Synthesis, in vitro antiplasmodial and antiproliferative activities of a series of quinoline-ferrocene hybrids

Article abstract of DOI:10.1007/s00044-013-0748-4

Series of quinoline-ferrocene hybrids containing various linkers were synthesized and evaluated for antimalarial and anticancer activities as well as cytotoxicity. The hybrids with rigid linkers were found to be inactive, while those with flexible spacers

Full text of DOI:10.1007/s00044-013-0748-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1214–1224
DOI 10.1007/s00044-013-0748-4
ORIGINAL RESEARCH
Synthesis, in vitro antiplasmodial and antiproliferative activities
of a series of quinoline–ferrocene hybrids
David D. N’Da Peter J. Smith
•
Received: 4 June 2013 / Accepted: 17 August 2013 / Published online: 28 August 2013
Ó Springer Science+Business Media New York 2013
Abstract Series of quinoline–ferrocene hybrids contain-
ing various linkers were synthesized and evaluated for
antimalarial and anticancer activities as well as cytotoxicity.
The hybrids with rigid linkers were found to be inactive,
while those with flexible spacers showed activity against
both the D10 and Dd2 strains of Plasmodium falciparum, and
demonstrated a good selectivity towards these parasitic cells
in comparison with emetine. The hybrid 16, featuring
Keywords Malaria ꢀ Cancer ꢀ Quinoline ꢀ Ferrocene ꢀ
Hybrids ꢀ Etoposide
Introduction
Overall, in the past decade, the incidence of malaria has
slightly decreased, such that in 2011, there were approxi-
mately 216 million cases worldwide with 655,000 deaths
(WHO, 2012). More than 80 % of reported cases and 90 %
of all deaths are estimated to be in Africa, most of which
are pregnant women and children under the age of 5 years
(WHO, 2011). These figures exceed by far those due to
other parasitic infections causing mortality in humans. The
underlying main cause is the development of resistance of
Plasmodium falciparum parasites against the widely used
antimalarial drugs such as chloroquine and pyrimethamine/
sulfadoxine (Petersen et al., 2011).
3
-aminopropyl methylamine linker, was the most antima-
larial active compound, exhibiting a significantly better
potency than chloroquine against the Dd2 strain
(
IC50 = 0.008 vs. 0.148 lM; 19-fold), and was also found to
be significantly more active than the equimolar chloroquine–
ferrocene combination (IC50 = 3.7 vs. 41 ng/ml, tenfold)
against the Dd2 strain. Anticancer activity screening showed
that all the antimalarial active hybrids also exhibited potent
cytostatic (GI50 = 0.6–3.3 lM) and had good cytotoxic
effects (LC50 = 6–8 lM) against all three cancer cell lines.
The hybrid 11 possessing 1,4-butanediamine linker was
distinctively the most antiproliferative of all. It was found to
be more cytostatic (GI : 0.7 vs. 5.9 lM, eightfold) and
The artemisinin class of compounds is currently the
basis favoured by the World Health Organization (WHO)
for the treatment of uncomplicated P. falciparum. Unfor-
tunately, a cluster of alarming reports have been appearing
over the increasing times taken for artemisinins to reduce
parasitaemia; that is, the parasites are becoming increas-
ingly tolerant to artemisinins (Dondorp et al., 2009; Muller
et al., 2009; Noedl et al., 2010; Phyo et al., 2012). The
emergence of resistance against the artemisinins will be
catastrophic for malarial control. The global impact of this
disease and the extraordinary ability of Plasmodia parasites
to circumvent most efforts to counter them through che-
motherapy justify the search for new drugs.
5
0
(
LC : 6.4 vs. 92.6 lM, 14-fold) more cytotoxic than eto-
5
0
poside against the TK10 (renal) cell line.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0748-4) contains supplementary
material, which is available to authorized users.
D. D. N’Da (&)
Department of Pharmaceutical Chemistry, School of Pharmacy,
North-West University, Private Bag X 6001, Internal Box 304,
Potchefstroom 2520, South Africa
e-mail: david.nda@nwu.ac.za
Cancer, accounting for ten millions of death worldwide,
is a major disease and projected to continue rising (Beqa
et al., 2011). Efficient treatment of cancerous diseases is of
crucial importance. Metal-based antitumour agents, such as
P. J. Smith
Department of Pharmacology, Groote Schuur Hospital,
University of Cape Town, Observatory 7925, South Africa
1
23

Med Chem Res (2014) 23:1214–1224
1215
cisplatin and carboplatin, are commonly used drugs for
of a series of quinoline–ferrocene hybrids in an attempt to
highlight the influence of the linker between the two phar-
macophores with regard to these activities.
cancer treatment (Kelland et al., 1999; Barnes and Lippard,
2
004). However, many metal-based drugs would lead to
serious side effects after administration. Thus, a numerous
effort has been made to explore more efficient anticancer
agents.
Results and discussion
Since the pioneering work of K o¨ pf-Maier and co-
workers (K o¨ pf-Maier et al., 1984) reporting the anticancer
activity of ferrocenium complex in Ehrlich ascites tumour,
there has been an increasing interest in metallocenes and
their potential use in the biomedical field. Ferrocene in
particular has been extensively studied for potential bio-
logical and medicinal applications. Many functionalized
ferrocenes or ferrocene-containing compounds have been
prepared and tested in many research areas including
cancer (Hottin et al., 2012; Lal et al., 2012; Zhou et al.,
Chemistry
The hybrids 10–16 were synthesized in moderate to good
yields (55–70 %) by reductive amination involving amino-
functionalized quinolines and ferrocenic aldehyde. The
two-step process started with the condensation of diamines
with commercially available 4,7-dichloroquinoline 1 to
afford the primary and secondary amino-functionalized
quinolines 2–8. Their IR spectra of the intermediates
2
012; Acevedo-Morantes et al., 2012), Plasmodium
commonly
showed
absorption
bands
in
the
-
3360–3250 cm region due to the NH and NH groups.
1
malaria (Quirante et al., 2011; Dubar et al., 2011; Biot
et al., 2011; Roux and Biot, 2012), tuberculosis therapy
2
The presence of these functional groups was also confirmed
(
Mahajan et al., 2011; Kumar et al., 2012, 2013) and
in the structures 8 by the singlet attributable to signal of the
exchangeable NH and NH protons ca * 3.5 ppm in the
H spectra.
pneumocystis infections (Matos et al., 2010). From the
extensive research carried out with the aim of generating
metallocene-based antimalarials, ferroquine (FQ), a ferro-
cene–quinoline conjugate stands as the most promising
organometallic drug candidate with a real potential to reach
clinic. FQ is found to, like chloroquine (CQ), inhibit hae-
mozoin formation (Biot et al., 2011; Supan et al., 2012) but
is also remarkably active against both CQ-susceptible and
CQ-resistant P. falciparum (Biot et al., 2011; Supan et al.,
2
1
The IR spectra of hybrids 10–12, 15 and 16 exhibited
-
stretching vibrational bands in the 3100–3000 cm region
1
1
due to the NH group (s) in the linkers. The H NMR spectra
commonly display a broad singlet in the 4.39–3.9 ppm
region assignable to protons of the ferrocenic moiety and a
distinctive doublet in the 8.70–8.40 ppm region attribut-
able to the heterocyclic H-2 protons of the quinoline ring.
Comparing the intensities of the ferrocenic singlet and the
doublet gives a 9:1 ratio. This indicates that one ferrocene
moiety was linked to one quinoline ring, which is further
confirmed by the presence of the broad singlet in the
spectra of the hybrids as a result of the resonance methy-
2
012), against Plasmodium vivax strains (Leimanis et al.,
010), isolates (Barends et al., 2007; Daher et al., 2006)
2
and has displayed efficiency for the treatment of uncom-
plicated malaria in humans in phase IIb of clinical trials
(
http://clinicaltrials.gov/ct2/show/ NCT00988507). How-
ever, as for CQ and artemisinin, the mechanism of action of
FQ is partially unknown.
lene protons (CH –Fc) in the d 3.75–3.50 ppm region. The
HRMS-APCI data are in accordance with the structures of
2
In an attempt to elucidate the mechanism of action of
ferroquine, Chavain et al. (2008) reported the ferrocene
skeleton present in its structure to be able to generate
reactive oxygen species under oxidizing conditions such as
those estimated in the food vacuole of the parasite while
other reports pointed out the presence of the intramolecular
hydrogen bond to have a strong influence upon the anti-
malarial activity (Biot et al., 2009, 2012).
the compounds 10–16.
