Journal of Molecular Structure 1156 (2018) 43e50

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Journal of Molecular Structure

journal homepage: http://www.elsevier.com/locate/molstruc

Synthesis, spectroscopic characterization, antimicrobial evaluation

and molecular docking study of novel triazine-quinazolinone based
hybrids
Mohammad Dinari a, *, Fateme Gharahi a, Parvin Asadi b
a
Department of Chemistry, Isfahan University of Technology, Isfahan, 84156-83111, Islamic Republic of Iran
b
Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, 81746-73461,
Islamic Republic of Iran

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

Article history: A new series of 1,3,5-triazine incorporating aromatic quinazolinone moieties as a potential antimicrobial
Received 15 June 2017 agents is reported. The first chlorine group of the cyanuric chloride (1) was replaced by aniline and the
Received in revised form second one was replaced by various aromatic amines. The prepared monochlorotriazine was allowed to
27 October 2017
react with hydrazine and subsequently it was reacted with 2-methyl-4H-benzo[1,3]oxazin-4-one to
Accepted 21 November 2017
Available online 21 November 2017
obtain novel triazine-quinazolinone based hybrids (9a-f). The chemical structure and purity of the hybrid
compounds were evaluated by different techniques such as thin layer chromatography, melting point,
Fourier-transform infrared (FTIR), 1H and 13C NMR spectra and elemental analysis. Antimicrobial activity
Keywords:
s-Triazine
of the hybrid compounds were study by three Gram-negative bacteria (Salmonella entritidis, Escherichia
Antimicrobial agents coli, Pseudomonas aeruginosa) and three Gram-positive bacteria (Staphylococcus aureus, Listeria mono-
Docking simulation citogenes, Bacillus subtilis) as well as Candida albicansas a yeast-like fungus using the serial broth
Quinazolinone dilution method. Among them, compound 9d with benzenesulfonamide group showed higher antimi-
Hybrids crobial activity with a minimum inhibitory concentration (MIC) value of 16 mg/mL. Furthermore, com-
pounds 5d, 9a and 9b showed good activity against several tested strains. In addition, docking simulation
was perform to position best antibacterial compounds in to the S. aureus dihydrofolate reductase (DHFR)
active site to determine the probable binding conformations.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction been already achieved significant attention because of their anti-

Heterocyclic compounds have a critical role in synthetic drugs
and biological processes. The tri-substituted 1,3,5-triazine hetero-
cyclic analogs is a very important pharmacophore due to its pres-
ence in an enormous amount of bioactive compounds [1].
Biologically active tri-substituted triazines showed different po-
tential such as anti malarial [2,3], antibacterial [4e6], anticancer
[7,8], antitubercular [9] and etc. Previous studies show that s-
triazine core molecules display excellent antimicrobial activity in
terms of antibacterial and antifungal [10].
On the other hand, quinazolinone scaffold and their derivatives
constitute an important class of biologically active compound.
Various classes of quinazolinones with different structures have
* Corresponding author.
E-mail addresses: dinari@cc.iut.ac.ir, mdinary@gmail.com (M. Dinari).
microbial [11,12], antiviral [13], antitubercular [14,15], anti-
inflammatory [16], and anticancer activities [17,18]. Study on
structure activity relationship of quinazolinone derivatives have
showed that substitution at positions 2 and 3 of the quinazolinone
ring can improve their antimicrobial activities [19]. In addition, the
presence of substituted aromatic ring at position 3 and amine,
methyl or thiol groups at position 2 are helpful for antimicrobial
activities [20e22].
Joining two or more biologically active pharmacophores in a
single molecular framework might result in pharmaceutically
important hybrid molecules which could address the active site of
different targets and/or offer the possibility of overcoming drug
resistance or reducing the unwanted side effects [23]. Recently,
novel hybrid compounds based on heterocyclic pharmacophores
have been synthesized and also proved to show different biological
activities [24,25]. Previously, a series of hybrid quinazolinee-
triazine derivatives were designed and synthesized from cyanuric

