Synthesis and in vitro toxicity of 4-MTA, its characteristic clandestine synthesis byproducts and related sulfur substituted α-alkylthioamphetamines

Article abstract of DOI:10.1016/j.bmc.2010.04.022

4-Methylthioamphetamine (4-MTA) is recognised as a 3,4-methylenedioxymethamphetamine (MDMA)-like drug of abuse. Such amphetamine-type drugs often contain byproducts of uncontrolled, illegal clandestine synthetic processes. We report the isolation and stru

Full text of DOI:10.1016/j.bmc.2010.04.022

Bioorganic & Medicinal Chemistry 18 (2010) 4009–4031
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro toxicity of 4-MTA, its characteristic clandestine
synthesis byproducts and related sulfur substituted a-alkylthioamphetamines
Suzanne M. Cloonan a, , John J. Keating b, ,à, Desmond Corrigan b, John E. O’Brien c, Pierce V. Kavanagh d,
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, Ireland
c School of Chemistry, Trinity College Dublin, Ireland
d Department of Pharmacology and Therapeutics, Trinity Centre for Health Sciences, St. James’s Hospital, Dublin 8, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 November 2009
Revised 5 April 2010
Accepted 7 April 2010
Available online 13 April 2010
4-Methylthioamphetamine (4-MTA) is recognised as a 3,4-methylenedioxymethamphetamine (MDMA)-
like drug of abuse. Such amphetamine-type drugs often contain byproducts of uncontrolled, illegal clan-
destine synthetic processes. We report the isolation and structural identification of a number of novel
pyridines, dihydropyridone and N,N-di(1-aryl-2-propyl) amines as route-specific byproducts associated
with clandestine synthesis of 4-MTA and related amphetamines. We report the in vitro cytotoxicity of
4-MTA, its synthesis byproducts together with some structurally related sulfur substituted a-alkyl phen-
Keywords:
4-Methylthioamphetamine (4-MTA)
Leuckart reaction
Clandestine synthesises
Impurity profiling
Toxicity profiling
ethylamines in cell lines overexpressing human monoamine transporters as well as in a primary neuronal
cell line model and a dopaminergic neuroblastoma cell line. 4-MTA along with a number of other struc-
turally related amphetamine derivatives and synthetic impurities were found to be cytotoxic to these
cells within pharmacologically defined concentrations implying that 4-MTA is
in vitro and therefore might have the potential to be a neurotoxic agent in vivo.
a cytotoxic agent
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
(1a) (Fig. 1) constantly appearing on the illicit drug market. As
these drugs arise, new drug legislation appears simultaneously in
an attempt to halt the manufacture, trafficking and sale of these
drugs. 4-MTA was first synthesised in 1963 as an anorectic agent1
and became the subject of abuse when, in 1997, the first under-
ground laboratory involved in its manufacture was identified in
the Netherlands. It is recognised as a 3,4-methylenedioxymetham-
phetamine (MDMA)-like drug of abuse2 and is classified as a Sche-
dule-1 controlled substance.3 4-MTA is commonly sold as tablets
marked as ‘Ecstasy’ or ‘flatliners’.4
Amphetamine samples often contain byproducts of uncon-
trolled, illegal and clandestine synthetic processes.5–7 Detailed
knowledge of the synthetic methods employed by clandestine
chemists towards the manufacture of illegal drugs such as 4-MTA
provides valuable information to drug enforcement agencies and
may assist in the detection and seizure of illegal underground lab-
oratories with subsequent reduction in supply. Further knowledge
of the impurities that are formed as byproducts of illicit drug man-
ufacture enhances the ability of determining the synthetic route
employed. Impurities are present in variable quantities, depending
on the reaction conditions and purification procedure used (if any).
Most amphetamines abused in Europe are believed to be synthes-
ised by the Leukart route5,8 which involves the condensation of
formamide with the benzylketone precursor (Scheme 1) at
Amphetamine misuse is a significant problem in Europe with
new ring-substituted amphetamines such as methylenedioxyeth-
amphetamine (MDEA), N-methyl-1-(3,4-methylenedioxyphenyl)-
2-butanamine (MBDB) and 4-methylthioamphetamine (4-MTA),
Abbreviations: 4-MTA, 4-methylthioamphetamine; 5-HT, 5-hydroxytryptamine;
ALEPH-2, 1-(2,5-dimethoxy-4-ethylthiophenyl)-2-aminopropane; CNS, central ner-
vous system; DAT, dopamine reuptake transporter; DEPT, distortionless enhance-
ment by polarisation transfer; EI, electron impact; FBS, fetal bovine serum; He,
helium; HEK, human embryonic kidney cells; HMQC, heteronuclear multiple
quantum coherence; HRMS, high resolution molecular ion determination; IR, infra
red; LRMS, low resolution mass spectra; MBDB, N-methyl-1-(3,4-methylenedioxy-
phenyl)-2-butanamine; MDA, 3,4-methylenedioxyamphetamine; MDEA, methy-
lenedioxyethamphetamine; MDMA, 3,4-methylenedioxymethamphetamine; NAT,
noradrenaline reuptake transporter; NGF, nerve growth factor; NMR, nuclear
magnetic resonance; NOE, nuclear overhauser effect; NR, neutral red; PCA,
p-chloroamphetamine; PMA, p-methoxyamphetamine; SAR, structure–activity
relationship; SEM, standard error of the mean; SERT, serotonin reuptake trans-
porter; SSRI, selective serotonin reuptake inhibitors; TCA, tricyclic antidepressant;
TMS,
tetramethylsilane;
TOCSY,
total
correlation
spectroscopy.
* Corresponding author. Tel.: +353 1 8962798; fax: +353 1 8962793.
E-mail address: mmeegan@tcd.ie (M.J. Meegan).
Authors contribute equally to this work.
à
Present address: School of Pharmacy, Cavanagh Pharmacy Building, College Road,
University College Cork, Cork, Ireland.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.022

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S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
Figure 1. MDMA, 4-MTA and related amphetamines.
(g)
R
R
(f)
CO2H
H3C
O
H3C
N
H3C
S
S
H3C
CHO
S
3a R = H
3b R = CH3
6
18 R = H
(h)
19 R = CH3
(f)
R
(d)
H3C
N
(c)
R
S
H
CHO
8a R = H
8b R = CH3
H3C
O
CH3
(b)
H3C
NOH
S
S
S
(i)
3a,3b
7
30a R = H
30b R = CH3
(i)
(e)
(k)
CHO
(a)
R
(j)
H3C
NO2
H3C
S
S
R
4a R = H
4b R = CH3
R'
5
H3C
N
H3C
NH2
S
H
CH3
S
1a R = R' = H
1b R = H, R' = CH3
2a R = H
2b R = CH3
Scheme 1. Synthetic routes for 2-amino and 2-N-methylamino-1-(4-methylthiophenyl)alkanes 1a, 1b, 2a, 2b. Reagents and conditions: (a) CH3CH2NO2 or CH3CH2CH2NO2,
cyclohexylamine, CH3CO2H, reflux; (b) Fe, CH3COOH, 100°, 2 h, (c) NaCNBH3, NH4OCOCH3, CH3OH, rt, 72 h; (d) NH2OHꢀHCl, pyridine, EtOH, reflux, 2 h; (e) LiAlH4, THF, reflux,
3 h; (f) Ac2O, pyridine, reflux, 7 h; (g) CH3NHCHO, HCOOH 150°, 7 h; (h) NH2CHO,190°, 5 h; (i) 30% aq HCl, MeOH, reflux, 7 h; (j) LiAlH4, THF, reflux, 8 h; (k) NaCNBH3,
CH3NH2ꢀHCl, CH3OH, rt, 72 h.
elevated temperatures, resulting in the formation of an intermedi-
ate N-formyl amphetamine derivative. Subsequent hydrolysis of
the N-formyl group affords the desired primary amphetamines.
In the present study, 4-MTA and its C-4 analogue are synthesised
by a number of routes including the Leuckart–Wallach, reductive
amination and oxime and nitrostyrene reduction routes. A compre-
hensive group of synthesis impurities specific to these routes are
isolated and identified.
Recent surveys show that there is an increase in the use of ille-
gal amphetamines with 5.4% of 15–34 year old Europeans admit-
ting to haven taken Ecstasy at some stage in their lives.3 In the
UK, from 1996 to 2006, there was an average of around 50 drug-re-
lated deaths per year involving Ecstasy and it was the sole drug
implicated in around 10 cases per year. There have been six re-
ported fatalities associated with 4-MTA in the UK and Netherlands
together with one death and seven non-fatal cases of 4-MTA intox-
ication in Belgium. Such fatalities are thought to be associated with
large doses (21.3–9800 mg/kg), the use of other drugs and to inter-
individual differences in metabolism.9–11 As 4-MTA is a relatively
new amphetamine derivative and the number of reported poison-
ings is limited, the pathology findings and the mechanism of death
due to this drug have not yet been fully evaluated.
4-MTA has previously been shown to be toxic to rat hypotha-
lamic cultures12 and to CYP2D6 expressing cells11 but has been
shown to be devoid of serotonin neurotoxic effects in rat models.13
MDMA and its related metabolites, including 3,4-methylenedioxy-
amphetamine (MDA) have been found to be toxic to numerous cell
lines14–18 while there is little information on the in vitro cellular
toxicity of p-methoxyamphetamine (PMA) or p-chloroamphet-
amine (PCA). MDMA synthesis byproducts have also been shown
to interact with monoamine transporters at a similar potency to
MDMA without displaying any neurotoxic effects.19 To the best
of our knowledge, there have been no reports on the identification
and potential toxicity of byproducts associated with 4-MTA syn-
thesis. In the second part of this study, we therefore wish to eval-
uate the in vitro monoamine transporter toxicity of 4-MTA along
with a representative group of synthetic impurities which may
arise from clandestine synthesis of 4-MTA together with some re-
lated sulfur substituted a-alkylthioamphetamines. This will be car-
ried out using an in vitro cytotoxicity assay using human
embryonic kidney (HEK) cells expressing the serotonin, noradren-
aline and dopamine transporters, in a primary neuronal cell line
model (PC-12) and in a dopaminergic neuroblastoma cell line
(SHSY-5Y). Any concentration-dependent in vitro cytotoxic effects

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4011
exerted by these compounds in the cell lines used in this study will
provide an insight into the potential of these compounds to behave
as neurotoxic agents in vivo. Such revelations could have implica-
tions for social amphetamine abuse, potentially identifying neuro-
toxic effects of 4-MTA related compounds present in street drugs.
the intensities of the fragment ions are significantly lower by com-
parison. The pyridines 11–15 can be considered as condensation
products of two molecules of ketone and one molecule of formam-
ide. The mechanism of formation of pyridines in Leuckart synthe-
ses of amphetamine has been proposed.23,25 Orientation of the
ketones upon ring cyclisation determines to a large extent the
structures of the resulting pyridines.
2. Results and discussion
2.1. Chemistry
As an example of the identification of the novel pyridines
11–15, the 1H NMR spectrum of 13 displays two singlets at d
6.99 and d 8.34, revealing that the pyridine ring is trisubstituted
at positions 2, 4 and 5. C-2 is substituted by a 4-methylthiobenzyl
group as indicated by the benzylic methylene singlet at d 4.09. A
series of NOE experiments provided the evidence for the proposed
substitution pattern. Irradiation of the C-2 benzyl singlet resulted
in a positive NOE to the pyridine proton singlet, located at d 6.99,
which corresponds to H-3. In addition, the irradiation also pro-
duces a partial enhancement of the aromatic multiplet, centered
at d 7.23 and represents the H-30 and H-50 protons of the C-2 aryl
ring system. Irradiation of H-3 afforded the expected reciprocal po-
sitive NOE to the benzyl singlet in addition to the methyl singlet at
d 2.21, which corresponds to the C-4 methyl group. Finally, when
both the C-4 methyl and H-6 proton signals were irradiated, an
enhancement of the aromatic doublet, centered at d 7.20 was
found. By elimination, the doublet must represent H-300 and H-500
of the C-5 aryl system.
Perhaps the most interesting of the characteristic byproducts
isolated from the Leuckart synthesis is the novel dihydropyridone
16 (obtained in 0.7% yield) which is formed from the reaction of
two molecules of propanone 3a and one molecule of formamide.
The 1H NMR spectrum of 16 exhibits three upfield signals at d
1.26, d 2.46 and d 2.47 corresponding to the methyl group at C-2
and both SCH3 moieties, respectively. The methylene benzyl group
at C-2 resonates at d 2.60 as a singlet. Both H-3 protons are diaste-
reotopic and so are represented as two distinct doublets at d 2.73
and d 3.00 exhibiting geminal coupling constants of J = 13.0 Hz
and J = 13.5 Hz, respectively. A broad doublet centered further
downfield at d 4.90 (J = 6.5 Hz) is assigned to the amino proton. A
total correlation spectroscopy experiment (TOCSY) shows coupling
of this proton to the deshielded doublet (J = 3.5 Hz) at d 7.25 which
corresponds to the vinylic H-6 proton. Addition of D2O to the NMR
sample collapses this signal to a singlet with the concomitant dis-
appearance of the amino proton resonance. The series of four dou-
blets, each integrating for two protons, at d 7.07 (J = 8.0 Hz), d 7.21
(J = 7.5 Hz), d 7.23 (J = 8.0 Hz), and d 7.32 (J = 8.5 Hz) correspond to
both A2B2 para substituted aromatic ring systems. The TOCSY
experiment confirmed that protons represented by the doublets
at d 7.07 and d 7.21 are on the same aromatic ring with the more
downfield doublets corresponding to protons of the second ring.
A HMQC experiment proved helpful in assigning the 13C NMR
spectrum. The C-2 methyl group resonates at 24.86 ppm while
the signal at 43.36 ppm, which is inverted in the DEPT 135° spec-
trum, corresponds to C-3. The signal at 56.77 ppm disappears in
the DEPT 135° spectrum and is identified as the aliphatic quater-
nary C-2 carbon. A further quaternary signal at 110.55 ppm is as-
signed to the vinylic C-5 of the dihydropyridine ring, while the
resonance at 147.34 ppm, which remains in the DEPT 135° spec-
trum is assigned to the C-6 vinyl carbon. Finally, the conjugated
C-4 carbonyl carbon is identified at 189.57 ppm. The IR spectrum
of 16, displays the NH absorbance at 3238 cmꢁ1, together with a
strong stretch at 1567 cmꢁ1, which is due to carbonyl stretching
involved in hydrogen bonding to the NH proton of a second mole-
cule of 16.
In the present study, 4-MTA (1a), the N-methyl analogue 2a,
2-amino-1-(4-methylthiophenyl) butane (1b) and corresponding
N-methyl analogue 2b were synthesised by a number of routes,
for example, Leuckart–Wallach and reductive amination, together
with oxime and nitrostyrene reduction routes, as illustrated in
Scheme 1. The major synthesis impurities specific to these routes
are now isolated and identified.
2.1.1. Route A: Leuckart–Wallach reaction
Most of the amphetamines abused in Europe are believed to be
synthesised by the Leuckart and reductive amination routes5,8 due
to the comparative ease of these synthetic processes. It would be
expected that similar routes used in the Leuckart manufacture of
amphetamine and the MDA series of amphetamines would also
be employed in the synthesis of 4-MTA (1a) and related derivative
2a. The b-nitrostyrene 4a was prepared via a Henry reaction of
4-methylthiobenzaldehyde (5), with an appropriate nitroalkane
and cyclohexylamine in a modification of the reported proce-
dure.20 The nitrostyrene 4a was converted to the corresponding
alkanone 3a using Fe powder as the reducing agent in glacial acetic
acid. Propanone 3a was also synthesised from phenylacetic acid 6
by refluxing in acetic anhydride and pyridine, with the ketone 7
isolated as a minor product (2%). The Leuckart reaction involves
two synthetic steps from the benzylketone 3a. In the first step, con-
densation of formamide with the benzylketone 3a at elevated tem-
peratures results in the formation of an intermediate N-formyl
amphetamine derivative 8a. Subsequent hydrolysis of the N-formyl
group affords the desired primary amine 1a (route A, Scheme 1).
Due to the many possibilities for condensation of the precursor
ketone with formamide, a large number of additional compounds
could be potentially formed in the first step of the Leuckart synthe-
sis. Van der Ark et al. have reported the isolation and identification
of a series of highly characteristic, route specific impurities which
arise from this synthesis of amphetamine.21–24 Route-specific pyr-
idines have been identified from the Leuckart MDMA syntheses.6,22
The tentative presence of pyridines in PMA was reported as de-
tected by GC–MS.25 Although the presence of two pyrimidines
was reported by Kirkbride,20 the comprehensive structural charac-
terisation of the forensically significant signature 4-MTA Leuckart
impurities and synthesis byproducts has not yet been reported.
In the present work, we have isolated from the Leuckart reac-
tion five novel pyridines 11–15, a pyridone 16, the 2-N-formylami-
no-1-(4-methylthiophenyl)propane 8a, N-formyl-N,N-di(1-(4-
methylthiophenyl)-2-propyl) amine 17 together with the previ-
ously reported pyrimidines 9, 1020 as route specific impurities.
The structures and isolated amount of the various Leuckart impu-
rities for the 4-MTA-synthesis route are illustrated in Table 1. The
mechanism of formation of the 4-methylthio-pyrimidine and pyr-
idine impurities 11–15 would be similar to that reported for corre-
sponding PMA and MDMA analogues.25 The compounds 11–17 are
previously unreported as 4-MTA impurities and are novel entities.
Identification of the isomeric pyridines 11–15 was achieved by
interpretation of the high and low resolution mass spectra, and
also by interpretation of the 1H and 13C NMR spectra. Due to the
polyaromatic nature of the isolated pyridines, their low resolution
mass spectra are dominated by a very intense molecular ion, while
The most likely mechanism of formation of the pyridone 16 is
outlined in Scheme 2, where R represents the 4-methylthiophe-
nyl substituent group. Base catalysed enolisation of propanone
3a by ammonia (formed by partial hydrolysis of formamide

