Synthesis and serotonin transporter activity of 1,3-bis(aryl)-2-nitro-1- propenes as a new class of anticancer agents

Bioorganic & Medicinal Chemistry 19 (2011) 1328–1348

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Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Synthesis and serotonin transporter activity of 1,3-bis(aryl)-2-nitro-1-propenes

as a new class of anticancer agents

Yvonne M. McNamara ^a,, Suzanne M. Cloonan ^b,, Andrew J. S. Knox ^b, John J. Keating ^a,à, Stephen G. Butler ^a,

Günther H. Peters ^c, Mary J. Meegan ^a, D. Clive Williams ^b,

⇑

^aSchool of Pharmacy and Pharmaceutical Sciences, Centre for Synthesis and Chemical Biology, Trinity College Dublin, Ireland

^bSchool of Biochemistry and Immunology, Trinity College Dublin, Ireland

^cEMPHYS-Center for Biomembrane Physics, Department of Chemistry, Technical University of Denmark, Building 206, 2800 Kgs. Lyngby, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history:

Structural derivatives of 4-MTA, an illegal amphetamine analogue have been previously shown to have

anticancer effects in vitro. In this study we report the synthesis of a series of novel 1,3-bis(aryl)-2-

nitro-1-propene derivatives related in structure to 4-MTA. A number of these compounds containing a

classic nitrostyrene structure are shown to have antiproliferative activities in vitro in a range of malig-

nant cell lines, particularly against Burkitt’s lymphoma derived cell lines, whilst having no effect on ‘nor-

mal’ peripheral blood mononuclear cells. Such effects appear to be independent of the serotonin

transporter, a high afﬁnity target for amphetamines and independent of protein tyrosine phosphatases

and tubulin dynamics both of which have been previously associated with nitrostyrene-induced cell

death. We demonstrate that a number of these compounds induce caspase activation, PARP cleavage,

chromatin condensation and membrane blebbing in a Burkitt’s lymphoma derived cell line, consistent

with these compounds inducing apoptosis in vitro. Although no speciﬁc target has yet been identiﬁed

for the action of these compounds, the cell death elicited is potent, selective and worthy of further

investigation.

Received 21 September 2010

Revised 22 November 2010

Accepted 23 November 2010

Available online 7 December 2010

Keywords:

4-Methylthioamphetamine (4-MTA)

Serotonin transporter (SERT)

Burkitt’s lymphoma

Apoptosis

Programmed cell death

Cancer

Nitrostyrene

1. Introduction

back into pre-synaptic neurons determining the duration and mag-

nitude of 5-HT responses. It is an important high afﬁnity target

The serotonin transporter (SERT) transports 5-hydroxytrypta-

mine (5-HT) from central and enteric nervous system synapses

in vivo for antidepressants and a non-selective target for stimu-

lants including cocaine and amphetamines. Despite its abundance

in the nervous system, SERT is also expressed in a wide range of

specialised non-neuronal cells as well as being expressed in a num-

ber of B cell malignancies including diffuse large B cell lymphoma,

multiple myeloma and Burkitt’s lymphoma (BL).¹Recently, a num-

ber of SERT-targeting ligands have been shown to induce apoptosis

in a number of malignant cell lines including Burkitt’s lym-

phoma.^2–6SERT has also been implicated in serotonin-mediated

apoptosis in Burkitt’s lymphoma⁷and in the mechanism of cyto-

toxicity associated with the amphetamine analogues, fenﬂura-

mine⁸and 3,4-methylenedioxymethamphetamine (MDMA).^9,10

Although these ligands can target the serotonin transporter, their

anticancer effects are thought to occur independently of SERT^1,2,11

suggesting that (a) molecular target(s) other than SERT may exist

on the lymphoma cell with the possibility of these ligands prefer-

entially targeting the proliferating malignant cell.¹

Abbreviations: 4-MTA, 4-methylthioamphetamine; 5-HT, 5-hydroxytryptamine;

BL, Burkitt’s lymphoma; BSA, bovine serum albumin; CML, chronic myelogenous

leukaemia; DAT, dopamine transporter; DMEM, Dulbecco’s modiﬁed Eagle’s me-

dium; ECACC, European collection of cell cultures; ER, estrogen receptor; ESI-MS,

electrospray-ionisation mass spectrometry; FACS, ﬂuorescence activated cell sorter;

FBS, foetal bovine serum; HBSS, Hanks balanced salt solution; HRMS, high

resolution mass spectrometry; IR, infra-red; LRMS, low resolution mass spectrom-

etry; MDA, 3,4-methylenedioxyamphetamine; MDMA, 3,4-methylenedioxymeth-

amphetamine; MOE, molecular operating environment; NAT, noradrenaline

transporter; NMR, nuclear magnetic resonance; NR, neutral red; PBMC, peripheral

blood mononuclear cells; PBS, phosphate buffered saline; PCA, para-chloroamphet-

amine; PCD, programmed cell death; PI, propidium iodide; PMA, para-methoxy-

amphetamine; PMK, 1-(3,4-methylenedioxyphenyl)-2-propanone; PTP, Protein

tyrosine phosphatase; SAR, structure–activity relationship; SERT, serotonin

transporter; SSRI, selective serotonin reuptake inhibitor; TBNS, trans-b-nitrosty-

rene; TCA, tricyclic antidepressant; TMS, tetramethylsilane; TLC, thin layer

chromatography.

Amphetamine analogues substituted at the 4-position with an

⇑

Corresponding author. Tel.: +353 1 8962596; fax: +353 1 8963130.

E-mail address: clive.williams@tcd.ie (D.C. Williams).

alkylthio group are

a pharmacologically important class of

compounds which demonstrate potent biochemical activity in

the central nervous system. The parent member of the series,

4-MTA (4-methylthioamphetamine) (Fig. 1) is recognised as a

Authors contributed equally to this work.

Present address: School of Pharmacy, Cavanagh Pharmacy Building, College Road,

à

University College Cork, Cork, Ireland.

doi:10.1016/j.bmc.2010.11.054

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1329

Leuckart–Wallach route of 4-MTA synthesis.¹²The compounds

chosen for the present study were designed to have modiﬁcations

to the aromatic substituents, and also include derivatives where

the amino group is alkylated and acylated. The complete panel of

compounds evaluated in this study are synthesized via multi-step

synthesis as illustrated in Scheme 1 based on modiﬁed literature

procedures.^19–21

O

NH₂

S

4-MTA

S

MDMA

S

NH

H

O

A Henry–Knovenagel reaction of the appropriate benzaldehyde

1a–1h and nitromethane is employed to produce the 1-aryl-2-

nitroethenes 2a–2h. The alkenes were reduced with NaBH₄and sil-

ica gel to give 1-aryl-1-nitroethanes 3a–3h. This was then followed

by a second Henry condensation of 1-aryl-1-nitroethane 3a–3h

with the appropriate benzaldehyde 1a–1h, potassium ﬂuoride

and N,N-dimethylamine hydrochloride to afford a series of 1,3-bi-

s(aryl)-2-nitro-1-propenes 4a–4i with variable yields (20–60%).

These nitrostyrene compounds 4a–4d were then reduced with

LiAlH₄to give the primary amines 5a–5d. 1,3-Bis(aryl)-2-nitro-1-

propenes 4a–4c and 1,3-bis(aryl)-2-propanamines 5a–5d can be

used as precursors for a variety of structurally related analogues.

Compounds 4a–4c were reduced using iron powder and acetic acid

to give 1,3-bis(aryl)-2-propanones 6a–6c. These propanone com-

pounds (6a, 6b) can then undergo the Leuckart reaction using N-

methylformamide, to generate tertiary amides 12a and 12b. When

reacted with formamide, propanones 6a–6c afforded the secondary

amides 7a–7c. Pyrimidine based impurities 14a and 14b were iso-

lated from this reaction in high yields (5% and 7%) compared to the

yields of the N-formyl compounds 7a and 7b (15% and 20%).

Numerous examples of pyrimidine impurities arising from the Leu-

ckart reactions of ketones and formamide have been documented

in the literature,^17,22however pyrimidines derived from dibenzylk-

etone impurities have not been documented. In the ¹H NMR spec-

trum for compound 14b, a singlet at d 4.04 integrating for two

protons is characteristic of the methylene protons of a benzyl

7a

Figure 1. Structure of 4-MTA (4-methylthioamphetamine), MDMA (3,4-methylen-

edioxymethamphetamine) and 1,3-bis(aryl)-2-propanamine compound 7a.

MDMA-like drug of abuse and is classiﬁed as a Schedule-1 con-

trolled substance.¹²4-MTA is a ligand for SERT, is a potent inhibitor

of serotonin reuptake in the rat brain, a serotonin releasing agent

in rat brain synaptosomes and has been shown to have inhibitory

effects on 5-HT-mediated vascular contraction in isolated rat aor-

tas.¹³Recently, our laboratory reported the isolation and study of

route-speciﬁc byproducts associated with the clandestine synthe-

sis of 4-MTA.¹²In our study, a N-formyl-1,3-bis(4-methylthiophe-

nyl)-2-propanamine compound (7a) (Fig. 1) was shown to have a

cytotoxic effect on a malignant neuroblastoma cell line, SHSY-5Y

(EC₅₀of 32 lM), and was found to be selectively toxic to SERT-

overexpressing human embryonic kidney cells (HEK).¹²

As compounds of type 7a are structurally related derivatives of

4-MTA and MDMA, it was proposed that these derivatives could

behave like amphetamines by binding to SERT and therefore may

have potential as antiproliferative agents against lymphomas, in

particular, Burkitt’s lymphoma. The initial objective of this study

is to therefore synthesize a number of structurally related ana-

logues of 7a and investigate their SERT-binding activity and anti-

proliferative potential against Burkitt’s lymphoma derived cell

lines. Burkitt’s lymphoma accounts for 30–50% of lymphomas in

children and remains a serious health problem in those areas

where it is endemic: namely the malarial belts of equatorial Africa,

north-eastern Brazil, and Papua New Guinea.⁷In addition, because

of its growing association with HIV infection, Burkitt’s lymphoma

is becoming a more common malignancy in adults, making up

the largest group of HIV-associated non-Hodgkin lymphomas

(35–50% of these neoplasms).¹⁴A variety of chemotherapeutic

drugs are used in the treatment of BL tumours including DNA inter-

calating agents and topoisomerase inhibitors. Such drugs are usu-

ally successfully supplemented with the monoclonal antibody

rituximab which sensitises B cell lymphomas and leukaemias to

chemotherapy¹⁵allowing for survival rates of up to 60% in children

affected with the disease. However, for older adults and HIV in-

fected patients the long term survival rates in response to chemo-

therapy is only 25%^7,16with reoccurrence and resistance common.

In addition, in parts of the developing world where BL is endemic,

access to chemotherapeutic regimens is limited. Consequently,

there is a need to develop more selective, potent, economical alter-

natives for the treatment of Burkitt’s lymphoma.

group positioned

a to a nitrogen of a heteroaromatic ring. Two

singlets, each integrating for two protons, at d 5.90 and d 6.05 cor-

respond to the OCH₂O protons of the methylenedioxyphenyl and

benzyl rings, respectively. Two 1,3,4-trisubstituted ring systems

are evident from the splitting patterns of six resonances between

d 6.50 and d 6.90, each integrating for one proton. At d 8.54, a sin-

glet integrating for one proton is characteristic of a proton

a to one

pyrimidine ring nitrogen, while a further singlet at d 9.13 is as-

signed to the proton located between both pyrimidine nitrogens.

Compounds 5b, 6b and 7b have been previously identiﬁed by Bohn

et al. as impurities arising from the synthesis of MDA from 3,4-

methylenedioxyphenylacetic acid by the Leuckart route.¹⁷In this

study these compounds were synthesized as previously reported

by Piﬂ et al..²¹We have previously reported the synthesis of com-

pounds 4a, 5a, 6a, 7a, 10a, 11a, 12a and 13a, 15a¹³and they have

been included in Scheme 1 for clarity.

Hydrolysis of the N-formyl amines 7a and 7b with methanolic

HCl affords the primary amine derivatives 5a and 5b in sufﬁcient

yields (84%), however when the same hydrolysis conditions were

applied to N-formyl-N-methyl propanamines 12a and 12b, only

trace amounts of N-methylamines 13a and 13b were formed. Reac-

tion conditions were changed to include a concentrated HCl/meth-

anol mixture, resulting in much higher yields of the hydrolysed

products 13a and 13b (76% and 84%). Compounds 12b and 13b

have previously been identiﬁed by Bohn et al., as impurities of

MDMA synthesized via the Leuckart reaction from PMK (1-(3,4-

methylenedioxyphenyl)-2-propanone), which itself arises as a syn-

thetic byproduct from the acetic acid route of MDMA synthesis

from 3,4-methylenedioxyphenylacetic acid.¹⁷Compounds 5a and

5b, when reacted with acetic anhydride and pyridine gives the

amides 10a and 10b, which are then reduced with LiAlH₄to the

N-ethyl derivatives 11a and 11b. The ketone 6b is reduced directly

with NaBH₄to give 1,3-(3,4-methylenedioxyphenyl)-2-propanol

In this study, we wish to therefore evaluate the potential activ-

ity of a series of 1,3-bis(aryl)-2-nitro-1-propenes as novel antipro-

liferative agents against Burkitt’s lymphoma.

