Synthesis and rational design of new appended 1,2,3-triazole-uracil ensembles as promising anti-tumor agents via in silico vegfr-2 transferase inhibition

Article abstract of DOI:10.3390/molecules26071952

Angiogenesis inhibition is a key step towards the designing of new chemotherapeutic agents. In a view to preparing new molecular entities for cancer treatment, eighteen 1,2,3-triazole-uracil ensembles 5a–r were designed and synthesized via the click reaction. The ligands were well characterized using¹ H-,¹³ C-NMR, elemental analysis and ESI-mass spectrometry. The in silico binding propinquities of the ligands were studied sequentially in the active region of VEGFR-2 using the Molegro virtual docker. All the compounds produced remarkable interactions and potentially inhibitory ligands against VEGFR-2 were obtained with high negative binding energies. Drug-likeness was assessed from the ADME properties. Cytotoxicity of the test compounds was measured against HeLa and HUH-7 tumor cells and NIH/3T3 normal cells by MTT assay. Compound 5h showed higher growth inhibition activity than the positive control, 5-fluorouracil (5-FU), against both HeLa and HUH-7 cells with IC₅₀ values of 4.5 and 7.7 μM respectively. Interestingly, the compounds 5a–r did not show any cytotoxicity towards the normal cell lines. The results advance the position of substituted triazoles in the area of drug design with no ambiguity.

Full text of DOI:10.3390/molecules26071952

molecules
Article
Synthesis and Rational Design of New Appended
1,2,3-Triazole-uracil Ensembles as Promising Anti-Tumor
Agents via In Silico VEGFR-2 Transferase Inhibition
Nadipolla Naresh Reddy 1,†, Sung-Jen Hung 2,3,†, Merugu Kumara Swamy 1, Ananthula Sanjeev 1,
Vankadari Srinivasa Rao 1, Rondla Rohini 1, Atcha Krishnam Raju 4, Kuthati Bhaskar 1, Anren Hu 3,5,
*
and Puchakayala Muralidhar Reddy 1,
*
1
Department of Chemistry, Osmania University, Hyderabad, Telangana 500007, India;
nareshnadipolla@gmail.com (N.N.R.); kumaraswamy.sci@gmail.com (M.K.S.);
sanjeev.ku610@gmail.com (A.S.); chemsrinu44@gmail.com (V.S.R.); prmnreddy@osmania.ac.in (R.R.);
kuthati18@osmania.ac.in (K.B.)
Department of Dermatology, Buddhist Tzu-Chi General Hospital, Hualien 97002, Taiwan;
md.hong@msa.hinet.net
Institute of Medical Sciences, Tzu-Chi University, Hualien 97002, Taiwan
Department of Chemistry, Nizam College, Osmania University, Hyderabad 500001, India;
krishnamrajua@osmania.ac.in
Department of Laboratory Medicine and Biotechnology, College of Medicine, Tzu-Chi University,
Hualien 97004, Taiwan
2
3
4
5
*
Correspondence: anren@gms.tcu.edu.tw (A.H.); pmdreddy@osmania.ac.in or pmdreddy@gmail.com (P.M.R.);
Tel.: +886-3-8565301 (ext. 2334 or 2335) (A.H.); Fax: +886-3-8571917 (A.H.); +91-9848792423 (P.M.R.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
†
Citation: Reddy, N.N.; Hung, S.-J.;
Swamy, M.K.; Sanjeev, A.; Rao, V.S.;
Rohini, R.; Raju, A.K.; Bhaskar, K.;
Hu, A.; Reddy, P.M. Synthesis and
Rational Design of New Appended
1,2,3-Triazole-uracil Ensembles as
Promising Anti-Tumor Agents via In
Silico VEGFR-2 Transferase Inhibition.
Molecules 2021, 26, 1952. https://
doi.org/10.3390/molecules26071952
Abstract: Angiogenesis inhibition is a key step towards the designing of new chemotherapeutic
agents. In a view to preparing new molecular entities for cancer treatment, eighteen 1,2,3-triazole-
uracil ensembles 5a
–
r
were designed and synthesized via the click reaction. The ligands were well
characterized using H-, 13C-NMR, elemental analysis and ESI-mass spectrometry. The in silico
binding propinquities of the ligands were studied sequentially in the active region of VEGFR-2 using
the Molegro virtual docker. All the compounds produced remarkable interactions and potentially
inhibitory ligands against VEGFR-2 were obtained with high negative binding energies. Drug-
likeness was assessed from the ADME properties. Cytotoxicity of the test compounds was measured
against HeLa and HUH-7 tumor cells and NIH/3T3 normal cells by MTT assay. Compound 5h
showed higher growth inhibition activity than the positive control, 5-fluorouracil (5-FU), against both
1
Academic Editor: Philippe Belmont
HeLa and HUH-7 cells with IC50 values of 4.5 and 7.7
µM respectively. Interestingly, the compounds
Received: 24 February 2021
Accepted: 25 March 2021
Published: 30 March 2021
5a did not show any cytotoxicity towards the normal cell lines. The results advance the position of
–
r
substituted triazoles in the area of drug design with no ambiguity.
Keywords: drug design; 1,2,3-triazole-uracil; VEGFR-2; in silico docking; MTT assay; anti-cancer agents
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Cancer is a disease with a high mortality rate that accounts for more than 9 million
deaths every year [
no side effects is of great importance in medicinal chemistry. To date, over 100 anti-
cancer agents are approved for chemotherapy [ ]. Due to the increasing resistance to
existing drugs, there is a need for the synthesis of new agents which can effectively treat
various cancers. The present investigation attempts to develop new therapeutic agents
assembled using 1,2,3-triazoles and uracil moieties. 1,2,3-Triazoles contain three nitrogens
in the ring system and are an important class of heterocyclic compounds with remarkable
1,2]. The discovery of molecules to treat cancer with minimal or
Copyright:
© 2021 by the authors.
3
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biological properties [4]. It is known that they possess promising antiviral, antifungal
and anticancer, antileishmanial, antituberculosis, anti-inflammatory, antimicrobial and
Molecules 2021, 26, 1952. https://doi.org/10.3390/molecules26071952
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1952
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antibacterial activity [
5–8
]. It is assumed that the presence of heteroatoms directly affects
their toxicity towards cancer cells [9]. Out of many synthetic routes for the preparation of
1,2,3 triazoles, the Hüisgens thermal 1,3 dipolar cycloaddition is the classical and traditional
approach that involves the interaction of terminal alkynes with organic azides [10]. The
reaction proceeds via a concerted mechanism yielding 1,4- and 1,5-disubstituted triazoles.
Cu(I) catalyzed reactions are reported to exclusively produce 1,4 disubstituted triazoles [10].
On the other hand, uracil derivatives were proved to display bactericidal, herbicidal and
insecticidal properties [11]. Uracil moiety in combination with 1,2,3-triazoles recently
proved to possess potent anticancer activities [12]. 5-Fluorouracil has long been known to
inhibit tumor growth and was used as a standard drug in the current study [13].
Rational drug design is an important and widely accepted approach in the discovery
of new drug candidates for various diseases. It includes the study of in silico docking
interactions followed by an in vitro verification of the synthesized compounds. To account
for the small molecule and target protein interactions, the Moldock program was utilized.
Moldock is a high accuracy technique to predict protein-ligand interactions. It operates
on a heuristic search algorithm that incorporates differential evolution along with a cavity
prediction tool. The Moldock scoring function is an extension of the piecewise linear
potential that includes new hydrogen-bonded and electrostatic terms [14]. The accuracy
of Moldock was further improved by introducing a new rerank scoring function. It is
established that the overall performance of the Moldock program is better than that of
Flexx, Gold and Surflex [15].
Angiogenesis is a crucial physiological process that regulates the formation of new
blood vessels [16]. This process is greatly exploited by genetically altered cancer cells for
their proliferation, progression and metastasis [17,18]. The unwanted tumor cell prolif-
eration can be blocked by employing suitable drug candidates that specifically hinder
angiogenesis by interfering with the VEGFR signalling pathway. The vascular endothelial
growth factor receptors (VEGFR1/VEGFR2/VEGFR3) are transmembrane proteins and
only VEGFR-2 triggers the sprouting of new capillary blood vessels through VEGFR re-
ceptor signalling [19
,20]. Disabling the functions of VEGFR-2 protein is a key step in drug
discovery and it has become an undisputed target for new chemotherapeutic agents [21
–24].
Recently, Heba et al., reported the docking interactions of 1,2-disubstituted benzimidazoles
in the active site of VEGFR-2. The results depicted sharp hydrogen bond or hydrophobic
interactions with the receptor protein and the ligands produced remarkable VEGFR-2 inhi-
bition activity in HepG2 cell line with IC50 values as low as 1.98 µM [25]. Novel thiazolidine
2,4-diones reported by Khaled et al., have also demonstrated excellent in vitro VEGFR-2
inhibition and MCF-7 tumor growth suppression. Moreover, in silico VEGFR-2 binding
results of synthesized ligands were very well correlated with in vitro antiproliferative
activities [26]. Figure 1 depicts some recently developed in silico VEGFR-2 inhibitors that
eventually evolved into potential in vitro anti-tumor agents [22,27–37]. These results justify
the usage of VEGFR-2 protein in a rational drug design approach for the identification of
new molecular agents for treating cancer.
Because of the synthesis accessibility, excellent biological properties of triazoles and
uracils and VEGFR-20s role in identifying potential anti-cancer agents, in this work we
designed and isolated eighteen new 1,2,3-triazole-uracil scaffolds. The synthesized ligands
were docked into the active site of VEGFR-2 receptor protein using the Molegro virtual
docking program to estimate their inhibition properties. Finally, the compounds were
screened for their anti-cancer activity against HeLa and HUH-7 tumor cell lines using
standard MTT assay.