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S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
Table 1
4-MTA synthesis related byproducts from Leuckart and related synthetic routes
Compound
Structure
Synthesis routea
A
Yieldb,c (%)
3.2
8a
9
A
0.5
10
11
A
A
2
0.3
12
13
14
15
16
17
18
A
A
A
A
A
A
A
0.4
0.5
0.3
0.4
0.7
2
>5

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4013
Table 1 (continued)
Compound
Structure
Synthesis routea
Yieldb,c (%)
29
B
19
30a
C,D
D
18, 0.6
31
3
a
Synthetic route A: Leuckart–Wallach reaction of propanone 3a with formamide, synthetic route A*: Leuckart–Wallach reaction of propanone 3a with N-methylforma-
mide. Synthetic route B: reductive amination of propanone 3a with sodium amalgam and sodium cyanoborohydride. Synthetic route C: reduction of oxime with lithium
aluminium hydride. Synthetic route D: reduction of nitroalkane with lithium aluminium hydride. 4-Methylthiotoluene was also isolated as a route specific impurity in this
synthesis (3.5%).
b
Isolated yields.
c
The presence of the route specific Leuckart impurities in the above formamide Leuckart reaction mixture before the hydrolysis step was confirmed by GC–MS analysis
where the impurities were identified at the following retention times 8a (m/z 209, 10.57 min), 9 (m/z 216, 10.38 min), 10 (m/z 216, 10.27 min), 11 (m/z 351, 27.98 min), 12
(m/z 351, 27.97 min), 13 (m/z 351, 30.67 min), 14 (m/z 365, 28.39 min), 15 m/z (m/z 351, 28.61 min), 17 (m/z 373, 29.84 min), 18 (m/z 369, 9.60 min) and 4-methylthiotoluene
(m/z 138, 4.18 min) by comparison with reference isolated samples of these compounds.
O
O
O
O
NH3
H+
R
H+
H
R
H
R
R
+
R
OH
3a
R
OH2
O
A
R
-H2O
O
O
O
R
H
R
H+
R
O
R
R
N
H
N
H
R
NH2
O
O
C
B
NH3
O
O
O
O
H
R
R
R
H+
R
-H2O
R
R
R
+
R
N
H
N
H
N
H
OH2
N
H
OH
O
16
D
Scheme 2. Mechanism of formation of dihydropyridone 16 (R = 4-methylthiophenyl) from 1-(4-methylthiophenyl)-2-propanone (3a) and formamide.
under the reaction conditions) affords a resonance stabilised
enolate, which nucleophilically attacks a second molecule of 3a
at the carbonyl carbon. The resulting alkoxide anion is proton-
ated by water or formic acid. The aldol product A then under-
goes dehydration, giving rise to the conjugated enone B which
undergoes a conjugate Michael-type addition of formamide, fol-
lowed by proton abstraction to afford amidoketone C. A further
base catalysed enolisation can occur, this time with participation
of the carbon a to the 4-methylthiophenyl ring. Nucleophilic at-
tack by the enolate on the formyl amide carbon results in the
formation of cyclic aldol D. A further dehydration then affords
dihydropyridone 17. A similar route specific impurity has been
identified in the synthesis of amphetamine.22 2,3-Dihydro-4-
pyridonesare usually obtained by hetero-Diels–Alder reactions
or from cyclisations of enamines derived from
a,b-unsaturated
1,3-diketones.26

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S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
The origin of route specific tertiary amide 17 (obtained in 2%
hydroxylamine hydrochloride (Scheme 1). Oxime 30a was ob-
tained as a 1/3 syn/anti isomer mixture, whereas oxime 30b was
obtained as a 3/2 syn/anti isomer mixture as determined from
the chemical shifts of the H-10 and C-10 atoms. In the 1H and 13C
NMR spectra of syn oximes, benzylic protons resonate at further
downfield and carbons further upfield than corresponding reso-
nances of their anti oxime isomers. Oxime 30a was reduced with
LiAlH4 to afford 4-MTA (1a) in 50% yield, together with unreacted
oxime 30a (18%), while reduction of 30b afforded the amine 1b
(30%) together with unreacted oxime (1%) (Tables 1 and 2).
yield) is most likely from a further Leuckart reaction between the
initially formed N-formyl-4-MTA (8a) and propanone 3a. The com-
pound was isolated as an inseparable 65:35 mixture of diastereo-
mers. 4-Methylthiotoluene was also isolated as a route specific
impurity in this synthesis (3.5%). The presence of the route specific
Leuckart impurities in the above formamide Leuckart reaction mix-
ture before hydrolysis was confirmed by GC–MS analysis where
the impurities were identified at the following retention times 8a
(m/z 209, 10.57 min), 9 (m/z 216, 10.38 min), 10 (m/z 216,
10.27 min), 11 (m/z 351, 27.98 min), 12 (m/z 351, 27.97 min), 13
(m/z 351, 30.67 min), 14 (m/z 365, 28.39 min), 15 (m/z 351,
28.61 min), 16 (m/z 373, 29.84 min), 17 (m/z 369, 9.60 min) and
4-methylthiotoluene (m/z 138, 4.18 min) by comparison with ref-
erence isolated samples of these compounds.
Leuckart–Wallach reaction of 1-(4-methylthiophenyl)-2-alka-
none 3a and N-methylformamide results in formation of the 2-
(N-formyl-N-methyl)aminoalkane 18. Subsequent hydrolysis of
the N-formyl bond from 2-N-formylaminoalkane 8a and 2-(N-for-
myl-N-methyl)aminoalkane 18 afforded the amine products 1a
and 2a, respectively (Scheme 1).
2.1.4. Route D: reduction of nitrostyrenes
Reduction of nitrostyrene 4a with LiAlH4 afforded 4-MTA 1a in
80% yield with the oxime 30a (0.6%) and aziridine 31 (3%) identi-
fied as the major impurities (Table 1). A similar reduction of the
nitrostyrene 4b afforded the amine 2a (74%) together with the azi-
ridine 32 (7%).30 The structures of the aziridines 31 and 32 (which
were found to be unstable and decomposed rapidly) were con-
firmed to be the cis configuration from 1H NMR spectrum in which
the H-2 proton was observed as a doublet, d 3.22, J2,3 = 5.5 Hz.
A similar study of the Leuckart–Wallach synthesis of the homol-
ogous amine 1b was investigated with the ketone 3b (Scheme 1).
This reaction also afforded a signature group of two purines 20
and 21, five pyridine reaction byproducts 22–26 (listed in Table
2), together with amines 27 and 28. The symmetrical amine 27 is
diastereomeric and it was possible to isolate each diastereomer
by flash chromatography. The formation of 27 can be rationalised
by two successive reductive aminations between ammonia
(formed from initial partial decomposition of formamide to formic
acid and ammonia) and two molecules of ketone 3b, with formic
acid acting as the reducing agent. Analogous amines has been de-
tected in the Leuckart reactions of 4-methoxyphenylacetone25 and
1-(3,4-methylenedioxyphenyl)-2-propanone.27 The origin of the
tertiary amine 28 is most likely from a further Leuckart reaction
between 1b and ketone 3b. The compound was isolated as an
inseparable 60:40 mixture of diastereomers. Leuckart–Wallach
reaction of 1-(4-methylthiophenyl)-2-alkanone 3b and N-methyl-
formamide results in formation of the 2-(N-formyl-N-
methyl)aminoalkane 19. Subsequent hydrolysis of the N-formyl
bond from 2-N-formylaminoalkane 8b and 2-(N-formyl-N-
methyl)aminoalkane 19 afforded the amine products 1b and 2b,
respectively (Scheme 1).
2.1.5. Synthesis of structurally related 1,3-bis(4-methylthiophe-
nyl)-2-propanamines
The synthesis of a number structurally related 1,3-bis(4-meth-
ylthiophenyl)-2-propanamines for investigation in the toxicity
study was also explored as illustrated in Scheme 3. These amines
are derived from the diaryl propanone compound 7, already iso-
lated as a minor product in the preparation of the propanone 3
from 4-methylthiophenylacetic acid,3 and therefore could be re-
garded as a possible source of further route specific impurities in
the synthesis of 4-MTA.19 The related MDMA 1,3-bis(3,4-methyl-
enedioxyphenyl)-2-propanamines were reported to be weakly
active at monoamine transporters, with uptake inhibitory poten-
cies similar to MDMA in low micromolar range.19 Reduction of
nitroethene 33, followed by Henry reaction of 34 with 4-methyl-
thiobenzaldehyde 5 gave the corresponding 2-nitro-1,3-diphenyl-
prop-1-ene 35 which was treated with Fe/AcOH to afford the
propanone 7. Subsequent reaction of ketone 7 with formamide
gave the 36 which on hydrolysis afforded primary amine 37 in
84% yield. LiAlH4 reduction of the oxime 38 also afforded the pri-
mary amine 37 in 17% yield together with the novel aziridine 39
as the major reaction product in 61% yield. The aziridine 39 was
isolated as the cis isomer determined by the H-2 and H-3 1H
NMR coupling constant of 6.5 Hz. Hydrolysis of 40 (produced from
7 on treatment with N-methylformamide) afforded the corre-
sponding N-methyl product 41. Compounds 37 and 41 are analo-
gous in structure to previously reported Leuckart derived MDA6
and MDMA impurities.24 The N-ethylamine analogue 43 was ob-
tained by LiAlH4 reduction of the N-acetyl compound 42.
2.1.2. Route B: reductive amination
Ketone 3a was subjected to reductive amination conditions,
with either ammonium acetate or methylamine HCl as nitrogen
sources and NaCNBH3 or aluminium amalgam as reducing agents
to afford the amines 1a and 2a, respectively. The novel symmetri-
cal secondary amine 29 was isolated as a significant reaction
byproduct in 19% overall yield as an inseparable mixture of diaste-
reomers (in a ratio of 75:25) from the NaCNBH3 reduction of 3a
with ammonium acetate, (Table 1) and was identified by 1H and
13C spectra and by comparison with a similar amine previously
identified as a Leuckart derived MDMA impurity.6 The formation
of this product is explained by condensation of the initially formed
amine 1a with propanone 3a, followed by subsequent reduction of
the imine under the reaction conditions. A similar product 27 was
isolated as a minor reaction byproduct (0.2% yield) in the reductive
amination of the ketone 3b (Table 2).
2.2. Biological activity
Using an in vitro screen for cytotoxicity on HEK293 cells stably
expressing each of the human monoamine transporters, SERT
(serotonin transporter), NAT (noradrenaline transporter) and DAT
(dopamine transporter) (Supplementary Fig. 1), on a dopaminergic
cell line model (SHSY-5Y) as well as on a primary differentiated
neuronal cell line (PC-12) model provides a quick and economical
method for assessing some aspects of the potential neurotoxicity of
amphetamines and their related synthetic derivatives and impuri-
ties. To classify a drug as ‘neurotoxic’ requires further experimen-
tation such as the use of animal models or glutamate and serotonin
release experiments. However, any evidence of selective in vitro
cytotoxicity toward either one or more of the monoamine trans-
porters compared to the control HEK cell line implies that such a
chemical may have an in vivo serotonergic, dopaminergic or norad-
2.1.3. Route C: oxime reduction
The production of primary amines such as amphetamine and
MDA is also possible by a two-step synthesis from an appropriate
ketone via an oxime intermediate.28,29 The oximes 30a, 30b were
prepared from ketones 3a, 3b, respectively by reaction with

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4015
Table 2
2-Amino-1-(4-methylthiophenyl)butane synthesis related byproducts from Leuckart and related synthetic routes
Compound
Structure
Synthesis routea
A
Yieldb (%)
8b
>5
20
A
1
21
22
A
A
2
0.2
23
24
25
A
A
A
0.2
0.2
0.6
26
27
A
0.2
A,B
0.4, 0.2
28
A
1
19
A*
C
>5
1
30b
32
D
7
a
Synthetic route A: Leuckart–Wallach reaction of propanone 3b with formamide, synthetic route A*: Leuckart–Wallach reaction of propanone 3a with N-methylforma-
mide; synthetic route B: reductive amination of propanone 3b with sodium amalgam and sodium cyanoborohydride; synthetic route C: reduction of oxime 19b with lithium
aluminium hydride; synthetic route D: reduction of nitroalkene 4b with lithium aluminium hydride.
b
Isolated yield.

4016
S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
CHO
(c)
(b)
(a)
H3C
NO2
H3C
H3C
NO2
S
S
S
5
34
33
(d)
H3C
NO2
35
CH3
H3C
O
CH3
S
S
S
S
7
(e),(f)
(h)
H3C
N
H3C
N
CH3
S
OH
S
S
R
CHO
S
38
(i)
36 R = H
40 R = CH3
(g)
H
NH
H
H3C
N
CH3
H3C
S
S
H
R
S
37 R = H
41 R = CH3
39
(j)
(k)
H3C
NH
CH3
H3C
NH
CH3
S
S
S
S
COCH3
CH3
42
43
Scheme 3. Synthesis of 2-amino-1,3-diphenylpropanamines and related compounds. Reagents and conditions: (a) CH3NO2, cyclohexylamine, AcOH; (b) NaBH4, IPA, CHCl3,
silica gel (c) 5, (CH3)2NHꢀHCl, KF, toluene (d) Fe, CH3COOH, 100°, 2 h, (e) NH2CHO,190°, 5 h; (f) CH3NHCHO, HCOOH 150°, 7 h; (g) 30% aq HCl, MeOH, reflux, 7 h; (h)
NH2OHꢀHCl, pyridine, EtOH, reflux, 2 h; (i) LiAlH4, THF, reflux, 3 h; (j) Ac2O, pyridine, 20°, 1 h, (k) LiAlH4, THF, reflux, 24 h.
renergic toxic effect and provides useful information for studying
further the possible mechanism of action of such compounds.
MDMA, with evidence of PMA decreasing synaptosomal 5-HT up-
take and content.32
Toxicity of 4-MTA in animal studies is thought to be related to
its ability to induce a hyperserotonergic state known as serotonin
syndrome which is characterised by a number of symptoms
including fever, confusion, shivering, diaphoresis, ataxia, hyperre-
flexia and diarrhoea.32 In humans adverse effects upon 4-MTA
ingestion include nausea, hyperthermia, memory loss, nystagmus,
thirst, shivering, confusion, sweating and amnesia. Metabolic path-
ways for 4-MTA have only been demonstrated in vivo in mice and
in vitro in primary hepatocytes.32 In humans, metabolism of
4-MTA is speculated to involve the following: (i) oxidative deami-
nation to a ketone metabolite which can be further reduced to the
corresponding alcohol or suffer degradation of the side chain to
produce the 4-methylthiobenzoic acid metabolite, (ii) ring hydrox-
ylation to a phenolic structure (iii) b-hydroxylation of the side
chain to 4-methylthioephedrine or (iv) oxidation of the thioether.32
The compounds selected for this study of 4-MTA(1a) were the
related amphetamines 1b, 2a and 2b together with some represen-
tative synthesis related byproducts 8a, 8b, 9, 25, 29 and 30a
2.2.1. In vitro cytotoxicity to monoamine transporters
It has been difficult to determine if amphetamines are genuine
substrates of the monoamine transporters, due to their lipophilic
nature and to the lack of a co-crystallised structural determination
with human SERT, NAT or DAT. However, amphetamines are
thought to release stores of catecholamines from nerve endings
by converting the respective molecular transporters into open
channels. 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.31 Amphetamine analogues such as 4-MTA, MDMA,
MDA and PCA have all been previously shown to inhibit SERT,
NAT and DAT activities with IC50 values of 74 nM, 2,375 nM,
3,073 nM (4-MTA), 425 nM, 405 nM, 1,442 nM (MDMA), 478 nM,
266 nM, 890 nM (MDA) and 182 nM 207 nM, 424 nM (PCA) for
SERT/NAT/DAT, respectively.32 PMA is another common substi-
tuted amphetamine that is thought to act in a similar way to

