Bioorganic & Medicinal Chemistry 17 (2009) 6054–6062

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Characterization and DNA-interaction studies of 1,1-dicyano-2,2-ethylene

dithiolate Ni(II) mixed-ligand complexes with 2-amino-5-methyl thiazole,
2-amino-2-thiazoline and imidazole. Crystal structure of [Ni(i-MNT)(2a-5mt)2]
Philip J. Cox a, George Psomas b, Christos A. Bolos b,*
a
School of Pharmacy, The Robert Gordon University, Schoolhill, Aberdeen AB10 1FR, Scotland, UK
b
Laboratory of Inorganic Chemistry, Chemistry Department, Aristotle University of Thessaloniki, GR-541 24, Greece

a r t i c l e i n f o a b s t r a c t

Article history: A series of mixed-ligand neutral nickel(II) complexes of the general formula [Ni(i-MNT)(2a-5mt)2] (1),
Received 14 April 2009 [Ni(i-MNT)(2a-2tzn)2] (2) and [Ni(i-MNT)(Im)2] (3), [where i-MNT2 = the dianion of 1,1-dicyano-2,2-
Revised 18 June 2009 ethylenedithiolate, 2a-5mt = 2-amino-5-methyl thiazole, 2a-2tzn = 2-amino-2-thiazoline and Im = imid-
Accepted 20 June 2009
azole] were prepared and characterized with elemental analyses, spectroscopic (IR, UV–vis) methods,
Available online 30 June 2009
magnetic susceptibility, molar conductivity and cyclic voltammetry measurements. The magnetic data,
the electronic spectra and the electrical conductivity measurements indicated mononuclear neutral com-
Keywords:
plexes with square-planar geometry. The X-ray analysis of [Ni(i-MNT)(2a-5mt)2] shows the nickel atom
Ni(II) complexes
i-MNT
being fourfold coordinated with the two sulfur atoms of the dithiolate (i-MNT) ligand and the endocyclic
2-Amino-5-methyl-thiazole nitrogen atoms from the two 2a-5mt ring giving rise to a slightly distorted square-planar arrangement.
2-Amino-2-thiazoline The cyclic voltammograms of the complexes have been recorded and the corresponding redox potentials
Imidazole have been estimated. The DNA-binding studies of the complexes has been evaluated by examining their
Interaction with calf-thymus DNA ability to bind to calf-thymus DNA (CT DNA) with UV spectroscopy and cyclic voltammetry. Both studies
Cyclic voltammetry have shown that the complexes can bind to CT-DNA by the intercalative and the electrostatic binding
mode. Competitive binding studies with ethidium bromide (EB) with fluorescence spectroscopy have also
shown that the complexes exhibit the ability to displace the DNA-bound EB indicating that they can bind
to DNA in strong competition with EB.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction In our previous studies a series of mixed-ligand Cu(II) coordina-

