Non–symmetrically p–nitrobenzyl–substituted N–heterocyclic carbene–silver(I) complexes as metallopharmaceutical agents

Article abstract of DOI:10.1002/aoc.3819

In the present work, a series of eight new imidazole, 4,5–dichloroimidazole, 4,5–diphenylimidazole and benzimidazole based nitro–functionalized mono–N–heterocyclic carbene (NHC)–silver(I) acetate (7a–d) and bis–NHC–silver(I) hexafluorophosphate complexes (8a–d) were synthesised by the reaction of the corresponding azolium hexafluorophosphate salts (6a–d) with silver(I) acetate and silver(I) oxide in methanol and acetonitrile, respectively. All the synthesised compounds were fully characterized by various spectroscopic techniques and elemental analyses. Additionally, the structure of bis–(1–benzyl–3–(p–nitrobenzyl)–4,5–dichloroimidazole–2–ylidene)silver(I) hexafluorophosphate complex (8b) was confirmed by single crystal X–ray diffraction analysis. Preliminary in vitro antibacterial evaluation was conducted for all the compounds (6a–d), (7a–d), and (8a–d) by Kirby–Bauer's disc diffusion method followed by the determination of Minimum Inhibitory Concentration (MIC) from broth macrodilution method against five standard bacteria; two Gram–positive bacterial strains (Staphylococcus aureus and Bacillus subtilis) and three Gram–negative bacterial strains (Escherichia coli, Shigella sonnei, and Salmonella typhi). All the hexafluorophosphate salts (6a–d) were found inactive against the tested bacterial strains and their corresponding mono– and bis–NHC–silver(I) complexes (7a–d and 8a–d) exhibited moderate to high antibacterial activity with MIC value in the range 8–128?μg/mL. In addition, preliminary in vitro anticancer potential of all the silver(I) complexes (7a–d and 8a–d) was determined against the human derived breast adenocarcinoma cells (MCF 7) by MTT assay. All the mono– and bis–NHC–silver(I) complexes (7a–d and 8a–d) orchestrated high anticancer potential with IC₅₀ values ranging from 10.39 to 59.56?nM. In comparison, mono– NHC–silver(I) complexes performed better than the bis–NHC–silver(I) complexes.

Full text of DOI:10.1002/aoc.3819

Received: 28 November 2016
DOI: 10.1002/aoc.3819
Revised: 5 March 2017
Accepted: 5 March 2017
F U L L PA P E R
Non–symmetrically p–nitrobenzyl–substituted N–heterocyclic
carbene–silver(I) complexes as metallopharmaceutical agents
C. R. Shahini1 | Gautam Achar1 | Srinivasa Budagumpi1 | Matthias Tacke2 | Siddappa A. Patil1
1 Centre for Nano and Material Sciences, Jain
University, Jain Global Campus,
Kanakapura, Ramanagaram, Bangalore
562112, India
2 School of Chemistry, University College
Dublin, Belfield, Dublin 4, Ireland
In the present work, a series of eight new imidazole, 4,5–dichloroimidazole,
4,5–diphenylimidazole and benzimidazole based nitro–functionalized mono–N–
heterocyclic carbene (NHC)–silver(I) acetate (7a–d) and bis–NHC–silver(I)
hexafluorophosphate complexes (8a–d) were synthesised by the reaction of the
corresponding azolium hexafluorophosphate salts (6a–d) with silver(I) acetate
and silver(I) oxide in methanol and acetonitrile, respectively. All the synthesised
Correspondence
Dr. Siddappa A. Patil, Centre for Nano and
Material Sciences, Jain University, Jain
compounds were fully characterized by various spectroscopic techniques and
Global Campus, Kanakapura, Ramanagaram,
Bangalore 562112, India.
Email: p.siddappa@jainuniversity.ac.in
elemental analyses. Additionally, the structure of bis–(1–benzyl–3–(p–
nitrobenzyl)–4,5–dichloroimidazole–2–ylidene)silver(I) hexafluorophosphate com-
plex (8b) was confirmed by single crystal X–ray diffraction analysis. Preliminary
in vitro antibacterial evaluation was conducted for all the compounds (6a–d), (7a–d),
and (8a–d) by Kirby–Bauer's disc diffusion method followed by the determination of
Minimum Inhibitory Concentration (MIC) from broth macrodilution method against
five standard bacteria; two Gram–positive bacterial strains (Staphylococcus aureus
and Bacillus subtilis) and three Gram–negative bacterial strains (Escherichia coli,
Shigella sonnei, and Salmonella typhi). All the hexafluorophosphate salts (6a–d)
were found inactive against the tested bacterial strains and their corresponding
mono– and bis–NHC–silver(I) complexes (7a–d and 8a–d) exhibited moderate to
high antibacterial activity with MIC value in the range 8–128 μg/mL. In addition,
preliminary in vitro anticancer potential of all the silver(I) complexes (7a–d and
8a–d) was determined against the human derived breast adenocarcinoma cells
(MCF 7) by MTT assay. All the mono– and bis–NHC–silver(I) complexes (7a–d
and 8a–d) orchestrated high anticancer potential with IC50 values ranging from
10.39 to 59.56 nM. In comparison, mono– NHC–silver(I) complexes performed
better than the bis–NHC–silver(I) complexes.
KEYWORDS
antibacterial drugs, anticancer agents, MCF 7, N–heterocyclic carbene, silver, synthesis and characterization
1 | INTRODUCTION
has devised diverse approaches to combat the death dealing
effects of cancer, among which chemotherapy is eminent
and is widely applied. Cisplatin, the most common chemo-
therapeutic agent and its derivative, carboplatin, treat ovarian,
lung, bladder, head and neck cancers.[2,3] In spite of being
Cancer is the second highest cause of death threatening
millions of lives worldwide with an alarming increase in
mortality each year.[1] With this incentive, recent research
Appl Organometal Chem. 2017;e3819.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 15
https://doi.org/10.1002/aoc.3819

