Bis-phenanthroline copper(ii) phthalate complexes are potent in vitro antitumour agents with 'self-activating' metallo-nuclease and DNA binding properties

Article abstract of DOI:10.1039/c0dt01607a

Three, structurally characterised, bis-phen Cu(ii) complexes of the phthalate isomers display rapid, low micromolar in vitro cytotoxicity against a range of epithelial tumour cells. The complexes induce relaxation of supercoiled plasmid DNA in the absence of external reducing agents and display efficient CT-DNA, Poly[d(A-T)]₂ and Poly[d(G-C)]₂ binding.

Full text of DOI:10.1039/c0dt01607a

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Dalton
Transactions
Cite this: Dalton Trans., 2011, 40, 1024
www.rsc.org/dalton
COMMUNICATION
Bis-phenanthroline copper(II) phthalate complexes are potent in vitro
antitumour agents with ‘self-activating’ metallo-nuclease and DNA binding
properties†
Andrew Kellett,*a Mark O’Connor,a Malachy McCann,b Mary McNamara,a Patrick Lynch,a Georgina Rosair,c
Vickie McKee,d Bernie Creaven,e Maureen Walsh,e Siobhan McClean,e Agnieszka Foltyn,e Denis O’Shea,a
Orla Howea and Michael Devereux*a
Received 18th November 2010, Accepted 30th November 2010
DOI: 10.1039/c0dt01607a
Three, structurally characterised, bis-phen Cu(II) complexes
of the phthalate isomers display rapid, low micromolar in vitro
cytotoxicity against a range of epithelial tumour cells. The
complexes induce relaxation of supercoiled plasmid DNA in
the absence of external reducing agents and display efficient
CT-DNA, Poly[d(A–T)]2 and Poly[d(G–C)]2 binding.
Fig. 1 Molecular structure of the phthalates and phen.
effectively limits their in vivo application. Only a few self-activating
DNA cleavage systems have been reported including Fe(BLM)
and Cu(BLM) (BLM = bleomycin) which require the presence of
molecular oxygen.3 Bis(hydroxysalicylidene)ethylenediamine, Cu-
tambjamine E† and its pyrrolidine derivatives, HPyramol† and
2-(2-pyridyl)benzthiazole have all been reported to cleave DNA in
the presence of copper(II) and molecular oxygen and a number of
them exhibit anticancer activity.6–9 To the best of our knowledge
no copper bis-phen complexes have been reported to cleave DNA
in the absence of added reductant. Karlin and co-workers and
recently Pitie´ and Reedijk have demonstrated that nuclearity is
an important factor in oxidative DNA cleavage.10,11 Copper bis-
phen complexes are among a relatively small group of metallo-
systems that can bind DNA efficiently and selectively activate
C–H deoxyribose bonds leading to strand scission.3,12
Phen and its substituted derivatives have high affinities for
copper ions13,14 and copper complexes of phen have been shown to
exhibit excellent antitumour,15 anti-candida16 and antibacterial17
activities. The tumour suppressor gene p53 is known to become
mutated in many human cancers and thus fails to function. It has
been demonstrated that phen enhances p53 activity in vitro and
can trigger apoptosis in p53 negative cell lines.18,19 Isomers of the
phthalates; phthalic (phH2), isopthalic (isophH2) and terephthalic
acid (terphH2) (Fig. 1) and derivatives thereof are ubiquitous
within the industrial plastics sector and extensive applications of
metal–phthalate scaffolds are prominent within metal–organic-
frameworks (MOFs). There are very few reports describing the
medicinal potential of the phthalates. Herein, we report the
synthesis of Cu(II) complexes of these versatile ligands along with
their chemotherapeutic potential, DNA binding and their ability
to act as ‘self-activating’ chemical nucleases.
The design of agents capable of controlled cleavage of DNA and
RNA is of paramount importance due to their potential as DNA-
targeted chemotherapeutic drugs and their potential applications
in biology.1,2 Complexes of metals such as iron, copper and
manganese have been shown to act as effective chemical nucleases
through oxidative-induced DNA damage.3 The oxidative DNA
cleavage activity of Cu2+-1,10-phenanthroline (phen, Fig. 1) in the
presence of a reductant was reported by Sigman et al. in 1979 and
the activity involves a succession of redox events with a copper-
‘oxo’ species believed to be the active agent.4 More recently the
ligands 2-clip-phen† and 3-clip-phen† were investigated as systems
that maintain the 2 : 1 phen : Cu stoichiometry at low concen-
trations. The oxidative nuclease activity of copper complexes of
these new ligands was found to be higher than that of phen itself
and it would appear that they induce a different DNA oxidation
profile to the parent [Cu(phen)2]2+.3,5 In these systems, the cleavage
reactions of the copper bis-phen and copper clip-phen require
initiation by an excess of exogeneous reducing agent and this
aThe inorganic pharmaceutical and biomimetic research centre, Focas
Research Institute, Dublin Institute of Technology, Camden Row, Dublin, 8,
Ireland. E-mail: andrew.kellett@dit.ie, michael.devereux@dit.ie; Tel: +353
1 4027986, +353 1 4024680
bChemistry Department, National University of Ireland, Maynooth, Co.