https://doi.org/10.1016/j.molstruc.2017.11.087
0022-2860/© 2017 Elsevier B.V. All rights reserved.
44 M. Dinari et al. / Journal of Molecular Structure 1156 (2018) 43e50
chloride and anthranilic acid through sequential reactions, which
contain different pharmacophores like quinazoline and substituted
diaryltriazine linked with ethylene diamine [26]. In vitro antimi-
crobial activity was evaluated against four bacteria (Staphylococcus
aureus, Bacillus cereus, Pseudomonas aeruginosa, Klebsiella pneu-
moniae) and two fungi (Aspergillus clavatus, Candida albicans). From
the bioassay results, it can be stated that all derivatives showed
appreciable antimicrobial activities. Generally the incorporation of
quinazoline moiety linked to triazine via ethylene diamine is
beneficial to antimicrobial activity [26]. Another s-triazine based
hybrid compounds have also been proved to show inhibitory ac-
tivity on Dihydrofolate Reductase (DHFR) metabolism [27]. This
enzyme has profound impact on the biosynthesis of biomolecules
that are essential for cell proliferation. It catalyzes the reduction of
dihydrofolate (DHF) to tetrahydrofolates (THF), using nicotinamide
adenine dinucleotide phosphate (NADPH) as a cofactor [28]. Thus,
the inhibition of DHFR can result in the depletion of the intracel-
lular THF pool and prevents biosynthesis of thymidine, DNA, RNA
and protein. Fortunately, differ structurally DHFR from species to
species have allowed the expansion of very DHFR inhibitors for
some bacteria and some parasitic [29].
Considering the antibacterial activities of s-triazine and quina-
zolinone derivatives, in this study we have combined these two
heterocyclic moieties in a novel chemical framework. The resulting
hybrid compounds were characterized by different techniques and
their antimicrobial activities against three Gram-positive bacteria,
three Gram-negative bacteria and also one yeast-like fungi was
investigated. To achieve insight of necessary key structural re-
quirements for antimicrobial activity, molecular docking study was
consummated.
2. Experimental
2.1. Materials
All chemicals and solvents used in this work were purchased
commercially from the Merck Chemical Co. and Aldrich Chemical Co.
2-Aminobenzoic acid or anthranilic acid, cyanuric chloride, 4-
chlorobenzenamine, 4-aminobenzonitrile, 4-methoxybenzenamine,
sulfanilamide, aniline, 4-nitrobenzenamine and hydrazine hydroxide
(NH2NH2$H2O) were used without any extra purification.
2.2. Characterization techniques
A JASCO 680 (Japan) Fourier transform infrared (FT-IR) spec-
trophotometer was used to study the chemical structure of the
materials. Proton nuclear magnetic resonance (1H NMR; 500 MHz)
and carbon nuclear magnetic resonance (13C NMR; 125 MHz)
spectra were recorded in DMSO-d6 on a BrukerAvance spectrom-
eter. The chemical shifts are reported in the d scale in ppm. Elec-
trothermal 9200 melting point apparatus was used to determine
the melting points (m.p.) of the compounds. Pre-coated silica gel 60
F254 aluminum plates (Merck, Germany) was performed on
analytical thin-layer chromatography.
2.3. Synthesis of 4,6-dichloro-1,3,5-triazin-2-ylphenylamine (2)
The first chlorine group of the cyanuric chloride (1) was
replaced by aniline according to the reported producer [30,31]. In
brief, to a solution of cyanuric chloride (9.2 g, 0.05 mol) in 150 mL of
acetic acid, 4.65 g (0.05 mol) of aniline was added slowly and 7.2 mL
(0.05 mol) of triethylamine (Et3N) was added to the mixture at 5
C.
Then, it was stirred for 1 h and diluted with 500 mL of brine. The
solid product was filtered, and recrystallized from 400 mL of n-
heptane. Yield: 67%; m.p. ¼ 136e137
C. FT-IR (KBr, cm1) n: 3363
(NH), 1563 (C]C), 1552 (C]N).
2.4. General procedure for the synthesis of compounds 4a-f
The second chlorine group of the cyanuric chloride was replaced
by different aromatic amine such as 4-chlorobenzenamine (3a), 4-
aminobenzonitrile (3b), 4-methoxybenzenamine (c), sulfanilamide
(3d), aniline (3e), and 4-nitrobenzenamine (3f) to formed com-
pounds 4a-f. First, 5.5 mmol of compounds 3a-f were added to a
stirred solution of compound 2 in the mixture of 50 mL of AcOH and
15 mL of H2O in the presence of sodium acetate (0.81 g; 6 mmol).
Then, the reaction mixture was stirred until the whole solids were
dissolved and it was contentious for 12 h. The progress of the re-
action was monitored by TLC. Upon completion, the solid product
were obtained by filtration, washed with boiling water and
recrystallized from isopropanol.
6-Chloro-N2-(4-chlorophenyl)-N4-phenyl-1,3,5-triazine-2,4-
diamine (4a): Yield: 82%, white solid, m.p. ¼ 210
C; IR (KBr, cm1)
n: 3397, 3259 (NH), 3179 (AreH), 1610 (C]C), 1570 (C]N); 1H NMR
(500 MHz, DMSO-d6) d: 10.1e10.4 (br, 2H, NH), 7.66 (m, 4H, AreCH),
