Synthesis, in vitro and in silico antimalarial activity of 7-chloroquinoline and 4H-chromene conjugates

Article abstract of DOI:10.1016/j.bmcl.2015.08.030

A new series of chloroquinoline-4H-chromene conjugates incorporating piperizine or azipane tethers were synthesized and their anti-malarial activity were evaluated against two Plasmodium falciparum strains namely 3D7 chloroquine sensitive (CQS) and K1 chloroquine resistant (CQR). Chloroquine was used as the standard and also reference for comparison. The conjugates exhibit intense UV absorption with λ_max located at 342 nm (log ε = 4.0), 254 nm (log ε = 4.2), 223 nm (log ε = 4.4) which can be used to spectrometrically track the molecules even in trace amounts. Among all the synthetic compounds, two molecules namely 6-nitro and N-piperazine groups incorporated 7d and 6-chloro and N-azapane incorporated 15b chloroquinoline-4H-chromene conjugates showed significant anti-malarial activity against two strains (3D7 and K1) of P. falciparum. These values are lesser than the values of standard antimalarial compound. Molecular docking results suggested that these two compounds showing strong binding affinity with P. falciparum lactate dehydrogenase (PfLDH) and also they occupy the co-factor position which indicated that they could be the potent inhibitors for dreadful disease malaria and specifically attack the glycolytic pathway in parasite for energy production.

Full text of DOI:10.1016/j.bmcl.2015.08.030

Accepted Manuscript
Synthesis, in vitro and in silico antimalarial activity of 7-chloroquinoline and
4H-chromene conjugates
A. Parthiban, J. Muthukumaran, Ashan Manhas, Kumkum Srivastava, R.
Krishna, H. Surya Prakash Rao
PII:
S0960-894X(15)00865-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.08.030
BMCL 23024
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 May 2015
23 July 2015
11 August 2015
Please cite this article as: Parthiban, A., Muthukumaran, J., Manhas, A., Srivastava, K., Krishna, R., Surya Prakash
Rao, H., Synthesis, in vitro and in silico antimalarial activity of 7-chloroquinoline and 4H-chromene conjugates,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.08.030
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A. Parthibana, J. Muthukumaranb, Ashan Manhasc, Kumkum Srivastavac,*R. Krishnad,*and H. Surya
Prakash Raoa,*
*Corresponding authors
aDepartment of Chemistry, School of Physical, Chemical and Applied Sciences, Pondicherry
University, Puducherry – 605 014, India. H. Surya Prakash Rao: Email: hspr.che@pondiuni.edu.in,
hspr@yahoo.com, Tel: +91-413-2654411, Fax: +91-413-2656230.
bUCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
cParasitology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India. Kumkum
Srivastava: Email: kumkum_srivastava@cdri.res.in, Tel: +91 9450917760.
dCentre for Bioinformatics, School of Life Sciences, Pondicherry University, Puducherry – 605 014,
India. R. Krishna: Email: krishstrucbio@gmail.com, Tel: +91 413 2654580, Fax: +91-413 2655211.

Synthesis, in vitro and in silico antimalarial activity of 7-chloroquinoline and 4H-
chromene conjugates
A. Parthibana, J. Muthukumaranb, Ashan Manhasc, Kumkum Srivastavad,*R. Krishnae,*and H.
Surya Prakash Raoa,*
*Corresponding authors
aH. Surya Prakash Rao, Department of Chemistry, School of Physical, Chemical and Applied
Sciences, Pondicherry University, Puducherry – 605 014, India, Email:
hspr.che@pondiuni.edu.in, hspr@yahoo.com, Tel: +91-413-2654411, Fax: +91-413-
2656230.
bUCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, Caparica 2829-516, Portugal.
cParasitology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India.
dR. Krishna, Centre for Bioinformatics, School of Life Sciences, Pondicherry University,
Puducherry – 605 014, India, Email: krishstrucbio@gmail.com, Tel: +91 413 2654580, Fax:
+91-413 2655211.
1