Kildare, Ireland
cSchool of Engineering and Physical Sciences, Heriot Watt University,
Edinburgh, UK, EH14 4AS
dDepartment of Chemistry, Loughborough University, Loughborough, Le-
icestershire, UK, LE11 3TU
eCentre of Applied Science for Health, Institute of Technology Tallaght
Dublin, Tallaght, Dublin, 24, Ireland
† Electronic supplementary information (ESI) available: Experimental
procedures, X-ray crystallographic data and biological evaluation studies.
CCDC reference numbers 783510–783512. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c0dt01607a
Complexes 1–6 were prepared (Scheme 1) and characterised
by IR and Raman spectroscopy, elemental analysis, magnetic
1024 | Dalton Trans., 2011, 40, 1024–1027
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 1 Synthetic routes toward complexes 1–6†.
Fig. 4 X-Ray structure of the cation in [{Cu(phen)2}2(terph)](terph) (6),
atoms with a prime (¢) in the atom labels are at equivalent position (1 - x,
1 - y, 1 - z).
moment, molar conductivity and solid/solution-state electronic
spectroscopy.† Crystals of 4–6, suitable for X-ray analysis, were
generated when 1–3 were treated with phen in ethanol (Fig. 2–4).†
The Cu(II) centres in complexes 4–6 have N4O ligation and all
have approximately square-pyramidal geometries. Mononuclear
complexes 4 and 5 are geometric isomers and both contain the ph
and isoph dianion bound to the metal through one carboxylate
group in a monodentate coordination mode. Complex 6 contains
a binuclear [{Cu(phen)2}2(terph)]2+ dication which is identical to
the cationic structure previously reported for the perchlorate salt,
[Cu(phen)2(terph)](ClO4)2.20 The complexes were either soluble or
partially soluble in DMF and DMSO. The molar conductivities
in DMSO are low and this, combined with essentially identical
solid and solution-state electronic spectroscopy suggests that the
complexes remain intact in DMSO solution.
sured, using a standard MTT assay against three human-derived
tumour cell lines (Table 1).† It should be noted that colorectal ade-
nocarcinoma (HT29) and prostate epithelial (DU145) carcinomas
are inherently resistant to cisplatin and they also contain mutant
versions of the p53 gene while MCF-7 contains a well characterised
and normal wild-type p53 gene.21–23 Complexes 4–6 display rapid,
low micromolar toxicities against all three tumour lines. Results
for this group are comparable to mitoxantrone against HT29,
however, significant improvements were noted in the DU145 line.
Complex 6 showed the best broad-spectrum activity and was the
most active complex against breast adenocarcinoma (MCF-7).
The fact that 6 is dinuclear, and so contains two active copper units,
may explain this heightened potency. It is also worth noting that
the free ligands, and the non-phen complexes 1–3, are essentially
inactive against all tumour lines.
As the complexes exhibited low molar extinction coefficients
(e) or were only partly soluble in DMF/DMSO an indirect
fluorometric method of examining their DNA binding capabilities
was chosen. Competitive ethidium bromide (EtBr) displacement
experiments were conducted with 1–6 along with the known
intercalator, actinomycin D, and the minor groove binder, pen-
tamidine, using calf thymus DNA (CT-DNA) (Fig. 5, Table 2).†
Et-bound DNA is highly fluorogenic and in the presence of
excess Et+, binding regions within the DNA polymer become
saturated. Thus, during competitive displacement, an exogenous
reagent must ‘compete’ at the binding site with Et+ resulting in a
Fig. 2 X-Ray structure of [Cu(ph)(phen)2] (4).
Table 1 IC50 values (mM) for complexes 1–6, the free ligands and the
antitumour agents, cisplatin and mitoxantrone, against breast cancer
(MCF-7), prostate cancer (DU145) and colon cancer cells (HT29) over
a period of 24 h
Activity IC50 (mM) 24 h
MCF-7
DU145
HT29
phen
phH2
337.7 64.7
>500
272.5 8.0
>500
376.8 9.2
>500
isophH2
terphH2
>500
>500
>500
>500
>500
>500
1
>500
>500
422.5 22.4
>500
11.6 4.5
10.6 2.2
5.7 0.2
27.3 5.6
>500
>500
>500
>500
6.0 0.4
5.8 0.2
5.4 0.3
2
401.2 54.1
414.3 26.0
44.9 7.0
41.2 1.4
7.9 0.4
7.5 0.2
90.8 3.5
3
4
5
Fig. 3 X-Ray structure of [Cu(isoph)(phen)2] (5).
