7-chloroquinoline-isatin conjugates: Antimalarial, antitubercular, and cytotoxic evaluation

Article abstract of DOI:10.1111/cbdd.12273

A series of twenty piperazine-tethered 7-chloroquinoline-isatin hybrids have been synthesized via either direct nucleophilic substitution or Cu(Ι)Cl-mediated Mannich reaction. These new conjugates were evaluated for their antimalarial and antitubercular efficacy against a chloroquine-resistant strain of Plasmodium falciparum and Mycobacterium tuberculosis, respectively, while the cytotoxic profiles were evaluated against 3T6 cell line, a permanent mouse embryonic fibroblast cell line. The most potent of the test compound with IC₅₀ of 0.22 μm against W2 strain of P. falciparum and 31.62 μm against the embryonic fibroblast cell line (cytotoxicity) displayed a high selective index of 143.73. Synthesis, antimalarial, anti-TB, and cytotoxic evaluation of piperazine-tethered 7-chloroquinoline-isatin conjugates.

Full text of DOI:10.1111/cbdd.12273

Chem Biol Drug Des 2014; 83: 622–629
Research Letter
7-Chloroquinoline–isatin Conjugates: Antimalarial,
Antitubercular, and Cytotoxic Evaluation
1.4 million deaths, each year, respectivelya,b. The statistics
showed an estimated 219 million cases of malaria with an
estimated 660 000 deaths in 2010. About 80% of the esti-
mated cases of malaria occur in sub-Saharan Africa and
86% of deaths occur in children < 5 years of age. Accord-
ing to the world malaria report 2012, resistance of parasite
to artemisinin (ART, 1 in Figure 1) has now been detected
in four countries of the Greater Mekong subregion viz.
Cambodia, Myanmar, Thailand, and Viet Nama.
Raghu Raj1, Christophe Biot2,3, Severine
ꢀ
4
Carrere-Kremer , Laurent Kremer4,5, Yann
ꢁ
Guerardel2,3, Jiri Gut6, Philip J. Rosenthal6,
ꢀ
Delphine Forge7 and Vipan Kumar1,*
1Department of Chemistry, Guru Nanak Dev University,
Amritsar, Punjab 143005, India
2
ꢀ
Unite de Glycobiologie Structurale et Fonctionnelle,
Universite Lille 1, F-59650, Villeneuve d’Ascq, France
ꢀ
3CNRS, UMR 8576, Villeneuve d’Ascq F-59650, France
4Laboratoire de Dynamique des Interactions Membranaires
In conjunction with malaria, TB ranks as the second leading
cause of death from a contagious disease after AIDSb.
One-third of the world’s population asymptomatically har-
bor a dormant or latent form of M. tuberculosis with a life-
long risk of disease reactivation (1). HIV-positive people are
prone to be infected with TB rendering latent TB to an
active condition, often with fatal consequences (2,3). The
use of the first-line antitubercular drugs isoniazid (INH) and
rifampicin (RIF) is considered inadequate due to the devel-
opment of multidrug-resistant strain of TB (MDR-TB),
requiring administration of more expensive second-line
agents (3,4). The problem is further exacerbated by the
development of extensively drug-resistant TB (XDR-TB) fol-
lowing previous treatment with second-line antitubercular
drugs contributing to the increased morbidity and mortality
(5). The widespread emergence of MDR strains of P. falci-
parum and M. tuberculosis to clinically available drugs has
generated a constant demand to develop novel, safe, highly
effective, and fast-acting antimalarial and anti-TB agents.
ꢀ
Normales et Pathologiques, UMR 5235 CNRS, Universite
Montpellier 2, Place Eugene Bataillon 34095, Montpellier
ꢁ
Cedex 05, France
INSERM, DIMNP, Place Eugene Bataillon 34095,
Montpellier Cedex 05, France
5
ꢁ
6Department of Medicine, University of California, San
Francisco, CA 94143, USA
7Laboratory of Organic Chemistry, Faculty of Sciences,
University of Mons-UMONS, 20 place du parc B-7000,
Mons, Belgium
*Corresponding author: Vipan Kumar,
vipan_org@yahoo.com
A series of twenty piperazine-tethered 7-chloroquino-
line–isatin hybrids have been synthesized via either
direct nucleophilic substitution or Cu()Cl-mediated Man-
nich reaction. These new conjugates were evaluated for
their antimalarial and antitubercular efficacy against a
chloroquine-resistant strain of Plasmodium falciparum
and Mycobacterium tuberculosis, respectively, while
the cytotoxic profiles were evaluated against 3T6 cell
line, a permanent mouse embryonic fibroblast cell line.
The most potent of the test compound with IC50 of
0.22 lM against W2 strain of P. falciparum and 31.62 lM
against the embryonic fibroblast cell line (cytotoxicity)
displayed a high selective index of 143.73.
