Azolium mediated N[sbnd]Heterocyclic carbene selenium adducts: Synthesis, cytotoxicity and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2021.130701

Ionic liquids (ILs) are remarkable for biological activities in numerous medical fields. With the aim of enhancing biological potential of azolium based ILs, four new selenium-N-Heterocyclic Carbene (Se-NHC) adducts were synthesized from bis-imidazolium and bis-benzimidazolium molten salts. Synthesized ILs (L₁-L₄) and Se-NHC adducts (C₁[sbnd]C₄) were confirmed through elemental analysis, chromatographic and spectroscopic techniques including UHPLC-PDA, FTIR, ¹H NMR & ¹³C NMR spectroscopy as well as mass spectrometry. The compounds were found stable in solution form for upto 96 h measured spectroscopically and showed partition coefficient with optimum lipophilicity measured through shake flask method. The simulation studies of these compounds for cancerous proteins indicated that there could be good to high anticancer potential due to their high affinity and less binding energies for COX-1, EGF, VEGF-A and HIF cancer protein targets. The In Vitro cytotoxicity study of compounds confirmed that these compounds were found highly active showeing IC₅₀ against HCT-116 (1.074–1.116 μg mL^?1), A549 (0.977–1.325 μg mL^?1) and MCF-7 (0.869–1.378 μg mL^?1) which is almost better than standard drug 5-Flourouracil but slightly lower than cisplatin and oxaliplatin. The interaction study of compounds with albumin proteins (BSA) and hemolysis assay assured their least toxicity.

Full text of DOI:10.1016/j.molstruc.2021.130701

Journal of Molecular Structure 1241 (2021) 130701
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Azolium mediated N–Heterocyclic carbene selenium adducts:
Synthesis, cytotoxicity and molecular docking studies
a,1,∗
a
a
a
Rizwan Ashraf
, Zohra Khalid , Ayesha Sarfraz , Haq Nawaz Bhatti ,
a
b
Muhammad Adnan Iqbal , Mansoureh Nazari V
a
Department of Chemistry, University of Agriculture Faisalabad, Pakistan
b
School of Pharmacy, University August 17, 1945 14350 Jakarta, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history:
Ionic liquids (ILs) are remarkable for biological activities in numerous medical fields. With the aim of
enhancing biological potential of azolium based ILs, four new selenium-N-Heterocyclic Carbene (Se-NHC)
adducts were synthesized from bis-imidazolium and bis-benzimidazolium molten salts. Synthesized ILs
Received 17 November 2020
Revised 21 April 2021
Accepted 8 May 2021
Available online 19 May 2021
(
L1-L4) and Se-NHC adducts (C1–C4) were confirmed through elemental analysis, chromatographic and
spectroscopic techniques including UHPLC-PDA, FTIR, 1H NMR & 13 C NMR spectroscopy as well as mass
spectrometry. The compounds were found stable in solution form for upto 96 h measured spectroscopi-
cally and showed partition coefficient with optimum lipophilicity measured through shake flask method.
The simulation studies of these compounds for cancerous proteins indicated that there could be good to
high anticancer potential due to their high affinity and less binding energies for COX-1, EGF, VEGF-A and
HIF cancer protein targets. The In Vitro cytotoxicity study of compounds confirmed that these compounds
Keywords:
Ionic Liquids
N-Heterocyclic Carbenes (NHCs)
Selenium compounds
Molecular docking
Anticancer studies
were found highly active showeing IC50 against HCT-116 (1.074–1.116 μg mL 1), A549 (0.977–1.325 μg
−
mL 1) and MCF-7 (0.869–1.378 μg mL ) which is almost better than standard drug 5-Flourouracil but
−
−1
slightly lower than cisplatin and oxaliplatin. The interaction study of compounds with albumin proteins
(BSA) and hemolysis assay assured their least toxicity.
©
2021 Elsevier B.V. All rights reserved.
Introduction
inreductase, glutathione peroxidase etc. involved in maintenance of
antioxidant functions [1,3,4].
Medicinal inorganic chemistry is a growing interdisciplinary
area of chemical research which showed substantial progress in di-
agnostics and cancer treatment [1]. In this continuation, develop-
ment of cis-platin and its analogues were a lead to combat various
types of cancers but its use was hampered due to growing resis-
tance and serious side effects [2]. Therefore, extensive efforts are
carried out in the search of new organometallics with minimum
side effects and interestingly, some non-platinum analogues of-
fered promising features to replace resistant class of chemo drugs.
In particular, there are reports indicating that organo-selenium
compounds are best fit having substantial ability to modulate mul-
tiple physiological pathways as well as being essential trace ele-
ment for various body mechanisms [3]. Although selenium could
be toxic in higher amount but its controlled amount is safe as well
as essential for cell metabolism of sleno-proteins like thioredox-
Although various In vitro studies indicated that selenium based
compounds showed good anticancer potential [5] but, its efficacy
is very much dependent upon bioavailability at its targeted site. Its
bioavailability at target site is very dependent on rate of ionization,
delivery route as well as its lipophilicity [6]. This entire scenario
calls for a class of ligands that could be tuned for these properties
in a wake of required drug development. Different researchers use
versatile approaches to synthesize selenium adducts and the recent
years have seen an emergence of N-heterocyclic carbenes (NHCs)
as class of ligands that can be structurally tuned to make fit for
drug design [7]. Imidazole and benzimidazole could results in ionic
liquids (ILs) with tunable structure to generate NHCs compound
[8]. The steric and electronic tunability is an additional feature of
these ILs that affect the stability while complexing through strong
σ-donor properties which can be customized by substituents at
the nitrogen atoms as well as at the carbon backbone of the hete-
rocyclic skeleton that optimized the coordination of ILs with metal
center and affects the lipophilicity, stability and ultimately efficacy
of drug [9]. The molten salts were reported for their widespread
applications including catalysis, extraction, biodiesel, solar cell as
∗
Corresponding author.
E-mail address: rizi_chem82@hotmail.com (R. Ashraf).
1
Dedications: Authors dedicate this article to the memories of Dr. Bushra Sultana
(
late).
https://doi.org/10.1016/j.molstruc.2021.130701
022-2860/© 2021 Elsevier B.V. All rights reserved.
0

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
well as biological significances [10,11]. These biological applications
of ILs can be improved through incorporation of elemental sele-
nium in their organic frameworks.
poured in cooled water and product was extracted through chlo-
roform. Yield:79.7%. FT-IR (ATR cm 1): 3246, 3237 (Carom-H str),
2869 (Caliph-H str), 1494 (C–C str), 1332, 1286 (C–N str), 974 (C–H
ben). UV-Visible (λmaxnm): 274.8, 254.6 212.0. MS (m/z): Predicted
−
Therefore, present study is planned for the synthesis of four ILs
+
+
+
(
L -L ) and development of selenium adducts (C –C ) from these
[M + 1] , [C H N ] = 137.12 Da, Found [M + 1] 137.14 Da.
1
4
1
4
8
13
2
molten salts and screened for their anticancer potentials. The sol-
ubility and stability of synthesized compounds were also investi-
gated which is an important parameter of drug safety and effi-
cacy. Furthermore, affinity of compunds (C1-C4) for serum albu-
mins was tested, since drug albumin affinity plays very important
role in drug distribution and its pharmacokinetics that influence In
vitro half-life and extravasation of drug at the target site.
