Amalgamating Isatin/Indole/Nitroimidazole with 7-chloroquinolines via azide-alkyne cycloaddition: Synthesis, anti-plasmodial, and cytotoxic evaluation

Article abstract of DOI:10.1111/cbdd.13738

The present paper describes the synthesis, anti-plasmodial, and cytotoxic evaluation of 7-chloroquinoline-based conjugates with isatins/indoles/ nitroimidazoles, obtained via Cu-promoted 1,3-dipolar cycloadditions. On contemplating SAR of the synthesized series, the inclusion of indole and nitroimidazole-core improved the anti-plasmodial activities while the isatin seemed to have a lesser effect. The conjugate with a nitroimidazole-core and hexyl chain length as a spacer between the two pharmacophores was found to be most potent among the synthesized series and displayed an IC₅₀ of 0.12?μM and a selectivity index of 1748.

Full text of DOI:10.1111/cbdd.13738

DR DR. RAGHU RAJ (Orcid ID : 0000-0001-8222-1314)
DR VIPAN KUMAR (Orcid ID : 0000-0002-6164-7161)
Article type : Research Letter
Amalgamating Isatin/Indole/Nitro-imidazole with 7-chloroquinolines via azide-
alkyne cycloaddition: Synthesis, anti-plasmodial and cytotoxic evaluation
1
2
3
4
2
1
Sumit Kumar, Anu Saini, Jenny Legac, Philip J. Rosenthal, Raghu Raj *, Vipan Kumar *
1
Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, Punjab, India
2
Department of Chemistry, DAV College, Amritsar, Punjab, India
2
Department of Medicine, University of California, San Francisco, CA, USA
*
Raghu Raj and Vipan Kumar should be considered as joint Corresponding Authors. Tel: +91 183 2553377 (R.R.), +91 183
2
258802-09*3320 (V.K.); Fax: +91 183 2258819-20, Email address: raghusharma4@gmail.com (R. Raj), vipan_org@yahoo.com
V. Kumar)
(
Abstract: The present paper describes the synthesis, anti-plasmodial and cytotoxic evaluation of 7-
chloroquinoline-based conjugates with isatins/indoles/ nitroimidazoles, obtained via Cu-promoted 1,3-
dipolar cycloadditions. On contemplating SAR of the synthesized series, the inclusion of indole and
nitroimidazole-core improved the anti-plasmodial activities while the isatin seemed to have a lesser
effect. The conjugate with a nitroimidazole-core and hexyl chain length as a spacer between the two
pharmacophores was found to be most potent among the synthesized series and displayed an IC of
5
0
0
.12 M and a selectivity index of 1748.
Keywords: 7-chloroquinoline-based Conjugates, Azide-Alkyne Cyclo-addition, Antiplasmodial
activity, Cytotoxicity
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
1
0.1111/CBDD.13738
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Introduction
Malaria is one of the most devastating and deadliest diseases caused by parasite Plasmodium
falciparum and transmitted via the bite of a female mosquito, anopheles in humans. Even after a
decade of progressive efforts, the disease remains most prevalent and pernicious. According to World
Malaria Report 2018, an estimated 219 million malaria cases were witnessed with 4,35,000 deaths in
2
017 with an increase in 3.5 million cases compared to the previous year in African countries (1). P.
falciparum, is responsible for more than 90% of malaria-related deaths annually despite the
accessibility of an arsenal of drugs (2). In the fight against malaria, the development of 4-
aminoquinoline (CQ) core had a deep impact because of its rapid onset of action, good tolerability,
limited toxicity and low cost (3). However, the emergence of CQ-resistance in the 1960s paved the
way for the development of Artemisinin Combination Therapy (ACT), worldwide (4,5).
Unfortunately, the development of resistance to ACTs in Southeast Asia along with the lack of
effective vaccine has called for urgent efforts to enrich the chemical libraries with new scaffolds with
potent anti-plasmodial activities and low incidence of resistance (6).
