Received: 1 June 2022 Revised: 30 June 2022 Accepted: 4 July 2022

DOI: 10.1002/aoc.6811

RESEARCH ARTICLE

Synthesis and biological evaluation of Au-NHC complexes

Orhan Ekinci1 | Mitat Akkoç2 | Siraj Khan3 | Sedat Yaşar1 |

Canbolat Gürses4 | Samir Noma1,5
lu1,6
| Sevgi Balcıog | Betül Sen7 |
Muhittin Aygün 7 _
| Ismet Yılmaz1

1
Faculty of Arts and Science, Department
_
of Chemistry, Inönü University, Malatya, New seven Au-N-heterocyclic carbene (NHC) complexes have been synthe-
Turkey sized via transmetalation from Ag-NHC complexes. NHC salts, Ag-NHC, and
2
Hekimhan Vocational College, Au-NHC complexes were fully characterized by widely used spectroscopic
Department of Property Protection and
Security, Malatya Turgut Özal University,
techniques. The molecular and crystal structures of 3b and 3f Au-NHC com-
Malatya, Turkey plexes were clarified through the single-crystal X-ray diffraction method.
3
Faculty of Biological Sciences, According to X-ray diffraction analysis results, the coordination geometry
Department of Pharmacy, Quaid-i-Azam
around Au(I) atoms in the complexes are revealed to be almost linear with
University, Islamabad, Pakistan
4
Faculty of Arts and Science, Department
C–Au–Cl angle. Anticancer activity, DNA binding, xanthine oxidase
_
of Molecular Biology and Genetics, Inönü (XO) inhibitory activity studies, and molecular docking studies were evalu-
University, Malatya, Turkey ated for all Au-NHC complexes to explore the binding mechanism at the
5
Faculty of Arts and Science, Department
active site. The IC50 value of Au-NHC complexes against human colorectal
of Chemistry, Bursa Uluda
g University,
Bursa, Turkey cancer (Caco-2) and breast cancer (MCF-7) cell lines was defined by MTT
6
Vocational School of Health Services at assay. The IC50 values for MCF-7 in the range of 5.2 ± 2 to 152.4 ± 1 μM and
Akyazı, Department of Medical Caco-2 5.2 ± 1 to 152.7 ± 2 μM showed that 3a, 3b, 3c, 3d, and 3g have better
Laboratory Techniques, Sakarya
University of Applied Sciences, Serdivan,
anticancer activity than Cisplatin incredibly complex 3a against both cancer
Sakarya, Turkey cell line. All Au-NHC complexes showed excellent antimicrobial activity
7
Faculty of Science, Department of against different bacteria and fungi. 3a was the complex that exhibited the
_
Physics, Dokuz Eylül University, Izmir,
best antimicrobial activity here as well. The XO inhibitory activity experimen-
Turkey
tal results indicated that all gold complexes showed remarkable inhibition
Correspondence activity against XO compared to the generally used standard, allopurinol. The
Mitat Akkoç, Malatya Turgut Özal
range of IC50 value was determined from 0.407 to 2.681 μM. 3d complex
University, Hekimhan Vocational College,
Department of Property Protection and showed the lowest IC50 value at 0.407 μM. DNA binding experiments were
Security, Hekimhan, Malatya 44400, performed using agarose gel electrophoresis to observe the ability of synthe-
Turkey.
Email: mitat.akkoc@ozal.edu.tr
sized Au-NHC complexes to interact with the supercoiled pUC19 plasmid
DNA. Molecular docking studies were performed to determine the binding
Funding information mode of all active compounds against the XO enzyme, antibacterial, antifun-
The authors declare that they have no
known competing financial support for gal, and MCF-7 cell lines.
this work.
KEYWORDS
anticancer activity, au-NHC, DNA binding, molecular docking, xanthine oxidase

Appl Organomet Chem. 2022;36:e6811. wileyonlinelibrary.com/journal/aoc © 2022 John Wiley & Sons Ltd. 1 of 19
https://doi.org/10.1002/aoc.6811

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1 | INTRODUCTION
Transition metal complexes are a beneficial resource for
drug development. The type of coordinated ligand on
metal plays a critical role in designing effective drugs.
For example, gold complexes are recognized as promising
compounds for cancer treatment.[1–4] Auranofin and its
chloro analog Et3PAuCl are two important anticancer
active gold complexes.
Lately, there has been considerable attention to
N-heterocyclic carbenes (NHC) due to their electron
richness with excellent stability.[5] Such features of
NHCs are provided by the delocalization of electrons
between C2 and N atoms in heterocycle rings via
mesomeric and inductive effects. NHCs are among the
most preferred all-purpose ligands for coordination
chemistry and catalysis because of their matchless
properties such as electron richness, excellent stability,
unique σ-donor property, and simple structural
modification.[5–7] Moreover, M-NHC bonds are more
robust than the bonds of other known ligands with
metals. M-NHC complexes are more stable toward the
air, moisture, and harsh reaction conditions in many
applications.[5,8–13] Effortless modification of the NHC
ligand via N1 and N3 positions is essential, while
N-substituents significantly affect the reactivity of
NHC and binding. Different functional groups as
N-substituents can be introduced depending on the
purpose to provide selectivity or reactivity. Different
M-NHC complexes have been studied, mainly catalysis
as a catalyst.[5,8–13] However, M-NHC complexes have
been used as antibacterial, antifungal, and especially
anticancer and arthritis agents.[3,4,6,7,14–20] In the begin-
ning, NHC ligands were used as auxiliary ligands to
replace toxic phosphines ligands.
The research on anticancer activity of NHC com-
plexes has been extensively explored after the millen-
nium. Willans et al., Pellei et al., Jin et al., Rashinkar
et al., Kühn et al., Razali et al., and our group reported
the synthesis of the different Ag-NHC complexes and
their anticancer activities against to different cancer cell
lines.[21–27] Studies showed that NHC complexes have
unique structural scaffolds for target-specific biomole-
cules relevant to cancer. Barnard et al.,[28] Hindi et al.,[29]
Teyssot et al.,[30] Lin et al.,[31] Bernard and Berners-
Price,[32] Dada et al.,[33] Hackenberg et al.,[33] Veenboer
et al.,[11] Budagumpi and Endud,[35] and our group[36–39]
have been investigated anticancer properties of NHC
complexes in various types of cancer. The non-platinum
anti-tumor drug such as gold-NHC in the different oxida-
tion states (+1 or +3) has emerged because of their
strong cytotoxic effects against broad cancer cell
lines.[1,32,40,41] Previous research showed that NHC-Au-Cl
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EKINCI ET AL.
and NHC-Au-thioglucoside exhibited good in vitro and
in vivo anticancer activity against a wide range of human
cancer cell lines with significant inhibition against mam-
malian thioredoxin reductase (TrxR).[34,42] Many studies
show that different Au-NHC complexes effectively inhibit
the TrXR enzyme.[10,43–46] Thus, they can serve as a
potential anticancer drug and may be an alternative to
Auronafin.
Considering the mechanism of action of gold-NHC
complexes as anticancer compounds, they exhibit simi-
lar behaviors to other anticancer drugs. Although the
exact action mechanism of Au-NHC complexes is not
precise, they could initiate the death of cancer cells by
targeting different cellular systems such as interaction
with DNA, effect on mitochondria, proteolysis, and sig-
nal transduction.[3,11,47,48] Drugs are transported inside
the cell by a couple of different transport mechanisms
via passive diffusion, carrier-mediated transport, ligand-
gated ion channels, active transport, and so forth. While
neutral NHC complexes enter cells by passive diffusion,
charged NHC complexes will be transported by organic
cation transporters (OCT6). SLC22A16 are the primary
transporters responsible for doxorubicin uptake into
cancer cells.[49] In the same way, the OCT transporter
family has involved in transporting M-MHC complexes
into the cancer cell as a cationic species. Au-NHC com-
plexes are transported into the cell by OCT transporters,
settle in the mitochondria, and cause ROS release. These
released ROS and other mitochondrial contents initiate
the process of cell death. This mechanism is essential in
the selectivity of Au-NHC complexes against cancer
cells.
As know from the studies, NHC ligands can interact
with different biomolecules. However, they can make
metals quite stable thanks to their sigma donor proper-
ties. For this reason, in this study, we wanted to synthe-
size different types of NHCs which are bearing different
R groups in aiming improve interactions with biomole-
cules and improve the stability of Au-NHC complex in
aqueous media. Starting from synthesized Ag-NHC com-
plexes, transmetallation of Ag with Au leads to novel sta-
ble Au-NHC complexes. We have reported in vitro
antimicrobial, anti-tumor activity against human cancer
cell lines, and molecular docking calculations of these
novels Au-NHC complexes.
2 | EXPERIMENTAL
2.1 | General considerations
Unless stated otherwise, Schlenk techniques were used
for all experiments. All chemicals were purchased from

