For CDCl3, 1H NMR spectra were assigned relative to the
hSERT. Despite this lack of correlation between SERT expression
and selective cytotoxicity, it was found that a number of the Type
IIa compounds showed strong cytotoxic effects (EC50 values of less
than 10 mM) toward the chemoresistant Burkitt’s lymphoma cell
line DG-75, which expresses SERT. These derivatives also had
a potent effect on the neuroblastoma cell line SHSY-5Y which does
not express SERT [64] further eliminating a pro-apoptotic role for
SERT.
Derivative 9e was subsequently investigated for anti-
proliferative activity and was found to show potent activity in two
Burkitt’s lymphoma and three breast cancer cell lines having an
TMS peak at 0.00
d
and 13C NMR spectra were assigned relative to
the middle CDCl3 triplet at 77.00 ppm. For CD3OD, 1H and 13C NMR
spectra were assigned relative to the center peaks of the CD3OD
multiplets at 3.30
d
and 49.00 ppm respectively. 19F NMR spectra
were not calibrated. Coupling constants are reported in Hertz. For
1H NMR assignments, chemical shifts are reported: shift value
(number of protons, description of absorption, coupling constant(s)
where applicable). Electrospray ionisation mass spectrometry (ESI-
MS) was performed in the positive ion mode on a liquid chroma-
tography time-of-flight mass spectrometer (Micromass LCT, Waters
Ltd., Manchester, UK). The samples were introduced into the ion
source by an LC system (Waters Alliance 2795, Waters Corporation,
effect at 10 mM and 50 mM. 9e and 25a were further found to induce
apoptosis in two BL cell lines and in leukaemia cell line with EC50
values for such an effect in the low micromolar range (Table 5).
These derivatives were also shown to have little effect on peripheral
blood mononuclear cells implying the potential selectivity of these
agents for cells of malignant origin.
Using the 4-MTA scaffold as a base to design new SERT-targeting
ligands could be disputed as 4-MTA is a known MDMA-like drug of
abuse and is classified as a Schedule 1 controlled substance [71].
There have been six reported fatalities associated with 4-MTA in
the UK and Netherlands together with one death and seven
non-fatal cases of 4-MTA intoxication in Belgium [72]. However,
such fatalities are thought to be associated with large doses
(21.3–9800 mg/kg) [25,72], the use of other drugs and to interin-
dividual differences in metabolism [54,55]. In vivo, reported fatal
4-MTA intoxications consist of blood concentrations of 4-MTA
USA) in acetonitrile/water (60:40% v/v) at 200 ml/min. The capillary
voltage of the mass spectrometer was at 3 kV. The sample cone
(de-clustering) voltage was set at 40 V. For exact mass determina-
tion, the instrument was externally calibrated for the mass range
m/z 100 to m/z 1000. A lock (reference) mass (m/z 556.2771) was
used. Mass measurement accuracies of <ꢁ5 ppm were obtained.
Low resolution mass spectra (LRMS) were acquired on a Hewlett–
Packard 5973 MSD GC–MS system in electron impact (EI) mode.
Elemental analyses were performed on an Exetor Analytical
CE4400 CHN analyser in the Microanalysis Laboratory, Department
of Chemistry, University College Dublin. Rf values are quoted for
thin layer chromatography on silica gel Merck F-254 plates, unless
otherwise stated. Flash column chromatography was carried out on
Merck Kieselgel 60 (particle size 0.040–0.063 mm), Aldrich
between 8.27 and 29.6
mM [73–76] whereas in tissue such as brain
or liver concentrations have been found as high as 170–202
mM
[74,77,78]. Assuming the derivatives used in this study behave in
a similar way to 4-MTA at the concentrations (EC50s in the low
micromolar range) used in this study, they could be compared to

4876
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
aluminium oxide (activated, neutral, Brockmann I, 50 mesh) or
Aldrich aluminium oxide (activated, acidic, Brockmann I, 50 mesh).
Compounds 3 [31], 4s [34], 17 [82] were prepared according to the
literature methods.
d, J ¼ 6.5 Hz, CHCH3), 1.18 (1H, br s, NH), 2.46 (3H, s, SCH3), 2.51 (1H,
dd, J ¼ 7.0 Hz, 13.5 Hz, ArCH2CH), 2.70 (1H, dd, J ¼ 6.5 Hz, 13.5 Hz,
ArCH2CH), 2.92 (1H, m, NCH(CH3)2), 2.98 (1H, m, ArCH2CH), 7.09
(2H, d, J ¼ 8.0 Hz, ArH), 7.19 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR ppm
(CDCl3) 15.9, 20.4, 22.7, 23.5, 43.0, 45.1, 51.0, 126.7, 129.6, 135.5,
136.4. HCl salt. Colourless solid, mp 147–148 ꢀC (ethanol/hexane).
Anal. Calculated for C13H22ClNS: C, 60.09; H, 8.53; N, 5.39; S, 12.34.
Found: C, 59.89; H, 8.41; N, 5.57; S, 12.17%.
4.1.2. General method A: reductive amination procedure
To a stirred mixture of 1-(4-methylthiophenyl)-2-alkanones 3
or 18 (11.45 mmol) in dry methanol (50 mL) was added either
ammonium acetate (primary amines) or an appropriate alkyl-
amine HCl salt (80 mmol) and sodium cyanoborohydride
(15.92 mmol, 1.00 g) and the reaction was stirred at 20 ꢀC for 72 h.
The pH of the reaction was occasionally adjusted to pH 5–6 by the
addition of 4 M methanolic HCl as determined by damp universal
pH paper. Excess hydride was decomposed by the addition of 10%
aq. HCl (150 mL) and resulting aqueous phase washed with
dichloromethane (3 ꢂ 50 mL). The aqueous phase was basified
with 15% aq. NaOH solution and extracted with dichloromethane
(3 ꢂ 50 mL). The organic phases were combined, dried over
anhydrous Na2SO4 and volatiles removed in vacuo leaving the
product as an oil.
4.1.2.5. 2-N-Cyclopropylamino-1-(4-methylthiophenyl)propane (4e).
Compound 4e was prepared from 3 and cyclopropylamine HCl
according to the general method A. The resulting residue was
purified by flash chromatography (eluent: hexane/ethyl acetate
60:40). Pale amber oil (21%). IR nmax (film) 3313,1599,1094 cmꢃ1. 1H
NMR
d
(CDCl3) 0.26–0.51 (4H, m, NCH(CH2)2), 1.09 (3H, d, J ¼ 6.5 Hz,
CHCH3), 1.43 (1H, br s, NH), 2.06 (1H, m, NCH(CH2)2), 2.47 (3H, s,
SCH3), 2.55 (1H, dd, J ¼ 6.5 Hz, 13.6 Hz, ArCH2CH), 2.75 (1H, dd,
J ¼ 7.0 Hz, 13.6 Hz, ArCH2CH), 3.01 (1H, m, ArCH2CH), 7.11 (2H, d,
J ¼ 8.0 Hz, ArH), 7.20 (2H, d, J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3)
5.8, 7.0, 16.1, 20.4, 28.6, 43.0, 55.3, 127.0, 129.7, 135.7, 136.6. HCl salt.
Colourless solid, mp 137–138 ꢀC (ethanol/hexane). Anal. Calculated
for C13H20ClNS: C, 60.56; H, 7.82; N, 5.43; S, 12.44. Found: C, 60.86;
H, 7.59; N, 5.26; S, 12.21%.
4.1.2.1. 2-Amino-1-(4-methylthiophenyl)
propane
(4-MTA)
(4a). Compound 4a was prepared from 3 and ammonium acetate
according to the general method A. Colourless oil (85%) (lit. [29] bp
4.1.2.6. 2-N-Allylamino-1-(4-methylthiophenyl)propane (4f). Compound
4f was prepared from 3 and allylamine HCl according to the general
method A. The resulting residue was purified by flash chromatog-
raphy (eluent: dichloromethane/methanol 90:10). Amber oil (23%).
108–118 ꢀC/0.6 mmHg). IR nmax (film) 3359, 3285, 1584, 1093 cmꢃ1
.
1H NMR
d
(CDCl3) 1.10 (3H, d, J ¼ 6.5 Hz, CHCH3),1.25 (2H, br s, NH2),
2.46 (3H, s, SCH3), 2.48 (1H, dd, J ¼ 8.0 Hz, J ¼ 13.3 Hz, ArCH2CH),
2.67 (1H, dd, J ¼ 5.0 Hz, J ¼ 13.3 Hz, ArCH2CH), 3.13 (1H, m,
ArCH2CH), 7.10 (2H, d, J ¼ 8.0 Hz, ArH), 7.21 (2H, d, J ¼ 8.0 Hz, ArH).
13C NMR ppm (CDCl3) 16.1, 23.4, 46.0, 48.3,127.0, 129.6, 135.7,136.7;
MS m/z 181 (Mþ). HCl salt. Colourless solid, mp 187–189 ꢀC
(ethanol/hexane) (lit. [27,29] 190–191 ꢀC).
IR nmax (film) 3310, 1600, 1092 cmꢃ1. 1H NMR
d (CDCl3) 1.06 (3H, d,
J ¼ 6.5 Hz, CHCH3), 2.37 (1H, br s, NH), 2.46 (3H, s, SCH3), 2.58 (1H,
dd, J ¼ 7.0 Hz, 13.3 Hz, ArCH2CH), 2.77 (1H, dd, J ¼ 6.5 Hz, 13.5 Hz,
ArCH2CH), 2.95 (1H, m, ArCH2CH), 3.23 (1H, dd, J ¼ 6.0 Hz, 14.0 Hz,
NCH2CHCH2), 3.34 (1H, dd, J ¼ 5.5 Hz, 14.0 Hz, NCH2CHCH2), 5.08
(1H, d, J ¼ 10.5 Hz, NCH2CHCH2), 5.15 (1H, d, J ¼ 17.0 Hz,
NCH2CHCH2), 5.90 (1H, m, NCH2CHCH2), 7.10 (2H, d, J ¼ 8.5 Hz,
ArH), 7.20 (2H, d, J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3) 16.0, 19.6,
42.5, 49.4, 53.7, 116.2, 126.9, 129.7, 135.8, 136.0, 136.1. HCl salt.
Colourless solid, mp 170–172 ꢀC (ethanol/hexane). Anal. Calculated
for C13H20ClNS: C, 60.56; H, 7.82; N, 5.38; S, 12.44. Found: C, 60.16;
H, 7.74; N, 5.45; S, 13.23%.
4.1.2.2. 2-N-Methylamino-1-(4-methylthiophenyl)
propane
(4b). Compound 4b was prepared from 3 and methylamine HCl
according to the general method A. Colourless oil (49%). IR nmax
(film) 3318, 2789, 1600, 1094 cmꢃ1. 1H NMR ppm (CDCl3) 1.04 (3H,
d, J ¼ 6.0 Hz, CHCH3), 1.35 (1H, br s, NH), 2.39 (3H, s, NCH3), 2.47
(3H, s, SCH3), 2.57 (1H, dd, J ¼ 6.5 Hz, 13.3 Hz, ArCH2CH), 2.67 (1H,
dd, J ¼ 7.0 Hz, 13.0 Hz, ArCH2CH), 2.76 (1H, m, ArCH2CH), 7.11 (2H, d,
J ¼ 8.6 Hz, ArH), 7.20 (2H, d, J ¼ 8.6 Hz, ArH). 13C NMR ppm (CDCl3)
16.1, 19.6, 33.9, 42.8, 56.2, 127.0, 129.7, 135.7, 136.5. MS m/z 195
(Mþ); HCl salt. Colourless solid, mp 161–162 ꢀC (ethanol/hexane)
[27]. Anal. Calculated for C11H18ClNS: C, 57.00; H, 7.83; N, 6.04; S,
13.83. Found: C, 56.81; H, 7.79; N, 6.05; S, 13.43%.
4.1.2.7. 2-N-Propargylamino-1-(4-methylthiophenyl)propane (4g).
Compound 4g was prepared from 3 and propargylamine HCl
according to the general method A. Colourless oil (31%). IR nmax
(film) 3291, 2104, 1600 cmꢃ1 1H NMR
. d (CDCl3) 1.05 (3H, d,
J ¼ 6.5 Hz, CHCH3), 1.42 (1H, br s, NH), 2.18 (1H, m, NCH2CCH), 2.47
(3H, s, SCH3), 2.59 (1H, dd, J ¼ 6.5 Hz, 13.6 Hz, ArCH2CH), 2.66
(1H, dd, J ¼ 7.0 Hz, 13.6 Hz, ArCH2CH), 3.13 (1H, m, ArCH2CH), 3.40
(1H, dd, J ¼ 2.0 Hz, 17.3 Hz, NCH2CCH), 3.47 (1H, dd, J ¼ 2.5 Hz,
17.3 Hz, NCH2CCH), 7.12 (2H, d, J ¼ 8.5 Hz, ArH), 7.20 (2H, d,
J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 16.0, 19.4, 35.4, 42.7, 52.3,
71.2, 81.8, 126.8, 129.7, 135.8, 135.9. HCl salt. Colourless solid, mp
173–177 ꢀC (dec.) (ethanol/hexane). Anal. Calculated for
C13H18ClNS: C, 61.04; H, 7.09; N, 5.48; S, 12.54. Found: C, 60.37; H,
7.05; N, 5.66; S, 12.56%.
4.1.2.3. 2-N-Ethylamino-1-(4-methylthiophenyl)propane
(4c).
Compound 4c was prepared from 3 and ethylamine HCl according
to the general method A. Colourless oil (35%). IR nmax (film) 3306,
1600, 1093 cmꢃ1
.
1H NMR ppm (CDCl3) 1.04 (3H, d, J ¼ 6.0 Hz,
CHCH3), 1.06 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 1.26 (1H, br s, NH), 2.47
(3H, s, SCH3), 2.53–2.75 (4H, m, CHCH3, NCH2CH3), 2.88 (1H, m,
ArCH2CH), 7.10 (2H, d, J ¼ 8.5 Hz, ArH), 7.20 (2H, d, J ¼ 8.0 Hz,
ArH). 13C NMR ppm (CDCl3) 15.3, 16.1, 20.1, 41.4, 43.0, 54.45, 126.9,
129.7, 135.6, 136.5. HCl salt. Colourless solid, mp 176–179 ꢀC
(ethanol/hexane) [27]. Anal. Calculated for C12H20ClNS: C, 58.63;
H, 8.20; N, 5.70; S, 13.04. Found: C, 58.48; H, 8.10; N, 5.82; S,
12.64%.
4.1.2.8. 2-N-(2-Hydroxyethyl)amino-1-(4-methylthiophenyl)propane
(4h). Compound 4h was prepared from 3 and 2-hydroxyethyl-
amine HCl according to the general method A. Colourless oil (68%).
IR nmax (film) 3286, 3121, 1597, 1060 cmꢃ1. 1H NMR
d (CDCl3) 1.05
4.1.2.4. 2-N-(Isopropyl)amino-1-(4-methylthiophenyl)propane (4d).
Compound 4d was prepared from 3 and n-propylamine HCl
according to the general method A. Colourless oil (36%). IR nmax
(3H, d, J ¼ 6.0 Hz, CHCH3), 2.36 (2H, br s, NH, NCH2CH2OH), 2.46
(3H, s, SCH3), 2.56 (1H, dd, J ¼ 7.0 Hz, 13.3 Hz, ArCH2CH), 2.68–2.82
(3H, m, ArCH2CH, NCH2CH2OH), 2.88 (1H, m, ArCH2CH), 3.59 (2H,
m, NCH2CH2OH), 7.09 (2H, d, J ¼ 8.5 Hz, ArH), 7.19 (2H, d, J ¼ 8.0 Hz,
ArH). 13C NMR ppm (CDCl3) 16.0, 20.1, 42.9, 48.4, 54.3, 61.1, 126.9,
(film) 3304, 1599, 1092 cmꢃ1
.