It is noteworthy indicating that the synthesis of metal-
locenes 10 and 16 has previously been reported elsewhere
(Biot et al., 2006).
In vitro antimalarial activity
The metallocenes 10–16 commonly possess both quinoline
and ferrocene skeletons and thus only differed structurally
by the spacers. They were screened against the CQ-sus-
ceptible (D10) and CQ-resistant (Dd2) strains of P. falci-
parum. Hybrids 10, 11, 15 and 16 containing flexible
hydrocarbon spacers and a 4-N proton were found to be
active against both P. falciparum strains (Table 1). On
contrary, compounds 13 and 14, which possess rigid/non-
flexible spacer and no 4-N proton, as well as ferrocene
aldehyde 9, were all inactive. Hybrid 12 also possesses a
Ferrocene nucleus has been directly linked to quinoline
pharmacophore through various spacers (Biot et al., 2004,
2
006;N’Daetal.,2010, 2011). Though, in general, noneof the
resulting compounds exhibited a global antimalarial either
similar or better than FQ, some among these compounds
displayed better activity than CQ especially against the CQ-
resistant strains of P. falciparum (Biot et al., 2004, 2006).
In this communication, we report the synthesis, in vitro
antimalarial and anticancer activities as well as cytotoxicity
123

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Med Chem Res (2014) 23:1214–1224
rigid spacer but has a 4-N proton and was also found active.
The equimolar combination M screened against the D10
and Dd2 strains also proved to be active. Overall, all the
active hybrids and the combination M showed potency
comparable to that of CQ against the CQS strains D10.
Against the CQR strains, however, these hybrids and
M were more potent than CQ. Compounds 10, 11, 15 and
hydrogen bonding between 4-N and the terminal N, which
is favoured by the flexibility of the spacer. Such is not the
case for the rigid or/and 4-N proton-depleted hybrids 12–
14. Thus, as for ferroquine, the antiplasmodial activity of
these quinoline–ferrocene hybrids seems to be greatly
conformation dependent.
Furthermore, all tested compounds (except 10, 11 and
M) showed good selectivity towards CHO plasmodia cell-
types. Hybrids 15 and 16 with SI values of 1 782 and 1 378,
respectively, appeared as the least cytotoxic of all the
active hybrids. Hybrid 16 possessed IC50 values of 43.2
and 8.2 nM against the D10 and Dd2 strains, which
translate into activities of 19.3 and 3.7 ng/ml, respectively,
on mass basis. Thus, it showed activity comparable to that
of the equimolar combination against the D10 strain (IC :
1
6 were more active against the CQR than the CQS strains
(
RI \ 1) while the combination showed a slight decrease in
activity against Dd2 in comparison with D10 (RI = 1.4).
Previous study identified common structural features for
active aminoquinolines against CQ-susceptible and CQ-
resistant P. falciparum strains. These include (1) an ami-
noquinoline (AQ) ring without alkyl substitutions, (2) a
halogen at position 7 of the AQ ring (Cl, Br or I but not F),
5
0
(
(
3) a protonatable nitrogen at position 1 of the AQ ring and
4) a second protonatable nitrogen at the other end of the
19.3 vs. 29 ng/ml) while possessing superior active than
M against the Dd2 strain (IC : 3.7 vs. 41 ng/ml).
5
0
side chain from the point of attachment to the AQ ring
through the 4-aminonitrogen. Additional considerations
such as latitude in the side chain (2–12 carbons) and ter-
minal amines (diethylamine, piperidine, etc.) are also
reported to influence antiparasitic activity of AQs (Hocart
et al., 2011). The current active hybrids, though addition-
ally contain a terminal ferrocenyl moiety, comply with
these reported structural features.
In vitro anticancer activity
The antiplasmodial active hybrids 10, 11, 15 and 16 were
selected for antiproliferative activity screening against
three different cell lines viz., renal (TK10), melanoma
(UACC62) and breast (MCF7). They all exhibited potent
growth inhibitory activity against the three cell-types as
seen from the GI50 values less than 10 lM (Table 2).
However, this was more pronounced against the TK10 and
Moreover, the structural characteristics of hybrids 10,
1, 15 and 16 confer the possibility of intramolecular
1
Table 1 In vitro antimalarial activity and cytotoxicity of compounds
d
Compound
D10
Dd2
CHO
a
n
b
b
c
a
b
e
SI
IC50 (lM)
SD
n
IC50 (lM)
SD
RI
n
IC50 (lM)
SD
1
1
1
1
1
1
1
0
1
2
3
4
5
6
3
3
3
3
3
3
3
3
3
3
0.09
0.13
0.96
0.01
0.01
0.04
2.89
1.07
0.00
0.00
3.27
0.00
0.00
3
3
3
3
3
3
3
3
3
3
0.01
0.01
0.24
0.00
0.00
0.01
0.12
0.25
0.00
0.00
2.82
0.01
0.00
0.1
0.1
0.3
0.2
0.4
0.3
0.2
0.7
2.7
1.4
3
3
3
3
3
3
3
3
2.25
0.51
25
1.30
0.19
10
33.75
58.43
85.10
81.95
59.26
378.92
nd
27.23
39.08
16.85
44.77
6.60
35
f
f
13.54
2.13
4
f
f
8.12
2.98
10
0.05
0.04
0.02
0.01
1 782
1 378
12
f
f
FcCHO
CQ
29.43
19.47
50.79
nd
0.05
0.15
nd
M
0.03 lg/ml
0.04 lg/ml
3
3
69.93 lg/
ml
14.97
2 412
Emetine
97.09
15.75
Cells were incubated with compounds at various concentrations for 48 h; antimalarial activity and cytotoxicity were determined using parasite
lactate dehydrogenase and MTT-assays, respectively
a
Number of replicates
b
Data represent the mean of three independent experiments, statistical significance p \ 0.05
Resistance index (RI) = IC50Dd2/IC50D10
c
d
Chinese hamster ovarian (CHO)
e
Selectivity index (SI) = IC50CHO/IC50D10
f
IC50 value above maximum tested concentration therefore compound is considered inactive; not determined (nd)
1
23

Med Chem Res (2014) 23:1214–1224
1217
MCF7 than against the UACC62 cells. Indeed, these
hybrids were found to be twofold to tenfold more growth
inhibitors of TK10 cell line than etoposide while compa-
rable activities, against the UACC62 cells. Against the
breast cancer cell line, hybrids 10 and 11 exhibited growth
inhibitory activity similar while 15 and 16 were found to be
up to threefold less active than etoposide. Hybrid 11 had
the best growth inhibitory activity of all TK10 and MCF7
cells.
strand scission (Mcfadyen et al., 1988) and the ferrocenyl
ring. In these hybrids, the spacers confer a region of flex-
ibility between the quinoline and ferrocene pharmaco-
phores, with the ionized secondary amines capable of
increasing van der Waals interactions with the highly polar
phosphate backbone and bases of DNA (Mcfadyen et al.,
1988; Blagbrough et al., 1997).
Better antiproliferative activity was observed against
TK10 and MCF7 than melanoma cells in comparison with
etoposide. Hybrid 11 had the smallest TGI values against
both tumour cell lines and thus appeared as the most active
of all screened hybrids. However, the toxicity towards the
CHO cells seems to militate against this fact. Indeed, this
good displayed antiproliferative activity rather than being
intrinsic, which is that, it solely emanates from its struc-
tural features, could well be the sum of both cytotoxicity
and intrinsic activity. The hybrids 15 and 16, on the other
hand, are nontoxic to CHO cells suggesting that their
anticancer activity may be intrinsic.
Comparison of the TGI concentrations revealed the
hybrids to possess potent activity against all cell lines while
etoposide showed potent, weak and inactivity against
UACC62, TK10 and MCF7 cell lines, respectively. Hybrid
1
1 again displayed the highest total growth inhibitory
power, being 15- and 50-fold more potent than etoposide
against the TK10 and MCF7 cell lines, respectively.