EKINCI ET AL.
Alpha easer or Sigma-Aldrich and used as obtained.
The Ag-NHC and Au-NHC complexes were synthesized
in the dark. 1H and 13C NMR spectra were recorded by
a Bruker Avance III HD 300 or 400 or 600 MHz NMR
spectrometer. The Q-TOF/LCMS analysis was recorded
by an Agilent 6530 Accurate-Mass Q-TOF LC/MS
spectrometer. The LCMS analysis was recorded on a
Shimadzu LCMS 8040 spectrometer. Rigaku-Oxford
Xcalibur diffractometer with an Eos CCD area detector
has been used to collect the single-crystal data of 3b
and 3f complexes.
2.2 | Synthesis
2.2.1 | Synthesis procedure for 1a-g
The 1a-g was synthesized according to the literature
(Schema 1).[50]
S C H E M E 1 General reaction SCHEME for
1a-g, 2a-g, and 3a-g
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3 of 19
1-methylpyridine-3-(4-methyl)benzylbenzimidazolium
chloride, 1a
The reaction of 1-(methyl)pyridinebenzimidazole
(2 mmol) and 4-methylbenzyl chloride (2.1 mmol) in
DMF led to the desired 1a as brown crystals (0.58 g,
85%). 1H NMR (400 MHz, CDCl3) δ = 11.66 (s, 1H,
NCHN), 8.41 (d, J = 4 Hz, 1H, CH2C6H4N), 7.83–7.09
(m, 11H, C6H4, CH2C6H4N ve C6H4(CH3)-4), 5.99 (s, 2H,
CH2C6H3(CH3)-4), 5.76 (s, 2H, CH2C6H4N), 2.24 (s, 3H,
C6H4(CH3)-4).13C NMR (75 MHz, CDCl3) δ = 152.2,
149.5, 143.3, 139.4, 137.9, 132, 130.1, 129.4, 128.3, 127.1,
124.0, 114.6, 113.8, 113.4, 51.4, 51.5, 21.2 ppm.
1-methylpyridine-3-(2-methoxyethyl)benzimidazolium
chloride, 1b
The reaction of 1-(methyl)pyridinebenzimidazole (2 mmol)
and 2-methoxyethyl chloride (2.1 mmol) in DMF led to
the desired 1b as brown crystals (0.48 g, 80%). 1H NMR
(400 MHz, CDCl3) δ = 11.23 (s, 1H, NCHN), 8.41

4 of 19
(d, J = 4 Hz 1H, C5H4N), 7.80–7.15 (m, 8H, C6H4 ve
C5H4N), 5.98 (s, 2H, CH2C6H4N), 4.77 (t, J = 4 Hz, 2H,
CH2CH2OCH3), 3.88 (t, J = 6 Hz, 2H, CH2CH2OCH3),
3.27 (s, 3H, C2H4OCH3).13C NMR (75 MHz, CDCl3)
δ = 162.6, 152.5, 149.6, 143.6, 137.7, 132.0, 131.4, 126.9,
123.8, 114.1, 113.8, 70.2, 59.1, 52.5, 47.8, 36.5, 31.4 ppm.
1-methylpyridine-3-(3,5-dimethyl)benzyl-
5,6-dimethylbenzimidazolium chloride, 1c
The reaction of 1-(methyl)pyridine-5,-
6-dimethylbenzimidazole (2 mmol) and
3,5-dimethylbenzyl chloride (2.1 mmol) in DMF led to
the desired 1c as brown crystals (0.58 g, 82%). 1H NMR
(400 MHz, CDCl3) δ = 11.64 (s, 1H, CNHN), 8.43–6.88
(m, 9H, C6H2, C6H3 ve C5H4N), 5.96 (s, 2H,
CH2C6H3(CH3)2–3,5), 5.59 (s, 2H, CH2C6H4N), 2.27 (d,
J = 6 Hz, 6H, C6H2(CH3)2–5,6), 2.20 (s, 6H,
CH2C6H3(CH3)2–3,5). 13C NMR (75 MHz, CDCl3)
δ = 152.8, 149.4, 142.7, 139, 137.7, 137.2, 132.6, 130.8,
129.7, 125.5, 123.8, 114, 113.7, 113, 52.2, 51.2, 21.2, 20.7,
20.6 ppm.
1-methylpyridine-3-(2,3,4,5,6-pentamethyl)
benzylbenzimidazolium chloride, 1d
The reaction of 1-(methyl)pyridinebenzimidazole
(2 mmol) and 2,3,4,5,6-pentamethylbenzyl chloride
(2.1 mmol) in DMF led to the desired 1d as brown crys-
tals (0.64 g, 80%). 1H NMR (400 MHz, CDCl3) δ = 10.76
(s, 1H, NCHN), 8.35 (d, J = 3 Hz, 1H, CH2C6H4N), 7.92–
7.13 (m, 7H, C6H4, CH2C6H4N), 6.11 (s, 2H,
CH2C6(CH3)5–2,3,4,5,6), 5.70 (s, 2H, CH2C6H4N), 2.23,
2.21, 2.19 (s, 15H, C6H(CH3)2–2,3,4,5,6).13C NMR
(75 MHz, CDCl3) δ = 152.7, 149.2, 143.4, 137.8, 137.5,
134.0, 133.6, 132.3, 131.3, 127.1, 127.0, 124.6, 124.1, 123.8,
114.7, 113.1, 52.2, 50.6, 47.9, 17.4, 17.1, 17.0 ppm.
1,3-Bis (ethylmorpholine)benzimidazolium chloride, 1e
The reaction of 1-(ethylmorpholine)benzimidazole
(2 mmol) and ethylmorpholine chloride (2.1 mmol) in
DMF led to the desired 1e as a white powder (0.66 g,
86%). 1H NMR (400 MHz, CDCl3) δ = 11.21 (s, 1H,
NCHN), 7.78–7.62 (m, 4H, C6H4), 4.76 (t, J = 8 Hz, 4H,
NCH2CH2N(CH2)2-(CH2)2O), 3.65 (t, J = 6 Hz, 8H,
NC2H4N(CH2)2-(CH2)2O), 2.97 (t, J = 8 Hz, 4H,
NCH2CH2N(CH2)2-(CH2)2O), 2.59 (t, J = 6 Hz, 8H,
NC2H4N(CH2)2-(CH2)2O). 13C NMR (75 MHz, CDCl3)
δ = 144.3, 131.4, 126.9, 113.1, 66.8, 60.8, 56.7, 53.6,
44.4 ppm.
1,3-bis(4-tert-butyl)benzyl-5,6-dimethylbenzimidazolium
chloride, 1f
The reaction of 1-(4-tert-butyl)benzyl-5,-
6-dimethylbenzimidazole (2 mmol) and 4-tert-
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EKINCI ET AL.
butylbenzyl chloride (2.1 mmol) in DMF led to the
desired 1f as white crystals (0.74 g, 77%). 1H NMR
(400 MHz, CDCl3) δ = 11.84 (s, 1H, NCHN), 7.37–7.25
(m, 10H, C6H2, CH2C6H4-(C4H9)-4), 5.69 (s, 4H,
CH2C6H4-(C4H9)-4), 2.27 (s, 6H, 5,6-(CH3)C6H2), 1.19 (s,
18H, CH2C6H4-(C4H9)-4). 13C NMR (75 MHz, CDCl3)
δ = 162.6, 152.2, 142.5, 137.2, 130.0, 129.9, 127.9, 126.2,
113.3, 50.9, 36.5, 34.6, 31.4, 20.7 ppm.
1-(2,3,4,5,6-pentamethyl)benzyl-3-(ethylphthalimide)
benzimidazolium bromide, 1g
The reaction of 1-(2,3,4,5,6-pentamethyl)benzylbenzimi-
dazole (2 mmol) and ethylphthalimide bromide
(2.1 mmol) in DMF led to the desired 1g as a white
powder (0.90 g, 85%). 1H NMR (300 MHz, CDCl3)
δ = 9.95 (s, 1H, NCHN), 8.23–7.63 (m, 8H, C6H4,
CH2CH2N[CO]2C6H4), 5.69 (s, 2H, CH2C6[CH3]5–
2,3,4,5,6), 4.84 (d, J = 4.8 Hz, 2H, CH2CH2N[CO]2C6H4),
4.10 (d, J = 4.8 Hz, 2H, CH2CH2N[CO]2C6H4), 2.90,
2.74, 2.29 (s, 15H, CH2C6[CH3]5–2,3,4,5,6). 13C NMR
(75 MHz, CDCl3) δ = 168.2, 162.6, 138.5, 135, 131.9,
131.8, 131.4, 131.1, 130, 129.7, 128.6, 128.5, 127.5, 127.3,
127.2, 123.7, 114.5, 114.1, 50.2, 46.4, 37.3, 34.7, 31.2,
21.2 ppm.
2.2.2 | Synthesis procedure for the 2a-g
The 2a-g complexes were synthesized according to the
literature.[50]
Chloro[1-methylpyridine-3-(4-methyl)
benzylbenzimidazol-2-yliden]silver(I), 2a
The reaction of 1a (0.70 g, 2 mmol) and Ag2O (0.46 g,
2 mmol) led to the desired 2a complex as a brown pow-
der (0.64 g, 70%). 1H NMR (400 MHz, CDCl3) δ = 8.52
(d, J = 4 Hz, 1H, C5H4N), 7.62–7.06 (m, 11H, C6H4,
CH2C5H4N), 5.67 (s, 2H, CH2C6H2(CH3)-4), 5.53 (s, 2H,
CH2C6H4N), 2.25 (s, 3H, CH2C6H2(CH3)-4).13C NMR
(75 MHz, CDCl3) δ = 192.1, 155.0, 149.6, 138.1, 137.2,
134.3, 134.0, 133.9, 132.2, 129.6, 127.3, 127.2, 124.1, 124.0,
123.2, 122.3, 112.4, 112.0, 111.9, 55.0, 53.2, 21.1 ppm.
Anal. calcd. for C21H19N3ClAg: C, 55.23; H, 4.19; N, 9.20.
Found: C, 55.12; H, 4.14; N, 9.26.
Chloro[1-methylpyridine-3-(2-methoxyethyl)
benzimidazol-2-yliden]silver(I), 2b
The reaction of 1b (0.68 g, 2 mmol) and Ag2O (0.46 g,
2 mmol) led to the desired 2b complex as a brown
powder (0.66 g, 80%). 1H NMR (400 MHz, CDCl3)
δ = 8.50 (d, J = 2 Hz, 1H, C5H4N), 7.59–7.14 (m, 8H,
C6H4 ve C5H4N), 5.65 (s, 2H, CH2C6H4N), 4.55 (t,
J = 6 Hz, 2H, CH2CH2OCH3), 3.76 (t, J = 6 Hz, 2H,