Literary survey on 4-aminoquinolines suggested the fact that the CQ-resistance is compound
specific and a large number of 4-aminoquinoline derivatives developed via synthetic modification of
CQ-side chain have shown improved anti-plasmodial activities against drug resistant strains (7,8). 4-
aminoquinoline-hybridization involving conjugation of various pharmacophores with 4-
aminoquinoline is one of the emerging strategy for the development of new molecular frameworks
with promising anti-plasmodial potential (9,10).
Indole, a five-membered pyrrole ring fused with benzene ring, is considered as promising drug
candidate due to diverse biological properties that include anti-inflammatory (11), anti-HIV (12), anti-
tubercular (13), anti-malarial (14), anti-convulsant (15), anti-diabetic (16), anti-microbial (17), anti-
cancer (18), anti-oxidant (19), and anti-fungal agents (20). Currently, an indole-based compound,
NITD609 (I) has been proposed as a promising candidate against Falciparum malaria and is currently
in Phase II clinical trial (21). Aspidocarpine (II), isolated from trunk bark of Aspidosperma vargasii, is
an indole based plant alkaloid with significant antiplasmodial efficacy against CQ-resistant K1 strain
of P. falciparum (22). Thakur et al has recently disclosed the promising anti-plasmodial activities of a
1
series of N -alkylated-3--C-glycoconjugate-oxopropylidene oxindoles (III) against 3D7 and K1
strains of P. falciparum (23). Recently we have disclosed the synthesis and antiplasmodial activities of
isatin-7-chloroquinoline and 3-hydroxy-oxindole-7-chloroquinoline conjugates. Both have shown good
activities against CQ-resistant W2 strain, while 3-hydroxy-oxindole based conjugate (IV) proved to
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have better anti-plasmodial activities than isatin-analogues (24). Kondaparla et al synthesized a series
of 4-aminoquinoline-imidazole derivatives (V) by utilizing Van Leusen multi-component approach
and assessed for their antiplasmodial activity against CQ-sensitive (3D7) and CQ-resistant (K1)
strains. Some of the potent compounds displayed comparable efficacy against K1 strain of P.
falciparum along high selectivity index (Figure 1) (25). Few representatives of isatin/indole based
molecules with significant antiplasmodial activities are depicted in Figure 1.
Thus, in continuation of our pursuit for synthesizing and evaluating new molecular conjugates
with promising anti-plasmodial potential (26), the present work describes the synthesis of 7-
chloroquinolines linked with different heterocyclic scaffolds viz. isatins, nitroimidazole, and indoles
via Cu-prompted azide-alkyne cycloaddition reaction along with their anti-plasmodial evaluation
against CQ-R W2 strain of P. falciparum as well as cytotoxic studies on mammalian Vero Kidney
cells.
Experimental Section
Materials and Methods
1
Instrumentation: H NMR spectra were recorded in deuterochloroform and dimethylsulfoxide-d with
6
a Jeol 400 (400 MHz) and Bruker 500 (500 MHz) spectrometer using TMS as an internal standard.
Chemical shift values are expressed as parts per million 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 spectra were recorded on Jeol 400
(100MHz) and Bruker 500 (125 MHz) spectrometer in dimethylsulfoxide using TMS as internal
standard. High resolution mass spectra were recorded on Bruker-micrOTOF-Q II spectrometer.
General procedure for the preparation of 7-Chloroquinoline-Isatin/Indole/nitroimidazole
conjugates (7-9): To the well stirred solution of
3
(1 mmol) and propargylated
isatin/indole/nitroimidazole 4-6 (1 mmol) in 20 mL EtOH:H O (9:1) mixture was added in succession
2
copper sulphate (0.055 mmol) and sodium ascorbate (0.143 mmol). The reaction mixture was stirred
for 7-8 h at room temperature. After completion of the reaction, as monitored by TLC, water and ethyl
acetate were added in the reaction mixture and organic layer was extracted and combined, dried over
sodium sulphate and concentrated under reduced pressure to result in the isolation of the crude
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product. The pure product was obtained via recrystallization using chloroform: methanol (90:10)
mixture.