1H NMR
d
(CDCl3) 0.97 (3H, d,
J ¼ 6.0 Hz, NCH(CH3)2), 1.00 (3H, d, J ¼ 6.0 Hz, NCH(CH3)2), 1.04 (3H,

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4877
129.7, 135.8, 136.2. HCl salt. HRMS Calculated for C12H20NOS:
(Mþ þ 1) 226.1266; Found: 226.1265.
J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 16.2, 30.3, 38.7, 44.0, 65.0,
127.0, 129.8, 135.6, 136.8. HCl salt. Colourless solid, mp 115–116 ꢀC
(ethanol/hexane). Anal. Calculated for C11H18ClNOS: C, 53.32; H,
7.32; N, 5.65; S, 12.94. Found: C, 53.06; H, 7.24; N, 5.72; S, 12.65%.
4.1.2.9. 2-N-(2-Methoxyethyl)amino-1-(4-methylthiophenyl)propane
(4i). Compound 4i was prepared from 3 and 2-methoxyethylamine
HCl according to the general method A. The resulting residue was
purified by flash chromatography (eluent: dichloromethane/meth-
4.1.2.14. 2-N-Methoxy-N-methylamino-1-(4-methylthiophenyl)
propane (4n). Compound 4n was prepared from 3 and N-methoxy-
N-methylamine HCl according to the general method A. Colourless
anol 90:10). Colourlessoil(28%). IR nmax (film) 3322,1600,1494 cmꢃ1
.
1H NMR
d
(CDCl3) 1.05 (3H, d, J ¼ 6.5 Hz, CHCH3), 2.39 (1H, br s, NH),
oil (73%). IR nmax (film) 3060, 2781, 1599, 1045 cmꢃ1 1H NMR
.
2.55 (1H, dd, J ¼ 7.0 Hz, 13.6 Hz, ArCH2CH), 2.47 (3H, s, SCH3), 2.74–
2.86 (3H, m, ArCH2CH, NCH2CH2O), 2.91 (1H, m, ArCH2CH), 3.31 (3H,
s, OCH3), 3.48 (2H, t, J ¼ 5.5 Hz, NCH2CH2O), 7.11 (2H, d, J ¼ 8.5 Hz,
ArH), 7.20 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 16.1, 19.7,
42.6, 46.5, 54.6, 58.6, 71.8, 126.9, 129.7, 135.7, 136.2. HCl salt. Col-
ourless solid, mp 156–158 ꢀC (ethanol/hexane). Anal. Calculated for
C13H22ClNOS: C, 56.61; H, 8.04; N, 5.08; S, 11.62. Found: C, 56.63; H,
8.09; N, 5.04; S, 11.25%.
d
(CDCl3) 0.95 (3H, d, J ¼ 6.5 Hz, CHCH3), 2.41 (1H, dd, J ¼ 9.5 Hz,
13.1 Hz, ArCH2CH), 2.46 (3H, s, SCH3), 2.61 (3H, s, NCH3), 2.87 (1H,
m, ArCH2CH), 3.10 (1H, dd, J ¼ 4.0 Hz,13.1 Hz, ArCH2CH), 3.54 (3H, s,
OCH3), 7.11 (2H, d, J ¼ 8.5 Hz, ArH), 7.19 (2H, d, J ¼ 8.0 Hz, ArH). 13
C
NMR ppm (CDCl3) 16.2, 38.9, 40.8, 60.1, 64.1, 127.0, 129.8, 135.4,
137.2. HCl salt. Colourless solid, mp 135–137 ꢀC (ethanol/hexane).
Anal. Calculated for C12H20ClNOS: C, 55.05; H, 7.70; N, 5.35; S, 12.25.
Found: C, 54.87; H, 7.67; N, 5.28; S, 11.93%.
4.1.2.10. 2-N-(2-Ethanethiol)amino-1-(4-methylthiophenyl)propane
(4j). Compound 4j was prepared from 3 and 2-ethanethiolamine
HCl according to the general method A. The resulting residue was
purified by flash chromatography (eluent: diethylether/methanol
50:50). Pale amber oil (27%). IR nmax (film) 3302, 2848, 1599,
4.1.3. General method B: HCl salt formation procedure
10 mmol of amine was dissolved in propan-2-ol (IPA) (3 mL).
Concentrated HCl (15 drops) was added and the resulting solution
diluted with diethylether until turbid. The mixture was allowed to
stand at room temperature until crystal formation had occurred
and resulting solids isolated by gravity or vacuum filtration. The
crude HCl salt was recrystallised from a mixture of ethanol and
hexane.
1093 cmꢃ1
.
1H NMR
d
(CDCl3) 1.05 (3H, d, J ¼ 6.0 Hz, CHCH3), 1.57
(1H, br s, NH), 2.46 (3H, s, SCH3), 2.57 (1H, dd, J ¼ 6.5 Hz, 13.0 Hz,
ArCH2CH), 2.66–2.96 (7H, m, ArCH2CH, ArCH2CH, NCH2CH2SH), 7.10
(2H, d, J ¼ 8.5 Hz, ArH), 7.19 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR ppm
(CDCl3) 16.0, 20.1, 38.8, 42.9, 45.2, 54.1, 126.8, 129.6, 135.7, 136.09.
HRMS Calculated for C12H18NS2: (Mþ) 240.0881, found: 240.0875.
4.1.4. General method C: synthesis of imines 5a–d, 20a–d
A solution of the appropriate ketone 3 or 18 (8.32 mmol) and
amine HCl salt (29.93 mmol) in pyridine (7.5 mL) and ethanol
(7.5 mL) was refluxed for 2 h. After cooling, the mixture was acid-
ified with dilute HCl and extracted with dichloromethane
(3 ꢂ 50 mL). The extracts were combined, dried over anhydrous
Na2SO4 and solvent removed in vacuo to provide the required
imines as pale amber oils requiring no further purification. Note.
The imines were isolated as mixtures of syn/anti isomers (as
determined by 1H NMR, major isomer is indicated by ‘‘*’’).
4.1.2.11. 2-N-(n-Propyl)amino-1-(4-methylthiophenyl)propane (4i).
Compound 4i was prepared from 3 and n-propylamine HCl
according to the general method A. Colourless oil (26%). IR nmax
(film) 3312, 1600, 1092 cmꢃ1 1H NMR ppm (CDCl3) 0.86 (3H, t,
.
J ¼ 7.5 Hz, NCH2CH2CH3), 1.04 (3H, d, J ¼ 6.0 Hz, CHCH3), 1.37–1.54
(3H, m, NH, NCH2CH2CH3), 2.46 (3H, s, SCH3), 2.50 (1H, m,
NCH2CH2CH3), 2.55 (1H, dd, J ¼ 7.0 Hz, 13.3 Hz, ArCH2CH), 2.62 (1H,
m, NCH2CH2CH3), 2.69 (1H, dd, J ¼ 7.0 Hz, 13.3 Hz, ArCH2CH), 2.85
(1H, m, ArCH2CH), 7.10 (2H, d, J ¼ 8.5 Hz, ArH), 7.19 (2H, d, J ¼ 8.0 Hz,
ArH). 13C NMR ppm (CDCl3) 11.6, 16.0, 20.0, 23.2, 42.9, 49.1, 54.3,
126.8, 129.6, 135.6, 136.4. HCl salt. Colourless solid, mp 160–162 ꢀC
(dec.) (ethanol/hexane). Anal. Calculated for C13H22ClNS: C, 60.09;
H, 8.53; N, 5.39; S, 12.34. Found: C, 60.35; H, 8.61; N, 5.08; S, 12.38%.
4.1.4.1. 1-(4-Methylthiophenyl)-2-propanoneoxime(5a). Compound
5a was prepared from 3 (2.50 g, 12.87 mmol scale) according to
general method C and chromatographed on silica gel (eluent:
diethylether/hexane 35:65), providing a 25:75 mixture of syn/anti
isomers. Colourless solid (85%), mp 57–59 ꢀC (ethanol/water). Rf
0.75 (diethylether/hexane 80:20). IR nmax (KBr) 3260 (OH), 1668
4.1.2.12. 2-N,N-Dimethylamino-1-(4-methylthiophenyl)propane (4l).
Compound 4l was prepared from 3 and dimethylamine HCl
according to the general method A. Colourless oil (48%). IR nmax
(C]N) cmꢃ1. anti-(5a). 1H NMR (CDCl3) 1.81 (2.25H, s, H-30), 2.46
d
(3H, s, SCH3), 3.45 (1.5H, s, H-10), 7.14, 7.20 (3H, 2d, J ¼ 8.0 Hz,
J ¼ 8.6 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3) 16.0, 19.6, 41.5,
(film) 3073, 1600, 1094 cmꢃ1
.
1H NMR
d
(CDCl3) 0.91 (3H, d,
127.0, 129.4, 133.6, 136.7, 157.4. syn-(5a). 1H NMR
d (CDCl3) 1.80
J ¼ 6.5 Hz, CHCH3), 2.31 (6H, s, N(CH3)2), 2.34 (1H, m, ArCH2CH),
2.45 (3H, s, SCH3), 2.74 (1H, m, ArCH2CH), 2.91 (1H, dd, J ¼ 4.5 Hz,
13.0 Hz, ArCH2CH), 7.09 (2H, d, J ¼ 8.0 Hz, ArH), 7.19 (2H, d,
J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3) 13.7, 16.2, 38.5, 40.6, 61.2,
126.9, 129.6, 135.2, 137.6. HCl salt. Colourless solid, mp 164–166 ꢀC
(ethanol/hexane) [27]. Anal. Calculated for C12H20ClNS: C, 58.63; H,
8.20; N, 5.70; S, 13.04. Found: C, 58.35; H, 8.09; N, 5.66; S, 13.37%.
(0.75H, s, H-30), SCH3 signal overlapping with anti-(5a), 3.70 (0.5H,
s, H-10), 7.15, 7.19 (1H, 2d, J ¼ 8.5 Hz, 9.5 Hz, H-2, H-3, H-5, H-6). 13C
NMR ppm (CDCl3) 16.0, 19.6, 34.2, 127.1, 129.6, 133.4, 136.3, 156.7.
m/z 195, Mþ (syn/anti isomer mixture), Anal. Calculated for
C10H13NOS: C, 61.50; H, 6.71; N, 7.17. Found: C, 61.40; H, 6.71; N,
7.08%.
4.1.4.2. 2-N-Methoxyimino-1-(4-methylthiophenyl)propane
4.1.2.13. 2-N-Hydroxy-N-methylamino-1-(4-methylthiophenyl)
propane (4m). Compound 4m was prepared from 3 and N-hydroxy-
N-methylamine HCl according to the general method A. The
resulting residue was purified by flash chromatography (eluent:
diethylether). Colourless oil (20%). IR nmax (film) 3220, 1600,
(5b). Compound 5b was prepared from 3 and methoxylamine HCl
according to general method C as a 70:30 mixture of isomers. Pale
amber oil (97%). IR nmax (film) 2898, 2814, 1636, 1598, 1093 cmꢃ1. 1H
NMR
d
(CDCl3) 1.72*, 1.77 (3H, 2s, CCH3), 2.47 (3H, s, SCH3), 3.42*,
3.62 (2H, 2s, ArCH2C), 3.87, 3.88* (3H, 2s, OCH3), 7.11, 7.14* (2H, 2d,
J ¼ 8.0 Hz, ArH), 7.19, 7.21* (2H, 2d, J ¼ 8.0 Hz, ArH). 13C NMR ppm
(CDCl3) 13.5*, 15.9*, 16.0, 19.6, 34.8, 41.5*, 61.1, 61.2*, 126.9*, 127.0,
129.4*, 129.5, 133.5, 133.8*, 136.2, 136.6*, 155.8, 156.3*. HRMS
Calculated for C11H15NOS (Mþ)208.0796; Found 208.0790.
1494 cmꢃ1. 1H NMR
d
(CDCl3) 0.92 (3H, d, J ¼ 6.5 Hz, CHCH3), 2.33–
2.38 (1H, m, ArCH2CH), 2.38 (3H, s, SCH3), 2.60 (3H, s, NCH3), 2.80–
2.88 (1H, m, ArCH2CH), 3.07 (1H, dd, J ¼ 4.0 Hz, 13.3 Hz, ArCH2CH),
6.36 (1H, br s, NOH), 7.02 (2H, d, J ¼ 8.5 Hz, ArH), 7.12 (2H, d,

4878
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4.1.4.3. 2-N-Ethoxyimino-1-(4-methylthiophenyl)propane
(5c).
4.1.5.3. 2-N-Ethoxyamino-1-(4-methylthiophenyl)propane
Compound 5c was prepared from and ethoxylamine HCl
3
(4q). Compound 4q was prepared from imine (5c) according to the
general method D. Colourless oil (93%). IR nmax (film) 3238, 2866,
according to general method C as a 70:30 mixture of isomers. Pale
amber oil (93%). IR nmax (film) 2878, 1638, 1597, 1092 cmꢃ1. 1H NMR
1599, 1092 cmꢃ1. 1H NMR
d
(CDCl3) 1.05 (3H, d, J ¼ 6.5 Hz, CHCH3),
d
(CDCl3) 1.27*, 1.28 (3H, 2 t, J ¼ 7.0 Hz, NOCH2CH3), 1.73*, 1.77 (3H,
1.16 (3H, t, J ¼ 7.0 Hz, NOCH2CH3), 2.46 (3H, s, SCH3), 2.55 (1H, dd,
J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 2.78 (1H, dd, J ¼ 7.0 Hz, 13.5 Hz,
ArCH2CH), 3.19 (1H, m, ArCH2CH), 3.73 (2H, q, J ¼ 7.0 Hz,
NOCH2CH3), 5.34 (1H, s, NH), 7.11 (2H, d, J ¼ 8.0 Hz, ArH), 7.20 (2H,
d, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 14.1, 16.1, 17.7, 39.7, 57.2,
69.7, 127.0, 139.7, 135.8, 135.8. HRMS Calculated for C12H20NOS:
(Mþ þ 1) 226.1266; found: 226.1266.
2s, CCH3), 2.46 (3H, s, SCH3), 3.42*, 3.63 (2H, 2s, ArCH2C), 4.12, 4.13*
(2H, 2q, J ¼ 7.0 Hz, NOCH2CH3), 7.12, 7.14* (2H, 2d, J ¼ 8.0 Hz, ArH),
13
7.19, 7.20* (2H, 2d, J ¼ 8.0 Hz, ArH). C NMR ppm (CDCl3) 13.6*,
14.6*, 14.6, 16.0*, 16.0, 19.6, 34.9, 41.6*, 68.8, 68.8*, 127.0*, 127.0,
129.3*, 129.5, 133.8, 134.0*, 136.1, 136.5*, 155.3, 155.9*. HRMS
Calculated for C12H18NOS: (Mþ þ 1) 224.1109: Found: 224.1120.
4.1.4.4. 2-(N-(O-Allyl))imino-1-(4-methylthiophenyl)propane (5d).
