Microwave assisted synthesis, docking and antimalarial evaluation of hybrid PABA-substituted 1,3,5-triazine derivatives

Article abstract of DOI:10.1002/jhet.3955

A series of novel PABA-substituted 1,3,5-triazine derivatives were developed via microwave assisted synthesis and subsequently tested for antimalarial activity against chloroquine sensitive 3D7 strain of Plasmodium falciparum using chloroquine as standard. Antimalarial screening result showed that synthesized compounds exhibited IC₅₀ in the range of 4.46 to 79.72 μg mL^?1. Among the tested compounds, 4c and 4f showed significant antimalarial activity with low binding energies (BE) -172.32 and 160.41 kcal mol^?1 via interacting with Arg122 through the involvement of COOH of the phenyl linked to 1,3,5-triazine. In conclusion, these core scaffolds can be used for future antimalarial drug development.

Full text of DOI:10.1002/jhet.3955

Received: 6 September 2019
DOI: 10.1002/jhet.3955
Revised: 13 February 2020
Accepted: 24 February 2020
A R T I C L E
Microwave assisted synthesis, docking and antimalarial
evaluation of hybrid PABA-substituted
1,3,5-triazine derivatives
Nayana Adhikari1 | Ankita Kashyap1 | Anshul Shakya1
|
Surajit Kumar Ghosh1 | Dibya Ranjan Bhattacharyya2 | Hans Raj Bhat1
Udaya Pratap Singh3
|
1Department of Pharmaceutical Sciences,
Abstract
Dibrugarh University, Dibrugarh, India
2Regional Medical Research Centre,
Indian Council of Medical Research
(ICMR), Dibrugarh, India
3Drug Design & Discovery Laboratory,
Department of Pharmaceutical Sciences,
Sam Higginbottom University of
Agriculture, Technology & Sciences,
Allahabad, India
A series of novel PABA-substituted 1,3,5-triazine derivatives were developed via
microwave assisted synthesis and subsequently tested for antimalarial activity
against chloroquine sensitive 3D7 strain of Plasmodium falciparum using chloro-
quine as standard. Antimalarial screening result showed that synthesized com-
pounds exhibited IC50 in the range of 4.46 to 79.72 μg mL−1. Among the tested
compounds, 4c and 4f showed significant antimalarial activity with low binding
energies (BE) -172.32 and 160.41 kcal mol−1 via interacting with Arg122 through
the involvement of COOH of the phenyl linked to 1,3,5-triazine. In conclusion,
these core scaffolds can be used for future antimalarial drug development.
Correspondence
Hans Raj Bhat, Department of
Pharmaceutical Sciences, Dibrugarh
University, Dibrugarh, Assam 786004,
India.
Email: pharmahans@gmail.com
Funding information
Department of Science and Technology
(DST), New Delhi, India, Grant/Award
Number: EMR/2017/000708
1 | INTRODUCTION
which demonstrated wide range of therapeutic activities,
such as, antifungal,[8] antibacterial[9,10] and anti-HIV,[11]
Malaria is a growing infectious disease and becoming a seri-
ous health concern around the developing world. The cur-
rent data on the incidence and mortality of malaria shows
that 90 countries especially from Africa, South-East Asia
and Eastern Mediterranean region are majorly affected by
it.[1] The disease affects around 219 million people globally
and accounted for almost 435 000 deaths in 2017 alone.[2]
The mutant form of Plasmodium falciparum currently
poses significant risk to currently available antimalarial
agents.[3–6] Therefore, it is worthwhile to discover newer
agents which can be able to arrest the growth of parasites.
Modified 2,4,6-trisubstituted 1,3,5-triazine derivatives
offer access to a diverse array of useful molecules[7]
anti-diabetic, anti-inflammatory, antitumor and against
cystic-fibrosis.[12,13] Moreover, substituted 1,3,5-triazine
derrivatives remain the attractive scaffolds for the genera-
tion of antimalarial drugs, such as, cycloguanil,
chlorcycloguanil,
clociguanil
and
WR99210
(diaminotriazine).[14–16]
1,3,5-Triazines have been identified as non-classical
antifolates and resulted in many potent DHFR inhibitors.
