Nuclease and anti-proliferative activities of copper(ii) complexes of N3O tripodal ligands involving a sterically hindered phenolate

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Nuclease and anti-proliferative activities of copper(II)

complexes of N₃O tripodal ligands involving a sterically

hindered phenolate†

Cite this: Dalton Trans., 2013, 42, 8468

Nathalie Berthet,^aVéronique Martel-Frachet,^bFabien Michel,^aChristian Philouze,^a

Sylvain Hamman,^aXavier Ronot^band Fabrice Thomas*^a

Copper(II) complexes 1²⁺–6 of a series of tripodal ligands involving a N₃O donor set, namely 2-[(bis-

pyridin-2-ylmethyl-amino)-methyl]-4-methoxy-phenol (1L), 2-tert-butyl-4-methoxy-6-[bis-pyridin-2-

ylmethyl-amino)-methyl]-phenol (2L), 2-tert-butyl-4-methoxy-6-{[(2-pyridin-2-yl-ethyl)-pyridin-2-ylmethyl-

amino]-methyl}-phenol (3L), 2-tert-butyl-4-methoxy-6-{[(6-methyl-pyridin-2-ylmethyl)-pyridin-2-ylmethyl-

amino]-methyl}-phenol (4L), 2-tert-butyl-4-ﬂuoro-6-{[(6-methyl-pyridin-2-ylmethyl)-pyridin-2-ylmethyl-

amino]-methyl}-phenol (5L) and 2-tert-butyl-4-methoxy-6-{bis[(6-methyl-pyridin-2-ylmethyl)-amino]-

methyl}-phenol (6L), respectively, were synthesized. Complexes 1²⁺, 3⁺and 4⁺were structurally character-

ized by X-ray diﬀraction. The structure of 1²⁺is dimeric, with an essentially trigonal bipyramidal geometry

around the copper(^II) ions and two bridging deprotonated phenolate moieties. The mononuclear com-

plexes 3⁺and 4⁺contain a square pyramidal copper ion, coordinated in axial position by the phenol

moiety. In the water–DMF (90 : 10) mixture at pH 7.3 all the copper(^II) complexes are mononuclear,

mainly under their phenolate neutral form (except 3⁺), with a coordinated solvent molecule. The DNA

cleavage activity of the complexes was tested towards the ϕX174 DNA plasmid. In the absence of an

exogenous agent 1²⁺does not show any cleavage activity, 2⁺and 3⁺are moderately active, while 4⁺, 5⁺

and 6⁺exhibit a high nuclease activity. Experiments in the presence of various scavengers reveal that

reactive oxygen species (ROS) are not involved in the strand scission mechanism. The cytotoxicity of the

complexes was evaluated on bladder cancer cell lines sensitive or resistant to cisplatin. The IC₅₀values of

the complexes 2⁺, 4⁺, 5⁺and 6⁺are lower than that of cisplatin (range from 6.3 to 3.1 µM against 9.1 µM

for cisplatin). Furthermore, complexes 2⁺, 4⁺, 5⁺and 6⁺are able to circumvent cisplatin cellular resistance.

Received 6th November 2012,

Accepted 5th April 2013

DOI: 10.1039/c3dt32659d

www.rsc.org/dalton

more, patients with drug-resistant breast, colon carcinoma or

lung cancer showed higher serum copper contents than

Introduction

Copper plays an important role in biological systems, as do patients whose tumors respond to treatment.⁵These essential

many other metals. It is involved in many primordial processes observations open new prospects in anticancer strategies.

such as dioxygen transport, oxidation processes, signalling, For example it has been recently suggested that the copper

etc. Recently, copper has also been shown to be a cofactor level could be used as a marker of drug resistance.⁵On the

essential for the tumour angiogenesis processes, in contrast to other hand, drugs harbouring a biologically relevant metal,

other transition metals.^1,2High serum and tissue levels of such as copper, were proposed to be more eﬀective and less

copper were, for instance, found in many types of human toxic.^2,6They could thus represent a valuable and cheaper

cancer (breast, prostate, colon, lung, and brain).^3,4Further- alternative to cisplatin ([cis-diamminedichloridoplatinum(^II)]),

which is so far the most used anti-cancer metallodrug,⁷in

spite of severe side-eﬀects and cellular resistance.⁸For

example, cisplatin-based chemotherapy is the first-line therapy

^aDépartement de Chimie Moléculaire, Chimie Inorganique Redox Biomimétique

(CIRE), UMR CNRS 5250, Université J. Fourier, B.P. 53, 38041 Grenoble cedex 9,

for metastatic bladder cancer with a response rate of approxi-

mately 50%.⁹For patients ineligible for cisplatin, there is a

continued need to identify new and more eﬀective drugs.

It has been known for decades that copper complexes could

cleave DNA eﬃciently in vitro (Fig. 1). In most of the cases they

are active only under specific conditions and DNA cleavage is

France. E-mail: Fabrice.Thomas@ujf-grenoble.fr; Fax: +33 476 514 836;

Tel: +33 476 514 373

^bAGing Imaging Modeling, FRE 3405, Université Joseph Fourier, CNRS, EPHE, UPMF,

38706 La Tronche cedex, France

†Electronic supplementary information (ESI) available. CCDC 795941, 795943

and 795944. For ESI and crystallographic data in CIF or other electronic format

see DOI: 10.1039/c3dt32659d

8468 | Dalton Trans., 2013, 42, 8468–8483

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Fig. 1 Copper complexes exhibiting a DNA cleavage activity: (a) in the pres-

ence of a reductant or H₂O₂;¹¹(b) upon irradiation,¹³(c, d, e) without an exter-

nal agent.^17,22,23

Fig. 2 Formula of the ligands used in this study.

promoted by reactive oxygen species (ROS).^10–14As a result, the

nuclease activity is observed upon the addition of a reductant

(mercaptan) or a co-oxidant (such as H₂O₂) to the copper(II)

A prototypical example is the dicopper(II)

complex of the bis(bipyridylamine) ligand shown in Fig. 1a.

proposed later by Ghosh et al. to account for the nuclease

activity of related copper(^II) and zinc(II) phenolate complexes.²³

In this study, we investigate the coordination chemistry, the

DNA cleavage activity (in the absence of an external agent) and

the anti-proliferative eﬀect against a bladder tumor cell line of

a series of copper(^II) complexes involving a sterically hindered

(and thus pro-phenoxyl radical) phenolate group (Fig. 2).

Variations in the phenolic ortho substituent, as well as in the

alkylpyridine arms, are shown to dramatically influence both

DNA cleavage and biological activity.

complex.^11,12

The mercaptan reduces the copper(^II) center into copper(I),

2−

which then reacts with dioxygen to form reduced ROS (O₂

,

O₂˙⁻and/or OH˙) responsible for DNA cleavage. The addition

of H₂O₂to similar copper(II) complexes also induces DNA clea-

vage, but in this case metal coordination facilitates deprotona-

tion of hydrogen peroxide, thereby dramatically enhancing its

reactivity.¹⁰An alternative strategy to generate toxic ROS is the

use of copper(^II) complexes involving a photo-sensitizer such

as dpq (Fig. 1). Upon irradiation the copper complex depicted

in Fig. 1b ¹³forms highly reactive singlet oxygen ¹O₂, which

damages DNA.^13,14

Results and discussion

Synthesis of the ligands and copper complexes

The ligands 1L, 2L, 4L, 5L and 6L (Fig. 2) were obtained by

condensation of the appropriate phenol with bis-pyridin-2-

Copper complexes that cleave DNA without the need for an

external agent are more interesting for an in cellulo use, but ylmethyl-amine derivatives in the presence of formaldehyde.

they are much rarer. Their development is a subject of current 3L was synthesized by reductive amination of 3-tert-butyl-2-

interest.^15–23The nuclease activity of copper(II) complexes in

hydroxy-5-methoxy-benzaldehyde in the presence of (2-pyridin-

the absence of an exogenous agent results from either hydro- 2-yl-ethyl)-pyridin-2-ylmethyl-amine; the complexes were syn-

lytic or oxidative catalysis. Neves et al.^15,16and Palaniandavar thesized by reacting the ligand with one molar equivalent of

et al.^17,18developed several copper complexes (Fig. 1c) able to

Cu(ClO₄)₂·6H₂O in methanol (1²⁺) or acetonitrile (2⁺–5⁺).

mediate hydrolytic DNA cleavage: The role of the metal ion is

The copper complex of 1L was isolated in the presence of

here to favour the nucleophilic attack at the DNA phosphates. base in dimeric form. Brown single crystals of 1·(ClO₄)₂·

The copper complexes of Tambjamine E and Doxycycline are

(CH₃CN)₄were grown by slow diﬀusion of diethyl ether into a

rare examples of oxidative cleavage whereby the ligand itself methanolic solution of the complex (Table 1). The copper com-

reduces Cu(^II) into Cu(I), which further reduces O₂into reactive plexes of 2L–5L, namely 2⁺–5⁺, were isolated as monomers due

O₂˙⁻.^19,20ROS were also identified as responsible for DNA

to the steric bulk at the ortho position of the phenol. We

cleavage in some other cases but their origin was unclear.²¹reported previously the crystal structure of 2·ClO₄.²⁴Blue crys-

Recently, a high DNA cleavage activity has been reported tals of 3·ClO₄, 4·ClO₄and 5·ClO₄were grown by slow diﬀusion

for the copper(^II)-phenolate complex depicted in Fig. 1d. This

of diethyl ether into a concentrated solution of the complex in

complex also exhibits a significant anti-proliferative activity acetonitrile. Only the former two gave crystals of suﬃcient

towards L1210 murine leukemia cancer cell line and quality for an X-ray diﬀraction analysis. Attempts to crystallize

A2780 human ovarian carcinoma cell line, with IC₅₀values

the copper complex of 6L in a similar manner were unsuccess-

similar to cisplatin (3.4–10.3 μM).²²Its reactivity towards DNA ful: mixing one equivalent of ligand with the copper salt

was quite original since the proposed reactive species is a Cu(^II)- in CH₃CN aﬀords a blue solution that quickly turns green and

phenoxyl radical formed during aerobic oxidation of the

then colourless. It is likely that the steric demand of two

phenolate complex.²²Similar phenoxyl radical species were α-methylpyridines decreases the copper binding constant.

