Synthesis of new 1, 3, 4-oxadiazole-incorporated 1, 2, 3-triazole moieties as potential anticancer agents targeting thymidylate synthase and their docking studies

Article abstract of DOI:10.3390/ph13110390

Thymidylate synthase (TS) has emerged as a hot spot in cancer treatment, as it is directly involved in DNA synthesis. In the present article, nine hybrids containing 1,2,3-triazole and 1,3,4-oxadiazole moieties (6–14) were synthesized and evaluated for anticancer and in vitro thymidylate synthase activities. According to in silico pharmacokinetic studies, the synthesized hybrids exhibited good drug likeness properties and bioavailability. The cytotoxicity results indicated that compounds 12 and 13 exhibited remarkable inhibition on the tested Michigan Cancer Foundation (MCF-7) and Human colorectal Carcinoma (HCT-116) cell lines. Compound 12 showed four-fold inhibition to a standard drug, 5-fluoruracil, and comparable inhibition to tamoxifen, whereas compound 13 exerted five-fold activity of tamoxifen and 24-fold activity of 5-fluorouracil for MCF-7 cells. Compounds 12 and 13 inhibited thymidylate synthase enzyme, with an half maximal inhibitory concentration, IC₅₀ of 2.52 μM and 4.38 μM, while a standard drug, pemetrexed, showed IC₅₀ = 6.75 μM. The molecular docking data of compounds 12 and 13 were found to be in support of biological activities data. In conclusion, hybrids (12 and 13) may inhibit thymidylate synthase enzyme, which could play a significant role as a chemotherapeutic agent.

Full text of DOI:10.3390/ph13110390

pharmaceuticals
Article
Synthesis of New 1, 3, 4-Oxadiazole-Incorporated 1, 2,
3-Triazole Moieties as Potential Anticancer Agents
Targeting Thymidylate Synthase and Their
Docking Studies
Mohammad Mahboob Alam 1, Abdulraheem SA Almalki 2 , Thikryat Neamatallah 3,†
Nada M. Ali 1, Azizah M. Malebari 4 and Syed Nazreen 1, ,†
,
*
1
2
3
4
Department of Chemistry, Faculty of Science, Albaha University, Albaha-1988, Saudi Arabia;
mmalamchem@gmail.com (M.M.A.); umyousee@gmail.com (N.M.A.)
Department of Chemistry, Faculty of Science, Taif University, Taif-21974, Saudi Arabia;
drasaalmalki1@gmail.com
Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah
21589, Saudi Arabia; Taneamatallah@kau.edu.sa
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589,
Saudi Arabia; amelibary@kau.edu.sa
*
Correspondence: syed.nazreen22885@gmail.com
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 22 October 2020; Accepted: 12 November 2020; Published: 14 November 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Thymidylate synthase (TS) has emerged as a hot spot in cancer treatment, as it is
directly involved in DNA synthesis. In the present article, nine hybrids containing 1,2,3-triazole
and 1,3,4-oxadiazole moieties (6–14) were synthesized and evaluated for anticancer and in vitro
thymidylate synthase activities. According to in silico pharmacokinetic studies, the synthesized
hybrids exhibited good drug likeness properties and bioavailability. The cytotoxicity results indicated
that compounds 12 and 13 exhibited remarkable inhibition on the tested Michigan Cancer Foundation
(MCF-7) and Human colorectal Carcinoma (HCT-116) cell lines. Compound 12 showed four-fold
inhibition to a standard drug, 5-fluoruracil, and comparable inhibition to tamoxifen, whereas
compound 13 exerted five-fold activity of tamoxifen and 24-fold activity of 5-fluorouracil for
MCF-7 cells. Compounds 12 and 13 inhibited thymidylate synthase enzyme, with an half maximal
inhibitory concentration, IC50 of 2.52 µM and 4.38 µM, while a standard drug, pemetrexed, showed
IC50 = 6.75 µM. The molecular docking data of compounds 12 and 13 were found to be in support
of biological activities data. In conclusion, hybrids (12 and 13) may inhibit thymidylate synthase
enzyme, which could play a significant role as a chemotherapeutic agent.
Keywords: thymidylate synthase; cytotoxicity; 1,2,3-triazole; 1,3,4-oxadiazole; 5-fluoruracil;
pemetrexed; docking
1. Introduction
Cancer is uncontrolled cell growth and cell proliferation, and remains a global burden despite the
advancements in cancer diagnosis and treatment [
develop resistance against the chemotherapeutic agents and toxicity to normal cells [
is the only effective treatment, causing the inhibition of cancer cell growth and induction of apoptosis,
as the DNA levels in tumor cells are significantly higher than in normal cells [ ]. Interestingly,
1
]. The available anticancer drugs on the market
2,3]. Chemotherapy
4,5
thymidylate synthase (TS) has now emerged as an important target for chemotherapy for cancer
treatment, as it is directly involved in DNA synthesis [6].
Pharmaceuticals 2020, 13, 390; doi:10.3390/ph13110390
www.mdpi.com/journal/pharmaceuticals