A large number of coordination compounds with metal ions
have been investigated for their anti-proliferative activity and used
in cancer therapy.1 However their several side-effects leave room
for the selection of other metals and additional ligands, known
for their bioactivity.2 A principal target of metals in the cancer cells
is their coordination with DNA, bound selectively to it either
through the oxygen of phosphates or to heterocyclic nitrogen
atoms (N1, N3 and N7) of DNA bases, or both.3 Nickel is recognized
as an essential trace element for bacteria and plants, but its role in
animal biochemistry is not well defined.4 Some of the Ni(II) com-
plexes with dimethyldithiophosphates ligands is reported to be ac-
tive against Walker 256 carcinosarkoma tumor system, but they
were found to be inactive against L1210 leukemia.5 Regarding
the DNA interaction with Ni(II) complexes showed intercalative
behavior and also DNA cleavage ability.6
* Corresponding author. Tel.: +30 2310 997705; fax: +30 2310 997738.
E-mail address: bolos@chem.auth.gr (C.A. Bolos).
tion compounds, with bidentate, tridentate and polydentate
amines and their Schiff bases,7,8 trihalides,9 1,2-dithiolates,10 and
1,3-thiazoles, thiazolines and imidazole11–14 have been utilized
as models to investigate their toxicity,10,12 anti-inflammatory
activity,12 antimicrobial activity against Gram(+) and Gram() bac-
teria9,11,14 and anti-proliferative activity in vitro9,11,13,14 and
in vivo10,12 towards various cancer cell lines, for example, HeLa,
T47D, HT-29, MCF7, MRC5 and P388 leukemia. A relationship be-
tween medicinal effectiveness and physicochemical, structural
properties of the ligands and their Cu(II) complexes, have been car-
ried out. Among the ligands characterized for individual anti-pro-
liferative activity are those of S,N-1,3-thiazoles and especially
their Cu(II) complexes with 2-amino-5-methyl thiazole (2a-5mt)
which were found to meet the necessary criteria established by Na-
tional Cancer Institute.15 It has also been demonstrated that these
Cu(II) complexes induce apoptosis via mitochondrial pathway. In
these studies, although the molar ratio of the reactants (Ligand/
Cu(II)/2a-5mt) was 1:1:2, the isolated complexes conform to
1:1:1 stoichiometries. Our efforts to insert one more 2a-5mt
molecule into the coordination sphere of Cu(II), which might

