1,2,4-Triazole and 1,3,4-oxadiazole analogues: Synthesis, MO studies, in silico molecular docking studies, antimalarial as DHFR inhibitor and antimicrobial activities

Article abstract of DOI:10.1016/j.bmc.2017.05.054

1,2,4-Triazole and 1,3,4-oxadiazole analogues are of interest due to their potential activity against microbial and malarial infections. In search of suitable antimicrobial and antimalarial compounds, we report here the synthesis, characterization and biological activities of 1,2,4-triazole and 1,3,4-oxadiazole analogues (SS 1-SS 10). The molecules were characterized by IR, mass, ¹H NMR, ¹³C NMR and elemental analysis. The in vitro antimicrobial activity was investigated against pathogenic strains, the results were explained with the help of DFT and PM6 molecular orbital calculations. In vitro cytotoxicity and genotoxicity of the molecules were studied against S. pombe cells. In vitro antimalarial activity was studied. The active compounds were further evaluated for enzyme inhibition efficacy against the receptor Pf-DHFR computationally as well as in vitro to prove their candidature as lead dihydrofolate reductase inhibitors.

Full text of DOI:10.1016/j.bmc.2017.05.054

Accepted Manuscript
1, 2, 4-triazole and 1, 3, 4-oxadiazole analogues: synthesis, MO studies, in sil-
ico molecular docking studies, antimalarial as DHFR inhibitor and antimicrobial
activities
Sampark S. Thakkar, Parth Thakor, Hiren Doshi, Arabinda Ray
PII:
S0968-0896(17)30831-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.054
BMC 13773
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 April 2017
23 May 2017
26 May 2017
Please cite this article as: Thakkar, S.S., Thakor, P., Doshi, H., Ray, A., 1, 2, 4-triazole and 1, 3, 4-oxadiazole
analogues: synthesis, MO studies, in silico molecular docking studies, antimalarial as DHFR inhibitor and
antimicrobial activities, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.05.054
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1, 2, 4-triazole and 1, 3, 4-oxadiazole analogues: synthesis, MO studies, in silico
molecular docking studies, antimalarial as DHFR inhibitor and antimicrobial activities
Sampark S. Thakkara, Parth Thakorb, Hiren Doshic, Arabinda Raya*
a
Advanced Organic Chemistry Department, P. D. Patel Institute of Applied Sciences,
CHARUSAT, Changa-388421, Gujarat, India
b
P. G. Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar-388120,
Gujarat, India
c Madhav University, Abu Road, Sirohi- 307026, Rajasthan, India
* Corresponding author: Prof. Arabinda Ray, Advanced Organic Chemistry Department, P.
D. Patel Institute of Applied Sciences, CHARUSAT, Changa-388421, Gujarat, India. Tel.:
+91-9426397409, E-mail: arabinda24@yahoo.co.in

Highlights
•
•
•
Design and synthesis of 1, 2, 4-triazole and 1, 3, 4-oxadiazole analogues.
In vitro antimicrobial and antimalarial activities.
Molecules were subjected to DFT and PM6 calculations to explain the anti-bacterial
activity.
•
In silico molecular docking and DHFR inhibitors assay.

Abstract
1, 2, 4-triazole and 1, 3, 4-oxadiazole analogues are of interest due to their potential activity
against microbial and malarial infections. In search of suitable antimicrobial and antimalarial
compounds, we report here the synthesis, characterization and biological activities of 1, 2, 4-
triazole and 1, 3, 4-oxadiazole analogues (SS 1-SS 10). The molecules were characterized by
1
IR, mass, H NMR, 13C NMR and elemental analysis. The in vitro antimicrobial activity was
investigated against pathogenic strains, the results were explained with the help of DFT and
PM6 molecular orbital calculations. In vitro cytotoxicity and genotoxicity of the molecules
were studied against S. pombe cells. In vitro antimalarial activity was studied. The active
compounds were further evaluated for enzyme inhibition efficacy against the receptor Pf-
DHFR computationally as well as in vitro to prove their candidature as lead dihydrofolate
reductase inhibitors.
Keywords 1, 2, 4-triazole; 1, 3, 4-oxadizaole; DFT; PM6; anti-microbial activity; S. pombe;
molecular docking; anti-malarial activity; DHFR
1. Introduction
Heterocyclic chemistry has become one of the most important fields of research in
pharmaceutical industry due to their many fold applications. Amongst all, heterocyclic
molecules containing nitrogen and oxygen have shown most potent biological activities [1]. It
follows from the literature that depending on the type of substituent, the analogues of 1, 2, 4-
triazole have a high potential for a wide range of biological activities such as antimicrobial
[2-4], analgesic [4], anti-tumor [5], anti-inflammatory [6], anti-hypertensive [7], anti-cancer
[8] and antiviral [9] activities. In a host of standard medicines 1, 2, 4-triazole moiety is
present [10]. 1, 3, 4-oxadiazole is an essential core in heterocyclic chemistry and represents a
key motif in medicinal chemistry due to their potential to exhibit bioactivities such as anti-
HIV [11], analgesic [12, 13], anti-inflammatory [12, 13], anti-cancer [14, 15], antimalarial
[16], antimicrobial [16] and anti-tuberculosis [16]. Medicines having 1, 3, 4-oxadiazole ring
are plenty [10]. Schiff bases also have gained importance in the field of medicine due to their
wide spectrum of biological activities such as antimicrobial [17-19], anti-tubercular [20-22],
anti-HIV [22], anti-cancer [22], anti-tumor [23], anti-inflammatory [24], analgesic [24] and
anticonvulsant [25, 26].
Considering the significant biological and medicinal importance of Schiff bases, 1, 2, 4-
triazole and 1, 3, 4-oxadiazole, Schiff bases derived from 1, 2, 4-triazole and 1, 3, 4-

oxadiazole were synthesized and efficacy of these compounds as bioactive material was
investigated. Different aldehydes having five members and six members (benzene) rings with
various electron withdrawing and electron donating groups have been used to prepare the
analogues. This was done to examine the effect of the ring size and substituent in the ring on
the efficacy of the biological activities of these compounds.
Data from FTIR, NMR, mass and elemental analysis were used to characterize the
compounds. The antibacterial and antifungal potency of these Schiff bases were investigated
against certain standard strains. The activities of these compounds were compared with
standard antibacterial and antifungal drugs. Molecular orbital calculations with DFT and PM6
have been done for the Schiff bases to correlate the antimicrobial activities with electronic
parameters. The fission yeast Schizosaccharomyces pombe is an important model organism
for the study of eukaryotic molecular and cellular biology [27]. As eukaryotes, these yeasts
can be used to study processes that are conserved from yeast to humans but absent from
bacteria, such as organelle biogenesis and cytoskeletal organization or to study mechanisms
such as transcription, translation and DNA replication, in which the eukaryotic components
and processes are significantly different from those of their bacterial counterparts [28]. Hence
we studied the cytotoxic and genotoxic behaviors of compounds (S 1- SS 10) on S. pombe
cells.
Malaria remains a major cause of public health problem in about 95 countries mainly located
in the tropical zone of the globe (notably Africa, South-East Asia and also Eastern
Mediterranean region) [29]. This parasitic disease is still estimated to affect over 212 million
people and accounted for 429,000 deaths in 2015 [29]. Five species of protozoan parasites
belonging to the Plasmodium genus, namely falciparum, malariae, vivax, ovale and knowlesi
cause malaria in human beings and Plasmodium falciparum is the most dangerous of these
species [30, 31]. The increasing prevalence of multiple drug resistant P. falciparum has
significantly reduced the efficacy of the current anti-malarial drugs. Also, the resistance
against P. falciparum is associated with mutations in the dihydrofolate reductase (DHFR)
domain [32]. Hence, DHFR enzyme has shown to be reliable and the best target to design
new antimalarial drugs. Accordingly, we studied antimalarial activity against P. falciparum
strain for the compounds (S 1-SS 10) and obtained IC50. Of these compounds, SS 2, SS 3, SS
4 and SS 9 were found to be significantly active. These compounds along with standard drugs
(Chloroquine & Pyrimethamine) were docked with wild-type Plasmodium falciparum

dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) complex (PDB ID: 4DPD) and their
in vitro DHFR enzyme inhibition activity was also studied.
2. Materials and methods
All starting materials and reagents were purchased from Sigma-Aldrich, Merck and Loba
Chemie. Thin layer chromatography (TLC) was performed on silica gel G60 F254 (Merck)
plates and eluted with the mobile phases n-hexane: ethyl acetate and methanol: chloroform
(70:30 % v/v). Melting points were recorded on automated melting point system and were
uncorrected. Nicolet 6700 spectrophotometer (Thermo Scientific) was employed to record IR
spectra using KBr pellet. NMR spectra were recorded on Bruker 400 MHz using DMSO-d6
as solvent and TMS as an internal standard. Chemical shifts are reported in parts per million
(δ in ppm). Mass spectra were recorded on Waters Micromass Q-Tof Micro. Elemental
analysis was performed on the Thermo Scientific Flash 2000.
2.1 Preparation and characterization of compounds (A 1-5, S 1, S 2 and SS 1-10)
2.1.1 Synthesis of methyl-4-aminobenzoate (A 1): Concentrated H2SO4 (0.131 mol) was
added dropwise to a solution of p-aminobenzoic acid (0.131 mol) in methanol (200 ml). The
contents were refluxed for 3 h (Scheme 1) and then cooled to room temperature. Excess
methanol was distilled out and the solution poured into cold water. The mixture was made
alkaline by adding 10% NaOH solution. The precipitate of A 1 obtained was filtered, washed
with cold water and recrystallized from 1:1 methanol: water. (17.13 g, 86.40%); white solid;
M.P.: 109 °C; IR (KBr) (cm-1):3466, 3370, 3069, 2902, 2845, 1681, 1657, 1566, 1458, 836;
1
EIMS (m/z): 152 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.696 (d, 2H, Ar-H),
13
6.625 (d, 2H, Ar-H), 5.945 (s, 2H, -NH2), 3.745 (s, 3H, -OCH3); C NMR (DMSO-d6, 100
MHz) δ (ppm): 166.4, 153.4, 131.0, 115.8, 112.6, 51.0; Anal Calcd. for C8H9NO2: C, 63.56;
H, 6.00; N, 9.27%. Found: C, 63.58; H, 6.01; N, 9.30%.
2.1.2 Synthesis of methyl-4-acetamidobenzoate (A 2): A mixture of acetic acid (0.093 mol)
and acetic anhydride (0.093 mol) was taken in a RBF and compound A 1 (0.093 mol) was
added pinch wise to the mixture with stirring (Scheme 1). After addition the content in the
RBF was refluxed for 2 h and then poured into crushed ice. The solid was filtered, washed
with cold water and recrystallized from methanol. (16.34 g, 91.32%); white solid; M.P.: 129
°C; IR (KBr) (cm-1): 3361, 3071, 2927, 2851, 1686, 1611, 1597, 1525, 1442, 1368, 833;
1
EIMS (m/z): 193.9 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.271 (s, 1H, -
NHCOCH3), 7.886 (d, 2H, Ar-H), 7.734 (d, 2H, Ar-H), 3.783 (s, 3H, -OCH3), 2.090 (s, 3H, -

NHCOCH3); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.8, 165.7, 143.6, 130.0, 123.6,
118.1, 51.5, 23.9; Anal Calcd. for C10H11NO3: C, 62.17; H, 5.74; N, 7.25%. Found: C, 62.08;
H, 5.76; N, 7.23%.
2.1.3 Synthesis of N-(4-(hydrazinecarbonyl) phenyl)acetamide (A 3): Compound A 2
(0.078 mol) was dissolved in a mixture of hydrazine hydrate (0.233 mol) and 50 ml methanol
taken in a 150 ml RBF. The contents were refluxed for 3 h (Scheme 1). During reflux the
solid residue of A 3 was obtained. It was filtered, washed with methanol and dried. (13.81 g,
92.07%); white solid; M.P.: 255 °C; IR (KBr) (cm-1): 3367, 3314, 3055, 2933, 2852, 1670,
1618, 1590, 1435, 1370, 835; EIMS (m/z): 194.1 (M+1); 1H NMR (DMSO-d6, 400 MHz) δ
(ppm): 10.129 (s, 1H, -NHCOCH3), 9.627 (S, 1H, -CONHNH2), 7.780 (d, 2H, Ar-H), 7.642
(d, 2H, Ar-H), 4.436 (s, 2H, -CONHNH2), 2.069 (s, 3H, -NHCOCH3); 13C NMR (DMSO-d6,
100 MHz) δ (ppm): 168.4, 165.6, 141.7, 127.6, 118.0, 112.5, 24.0; Anal Calcd. for
C9H11N3O2: C, 55.95; H, 5.74; N, 21.75%. Found: C, 56.04; H, 5.72; N, 21.73%.
2.1.4 Synthesis of N-(4-(4-amino-5-mercapto-4H-1, 2, 4-triazol-3-yl) phenyl)acetamide
(A 4): The compound A 3 (0.031 mol) and carbon disulphide (0.031 mol) were added to
solution of sodium hydroxide (0.031 mol) in methanol (30 ml) with constant stirring. The
reaction mixture was stirred for 6 h at room temperature (Scheme 1). The precipitate of
sodium dithiocarbazinate was collected by filtration, washed with ether and dried under
vacuum. The sodium salt was obtained in quantitative yield and was used in the next step
without further purification.
A suspension of the sodium salt, hydrazine hydrate (0.062 mol) and water (30 ml) was
refluxed for 2 h. Hydrogen sulphide evolved and homogenous solution resulted, which was
diluted with ice cold water and subsequent acidification with dilute HCl gave a white
precipitate of A 4, which was filtered, washed with water and recrystallized from methanol.
(6.95 g, 89.85%); white solid; M.P.: 207 °C; IR (KBr) (cm-1): 3394, 3314, 3055, 2933, 2852,
1
1670, 1618, 1590, 1435, 1370, 835; EIMS (m/z): 250.1 (M+1); H NMR (DMSO-d6, 400
MHz) δ (ppm): 13.772 (s, 1H, -SH), 10.132 (s, 1H, -NHCOCH3), 7.958 (d, 2H, Ar-H), 7.706
(d, 2H, Ar-H), 5.702 (s, 2H, -NH2), 2.070 (s, 3H, -NHCOCH3); 13C NMR (DMSO-d6, 100
MHz) δ (ppm): 168.8, 166.2, 148.9, 141.0, 128.2, 120.0, 118.4, 23.9; Anal Calcd. for
C10H11N5OS: C, 48.18; H, 4.45; N, 28.09; S, 12.86%. Found: C, 48.20; H, 4.44; N, 28.02; S,
12.90%.