The LC50 concentrations of the hybrids were also
compared to that of etoposide to get a glimpse of the
cytotoxic effects of these compounds against the three cell
lines. The hybrids were found to possess equipotent cyto-
toxic effects against all cell lines (LC50 = 6–8 lM),
whereas etoposide showed inactivity against both UACC62
and MCF7 while being 15-fold less cytotoxic to the TK10
line. Yet again, the compound 11 distinguished itself with
lower LC50 values, an indication of slightly superior
though negligible cytotoxicity over the other compounds.
Thus, overall, hybrid 11 featuring 1,4-butanediamine linker
exhibited better cytostatic (lower TGI concentration) and
cytotoxic (lower LC50 concentration) effects as compared
to all other compounds.
Overall, compounds 15 and 16 exhibited the most potent
cytostatic and cytotoxic effects on the proliferation of all
three cancer cell lines. These potent activities reveal an
optimal distance of 3C atoms between two consecutive N
atoms in the spacer, possibly allowing optimal interaction
with the negatively charged phosphate groups of the DNA
backbone. This ultimately may induce the external binding
of these compounds to DNA and pull the AT-rich strains
apart and result in the disruption of DNA transcription
leading to tumour cell death.
Small quinoline pharmacophore lowers DNA binding
strength, which reduces nonspecific binding to other cell
macromolecules like proteins and lipids (Atwell et al.,
Methods and materials
1
988). Ferrocenyl moiety, on the other hand, inhibits cell
growth through ROS generation (Acevedo-Morantes et al.,
012). Thus, the potent anticancer activity of these hybrids
Materials
2
The reagents ethylenediamine and 1,3-diaminopropane,
and the anhydrous solvents dichloromethane (DCM) and
tetrahydrofuran (THF) and acetonitrile (ACN) were all
purchased from Fluka (Johannesburg, South Africa).
may be the result of synergism between the 4-aminoquin-
oline pharmacophore interacting favourably with the nar-
row minor groove of AT-rich sequences leading to DNA
Table 2 In vitro anticancer activity screening of selected hybrids against TK10 (renal), UACC62 (melanoma) and breast (MCF7) cancer cells
expressed as GI50, TGI and LC50 values (lM)
Compound TK10 (Renal)
UACC62 (Melanoma)
MCF7 (Breast)
GI50 Activity TGI Activity LC50 GI50 Activity TGI Activity LC50
GI50 Activity TGI
Activity LC50
1
1
1
1
0
1
5
6
1.5
0.7
2.6
1.7
p
p
p
p
p
4.4
2.8
p
p
p
p
w
7.3 2.6
6.4 2.4
7.7 3.3
7.4 2.5
92.6 3.4
p
p
p
p
p
5.1
4.9
5.6
5.1
4
p
p
p
p
p
7.6 0.8
p
p
p
p
p
3.5
1.7
5.3
5.3
p
p
p
p
i
7.1
7.5 0.6
7.9 2.7
7.6 2.7
6.2
8.2
8.0
5.2
4.6
Etoposide 5.9
43.3
[100
0.8
[100
[100
Inactive, i GI50 or TGI [ 100 lM; weak activity, w 30 \ GI50 or TGI \ 100 lM; moderate activity, m 10 \ GI50 or TGI \ 30 lM; potent
activity, p GI50 or TGI \ 10 lM
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Med Chem Res (2014) 23:1214–1224
Piperazine, para-phenylenediamine, 1,4-butanediamine,
For the synthesis of compounds 4–6, each diamine and
4,7-dichloroquinoline in 4:1 molar ratio were dissolved in
acetonitrile and refluxed for 4 h. Afterwards, the precipi-
tate that formed during the reaction was filtered off. The
filtrate was evaporated to dryness. The resulting residue
was redissolved in boiling ethyl acetate and was succes-
sively washed with 1 M NaOH (3 9 150 ml) and with
distilled water (4 9 150 ml). The organic layer was dried
1
-methylpiperazine,1-(2-aminoethyl)piperazine, N-methyl-
1
,3-diaminopropane, sodium borohydride and sodium tri-
acetoxyborohydride were purchased from Sigma-Aldrich
Johannesburg, South Africa). Ferrocene carboxyaldehyde
(
was obtained from Dayangchem (Hangzhou, China). 4,7-
Dichloroquinoline (CAS number: 86-98-6) was obtained
from Leapchem (Hangzhou, China). All the reagents and
chemicals were of analytical grade.
over MgSO , concentrated to half-volume and allowed to
4
recrystallize at 0–5 °C to afford the target compound.
General procedures
N-(3-aminopropyl)-7-chloroquinolin-4-amine 2
Thin-layer chromatography (TLC) was performed using
silica gel plates (60 F254), and purification of compounds
was done by preparative flash column chromatography on
silica gel (230–240 mesh, G60), all purchased from Merck
Yield: 65 %; off-yellow powder; mp: 125–129 °C; IR
-
ATR)/cm : 3269, 1921, 1728; H NMR (600 MHz,
1
1
(
DMSO-d ): d (ppm) 8.34 (d, J = 5.2 Hz, 1H, H-2), 8.10
6
(
Johannesburg, South Africa).
0
1
13
The H and C NMR spectra were recorded on a Bruker
(d, J = 8.6 Hz, 1H, H-5), 7.76 (s, 1H, H-5 ), 7.38 (d,
J = 2.8 Hz, 1H, H-8), 7.15 (dd, J = 2.8, 8.6 Hz, 1H, H-6),
Avance III 600 spectrometer, cheIC50al shifts are reported
in parts per million (ppm) from internal TMS and coupling
constants (J) are measured in hertz. The splitting pattern
abbreviations are as follows: s (singlet), bs (broad singlet),
d (doublet), dd (doublet of doublets), t (triplet), dt (double
of triplets) and m (multiplet).
6
.51 (d, J = 5.2 Hz, 1H, H-3), 3.38 (t, J = 7.5 Hz, 2H,
0
0
0
H-2 ), 3.15 (s, 2H, H-4 ), 2.82 (t, J = 6.5 Hz, 2H, H-4 ),
0
13
.2–1.85 (m, 2H, H-3 ); C NMR (151 MHz, DMSO-d6):
2
d (ppm) 152.68 (C-2), 149.67 (C-4), 136.29 (C-7), 127.61
(
(
C-8), 125.96 (C-5), 124.28 (C-6), 99.65 (C-3), 41.73
C-11), 40.28 (C-9), 32.03 (C-10).
High resolution mass spectral measurements were per-
formed on a Bruker microTOF-Q II 10390 instrument in
?
?
the APCI mode. Positive ions M and [M ? H] were
recorded. IR spectra were recorded on a Bruker alpha-
PFTIR Spectrometer. The melting points (mp) were
N-(4-aminobutyl)-7-chloroquinolin-4-amine 3
Yield: 68 %, off-white powder; mp: 115–120 °C; IR
¨
determined in triplicate on a BUCHI melting point B-545
-1 1
ATR)/cm : 3300, 1910, 1881, H NMR (600 MHz,
(
instrument and are reported in degree Celsius (°C)
uncorrected.
DMSO): d (ppm) 8.37 (d, J = 5.3 Hz, 1H, H-2), 8.26 (d,
J = 9.0 Hz, 1H, H-5), 7.76 (d, J = 1.5 Hz, 1H, H-8), 7.44
(
dd, J = 2.8, 9.7 Hz, 1H, H-6), 6.43 (d, J = 5.3 Hz, 1H,
Experimental
0
13
H-3), 3.23 (t, J = 25.72 Hz, 2H, H-2 ), 2.63–2.52 (m, 4H,
0
0
0
H-3 , -5 ), 1.77-157 (m, 2H, H-4 ). C NMR (600 MHz,
Synthetic procedure for amine-functionalized quinolines
DMSO): d (ppm) 151.92 (C-2), 150.09 (C-4), 133.30 (C-7),
1
4
27.47 (C-8), 124.13 (C-5), 123.93 (C-6), 98.59 (C-3),
0
The amine-functionalized quinolines 2, 3, 7 and 8 were
brought about following a general procedure (N’Da et al.,
0
0
0
2.40 (C-2 ), 41.45 (C-5 ), 30.90 (C-3 ), 25.34 (C-4 ).
