Cytotoxicity of cationic NHC copper(I)complexes coordinated to 2,2'-bis-pyridyl ligands

Article abstract of DOI:10.1016/j.jorganchem.2019.04.003

Cytotoxicity of cationic (NHC)Cu(I)complexes bearing 2,2′-dipyridylamine (dpa)type ligands has been evaluated toward 4 cancer cell lines, and compared to the one of neutral (NHC)Cu(I)complexes. The high cytotoxicity of these novel cationic (NHC)Cu(I)complexes, combined with the straightforward synthesis, and versatility of dpa type ligands may offer new prospects in cancer research, toward the development of novel carrier linked prodrugs.

Full text of DOI:10.1016/j.jorganchem.2019.04.003

Accepted Manuscript
Cytotoxicity of cationic NHC copper(I) complexes coordinated to 2,2'-bis-pyridyl
ligands
Margaux Elie, Gilbert Umuhire Mahoro, Eric Duverger, Jean-Luc Renaud, Richard
Daniellou, Sylvain Gaillard
PII:
S0022-328X(19)30129-9
DOI:
https://doi.org/10.1016/j.jorganchem.2019.04.003
JOM 20753
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 11 March 2019
Revised Date: 3 April 2019
Accepted Date: 4 April 2019
Please cite this article as: M. Elie, G.U. Mahoro, E. Duverger, J.-L. Renaud, R. Daniellou, S. Gaillard,
Cytotoxicity of cationic NHC copper(I) complexes coordinated to 2,2'-bis-pyridyl ligands, Journal of
Organometallic Chemistry (2019), doi: https://doi.org/10.1016/j.jorganchem.2019.04.003.
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ACCEPTED MANUSCRIPT
Cytotoxicity of cationic NHC copper(I) complexes
coordinated to 2,2’-bis-pyridyl ligands
Margaux Elie,a Gilbert Umuhire Mahoro,a Eric Duverger,b,c Jean-Luc Renaud,a Richard Daniellou,b
Sylvain Gaillarda,*
a Normandie University, LCMT, ENSICAEN, UNICAEN, CNRS, 14000 Caen, France.
b
Institut de Chimie Organique et Analytique (ICOA) – UMR CNRS 7311, Université d’Orléans, rue de
Chartres, BP 6759, 45067 Orléans Cedex 2, France.
c GLYcoDIAG, Université d’Orléans, Orléans, France.
* Corresponding authors: (S. Gaillard) E-mail: sylvain.gaillard@ensicaen.fr
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript.
ABSTRACT
Cytotoxicity of cationic (NHC)Cu(I) complexes bearing 2,2’-dipyridylamine (dpa) type ligands has
been evaluated toward 4 cancer cell lines, and compared to the one of neutral (NHC)Cu(I) complexes.
The high cytotoxicity of these novel cationic (NHC)Cu(I) complexes, combined with the
straightforward synthesis, and versatility of dpa type ligands may offer new prospects in cancer
research,
toward
the
development
of
novel
carrier
linked
prodrugs.

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INTRODUCTION
Cancer therapy research was strongly impacted by the discovery of the cytotoxicity of cis-platin against
different cancer cell lines,[1] and the development of its congeners.[2, 3] Transition metal complexes
have been then extensively studied for anticancer applications.[4-9] An important attention was focused
on gold complexes which were considered as alternative to cis-platin because of their different
biological mechanisms. Indeed, many research groups have demonstrated that the cytotoxicity of gold(I)
and gold(III) complexes[10-12] is mainly driven by the metal center which binds cysteine or
selenocysteine protein fragments, inhibits thioredoxin reductase (TrxR) or leads to mitochondria
swelling.[13-18] These effects on cancer cells would allow to overcome some cell-line resistance to cis-
platin. The NHC complexes based on isoelectronic less expensive and more abundant transition metals
like silver(I) and copper(I) have been also investigated because they might behave similarly from an
organometallic point of view.[19-24] Indeed, gold(I) and copper(I) complexes present similar affinities
toward selenium and could be considered to have similar biological mechanism.[25] However,
regarding the biological mechanisms proposed in the literature for different organometallics congeners
of the column 11, they seems to act differently and the simple previous assumption appears to be an
incorrect hypothesis. Indeed, as example, the group of Hecht has reported that the cleavage of DNA by
bleomycin depends on the presence of copper(I) as metal cofactor.[26] Gautier et al. assumed that NHC
copper complexes could generate reactive oxygen species (ROS) leading to a DNA strand break,
responsible of the observed cytotoxicity.[27] Consequently, the development of new cytotoxic transition
metal complexes and more specifically Earth-abundant metal based complexes, might increase the panel
of anticancer agents and furnish more drugs to overcome some cancer cell line resistance.
In the literature, several copper(I) complexes have been reported to exhibit IC50 micromolar (µM) range
against different cancer cell lines. These copper(I) complexes are generally coordinated to phosphane
and/or nitrogen ligands.[28-33] Unlike its group 11 congeners, (NHC) copper(I) complexes are scarcely
reported and are based on two families of general formula [CuX(NHC)] and [Cu(NHC)2][X].[19-24]
Gautier et al. have firstly selected the [CuX(NHC)] complexes and compared their activities on different