In crystallographic studies of WR99210 with M tuberculo-
sis DHFR or human DHFR with their cofactor NADPH
suggested that the triazine core oriented as a pseudo
chair confirmation with C2 moving out of the plane of
the ring. The two amino groups form four hydrogen
J Heterocyclic Chem. 2020;1–11.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

2
ADHIKARI ET AL.
bonds with protein residues Ile5, Asp27, Ile94 and
Tyr100, which is similar to the binding between triazines
with chicken liver DHFR.[17] This leads to the discovery
of various 1,3,5-triazine derivatives as potent inhibitors of
DHFR against malaria as well as cancer. On the other
hand, numerous sulphonamides and sulphones were
identified as potent antifolate antimalarial drugs belong-
ing to the category of p-amino benzoic acid (PABA), an
essential precursor for the de novo synthesis of folic
acid.[18] However, the clinical utility of above moieties
against malaria parasite is jeopardized due to drug resis-
tance, poor pharmacokinetics and toxic side-effect. Con-
cerning this and previous studies which suggests
hybridization of two distinct moieties improve pharmaco-
kinetics as well as pharmacological activity via dual
action,[19] we intended to develop novel series of PABA-
substituted 1,3,5-triazine derivatives was developed via
microwave assisted synthesis and subsequently tested for
antimalarial activity against 3D7strain of P falciparum
(Pf). The molecules were also docked in the active site of
P falciparum dihydrofolate reductase thymidylate synthe-
tase (Pf-DHFR-TS) to explain the interaction vital for bio-
activity generation.
showed all the test compounds have good human intesti-
nal absorption.
Entire compounds showed binding energies within
the range of −153.10 to −224.14 (kcal mol−1) which
found greater than the binding energy of WR99210, the
originally bound ligand with 1J3K (−144.15 kcal mol−1
)
as shown in Table 3. Docking results showed that synthe-
sized compounds have considerable and diverse binding
affinities against mutant type (1J3K pdb) Pf-DHFR-TS via
formation of H bonds, pi-pi and pi-sigma interactions as
shown in Figure 1. Compounds 4c and 4f showed higher
antimalarial activity with low binding energies −172.32
and −160.41 (kcal mol−1) respectively due to the forma-
tion of H bond with Arg122 on interacting with COOH
group of phenyl ring linked to 1,3,5-triazine. Along with
compound 4h showed sigma-cation interaction with
COOH group at phenyl linkage with 1,3,5–triazine ring.
Compounds 4d, 4e, 4i and 4j showed pi-pi interaction
with Arg122 through the involvement of COOH group
attached on phenyl linkage with 1,3,5-triazine. Further-
more, compounds 4a and 4d showed sigma-pi interaction
with the involvement of 1,3,5-triazine ring. Compounds
4d, 4e and 4i showed mild to moderate activity by for-
ming H bond with Phe58 through COOH group of phe-
nyl linkage with 1,3,5-triazine ring. All the synthesized
compounds showed strong H-bond interaction with
Arg122, which was suggested to be responsible for the
generation mutation in Pf-DHFR-TS.[20]
2 | RESULTS AND DISCUSSION
2.1 | In-silico screening and docking
The compounds 4(a-j) were analyzed through prelimi-
nary screening filters and the results were presented in
Tables 1 and 2. All the title compounds followed
Lipinski's rule of five reflecting good oral bioactivity and
indicated no risk in case of carcinogenicity, Ames muta-
genicity and skin irritancy. The ADMET screening results
2.2 | Chemistry
The synthesis of the title compounds was achieved as
shown in Scheme 1. Initially, the synthesis was started with
the development of 4,6-dichloro-1,3,5-triazine-2-amine (2)
TABLE 1 Predicted molecular properties of the compounds using “Molinspiration cheminformatics” software
No. of
H-bond
acceptor
No. of
H-bond
donor
No. of
rotatable
bonds
Compound
code
No. of
atoms
Molecular
weight
No. of
violation
mi LogP
1.93
TPSA
126.05
126.05
126.05
126.05
126.05
126.05
126.05
126.05
126.05
126.05
Volume
222.64
256.24
273.04
282.42
282.42
297.37
294.05
294.05
294.05
291.02
4a
4b
4c
4d
4e
4f
19
21
22
25
25
25
25
25
25
25
260.26
288.31
302.34
340.32
340.32
401.22
336.36
336.36
336.36
356.77
8
8
8
8
8
8
8
8
8
8
5
5
5
5
5
5
5
5
5
5
0
0
0
0
0
0
0
0
0
0
4
6
7
5
5
5
5
5
5
5
2.81
3.37
3.76
3.81
4.45
4g
4h
4i
4.07
4.09
4.04
4j
4.30

ADHIKARI ET AL.
3
TABLE 2 Predicted toxicity risks of designed compounds using “TOPKAT” module
Mouse female
NTP carcinogen
Mouse male
NTP carcinogen
Rat male NTP
carcinogen
Compound code
Ames mutagen
Skin irritancy
4a
4b
4c
4d
4e
4f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4g
4h
4i
4j
Note: (-) indicates no toxicity risk.
