Copper(ii) complexes based on tripodal pyridyl amine derivatives as efficient anticancer agents

Article abstract of DOI:10.1039/c9nj00061e

The complexes [Cu(TPA)Cl]ClO₄·?H₂O (1-ClO₄), [Cu(6-MeTPA)Cl]ClO₄/PF₆ (2-ClO₄/2-PF₆), [Cu(6-Me₂TPA)Cl]PF₆ (3-PF₆), [Cu(BPQA)Cl]ClO₄/PF₆ (4-ClO₄/4-PF₆), [Cu(BPQA)Cl]ClO₄/PF₆ (4-ClO₄/4-PF₆), [Cu(BQPA)Cl]ClO₄/PF₆ (5-ClO₄/PF₆), [Cu(L¹)Cl]ClO₄/PF₆ (6-ClO₄/6-PF₆), [Cu(L²)Cl]ClO₄ (7-ClO₄) and [Cu(L³)Cl]ClO₄ (8-ClO₄) have been synthesized and structurally characterized by spectroscopic techniques and single X-ray crystallography. The in vitro cytotoxicity of the prepared Cu(ii) complexes was evaluated against A2780 (ovarian), A2780R (cisplatin-resistant variant) and MCF7 (breast cancer) human cancer cell lines. Overall, the complexes revealed significant-to-moderate cytotoxicity, with the best results obtained for the complexes [Cu(BQPA)Cl]ClO₄ (5-ClO₄) and [Cu(BQPA)Cl]PF₆ (5-PF₆), showing IC₅₀ values within the range of 4.7-10.8 μM. The ability of the most cytotoxic complexes to cleave DNA under different conditions and the mechanisms underlying this activity were assessed by means of agarose gel electrophoresis.

Full text of DOI:10.1039/c9nj00061e

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Travnicek, New J. Chem., 2019, DOI: 10.1039/C9NJ00061E.
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Page 1 of 30
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DOI: 10.1039/C9NJ00061E
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Copper(II) complexes based on tripodal pyridyl amine
derivatives as efficient anticancer agents
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Salah S. Massoud,*a Febee R. Louka,a Ada F. Tusa,a Nicole E. Bordelon,a Roland
C. Fischer,b Franz A. Mautner,*c Ján Vančo,d Jan Hošek,d Zdeněk Dvořákd and
Zdeněk Trávníček*d
a Department of Chemistry, University of Louisiana at Lafayette, Lafayette, LA 70504,
U. S. A.
b Institut für Anorganische Chemische, Technische Universität Graz, Stremayrgasse 9/V, A-
8010 Graz, Austria
c Institut für Physikalische and Theoretische Chemie, Technische Universität Graz,
Stremayrgasse 9/II, A-8010, Graz, Austria
d Division of Biologically Active Complexes and Molecular Magnets, Regional Centre of
Advanced Technologies and Materials, Faculty of Science, Palacký University, Šlechtitelů 27,
CZ-783 71 Olomouc, Czech Republic
(Received:
Accepted:
)
Keywords Copper; Pyridyl compounds; Crystal structure; Anticancer activity; Cytotoxicity
Electronic supplementary information (ESI) available. CCDC-1885301-1885307 contain the
crystallographic data in CIF format for 2-PF6, 4-ClO4, 4-PF6, 5-PF6, 7-ClO4, 8-ClO4 and 6-PF6,
respectively. These data can be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif. Selected bond and angles are summarized in
Table S1. ESI materials associated with the gel electrophoresis results (Figs S1-S6) of this article can
be found, in the online version at http://dx.doi.org/??????????????????????.
________________________________
* Corresponding authors: E-mail: ssmassoud@louisiana.edu, Tel. +01 337-482-5672, Fax: +01 337-
482-5676 (S.S. Massoud); E-mail: mautner@tugraz.at, Tel. ++43 316-873-32270, Fax: ++43 316-
873-8225 (F.A. Mautner); E-mail: zdenek.travnicek@upol.cz, Tel. +420 585-634-352, Fax: +420 585-
634-954 (Z. Trávníček).
1

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Abstract
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The complexes [Cu(TPA)Cl]ClO4.½H2O (1-ClO4), [Cu(6-MeTPA)Cl]ClO4/PF6 (2-ClO4/2-PF6),
[Cu(6-Me2TPA)Cl]PF6 (3-PF6), [Cu(BPQA)Cl]ClO4/PF6 (4-ClO4/4-PF6), [Cu(BPQA)Cl]ClO4/PF6
(4- ClO4/4-PF6), [Cu(BQPA)Cl]ClO4/PF6 (5-ClO4/PF6), [Cu(L1)Cl]ClO4/PF6 (6-ClO4/6-PF6),
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[Cu(L2)Cl]ClO4
(7-ClO4) and [Cu(L3)Cl]ClO4 (8-ClO4) have been synthesized and structurally
characterized by spectroscopic techniques and single X-ray crystallography. The in vitro cytotoxicity
of the prepared Cu(II) complexes were evaluated against A2780 (ovarian), A2780R (cisplatin-resistant
variant) and MCF7 (breast cancer) human cancer cell lines. Overall, the complexes revealed
significant-to-moderate cytotoxicity, with the best results obtained for the complexes
[Cu(BQPA)Cl]ClO4 (5-ClO4) and [Cu(BQPA)Cl]PF6 (5-PF6) showing IC50 values within the range of 4.7–10.8
M. The ability of the most cytotoxic complexes to cleave the DNA at different conditions and the
mechanisms underlying this activity were assessed by means of the agarose gel electrophoresis.
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Introduction
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According to World Health Organization cancer is considered as the second most common cause of
death worldwide after cardiovascular diseases. In 2015, cancer diseases were responsible for 8.8
million deaths around the world. Sadly, this figure is expected to rise to 15.1 million people in 2030.1,2
Moreover, in the developed countries, it was statistically pointed out that two in every five born
people will be diagnosed with cancer during their life time. The mortality data which were collected in
2016 by the National Center for Health Statistics predicted 1,685,210 new cancer cases and 595,690
cancer deaths were projected to occur in the United States.1,2
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In the design of anticancer agents, the transition metal complexes provide some interesting
properties which may not exist in the organic compounds. These include the nature of the central
metal ion, its coordination number, oxidation state, redox properties, the geometry of the coordination
compound, its thermodynamic and kinetic stability as well as the wide variety in the co-ligand(s)
skeleton coordinated to the central metal ion. The great success of cisplatin and the second and third
generation of its derivatives, used as clinically effective therapeutic agents in the fight against a wide
spectrum of cancers,3,4 was confronted by some major problems associated with the serious side
effects of these drugs and the resistance exhibited by some cancer cells.5 Extensive attempts have
been directed into this area to develop alternative therapeutic agents that can improve the effectivity
(i.e. cytotoxicity against cancer cells) of the drug, while reducing its dose and its toxicity.4 Many metal
complexes, derived from ruthenium(II/III), gold(I/III), gallium(III), titanium((III/IV), iron(II/III) and
cobalt(II/III) with a variety of ligand structures, have been launched and investigated as anticancer
therapeutic agents.6-11
In the search for developing of effective antitumor agents copper(I/II) complexes constructed
from polydentate linear, Schiff bases, macrocycles or tripodal-tetradentate ligands with X-donor
atoms (X = N; O; S; N, O; N, O, S) were designed and tested for their anticancer activities against
different human cancer cell lines such as MCF7 (breast cancer), A2780 (ovarian), A2780R (cisplatin-
resistant variant), HOS (aggressive bone tumors), CaCo2 (epithelial colorectal adenocarcinoma),
AS49 (lung), DU-145 (prostate), HCT-15 (Colon), HeLa (human cervix epithelial carcinoma) and
hepatocytes.12-35 There are also some recent review papers taking together the knowledge about the
6,12, 13
anticancer activities and the underlying mechanisms of action regarding the copper complexes.