EKINCI ET AL.
CH2CH2OCH3), 3.24 (s, 3H, CH2CH2OCH3).13C NMR
(75 MHz, CDCl3) δ = 189.2, 154.7, 149.8, 137.3, 134.6,
133.7, 124.4, 124.3, 123.4, 122.1, 112.3, 112.2, 71.9, 59.2,
55.2, 49.6 ppm. Q-TOF/LCMS (ESI-TOF) m/z:
[M + 2H-AgCl2]+ calcd for C32H34N6O2Ag, 643.17908,
found 643.17602. Anal. calcd. for C16H17N3OClAg: C,
46.80; H, 4.17; N, 10.23. Found: C, 46.92; H, 4.15; N,
10.55.
Chloro[1-methylpyridine-3-(3,5-dimethyl)benzyl-
5,6-dimethylbenzimidazol-2-yliden] silver(I), 2c
The reaction of 1c (0.72 g, 2 mmol) and Ag2O (0.46 g,
2 mmol) led to the desired 2c complex as a brown powder
(0.74 g, 75%). 1H NMR (400 MHz, CDCl3) δ = 8.62
(d, J = 2 Hz, 1H, C5H4N), 7.69–6.86 (m, 8H, C6H2 ve
C5H4N), 5.71 (s, 2H, CH2C6H3-(CH3)2), 5.51 (s, 2H,
CH2C5H4N), 2.32–2.29 (s, 12 H, CH2C6H3-(CH3)2, C6H2-
(CH3)2). 13C NMR (75 MHz, CDCl3) δ = 155.0, 149.8,
138.8, 137.3, 134.9, 134.0, 132.5, 130.2, 124.8, 123.3, 121.7,
112.4, 112.2, 55.0, 53.2, 21.3, 20.4 ppm. Q-TOF/LCMS
(ESI-TOF) m/z: [M + H-AgCl2]+ calcd for C48H50N6Ag,
819.31445, found 819.31069. Anal. calcd. for
C24H25N3ClAg: C, 57.79; H, 5.05; N, 8.42. Found: C,
57.87; H, 5.20; N, 8.67.
Chloro[1-methylpyridine-3-(2,3,4,5,6-pentamethyl)
benzylbenzimidazol-2-yliden] silver(I), 2d
The reaction of 1d (0.82 g, 2 mmol) and Ag2O (0.46 g,
2 mmol) led to the desired 2d complex as a brown pow-
der (0.88 g, 85%). 1H NMR (400 MHz, CDCl3) δ = 8.49
(d, = 2 Hz, 1H, C5H4N)-7.12 (m, 7H, C6H4 ve C5H4N),
5.59 (s, 2H, CH2C6(CH3)5–2,3,4,5,6), 5.43 (s, 2H,
CH2C6H4N), 2.27, 2.22, 2.14 (s, 15H, CH2C6(CH3)5–
2,3,4,5,6).13C NMR (75 MHz, CDCl3) δ = 136.2, 136.1,
135.0, 134.6, 134.5, 134.3, 133.3, 133.0, 128.5, 128.3, 128.0,
127.9, 127.8, 127.1, 123.2, 122.4, 111.9, 111.3, 51.7, 50.9,
17.6, 17.5, 17.2, 16.9, 16.8 ppm. Anal. calcd. for
C25H27N3ClAg: C, 58.55; H, 5.31; N, 8.19. Found: C,
58.47; H, 5.39; N, 8.13.
Chloro[1,3-Bis (ethylmorpholine)benzimidazol-2-yliden]
silver(I), 2e
The reaction of 1e (0.76 g, 2 mmol) and Ag2O (0.46 g,
2 mmol) led to the desired 2e complex as a white powder
(0.72 g, 75%). 1H NMR (400 MHz, CDCl3) δ = 7.47–7.34
(m, 4H, C6H4), 4.43 (t, J = 6 Hz, 4H, NCH2CH2N(CH2)2-
(CH2)2O), 3.63 (t, J = 4 Hz, 8H, NCH2CH2N(CH2)2-
(CH2)2O), 2.75 (t, J = 6 Hz, 4H, NCH2CH2N(CH2)2-
(CH2)2O), 2.47 (t, J = 6 Hz, 8H, NCH2CH2N(CH2)2-
(CH2)2O). 13C NMR (75 MHz, CDCl3) δ = 133.7, 124.2,
111.5, 66.9, 58.3, 54.1, 47.0 ppm. Anal. calcd. for
C19H28N4O2ClAg: C, 46.79; H, 5.79; N, 11.49. Found: C,
46.85; H, 5.90; N, 11.35.
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5 of 19
Chloro[1,3-bis(4-tert-butyl)benzyl-
5,6-dimethylbenzimidazol-2-yliden] silver(I), 2f
The reaction of 1f (0.96 g, 2 mmol) and Ag2O (0.46 g,
2 mmol) led to the desired 2f complex as a white powder
(0.98 g, 85%). 1H NMR (400 MHz, CDCl3) δ = 7.27–7.05
(m, 10H, C6H2, CH2C6H4-(C4H9)-4), 5.47 (s, 4H,
CH2C6H4-(C4H9)-4), 2.72 (s, 6H, 5,6-(CH3)C6H2), 1.20 (s,
18H, CH2C6H4-(C4H9)-4). 13C NMR (75 MHz, CDCl3)
δ = 151.4, 133.7, 132.6, 132.2, 127.1, 126.8, 126, 125.7,
112.2, 52.9, 34.6, 31.3, 20.5 ppm. Q-TOF/LCMS (ESI-
TOF) m/z: [M + H-AgCl2]+ calcd for C62H76N4Ag,
985.51175, found 985.50883. Anal. calcd. for
C31H38N2ClAg: C, 63.98; H, 6.58; N, 4.81. Found: C,
64.02; H, 6.55; N, 4.84.
Bromo[1-(2,3,4,5,6-pentamethyl)
benzyl-3-(ethylphthalimide)benzimidazol-2-yliden]
silver(I), 2g
The reaction of 1g (0.106 g, 1 mmol) and Ag2O (0.46 g,
2 mmol) led to the desired 2g complex 2g as a white
powder (0.1 g, 80%). 1H NMR (300 MHz, CDCl3)
δ = 7.93–7.49 (m, 8H, C6H4, CH2CH2N(CO)2C6H4), 5.38
(s, 2H, CH2C6(CH3)5–2,3,4,5,6), 4.60 (s, 2H, CH2CH2N
(CO)2C6H4), 3.96 (s, CH2CH2N(CO)2C6H4), 2.51, 2.12,
1.93 (s, 15H, CH2C6(CH3)5–2,3,4,5,6). 13C NMR
(75 MHz, CDCl3) δ = 167.7, 136.2, 134.7, 133.7, 133.4,
133, 131.7, 127.4, 124.4, 136.2, 123.8, 112.5, 112.2, 65.8,
46.9, 37.5, 17.3, 17, 15.6 ppm. Anal. calcd. for
C29H29N3O2BrAg: C, 54.48; H, 4.57; N, 6.57. Found: C,
54.56; H, 4.53; N, 6.52.
2.2.3 | Synthesis procedure for the 3a-g
The 3a-g complexes were synthesized according to the
literature.[50]
Chloro[1-methylpyridine-3-(4-methyl)
benzylbenzimidazol-2-yliden] gold(I), 3a
The transmetallation reaction of 2a (0.64 g, 1 mmol)
and (CH3)2SAuCl (0.29 g, 1 mmol) led to the desired
3a complex as a white powder (0.38 g, 70%): 1H NMR
(400 MHz, CDCl3) δ = 8.58 (d, J = 2 Hz, 1H, C5H4N),
7.68–7.13 (m, 11H, C6H4, CH2C5H4N), 5.84
(s, 2H, CH2C6H2(CH3)-4), 5.70 (s, 2H, CH2C6H4N),
2.31 (s, 3H, CH2C6H2(CH3)-4).13C NMR (75 MHz,
CDCl3) δ = 179.1, 154.5, 149.6, 138.5, 137.3, 133.6,
133.1, 129.7, 127.5, 124.7, 123.4, 122.3, 112.8, 112.1,
54.6, 52.8, 21.2 ppm. LC–MS (ESI) 1m/z = [M-Cl + 3
K + 2Na]+ was calculated for [C21H21N3K3Na2Au]:
675.01 and found 675.25; m/z = [M + 2 K + 3Na]+
was calculated for [C21H16N3K2Na3ClAu]: 688.97 and
found 689.00.