Abstract
A new series of chloroquinoline-4H-chromene conjugates incorporating piperizine or
azipane tethers were synthesized and their anti-malarial activity were evaluated against two
Plasmodium falciparum strains namely 3D7 chloroquine sensitive (CQS) and K1 chloroquine
resistant (CQR). Chloroquine was used as the standard and also reference for comparison.
The conjugates exhibit intense UV absorption with λmax located at 342 nm (log ε = 4.0), 254
nm (log ε = 4.2), 223 nm (log ε = 4.4) which can be used to spectrometrically track the
molecules even in trace amounts. Among all the synthetic compounds, two molecules namely
6-nitro and N-piperazine groups incorporated 7d and 6-chloro and N-azapane incorporated
15b chloroquinoline-4H-chromene conjugates showed significant anti-malarial activity
against two strains (3D7 and K1) of P. falciparum. These values are lesser than the values of
standard antimalarial compound. Molecular docking results suggested that these two
compounds showing strong binding affinity with P. falciparum Lactate dehydrogenase
(PfLDH) and also they occupy the co-factor position which indicated that they could be the
potent inhibitors for dreadful disease malaria and specifically attack the glycolytic pathway in
parasite for energy production.
Key words: Chloroquinoline-4H-chromene conjugates, in vitro antimalarial activity and in
silico analysis.
Malaria is one of the most infectious and parasitic disease affecting about 40% of the
human population, particularly those living in tropical and poorer countries.1 According to
World Health Organization (WHO) World Malaria Report 2014 out of an estimated 198
million cases nearly 584,000 malaria deaths occurred in 2013.2 Because of the devastating
effects on human population, WHO rates malaria as one of the top three of the infectious
2

diseases.3 Malarial disease is caused by any one of the species of the Plasmodium parasite,
namely P. falciparum, P. malariae, P. ovale, P. vivax and P.knowlesi.4 Among all the
species, P. falciparum is the most severe pathogenic.5
Treatment of malaria began in earlier days by administering decoction made from
cinchona (Cinchona rubra, Rubiaceae) bark. Subsequently, quinine 1 was isolated from this
tree and shown to have anti-malarial properties.6 Medicinal chemists developed simpler
synthetic analogues of quinine 1 like pamaquine 2, mepacrine 3, chloroquine 4, amodiaquine
5 and piperaquine 6 (Fig. 1), all of which had pharmacopore of quinine, but did not have
multiple stereogenic centers. Among these, chloroquine 4 is the most efficient and important
drug and served humanity for over five decades.7 Although in recent years artemisinin-based
combination therapy (ACT) has been employed for treatment of most cases of malaria, owing
to being inexpensive and efficacious, chloroquine 4 is still used as a first-line drug in some
Central American and Caribbean countries.8 Moreover it remains as a first-line drug for
treatment of malaria caused by P. vivax.9 Extensive use of chloroquine over the years lead to
emergence of the drug resistance in parasite and this forced medicinal chemists to look for
alternate molecules.10 Since, artemisinin and its close analogs have the problem of low bio-
availability, tedious isolation from natural sources or difficult to synthesize in industrial scale,
reliance of chloroquine continues with room for further development in its structural
features.11
3

Figure 1. Structure of quinoline based antimalarial drugs. Quinine 1, pamaquine 2,
mepacrine 3, chloroquine 4, amodiaquine 5 and piperaquine 6.
One of the major issues with chloroquine and its analogues is the hydrophobic nature
of the molecule, which allows it to cross the blood brain barrier (BBB). To restrict crossing of
BBB it is desirable to attach a polar group to the chloroquine pharmacophore. Our experience
in synthesis of 4-aryl-4H-chromenes12 prompted us link 6-chloroquineand 4H-chromene
through electron rich aromatic ring tether to generate chloroquinoline-4H-chromene
conjugates 7 (Fig. 2). The chloroquinoline-4H-chromene conjugates 7 possess medicinally
important rigid and nearly flat 4H-chromene framework with hydrogen bond acceptor C1
oxygen, hydrogen bond donor and acceptor C2-amino group and highly polarized C3 nitro
group and extremely stable push-pull alkene located between C2 and C3.13 For structural
diversity there are several possibilities of installation of different substitution in the aromatic
ring in the 4H-chromene. There are also possibilities for changes in the N-phenylpiperazine
moiety in 7. The 4Hchromene-chloroquinoline conjugate 7 possess highly polarized
nitroketene-N,O-acetal substructure which is not expected to allow 7 to cross BBB.
Moreover, nitroketene-N,O-acetal moiety in 7 is expected to be responsible for intense UV
absorption with λmax at about 345 nm and 255 nm (log ε = 4.0) which should allow detection
4