Copper complexes were introduced into this area based on the assumption that as it is an essential
element for life, it may be less toxic compared to other used metal complexes.5,12,13,35 Copper plays a
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crucial role not only in biochemistry but also in medicine.5,12,36 In contrast, while the metal has been
used in the treatment of copper deficiency in Menkes disease, excess copper should be removed from
the body with a chelating therapeutic agents in Wilson disease.5,12,36
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Recently, a series of copper(II) complexes with tripodal tris(pyridin-2-yl)amine ligands were
investigated for their in vitro anticancer potential against some tumor cell lines.26 This study did not
reveal any systematic behavior between the pyridyl copper compounds and the observed cancer
activity.26 However, this result motivated us to perform a screening focused on the effect of
substituents in the parent tris(2-pyridylmethyl)amine (TPA) ligand (Scheme 1) and the in vitro
cytotoxicity of their corresponding Cu(II) complexes, [Cu(L)X]ClO4/PF6 (L = any ligand shown in
Scheme 1), against some cancer cells in order to evaluate their cytotoxic effectiveness compared to the
standard cisplatin.
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Scheme 1 Structural formulas of the tripod pyridyl-based ligands used in this study.
Results and discussion
Synthetic aspects
The tripod tetradentate N-donor atoms based pyridyl groups, shown in scheme 1 (6-MeTPA, 6-
Me2TPA, BPQA, BQPA, L1, L2 and L3), with the exception of TPA,37,38 were synthesized by gentle
reflux and stirring (48-72 h) of a slurry mixture containing 2-aminomethylpyridine or di(2-
pyridyl)amine with the appropriate 2-chloromethylpyridine hydrochloride derivatives or 2-
chloromethylquinoline hydrochloride under anhydrous conditions of CH3CN or THF in the presence
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of anhydrous Na2CO3/Cs2CO3 or Et3N, which is used in slight excess used more than the
stoichiometric amounts under nitrogen.39,40 These compounds, which solidified upon standing at room
temperature require several recrystallization from Et2O with the aid of activated charcoal were
characterized by elemental microanalysis, IR, 1H and 13C NMR spectroscopy as well by ESI-MS.
The syntheses of the chlorido copper(II) complexes: [Cu(TPA)Cl]ClO4.½H2O (1-ClO4),41
[Cu(6-MeTPA)Cl]ClO4 (2-ClO4), [Cu(6-MeTPA)Cl]PF6 (2-PF6), [Cu(6-Me2TPA)Cl]PF6 (3-PF6),
[Cu(BPQA)Cl]ClO4 (4-ClO4), [Cu(BPQA)Cl]PF6 (4-PF6), [Cu(BQPA)Cl]ClO4 (5-ClO4),
[Cu(BQPA)Cl]PF6 (5-PF6), [Cu(L1)Cl]PF6 (6-PF6), [Cu(L1)Cl]ClO4 (6-ClO4), [Cu(L2)Cl]ClO4 (7-
ClO4) and [Cu(L3)Cl]ClO4 (8-ClO4) were achieved by the reaction of a methanolic solution of the
ligand and CuCl2·2H2O in a 1:1 molar ratio, followed by addition of slight excess of NaClO4 or
NH4PF6. The isolated complexes, were crystallized from CH3CN to remove the chloride salt and
unreacted NaClO4/NH4PF6. In most cases further recrysallization of the products from MeOH resulted
in the isolation of single crystals of suitable for X-ray structure determination. These complexes which
were produced in pure form with reasonable to good yields (51-93%) were characterized by elemental
microanalyses, molar conductivity measurements, IR, UV-Vis and single crystal X-ray
crystallography as well as by ESI-MS in some cases.
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Description of the crystal structures
The structures of the seven complexes: 2-PF6, 4-PF6, 4-ClO4, 5-PF6, 6-PF6, 7-ClO4 and 8-ClO4 were
determined and selected interatomic parameters in the vicinity of the Cu(II) centers are listed in Table
1. In these complexes, the copper(II) centers of the mononuclear complex cations are penta-
coordinated by four N-donor atoms of the tripod tetraamine ligand and one terminal chloride anion as
illustrated in Fig. 1. The CuN4Cl chromophores form distorted trigonal bipyramids (TBP) in 5-PF6, 6-
PF6, 7-ClO4 and 8-ClO4 with -values of 0.64, 0.96 (mean), 0.86 and 0.80, respectively (-values of 0
and 1 refer to ideal geometries of square pyramid (SP) and trigonal bipyramid, respectively).42 The
axial sites are occupied by terminal chloride anion and N(amine), whereas the three equatorial sites are
occupied by the three pyridyl nitrogen donor atoms. In 2-PF6, 4-PF6, 4-ClO4 the CuN4Cl
chromophores form distorted square pyramids with -values of 0.12, 0.16, and 0.13, respectively. The
Cu-Cl bond distances vary from 2.222 to 2.2528 Å, the Cu-N(amine) from 2.004 to 2.0612 Å, and the
Cu-N(pyridyl) from 1.9831 to 2.3616 Å. For the CuN4Cl chromophores in penta-coordinated
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[Cu(TPA)Cl]ClO4·½H2O (1-ClO4), [Cu(6-MeTPA)Cl]ClO4 (2-ClO4) and [Cu(6-Me2TPA)Cl]ClO4 (3-
ClO4) -values of 0.98 and 0.94; 0.16 and 0.24; 0.07, respectively, were reported.41,43
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Fig. 1 Representative plots of coordination spheres of 2-PF6, 4-PF6, 4-ClO4, 5-PF6, 6-PF6, 7-ClO4
and 8-ClO4 together with partial atom numbering scheme.
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Table 1 Selected bond distances (Å) and bond angles (°) of 2-PF6, 4-ClO4 and 4-PF6
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2-PF6
4-ClO4
4-PF6
Cu1-N2
1.9885(16)
2.0026(16)
2.3616(16)
2.0585(16)
2.2480(5)
170.48(5)
162.86(7)
100.46(6)
86.04(6)
1.9934(12)
2.0099(12)
2.3572(12)
2.0612(11)
2.2528(4)
172.00(3)
162.57(5)
101.92(4)
83.90(4)
1.9831(13)
2.0034(13)
2.3473(14)
2.0606(12)
2.2453(4)
170.82(4)
163.03(5)
100.46(5)
85.12(5)
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Cu1-N3
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Cu1-N4
Cu1-N1
Cu1-Cl1
N1-Cu1-Cl1
N2-Cu1-N3
N2-Cu1-N4
N3-Cu1-N4
-value
0.119
0.157
0.130
Table 1 Cont. Selected bond distances (Å) and bond angles (°) of 5-PF6, 7-ClO4 and 8-ClO4
.