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Thymidylate synthase enzyme catalyzes the reductive methylation of deoxyuridine monophosphate
(dUMP) to deoxythymidine monophosphate (dTMP) and 5,10-methylenetetrahydrofolate (CH2THF) [
]. This dTMP upon phosphorylation changes into thymine triphospate (dTTP), which acts as a direct
precursor for the synthesis of DNA [ 10]. The blocking of dTMP causes a decrease in deoxythymidine
7,
8
9,
triphosphate (dTTP), thereby interrupting DNA biosynthesis causing DNA damage [11]. In addition,
TS inhibition causes an increase in dUMP, leading to surge in the deoxyuridine triphosphate (dUTP)
level [12]. One of the anticancer drugs, 5-fluoruracil (5-FU), acts as a strong thymidylate inhibitor for
various cancers [13]. Also, a 5-FU metabolite, fluorodeoxyuridine monophosphate (FdUMP), binds
with a TS active site to form a stable ternary complex, thus blocking the binding of the dUMP with TS
and leading to the inhibition of dTMP synthesis [14].
Heterocycle has been a main pharmacophore in drug discovery and development. In the last few
years, there is an emergence in the development of different 1,2,3-triazole-linked heterocycles, due to
their excellent pharmacological properties, including anticancer [15], antiviral [16], antidiabetic [17],
antimicrobial [18], anti-inflammatory [19], and antitubercular [20]. Compounds containing
1,2,3-triazoles have been reported to exert anticancer effect by inhibiting TS enzymes [21–23]. On the
other hand, 1,3,4-oxadiazole plays a crucial part in medicinal chemistry. Zibotentan, an anticancer
drug containing 1,3,4-oxadiazole as a pharmacophore, is used for various cancers [24]. Furthermore,
1,3,4-oxadiazole-linked heterocycles have also been reported to act as potential TS inhibitors [25,26].
Therefore, combining these two heterocycles in one molecule may lead to development of effective TS
inhibitor (Figure 1).
O
O
N
S
NO2
HN
NH
O
HN
NH
O
H3C
O
N
O
O
O
O
O
O
S
HN
O
S
HN
F
HN
O
N
F
N
N
N
N
F
N
O
Cl
NO2
N
N
N
IC50 = 0.057 M
N
N
IC50 = 0.62 M
Cl
IC50 = 0.03 M
IC50 = 0.11 M
F
O
HN
H3C
N
O
O
O
N
O
O
F
N
N
S
HN
N
HO
O
N
N
O
O
O
N
S
N
O
N
O
N
N
N
IC50 = 0.11 M
OH
Cl
IC50 = 1.67 M
R
Target compounds
Figure 1. Reported thymidylate synthase inhibitors containing 1,3,4-oxadiazole and 1,2,3-triazoles.
In our previous work, we have reported thiazolidinedione-linked 1,3,4-oxadiazole as a promising
TS inhibitor [27]. In continuation of our work to develop an effective TS inhibitor, we tried to conjugate
1,3,4-oxadiazole and 1,2,3-triazole pharmacophore in a single hybrid, which can inhibit the TS enzyme
effectively. In this work, we describe the synthesis, pharmacokinetic study, anticancer, and TS inhibitory
activities. The active compounds were docked against the thymidylate synthase enzymes to see the
molecular interactions of active compounds with binding proteins.

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2. Results and Discussion
2.1. Chemistry
Esterification of benzoic acid
1
in the presence of MeOH and concentrated H2SO4 yielded methyl
benzoate
hydrazide
2
, which further reacted with hydrazine monohydrate in absolute ethanol to give acid
. To this acid hydrazide were added ethanol, carbon disulphide, and KOH, and the
3
3
mixture was stirred for 24 h and then refluxed for 14 h. After completion of the reaction, monitored by
Thin Layer Chromatography (TLC), the reaction mixture was concentrated, and 100 mL cold water
was added. The acidification of aqueous solution with concentrated HCl (pH 3–4) produced a white
precipitate, which was filtered and recrystalized in ethanol to afford compound 4. The propargylation
of compound
intermediate 5. Finally, the key intermediate
4
in the presence of potassium carbonate and propargyl bromide yielded the key
, using a click chemistry approach with different aromatic
5
azides in the presence of copper sulphate pentahydrate and sodium ascorbate, yielded final compounds
6–14 (Figure 2).
O
O
(b)
O
(a)
OH
O CH3
HN NH2
3
2
1
(c, d)
N
N
O
(e)
Ar-N3
(f)
N
N
N
N
S
O
O
SH
S
Ar
N
N
N
6-14
4
5
CH3
NO2
Cl
H3C
6
Cl
Br
10
8
9
7
O
O
Ar =
O
HO
H3C
11
13
12
14
Figure 2. Synthesis of 1,3,4-oxadiazole linked 1,2,3-triazole hybrids. Reagents and conditions:
) Methanol, Conc. H2SO4, reflux, 6h; ( ) Absolute ethanol, H2N-NH2.H2O, reflux 4h; ( ) Absolute
ethanol, CS2, KOH, stir, 24 h; ( ) Acetone, K2CO3, Propargyl bromide, stir,
(
a
b
c
d
) reflux, 14 h, Conc. HCl; (e
50–60 ◦C, 6h; (f) tert.butanol:water (1:1), CuSO4.5H2O, sodium ascorbate, stir, 6–12 h.
All the synthesized conjugates have been confirmed using different analytical techniques, such as
IR, NMR, and mass spectrometry. Formation of the compounds
the molecular ion peak at 137 in Electron Spray Ionisation (ESI ) mass spectra and from their boiling
point and melting point, respectively [28]. Formation of compound was done by the presence of
stretching frequency of an oxadiazole ring at 1500–1600 cm−1,–SH bond stretching at 2400 cm−1 in the
IR spectrum, presence of an S–H singlet at
10.77 ppm in the 1H NMR spectrum, and the molecular
ion signal at to compound was
179 [M + H]+ in mass spectrum. The propargylation of compound
confirmed by the absence of chemical shift of an –SH proton at 10.77 ppm, as well as the presence
2 and 3 were done by the presence of
4
δ
δ
4
5
δ