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Table 1 Crystallographic data for the copper complexes 1·(ClO₄)₂·(CH₃CN)₄, 3·ClO₄and 4·ClO₄

1·(ClO₄)₂·(CH₃CN)₄

3·ClO₄

4·ClO₄

Formula

M

C₄₈H₅₂Cu₂N₁₀O₁₂Cl₂

1158.98

C₂₇H₃₄CuN₄O₁₀Cl₂

709.02

C₂₇H₃₄CuN₄O₁₀Cl₂

709.02

Crystal system

Space group

a/Å

b/Å

c/Å

α/°

β/°

Monoclinic

P121/c1

11.731(2)

19.464(4)

10.997(2)

90

93.31(3)

90

2506.6(9)

2

Monoclinic

P121/n1

15.395(3)

10.905(2)

18.813(4)

90

90.53(3)

90

3158(1)

Triclinic

P1

ˉ

8.792(2)

9.363(2)

19.591(4)

83.90(3)

78.29(3)

83.83(3)

1564.2(5)

2

γ/°

V/Å³

Z

4

T/K

150

1.536

10.28

293

1.491

9.21

293

1.505

9.30

D_c/g cm⁻³

μ (cm⁻¹

)

Monochromator

Wavelength

Reflections collected

Independent reflections (R_int

Observed reflections

R

Graphite

MoKα (0.71073 Å)

30 584

10 407 (0.0437)

7267 [I > 2σ(I)]

0.0433

Graphite

MoKα (0.71073 Å)

34 696

6544 (0.0622)

4950 [I > 2σ(I)]

0.0564

Graphite

MoKα (0.71073 Å)

47 781

9782 (0.0381)

7148 [I > 2σ(I)]

0.0457

)

R_w

0.1045

0.1451

0.1081

Since unchelated copper(II) is known to be a strong oxidizer

in CH₃CN it may slowly oxidize the phenol into a phenoxyl

radical (well-known green colour), which further decom-

poses.²⁵This assumption is confirmed by the fact that repla-

cing CH₃CN by DMF prevents this phenomenon. The desired

complex 6⁺was thus generated in situ in DMF for further analy-

sis by adding one molar equivalent of Cu(ClO₄)₂·6H₂O to the

ligand 6L. Noteworthy, 6⁺is mainly in its phenolate form

(see ESI†) in this solvent, due to the basic character of DMF.

Fig. 4 X-ray crystal structures of 3⁺and 4⁺shown with 30% thermal ellipsoids.

X-ray crystal structures of the copper complexes

H atoms are omitted for clarity, except H1.

The ORTEP views of 1²⁺, 3⁺, 4⁺are shown in Fig. 3 and 4, and

selected bond lengths and angles are listed in Table 2.

In 1²⁺the two copper atoms have similar pentacoordinate

environments that share the oxygen atoms O1 and O1* from

two ligands. The geometry around the metal ion is intermedi-

ate between trigonal-bipyramidal and square-pyramidal. The

Table 2 Selected bond lengths (Å) and angles (°) for the copper complexes

1²⁺, 3⁺and 4⁺

1²⁺

Cu–O1 2.110(1)

Cu–N2 2.022(2)

O1–Cu–O1* 82.1(1)

O1–Cu–N3 117.5(1)

O1*–Cu–N3 95.5(1)

N2–Cu–N3 140.1(1)

3⁺

Cu–O1* 1.944(1)

Cu–N3 2.000(2)

O1–Cu–N1 93.4(1)

O1*–Cu–N1 174.5(1)

N1–Cu–N2 82.6(1)

Cu–N1 2.026(2)

O1–Cu–N2 100.8(1)

O1*–Cu–N2 101.2(1)

N1–Cu–N3 84.0(1)

Cu–O1 2.460(4)

Cu–N2 2.001(3)

O1–Cu–O10 170.7(2)

O1–Cu–N3 99.7(2)

N1–Cu–N3 93.8(2)

N2–Cu–N4 92.3(2)

4⁺

Cu–O1 2.444(2)

Cu–N2 2.016(2)

O1–Cu–O6 176.5(1)

O1–Cu–N3 95.4(1)

N1–Cu–N3 82.3(1)

N2–Cu–N4 92.7(1)

Cu–O10 2.826(6)

Cu–N3 2.008(3)

O1–Cu–N1 87.9(1)

O1–Cu–N4 87.4(2)

N1–Cu–N4 173.3(2)

N3–Cu–N4 91.7(2)

Cu–N1 2.062(3)

Cu–N4 2.014(3)

O1–Cu–N2 92.6(1)

N1–Cu–N2 83.1(2)

N2–Cu–N3 167.3(2)

Cu–O6 2.495(2)

Cu–N3 2.066(2)

O1–Cu–N1 89.5(1)

O1–Cu–N4 91.6(1)

N1–Cu–N4 175.4(1)

N3–Cu–N4 102.0(1)

Cu–N1 2.013(2)

Cu–N4 1.985(2)

O1–Cu–N2 90.0(1)

N1–Cu–N2 82.9(1)

N2–Cu–N3 164.5(1)

Fig. 3 X-ray crystal structures of 1²⁺shown with 30% thermal ellipsoids. H

atoms are omitted for clarity.

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relative extent of the trigonal-bipyramidal distortion is indi-

The geometry of 4⁺is roughly similar to that observed for

cated by an index τ representing the degree of trigonality 3⁺, with the metal ion lying within an axially elongated octa-

within the structural continuum between square-planar hedral geometry. The copper atom is coordinated in the equa-

and trigonal-bipyramidal structures.²⁶The τ index calculated torial plane by one tertiary nitrogen N1, two pyridine nitrogens

according to the equation τ = (β − α)/60 (where β = O1*–Cu–N1 N2 and N3 and one acetonitrile nitrogen N4. The phenol

and α = N2–Cu–N3) is 0.58. Assuming that 0 corresponds to a oxygen O1 and one perchlorate oxygen O5 bind weakly in axial

perfect square pyramidal geometry and 1 a perfect trigonal position with Cu–O1 and Cu–O5 bond distances of 2.444(2)

bipyramid, the geometry around the metal center in 1²⁺could and 2.495(2) Å respectively. The copper–nitrogen bond dis-

be thus better described as distorted trigonal-bipyramidal. tances Cu–N1, Cu–N2, Cu–N3 and Cu–N4 are 2.013(2),

Each copper atom is coordinated to three nitrogen and two 2.016(2), 2.066(2) and 1.985(2) Å respectively. It is noteworthy

oxygen atoms: the pyridine nitrogens N2 and N3, the tertiary that the Cu–N3 bond is the weakest Cu–N bond in the series,

amino nitrogen N1 and two μ-phenolato oxygens O1, O1*. as a result of the incorporation of a steric bulk at the α-posi-

The Cu–O1 bond (2.110(1) Å) is longer than the Cu–O1* one tion of the pyridine.²⁹The angle between the two planes

(1.944(1) Å) and the O1–Cu–O1* angle (82.1(1)°) is significantly defined by the N1–Cu–N3 and N2–Cu–N4 atoms is rather

diﬀerent from the Cu–O1–Cu one (97.9(1)°). The CuO1CuO1* small (only 4.8°), in contrast with 2⁺and 3⁺. The weakening of

unit is found to be completely flat, in contrast with butterfly one of the pyridine–metal bonds thus favours an almost

shapes commonly observed in dimers of tripodal ligands invol- square planar equatorial geometry around the copper ion.

ving two phenolato donors (N₂O₂donor set).^24,27As a con-

EPR properties of the complexes

sequence the intermetallic distance is longer, the Cu–Cu bond

length being 3.060(1) Å.

The EPR spectra of the copper(^II) complexes were recorded in a

water–DMF (90 : 10) mixture due to their low solubility in pure

water. The 100 K EPR spectra recorded at pH 7.3 are shown in

Fig. 5. They all consist of an (S = 1/2) signal with copper hyper-

fine structure, which is typical of mononuclear complexes.

This result is not surprising for complexes 2⁺–5⁺since they

were isolated as monomers in the solid state. Regarding 1²⁺, a

water or DMF molecule likely acts as a fifth ligand, thereby

replacing one μ-phenolate moiety in the copper coordination

sphere (Scheme 1).

In order to confirm this hypothesis we recorded the EPR

spectrum of 1²⁺in the less coordinating solvent CH₃CN. No

signal could be evidenced at 100 K, showing that 1²⁺cannot be

described as two isolated (S = 1/2) spins. Clearly, the two

copper(^II) ions are magnetically exchange coupled. This result

3⁺contains a copper atom within an axially elongated octa-

hedral geometry. One tertiary amine N1 and two pyridine N2,

N3 nitrogen donors from the ligand, as well as one nitrogen

N4 from an exogenous acetonitrile molecule coordinate in the

equatorial positions. The axial positions are occupied by two

oxygen donors, one from the phenol moiety (O1) and the

other (O10) from an exogenous perchlorate ion. The copper–

nitrogen bond distances Cu–N1, Cu–N2, Cu–N3 and Cu–N4 are

2.062(3), 2.001(3), 2.008(3) and 2.014(3) Å respectively, whereas

the axial copper–oxygen bond distances Cu–O1 and Cu–O10

are found to be much longer, 2.460(4) and 2.826(6) Å, with a

copper displaced 0.068 Å above the mean plane formed by the

four nitrogen donors, toward the axial O1 atom. The large

axial bonds reflect the very weak donor ability of the phenol

and perchlorate ligands, and the sensitivity of the d⁹electronic

configuration to Jahn–Teller distortion. Interestingly,

a

hydrogen bond exists between the phenol hydrogen H1 and

the oxygen of one perchlorate molecule (O1–O4 bond distance

of 2.76 Å). It is noticeable that the two copper–pyridine nitro-

gen bond distances Cu–N2 and Cu–N3 were found to be short,

nearly equivalent in the previously described complex 2⁺

(1.974(3) and 1.983(3) Å). The longer Cu–N2 and Cu–N3 bond

distances observed in the case of 3⁺(2.001(3) and 2.008(3) Å)

likely originate from the formation of an in-plane 6-membered

rather than 5-membered coordination ring.²⁸The two pyridine

nitrogen atoms N2 and N3 are located 0.153 and 0.138 Å below

the mean plane defined by the N1,N2,N3,N4 atoms, whereas

the tertiary amine and acetonitrile nitrogens are located 0.149

and 0.142 Å above this mean plane, with an angle between the

two N1–Cu–N3 and N2–Cu–N4 planes of 12.8°. In the case of

2⁺, we have reported that the mean displacement of the nitro-

gens with respect to the four nitrogens plane was only 0.055 Å,

whereas the angle between the two opposite N–Cu–N planes

was 10.2°. Therefore, an increase in the length of the linker

between the tertiary amine nitrogen and one pyridine results

in a marked tetrahedral distortion of the copper ion.