Compound 5d was prepared from 3 and allyloxyamine HCl
according to general method C as a 70:30 mixture of isomers. Pale
amber oil (92%). IR nmax (film) 2862, 1644, 1598, 1093 cmꢃ1. 1H NMR
4.1.5.4. 2-(N-(O-Allyl))amino-1-(4-methylthiophenyl)propane
(4r). Compound 4r was prepared from imine 5d according to the
general method D. Colourless oil (89%). IR nmax (film) 3243, 2856,
1599, 1094 cmꢃ1. 1H NMR
d
(CDCl3) 1.06 (3H, d, J ¼ 6.5 Hz, CHCH3),
d
(CDCl3) 1.75*, 1.78 (3H, 2s, CCH3), 2.46 (3H, s, SCH3), 3.42*, 3.65
2.46 (3H, s, SCH3), 2.56 (1H, dd, J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 2.80
(1H, dd, J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 3.22 (1H, m, ArCH2CH),
4.20 (2H, d, J ¼ 6.0 Hz, NOCH2CHCH2), 5.17 (1H, d, J ¼ 10.6 Hz,
NOCH2CHCH2), 5.26 (1H, dd, J ¼ 1.5 Hz, J ¼ 17.3 Hz, NOCH2CHCH2),
5.43 (1H, s, NH), 5.93 (1H, m, NOCH2CHCH2), 7.11 (2H, d,
J ¼ 8.0 Hz, ArH), 7.20 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3)
16.1, 17.7, 39.6, 57.3, 75.5, 117.3, 127.0, 129.8, 134.4, 135.8, 135.9.
HRMS Calculated for C13H20NOS: (Mþ þ 1) 238.1266; Found:
238.1261.
(2H, 2s, ArCH2C), 4.58–4.61 (2H, m, NOCH2CHCH2), 5.20*, 5.21 (1H,
2d, J ¼ 10.3 Hz, J ¼ 10.6 Hz, NOCH2CHCH2), 5.29, 5.30* (1H, 2d,
J ¼ 17.6 Hz, J ¼ 17.1 Hz, NOCH2CHCH2), 5.97–6.07 (1H, m,
NOCH2CHCH2), 7.13, 7.14* (2H, 2d, J ¼ 8.0 Hz, J ¼ 8.5 Hz, ArH), 7.19,
7.20* (2H, 2d, J ¼ 8.0 Hz, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3)
13.7*, 15.9*, 16.0, 19.6, 34.9, 41.5*, 74.2*, 74.2, 116.9*, 126.9*, 117.0,
127.0, 129.3*, 129.5, 133.5, 133.8*, 134.5*, 134.5, 136.2, 136.6*, 156.0,
156.6*. HRMS Calculated for C13H18NOS: (Mþ þ 1) 236.1109. Found:
236.1104.
4.1.6. General method E: synthesis of nitrostyrenes 8b–e
4.1.5. General method D: reduction of imines: preparation of 4o–r
To a stirred mixture of an appropriate imine 5a–d, 20a–d
(4.78 mmol) in dry methanol (40 mL) was added sodium cyanobor-
ohydride (15.92 mmol, 1.00 g) and the reaction was stirred at 20 ꢀC
for 72 h. The pH of the reaction was occasionally adjusted to pH 3 by
the addition of 4 M methanolic HCl as determined by damp universal
pH paper. Excess hydride was decomposed by the addition of 10% aq.
HCl (150 mL) and resulting aqueous phase washed with dichloro-
methane (3 ꢂ 50 mL). The aqueous phase was basified with 15% aq.
NaOH solution and extracted with dichloromethane (3 ꢂ 50 mL). The
organic phases were combined, dried over anhydrous Na2SO4, and
volatiles removed in vacuo leaving the product as an oil.
The appropriate aldehyde 7a–e (6.340 mmol) was dissolved in
absolute ethanol (20 mL). Nitroethane (0.456 mL, 6.340 mmol) was
added followed by N,N-dimethylamine HCl or cyclohexylamine (8
drops) and potassium fluoride(1 mmol). The solution was heated at
reflux for 18 h, concentrated and the residue was chromatographed
on silica gel (eluent: 4:1 diethylether/hexane).
4.1.6.1. 1-tert-Butylsulfanyl-4-(2-nitropropenyl)-benzene (8b). The
product was obtained from 7b according to the general method E as
a yellow solid was purified on silica gel using a 4:1 hexane/dieth-
ylether mobile phase. The solid isolated was recrystallised from hot
ethanol to give the title compound as yellow needles (75%), mp
60–62 ꢀC. IR nmax (KBr) cmꢃ1: 1511, 1318 (conj-NO2). 1H NMR
4.1.5.1. 2-N-Hydroxyamino-1-(4-methylthiophenyl)propane
(CDCl3)
J ¼ 8.0 Hz, ArH), 7.58–7.60 (d, J ¼ 8.0 Hz, ArH), 8.05 (s, 1H, CH]C).
13C NMR (CDCl3)
: 13.7, 30.8, 46.5, 129.7, 132.3, 132.5, 135.3, 137.2,
147.8. Anal. Calculated for C13H17NO2S: C, 62.12; H, 6.82; N, 5.57.
Found C, 62.05; H, 6.78; N, 5.51%.
d: 1.30 (s, 9H, C(CH3)3), 2.45 (s, 3H, CH3), 7.37–7.39 (d,
(4o). Compound 4o was prepared from the oxime 5a according to
the general method D. Colourless solid (68%), mp 73–74 ꢀC. IR nmax
d
(film) 3243, 3126, 1493, 1093 cmꢃ1. 1H NMR
d
(CDCl3) 1.08 (3H, d,
J ¼ 6.0 Hz, CHCH3), 2.47 (3H, s, SCH3), 2.58 (1H, dd, J ¼ 6.5 Hz,
13.5 Hz, ArCH2CH), 2.82 (1H, dd, J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 3.17
(1H, m, ArCH2CH), 5.15 (1H, br s, NH), 6.82 (1H, br s, NOH), 7.12 (2H,
d, J ¼ 8.0 Hz, ArH), 7.21 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR ppm
(CDCl3) 16.1, 17.4, 39.3, 58.3, 127.0, 129.8, 135.5, 136.0. Anal. Calcu-
lated for C10H10NOS: C, 60.88; H, 7.66; N, 7.10. Found: C, 60.89; H,
7.57; N, 7.05%. HCl salt. Colourless solid, mp 124–126 ꢀC (ethanol/
hexane). Anal. Calculated for C10H16ClNOS: C, 51.38; H, 6.90; N,
5.99; S, 13.72. Found: C, 51.39; H, 6.83; N, 6.10; S, 13.30%.
4.1.6.2. 1-Benzylsulfanyl-4-(2-nitropropenyl)-benzene
(8c). The
product was obtained from 7c according to the general method E in
23% yield, yellow needles, (72%), mp 84 ꢀC (lit. 74–75 ꢀC). IR nmax
(KBr) cmꢃ1: 1518, 1316 (NO2). 1H NMR (CDCl3)
d
: 2.47 (s, 3H, CH3),
4.22 (s, 2H, SCH2), 7.28–7.39 (m, 10H, SCH2C6H5, ArH), 8.06 (s, 1H,
CH]C). 13C NMR (CDCl3)
: 14.1, 37.6, 127.4, 128.7, 128.7, 128.7, 129.2,
d
130.4, 133.0, 135.0, 136.4, 140.2, 147.1. Anal. Calculated for
C16H15NO2S: C, 67.34; H, 5.30; N, 4.91. Found C, 67.71; H, 5.14; N,
4.68%.
4.1.5.2. 2-N-Methoxyamino-1-(4-methylthiophenyl)propane
(4p). Compound 4p was prepared from 5b according to the general
method D. Colourless oil (81%). IR nmax (film) 3239, 2808, 1598,
4.1.6.3. 1-(2-Dimethylamino-ethylsulfanyl)-4-(2-nitropropenyl)-
benzene (8d). The product was obtained from 7d according to the
general method E as a yellow oil (73%), which was used in the
subsequent reaction without further purification. IR nmax (KBr)
1093 cmꢃ1. 1H NMR
d
(CDCl3) 1.07 (3H, d, J ¼ 6.5 Hz, CHCH3), 2.47
(3H, s, SCH3), 2.57 (1H, dd, J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 2.79 (1H,
dd, J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 3.20 (1H, m, ArCH2CH), 5.47 (1H,
br s, NH), 7.13 (2H, d, J ¼ 8.0 Hz, ArH), 7.21 (2H, d, J ¼ 8.0 Hz, ArH).
13C NMR ppm (CDCl3) 15.9, 17.6, 39.5, 57.1, 62.3, 126.8, 129.7, 135.5,
135.8. HCl salt. Colourless solid, mp 106–107 ꢀC (ethanol/hexane).
Anal. Calculated for C11H18ClNOS: C, 53.32; H, 7.32; N, 5.65; S, 12.94.
Found: C, 53.13; H, 7.16; N, 5.66; S, 12.99%.
cmꢃ1: 1523, 1311 (conj-NO2). 1H NMR (CDCl3)
d: 2.28 (s, 6H,
N(CH3)2), 2.44 (s, 3H, CH3), 2.58–2.62 (t, J ¼ 7.0 Hz, 2H, NCH2), 3.07–
3.11(t, J ¼ 7.0 Hz, 2H, SCH2), 7.28–7.36 (m, 4H, C6H4), 8.02 (s, 1H,
CH]C). 13C NMR (CDCl3)
d: 14.0, 30.2, 45.1, 57.94, 127.1, 128.9, 130.4,
132.9, 140.4, 146.8. MS m/z 267 (Mþ þ 1).

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4879
4.1.6.4. 1-Phenylsulfanyl-4-(2-nitropropenyl)-benzene
(8e). The
product was obtained from 7e according to the general method E as
a yellow oil (46%), which was used in the subsequent reaction
without further purification. IR nmax (KBr) cmꢃ1: 1584, 1300 (NO2).
132.8, 136.1, 138.7. HRMS Calculated for
244.1160, found 244.1172.
C
15H17NS: (Mþ þ 1)
4.1.8. General preparation G: 1-(4-bromophenyl)-2-(1,3-dioxyl)
alkanes (13a–b)
1H NMR (CDCl3)
d: 2.47 (s, 3H, CH3), 7.27–7.29 (m, 2H, ArH), 7.34–
7.36 (m, 2H, ArH), 7.41–7.44 (m, 3H, ArH), 7.50–7.52 (m, 2H, ArH),
8.05 (s, 1H, CH]C). 13C NMR (CDCl3) ppm: 14.1, 128.5, 129.6, 129.8,
130.6, 132.6, 132.9, 133.3, 140.8.147.2.
A mixture of the appropriate 1-(4-bromophenyl)-2-alkanone
12a–b (92.67 mmol) and ethylene glycol (832 mmol, 67 mL) in
toluene (350 mL) was vigorously stirred. To this mixture was added
p-TSA (2.50 mmol, 0.50 g) and the reaction was refluxed with
a Dean–Stark trap for 18 h. After cooling, the reaction phases were
separated. The toluene phase was washed with satd. aq. NaHCO3
(3 ꢂ 75 mL). The ethylene glycol phase was diluted with water
(450 mL) and extracted with toluene (3 ꢂ 50 mL). All toluene pha-
ses were combined and washed with satd. aq. NaHCO3 (3 ꢂ 50 mL).
The organic phases were dried over anhydrous Na2SO4, and
concentrated in vacuo, providing the product as a colourless oil.
4.1.7. General method F: reduction of nitrostyrenes 8b–e
To a stirred suspension of LAlH4 (0.655 g,17.26 mmol) in dry THF
(20 mL) was added very slowly the appropriate nitrostyrene
(3.45 mmol) in dry THF (10 mL) under a nitrogen atmosphere. The
suspension was heated to reflux for 12 h and then quenched by the
careful dropwise addition at 0 ꢀC of 10 mL of 20:1 methanol/H2O
followed by the addition of 15% NaOH (50 mL). The inorganic salts
were removed by filtration and the filtrate was extracted with ethyl
acetate (3 ꢂ 75 mL). The organic layer was washed with 10% HCl
(4 ꢂ 50 mL), this acidic extract was basified with 15% NaOH to pH 8
and extracted with ethyl acetate (3 ꢂ100 mL). The solvent was
removed in vacuo to leave a yellow oil that was further purified on
a short silica column using a 100% methanol eluent.
4.1.8.1. 1-(4-Bromophenyl)-2-(1,3-dioxyl)propane (13a). Compound
13a was prepared from 12a (19.75 g, 92.67 mmol scale) according
to the general method G. Colourless oil (100%). IR nmax (film) 1480
(ArH) cmꢃ1. 1H NMR
d
(CDCl3) 1.30 (3H, s, H-30), 2.87 (2H, s, H-10),
3.71 (2H, m, OCH2CH2O), 3.87 (2H, m, OCH2CH2O), 7.14, 7.39 (4H, 2d,
J ¼ 8.5 Hz, 8.0 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3) 24.3,
44.7, 64.8, 109.3, 120.4, 130.9, 132.2, 135.8. m/z 257 (Mþ, 1%). HRMS
Calculated for C11H13BrO2: (Mþ) 256.00989, found: 256.00958.
4.1.7.1. 2-(4-tert-Butylsulfanyl-phenyl)-1-methyl-ethylamine
(9b). The product was obtained from 8b according to the general
method F as a pale yellow oil, (58%). IR nmax (KBr–HCl salt) cmꢃ1
:
2956 (N–H), 1505, 797 (ArCH). 1H NMR (CDCl3)
d: 1.06–1.08 (d,
4.1.8.2. 1-(4-Bromophenyl)-2-(1,3-dioxyl)butane (13b). Compound
13b was prepared from 12b (23.30 g, 102.60 mmol scale) according
to the general method G. Colourless oil (100%). IR nmax (film0) 1481
J ¼ 6.0 Hz, 3H, CHCH3), 1.23 (s, 9H, C(CH3)3), 1.39 (s, 2H, NH2), 2.47–
2.68 (2 ꢂ dd, Jgem ¼ 13 Hz, Jvic ¼ 8.0 Hz (CH2CH), Jgem ¼ 13 Hz,
Jvic ¼ 5.5 Hz (CH2CH), 2H, CH2CH), 3.09–3.17 (m, 1H, CH2CH), 7.10–
7.12 (d, J ¼ 8.0 Hz, 2H, ArH), 7.41–7.43 (d, J ¼ 8.0 Hz, 2H, ArH). 13C
(ArH) cmꢃ1. 1H NMR
d
(CDCl3) 0.93 (3H, t, J4 ,3 ¼ 7.5 Hz, H-4 ), 1.62
0
0
0
0
0
0
(2H, q, J3 ,4 ¼7.5 Hz, H-3 ), 2.84 (2H, s, H-1 ), 3.65 (2H, m, OCH2
-
NMR (CDCl3)
d
: 23.3, 30.7, 45.4, 46.0, 48.1, 129.1, 129.9, 137.3, 140.2.
CH2O), 3.85 (2H, m, OCH2CH2O), 7.15, 7.38 (4H, 2d, J ¼ 8.0 Hz, 8.0 Hz,
H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3) 7.8, 30.7, 42.7, 65.2, 120.2,
130.8, 132.2, 135.8. m/z 271 (Mþ þ 1, 1%). HRMS Calculated for
C12H15BrO2: (Mþ) 270.02554, found: 270.02389.
HRMS Calculated for C13H22NS: (Mþ þ 1) 224.1473, found 224.1479.
4.1.7.2. 1-(4-Benzylsulfanyl-phenyl)-propylamine (9c). The product
was obtained from 8c according to the general method F as a pale
yellow oil [29] (71%). IR nmax (KBr–HCl salt) cmꢃ1: 2936 (N–H). 1H
4.1.9. General preparation H: 1-(4-ethylthiophenyl)-2-alkanones
(14a–b)
NMR (CDCl3)
d
: 1.12 (d, J ¼ 6.2 Hz, 3H, CHCH3), 1.58 (s, 2H, NH2),
2.47–2.52 (dd, 1H, Jgem ¼ 13.3 Hz, Jvic ¼ 8.0 Hz (CH2CH)), 3.11–3.19
A solution of the appropriate 1-(4-bromophenyl)-2-(1,3-diox-
yl)alkane (13a–b) (33.38 mmol) in dry THF (60 mL) was stirred
and cooled to ꢃ78 ꢀC under nitrogen. After 30 min at ꢃ78 ꢀC, n-
BuLi (2.5 M in hexanes, 36.00 mmol, 14.40 mL) was added and the
reaction was stirred for a further 1 h at ꢃ78 ꢀC. Diethyl disulphide
(36.00 mmol, 4.40 g, 4.13 mL) was then added and the reaction
was stirred for a further 1 h at ꢃ78 ꢀC, then at 20 ꢀC overnight. The
solution was then diluted with water (250 mL) and extracted with
dichloromethane (3 ꢂ 50 mL). The organic phases were combined
and volatiles removed in vacuo leaving an oil which was dissolved
in a mixture of ethanol (100 mL) and 20% aq. HCl (50 mL) and
refluxed for 2 h. After cooling the reaction was diluted with water
(250 mL) and extracted with dichloromethane (3 ꢂ 50 mL). The
organic phases were combined, dried over anhydrous Na2SO4 and
volatiles removed in vacuo leaving an oil. This was purified by
flash chromatography on silica gel followed by vacuum
distillation.
