Green synthesis of selenium-N-heterocyclic carbene compounds: Evaluation of antimicrobial and anticancer potential

Article abstract of DOI:10.1016/j.bioorg.2019.103042

Three benzimidazolium salts (III-V) and respective selenium adducts (VI-VIII) were designed, synthesized and characterized by various analytical techniques (FT-IR and NMR ¹H, ¹³C). Selected salts and respective selenium N-Heterocycli

Full text of DOI:10.1016/j.bioorg.2019.103042

Accepted Manuscript
Green Synthesis of Selenium-N-Heterocyclic Carbene Compounds: Evaluation
of Antimicrobial and Anticancer Potential
Amna Kamal, Mansoureh Nazari V., Muhammad Yaseen, Muhammad Adnan
Iqbal, Muhammad B. Ahmed Khadeer, Aman Shah Abdul Majid, Haq Nawaz
Bhatti
PII:
S0045-2068(19)30218-4
https://doi.org/10.1016/j.bioorg.2019.103042
103042
DOI:
Article Number:
Reference:
YBIOO 103042
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
11 February 2019
22 May 2019
3 June 2019
Please cite this article as: A. Kamal, M. Nazari V., M. Yaseen, M. Adnan Iqbal, M.B. Ahmed Khadeer, A. Shah
Abdul Majid, H. Nawaz Bhatti, Green Synthesis of Selenium-N-Heterocyclic Carbene Compounds: Evaluation of
Antimicrobial and Anticancer Potential, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103042
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Green Synthesis of Selenium-N-Heterocyclic Carbene Compounds: Evaluation of
Antimicrobial and Anticancer Potential
Amna Kamal1, Mansoureh Nazari V.2, Muhammad Yaseen4, Muhammad Adnan Iqbal1,3,Ϯ
Muhammad B. Ahmed Khadeer5, Aman Shah Abdul Majid6, Haq Nawaz Bhatti1,*
,
1Department of Chemistry, University of Agriculture, Faisalabad-38040, Pakistan
2Department of Pharmacology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, 11800-Pulau
Penang, Malaysia
3Organometallic and Coordination Chemistry Laboratory, Department of Chemistry, University of Agriculture
Faisalabad-38040, Pakistan
4Department of Chemistry, University of Education (Lahore), Faisalabad Campus, Faisalabad, Pakistan
5EMAN Testing and Research Laboratories, Department of Pharmacology, School of Pharmaceutical Sciences,
Universiti Sains Malaysia, Penang 11800, Malaysia
6Faculty of Medicine, Quest International University Perak, Ipoh, Perak, Malaysia
Corresponding Authors: † adnan.iqbal@uaf.edu.pk
* hnbhatti2005@yahoo.com
Abstract
Three benzimidazolium salts (III-V) and respective selenium adducts (VI-VIII) were designed,
1
synthesized and characterized by various analytical techniques (FT-IR and NMR H, 13C).
Selected salts and respective selenium N-Heterocyclic carbenes (selenium-NHC) adducts were
tested in vitro against Cervical Cancer Cell line (Hela), Breast Adenocarcinoma cell line (MCF-
7), Retinal Ganglion Cell line (RGC-5) and Mouse Melanoma Cell line (B16F10) using MTT
assay and the results were compared with standard drug 5-Fluorouracil. Se-NHC compounds and
azolium salts showed significant anticancer potential. Molecular docking studies of compounds
(VI, VII and VIII) showed strong binding energies and ligand affinity toward following
angiogenic factors: VEGF-A (vascular endothelial growth factor A), EGF (human epidermal
growth factor), HIF (Hypoxia-inducible factor) and COX-1 (Cyclooxygenase-1) suggesting that
the anticancer activity of adducts (VI, VII and VIII) may be due to their strong anti-angiogenic
effect. In addition, compounds III-VIII were screened for their antibacterial and antifungal
potential. Adduct VI was found to be potent anti-fungal agent against A. Niger with zone of
inhibition (ZI) value 27.01±0.251 mm which is better than standard drug Clotrimazole tested in
parallel.
Keywords: Selenium, N-Heterocyclic Carbenes, NHC, Se-NHC, breast cancer (MCF-7),
Cervical cancer (Hela), Retinal Ganglion cancer (RGC-5)
1