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Figure 1. Recently reported drug candidates that proved to inhibit VEGFR-2 and emerged as effective molecules against
various cancers.
2. Results and Discussion
2.1. Chemistry
The target compounds 5a
–r
(Scheme 1) were synthesized using previously reported
literature methods [38,39]. The required aromatic azides 2a–e and benzyl or alkyl azides
2f–r were first synthesized by the reaction of aromatic anilines 1a
NaNO2 in water at 0–5 C, followed by the addition of sodium azide in water at room
–
e
with dil. HCl and
◦
temperature. The benzyl and other alkyl bromides 1f
azide to give azide reactants 2f by the replacement of the terminal bromide. Finally, Cu(I)
catalyzed coupling reactions between 6-chloro-3-methyl-1-(prop-2-yn-1-yl)pyrimidine-
–r were made to react with sodium
–r
2,4(1H,3H)-dione (
4
) and the aromatic/benzyl/alkyl azides 2a
–
r
was achieved by refluxing
in DMF at 80 ◦C for 12 h to produce the target compounds 5a
the spectral analysis of compound 5e is discussed below.
–r. As a representative case,
The formation of the substituted triazole-uracil compound 5e was confirmed by
analyzing its 400 MHz 1H-NMR spectrum (Supplementary Materials, Figure S10) which
was recorded in CDCl3. The protons of the –CH2– group bridging between the uracil and
triazole rings appeared at
(s, 1H) and phenyl ring protons appeared at
protons of the phenyl ring and N–CH3 protons of uracil appeared at 3.94 (s, 3H) and 3.34
(s, 3H), respectively. The triazole ring proton appeared at
8.08 (s, 1H). The 100.6 MHz 13C-
NMR spectrum of the compound was also recorded in CDCl3, (Supplementary Materials,
Figure S11). The uracil carbonyl carbons appeared at 164.3 (C2) and 150.5 (C5). The
triazole ring carbons appeared at 134.0 and 126.9. The four phenyl carbons were assigned
to the peaks at
160.5 (C40), 131.9 (C10), 131.3 (C20), 114.7 (C30). The carbon of the –CH2–
group between the uracil and triazole rings was observed at 36.1. The methoxy and
δ
(ppm) 5.44 (s, 2H). The uracil ring proton was present at
δ 6.02
δ
7.65 (m, 2H) and 7.54 (m, 2H). The methoxy
δ
δ
δ
δ
δ
δ
N–CH3 carbons appeared at
δ 66.6 and δ 28.4, respectively. The peaks that appeared at m/z
348 and 349 in the mass spectrum of 5e (Figure S37) were assigned to the [M + H]+ and
[M + 2]+ molecular ion peaks, respectively.

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Scheme 1. Synthetic route for the preparation of new 1,2,3-triazole-uracil scaffolds 5a–r.
2.2. In Silico Docking Study
Molecular docking is an essential tool to screen the binding affinities and mode of
binding of protein-ligand interactions [40]. The docking analysis of the synthesized ligands
(Figure 2) 5a–r was performed using the Molegro virtual docker. A well-established
molecular target, VEGFR-2 (PDB ID: 4AGD), was selected to study the docking interactions.
The active site pocket of the receptor protein consists of Leu840, Gly922, Leu1038, Phe1047,
Asp1046, Glu885, Val916, Glu917, Phe918, Cys919, Val848, Val899, Ile392, Leu889, Val916,
Lys868, Gly841, Cys1045, Asp1052, Lys1055, Tyr1082, Arg1027, Leu802, Ser803, Leu1053,
Pro1068, Tyr1054 and Arg842 amino acid residues. The results (Table 1) indicated that all
the compounds bound to VEGFR-2 through hydrogen bonds and Van der Waal interactions
with high binding affinities ranging from
best binding poses for the reference drug 5-FU and compounds 5a
−
108 to
−
198.7 kcal/mol. Figures 3–10 depict the
5f 5h 5i 5j 5n and 5r.
,
,
,
,
,

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Figure 2. Picture showing all the substituted 1,2,3-triazole-uracil ensembles that were docked into the active site of the
VEGFR-2 receptor protein.
Table 1. Moldock and rerank scores for compounds 5a–r
Ligand
Moldock Score [Grid] (Kcal/mol)
Moldock Score
Rerank Score
RMSD
5a
5b
5c
5d
5e
−108.00
−118.04
−113.87
−126.20
−118.97
−112.05
−122.27
−124.79
−118.52
−152.32
−141.58
−151.01
−124.53
−125.00
−110.97
−129.45
−142.92
−198.07
−65.73
−111.498
−121.41
−117.85
−129.04
−118.2
−91.02
−48.81
−97.03
−71.39
−53.30
−75.16
−95.98
−100.77
−99.87
−44.55
−123.80
−113.42
−92.41
−59.54
−64.34
−48.51
−42.75
−137.60
−59.28
43.22
43.58
43.51
46.09
42.54
46.45
50.76
43.38
51.82
48.89
52.33
41.92
54.27
51.16
47.66
47.25
54.60
51.45
40.95
5f
−113.68
−117.88
−126.60
−119.90
−155.11
−147.40
−155.75
−124.14
−112.56
−111.43
−126.51
−128.58
−204.56
−65.92
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5-FU
The reference drug, 5-FU produced a binding affinity of
−
65.73 kcal/mol. The inter-
actions of the reference drug with the target protein are depicted in Figure 3. The –NH
and carbonyl groups of the uracil moiety were stabilized by conventional hydrogen bonds
with amino acid residues Glu885 (2.37 Å) and Asp1046 (2.03 Å), respectively. The fluorine
atom of the uracil core interacted with the amino acid Val899. Further, the uracil nucleus is
anchored by π-alkyl interactions with the amino acids Ile888, Ile804 and Leu889.