Fig. 5 X-band EPR spectra of 0.5 mM solutions of (a) 1²⁺, (b) 2⁺, (c) 3⁺, (d) 4⁺,

(e) 5⁺and (f) 6⁺in a water–DMF 90 : 10 mixture at pH 7.3 ([Tris] = 0.05 M,

[NaCl] = 0.02 M). Solid lines represent experimental spectra, and dotted lines

represent simulations using parameters given in the text. T = 100 K, microwave

frequency 9.34 GHz (a, c, d, f) or 9.44 GHz (b, e), power 10 mW, modulation

frequency 100 kHz, amplitude 0.3 mT.

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Scheme 1 Dissociation of the dimer 1²⁺in aqueous solution (L = H₂O or DMF).

Table 3 Spin Hamiltonian parameters for mononuclear copper complexes^a

g values

g_xx, g_xy, g_zz

A_Cu(mT)

A_xx, A_yy, A_zz

Complex

Fig. 6 UV-Vis spectra of 0.5 mM solutions (except (e): 0.25 mM) of the com-

plexes in a water–DMF 90 : 10 mixture at pH 7.3 ([Tris] = 0.05 M, [NaCl] =

0.02 M): (a) 1²⁺, (b) 2⁺, (c) 3⁺, (d) 4⁺, (e) 5⁺, (f) 6⁺. T = 298 K, l = 1.000 cm.

1²⁺

2⁺

3⁺

4⁺

5⁺

6⁺

2.053, 2.053, 2.248

2.057, 2.057, 2.253

2.055, 2.055, 2.262

2.055, 2.055, 2.255

2.057, 2.057, 2.245

2.053, 2.053, 2.265

1.0, 1.0, 17.0

1.5, 1.5, 16.3

1.0, 1.0, 15.5

1.5, 1.5, 16.1

1.5, 1.5, 17.1

1.0, 1.0, 15.3

Table 4 UV-Vis properties of the complexes at pH 7.3 in water–DMF (90 : 10)

and thermodynamic constants

^aConcentration of the complexes: 0.5 mM; medium: water + 0.05 M

Tris HCl buﬀer pH 7.5 + 10% (vol) DMF. T = 100 K.

Complex

λ_max/nm (ε/M⁻¹cm^{−1 a}

)

pK_a(phenol)^c

K_b^e(M⁻¹

)

d

1²⁺

2⁺

3⁺

4⁺

5⁺

6⁺

470 (880), 690br (340)

501 (620)/501 (890)^b

496 (970)

500 (880)

468 (1070)

—

7770 1500

2580 300

1940 550

6.98 0.02

5.80 0.03

5.49 0.02

is not surprising if one considers 1²⁺as dimer. Both the short

intermetallic distance and the favorable orbital overlap lead to

a significant magnetic interaction between the paramagnetic

(S = 1/2) metal ions. The resulting system is diamagnetic or

paramagnetic (S = 1).

From the simulation of the spectra in the water–DMF

(90 : 10) mixture, we obtained the spin Hamiltonian para-

meters listed in Table 3. They are consistent with an essentially

square pyramidal geometry for the copper center. Interestingly,

slight diﬀerences of g_zzare observed in the complexes, reflect-

ing distortions in the metal ion geometry within the series.

f

—

d

f

—

g

521 (950)

4.44 0.04

—

^a0.05 M Tris buﬀer + 0.02 M NaCl at 298 K. ^bThe first value at pH 7.3

in 0.05 M Tris + 0.02 M NaCl, and the second value at pH 9 in NaClO₄

0.1 M medium. ^c0.1 M NaClO₄. ^dPrecipitation is observed at neutral

pH in the presence of 0.1 M NaClO₄. ^eBinding constant for CT DNA in

a 0.05 M Tris buﬀer + 0.02 M NaCl at 298 K. ^fThe spectral variations

are too small to determine a binding constant. ^gSignificant DNA

digestion occurs during titration.

whereas a low-energy shoulder is rather indicative of square

pyramidal geometry. The significant overlap between the CT

transitions and the d–d bands precludes such a detailed analy-

Electronic spectra of the complexes

The electronic spectra of the copper complexes were recorded sis. Nevertheless the observed NIR-tailoring, together with the

in a water–DMF (90 : 10) mixture at pH 7.3 (Fig. 6, Table 4). EPR results, is fully consistent with a square pyramidal geome-

The electronic spectrum of 1²⁺displays two main transitions at try of the copper ion. This assumption is further confirmed by

470 nm (ε = 880 M⁻¹cm⁻¹) and 690 nm (ε = 340 M⁻¹cm⁻¹). the close similarity between the spectra of 2⁺, 3⁺, 4⁺, 5⁺, 6⁺and

Based on its low intensity the latter is attributed to d–d tran- some structurally characterized copper(^II) phenolate complexes

sitions. The higher energy band, which is absent in the spec- where the metal ion lies in a square pyramidal geometry.^28–32

trum of the free ligand, arises from a phenolate-to-copper CT On the basis of the reported ε values for phenolate-to-copper

transition. The spectra of 2⁺, 3⁺and 4⁺exhibit similar shapes, CT (ca. 1000 M⁻¹cm⁻¹) for complexes having the same geome-

with a dominating band at ca. 500 nm and a less intense try^24,28,29it is reasonable to assume that 3⁺and 4⁺exist almost

shoulder above 700 nm (Table 4). Comparison with the litera- exclusively in their deprotonated phenolate form at pH 7.3

ture data²⁴shows that the former band arises from a pheno- (in other words, their phenol’s pK_ais lower than 6) (Scheme 2).

late-to-copper(^II) charge transfer (CT), while the latter is

Of interest is the lower intensity of the LMCT band of 2⁺,

assigned to d–d transitions. For copper(^II) complexes of tri- which suggests that the phenol’s pK_ais much higher for this

podal ligands it has been proposed that the shape of the d–d compound (see below). We previously reported the spectrum

band, and especially the presence of a high or low-energy of 2⁺in its deprotonated phenolate form in CH₃CN, which

shoulder, reflects the geometry of the metal ion.²⁶A high- is characterized by an absorption maximum at ca. 530 nm.²⁹

energy shoulder is indicative of trigonal bipyramidal geometry, The shift of 30 nm resulting from the change of solvent

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phenolate-to-copper CT band, consistent with protonation of

the phenolate moiety (Scheme 2). The titration data were

refined with SPECFIT software in order to extract the phenol’s

pK_as. The computed pK_as are found to be 6.98 0.02, 5.80

0.03, 5.49 0.02 and 4.44 0.04 for 2⁺, 3⁺, 4⁺and 6⁺respect-

ively. Noteworthy, the pK_avalue measured for 2⁺is close to

that reported for the axial tyrosine in the copper active site of

Galactose Oxidase (ca. 8)³⁰and structurally characterized

copper complexes involving axial phenol/phenolates.³¹

Scheme 2 Acid–base equilibrium for 2⁺in H₂O–DMF solution (L = H₂O or

DMF).

The trend of pK_a2⁺> 4⁺> 6⁺indicates that substitution of

the pyridines by methylpyridines stabilizes the phenolate

forms. This feature likely arises from geometrical rearrange-

ments associated with deprotonation.³²Indeed, phenols are

weak donors that systematically coordinate the copper in axial

position. Exhaustive structural characterizations on copper(^II)-

phenolate complexes of N₃O tripodal ligands related to 2⁺

(i.e. involving exclusively two pyridin-2-ylmethyl N-donors)

revealed that the phenolate moiety usually coordinates in axial

position.^24,33,34On the other hand, Shimazaki et al. investi-

gated copper(^II)-phenolate complexes of N₃O tripodal ligands

involving one or two 6-methylpyridin-2-ylmethyl N-donors.²⁹

They reported that the phenolate group occupies systematically

the equatorial position. This behaviour is ascribed to the steric

hindrance of the methyl group, which forces the methylpyri-

dine to coordinate in an axial position. A noticeable lowering

of pK_ais also observed on increasing the size of the chelate

ring involving the pyridine nitrogen, as reflected by the diﬀer-

ence in pK_abetween 2⁺and 3⁺. Previously, it was found that

the nature of the chelate rings could influence the positioning

of the phenolate (the steric constraint at the copper center

diﬀers on replacing the five-membered chelate rings by six-

membered ones), but the phenomenon is less clear. For

example the copper(^II) complex of the 4-nitro-2-{[(2-pyridin-

2-yl-ethyl)-pyridin-2-ylmethyl-amino]-methyl}-phenolate ligand

exhibits an axially bound phenolate,³³in contrast with the

complex of the 2,4-di-tert-butyl-6-{N,N-bis[2-(2-pyrid-2-ylethyl]-

amino]-methyl}-phenolate²⁸and 4-nitro-2-{N,N-bis[2-(2-pyrid-2-

ylethyl]amino]-methyl}-phenolate ligands,³⁵where the pheno-

late coordinates in equatorial position. We will therefore limit

our discussion to 2⁺, 4⁺and 6⁺and interpret the lower pK_as of

4⁺and 6⁺, when compared to 2⁺, by a structural rearrangement

induced by deprotonation of the phenol moiety.³²

The UV-Vis spectra of the copper complexes were recorded

after 10 min incubation with calf thymus DNA (concentrations

up to 500 μM in mole base pairs). On increasing the DNA

concentration a decrease in the intensity of the CT band is

observed for 1²⁺, 2⁺and 3⁺, while very little change was

observed for 4⁺and 5⁺. Noteworthy, we did not observe a sig-

nificant red shift of the CT band. A plot of [DNA]/(ε_a− ε_f)

(where ε_ais the apparent molar extinction coeﬃcient at a given

DNA concentration and ε_fis the molar extinction coeﬃcient of

the free complex) as a function of DNA concentration gives a

straight line (Fig. 8) for 1²⁺and 2⁺from which the intrinsic

binding constants K_bcould be calculated (see the Experimen-

tal section). In the case of 3⁺the absorbance change resulting

from DNA binding is smaller than that for 2⁺and 3⁺;