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cancer cell lines such as MCF-7, MCF-7R, LNCaP, HL60 and KB. They demonstrated that the copper
complexes stopped the cell cycle progression at the G1 phase.[27] Then, the group of Tacke has
synthesized copper complexes bearing benzyl-substituted NHC ligands and studied their cytotoxicity
against CAKI-1.[34-36] In-vivo studies on nude mice with these aforementioned complexes showed
encouraging results as strong inhibition growth of the tumor was noticed.[35, 36]
We have recently reported a series of cationic (NHC)copper(I) complexes bearing 2,2’-dipyridylamine
ligands (dpa) and exhibiting highly blue emissive properties.[37-39] We anticipated that such complexes
would be of interest as cytotoxic agents. Indeed, their straightforward synthesis[40] allows rapid
modulation of the electronic and steric properties of the dpa ligands. Moreover, the functionalization via
alkylation of the central nitrogen atom of the dpa ligand may open a route to their potential
vectorization.[41-43] Adding a recognized biological framework to cytotoxic complexes would also
allow their preferential accumulation in the targeted cells, and consequently, reduce the quantity of
prodrug absorbed by the patient.[44] Before going further with the development of such sophisticated
ligands, the influence of different functionalized ligands (NHC and dpa) and anions on the cytotoxicity
against cancer cells had to be evaluated. This study aimed to demonstrate (i) the influence of the
substitution on dpa ligand or the nature of the anion on the anticancer activity and (ii) whether a well-
functionalized dpa moiety could open a route to vectorization.[43] Moreover, the emissive properties of
our cationic copper(I) complexes may also be interesting as potential fluorescent probes to detect the
copper accumulation in the cell as their photophysical design is now understood.[45, 46]
Herein, we present a comparative study of the cytotoxicity of various cationic (NHC) (dpa) copper(I)
complexes and neutral (NHC) copper complexes having different anionic ligands such as chloride,
iodide, hydroxide and cyanide (Fig. 1).

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Figure 1. NHC copper complexes used in this study
MATERIALAND METHODS
Synthesis of the copper(I) complexes.
General Considerations. All reactions were carried out using standard Schlenk technique under an
atmosphere of dry argon. Solvents were purchased from Carlo Erba and degassed prior to use by
bubbling argon gas directly in the solvent. Solvents for NMR spectroscopy were dried over molecular
sieves. NMR spectra were recorded on 400 MHz and 500 MHz Bruker spectrometers. Proton (1H) NMR
information is given in the following format: multiplicity, coupling constant(s) (J) in Hertz (Hz),

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number of protons. Carbon (13C) NMR spectra are reported in ppm (δ) relative to residual CHCl3 (δ
77.0) unless noted otherwise. HRMS were performed by LCMT analytical services. NMR solvent was
passed through a pad of basic alumina before uses. 2,2’-dipyridylamine (dpa) was purchased from
Sigma-Aldrich and used without prior purification. The complexes [CuCl(SIPr)] (9), [CuCl(IMes)] (10)
and [CuCl(SIMes)] (11) were purchased from Strem and used without prior purification. The
imidazolium salts used for the preparation of the N-Heterocyclic Carbenes[47-50] and the 2,2’-
dipyridylamine derivatives[38, 40] were synthesized as previously reported. Complexes [CuCl(NHC)]
(1, 5-8) where synthesized according to reported procedures.[51, 52] Complexes [CuI(IPr)] (2)[53]
[CuOH(IPr)] (3)[54] were prepared by the reported methods. Complexes [Cu(NHC)(dpa)][PF6] 12,
18-26 were prepared following our previously reported procedure.[38]
Complex [CuCN(IPr)] (4). In a flame-dried Schlenk tube under argon atmosphere, [CuCl(IPr)] (1) (0.3
mmol, 150 mg, 1 equiv.) and KCN (0.3 mmol, 19 mg, 1 equiv.) were introduced in degassed MeOH (5
mL) and the reaction mixture was stirred under reflux (50 °C) for 4 hours. After returning to room
temperature, the reaction mixture was concentrated to dryness under vacuum. The complex was then
dissolved in dichloromethane and filtered through a pad of Celite® and concentrated again under
vacuum. A purification by recrystallization by slow diffusion of pentane in a THF solution of the
1
complex led to the pure complex (4) as a white powder (143 mg, 97 % yield). H-NMR (CDCl3, 400
MHz): δ 1.22 (d, J = 6.9 Hz, 12H), 1.27 (d, J = 6.9 Hz, 12H), 2.50 (sept, J = 6.9 Hz, 4H), 7.14 (s, 2H),
7.30 (d, J = 7.8 Hz, 4H), 7.50 (t, J = 7.8 Hz, 4H) ppm. (spectroscopic data in good agreement with the
literature).[54]
General procedure for the synthesis of complexes having general formula of [Cu(IPr)(dpa)][X] 13-17.
Following the general procedure described previously for the synthesis of complexes 12,[38] the KPF6
salt of the aqueous treatment at the end of the reaction was replaced by the appropriate salt to give
complexes 13-17.
[Cu(IPr)(dpa)][BF4] complex 13. Following the general procedure with copper complex 1 (162.5 mg,
0.33 mmol), dpa ligand (59.9 mg, 0.35 mmol) and NaBF4 as salt for the final aqueous solution, complex

