Synthesis, characterization, antiparasitic and cytotoxic evaluation of thioureas conjugated to polyamine scaffolds

Article abstract of DOI:10.1016/j.ejmech.2013.08.004

A series of mono- and multimeric 4-amino-7-chloroquinoline and ferrocenyl thioureas have been prepared by the reaction of a 7-chloroquinoline methyl ester and a ferrocenylimine methyl ester with various amines. These compounds were characterized using standard spectroscopic and analytical techniques. The compounds were evaluated against the NF54 (CQ-sensitive) and Dd2 (CQ-resistant) strains of Plasmodiumfalciparum. The quinoline compounds show enhanced activity compared to the ferrocene compounds against this parasite. Compound 5 displays the most promising activity against the NF54 strain. Compounds 5 and 6 are effective at inhibiting β-hematin formation perhaps due to an increased number of quinoline moieties. The trimeric (12) and tetrameric (13) ferrocenyl compounds also inhibit β-hematin formation, albeit to a lesser degree compared to the quinoline thioureas. The compounds were also screened against the G3 strain of Trichomonasvaginalis and here the ferrocene-containing compounds show a slightly higher parasite growth inhibition compared to the quinoline thioureas. The quinoline compounds were also found to be more cytotoxic compared to the ferrocenyl compounds. Compound 6 displays good cytotoxicity against WHCO1 oesophageal cancer cells.

Full text of DOI:10.1016/j.ejmech.2013.08.004

European Journal of Medicinal Chemistry 69 (2013) 90e98
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization, antiparasitic and cytotoxic evaluation
of thioureas conjugated to polyamine scaffolds
Tameryn Stringer a, Dale Taylor b, Carmen de Kock b, Hajira Guzgay c, Aaron Au d,
Seung Hwan An d, Benjamin Sanchez d, Raquel O’Connor d, Neal Patel d,
Kirkwood M. Land d, Peter J. Smith b, Denver T. Hendricks c, Timothy J. Egan a,
,
Gregory S. Smith a
*
a Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
b Division of Pharmacology, Department of Medicine, University of Cape Town Medical School, Observatory 7925, South Africa
c Division of Medical Biochemistry, University of Cape Town, Rondebosch 7701, South Africa
d Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of mono- and multimeric 4-amino-7-chloroquinoline and ferrocenyl thioureas have been pre-
pared by the reaction of a 7-chloroquinoline methyl ester and a ferrocenylimine methyl ester with
various amines. These compounds were characterized using standard spectroscopic and analytical
techniques. The compounds were evaluated against the NF54 (CQ-sensitive) and Dd2 (CQ-resistant)
strains of Plasmodium falciparum. The quinoline compounds show enhanced activity compared to the
ferrocene compounds against this parasite. Compound 5 displays the most promising activity against the
Received 20 March 2013
Received in revised form
10 July 2013
Accepted 2 August 2013
Available online 11 August 2013
NF54 strain. Compounds 5 and 6 are effective at inhibiting
increased number of quinoline moieties. The trimeric (12) and tetrameric (13) ferrocenyl compounds also
inhibit -hematin formation, albeit to a lesser degree compared to the quinoline thioureas. The com-
b-hematin formation perhaps due to an
Keywords:
Quinoline
Ferrocene
b
pounds were also screened against the G3 strain of Trichomonas vaginalis and here the ferrocene-
containing compounds show a slightly higher parasite growth inhibition compared to the quinoline
thioureas. The quinoline compounds were also found to be more cytotoxic compared to the ferrocenyl
compounds. Compound 6 displays good cytotoxicity against WHCO1 oesophageal cancer cells.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Thioureas
Polyamines
Antiparasitic activity
Antitumor activity
b-Hematin inhibition
1. Introduction
Malaria is an infectious disease that affects humans and is
considered to be the most common parasitic disease in the world
Polyamines are compounds that play a vital role in all living
systems. These compounds are naturally occurring agents that are
found in large amounts in mammalian cells and are mainly
responsible for maintaining cell viability [1]. Previous studies
reveal that these systems are able to transport cytotoxic drugs into
tumor cells [2]. The tumor cells import polyamines by means of a
polyamine transporter (PAT) in order to sustain their growth. The
transporter is able to tolerate modified polyamines, therefore drug
incorporated polyamines are able to penetrate the tumor cells [3].
Although polyamine research has been aimed mainly towards the
discovery of selective drugs targeting human tumor cells, these
compounds have also been found to exhibit selective uptake by
Plasmodium infected erythrocytes [4].
[5]. This disease is caused by protozoans of the genus Plasmodium.
The most deadly species of the parasite is Plasmodium falciparum
[6]. According to the World Health Organization (WHO), in 2010,
216 million cases of malaria were reported worldwide. In 2010,
655,000 cases of malaria-related deaths were documented [7].
Many treatments are available to date to combat this disease, his-
torically the most widely used being chloroquine (CQ), (Fig. 1). This
drug has been a successful treatment against this disease for many
years. The onset of resistance against CQ has led to increased
research towards the attainment of more potent compounds that
are able to overcome drug resistance. Artemisinin-based combi-
nation therapies (ACTs) are the current treatment for this disease
and involve treatment using an artemisinin derivative in conjunc-
tion with a 4-aminoquinoline or an amino alcohol, in order to
delay the onset of resistance [8]. Resistance in many strains
of P. falciparum is mainly due to mutations in the P. falciparum
* Corresponding author. Tel.: þ27 (0)21 650 5279; fax: þ27 (0)21 650 5195.
