Synthesis and cytotoxicity studies of p-benzyl substituted NHC-copper(I) bromide derivatives

Article abstract of DOI:10.1016/j.poly.2013.11.039

Two series of newly synthesised N-heterocyclic carbene-copper(I) bromide derivatives (1a-e and 2a-e) have been isolated and characterised. A series of highly biologically active symmetrically and un-symmetrically p-benzyl substituted imidazolium halides,

Full text of DOI:10.1016/j.poly.2013.11.039

Polyhedron xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis and cytotoxicity studies of p-benzyl substituted
NHC–copper(I) bromide derivatives
⇑
Wojciech Streciwilk, Frauke Hackenberg, Helge Müller-Bunz, Matthias Tacke
UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of newly synthesised N-heterocyclic carbene–copper(I) bromide derivatives (1a–e and 2a–e)
have been isolated and characterised.
A series of highly biologically active symmetrically and un-symmetrically p-benzyl substituted
imidazolium halides, reported previously by our group, was chosen as precursors for the series 2a–e.
The imidazolium halides were reacted with silver oxide and then transmetallated to copper(I) to yield
the corresponding N-heterocyclic carbene (NHC) copper(I) bromide complexes.
Received 13 September 2013
Accepted 25 November 2013
Available online xxxx
Keywords:
Anticancer drugs
Cytotoxicity
N-Heterocyclic carbene
Copper(I) bromide
MCF-7
The NHC–copper(I) bromide complexes 1a–e were found to be cytotoxic, giving IC50 values of 3.8
( 0.9), 7.4 ( 2.4), 0.60 ( 0.09), 13 ( 4) and 42 ( 7) lM, while NHC–copper(I) bromide complexes 2a–e
showed IC50 values of 17 ( 2), 21 ( 3), 26 ( 5), 68 ( 7) and 61 ( 10)
lM against the breast cancer cell line
MCF-7, respectively.
The same NHC–copper(I) complexes 1a–e exhibited IC50 values of 5.6 ( 0.9), 3.3 ( 0.6), 0.65 ( 0.08),
18 ( 3) and 126 ( 10) M, while NHC–copper(I) bromide complexes 2a–e gave IC50 values of 17 ( 2),
19 ( 1), 18 ( 1), 47 ( 3) and 134 ( 16)
Caki-1
l
l
M against the renal cancer cell-line Caki-1, respectively.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
First reports on the biological activity of metal NHC complexes
date back to 1996–1999 when Cetinkaya et al. described the
antibacterial properties of ruthenium(II) and rhodium(I) NHC
complexes [29,30]. Since then, Ghosh and co-workers described
antimicrobial activity of NHC–silver(I) complexes against a panel
of highly resistant pathogens [31]. Very recently, Youngs and
co-workers have reported the properties of the first anticancer
silver(I)–NHC, which displayed IC50 values similar to cisplatin on
OVCAR-3 (ovarian) and MB157 (breast) cancerous cell lines [32].
The work of our group produced promising cytotoxic results of
NHC–silver(I) acetate complexes against the breast cancer cell line
MCF-7 and renal cancer cell line CAKI-1 [33,34].
Following the assumption that endogenous metals may be less
toxic, considerable efforts have been devoted to copper(I) com-
plexes. Copper is an essential trace nutrient. In mammals it is
mainly found in the bloodstream as a co-factor in various enzymes.
Since copper is only toxic when outside its normal metabolic path-
ways, complexes of Cu(I/II) may serve as Fenton-type reagents and
effective cytotoxic agents [35–37]. Recently published reports of
anticancer properties of copper complexes may be roughly divided
into two sets. The main set contains complexes of copper(II)
[38–43] which sometimes are reduced in vivo to yield more toxic
copper(I) species. Yet it seems more logical to use copper(I) and
omit the in vivo reduction phase. Santini and co-workers reported
promising activity of phosphine–copper(I) complexes against a
panel of tumor cell lines [44]. Since phosphine ligands can be
Modern medicinal chemistry makes use of a broad variety of
structurally different compounds in order to improve cancer
treatment [1–6]. Cisplatin and its further generation analogues
are often successful against a specific type of cancer [7] although
several side effects including emetogenesis and neurotoxicity
drastically limit their applications [8,9]. Therefore, an interest in
research and development of novel non-Pt based anticancer drugs
has emerged [10–16].
Since the discovery of stable ‘‘Arduengo-type’’ carbenes [17], a
continuously growing interest has started in the preparation and
application of these compounds [18,19]. Despite the fact of their
excellent catalytic activities in metal-free organocatalysis [20,21],
the reports of their
r-donor character, displaying similar ligand
properties as trialkylphosphines [22], allowed to assume that NHCs
can act as ideal coordination ligands to form stable and active
transition metal complexes [23–26]. This assumption proposal
was quickly confirmed and lead to the discovery of outstanding
NHC–metal catalysts such as Grubb’s catalyst [27,28]. Yet with
focus on their catalytic properties, the investigation of use of the
NHCs in the therapeutic field remained limited.
⇑
Corresponding author. Tel.: +353 1 7168428.
E-mail address: matthias.tacke@ucd.ie (M. Tacke).
0277-5387/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2013.11.039
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2
W. Streciwilk et al. / Polyhedron xxx (2013) xxx–xxx
successfully replaced with NHCs to stabilise the sensitive copper(I)
ion, this idea was quickly picked up. The majority of attention on
these new complexes was focused onto establishing their catalytic
properties [45–48], but studies of their anticancer properties have
recently been reported. Gautier and co-workers [49] showed that
the cytotoxicity of copper(I) complexes against human cancer cells
lies well with that of cisplatin and other metal–NHCs. Therefore,
the use of copper(I) against human cancer cells is, in our opinion,
a valuable strategy .
