Experimental Parasitology 163 (2016) 38e45

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Experimental Parasitology
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Full length article

Design, synthesis and antimalarial screening of some hybrid 4-

aminoquinoline-triazine derivatives against pf-DHFR-TS
Supriya Sahu a, *, Surajit Kumar Ghosh a, Junmoni Kalita a, Mayurakhi Dutta b,
Hans Raj Bhat c
a
Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam 786004, India
b
Department of Pharmacy, Assam University, Silchar, Assam 788011, India
c
Department of Pharmaceutical Sciences, Sam Higginbottom Institute of Agriculture, Technology & Science, Deemed University, Allahabad 211007 India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Compound S1 showed encouraging

IC50 value against 3D7 strain of Plas-
modium falciparum.

Presence of primary, secondary and
tertiary amine in S1 made it most
active.

S1 showed favourable interaction
with Met55, Phe58 and Leu164.

a r t i c l e i n f o a b s t r a c t

Article history: Existing antifolate antimalarial drugs have shown resistance due to the mutations at some amino acid
Received 21 May 2015 positions of Plasmodium falciparum DHFR-TS. In the present study, to overcome this resistance, a new
Received in revised form series of hybrid 4-aminoquinoline-triazine derivatives were designed and docked into the active site of
15 January 2016
Pf-DHFR-TS (PDB i.d. 1J3K) using validated CDOCKER protocol. Binding energy was calculated by applying
Accepted 20 January 2016
Available online 25 January 2016
CHARMm forcefield. Binding energy and the pattern of interaction of the docked compounds were
analysed. Fifteen compounds were selected for synthesis based on their binding energy values and
docking poses. Synthesized compounds were characterised by FTIR, 1H NMR, 13C NMR, mass spectros-
Keywords:
Antimalarial
copy and were screened for antimalarial activity against 3D7 strain of Plasmodium falciparum.
Docking © 2016 Elsevier Inc. All rights reserved.
Hybridisation
Triazine
Aminoquinoline

1. Introduction
Malaria, the most threatening parasitic infection in human has
been a real concern for centuries. 300e500 million new clinical
* Corresponding author.
E-mail address: supsjrt@gmail.com (S. Sahu).
cases are reported by the World Health Organization (WHO) every
year, resulting in annual deaths of about one million people. Ac-
cording to the World Malaria Report 2014, there are 104 countries
and territories in total where malaria is presently considered
endemic. Globally, an estimated 3.4 billion people are at risk of
malaria. WHO estimates that there are 198 million cases of malaria
leading to 584,000 deaths globally. Most cases (80%) and deaths
(90%) occurred in Africa and most deaths (77%) were in children