5-PF6
7-ClO4
8-ClO4
Cu1-N2
2.0733(9)
2.2512(9)
2.1115(9)
2.0251(9)
2.2264(3)
171.07(3)
114.50(3)
132.69(4)
103.41(3)
0.640
2.0291(17)
2.0973(18)
2.0542(17)
2.0505(16)
2.2250(5)
179.65(5)
122.48(7)
128.22(7)
103.11(7)
0.857
2.1022(12)
2.0394(12)
2.0464(12)
2.0317(12)
2.2411(4)
178.34(4)
113.44(5)
109.12(5)
130.64(5)
0.795
Cu1-N3
Cu1-N4
Cu1-N1
Cu1-Cl1
N1-Cu1-Cl1
N2-Cu1-N3
N2-Cu1-N4
N3-Cu1-N4
-value
Table 1 Cont. Selected bond distances (Å) and bond angles (°) of 6-PF6.
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6-PF6
Cu1-N2
Cu1-N3
2.049(4)
2.072(5)
Cu2-N8
Cu2-N7
2.045(4)
2.074(5)
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Cu1-N4
2.067(5)
2.022(4)
2.2386(18)
177.40(14)
121.8(2)
0.927
Cu2-N6
2.047(5)
Cu1-N1
Cu2-N5
2.042(4)
2.2384(15)
178.01(14)
119.2(2)
0.980
Cu1-Cl1
N1-Cu1-Cl1
N2-Cu1-N4
-value
Cu2-Cl2
N5-Cu2-Cl2
N6-Cu1-N7
-value
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Cu3-N9
2.043(5)
2.098(5)
2.029(4)
2.046(5)
2.2399(16)
178.65(14)
121.56(19)
0.952
Cu4-N13
Cu4-N15
Cu4-N16
Cu4-N14
Cu4-Cl4
2.031(5)
2.062(5)
2.073(5)
2.092(3)
2.2124(18)
178.15(16)
118.75(16)
0.990
Cu3-N11
Cu3-N12
Cu3-N10
Cu3-Cl3
N9-Cu3-Cl3
N10-Cu3-N12
-value
N13-Cu4-Cl4
N15-Cu4-N14
-value
Characterization of the compounds
The acetonitrile molar conductivity of the chlorido-Cu(II) complexes under investigation gave ΛM
values in the range of 137 to 149 Ω-1cm2mol-1 which are typical for 1:1 electrolytic behavior.44 The
ESI-MS of the complexes 2-ClO4 and 8-ClO4 in CH3CN revealed the presence of the molecular ion
[Cu(L)Cl]+, where L = MeTPA or L3, respectively with perfect agreement between the calculated and
experimental m/z values (see experimental section). Similar results were produced for corresponding
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counter anions ClO4 or PF6 .
The ATR-IR spectra of complex 1-ClO4, 4-ClO4, 5-ClO4, 6-ClO4, 7-ClO4 and 8-ClO4 display
a very strong band at 1074-1093 cm-1, which is assigned to stretching vibration of (Cl-O) of the
perchlorate counter ion. The split for this band into 1114 and 1093 cm-1 in the later complex is most
likely attributed to the distortion of the perchlorate counter ion and reducing its symmetry from Td to
C3ʋ or C2ʋ. On the other hand, the hexafluoroammonium phosphate compounds 3-PF6 , 4-PF6, 5-PF6
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and 6-PF6 revealed a strong band over the vibration range 827-854 cm-1 due to (P-F) of the PF6
counter ion. The very weak bands observed above 3000±100 cm-1 are attributed to the (C-H)
strtching vibration, of the pyridyl/quinoly and the aliphatic C-H vibrations, respectively. Whereas, the
medium to weak intensity vibrational band over the range 1610-1400 cm-1 are characteristic to the
pyridyl moieties.38-41
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Table 2 The UV-Vis spectral and molar conductivity, ΛM data of the complexes under investigation
in CH3CN solutions.
Complex
UV-Vis
Assigned Geometry ΛM
(dist TBP vs SP) a)
λmax, nm (εmax , M-1cm-1)
(Ω-1cm2mol-1)
[Cu(TPA)Cl]ClO4·½H2O b) (1-ClO4) ~730 (sh), 950 (b)
dist TBP
dist TBP
dist TBP
177 c)
142
[Cu(6-MeTPA)Cl]ClO4
[Cu(6-MeTPA)Cl]PF6
(2-ClO4) ~660 (sh), 900 (112, b)
(2-PF6)
(3-PF6)
143
~670 (sh), 885 (132, b)
685 (148), ~860 (sh)
[Cu(6-Me2TPA)Cl]PF6
[Cu(BPQA)Cl]ClO4
[Cu(BPQA)Cl]PF6
[Cu(BQPA)Cl]ClO4
[Cu(BQPA)Cl]PF6
[Cu(L1)Cl]ClO4
dist SP
146
141
147
145
146
143
147
149
137
(4-ClO4) ~640, 880 (151, b)
dist TBP
dist TBP
intermediate
dist TBP
dist TBP
(4-PF6)
(5-ClO4)
(5-PF6)
~700 (sh), 900 (159, b)
730 (138, b), ~880 (143, b)
~660 (sh), 880 (141, b)
(6-ClO4) ~725 (sh), 955 (221)
[Cu(L1)Cl]PF6
(6-PF6)
~700 (sh), ~ 850 (sh), 970 (355) dist TBP
[Cu(L2)Cl]ClO4
(7-ClO4) ~715 (sh), 960 (230, b)
dist TBP
[Cu(L3)Cl]ClO4
(8-ClO4) ~710 (sh), ~880 (sh), 970 (371) dist TBP
a) dist = distorted, TBP = trigonal bipyramid, SP = square pyramid
b) Ref 41
c) Determined in aqueous solution
The visible spectra of the complexes in CH3CN are summarized in Table 2 together with their
molar conductivity. Inspection of this data reveal that the complexes have similar spectral features.
With the exception of 3-PF6, the complexes in general exhibit a shoulder over the wavelength range
(640-730 nm) and a broad band at λmax ≥ 850 nm.45 These spectral features are fully consistent with
five-coordinate species with distorted trigonal bipyramidal geometry (TBP) around the central Cu(II)
ion. In contrast, the more sterically hindered complex [Cu(6-Me2TPA)Cl]PF6 (3-PF6) showed an
intense broad band at 685 and a shoulder at around 860 nm where this feature is in an agreement with
a distorted square pyramidal geometry (SP). On the other hand, the appearance of two broad bands
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around 730 and 880 nm of almost the same intensity in [Cu(BQPA)Cl]ClO4 (5-ClO4) may indicate an
intermediate geometry between TBP and SP around the central Cu(II) ion with a slightly increased
distortion towards SP geometry.45-49 The TBP geometrical assignments in acetonitrile solution were in
full agreements with those obtained from X-ray structural determinations in complexes 5-PF6, 6-PF6,
7-ClO4 and 8-ClO4, whereas the complexes 2-PF6, 4-PF6 and 4-ClO4 revealed a pronounced
tendency toward SP geometry in the solid state (see X-ray section).