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Figure 3. Docking interactions (3D & 2D) of reference drug 5-FU with the receptor protein, VEGFR-2 transferase. Critical
amino acid residues are shown in the stick model and pink colour.
The synthesized compound 5a showed a binding affinity of
the corresponding interactions are presented in Figure 4. The uracil core is anchored by
-alkyl interactions with the Ile804, Val898, Ile892 and Leu889 residues. The chlorine atom
−
108.00 kcal/mol and
π
of the uracil ring interacted with the carbonyl backbone of the Ile1044 amino acid. The
methyl group present on the nitrogen of the uracil ring interacted with the carbonyl oxygen
of Ile888. However, the carbonyl oxygens of the uracil ring did not form any kind of
interactions with the target protein. The triazole ring is located in the hydrophobic pocket
formed by Val899, Cys1045, Lys868 and Leu889 with
atom of the triazole ring is bonded to the carbonyl oxygen of the Asp1046 residue. Further,
the triazole moiety is stabilized by -cation and -anion interactions with the amino acids
Lys868 and Glu885, respectively. The appended phenyl ring of 5a showed interactions
with Phe1047 and -alkyl interactions with the Val848, Val916, Val899, Leu1035 and Cys1045
amino acid residues.
π-alkyl interactions. The hydrogen
π
π
π-π
π
Figure 4. Docking interactions (3D & 2D) of 5a with the receptor protein, VEGFR-2 transferase. Critical amino acid residues
are shown in the stick model and pink colour.
Compound 5f produced a binding affinity of
interactions are presented in Figure 5. The uracil moiety is anchored by
−
112.05 kcal/mol and the corresponding
-alkyl interactions
π
with the Leu889, Ile804, Ile888 and Ile892 residues. The chlorine atom of the uracil ring
interacted with the carboxyl group of the Glu885 amino acid. The methyl group present
on the nitrogen of the uracil ring settled in the hydrophobic pocket formed by the Ile892,
Val898 and Leu1019 residues. The triazole ring is located in the hydrophobic pocket formed

Molecules 2021, 26, 1952
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by Val916, Leu889, Val899 and Cys1045 with
π-alkyl interactions. The hydrogen atom of the
triazole ring is bonded to the carbonyl oxygen of the Asp1046 residue. Further, the triazole
moiety is stabilized by -cation and -anion interactions with the amino acids Lys868 and
Glu885, respectively. The appended benzyl ring of 5f is stabilized via interactions with
-alkyl interactions the with amino acid residues Val848, Val899, Leu1035
π
π
π-π
Phe1047 and
and Cys1045.
π
Figure 5. Docking interactions (3D & 2D) of 5f with the receptor protein, VEGFR-2 transferase. Critical amino acid residues
are shown in the stick model and pink colour.
Compound 5h showed a binding affinity of
−
124.79 kcal/mol and the corresponding
interactions are presented in Figure 6. It produced three conventional hydrogen bonds
in the active region of the target protein. The carbonyl oxygen of the uracil ring formed
a hydrogen bond with Cys919 with a bond distance of 1.97 Å. The two nitrogens at the
2 and 3-positons of the triazole ring formed hydrogen bonds with Asp1046 (2.06 Å) and
Lys868 (2.54 Å), respectively. The uracil core is anchored by π-alkyl interactions with the
Ala868, Val848, Cys919 and Leu1035 residues. The methyl group present on the nitrogen of
the uracil ring interacted with the alkyl groups of the amino acids Leu840 and Leu1035
and it also formed a
anchored by -alkyl interactions with Val916, Cys1045 and Val899. Further, the triazole
core is stabilized by and -cation interactions with the amino acids Phe1047 and Lys868,
respectively. The appended benzyl ring of 5h showed -alkyl interactions with the amino
π-sigma interaction with the Phe1047 residue. The triazole ring is
π
π-π
π
π
acids Ile804, Leu889 and Val899. The methyl group present on this phenyl ring is located in
the hydrophobic pocket formed by the Ile888, Ile804 and Leu889 residues.
Figure 6. Docking interactions (3D & 2D) of 5h with the receptor protein, VEGFR-2 transferase. Critical amino acid residues
are shown in the stick model and pink colour.

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Compound 5i showed a binding affinity of
−
118.52 kcal/mol and the corresponding
interactions are depicted in Figure 7. It showed three conventional hydrogen bonds in
the active site of the receptor protein. The nitrogen at the 2-positon of the triazole ring
formed a hydrogen bond with Asn923 with a bond distance of 2.48 Å. The oxygen of the
appended benzyl group produced two hydrogen bonds with Arg1051 forming a stable
six-membered ring with bond distances of 2.16 and 2.37 Å. The chlorine atom of the uracil
moiety produced four hydrophobic interactions with the amino acids Leu840, Cys919,
Phe918 and Ala866. The uracil core is anchored by
Ala866 and Val848. N-methyl group of uracil ring interacted with Val848 and Phe1047
via alkyl and -alkyl interactions. The triazole ring is stabilized by Leu1035 and Leu840
though -alkyl interactions and Phe1047 with interactions. The appended benzyl ring
showed a π-π interaction with the amino acid Phe1047.
π-alkyl interactions with Leu1035,
π
π
π-π
Figure 7. Docking interactions (3D & 2D) of 5i with the receptor protein, VEGFR-2 transferase. Critical amino acid residues
are shown in the stick model and pink colour.
Compound 5j produced a binding affinity of
−
152.32 kcal/mol and the corresponding
interactions are presented in Figure 8. It produced two hydrogen bonds in the active region
of the target protein. The carbonyl oxygen of the uracil ring formed two hydrogen bonds
with Cys919 and Phe918 with bond distances of 1.61 and 2.93 Å, respectively. The uracil
core is anchored by π-alkyl interactions with Ala866, Val848, leu1035, Cys919 and Lue840.
The N-methyl group of the uracil ring interacted with the Leu840 and Cys919 residues via
alkyl interactions. The triazole ring is stabilized by π-alkyl interactions with Cys1045 and
Val848 and
interaction with the amino acids Val916, Val899 and Leu889. Further, the benzyl ring is
stabilized by -cation and -anion interactions with the amino acids Lys868 and Glu885,
π-π interactions with Phe1047. The appended benzyl ring showed a π-alkyl
π
π
respectively. The phenoxy ring produced
Leu889, Ile888 and Ile892.
π-alkyl interactions with the amino acids Ile804,

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Figure 8. Docking interactions (3D & 2D) of 5j with the receptor protein, VEGFR-2 transferase. Critical amino acid residues
are shown in the stick model and pink colour.
Compound 5n produced a binding affinity of −125 kcal/mol and the corresponding
interactions are presented in Figure 9. It produced one conventional hydrogen bond with
the target protein. The carbonyl oxygen of the isoquinoline ring formed one hydrogen
bond with Leu802 with a bond distance of 1.96 Å. The uracil core is stabilized by a π-alkyl
interaction with the amino acid Leu1067. The uracil chlorine atom produced two hydropho-
bic interactions with the Met1072 and Pro1068 amino acids and it also interacted with the
carbonyl backbone of Arg1027. The triazole ring is stabilized by π-anionic interactions
with the carboxylic acid group of Asp1028. The isoquinoline ring is anchored by
interactions with the amino acids Ile1053, Leu1049 and Ala844.
π-alkyl
Figure 9. Docking interactions (3D & 2D) of 5n with the receptor protein, VEGFR-2 transferase. Critical amino acid residues
are shown in the stick model and pink colour.
Of all the docked compounds, 5r showed the highest binding affinity (
with the receptor protein. The interactions of the molecule 5r with the target protein are
depicted in Figure 10. In this molecule, the uracil and triazole rings are anchored by π-π
−
198 kcal/mol)
stacking interactions between uracil-triazole, triazole-triazole rings and uracil-Phe1047
amino acid residue of the receptor protein. The carbonyl oxygen of two uracil rings
interacted with the side chain hydrogens of the amino acids Gly922 and Gly841. Further,
the uracil and triazole rings are stabilized by the amino acids Leu1035, Leu840, Val848,
Lys868, Val916 and Val899 through π-alkyl interactions.