http://dx.doi.org/10.1016/j.exppara.2016.01.010
0014-4894/© 2016 Elsevier Inc. All rights reserved.
 S. Sahu et al. / Experimental Parasitology 163 (2016) 38e45
below 5 years of age (World Health Organi, 2014).
For several decades, the most wonderful drug for the treatment
of malaria was chloroquine (Fidock et al., 2004). Chloroquine (CQ)
was introduced in 1944e1945 and because of its cheapness, non-
toxicity and activity against all strains of malaria parasite, soon it
became the mainstay of therapy and prevention. The main classes
of active antimalarials are 4-aminoquinolines, aryl-alcohols
including quinoline alcohols, antifolates that inhibit the synthesis
of parasitic pyrimidines, artemisinin and its semisynthetic and
synthetic analogs which cause parasite death due to oxidative
stress produced by breakage of the endoperoxide ring present
therein (Robert et al., 2001). Amongst the currently used clinical
antimalarial drugs, the antifolates have the best defined molecular
targets: enzymes dihydrofolate reductase (DHFR) and dihy-
dropteroate synthase (DHPS), functioning in the folate metabolic
pathway (Hyde, 2005). Folate metabolism is important for the
viability of malaria parasites. These two pathways have been tar-
geted in both treatment and prophylaxis of the disease. The most
widely used antifolate antimalarial drugs include pyrimethamine
(PYR), proguanil, sulfadoxine (SDX) and dapsone, which have long
provided chemotherapy at an affordable price to the poorer nations
(Sibley et al., 2001).
4-aminoquinoline, the nucleus of CQ and triazine which is the
nucleus of clociguanil when joined together with a linker meets the
entire structural requirement such as the presence of a hydro-
phobic tail and hydrogen bond donor head group respectively for
inhibition of pf-DHFR-TS (Legesse and Prasad, 2011). Recently,
conjugates of 4-aminoquinoline and 1,3,5-triazine have been
widely studied as novel pf-DHFR-TS inhibitors (Bhat et al., 2013a;
Kumar et al., 2008; Kumar et al., 2011; Manohar et al., 2010;
Sharma et al., 2012). As a part of our ongoing research work to
develop hybrid antimalarial molecules, we have designed a new
series of hybrid 4-aminoquinolne-triazine derivatives. Based on the
in-silico results, some selected molecules were tested for antima-
larial activity against 3D7 strain of Plasmodium falciparum.
2. Materials and methods
2.1. Receptor preparation and docking study
The crystal structure of quadruple mutant Pf-DHFR-TS complex
was obtained from Protein Data Bank. In the protein workspace of
Accelrys Discovery Studio Version 2.5, water molecules, co-
crystallized ligand WR99210 were removed and cofactors NADPH,
dUMP were retained. Pf-DHFR-TS consist of four chains A, chain B,
chain C and chain D out of which chain C & chain D are of TS domain
and chain A & chain B are of DHFR domain. Since the prototype
WR99210 was bound to chain A, so only chain A of the protein was
used in the present work. This refined protein was simulated in the
workspace by applying CHARMm forcefield and finally binding site
was defined as sphere (28.0015, 5.89121, 59.8342, 16.1) around the
active site of chain A.
A virtual library of hybrid 4-aminoquinolne-triazine was
designed (Table 2) by joining both the nuclei with ethylenediamine
Table 1
Heavy Atom RMSD to WR99210 X-ray pose.
Pose RMSD (Å)
1 0.9770
2 0.9711
3 1.5187
4 1.3163
5 1.1196
WR99210 0.0000
39
and substituting the chlorines of cyanuric chloride (2,4,6-trichloro-
1,3,5-triazine) with different amines for improved H-bonding with
target protein.
Docking was done using CDOCKER of Accelrys Discovery Studio
version 2.5. The centre of co-crystallized ligand WR99210 was
selected as the binding site for all calculations. The protocol was
validated by calculating RMSD between five docked poses of
WR99210 and ligand's X-ray docking pose. The X-ray pose of
WR99210 was taken as reference. Docking is considered to be
successful if the RMSD value is less than 2 Å (Guosheng et al., 2003).
Ligands prepared were docked at the active site of the prepared
protein and finally binding energy of the ligandeprotein complex
was calculated by using ‘Calculate Binding Energy’ protocol which
uses CHARMm implicit solvent model.
2.2. Chemistry
All the chemicals and solvents used for synthesis, recrystalli-
zation and analysis were of AR grade and used without further
purification. Melting point of the synthesized compounds was
determined by Melting Point apparatus (BUCHI Melting Point
M560) at 10  C/min temperature gradient. The UV-Spectra (lmax)
of the synthesized compounds were recorded on Shimadzu, UV-
1800, UV-VIS spectrophotometer instrument. The FTIR spectra of
the synthesized compounds were recorded on Bruker ALPHA FTIR
spectrometer. Infrared spectra of compounds showed absorption
bands which are characteristic of the anticipated structure of the
synthesized compounds. The 1H NMR spectra of the synthesized
compounds were recorded in DMSO at 300 MHz by Bruker Avance
DPX 300 NMR spectrometer and 13C NMR was also recorded in
DMSO at 100 MHz by Bruker Avance DPX 100 NMR spectrometer.
The mass spectra of the synthesized compounds were recorded on
ZQ-4000 equipped with an Electrospray Ionizer as an ionization
method.
The synthesis of the intermediates and the final compounds was
achieved by the protocol shown in Scheme 1. Compound 3 was
synthesized by substituting the chlorine in fourth position of 4,7-
dicholoroquinoline ring by ethylenediamine under neat condi-
tion. First and second chlorine of cyanuric chloride was substituted
by different amines at 0e5  C and at room temperature respec-
tively. Final compounds were obtained by nucleophilic substitution
of disubstituted cyanuric chloride with compound 3 at 100e120  C.
2.2.1. Synthesis of N-(2-aminoethyl)-7-chloroquinolin-4-amine
The compound was prepared by adding ethylenediamine to
melted 4,7-dichloroquinoline. This was first heated at 80  C for 1 h
and then refluxed at 120e130  C for 2e3 h. The reaction mixture
was then cooled to room temperature over a long period of time. It
was then dissolved in dichloromethane and cold water was added
to it. Brine solution was also added. The mixture was stirred with a
glass rod and kept overnight without disturbance for settling the
solid product in between the two solvent layers (colour of the so-
lution changes from yellow to dark pink over night).The solid
product was separated out by filtration. It was then dried in hot air
oven at 45  C. The dried powder was then dissolved in n-butanol
and filtered. The filtrate was poured in a petridish and n-butanol
was evaporated. After complete evaporation of the solvent, pale
yellow crystals appeared.
2.2.2. General procedure for synthesis of mono amino-substituted
1,3,5-triazine derivatives
Cyanuric chloride was dissolved in quantity sufficient diethyl
ether. It was then cooled to 0e5  C. To this cold solution, ammonia
was added and stirred for 3 h till the solution turned milky. It was
then filtered and the filtrate was kept for solvent evaporation.
40 S. Sahu et al. / Experimental Parasitology 163 (2016) 38e45

Table 2
Designed compounds with their CDOCKER binding energies (*Compounds selected for synthesis).