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Solution studies of the complexes
The visible spectra of the complexes under investigation in CH3CN, CH3OH and aqueous
acetonitrile solutions produced the same spectra and did not show any sign of spectral changes over a
period of five days. Moreover, the molar conductivities of the complexes in these media and over the
tested time period were eventually unchanged within the experimental error and fully consistent with
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1:1 electrolyte ([Cu(L)Cl]+ + ClO4 /PF6 ) as determined before in CH3CN solution. These indicate that
the complexes in these media are very stable and do not undergo any hydrolysis and/or solvolysis
reactions as well as their configurations are retained in these media as the chlorido, [Cu(L)Cl]+
complex ion. The corresponding Co(II) complexes, [Co(L)Cl]ClO4/PF6 with L = TPA, 6-MeTPA, 6-
Me2TPA, BPQA, BQPA, L1, L2 and L3, were reported to undergo aquation and/or solvolysis through
the chloride displacement reactions ([Co(L)(H2O/CH3CN)]2+) and this process was accompanied by
dramatic color, spectral and conductivity changes.39,40
In vitro cytotoxicity
The in vitro cytotoxicity of the complexes against a series of human cancer cell lines (A2780, A2780R
and MCF7) was studied using the MTT cell viability assay. The cytotoxicity of the complexes under
investigation together with cisplatin used as a reference drug was evaluated under the same
experimental conditions. The IC50 values, calculated from the dose-survival curves obtained after 24 h
incubation with the complexes, are shown in Table 3.
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Table 3 The cytotoxicity of the studied complexes and cisplatin against a series of human cancer cell
lines (ovarian cancer A2780, ovarian cancer resistant to cisplatin A2780R, and breast
adenocarcinoma MCF7). The values of half maximal inhibitory concentration (IC50) of cells viability
were calculated from the corresponding dose-response curves.
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Complex
A2780
> 100
A2780R
MCF7
[Cu(TPA)Cl]ClO4·½H2O (1-ClO4)
Not tested 52.3 ± 1.4
[Cu(6-MeTPA)Cl]ClO4
[Cu(6-MeTPA)Cl]PF6
[Cu(6-Me2TPA)Cl]PF6
[Cu(BPQA)Cl]ClO4
[Cu(BPQA)Cl]PF6
[Cu(BQPA)Cl]ClO4
[Cu(BQPA)Cl]PF6
[Cu(L1)Cl]ClO4
(2-ClO4)
(2-PF6)
83.2 ± 2.1 > 100
23.2 ± 2.5 > 100
23.7 ± 0.6
57.9 ± 4.5
(3-PF6)
21.1 ± 3.3 54.2 ± 1.9 17.4 ± 0.4
18.2 ± 3.0 28.5 ± 3.0 25.9 ± 3.0
20.5 ± 1.0 47.3 ± 0.5 29.2 ± 1.3
(4-ClO4)
(4-PF6)
(5-ClO4)
(5-PF6)
4.9 ± 0.8
4.7 ± 0.1
9.4 ± 1.1
8.0 ± 2.0
5.5 ± 0.3
10.8 ± 0.3
(6-ClO4)
(6-PF6)
23.6 ± 3.7 36.7 ± 1.1 31.9 ± 3.1
28.5 ± 4.0 36.2 ± 2.8 31.0 ± 0.6
[Cu(L1)Cl]PF6
[Cu(L2)Cl]ClO4
(7-ClO4)
(8-ClO4)
81.7 ± 5.6 > 100
11.3 ± 0.8 > 100
15.7 ± 1.8 > 50
26.9 ± 2.7
25.5 ± 2.1
21.5 ± 0.5
[Cu(L3)Cl]ClO4
cisplatin
Complexes 5-ClO4 and 5-PF6 showed the best cytotoxicity against all three cancer cell lines,
significantly better than that of the reference cisplatin. Moreover, the cytotoxic effects of these
complexes were not influenced by the intrinsic resistance of A2780R cell line against cisplatin, which
might indicate that another mechanism of action (as compared with cisplatin) is responsible for this
biological activity. These two complexes are about three times more effective than cisplatin towards
A2780, and they also revealed significant effectiveness against A2780R and MCF7 cancer cells. . The
cytotoxicity of 4-ClO4 and 4-PF6 compounds is comparable to that observed for cisplatin. Overall, it
may be concluded that the complexes revealed significant-to-moderate cytotoxicity against the human
cancer cell lines used. These results are comparable with previously reported cytotoxicity data
obtained for copper(II) complexes involving tris(pyridin-2-yl-alkyl)amine ligands.26 In both cases, the
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complexes differed in their counter-ions only (ClO4 vs. PF6 ), which in most cases do not have a
substantial impact on the resulting cytotoxicity of the complexes, as previously demonstrated.50
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Moreover, the results of cytotoxicity pointed out the trend of increasing cytotoxicity (i.e.
decreasing IC50 values) depending on the increasing lipophilicity of the tripodal ligands. This trend is
evident either in the series of the complexes with increasing number of binuclear aryl substitution in
the side-arms (quinolin-2-ylmethyl substitution) following the order of increasing lipophilicity and
cztotoxicity: 1-ClO4 < 4-ClO4 ≈ 4-PF6 < 5-ClO4 ≈ 5-PF6, so in the case of the number of lipophilic
substituents on pyridine rings in the following order: no substitution (1-ClO4) < monomethyl-
substitution (2-ClO4, 2-PF6) < disubstitution (dimethyl-substitution 3-PF6 and dimethoxy-substitution
7-ClO4) ≈ trisubstitution (6-ClO4, 6-PF6) < hexasubstitution (8-ClO4).
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DNA cleavage
One of the most relevant mechanisms, by which the copper(II) complexes can influence the processes
of cell survival and cell death in the cancer cells, is their participation in the initiation and progression
of the oxidative stress, having a strong damaging effect on various cellular targets and biomolecules.50
The tested complexes, containing the tetradentate N-donor ligands, showed mild nuclease effects in
the concentration-dependent manner in aqueous solutions (Fig. 2A), producing up to ca. 20 % of
single-strand cleaved OC-form at the 300 µM concentration in case of 5-ClO4 complex. In order to
reveal whether the observed nuclease activity is based on hydrolytic or oxidative mechanism, the same
experiments were performed in the absence of oxygen under inert atmosphere of helium (Fig. S1 in
the ESI section). The results showed that the complexes were able to cleave the DNA in the same
magnitude in the absence of oxygen (under He atmosphere) as under the normal conditions. This
finding indicates that the observed small portion of DNA damage can be attributed to the direct
hydrolytic mechanism.
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Fig. 2 Nuclease-like effect of the selected complexes (5-ClO4, 5-PF6, 1-ClO4). Supercoiled plasmid
DNA (CCC) was incubated with the complexes at different concentrations or pure solvent (blank) in
the water at 37 °C for 1 h (A) or with the addition of 0.66 mM hydrogen peroxide (B). After incubation,
the relative amount of open circle (OC) and linear (L) forms of plasmid DNA was evaluated by
densitometric analysis. Graphs indicate means ± SEM of three independent experiments
electrophoretograms show representative results of agarose electrophoresis. * indicates statistical
difference to blank (p < 0.05); ** indicates statistical difference to blank (p < 0.01); **** indicates
statistical difference to blank (p < 0.0001).