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4017
selected from the Leuckart, reductive amination and oxime routes.
The structurally related 1,3-bis(4-methylthiophenyl)-2-propanam-
ines 36, 37, 42 and 43 were also included in the study. Cytotoxicity
was assessed in HEK293 cells stably expressing the human mono-
Leuckart route (A), and is obtained in 3.2% yield. Analysis of the
toxicity of this impurity revealed that it had little effect on all of
the monoamine transporter-expressing cell lines with approxi-
mate EC50 values of between 500 lM and 1 mM (Fig. 2). 8a features
amine transporters SERT, NAT and DAT as well as in
a
the N-formyl group rather than the primary amine which is found
in the other members of the 4-thiomethylamphetamine series, for
example, 1a that were found by this study to be cytotoxic. Further
SAR analysis is needed to confirm if the primary or secondary
amine is a pre-requisite for toxic activity in these compounds as
the observation that compound 8b has a similar cytotoxic effect
to 4-MTA and 1b, 2a, 2b, despite the presence of the N-formyl
group rather than a primary or secondary amine. The N-formyl
compounds 8a and 8b, because of amide resonance effects have re-
duced availability of the lone pair of electrons on the nitrogen and
may have altered target binding and reactivity when compared
with parent amine 1a.
dopaminergic (SHSY-5Y) and a primary neuronal cell line model
(PC-12). The results are displayed in Tables 3 and 4. As EC50 values
were estimated from log concentration sigmoidal dose response
curves, the cytotoxic potency of each compound was quantified
by a pEC50 value, where pEC50 is ꢁ[ꢁLog EC50
]
S.E (log EC50 is
the log [Dose] when response is equal to 50% cell viability). To
determine if any of the derivatives had a selective cytotoxic effect
to any of the cells lines, the effects of each derivative in each of the
cell lines was compared in a two-way ANOVA test (GRAPH pad
Prism 4) with no matching followed by a Bonferroni Post Test.
P values of <0.05 were considered to reflect a significant difference.
2.2.1.1. 4-MTA and a number of related compounds showed no
transporter-selective cytotoxic effects. In this study, 4-MTA (1a)
was found to be toxic to all of the human monoamine transporter
expressing cell lines as well as to the control HEK293 cell line with
2.2.1.3. Derivative selectively cytotoxic to monoamine trans-
porters only. Compound 36 is an N-formyl amine derivative of
1,3-bis(4-methylthiophenyl)-2-propanamine, and is structurally
related to 4-MTA. 36 was the only impurity found to be selectively
toxic to all of the human monoamine transporter-expressing cell
EC50 values in the low micromolar range (10–40 lM) (Table 3 and
Fig. 2). 4-MTA (pEC50 values from 4.38 to 4.73) was also found to be
more toxic than MDMA (pEC50 values from 2.71 to 3.61) and MDA
(pEC50 values from 2.23 to 3.49) in these cell lines (P <0.05) (Table
3).
lines (P <0.05) with an average estimated EC50 of between 10
lM
and 50 M (Table 3 and Fig. 2). The activity of 36 therefore differs
l
from 8a (N-formyl derivative of 4-MTA) which was not toxic to all
cell lines.
Many of the compounds presented in Table 3 were found by
this study to have a similar effect to 4-MTA. One such compound,
the secondary amine (29) is the most abundant synthetic byprod-
uct from the reductive amination route for synthesis of 4-MTA,
(obtained in 19% yield) which also displayed a general toxic effect
on all of the catecholamine expressing cell lines as well as the con-
trol HEK293 cell line. As the amine 29 occurs in significant amount
in 4-MTA obtained by the reductive amination route, it would be
interesting to investigate the levels of this compound in seized 4-
MTA street samples.
The 4-MTA analogue (2a), two representative heterocyclic Leu-
ckart-type impurities including the pyrimidine (9) and the pyridine
(25), as well as the N-acetylamine derivative (42) all had a similar
general toxic effect to 4-MTA (Table 3 and Fig. 2). Although no
extensive SAR (structure–activity relationship) analysis was car-
ried out, it appears that the introduction of a methylthio group
at position 4 on the amphetamine aryl ring as in 4-MTA and 1b
could be responsible for the increased cytotoxic effects of these
compounds, when compared to MDMA which contains the
3,4-methylenedioxy substitution in aryl ring.
4-MTA is known to be highly selective for SERT compared to
DAT (ratio of IC50 value for serotonin versus dopamine:
3.07 ꢂ 103)13 and NAT (ratio IC50 value for serotonin versus nor-
adrenaline: 2.37 ꢂ 103).13 Although the SERT, NAT and DAT bind-
ing activities of these compounds were not determined by this
study, it appears that SERT, NAT or DAT expression alone may
not be sufficient to confer 4-MTA- (and the above associated com-
pounds) toxicity to HEK cell lines. This is consistent with previous
reports of 4-MTA having a SERT-independent in vitro cytotoxic ef-
fect.32 To the best of our knowledge this study is the first report
comparing the cytotoxic effects of 4-MTA as well as the cytotoxic-
ities of a range of different 4-MTA synthetic derivatives and sulfur
2.2.1.4. Compounds not cytotoxic to the hDAT cell line. A num-
ber of compounds presented in Table 3 were found by this study to
have little effect on the dopamine transporter-expressing cell line
(P <0.05). This cell line was found to be insensitive to the toxic
effects of the N-methyl analogue of 4-MTA (1b), the N-methyl-1-
(4-methylthiophenyl) butane (2b) and 2-N-formylamino-1-(4-
methylthiophenyl) butane (8b),which is a significant synthetic
byproduct of the Leuckart synthetic route for the butane analogue
2a. Each of these derivatives had a cytotoxic effect on the SERT,
NAT and control HEK cell lines with EC50 values in the low micro-
molar range (Fig. 2). The above data suggests that hDAT may confer
a protective effect to HEK293 cells against the above compounds.
This maybe a direct result of these compounds preferentially bind-
ing to hDAT and consequently sequestering the cytotoxic activity.
As these compounds appear to be having an equipotent effect on
HEK control cells and on HEK cells expressing SERT and NAT, it is
unlikely that these compounds elicit their toxic effects through
SERT or NAT. Investigating the SERT/NAT/DAT binding of these
compounds will provide further information into the mechanism
of action of these toxic compounds.
Interestingly, both MDMA and MDA were also less toxic to the
hDAT cell line compared to the other cell lines (P <0.05), with
pEC50 values of 2.71 0.15 and 2.23 0.38, respectively (Table 3).
MDMA and MDA have a lower binding affinity for hDAT, binding
to SERT/NAT/DAT with IC50 values of 425 nM, 405 nM, 1,442 nM
(MDMA)33 and 478 nM, 266 nM, 890 nM (MDA),13 respectively.
Therefore, hDAT expression may confer a protective effect to HEK
cells upon MDMA or MDA treatment, perhaps due to the lack of
MDMA/MDA binding to these cells or through a yet undefined
mechanism involving another target.
It is also of interest that the oxime compound 30a which is a
significant synthesis impurity in 4-MTA derived from the oxime
reduction route, had the opposite effect on the hDAT cell line.
This compound was found to have little effect on the hSERT,
substituted a-alkylthioamphetamines on cell lines overexpressing
human monoamine transporters.
2.2.1.2. Compounds not toxic to any of the monoamine trans-
porter-expressing cell lines. In this study, the only amphet-
amine-type compound investigated that had no cytotoxic effect
to any of the cell lines was 8a, which is the N-formyl derivative
of 4-MTA. 8a is one of the major impurities of 4-MTA from the
hNAT and HEK293 cell lines (EC50 value of 500
displayed a selective cytotoxic effect to the hDAT (EC50 value
of 50–100 M) cell line (P <0.05). This result may imply that
this oxime impurity is selectively neurotoxic to dopaminergic
cells.
lM–1 mM) but
l

4018
S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
Table 3
In vitro cytotoxic effects (pEC50) of the 4-MTA synthetic derivatives, on SERT, NAT and DAT expressing HEK cells
Compound structure
Compound number
HEK293
pEC50 SE
HEK293hSERT
pEC50 SE
HEK293 hNAT
pEC50 SE
HEK293 hDAT
pEC50 SE
a
a
a
a
1a
4-MTA
4.73 0.14
4.71 0.1
4.65 0.15
4.56 0.22
4.65 0.14
4.52 0.16
4.38 0.17
3.67 0.22*
1b
2a
2b
8a
8b
4.83 0.11
4.30 0.15
2.54 0.01
4.21 0.18
4.75 0.13
4.26 0.10
2.87 0.19
4.19 0.21
4.37 0.13
4.18 0.21
3.43 0.17
4.10 0.15
4.14 0.18
3.38 0.14*
2.64 0.18
2.95 0.16*
9
5.03 0.16
4.78 0.17
4.20 0.18
4.93 0.19
25
29
4.73 0.1
4.46 0.21
4.59 0.19
4.53 0.20
4.60 0.15
4.57 0.22
4.58 0.17
4.79 0.14
30a
37
2.52 0.25
4.65 0.20
3.67 0.17
4.61 0.15
2.76 0.26
3.90 0.18
4.17 0.15
4.60 0.25
36
43
42
<1
4.30 0.20
4.59 0.17
4.41 0.26
3.34 0.17
3.58 0.16
4.59 0.23
4.06 0.24
4.57 0.19
4.08 0.34
4.77 0.16
4.30 0.18

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4019
Table 3 (continued)
Compound structure
Compound number
HEK293
pEC50 SE
HEK293hSERT
pEC50 SE
HEK293 hNAT
pEC50 SE
HEK293 hDAT
pEC50 SE
a
a
a
a
MDMA
MDA
3.61 0.19
3.49 0.18
3.31 0.21
3.16 0.34
2.81 0.23*
3.23 0.22*
2.71 0.15*
2.23 0.38*
a
pEC50 (ꢁ(ꢁLog EC50 is the log [Dose] when response = 50%)) values were calculated from % cell viability versus ꢁlog 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 = 1) using GRAPHPAD Prism4 software
(Graphpad software Inc., San Diego, CA). Sodium azide (30 mM) and Triton-X (2%) acted as positive controls for cytotoxicity resulting in 80–90% cytotoxicity to all cells.
*
P <0.05. The effect of each compound in the HEK293 cell line was compared to the effect of each compound in all other cell lines using a two-way ANOVA test (GRAPH pad
Prism 4) with no matching followed by a Bonferroni Multiple comparison Post test, comparing all cell lines to the HEK293 cell line.
Table 4
In vitro cytotoxic effects (pEC50) of the 4-MTA synthetic derivatives, on the primary neuronal PC-12 and dopaminergic, SHSY-5Y cell lines
Compound structure
Compound number
PC-12
pEC50 SE
SHSY-5Y
pEC50 SE
a
a
1a
4-MTA
3.53 0.13
3.29 0.16
2.81 0.21
1b
3.26 0.15
8a
8b
<2
<2
<2
3.13 0.16
9
4.17 0.25*
2.53 0.11
25
4.53 0.15*
3.75 0.09*
29
4.56 0.16*
3.11 0.20
4.65 0.14*
3.47 0.06
30a
37
36
3.20 0.22
4.42 0.20*
4.73 0.12*
4.49 0.15*
43
42
3.64 0.15
2.38 0.30
4.86 0.13*
3.22 0.12
a
pEC50 (ꢁ(ꢁLog EC50 is the log [Dose] when response = 50%)) values were calculated from% cell viability versus ꢁlog 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 = 1) using GRAPHPAD Prism4 software
(Graphpad software Inc., San Diego, CA). Sodium azide (30 mM) and Triton-X (2%) acted as positive controls for cytotoxicity resulting in 80–90% cytotoxicity to all cells.
*
P <0.05. The effect of each compound was compared to the effect of 4-MTA in each cell line using an unpaired T-test (GRAPH pad Prism 4).

4020
S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
*
HEK293
hSERT
hNAT
*
*
*
*
3100
2100
1100
hDAT
100
100
50
0
1a
1b
2a
2b
8a
8b
9
25
29
30a
36
42
43
37
H3CS
CH3
CH3
S
SCH3
CH3
NH2
CH3
S
CH3
S
N
CH3
S
S
H2N
S
CH3
S
H3
C
N
S
N
H
H
N
OHC
CH3
S
H
N
H2N
H3COC
N
H
CH3
H2N
H3C
OHC
H3C
HN
S
CH3
H3
C
N
H3CS
S
H
CHO
S
CH3
H3CS
S
CH3
S
CH3
HN
H3C
CH2CH3
CH3
CH3
SCH3
H3C
H3
C
N
N
S
OH
Figure 2. The majority of representative 4-MTA synthetic impurities are cytotoxic to hSERT, hNAT, hDAT and wild type HEK cells with EC50 values in the low micromolar
range. 5 ꢂ 104 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. The effect of each compound on the HEK293 cell line was
compared to the effect of the compound on each of the transporter-overexpressing cell lines using two-way ANOVA statistical analysis, where p <0.05 (*) represented a
significant difference.
2.2.1.5. Compounds not toxic to hNAT. The primary amine 37,
which is structurally related to 4-MTA, together with the related
N-ethylamine 43 were found to be less toxic to the hNAT cell line
(pEC50 values of 3.90 0.18 (37) and 3.58 0.16 (43)) compared to
the hSERT, HEK293 and hDAT cell lines (P <0.05). The above data
suggests that hNAT may confer a protective effect to HEK293 cells
against the above compounds. This maybe a direct result of these
compounds preferentially binding to hNAT and consequently
sequestering the cytotoxic activity. As these compounds appear
to be having an equipotent effect on HEK control cells and on
HEK cells expressing SERT and DAT, it is unlikely that these com-
pounds elicit their toxic effects through SERT or DAT. Again, inves-
tigating the SERT/NAT/DAT binding of these compounds will
provide further information into the mechanism of action of these
toxic compounds.
neuroendocrine cell line model,34 that has been previously used
to test the potential neurotoxicity of a range of drugs.35 Using this
model, 4-MTA (1a) was found to be cytotoxic to the PC-12 cell line
at high concentrations (Fig. 3) with an approximate pEC50 value of
3.53 0.13 (corresponds to an approximate EC50 range of between
160 and 600 lM) (Table 4). This data is consistent with previous
reports showing that 4-MTA is neurotoxic particularly at high con-
centrations.11,12,36–39 These results also show that 4-MTA is more
toxic to PC-12 cells than MDA and MDMA which have been previ-
ously shown to have little effect on the PC-12 cell line (IC50 values
of 3365 lM (MDA) and 4464 l
M (MDMA), respectively).15
The N-methyl-4-MTA derivative 1b, together with significant
reductive amination synthesis byproduct 29 were also found to be-
have in a similar way to 4-MTA having a potent cytotoxic effect at
high concentrations (Table 4 andFig. 3). Derivative 29 was found to
have a greater pEC50 value than 4-MTA (4.56 versus 3.53) (P <005)
implying that this derivative is more cytotoxic to the PC-12 cell
line than 4-MTA itself. The pyridine 25 and the N-ethylamine 43
also demonstrated a similar toxicity profile, showing more toxicity
2.2.1.6. The effects of representative compounds on the PC-12
cell line. 4-MTA together with some relevant synthesis byprod-
ucts and structurally related amines were then tested on the nerve
growth factor (NGF) differentiated PC-12 cell line, (Table 4, Fig. 3).
The PC-12 cell line is derived from a transplantable rat pheochom-
ocytoma34 that when supplemented with NGF displays a differen-
tiated morphology from a proliferating cell to a post-mitotic,
neurite bearing neuron. The PC-12 cell line is a classical in vitro
than 4-MTA at 40 lM. Derivative 25 was also found to have a sig-
nificantly greater pEC50 value than 4-MTA (P <0.005) implying it is
more toxic to the PC-12 cell line than 4-MTA (Table 4). These com-
pounds were also found to have a general toxic effect to HEK293
and to HEK cells overexpressing hSERT, hNAT and hDAT (Section

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4021
1a
1b
130
130
120
110
100
90
PC-12
SHSY-5Y
120
110
100
90
80
70
60
50
40
30
20
10
0
PC-12
SHSY-5Y
80
70
***
60
***
50
40
***
30
20
10
0
Control
1
10
40
1000
Control
1
10
40
1000
Concentration (µM)
Concentration (µM)
8a
8b
130
120
110
100
90
130
120
110
100
90
PC-12
SHSY-5Y
PC-12
SHSY-5Y
80
80
70
70
60
60
***
50
50
40
40
30
30
20
20
10
10
0
0
Control
1
10
40
1000
Control
1
10
40
1000
Concentration (µM)
Concentration (µM)
9
25
130
120
110
100
90
130
120
110
100
90
PC-12
SHSY5Y
Pc-12
SHSY-5Y
80
**
**
**
80
70
**
70
**
60
60
50
50
40
40
***
30
30
***
20
20
10
10
0
0
Control
1
10
40
1000
Control
1
10
40
1000
Concentration (µM)
Concentration (µM)
29
30a
130
120
110
100
90
130
120
110
100
90
PC-12
SHSY-5Y
Pc-12
SHSY-5Y
80
80
70
*
**
70
60
60
50
50
40
40
30
***
30
20
20
***
*** ***
10
10
0
0
Control
1
10
40
1000
Control
1
10
40
1000
Concentration (µM)
Concentration (µM)
130
120
110
100
90
37
36
PC-12
SHSY-5Y
130
120
110
100
90
Pc-12
SHSY-5Y
80
80
70
70
60
*
*
60
*
50
50
*
40
***
40
30
30
20
20
10
***
***
10
0
0
Control
1
10
40
1000
Control
1
10
40
1000
Concentration (µM)
Concentration (µM)
43
42
130
120
110
100
90
130
PC-12
SHSY-5Y
PC-12
SHSY-5Y
120
110
100
90
80
70
60
50
40
30
20
10
0
80
70
60
50
***
40
30
***
***
20
***
10
0
Control
1
10
40
1000
Control
1
10
40
1000
Concentration (µM)
Concentration (µM)
Figure 3. A number of impurities associated with clandestine 4-MTA synthesis are toxic to the primary neuronal PC-12 and dopaminergic SHSY-5Y cell lines at high
concentrations. 5 ꢂ 104 undifferentiated PC-12 cells were seeded and cultured for seven days in media supplemented with 100 ng/mL NGF. After seven days, all cells
displayed a differentiated morphology from a proliferating cell to a post-mitotic, neurite bearing neuron. Cells (including 5 ꢂ 104 SHSY-5Y) were then treated for 48 h with
each impurity at the desired concentration before incubating 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 concentration of compound was compared to untreated control cells using one-way
ANOVA statistical analysis where (***) represents p < 0.001, (**) represents p < 0.005 and (*) represents p < 0.05.