Within this paper we present two new series of symmetrically
and un-symmetrically p-benzyl substituted N-heterocyclic carbene
copper(I) bromide complexes, their synthesis and cytotoxic
evaluation.
J = 2.3 Hz, 4H), 5.31 (s, 4HCH benzyl), 2.92–2.81 (p, J = 6.9 Hz, 2HCHiso-
2
propyl), 1.22 (d, J = 6.9 Hz, 12HCH isopropyl).
3
13C NMR (d ppm, CDCl3, 101 MHz): 149.89 (NCN), 136.24,
131.91, 130.59, 128.29, 127.95, 127.61, 127.03, 126.56 (Cphenyl
+
Cbenzyl), 125.03 (Cimidazole pos: 4,5), 53.00 (CH2benzyl), 33.80 (CHisopropyl),
23.78 (CH3isopropyl).
IR absorptions (KBr, cmÀ1): 2960 (s), 1497 (m), 1456 (m), 1341
(m), 832 (m), 701 (s).
MS (m/z, QMS-MS/MS): 485.34 [M+ÀCuBr].
Micro Anal. Calc. for C35H36N2CuBr (628.12 g/mol): C, 66.91; H,
5.78; N, 4.46; Cu, 10.12. Found: C, 64.11; H, 5.16; N, 4.40; Cu,
8.94%.
Melting point: 164–167 °C.
2.2.2. 1,3-Di(p-methylbenzyl)-4,5-di(p-isopropylphenyl)-imidazol-2-
ylidene copper(I) bromide (1b)
2. Material and methods
Yield: 45% (0.076 mmol, 50 mg).
1H NMR (d ppm, CDCl3, 300 MHz): 7.12 (d, J = 8.0 Hz, 4H), 7.04
(d, J = 7.5 Hz, 4H), 6.96 (d, J = 7.4 Hz, 4H), 6.00 (d, J = 7.9 Hz 4H),
2.1. General conditions
The metallation reactions of imidazolium halides with silver(I)
oxide and transmetallation with copper(I) bromide dimethylsul-
fide were carried out under nitrogen atmosphere and with exclu-
sion of light. All solvents and reagents were purchased from
commercial suppliers and were of analytical grade. Dichlorometh-
ane was further dried over calcium dihydride and collected by dis-
tillation. The remaining solvents and all reagents were used
without further purification. Celite S purchased From Sigma–Al-
drich was used for filtration.
5.26 (s, 4HCH benzyl), 2.88 (p, J = 7.0 Hz, 2HCHisopropyl), 2.31 (s,
2
6HCH benzyl), 1.24 (d, J = 1.2 Hz, 6HCH isopropyl), 1.22 (d, J = 1.1 Hz,
3
3
6HCH isopropyl).
3
13C NMR (d ppm, CDCl3, 101 MHz): 149.69 (NCN), 137.47,
133.56, 131.71, 130.62, 129.64, 128.95, 127.52, 126.20 (Cphenyl
+
Cbenzyl), 125.28 (Cimidazole pos: 4,5), 52.57 (CH3benzyl), 33.79 (CHisopropyl),
23.69 (CH3isopropyl), 21.08 (CH3benzyl).
IR absorptions (KBr, cmÀ1): 3434 (s), 3050 (w), 2961 (s), 1516
(m), 1445 (m), 832 (m).
MS (m/z, QMS-MS/MS): 513.44 [M+ÀCuBr].
Micro Anal. Calc. for C37H40N2CuBr (656.18 g/mol): C, 67.73; H,
5.93; N, 4.12; Cu, 9.35. Found: C, 67.13; H, 5.90; N, 3.89, Cu, 8.05%.
Melting point: 190 °C.
The imidazolium halide derivatives, acting as precursors for
NHC–copper(I) complexes were synthesised by following previ-
ously reported methods [33,34,50,51].
NMR spectra were measured on either Varian 300 MHz or
Varian 101 MHz spectrometer. Chemical shifts are reported in
ppm and are referenced to TMS. IR spectra were recorded on a
Perkin Elmer Paragon 1000 FT-IR Spectrometer employing a KBr
disc. ESI MS was performed on a quadrupole tandem mass
spectrometer (Quattro Micro, Micromass/Water’s Corp., USA),
using solutions in 100% MeOH. MS spectra were obtained in the
ESþ (electron spray positive ionisation) mode for all compounds.
C,H,N,Cu analysis was carried out in an Exeter Analytical CE-440
elemental analyzer.
2.2.3. 1,3-Di(p-methoxybenzyl)-4,5-di(p-isopropylphenyl)-imidazol-
2-ylidene copper(I) bromide (1c)
Yield: 74% (87.6 mmol, 0.127 mg).
1H NMR (d ppm, CDCl3, 300 MHz): 7.13 (d, J = 8.1 Hz, 4H), 6.94
(t, 8H), 6.75 (d, J = 8.7 Hz, 4H), 5.24 (s, 4HCH benzyl), 3.79 (s,
2
6HOCH benzyl), 2.89 (p, J = 7.0 Hz, 2HCHisopropyl), 1.23 (d, J = 6.9 Hz,
3
12HCH isopropyl).
3
13C NMR (d ppm, CDCl3, 101 MHz): 159.23 (NCN), 149.82,
131.76, 130.50, 130.29, 129.12, 128.93, 126.55 (Cphenyl + Cbenzyl),
125.21 (Cimidazole pos. 4,5), 113.92 (Cpos. 2 benzyl), 55.23 (OCH3benzyl),
52.49 (CH2benzyl), 33.81 (CHisopropyl), 23.80 (CH3isopropyl).