Compounds R1 R2 Binding energy (kCal/mol)

S1* Amino Dimethylamine 51.08

S2* Amino Amino 102.30
S3* Amino Propylamine 96.71
S4* Amino Cyclopropylamine 41.98
S5 Amino Piperazine 187.63
S6* Cyclopropylamine Cyclopropylamine 23.59
S7* Amino Piperidine 75.07
S8 Amino Methylpiperazine 99.46
S9* Amino Morpholine 76.32
S10 Amino Methylamine 110.71
S11* Methylamine Propylamine 90.91
S12* Methylamine Cyclopropylamine 171.56
S13 Propylamine Piperazine 260.94
S14 Propylamine Morpholine 70.90
S15 Propylamine Dimethylamine 5.14
S16 Piperazine Piperazine 285.51
S17* Morpholine Methylamine 65.02
S18 Propylamine Piperidine 156.60
S19 Propylamine Methylpiperazine 122.49
S20 Methylpiperazine Methylpiperazine 22.62
S21 Morpholine Morpholine 8.69
S22 Piperidine Piperidine 2.12
S23 Methylpiperazine Morpholine 54.56
S24 Piperazine Dimethylamine 140.98
S25 Cyclopropylamine Morpholine 37.10
S26 Cyclopropylamine Methylpiperazine 4.28
S27 Piperazine Piperidine 120.41
S28 Piperazine Morpholine 3.51
S29 Piperazine Methylpiperazine 110.40
S30 Piperazine Methylamine 31.26
S31 Morpholine Piperidine 6.54
S32* Propylamine Propylamine 48.44
S33* Dimethylamine Dimethylamine 32.54
S34 Dimethylamine Methylpiperazine 135.56
S35 Dimethylamine Morpholine 5.28
S36 Dimethylamine Piperidine 71.29
S37 Cyclopropylamine Propylamine 52.26
S38 Cyclopropylamine Dimethylamine 54.04
S39 Cyclopropylamine Piperidine 30.89
S40 Cyclopropylamine Amino 140.56
S41* Ethylenediamine Ethylenediamine 159.24
S42* Methylamine Methylamine 98.16
S43* Methylamine Piperidine 122.85

White, needle shaped crystals appeared.

2.2.3. General procedure for second substitution in mono

substituted 1,3,5-triazine derivatives
Monosubstituted triazine derivatives were dissolved in quantity
sufficient acetone and different amines in equimolar quantity was
added to it at 30e40  C separately with continuous two-three
hours stirring. The reaction mixture was then filtered and the
filtrate was kept for evaporation of solvent. Solid, dried product was
collected from the filtrate.

2.2.4. General procedure for synthesis of di-substituted 1,3,5-

triazine derivatives
Cyanuric chloride was dissolved in 1,4-dioxane. Amine in double
molar that of cyanuric chloride was added to it at 40e50  C and the
solution was stirred for 3 h. The reaction mixture was then filtered
and filtrate was kept for solvent evaporation. After evaporation of
solvent solid product was collected. It was then recrystallized with
ethanol.