4022
S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
2.2.1.1 above). Compound 36 was also found to have a significantly
greater (p <0.05) pEC50 value than 4-MTA in the PC-12 cell line,
tative derivatives investigated by this study, the majority had a
similar effect to 4-MTA displaying cytotoxic EC50 values in the
having a cytotoxic effect at 40
lM ( Fig. 3) again implying this
low micromolar range (1 lM–50 lM). A number of the derivatives
derivative is more toxic to the PC-12 cells than 4-MTA (Fig. 3 and
Table 4). This derivative also had an effect against the hSERT, hNAT
and hDAT overexpressing cell lines but had little effect on the
HEK293 cell line (Table 3).
Of the other compounds screened in the PC-12 cell line, the
majority also displayed cytotoxic activity in the PC-12 cell line
with pEC50 values in the range of 2.38–4 (corresponding to an
used in this study, including 4-MTA itself were found to be more
toxic than MDA and MDMA to the primary neuronal PC-12 cell line
model and to the dopaminergic neuroblastoma SHSY-5Y cell line.
Such a concentration dependent effect by 4-MTA and its related
synthetic derivatives in these cell lines implies that these deriva-
tives are cytotoxic agents in vitro and therefore might have the po-
tential to act as neurotoxic agents in vivo. Future investigations
utilising a variety of in vitro and in vivo models will be required
to gain a better understanding of their possible in vivo neurotoxic
potential and mechanism of action.
approximate EC50 range of between 200 lM and 1 mM). Com-
pounds 8b, 30a, 37 and 43 all had a potent cytotoxic effect in the
PC-12 cell line at 1 mM (Fig. 3), comparable to the effect induced
by 4-MTA at 1 mM. Compounds 8a and 42 were found to have
no effect on the PC-12 cell line.
4. Experimental
4.1. Chemistry
2.2.1.7. The effects of representative compounds on the SHSY-
5Y cell line. The SHSY-5Y cell line SH-SY5Y is a thrice cloned (SK-
N-SH?SH-SY?SH-SY5?SH-SY5Y) subline of the human neuro-
blastoma cell line SK-N-SH established from a metastatic bone tu-
mour.40 This cell line was chosen for use in this screen as is widely
used as a model for dopaminergic cells and it has been shown not to
express SERT.40 This line has been previously used to test the poten-
tial neurotoxicity of a range of drugs41 including a number of cate-
cholamine derivatives.42 We have previously reported that 4-MTA
demonstrated cytotoxicity for the SHSY-5Y cell line32 (pEC50 of
3.29). In the present study a number of the compounds in particular
25, 29, 36, 37 and 43 showed significant cytotoxicity to the SHSY-5Y
cell line with pEC50 values in the range 4.65, 4.49, 4.73 and 4.86,
respectively, correlating to an EC50 range of approximately 10–33
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. Low resolution mass spectra (LRMS) were acquired on a
Hewlett–Packard 5973 MSD GC–MS system in electron impact
(EI) mode. GC–MS analysis was performed using an Agilent 6890
gas chromatograph with split–splitless injection (2 mL injected)
and a HP-5MS column (30 m ꢂ 0.25 mm, 0.25 mm film thickness).
Helium (He) was used as the carrier gas at a flow rate of 1.0 mL/
min. The GC was coupled to an Agilent 5973 MSD (EI, 70 eV, TIC
mode scanning m/z 50–800). The following temperature program
was used: 90 °C for 0.1 min, 15 °C/min to 250 °C, 250 °C for
1 min, 2 °C/min to 300 °C, 300 °C for 5 min, injector port 270 °C,
transfer line, 280 °C. High resolution molecular ion determinations
(HRMS) were acquired on either a Kratos Profile HV-4 mass spec-
trometer using a direct insertion probe and EI mode (Department
of Chemistry, University College Cork) or a Micromass mass spec-
trometer (EI mode) at the Department of Chemistry, Trinity Col-
lege, Dublin. 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 aluminium oxide, (activated, neutral, Brock-
mann I, 50 mesh) or Aldrich aluminium oxide, (activated, acidic,
Brockmann I, 50 mesh).
lM. (Table 4 and Fig. 3). These compounds were also significantly
morecytotoxic to theSHSY-5Y cells than4-MTA (p <0.05). This result
is in contrast to the effects obtained for MDMA and MDA which were
shown not to have any significant effect on SHSY-5Y cell line (pEC50
<2). Compounds 1b, 30a and 42 were found to behave in a similar
wayto4-MTAhavingapotentcytotoxiceffectat highconcentrations
(Table 4 andFig. 3). These compounds were also found to have a gen-
eral toxic effect to HEK293 and to HEK cells overexpressing hSERT,
hNAT and hDAT (Section 2.2.1.1). Compounds 8a, 8b and 9 were
found to have no effect on the SHSY-5Y cell line. Although many of
the compounds did not appear to be selective in toxicity for the
monoamine transporters, there appears to be some correlation of
activity between HEK293hDAT cytotoxic effects and SHSY-5Y cyto-
toxicity for compounds 29, 37 and 43.
3. Conclusion
4-MTA is known to be more dangerous than other designer
drugs of abuse because of its slow onset of action encouraging
abusers to administer further doses assuming that the first dose
was inadequate thus leading to fatally high concentrations of 4-
MTA and its derivatives in vivo.3 In this study we report for the first
time that 4-MTA is toxic to monoamine transporter-expressing
cells in vitro having a more potent effect than MDA and MDMA.
Such concentrations are pharmacologically comparable to in vivo
reported fatal 4-MTA intoxications, which have consisted of blood
4.1.1. 1-(4-Methylthiophenyl)-2-propanone (3a)
A suspension of iron powder (573 mmol, 32.0 g) in glacial acetic
acid (150 mL) was heated on a steam bath for 20 min, stirring occa-
sionally. To this mixture, a solution of nitrostyrene 4a (126.0 mmol)
in glacial acetic acid (150 mL) was added over 20 min, while stirring
the reaction occasionally. The reaction was heated for a further 2 h
and the added to a mixture of ice/water (1200 mL). The product
was extracted with dichloromethane (4 ꢂ 100 mL) and organic ex-
tracts 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 to afford the product as a col-
ourless oil (82%) (lit.20 bp 133 °C/0.4 mmHg).
concentrations of 4-MTA between 7.7 and 29.6
sue (brain or liver) concentrations of 170–201.6
l
M9,38,39,43 and tis-
M.38,44
l
In this study, the synthetic methods employed by clandestine
chemists towards the manufacture of ring-substituted ampheta-
mines were used to create a library of 4-MTA-like-compounds. This
study also reports, for the first time the cytotoxic activities of some
representative 4-MTA synthetic byproducts and derivatives on the
human monoamine transporters, SERT, NAT and DAT and on the
PC-12 and SHSY-5Y neuronal cell line models. Out of 14 represen-
4.1.2. 1-(4-Methylthiophenyl)-2-butanone (3b)
A suspension of iron powder (57.3 mmol, 3.20 g) in glacial ace-
tic acid (15 mL) was heated on a steam bath for 20 min, stirring

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4023
occasionally. To this mixture, a solution of the nitrostyrene 4b45
(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 ex-
tracted with dichloromethane (4 ꢂ 100 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 ke-
tone as a colourless solid (96%), mp 38–39 °C, lit. bp 116 °C/
the reaction was diluted with water (150 mL) and extracted with
ethylacetate (3 ꢂ 75 mL). The extracts were combined, washed with
water (3 ꢂ 50 mL) and dried over anhydrous Na2SO4. After remov-
ing all volatiles in vacuo the resulting dark brown oil was dissolved
in methanol (30 mL) and 30% aq HCl (150 mL) an the mixture was
heated under reflux for 7 h. Upon cooling, the reaction was diluted
with water (100 mL), made basic (pH 14), with 15% aq NaOH and ex-
tracted with dichloromethane (4 ꢂ 100 mL). The extracts were
combined and dried over anhydrous Na2SO4. All volatiles were re-
moved in vacuo, affording a dark brown oil. Isolation of the desired
product 1a, together with the following reaction byproducts was
performed by flash column chromatography over silica gel.
0.25 mm Hg.46 IR mmax (KBr) 1711 (C@O) cmꢁ1 1H NMR d (CDCl3)
.
1.02 (3H, t, J4 ,3 = 7.3 Hz, H-40), 2.26 (2H, q, J3 ,4 = 7.3 Hz, H-30),
2.46 (3H, s, SCH3), 3.63 (2H, s, H-10), 7.12, 7.22 (4H, 2ꢂd, J = 8.5,
J = 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
(M+). Anal. Calcd for C11H14OS; C, 68.00; H, 7.26. Found: C, 68.24;
H, 7.40.
0
0
0
0
4.1.7. 2-Amino-1-(4-methylthiophenyl)propane, 4-MTA (1a)
4-MTA (1a) was obtained as an amber oil (48%) (lit. bp 108–
118 °C/0.6 mm Hg48). IR mmax (film) 3359, 3285 cmꢁ1 1H NMR d
.
(CDCl3) 1.10 (3H, d, J3 ,2 = 6.5 Hz, H-30), 1.25 (2H, br s, NH2), 2.46
0
0
(3H, s, SCH3), 2.48 (1H, dd, Jgem = 13.3 Hz, J1 ,2 = 8.0 Hz, H-10), 2.67
0
0
(1H, dd, Jgem = 13.3 Hz, J1 ,2 = 5.0 Hz, H-10), 3.13 (1H, m, H-20),
7.10, 7.21 (4H, 2ꢂd, J = 8.0 Hz, J = 8.0 Hz, H-2, H-3, H-5, H-6). 13C
NMR ppm (CDCl3) 16.13, 23.48, 46.04, 48.36, 127.01, 129.68,
135.76, 136.71. HCl salt. Colourless solid. Mp 187–189 °C (etha-
nol/hexane), (lit. mp 190–191 °C49). MS m/z 181 (M+) The following
synthesis impurities were also isolated.
0
0
4.1.3. 1-(4-Methylthiophenyl)-2-nitro-1-propene (4a)
A
solution of 4-methylthio benzaldehyde (5) (300 mmol,
45.66 g), nitroethane (600 mmol, 45.04 g, 43.10 mL), cyclohexyl-
amine (300 mmol, 29.76 g, 34.30 mL) in glacial acetic acid
(240 mL) was heated on a steam bath for 6 h. Water (125 mL)
was then added and the mixture was then allowed to stand at
room temperature overnight. The resulting crystalline precipitate
was isolated by vacuum filtration and washed with water
(250 mL). Recrystallisation from ethanol afforded the pure product
as yellow crystals (53.79 g, 257.04 mmol, 86%), mp 72–73 °C (eth-
anol), (lit. mp 71–73 °C20). Rf 0.53 (hexane/diethylether: 80/20). IR
4.1.8. 2-N-Formylamino-1-(4-methylthiophenyl)propane (8a)
2-N-Formylamino-1-(4-methylthiophenyl)propane (8a) was
isolated by flash chromatography on silica gel (eluent: ethyl ace-
tate). Pale brown solid (3.2%), mp 70–72 °C. IR mmax (KBr) 3333,
1660 cmꢁ1 1H NMR d (CDCl3) 1.11 (2.4H, d, J3 ,2 = 6.5 Hz, H-30),
.
0
0
0
0
m
max (KBr) 1648, 1509, 1312 cmꢁ1. 1H NMR d (CDCl3) 2.46 (3H, s, H-
1.23 (0.6H, d, J3 ,2 = 6.5 Hz, H-30), 2.43 (3H, s, SCH3), 2.60–2.81
30), 2.52 (3H, s, SCH3), 7.29, 7.37 (4H, 2ꢂd, J = 8.5 Hz, J = 8.5 Hz, H-2,
H-3, H-5, H-6), 8.04 (1H, s, H-10). 13C NMR ppm (CDCl3) 14.11,
15.00, 125.89, 130.46, 128.58, 133.15, 142.14, 146.87. MS m/z
209 (M+).
(0.4H, m, H-10), 2.66 (0.8H, dd, Jgem = 13.6 Hz, J1 ,2 = 7.0 Hz, H-10),
0
0
2.79 (0.8H, dd, Jgem = 13.6 Hz, J1 ,2 = 6.5 Hz, H-10), 3.64 (0.2H, m,
H-20), 4.26 (0.8H, m, H-20), 6.30 (0.8H, d, J = 7.6 Hz, NH), 6.35
(0.2H, d, J = 10.6 Hz, NH), 7.05, 7.18 (0.8H, 2d, J = 8.0 Hz,
J = 8.6 Hz, H-2, H-3, H-5, H-6), 7.09, 7.17 (3.2H, 2ꢂd, J = 8.5 Hz,
J = 8.5 Hz, H-2, H-3, H-5, H-6), 7.73 (0.2H, d, J = 11.5 Hz, CHO),
8.00 (0.8H, s, CHO). 13C NMR ppm (CDCl3) 15.58, 15.66*, 19.66*,
21.57, 41.48*, 43.52, 44.80*, 49.74, 126.52*, 126.62, 129.58*,
129.64, 134.12, 134.57*, 135.98, 136.43*, 160.42*, 163.52. MS m/z
209 (M+). Anal. Calcd for C11H15NOS: C, 63.12; H, 7.22; N, 6.69.
Found: C, 63.13; H 7.09; N, 6.58.
0
0
4.1.4. 1-(4-Methylthiophenyl)-2-propanone (3a) via 4-
methylthiophenylacetic acid (6)
A
stirred solution of 4-methylthiophenylacetic acid (6)
(8.23 mmol, 1.50 g), pyridine (4.15 mL) and acetic anhydride
(4.15 mL) was refluxed for 7 h. After cooling the reaction was di-
luted with 10% aq HCl (100 mL) and extracted with diethylether
(3 ꢂ 50 mL). The organic phases were combined, dried over anhy-
drous Na2SO4 and solvent removed in vacuo. The residue was puri-
fied by flash chromatography, providing the desired ketone (20%)
(3a), and 1,3-di(4-methylthiophenyl)-2-propanone (7) as colour-
less needles (2%), mp 80–81 °C (diethylether/hexane), (lit. mp
79–80 °C47).
4.1.9. 4-(4-Methylthiobenzyl)pyrimidine (9)
4-(4-Methylthiobenzyl)pyrimidine (9) was isolated by flash
chromatography on silica gel (eluent: diethylether/ethylacetate
50/50) as an amber oil (0.5%).20 IR mmax (film) 2856, 1577 cmꢁ1
.
1H NMR d (CDCl3) 2.46 (3H, s, SCH3), 4.07 (2H, s, CH2), 7.10 (1H,
dd, J5,6 = 5.0 Hz, H-5), 7.18, 7.23 (4H, 2ꢂd, J = 8.0 Hz, J = 8.0 Hz, H-
20, H-30, H-50, H-60), 8.58 (1H, d, J6,5 = 5.0 Hz, H-6), 9.13 (1H, s, H-
2). 13C NMR ppm (CDCl3) 15.92, 43.58, 127.15, 129.67, 134.14,
137.16, 157.00, 158.73, 169.22. MS m/z 216 (M+).
4.1.5. 1-(4-Methylthiophenyl)-2-nitro-1-butene (4b)
Compound 4b was prepared from 4-methylthiobenzaldehyde
(5), (40 mmol, 6.09 g) and nitropropane (80 mmol, 7.12 g,
7.13 mL) according to general procedure for 4a. The crude product
was purified by flash chromatography on silica (eluent: hexane/
diethylether: 80/20) and obtained as a yellow oil (82%).45 Rf 0.56
(hexane/diethylether: 70/30). IR mmax (film) 1647, 1508, 1324
4.1.10. 4-Methyl-5-(3,4-methylthiophenyl)pyrimidine (10)
4-Methyl-5-(3,4-methylthiophenyl)pyrimidine (10) was iso-
lated by flash chromatography on silica gel (eluent: diethylether/
hexane 80/20) as an amber oil (2%).20 IR mmax (film) 2856,
cmꢁ1 1H NMR d (CDCl3) 1.28 (3H, t, J4 ,3 = 7.5 Hz, H-40), 2.52 (3H,
.
0
0
s, SCH3), 2.88 (2H, q, J3 ,4 = 7.5 Hz, H-30), 7.29, 7.36 (4H, 2d,
J = 8.5 Hz, J = 8.0 Hz, H-2, H-3, H-5, H-6), 7.98 (1H, s, H-10). 13C
NMR ppm (CDCl3) 12.34, 15.00, 20.81, 125.99, 130.17, 128.51,
132.73, 142.21, 152.47. MS m/z 223 (M+).
1598 cmꢁ1 1H NMR d (CDCl3) 2.52 (3H, s, CH3), 2.54 (3H, s,
.
0
0
SCH3), 7.25, 7.36 (4H, 2ꢂd, J = 8.0 Hz, J = 8.5 Hz, H-20, H-30, H-50,
H-60), 8.51 (1H, s, H-6), 9.06 (1H, s, H-2). 13C NMR ppm (CDCl3)
15.44, 22.78, 126.40, 129.28, 132.18, 134.23, 139.32, 156.18,
157.02, 164.32. MS m/z 216 (M+).
4.1.6. Preparation of 4-methylthioamphetamine (1a) via
Leuckart–Wallach reaction
4.1.11. 2,4-Dimethyl-3,5-di(4-methylthiophenyl)pyridine (11)
2,4-Dimethyl-3,5-di(4-methylthiophenyl)pyridine (11) was iso-
lated by flash chromatography on silica gel (eluent: diethylether/
A
mixture of formamide (70 g) and the ketone (3a)
(117.88 mmol, 21.01 g) was heated at 190 °C for 5 h. After cooling