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of a triplet at
δ
2.34 ppm and a singlet at
δ
4.5 ppm for terminal alkyne proton and S–CH2- protons,
respectively. Finally, the formation of the compound 5 was supported by the presence of a molecular
ion signal at 217 [M + H] + in the ESI mass spectrum. Structural confirmation of final hybrids
14 was
observed by disappearance of terminal alkyne proton peak at 2.34 ppm, as well as the appearance of
a 1,2,3-triazole ring proton in the range of 8.19–8.89 ppm with additional aromatic protons. Finally,
6
–
δ
δ
the presence of molecular ion peaks in the mass spectrum confirmed the formation of all the final
compounds (The spectra are provided in Supplementary Materials).
2.2. Pharmacokinetics Studies/ADME Predictions
Nowadays, in silico pharmacokinetic predictions are extensively used in drug discovery to reduce
the cost and time. To be orally available, the molecule is not only required to have excellent biological
activity, but it must follow the desired pharmacokinetic properties. The in silico studies that have been
carried out on synthesized molecules 6–14 have been to check whether these molecules satisfy the
desirable pharmacokinetics or not, which plays a crucial role in drug discovery. The physicochemical
properties directly affect the pharmacokinetic behavior. The synthesized molecules should obey the
Lipinski [29] and Veber [30] rules for an orally available drug. The molecule must follow the following
paremeters: molecular weight (MW) should be less than 500, the number of hydrogen bond acceptors
(HBAs) should be fewer than 10, the number of hydrogen bond donors (HBD) should be fewer than 5,
the partition coefficient (logP) should be less than 5, and the number of rotatable bonds should be 10
≤
for drug likeness and good bioavailability (Table 1).
Table 1. Pharmacokinetic/Absorption, Distribution, Metabolism and Elimination (ADME) predictions
of the target compounds 6–14.
Lipinski Parameters
GI
nROTB e
%ABS g
TPSA f
BBB h
No.
ABS i
MW a
HBDs c
Violations
HBAs b
LogP d
6
7
8
349.41
349.41
369.83
369.83
414.28
380.38
335.38
393.42
379.39
5
5
5
5
5
7
5
7
7
0
0
0
0
0
0
0
0
1
3.54
3.5
3.33
3.5
3.62
2.97
3.21
3.39
2.48
0
0
0
0
0
0
0
0
0
5
5
5
5
5
6
5
7
6
94.93
94.93
94.93
94.93
94.93
140.75
94.93
121.23
132.23
76.24
76.24
76.24
76.24
76.24
60.44
76.24
67.17
63.38
No
No
No
No
No
No
No
No
No
High
High
High
High
High
Low
High
High
High
9
10
11
12
13
14
a Molecular weight; b hydrogen bond acceptors; c hydrogen bond donors; d partition coefficient; e number of rotatable
bonds; f topological polar surface area; g absorption (%); h blood–brain barrier; i gastro-intestinal absorption.
From the above data, it was noticed that the synthesized molecules 6–14 follow the desired
pharmacokinetic properties. All the final compounds, except compound 11, showed high
gastrointestinal absorption. The % absorption of the molecules was found to be in the range 60.44–76.24.
Compounds 6, 7, 8, 9, 10, and 12 showed the highest absorption of 76.24%, while compound 11
showed the lowest absorption at 60.44%. All the compounds followed Lipinki and Veber rules, i.e.,
molecular weight ranging from 335–414, hydrogen bond acceptors ranging from 5–7, hydrogen bond
donors ranging from 0–1, lipophilicity appearing in the range 2.48–3.62, and the number of rotatable
bonds between 5 and 7. The synthesized compounds exhibited higher logP values, in the range
2.48–3.62, suggesting higher cell membrane permeability. The final hybrids exhibited topological
polar surface areas (TPSAs) between 94.93–140.75 A2, which suggests good intestinal absorption.
The pharmacokinetic results indicate that these compounds satisfy the criteria for good drug likeness
parameters and good bioavailability.