7.37 (m, 4H, AreCH) and 6.9 (m, 1H, AreCH).
4-((4-Chloro-6-(phenylamino)-1,3,5-triazin-2-yl)amino)benzoni-
trile (4b): Yield: 83%, white solid, m.p. ¼ 217
C; IR (KBr, cm1) n:
3329 (NH), 3065 (AreH), 2218 (CN), 1615 (C]C), and 1571 (C]N).
6-Chloro-N2-(4-methoxyphenyl)-N4-phenyl-1,3,5-triazine-2,4-
diamine (4c): Yield: 91%, white solid, m.p. ¼ 167
C; IR (KBr, cm1) n:
3370, 3262 (NH), 3113 (AreH), 2839 (CH3), 1612 (C]C), 1577 (C]N)
and 1178 (CeO).
4-((4-Chloro-6-(phenylamino)-1,3,5-triazin-2-yl)amino)benzene-
sulfonamide (4d): Yield: 92%, white solid, m.p. ¼ 268
C; IR (KBr,
cm1) n: 3398, 3209 (NH), 3358e3282 (NH2), 3060 (AreH), 1612
(C]C), 1571 (C]N), and 1326, 1152 (SO2).
6-Chloro-N2,N4-diphenyl-1,3,5-triazine-2,4-diamine (4e): Yield:
90%, white solid, m.p. ¼ 200
C; IR (KBr, cm1) n: 3267 (NH), 3107
(AreH), 1615 (C]C), and 1574 (C]N).
6-Chloro-N2-(4-nitrophenyl)-N4-phenyl-1,3,5-triazine-2,4-
diamine (4f): Yield: 79%, yellow solid, mp. ¼ 221
C; IR (KBr, cm1)
n: 3374e3338 (NH), 3065, 3159 (AreH), 1619 (C]C), 1588 (C]N),
and 1533, 1331 (NO2).
2.5. General procedure for the synthesis of compounds 5a-f
The last chlorine group of the 1 was replaced by hydrazine
molecule. In this regards, a suspension of compounds 4a-f in hy-
drazine hydrate was refluxed for 10e12 h. The excess hydrazine
hydrate was evaporated under vacuum and the residue was washed
with water to give compounds 5a-f.
N2-(4-Chlorophenyl)-6-hydrazinyl-N4-phenyl-1,3,5-triazine-2,4-
diamine (5a): Yield: 87%, white solid, m.p. ¼ 130
C; FT-IR (KBr,
cm1) n: 3038e3400 (NH, NH2, CH Aromatic); 1H NMR (500 MHz,
DMSO-d6) d: 9.0e9.55 (br, 2H, NH), 8.2 (s, 1H, NH), 7.7 (m, 4H,
AreH), 7.24 (m, 4H, AreH), 6.9 (m, 1H, AreCH), 4.25e4.7 (s, 2H,
NH2); Anal. Calcd. For C15H14ClN7: C, 54.97; H, 4.31; N, 29.91. Found:
C, 54.28; H, 4.20; N, 29.47.
4-((4-Hydrazinyl-6-(phenylamino)-1,3,5-triazin-2-yl)amino)ben-
zonitrile (5b): Yield: 83%, orange solid, m.p. ¼ 102
C; FT-IR (KBr,
cm1) n: 3050e3390 (NH, NH2, CH Aromatic), 2214 (CN); 1H NMR
(500 MHz, DMSO-d6) d: 8.9e9.5 (br, 2H, NH), 8.3 (s, 1H, NH), 7.3e7.9
(m, 8H, AreH), 6.97 (m, 1H, AreCH), 4.1e4.5 (s, 2H, NH2); Anal.
Calcd. For C16H14N8: C, 60.37; H, 4.43; N, 35.20. Found: C, 59.71; H,
4.69; N, 35.07.
6-Hydrazinyl-N2-(4-methoxyphenyl)-N4-phenyl-1,3,5-triazine-
2,4-diamine (5c): Yield: 89%, white solid, m.p. ¼ 126
C; FT-IR (KBr,
cm1) n: 3034e3480 (NH, NH2, CH-Aromatic), 2830 (CH-Aliphatic),
1073 (OeCH3); 1H NMR (500 MHz, DMSO-d6) d: 8.7e9.35 (br, 2H,
 M. Dinari et al. / Journal of Molecular Structure 1156 (2018) 43e50
NH), 8.05 (s, 1H, NH), 7.77 (m, 2H, AreH), 7.64 (m, 2H, AreH), 7.2 (m,
2H, AreH), 6.9 (m, 1H, AreCH), 6.7 (d, 2H, AreH), 4.1e4.4 (s, 2H,
NH2), 3.7 (s, 3H, OCH3); Anal. Calcd. For C16H17N7O: C, 59.43; H,
5.30; N, 30.32. Found: C, 59.37; H, 5.37; N, 30.12.
4-((4-Hydrazinyl-6-(phenylamino)-1,3,5-triazin-2-yl)amino)ben-
zenesulfonamide (5d): Yield: 89%, white solid, m.p. ¼ 161
C; FT-IR
(KBr, cm1) n: 3035e3446 (NH, NH2, CH Aromatic), 1152, 1358
(SO2); 1H NMR (500 MHz, DMSO-d6) d: 8.9e9.6 (br, 2H, NH), 8.25 (s,
1H, NH), 8.0 (m, 2H, AreH), 7.8 (m, 2H, AreH), 7.66 (m, 2H, AreH),
7.26 (m, 2H, AreH), 7.17 (s, 2H, NH2), 6.98 (m, 1H, AreCH), 4.0e4.9
(s, 2H, NH2); Anal. Calcd. For C15H16N8O2S: C, 48.38; H, 4.33; N,
30.09; S, 8.61. Found: C, 47.35; H, 4.48; N, 29.41; S, 7.96.
6-Hydrazinyl-N2,N4-diphenyl-1,3,5-triazine-2,4-diamine (5e):
Yield: 90%, white solid, m.p. ¼ 145
C; FT-IR (KBr, cm1) n:
3030e3324, 3400 (NH, NH2, CH-Aromatic), 1H NMR (500 MHz,
DMSO-d6) d: 8.9e9.2 (br, 2H, NH), 8.1 (s, 1H, NH), 7.8 (m, 4H, AreH),
7.2 (m, 4H, AreH), 6.9 (m, 1H, AreCH), 4.2e4.35 (s, 2H, NH2); Anal.
Calcd. For C15H15N7: C, 61.42; H, 5.15; N, 33.43. Found: C, 61.34; H,
5.07; N, 33.37.
6-Hydrazinyl-N2-(4-nitrophenyl)-N4-phenyl-1,3,5-triazine-2,4-
diamine (5f): Yield: 90%, Brown solid, m.p. ¼ 150
C; FT-IR (KBr,
cm1) n: 3029e3393 (NH, NH2, CH-Aromatic), 1305, 1507 (NO2); 1H
NMR (500 MHz, DMSO-d6) d: 9.4 (s, 1H, NH), 9.1 (s, 1H, NH), 8.8 (s,
1H, NH), 7.8e8.1 (m, 2H, AreH), 7.55 (m, 2H, AreH), 7.35 (m, 4H,
AreH), 6.9 (m, 1H, AreCH), 4.1e4.3 (s, 2H, NH2); Anal. Calcd. For
C15H14N8O2: C, 53.25; H, 4.17; N, 33.12. Found: C, 52.86; H, 4.21; N,
33.01.
2.6. Synthesis of 2-methyl-4H-benzo[1,3]oxazin-4-one
According to reported procedure [22], a mixture of anthranilic
acid (6, 20 mmol) and acetic anhydride (7, 40 mmol) was refluxed
for 3 h. After the completion of the reaction, the excess acetic an-
hydride was evaporated under vacuum and the collected solid, 2-
methyl-4H-benzo[1,3]oxazin-4-one (8), was washed with petro-
leum ether. Yield: 80%, yellow powder, m.p. ¼ 80e81
C; FT-IR (KBr,
cm1) n: 1665 (C]O), 1605 (C]N), 1169 (CeO); 1H NMR (DMSO-d6)
d (ppm): 2.39 (3H, s, CH3), 6.91e8.11 (4H aromatic).
2.7. General procedure for the synthesis of compounds 9a-f