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SHAHINI ET AL.
excessively efficient, they pose disturbing side effects[4,5] and
the recent reports of chemo–resistance is of growing
concern.[6] On the other hand, an upswing in drug resistance
in bacterial strains are unsettling, to overcome which is the
dire need of an equally competitive alternate. Among the best
alternatives counted are the NHC–metal complexes that have
been in the foreground since the discovery and isolation of
free NHCs.[7–12] NHC–metal complexes are being explored
for numerous applications like pharmaceutical, catalysis,
luminescence, functional materials, etc. till date[13,14] and
they have been exhibiting favourable results in preclinical
investigations and are attaining prominence for the ease of
synthesis, stability and diversity that can be achieved by
varying the N–heterocyclic core, the substituents, and the
metal centre.[14–17]
Since the first reports of biological importance of
NHC–silver(I) compounds by Youngs et al., researchers
have delved into designing an effective prescription for
cancer as well as bacterial infections utilising silver, owing
to the employment of silver in medicine since antique
times and its low toxicity.[18–22] In addition to the metal
centre, the other components of an effective NHC–metal
complex are the N–heterocyclic core and the N,N′–substit-
uents, whose right combination results in an excellent drug
with precise lipophilicity to penetrate in to an infected cell
and the ability to release the metal ions at a controlled
rate.[23–25] Further, the variety of drug design employed
in NHC‐silver complexes for biological applications has
been thoroughly reviewed[2,26–28] which served as a motiva-
tion to accomplish the present work that examines and
compares the effect of different cores viz., imidazole, 4,5–
dichloroimidazole, 4,5–diphenylimidazole and benzimid-
azole on the antibacterial and anticancer activity of the
corresponding mono– and bis–NHC–silver(I) complexes
against both Gram–positive and Gram–negative bacterial
species and the human derived breast adenocarcinoma cells
(MCF 7), respectively. However, as the N,N′–substituents
play a crucial role in the biological relevance of the com-
plexes, the study involves p–nitrobenzyl substitution on
the aforementioned core molecules as it is well known for
being utilised in the synthesis of medically relevant
molecules ranging from antipyretics and analgesics to
antipsychotic drugs.[29–32] Having the symmetric substitu-
tion previously reported complex with silver(I) acetate,[33]
the current investigation deals with asymmetric compounds
of mono– and bis–NHC–silver(I) complexes. Moreover, a
reported 1–benzyl–3–p–nitrobenzyl benzimidazolium salt
similar to (5d) did not deal with the targeted biological
studies.[34] Therefore, the core of the study is to compare
mono– and bis–NHC–silver(I) complexes, which facilitates
to establish a structure activity relationship.
We hereby report the synthesis, characterization and
preliminary in vitro antibacterial and anticancer activities of
a series of non–symmetrically p–nitrobenzyl–substituted
mono– and bis–NHC–silver(I) complexes.
2 | EXPERIMENTAL PROTOCOL
2.1 | Materials and methods
All the reactions were carried out under aerobic condi-
tions in oven–dried glassware with magnetic stirring.
Imidazole, benzimidazole, 4,5–diphenylimidazole, 4,5–
dichloroimidazole, benzyl bromide, p–nitrobenzyl bromide,
potassium hydroxide, potassium hexafluorophosphate,
silver(I) acetate and silver(I) oxide were procured commer-
cially from Sigma–Aldrich chemical company and were
used without further purification. Heating was accomplished
by either a heating mantle or silicone oil bath. Reactions were
monitored by thin–layer chromatography (TLC) performed on
0.25 mm Merck TLC silica gel plates, using UV light as a
visualizing agent. Concentration under vacuum refers to the
removal of volatile solvent using a rotary evaporator attached
to a dry diaphragm pump (10–15 mm Hg) followed by
pumping to a constant weight with an oil pump (<300 mTorr).
1H NMR spectra were recorded on Bruker 300 MHz or
Bruker AVANCE III 400 MHz spectrometer, and are
reported relative to DMSO–d6 (δ 2.50 ppm). 1H NMR
coupling constants (J) are reported in Hertz (Hz) and multi-
plicities are designated as, s (singlet), d (doublet), t (triplet),
m (multiplet). Proton–decoupled 13C NMR spectra were
recorded at 75 MHz or 100 MHz and reported relative to
DMSO–d6 (δ 39.5 ppm). ATR–IR spectra were recorded on
a Bruker ECO–ATR spectrophotometer in the range
600–4000 cm−1. Elemental analyses were performed using
a PerkinElmer 2400 Series II CHN/S microanalyzer. X–ray
intensity data of 6686 reflections (of which 3027 unique)
were collected at room temperature on a Bruker X8 Proteum
diffractometer equipped with graphite monochromated
CuK\a radiation (k = 1.54178 Å). The crystal used for data
collection was of dimensions 0.30 x 0.29 x 0.27 mm. The
intensities were measured by ϕ and ω scan mode for θ ranges
5.38–64.45. The structure was solved by direct methods
using SHELXS97.[35] Full–matrix least squares refinement
was carried out using SHELXL97.[35] The Ag(1), P(1),
F(1), F(2), F(3), F(4), F(5) and F(6) atoms lie on a special
position, and the occupancy factors are all 0.5. All the
hydrogen atoms bound to carbon atoms were placed at ideal-
ized positions and refined as riding atoms. All non‐hydrogen
atoms were refined anisotropically on F2 by full‐matrix
least–square using all unique data; the goodness of fit on F2
was 1.023. The final refinement cycles converged to an
the
preliminary
biological
activities
of
the
hexafluorophosphate salts (6a–d) and their corresponding
NHC–silver(I) complexes (7a–d and 8a–d). Further, a
comparison can be made on the biological efficacies of

SHAHINI ET AL.
3 of 15
R = 0.0741 and wR (F2) = 0.2037 for the observed data.
Residual electron densities ranged from −2.453 to 2.316 e
Å
article was deposited with the Cambridge Crystallographic
Data Center with the deposition number 1517151. A copy
of the data can be obtained free of charge from the Director,
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44
1223 336–033; e–mail: deposit@ccdc.cam.ac.uk or www.
ccdc.cam.ac.uk/deposit].
3.5 | Synthesis of 1–benzyl–1H–benzimidazole
(3d)
−3. Crystallographic data of the structure reported in this
Compound (3d) was synthesized from benzimidazole (1d)
(0.11 g, 1.0 mmol), according to the general procedure. Pale
yellow solid. Yield: 88.35% (0.18 g).
3.6 | General experimental procedure for the
synthesis of 1–benzyl–3–(4–nitrobenzyl)–
azolium bromides (5a–d)
A mixture of 1–benzyl azole (1.0 mmol) and p–nitrobenzyl
bromide (4) (0.21 g, 1.0 mmol) in dioxane (30 mL) was stirred
at100°Cfor24h.Thenthereactionmixturewascooledtoroom
temperature. The precipitate was filtered, washed with fresh
dioxane (30 mL) and dried under vacuum to get pure 5a–d.
3 | SYNTHESIS
3.1 | General experimental procedure for the
synthesis of 1–benzyl azoles (3a–d)
1–Benzyl azoles were synthesized according to the liter-
ature procedure with slight modifications.[36] A mixture
of imidazole/4,5–dichloroimidazole/4,5–diphenylimidazole/
benzimidazole (1 mmol) and potassium hydroxide (0.08 g,
1.5 mmol) were stirred at 100 °C for 2 h in minimum amount
of DMSO. The temperature of the reaction mixture was
reduced to 40 °C and then benzyl bromide (2) (0.11 mL,
1.0 mmol) was added in one portion, and stirring was contin-
ued for further 2 h. Later, the reaction mixture was cooled to
room temperature and ice cold water was added. The
precipitate was separated by decantation and washed with
water (3 x 10 mL). The product was then extracted using
chloroform and reduced under vacuum to obtain pure (3a–d).
3.7 | Synthesis of 1–benzyl–3–(4–nitrobenzyl)
imidazolium bromide (5a)
Compound (5a) was synthesized from 1–benzyl–1H–
imidazole (3a) (0.15 g, 1.0 mmol), according to the general
procedure. Pale yellow viscous liquid. Yield: 90.12% (0.33 g).
3.8 | Synthesis of 1–benzyl–3–(4–nitrobenzyl)–
4,5–dichloroimidazolium bromide (5b)
Compound (5b) was synthesized from 1–benzyl–4,5–
dichloro–1H–imidazole (3b) (0.22 g, 1.0 mmol), according
to the general procedure. White solid. Yield: 72.58% (0.32 g).
3.9 | Synthesis of 1–benzyl–3–(4–nitrobenzyl)–
4,5–diphenylimidazolium bromide (5c)
Compound (5c) was synthesized from 1–benzyl–4,5–
diphenyl–1H–imidazole (3c) (0.31 g, 1.0 mmol), according
to the general procedure. White solid. Yield: 73.48% (0.38 g).
3.2 | Synthesis of 1–benzyl–1H–imidazole (3a)
Compound (3a) was synthesized from imidazole (1a)
(0.06 g, 1.0 mmol), according to the general procedure. Pale
yellow solid. Yield: 89.92% (0.14 g).
3.10 | Synthesis of 1–benzyl–3–(4–nitrobenzyl)
3.3 | Synthesis of 1–benzyl–4,5–dichloro–1H–
benzimidazolium bromide (5d)
imidazole (3b)
Compound (5d) was synthesized from 1–benzyl–1H–
benzimidazole (3d) (0.20 g, 1.0 mmol), according to the
general procedure. White solid. Yield: 83.32% (0.35 g).
Compound (3b) was synthesized from 4,5–dichloroimidazole
(1b) (0.13 g, 1.0 mmol), according to the general procedure.
White solid. Yield: 71.54% (0.16 g).
3.11 | General experimental procedure for the
synthesis of 1–benzyl–3–(4–nitrobenzyl)azolium
hexaflourophosphates (6a–d)
The bromide salts (5a–d) (1.0 mmol) were directly converted
into their hexafluorophosphate counterpart by salt metathesis
reaction using KPF6 (0.27 g, 1.5 mmol) in a mixture of meth-
anol (20 mL) and water (5 mL). The resultant mixture was
3.4 | Synthesis of 1–benzyl–4,5–diphenyl–1H–
imidazole (3c)
Compound (3c) was synthesized from 4,5–diphenylimidazole
(1c) (0.22 g, 1.0 mmol), according to the general procedure.
White solid. Yield: 74.93% (0.23 g).