Scheme 1. Synthesis of intermediates and final compounds. Reagents and conditions:
(a) in neat condition first at 80  C for 1 h then reflux at 120e130  C for 2e3hrs (Katti
et al., 2005) (b) Diethyl ether, 0e5  C, 1e3 h (Thurston et al., 1951) (c) Acetone,
30e40  C, 4e5 h (Menicagli, 2006) (d) 1,4-Dioxane, 30e40  C, 3e4hrs (Thurston et al.,
1951) (e) 1,4-Dioxane, reflux, 5e6 h (Afonso et al., 2006).
2.2.5. General procedure for the synthesis of final compounds
Disubstituted triazine derivative was dissolved in quantity suf-
ficient 1,4-Dioxane and the temperature was raised to about
100e120  C. To this equimolar quantity of N-(2-aminoethyl)-7-
 S. Sahu et al. / Experimental Parasitology 163 (2016) 38e45
chloroquinolin-4-amine was added and the solution was refluxed
for 5 h. The reaction mixture was then kept overnight for solvent
evaporation. Dried product was collected after complete removal of
the solvent.
2.3. Antimalarial screening
3D7 strain of P. falciparum was maintained routinely in stock
cultures in medium RPMI-1640 supplemented with 25 mmol
HEPES, 1% D-glucose, 0.23% sodium bicarbonate and 10% heat
inactivated human serum. The asynchronous parasites of
P. falciparum were synchronized after 5% D-sorbitol treatment to
obtain only the ring stage parasitized cells. For carrying out the
assay, the initial ring stage parasitaemia of 0.8e1.5% at 3% hemat-
ocrit in a total volume of 200 mL of medium RPMI-1640 was uni-
formly maintained.
2.3.1. In-vitro antimalarial efficacy test
The in-vitro antimalarial assay was carried out according to the
microassay of Rieckmann et al. (1978) in 96 well-microtitre plates
(Rieckmann et al., 1978), with minor modifications. A stock solution
of 5 mg mL1 of each of the test samples was prepared in DMSO
and subsequent dilutions were prepared with the culture medium.
The test compounds in 20 mL volume concentration at 50 mg/ml in a
duplicate well were incubated with parasitized cell preparation at
37  C in a candle jar. After 36e40 h of incubation, the blood smears
were prepared from each well and stained with Giemsa stain. The
level of parasitemia in terms of % dead rings along with tropho-
zoites and schizonts was determined by counting a total of 100
asexual parasites (both live and alive) microscopically using chlo-
roquine as the reference drug at its IC50 dose. IC50 of the compound
which had shown highest level of parasitemia was determined by
acute toxicity method against 3D7 strain.
3. Results and discussion
3.1. Docking studies
WR99210's X-ray crystallographic docking pose was overlapped
with five different docking poses of the same (outputs of the
docking protocol used in this study) to get the RMSD. RMSD values
were found between 0.9711 and 1.5187 Å (Table 1) which was
sufficiently below 2 Å. This implies that the CDOCKER docking
protocol was validated. Binding energies of the designed com-
pounds with the target protein are given in Table 2. Docked poses of
all the compounds were studied carefully and it was observed that
compounds having very low binding energy (þ10 to 10) had not
formed any interaction with the target. Such compounds were kept
out of the scope of present study. Most of the compounds fall
within the binding energy range of 20 to 80 kCal/mol. Eight
compounds from this range and seven compounds from the higher
binding energy range of 80 to e170 kCal/mol were selected for
synthesis by observing their interactions (Fig. 1) with the target
protein. Compound S13 and S16 showed H-bonded interaction with
cofactor NADPH along with piepi/cationic interactions with Phe116
and Phe58 which also might be the reason of their large binding
energy. The presence of the NADPH in the binding pocket of S13
and S16 opened up the possibility of synthesis of pyrimidine bases
required for the normal growth of pf-DHFR-TS and thus eliminating
itself from becoming a possible inhibitor of the same.
Among these compounds, S1, S7, S17 and S43 have shown better
binding pose with 1J3K, showing H-bond, piepi & pi-cationic in-
teractions respectively. The presence of tertiary amino group in S1
and S43 is common. The tertiary amino group is cyclic in nature in
S43. It forms pi-cationic type of interaction with amino acid Phe58
41
and H-bond with Asn108 of the target protein. This might be the
reason of S43 having good binding energy value of 122.85 kcal/
mol with the target. Surprisingly, instead of the presence of two H-
bonds with Met55 and Leu164 and piepi interaction with Phe58,
compound S1 has binding energy of 51.08 kcal/mol. The ethyl-
enediamine part is also common in S1 and S43 which helps in
forming H-bonding with amino acid Met55 and Asn108 respec-
tively. Tertiary amine-containing group piperidine is also present in
S7. Here it forms piepi interaction with Phe58. S7 also forms H-
bond with Ser111 but has a comparatively less binding energy value
of 75.07. Other compounds with tertiary amine (morpholine)
group are S9 and S17. S9 shows the presence of piepi interaction
with Phe116. S17 exhibits all possible interactions. It forms H-bond
with Ser111, piepi interaction with Phe116 and pi-cationic inter-
action with Phe58. Compound S33 is substituted with two dimethyl
amino groups, showing the formation of piepi and pi-cationic in-
teractions with Phe116 and Arg59 respectively.
.
3.2. Chemistry
The synthesis of the intermediates and the final compounds
were achieved by the synthetic protocol as shown in Scheme 1.
These are all nucleophilic substitution reactions of the chlorine
atoms of cyanuric chloride by different amines and intermediate 3.
First chlorine atom of cyanuric chloride was substituted at 0e5  C,
second at 30e40  C and third at 100e120  C. Completion of the
reactions was ascertained by TLC.
Characteristic single FTIR peak was observed in the range of
3200e3400 cm1 due to presence of secondary amine as ethyl-
enediamino substitution, which is common in all the synthesized
compounds, while two peaks were observed within the same range
due to the presence of primary amine substitution. Peaks between
2800 and 3000 cm1 is due to CeH stretching present in ethyl-
enediamine, methylamine, dimethylamine, propylamine, cyclo-
propylamine, morpholine and piperidine. Broad peak near
3200 cm1 is due to the presence of OeH group. A characteristic
strong band at 1650e1683 cm1 is attributed to the C]N stretching
vibrations of hybrid derivatives. The 1H NMR spectra shows a
doublet at 8.65e7.57 ppm corresponding to the presence of the
quinoline ring. A distinct resonance at 2.14 ppm is due to the
presence of the NeH group situated on the carbon atom of the s-
triazine ring. The resonance due to AreH is observed at
6.83e8.30 ppm for the disubstituted 1,3,5-triazine derivatives. Final
structures of the compounds were confirmed from their mass
spectra.
3.2.1. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4,N4-dimethyl-
1,3,5-triazine-2,4,6-triamine (S1)
Yield: 74%; M.p: 235e240  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3400.51, 3339.00 (NeH primary), 3179.58 (NeH second-
ary), 3055.14 (aromatic CeH stretch), 1659.32 (C]N stretch),
1606.49 (C]C aromatic stretch), 1367.74 (CeN aromatic ring),
1184.86 (aliphatic CeN stretch), 1033.02 (aliphatic CeC stretch),
808.86 (CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm: 2.51 (s,
6H, CH3), 3.39 (s, 2H, NH2), 6.14 (s, 2H, NH), 3.00 (t, 4H, CH2), 8.90 (s,
1H, CH, quinoline), 8.19e8.22 (d, 4H, CH, quinoline); 13C NMR
(100 MHz, DMSO): d, ppm: 46.70e50.68, 122.60e129.35, 136.09,
142.33e152.09, 155.60, 166.62e169.63, 180.10, 183.22; Mass:
359.02 (MþH)þ.
42 S. Sahu et al. / Experimental Parasitology 163 (2016) 38e45

Fig. 1. Docking poses of some of the selected compounds for synthesis.