Introduction
Selenium containing compounds are promising candidates for cancer therapy due to their ability
to alter various physiological functions involved in cancer development, presenting either
anticancer, antimicrobial, antioxidant, anti-inflammatory, anti-viral, anti-neurodegenerative, anti-
depressant, anti-neoplastic and chemo preventive activities [1-18]. Selenium is essential
micronutrient [9, 19-21] which is non-toxic to humans in low concentrations, therefore an adduct
that releases selenium to the biological system in a steady rate act as an effective pharmaceutical
agent [22]. The effectiveness of selenium adducts as an anti-infective agent depends on the
bioavailability of selenium at the site of action [23]. Different factors such as route of delivery,
ionization and solubility of adducts can affect the bioavailability of Selenium. The application of
selenium containing adducts in cancer treatment and prevention is a captivating field for
selenium research as they have been proven as effective anti-carcinogenic agents in different
models, such as chemically induced, spontaneous and culture or transplanted tumors [24-29].
Different researchers use versatile approaches to synthesize selenium adducts. Among them,
ebselen, 1,4-anhydro-5-seleno-D-talitol (SeTal), diphenyl diselenide, m-trifluoromethyl-diphenyl
diselenide, 3-(4-fluorophenylselenyl)-2,5-diphenylselenophene, selenomethionine (SeMet) and
bis-selenide have been recognized as promising pharmacological agents that is helpful to reduce
the risk of various diseases like cancer, alzheimer, oxidative stress, HIV and depression [30-32].
Beyond these it also prevents ferroptosis which is induced by hydroperoxide [33]. Some
selenium containing compounds e.g., ebselen is under the clinical trial of phase II for the
treatment and prevention of noise and chemotherapy-induced hearing loss and advance stage
cancers and its derivative ethaselen is under clinical trials of 1st phase [34, 35]. The use of
selenium in medicinal chemistry is helpful to cure many kinds of illness due to compatibility
with the biological system as compared to the other already used elements of periodic table e.g.,
Selenium supplements are given to repay its deficiency while selenium sulphide is used in
shampoos for treatment of dandruff [36, 37]. Keeping in view these observations and in
continuation of our previous work to synthesize new bioactive selenium N-heterocyclic
derivatives by facile and greener methods using water as solvent (Iqbal et al., 2016b), the current
study has been carried out with the hope to get better anti-cancer and anti-microbial agents.
Results and Discussion
Synthesis and characterization
The syntheses of azolium salts (III-V) and respective Se-NHC adducts (VI-VIII) were carried
out according to our previously reported work [38]. The preliminary indications for the
successful synthesis of desired compounds (III-VIII) were the solubility, physical states, and a
difference in the melting points (mp) of benzimidazolium salts and their respective selenium
adducts. For example, the Se-NHC adducts were found as sticky brown material which on
recrystallization gave either light yellow thick fluid which turned colorless on recrystallization
compared to the benzimidazolium salts which appeared as white solids in the reaction medium
depending upon the substitution of alkyl chain on nitrogen atoms of benzimidazole group.
Furthermore, the selenium adducts (VI-VIII) were found to be soluble in non-polar solvents like
chloroform, dichloromethane, n-hexane and diethyl ether compared to the benzimidazolium salts
2

which are soluble in polar solvents like water, methanol and ethanol. Moreover, the difference in
melting points (mp) of the benzimidazolium salts (III-VI) and their respective Se-NHC adducts
(VI-VIII) provided a clear indication of successful synthesis of VI-VIII (Scheme-I). As the
melting points of all the synthesized salts were found in the range of 110-235 °C and their
respective Se-NHC adducts (VI-VIII) were found in 90-135 °C, which distinguish the inorganic
and organic nature of synthesized compounds, respectively. The percentage yields of these
compounds were found in the range of 69-85% and remain stable in the presence of moisture and
air.
KN
N
HN
R1-X
N
R1
1
.
DMSO, KOH(1.5 eq)
N
N
RT, stirring for 30 min
H2O
2. RT, stirring for 2.5 h
-KX
I-II
R2-X
H
Se
R2
1
,4
d
io
x
a
n
e
R2
R1
R1
reflux, 12 h
N
N
N
N
O
a C
eq)+N
Se(1.5
eq)
3(2
2
h
6
,
x
u
fl
e
R
,
O
H2
VI-VIII
III-V
Alkyl (R) group
R2
-
R1
Compound
I
Benzhydryl
Octyl
-
II
2-ethyl phenyl
Benzhydryl
Octyl
III & VI
Octyl
Octyl
IV & VII
V & VIII
Benzhydryl
Scheme 1: General Synthesis of Benzimidazolium salts (III-V) and their respective selenium adducts
(VI-VIII).
Spectroscopic Studies
The synthesized compounds were characterized by FT-IR, 1H and 13C NMR. To find the spectral
changes of benzimidazolium salts and their respective selenium adducts before and after the
predicted bonding of elemental selenium, show some specific changes which could be used as
prelude information for the successful insertion of elemental selenium, to the organic framework.
In our previous work on Se-NHC adducts [39-41], we have reported that after the incorporation
of selenium element to carbene carbon (C=Se) bond for imidazole has a vibrational band at 1222
cm-1 and a distinct spectral change appears in the region 1100-1600 cm-1. Some prominent peaks
in azolium salts suppressed in selenium adducts and some of them enhanced (see supplementary
file, Figs. S5-S7). In general, the vibrational bands due to N-C=Se and aromatic C=C disturb
prominently providing a preliminary indication of successful synthesis. Same behavior was
3