2
011) and described as follows to exemplify the prepara-
tion of compound 2: A mixture of 4,7-dichloroquinoline 1
0.03 mol) and 1,3-propanediamine (0.13 mol, 5.2 eq.)
1-N-(7-chloroquinolinyl-yl) benzene-1,4-diamine 4
(
were heated with stirring at 120 °C for 2 h. The mixture
was allowed to cool to 50 °C, 1 M aqueous solution of
NaOH (50 ml) was added, and then, stirring was continued
until the mixture cooled to room temperature. The product
was extracted with dichloromethane (3 9 150 ml). The
combined organic fractions were washed with distilled
Yield: 55 %; dark-brown powder; mp: 183–185 °C; IR
(ATR)/cm : 3377, 1927, 1883; H NMR (600 MHz,
-
1
1
DMSO-d ): d (ppm) 8.79 (d, J = 5.9 Hz, 1H, H-2), 8.39
6
(d, J = 8.2 Hz, 1H, H-5), 8.31 (d, J = 1.5 Hz, 1H, H-8),
7.81 (dd, J = 1.5, 8.2 Hz, 1H, H-6), 7.48 (d, J = 8.8 Hz,
0
2H, H-4 ), 6.98 (d, J = 5.9 Hz, 2 H, H-3), 6.64 (d,
0 0
water (4 9 150 ml) and dried over MgSO , and the solvent
4
J = 8.8 Hz, 2H, H-3 ), 5.13 (s, 1H, H-6 ), 3.37 (s, 2H,
0
13
H-1 ); C NMR (151 MHz, DMSO-d
was evaporated under reduced pressure. The resulting
residue was redissolved in boiling ethyl acetate and
allowed to recrystallize at 0–5 °C to afford the target
compound 2.
6
): d (ppm) 151.80
0
0
(H-2 ), 150.05 (C-2), 149.46.10 (C-4), 133.60 (C-5 ),
127.53 (C-7), 126.37 (C-8), 124.33 (C-5), 124.22 (C-6),
0
0
117.58 (C-3 ), 114.54 (C-4 ), 100.24 (C-3).
1
23

Med Chem Res (2014) 23:1214–1224
1219
0
0
13
2H, H-2 ), 1.71 (dt, J = 4.0, 5.3 Hz, 2H, H-3 ); C NMR
7
-Chloro-4-(piperazin-1-yl) quinoline 5
(
151 MHz, DMSO-d ): d (ppm) 151.92 (C-2), 150.13 (C-4),
6
Yield: 66 %; off-white powder; mp: 118–120 °C; IR
133.31 (C-7), 127.50 (C-8), 124.00 (C-5), 123.85 (C-6),
0
-
ATR)/cm : 3253, 2939, 2823; H NMR (600 MHz,
1
1
0
0
(
98.73 (C-3), 72.28 (C-4 ), 51.00 (C-2 ), 36.15 (C-6 ), 22.29
0
DMSO-d ): d (ppm)8.67 (d, J = 5.2 Hz, 1H, H-2), 7.79 (d,
(C-3 ).
6
J = 9.0 Hz, 1H, H-5), 7.52 (d, J = 2.0 Hz, 1H, H-8), 7.45
(
dd, J = 2.0, 9.0 Hz, 1H, H-6), 6.94 (d, J = 5.2 Hz, 1H,
0
Synthesis of quinoline–ferrocene hybrids
0
H-3), 3.5 (s, 1H, H-1 ), 3.06 (t, J = 8.3 Hz, 4H, H-2 ), 2.94
0
13
4H, t, J = 8.3 Hz, H-3 ); C NMR (151 MHz, DMSO-
(
The quinoline–ferrocene hybrids 10–12 were synthesized
using a method reported (Biot et al., 2006) with slight
modifications (Scheme 1) and described as follows: a
mixture of primary aminoquinoline (2–3) (1 mmol) and
ferrocenecarboxyaldehyde 9 (3 mmol, 3 equiv.) were
thoroughly dissolved in 30 ml of anhydrous methanol and
stirred for 4 h at room temperature. An excess of sodium
borohydride (10 mmol, 20 eq.) was added portion wise,
and the reaction mixture was stirred for an additional 2 h.
After addition of 0.6 M hydrochloric acid (100 ml) and
distilled water (100 ml), the aqueous layer was washed
with diethyl ether (3 9 100 ml) then basified with satu-
d6): d (ppm) 156.88 (C-2), 152.18 (C-4), 133.46 (C-7),
1
5
28.03 (C-8), 126.10 (C-5), 125.58 (C-6), 109.22 (C-3),
0
0
4.21 (C-2 ), 45.51 (C-3 ).
7
-Chloro-4-(3-methylpiperazin-1-yl) quinoline 6
Yield: 62 %; off-white powder; mp: 125–130 °C; IR
-
ATR)/cm : 3255, 2960, 2818; H NMR (600 MHz,
1
1
(
CDCl ): d (ppm) 8.66 (d, J = 5.0 Hz, 1H, H-2), 7.98 (d,
3
J = 9.0 Hz, 1H, H-5), 7.89 (d, J = 2.0 Hz, 1H, H-8), 7.45
(
dd, J = 2.0, 9.0 Hz, 1H, H-6), 6.77 (d, J = 5.0 Hz, 1H,
0
0
H-3), 3.75 (s, 1H, H-1 ), 3.52 (t, J = 7.5 Hz, 2H, H-2 ),
rated NaHCO (pH 7) and extracted with dichloromethane
3
0
3
1
1
.27 (t, J = 7.5 Hz, 2H, H-3 ), 2.49 (dt, J = 2.8, 11.5 Hz,
0
(3 9 150 ml). The combined organic layers were dried
0
13
H, H-4 a), 2.49 (dt, J = 10.5, 11.5 Hz, 1H, H-4 b),
over anhydrous MgSO . Afterwards, the solvent was
4
0
0
.92–1.61 (m, 1H, H-5 ), 1.1 (d, J = 6.4 Hz, 3H, H-6 );
C
evaporated in vacuo, and the residue was purified by flash
chromatography on silica gel eluting with DCM–MeOH
(4:1) to afford the desired compounds 10–12.
NMR (151 MHz, CDCl ): d (ppm) 157.05 (C-2), 151.89
3
(
C-4), 134.74 (C-7), 128.79 (C-8), 126.01 (C-5), 125.16
0
0
0
(
C-6), 108.97 (C-3), 77.22 (C-4 ), 77.00 (C-2 & C-3 ),
0
For the synthesis of hybrids 13–16, amine 5–8
(1 mmol), ferrocenecarboxyaldehyde 9 (3 mmol, 3 equiv.)
and NaHB(OAc) (5 mmol, 5 eq.) were dissolved in 30 ml
0
5
8.00 (C-5 ), 19.61 (C-6 ).
3
7
-Chloro-N-[2-(piperazin-1-yl)ethyl] quinolin-4-amine 7
of anhydrous tetrahydrofuran (THF), and the solution was
stirred at room temperature for 24 h. After evaporation of
the solvent in vacuo, the residue was dissolved in 50 ml of
anhydrous dichloromethane and 15 ml of 1 M NaOH
solution was added and left for 20 min. The organic layer
was washed with distilled water (3 9 20 ml) and was dried
Yield: 60 %; off-yellow powder; mp: 128–132 °C; IR
-
1
1
ATR)/cm : 3210, 2939, 2424; H NMR (600 MHz,
(
DMSO-d ): d (ppm) 8.70 (d, J = 5.2 Hz, 1H, H-2), 8.45
6
(
d, J = 9.0 Hz, 1H, H-5), 7.77 (d, J = 2.1 Hz, 1H, H-8),
7
1
2
.38 (dd, J = 2.1, 9.0 Hz, 1H, H-6), 6.74 (d, J = 5.2 Hz,
0
over MgSO . The solvent was evaporated, and the residue
4
0
H, H-3), 3.82 (s, 1H, H-6 ), 3.42 (t, J = 6.5 Hz, H-4 ),
0
was purified by flash chromatography on silica gel eluting
with DCM–MeOH (4:1) to afford the desired hybrids.