0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.058
 P. J. Cox et al. / Bioorg. Med. Chem. 17 (2009) 6054–6062
differentiate further the bioactivity of the compounds under study,
failed. This disadvantage of Cu(II) led us to carry out similar reac-
tions with the Ni(II) since this metal adopts more easily various
geometries and characteristic magnetic and redox properties. In
spite of the toxicity of Ni(II) complexes, ternary Ni(II) complexes
are formed with DNA, oligopeptides and proteins under physiolog-
ical conditions.16,17 The latter had reversible Ni(III, II) redox behav-
ior, whereas Ni(II) complexes with bases were able to induce,
cooperatively, conformational changes in synthetic DNA.18 In ef-
fect, what we wanted to address in this project was the synthesis
and characterization of neutral mixed-ligand dithiolato Ni(II) com-
plexes with 2a-5mt, 2a-2tzn and imidazole, with the aim of exam-
ining (i) the possible coordination of a second 2a-5mt molecule
with Ni(II), (ii) the resultant geometry and (iii) the biological activ-
ity of the new Ni(II) compounds. Prior to the examination of their
possible bioactivity we thought that it would be important to per-
form various studies of the compounds, such as reactivity with
calf-thymus (CT) DNA with cyclic voltammetry (CV) and UV and
fluorescence spectroscopy. The results of these studies are dis-
cussed in details and found to be promising for biological studies.
2. Results and discussion
The reaction of [Ni(L)4Cl2] with Na2(i-MNT)3H2O affords brown
mixed-ligand complexes corresponding to the general formulas
[Ni(i-MNT)(2a-5mt)2], [Ni(i-MNT)(2a-2tzn)2] and [Ni(i-MNT)-
(Im)2]. The new chelates are slightly soluble in methanol, soluble
in acetonitrile and are very soluble in acetone, DMSO and DMF.
The analytical data C, H, N and Ni confirm the composition of the
complexes and the stoichiometry proposed. Evidence of the mode
of bonding of the ligands was gathered from the IR spectra, while
their geometry was proposed by electronic excitation spectra and
confirmed with X-ray determination of [Ni(i-MNT)(2a-5mt)2].
2.1. Spectroscopic data
2.1.1. IR spectral studies
The most significant bands emerging from the functional group
of i-MNT and from the 2a-5mt, 2a-2tzn and imidazole and their
complexes are given in the experimental section. The dianion of
the 1,1-dicyano-2,2-ethylene dithiolate contain the C„N, the
C@CS2, and the C-SS functional groups and while the absorption
due to the multiple bond in each group is easily distinguishable.
The very strong bands at 2175 cm1 and in the region 1356–
1436 cm1 have been assigned to m(C„N) and m(C@C), respec-
tively, of the i-MNT ligand.19 Upon coordination, these bands are
shifted to higher wavenumbers: the former by 28–53 cm1 and
the latter by 35–93 cm1 compared to the free i-MNT ligand. These
shifts reveal an increase of the order of the C„N and ˜C@C— bonds,
since the planar extensive p-conjugated system of the i-MNT li-
gand facilitate a drift of electron density towards the sulfur atoms
that results in a shortening of the C„N and ˜C@C— bonds.20 The
medium intensity bands at 943–989 cm1 and 794–896 cm1 in
the IR spectra are assigned to mas(CS2) and msym(CS2), respectively.
These two bands are shifted to lower (13–38 cm1) or higher (9–
28 cm1) wavenumbers in the infrared spectra of the complexes.
The variation in these shifts could be due to the different donation
ability of the 2a-5mt, 2a-2tzn and imidazole ligands. Other infrared
data further confirming the coordination of the ligands, localized in
the S–Ni–S and the Ni–N groups are the new medium or weak
intensity peaks appearing at 478–486 cm1 and 387–396 cm1,
which may be attributed to the m(Ni–N) and m(Ni–S), respectively.
The bands in the range 1631–1531 cm1 in the spectra of the free
thiazole, thiazoline or imidazole ligands can be assigned to m(C@N)
and m(C@C) of their rings. In the spectra of the complexes this re-
gion gives rise to a number of bands located in the range 1532–
6055
1630 cm1. It can be observed that the lower limit is slightly
shifted to higher frequencies upon coordination, while the upper
limit is shifted to lower frequencies by 1–17 cm1. This picture
may be tentatively explained in terms of the highly mobile p-elec-
trons within the thiazole and imidazole conjugated systems upon
the coordination through the endocyclic nitrogen.8,21 The IR peaks
of all complexes are almost similar, indicating that the complexes
may have similar structures.
2.1.2. UV–vis spectral studies
The band maxima of the electronic excitation spectra of the
complexes in DMSO solution were studied in the visible and UV re-
gions and are given in the experimental part. The spectra of com-
plexes 1–3 exhibit a broad absorption band of low intensity
(e = 38–45 M1 cm1) in the range 622–634 nm. This band could
be attributed to d?d transition for the central Ni(II), probably of
2
the dz ?dx2 y2 type.22 The band or shoulder at ca. 457 nm for all
complexes could be characterized as charge transfer band. The
transfer could take place from the metal to the dithiolate ligand
(metal-to-ligand charge transfer) a typically d(M)?p* (S2C@) tran-
sition (most probable) or, on the contrary, from the ligand to the
metal (ligand-to-metal charge transfer). This band is due to the
extensive conjugation of the dithiolate ligand and is red-shifted
in the spectra of the complexes, compared to that of the free ligand
(410 nm). Credence to the above arguments is given by the ob-
served strong shift of the IR m(CS2) band at higher wavenumbers
(vide supra). Finally, the band at 340 nm is due to internal transi-
tions of the ligands and can be attributed to n?p* or p?p* transi-
tions.23 The similarity of the position of these bands in the spectra
of the complexes should be noted, indicating similar chromophore
with different N-coordinated ligands.
2.1.3. Molar conductivity measurements
The molar conductivity KV values of the complexes in 103 V
DMSO solutions vary from 4 to 8 lS cm1, thus revealing their
non-electrolytic character.24
2.1.4. Magnetic measurements
The low magnetic measurement values (0.2 BM) of the Ni(II)
complexes shows that they are diamagnetic giving rise to a slightly
distorted square-planar arrangement. This is a natural conse-
quence of the low spin Ni(II) d8 configuration and the eight elec-
trons being paired, can occupy the four low lying d orbitals.
2.2. Description of the structure of [Ni(i-MNT)(2a-5mt)2]
The molecular structure of the [Ni(i-MNT)(2a-5mt)2] complex
along with the atomic numbering scheme is shown in Figure 1, se-
lected bond lengths and angles are given in Table 1.
The nickel atom is four-coordinated with the two sulfur atoms
of the dithiolate ligand (i-MNT) and the ring-nitrogen atoms of
the two 2a-5mt ligands giving rise to a slightly distorted square-
planar arrangement with the maximum deviation of the fitted
atoms from the mean S2N2 plane being 0.084(2) Å. The central
nickel atom lies in this plane within experimental error, its out-
of-plane distance being 0.011(1) Å. Severe distortion with respect
to the idealized square-planar geometry is observed in the coordi-
nation angles. The small S–Ni–S chelation angle of 78.85(2)° indi-
cates that it is determined by the geometry of the rigid i-MNT
ligand.19,20
On the other hand, as a result of the relatively small separation
of the coordinated nitrogen atoms of the flexible 2a-5mt ligands
(2.851 Å in comparison to 3.10 Å, which is the sum of their van
der Waals radii) the opposite N–Ni–N angle appears greater than
the ideal value of 90° [95.26(7)°]. The two Ni–S distances are very
close to each other and have a mean value of 2.189(1) Å, which is in
6056 P. J. Cox et al. / Bioorg. Med. Chem. 17 (2009) 6054–6062