observed in our newly synthesized compounds (III-VIII). But our work on silver showed four
finger pattern in a region between 1200-1500 cm-1 which is completely different from selenium
compounds [42]. After successful preliminary indications from physical properties and FT-IR,
synthesized compounds (III-VIII) were also confirmed by 1H and 13C NMR. 1H NMR spectra of
benzimidazolium salts (III-V) have a distinctive proton signal due to NCHN at 9.81-10.1 δ ppm.
However, this peak (NCHN) disappeared in Se-NHC compounds VI-VIII due to replacement of
proton with selenium. This result further verified the successful synthesis of Se–NHC adducts.
13C NMR spectra of salts (III-V) indicated that the NCN carbon remained in up field region
(142-148 δ ppm) in azolium salts as compared to selenium adducts (V-VIII) which appeared in
the range ~165-168 δ ppm which is according to the previous reports [34, 43-45] and is another
significant indication (other than FT-IR) for the successful synthesis of desired compounds (see
supplementary data for details figures S1-S7). Chemical shift of C=Se varies and becomes more
versatile with the change of substitutions over imidazole/benzimidazole ring and ring size
(Figure 1) [39, 44].
Figure 1: Shows NMR (13C) chemical shifts of Se-NHC adducts reported by other researchers
highlighting variation in chemical shifts indicating the effect of ring size and substitutions on 13C NMR
Chemical shifts.
Antimicrobial activity
The synthesized compounds were tested against different micro-organisms (bacterial and fungal
strains) in addition to the cytotoxicity against targeted cancer cell lines. Thus the antimicrobial
activity of the synthesized salts (III-V) and respective Se-NHC adducts (VI-VIII) were
evaluated against E.coli (Escherichia coli), B.subtilis (Bacillus subtilis)¸ S. aureus
(Staphylococcus aureus) and A.niger (Aspergilius Niger) by a disc diffusion method [46]. Zones
of inhibition (mm) are summarized in Figure 3 (see supplementary file for tabulated values). In
4

general, most of the compounds showed good to moderate activity against all tested strains.
Comparing the zone of inhibition (ZI) values of synthesized compounds (III-VIII) with the
previously published compounds (1-9, Figure 2), it could be noticed that the selenium adduct
VIII showed ZI value (20.01±0.32 mm) better than most of the previously published selenium
adducts against E.coli. Similarly, compounds III, VI & VII were active against B. Subtilis. On
the other hand, compounds III, V, VI and VIII were the most active against A.niger (Figure 3).
R
N
2P
F6
R
N
N
2B
r
R
N
N
2P
F6
N
N
N
O
N
N
n
1:
2:
R=Propargyl, R= Pentyl
7:
9:
8:
R=Propyl, R= Pentyl
5:
n=2,
6:
n=3
3
4:
:R=Methoxyethyl, R=Hexyl
R=Butyl
Figure 2: Chemical structures of some previously reported compounds representing different ZI value
against A. Niger, E.coli, B. subtilis and S.aureus depending on the substitutions[32].
Figure 3: In vitro antimicrobial activity of synthesized compounds against different pathological strains
using disc diffusion method.
In the current study, it was also observed that some selenium adducts are active than their
respective salts. However, previous reports demonstrated that selenium compounds showed poor
antibacterial activity as compared to their salts. Pronounced activity of salts might be due to
presence of electronegative element like chlorine and bromine. From previous experience it was
noticed that presence of electronegative element is responsible for increasing antimicrobial
activity [19, 45, 47]. The good activity of our salts may be due to same reason. In addition,
selenium compounds have also shown moderate to good activity.
Anticancer Studies
The synthesized salts (III-V) and their respective selenium adducts (VI-VIII) were preliminarily
tested against Cervical Cancer Cell line (Hela), breast adenocarcinoma (MCF-7), Retinal
Ganglion Cell line (RGC-5) and Mouse Melanoma Cell line (B16F10) using MTT assay and
5

compared their cytotoxicity with standard drug 5-Fluorouracil in vitro. IC50 values were
calculated for selected compounds (Table 1).
Table 1: IC50 value of tested compound against different cell lines.
Compounds
MCF-7
(µM)
Hela
(µM)
RGC-5
(µM)
B16F10
(µM)
Benzimidazolium III
salts
47.67±0.21
8.39±0.18
NT
0.04±0.31
11.67±0.18
32.22±0.30
V
0.24±0.22
0.11±0.20
4.30 ±0.11
4.9±0.10
11.63±0.34
09.16±0.27
11.61 ±0.15
16.5 ±0.12
68.86±0.14
386.21±0.21
62.9±0.26
0.87±0.15
Se-NHC adducts VI
VIII
5-Fu
66.60±0.01
11±0.09
Standard drug
*NT=Not tested
Synthesized compounds demonstrated different levels of cytotoxicity against different cell lines.
Percentage inhibition (% inhibition) of cell proliferation was evaluated at different
concentrations of compounds (III-VIII) ranging from 6.125 to 100 μm [34]. From the results
obtained through Hela, MCF-7, RGC-5 and B16F10 cancer cell lines, it is possible to compare
the anticancer activity of salt (III-V) and its selenium adducts (VII-VIII), and to learn from
factors Such as specificity, selectivity and of their cytotoxic and/or anti-proliferation effects. Fig.
4 depict the dose-dependent anticancer effects of the salts (III-V) and selenium adducts (VII-
VIII) against all tested cancer cell lines. It is clearly shown in fig 4, as the concentration of tested
sample increases, percentage inhibition of cancer cells increases. As treatment of Hela and RGC-
5 cells with Salt III exhibited the most profound activity with IC50 = 0.04±0.31 µM &
11.67±0.18 µM, respectively. From Fig 5, it is clearly demonstrated that the population of Hela
cells reduced significantly with almost complete cell inhibition in salt III.
Figure 4: Dose dependent effects of ascending amounts of selected salts and selenium adducts vs Hela,
MCF-7, RGC-5 and B16F10 cancer cell lines on the percentage inhibition of cell proliferation.
6