.64 (t, J = 4.1 Hz, 2H, H-3 ), 2.57 (t, J = 4.1 Hz, 2H,
13
0
0
H-2 ), 2.57 (t, J = 6.5 Hz, 2H, H-5 ); C NMR (151 MHz,
DMSO-d ): d (ppm) 151.96 (C-2), 150.00 (C-4), 133.41
{3-[(7-Chloroquinolin-4-yl)amino]propyl}
6
(
C-7), 127.53 (C-8), 127.53 (C-5), 124.14 (C-6), 98.73 (C-
0 0 0 0
(ferrocenylmethyl) amine 10
3
), 53.21 (C-2 ), 46.37 (C-3 ), 45.51 (C-4 ), 40.88 (C-5 ).
Yield: 62 %, yellow powder; mp: 100–104 °C; IR (ATR)/
cm : 3309, 3079, 2928, 1733,1578; H NMR (600 MHz,
-
1
1
{3-[(7-Chloroquinolin-4-yl)amino] propyl} (methyl)
amine 8
CDCl ): d (ppm) 8.43 (d, J = 5.0 Hz, 1H, H-2), 7.93 (d,
J = 8.8 Hz, 1H, H-5), 7.70 (s, 1H, H-5 ), 7.59 (d,
3
0
Yield: 60 %; off-white powder; mp: 115–119 °C; IR
J = 2.1 Hz, 1H, H-8), 7.18 (dd, J = 2.1, 8.8 Hz, 1H, H-6),
-
1
:
1
(
ATR)/cm
3220, 3103, 3060, 2947;
H
NMR
6.23 (d, J = 5.0 Hz, 1H, H-3), 4.35–3.95 (bs, 9H, H-Cp, -
00
0
(
600 MHz, DMSO-d ): d (ppm) 8.38 (d, J = 5.06 Hz, 1H,
Cp’), 3.80 (s, 1H, H-1 ), 3.59 (s, 2H, H-1 ), 3.21 (t,
6
0
0
H-2), 8.25 (d, J = 9.2 Hz, 1H, H-5), 7.73 (d, J = 3.4 Hz,
J = 7.0 Hz, 2H, H-2 ), 3.06 (t, J = 7.0 Hz, 2H, H-4 ), 2.02
13
0
1
H, H-8), 7.39 (dd, J = 3.4, 9.2 Hz, 1H, H-6), 6.44 (d,
0
(m, 2H, H-3 ); C NMR (151 MHz, CDCl ): d (ppm)
3
J = 5.06 Hz, 1H, H-3), 3.8 (s, 1H, H-1 ), 3.42 (t,
0
151.86 (C-2), 150.62 (C-4), 134.61 (C-7), 128.15 (C-8),
0
0
J = 5.3 Hz, H-4 ), 3.26 (s, 3H, H-6 ), 2.57 (t, J = 4.0 Hz,
124.92 (C-5), 122.57 (C-6), 98.06 (C-3), 48.27 (C-4 ), 46.32
123

1
220
Med Chem Res (2014) 23:1214–1224
1''
3''
2''
L
4''
Cl
N
LH
Cp
5
8
5
4
4
6''
5''
Fe
(a)
6
3
2
(b)
6
3
7
Cp'
7
2
Cl
N
1
Cl
N
1
Cl
8
1
2
- 8
10 - 16
4
-Aminoquinoline
L
2'
Hybrids
10
L
4
'
4'
5'
2'
1'
5
'
1'
1'
2
NH
NH
NH
NH
3
'
3'
6
'
4'
2' 1'
6
'
4'
2'
NH
3
11
NH
NH
5
'
3'
NH
5
'
3'
4'
4
'
3'
3'
6
NH
'
2' 1'
6'
NH
2' 1'
5
'
5'
4
5
6
12
13
14
NH
NH
3
'
2'
2'
3' 2'
1
N
'
1'
N
N
N
N
3
'
3' 2'
1
'
1'
N
N
N
4
'
5'
4' 5'
6
'
6'
3
'
2'
3' 2'
6
HN
'
6'
HN
4
'
4'
5'
1
'
1'
5
'
7
8
15
16
N
N
N
N
6
'
6'
5
'
3'
5'
3'
HN
N
HN
N
1
'
1'
4
'
2'
4'
2'
Scheme 1 A general reaction illustrating the synthesis of amino-
quinolines 2–8 and of quinoline–ferrocene hybrids 10–16. Reagents
and conditions: (a) 4,7-dichloroquinoline (1), diamine (liquid), neat,
120 °C, 2 h or (1), diamine (powder), acetonitrile, reflux, 4 h;
(b) aminoquinoline (2–4), ferrocenecarboxyaldehyde 9, MeOH, 4 h,
rt then NaBH , 2 h, rt or (5–8), 9, THF, 24 h, rt
4
0
0
0
0
00
0
(
C-1 ), 41.57 (C-2 ), 27.00 (C-3 ); HRMS-APCI m/z:
2H, H-1 ), 3.25 (t, J = 6.15 Hz, 2H, H-5 ), 2.75 (t,
?
34.1066 [M ? H] ([FeC H ClN , [M ? H] 434.1535
?
0
0
0
0
4
J = 7.5 Hz, 2H, H-2 ), 1.9–1.65 (m, 2H, H-1 , -3 and 4 );
1
C NMR (151 MHz, CDCl ): d (ppm) 152.04 (C-2),
2
3
25
3
3
calcd).
3
1
50.00 (C-4), 134.67 (C-7), 124.99 (C-8), 121.52 (C-5),
0 00
{4-[(7-Chloroquinolin-4-yl)amino]butyl}
117.31 (C-6), 98.80 (C-3), 49.04 (C-5 ), 48.47 (C-1 ),
0 0 0
43.16 (C-2 ); 27.69 (C-4 ), 26.18 (C-3 ); HRMS-APCI m/z:
(
ferrocenylmethyl) amine 11
?
47.1166 M [FeC H ClN , M 447.1500 calcd].
?
4
24 26 3
Yield: 58 %, yellow powder; mp: 90–95 °C; IR (ATR)/
cm : 3213, 3066, 2925, 1609, 1578; H NMR (600 MHz,
-
1
1
1-N-(7-Chloroquinolin-4-yl)-4-N-(ferrocenylmethyl)
CDCl ): d (ppm) 8.50 (d, J = 5.5 Hz, 1H, H-2), 7.90 (d,
benzene-1,4-diamine 12
3
J = 8.4 Hz, 1H, H-5), 7.70 (d, J = 2.5 Hz, 1H, H-8), 7.32
(
dd, J = 2.5, 8.4 Hz, 1H, H-6), 6.28 (d, J = 5.5 Hz, H-3),
0
Yield: 69 %, brown powder; mp: 104–107 °C; IR (ATR)/
cm : 3374, 3194, 2954, 1885, 1572; H NMR (600 MHz,
-1
1
6
.10 (s, 1H, H-6 ), 4.35–4.1 (bs, 9H, H-Cp, -Cp’), 3.55 (s,
1
23

Med Chem Res (2014) 23:1214–1224
1221
DMSO-d ): d (ppm)8.40 (d, J = 4.8 Hz, 1H, H-2), 7.95 (d,
J = 9.0 Hz, 1H, H-5), 7.82 (d, J = 1.8 Hz, 1H, H-8), 7.68
J = 9.0 Hz, 1H, H-5), 7.64 (d, J = 2.1 Hz, 1H, H-8), 7.36
0
6
(dd, J = 2.1, 9.0 Hz, 1H, H-6), 7.12 (s, 1H, H-6 ), 6.30 (d,
(
dd, J = 1.8, 9.0 Hz, 1H, H-6), 7.51 (d, J = 8.5 Hz, 1H,
0
J = 5.2 Hz, 1H, H-3), 4.24–4.03 (bs, 9H, H-Cp, -Cp’),
00 0
0
0
H-3 ), 7.39 (d, J = 8.5 Hz, 2H, H-4 ), 7.15 (s, 1H, H-6 ),
0
3.37 (s, 2H, H-1 ), 3.27 (t, J = 7.2 Hz, 2H, H-5 ), 2.73 (t,
0 0
J = 7.2 Hz, 2H, H-4 ), 2.55 (t, J = 5.7 Hz, 4H, H-2 ), 2.45
6
.75 (d, J = 4.8 Hz, 1H, H-3), 5.2 (s, 1H, H-1 ), 4.5–4.20
00
13
bs, 9H, H-Cp, -Cp’), 3.73 (2H, s, H-1 ); C NMR
0
13
(t, J = 5.7 Hz, 4H, H-3 ); C NMR (151 MHz, CDCl ): d
(
(
3
0
151 MHz, CDCl ): d (ppm) 151.80 (C-1 ), 150.05 (C-2),
(ppm) 151.76 (C-2), 149.92 (C-4), 134.97 (C-7), 128.46
3
0
1
1
1
44.77 (C-4), 132.42 (C-4 ), 128.79 (C-7), 126.54 (C-8),
0
(C-8), 125.41 (C-5), 121.21 (C-6), 99.22 (C-3), 77.22 (C-
00
0
0
0
0
0
26.54 (C-5), 125.67 (C-6), 117.35 (C-4 ), 116.03 (C-3 ),
1 ), 76.79 (C-2 ), 70.32 (C-3 ), 45.51 (C-4 ), 40.88 (C-5 );
00
?