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Figure 10. Docking interactions (3D & 2D) of 5r with the receptor protein, VEGFR-2 Transferase. Critical amino acid residues
are shown in the stick model and pink colour.
2.3. ADME Properties
A favorable absorption, distribution, metabolism and elimination profile is a prereq-
uisite for any drug candidate to conclude its drug-likeness. The ADME properties for
the reported ligands, were predicted using the Lipinski filters from the Molinspiration
online tool kit. The properties like molecular weight, molecular volume, the logarithm of
partition coefficient, hydrogen bond acceptors and donors, polar surface area, the number
of rotatable bonds and Lipinski’s rule of five were determined by using the Molinspi-
ration online property calculation toolkit [41]. Absorption percentage was obtained by:
%ABS = 109 − (0.345 × TPSA) [42].
The Molsoft software was utilized to calculate properties like physico-chemical pa-
rameters, pharmacokinetics, pharmacodynamics (drug-likeness model score) and was
represented by a numerical value [43]. The pharmacokinetic parameters for the synthe-
sized ligands are presented in Table 2.
The results indicated good %Abs values for the triazole ligands ranging from 57.43% to
83.21%. Furthermore, compounds 5a–r obeyed Lipinski’s rule of five (number of hydrogen
bond acceptors (n-ON) 10) and followed the criteria for orally active drug and therefore,
≤
these compounds may have a good potential for eventual development as oral agents. A
molecule likely to be developed as an orally active drug candidate should not produce more
than one violation of the following four criteria: miLogP (octanol-water partition coefficient)
molecular weight 500, number of hydrogen bond acceptors 10 and number of hydrogen
bond donors 5 [44]. All the synthesized triazole-uracil analogues 5a obeyed the criteria and
hence, can be considered for the development as orally active drugs.
≤
5,
≤
≤
≤
–r

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Table 2. Pharmacokinetic parameters of triazole-uracil scaffolds (5a–r).
Gpcr
Ligand
Ion Channel
Modulator
Kinase
Inhibitor
Nuclear
Receptor Ligand
Protease
Inhibitor
Enzyme
Inhibitor
C 1
milogP 2
TPSA (A2) 3
n Violation 4
M.wt 5
nO N 6
nOHNH 7
MV 8
%ABS
Rule
≤5
—
≤1
<500
<10
<5
5a
5b
5c
0.11
0.13
−0.15
−0.16
−0.15
−0.19
−0.21
−0.19
−0.26
−0.25
−0.15
−0.15
−0.24
−0.28
−0.60
−0.29
−0.28
−0.26
−0.20
−0.23
−1.95
−0.21
−0.15
−0.20
−0.32
−0.21
−0.21
−0.24
−0.24
−0.11
−0.11
−0.34
−0.35
−0.36
−0.14
−0.29
−0.22
−0.12
−0.12
−2.62
−0.22
−0.17
−0.21
−0.28
−0.19
−0.26
−0.36
−0.28
−0.10
−0.10
−0.15
−0.22
−0.19
−0.32
−0.36
−0.26
−0.20
−0.19
−3.04
−0.28
−0.27
−0.27
−0.35
−0.27
−0.21
−0.31
−0.24
−0.24
−0.10
−0.19
−0.24
−0.34
−0.16
−0.18
−0.12
−0.09
−0.08
−3.15
0.09
0.06
1.28
1.45
1.96
1.24
1.34
1.60
2.41
2.05
1.64
3.33
1.45
1.26
2.66
2.02
2.46
3.47
0.24
0.75
−0.59
74.73
74.73
74.73
120.55
83.96
74.73
74.73
74.73
83.96
83.96
122.89
132.12
114.17
113.80
74.73
74.73
149.46
149.46
65.72
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
2
2
0
317.74
335.73
352.18
362.73
347.76
331.76
410.66
345.79
361.79
423.86
453.85
483.87
471.90
478.90
325.80
353.85
551.39
551.39
130.08
7
7
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
83.21
83.21
83.21
67.41
80.03
83.21
83.21
83.21
80.03
80.03
66.60
63.41
69.63
69.73
83.21
83.21
57.43
57.43
86.32
259.51
264.45
273.05
282.85
285.06
276.32
294.20
292.88
301.86
356.71
363.34
388.89
396.81
393.82
288.68
322.28
430.79
447.59
96.91
0.12
0.06
5d
5e
−0.04
−0.05
10
8
0.07
0.03
5f
0.07
0.07
7
5g
5h
5i
−0.04
−0.01
7
0.02
0.01
7
0.07
0.01
8
5j
0.07
0.07
8
5k
5l
0.06
0.01
11
12
10
10
7
0.01
−0.03
5m
5n
5o
5p
5q
5r
−0.20
0.02
0.03
0.11
0.12
0.16
0.13
0.14
7
0.07
0.08
14
14
4
0.07
0.08
5-FU
−2.60
−1.56
1
8
Compounds; 2 Measured lipophilicity; 3 Total polar surface area; 4 No of violations from Lipinski’s rule of five; 5 Molecular weight; 6 No. of hydrogen bond acceptors; 7 No. of hydrogen bond donors;
Molar volume.

Molecules 2021, 26, 1952
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2.4. In Vitro Cytotoxicity
The cytotoxic potency for 5a
–
r
was assayed against HeLa and HUH-7 tumor cells
by the standard MTT protocol. The ligands significantly inhibited the proliferation of the
selected tumor cells and the corresponding viability profiles are presented in Figure 11.
Figure 11. Cell viability profiles of HeLa and HUH-7 tumor cell lines against the treatment of
substituted 1,2,3-triazole-uracil analogues for 72 h.
The half-maximal inhibitory concentrations (IC50) for compounds 5a
in Table 3. The ligands 5a 5h 5i 5j 5n and 5r produced higher inhibition effect against
HeLa cells, with IC50 values ranging from 4.5 M to 11.2 M. HUH-7 cells were found to
be more sensitive towards the treatment of 5f 5h and 5i with IC50 values 10.8 M, 7.7
and 9 M, respectively, and the remaining compounds failed to hamper the proliferation of
HUH-7 tumor cells. Compound 5h emerged as a hit compound and produced IC50 values
of 4.5 M and 7.7 M, respectively, against both the cell lines. The obtained values for 5h
are better than those of the standard drug, 5-FU (4.6 M and 30 M) and it has the potential
to be developed as a new chemotherapeutic agent for cervical and liver carcinoma.
–r are presented
,
,
,
,
µ
µ
,
µ
µM
µ
µ
µ
µ
µ