 S. Sahu et al. / Experimental Parasitology 163 (2016) 38e45
3.2.2. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-1,3,5-triazine-
2,4,6-triamine (S2)
Yield: 78%; M.p: infusible; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3488.91, 3295.94 (NeH primary), 3109.32 (NeH second-
ary), 1680.18 (C]N stretch), 1608.96 (C]C aromatic stretch),
1292.37 (CeN aromatic ring), 1075.54 (aliphatic CeN stretch),
1016.18 (aliphatic CeC stretch), 794.42 (CeCl stretch); 1H NMR
(300 MHz, DMSO): d, ppm: 3.09 (s, 4H, NH2), 6.59 (s, 2H, NH),
3.35e3.43 (t, 4H, CH2), 8.74 (s, 1H, CH, quinoline), 7.23e7.28,
7.46e7.49, 7.51e7.53, 8.88e8.90 (d, 4H, CH, quinoline); 13C NMR
(100 MHz, DMSO): d, ppm: 39.33e40.92, 122.04e129.88, 136.09,
142.34e152.10, 166.36e169.29; Mass: 331.51 (MþH)þ.
3.2.3. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4-propyl-1,3,5-
triazine-2,4,6-triamine (S3)
Yield: 63%; M.p: 95e97  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3324.95, 3274.35 (NeH primary), 3173.41 (NeH second-
ary), 3119.20 (aromatic CeH stretch), 1658.83 (C]N stretch),
1553.35 (C]C aromatic stretch), 1289.92 (CeN aromatic ring),
1071.21 (aliphatic CeN stretch), 973.90 (aliphatic CeC stretch),
813.59 (CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm:
0.84e1.46 (t, 3H, CH3), 2.51e3.86 (sextet, 2H, CH2), 6.69 (s, 3H, NH),
7.07e7.29 (t, 6H, CH2), 8.88 (s, 1H, CH, quinoline), 6.18e6.34,
7.64e7.80, 8.42e8.54, 9.89e9.92 (d, 4H, CH, quinoline); 13C NMR
(100 MHz, DMSO): d, ppm: 46.24e50.81, 122.35e130.73,
142.74e152.60, 158.52, 166.63e169.62, 180.26, 183.09; Mass: 373.13
(MþH)þ.
3.2.4. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4-cyclopropyl-
1,3,5-triazine-2,4,6-triamine (S4)
Yield: 85%; M.p: 109  C; UV lmax (DMSO): 323 nm; FTIR (cm1):
3436.11, 3262.72 (NeH primary), 3133.27 (NeH secondary),
2975.04 (aromatic CeH stretch), 1658.38 (C]N stretch), 1553.34
(C]C aromatic stretch), 1344.61 (CeN aromatic ring), 1184.44
(aliphatic CeN stretch), 1071.82 (aliphatic CeC stretch), 875.71
(CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm: 0.48e1.74 (p,
1H, CH), 2.51e2.83 (t, 4H, CH2), 4.99 (s, 2H, NH2), 6.41 (s, 3H, NH),
9.33 (s, 1H, CH, quinoline), 7.31e7.44, 7.83e7.96, 8.19e8.40,
8.90e9.04 (d, 4H, CH, quinoline); 13C NMR (100 MHz, DMSO): d,
ppm: 49.35e50.80, 122.41e129.50, 136.25, 142.60e152.33, 158.63,
166.69e169.81, 183.67; Mass: 371.42 (MþH)þ.
3.2.5. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4,N6-
dicyclopropyl-1,3,5-triazine-2,4,6-triamine (S6)
Yield: 81%; M.p: 120  C; UV lmax (DMSO): 323 nm; FTIR (cm1):
3334.90 (NeH secondary), 3008.77 (aromatic CeH stretch), 1605.82
(C]N stretch), 1579.44 (C]C aromatic stretch), 1343.84 (CeN ar-
omatic ring), 1217.01 (aliphatic CeN stretch), 1014.96 (aliphatic CeC
stretch), 805.55 (CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm:
0.43e0.59 (q, 4H, CH2), 2.45e2.69 (p, 1H, CH), 3.29e3.52 (t, 4H,
CH2), 7.73 (s, 4H, NH), 8.09 (s, 1H, CH, quinoline), 8.81e8.84 (d, 4H,
CH, quinoline); 13C NMR (100 MHz, DMSO): d, ppm: 39.83e40.46,
122.58e129.22, 141.85, 149.32e152.42, 166.59e167.09; Mass:
411.31 (MþH)þ.
3.2.6. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-6-(piperidin-1-
yl)-1,3,5-triazine-2,4-diamine (S7)
Yield: 77%; M.p: 115  C; UV lmax (DMSO): 323 nm; FTIR (cm1):
3424.21, 3329.66 (NeH primary), 3177.68 (NeH secondary),
2934.92 (aromatic CeH stretch), 1608.17 (C]N stretch), 1548.80
(C]C aromatic stretch), 1345.15 (CeN aromatic ring), 1226.10
(aliphatic CeN stretch), 1020.54 (aliphatic CeC stretch), 798.24
(CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm: 0.82e1.56 (p,
6H, CH2), 3.29 (s, 2H, NH2), 6.00 (s, 2H, NH), 3.62e3.97 (t, 4H, CH2),
7.16 (s, 1H, CH, quinoline), 7.76, 8.09e8.13, 8.17e8.20, 8.84 (d, 4H,
43