4 of 15
SHAHINI ET AL.
stirred for 4 h at room temperature and poured into water
(100 mL). The separated precipitate was filtered and washed
with water (100 mL) and dried under vacuum to obtain
(6a–d) in high purity.
DMSO–d6, 400 MHz): 9.04 (s, 1H, NCHN), 7.99
(d, J = 8.4 Hz, 2H, CHNitrobenzyl), 7.38–6.98 (m, 17H,
CHNitrobenzyl + CHBenzyl + CHImid), 5.41 (s, 2H, CH2), 5.27
(s, 2H, CH2). 13C NMR (δ ppm DMSO–d6, 100 MHz, proton
decoupled): 147.7, 141.9, 137.5, 134.6, 132.4, 132.2, 131.2,
131.2, 130.6, 129.5, 129.2, 128.9, 128.4, 125.3, 125.2, 124.1
(CImid + CNitrobenzyl + CBenzyl), 51.12 (CH2), 50.34 (CH2).
ATR–IR (cm−1): 2896.6 (w), 1555.3 (m), 1445.7 (m),
1344.8 (s), 1181.3 (m), 1106.7 (m), 1070.6 (m), 1022.4
(m), 972.3 (m), 849.66 (m), 766.5 (s). Micro Analysis
Calculated for C29H24N3O2PF6: C, 58.9; H, 4.1; N, 7.1%;
Found: C, 60.1; H, 4.3; N, 7.7%.
3.12 | Synthesis of 1–benzyl–3–(4–nitrobenzyl)
imidazolium hexafluorophosphate (6a)
Compound (6a) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)imidazolium bromide (5a) (0.32 g, 1.0 mmol),
according to the general procedure. White solid. Yield:
1
74.51% (0.32 g). H NMR (δ ppm, DMSO–d6, 300 MHz):
9.39 (s, 1H, NCHN), 8.31 (d, J = 8.7 Hz, 2H, CHNitrobenzyl),
7.86 (s, 2H, CHNitrobenzyl), 7.68 (d, J = 8.7 Hz, 2H, CHImid),
7.43 (m, 5H, CHBenzyl), 5.59 (s, 2H, CH2), 5.44 (s, 2H, CH2).
13C NMR (δ ppm DMSO–d6, 75 MHz, proton decoupled):
142.4, 137.2, 135.0, 130.0, 129.5, 129.3, 128.8, 124.5, 123.6
(NCN + CImid + CNitrobenzyl), 52.6 (CH2), 51.6 (CH2). ATR–IR
(cm−1): 2954.4 (w), 1566.7 (m), 1516.8 (s), 1343.0 (s), 1207.3
(m), 1160.4 (m), 1108.3 (m), 1035.9 (m), 955.2 (m), 840.6 (s),
804.9 (s), 737.3 (s), 708.6 (s), 660.0 (m), 629.8 (m). Micro
Analysis Calculated for C17H16N3O2PF6: C, 46.5; H, 3.7; N,
9.6%; Found: C, 46.5; H, 3.7; N, 9.6%.
3.15 | Synthesis of 1–benzyl–3–(4–nitrobenzyl)
benzimidazolium hexafluorophosphate (6d)
Compound (6d) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)benzimidazolium bromide (5d) (0.37 g,
1.0 mmol), according to the general procedure. White solid.
Yield: 81.30% (0.39 g). 1H NMR (δ ppm DMSO–d6,
300 MHz): 10.14 (m, 1H, NCHN), 8.30 − 8.28 (m, 2H,
CHNitrobenzyl), 8.00–7.98 (m, 2H, CHNitrobenzyl), 7.94–7.91
(m, 2H, CHBenzimid), 7.81–7.77 (m, 2H, CHBenzimid), 7.67–
7.62 (m, 5H, CHBenzyl), 5.98 (s, 2H, CH2), 5.82 (s, 2H,
CH2). 13C NMR (δ ppm DMSO–d6, 75 MHz, proton
decoupled): 148.0, 143.6, 141.7, 134.2, 131.5, 131.5, 129.9,
129.4, 129.25, 128.8, 127.4, 127.3, 124.4, 114.5, 114.3
(NCN + CBenzmid + CNitrobenzyl + CBenzyl), 50.62 (CH2),
49.70 (CH2). ATR–IR (cm−1): 2950.8 (w), 1553.8 (m),
1485.4 (m), 1423.2 (m), 1338.9 (s), 1217.0 (m), 1183.8 (m),
1106.3 (m), 1015.8 (m), 854.7 (m), 802.1 (m), 750.4 (s),
705.2 (s). Micro Analysis Calculated for C21H18N3O2PF6: C,
51.5; H, 3.7; N, 8.6%; Found: C, 52.6; H, 4.3; N, 8.9%.
3.13 | Synthesis of 1–benzyl–3–
(4–nitrobenzyl)–4,5–dichloroimidazolium
hexafluorophosphate (6b)
Compound (6b) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)–4,5–dichloroimidazolium
bromide
(5b)
(0.39 g, 1.0 mmol), according to the general procedure.
White solid. Yield: 64.76% (0.32 g). 1H NMR (δ ppm,
DMSO–d6, 300 MHz): 9.68 (NCHN), 8.33 (d, J = 8.7 Hz,
2H, CHNitrobenzyl), 7.73 (d, J = 8.7 Hz, 2H, CHNitrobenzyl),
7.48–7.45 (m, 5H, CHBenzyl), 5.71 (CH2), 5.54 (CH2). 13C
NMR (δ ppm DMSO–d6, 75 MHz, proton decoupled):
148.1, 140.54, 137.6, 132.9, 129.9, 129.4, 219.4, 128.8,
124.4, 119.9, 119.7 (CCl + CBenzyl + CNitrobenzyl), 52.1
(CH2), 51.2 (CH2). ATR–IR (cm−1): 2851.0 (w), 1551.5
(m), 1451.6 (m), 1425.1 (m), 1344.1 (s), 1185.0 (m),
1143.9 (m), 1115.9 (m), 1083.1 (m), 1006.1 (m), 961.8
(m), 931.7 (m), 848.5 (m), 799.8 (s). Micro Analysis
Calculated for C17H14N3O2Cl2PF6: C, 40.2; H, 2.8; N,
8.3%; Found: C, 41.26; H, 3.54; N, 8.39%.
3.16 | General experimental procedure for the
synthesis of (1–benzyl–3–(4–nitrobenzyl)azol–
2–ylidene)silver(I) acetates (7a–d)
A
mixture
of
1–benzyl–3–(4–nitrobenzyl)azolium
hexaflourophosphate (1.0 mmol) and silver(I) acetate
(0.33 g, 2.0 mmol) in methanol (20 mL) was stirred at room
temperature for 24 h under dark in round bottom flask
wrapped with aluminium foil. Then the reaction mixture
was filtered through a Celite bed and the filtrate was reduced
under vacuum to afford an off–white sticky solid. The solid
was washed with excess diethyl ether and dried under
vacuum to obtain pure (7a–d).
3.14 | Synthesis of 1–benzyl–3–
(4–nitrobenzyl)–4,5–diphenylimidazolium
hexafluorophosphate (6c)
3.17 | Synthesis of (1–benzyl–3–
(4–nitrobenzyl)imidazole–2–ylidene)silver(I)
acetate (7a)
Compound (6c) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)–4,5–diphenylimidazolium
bromide
(5c)
(0.48 g, 1.0 mmol), according to the general procedure.
Compound (7a) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)imidazolium hexafluorophosphate (6a) (0.43 g,
White solid. Yield: 68.03% (0.40 g). 1H NMR (δ ppm