Molecules 2021, 26, 1952
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Table 3. IC50 values (µM) for the new 1,2,3-triazole-uracil scaffolds 5a
–
r
against the treatment of
HeLa and HUH-7 tumor cells and NIH/3T3 normal cells after 72 h.
Compound
HeLa
HUH-7
NIH/3T3
ND 1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NA 2
5a
5b
5c
5d
5e
11.2 ± 0.1
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
5f
ꢀ10
10.8 ± 0.5
ꢀ10
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
ꢀ10
4.5 ± 0.5
ꢀ10
7.7 ± 0.3
9.0 ± 0.8
ꢀ10
10 ± 1.5
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
9.6 ± 1.2
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
ꢀ10
9.6 ± 0.9
4.6 ± 0.2
ꢀ10
5-FU
30 ± 2.5
No considerable cytotoxicity was determined; 2 Cells were not treated with 5-FU.
1
For all the compounds 5a
hence some structure-activity relationship inferences can be drawn based on the differ-
ent substituents. The compounds 5b bearing a phenyl ring with electron-donating or
withdrawing groups at the para position showed lower activity than the unsubstituted
compound 5a. The compounds 5f (except 5g) bearing a benzyl ring and with various
electron-donating groups produced higher activity against HeLa or HUH-7 cells. The
compounds 5k with chromene rings failed to show any remarkable activity against
–r, the 1,2,3-triazole and uracil rings are common, and
–
e
–
j
–
m
the selected tumor cell lines. The compound 5n with an isoquinoline ring showed good
activity against HeLa cells. Compounds 5o and 5p with aliphatic hydrocarbon chains did
not induce considerable activity against either of the cancer cell lines. The compound 5r
produced a higher inhibition effect than its dimer analogue 5q. Interestingly, the com-
pounds 5a–r did not show any cytotoxicity towards the normal NIH/3T3 cells and this
offer a great future for the reported ligands in cancer research.
On the other hand, the approved drugs for the treatment of liver cancer are sparse
and sorafenib (VEGFR inhibitor) is the only drug approved to treat patients with advanced
hepatocellular carcinoma [45
patients are reported to develop resistance within six months and no alternative treatment is
currently available [48 49]. Thus, the current results, that are close to the in vitro cytotoxicity
of sorafenib [22 28] against liver carcinoma, are exciting and the reported ligands 5f 5h
–47]. Even though it is effective in treating liver cancer,
,
,
,
and 5i could be developed as new alternatives for treating liver cancer.
3. Materials and Methods
3.1. General Information
All the chemicals used in this study were obtained from Sigma Chemicals (St. Louis,
MO, USA). Solvents of reagent grade were purchased from nearby commercial sources
and distilled before the usage. DMEM media and the serum supplement FBS for the
cytotoxicity assay were obtained from Sigma Chemicals and Gibco (Hyderabad, TS, India),
respectively. The melting points of the ligands were measured in open capillaries using
1
the electrothemal melting point apparatus. H- and 13C-NMR spectra were recorded using
Avance II 400 and 100 MHz spectrometers (Bruker, Zurich, Switzerland) in 99.99% DMSO-
d6 and 99.82% CDCl3 (2 mg/mL) using TMS as the standard solvent. ESI-MS spectra were
measured on a LCMS 2010 VG mass spectrometer (Thermo Finnigan, San Jose, CA, USA)

Molecules 2021, 26, 1952
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in methanol/DCM solvents (10
µg/mL). Elemental analysis was carried out using a Carlo
Erba 1108 analyzer (Heraeus, Hanau, Germany).
3.2. Syntheses
3.2.1. Synthesis of 6-Chloro-3-methyl-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione (4)
A DMF (50 mL) solution of
bromide (1.33 mL, 0.015 mol) and anhydrous K2CO3 (2.56 g, 0.0186 mol). The resulting
mixture was allowed to reflux for h and the course of the reaction was monitored by
3 (1 g, 0.0062 mol) was slowly added with propargyl
4
TLC until its completion. When the reaction was complete, the remaining solvent was
evaporated under vacuum. Excess propargyl bromide was removed by washing the
reaction mixture with hexane and then poured into ice-water (100 mL) and extracted with
ethyl acetate. Then, the organic layer was added with the brine solution and residual water
content after workup was removed by sodium sulphate. Final evaporation under high
vacuum and purification by column chromatography produced the desired compound 4.
White solid; 92% yield; MP 120.3–121.5 ◦C; 1H-NMR (400 MHz, CDCl3),
δ, ppm: 5.99
(s, 1H), 4.86 (d, 2H, J = 2.0 Hz), 3.35 (s, 3H), 2.39 (s, 1H). M.F. C8H7ClN2O2; M. Wt. Cal: 198.
Elemental analysis: Cal: C, 48.38; H, 3.55; N, 14.11; Found: C, 48.35; H, 3.50; N, 14.05.
3.2.2. Synthesis of Substituted 1,2,3-Triazole-uracil Scaffolds 5a–r
To a mixture of 6-chloro-3-methyl-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione (
4)
and alkyl/benzyl/aryl azides 2a in DMF and water (1:1) solution, copper sulphate
–
r
pentahydrate (2 mol%) and sodium ascorbate (10 mol%) were added. The reaction mixture
was stirred for 1–1.5 h to afford 5a–r.
6-Chloro-3-methyl-1-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione (5a):
◦
Yellow solid; 87% yield; M.P. 120–121 C; 1H-NMR (400 MHz, CDCl3),
δ
, ppm: 8.11 (s, 1H),
7.74–7.72 (d, 2H, J = 7.2 Hz), 7.55–7.49 (m, 3H), 5.97 (s, 1H), 5.44 (s, 2H), 3.34 (s, 3H). 13C-
NMR (100.6 MHz, CDCl3), δ, ppm: 160.6, 151.1, 145.3, 136.8, 129.8, 129.0, 120.6, 102.4, 41.9,
28.3. M.F. C14H12ClN5O2; M. Wt. Cal: 317; Found: ESI-Ms m/z, 318 [M + H]+. Elemental
analysis: Cal: C, 52.92; H, 3.81; N, 22.04; Found: C, 52.86; H, 3.93; N, 22.15.
6-Chloro-1-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpyrimidine-2,4(1H,3H)-
dione (5b): Light white solid; 87% yield; M.P. 97 ◦C; 1H-NMR (400 MHz, CDCl3),
δ
, ppm:
8.08 (s, 1H), 7.69–7.66 (d, 2H, J = 8.8 Hz), 7.52–7.49 (d, 2H, J = 8.8 Hz), 5.98 (s, 1H), 5.43
(s, 2H), 3.34 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
, ppm: 160.6, 151.1, 145.2, 142.9, 137.6,
δ
135.6, 130.9, 129.1, 121.8, 120.8, 118.5, 102.5, 41.8, 28.3. M.F. C14H11ClFN5O2; M. Wt. Cal:
335; Found: ESI-Ms m/z, 358 [M + Na]+. Elemental analysis: Cal: C, 50.09; H, 3.30; N, 20.86;
Found: C, 50.18; H, 3.23; N, 20.72.
6-Chloro-1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpyrimidine-2,4(1H,3H)-
dione (5c): White solid; 85% yield; M.P. 100 ◦C; 1H-NMR (400 MHz, CDCl3),
δ
, ppm: 8.08
(s, 1H), 7.69–7.67 (d, 2H, J = 8.8 Hz), 7.51–7.49 (d, 2H, J = 8.8 Hz), 5.98 (s, 1H), 5.44 (s, 2H),
3.34 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
, ppm: 160.6, 151.1, 145.2, 135.2, 134.8, 130.0,
δ
121.3, 118.5, 102.5, 96.1, 41.8, 28.3. M.F. C14H11Cl2N5O2; M. Wt. Cal: 351; Found: ESI-Ms
m/z, 352 [M + H]+. Elemental analysis: Cal: C, 47.75; H, 3.15; N, 19.89; Found: C, 47.89;
H, 3.09; N, 19.82.
6-Chloro-3-methyl-1-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-
◦
dione (5d): Brick red; 75% yield; M.P. 110–112 C; 1H-NMR (400 MHz, CDCl3),
δ
, ppm:
8.43–8.41 (d, J = 9.1 Hz, 2H), 8.23 (s, 1H), 7.99–7.97 (d, J = 9.2 Hz, 2H), 6.00 (s, 1H), 5.46
(s, 2H), 3.35 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
, ppm: 194.6, 160.5, 145.1, 143.5, 140.8,
δ
126.5, 125.9, 121.8, 120.6, 102.6, 41.7, 28.3. M.F. C14H11ClN6O4; M. Wt. Cal: 362; Found:
ESI-Ms m/z, 363 [M + H]+. Elemental analysis: Cal: C, 46.36; H, 3.06; N, 23.17; Found: C,
46.45; H, 3.14; N, 23.17.
6-Chloro-1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpyrimidine-2,4-
◦
(1H,3H)-dione (5e): Yellow solid; 80% yield; M.P. 145–148 C; 1H NMR (400 MHz, CDCl3),
δ,
ppm: 8.08 (s, 1H), 7.65 (d, 2H, J = 8.2 Hz), 7.54 (d, 2H, J = 8.4 Hz), 6.02 (s, 1H), 5.44 (s, 2H),
3.94 (s, 3H), 3.34 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
δ
, ppm: 164.3, 160.5, 150.5, 144.7,