A solution of compounds 5a-f (5.0 mmol) and the 2-methyl-4H-
1,3-benzoxazin-4-one (5 mmol) was refluxed in ethanol for
10e12 h and then it was cooled to R.T. furthermore concentrated
under reduced pressure. The resulting products were washed with
hot water, dried, and crystallized or purified by silica gel column
chromatography to get the desired compounds 9a-f.
3-((4-((4-Chlorophenyl)amino)-6-(phenylamino)-1,3,5-triazin-2-
yl)amino)-2-methylquinazolin-4(3H)-one (9a): Yield: 70%, white
solid, m.p. ¼ 170
C; FT-IR (KBr, cm1) n: 2917 (CeH, aliphatic), 1685
(C]O, ketone); 1H NMR (500 MHz, DMSO-d6) d: 10.4e10.1 (br, 1H,
NH), 9.7 (s, 1H, NH), 9.5 (s, 1H, NH), 8.2 (d, 1H, AreH), 7.9e7.0 (m,
12H, AreH); 13C NMR (125 MHz, DMSO-d6) d: 22.0, 110e135,
147,151.9, 158, 164.8, 167, 170; Anal. Calcd. For C24H19ClN8O: C,
61.21; H, 4.07; N, 23.80. Found: C, 59.94; H, 4.31; N, 23.13.
4-((4-((2-Methyl-4-oxoquinazolin-3(4H)-yl)amino)-6-(phenyl-
amino)-1,3,5-triazin-2-yl)amino) benzonitrile (9b): Yield: 60%, white
solid, m.p. ¼ 140
C; FT-IR (KBr, cm1) n: 2928 (CeH, aliphatic),
2220 (CN), 1678 (C]O, ketone); 1H NMR (500 MHz, DMSO-d6) d:
10.5e10.0 (br, 2H, NH), 9.9e9.2 (s, 1H, NH), 8.2e6.9 (m, 13H, AreH);
13
C NMR (125 MHz, DMSO-d6) d: 22.0, 110, 115, 117, 122e134.2, 147,
153,164.5, 170; Anal. Calcd. For C25H19N9O: C, 65.07; H, 4.15; N,
27.32. Found: C, 64.13; H, 4.27; N, 26.54.
3-((4-((4-Methoxyphenyl)amino)-6-(phenylamino)-1,3,5-triazin-
2-yl)amino)-2-methylquinazolin-4(3H)-one (9c): Yield: 73%, white
45
solid, m.p. ¼ 160
C; FT-IR (KBr, cm1) n: 2932 (CeH, aliphatic),
2832 (CeH, aliphatic), 1675 (C]O, ketone); 1H NMR (500 MHz,
DMSO-d6) d: 9.4e9.2 (s, 1H, NH), 8.9 (s, 1H, NH), 8.6 (s, 1H, NH), 8.2
(d, 1H, AreH), 7.9e6.9 (m, 12H, AreH), 3.7 (s, 3H, OeCH3); Anal.
Calcd. For C25H22N8O2: C, 64.37; H, 4.75; N, 24.02. Found: C, 63.89;
H, 4.83; N, 23.44.
4-((4-((2-Methyl-4-oxoquinazolin-3(4H)-yl)amino)-6-(phenyl-
amino)-1,3,5-triazin-2-yl)amino) benzenesulfonamide (9d): Yield:
67%, white solid, m.p. ¼ 200
C; FT-IR (KBr, cm1) n: 2905 (CeH,
aliphatic), 1678 (C]O, ketone), 1330,1145 (SO2); 1H NMR (500 MHz,
DMSO-d6) d: 10.0e10.3 (s, 1H, NH), 9.7 (s, 1H, NH), 9.3 (s, 1H, NH),
8.3 (d, 1H, AreH), 7.0e8.1 (m, 15H, AreH, NH2), 3.7 (s, 3H, CH3);
Anal. Calcd. For C24H21N9O3S: C, 55.91; H, 4.11; N, 24.45; S, 6.22.
Found: C, 55.31; H, 4.31; N, 24.12; S, 5.77.
3-((4,6-bis(Phenylamino)-1,3,5-triazin-2-yl)amino)-2-
methylquinazolin-4(3H)-one (9e): Yield: 73%, white solid,
m.p. ¼ 160
C; FT-IR (KBr, cm1) n: 2920 (CeH, aliphatic), 1685 (C]
O, ketone); 1H NMR (500 MHz, DMSO-d6) d: 10.0e10.4 (s, 1H, NH),
9.4e9.6 (s, 1H, NH), 9.7 (s, 1H, NH), 6.95e8.15 (m, 14H, AreH); Anal.
Calcd. For C24H20N8O: C, 66.04; H, 4.62; N, 25.67. Found: C, 65.68; H,
4.42; N, 25.08.
2-Methyl-3-((4-((4-nitrophenyl)amino)-6-(phenylamino)-1,3,5-
triazin-2-l)amino)quinazolin-4(3H)-one (9f): Yield: 55%, white solid,
m.p. ¼ 176
C; FT-IR (KBr, cm1) n: 2923 (CeH, aliphatic), 1678 (C]
O, ketone), 1509,1302 (NO2); 1H NMR (500 MHz, DMSO-d6) d:
9.65e10.0 (s, 1H, NH), 9.35 (s, 1H, NH), 9.2 (s, 1H, NH), 6.9e8.1 (m,
13H, AreH); Anal. Calcd. For C24H19N9O3: C, 59.87; H, 3.98; N, 26.18.
Found: C, 59.09; H, 4.11; N, 25.63.
2.8. Biological activity
Minimum inhibitory concentration (MIC) of the synthesized
hybrids was obtained by micro plate alamar blue assay (MABA)
method [22]. Sabouraud dextrose agar (Scharlo) was used to cul-
ture fungi strain and Mueller Hinton agar (Scharlo) was used for the
bacterial strains. The inocula of bacteria (1.5  104 CFU/mL) and
fungi (1.5  105 CFU/mL) were used as microbial suspension. The
stock solutions with concentration of 5120 mg/mL were obtained by
dissolution of the hybrid compounds in 0.1 mL of DMSO and diluted
with water up to 1.0 mL. To obtain 2560 to 320 mg/mL concentra-
tions, the serial dilution method was used. Except negative control
wells which should containing 160 ml of culture, 140 mL of culture
poured in each well of 96-well plates. Afterward, 20 ml of each
bacterial dilution was distributed in all 96 wells of microplate
including positive control (encloses microorganisms and standard
antibiotic) and growth control having microorganisms and culture.
Then, 20 ml of each concentration of the synthesized compounds
were added to each well of 96-well plate except for positive and
growth control wells. After adding 20 ml of alamar blue to all of 96
wells, the total volume in each well reach to 200 ml. The tested
hybrids have the final concentrations of 512, 256, 128, 64 and 32 mg/
mL. After incubation, the lowest concentration, which prevented a
color change from blue to pink, was defined as MIC. Ketoconazole
and Ciprofloxacin were used as standard antifungal and antibac-
terial drug, respectively.
2.9. Docking study
Computational docking studies were carried out using Auto-