Quinolines are considered as important and established
scaffolds for antimalarial drug discovery. Among the quino-
line derivatives, 4-aminoquinoline especially chloroquine
(CQ, 2 in Figure 1) was considered as prime therapy for
malaria treatment due to its clinical efficacy, low toxicity,
cheap synthesis, and ease of administration (6). CQ acts by
the inhibition of hemozoin formation within the acidic food
vacuole of parasite (7,8), but the development of resistant
strains of P. falciparum to CQ has limited its use (9). Despite
the development of resistance, literary rationale has
revealed a number of reports appearing on the synthesis of
a number of new 4-aminoquinoline analogs with enhanced
activity against chloroquine-resistant (CQR) strains devel-
oped via synthetic modifications of the CQ side chain
(10–14). Chibale et al. in a recent communication explored
the synthesis of a series of triazole-tethered chalcone and
dienone hybrid compounds containing aminoquinoline and
Key words: 7-chloroquinoline–isatin scaffolds, antimalarial
activity, antitubercular activity, cytotoxicity, structure activity
relationship
Received 18 October 2013, revised 28 November 2013 and
accepted for publication 9 December 2013
Malaria and tuberculosis (TB) are infectious diseases that
continue to be global health problems with devastating
impact on mankind. According to World Health Organiza-
tion (WHO), they are responsible for one million and
622
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12273

7-Chloroquinoline–isatin Hybrids and Bio-evaluation
CH3
CH3
Me
N
N
CH3
H
N
N
N
O
HN
Me
O
N
N
O
H
Me
Cl
N
O
Cl
Cl
1
2
3
Figure 1: Antimalarials: Artemisinin (ART) (1), chloroquine (CQ) (2), piperaquine (3).
azidothymidine (AZT) leading to the identification of highly
active hybrids having submicromolar IC50 values against
D10, Dd2, and W2 strains of P. falciparum (15). Further, a
series of urea-/oxalamide-tethered b-lactam-7-chloroquino-
line hybrids as potential antimalarial agents has been
reported by our group. Their antiplasmodial profiles against
CQ-resistant W2 strain revealed the dependency of activity
profile on the N-1 substituent of the b-lactam ring, the nat-
ure of the linker as well as the length of the alkyl chain (16).
The exploration of novel 4-aminoquinoline analogs thus has
emerged as a promising strategy to construct the molecules
with enhanced activity against drug-resistant P. falciparum.
Apart from its significance as an antimalarial agent, quino-
line nucleus has also shown its potential against TB, which
can be deduced from the synthesis of diarylquinoline TMC
207 (ex R207910) (4 in Figure 2). This analog was shown to
possess a new mechanism of action based on interaction
with the mycobacterial adenosine triphosphate (ATP)
synthase (17).
hybrids along with their antimalarial profile. The activity
profiles showed dependence on the substituents at the
C-5 position of isatin as well as the length of the alkyl
chain, with the most potent compound having an IC50
value of 1.21 lM against cultured chloroquine-resistant W2
strain of P. falciparum (31). With our efforts in designing
molecular scaffolds using hybridization protocols (32–35),
we report herein the extension of the above approach
comprising the synthesis of 7-chloroquinoline–isatin conju-
gates tethered via piperazine functionality and non-hydro-
lyzable alkyl chain (5 and 6 in Figure 3) along with their in
vitro antimalarial and anti-TB evaluation. The piperazine
core has been introduced because of its well-established
profile in improving the biological activities as evidenced by
good antimalarial profiles of piperaquine (3 in Figure 1) and
anti-TB profiles of piperazine-tethered benzamidines
(36–39). Interestingly, piperazine nucleus also constitutes
an integral part of rifampicin, the first-line anti-TB drug
(40). It was further envisaged that a non-hydrolyzable
alkane spacer (linker) would augment the lipophilicity of the
target compounds, a property considered important for
antitubercular activity because of its contribution toward
improving the membrane permeability (41–46).
Another privileged heterocyclic scaffold, isatin (1H-
Indole-2,3-dione), has many promising biological activities
such as anti-HIV (18), antiviral (19), antitumor (20–22), anti-
fungal (23,24), antiangiogenic (25), anticonvulsants (26),
anti-Parkinson’s disease therapeutic (27), antimalarial
(28,29), along with good tolerance in humans and its ability
to bind with the hydrophobic sites of the target. Recently,
Chibale and co-workers have described the synthesis of
thiolactone–isatin conjugates and their tetracyclic by-prod-
ucts which exhibited superior antiplasmodial activity. The
most potent of the synthesized tetracyclic conjugate from
the series displayed an IC50 of 6.92 lM against the chloro-
quine-resistant (W2) strain of P. falciparum (30).
Experimental Section
Materials and methods
Melting points were determined by open capillary using a
Veego Precision Digital Melting Point apparatus (MP-D)
and are uncorrected. IR spectra were recorded on a
R
R
O
O
Recent report from our laboratory has described the syn-
thesis of 1H-1,2,3-triazole-tethered isatin-7-chloroquinoline
O
N
O
N
n
N
N
N
N
N
OH
Br
N
O
Cl
N
Cl
N
6
4
5
Figure 2: Antitubercular TMC 207 (4).
Figure 3: General structure of the target hybrids 5 and 6.
Chem Biol Drug Des 2014; 83: 622–629
623

Raj et al.