(m, 1H, CH2CH), 4.11 (s, 2H, SCH2), 7.08–7.10 (d, J ¼ 8.1 Hz, 2H, ArH),
7.25–7.32 (m, 7H, ArH). 13C NMR (CDCl3)
d: 23.3, 39.3, 46.0, 48.3,
127.0, 128.3, 128.7, 129.9, 130.2, 133.6, 137.4, 138.0. HRMS Calculated
for C16H19NS: (Mþ þ 1) 257.1238, found 257.1242.
4.1.7.3. 2-[4-(2-Dimethylamino-ethylsulfanyl)-phenyl]-1-methyl-
ethylamine (9d). The product was obtained from 8d according to
the general method F as a pale yellow oil, (52%). IR nmax (film) cmꢃ1
:
3438 (N–H). 1H NMR (CDCl3)
d
: 1.10–1.12 (d, J ¼ 6.5 Hz, 3H, CHCH3),
1.26 (s, 2H, NH2), 2.26 (s, 6H, N(CH3)2), 2.45–2.51 (dd,
Jgem ¼ 13.0 Hz, Jvic ¼ 8.0 Hz, 1H, (CH2CH)), 2.53–2.57 (t, J ¼ 7.0 Hz,
2H, (NCH2)), 2.64–2.69 (dd, Jgem ¼ 13.0 Hz, Jvic ¼ 5.5 Hz, 1H,
(CH2CH)), 2.98–3.03 (t, J ¼ 7.0 Hz, 2H, (SCH2)), 3.09–3.18 (m, 1H,
CH2CH), 7.10–7.12 (d, J ¼ 8.0 Hz, 2H, ArH), 7.28–7.30 (d, J ¼ 8.0 Hz,
2H, ArH). 13C NMR (CDCl3)
d: 23.5, 31.7, 45.2, 46.0, 48.3, 58.5, 129.2,
129.7, 133.7, 137.6. HRMS Calculated for C13H22N2S: (Mþ þ 1)
239.1582, found 239.1580.
4.1.9.1. 1-(4-Ethylthiophenyl)-2-propanone (14a). Compound 14a
was prepared from 13a according to the general method H and
chromatographed on silica gel (eluent: hexane/diethylether 60:40).
4.1.7.4. 1-Methyl-2-(4-phenylsulfanyl-phenyl)-ethylamine (9e). The
product was obtained from 8e according to the general method H
as a pale yellow oil (47%). IR nmax (KBr–HCl salt) cmꢃ1: 2905 (N–H).
Amber oil (71%). IR nmax (film) 1713 (C]O) cmꢃ1. 1H NMR
d (CDCl3)
1H NMR (CDCl3)
d
: 1.13–1.15 (d, J ¼ 6.5 Hz, 3H, CHCH3), 1.95 (s, 2H,
1.30 (3H, t, J ¼ 7.5 Hz, SCH2CH3), 2.14 (3H, s, H-30), 2.93 (2H, q,
J ¼ 7.6 Hz, SCH2CH3), 3.65 (2H, s, H-10), 7.11, 7.29 (4H, 2d, J ¼ 8.5 Hz,
8.0 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3) 14.3, 27.6, 29.1,
50.3, 129.3, 129.8, 131.7, 135.4, 205.9. m/z 194 (Mþ, 59%). HRMS
Calculated for C11H14OS: (Mþ) 194.07654; found: 194.07631.
NH2), 2.52–2.73 (2dd, Jgem ¼ 13.5 Hz, Jvic ¼ 7.5 Hz (CH2CH),
Jgem ¼ 13.5 Hz, Jvic ¼ 5.5 Hz (CH2CH), 2H, CH2CH), 3.14–3.20 (m, 1H,
CH2CH), 7.14–7.16 (d, J ¼ 8.0 Hz, 2H, ArH), 7.25–7.32 (m, 7H, ArH).
13C NMR (CDCl3)
d: 23.2, 45.8, 48.2, 126.7, 129.0, 130.0, 130.4, 131.4,

4880
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4.1.9.2. 1-(4-Ethylthiophenyl)-2-butanone (14b). Compound 14b
was prepared from 13b according to the general method H. Col-
28.0, 38.9, 44.2, 65.0, 129.6, 129.8, 133.7, 137.8. m/z 225 (Mþ). HCl
salt. Colourless solid, mp 111–112 ꢀC (ethanol/hexane). IR nmax
(KBr) 2546, 2509 (NHþ) cmꢃ1. Anal. Calculated for C12H20ClNOS: C,
55.05; H, 7.70; N, 5.35; S, 12.25. Found: C, 54.86; H, 7.58; N, 5.21; S,
12.46%.
ourless oil (68%). IR nmax (film) 1712 (C]O) cmꢃ1. 1H NMR
d (CDCl3)
0
0
0
1.03 (3H, t, J4 ,3 ¼ 7.3 Hz, H-4 ), 1.30 (3H, t, J ¼ 7.5 Hz, SCH2CH3), 2.46
0
0
0
(2H, q, J3 ,4 ¼ 7.5 Hz, H-3 ), 2.92 (2H, q, J ¼ 7.5 Hz, SCH2CH3), 3.64
(2H, s, H-10), 7.12, 7.28 (4H, 2d, J ¼ 8.6 Hz, 8.0 Hz, H-2, H-3, H-5, H-
6). 13C NMR ppm (CDCl3) 7.7, 14.3, 27.7, 35.2, 49.1, 129.3, 129.8, 132.0,
135.2, 208.5. m/z 208 (Mþ). HRMS Calculated for C12H16OS: (Mþ)
208.09219; found: 208.09247.
4.1.9.7. 2-Amino-1-(4-ethylthiophenyl)butane
(15e). Compound
15e was prepared from 14b (1.50 g, 7.20 mmol scale) and
ammonium acetate according to the general method A. Colour-
less oil (73%). IR nmax (film) 3367, 3293 (NH2) cmꢃ1 1H NMR
.
0
0
0
4.1.9.3. 2-Amino-1-(4-ethylthiophenyl)propane
(4-ETA)
(9a).
d
(CDCl3) 0.97 (3H, t, J4 ,3 ¼ 7.3 Hz, H-4 ), 1.16 (2H, br s, NH2), 1.30
Compound 9a was prepared from ketone 14a (1.50 g, 7.80 mmol
scale) and ammonium acetate according to the general method A.
Colourless oil (75%). IR nmax (film) 3357, 3282 (NH2) cmꢃ1. 1H NMR
(3H, t, J ¼ 7.3 Hz, SCH2CH3), 1.35 (1H, m, H-30), 1.50 (1H, m, H-30),
0
0
0
2.42 (1H, dd, Jgem ¼ 13.3 Hz, J10,2 ¼ 8.7 Hz, H-1 ), 2.76 (1H, dd,
0
0
0
Jgem ¼ 13.6 Hz, J1 ,2 ¼ 4.5 Hz, H-1 ), 2.88 (1H, m, H-2 ), 2.92 (2H, q,
J ¼ 7.5 Hz, SCH2CH3), 7.11, 7.27 (2H, 2s, J ¼ 8.5 Hz, J ¼ 8.0 Hz, H-2,
H-3, H-5, H-6). 13C NMR ppm (CDCl3) 10.5, 14.4, 28.0, 30.3, 43.7,
54.1, 129.5, 129.7, 133.9, 137.6. MS m/z 209 (Mþ). HCl salt. Col-
ourless solid, mp 130–132 ꢀC (ethanol/hexane). IR nmax (KBr)
2617, 2526 (NHþ) cmꢃ1. Anal. Calculated for C12H20ClNS: C, 58.65;
H, 8.20; N, 5.70; S, 13.04. Found: C, 58.67; H, 8.14; N, 5.64; S,
12.70%.
0
0
0
d
(CDCl3) 1.10 (3H, d, J3 ,2 ¼ 6.5 Hz, H-3 ), 1.30 (3H, t, J ¼ 7.5 Hz,
SCH2CH3), 1.33 (2H, br s, NH2), 2.49 (1H, dd, Jgem ¼ 13.6 Hz,
0
0
0
0
0
0
J1 ,2 ¼ 8.0 Hz, H-1 ), 2.66 (1H, dd, Jgem ¼ 13.0 Hz, J1 ,2 ¼ 5.5 Hz, H-1 ),
2.91 (2H, q, J ¼ 7.5 Hz, SCH2CH3), 3.14 (1H, m, H-20), 7.10, 7.27 (2H,
2s, J ¼ 8.0 Hz, J ¼ 8.0 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3)
14.3 (SCH2CH3), 23.4, 27.9, 46.1, 48.3, 129.5, 129.6, 133.9, 137.5. m/z
195 (Mþ). HCl salt. Colourless solid, mp 176–178 ꢀC (ethanol/
hexane) [27]. IR nmax (KBr) 2515 (NHþ) cmꢃ1. Anal. Calculated for
C11H18ClNS: C, 57.00; H, 7.83; N, 6.04; S, 13.83. Found: C, 56.82; H,
7.73; N, 6.05; S, 13.52%.
4.1.9.8. 2-N-Methylamino-1-(4-ethylthiophenyl)butane
(15f).
Compound 15f was prepared from 14b (1.50 g, 7.20 mmol scale)
and methylamine HCl according to the general method A. Colour-
less oil (27%). IR nmax (film) 3333 (NH), 2789 (NCH3) cmꢃ1. 1H NMR
4.1.9.4. 2-N-Methylamino-1-(4-ethylthiophenyl)propane
(15a).
0
0
0
Compound 15a was prepared from ketone 14a (1.50 g, 7.80 mmol
scale) and methylamine HCl according to the general method A.
Colourless oil (34%). IR nmax (film) 3321 (NH), 2788 (NCH3) cmꢃ1. 1H
d
(CDCl3) 0.93 (3H, t, J4 ,3 ¼ 7.5 Hz, H-4 ),1.18 (1H, br s, NH),1.30 (3H,
t, J ¼ 7.3 Hz, SCH2CH3), 1.35–1.52 (2H, m, H-30), 2.37 (3H, s, NCH3),
2.53–2.71 (3H, m, H-10, H-20), 2.92 (2H, q, J ¼ 7.5 Hz, SCH2CH3), 7.11,
7.26 (2H, 2s, J ¼ 8.0 Hz, J ¼ 8.0 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm
(CDCl3) 9.7, 14.4, 25.5, 28.0, 33.7, 39.3, 62.0, 129.5, 129.7, 133.8, 137.6.
MS m/z 223 (Mþ). HCl salt. Colourless solid, mp 137–139 ꢀC
(ethanol/hexane). IR nmax (KBr) 2463 (NHþ) cmꢃ1. Anal. Calculated
for C13H22ClNS: C, 60.09; H, 8.53; N, 5.39; S, 12.34. Found: C, 59.90;
H, 8.49; N, 5.30; S, 11.94%.
0
0
0
NMR
d
(CDCl3) 1.04 (3H, d, J3 ,2 ¼ 6.5 Hz, H-3 ), 1.28 (1H, br s, NH),
1.30 (3H, t, J ¼ 7.5 Hz, SCH2CH3), 2.39 (3H, s, NCH3), 2.57 (1H, dd,
0
0
0
Jgem ¼ 13.3 Hz, J1 ,2 ¼ 6.5 Hz, H-1 ), 2.68 (1H, dd, Jgem ¼ 13.3 Hz,
0
0
0
0
J1 ,2 ¼ 7.0 Hz, H-1 ), 2.77 (1H, m, H-2 ), 2.92 (2H, q, J ¼ 7.5 Hz,
SCH2CH3), 7.10, 7.27 (2H, 2s, J ¼ 8.0 Hz, J ¼ 8.0 Hz, H-2, H-3, H-5, H-
6). 13C NMR ppm (CDCl3) 14.4, 19.6, 27.9, 33.9, 42.9, 56.24, 129.4,
129.7,133.9, 137.3. m/z 209 (Mþ). HCl salt. Colourless solid, mp 130–
132 ꢀC (ethanol/hexane). IR nmax (KBr) 2465 (NHþ) cmꢃ1. Anal.
Calculated for C12H20ClNS: C, 58.63; H, 8.20; N, 5.70; S, 13.04.
Found: C, 58.57; H, 8.14; N, 5.79; S, 13.11%.
4.1.9.9. 2-N-Ethylamino-1-(4-ethylthiophenyl)butane
(15g).
Compound 15g was prepared from 14b (1.50 g, 7.20 mmol scale)
and ethylamine HCl according to the general method A. Colourless
oil (28%). IR nmax (film) 3317 (NH) cmꢃ1. 1H NMR
d (CDCl3) 0.92 (3H,
0
0
0
4.1.9.5. 2-N-Ethylamino-1-(4-ethylthiophenyl)propane
(15b).
t, J4 ,3 ¼ 7.3 Hz, H-4 ), 1.02 (1H, br s, NH), 1.03 (3H, t, J ¼ 7.0 Hz,
NCH2CH3), 1.30 (3H, t, J ¼ 7.3 Hz, SCH2CH3), 1.37–1.46 (2H, m, H-30),
2.52–2.73 (5H, m, H-10, H-20, NCH2CH3), 2.92 (2H, q, J ¼ 7.5 Hz,
SCH2CH3), 7.11, 7.26 (2H, 2s, J ¼ 8.0 Hz, J ¼ 8.0 Hz, H-2, H-3, H-5,
H-6). 13C NMR ppm (CDCl3) 9.9,14.4,15.4, 26.2, 28.0, 39.8, 41.3, 60.3,
129.5, 129.7, 133.7, 137.7. MS m/z 237 (Mþ). HCl salt. Colourless
solid, mp 144–146 ꢀC (ethanol/hexane). IR nmax (KBr) 2476, 2378
(NHþ) cmꢃ1. Anal. Calculated for C14H24ClNS: C, 61.40; H, 8.83; N,
5.11. Found: C, 59.79; H, 8.53; N, 5.45%.
Compound 15b was prepared from 14a (1.50 g, 7.80 mmol scale)
and ethylamine HCl according to the general method A. Colourless
oil (17%). IR nmax (film) 3307 (NH) cmꢃ1. 1H NMR
d (CDCl3) 1.04 (3H,
0
0
0
d, J3 ,2 ¼ 6.0 Hz, H-3 ), 1.06 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 1.28 (1H, br
s, NH), 1.30 (3H, t, J ¼ 7.5 Hz, SCH2CH3), 2.55 (1H, dd,0Jgem ¼ 13.3 Hz,
0
0
0
J1 ,2 ¼ 6.5 Hz, H-1 ), 2.57–2.74 (3H, m, NCH2CH3, H-1 ), 2.89 (1H, m,
H-20), 2.92 (2H, q, J ¼ 7.5 Hz, SCH2CH3), 7.10, 7.27 (2H, 2s, J ¼ 8.0 Hz,
J ¼ 8.0 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3) 14.4,15.4, 20.2,
28.0, 41.4, 43.1, 54.4, 129.5, 129.7, 133.9, 137.4. m/z 223 (Mþ). HCl
salt. Colourless solid, mp 164–166 ꢀC (ethanol/hexane). IR nmax
(KBr) 2481, 2369 (NHþ) cmꢃ1. Anal. Calculated for C13H22ClNS: C,
60.09; H, 8.53; N, 5.39; S, 12.34. Found: C, 59.84; H, 8.45; N, 5.36; S,
11.99%.