Dock 4.2 [32e34] to understand the types of interactions and
binding orientations. S. aureus DHFR was chosen as a bacterial
target to deduce the interactions and binding mode of synthesized
derivatives. X-ray structure of DHFR from S. aureus was retrieved
from PDB (PDB ID 2W9S) with trimethoprim as co-crystallized
ligand and then needless parts (ligand, non-important chain, co-
46 M. Dinari et al. / Journal of Molecular Structure 1156 (2018) 43e50
enzymes, and crystallographic waters) were removed. The struc-
tures of the synthesized compounds were drawn using ChemDrow
8.0 and optimized by HyperChem software with PM3 semi-
empirical method. Based on the tutorial of AutoDockTools, the li-
gands and protein target were prepared [28e30]. A grid map was
built on the ligand's binding site with 40 grid points in X, Y, and Z
directions. For analysis of the docking interactions, AutoDockTools
1.5.6 soft ware was used in the complex protein-ligand conforma-
tions [35]. To estimate the strength of the docking process,
trimethoprim as the substrate of enzyme, was removed from the
binding cavity and re-docked to the S. aureus DHFR enzyme. This
produced a good agreement between the docked and crystal
structures as it further confirmed the interaction region of the
ligand with DHFR. At last, the main interactions between the ligand
and the protein were examined with selection of the best interac-
tion according to its cluster and binding energy.
3. Results and discussion
3.1. Preparation of novel 1,3,5-triazine-quinazolinone based hybrids
Triazine-quinazolinones hybrids were synthesized from the
stepwise condensation reaction of cyanuric chloride (1) and
different aromatic amines as shown in Scheme 1. Due to the
different reactivity of trichlorotriazines, the reaction was arising at
different temperatures. Only one site of triazine can be substituted
at low temperature, while at room temperature two sites can
participate in substitution reactions, and under high temperatures,
all three sites can react [31]. In this regards, first, 4,6-dichloro-
[1,3,5]triazin-2-yl phenylamine (2) was synthesized through
nucleophilic attack of aniline to 1 in the presence of triethyleamine.
The disubstituted s-triazine compounds (4a-f) were obtained by
the reaction of 4,6-dichloro-[1,3,5]triazin-2-yl phenylamine (2)
with different aniline derivatives (3a-f). The replacement of the last
Cl
Cl N Cl
N N i N N ii
+ R NH2
Cl N Cl NH
3a-f
1 2
O O
O O
OH
+ H3C
NH2
6 7
a: Cl b:
d: SO2NH2 e:
Scheme 1. Reagents and conditions: i) Aniline/acetic acid: water/triethyleamine, 5
C; ii) arylamine/acetic acid: water/CH3COONa, r.t.; iii) NHNH2/reflux, iv) acetic anhydride,
140
C; v) EtOH/reflux.
chlorine by hydrazine group was accomplished under heating
conditions, and afforded the compounds 5a-f in good yields. Finally,
the coupling of benzoxazinone (8) and triazine intermediates (5a-f)
afforded novel target hybrids (9a-f) in moderate yields.
3.2. Characterizations of triazine-quinazolinone hybrids
Characterization of the novel synthesized compounds was
accomplished by various spectral techniques such as 1H and 13C
NMR, FT-IR and elemental analysis techniques. The experimental
results in elemental analyses of the synthesized hybrids were
closely matched to the calculated ones, indicating that the expected
compounds were obtained (the results are presented in experi-
mental part).
From the structural investigation, FT-IR spectrum of compound
2 showed absorptions at 3375 and 1552 cm1 which indicate the
presence of NH and C]N groups, respectively. The FT-IR spectra of
compounds4a-f showed NH and C]C group vibrations in the range
of 3070e3350 cm1 and 1605-1618 cm1, respectively. The bands at
2219, 1333, 1532, 1152, 1329 and 1080 cm1 confirm the presence of
C^N, NO2, SO2 and OeCH3 groups, respectively. Fig. 1 shows the FT-
IR spectra of compounds 2, 4a, 7a and 9a.
The main absorption bands of NH amine, C]N, C]C and CeCl
are observed for compound 2. In the FT-IR spectrum of 4a, two
special peaks were appeared for different NH groups. Also, in the
FT-IR spectrum of 7a, the absorption band of CeCl was disappeared
and a broad absorption band related to amine groups in hydrazine
linkages due to the hydrogen bonding was observed at 3030-
3400 cm1. These results confirmed the successful formation of
compound 7a. The formation of 1,3,5-triazine-quinazolinon hybrid
product (9a) was confirmed by the appearance of a new band in
1686 cm1 which was assigned to the vibration of heterocyclic C]
O of amide group.
The chemical structure of the synthesized compounds was
H2NHN N NHR
Cl N NHR
N N
N N iii
NH
NH
O
v
N N
4a-f 5a-f HN N NHR
N N
NH
iv O
O CH3
N CH3
8 9a-f
CN
c: OCH3
f: NO2
 M. Dinari et al. / Journal of Molecular Structure 1156 (2018) 43e50 47