E-mail address: gregory.smith@uct.ac.za (G.S. Smith).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.004

T. Stringer et al. / European Journal of Medicinal Chemistry 69 (2013) 90e98
91
HN
N
N
HN
Fe
Cl
N
Cl
N
Fig. 1. CQ and FQ, potent 7-chloroquinoline compounds.
chloroquine resistant transporter (PfCRT), a protein involved in
drug efflux and proton equilibrium across the membrane of the
digestive vacuole. This mutation prevents accumulation of lethal
concentrations of chloroquine [9,10]. It is widely accepted that
many quinoline-based antimalarials disturb the detoxification of
free heme, which is generated upon hemoglobin (Hb) degradation.
The oxidized by-product of Hb degradation is ferriprotoporphyrin-
IX (hematin), which is toxic towards the parasite. The parasite
converts this compound into a less toxic form known as hemozoin
(malarial pigment). CQ is believed to form a complex with heme
and in turn inhibits hemozoin formation, causing a build up of toxic
hematin (Fe(III)PPIX) [11e14]. Hematin is therefore believed to be
the target of many quinoline antimalarials, but a study conducted
by Buller et al. suggests that hemozoin may be the target. In this
diamine (1) and 3-(7-chloro-quinolin-4-ylamino)-propyl-dithio-
carbamic acid methyl ester (2) were prepared according to literature
procedures [26,27]. The first step involves reacting 4,7-
dichloroquinoline and 1,3-diaminopropane to yield compound 1.
Subsequent reaction of compound 1 with carbon disulfide in the
presence of KOH, followed by addition of methyl iodide drop-wise at
0
ꢀC gives rise to compound 2. Compound 2 was refluxed with n-
propylamine, 1,3-diaminopropane, tris(2-amino)ethyl amine and
N,N,N0,N0-tetrakis(3-aminopropyl)-1,4-butanediamine (DAB-Am-4)
in MeOH to afford compounds 3e6, respectively, as cream powders.
Three ferrocenylimine thioureas were prepared by the procedures
outlined in Scheme 2. Methyl hydrazinecarbodithioate (7) was pre-
pared using the method described by Klayman et al. [16]. Subsequent
reaction of compound 7 with ferrocenecarboxaldehyde in i-PrOH
gives rise to thioester 9 [28]. Compounds 10 and 12 were prepared by
refluxing compound 9 with n-propylamine and tris(2-amino)ethyl
amine in EtOH, respectively. Compound 11 was prepared by modi-
fication of the method outlined by D.M. Wiles et al. [29]. Compound
11 was prepared by reacting ethane-1,2-dithiosemicarbazide (8) [30]
and ferrocenecarboxaldehyde in water. The ferrocenyl compounds
were isolated as orangeered powders.
study they found that the fastest growing crystal face of b-hematin
(synthetic hemozoin) is the binding site for quinoline-based drugs.
This in turn leads to inhibition of crystal growth [15].
Thioureas are attractive for the design of antiplasmodial agents.
Thiosemicarbazones are a class of thioureas that possess a wide
range of biological properties including antimalarial activity [16e
20]. Many thiosemicarbazones display activity against protozoans
by inhibition of various cysteine proteases as well as other targets
[20]. Ferrocenyl compounds have also been shown to possess ac-
tivity including anticancer, antimalarial and antifungal [21]. In
addition to this, thiosemicarbazones (TSCs) containing ferrocenyl
moieties have been found to be active against protozoa as well as
certain tumors [22,23]. More recently ferrocenyl thio-
semicarbazones conjugated to a poly(propyleneimine) dendrimer
have been evaluated in vitro and display moderate antiplasmodial
activity [24]. Incorporation of the ferrocenyl moiety may alter the
lipophilic nature of molecules and therefore may enhance the ac-
tivity of certain compounds. Ferroquine (Fig. 1) is an excellent
example of a ferrocenyl-containing molecule that exhibits potent
antiplasmodial activity against CQ resistant strains of P. falciparum
[25]. This study investigates the synthesis and characterization of
polyamineethiourea conjugates. The aims were to (i) investigate
the effect of increasing the number of active pharmacophores (and
molecule size) on biological activity and (ii) to evaluate whether
conjugation to the various polyamines enhances the biological ac-
tivity of these compounds. A preliminary evaluation of the in vitro
antiparasitic and antitumor activity is described. In addition to this,
2.2. Characterization
The structures of all the compounds were confirmed by 1H NMR,
13C{1H} NMR and FTIR spectroscopy, ESI-mass spectrometry and
elemental analysis. The lack of a singlet at 2.46 ppm in the 1H NMR
spectra of compounds 3e6 confirms successful displacement of the
eSCH3 group of compound 2 in each case. For compounds
3e6, signals for the newly formed NH group occur at approximately
7.19 ppm. Signals for the imine protons of compounds 10e12 are
observed as sharp singlets at 7.89, 7.90 and 7.90 ppm for 10e12
respectively. These shifts are upfield in comparison to the same
signal observed for compound 9. Signals for the hydrazinic protons
are observed between 11.11 and 11.29 ppm. Singlets for the newly
formed amino group are observed between 8.12 and 8.36 ppm.