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1
13 was obtained as a white powder (189 mg, 80% yield). H-NMR (CDCl3, 400 MHz): δ 1.07 (d, J =
6.9 Hz, 12H), 1.22 (d, J = 6.9 Hz, 12H), 2.65 (sept, J = 6.9 Hz, 4H), 6.13 (dd, J = 5.5 and 1.5 Hz, 2H),
6.28 (t, J = 6.3 Hz, 2H), 7.20-7.23 (m, 4H), 7.33 (d, J = 7.8 Hz, 4H), 7.44 (dt, J = 1.8 and 6.9 Hz, 2H),
7.58 (t, J = 7.8 Hz, 2H), 8.81 (s, 1H) ppm. 13C-NMR (CDCl3, 100 MHz): δ 24.0 (4xCH3), 24.2
(4xCH3), 28.3 (4xCH), 115.3 (2xCH), 116.1 (2xCH), 123.4 (2xCH), 124.7 (4xCH), 130.61 (2xCH),
136.0 (2xC), 138.9 (2xCH), 146.0 (4xC), 147.4 (2xCH), 153.2 (2xC), 183.2 (C) ppm. 19F-NMR
(CDCl3, 377 MHz): δ 151.27 ppm. 11B-NMR (CDCl3, 100 MHz): δ -0.76 ppm. IR (neat): ν 2964,
1634, 1582, 1478, 1228, 1072, 761 cm-1. HRMS (ESI): m/z calcd for C37H45CuN5 [M-BF4]+: 622.2971;
found: 622.2989. Elemental Anal. Calcd for C37H45BCuF4N5: C, 62.58; H, 6.39; N, 9.86; found: C,
62.31; H, 6.52; N, 9.89.
[Cu(IPr)(dpa)][BPh4] complex 14. Following the general procedure with copper complex 1 (162.5 mg,
0.33 mmol), dpa ligand (59.9 mg, 0.35 mmol) and NaBPh4 as salt for the final aqueous solution,
complex 14 was obtained as a white powder (293 mg, 94% yield). 1H-NMR (CDCl3, 400 MHz): δ 1.06
(d, J = 6.8 Hz, 12H), 1.23 (d, J = 6.8 Hz, 12H), 2.62 (sept, J = 6.8 Hz, 4H), 5.67 (d, J = 8.4 Hz, 2H),
5.91 (s, 1H), 6.07 (d, J = 4.4 Hz, 2H), 6.22 (t, J = 6.2 Hz, 2H), 6.79 (t, J = 6.8 Hz, 4H, BPh4), 6.97 (t, J
= 7.3 Hz, 8H, BPh4), 7.13 (s, 2H), 7.16 (dd, J = 11.4 and 4.4 Hz, 2H), 7.32 (d, J = 7.8 Hz, 4H), 7.49 (br
s, 8H, BPh4), 7.56 (t, J = 7.8 Hz, 2H) ppm. 13C-NMR (CDCl3, 125MHz): δ 24.0 (4xCH3), 24.2
(4xCH3), 28.7 (4xCH), 115.2 (2xCH), 116.3 (2xCH), 123.4 (4xCH, BPh4), 123.4 (2xCH), 124.7
2
(4xCH), 125.8 (q, J13C-11B = 2.8 Hz, 8xCH, BPh4), 130.6 (2xCH), 135.9 (2xC), 136.2 (8xCH, BPh4),
138.7 (2xCH), 146.0 (4xC), 147.3 (2xCH), 152.0 (2xC), 163.1 (q, 1J13C-11B = 49.3 Hz, 4xC, BPh4), 181.9
(C) ppm. 11B-NMR (CDCl3, 100MHz): δ -6.40 ppm. IR (neat): ν 3339, 2961, 1619, 1578, 1466, 1157,
731, 705 cm-1. HRMS (ESI): m/z calcd for C37H45CuN5 [M-BPh4]+: 622.2971; found: 622.2989.
Elemental Anal. Calcd for C61H65BCuN5: C, 77.73; H, 6.95; N, 7.43; found: C, 77.53; H, 7.12; N, 7.49.
[Cu(IPr)(dpa)][NTf2] complex 15. Following the general procedure with copper complex 1 (146.3 mg,
0.30 mmol), dpa ligand (53.6 mg, 0.32 mmol) and LiNTf2 as salt for the final aqueous solution,
complex 15 was obtained as a white powder (269 mg, 99% yield). 1H-NMR (CDCl3, 500 MHz): δ 1.08

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(d, J = 6.7 Hz, 12H), 1.23 (d, J = 6.7 Hz, 12H), 2.65 (sept, J = 6.7 Hz, 4H), 6.17 (d, J = 4.4 Hz, 2H),
6.33 (t, J = 6.3 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 7.22 (s, 2H), 7.33 (d, J = 7.8 Hz, 4H), 7.48 (t, J = 7.2
Hz, 2H), 7.58 (t, J = 7.8 Hz, 2H), 8.49 (br s, 1H) ppm. 13C-NMR (CDCl3, 100 MHz): δ 23.9 (s,
4xCH3), 24.1 (s, 4xCH3), 28.7 (s, 4xCH), 114.9 (s, 2xCH), 116.3 (s, 2xCH), 119.9 (q, J = 321 Hz, 2xC,
2xCF3 from NTf2 anion), 123.5 (s, 2xCH), 124.7 (s, 4xCH), 130.6 (s, 2xCH), 135.9 (s, 2xC), 139.0 (s,
2xCH), 146.0 (s, 4xC), 147.5 (s, 2xCH), 153.0 (s, 2xC), 183.0 (s, C) ppm. 19F-NMR (CDCl3, 400
MHz): δ -78.80 (s, 2xF, 2xCF3 from NTf2 anion) ppm. IR (neat) ν 3357, 1626, 1581, 1469, 1356, 1197,
1139, 1050, 806, 762, 740, 696 cm-1. HRMS (ESI): m/z calcd for C37H45CuN5 [M-NTf2]+: 622.2971;
found: 622.2973. Elemental Anal. Calcd for C39H45CuF6N6O2S2: C, 51.85; H, 5.02; N, 9.30; found: C,
51.67; H, 5.21; N, 9.33.
[Cu(IPr)(dpa)][B(3,5-(CF3)2Ph)4] complex 16. Following the general procedure with copper complex
1 (87.8 mg, 0.18 mmol), dpa ligand (32.5 mg, 0.19 mmol) and NaB(3,5-(CF3)2Ph)4 as salt for the final
aqueous solution, complex 16 was obtained as a white powder (208 mg, 78% yield). 1H-NMR (CDCl3,
400 MHz): δ 1.05 (d, J = 6.9 Hz, 12H), 1.22 (d, J = 6.9 Hz, 12H), 2.63 (sept, J = 6.9 Hz, 4H), 6.19 (dd,
J = 5.5 and 1.3 Hz, 2H), 6.39 (t, J = 6.9 Hz, 2H), 6.49 (d, J = 8.4 Hz, 2H), 6.59 (s, 1H), 7.24 (s, 2H),
7.33 (d, J = 7.8 Hz, 4H), 7.42 (dt, J = 1.8 and 6.9 Hz, 2H), 7.49 (br s, 4H, B(3,5-(CF3)2Ph)4), 7.57 (t, J =
7.8 Hz, 2H), 7.70 (br s, 8H, B(3,5-(CF3)2Ph)4) ppm. 13C-NMR (CDCl3, 100 MHz): δ 23.8 (4xCH3),
24.1 (4xCH3),28.7 (2xCH), 113.4 (2xCH), 117.3 (2xCH), 117.4-117.5 (m, 4xCH, B(3,5-(CF3)2Ph)4),
123.5 (2xCH), 124.5 (q, 1J13C-19F = 272.5 Hz, 8xCF3, B(3,5-(CF3)2Ph)4), 124.7 (4xCH), 128.8 (qq, 2J13C-
4
= 31.4 Hz and J13C-19F = 2.8 Hz, 8xC, B(3,5-(CF3)2Ph)4), 130.7 (2xCH), 134.8 (8xCH, B(3,5-
19F
1
(CF3)2Ph)4), 135.8 (2xC), 139.5 (2xCH), 146.0 (4xC), 148.4 (2xCH), 152.0 (2xC), 161.7 (q, J13C-11B
=
49.8 Hz, 4xC, B(3,5-(CF3)2Ph)4), 182.5 (C). 19F-NMR (CDCl3, 377 MHz): δ -60.41 ppm. 11B-NMR
(CDCl3, 100 MHz): δ -6.60 ppm. IR (neat): ν 2967, 1625, 1585, 1473, 1352, 1272, 1156, 1124, 887,
681, 668 cm-1. HRMS (ESI): m/z calcd for C37H45CuN5 [M-B(3,5-(CF3)2Ph)4]+: 622.2971; found:
622.2959. Elemental Anal. Calcd for C69H57BCuF24N5: C, 55.75; H, 3.87; N, 4.71; found: C, 55.62; H,
4.02; N, 4.72.