Shimadzu D-8001 spectrophotometer. 1H NMR spectra
were recorded in deuterochloroform with a Jeol 300
(300 MHz) spectrometer using TMS as an internal stan-
dard. Chemical shift values are expressed as parts per mil-
lion downfield from TMS and J values are in Hertz.
Splitting patterns are indicated as s: singlet, d: doublet, t:
triplet, m: multiplet, dd: double doublet, ddd: doublet of a
doublet of a doublet, and br: broad peak. 13C NMR spec-
tra were recorded on Jeol 300 (75 MHz) and BRUKER
AVANCE II (400 MHz) spectrometers in deuterochloroform
using TMS as internal standard. High-resolution mass
spectra were recorded on Bruker-micrOTOF-Q II spec-
trometer.
the maximum concentration of DMSO used was 0.1%.
Controls without inhibitors included 0.1% DMSO. After
48 h when control cultures had progressed to new rings,
the culture medium was removed, and cultures were incu-
bated for 48 h with 1% formaldehyde in PBS, pH 7.4, at
room temperature. Fixed parasites were then transferred
to 0.1% Triton X-100 in PBS containing 1 nM YOYO-1 dye
(Molecular Probes). Parasitemia was determined from dot
plots (forward scatter versus fluorescence) acquired on a
FACSort flow cytometer using Cell Quest software (Beck-
ton Dickinson, San Jose, CA, USA). IC50 values for growth
inhibition were determined from plots of percent control
parasitemia over inhibitor concentration using the Prism
3.0 program, (GraphPad Software, San Diego, CA, USA),
with data from duplicate experiments fitted by nonlinear
regression (48).
General procedure for the synthesis of
conjugates, 5a–5p
To a stirred solution of 7-chloro-4-piperazin-1-yl-quinoline
9 (1 mmol) in 15 mL DMF, K2CO3 (3 mmol) and catalytic
amount of KI was added and the resulting solution was
stirred for 10 min; this was followed by the addition of
N-alkylated isatin 11 (1 mmol) and the stirring was contin-
ued for 10–12 h at 85 °C. Progress of the reaction was
monitored by TLC, and on completion, the solvent was
evaporated and water (15 mL) was added to it. The reac-
tion mixture was then extracted with ethyl acetate
(3 9 50 mL), and the oragnic layer was dried over anhy-
drous sodium sulfate. The organic layer was then filtered
and concentrated under reduced pressure resulting into
the crude product which was purified by column chroma-
tography using chloroform/methanol (95:5) mixture.
Methods for assessment of In vitro antitubercular
activity of test compounds
Bacterial strains and growth conditions. Mycobacterium
tuberculosis mc26230 was grown at 37 °C in Sauton’s
medium supplemented with 24 lg/mL of pantothenic acid.
Drug susceptibility testing. The susceptibility of
M. tuberculosis to the various compounds was determined
as reported previously (49). In brief, Middlebrook 7H10
solid medium containing oleic-albumin-dextrose-catalase
enrichment (OADC) and 24 lg/mL of pantothenic acid
was supplemented with increasing concentrations of the
chemical analogs. Serial 10-fold dilutions of each actively
growing culture were plated and incubated at 37 °C for
2–3 weeks. The MIC was defined as the minimum con-
centration required to inhibit 99% of the growth.
General procedure for the synthesis of Mannich
adducts, 6a–6d
A mixture of N-propargylated isatin 12 (1 mmol), parafor-
maldehyde (2.5 mmol), 7-chloro-4-piperazin-1-yl-quinoline
9 (1 mmol), and anhydrous cupric chloride (0.5 mmol) in
dry 1,4-dioxane was heated at 100 °C for 3 h. After com-
pletion of reaction as evidenced by TLC, the reaction mix-
ture was filtered and the filtrate was treated with brine
solution and extracted with ethyl acetate (3 9 60 mL).
Combined organic layers were dried over Na2SO4, and the
solvent was evaporated under reduced pressure resulting
in a crude product which was purified by column chroma-
tography using a chloroform/methanol (95:5) mixture.
Method for assessment of in vitro cytotoxicity of
test compounds
The cytotoxicity of compounds was evaluated using 3T6
fibroblast cells (kindly provided by the Laboratory of Biology
and Embryology of the University of Mons). These cells are
a permanent mouse embryonic fibroblast cell line. They
were cultured in DMEM supplemented with 10% heat-
inactivated FBS (InvitroGen, Merelbeke, Belgium), in a CO2
incubator (37 °C, 5% CO2). 200 lL of 5 9 103 trypsinized
3T6 cells was seeded in each well (except for the wells on
the edge) of a 96-well plate. Under sterile conditions, 8
successive dilutions of each compound were realized
(starting at 100 lM to reach 0.05 lM). After 24 h of incuba-
tion, the culture medium was eliminated and replaced by
198 lL of culture medium and 2 lL of each dilution. Also
200 lL of culture medium was added to the cell growth
control rows, and the solvent control wells received 200 lL
of a mixture of solvent (DMSO; Sigma Aldrich, Bornem,
Belgium) and culture medium. For the blanks, the same
mixture was added to the wells without cells. After 48 h of
incubation, the cells were carefully washed with PBS, and
100 lL of fresh culture medium was added into each well.