Signals for the unsubstituted Cp ring of the ferrocenyl moieties are
observed as singlets at approximately 4.2 ppm for these com-
pounds. The 13C NMR spectral data displays signals for the thio-
carbonyl of compounds 3e6 between 181.68 and 182.23 ppm,
while these signals appear at 176.73e177.23 ppm for compounds
10e12. The imine carbon signals appear at 143.45e143.96 ppm for
the ferrocenyl derivatives. The IR spectra of compounds 3e6
display absorption bands for the quinoline C]N between 1611 and
1613 cmꢁ1. Absorption bands attributed to the C]N are observed
between 1604 and 1611 cmꢁ1 for compounds 10e12. Further
confirmation of the attainment of these compounds can be
observed by the data obtained from ESI mass spectrometry. The
mass spectra of the compounds gave peaks corresponding to
[M þ H]þ. Peaks corresponding to [M þ 2H]2þ and [M þ 2Na]2þ
were also observed for compounds 4 and 5, respectively.
the inhibition of
studied.
b-hematin formation by these compounds is also
2. Results and discussion
2.1. Chemistry
Four 4-amino-7-chloroquinolines were synthesized and charac-
terized using template reactions. These compounds were prepared
as outlined in Scheme 1. N0-(7-Chloroquinolin-4-yl)-propane-1,3-

92
T. Stringer et al. / European Journal of Medicinal Chemistry 69 (2013) 90e98
Cl
HN
NH2
H2N
NH2
a
1
Cl
N
Cl
N
b
S
HN
S
Cl
S
HN
HN
N
H
S
HN
N
HN
N
N
H
N
H
S
HN
HN
4
c
f
2
3
NH
Cl
Cl
N
N
Cl
Cl
H
e
N
d
N
NH
Cl
S
S
NH
NH
S
NH
N
HN
HN
N
N
N
H
H
S
N
S
N
N
S
HN
N
N
N
H
Cl
N
NH
NH
S
H
HN
H
HN
HN
HN
6
N
Cl
NH
Cl
NH
5
N
N
Cl
Cl
Scheme 1. Synthesis of 4-amino-7-chloroquinoline thioureas. Reagents and conditions: (a) neat; (b) KOH (1 eq.), CS2 (1 eq.), CH3I (1 eq.) 0e5 ꢀC; (c) n-propylamine, (d) tris(2-
amino)ethyl amine; (e) DAB-Am-4; (f) 1,3-diaminopropane MeOH, reflux.
2.3. In vitro antiplasmodial activity
compound (12), shows the lowest activity. Comparing the activity
of compounds 3, 4 and 5, the enhanced activity of the latter may be
due to incorporation of a basic tertiary amino group (not found in 3
and 4) that may aid in accumulation of the compound in the
digestive vacuole of the parasite. In the case of 6, conjugation to the
1st generation DAB dendrimer leads to a loss of activity compared
to 3 and 5, however this particular compound is more active than 4.
The slightly lower activity of compound 6, which also contain ter-
tiary amines, may be attributed to its lipophilicity. Predicted log P
and log D values are shown in Table 1. In the sensitive strain, 5
shows the best activity, suggesting sufficient vacuolar accumulation
of this compound which may be a consequence of the log D at pH
7.4. Compounds 3 and 6 exhibit similar activity in the sensitive
strain but are less active than 5. This suggests less accumulation
which may be a consequence of the lower log D7.4 values compared
to the rest of the compounds. Compound 4 has a higher log D7.4
value than 3 and 6 but appears to accumulate less based on the
larger IC50 value. The estimated log P value of compound 6 indicates
it is more lipophilic than the other members of this series (when
uncharged) and this factor may hinder its transport as it may be
trapped in the lipid membrane and may not accumulate sufficiently
inside of the digestive vacuole [38] which may be the reason for the
low activity in the resistant strain. The mono- and trimeric ferro-
cenyl compounds (10 and 12) exhibited the lowest activity at the
tested concentration, while the dimeric ferrocenyl thiourea (11)
displayed moderate activity in the CQ sensitive strain. This partic-
ular compound displays a log P value comparable to chloroquine.
The low activity of 12 in the sensitive strain may be a combination
of its lipophilic character and the lack of the active quinoline
Thiourea moieties are active pharmacophores that form part of
many biologically active systems [31,32]. More recently, researchers
have introduced thiourea moieties as part of the side chain of some
4-aminoquinoline systems. These compounds have been shown to
exhibit antiplasmodial activity [26,33]. In addition to this, poly-
amine conjugates have attracted attention due to their pharma-
ceutical application [34]. These polyamines may alter the lipophilic
nature of compounds which may aid in the transport of drugs
across membranes, which is a specifically useful trait for antima-
larial agents. In addition to this, incorporation of pharmacophores
onto polyamines may result in the attainment of multifunctional
systems which may show enhanced activity in comparison to
monofunctional derivatives. This has been observed with ruthe-
niumearene complexes and ferrocenyl thiosemicarbazones
conjugated to poly(propyleneimine) dendrimers [24,35,36]. Com-
pounds 3e6 and 10e12 were evaluated for their in vitro anti-
plasmodial activity against the NF54 CQ sensitive and Dd2 CQ
resistant strains of P. falciparum. A doseeresponse experiment was
performed on the compounds to determine the IC50 or the con-
centration inhibiting 50% of parasite growth. The results are shown
in Table 1.