Comparison of IC50 values of synthesized salts and their corresponding adducts with standard
drug, 5-fluorouracil (5-Fu) showed that salt III, V and their respective Se-NHC adducts VI
&VIII showed the cytotoxic results even better than 5-Fu against Hela and RGC-5 cell lines.
Figures 5 shows the selected cell images of Hela, MCF-7, RGC-5 and B16F10 cell lines with 48
h control without and with test drugs. It can be observed that all the cell lines have confluent
growth of cancerous cells under negative control whereas under the influence of standard drug
(5-Fu) a drastic decrease in cancerous cell concentration occurred indicating the death of cancer
cells under standard drug tested in parallel to the test compounds. The test compound III
significantly inhibited the cell proliferation comparted to the standard drug however the figure
indicates different patterns of MCF-7 cell death by III compared to 5-Fu. Similar effects may be
observed for cell line B16F10 and RGC-5 insisting the speculation of cell death under different
mechanisms of action by test compounds and standard drug in the selected cell lines. However,
the conditions are different for HeLa cell line where the cell death by standard drug and test
compounds III and VI seems identical indicating the similar mechanism of action. However,
such observations are merely speculations without detailed mechanistic assays.
Figure 5: MCF-7, B16F10, RGC-5 and Hela cell images were taken with a digital camera under an
inverted phase contrast microscope at * 200 magnification at 48 hours after treatment with the standard
drug (5-Fu) and samples.
Molecular Docking
Angiogenesis is necessary for metastasis and growth of cancer cells. It is initiated by imbalance
between negative and positive angiogenic factors produced by both cancer and normal cells. A
currently successful strategy for the cancer treatment is targeting angiogenesis by suppressing
7

angiogenic factors. In most of the cancers, the level of VEGF-A (vascular endothelial growth
factor A), EGF (human epidermal growth factor), HIF (Hypoxia-inducible factor) and COX-1
(Cyclooxygenase-1) have been correlated with metastasis and tumorigenesis [48-52]. Automated
docking systems and specific active site of target were used to explore the binding affinity and
ligand efficiency of organoselenium adducts (VI-VIII) on VEGF-A, EGF, COX-1 and HIF. The
activity of adducts was compared with that of the standard reference (5-Fluorouracil and
sunitinib). The docked conformation of VEGF-A, EGF, COX-1 and HIF with active
conformation of each compounds VI, VII, VIII, sunitinib and 5-FU (See Fig. 6 for chemical
formula) clearly revealed that numerous potential interactions were present (Figure 7-10). Free
binding energies of VI, VII and VIII are lower than sunitinib (Table 2) which have been
selected as positive control. However, compound VII showed higher free binding energy than 5-
FU (Figure 7). Sutent because of having two conventional hydrogen bind by GLU38 and ASP41
with VEGF, has shown strongest affinity to this target among all the tested samples.
Figure 6: 2D view of VI, VII, VIII, IX (5-Fu) and X (sunitinib).
Even though, compound VI because of containing four aromatic groups involved in interaction
with VEGF but its affinity was still less than sutent. This was probably because of the halogen
bond occurring between sutent and VEGF via SER95 strengthens best conformation between
seren 50 and ASP34 this substrate and target molecule [53].
8

Insert Fig. 7 Here
Figure 7: Visualization of ligands and protein interaction profile: VI: VEGF with - VI surface, VII:
VEGF with – VII, VIII: VEGF with - VIII surface, IX: VEGF with - IX surface and their respective
active site residue interaction.
5-FU contained four hydrogens that bind with VEGFA via PHE47, PHE36, and ASP34 that
made it to cause stronger affinity towards VEGFA than compound VII. However due to
presence of one aromatic group in compound VII, it showed similar affinity to the target
molecule as 5-FU with VEGFA. Compound VI and VII showed almost same affinity towards
VEGFA as sutent even though they do not contain any conventional hydrogen bond in
interaction active pocket but because of containing four and three aromatic compounds in
9

interaction with VEGFA could stabilize it and cause lower affinity than compound VII and 5-FU
[54]. However, compound VI contains Pi-sufur bond involved in binding with VEGFA protein
by CYS104 and CYS26. This causes the binding pocket to be stabilized more than compounds
VI-VII and 5-FU [54]. The free binding energy of compounds VI, VII and VII are lower than 5-
FU which has been selected as positive control (Figure 8). Although 5-FU involves seven
conventional hydrogen binds through LEU8, CYS14, ASP11, PRO7, GLY12, TYR13 and
CYS14 but compounds VI, VII and VIII can be involved in Pi-binding and contain four, one
and three aromatic groups involve in Pi binding respectively which can stabilize the active
pocket and cause lower binding energy in it compare to the positive control [54].
10

Figure 8 here
Figure 8: Visualization of ligands and protein interaction profile: VI: EGF with – VI surface, VII: EGF
with – VII, VIII: EGF with – VIII surface, IX: EGF with – IX surface, X: EGF with – X surface and their
respective active site residue interaction.
11