IR absorptions (KBr, cmÀ1): 2958 (m), 2869 (w), 2835 (w), 1514
(s), 1252 (s), 1176 (m), 1030 (m), 833 (m).
2.2. Synthesis
General procedure for the synthesis of NHC–copper(I) bromide
complexes 1a–e and 2a–e.
MS (m/z, QMS-MS/MS): 545.43 [M+ÀCuBr].
The NHC–imidazolium halide derivatives, which were used as
precursors for the NHC–copper(I) bromide complexes 1a–e and
2a–e, were synthesised by methods reported previously (1a–e,
2d [50], 2a, 2e [51], 2b [34] and 2c [33]) in comparable yields.
100 mg of the appropriately p-benzyl substituted imidazolium
halide precursor and 0.6 eq. of silver(I) oxide were dissolved in
dry dichloromethane and stirred under nitrogen atmosphere for
12 h at RT. Upon formation of black precipitate (silver(I) bromide),
the copper(I) bromide dimethylsulfide (1 eq.) was added and the
reaction was stirred for another 10 h at RT under nitrogen atmo-
sphere. After filtering the reaction mixture through celite and
reducing the solvent under reduced pressure, the obtained yellow
liquid was washed with pentane (2 Â 20 mL) to yield a white solid.
Micro Anal. Calc. for C35H36N2O2CuBr (688.17 g/mol): C, 63.68;
H, 5.50; N, 4.24, Cu, 9.63. Found: C, 67.31; H, 5.78; N, 4.06, Cu,
9.14%.
Melting point: 123–125 °C.
2.2.4. 1,3-Di(p-methoxycarbonylbenzyl)-4,5-di(p-isopropylphenyl)-
imidazol-2-ylidene copper(I) bromide (1d)
Yield: 40% (0.058 mmol, 43.7 mg).
1H NMR (d ppm, CDCl3, 300 MHz): 7.92 (d, J = 7.9 Hz, 4H), 7.10
(d, J = 8.1 Hz, 4H), 7.05 (d, J = 8.2 Hz, 4H), 6.91 (d, J = 7.9 Hz, 4H),
5.39 (s, 4HCH benzyl), 3.93 (s, 6HCH methoxycarbonyl), 2.94–2.80 (p,
2
3
2HCHisopropyl), 1.22 (d, J = 6.9 Hz, 12HCH isopropyl).
3
13C NMR (d ppm, CDCl3, 101 MHz): 166.53 (CO), 150.24 (NCN),
141.02, 130.47, 129.91, 129.85, 129.73, 127.40, 127.32, 126.70
(Cphenyl + Cbenzyl), 126.39 (Cimidazole pos. 4,5), 52.04 (CH2benzyl), 45.18
(OCH3benzyl), 33.73 (CHisopropyl), 23.75 (CH3isopropyl).
2.2.1. 1,3-Dibenzyl-4,5-di(p-isopropylphenyl)-imidazol-2-ylidene
copper(I) bromide (1a)
Yield: 52% (0.091 mmol, 57.7 mg).
IR absorptions (KBr, cmÀ1): 2960 (m), 2872 (w), 1723 (s)
(COcarbonyl), 1613 (m), 1435 (m), 1280 (s), 1109 (m), 837 (w).
MS (m/z, QMS-MS/MS): 601.45 [M+ÀCuBr].
1H NMR (d ppm, CDCl3, 300 MHz): 7.23 (d, J = 2.4 Hz, 6H), 7.10
(d, J = 8.2 Hz, 4H), 7.01–6.96 (d, J = 8.1 Hz, 4H), 6.95–6.91 (d,
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W. Streciwilk et al. / Polyhedron xxx (2013) xxx–xxx
3
Micro Anal. Calc. for C39H40N2O4CuBr (744.20 g/mol): C, 62.92;
H, 5.42; N, 3.77; Cu, 9.54. Found: C, 61.31; H, 4.77; N, 4.16; Cu,
9.21%.
13C NMR (d ppm, CDCl3, 101 MHz): 141.02 (NCN), 137.99,
132.37, 131.97, 130.32, 129.32, 129.00, 127.53 (Cpheny + Cbenzyl),
123.96 (Cimidazole
4,5), 113.65 (Cphenyl), 55.18 (CH2benzyl), 52.62
pos.
Melting point: 167–170 °C.
(OCH3phenyl), 21.09 (CH3benzyl).
IR absorptions (KBr, cmÀ1): 3446 (s), 2933 (m), 1611 (m), 1506
(s), 1251 (s), 833 (m).
2.2.5. 1,3-Di(p-cyanobenzyl)-4,5-di(p-isopropylphenyl)-imidazol-2-
ylidene copper(I) bromide (1e)
MS (m/z, QMS-MS/MS): 489.32 [M+ÀCuBr], 569.16
Yield: 54% (0.087 mmol, 59.5 mg).
[M+ÀBr + H2O].
1H NMR (d ppm, CDCl3, 300 MHz): 7.563 (d, J = 8.0 Hz, 4H), 7.11
Micro Anal. Calc. for C33H32N2O2CuBr (632.07 g/mol): C, 62.71;
H, 5.10; N, 4.43; Cu, 10.05. Found: C, 62.32; H, 4.81; N, 3.87; Cu,
9.92%.
(dd, J = 10.1, 8.0 Hz, 8H), 6.92 (d, J = 7.9 Hz, 4H), 5.40 (s, 4HCH benzyl),
2.88 (m, J = 13.2, 6.7 Hz, 2HCHisopropyl), 1.23 (d, J = 6.9 Hz,
2
12HCH isopropyl).
Melting point: 90–92 °C.