4.1.9.10. 2-(N-Methyl-N-hydroxy)amino-1-(4-ethylthiophenyl)
butane (15i). Compound 15i was prepared from 14b (1.50 g,
7.20 mmol scale) and N-methylhydroxylamine HCl according to the
general method A. Colourless oil (62%). IR nmax (film) 3226 (OH),
2784 (NCH3) cmꢃ1. 1H NMR
d
(CDCl3) 0.90 (3H, t, J4 ,3 ¼ 7.3 Hz, H-4 ),
0
0
0
4.1.9.6. 2-(N-Methyl-N-hydroxy)amino-1-(4-ethylthiophenyl)
propane (15d). Compound 15d was prepared from 14a (1.50 g,
7.80 mmol scale) and N-methylhydroxylamine HCl according to the
general method A. Colourless oil (35%). IR nmax (film) 3206 (OH),
1.32 (3H, t, J ¼ 7.5 Hz, SCH2CH3), 1.43–1.57 (2H, m, H-30), 2.55 (1H,
0
0
0
dd, Jgem ¼ 13.6 Hz, J1 ,2 ¼ 9.0 Hz, H-1 ), 2.69 (3H, s, NCH3), 2.80 (1H,
m, H-20), 2.94 (2H, q, J ¼ 7.5 Hz, SCH2CH3), 3.12 (1H, dd,
0
0
0
Jgem ¼ 13.5 Hz, J1 ,2 ¼ 4.5 Hz, H-1 ), 7.14 (1H, br s, OH), 7.15, 7.29 (2H,
2s, J ¼ 8.0 Hz, J ¼ 8.0 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3)
10.5, 14.4, 22.6, 28.1, 34.8, 43.3, 70.8, 129.6, 129.7, 133.5, 138.4. m/z
225 (Mþ). HCl salt. Colourless solid, mp 87–90 ꢀC (ethanol/hexane).
2792 (NCH3) cmꢃ1. 1H NMR
d
(CDCl3) 0.99 (3H, d, J3 ,2 ¼ 6.5 Hz, H-
0
0
30), 1.30 (3H, t, J ¼ 7.5 Hz, SCH2CH3), 2.45 (1H, dd, Jgem ¼ 13.0 Hz,
0
0
0
J1 ,2 ¼ 9.5 Hz, H-1 ), 2.68 (3H, s, NCH3), 2.91 (2H, q, J ¼ 7.5 Hz,
SCH2CH3), 2.92 0(1H, m, H-20), 3.13 (1H, dd, Jgem ¼ 13.3 Hz,
IR nmax (KBr) 2597, 2524 (NHþ) cmꢃ1
. Anal. Calculated for
0
0
J1 ,2 ¼ 4.5 Hz, H-1 ), 6.76 (1H, br s, OH), 7.10, 7.26 (2H, 2s, J ¼ 8.0 Hz,
C13H22ClNOS: C, 56.61; H, 8.04; N, 5.08; S, 11.62. Found: C, 56.87; H,
8.03; N, 5.07; S, 11.33%.
J ¼ 8.6 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3) 14.4, 23.4,

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4881
4.1.9.11. 1-(4-Ethylthiophenyl)-2-propanone
oxime
(16a).
101 ꢀC (ethanol/hexane). IR nmax (KBr) 2519 (NHþ) cmꢃ1. Anal.
Calculated for C12H20ClNOS; C, 55.05; H, 7.70; N, 5.35; S, 12.25.
Found: C, 55.30; H, 7.73; N, 5.35; S, 12.11%.
Compound 16a was prepared from 14a (2.50 g, 13.00 mmol scale)
according to the general method C and chromatographed on silica
gel (eluent: hexane/diethylether 65:35), providing a 30:70 mixture
of syn/anti isomers. Pale amber solid (90%), mp 57–60 ꢀC (hexane).
4.1.10. 1-(4-Methylthiophenyl)-2-butanone (18)
IR nmax (KBr) 3250 (OH) cmꢃ1. anti-(608) 1H NMR
d
(CDCl3) 1.80
A suspension of iron powder (57.3 mmol, 3.20 g) in glacial
acetic acid (15 mL) was heated on a steam bath for 20 min, stirring
occasionally. To this mixture, a solution of the nitrostyrene 17 [82]
(3.19 g, 14.30 mmol) in glacial acetic acid (15 mL) was added over
20 min, while stirring the reaction occasionally. The reaction was
heated for a further 2 h when the mixture became a grey-white
colour. The reaction was allowed to cool to ambient temperature
and added to a mixture of ice/water (120 mL). The product was
extracted with dichloromethane (4 ꢂ100 mL) and organic extracts
washed with 15% aq. NaOH (3 ꢂ 100 mL). The organic layer was
then dried over anhydrous Na2SO4, concentrated in vacuo, and the
resulting residue vacuum distilled, producing the desired ketone as
a colourless solid (96%), mp 38–39 ꢀC; bp 116 ꢀC/0.25 mmHg [83].
(2.1H, s, H-30), 1.30 (3H, t, J ¼ 7.3 Hz, SCH2CH3), 2.91 (2H, q,
J ¼ 7.6 Hz, SCH2CH3), 3.46 (1.4H, s, H-10), 7.14, 7.27 (2.8H, 2s, H-2,
H-3, H-5, H-6), 7.27 (1.4H, s, ArH). 13C NMR ppm (CDCl3) 14.0, 27.8,
41.6, 129.4, 129.5, 134.4, 134.9, 157.4. m/z 209 (Mþ). syn-(16a) 1H
NMR
d
(CDCl3) 1.81 (0.9H, s, H-30), (SCH2CH3 signals overlap with
anti-(16a)), 3.70 (0.6H, s, H-10), 7.15, 7.26 (1.2H, 2s, H-2, H-3, H-5,
H-6). 13C NMR ppm (CDCl3) 13.1, 27.9, 34.2,129.5,129.6,134.2,134.5,
156.6. m/z 209 (Mþ). Anal. (syn/anti isomer mixture) calculated for
C11H15NOS: C, 63.12; H, 7.22; N, 6.69. Found: C, 63.41; H, 7.27; N,
6.69%.
4.1.9.12. 1-(4-Ethylthiophenyl)-2-butanone oxime (16b). Compound
16b was prepared from 14b (2.50 g, 12.00 mmol scale) according to
the general method C and chromatographed on silica gel (eluent:
hexane/diethylether 70:30), providing a 45:55 mixture of syn/anti
isomers. Colourless solid (86%). IR nmax (film) 3246 (OH) cmꢃ1. anti-
IR nmax (KBr) 1711 (C]O) cmꢃ1
.
1H NMR
d
(CDCl3) 1.02 (3H, t,
0
0
0
0
0
0
J4 ,3 ¼ 7.3 Hz, H-4 ), 2.26 (2H, q, J3 ,4 ¼ 7.3 Hz, H-3 ), 2.46 (3H, s,
SCH3), 3.63 (2H, s, H-10), 7.12, 7.22 (4H, 2d, J ¼ 8.5, J ¼ 8.5 Hz, H-2,
H-3, H-5, H-6). 13C NMR ppm (CDCl3) 7.73, 15.95, 35.18, 49.11,
127.05, 129.81, 131.29, 137.04, 208.70. MS m/z 194 (Mþ). Anal.
Calculated for C11H14OS; C, 68.00; H, 7.26. Found: C, 68.24; H,
7.40%.
(16b) 1H NMR
d
(CDCl3) 1.05 (1.65H, t, J4 ,3 ¼ 7.0 Hz, H-4 ), 1.30
0
0
0
0
0
0
(1.65H, t, J ¼ 7.3 Hz, SCH2CH3), 2.18 (1.65H, q, J3 ,4 ¼7.5 Hz, H-3 ),
2.90 (2H, q, J ¼ 7.5 Hz, SCH2CH3), 3.47 (1.3H, s, H-10), 7.16, 7.12
(2.20H, 2d, J ¼ 8.0 Hz, J ¼ 8.0 Hz, H-2, H-3, H-5, H-6), 9.29 (0.55H, s,
OH). 13C NMR ppm (CDCl3) 10.0, 14.3, 20.5, 27.9, 39.5, 129.5*, 134.3,
4.1.10.1. 2-Amino-1-(4-methylthiophenyl)butane (19a). Compound
19a was prepared from 18 and ammonium acetate according to the
general method A. Colourless solid (53%), mp 109–110 ꢀC. IR nmax
134.7, 160.2. MS m/z 223 (Mþ). Syn–(16b) 1H NMR
d (CDCl3) 0.99
0
0
0
(1.35H, t, J4 ,3 ¼ 7.5 Hz, H-4 ), 1.29 (1.35H, t, J ¼ 7.3 Hz, SCH2CH3),
2.31 (1.35H, q, J3 ,4 ¼ 7.7 Hz, H-3 ), (SCH2CH3 signal overlaps with
anti-(16b)), 3.70 (0.7H, s, H-10) 7.15, 7.27 (1.80H, 2d, J ¼ 8.5 Hz,
J ¼ 8.0 Hz, H-2, H-3, H-5, H-6), 9.20 (1H, s, OH). 13C NMR ppm
(CDCl3) 10.5, 14.3, 20.5, 27.0, 33.0, 129.5, 134.4, 134.5, 161.6. MS m/z
223 (Mþ). Anal. (syn/anti isomer mixture) calculated for C12H17NOS:
C, 64.53; H, 7.67; N, 6.27. Found: C, 64.75; H, 7.73; N, 6.33%.
(film) 3360, 3292, 1597, 1094 cmꢃ1 1H NMR
. d (CDCl3) 0.97 (3H, t,
0
0
0
J ¼ 7.5 Hz, CHCH2CH3), 1.17 (2H, br s, NH2), 1.36 (1H, m, CHCH2CH3),
1.52 (1H, m, CHCH2CH3), 2.41 (1H, dd, J ¼ 8.5 Hz, J ¼ 13.6 Hz,
ArCH2CH), 2.47 (3H, s, SCH3), 2.75 (1H, dd, J ¼ 4.5 Hz, J ¼ 13.5 Hz,
ArCH2CH), 2.88 (1H, m, ArCH2CH), 7.12 (2H, d, J ¼ 8.0 Hz, ArH), 7.21
(2H, d, J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3) 10.5, 16.2, 30.3, 43.6,
54.1, 127.1, 129.7, 135.7, 136.8. MS m/z 195 (Mþ). HCl salt. Colourless
solid, mp 154–155 ꢀC (ethanol/hexane) [27]. Anal. Calculated for
C11H18ClNS: C, 57.00; H, 7.83; N, 6.04; S, 13.83. Found: C, 56.90; H,
7.69; N, 5.78; S, 12.88%.
4.1.9.13. 2-N-Hydroxyamino-1-(4-ethylthiophenyl)propane (15c).
Compound 15c was prepared from imine 16a (0.80 g, 3.82 mmol
scale) according to the general method D and chromatographed on
silica gel (eluent: hexane/diethylether 60:40). Colourless solid
(86%), mp 78–79 ꢀC (ethylacetate/hexane). IR nmax (KBr) 3254, 3120
4.1.10.2. 2-N-Methylamino-1-(4-methylthiophenyl)butane
(19b).
(NH, OH) cmꢃ1. 1H NMR
d
(CDCl3) 1.08 (3H, d, J3 ,2 ¼ 6.5 Hz, H-3 ),
Compound 19b was prepared from 18 and methylamine HCl
according to the general method A. Colourless oil (77%). IR nmax
0
0
0
1.30 (3H, t, J ¼ 7.5 Hz, SCH2CH3), 2.59 (1H, dd, Jgem ¼ 13.3 Hz,
J1 ,2 ¼ 6.5 Hz, H-1 ), 2.92 (2H, q, 0J ¼ 7.5 Hz, SCH2CH3), 2.83 (1H, dd,
(film) 3331, 2788, 1599, 1091 cmꢃ1 1H NMR
. d (CDCl3) 0.93 (3H, t,
0
0
0
0
0
0
Jgem ¼ 13.3 Hz, J1 ,2 ¼ 6.8 Hz, H-1 ), 3.17 (1H, m, H-2 ), 5.18, 6.87 (2H,
2br s, OH, NH), 7.11, 7.27 (4H, 2d, J ¼ 8.0 Hz, J ¼ 7.5 Hz, H-2, H-3, H-5,
H-6). 13C NMR ppm (CDCl3) 14.8, 24.2, 28.1, 37.2, 64.3, 129.6, 129.8,
134.2, 136.9. m/z 211 (Mþ). Anal. Calculated for C11H17NOS: C, 62.52;
H, 8.11; N, 6.63. Found: C, 62.22; H, 7.82; N, 6.54%. HCl salt.
Colourless solid, mp 100–101 ꢀC (ethanol/hexane). IR nmax (KBr)
2542 (NHþ) cmꢃ1. Anal. Calculated for C11H18ClNOS: C, 53.22; H,
7.32; N, 5.65; S, 12.94. Found: C, 53.27; H, 7.17; N, 5.60; S, 13.09%.
J ¼ 7.5 Hz, CHCH2CH3), 1.36–1.52 (2H, m, CHCH2CH3), 1.55 (1H, br s,
NH), 2.37 (3H, s, NCH3), 2.47 (3H, s, SCH3), 2.53–2.72 (3H, m,
ArCH2CH), 7.11 (2H, d, J ¼ 8.0 Hz, ArH), 7.20 (2H, d, J ¼ 8.0 Hz, ArH).
13C NMR ppm (CDCl3) 9.75, 16.14, 25.48, 33.69, 39.23, 62.05, 127.04,
129.74, 135.67, 136.70. MS m/z 209 (Mþ þ 1). HCl salt. Colourless
solid, mp 128–129 ꢀC (ethanol/hexane). Anal. calculated for
C12H20ClNS: C, 58.53; H, 8.20; N, 5.70; S, 13.04. Found: C, 58.50; H,
8.22; N, 5.78; S, 12.75%.
4.1.9.14. 2-N-Hydroxyamino-1-(4-ethylthiophenyl)butane
(15h).
4.1.10.3. 2-N-Ethylamino-1-(4-methylthiophenyl)butane
(19c).
Compound 15h was prepared from imine 16b (0.80 g, 3.58 mmol
scale) according to the general method D and chromatographed on
silica gel (eluent: hexane/diethylether 65:35). Colourless oil (73%).
Compound 19c was prepared from 18 and ethylamine HCl
according to the general method A. Colourless oil (28%). IR nmax
(film) 3266, 2872, 1602 cmꢃ1 1H NMR
. d (CDCl3) 0.92 (3H, t,
IR nmax (film) 3249 (NH, OH) cmꢃ1
.
1H NMR
d
(CDCl3) 0.96 (3H, t,
J ¼ 7.5 Hz, CHCH2CH3), 1.04 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 1.26 (1H, br
s, NH), 1.37–1.49 (2H, m, CHCH2CH3), 2.47 (3H, s, SCH3), 2.53–2.70
(5H, m, NCH2CH3, ArCH2CH), 7.11 (2H, d, J ¼ 8.0 Hz, ArH), 7.20 (2H,
d, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 9.8, 15.4, 16.1, 26.1, 39.7,
41.3, 60.3, 127.0, 129.7, 135.5, 136.8. HCl salt. Colourless solid, mp
130–132 ꢀC (ethanol/hexane). Anal. Calculated for C13H22ClNS: C,
60.09; H, 8.53; N, 5.39; S, 12.34. Found: C, 59.79; H, 8.53; N, 5.45; S,
12.28%.