2.1.5 Synthesis of N-(4-(5-mercapto-1, 3, 4-oxadiazol-2-yl)phenyl)acetamide (A 5): To a
mixture of NaOH (0.031 mol) and methanol (30 ml), A 3 (0.031 mol) was added pinch wise
with stirring. After complete addition, CS2 (0.031 mol) was poured in reaction mixture and
stirred for 6 h at room temperature (Scheme 1).The contents were then poured in crushed ice
and acidified with dilute HCl. Precipitate of A 5 thus obtained was filtered, recrystallized
from methanol and dried (6.77 g, 92.70%); white solid; M.P.: 230 °C; IR (KBr) (cm-1): 3307,
1
3052, 2930, 2573, 1679, 1613, 1588, 1351, 838; EIMS (m/z): 236.1 (M+1); H NMR
(DMSO-d6, 400 MHz) δ (ppm): 14.649 (s, 1H,-SH), 10.331 (s, 1H, -NHCOCH3), 7.881 (m,
4H, Ar-H), 2.091 (s, 3H, -NHCOCH3); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 177.1,
168.7, 160.3, 142.7, 126.8, 118.9, 116.5, 24.0; Anal Calcd. for C10H9N3O2S: C, 51.05; H,
3.86; N, 17.86; S, 13.63%. Found: C, 50.89; H, 3.87; N, 17.92; S, 13.67%.
2.1.6 Synthesis of 4-amino-5-(4-aminophenyl)-4H-1, 2, 4-triazole-3-thiol (S 1) and 5-(4-
aminophenyl)-1, 3, 4-oxadiazole-2-thiol (S 2): Compound A 4 (0.020 mol) in 15 ml 2.5 N
HCl was refluxed for 1 h in round bottom flask. The refluxed mixture was poured in finely
crushed ice and made alkaline using 10% NaHCO3 solution; the residue of S 1 was filtered,
washed with cold water and recrystallized from methanol to get final product (Scheme 1).
The same procedure was repeated with A 5 (0.020 mol) to obtain S 2 compound (Scheme 1).
4-amino-5-(4-aminophenyl)-4H-1, 2, 4-triazole-3-thiol (S 1): (3.46 g, 83.20%); white solid;
M.P.: 218 °C; IR (KBr) (cm-1): 3351, 3269, 3095, 2592, 1614, 1582, 1451, 846; EIMS (m/z):
1
208.1 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 13.577 (s, 1H, -SH), 7.742 (d, 2H,
Ar-H), 6.640 (d, 2H, Ar-H), 5.665 (s, 2H, -NH2), 5.543 (s, 2H, -NH2); 13C NMR (DMSO-d6,
100 MHz) δ (ppm): 165.7, 150.7, 149.8, 128.9, 113.0, 112.5; Anal Calcd. for C8H9N5S: C,
46.36; H, 4.38; N, 33.79; S, 15.47%. Found: C, 46.41; H, 4.39; N, 33.70; S, 15.50%.
5-(4-aminophenyl)-1, 3, 4-oxadiazole-2-thiol (S 2): (3.26 g, 79.30%); white solid; M.P.: 215
°C; IR (KBr) (cm-1): 3447, 3351, 3093, 2587, 1629, 1605, 1565, 1440, 836; EIMS (m/z):
1
194.1 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 14.375 (s, 1H,-SH), 7.542 (d, 2H,
Ar-H), 6.670 (d, 2H, Ar-H), 6.021 (s, 2H, -NH2); 13C NMR (DMSO-d6, 100 MHz) δ (ppm):
176.5, 161.5, 152.5, 127.5, 113.4, 108.4; Anal Calcd. for C8H7N3OS: C, 49.73; H, 3.65; N,
21.75; S, 16.59%. Found: C, 48.78; H, 3.66; N, 21.72; S, 16.55%.
2.1.7 General procedure for the synthesis of Schiff Bases (SS 1-10): Compounds S 1
(0.002 mol)/ S 2 (0.003 mol) were dissolved in 5 ml DMF followed by addition of different

aldehydes (0.005 mol for S 1 and 0.003 mol for S 2) (Schemes 2 and 3) and catalytic amount
of glacial acetic acid. The mixture was stirred at room temperature for 4 h. The reaction
mixture was then poured into crushed ice, the residues of SS 1-10 were obtained, which were
filtered, washed with cold water and recrystallized from methanol to get purified products.
4-((thiophen-2-ylmethylene)amino)-5-(4-((-thiophen-2-ylmethylene)amino)phenyl)-4H-1, 2,
4-triazole-3-thiol (SS 1): (0.70 g, 73.62%); light yellow solid; M.P.: 181 °C; IR (KBr) (cm-1):
3100, 2565, 1616, 1588, 1425, 838, 647; EIMS (m/z): 396 (M+1); 1H NMR (DMSO-d6, 400
MHz) δ (ppm): 14.132 (s, 1H, -SH), 10.036 (s, 1H, Schiff Base), 9.948 (s, 1H, Schiff Base),
7.928 (m, 2H, thiophene-H), 7.879 (m, 2H, thiophene-H), 7.689 (m, 2H, thiophene-H), 7.578
(d, 2H, Ar-H), 7.363 (d, 2H, Ar-H); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 165.8, 162.3,
161.5, 152.5, 149.0, 136.4, 136.3, 132.5, 132.3, 131.6, 131.4, 129.2, 129.0, 128.0, 122.7,
121.1; Anal Calcd. for C18H13N5S3: C, 54.66; H, 3.31; N, 17.71; S, 24.32%. Found: C, 54.70;
H, 3.30; N, 17.76; S, 24.24%.
4-(((4-(4-((4-hydroxybenzylidene)amino)-5-mercapto-4H-1, 2, 4-triazol-3-yl)phenyl)imino)
methyl) phenol (SS 2): (0.72 g, 71.97%); light yellow solid; M.P.: 172 °C; IR (KBr) (cm-1):
1
3318, 3019, 2598, 1601, 1580, 1437, 827; EIMS (m/z): 416 (M+1); H NMR (DMSO-d6,
400 MHz) δ (ppm): 14.212 (s, 1H, -SH), 10.447 (b, 2H, -OH), 9.994 (s, 1H, Schiff Base),
9.810 (s, 1H, Schiff Base), 7.916 (m, 4H, Ar-H), 7.508 (m, 4H, Ar-H), 7.327 (d, 2H, Ar-H),
6.875 (d, 2H, Ar-H); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 167.3, 163.9, 162.7, 159.39,
159.31, 150.8, 148.6, 132.4, 132.2, 130.8, 130.7, 128.3, 126.3, 126.2, 122.6, 121.4; Anal
Calcd. for C22H17N5O2S: C, 63.60; H, 4.12; N, 16.86; S, 7.72%. Found: C, 63.70; H, 4.11; N,
16.80; S, 7.71%.
4-((3-nitrobenzylidene)amino)-5-(4-((3-nitrobenzylidene)amino)phenyl)-4H-1, 2, 4-triazole-
3-thiol (SS 3): (0.86 g, 75.57%); light yellow solid; M.P.: 151 °C; IR (KBr) (cm-1): 3030,
1
2605, 1614, 1602, 1580, 1532, 1443, 1313, 827, 740; EIMS (m/z): 474 (M+1); H NMR
(DMSO-d6, 400 MHz) δ (ppm): 14.300 (s, 1H, -SH), 10.159 (s, 1H, Schiff Base), 10.081 (s,
1H, Schiff Base), 8.860 (d, 2H, Ar-H), 8.534 (m, 2H, Ar-H), 8.441 (m, 2H, Ar-H), 7.985 (m,
2H, Ar-H), 7.560 (d, 2H, Ar-H), 7.468 (d, 2H, Ar-H); 13C NMR (DMSO-d6, 100 MHz) δ
(ppm): 168.1, 163.0, 162.3, 151.0, 148.2, 138.8, 138.7, 137.1, 136.9, 134.77, 134.71, 130.4,
130.3, 129. 5, 129.4, 128.7, 125.6, 125.5, 122.9, 122.0; Anal Calcd. for C22H15N7O4S: C,
55.81; H, 3.19; N, 20.71; S, 6.77%. Found: C, 55.93; H, 3.20; N, 20.63; S, 6.75%.