TABLE 3 Docking interactions and binding energy of title
compounds 4(a-j) with 1J3K
were characterized by different physicochemical properties
like solubility, physical state, color, melting point and deter-
mination of Rf value by TLC using solvent system of metha-
nol: ethylacetate: chloroform (0.5:0.5:1).
(-) Binding
Compounds Docking
energy
(kcal mol−1
code
interaction
)
Ki (mM)
The title compounds were characterized by FT-IR, MS,
1H NMR, 13C NMR spectroscopy. The mass spectrometry
showed an ion as M+H for each compound. In the
1H NMR spectra of 4(a-j), singlet between δ 9 and 12 ppm
was assignable to the acidic proton at para position, and
singlet at δ 2.5/4.8 ppm corresponded to the C NH pro-
tons, and the protons in the aromatic ring appeared in the
range of δ 6 to 7.8 ppm as characteristic doublet signal.
13C-NMR showed the appearance of carbon signal at about
δ 166 to 185 ppm for carboxyl and at about δ 150 to
165 ppm for triazine nucleus. Characteristic FT-IR peaks
were observed in the region 3400 to 3200 cm−1 due to sec-
ondary N H stretching at 1,3,5-triazine ring, another
peak at 3000 to 2500 cm−1 (broad) due to OH group pre-
sent in COOH at phenyl ring and 1600 to 1150 cm−1 due to
C C, C N stretching of 1,3,5-triazine ring. But in some
cases, the OH stretching of COOH overlapped on C H
4a
Ser111, Ile112,
Arg122
184.31
0.392
4b
4c
4d
Arg59, Arg122
Arg122
153.10
172.32
224.14
8.450
0.868
Phe58, Ser111,
Ile112, Arg122
13.840
4e
Phe58, Phe116,
Arg122
178.13
123.630
4f
Arg122
160.41
160.55
0.205
7.260
4g
Arg59, Ser120,
Arg122
4h
4i
Arg59, Ser120,
Arg122
199.05
184.89
201.72
7.380
0.673
0.169
Phe58, Phe116,
Arg122
stretching in the region of 3000 to 2400 cm−1
.
4j
Ser111, Ile112,
Phe116, Arg122
obtained by the reaction between 2,4,6-trichloro-1,3,5-
triazine (1) with aqueous ammonia at 0ꢀC to 5ꢀC for 2 hours.
The di-substituted triazine 3(a-j) was achieved by reaction
between 4,6-dichloro-1,3,5-triazin-2-amine (2) with differ-
ent aliphatic and aromatic amines (a-j) at 27ꢀC to 31ꢀC,
1 bar pressure, 15 W power for 10 minutes in the presence
of a base. Whereas, the synthesis of title compounds 4(a-j)
was achieved by reacting di-substituted triazine 3(a-j) with
excess amount of p-aminobenzoic acid (PABA) in 10 mL
closed microwave vessel at 100ꢀC, 15 bars pressure and
40 W power for 20 minutes. The synthesized compounds
2.3 | In-vitro antimalarial activity
The concept of hybrid drugs/compounds has revolutionized
the area of antimalarial drug discovery. These compounds
are designed and developed by adjoining two or different
chemical entities with different mechanism of action/phar-
macological action via covalent linker into a single mole-
cule. Particularly, in the present study, two different
pharmacophores, for instance PABA and 1,3,5-triazine
have own antimalarial activity via different mechanisms
which results in enhanced activity.[21] The results of In-vitro

4
ADHIKARI ET AL.
FIGURE 1 Docking poses of
p-aminobenzoic acid-substituted
1,3,5-triazine derivatives 4(a-j) in
mutant type Pf-DHFR-TS
antimalarial activity of synthesized compounds 4(a-j) were
identified against chloroquine-sensitive (3D7) strains at
5 μg mL−1 using chloroquine as standard and the results
are shown in Table 4. All synthesized derivatives 4(a-j)
showed IC50 in the range of 4.46 to 79.72 μg mL−1 along
with inhibition of parasitemia in terms of % dead rings and
trophozoites from 3% to 47% at 5 μg mL−1 dose. Compound
4f having a para-bromo showed high antimalarial activity

ADHIKARI ET AL.