cule in one layer and the N atoms of the i-MNT ligands of two other
molecules, lying either side of this layer.

2.3. Cyclic voltammetry for the complexes

The complete scan in the range 1.5 V to +1.0 V of complex 1 in

0.4 mM DMSO solution shows two cathodic waves at 720 mV and
1360 mV and three anodic waves at +90 mV, +565 mV and
+850 mV (Fig. 2A). These two one-electron cathodic waves suggest
that the reduction of the mononuclear complex 1 takes place in
two steps.31 In the reverse scan, one new cathodic wave at
+10 mV appears. Scanning in the range +1.0 to 1.0 V the anodic
wave at +80 mV disappears, confirming that this wave can be as-
signed to the process [Ni0]?[NiI]. Scanning in the range 1.0 to
+0.5 V the cathodic wave at +10 mV and the anodic wave at
+880 mV disappear, confirming that these waves can be assigned
to the couple Ni(II)/Ni(III).
Figure 1. The molecular structure and atom labeling of [Ni(i-MNT)(2a-5mt)2].
In 0.4 mM 1/2 DMSO/buffer solution, the waves are not distin-
guished as clear as in DMSO solution. The cyclic voltammogram
Table 1
Selective bond lengths (Å) and angles (°) for complex 1

Bond lengths (Å) Bond angles (°) A -200

Ni(1)–N(3) 1.9225(15) N(3)–Ni(1)–N(1) 95.26(7)
Ni(1)–N(1) 1.9368(15) N(3)–Ni(1)–S(3) 170.35(5) 0
Ni(1)–S(3) 2.1839(5) N(1)–Ni(1)–S(3) 93.76(5)
Ni(1)–S(4) 2.1934(5) N(3)–Ni(1)–S(4) 92.30(5)
S(3)–C(9) 1.7140(18) N(1)–Ni(1)–S(4) 172.07(5)
I (μA) 200
S(4)–C(9) 1.7200(18) S(3)–Ni(1)–S(4) 78.847(18)
N(5)–C(11) 1.148(2) C(9)–S(3)–Ni(1) 86.50(6) 400
N(6)–C(12) 1.149(2) C(9)–S(4)–Ni(1) 86.05(6)
C(9)–C(10) 1.383(3) C(10)–C(9)–S(3) 125.17(14)
C(10)–C(12) 1.422(3) C(10)–C(9)–S(4) 126.71(14) 600
C(10)–C(11) 1.424(3) S(3)–C(9)–S(4) 108.09(10)
800
1.0 0.5 0.0 -0.5 -1.0 -1.5

very good agreement with the corresponding ones observed in E (V)

