Polynuclear Ag(I)-N-heterocyclic carbene complexes: synthesis, electrochemical and in vitro anticancer study against human breast cancer and colon cancer

Article abstract of DOI:10.1080/00958972.2019.1632837

Four new bis-imidazolium salts 3–6 and their di- and polynuclear silver(I)–N-heterocyclic carbene (Ag(I)–NHC) complexes 7–10 were synthesized and characterized by using various analytical techniques. Single-crystal studies of 8 revealed a dinuclear struct

Full text of DOI:10.1080/00958972.2019.1632837

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Polynuclear Ag(I)-N-heterocyclic carbene
complexes: synthesis, electrochemical and in vitro
anticancer study against human breast cancer and
colon cancer
Aqsa Habib, Muhammad Adnan Iqbal & Haq Nawaz Bhatti
To cite this article: Aqsa Habib, Muhammad Adnan Iqbal & Haq Nawaz Bhatti (2019): Polynuclear
Ag(I)-N-heterocyclic carbene complexes: synthesis, electrochemical and inꢀvitro anticancer
study against human breast cancer and colon cancer, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2019.1632837
To link to this article: https://doi.org/10.1080/00958972.2019.1632837
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2019.1632837
Polynuclear Ag(I)-N-heterocyclic carbene complexes:
synthesis, electrochemical and in vitro anticancer study
against human breast cancer and colon cancer
Aqsa Habib, Muhammad Adnan Iqbal and Haq Nawaz Bhatti
Department of Chemistry, University of Agriculture Faisalabad, Punjab, Pakistan
ABSTRACT
ARTICLE HISTORY
Received 8 February 2019
Accepted 1 June 2019
Four new bis-imidazolium salts 3–6 and their di- and polynuclear
silver(I)–N-heterocyclic carbene (Ag(I)–NHC) complexes 7–10 were
synthesized and characterized by using various analytical techni-
ques. Single-crystal studies of 8 revealed a dinuclear structure
whereas that of 9 revealed an interesting polynuclear structure.
The quasi-reversibility of redox events in electrochemical study of
9 indicated that reduction produces unstable decomposed spe-
cies and oxidation may be ascribed to these decomposed species.
All the compounds were studied for their in vitro anticancer study
against human breast cancer (MDA-MB-231) and colon cancer
(HCT-116) cells. The study revealed that Ag(I)–NHC complexes are
comparatively more potent than NHC precursors (salts). MDA-MB-
231 cells appeared more sensitive to complexes than HCT-116
cells while reverse is the case for NHC salts.
KEYWORDS
Polymeric; quasi-reversible;
redox process; HCT-116
CONTACT Muhammad Adnan Iqbal
adnan.iqbal@uaf.edu.pk; Haq Nawaz Bhatti
hnbhatti2005@yahoo.com
Supplemental data for this article is available online at https://doi.org/10.1080/00958972.2019.1632837.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
A. HABIB ET AL.
1. Introduction
Ag(I)–N-heterocyclic carbene complexes (Ag(I)–NHCs) are moisture and air stable clas
of compounds which can be easily synthesized by simple reported protocols including
(1) the reaction of free carbenes with suitable sources of silver, (2) the reaction of sil-
ver sources with azolium salts in alkaline medium to generate alkaline phase-transfer
conditions and (3) the reaction of basic silver sources with azolium salts [1–3].
The striking features of NHC ligands make them suitable for the formation of metal-
based polymeric frameworks which is still least studied area [4]. Polydentate-NHC ligands,
on the account of their chelating or pincer effects, have become the pronounced species
to target mixed-valence, polymeric or high-oxidation state complexes by producing stable
metal–ligand system with a variety of coordination spheres and remarkable chemical and
medicinal features [5–7]. Polymeric silver(I)–NHC systems with aliphatic or aromatic moi-
eties as linkers have become a novel class of silver-based drugs with pronounced anti-
cancer potential on the account of their biocompatibility [8] but only few examples of
silver-based multinuclear/supramolecular coordination motifs having weak Ag–Ag interac-
tions (3.1–3.4 Å) or metal–carbon/metal–heteroatom interactions have been reported [9].
Breast cancer is the most substantial sort of human non-skin malignancy and the second
major cause of women mortality in western countries, while human colon cancer is among
the most common types of cancer and major cause of deaths all over the world [10, 11].
Numerous silver-based drugs are already in the market for treatment of both cancer types,
but almost all have several side effects including allergy, fatigue, seizures, bleeding,
thrombocytopenia, renal damage, alopecia, peripheral neuropathy, blood dyscrasias, hepato-
toxicity and nephrotoxicity, destruction of central nervous system and oligodendrocytes
[12]. The major cause of these side-effects is the fast release of Agþ ions which in turn
destroys the normal cells in addition to diseased cells during their activity [13]. The disad-
vantages of these market drugs have attracted the attention of researchers to introduce
new metallodrugs having little or no side-effects [14–21]. Recently, gold– and silver(I)–NHC
complexes have been examined for their anticancer potential against human breast cancer
and colon cancer cell lines [22, 23]. Ag(I)–NHC complexes are surpassing some already avail-
able market silver-based drugs [24]. The complexes bearing various substituents have been
observed to pose strong biopotential due to sustained release of Agþ ions which depends
on their electronic and steric features influencing their lipophilicity [25].
In previous studies, we have explored the anticancer potential of substituted benzi-
midazolium-based silver complexes [2, 3]. This manuscript describes augmentation of
the previous studies. We report the synthesis of three dinuclear and one unique poly-
meric Ag(I)–NHC complex. To best of our knowledge, such a polymeric structure with-
out silver–silver bonding as we document for 9 has not yet been reported. We have
afforded these complexes from bis-imidazolium based NHC ligands and studied their
electrochemical and anticancer properties.
2. Materials and methods
2.1. Chemicals
Imidazole, 1-methyl imidazole, alkyl halides (1,5-dibromopentane, bromodiphenylme-
thane, 1,3-bis(bromomethyl)benzene, 1,4-bis(bromomethyl)benzene, 1,4-dibromobutane)