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2.3. Biological Studies
2.3.1. Cytotoxicity Assay
The final compounds
6
–
14 were screened for their antiproliferative effect using an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. These compounds were tested
against breast MCF-7 and colorectal HCT-116 cell lines. These compounds exhibited moderate to
significant activity compared to standard drugs tamoxifen and 5-fluorouracil (5-FU). The results are
presented in Table 2.
Table 2. The IC50
(
µ
M) of the synthesized compounds (
6–
14) against tested human cancer cell lines
(MCF-7 and HCT-116).a
MCF-7 b
Compound
HCT-116 c
6
7
8
9
10
11
12
13
14
79.80
30.70
73.30
34.40
25.90
98.20
5.80
1.26
40.60
5.12
24.74
89.20
34.30
107.50
36.70
32.70
102.30
14.80
17.30
46.80
26.41
32.68
Tam d
5-FU e
a
IC50 values are the concentrations that cause 50% inhibition of cancer cell growth. Data represent the mean values
standard deviation of three independent experiments, performed in triplicate; b breast cancer (MCF-7); c colorectal
±
cancer (HCT-116); d Tam: tamoxifen; e 5-FU: 5-florouracil, which was used as a reference drug (positive control).
Among the tested compounds, compounds 12 and 13 showed significant inhibitory effects on the
viability of MCF-7 and HCT-116 cells (Figure 3). Compound 12 (IC50 = 5.8
inhibition to the standard drug tamoxifen (IC50 = 5.12) M and four-fold inhibition compared to
the standard drug 5-fluoruracil (IC50 = 24.74 M). Compound 13, however, was found to exhibit
promising inhibition against MCF-7, with IC50 = 1.26 M, while the two standard drugs, tamoxifen
and 5-florouracil, showed IC50 = 5.12 M and 24.74 M, respectively. It is clear from the results that
µM) showed comparable
µ
µ
µ
µ
µ
compound 13 was more potent in exerting the anticancer effect, with five times the activity of tamoxifen
and 24 times the activity of 5-fluorouracil for MCF-7 (Figure 4).
The same two compounds (12 and 13) also displayed significant inhibition against HCT-116 cells.
Compound 12 (IC50 = 14.8
and 2.2-fold activity compared to 5-fluorouracil (IC50 = 32.68
13 (IC50 = 17.3 M) displayed 1.9-fold activity compared to 5-FU and 1.5-fold the activity of tamoxifen.
Other compounds, 6, 7, 8, 9, 10, 11, and 14, exhibited moderate cytotoxicity with the MCF-7, with IC50
values in the range 25.9–98.28 M. On the other hand, compounds 6, 7, 9, 10, and 14 displayed
µM) revealed 1.7-fold activity compared to tamoxifen (IC50 = 25.41
µM)
µM) against HCT-116, while compound
µ
µ
inhibition with IC50 values in the range of 32.7–89.2 µM, whereas compounds 8 and 11 were found to
be inactive (IC50 > 100) against the HCT-116 cells.

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Figure 3. Antiproliferative effects of compound 12 in HCT-116 and MCF-7 cells. Cell viability
was expressed as a percentage of vehicle control (ethanol 1% (v/v)) and was measured by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The values represent the mean
± standard error of the mean (SEM) for three independent experiments performed in triplicate.
Figure 4. Antiproliferative effects of compound 13 in HCT-116 and MCF-7 cells. Cell viability was
expressed as a percentage of vehicle control (ethanol 1% (v/v)) and was measured by MTT assay.
The values represent the mean ± SEM for three independent experiments performed in triplicate.
The most potent compounds, 12 and 13, were also tested on non-tumorigenic cell line HEK 293
(normal human embryonic kidney) cells, in order to investigate the toxicity and selectivity of these
two potent compounds (Figures 5 and 6). It was found that these two compounds (12 and 13) showed
IC50 values above 50
cancer cell lines—MCF-7: IC50 = 5.8
13)—indicating that these molecules was less toxic to human normal cells and selective to cancerous
µM on HEK 293 cells, which was higher than those observed against the two
µ
M (12), 1.26 M (13); HCT-116: IC50 = 14.8 M (12), 17.3 µM
µ
µ
(
cells. From these results, it is clear that compound 12 with no substituents and compound 13 with a
–COOMe group at the ortho position exerted promising antiproliferative effects on both the tested cells,
whereas the presence of halogens and electron withdrawing groups (nitro) on the aromatic ring did
not played a significant role in exerting the anticancer effect.

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MCF-7
*
*
HCT-116
HEK-293
100
*
*
80
60
40
20
0
*
*
Concentration (μM)
Figure 5. Antiproliferative effects of compound 12 on the viability of cancerous MCF-7 and HCT-116
cells, as well as non-tumorigenic HEK-293 cells. The values represent the mean SEM for three
±
independent experiments performed in triplicate. * p < 0.05 between the indicated groups, via two-way
ANOVA (Bonferroni post-test).
MCF-7
*
*
100
80
60
40
20
0
HCT-116
HEK-293
*
*
*
*
Concentration (μM)
Figure 6. Antiproliferative effects of compound 13 on the viability of cancerous MCF-7 and HCT-116
cells, as well as non-tumorigenic HEK-293 cells. The values represent the mean SEM for three
±
independent experiments performed in triplicate. * p < 0.05 between indicate groups, via two-way
ANOVA (Bonferroni post-test).
2.3.2. In Vitro Thymidylate Synthase Activity
Thymidylate synthase (TS) has become an important target for cancer treatment, as it is involved in
DNA synthesis. The inhibition of this enzyme plays a vital role in chemotherapy treatment. The active
compounds (12 and 13) from the MTT assay were screened for in vitro thymidylate synthase, to confirm
its mechanism of action. These compounds inhibited the TS enzyme significantly compared to the
standard drug pemetrexed. Compound 12 inhibited TS with IC50 = 2.52
IC50 = 4.38 (Table 3). It was observed that compounds 12 and 13 showed 2.6- and 1.5-fold activity
compared to pemetrexed (IC50 = 6.75 M). From these results, it can be inferred that compounds 12
µM, and 13 inhibited it with
µ
and 13 inhibit TS by binding with the active binding site of the enzyme, which results in the blocking
of dUMP with TS, leading to inactivation of dTMP. This inactivation of dTMP results in a reduction of
deoxythymidine triphosphate (dTTP), leading to the disruption of DNA synthesis and cessation of cell
growth and proliferation (Figure 7). These results support our antiproliferative results.