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[Cu(IPr)(dpa)][B(F5Ph)4] complex 17. Following the general procedure with copper complex 1 (97.5
mg, 0.20 mmol), dpa ligand (36 mg, 0.21 mmol) and NaB(F5Ph)4 as salt of the final aqueous solution,
complex 17 was obtained as a white powder (198 mg, 76% yield). 1H-NMR (CDCl3, 400 MHz): δ 1.07
(d, J = 6.7 Hz, 12H), 1.23 (d, J = 6.7 Hz, 12H), 2.65 (sept, J = 6.7 Hz, 4H), 6.21 (d, J = 4.8 Hz, 2H),
6.42 (t, J = 6.3 Hz, 2H), 6.61 (d, J = 8.3 Hz, 2H), 6.89 (br s, 1H), 7.24 (s, 2H), 7.35 (d, J = 7.8 Hz, 4H),
7.50 (t, J = 7.9 Hz, 2H), 7.59 (t, J = 7.8 Hz, 2H) ppm. 13C-NMR (CDCl3, 100 MHz): δ 23.8 (s, 4xCH3),
24.1 (s, 4xCH3), 28.7 (s, 4xCH), 113.62 (s, 2xCH), 117.2 (s, 2xCH), 123.5 (s, 2xCH), 124.8 (s, 4xCH),
130.7 (s, 2xCH), 135.0 (m, 4xC, B(F5Ph)4), 135.9 (s, 2xC), 137.5 (m, 8xC, B(F5Ph)4), 139.5 (s, 2xCH),
146.0 (s, 4xC), 147.0 (m, 4xC, B(F5Ph)4), 148.3 (s, 2xCH), 149.3 (m, 8xC, B(F5Ph)4), 152.2 (s, 2xC),
182.7 (s, C) ppm. 19F-NMR (CDCl3, 400 MHz): δ -166.8 (t, J = 17.5 Hz, 8xF, B(F5Ph)4), -163.0 (t, J =
11
20.5 Hz, 8xF, B(F5Ph)4), -132.7 (br s, 4xF, B(F5Ph)4) ppm. B-NMR (CDCl3, 100 MHz): δ -16.67 (s,
B(F5Ph)4) ppm. IR (neat): ν 2966, 1638, 1515, 1462, 1088, 979, 773 cm-1. HRMS (ESI): m/z calcd for
C37H45CuN5 [M-B(F5Ph)4]+: 622.2971; found: 622.2974. Elemental Anal. Calcd for C61H45BCuF20N5:
C, 56.26; H, 3.48; N, 5.38; found: C, 56.02; H, 3.65; N, 5.41.
Biological study details
The growth level of four cancer cell lines was determined using a colorimetric MTT (thiazolyl blue
tetrazolium bromide, Sigma) assay. Cancer cell lines and growth medium were obtained from CLS Cell
Line Service GmbH, Eppelheim, Germany). Human skin melanoma SK-Mel-28 and human brain
glioma HS-683 were grown in DMEM supplemented with 4.5 g/l glucose, L-glutamine and 10% FBS.
The human lung carcinoma cell line A549 was grown in DMEM:Ham’s F12 (1:1) supplemented with L-
glutamine and 5% FBS and human breast adenoma carcinoma MCF-7 in EMEM supplemented with L-
glutamine, sodium pyruvate, NEAA and 10 % FBS. MTT assay is based on the reduction of the yellow
product thiazolyl blue tetrazolium bromide (MTT) to purple-blue formazan by mitochondrial
dehydrogenase of metabolically active cells. The number of living cells after incubation in the presence
(or absence, control) of the tested molecule is directly proportional to the blue color which was
measured by spectrophotometry. Briefly, cells were seeded (200 µl of a 5.104 cells/ml suspension) in