CH, quinoline); 13C NMR (100 MHz, DMSO): d, ppm: 24.55e25.78,
39.87e40.49, 122.62e129.30, 152.47; Mass: 399.42 (MþH)þ.
3.2.7. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-6-morpholino-
1,3,5-triazine-2,4-diamine (S9)
Yield: 83%; M.p: 142e145  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3386.56, 3315.12 (NeH primary), 3215.89 (NeH second-
ary), 2999.05 (aromatic CeH stretch), 3002.94 (aliphatic CeH
stretch), 1625.26 (C]N stretch), 1580.33 (C]C aromatic stretch),
1322.96 (CeN aromatic ring), 1279.34 (aliphatic CeN stretch),
1188.10 (aliphatic CeC stretch), 1215.89 (CeO stretch), 824.35 (CeCl
stretch); 1H NMR (300 MHz, DMSO): d, ppm: 2.90e3.67 (t, 8H, CH2),
3.29 (t, 4H, CH2), 4.02 (s, 4H, NH), 8.09 (s, 1H, CH, quinoline),
8.82e8.84 (d, 4H, CH, quinoline); 13C NMR (100 MHz, DMSO): d,
ppm: 46.34e49.50, 66.41, 113.34e134.90, 148.23e162.60,
176.00e182.43; Mass: 401.11 (MþH)þ.
3.2.8. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4-methyl-N6-
propyl-1,3,5-triazine-2,4,6-triamine (S11)
Yield: 76%; M.p: 103e107  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3275.43 (NeH secondary), 2914.12 (aromatic CeH stretch),
3012.16 (aromatic CeH stretch), 1705.68 (C]N stretch), 1588.05
(C]C aromatic stretch), 1358.13 (CeN aromatic ring), 1286.34
(aliphatic CeN stretch), 1112.04 (aliphatic CeC stretch), 799.49
(CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm: 0.96 (t, 3H, CH3),
2.47 (s, 3H, CH3), 1.56e1.57 (sextet, 2H, CH2), 3.06e3.29 (t, 6H, CH2),
4.00 (s, 4H, NH), 8.01 (s, 1H, CH, quinoline), 6.49, 7.43, 7.62, 8.65870
(d, 4H, CH, quinoline); 13C NMR (100 MHz, DMSO): d, ppm:
11.50e29.72, 47.31e49.55, 113.00e129.05, 134.90e148.23,
151.4e159.90, 162.60e165.87; Mass: 387.92 (MþH)þ.
3.2.9. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4-cyclopropyl-
N6-methyl-1,3,5-triazine-2,4,6-triamine (S12)
Yield: 62%; M.p: 112e115  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3395.14 (NeH secondary), 2942.20 (aromatic CeH stretch),
3002.60 (aromatic CeH stretch), 1715.11(C]N stretch), 1598.12(C]
C aromatic stretch), 1328.92(CeN aromatic ring), 1265.48 (aliphatic
CeN stretch), 1192.10 (aliphatic CeC stretch), 839.19 (CeCl stretch);
1
H NMR (300 MHz, DMSO): d, ppm: 2.47 (s, 3H, CH3), 0.53e1.53 (p,
6H, CH2), 3.29 (t, 4H, CH2), 4.00 (s, 4H, NH), 8.03 (s, 1H, CH, quin-
oline), 6.40, 7.41, 7.64, 8.66 (d, 4H, CH, quinoline); 13C NMR
(100 MHz, DMSO): d, ppm: 11.55e29.42, 47.71e49.35,
113.80e129.53, 134.88e148.56, 151.45e159.69, 162.36e165.45;
Mass: 385.65 (MþH)þ.
3.2.10. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4-methyl-6-
morpholino-1,3,5-triazine-2,4-diamine (S17)
Yield: 65%; M.p: 90e94  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3345.90 (NeH secondary), 2982.17 (aromatic CeH stretch),
3012.32 (aliphatic CeH stretch), 1625.26 (C]N stretch), 1585.27
(C]C aromatic stretch), 1389.56 (CeN aromatic ring), 1217.19
(aliphatic CeN stretch), 1088.16 (aliphatic CeC stretch), 1310.24
(CeO stretch), 816.45 (CeCl stretch); 1H NMR (300 MHz, DMSO): d,
ppm: 2.90e3.67 (t, 8H, CH2), 2.47 (s, 3H, CH3), 3.29 (t, 4H, CH2), 4.12
(s, 3H, NH), 8.09 (s, 1H, CH, quinoline), 8.82e8.84 (d, 4H, CH,
quinoline); 13C NMR (100 MHz, DMSO): d, ppm: 29.80e49.56, 66.4,
113.34e134.90, 148.23e162.60, 182.43; Mass: 415.09 (MþH)þ.
3.2.11. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4,N6-dipropyl-
1,3,5-triazine-2,4,6-triamine (S32)
Yield: 81%; M.p: 74e76  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3294.98 (NeH secondary), 3108.45 (aromatic CeH stretch),
1595.12 (C]N stretch), 1555.82 (C]C aromatic stretch), 1378.42
(CeN aromatic ring), 1219.23 (aliphatic CeN stretch), 1032.60
(aliphatic CeC stretch), 897.34 (CeCl stretch); 1H NMR (300 MHz,
44 S. Sahu et al. / Experimental Parasitology 163 (2016) 38e45