4024
S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
hexane: 65/35). Amber oil (0.3%). IR mmax (film) 2851, 1597 cmꢁ1
.
4.1.16. 2-Methyl-2-methylthiobenzyl)-5-(4-methylthiophenyl)-
2,3-dihydropyrid-4-one (16)
1H NMR d (CDCl3) 1.94 (3H, s, CH3), 2.31 (3H, s, CH3), 2.52 (3H, s,
SCH3), 2.54 (3H, s, SCH3), 7.11, 7.25, 7.32, 7.35 (8H, 4ꢂd,
J = 8.5 Hz, J = 8.5 Hz, J = 8.5 Hz, J = 8.0 Hz, H-20, H-200, H-30, H-300,
H-50, H-500, H-60, H-600), 8.31 (1H, s, H-6). 13C NMR ppm (CDCl3)
15.62, 15.72, 18.09, 23.71, 126.40, 126.73, 129.40, 129.90, 134.97,
135.18, 135.80, 136.39, 137.66, 137.90, 142.94, 147.96, 155.10.
MS m/z 351 (M+), HRMS Calcd for C21H22NS2: (M++H) 352.1194.
Found: 352.1174.
2-Methyl-2–methylthiobenzyl)-5-(4-methylthiophenyl)-2,3-
dihydropyrid-4-one (16) was isolated by flash chromatography on
silica gel (eluent: diethylether). Colourless solid (0.7%) (ethylace-
tate/hexane), mp 148.5–149.5 °C. IR mmax (KBr) 3238, 1567 cmꢁ1
.
1H NMR d (CDCl3) 1.26 (3H, s, CH3), 2.46 (3H, s, SCH3), 2.47 (3H,
s, SCH3), 2.60 (2H, s, CH2), 2.73 (1H, d, Jgem = 13.0 Hz, H-3), 3.00
(1H, d, Jgem = 13.5 Hz, H-3), 4.90 (1H, d, J = 6.5 Hz, NH), 7.07, 7.21,
7.23, 7.32 (8H, 4ꢂd, J = 8.0 Hz, J = 7.5 Hz, J = 8.0 Hz, J = 8.5 Hz,
H-20, H-30, H-50, H-60), 7.25 (1H, d, J = 3.5 Hz, H-6). 13C NMR ppm
(CDCl3) 15.85 (SCH3), 16.46, 24.86, 43.36, 48.78, 56.77, 110.55,
126.66, 127.18, 128.03, 130.97, 132.61, 133.09, 135.22, 137.31,
147.34, 189.57. MS m/z 369 (M+). Anal. Calcd for C21H23NOS2: C,
68.25; H, 6.27; N, 3.79; S, 17.35. Found: C, 68.08; H, 6.19; N,
3.67; S, 17.67. HRMS Calcd for C21H23NOS2: (M+) 369.12210.
Found: 369.12215.
4.1.12. 2,6-Dimethyl-3,5-di(4-methylthiophenyl)pyridine (12)
2,6-Dimethyl-3,5-di(4-methylthiophenyl)pyridine (12) was iso-
lated by flash chromatography on silica gel (eluent: diethylether/
hexane: 60/40) followed by flash chromatography on silica gel (elu-
ent: hexane/ethylacetate: 88/12). Amber oil (0.4%). IR mmax (film)
2852, 1596 cmꢁ1 1H NMR d (CDCl3) 2.52 (6H, s, SCH3), 2.53 (6H,
.
s, CH3), 7.24–7.35 (8H, m, H-20, H-30, H-50, H-60), 7.30 (1H, s, H-4).
13C NMR ppm (CDCl3) 15.74, 22.99, 126.42, 129.50, 133.74,
136.43, 137.79, 138.31, 153.75. MS m/z 351 (M+). HRMS Calcd for
C21H22NS4: (M++1) 352.1194. Found: 352.1173.
4.1.17. N-Formyl-N,N-di(1-(4-methylthiophenyl)-2-propyl)-
amine (17)
N-Formyl-N,N-di(1-(4-methylthiophenyl)-2-propyl)amine (17)
was isolated by flash chromatography on silica gel as a 65/35 mix-
ture of diastereomers (eluent: diethylether/hexane: 75/25). Amber
4.1.13. 4-Methyl-2-(4-methylthiobenzyl)-5-(4-methylthiophe-
nyl)pyridine (13)
oil (2%). IR mmax (film) 1664, 1599 cmꢁ1 1H NMR d (CDCl3) 1.00
.
(1.05H, d, J3 ,2 = 7.0 Hz, H-30), 1.16 (1.95H, d, J3 ,2 = 6.5 Hz, H-30),
0
0
0
0
4-Methyl-2-(4-methylthiobenzyl)-5-(4-methylthiophenyl)pyri-
dine (13) was isolated by flash chromatography on silica gel (elu-
ent: diethylether/hexane: 60/40). Amber oil, (0.5%). IR mmax (film)
1.21 (1.05H, d, J3 ,2 = 7.0 Hz, H-30), 1.24 (1.95H, d, J3 ,2 = 7.0 Hz,
0
0
0
0
H-30), 2.44 (1.95H, s, SCH3), 2.45 (4.05H, s, SCH3), 2.59 (1.3H, d,
2853, 1594 cmꢁ1 1H NMR d (CDCl3) 2.21 (3H, s, CH3), 2.45 (3H, s,
.
J1 ,2 = 7.5 Hz, H-10), 2.68 (0.35H, dd, Jgem = 13.6 Hz, J1 ,2 = 8.0 Hz,
0
0
0
0
H-10), 2.80 (0.35H, dd, Jgem = 13.6 Hz, J1 ,2 = 7.0 Hz, H-10), 2.83
0
0
SCH3), 2.51 (3H, s, SCH3), 4.09 (2H, s, CH2), 6.99 (1H, s, H-3), 7.20
(2H, d, J = 8.5 Hz, ArH), 7.21–7.23 (4H, m, ArH), 7.31 (2H, d,
J = 8.5 Hz, ArH), 8.34 (1H, s, H-6). 13C NMR ppm (CDCl3) 15.66,
16.10, 19.83, 43.69, 124.32, 126.35, 127.16, 129.55, 129.65,
134.51, 134.82, 136.13, 136.61, 137.94, 145.00, 149.41, 159.32.
MS m/z 351 (M+). HRMS Calcd for C21H22NS2: (M++H) 352.1194.
Found: 352.1179.
(0.65H, dd, Jgem = 13.8 Hz, J1 ,2 = 7.8 Hz, H-10), 2.87 (0.35H, dd,
0
0
Jgem = 14.0 Hz, J1 ,2 = 8.0 Hz, H-10), 2.95 (0.65H, dd, Jgem = 13.6 Hz,
0
0
J1 ,2 = 7.0 Hz, H-10), 3.06 (0.35H, dd, Jgem = 13.8 Hz, J1 ,2 = 7.3 Hz,
H-10), 3.52 (1H, m, H-20), 3.98 (1H, m, H-20), 7.00–7.21 (8H, m,
H-2, H-3, H-5, H-6), 8.16 (0.35H, s, CHO), 8.18 (0.65H, s, CHO).
13C NMR ppm (CDCl3) 15.76*, 15.93, 16.05, 16.10*, 17.69, 17.81*,
20.68*, 20.93, 39.50*, 39.80, 42.51*, 42.80*, 51.47, 52.00*, 54.39,
54.59*, 126.91, 126.95, 126.99*, 127.02*, 129.47*, 129.60, 129.67,
134.78, 134.82*, 136.10, 136.16*, 136.18*, 136.76*, 162.17*,
162.23. MS m/z 373 (M+); HRMS Calcd for C21H28NOS2: (M++H)
374.1612. Found: 374.1611.
0
0
0
0
4.1.14. 2,4-Dimethyl-6-(4-methylthiobenzyl)-3-(4-methylthi-
ophenyl)pyridine (14)
2,4-Dimethyl-6-(4-methylthiobenzyl)-3-(4-methylthiophe-
nyl)pyridine (14) was isolated by flash chromatography on silica
gel (eluent: diethylether/hexane: 60/40). Amber oil (0.3%). IR mmax
4.1.18. 4-Methylthiotoluene
(film) 2853, 1587 cmꢁ1 1H NMR d (CDCl3) 1.95 (3H, s, CH3), 2.27
.
4-Methylthiotoluene was isolated as an impurity from the prep-
aration of (8a) by flash chromatography on silica gel (eluent: hex-
ane)as an amber oil (3.5%) (bp 52–54 °C/1 mmHg), identified by
comparison with authentic sample purchased from Sigma–Aldrich.
Rf 0.96 (diethylether/hexane: 80/20). IR mmax (film) 2854,
(3H, s, CH3), 2.46 (3H, s, SCH3), 2.51 (3H, s, SCH3), 4.07 (2H, s,
CH2), 6.79 (1H, s, H-5), 7.04 (2H, d, J = 8.0 Hz, ArH), 7.21–7.24
(4H, m, ArH), 7.30 (2H, d, J = 8.6 Hz, ArH). 13C NMR ppm (CDCl3)
15.63, 16.10, 20.24, 23.56, 43.81, 121.67, 126.58, 127.09, 129.51,
129.66, 133.96, 135.56, 135.99, 136.74, 137.35, 145.86, 155.57,
158.48. MS m/z 365 (M+). HRMS Calcd for C22H24NS4: (M++H)
366.1350. Found: 366.1346.
1598 cmꢁ1 1H NMR d (CDCl3) 2.35 (3H, s, CH3), 2.49 (3H, s,
.
SCH3), 7.13, 7.22 (4H, 2ꢂd, H-2, H-3, H-5, H-6). d 13C NMR ppm
(CDCl3) 16.56, 20.87, 127.37, 129.58, 134.71, 135.06. MS m/z 138
(M+, 100%).
4.1.15. 2-Methyl-6-(4-methylthiobenzyl)-3-(4-methylthiophe-
nyl)pyridine (15)
4.1.19. 2-(N-Formyl-N-methyl)amino-1-(4-methylthiophenyl)-
propane (18)
2-Methyl-6-(4-methylthiobenzyl)-3-(4-methylthiophenyl)pyri-
dine (15) was isolated by flash chromatography on silica gel (elu-
ent: diethylether/hexane: 60/40) followed by an additional flash
chromatography on silica gel (eluent: hexane/ethylacetate 90/
A solution of the ketone (3a) (25.32 mmol) in N-methylforma-
mide (64.41 mmol, 3.81 g) and 96% formic acid (1.84 g) was stirred
and refluxed at 150 °C for 7 h. After cooling the reaction was di-
luted with water (50 mL) and extracted with dichloromethane
(3 ꢂ 25 mL). The extracts were combined, washed with water
(2 ꢂ 50 mL) and satd aq NaCHO3 (2 ꢂ 25 mL), followed by drying
over anhydrous Na2SO4. The volatiles were removed in vacuo,
yielding an oil which was purified by flash chromatography over
10). Amber oil (0.4%). IR mmax (film) 2851, 1585 cmꢁ1 1H NMR d
.
(CDCl3) 2.46 (3H, s, SCH3), 2.50 (3H, s, SCH3), 2.52 (3H, s, CH3),
4.12 (2H, s, CH2), 6.93 (1H, d, J5,4 = 7.5 Hz, H-5), 7.21 (2H, d,
J = 8.5 Hz, ArH), 7.23 (4H, s, ArH), 7.30 (2H, d, J = 8.5 Hz, ArH),
7.37 (1H, d, J4,5 = 8.0 Hz, H-4). 13C NMR ppm (CDCl3) 15.76, 16.16,
23.43, 43.92, 120.20, 126.41, 127.18, 129.50, 129.70, 133.91,
136.16, 136.63, 136.73, 137.69, 137.75, 155.22, 159.06. MS m/z
351 (M+). HRMS Calcd for C21H22NS2: (M++H) 352.1194. Found:
352.1200.
silica gel (eluent: diethylether). Pale amber oil (73%). IR mmax (film)
2858, 1667 cmꢁ1. 1H NMR d (CDCl3) 1.16 (0.75H, d, J3 ,2 = 6.5 Hz, H-
0
0
30), 1.29 (2.25H, d, J3 ,2 = 6.5 Hz, H-30), 2.45 (3H, s, SCH3), 2.69–2.79
0
0
(2H, m, H-10), 2.77 (0.75H, s, NCH3), 2.79 (2.25H, s, NCH3), 3.75

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4025
(0.75H, m, H-20), 4.73 (0.25H, m, H-20), 7.00, 7.18 (3H, 2ꢂd,
J = 8.0 Hz, J = 8.5 Hz, H-2, H-3, H-5, H-6), 7.12, 7.18 (1H, 2ꢂd,
J = 8.0 Hz, J = 8.5 Hz, H-2, H-3, H-5, H-6), 7.80 (0.75H, s, CHO),
7.94 (0.25H, s, CHO). 13C NMR ppm (CDCl3) 15.85*, 15.94, 16.74,
18.91*, 24.85*, 29.69, 39.01, 40.07*, 47.83, 55.79*, 126.83,
127.02*, 129.19*, 129.30, 134.58*, 134.96, 136.15, 136.73*,
162.37*, 162.56. MS m/z 223 (M+); HRMS Calcd for C12H18NOS:
(M++H) 224.1109. Found: 224.1098.
(50 mL) and the solution was refluxed for 7 h. After cooling the
reaction was diluted with water (100 mL) and washed with dichlo-
romethane (3 ꢂ 30 mL). The aqueous phase was basified with 15%
aq NaOH and extracted with dichloromethane (3 ꢂ 50 mL). The or-
ganic phases were combined, dried over anhydrous Na2SO4 and
solvent removed in vacuo, yielding the product 1a as an amber
oil, (85%), confirmed by comparison with an authentic sample.
4.1.24. Preparation of 2-N-methylamino-1-(4-methylthiophe-
nyl)propane (2a) via N-formyl hydrolysis of 18
4.1.20. 2-(N-Formyl-N-methyl)amino-1-(4-methylthiophe-
nyl)butane (19)
To a stirred solution of N-formyl-N-methylaminoalkane (18)
(11.68 mmol) in methanol (15 mL) was added 30% aq HCl
(50 mL) and the solution was refluxed for 7 h. After cooling the
reaction was diluted with water (100 mL) and washed with dichlo-
romethane (3 ꢂ 30 mL). The aqueous phase was basified with 15%
aq NaOH and extracted with dichloromethane (3 ꢂ 50 mL). The or-
ganic phases were combined, dried over anhydrous Na2SO4 and
solvent removed in vacuo, yielding the product 2a as a pale amber
2-(N-Formyl-N-methyl)amino-1-(4-methylthiophenyl)butane
(19) was prepared from (3b) (2.00 g, 10.29 mmol scale) according
to general procedure for 18 above and isolated by flash chromatog-
raphy on silica gel (eluent: diethylether). Pale amber oil (67%). Rf
0.52 (diethylether). IR mmax (film) 2874, 1671 cmꢁ1 1H NMR d
.
(CDCl3) 0.87 (3H, t, J4 ,3 = 7.3 Hz, H-40), 1.57 (0.4H, m, H-30), 1.64
(1.6H, m, H-30), 2.45 (3H, s, SCH3), 2.72–2.83 (2H, m, H-10), 2.72
(2.4H, s, NCH3), 2.75 (0.6H, s, NCH3), 3.40 (0.8H, m, H-20), 4.55
(0.2H, m, H-20), 7.00, 7.18 (3.2H, 2ꢂd, J = 8.0 Hz, J = 8.6 Hz, H-6),
7.12, 7.19 (0.8H, 2ꢂd, J = 8.0 Hz, J = 8.6 Hz, H-6), 7.74 (0.8H, s,
CHO), 8.00 (0.2H, s, CHO). 13C NMR ppm (CDCl3) 10.62, 10.69*,
15.87*, 15.94, 23.89, 24.67*, 24.98*, 29.72, 37.66, 38.65*, 54.83,
62.24*, 126.83, 127.03*, 129.17*, 129.30, 134.66*, 134.96, 136.05,
136.64*, 163.02*, 163.24. MS m/z 237 (M+). HRMS Calcd for
C13H19NOSNa: (M++Na) 260.1085. Found: 260.1109.
0
0
oil (66%). IR mmax (film) 3321, 2788 cmꢁ1
.
1H NMR d (CDCl3) 1.04
(3H, d, J3 ,2 = 6.0 Hz, H-30), 1.35 (1H, br s, NH), 2.39 (3H, s, NCH3),
0
0
2.47 (3H, s, SCH3), 2.57 (1H, dd, Jgem = 13.3 Hz, J1 ,2 = 6.5 Hz, H-10),
0
0
2.67 (1H, dd, Jgem = 13.0 Hz, J1 ,2 = 7.0 Hz, H-10), 2.76 (1H, m,
H-20), 7.11, 7.20 (4H, 2ꢂd, J = 8.6 Hz, J = 8.6 Hz, H-2, H-3, H-5,
H-6). 13C NMR ppm (CDCl3) 16.13, 19.64, 33.94, 42.83, 56.26,
127.02, 129.76, 135.76, 136.50. MS m/z 195 (M++1). HCl salt. Col-
ourless solid. Mp 161–162 °C (ethanol/hexane).12,49 IR mmax (KBr)
2449 (NH+) cmꢁ1. Anal. Calcd 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%.
0
0
4.1.21. 2-(N-Formyl)amino-1-(4-methylthiophenyl)propane (8a)
A mixture of formamide (70 g) and the ketone 3a (117.88 mmol,
21.01 g) was heated at 190 °C for 5 h. After cooling the reaction
was diluted with water (150 mL) and extracted with ethylacetate
(3 ꢂ 75 mL). The extracts were combined, washed with water
(3 ꢂ 50 mL) and dried over anhydrous Na2SO4. After removing all
volatiles in vacuo the resulting black oil was purified by flash chro-
matography (eluent: ethyl acetate), providing the expected prod-
uct 8a, as a pale brown solid (52%) identified by comparison with
authentic sample, together with the impurities and reaction
byproducts 9–17 as reported above for direct preparation of 1a
by Leuckart route.
4.1.25. 2-Amino-1-(4-methylthiophenyl)butane (1b)
2-Amino-1-(4-methylthiophenyl)butane (1b) was prepared
from (8b) (0.54 g, 2.42 mmol scale) by hydrolysis in HCl according
to general procedure for hydrolysis of 8a above. Colourless oil
(53%). Rf 0.26 (methanol). IR mmax (film) 3360, 3292 cmꢁ1 1H
.
NMR d (CDCl3) 0.97 (3H, t, J4 ,3 = 7.5 Hz, H-40), 1.17 (2H, br s,
0
0
NH2), 1.36 (1H, m, H-30), 1.52 (1H, m, H-30), 2.41 (1H, dd,
Jgem = 13.6 Hz, J1 ,2 = 8.5 Hz, H-10), 2.47 (3H, s, SCH3), 2.75 (1H, dd,
0
0
Jgem = 13.5 Hz, J1 ,2 = 4.5 Hz, H-10), 2.88 (1H, m, H-20), 7.12, 7.21
(4H, 2ꢂd, J = 8.0 Hz, J = 8.5 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm
(CDCl3) 10.54, 16.21, 30.30, 43.69, 54.18, 127.11, 129.78, 135.75,
136.85. MS m/z 195 (M+). HCl salt. Colourless solid, mp 154–
155 °C (ethanol/hexane).49 IR mmax (KBr) 2600, 2531, 2449,
2362 cmꢁ1. Anal. Calcd for C11H18ClNS: C, 57.00; H, 7.83; N, 6.04.
Found: C, 56.90; H, 7.69; N, 5.78%.
0
0
4.1.22. 2-(N-Formyl)amino-1-(4-methylthiophenyl)butane (8b)
Compound 8b was prepared from 3b (2.00 g, 10.29 mmol scale)
according to general procedure for compound 8a above and isolated
by flash chromatography on silica gel (eluent: diethylether). Colour-
less needles (37%), mp 67–69 °C (diethylether/hexane). Rf 0.05
(diethylether/hexane: 50/50). IR mmax (KBr) 3300, 1656 cmꢁ1
.
1H
4.1.26. 2-N-Methylamino-1-(4-methylthiophenyl)butane (2b)
Compound 2b was prepared from 19 (1.50 g, 6.32 mmol scale)
by hydrolysis in HCl according to general procedure for the hydro-
lysis of 18 above. The product was obtained as a pale amber oil
(77%). Rf 0.16 (methanol), together with unreacted 19 (5%). IR mmax
NMR d (CDCl3) 0.93 (2.25H, t, J4 ,3 = 7.5 Hz, H-40), 0.96 (0.75H, t,
0
0
J4 ,3 = 7.5 Hz, H-40), 1.39 (1H, m, H-30), 1.62 (1H, m, H-30), 2.45 (3H,
0
0
s, SCH3), 2.62 (0.25H, dd, Jgem = 13.8 Hz, J1 ,2 = 8.3 Hz, H-10), 2.74
0
0
(0.75H, dd, Jgem = 15.0 Hz, J1 ,2 = 6.5 Hz, H-10), 2.77 (0.75H, dd,
0
0
Jgem = 15.0 Hz, J1 ,2 = 6.5 Hz, H-10), 2.80 (0.25H, dd, Jgem = 13.5 Hz,
(film) 3324, 2789 cmꢁ1. 1H NMR d (CDCl3) 0.93 (3H, t, J4 ,3 = 7.5 Hz,
H-40), 1.36–1.52 (2H, m, H-30), 1.55 (1H, br s, NH), 2.37 (3H, s,
NCH3), 2.47 (3H, s, SCH3), 2.53–2.72 (3H, m, H-10, H-20), 7.11,
7.20 (4H, 2ꢂd, J = 8.0 Hz, J = 8.0 Hz, H-2, H-3, H-5, H-6). 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 (M++1). HCl salt. Colourless so-
lid, mp 128–129 °C (ethanol/hexane). IR mmax (KBr) 2505,
2462 cmꢁ1. Anal. Calcd for C12H20ClNS: C, 58.63; H, 8.20; N 5.70;
S, 13.04. Found: C, 58.50; H, 8.22; N, 5.78; S, 12.75.
0
0
0
0
J1 ,2 = 5.0 Hz, H-10), 3.38 (0.25H, m, H-20), 4.14 (0.75H, m, H-20),
5.93 (0.25H, d, J = 10.5 Hz, NH), 5.60 (0.75H, d, J = 7.5 Hz, NH), 5.93
(0.25H, t, J = 10.5 Hz, NH), 7.05, 7.19 (1H, 2ꢂd, J = 8.0 Hz,
J = 8.0 Hz, H-2, H-3, H-5, H-6), 7.10, 7.18 (3H, 2ꢂd, J = 8.5 Hz,
J = 8.5 Hz, H-2, H-3, H-5, H-6), 7.72 (0.25H, d, J = 11.5 Hz, CHO),
8.10 (0.75H, s, CHO). 13C NMR ppm (CDCl3) 10.24*, 10.37, 15.84,
15.91*, 26.71*, 28.26, 39.74*, 41.94, 50.38*, 55.97, 126.78*, 126.91,
129.78*, 129.81, 134.26, 134.66*, 136.20*, 136.64, 160.73*, 164.07.
MS m/z 223 (M+). Anal. Calcd for C12H17NOS: C, 64.53; H, 7.67; N,
6.27. Found: C, 64.51; H, 7.58; N, 6.20.
0
0
4.1.27. Preparation of 2-N-methylamino-1-(4-methylthiophe-
nyl)butane (2b) via Leuckart–Wallach reaction
4.1.23. Preparation of 2-amino-1-(4-methylthiophenyl)propane
(4-MTA) (1a) via N-formyl hydrolysis of 8a
To a stirred solution of an appropriate N-formylaminoalkane
(8a) (11.68 mmol) in methanol (15 mL) was added 30% aq HCl
A mixture of formamide (60 g) and the ketone 3b (16.74 g,
86.16 mmol scale) was heated at 190° for 5 h according to the pro-
cedure used in the Leuckart–Wallach preparation of 1a above.
After cooling the reaction was diluted with water (150 mL) and