Compounds 3e6 (quinoline derivatives) display better activity
against both strains of P. falciparum compared to compounds
10e12. This result is comparable to other compounds containing
the 4-amino-7-chloroquinoline motif that have been used suc-
cessfully to combat malaria. The trimeric quinoline, 5, shows the
best activity in the CQ sensitive strain, while the trimeric ferrocenyl

T. Stringer et al. / European Journal of Medicinal Chemistry 69 (2013) 90e98
93
H
N
H
S
a
N
S
b
NH2NH2.H2O
N
H2N
S
Fe
S
9
7
c
H
N
H
N
d
N
S
Fe
H
N
H
H
N
N
H
N
10
N
N
N
Fe
S
Fe
S
HN
12
HN
N
S
Fe
Fe
S
S
H
N
H
H
H
N
N
N
e
N
NH2
f
N
N
N
H
NH2
H2N
N
H
N
H
H
S
H2N
Fe
S
8
11
Scheme 2. Synthesis of ferrocenyl thioureas. Reagents and conditions: (a) KOH (1 eq.), CS2 (1 eq.), CH3I (1 eq.) 0e5 ꢀC; (b) ferrocenecarboxaldehyde i-PrOH, reflux; (c) n-pro-
pylamine EtOH reflux; (d) tris(2-amino)ethyl amine EtOH reflux; (e) NaOH, CS2, NH2NH2$H2O; (f) ferrocenecarboxaldehyde H2O r.t.
moiety. The ferrocenyl precursor (9), was also tested, but was found
to be inactive (not shown). This suggests functionalization with the
various amines leads to enhanced activity.
Lipophilicity appears to have little effect on the activity of these
compounds in the resistant strain. Compounds 3 and 4 show
comparable activity, while compounds 5 and 6 exhibit similar ac-
tivity in the Dd2 strain. Compounds 3, 4 and 10 show similar
activity in both the sensitive and resistant strains, while com-
pounds 5 and 6 show slightly lower activity in the resistant strain
compared to NF54. The activity of compound 11 decreased signif-
icantly against Dd2 compared to NF54 which is confirmed by the
large Resistance Index (RI) of 27.7. The RI is valuable for the analysis
of the potential of compounds as antimalarial drugs. RI values are
defined as the quotient of the IC50 value obtained in the resistant
strain and the IC50 value obtained in the sensitive strain. Smaller RI
values (<1) indicate that the compound is more active in the
resistant strain than in the sensitive strain, while larger values (>1)
indicate the opposite. Ideally, the compound should exhibit similar
activity in both strains. The activity of compound 12 increased
significantly; this particular compound gave an RI of 0.1. In most
cases, with the exception of compound 11, the compounds show a
lower RI compared to CQ.
Table 1
In vitro antiplasmodial activity for thioureas 3e6 and 10e12 against NF54 and Dd2
strains of P. falciparum. Selected log P and log D values are also given.
Compound IC50
(m
M) ꢂ
IC50
(
m
M) ꢂ
RId
Log Pe Log D7.4 Log D4.8
SEa (NF54)b
SE (Dd2)c
3
4
0.16 ꢂ 0.03
0.34 ꢂ 0.03
(n ¼ 6)f
0.26 ꢂ 0.01
0.23 ꢂ 0.01
1.6 2.95
0.7 4.03
2.71
3.54
1.63
1.40
2.4.
b-Hematin inhibition study
5
6
10
11
12
CQ
0.09 ꢂ 0.01
0.20 ꢂ 0.01
0.52 ꢂ 0.06
0.51 ꢂ 0.03
5.8 5.88
2.5 8.62
1.0 2.99g
5.02
2.78
e
ꢁ0.22
ꢁ3.62
In order to design new antimalarials, the ability of the com-
pounds to inhibit heme aggregation should also be considered
15.79 ꢂ 1.28 16.22 ꢂ 1.59
e
e
e
e
1.15 ꢂ 0.07
71.71 ꢂ 9.93
0.02 ꢂ 0.01
31.88 ꢂ 5.89 27.7 4.78
e
5.63 ꢂ 0.19
0.14 ꢂ 0.01
0.1 7.66
4.63h
e
(Table 2). The ability of these compounds to inhibit
b-hematin
7
e
(synthetic hemozoin) formation was investigated using an NP-40
detergent mediated assay [39]. In addition to the above-
mentioned compounds, a tetrameric ferrocenyl derivative (13)
shown in Fig. 2, was also evaluated. Compound 13 showed activity
against the W2 CQ-resistant strain of P. falciparum in a previous
a
IC50 represents the micromolar equivalents of test compounds required to
inhibit parasite growth by 50%.
b
NF54 chloroquine-sensitive strain of P. falciparum.
Dd2 chloroquine-resistant strain of P. falciparum.
c
d
Resistance index (RI) ¼ IC50Dd2/IC50NF54.
e
Log P and log D values calculated using MarvinSketch V5.9.4.
study [24]. Compounds 12 and 13 appear to inhibit
formation giving IC50 values of 30.70 and 15.20 M, respectively.
The quinoline compounds inhibit -hematin formation better than
the ferrocenyl thioureas, specifically compound 6 (IC50 < 4.5 M).
This may be attributed to an increased number of planar aromatic
b-hematin
f
n ¼ number of data sets averaged, n ¼ 3 (for NF54) and n ¼ 2 (for Dd2), ex-
m
ceptions are specified.
b
g
Log P of ferrocenyl compounds estimated using methods described in the
m
Supporting information.
h
Literature log P value [37].