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96-well culture plates (TPP, Trasadingen, Switzerland) and incubated for 24h. Each compound (starting
from DMSO solutions, stable for months) was assessed in serial dilution (four concentrations in 0.5%
DMSO at the highest concentration) in six replicates (n=6) and incubated for 72 h. Thereafter, MTT (5
mg/ml solution in PBS) was added to each well (10% v/v) and cells were further incubated for 4 hours.
Then, after removing the culture medium, the blue crystals were dissolved in 100 µL SDS-acidic-
isopropanol solution (0.5% SDS; 80 mM HCl) and absorbance measured at 540 nm using a 620 nm
reference. Absorbance of the serial dilution of each cell line treated under the same conditions but
without the tested compounds was measured to generate a standard curve allowing IC50 determination
(IC50 is defined as the concentration reducing cell growth by 50%).
RESULTS AND DISCUSSION
Synthesis and characterization
The imidazolium salts[47-50] and 2,2’-dipyridylamine derivatives[38, 40] were prepared according to
reported procedures.
The [CuCl(NHC)] precursors 1 and 5-8 were synthesized following the Nolan-Cazin procedure using
Cu2O as metal precursor and the appropriate imidazolium salt and complexes 1, 5-8 were isolated in 40
to 94% yield (Scheme 1, i).[51, 52] Then, complex 2 was prepared in 53% isolated yield by reacting
1,3-bis(-2,6-di-iso-propylphenyl)-4,5-dichloroimidazolium chloride in the presence of KOtBu and CuI
(Scheme 1, ii).[53] The other NHC copper(I) complexes 9-11 were commercially available and were
introduced in order to evaluate other N-aromatic substituent on the NHC ligand. The (NHC)copper
hydroxide complex 3 was synthesized via a known procedure[54] and was also used as precursor for the
synthesis of some cationic complexes described below (Scheme 1, iii). The exchange of the chlorine
ligand by a cyano ligand in degassed methanol furnished the complex 4 in 97% isolated yield (Scheme
1, iv).

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Scheme 1. General scheme for the synthesis of neutral complexes 1-8.
Then, complexes 1, 3 and 5-8 were used as precursors for the synthesis of cationic copper complexes
bearing 2,2’-dipyridylamine (dpa) ligands. Complexes 12-21, 23, 24 and 26 were obtained from 1, or 5-
8 via the exchange of the chloride atom by a dpa ligand in refluxed methanol, followed by an anion
metathesis (Scheme 2, i).[38] The expected cationic (NHC)copper(I) complexes were isolated in 76 to
99% yields. In details, the synthesis of complexes 12 to 17 were carried out with KPF6, NaBF4, NaBPh4,
LiNTf2, NaB((3,5-CF3)2-Ph)4, NaB(F5Ph)4 salts, respectively.

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Scheme 2. General scheme for the synthesis of cationic complexes 12-26.
-
Complexes 18-21, 23, 24, and 26, having different substituted dpa, were obtained with the PF6 anion.
As previously noticed,[38] the synthesis of (NHC) copper(I) complexes coordinated to electron-
deficient dpa ligands required a different procedure involving [CuOH(NHC)] 3 as precursor. In details,
the cationic copper complexes 22 and 25 were obtained from complex 4 by addition of 1 equivalent of
.
HBF4 OEt2 in the presence of 1 equivalent of dpa ligand. Complexes 22 and 25 were isolated after an
anion metathesis in 77 and 70 % overall yields, respectively (Scheme 1, ii).
Cytotoxicity studies.
Half growth inhibition concentration (IC50) of all the copper(I) complexes were measured on four cancer
lines, namely MCF-7 (breast cancer), A-549 (lung carcimona), SK-Mel-28 (melanoma) and HS-683
(glioma) in order to establish structure/cytotoxicity relationships toward each cancer cell lines
independently. The choice for the three first cancer lines was directed by the previous studies with gold
complexes.[19-24] In addition, the cytotoxicity of [(NHC)CuCl] complexes against MCF-7 was also
reported by Gautier and this cell line was used as reference for this work.[27]

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The growth level of the four cancer cell lines was determined using a colorimetric MTT (thiazolyl blue
tetrazolium bromide, Sigma) assay. MTT assay is based on the reduction of the yellow product thiazolyl
blue tetrazolium bromide (MTT) to purple-blue formazan by mitochondrial dehydrogenase of
metabolically active cells. The number of living cells after incubation in the presence (or absence,
control) of the tested molecule is directly proportional to the blue color, which was measured by
spectrophotometry. Absorbance spectra were measured to generate a standard curve allowing IC50
determination (IC50 is defined as the concentration reducing cell growth by 50 %). All active complexes
were then subjected to their IC50 evaluation on a 10 000 cells population.
Results on the MCF-7 are presented in table 1 and chart 1. Etoposide and 5-fluorouracil, two
commercially available generic drugs classically used to treat cancer, were used as positive controls. We
were pleased to observe cytotoxicity toward the breast cancer MCF-7 cell lines for all copper complexes
with IC50 around 100 to 1000 fold lower than the two reference compounds i.e. Etoposide and 5-
fluorouracil (Table 1, Chart 1).
Chart 1. IC50 of neutral (NHC)copper(I) complexes 1-11 (red) and [Cu(NHC)(dpa)][X] 12-26 (blue) on
MCF-7.
Concerning the neutral copper complexes 1-11, the IC50 were found between 0.015 ± 0.020 µM (for
complex 4 and 9, entries 4 and 9, Table 1) and 0.30 ± 0.05 µM (for complexes 2 and 7, entries 2 and 7,
Table 1) except for complex 11 bearing SIMes ligand with a much higher IC50 of 1.45 ± 0.10 than other
neutral (NHC)copper(I) complexes. This surprising IC50 for complex 11 is 20 times higher than the IC50
previously reported by Gautier et al. (0.075 ± 0.002 µM on a 3 000 cells population).[27] Nevertheless,