Biological evaluations
Methods for assessment of antimalarial activity of
test compounds
The W2 strain of P. falciparum was cultured in RPMI-1640
medium with 10% human serum, following standard meth-
ods, and parasites were synchronized with 5% D-sorbitol
(47). Beginning at the ring stage, microwell cultures were
incubated with different concentrations of compounds for
48 h. The compounds were added from DMSO stocks;
624
Chem Biol Drug Des 2014; 83: 622–629

7-Chloroquinoline–isatin Hybrids and Bio-evaluation
10 lL of MTT (In vitro toxicology assay kit, MTT based;
Sigma Aldrich) was then added to all wells and incubated
for 3 h. The MTT solubilization reagent was also added into
each well to solubilize formazan crystals. The plate was
incubated overnight at 37 °C. With a microplate reader, the
optical density of each well was measured at 540 nm
against the background absorbance whose reference filter
was set at 690 nm. Eight concentrations of each test com-
pound (100–0.05 lM) were verified in triplicate. Blank
controls (culture medium), free-drug controls, and solvent
(DMSO) controls were included in each assay.
H
N
Cl
N
N
H
N
Et3N, 120 0
overnight
C
+
Cl
N
N
H
Cl
7
8
9
Scheme 1: Synthesis of 7-chloro-4-piperazin-1-yl-quinoline 9.
The structure to 5 was assigned on the basis of spectral
data and analytical evidence. For example, 5b, showed a
molecular ion peak [M]+ at 466.1580, along with charac-
teristic peaks in 1H and 13C NMR spectra. The 1H NMR
spectrum displayed peaks at d 1.57, 1.72, 2.46, and 3.72
corresponding to methylene protons of the alkyl chain
along with the characteristic protons of 7-chloroquinoline
ring. The requisite number of carbons in the 13C NMR
spectrum along with the two characteristic peaks at d
157.8 and 182.8 assigned to isatin ring carbonyls further
attested the assigned structure.
The % inhibition (% IC) of cell proliferation was calculated
using the formula:% IC = 100 – [corrected mean OD
sample 9 100/corrected mean OD solvent controls]
where corrected mean OD sample/solvent = mean
OD540–690 of samples/controls
– mean OD540–690 of
blanks. The concentration inhibiting 50% of cell prolifera-
tion (IC50) of each compound was obtained by plotting
the% inhibition of activity against the compound concen-
tration scale.
The precursor for 7-chloroquinoline–isatin Mannich
adducts viz. N-propargylated isatin 12 was prepared by an
initial treatment of isatin with sodium hydride with
subsequent addition of propargyl bromide at 60 °C. The
N-propargylated isatins 12 were then utilized in Cu(Ι)Cl
catalyzed Mannich reaction with 9 in the presence of form-
aldehyde in dry 1,4-dioxane (Scheme 3) (50). Mechanisti-
cally, the reaction is thought to proceed via an initial
formation of Cu-acetylide, which undergoes a nucleophilic
addition to the iminium ion formed by the condensation
reaction between 9 and formaldehyde. The progress of
reactions was monitored by TLC. On completion and after
workup with ethyl acetate and water, the organic layer
was separated and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure resulting
into the crude product that was purified by column chro-
matography using a chloroform/methanol (95:5) mixture.
The structure of the synthesized Mannich adducts has
been assigned on the basis of spectral data and analytical
data, the details of which are provided in the experimental
section.
Result and Discussions
Chemistry
7-Chloro-4-piperazin-1-yl-quinoline 9, required for the syn-
thesis of desired scaffolds, was synthesized by heating
overnight 4,7-dichloroquinoline 7 with an excess of pipera-
zine 8 and triethylamine at 120 °C (Scheme 1). The other
precursor, N-alkylated isatins 11, was prepared by react-
ing a solution of isatin in DMF with sodium hydride with
subsequent treatment with dibromoalkanes. The target
conjugates 5a–5p were synthesized by initial stirring of a
solution of K2CO3 and KI with 7-chloro-4-piperazin-1-yl-
quinoline 9 in dry DMF for 5–10 min, followed by subse-
quent addition of N-alkylated isatin 11 (Scheme 2). The
reaction mixture was stirred at 85 °C for 10–12 h, and the
progress of reactions was monitored by TLC. After usual
workup on the completion of reaction, solvent was evapo-
rated and the desired product was obtained after purifica-
tion via column chromatography using
methanol (95:5) mixture.
a
chloroform/
R
O
O
N
O
O
O
Br
Br
R
R
n
9, K2CO3, KI,
DMF, 85 0C,
10-12 h
O
n
NaH, 60 0
5-6 h
C
N
N
N
N
H
11
10
n
Br
R = H, F, Cl, CH3
Scheme 2: Synthesis of 7-
chloroquinoline–isatin conjugates
5a–5p.
Cl
N
5
n = 1, 2, 3, 5
Chem Biol Drug Des 2014; 83: 622–629
625

Raj et al.