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Previous experience on this class of compounds52,53 showed that these complexes cleave the
DNA most effectively in the presence of hydrogen peroxide via an oxidative mechanism by taking part
in the Fenton reaction.54 In fact, dramatic increase in the nuclease activity was observed in all
complexes when the reactions were performed in the presence of hydrogen peroxide (Fig. 2B). These
results clearly revealed that all complexes were able not only to cleave both the single and double
stranded DNA, but also were able to cleave the native CCC-form of the plasmid DNA completely
even at 10 µM concentration. At higher concentrations, complete disintegration of all DNA forms to
small fragments occurred (a smear was detected in the electrophoretograms) with most effective
reactivity pattern determined in 1-ClO4 followed by 5-ClO4 and 5-PF6.
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To pinpoint the participation of different reactive oxygen species (ROS) in the DNA cleavage
process, the free-radical inhibitors (NaN3, KI, and DMSO) were added into the reaction mixtures in
equimolar concentrations to the applied complexes. Surprisingly, the radical scavengers produced no
changes under these conditions (Figs. S2–S4). Furthermore, we checked also the effect of using high
concentrations of DMSO on the cleavage process. Only when the DMSO was about 10 000-times
more excess compared to the complex concentration, the cleavage process was attenuated significantly
(Fig. S5). This indicates, that the hydroxyl radicals partially participate in the cleavage mechanism
induced by the complexes but the major species responsible for the oxidative damage of DNA are
probably metal-based intermediates which are formed in the Fenton reaction or in the mechanism of
enzymatic activation of mono-copper monooxygenases such as CuO+, CuO+• or [CuOOH]+.54–57 This
hypothesis has been indirectly confirmed by the addition of the highly efficient metal chelator EDTA
to the reaction mixtures and resulted in the prevention of plasmid DNA destruction (Fig. S6).
Experimental
Materials and physical measurements
Bis(2-pyridylmethyl)amine (DPA) and 2-aminomethylpyridine as well as the hydrochloride salts of 2-
chloromethylquinoline, 2-chloromethylpyridine, 3,5-dimethyl-4-methoxy-2-chloromethyl-pyridine and
3,4-dimethoxy-2-chloromethylpyridine were purchased from TCI-America, whereas 6-methyl-2-
pyridinemethanol was purchased from Alfaaesar. All other chemicals were commercially available and
used without further purification. Infrared spectra were recorded on a JASCO FTIR-480 plus
spectrometer as KBr pellets or on a Cary 630 (ATR-IR) spectrometer. Electronic spectra were
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recorded using an Agilent 8453 HP diode array UV-Vis spectrophotometer. H and C NMR spectra
were obtained at room temperature on a Varian 400 NMR spectrometer operating at 400 MHz (1H)
and 100 MHz (13C). H and C NMR chemical shifts (δ) are reported in ppm and were referenced
internally to residual solvent resonances (DMSO-d6: δH = 2.49, δC = 39.4 ppm). ESI-MS were
measured on Agilent 6210 electrospray TOF mass spectrometer. The conductivity measurements were
performed using Mettler Toledo Seven Easy conductivity meter and calibrated by the aid of 1413
μS/cm conductivity standard. The molar conductivities of the complexes were determined from ΛM =
(1.0 x103 κ)/[Cu], where κ = specific conductance and [Cu] is the molar concentration of the complex.
Elemental microanalyses were carried out by Atlantic Microlaboratory, Norcross, Georgia U.S.A.
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Caution: Salts of perchlorate and their metal complexes are potentially explosive and should be handled with
great care and in small quantities.
Synthesis of the ligands
Tris(2-pyridymethyl)amine (TPA), [(6-methyl-2-pyridyl)methyl)-bis(2-pyridylmethyl)]amine (6-
MeTPA),
pyridylmethyl)-(2-quinolylmethyl)]amine (BPQA), [bis(2-quinolylmethyl)-(2-pyridylmethyl)]amine
(BQPA), [(3,5-dimethyl-4-methoxy-2-pyridylmethyl)-bis(2-pyridylmethyl)]amine [(3,4-
(L1),
[bis(6-methyl-2-pyridyl)methyl)-(2-pyridylmethyl)]amine
(6-Me2TPA),
[bis(2-
dimethoxy-2-pyridylmethyl)-bis(2-pyridylmethyl)]amine (L2) and [bis(3,5-dimethyl-4-methoxy-2-
pyridylmethyl)-(2-pyridylmethyl)]amine (L3) were synthesized according to the published procedure
[39,40]. The conversion of 6-methyl-2-pyridinemethanol into 6-methyl-2-chloromethylpyridine
hydrochloride was achieved in CCl4 by SOCl2, followed by recrystallization from ethanol.
Synthesis of the complexes
The complex [Cu(TPA)Cl]ClO4·½H2O (1-ClO4) was synthesized as described.41
[Cu(6-MeTPA)Cl]ClO4 (2-ClO4). Although this complex was previously synthesized and
structurally characterized,43 here in we described its synthesis which is slightly modified from the
reported one. A mixture containing [(6-methyl-2-pyridyl)-methyl)-bis(2-pyridylmethyl)]amine (6-
MeTPA), (0.152 g, 0.50 mmol) and CuCl2·2H2O (0.090 g, 0.5 mmol) dissolved in MeOH (20 mL) was
heated on a steam-bath for 5 min. To this solution NaClO4 (0.070 g, 0.57 mmol) was followed by
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filtration through celite. The resulting solution was allowed to stand at room temperature. After one
day, the blue precipitate, which separated was collected by filtration, recrystallized from CH3CN to
remove unreacted NaClO4 and allowed to evaporate at room temperature. The resulting blue
precipitate was collected and further recrystallization of the product from MeOH afforded good single
crystals suitable for X-ray analysis. These were washed with propan-2-ol and Et2O, and dried in air
(yield: 0.210 g, 84%). Characterization: Anal. Calcd for C19H20CuCl2N4O4 (502.84 g/mol): C, 45.38;
H, 4.01; N, 11.14. Found: C, 45.39; H, 4.06; N, 11.10%. ESI-MS (CH3CN): m/z = 402.07 (Calcd for
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-
[C19H20ClCuN4]+ = 402.08, 100%) and 98.95 (Calcd for ClO4 = 98.95, 100%). Selected IR bands
(ATR-IR, cm-1): 1604 (s), 1438 (s), 1372 (w), 1261 (w), 1167 (w), 1078 (vs), 1023 (vs), 1007 (w), 955
(m), 768 (m). UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): ~660 (sh), 900 (112, b). ΛM (CH3CN) =
142 Ω-1cm2mol-1.
[Cu(6-Me2TPA)Cl]PF6 (3-PF6). To a mixture containing [bis(6-methyl-2-pyridyl)methyl)-(2-
pyridylmethyl)]amine (6-Me2TPA) (0.152 g, 0.50 mmol) and CuCl2·2H2O (0.090 g, 0.5mmol)
dissolved in MeOH (20 mL) NH4PF6 (0.098 g, 0.60 mmol) was added. The bluish-torques solution
was heated on a steam-bath for 5 min, filtered through celite and then was allowed to stand at room
temperature. The crude product which separated after one day was collected by filtration,
recrystallized from CH3CN to remove unreacted NH4PF6 and allowed to evaporate at room
temperature. Recrystallization from MeOH afforded good blue single crystals suitable for X-ray
analysis. These were collected by filtration, washed with propan-2-ol and Et2O, and dried in air (yield:
0.240 g, 85%). Characterization: Anal. Calcd for C20H22ClCuF6N4P (562.38 g/mol): C, 42.71; H, 3.94;
N, 9.96. Found: C, 42.61; H, 3.97; N, 9.86%. Selected IR bands (ATR-IR, cm-1): 3100 (vw), 2954
(vw), 1600 (m), 1574 (w), 1467 (w), 1443 (m), 1284 (m), 1167 (m), 1092 (m), 1007 (m), 970 (w), 827
(vs). UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): 685 (148), ~860 (sh). ΛM (CH3CN) = 146 Ω-
1cm2mol-1.