HRMS-APCI m/z: 489.1498 [M ? H] [FeC H ClN ,
?
[M ? H] 489.1318 calcd].
01.26 (C-3), 46.32 (C-1 ); HRMS-APCI m/z: 468.0907
2
6
30
4
?
M ? H] [FeC H ClN , [M ? H] 468.1078 calcd].
?
[
2
6
23
3
7
-Chloro-4-[4-(ferrocenylmethyl)piperazin-1-yl]
{3-[(7-Chloroquinolin-4-yl)amino]propyl}(ferrocenylm
quinoline 13
ethyl)methylamine 16
Yield: 55 %, brown powder; mp: 135–138 °C; IR (ATR)/
cm : 3215, 3061, 2950, 1709, 1575; H NMR (600 MHz,
Yield: 70 %, brown powder; mp: 112–115 °C; IR (ATR)/
-1 1
cm : 3338, 3087, 2986, 1658, 1538; H NMR (600 MHz,
-
1
1
CDCl ): d (ppm) 8.66 (d, J = 4.9 Hz, 1H, H-2), 8.0 (d,
CDCl ): d (ppm) 8.43 (d, J = 5.1 Hz, 1H, H-2), 8.11 (d,
3
3
J = 8.9 Hz, 1H, H-5), 7.87 (d, J = 1.5 Hz, 1H, H-8), 7.37
J = 9.2 Hz, 1H, H-5), 7.89 (d, J = 1.9 Hz, 1H, H-8), 7.36
0
(
dd, J = 1.5, 8.9 Hz, 1H, H-6), 6.77 (d, J = 4.9 Hz, 1H,
0
(dd, J = 1.9, 9.2 Hz, 1H, H-6), 7.05 (s, 1H, H-5 ), 6.24 (d,
0
H-3), 4.34–3.92 (bs, 9H, H-Cp, -Cp’), 3.52 (s, 2H, H-1 ),
0
J = 5.1 Hz, 1H, H-3), 4.39–3.9 (bs, 9H, H-Cp, -Cp’), 3.45
00
0
0
3
.19 (t, J = 8.0 Hz, 4H, H-2 ), 2.69 (t, J = 8.0 Hz, 4H,
13
(s, 2H, H-1 ), 3.39 (t, J = 12.5 Hz, H-4 ), 3.25 (s, 3H, H-6 ),
0
0
H-3 ); C NMR (151 MHz, CDCl ): d (ppm) 156.88 (C-2),
2.65 (t, J = 9.0 Hz, 2H, H-2 ), 1.88 (dt, J = 9.0, 12.5 Hz,
13
2H, H-3 ); C NMR (151 MHz, CDCl ): d (ppm) 151.54
3
0
1
51.89 (C-4), 134.82 (C-7), 128.82 (C-8), 126.04 (C-5),
0
3
00
1
25.17 (C-6), 108.95 (C-3), 58.00 (C-2 ), 53.27 (C-1 ) and
?
ppm 51.88 (C-3 ); HRMS-APCI m/z: 446.1080 [M ? H]
(C-2), 149.52 (C-4), 134.76 (C-7), 127.89 (C-8), 125.06 (C-
00 0
0
5), 122.51 (C-6), 98.05 (C-3), 77. 22 (C-1 ), 77.00 (C-4 ),
0 0 0
76.79 (C-2 ), 70.37 (C-5 ) and ppm 24.00 (C-3 ); HRMS-
?
FeC H ClN , [M ? H] 446.1642 calcd].
[
2
4
25
3
?
APCI m/z: 448.1233 [M ? H] [FeC H ClN , [M ? H]
?
2
4
26
3
7
-Chloro-4-[4-(ferrocenylmethyl)-3-methylpiperazin-1-yl]
448.1879 calcd].
quinoline 14
In vitro biological evaluation
Antimalarial activity
Yield: 58 %, brown powder; mp: 142–145 °C; IR (ATR)/
cm : 3071, 2971, 2876, 1724, 1563; H NMR (600 MHz,
-
1
1
CDCl ): d (ppm) 8.65 (d, J = 4.7 Hz, 1H, H-2), 7.99 (d,
3
J = 8.9 Hz, 1H, H-5), 7.84 (d, J = 1.9 Hz, 1H, H-8), 7.35
The hybrids 10–16 were screened in triplicate alongside
ferrocenecarboxyaldehyde 9 and the equimolar mixture of
chloroquine and 9, on one occasion against chloroquine-
sensitive (CQS) D10 and chloroquine-resistant (CQR) Dd2
strains 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
(1976). The quantitative assessment of in vitro antimalarial
activity was determined via the parasite lactate dehydro-
genase assay using a modified method (Makler et al., 1993)
reported elsewhere (van Heerden et al., 2012). The test
samples were prepared from a 20 mg/ml stock solution in
(
dd, J = 1.9, 8.9 Hz, 1H, H-6), 6.75 (d, J = 4.7 Hz, 1H,
0
0
H-3), 4.34–3.94 (bs, 9H, H-Cp, -Cp’), 3.75 (2H, s, H-1 ),
0
3
.61 (t, J = 6.3 Hz, 2H, H-3 ), 3.27 (t, J = 6.3 Hz, 2H,
0
0
H-4 ), 3.08 (dt, J = 2.5, 11.5 Hz, 1H, H-5 a), 2.97 (dt,
0
J = 2.5, 10.5 Hz, 1H, H-5 b), 2.64 (d, J = 7.5 Hz, 3H,
13
0
H-5 ); C NMR (151 MHz, CDCl ): d (ppm) 156.67 (C-
3
2
), 151.86 (C-4), 134.81 (C-7), 128.76 (C-8), 126.03 (C-5),
0
0
1
25.16 (C-6), 108.89 (C-3), 77.22 (C-3 ), 77.00 (C-4 and
0
0
00
0
C-5 ), 58.97 (C-1 ), 50.55 (C-2 ), 19.61 (C-6 ); HRMS-
?
APCI m/z: 460.1241 [M ? H]
?
M ? H] 460.1907 calcd].
[FeC H ClN ,
25 27 3
[
10 % dimethylsulphoxide (DMSO) and sonicated to
7
-Chloro-N-{2-[4-(ferrocenylmethyl)piperazin-1-yl]ethyl}
enhance solubility. Stock solutions were stored at -20 °C.
Further dilutions were done on the day of the experiment.