SHAHINI ET AL.
5 of 15
1.0 mmol), according to the general procedure. White solid.
Yield: 71.39% (0.33 g). 1H NMR (δ ppm, DMSO–d6,
400 MHz): 8.27 (d, J = 8.4 Hz, 2H, CHNitrobenzyl), 7.83 (s,
2H, CHNitrobenzyl), 7.66 (d, J = 8.8 Hz, 2H, CHImid), 7.422–
7.37 (m, 5H, CBenzyl), 5.58 (s, 2H, CH2), 5.42 (s, 2H, CH2),
1.86 (s, 3H, COCH3). 13C NMR (δ ppm DMSO–d6,
100 MHz, proton decoupled): 180.8 (NCN), 173.1 (C = O),
148.1, 142.3, 137.2, 134.9, 130.0, 129.4, 129.2, 128.8,
124.4, 123.5, 123.5 (CImid + CBenzyl + CNitrobenzyl), 52.6
(CH2), 51.6 (CH2), 21.9 (COCH3). ATR–IR (cm−1): 2950.2
(w), 1604.5 (m), 1566.4 (m), 1515.9 (s), 1447.8 (m),
1398.7 (m), 1342.7 (s), 1160.9 (m), 1108.8 (m), 1030.6
(m), 842.9 (s), 807.6 (s), 712.6 (s), 663.9 (m). Micro Analysis
Calculated for C19H18N3O4Ag: C, 49.6; H, 3.9; N, 9.1%;
Found: C, 50.1; H, 4.6; N, 9.9%.
127.0, 123.9 (CImid + CBenzyl + CNitrobenzyl), 52.9 (CH2), 52.3
(CH2), 22.9 (COCH3). ATR–IR (cm−1): 2913.1 (w), 1620.1
(s), 1561.0 (m), 1519.2 (s), 1445.1 (m), 1375.0 (m), 1339.3
(s), 1216.1 (m), 1160.5 (m), 1105.1 (m), 1074.7 (m), 1014.1
(m), 977.8 (m), 927.8 (m), 847.3 (m), 800.8 (m), 755.3 (m),
729.4 (s). Micro Analysis Calculated for C31H26N3O4Ag: C,
60.8; H, 4.3; N, 6.9%; Found: C, 61.0; H, 4.4; N, 6.6%.
3.20 | Synthesis of (1–benzyl–3–
(4–nitrobenzyl)benzimidazole–2–ylidene)
silver(I) acetate (7d)
Compound (7d) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)benzimidazolium hexafluorophosphate (6d)
(0.48 g, 1.0 mmol), according to the general procedure. White
1
solid. Yield: 71.52% (0.36 g). H NMR (δ ppm DMSO–d6,
400 MHz): 8.23 (m, 2H, CHNitrobenzyl), 8.09–8.04 (m, 2H,
CHNitrobenzyl), 7.87 (d, J = 7.6 Hz, 2H, CHBenzimid), 7.76
(d, J = 8.8 Hz, 2H, CHBenzimid), 7.68–7.55 (m, 3H, CBenzyl),
7.48–7.39 (m, 2H, CBenzyl), 5.96 (s, 2H, CH2), 4.57–4.51
(s, 2H, CH2), 1.70 (s, 3H, COCH3). 13C NMR (δ ppm
DMSO–d6, 100 MHz, proton decoupled): 190.9 (NCN),
172.0 (C = O), 147.5, 144.3, 136.6, 133.9, 133.9, 129.2,
128.5, 128.4, 127.6, 124.8, 124.2, 113.0, 112.6
(CBenzmid + CNitrobenzyl + CBenzyl), 52.4 (CH2), 51.4 (CH2),
22.9 (COCH3). ATR–IR (cm−1): 2847.5 (w), 1652.9 (s),
1519.5 (m), 1514.6 (s), 1481.9 (m), 1441.2 (m), 1395.7 (m),
1342.0 (s), 1263.3 (m), 1203.7 (m), 1175.5 (m), 1106.0 (m),
1081.2 (m), 1021.2 (m), 857.6 (m), 807.6 (m), 743.9 (s),
706.2 (s). Micro Analysis Calculated for C23H20N3O4Ag: C,
54.1; H, 3.9; N, 8.2%; Found: C, 54.9; H, 4.0; N, 9.1%.
3.18 | Synthesis of (1–benzyl–3–
(4–nitrobenzyl)–4,5–dichloroimidazole–2–
ylidene)silver(I) acetate (7b)
Compound (7b) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)–4,5–dichloroimidazolium hexafluorophosphate
(6b) (0.50 g, 1.0 mmol), according to the general procedure.
White solid. Yield: 60.13% (0.31 g). 1H NMR (δ ppm
DMSO–d6, 400 MHz): 8.02 (d, J = 8.8 Hz, 2H, CHNitrobenzyl),
7.36 (d, J = 8.4 Hz, 2H, CHNitrobenzyl), 7.24–7.16 (m, 3H,
CHBenzyl), 7.17 (m, 2H, CHBenzyl), 5.58 (s, 2H, CH2), 5.40
(s, 2H, CH2), 1.62 (s, 3H, OCH3). 13C NMR (δ ppm
DMSO–d6, 100 MHz, proton decoupled): 183.3 (NCN),
171.9 (C = O), 147.6, 143.1, 135.5, 129.2, 128.6, 128.3,
127.4, 124.2, 118.3, 118.0 (CImid + CBenzyl + CNitrobenzyl),
54.0 (CH2), 53.1 (CH2), 22.9 (COCH3). ATR–IR (cm−1):
2845.9 (w), 1652.3(s), 1572.3 (m), 1519.2 (s), 1435.9 (m),
1387.1 (m), 1342.4 (m), 1315.3 (s), 1263.7 (m), 1108.4 (m),
1013.7 (m), 979.5 (m), 836.4 (m), 797.8 (m), 732.5 (s). Micro
Analysis Calculated for C19H16N3O4Cl2Ag: C, 43.1; H, 3.0;
N, 8.3%; Found: C, 41.3; H, 3.5; N, 7.9%.
3.21 | General experimental procedure for the
synthesis of bis–(1–benzyl–3–(4–nitrobenzyl)
azol–2–ylidene)silver(I) hexafluorophosphates
(8a–d)
A
mixture
of
1–benzyl–3–(4–nitrobenzyl)–azolium
hexafluorophosphate (1.0 mmol) and silver(I) oxide (0.27 g,
1.2 mmol) in acetonitrile (20 mL) was stirred at 45 °C for
24 h. Light was eliminated during the reaction by wrapping
the reaction vessel with aluminium foil. Then the reaction
mixture was filtered through a Celite bed and the resulted
filtrate was concentrated to 3 mL under vacuum. The fine
white precipitate was afforded by addition of diethyl ether
(30 mL). The precipitate was filtered, washed with diethyl
ether (30 mL), and dried under vacuum to yield pure (8a–d).
3.19 | Synthesis of (1–benzyl–3–
(4–nitrobenzyl)–4,5–diphenylimidazole–2–
ylidene)silver(I) acetate (7c)
Compound (7c) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)–4,5–diphenylimidazolium hexafluorophosphate
(6c) (0.59 g, 1.0 mmol), according to the general procedure.
White solid. Yield: 68.91% (0.42 g). 1H NMR (δ ppm
DMSO–d6, 400 MHz): 8.12 (d, J = 8.0 Hz, 2H, CHNitrobenzyl),
7.39–7.24 (m, 15H, CHImid + CHBenzyl), 7.126–7.09 (m, 2H,
CHNitrobenzyl), 5.63 (s, 2H, CH2), 5.44 (s, 3H, CH2), 1.63
(s, 3H, OCH3). 13C NMR (δ ppm DMSO–d6, 100 MHz,
proton decoupled): 181.9 (NCN), 171.9 (C = O), 147.2,
144.7, 137.1, 133.0, 132.7, 131.0, 130.9, 129.7, 129.7,
129.2, 129.0, 128.9, 128.3, 128.1, 128.0, 127.6, 127.5,
3.22 | Synthesis of bis–(1–benzyl–3–
(4–nitrobenzyl)imidazole–2–ylidene)silver(I)
hexafluorophosphate complex (8a)
Compound (8a) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)imidazolium hexafluorophosphate (6a) (0.43 g,