of the sample present in trace amounts. We report, herein, synthesis of a small library of
chloroquinoline-4H-chromene conjugates 7 and their antimalarial activity was tested against
drug resistant strains of P. falciparum. Furthermore, we report on in silico studies performed
with PfLDH for all synthetic compounds to delineate structure activity relationships. The
experimental part of the chemistry, in vitro and in silico studies are given in supplementary
section.
Figure 2. Structures of present target chloroquinoline-4H-chromene conjugates 7.
In the first step of the two-step sequence towards 7a, we subjected commercially
available 4,7-dichloroquinoline 8 to reaction with N-phenylpiperizine 9 in EtOH reflux. In
this reaction, regio-selective nucleophilic displacement of C(4)Cl in 8 took place readily to
provide N-phenylpiperazine substituted chloroquinoline 10 in near quantitative yield (Scheme
1). The nucleophilic displacement of C(4)SMe in 11 took place readily on treatment with 10
in ethanol reflux to provide chloroquinoline-4-aryl-4H-chromene conjugate in near
quantitative yield. Mechanistically, the displacement of SMe in 11 went through aromatic
electrophilic substitution on electron rich and sterically less crowded C(4) of N-
phenylpiperizine moiety in 10.14 The product 7a generated as faintly yellow colored solid
precipitated out of the reaction and thus no work-up and column chromatographic
5

purification was necessary. The UV-Vis spectrum of 7a displayed three intense bands with
max located at 342 nm (log ε = 4.0), 254 nm (log ε = 4.2), 223 nm (log ε = 4.4). Of the three
λ
one with λmax at 223 nm was due to 6-chloroquinoline chromophore since chloroquine
displayed λmax at 228 nm (log ε = 3.8). The other two high intensity bands at 342 nm (log ε =
4.0) and 254 nm (log ε = 4.2) are clearly due to nitroketene-N,O-acetal moiety and these
bands can be used to track the molecule 7a. The 1H NMR spectrum of 7a exhibited a signal
for C(4)H of 4-aryl-4H-chromene portion at 5.40 ppm. The doublet at 3.26 ppm for NH-Me
and a broad singlet at 10.46 ppm were the other characteristic peaks for the 4-aryl-4H-
chromene portion. Two types of CH2 protons of the piperazinyl moiety gave rise to two
triplets located at 3.33 and 3.36 ppm. Two doublets located at 8.72 and 7.96 ppm were due to
C(2)H and C(8)H of quinoline moiety. The 13C NMR spectrum of 7 showed four signals at δ
27.9, 40.9, 49.2 and 52.2 ppm in the aliphatic region which were assigned to N-Me, C4, two
methylene carbons of piperazine ring.
Scheme 1. Synthesis of chloroquinoline 4-aryl-4H-chromene conjugate 7a.
After having established conditions for the synthesis of chloroquinoline-4H-chromene
conjugate 7a the sequence was repeated for synthesis of twelve conjugates 7b-m by varying
substitutions in the aromatic ring and the alkyl group on C(2) nitrogen of the 4H-chromene
6

(Scheme 2). Spectral (UV-Vis, IR, 1H NMR, 13C NMR and DEPT) and analytical (ESI-MS
HRMS) data of the chloroquinoline conjugates 7b-m agreed well with the assigned
structures. Surprisingly the reaction of 10 did not take place with 4H-chromene having C(2)N
phenyl group. It is possible that due to electron withdrawing effect of the phenyl group makes
C(2) nitrogen less electron donating for ejection of C(4)SMe group.
Scheme 2: Synthesis of chloroquinolin-4-ylpiperazin substituted 4H-chromene conjugates
7a-m.
Next, our attention was focused on changing six member piperazinyl ring in 10 to
seven-member 1,4-diazepane ring (homopiperazine) 14 to evaluate if the ring size and
concomitant change of orientation would affect antimalarial activity. Synthesis of 4-(4-(4-(7-
7

chloroquinolin-4-yl)-1,4-diazepan-1-yl)phenyl)-N-methyl-3-nitro-4H-chromen-2-amine
derivatives 15a-c is given in Schemes 3 and 4.
Scheme 3. Synthesis 7-chloro-4-(4-phenyl-1,4-diazepan-1-yl)quinoline 14.
The synthesis started with commercially available 4,7-dichloroquinoline 8 and 1,4-
diazapine (homopiperazine) 12. The nuclophilic displacement of C(4)Cl in 8 with secondary
amine in 12 worked well to provide 13. The Buchwald–Hartwigamination15 with
iodobenzene in the presence of catalytic amount of copper (I) iodide afforded N-
phenyldiazepan substituted chloroquinoline 14 in good yield (Scheme 3).
NHMe
O
NO
2
R
N
N
N
SMe
N
R
NO
2
Ethanol reflux
5-15 h, 80-85%
N
O
11a-c
NHMe
Cl
Cl
N
14
15a-c
11a: R = H;
11b: R = Cl;
11c: R = Br.
15a: R = H (5 h, 85%);
15b: R = Cl (12 h, 80%);
15c: R = Br (15 h, 84%).
Scheme 4. Synthesis of chloroquinoline-4H-chromene conjugates 15a-c.
The reaction of 4-methylsulfanyl-4H-chromene derivatives 11a-c and N-
phenyldiazepan substituted chloroquinoline14 furnished diazepan incorporated
8