4-((4-chlorobenzylidene)amino)-5-(4-((4-chlorobenzylidene)amino)phenyl)-4H-1,
2,
4-
triazole-3-thiol (SS 4): (0.69 g, 63.16%); light yellow solid; M.P.: 185 °C; IR (KBr) (cm-1):
3095, 2568, 1610, 1592, 1425, 825, 728; EIMS (m/z): 452 (M+1); 1H NMR (DMSO-d6, 400
MHz) δ (ppm): 14.192 (s, 1H, -SH), 10.001 (s, 1H, Schiff Base), 9.915 (s, 1H, Schiff Base),
7.903 (m, 4H, Ar-H), 7.558 (m, 4H, Ar-H), 7.378 (d, 2H, Ar-H), 6.644 (d, 2H, Ar-H); 13C
NMR (DMSO-d6, 100 MHz) δ (ppm): 169.6, 163.7, 162.2, 150.9, 148.3, 136.6, 136.5, 134.4,
134.2, 130.9, 130.7, 129.2, 129.1, 128.8, 122.9, 121.1; Anal Calcd. for C22H15Cl2N5S: C,
58.41; H, 3.34; N, 15.48; S, 7.09%. Found: C, 58.30; H, 3.33; N, 15.54; S, 7.11%.
4-((4-methoxybenzylidene)amino)-5-(4-((4-methoxybenzylidene)amino)phenyl)-4H-1, 2, 4-
triazole-3-thiol (SS 5): (0.72 g, 67.39%); light yellow solid; M.P.: 173 °C; IR (KBr) (cm-1):
1
3002, 2931, 2836, 2565, 1617, 1571, 1440, 1355, 1255, 831; EIMS (m/z): 444 (M+1); H
NMR (DMSO-d6, 400 MHz) δ (ppm): 14.097 (s, 1H, -SH), 9.862 (s, 1H, Schiff Base), 9.612
(s, 1H, Schiff Base), 7.945 (m, 4H, Ar-H), 7.575 (m, 4H, Ar-H), 7.321 (d, 2H, Ar-H), 6.637
(d, 2H, Ar-H), 3.850 (s, 6H, -OCH3); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 169.2, 162.9,
161.5, 150.8, 148.1, 131.6, 131.5, 130.6, 130.9, 129.0, 128.9, 128.6, 124.7, 124.3, 122.4,
121.0, 55.35, 55.31; Anal Calcd. for C24H21N5O2S: C, 64.99; H, 4.77; N, 15.79; S, 7.23%.
Found: C, 65.02; H, 4.76; N, 15.82; S, 7.21%.
5-(4-((thiophen-2-ylmethylene)amino)phenyl)-1, 3, 4-oxadiazole-2-thiol (SS 6): (0.57 g,
77.24%); light yellow solid; M.P.: 160 °C; IR (KBr) (cm-1): 3080, 3060, 2577, 1621, 1601,
1
1592, 1426, 1170, 838, 694; EIMS (m/z): 288 (M+1); H NMR (DMSO-d6, 400 MHz) δ
(ppm): 14.517 (s, 1H, -SH), 9.965 (s, 1H, Schiff Base), 8.156 (d, 1H, thiophene-H), 8.046 (d,
1H, thiophene-H), 7.922 (t, 1H, thiophene-H), 7.554 (d, 2H, Ar-H), 6.687 (d, 2H, Ar-H); 13C
NMR (DMSO-d6, 100 MHz) δ (ppm): 177.3, 161.5, 160.3, 152.6, 137.8, 134.6, 132.1, 128.9,
127.6, 113.8, 108.4; Anal Calcd. for C13H9N3OS2: C, 54.34; H, 3.16; N, 14.62; S, 22.31%.
Found: C, 54.27; H, 3.17; N, 14.59; S, 22.38%.
4-(((4-(5-mercapto-1, 3, 4-oxadiazol-2-yl)phenyl)imino)methyl)phenol (SS 7): (0.54 g,
70.73%); light yellow solid; M.P.: 178 °C; IR (KBr) (cm-1): 3381, 3096, 2589, 1631, 1606,
1575, 1440, 1175, 836, ; EIMS (m/z): 297 (M+1); 1H NMR (DMSO-d6, 400 MHz) δ (ppm):
14.343 (s, 1H, -SH), 10.420 (s, 1H, -OH), 9.783 (s, 1H, Schiff Base), 7.792 (d, 2H, Ar-H),
7.548 (d, 2H, Ar-H), 6.929 (d, 2H, Ar-H), 6.687 (d, 2H, Ar-H); 13C NMR (DMSO-d6, 100
MHz) δ (ppm): 176.6, 163.2, 161.4, 160.6, 152.3, 131.7, 128.2, 127.3, 113.4, 108.7; Anal

Calcd. for C15H11N3O2S: C, 60.59; H, 3.73; N, 14.13; S, 10.78%. Found: C, 60.65; H, 3.72;
N, 14.18; S, 10.74%.
5-(4-((3-nitrobenzylidene)amino)phenyl)-1, 3, 4-oxadiazole-2-thiol (SS 8): (0.64 g, 76.22%);
light yellow solid; M.P.: 189 °C; IR (KBr) (cm-1): 3081, 2577, 1611, 1559, 1528, 1445,
1
1350, 1188, 833, 787; EIMS (m/z): 327 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm):
14.391 (s, 1H, -SH), 10.158 (s, 1H, Schiff Base), 8.705 (s, 1H, Ar-H), 8.517 (d, 1H, Ar-H),
8.364 (d, 1H, Ar-H), 7.876 (t, 1H, Ar-H), 7.542 (d, 2H, Ar-H), 7.467 (d, 2H, Ar-H); 13C
NMR (DMSO-d6, 100 MHz) δ (ppm): 176.6, 161.4, 159.8, 152.2, 137.8, 134.8, 134.6, 130.5,
128.2, 123.7, 113.4, 108.8; Anal Calcd. for C15H10N4O3S: C, 55.21; H, 3.09; N, 17.17; S,
9.82%. Found: C, 55.11; H, 3.10; N, 17.23; S, 9.79%.
5-(4-((4-chlorobenzylidene)amino)phenyl)-1, 3, 4-oxadiazole-2-thiol (SS 9): (0.57 g,
69.78%); light yellow solid; M.P.: 193 °C; IR (KBr) (cm-1): 3057, 2593, 1651, 1611, 1560,
1
1491, 1185, 830, 729, ; EIMS (m/z): 316 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm):
14.439 (s, 1H, -SH), 10.015 (s, 1H, Schiff Base), 7.957 (d, 2H, Ar-H), 7.696 (d, 2H, Ar-H),
7.552 (d, 2H, Ar-H), 6.691 (d, 2H, Ar-H); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 176.5,
162.2, 161.5, 152.6, 139.3, 134.7, 130.5, 129.3, 113.5, 108.4; Anal Calcd. for C15H10ClN3OS:
C, 57.05; H, 3.19; N, 13.31; S, 10.15%. Found: C, 56.94; H, 3.18; N, 13.36; S, 10.19%.
5-(4-((4-methoxybenzylidene)amino)phenyl)-1, 3, 4-oxadiazole-2-thiol (SS 10): (0.56 g,
69.03%); light yellow solid; M.P.: 165 °C; IR (KBr) (cm-1): 3096, 2953, 2834, 2588, 1629,
1
1607, 1574, 1440, 1353, 1212, 1195, 832, ; EIMS (m/z): 312 (M+1); H NMR (DMSO-d6,
400 MHz) δ (ppm): 14.349 (s, 1H, -SH), 9.867 (s, 1H, Schiff Base), 7.870 (d, 2H, Ar-H),
7.537 (d, 2H, Ar-H), 7.113 (d, 2H, Ar-H), 6.672 (d, 2H, Ar-H), 3.875 (s, 3H, -OCH3); 13C
NMR (DMSO-d6, 100 MHz) δ (ppm): 176.6, 164.1, 161.4, 152.3, 131.5, 129.5, 127.3, 114.1,
114.0, 113.4, 108.7, 55.4; Anal Calcd. for C16H13N3O2S: C, 61.72; H, 4.21; N, 13.50; S,
10.30%. Found: C, 61.82; H, 4.22; N, 13.45; S, 10.26%.
3. Results and Discussion
3.1 Chemistry
The syntheses of molecules (A 1-5, S 1, S 2 and SS 1-10) are summarized in Schemes 1-3.
Table 1 shows the type of substituent present on the target molecules SS 1-10. The starting
material methyl-4-aminobenzoate (A 1) was prepared according to Fischer-Speier
esterification reaction. Methyl-4-aminobenzoate (A 1) was refluxed for 2 h with the mixture