The rest of the complexes were typically synthesized using the appropriate ligand and a
procedure similar to that described either for the perchlorate complex, [Cu(6-MeTPA)Cl]ClO4 (2-
ClO4)43 or the hexafluorophosphate, [Cu(6-Me2TPA)Cl]PF6 (3-PF6).
[Cu(6-MeTPA)Cl]PF6 (2-PF6). Blue crystals were obtained from a green solution (yield:
0.200 g, 73%). Characterization: Anal. Calcd. for C19H20ClCuF6N4P (548.35 g/mol): C, 41.62; H, 3.68;
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N, 10.22. Found: C, 41.68; H, 3.80; N, 10.17%. ESI-MS (CH3CN): m/z = 402.07 (Calcd for
-
[C19H20ClCuN4]+ = 402.08, 100%) and 144.97 (Calcd for PF6 = 144.96, 100%). Selected IR bands
(ATR-IR, cm-1): 1611 (s), 1576 (m), 1482 (m), 1459 (m), 1446 (s), 1161 (m), 1105 (m), 1056 (m),
1031 (m), 840 (vs), 770 (w). UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): ~670 (sh), 885 (132, b). ΛM
(CH3CN) = 143 Ω-1cm2mol-1.
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[Cu(BPQA)Cl]ClO4 (4-ClO4). Blue crystalline compound (yield: 0.180 g, 67%).
Characterization: Anal. Calcd for C22H20Cl2CuN4O4 (538.87 g/mol): C, 49.03; H, 3.74; N, 10.40.
Found: C, 49.08; H, 3.90; N, 10.29%. Selected IR bands (ATR-IR, cm-1): 3060 (w), 2965 (vw), 1600
(s), 1571 (m), 1507 (m) 1472 (m), 1433 (s), 1284 (m), 1156 (m), 1078 (vs), 906 (m), 833 (s), 779 (s),
768 (s), 728 (m). UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): ~640, 880 (151, b). ΛM (CH3CN) = 141
Ω-1cm2mol-1.
[Cu(BPQA)Cl]PF6 (4-PF6). Long green needles (yield: 0.210 g, 72%). Characterization:
Anal. Calcd. for C22H20ClCuF6N4P (584.38 g/mol): C, 45.22; H, 3.45; N, 9.59. Found: C, 45.55; H,
3.36; N, 9.59%. Selected IR bands (ATR-IR, cm-1): 3080 vw), 3063 (vw), 2968 (vw), 2923 (vw), 1610
(s), 1599 (m), 1509 (w), 1482 (w), 1446 (m), 1286 (m), 1158 (m), 1102 (w), 1054 (m), 1030 (w), 964
(m), 891 (m), 830 (vs), 769 (s). UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): ~700 (sh), 900 (159, b).
ΛM (CH3CN) = 147 Ω-1cm2mol-1.
[Cu(BQPA)Cl]ClO4 (5-ClO4). Blue crystalline compound (yield: 0.24 g, 75%).
Characterization: Anal. Calcd for C26H22Cl2CuN4O4 (588.93 g/mol): C, 53.02; H, 3.77; N, 9.51.
Found: C, 52.93; H, 3.63; N, 9.41%. Selected IR bands (ATR-IR, cm-1): 3072 (w), 2932 (vw), 1600
(s), 1570 (m), 1513 (s), 1474 (m), 1434 (m), 1370 (w), 1344 (w), 1299 (m), 1208 (m), 1074 (vs), 1017
(s), 955 (s), 908 (w), 838 (s), 829 (s), 780 (vs), 761 (s), 748 (m). UV-Vis (CH3CN) λmax, nm (εmax, cm-1
M-1): 730 (138, b), ~880 (143, b). ΛM (CH3CN) = 145 Ω-1cm2mol-1.
[Cu(BQPA)Cl]PF6 (5-PF6). Olive green crystalline compound (yield: 0.220 g, 69%).
Characterization: Anal. Calcd for C26H22Cl2CuN4O4 (634.44 g/mol): C, 49.22; H, 3.50; N, 8.83.
Found: C, 49.56; H, 3.53; N, 8.96%. Selected IR bands (ATR-IR, cm-1): 3072 (w), 2934 (vw), 2871
(vw), 1601 (s), 1571 (m), 1515 (m), 1470 (m), 1446 (s), 1374 (m), 1312 (m), 1209 (m), 1022 (m), 967
(m), 847 (vs), 779 (s), 558 (s). UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): ~660 (sh), 880 (141, b).
ΛM (CH3CN) = 146 Ω-1cm2mol-1.
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[Cu(L1)Cl]ClO4 (6-ClO4). Green crystalline compound (yield: 0.160 g, 59%).
Characterization: Anal. Calcd for C21H24Cl2CuN4O5 (546.89 g/mol): C, 46.12; H, 4.42; N, 10.24.
Found: C, 45.93; H, 4.53; N, 10.41%. ESI-MS (CH3CN): m/z = 446.09 (Calcd for [C21H24ClCuN4O]+
9
-
= 446.10, 100%), 411.12 (Calcd for C21H24CuN4O = 411.13), 98.95 (Calcd for ClO4 = 98.95, 100%).
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Selected IR bands (ATR-IR, cm-1): 3068 (vw), 3027 (vw), 2942 (w), 1607 (s), 1478 (s), 1445 (s), 1405
(m), 1267 (s), 1093 (vs), 957 (w), 878 (w), 768 (s). UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): ~725
(sh), 955 (221). ΛM (CH3CN) = 143 Ω-1cm2mol-1.
[Cu(L1)Cl]PF6 (6-PF6). Green crystalline compound (yield: 0.150 g, 51%). Characterization:
Anal. Calcd for C21H24ClCuF6N4OP (592.41 g/mol): C, 42.58; H, 4.08; N, 9.46. Found: C, 42.52; H,
4.02; N, 9.37%. Selected IR bands (ATR-IR, cm-1): 3103 (vw), 3089 (vw), 3030 (vw), 1607 (s), 1576
(m), 1479 (s), 1445 (s), 1267 (s), 1081 (s), 1023 (m), 996 (m), 958 (m), 835 (vs), 557 (s). UV-Vis
(CH3CN), λmax, nm (εmax, cm-1 M-1): ~700 (sh), ~ 850 (sh), 970 (355). ΛM (CH3CN) = 147 Ω-1cm2mol-
1
.
[Cu(L2)Cl]ClO4 (7-ClO4). Green crystalline compound (yield: 0.170 g, 62%).
Characterization: Anal. Calcd for C20H22Cl2CuN4O6 (548.86 g/mol): C, 43.77; H, 4.04; N, 10.21.