JOURNAL OF COORDINATION CHEMISTRY
3
and Ag2O were purchased from Sigma Aldrich. Phosphate buffered saline (PBS), penicil-
lin/streptomycin solution (PS), 5-fluorouracil and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) reagent were purchased from Sigma-Aldrich. Human colon
cancer (HCT-116) and breast cancer (MDA-MB-231) cells were obtained from the
American type culture collection (Rockville, MD, USA).
2.2. Instrumentation
The melting points of the synthesized compounds were evaluated with the help of a
Stuart Scientific SMP-1 (UK) instrument. Low-boiling point solvents were removed by
an EYELA 1L Rotary Evaporator N-1001V-WD. To monitor the alkylation reactions,
Agilent Technologies 7890A GC/MS (Gas chromatography/Mass spectrometry) was
used. The FTIR (Fourier transform infrared spectroscopy) spectra were obtained with
1
an ALPHA-P, compact FTIR spectrometer. H, 13C, 31P and 19F NMR spectra were taken
on a Bruker Avance-300 and Avance-400 or on a Varian Inova-300 spectrometer in
deuterated solvents. Chemical shifts are given in ppm while J values are given in
Hertz. Spectral assignments were achieved by 1H–13C HSQC (Heteronuclear single
quantum coherence) experiments. The elemental analysis of the synthesized com-
pounds was carried out by 180508E; Fisons Instruments EA 1108 CHNS analyzer. The
crystallographic data for 8 and 9 were collected on a Bruker APEX II as reported [2, 3].
The electrochemical measurements were performed to investigate the redox proper-
ties of compounds by testing their reactivity toward chemical oxidant using a BASI-
Epsilon potentiostat, at a scan rate of 100 mV s–1 under N2 at room temperature.
Tetrabutyl ammonium hexaflouro phosphate [nBu4N][PF6] (1 mM in CH3CN) was used
as a supporting electrolyte and the sample was 1 mM. Ag/AgCl was used as reference,
platinum disk as working and platinum wire as counter electrodes. For calibration,
ferrocene (1 mmol) was added as an internal standard. IC50 values were determined
with a TECAN Multi-mode microplate reader obtained from USA.
2.3. Synthesis of pre-ligands (1 and 2)
2.3.1. Methyl imidazole (1)
Compound 1 was purchased from Sigma Aldrich.
2.3.2. 1-Benzhydryl 1H-imidazole (2)
A mixture of imidazole (0.68 g, 10 mmol), bromodiphenylmethane (2.47 g, 10 mmol)
and KOH (0.84 g, 15 mmol) in DMSO (20 mL) was stirred at room temperature until the
reaction was complete (as monitored by GC-MS). To remove DMSO (Dimethyl
Sulfoxide), the mixture was poured in cold distilled water (200 mL) and extracted the
product with a mixture of ether and hexane (25 mL, 1:1) and washed it with cold
water. The product contained in organic layer was isolated and purified by adding
MgSO4 to remove residual water. After solvent removal, oily product was obtained.
Yield 1.52 g (65%). GC-MS, m/z, (%): ([C16H14N2]þ 234.3 (100). FTIR (cmꢀ1): 2958, 2872
(Caliph-H str), 1494, 1457, 1364 (C[dbond]Nimid str), 1285, 1259, 1150 (Carom-Nimid str),
1
841, 738, 632 (Carom-Himid ben). H NMR (400 MHz, CDCl3), d (ppm): 6.26 (1H, s, 1 ꢁ CH),