0
0
0
J4 ,3 ¼ 7.3 Hz, H-4 ), 1.30 (3H, t, J ¼ 7.0 Hz, SCH2CH3), 1.45 (1H, m, H-
30), 1.56 (1H, m, H-30), 2.71 (1H, dd, Jgem ¼ 13.5 Hz, J1 ,2 ¼ 6.0 Hz, H-
0
0
0
10), 2.76 (1H, dd, Jgem ¼ 13.5 Hz, J1 ,2 ¼ 7.5 Hz, H-1 ), 2.92 (2H, q,
J ¼ 7.0 Hz, SCH2CH3), 2.90 (1H, m, H-20), 5.24, 6.23 (2H, 2br s, OH,
NH), 7.13, 7.27 (4H, 2d, J ¼ 8.5 Hz, J ¼ 8.0 Hz, H-2, H-3, H-5, H-6). 13C
NMR ppm (CDCl3) 10.4, 14.4, 24.1, 28.0, 36.9, 64.2, 129.6, 129.8,
134.1, 136.7. MS m/z 225 (Mþ). HCl salt. Colourless solid, mp 100–
0
0

4882
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4.1.10.4. 2-N-(n-Propyl)amino-1-(4-methylthiophenyl)butane
(19d). Compound 19d was prepared from 18 and n-propylamine
HCl according to the general method A. Colourless oil (26%). IR nmax
7.12 (2H, d, J ¼ 8.5 Hz, ArH), 7.20 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR
ppm (CDCl3) 9.8, 16.2, 26.0, 39.7, 46.5, 58.6, 60.4, 72.1, 127.0, 129.7,
135.6, 136.7. HCl salt. Colourless solid, mp 112–114 ꢀC (ethanol/
hexane). Anal. Calculated for C14H24ClNOS: C, 58.01; H, 8.35; N,
4.83; S, 11.06. Found: C, 57.98; H, 8.20; N, 4.75; S, 10.69%.
(film) 3323, 1600, 1093 cmꢃ1 1H NMR
. d (CDCl3) 0.92 (3H, t,
J ¼ 7.5 Hz, CHCH2CH3), 1.04 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 1.26 (1H, br
s, NH), 1.37–1.49 (2H, m, CHCH2CH3), 2.47 (3H, s, SCH3), 2.53–2.70
(5H, m, NCH2CH3, ArCH2CH), 7.11 (2H, d, J ¼ 8.0 Hz, ArH), 7.20 (2H, d,
J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 9.8, 15.4, 16.1, 26.1, 39.7, 41.3,
60.3, 127.0, 129.7, 135.5, 136.8. HCl salt. Colourless solid, mp 134–
135 ꢀC (ethanol/hexane). Anal. Calculated for C14H24ClNS: C, 61.40;
H, 8.83; N, 5.11; S, 11.71. Found: C, 60.90; H, 8.69; N, 5.00; S, 12.30%.
4.1.10.9. 2-N-Allylamino-1-(4-methylthiophenyl)butane
(19i).
Compound 19i was prepared from 18 and allylamine HCl
according to the general method A. Colourless oil (33%). IR nmax
(film) 3342, 1641, 1493, 1092 cmꢃ1 1H NMR
. d (CDCl3) 0.93 (3H, t,
J ¼ 7.5 Hz, CHCH2CH3), 1.37–1.48 (2H, m, CHCH2CH3), 1.56 (1H, br
s, NH), 2.47 (3H, s, SCH3), 2.61–2.76 (3H, m, ArCH2CH), 3.20 (1H,
dd, J ¼ 6.0 Hz, 14.0 Hz, NCH2CHCH2), 3.27 (1H, dd, J ¼ 5.5 Hz,
14.4 Hz, NCH2CHCH2), 5.05 (1H, dd, J ¼ 1.5 Hz, J ¼ 10.3 Hz,
NCH2CHCH2), 5.11 (1H, d, J ¼ 1.5 Hz, J ¼ 17.0 Hz, NCH2CHCH2), 5.83
(1H, m, NCH2CHCH2), 7.11 (2H, d, J ¼ 8.5 Hz, ArH), 7.20 (2H, d,
J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3) 9.8, 16.1, 25.9, 39.5, 49.6,
59.5, 115.7, 126.9, 129.8, 135.6, 136.6, 136.9. HCl salt. Colourless
solid, mp 127–128 ꢀC (ethanol/hexane). Anal. Calculated for
C14H22ClNS: C, 61.85; H, 8.16; N, 5.15; S, 11.80. Found: C, 61.81; H,
8.12; N, 5.20; S, 11.73%.
4.1.10.5. 2-N-(Isopropyl)amino-1-(4-methylthiophenyl)butane
(19e). Compound 19e was prepared from 18 and isopropylamine
HCl according to the general method A. Colourless oil (15%). IR nmax
(film) 3323, 1600, 1093 cmꢃ1 1H NMR
. d (CDCl3) 0.91 (3H, t,
J ¼ 7.5 Hz, CHCH2CH3), 0.94 (3H, d, J ¼ 6.5 Hz, NCH(CH3)2), 1.02 (3H,
d, J ¼ 6.5 Hz, NCH(CH3)2), 1.11 (1H, br s, NH), 1.28–1.43 (2H, m,
CHCH2CH3), 2.47 (3H, s, SCH3), 2.59 (1H, dd, J ¼ 7.5 Hz, J ¼ 13.8 Hz,
ArCH2CH), 2.65 (1H, dd, J ¼ 7.0 Hz, J ¼ 13.8 Hz, ArCH2CH), 2.73 (1H,
m, ArCH2CH), 2.85 (1H, m, NCH(CH3)2), 7.10 (2H, d, J ¼ 8.0 Hz, ArH),
7.19 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 9.8, 16.1, 23.1,
23.5, 26.6, 40.1, 45.6, 57.2, 126.8, 129.7, 135.4, 136.7. HCl salt. Col-
ourless crystals, mp 150–151 ꢀC (ethanol/hexane). Anal. Calculated
for C14H24ClNS: C, 61.40; H, 8.83; N, 5.11; S, 11.71. Found: C, 61.28; H,
8.93; N, 5.08; S, 12.04%.
4.1.10.10. 2-(N-2-Hydroxyethyl)amino-1-(4-methylthiophenyl)butane
(19j). Compound 19j was prepared from 18 and 2-hydroxyethyl-
amine HCl according to the general method A. Colourless oil (43%). IR
nmax (film) 3304, 2873, 1092 cmꢃ1 1H NMR
. d (CDCl3) 0.93 (3H, t,
J ¼ 7.5 Hz, CHCH2CH3), 1.40–1.47 (2H, m, CHCH2CH3), 2.14 (2H, br s,
NH, NCH2CH2OH), 2.46 (3H, s, SCH3), 2.58–2.78 (5H, m, ArCH2CH,
ArCH2CH, NCH2CH2OH), 3.54 (2H, m, NCH2CH2OH), 7.10 (2H, d,
J ¼ 8.0 Hz, ArH), 7.19 (2H, d, J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3)
9.9, 16.1, 26.3, 39.8, 48.2, 60.0, 61.1, 126.9, 129.6, 135.7, 136.5. HRMS
Calculated for C13H22NOS: (Mþ) 240.1422, Found 240.1432.
4.1.10.6. 2-N-Cyclopropylamino-1-(4-methylthiophenyl)butane
(19f). Compound 19f was prepared from 18 and cyclopropylamine
HCl according to the general method A. The resulting residue was
purified by flash chromatography (eluent: diethylether). Colourless
oil (30%). IR nmax (film) 3290, 1493, 1093 cmꢃ1 1H NMR
. d (CDCl3)
0.26 (2H, m, NCH(CH2)2), 0.42 (2H, m, NCH(CH2)2), 0.92 (3H, t,
J ¼ 7.5 Hz, CHCH2CH3), 1.38–1.57 (2H, m, CHCH2CH3), 1.63 (1H, br s,
NH), 2.03 (1H, m, NCH(CH2)2), 2.47 (3H, s, SCH3), 2.68 (2H, d,
J ¼ 7.0 Hz, ArCH2CH), 2.80 (1H, m, ArCH2CH), 7.12 (2H, d, J ¼ 8.5 Hz,
ArH), 7.20 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 6.1, 7.0, 9.9,
16.1, 26.2, 28.6, 39.8, 61.0, 126.9, 129.7, 135.5, 136.9. HCl salt. Col-
ourless solid, mp 136–137 ꢀC (ethanol/hexane). Anal. Calculated for
C14H22ClNS: C, 61.85; H, 8.16; N, 5.15; S, 11.80. Found: C, 61.87; H,
8.11; N, 5.31; S, 12.19%.
4.1.10.11. 2-N,N-Dimethylamino-1-(4-methylthiophenyl)butane (19k).
Compound 19k was prepared from 18 and dimethylamine HCl
according to the general method A. Colourless oil (16%). IR nmax
(film) 3072, 2817, 1600, 1094 cmꢃ1 1H NMR
. d (CDCl3) 0.84 (3H, t,
J ¼ 7.5 Hz, CHCH2CH3), 1.28–1.47 (2H, m, CHCH2CH3), 2.30 (6H, s,
N(CH3)2), 2.32 (1H, dd, J ¼ 9.0 Hz, J ¼ 13.3 Hz, ArCH2CH), 2.46 (3H,
s, SCH3), 2.50 (1H, m, ArCH2CH), 2.86 (1H, dd, J ¼ 4.5 Hz,
J ¼ 13.3 Hz, ArCH2CH), 7.09 (2H, d, J ¼ 8.5 Hz, ArH), 7.19 (2H, d,
J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3) 11.4, 16.3, 23.0, 34.0, 40.5,
67.7, 127.1, 129.6, 138.4, 139.4. HCl salt. Colourless solid, mp
150–153 ꢀC (ethanol/hexane). Anal. Calculated for C13H22ClNS; C,
60.09; H, 8.53; N, 5.39; S, 12.34. Found: C, 59.57; H, 8.42; N, 5.26;
S, 12.00%.
4.1.10.7. 2-N-Propargylamino-1-(4-methylthiophenyl)butane (19g).
Compound 19g was prepared from 18 and propargylamine HCl
according tothe general method A. Colourless oil (26%). IR nmax (film)
3290, 1599, 1091 cmꢃ1
.
1H NMR
d
(CDCl3) 0.93 (3H, t, J ¼ 7.5 Hz,
CHCH2CH3), 1.40–1.48 (2H, m, CHCH2CH3), 1.49 (1H, br s, NH), 2.16
(1H, m, NCH2CCH), 2.47 (3H, s, SCH3), 2.60 (1H, dd, J ¼ 7.5 Hz,
J ¼ 13.6 Hz, ArCH2CH), 2.70 (1H, dd, J ¼ 6.0 Hz, J ¼ 13.6 Hz,
ArCH2CH), 2.93 (1H, m, ArCH2CH), 3.40 (2H, d, J ¼ 2.5 Hz, NCH2CCH),
7.13 (2H, d, J ¼ 8.5 Hz, ArH), 7.20 (2H, d, J ¼ 8.5 Hz, ArH). 13C NMR
ppm (CDCl3) 9.5, 16.1, 25.6, 35.6, 39.4, 58.2, 71.1, 82.1, 127.0, 129.7,
135.8, 136.2. HCl salt. Orange solid, mp 111–113 ꢀC (dec.) (ethanol/
hexane). Anal. Calculated for C14H20ClNS: C, 62.32; H, 7.47; N, 5.19; S,
11.88. Found: C, 61.23; H, 7.35; N, 5.53; S, 12.33%.
4.1.10.12. 2-(N-Methoxy-N-methyl)amino-1-(4-methylthiophenyl)
butane(19l). Compound 19l was prepared from 18 and N-methoxy-
N-methylamine HCl according to the general method A. The
resulting residue was purified by flash chromatography (eluent:
hexane/diethylether 60:40). Colourless oil (68%). IR nmax (film)
3073, 1600, 1493, 1096 cmꢃ1 1H NMR
. d (CDCl3) 0.88 (3H, t,
J ¼ 7.5 Hz, CHCH2CH3), 1.37–1.49 (2H, m, CHCH2CH3), 2.46 (3H,
s, SCH3), 2.49 (1H, dd, J ¼ 8.5 Hz, J ¼ 13.6 Hz, ArCH2CH), 2.60 (3H, s,
NCH3), 2.72 (1H, m, ArCH2CH), 3.01 (1H, dd, J ¼ 4.5 Hz, J ¼ 13.6 Hz,
ArCH2CH), 2.50 (3H, s, OCH3), 7.12 (2H, d, J ¼ 8.0 Hz, ArH), 7.19 (2H,
d, J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3) 10.7, 16.2, 22.7, 34.6, 40.1,
59.7, 69.5, 127.0, 129.7, 135.2, 137.9. HCl salt. Colourless solid, mp
115–116 ꢀC (ethanol/hexane). Anal. Calculated for C13H22ClNOS: C,
56.61; H, 8.04; N, 5.08; S, 11.62. Found: C, 56.91; H, 8.03; N, 5.05; S,
11.56%.
4.1.10.8. 2-N-(2-Methoxyethyl)amino-1-(4-methylthiophenyl)butane
(19h). Compound 19h was prepared from 18 and 2-methox-
yethylamine HCl according to the general method A. The resulting
residue was purified by flash chromatography (eluent: dieth-
ylether/methanol 90:10). Pale amber oil (39%). IR nmax (film) 3327,
1599, 1092 cmꢃ1
.
1H NMR
d
(CDCl3) 0.93 (3H, t, J ¼ 7.5 Hz,
CHCH2CH3), 1.37–1.45 (2H, m, CHCH2CH3), 1.74 (1H, br s, NH), 2.47
(3H, s, SCH3), 2.63–2.69 (2H, m, NCH2CH2O), 2.70–2.81 (3H, m,
ArCH2CH), 3.29 (3H, s, OCH3), 3.44 (2H, t, J ¼ 5.5 Hz, NCH2CH2O),
4.1.10.13. 2-N-hydroxy-N-methylamino-1-(4-methylthiophenyl)
butane (19n). Compound19n was prepared from18 andN-hydroxy-

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4883
N-methylamine HCl according to the general method A. The
resulting residue was purified by flash chromatography (eluent:
hexane/diethylether 60:40). Colourless oil (51%). IR nmax (film) 3090,
ArCH2C), 4.59 (2H, d, J ¼ 5.5 Hz, NOCH2CHCH2), 5.20 (1H,
m, NOCH2CHCH2), 5.30 (1H, m, NOCH2CHCH2), 6.01 (1H, m,
NOCH2CHCH2), 7.13*, 7.16 (2H, 2d, J ¼ 8.5 Hz, J ¼ 8.5 Hz, ArH), 7.18*,
7.20 (2H, d, J ¼ 8.0 Hz, J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3) 10.2,
11.04*, 16.0*, 16.0, 21.0, 26.9*, 33.3*, 39.4, 74.2*, 74.2, 116.7, 116.9*,
126.9, 127.0*, 129.4*, 129.4, 133.8*, 133.9, 134.5, 134.6*, 136.0*,
136.5, 159.9*, 161.2. HRMS Calculated for C14H20NOS: (Mþ þ 1)
250.1266: Found: 250.1260.
1599, 1493 cmꢃ1
.
1H NMR
d
(CDCl3) 0.87 (3H, t, J ¼ 7.5 Hz,
CHCH2CH3), 1.38–1.55 (2H, m, CHCH2CH3), 2.46 (3H, s, SCH3), 2.51
(1H, dd, J ¼ 9.0 Hz, J ¼ 13.6 Hz, ArCH2CH), 2.66 (3H, s, NCH3), 2.78
(1H, m, ArCH2CH), 3.11 (1H, dd, J ¼ 4.5 Hz, J ¼ 13.6 Hz, ArCH2CH), 7.11
(2H, d, J ¼ 8.0 Hz, ArH), 7.19 (2H, d, J ¼ 8.0 Hz, ArH), 7.66 (1H, s, NOH).