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the IC50 value of complex 11 is still lower than the IC50 of cis-platin in similar conditions.[55] Then,
when neutral complexes 1-4 are now compared, the best X ligand appeared to be the cyanide present in
complex 4. Indeed, 4 exhibited a IC50 of at least 10 fold lower than the complexes 1-3 (Entries 1-4,
Table 1). Gratefully, cationic copper(I) complexes 12-26 also presented a cytotoxic activity against
MCF-7 cell-line. To the best of our knowledge, no cytotoxicity against breast cancer MCF-7 cell lines
has ever been reported for cationic (NHC)copper(I) complexes. Most of the IC50 ranged from 0.03 ±
0.01 to 0.28 ± 0.05 excepted for complexes 16, 17 and 25 (Entries 20, 21 and 29, Table 1) and were
generally slightly higher than those determined for neutral copper(I) complexes 1-10. These values seem
to indicate that large anions (complexes 16 and 17) and electron deficient substituent at the 5 position of
the pyridine ring in dpa ligand (complex 25) are not suitable for a high cytotoxicity. The best activities
were found for complexes 15 and 20, both having an unsubstituted dpa, small anion and electron rich
NHC (complex 20), with a IC50 of 0.03 ± 0.01 and 0.04 ± 0.03 µM, respectively (Entries 19 and 24,
Table 1). The comparison of these IC50 gave us some trends on the structure/cytotoxicity property
relationships, (i) the presence of electron donor or withdrawing substituent on the pyridine ring of the
dpa skeleton in these cationic copper(I) complexes led to higher IC50 and unsubstituted dpa should be
-
-
favoured, and (ii) large fluorinated tetraarylborate anions B(3,5-F3C-C6H3)4 and B(C6F5)4 have a
negative effect on the cytotoxicity against MCF-7. The latter might be due to solubility or lipophilicity
issues. Very interestingly, the
N-alkylated dpa complex 26 presents a higher IC50 than the most active
cationic copper(I) complex but still below 1 µM (0.19 ± 0.17 µM, Entry 30, Table 1). Nevertheless, this
complex appears more active than Etoposide, 5F-uracil and the cis-platin.

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Table 1. In vitro IC50 growth inhibitory concentrations (µM) for breast cancer MCF-7.
Neutral
Entry
IC50 (µM)a
Entry
Cationic complex
IC50 (µM)a
complex
1
1
0.15 ± 0.03
0.30 ± 0.05
0.22 ± 0.05
16
17
18
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
0.16 ± 0.10
0.08 ± 0.02
0.10 ± 0.01
0.03 ± 0.01
1.6 ± 0.4
2
2
3
3
4
4
0.015 ± 0.020 19
5
5
0.14 ± 0.02
0.03 ± 0.03
0.30 ± 0.05
0.03 ± 0.04
20
21
22
23
6
6
0.6 ± 0.1
7
7
0.22 ± 0.09
0.15 ± 0.90
0.04 ± 0.03
0.28 ± 0.05
0.20 ± 0.10
0.15 ± 0.07
0.22 ± 0.12
1.00 ± 0.80
0.19 ± 0.17
8
8
9
9
0.015 ± 0.020 24
10
11
12
13
14
10
11
11b
0.10 ± 0.01
1.45 ± 0.10
25
26
0.075 ± 0.002 27
Cis-platin[55] 8.8 ± 1.3
28
29
30
Etoposide
5F-Uracil
25.0 ± 0.8
19.5 ± 2.0
15
a
b
± values, means standard error of at least 6 determinations. IC50 of complex 11 previously reported by
Gautier et al. on 3000 cells populations.[27]
In the second survey, all the copper complexes were studied toward lung carcimona A-549. All
complexes were found active, the IC50 were determined and reported in table 2 and chart 2. In the
neutral complexes series, complexes 1-3, 5-6 and 8-10 exhibited low IC50 between 0.04 ± 0.01 µM
(complex 1) and 0.15 ± 0.03 µM (complex 10) (Entries 1-3, 5-6, 8-10, Table 2). Then, complexes 4 and
7 presented a higher IC50 of 0.6 ± 0.2 and 0.30 ± 0.06 µM respectively, but still lower than Etoposide
and 5-fluorouracil (Entries 4 and 7 vs. 12 and 13). Noteworthy, because of solubility issues above 3 µM
of complex 11 its IC50 was not determined (Entry 11, Table 2). In details, the copper complexes of
general formula [CuX(NHC)], 1 and 2 appeared to be more active than other inner sphere anion such as

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HO- (complex 3) and NC- (complex 4) (Entries 1-4, Table 2). In sharp contrast with the results on the
MCF-7 cell line, the complex 4 bearing a cyanide ligand showed the lowest activity with a IC50 of 0.6 ±
0.2 µM (Entry 4, Table 2). Cationic copper(I) complexes presented much higher IC50 than neutral
(NHC)copper complexes, except for complexes 13-15 having unsubstituted dpa and IPr ligands (Entries
15-17, Table 2). However, these copper complexes remained more active than the reference drugs
(Entries 12-13, Table 2). In this cationic copper(I) complexes series, the non-coordinating anion
-
-
-
appeared to be structurally important as complexes 13-15 with BF4 , BPh4 and NTf2 anions exhibited
IC50 (at maximum 0.06 ± 0.02 µM) similar to the most active neutral copper complexes 1 and 8 (Entries
1, 8, 15-18, Table 2). Then, adding substituents on the IPr motif decreased the IC50 (Complex 12 vs.
complexes 18-21, entries 14 and 20-23, Table 2) and, when the dpa substitution is considered, the N-
substituted dpa complex presented the lowest IC50 (complex 26 vs. complexes 12, 22-25), with a value
of 0.78 ± 0.10 µM for complex 26 (Entry 28, Table 2).
Chart 2. IC50 of neutral (NHC)copper(I) complexes 1-11 (red) and [Cu(NHC)(dpa)][X] 12-26 (blue) on
A-549.