4
A. HABIB ET AL.
6.86 (1H, d, Himid, J ¼ 7.53 Hz), 7.23 (4H, d, Himid, J¼ 7.5 Hz), 7.26 (1H, d, Himid, J¼ 7.81 Hz),
7.33 (4H, t, Himid, J¼ 7.3 Hz), 7.96 (1H, N-CH-Nimid). Anal. Calcd for C16H14N2 (%): C, 80.02;
H, 6.02; N, 11.96. Found C, 80.61; H, 6.31; N, 11.56.
2.4. Synthesis of ligands (3–6)
2.4.1. 1-Methyl-3-(4-(3-methyl-1H-imidazol-3-ium-1-yl) butyl)-1H-imidazol-3-ium
bromide (3)
A mixture of 1 (1.6 mL, 20 mmol) and 1,4-dibromobutane (6 mL, 50 mmol, excess) was
refluxed at 120 ꢂC for 48 h and monitored the reaction by GC-MS. Upon completion of
reaction, oily layer settled down, decanted, washed the residue with ether (3 ꢁ 3 mL)
and dried at 40 ꢂC. Compound 3 was formed as white powder. Yield 1.5 g (75%); m.p.
113–115 ꢂC. FTIR (cmꢀ1): 2936, 2873 (Caliph-H str), 1566, 1456, 1400, 1363
1
(C[dbond]Nimid str)), 1163 (Carom-Nimid str), 823, 788, 744, 623 (C-Himid ben). H NMR
(400 MHz, CDCl3), d (ppm): 1.76 (4H, t, CH2, J ¼ 3.02 Hz), 3.83 (6H, s, 2ꢁ CH3), 4.21 (4H,
s, 2ꢁ R-CH2-N, J ¼ 6.65 Hz), 7.73 (4H, d.t, Himid, J ¼ 7.92 Hz), 9.11 (2H, s, N-CH-N). 13C
NMR (125.72 MHz, CDCl3), d (ppm): 26.5 (CH3-N), 36.2 (R-CH2-R), 48.4 (R-CH2-N), 122.6,
124.2 (Cimid), 137.0 (N[dbond]C[dbond]N). 19F (470.4 MHz, CDCl3), d (ppm): –72.2 (d,
6F). 31P (202.4 MHz, CDCl3), d (ppm): –145.1 (h, 1P). Anal. Calcd for C12H20N4Br2 (%): C,
37.92; H, 5.30; N, 14.74. Found C, 37.21; H, 5.12; N, 14.87.
2.4.2. 1-Methyl-3-(5-(3-methyl-1H-imidazol-3-ium-1-pentyl)-1H-imidazol-3-ium brom-
ide (4)
The same procedure for formation of 3 was followed to synthesize 4 using 1,5-dibro-
mopentane (4 mL, 29.3 mmol, excess). After work up, 4 was obtained as white powder.
Yield 2.16 g (55%); m.p. 120–122 ꢂC. FTIR (cmꢀ1): 3420 (Caliph-Nimid), 2935, 2862 (Caliph-H
str), 1576, 1561, 1463, 1384, 1365 (C[dbond]Nimid str), 1184 (Carom-Nimid), 875, 840, 771,
1
756, 621 (C-Himid ben). H NMR (400 MHz, CDCl3), d (ppm): 1.63 (2H, m, CH2), 2.11 (4H,
m, 2ꢁ CH2), 4.12 (6H, s, 2ꢁ CH3), 4.50 (4H, t, R-CH2-N), 7.24 (2H, s, Himid), 7.81 (2H, s,
Himid), 10.50 (2H, s, N-CH-N). 13C NMR (125.72 MHz, CDCl3), d (ppm): 28.9 (CH2), 30.9
(CH2), 36.8 (CH2), 49.2 (CH3-N), 65.8 (CH2-N), 122.8 (Nimid[dbond]C[dbond]Nimid). Anal.
Calcd for C13H22N4Br2 (%): C, 39.61; H, 5.63; N, 14.21. Found C, 39.21; H, 5.69; N, 14.99.
2.4.3. 1-Methyl-3-(3-((3-methyl-1H-imidazole-3-ium-1-methyl) benzyl)-1H-imidazol-
3-ium chloride (5)
The same procedure for formation of 3 was followed to synthesize 5 using 1,3-bis(bro-
momethyl)benzene (1.75 g, 10 mmol). After work up, 5 was obtained as white powder.
Yield 2.4 g (72%); m.p. 170 ꢂC. FTIR (cmꢀ1): 3365 (Caliph-Nimid), 2983, 2870 (Caliph-H str),
1
1563, 1455 (C[dbond]Nimid str), 1172 (Carom-Nimid), 809, 789, 754, 623 (C-Himid ben). H
NMR (400 MHz, CDCl3), d (ppm): 3.87 (6H, s, 2ꢁ CH3), 5.42 (4H, s, 2ꢁ CH2), 7.39 (2H, d,
Himid, J ¼ 7.29 Hz), 7.48 (2H, m, Himid), 7.74 (4H, s, Himid), 9.21 (2H, s, N-CH-N). 13C NMR
(125.72 MHz, CDCl3), d (ppm): 35.9 (CH3), 51.53 (CH2), 122.4, 124.1, 128.6, 128.7, 129.7,
135.7 (Ar-C), 136.9 (N[dbond]C[dbond]N). Anal. Calcd for C16H20N4Cl2(%): C, 56.64; H,
5.94; N, 16.51. Found C, 56.21; H, 5.69; N, 16.99.