6 of 15
SHAHINI ET AL.
1.0 mmol), according to the general procedure. White solid.
Yield: 73.54% (0.61 g). 1H NMR (δ ppm DMSO–d6,
400 MHz): 8.11 (d, J = 8.4 Hz, 2H, CHNitrobenzyl), 7.56 (s,
2H, CHNitrobenzyl), 7.37–7.18 (m, 7H, CHNitrobenzyl + CBenzyl),
5.45 (s, 2H, CH2), 5.30 (s, 2H, CH2). 13C NMR (δ ppm
DMSO–d6, 100 MHz, proton decoupled): 181.0 (NCN),
147.4, 145.1, 137.5, 129.1, 128.6, 128.4, 127.7, 124.2,
123.4, 123.3 (CImid + CBenzyl + CNitrobenzyl), 54.7 (CH2),
53.7 (CH2). ATR–IR (cm−1): 2945.1 (w), 1527.2 (m),
1412.6 (m), 1345.9 (s), 1233.3 (m), 1205.8 (m), 1159.3
(m), 1109.7 (m), 1037.6 (m), 828.8 (s), 725.8 (s). Micro
Analysis Calculated for C34H30N6O4PF6Ag: C, 48.6; H,
3.6; N, 10.0%; Found: C, 48.8; H, 3.8; N, 10.0%.
C58H46N6O4PF6Ag (1143.85): C, 60.9; H, 4.1; N, 7.4%;
Found: C, 61.0; H, 4.2; N, 7.5%.
3.25 | Synthesis of bis–(1–benzyl–3–
(4–nitrobenzyl)benzimidazole–2–ylidene)silver
(I) hexafluorophosphate complex (8d)
Compound (8d) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)benzimidazolium hexafluorophosphate (6d)
(0.48 g, 1.0 mmol), according to the general procedure. White
1
solid. Yield: 78.80% (0.74 g). H NMR (δ ppm DMSO–d6,
400 MHz): 8.09 (d, J = 8.8 Hz, 2H, CHNitrobenzyl), 7.71–7.68
(m, 2H, CHNitrobenzyl), 7.47–7.45 (m, 2H, CHNitrobenzyl),
7.39–7.34 (m, 2H, CHNitrobenzyl), 7.32–7.22 (m, 5H,
CHBenzyl), 5.91 (s, 2H, CH2), 5.75 (s, 2H, CH2). 13C NMR
(δ ppm DMSO–d6, 100 MHz, proton decoupled): 173.7
(NCN), 148.0, 143.9, 143.6, 141.9, 131.6, 130.2, 129.8,
128.5, 127.2, 124.3, 122.8, 122.5, 121.7, 119.8, 114.3,
114.1, 112.5, 110.7 (CBenzmid + CNitrobenzyl + CBenzyl), 49.4
(CH2), 42.7 (CH2). ATR–IR (cm−1): 2830.6 (w), 1551.2
(m), 1435.2 (m), 1339.5 (s), 1181.3 (m), 1109.1 (m), 1032.6
(m), 824.2 (s), 745.8 (s). Micro Analysis Calculated for
C42H34N6O4PF6Ag: C, 53.7; H, 3.7; N, 8.9%; Found: C,
54.0; H, 3.8; N, 9.0%.
3.23 | Synthesis of bis–(1–benzyl–3–(4–
nitrobenzyl)–4,5–dichloroimidazole–2–ylidene)
silver(I) hexafluorophosphate complex (8b)
Compound (8b) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)–4,5–dichloroimidazolium hexafluorophosphate
(6b) (0.50 g, 1.0 mmol), according to the general procedure.
White solid. Yield: 62.39% (0.56 g). 1H NMR (δ ppm
DMSO–d6, 400 MHz): 8.11 (d, J = 8.8 Hz, 2H, CHNitrobenzyl),
7.41 (d, J = 8.8 Hz, 2H, CHNitrobenzyl), 7.31–7.20 (m, 5H,
CHBenzyl), 5.60 (s, 2H, CH2), 5.43 (s, 2H, CH2). 13C NMR
(δ ppm DMSO–d6, 100 MHz, proton decoupled): 183.3
(NCN), 147.5, 143.1, 135.6, 129.2, 128.6, 128.3, 127.4,
124.2, 118.3, 118.0 (CImid + CBenzyl + CNitrobenzyl), 54.0
(CH2), 53.1 (CH2). ATR–IR (cm−1): 2830.8 (w), 1565.1
(m), 1515.9 (s), 1437.2 (m), 1386.8 (m), 1337.5 (s), 1197.7
(m), 1118.4 (m), 822.7 (s), 741.6 (s). Micro Analysis
Calculated for C34H26N6O4Cl2PF6Ag: C, 45.1; H, 2.9; N,
9.3%; Found: C, 45.2; H, 3.1; N, 9.6%.
4 | BIOLOGICAL EVALUATION
4.1 | Antibacterial screening
Preliminary in vitro antibacterial activity of NHC–precursors
(6a–d) and their corresponding mono– and bis–NHC–
silver(I) complexes (7a–d and 8a–d) were evaluated by
Kirby–Bauer's disc diffusion method followed by the
determination of Minimum Inhibitory Concentration from
broth macrodilution method against standard five bacterial
strains Staphylococcus aureus (S. aureus), Bacillus subtilis
(B. subtilis), Escherichia coli (E. coli), Shigella sonnei
(S. sonnei), and Salmonella typhi (S. typhi) with standard
Ampicillin.
3.24 | Synthesis of bis–(1–benzyl–3–(4–
nitrobenzyl)–4,5–diphenylimidazole–2–ylidene)
silver(I) hexafluorophosphate complex (8c)
Compound (8c) was synthesized from 1–benzyl–3–(4–
nitrobenzyl)–4,5–diphenylimidazolium hexafluorophosphate
(6c) (0.27 g, 1.0 mmol), according to the general procedure.
White solid. Yield: 69.33% (0.79 g). 1H NMR (δ ppm
DMSO–d6, 400 MHz): 7.99 (d, J = 8.8 Hz, 2H,
CHNitrobenzyl), 7.31–7.11 (m, 15H, CHImid + CHBenzyl),
6.90–6.88 (m, 2H, CHNitrobenzyl), 5.40 (s, 2H, CH2), 5.26
(s, 2H, CH2). 13C NMR (δ ppm DMSO–d6, 100 MHz, proton
decoupled): 151.9 (NCN), 149.5, 141.8, 137.7, 137.4, 135.7,
135.7, 134.4, 133.7, 132.8, 132.8, 132.4, 132.2, 131.8, 128.7
(CImid + CBenzyl + CNitrobenzyl), 57.6 (CH2), 57.0 (CH2).
ATR–IR (cm−1): 2905.7 (w), 1523.5 (m), 1342.5 (s),
1179.0 (m), 1108.5 (m), 1074.3 (m), 1019.0 (m), 831.5 (s),
765.3 (s), 730.1 (s). Micro Analysis Calculated for
4.2 | Kirby–Bauer's disc diffusion method
The bacterial strains were individually cultured from a single
colony in sterile Muller–Hinton agar medium overnight at
37 °C. Then, a single colony was picked and introduced to
nutrient broth and the absorbance was adjusted to McFarland
standard 0.5. For each strain, 100 μL of culture were spread
evenly on nutrient agar medium. Five 6 mm diametre
Whatman paper discs were placed evenly separated on each
plate. Stock solution of every compound was prepared at
800.0 μg/mL in DMSO. Each plate was tested with control,
3.0 μL, 6.0 μL, 9.0 μL and 12.0 μL of stock solution. The
plates were covered and placed in an incubator at 37 °C for

SHAHINI ET AL.
7 of 15
24 h. The plates were then removed and the diameter of zone
of inhibition for each sample was measured in millimetres
against the standard Amphicillin.
precursors (6a–d) and their corresponding silver(I) com-
plexes (7a–d and 8a–d) depicted in the present work is
outlined in Schemes 1 and 2, respectively. 1–Benzyl azoles
(3a–d) were prepared by stirring imidazoles [imidazole
(1a), 4,5–dichloroimidazole (1b), 4,5–diphenylimidazole
(1c) and benzimidazole (1d)] with one equivalent of benzyl
bromide (2) in the presence of potassium hydroxide as a
base in DMSO at 100–110 °C for 4 h. The bromide salts
(5a–d) were prepared by reacting one equivalent of p–
nitrobenzyl bromide (4) with 1–benzyl azoles [1–benzyl–
1H–imidazole (3a), 1–benzyl–4,5–dichloro–1H–imidazole
(3b), 1–benzyl–4,5–diphenyl–1H–imidazole (3c), and 1–
benzyl–1H–benzimidazole (3d)] in dioxane at 100 °C for
24 h, with variable yields between 71.54%–89.92%. All the
hexafluorophoaphate salts (6a–d) were prepared by the
reaction of their corresponding bromide salts (5a–d) with
KPF6 in a mixture of methanol and water at room
temperature for 4 h as an air and moisture stable white
powders, with moderate to good yields (64.76%–81.30%).
The prepared NHC hexafluorophosphate salts (6a–d) were
easily soluble in organic solvents such as acetone,
acetonitrile, DMF and DMSO and insoluble in common
organic solvents such as pentane, hexane, toluene, and
diethyl ether. Structures of all the newly synthesized
hexafluorophosphate salts (6a–d) were confirmed by 1H,
13C NMR and ATR–IR spectroscopic techniques and
elemental analysis.
4.3 | Determination of MIC by broth
macrodilution method
A series of concentrations ranging from 1.0 μg/mL to
256.0 μg/mL of all the test compounds were obtained by
serial dilution. Then, 1.0 mL of bacterial suspension was
added to the series after adjusting the absorbance to
McFarland standard 0.5 to derive a concentration range of
0.5–128.0 μg/mL. The sample tubes, along with the control
and standard tubes were incubated at 37 °C for 16 h. MIC
was determined visually as the lowest concentration of the
test compound that inhibited bacterial growth.
4.4 | Anticancer screening
Preliminary in vitro anticancer potential of mono– and bis–
NHC–silver(I) complexes (7a–d and 8a–d) was performed
on human breast adenocarcinoma (MCF–7) cell line (pro-
cured from NCCS, Pune). The cell line was maintained in
96 wells micro titre plate containing Minimum Essential
Medium (MEM) supplemented with 10% heat inactivated
fetal calf serum (FCS) containing 5% of mixture of
Gentamicin (10.0 μg), Penicillin (100 units/mL) and
Streptomycin (100.0 μg/mL) in the presence of 5% CO2 at
37 °C for 48–72 h. Preliminary in vitro growth inhibition
effect of test compounds was assessed by calorimetric or
spectrophotometric determination of conversion of MTT into
Formazan blue by living cells. The supernatant from the plate
was removed and added fresh MEM solution followed by
treatment with different concentrations of the compound
appropriately diluted with DMSO to respective wells
containing 100.0 μL of the medium in order to obtain final
concentrations of 10.0, 20.0, 25.0, 30.0 and 50.0 μg/mL.
Control group contained only DMSO. After 48 h of incuba-
tion at 37 °C in a humidified atmosphere of 5% CO2, stock
solution of MTT was added to each well (20.0 μL, 5.0 mg/mL
in sterile PBS) for further incubation of 4 h. The supernatant
was then carefully aspirated and the precipitated crystals of
Formazan blue were solubilised by adding DMSO
(100.0 μL) and optical density was measured at wavelength
of 570 nm by using LISA plus. The results represent the
mean of five readings.
The 1H NMR spectra of all the newly synthesised
hexafluorophosphate salts (6a–d) displayed a characteristic
downfield resonance in the range δ = 9.04–10.14 ppm
attributed to the resonance of NCHN protons indicated the
successful formation of desired hexafluorophosphate salts
(6a–d). The spectra also evidenced the presence of a set of
peaks in the range δ = 8.33–6.98 ppm ascribed to the reso-
nanceofaromaticprotons(CHAzole +CHBenzyl+CHNitrobenzyl).
In addition, two singlet peaks were observed in the range
δ = 5.98–5.27 ppm which are due to the methylene (CH2)
protons of p–nitrobenzyl and benzyl module attached to the
nitrogen atoms of the N–heterocyclic core. The 13C NMR
spectra of all the hexafluorophosphate salts (6a–d) revealed
the presence of peaks in the range δ = 148.18–114.38 ppm
attributed to the aromatic carbon atoms (NCN,
CAzole + CBenzyl + CNitrobenzyl). Also, two peaks were
observed in the range δ = 52.66–49.70 ppm ascribed to the
methylene (CH2) carbon atoms of p–nitrobenzyl and benzyl
module attached to the nitrogen atoms of the N–heterocyclic
core. In the ATR–IR spectra of hexafluorophosphate salts
(6a–d), stretching vibrational bands of nitro functionality
was observed as two medium intensity bands at ca.
5 | RESULTS AND DISCUSSION
5.1 | Synthesis and characterization
1566–1551 and 1344–1338 cm−1 [33,34]
Further, the ATR–
.
IR spectra of (6a–d) displayed a sharp band of medium
intensity in the range 2954–2851 cm−1, attributed to the
aromatic ν(C–H) stretching vibrations. In addition, the results
The synthetic route for novel non–symmetrically p–
nitrobenzyl–substituted N–heterocyclic carbenes as ligand