O
O
O
R
R
R
Br
9, HCHO, CuCl,
1,4-dioxane
O
O
O
NaH, 60 0
5-6 h
C
N
H
N
N
100 0C, 2-3 h
CCu
12
10
R
O
O
N
N
N
Cl
N
Scheme 3: Synthesis of
7-chloroquinoline–isatin Mannich
adducts 6a–6d.
6
R = H, F, Cl, CH3
In vitro antimalarial, antitubercular, and cytotoxic
evaluation
The test compounds were evaluated for their antimalarial
profiles against the CQ-resistant W2 strain of P. falcipa-
rum. Analysis of Table 1 revealed that these were not as
active as standard drug viz. chloroquine (CQ), although
compounds 5c, 5f, 5h, and 5j have shown good anti-
malarial activity among the test compounds with IC50
values ranging from 0.22 to 0.27 lM. The observed
activity profiles were found to be independent of the
length of alkyl chain especially among butyl, pentyl, and
hexyl spacer, while a fifteen times decrease (compare 5c
Table 1: Antiplasmodial activity against a chloroquine-resistant strain, anti-TB activity against Mycobacterium tuberculosis mc26230, and
cytotoxicity of compounds 5a–5p and 6a–6d
Antiplasmodial activity
Anti-TB activity
MIC (lm)
Cytotoxicity
IC50 (lM) Æ Standard error
Code
R
n
W2 (CQ-R) IC50 (lM)
SIa
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
H
F
Cl
CH3
H
F
Cl
CH3
H
F
Cl
CH3
H
F
Cl
CH3
H
F
Cl
CH3
1
1
1
1
2
2
2
2
3
3
3
3
5
5
5
5
–
–
–
–
1.12
1.17
0.27
0.81
0.46
0.27
0.42
0.25
0.54
0.22
0.92
0.62
1.39
0.90
3.79
7.52
1.01
0.58
0.42
1.12
0.036
1
55.80
107.29
10.35
54.11
43.29
<10.41
20.12
52.52
52.52
<10.12
19.56
ND
39.60
38.24
166.97
>192.67
>225
1.81 Æ 0.01
6.77 Æ 0.09
7.57 Æ 0.01
5.82 Æ 0.06
2.01 Æ 0.02
6.35 Æ 0.10
15.40 Æ 0.10
6.79 Æ 0.02
7.55 Æ 0.03
31.62 Æ 0.13
14.72 Æ 0.08
8.35 Æ 0.01
1.22 Æ 0.07
ND
5
28
7
4
23
36
27
13
143
16
13
0.8
ND
ND
ND
4
ND
ND
6a
6b
6c
6d
4.81 Æ 0.01
15.56 Æ 0.03
7.32 Æ 0.07
11.04 Æ 0.12
26
17
9
>216
135.69
54.58
Chloroquine
Cephalexin
Isoniazid (INH)
68.41
0.18
ND, Not determined.
aSI: Selective index is ratio of IC50 of 3T6 cell line to that of chloroquine-resistant (W2) strain.
626
Chem Biol Drug Des 2014; 83: 622–629

7-Chloroquinoline–isatin Hybrids and Bio-evaluation
and 5o) in antimalarial efficacy has been observed in
octyl spacers. The activity profiles at shorter alkyl chain
length viz. n = 1, 2, and 3 seemed to be independent
over the nature of substituent at C-5 position of the isat-
in ring, while a preference for flouro substituent for good
activity has been observed at n = 5. Similar preference
for the halogen substituent for good activity has been
observed in case of 7-chloroquinoline–isatin Mannich
adducts with compounds 6c having chloro substituent
showed an IC50 of 0.42 lM. The test compounds were
further evaluated for their antitubercular profiles, and the
results are listed in Table 1. As evident, compounds are
not as active as that of isoniazid. Analysis of Table 1
explicated that the presence of substituent at C-5
position of the isatin ring tends to influence the activity
profile, while longer alkyl chain lengths adversely affect
the anti-TB efficacy of the test compounds. A clear pref-
erence for fluoro substituent is evident for good anti-TB
efficacy as observed in case of 5f and 5j, being six
times more potent than cephalexin. The Mannich
adducts on the other hand proved to be inefficient in
inhibiting the growth of M. tuberculosis.
References
1. Pasqualoto K.F.M., Ferreira E.I., Santos-Filho O.A.,
Hopfinger A.J. (2004) Rational design of new antituber-
culosis agents: receptor-independent four-dimensional
quantitative structure-activity relationship analysis of a
set of isoniazid derivatives. J Med Chem;47:3755–3764.
2. Jones D. (2005) Antibacterial drugs: tackling tuberculo-
sis. Nat Rev Drug Disc;4:103.
3. Gandhi N.R., Moll A., Sturm A.W., Pawinski R., Gov-
ender T., Lalloo U., Zeller K., Andrews J., Friedland G.
(2006) Extensively drug-resistant tuberculosis as
cause of death in patients co-infected with tuberculosis
and HIV in rural area of South Africa.
a
a
Lancet;368:1575–1580.
4. Veziris N., Truffot-Pernot C., Aubry A., Jarlier V., Lounis
N. (2003) Fluoroquinolone-containing third-line regimen
against Mycobacterium tuberculosis in vivo. Antimicrob
Agents Chemother;47:3117–3122.