Compound VI contains Pi-anion binding through ASP11 and ASP17 with EGF protein (Figure
8). This causes the binding pocket to be stabilized more than 5FU. It revealed that because it
involved in pi-sulfur binding with CYS20 and CYS6 residues of EGF and stabilized and the free
binding energy is less than 5FU, VII and VIII. Moreover, sutent in interaction with EGF could
have only four conventional hydrogen bonds but because of stabilizing the binding HIF and 5-
Fluorouracil surface pocket with Pi-Lone pair, Pi-anion and Pi sigma with EGF demonstrated
less free binding energy than all other tested samples [55].
12

13

Figure 9 here
Figure 9: Visualization of ligands and protein interaction profile: VI: COX1 with - VI surface, VII:
COX1 with – VII, VIII: COX1 with - VIII surface, IX: COX1 with - IX surface, X: COX1 with – X
surface and their respective active site residue.
Free binding energy of compound VI is lower than VII, VIII, 5-FU and sutent with COX1.
Even though sutent has made four conventional hydrogen bonds with CYS47, GLN461,
GLU465 and GLN44 of COX1 but compound VI showed more affinity to interact with COX1
than all other tested samples. This is due to stabilization of CYS36 in COX1 that made such
interactions stronger than all (Figure 9). Although, Compounds VII and VIII also don not
contain hydrogen binding in interaction with COX1 but because of containing one and three
aromatic groups, respectively showed higher affinity than sutent and 5-FU (Figure 9). 5-FU
binds to LEU101, SER118 and GLN147 residues of HIF through conventional hydrogen bonds.
Compound VI has shown pi-sigma interaction with VAL336 and LEU340 and because of
containing four aromatic groups in its active pocket, VI was the strongest sample from the point
of affinity towards HIF. However, compound VIII because of containing three aromatic groups
in interaction pocket was stabilized and caused to have less free binding energy than 5-FU.
14

Figure 10 here
Figure 10: Visualization of ligands and protein interaction profile: VI: HIF with - VI surface, VII: HIF
with – VII, VIII: HIF with - VIII surface, IX: HIF with - IX surface, X: HIF with – X surface and their
respective active site residue interaction.
15

None of the studied samples in interaction with HIF were as strong as sutent because not only it
could make hydrogen bond with THR39, PHE37, ARG215, ALA10 and SER11 but also
obtained halogen bond with ARG33. This made very stable conformation of sutent in interaction
with HIF and caused it to have the highest affinity towards this target (Figure.10)
Table 2: Binding energy and inhibition constant of compounds VI-VIII
Binding energy
VI
-10.53
-8.55
-6.79
-4.64
-6.36
-8.11
-4.97
5.84
-6.32
-4.05
-5.25
-4.97
-6.71
4.15
-7.09
-6.46
(kJmole-1)
VII.
VIII
-9.24
-8.21
Standard drugs
5-Fluorouracil
Sunitinib
VI
-10.3
-8.21
-5.09
-5.97
Inhibition constant
19.09*103
1.50
(µM)
VII
543.61*103 220.01
391.83
64.11
229.15
12.12
25.16
4.08
VIII
76.84*103
28.04
21.93
1.14
Standard drugs
5-Fluorouracil
Sunitinib
964.34
138.96*103
187.11*103 227.34
Experimental
Chemicals and Instruments
All chemicals were purchased from Merck and Sigma Aldrich Chemicals and were used as such
without further purifications. The melting points of adducts were determined by Stuart Melting
Point SMP11. FT-IR (Fourier transform Infrared) spectra were recorded using Perkin-Elmer
2000 spectrophotometer. NMR (Nuclear magnetic resonance) spectra were collected using
Bruker Avance 500. Cell culture reagents were bought from Gibco, USA; Eagle Medium, RPMI
1640 medium; Trypsin, Dulbecco’s modified, and HIFBS (heat inactivated foetal bovine serum)
were purchased from GIBCO, UK. Cervical Cancer Cell line (Hela), breast adenocarcinom
(MCF-7), Retinal Ganglion Cell line (RGC-5) and Mouse Melanoma Cell line (B16F10) were
purchased from ATCC, USA. MTT was purchased from Sigma Aldrich, Germany. DMSO
(Dimethyl sulfoxide) was purchased from Fluka, USA. Stock solution (10 mM) was prepared by
o
dissolving synthesized compounds in DMSO and stored at 4 C. In each experiment different
concentrations of solutions for different culture medium were made.
Synthesis of 1-benzhydryl-3, 2-ethyl benzene benzimidazolium bromide (III)
N-benzhydryl benzimidazole (I) (1.0 g, 3.52mM) was dissolved in 1,4-dioxane (40 mL) on
heating and 2-bromo ethyl benzene (0.478 mL, 3.51mM) was then added and reflux the reaction
o
mixture at 100 C with consistent stirring, After 24 hr the reaction mixture was cooled to room
temperature slowly and the precipitates were filtered, washed with distilled water. Yield: 1.34 g
o
(81 %). M.P.: 233-235 C FT-IR (KBr, ν cm-1): 3210, 3120, 3011 (C-Harom stretch), 2974, 2879
(C-Haliph stretch), 1660, 1598 (C=Carom stretch), 1476, 1336, 1314 (CH2 bendings). 1H NMR (300
MHz, DMSO-d6, δppm): 3.25(t, 2H, 1 × CH2, J = 6 Hz), 4.85 (t, 2H, 1 × CH2, J =9 Hz), 5.70 (s,
16