8 of 15
SHAHINI ET AL.
SCHEME 1 General reaction scheme for the synthesis of nitro–functionalized NHC precursors (6a–d) and their corresponding mono–NHC–
silver(I) acetate complexes (7a–d)
SCHEME 2 General reaction scheme for the synthesis of nitro–functionalized NHC precursors (6a–d) and their corresponding bis–NHC–
silver(I) complexes (8a–d)
obtained from the elemental analysis are in good agreement
with the proposed structures of the hexafluorophosphate salts
(6a–d).
The complete disappearance of a downfield NCHN signal
and appearance of new signals at δ = 1.62 to 1.86 ppm for the
1
acetate (O2CCH3) protons in all the H NMR spectra for
The mono–NHC–silver(I) acetate complexes (7a–d)
were synthesized by the reaction of the hexafluorophosphate
salts (6a–d) with two equivalents of silver(I) acetate in
methanol. The reaction mixture was stirred for 24 h at room
temperature in dark to afford the mono NHC–silver(I)
acetate complexes (7a–d) as off white solids in 60.13 to
71.52% yield. All the newly synthesized mono NHC–
(7a–d), indicates a successful NHC–silver(I) acetate complex
formation. The 13C NMR resonances of the carbene carbon
atoms in complexes (7a–d) observed in the range
δ = 180.82–190.92 ppm. These resonances are shifted to
downfield region compared to their corresponding
hexafluorophosphate salts (6a–d) carbene carbons resonance
at the range 148.1–142.4 ppm, which further reveals the
formation of expected mono–NHC–silver(I) acetate
complexes. Furthermore, the presence of the 13C NMR reso-
nances for the carbonyl and methyl carbons of the acetate
1
silver(I) acetate complexes were fully characterized by H,
13C NMR, ATR–IR spectroscopic techniques and elemental
analyses.

SHAHINI ET AL.
9 of 15
group of NHC–silver(I) acetate complexes (7a–d) in the
range δ = 171.9–173.1 and 21.9–22.9 ppm, respectively
demonstrated the formation of the targeted mono–NHC–
silver(I) acetate complexes. The ATR–IR spectra of all
mono–NHC–silver(I) acetate complexes (7a–d) exhibit
strong bands in the region 1604–1652 and 1263–1160 cm−1
respectively, attributed to the acetate ν(C = O) and ν(C–O)
stretching vibrations, which further confirms the successful
NHC–silver(I) acetate complex formation. Medium intensi-
ties stretching vibrational bands in the region 1566–1519
and 1342–1339 cm−1, indicate the presence of nitro moiety.
Further, the ATR–IR spectra of all the mono–NHC–silver(I)
acetate complexes (7a–d) displayed one sharp band of
medium intensity around 2950–2845 cm−1, attributed to the
aromatic ν(C–H) stretching vibrations. Additionally, elemen-
tal analysis results are in good agreement with the proposed
structures of the mono–NHC–silver(I) acetate complexes
(7a–d).
5.2 | Structural discussion
Suitable single crystals for X‐ray diffraction analysis of
bis‐NHC‐silver(I) complex (8b) were grown in saturated ace-
tonitrile solution. A perspective view of compound (8b) is
depicted in Figure 1. The crystal data and details concerning
data collection and structure refinement are given in Table 1
and bond distances and bond angles are given inTable 2. Com-
pound (8b) crystallized in the triclinic space group P‐1 with
two motifs in a unit cell. The crystal structure consists of
cation [NHC‐Ag‐NHC]+ and PF6 anion. Consequently, the
−
charge on complex (8b) is neutralised by the presence of PF6
anion in the crystal lattice. Further, compound (8b) does not
possess any lattice held water molecules or organic solvent
molecules in the unit cell of the determined structure. The
X‐ray structure of (8b) revealed that the molecule is nonpla-
nar in nature and the metal centre lies on the inversion centre.
The silver(I) ion is coordinated to two carbene carbons of the
4,5‐dichloroimidazolium rings in an anti–arrangement, in a
perfect linear fashion with C(1)‐Ag(1)‐C(1′) (′ is reported
by the symmetry operation ‐x, −y, −z) bond angle of 180°
probably indicating that the steric bulk offered by the ligand
does not hinder coordination geometry around the silver(I)
centre while the bond distance between Ag(1)‐C(1) is 2.093
(5) Å. Also, the bond distances and bond angles in the five‐
membered ring (NCNCC) compare well with those found
in similar compounds bis‐[1‐benzyl‐3‐(4‐nitrobenzyl)
benzimidazolium]‐silver(I) tetrafluoroborate and 1‐hexyl‐3‐
methyl‐4,5‐dichloroimidazolium iodide.[34,37] The C‐C bond
distances in aromatic rings are in the normal range of
1.328(8)‐1.510(7) Å, which is characteristic of delocalized
aromatic rings. The C‐C‐C bond angles in aromatic rings
are close to 120°, it suggests that carbon atoms are sp2
hybridized. The NO2 [N(3) O(1) and N(3) O(2)] group
On
the
other
hand,
the
bis–NHC–silver(I)
hexafluorophosphate complexes (8a–d) were synthesized by
treating the hexafluorophosphate salts (6a–d) with one
equivalent of silver(I) oxide in acetonitrile at 45 °C for
24 h. All the newly synthesized bis–NHC–silver(I) hexafluo-
rophosphate complexes (8a–d) were obtained in good
yields ranging from 62.39 to 78.80%, and were character-
ized by 1H, 13C NMR, ATR–IR spectroscopic methods
and elemental analyses.
1
The absence of a downfield NCHN signal in all the H
NMR spectra for (8a–d), indicates a successful bis–NHC–
silver(I) hexafluorophosphate complex formation.[33,34]
However, the resonances of aromatic and methylene
protons of NHC–silver(I) complexes (8a–d) were observed
with no or a negligible change with their respective
hexafluorophosphate salts (6a–d). The 13C NMR reso-
nances of the carbene carbon atoms in complexes (8a–d)
occur in the range δ = 183.3–151.9 ppm, respectively.
These signals are shifted downfield compared to the
corresponding precursors of (6a–d) carbene carbons reso-
nance at δ = 148.1–142.4 ppm respectively, which further
supports the formation of expected bis–NHC–silver(I)
hexafluorophosphate complexes (8a–d). In the ATR–IR
spectra
of
bis–NHC–silver(I)
hexafluorophosphate
complexes (8a–d), stretching vibrational bands of nitro
moiety was observed as a medium intensities bands at
1565–1523 and 1345–1337 cm−1. Further, all the bis–
NHC–silver(I) complexes (8a–d) displayed one sharp band
of medium intensity around 2945–2830 cm−1, attributed to
the aromatic ν(C–H) stretching vibrations. These spectral
assignments are consistent with the similar reported
compounds available in the literature.[34] In addition, the
proposed structures of all the bis–NHC–silver(I) complexes
(8a–d) were further confirmed from the elemental analysis
results.
FIGURE 1 X‐ray diffraction structure of compound (8b); thermal
ellipsoids are drawn on the 50% probability level. Hydrogen atoms
and hexafluorophosphate counterion are excluded for clarity