5
R
N
Cl
Cl
R
N
N
N
N
i
ii
N
N
iii
N
N
HN
a-j
NH2
Cl
N
Cl
N
1
Cl
NH2
Cl
N
NH2
2
3(a-j)
COOH
where R=
4(a-j)
H
N
4a
4b
4g
N
H
H
N
H
N
4c
N
H
4h
F
H
N
4d
H
4i
4j
N
H
N
4e
4f
F
N
Cl
H
H
N
Br
SCHEME 1 Reagents and conditions: (i) Aq. Ammonia, Diethyl ether, 0ꢀC to 5ꢀC, stirring for 2 hours. (ii) Various amines (a-j),
1,4-dioxane, 27ꢀC to 31ꢀC, 1 bar, 15 W, 10 minutes. (iii) PABA, Dimethyl sulfoxide (DMSO), 100ꢀC, 15 bars, 40 W, 20 minutes
with IC50 4.46 μg mL−1 along with 47% parasitaemia at
5 μg mL−1 dose level. Whereas, the replacement of aromatic
with aliphatic amine, for instance compound 4a and 4b, the
activity was reduced considerably. The butylamino
substituted compound 4c showed good antimalarial activity
with IC50 6.69 μg mL−1. The presence of propylamine (4b)
in place of butylamine (4c) showed mild antimalarial activ-
ity. The 3-chlorophenyl substituted compound 4j showed
moderate antimalarial activity with IC50 7.09 μg mL−1
along with 31% of parasitaemia at 5 μg mL−1 dose. Com-
pounds 4g, 4h and 4i containing electron-donating groups,
such as meta-CH3, para-CH3, and ortho CH3, respectively,
showed mild to moderate antimalarial activity with
parasitaemia inhibition 2% to 6.5%. Compounds 4d and 4e
containing electron-withdrawing groups, such as, ortho-
fluoro and para-fluoro, respectively, also showed mild anti-
malarial activity with 7% to 10% of parasitaemia at
5 μg mL−1 dose level. All compounds were tested for cyto-
toxicity against MCF 12A (normal epithelial cell) by the
MTT assay method and found non-toxic at the highest test
dose of 150 μg mL−1.
SAR study indicated that substitution of aliphatic
amine such as propyl amine (4b) and butyl amine (4c)
at 1,3,5-triazine appears to influence the antimalarial
activity, whereas on replacing aliphatic amine by aro-
matic amine viz 2-fluoroaniline (4d), 4-fluoroaniline
(4e), para-toluidine (4h), meta-toluidine (4g), ortho-
toluidine (4i) showed reduction in the activity. These
results were found in agreement with Redley et al[22]
which showed that, presence of aliphatic chain on
4-aminoquinoline from carbon chain length from two
to six increase the antimalarial activity. The above
result indicated that smaller group favors antimalarial
activity while bigger groups were detrimental for the
activity. This might be due to steric hindrance which
interferes with the binding of ligand to the receptor. On
the other hand, compounds with electron-withdrawing
group (para-Br, 4f) and (meta-Cl, 4j) at phenyl ring
of 1,3,5-triazine showed good antimalarial activity.
These results were further found in agreement with our
earlier study[23] and Pathak et al,[24] where electron
withdrawing group enhances antimalarial activity. The

6
ADHIKARI ET AL.
activity showed to be reduced on introduction of
electron-donating groups, such as, 4g, 4h and 4i having
meta-CH3, para-CH3 and ortho-CH3, respectively. Based
on the above observation the SAR could be summarized
in Figure 2.
3 | EXPERIMENTAL
3.1 | In-silico screening
The in-silico study included design and screening of virtual
library, validation of the docking protocol by root mean
square deviation (RMSD) calculation and docking of
designed compounds at the active binding sites of the target
protein. Preliminary screening of the compounds was done
using software “Molinspiration cheminformatics”[25] and
“Accelrys Discovery Studio version 3.0.” Molinspiration che-
minformatics provides prediction of different molecular
properties (Table 1) while TOPKAT module of Accelrys Dis-
covery Studio version 3.0 predicts toxicity risks of the com-
pounds based on their molecular structure (Table 2).
According to Accelrys Discovery Studio tutorial, the three
models (Table 2) for determining carcinogenicity on rodents
were based on the critical review of technical reports on
rodent carcinogenicity conducted by National Cancer Insti-
tute and National Toxicology Program (NTP) utilizing
hybrid mice and inbred rats.
TABLE 4 In-vitro antimalarial evaluation of title compounds
4(a-j) against chloroquine-sensitive (3D7) strains
% Dead rings, trophozoitesa
Serial
No.
