Chem Biol Drug Des 2014; 83: 622–629

Research Letter

7-Chloroquinoline–isatin Conjugates: Antimalarial,

Antitubercular, and Cytotoxic Evaluation
Raghu Raj1, Christophe Biot2,3, Se verine 1.4 million deaths, each year, respectivelya,b. The statistics
 4 4,5
Carrere-Kremer , Laurent Kremer , Yann showed an estimated 219 million cases of malaria with an
rardel2,3, Jiri Gut6, Philip J. Rosenthal6,
Gue estimated 660 000 deaths in 2010. About 80% of the esti-
Delphine Forge7 and Vipan Kumar1,* mated cases of malaria occur in sub-Saharan Africa and
86% of deaths occur in children < 5 years of age. Accord-
1
Department of Chemistry, Guru Nanak Dev University, ing to the world malaria report 2012, resistance of parasite
Amritsar, Punjab 143005, India to artemisinin (ART, 1 in Figure 1) has now been detected
2
 de Glycobiologie Structurale et Fonctionnelle,
Unite in four countries of the Greater Mekong subregion viz.
 Lille 1, F-59650, Villeneuve d’Ascq, France
Universite Cambodia, Myanmar, Thailand, and Viet Nama.
3
CNRS, UMR 8576, Villeneuve d’Ascq F-59650, France
4
Laboratoire de Dynamique des Interactions Membranaires In conjunction with malaria, TB ranks as the second leading
Normales et Pathologiques, UMR 5235 CNRS, Universite 
Montpellier 2, Place Euge ne Bataillon 34095, Montpellier cause of death from a contagious disease after AIDSb.
Cedex 05, France One-third of the world’s population asymptomatically har-
5
INSERM, DIMNP, Place Euge ne Bataillon 34095, bor a dormant or latent form of M. tuberculosis with a life-
Montpellier Cedex 05, France long risk of disease reactivation (1). HIV-positive people are
6
Department of Medicine, University of California, San prone to be infected with TB rendering latent TB to an
Francisco, CA 94143, USA active condition, often with fatal consequences (2,3). The
7
Laboratory of Organic Chemistry, Faculty of Sciences, use of the first-line antitubercular drugs isoniazid (INH) and
University of Mons-UMONS, 20 place du parc B-7000,
rifampicin (RIF) is considered inadequate due to the devel-
Mons, Belgium
*Corresponding author: Vipan Kumar, opment of multidrug-resistant strain of TB (MDR-TB),
[email protected] requiring administration of more expensive second-line
agents (3,4). The problem is further exacerbated by the
A series of twenty piperazine-tethered 7-chloroquino- development of extensively drug-resistant TB (XDR-TB) fol-
line–isatin hybrids have been synthesized via either lowing previous treatment with second-line antitubercular
direct nucleophilic substitution or Cu()Cl-mediated Man- drugs contributing to the increased morbidity and mortality
nich reaction. These new conjugates were evaluated for (5). The widespread emergence of MDR strains of P. falci-
their antimalarial and antitubercular efficacy against a parum and M. tuberculosis to clinically available drugs has
chloroquine-resistant strain of Plasmodium falciparum generated a constant demand to develop novel, safe, highly
and Mycobacterium tuberculosis, respectively, while effective, and fast-acting antimalarial and anti-TB agents.
the cytotoxic profiles were evaluated against 3T6 cell
line, a permanent mouse embryonic fibroblast cell line. Quinolines are considered as important and established
The most potent of the test compound with IC50 of
scaffolds for antimalarial drug discovery. Among the quino-
0.22 lM against W2 strain of P. falciparum and 31.62 lM
line derivatives, 4-aminoquinoline especially chloroquine
against the embryonic fibroblast cell line (cytotoxicity)
displayed a high selective index of 143.73. (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
Key words: 7-chloroquinoline–isatin scaffolds, antimalarial
the inhibition of hemozoin formation within the acidic food
activity, antitubercular activity, cytotoxicity, structure activity
relationship vacuole of parasite (7,8), but the development of resistant
strains of P. falciparum to CQ has limited its use (9). Despite
Received 18 October 2013, revised 28 November 2013 and the development of resistance, literary rationale has
accepted for publication 9 December 2013 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-
Malaria and tuberculosis (TB) are infectious diseases that oped via synthetic modifications of the CQ side chain
continue to be global health problems with devastating (10–14). Chibale et al. in a recent communication explored
impact on mankind. According to World Health Organiza- the synthesis of a series of triazole-tethered chalcone and
tion (WHO), they are responsible for one million and dienone hybrid compounds containing aminoquinoline and