10 of 15
SHAHINI ET AL.
TABLE 1 The crystal data and structure refinement data of com-
pound (8b)
TABLE 2 Selected bond lengths (Å) and bond angles (°) of com-
pound (8b)
Identification code
8b
Bond lengths
[Å]
Bond angle
[°]
Empirical formula
C17 H13 Ag0.50 Cl2 F3
N3 O2 P0.50
Ag(1)–C(1)
N(1)–C(1)
N(2)–C(1)
N(1)–C(2)
N(2)–C(3)
C(2)–C(3)
Cl(1)–C(2)
Cl(2)–C(3)
N(1)–C(4)
N(2)–C(11)
N(3)–O(1)
N(3)–O(2)
2.093 (5)
1.365 (7)
1.361(6)
1.392(6)
1.388 (7)
1.328(8)
1.703(5)
1.704(5)
1.461(7)
1.461(7)
1.229(6)
1.235(6)
C(1)–ag(1)–C(1′)a
N(1)–C(1)–ag(1)
N(2)–C(1)–ag(1)
N(1)–C(1)–N(2)
C(1)–N(1)–C(2)
C(3)–C(2)–N(1)
C(2)–C(3)–N(2)
C(1)–N(2)–C(3)
N(1)–C(2)–Cl(1)
C(3)–C(2)–Cl(1)
C(2)–C(3)–Cl(2)
N(2)–C(3)–Cl(2)
180.00
127.1 (4)
127.5 (4)
105.0 (4)
109.8 (4)
107.5 (5)
107.4 (5)
110.2 (4)
122.4 (4)
130.1 (4)
129.1 (4)
123.5 (4)
Formula weight
Temperature (K)
Wavelength
Crystal system
Space group
Unit cell dimensions
a (Å)
488.62
296(2)
1.54178
Triclinic
P–1
8.0304(4)
9.7449(6)
12.3369(7)
78.110(2)
85.095(2)
78.871(2)
925.90(9)
2
b (Å)
c (Å)
α (°)
β (°)
ais reported by the symmetry operation ‐x,‐ y,‐ z
γ (°)
Volume (Å3)
[C(4) C(5) C(6) C(7) C(8) C(9), and C(10)], whereas the
plane II forms a dihedral angle of 65.44° with plane III.
However, the C(1)‐Ag(1)‐C(1′) plane is almost perpendicu-
lar to the plane of the aryl rings. The molecular packing
of (8b) reveals a number of intermolecular and intramolecu-
lar hydrogen bond interactions where the fluoride of the
hexafluorophosphate anion participates in a strong interac-
tion with the silver(I) centre with an interaction distance of
3.07 Å which is followed in a diagonal pattern that results
in a step‐like arrangement (Figure 2). Additionally, the
fluoride atoms are involved in strong interactions with N
(2), C(1) and C(17) with interaction distances of 2.98,
2.97 and 2.99 Å respectively along with strong intramolecu-
lar hydrogen bonds between F(1) and H(6), F(2) and H(6),
and F(2) and H(17) at distances of 2.52, 2.50 and 2.27 Å
respectively.
Z
Density(calcd) (g/cm3)
Absorption coefficient (mm−1
F000
1.753
)
8.139
488
Crystal size
θ range for data collection
Index ranges
0.30 x 0.29 x 0.27
5.38–64.45o
–8 ≤ h ≤ 9, −11 ≤ k ≤ 11,
−14 ≤ l ≤ 14
Reflections collected
Independent reflections
Refinement method
6686
3027[R(int) = 0.0518]
Full–matrix least–squares
on F2
Completeness to θmax
Max. And min. Transmission
Data/ restrains/parameters
Radiation (Å)
97.0%
0.165 and 0.111
3027/0/256
5.3 | Pharmacological screening
1.54178
Owing to the employment of nitro–aromatic compounds
in the synthesis of effective pharmaceutical agents such
as analgesics, antipyretics and antipsychotic drugs,[29–32]
p–nitrobenzyl substituents were investigated as N–
substitutents in different azolium compounds for antibacterial
and anticancer properties against both Gram–positive and
Gram–negative bacterial strains as well as human breast
adenocarcinoma cell line (MCF 7).
θ min, max (°)
5.38–64.45
Goodness–of–fit on F2
Final R indices [1 > 2σ(1)]
R indices (all data)
1.039
R = 0.0741, wR = 0.2040
R = 0.0741, wR = 0.2037
2.316 and −2.453
Largest diff. Peak
and hole (e Å−3
)
present in the complex is trans to methylene group. The
compound (8b) lies in three planes with plane I [C(1) N
(2) N(1) C(2) C(3) Cl(2) Cl(1), and Ag(1)] making a dihe-
dral angle of 80.39° and 78.65° with plane II [C(11) C(17)
C(12) C(13) C(14) C(15) C(16), and N(3)], and plane III
5.4 | Antibacterial activity
With the freshly prepared compounds on hand, we then
proceeded to examine their antibacterial activity from
Kirby–Bauer's disc diffusion method and MIC was

SHAHINI ET AL.
11 of 15
FIGURE 2 Interaction of the hexafluorophosphate anion with the metal centre. Hydrogen atoms are excluded for clarity
determined by broth macrodilution method towards
S. aureus, B. subtilis, E. coli, S. sonnei and S. typhi. The
solvent (DMSO) played no role in the inhibition of bacterial
growth as reported earlier.[38,39] The outcome of antibacterial
activities of hexafluorophosphate salts (6a–d) and their cor-
responding mono–NHC–silver(I) acetate (7a–d) and bis–
NHC–silver(I) hexafluorophosphate complexes (8a–d) are
depicted in Figures 3–7. The results are tabulated in Table 3.
In general the hexafluorophosphate salts (6a–d) showed
no antibacterial activity. On the other hand, mono– and bis–
NHC–silver(I) complexes (7a–d and 8a–d) exhibited promis-
ing antibacterial activity. Bis–NHC–silver(I) complex (8c)
orchestrated impressive antibacterial potential against E. coli
at a concentration of 8 μg/mL. Similarly, mono–NHC–
silver(I) complex (7d) displayed a high activity against
S. aureus and E. coli at 8 μg/mL. Overall, mono– and bis–
NHC–silver(I) complexes (7a–d and 8a–d) showed high
FIGURE 5 Area of clearance on E. coli by (6a–d), (7a–d), and
(8a–d)
FIGURE 6 Area of clearance on S. sonnei by (6a–d), (7a–d), and
(8a–d)
FIGURE 3 Area of clearance on S. aureus by (6a–d), (7a–d), and
(8a–d)
FIGURE 7 Area of clearance on S. typhi by (6a–d), (7a–d), and
(8a–d)
antibacterial activity against S. aureus, good to moderate
activity against E. coli and S. sonnei and moderate to
poor activity against B. subtilis and S. typhi. Though
the results obtained by Kirby Bauer's disc diffusion
FIGURE 4 Area of clearance on B. subtilis by (6a–d), (7a–d), and
(8a–d)