DMSO): d, ppm: 0.96 (t, 6H, CH2), 1.56 (sextet, 4H, CH2), 3.06 (t, 4H,
CH2), 3.29 (t, 4H, CH2), 4.09 (s, 4H, NH), 8.10 (s, 1H, CH, quinoline),
8.80e8.84 (d, 4H, CH, quinoline); 13C NMR (100 MHz, DMSO): d,
ppm: 11.50e49.50, 113.00e134.90, 148.20e162.61; Mass: 415.41
(MþH)þ.
3.2.12. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4,N4,N6,N6-
tetramethyl-1,3,5-triazine-2,4,6-triamine (S33)
Yield: 56%; M.p: 172e175  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3222.37 (NeH secondary), 2990.98 (aromatic CeH stretch),
1670.87 (C]N stretch), 1596.42 (C]C aromatic stretch), 1377.46
(CeN aromatic ring), 1111.96 (aliphatic CeN stretch), 1070.34
(aliphatic CeC stretch), 798.65 (CeCl stretch); 1H NMR (300 MHz,
DMSO): d, ppm: 2.47 (s, 12H, CH3), 3.29 (t, 2H, CH2), 4.00 (s, 2H, NH),
8.90 (s, 1H, CH, quinoline), 8.19e8.23 (d, 4H, CH, quinoline); 13C
NMR (100 MHz, DMSO): d, ppm: 40.30e49.54, 113.00e134.90,
148.21e155.41, 162.6e179.2; Mass: 387.11 (MþH)þ.
3.2.13. N2,N4-bis(2-aminoethyl)-N6-(2-(7-chloroquinolin-4-
ylamino)ethyl)-1,3,5-triazine-2,4,6-triamine (S41)
Yield: 59%; M.p:130e132  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3390.46, 3223.45 (NeH primary), 3210.90 (NeH second-
ary), 3112.31 (aromatic CeH stretch), 1696.76 (C]N stretch),
1600.12 (C]C aromatic stretch), 1398.43 (CeN aromatic ring),
1155.32 (aliphatic CeN stretch), 1006.92 (aliphatic CeC stretch),
789.18 (CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm:
2.88e3.32 (t, 8H, CH2), 2.00 (s, 4H, NH2), 4.06 (s, 4H, NH), 3.29 (t,
4H, CH2), 8.88 (s, 1H, CH, quinoline), 8.19e8.23 (d, 4H, CH, quino-
line); 13C NMR (100 MHz, DMSO): d, ppm: 40.70e51.71, 162.60,
148.18e154.54, 113.00e139.51; Mass: 417.12 (MþH)þ.
3.2.14. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4,N6-dimethyl-
1,3,5-triazine-2,4,6-triamine (S42)
Yield: 64%; M.p: 102e106  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3310.97 (NeH secondary), 3003.23 (aromatic CeH stretch),
2900.35 (aliphatic CeH stretch), 1656.77 (C]N stretch), 1598.02
(C]C aromatic stretch), 1402.13 (CeN aromatic ring), 1212.45
(aliphatic CeN stretch), 1016.25 (aliphatic CeC stretch), 819.14
(CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm: 2.47 (s, 6H,
CH3), 4.00 (s, 4H, NH), 3.29 (t, 4H, CH2), 8.01 (s, 1H, CH, quinoline),
6.49e8.64 (d, 4H, CH, quinoline); 13C NMR (100 MHz, DMSO): d,
ppm: 29.70e49.51, 162.60, 148.20e151.40, 113.00e134.50; Mass:
359.11 (MþH)þ.
3.2.15. N2-(2-(7-chloroquinolin-4-ylamino)ethyl)-N4-methyl-6-
(piperidin-1-yl)-1,3,5-triazine-2,4-diamine (S43)
Yield: 67%; M.p: 113e116  C; UV lmax (DMSO): 323 nm; FTIR
(cm1): 3250.52 (NeH secondary), 3012.26 (aromatic CeH stretch),
2990.13 (aliphatic CeH stretch), 1697.54 (C]N stretch), 1600.21
(C]C aromatic stretch), 1414.17 (CeN aromatic ring), 1229.43
(aliphatic CeN stretch), 1016.59 (aliphatic CeC stretch), 838.91
(CeCl stretch); 1H NMR (300 MHz, DMSO): d, ppm: 1.50 (p, 6H,
CH2), 2.70 (t, 4H, CH2), 2.47 (s, 6H, CH3), 4.00 (s, 3H, NH), 3.29 (t, 4H,
CH2), 8.00 (s, 1H, CH, quinoline), 6.49e8.64 (d, 4H, CH, quinoline);
13
C NMR (100 MHz, DMSO): d, ppm: 25.90e52.40, 182.40,
148.20e162.60, 113.00e134.90; Mass: 413.26(MþH)þ.
3.3. Antimalarial activity and SAR
The antimalarial activity of the designed compounds was
determined in terms of % dead rings, trophozoites and schizonts.
The % dead ring values of the series of the compounds had shown
activity in wide range (Table 3). Compounds S1 and S43 have shown
good activity against 3D7 strain of P. falciparum at both 5 mg/ml and
50 mg/ml doses. At 5 mg/ml concentration, compound S11 and at
50 mg/ml, compound S41 exhibited weakest activity.
After carrying out the in-vitro antimalarial screening of the
synthesized compounds, it is confirmed that some of the in-silico
active moieties are biologically potent as well. As mentioned in
Table 3, compound S1 has shown maximum number of dead rings,
trophozoites and schizonts at 5 mg/ml (39.5%) and 50 mg/ml (48.5%)
respectively. Compound S43 has shown 35% dead rings, tropho-
zoites and schizonts at both 5 mg/ml and 50 mg/ml dose level.
It is clear from Table 3 that the presence of primary, secondary
and tertiary amine in the same structure increases the antimalarial
activity of the compound. Compound 6 h of previously synthesized
4-aminoquinoline-triazine hybrids having same set of amines had
shown highest number of dead rings, which supports this claim
(Bhat et al., 2013b). S1 have amino, ethylenediamino and dime-
thylamino as primary, secondary and tertiary amine respectively,
has shown encouraging IC50 value of 46.33 mg/ml. Combination of
primary, secondary and tertiary amines is also present in S7 and S9.
They are also found to be active against the strain but their activity
is less than S1. Cyclic tertiary amine piperidino group is present in
S7 and morpholino group is present in S9. Besides S1, compound
S43 has also shown good activity. It contains secondary amine
methyl amino and tertiary amine piperidino substituent. The
combination of secondary and tertiary amine is also present in S17,
but the presence of oxygen in morpholino substituent of S17 might