Fig. 7 Evolution of the UV-Vis spectra as a function of pH of a 0.5 mM solution

of 2⁺in a water–DMF 90 : 10 mixture ([NaClO₄] = 0.1 M): T = 298 K, l =

1.000 cm. Arrows indicate spectral changes upon an increase of the pH from

3.54 to 8.84.

confirms that the coordinating CH₃CN molecule (observed in

the X-ray crystal structure) is substituted in the water–DMF

medium (Scheme 2). The spectrum of 5⁺resembles that of 4⁺,

although the CT band is blue-shifted by 32 nm. This behaviour

is easily explained by the weaker electron-donating ability of

the fluorine group, when compared to the methoxy substitu-

ent.²⁴More surprising, the phenolate-to-copper CT is observed

at 521 nm for 6⁺while it appeared at 500 nm for 4⁺, which

exhibits a similar para phenolate substituent. Such a trend was

also reported by Shimazaki et al. for related copper com-

plexes.²⁹A possible explanation is a diﬀerence in the geometry

of the phenolate complexes, which aﬀects the strength of the

Cu–O bond.²⁹

We investigated the pH-dependence of the UV-Vis spectra

(Fig. 7) in the pH range 3.5–9 in a water–DMF (90 : 10) mixture

(+0.1 M NaClO₄). Except for 1²⁺and 5⁺, which were found to

be insuﬃciently soluble at neutral pH in this medium, we

observed perfect agreement between the spectra at pH 7.3 in

the 0.1 M NaClO₄and the 0.05 M Tris media. This shows that

the buﬀer or supporting electrolyte does not interfere with

complexes 2⁺, 3⁺, 4⁺and 6⁺. For these complexes an increase in

pH from 7 to 9 does not induce a significant shift of λ_max, indi-

cating that the pK_aof the coordinated water molecule is rela-

tively high (>10) or DMF binds instead. In contrast, a decrease

in pH down to 3.5 results in the disappearance of the

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consequently the resolution is poorer and the uncertainty with time, with a progressive decrease of the CT band. This

higher. Based on structural similarities between 1²⁺, 2⁺and 3⁺suggests that significant DNA digestion occurs during the titra-

we assumed that the dependence of [DNA]/(ε_a− ε_f)] vs. [DNA] tion experiment, thus precluding refinement of the data to

was linear in the latter case and treat the data accordingly. The extract binding constants.

binding constants K_b(Table 4) determined from the spectral

change are 7770 1500, 2580 300 and 1940 550 M⁻¹for Electrochemical properties of the complexes

1²⁺, 2⁺and 3⁺, respectively. As expected, the K_bvalue of 1²⁺is

the highest, due to the lack of steric hindrance at the ortho

The electrochemical behaviour of the copper complexes was

investigated both at pH 7.3 (phenolate form) and 3.5 (phenol

form) by cyclic voltammetry in a water–DMF (90 : 10) medium

(+0.1 M NaClO₄). The potentials are referenced vs. SCE

(Table 5).

At pH 7.3 all the compounds exhibit an oxidation wave in

the range 0.30–0.65 V (Table 5, Fig. 9). It is found to be irre-

versible for both 1²⁺and 5⁺(E^a_p= 0.65 and 0.59 V respectively),

while it is reversible for 2⁺, 3⁺, 4⁺and 6⁺, i.e. for complexes

involving an electron-rich and o,p-protected phenolate moiety

(E_1/2= 0.30, 0.32, 0.35, 0.36 V, respectively). This behaviour is

strong evidence that the redox process is ligand-centered,

aﬀording phenoxyl radical species. Indeed, the potential

values measured for 2⁺, 3⁺, 4⁺and 6⁺are very similar to those

reported for 2⁺in CH₃CN (+0.38 V vs. SCE)²⁴and other related

position of the phenolate group. The K_bvalues are lower than

those of typical intercalators such as EthBr (K_b= 1.4 × 10⁶M⁻¹

)

and partially intercalating complexes such as heteroleptic

[Cu(tdp)(dipyrido-[3,2-d2′,3′-f]-quinoxaline)]⁺(9 × 10⁵M⁻¹).¹⁷

They are typical of weak DNA binding interactions, as observed

in the [Cu(tdp)(bipyridine)]⁺complex (K_b= 7100 M⁻¹) and

derivatives¹⁷and very close to that recently reported for the

copper complex of the [(2-pyridin-2-yl-ethyl)-pyridin-2-

ylmethyl-amino]-methyl ligand (roughly 10⁴).³⁶The eﬃcient

nuclease 6⁺(see below) exhibits a dramatically diﬀerent behav-

iour. Indeed, the spectra were found to evolve significantly

Fig. 8 Plot of the absorbance at 500 nm as a function of DNA concentration

(expressed in mole base pairs) for a 0.22 mM solution of 2⁺in a water–DMF

90 : 10 mixture at pH 7.3 ([Tris] = 0.05 M, [NaCl] = 0.02 M). Inset: plot of [DNA]/

(ε_a− ε_f) as a function of the DNA concentration (ε_ais the apparent molar extinc-

tion coeﬃcient at a given DNA concentration, and ε_fis the molar extinction

coeﬃcient of the free complex). T = 298 K, l = 1.000 cm.

Fig. 9 Cyclic voltammetry curves of 0.5 mM solutions of the phenolate com-

plexes in a water–DMF 90 : 10 mixture at pH 7.3: (a) 1²⁺in 0.05 M Tris contain-

ing 0.02 M NaCl, (b) 2⁺and (c) 3⁺in 0.1 M NaClO₄. Scan rate = 0.1 V s⁻¹

Potentials are given vs. the SCE reference.

.

Table 5 Electrochemical properties of the complexes^a

pH = 3.5

pH = 7.3^a

Complexes

Red^b

Ox^c

Red^b

Ox

1²⁺

2⁺

3⁺

4⁺

5⁺

6⁺

−0.35; −0.31

−0.20; −0.10

−0.22; −0.14

−0.23; −0.12

−0.06; 0.22

0.85

0.64

0.58

−0.40; −0.32^d

−0.56; −0.24

−0.42; −0.29

−0.48; −0.24

−0.35; −0.17^d

−0.35; −0.16

E^a_p= 0.65^c,d

E¹_1/2= 0.30; E_p^a,2= 0.65

E¹_1/2= 0.32; E_p^a,2= 0.57

E¹_1/2= 0.35; E_p^a,2= 0.63

E^a_p^,1= 0.59^c,d

e

—

0.64

0.53

E¹_1/2= 0.36; E_p^a,2= 0.56

^aIn water–DMF (90 : 10) + 0.1 M NaClO₄at 298 K, except 1 and 5 at high pH. All potentials are given vs. the SCE reference. ^bE_p^cand E^a_pare given.

^cIrreversible processes, E^a_pis given. ^dIn water–DMF (90 : 10) medium containing [Tris] = 0.05 M, [NaCl] = 0.02 M at pH 7.3 due to the low

solubility of the complexes in the water–DMF (90 : 10) + 0.1 M NaClO₄mixture at neutral pH. ^eIll-defined wave.

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complexes for which the copper(II)-phenoxyl radical form

was electrogenerated and unambiguously identified by spec-

troscopy. A slight increase in oxidation potential is observed

upon substitution of the pyridines by methylpyridines. The

decreased N-donating ability of the methylpyridine thus desta-

bilizes the radical form, as previously observed by Shimazaki

et al.²⁹When scanning towards the negative region of poten-

tials a metal-centered reduction peak E^c_pis observed in the

potential range −0.35 to −0.56 V (Table 5). An enhancement of

the anodic peak current was observed in the reverse scan of

1²⁺and 2⁺,³⁷suggesting that the electron transfer involves

adsorbed copper(^I)-phenolate material at nearly the same

potential as the “normal” electron transfer.³⁸As shown in

Table 4 the cathodic wave shifts to positive potentials in the

order 2⁺< 4⁺< 6⁺. The electron density of the copper center

thus decreases with an increase in the number of methylpyri-

dine groups in the ligand.³⁹A similar anodic shift of the

reduction wave is observed on going from 2⁺to 3⁺(substitution

of one pyridylmethyl by a longer pyridylethyl arm). The influ-

ence of the ligand arm’s length on the Cu(^II)/Cu(I) redox poten-

tial in copper(^II) tris(pyridyl)alkylamine complexes has been

investigated recently.⁴⁰In this case the diﬀerence in oxidation

Fig. 10 Agarose gel electrophoresis patterns of the cleavage reaction of

ϕX174 supercoiled DNA (20 μM base pairs) mediated by the copper complexes

in a phosphate buﬀer 10 mM, pH 7.2 (+10% DMF) at 37 °C for 1 h. Abbrevi-

ations: NC: nicked circular, L: Linear, SC: supercoiled. (a) Complex concentration

= 300 μM (constant); lane 0 is the DNA control without the copper complexes;

lane 1, complex 1²⁺; lane 2, complex 2⁺; lane 3, complex 3⁺; lane 4, complex 4⁺;

lane 5, complex 5⁺; lane 6, complex 6⁺. (b, c, d): Dose dependent activity

(complex concentrations are indicated in μM): (b), 4⁺; (c), 5⁺; and (d), 6⁺.

potential originates from structural rather than electronic 300 μM. The reaction was conducted on ϕX174 DNA (20 μM, in

eﬀects: Cu(^II) favours five-membered coordination rings; there- base pairs) in phosphate buﬀer for 1 h at 37 °C (Fig. 10a).

fore compounds containing longer arms are stabilized in lower

In the absence of complex, DNA was found to be mainly in

oxidation states and the Cu(^II)/Cu(I) redox couple increases. the supercoiled form (82%) both at pH 7.2 (Fig. 10a, lane 0)

Finally, the trend of E^c_p4 < 5 very likely reflects the lower elec- and 8.2. Cleavage eﬃciency of the complexes was next evalu-

tron-donating ability of the fluorine substituent, which ated by subtracting this starting percentage of DNA in SC

decreases the electron density at the metal.

form. In the presence of 300 μM of 1²⁺, no significant change

At pH 3.5 the six compounds exhibit an irreversible oxi- in the proportion of the SC and cleaved forms of DNA was

dation wave above 0.53 V assigned to the oxidation of the observed (lane 1), indicating that this complex is not an

phenol moiety. It is well known that oxidation of phenols eﬃcient nuclease.