JOURNAL OF COORDINATION CHEMISTRY
5
2.4.4. 1-Benzhydryl-3-(4-(3-benzhydryl-1H-imidazol-3-ium-1-methyl)benzyl)-1H-imi-
dazol-3-ium bromide (6)
The same procedure for formation of 3 was followed to synthesize 6 using 2 (1.15 mL,
5 mmol) and 1,4-bis(bromomethyl)benzene (0.66 g, 2.5 mmol). After work up, 6 was
obtained as white powder. Yield 1.19 g (65%); m.p. 120–125 ꢂC. FTIR (cmꢀ1): 2987
(Caliph-H str), 1543, 1448, 1418 (C[dbond]Nimid str), 1189, 1008 (Carom-Nimid str), 754.8,
1
702.6 (C-Himid ben). H NMR (400 MHz, CDCl3), d (ppm): 3.56 (4H, s, N-CH2-Ar), 5.81 (2H,
s, Ph2-CH-N), 7.52 (14H, m, Ar-H), 7.80 (8H, d, Ar-H, J ¼ 7.31 Hz), 9.71 (2H, s, N-CH-N).
13C NMR (125.72 MHz, CDCl3), d (ppm): 50.2 (Ar-CH2-N), 64.7 (Ph2-CH-N), 127.3, 127.4,
128.93, 129.8, 131.8, 134.9 (Ar-C), 136.5 (Ph2-CH-N), 142.8 (N[dbond]C[dbond]Nimid).
Anal. Calcd for C40H36N4Br2(%): C, 65.58; H, 4.95; N, 7.65. Found C, 65.65; H, 4.69;
N, 7.69.
2.5. Synthesis of Ag(I)–NHC complexes (7–10)
2.5.1.
Bis(1-methyl-3-(5-(3-methyl-1H-imidazol-3-ium-1-butyl)-1H-imidazol-3-ium
silver) bis(hexafluorophosphate) (7)
A mixture of 3 (2.6 g, 7 mmol) and silver oxide (3.5 g, 14 mmol) in methanol (120 mL)
was stirred for 48 h at room temperature in complete darkness. After filtration of black
suspension, aqueous solution of potassium hexafluorophosphate (2.54 g, 14 mmol) was
added to exchange the counterions as the complex with halide counterions was
highly photosensitive/unstable in solution form and decomposed during isolation
attempts. The mixture was allowed to stir for 4 h at room temperature. The precipi-
tates obtained were filtered and dried at room temperature. Compound 7 was
obtained as beige powder. Yield 3.8 g (62%); m.p. 240–242 ꢂC. FTIR (cmꢀ1): 2857,
(Caliph-H str), 1523, 1414, 1401, 1313 (C[dbond]Nimid str), 821, 718 (C-Himid ben). 1H
NMR (400 MHz, DMSO-d6), d (ppm): 1.91 (8H, m, 4ꢁ CH2), 3.69 (12H, s, 4ꢁ CH3), 4.31
(8H, t, 4ꢁ R-CH2-N, J ¼ 6.75 Hz), 7.50 (8H, s, Ar-H). 13C NMR (125.72 MHz, DMSO-d6), d
(ppm): 31.0 (CH2), 38.3 (CH2), 51.7 (CH3-N), 122.1, 123.8 (Cimid), 179.2, 180.7 (d, Ag-C-
Ag, J ¼ 190.6 Hz). 19F (470.4 MHz, DMSO-d6, d ppm) –72.5 (d, 6F). 31P (202.4 MHz,
DMSO-d6, d ppm) –144.3 (h, 1P). Anal. Calcd for C24H36Ag2F12N8P2 (%): C, 30.59; H,
3.85; N, 11.89. Found C, 31.10; H, 3.90; N, 11.56.
2.5.2. Bis(1-methyl-3-(5-(3-methyl-1H-imidazol-3-ium-1-pentyl)-1H-imidazol-3-ium
silver) bis(hexafluorophosphate) (8)
The same procedure for the formation of 7 was followed to synthesize 8 using 4
(2.7 g, 7 mmol). Compound 8 was obtained as beige powder. Yield 3.5 g (57%); m.p.
240 ꢂC. FTIR (cmꢀ1): 2957, 2872 (Caliph-H str), 1567, 1457, 1404, 1398 (C[dbond]Nimid
1
str), 862, 740 (C-Himid ben). H NMR (400 MHz, DMSO-d6), d (ppm): 1.21 (4H, m, CH2),
1.85 (8H, m, 2ꢁ CH2), 3.67 (6H, s, 2ꢁ CH3), 4.11 (8H, t, R-CH2-N, J ¼ 6.79 Hz), 7.42 (4H,
s, Ar-H), 7.50 (4H, s, Ar-H). 13C NMR (125.72 MHz, DMSO-d6), d (ppm): 23.6 (CH2), 31.0
(CH2), 38.2 (CH2), 51.5 (CH3-N), 122.2, 123.5 (Cimid), 179.0, 180.9 (d, Ag-C-Ag,
J ¼ 190.6 Hz). 19F (470.4 MHz, DMSO-d6), d (ppm): –72.5 (d, 6F). 31P (202.4 MHz, DMSO-
d6), d (ppm): –144.3 (h, 1P). Anal. Calcd for C26H40Ag2F12N8P2 (%): C, 32.18; H, 4.16; N,
11.55. Found C, 32.18; H, 4.15; N, 11.41.