Fig. 1. FT-IR spectra of the a) 4,6-dichloro-1,3,5-triazin-2-ylphenylamine (2), b) 6-

chloro-N2-(4-chlorophenyl)-N4-phenyl-1,3,5-triazine-2,4-diamine (4a), c) N2-(4-
chlorophenyl)-6-hydrazinyl-N4-phenyl-1,3,5-triazine-2,4-diamine (5a) and d) 3-((4-
((4-chlorophenyl)amino)-6-(phenylamino)-1,3,5-triazin-2-yl)amino)-2-
methylquinazolin-4(3H)-one (9a).
further confirmed by NMR technique. Fig. 2 demonstrated the 1H
NMR spectra of the compounds 4a, 7a and 9a. In the 1H NMR
Fig. 2. 1H NMR (500 MHz) spectrum of A) 6-chloro-N2-(4-chlorophenyl)eN4-phenyl-
1,3,5-triazine-2,4-diamine (4a), B) N2-(4-Chlorophenyl)-6-hydrazinyl-N4-phenyl-
1,3,5-triazine-2,4-diamine (5a) and C) 3-((4-((4-Chlorophenyl)amino)-6-(phenyl-
amino)-1,3,5-triazin-2-yl)amino)-2-methylquinazolin-4(3H)-one (9a) in DMSO-d6 at
R.T.