4026
S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
extracted with ethylacetate (3 ꢂ 75 mL). The extracts were com-
bined, washed with water (3 ꢂ 50 mL) and dried over anhydrous
Na2SO4. After removing all volatiles in vacuo the resulting dark
brown oil was dissolved in methanol (30 mL) and 30% aq HCl
(150 mL) an the mixture was heated under reflux for 7 h. Upon
cooling, the reaction was diluted with water (100 mL), made basic
(pH 14), with 15% aq NaOH and extracted with dichloromethane
(4 ꢂ 100 mL). The extracts were combined and dried over anhy-
drous Na2SO4. All volatiles were removed in vacuo, affording a dark
brown oil. Isolation of the desired product 2b, together with the
following reaction byproducts was performed by flash column
chromatography over silica gel.
chromatography on silica gel (eluent: hexane/diethylether:
70/30) followed by flash chromatography on acidic alumina (elu-
ent: diethylether). Amber oil (0.2%). Rf 0.88 (diethylether/hexane:
50/50). IR mmax (film) 2852, 1597 cmꢁ1 1H NMR d (CDCl3) 1.24
.
(6H, t, J = 7.5 Hz, CH2CH3), 2.52 (6H, s, SCH3), 2.81 (4H, q,
J = 7.5 Hz, CH2CH3), 7.26, 7.31 (8H, 2ꢂd, J = 8.5 Hz, J = 8.0 Hz, H-20,
H-30, H-50, H-60), 7.29 (1H, s, H-4). 13C NMR ppm (CDCl3) 14.23,
15.78, 28.45, 126.39, 129.57, 133.02, 136.66, 137.60, 138.72,
158.91. MS m/z 379 (M+ꢁ1). HRMS Calcd for C23H26NS2: (M++H)
380.1507. Found: 380.1496.
4.1.33. 4-Ethyl-3-methyl-2-(4-methylthiobenzyl)-5-(4-meth-
ylthiophenyl)pyridine (24)
4.1.28. 2-N-Formylamino-1-(4-methylthiophenyl)butane (8b)
2-N-Formylamino-1-(4-methylthiophenyl)butane (8b) was iso-
lated by flash chromatography on silica gel (eluent: ethyl acetate/
diethylether: 50/50). Colourless needles (5%), mp 67–69 °C (dieth-
ylether/hexane). Rf 0.05 (diethylether/hexane: 50/50); identified
by comparison with an authentic sample.
4-Ethyl-3-methyl-2-(4-methylthiobenzyl)-5-(4-methylthi-
ophenyl)pyridine (24) was isolated as an impurity from the prepa-
ration of (1b) by flash chromatography on silica gel (eluent:
hexane/diethylether: 60/40) followed by flash chromatography
on neutral alumina (eluent: hexane/ethylacetate: 80/20). Amber
oil (0.2%). Rf 0.46 on neutral alumina (diethylether/hexane: 50/
50). IR mmax (film) 2853, 1599 cmꢁ1 1H NMR d (CDCl3) 0.92 (3H,
.
4.1.29. 5-Methyl-4-(4-methylthiobenzyl)pyrimidine (20)
5-Methyl-4-(4-methylthiobenzyl)pyrimidine (20) was isolated
as an impurity from the preparation of (1b) by flash chromatogra-
phy on silica gel (eluent: diethylether). Amber oil (1%). Rf 0.13
t, J = 7.5 Hz, CH2CH3), 2.26 (3H, s, CH3), 2.45 (3H, s, SCH3), 2.53
(3H, s, SCH3), 2.57 (2H, q, J = 7.5 Hz, CH2CH3), 4.20 (2H, s, CH2),
7.17–7.19 (4H, m, ArH), 7.20, 7.31 (4H, 2ꢂd, J = 8.5 Hz, J = 8.5 Hz,
ArH), 8.20 (1H, s, H-6). 13C NMR ppm (CDCl3) 13.63, 14.08, 15.28,
15.74, 23.32, 41.74, 125.81, 126.67, 128.71, 129.42, 135.01,
135.19, 135.29, 136.00, 137.29, 138.80, 145.00, 146.48, 157.24.
MS m/z 379 (M+ꢁ1). HRMS Calcd for C23H26NS2: (M++H)
380.1507. Found: 380.1474.
(diethylether/hexane: 50/50). IR mmax (film) 2858, 1576 cmꢁ1 1H
.
NMR d (CDCl3) 2.24 (3H, s, CH3), 2.44 (3H, s, SCH3), 4.08 (2H, s,
CH2), 7.13, 7.18 (4H, 2ꢂd, J = 8.0 Hz, J = 8.5 Hz, H-20, H-30, H-50, H-
60), 8.42 (1H, s, H-6), 9.00 (1H, s, H-2). 13C NMR ppm (CDCl3)
15.62, 15.94, 40.97, 127.02, 129.29, 129.04, 133.83, 136.72,
156.63, 157.53, 166.71. MS m/z 230 (M+). HRMS Calcd for
C13H15N2S: (M++H) 231.0956. Found: 231.0948.
4.1.34. 5-Methyl-2-ethyl-6-(4-methylthiobenzyl)-3-(4-methyl-
thiophenyl)pyridine (25)
5-Methyl-2-ethyl-6-(4-methylthiobenzyl)-3-(4-methylthiophe-
nyl)pyridine (25) was isolated as an impurity from the preparation
of (1b) by flash chromatography on silica gel (eluent: hexane/
diethylether: 70/30) followed by flash chromatography on silica
gel (eluent: hexane/acetone 90/10). Amber oil (0.6%). Rf 0.84
4.1.30. 4-Ethyl-5-(3,4-methylthiophenyl)pyrimidine (21)
4-Ethyl-5-(3,4-methylthiophenyl)pyrimidine (21) was isolated
as an impurity from the preparation of (1b) by flash chromatogra-
phy on silica gel (eluent: diethylether/hexane 70/30) followed by
flash chromatography on neutral alumina (eluent: ethylacetate).
(diethylether/hexane: 50/50). IR mmax (film) 2869, 1594 cmꢁ1 1H
.
Amber oil (2%). Rf 0.34 (diethylether/hexane: 50/50). IR
m
max (film)
NMR d (CDCl3) 1.19 (3H, t, J = 7.3 Hz, CH2CH3), 2.20 (3H, s, CH3),
2.44 (3H, s, SCH3), 2.51 (3H, s, SCH3), 2.76 (2H, q, J = 7.5 Hz,
CH2CH3), 4.15 (2H, s, CH2), 7.18 (1H, s, H-4), 7.21 (2H, d,
J = 8.5 Hz, ArH), 7.18–7.20 (4H, m, ArH), 7.29 (2H, d, J = 8.5 Hz,
ArH). 13C NMR ppm (CDCl3) 14.38, 15.74, 16.15, 18.24, 28.15,
41.44, 126.31, 126.98, 129.21, 129.48, 128.32, 133.81, 135.60,
136.37, 136.80, 137.45, 139.50, 156.85, 157.35. MS m/z 379 (M+).
HRMS Calcd for C23H26NS2: (M++H) 380.1507. Found: 380.1525.
2858, 1576 cmꢁ1. 1H NMR d (CDCl3) 1.24 (3H, t, J = 7.5 Hz, CH2CH3),
2.54 (3H, s, SCH3), 2.78 (2H, q, J = 7.5 Hz, CH2CH3), 7.23, 7.35 (4H,
2ꢂd, J = 8.5 Hz, J = 8.0 Hz, H-20, H-30, H-50, H-60), 8.50 (1H, s, H-6),
9.11 (1H, s, H-2). 13C NMR ppm (CDCl3) 15.86, 15.52, 28.18,
126.45, 129.39, 132.30, 133.94, 139.26, 156.47, 157.40, 168.92.
MS m/z 230 (M+–1). HRMS Calcd for C13H15N2S: (M++H)
231.0956. Found: 231.0958.
4.1.31. 2,4-Diethyl-3,5-di(4-methylthiophenyl)pyridine (22)
2,4-Diethyl-3,5-di(4-methylthiophenyl)pyridine (22) was iso-
lated as an impurity from the preparation of (1b) by flash chroma-
tography on silica gel (eluent: hexane/diethylether: 40/60)
followed by flash chromatography on neutral alumina (eluent: hex-
ane/ethylacetate: 80/20). Amber oil (0.2%). Rf 0.52 on neutral alu-
mina (diethylether/hexane: 50/50). IR mmax (film) 2853, 1598
4.1.35. 3,5-Dimethyl-2,6-di(4-methylthiobenzyl)pyridine (26)
3,5-Dimethyl-2,6-di(4-methylthiobenzyl)pyridine (26) was iso-
lated as an impurity from the preparation of (1b) by flash chroma-
tography on silica gel (eluent: hexane/diethylether: 70/30)
followed by flash chromatography on silica gel (eluent: hexane/
acetone: 90/10). Amber oil (0.2%). Rf 0.75 (diethylether/hexane:
50/50). IR mmax (film) 2854, 1596 cmꢁ1 1H NMR d (CDCl3) 2.16
.
cmꢁ1
.
1H NMR d (CDCl3) 0.71 (3H, t, J = 7.5 Hz, CH2CH3), 1.15 (3H,
(6H, s, CH3), 2.50 (6H, s, SCH3), 4.11 (4H, s, CH2), 7.11, 7.15 (8H,
2ꢂd, J = 8.0 Hz, J = 8.5 Hz, H-20, H-30, H-50, H-60), 7.13 (1H, s, H-4).
13C NMR ppm (CDCl3) 16.25, 18.21, 41.12, 127.03, 129.14, 129.31,
135.45, 136.72, 140.34, 155.30. MS m/z 379 (M+). HRMS Calcd for
C23H26NS2: (M++H) 380.1507. Found: 380.1522.
t, J = 7.5 Hz, CH2CH3), 2.35 (2H, q, J = 7.5 Hz, CH2CH3), 2.52 (2H, q,
J = 7.5 Hz, CH2CH3), 2.53 (3H, s, SCH3), 2.55 (3H, s, SCH3), 7.15,
7.26, 7.32, 7.33 (8H, 4ꢂd, J = 8.0 Hz, J = 8.0 Hz, J = 8.0 Hz, J = 8.5 Hz,
H-20, H-200, H-30, H-300, H-50, H-500, H-60, H-600), 8.32 (1H, s, H-6). 13C
NMR ppm (CDCl3) 13.89, 14.59, 15.60, 15.73, 23.28, 29.35, 126.24,
126.32, 129.93, 129.95, 134.53, 135.12, 135.24, 135.39, 137.61,
137.80, 148.90, 149.13. MS m/z 379 (M+–1). HRMS Calcd for
C23H26NS2: (M++H) 380.1507. Found: 380.1499.
4.1.36. N,N-Di(1-(4-methylthiophenyl)-2-butyl)amine (27a)
N,N-Di(1-(4-methylthiophenyl)-2-butyl)amine (27a) was iso-
lated as an impurity from the preparation of (1b) by flash chromatog-
raphy on silica gel as a single diastereomer (eluent: diethylether/
hexane: 80/20). Amber oil (0.2%). Rf 0.18 (diethylether/hexane:
4.1.32. 2,6-Diethyl-3,5-di(4-methylthiophenyl)pyridine (23)
2,6-Diethyl-3,5-di(4-methylthiophenyl)pyridine
(23)
was
50/50). IR m
max (film) 3317, 1598 cmꢁ1. 1H NMR d (CDCl3) 0.75 (6H,
t, J4 ,3 = 7.3 Hz, H-40), 1.28–1.37 (5H, m, H-30, NH), 2.45 (8H, m,
0
0
isolated as an impurity from the preparation of (1b) by flash