other dithiolato complexes, having the Ni(II) atom in a square-pla-
nar coordination.25–28 The first 2a-5mt ring, comprising the atoms B -100
N1,C1,C2,C3 and S1 is strictly planar within the experimental error
(3r), the maximum deviation from the mean plane being -50
0.005(2) Å. On the other hand, the second thiazole ring, comprising
I (μA)

the atoms N3, C5, C6, C7 and S2, exhibits a small puckering, the 0
maximum deviation from the mean plane being 0.013(2) Å.
The i-MNT ligand is planar [maximum deviation of the fitted 50
atoms from their best plane: 0.087(2) Å], having the approximate
symmetry C2v and forming with the plane of the chromophore 100
NiS2N2 an angle of 10.20(3)°. The nickel—i-MNT ligand bond for-
mation does not affect the average C–S bond length or influences 150
0.5 0.0 -0.5 -1.0 -1.5
appreciably the C@CS2 interatomic distance in comparison with
E (V)
the observed ones in the free [S2C@C(CN)2]2 dianion.21,29,30 The
molecules are arranged in the unit cell having the approximate
mirror plane of the i-MNT ligand almost normal to the c-axis and C -200
are stacked almost normal to this axis. These layers are held to- -100
gether mainly through H-bonds (Table 2), formed between the H
atoms of the two amino groups of a [Ni(i-MNT)(2a-5mt)2] mole- 0
I (μA)

100

200
Table 2
300
Hydrogen bonds for complex 1 (Å and °)

D–HA d(D–H) (Å) d(HA) (Å) d(DA) (Å) \(DHA) (°) 400

N(2)–H(2A)N(6)#1 0.88 2.16 3.010(3) 163.3 500

N(2)–H(2B)N(5)#2 0.88 2.19 3.057(2) 167.3 1.0 0.5 0.0 -0.5 -1.0 -1.5
N(4)–H(4A)N(5)#3 0.88 2.24 3.018(3) 146.9 E (V)
N(4)–H(4B)N(6)#4 0.88 2.17 3.009(2) 158.0
Figure 2. Cyclic voltammogram of 0.4 mM DMSO solution of (A) [Ni(i-MNT)(2a-
Symmetry transformations used to generate equivalent atoms: #1 x, y, z + 2, 5mt)2], 1, (B) [Ni(i-MNT)(2a-2tzn)2], 2 and (C) [Ni(i-MNT)(Im)2], 3. Scan rate = 100
#2 x + 1/2, y + 1/2, z + 5/2, #3 x + 1, y, z + 2, #4 x + 1/2, y + 1/2, z + 3/2. mV s1. Supporting electrolyte = TEAP, 0.1 M.
 P. J. Cox et al. / Bioorg. Med. Chem. 17 (2009) 6054–6062 6057

of complex 1 shows the existence of two cathodic waves at Table 3

850 mV ([NiII]?[NiI]) and 1400 mV ([NiI]?[Ni0]) and three ano- Redox couples of the complexes and their cathodic and anodic potentials (in mV) in
DMSO and in 1/2 DMSO/buffer solution in the absence and presence of CT DNA
dic waves at 670 mV ([Ni0]?[NiI]), 290 mV ([NiI]?[NiII]) and
+770 mV ([NiII]?[NiIII]), while a third cathodic wave attributed to Ni(II)/Ni(I) Ni(I)/Ni(0) Ni(II)/Ni(III)
[NiIII]?[NiII] process appears at +20 mV. However, the current Epc1 Epa1 Epc2 Epa2 Epc3 Epa3
intensities attributed to the couple Ni(II)/Ni(I) are much higher 1 720a +565a 1360a +90a +850a +10a
than the others which are sometimes difficult to distinguish. Scan- 850b 290b 1400b 670b +770b +20b
ning in the range +1.0 to 1.0 V, the waves attributed to the pro- 825c 360c
I 0
cess ½Ni ¡½Ni  disappear once again (inset in Fig. 3A). +25d 70d