occurs at potentials well above those required for oxidation of

Complexes 2⁺and 3⁺were found to exhibit a moderate DNA

the corresponding phenolates into phenoxyl radicals.⁴¹The cleavage activity, as judged by the appearance of the nicked

metal-centered reduction wave is observed within the potential circular form in 15 and 22% ratio, respectively, at pH = 7.2

range −0.35 to +0.32 V. Again, an abnormally high I^a_p/I_p^cratio is (lanes 2 and 3, respectively). Similar moderate activities were

observed, suggesting that the reduced complexes easily adsorb reported for copper(^II) complexes of bis(pyridyl)alkylamine

at the surface of the electrode. Interestingly, low E_p^cvalues were ligands that do not involve an intercalating moiety.^42–44The

observed for 1²⁺and 2⁺(−0.31 V), while intermediate values better activity of 2⁺and 3⁺, when compared to 1²⁺, suggests

were measured for 3⁺, 4⁺and 5⁺(−0.20, −0.22, −0.23 respect- that a hydrophobic tert-butyl substituent in ortho position of

ively), and a relatively high potential (−0.06 V) was obtained the phenol enhances the nuclease activity. Since tert-butyl

for 6⁺. This trend follows that observed for the phenolate com- groups are expected to increase the steric protection and thus

pounds (see above) and thus will not be commented on further. perturb the access to the metal center, the enhanced activity

may result from either electronic eﬀects, stronger hydrophobic

interactions of the compound with DNA and/or diﬀerences in

the complex geometry. Finally, we investigated the nuclease

Nuclease activity of the complexes

The ability of the copper(^II) complexes to cleave DNA has been activity of these compounds at pH 8.2 since 2 was found to

investigated by gel electrophoresis using ϕX174 RF1 super- exist as a mixture of phenol and phenolate forms at physiologi-

coiled DNA in phosphate buﬀer (10 mM, pH 7.2 or 8.2 with cal pH. The extent of DNA damage was found to be essentially

10% DMF). Cleavage was evidenced by monitoring the conver- the same within the error margin at both pH (data not shown).

sion of the covalently closed circular supercoiled plasmid DNA Interestingly, the cleavage activity of a copper(^II) complex of a

form (SC) into the nicked circular form (NC, single strand tripodal N₃O ligand involving a 2-hydroxy-5-methyl-benzal-

breakage) and then to the linear form (L, double strand dehyde moiety was recently investigated by Neves et al. In con-

breakage).

trast with our results they did not detect nuclease activity at

The nuclease activity of the complexes was first investigated pH 7, but observed significant DNA cleavage at pH 8.¹⁶It has

at pH 7.2 and 8.2 using a constant complex concentration of to be emphasized that there are also some examples in the

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literature of a drop in cleavage activity that results from an

increase in pH.⁴⁵

Complexes involving methylpyridines exhibit a remarkable

nuclease activity: in the case of 4⁺total disappearance of the

SC form was observed at pH 7.2 (lane 4) to the detriment of

the formation of 94% of NC and 6% of L forms. Thus 4⁺pro-

motes both single strand and double strand DNA cleavage

without the need for external agents. For comparison, the

recently described copper nuclease [Cu(tdp)(dpq)]⁺promotes

only single strand cleavage under comparable conditions,¹⁷

while the copper(II)-xylylguanidine TACN complex exhibits

a similar activity.⁴⁶The nuclease activity of 4⁺is slightly lower

than that reported for the heteroleptic complex [Cu(Phen)-

(maltol)]^{+ 47}and other complexes involving intercalating

groups.^18,45Complexes 5⁺and 6⁺exhibit a slightly lower

activity (lanes 5 and 6 respectively) at 300 μM since the SC

DNA form was not fully converted to the NC form (17% and

29% of the SC form remains for 83% and 71% of the NC form

produced, for compounds 5⁺and 6⁺, respectively), while

double strand cleavage (L form) was not observed. For the

latter compounds a moderate enhancement of the nuclease

activity was observed at higher pH (full digestion), thus sup-

porting a hydrolytic pathway (data not shown).

Fig. 12 Agarose gel electrophoresis patterns of supercoiled ϕX174 DNA

(20 μM base pairs) incubated with the copper complexes (300 μM; excepted in

lane 1) in a phosphate buﬀer 10 mM pH 7.2 (+10% DMF) at 37 °C for 1 h; (a)

4⁺, (b) 5⁺, (c) 6⁺. Lane 1, DNA control; lane 2, DNA + complex (300 μM) without

scavenger; lanes 3–11, DNA + complex (300 μM) in the presence of agents: lane

3, NaN₃(100 μM); lane 4, Superoxide Dismutase (0.5 unit); lane 5, Catalase (0.1

unit); lane 6, DMSO (2 μL); lane 7, EtOH; lane 8, mercaptoethanol (0.71 mM);

lane 9, Hoechst 33258 (100 μM); lane 10, NaCl (350 μM); lane 11, EDTA

(10 mM).

significant amounts of double strand breakage could be

evidenced for 500 μM concentrations of 5⁺and 6⁺.

Examination of the time course for the reaction (Fig. 11a–c)

reveals that more than 80% of plasmid is cleaved after two

hours for 4⁺and 6⁺at 300 μM. The rate constants were found

For the most active compounds 4⁺, 5⁺and 6⁺we investi-

gated the dependence of the DNA cleavage on both the concen-

tration of the copper complexes (Fig. 10b–d) and the reaction

time at a given complex concentration (300 μM, Fig. 11a–c).

Not surprisingly, the higher the concentration, the greater the

yield in cleavage product (Fig. 10b–d). For 5⁺and 6⁺the NC

form could be detected for complex concentrations within the

30–50 μM range, while >90% cleavage is observed in the

300–500 μM range. There is a progressive increase of the reac-

tion yield over a wide concentration range for 5⁺and 6⁺, as

shown in Fig. 10b–d. In contrast, 4⁺exhibits a dramatic

enhancement of cleavage activity from 100 μM (no significant

activity) to 300 μM (full digestion of the SC form). Such a dose-

dependent drop in activity is rather uncommon, but it has

been recently reported for the copper(^II) complex of a bis

(pyridyl)alkyl-acridine conjugate.⁴⁸

to be 0.49

0.05 min⁻¹, 0.09

0.01 min⁻¹and 0.50

0.05 min⁻¹for 4⁺, 5⁺and 6⁺, respectively. While 4⁺and 6⁺

exhibit comparable rate constants, 5⁺, which exhibits a less

electron-donating fluorine substituent, was found to react

much slower with DNA.

In order to get insight into the reaction mechanism we evalu-

ated the eﬀect of various scavengers and exogenous agents on

the cleavage activity (Fig. 12). For all the complexes the

cleavage is fully inhibited by the addition of EDTA (lane 11),

showing that the metal ion is essential for the activity. For the

three compounds neither NaN₃(singlet oxygen scavenger),

superoxide dismutase (superoxide scavenger) nor Calatase

(H₂O₂scavenger) inhibit cleavage (lanes 3, 4, 5), indicating

that ROS are not responsible for the activity. DMSO and EtOH

also did not aﬀect the strand scission by 4⁺(lanes 6, 7),

showing that the freely diﬀusible OH˙ species is not respon-

sible for the cleavage. Surprisingly, DMSO was found to

enhance the cleavage activity in the case of 5⁺, while both

DMSO and EtOH improve cleavage by 6⁺. We interpret these

unexpected results by a higher solubilization of the complexes

in the presence of these co-solvents.

For 4⁺, 5⁺and 6⁺50% cleavage of the SC form is observed

for concentrations of 120

50 μM. It is noteworthy that

The addition of a reductant (mercaptoethanol) in the

medium enhances DNA cleavage in the case of 6⁺(lane 8).

This result may be understood by considering an alternative

oxidative cleavage pathway,^17,18which is initiated by reduction

of the copper(^II) into copper(I). This fact is consistent with elec-

trochemical measurements, which showed that 6⁺exhibits the

highest Cu(^II)/Cu(I) redox potential. Mercaptoethanol slightly

inhibits the activity of 4⁺, while it does not aﬀect the cleavage

by 5⁺. The main cleavage pathway, thus, likely remains hydro-

lytic for both complexes.

Fig. 11 Agarose gel electrophoresis patterns of supercoiled ϕX174 DNA

(20 μM base pairs) incubated for diﬀerent times at 37 °C with 300 μM of the

copper complexes in a phosphate buﬀer 10 mM pH 7.2 (+10% DMF): (a) 4⁺, (b)

5⁺, (c) 6⁺.

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Table 6 Anti-proliferative activity of the complexes^a

IC₅₀(μM)

In the presence of a minor groove binder (Hoechst 33258)

the cleavage activity of 4⁺and 5⁺was significantly inhibited

(lane 9). This experiment shows that both complexes bind to

DNA in the minor groove. In addition, excess of sodium chlor-

ide does not inhibit the cleavage reaction (lane 10), which

rules out a simple electrostatic interaction of copper complexes

4⁺and 5⁺with DNA.

Similarly to previous studies we performed religation assays

with T4 ligase on the ϕX174 DNA plasmid after incubation

(1 h) with the three most active complexes 4⁺, 5⁺, 6⁺(300 μM)

or restriction nuclease Xho I (blank).²²For complexes 4⁺, 5⁺, 6⁺

the initial band corresponding to the NC form of DNA is still

present after religation, which would at first glance suggest an

oxidative rather than hydrolytic cleavage. Nevertheless, the

latter results should be used with special care since DNA

hydrolysis was also assumed to be responsible for other DNA

damage which could not be enzymatically religated. For

instance hydrolytic cleavage products may not possess strictly

matched ends suitable for religation.⁵⁰In addition, the DNA

phosphate group could remain bound to the complex after

hydrolysis, and thus is inaccessible for the ligase.¹⁵Because

the relevance of the latter experiment is not particularly

strong, we still entertain the hydrolytic mechanism as the

most serious option.

Complexes

RT112

RT112-CP

RF

Cisplatin

9.1 1.2

99.1 5.8

3.1 0.2

20.8 2.5

6.3 0.6

3.7 0.2

3.9 0.1

23.8 1.2

ND

5.5 0.4

ND

5.2 0.4

5.0 0.3

4.7 0.5

2.6

ND

1.8

ND

0.8

1.4

1.2

1²⁺

2⁺

3⁺

4⁺

5⁺

6⁺

^aThe concentration resulting in 50% loss of cell viability relative to

untreated cells (IC₅₀) was determined from dose–response curves.

Results represent the means SD of three independent experiments.

