Docking, synthesis and antimalarial activity of novel 4-anilinoquinoline derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.03.005

A series of 4-anilinoquinoline triazine derivatives were designed, synthesized and screened for in vivo antimalarial activity against a chloroquine-sensitive strain of Plasmodium berghei. The compounds were further subjected to in vitro antimalarial activity against chloroquine-resistant W2 strain of Plasmodium falciparum and β-haematin inhibition studies. All the compounds exhibited in vivo antimalarial activity better than that shown by the standard drug, chloroquine. Twelve out of fifteen compounds showed better inhibition than that of chloroquine against chloroquine-resistant W2 strain of Plasmodium falciparum. Ten compounds showed β-haematin inhibition, better than that of chloroquine, with IC₅₀ values in the range of 18–25?μM. One compound, 3k, was found to be better than artemisinin against W2 strain of Plasmodium falciparum and also displayed the best β-haematin inhibitory activity, thereby becoming eligible to be explored as a potential lead for antimalarial chemotherapy.

Full text of DOI:10.1016/j.bmcl.2017.03.005

Accepted Manuscript
Docking, synthesis and antimalarial activity of novel 4-anilinoquinoline deriv-
atives
Shilpa Vijayaraghavan, Supriya Mahajan
PII:
S0960-894X(17)30226-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.005
BMCL 24757
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
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Revised Date:
Accepted Date:
30 September 2016
27 February 2017
2 March 2017
Please cite this article as: Vijayaraghavan, S., Mahajan, S., Docking, synthesis and antimalarial activity of novel 4-
anilinoquinoline derivatives, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.03.005
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Docking, synthesis and antimalarial activity of novel 4-anilinoquinoline derivatives
Shilpa Vijayaraghavana, * and Supriya Mahajana
a, *Department of Pharmaceutical Chemistry, C. U. Shah College of Pharmacy, S. N. D. T.
Women’s University, Sir Vithaldas Vidyavihar, Santacruz (W), Mumbai – 400049, India
Author for correspondence: Tel.: +919819480211
Email: shilpa_vijayaraghavan@yahoo.co.in
1

Abstract
A series of 4-anilinoquinoline triazine derivatives were designed, synthesized and screened for in
vivo antimalarial activity against a chloroquine-sensitive strain of Plasmodium berghei. The
compounds were further subjected to in vitro antimalarial activity against chloroquine-resistant
W2 strain of Plasmodium falciparum and β-haematin inhibition studies. All the compounds
exhibited in vivo antimalarial activity better than that shown by the standard drug, chloroquine.
Twelve out of fifteen compounds showed better inhibition than that of chloroquine against
chloroquine-resistant W2 strain of Plasmodium falciparum. Ten compounds showed β-haematin
inhibition, better than that of chloroquine, with IC50 values in the range of 18 – 25 µM. One
compound, 3k, was found to be better than artemisinin against W2 strain of Plasmodium
falciparum and also displayed the best β-haematin inhibitory activity, thereby becoming eligible
to be explored as a potential lead for antimalarial chemotherapy.
Keywords: Malaria, Plasmodium falciparum, falcipain-2, cysteine protease, 4-anilinoquinoline
triazine, β-haematin.
2