of acetic acid and acetic anhydride to get methyl-4-acetamidobenzoate (A 2). N-(4-(hydrazine
carbonyl)phenyl)acetamide (A 3) was prepared by refluxing compound A 2 and hydrazine
hydrate in methanol. The compound A 3 undergo cyclization reaction to form compounds A
4 and A 5 (Scheme 1). The parent molecules 1, 2, 4-triazole (S 1) and 1, 3, 4-oxadizaole (S 2)
were obtained by hydrolysis of compounds A 4 and A 5 respectively. The analogues of 1, 2,
4-triazole and 1, 3, 4-oxadiazole (SS 1-10) were prepared by reacting S 1 (Scheme 2) and S 2
(Scheme 3) with different aldehydes in the presence of acetic acid as a catalyst.
1
13
The formation of synthesized compounds was confirmed by IR, mass spectra, H NMR, C
NMR and elemental analysis. The IR spectra of all synthesized compounds (A 1-5, S 1, S 2
and SS 1-10) showed peaks at ~3070 cm-1, ~1570 cm-1 and ~1440 cm-1 were due to aromatic
C-H, and C=C stretching. The peak at ~2570 cm-1 in A 4, A 5, S 1, S 2 and SS 1-10 was
attributed to stretching of -SH group. The molecules SS 2 and SS 7 showed a broad peak at
~3350 cm-1 due to -OH stretching. The compounds SS 3 and SS 8 showed two bands near
~1530 cm-1 and ~1340 cm-1 due to asymmetric and symmetric stretching of -NO2 group. The
peak at ~730 cm-1 in SS 4 and SS 9 was attributed to stretching of C-Cl group. The
compounds SS 5 and SS 10 showed a peak at ~1230 cm-1 due to C-O-CH3 stretching.
The mass spectrum of all the compounds showed molecular ion peak (M+1) corresponding to
their respective molecular weights, which additionally confirmed by the molecular
framework. The 1H NMR resonances due to aromatic hydrogen in all synthesized compounds
(A 1-5, S 1, S 2 and SS 1-10) appeared between ~6.6-8.8 ppm. For compounds (A 4, A 5, S
1, S 2 and SS 1-10), the thiol -SH proton appeared at ~13.8 ppm as a singlet. The two new
peaks at ~9.8 ppm (singlet) and ~10.0 ppm (singlet) in the Schiff bases is due to -N=CH- for
compounds SS 1-5. The peak at ~9.9 ppm (singlet) is attributed to Schiff base proton in
compounds SS 6-10. The 13C NMR of compounds (A 1-5, S 1, S 2 and SS 1-10) showed
peaks in the range of ~118-150 ppm due to the presence of aromatic C-atoms. The C-atoms
of -N=CH- moiety in Schiff bases are seen between ~160-170 ppm for molecules SS 1-10.

O
COOCH3
COOCH3
(i)
(ii)
OH
H3COCHN
H2N
H2N
A 1
A 2
(iii)
N
N
N
N
(vi)
SH
SH
(iv)
N
N
CONHNH2
NH2
NH2
H2N
H3COCHN
S 1
A 4
H3COCHN
A 3
N
O
N
N
N
(vi)
SH
SH
(v)
O
H2N
H3COCHN
S 2
A 5
Scheme 1. Method for the preparation of parent moieties i.e. 4-amino-5-(4-aminophenyl)-
4H-1, 2, 4-triazole-3-thiol (S 1) and 5-(4-aminophenyl)-1, 3, 4-oxadiazole-2-thiol (S 2). (i)
CH3OH, conc. H2SO4, Reflux 3 h, 10% NaOH solution; (ii) (CH3CO)2O, CH3COOH, Reflux
2 h; (iii) NH2NH2 H2O, CH3OH, Reflux 3 h; (iv) CS2, NaOH, CH3OH, RT for 6 h, NH2NH2
H2O, Reflux 2 h, dil. HCl; (v) CS2, 1 N NaOH, CH3OH, RT for 6 h, dil. HCl; (vi) dil. HCl,
Reflux 1 h, 10% NaHCO3.
N
N
N
N
SH
O
CH3COOH,
RT
H
SH
N
N
+
N
R
NH2
R
N
H2N
R
S 1
SS 1-5
Scheme 2. Method for the preparation of Schiff Bases (SS 1-5)
Scheme 3. Method for the preparation of Schiff Bases (SS 6-10)
Table 1. Scheme followed to designate the molecules synthesized
Sample
R
ID(s)
SS 1, SS 6
SS 2, SS 7

SS 3, SS 8
SS 4, SS 9
SS 5, SS 10
3.2 In silico pharmacokinetic evaluation
The pharmacokinetic phase is important for an orally administrated drug. This phase includes
absorption from gastrointestinal tract into the blood supply and various factors that affect
drug’s survival and processes as it travels to the target. The in silico study was performed by
VlifeMDS 4.6 and ADMET software and the data obtained are presented in Table 2 and
Table 3.
Table 2. Prediction of Lipinski’s Rule of Five and molecular properties of compounds
Compound Mol
logSf
PSAg
Volume
(°A3)
HBAb HBDc RotBd ClogPe
ID(s)
S 1
Wt.a
log(mol/l) (°A2)
207.06
193.03
395.03
415.11
473.09
451.04
443.14
287.02
297.06
326.05
315.02
311.07
3
4
7
7
9
5
7
6
6
7
5
6
5
3
1
3
1
1
1
1
2
1
1
1
1
1
5
5
7
5
7
3
3
4
3
4
1.06
1.68
4.83
4.56
4.53
6.51
5.26
3.44
3.31
3.29
4.28
3.66
-3.67
-3.60
-7.99
-8.39
-9.51
-10.59
-9.19
-5.94
-6.14
-6.70
-7.24
-6.54
61.69
50.44
44.24
77.44
118.73
42.21
57.29
38.78
55.37
76.02
37.76
45.30
156.66
148.97
349.21
373.59
404.21
387.18
416.48
243.74
256.08
271.24
262.73
277.38
S 2
SS 1
SS 2
SS 3
SS 4
SS 5
SS 6
SS 7
SS 8
SS 9
SS 10
a = Molecular Weight ≤ 500 (gm/mol) [33]; b = Hydrogen Bond Acceptor ≤ 10 [33]; c =
Hydrogen Bond Donor ≤ 5 [33]; d = Rotatable Bonds ≤ 10 [33]; e = ClogP ≤ 5 [33]; f; Water
Solubility range -0.5 to -6.5 (mol/l) [33]; g = Polar Surface Area ≤ 140°A2 [33].
The values of physicochemical parameters (Table 2) such as molecular weight, HBA, HBD,
RotB, PSA, volume and logP except for SS 4 and SS 5 analogues suggested that all of the