2. Results and discussion

2.1. Chemistry

Synthetic routes available for the production of amphetamines,

together with their characteristic impurities have been extensively

reported.^17–19We have previously examined the synthesis of a

series of 1,3-bis(aryl)-2-propanamine compounds structurally

similar to 7a, which are related to synthetic byproducts in the

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Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

Scheme 1. Reagents and conditions: (a) CH₃NO₂, cyclohexylamine, AcOH, 6 h reﬂux; (b) NaBH₄silica gel, propan-2-ol, CH₂Cl₂, room temperature; (c) HN(CH₃)₂ÁHCl, KF,

toluene, 24 h reﬂux; (d) LiAlH₄, benzene, THF, 3 h reﬂux; (e) Fe, AcOH; (f) NH₂CHO 190 °C, 5 h; (g) NH₂OHÁHCl, pyridine, 2 h reﬂux; (h) NaBH₄, propan-2-ol; (i) CH₃NHCHO,

HCOOH, 150 °C, 7 h; (j) Ac₂O, pyridine, 1 h, room temperature; (k) MeOH, concd HCl, 7 h reﬂux; (l) 30% aq HCl, MeOH, reﬂux, 7 h. Compounds 14a, 14b, 15a and 15b are

formed as route-speciﬁc byproducts during the synthesis of compounds 7a, 7b, 5a and 5b, respectively.

9b. The oxime 8b is obtained from 6b via reaction with hydroxyl-

amine hydrochloride and pyridine. Oxime 8b was subsequently

reduced with LiAlH₄to afford the 1,3-bis(3,4-methylenedioxy-

phenyl)-2-propanamine 5b, along with a novel aziridine based

byproduct 15b. The aziridine derivative was formed in high yields

(62%) compared to the desired propanamine 5b (27%).

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1331

2.2. SERT-dependent cytotoxicity

kitt’s lymphoma cells (Fig. 2D). Dose–response curve analysis of the

apoptotic effect of 7a in DG-75 and MUTU-I cell lines estimated the

A number of novel pyridines, dihydropyridone and N,N-di(1-

aryl-2-propyl) amines described as route-speciﬁc byproducts

associated with clandestine synthesis of 4-MTA and related

amphetamines¹²were previously evaluated for SERT-selective

cytotoxicity using human embryonic kidney cells overexpressing

SERT, and two malignant cell lines; the DG-75 cell line, which

has been previously shown by our laboratory to express SERT^2,13

and to the SHSY-5Y neuroblastoma cell line, which does not

express SERT.²³As previously described, the compound of most

interest from this study was the N-formyl-1,3-bis(4-methylthi-

ophenyl)-2-propanamine (7a) (Fig. 1) which was found to be cyto-

toxic to the hSERT-overexpressing cell line and not to the HEK293

cell line implying a selective-SERT cytotoxic effect.¹²

Compound 7a was therefore selected as a lead compound that

may have potential to target SERT-expressing malignancies such

as BL. Further investigations by this study found compound 7a to

be selectively antiproliferative towards two Burkitt’s lymphoma

cell lines (DG-75, a chemoresistant cell line and MUTU-1, a chemo-

sensitive cell line²⁴), (Fig. 2A), having little effect on a number of

breast-cancer derived cell lines (Fig. 2B) and having no effect on

peripheral blood mononuclear cells (Fig. 2C). It was also found to in-

duce the formation of apoptotic bodies, as detected by propidium

iodide (PI) FACS (ﬂuorescence activated cell sorter) analysis in Bur-

EC₅₀values to be approximately 10 lM and 13 lM, respectively

(data not shown). These results show that compound 7a is a novel

antiproliferative apoptotic agent selective for B cell malignancies.

In this study, the cytotoxic activity of the series of 1,3-bis(aryl)-2-

nitro-1-propenes based on the structure of 7a and described in Sec-

tion 2.1, were evaluated in the HEK, HEK-SERT, DG-75 and SHSY-5Y

cell lines (presented in Table 1). Again, compound 7a had a potent

effect on the DG-75, Burkitt’s lymphoma cell line (pEC₅₀4.5) as well

as to SHSY-5Y neuroblastoma cell line (pEC₅₀4.5) (Table 1). In gen-

eral, this study found that the majority of compounds had cytotoxic

effects in the low micromolar range (pEC₅₀values of between 4 and

5, correlating to an EC₅₀value of between 1 and 20 lM) to HEK cells

and to hSERT-overexpressing cells consistentwith these compounds

having no selective cytotoxic effects towards SERT (P <0.05). The sul-

fur-substituted 4-MTA compounds 5a, 7a, 10a and 11a have previ-

ously been reported to be cytotoxic to monoamine-expressing cell

lines as well as to the PC-12 neuronal cell line model.¹²

Compounds 5a and 11a (previously found to have cytotoxic ef-

fects to a SERT-over expressing cell line, a dopamine transporter

(DAT)-over expressing cell line as well as to the SHSY-5Y cell line

but having little effect on a noradrenaline transporter (NAT) over-

expressing cell line¹²) were found by this study to have potent

cytotoxic effects (pEC₅₀4.6–4.9) to all of the cell lines including

Figure 2. Compound 7a is a potent antiproliferative pro-apoptotic agent against Burkitt’s lymphoma but has no effect on PBMC’s. (A, B and C) 1–5 Â 10⁴cells/200

ll were

seeded and treated with the 10 lM or 50 lM 7a for 24 h (MUTU-I, 4TI, MDA-MB231 and MCF-7) or 72 h (DG-75). 10 ll of Alamar Blue reagent was added to each well and

ﬂuorescence was read at 544 nm (excitation 590 nm) and values represent the mean value the SEM of six data points (recording in triplicate on two independent days).

Large error bars may indicate low solubility of 7a at high concentrations. (D) 7 Â 10⁵cells/5 mL Cells were treated with 50

lM 7a for 24 h (MUTU-I) and 72 h (DG-75),

harvested by centrifugation and ﬁxed overnight in 70% ethanol. FACS analysis was carried out upon incubation with propidium iodide and RNase A. 10,000 cells were counted

using appropriate gates. Values represent the mean SEM of three independent experiments.

1332

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

the Burkitt’s lymphoma cell line, DG-75 (Table 1). Compounds 5a

and 11a both contain methylthio-substituents at the 6-position

of the aromatic rings, which is similar in structure to 4-MTA (also

been shown to have a general cytotoxic effect in vitro¹²).

a crystal structure of human SERT. However, amphetamines in

general 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.²⁷The amphetamine analogues, 4-MTA, MDMA,

3,4-methylenedioxyamphetamine (MDA) and para-chloroamphet-

amine (PCA) have all been previously shown to inhibit SERT activ-

ity with IC₅₀values of 74 nM (4-MTA), 425 nM (MDMA), 478 nM

(MDA) and 182 nM (PCA), respectively.¹³These amphetamines also

inhibit the activity of the noradrenaline transporter (2,375 nM

(4-MTA), 405 nM (MDMA), 266 nM (MDA) and 207 nM (PCA))

and inhibit the activity of the dopamine transporter (3,073 nM

(4-MTA), 1,442 nM (MDMA), 890 nM (MDA) and 424 nM (PCA)).¹³

In this study, a subset of the 1,3-bis(aryl)-2-nitro-1-propene

and 1,3-bis(aryl)-2-propanamines were synthesized and evaluated

for their ability to bind to and inhibit SERT (Table 2). The percent-

age reuptake for the compounds was initially determined at

Compounds 5c and 5b were also found to be cytotoxic to all the

cell lines used in this study. Interestingly derivatives 5b, 4b, 7b and

6b have been previously reported as synthetic byproducts of clan-

destine MDMA synthesis²¹and when investigated for interaction

with human monoamine transporters were found to interact with

SERT at a similar potency to MDMA without displaying any neuro-

toxic effects (carrier-mediated release experiments).²¹In this study

we report that the 1,3-bis(3,4-methylenedioxyphenyl)-2-prop-

amine (5b) and the 1,3-bis(3,4-methylenedioxyphenyl)-2-nitro-1-

propene (4b) are cytotoxic to HEK cells and to HEK cells over-

expressing SERT with EC₅₀values in the low micromolar range

(pEC₅₀values of 4–5 correlating to an approximate EC₅₀value of

1–10

effect on the two malignant cell lines, DG-75 and SHSY-5Y (pEC₅₀

4.7–6.5, EC₅₀values 0.5–10 M). Compounds 7b and 6b were

lM) (Table 1). These derivatives also had a potent cytotoxic

l

found to have little effect on the hSERT overexpressing cell line

in agreement with Piﬂ et al.²¹and were also found to have little ef-

fect on the malignant cell lines. These results suggest that the free

amine and nitroethene functional groups may be involved in the

cytotoxic ability of these compounds, with no signiﬁcant difference

between the toxicity of compounds with 3,4-methylenedioxy- and

4-methoxy- aromatic substituents. Compounds 10a (previously

found to have a general cytotoxic effect to all monoamine trans-

porter-expressing cell lines and to the SHSY-5Y cell line¹²), 10b

and compound 7c (which was more cytotoxic to the HEK cell line

compared to the hSERT-overexpressing HEK cell line) (Table 1)

were found to have little effect to the DG-75 cell line and the

SHSY-5Y cell line.

We conclude that the majority of these 1,3-bis(aryl)-2-propan-

amines have signiﬁcant cytotoxic effects against the BL cell line

DG-75 and the neuroblastoma cell line SHSY-5Y. We ﬁnd that com-

pound 7a had a potent effect on the SHSY-5Y neuroblastoma and

on the DG-75 Burkitt’s lymphoma cell line, implying that SERT

may not be involved in its cytotoxic mechanism of action (Table

1). By contrast, the methoxy analogue of 7a, compound 7b an N-

formyl-1,3-bis(aryl)2-propanamine had little effect on the SHSY-

5Y cell line but was found to be cytotoxic to the BL cell line imply-

ing that the aldehyde is possibly partly responsible for the selective

activity of these derivatives against BL and the cytotoxic effects of

compound 7b may be mediated through SERT. To address this

hypothesis, compounds will be evaluated for SERT inhibitory activ-

ity in Section 2.3.

concentrations of 1 and 100

bition of the more potent compounds (>80% uptake inhibition at

100 M) was then determined from the appropriate sigmoidal

dose-dependent curves (data not shown). The selective serotonin

reuptake inhibitor (SSRI antidepressant) citalopram (10 M) was

used as a positive control, resulting in >97% SERT inhibition. The

IC₅₀for citalopram was determined as 19.3 nM (compared to a

reported K_ivalue of 8.9 nM²⁸).

The lead compound 7a was found to inhibit SERT with an IC₅₀of

15.7 lM, implying that the cytotoxic activity of 7a is unlikely

mediated through SERT, as the IC₅₀value for SERT binding is high

compared to amphetamines 4-MTA and MDMA (74 nM and

425 nM, respectively). Compound 7b, the methoxy analogue of

7a also showed little SERT inhibition, again implying that that

the cytotoxic activity of 7b may not be mediated through SERT.

lM. The IC₅₀value for reuptake inhi-

l

At concentrations of 100 and 1

enes 4a–4d and 1,3-bis(aryl)-2-propanamines 5c, 7c, 10a, did not

bind to SERT with <20% inhibition observed at 1 M. Compounds

lM, 1,3-bis(aryl)-2-nitro-1-prop-

l

5a, 5b, 5d, 7a and 11a displayed SERT inhibition in a low micromo-

lar range, with the most active compound 5b, having an IC₅₀of

1.6

l

M. Compounds 5a, 5d and 11a showed a similar afﬁnity for

M,

SERT compared to 5b, with IC₅₀values of 2.5, 1.9 and 2.2

l

respectively. Previous studies investigating the SERT inhibition of

compounds 5b and 7b found that while a similar SERT inhibition

activities were found for compound 5b (IC₅₀of 7.7 1.5

pared to 2.2 M in our study), contrasting activities were found

for compound 7b, as no SERT inhibition was observed in this study

(compared to 8.0 2.0 M).

lM com-

l

Lastly, overall the EC₅₀values for the antiproliferative effect of 7a

and related 1,3-bis(aryl)-2-propanamines is high compared to other

chemotherapeutic agents, such as the microtubule targeting agent,

Paclitaxel (Taxol), which has been shown to have effects in BL cell

lines with IC₅₀values of between 10 and 40 nM.²⁵It was therefore

decided to synthesize and examine a range of novel 1,3-bis(aryl)-

2-nitro-1-propenes (4a–4i) and 1,3-bis-(aryl)-2-propanamines (

5a–5d) related in structure to 7a with a view to identifying a more

potent range of antiproliferative compounds (Section 2.1).

l

Compared to SERT inhibition values for amphetamines 4-MTA

(74 nM),²⁹MDMA (425 nM)³⁰and PCA (173 nM),³¹which are in

the low nanomolar range, observed IC₅₀values for the compounds

in this study are high, indicating that they most likely do not bind

strongly to SERT. This is probably due to the presence of the second

aromatic group, which may hinder the binding of the free amine to

the appropriate residues in the 5-HT binding site. We can therefore

hypothesise that while displaying low micromolar binding to SERT,

compounds, 5a, 5b, 5d, 7a and 11a, may not be interacting in the

same way as amphetamines at SERT. Although these compounds

may not be directly binding to SERT in the same manner as

amphetamines, it is possible that there may be some other indirect

interaction with the protein, however; further binding studies

would be required to determine this.

2.3. Inhibition of SERT activity

To determine if these new compounds synthesized as described

in Section 2.1, along with the previously described derivative 7a

could behave like other amphetamines and inhibit the activity of

SERT, the effects of these derivatives on 5-HT reuptake was as-

sessed using a novel ﬂuorescence neurotransmitter transporter as-

say²⁶in HEK cells stably expressing hSERT.¹³

2.4. Molecular modelling

It has been difﬁcult to determine if amphetamines are genuine

substrates of SERT, due to their lipophilic nature and to the lack of

A molecular modelling study was carried out to investigate pos-

sible binding orientations for compounds 4a, 5a, 5b, 7a, 5-HT and

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1333

Table 1

The effects of 1,3-bis(aryl)-2-propanamine compounds on SERT-expressing HEK cells and on the malignant cell lines, DG-75 and SHSY-5Y.