Malaria still remains one of the major threats in the developing countries. Globally, an
estimated 3.2 billion people in 97 countries and territories are at risk of being infected with
malaria and 1.2 billion are at high risk (>1 in 1000 chance of getting malaria in a year).
According to the latest estimates by WHO, released in September 2015 and updated on October
2015 (WHO World Malaria Report, 2014), 214 million cases of malaria occurred globally in
2014 and the disease led to 4,80,000 deaths. Approximately, 78% deaths were mostly in children
younger than five years old. In India, around 4,00,000 malarial deaths were reported in 2013.
Malaria caused by Plasmodium falciparum (P. falciparum) is more prevalent than the
other less virulent Plasmodium species, which include Plasmodium vivax (P. vivax), Plasmodium
malariae (P. malariae) and Plasmodium ovale (P. ovale).1 The WHO has advocated the use of
artemisinin and its analogues in combination with 4-aminoquinoline antimalarials like
3
lumefantrine and mefloquine.2, However, artemisinin based combination therapy (ACT)
possesses limitations due to some serious issues like higher cost of treatment, safety in
pregnancy and imbalance in demand over supply.4, 5 Other conventional methods of malaria
treatment and control have been hampered by increasing resistance to all antimalarial drugs and
mosquito insecticides.6, 7 In some parts of the world, ACT is used indiscriminately for self
treatment of suspected uncomplicated malaria, which ultimately has lead to the emergence of
resistance to artemisinin derivatives in some of the Southeast Asian countries namely, Laos,
Cambodia, Myanmar, Vietnam and Thailand.8 This resistance has the potential to continue to
develop and spread, subsequently making malaria chemotherapy more difficult. With the
absence of an effective vaccine9 and also, in view of the widespread of resistant strains, there is
an urgent need for development of new, cost-effective and efficacious antimalarial agents with
low potential of triggering resistance.
Since the discovery of natural product quinine, structural modifications of its quinoline
pharmacophore gave rise to the most effective antimalarial agents namely, chloroquine (CQ)
[Figure 1 (a)], amodiaquine (AQ) [Figure 1(b)] and mefloquine [Figure 1 (c)].10, 11 After
chloroquine, compounds such as aminoquinoline derivatives, sulphadoxine, pyrimethamine,
cycloguanil, etc. were developed for the treatment of malaria.12, 13 Although the resistance to
chloroquine and related 4-aminoquinoline antimalarial drugs has already evolved and spread;
designing novel antimalarials based on the quinoline pharmacophore has discrete benefits due to
the exclusive pharmacological effect of 4-aminoquinoline drugs.14 The 1, 3, 5-triazine
derivatives are also quite attractive scaffolds for the production of antimalarial drugs, e.g.,
cycloguanil, chlorcycloguanil, clociguanil and WRA99210.15 Therefore, it was planned to
synthesize the compounds possessing the combination of both 1, 3, 5-triazine and 4-
anilinoquinoline moieties with a view to expect synergistic antimalarial activity and improved
efficacy compared to the traditional marketed antimalarial drugs.
3

CH3
CH3
OH
CH3
N
N
HN
N
HN
N
CH3
CH3
(b)
(a)
Cl
Cl
HN
HO
F
N
F
F
F
F
(c)
F
Figure 1. Structures of (a) chloroquine, (b) amodiaquine and (c) mefloquine.
Even if the novel compounds do exhibit antimalarial activity, it is also essential to
determine their mechanism of action. Chloroquine and other aminoquinoline drugs act by
inhibiting β-haematin, which converts toxic haem into haemozoin in the food vacuole of the
parasite, thereby leading to substantial accumulation of toxic haem, causing the death of the
parasite.16, 17, 18 Therefore, the novel synthesized compounds were evaluated for β-haematin
inhibition activity. Another target, which is new and promising, is malarial parasitic cysteine
protease enzyme. Falcipain-2 (FP-2) is a major cysteine protease that plays a key role in parasite
food assimilation by its ability to degrade haemoglobin. Falcipain-2 has been known to cleave
denatured and native haemoglobin in the acidic food vacuole of the parasite to produce globin
and haem.19 Globin is then degraded to various proteins and subsequently to amino acids, which
are a major requirement for parasite protein synthesis. Inhibition of falcipain-2 will thereby block
the degradation of haemoglobin and consequently, parasite growth and development [20].
Falcipain-2 inhibitors have proved to block parasite development in vitro21, 22 and also in murine
models.23, 24 However, all these inhibitors are derived from peptide analogues25 and show side
effects, display susceptibility to host proteases, have poor pharmacological profiles and low
absorption rates through cell membranes and therefore, have not been further explored and
developed into marketed preparations. Moreover, covalent interactions are formed between the
electrophilic groups, such as aldehydes, nitriles, vinyl sulfones and epoxides with thiolate of the
catalytic cysteine amino acid, thereby leading to increased toxicity of the molecules. Therefore, it
was planned to target the falcipain-2 enzyme by designing non-peptidic molecules in such a way
so that they exhibit antimalarial activity by inhibiting the enzyme through non-covalent binding,
in order to minimize toxicity while retaining the potential for high in vivo and in vitro activity
and selectivity. A library of structures of 4-anilinoquinoline triazine were docked on the X-ray
4