chloroquinoline-4H-chromene conjugates 15a-c in high yields (Scheme 4). The structures of
chloroquinoline-conjugates 15a-c were confirmed on the basis of spectral (UV-Vis, IR, 1H
NMR, 13C NMR and DEPT) and analytical (ESI-MS HRMS) data and they compared well
with the those having piperazine moiety 7a-m.The structures of chloroquinoline-4H-
chromene conjugates 7a-m and 15a-c prepared in present study along with yield of the
product obtained are gathered in Fig. 3.
Figure 3. Library synthesis of chloroquinoline-4H-chromene conjugates 7a-m and 15a-c.
After the successful synthesis and characterization, the chloroquinoline-4H-chromene
conjugates (7a-m and15a-c) were evaluated for their anti-malarial activity against two
9

pathogenic strains of P. falciparum namely chloroquine sensitive (CQS; 3D7) and
chloroquinine resistant (CQR; K1). Chloroquine was used as the standard and reference drug
for comparison. Table 1 shows 50% inhibitory concentrations (IC50) against 3D7 & K1
parasite strains, 50% cytotoxic concentration (CC50) against Vero cell line (C1008; monkey
kidney fibroblast) and selectivity index SI (SI = CC50/IC50) of all the newly made
chloroquinoline-4H-chromene conjugates (7a-m and 15a-c).
Table 1. In vitro antimalarial activity and cytotoxicity of the chloroquinoline-4H-chromene
conjugates 7a-m and 15a-c against 3D7 and K1 strains of P. falciparum.
Compounds
IC50 (µM)
[3D7,CQS]
1.11
IC50 (µM)
[K1,CQR]
3.38
CC50 (µM)
SI
SI
[3D7,CQS] [K1, CQR]
7a
7b
7c
>200
74.46
88.56
31.87
81.92
40.38
21.84
97.77
>200
>180.2
54.3
47.1
51.4
33.0
13.7
8.8
>59.2
24.9
50.6
17.9
19.3
13.6
9.1
1.37
2.99
1.88
1.75
7d
7e
0.62
1.78
2.48
4.25
7f
2.93
2.95
7g
7h
7i
2.47
2.40
3.92
1.45
24.9
~40.0
35.8
13.3
39.6
34.6
<18.3
265.4
67.4
>68.2
96.5
13.3
75.5
29.9
<18.3
155.2
>5.0
2.93
7j
2.34
0.87
83.98
66.45
131.34
106.91
91.41
76.97
7k
7l
>5.0
>5.0
3.31
1.74
7m
15a
15b
3.09
3.57
>5.0
>5.0
0.29
0.496
10

15c
10
2.24
2.69
3.98
1.27
2.49
4.47
>200
178.57
147.93
125
89.3
66.3
157.5
2.5
14
37.1
33.1
Chloroquine 0.005 0.0009 0.254 0.020
25000
492.13
CQS- chloroquine sensitive and CQR - chloroquine resistant
CC50- 50% Cytotoxic concentration, SI -selectivity index (CC50/ IC50).
Out of thirteen conjugates (7a-7m) which incorporated N-phenylpiperazine tether, one
(7d) with C(6)-nitro group exhibited IC50 value 0.62 µM against 3D7 (CQS) strain and 1.78
µM in case of K1 (CQR) strain. Interestingly, the conjugate 15b which has seven-member
1,4-diazepane ring in the tether was most potent in the set of all sixteen compounds. It
displayed significant anti-malarial activity with the IC50 values of 0.29 µM against 3D7 strain
and 0.49 µM against K1 strain. This result indicates that bent structure and chloro-group in
4H-portion were responsible for enhanced activity. Although none of the newly synthesized
compounds exhibited antimalarial activity better than chloroquine, they have the advantage of
being polar and resistant to crossing bbb.
PfLDH is an important enzyme that involves the inter-conversion of pyruvate to
lactate and vice-versa.16 For the conversion of pyruvate to lactate NADH is employed as a
cofactor, which in turn gets converted into NAD+. Being an essential enzyme for glycolysis
in the parasite, it is a good target for anti-malarial drugs. The amino acid sequences for
human and PfLDH are very different (sequence homology: 30.7 %), hence this implies that
the synthetic compounds specifically interact with glycolytic enzyme of parasite.17
Chloroquine is known to interact with PfLDH effectively through competitive inhibition of
binding of NADH at the active site.18 We reasoned that our newly synthesized molecules
which possess both chloroquine and 4H-chromene portions could bind to the active site more
11