Compound structure

Compound number

HEK293

pEC₅₀SE

HEK293 hSERT

pEC₅₀SE

DG-75

pEC₅₀SE

SHSY-5Y

pEC₅₀SE

a

O

4b

5.1 0.2

4.7 0.2

4.6 0.2

4.5 0.2

2.9 0.2

4.9 0.1

4.5 0.2

4.6 0.2

2.3 0.3

6.0 0.1

4.6 0.1

4.8 0.2

4.7 0.1

2.5 0.2

4.8 0.1

NO₂

O

5a^b

5b

5c

4.7 0.1

4.5 0.2

4.7 0.1

<2

NH₂

S

O

NH₂

O

6b

O

4.0 0.3^*

2.8 0.2

2.4 0.3^*

4.4 0.3

4.5 0.2

4.8 0.2

2.8 0.3

2.4 0.3

4.5 0.1

NH

O

7a

<2

S

H

O

NH

O

7b

7c

<2

H

NH

O

4.5 0.1

<2

O

S

H

NH

O

10a

4.3 0.2

2.4 0.5

S

O

NH

10b

11a

2.4 0.4

2.9 0.4

<2

2.9 0.2

<2

O

4.6 0.2

4.9 0.1

4.8 0.1

NH

S

4-MTA

MDMA

MDA

4.7 0.1

3.6 0.1

3.5 0.2

4.6 0.1

3.3 0.2

3.2 0.3

3.5 0.7

4.5 0.2

2.7 0.2

3.2 0.5

NH₂

S

O

<2

HN

O

<2

NH₂

O

(continued on next page)

1334

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

Table 1 (continued)

Compound structure

Compound number

HEK293

pEC₅₀SE

HEK293 hSERT

pEC₅₀SE

DG-75

pEC₅₀SE

SHSY-5Y

pEC₅₀SE

a

NC

O

Citalopram

4.8 0.1

468 0.1

4.9 0.1

4.6 0.2

F

N

5 Â 10⁴cells/well 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 a pEC₅₀value determined by non-linear

regression analysis of sigmoidal log concentration dependence curves whereby pEC₅₀is À[Àlog EC₅₀

]

SE (log EC₅₀is the log [Dose] when response is equal to 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.

Sodium azide (30 mM) and Triton-X (2%) acted as positive controls for cytotoxicity resulting in 80–90% cytotoxicity to all cells.

a

pEC₅₀(À(Àlog EC₅₀is the log [Dose] when response = 50%)) values were calculated from % cell viability versus Àlog concentration curves, using four concentrations in

triplicate on two independent days. Data was subjected to non-linear regression analysis using a sigmoidal dose–response (Hill slope = 1) using ^GRAPHPADPrism4 software

(Graphpad software Inc., San Diego, CA).

b

Compound evaluated as the hydrochloride salt.

*

P <0.05. hSERT cells were compared to HEK293 wildtype cells using a two-way ANOVA test (GRAPHPAD Prism 4) with no matching followed by a Bonferroni Post Test. ND: not

determined.

4-MTA within the 5-HT binding site of SERT to allow us to rational-

ise results of SERT uptake experiments. Currently, there is no exist-

ing crystal structure of SERT; however, a number of homology

models of hSERT are available which were constructed using the

bacterial leucine transporter (LeuT) as a template.^32,33For this

study the homology model of hSERT constructed by Jørgensen et

al. was used.³³A number of previous studies have identiﬁed key

binding residues involved in the binding of SERT ligands.^33–36

5-HT, the natural substrate of SERT, and amphetamines such as

4-MTA interact with residues Ala96, Asp98 and Phe335 via binding

of the protonated amine (Fig. 3A), while the tricyclic antidepres-

sant imipramine is also thought to interact strongly with the

Asp98 residue.^13,32,37A study by Koldsø et al. discovered that the

two enantiomers of the SSRI citalopram bind to a central binding

site of the hSERT homology model with reversed orientations.³⁸

It has also been previously shown that the protonated amine of a

tions. Signiﬁcantly, 7a contains an amide which does not become

protonated like the amine groups of compounds 5a–5d, preventing

strong acid/base interactions and therefore explains the reduction

in SERT binding, as indicated by the high IC₅₀determined from

SERT uptake experiments (Section 2.3).

Figure 4 highlights the key interactions made by compound 5b,

which was selected for docking analysis based on having the

lowest determined IC₅₀of 1.6

observed with Phe341 and from visual analysis, possible T-shaped

-stacking with Tyr176 may occur in addition to H-bonding with

lM. Parallel displaced p-stacking is

p

Tyr95, Ala96, Asp98 and Leu337. Overall it is clear from the molec-

ular modelling studies that for minimal SERT binding within the 5-

HT site, H-bonding interactions to Ala96 and Asp98 are necessary.

2.5. Biological activity

series of sulfur-substituted

a-alkyl phenethylamines is essential

2.5.1. Antiproliferative activity

for protein–ligand binding. Other commonly annotated signiﬁcant

binding residues include Ile172 and Tyr95, which allow for hydro-

phobic and aromatic interactions, respectively.³⁴

To assess if the observed cytotoxicity of each compound was

due to an antiproliferative effect, each compound (4a–4i, 5a–5d,

6b, 7a–7c, 8b, 9b, 10a) was evaluated using the Alamar Blue assay

in a range of malignant cell lines; two Burkitt’s lymphoma cell lines

(MUTU-I and DG-75) and the human dopaminergic neuroblastoma

cell line, (SHSY-5Y). EC₅₀values for antiproliferative activity were

estimated from log concentration sigmoidal dose–response curves

where the cytotoxic potency of each compound was quantiﬁed by

In this study, ﬁrstly as validation, both 5-HT and 4-MTA were

docked with both receptor and ligand ﬂexibility using Molegro Vir-

tual Docker (http://www.molegro.com). Figure 3A and B clearly

illustrate the key interactions (H-bonding to Ala96, Asp98 and

Phe335 are common) made by both compounds via the protonated

primary amine. 5-HT makes additional H-bonding interactions

with Ser336 and also Thr439 (Fig. 3A). Subsequent docking of a

representative series 4a, 5a and 7a was undertaken.

a pEC₅₀value, where pEC₅₀is À[Àlog EC₅₀

]

SE (log EC₅₀is the log

[Dose] when response is equal to 50% cell viability) (Table 3 and

Fig. 5).

The nitrostyrene compound 4a was found to have no signiﬁcant

protein–ligand binding interactions with the 5-HT binding site of

SERT (Fig. 3C). This result is consistent with our SERT uptake exper-

iments, in which none of the nitrostyrene compounds 4a–4d

showed SERT inhibition.

Compound 5d was found to have the most potent antiprolifer-

ative effect on the MUTU-1 BL cell line (pEC₅₀of 6.8 0.4), with

an approximate EC₅₀value of 160 nM. The slightly more lipophilic

nitrostyrene analogue of 5d, compound 4d (c log P values of 4.5

and 5.3, respectively, as shown in Table 3) also displayed a potent

effect against the MUTU-1 cells (pEC₅₀of 6.0 0.1), with an

As previously discussed, derivatives 5a, 5b and 5d were found to

haveamoderateinhibitoryeffectonSERT(EC₅₀approx. 1.6–2.5

lM).

approximate EC₅₀value of 1 lM. The other 1,3-bis(aryl)-2-propan-

Amine 5a is shown to have similar critical H-bonding interactions

between the protonated amine group and residues, Ala96, Asp98

and Phe335, as observed with both 5-HT and 4-MTA (Fig. 3C).

amine compounds 5a–5c exhibited only moderate antiproliferative

effects on this cell line (pEC₅₀range of 3.1–4.9). This is in contrast

to the other 1,3-bis(aryl)-2-nitro-1-propenes (4a–4f), which all

showed similarly potent effects on the MUTU-1 cell line (pEC₅₀

range of 5.5–5.6) (Table 3 and Fig. 5A). This suggests that the pres-

ence of the slightly more lipophilic nitropropene structure may be

crucial for a potent antiproliferative effect in MUTU-1 cells.

Additional H-bonding with Leu337 is also observed as well as

p-

stacking with Phe341 to stabilise the compound in the binding site.

Compound 7a, illustrated in Figure 3E, is shown to make a sin-

gle

p-stack with Phe341 without any added H-bonding interac-

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1335

M)

Table 2

Inhibition of SERT by 1,3-bis(aryl)-2-nitro-1-propenes and related compounds

Compound structure

Compound number

% SERT inhibition (100

18

lM)

% SERT inhibition (1

25

lM)

IC₅₀

ND

(l

4a

NO₂

S

O

4b

4c

36

10

23

98

77

65

94

15

25

0

ND

2.5

1.6

ND

1.9

NO₂

O

4d

5a^a

5b

5c

NO₂

NH₂

Cl

50

44

11

39

S

O

NH₂

O

5d

NH₂

Cl

NH

O

7a

7b

7c

85

55

18

5

18

8

15.7

ND

S

H

O

NH

O

H

NH

O

ND

O

S

H

NH

O

10a

11a

28

92

0

ND

2.2

S

31

NH

NC

O

Citalopram

n/a

89

0.019

F

N

4-MTA

n/a

0.2^*

NH₂

S

(continued on next page)

1336

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

Table 2 (continued)

Compound structure

Compound number

MDMA

% SERT inhibition (100

n/a

l

M)

% SERT inhibition (1

n/a

lM)

IC₅₀

1.1^*

(lM)

O

HN

O

MDA

n/a

0.9^*

NH₂

O

Cells were seeded at a density of 2 Â 10⁴in black, clear bottomed, poly-

L

-lysine coated plates. After 24 h, the media was aspirated cells were washed with 100

ll of 1Â HBSS.

Cells were incubated for 15–20 min with 100

ll of compound dissolved in 1Â HBSS + 0.1% BSA, before 100 l

l of the ﬂuorescent dye was added. Plates were read at 520 nm

(excitation 440 nm). The background ﬂuorescence of the cells and 1Â HBSS buffer was taken away from each group. Untreated cells represented 100% ﬂuorescence/0%

inhibition. Citalopram (10 M) was used as a positive control resulting in >98% inhibition. Fluorescence for each compound was calculated as a percent of untreated cell

l

response. This value was then subtracted from 100% to determine the percent inhibition. IC₅₀values were calculated for compounds having >80% inhibition at 100

lM. IC₅₀

for citalopram was calculated as 19.7 nM.

Citalopram (10 lM) was used as a positive control for SERT inhibition, resulting in >98% inhibition. IC₅₀values were calculated for compounds having >80% inhibition at

100

lM.

a

Compound evaluated as free base. ND: not determined.

*

Evaluated using [³H] 5-HT as previously described by Cloonan et al.¹³

1,3-Bis(aryl)-2-propanamines 5a–5d did not show any signiﬁ-

cant antiproliferative effect on the resistant BL cell line, DG-75

(pEC₅₀of 3.4–4.6) compared to 1,3-bis(aryl)-2-nitro-1-propene

compounds 4a–4d, which displayed a moderate antiproliferative

effect in this cell line (pEC₅₀range of 4.3–4.9) with an approximate

dence that the antiproliferative abilities of these compounds are

not related to their SERT binding ability, as the nitrostyrene com-

pounds do not bind to SERT (Table 2).

2.5.2. Potential anticancer activity

EC₅₀range of 11–37

l

M (Table 3 and Fig. 5A). Interestingly, haloge-

Designing drugs that can induce programmed cell death (PCD),

namely apoptosis, of a cancer cell, whilst ignoring the ‘normal cells’

of the body is imperative to the future development of safe effec-

tive anticancer agents. A drug that can induce PCD in vitro in

malignant cell lines has the potential to become an anticancer

agent. Propidium iodide FACS analysis was carried out on a repre-

sentative subset of compounds in the MUTU-I cell line, initially

nated compounds 4e and 4f were found to have a much more po-

tent antiproliferative effect on the resistant DG-75 cell line (pEC₅₀

of 5.5 0.1 and 5.1 0.1, respectively), with approximate EC₅₀val-

ues of 3.3 lM and 9.3 lM, respectively (Table 3 and Fig. 5A). Other

halogenated analogues 4g and 4h, displayed potent effects on both

the MUTU-1 and DG-75 cell lines along with compound 4i, a struc-

tural isomer of compound 4f which has approximate EC₅₀values of

using 1 and 10

lM for potent compounds (EC₅₀<20

lM in

2.6

lM and 1.5

lM in MUTU-1 and DG-75 cell lines, respectively

MUTU-1 cells) in the Alamar Blue assay. Taxol (10

lM and 1 lM)

(data not shown). When evaluated for their antiproliferative effect

on the SHSY-5Y cell line, compounds 4d and 5d showed a potent

effect (pEC₅₀of 5.6 0.1 and 5.3 0.0, respectively) with approxi-

was used as positive control, showing 58–77% cell death at both

concentrations (Fig. 6A).

At 10 lM, compounds 4a, 4b, 4c and 4d showed 50%, 30%, 32%

mate EC₅₀values of 2.5

anamine and nitropropene compounds 4a–4c and 5a–5c showed

only moderate effects against the SHSY-5Y cell line (pEC₅₀of 4.5–

4.9) with an approximate EC₅₀range of 11–33

Fig. 5A).

All other compounds were found not to possess a signiﬁcant anti-

proliferative effect on each of the cell lines (Table 3 and Fig. 5A).