Found: C, 43.57; H, 4.11; N, 10.15%. Selected IR bands (ATR-IR, cm-1): 3105 (vw), 3064 (vw), 2926
(w), 2854 (vw), 1604 (s), 1498 (s), 1442 (m), 1305 (s), 2060 (vs), 1451 (m), 1393 (m), 1092 (vs), 1063
(vs), 1007 (w), 845 (m), 772 (m). UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): ~715 (sh), 960 (230, b).
ΛM (CH3CN) = 149 Ω-1cm2mol-1.
[Cu(L3)Cl]ClO4 (8-ClO4). Aquamarine crystalline compound (yield: 0.280 g, 93%) was
obtained upon recrystallization from CH3CN. Characterization: Anal. Calcd for C24H30Cl2CuN4O6
(604.97 g/mol): C, 47.65; H, 5.00; N, 9.26. Found: C, 47.37; H, 4.93; N, 9.22%. ESI-MS (CH3CN):
m/z = 504.13 (Calcd for [C24H30ClCuN4O2]+ = 504.15, 100%), 469.17 (Calcd for C24H30CuN4O2 =
-
469.18), 98.95 (Calcd for ClO4 = 98.95, 100%). Selected IR bands (ATR-IR, cm-1): 3166 (vw), 3132
(vw), 2926 (w), 2860 (w), 1552 (m), 1470 (m), 1419 (m), 1272 (m), 1143 (s), 1114 (vs), 1090 (vs).
UV-Vis (CH3CN), λmax, nm (εmax, cm-1 M-1): ~710 (sh), ~880 (sh), 970 (371). ΛM (CH3CN) = 137 Ω-
1cm2mol-1.
X-Ray crystallography
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The X-ray single-crystal data of compounds were collected on a Bruker-AXS APEX-II CCD
diffractometer at 100(2) K. The crystallographic data, conditions retained for the intensity data
collection and some features of the structure refinements are listed in Table 4. The intensities were
collected with Mo-K radiation (= 0.71073 Å). Data processing, Lorentz-polarization and absorption
corrections were performed using SAINT, APEX and the SADABS computer programs.58,59 The
structures were solved by direct methods and refined by full-matrix least-squares methods on F2, using
the SHELX program package.60,61 All non-hydrogen atoms were refined anisotropically. The hydrogen
atoms were located from difference Fourier maps, assigned with isotropic displacement factors and
included in the final refinement cycles by use of geometrical constraints. Complex 6-PF6 was refined
as a 2-component inversion twin. Molecular plots were performed with the Mercury program.62
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Table 4 Crystallographic data and processing parameters
5
6
7
Compound
2-PF6
4-ClO4
4-PF6
5-PF6
8
9
Empirical formula
Formula mass
System
C19H20ClCuF6N4P C22H20Cl2CuN4O4 C22H20ClCuF6N4P C26H22ClCuF6N4P
548.36
538.87
584.39
634.45
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Monoclinic
P21/c
Monoclinic
P21/n
Monoclinic
P21/n
Monoclinic
C2/c
Space group
a (Å)
11.8757(5)
12.3694(5)
12.7761(4)
33.4625(10)
b (Å)
c (Å)
 (°)
 (°)
 (°)
13.1776(6)
15.2002(6)
90
12.6452(5)
14.7677(6)
90
12.9219(4)
14.6930(5)
90
9.7014(3)
15.7910(5)
90
112.138(2)
90
106.807(2)
90
106.900(2)
90
91.518(1)
90
3
2203.37(16)
4
2211.20(16)
4
2320.93(13)
4
5124.5(3)
8
V (Å )
Z
T (K)
100(2)
1.250
100(2)
1.268
100(2)
1.193
100(2)
1.088
-1
 (mm )
3
1.653
1.619
1.673
1.645
D
(Mg/m )
calc
Crystal size (mm)
 max (°)
0.22 x 0.19 x 0.14 0.29 x 0.24 x 0.13 0.18 x 0.17 x 0.08 0.25 x 0.21 x 0.14
30.104
30.082
27.996
29.998
Data collected
77316
103512
49562
137606
Unique refl. / R
int
6437 / 0.0611
6468 / 0.0603
344 / 25
5588 / 0.0340
353 / 0
7473 / 0.0351
352 / 0
Parameters / Restraints 290 / 0
2
1.006
1.068
1.017
1.019
Goodness-of-Fit on F
R1 / wR2 (all data)
0.0362 / 0.0921
1.09 / -0.63
0.0287 / 0.0660
0.44 / -0.43
0.0240 / 0.0681
0.38 / -0.30
0.0228 / 0.0644
0.50 / -0.37
3
Residual extrema (e/Å )
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Table 4 Crystallographic data and processing parameters
5
6
7
.
Compound
7-ClO4
8-ClO4
6-PF6
8
9
Empirical formula
Formula mass
System
C20H22Cl2CuN4O6 C24H30Cl2CuN4O6 C21H24ClCuF6N4OP
548.87
604.98
592.41
Triclinic
P-1
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Orthorhombic
P212121
Monoclinic
P21/c
Space group
a (Å)
9.2019(5)
16.9159(6)
14.5459(10)
b (Å)
c (Å)
 (°)
 (°)
 (°)
11.2119(6)
21.9518(11)
90
8.7895(3)
19.0235(7)
90
18.4758(12)
18.4808(3)
97.653(3)
93.831(3)
93.870(3)
4896.8(6)
8
90
112.756(2)
90
90
3
2264.8(2)
4
2608.29(16)
4
V (Å )
Z
T (K)
100(2)
1.246
1.610
100(2)
1.089
100(2)
-1
1.135
 (mm )
3
1.541
1.607
D
(Mg/m )
calc
Crystal size (mm)
 max (°)
0.17 x 0.16 x 0.09 0.26 x 0.20 x 0.15 0.14 x 0.11 x 0.08
30.089
29.998
27.000
Data collected
55536
132850
305982
Unique refl. / R
int
6635 / 0.0559
346 / 48
7607 / 0.0718
340 / 0
21360 / 0.0990
1262 / 12
1.042
Parameters / Restraints
2
1.018
1.010
Goodness-of-Fit on F
R1 / wR2 (all data)
0.0219 / 0.0537
0.31 / -0.24
0.0301 / 0.0766
0.54 / -0.52
0.0648 / 0.1852
1.42 / -0.99
3
Residual extrema (e/Å )
CCDC 1885301-1885307 contain the crystallographic data in CIF format for 2-PF6,
4-ClO4, 4-PF6, 5-PF6, 7-ClO4, 8-ClO4 and 6-PF6, respectively.
MTT Cell viability assay
The human cancer cell lines A2780, A2780R and MCF7 were obtained from ECACC and cultivated
according to the producer’s protocols. The cells were treated with the tested compounds and a
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reference drug cisplatin in the concentrations up to 100 µM for 24 h, using the 96-well culture plates.
In parallel, the cells were treated with the vehicle (0.1% v/v DMF) and Triton X-100 (1%, v/v) to
assess the minimal, and maximal cell damage, respectively. MTT assay was performed and absorbance
was measured spectrophotometrically at 570 nm on an Infinite M200 (Schoeller Instruments, Prague,
Czech Republic). The data were expressed as the percentage of cell viability, where 100% and 0%
represent the treatments with the negative control (DMF), and positive control (Triton X-100),
respectively. Half-maximal inhibitory concentrations (IC50) were calculated using GraphPad Prism 6
software (GraphPad Software, San Diego, USA).