Synthesis of 1-isopentyl-1H-imidazole (IV)
Imidazole (1.0 g, 14.7 mmol) and KOH (1.2 g, 22 mmol) in 25 ml
of DMSO stirred for 30 min followed by addition benzyl bromide
(
2.2 g, 14.7 mmol) and stirred for 5 h at room temperature. Product
was isolated by following the same method of III. Yield: 88.5%. FT-
IR (ATR cm 1) 3089, 2935 (C–H alip str), 1462 (C–C str), 1382 (C–H
−
Materials and methods
ben), 1282 (C–N str), 877, 775 (C–H ben). UV–Vis (λmax nm). 252.3,
+
+
2
34.6. MS (m/z): Predicted [M + 1] , [C14 H11 N ] = 189.13 Da,
2
All the chemicals including imidazole, Benzimidazole, Benzyl
Bromide, 3-methyl butyl bromide and 1,2-dibromoethane were
procured from Alfa Aesar, Ward Hill, Massachusetts, United States
through local supplier Musaji Adam & sons Pakistan. Elemental se-
lenium powder was purchased from Avonchem, United Kingdom
through local supplier UH-Analytics Pakistan. The solvents used,
Dimethyl sulfoxide (DMSO), Methanol, 1, 4-dioxane, n-hexane,
petroleum ether were of analytical grade and used as they were
procured from Merck (Darmstadt, Germany). Synthesized Pre-
cursors, ILs and their selenium compounds were characterized
through FTIR (IRTracer-100, Shimadzu Japan) with post run pro-
cessing software Lab solutions. UV–visible spectra of salts and
their selenium compounds were measured through Shimadzu UV-
+
Found [M + 1] 189.11 Da.
Synthesis of
1
,2-bis(3-benzyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl)ethane
bromide salt (L1)
A mixture of I (1.0 g, 6.25 mmol) and 1, 2-dibromoethane (5 ml,
5
7.7 mmol) refluxed (at 90 °C) in 1,4-dioxane for 18 h. Yellowish
product was settled down, washed with petroleum ether. Yields:
−1
7
7%. FTIR (ATR cm ): 2955, 2918 (C–H str), 1481, 1444 (C–C str),
211, 1171 (C–N str), 739, 652 (C–H bend). 1H NMR (400 MHz
1
DMSO–d , δppm), 3.03 (s, 4H, 2×CH ), 5.61 (s, 4H, 2 × N–CH ),
6
2
2
1800 UV/Visible Scanning Spectrophotometer. All precursors and
6
.62 (m, 4H, Ar-H), 6.91 (m, 4H, Ar-H), 7.40 (m, 10H, Ar-H), 9.11
ligands were characterized through UHPLC-PDA/ESI-MS (Waters,
USA) equipped with Empower 3.0 software to acquire UV–visible
and mass spectra. 1H & 13C NMR spectra were acquired on Bruker–
Avance NMR spectrometer using internal standard (TMS).
s, 2H, NCHN). 13C NMR (110 MHz, DMSO–d , δppm), 49.4 (2C,
(
6
N–CH ), 77.6 (4C, Ar-C), 110.4 (4C, Ar-C), 120.0 (2C, Ar-C), 126.3 (4C,
2
Ar-C), 127.6 (4C, Ar-C), 128.7 (2C, Ar-C-CH ), 128.9 (4C, N–CH ),
2
2
144.8 (2C, NCN). UV–Vis (λmax nm): 261.7, 217.9. Anal. Calc. for
C30H30Br N : C, 59.42; H, 4.99; N, 9.24. Found: C, 59.73, H, 4.89;
2
4
Synthesis of 1-benzyl-1H-benzo[d]imidazole (I)
N, 9.29.
Benzimidazole (2 g, 6.9 mmol) and KOH (1.4 g, 10.3 mmol) in
Synthesis of 1,2-bis(3-benzyl-2,3-dihydro-1H-imidazol-1-yl)ethane
bromide salt (L2)
2
5 ml of DMSO stirred for 30 min followed by the addition of ben-
zyl bromide (1.2 g, 6.9 mmol) and continued stirring for 5 h at
room temperature. Reaction mixture was poured in chilled water
and product was extracted with chloroform and solvent was evap-
A mixture of II (1.0 g, 6.33 mmol) and 1, 2-dibromoethane
orated through rotary evaporator. Yield: 89.3%. FT-IR (ATR cm 1):
−
(5 ml, 57.7 mmol) was reacted by following the same procedure
of L1. Yields: 63%. FT-IR (ATR cm 1): 3047 (C arom-H str), 2999,
−
3
075 (C–H str), 1624 (C = C str), 1429 (C–C str), 1203, 1188 (C–N
2
864 (Caliph-H str), 1215, 1157 (C–N str), 1583 (C = C str), 1496,
str), 935 (C = C str), 673, 669 (C–H ben). UV-Visible (λmaxnm):
1425 (C–H bend). 1H NMR (400 MHz DMSO–d , δppm), 2.52 (s,
+
+
2
48.6, 212.0. MS (m/z): Predicted [M-1] , [C
H
N ] = 207.12 Da,
6
14
11
2
4
H, 2×CH ), 4.73 (s, 4H, 2 × N–CH ), 5.50 (s, 4H), 7.51 (m, 10H,
Found 207.10 Da.
2
2
Ar-H), 9.42 (s, 2H, NCHN).13C NMR (110 MHz, DMSO–d , δppm),
6
3
8.2 (2C, N–CH ), 48.9 (2C, N–CH ), 122.9 (4C, N–CH ), 129.3 (2C,
2 2 2
Synthesis of 1-benzyl-1H-imidazole (II)
Ar-C), 134.9 (4C, Ar-C), 136.8 (2C, Ar-C-CH ), 144.7 (2C, NCN). UV–
2
Vis (λmax nm): 247.5, 219.7. Anal. Calc. for C22H26Br N : C, 52.19;
H, 5.18; N, 11.07. Found: C, 52.55; H, 5.27; N, 10.93.
2
4
Imidazole (1.0 g, 14.7 mmol) and KOH (1.2 g, 22 mmol) in 25 ml
of DMSO stirred for 30 min followed by addition of benzyl bro-
mide (2.5 g, 14.7 mmol) and stirred for 5 h at room tempera-
ture. Product was separated by following he same method as fol-
Synthesis of
lowed for I. Yield: 82.7%. FT-IR (ATR cm 1): 3082 (C–H str), 1614
−
1,2-bis(3-isopentyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl) ethane
(
(
C = C str), 1493, 1452 (C–C str), 1286, 1265 (C–N str), 974, 935
C = C str), 775, 745 (C–H ben). UV-Visible (λmaxnm): 258.1, 223.8.
bromide salt (L3)
+
+
MS (m/z): Predicted [M + 1] , [C
H
N ] = 159.05 Da, Found
14
11
2
A mixture of III (1 g, 5.32 mmol) and 1, 2-dibromoethane
+
[
M + 1] 158.97 Da.
(
5 ml, 57.7 mmol) processed by the same procedure of L1. Yield:
5%. FTIR (ATR cm 1), 2961 (C–H str), 1480, 1400 (C–C str), 829,
−
6
747 (C–H ben). 1H NMR (400 MHz, DMSO–d , δppm), 0.92 (d,
Synthesis of 1-isopentyl-1H-benzo[d]imidazole (III)
6
J = 4.0 Hz, 12H, 4×CH ), 1.49 (q, 4H, 2×CH ), 1.69 (m, 2H, 2×CH),
3
2
Benzimidazole (2.0 g, 6.9 mmol) and KOH (1.4 g, 10.3 mmol)
in 25 ml of DMSO stirred for 30 min followed by the addi-
tion of 1–bromo-3-methylbutane (1.0 g, 6.9 mmol) and stirring
continued for 5 h at room temperature. Reaction mixture was
2.50 (s, 4H, 2×CH ), 4.43 (t, 4H, 2 × N–CH ), 7.60 (m, 4H, Ar-H),
2
2
7.67, 7.84, 8.09 (m, 4H, Ar-H), 10.07 (s, NCHN). UV–Vis (λmax, nm):
276.3, 270.0. Anal. Calc. for C26H38Br N : C, 55.13; H, 6.76; N, 9.89.