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Table 3. In vitro thymidylate synthase (TS) activity of the active compounds 12 and 13, as well as PTX.
Compounds
IC50 (µM)
12
13
PTX
2.52
4.38
6.75
IC50 values are the mean ± SD of three separate experiments. PTX: pemetrexed.
Figure 7. Mechanism of action of compound 12 on thymidylate synthase enzyme.
2.4. Molecular Docking
It is a computational technique used frequently to know the possible interactions of a molecule with
a receptor. The active compounds from in vitro studies were docked against TS proteins (PDB = 6QXG)
to support our in vitro thymidylate synthase results, as well as to know the possible binding modes
with the protein. The 5-fluorouracil has been reported as a TS inhibitor that interacts with the active
binding site of the protein through different residues [31,32]. Therefore, we have docked our active
compounds 12 and 13 against this protein, in order to support our in vitro findings.The results are
presented in Figure 8 and Table 4.
Table 4. Docking scores of active compounds 12 and 13 against human thymidylate synthase
protein 6QXG.
Compound
Docking Score
Amino Acid Residue
12
13
5-FU
−3.81
−4.25
−3.5
ASN 226
ASN 226, PHE 225, HIE 196
ASP 218, ASN 226

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Figure 8. Molecular docking of the active compounds 12 and 13, as well as 5-FU, against thimidylate
synthase (TS) protein 6QXG. ( ) Binding mode of 12 13, and 5-FU at the TS binding site, with a
three-dimensional (3D) plot. ( ) Binding mode of 12 13, and 5-FU at the TS binding site on a
two-dimensional (2D) plot. 5-FU: 5-fluorouracil.
A
,
B
,
From the docking results, it was observed that compound 12
(
−
3.81) and 13
( 4.25) showed higher
−
dock scores than the standard drug 5-fluorouracil ( 3.5). The nitrogen of the 1,2,3-triazole ring in
−
compounds 12 and 13 showed hydrogen bonding interactions with ASN 226 residues, while two pi–pi
interactions were also observed in compound 13. One pi interaction was between a 1,2,3-oxadiazole
ring and HIE 196, and another was the interaction of a 1,2,3-triazole ring with a PHE 225 residue.
The standard drug 5-FU showed H-bonding interactions of ASP 218 with a C=O group at the 2 position,
and ASN 226 with N–H and a C=O group at the 3 and 4 positions. The binding pattern of compounds
12 and 13, as well as 5-FU, was found to be similar to the thymidylate synthase protein. These finding
suggests that these two compounds (12 and 13) exert anticancer effects by inhibiting the thymidylate
synthase enzyme, which supports the in vitro TS results of hybrids 12 and 13, with IC50 values of 2.52
and 4.38 µM, respectively.

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3. Experimental
3.1. Chemistry
The chemicals and other reagents were procured from Sigma Aldrich (Germany) and Loba Chem
(India), and were used without further purification. FTIR spectra were recorded on a Thermo Scientific
iS 50 by ATR method. Nuclear magnetic resonance (NMR) analysis was performed on Bruker 300 and
850 MHz instruments in CDCl3 or DMSO-d6 solvents; tetramethylsilane (TMS) was used as an internal
reference. Chemical shift and coupling constant are provided in parts per million (ppm) and Hertz,
respectively. All the samples were analyzed by mass spectrometry on a Thermo Scientific-LCQ Fleet
(LCF10605) using the electron spray ionization method, and are provided in m/z. The melting points
of the compounds were determined on the electro-thermal melting point apparatus (Stuart SMP40).
A LEECO Elementar Analyzer was used for elemental analysis of the synthesized compounds, which
was reported in % standard; these were 0.4% of the calculated values. Monitoring of the reaction and
±
purity of the compounds were checked on a silica gel G plate (Merck, Germany).
3.2. General Procedure for the Synthesis of 1,3,4-Oxadiazole Linked 1,2,3-Triazole Hybrids (6–14)
Compound 5 (0.01 mmole) was charged in a 100 mL round-bottom flask, followed by addition
of tert-BuOH–H2O (1:1, 30–50 mL). The reaction mixture was heated to get a clear solution and
cooled to room temperature. To this reaction mixture, copper sulphate pentahydrate (0.0013 mmol)
and sodium ascorbate (0.0013 mmol) was added and stirred for 30 mins, followed by drop-wise
addition of aromatic azides (0.15 mmole). The reaction mixture was stirred for 5–12 hrs at room
temperature, and the progress of the reaction was monitored by TLC, using n-hexane/ethylacetate (6:4)
as eluents. After completion of the reaction, water (50 mL) was added to the reaction mixture and the
products were extracted with dichloromethane (100 mL
dried over anhydrous sodium sulphate, concentrated, and recrystallized using dichloromethane and
×
2). The dichloromethane layer was combined,
cyclohexane to get target compounds 6–14 in 65–85% yield.
3.2.1. 4-((5-phenyl–1,3,4-oxadiazol–2-ylthio)methyl)-1-p-tolyl–1H-1,2,3-triazole (6)
Grey crystals, yield = 80%, melting point (MP) = 146–147 ◦C. IR (ATR)
(C=C), 1518 1468, 1340, 1257, 1232 (C=N, C–N), 1192, 1079 (C–O), 700, 688 (C–S) cm−1
(300 MHz, CDCl3): 2.40 (s, 3H), 4.69 (s, 2H), 7.28 (d, J = 8.1 Hz, 2H), 7.46–7.59 (m, 5H), 7.98 (d,
J = 7.2 Hz, 2H), 8.19 (s, 1H). 13C NMR (75 MHz, CDCl3):
21.01, 27.25, 120.41, 122.48, 123.49, 126.87,
ν
max: 3086 (C–H), 1559
1H NMR
.
δ
δ
129.86, 130.68, 132.49, 134.68, 138.86, 143.91, 163.40, 165.84. ESI MS: 350 (M+ + H). C18H15N5OS
(calculated): C = 61.87; H = 4.33; N = 20.04; O = 4.58; S = 9.18; observed: C = 61.42; H = 4.35; N = 20.01;
O = 4.60; S = 9.16.
3.2.2. 4-((5-phenyl–1,3,4-oxadiazol-2-ylthio)methyl)–1-o-tolyl-1H–1,2,3-triazole (7)
White powder, yield = 75%, MP = 112–113 ◦C. IR (ATR)
νmax: 3123 (C–H aromatic), (C–H), 2920
(C–H), 1606, 1557 (C=C), 1505, 1491, 1466, 1382, 1341 (C=N, C–N), 1185, 1076 (C–O), 701, 683 (C–S)
1
cm−1. H NMR (300 MHz, CDCl3):
δ
2.52 (s, 3H), 4.75 (s, 2H), 7.11–7.14 (m, 2H), 7.57–7.66 (m, 3H),
17.75, 27.33, 123.51, 125.94, 126.41, 126.89,
7.77–8.0 (3, 4H), 8.72 (s, 1H). 13C NMR (75 MHz, CDCl3):
δ
127.43, 129.86, 130.31, 131.80, 132.50, 133.47, 136.57, 142.86, 163.36, 165.87. ESI MS: 350 (M+ + H).
C18H15N5OS (calculated): C = 61.87; H = 4.33; N = 20.04; O = 4.58; S = 9.18; observed: C = 61.82;
H = 4.36; N = 20.01; O = 4.60; S = 9.17.
3.2.3. 4-((5-phenyl–1,3,4-oxadiazol–2-ylthio)methyl)–1-(2-chlorophenyl)-1H–1,2,3-triazole (8)
White crystals, yield = 65%, MP = 111–112 ◦C. (ATR)
ν
max: 3142, 3089 (C–H aromatic), 1557 (C=C),
1490, 1470, 1342 (C=N, C–N), 1192, 1073, 1064 (C–O), 769, 717 (C–Cl), 704, 686 (C–S) cm−1. H NMR
1
(300 MHz, CDCl3):
δ
4.71 (s, 1H), 7.39–7.61 (m, 7H), 7.97–8.00 (m, 2H), 8.23 (s, 1H). 13C NMR (75 MHz,