12 of 15
SHAHINI ET AL.
TABLE 3 Preliminary in vitro antimicrobial activity data of compounds (6a–d), (7a–d), and (8a–d) towards gram–positive and gram–negative
bacterial strainsa
Gram–positive bacteria
S. aureus B. subtilis
Gram–negative bacteria
Test
volume
(μL)
E. coli
S. Sonnei
S. Typhi
Compounds (#)
ZOI
MIC
ZOI
MIC
ZOI
MIC
ZOI
MIC
ZOI
MIC
6a
3
6
9
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
>128
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
>128
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
>128
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
>128
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
>128
12
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
3
6
9
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
10 Æ 1
12 Æ 1
14 Æ 1
16 Æ 1
13 Æ 1
17 Æ 1
20 Æ 1
20 Æ 1
19 Æ 1
24 Æ 1
26 Æ 1
27 Æ 1
12 Æ 1
16 Æ 1
16 Æ 1
19 Æ 1
8 Æ 1
10 Æ 1
13 Æ 1
15 Æ 1
>128
>128
>128
16
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
>128
>128
>128
128
128
64
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
>128
>128
>128
32
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
9 Æ 1
10 Æ 1
10 Æ 1
11 Æ 1
9 Æ 1
10 Æ 1
10 Æ 1
11 Æ 1
10 Æ 1
11 Æ 1
12 Æ 1
14 Æ 1
>128
>128
>128
64
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
6 Æ 1
>128
>128
>128
128
128
64
12
3
6
9
12
3
6
9
12
3
6
9
9 Æ 1
8 Æ 1
7 Æ 1
9 Æ 1
9 Æ 1
9 Æ 1
11 Æ 1
12 Æ 1
13 Æ 1
15 Æ 1
9 Æ 1
12
10 Æ 1
3
6
9
16
12 Æ 1
13 Æ 1
14 Æ 1
17 Æ 1
11 Æ 1
13 Æ 1
13 Æ 1
15 Æ 1
11 Æ 1
12 Æ 1
14 Æ 1
16 Æ 1
16 Æ 1
19 Æ 1
20 Æ 1
22 Æ 1
11 Æ 1
12 Æ 1
14 Æ 1
14 Æ 1
15 Æ 1
19 Æ 1
21 Æ 1
22 Æ 1
16
128
64
11 Æ 1
12 Æ 1
14 Æ 1
15 Æ 1
12 Æ 1
12 Æ 1
13 Æ 1
15 Æ 1
11 Æ 1
11 Æ 1
12 Æ 1
13 Æ 1
12
3
6
9
16
16
12
3
6
9
8
128
128
64
8
8 Æ 1
128
64
128
128
128
64
8 Æ 1
9 Æ 1
12
10 Æ 1
3
6
9
16
9 Æ 1
9 Æ 1
64
11 Æ 1
12 Æ 1
13 Æ 1
13 Æ 1
9 Æ 1
10 Æ 1
11 Æ 1
13 Æ 1
10 Æ 1
10 Æ 1
11 Æ 1
13 Æ 1
11 Æ 1
13 Æ 1
14 Æ 1
8 Æ 1
9 Æ 1
9 Æ 1
8 Æ 1
10 Æ 1
11 Æ 1
10 Æ 1
13 Æ 1
10 Æ 1
12 Æ 1
12
3
6
9
15 Æ 1
17 Æ 1
18 Æ 1
19 Æ 1
14 Æ 1
16 Æ 1
17 Æ 1
18 Æ 1
16
11 Æ 1
13 Æ 1
13 Æ 1
15 Æ 1
12 Æ 1
15 Æ 1
16 Æ 1
17 Æ 1
17 Æ 1
18 Æ 1
19 Æ 1
21 Æ 1
16 Æ 1
19 Æ 1
21 Æ 1
24 Æ 1
16
64
9 Æ 1
11 Æ 1
12 Æ 1
15 Æ 1
11 Æ 1
12 Æ 1
13 Æ 1
16 Æ 1
12
3
6
9
16
64
8
64
12
3
6
9
11 Æ 1
15 Æ 1
16 Æ 1
16
10 Æ 1
11 Æ 1
12 Æ 1
128
10 Æ 1
12 Æ 1
14 Æ 1
64
64
11 Æ 1
12 Æ 1
14 Æ 1
128
(Continues)

SHAHINI ET AL.
13 of 15
TABLE 3 (Continued)
Gram–positive bacteria
S. aureus B. subtilis
Gram–negative bacteria
Test
volume
E. coli
S. Sonnei
S. Typhi
Compounds (#)
(μL)
ZOI
MIC
ZOI
MIC
ZOI
MIC
ZOI
MIC
ZOI
MIC
12
17 Æ 1
13 Æ 1
15 Æ 1
16 Æ 1
15 Æ 1
Ampicillin
3
6
9
23 Æ 1
24 Æ 1
25 Æ 1
27 Æ 1
<0.5
CI
CI
CI
CI
<0.5
16 Æ 1
19 Æ 1
21 Æ 1
24 Æ 1
<0.5
CI
CI
CI
CI
<0.5
CI
CI
CI
CI
<0.5
12
aZOI: Zone of inhibition (mm); MIC: Minimal bacterial inhibitory concentration (μg/mL); CI: Complete inhibition.
method is comparable with that of MIC values, slight
deviations could be attributed to the nature of medium
employed for the purpose, where the compounds diffuse
through an agar based medium in Kirby Bauer's disc
diffusion method, whereas the components are easily
available in a broth medium during MIC determination.
Also, an evident amount of difference could not be
drawn between the activity of mono–NHC–silver(I)
acetate complexes (7a–d) and bis–NHC–silver(I)
hexafluorophosphate complexes (8a–d), which reflects
that more number of NHC ligands do not add to the
targeted biological activity.
Comparing the overall activity of the N–heterocyclic cores
in the corresponding complexes, 4,5–diphenylimidazole
based complexes exhibit higher activity than benzimid-
azole based complexes that were better than imidazole
and 4,5–dichloroimidazole based complexes that are
comparable. It is also noteworthy that the difference in
activity brought about by different N–heterocyclic cores
is not bold.
FIGURE 8 Cytotoxicity curves from typical MTT assays showing
the effect of compounds (7a–d) on the cell proliferation of MCF 7 cells
5.5 | Anticancer activity
The anticancer study of the hexafluorophosphate salts (6a–d)
was excluded as the focus of the study was on the effective-
ness of silver upon complexation with the rationally designed
NHC ligands. Hence, anticancer potential of the mono– and
bis–NHC–silver(I) complexes (7a–d and 8a–d) was evalu-
ated by MTT assay against human derived breast adenocarci-
noma cell line (MCF 7). The inhibition of cell proliferation
along varying concentrations of the mono– and bis–NHC–
silver(I) complexes (7a–d and 8a–d) is depicted in Figures 8
and 9. The compounds displayed excellent activity with IC50
values at nanomolar level. Bis–NHC silver(I) complex (8b)
exhibited a high anticancer potential with an IC50 value of
10.39 nM, whereas complexes (7a–d) presented with appre-
ciable IC50 values ranging from 13.28 to 16.74 nM. While
complexes (8c and 8d) illustrated IC50 values of 33.18 and
36.56 nM respectively, that of (8a) exceeded the tested range.
On the whole, mono–NHC complexes surfaced as better
FIGURE 9 Cytotoxicity curves from typical MTT assays showing
the effect of compounds (8a–d) on the cell proliferation of MCF 7 cells
candidates than the bis–NHC analogues, with an exception
of (8b). Hence, it can be deduced that possession of two
NHC based ligands do not result in a better drug candidate

14 of 15
SHAHINI ET AL.
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6 | CONCLUSION AND OUTLOOK
Several new non–symmetrically p–nitrobenzyl–substituted
mono–NHC–silver(I) acetate and bis–NHC–silver(I)
hexafluorophosphate derivatives (7a–d and 8a–d) were
synthesised through the reaction of corresponding non–
symmetrically p–nitrobenzyl–substituted azolium salts
(6a–d) with silver(I) acetate and silver(I) oxide, and appro-
priately characterized using various spectroscopic
techniques as well as elemental analysis. Additionally, the
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tions. The compound (7d) exhibited high antibacterial
activity towards S. aureus and E. coli, whereas (8c) was
equally effective against E. coli, at 8 μg/mL. Furthermore,
all the NHC–silver(I) complexes (7a–d and 8a–d) demon-
strated exceptional anticancer activity with IC50 values at
nano molar range. While the bis–NHC–silver(I) complex
(8b) demonstrated the best anticancer potential with an
IC50 value of 10.39 nM, other complexes (7a–d, 8c–d)
exhibited proficient activity with IC50 values ranging
from 13.28 to 36.56 nM against human breast adenocar-
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bacterial activity between mono and bis–NHC–silver(I)
complexes (7a–d and 8a–d) was not pronounced,
whereas the former (7a–d) possessed better anticancer
potential than the latter (8a, 8c and 8d). Therefore, the
outcomes obtained from this study could afford valuable
hints for the design and discovery of new NHC–silver
(I) based antimicrobial and anticancer agents.
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