2
4
Found: C, 55.53; H, 6.84; N, 9.76.
2

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
2
.1.8. Synthesis of
,3-bis(3-isopentyl-2,3-dihydro-1H-imidazol-1-yl)propane, bromide
salt (L4)
1
Mixture of IV (1.5 g, 10.86 mmol) and 1, 2-dibromoethane
5.5 ml, 63.5 mmol) reacted by following similar protocols of L1.
(
−
1
Yields: 63%. FTIR (ATR cm ): 2958, 2872 (C–H str), 1564 (C = C
str), 756 (C–H bend). 1H NMR (400 MHz DMSO–d , δppm), 0.91
6
(
d, J = 4.00 Hz, 12H, 4×CH ), 1.52 (q, 4H, 2×CH ), 1.81 (m, 2H,
3
2
2
×CH), 4.73 (t, 8H, 4 × N–CH ), 7.70 (s, 4H), 9.3 (s, 2H, NCHN).
2
UV–Vis (λmax, nm): 270.0, 219.1. Anal. Calc. for C19
H Br N : C,
36 2 4
47.51; H, 7.55; N, 11.66. Found: C, 47.94; H, 7.63; N, 11.51.
ꢀ
Synthesis of 3,3 -(ethane-1,2-diyl)bis(1-benzyl-1H-benzo[d]imidazole-
(3H)-selenone)
C1)
2
Fig. 1. A: VEGFA protein from RCSB protein data bank (4KZN), B: COX1 protein from
RCSB protein data bank (1EQH), C: EGF protein from RCSB protein data bank (1JL9),
D: HIFprotein from RCSB protein data bank (1YCI).
(
A mixture of L1 (1.0 g, 1.7 mmol), K CO (0.47 g, 3.4 mmol)
2
3
and selenium powder (0.27 g, 3.5 mmol) was refluxed at 110 o
C
Synthesis of
for 8 h in water. A blackish thick material was found attached with
magnet bar and on the walls of the flask. Hot mixture was filtered
through Whatmann filter paper and residue was left for drying. Af-
ter drying the material was solubilized in DCM and again filtered
through celite column and obtained crystal clear filtrate. This fil-
trate was placed in fumehood for overnight evaporation.Yield:62%.
ꢀ
C4)
3
,3 -(propane-1,3-diyl)bis(1-isopentyl-1H-imidazole-2(3H)-selenone)
(
A mixture of L4 (1.0 g, 2.12 mmol), K CO3 (0.58 g, 4.25 mmol)
2
and selenium powder (0.35 g, 5.0 mmol) was refluxed at 110 °C
for 8 h by following the procedure of C1. Yield: 68.7%. FT-IR(ATR
FTIR (ATR cm 1
− ): 3177, 3156 (Carom-H str), 2955 (Calip
-H str), 1669
−1
cm ): 2956, 2870 (C–H str), 1643 (C = C str), 1456 (C–C str), 1284
C = C str), 1494, 1479 (C–C str), 1076, 1028 (C–H bend). 1H
(C–N str), 970 (C–H bend). 1H NMR (400 MHz, CDCl , δppm), 0.99
(
3
NMR (400 MHz CDCl , δppm), 3.03 (s, 4H, 2×CH ), 5.51 (s, 4H,
(d, J = 5.18 Hz, 12H, 4×CH ), 1.52 (q, 4H, 2×CH ), 1.62 (m, 2H,
3
2
3
2
13
2
×CH ), 7.11 (m, 18H, Ar-H). C NMR (110 MHz, CDCl , δppm), 47.8
2×CH), 1.75 (m, 2H, 1×CH ), 3.91 (t, 8H, 4 × N–CH ), 7.52 (s, 4H).
2
3
2
2
(
2C, N–CH ), 50.1 (2C, N–CH ), 107.7 (4C, Ar-C), 111.6 (4C, Ar-C),
UV–Vis (λ
, nm): 277.3, 271.2. Anal. Calc. for C H N Se : C,
19 32 4 2
2
2
max
1
23.9 (6C, Ar-C), 127.6 (4C, Ar-C), 128.6 (4C, N–CH ), 134.6 (2C,
48.10; H, 6.80; N, 11.81. Found: C, 48.44; H, 6.93; N, 11.73. MS (m/z,
Da): [M-1]+, [C19 H32N Se ]+, Predicted = 474.11, Found = 474.21.
2
Ar-C-CH ), 154.4 (2C, NCNSe). UV–Vis (λmax, nm): 286.12, 277.61.
2
4
2
Anal. Calc. for C30H26N Se : C, 60.01; H, 4.36; N, 9.33. Found: C,
4
2
+
+
6
0.38; H, 4.41; N, 9.28. MS (m/z, Da): [M-1] , [C30H25N Se ] , Pre-
Molecular docking of compound C –C
4
2
1
4
dicted = 601.05, Found = 601.18.
Protein preparation
ꢀ
Synthesis of 3, 3 -(ethane-1,
2
-diyl)bis(1-benzyl-1H-imidazole-2(3H)-selenone) (C2)
For molecular docking of compounds C1–C4 with selected
protein targets, Molecular graphics laboratory(MGL) tools were
downloaded from http://mgltools.scripps.edu, Python language was
downloaded from www.python.com and AutoDock4.2 was down-
loaded from http://autodock.scripps.edu, BioVia draw was down-
loaded from http://accelrys.com, Discovery studio visualizer 2017
downloaded from http://accelrys.com and Chem3D was down-
loaded from https://acms.ucsd.edu.The three dimensional crystal
structure of anticancer targets VEGFA with PDB ID: 4KZN, COX1
with PDB ID: 1EQH, EGF with PDB ID: 1JL9, HIF with PDB ID:
1YCI were selected and downloaded from Protein Data Bank (www.
rvcsb.org/pdb) (Fig. 1) [12]. The complexes bound to the receptor-
molecule, all the non-essential watermolecules and heteroatoms
were deleted and ultimately hydrogen atoms were added to the
target receptor molecule using Argus Lab.
A mixture of L2 (1 g, 1.98 mmol), K CO (0.52 g, 3.99 mmol)
2
3
and selenium powder (0.50 g, 6.2 mmol) was refluxed at 110 °C for
−1
8
h. Yield: 51.8%. FTIR (ATR cm ): 2925 (C–H str), 1654 (C = C str),
484 (C–C str), 1073 (C–H bend). 1H NMR (400 MHz, CDCl , δppm),
1
3
1
.12 (s, 4H, 2×CH ), 2.53 (s, 4H, 2 × N–CH ), 4.01 (s, 4H), 7.30
2
2
m, 10H, Ar-H), 1 C NMR (110 MHz, CDCl , δppm), 39.6 (2C,CH ),
3
(
3
2
4
8.9 (2C, N–CH ), 123.6 (4C, N–CH ), 129.4 (6C, Ar-C), 134.9 (4C,
2 2
Ar-C), 137.2 (2C, Ar-C-CH ), 154.3 (NCNSe). UV–Vis (λmax, nm):
2
2
83.5, 275.21. Anal. Calc. for C22H22N Se : C, 52.81; H, 4.43; N,
4 2
1
+
1.20. Found: C, 53.05; H, 4.55; N, 11.09. MS (m/z, Da): [M + 1]
+
,
[C
H
N Se ] , Predicted = 501.02, Found = 501.028,
2
2
23
4
2
ꢀ
Synthesis of 3,3 -(ethane-1,2-diyl)bis(1-isopentyl-1H-
benzo[d]imidazole-2(3H)-selenone)
(
C3)
A mixture of L3 (1.0 g, 1.78 mmol), K CO (0.52 g, 3.89 mmol)
Ligand preparation
For the computational targets four synthetic active compounds
(available with identified structure of ligands from crystallography)
were used from Pubchem to make sdf format and converted to PDB
format using Pymol which was further used for docking studies.