3
13C NMR (d ppm, CDCl3, 101 MHz): 150.38 (NCN), 141.47,
132.29, 130.39, 128.14, 126.77, 125.58, 121.23 (Cphenyl + Cbenzyl),
2.2.9. 1,3-Di(p-methoxybenzyl)-4,5-di(p-chlorophenyl)-imidazol-2-
ylidene copper(I) bromide (2d)
118.34 (Cimidazole
4,5), 111.82 (Cbenzyl
2), 52.45 (CH2benzyl),
pos
pos.
45.12 (CNbenzyl), 33.74 (CHisopropyl), 23.74 (CH3isopropyl).
IR absorptions (KBr, cmÀ1): 3446 (m), 2961 (s), 2230 (s) (CN),
1600 (m), 1503 (m), 1342 (m), 835 (m).
Yield: 45% (0.074 mmol, 50.2 mg).
1H NMR (d ppm, CDCl3, 300 MHz): 7.28 (d, J = 2.1 Hz, 4H), 6.94
(t, J = 7.9 Hz, 8H), 6.78 (d, J = 8.7 Hz, 4H), 5.26 (s;CH benzyl), 3.79 (s,
2
MS (m/z, QMS-MS/MS): 535.36 [M+ÀCuBr].
6HOCH benzyl).
3
Micro Anal. Calc. for C37H34N4CuBr (678.14 g/mol): C, 65.51; H,
5.06; N, 8.26; Cu, 9.38. Found: C, 65.16; H, 4.96; N, 7.87; Cu, 8.65%.
Melting point: 170–172 °C.
13C NMR (d ppm, CDCl3, 101 MHz): 159.47 (NCN), 135.67,
132.12, 131.67, 129.71, 128.88, 128.69, 127.26, 114.15 (Cphenyl + -
C
benzyl),
113.84
(Cimidazole
4,5),
55.21
(CH2benzyl),
pos.
44.91(OCH3benzyl).
2.2.6. 1,3-Dibenzyl-4,5-diphenyl-imidazol-2-ylidene copper(I)
bromide (2a)
IR absorptions (KBr, cmÀ1): 3468 (s), 2957 (w), 1612 (m), 1513
(s), 1249 (s), 835 (m).
Yield: 72% (0.150 mmol, 81.5 mg).
MS (m/z, QMS-MS/MS): 529.24 [M+ÀCuBr].
Micro Anal. Calc. for C31H26N2O2Cl2CuBr (672.90 g/mol): C,
55.33; H, 3.89; N, 4.16; Cu, 9.44. Found: C, 54.94; H, 3.85; N,
4.12; Cu, 9.20%.
1H NMR (d ppm, CDCl3, 300 MHz): 7.25 (d, J = 10.1 Hz 10H), 7.01
(d, J = 6.7 Hz, 10H), 5.35 (s, 4HCH isopropyl).
2
13C NMR (d ppm, DMSO, 101 MHz): 178.39 (NCN), 138.30,
137.22 134.42, 132.27, 131.03, 130.65, 129.23 (Cphenyl + Cbenzyl),
127.99 (Cimidazole pos. 4,5), 125.35, 52.42 (CH2benzyl).
IR absorptions (KBr, cmÀ1): 3436 (s), 3032 (w), 286 (w), 1635
(m), 1496 (m), 1448 (s), 700 (s).
Melting point: 110–112 °C.
2.2.10. (1-Methyl-3-(p-cyanobenzyl)benzimidazole-2-ylidene)
copper(I) bromide (2e)
MS (m/z, QMS-MS/MS): 401.22 [M+ -CuBr], 481.03
[M+ÀBr+H2O].
Yield: 32% (0.097 mmol, 38.1 mg).
1H NMR (d ppm, CDCl3, 300 MHz): 7.66 (d, J = 8.2 Hz, 2HCHben-
zyl), 7.50 (dd, J = 7.6 Hz, 3HCHbenzimidazole), 7.43 (d, J = 8.4 Hz, 2HCHb-
Micro Anal. Calc. for C29H24N2CuBr (543.96 g/mol): C, 64.03; H,
4.45; N, 5.15; Cu, 11.68. Found: C, 63.90; H, 4.40; N, 4.47; Cu,
10.95%.
enzyl), 7.37 (d, J = 7.1 Hz, 1HCHbenzimidazole), 5.72 (s, 2HCH benzyl), 4.12
2
(s, 3HN—CH ).
3
Melting point: 88–91 °C.
13C NMR (d ppm, DMSO, 101 MHz): 143.20 (NCN), 133.01,
128.57, 124.62, 121.75, 121.49, 118.56, 112,23, 111.21 108.45,
106.41 (CN + Cbenzyl + Cphenyl), 43.80 (CH2benzyl), 27.50 (N–CH3).
IR absorptions (KBr, cmÀ1): 3447 (s), 2229 (m) (CN), 1703 (s),
1498 (m), 1443 (m), 1099 (m), 756 (m).
2.2.7. 1,3-Di(p-methylbenzyl)-4,5-di(p-methylphenyl)-imidazol-2-
ylidene copper(I) bromide (2b)
Yield: 56% (0.105 mmol, 63.2 mg).
1H NMR (d ppm, CDCl3, 300 MHz): 7.06 (d, J = 6.6 Hz 8H), 6.93 (t,
MS (m/z, QMS-MS/MS): 247.05 [M+ÀCuBr].
J = 8.4 Hz, 8H), 5.26 (s, 4HCH isopropyl), 2.32 (m, J = 2.0 Hz 12HCH3benzyl
Micro Anal. Calc. for C16H13N3CuBr (390.74 g/mol): C, 49.18; H,
3.35; N, 10.75; Cu, 16.26. Found: C, 48.55; H, 2.90; N, 9.99; Cu,
15.51%.