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CDCl3):
δ 27.19, 123.49, 126.51, 126.89, 128.77, 128.85, 128.92, 129.86, 130.99, 132.13, 132.50, 134.80,
142.98, 163.31, 165.87. ESI MS: 370 (M+ + H), 372 (M+ + 2 + H). C17H12N5OS (calculated): C = 55.21;
H = 3.27; N = 18.94; O = 4.33; S = 8.67; observed: C = 55.16; H = 3.30; N = 18.89; O = 4.35; S = 8.64.
3.2.4. 4-((5-phenyl–1,3,4-oxadiazol-2-ylthio)methyl)–1-(4-chlorophenyl)-1H–1,2,3-triazole (9)
Brown powder, yield = 75%, MP = 200–202 ◦C. (ATR)
νmax: 3082 (C–H, aromatic), 2920 (C–H),
1558 (C=C), 1490, 1473, 1448, 1427, 1339 (C=N, C–N), 1195, 1093, 1077, 1051 (C–O), 846, 827 (C–Cl), 702,
1
686 (C–S) cm−1. H NMR (300 MHz, CDCl3):
δ 4.68 (s, 2H), 7.26–7.28 (m, 5H), 7.49 (t, J = 7.8 Hz, 2H),
7.67 (d, J = 8.7 Hz, 2H), 7.87 (d, J = 6.6 Hz, 2H), 8.24 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 27.18, 119.06,
120.30, 122.77, 123.47, 126.83, 128.97, 129.81, 132.00, 132.45, 134.64, 137.92, 144.23, 163.33, 165.82. ESI MS:
370 (M++H), 372 (M++2+H). C17H12N5OS (calculated): C = 55.21; H = 3.27; N = 18.94; O = 4.33;
S = 8.67; observed: C = 55.16; H = 3.30; N = 18.93; O = 4.37; S = 8.63.
3.2.5. 4-((5-phenyl–1,3,4-oxadiazol–2-ylthio)methyl)-1-(3-bromophenyl)-1H–1,2,3-triazole (10)
White flakes, yield = 70%, MP = 146–148 ◦C. IR (ATR)
νmax: 3137, 3083 (C–H, aromatic), 1607,
1588 (C=C), 1558, 1486, 1471, 1461, 1341, 1289, 1255 (C=N, C–N), 1191, 1077, 1046 (C–O), 702 (C–S), 691
1
(C–Br) cm−1. H NMR (300 MHz, CDCl3):
δ
4.67 (s, 2H), 7.35–7.68 (m, 6H), 7.92–8.00 (m, 3H), 8.24 (s,
1H). 13C NMR (75 MHz, CDCl3):
δ 27.19, 119.53, 122.79, 122.89, 123.09, 123.50, 126.87, 129.86, 131.95,
132.28, 132.49, 138.02, 144.22, 163.32, 165.85. ESI MS: 414 (M+ + H), 416 (M+ + 2 + H). C17H12N5OS:
(calculated): C = 49.29; H = 2.92; N = 16.90; O = 3.86; S = 7.74; observed: C = 49.31; H = 2.94; N = 16.87;
O = 3.83; S = 7.76.
3.2.6. 4-((5-phenyl–1,3,4-oxadiazol–2-ylthio)methyl)–1-(4-nitrophenyl)-1H–1,2,3-triazole (11)
Light orange solid, yield = 65%, MP = 202–204 ◦C. IR (ATR)
νmax: 3127, 3079 (C–H, aromatic),
1596 (C=C), 1558 (N–O) 1523, 1505, 1473, 1388 (C=N, C–N), 1260, 1232, 1191, 1109 (C–O), 703, 686
1
(C–S) cm−1. H NMR (850 MHz, DMSO–d6)
δ
: 4.79 (s, 2H), 7.27–8.01 (m, 9H), 8.26 (s, 1H). 13C NMR
(213 MHz, DMSO) δ: 27.25, 120.17, 12.87, 123.38, 126.60, 127.47, 127.66, 128.54, 128.70, 131.98, 132.22,
137.11, 147.54, 163.52, 165.58. ESI MS: 381 (M + H). C17H12N6O3S (calculated): C = 53.68; H = 3.18; N =
22.09; O = 12.62; S = 8.43; observed: C = 53.69; H = 3.20; N = 22.07; O = 12.62; S = 8.42.
3.2.7. 4-((5-phenyl–1,3,4-oxadiazol–2-ylthio)methyl)-1-phenyl-1H–1,2,3-triazole (12)
White flakes, yield = 75%, MP = 130–131 ◦C. IR (ATR)
ν
max: 3144 (C–H), 1594 (C=C), 1506, 1466,
1344, 1290, 1256 (C=N, C–N), 1191, 1173, 1077, 1064 (C–O), 702, 683 (C–S) cm−1. H NMR (300 MHz,
1
CDCl3):
δ δ 27.26, 120.55,
4.69 (s, 2H), 7.36–8.24 (m, 10H), 8.35 (s, 1H).13C NMR (75 MHz, CDCl3):
122.58, 123.51, 126.86, 129.21, 129.85, 130.34, 132.48, 136.92, 143.99, 163.37, 165.85; ESI MS: 336 (M+ +
H). C17H13N5OS (calculated): C = 60.88; H = 3.91; N = 20.88; O = 4.77; S = 9.56; observed: C = 60.81;
H = 3.95; N = 20.85; O = 4.80; S = 9.55.
3.2.8. Methyl 2-(4-((5-phenyl–1,3,4-oxadiazol-2-ylthio)methyl)-1H–1,2,3-triazol–1-yl)benzoate (13)
Brown flakes, yield = 70%, MP = 119–120 ◦C. IR (ATR)
νmax: 3127, 3083 (C–H, aromatic), 2953
(C–H), 1727 (C=O), 1602, 1558 (C=C), 1506, 1472, 1450, 1340, 1272 (C=N, C–N), 1192, 1134, 1053 (C–O),
1
702, 688 (C–S) cm−1. H NMR (300 MHz, CDCl3):
δ
3.85 (s, 3H), 4.68 (s, 2H), 6.99 (d, J = 9.0 Hz, 2H),
:27.30, 56.00, 115.32,
7.21–7.75 (m, 5H), 7.93–8.44 (m, 2H), 8.45 (s, 1H). 13C NMR (75 MHz, DMSO)
δ
122.21, 122.51, 122.83, 123.51, 126.86, 129.84, 130.34, 132.46, 143.66, 163.40, 165.84, 169.77. ESI MS: 394
(M+ + H). C19H15N5O3S (calculated): C = 58.01; H = 3.84; N = 17.80; O = 12.20; S = 8.15; observed:
C = 58.02; H = 3.82; N = 17.81; O = 12.21; S = 8.14.