Molecules 2021, 26, 1952
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134.0, 131.9, 131.3, 126.9, 114.7, 102.6, 66.6, 36.1, 28.4. M.F. C15H14ClN5O3; M. Wt. Cal: 347;
Found: ESI-Ms m/z, 348 [M + H]+. Elemental analysis: Cal: C, 51.81; H, 4.06; N, 20.14;
Found: C, 51.79; H, 4.01; N, 20.10.
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6-chloro-3-methylpyrimidine-2,4(1H,3H)-dione (5f):
Light yellow solid; 92% yield; M.P. 115–118 ◦C; 1H-NMR (400 MHz, CDCl3),
δ, ppm: 8.59
(dd, J = 7.3, 0.9 Hz, 1H), 8.24 (dd, J = 8.3, 0.9 Hz, 2H), 7.81–7.72 (m, 2H), 6.91 (d, J = 8.7 Hz,
1H), 5.98 (s, 1H), 4.85 (d, J = 2.5 Hz, 2H), 4.43 (s, 2H), 3.35 (s, 3H). 13C-NMR (100.6 MHz,
CDCl3), δ, ppm: 190.8, 164.3, 160.5, 144.7, 134.0, 131.9, 131.3, 126.9, 114.7, 102.6, 73.3, 36.1,
28.4. M.F. C15H14ClN5O2; M. Wt. Cal: 331; Found: ESI-Ms m/z, 332 [M + H]+. Elemental
analysis: Cal: C, 54.30; H, 4.25; N, 21.11; Found: C, 54.36; H, 4.30; N, 21.18.
1-((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-chloro-3-methylpyrimidine-2,4(1H,3H)-
dione (5g): Pale green; 81% yield; M.P. 138–140 ◦C; 1H-NMR (400 MHz, CDCl3),
δ
, ppm:
7.58 (s, 1H), 7.51 (dd, J = 8.5, 2.4 Hz, 2H), 7.18 (dd, J = 15.3, 8.4 Hz, 2H), 5.93 (s, 1H), 5.46
(s, 2H), 5.32 (s, 2H), 3.30 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
, ppm: 160.6, 151.0, 145.3,
δ
142.5, 134.4, 132.3, 131.9, 129.8, 123.3, 102.3, 53.5, 41.9, 28.3. M.F. C15H13ClBrN5O2; M. Wt.
Cal: 409; Found: ESI-Ms m/z, 410 [M + H]+. Elemental analysis: Cal: C, 43.87; H, 3.19; N,
17.05; Found: C, 43.80; H, 3.15; N, 17.01.
6-Chloro-3-methyl-1-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-
◦
dione (5h): Off white solid; 78% yield; M.P. 110–112 C; 1H-NMR (400 MHz, CDCl3),
δ,
ppm: 7.53 (s, 1H), 7.18 (d, J = 7.2 Hz, 4H), 5.93 (s, 1H), 5.46 (s, 2H), 5.31 (s, 2H), 3.30 (s, 3H),
2.35 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
δ, ppm: 160.7, 151.0, 145.4, 138.8, 131.2, 130.5,
129.8, 129.5, 128.5, 128.2, 102.3, 54.1, 41.9, 28.3, 21.1. M.F. C16H16ClN5O2; M. Wt. Cal: 345.
Elemental analysis: Cal: C, 55.58; H, 4.66; N, 20.25; Found: C, 55.50; H, 4.60; N, 20.20.
6-Chloro-1-((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpyrimidine-2,4(1H,3H)-
dione (5i): Light yellow solid; 80% yield; M.P. 98–100 ◦C; 1H-NMR (400 MHz, CDCl3),
δ,
ppm: 7.57 (s, 1H), 7.29 (m, 2H, J = 7.2 Hz), 6.86 (m, 2H, J = 7.1 Hz), 5.93 (s, 1H), 5.46 (s, 2H),
5.32 (s, 2H), 3.81 (d, J = 2.4 Hz, 3H), 3.30 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
δ
, ppm:
190.8, 164.3, 160.5, 144.7, 134.0, 131.9, 131.3, 126.9, 114.7, 102.6, 73.3, 66.6, 37.7, 36.1, 35.2,
28.4. M.F. C16H16ClN5O3; M. Wt. Cal: 361. Elemental analysis: Cal: C, 53.12; H, 4.46; N,
19.36; Found: C, 53.08; H, 4.40; N, 19.30.
6-Chloro-3-methyl-1-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-
dione (5j): Cream yellow solid; 86% yield; M.P. 101–105 ◦C; 1H-NMR (400 MHz,
CDCl3 + DMSO), δ, ppm: 8.57 (d, J = 7.3 Hz, 2H), 8.43 (d, J = 8.2 Hz, 2H), 8.07 (s, 1H),
7.86 (t, J = 7.8 Hz, 2H), 7.46 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 4.84 (s, 4H), 3.34
(s, 3H). M.F. C21H18ClN5O3; M. Wt. Cal: 423; Found: ESI-Ms m/z, 424 [M + H]+. Elemental
analysis: Cal: C, 59.51; H, 4.28; N, 16.52; Found: C, 59.50; H, 4.21; N, 16.45.
1-((1-((3-(2H-Chromen-3-yl)-1,2,4-oxadiazol-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-6-
chloro-3-methylpyrimidine-2,4(1H,3H)-dione (5k): Off white solid; 86% yield; M.P. 125–128 ◦C
;
1H-NMR (400 MHz, CDCl3),
7.16–6.93 (t, J = 7.5 Hz, 2H), 6.89 (d, 2H, J = 8.2 Hz), 5.97 (s, 1H), 5.84 (s, 2H), 5.40 (s, 2H),
5.16 (s, 2H), 3.32 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
, ppm: 171.4, 166.2, 160.6, 154.6,
δ, ppm: 7.97 (s, 1H), 7.44 (s, 1H), 7.25 (d, 1H, J = 5.7 Hz),
δ
151.1, 145.2, 143.0, 131.4, 129.5, 128.4, 124.6, 121.9, 120.9, 118.1, 116.1, 102.4, 64.1, 45.1, 41.7,
28.3. M.F. C20H16ClN7O4; M. Wt. Cal: 453. Elemental analysis: Cal: C, 52.93; H, 3.55; N,
21.60; Found: C, 52.89; H, 3.50; N, 21.51.
6-Chloro-1-((1-((3-(8-methoxy-2H-chromen-3-yl)-1,2,4-oxadiazol-5-yl)methyl)-1H-1,2,3-
triazol-4-yl)methyl)-3-methylpyrimidine-2,4(1H,3H)-dione (5l): Off white solid; 78% yield;
M.P. 122–125 ◦C; 1H-NMR (400 MHz, CDCl3),
δ
, ppm: 7.96 (s, 1H), 7.44 (s, 1H), 6.90–6.75
(m, 3H), 5.97 (s, 1H), 5.84 (s, 2H), 5.39 (s, 2H), 5.22 (s, 2H), 3.90 (s, 3H), 3.32 (s, 3H). 13C-NMR
(100.6 MHz, CDCl3), , ppm: 171.4, 166.1, 160.6, 151.1, 147.9, 146.3, 145.2, 143.5, 143.0, 129.5,
δ
124.5, 121.6, 120.5, 118.2, 114.0, 102.4, 64.4, 56.1, 45.0, 41.7, 28.3. M.F. C21H18ClN7O5; M. Wt.
Cal: 483; Found: ESI-Ms m/z, 484 [M + H]+. Elemental analysis: Cal: C, 52.13; H, 3.75; N,
20.26; Found: C, 52.07; H, 3.69; N, 20.19.
6-Chloro-3-methyl-1-((1-(4-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)butyl)-1H-1,2,3-triazol-4-
yl)methyl)pyrimidine-2,4(1H,3H)-dione (5m): Off white solid; 80% yield; M.P. 145–148 ◦C;