6 of 19
Chloro[1-methylpyridine-3-(2-methoxyethyl)
benzimidazol-2-yliden] gold(I), 3b
The 3b complex was synthesized as a brown powder
(0.46 g, 92%) as a result of the transmetalation reaction
of (0.41 g, 1 mmol) 2b and (0.29 g, 1 mmol)
(CH3)2SAuCl. 1H NMR (400 MHz, CDCl3) δ = 8.51 (d,
J = 2 Hz, 1H, C5H4N), 7.60–7.15 (m, 8H, C6H4 ve
C5H4N), 5.74 (s, 2H, CH2C6H4N), 4.59 (t, J = 6 Hz 2H,
CH2CH2OCH3), 3.83 (t, J = 4 Hz, 2H, CH2CH2OCH3),
3.25 (s, 3H, CH2CH2OCH3).13C NMR (75 MHz, CDCl3)
δ = 178.7, 154.5, 149.6, 137.3, 134.2, 133.1, 124.7, 123.4,
122.3, 71.9, 59.2, 54.7, 48.9 ppm. LC-MS (ESI) m/z =
[M + Cl + K]+ was calculated for [C16H19N3OKAu]:
505.08 and found 505.00; m/z = [M-Cl + 3Na]+ was cal-
culated for [C16H22N3ONa3Au]: 538.11 and found
538.10.
Chloro[1-methylpyridine-3-(3,5-dimethyl)
benzyl-5,6-dimethylbenzimidazol-2-yliden] gold(I), 3c
The 3c complex was synthesized as a brown powder
(0.52 g, 88%) as a result of the transmetalation reaction
of 2c (0.50 g, 1 mmol) and (CH3)2SAuCl (0.29 g,
1 mmol). 1H NMR (400 MHz, CDCl3) δ = 8.53–6.85
(m, 9H, C6H2, C6H3 ve C5H4N), 5.74 (s, 2H,
CH2C6H3(CH3)2–3,5), 5.54 (s, 2H, CH2C6H4N), 2.22 (s,
6H, C6H2(CH3)2
5,6), 2.20 (s, 6H, CH2C6H3(CH3)2–
3,5). 13C NMR (75 MHz, CDCl3) δ = 177, 153.7, 148.3,
137.6, 136.5, 133.3, 133.2, 131, 130.7, 129.1, 123.9,
122.4, 121.2, 111.6, 111.2, 53.2, 51.7, 20.3, 19.4,
19.3 ppm. LC–MS (ESI) m/z = [M + Cl + K]+ was cal-
culated for [C24H27N3KAu]: 593.15 and found 593.20;
m/z = [M-Cl + 2 K + 2Na]+ was calculated for
[C24H25N3K2Na2Au]: 676.08 and found 676.30; m/z =
[M-Cl]+ was calculated for [C24H25N3Au]: 552.17 and
found 552.00.
Chloro[1-methylpyridine-3-(2,3,4,5,6-pentamethyl)
benzylbenzimidazol-2-yliden] gold(I), 3d
The 3d complex was synthesized as a brown
powder (0.54 g, 90%) as a result of the transmetalation
reaction of 2d (0.51 g, 1 mmol) and (CH3)2SAuCl
(0.29 g, 1 mmol). 1H NMR (400 MHz, CDCl3)
δ = 8.49 (d, J = 2 Hz, 1H, C5H4N), 7.83–7.31 (m, 7H,
C6H4 ve C5H3N), 5.83, 5.75 (s, 2H, CH2C6(CH3)5–
2,3,4,5,6), 5.76 (s, 2H, CH2C6H4N), 2.22, 2.20, 2.17 (s,
15H, CH2C6(CH3)5–2,3,4,5,6).13C NMR (75 MHz,
CDCl3) δ = 179.1, 155.5, 149.9, 137.7, 135.5, 133.8,
133.5, 133.1, 129.0, 124.9, 124.8, 123.6, 122.1, 113.0,
112.6, 55.4, 53.9, 48.8, 17.7, 17.5, 17.3 ppm. LC–MS
(ESI) m/z = [M + Cl + K]+ was calculated for
[C25H29N3KAu]: 607.17 and found 607.20; m/z = [M-
Cl]+ was calculated for [C25H27N3Au]: 566.19 and
found 566.05.
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EKINCI ET AL.
Chloro[1,3-Bis (ethylmorpholine)benzimidazol-2-yliden]
gold(I), 3e
The 3e complex was synthesized as a white powder
(0.49 g, 85%) as a result of the transmetalation reaction
of 2e (0.49 g, 1 mmol) and (CH3)2SAuCl (0.29 g,
1 mmol). 1H NMR (400 MHz, CDCl3) δ = 7.85–7.45
(m, 4H, C6H4), 4.59 (t, J = 6 Hz, 4H, NCH2CH2N
(CH2)2-(CH2)2O), 3.50 (t, J = 4 Hz, 8H, NCH2CH2N
(CH2)2-(CH2)2O), 2.80 (t, J = 6 Hz, 4H, NCH2CH2N
(CH2)2-(CH2)2O), 2.52 (t, J = 4 Hz, 8H, NCH2CH2N
(CH2)2-(CH2)2O). 13
C NMR (75 MHz, CDCl3)
δ = 178.1, 133.2, 124.7, 112.8, 66.6, 57.8, 53.9,
45.9 ppm. LC–MS (ESI) m/z = [M]+ was calculated for
[C19H29N4O2KAu]: 582.17 and found 577.15; m/z = [M-
Cl + K]+ was calculated for [C19H30N4O2KAu]: 582.17
and found 582.20.
Chloro[1,3-bis(4-tert-butyl)benzyl-
5,6-dimethylbenzimidazol-2-yliden] gold(I), 3f
The 3f complex was synthesized as a white
powder (0.60 g, 90%) as a result of the transmetalation
reaction of 2f (0.58 g, 1 mmol) and (CH3)2SAuCl
(0.29 g, 1 mmol). 1H NMR (400 MHz, CDCl3)
δ = 7.36–7.26 (m, 8H, CH2C6H4-(C4H9)-4), 7.11
(s, 2H, 5,6-(CH3)C6H2), 5.65 (s, 4H, CH2C6H4-(C4H9)-
4), 2.28 (s, 6H, 5,6-(CH3)C6H2), 1.28 (s, 18H,
CH2C6H4-(C4H9)-4). 13C NMR (75 MHz, CDCl3)
δ = 177.8, 151.4, 134.1, 131.9, 131.8, 127, 125.9, 112.3,
53.4, 52.3, 34.6, 31.3, 20.4 ppm. LC-MS (ESI) m/z =
[M + Cl + K]+ was calculated for [C31H40N2KAu]:
676.25 and found 676.25; m/z = [M-Cl + K + 2Na]+
was calculated for [C31H36N2KNaAu]: 718.20 and
found 718.30.
Bromo[1-(2,3,4,5,6-pentamethyl)benzyl-
3-(ethylphthalimide)benzimidazol-2-yliden] gold(I), 3g
The 3g complex was synthesized as a white powder
(0.58 g, 80%) as a result of the transmetalation reaction
of 2g (0.64 g, 1 mmol) and (CH3)2SAuCl (0.29 g, 1 mmol).
1
H NMR (400 MHz, CDCl3) δ = 7.71–6.76 (m, 8H, C6H4,
CH2CH2N(CO)2C6H4), 5.62 (s, 2H, CH2C6(CH3)5-
2,3,4,5,6), 4.71 (t, J =6 Hz, 2H, CH2CH2N(CO)2C6H4),
3.96 (t, J = 6 Hz, 2H, CH2CH2N(CO)2C6H4), 2.21, 2.15,
2.12 (s, 15H, CH2C6(CH3)5–2,3,4,5,6). 13C NMR
(75 MHz, CDCl3) δ = 179.4, 167.8, 136.4, 134.2, 133.6,
133.4, 133.2, 131.7, 127.0, 124.5, 124.4, 123.6, 112.6,
110.4, 53.5, 50.4, 47.1, 37.2, 17.4, 17.3, 17.0 ppm. LC–MS
(ESI) m/z = [M + Cl + K]+ was calculated for
[C29H31N3O2KAu]: 689.17 and found 689.00; m/z =
[M + 2Na]+ was calculated for [C29H31N3O2Na2AuCl]:
731.16 and found 731.25; m/z = [M + Na + K]+ was
calculated for [C29H31N3O2NaKAu]: 747.13 and found
747.15.