13C NMR ppm (CDCl3) 10.5, 16.2, 22.6, 34.3, 43.1, 70.8, 127.0, 129.6,
135.3, 137.5. HCl salt. Colourless solid, mp 115–116 ꢀC (ethanol/
hexane). Anal. Calculated for C12H20ClNOS; C, 55.05; H, 7.70; N, 5.35;
S, 12.25. Found: C, 54.98; H, 7.67; N, 5.42; S, 13.37%.
4.1.10.18. 2-N-Hydroxyamino-1-(4-methylthiophenyl)butane
(19m). Compound 19m was prepared from oxime 20a according to
the general method D. Colourless solid (30%), mp 50–51 ꢀC
(hexane). IR nmax (film) 3253, 2863, 1493, 1092 cmꢃ1 1H NMR
.
4.1.10.14. 1-(4-Methylthiophenyl)-2-butanone
oxime
(20a).
d
(CDCl3) 0.95 (3H, t, J ¼ 7.5 Hz, CHCH2CH3), 1.38–1.62 (2H, m,
Compound 20a was prepared from 18 (2.50 g, 12.87 mmol scale)
according to the general method C without the need for flash
chromatography, generating a 60:40 mixture of syn/anti isomers.
Colourless solid (86%), mp 77–79 ꢀC (ethanol/water). Rf 0.36
(dichloromethane/hexane 80:20). IR nmax (KBr) 3220 (OH), 1669
CHCH2CH3), 2.47 (3H, s, SCH3), 2.70 (1H, dd, J ¼ 6.0 Hz, J ¼ 13.6 Hz,
ArCH2CH), 2.76 (1H, dd, J ¼ 7.0 Hz, J ¼ 13.6 Hz, ArCH2CH), 2.92 (1H,
m, ArCH2CH), 5.27 (1H, s, NH), 6.52 (1H, s, NOH), 7.12 (2H, d,
J ¼ 8.0 Hz, ArH), 7.20 (2H, d, J ¼ 8.5 Hz, ArH). 13C NMR ppm (CDCl3)
10.4, 16.1, 24.0, 36.8, 64.2, 127.1, 129.8, 135.8, 135.9. HCl salt. Col-
ourless crystals, mp 114–115 ꢀC (ethanol/hexane). Anal. Calculated
for C11H18ClNOS: C, 53.22; H, 7.32; N, 5.65; S, 12.94. Found: C, 53.18;
H, 7.11; N, 5.69; S, 12.88%.
(C]N) cmꢃ1. syn-(20a). 1H NMR
d
(CDCl3) 1.04 (1.8H, t, J4 ,3 ¼ 7.5 Hz,
0
0
H-40), 2.18 (1.2H, q, J3 ,4 ¼ 7.5 Hz, H-3 ), 2.45 (3H, s, SCH3), 3.70 (1.2H,
s, H-10), 7.13–7.21 (4H, m, H-2, H-3, H-5, H-6), OH signal not visible.
13C NMR ppm (CDCl3) 10.6, 16.0, 27.0, 32.9, 127.1, 128.5, 133.6, 136.1,
0
0
0
160.4. anti-(20a). 1H NMR
d
(CDCl3) 0.99 (1.2H, t, J4 ,3 ¼ 7.5 Hz, H-4 ),
4.1.10.19. 2-N-Methoxyamino-1-(4-methylthiophenyl)butane (19o).
Compound 19o was prepared from imine (20b) according to
the general method D. Colourless oil (87%). IR nmax (film) 3241,
0
0
0
0
0
0
2.31 (0.8H, q, J3 ,4 ¼ 7.7 Hz, H-3 ), SCH3 signal overlapping with syn-
(20a), 3.46 (0.8H, s, H-10), H-2, H-3, H-5, H-6 signals overlapping
with syn-(20a), OH signal not visible. 13C NMR ppm (CDCl3) 10.0,
16.0, 20.4, 39.4, 127.0, 129.5, 133.6, 136.6, 161.7. m/z (syn/anti isomer
mixture) 209 (Mþ, 69%). Anal. calculated for C11H15NOS: C, 63.12; H,
7.22; N, 6.69. Found: C, 63.46; H, 7.11; N, 6.50%.
2806, 1600, 1095 cmꢃ1
. d (CDCl3) 0.97 (3H, t,
1H NMR
J ¼ 7.5 Hz, CHCH2CH3), 1.38–1.59 (2H, m, CHCH2CH3), 2.46 (3H,
s, SCH3), 2.67 (1H, dd, J ¼ 6.0 Hz, J ¼ 13.6 Hz, ArCH2CH), 2.71
(1H, dd, J ¼ 7.0 Hz, J ¼ 13.6 Hz, ArCH2CH), 2.93 (1H, m,
ArCH2CH), 3.52 (3H, s, OCH3), 5.50 (1H, br s, NH), 7.13 (2H, d,
J ¼ 8.5 Hz, ArH), 7.20 (2H, d, J ¼ 8.5 Hz, ArH). 13C NMR ppm
(CDCl3) 10.4, 16.1, 24.3, 37.1, 62.2, 63.2, 127.0, 129.8, 135.8,
136.0. HRMS Calculated for C12H20NOS: (Mþ þ 1) 226.1266:
Found: 226.1274.
4.1.10.15. 2-N-Methoxyimino-1-(4-methylthiophenyl)butane (20b).
Compound 20b was prepared from 18 and methoxylamine HCl
according to the general method C as a 60:40 mixture of isomers.
Pale amber oil (96%). IR nmax (film) 2897, 2814, 1598, 1093 cmꢃ1. 1H
NMR
d
(CDCl3) 0.93, 1.03* (3H, 2t, J ¼ 7.5 Hz, J ¼ 7.5 Hz, CCH2CH3),
2.13*, 2.20 (2H, 2q, J ¼ 7.5 Hz, J ¼ 7.5 Hz, CCH2CH3), 2.45*, 2.46 (3H,
2s, SCH3), 3.42, 3.63* (2H, 2s, ArCH2C), 3.87, 3.88* (3H, 2s, OCH3),
7.11*, 7.16 (2H, 2d, J ¼ 8.0 Hz, J ¼ 8.0 Hz, ArH), 7.18, 7.20* (2H, 2d,
4.1.10.20. 2-N-Ethoxyamino-1-(4-methylthiophenyl)propane (19p).
Compound 19p was prepared from imine 20c according to the
general method D. Colourless oil (93%). IR nmax (film) 3238, 2866,
13
J ¼ 8.0 Hz, J ¼ 8.0 Hz, ArH). C NMR ppm (CDCl3) 10.2, 11.0*, 15.9,
1599, 1092 cmꢃ1. 1H NMR
d
(CDCl3) 1.05 (3H, d, J ¼ 6.5 Hz, CHCH3),
16.0*, 20.7, 26.9*, 33.1*, 39.4, 61.1*, 61.2, 126.8, 127.0*, 129.4*, 129.4,
133.7, 133.9*, 136.0*, 136.5, 159.6*, 160.9. HRMS Calculated for
C12H18NOS: (Mþ þ 1) 224.1109: Found: 224.1105.
1.16 (3H, t, J ¼ 7.0 Hz, NOCH2CH3), 2.46 (3H, s, SCH3), 2.55 (1H, dd,
J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 2.78 (1H, dd, J ¼ 7.0 Hz, 13.5 Hz,
ArCH2CH), 3.19 (1H, m, ArCH2CH), 3.73 (2H, q, J ¼ 7.0 Hz,
NOCH2CH3), 5.34 (1H, s, NH), 7.11 (2H, d, J ¼ 8.0 Hz, ArH), 7.20 (2H,
d, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 14.1, 16.1, 17.7, 39.7, 57.2,
69.7, 127.0, 139.7, 135.8, 135.8. HRMS Calculated for C13H22NOS:
(Mþ þ 1) 240.1422, Found: 240.1430.
4.1.10.16. 2-N-Ethoxyimino-1-(4-methylthiophenyl)butane
(20c).
Compound 20c was prepared from 18 and ethoxylamine HCl
according to the general method C as a 60:40 mixture of isomers.
Pale amber oil (95%). IR nmax (film) 2876, 1630, 1493, 1091 cmꢃ1. 1H
NMR
d
(CDCl3) 0.93, 1.03* (3H, 2t, J ¼ 7.5 Hz, J ¼ 7.5 Hz, CCH2CH3),
4.1.10.21. 2-(N-(O-Allyl))amino-1-(4-methylthiophenyl)propane (19q).
Compound 19q was prepared from imine 20d according to the
general method D. Colourless oil (89%). IR nmax (film) 3243, 2856,
1.27 (3H, t, J ¼ 7.0 Hz, NOCH2CH3), 2.14*, 2.20 (2H, q, J ¼ 7.5 Hz,
J ¼ 7.5 Hz, CCH2CH3), 2.45, 2.46* (3H, 2s, SCH3), 3.42, 3.64* (2H, 2s,
ArCH2C), 4.11, 4.12* (2H, 2q, J ¼ 7.0 Hz, NOCH2CH3), 7.12*, 7.15 (2H, 2d,
J ¼ 8.0 Hz, J ¼ 8.5 Hz, ArH), 7.18*, 7.20 (2H, d, J ¼ 8.0 Hz, J ¼ 8.5 Hz,
ArH). 13C NMR ppm (CDCl3) 10.2, 11.0*, 14.6, 14.6*, 16.0, 16.1*, 20.9,
27.0*, 33.2*, 39.5, 68.7, 68.8*,126.9,127.0*,129.4,129.4*,134.0*,134.1,
136.0*, 136.4, 159.2*, 160.5. HRMS Calculated for C13H20NOS:
(Mþ þ 1) 238.1266: found: 238.1267.
1599, 1094 cmꢃ1. 1H NMR
d
(CDCl3) 1.06 (3H, d, J ¼ 6.5 Hz, CHCH3),
2.46 (3H, s, SCH3), 2.56 (1H, dd, J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 2.80
(1H, dd, J ¼ 6.5 Hz, 13.5 Hz, ArCH2CH), 3.22 (1H, m, ArCH2CH), 4.20
(2H, d, J ¼ 6.0 Hz, NOCH2CHCH2), 5.17 (1H, d, J ¼ 10.6 Hz,
NOCH2CHCH2), 5.26 (1H, dd, J ¼ 1.5 Hz, J ¼ 17.3 Hz, NOCH2CHCH2),
5.43 (1H, s, NH), 5.93 (1H, m, NOCH2CHCH2), 7.11 (2H, d, J ¼ 8.0 Hz,
ArH), 7.20 (2H, d, J ¼ 8.0 Hz, ArH). 13C NMR ppm (CDCl3) 16.1, 17.7,
39.6, 57.3, 75.5, 117.3, 127.0, 129.8, 134.4, 135.8, 135.9. HRMS Calcu-
lated for C14H22NOS: (Mþ þ 1) 252.1422: Found: 252.1411.
4.1.10.17. 2-(N-(O-Allyl))imino-1-(4-methylthiophenyl)butane
(20d). Compound 20d was prepared from 18 and allyloxyamine
HCl according to the general method C as a 60:40 mixture of
isomers. Pale amber oil (91%). IR nmax (film) 2875, 1640, 1493,
4.1.11. 1-(4-Methylsulfanyl-phenyl)-propylamine 25a
1093 cmꢃ1
.
1H NMR
d
(CDCl3) 0.94, 1.03* (3H, 2 t, J ¼ 7.5 Hz,
(i) A solution of 4-methylthiobenzaldehyde (10 g) in dry dieth-
ylether (20 mL) was added dropwise to a solution of ethyl-
magnesium bromide (prepared by the dropwise addition of
J ¼ 7.5 Hz, CCH2CH3), 2.14*, 2.23 (2H, 2q, J ¼ 7.5 Hz, J ¼ 7.5 Hz,
CHCH2CH3), 2.45*, 2.46 (3H, 2s, SCH3), 3.43, 3.67* (2H, 2s,

4884
S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
bromoethane (7.30 mL) to stirred Mg turnings (2.588 g) in 75 mL of
dry diethylether under N2 atmosphere), and the mixture was
subsequently refluxed for 3 h. The reaction was quenched by the
addition of saturated ammonium chloride (200 mL). The organic
phase was separated and washed with 10% HCl (3 ꢂ 75 mL). The
solvent was dried over anhydrous sodium sulphate and removed
under vacuum to give 1-(4-methylsulfanyl-phenyl)-propan-1-ol 21
a clear yellow oil (29%) which was used without further purification
dropwise addition of propionyl chloride (0.361 mL, 4.16 mmol).
After the addition of the acid chloride the reaction was allowed to
warm to ambient temperature and stirred for a further 3 h. The
reaction mixture was diluted with DCM (200 mL) and poured
slowly into 100 mL of ice water. The organic phase was washed
with water (6 ꢂ 100 mL). The organic layer was dried over anhy-
drous Na2SO4, filtered and the solvent removed under vacuum. The
solid formed was recrystallised from hexane/ether to yield 1-(4-
pentylsulfanyl-phenyl)-propan-1-one 23 as colourless needles
(94%), which was used without further purification in the next
in the next reaction. m/z 183 (Mþ þ 1); 1H NMR (CDCl3)
d: 0.91–0.94
(t, J ¼ 7.5 Hz, 3H, CH2CH3), 1.65–1.90 (m, 2H, CH2CH3), 2.50 (s, 3H,
SCH3), 4.56–4.60 (t, J ¼ 7.0 Hz, 1H, CHOH), 7.25–7.30 (m, 4H, ArH).
reaction. IR nmax film: 2931,1677,1592, 822 cmꢃ1. 1H NMR (CDCl3)
d:
13C NMR (CDCl3)
d
: 10.21, 30.11, 33.18, 72.80, 126.21, 127.41, 133.42,
0.90–0.94 (t, J ¼ 7.0 Hz, 3H, COCH2CH3), 1.21–1.24 (t, J ¼ 7.5 Hz, 3H,
CH3C4H8), 1.32–1.48 (m, 4H, CH3CH2CH2) 1.68–1.75 (m, 2H,
CH2CH2S), 2.94–3.01 (m, 4H, CH2SAr, COCH2), 7.29–7.31 (d,
J ¼ 8.5 Hz, 2H, ArH), 7.86–7.89 (d, J ¼ 8.5 Hz, 2H, ArH). 13C NMR
143.39. (ii) To a solution of 21 (18.5 mmol) in dry DCM (50 mL) and
added in one portion a suspension of pyridinium chlorochromate
(33.20 g) in dry dichloromethane (50 mL). The reaction mixture
was heated to ensure reflux for 3 h. The solvent was decanted from
the black gum which was triturated with diethylether (5 ꢂ100 mL).
The ethereal portions combined with DCM extract and solvent was
removed under vacuum leaving a dark brown oil which was puri-
fied by flash column chromatography (65:35 hexane/diethylether)
to afford the product 22 as colourless solid (ethanol/hexane, 90%)
which was used without further purification in the next reaction.
m/z: 180, (Mþ); IR nmax film: 1672, 1096, 792 cmꢃ1. 1H NMR (CDCl3)
(CDCl3)
d: 8.25, 13.89, 22.18, 28.38, 30.99, 31.50, 31.87, 126.19,
128.34, 133.36, 144.53, 199.81. (iii) To a solution of 24 (0.270 g,
1.139 mmol) in dry DCM (50 mL) under a nitrogen atmosphere was
added hexamethyldisilazane (0.95 mL, 4.50 mmol) followed by
TiCl4 (0.25 mL, 2.278 mmol) and the reaction mixture was allowed
to stir for 20 h after which time NaCNBH3 (0.50 g, 8.1 mmol) dis-
solved in freshly dried methanol was added dropwise. The reaction
mixture was poured slowly over crushed ice. 200 mL of 15% NaOH
was added and the mixture was extracted with DCM (5 ꢂ100 mL).