94
T. Stringer et al. / European Journal of Medicinal Chemistry 69 (2013) 90e98
Table 2
IC50
M)a values obtained for compounds 3e6 and 10e13
Table 3
(
m
Percentage parasite growth inhibition (%) obtained for 3e6 and
9e13 against T. vaginalis.
using an NP-40 detergent mediated
b-hematin inhibition
assay.b
Compound
% Inhibition ꢂ SE
Compound
IC50
(
m
M)a ꢂ SE
3
4
5
6
9
10
11
12
34.9 ꢂ 11
55.9 ꢂ 9
51.4 ꢂ 11
58.7 ꢂ 9
43.1 ꢂ 9
46.2 ꢂ 10
59.6 ꢂ 11
53.2 ꢂ 10
61.5 ꢂ 11
100
3
4
5
6
58.17 ꢂ 1.01
6.52 ꢂ 1.06
4.48 ꢂ 1.04
<4.5
10
11
12
13
CQ
Inactive
Inactive
30.70 ꢂ 1.06
15.20 ꢂ 1.03
73.76 ꢂ 1.02
13
Metronidazole
n ¼ number of data sets averaged, n ¼ 4.
a
IC50 represents the micromolar equivalents of test
compounds required to inhibit
b-hematin formation by
percentage growth inhibition. The tetrameric compound 6, which
was functionalised with DAB-Am-4, showed the best inhibition of the
50%.
b
Sigmoidal doseeresponse curves (variable slope) were
plotted using GraphPad Prism V. 5.
series, followed by compound
diaminopropane) and then compound
4
(functionalized with 1,3-
(functionalized with
5
tris(2-aminoethyl)amine). A similar trend is observed for the ferro-
cenyl compounds. The tetrameric compound (13) shows the highest
percentage inhibition, followed by the dimeric and then the trimeric
derivative. The monomeric compounds (9 and 10) display the lowest
inhibition of the series. Overall, the largest compounds show the
highest inhibition. On average the ferrocenyl compounds inhibit
parasite growth slightly better than the quinoline compounds. The
lipophilic nature of the ferrocene moiety may be a factor contributing
to the slightly enhanced activity of the organometallic derivatives.
Although many of these compounds are able to inhibit more than 50%
of parasite growth, the activity is not comparable to metronidazole.
moieties that are able to interact with heme and thus preventing
biocrystallization into -hematin [13]. There appears to be a rela-
tionship between the size of these molecules and their ability to
inhibit heme aggregation. The larger, tetrameric molecules are able
b
to inhibit
monomeric derivatives. No clear correlation is observed between
-hematin inhibition and the in vitro antiplasmodial activity. This is
b-hematin formation to a greater extent than the
b
probably due to differences in accumulation of these compounds
inside the parasite.
2.5. T. vaginalis growth inhibition
2.6. In vitro cytotoxicity
Trichomonas vaginalis (T. vaginalis) is a protozoan responsible for
the sexually transmitted disease trichomoniasis. The current FDA-
approved drug, metronidazole (Fig. 2), currently experiences
resistance in some patients [40,41]. There has recently been an
interest in obtaining a suitable alternative treatment for this
infection [41e44]. We have conducted a preliminary study to
evaluate the activity of our synthesized compounds against this
particular parasite. The compounds were screened against the G3
strain of T. vaginalis at a drug concentration of 10 mM. Table 3 shows
the data obtained from this screen.
In the case of the quinoline compounds, the monomeric deriva-
tive 3 (functionalised with n-propylamine) showed the lowest
The in vitro antitumor activity of the compounds was also
evaluated against WHCO1 oesophageal cancer. This was to eluci-
date information regarding the selectivity of these compounds. A
promising antimalarial candidate should ideally exhibit good
antiplasmodial activity, but low cytotoxicity. It has been found that
both quinoline and ferrocene containing compounds have been
studied as anticancer agents [45,46]. Some ferrocene compounds
have been found to be effective against certain cancers [46].
The compounds were evaluated for their antitumor activity using
a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
(MTT) assay. With the exception of compounds 11 and 12, the rest
Fig. 2. Tetrameric ferrocenyl thiourea (13) and metronidazole, the current treatment for T. vaginalis.

T. Stringer et al. / European Journal of Medicinal Chemistry 69 (2013) 90e98
95
Table 4
promising cytotoxic behavior, better than cisplatin against this
particular cell-line. There appears to be a relationship between the
size of the molecule and cytotoxicity in the case of the quinoline
derivatives. The ferrocenyl thioureas show lower antitumor activity
compared to the quinolines. In the case of T. vaginalis growth, it
appears that the ferrocenyl compounds show slightly enhanced
activity compared to the quinolines although not comparable to
metronidazole. All of the quinoline compounds were able to inhibit
IC50 values obtained for 3e6 and 10e12 against WHCO1
oesophageal cancer cells.a
Compound
IC50
(
m
M) ꢂ SE
3
4
5
6
10
11
43.75 ꢂ 1.09
38.35 ꢂ 1.12
6.21 ꢂ 1.09
0.99 ꢂ 1.05
55.73 ꢂ 1.28
Inactive
b-hematin formation, but the tetrameric derivative (6) displays the
best activity which is of interest for further investigation. A size
dependant-activity was observed for all compounds for this assay.
12
Cisplatin
Inactive
13.00b
No clear correlation between the b-hematin inhibition activity and
n ¼ number of data sets averaged, n ¼ 3.
a
Values were determined from a dose response curve
(assayed with MTT), IC50 represents the micromolar
equivalents of test compounds required to inhibit 50% cell
viability.
the antiplasmodial activity was observed. However, the data does
suggest that 4-amino-7-chloroquinolines remain a potent and
important class of compounds towards malaria.
b
IC50 value obtained for cisplatin against WHCO1 can-
4. Experimental
cer cells [47].
4.1. General
of the compounds appeared to cause decreased cell viability at the
tested concentration. Nevertheless, some trends with regards to the
antitumor activity are observed and the IC50 values are shown in
Table 4.