6
A. HABIB ET AL.
2.5.3. Bis(1-methyl-3-(3-((3-methyl-1H-imidazole-3-ium-1-methyl)benzene)-1H-imi-
dazol-3-ium silver) bis(hexafluorophosphate) (9)
The same procedure for the formation of 7 was followed to synthesize 9 using 5
(2.3 g, 7 mmol). Compound 9 was obtained as beige powder. Yield 2.4 g (53%); m.p.
175–180 ꢂC. FTIR (cmꢀ1): 2936, 2873 (Caliph-H str), 1566, 1460, 1400, 1360
1
(C[dbond]Nimid str), 827, 742 (C-Himid ben). H NMR (400 MHz, DMSO-d6), d (ppm): 3.67
(12H, s, 4ꢁ CH3), 5.21 (8H, s, 4ꢁ CH2), 7.12 (6H, d, Ar-H, J ¼ 37.58 Hz), 7.31 (2H, m, Ar-
H), 7.49 (8H, d, Ar-H, J ¼ 16.55 Hz). 13C NMR (125.72 MHz, DMSO-d6), d (ppm): 31.1
(CH3), 54.0 (CH2), 118.5 (N-CHimid), 125.9, 127.2, 129.7, 138.3 (Ar-C), 179.9,181.8 (s,
N[dbond]C[dbond]N, J ¼ 190.5 Hz). 19F (470.4 MHz, DMSO-d6), d (ppm): –73.2 (d, 6F).
31P (202.4 MHz, DMSO-d6, d ppm) –144.2 (h, 1P). Anal. Calcd for C32H36Ag2F12N8P2 (%):
C, 37.01; H, 3.49; N, 10.79. Found C, 37.12; H, 3.51; N, 10.85.
2.5.4. Bis(1-benzhydryl-3-(4-((3-benzhydryl-1H-imidazol-3-ium-1-methyl) benzene)-
1H-imidazol-3-ium silver) bis(hexafluorophosphate) (10)
The same procedure for the formation of 7 was followed to synthesize 10 using 6
(2.1 g, 3 mmol) and silver oxide (1.6 g, 6 mmol). Complex 10 was obtained as beige
powder. Yield 1.1 g (55%); m.p. 120–125 ꢂC. FTIR (cmꢀ1): 2905 (Caliph-H str), 1492, 1448
1
(C[dbond]Nimid str), 1183, 1259, 1049 (Carom-Nimid str), 772, 738 (C-Himid ben). H NMR
(400 MHz, DMSO-d6), d (ppm): 3.52 (8H, s, N-CH2-Ar), 5.41 (4H, s, Ph2-CH-N), 7.21 (8H, t,
Ar-H, J ¼ 7.3 Hz), 7.33 (24H, t, Ar-H, J ¼ 7.7 Hz), 7.377 (16H, d, 7.33 Hz). 13C NMR
(125.72 MHz, DMSO-d6), d (ppm): 49.1 (Ar-CH2-N), 63.2 (Ph2-CH-N), 127.1, 127.8, 128.9,
133.1, 137.4 (Ar-C), 142.6 (Ph2-CH-N), 193.2, 196.3 (s, C-Ag-C). 19F (470.4 MHz, DMSO-
d6), d (ppm): –72.6 (d, 6F). 31P (202.4 MHz, DMSO-d6), d (ppm): –143.8 (h, 1P). Anal.
Calcd for C80H68Ag2F12N8P2 (%): C, 58.34; H, 4.16; N, 6.8. Found C, 57.99; H, 4.56; N, 7.1.
MTT assay was performed according to reported method [2, 3].
3. Results and discussion
The syntheses of the NHC precursors and their silver complexes were successfully
achieved by following our reported protocol [2, 13, 22]. The complexes with halide
counterions are highly photosensitive/unstable in solution form and usually decom-
pose during isolation attempts, so as to get stable end product, the counterions were
exchanged in situ with hexafluorophosphate ions by adding two equivalent aqueous
solution of KPF6 after metallation step. Scheme 1 shows the simple steps of the syn-
theses of precursors and complexes. Fully sketched chemical structures of all the syn-
thesized compounds have been given in the Supplementary data file (Figure S1).
3.1. Characterization
The preliminary structural elucidation of the synthesized compounds was obtained
from FTIR analysis. Strong vibrational bands appeared at 3402–3424 cmꢀ1 due to
Caliph-Nimi stretching vibrations of the preligands 1 and 2 and ligands 3–6 [8, 13]
(Figures S2–S5). C[dbond]Nimidazole stretching vibrations appeared at 1350–1500 cmꢀ1
for 1 and 2 and 3–6, however, in the case of 7–10 these signals were observed in

JOURNAL OF COORDINATION CHEMISTRY
7
Scheme 1. Synthesis of N-alkylated-imidazoles (1 and 2), N,N0-dialkylated-imidazolium salts (3–6)
and Ag(I)–NHC complexes (7–10).
specific “four fingers (f.fs)” pattern [26] which provided evidence of complex formation
[27] (Figures S2–S7).
NMR studies further supported the evidences of synthesis. Ligands 3–6 displayed
1
alkyl group resonances at 6.0–8.0 ppm in H NMR spectra and a signal for the most
1
de-shielded acidic proton and carbon (NCHN) between 9.0 and 12.0 ppm in H NMR
and 140 and 150 ppm in 13C NMR spectra, respectively [13] (Figures S8–S14). The pro-
gress of the metalation reaction of 3–6 to form silver complexes 7–10 was evident
from disappearance of NCHN proton signal (at 9.0–12.0 ppm) which indicated
Ag(I)–NHC bonding [28]. This bonding was also confirmed from 13C NMR spectra
where these resonances (Ccarbene-Ag-Ccarbene) fall between 180 and 200 ppm [29] in
the form of doublet of doublet or in some cases singlets [30]. This behavior is evi-
dent in Figures S9–S13 where two singlets appeared in 13C NMR spectra of com-
plexes. Two-dimensional (HSQC) 1H–13C correlation NMR spectra were helpful to
1
assign signals of H and 13C spectra (Figures S15–S17) [31]. Appearance of septet
and doublet in 31P and 19F NMR spectra at –141.2 to –144.24 ppm and –71.3 to
–
–72.5 ppm, respectively, indicated the presence of PF6 counterions with complexes
(Figures S18 and S19).