5. Caminero J.A., Sotgiu G., Zumla A., Migliori G.B.
(2010) Best drug treatment for multidrug-resistant and
extensively drug-resistant tuberculosis. Lancet Infect
Dis;10:621–629.
The synthesized scaffolds have also been evaluated for
their cytotoxic profiles against 3T6 cell line, a permanent
mouse embryonic fibroblast cell line. As evident, the
compounds viz. 5c, 5f, and 5j with good antimalarial effi-
cacy also possess higher cytotoxicity against the embry-
onic fibroblast cell line. However, the compound 5j with
an IC50 of 0.22 lM against W2 strain of P. falciparum
and 31.62 lM against the embryonic fibroblast cell line
6. Ridley R.G. (2002) Medical need, scientific opportunity
and the drive for antimalarial drugs. Nature;415:686–
693.
7. Egan T.J., Ncokazi K.K. (2005) Quinoline antimalarials
decrease the rate of beta-hematin formation. J Inorg
Biochem;99:1532–1539.
8. Joshi A.A., Viswanathan C.L. (2006) Recent develop-
ments in antimalarial drug discovery. Anti Infect Agents
Med Chem;5:105–122.
9. Hyde J.E. (2007) Drug-resistant malaria- An insight.
FEBS J;274:4688–4698.
(cytotoxicity) also possess
143.73.
a high selective index of
10. Hwang J.Y., Kawasuji T., Lowes D.J., Clark J.A., Con-
nelly M.C., Zhu F., Guiguemde W.A., Sigal M.S., Wil-
son E.B., DeRisi J.L., Guy R.K. (2011) Synthesis and
evaluation of 7-substituted 4-aminoquinoline analogues
for antimalarial activity. J Med Chem;54:7084–7093.
11. Manohar S., Rajesh U.C., Khan S.I., Tekwani B.L.,
Rawat D.S. (2012) Novel 4-aminoquinoline-pyrimidine
based hybrids with improved in Vitro and in vivo anti-
malarial activity. ACS Med Chem Lett;3:555–559.
12. Ekoue-Kovi K.A., Yearick K., Iwaniuk D.P., Natarajan
J.K., Alumasa J., de Dios A.C., Roepe P.D., Wolf C.
(2009) Synthesis and antimalarial activity of new
4-amino-7-chloroquinolyl amides, sulfonamides, ureas
and thioureas. Bioorg Med Chem;17:270–283.
13. Natarajan J.K., Alumasa J.N., Yearick K., Ekoue-Kovi
K.A., Casabianca L.B., de Rios A.C., Wolf C., Roepe
P.D. (2008) 4-N-, 4-S-, and 4-O-chloroquine ana-
logues: influence of side chain length and quinolyl
nitrogen pKa on activity vs chloroquine resistant
malaria. J Med Chem;51:3466–3479.
Conclusion
In conclusion, the present manuscript describes the syn-
thesis of piperazine-tethered 7-chloroquinoline–isatin
conjugates prepared via nucleophilic substitution and
Cu-catalyzed Mannich addition protocols along with their
antimalarial, antitubercular, and cytotoxic evaluation. SAR
studies showed a preference for halogen substituent at
C-5 position of isatin ring for good activity, while longer
alkyl chain lengths adversely affected the activity profiles.
Acknowledgment
Financial assistance from Board of Research in Nuclear
Sciences under DAE Research Award for Young Scientist
Scheme (VK) is gratefully acknowledged. We thank Eliane
Rubo for excellent technical assistance.
14. Biot C., Nosten F., Fraisse L., Ter-Minassian D., Kha-
life J., Drive D. (2011) The antimalarial ferroquine: from
bench to clinic. Parasite;18:207–214.
Conflict of Interest
The authors declare no competing financial interest.
Chem Biol Drug Des 2014; 83: 622–629
627

Raj et al.
15. Guantai E.M., Ncokazi K., Egan T.J., Gut J., Rosenthal
P.J., Smith P.J., Chibale K. (2010) Design, synthesis
and in vitro antimalarial evaluation of triazole-linked
chalcone and dienone hybrid compounds. Bioorg Med
Chem;18:8243–8256.
inhibitor, triggers a dose- and time-dependent switch
from apoptosis to necrosis in human neuroblastoma
cells. Neurochem Int;47:216–224.
28. Yeung B.K.S., Zou B., Rottmann M., Lakshminarayana
S.B., Ang S.H., Leong S.Y., Tan J. et al. (2010) Spiro-
16. Singh P., Raj R., Singh P., Gut J., Rosenthal P.J.,
Kumar V. (2014) Urea/oxalamide tethered b-lactam-7-
chloroquinoline conjugates: synthesis and in vitro anti-
malarial evaluation. Eur J Med Chem;71:128–134.
17. Diacon A.H., Pym A., Grobush M., Patienta R.,
Rustomjee R., Page-Shipp L., Pistorius C. et al. (2009)
The diarylquinoline TMC207 for multidrug-resistant
tuberculosis. N Eng J Med;360:2397–2405.