molecules (S 1-SS 10) had favorable Lipinski’s rule of five (RO5) [33, 34]. The logS value
for compounds SS 1 to SS 5 and SS 7 to SS 10 violated the RO5 suggested that these
molecules were poorly absorbed and distributed.
Table 3. Prediction of ADMET parameters of compounds
Compound
ID(s)
S 1
Caco2
MDCK
(nm/sec)
4.9436
32.3434
3.2930
0.0688
0.0567
2.5059
0.0624
3.9198
10.4598
0.4744
0.5745
1.1258
hERG
BBB
% HIA
%PPB
25.494
(nm/sec)
inhibition
0.1419 20.043
0.2459 20.456
0.2234 49.273
0.0247 21.075
0.1409 19.794
0.3170 45.947
0.7745 34.712
0.5181 44.420
0.3553 7.7060
0.2260 20.431
1.7178 33.559
0.6107 25.829
86.124
89.611
99.156
96.446
87.980
97.704
99.572
97.115
96.357
90.218
99.318
98.914
Medium Risk
Low Risk
67.574
96.047
95.165
95.354
99.999
92.199
94.517
95.479
92.804
97.995
92.027
S 2
Medium Risk
Medium Risk
Low Risk
SS 1
SS 2
SS 3
Medium Risk
Medium Risk
Medium Risk
Medium Risk
Medium Risk
Medium Risk
Medium Risk
SS 4
SS 5
SS 6
SS 7
SS 8
SS 9
SS 10
BBB (Blood Brain Barrier) [35]: High absorption CNS >2.0, Middle absorption CNS 2.0-0.1,
Low absorption to CNS <0.1; Caco2 [35]: High permeability >70, Middle permeability 4-70,
Low permeability <4; %HIA (Human Intestinal Absorbance) [35]: Well absorbed
compounds 70-100%, Moderately absorbed compounds 20-70%, Poorly absorbed compounds
0-20%; %PPB (Plasma Protein Binding) [35]: Strongly Bound >90%, Weakly Bound <90%,
MDCK [35]: Higher permeability >500, Medium Permeability 25-500, lower permeability
<25.
The parameters obtained from ADMET software are compared with the results reported in
earlier work [35]. In comparison with 1, 2, 4-triazole (S 1) and 1, 3, 4-oxadiazole (S 2), S 1
had lower absorption to CNS (Table 3). Also the analogues of triazole SS 1-SS 5 had less
absorption to CNS compared to oxadiazole SS 6-SS 10 analogues, suggested that 1, 2, 4-
triazole ring was more fit than 1, 3, 4-oxaadizole ring.

Compared with parent molecules S 1 and S 2, the analogues SS 1-SS 10 had good Caco2
permeability except SS 2, SS 3, SS 7 and SS 8. Also all analogues SS 1-SS 10 had higher
human intestinal absorption compared to S 1 and S 2 (Table 3). In case of %PPB (Plasma
protein binding), the parent molecule S 1 weakly bound compared to all other molecules S 2-
SS 10. Moreover all molecules S 1-SS 10 had more or less MDCK permeability except S 2
and SS 7. Additionally the lower to moderate risk to hERG inhibition of all molecules S 1-SS
10 suggested that these molecules might be a good contender as a drug.
3.3 In vitro antimicrobial activity
The antibacterial and antifungal activities of the Schiff bases against certain bacterial and
fungal strains were investigated. The activities of these compounds are compared with
standard antibacterial and antifungal drugs. It is to be noted that all the compounds
synthesized S 1 - SS 10 in this study did not show any appreciable anti-fungal activities.
Molecular orbital calculations have been done to correlate the anti-bacterial potency with
electronic parameters.
The compound (S 1) is relatively a poor electron acceptor (energy of LUMO = -0.4870 eV
from PM6 and = -1.0805 eV from DFT). When this is anchored to the Schiff bases, the
resultant compounds become better electron acceptors. The compounds SS 1-SS 5 are
derivatives of S 1 and as these molecules have stable LUMOs, we try to explain the anti-
bacterial potency of these as electron acceptors (Table 4).
Amongst these compounds SS 3 has most stable LUMO (energy = -1.9493 eV from PM6 and
= -3.2750 eV from DFT) and therefore most potent electron acceptor in this series. It is
gratifying to note that this molecule is the strongest antibacterial agent in the series. Of the
two molecules SS 4 (LUMO = -1.4605 eV from PM6 and = -2.5465 eV from DFT) and SS 5
(LUMO=-1.0612 eV from PM6 and = -2.0412 eV from DFT), latter is a relatively poor
electron acceptor, as expected SS 4 is a slightly better antibacterial agent. Although, the
compound SS 1 (LUMO = -1.5232 eV from PM6 and = -2.5798 eV from DFT) appears to be
a better acceptor than SS 4 and SS 5, but is a less potent anti-bacterial agent than SS 4 and SS
5. It is interesting to note that highest LUMO electron density in SS 1 is 0.2031 (PM6) and
0.5144 (DFT), while the same in SS 4 is 0.4017 (PM6) and 0.6930 (DFT) and in SS 5 it is
0.4657 (PM6) and 0.7729 (DFT) (Table 4). The molecule SS 2 (LUMO= -1.1897 eV from
PM6 and = -1.8818 eV from DFT), although is a little more potent antibacterial agent than SS

1, is a poor electron acceptor compared to SS 1. Interestingly, the highest LUMO electron
density in SS 2 is 0.4530 (PM6).
Table 4. Energies of frontier orbitals from PM6 and DFT calculations
Energies (eV) from PM6 Energies (eV) from DFT
Compound
ID(s)
S 1
HOMO
-8.4879
-8.8557
-8.7632
-9.1780
-8.9797
-8.6617
-8.6846
-9.0245
-8.9718
-9.2490
-9.0791
-8.9176
LUMO
-0.48707
-1.5232
-1.1897
-1.9493
-1.4603
-1.0612
-0.8372
-1.1450
-1.0325
-1.7531
-1.1259
-0.9810
HOMO
-5.6950
-5.7351
-5.2632
-6.1949
-5.9242
-5.5772
-5.9138
-6.1237
-5.9863
-6.3546
-6.1464
-5.9411
LUMO
-1.0805
-2.5798
-1.8818
-3.2750
-2.5465
-2.0412
-1.4285
-2.1926
-1.8142
-3.2811
-2.2734
-1.7684
SS 1
SS 2
SS 3
SS 4
SS 5
S 2
SS 6
SS 7
SS 8
SS 9
SS 10
It appears that for a good antibacterial agent, electron accepting ability and easy
accommodation (high LUMO electron density) of an electron at the certain centre of the
molecule are important. The compound S 2 has LUMO energy (= -0.8370 eV from PM6 and
= -1.4285 eV from DFT) and when this moiety is incorporated in Schiff bases (SS 6-SS 10),
the LUMOs become more stable and consequently the analogues become better electron
acceptor compared to S 2. In the series SS 6-SS 10, the molecules SS 6, SS 8 and SS 9 are
relatively better anti-bacterial agents, compared to others in this series. The compound SS 8
appears to be most potent anti-bacterial agent and a better electron acceptor (LUMO energy =
-1.7531 eV, PM6 and = -3.2811 eV, DFT) in this series. The highest LUMO electron density
in SS 8 is 0.4706 from PM6 and 0.4763 from DFT. SS 6 and SS 9 are relatively poor electron
acceptors as seen from their LUMO energies (Table 4).
Table 5. In vitro antimicrobial activity of compounds S 1-SS 10 (MICs, mM)
Compound
Gram-positive bacteria Gram-negative bacteria Fungi