1H, CH-), 6.94 (q, 1H, Ar-H), 7.21 (m, 10H, Ar-H), 7.31 (m, 4H, Ar-H, d), 7.80 (d, 2H, J =30
Hz Ar-H), 9.18 (s, 1H, NCHN). 13C NMR (125.1 MHz, DMSO-d6, δppm): 34.7 (CH3), 62.5
(CH2), 64.3 (N-CH2), 74.7, (Ar-CH-N), 114.7, 114.8, 120.1, 122.1, 123.7, 127.4, 128.2, 129.1,
129.7, 131.4, 136.3, 137.1, 139.1, 142, 148 (NCHN). Anal. Calcd for C28H25BrN2: C, 71.64; H,
5.37; N, 5.97; Found: C, 71.52; H, 5.47; N, 5.83.
Synthesis of 1,3-dioctyl-benzimidazolium bromide (IV)
N-octyl benzimidazole (II) (1g, 4.34mM) was dissolved in 1, 4-dioxane (20 mL) and n-octyl
bromide (1.62 g, 1.46 mL) was then added. The reaction mixture was refluxed for 24 hr
continuously. Shiny white crystals were obtained within few hours. Yield: 1.2g (83 %). M.P:
111-113oC. FT-IR (KBr, ν cm-1): 3475, 3401, (C-Harom stretch), 2971, 2936, 2875 (C-Haliph
1
stretch), 1615, 1557, 1458 (C=Carom stretch), 1377 (CH2 bending). H NMR (500 MHz,
DMSO-d6, δ ppm): 0.80 (6H, t, 2 × CH3, J = 7.1 Hz), 1.24 (20H, m, 10 × CH2), 1.90 (4H, t, , 2 ×
CH2, J = 6.94 Hz ), 4.51(4H, t, Ar-CH2-N, J = 7.7 Hz), 7.68(2H, m, Ar-H), 8.10 (2H, m, Ar-H),
13
10.1 (1H, s, NCHN). C NMR (125.1 MHz, DMSO-d6, δ ppm): 14.3 (CH3), 22.5, 26.2, 28.8,
31.6 (R-CH2), 47.2 (N-CH2-R), 114.2, 126.9, 131.5, (Ar-C), 142.5 (NCHN). Anal. Calcd for
C23H39BrN2: C, 65.23; H, 9.28; N, 6.62; Found: C, 65.15; H, 9.39; N, 6.72.
Synthesis of 1-benzhydryl-3-octyl benzimidazolium bromide (V)
N-benzhydryl benzimidazole (I) (0.51 g, 1.76mM) was dissolved in 1, 4-dioxane (20 mL) and n-
octyl bromide (1.62 g, 1.46 mL) was then added. The reaction mixture was heated to reflux (100
oC). A brown liquid is obtained. Solvent extraction was done using chloroform. Pass the reaction
mixture from six plies of wattman filter paper. After that pass the filtrate through celite, a light
brown thick liquid was obtained. Yield: 1.1g (79 %). M.P: 113-115oC. FT-IR (KBr, ν cm-1):
3022 (C-Harom stretch), 2972, 2936, (C-Haliph stretch), 1498, 1458 (C=Carom stretch), 1377
1
(CH2 bending). H NMR (500 MHz, DMSO-d6, δ ppm): 0.82 (3H, t, CH3, J = 7.16 Hz), 1.22
(10H, m, 10), 1.89 (2H, t, CH2, J = 7.45 Hz ), 4.56(2H, t, Ar-CH2-N, J = 7.43 Hz), 7.45(10H, m,
Ar-H), 7.6 (1H, t, CH2, J = 7.53 Hz), 7.69 (2H, m, Ar-H), 7.7 (1H, d, Ar-H, J = 8.45 Hz), 8.2
(1H, d, Ar-H, J = 8.7 Hz) ,9.6 (1H, s, NCHN), 21.6 (CH2), 48.1 (N-CH2), 50.1 (Ar-CH-N),
113.7, 127.2, 128.2, 129.9, 130.1, 131.7, 134.9 (Ar-C), 142.2 (NCHN). 13C NMR (125.1 MHz,
DMSO-d6, δ ppm): 14.4 (CH3), 22.5, 26.2, 28.8, 39.1, 31.5 (R-CH2), 47.5, 64.4 (N-CH2-R),
114.6, 114.9, 127.2, 128.8, 129.7, 131.6, 131.9, 136.6 (Ar-C), 142.2 (NCHN). Anal. Calcd for
C28H33BrN2: C, 70.43; H, 6.97; N, 5.87; Found: C, 70.27; H, 7.13; N, 5.97.
Synthesis of 1-benzhydryl-3-2 ethyl benzene-benzimidazole-2-selenone (VI)
Salt III (1g, 2.51mM) was dissolved in deionized water (50 mL) on heating using round bottom
flask (100 mL). Selenium powder (0.19 g, 2.49 mM) along with Na2CO3 (0.46g, 3.32 mM) were
then dissolved and the reaction mixture was heated to reflux for 5 h. Oily layer appeared above
the water surface along with adhered sticky material to the magnetic stirrer and unreacted
selenium metal remained remains settled at the bottom of the flask. The reaction mixture was
filtered through celite, washed with distilled water (3 × 5 mL) and was extracted using
chloroform. A White powder was obtained which on recrystallization gives white shiny needle
like crystals. Yield: 0.33 g (78 %). M.P.: 133-135 oC. FT-R (KBr, ν cm-1): 3294, 3060, 3027 (C-
17