8
A. HABIB ET AL.
Table 1. Crystal data and structure refinement details for 8 and 9.
Complex
8
9
Formula
Formula weight
C26H40Ag2F12N8P2ꢃCH3CN
C32H36Ag2N8F12P2
1038.35
1011.39
Crystal system
Monoclinic
Triclinic
Space group
P 21/n (14)
P1(2)
Unit cell dimensions
a (Å)
b (Å)
7.5193(11)
43.322(6)
8.8761(2)
11.3841(2)
c (Å)
12.1065(17)
90
101.872
90
3859.35
11.3846(2)
62.8050(10)
67.0390(10)
67.0610(10)
a (ꢂ)
b (ꢂ)
c (ꢂ)
V (Å3)
910.191
R-factor (%)
–
3.71
Z
4
2
Density (calcd) (g cm–3
)
1.741
1.190
1.894
10.374
516.0
Abs. coeff. (mm–1
F(000)
)
2024.0
0.23 ꢁ 0.19 ꢁ 0.17
100
CuK/a 1.54178
3.868, 73.046
–9:9; –53:51; –4:15
7653
Crystal size (mm)
Temperature (K)
Radiation (Å)
0.1 ꢁ 0.07 ꢁ 0.03
100
CuK/a 1.54178
4.520, 71.692
–9:10; –14:14; –14:14
9927
theta Min, max (ꢂ)
Data set
Tot.; Uniq. data
R (int)
2842
4059, 3.71
Nref, Npar
7762, 483
3565, 325
R, wR2, S
0.0537 (7423), 0.1416 (7653), 1.043
0.0371 (348), 0.0992 (3464), 1.089
3.2. X-ray crystallographic studies
Single-crystals of 8 and 9 were obtained by evaporation of their acetonitrile solution
at room temperature. Crystal data are given in Tables 1–3.
X-ray crystal data for 8 indicate that it is a dinuclear compound with molecular for-
mula C26H40Ag2F12N8P2, which crystallizes in monoclinic space group P-1(2) having
one cationic imidazolium core, two hexafluorophosphate anions and one acetonitrile
solvent molecule. Imidazolyl units were found on either side of the central pentyl
core. The two NHC moieties attached to one silver center are twisted by 5ꢂ. Ag–Ag
distances (4.21 Å) are longer than van der Waals radii (3.44 Å) of two silver atoms
which is an indication of lack of interaction between Ag–Ag centers of successive
dinuclear units [32], hence the molecule which appears as polymer is actually a dinu-
clear silver complex. Perspective view of 8 is shown in Figure 1.
X-ray crystal data of 9 with molecular formula C16H18AgN4F6P indicated that the
compound crystallizes in triclinic space group P-1(2) having one cationic bis-imidazo-
lium core and two hexafluorophosphate counter anions. The imidazolyl units were
found on either side of the central meta-xylyl core. The structure appeared as a unique
polymer (as shown in Figure 2). Various multinuclear and supramolecular motifs hav-
ing weak Ag–Ag interactions (3.1–3.4 Å) have been reported [6, 33–35] but according
to the best of our knowledge, polymeric structure without silver–silver interaction as
we document for 9 is novel in its nature. Perspective view of 9 is shown in Figure 3.
The internal imidazole-ring angles (N-C-N) at the carbene center were found between
104.3(4)ꢂ to 105.0(4)ꢂ in crystal structures of both complexes which are in agreement
with the reported values [22]. The bond angle between imidazole ring and pentyl