6
Mitoxantrone
Cisplatin
8
0.6
The cytotoxicity of 1–6 along with their free ligands and the
clinical antitumour agents cisplatin and mitoxantrone, were mea-
348.6 80.6
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 1024–1027 | 1025
©

View Article Online
Table 2 Apparent DNA binding constants (Kapp) of 4–6 along with
actinomycin D and pentamidine. Assay conditions: final volume 2 mL,
1.2 mM EtBr, 1 mM CT-DNAp, 10 mM TES, 0.1 mM Na2EDTA, pH 7.0
within poly[d(G–C)]2, possibly indicating flexible or multimodal
binding.
Relaxation of supercoiled (SC) pUC18 DNA (Form I) into open
circular (OC, Form II) and linear (LC, Form III) conformations
was used to quantify the relative cleavage efficiency of complexes
4–6. SC DNA was exposed to 4–6 over a concentration range
of 1–100 mM for 5 h in the absence of added H2O2 or reductant
(Fig. 6). All complexes show concentration-dependant relaxation
of SC (Form I) DNA to OC (Form II) while some LC (Form III)
activity is evident but only at lower concentrations. The overall
trend in nuclease activity is 6 ꢀ 4 > 5. Indeed, 6 displayed some
activity at lower concentrations of 1–5 mM (lanes 11 and 12) with
almost complete depletion of the parent SC band (I→II) being
witnessed at higher concentrations (lanes 15 and 16).
a
b
Drug
C50 (mM)
Kapp
Actinomycin D
Pentamidine
4
5
6
12.35
19.72
99.06
99.79
39.36
9.69 ¥ 105
6.07 ¥ 105
1.21 ¥ 105
1.20 ¥ 105
3.04 ¥ 105
a C50 = concentration required to reduce fluorescence by 50%. b Kapp = Ke
¥ 1.26/C50 where Ke = 9.5 ¥ 106 M(bp)-1
Fig. 6 Relaxation of pUC18 by 4–6. Cleavage was carried out at 37 ◦C
for 5 h and then analyzed by agarose gel electrophoresis.† Lane 1: DNA
alone; lanes 2–6: 5, 10, 20, 50, 100 mM 4; lanes 7–10: 10, 20, 50, 100 mM 5;
lanes 11–16: 1, 5, 10, 20, 50, 100 mM 6.
Fig. 5 Competitive ethidium bromide displacement for actinomycin D,
pentamidine and complexes 4–6 with CT-DNA.
In conclusion, copper(II) bis-phen complexes of the phthalates
are the first ‘self-activating’ chemical nucleases of their class.
Their potential application as DNA-targeted chemotherapeutics
is significant given their binding affinities, efficient DNA cleav-
age and potent broad-spectrum cytotoxicity. Interestingly these
complexes do not appear to induce cytotoxicity in a similar
manner to the clinical Pt(II) drugs as evidenced by results against
cisplatin resistant colorectal (HT29) and prostate (DU145) cell
lines which lack the endogenous p53 tumour suppressor gene.
Detailed mechanistic investigations, to help elucidate the nature
of the excellent copper-mediated DNA cleavage activity in these
novel bis-phen systems are currently in progress.
sequential reduction in fluorescence. Complexes 1–3 are devoid of
binding ability but thephen-containingcomplexes, 4–6, do interact
with DNA. Actinomycin D and pentamidine are highly efficient
in the displacement of Et+ bound DNA and, as expected, their
apparent binding constants (Kapp) are high (Table 2). Comparably,
complexes 4–6 have lower, but notable, Kapp constants with
the cationic dinuclear 6 showing superiority over the neutral,
mononuclear complexes 4 and 5. Thus the advancement of DNA
binding in 6 may be explained by its expected stronger electrostatic
interaction toward DNA.
The authors wish to acknowledge financial support from
the Irish Technological Sector Research Strand III programme
(Project No. CRS02-TA01) and from the Dublin Institute of
Technology Capacity Building Scheme for Strategic Research
programme (CaBS). The Centre of Applied Science for Health,
ITT Dublin is funded under the Higher Education Authority of
Ireland Programme for Research in Third Level Institutions. This
work has been carried out (in part) within the structures of the
Focas Research Institute, DIT, funded under The Irish National
Development Plan with assistance from the European Regional
Development Fund.