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3.2.9. 2-(4-((5-phenyl–1,3,4-oxadiazol–2-ylthio)methyl)-1H–1,2,3-triazol–1-yl)benzoic acid (14)
◦
White solid, yield = 72%, MP = 119–120 C. IR (ATR)
ν
max: 3127 (brs–OH) 3083, 2953 (C–H),
1727 (C = O), 1602, 1558 (C=C), 1506, 1472, 1450, 1340, 1272 (C=N, C–N), 1260, 1192, 1134, 1053
(C–O), 702, 688 (C–S) cm−1.1H NMR (400 MHz, DMSO):
4.69 (s, 2H), 7.13–7.24 (m, 2H), 7.41–7.99
(m, 7H), 8.89 (s, 1H), 10.66 (s, 1H).13C NMR (213 MHz, DMSO):
27.26, 120.01, 125.48, 126.76, 132.57,
134.09, 135.61, 138.57, 143.77, 163.92, 165.21, 167.27; ESI MS: 380 (M+ + H). C18H13N5O3S (calculated):
δ
δ
C = 56.98
S = 8.45.
; H = 3.45; N = 18.46; O = 12.65; S = 8.45; observed: C = 56.99; H = 3.47; N = 18.43; O = 12.64;
3.3. Anticancer Activity
3.3.1. Cell Lines and Culture Medium
The human breast cancer cell line (MCF-7) used in the present study was obtained from
Dr. Neamatallah’s lab. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% (v/v) fetal bovine serum (FBS), 10,000 units/mL penicillin/streptomycin, and 1%
(v/v) L-glutamine at 37 ◦C in humidified 5% CO2 incubator.
3.3.2. Cytotoxicity Assay
The cytotoxicity activity was done by MTT assay [33]. Breast MCF-7 and colorectal HCT-116
cancer cells were added at 1
overnight for attachment at 37 C in a 5% CO2 humidified atmosphere. Drug concentrations at
×
105 cells/mL into a 96-well plate with three replicates, and incubated
◦
six se◦rial dilutions (100.0, 50.0, 10.0, 1.0, 0.5, and 0.1
µM) were added in triplicate and incubated
at 37 C and 5% CO2 for 72 h. Drugs were dissolved in 0.1% DMSO as a vehicle. Untreated cells
were used as control. Tamoxifen and 5-fluorouracil (5-FU) was used as positive controls. Thereafter,
each well for each time point was removed and replaced with 100
3-(4,5- dimethylthiaxolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) (10 mg/mL). Then the media was
removed and 100
l of DMSO was added, and cells were incubated for a further 5 mins at 37 ◦C and 5%
CO2. Plates were quantified using the SpectraMax M3 plate reader at 570 nm. The percentage inhibition
was calculated as 100 ((mean OD of treated cell 100)/Mean OD of vehicle treated cells (DMSO)).
µM of full medium containing 10%
µ
−
×
All the experiments were repeated in at least three independent experiments (Table 2, Figures 3–6).
3.4. In Vitro Thymidylate Synthase Enzyme Assay
A thymidylate synthase enzymatic assay was carried out according to the reported method [34,35]
.
It involves a mixture containing 2-mercaptoethanol (0.1 M), (6R,S)-tetrahydrofolate (0.0003 M),
formaldehyde (0.012 M), MgCl2 (0.02 M), dUMP (0.001 M), TrisHCl (0.04 M), and NaEDTA (0.00075
M).This assay was done spectrophotometrically at 30◦ C and pH 7.4. The reaction was initiated by the
addition of an amount of enzyme, giving a change in absorbance at 340 nm of 0.016/min in the absence
of inhibitor. The percent inhibition was determined at a minimum of four inhibitor concentrations
within 20% of the 50% point. The standard deviations for determination of the 50% points were within
± 10% of the values given. The results are presented in Table 3 and Figure 7.
3.5. Molecular Docking
Molecular docking studies involve mainly protein selection and preparation, grid generation,
ligand preparation, docking, and further analysis of docking studies. A protein with accession number
6QXG was selected and downloaded from Protein Data Bank. This protein is reported to act as a
thymidylate synthase inhibitor. The protein was imported, optimized, and minimized by removing
unwanted molecules and other defects reported by the software. The minimized protein was used for
grid generation, which involves the selected ligand as the reference, as it signifies the binding sites of
the drug with respect to the target. Molecules drawn in 3D form were refined by the LigPrep module.