ID(s)
S.A.
S.P.
E.C.
P.A.
C.A.
MTCC
227
A.C.
MTCC
1323
2.415
1.295
NA
MTCC
96
MTCC
442
MTCC
443
MTCC
1688
0.966
1.295
0.633
0.602
0.528
0.222
0.564
0.871
0.842
0.613
0.397
0.402
0.286
0.155
0.075
0.031
NT
1.207
0.324
0.633
0.602
0.264
0.554
0.564
0.697
0.673
0.383
0.794
0.804
0.716
0.155
0.151
0.031
NT
1.207
0.518
0.506
0.482
0.132
0.222
0.226
0.348
0.673
0.307
0.397
0.804
0.286
0.155
0.151
0.031
NT
1.207
0.518
0.316
0.241
0.423
0.554
0.282
0.436
0.421
0.613
0.635
0.643
0.286
0.155
0.075
0.031
NT
4.830
2.590
2.531
NA
S 1
S 2
SS 1
SS 2
SS 3
SS 4
SS 5
SS 6
SS 7
SS 8
SS 9
SS 10
A
NA
1.057
NA
2.114
NA
1.128
NA
1.128
NA
NA
NA
1.534
NA
1.534
NA
3.215
NT
1.607
NT
NT
NT
B
NT
NT
C
NT
NT
D
0.108
1.420
0.108
0.284
E
NT
NT
NT
NT
F
S.A.: Staphylococcus aureus; S.P.: Streptococcus pyogenes; E.C.: Escherichia coli; P.A.:
Pseudomonas aeruginosa; C.A.: Candida albicans; A.C.: Aspergillus clavatus; A:
Ampicillin; B: Chloramphenicol; C: Ciprofloxacin; D: Norfloxacin; E: Nystatin; F:
Greseofulvin; NA: Not active; NT: Not tested
3.4 Cytotoxicity study
The cytotoxicity of compounds (S 1-SS 10) was tested using bioassay on S. pombe cells at
the cellular level. The results obtained are shown in Table 6. From the result, it was easily
monitored that cell death caused by toxic nature of the compounds (S 1-SS 10) by vital
staining. The toxicity was found to vary with the type of substituent present and different
concentrations of compounds (S 1-SS 10). It has been observed that cytotoxicity increases
with increasing the concentration of compounds (S 1-SS 10). The incorporation of S 1 /S 2 in

the Schiff bases (SS 1-SS 10) increased the cytotoxicity appreciably, except for SS 2 having
1, 2, 4-triazole (S 1) moiety. The compound SS 5 containing 1, 2, 4-triazole (S 1) moiety and
-OCH3 group on p-position in benzene ring has maximum cytotoxicity against S. pombe cells.
Most of the compounds can cause about 50% cell death.
Table 6. Effect of compounds on percentage cell viability of S. pombe at different
concentrations with standard deviation for three independent experiments
Percentage cell viability at different concentrations
Compound
ID (s)
2 µg/ml
91.33
0.58
4 µg/ml
85.67
0.58
6 µg/ml
82.00
1.00
8 µg/ml
75.67
1.53
10 µg/ml
67.00
1.00
-
-
-
-
-
-
-
-
-
-
S 1
S 2
87.33
1.15
83.00
1.00
76.00
1.00
71.00
1.00
68.33
0.58
87.67
0.58
82.00
2.00
75.33
0.58
70.67
1.15
63.33
1.15
SS 1
SS 2
SS 3
SS 4
SS 5
SS 6
SS 7
SS 8
SS 9
SS 10
84.67
1.15
79.67
0.58
78.67
0.58
77.00
1.00
72.33
0.58
87.00
1.00
80.00
1.00
73.67
0.58
65.33
1.53
59.67
0.58
83.33
1.53
72.33
0.58
62.33
1.53
55.67
1.53
47.00
1.00
79.00
1.00
64.33
0.58
59.00
1.00
52.67
1.53
38.67
1.53
74.33
0.58
70.67
0.58
66.33
0.58
61.00
1.00
52.67
2.08
82.33
0.58
78.33
0.58
72.00
1.00
67.00
1.00
45.33
3.06
78.33
1.15
74.33
0.58
65.67
0.58
56.67
1.15
46.33
1.53
81.33
1.15
73.33
2.52
64.67
1.15
54.67
0.58
47.00
2.00
80.33
1.15
72.33
0.58
68.00
1.00
63.67
1.53
49.00
1.00

94.67
1.53
-
-
-
-
-
-
-
-
-
-
Untreated
DMSO
93.67
1.53
3.5 Genotoxicity study
Results of cytotoxicity encouraged us to find out whether compounds (S 1-SS 10) have any
effect on the integrity of DNA or not. The effect of compounds (S 1-SS 10) on the DNA
integrity of S. pombe cells was carried out by the isolation of DNA from treated and untreated
S. pombe cells and isolated DNA was electrophoresed on 1% agarose gel followed by
visualization. DNA damage was found as smeared in triazole and oxadiazole analogues (S 1-
SS 10) treated S. pombe cells while in untreated S. pombe cells it was observed as an intact
band without any smear (Fig. 1). The results showed that compounds (S 1-SS 10) affected the
integrity of DNA of S. pombe cells. Smearing of DNA suggested that the damage has
occurred due to the toxic nature of the compounds (S 1-SS 10). This result is in agreement
with the results of cytotoxicity that triazole and oxadiazole analogues (S 1-SS 10) have
entered into the cell and affected the integrity of DNA.

Fig. 1 Effect of the chemically synthesized compounds (S 1-2 and SS 1-10) on the integrity
of DNA isolated from S. pombe cells.
3.6 In vitro antimalarial activity
The compounds S 1-SS 10, Chloroquine and Pyrimethamine were evaluated for their
antimalarial screening against P. falciparum strain. All the experiments were performed in
duplicate and a mean value of IC50 is reported in Table 7.
Table 7. In vitro antimalarial activity of triazole and oxadiazole analogues (S 1-SS 10) (IC50,
µM)
Compound ID(s)
IC50 (µM)
0.960 0.031
0.810 0.043
0.815 0.026
0.282 0.020
0.245 0.030
0.230 0.029
0.835 0.055
0.802 0.041
0.822 0.035
0.806 0.039
0.301 0.021
0.790 0.030
0.063 0.009
1.005 0.053
S 1
S 2
SS 1
SS 2
SS 3
SS 4
SS 5
SS 6
SS 7
SS 8
SS 9
SS 10
Chloroquine
Pyrimethamine

The IC50 values of all compounds (S 1-SS 10) showed improved potency, even better than
reference compound Pyrimethamine. The result of in vitro assay suggested that the presence
of 4-OH (SS 2), 3-NO2 (SS 3) and 4-Cl (SS 4 and SS 9) in phenyl ring exhibited best
antimalarial activity (Table 7). The compounds containing thiophene ring (SS 1 and SS 6), p-
hydroxy benzene (SS 7), m-nitrobenzene (SS 8) and p-methoxybenzene (SS 10) were found
to have enhanced potency, while S 1 (1, 2, 4-triazole; IC50 = 0.960 0.031 µM) and SS 5 (p-
methoxybenzene; IC50 = 0.835 0.055 µM) showed poor activity against P. falciparum.
3.7 In silico docking study
To rationalize the potency of the active compounds (SS 2, SS 3, SS 4, SS 9) as antimalarial
agents, these compounds were docked against P. falciparum dihydrofolate reductase (PDB
ID: 4DPD) obtained from protein data bank (RCSB). The binding energies of drug-receptor
complex were obtained for ligands (SS 2, SS 3, SS 4, SS 9) and standard drugs Chloroquine
& Pyrimethamine are presented in Table 8.
Table 8. Docking results of triazole and oxadiazole analogues (S 1-SS 10) against P.
falciparum (PDB ID: 4DPD)
Binding
Energy
Intermol
Energy
Internal
Energy
Torsional Unbound
Energy Energy
Compound
ID(s)
(kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)
-7.70
-9.06
-8.78
-7.20
-4.24
-6.10
-10.09
-11.45
-10.57
-8.39
-0.93
-1.02
-0.95
-0.64
-1.06
-0.26
2.39
2.39
1.79
1.19
2.39
0.89
-0.93
-1.02
-0.95
-0.64
-1.06
-0.26
SS 2
SS 3
SS 4
SS 9
-6.63
Chloroquine
Pyrimethamine
-7.00
A potential interaction was observed between the active molecules and the Pf-DHFR enzyme.
The 3D diagrams (Fig. 2a-7a) showed the binding sites of all the ligands within the receptor
4DPD. The molecules interacted with the active sites (amino acids) of receptor 4DPD
through conventional hydrogen bonds, van der Waals, π-cation, π-anion, π-alkyl, π-donor
hydrogen bond, π-sulphur, carbon-hydrogen bond, π-sigma and much more (Fig. 2b-7b). As
shown in Table 8, binding energies of the molecules against the active sites indicated higher
affinity of the molecules to bind to the protein leading to its inhibition. The binding energies