3D7
IC50
Compound code (5 μg mL−1 dose) (μg mL−1
)
1
4a
4b
4c
4d
4e
4f
3.12 0.16
22.38 0.72
37.24 0.88
7.56 0.20
10.62 0.44
47.48 1.02
2.08 0.09
6.56 0.32
4.06 0.12
31.62 0.22
16.82 0.54
2
9.89 0.36
6.69 0.28
11.61 0.42
10.06 0.21
4.46 0.18
79.72 1.66
12.98 0.12
14.85 0.48
7.09 0.22
NIL
3
4
5
6
7
4g
4h
4i
8
9
3.2 | ADME prediction
10
11
4j
Chloroquine (CQ) 50.54 0.48
(0.7 μg mL−1
The in-built ADMET model of Accelrys Discovery Studio
version 3.0 for predicting human intestinal absorption of
the compounds was developed using descriptors that
)
aData are the means SD of n = 3.
FIGURE 2 Structure–activity
relationship of hybrid PABA
substituted–1,3,5-triazine derivatives

ADHIKARI ET AL.
7
FIGURE 3 Confidence ellipses
of human intestinal absorption for
selected compounds
include AlogP98 and 2D polar surface area (PSA_2D)
which was used in plotting the confidence ellipses. Com-
pounds that were outside 95% and 99% ellipse region
(Figure 3) are poorly absorbed compounds (<30%
absorbed).[26,27]
3.3.4 | Binding energy calculation
The average of the binding energies of the five poses of
each complex was calculated.
3.4 | Chemistry
3.3 | Molecular docking studies
3.3.1 | Protein preparation
The melting points were determined using melting point
instrument M-560: Buchi Switzerland, FT-IR spectra
were recorded using Bruker ALPHA ECO-ATR, UV spec-
tra were recorded using UV/Visible Spectrophotometer
The compounds that were passed through both the pre-
liminary screening filters were docked at the binding sites
of the mutant Pf-DHFR protein using CDOCKER dock-
ing protocol of Accelrys Discovery Studio version 3.0. The
crystal structure of quadruple mutant Pf-DHFR-TS com-
plex was obtained from RCSB Protein Data Bank as PDB
entry code: 1J3K. CDOCKER docking protocol was vali-
dated by RMSD calculation, which was less than 2 Å in
all cases. Co-crystallized ligand WR99210 and water mol-
ecules were removed and cofactor NADPH was allowed
to retain. Finally, receptor was prepared according to the
requirements of the docking protocol.
(Lab India analytical: UV 3200), H NMR and 13C NMR
1
spectra were recorded respectively at 400 MHz and
100 MHz in DMSO-d6 using Bruker Avance II 400 NMR
Spectrometer along with TMS as the internal standard
where chemical shift (δ) values were given in parts per
million (ppm). Molecular mass was recorded on Waters
Q-TOF Micromass Spectrometer. Compounds were syn-
thesized using microwave synthesizer (CEM, Discover
System Model No. 908010). All commercially available
chemicals and reagents with specifications were directly
used without further purification.
The general synthetic procedure of the desired com-
pounds 2, 3(a-j) and 4(a-j) were accomplished by nucleo-
philic substitution according to the reaction Scheme 1.
3.3.2 | Ligand preparation
The structures of the ligands were drawn in ChemDraw
ultra 8.0 and energy minimization was done by applying
CHARMm forcefield to create a stable structure.
3.4.1 | Synthetic procedure for
4,6-dichloro-1,3,5-triazin-2-amine (2)
0.02 mol of 2,4,6-trichloro-1,3,5-triazine (1) was dissolved
in 30 mL of ice-cold diethyl ether in a round bottom flask
followed by addition of equimolar ice-cold aq. ammonia
solution (25%). The reaction mixture was stirred for
2 hours in presence of NaHCO3 as a base maintaining at
0ꢀC to 5ꢀC. The product was obtained as (2) white needle
3.3.3 | Docking
The most stable conformer of ligands was docked into
the active site of protein 1J3K, which uses CHARMm
based molecular dynamics program.

8
ADHIKARI ET AL.
shaped crystals (84% yield) with melting point of 229ꢀC
to 231ꢀC.
4H, NH), 3.07 to 3.03 (t, J = 16 Hz, 2H, CH2CH2), 6.31 to
6.29 (d, J = 8 Hz, 2H, Ar H), 7.35 to 7.33 (d, J = 8 Hz,
2H, Ar H), 10.14 (s, 1H, COOH); 13C NMR (100 MHz,
DMSO-d6) δ: 167.55, 166.88, 152.76, 131.05, 129.87,
117.06, 112.63, 78.82, 21.95, 20.30, 11.07; MS For
C13H16N6O2 (M+H)+: 289.3.