crystallized structure of falcipain-2 enzyme before their synthesis and only those compounds
showing good interactions with the enzyme were taken up for the synthesis.
Docking studies of the compounds on falcipain-2 enzyme26 and the validation of the
docking protocol27 were done prior to their synthesis with a view to study the in silico interaction
of the ligands with the enzyme, giving way to the synthesis of only those best fit lead
compounds, which are proposed to exhibit improved antimalarial activity by the mechanism of
falcipain-2 inhibition. The procedures for docking and validation have been mentioned in the
Supplementary data section. The docking results were expressed in terms of G-Score. Figures 2
and 3 display the images of E-64 and compound 3a, respectively, interacting with the enzyme
falcipain-2.
Figure 2. Chloroquine docked on falcipain-2 enzyme.
Figure 3. Compound 3a docked on falcipain-2 enzyme.
5

After docking a series of 4-anilinoquinoline triazine derivatives on falcipain-2 enzyme,
those compounds displaying G-score more than that shown by E-64 were successfully
synthesized using inexpensive reactants and were produced with good yields of more than 60%.
1
The structures of the compounds were characterized and confirmed by IR, H NMR and mass
spectral analysis (see Supplementary data). Table 2 displays the R and R1 substituents of the
compounds 3a-3n.
Converting the acidic pH of the food vacuole to the basic pH would disrupt the process of
haemoglobin degradation and thereby, improve the activity of the compounds. Therefore,
compounds were synthesized in such a way that the basicity of the increased number of nitrogen
atoms in the derivatives would help in their accumulation within the acidic food vacuole of the
parasite. Linking both the quinoline and 1, 3, 5-triazine moieties with an anilino group at the 4th
position of the quinoline ring improved the hydrophobicity of the molecules, thereby displaying
good hydrophobic interactions with the required amino acid residues of the falcipain-2 enzyme.
Increasing the side chain of the anilino group with aromatic groups and adding secondary amines
to the 1, 3, 5-triazine moiety also showed improvement in the G-score and in the in vivo and in
vitro antimalarial activity.
The fifteen derivatives were synthesized in three steps using nucleophilic aromatic
substitution reaction as in Figure 4. In the first step, first chlorine of cyanuric chloride was
substituted with aryl and alkyl amines by keeping the reaction mixture at 0 ºC for the first 20 min
and then, overnight at room temperature, to yield monosubstituted triazines.28 The second step
involved substitution of chlorine of 4, 7 - dichloroquinoline with paraphenylene diamine at
reflux. The third step included the reflux of the first and second step products to give 4 –
anilinoquinoline triazine derivatives. The physical data are included in the Supplementary data.
R
Cl
N
Cl
N
Cl
N
R1
NHRR1, THF
N
N
N
N
Addition at 00C for 20 min; then overnight
stirring at RT
NH2
Cl
1
Cl
HN
Cl
PPD, p TSA EtOH
reflux for 3 h
Cl
N
N
Cl
R
2
NH
N
N
R1
N
N
HN
N
Cl
THF, DMSO
1 + 2
reflux for 14 h
Cl
3a-3n
Figure 4. Procedure for the synthesis of 4-anilinoquinoline triazine derivatives.
6

Table 1: R and R1 substituents of the titled compounds 3a-3n
R
g
NH
N
N
f
R1
N
N
h
NH
N
i
a
c
Cl
b
e
d
Cl
Compound
3a
R
R1
H
j
n
k
m
l
3b
o
p
s
j
n
r
k
m
q
l
3c
3d
3e
-
N
-
NH
N
H
O
n
CH3
k
m
l
k
3f
H
j
m
7
N
n
O
O