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The molecules were subjected to an OPLS-2005 force field to generate a single, low-energy 3D structure
for each input structure. Docking studies was carried using Glide software. It was carried out using
extra precision and write XP descriptor information. This generates favorable ligand poses, which
are further screened through filters to examine the spatial fit of the ligand in the active site. Ligand
poses, which pass through an initial screening, are subjected to evaluation and minimization of grid
approximation. Scoring was then carried on energy-minimized poses to generate a Glide score [36
(Table 4, Figure 8).
]
4. Conclusions
In the present article, a series of nine hybrids of 1,2,3-triazole and 1,3,4-oxadiazole moieties
(6–14) have been described. The final compounds have been characterized using different analytical
techniques. These hybrids have been tested for in vitro anticancer and thymidylate synthase activities.
According to in silico pharmacokinetic studies, the synthesized hybrids exhibited good drug likeness
properties and bioavailability. The cytotoxicity results indicated that compounds 12 and 13 exhibited
remarkable inhibition on the tested MCF-7 and HCT-116 cell lines. Compound 12 showed four-fold
inhibition compared to the standard drug 5-fluoruracil, and comparable inhibition to tamoxifen,
whereas compound 13 exerted five-fold and 24 times the activity of tamoxifen and 5-fluorouracil,
respectively, for MCF-7 cells. The same compounds (12 and 13) also revealed significant inhibition
against HCT-116 cells. Compound 12 revealed 1.7-fold activity of tamoxifen and 2.2-fold activity of
5-fluorouracil, while compound 13 displayed 1.9-fold the activity of 5-FU and 1.5-fold that of tamoxifen
against HCT-116. The in vitro thymidylate synthase activity results supported our cytotoxicity results.
Compounds 12 and 13 inhibited thymidylate synthase enzyme with IC50 values of 2.52
respectively, while the standard drug pemetrexed showed an IC50 of 6.75 M. The molecular docking
data of compounds 12 and 13 supported the in vitro biological activity data. In conclusion, hybrids
12 and 13) may inhibit the thymidylate synthase enzyme, which could play a significant role as a
µM and 4.38 µM,
µ
(
chemotherapeutic agent.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/13/11/390/s1,
Figures S1–S9: 1H NMR spectra of all final compounds; Figures S10–S18: 13C NMR spectra of all final compounds;
Figures S19–S27: Mass spectra of all final compounds.
Author Contributions: M.M.A. and S.N. designed the experiment and supervised. T.N. and N.M.A. carried out
the pharmacology work, M.M.A and A.S.A.A. performed the synthetic work, A.M.M. and S.N. analyzed the data,
characterized the compounds, and wrote the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding: Taif University Researchers Supporting Project (Number TURSP-2020/44) for financial assistance for the
project work.
Acknowledgments: A.S.A.A. acknowledges the Taif University Researchers Supporting Project (Number
TURSP-2020/44) for financial assistance. M.M.A and S.N. thanks the Chemistry department, Albaha University,
for providing the necessary facilities to carry out the project work. We also thank Safaa S. Bayashut, King Abdulaziz
University (KAU) for helping perform the pharmacological activities. The non-tumorigenic HEK-293 cells (normal
human embryonic kidney) were generously provided by Anwar Hashem, Vaccine and Immunotherapy Unit,
King Fahd Medical Research Centre, KAU.
Conflicts of Interest: The authors declare no conflict of interest.
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