RF is the relative ratio between IC₅₀of the cisplatin-resistant cells

against IC₅₀of the sensitive cell lines. ND: not determined.

to replace cisplatin in patients where cisplatin therapy failed

due to chemoresistance, the four more active complexes 2⁺, 4⁺,

5⁺and 6⁺were tested on a bladder cancer cell line resistant to

cisplatin (RT112-CP).⁵⁷As shown in Table 6, RT112-CP cells

are 2.6 times more resistant to cisplatin than the RT112

parental cell line. Interestingly, all four tested complexes (com-

pounds 2⁺, 4⁺, 5⁺and 6⁺) overcame the resistance observed

with cisplatin as expressed by the resistance factors (RF),²²

which are lower for our complexes compared to cisplatin. Com-

pared to the copper pyrimol complex (the structure shown in

Fig. 1d)²²the RF values are much lower and even close to 1 for

4⁺, 5⁺and 6⁺. So, these complexes have not only higher anti-

tumour activity than cisplatin against a bladder cancer cell line,

but also they are very powerful in circumventing the cellular

resistance of RT112-CP cells.

Anti-proliferative activity against bladder tumor cells

Some anticancer agents approved for clinical uses trigger cell

death by damaging DNA. For example, platinum-containing

drugs cause crosslinking of DNA,⁵¹Melphalan and Chloram-

bucil are alkylating agents^52,53while Bleomycin acts by produ-

cing single-strand and double-strands breaks in DNA.⁵⁴Since

our molecules induced DNA cleavage, we have carried out

in vitro assays to evaluate the cytotoxic and anti-proliferative

eﬀects of the complexes 1²⁺–6⁺.

Eﬀect of complexes on the cell cycle progression

In order to identify more potent and safe molecules for the In order to further characterize the mechanism of action of

treatment of bladder cancer, the biological activity of our complexes 2⁺, 4⁺, 5⁺and 6⁺, cell cycle analysis was performed

copper complexes was studied on the RT112 cell line, derived to determine the eﬀect of these molecules on cell cycle pro-

from a human bladder tumor. Cells were incubated with gression. RT112 cells were incubated with vehicle alone

various concentrations of the complexes 1²⁺–6⁺and their pro- (DMSO) as the control, or with IC₅₀concentrations of com-

liferation was monitored by a MTT assay.⁵⁵Our results indicate plexes. After 24 or 48 h, cells in suspension in the medium

that it was necessary to treat cells for at least 48 h to observe a and attached cells were harvested and analyzed by flow cyto-

massive cell death. The anti-proliferative activities of tested metry. As shown in Fig. 13A, after 24 h the majority of control

compounds after 48 h of treatment, expressed as IC₅₀values cells (55%) are in G0/G1 phases, whereas the rest of the cells

(the concentration resulting in 50% loss of cell viability relative are approximately equally distributed between the S and

to untreated cells), are summarized in Table 6. Cisplatin, G2 + M phases (26% and 19% respectively). A 24 h treatment

which is currently used in the management of advanced of RT112 cells with complex 2⁺results in a significant accumu-

bladder cancer, was applied as a test referential agent.⁵⁶lation of cells in the G0/G1 phase (73%), with a concomitant

Complex 1²⁺presented a low cytotoxic activity against RT112 loss from the S phase (9%). This G0/G1 phase accumulation

cells (IC₅₀= 99.1 µM), which could be correlated to its inability increases with incubation time, since it reaches 82% after 48 h

to cleave DNA. For complexes 2⁺and 3⁺there seems to be no against 70% in the control (Fig. 13B). The results obtained

direct correlation between their moderate nuclease activity and with the other complexes (4⁺, 5⁺and 6⁺) are diﬀerent. After

their cytotoxicity, since IC₅₀of complex 3⁺is 6.7 times more 24 h of incubation with the IC₅₀concentration of the mole-

than that of complex 2⁺. Finally, complexes 4⁺, 5⁺and 6⁺, cules, the G2 + M accumulation of cells increases from 19% in

which have higher DNA cleavage activity, were about 1.4–2.4 the control to 39% with complex 4⁺, 34% with complex 5⁺and

times more active than cisplatin against the RT112 cell line. In 44% with complex 6⁺(Fig. 13A). This is accompanied by a

order to determine if our complexes could be valuable agents decrease of the number of cells in the remaining phases of the

This journal is © The Royal Society of Chemistry 2013

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changes in DNA binding constants do not explain directly the

trend of cleavage activity in the series since 3⁺exhibits a lower

aﬃnity for DNA than 1²⁺, although it is more active.

A hydrolytic pathway is often proposed for copper-catalyzed

DNA cleavage in the absence of a reductant or oxidants.

Recently, oxidative pathways were proposed to account for DNA

cleavage by some copper(^II) complexes involving sterically

hindered phenolate or aminophenolate coordinating groups

(Fig. 1d). They imply the formation of a reactive phenoxyl

radical by aerobic oxidation of the coordinated phenolate. The

phenoxyl/phenolate potential for the complex shown in Fig. 1d

is +1.11 V vs. Ag/AgCl (roughly +1.07 V vs. SCE), i.e. much

higher than the compounds investigated there.²²Therefore,

from a thermodynamic point of view 1²⁺–6⁺might form phe-

noxyl radicals more easily than Reedijk’s complex, rendering

an oxidative mechanism promoted by phenoxyl species plaus-

ible.²²Nevertheless, large diﬀerences in chemical stabilities

for the radical species (estimated via the I^a_p/I_p^cratio of the phe-

noxyl/phenolate redox couple) were observed within our series,

without any evident correlation between DNA cleavage activity

and phenoxyl radical stabilities. In addition, attempts to trap a

phenoxyl radical intermediate by UV-Vis spectroscopy upon

reaction with DNA (phenoxyl radicals exhibit an intense π–π*

transition at around 420 nm)²⁴were unsuccessful. These

experimental results thus argue against the involvement of a

phenoxyl radical as reactive species for DNA cleavage and

rather support a hydrolytic pathway. Hydrolytic DNA cleavage

may be initiated by substitution of the labile coordinated

solvent by a phosphate group of the DNA backbone, which is

activated by the Lewis acidity of the copper ion. Methylpyridine

N-donors could facilitate this activation and subsequent

nucleophilic attack. A detailed investigation on the cleavage

mechanism is currently under way in our laboratories.

Fig. 13 Cell cycle distribution of RT112 cells treated with complexes 2⁺, 4⁺, 5⁺

and 6⁺. RT112 cells were treated with vehicle alone (Control, CT) or with the

IC₅₀value of each compound for 24 h (A) or 48 h (B). Cells were then ﬁxed,

stained with PI and cell cycle was analyzed by ﬂow cytometry. Each value is the

mean SD of three independent determinations.

cell cycle. This G2 + M accumulation is sustained after 48 h of

incubation with complex 4⁺(36% against 14% in the control)

and even increased with complexes 5⁺and 6⁺, which led to 43

and 48% respectively of cells in G2 + M phases (Fig. 13B).

Complexes 4⁺, 5⁺and 6⁺thus clearly alter the cell cycle pro-

gression. This is most likely owing to DNA damage which

results in the activation of cell cycle checkpoints in dividing

cells. If cells undergo DNA damage in G2, the G2/M check-

point is activated and stops the cell cycle to prevent cells from

starting mitosis.^58,59So, complexes 2⁺, 4⁺, 5⁺and 6⁺promote

cell cycle arrest in G2 + M phases, which probably reflects their

activity on DNA.

The anti-proliferative activity of the four more active

nucleases 2⁺, 4⁺, 5⁺and 6⁺was tested against RT112 bladder

cancer cell lines. The four investigated compounds exhibit IC₅₀

values lower than cisplatin (3.1–6.3 μM vs. 9.1 μM). They are

also able to circumvent the cellular resistance of the RT112CP

cell line, as reflected by RF up to three times lower for 2⁺, 4⁺,

5⁺and 6⁺than for cisplatin. Finally, we show here that the

most active compounds alter cell cycle progression, which

probably reflects their activity on DNA. Further analysis will be

conducted to determine molecular alterations induced by our

complexes and to understand how they could block the cell

Conclusion

The present results point to a very simple way to dramatically cycle at diﬀerent levels.

enhance the nuclease activity, in the absence of an exogenous

agent, of copper-phenolate complexes involving tripodal

ligands: when at least one pyridine is α-methylated the yield of

the reaction is significantly improved, with 75 to 95% DNA

digestion after 2 hours for complex concentrations of 300 μM

Experimental section

Materials and methods

(vs. <22% for complexes involving exclusively non-methylated All chemicals were of reagent grade and were used without

pyridine donors). We hypothesize that this result is correlated purification. NMR spectra were recorded on a Bruker AM 300

to the weaker N-donating ability of methylpyridines, which (¹H at 300 MHz) spectrometer. Chemical shifts are quoted rela-

facilitates exchange of the coordinated exogenous ligand and tive to tetramethylsilane (TMS). Mass spectra were recorded on

modifies significantly the structure of the phenolate complex a Thermofinningen (EI/DCI) or a Bruker Esquire ESI-MS appar-

(equatorial positioning of the phenolate moiety). Noteworthy, atus. For pK_adeterminations UV/Vis spectra were recorded on

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Paper

a Cary Varian 50 spectrophotometer equipped with a Hellma NaCl solution and dried over Na₂SO₄. The solution was evapor-

immersion probe (1.000 cm path length). The temperature in ated under vacuum to aﬀord yellow oil. Column chromato-

the cell was controlled using a Lauda M3 circulating bath and graphy on silica gel (ethyl acetate–pentane (1/5) yielded 3L

1

the pH was monitored using a Methrom 716 DMS Titrino (263 mg, 65%) as a yellow solid. H NMR (300 MHz, CDCl₃): δ

apparatus. A least square fit of the titration data was realized = 1.40 (9H, s), 2.97–3.09 (4H, m), 3.73 (3H, s), 3.83 (2H, s), 3.85

with SPECFIT software. For UV/Vis measurements at neutral (2H, s), 7.03–7.20 (4H, m), 7.52 (1H, td, ³J = 7.6 Hz, ⁴J =

3

4

3

pH (buﬀered media) a Perkin Elmer Lambda 2 spectropho- 1.9 Hz), 7.57 (1H, td, J = 7.6 Hz, J = 1.8 Hz), 8.47 (1H, d, J =

tometer was used. X-band EPR spectra were recorded on either 4.8 Hz), 8.52 (1H, d, ³J = 5.9 Hz). ¹³C NMR (75 MHz, d₆-DMSO):

a Bruker ESP 300E or a Bruker EMX Plus spectrometer δ = 29.8 (3 CH₃), 35.2 (C), 35.6 (CH₂), 54.0 (CH₂), 56.0 (CH₃),

equipped with a Bruker nitrogen flow cryostat. Spectra were 59.0 (CH₂), 59.8 (CH₂), 112.0 (CH), 113.3 (CH), 121.7 (CH),

simulated using Bruker SIMFONIA software. Electrochemical 122.6 (CH), 123.1 (C), 123.7 (CH), 123.9 (CH), 136.8 (CH), 136.9

measurements were carried out using a CHI 620 potentiostat. (CH), 138.3 (C), 149.4 (CH), 149.6 (CH), 150.7 (C), 151.8 (C),

Experiments were performed in a standard three-electrode cell 152.1 (C), 158.2 (C), 158.4 (C), 160.0 (C). MS (DCI, NH₃/

under an argon atmosphere. A glassy carbon disc electrode isobutane): m/z (%) 406 (100) [M + H]⁺. M.p. 70 °C. Anal. Calcd

(3 mm diameter) or a platinum disc electrode (1 mm dia- for C₂₅H₃₁N₃O₂: C, 74.04; H, 7.70; N, 10.36. Found: C, 73.82;

meter), which was polished with 1 mm diamond paste, was H, 7.75; N, 10.31%.

used as the working electrode. An SCE was used as the

reference.