Harom stretch), 2927, 2857 (C-Haliph stretch), 1601, 1582, 1482 (C=Carom stretch), 1453, 1401,
1
1339, 1358 (CH2 bending). H NMR (500 MHz, CDCl3, δ ppm): 3.14 (2H, t, 1 × CH2), 4.65-
13
4.68 (2H, t, 1 × CH2, J = 9 Hz), 7.29-7.33 (19H, m, Ar-C) 8.14(1H, s) C NMR (125.1 MHz,
CDCl3, δ ppm): 34.3 (R-CH2), 57.1(N-CH2-R), 72.0 (Ar-CH-N), 118.6, 125.9, 126.2, 128.2, (Ar-
C), 130.5(R-CH-N), 139.4(R-CH-R), 142.7(Ar-CH), 167.8 (C=Se). Anal. Calcd for C28H24N2Se:
C, 71.94; H, 5.17; N, 5.99; Found: C, 71.81; H, 5.25; N, 5.79.
Synthesis of 1,3-dioctyl-benzimidazole-2-selenone (VII)
Salt III (1g, 2.09mM) was dissolved in 50 mL distilled water on heating using 100 mL two neck
round bottom flask. Selenium metal powder (0.24 g, 3.04mM) along with Na2CO3 (0.44g, 4.15
mM) were then added and refluxed for 4 h. After 4 h dark brown oily layer appeared above the
reaction mixture along with adhered sticky material to the magnetic stirrer and unreacted
selenium metal remained settled with magnetic stirrer at the bottom of the flask. To remove
unreacted selenium metal filter the reaction mixture by using celite. Extarction of oily layer was
done by solvent extraction method using chloroform as solvent. Washing was done by
acetonitrile (3 × 5 mL). A thich dark brown liquid was obtained. Yield: 0.78 g (82 %). M.P.: 99-
o
101 C. FT-R (KBr, ν cm-1): 2927, 2857 (C-Haliph stretch), 1705, 1660, 1596, 1476 (C=Carom
stretch), 1492, 1448, 1406, 1392 (CH2 bending) 1H NMR (500 MHz, CDCl3, δ ppm): 0.86( 6H,t,
J=6.8Hz), 1.20(20H, m, 10× CH2), 1.71(2H, t, CH2, J=7.16 Hz), 1.85(2H, t, CH2, J=7.52 Hz)
4.40 (2H, t, N-CH2, J =7.70 Hz), 6.90(2H, m, Ar-H), 7.07(2H, m, Ar-H), 7.21(2H, m, Ar-H). 13C
NMR (125.1 MHz, CDCl3, δ ppm): 13.6 (CH3), 22.2, 26.4, 28.7, 31.3(R-CH2), 46.2, 47.2 (Ar-
CH-N), 112.6, 120.4, 122.6, 129, (Ar-C), 165.01 (C=Se). Anal. Calcd for C23H38N2Se: C, 65.54;
H, 9.09; N, 6.65; Found: C, 65.63; H, 9.18; N, 6.71.
Synthesis of 1-benzhydryl-3-octyl-benzimidazole-2-selenone (VIII)
II (0.55g, 1.66 mM) was dissolved in distilled water (20 mL) on heat using round bottom flask
(100 mL). Selenium powder (0.2 g, 2.49 mM) along with Na2CO3 (0.46g, 3.32 mM) were then
added and the reaction medium was heated to reflux for 5 h. Oily layer formed above the water
surface along with sticky black solid attached to the magnetic stirrer and the unreacted selenium
powder remained settled at the bottom of the flask. The reaction mixture was filtered, washed
with distilled water (3 × 5 mL) and was extracted using dichloromethane. A thick brown liquid is
o
obtained. Yield: 0.61 g (69 %). M.P.: 96-98 C. FT-IR (KBr, ν cm-1): 3084, 3055, 3027 (C-
Harom stretch), 1482 (C=Carom stretch), 1453, 1405, 1337, 1358 (CH2 bending). `1H NMR (500
MHz, CDCl3, δ ppm): 0.82( 3H,t, J=7.2Hz), 1.27(10H, m, CH2), 1.74(2H, m, CH2),) 4.34 (2H,
13
m), 6.7-8.47(14H, m, Ar-H) C NMR (125.1 MHz, CDCl3, δ ppm): 14.3 (CH3), 22.5, 26.6, 29,
31.6(R-CH2), 46.1(R-CH2-N) 64.9 (Ar-CH-N), 110.9, 112.4, 126.7, 128.4, 128.5, 129.1,137.5,
146.2 (Ar-C), 167.8 (C=Se). Anal. Calcd for C28H32N2Se: C, 70.72; H, 6.78; N, 5.89; Se, 16.60;
Found: C, 70.89; H, 6.69; N, 5.98.
In vitro Antimicrobial activity
Agar disc diffusion method was used to evaluate the synthesized compounds (III-VIII)
individually against gram positive (Staphylococcus aureus & Bacillus subtilis) and gram
negative (Escherichia coli) bacterial strains as well as fungal strain A. Niger (Aspergillus Niger).
18