18. Ratan B.T., Anand B., Yogeeswari P., Sriram D.H.
(2005) Synthesis and evaluation of anti-HIV activity of
isatin beta-thiosemicarbazone derivatives. Bioorg Med
Chem Lett;15:4451–4455.
tetrahydro beta-carbolines (spiroindolones): a new
class of potent and orally efficacious compounds for
the treatment of malaria. J Med Chem;53:5155–5164.
29. Rottmann M., McNamara C., Yeung B.K.S., Lee
M.C.S., Zhou B., Russell B., Seitz P. et al. (2010) Spir-
oindolones, a potent compound class for the treatment
of malaria. Science;329:1175–1180.
30. Hans R.H., Wiid I.J.F., Helden P.D.V., Wan B., Franzb-
lau S.G., Gut J., Rosenthal P.J., Chibale K. (2011)
Novel thiolactone-isatin hybrids as potential antimalarial
and antitubercular agents. Bioorg Med Chem
Lett;21:2055–2058.
19. Jiang T., Kuhen K.L., Wolff K., Yin H., Bieza K., Cald-
well J., Bursulaya B., Tuntland T., Zhang K., Karanew-
sky D., He Y. (2006) Design, synthesis, and biological
evaluations of novel oxindoles as HIV-1 non-nucleoside
reverse transcriptase inhibitors. Part 2. Bioorg Med
Chem Lett;16:2109–2112.
31. Raj R., Singh P., Singh P., Gut J., Rosenthal P.J.,
Kumar V. (2013) Azide-alkyne cycloaddition en route to
1H-1,2,3-triazole-tethered 7-chloroquinoline-isatin chi-
meras: synthesis and antimalarial evaluation. Eur
J Med Chem;62:590–596.
ꢁ
ꢀ
32. Raj R., Biot C., Carrere-Kremer S., Kremer L., Guerar-
del Y., Gut J., Rosenthal P.J., Kumar V. (2013) 4-ami-
20. Tripathy R., Reiboldt A., Messina P.A., Iqbal M., Singh
J., Bacon E.R., Angeles Th.S., Yang Sh.X., Albom
M.S., Robinson C., Chang H., Ruggeri B.A., Mallamo
J.P. (2006) Structure-guided identification of novel
VEGFR-2 kinase inhibitors via solution phase parallel
synthesis. Bioorg Med Chem Lett;16:2158–2162.
21. Cane A., Tournaire M.C., Barritault D., Crumeyrolle-
Arias M. (2000) The endogenous oxindoles 5-hydroxy-
oxindole and isatin are antiproliferative and proapoptot-
ic. Biochem Biophys Res Commun;276:379–384.
22. Silveira V.Ch., Luz J.S., Oliveira C.C., Graziani I., Ciriol-
o M.R., Costa-Ferreira A.M. (2008) Double-strand DNA
cleavage induced by oxindole-Schiff base copper(II)
complexes with potential antitumor activity. J Inorg
Biochem;102:1090–1103.
23. Amal R.A., Raghunathan R., Sridevikumaria M.R.,
Raman N. (2003) Synthesis, antimicrobial and antifun-
gal activity of a new class of spiro pyrrolidines. Bioorg
Med Chem;11:407–419.
24. Rodriguez-Arguelles M.C., Mosquera-Vazaquez S.,
Touron-Touceda P., Sanmartin- Matalobos J., Garcia-
Deibe A.M., Belicchi-Ferraris M., Pelosi G., Pelizzi C.,
Zani F. (2007) Complexes of 2-thiophenecarbonyl and
isonicotinoyl hydrazones of 3-(N-methyl)isatin. A study
noquinoline-b-lactam
conjugates:
synthesis,
antimalarial and anti-tubercular evaluation. Chem Biol
Drug Des (Accepted manuscript): doi: 10.1111/cbdd.
12225
33. Raj R., Singh P., Haberkern N.T., Faucher R.M., Patel
N., Land K.M., Kumar V. (2013) Synthesis of 1H-1,2,3-
triazole linked b-lactam-isatin bi-functional hybrids and
preliminary analysis of in vitro activity against the proto-
zoal parasite Trichomonas vaginalis. Eur
Chem;63:897–906.
J Med
34. Nisha, Mehra V., Hopper M., Patel N., Hall D., Wrisch-
nik L.A., Land K.M., Kumar V. (2013) Design and syn-
thesis of b-amino alcohol based b-lactam-
isatin chimeras and preliminary analysis of in vitro
activity against the protozoal pathogen Trichomonas
vaginalis. MedChemComm;4:1018–1024.
ꢁ
ꢀ
35. Kumar K., Carrere-Kremer S., Kremer L., Guerardel Y.,
Biot C., Kumar V. (2013) 1H-1,2,3-triazole-tethered isat-
in-ferrocene and isatin-ferrocenylchalcone conjugates:
synthesis and in vitro anti-tubercular evaluation. Organo-
metallics; 32: 5713–5719.
36. Biamonte M.A., Wanner J., Le Roch K.G. (2013)
Recent advances in malaria drug discovery. Bioorg
Med Chem Lett;23:2829–2843.
of their antimicrobial activity.