EKINCI ET AL.
2.3 | In vitro assay of xanthine oxidase
inhibitory activity
The enzyme activity was assayed spectrophotometrically
by measuring the uric acid formation at 294 nm and
37 C. The xanthine oxidase (XO) inhibition determina-
tion in IC50 values for different concentrations of 3a-g
was performed by adding them into the reaction mixture
and measuring the absorbance. Briefly, the procedure
used for the enzyme assay contains phosphate buffer
(50 mM, pH = 7.4), XO (0.2 U), and xanthine (1 mM).
The enzyme was pre-incubated for 10 min with tested
compounds, and then the reaction was started by adding
xanthine into the reaction mixture. The tested com-
pounds were dissolved in DMSO and then diluted with
phosphate buffer. The final concentration of DMSO in
the reaction mixture was less than 0.01% (v/v), and this
ratio does not interfere with the enzyme assay.[51] All the
experiments were performed in triplicates, and the values
were expressed as means of three experiments. Allopuri-
nol was used as a positive control. The IC50 values were
determined for all compounds and measured as percent
inhibition of XO in terms of decreasing the formation of
uric acid compared to the product formation in the
absence of an inhibitor. The percent inhibition (IC50) of
XO activity was calculated as the following:
Inhibition ð%Þ ¼ ðA
BÞ=A  100,
where A = the absorbance at 294 nm without the tested
compound and B = the absorbance at 294 nm with the
tested compound.
2.4 | Antimicrobial activity studies
The antimicrobial activities of 3a-g were examined using
96-well plates following the micro-dilution method. In
order to obtain the minimum inhibition concentration
(MIC) values of each complex, Escherichia coli (ATCC
25922) [Gram (
)], Bacillus subtilis (ATCC 21332) [Gram
(+)] and Candida albicans (ATCC 60193) were used as
the two bacterial and one fungal species. The bacterial
and fungal microorganisms were cultured in Luria-
Bertani (LB) medium (Sigma-Aldrich Chemie GmbH,
Taufkirchen/Germany) until 0.5 of McFarland standard
turbidity value was reached at 37 C. Besides, after the
Au-NHC complexes were dissolved in DMSO at 1 mg/ml
concentrations, they were diluted with distilled water to
a final concentration of 10% (v/v) to prepare different
concentrations of the synthesized gold complexes. With
the addition of bacterial and fungal cultures, the final
concentrations of the tested complexes were adjusted as
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7 of 19
1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg/ml. As
an antibacterial and antifungal standard, cefotaxime was
used with the final concentrations as 0.0625, 0.125, 0.25,
0.50, 0.75, 1.0, 1.125, and 1.250 mg/ml. After incubating
cultures, 100 μl of the liquid microorganisms and 100 μl
of the different concentrations of each gold complex were
added to each well of 96-well plates. At the end of the
24-h incubation period, to determine the antimicrobial
activities of the synthesized gold complexes against the
cultured microorganisms, MIC values at which the
growth of the microorganisms stopped were determined
by reading the absorbance values of the 96-well plates at
625 nm.
2.5 | MTT assay
Anticancer activities of the 3a-g were determined by MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide) assay on MCF-7 (human breast adenocarcinoma)
and Caco-2 (human colorectal adenocarcinoma) cell
lines. The cells were first grown with a DMEM
medium containing 10% fetal bovine serum (FBS) and 1%
Penicillin/Streptomycin until they were 80% confluent in
a CO2 incubator at 37 C. The cells were then removed
from the surface with trypsin and seeded into 96-well plates
as 104 cells/well. The medium was removed from the
cells, incubated under the same conditions for 24 h,
and exposed to different concentrations of compound
solutions (3.125, 6.25, 12.5, 25, 50, 100, and 200 μM).
After 24 h incubation, the solutions were removed,
and 10 μl of MTT (5 mg/ml in PBS) and 90 μl of DMEM
were added. After 4 h of incubation in the dark,
absorbances were taken in the ELISA microplate reader
at 550 nm, and the control wells were considered 100%
viable.
2.6 | DNA binding studies
The DNA binding activities of the 3a-g were investigated
by the agarose gel electrophoresis method using pUC19
plasmid DNA. DMSO was used as the solvent because 3a,
3b, 3c, 3d, 3e, 3f, and 3g complexes were not soluble in
water. The obtained complexes were prepared in different
final concentrations as 75 and 150 μg/ml, adding pUC19
plasmid DNA. After adding 4 μl of pUC19 (0.05 μg/ml)
into each tube containing 4 μl of Cisplatin (final concen-
trations as 75 and 150 μg/ml) as controls and the synthe-
sized complexes in different concentrations, all of them
were incubated for 24 h at 37 C. After incubation, the
plasmid DNA sample was mixed with gel loading dye
and loaded into the electrophoresis chamber wells and

8 of 19
the control DNA (0.05 μg/μl pUC19). The electrophoresis
procedure was initiated at 80 V for 45 min using 1% aga-
rose gel. After electrophoresis was finished, the gel was
removed and stained with ethidium bromide solution
and the image was taken with Syngene G: BOX gel docu-
mentation system.
2.7 | Molecular docking
Molecular docking studies were done on the synthesized
complexes to explore the binding mechanism at the
active site. All the synthesized complexes were evaluated
for their XO inhibitory, antifungal, antibacterial, and
anticancer activities. The 3D structures of XO (PDB ID:
3NVY), antibacterial (PDB ID: 4QGG), antifungal (PDB
ID: 6F0E), and MCF-7 cell line (PDB ID: 1YWN) were
downloaded from Protein Data Bank (PDB) (www.rcsb.
org). The maximum grid box was adjusted as center X, Y,
and Z. The default exhaustiveness value eight was
adjusted in both docking to maximize the binding confor-
mational analysis. The docking poses for each were
adjusted to obtain the best docking results. The docked
3a-g complexes were evaluated on the lowest binding
energy (Kcal/mol) values and structure-activity relation-
ship analyses. The ligand and protein 2D and 3D interac-
tion were visualized using Discovery Studio (2.1.0).[52]
The binding affinity of all synthesized complexes was
calculated using the PyRx docking tool.[53] In order to
confirm the docking results in the term of binding con-
formations, standard drugs were also docked to the same
proteins.
2.8 | Crystal structure studies
The single-crystal X-ray diffraction method elucidated
the molecular and crystal structures of compounds 3b
and 3f. The measurements were performed by an ω-scan
technique using graphite–monochromated MoKα radia-
tion (λ = 0.71073 Å) from an enhanced X-ray source.
CrysAlisPro program[54] has been utilized to collect and
reduce the data and handle the cell refinement. For the
solution of the crystal structure, we have used the
ShelXT[55] structure solution program with Intrinsic
Phasing. The full-matrix least-squares method based on
F2 against all reflections using the SHELXL[56] has been
employed to refine the coordinates and anisotropic ther-
mal parameters of non-hydrogen atoms. These calcula-
tions are carried out under the crystal structure
crystallographic software package OLEX2 system.[57]
Anisotropic thermal parameters were applied to all non-
hydrogen atoms. PLATON software was used to calculate
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EKINCI ET AL.
and analyze the geometrical results.[58] OLEX2 tools have
created the pictures.[57] The crystal structure of I was
determined as a two-component non-merohedral twin,
with the final ratio of the twin domains being 0.56
(3):0.44(3). The non-merohedral twinning was considered
during the data reduction and structure refinement using
the data in HKLF5. The Rint value is not available in
Table 1 due to merging twin components with MERGE
0. A rotational disorder was noted for the methyl C atoms
of two methylbenzyl groups and related C atoms were set
to equal each other by an EADP constraint instruction.
The C-Cl bond distances of the dichloromethane solvent
molecule were also restrained from being equal using the
SADI command. Besides, the rigid-bond restraint RIGU
was applied for the C11 and C16 atoms of the aromatic
ring. Table 1 contains details of the crystallographic data
and some refinement parameters for complexes.
3 | RESULTS A ND DISCUSSION
3.1 | Synthesis and characterization
The synthesis of NHC salts Ag-NHC and Au-NHC com-
plexes is presented in Scheme 1. The synthesis of 1a-g
and 2a-g reported in the literature was followed.[50] NHC
salts were successfully transformed into Ag-NHC com-
plexes in good yields (70–85%). The 3a-g was synthesized
in good to high yields (70–92%) via transmetallation reac-
tion of the Ag-NHC complexes as carben transfer agents.
Spectroscopic techniques fully characterized 2a-g. The
resonance of NCHN protons of 1a-g in 1H NMR reso-
nates at 11.66, 11.23, 11.64, 10.76, 11.21, 11.84, and
9.95 ppm, respectively. In addition, the NCHN carbons of
1a-g resonate in the 13C{1H} NMR spectra at 152.2, 162.6,
152.8, 152.7, 144.3, 162.6, and 162.6 ppm, respectively.
The synthesis of Ag-NHC complexes was proved in the
1
H NMR by the disappearance of NCHN protons of NHC
salts (9.95–11.84 ppm) and a downfield shifts of the C2
carbons of Ag-NHC complexes in 13C NMR spectra
(192.1, 189.2 and 151.39 ppm for silver complexes 2a, 2b,
2f and C2 carbons of 2c, 2d, 2e, 2g not observed). After
transmetalation, the formation of 3a-g complexes was
approved by a highfield shift of the C2 carbons of Au-
NHC to 179.1, 178.7, 177.0, 179.1, 178.1, 177.8, and
179.4 ppm, respectively. The Q-TOF/LCMS analysis of
2b, 2c, and 2f and LC/MS analysis of 3a-g complexes
verified the proposed structures for the Ag-NHC and
Au-NHC complexes. According to Q-TOF/LCMS analy-
sis, Ag-NHC complexes (2b, 2c, and 2f) have a dimeric
structure in solution due to observation of the [NHC-Ag-
NHC]+ molecular ion fragment. However, X-ray analysis
of Ag-NHC complexes in the literature showed that
 10990739, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/aoc.6811 by Zhejiang University, Wiley Online Library on [17/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
EKINCI ET AL. 9 of 19

TABLE 1 Crystal data and structure refinement parameters for complexes 3b and 3f

Complex 3b Complex 3f
Chemical formula C16H17AuClN3O C32H40AuCl3N2
Formula weight 499.74 755.97
Temperature (K) 293(2) 293(2)
Space group P-1 P-1
Crystal system triclinic triclinic
a (Å) 8.6775(5) 9.8598(4)
b (Å) 9.7362(6) 10.2044(7)
c (Å) 11.3505(6) 18.2321(9)
  