All reagents and solvents were obtained from commercial sources
(SigmaeAldrich, Merck, and Kimix) and, unless otherwise stated, were
used as received. N0-(7-Chloroquinolin-4-yl)-propane-1,3-diamine (1)
[24] and 3-(7-chloro-quinolin-4-ylamino)-propyl-dithiocarbamic
acid methyl ester (2) [26], ethane-1,2-dithiosemicarbazide (8) [27],
ferrocenylthioester (9) [28] and the tetrameric thiourea (13) [30] were
prepared by modification of literature procedures. Nuclear magnetic
resonance (NMR) spectra were recorded using a Varian Mercury 300
spectrometer (1H at 300.077 MHz, 13C at 75.454 MHz), a Varian Unity
400 spectrometer (1H at 399.953 MHz,13C at 100.577 MHz) ora Bruker
400 FT spectrometer (1H at 400.200 MHz, 13C at 100.600 MHz).
Coupling constants are reported in Hz. Infrared (IR) spectra were
determined using a Perkin Elmer Spectrum 100 FT-IR spectrometer
and was carried out in the solid state using KBr pellets. Electrospray
Ionization (ESI) mass spectrometry was used to further characterize
compounds and determinations were carried out using a Waters API
Quattro instrument in the positive mode. Elemental analyses of these
compounds were performed using a Thermo Flash1112 Series CHNS-O
Analyser.
The quinoline compounds show enhanced activity compared to
the ferrocenyl thioureas. Compound 6 exhibits promising activity
compared to the rest of the series. There appears to be an increase
in activity when going from the monomeric quinoline derivative
to the tetrameric compound. This phenomenon is also observed
for certain dendritic compounds whereby cytotoxicity is often
generation-dependant, with higher generation dendrimers
showing the best activity [35,36,48]. Studies carried out on natu-
rally occurring polyamines show that these systems are able to
transport cytotoxic drugs into tumor cells [2]. In our case, for the
synthetic polyamines are under investigation, it has been shown
that the polyamine transporter is able to tolerate modified poly-
amines therefore this compound may be taken up by the cell [3].
This factor in combination with the ability of these quinolines to
interact with DNA, possibly by intercalation, may be a reason for the
enhanced activity [49]. The activity of the compounds was also
compared to cisplatin, a potent metal-containing anticancer agent,
compounds 5 and 6 exhibit better activity than this drug. Com-
pound 10 exhibits low cytotoxicity in this cell-line, while com-
pounds 11 and 12 were not active at the concentration tested.
4.2. Synthetic methods and characterization
4.2.1. Compound 3
Compound 2 (0.354 g, 1.09 mmol) was dissolved in MeOH
(20 ml), to this n-propylamine (0.108 g, 1.82 mmol) was added
drop-wise with stirring. The solution was refluxed for 24 h and
cooled to room temperature. The solvent was removed in vacuo and
diethyl ether added to the residue. The mixture was washed with
diethyl ether and the supernatant decanted. This was repeated until
the supernatant remained clear. The oily residue was dried under
vacuum giving rise to a cream solid. Yield: 0.220 g, 60%; IR (cmꢁ1):
3. Conclusions
A series of 4-amino-7-chloroquinoline (3e6) and ferrocenyl
(10e12) thioureas have been prepared. These compounds were
characterized using standard spectroscopic and spectrometric
techniques, confirming the integrity of these molecules. The com-
pounds were evaluated for their in vitro antiplasmodial activity
against the NF54 CQ sensitive and Dd2 CQ resistant strain of
P. falciparum, in vitro antitumor activity against WHCO1 oesopha-
3435 (NeH); 1613 (C]N); 1136 (C]S); 1H NMR (DMSO-d6):
d ppm
0.86 (3H, t, 3JHeH ¼ 7.4, CH3); 1.49 (2H, m, CH2); 1.90 (2H, m, CH2);
3
geal cancer cells and in vitro inhibition of T. vaginalis growth. The
b
-
3.30 (4H, m, CH2); 3.52 (2H, m, CH2); 6.47 (1H, d, JHeH ¼ 5.4, Are
hematin inhibition activity of these compounds was also evaluated.
The quinoline compounds 3e6 display moderate to good activity
against both strains of P. falciparum, although not as good as chlo-
roquine. There does not appear to be a clear correlation between
increasing the number of active moieties and antiplasmodial ac-
tivity. Various other factors including vacuolar accumulation
appear to be a contributing factor. In the case of the ferrocenyl
thioureas, functionalization with the various amines does lead to
enhanced activity compared to the methyl ester precursor. With
regard to cytotoxicity, the monomeric thiourea shows the lowest
activity while compound 6, the tetrameric compound shows
H); 7.19 (1H, br s, NH); 7.35 (2H, m, NH); 7.42 (1H, dd, 3JHeH ¼ 8.9,
4JHeH ¼ 2.2, AreH); 7.80 (1H, d, 4JHeH ¼ 2.2, AreH); 8.24 (1H, d, 3JHe
3
¼ 9.0, AreH); 8.40 (1H, d, JHeH ¼ 5.4, AreH); 13C NMR (DMSO-
H
d6):
d ppm 11.1; 21.8; 27.6; 40.3; 41.1; 45.1; 98.5; 117.4; 123.8 (2C);
127.4; 133.2; 148.9; 149.9; 151.7; 181.99. ESI MS: m/z 337.1248
(100%, [M þ H]þ). C16H21N4SCl$1.5H2O calculated: C 52.81, H 6.65, N
15.40%; found: C 53.06; H 6.96; N 16.21%.
By following the same synthetic procedure as that for 3, the
following compounds were synthesized, with the exception that
these compounds were washed repeatedly with acetone (until
supernatant remained clear) followed by diethyl ether.