2
+ CH3phenyl).
13C NMR (d ppm, CDCl3, 101 MHz): 151.69 (NCN), 138.97,
137.73, 133.34, 131.84, 130.53, 129.31, 128.99, 127.58 (Cphenyl
+
Melting point: 187–189 °C.
Cbenzyl), 124.84 (Cimidazole pos. 4,5), 52.60 (CH2benzyl), 21.27 (CH3tolyl),
21.10 (CH3benzyl).
2.3. Cytotoxicity studies
IR absorptions (KBr, cmÀ1): 3436 (s), 3026 (w), 2920 (m), 1635
(m), 1516 (s), 1442 (s), 823 (s).
Preliminary in vitro cell tests were performed on the human
cancerous renal cell line Caki-1 and the human cancerous breast
cell line MCF-7 in order to compare the cytotoxicity of the com-
pounds presented in this paper. These cell lines were chosen based
on their regular and long-lasting growth behaviour. The cells were
obtained from the ATCC (American Tissue Cell Culture Collection)
and maintained in Dulbecco’s Modified Eagle Medium containing
5% (v/v) FCS (fetal calf serum), 1% (v/v) penicillin streptomycin
MS (m/z, QMS-MS/MS): 457.31 [M+ÀCuBr], 537.22
[M+ÀBr+H2O].
Micro Anal. Calc. for C33H32N2CuBr (600.07.14 g/mol): C, 66.05;
H, 5.37; N, 4.67; Cu, 10.59. Found: C, 65.80; H, 5.07; N, 4.18; Cu,
10.22%.
Melting point: 160–161 °C.
2.2.8. 1,3-Di(p-methylbenzyl)-4,5-di(p-methoxyphenyl)-imidazol-2-
ylidene copper(I) bromide (2c)
and 1% (v/v)
taining 200
L
-glutamine. Cells were seeded in 96-well plates con-
lL microlitre wells at a density of 3000-cells/200 L of
l
Yield: 58% (0.101 mmol, 64.2 mg).
medium and were incubated at 37 °C for 24 h to allow for expo-
nential growth. Then the compounds used for the testing were dis-
1H NMR (d ppm, CDCl3, 300 MHz): 7.07 (d, J = 7.8 Hz, 4H), 6.96
(d, J = 4.6 Hz, 4H), 6.93 (d, J = 5.7 Hz, 4H), 6.78 (d, J = 8.6 Hz, 4H),
5.26 (s, 4HCH benzyl), 3.78 (d, J = 3.7 Hz, 6HOCH phenyl), 2.32 (s,
solved in the 70 lL of DMSO (dimethylsulfoxide) and diluted with
medium to obtain stock solutions of 5 Â 10À4 M in concentration
2
3
6HCH benzyl).
and less than 0.7% of DMSO. The cells were then treated with
3
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4
W. Streciwilk et al. / Polyhedron xxx (2013) xxx–xxx
varying concentrations (5 Â 10À4–5 Â 10À10 mol/l) of the com-
pounds and incubated for 48 h at 37 °C. Then, the solutions were
removed from the wells and the cells were washed with PBS (phos-
phate buffer solution) and fresh medium was added to the wells.
Following a recovery period of 24 h incubation at 37 °C, individual
spectrum and tend to yield colourless complexes. In copper(II)
ion (3d9), the unpaired electronic can be promoted to a higher orbi-
tal and upon relaxation emits at a wavelength of approx. 550 nm,
thus causing the complex to be green in appearance).
The 13C NMR resonances of the carbene carbon atoms occur in
the range d = 159–149 ppm for series 1a–e and d = 178–141 ppm
for series 2a–e respectively. These signals are slightly shifted
downfield compared to the NCN signal values reported for their
corresponding NHC imidazolium halide precursors. Only in case
of NHC–copper(I) bromide 2a the C2 carbon shift is indicating
the formation of carbon-copper bond formation. Furthermore, the
absence of strongly shifted NCN signals, as observed for NHC–sil-
ver(I) derivatives [33,34,50,51], indicates the successful formation
of the NHC–copper(I) complexes.
wells were treated with 200 lL of a solution of MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in medium.
The solution consisted of 22 mg of MTT in 40 mL of medium.
The cells were incubated for 3 h at 37 °C. The medium was then re-
moved and the purple formazan crystals were dissolved in 200
DMSO per well. As a control for each plate, 9 cell-containing wells
were treated only with medium and another wells with
lL
9
medium + DMSO. For all tests cells with low passage numbers
were used. A Wallac Victor (Multilabel HTS Counter) Plate Reader
was used to measure absorbance at 540 nm. Cell viability was
expressed as a percentage of the absorbance recorded for control
wells. The values used for the dose response curves represent the
values obtained from four consistent MTT-based assays for each
compound tested. [53].
Additionally, positive mode ESI mass spectra of NHCs-copper(I)
bromide complexes 1a–e are dominated by [M+ÀCuBr] fragment
peaks arising from the loss of copper(I) bromide. For compounds
2a–e, apart from the standard [M+ÀCuBr] fragment,
a
[M+-
À(Br + H2O)] fragment was often observed. This fragment arises
from loss of bromine and coordination of a water molecule to the
positively charged complex ion.
3. Results and discussion
The melting points of the NHC copper(I) bromide complexes
1a–e and 2a–e range between 88 and 190 °C while their
corresponding NHC imidazolium bromide precursors melt at
significantly higher temperatures of 212–274 °C with exception
of an imidazolium chloride precursor for complex 1c, which melts
at 157–160 °C. In case of both series of compounds a trend of
decreased thermal stability of NHC–copper(I) bromide compared
to its imidazolium bromide/chloride derivative precursor has been
observed. Therefore the formation of the copper carbene–carbon
bond has to have significant influence on the thermal stability. This
trend was also noticed previously in case of NHC–silver(I) acetate
complexes [33,34].