Biological evaluations
Methods for assessment of anti-malarial activity of test compounds
The W2 strain of P. falciparum was cultured at 37°C in human red blood cells at 2% hematocrit in
medium RPMI-1640 supplemented with 0.5% Albumax®, 100 uM hypoxanthine, 2 M L-
glutamine and 25 M HEPES pH 7.4 under atmosphere of 3% O , 5% CO balance nitrogen. The
2
2
parasites were synchronized with 5% D-sorbitol at ring stage (27) and incubated with different
concentrations of compounds for 48 h. The compounds were added from DMSO stocks; 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 was fixed for
4
8 h by adding equal volume of 2% formaldehyde in PBS, pH 7.4, at room temperature. Fixed
parasites were then transferred to 0.1% Triton X-100 in PBS containing 100 M ammonium
chloride and 1 nM YOYO-1 dye (Molecular Probes). Parasitemia was determined from dot plots
(forward scatter vs. fluorescence) acquired on a FACSort flow cytometer using Cell Quest
software (Beckton Dickinson). IC values for growth inhibition were determined from plots of
5
0
percent control parasitemia over inhibitor concentration using the Prism 3.0 program, (GraphPad
Software), with data from duplicate experiments fitted by non linear regression (28).
Method for assessment of in vitro cytotoxicity of test compounds
Cell viability was determined using Vero cells (ATCC, Sigma,Germany) grown in RPMI medium
(Gibco, USA), supplemented with 10% decomplemented fetal calf serum, under a 5% CO2
atmosphere. Cells were seeded in 96-well plates at a density of 2104 cells per well in 160 mL
medium and incubated overnight at 37°C to allow cells to adhere. Compounds (dissolved in
DMSO) were freshly diluted to appropriate concentrations in RPMI, so as to allow addition of 20
mL volumes of the diluted compounds to the cells that resulted in final compound concentrations
ranging from 100 g/mL to 0.78 g/mL. The maximum final concentration of DMSO was 1%
(v/v) and nocytotoxic effect of DMSO was observed at this concentration. After 24 h incubation at
3
7°C, 20 mL of 1 g/mL resazurin (Sigma, Germany) was added to each well and the cells were
incubated for an additional 3 hours at 37°C. Fluorescence was measured in a Polarstar Omega
fluorometer using appropriate filters (540 nm excitation and 590 nm emission wave length).
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Percentage survival was determined by dividing fluorescence values obtained in the compound
containing wells by values obtained for control wells containing cells incubated with a dilution
series of DMSO and multiplying this value by 100. SDS (20%) was included as a positive control.
Cytotoxic evaluation was completed twice, with each compound tested induplicate. The IC is
5
0
defined as the lowest concentration of compound tested at which exactly 50% cell viability was
observed and was calculated using a non-linear regression curve using Graphpad Prism 5. The SI
values were determined as a function of IC /MIC .
5
0
99
Result and Discussions
Chemistry
The synthesis of desired conjugates involved an initial preparation of precursor 3 obtained by reacting
,7-dichloroquinoline 1 with an excess of different diamino alkanes at 120°C for 10-12 h to yield
4
intermediate 4-amino-7-chloroquinolines 2. The coupling of 2 with azido acetic acid using HOBt-EDC
afforded 2-azido-N-(2-((7-chloroquinolin-4-yl)amino)alkyl)acetamide 3 (Scheme 1).
Another precursor viz. N-propargylated isatins/indole (4/5) was prepared via base assisted
propargylation of isatin/indole with propargyl bromide in the presence of sodium hydride in DMF for
2
-3 h at 60°C. Similar base-promoted (K CO ) propargylation of 2-methyl-4/5-nitroimidazole yielded
2 3
N-propargylated-2-methyl-4-nitroimidazole 6 as shown in Scheme 2 (29).
Cu-promoted azide-alkyne cycloaddition reaction of
3
with N-propargylated
isatin/indole/nitroimidazole (4/5/6) in EtOH:H O mixture for 7 h afforded 1H-1,2,3-triazole linked 4-
2
aminoquinoline-isatin/indole/imidazole conjugates 7-9 (Scheme 3).