Compounds were also evaluated for their antiproliferative activity

on the K562 cell line, a chemoresistant chronic myelogenous leu-

kaemia (CML) cell line. Only one compound, compound 5a (pEC₅₀

l

M and 4.6

l

M, respectively. Other prop-

and 41% cells in pre-G1 phase of the cell cycle, respectively; indi-

cating the formation of apoptotic bodies in MUTU-1 cell line

(Fig. 5A). These compounds were also able to activate caspases 3

and 7 (Fig. 6B), induce PARP cleavage (Fig. 6C) and elicit morpho-

logical features of apoptosis such as chromatin condensation and

membrane blebbing (Fig. 6D) in the MUTU-I cell line all conclusive

with these compounds inducing Type-I programmed cell death

(apoptosis) in Burkitt’s lymphoma cells. It is also worth noting that

after 4 h of treatment with these representative compounds, the

majority of MUTU-I cells displayed all of the features of apoptotic

lM (Table 3 and

of 4.4, approximate EC₅₀value of 50.1

lM) was found to have signif-

PCD, whereas Taxol at the same concentration (10 lM) did not dis-

icant activity on this cell line (data not shown). From these results it

appears that these 1,3-bis(aryl)-2-nitro-1-propenes (4a–4i) appear

to be selective to Burkitt’s lymphoma derived cell lines.

In order to assess the selectivity of these derivatives towards

proliferating malignant cells over the ‘normal’ cells of the body,

the cytotoxic effects of a representative group of these derivatives

were assessed in peripheral blood mononuclear cells (PBMCs).

play all of features (just morphological features associated with

G2/M arrest). While compound 5d displayed the most potent activ-

ity in MUTU-1 cells as shown in the cell viability assay, no apopto-

tic bodies were observed by FACS analysis and there was no

evidence of PARP cleavage (Fig. 6C), perhaps indicative of this com-

pound eliciting a non-speciﬁc toxic effect or inducing an alterna-

tive form of programmed cell death such as Type-II PCD.

Compound 5d was also found to have a potent toxic effect in the

PBMC cell line, further suggesting a non-selective cytotoxicity.

These results indicate that the nitroethene structure may be essen-

tial for induction of apoptosis. Further studies on the structure–

activity relationship of the nitroethene functional group are cur-

rently ongoing to identify structural requirements important for

inducing the antiproliferative and pro-apoptotic effects observed

in this study.

After 24 h, it was found that at 10 lM, most of the derivatives

had little effect on the viability of PBMCs compared to their effect

on a range of malignant cells lines (Fig. 6B). Such results are consis-

tent with these agents selectively targeting cells of malignant ori-

gin over ‘normal cells’ of the body. However, 1,3-bis(aryl)-2-

propanamines 5a and 5d were found to have a potent toxic effect

in PBMCs, implying a non-speciﬁc toxicity toward malignant cell

lines as well as ‘normal’ PBMCs. From the results of the cell viabil-

ity studies, it appears that the nitrostyrene compounds (4a–4i), in

general, have a more potent, selective antiproliferative activity

than their propanamine analogues (5a–5d), suggesting further evi-

As a number of compounds (4e, 4g, 4h, 4i) in this screen also

had an antiproliferative effect in chemoresistant BL cell line DG-

75 (which cannot die by classical apoptosis²⁴but dies by Type-II

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1337

Figure 3. 2D representation of the interactions produced by 5-HT, 4-MTA, 4a, 5a and 7a in the 5-HT SERT binding site. It has been previously shown that the binding of the

protonated amine of a series of sulfur-substituted

-alkyl phenethylamines and amphetamines to Asp98 is essential for protein-ligand binding.¹³Nitrostyrene compound 4a

shows no binding interactions with the 5-HT binding site of SERT (Fig. 3C). Compound 5a is shown to have similar critical H-bonding interactions between the protonated

amine group and residues, Ala96, Asp98 and Phe335, as observed with both 5-HT and 4-MTA (Fig. 3A, B and D). Compound 7a is shown to make a single -stack with Phe341

a

p

without any added H-bonding interactions (Fig. 3E). Docking was carried out using Molegro Virtual Docker (www.molegro.com), using a recent homology model of hSERT,

constructed by Jørgensen et al.³³containing 5-HT as a bound ligand.¹³

autophagic PCD), investigations are currently underway to evalu-

ate if these compounds can act as pro-autophagic agents in the

treatment of chemoresistant cancers (cancer cells that cannot die

by the archetypical Type-I apoptotic response). In addition, since

these active compounds were also found from an initial screen,

with little information regarding their suspected target-based

1338

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

Figure 4. 3D image of the docked pose of compound 5b in the 5-HT binding site of SERT with isolated important residues for binding. Key H-bonding interactions are

highlighted (purple) with distances annotated. -Stacking between Phe341 and 5b is also highlighted (green). Docking was carried out using Molegro Virtual Docker

(www.molegro.com), using a recent homology model of hSERT, constructed by Jørgensen et al.³³containing 5-HT as a bound ligand.¹³

p

mechanism of action, extensive further investigations are required

to develop more potent analogues and to identify their mechanism

of action.

(Fig. 7B) revealed that at 2, 4, 6 and 8 h compounds 4a–4d had little

effect on the G2/M phase of the cell cycle compared to Taxol. Inter-

estingly, the morphology of cell death elicited by compounds 4a–

4d after 4 h of treatment shows some evidence of these com-

pounds inducing apoptotic cell death (Fig. 7C) and affecting cell cy-

cle dynamics as indicated by the apparent arrest of chromosomal

alignment in the nucleus (Fig. 6D). This maybe an indirect effect

of the action of these drugs or may be as a result of these com-

pounds targeting a yet undeﬁned cell cycle target. Further investi-

gations are required to evaluate such possibilities.

Interestingly, a number of these compounds (4a–4i) are struc-

turally related to simple nitrostyrene compounds which have been

previously shown to have a variety of biological effects including

anti-tumour and pro-apoptotic effects.^39–41In recent years, there

have been many studies investigating the biological effects and

functions of these compounds, with nitrostyrenes having been

shown to have antimicrobial^39,41and insecticidal⁴²properties.

Some have even been shown to affect the down regulation of inter-

leukins, suggesting an immunological role.⁴³Some b-nitrostyrenes

and related compounds have been found to possess anti-tumour

capabilities via the inhibition of tubulin polymerisation⁴⁴and pro-

tein tyrosine phosphatases (PTP). trans-b-Nitrostyrene (TBNS) is a

byproduct in the synthesis of methamphetamine and there is evi-

dence to suggest that it induces apoptosis in cancer cell lines via

inhibition of PTP.^40,45

3. Conclusion

The initial rationale for this study was to create a library of no-

vel potential SERT ligands and assess their antiproliferative activi-

ties against Burkitt’s lymphoma. This was based on previous

literature documenting the ability of SERT ligands to induce potent

selective programmed cell death in Burkitt’s lymphoma^1,5,7and

from preliminary work arising from a screen of synthesis byprod-

ucts associated with the clandestine synthesis of 4-MTA which

identiﬁed one lead compound 7a¹²as having SERT-selective toxic-

ity and antiproliferative and pro-apoptotic activities (Fig. 2) against

Burkitt’s lymphoma cell lines.

In the current study, we report the synthesis and SERT-inhibi-

tory activity of a number of structurally related 1,3-bis(aryl)-2-ni-

tro-1-propene and 1,3-bis(aryl)-2-propanamine compounds. We

ﬁnd that despite the lack of SERT binding activity of the majority

of these derivatives, a number of compounds showed antiprolifer-

ative activity against malignant cell lines DG-75, MUTU-I and

SHSY-5Y showing apparent selectivity for BL-derived cell lines.

Nitrostyrene-like derivatives had more potent antiproliferative

activity than their propanamine equivalents with the nitrostyrene

derivatives 4a–4d inducing the formation of apoptotic bodies,

(Fig. 6A), activating caspases (3 and 7) (Fig. 6B), inducing chroma-

Work carried out by Park and Pei, found that TBNS and a 4-

methoxy-TBNS derivative inhibited three PTPs (Yop, PTP1B, SHP-

1) at concentrations in the low micromolar range (2.5 and

4.5 l

M, respectively),⁴⁰which is similar to the concentration at

which our 1,3-bis-(4-methoxyphenyl)-2-nitro-propene 4c induces

cell death in the MUTU-1 cell line. Due to the structural similarity

between TBNS and the 1,3-bis(aryl)-2-nitro-1-propenes synthe-

sized in this study, we thought it possible that they are targeting

PTP in order to cause an apoptotic effect. However, further investi-

gation into the ability of these compounds to inhibit protein tyro-

sine phosphatases (PTP1B) revealed that compared to the known

PTP1B inhibitor Suramin, compounds 4a, 4c and 4d had no effect

on the activity of PTP1B (Fig. 7A). Investigations into the ability

of these compounds to affect microtubules was investigated using

PI FACS analysis, assessing the amount of cells in the G2/M phase

(characteristic of microtubule disrupting drugs) of the cell cycle

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1339

Table 3

In vitro, antiproliferative effects (pEC₅₀) of 1,3-bis(aryl)-2-nitro-1-propenes and related compounds in various cell lines

a

Compound structure

Compound number

pEC₅₀SE cell viability

DG-75

c log P^c

MUTU-1

5.5 0.1

SHSY-5Y

4.7 0.1

4a

4b

4c

4d

4.4 0.0

4.3 0.0

4.9 0.0

5.0

4.0

4.3

5.3

NO₂

S

O

5.5 0.1

5.6 0.1

6.0 0.1

4.5 0.1

4.9 0.1

5.6 0.1

NO₂

O

NO₂

Cl

4e

5.5 0.1

4.6 0.1

6.8

NO₂

NH₂

Cl

S

4f

5.5 0.1

4.9 0.0

4.3 0.1

3.1 0.6

6.8 0.4

5.1 0.1

4.6 0.0

3.6 0.1

3.4 0.1

4.6 0.0

4.7 0.1

4.9 0.1

4.5 0.2^d

4.7 0.1^d

5.3 0.0

5.3

4.9

3.8

4.1

4.5

5a^b

5b

5c

S

O

NH₂

O

5d

NH₂

Cl

S

7a

0.9 1.8

4.2 0.1

3.7 0.2

3.8 0.1

2.3 1.1

3.8 0.1

3.7 0.1

3.4 0.1

4.5 0.1^d

3.65 0.2

3.5 0.1

3.9

2.7

2.6

NH

O

S

H

O

NH

O

7b

7c

O

H

NH

O

H

NH

O

10a

1.8 1.4

3.8

3.1

S

O

6b

9b

4.0 0.1

4.4 0.1

3.8 0.1

3.9 0.1

3.6 0.1

3.8 0.1

O

2.9

OH

O

(continued on next page)

1340

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

Table 3 (continued)

a

Compound structure

Compound number

pEC₅₀SE cell viability

c log P^c

MUTU-1

4.6 0.1

DG-75

SHSY-5Y

4.2 0.1

O

8b

4.3 0.1

3.5

N

O

OH

5 Â 10⁴cells/well were treated with the respective drug for 24 h. Each well was treated with 20

ll of Alamar Blue (pre-warmed to 37 °C) and left to incubate at 37 °C in the

dark for 4–6 h. Fluorescence was read at 590 nM (excitation 544 nm). Background ﬂuorescence of the media without cells + Alamar Blue was taken away from each group,

and the control untreated cells represented 100% cell viability. The antifungal agent miconazole (10

in 90% cytotoxicity. The cytotoxic potency of each compound was quantiﬁed by a pEC₅₀value determined by non-linear regression analysis of sigmoidal log concentration

SE (log EC₅₀is the log [Dose] when response is equal to 50% cell viability).

lM) was used as a positive control for cell death in all cell lines, resulting

dependence curves whereby pEC₅₀is À[Àlog EC₅₀

]

a

pEC₅₀(À(Àlog EC₅₀is the log [Dose] when response = 50%)) values were calculated from % cell viability versus Àlog concentration curves, using four concentrations in

triplicate on two independent days. Data was subjected to non-linear regression analysis using a sigmoidal dose–response (Hill slope = 1) using ^GRAPHPADPrism4 software

(Graphpad software Inc., San Diego, CA). Miconazole (10 mM) acted as positive control for cytotoxicity resulting in 90% cytotoxicity to all cells.

b

Compound evaluated as the free base.

c log P was determined using ChemDraw Ultra version 12.

Evaluated using the neutral red assay.

c

d

tin condensation, membrane blebbing and PARP cleavage (Fig. 6C

and D) in the MUTU-I cell line, all conclusive with these com-

pounds inducing Type-I programmed cell death (apoptosis). From

this study it appears that the nitroethene structure may be essen-

tial for induction of apoptosis, as derivative 5d 1,3-bis(4-chloro-

phenyl)-2-propanamine, although having potent antiproliferative

activity in MUTU-1 cells did not induce apoptosis in these cells,

however further SAR are required to conﬁrm this observation. Fur-

ther studies will also examine the ability of these derivatives to in-

duce Type-II programmed cell death in chemoresistant BL, as a

number of the nitrostyrene compounds showed potent antiprolif-

erative activity in the DG-75 cell line, a known chemoresistant cell

line previously shown to be susceptible to antidepressant induced-

Type-II PCD.²⁴

Investigations into the mechanism of action of these compounds

eliminated a possible role for protein tyrosine phosphatases and for

tubulin polymerisation effects. Examination of the morphology of

MUTU-I cells treated with compounds 4a–4d (Fig. 6D) for 2 and

4 h reveals that the mechanism of cell death is fast and potent

and may involve the cell cycle and/or mitosis, implying that these

derivatives most likely target a fundamental part of the BL survival

pathway. Further investigations into this mechanism of action and

the identiﬁcation of a possible target for these nitrostyrene

derivatives is of paramount importance to the potential develop-

ment of these compounds as novel anticancer agents.

LC system (Waters Alliance 2795, Waters Corporation, USA) in ace-

tonitrile/water (60:40% v/v) at 200 l/min. The capillary voltage of

l

the mass spectrometer was at 3 kV. The sample cone (de-clustering)

voltage was set at 40 V. For exact mass determination, the instru-

ment was externally calibrated for the mass range m/z 100–1000.