JOURNAL OF COORDINATION CHEMISTRY
9
Table 2. Selected bond lengths and angles of 8.
Bond lengths (Å)
Bond angles (ꢂ)
Dihedral angles (ꢂ)
Ag1-C1
Ag1-C14
Ag2-C23
C3-C10
C5-C6
C7-C8
C8-C9
C19-C20
2.087(5)
2.081(5)
2.084(5)
1.342(7)
1.522(8)
1.495(8)
1.572(9)
1.513(8)
C1-Ag1-C14
176.8(2)
C14-Ag1-C1-N1
C1-Ag1-C14-N5
C4-N1-C1-Ag1
C5-N2-C1-N1
C5-N2-C1-Ag1
C11-N3-C10-Ag2
C13-N4-C10-Ag2
C6-C7-C8-C9
84(4)
85(4)
–178.5(5)
–1.7(8)
C10-Ag2-C23
C14-N5-C15
C14-N6-C18
C23-N8-C26
Ag1-C1-N2
N1-C1-N2
175.4(2)
111.4(5)
124.8(5)
124.5(5)
129.0(4)
105.0(4)
104.8(4)
–1.7(8)
178.0(4)
178.6(5)
–163.0(5)
N3-C10-N4
Table 3. Selected bond lengths and angles of 9.
Bond lengths (Å)
Bond angles (ꢂ)
Dihedral angles (ꢂ)
Ag1-C13
N3-C12
N3-C14
N4-C15
C16-C18
C17-C18
C17-C22
C18-C19
C20-C21
2.082(3)
1.459(8)
1.386(5)
1.383(5)
1.496(8)
1.41(2)
1.41(2)
1.38(1)
1.39(1)
C13-Ag1-C2
C13-N3-C14
Ag1-C13-N3
Ag1-C13-N4
N3-C13-Ag1
N4-C13-Ag1
Ag1-C2-N1
Ag1-C2-N2
C13-Ag1-C2
176.7(2)
111.1(4)
128.8(3)
126.6(3)
128.8(3)
126.6(3)
128.7(3)
126.6(3)
176.7(2)
C1-N1-C2-Ag1
C2-N2-C4-C3
C5-N2-C2-N1
C5-N2-C2-Ag1
C4-N2-C2-Ag1
C2-N1-C3-C4
C1-N1-C3-C4
C3-N1-C2-Ag1
C13-Ag1-C2-N2
5.9(7)
0.1(6)
179.4(4)
5.8(7)
175.0(3)
0.0(6)
179.4(4)
174.8(4)
46(4)
moiety in 8 was 125.8(4)ꢂ for C1-N2-C5 and 124.7(5)ꢂ for C22-N7-C23 while between
imidazole ring and xylyl moiety of 9 was 111.6(6)ꢂ for N4-C16-C18 and 121.3(6)ꢂ for
N4-C16-C22. N-C and P-F bond distances in both structures were found in the range
1.350(7)–1.47(1) Å and 1.566(4)–1.609 (3) Å, respectively. Agþ ions were observed in
approximately linear coordination geometry between 175.4(2)ꢂ and 176.8(2)ꢂ for C-Ag-
–
C in both structures. PF6 ions bind to cationic core by feeble electrostatic C … F inter-
action (3.140 to 6.1 Å) in a 3-D network [8].
3.3. Cyclic voltammetry
In cyclic voltammetry experiments, we studied the redox behavior of 8 as a represen-
tative and the results are in close agreement with our previous study [3]. The cyclic
voltammogram for 8 is shown in Figure 4. As the voltammogram is more drawn out
and displays a greater separation in peak potentials (i.e. 400 mV) as compared to a
reversible system (separation in peak potentials should be less than 59 mV), the infer-
ence could be drawn that a quasi-reversible one-electron redox process (Ag(I)/Ag(0) or
Ag(0)/(Ag(I)) is occurring [36]. It can be observed from Figure 4 that during redox
cycle, at applied potential in the range of –1.5 to 0 eV, irreversible one-electron redox
processes occur at E1o=2 –850 mV (at a scan rate of 100 mV s–1). The current is first
observed to peak at Epc –1010 mV (with Ipc –0.01 mA) indicating that a reduction
(Ag(I)/Ag(0) is taking place and then the current rises to peak at Epa –650 mV (with Ipa
0.0035 mA) indicating oxidation process Ag(0)/Ag(I) but not of previously reduced spe-
cies. This quasi-reversibility indicates that the species produced as a result of reduction
of complex is unstable and it decomposes before the Ag(0)/Ag(I) oxidation. So the
occurrence of oxidation may be attributed to Ag(0)/Ag(I) in different species instead of
silver complex, i.e. silver complex decomposes during reduction and during oxidation
merely decomposed species are oxidized (Figure 4).

10
A. HABIB ET AL.
Figure 1. ORTEP view of 8 (50% probability level).
Figure 2. Polymeric structure of 9 revealed from X-ray crystallographic study.
3.4. Anticancer activity
Several research groups have tested a number of NHC salts and Ag(I)–NHC complexes
derived from imidazoles (with structural diversity) against various types of cancer cells
[22, 37–39]. Herein, we report the cytotoxic activity of newly synthesized imidazolium-
based compounds against breast cancer cell line (MDA-MB-231) and human colon can-
cer cell line (HCT-116) to study their antiproliferative potential.
IC50 values of all NHC salts (3–6) and Ag(I)–NHC complexes (7–10) are given in
Table 4. NHC salts exhibited lower cytotoxic activities against MDA-MB-231 and HCT-
116 cells (with IC50 values in the range of 60.24–155.30 lM) as compared to Ag(I)–NHC
complexes (7–10) which showed pronounced cytotoxic activities (with IC50 values
ranging from 4.41 to 15.45 lM). It is also evident from the IC50 values that the com-
plexes are more cytotoxic to MDA-MB-231 cells while NHC salts are more cytotoxic to
HCT-116 cells but almost non-toxic to MDA-MB-231 cells (very high IC50 values).
Reduced activities of salts compared to respective silver complexes can be attributed
to silver–ligand coordination, resulting in increased antiproliferative characteristic of
complexes due to involvement of silver ions in the cancer cell death mechanism. On
the other hand, lower cytotoxic activities of these imidazole-based complexes com-
pared to that of reported benzimidazole-based compounds [2, 13] is due to lack of
benzene ring (as benzene ring increases the lipophilic feature and facilitates the

JOURNAL OF COORDINATION CHEMISTRY
11
Figure 3. ORTEP view of crystal of 9 (50% probability level). The crystal structure reveals the poly-
meric nature of compound.
Figure 4. Cyclic voltammogram of 8 indicating the redox events. The measurements were carried out
at 298 K on acetonitrile solutions containing a concentration of 0.1 M of [Bu4N] [PF6] and 1mM sample.
passage of compound through cell membranes). It is also evident from IC50 values
that aryl group as a linker decreases the cytotoxicity of ligands (5 and 6) (IC50 126.14,
79.48 lM and 135.67, 60.24 lM, respectively, against both cell lines) while increases
that of silver complexes (9 and 10) (IC50 4.41, 7.82 lM and 7.91–10.34 lM, respectively,
against both cell lines), which is attributed to enhanced lipophilicity that facilitates the
smooth passage of Agþ ions from cell membrane into the cell where they interrupt