The structures to the conjugates were assigned on the basis of spectral data and analytical
+
evidence. The compound 9b, for example, showed a molecular ion peak M at 483.1545 in its High
1
Resolution Mass Spectrum (HRMS) while its H NMR spectrum showed the presence of a singlet at

2.37 due to the presence of methyl group, two singlets at  5.08 and 5.33 due to presence of two
methylenes, and characteristic singlets at  8.13 and 8.32 corresponding to a triazole and 4-
nitroimidazole ring protons. Further, the appearance of characteristic signal at  165.8 due to carbonyl
1
3
carbon along with the required number of carbons in its C NMR spectrum confirmed the assigned
structure.
In vitro anti-malarial and cytotoxic evaluation
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The synthesized compounds were assessed for their anti-plasmodial activities against CQ-resistant W2
strain of P. falciparum and results are enlisted in Table 1. As evident, most of the synthesized
compounds have shown activities in the sub-micromolar concentration and exhibit an interesting
Structure Activity Relationship (SAR). Among 7-chloroquinoline-isatin conjugates 7a-c, the
compounds displayed activities in the range of 0.33-1.25 M. The conjugates with shorter alkyl chain
length 7a and 7c, having ethyl and butyl as spacer, exhibited better anti-plasmodial activities compared
to the ones with longer chain lengths i.e. 7d (hexyl) and 7e (octyl); exception being 7b (propyl) which
exhibited lowest anti-plasmodial activity among the series. SAR analysis among 7-chloroquinoline-
indole conjugates 8a-e revealed reversal in the anti-plasmodial data with conjugates exhibiting good
activities at longer alkyl chain length as evident by 8d (hexyl) and 8e (octyl) with IC50s of 0.18 and
0
.36 M, respectively. The inclusion of nitroimidazole on the side chain of 7-chloroquinoline-core
substantially enhanced the activity of the synthesized conjugates as shown by 9b, 9d and 9e. SAR
analysis of this series revealed that the observed activities were independent upon the length of alkyl
chain used as spacer and the activities are in the range of 0.12-0.26 M. A generalized SAR of the
synthesized conjugates is depicted in Figure 2.
In order to confirm if the observed activities of the synthesized conjugates were because of
their inherent anti-plasmodial activities, the cytotoxicity of most promising compounds viz. 8d, 9b, 9d,
9
e was determined on mammalian Vero Kidney cells (Table 2). As evident, the promising conjugates
were proved to be non-cytotoxic against mammalian Vero cells.
Conclusion
A series of 7-chloroquinoline-based conjugates with isatins/indoles/ nitroimidazoles were synthesized
via Cu-promoted azide-alkyne cycloaddition reactions. The anti-plasmodial activities of the
synthesized conjugates on the CQ-resistant W2 strain of P. falciparum indicated modest to good
activities. Structure-Activity Relationship studies revealed the dependence of anti-plasmodial activities
on alkyl chain length among quinoline-isatins and quinoline-indoles while no such role was noticed
among quinoline-indoles. The most potent and non-cytotoxic conjugate 9d with an optimum
combination of a hexyl chain length as spacer along with a nitroimidazole pharmacophore proved to be
most potent and non-cytotoxic and displayed an IC value of 0.12 M along with a selectivity index
5
0
of 1748.
Conflicts of interest
There are no conflicts to declare.
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Acknowledgment:
Financial assistance from SERB (Science and Engineering Research Board) under Start-Up Research
grant Scheme No. YSS/2015/000879/CS (RR) is gratefully acknowledged.
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Data Availability Statement
1
13
The details of characterisation of compounds 7a-7e, 8a-8e and 9a-9e, along with scanned H and C
spectra of 7c, 8c, 8d, 8e and 9b are given in supplementary file.