3.4.2 | General procedure for synthesis
of disubstituted 1,3,5-triazine derivatives 3
(a-j)
4-(4-amino-6-(butylamino)-1,3,5-triazin-2-ylamino)
benzoic acid (4c)
0.002 mol of 4,6-dichloro-1,3,5-triazin-2-amine (2) was
dissolved in 5 mL of 1,4-dioxane followed by addition of
0.004 mol of various amines (a-j) and stirred in closed
vessel in microwave synthesizer at 27ꢀC to 31ꢀC, 1 bar
pressure, 15 W power for 10 minutes in the presence of
NaHCO3 as a base. The reaction mixture was dried and
the product was recrystallized with suitable solvent to
afford compounds 3(a-j).
Off-white solid; Yield 66%; Rf 0.5; mp 181ꢀC; FTIR
(cm−1): 3248 (N H stretch, secondary), 3091
(OH stretch, COOH), 2951, 2861 (C H stretch), 1626
(C C stretch), 1536, 1406 (C C aromatic stretch), 1358
(C H aromatic stretch), 1282, 1166 (C N aromatic
1
stretch); λmax 240 nm; H NMR (400 MHz, DMSO-d6) δ:
0.91 to 0.88 (t, J = 12 Hz, 3H, CH2CH3), 1.34 to 1.30 (t,
J = 16 Hz, 2H, CH2CH2), 1.53 to 1.46 (m, 2H,
CH2CH2CH2), 3.29 to 3.20 (m, 2H, CH2CH2CH3), 4.84 (s,
4H, NH), 6.57 to 6.55 (d, J = 8 Hz, 2H, Ar H), 7.65 to
7.63 (d, J = 8 Hz, 2H, Ar H), 9.28 (s, 1H, COOH); 13C
NMR (100 MHz, DMSO-d6) δ: 167.58, 165.29, 152.89,
131.10, 129.85, 117.04, 112.51, 78.74, 30.81, 19.56, 13.71;
MS For C14H18N6O2 (M+H)+: 302.61.
3.4.3 | General synthetic procedure for
title compounds 4(a-j)
In the final step, corresponding to the synthesis of
2,4,6-trisubstituted-1,3,5-triazine derivatives 4(a-j) using
microwave irradiation. To this 0.001 mol of 3(a-j) was
dissolved in 2 mL DMSO followed by addition of excess
of p-aminobenzoic acid (PABA) and was stirred in 10 mL
closed microwave vessel at 100ꢀC, 15 bars pressure and
40 W power for 20 minutes. The desired products 4(a-j)
were recrystallized.
4-(4-amino-6-(2-fluorophenylamino)-1,3,5-triazin-
2-ylamino)benzoic acid (4d)
Off-white solid; Yield 64%; Rf 0.72; mp 204ꢀC; FTIR
(cm−1): 3359 (N H stretch, secondary), 3065
(OH stretch, COOH), 2933, 2833, 2527 (C H stretch),
1596, 1505, 1447, 1413 (C C aromatic stretch), 1247,
1166, 1108 (C N aromatic stretch); λmax 290 nm; 1H
NMR (400 MHz, DMSO-d6) δ: 2.56 (s, 4H, NH), 6.59 to
6.58 (d, J = 4 Hz, 2H, Ar H), 7.22 to 7.06 (m, 4H, Ar H),
7.66 to 7.64 (d, J = 8 Hz, 2H, Ar H), 9.55 (s, 1H, COOH);
13C NMR (100 MHz, DMSO-d6) δ: 167.60, 167.19, 164.80,
164.13, 152.86, 144.23, 131, 129.87, 125.53, 123.78, 118.60,
117.08, 115.14, 112.54; MS For C16H13FN6O2 (M+H)+:
340.31.
4-(4-amino-6-(methylamino)-1,3,5-triazin-2-ylamino)
benzoic acid (4a)
Off-white solid; Yield 59%; Rf 0.46; mp 260ꢀC; FTIR
(cm−1): 3246 (N H stretch, secondary), 3100
(OH stretch, COOH, broad), 2937 (C H stretch), 1590
(N H bend), 1559, 1494 (C C stretch, aromatic), 1341,
1
1241, 1167 (C N stretch, aromatic); λmax 296 nm; H
NMR (400 MHz, DMSO-d6) δ: 2.57 (s, 3H, CH3), 4.65 (s,
2H, 2NH), 4.84 (s, 2H, NH2), 6.53 to 6.50 (d, J = 12 Hz,
2H, Ar H), 7.10 to 7.08 (d, J = 8 Hz, 2H, Ar H), 11.36
(s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6) δ: 167.60,
165.75, 163.51, 152.88, 147.73, 129.90, 114.95, 112.52,
27.26; MS For C11H12N6O2 (M+H)+: 260.65.