spectrum of compound 4a, the eNH group of 4-cholro aniline and

aniline are resented at 10.1e10.5 ppm (Fig. 2a). Also, the protons of
aromatic rings are resented in the range of 7.2e7.7 ppm. In the 1H
NMR spectrum of the 7a, the signals of NH and NH2 for hydrazine
group are presented at around 8.0e8.3 and 4.2e4.6 ppm, respec-
tively. Their 1H NMR spectra provided the disappearance of the NH2
proton and the peaks of the rest of the molecule have emerged in
their region (Fig. 2b). After incorporating of quinazolinone moiety
into the tiazine ring, the 1H NMR spectrum also showed the three
methyl protons of quinazolinone moiety at 2.6 ppm and the aro-
matic hydrogen are presented in the range of 7.3e8.1 ppm. These
results in sum, established the formation of the final hybrid (Fig. 2).
Also, these results were confirmed by 13C NMR spectrum in which
the presences of aromatic and aliphatic carbon atoms were
approved.
3.3. Antibacterial activity
Hybridization approach has already been applied in developing
novel antibacterial and antimalarial agents to overcome drug
resistance [4,22,36,37]. In this study novel hybrid compounds
based on quinazolinone and s-triazine moieties were synthesized.
Since each of these heterocyclic rings has been reported as anti-
microbial agents with potential activity against a wide spectrum
of significant bacterial pathogens [5,6,10e12,22], it is thought that
the hybrid structure resulting from these heterocyclic pharmaco-
phores also provides a good antimicrobial property. The synthe-
sized compounds were screened for antibacterial and anti-fungal
activities by MABA method, against Salmonella entritidis, Pseudo-
monas aeruginosa, Escherichiacoli as Gram-negative bacteria as well
as Bacillus subtilis, Staphylococcus aureus, Listeria monocitogenes as
Gram positive bacteria and also Candida albicansas a yeast-like
fungi strain. In vitro antimicrobial activities were showed in
Table 1 and resulted in the following observations:
Compounds 5a, 5b, 5c, 5e and 5f containing 4-chloroaniline, 4-
aminobenzonitrile, p-anisidine, aniline and 4-nitroaniline moiety
attached to s-triazine scaffolds displayed moderate inhibitory ac-
tion against all the fungal and bacterial strains, respectively.
Nevertheless, 4-((4-hydrazinyl-6-(phenylamino)-1,3,5-triazin-2-
yl)amino)benzenesulfonamide (5d) demonstrate a major growth
inhibitory against both the Gram-negative strains (Escherichia coli
and Pseudomonas aeruginosa) with an MIC value of 64 mg/mL and
against Gram-positive bacteria with an MIC value of 32e64 mg/mL.
The results indicated that incorporation of quinazolinone group on
s-triazine ring increased antimicrobial activity compare to hydra-
zine group. Compounds 9a and 9b proved good antimicrobial ac-
tivity against S. aureus, B.subtilis, E. coli, L. monocitogenes with an
MIC value of 32e64 mg/mL. Compound 9c and 9f bearing p-
48 M. Dinari et al. / Journal of Molecular Structure 1156 (2018) 43e50

Table 1
Results of in vitro antimicrobial screening of analogs 5a-f and 9a-f.

Compounds MIC (mg/Ml)

Gram-negative bacteria Gram-positive bacteria Fungal

E.coli P.aeruginosa S.entritidis S.aureus B.subtilis L.monocitogenes C.albicans

5a 256 128 512 128 128 256 256

5b 256 256 512 512 512 512 256
5c 512 512 512 256 512 512 512
5d 64 64 256 32 64 64 128
5e 256 512 G 256 512 512 512
5f 512 512 512 128 256 512 512
9a 64 128 256 32 32 128 128
9b 64 128 128 32 32 64 128
9c 256 512 512 256 128 256 256
9d 32 128 128 16 32 32 128
9e 128 128 512 64 128 512 256
9f 256 512 256 128 128 128 256
Ciprofloxacin 8 4 4 4 4 8 e
Ketoconazol e e e e e e 8

anisidine and 4-nitroaniline, respectively indicate moderate
inhibitory against all the fungal and bacterial strains. Compound 9d
also showed highest activity against E. coli and Gram-positive
bacteria with an MIC value of 16 and 32 mg/mL. Compound 9e
demonstrated to have moderate activity against all the bacterial
except S. aureus with an MIC value of 64 mg/mL. In case of antifungal
activity against Candida albicans, the synthesized compounds pro-
duced moderate activity and compounds 5d, 9a, 9b and 9d were
found to be most potent members with MIC ¼ 128 mg/mL. In gen-
eral, antifungal activity was lower than antibacterial activity.
3.4. Docking study