CAPTIONS:
Figure 1: Structures of some Isatin/Indole based antiplasmodials
Figure 2: Generalized SAR of the synthesized conjugates
Scheme 1. Synthesis of 2-azido-N-(2-((7-chloroquinolin-4-yl)amino)alkyl)acetamide 3
Scheme 2. Synthesis of precursor’s viz. N-propargylated isatins/indole (4/5) and N-propargylated-2-
methyl-4-nitroimidazole 6
Scheme 3. Synthesis of desired conjugates viz. 1H-1,2,3-triazole linked 4-aminoquinoline based
isatin/indole/imidazole conjugates 7-9
Table 1. Anti-malarial activities of synthesized compounds against CQ-R W2 strains of P. falciparum
Table 2. Cytotoxicity and Selectivity index of potent conjugates
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N
OH
N
Cl
H
O
F
NH
N
H
MeO
N
Cl
H
N
H
OH COCH3
O
N
NITD609
IC50 = 0.7 nM
Pf3D7 strain
Aspidocarpine (Plant alkaloid)
IC50 = 19 nM
N
O
N
H
Pf3K1 strain
Br
N
(I)
NH
N
(II)
N
N
Cl
HN
N
N
3
-Hydroxy-oxindole-7-chloroquinoline
O
N
Cl
conjugates
IC50 = 69 nM
(IV)
N
O
N
O
O
O
N
Imidazole-7-chloroquinoline derivative
IC50 = 290 nM
Glucoconjugated-
oxopropylidene
oxindoles
IC50 = 1.61 M
Pf3K1 strain
O
Pf3K1 strain
(V)
(III)
Figure 1: Structures of some Isatin/Indole based antiplasmodials
N N
Spacer
N
HN
N
O
Cl
Isatins, modest activity at n=1,3
Indoles, good activity at n= 5,7
Nitroimidazole, promising activity
independent of chain length
Figure 2: Generalized SAR of the synthesized conjugates
H
N
NH2
O
Cl
N
HN
N
HN
N
N3
n
n
N3
O
HO
(a)
(b)
Cl
Cl
Cl
3
1
2
n = 1,2,3,5,7
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Scheme 1: Reagents and conditions: (a) Diamines, 120 °C, 10-12 h; (b) HOBt, EDC, Et N, Dry DCM,
3
rt, 10 h
O
O
Br
O
O
(a)
N
N
H
Isatin
4
Br
Br
N
(a)
(b)
N
H
5
Indole
N
NO2
N
NO2
H C
3
N
H
N
H C
3
6
Nitroimidazole
Scheme 2: Reagents and conditions: (a) NaH, Dry DMF, rt-60°C, 2-3 h (b) K CO , Dry DMF, rt
2
3
O
H
N
O
N
N
HN
N
n
N
N O
O
4
O
N
(a)
Cl
7
H
H
N
N
N
N
HN
N
N3
N
HN
n
n
N
O
5
O
N
(
a)
Cl
Cl
N
n
3
8
n = 1,2,3,5,7
O2N
N
H
CH3
N
N
N
N
HN
N
N
O
NO2
6
N
N
Cl
(
a)
9
H3C
Scheme 3: Reagents and conditions: (a) CuSO , Sodium Ascorbate, EtOH:H O, rt, 7-8 h
4
2
Table 1: Anti-malarial activities of synthesized compounds against CQ-R W2 strains of P. falciparum
Compound
n
IC (M)±SD
5
0
7
7
7
7
7
a
b
c
1
2
3
5
7
0.33± 0.00
1.25± 0.03
0.39± 0.01
1.09± 0.08
1.12± 0.06
d
e
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8
8
8
8
8
9
9
9
9
9
a
b
c
1
2
3
5
7
1
2
3
5
7
1.24± 0.12
1.20± 0.07
0.29± 0.13
0.18± 0.04
0.36± 0.01
ND
d
e
a
b
c
0.18± 0.02
ND
d
e
0.12± 0.01
0.26± 0.01
0.07± 0.00
CQ
Table 2: Cytotoxicity and Selectivity index of potent conjugates
Compound
IC (M)
IC (M)
SI
5
0
50
(Cytotoxicity)
(W2 strain P. falciparum)
8d
9b
9d
9e
260.5
141.5
209.8
151.3
0.18
0.18
0.12
0.26
1447.2
786.11
1748.33
581.92
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