4L. 2-tert-Butyl-4-methoxy-phenol (1.8 g, 10 mmol),

(6-methyl-pyridin-2-ylmethyl)-pyridin-2-ylmethyl-amine (2.13 g,

10 mmol)²⁹and formaldehyde (0.75 ml of a 37% aqueous solu-

tion) in ethanol–H₂O (4 : 6, 10 ml) were refluxed for 24 h. The

Crystal structure analysis

The collected reflections were corrected for absorption reaction mixture was then extracted with dichloromethane,

(SADABS) and solved by direct methods and refined with Olex washed with a saturated NaCl solution and dried over Na₂SO₄.

software.⁶⁰All non-hydrogen atoms were refined with anisotro- The solution was evaporated under vacuum to aﬀord orange

pic thermal parameters. Hydrogen atoms were generated in oil. Column chromatography on silica gel (ethyl acetate–

idealized positions, riding on the carrier atoms, with isotropic pentane (1/1)) yielded 4L (1.82 g, 45%) as a pale pink oil.

thermal parameters.

¹H NMR (200 MHz, CDCl₃): δ = 1.45 (9H, s), 2.58 (3H, s), 3.73

4

(3H, s), 3.76 (2H, s), 3.83 (2H, s), 3.85 (2H, s), 6.49 (1H, d, J =

Preparation of the ligands and complexes

3.0 Hz), 6.80 (1H, d, ⁴J = 3.0 Hz), 7.00 (2H, d, ³J = 7.5 Hz),

1L. 4-Methoxyphenol (1.24 g, 10 mmol), bis-pyridin-2- 7.00–7.16 (2H, m), 7.38 (2H, d, ³J = 7.8 Hz), 7.50 (1H, t, ³J =

ylmethyl-amine (1.99 g, 10 mmol) and formaldehyde (0.75 ml 7.54 Hz), 7.61 (1H, td, ³J = 7.8 Hz, ⁴J = 1.7 Hz), 8.52 (2H, d, ³J =

of a 37% aqueous solution) were refluxed for 24 h in ethanol– 5.1 Hz), 10.40 (1H, s, broad). ¹³C NMR (75 MHz, CDCl₃): δ =

H₂O (1 : 1, 12 ml). The reaction mixture was then extracted 24.5 (CH₃), 29.9 (3 CH₃), 35.4 (C), 56.3 (CH₃), 58.2 (CH₂), 59.7

with dichloromethane, washed with a saturated NaCl solution (2 CH₂), 112.7 (CH), 1313.4 (CH), 120.5 (CH), 122.1 (CH), 122.5

and dried over Na₂SO₄. The solution was evaporated under (CH), 123.6 (C), 123.9 (CH), 136.9 (CH), 137.1 (CH), 138.2 (C),

vacuum to aﬀord brown oil. Column chromatography on silica 149.3 (CH), 150.8 (C), 151.8 (C), 157.8 (C), 158.4 (C), 158.8 (C).

gel with CH₂Cl₂–methanol (10/0 to 5/5) as the eluent yielded MS (DCI, NH₃/isobutane): m/z (%) 406 (100) [M + H]⁺. Anal.

1L (0.67 g, 20%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃): Calcd for C₂₅H₃₁N₃O₂: C, 74.04; H, 7.70; N, 10.36. Found:

4

δ = 3.74 (2H, s), 3.77 (3H, s), 3.87 (4H, s), 6.64 (1H, d, J = 3.0 C, 72.26; H, 7.63; N, 10.36%.

Hz), 6.74 (1H, dd, ³J = 8.8 Hz, ⁴J = 3.0 Hz), 6.83 (1H, d, ³J =

5L. This ligand was prepared in a similar way as 4H, by

3

8.8 Hz), 7.16 (2H, dd, J₁= 7.3 Hz, J₂= 5.1 Hz), 7.34 (2H, d, using the 2-tert-butyl-4-fluoro-phenol instead of the 2-tert-

3

4

³J = 7.9 Hz), 7.62 (2H, td, J = 7.8 Hz, J = 1.8 Hz), 8.57 (2H, d, butyl-4-methoxy-phenol. Column chromatography on silica gel

³J = 4.9 Hz). 13C NMR (100 MHz, CDCl₃): δ = 55.9 (CH₃), 57.1 (ethyl acetate–pentane (2/8)) yielded 5L (40%) as a pale yellow

1

(CH₂, 59.1 (2 CH₂), 114.0 (CH), 115.9 (CH), 116.9 (CH), 122.9 oil. H NMR (400 MHz, CDCl₃): δ = 1.44 (9H, s), 2.59 (3H, s),

(CH), 123.3 (CH), 123.4 (C), 136.8 (CH), 149.0 (CH), 151.4 (C), 3.74 (2H, s), 3.83 (2H, s), 3.84 (2H, s), 6.63 (1H, dd, ³J = 8.1 Hz,

152.3 (C), 158.2 (C). MS (DCI, NH₃/isobutane): m/z (%) 336.2 ⁴J = 3.1 Hz), 6.90 (1H, dd, ³J = 11.1 Hz, ⁴J = 3.1 Hz), 7.01 (1H, d,

(100) [M + H]⁺. M.p. 70 °C. Anal. Calcd for C₂₁H₂₅N₃O₂: ³J = 7.6 Hz), 7.07 (1H, d, J = 7.6 Hz), 7.13 (1H, dd, J = 7.0 Hz,

3

C, 71.77; H, 7.17; N, 11.96. Found: C, 71.73; H, 6.45; N, ⁴J = 5.0 Hz), 7.36 (1H, d, J = 7.8 Hz), 7.50 (1H, t, J = 7.8 Hz),

3

4

3

12.42%.

7.60 (1H, td, J = 7.8 Hz, J = 1.8 Hz), 8.53 (1H, d, J = 4.7 Hz),

3L. 3-tert-Butyl-2-hydroxy-5-methoxy-benzaldehyde (208 mg, 10.70 (1H, s, broad). ¹³C NMR (100 MHz, CDCl₃): δ = 24.4

1

mmol), (2-pyridin-2-yl-ethyl)-pyridin-2-ylmethyl-amine (CH₃), 29.7 (3 CH₃), 35.3 (C), 57.6 (CH₂), 59.6 (CH₂), 59.8

(213 mg, 1 mmol)³⁵and glacial acetic acid (0.2 ml) were dis- (CH₂), 113.1 (CH), 114.1 (CH), 120.4 (CH), 122.1 (CH), 122.5

solved in ethanol (20 ml). After 30 min stirring, 62 mg of (CH), 123.8 (CH), 124.0 (C), 136.9 (CH), 137.2 (CH), 138.6 (C),

sodium cyanoborohydride (62 mg, 1 mmol) were slowly added 149.3 (CH), 156.9 (C), 157.5 (C), 158.4 (C), 158.6 (C). MS (DCI,

(over 6 hours). The mixture was stirred for 12 hours and then NH₃/isobutane): m/z (%) 394 (100) [M + H]⁺. Anal. Calcd for

neutralized with concentrated HCl (2 M). The reaction mixture C₂₄H₂₈FN₃O: C, 73.26; H, 7.17; N, 10.68. Found: C, 74.01; H,

was extracted with dichloromethane, washed with a saturated 7.22; N, 10.52%.

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6L. This ligand was prepared in a similar way as 4L, by Determination of the DNA binding constants

using bis-(6-methyl-pyridin-2-ylmethyl)-amine⁶¹instead of

(6-methyl-pyridin-2-ylmethyl)-pyridin-2-ylmethyl-amine. Column

chromatography on silica gel (ethyl acetate–pentane (2/8))

yielded 6L (40%) as a pale yellow oil. ¹H NMR (400 MHz,

CDCl₃): δ = 1.45 (9H, s), 2.56 (6H, s), 3.73 (3H, s), 3.74 (2H, s),

The DNA binding constants were obtained from titration of

solutions of the complexes with CT DNA. DNA (type I,

fibers, from Sigma Aldrich) was first purified by four succes-

sive extractions with the phenol–CHCl₃–isoamyl alcohol

25–24–1 mixture. Then 0.1 volume (with respect to the

aqueous phase) of a 3 M sodium acetate solution and 2

volumes of ethanol were added and the solution was stored

overnight at 253 K. The precipitated DNA was solubilised and

analyzed by UV-Vis to ensure that the A₂₆₀/A₂₈₀ratio was ≥2. In

a typical titration experiment 50 μl of a stock solution of the

complex in DMF (0.5–1 mM) was mixed with 20 μl of phos-

phate buﬀer (50 mM) at pH 7.3, and 430 μl of an aqueous solu-

tion of CT DNA (concentration ranging from 0 to 0.5 mM) was

added. After the equilibrium was reached (10 min) at 298 K

the UV-Vis spectrum of the solution was recorded. The DNA

binding constants K_bwere calculated using the equation:

4

3.82 (4H, s), 6.48 (1H, d, J = 3.1 Hz), 6.79 (1H, d, J = 3.2 Hz),

3

7.00 (2H, d, J = 7.6 Hz), 7.15 (2H, d, J = 7.6 Hz), 7.49 (1H, t,

³J = 7.9 Hz), 10.40 (1H, s, broad). ¹³C NMR (100 MHz, CDCl₃):

δ = 24.5 (CH₃), 29.7 (3 CH₃), 35.2 (C), 56.0 (CH₃), 58.0 (CH₂),

59.7 (2 CH₂), 112.6 (CH), 113.3 (CH), 120.4 (CH), 121.9 (CH),

123.6 (C), 137.0 (CH), 138.0 (C), 150.7 (C), 151.8 (C), 157.9 (C),

158.0 (C). MS (DCI, NH₃/isobutane): m/z (%) 420 (100)

[M + H]⁺. Anal. Calcd for C₂₆H₃₃N₃O₂: C, 74.43; H, 7.93; N,

10.02. Found: C, 74.36; H, 7.81; N, 10.06%.

1·(ClO₄)₂. 1L (54.6 mg, 0.163 mmol) was dissolved in

methanol (3 mL) and then Cu(ClO₄)₂·6H₂O (60.3 mg,

0.163 mmol in a methanolic solution) and NEt₃(22.8 μl,

0.163 mmol ml) were added. The solution was stirred for

10 min at room temperature, left to evaporate overnight, and

½DNAꢀ=ðε_aꢁ ε_fÞ ¼ ½DNAꢀ=ðε_bꢁ ε_fÞ þ 1=K_b

then stored for 10 h at 253 K. A dark brown precipitate of where ε_ais the apparent molar extinction coeﬃcient of the

1·(ClO₄)₂was formed, which was collected by filtration (50 mg, complex at a given DNA concentration, ε_fis the molar extinc-

31% yield). Slow diﬀusion of diethyl ether into a concentrated tion coeﬃcient of the free complex, and ε_bis the molar extinc-

CH₃CN solution of the complex aﬀords brown single crystals tion coeﬃcient of fully bound complex. A plot of [DNA]/(ε_a−

of 1·(ClO₄)₂·(CH₃CN)₄. MS (ESI, CH₃CN): m/z (%) 396 (100) ε_f) as a function of DNA concentration gives a straight line

[M_0.5− 2ClO₄]⁺. Anal. Calcd for C₄₀H₄₀Cl₂Cu₂N₆O₁₂: C, 48.30; whose slope is 1/(ε_b− ε_f) and the Y-intercept is equal to

H, 4.05; N, 8.45. Found: C, 48.52; H, 4.03; N, 8.35. UV-Vis (λ_max1/K_b(ε_b− ε_f). Each binding constant was obtained from a tripli-

[nm] [ε/M⁻¹cm⁻¹] in CH₃CN, 298 K): 466 [1015], 853 [670].

3·ClO₄. 3L (170 mg, 0.419 mmol) was dissolved in CH₃CN

(3 mL) and then Cu(ClO₄)₂·6H₂O (156 mg, 0.412 mmol) was

added. The solution was stirred for 30 min. Slow diﬀusion of

diisopropyl ether into the resulting solution aﬀords blue

cate experiment.

Procedure for DNA cleavage experiments

single crystals of 3·ClO₄(237 mg, 80% yield). MS (ESI, CH₃CN): ϕX174 RF1 DNA was purchased from Fermentas and was

m/z (%) 467 (100) [M − H − 2ClO₄− CH₃CN]⁺. Anal. Calcd for stored at −20 °C. The typical reaction mixture, containing

C₂₇H₃₄Cl₂CuN₄O₁₀: C, 45.74; H, 4.83; N, 7.90. Found: C, 45.82; double-stranded DNA and the Cu^IIcomplexes in a 10 mM

H, 4.83; N, 7.90. UV-Vis (λ_max[nm] [ε/M⁻¹cm⁻¹] in CH₃CN, phosphate solution (pH 7.2 or 8.2) with 10% DMF, was incu-

298 K): 573 [230].

bated at 37 °C for the required time, with or without additives.

Complexes 4·ClO₄and 5·ClO₄were prepared in an identical After the incubation period, the reaction was quenched at

manner in 80 and 63% yield respectively. 6⁺was prepared −20 °C, followed by the addition of loading buﬀer (6× loading

in situ in its phenolate form by mixing equimolar amounts of dye solution, Fermentas). The reaction mixture was loaded on

6L and Cu(ClO₄)₂·6H₂O in DMF.

a 0.8% agarose gel in Tris–Boric acid–EDTA buﬀer (pH 8.2)

4·ClO₄. MS (ESI, CH₃CN): m/z (%) 467 (100) [M − H − 2ClO₄(0.5× TBE) and electrophoresis was performed at 70 V for 2 h

− CH₃CN]⁺. Anal. Calcd for C₂₇H₃₄Cl₂CuN₄O₁₀: C, 45.74; H, 30 min–3 h. After DNA migration, the gels were stained by

4.83; N, 7.90. Found: C, 45.50; H, 4.77; N, 7.86. UV-Vis (λ_maxincubation for 10 min with a 1 μg ml⁻¹ethidium bromide (EB)

[nm] [ε/M⁻¹cm⁻¹] in CH₃CN, 298 K): 590 [210].

solution and then washed with distilled water. The gels were

5·ClO₄. MS (ESI, CH₃CN): m/z (%) 455 (100) [M − H − 2ClO₄visualised and the fluorescence was quantified using Imager

− CH₃CN]⁺. Anal. Calcd for C₂₆H₃₁Cl₂CuFN₄O₉: C, 44.80; H, Typhoon 9400 and Image Quant software. The cleavage

4.48; N, 8.04. Found: C, 44.51; H, 4.39; N, 7.90. UV-Vis (λ_maxeﬃciency was measured by determining the ability of the

[nm] [ε/M⁻¹cm⁻¹] in CH₃CN, 298 K): 622 [100].

complex to convert the supercoiled DNA (SC) to nicked circular

6⁺in DMF solution (phenolate cationic form with a coordi- form (NC) and linear form (L). For kinetic analysis we deter-

nated DMF molecule). MS (ESI, DMF–H₂O): m/z (%) 481.2 (30) mined the amounts of SC and NC forms on the agarose gel by

[M − DMF]⁺, 482.2 (70) [M + H − DMF]⁺(this copper(I) form of image processing with imageJ 1.46r freeware (Wayne Rasband

the complex results from the reduction of 6⁺in the gas National Institute of Health, USA). The data were then refined

phase).⁶²EPR (100 K, DMF): g_xx= g_yy= 2.057, g_zz= 2.265, A_{xx (Cu)}

=

by simplex algorithm by considering a mono-exponential

A_yy

= 1.5 mT, A_zz

(Cu)

= 15.7 mT; UV-Vis (λ_max[nm] function (commercial Bio-kine 4.44 software, Biologic Co., Le

(Cu)

[ε/M⁻¹cm⁻¹] in DMF, 298 K): 543 [975].

Pont-de-Claix, France).

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Enzymatic religation assays

585 44 nm filter. Parameters from 2 × 10⁴cells were acquired

using Cell Quest Pro software (Becton Dickinson). The percen-

tage of cell cycle distribution in the G1, S and G2 + M phases

was determined using FCS Express 3 software (De Novo

Sofware, Los Angeles, CA).

The supercoiled DNA was treated with the copper complexes at

37 °C and pH 7.2. DNA samples were purified over QIAquick

PCR purification columns (Qiagen) and were used for religa-

tion experiments. Ligation was performed in 25 μL with 2.5 μL

of 10× ligation buﬀer and 2 units of T4 ligase (Fermentas) for

16 h at 16 °C. Similarly, and for comparison, the supercoiled

DNA was digested using XhoI restriction enzyme (Fermentas) Acknowledgements

and religated using the above procedure, and found 100%

religation.

We thank the Labex Arcane (Université Joseph Fourier,

Grenoble) and the Association Espoir and GEFLUC (Délégation

de l’Isère) for financial support.

Cell culture

Human bladder cancer cell line RT112 was obtained from Cell

Lines Service (Eppelheim, Germany). Cisplatin resistant RT112

cells (RT112-CP) were kindly provided by B. Köberle (Institute

of Toxicology, Clinical Centre of University of Mainz, Mainz,

Germany). RT112 and RT112-CP cells were cultured in RPMI

1640 medium supplemented with 10% (v/v) fetal calf serum

(FCS) and 2 mM glutamine (Invitrogen Life Technologies,

Paisley, UK). Cells were maintained at 37 °C in a 5% CO₂-

humidified atmosphere and tested to ensure freedom from

mycoplasma contamination.

Notes and references

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2 T. Theophanides and J. Anastassopoulou, Crit. Rev. Oncol.

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D. Chen, Y. Xiao and Q. Ping Dou, Int. J. Mol. Med., 2008,

22, 677; C. Marzano, M. Pellei, F. Tisano and C. Santini,

Anti-Cancer Med. Chem., 2009, 9, 185; S. Tardito and

L. Marchio, Curr. Med. Chem., 2009, 16, 1325.

3 A. Chan, F. Wong and M. Arumanayagam, Ann. Clin.

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Cell proliferation assay

Inhibition of cell proliferation by copper complexes was

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seeded into 96-well plates (5 × 10³cells per well) in 100 µl of

culture medium. After 24 h, cells were treated with cisplatin

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concentrations. In parallel, a control with DMSO (vehicle

alone) at the same dilutions was done. Following incubation

for 48 h, 10 µl of a MTT (Euromedex, Mundolsheim, France)

stock solution in PBS at 5 mg ml⁻¹was added in each well and

the plates were incubated at 37 °C for 3 h. Plates were then

centrifugated for 5 min at 400 g before the medium was

discarded and replaced with DMSO (100 µl per well) to solubil-

ize water-insoluble purple formazan crystals. After 15 min

under shaking, absorbance was measured on an ELISA reader

(Tecan, Männedorf, Switzerland) at a test wavelength of

570 nm and a reference wavelength of 650 nm. Absorbance

obtained by cells treated with the same dilution of the vehicle

alone (DMSO) was rated as 100% cell survival. Each data point

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a

FACScan (Becton

Dickinson France, Le Pont-de-Claix, France) equipped with a

488 nm argon laser and PI fluorescence was collected with a

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Nuclease and anti-proliferative activities of copper(ii) complexes of N3O tripodal ligands involving a sterically hindered phenolate

DOI: 10.1039/c3dt32659d

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