α ( ), β ( ), γ ( ) 68.696(5), 98.168(5),
82.177(4), 97.689(4),
67.609(5) 110.819(5)
3
Cell volume (Å ) 826.07(9) 1663.22(17)
Formula unit cell Z 2 2
ρcalc (g/cm3) 2.009 1.510
μ (mm
1) 9.070 4.686
F(000) 476.0 752.0
Crystal size (mm ) 3
0.35  0.27  0.2 0.57  0.27  0.23
Diffractometer Xcalibur, Eos Xcalibur, Eos
Radiation/wavelength (Å) Mo Kα (λ = 0.71073) Mo Kα (λ = 0.71073)
Reflections measured 5,502 6,171
Independent reflections 3,323 [Rint = 0.0264, Rsigma = 0.0527] 6,171 [Rint = n/a, Rsigma = 0.1653]
Range of h, k, l
9 ≤ h ≤ 10,
9 ≤ h ≤ 12,

12 ≤ k ≤ 11,
12 ≤ k ≤ 12,

14 ≤ l ≤ 14
22 ≤ l ≤ 16
Data/restraints/parameters 3,323/0/200 6,171/76/354
Final R indexes [I ≥ 2σ (I)] R1 = 0.0348, wR2 = 0.0551 R1 = 0.0453, wR2 = 0.0667
Final R indexes [all data] R1 = 0.0467, wR2 = 0.0600 R1 = 0.0892, wR2 = 0.0732
2
Goodness-of-fit on F 1.058 0.840

3
Largest diff. peak/hole (e Å ) 0.77/
0.80 1.20/
1.03

Ag-NHC complexes are in monomeric form as NHC-Ag-
X.[59] In the LCMS spectra of 3a-g, the most intense
peaks correspond to molecular fragments ionized by the
loss of one or more ions (Cl
or Br
). The LCMS analysis
displays gold-containing molecular fragment peaks. The
obtained results prove that the theory and experimental
results are compatible for 2b, 2c, 2f, and 3a-g complexes.
3.2 | Descriptions of the crystal
structures of 3b and 3f
The molecular and crystal structures of compounds 3b
and 3f are depicted in Figures 1 and 2, and selected bond
distances and bond angles are given in the figure caption.
The complexes crystallize in the same triclinic unit cell in
the space group type P-1. The 3b possesses one indepen-
dent molecule per asymmetric unit. The asymmetric unit
of complex 3f, unlike complex 3b, is composed of the
entire molecule and a disordered dichloromethane solvent
molecule. The two-coordinate gold atom in 3b and 3f
adopts a slightly distorted linear coordination geometry
with C–Au–Cl bond angles of 179.55 (15) and 178.56
(19) , respectively. Complex 3b contains a contact between
two adjacent gold(I) atoms Au and Aui (symmetry
code = i: 1-x, 1-y, 1-z) with a distance of 3.7657(5) Å due
to the deviation of the angle around gold from the ideal
180 . Similar behavior is observed in complex 3f with
AuAuj distance of 3.8282(5) Å (symmetry code = j: 1-x,
1-y, 1-z). These metal–metal distances are rather long for

10 of 19
F I G U R E 1 The molecular structure of complex 3b, with atom
labeling and 50% probability displacement ellipsoids. H atoms are
shown as spheres of arbitrary radius. Selected bond parameters (Å,

): Au1–Cl1 2.2765(16); N1–C1 1.341(7); N1–C2 1.389(6); N2–C1
1.356(6); N2–C7 1.383(7); C2–C7 1.372(7); C3–C4 1.375(7); C1–N1–
C2 109.9(4); C1–N1–C14 125.1(5); C1–N2–C7 109.6(5); C1–N2–C8
125.0(5); N1–C2–C3–C4–179.8(5); N1–C14–C15–O1–66.7(6)
F I G U R E 2 Plot showing 50% probability ellipsoids for non-H
atoms for 3f. The minor components of the disordered methyl C
atoms are shown with a gray color. For simplicity, hydrogen atoms
are omitted. Selected bond parameters (Å,  ): Au1–Cl1 2.2870(17);
N1–C1 1.366(7); N1–C7 1.393(7); N2–C1 1.351(8); N2–C2 1.403(7);
C2–C7 1.364(8); C3–C4 1.408 (7); C1–N1–C7 110.1(5); C1–N1–C21
125.4(6); C1–N2–C2 110.4(6); C1–N2–C10 123.8(6); N1–C21–C22–
C23–33.8(8); N1–C21–C22–C27 150.8(6); N2–C10–C11–C12 81.7(8);
N2–C10–C11–C16–98.2(7)
an aurophilic interaction defined as 2.7 and 3.4 Å by
Wiberg and Holleman.[60] The Au–C bond length is 1.981
(6) and 1.979(7) Å in complexes 3b and 3f, respectively.
These distances confirm a single-bond character, conso-
nant with their strong σ-donor characteristic, and similar
values are related to Au(I) complexes.[61–67] These
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EKINCI ET AL.
distances also correspond with the relatively longer Au–Cl
bond lengths, indicating that the carbene ligand has a
stronger trans-influence than the chloride ligand, as men-
tioned in the literature.[63–67] No exceptional values are
found for the bond lengths and angles in the benzimid-
azole ring for both complexes, and these values agree well
with those reported for carbene complexes.[63,65,68–70]
In compound 3b, the benzimidazole ring links to the
pyridine ring via –CH2– bridge and methoxyethyl at the
N2 and N1 atoms, respectively. The pyridine ring is
rotated from the benzimidazole ring plane by a dihedral
angle of 97.97(5) . The torsion angles C16 can define the
orientation of the methoxyethyl unit–O1–C15–
C14 =
172.1(5) . In the crystal structure of complex 3b,
the methoxyethyl oxygen atom O1 participates in form-
ing a C6–H6O1ii hydrogen bond producing an
inversion-symmetric motif with graph-set R22 ð16Þ. Thus,
these dimers are arranged in the crystallographic c-axis
direction. Intermolecular C5–H5 Cg1iii interactions are
present between the hydrogen atom of the aromatic ring
(C2–C7) and the pyridine ring, which cause the stacking
of molecules along the a-axis (see the supporting
information). The AuAui interactions play a significant
role in crystal structure stability, although they are longer
than 3.6 Å, which is twice the van der Waals radius of an
Au atom. Thus, molecules are connected in a head-to-tail
configuration to form pairs of molecules through these
metal–metal interactions. The crystal structure shows
π–π stacking interactions between adjacent benzene and
imidazole rings with centroid–centroid distances of 3.818
(4) Å (Symmetry code: 1-x, 1-y, -z). Compound 3b con-
tains a weak intramolecular C10—H10N2 interaction
consolidating the molecular conformation. The details of
the whole intramolecular and intermolecular interactions
are given in the supporting information.
In compound 3f, the two benzyl rings are oriented
oppositely, forming dihedral angles of 114.92(4) and
92.73(5) with the mean plane through the benzimid-
azole ring. The methyl C atoms of two methylbenzyl moi-
eties have a rotational disorder, with site occupancies of
0.724(7) and 0.572(6) for the major components. The
analogous values in the minor component are 0.276
(7) and 0.428(6). There are no significant directed inter-
molecular hydrogen bonds in the crystal structure of
complex 3f. The molecules are stacked by intermolecular
C9–H9BCg1 interactions involving the aromatic ring
(C11–C16) as acceptor. Additionally, π–π interactions of a
weak nature between benzene and imidazole rings help
establish supramolecular aggregation [centroid–centroid
distance: 3.864(4) Å, symmetry code: -x, -y, 1-z]. Thus,
the molecules are assembled by C–H  π and π–π interac-
tions along the diagonal line of the a and b axes (see the
supporting information). As observed for complex 3b,
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EKINCI ET AL. 11 of 19

molecules of 3f are arranged in head-to-tail orientations standard, allopurinol. The range of IC50 value was deter-
based on AuAuj interactions, which strongly contrib- mined from 0.407 to 2.681 μM. 3d complex showed the
uted to the stability of crystal structure. lowest IC50 value at 0.407 μM; on the other hand, the 3e
complex showed the highest IC50 value at 2.681 μM. The
IC50 value for allopurinol as a positive control was deter-
3.3 | Biological evaluation mined as 3.429 μM. In the previous study of Al-Majid
et al.[71] They synthesized gold-NHC complexes and inves-
3.3.1 | In vitro assay of XO inhibitory tigated their inhibitory effects against the XO enzyme.
activity studies They had reported that the IC50 value range was from 4.4–
48.1 μM. In a recent study, 5-benzyl-3-pyridyl-1H-1,-
XO catalyzes hypoxanthine to xanthine and xanthine to 2,4-triazole exhibited IC50 for XO from 0.16 to
uric acid. Accumulation of uric acid leads to hydrogen per- 12.70 μM.[72] In another study, 2-hydroxyethyl substituted
oxide formation and then conversion to superoxide. Super- NHC complexes showed good inhibition on XO (inhibition
oxide ion is an attacker as a free radical, and it is on XO IC50 in the range from 1.253 to 5.342 μM).[73] Our
responsible for DNA damaging and changing protein results showed that 3a-g complexes exhibited excellent
structures and functions. The inhibition of XO may be inhibition activity than allopurinol. The mechanism of
identified in various ways, showing the anticancer effect. action of Au-NHC complexes could efficiently react with
3a-g gold complexes were tested against the XO inhibitory biological molecules in the enzyme structure, such as
effect, and IC50 values were given in Table 2. The experi- amino acids.[74] However, due to their nucleophilic charac-
mental results indicated that all gold complexes showed ter, the Au–NHC complexes perform electronic interac-
remarkable inhibition activity against XO compared to the tions with organic molecules. Also, Au–NHC can form
hydrogen bond interaction with amino acid molecules.
Furthermore, these complexes are important in secondary
TABLE 2 The range of IC50 values of gold complexes on XO interaction such as electronic inductive effect and steric
activity bulky enzyme inhibition activity.
Compound IC50 (μM) r2
3a 1.170 ± 0.052 0.998
3.3.2 | Antimicrobial activity studies
3b 1.131 ± 0.071 0.995
3c 0.728 ± 0.028 0.999 As can be seen from Table 3, the synthesized complexes
3d 0.409 ± 0.033 0.944 showed high antibacterial activities against both E. coli
3e 2.681 ± 0.091 0.982 (ATCC 25922) [Gram (
)] and B. subtilis (ATCC 21332)
3f 0.987 ± 0.040 0.988 [Gram (+)] species and antifungal activity against
C. albicans (ATCC 60193). Compared to the standard
3g 0.747 ± 0.031 0.997
cefotaxime, all synthesized gold complexes showed anti-
Allopurinol 3.429 ± 0.029 0.986
microbial effects. In addition, it is clearly understood