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Table 2. In vitro IC50 growth inhibitory concentrations (µM) for lung carcinoma A-549.
Neutral
Entry
IC50 (µM)a
Entry
Cationic complex IC50 (µM)a
complex
1
1
0.04 ± 0.01
0.07 ± 0.03
0.10 ± 0.02
0.6 ± 0.2
14
15
16
17
18
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
1.60 ± 0.10
0.05 ± 0.02
0.06 ± 0.02
0.06 ± 0.01
2.0 ± 0.2
2
2
3
3
4
4
5
5
0.07 ± 0.01
6
6
0.050 ± 0.005 19
0.30 ± 0.06 20
0.050 ± 0.005 21
0.60 ± 0.10
0.56 ± 0.17
0.66 ± 0.13
0.63 ± 0.20
0.70 ± 0.10
1.70 ± 0.40
2.90 ± 0.40
1.47 ± 0.50
1.30 ± 0.10
0.78 ± 0.10
7
7
8
8
9
9
0.08 ± 0.02
0.15 ± 0.03
> 3b
22
23
24
25
26
27
28
10
11
12
13
10
11
Etoposide
5F-Uracil
3.0 ± 0.2
3.5 ± 0.3
a ± values, means standard error of at least 6 determinations. b higher concentrations were not considered due to
solubility issue and IC50 was not determined.
The cytotoxicity of the copper(I) complexes toward the Melanoma SK-Mel-28 cancer cells was then
evaluated (Table 3 and Chart 3). For neutral complexes 1-11, all the IC50 were in a smaller range (0.04 ±
0.002 for 5 to 0.42 ± 0.04 for 11) compared to the two previous cancer cell lines. In addition, complexes
1-11 have IC50 at least 15 fold lower than etoposide (IC50 of 6.5 ± 0.8), the most active commercially
available reference used in this study (Entries 1-13, Table 3).

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Chart 3. IC50 of neutral (NHC)copper(I) complexes 1-11 (red) and [Cu(NHC)(dpa)][X] 12-26 (blue) on
SK-Mel-28.
Concerning the cationic copper complexes, the range of IC50 was found between 0.09 ± 0.02 for
complex 20 and 1.80 ± 0.60 µM for complex 23 (Entries 14-28, Table 3). The role of the anion seemed
to have a little impact on the activity and its nature was not as important as observed with the MCF-7
and A-549 cell lines (Entries 14-19, Table 3). Then, it appeared that electron donating substituent on the
IPr moiety decreased the IC50 (Complex 12 vs complexes 18-20, Entries 14 and 20-22, Table 3), while,
in sharp contrast, the electron withdrawing chloride substituent on the C4 and C5 positions of the IPr
ligand led to an increase of the IC50 (Complex 12 vs complex 21, Entries 14 and 23, Table 3).

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Table 3. In vitro IC50 growth inhibitory concentrations (µM) for melanoma SK-Mel-28.
Neutral
Cationic
complex
Entry
IC50 (µM)a
Entry
IC50 (µM)a
complex
1
1
0.045 ± 0.005
0.16 ± 0.03
14
15
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
0.47 ± 0.10
0.29 ± 0.03
0.22 ± 0.12
0.24 ± 0.02
0.95 ± 0.05
0.52 ± 0.03
0.13 ± 0.05
0.12 ± 0.02
0.09 ± 0.02
1.30 ± 0.30
0.90 ± 0.40
1.80 ± 0.60
0.50 ± 0.20
1.20 ± 0.40
0.60 ± 0.20
2
2
3
3
0.100 ± 0.025 16
4
4
0.16 ± 0.02
0.040 ± 0.002
0.18 ± 0.40
0.13 ± 0.01
0.25 ± 0.04
0.21 ± 0.01
0.35 ± 0.25
0.42 ± 0.04
6.5 ± 0.8
17
18
19
20
21
22
23
24
25
26
27
28
5
5
6
6
7
7
8
8
9
9
10
11
12
13
10
11
Etoposide
5F-Uracil
13.4 ± 2.0
a
±
values,
means
standard
error
of
at
least
6
determinations.
The fourth cancer cell line we studied in this work was Glioma HS-683. The IC50 were presented in
table 4 and chart 4. Fortunately, all the copper(I) complexes presented a higher cytotoxicity toward
Glioma HS-683 than the commercially available drugs etoposide and 5-fluorouracil, which exhibited
IC50 of 4.00 ± 0.52 and 16.7 ± 12.2, respectively (Entries 12 and 13, Table 4). And even more interesting
was the micromolar range of the IC50 of some (NHC) copper(I) complexes (Entries 1-3, 5-6, 8-11, 16-
17, 20, 22 and 24, Table 4). As example, in the neutral copper complex family, the most active (NHC)
copper(I) complexes were 5 and 10 with IC50 of 0.21 ± 0.01 and 0.20 ± 0.05, respectively (Entries 5 and
10, Table 4). Cationic copper(I) complexes exhibited a lower cytotoxicity than neutral copper(I)

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complexes 1-11 except complex 14, for which an IC50 of 0.22 ± 0.01 µM was determined, similarly to
complexes 5 and 10 (Entry 16 vs entries 5 and 10, Table 4).
Chart 4. IC50 of neutral (NHC)copper(I) complexes 1-11 (red) and [Cu(NHC)(dpa)][X] 12-26 (blue) on
HS-683.
In the cationic complex series, IC50 were not determined for 16 and 17 due to solubility issues above 3
µM (Entries 16 and 17, Table 4). Complexes 23 and 25 presented the lowest cytotoxicity with IC50 of
3.60 ± 0.40 and 3.50 ± 0.70, respectively (Entries 25 and 27, Table 4). Concerning complex 26, having
the N-alkylated dpa ligand, its IC50 was found at 1.8 ± 0,1 µM representing a cytotoxicity 10 fold lower
-
than the one of 14 (Entries 16 and 28, Table 4). Even if BPh4 anion is very hydrophobic, this anion
appeared to bring the highest cytotoxicity to cationic NHC copper complexes as 14 presented the lowest
IC50 compared to complexes 12-17 (Entry 16, Table 4). Therefore, an eventual anion metathesis on
complex 26 might lead to more active complexes and balance the negative effect of the N-alkyl
substitution on the dpa ligand.