Molecules 2021, 26, 1952
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1H-NMR (400 MHz, CDCl3),
δ
, ppm: 8.60 (dd, J = 7.3, 1.1 Hz, 2H), 8.25 (dd, J = 8.3, 1.0 Hz,
2H), 7.85 (s, 1H), 7.78 (dd, J = 8.2, 7.4 Hz, 2H), 5.94 (s, 1H), 5.35 (s, 2H), 4.48 (t, J = 7.2 Hz,
2H), 4.26 (t, J = 6.8 Hz, 3H), 3.33 (s, 3H), 2.46–2.35 (m, 2H), 2.05 (t, J = 3.5 Hz, 2H). 13C-NMR
(100.6 MHz, CDCl3), δ, ppm: 164.3, 160.7, 151.1, 145.4, 141.9, 134.3, 134.1, 131.5, 127.0, 123.6,
122.3, 102.3, 48.3, 41.9, 37.4, 29.7, 29.1, 28.9, 28.3. M.F. C22H22ClN5O5; M. Wt. Cal: 471.
Elemental analysis: Cal: C, 55.99; H, 4.70; N, 14.84; Found: C, 55.87; H, 4.64; N, 14.75.
2-(3-(4-((6-Chloro-3-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (5n): White solid; 67% yield; M.P. 96–98 ◦C
;
1H-NMR (400 MHz, CDCl3),
δ, ppm: 8.60 (dd, J = 7.3, 0.9 Hz, 2H), 8.23 (dd, J = 8.3, 0.8 Hz,
2H), 7.82–7.72 (m, 2H), 7.67 (s, 1H), 5.96 (d, J = 15.1 Hz, 1H), 5.34 (s, 2H), 4.85 (d, J = 2.5 Hz,
1H), 4.37 (t, J = 7.3 Hz, 2H), 4.25–4.12 (m, 2H), 3.33 (s, 3H). 13C-NMR (100.6MHz, CDCl3),
δ, ppm: 164.2, 160.7, 151.1, 145.4, 134.0, 131.6, 131.3, 128.1, 126.9, 123.3, 122.5, 102.6, 102.3,
73.3, 50.2, 42.0, 39.8, 36.1, 29.8, 28.3, 27.3, 23.9. M.F. C23H19ClN6O4; M. Wt. Cal: 478; Found:
ESI-Ms m/z, 479 [M + H]+. Elemental analysis: Cal: C, 57.68; H, 4.00; N, 17.55; Found: C,
57.60; H, 4.09; N, 17.48.
6-Chloro-1-((1-hexyl-1H-1,2,3-triazol-4-yl)methyl)-3-methylpyrimidine-2,4(1H,3H)-dione (5o):
◦
Brown solid; 80% yield; M.P 100–102 C; 1H-NMR (400 MHz, CDCl3),
δ
, ppm: 7.64 (s, 1H),
5.95 (s, 1H), 5.35 (s, 2H), 4.37–4.29 (m, 2H), 3.33 (s, 3H), 2.10–1.68 (m, 2H), 1.31 (d, J = 1.9
Hz, 6H), 0.88 (s, 3H). 13C-NMR (100.6 MHz, CDCl3),
, ppm: 160.7, 152.5, 151.1, 145.4,
δ
123.3, 102.3, 50.5, 42.0, 31.0, 30.1, 28.3, 26.1, 22.3, 13.9. M.F. C14H20ClN5O2; M. Wt. Cal: 325.
Elemental analysis: Cal: C, 51.61; H, 6.19; N, 21.50; Found: C, 51.57; H, 6.12; N, 21.46.
6-Chloro-3-methyl-1-((1-octyl-1H-1,◦2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione (5p):
Brown solid; 68% yield; M.P. 120–121 C; 1H-NMR (400 MHz, CDCl3),
δ, ppm: 7.62 (s, 1H),
5.95 (s, 1H), 5.35 (s, 2H), 4.32 (t, J = 7.3 Hz, 2H), 3.33 (s, 3H), 3.25 (t, J = 7.0 Hz, 3H), 1.63
(d, J = 4.0 Hz, 6H), 1.44 (d, J = 6.3 Hz, 2H), 0.88 (d, J = 7.1 Hz, 4H). 13C-NMR (100.6 MHz,
CDCl3), δ, ppm: 160.7, 151.1, 145.4, 141.8, 123.2, 102.3, 51.5, 50.5, 42.0, 31.7, 30.2, 28.3, 26.7,
26.4, 22.6, 14.0. M.F. C16H24ClN5O2; M. Wt. Cal: 353. Elemental analysis: Cal: C, 54.31; H,
6.84; N, 19.79; Found: C, 54.36; H, 6.94; N, 19.71.
1,1’-((Butane-1,4-diylbis(1H-1,2,3-triazole-1,4-diyl))bis(methylene))bis(6-chloro-3-methyl
pyrimidine-2,4(1H,3H)-dione) (5q): Pale yellow solid; 83% yield; M.P. 115–118 ◦C; 1H-NMR
(400 MHz, CDCl3),
δ
, ppm: 7.66 (s, 2H), 5.96 (s, 2H), 5.35 (s, 4H), 4.39 (t, J = 7.0 Hz, 4H),
3.42 (t, J = 6.4 Hz, 4H), 3.33 (s, 6H). 13C-NMR (100.6 MHz, CDCl3),
δ, ppm: 160.6, 151.1,
145.4, 142.0, 123.3, 102.3, 51.0, 50.2, 41.9, 29.7, 28.2, 23.7. M.F. C20H22Cl2N10O4; M. Wt. Cal:
536; Found: ESI-Ms m/z, 537 [M + H]+. Elemental analysis: Cal: C, 44.70; H, 4.13; N, 26.07.
Found: C, 44.65; H, 4.08; N, 26.01.
1,1’-((Pentane-1,5-diylbis(1H-1,2,3-triazole-1,4-diyl))bis(methylene))bis(6-chloro-3-methyl
pyrimidine-2,4(1H,3H)-dione) (5r): White solid; 90% yield; M.P. 114–115 ◦C; 1H-NMR (400
MHz, CDCl3),
3.33 (s, 6H), 3.28 (t, J = 6.7 Hz, 4H), 1.98–1.93 (m, 2H). 13C-NMR (100.6 MHz, CDCl3),
, ppm: 160.6, 151.1, 145.4, 142.0, 123.3, 102.3, 63.3, 51.0, 50.2, 41.9, 29.7, 28.2, 23.7. M.F.
δ, ppm: 7.64 (s, 2H), 5.95 (s, 2H), 5.35 (s, 4H), 4.35 (t, J = 7.2 Hz, 4H),
δ
C21H24Cl2N10O4; M. Wt. Cal: 551. Elemental analysis: Cal: C, 45.74; H, 4.39; N, 25.40;
Found: C, 45.69; H, 4.32; N, 25.34
3.3. In Silico Docking Simulations
3.3.1. Preparation of Ligands and Target Protein
The structures of all the ligands 5a–r were drawn using the ChemDraw suite
(Ver.14.0.0.117, PerkinElmer Inc., Waltham, MA, USA) [50] and energy minimization
was accomplished by OPLS 2005 through the Ligprep module of the Schrodinger suite
(Schrödinger, New York, NY, USA) [51]. Ionization states for ligands were retained at their
original values and saved in .sdf format for docking simulations. The crystal structure of
the VEGFR-2 protein was retrieved from the protein data bank (PDB ID: 4AGD). Molecular
docking operations in the active site of receptor protein were executed by the Molegro
virtual docking program (MVD 6.0, 2013, Molexus, Odder, Denmark). All the solvent
molecules attached to the receptor protein were removed and structural parameters like