TABLE 3 Minimum inhibition concentration (MIC) values of the complexes

Minimum Inhibition Concentration (μg/ml)

Bacteria Fungus

Complexes Escherichia coli (ATCC 25922) Bacillus subtilis (ATCC 21332) Candida albicans (ATCC 60193)
3a 3.125 μg/ml 100 μg/ml 100 μg/ml
3b 6.25 μg/ml 250 μg/ml 200 μg/ml
3c 25 μg/ml 50 μg/ml 100 μg/ml
3d 3.125 μg/ml 6.25 μg/ml 100 μg/ml
3e 18.75 μg/ml 50 μg/ml 50 μg/ml
3f 200 μg/ml 200 μg/ml 200 μg/ml
3g 12.5 μg/ml 50 μg/ml 100 μg/ml
Cefotaxime 0.25 mg/ml 0.25 mg/ml 0.125 mg/ml
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12 of 19 EKINCI ET AL.

from the results that although small doses of synthesized
complexes exhibited antibacterial activity against bacteria
in general, higher doses of the same complexes were
needed for the antifungal activity. Therefore, it was deter-
mined that 3d is the most effective complex in antibacter-
ial activity, and for the antifungal activity, it is 3e. In
bacterial strains, Bacillus subtilis is more active than
Escherichia coli because MIC values of the complexes for
E. coli are lower than the MIC values of the complexes
for B. subtilis, except 3f.
complexes may be the main reason for the differences in
the anticancer activity. However, a molecular docking
study was performed to determine the activation mecha-
nism of the Au-NHC complexes.
3.3.4 | DNA binding studies
Using pUC19 plasmid DNA, the DNA binding properties
of the produced gold complexes were studied. When

3.3.3 | Anticancer activity studies

The in vitro anticancer activity of 3a-g complexes was

tested against MCF-7 and Caco-2 cancer cell lines using
different concentrations of 3a-g. According to the results
in Table 4, the compounds 3c, 3g, 3b, 3d, and 3a showed
higher activity than Cisplatin in both cell types. In particu-
lar, 3a exhibited the highest anticancer property in both
cell types and showed 15–16 times higher anticancer activ-
ity (IC50 = 5.2 μM) than Cisplatin. The activity of the com-
pounds was generally similar in both cell lines. However,
complex 3e and 3f showed less activity than Cisplatin. The
F I G U R E 3 The electrophoretic mobility of the supercoiled
difference between these activities of complexes may be form (I), the open circular form (II), and the linear form (III) of
due to the difference in NHC ligands carried by the metal plasmid pUC19 is shown in the above agarose gel electrophoresis
center. The diversity in the lipophilic and hydrophilic image. pUC19 (0.025 μg/μl final concentration) (1–2) after
properties of the complexes can directly affect the antican- incubation for 24 h at 37 C with final concentrations of 75 and
cer activity. When the structures of the complexes were 150 μg/ml cisplatin controls (3–4), 3a complex (5–6), 3b complex
examined, it was observed that gold complexes with NHC (7–8), 3c complex (9–10), and 3d complex (11–12), respectively
containing pyridine and phthalimide groups as N-
substituents exhibited high anticancer activity in low con-
centrations in both cancer cell lines. The 3e and 3f com-
plexes, which can show the inhibition value shown by the
3a-d complexes only at high concentrations, contain
straight aromatic and morpholine groups as N-substituents
on NHCs. We believe that this structural difference in the

TABLE 4 IC50 values of the complexes on MCF-7 and Caco-2

cell lines

IC50 (μM)

Complexes MCF-7 Caco-2

3a 5.2 ± 2.0 5.2 ± 0.9
3b 14.6 ± 3.0 28.3 ± 0.8
3c 14.3 ± 0.2 16.7 ± 0.3
F I G U R E 4 The electrophoretic mobility of the supercoiled
3d 16.6 ± 0.4 25.0 ± 0.2
form (I), the open circular form (II), and the linear form (III) of
3e 152.4 ± 1.5 152.7 ± 2.2 plasmid pUC19 are shown in the above agarose gel electrophoresis
3f 107.8 ± 6.4 142.3 ± 6.5 image. pUC19 (0.025 μg/μl final concentration) (1–2) after
3g 16.8 ± 0.2 20.9 ± 0.5 incubation for 24 h at 37 C with final concentrations of 75 and
150 μg/ml cisplatin controls (3–4), 3e complex (5–6), 3f complex
Cisplatin 81.1 ± 1.0 76.6 ± 6.0
(7–8), and 3g complex (9–10), respectively
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EKINCI ET AL. 13 of 19

circular plasmid DNA was used in the gel electrophoresis to the gold complex, the supercoiled form of DNA
procedure, the supercoiled form of DNA (I) in the (I) moves slowly compared to pUC19 not binding to the
agarose gel moved quickly. However, if DNA binds complex.

FIGURE 5 The 2D and 3D binding interactions of the 3c, 3d, 3g and allopurinol within the active pocket of the xanthine oxidase enzyme
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14 of 19 EKINCI ET AL.

DNA binding studies were performed using agarose
gel electrophoresis to determine the interaction ability of
Au-NHC complexes with supercoiled pUC19 plasmid
DNA. The DNA-binding properties of the Au-NHC com-
plexes can be examined thanks to their DNA-binding
abilities. Figures 3 and 4 show the movement behaviors
of pure plasmid DNA, plasmid DNA with Cisplatin as
controls, and plasmid DNA treated with 3a, 3b, 3c, 3d,
3e, 3f, and 3g in increasing amounts as 75 and 150 μg/ml
final concentrations of the Au-NHC complexes with
pUC19 plasmid DNA.
The slightly binding of 3a, 3b, 3c, 3d, 3e, 3f, and 3g
complexes at varying concentrations causes a
concentration-dependent delay in the supercoiled plas-
mid DNA motility. Compared to lower concentrations of
each complex, supercoiled form (I) of the plasmid moves
fast with increasing concentrations of 3b, 3c, 3d from
Figure 3 and 3e, 3f, and 3g from Figure 4, respectively.
The band patterns for 3a, on the other hand, are distinct
from those of other gold complexes. For 150 μg/ml final
concentrations of 3c and 75 and 150 μg/ml final concen-
trations of 3d, there are no open circular form (II) and

T A B L E 5 Binding energy (kcal/mol), interaction sites of the studied compounds with xanthine oxidase, antibacterial, antifungal, and
MCF-7 cell lines