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Table 4. In vitro IC50 growth inhibitory concentrations (µM) for Glioma HS-683.
Neutral
Cationic
complex
Entry
IC50 (µM)
Entry
IC50 (µM)
complex
1
1
0.65 ± 0.01
0.58 ± 0.03
0.77 ± 0.01
1.5 ± 0.2
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
1.30 ± 0.10
1.07 ± 0.70
0.22 ± 0.01
0.89 ± 0.18
> 3b
2
2
3
3
4
4
5
5
0.21 ± 0.01
0.39 ± 0.12
1.1 ± 0.2
6
6
> 3b
7
7
0.63 ± 0.20
1.10 ± 0.10
0.80 ± 0.20
1.40 ± 0.20
0.70 ± 0.10
3.60 ± 0.40
2.10 ± 0.10
3.50 ± 0.70
1.80 ± 0.10
8
8
0.39 ± 0.01
0.81 ± 0.13
0.20 ± 0.05
0.41 ± 0.02
4.00 ± 0.52
16.7 ± 12.2
9
9
10
11
12
13
10
11
Etoposide
5F-Uracil
a
b
± values, means standard error of at least 6 determinations. higher concentrations was not
considered due to solubility issue and IC50 was not determined.
Finally, the evolution and stability of complexes 1 and 12 in DMSO-d6 solution was
1
evaluated by H NMR spectroscopy (Figure S42 and S43 in supplementary information).
After 14 h, no evolution of both complexes was noticed. Then, the stability study was
assessed for complex 12 in biological medium (1%DMSO/DMEM+GlutaMAX-ITM
solution) in absence of cells and was monitored by UV-visible absorption spectroscopy for 25
hours (Figure 2). As control, the same experiment was performed in the biological medium.
Surprisingly, the absorption spectra of the medium presented important changes in both UV

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(274 nm) and visible region (560 nm) (See Supporting Information, Figure S1). As the
ligands (dpa and NHC) and complex 12 have only their maximal absorption wavelength in
the UV region, our analysis of the evolution, in function of time, of the absorption spectra of
complex 12, in 1%DMSO/DMEM+GlutaMAX-ITM solution, was only focused in the 250 to
380 nm region. The maxima of the absorption wavelength in a dichloromethane solution were
previously measured at 260 and 315 nm, assigned to π-π* and d-π* transitions, respectively
(Figure 2, inset).[37]
Figure 2. Stability of 12 followed by UV-vis spectroscopy over 25 h in 1%DMSO/
DMEM+GlutaMAX-I™ solution (c = 50 µM) and UV-visible spectra of 12 in
dichloromethane solution (c = 100 µM) (red line, inset) and in 1%DMSO/
DMEM+GlutaMAX-I™ solution (c = 50 µM) in absence of cell line (dark line, inset).
Encouragingly, the UV-visible spectra in the 1%DMSO/DMEM+GlutaMAX-ITM solution did
not dramatically change in the 260 and 315 nm region within 4 to 5 h. After this period of
time, a slow decrease of the absorbance was observed: 13 and 26% at 260 nm, 17 and 39% at
312 nm after 8 and 25 h, respectively. In a few words, the integrity of the cationic complex 12
seems to be maintained during the first 4 to 5 h in the biological medium and such stability
might open a door to vectorizable copper complexes bearing a dpa ligand.

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CONCLUSION
We reported the synthesis of several new neutral and cationic (NHC)copper(I) complexes.
The cytotoxicity of all these copper(I) complexes was studied on three cancer lines, breast
cancer MCF-7, lung carcimona A-549 and Melanoma SK-Mel-28 which were previously
reported to be sensitive to (NHC)gold(I) complexes. In addition, we also reported
cytotoxicity of these (NHC)copper(I) complexes toward Glioma HS-683. This work further
demonstrates that copper complexes can act as anticancer agents like their group 11
congeners and are more active than commercially available anti-cancer agents. Some of the
cationic (NHC)copper(I) complexes 12-26, bearing NHC and dpa ligands exhibit similar sub-
micromolar IC50 than neutral [CuX(NHC)] complexes 1-11 and, in all cases, lower than the
commercial Etoposide and 5-fluorouracil drugs.
Chart 5 depicts the whole IC50, on the four cell lines determined in this study. This overview
highlights that neutral (NHC) copper(I) complexes are in general more active than the
cationic (NHC) copper(I) analogs bearing dpa ligands. Nevertheless, in few cases, the
cytotoxicity of some cationic copper(I) complexes is comparable to the one observed with the
neutral copper(I) complexes and depends on the nature of the anion, the electronic property
or the steric hindrance of their ligands. Complexes 13-15 having a small anion, present
similar cytotoxicity to neutral copper(I) complexes toward MCF-7 and lung carcimona A-
549. Complexes 18-20, having electron rich substituent on the NHC moiety, present also IC50
in the range of the most active neutral (NHC)copper(I) complexes toward MCF-7 and SK-
Mel-28. Finally, complexes bearing substituted dpa present higher IC50, whatever the electron
donating or electron withdrawing properties of the substituent. Such results might be due to
steric hindrance issues. Finally, the complex 26, which could be seen as a model of a future
vectorizable complex if the dpa is substituted by a biologically recognized framework, has
higher IC50 than its non-substituted congeners. However, its cytotoxicity against the four cell

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lines is higher than commercially available anticancer agents. All these observations might
give some guidelines for the future design of more efficient cationic copper(I) complexes.
Chart 5. IC50 of complexes 1-26 on the four cancer cell lines (MCF-7, A-549, SK-Mel-28
and HS-68)
Table of Abbreviation
dpa
2,2’-dipyridylamine derivative.
IC50 half growth inhibitory concentration
NHC N-Heterocyclic Carbene
Acknowledgements
This work was supported by the "Ministère de la Recherche et des Nouvelles Technologies",
CNRS (Centre National de la Recherche Scientifique) and the LABEX SynOrg (ANR-11-
LABX-0029). We thank the “Agence Nationale de la Recherche”, within the CSOSG
program (ANR-12-SECU-0002-02), the ANR program (ANR-15-CE39-0006), the “Région
Basse-Normandie” (M.E.) and Normandie University (G.U.M.) for funding. SG thanks
Johnson Matthey for the gift of metals.
Supporting Information.

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Supporting information available: experimental procedures for the synthesis of the iridium (III)
complexes, their characterization, and the biological evaluation of the inhibition. See
DOI: 10.1039/c000000x/
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