Chloroquine (CQ) was used as the reference drug in all
experiments. A full dose–response was performed for all
compounds to determine the concentration inhibiting 50 %
quinolin-4-amine 15
Yield: 68 %, brown powder; mp: 139–144 °C; IR (ATR)/
cm : 3211, 3078, 2939, 1737, 1572; H NMR (600 MHz,
-
1
1
CDCl ): d (ppm) 8.49 (d, J = 5.2 Hz, 1H, H-2), 7.91 (d,
of parasite growth (IC -value). The samples were tested at
50
3
123

1
222
Med Chem Res (2014) 23:1214–1224
a starting concentration of 100 ng/ml, which was then
serially diluted twofold in complete medium to give 10
concentrations, with the lowest being 0.2 ng/ml. The same
dilution technique was used for all samples. The IC -
Conclusion
A series of seven quinoline–ferrocene hybrids were syn-
thesized, and their structures confirmed by NMR and MS
analyses. The hybrids 10, 11, 15 and 16 featuring flexible
linkers and 4-N proton were active against both strains of
P. falciparum, demonstrated good selectivity towards the
parasitic cells and displayed potent anticancer activity
against all three cell lines. The remaining three hybris with
rigid or/and 4-N proton-depleted structures were found to
be antimalarial inactive. This study confirms the antima-
larial activity of this hybrid-type to be conformation
dependent. Compound 16, the most active hybrid, exhib-
ited activity comparable to that of CQ against the CQ-
susceptible strain D10 while being significantly more
potent than CQ and the equimolar combination against the
resistant strain Dd2 of P. falciparum. Hybrid 11, on the
other hand, was the most antiproliferative compound of all
against the three human tumours cell lines.
5
0
values were obtained using a nonlinear dose–response
curve fitting analysis via GraphPad Prism v.4.0 software.
Cytotoxicity
The MTT-assay, a colorimetric assay, was used for cellular
growth and survival and compares well with other avail-
able assays (Mosmann, 1983; Rubinstein et al., 1990).
Mammalian cell line viz. CHO (Chinese Hamster Ovarian)
was treated with the compounds, and the assay was used to
measure all growth and chemosensitivity. The test samples
were tested in triplicate on one occasion. The same stock
solutions were used for both antimalarial activity and
cytotoxicity tests, and dilutions were made on the day of
the experiment. Emetine (EM) was used as the reference
drug. The initial concentration of emetine was 100 lg/ml,
which was serially diluted in complete medium with ten-
fold dilutions to give 6 concentrations, the lowest being
On account of both the activity and selectivity, hybrids
11 and 16 stand as a good drug candidates to be further
investigated in the search for safe and new therapies
against malaria and renal cancer diseases, respectively.
0
.001 lg/ml. The same dilution technique was applied to
the all test samples. The highest concentration of solvent to
which the cells were exposed to had no measurable effect
on the cell viability. The 50 % inhibitory concentration
Acknowledgments This work is based upon research supported by
the National Research Foundation (NRF). The author N’Da DD
thanks the North-West University for the financial support, Mrs.
Kolesnikova N. from the CSIR for in vitro cancer activity screening,
the short-term exchange students Meijler LE (University of Applied
Science, Breda, Netherlands) and van Ryn SHC (Hogeschool Zuyd,
Netherlands) for their involvement in the synthesis of the hybrids.
values (IC -value) were obtained from full dose–response
5
0
curves, using a nonlinear dose–response curve fitting
analysis via GraphPad Prism v.4 software. All the anti-
malarial activity and cytotoxicity results are summarized in
Table 1.
Conflict of interest The authors declare that they have no conflicts
of interest.
Antiproliferative activity
Disclaimer Any opinion, findings and conclusions or recommen-
dations expressed in this material are those of the author(s), and
therefore the NRF do not accept any liability in regard thereto.
The growth inhibitory effects of the 10, 11, 15 and 16 were
assessed in a panel of three cell lines recommended by the
National Cancer Institute (NCI) for preliminary screens,
using a sulforhodamine B (SRB) assay (Skehan et al., 1990).
Screening was performed as previously reported (van
Heerden et al., 2012). Etoposide, an anticancer agent, known
to be an inhibitor of topoisomerase II and aids in DNA
unwinding, which causes DNA strands to break, was used as
a positive control (Wu et al., 2011). Three parameters were
determined during the screening process. These included
References
Acevedo-Morantes CY, Mel e´ ndez E, Singh SP, Ram ´ı rez-Vick JE
(
2012) Cytotoxicity and Reactive Oxygen Species Generated by
Ferrocenium and Ferrocene on MCF7 and MCF10A Cell Lines.
J Cancer Sci Ther 4:271–275
Atwell GJ, Bos CD, Baguley BC, Denny WA (1988) Potential
antitumor agents. 56. ‘‘Minimal’’ DNA-intercalating ligands as
antitumor drugs: phenylquinoline-8-carboxamides. J Med Chem
5
0 % cell growth inhibition (GI ), total cell growth inhibi-
50
tion (TGI) and the lethal concentration that kills 50 % of
cells (LC ). The TGI signifies a cytostatic effect, while LC
50
3
1:1048–1052
Barends M, Jaidee A, Khaohirun N, Singhasivanon P, Nosten F
2007) In vitro activity of ferroquine (SSR 97193) against
5
0
indicates the cytotoxic effect of the tested agent. The bio-
logical activities were separated into 4 categories: inactive,
i GI or TGI [ 100 lM), weak activity, w 30 lM \ GI or
(
Plasmodium falciparum isolates from the Thai-Burmese border.
Malar J 6:81
5
0
50
Barnes KR, Lippard SJ (2004) Cisplatin and related anticancer drugs:
recent advances and insights. Met Ions Biol Syst 42:143–177
Beqa L, Fan Z, Singh AK, Senapati D, Ray PC (2011) Gold nano
popcorn attached SWCNT hybrid nanomaterial for targeted
TGI \ 100 lM, moderate activity, m 10 lM \ GI50 or
TGI \ 30 lM and potent activity, p TGI \ 10 lM. The
results are reported in Table 2.
1
23

Med Chem Res (2014) 23:1214–1224
1223
diagnosis and photothermal therapy of human breast cancer
cells. ACS Appl Mater Interfaces 3:3316–3324
tethered b-lactam-ferrocene and b-lactam-ferrocenylchalcone
chimeric scaffolds. Dalton Trans 41:5778–5781
Biot C, Dessolin J, Ricard I, Dive D (2004) Easily synthesized
antimalarial ferrocene triazacyclononane quinoline conjugates.
J Organomet Chem 689:4678–4682
Biot C, Daher W, N’Diaye CM, Melnyk P, Pradines B, Chauvain N,
Pellet A, Fraisse L, Pelinski L, Jarry C, Brocard J, Khalife J,
Forfar-Bares I, Dive D (2006) Probing the role of the covalent
linkage of ferrocene into a chloroquine template. J Med Chem
Kumar K, Carr e` re-Kremer S, Kremer L, Gu e´ rardel Y, Biot C, Kumar
V (2013) Azide-alkyne cycloaddition en route towards 1H-1,2,3-
triazole-tethered b-lactam-ferrocene and b-lactam-ferrocenyl-
chalcone conjugates: synthesis and in vitro anti-tubercular
evaluation. Dalton Trans 42:1492–1500
Lal B, Badshah A, Altaf AA, Tahir MN, Ullah S, Huq F (2012)
Synthesis, characterization and antitumor activity of new
ferrocene incorporated N, N’-disubstituted thioureas. Dalton
Trans 41:14643–14650
Leimanis ML, Jaidee A, Sriprawat K, Kaewpongsri S, Suwanarusk R,
Barends M, Phyo AP, Russell B, Renia L, Nosten F (2010)
Plasmodium vivax susceptibility to ferroquine. Antimicrob
Agents Chemother 54:2228–2230
Mahajan A, Kremer L, Louw S, Gu e´ rardel Y, Chibale K, Biot C
(2011) Synthesis and in vitro antitubercular activity of ferrocene-
based hydrazones. Bioorg Med Chem Lett 21:2866–2868
Makler MT, Ries JM, Williams JA, Bancroft JE, Peper RC, Gibbins
BL, Hinrichs DJ (1993) Parasite lactate dehydrogenase as an
assay for Plasmodium falciparum drug sensitivity. Am Soc Trop
Med Hyg 48:739–741
Matos J, Vale N, Collins MS, Gut J, Rosenthal PJ, Cushion MT,
Moreira R, Paula Gomes (2010) PRIMACENES: novel non-
cytotoxic primaquine-ferrocene conjugates with anti-Pneumo-
cystis carinii activity. MedChemComm 1:199–201
Mcfadyen WD, Denny WA, Wakelin LPG (1988) Alkyl-linked
diquinolines are monofunctional AT-selective DNA-intercalat-
ing agents. FEBS Lett 228:235–240
Mosmann TJ (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
Immunol 65:55–63
Muller O, Sie A, Meissner P, Schirmer RH, Kouyate B (2009)
Artemisinin resistance on the Thai-Cambodian border. Lancet
374:1419
4
9:4707–4714
Biot C, Chavain N, Dubar F, Pradines B, Trivelli X, Brocard J, Forfar
I, Dive D (2009) Structure-activity relationships of 4-N-substi-
tuted ferroquine analogues. Time to re-evaluate the mechanism
of action of ferroquine. J Organomet Chem 694:845–854
Biot C, Nosten F, Fraisse L, Ter-Minassian D, Khalife J, Dive D
(2011) The antimalarial ferroquine: from bench to clinic.