96
T. Stringer et al. / European Journal of Medicinal Chemistry 69 (2013) 90e98
4.2.2. Compound 4
few drops of 10.1 M HCl was added. Ferrocenecarboxaldehyde
(0.331 g, 1.55 mmol) was added to the suspension and the reaction
mixture stirred for 24 h at room temperature. The precipitate was
filtered, washed with water and dried in vacuo. Yield: 0.453 g, 98%. IR
(cmꢁ1): 3437 (NeH); 3267 (NeH); 1611 (C]N); 816 (C]S). 1H NMR
Compound 4 was prepared from 2 (1.49 g, 4.56 mmol) and 1,3-
propanediamine (0.117 g, 1.56 mmol). Yield: 0.818 g, 85%; IR
(cmꢁ1): 3258 (NeH); 1613 (C]N); 1138 (C]S); 1H NMR (DMSO-
d6):
d ppm 1.73 (2H, m, CH2); 1.90 (4H, m, CH2); 3.30 (4H, m, CH2);
3
3.40 (4H, m, CH2); 3.51 (4H, m, CH2); 6.46 (2H, d, JHeH ¼ 5.4, Are
(DMSO-d6): d
ppm 3.78 (4H, s, CH2); 4.17 (10H, s, Cp); 4.39 (4H, t, 3JHe
H); 7.19 (2H, m, NH); 7.37e7.47 (6H, m, NH, AreH); 7.77 (2H, d, 4JHe
¼ 1.8, Cp); 4.74 (4H, t, 3JHeH ¼ 1.8, Cp); 7.90 (2H, s, HC]N); 8.36 (2H,
H
3
3
¼ 2.1, AreH); 8.24 (2H, d, JHeH ¼ 9.0, AreH); 8.40 (2H, d, JHe
br s, NH); 11.29 (2H, s, NH) ppm. 13C NMR (DMSO-d6):
d ppm 44.0;
H
H
¼ 5.4, AreH); 13C NMR (DMSO-d6):
d
ppm 27.4; 28.5; 40.9e40.9
68.2; 69.4; 70.5; 79.4; 143.9; 177.2. ESI MS: m/z 601.0582 (100%,
[M þ H]þ). C26H28N6Fe2S2$H2O calculated: C 50.50, H 4.89, N 13.59%;
found: C 50.67, H 4.71, N 13.85%.
(6C); 98.4; 117.2; 123.7; 123.9; 127.0; 133.3; 148.7; 149.9; 151.6;
181.7; ESI MS: m/z 629.1812 (20%, [M þ H]þ); 315.0951 (100%,
[M þ 2H]2þ). C29H34N8S2Cl2$2H2O calculated: C 52.32, H 5.75, N
16.83%; found: C 52.51; H 5.95; N 15.13%.
4.2.7. Compound 12
Compound 12 was prepared from compound 9 (0.506 g,
1.59 mmol) and tris(2-aminoethyl)amine (0.0780 g, 0.534 mmol).
Yield: 0.226 g, 46%. IR (cmꢁ1): 3431 (NeH); 3366 (NeH); 1606
4.2.3. Compound 5
Compound
5 was prepared from Compound 2 (0.415 g,
1.27 mmol) and tris(2-aminoethyl)amine (0.0586 g, 0.400 mmol).
(C]N); 818 (C]S); 1103 (ferrocene). 1H NMR (DMSO-d6):
d ppm
Yield: 0.242 g, 58%. IR (cmꢁ1): 3280 (NeH); 1611 (C]N); 1138
2.83 (6H, m, CH2); 3.71 (6H, m, CH2); 4.18 (15H, s, Cp); 4.41 (6H, m,
(C]S). 1H NMR (CD3OD-d4):
d
ppm 1.96 (6H, m, CH2); 2.66 (12H, m,
Cp); 4.69 (6H, m, Cp); 7.90 (3H, s, HC]N); 8.15 (3H, br s, NH); 11.22
CH2); 3.44e3.74 (12H, m, CH2); 6.44 (3H, m, AreH); 7.31 (3H, m,
AreH); 7.71 (3H, m, AreH); 7.99 (3H, m, AreH); 8.28 (3H, m, AreH).
(3H, s, NH) ppm. 13C NMR (DMSO-d6):
d ppm 43.4; 53.3; 68.0; 69.4;
70.5; 79.4; 143.7; 176.7. ESI-MS: m/z 957.1362 (70%, [M þ H]þ).
13C NMR (DMSO-d6):
d
ppm 27.5; 41.1; 41.6; 52.9; 54.4; 98.5; 117.3;
C42H48N10Fe3S3 calculated: C 52.73, H 5.06, N 14.64%; found: C
123.8 (6C); 127.3; 133.1; 148.9; 149.8; 151.6; 182.2. ESI-MS: m/z 980
(5%, [M þ H]þ); 513 (60%, [M þ 2Na]2þ). C45H54N13S3Cl3$3H2O
calculated: C 52.29, H 5.85, N 17.62%; found: C 52.00; H 6.38; N
16.60%.
52.63, H 5.54, N 13.98%.