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4027
SCH3, H-10), 2.52 (2H, dd, Jgem = 13.5 Hz, J1 ,2 = 5.0 Hz, H-10), 2.72 (2H,
m, H-20), 7.08, 7.18 (8H, 2ꢂd, J = 8.5 Hz, J = 8.0 Hz, H-2, H-3, H-5, H-6).
13C NMR ppm (CDCl3) 9.40, 16.29, 26.17, 40.57, 57.61, 127.02, 129.81,
135.48, 137.12. MS m/z 373 (M+–1). HRMS Calcd for C22H32NS2:
(M++H) 374.1976. Found: 374.1964.
75/25 mixture of diastereomers (eluent: ethylacetate). Amber oil
0
0
(19%). IR mmax (film) 3284 cmꢁ1
.
1H NMR d (CDCl3) 0.93 (3H, d,
J3 ,2 = 6.0 Hz, H-30), 1.02 (1H, d, J3 ,2 = 6.5 Hz, H-30), 1.12 (1H, br s,
0
0
0
0
NH), 2.45 (4H, s, SCH3), 2.46 (1H, s, SCH3), 2.45–2.52 (2H, m, H-
10), 2.58 (0.5H, dd, Jgem = 13.3 Hz, J1 ,2 = 6.8 Hz, H-10), 2.71 (1.5H,
0
0
dd, Jgem = 13.6 Hz, J1 ,2 = 6.0 Hz, H-10), 2.96 (2H, m, H-20), 6.94,
7.13 (2H, 2ꢂd, J = 8.0 Hz, J = 8.0 Hz, H-2, H-3, H-5, H-6), 7.06, 7.17
(6H, 2ꢂd, J = 8.6 Hz, J = 8.5 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm
(CDCl3) 16.01, 16.11*, 20.07*, 21.28, 42.65, 43.54*, 51.10*, 51.55,
126.78, 126.86*, 129.68, 129.75*, 135.62*, 135.63, 136.63, 136.48.
MS m/z 346 (M+). HRMS Calcd for C20H27NS2: (M+) 345.15849.
Found: 345.15922.
0
0
4.1.37. N,N-Di(1-(4-methylthiophenyl)-2-butyl)amine (27b)
N,N-Di(1-(4-methylthiophenyl)-2-butyl)amine (27b) was iso-
lated from the preparation of (1b) by flash chromatography on sil-
ica gel as a single diastereomer (eluent: diethylether/hexane: 80/
20). Amber oil (0.2%). Rf 0.27 (diethylether/hexane: 50/50). IR mmax
(film) 3317, 1598 cmꢁ1. 1H NMR d (CDCl3) 0.91 (6H, t, J4 ,3 = 7.5 Hz,
H-40), 1.29–1.43 (5H, m, H-30, NH), 2.46 (2H, dd, Jgem = 13.5 Hz,
0
0
J1 ,2 = 6.5 Hz, H-10), 2.47 (6H, s, SCH3), 2.54 (2H, dd, Jgem = 13.5 Hz,
4.1.42. Preparation of 2-amino-1-(4-methylthiophenyl)butane
(1b) by reductive amination (route B)
0
0
J1 ,2 = 6.0 Hz, H-10), 2.70 (2H, m, H-20), 6.93, 7.13 (8H, 2ꢂd,
J = 8.0 Hz, J = 8.5 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3)
9.86, 16.16, 27.04, 39.79, 57.54, 126.89, 129.81, 135.49, 136.71.
m/z 373 (M+–1). HRMS Calcd for C22H32NS2: (M++H) 374.1976.
Found: 374.1979.
0
0
2-Amino-1-(4-methylthiophenyl)butane (1b) was prepared
from 3b and ammonium acetate as described for 1a above. Colour-
less solid (53%), mp 109–110 °C. HCl salt. Colourless solid. mp 154–
155 °C (ethanol/hexane),49 identified by comparison with an
authentic sample.
4.1.38. N-Methyl-N,N-di(1-(4-methylthiophenyl)-2-butyl)amine
(28)
N-Methyl-N,N-di(1-(4-methylthiophenyl)-2-butyl)amine (28)
was isolated as an impurity from the preparation of (1b) by flash
chromatography on silica gel (eluent: hexane/acetone 85/15) as a
60/40 mixture of diastereomers. Amber oil (1%). Rf 0.67 (diethyl-
4.1.43. N,N-Di(1-(4-methylthiophenyl)-2-butyl)amine (27a)
N,N-Di(1-(4-methylthiophenyl)-2-butyl)amine (27a) was iso-
lated by flash chromatography on silica gel as a single diastereo-
mer (eluent: diethylether/hexane: 80/20) as an impurity from the
preparation of 1b by reductive amination of ketone 3b with ammo-
nium acetate. The product was isolated as an amber oil (0.2%), and
identified by comparison with an authentic sample.
ether/hexane: 50/50). IR mmax (film) 1664, 1599 cmꢁ1
.
1H NMR d
0
(CDCl3) 0.75 (2.4H, t, J4 ,3 = 7.3 Hz, H-40), 0.78 (3.6H, t, J4 ,3 = 7.3 Hz,
H-40), 1.20–1.43 (4H, m, H-30), 2.28 (1.2H, s, NCH3), 2.29 (1.8H, s,
NCH3), 2.45 (6H, s, SCH3), 2.47 (0.8H, m, H-10), 2.49 (1.2H, dd,
0
0
0
4.1.44. 2-N-Methylamino-1-(4-methylthiophenyl)propane (2a)
2-N-Methylamino-1-(4-methylthiophenyl)propane (2a) was
prepared from 3a and methylamine HCl as described for 1a above
and obtained as a colourless oil, (39%), which was identified by
comparison with an authentic sample.
Jgem = 14.5 Hz, J1 ,2 = 6.5 Hz, H-10), 2.69 (2H, m, H-20), 2.80 (0.8H,
0
0
dd, Jgem = 13.1 Hz, J1 ,2 = 5.5 Hz, H-10), 2.86 (1.2H, dd, Jgem = 13.3 Hz,
0
0
J1 ,2 = 4.8 Hz, H-10), 7.05, 7.16 (8H, 2ꢂd, J = 8.0 Hz, J = 8.0 Hz, H-2,
H-3, H-5, H-6). 13C NMR ppm (CDCl3) 11.41*, 16.40*, 16.45,
24.32, 24.42*, 29.50*, 30.08, 37.74, 37.81*, 65.67, 66.21*, 127.11*,
129.72*, 129.74, 134.90, 134.98*, 138.58*, 138.64. m/z 387 (60%
diastereomer M+–1), MS m/z 387 (40% diastereomer M+–1). HRMS
Calcd for C23H34NS2: (M++H) 388.2133. Found: 388.2147.
0
0
4.1.45. 2-N-Methylamino-1-(4-methylthiophenyl)butane (2b)
2-N-Methylamino-1-(4-methylthiophenyl)butane (2b) was
prepared from 3b and N-methylamine HCl as described for 1a
above. The product was isolated as a colourless oil (77%) and iden-
tified by comparison with an authentic sample.
4.1.39. Preparation of 2-amino- and 2-N-alkylamino-1-(4-meth-
ylthiophenyl) alkanes (1a,1b) and (2a,2b) via reductive amina-
tion (route B)
4.1.46. 1-(4-Methylthiophenyl)-2-propanone oxime (30a)
A stirred solution of the ketone (3a) (15.23 mmol) and hydrox-
ylamine HCl (36.00 mmol, 2.51 g) in pyridine (12.5 mL) and etha-
nol (12.5 mL) was refluxed for 2 h. After cooling the reaction was
acidified with 10% aq HCl and extracted with dichloromethane
(3 ꢂ 50 mL). The organic phases were combined, dried over anhy-
drous Na2SO4, and solvent removed in vacuo. The crude product
was purified by flash chromatography on silica gel (eluent: dieth-
ylether/hexane: 35/65), providing a 25/75 mixture of syn/anti iso-
mers. Colourless solid (85%), mp 57–59 °C (ethanol/water). IR mmax
(KBr) 3260, 1668 cmꢁ1. anti-(30a). 1H NMR d (CDCl3) 1.81 (2.25H,
s, H-30), 2.46 (3H, s, SCH3), 3.45 (1.5H, s, H-10), 7.14, 7.20 (3H,
2ꢂd, J = 8.0 Hz, J = 8.6 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm
(CDCl3) 16.02, 19.61, 41.51, 127.05, 129.45, 133.61, 136.72,
157.49. syn-(30a). 1H NMR d (CDCl3) 1.80 (0.75H, s, H-30), SCH3 sig-
nal overlapping with anti-(30a), 3.70 (0.5H, s, H-10), 7.15, 7.19 (1H,
2ꢂd, J = 8.5 Hz, 9.5 Hz, H-2, H-3, H-5, H-6). 13C NMR ppm (CDCl3)
16.06, 19.61, 34.25, 127.11, 129.62, 133.42, 136.34, 156.75. MS
m/z 195 (syn/anti isomer mixture, M+). Anal. Calcd for C10H13NOS:
C, 61.50; H, 6.71; N, 7.17. Found: C, 61.40; H, 6.71; N, 7.08.
To
a stirred mixture of the appropriate ketone (3a, 3b)
(22.45 mmol) in dry methanol (100 mL) there was added either
ammonium acetate or methylamine HCl salt (179.60 mmol) and so-
dium cyanoborohydride (31.84 mmol, 2.00 g) and the reaction was
stirred at ambient temperature 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 result-
ing aqueous phase washed with dichloromethane (3 ꢂ 50 mL). The
aqueous phase basified with 15% aq NaOH solution and extracted
with dichloromethane (3 ꢂ 50 mL). The organic phases were com-
bined, dried over anhydrous Na2SO4, and volatiles removed in vacuo
leaving the product as an oil. The acidic phase organic washes were
each combined, washed with 15% aq NaOH (5 mL) and the residue
chromatographed to afford synthesis related impurities.
4.1.40. 2-Amino-1-(4-methylthiophenyl)propane (1a)
2-Amino-1-(4-methylthiophenyl)propane (1a) was obtained as
a colourless oil (80%), which was identified by comparison with
an authentic sample.
4.1.47. 1-(4-Methylthiophenyl)-2-butanone oxime (30b)
1-(4-Methylthiophenyl)-2-butanone oxime (30b) was prepared
from (3b) (2.50 g, 12.87 mmol scale) according to general proce-
dure for 30a above without the need for flash chromatography,
4.1.41. N,N-Di(1-(4-methylthiophenyl)-2-propyl)amine (29)
N,N-Di(1-(4-methylthiophenyl)-2-propyl)amine (29) was iso-
lated as an impurity by flash chromatography on silica gel as a

4028
S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
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 mmax (KBr) 3220, 1669 cmꢁ1. syn-(30b). 1H
methylthiophenyl)aziridine (31) isolated by flash chromatography
on silica gel (eluent: ethylacetate) as an amber oil (3%). Rf 0.21
(diethylether/hexane: 80/20). IR mmax (KBr) 3302, 2869 cmꢁ1 1H
.
NMR d (CDCl3) 1.04 (1.8H, t, J4 ,3 = 7.5 Hz, H-40), 2.18 (1.2H, q,
NMR d (CDCl3) 0.89 (3H, d, J = 6.0 Hz, CH3), 1.06 (1H, br s, NH),
2.38 (1H, m, H-3), 2.46 (3H, s, SCH3), 3.18 (1H, m, H-2), 7.21,
7.25 (4H, 2ꢂd, J = 8.5 Hz, J = 8.0 Hz, H-20, H-30, H-50, H-60). 13C
NMR ppm (CDCl3) 13.48, 15.97, 32.11, 36.60, 126.34, 128.21,
134.69, 136.23. m/z 179 (M+ꢁ1). HRMS Calcd for C10H14NS:
(M++H) 180.0847. Found: 180.0856.
0
0
J3 ,4 = 7.5 Hz, H-30), 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 observed. 13C
NMR ppm (CDCl3) 10.62, 16.04, 27.00, 32.90, 127.10, 128.54,
133.62, 136.19, 160.42. anti-(30b). 1H NMR d (CDCl3) 0.99 (1.2H,
0
0
t, J4 ,3 = 7.5 Hz, H-40), 2.31 (0.8H, q, J3 ,4 = 7.7 Hz, H-30), SCH3 signal
overlapping with syn-(30b), 3.46 (0.8H, s, H-10), H-2, H-3, H-5, H-6
signals overlapping with syn-(30b), OH signal not observed. 13C
NMR ppm (CDCl3) 10.00, 16.08, 20.49, 39.43, 127.03, 129.51,
133.69, 136.62, 161.78. MS m/z (syn/anti isomer mixture) 209
(M+). Anal. Calcd for C11H15NOS: C, 63.12; H, 7.22; N, 6.69. Found:
C, 63.46; H, 7.11; N, 6.50.
0
0
0
0
4.1.51. Preparation of 2-amino-1-(4-methylthiophenyl)butane
(1b) via reduction of nitrostyrene 4b (route D)
Compound 1b was prepared from 4a as described for 1a
above. Purification of the crude product by flash chromatography
over silica gel (eluent: ethylacetate) provided the product 2a,
(74%), identified by comparison with an authentic sample. The
following synthesis byproducts was also obtained: 3-Ethyl-2-(4-
methylthiophenyl) aziridine (32) was isolated by flash chroma-
tography on silica gel (eluent: diethylether/hexane: 80/20) as a
colourless oil, (7%). Rf 0.51 (diethylether/hexane: 80/20). IR mmax
4.1.48. Preparation of 2-amino-1-(4-methylthiophenyl)alkanes
(1a and 1b) via oxime reduction (route C)
To a stirred and refluxing suspension of LiAlH4 under nitrogen
(52.70 mmol, 2.00 g) in the dry THF (100 mL) there was added
dropwise over 20 min a solution of the appropriate 1-(4-methyl-
thiophenyl)-2-alkanone oxime (30a) (10.24 mmol) in dry THF
(50 mL). The reaction was maintained at the specified temperature
for 3 h. After cooling in an ice bath the excess LiAlH4 was quenched
by the successive dropwise additions of propan-2-ol (2 mL), 15% aq
NaOH (2 mL) and water (15 mL). The aluminium salts were re-
moved by filtration and washed with THF (100 mL). All organic
phases were combined and volatiles removed in vacuo, leaving a
residue which was diluted with dichloromethane (100 mL). This
was dried over anhydrous Na2SO4 and volatiles again removed in
vacuo leaving an oil which was purified by flash chromatography
over silica gel (diethylether/hexane:50/50) to afford the amine
products: 2-Amino-1-(4-methylthiophenyl)propane (1a) was iso-
lated by flash chromatography on silica gel (eluent: methanol)
and was obtained as an amber oil(50%), identified by comparison
with an authentic sample. 1-(4-Methylthiophenyl)-2-propanone
oxime (30a) was also recovered by flash chromatography on silica
gel (eluent: diethylether/hexane: 50/50) as an amber solid, 18%.
(film) 3308, 2871 cmꢁ1 1H NMR d (CDCl3) 0.85 (3H, t, J = 7.3 Hz,
.
CH2CH3), 1.17 (1H, m, CH2CH3), 1.21 (1H, br s, NH), 1.24 (1H,
m, CH2CH3), 2.24 (1H, m, H-3), 2.47 (3H, s, SCH3), 3.22 (1H, d,
J2,3=5.5 Hz, H-2), 7.20, 7.26 (4H, 2ꢂd, J = 8.0 Hz, J = 8.5 Hz, H-20,
H-30, H-50, H-60). 13C NMR ppm (CDCl3) 11.32, 16.04, 21.43,
36.59, 38.97, 126.37, 128.18, 134.89, 136.22. MS m/z 193 (diaste-
reomer M+).
4.1.52. 1-(4-Methylthiophenyl)-1-nitroethane (34)
To a vigorously stirred mixture of 1-(4-methylthiophenyl)-1-
nitroethene (33)50 (15.00 mmol), silica gel (30 g) and propan-2-ol
(45 mL) in dichloromethane (240 mL) at room temperature there
was added, over a period of 5 min, NaBH4 (61.50 mmol, 2.38 g).
The mixture was stirred for an additional 15 min or until the reac-
tion has turned from yellow to colourless. Excess NaBH4 was
quenched by the addition of dilute HCl (50 mL). The mixture was
filtered and silica gel washed with dichloromethane (100 mL). All
organic phases were combined and washed successively with brine
(3 ꢂ 50 mL) and water (3 ꢂ 50 mL). The organic phase was dried
over anhydrous Na2SO4 and solvent removed in vacuo, leaving a
brown oil. This was purified by flash chromatography on silica
gel. (2.96 g, 15.00 mmol scale) and chromatographed on silica gel
(eluent: hexane/diethylether: 65/35). Amber oil (89%). IR mmax
4.1.49. Preparation of 2-amino-1-(4-methylthiophenyl)butane
(1b) via oxime reduction (route C)
2-Amino-1-(4-methylthiophenyl)butane (1b) was obtained
from oxime (30b) following the method above and was isolated
by flash chromatography over silica gel 30% yield and identified
by comparison with an authentic sample, together with unreacted
oxime 30b, (1%).
(film) 1549, 1378 cmꢁ1
.
1H NMR d (CDCl3) 2.46 (3H, s, SCH3),
3.27 (2H, t, J1 ,2 = 7.3 Hz, H-10), 4.57 (1H, t, J2 ,1 =7.3 Hz, H-2 ).
7.12, 7.21 (4H, 2d, J = 8.0 Hz, J = 8.0 Hz, H-2, H-3, H-5, H-6). 13C
NMR ppm (CDCl3) 15.81, 32.85, 76.16, 127.12, 128.99, 132.38,
137.73. MS m/z 197 (M+). HRMS Calcd for C9H11NO2S: (M+)
197.05105. Found: 197.05167.
0
0
0
0
0
4.1.50. Preparation of 2-amino-1-(4-methylthiophenyl)alkane
4-MTA (1a) via reduction of nitrostyrene 4a (route D)
To a stirred solution of LiAlH4 (184.45 mmol, 7.50 g) in dry THF
(150 mL) under nitrogen there was added dropwise over 30 min, a
solution of 4a, (37.78 mmol) in dry THF (100 mL). The reaction was
then refluxed for 8 h. After cooling in an ice bath the excess LiAlH4
was quenched by the successive dropwise additions of propanol-2-
ol (7.5 mL), 15% aq NaOH (7.5 mL) and water (20 mL). The alumin-
ium salts were removed by filtration and washed with THF
(100 mL). All organic phases were combined and reduced in vacuo.
The resulting residue was dissolved in dichloromethane (100 mL),
dried over anhydrous Na2SO4 and solvent removed in vacuo, leav-
ing an oil. The residue was purified by flash chromatography on sil-
ica gel (eluent: ethylacetate) providing the product 1a, (80%),
identified by comparison with an authentic sample. The following
synthesis byproducts were also obtained: 1-(4-Methylthiophenyl)-
2-propanone oxime (30a), isolated by flash chromatography on sil-
ica gel (eluent: ethylacetate) as a colourless solid (0.6%) and iden-
tified by comparison with an authentic sample. 3-Methyl-2-(4-
4.1.53. 1,3-Di(4-methylthiophenyl)-2-nitro-1-propene (35)
A mixture 4-methylthiobenzaldehyde (5) (3.20 g, 21.00 mmol)
the nitroalkane (34) (21 mmol), dimethylamine HCl (44.36 mmol,
3.61 g) and potassium fluoride (3.66 mmol, 0.19 g) in toluene
(30 mL) was refluxed with a Dean–Stark trap for 24 h. The reaction
was then diluted with toluene (150 mL) and the organic phase
washed with 10% aq HCl (3 ꢂ 75 mL). The organic phase was dried
over anhydrous Na2SO4, and volatiles were removed in vacuo leav-
ing an orange oil which was chromatographed on silica gel (eluent:
hexane/diethylether: 70/30). Yellow crystals (73%), mp 81–82 °C
(diethylether/hexane). IR mmax (KBr) 1636, 1506, 1312 cmꢁ1 1H
.
NMR d (CDCl3) 2.46 (3H, s, SCH3), 2.49 (3H, s, SCH3), 4.22 (2H, s,
H-3), 7.12, 7.22, 7.24, 7.35 (8H, 4ꢂd, J = 8.6 Hz, J = 8.6 Hz,
J = 8.5 Hz, J = 8.0 Hz, H-20, H-200, H-30, H-300, H-50, H-500, H-60, H-600),
8.25 (1H, s, H-1). 13C NMR ppm (CDCl3) 14.92, 15.97, 32.58,
125.98, 127.33, 128.08, 130.24, 127.98, 133.50, 137.07, 148.52,