Molecules 2021, 26, 1952
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hybridization state, bond order, explicit hydrogen atoms and relevant charges for ligands
were assigned according to the docking software instructions. Five potential cavities for
binding were obtained by using the detect cavities option from the preparation tools. The
cavity, surrounding the anion binding site with a volume of 177 Å3, was further modified
by side-chain minimization and used for virtual screening. Moldock score (grid-based)
with a grid resolution of 0.20 Å was used as a scoring function for docking analysis [52].
Moldock and Rerank scores estimated the best binding poses for ligands. ALIGN program
from COOT graphical program was used for molecular alignments [53].
3.3.2. Molecular Docking
MVD is a convenient and fast algorithm to estimate protein and ligand interactions.
The virtual screening of synthesized ligands depends on Rerank score which is a mathe-
matical expression for protein-ligand binding affinity and is based on the Moldock score
derived from PLP (piecewise linear potential) [14]. Total energy for the protein-ligand
system was minimized by Medler Mead simplex minimization with H bond directionality
and non-grid force field [54]. Binding affinities of ligands with protein were computed
based on internal electrostatic, hydrogen bond interactions and sp2-sp2 tortions. All the
docking interactions of the protein-ligand system were analyzed using Accelrys Discovery
Studio v3.5 (Waltham, MA, USA) [55].
3.4. ADME Profiles
Drug-likeness of the derived ligands was assessed by Lipinski filters in the Molinspi-
ration webserver (©Molinspiration Cheminformatics 2018, Nova Ulica, Slovensky Grob,
Slovak Republic).
3.5. Softwares, Suites and Webservers
The ligands were designed by Marvin Sketch 5.6.0.2 (1998–2011; Copyright Chem
Axon Ltd., San Diego, CA, USA). Docking interactions were obtained from Molegro Virtual
Docker (2010.4.0.0). Accelrys Discovery studio visualizer 3.5.0.12158 (©2005–12; Accelrys
Software Inc., San Diego, CA, USA) was utilized for molecular visualizations. Solubility
parameters were calculated by the Qikprop module from Schrodinger Suite 2013.
3.6. In Vitro Cytotoxicity
The cell viability was measured by MTT colourimetric assay. The cell lines viz., HeLa
(human cervical adenocarcinoma), HUH-7 (human hepatoma cancer cells) and NIH/3T3
(normal murine fibroblast cells) were supplemented with 10% fetal bovine serum and
◦
maintained in an environment of 5% CO2 at 37 C temperature. Cell lines at a density
of 2 × 103 cells/well were seeded onto 96 well plates for 16 h and maintained in DMEM
culture media. Five different concentrations of synthesized ligands and 5-fluorouracil
(except for normal cells) i.e., 0.1, 0.5, 1, 5 and 10
and incubated for 3 days at 37 ◦C [56]. Then, the solutions were discarded and an MTT
reagent with a final concentration of 500 g/mL was added to each well and plate and
incubated for another 4–6 h [57 59]. 200 L of DMSO was added to solubilize purple
µM were treated against each cell line
µ
–
µ
precipitates (formazan product) and absorbance (optical density) was recorded at 570 nm
on ELISA reader. IC50 values were calculated using dose-response data by applying the
linear regression method. The experiments were done in triplicate for each concentration
of ligand.
4. Conclusions
Herein, eighteen new 1,2,3-triazole-uracil scaffolds have been designed, synthesized
and tested as anti-cancer agents. The structures of the compounds were established by
1
standard analytical techniques viz., H-NMR, 13C-NMR, elemental analysis and mass
spectrometry. The analytical data were in good agreement with the molecular formulae
of the compounds. A rational approach of drug design was adopted to present the anti-

Molecules 2021, 26, 1952
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tumor efficacies of the synthesized ligands 5a–r. To realize the objective, the ligands 5a–r
were sequentially screened in the active site of VEGFR-2 protein and the results depicted
remarkable interactions with the receptor protein. The molecular docking simulations
by Molegro virtual docker produced high binding affinities for all the compounds (
to −198.7 kcal/mol) with various hydrophobic, electrostatic and hydrogen bond interac-
tions. The interactions observed for the compounds 5a 5f 5h 5i 5j 5n and 5r determine
−
108
,
,
,
,
,
proximity with receptor protein with different binding modes and justify their remarkable
cytotoxic activity among all the synthesized compounds. The propinquity of ligands with
receptor protein presumed the ability to inhibit in vitro cell proliferation and hence the
ligands were screened for cell viability by the MTT method. Six compounds 5a
5j 5n and 5r were found to inhibit cell proliferation with low half-maximal inhibitory
concentrations (IC50) of 11.2 M, 4.5 9.0 M, 10 M, 9.6 M, 9.6 M, respectively.
Compound 5h emerged as a hit compound by producing higher inhibition activity than
5-FU against both the HeLa and HUH-7 tumor cell lines with IC50 values 4.5 and 7.7 M,
, 5h, 5i,
,
µ
µM
,
µ
µ
µ
µ
µ
respectively. The docking results were very well correlated to the experimental results.
Interestingly, the compounds didn’t show any cytotoxicity towards normal cells, paving
the way for the reported ligands to be developed as new alternatives to existing drugs for
cervical and liver cancers.
Supplementary Materials: The following are available online at, Figures S1–S36: 1H-NMR, 13C-NMR
for
4 & 5a–r; Figures S37–S47: Mass spectra of 5a, 5b, 5c, 5d, 5e, 5f, 5g, 5j, 5l, 5n, 5q; Figures S48–S57:
Molecular docking figures of 5b, 5c, 5d, 5e, 5g, 5k, 5l, 5m, 5o, 5q.
Author Contributions: Conceptualization, P.M.R., A.K.R., K.B. and A.H.; methodology, investigation
and data curation N.N.R. and S.-J.H.; software, V.S.R. and R.R.; writing—original draft preparation,
M.K.S.; writing—review and editing, M.K.S., S.-J.H. and N.N.R.; formal analysis, A.S.; supervision,
A.H. and P.M.R. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Data is contained within the article and supplementary materials.
Acknowledgments: One of the authors N. Naresh Reddy is thankful to UGC-New Delhi, India for
financial support in the form of UGC-SRF [Ref. no: 22/12/2013(II)EU-V] and Head, Department
of Chemistry, Osmania University, Hyderabad, India for providing laboratory facilities and CFRD,
O.U, IICT, Hyderabad, India for providing spectral data. PMR is grateful to UGC for a Start-up grant.
The authors AH and SH are thankful to Buddhist Tzu-Chi General Hospital (TCRD109-77), Tzu-Chi
University (TCMRC-P-109006) (TCRPP109008), and the Ministry of Science and Technology (MOST
108-2113-M-320-001), Taiwan for financial support.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 5a–r are available from the authors.
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