A lock (reference) mass (m/z 556.2771) was used. Mass measure-

ment accuracies of < 5 ppm were obtained. Low resolution mass

spectra (LRMS) were acquired on a Hewlett–Packard 5973 MSD

GC–MS system in electron impact (ESI) mode. Elemental analyses

were performed on an Exetor Analytical CE4400 CHN analyser in

the microanalysis laboratory, Department of Chemistry, University

College Dublin. High resolution mass spectra (HRMS) were acquired

on a Thermo Scientiﬁc LTQ Orbitrap Discovery system in EI mode.

Flash column chromatography was carried out on Merck Kieselgel

60 (particle size 0.040–0.063 mm), Aldrich aluminium oxide, (acti-

vated, neutral, Brockmann I, 50 mesh) or Aldrich aluminium oxide,

(activated, acidic, Brockmann I, 50 mesh). Preparation of com-

pounds 2a–2f, 3a–3c, 3e, 4a, 4b, 4c, 5a, 5b, 6b, 7b, 12b was carried

out as described in previously reported literature.^{12,19–21,46–48}

4.1.1. Preparation of 1-(4-methoxyphenyl)-2-nitroethene (2c)

Compound 2c was synthesized according to the literature pro-

cedure¹³(eluent: 4:1 dichloromethane/hexane). Yellow crystals

(70%). Mp 83–85 °C. IR m_max(KBr) 1602, 1174, 1493, 1338 cm^À1

.

¹H NMR d (CDCl₃) 3.89 (3H, s, OCH₃), 6.98 (2H, d, J = 9.0 Hz, ArH),

7.52–7.57 (3H, 2d, J = 9.0 Hz, ArH J = 13.5 Hz, ArCHCH), 8.00 (1H,

d, J = 13.5 Hz, ArCHCH). ¹³C NMR ppm (CDCl₃) 55.57, 114.95,

122.58, 131.19, 135.07, 139.08, 162.96. HRMS (ESI) calculated for

C₉H₁₀NO₃: (M⁺+H) 180.0661: found 180.0664.

4. Experimental

4.1. Chemistry

4.1.2. Preparation of 1-(2,4-dichlorophenyl)-2-nitroethene (2e)

Compound 2e was prepared according to the literature proce-

dure¹²(eluent: 7:3, hexane/diethyl ether). Yellow crystals (91%).

Uncorrected melting points were measured on a Gallenkamp

apparatus. Infra-red (IR) spectra were recorded on a Perkin–Elmer

FT-IR Paragon 1000 spectrometer. ¹H, ¹³C and ¹⁹F nuclear magnetic

resonance (NMR) spectra were recorded at 27 °C on a Brucker DPX

400 spectrometer (400.13 MHz, ¹H; 100.61 MHz, ¹³C; 376.47 MHz,

¹⁹F) in either CDCl₃(internal standard tetramethylsilane (TMS)) or

CD₃OD. For CDCl₃, ¹H NMR spectra were assigned relative to the

TMS peak at d 0.00 and ¹³C NMR spectra were assigned relative to

the middle CDCl₃triplet at 77.00 ppm. For CD₃OD, ¹H and ¹³C NMR

spectra were assigned relative to the centre peaks of the CD₃OD mul-

tiplets at d 3.30 and 49.00 ppm, respectively. ¹⁹F NMR spectra were

not calibrated. Coupling constants are reported in Hertz. For ¹H NMR

assignments, chemical shifts are reported: shift value (number of

protons, description of absorption, coupling constant(s) where

applicable). Electrospray ionisation mass spectrometry (ESI-MS)

was performed in the positive ion mode on a liquid chromatography

time-of-ﬂight mass spectrometer (Micromass LCT, Waters Ltd, Man-

chester, UK). The samples were introduced into the ion source by an

Mp 100–102 °C. IR m_max(KBr) 1337, 1587, 1634 cm^{À1 1}H NMR d

.

(CDCl₃) 7.34 (1H, d, J = 8.0 Hz, ArH), 7.42 (1H, s, ArH), 7.45 (1H, d,

J = 8.0 Hz, ArH), 7.87 (1H, d, J = 14.0 Hz, ArCHCH), 8.27 (1H, d,

J = 14.0 Hz, ArCHCH). ¹³C NMR ppm (CDCl₃) 128.15, 129.32,

131.14, 131.60, 133.16, 135.60, 136.42, 142.50. HRMS (ESI) calcu-

lated for C₈H₅NO₂Cl₂: (M⁺) 216.9697: found 216.9694.

4.1.3. General procedure A: preparation of 1-aryl-1-

nitroethanes

Sodium borohydride (15.00 mmol, 5.70 g) was added to a vigor-

ously stirred mixture of the appropriate nitroethene (3.68 mmol),

silica gel (9.30 g) and propan-2-ol (100 mL) in dichloromethane

(350 mL) at room temperature. The mixture was stirred at room

temperature for 20 min until the solution turned colourless. Excess

NaBH₄was quenched with the addition of dilute HCl (50 mL). The

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1341

A

470

420

370

320

270

220

170

120

70

MUTU-1

DG-75

SHSY-5Y

20

18

16

14

12

10

8

6

4

2

0

4a

4b

4c

4d

4e

4f

5a

5b

5c

5d

6b

7b

7c

8b

9b 10a

Cl

H₂

N

Cl

90

B

PBMC

MUTU-1

80

70

60

50

40

30

20

10

0

4a 4b 4c 4d 4e

4f 5a 5d

Figure 5. 1,3-Bis(aryl)-2-nitro-1-propenes and related compounds show selective toxicity toward the MUTU-1 cell line. (A and B) 1–5 Â 10⁴cells/well were treated with the

respective drug for 24 h. 20 ll of Alamar Blue was added and left to incubate at 37 °C in the dark for 4–6 h. Fluorescence was read using at 590 nm (excitation 544 nm). The

background ﬂuorescence of the media without cells + Alamar Blue was taken away from each group, and the control untreated cells represented 100% cell viability.

The antifungal agent miconazole (10 M) was used as a positive control for cell death in each of the other cell lines, resulting in 90% cytotoxicity. Each compound was

screened over a 1 M–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 pEC₅₀value determined by non-linear regression analysis of sigmoidal log concentration dependence

l

curves, whereby approx. EC₅₀values were estimated from log concentration sigmoidal dose–response curves; pEC₅₀value corresponds to À[Àlog EC₅₀

]

SE (log EC₅₀is the log

[Dose] when response is equal to 50% cell viability).

mixture was ﬁltered and the silica gel washed with dichlorometh-

ane (100 mL). All organic phases were combined and washed with

brine (3 Â 50 mL) and water (3 Â 50 mL). The organic phases were

then dried over anhydrous Mg₂SO₄and the solvent removed in va-

cuo. The crude product was puriﬁed by column chromatography

over silica gel.

over silica gel (eluent: 7:3 hexane/diethyl ether). Dark brown oil

(82%). IR m_max(ﬁlm) 1552, 1376 cm^{À1 1}H NMR d (CDCl₃) 3.31

(2H, t, J = 7.3 Hz, ArCH₂CH₂), 4.62 (2H, t, J = 7.3 Hz, ArCH₂CH₂),

7.17 (2H, d, J = 8.5 Hz, ArH), 7.32 (2H, d, J = 8.5 Hz, ArH). ¹³C NMR

ppm (CDCl₃) 32.71, 76.03, 129.15, 129.97, 133.42, 134.12. HRMS

(ESI) calculated for C₈H₈ClNO₂: (M⁺) 185.0244: found 185.0250.

.

4.1.3.1. 1-(4-Chlorophenyl)-1-nitroethane (3d). Compound 3d

was prepared from 1-(4-chlorophenyl)-2-nitroethene (2d)

(36.80 mmol, 6.76 g) according to general procedure A (Section

4.1.3). The crude product was puriﬁed by column chromatography

4.1.3.2. 1-(2,4-Dichlorophenyl)-1-nitroethane (3e). Compound

3e was prepared from 1-(dichlorophenyl)-2-nitroethene (2e)

(1.38 mmol, 0.30 g) according to general procedure A (Section

4.1.3). The crude product was puriﬁed by column chromatography

1342

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

Figure 6. Compounds 4a, 4b, 4c and 4d induce apoptosis in the MUTU-I cell line. (A) 750,000 MUTU-I cells were treated with the appropriate amount of compound and

incubated for 24 h. Cells were harvested, washed and ﬁxed overnight at 4 °C. RNAse A and PI were added prior to cell cycle analysis counting 10,000 cells and analysed using

CELLQUEST software package. (B) 0.5 Â 10⁵cells were treated with compound (10

lM) for 5 h before the addition of 100 ll caspase substrate Z-DEVD-R110. Caspase 3/7 was

quantiﬁed by measuring the amount of cleaved substrate at 521 nm (excitation 499 nm). (C) Samples were resolved by SDS–PAGE, transferred onto PVDF membranes and

were probed with anti-PARP (recognises full length 113 kDa PARP as well as the 85 kDa cleaved form). (D) 3 Â 10⁵cells were treated with the indicated compounds for 4 h or

8 h. Cells (150

ll) were cytocentrifuged onto microscopy slides, stained Eosin Y and methylene blue and examined under a light microscope using a 60Â magniﬁcation.

over silica gel (eluent: 7:3 hexane/diethyl ether). Pale yellow oil

(83%). IR m_max1549, 1369 cm^{À1 1}H NMR d (CDCl₃) 3.24 (2H, t,

J = 7.0 Hz, ArCH₂CH₂), 4.65 (2H, t, J = 7.0 Hz, ArCH₂CH₂), 7.20 (2H,

d, J = 8.0 Hz, ArH), 7.42 (1H, s, ArH). ¹³C NMR ppm (CDCl₃) 30.42,

73.53, 127.21, 129.27, 131.49, 133.82, 134.21. HRMS (ESI) calcu-

lated for C₈H₇Cl₂NO₂: (M⁺) 218.9854: found 218.9848.

4.1.3.3. 1-(4-Fluorophenyl)-1-nitroethane (3h). Compound 3h

was prepared from 1-(4-ﬂuorophenyl)-2-nitroethene (2h)

(10.00 mmol, 1.67 g) according to general procedure A (Section

4.1.3). No further puriﬁcation was necessary. Orange oil (85%). IR

.

m_max(ﬁlm) 1378, 1552 cm^{À1 1}H NMR d (CDCl₃) 3.21 (2H, t,

.

J = 7.0 Hz, ArCH₂CH₂), 4.51 (2H, t, J = 7.0 Hz, ArCH₂CH₂), 7.01 (2H,

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1343

110

100

90

80

70

60

50

40

30

20

10

0

A

50

40

30

20

10

0

B

Control

taxol

4a

4b

4c

4d

0

2

4

6

8

Time (h)

100

90

80

70

60

50

40

30

20

10

0

C

Control

taxol

4a

4b

4c

4d

0

2

4

6

8

Time (h)

Figure 7. 1,3-Bis(aryl)-2-nitro-1-propenes (4a–4d) do not inhibit PTP1B or affect tubulin polymerisation in the Burkitt’s lymphoma derived cell line MUTU-I. (A)

Recombinant PTP1B was incubated with the test compound and a phosphopeptide substrate for 30 min at 30 °C. Free phosphate released was detected at 620 nm. (B) 750,000

MUTU-I cells were treated with the appropriate amount of compound and incubated for 24 h. Cells were harvested, washed and ﬁxed overnight at 4 °C. RNAse A and PI were

added prior to cell cycle analysis counting 10,000 cells and analysed using ^CELLQUESTsoftware package.

d, J = 8.0 Hz, ArH), 7.38 (2H, d, J = 8.0 Hz, ArH). ¹³C NMR ppm

(CDCl₃) 32.53, 76.27, 115.68 (d J_CF= 11.6 Hz), 115.89 (d,

J_CF= 11.6 Hz), 130.23, 131.54 (³J = 9.0 Hz, ⁴J = 3.5 Hz), 160.87,

163.31 (J_CF= 250 Hz). HRMS (ESI) calculated for C₈H₈FNO₂: (M⁺)

169.0539: found 169.0544.

with 10% aq HCl (3 Â 30 mL). The organic phase was dried over

anhydrous Mg₂SO₄and solvent evaporated in vacuo. The product

was puriﬁed by column chromatography over silica gel.

4.1.4.1. 1,3-Bis(4-chlorophenyl)-2-nitro-1-propene (4d). Com-

pound 4d was prepared from 1-(4-chlorophenyl)-1-nitroethane

(3d) and 4-chlorobenzaldehyde (15 mmol, 2.11 g) according to

general procedure B (Section 4.1.4) and puriﬁed by column chro-

matography over silica gel (eluent: 10:1 hexane/ethyl acetate)

and recrystallized from methanol. Yellow crystals (50%). Mp 74–

4.1.4. General procedure B: preparation of 1,3-bis(aryl)-2-nitro-

1-propenes

A

mixture of the appropriate 1-aryl-1-nitroethane (15.00

mmol), dimethylamine hydrochloride (30.00 mmol), potassium

ﬂuoride (2.25 mmol) and the appropriate aldehyde (15.00 mmol)

in toluene (20 mL) was reﬂuxed with a Dean–Stark trap for 24 h.

The reaction was then diluted with toluene (100 mL) and washed

76 °C. IR m_max(KBr) 1520, 1312 cm^{À1 1}H NMR d (CDCl₃) 4.22 (2H,

.

s, ArCH₂), 7.14 (2H, d, J = 8.5 Hz, ArH), 7.32, 7.37 (4H, 2d,

J = 8.5 Hz, ArH), 7.43 (2H, d, J = 8.5 Hz, ArH), 8.28 (1H, s, ArCH).