12
A. HABIB ET AL.
Table 4. IC50 (lM) values of NHC-ligands (3–6) and respective silver complexes (7–10) against
MDA-MB-231 and HCT-116 cell lines.
IC50 (lM)
IC50 (lM)
MDA-MB-231
Compounds
(complexes)
Compounds (ligands)
MDA-MB-231
HCT-116
HCT-116
Control (5FU)
7.50 1.1
155.32 1.3
152.12 1.4
126.14 0.9
79.48 0.7
5.5 0.8
102.34 1.1
80.45 1.4
Control (5FU)
7.50 0.1
8.23 0.2
7.42 0.1
4.41 0.2
7.82 0.1
5.5 0.3
17.45 1.6
8.35 1.2
7.91 1.1
10.34 0.4
3
4
5
6
7
8
9
135.67
1
60.24 0.8
10
Figure 5. Dose-dependent antiproliferative effect of synthesized ligands (3–6) and Ag(I)–NHC com-
plexes (7–10) on MDA-MB-231 cells.
the cellular metabolic functionalities. Compound 9 showed enhanced activity (IC50
4.41 and 7.91 lM) against MDA-MB-231 and HCT-116 cells, respectively, due to its
polymeric nature (as discussed in XRD section) which may be ascribed to the presence
of more than two silver centers. Thus, the presence of imidazole moiety (instead of
benzimidazole) decreases the biopotency, while longer side chains, presence of aryl
linker as well as greater number of silver centers enhance the biopotency of imid-
azole-based Ag(I)–NHC complexes. Figures 5 and 6 depict dose-dependent cytotoxic
effect of ligands and 3–10 against both cancer cell lines. It was observed that all com-
pounds showed dose-dependent cytotoxic activities.
3.5. Anticancer mechanism of action and structure–activity relationship (SAR)
The mechanistic study during anticancer activity of NHC ligands and silver complexes
has revealed that silver–ligand bonding results in increased biopotential by hindering
quick release of Agþ ions into biological system [38, 40]. Gradual release of Agþ ions

JOURNAL OF COORDINATION CHEMISTRY
13
Figure 6. Dose-dependent antiproliferative effect of synthesized ligands (3–6) and Ag(I)–NHC com-
plexes (7–10) on HCT-116 cells.
may be ascribed to the weak p-acceptor and strong r-donor nature of Ag(I)–NHC
complexes [38, 40]. Our previous studies [2, 26] have demonstrated that anticancer
phenomenon mostly occurs through apoptotic pathway [8, 13, 26]. It has been estab-
lished that lipophilic character depends on the chain length that facilitates the
silver(I)–NHC compounds to penetrate the cell membranes, where they release silver
ions which interact with cellular organelles and affect their functionalities [13, 41, 42].
Thus, ionization, delivery approaches and solubility of the silver sources are main
parameters which control the interaction of silver with biological systems [41]. Hence,
concluding the discussion, it may be generalized that longer side chains, presence of
aryl linker as well as greater number of silver centers enhance the biopotency of
Ag(I)–NHC complexes.
4. Conclusion
A new series of dinuclear N-heterocyclic carbene-based ligands and silver complexes
were synthesized. An interesting complex with polymeric structure (quite different
from reported structures) was afforded as revealed from solid-state structure. The
quasi-reversibility of redox events in electrochemical study of synthesized complexes
indicated that reduction event produces unstable decomposed species and oxidation
event may be ascribed to decomposed species. All ligands and complexes were
studied for their in vitro antiproliferative potential against human breast cancer and
colon cancer cells. Silver complexes were observed comparatively more cytotoxic than
ligands. It was observed that breast cancer cells (MDA-MB-231) are more sensitive to
complexes than colon cancer cells (HCT-116) while reverse is the case for NHC salts.

14
A. HABIB ET AL.
Increased chain length of NHC salts decreases its cytotoxicity while increases that of
respective complexes. Hence, the presence of aryl linker, increased chain length, as
well as multiple silver centers increase the anticancer potential of Ag(I)–NHC com-
plexes. Further preclinical trials of synthesized drugs are under process.
Additional information
Crystallographic data of the structures have been deposited with the Cambridge
Crystallographic Data Center, CCDC 1890743 for 8 and CCDC 1890744 for 9. This data
can be obtained free of charge from CCDC via www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgement
The authors are obliged to Prof. Davit Zargarian for his assistance during IRSIP at Chemistry
Department, University de Montreal, and to Dr. Loic Mangin for solving crystal structures and
furnishing useful guidance to write this manuscript.
Funding
The authors are grateful to Higher Education Commission (HEC), Pakistan for funding during
IRSIP Vide Letter No. 1-8/HEC/HRD/2017/6940 and for the National Research Program for
Universities Vide Letter No. 8396/Punjab/R&D/HEC/2017.
Disclosure statement
No potential conflict of interest was reported by the authors.
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