According to antimicrobial results, some of the novel synthe-

Table 2
sized compounds such as 5a, 5b, 5d, 9a, 9b and 9d were selected for
The docking study results of some synthesized compounds which showed good docking to S. aureus DHFR. According to the mode of orientation of
antibacterial activity. these compounds and their binding energy, it could be said that
Compound Binding energy Inhibitory H-Bonding(distance, Å)
these compounds fit well in the binding cavity of DHFR (Table 2). All
(kcal/mol) constant (Ki) the docked compounds showed desirable interaction specially
hydrogen bond with protein target (Table 2). Visual inspection of
5a 7.19 5.36 mm NeH hydrazine with Ser49:
2.20 the docked poses of compounds 9a, 9b and 9d, substituted with
NeH triazine with Ser49: amino quinazolinone, showed that hydrogen bonds of carbonyl
1.43 group of quinazolinone could help in stabilization of these com-
NeH bridge with Thr46: pounds in the active site. On the other hand, compound 5a, 5b and
2.18
5b 7.16 5.63 mm NeH hydrazine with Ser49:
5d which didn't have quinazolinone group are also able to exhibi-
1.77 ted hydrogen bonds through their hydrazine groups (Table 2), but
NeH triazine with Ser49: they showed less affinity and binding energy. This desirable effect
2.34 of quinazolinone ring could be attributed to additional Van der
NeH bridge with Leu20:
Waals interaction which stabilizes the ligandeenzyme complex.
1.88
5d 7.12 6.06 mm NeH hydrazine with Ser49: Compound 9d with the lowest binding energy of 9.09 (Kcal/mol)
2.10 was considered as the best compound into the binding site of
NeH triazine with Ser49: S. aureus DHFR. This compound with quinazolinone and sol-
2.15 funamide groups oriented itself in such a way that it forms four
NeH bridge with Leu20:
1.96
hydrogen bonds with Ser49, Ileu50, Gln19 and Phe92 (Fig. 3 and
9a 8.34 650.98 nm C¼O quinazolinone with Table 2). Compound 9b also exhibited three conventional hydrogen
Leu29: 1.79 bonds with Ser49 (2.20, 2.35 and 2.28 Å). In this compound, ac-
NeH quinazolinone with cording to docking results, cyano group didn't take part in forma-
Ser49: 2.00
tion of hydrogen bond. Compound 9a (Fig. 3 and Table 2), also
NeH triazine with Ser49:
2.19 exhibited three conventional hydrogen bonds with Ser49 (2.00 and
9b 8.21 829.97 nm C¼O quinazolinone with 2.19 Å) and Leu29 (1.79 Å). Docking results show that these novel
Ser49: 2.20 hybrids were assumed accurately within the DHFR binding site,
NeH triazine with Ser49: suggesting that they could be potential antimicrobial agents.
2.35
NeH bridge with Ser49:
2.28 4. Conclusions
9d 9.09 217 nm C¼O quinazolinone with
Gln49: 2.12
In this study, a series of novel hybrid derivatives bearing s-
NeH triazine with Ileu50:
1.74 triazine and quinazolinone moieties was synthesized through
NeH bridge with Ser49: multistep chemical reactions. Structures of the new compounds
2.00 were characterized by elemental analysis, NMR and FT-IR spectra.
NeH sulfonamide with Since different derivatives of each heterocyclic rings have been
Phe92: 2.35
reported as anti-microbial agents with potential activity against a
 M. Dinari et al. / Journal of Molecular Structure 1156 (2018) 43e50
Fig. 3. The binding modes and hydrogen bonds interactions of some synthesized compounds which showed good antibacterial activity, in the DHFR active site.
wide spectrum of significant bacterial pathogens, the synthesized
compounds were screened for antibacterial and anti-fungal activ-
ities by MABA method, against Salmonella entritidis, Pseudomonas
aeruginosa, Escherichiacoli as Gram-negative bacteria as well as
Bacillus subtilis, Staphylococcus aureus, Listeria monocitogenes as
gram positive bacteria and also Candida albicansas a yeast-like
fungistrain. The in vitro biological evaluation data revealed that
some of the new compounds showed good antibacterial activity.
Among all the tested compounds, 4-((4-((2-methyl-4-
oxoquinazolin-3(4H)-yl)amino)-6-(phenylamino)-1,3,5-triazin-2-
yl)amino) benzenesulfonamide 9d exhibited exclusively strong
antibacterial activity against four bacterial strains with low MICs
ranging from 16 to 32 mg/Ml. Docking study of the synthesized
compounds with S. aureus DHFR revealed that compound 9d
bearing the quinazolinone and solfunamide groups oriented itself
in such a way that it forms four hydrogen bonds with Ser49, Ileu50,
Gln19 and Phe92 in the binding site and has lowest binding energy
of 9.09 kcal/mol.
Acknowledgment
We wish to express our gratitude to the Research Affairs Divi-
sion at Isfahan University of Technology (IUT), Isfahan, for the
partial financial support.
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