The starting structures of the proteins were prepared using
AutoDock tools. Water molecule was deleted, polar hydrogen and
Kollman charges were added to the protein starting structure.Grid
2
3
and selenium powder (0.50 g, 6.2 mmol) was refluxed at 110 °C
for 8 h by following the same procedure of C1. Yield: 46.3%. FT-IR
ATR cm 1): 2956 (C–H str), 1656 (C=Cstr), 1481 (C–C str), 1259
−
(
(
1
C–N str), 738, 651 (C–H ben). H NMR (400 MHz, CDCl , δppm),
3
1
2
4
.12 (d, J = 5.00 MHz 12H, 4×CH ), 1.51 (q, 4H, 2×CH ), 1.63 (m,
3
2
H, 2×CH), 4.42 (t, 4H, 2 × N–CH ), 4.95 (s, 4H, 2×CH ), 7.25 (m,
2
2
˚
H, Ar-H), 7.5 (m, 4H, Ar-H). UV–Vis (λmax, nm): 278.1, 272.0. Anal.
box was set with the size of 126×126×126 A with the grid spac-
˚
Calc. for C26H34N Se : C, 55.71; H, 6.11; N, 10.00. Found: C, 55.97;
ing of 0.375 A at the binding site. The starting structure for all
4
2
+
+
H, 6.27; N, 10.08. MS (m/z, Da): [M + 1] , [C26H35N Se ] , Pre-
the ligands namely 5 s, 6 s, C2 and C4 constructed using BioVia
draw. 5FU was selected as positive control. Their structures were
4
2
dicted = 561.11, Found = 561.15.
3

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
provided from Pubchemwebsite Gasteiger charges were assigned
into optimized ligand using Autodock Tools. 100 docking runs were
conducted with mutation rate of 0.02 and crossover rate of 0.8.
The population size was set to use 250 randomly placed individual.
Lamarckian Genetic algorithm was used as the searching algorithm
at 570 nm and% viability of cells were calculated through following
formula
ODsmp
%
cell viability =
× 100
ODcontrol
OD is the optical density measured at 570 nm.
˚
˚
with a translational step of 0.2 A, a quaternion step of 5 A and a
˚
torsion step of 5 A.
Results and discussions
Stability assay
Synthesis
The stability of synthesized compounds was studied through
spectral studies by monitoring spectral behavior for specific period
through UV-Visible spectroscopy. The studied compounds were sol-
ubilized in minimum amount of Acetonitrile diluted further to
Synthesis of precursors (I-IV), bis-azolium molten salts (L -L )
1
4
and their selenium adducts (C –C ) was accomplished through
1
4
procedures of [17] with minor modifications that described in
Scheme 1. Synthesis of precursors (I-IV) was achieved in first step
through N-alkylation that involved deprotonation from terminal
Nitrogen of azolium (imidazole and benzimidazole) moiety with
−
4
10
M with PBS and their UV–Vis spectra were monitored for 0,
16, 24, 48, 96 h, respectively, by following the protocol of [13].
the aid of strong base (KOH) followed by alkylation (R ) at N
1
1
Lipophilicity assay
with selected alkyl halides. In the second step, alkylation on sec-
ond Nitrogen (N ) of precursors was made by refluxing precursor
2
Lipophilic values of synthesized compounds in terms of log P
was measured through ability of prinpartitioning of studied com-
pound between octanol and aqueous layers. UV–vis spectroscopy
based method was used to estimate the concentration of studied
compounds in each layer/phase. Briefly, 0.5 mg of each compound
(
I-IV) individually with 1,2-dibromoethane in 1,4-dioxan for 24 h
which yields Ionic liquids L -L with bromide as counter ion and
1
4
became readily water soluble due to their ionic nature. These ILs
L1-L4 were refluxed with potassium carbonate and selenium pow-
der in water medium for 8 h by following simple procedure of
[18] which yields selenium adducts (C –C ). The replacement of
(
L1-L4) in distilled water and (C –C ) in Acetonitrile respectively
1 4
1
4
and Log P value was measured through logarithm of ratio of con-
organic solvent with water for sysnthsis of selenium compounds
was termed as green synthesis [19]. A sticky blackish fluid insolu-
ble in water attached with magnet bar as well as walls of the flask,
which was later separated, dried and dissolved in chloroform and
passed through celite column which produced light yellowish fluid
product.
centration in octanol layer to aqueous layer by formula (Log P=Log
(
[org]/[Aq]).
Interaction with BSA
UV-visible spectrophotometric method was used for interaction
study of increasing concentration of synthesized molecules with
constant concentration(0.01 mM) of BSA by following the proto-
col of [14]. Shift in spectrum of BSA-drug solutions was measured
as an indication of drug-BSA binding compared to both BSA and
compound solutions individually. FTIR-ATR based study was also
conducted to investigated the mode of affinity among synthesized
compounds and protiens [15].
Characterization
The imidazolium and benizimidazolium based precursors (I-IV)
were confirmed through Ultra high pressure Liquid chromatogra-
phy (UHPLC) hyphenated with spectroscopy (PDA) and mass spec-
trometry (ESI-MS). Synthesized molten salts (L -L ) were charac-
1
4
terized by spectroscopic techniques (UV-spectrophotometer, FTIR),
1H & 13 C NMR and mass spectrometry. The selenium adducts
(C1
–
C4) were characterized through distinguished behavior ob-
served in FTIR, 1H & 13 C NMR as well as confirmed through mass
Cytotoxicity
Cytotoxicity of synthesized compounds against three cancer
strains was measured through MTT assay by following the method
of [16]. The amount of formazan product is directly proportional
to number of living cells present. Synthesized compounds C1-
C4 and L1-L4 were prepared for a series dilution (10–0.001 μg
spectrometry.
UHPLC-PDA/MS
Syntheses of precursors I-IV were assured through UHPLC-PDA
by measuring their retention time (RT) as well as UV-Visible spec-
tra in comparison to azolium base. The purity of precursors was
also assured through single peak as well their 3D spectra thorugh
Photodiod array detector (PDA). When precursor I was analyzed
through UHPLC-PDA, a prominent peak at RT 2.388 min was ob-
served which was different from benzimidazole (RT: 2.154 min)
confirming the different chemical nature of precursor I from ben-
zimidazole. Additionally, Precursor I showed UV-visible spectrum
with absorption maxima at 212.0 and 248.8 nm which was dif-
ferent from its reactant. The synthesis of I was further assured
through mass spectrometry which recorded major peak at 207.21
mL 1) in Methanol and water respectively and standard drugs
−
5
-Florourouracil, oxaliplatin and cisplatin (1–0.0001 μg mL 1) in
−
methanol. Human cancer cell lines MCF-7 (1000 cell/well), lung
cancer cell A549 (1000 cell/well) and breast cancer cell line HCT-
1
16 (1000 cell/well) were cultured in DMEM medium (Dubecco’s
modified Eagle medium) in the presence of 5% CO2 continuous aer-
ation at 37 °C in 96 well plate with 100 μL medium and incubated
for 24 h. After 24 h, replaced the DMEM with freshly prepared
DMEM with 10 μL of each concentration of samples were added.