The solvent was dried over anhydrous sodium sulphate and
removed under vacuum resulting in a residue that was purified by
flash chromatography over silica gel (50:50 methanol/diethylether)
to afford the product as an oil which solidified (48%). IR nmax (film)
d
: 1.22–1.26 (t, J ¼ 7.0 Hz, 3H, CH2CH3), 1.65–1.90 (m, 2H, CH2CH3),
2.53 (s, 3H, SCH3), 7.26–7.29 (d, J ¼ 8.0 Hz, 2H, ArH), 7.88–7.90 (d,
J ¼ 8.0 Hz, 2H, ArH). 13C NMR (CDCl3)
d: 8.28, 14.74, 31.53, 124.93,
128.36, 133.17, 145.44, 199.85. (iii) To a solution of 24 (0.435 g,
2.414 mmol) in dry DCM (50 mL) under a nitrogen atmosphere,
were added hexamethyldisilazane (1.273 mL, 6.035 mmol) and
TiCl4 (0.265 mL, 2.414 mmol) dropwise and the reaction mixture
was allowed to stir for 20 h after which time NaCNBH3 (0.80 g)
dissolved in freshly dried methanol was added dropwise. The
reaction mixture was poured slowly over crushed ice. 200 mL of
15% NaOH was added and the mixture was extracted with DCM
(5 ꢂ100 mL). The solvent was dried over anhydrous sodium
sulphate and removed under vacuum resulting in a residue that
was purified by flash chromatography over silica gel (50:50
methanol/diethylether) to afford the product 1-(4-methylsulfanyl-
cmꢃ1: 3433, 1528, 801. 1H NMR (CDCl3)
d
: 0.78–0.83 (t, J ¼ 7.5 Hz,
3H, CH3), 0.88–0.91 (t, J ¼ 7.0 Hz, 3H, CH3), 1.26–1.44 (m, 4H,
CH2CH2–), 1.61–1.72 (m, 4H, CH2, CH2), 2.81 (s, 2H, NH2) 2.88–2.92
(t, J ¼ 7.5 Hz, 2H, SCH2), 3.73–3.76 (t, J ¼ 7.0 Hz, 1H, CH), 7.21–7.23
(d, J ¼ 8.0 Hz, 2H, ArH), 7.26–7.29 (d, J ¼ 8.0 Hz, 2H, ArH). 13C NMR
(CDCl3)
d: 10.73, 13.87, 22.14, 28.75, 30.89, 31.76, 33.59, 57.12,
126.94, 128.87, 135.27, 142.92. HRMS (EI) found 239.1597; C14H25NS
requires 239.1707.
4.2. Biochemistry
phenyl)-propylamine 25a as a waxy solid (46%). 1H NMR (CDCl3)
d:
0.81 (t, J ¼ 8 Hz, 3H, CH2CH3), 1.80–2.10 (m, 2H, CH2CH3), 2.10 (bs,
2H, NH2), 4.26 (s, 3H, SCH3), 4.00–4.01 (1H, m, CHCH2), 7.21 (d,
J ¼ 6 Hz, 2H, H-2,H-6), 7.39 (d, J ¼ 6 Hz, 2H, H-3, H-5), 13C NMR
4.2.1. Materials
DG-75 and MUTU-I (c179) BL cell lines were gifts from Dr.
Dermot Walls (School of Biotechnology, Dublin City University,
Ireland) and Prof. Martin Rowe (Division of Cancer Studies, The
University of Birmingham, UK) respectively. The SHSY-5Y cell line
was purchased from the European Collection of Cell Cultures
(ECACC Lot04/c/011p17) and the TREx (þrSERT) cell line was
created as in Ref. [84] and a gift from by Dr. Chris Tate (MRC
Laboratory of Molecular Biology, Cambridge, UK). The HEK293 cell
line stably overexpressing the human SERT was a gift from Dr.
Patrick Schloss (Central Institute for Mental Health, Mannheim,
Germany) and the HL-60 cell line was a gift from Dr. D. Zisterer
(School of Biochemistry and Immunology, Trinity College, Dublin).
RPMI-1640, DMEM, FBS, HEPES, sodium pyruvate, gentamycin
(G418) and glutamine were from Gibco (Invitrogen, Biosciences
Ltd., Ireland). Alamar Blue and LymphoPrep were from Biosciences
Ltd. and all other chemicals were purchased through Sigma–
Aldrich Inc., Ireland.
(CDCl3) d: 9.85, 14.84, 27.03, 56.82, 125.94, 127.75, 132.26, 139.21.
HRMS (EI) Found 165.0736; C10H13S requires 165.0738.
4.1.12. 1-(4-Pentylsulfanyl-phenyl)propylamine 25b
(i) A solution of thioanisole (0.497 g, 4.00 mmol) in dry THF
(20 mL) was cooled to ꢃ57 ꢀC and sec-BuLi (5.714 mL (1.4 M in
C6H12), 4.10 mmol) was added dropwise. The solution was stirred
for 1.5 h after which, iodobutane (0.478 mL, 4.00 mmol) was added
dropwise. The reaction mixture was warmed to room temperature
and was stirred for 3.5 h before being poured slowly onto crushed
ice and extracted with DCM (4 ꢂ100 mL). The organic extracts were
combined, dried over anhydrous Na2SO4, filtered and removed in
vacuo. The resulting oil was purified by flash chromatography on
silica gel (95:5 hexane/diethylether) to afford the product 23 as an
oil (76%), which was used without further purification in the next
reaction. m/z 180 (Mþ); IR nmax (film) cmꢃ1: 1514, 729. 1H NMR
(CDCl3)
d
: 0.92–0.96 (t, J ¼ 7.0 Hz, 3H, CH3CH2), 1.31–1.49 (m, 4H,
4.2.2. Cell culture
CH3C2CH2) 1.66–1.73 (m, 2H, CH2CH2S), 2.94–2.97 (t, J ¼ 7.5 Hz, 2H,
The DG-75 cell line is a B-lymphocyte, Burkitt’s lymphoma line
derived from a metastatic pleural effusion (lung) of a sporadic case
of EBV negative Burkitt’s lymphoma and has been that expresses
SERT [62] (Supplemental Fig. 1). The MUTU-I (c179) cell line is an
isogenic stable group I BL cell line derived from a BL biopsy that was
EBV positive and expresses SERT [85] (Supplemental Fig. 1). The
CH2SAr), 7.17–7.21 (m, 1H, ArH), 7.28–7.38 (m, 4H, ArH). 13C NMR
(CDCl3) d: 13.9, 22.2, 28.7, 30.9, 33.4, 125.5, 128.7, 128.7, 137.0. (ii)
Pentylsulfanylbenzene 23 (0.415 g, 2.31 mmol) was dissolved in dry
DCM (30 mL) and the solution was cooled to 0 ꢀC in an ice bath.
AlCl3 (0.401 g, 3.00 mmol) was added, followed by the slow

S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
4885
SHSY-5Y cell line SH-SY5Y is a thrice cloned (SK-N-SH / SH-SY /
SH-SY5 / SH-SY5Y) subline of the human neuroblastoma cell line
SK-N-SH established from a metastatic bone tumour [63]. The
HL-60 cell line is a promyelocytic cell line derived from a 36-year-
old Caucasian female with acute promyelocytic leukaemia [86]. The
above cell lines were cultured in RPMI-1640 medium containing
phenol red and supplemented with 10% (v/v) foetal bovine serum
4.2.5. Neutral Red assay for in vitro cytotoxicity
The Neutral Red assay was carried out as previously described
in Ref. [87]. Briefly, 5 ꢂ104 cells per well (200
ml) were seeded in
a 96-well plate until sub-confluent (24–36 h) and treated with the
appropriate compound for 48 h. Following exposure of cells to
drug the supernatant was removed and the cells incubated for
3 ꢁ 1 h with 250
tions. NR solution was removed carefully and the cells washed
before the addition of 100 l of NR assay solubilisation solution
ml Neutral Red dye solution under sterile condi-
(FBS),
mL). The MUTU-I c179 cell line required the additional supplements
of alpha-thioglycerol (5 mM in PBS with 20 M bathocuprione
L-glutamine (2 mM), penicillin and streptomycin (100 mg/
m
m
(50% ethanol–1% acetic acid solution in dH20). Plates were left to
incubate in the dark for 20–30 min at room temperature with
gentle shaking. The absorbance of each plate was read at 540 nm
and at 690 nm (background) within 1 h. Relative cell viability was
expressed as percent of vehicle treated cells. Sodium azide and
Triton-X were used as positive controls for cytotoxicity, where
30 mM sodium azide and 2% Triton-X resulted in 80–90% cyto-
toxicity on all cell lines. Untreated cells represented 0% cytotoxicity
(100% viability).
disulphonic acid), sodium pyruvate (100 mM) and HEPES (1 mM).
The TREx (ꢃ) and TREx (SERT) cell lines were derived from HEK293
cells stably expressing the TetR protein [84] that turns off rat SERT-
FLAG expression. TREx cells were cultured in DMEM containing 10%
(v/v) Tet-System approved FBS,
(100 g/mL), blasticidin (5 g/mL) and zeocin (200
FLAG expression was induced by adding tetracycline (1
L
-glutamine (2 mM), gentamycin
g/mL). SERT-
g/mL) and
m
m
m
m
incubating for 16–24 h. The HEK293 cells lines stably over-
expressing human SERT were cultured in DMEM supplemented
with 10% (v/v) FBS,
L-glutamine (2 mM), penicillin/streptomycin
4.2.6. Data analysis of NR assay
(100 mg/mL) and Geneticin (500 mg/mL). Stable expression was
valid up to 30 passages. Cells were maintained in a 72 cm2 tissue
culture flasks at 37 ꢀC in a humidified atmosphere of 95% oxygen,
and 5% carbon dioxide.
Each compound was screened over a 1 mM to 1 mM concen-
tration range in triplicate on two independent days with activity
expressed as percentage cell viability compared to vehicle treated
controls. The cytotoxic potency of each compound was quantified
by a pEC50 value determined by non-linear regression analysis of
sigmoidal log concentration dependence curves whereby pEC50 is
ꢃ[ꢃlog EC50] ꢁ SE (log EC50 is the log[Dose] when response is
equal to 50% cell viability) (Tables 1–4). All data points (expressed
as means ꢁ S.E.M.) were analysed using GRAPHPAD Prism (version
4) software (Graphpad software Inc., San Diego, CA). To determine
if the pEC50s calculated for each drug differed significantly in each
cell line, statistical analysis was carried out using a one way
ANOVA test comparing each pEC50 value. A P value of <0.05 was
considered to reflect a significant difference. The means for
different treatment groups were then compared using a two-way
ANOVA test with no matching followed by a Bonferroni Post Test
to compare replicate means by row to the control cell line
HEK293. P values of <0.05 were considered to reflect a significant
difference.
4.2.3. Generation of human peripheral blood mononuclear cells
Blood was obtained from a healthy donor, transferred into
a 50 mL falcon tube and diluted 1:2 with phosphate buffered saline
(PBS). LymphoPrep was used to separate the blood into red blood
cells, white blood cell ring and serum. The blood was slowly added
to 20 mL of Ficoll Pague plus. The tubes were centrifuged at 1700g
for 30 min. The white blood cell ring was transferred into a new
50 mL tube. The volume was adjusted to 50 mL and the samples
were centrifuged again at 1700g for 10 min. The supernatant was
removed. This step was repeated again, the pellet was then resus-
pended in 10 mL of complete IMDM media (1FCS, penicillin/
streptomycin (100 mg/mL)). Cells were counted and seeded at an
appropriate concentration of cells/mL.
4.2.4. 5-HT uptake assay
SERT inhibition was tested as previously described [84]. Briefly,
4.2.7. Alamar Blue assay
TREx cells (1 ꢂ105) were plated on poly-
L-lysine coated (0.1 mg/
1–5 ꢂ104 cells/well were seeded in a 96-well plate and treated
mL) plates. SERT-FLAG expression was induced by adding tetra-
with the respective drug for the desired length of time. Each well
cycline (1
m
g/mL) and incubating for 24 h. When confluent,
was then treated with 10 ml of Alamar Blue and left to incubate at
medium was removed from the cells and washed twice with
0.5 mL (per well) of TB buffer (10 mM HEPES, 150 mM NaCl, 2 mM
KCl, 1 mM CaCl2, 1 mM MgCl2) pre-warmed to 37 ꢀC. Cells were
incubated in TB buffer containing the drug of interest at various
concentrations or equal volumes of vehicle control for 5 min at
37 ꢀC. Specific activity of [3H] 5-HT was diluted with unlabelled 5-
HT. Cells were incubated with various concentrations of 5-HT
containing approximately 1 Ci/mmol [3H] 5-HT at room tempera-
ture. After 2 min uptake was stopped by removing the 5-HT
solution and immediately adding cold TB buffer containing 1 mM
paroxetine (to inhibit 5-HT transport). Cells were washed twice
with cold TB and then lysed in 2% SDS. Radioactivity in the lysates
was determined by liquid scintillation counting. The 3H isotope
was measured using a Packard Tricarb 2100 TR liquid scintillation
analyser. The cocktail used was the commercial scintillant Eco-
scintÔ, for aqueous samples. Non-specific uptake was defined as
37 ꢀC in the dark for 4–6 h. Fluorescence was read using an
emission of 590 nM and excitation 544 nm. The background
fluorescence of the media without cells þ Alamar Blue was taken
away from each group, and the control untreated cells repre-
sented 100% cell viability and 10
control.
mM Taxol was used as a positive
4.2.8. Quantification of apoptosis: propidium iodide FACS analysis
750,000 cells were seeded in 5 mL, treated with the appropriate
amount of compound and incubated for a specified time. Cells were
harvested by centrifugation at 300g for 5 min and washed with
5 mL of ice-cold PBS. The pellet was resuspended in 200 ml PBS and
2 mL of ice-cold 70% ethanol and cells were fixed overnight at 4 ꢀC.
Cells were pelleted by centrifugation at 300g for 5 min and resus-
pended in PBS with 25 ml of RNAse A (10 mg/mL stock) and 75 ml of
propidium iodide (1 mg/mL). The tubes were incubated in the dark
at 37 ꢀC for 30 min. Cell cycle analysis was performed using
appropriate gates counting 10,000 cells and analysed using CELL-
QUEST software package. Untreated cells had <5% cells in the pre-
the uptake in the presence of 10 mM paroxetine subtracted from
the total uptake to obtain specific uptake. Cells in the absence of
any test compound (untreated cells) represented the 100% reup-
take and the SSRI, citalopram was used as a positive control for
SERT inhibition.
G1 phase of the cell cycle and 10
control for cell death.
mM Taxol was used as a positive

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S.M. Cloonan et al. / European Journal of Medicinal Chemistry 44 (2009) 4862–4888
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Acknowledgements
The authors sincerely thank Dr. Georgia Golfis for her contri-
butions to the Molecular Modelling Study and thank Prof. Martin
Rowe, Dr. Dermot Walls and Dr. Patrick Schloss for kindly donating
cells used in this study. This work was supported through funding
from the Trinity College Postgraduate award, Centre for Synthesis
and Chemical Biology (HEA PRTLI, Cycle 3), Enterprise Ireland Basic
Research Award and Enterprise Ireland, Science and Technology
against Drugs with additional support for computational facilities
from the Wellcome Trust. The authors also thank the software
vendors for their continuing support of such academic research
efforts, in particular contributions from BiosolveIT, Openeye
Scientific, Accelrys/Scitegic and Chemical Computing Group.
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Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2009.07.027.
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