4-(4-amino-6-(4-fluorophenylamino)-1,3,5-triazin-
2-ylamino)benzoic acid (4e)
Green solid; Yield 64%; Rf 0.52; mp 150ꢀC; FTIR (cm−1):
3221 (N H stretch, secondary), 3086 (OH stretch,
COOH), 2864 (C H stretch), 1599, 1497, 1413 (C C aro-
matic stretch), 1315 (C H aromatic stretch), 1283, 1217,
1168 (C N aromatic stretch); λmax 230 nm; 1H NMR
(400 MHz, DMSO-d6) δ: 2.57 (s, 4H, NH), 6.63 to 6.61 (d,
J = 8 Hz, 2H, Ar H), 7.16 to 7.04 (m, 4H, Ar H), 7.69 to
7.66 (d, J = 12 Hz, 2H, Ar H), 9.58 (s, 1H, COOH); 13C
NMR (100 MHz, DMSO-d6) δ: 167.53, 167.19, 163.61,
158.95, 151.99, 144.15, 131.08, 129.92, 118.86, 117.76,
114.75, 113.08, 111.09; MS For C16H13FN6O2 (M+H)+:
340.31.
4-(4-amino-6-(propylamino)-1,3,5-triazin-2-ylamino)
benzoic acid (4b)
Off-white solid; Yield 67%; Rf 0.48; mp 154ꢀC; FTIR
(cm−1): 3345 (N H stretch), 3219 (OH stretch, COOH),
2963 (C H stretch), 1597 (N H bend), 1554, 1514 (C C
aromatic stretch), 1372 (C H aromatic stretch), 1242,
1167 (C N aromatic stretch); λmax 273 nm; 1H NMR
(400 MHz, DMSO-d6) δ: 0.65 to 0.62 (t, J = 12 Hz, 3H,
CH2 CH3), 1.37 to 1.27 (m, 2H, CH2CH2CH3), 2.25 (s,

ADHIKARI ET AL.
9
4-(4-amino-6-(4-bromophenylamino)-1,3,5-triazin-2-
ylamino)benzoic acid (4f)
Ar H), 9.27 (s, 1H, COOH); 13C NMR (100 MHz, DMSO-
d6) δ: 167.63, 152.60, 136.58, 133.52, 131.12, 128.67,
121.95, 120.75, 117.31, 112.71, 79, 20.47; MS For
C17N16N6O2 (M+H)+: 336.35.
Off-white solid; Yield 64%; Rf 0.76; mp 277ꢀC; FTIR
(cm−1): 3355 (N H stretch, secondary), 2914
(OH stretch, COOH), 2619 (C H stretch), 1669 (C C
stretch), 1593, 1562, 1483, 1401 (C C aromatic stretch),
1309 (C H aromatic stretch), 1239, 1163, 1110 (C N aro-
matic stretch); λmax 233 nm; 1H NMR (400 MHz,
DMSO-d6) δ: 2.57 (s, 4H, NH), 6.61 to 6.58 (d, J = 12 Hz,
2H, Ar H), 7.47 to 7.38 (m, 4H, Ar H), 7.68 to 7.66 (d,
J = 8 Hz, 2H, Ar H), 11.24 (s, 1H, COOH); 13C NMR
(400 MHz, DMSO-d6) δ: 167.56, 167.13, 163.83, 152.73,
149.85, 139.12, 130.90, 129.94, 122.10, 117.16, 112.60; MS
For C16H13BrN6O2 (M+H)+: 401.19.
4-(4-(o-toluidino)-6-amino-1,3,5-triazin-2-ylamino)
benzoic acid (4i)
White solid; Yield 62%; Rf 0.55; mp 180ꢀC; FTIR (cm−1):
3455, 3365 (N H stretch), 2913 (OH stretch, COOH),
2534 (C H sretch), 1665 (C C stretch), 1593, 1505, 1411
(C C aromatic stretch), 1278, 1164, 1118 (C N aromatic
1
stretch); λmax 295 nm; H NMR (400 MHz, DMSO-d6) δ:
2.21 (s, 3H, CH3), 5.80 (s, 4H, NH), 6.56 to 6.54 (d,
J = 8 Hz, 2H, Ar H), 6.72 to 6.70 (d, J = 8 Hz, 2H,
Ar H), 7.00 to 6.98 (d, J = 8 Hz, 2H, Ar H), 7.71 to 7.62
(m, 2H, Ar H), 11.84 (s, 1H, COOH); 13C NMR:
(100 MHz, DMSO-d6) δ: 167.55, 165.01, 153, 137.30,
131.15, 129.91, 125.57, 124.23, 116.94, 112.54, 111.41,
78.83, 18.03; MS For C17H16N6O2 (M+H)+: 336.35.