4.3. Biological and biophysical assays
4.3.1. In vitro antiplasmodial assay
4.2.4. Compound 6
The test samples were tested in triplicate on one occasion
against chloroquine sensitive (CQS) NF54 strain and chloroquine-
resistant (CQR) Dd2 strain of P. falciparum. Continuous in vitro
cultures of asexual erythrocyte stages of P. falciparum were main-
tained using a modified method of Trager and Jensen [50]. Quan-
titative assessment of antiplasmodial activity in vitro was
determined via the parasite lactate dehydrogenase assay using a
modified method described by Makler et al. [51]. The test samples
were prepared as a 20 mg/ml stock solution in 100% DMSO and
sonicated to enhance solubility. Samples were tested as a suspen-
sion if not completely dissolved. Stock solutions were stored
at ꢁ20 ꢀC. Further dilutions were prepared on the day of the
experiment. Chloroquine (CQ) was used as the reference drug in all
experiments. A full doseeresponse measurement was performed
for all compounds to determine the concentration inhibiting 50% of
parasite growth (IC50 value). Test samples were tested at a starting
concentration of 1000 ng/ml, which was then serially diluted 2-fold
in complete medium to give 10 concentrations; with the lowest
concentration being 2 ng/ml. The same dilution technique was used
for all samples. The highest concentration of solvent to which
the parasites were exposed to had no measurable effect on the
parasite viability. The IC50 values were obtained using a non-linear
doseeresponse curve fitting analysis via GraphPad Prism v.4.0
software. Evaluation is based on means from three independent
experiments.
Compound
6 was prepared from compound 2 (0.888 g,
2.725 mmol) and G1 DAB dendrimer (0.164 g, 0.518 mmol). Yield:
0.092 g, 12%; IR (cmꢁ1): 3272 (NeH); 1611 (C]N); 1138 (C]S). 1H
NMR (DMSO-d6):
d ppm 1.32 (4H, m, CH2); 1.59 (8H, m, CH2); 1.89
(8H, m, CH2); 2.35 (12H, m, CH2); 3.21e3.45 (16H, m, CH2); 3.50
(8H, m, CH2); 6.44 (4H, br d, 3JHeH ¼ 5.4, AreH); 7.19 (4H, br s, NH);
7.41 (12H, m, NH, AreH); 7.77 (4H, d, 4JHeH ¼ 2.2, AreH); 8.25 (4H,
m, AreH); 8.38 (4H, d, JHeH ¼ 5.3, AreH); 13C NMR (DMSO-d6):
3
d
ppm 24.7; 26.8; 28.1; 41.4e42.9 (12C); 51.6; 53.8; 99.1; 117.9;
124.5 (8C); 127.9; 133.8; 149.5; 150.4; 152.3; 182.3; ESI MS: m/z
1429 (70%, [M þ H]þ); C68H88N18S4Cl4$5H2O calculated: C 53.81, H
6.51, N 16.61%; found: C 53.90; H 6.42; N 15.02%.
4.2.5. Compound 10
Compound 9 (0.197 g, 0.618 mmol) was suspended in EtOH
(20 ml). n-Propylamine (0.0430 g, 0.730 mmol) was added to the
suspension and the resulting solution was refluxed under Argon
for 12 h. The solvent was removed in vacuo and the residue dis-
solved in DCM (15 ml). The organic layer was washed with water
until the water layer remained clear. The organic extracts were
dried over NaSO4. The solvent was removed in vacuo, to the res-
idue diethyl ether and petroleum ether was added and the product
was allowed to precipitate. Yield: 0.062 g, 30%. IR (cmꢁ1): 3371
(NeH); 3129 (NeH); 1604 (C]N); 816 (C]S); 1090 (ferrocene). 1H
NMR (DMSO-d6):
d
ppm 0.88 (3H, t, 3JHeH ¼ 7.5, CH3); 1.59 (2H, m,
CH2); 3.49 (2H, m, CH2); 4.21 (5H, s, Cp); 4.42 (2H, m Cp); 4.72
4.3.2.
b
-Hematin inhibition assay
b-hematin formation assay was adapted from the method
(2H, m, Cp); 7.89 (1H, s, HC]N); 8.12 (1H, br s, NH); 11.11 (1H, s,
The
NH); 13C NMR (DMSO-d6):
d
ppm 11.7; 22.7; 45.5; 68.0; 69.4; 70.4;
described by Wright and co-workers [39]. Test compounds were
prepared as a 10 mM stock solution in 100% DMSO. Test samples
79.6; 143.4; 176.8. ESI-MS: m/z 330.0724 (100%, [M þ H]þ).
C
15H19N3FeS calculated: C 54.72, H 5.82, N 12.76%; found: C 53.99;
were tested at a starting concentration of 500
mM and the lowest
H 5.73; N 10.84%.
drug concentration being 5 M. The stock solution was serially
m
By following the same synthetic procedure as that for 10,
compound 12 was synthesized.
diluted to give 12 concentrations in a 96 well flat-bottom assay
plate. NP-40 detergent was then added to mediate the formation of
b
-hematin (30.55
of hematin was prepared by dissolving hemin (16.3 mg) in dimethyl
sulfoxide (DMSO) (1 ml). A 177.76 l aliquot of hematin stock was
suspended in 20 ml of a 2 M acetate buffer, pH 4.7. The hematin
mM, final concentration). A 25 mM stock solution
4.2.6. Compound 11
Compound 11 wasprepared fromethane-1,2-dithiosemicarbazide
(0.160 g, 0.767 mmol) which was suspended in H2O (25 ml). To this, a
m

T. Stringer et al. / European Journal of Medicinal Chemistry 69 (2013) 90e98
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Professor Rob Veale (University of the Witwatersrand, Johannes-
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Financial support from the University of Cape Town, the Na-
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African Medical Research Council (MRC) is greatly acknowledged.
Even though the work is supported by the MRC, the views and
opinions expressed are not those of the MRC but of the authors of
the material produced or published.
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