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DNA cleavage studies
Isolation of plasmid DNA
The ability of metal complexes to act as chemical nucleases was evaluated using the pUC19 plasmid
(2686 bp). Plasmids were isolated from the transformed bacteria Escherichia coli TOP10F’ by the
QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) according to the manufacturer`s instruction.
Plasmid DNA was eluted from the column by nuclease-free ultrapure water and it was used in the
DNA cleavage assays within 96 h after isolation. The quantity and purity of isolated DNA was
measured spectrophotometrically at 230, 260, 280, and 320 nm. The absorbance ratio A260/A280 was in
the range between 1.75 and 1.85, and the absorbance ratio A260/A230 was in the range between 2.00 and
3.00 which confirmed that the DNA was free of proteins, RNA and other impurities.
Interactions of the complexes with the plasmid DNA
The nuclease activity of the two most cytotoxic complexes 5-ClO4 and 5-PF6 as well as the less active
complex 1-ClO4 was determined using the previously published method with small modifications.52
The 300 ng of the native supercoiled pUC19 plasmid DNA (i.e. 23.1 µM of base pairs in the final
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volume of 20 µL of reaction mixture) was incubated either in the presence or in the absence of 0.66
mM hydrogen peroxide, and together with different concentrations of the tested complexes dissolved
in acetonitrile (at the concentration levels of 0, 10, 100, and 300 µM, while the final concentration of
acetonitrile was 10 % (v/v) in the reaction mixture) at 37° C for 1 h. Immediately after this, the
samples were thoroughly mixed with 6X gel loading buffer (containing 60 mM EDTA, 60% (v/v)
glycerol and 0.03% (w/v) bromphenol blue) and subsequently loaded on a 0.8% (w/v) agarose gel
prepared in TBE buffer (containing 89 mM Tris-borate buffer and 2 mM EDTA; Sigma-Aldrich)
impregnated with 0.15 µg/mL of ethidium bromide (EtBr). The electrophoreograms were analysed by
the AlphaEaseFC version 4.0.0.34 software (Alpha Innotech, USA) and the relative amounts of the
supercoiled circular (CCC-form), single-strand nicked (OC-form) and linear (L-form) forms were
evaluated. The quantification of CCC-form of plasmid DNA was corrected by a factor of 1.47.39 The
relative amount of each plasmid form was calculated as a percentage of total amounts of DNA in the
negative control containing the native form of plasmid DNA only. To clarify if the DNA cleavage is
caused by oxidative or hydrolytic mechanisms, the reactions of complexes and plasmid DNA in
aqueous solutions were performed under the inert atmosphere (solutions were bubbled with helium
before use). The other reaction conditions remained unchanged (see above).
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Effect of the antioxidants and other inhibitors on the plasmid DNA cleavage
To understand the role of reactive oxygen species or other mechanisms in the DNA cleavage process,
the reaction mixtures were also supplemented with the various ROS scavengers, specifically NaN3 (a
selective quencher of reactive singlet oxygen),63 DMSO and KI (very effective scavengers for
hydroxyl radicals),64 and highly efficient EDTA metal chelator. All antioxidants and inhibitors, except
DMSO, were added to the reaction mixtures in the molar ratio of 1:1 with 300 µM and 10 µM
concentrations of the complexes and these were incubated with the plasmid DNA in a similar manner
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as that described above. In the case of DMSO, molar ratios 1:100 and 1:10 000 were also used instead.
After densitometric analysis of the obtained electrophoreograms, the relative amount of each plasmid
form was calculated as a percentage of total amounts of DNA.
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Statistical evaluation
All experiments were performed at least in three independent repetitions. The statistical analysis of the
obtained data was performed using the GraphPad Prism 6.01 software (GraphPad Software, Inc., La
Jolla, CA, USA) and ANOVA test with multiple comparisons followed by Bonferroni post-hoc test
was applied.
Conclusions
A series of five-coordinate chlorido-Cu(II) complexes has been synthesezed, using tetradentate N-
donor tripod pyridyl amine derivatives, and structurally characterized: [Cu(TPA)Cl]ClO4.½H2O (1-
ClO4),
[Cu(6-MeTPA)Cl]ClO4/PF6
(2-ClO4/2-PF6),
[Cu(6-Me2TPA)Cl]PF6
(3-PF6),
[Cu(BPQA)Cl]ClO4/PF6
(4-ClO4/4-PF6),
[Cu(BPQA)Cl]ClO4/PF6
(4-ClO4/4-PF6),
[Cu(BQPA)Cl]ClO4/PF6 (5-ClO4/PF6), [Cu(L1)Cl]ClO4/PF6 (6-ClO4/6-PF6), [Cu(L2)Cl]ClO4 (7-
ClO4) and [Cu(L3)Cl]ClO4 (8-ClO4). In acetonitrile or aqueous acetonitrile solution, all the complexes
display TBP with the exception of 3-PF6 which exhibits SP and 5-ClO4 which has an intermediate
geometry. The in vitro cytotoxicity studies of the complexes against A2780 (ovarian), A2780R
(cisplatin-resistant variant) and MCF7 (breast cancer) human cancer cell lines revealed moderate-to-
significant effects compared to the reference cisplatin drug. Interestingly, 5-ClO4 and 5-PF6
compounds showed very high anticancer activities in the three tested cancer cell lines, with the best
IC50 values about 8–10 M). The DNA cleavage studies demonstrated that the studied complexes are effective
in causing the DNA damage by means of the minor direct hydrolytic cleavage and major oxidative mechanism
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(associated probably with the formation of oxidative metal-based intermediates, similar to those formed in
the enzymatic mechanism of mono-copper monooxygenases, such as CuO+, CuO+• or [CuOOH]+).
7
8
9
10
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Abbreviations. TPA = tris(2-pyridylmethyl)amine, 6-MeTPA = [(6-Methyl-2-pyridyl)methyl)bis(2-
pyridylmethyl)]amine, 6-Me2TPA = [bis(6-Methyl-2-pyridyl)methyl)-(2-pyridylmethyl)]amine,
BPQA = [bis(2-pyridylmethyl)-(2-quinolylmethyl)]amine, BQPA
=
[bis(2-quinolylmethyl)-(2-
pyridylmethyl)]amine, L1 = [(3,5-dimethyl-4-methoxy-2-pyridylmethyl)-bis(2-pyridylmethyl)]amine,
L2 = [(3,4-dimethoxy-2-pyridylmethyl)-bis(2-pyridylmethyl)]amine, L3 = [bis(3,5-dimethyl-4-
methoxy-2-pyridylmethyl)-(2-pyridylmethyl)]amine, MTT
=
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, A2780: ovarian cancer, A2780R: cisplatin-resistant variant, MCF7:
breast cancer cell line
Acknowledgments This research was financially supported by the Department of Chemistry-UL
Lafayette. FAM thanks Dr. J. Baumgartner (TU Graz) for assistance and NAWI Graz for support. JV,
JH, ZD and ZT acknowledge the financial support from Ministry of Education, Youth and Sports of
the Czech Republic, NPU I (a grant no. LO1305).
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