In this method 100 µL of suspension containing 106 CFU/mL and 104 spores/mL of tested
bacteria and fungi spread on NA (nutrient agar), and PDA (potato dextrose agar) medium
respectively. Media was allowed to cool and solidified. After solidification paper discs of 6 mm
diameter soaked with 80 µL of the test compounds to the agar plates and incubated at 30°C.
After one day, zone of inhibition (ZI) was measured against all the tested micro-organisms and
compared with that of the standard (ampicillin and Clotrimazole).
In vitro Anti-cancer effect of synthesized compounds
The cytotoxicity effect of the compounds (III-VIII) was evaluated using MTT assay. micro-titer
plate reader was used to read the assay plates. 5-fluorouracil (5-Fu) was used as reference
(standard).
Preparation of cell culture
Initially, Hela, MCF-7, RGC-5 and B16F10 cells were grown under maximum incubated
conditions. Only those cells were selected for cell plating that had reached a confluency of 75–
80% Discard the old medium from plate and cells were washed twice or thrice with 7.4 pH of
PBS (phosphate-buffer saline), after washing PBS was completely discarded. Now, trypsin was
added and evenly distributed on the cell surfaces. Cells were incubated for 1 min at 37°C in 5%
CO2. Then, the flasks containing the cells were gently tapped to help cell segregation and
observed under inverted microscopeAfter that, trypsins were added and cells were incubated at
37°C in 5% CO2 for 1 min. Then, the cell segregation was observed using inverted microscope.
5mL of fresh media (10% Fetal Bovine Serum) was added to observe trypsin activity. Finally,
Added 100mL cells per well with concentration of 2.5*105 cells per mL and incubated with 5%
CO2 as internal atmosphere at 37°C
MTT assay
MTT assay was performed according to previously reported method of our research group [38]
Molecular Docking study of Compounds VI-VIII
Protein preparation
Software
Python language was downloaded from www.python.com, Molecular graphics laboratory(MGL)
tools was downloaded from http://mgltools.scripps.edu and AutoDock4.2 was downloaded from
http://autodock.scripps.edu, Bio Via draw was downloaded from http://accelrys.com, Discovery
studio visualizer 2017 downloaded from http://accelrys.com and Chem3D was downloaded from
https://acms.ucsd.edu.
Methods
The three dimensional X-ray crystallographic structures of anticancer targets VEGF-A with PDB
ID: 4KZN, COX1 with PDB ID: 1EQH, EGF with PDB ID: 1JL9, HIF with PDB ID: 1YCI were
19

selected and downloaded from Protein Data Bank (www.rvcsb.org/pdb) (Figure.11)[57]. To
make the selected proteins for molecular docking, all non-essential water molecules, small
molecules, heteroatoms, nonpolar hydrogens, nonstandard residues and lone pairs were deleted
and hydrogens were added to the target receptor molecule. Optimization of geometry and
minimum energy of all structures were performed using Dock Prep, a built-in tool for structures
preparation before docking.
A
B
C
D
Figure 11: 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: HIF protein from RCSB protein data bank
(1YCI).
Ligand preparation
Four synthetic active compounds available with identified structure of salts from crystallography
were used Pubchem to make sdf format and converted to PDB format using Pymol and further
used for docking studies towards three different cancer cells and EA.hy 926 cell line as normal
cell line. 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 box was set with the size of 126×126×126Å with the grid spacing of 0.375 Å at
the binding site. The starting structure for all the salts namely VI, VII and VIII were constructed
using BioVia draw While Sunitinib and 5FU were selected as positive control. Their structures
were provided from Pubchem website 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 with a translational step of 0.2 Å, a
quaternion step of 5 Å and a torsion step of 5 Å. Most populated and lowest binding free energy
Conclusion
Three novel benzimidazolium salts and their respective selenium-NHC adducts were designed,
synthesized and tested in vitro against a panel of non-fastidious fungus, bacteria and various
cancerous cell lines. Adduct VII was particularly effective against B. subtilis and A. Niger,
having the highest antimicrobial activity across the panel of microorganisms in comparison to
the other adducts and their standard drugs. From MTT assay it was concluded that compounds
(III, V, VI and VIII) showed better cytotoxicity than standard drug against Hela and RGC-5
while results of molecular docking study showed that, all the designed and synthesized
compounds had good affinity toward the active pocket and minimum binding energy.
Conflict of Interest
The authors have declared no Conflict of Interest.
Acknowledgements
20

Dr. Muhammad Adnan Iqbal and Prof. Haq Nawaz Bhatti are thankful to the HEC-Pakistan for
the startup research grant Vide Letter No. 21-1085/SRGP/R&D/HEC/2016 to establish the
organometallic and coordination chemistry laboratory at University of Agriculture, Faisalabad
where a major part of this research was accomplished. Dr. MAI is also thankful to HEC-Pak for
awarding research grant NRPU-8396 vide letter No. 8396/Punjab/NRPU/R&D/HEC/2017.
Supplementary Material
The article contains supplementary material file.
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23

Graphical abstract
24

Highlights
 Green Synthesis of Selenium-N-Heterocyclic Carbene (Se-NHC) Adducts.
 Anticancer and Antimicrobial Potential.
 Molecular docking studies using angiogenic factor VEGF-A, EGF, HIF and COX-1.
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