For the accuracy of the experiment, treatments were performed in
triplicate. Control was set as to grow cell line without drug treat-
ment which holds only living cells in well and triplicate of blank
DMEM was also adjusted on 96 well plates. After 48 h incuba-
tion, 20μL of MTT dye (5 mg/ml in PBS) was added in each well
and allowed to incubate at 37 °C for four hours. Contents of wells
were removed and frozen product was solubilized in 100μL DMSO
hence, the purple color appeared. The absorbance of each well was
measured on Biobase multiplate reader through Gen 2.0 software
+
for their parent ion [M-1] Fig. 2. Precursor II showed RT 2.55 min
having unique UV-visible spectrum different from imidazole which
indicated the difference in chemical nature as an indication of sub-
stitution at imidazole for precursor II and detailed spectra are pro-
vided in supplemantry data Fig. S1. UHPLC-ESI-MS spectrum fur-
ther confirmed the successful synthesis of Precursor II by showing
prominent parent ion peak [M + 1]+ for substituted imidazole Pre-
4

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
Scheme 1. Synthesis of precursors (I-IV), salts (L1-L4), selenium adducts (C1–C4).
Fig. 2. A) UHPLC-PDA (UV–Vis.) chromatogram and spectrum at specific peak RT 2.38 min of precursor I, B) UHPLC-QDa (ESI-MS) at RT 2.45 min at negative mode with
spectrum and 3D imaging of precursor I.
cursor II coincide with its theoretical expected mass supplemantry
data Fig.S1. The Preucrsors III & IV were also measured through
UHPLC-PDA/ESI-MS at both positive and negative ion mode at var-
ied capillary voltage (9–15 V) and observed their molecular ion
peaks at 189.20 and 137.21 Da for Precursor III &IV, respectively
which is in accordance with parent ion peak of mass spectra of
III & IV predicted through computational software (ChemDrawUlta
When ILs (L -L ) studied through UHPLC-PDA, a far difference
1 4
in absorption spectra from than that of respective precursors I-IV
and shift in retention time was observed, results are provided in
supplemantry data Fig. S3-S6. This difference in absorption spectra
could be attributed to formation of molten salts by combining pre-
cursor’s I-IV. However, very minor difference in absorption spectra
of selenium adducts (C –C ) was observed in comparison to their
1
4
12.0) and also measured for their UHPLC-PDA chromatograms and
respective ligands, supplemantry data Fig.S6a. This minimal differ-
spectra that confirmed the purity as well as successful synthesis of
Precursors, chromatrographic results are provided in supplemantry
data Fig. S1-S2.
ence among the spectral behavior could be due to minor modifi-
caton of molecular structure of binuclear salts L -L and addition
1
4
of unsaturation by replacing proton of carbene carbon through el-
5

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
Fig. 3. overlay FTIR spectra of precursors I-IV, binuclear salts L1-L4 and selenone compounds C1–C4 from bottom to top respectively.
emental selenium. Selenium adducts were further assured through
mass spectrometery (HRMS) which confirmed the successful con-
version of ILs into respective adducts and results are provided in
supplemantry data Fig.S19-S22.
observed by Iqbal, Haque, Ng, Hassan, Majid and Razali [19] who
reported the suppression of this region upon incorporation of
elemental selenium in organic framework of imidazolium salts.
The comparable characteristic pattern was observed by [18] for
mononuclear selenium adducts showing suppression in stretching
frequency of this functional group, Fig. 4.
FTIR spectroscopy
FTIR spectroscopy was applied for the investigation of Precur-
NMR spectroscopy
sors (I-IV) and their conversions into ILs (L -L ) through peculiar
1
4
behavior observed in spectra, Fig. 3. An intense peak pattern in the
Successful synthesis of salts (L -L ) and compounds (C –C )
1
4
1
4
−
1
were also confirmed through 1H & 13C NMR spectroscopy, and de-
tailed NMR description is given in experimental section. Resonance
of aromatic proton of imidazolium and benzimidazolium salts (L1-
L4) was observed in 7.0 - 8.3 ppm, which is in line agreement with
previous reports showed similar region of resonance for aromatic
proton of azolium salts [22]. However, the peculiar behavior ob-
served for bis-azolium salts (L -L ) was the appearance of char-
range of 800–650 cm of =C–H bending vibrations was observed
in all spectra of precursors and salts irrespective of the nature of
substitution. A sharp peak pattern of aromatic C = C stretching
−1
in the region of 1650–1500 cm
was observed for I & III but
weak for II & IV which differentiates imidazolium and benzimi-
dazolium precursors [20]. Similarly, an intense peak at 1300–1450
cm 1 for precursor I & III and weak for II & IV marked the dif-
−
1
4
ference among N-alkylated benzimidazolium and N-alkylated imi-
acteristic singlet resonance peak at aroud 9.0–12.0 ppm appeared
in 1H NMR spectra that diminished with incorporation of elemen-
tal selenium in the organic framework of salts, detailed spectra are
provided in supplemantry data Fig S7-S13. The appearance of this
dazolium precursors. A prominent peak region (3250–3140 cm 1)
could be attributed to substitution at N1 position through replace-
ment of proton with alkyl and aryl groups [21]. The successful con-
−
version of precursors into ILs L -L4 was marked through shift in
characteristic peak in salts L -L at 9.1, 9.4, 10.0 and 9.3 ppm re-
1 4
1
spectral behavior from their precursors, Fig. 3. The distinguished
spectively is associated with resonance of carbene proton (NCHN)
supplemantry data Fig S7-S10. These observations were consistent
with literature which showed presence of carbene proton in this
region [23,24]. This peculiar peak of carbene proton disappeared
in selenium adducts C –C due to replacement of carbene proton
spectral patterns appeared in spectra of bis-azolium salts (L -L )
1
4
−
1
at around 2890–3010 cm
(C–Haliph) due to attachment of alkyl
linker that differentiates ligands from the respective precursors.
The conversion of ligands into selenium adducts (C –C ) were
1
4
1
4
measured through changes in FTIR spectra in comparison to re-
with elemental selenium, an indication of successful conversion of
spective ILs (L -L ) by highlighting typical changes appeared in
carbene salts into their respective compounds (C –C ) [25]. The
1
4
1
4
comparative spectra Fig. 3. Some distinguished pattern of FTIR
spectra of Se-NHC adducts comparing to their azolium salts at
successful synthesis of salts (L -L ) was also confirmed through
1 4
13C NMR spectra where a peculiar peak at 140–150 ppm is asso-
ciated with carbene carbon (NCHN), supplemantry data Fig S14-
S17. The similar observations were reported with chemical shift
(13C, ppm) at 143.0, 142.6, 143.0 [22] and 144.7, 142.1 [26] for car-
bene carbon (NCHN) of azolium salts. Interestingly this characteris-
tic peak of carbene carbon shifted to low field (152.5–160.5 ppm)
when proton of carbene carbon is replaced with elemental sele-
nium, described in Fig. 4. The other detailed NMR description is
1
450–1100 cm 1 was observed that could be linked with vibra-
tional frequency of carbon selenium (C=Se) bonds. The same be-
havior was confirmed through literature showing vibrational fre-
−
−
1
quency at 1222 cm
prominent region at 1560–1500 cm
suppressed in selenium adducts (C –C ) an indication of success-
for C=Se of imidazolium adducts [19].The
−
1
for molten salts L -L was
1
4
1
4
ful conversion of ligands into adducts. The similar trend was also
6

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
Fig. 4. overlay spectra of 13 C NMR of ligands L1-L2 and compounds C1-C2.