1344

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

¹³C NMR ppm (CDCl₃) 32.37, 128.90, 129.20, 129.57, 130.87,

133.06, 134.56, 136.88, 149.46. HRMS (ESI) calculated for

C

B (Section 4.1.4). The product was puriﬁed by column chromatogra-

phy over silica gel (eluent: 7:3 hexane/diethyl ether) and recrystal-

lized from methanol. Yellow needles (30%). Mp 115–117 °C. IR m_max

₁₅H₁₀Cl₂NO₂: (M^ÀÀH) 306.0089: found 306.0099.

(KBr) 1525, 3091, 1654 cm^{À1 1}H NMR d (CDCl₃) 2.53 (3H, s, SCH₃),

.

4.1.4.2. 1-(4-Chlorophenyl)-3-(2,4-dichlorophenyl)-2-nitro-1-

propene (4e). Compound 4e was prepared from 1-(4-chloro-

phenyl)-1-nitroethane (3d) (2.69 mmol, 0.50 g) and 2,4-dichloro-

benzaldehyde (1e) (2.69 mmol, 0.47 g) according to general

procedure B. The product was puriﬁed by column chromatography

over silica gel (eluent: 7:3 hexane/diethyl ether) and recrystallized

from methanol. Yellow solid (20%). Mp 90–92 °C. IR m_max(KBr)

4.27 (2H, s, ArCH₂), 7.16 (2H, d, J = 8.5 Hz, ArH), 7.26 (2H, d,

J = 8.5 Hz, ArH), 7.31 (2H, d, J = 8.5 Hz, ArH), 7.36 (2H, d, J = 8.5 Hz,

ArH), 8.31 (1H, s, ArCH). ¹³C NMR ppm (CDCl₃) 144.44, 32.11,

125.51, 127.31, 128.50, 128.65, 129.78, 132.43, 135.15, 142.84.

HRMS (ESI) calculated for C₁₆H₁₄NO₂SCl: 319.0434 (M⁺+H): found:

319.0432.

1508, 1641 cm^À^{1 1}H NMR d (CDCl₃) 4.09 (3H, s, ArCH₂), 7.09 (2H,

.

4.1.5. General procedure C: preparation of 1,3-bis(aryl)-2-

propanamines

d, J = 8.5 Hz ArH), 7.23 (1H, d, J = 8.5 Hz, ArH), 7.30 (3H, d,

J = 8.5 Hz, ArH), 7.55 (1H, d, J = 1.5 Hz, ArH), 8.32 (1H, s, ArCH).

¹³C NMR ppm (CDCl₃) 31.83, 127.25, 128.57, 128.69, 129.73,

129.75, 131.21, 128.88, 132.66, 133.74, 135.18, 136.46, 150.68.

HRMS (ESI) calculated for C₁₅H₉NO₂Cl₃: (M⁺ÀH) 339.9699: found

339.9696.

To a solution of LiAlH₄(15.00 mmol) in dry tetrahydrofuran

(25 mL), the appropriate nitroalkene (5.00 mmol) in anhydrous

benzene (20 mL) was added dropwise. After the addition, the mix-

ture was heated at reﬂux for 3 h. The excess LiAlH₄was quenched

by addition of 1–2 mL of water, and the mixture ﬁltered through

Celite. The organic ﬁltrate was extracted using 2 N HCl (3 Â

30 mL), and the combined aqueous extracts were basiﬁed with

2 M NaOH. The free base was then extracted with ethyl acetate,

dried over anhydrous Mg₂SO₄and the solvent evaporated in vacuo.

No further puriﬁcation was required.

4.1.4.3. 1-(4-Methylthiophenyl)-3-(4-chlorophenyl)-2-nitro-1-

propene (4f). Compound 4f was prepared from 1-(4-methylthi-

ophenyl)-1-nitroethane (3a) (2.53 mmol, 0.50 g) and 4-chlorobenz-

aldehyde (2.53 mmol, 0.35 g) were reacted together according to

general procedure B (Section 4.1.4). The product was puriﬁed by

column chromatography over silica gel (eluent: 10:1 petroleum

ether/ethyl acetate) and recrystallized from methanol. Yellow solid

4.1.5.1. 1,3-Bis(4-methoxyphenyl)-2-propanamine (5c). Com-

pound 5c was prepared from 1,3-bis(4-methoxyphenyl)-2-nitro-

1-propene (4c) (5.00 mmol, 1.49 g) according to general procedure

C (Section 4.1.5). Pale yellow oil (66%). IR m_max(ﬁlm) 3341,

(55%). Mp 76–78 °C. IR m

_max(ﬁlm) 1517, 1316 cm^À1. ¹H NMR (CDCl₃)

d 2.49 (3H, s, SCH₃), 4.22 (2H, s, ArCH₂), 7.13 (2H, d, J = 8.5 Hz, ArH),

7.23 (2H, d, J = 8.5 Hz, ArH), 7.40 (4H, dd, J = 15.0 Hz, J = 8.5 Hz, ArH),

8.26 (1H, s, ArCH). ¹³C NMR ppm (CDCl₃) 15.44, 31.96, 126.74,

127.62, 129.04, 129.87, 130.48, 132.17, 133.80, 136.27, 136.85,

1179 cm^À^{1 1}H NMR d (CDCl₃) 1.40 (2H, br s, NH₂), 2.47 (2H, dd,

.

J = 9.0 Hz, J_gem= 13.5 Hz, ArCH₂), 2.78 (2H, dd, J = 4.5 Hz, J_gem

=

13.3 Hz, ArCH₂), 3.20 (1H, m, NH₂CH), 3.81 (6H, s, OCH₂), 6.87

(4H, d, J = 8.5 Hz, ArH), 7.15 (4H, d, J = 8.5 Hz, ArH). ¹³C NMR ppm

(CDCl₃) 42.50, 53.93, 54.81, 113.42, 129.54, 129.74, 130.99,

157.66. HRMS (ESI) calculated for C₁₇H₂₂NO₂: 271.1572 (M⁺+H):

found 272.1649.

149.34. HRMS (ESI) calculated for

C₁₆H₁₄NO₂SCl: 358.0071

(M⁺+K): found: 358.0321.

4.1.4.4. 1,3-Bis(4-bromophenyl)-2-nitro-1-propene (4g). Com-

pound 4g was prepared from 1-(4-bromophenyl)-1-nitroethane

(3g) (1.62 mmol, 0.30 g) according to general procedure B (Section

4.1.4). The product was puriﬁed by column chromatography over

silica gel (eluent: 6:1 dichloromethane/hexane) and recrystallized

from methanol. Yellow solid (35%). Mp 91–93 °C. IR m_max(ﬁlm)

4.1.5.2. 1,3-Bis(4-chlorophenyl)-2-propanamine (5d). Com-

pound 5d was prepared from 1-(4-chlorophenyl)-1-nitroethane

(4d) (5.00 mmol, 1.40 g) according to general procedure C (Section

1520, 1325 cm^{À1 1}H NMR d (CDCl₃) 4.19 (2H, s, ArCH₂), 7.07 (2H,

.

4.1.5). Pale resin (30%). IR m_max(ﬁlm) 3307, 810 cm^{À1 1}H NMR d

.

d, J = 8.0 Hz, ArH), 7.28 (2H, d, J = 8.5 Hz, ArH), 7.46 (2H, d,

J = 8.5 Hz, ArH), 7.57 (2H, d, J = 8.5 Hz, ArH), 8.25 (1H, s, ArCH).

¹³C NMR ppm (CDCl₃) 32.00, 128.86, 129.85, 130.57, 131.67,

132.08, 134.23, 134.44, 148.93. HRMS (ESI) calculated for

(CDCl₃) 1.32 (2H, br s, NH₂), 2.55 (2H, dd, J = 8.5 Hz, J_gem= 13.5 Hz,

ArCH₂), 2.82 (2H, dd, J = 4.5 Hz, J_gem= 13.5 Hz, ArCH₂), 3.24 (1H, m,

NH₂CH), 7.15 (4H, d, J = 8.0 Hz, ArH), 7.30 (4H, d, J = 8.0 Hz, ArH).

¹³C NMR ppm (CDCl₃) 42.97, 53.54, 128.21, 130.13, 131.77,

137.16. HRMS (ESI) calculated for C₁₅H₁₆NCl₂: (M⁺+H) 280.0660:

found 280.0668.

C

₁₅H₁₀Br₂NO₂: (M^ÀÀH) 393.9078: found 393.9076.

4.1.4.5. 1,3-Bis(4-ﬂuorophenyl)-2-nitro-1-propene (4h). Com-

pound 4h was prepared from 1-(4-ﬂuorophenyl)-1-nitroethane

(3h) (2.99 mmol, 0.50 g) according to general procedure B (Section

4.1.4). The product was puriﬁed by column chromatography over

silica gel (eluent: 6:1 dichloromethane/hexane) and recrystallized

from methanol. Yellow solid (30%). Mp 73–75 °C. IR m_max(ﬁlm)

4.1.6. 4-(3,4-Methylenedioxybenzyl)-5-(3,4-methylenedioxy-

phenyl)pyrimidine (14b)

Compound 14b was isolated as a byproduct from the prepara-

tion of 2-N-formylamino-1,3-bis(3,4-methylenedioxyphenyl)pro-

pane (7b) from 1,3-bis(3,4-methylenedioxyphenyl)-2-propanone

(6b) by column chromatography over silica gel (eluent: 80:20

diethylether/hexane). Amber crystals (20%). Mp 88–90 °C. IR m_max

1519, 1333 cm^À^{1 1}H NMR d (CDCl₃) 4.24 (2H, s, ArCH₂) 7.04 (2H,

.

dd, J_HH= 9.0 Hz, J_HF= 5.5 Hz, ArH), 7.13–7.19 (4H, m, ArH), 7.45

(2H, dd, J_HH= 9.0 Hz, J_HF= 5.5 Hz, ArH), 8.29 (1H, s, ArCH). ¹³C NMR

ppm (CDCl₃) 32.15, 115.80, 116.01, 116.41, 116.64 (J_CF= 22.0 Hz),

127.94, 128.62, 128.70 (³J = 9.0 Hz, ⁴J = 3.5 Hz), 131.31, 131.40

(KBr) 2779, 1609 cm^{À1 1}H NMR d (CDCl₃) 4.04 (2H, s, CH₂), 5.90

.

(2H, s, OCH₂O), 6.05 (2H, s, OCH₂O), 6.50 (1H, dd, J = 8.3 Hz,

J = 1.3 Hz, ArH), 6.61 (1H, d, J = 1.5 Hz, ArH), 6.68 (1H, d,

J = 8.0 Hz, ArH), 6.71 (1H, dd, J = 7.8 Hz, J = 1.8 Hz, ArH), 6.73 (1H,

d, J = 1.5 Hz, ArH), 6.90 (1H, d, J = 8.0 Hz, ArH), 8.54 (1H, s, NCHC),

9.13 (1H, s, NCHN). ¹³C NMR ppm (CDCl₃) 40.17, 100.42, 100.98,

107.72, 108.13, 108.93, 109.15, 121.47, 122.53, 128.71, 131.20,

134.21, 145.78, 147.21, 147.44, 147.55, 156.70, 156.98, 165.69.

LRMS: m/z 334 (M⁺). Anal. Calcd for C₁₉H₁₄N₂O₄: C, 68.26; H,

4.22; N, 8.38. Found: C, 68.02; H, 4.31; N, 8.16.

(³J = 9 Hz, ⁴J = 3.5 Hz), 131.84, 134.07, 160.70, 163.14 (J_CF

=

253.00 Hz), 162.58, 165.10 (J_CF= 247.00 Hz, CF). HRMS (ESI) calcu-

lated for C₁₅H₁₀F₂NO₂: (M^ÀÀH) 274.0680: found 274.0667.

4.1.4.6. 1-(4-Chlorophenyl)-3-(4-methylthiophenyl)-2-nitro-1-

propene (4i). Compound 4i was prepared from 1-(4-chloro-

phenyl)-1-nitroethane (3d) (2.69 mmol, 0.50 g) and 4-methylthio-

benzaldehyde (2.69 mmol, 0.41 g) according to general procedure

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1345

4.1.7. 4-(4-Methylthiobenzyl)-5-(4-methylthiophenyl)pyrimi-

dine (14a)

LRMS: m/z 297 (M⁺), 135. Anal. Calcd for C₁₇H₁₅NO₄: C, 68.68; H,

5.09; N, 4.71. Found: C, 68.62; H, 5.04; N, 4.56.

Compound 14a was isolated as an impurity from the prepara-

tion of 2-N-formylamino-1,3-bis(3,4-methylenedioxyphenyl)pro-

pane (7a) from 1,3-bis(3,4-methylenedioxyphenyl)-2-propanone

(6a) by column chromatography over silica gel (eluent: 90:10

diethyl ether/hexane). Amber solid (5%). Mp 111–112 °C (diethyl

4.1.11. 1,3-Bis(3,4-methylenedioxy)-2-propanol (9b)

1,3-Bis(3,4-methylenedioxyphenyl)-2-propanone (6b) (0.67

mmol, 0.20 g) was stirred in dry MeOH (20 mL) and dichlorometh-

ane (5 mL) while NaBH₄(2.74 mmol, 1.04 g) was added slowly. The

reaction mixture was stirred for 2 h, checking progress by TLC.

When the reaction was complete, the solvent was evaporated in

vacuo. The residue was dissolved in dichloromethane and washed

with water (3 Â 20 mL). The solvent was evaporated in vacuo. No

further puriﬁcation was required. Colourless solid (90%). Mp

ether/hexane). IR m_max(KBr) 1653, 1595 cm^{À1 1}H NMR d (CDCl₃)

.