S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
4029
143.02. MS m/z 331 (M+). Anal. Calcd for C17H17NO2S2: C, 61.60; H,
4.1.58. 1,3-Di(4-methylthiophenyl)-2-propanone oxime (38)
5.17; N, 4.23. Found: C, 61.56; H, 5.13; N, 4.13.
1,3-Di(4-methylthiophenyl)-2-propanone oxime (38) was pre-
pared from 7 (0.80 g, 2.64 mmol scale) according to general proce-
dure for compound (19a) above, and was isolated as colourless
needles (73%), mp 109–110 °C (ethanol/water). IR mmax (KBr)
4.1.54. 1,3-Di(4-methylthiophenyl)-2-propanone (7)
1,3-Di(4-methylthiophenyl)-2-propanone (7) was prepared
from 35 (2.38 g, 7.54 mmol scale) as described for (3a) above.
The product was obtained as colourless needles (72%) following
purification by flash chromatography on silica gel (eluent: hex-
ane/diethylether: 90/10). mp 80–81 °C (diethylether/hexane)
3229 cmꢁ1 1H NMR d (CDCl3) 2.50 (3H, s, SCH3), 2.51 (3H, s,
.
SCH3), 3.42 (2H, s, H-1), 3.63 (2H, s, H-3), 7.05, 7.07, 7.17, 7.18
(8H, 4ꢂd, J = 8.0 Hz, J = 8.0 Hz, J = 8.0 Hz, J = 8.0 Hz H-20, H-30, H-
50, H-60). 13C NMR ppm (CDCl3) 16.06, 16.07, 32.04, 39.06, 127.04,
127.09, 129.62, 129.70, 133.18, 133.36, 136.38, 136.78, 158.83.
MS m/z 317 (M+). Anal. Calcd for C17H19NOS2: C, 64.32; H, 6.03;
N, 4.41. Found: C, 64.33; H, 5.96; N, 4.42.
(lit.47 mp 79–80 °C). IR mmax (KBr) 1698 cmꢁ1 1H NMR d (CDCl3)
.
2.46 (6H, s, SCH3), 3.66 (4H, s, H-1), 7.06, 7.20 (8H, 2ꢂd,
J = 8.5 Hz, H-20, H-30, H-50, H-60). 13C NMR ppm (CDCl3) 15.91,
48.46, 127.00, 129.90, 130.69, 137.25, 205.31. MS m/z 302 (M+).
4.1.59. Preparation of 1,3-di(phenyl substituted)-2-aminopro-
pane (37) via oxime reduction
4.1.55. 2-N-Formylamino-1,3-di(4-methylthiophenyl)propane
(36)
Compound 37 was prepared by LiAlH4 reduction of 38 (0.45 g,
1.42 mmol scale) according to general procedure for reduction of
compound 30a above. The product was obtained as a pale amber
solid (17%), mp 52–53 °C. Spectroscopic analyses were comparable
to an authentic sample.
2-N-Formylamino-1,3-di(4-methylthiophenyl)propane
(36)
was prepared from 7 (1.50 g, 4.96 mmol scale) according to the
procedure for compound (8a) above following chromatography
on silica gel (eluent: diethylether) as colourless needles (15%),
mp 132–133 °C (diethylether/hexane). IR mmax (KBr) 3326,
1658 cmꢁ1
.
1H NMR d (CDCl3) 2.46 (6H, s, SCH3), 2.66 (0.5H, dd,
4.1.60. 2-(4-Methylthiobenzyl)-3-(4-methylthiophenyl)aziri-
dine (39)
Jgem = 14.0 Hz, J1,2 = 8.5 Hz, H-1), 2.74 (1.5H, dd, Jgem = 14.1 Hz,
J1,2 = 7.0 Hz, H-1), 2.82 (1.5H, dd, Jgem = 13.8 Hz, J1,2 = 6.5 Hz, H-1),
2.87 (0.5H, dd, Jgem = 13.8 Hz, J1,2 = 4.5 Hz, H-1), 3.70 (0.25H, m,
H-2), 4.48 (0.75H, m, H-2), 5.35 (0.75H, d, J = 8.0 Hz, NH), 5.60
(0.25H, t, J = 10.8 Hz, NH), 7.05, 7.20 (2H, 2ꢂd, J = 8.5 Hz,
J = 8.5 Hz, H-20, H-30, H-50, H-60), 7.10, 7.19 (6H, 2ꢂd, J = 8.5 Hz,
J = 8.0 Hz, H-20, H-30, H-50, H-60), 7.48 (0.25H, d, J = 12.0 Hz, CHO),
8.01 (0.75H, s, CHO). 13C NMR ppm (CDCl3) 15.87, 15.95*, 39.23*,
41.64, 50.16*, 55.68, 126.92*, 127.04, 129.76*, 129.83, 133.82,
134.38*, 137.06*, 160.60*, 163.68. MS m/z 331 (M+). Anal. Calcd
for C18H21NOS2: C, 65.22; H, 6.39; N, 4.23. Found: C, 65.29; H,
6.34; N, 4.15. HRMS Calcd for C19H24NOS2: (M++H) 346.1299.
Found: 346.1314.
2-(4-Methylthiobenzyl)-3-(4-methylthiophenyl)aziridine (39)
was isolated as an impurity by flash chromatography on silica gel
(eluent: diethylether) from the preparation of 37 by LiAlH4 reduc-
tion of 38. Colourless needles (61%), mp 97–99 °C (diethylether/
hexane). Rf 0.64 (methanol). IR mmax (KBr) 3174 cmꢁ1 1H NMR d
.
(CDCl3) 1.13 (1H, s, NH), 2.35 (1H, dd, Jgem = 14.3 Hz, J = 6.3 Hz,
CH2), 2.44 (3H, s, SCH3), 2.48 (1H, dd, Jgem = 12.6 Hz, J = 1.5 Hz,
CH2), 2.49 (3H, s, SCH3), 2.52 (1H, m, H-2), 3.30 (1H, d,
J3,2=6.5 Hz, H-3), 6.98, 7.15, 7.24, 7.31 (8H, 4ꢂd, J = 8.5 Hz,
J = 8.5 Hz, J = 8.0 Hz, J = 8.0 Hz, H-20, H-30, H-50, H-60). 13C NMR
ppm (CDCl3) 16.06, 16.26, 33.75, 36.77, 38.53, 126.46, 127.07,
128.30, 129.23, 134.50, 135.70, 136.69, 136.77. MS m/z 301 (M+).
Anal. Calcd for C17H19NS2: C, 67.73; H, 6.35; N, 4.65. Found: C,
67.70; H, 6.30; N, 4.68%.
4.1.56. 2-(N-Formyl-N-methyl)amino-1,3-di(4-methylthiophe-
nyl)propane (40)
2-(N-Formyl-N-methyl)amino-1,3-di(4-methylthiophenyl)pro-
pane (40) was prepared from 7 (1.00 g, 3.31 mmol scale) according
to procedure for compound 19a above and isolated as an amber oil
(29%), which was used without further purification for the next
4.1.61. 2-N-Methylamino-1,3-di(4-methylthiophenyl)propane
(41)
Compound 41 was prepared from 40 as described above for 2a
and obtained as a pale amber oil (76%). IR mmax (film) 3310,
reaction. IR mmax (film) 1666 cmꢁ1
.
1H NMR d (CDCl3) 2.45 (6H, s,
2797 cmꢁ1 1H NMR d (CDCl3) 1.68 (1H, br s, NH), 2.41 (3H, s,
.
SCH3), 2.63 (0.6H, s, NCH3), 2.80 (2.4H, s, NCH3), 2.83–2.94 (4H,
m, H-1), 3.47 (0.8H, m, H-2), 4.75 (0.2H, m, H-2), 6.99, 7.12
(1.6H, 2ꢂd, J = 9.0 Hz, J = 8.5 Hz, H-20, H-30, H-50, H-60), 7.00, 7.18
(6.4H, 2ꢂd, J = 8.6 Hz, J = 8.6 Hz, H-20, H-30, H-50, H-60), 7.51 (0.8H,
s, CHO), 7.85 (0.2H, s, CHO). 13C NMR ppm (CDCl3) 15.89, 15.90*,
25.44*, 30.31, 37.10, 38.26*, 62.37*, 126.93, 127.14*, 129.18*,
129.34, 134.29*, 134.83, 136.34, 137.00*, 162.88*, 163.03. MS m/z
345 (M+).
NCH3), 2.49 (6H, s, SCH3), 2.63 (2H, dd, Jgem = 13.8 Hz, J1,2 =
6.3 Hz, H-1), 2.70 (2H, dd, Jgem = 13.8 Hz, J1,2 = 6.8 Hz, H-1), 2.90
(1H, m, H-2), 7.12, 7.22 (8H, 2ꢂd, J = 8.0 Hz, J = 8.5 Hz, H-20, H-30,
H-50, H-60). 13C NMR ppm (CDCl3) 16.12, 34.12, 39.56, 62.58,
127.04, 129.79, 135.86, 136.35. MS m/z 317 (M+). HRMS Calcd for
C18H24NS2: (M++H) 318.1350. Found: 318.1355.
4.1.62. 2-N-Acetylamino-1,3-di(4-methylthiophenyl)-propane
(42)
4.1.57. 2-Amino-1,3-di(4-methylthiophenyl)propane via
N-formyl hydrolysis (37)
To a stirred solution of 37 (2.92 mmol) in pyridine (6 mL) was
added acetic anhydride (5.84 mmol, 0.60 g, 0.55 mL) and the mix-
ture was stirred at ambient temperature for 1 h. The reaction was
acidified with 10% aq HCl (100 mL) and extracted with dichloro-
methane (3 ꢂ 25 mL). The extracts were combined, dried over
anhydrous Na2SO4 and volatiles removed in vacuo, generating an
amber oil which slowly solidified. Recrystallisation provided the
pure product as pale amber needles (64%),mp 137–138 °C (diethyl-
Compound 37 was prepared from 36 as described above for 1a
and obtained as a pale amber solid (84%), mp 52–53 °C. IR mmax
(KBr) 3340 cmꢁ1 1H NMR d (CDCl3) 1.45 (2H, s, NH2), 2.47 (6H, s,
.
SCH3), 2.51 (2H, dd, Jgem = 13.5 Hz, J1,2=8.5 Hz, H-1), 2.79 (2H, dd,
Jgem = 13.3 Hz, J1,2=5.0 Hz, H-1), 3.22 (1H, m, H-2), 7.13, 7.21 (8H,
2ꢂd, J = 8.5 Hz, J = 8.5 Hz, H-20, H-30, H-50, H-60). 13C NMR ppm
(CDCl3) 16.15, 43.59, 54.09, 127.12, 129.77, 136.06, 136.35. MS
m/z 303 (M+). Anal. Calcd for C17H21NS2: C, 67.28; H, 6.97; N,
4.62. Found: C, 67.36; H, 7.02; N, 4.67. HCl salt. Colourless solid.
Mp 248–252 °C (dec.). IR mmax (KBr) 2579, 2478 cmꢁ1. Anal. Calcd
for C17H22ClNS2: C, 60.06; H, 6.52; N, 4.12. Found: C, 59.98; H,
6.39; N, 4.11.
ether/dichloromethane). IR
(CDCl3) 1.85 (3H, s, COCH3), 2.46 (6H, s, SCH3), 2.70 (2H, dd,
m .
max (KBr) 3294, 1647 cmꢁ1 1H NMR d
0
0
Jgem = 14.0 Hz, J1 ,2 = 7.0 Hz, H-1), 2.79 (2H, dd, Jgem = 14.0 Hz,
0
0
J1 ,2 = 6.5 Hz, H-1), 4.41 (1H, m, H-2), 5.24 (1H, d, J = 8.0 Hz, NH),
7.09, 7.19 (8H, 2ꢂd, J = 8.0 Hz, J = 8.0 Hz, H-20, H-30, H-50, H-60).
13C NMR ppm (CDCl3) 15.98, 23.37, 39.14, 50.99, 126.87, 129.76,

4030
S. M. Cloonan et al. / Bioorg. Med. Chem. 18 (2010) 4009–4031
136.35, 169.46. MS m/z 345 (M+). Anal. Calcd for C19H23NOS2: C,
66.05; H, 6.71; N, 4.05. Found: C, 65.75; H, 6.69; N, 4.01.
(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% cytotoxicity on all cell
lines. Untreated cells represented 0% cytotoxicity (100% viability).
4.1.63. 2-N-Ethylamino-1,3-di(4-methylthiophenyl)propane
(43)
To a stirred suspension of LiAlH4 (32.94 mmol, 1.25 g) in dry
THF (100 mL) under nitrogen was added dropwise over 15 min a
solution of 42 (7.50 mmol) in dry THF (100 mL) and the reaction
was heated at reflux for 24 h. After cooling the reaction in an ice
bath, excess LiAlH4 was decomposed by the successive dropwise
additions of propan-2-ol (1.5 mL), 15% aq NaOH (1.5 mL) and water
(12 mL). All organic phases were combined and volatiles removed
in vacuo, leaving a residue that was dissolved in dichloromethane
(100 mL). This was dried over anhydrous Na2SO4 and solvent again
removed in vacuo, leaving a residue that was purified by flash
chromatography on silica gel. (eluent: methanol). Pale amber oil
4.2.3. Statistical analysis
4.2.3.1. Non-linear regression analysis. Each compound was
screened over a 1 lM–1 mM concentration 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 deter-
mined by non-linear regression analysis of sigmoidal log concen-
tration dependence curves whereby pEC50 is ꢁ[ꢁLog EC50
]
SE
(log EC50 is the log [Dose] when response is equal to 50% cell viabil-
ity) (Tables 3 and 4). This was carried out as in order to express the
EC50 values as EC50 SEM (standard error of the mean) as the
EC50 SEM cannot be calculated from a Log scale. All data points
(expressed as means S.E.M.) were analysed using GRAPHPAD
Prism (version 4) software (Graphpad software Inc., San Diego, CA).
(30%). IR mmax (film) 3312 cmꢁ1 1H NMR d (CDCl3) 0.99 (3H, t,
.
J = 7.0 Hz, NCH2CH3), 1.31 (1H, br s, NH), 2.46 (6H, s, SCH3), 2.59–
2.69 (6H, m, H-1, NCH2CH3), 2.98 (1H, m, H-2), 7.09, 7.19 (8H,
2ꢂd, J = 8.0 Hz, J = 8.0 Hz, H-20, H-30, H-50, H-60). 13C NMR ppm
(CDCl3) 15.28, 16.07, 39.99, 41.53, 60.72, 126.96, 129.73, 135.78,
136.36. MS m/z 331 (M++1). HCl salt. Colourless solid. mp 156–
4.2.3.2. Comparison of pEC50 values in all cell lines. To deter-
mine if the pEC50 value calculated for each drug differed signifi-
cantly 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.
158 °C (ethanol/hexane). IR mmax (KBr) 2618, 2480, 2353 cmꢁ1
.
Anal. Calcd for C19H26ClNS2: C, 62.01; H, 7.12; N, 3.81. Found: C,
62.05; H, 7.05; N, 3.80%.
4.2. Biochemistry
4.2.1. Cell culture
HEK293 cells lines stably overexpressing SERT, NAT and DAT
were obtained from Dr. Patrick Schloss (Central Institute for Men-
tal Health, Mannheim, Germany) and were cultured in DMEM sup-
plemented with 10% (v/v) fetal bovine serum (FBS),
L
-glutamine
4.2.3.3. Comparison of pEC50 values to 4-MTA. To determine if
the pEC50 value calculated for each drug differed significantly to
4-MTA, statistical analysis was carried out using an unpaired
T-test, comparing each pEC50 value to the pEC50 value of 4-MTA.
A P value of <0.05 was considered to reflect a significant difference.
(2 mM), penicillin/streptomycin (100 mg/mL) and Geneticin
(500 mg/mL). Stable expression was valid up to 30 passages. The
PC-12 cell line was obtained from Dr. Gabriele Schmuck (Bayer
HealthCare AG). PC-12 cells are derived from a transplantable rat
pheochromocytoma. Undifferentiated PC-12 cells were cultured
in DMEM/F12 (1:1) supplemented with 10% (v/v) horse serum,
Acknowledgements
5% (v/v) FBS,
(100 g/mL) and were grown on collagen coated plates. Cells were
cultured for seven days in DMEM/F12 (1:1) supplemented with 1%
(v/v) Horse serum, 5% (v/v) FBS, -glutamine (2 mM), penicillin/
streptomycin (100 g/mL) supplemented with 100 ng/mL of nerve
growth factor (NGF) with media changed every two days. After se-
ven days, all cells displayed a differentiated morphology and a sta-
tic population was obtained for an additional seven days
thereafter. SHSY-5Y cells were purchased from the European Col-
lection of Cell Cultures (ECACC Lot04/c/011p17) and were cultured
in DMEM/F12 (1:1) supplemented with 10% (v/v) FBS, sodium
L-glutamine (2 mM) and penicillin/streptomycin
l
The authors sincerely thank Dr. Patrick Schloss (Central Insti-
tute for Mental Health, Mannheim, Germany) and Dr. Gabriele
Schmuck (Bayer HealthCare AG) for kindly donating cells used in
this study and the staff of the Forensic Laboratory, Phoenix Park,
Dublin. 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.
L
l
pyruvate (1 mM), L-glutamine (2 mM), non-essential amino acids
(0.1 mM) and penicillin/streptomycin (100 lg/mL). Cells were
Supplementary data
maintained in a 72 cm2 tissue culture flasks at 37 °C in a humidi-
fied atmosphere of 95% oxygen, and 5% carbon dioxide.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.022.
4.2.2. Neutral red assay
5 ꢂ 104 cells per well (200
lL) were seeded in a 96 well plate
until sub-confluent (24–36 h) and treated with the appropriate
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