J Inorg Biochem;
101:138–147.
37. Kumar V., Mahajan A., Chibale K. (2009) Synthetic
medicinal chemistry of selected antimalarial natural
products. Bioorg Med Chem;17:2236–2275.
38. Forge D., Cappoen D., Laurent J., Stanicki D., May-
ence A., Huang T.L., Verschaeve L., Huygen K., Eynde
J.J.V. (2012) 1,4-diarylpiperazines and analogs as anti-
tubercular agents: synthesis and biological evaluation.
Eur J Med Chem;49:95–101.
25. Maskell L., Blanche E.A., Colucci M.A., Whatmore
J.L., Moody Ch.J. (2007) Synthesis and evaluation of
prodrugs for anti-angiogenic pyrrolylmethylidenyl oxin-
doles. Bioorg Med Chem Lett;17:1575–1578.
26. Verma M., Nath P.S., Nand S.K., Stables J.P. (2004)
Anticonvulsant activity of Schiff bases of isatin deriva-
tives. Acta Pharm;54:49–56.
27. Igosheva N., Lorz C., O’Conner E., Glover V., Mehmet
H. (2005) Isatin, an endogenous monoamine oxidase
39. Eynde J.J.V., Mayence A., Lecour L. Jr, Huang T.L.
(2003) Synthesis, antituberculosis activity, and DNA
628
Chem Biol Drug Des 2014; 83: 622–629

7-Chloroquinoline–isatin Hybrids and Bio-evaluation
binding affinity of a highly diverse library of 1,4-diaryl-
piperazines. Med Chem Res;12:401–414.
48. Singh A., Rosenthal P.J. (2001) Comparison of effica-
cies of cysteine protease inhibitors against five strains
of Plasmodium falciparum. Antimicrob Agents Chemo-
ther;45:949–951.
49. Kremer L., Douglas J.D., Baulard A.R., Morehouse C.,
Guy M.R., Alland D., Dover L.G., Lakey J.H., Jacobs
W.R. Jr, Brennan P.J., Minnikin D.E., Besra G.S.
(2000) Thiolactomycin and related analogues as novel
anti-mycobacterial agents targeting KasA and KasB
condensing enzymes in Mycobacterium tuberculosis.
J Biol Chem;275:16857–16864.
40. Figueiredo R., Ramos D.F., Moiteiro C., Medeiros
M.A., Curto M.J.M., de Menezes J.C., Pando R.H.,
Silva P.E.A., Costa M.D.C. (2012) Pharmacophore
insights into rpoB gene mutations in Mycobacterium
tuberculosis rifampicin resistant isolates. Eur J Med
Chem;47:186–193.
41. Krishnan S.S., Pandeya N.S., Stables J.P., Ramesh A.
(2002) Anticonvulsant activity of hydrazones, Schiff and
Mannich bases of isatin derivatives. Eur J Pharm
Sci;16:129–132.
50. Kumar V., Chipeleme A., Chibale K. (2008) Effect of
varying the anionic component of a copper (I) catalyst
on homologation of arylacetylenes to allenes by the
Mannich reaction. Eur J Org Chem; 43–46.
42. Sridhar S.K., Ramesh A. (2001) Synthesis and phar-
macological activities of hydrazones, Schiff and Man-
nich bases of isatin derivatives. Biol Pharm
Bull;24:1149–1152.
43. Sriram D., Yogeeswari P., Gopal G. (2005) Synthesis,
anti-HIV and antitubercular activities of lamivudine pro-
drugs. Eur J Med Chem;40:1373–1376.
44. Karali N., Gursoy A., Kandemirli F., Shvets N., Kaynak
F.B., Ozbey S., Kovalishyn V., Dimoglo A. (2007) Syn-
thesis and structure-antituberculosis activity relation-
ship of 1H-indole-2,3-dione derivatives. Bioorg Med
Chem;15:5888–5904.
Notes
aWHO, World Malaria Report (2012), http://www.who.int/
malaria/publications/world malaria report 2012/en/index
.html.
bWorld Health Organization, Global Tuberculosis Report
(2012), http://www.who.int/tb/publications/global_report/
en/.
45. Karali N. (2002) Synthesis and primary cytotoxicity
evaluation of new 5-nitroindole-2,3-dione derivatives.
Eur J Med Chem;37:909–918.
Supporting Information
46. Terzioglu N., Karali N., Gursoy A., Pannecouque C.,
Leysen P., Paeshuyse J., Neyts J., De Clercq E.
(2006) Synthesis and primary antiviral activity evalua-
tion of 3-hydrazono-5-nitro-2-indolinone derivatives.
ARKIVOC;1:109–118.
47. Jensen J.B. (2002) In vitro culture of plasmodium para-
sites. In: Doolan D.L., editor. Malaria Methods and
Protocols. Totowa, NJ: Humana; p. 477–488.
Additional Supporting Information may be found in the
online version of this article:
Data S1. The details of characterisation of compounds
5a–5p and 6a–6d, along with scanned 1H and 13C spec-
tra of 5a, 5b, 5l, and 6a are given in supplementary file.
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