3.1. Synthesis
Copper(I) bromide complexes reported in this publication were
prepared by ‘‘one pot’’ transmetallation firstly reported by Arnold
et al. [52]. The synthetic routes for the symmetrically substituted
NHC–copper(I) bromide complexes 1a–e, 2a–d and un-symmetri-
cally substituted NHC–copper(I) carbene complex 2e are given in
Schemes 1 and 2.
The symmetrically substituted NHC–copper(I) bromide com-
plexes were fully characterized by spectral (1H NMR, 13C NMR,
IR, MS), elemental analysis studies and melting point determina-
tions. Due to the positive charge of the molecule, all imidazolium
halide precursors show a characteristic downfield shift in the range
d = 9.63–11.63 ppm for the NCHN proton [50,51]. A successful for-
mation of the NHC–metal complex was indicated by absence of
this characteristic chemical shift in the NMR spectrum. Final
confirmation of the synthesised compound (whether it is truly
NHC–copper(I) bromide and not unreacted NHC–silver(I) bromide
intermediate) was determined by elemental analysis. The
oxidation state of the copper ion in the complex was determined
by observation (copper(I) does not absorb energy in the visible
3.2. Cytotoxicity studies
In previous studies [33,34,50,51], we synthesised and biologi-
cally evaluated the NHC–silver acetate equivalents of compounds
1a–e and 2a–e. Compounds 1a–e represent a library of similar
NHC–copper(I) bromide derivatives that differ only by substitution
patter of the benzyl rings. The aim of the screening was to evaluate
the direct influence of the different p-benzyl substituents
(–H, –CH3, –OCH3, COOCH3, –CN) on the cytotoxic activity.
Scheme 1. General reaction scheme for the synthesis of symmetrically substituted NHC–copper(I) bromide complexes 1a–e and 2a–d.
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W. Streciwilk et al. / Polyhedron xxx (2013) xxx–xxx
5
Scheme 2. General reaction scheme for the synthesis of un-symmetrically substituted NHC–copper(I) bromide complex 2e.
For the family of compounds 2a–e, only the most active NHC
imidazolium halide reported previously were chosen. They acted
as precursor compounds for silver-to-copper exchange in order
to optimise and enhance cytotoxic activity.
The log dose response curves for complexes 1a–e and 2a–e
against the human renal cancer cell line Caki-1 are shown in Figs. 1
and 2 respectively. The log dose response curves for the same
compounds against the human breast cancer cell line MCF-7 are
shown in Figs. 3 and 4 respectively.
All NHC–copper(I) bromide complexes exhibit unique IC50 val-
ues against both cell lines which indicates that the cytotoxic activ-
ity of the compounds is directly influenced either by the different
p-substituents on the benzyl rings (1a–e) or by influence of both
p-substitution patterns on the phenyl and benzyl rings (2a–e).
The highest activity was observed for compound 1c with an IC50
value of 0.60 ( 0.09) lM against breast cancerous cell line MCF-7
and an IC50 = 0.65 ( 0.08)
lM against renal cancerous cell line
Caki-1.
Compared to its previously reported NHC–silver(I) acetate
derivative [53] (IC50 of 3.4 ( 0.6) M against MCF-7 and an
IC50 = 5.5 ( 0.6) M against Caki-1), the newly synthesised NHC–
l
l
copper(I) bromide complex 1c shows significant 10 fold increase
in cytotoxic activity against both cancerous cell lines. Absence or
replacement of the methoxy functional groups decreases the cyto-
toxicity of the compound significantly (even up to hundred times
less active e.g. NHC–copper(I) bromide compound 1e against
Caki-1).
Fig. 2. Cytotoxicity curves from typical MTT assays showing the effect of
compounds 2a–e on the viability of Caki-1 cells.
The NHC–copper(I) complexes 2a–e were observed to have
promising IC50 values but unfortunately, none of them showed
better cytotoxic properties than 1c and their corresponding
NHC–silver(I) acetate derivatives.
Fig. 3. Cytotoxicity curves from typical MTT assays showing the effect of
compounds 1a–e on the viability of MCF-7 cells.
The comparison of the results of the biological evaluation of 1c
and its NHC–silver(I) acetate equivalent allows to visualise the
effect of metal ion exchange on the overall cytotoxic potential of
the complex. NHC–copper(I) bromide 1c exhibits a remarkable
increase in cytotoxic activity compared to its silver(I) equivalent.
Fig. 1. Cytotoxicity curves from typical MTT assays showing the effect of
compounds 1a–e on the viability of Caki-1 cells.
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6
W. Streciwilk et al. / Polyhedron xxx (2013) xxx–xxx
library of the candidate compounds, no predictions are to be made
based on the activity of the precursor’s analogues with other
transition metals. This indicates that certain previously reported
substitution patterns of the imidazolium halide precursors, that
showed no particular biological activity combined with silver,
may still become an interesting cytotoxic agents if transmetallated
to copper and tested against appropriate cancerous cell lines.
With its low IC50 values against both cancer cell lines,
compound 1c belongs to the group of highly biologically active
NHC–metal complexes synthesised by our research group. Future
studies will focus on establishing the potential of 1c as an
anticancer drug, as well as further investigation and structure
optimisation of biologically active NHC–copper(I) complexes.
References
[1] T.W. Hambley, Science 318 (2007) 1392.
[2] B.K. Keppler, Metal Complexes in Cancer Chemotherapy, ed., VCH, Weinheim
and New York, 1993.