In an effort to elucidate the binding mode of complexes
4–6, fluorescence quenching (Q values) of duplex adenine–thymine
(A–T) and guanine–cytosine (G–C) polymers were conducted
(Table 3). Under conditions of limited Et bound to an excess
of DNA, exogenous intercalating agents (e.g. actinomycin D)
demonstrate high affinities toward G–C base pairs, while mi-
nor groove binding species (e.g. pentamidine) prefer A–T base
paired regions. Complexes 4–6 display greater affinities toward
poly[d(A–T)]2 than pentamidine but low Q values are also evident
Table 3 Q values of poly[d(A–T)]2 and poly[d(G–C)]2 for actinomycin
D, pentamidine and complexes 4–6. Assay conditions: final volume 2 mL,
2.0 mM EtBr, 20 mM DNAp, 2 mM NaOAc buffer, 9.3 mM NaCl, 0.1 mM
Na2EDTA, pH 5.0
Notes and references
1 E. L. Hegg and J. N. Burstyn, Coord. Chem. Rev., 1998, 173, 133–165.
2 Q. Jiang, N. Xiao, P. Shi, Y. Zhu and Z. Guo, Coord. Chem. Rev., 2007,
251, 1951–1972.
3 M. Pitie and G. Pratviel, Chem. Rev., 2010, 110, 1018–1059.
4 D. S. Sigman, D. R. Graham, V. D’Aurora and A. M. Stern, J. Biol.
Chem., 1979, 254, 12269–12272.
5 M. Pitie, C. J. Burrows and B. Meunier, Nucleic Acids Res., 2000, 28,
4856–4864.
6 E. Lamour, S. Routier, J.-L. Bernier, J.-P. Catteau, C. Bailly and H.
Vezin, J. Am. Chem. Soc., 1999, 121, 1862–1869.
Drug
Qa poly[d(A–T)2] (mM)
Q poly[d(G–C)2] (mM)
Actinomycin D
Pentamidine
4
5
6
314.0
44.9
26.2
20.9
8.6
4.2
203.4
69.8
37.3
10.3
a Q = equivalent concentration required to reduce fluorescence by 50%
1026 | Dalton Trans., 2011, 40, 1024–1027
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
7 M. S. Melvin, J. T. Tomlinson, G. R. Saluta, G. L. Kucera, N.
Lindquist and R. A. Manderville, J. Am. Chem. Soc., 2000, 122, 6333–
6334.
8 M. S. Melvin, K. E. Wooton, C. C. Rich, G. R. Saluta, G. L. Kucera,
N. Lindquist and R. A. Manderville, J. Inorg. Biochem., 2001, 87, 129–
135.
15 A. D. Randford and P. J. Sadler, J. Chem. Soc., Dalton Trans., 1993,
3393–3399.
16 M. Geraghty, V. Sheridan, M. McCann, M. Devereux and V. McKee,
Polyhedron, 1999, 18, 2931–2939.
17 M. A. Zoroddu, S. Zanetti, R. Pogni and R. Basosi, J. Inorg. Biochem.,
1996, 63, 291–300.
18 Y. Sun, FEBS Lett., 1997, 408, 16–20.
19 Y. Sun, J. Bian, Y. Wang and C. Jacobs, Oncogene, 1997, 14, 385–393.
20 D. Sun, R. Cao, Y. Liang, M. Hong, W. Su and J. Weng, Acta
Crystallogr., Sect. C, 2000, 56, e240–e241.
21 N. R. Rodrigues, A. Rowan, M. E. Smith, I. B. Kerr, W. F. Bodmer, J.
V. Gannon and D. P. Lane, Proc. Natl. Acad. Sci. U. S. A., 1990, 87,
7555–7559.
22 G. Pratesi, P. Perego, D. Polizzi, S. C. Righetti, R. Supino, C. Caserini,
C. Manzotti, F. C. Giuliani, G. Pezzoni, S. Tognella, S. Spinelli, N.
Farrell and F. Zunino, Br. J. Cancer, 1999, 80, 1912–1919.
23 R. R. Chhipa, R. Kumari, A. K. Upadhyay and M. K. Bhat, Exp. Cell
Res., 2007, 313, 3945–3958.
9 P. U. Maheswari, M. v. d. Ster, S. Smulders, S. Barends, G. P. v. Wezel,
C. Massera, S. Roy, H. d. Dulk, P. Gamez and J. Reedijk, Inorg. Chem.,
2008, 47, 3719–3727.
10 L. Li, K. D. Karlin and S. E. Rokita, J. Am. Chem. Soc., 2004, 127,
520–521.
11 S. Van Der Steen, P. de Hoog, K. Van Der Schilden, P. Gamez, M. Piti,
R. Kiss and J. Reedijk, Chem. Commun., 2010, 46, 3568–3570.
12 M. Pitie, B. Donnadieu and B. Meunier, Inorg. Chem., 1998, 37, 3486–
3489.
13 W. A. E. McBryde, D. A. Brisbin and H. Irving, J. Chem. Soc., 1962,
5245–5253.
14 H. Irving and D. H. Mellor, J. Chem. Soc., 1962, 5237–5245.
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 1024–1027 | 1027
©