2.43 (3H, s, SCH₃), 2.54 (3H, s, SCH₃), 4.06 (2H, s, CH₂), 6.98, 7.12,

7.15, 7.32 (8H, 4d, J = 8.0 Hz, J = 8.0 Hz, J = 8.5 Hz, J = 8.6 Hz, ArH),

8.54 (1H, s, NCHC), 9.13 (1H, s, NCHN). ¹³C NMR ppm (CDCl₃)

15.49, 15.98, 40.49, 126.36, 126.90, 129.33, 129.54, 131.94,

134.50, 134.84, 136.53, 139.54, 157.09, 157.50, 165.83. LRMS: m/

z 338 (M⁺). Anal. Calcd for C₁₉H₁₈NS₂: C, 67.42; H, 5.36; N, 8.28;

S, 18.95. Found: C, 67.13; H, 5.38; N, 8.08; S, 18.89.

141–144 °C. IR m_max(KBr) 3397 cm^{À1 1}H NMR d (CDCl₃) 1.75 (1H,

.

br s, OH), 2.63 (2H, dd, J = 4.0 Hz, J = 14.0 Hz, ArCH₂), 2.75 (2H,

dd, J = 8.0 Hz, J = 14.0 Hz, ArCH₂), 3.95 (1H, m, CHOH), 5.95 (2H, s,

OCH₂O), 6.68 (2H, dd, J = 8.0 Hz, J = 1.5 Hz, ArH), 6.74 (2H, d,

J = 1.5 Hz, ArH), 6.76 (2H, d, J = 8.0 Hz, ArH). ¹³C NMR ppm (CDCl₃)

42.49, 73.23, 100.45, 107.85, 109.24, 121.84, 131.65, 145.74,

147.29. HRMS (ESI) calculated for C₁₇H₁₆O₅Na: (M+Na) 323.0895:

found 323.0899

4.1.8. 2-N-Formylamino-1,3-bis(3,4-methoxyphenyl)propane

(7c)

1,3-(3,4-Methyoxyphenyl)-2-propanone

(6c)

(5.03 mmol,

1.51 g) and formamide (3.00 g) were heated at 190 °C for 5 h. After

cooling the reaction was diluted with water (50 mL) and extracted

with ethyl acetate (3 Â 25 mL). The extracts were combined,

washed with water (3 Â 25 mL) and dried over anhydrous Na₂SO₄.

Volatiles were removed in vacuo and the residue was puriﬁed by

column chromatography over silica gel (eluent: diethylether). Col-

ourless crystals (30%). Mp 134–135 °C. IR m_max(KBr) 3341,

4.1.12. 2-N-Acetylamino-1,3-bis(3,4-methylenedioxyphenyl)-

propane (10b)

To a solution of 1,3-bis(3,4-methylenedioxyphenyl)-2-propan-

amine (5b) (2.92 mmol, 0.87 g) in pyridine (6 mL), was added ace-

tic anhydride (5.84 mmol, 0.59 g) and the mixture stirred at room

temperature for 1 h. The reaction was acidiﬁed with 10% aq HCl

(100 mL) and extracted with dichloromethane (3 Â 25 mL). The ex-

tracts were combined, dried over anhydrous Na₂SO₄and volatiles

removed in vacuo, generating an amber oil which slowly solidiﬁed.

Recrystallisation from methanol provided the pure product. Col-

ourless solid (65%). Mp 133–134 °C. IR m_max(KBr) 3288, 2789,

1660 cm^À^{1 1}H NMR d (CDCl₃) 2.59 (2H, m, ArCH₂), 2.75 (2H, m,

.

ArCH₂), 3.58 (0.3H, m, NHCH), 3.75 (6H, s, OCH₃), 4.38 (0.7H, dd,

J = 14.6 Hz, J = 7.0 Hz, NHCH), 5.70 (0.72H, br s, NH), 6.00 (0.27, br

s, NH), 6.80 (4H, d, J = 8.5 Hz, ArH), 7.03, 7.07 (4H, 2 d, J = 8.5 Hz,

J = 8.5 Hz, ArH), 7.37 (0.27H, d, J = 11.5 Hz, CHO), 7.96 (0.72H, s,

CHO). ¹³C NMR ppm (CDCl₃) 38.80, 41.12^⁄, 50.41, 55.07, 56.27^⁄,

113.79, 114.01^⁄, 129.24, 129.65^⁄, 130.12, 130.24^⁄, 158.18,

158.35^⁄, 160.61, 163.91. HRMS (ESI) calculated for C₁₈H₂₁O₃Na :

(M⁺+Na) 322.1419: found 322.1424.

1643 cm^À^{1 1}H NMR d (CDCl₃) 1.87 (3H, s, COCH₃), 2.66 (2H, dd,

.

J = 14.1 Hz, J = 7.0 Hz, ArCH₂), 2.74 (2H, dd, J = 14.0 Hz, J = 6.0 Hz,

ArCH₂), 4.31 (1H, m, NHCH), 5.17 (1H, d, J = 7.5 Hz, NH), 5.93 (4H,

s, OCH₂O), 6.61 (2H, dd, J = 7.8 Hz, J = 1.5 Hz, ArH), 6.67 (2H, d,

H = 1.5 Hz, ArH), 6.73 (2H, d, J = 7.5 Hz, ArH). ¹³C NMR ppm (CDCl₃)

22.97, 39.01, 51.06, 100.43, 107.74, 109.13, 121.75, 131.14, 145.77,

147.28, 169.00. LRMS: m/z 341 (M⁺+H) 282. Anal. Calcd for

4.1.9. 1,3-Bis(3,4-methylenedioxy)-2-propanone oxime (8b)

1,3-Bis(3,4-methylenedioxyphenyl)-2-nitro-1-propene

(4b)

(1.00 mmol, 0.31 g), hydroxylamine HCl (2.36 mmol, 0.16 g), pyri-

dine (3 mL) and ethanol (5 mL) were reﬂuxed for 2 h. After cooling,

the reaction was acidiﬁed using 10% aq HCl and extracted with

dichloromethane (3 Â 20 mL). The organic phases were combined,

dried over anhydrous Na₂SO₄and the solvent evaporated in vacuo.

No further puriﬁcation was required. Dark brown crystals (83%).

C₁₉H₁₉NO₅: C, 66.85; H, 5.61; N, 4.10. Found: C, 66.56; H, 5.70;

N, 4.07.

4.1.13. 2-N-Ethylamino-1,3-bis(3,4-methylenedioxyphenyl)-

propane (11b)

Mp 106–107 °C. IR m_max(KBr) 3251 cm^{À1 1}H NMR d (CDCl₃) 3.37

.

Compound 11b was prepared from 2-N-acetylamino-1,3-di(3,4-

methylenedioxyphenyl)propane (10b) (7.50 mmol, 2.56 g) accord-

ing to general procedure C and puriﬁed by column chromatogra-

phy over silica gel (eluent: methanol). Pale amber oil (33%). IR

(2H, s, ArCH₂), 3.58 (2H, s, ArCH₂), 5.95 (4H, s, OCH₂O), 6.63–6.77

(6H, m, ArH), 8.60 (1H, br s, OH). ¹³C NMR ppm (CDCl₃) 31.81,

38.93, 101.15, 108.47, 109.73, 109.94, 122.53, 130.36, 130.45,

146.64, 146.90, 148.25, 159.83. HRMS (ESI) calculated for

m_max(ﬁlm) 3283 cm^{À1 1}H NMR d (CDCl₃) 1.02 (3H, t, J = 7.0 Hz,

.

C

₁₇H₁₆NO₅: (M⁺+H) 314.1028: found 314.1032.

NCH₂CH₃), 2.57–2.68 (6H, m, NCH₂CH₃, ArCH₂), 2.93 (1H, m,

NHCH), 3.73 (1H, br s, NH), 5.90 (4H, s, OCH₂O), 6.61 (2H, dd,

J = 7.8 Hz, J = 2.0 Hz, ArH), 6.65 (2H, d, H = 2.0 Hz, ArH), 6.72 (2H,

d, J = 8.0 Hz, ArH). ¹³C NMR ppm (CDCl₃) 15.14, 40.06, 41.46,

61.17, 100.75, 108.12, 109.37, 122.11, 130.19, 145.93, 147.64.

LRMS: m/z 327 (M⁺+H) 192. HCl salt. Colourless solid (90%). Mp

4.1.10. 2-(3,4-Methylenedioxybenzyl)-3-(3,4-methylenedioxy-

phenyl)aziridine (15b)

Compound 15b was isolated as a byproduct from the prepara-

tion of 1,3-bis(3,4-methylenedioxyphenyl)-2-propanamine (5b)

from 1,3-bis(3,4-methylenedioxy)-2-propanone oxime (8b) by col-

umn chromatography over silica gel (eluent: 90:10 diethylether/

methanol). Colourless needles (62%). Mp 78–79 °C. IR m_max(KBr)

164–167 °C. IR m

_max(KBr) 2481 cm^À1. Anal. Calcd for C₁₉H₂₂ClNO₄:

C, 62.72; H, 6.09; N, 3.85. Found: C, 62.89; H, 6.11; N, 4.04.

3166, 2770 cm^À^{1 1}H NMR d (CDCl₃) 1.08 (1H, s, NH), 2.36–2.43

.

4.1.14. 2-(N-Formyl-N-methyl)amino-1,3-bis(3,4-methylenedi-

oxyphenyl)propane (12b)

(3H, m, NHCHCH₂, CH₂), 3.26 (1H, d, J = 6.0 Hz, ArCHNH), 5.88

(2H, m, OCH₂O), 5.94 (2H, s, OCH₂O), 6.52 (1H, dd, J = 8.0 Hz,

J = 1.5 Hz, ArH), 6.60 (1H, d, J = 1.5 Hz, ArH), 6.68 (1H, d,

J = 8.0 Hz, ArH), 6.78 (1H, d, J = 8.0 Hz, ArH), 6.86 (1H, d,

J = 8.0 Hz, ArH), 6.90 (1H, s, ArH). ¹³C NMR ppm (CDCl₃) 33.93,

36.83, 38.61, 100.68, 100.85, 107.89, 108.05, 108.23, 109.18,

120.91, 121.42, 131.46, 133.56, 145.79, 146.44, 147.43, 147.46.

A solution of 1,3-bis(3,4-methylenedioxyphenyl)-2-propanone

(6b) (2.51 mmol, 0.75 g) in N-methylformamide (6.44 mmol,

0.38 g) and 96% formic acid (0.18 g) was stirred and heated at re-

ﬂux at 150 °C for 7 h. After cooling the reaction was diluted with

water (25 mL) and extracted with dichloromethane (3 Â 25 mL).

1346

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

The extracts were combined, washed with water (2 Â 25 mL) and

satd aq NaHCO₃(2 Â 25 mL), followed by drying over anhydrous

Na₂SO₄. Volatiles were removed in vacuo and resulting residue

was puriﬁed by ﬂash chromatography over silica gel (eluent: 7:3

hexane/diethyl ether). Amber solid (58%). Mp 118–120 °C. IR m_max

MDA-MB231 and 4TI cell lines were purchased from the European

Collection of Cell Cultures (ECACC). The HEK293 cell line stably

overexpressing the human SERT was a gift from Dr. Patrick Schloss

(Central Institute for Mental Health, Mannheim, Germany). RPMI-

1640, DMEM, FBS, HEPES, sodium pyruvate, gentamycin (G418)

and glutamine were from Gibco (Invitrogen. Biosciences Ltd, Ire-

land). Alamar Blue and LymphoPrep were from Biosciences Ltd.

The neurotransmitter transporter uptake assay was purchased

from Molecular Devises Inc, the Apo-ONE^Òhomogenous caspase

3/7 assay kit was from Promega and the protein tyrosine phospha-

tase 1B activity kit was purchased Calbiochem. All other chemicals

were purchased through Sigma–Aldrich Inc., Ireland.

(KBr) 2781, 1661 cm^{À1 1}H NMR d (CDCl₃) 2.67 (0.6H, s, NCH₃),

.

2.79 (2.4H, s, NCH₃), 2.73–2.84 (4H, m, ArCH₂), 3.70 (0.8H, m,

NCH), 4.63 (0.2H, m, NCH), 5.90 (0.6H, s, OCH₂O), 5.91 (2.4H, s,

OCH₂O), 6.55 (1.6H, dd, J = 8.0 Hz, J = 1.5 Hz, ArH), 6.58 (1.6H, d,

J = 1.5 Hz, ArH), 6.65 (0.4H, dd, J = 7.8 Hz, J = 1.8 Hz, ArH), 6.68–

6.75 (2.4H, m, ArH), 7.55 (0.8H, s, CHO), 7.89 (0.2H, s, CHO). ¹³C

NMR ppm (CDCl₃) 24.86^⁄, 36.77, 37.95^⁄, 62.31^⁄, 100.40, 100.54^⁄,

107.74, 108.02^⁄, 108.43^⁄, 108.75, 121.31^⁄, 130.77^⁄, 131.30,

145.71, 146.00^⁄, 147.24, 147.50^⁄, 162.50^⁄, 162.58 (CHO). LRMS:

m/z 341 (M⁺), 282. Anal. Calcd for C₁₉H₁₉NO₅: C, 66.85; H, 5.61;

N, 4.10. Found: C, 66.78; H, 5.66; N, 4.02.

4.3.2. Cell culture

The DG-75 cell line is a B-lymphocyte, Burkitt’s lymphoma line

derived from a metastatic pleural effusion (lung) of a sporadic case

of Burkitt’s lymphoma.²⁴The MUTU-I (c179) cell line is an isogenic

stable group I BL cell line derived from a BL biopsy.²⁴The above cell

lines were cultured in RPMI-1640 medium containing phenol red

4.1.15. 2-N-Methylamino-1,3-bis(3,4-methylenedioxyphenyl)-

propane (13b)

To a stirred solution of 2-(N-formyl-N-methyl)amino-1,3-bis-

(3,4-methylenedioxyphenyl)propane (12b) (0.73 mmol, 0.25 g) in

methanol (25 mL), was added concd HCl (2.5 mL) and the mixture

heated at reﬂux for 7 h. After cooling the reaction was diluted with

water (100 mL) and washed with dichloromethane (3 Â 30 mL).