[3] P.C.A. Bruijnincx, P.J. Sadler, Curr. Opin. Chem. Biol. 12 (2008) 197.
[4] M. Gielen, E.R.T. Tiekink, Metallotherapeutic Drugs and Metal-Based
Diagnostic Agents: The Use of Metals in Medicine, ed., Wiley, Chichester, 2005.
[5] C.X. Zhang, S.J. Lippard, Curr. Opin. Chem. Biol. 7 (4) (2003) 481.
[6] J.L. Sessler, S.R. Doctrow, T.J. McMurry, S.J. Lippard, Med. Inorg. Chem., ACS,
Washington D.C., 2003.
Fig. 4. Cytotoxicity curves from typical MTT assays showing the effect of
compounds 2a–e on the viability of MCF-7 cells.
[7] L.H. Einhorn, J. Clin. Oncol. 8 (1990) 1777.
[8] L.X. Cubeddu, I.S. Hoffmann, N.T. Fuenmayor, A.L. Finn, New Engl. J. Med. 322
(1990) 810.
[9] L.R. Kelland, Drugs 59 (2000) 1.
[10] D. Wallis, J. Claffey, B. Gleeson, M. Hogan, H. Müller-Bunz, M. Tacke, J.
Organomet. Chem. 694 (2009) 828.
3.3. Conclusion and further studies
[11] B. Gleeson, J. Claffey, M. Hogan, H. Müller-Bunz, D. Wallis, M. Tacke, J.
Organomet. Chem. 694 (2009) 1369.
[12] M. Hogan, B. Gleeson, M. Tacke, Lett. Drug Des. Discov. 7 (2010) 310.
[13] I. Fichtner, D. Behrens, J. Claffey, A. Deally, S. Patil, H. Weber, M. Tacke, Lett.
Drug Des. Discov. 8 (2011) 302.
Nine new symmetrically substituted (1a–e, 2a–d) and one un-
symmetrically substituted (2e) NHC–copper(I) bromide complexes
were synthesised in the transmetallation reaction of p-benzyl
substituted imidazolium halides with copper(I) bromide dimethyl-
sulfide over silver(I) oxide [52].
[14] I. Fichtner, C. Pampillón, N.J. Sweeney, K. Strohfeldt, M. Tacke, Anticancer
Drugs 17 (2006) 333.
The newly synthesised NHC–copper(I) bromide compounds
show relatively high biological activity with compound 1c being
[15] P. Beckhove, O. Oberschmidt, A.R. Hanauske, C. Pampillón, V. Schirrmacher, N.J.
Sweeney, K. Strohfeldt, M. Tacke, Anticancer Drugs 18 (2007) 311.
[16] O. Oberschmidt, A.R. Hanauske, C. Pampillón, N.J. Sweeney, K. Strohfeldt, M.
Tacke, Anticancer Drugs 18 (2007) 317.
[17] A.J. Arduengo, R.L. Harlow, M. Kline, J. Am. Chem. Soc. 113 (1991) 361.
[18] A.J. Arduengo, H.V.R. Dias, J.C. Calabrese, F. Davidson, Organometallics 12
(1993) 3405.
[19] A. Krüger, M. Albrecht, Non-classical N-heterocyclic carbenes, in: S. Diez-
Gonzalez (Ed.), N-Heterocyclic Carbenes: From Laboratory Curiosities to
Efficient Synthetic Tools, RSC, Cambridge, 2010.
[20] N. Marion, S. Díez-Gonzlez, S.P. Nolan, Angew. Chem. Int. Ed. 46 (2007) 2988.
[21] D. Enders, O. Niemeier, A. Henseler, Chem. Rev. 107 (2007) 5606.
[22] K. Öfele, W.A. Herrmann, D. Mihalios, M. Elison, E. Herdtweck, W. Scherer, J.
Mink, J. Organomet. Chem. 459 (1993) 177.
[23] W.A. Herrmann, Angew. Chem. Int. Ed. 41 (2002) 1290.
[24] D. Bourissou, O. Guerret, F.P. Gabbai, G. Bertrand, Chem. Rev. 100 (2000) 39.
[25] H. Jacobsen, A. Correa, A. Poater, C. Costabile, L. Cavallo, Coord. Chem. Rev. 253
(2009) 687.
[26] T. Kösterke, J. Kösters, E.U. Würthwein, C. Mück-Lichtenfeld, C. Schulte to
Brinke, F. Lahoz, F.E. Hahn, Chem. Eur. J. 18 (2012) 14594.
[27] R.H. Grubbs, T.M. Trnka, Ruthenium-catalyzed olefin metathesis, in: S.-I.
Murahashi (Ed.), Ruthenium in Organic Synthesis, Wiley-VCH, Germany, 2004.
[28] L. Oehninger, H. Alborzinia, S. Ludewig, K. Baumann, S. Wölfl, I. Ott, Chem.
Med. Chem. 6 (2011) 2142.
the most active. With an IC50 value of 0.60 ( 0.09)
man breast carcinoma MCF-7 and an IC50 = 0.65 ( 0.08)
l
M against hu-
M against
l
human renal cell carcinoma Caki-1, it is a promising candidate for
further biological studies. Interestingly, the IC50 values of 1c are
approximately ten times better compared to its previously
reported NHC–silver(I) acetate derivative [50] (IC50 of 3.4
( 0.6) lM against MCF-7 and IC50 = 5.5 ( 0.6) lM against Caki-1).
At the same time compound 2b is almost twenty times less active
against MCF-7 cell line and 40 times less cytotoxic when tested
against Caki-1 renal cell carcinoma than its previously reported
silver(I) acetate equivalent [34].