Table 1
Log P values of compounds C1–C4 in aqueous-octanol sys-
tem.
Interaction studies with BSA (Bovine serum albumin)
Human serum albumins (HSA) are principle proteins responsi-
ble for binding and carrier of diverse drug entities. However, BSA
(bovine serum albumin) is the most studied conjugate protein due
to high similarities with HSA that mimics in reactivity to study
various metal–based complexes for their potential selective target-
−
Cw (mg L 1)
−
Compound
Co (mg L 1)
Log P
C1
C2
C3
C4
9.32
8.45
9.57
8.59
0.68
1.55
0.43
1.31
1.13
0.74
1.34
0.82
ing to HSA [27]. A large number of metals, be it Pt2 , Cu , Zn
2+
,
+
2+
Ni2 and Co
+
2+
complexes have been investigated for their inter-
action with BSA in relationship to their bioactivity. However sele-
nium based NHC adducts were not studied for their potential inter-
action with albumins. Therefore, organ-selenium adducts (C –C )
provided in the experimental section and spectra are provided in
supplementary files Fig S7-S17.
1
4
were studied for their intrinsic binding affinity with albumin pro-
teins in terms of binding constant Kb through spectroscopic titra-
tions.
Stability in solution
The stability of compounds (C –C ) were studied through UV-
1
4
The absorption spectrum of BSA showed characteristic absorp-
tion maximum at 280 nm due to aromatic amino acid residues.
On sequential addition of incremental aliquots of drug C1 (0.647–
Visible spectroscopy in solution form. The studied compouds 0.2 g
dissolved individually in Acetonitrile (ACN) and diluted with PBS
solution 0.02 g/mL. The time dependent (16, 24, 36, 48, 96 h) UV–
Vis spectra of solutions exhibit no apparent spectral changes over
the studied time course given in supplemantry data Fig.S22. This
repetitive behavior of compounds with time course showed that
compounds are stable at ambient temperature in solution form for
studied time period. These results were further confirmed through
HPLC-PDA which showed repetitive chromatographic behavior over
studied time course, which showed that studied compounds are
stable at physiological conditions for studied time course.
2
.59 μM) to a fixed concentration of BSA, a sharp increase in
absorption intensity with slight shift in absorption pattern from
80 nm to 274 nm was observed, supplemantry data Fig.S23 The
2
shift in absorption pattern could be linked to binding of drug with
studied proteins and furthermore, increase in intensity with re-
spective increase in concentration of C1 was observed. The quanti-
tative assessment of binding interactions of complex C1 with BSA
5
−
1.
ascertained the intrinsic binding (Kb 2 × 10
±
0.06) M
The profound change in absorbance towards blue shift is an in-
dication of formation of adducts of studied complexes with BSA
Lipophilicity determination
[
28] that showed concentration dependent response. In similar
protocol compound C –C were also investigated for their po-
2
4
Hydrophilicity/lipophilicity is very important parameter speci-
fied by Lipinski’s rule for drug development, it exhibits the ability
of drug to cross biological membrane and determin druggability.
tential binding for BSA through intrinsic binding constants (Kb)
with increasing concentrations of aliquots while concentration of
BSA constant. The binding ability of studied compound is very
much dependent on nature ofdrug that affects the binding strength
with albumin proteins [29]. Interestingly, overall binding affinity
of all drugs was far superior to reported literature for cisplatin
The lipophilic character of selenium adducts C –C4 was measured
1
through flask shake method and the values are presented in Po/w
given in Table 1. The lipophilic values of compound C –C indicate
1
4
that these compounds are sufficiently lipophilic to satisfy the gen-
eral lipophilic requirement of drug. Among the studied compounds
C1 and C3 are more lipophlic than C2 and C4 which could be due
to nature of substituent at azolium moiety. The previous reports
suggest that cell membrane permeability and cytotoxicity of com-
pounds is very much dependent upon lipophilicity of compounds
2
−
1). Cisplatin is an FDA approved chemothera-
(
K = 8.52 × 10
M
peutic agent but notorious for nonselective in action [30].
The interaction of BSA was further investigated through FTIR
spectroscopy based on the assessment of changes in amide bands
of BSA upon interaction with C . The most prominent bands ap-
1
peared in the spectrum of BSA at 1750–1650 cm 1 and 1550–1500
−
[
13]
7

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
Table 2
Binding constant values (Kb) and equation parameters of compound
C1–C4.
Kb (M 1)
−
Compound
Equation
Linear cofficient
4
C1
C2
C3
C4
Y = 0.025X +0.362
Y = 0.387X +0.016
Y = 0.032X+0.596
Y = 0.283X +0.031
0.948
0.977
0.813
0.979
6.91×10
6
2.42×10
3
5.62×10
5
9.13×10
cm 1 could be attributed to C = O streching and C–N streching
of amide I and amide II of BSA respectively. Graphical results pro-
vided in supplemantry data Fig.S24 showed a clear difference in
spectrum of BSA-C1 from BSA with slight shift in frequency bands
as well appearance of new bands that could be associated with in-
teraction of selenium compounds with BSA. The slight shift in BSA
−
Fig. 5. % hemolysis of compound C1–C4, Triton X at concentration 0.1 mg mL 1 and
−
PBS against humane RBCs.
Table 3
Inhibitory Concentrations (IC50) of C1- C4.
−
1
spectra from 1740 to 1732 cm
could be due to engagement of
amide I band with the drug because it is most vulnerable for the
structual changes of protein. In the BSA-C1 spectra, appearnace of
new bands with three disntint peaks in the region of 1250–750
Compound
IC50 (μg/mL)
MCF-7
HCT116
A549
C1
C2
C3
C4
1.2768± 0.15
0.8691± 0.12
0.9813± 0.09
1.3787± 0.17
0.9978± 0.06
–
1.074± 0.08
1.116± 0.09
1.101± 0.14
1.099± 0.13
0.9981± 0.11
–
1.3251± 0.05
0.9850± 0.08
0.9970± 0.12
0.9773± 0.17
–
cm 1 may be an indication of binding of drug with BSA and gen-
erated new function groups. Interstingly intensity of this region
regularly changed with respective concentration of drug which in-
dicates that interaction among them is concentration dependent
phenomenon. The similar findings was also reported in literature
when interaction of mimosine with BSA was studied through FTIR
spectroscopy [14].
−
5
-FU
Oxaliplatin
Cisplatin
0.808
–
–
0.812
IC50 (μg/mL) values of compounds C1-C4, standard drug 5-
flourouracil (5-FU), oxaliplatin and cisplatin for cancer cell lines
Mcf-7, HCT116 and A549 was measured in triplicates and presented
Mean ± SD.
When compounds C –C4 were studied through UV-Visible spec-
2
troscopy for their binding affinity with BSA, no specific trends were
observed as described in Table 2. Compounds C1 & C3 showed
4
4
affinity for BSA molecules with Kb values 6.91×10 and 6.91×10
2
far better than Cisplatin 8.52 × 10 [31] but slightly lower than C
Cytotoxicity
2
6
5
&
C
having Kb 2.42×10 and 9.03×10 respectively.This change
4
in affinity trends may be attributed to the lipophilic character
which helps in intrinsic binding of drug with biological molecule.