O
3g
3h
-
N
N
-
H3C
H3C
H3C
3i
3j
H3C
H3C
H3C
H3C
H3C
3k
3l
H3C
H3C
H3C
3m
j
n
k
m
l
H3C
j
n
3n
k
m
l
______________________________________________________________________________
Note: The sign shows the position of attachment of the R and R1 groups to the “N” atom of the
triazine ring. Compounds 3c, 3d, 3g and 3h already have the “N” atom in their rings, which is the
the “N” atom of the triazine moiety.
Before subjecting the synthesized compounds to in vivo antimalarial evaluation, they
were observed for their toxic effects by performing acute toxicity studies as per the Organization
for Economic Co-operation and Development (OECD) guidelines for the testing of chemicals,
Test No. 423, 2008. This method helped to determine the lethal dose and whether the synthesized
8

compounds were toxic or not. From this information, the dose of oral administration of
compounds was determined.
The in vivo antimalarial testing of compounds was performed by Peter’s four days test29
on Swiss albino mice using chloroquine-sensitive Plasmodium berghei strain. It is a highly
reliable method as it involves inducing parasitemia in mice, treating them with a single oral
dosage of compounds, continuously for 4 days and observing their blood smears. The per cent
inhibition of parasitemia was calculated in terms of per cent chemosuppression. The results were
subjected to the statistical analysis, calculating mean and standard error of mean (SEM).
In order to determine the IC50 values of inhibition of malaria, the compounds were further
screened for in vitro antimalarial activity against chloroquine resistant W2 strain of P.
falciparum using human erythrocytes30 and β-haematin inhibition31 as per the procedures
reported. The IC50 values obtained were well corroborated with the G-scores and the data
obtained in in vivo studies and this helped to determine the most active compounds displaying
good therapeutic efficacy. It is observed that better the G-scores, better is the in vivo and the in
vitro activities of the compounds.
In the docking study, the derivatives showed good van der Waal (vdw) interaction with
Cys 42, Trp 43, Leu 84, Ile 85, Val 152, Leu 172, Asn 173, His 174 and Ala 175 and formed
hydrogen bond with Trp 206. The docking results revealed that the G-scores of all the 15
compounds, as revealed in Table 2, were found to be more than -6.73 compared to the score of -
6.65 of E-64, -5.78 of chloroquine and -6.70 of artemisinin, which proved that all the synthesized
compounds interacted with falcipain-2 enzyme better than the standard drug in silico.
Table 2: G- score, % chemosuppression and IC50 values of the titled compounds 3a-3n
against W2 resistant strain and β-haematin
Compound G-score
% Chemosuppression
(Mean+SEM)
W2 in vitro β-haematin inhibition
IC50 (µM)
IC50 (µM)
2
-6.74
-9.84
-9.60
-9.11
-9.22
-9.23
-9.54
-8.43
-9.20
-9.33
-9.05
-9.52
-9.06
-9.60
-9.40
-5.78
85.92 + 0.3700
97.03 + 0.3700
97.4 + 0.3700
94.07 + 0.3700
94.81 + 0.3700
94.81 + 0.3700
97.4 + 0.3700
97.03 + 0.3700
97.03 + 0.3700
95.55 + 0.6409
95.55 + 0.6409
91.48 + 0.3700
95.18 + 0.3700
91.11 + 0.6409
97.03 + 0.3700
82.96 + 0.3700
-
0.807
0.123
0.255
0.118
0.167
0.145
0.145
0.138
0.119
0.268
0.902
0.008
0.380
0.269
0.220
0.282
0.015
32.5 ND
21.8 2.3
24.9 4.3
20.6 4.5
23.7 5.6
21.0 3.0
23.0 5.7
21.1 2.4
28.1 4.6
21.6 3.1
> 500
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
18.6 2.0
29.7 1.1
23.7 4.2
29.2 4.7
27.2 3.0
-
3m
3n
CQ
Artemisinin -6.20
9