efficiently and effectively. Hence, we have used PfLDH as a drug target for our newly
synthesized compounds and chloroquine was used as a reference. Molecular docking results
suggested that out of sixteen compounds screened, (evaluated independently in both R and S
configuration) and the reference chloroquine (-6.61 kcal/mol), the R form of the conjugates
7d and 15b displayed good docking score (7d -10.91 kcal/mol and 15b -10.96 kcal/mol) and
strong binding affinity with PfLDH. The conjugates occupied the co-factor position at the
binding site which indicated that they could be the competitive inhibitors. In silico interaction
results of 7d and 15b with target enzyme is presented here (Fig. 4) and interactions of the
remaining molecules are given in supplementary information. The ligand 7d interacted with
the target enzyme mainly through hydrogen bonding interaction and hydrophobic contacts.
However, the ligand 15b made the hydrophobic contacts with the target enzyme. The
terminal oxygen atom of nitro group in 7d formed hydrogen bond with Ile31 residue of target
enzyme separated by an ideal distance of 2.94 Å. As 7d, the following amino acid residues
Gly27, Gly29, Met30, Phe52, Asp53, Ile54, Tyr85, Ala98, Gly99, Thr101 and Ile119were
formed significant hydrophobic contacts with the ligand 15b. Aforementioned interactions
are essential to stabilize the ligands in the active site of PfLDH.
12

Figure 4. Orientation of 1,4-Dihydronicotinamide adenine dinucleotide (Co-factor), 7d and
15b with PfLDH (PDB Accession Code: 1LDG). Two-dimensional representation of PfLDH
with ligand 7d and 15b. The three-dimensional representation of protein-ligand complex was
prepared by PyMOL. The LigPlot shows the amino acid residues of target enzyme around the
ligand molecule with hydrogen bond and hydrophobic contacts.
13

In conclusion, we have synthesized sixteen derivatives of chloroquinoline-4H-
chromene conjugates and evaluated in vitro antimalarial activity against P. falciparum
strains. Of all conjugates, 6-nitro and N-piperazine incorporated 7d and 6-chloro-N-azapane
incorporated 15b chloroquinoline-4H-chromene conjugates showed good anti-malarial
activity against P. falciparum strains (3D7 and K1) and their efficiency was almost equal to
that of chloroquine. Newly prepared molecules have the added advantage of possessing polar
4H-chromene to make the drug candidates brain impregnable. The conjugates display strong
bands at λmax 345, 254 and 223 nm which can be the used for their detection even in trace
quantities. The theoretical calculations corroborated experimental results. Overall, the
molecular docking results, along with experimental data, suggest that two molecules (7d and
15b) are potent inhibitors for malarial parasite by attacking the parasite specific glycolytic
pathway.
Acknowledgement
AP thanks Pondicherry University for fellowship. JM thanks UCIBIO
(UID/Multi/04378/2013), Fundação para a Ciência e a Tecnologia (FCT) (PTDC/QUI-
BIQ/117799/2010), (SFRH/BPD/97719/2013) Portugal for providing support to carry out in
silico part of the research work. RK thank the Centre for Bioinformatics (Funded by
Department of Biotechnology and Department of Information Technology, New Delhi,
India), Pondicherry University, India. AP thanks Central Instrumentation Facility,
Pondicherry University for providing the spectroscopic instruments to carry out the research
work. We thank Open Source Drug Discovery (OSDD) program of CDRI, Lucknow for
testing antimalarial activity. HSPR thanks the UGC for financial support under major
research project (MRP) and the Special Assistance Program (UGC SAP). He also thanks the
Department of Science and Technology (DST) for the Fund for Improvement of S&T
Infrastructure in Higher Educational Institutions (FIST).
14

Graphical Abstract
Synthesis, in vitro and in silico antimalarial activity of 7-chloroquinoline and 4H-
chromene conjugates
A. Parthiban1, J. Muthukumaran2, Ashan Manhas3, Kumkum Srivastava3 * R. Krishna4*and H.
Surya Prakash Rao1,*
IC50 values: 7d: 0.62 µM CQS strain & 1.78 µM CQR strain and 15b: 0.29 µM CQS strain &
0.496 µM CQR strain
15

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