Table 3
In-vitro antimalarial activity of the synthesized compounds against 3D7 strain.

Compounds Percent dead rings, Trophozoites and schizonts at dose

5 mg/ml 50 mg/ml

S1 39.5 48.5
S2 16 15
S3 9 18.5
S4 10 18.5
S6 17 25
S7 24 29.5
S9 18.5 19.5
S11 0 19.5
S12 19.5 20
S17 12.5 20
S32 32.5 24
S33 14 17
S41 27.5 13.5
S42 28 29.5
S43 35 35
Chloroquine(0.7 mg/ml) 50 e
 S. Sahu et al. / Experimental Parasitology 163 (2016) 38e45
decrease its activity. This finding is supported by our previous
antimalarial screening data of same hybrid nuclei carried out
against 3D7 strain of P. falciparum (Bhat et al., 2015). It is also
observed from Table 3 that the presence of same type of amino
substitution decreases the antimalarial activity. In compounds S6,
S11, S12, S32, S41, S42 both the substituents are secondary amine
like cyclopropylamine, methylamine, propylamine, ethylenedi-
amine. The combination of primary and secondary amines in S2, S3
and S4 has shown weak activity at 50 mg/ml dose level. From this
data the decreasing order of activity can be attributed to the
following combination of amines:
10, 20 & 30 > 20 & 30 > 20 only > 10 & 20
It can also be said that the groups which shows þ I effect
attached to the amino part, like methyl, dimethyl, propyl, cyclo-
propyl etc. increases activity while the groups showing eI effect
like oxygen present in morpholine declines antimalarial activity.
4. Conclusions
The present study describes the utility of molecular docking in
getting an in-depth idea of the interaction of designed compounds
with their target protein. Binding energy and docking pose helps in
selecting some potent inhibitors of P. falciparum. It also gives us an
idea of structure activity relationship of the various functional
groups attached to the 4-aminoquinoline clubbed triazine de-
rivatives and their potency as antimalarial agent. It was observed
from the results that compound S1 exhibited good activity against
3D7 strain of P. falciparum. Thus, the present study provides an
encouraging result to the new hybrid antimalarial drug molecules.
Conflict of interest
None.
Acknowledgement
Authors are thankful to Regional Medical Research Centre
(RMRC), North-East Region, Indian Council of Medical Research
(ICMR), Lahoal, Dibrugarh, Assam for providing the facility to carry
out the antimalarial activity screening of the synthesized com-
pounds mentioned in the work.
45
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