E-64
-6.65
-
-
From the toxicity studies, the data revealed that all the synthesized compounds were
confirmed to be non-toxic up to 2000 mg/kg dose level and were well tolerated by the
experimental animals. Therefore, the dose selected and fixed for pharmacological evaluation was
1/10th of 2000 mg/kg i. e. 200 mg/kg.
All the 15 compounds were tested in vivo at 200 mg/kg dose and the results were
compared with that of chloroquine as the standard drug. The in vivo antimalarial activity results
revealed that all the compounds displayed % chemosuppression more than 85, which was better
than that shown by the standard drug, chloroquine. Compounds 3a-3g, 3i-3j, 3l and 3n exhibited
more than 94% chemosuppression, as shown in Table 1.
As displayed in Table 1, the in vitro antimalarial activity of all the 15 compounds on
chloroquine-resistant Plasmodium falciparum W2 strain revealed that compounds 3a-3i and 3m-
3n showed inhibition against the resistant parasite better compared to chloroquine and moderate
compared to artemisinin. Compound 3k (IC50 value = 0.008 µM) displayed the highest level of
inhibition compared to that of the other derivatives, as well as chloroquine and artemisinin. The
IC50 values of all the compounds were found to be less than 1 µM.
In β-haematin inhibition, as revealed in Table 1, amongst all the compounds, ten
compounds 3a-3g, 3i, 3k and 3m showed β-haematin inhibitory activity with the IC50 values in
the range of 18 – 25 µM, better than that shown by chloroquine (IC50 value 27.2 µM).
Compound 3k displayed the best β-haematin inhibitory activity, with the IC50 value of 18.6 µM.
These results showed that using heterocyclic and aromatic amines enhanced the β-haematin
inhibitory activity of 4 – anilinoquinoline derivatives. Attaching single chain aliphatic amines to
cyanuric chloride did not help much in improving the β-haematin inhibitory activity of the
derivatives. However, attachment of a branched chain aliphatic amine to the same yielded the
best activity.
In conclusion, 4-anilinoquinoline derivatives were successfully designed, synthesized and
evaluated for both in vivo and in vitro antimalarial activity including β-haematin inhibitory
activity. These derivatives have displayed good antimalarial activity in vivo as well as in vitro by
the mechanism of β-haematin inhibition. One compound, 3k, was found to be better than
artemisinin against W2 strain of Plasmodium falciparum and also displayed the best β-haematin
inhibitory activity, thereby becoming eligible to be explored as a potential lead for antimalarial
chemotherapy. As the compounds exhibited good in silico interaction with falcipain-2 enzyme,
they may also act by inhibition of falcipain-2 enzyme in Plasmodium falciparum. Study on
falcipain-2 inhibition of these derivatives is underway and shall be published in due course of
time.
Acknowledgements
The authors wish to thank the Indian Council of Medical Research (ICMR), New Delhi,
India, for providing funds for the research work. The authors are also grateful to Dr. Jiri Gut and
Philip Rosenthal from the Department of Medicine, San Francisco General Hospital, University
10

of California San Francisco, USA, for carrying out the in vitro antimalarial activity against W2
strain of P. falciparum and falcipain-2 enzyme inhibition study.
Supplementary data
Supplementary data include the docking and validation procedures, physical and spectral
data of 4 – anilinoquinoline derivatives.
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Highlights
ꢀ Falcipain-2 and β-haematin are the promising targets for antimalarial activity.
ꢀ All compounds showed better G-score and in vivo activity compared to chloroquine.
ꢀ Twelve compounds exhibited in vitro inhibition better than that of chloroquine.
ꢀ Ten compounds exhibited β-haematin inhibition better than that of chloroquine.
ꢀ Compound 3k showed excellent in vitro and β-haematin inhibitory activity.
13