Parasite 18:207–214
Biot C, Castro W, Bott e´ CY, Navarro M (2012) The therapeutic
potential of metal-based antimalarial agents: implications for the
mechanism of action. Dalton Trans 41:6335–6349
Blagbrough IS, Carrington S, Geall AJ (1997) Polyamines and
polyamine amides as potent selective receptor probes, novel
therapeutic lead compounds and synthetic vectors in gene
therapy. Pharm Sci 3:223–233
Chavain N, Vezin H, Dive D, Touati N, Paul JP, Buisine E, Biot C
(2008) Investigation of the redox behavior of ferroquine, a new
antimalarial. Mol Pharm 5:710–716
Cloete TT, Krebs HJ, Clark JA, Connelly MC, Orcutt A, Sigal MS,
Guy RK, N’Da DD (2013) Synthesis, antimalarial activity and
cytotoxicity of 10-aminoethylether derivatives of artemisinin.
Bioorg Chem 46:10–16
Daher W, Biot C, Fandeur T, Jouin H, Pelinski L, Viscogliosi E,
Fraisse L, Pradines B, Brocard J, Khalife J, Dive D (2006)
Assessment of Plasmodium falciparum resistance to ferroquine
(SSR97193) in field isolates and in W2 strain under pressure.
Malar J 5:11
Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM,
Ariey F, Hanpithakpong W, Lee SJ, Ringwald P, Silamut K,
Imwong M, Chotivanich K, Lim P, Herdman T, An SS, Yeung S,
Singhasivanon P, Day NP, Lindegardh N, Socheat D, White NJ
N’Da DD, Breytenbach JC, Smith PJ, Lategan C (2010) Synthesis,
cytotoxicity and antimalarial activity of ferrocenyl amides of
4-aminoquinolines. ArzneimForsch-DrugRes 60:627–635
N’Da DD, Breytenbach JC, Smith PJ, Lategan C (2011) Synthesis and
antiplasmodial activity of quinoline-ferrocene esters. Ar-
zneimForsch-DrugRes 61:358–365
Noedl H, Se Y, Sriwichai S, Schaecher K, Teja-Isavadharm P, Smith
B, Rutvisuttinunt W, Bethell D, Surasri S, Fukuda MM, Socheat
D, Chan TL (2010) Artemisinin resistance in Cambodia: a
clinical trial designed to address an emerging problem in
Southeast Asia. Clin Infect Dis 51:e82–e89
(2009) Artemisinin resistance in Plasmodium falciparum
malaria. N Engl J Med 361:455–467
Dubar F, Egan TJ, Pradines B, Kuter D, Ncokazi KK, Forge D, Paul
JF, Pierrot C, Kalamou H, Khalife J, Buisine E, Rogier C, Vezin
H, Forfar I, Slomianny C, Trivelli X, Kapishnikov S, Leiserowitz
L, Dive D, Biot C (2011) The antimalarial ferroquine: role of the
metal and intramolecular hydrogen bond in activity and resis-
tance. ACS Chem Biol 6:275–287
Petersen I, Eastman R, Lanzer M (2011) Drug-resistant malaria:
molecular mechanisms and implications for public health. FEBS
Lett 585:1551–1562
Hocart SJ, Liu H, Deng H, De D, Krogstad FM, Krogstad DJ (2011)
4
-Aminoquinolines active against chloroquine-resistant Plasmo-
Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, Mcgready
R, Moo CL, Al-saai S, Dondorp AM, Lwin KM, Singhasivanon
P, Day NPJ, White NJ, Anderson TJC, Nosten F (2012)
Emergence of artemisinin-resistant malaria on the western
border of Thailand: a longitudinal study. Lancet 379:1960–1966
Quirante J, Dubar F, Gonz a´ lez A, Lopez C, Cascante M, Cort e´ s R,
Forfar I, Pradines B, Biot C (2011) Ferrocene–indole hybrids for
cancer and malaria therapy. J Organometal Chem 696:1011–1017
Roux C, Biot C (2012) Ferrocene-based antimalarials. Future. Med
Chem 4:783–797
Rubinstein LV, Shoemaker RH, Paull KD, Simon RM, Tosini S,
Skehan P, Scudiero DA, Monks A, Boyd MRJ (1990) Compar-
ison of in vitro anticancer-drug-screening data generated with a
tetrazolium assay versus a protein assay against a diverse panel
of human tumor cell lines. Natl Cancer Inst 82:1113–1117
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D,
Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New
dium falciparum: basis of antiparasite activity and quantitative
structure-activity relationship analyses. Antimicrob Agents
Chemother 55:2233–2244
Hottin A, Dubar F, Steenackers A, Delannoy P, Biot C, Behr JB
(2012) Iminosugar-ferrocene conjugates as potential anticancer
agents. Org Biomol Chem 10:5592-5597 http://clinicaltrials.gov/
ct2/show/ NCT00988507
Kelland LR, Sharp SY, O’Neill CF, Raynaud FI, Beale PJ, Judson IR
(
1999) Glutathione induces cellular resistance against cationic
dinuclear platinum anticancer drugs. Inorg Biochem
7:111–115
J
7
K o¨ pf-Maier P, K o¨ pf H, Neuse EW (1984) Ferricenium complexes: a
new type of water-soluble antitumor agent. J Cancer Res Clin
Oncol 108:336–340
Kumar K, Singh P, Kremer L, Gu e´ rardel Y, Biot C, Kumar V (2012)
Synthesis and in vitro anti-tubercular evaluation of 1,2,3-triazole
123

1
224
Med Chem Res (2014) 23:1214–1224
colorimetric cytotoxicity assay for anticancer-drug screening.
J Natl Cancer Inst 82:1107–1112
antimalarial activity of a series of bisquinoline and bispyrrol-
o[1,2a]quinoxaline compounds. Eur J Med Chem 55:335–345
WHO (2012); http://www.who.int/mediacentre/factsheets/fs094/en/
index.html
WHO (World Health Organization) (2011) World malaria report
Wu C–C, Li T-K, Farh L, Lin L-Y, Lin T-S, Yu Y-J, Yen T-J, Chiang
C-W, Chan N-L (2011) Structural basis of type II topoisomerase
inhibition by the anticancer drug etoposide. Science 333:
459–462
Supan C, Mombo-Ngoma G, Dal-Bianco MP, Ospina-Salazar CL,
Issifou S, Mazuir F, Filali-Ansary A, Biot C, Ter-Minassian D,
Ramharter M, Kremsner PG, Lell B (2012) Pharmacokinetics of
ferroquine, a novel 4-aminoquinoline, in asymptomatic carriers
of Plasmodium falciparum infections. Antimicrob Agents Che-
mother 56:3165–3173
Trager W, Jensen JB (1976) Human malaria parasites in continuous
culture. Science 193:673–675
van Heerden L, Cloete TT, Breytenbach JW, de Kock C, Smith PJ,
Breytenbach JC, N’Da DD (2012) Synthesis and in vitro
Zhou B, Li J, Feng BJ, Ouyang Y, Liu YN, Zhou F (2012) Syntheses
and in vitro antitumor activities of ferrocene-conjugated Arg-
Gly-Asp peptides. J Inorg Biochem 116:19–25
1
23