of the molecules (SS 2, SS 3, SS 4, SS 9) were found to be ranging from -7.20 to -9.06
kcal/mol and were better compared to standard drugs Chloroquine and Pyrimethamine
(Table 8). The H-atoms of -OH and -SH in SS 2 interacted with active sites of enzyme by
forming H-bond with ASP A:54, ILE A:164 and TYR A:70 at distances of 2.46 °A, 2.05 °A
and 2.57 °A respectively (Fig. 2b). Also, π-alkyl type of interaction observed between
aromatic rings of SS 2 and ALA A:16, LEU A:46, PRO A:113 and MET A:55 with distances
4.08 °A, 5.05 °A, 5.35 °A & 4.72 °A respectively. Hydrogen bonds were observed between
the O-atom of -NO2 group of SS 3 and ARG A:345, SER A:511 & ARG B:470 with bond
lengths of 3.14 °A, 3.20 °A & 3.08 °A respectively (Fig. 3b). In ligand SS 4, aromatic rings
interacted with receptor by forming π-alkyl and alkyl bonds with LEU B:40 (4.84 °A), MET
B:55 (5.31 °A), LEU B:46 (4.88 °A), ILE B:112 (4.64 °A) & PRO B:113 (5.27 °A) (Fig. 4b).
Also, van der Waals, H-bond & π-suplhur type of interactions were indicated with different
amino acids of chain A & B with SS 4 (Fig. 4b). The oxadiazole ring in SS 9 formed π-sigma
& π-alkyl bonds with ILE B:567 (3.93 °A) & ILE B:314 (4.61 °A) respectively (Fig. 5b).
The Cl-atom in Chloroquine (Fig. 6b) interacted through conventional H-bond with THR
A:391 at distance 2.90 °A. Furthermore this Cl-atom formed π-alkyl bond with PHE A:42
(4.95 °A), TYR A:441 (5.49 °A) & TYR A:448 (4.59 °A). In the case of Pyrimethamine,
van der Waals, carbon-hydrogen bond, π-alkyl, alkyl etc. type of interactions were observed
between different amino acids present in chain A of 4DPD receptor and ligand as shown in
Fig. 7b. The results of docking study indicated the selectivity of the potent ligand molecules
as DHFR inhibitors.
(b)
(a)
Fig. 2 (a) 3D model of ligand SS 2 bonded with active site of 4DPD receptor. (b) 2D model
of interaction between amino acid residues and SS 2 with types of bond and bond lengths.

(b)
(a)
Fig. 3 (a) 3D model of ligand SS 3 bonded with active site of 4DPD receptor. (b) 2D model
of interaction between amino acid residues and SS 3 with types of bond and bond lengths.
(a) (b)
Fig. 4 (a) 3D model of ligand SS 4 bonded with active site of 4DPD receptor. (b) 2D model
of interaction between amino acid residues and SS 4 with types of bond and bond lengths.
(b)
(a)
Fig. 5 (a) 3D model of ligand SS 9 bonded with active site of 4DPD receptor. (b) 2D model
of interaction between amino acid residues and SS 9 with types of bond and bond lengths.

(b)
(a)
Fig. 6 (a) 3D model of ligand Chloroquine bonded with active site of 4DPD receptor. (b) 2D
model of interaction between amino acid residues and Chloroquine with types of bond and
bond lengths.
(a)
(b)
Fig. 7 (a) 3D model of ligand Pyrimethamine bonded with active site of 4DPD receptor. (b)
2D model of interaction between amino acid residues and Pyrimethamine with types of
bond and bond lengths.
3.8 DHFR Inhibition assay:
The potent entities (SS 2, SS 3, SS 4, SS 9) found to be active against P. falciparum strain
and standard drugs (Chloroquine & Pyrimethamine) were further evaluated for inhibitory
efficacy against bovine liver DHFR enzyme. The average IC50 (µM) values are presented
with standard deviation for three independent experiments in Table 9.
Table 9. DHFR enzyme inhibition assay
Compound ID(s)
IC50 (µM)
0.0495 0.009
0.0259 0.006
0.0349 0.004
SS 2
SS 3
SS 4

0.0450 0.005
0.0301 0.005
SS 9
Chloroquine
Pyrimethamine 0.1007 0.012
The IC50 values of SS 2, SS 3, SS 4 & SS 9 indicated that all compounds were potent DHFR
inhibitors when compared to standard drugs (Table 9). The triazole motif attached with two
m-nitrobenzene groups (SS 3; IC50 = 0.0259 0.006 µM) was found to be most potent
inhibitor, while the compound SS 4 (IC50 = 0.0349 0.004 µM) having two p-chlorobenzene
ring attached to triazole motif had more or less inhibitory property similar to Chloroquine
(IC50 = 0.0301 0.005 µM). The presence of 4-OH (SS 2) and 4-Cl (SS 9) in phenyl ring
were found to be poor inhibitors of enzyme compared to Chloroquine (Table 9). The results
suggested that the analogues were active anti-malarial compounds inhibiting dihydrofolate
reductase enzyme.
4. Conclusion
Analogues of 1, 2, 4-triazole and 1, 3, 4-oxadiazole have been designed, synthesized and
characterized. To avoid late stage failure, it is important to study the preliminary
pharmacokinetic parameters. The results of pharmacokinetic data suggested that, all
molecules have tendency to be considered as drugs. So in vitro antimicrobial, cytotoxicity
and genotoxicity on S. pombe cells and antimalarial on P. falciparum of these analogues have
been investigated. The molecules were subjected to DFT and PM6 calculations to explain the
anti-bacterial activity. It was found that the antibacterial activity could be correlated to
energies of the LUMO and maximum LUMO electron density. All the compounds possessed
moderate to good inhibitory potency against bacterial strains and did not show any anti-
fungal activities. The compounds SS 4, SS 5 and SS 7-SS 10 were found to possess
maximum toxicity against S. pombe cells at cellular level. All the compounds S 1-SS 10
showed smearing type pattern on agarose gel due to toxic nature of these compounds. The
anti-malarial activity of these compounds were studied. The compounds SS 2 (two p-hydroxy
benzene groups), SS 3 (two m-nitrobenzene groups) and SS 4 (two p-chlorobenzene groups)
attached to 1, 2, 4-triazole ring and SS 9 (p-chlorobenzene) attached to 1, 3, 4-oxadiazole ring
were found to be potent against P. falciparum strain. The in silico docking showed that the
potent molecules synthesized and the standard drugs inhibit the enzyme P. falciparum
dihydrofolate reductase occupying the active binding pocket with great ease. The antimalarial
efficacy was further proved by in vitro DHFR enzyme inhibition study. Hence, this study

identified new structural type antibacterial and antimalarial agents which could be used as
lead molecules for further research and development of antibacterial and antimalarial agents.
Conflict of interest We wish to confirm that there are no known conflicts of interest
associated with this publication.
Acknowledgement The authors thank the authorities of Charotar University of Science &
Technology (CHARUSAT), Changa, Gujarat, India for providing the facilities to carry out
this investigation. They also thank Dr. Devendra Tiwari, School of Chemistry, University of
Bristol, U.K. for his help in the molecular orbital study. Mr. Parth Thakor is thankful to DST
INSPIRE program for the fellowship.
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