This fact was supported by Chen, Wang, Sun, Zhang, Sun, Sun,
Shang and Luo [32] who claimed that lipophilic character may in-
crease the effect on conformational changes in BSA that ultimately
played a role in protein-drug binding phenomenon. Among the
possible interaction forces, lipophilic interaction is considered as
more prominent role in the intrinsic binding affinities with pro-
tein molecule [33]. Although no trend was reported among binding
affinity of drug with BSA and bioactivity of drug but an interesting
The synthesized compounds L1-L4 and C1–C4 were tested for
their cytotoxicity against three cancer cell line, human colon can-
cer HCT 116, lung cancer A549 and breast cancer cell line MCF-7
through MTT assay. Reuslts of the studied compounds were com-
pared with standard drugs 5-flourouracil(5-FU) and platin com-
pounds cisplatin and oxiplatin and results are presented in Fig. 6.
The ILs L1-L4 showed IC50 values in the range of 8.6–15.4 μg
mL 1 for cancer cell line HCT-116 which seems very poor than
standard drug 5-FU (0.9981± 0.11 μg mL 1). But cytotoxicity of
these salts was surprisigly changed upon conversion to compounds
C1–C4 which showed very good IC50 value in the range of (1.074–
−
−
trend was observed in compounds C –C4 that compounds having
1
1.116 μg mL 1) and very near to standard drug 5-FU. Then sele-
nium compounds C1–C4 were further investigated for cancer cell
line A459 where they showed exceptionally good cytotoxicity but
slightly poor than standard drugs cisplatin and oxaliplatin, detailed
results are described in Fig. 6 and Table 3. The results showed that
studied compounds showed concentration dependent response to
inhibit the all cancerous cell lines which showed very good ac-
tivity when compared with control. The most potent activity of
compound C1 (0.987± 0.05 μg mL 1) was observed against A549
−
lower Kb values showed more cytotoxicity.
Compounds C –C4 titrated with BSA and a curve is drawn in-
1
dividually among 1/Conc. and Log of response factor. Equations of
best fit curve is measured and Kb value is slop to intecept ratio
Hemolytic assay
−
Synthesized selenium compounds C –C4 were studied for their
1
−1
hemolysis activity by measuring the destruction ability of com-
pounds for RBCs through spectroscopic method and compared
with possitive control Triton X. It was observed that all studied
selenium compounds were found very least toxic thant possitve
control Triton X, Fig. 5. The intra comparison of compounds for
their hemolysis against RBCs showed that imidazolium based com-
and C2 (0.8691 μg mL ) against MCF-7 which showed even better
potential than standard drug 5-FU (0.9978± 0.06 μg mL 1) while
other compounds also showed good activity but slightly less than
the standard drug.
−
Overall it was observed that selenium adducts C –C are more
1
4
potent than their binuclear salts L -L4 which indicates the possible
1
pounds (C2 & C ) were more inocuous for RBCs than that of ben-
contribution of selenium center in the organic framwork of hete-
rocyclic compound to exhibit bioactivity agaisnt cancer strain. The
4
zimidazolium (C1 & C ). The overall hemolysis potential of com-
3
pounds C –C4 was found in the range of (09.8–18.6)% hemolysis
studied compounds C –C4 showed better anticancer activity than
1
1
which is in the safer range if used as drug. The lower hemolytic
activity of selenium compounds (C –C ) than Triton X showed that
reported mononuclear Se-NHC adducts [18] and similar in level in
activity with silver complexes [23]. When anticancer potential of
compounds was observed in correlation with lipophilicity of stud-
ied compouds, a very keen relationship was observed; which indi-
1
4
these compounds are almost safer and could proceed for further
studies.
8

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
Fig. 6. In vitro anticancer activity of synthesized compounds C1–C4 and salts L1-L4 against cancer cell line HCT-116, A549 and MCF-7 thorugh MTT assay in comparison to
standard drugs 5-Flourouracil (5-FU), Oxaliplatin (oxi pt) and cisplatin (cis pt). All the experiments were conducted in triplicate and presented in mean± SD.
cates that lipophilicty has direct effect on the bioactivity of com-
pounds [34]. Similar observations were also reporeted in literature
showing a sucessful correlation among lipophilicity and cytotoxic-
ity of studied drugs [35].
clearly demonstrate that there is numerous interaction potential of
compounds was found with selected targets and results are sum-
marized in Table 4.
Docking with COX1
Molecular docking
The Interaction of selenium compounds with COX1 is shown in
Fig. 7 which revealed that free binding energy of compound C1,C3
& C4 are very lower than that of FU. Free binding energies of all
selenium compounds except C2 was lower than positive control FU
for growing factor COX1 while C2 showed almost equal response
to FU Table 4. This difference in free energy of compounds while
interacting with COX1 could be linked with difference in bonding
natures of active sites of compounds with protein target and de-
fined the strength of bonding as well as free energy. Compound
C2 showed C–H bonding with GLN461 bond length (BL) of 3.23
while C1 and C4 interacts with COX1 through C–H bonding with
ASP135 having BL 3.17 and 3.01 respectively.C4 also showed some
alkyl interaction with COX1 with ARG49, TRP323, ILE46, TYR130,
LEU152, LYS46, VAL48, PRO153, CYS47, CYS36 and CYS41. Moreover,
Compound C3 interacts with COX1 making pi sulfur bond with
CYS36 and pi anion bond with GLU465 and pi sigma with LEU152.
These differences in interaction sites for different compounds may
affect the drug efficacy. Overall it was observed that aromatic
Angiogensis is a necessary factor for cancer growth, which
ususally results by imbalancing of positive and negative angiogenic
factors produced by cancer cell funtioning. Targeting these specific
factors could be a selective anti-angiogensis approach which ap-
peals the attention of medical scientists to investigate potential of
drugs to hamper these factors. Among the growing factors in can-
cer cell, level of COX-1 (Cyclooxygenase-1), EGF(humanepidermal),
VEGF-A(vascularendothelial, FactorA) and HIF (Hypoxia-inducible
growth factor) have been found in direct correlation with tumori-
gensis. Therefore simulation studies for binding efficiency of active
sites of selenium compounds C –C with COX-1, EGF, VEGF-A and-
1
4
HIF were studied through automated docking systems. The simula-
tive potential of selenium compounds was measured through two
parameters i.e. free binding energies as well as interaction bind-
ing constant (Ki) and results were compared with standard drug 5-
Flourouracil (FU). The docked conformation of active sites of stud-
ied compounds with protein active factors COX-1, EGF, VEGF-A, HIF
9

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
Table 4
Bindingenergy and inhibition constant of compounds C1–C4.
COX1
EGF
VEGF
HIF
Binding
C1
−12.95
−5.15
−7.56
−4.51
−6.89
−7.36
−4.97
2.90
−6.35
−4.99
−5.85
−6.24
−6.71
3.31
−10.12
−3.69
−7.57
−7.88
−5.26
3.8 × 10
1.96
energy (KJ C2
mol 1
−
)
C3
C4
−11.02
−10.09
−5.09
5
FU
4
4
5
Inhibition C1
3.2 × 10
166.71
3.9 × 10
4.4 × 10
187.11
constant
μM)
C2
C3
C4
49
221.6
19.40
26.56
12.12
3
5
(
8.96
5.6 × 10
1.67
4
5
FU
227.34
138.96
Fig. 7. Molecular docking of selenon compounds with protein target COX 1 with I) compunds C1, II) Compound C2, III) standard drug 5-FU.
groups helped more to lower the free binding energy while in-
teracting with COX1. Since C2 has shown some pi sigma binding
with LEU152 BL 5.35 while C4 has shown pi sigma binding with
VAL48 BL 3.74 which make difference in binding energies of inter-
action of these compounds with targeted protein. In addition C4has
shown pi-pi T shaped binding with HIS43 and few alkyl bindings
with CYS67, CYS36, LEU152, PRO153, LEU46 and LYS468. This phe-
nomenon’s reason for stabilization of active pocket of COX after be-
ing in interaction with C1, C3 and C4 ended in less free binding en-
ergy than 5FU. 5FU has shown 3 H-bondings with LEU101, SER118
and GLN147 and showed free binding energy of −5.09 Kcal/mol.