The aqueous phase was made basic with 15% aq NaOH and ex-

tracted with dichloromethane (3 Â 25 mL). The organic phases

were combined, dried over anhydrous Na₂SO₄and solvent re-

moved in vacuo. The product was puriﬁed by column chromatog-

raphy over silica gel (eluent: 60/40 diethyl ether/hexane). Pale

and supplemented with 10% (v/v) foetal bovine serum (FBS),

L-glu-

tamine (2 mM), penicillin and streptomycin (100 g/mL). The

l

MUTU-I c179 cell line required the additional supplements of al-

pha-thioglycerol (5 mM in phosphate buffered saline (PBS) with

20

l

M

bathocuprione disulfonic acid), sodium pyruvate

(100 mM) and HEPES (1 mM). The SHSY-5Y cell line SH-SY5Y is a

thrice cloned (SK-N-SH?SH-SY?SH-SY5?SH-SY5Y) subline of

the human neuroblastoma cell line SK-N-SH established from a

metastatic bone tumour.¹²These were cultured in DMEM: F12

(1:1) supplemented with 10% (v/v) (FBS),

icillin and streptomycin (100 g/mL).

HEK293 cells lines stably overexpressing human SERT was cul-

tured in DMEM supplemented with 10% (v/v) FBS, -glutamine

L-glutamine (2 mM), pen-

amber oil (84%). IR

m

_max(ﬁlm) 3336, 2791 cm^À1

.

¹H NMR d (CDCl₃)

l

1.72 (1H, br s, NH), 2.39 (3H, s, CH₃), 2.57 (2H, dd, J = 13.8 Hz,

J = 6.3 Hz, ArCH₂), 2.64 (2H, dd, J = 13.8 Hz, J = 6.8 Hz, ArCH₂), 2.79

(1H, m, NCH), 5.92 (4H, s, OCH₂O), 6.62 (2H, dd, J = 7.5 Hz,

J = 1.5 Hz, ArH), 6.66 (2H, d, H = 1.5 Hz, ArH), 6.73 (1H, d,

J = 8.0 Hz, ArH). ¹³C NMR ppm (CDCl₃) 34.09, 39.78, 62.94,

100.80, 108.16, 109.46, 122.17, 133.06, 145.95, 147.67. LRMS: m/

z 313 (M^ÀÀ1) 178. HRMS calculated for C₁₈H₂₀NO₄: (M⁺+H)

314.1392: found: 314.1374.

L

(2 mM), penicillin/streptomycin (100 mg/mL) and Geneticin

(500 mg/mL). Stable expression was valid up to 30 passages.

The MDA-MB231 cell line is an estrogen receptor (ER) negative

breast adenocarcinoma derived from a metastatic pleural effu-

sion.⁵⁰MDA-MB231 cells were cultured in DMEM, 10% (v/v) FBS,

L-glutamine (2 mM) with penicillin and streptomycin (100 lg/

mL). The MCF-7 cell line is an ER positive breast adenocarcinoma

4.2. Computational studies

derived from a metastatic pleural effusion.⁵¹MCF-7 cells were cul-

tured in MEM supplemented with 10% (v/v) FBS,

L-glutamine

To date no crystal structure of hSERT exists to avail of in the

docking process. A recent homology model of hSERT was con-

structed using LeuT as a template by Jørgensen et al.³³Previous

investigation of the ﬂexibility of the binding site in complex with

the natural substrate (5-HT) determined key protein–ligand inter-

actions.¹³The optimal 5-HT bound SERT complex was utilised as

our starting point for the series of docking studies. For ligand prep-

aration, all compounds were built and minimised using ^MOE

v2009.10.⁴⁹Docking was carried out using Molegro Virtual Docker

(www.molegro.com), which enables both ligand and receptor ﬂex-

ibility to be accounted for in the docking process. All side-chains

within 5Å of bound 5-HT were chosen as ﬂexible and a search

space with grid radius of 15 Å was created. No template was used

in the docking process to prevent any bias, and all other parame-

ters were retained as default. The highest ranking solutions ranked

by MolDock Score with appropriate H-bonding interactions were

then selected for analysis using ^MOEv2009.10.

(2 mM), penicillin and streptomycin (100 g/mL) and 1% (v/v)

l

non-essential amino acids. The 4TI is a cell line derived from a

mouse mammary gland tumour⁵²that is commonly xenografted

to create disease models for breast carcinomas. 4TI’s were cultured

in DMEM supplemented with 10% (v/v) FBS, L-glutamine (2 mM),

penicillin and streptomycin (100 lg/mL) and HEPES (1 mM). Cells

were maintained in a 72 cm²tissue culture ﬂasks at 37 °C in a

humidiﬁed atmosphere of 95% oxygen, and 5% carbon dioxide.

4.3.3. Generation of human peripheral blood mononuclear cells

Blood was obtained from a healthy donor, transferred into a

50 mL falcon tube and diluted 1:2 with PBS. LymphoPrep was used

to separate the blood into red blood cells, white blood cell ring and

serum. The blood was slowly added to 20 mL of ﬁcoll pague plus.

The tubes were centrifuged at 1700g for 30 min. The white blood

cell ring was transferred into a new 50 mL tube. The volume was

adjusted to 50 mL and the samples were centrifuged again at

1700g for 10 min. The supernatant was removed. This step was re-

peated again, the pellet was then resuspended in 10 mL of com-

plete IMDM media (10% FBS, 0.1% Ciproﬂoxacin (10 mg/mL)).

4.3. Biochemistry

4.3.1. Materials

DG-75 and MUTU-I (c179) BL cell lines were gifts from Dr. Der-

mot Walls (School of Biotechnology, Dublin City University, Ire-

land) and Professor Martin Rowe (Division of Cancer Studies, The

University of Birmingham, UK) respectively. The SHSY-5Y, MCF-7,

4.3.4. Neurotransmitter transporter uptake assay

A novel ﬂuorescence neurotransmitter transporter uptake assay

which actively transports a biogenic amine mimic into the cell

through the transporters SERT, NAT and DAT was used. The mi-

Y. M. McNamara et al. / Bioorg. Med. Chem. 19 (2011) 1328–1348

1347

metic ﬂuoresces once inside the cell and any external ﬂuorescence

is extinguished by a membrane impermeable masking dye. This al-

lows the ﬂuorescence of the cell to be measured and the SERT inhi-

bition of the compound determined.²⁶

a one way ANOVA Test comparing each pEC₅₀value. 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.

Cells were seeded at a density of 2 Â 10⁴/well in black, clear

bottomed, poly-L-lysine coated plates. After 24 h, the media was

aspirated and cells were washed with 100

were incubated for 15–20 min with 100 l of compound dissolved

in 1Â HBSS (Hanks balanced salt solution) + 0.1% BSA (bovine ser-

um albumin), before 100 l of the ﬂuorescent dye was added. The

ﬂuorescence of each plate was read at 520 nm (excitation 440 nm)

on a bottom up plate reader. The background ﬂuorescence of the

cells and 1Â HBSS buffer was taken away from each group. Un-

treated cells represented 100% ﬂuorescence/0% inhibition. Citalo-

l

l of 1Â HBSS. Cells

l

4.3.8. Quantiﬁcation of apoptosis

4.3.8.1. Propidium iodide FACS analysis. 7.5 Â 10⁵cells/5mL

were treated with the appropriate amount of compound and incu-

bated for a speciﬁed time. Cells were harvested by centrifugation at

300g for 5 min and washed with 5 mL of ice-cold PBS. The pellet

l

was resuspended in 200 ll PBS and 2 mL of ice-cold 70% ethanol

and cells were ﬁxed overnight at 4 °C. After ﬁxation, the cells were

pram (10 lM) was used as a positive control resulting in >98%

pelleted by centrifugation at 300g for 5 min and the ethanol was

inhibition. Fluorescence for each compound was calculated as a

carefully removed. The pellet was resuspended in 400

ll of PBS

percent of untreated cell response.

with 25 l of RNAse A (10 mg/mL stock) and 75 l of propidium io-

l

dide (1 mg/mL). The tubes were incubated in the dark at 37 °C for

30 min. Cell cycle analysis was performed using appropriate gates

counting 10,000 cells and analysed using ^CELLQUESTsoftware pack-

age. Untreated cells had <5% cells in the pre-G1 phase of the cell

4.3.5. Neutral red assay for in vitro cytotoxicity

The Neutral red (NR) assay was carried out as previously de-

scribed.⁵³Brieﬂy, 5 Â 10⁴cells per well (200

ll) were seeded in a

96-well plate until sub-conﬂuent (24–36 h) and treated with the

appropriate compound for 48 h. Following exposure of cells to drug

the supernatant was removed and the cells incubated for 3 1 h

cycle and 10 lM Taxol was used as a positive control for cell death.

4.3.8.2. Caspase 3/7 activity. Caspase 3/7 activity was assessed

with 250

warmed to 37 °C in media) under sterile conditions. NR solution

was removed carefully and the cells washed with 200 l of pre-

warmed PBS. Upon removal of all the PBS 100 l of NR assay Solu-

l

neutral red dye solution (stock 3.3 mg/mL-pre-

using the Apo-ONE^Òhomogenous caspase 3/7 assay. Brieﬂy,

0.5 Â 10⁵cells were treated with compound (10

lM) for 5 h before

the addition of 100 ll caspase substrate Z-DEVD-R110. After 2 h

l

the activity of caspase 3/7 was quantiﬁed by measuring the

bilisation solution (50% ethanol–1% acetic acid solution in deion-

ized water) was added to each well, the plate left to incubate in

the dark for 20–30 min at room temperature with gentle shaking.

The absorbance of each plate was read at 540 nm and at 690 nm

(background) within 1 h. Relative cell viability was expressed as

percent of vehicle treated cells. Sodium azide and Triton-X were

used as positive controls for cytotoxicity, where 30 mM sodium

azide and 2% Triton-X resulted in 80–90% cytotoxicity on all cell

lines. Untreated cells represented 0% cytotoxicity (100% viability).

amount of cleaved substrate at 521 nm (excitation 499 nm).

4.3.8.3. Detection of PARP cleavage by Western blot analy-

sis. 5 Â 10⁶cells were harvested by centrifugation at 500g for

5 min and the pellet washed with ice-cold PBS. Cells were resus-

pended in 60 ll PBS and 60 ll lysis buffer (Laemmli buffer;

62.5 mM Tris–HCl, 2% w/v SDS, 10% glycerol, 0.1% w/v bromophe-

nol blue supplemented with protease inhibitors). Samples were

prepared for SDS–PAGE resolved on a 8% loading gel and trans-

ferred onto PVDF membranes. Membranes were probed with

anti-PARP (recognises full length 113 kDa PARP as well as the

85 kDa cleaved form) primary antibody followed by incubation

with the corresponding IgG HRP conjugated secondary antibody.

Membranes were developed using electrochemiluminescence

detection.

4.3.6. Alamar Blue assay for antiproliferative activity

1–5 Â 10⁴cells/well were seeded in a 96-well plate and treated

with the respective drug for the desired length of time. Each well

was then treated with 20 ll of Alamar Blue (pre-warmed to

37 °C) and left to incubate at 37 °C in the dark for 4–6 h. Fluores-

cence was read using at 590 nm (excitation 544 nm). The back-

ground ﬂuorescence of the media without cells + Alamar Blue

was taken away from each group, and the control untreated cells

represented 100% cell viability. The antifungal agent miconazole

4.3.8.4. Staining and visualisation of cells to assess programmed

cell death morphology. Cells were seeded at

a density of

3 Â 10⁵cells/mL treated with the indicated compounds for the rel-

(10

lM) was used as a positive control for cell death in each of

evant time. An aliquot of cells (150 ll) was cytocentrifuged onto

the cell lines, resulting in 90% cytotoxicity.

microscopy slides at 500g for 2 min using a Cytospin 3 (Shandon).

The slides were removed and left to air-dry at room temperature

for 2 min. Staining was carried out using the RapiDiff kit (Sigma–

Aldrich) containing solution A (100% methanol), solution B (Eosin

Y) and solution C (methylene blue). The cells were ﬁxed by dipping

them 10 times in solution A. The nucleus of the cells was stained

pink by dipping the slide ten times in solution B and the cytoplasm

stained blue by dipping eight times in solution C. Excess dye was

washed off with deionized water. After allowing the slides to air

dry cells were examined under a light microscope using a 60Â

magniﬁcation

4.3.7. Statistical analysis

4.3.7.1. 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 pEC₅₀value deter-

mined by non-linear regression analysis of sigmoidal log concentra-

tion dependence curves whereby pEC₅₀is À[Àlog EC₅₀

]

SE

(log EC₅₀is the log [Dose] when response is equal to 50% cell viabil-

ity) (Table 1). All data points (expressed as means SEM) were ana-

lysed using ^GRAPHPADPrism (version 4) software (Graphpad software

Inc., San Diego, CA).

4.3.9. Assessment of PTP1B inhibition

The ability of compounds to inhibit protein tyrosine phospha-

tise 1B activity was assessed using a PTP1B Assay Kit (Calbiochem).

Brieﬂy, recombinant PTP1B expressed in Escherichia coli was incu-

bated with the test compound and a phosphopeptide substrate for

30 min at 30 °C. The detection of free phosphate released is de-

4.3.7.2. Comparison of pEC₅₀values in all cell lines. To deter-

mine if the pEC₅₀value calculated for each drug differed signiﬁ-

cantly in each cell line, statistical analysis was carried out using

1348

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Synthesis and serotonin transporter activity of 1,3-bis(aryl)-2-nitro-1- propenes as a new class of anticancer agents

DOI: 10.1016/j.bmc.2010.11.054

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