Compared to cisplatin (IC50 values of 10.4 ( 0.2)
lM against
MCF-7 and 3.3 ( 0.2) M against Caki-1), 1c shows significant
l
improved activity and 1c compares well with the most active
NHC–Cu(I)Cl complex (IC50 value of 0.075 ( 0.002) against
MCF-7) reported by Gautier and co-workers [49].
It is therefore visible that the influence of the metal ion on the
toxicity of the overall complex is significant. Compounds based on
both transition metals can reach the single digit micromolar cyto-
toxicity level, provided the substitution pattern of the carbene li-
gand is appropriate. Additionally, no metal-to-activity
relationship pattern can be clearly concluded as in our opinion,
both NHC–silver(I) and NHC–copper(I) complexes induce cell
death via different mechanisms. Therefore, a particular substitu-
tion pattern of the carbene ligand can prove itself to be very active
for the copper complex (as shown above for NHC–copper(I)
bromide complex 1c) while exhibiting average cytotoxic activity
in the form of NHC–silver(I) acetate and vice versa. Most active
NHC–silver(I) acetate complexes reported previously, showed
average biological activity when transmetallated to NHC–copper(I)
bromide. It is therefore recommended that when screening a
[29] B. Cetinkaya, E. Cetinkaya, H. Kucubay, R. Durmaz, Drug Res. 46 (1996) 821.
[30] R. Durmaz, H. Kücükbay, E. Cetinkaya, B. Cetinkaya, Turk. J. Med. Sci. 27 (1997)
59.
[31] S. Ray, R. Mohan, J.K. Singh, M.K. Samantaray, M.M. Shaikh, D. Panda, P. Ghosh,
J. Am. Chem. Soc. 129 (2007) 15042.
[32] D.A. Medvetz, K.M. Hindi, M.J. Panzner, A.J. Ditto, Y.H. Yun, W.J. Youngs, Met.-
Based Drugs (2008) 7.
[33] F. Hackenberg, G. Lally, H. Müller-Bunz, F. Paradisi, D. Quaglia, W. Streciwilk,
M. Tacke, J. Organomet. Chem. 717 (2012) 123.
[34] F. Hackenberg, G. Lally, H. Müller-Bunz, F. Paradisi, D. Quaglia, W. Streciwilk,
M. Tacke, Inorg. Chim. Acta 395 (2013) 135.
[35] D.S. Sigman, A. Mazumder, D.M. Perrin, Chem. Rev. 93 (1993) 2295.
[36] D.S. Sigman, Acc. Chem. Res. 19 (1986) 180.
[37] D.S. Sigman, T.W. Bruice, A. Mazumder, C.L. Sutton, Acc. Chem. Res. 32 (1999)
2797.
[38] C. Marzano, M. Pellei, F. Tisato, C. Santini, Med. Chem. 9 (2009) 185.
[39] G. Cerchiaro, A.M. da Costa, J. Braz. Chem. Soc. 17 (2006) 1473.
[40] F. Saczewski, E. Dziemidowicz-Borys, P.J. Bednarski, R. Grünert, M. Daniec, P.
Tabin, J. Inorg. Biochem. 100 (2006) 1389.
Please cite this article in press as: W. Streciwilk et al., Polyhedron (2013), http://dx.doi.org/10.1016/j.poly.2013.11.039

W. Streciwilk et al. / Polyhedron xxx (2013) xxx–xxx
7
[41] Q. Xin, M. Zhong-Ying, X. Cheng-Zhi, X. Fei, Z. Yan-Wen, X. Jing-Yuan, Q. Zhao-
Yan, L. Jian-Shi, C. Gong-Jun, Y. Shi-Ping, J. Inorg. Biochem. 105 (2011) 728.
[42] M.P. Sathisha, V.K. Revankar, K.S.R. Pai, Met. Based Drugs, Vol. 2008, Article ID
362105, 2008, p. 11, doi: http://dx.doi.org/10.1155/2008/362105.
[43] S. Majumder, G.S. Panda, S.K. Choudhuri, Eur. J. Med. Chem. 38 (2003) 893.
[44] C. Marzano, V. Gandin, M. Pellei, D. Colavito, G. Papini, G.G. Lobbia, E. Del
Giudice, M. Porchia, F. Tisato, C. Santini, J. Med. Chem. 51 (2008) 798.
[45] L. Kang-sang, M.K. Brown, A.W. Hird, A.H. Hoveyda, J. Am. Chem. Soc. 128
(2006) 7182.
[48] N.P. Mankad, T.G. Gray, D.S. Laitar, J.P. Sadighi, Organometallics 23 (2004)
1191.
[49] M.-L. Teyssot, A.-S. Jarrousse, A. Chevry, A. De Haze, C. Beaudoin, M. Manin, S.P.
Nolan, S. Díez-González, L. Morel, A. Gautier, Chem. Eur. J. 15 (2009) 314.
[50] W. Streciwilk, J. Cassidy, F. Hackenberg, H. Müller-Bunz, F. Paradisi, M. Tacke, J.
Organomet. Chem. 749 (2014) 88.
[51] S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, M. Tacke, Metallomics
3 (2011) 74.
[52] P.L. Arnold, A.C. Scarisbrick, A.J. Blake, C. Wilson, Chem. Commun. (2001) 2340.
[53] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
[46] H. Lebel, M. Davi, S. Díez-González, S.P. Nolan, J. Org. Chem. 72 (2007) 144.
[47] P.L. Arnold, Heteroat. Chem. 13 (2002) 534.
Please cite this article in press as: W. Streciwilk et al., Polyhedron (2013), http://dx.doi.org/10.1016/j.poly.2013.11.039