Binding energy
(kcal/mol) Interaction site
Xanthine oxidase inhibitory activity
3C
8.3 SER 307, SER 344, ALA 302, THR 79, GLY 260, PRO 76, ASN 288, ILE 66, ALA 304, VAL 345,
VAL 259, LEU 55, VAL 83, PHE 68, LEU 80, ALA 282, TRP 283
3D
10.8 ALA 301, LYS 256, ILE 353, ILE 264, GLY 349, VAL 259, LEU 404, GLY 350, VAL 258, GLY 260,
ASN 261, GLU 263, THR 354, GLU 267, ARG 394,
3G
9.6 LYS 340, LEU 127, ILE 66, PHE 68, LEU 313, LYS 310, HIS 67, GLU 309, GLU 138, THR 126,
ALA 142, SER 69, SER 344, SER 306
Allopurinol
6.1 LEU 998, PHE 1272, ALA 1273, ASP 1276, CYS 999, TYR 977, ALA 938, VAL 932, ALA 931, ARG
1279, SER 928, LYS 1275
Antibacterial activities
3D
8.4 TYR 100, LYS 15, ARG 92, PHE 159, GLN 101, SER 96, VAL 51, GLU 37, SER 69, PRO38, ARG
48, ASP 91, ARG 36, THR 16, GLU 11, PHE 66
Cefotaxime
7.1 ARG 92, LYS 15, THR 16, GLY 14, SER 13, GLU 11, TYR 100, PHE 66, SER 96, ARG 70, PRO 10,
GLY 12, ARG 141
Antifungal activities
3E
7.3 ARG 208, THR 57, ASP 54, VAL 155, ARG 52, PHE 9, LEU 10, SER 56, GLU 6, GLN 156, LEU 92,
ASP 89, GLU 152, ARG 83, ARG 60, TYR 13, LEU 159
Cefotaxime
7.3 SER 201, TYR 205, PHE 212, MET 209, THR 175, MET 177, TYR 122, LEU 147, GLU 124, TYR
151, VAL 194, ALA 197, SER 198, GLN 202, PRO 206, GLU 207, LYS 239, ARG 208
Anticancer activities (MCF-7)
3A
7.3 CYS 1043, VAL 914, LEU 1033, LEU 838, VAL 846, ALA 864, VAL 912, LYS 866, GLU 833, GLY
839, VAL 897
3B
6.1 ASN 1031, ARG 1030, LEU 1033, ALA 864, LEU 838, VAL 846, GLY 839, LYS 866, GLU 883, VAL
897, VAL 914, PHE 916, CYS 917, CYS 1043, GLY 920
3C
7.5 VAL 914, CYS 1043, LEU 887, VAL 897, VAL 846, LYS 866, ALA 864, LEU 838, LEU 1033, GLY
839, ASP 1044, GLU 833, CYS 917, PHE 916
3D
8.6 VAL 846, PHE 916, LEU 838, ALA 864, LYS 866, VAL 914, LEU 1033, GLY 644, GLY 839, CYS
917, CYS 920
3G
9.1 ARG 1025, ASP 1044, VAL 897, LEU 887, VAL 896, CYS 1022, ILE 890, LEU 1017, ASP 1026, HIS
1024, CYS 1043, ILE 1042
Cisplatin
3.4 ASN 1038, SER 1035, PHE 919, GLU 1036, LYS 1037, LEU 1034, TYR 925, LEU 998, HIS 1002,
TYR 1006
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15 of 19

The 2D and 3D binding interactions of the 3d and cefotaxime within the active pocket of the antibacterial enzyme.

The 2D and 3D binding interactions of the 3e and cefotaxime within the active pocket of the antifungal enzyme
FIGURE 6

FIGURE 7
EKINCI ET AL.
 10990739, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/aoc.6811 by Zhejiang University, Wiley Online Library on [17/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
16 of 19 EKINCI ET AL.

linear form (III) of the plasmid, as shown in Figure 3. controls impacted all kinds of pUC19 plasmids (3–4)
The oxidizing actions of 3c and 3d complexes may be in both Figures 3 and 4, compared to pure pUC19
responsible for this condition. Furthermore, Cisplatin plasmids (1–2).

F I G U R E 8 The 2D and 3D binding

interactions of the 3a, 3b, 3c, 3g, and
Cisplatin within the active pocket of the
MCF-7 cell line

EKINCI ET AL.
3.4 | Molecular docking studies
Molecular studies were performed to determine the
binding mode of all active compounds against the XO
enzyme, antibacterial, antifungal, and MCF-7 cell lines.
In this study, the highest binding energy was calculated
for 3d with
10.8 kcal/mol, while the lowest binding
energy was determined for 3c with
8.3 kcal/mol. Car-
bon hydrogen interaction was observed with Ala 301, π-
cation with Lys 256, π-σ bond interaction with Ile 353, Ile
264 and many alkyls/π-alkyl and Van der Waals interac-
tions have been identified between 3d and amino acid
residue. The binding energies of 3g and allopurinol
were calculated as
9.6 and
6.1 kcal/mol, respectively.
It was observed that the binding energy of Au-complexes
is lower than the standard drug (allopurinol). The bind-
ing energy of the complexes with protein is shown in
Figure 5 and Table 5.
The docking studies were also performed to evaluate
the antibacterial and antifungal activities of the active
Au-complexes and compared the results with in vitro
studies. The detailed 2D and 3D binding interactions of
Au-complex (3d) within the active site of the bacterial
protein as shown in Figure 6 and Table 5. The binding
energy was calculated for 3d at
8.4 kcal/mol and cefo-
taxime at
7.1 kcal/mol. The interacting amino acid resi-
due with 3d is Tyr 100, Lys 15, Arg 92, Phe 159, Gln
101, Ser 96, Val 51, Glu 37, Ser 69, Pro38, Arg 48, Asp
91, Arg 36, Thr 16, Glu 11, Phe 66. π-cation, π-alkyl, and
Vaan der Waals interactions have been observed between
the Au-complex (3d) and amino acid residues. The chlo-
rine group in 3d complexes showed alkyl interaction with
Phe 366 amino acid residue. The chlorine has electron-
withdrawing properties, destabilizing the bacterial cell
membrane and leading to cell death. So it might be
assumed that the chlorine interaction with amino acid
plays an essential role in antibacterial activity.
The antifungal activity of the complex 3e was also cal-
culated. The 2D and 3D binding interactions within the
active site are shown in Figure 7 and Table 5. The bind-
ing energy was calculated for 3d at
7.3 kcal/mol and
cefotaxime at
7.3 kcal/mol. As shown in Figure 7, the
3e showed hydrogen bond interaction with Arg 208, car-
bon-hydrogen bond with Asp 54, Thr 57, π-σ with Val
155, alkyl, π-alkyl, and many Vaan der Waals interactions
had identified between the Au-complex (3e) and amino
acid residue. The chlorine group in the 3e complex has
also shown interaction, indicating that the electron-
withdrawing property of the chlorine makes them a
potential antifungal compound.
The docking study was performed against the MCF-7
cell line. The docking study was performed for those
compounds which showed in vitro anticancer activity.
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17 of 19
All the compounds showed good anticancer activity. The
binding energies of 3a, 3b, 3c, 3d, 3g, and Cisplatin were
calculated as
7.3,
6.1,
7.5,
8.6,
9.1, and

3.4 kcal/mol, respectively. The highest binding energy
was observed with compounds 3g and 3d, while low
binding energy was observed with 3b and Cisplatin. The
2D and 3D structures of the active Au-complexes are
shown in Figure 8 and Table 5.
4 | CONCLUSIONS
Novel Ag-NHC and Au-NHC complexes have been syn-
thesized and successfully employed for the in vitro assay
of XO inhibitory activity, antimicrobial activity, antican-
cer activity, and DNA binding experiments. The molecu-
lar docking calculation was performed for Au-NHC
complexes to determine the binding mode of all active
Au-NHC complexes against the XO enzyme, antibacter-
ial, antifungal, and MCF-7 cell lines. The crystal struc-
tures of 3b and 3f were obtained using the single-crystal
X-ray diffraction method. Structural analysis indicated
that the bond angles around the Au atom of the com-
pounds differ only slightly from their expected value of
180 for linear geometry. The intermolecular AuAu
interactions play a significant role in stabilizing and
packing the crystal structures of both complexes. Withal,
the crystal structures of both compounds are character-
ized by the presence of intermolecular C–Hπ and ππ
interactions. Anticancer activity test results showed that
all gold complexes exhibited more inhibition activity than
allopurinol. It was determined that 3d is the most effec-
tive complex in antibacterial activity, and for the antifun-
gal activity. In particular, 3a exhibited the highest
anticancer property in both cell types and showed 15–16
times higher anticancer activity than Cisplatin. Gel elec-
trophoresis experiments also showed that 3a interacts
strongly with the DNA. These results show that the anti-
cancer activity potential of Au(I)-NHC complexes with
high cancer cell selectivity is closely related to the struc-
ture of NHC ligands.
ACKNOWLEDGMENTS
This work was financially supported by Inönü Üniversi-
tesi (Project No: FBG-2021-2562) and thanks to Dokuz
Eylül University for the use of the Oxford Rigaku Xcali-
bur Eos Diffractometer (purchased under University
Research Grant No: 2010.KB.FEN.13).
CONFLICT OF INTEREST
The authors declare that they have no known competing
financial interest or personal relationships that could have
appeared to influence the work reported in this paper.

18 of 19
A U T H O R C ON T R I B U T I O NS
Orhan Ekinci: Investigation; methodology; resources.
Mitat Akkoç: Formal analysis; investigation; methodol-
ogy; project administration; resources; validation.
Siraj Khan: Software; validation; visualization. Sedat
Yasar: Data curation; formal analysis; funding acquisi-
tion; investigation; methodology; project administration;
resources; supervision. Canbolat Gürses: Investigation;
methodology. Samir Abbas Ali Noma: Formal analysis;
methodology. Sevgi Balcıog
lu: Investigation; methodol-
ogy. Betül Sen: Investigation; resources; software.
Muhittin Aygün: Investigation; methodology; software.
_
Ismet Yılmaz: Project administration.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are avail-
able from the corresponding author upon reasonable
request.
ORCID
Orhan Ekinci https://orcid.org/0000-0003-2883-6013
Mitat Akkoç https://orcid.org/0000-0001-8641-8958
Siraj Khan https://orcid.org/0000-0003-1948-452X
Sedat Yaşar https://orcid.org/0000-0001-7285-2761
Canbolat Gürses https://orcid.org/0000-0002-4085-0224
Samir Noma https://orcid.org/0000-0003-4165-0045
Sevgi Balcıo
glu https://orcid.org/0000-0003-0724-4772
Betül Sen https://orcid.org/0000-0001-7846-8090
Muhittin Aygün https://orcid.org/0000-0001-9670-9062
_
Ismet Yılmaz https://orcid.org/0000-0002-7204-1050
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