The compound C2 showed −5.15 Kcal/mol and other compounds
C1, C3 and C4 showed-12.95, −11.02 and −10.09 Kcal/mol respec-
tively.
10

R. Ashraf, Z. Khalid, A. Sarfraz et al.
Journal of Molecular Structure 1241 (2021) 130701
Docking with EGF
plemantry data, the free binding energy of 5FU binds to LEU101,
SER118 and GLN147 residues of HIF through conventional hydro-
gen bonds while compound C3 has shown pi-sigma interaction
with PRO231 and VAL336. It can interact as carbon hydrogen
bond with GLU323 and GLN241. Compound C3 can make alkyl
bond with TYR228, PHE224, PRO229 and PHE111. Even though 5FU
which has been selected as positive control has shown three hy-
drogen bindingswheareasneither C1 nor C3 have shown such con-
ventional hydrogen binding;but their free binding energy is less
than 5FU because of containing four and two aromatic groups re-
spectively.Compound C2 has shown carbon hydrogen binding with
PRO333 and GLU321 with length of 3.06 and 3.40 respectively
while C4 has shown such binding with PRO33 with the length of
2.72, which is a shorter bond.
The Interaction of selenium compounds with EGF is shown in
supplemantry data Fig. S25 which revealed that free binding en-
ergy of compound C1&C4 much lower than other competing com-
pounds as well as from standard drug FU. Compound C4 has shown
Pi lone pair with TYR37 and GLY18 BL of 2.96 and 2.91 respec-
tively,Pi anion binding with ASP11 while C–H boding with CYS14
and ASP17. Ithas also demonstrated some alkyl bonds with LEU15,
CYS6 and CYS20. Although 5FU involved 6 conventional hydrogen
bonds through LEU8, CYS14, ASP11, PRO7, GLY12, TYR13 and CYS14
but compounds C1 and C3 contain 4 and 2 aromatic groups that
are involvedin Pi binding which stabilized the active pocket more
than FU and cause lower binding energy in them as compared to
the positive control [36]. Moreover, C3 also showed C–H bond-
ing with CYS31 and TYR37 and also Pi- alkyl bindings with ILE23,
CYS33, TRP49, ALA30 and ILE38 from EGF which helps C3 to bind
pocket and get stabilized more than 5FU [36]. On contrary Com-
pound C2 demonstrated C–H bondings with CYS14 and ASP17 and
few alkyl bondings with LEU8, CYS20, HIS16 and LEU15. Free bind-
ing energy of C2 was detected at −4.51 Kcal/mol while it has been
Conclusion
Eight N-heterocyclic compounds among them four ionic salts
(L -L ) and four selenium compounds (C –C ) were synthesized
1
4
1
4
and fully characterized through spectroscopic and chromatographic
techniques. The stability studies of compounds for time period of
0, 24, 48 and 96 h in solution forms through UV-Visible spec-
troscopy confirmed that compounds are stable in solution form
for studied period of time. Lipophilicity of compounds was in the
range of log P (0.74–1.34) measured through shake flask method
and their effect on biological activities was evaluated. The binding
affinity of compounds with BSA measured in terms of Kb which
found better than FDA approved antiproliferative drug cisplatin.
−
7.56 Kcal/ mol for C1. This significant difference maybe due to
stability of the bonding pocket [36]. Although 5FU involves in 6
conventional hydrogen binds through LEU8, CYS14, ASP11, PRO7,
GLY12, TYR13 and CYS14. But due to aromatic compound in struc-
ture of C1 as well as short bindings which happened in interaction
with EGF, it stabilizes more than 5FU and C2, So it has more affin-
ity towards EGF than other compounds.
The hemolysis of studied compounds C –C4 was very less from
1
Docking with VEGFA
their positive control Triton-x 100 which was an indication of
safety of compounds for normal cells. The anticancer activity of
compounds was measured through molecular docking studies for
cancer proteins targets (COX-1, EGF, VEGF-A and HIF) as well as In
Vitro studies for HCT-116, MCF-7 and A549 cancer cell lines. Both
studies were supportive to each other for the conclusion that com-
pounds are good cytotoxic agents.
The selenium compounds showed interaction with VEGFA as
is shown in supplemantry data Fig. S26, but binding energy of
all studied compounds was higher than standard drug FU. How-
ever among the selenium compounds,free binding energy of C1 is
lower than which may be due to 4 aromatic rings in its structure
that caused pi-pi T shaped bond [36]. On the other hand benzim-
idazolium compound C3 showed higher free energywhen interacts
with VEGFA by making pi sulfur bond with MET78, and making pi
sigma with ILE91. This compound contains 2 aromatic compounds
so it needs to consume more energy to react with VEGFA com-
pared with 6S or 5FU [37]. 5FU interacted 4hydrogen bonds with
VEGFA via PHE47, PHE36, and ASP34 that made it to cause stronger
affinity towards VEGFA than compound C1 & C3[36]. As it is shown
in Fig. S26, compound C2 has shown carbon hydrogen bonds with
GLN22 and CYS26. It has shown pi sigma bindings with TYR25 and
TYR21, alkyl bindings with HIS27 and MET18. However, compound
C4 has shown pi sigma binding with MET78 and pi sulfur with
MET81 and MET55. It has shown few alkyl bindings with PRO53,
LYS48, PRO49 and ILE80. But it has shown less free binding energy
than C2, because of being stabilised more by aromatic compounds
in active pocket of enzyme. Free binding energy of all the studied
compounds are more than 5FU which has been selected as positive
control. 5FU interacted 4 hydrogen bonds with VEGFA via PHE47,
PHE36, and ASP34 that made it to cause stronger affinity towards
VEGFA than other compounds [36].
Declaration of Competing Interest
Authors decleared no conflict of intrest on this article.
CRediT authorship contribution statement
Rizwan Ashraf: Investigation, Data curation, Writing - original
draft. Zohra Khalid: Writing - review & editing. Ayesha Sarfraz:
Investigation. Haq Nawaz Bhatti: Supervision. Muhammad Adnan
Iqbal: Conceptualization, Methodology. Mansoureh Nazari V: For-
mal analysis, Resources.
Acknowldgment
Authors aknowldged Higher Education commission (HEC) of
Pakistan for funding of project through National research program
for universities-HEC via funding # NRPU-8198.
Supplementary materials
Docking with HIF
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130701.
When selenium compounds interaction was studied with HIF,
all compounds showed lower binding energy than standard drug
FU while C2 showed higher energy. Compound C1 contains pi-
sulfur bond with MET319 and pi-sigma with VAL232. It can re-
act and make alkyl binding with HIF in PRO229, PRO231, ARG320,
VAL336 and LEU340. Compound C1 from aromatic group inter-
acts with PRO231 and VAL336. As it is shown in Fig. S27 of sup-
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