622 ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12273

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7-Chloroquinoline–isatin Hybrids and Bio-evaluation

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

n
N N N
OH
Br N N

N O
Cl N Cl N
4 5 6

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

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Raj et al.

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

624 Chem Biol Drug Des 2014; 83: 622–629

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7-Chloroquinoline–isatin Hybrids and Bio-evaluation

10 lL of MTT (In vitro toxicology assay kit, MTT based; H

N
Sigma Aldrich) was then added to all wells and incubated
for 3 h. The MTT solubilization reagent was also added into Cl
H N
each well to solubilize formazan crystals. The plate was N
Et3N, 120 0C
incubated overnight at 37 °C. With a microplate reader, the +
Cl N overnight
optical density of each well was measured at 540 nm N
H Cl N
against the background absorbance whose reference filter 7 8 9
was set at 690 nm. Eight concentrations of each test com-
pound (100–0.05 lM) were verified in triplicate. Blank Scheme 1: Synthesis of 7-chloro-4-piperazin-1-yl-quinoline 9.
controls (culture medium), free-drug controls, and solvent
(DMSO) controls were included in each assay. The structure to 5 was assigned on the basis of spectral
data and analytical evidence. For example, 5b, showed a
The % inhibition (% IC) of cell proliferation was calculated molecular ion peak [M]+ at 466.1580, along with charac-
using the formula:% IC = 100 – [corrected mean OD teristic peaks in 1H and 13C NMR spectra. The 1H NMR
sample 9 100/corrected mean OD solvent controls] spectrum displayed peaks at d 1.57, 1.72, 2.46, and 3.72
where corrected mean OD sample/solvent = mean corresponding to methylene protons of the alkyl chain
OD540–690 of samples/controls – mean OD540–690 of along with the characteristic protons of 7-chloroquinoline
blanks. The concentration inhibiting 50% of cell prolifera- ring. The requisite number of carbons in the 13C NMR
tion (IC50) of each compound was obtained by plotting spectrum along with the two characteristic peaks at d
the% inhibition of activity against the compound concen- 157.8 and 182.8 assigned to isatin ring carbonyls further
tration scale. attested the assigned structure.

The precursor for 7-chloroquinoline–isatin Mannich

Result and Discussions adducts viz. N-propargylated isatin 12 was prepared by an
initial treatment of isatin with sodium hydride with
Chemistry subsequent addition of propargyl bromide at 60 °C. The
7-Chloro-4-piperazin-1-yl-quinoline 9, required for the syn- N-propargylated isatins 12 were then utilized in Cu(Ι)Cl
thesis of desired scaffolds, was synthesized by heating catalyzed Mannich reaction with 9 in the presence of form-
overnight 4,7-dichloroquinoline 7 with an excess of pipera- aldehyde in dry 1,4-dioxane (Scheme 3) (50). Mechanisti-
zine 8 and triethylamine at 120 °C (Scheme 1). The other cally, the reaction is thought to proceed via an initial
precursor, N-alkylated isatins 11, was prepared by react- formation of Cu-acetylide, which undergoes a nucleophilic
ing a solution of isatin in DMF with sodium hydride with addition to the iminium ion formed by the condensation
subsequent treatment with dibromoalkanes. The target reaction between 9 and formaldehyde. The progress of
conjugates 5a–5p were synthesized by initial stirring of a reactions was monitored by TLC. On completion and after
solution of K2CO3 and KI with 7-chloro-4-piperazin-1-yl- workup with ethyl acetate and water, the organic layer
quinoline 9 in dry DMF for 5–10 min, followed by subse- was separated and dried over anhydrous Na2SO4. The
quent addition of N-alkylated isatin 11 (Scheme 2). The solvent was evaporated under reduced pressure resulting
reaction mixture was stirred at 85 °C for 10–12 h, and the into the crude product that was purified by column chro-
progress of reactions was monitored by TLC. After usual matography using a chloroform/methanol (95:5) mixture.
workup on the completion of reaction, solvent was evapo- The structure of the synthesized Mannich adducts has
rated and the desired product was obtained after purifica- been assigned on the basis of spectral data and analytical
tion via column chromatography using a chloroform/ data, the details of which are provided in the experimental
methanol (95:5) mixture. section.