4-(4-(m-toluidino)-6-amino-1,3,5-triazin-2-ylamino)
benzoic acid (4g)
Off-white solid; Yield 65%; Rf 0.65; mp 173ꢀC; FTIR
(cm−1): 3269 (N H stretch, secondary), 2921
(OH stretch, COOH), 2853, 2584 (C H stretch), 1599,
1479, 1414 (C C aromatic stretch), 1360 (C H aromatic
stretch), 1254, 1170, 1122 (C N aromatic stretch); λmax
4-(4-amino-6-(3-chlorophenylamino)-1,3,5-triazin-2-
ylamino)benzoic acid (4j)
1
(nm) 290; H NMR (400 MHz, DMSO-d6) δ: 2.34 (s, 3H,
Off-white solid; Yield 64%; Rf 0.55; mp 196ꢀC; FTIR
(cm−1): 3354 (N H stretch), 2890 (OH stretch, COOH),
2617 (C H stretch), 1632 (C C stretch), 1596, 1574,
1514, 1420 (C C aromatic stretch), 1243, 1170, 1109
(C N aromatic); λmax 248 nm; 1H NMR (400 MHz,
DMSO-d6) δ: 2.53 (s, 4H, NH), 6.92 to 6.90 (d, J = 8 Hz,
2H, Ar H), 7.03 to 7.01 (d, J = 8 Hz, 1H, Ar H), 7.16 to
7.14 (d, J = 8 Hz, 1H, Ar H), 7.31 to 7.27 (t, J = 16 Hz,
1H, Ar H), 7.79 to 7.75 (d, J = 16 Hz, 1H, Ar H), 7.93 to
7.88 (d, J = 20 Hz, 2H, Ar H), 9.64 (s, 1H, COOH); 13C
NMR (100 MHz, DMSO-d6) δ: 167.14, 163.23, 149.83,
147.12, 140.86, 133.05, 130.95, 121.58, 119.87, 118.70,
116.20, 78.79; MS For C16H13ClN6O2 (M+H)+: 356.77.
CH3), 2.55 (s, 4H, NH), 6.58 to 6.56 (d, J = 8 Hz, 2H,
Ar H), 6.82 to 6.79 (t, J = 12 Hz, 1H, Ar H), 7.20 to 7.14
(m, 3H, Ar H), 7.68 to 7.66 (d, J = 8 Hz, 2H, Ar H),
9.33 (s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6) δ:
167.58, 163.98, 152.89, 139.66, 139.03, 137.43, 131.12,
128.09, 123.22, 122.69, 117.05, 112.55, 78.77, 21.20; MS
For C17H16N6O2 (M+H)+: 336.35.
4-(4-(p-toluidino)-6-amino-1,3,5-triazin-2-ylamino)
benzoic acid (4h)
Off-white solid; Yield 66%; Rf 0.55; mp 182ꢀC; FTIR
(cm−1): 3460, 3360 (N H stretch), 2827 (OH stretch,
COOH, broad) (C H stretch), 2539, 1657 (C C stretch),
1589, 1495, 1408 (C C aromatic stretch), 1279, 1240,
1164 (C N aromatic stretch); λmax 242 nm; 1H NMR
(400 MHz, DMSO-d6) δ: 2.29 (s, 3H, CH3), 2.56 (s, 4H,
NH), 6.60 to 6.58 (d, J = 8 Hz, 1H, Ar H), 7.09 to 7.07 (d,
J = 8 Hz, 2H, Ar H), 7.68 to 7.65 (d, J = 12 Hz, 2H,
3.5 | In-vitro antimalarial activity
In vitro antimalarial screening of synthesized compounds
was performed against chloroquine-sensitive P falciparum
FIGURE 4 A, Negative control
(3D7 strain) and B, Drug treated
parasite (3D7 strain)

10
ADHIKARI ET AL.
strain (3D7) using Giemsa stained slide method (Light
microscopy).
ORCID
Hans Raj Bhat https://orcid.org/0000-0002-9643-4916
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ACKNOWLEDGMENTS
The financial support from the Department of Science
and Technology (DST), New Delhi, India is gratefully
acknowledged (Grant No. EMR/2017/000708) and the
authors also are thankful to S.A.I.F., Panjab University,
Chandigarh, India for providing spectroscopic data.
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11
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How to cite this article: Adhikari N, Kashyap A,
Shakya A, et al. Microwave assisted synthesis,
docking and antimalarial evaluation of hybrid
PABA-substituted 1,3,5-triazine derivatives.
J Heterocyclic Chem. 2020;1–11. https://doi.org/10.
1002/jhet.3955
SUPPORTING INFORMATION
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