R
O

O N
O Br O
R Br R
n 9, K2CO3, KI,
O O n
NaH, 60 0C N DMF, 85 0C,
N N
H 5-6 h 10-12 h
10 11
N
n
Br
R = H, F, Cl, CH3

Scheme 2: Synthesis of 7- Cl N
chloroquinoline–isatin conjugates 5
5a–5p. n = 1, 2, 3, 5

Chem Biol Drug Des 2014; 83: 622–629 625

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Raj et al.

O O O
R R R
Br 9, HCHO, CuCl,
O O O
N 1,4-dioxane
NaH, 60 0C N N
H 100 0C, 2-3 h
5-6 h
CCu
10 12

R
O

O N

Cl N
Scheme 3: Synthesis of
6
7-chloroquinoline–isatin Mannich
R = H, F, Cl, CH3 adducts 6a–6d.

In vitro antimalarial, antitubercular, and cytotoxic compounds 5c, 5f, 5h, and 5j have shown good anti-
evaluation malarial activity among the test compounds with IC50
The test compounds were evaluated for their antimalarial values ranging from 0.22 to 0.27 lM. The observed
profiles against the CQ-resistant W2 strain of P. falcipa- activity profiles were found to be independent of the
rum. Analysis of Table 1 revealed that these were not as length of alkyl chain especially among butyl, pentyl, and
active as standard drug viz. chloroquine (CQ), although 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 Cytotoxicity
Code R n W2 (CQ-R) IC50 (lM) SIa MIC (lm) IC50 (lM)  Standard error

5a H 1 1.12 1 55.80 1.81  0.01

5b F 1 1.17 5 107.29 6.77  0.09
5c Cl 1 0.27 28 10.35 7.57  0.01
5d CH3 1 0.81 7 54.11 5.82  0.06
5e H 2 0.46 4 43.29 2.01  0.02
5f F 2 0.27 23 <10.41 6.35  0.10
5g Cl 2 0.42 36 20.12 15.40  0.10
5h CH3 2 0.25 27 52.52 6.79  0.02
5i H 3 0.54 13 52.52 7.55  0.03
5j F 3 0.22 143 <10.12 31.62  0.13
5k Cl 3 0.92 16 19.56 14.72  0.08
5l CH3 3 0.62 13 ND 8.35  0.01
5m H 5 1.39 0.8 39.60 1.22  0.07
5n F 5 0.90 ND 38.24 ND
5o Cl 5 3.79 ND 166.97 ND
5p CH3 5 7.52 ND >192.67 ND
6a H – 1.01 4 >225 4.81  0.01
6b F – 0.58 26 >216 15.56  0.03
6c Cl – 0.42 17 135.69 7.32  0.07
6d CH3 – 1.12 9 54.58 11.04  0.12
Chloroquine 0.036
Cephalexin 68.41
Isoniazid (INH) 0.18

ND, Not determined.

a
SI: 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

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7-Chloroquinoline–isatin Hybrids and Bio-evaluation

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