Quite similar is the redox behavior of complexes 2 and 3 2 850a +570a 1030a +195a +1260a +30a
(Fig. 2B–C and insets in Fig. 3B–C) and the corresponding redox 870b 410b n.d.b,e n.d.b,e +420b +40b
750c 485c
potentials are given in Table 3. Considering all the above processes
+120d 75d
3 930a +540a 1340a +130a +1430a +10a
705b 480b 1480b 640b n.d.b,e n.d.b,e
700c 510c
A -200
+5d 30d
a
-200 In 0.4 mM DMSO solution.
I (μA)

0 b
In 0.4 mM DMSO/buffer (1/2) solution (Efpc=a ).
c
In presence of CT DNA (Ebpc=a ) in 0.4 mM DMSO/buffer (1/2) solution.
-150 200 d
DEpc=a ¼ Ebpc=a  Efpc=a .
e
n.d. = not determined.
-100 400
I (μA)

1.0 0.5 0.0 -0.5 -1.0

E(V)
-50
and taking into account the literature,32–34 the complete pattern of
0
the redox behavior of complexes 1–3 could be described in the fol-
lowing equations:
50
0.0 -0.2 -0.4 -0.6 -0.8 -1.0
Epc1 Epc2
E (V)
[NiII] + e [NiI] [Ni0]
-400
B e
-200
Epa1 Epa3
I (μA)

-200 0 Epa2
[Ni0] [NiI] [NiII] [NiIII]
200

-100 400 +e +e +e
I (μA)

1.0 0.5 0.0 -0.5 -1.0

E (V)
Epc3
0 [NiIII] + e [NiII]

100
2.4. Interaction with DNA
0.0 -0.2 -0.4 -0.6 -0.8 -1.0
E (V) Transition metal complexes can bind to DNA via covalent and/
or noncovalent interactions.35,36 In covalent binding mode, a labile
C -400 ligand of the complexes is usually replaced by a nitrogen base of
-200 DNA such as guanine N7. On the other hand, the noncovalent
-200
DNA interactions include intercalative, electrostatic and groove
I (μA)

0
(surface) binding of metal complexes along outside of DNA helix,
200 along major or minor groove.36
-100
400
1.0 0.5 0.0 -0.5 -1.0
I (μA)

E (V)
2.4.1. Spectroscopic study of the DNA-binding
0 Electronic absorption spectroscopy is an effective method to
examine the binding mode of DNA with metal complexes.37 The
absorption spectra of the interaction of CT DNA with the com-
100 plexes have been recorded for a constant CT DNA concentration
in different [complex]/[DNA] mixing ratios (r) values and they
are shown in Figure 4. The changes observed in the absorption
0.0 -0.2 -0.4 -0.6 -0.8 -1.0
E (V) spectra of CT DNA in the presence of complexes 1–3, that is, the de-
crease of the intensity at kmax = 258 nm, which, for complexes 2
Figure 3. Cyclic voltammogram of 0.4 mM 1/2 DMSO/buffer (containing 150 mM and 3, is accompanied with a red-shift of the kmax up to 266 nm,
NaCl and 15 mM trisodium citrate at pH 7.0) solution of (A) [Ni(i-MNT)(2a-5mt)2] 1, indicate that the interaction with CT DNA results in the direct for-
(B) [Ni(i-MNT)(2a-2tzn)2] 2 and (C) [Ni(i-MNT)(Im)2], 3 in the absence (insets) or
presence of CT DNA in increasing amounts. The arrows show the changes upon
mation of a new complex with double-helical CT DNA,38 which
increasing amounts of CT DNA. Scan rate = 100 mV s1. Supporting electrolyte = simultaneously may cause the slight change of the conformation
buffer solution. of DNA39 in the case of complexes 2 and 3. The observed hypochro-
6058 P. J. Cox et al. / Bioorg. Med. Chem. 17 (2009) 6054–6062

A A

{[DNA]/(εa-εf)} x 10 , (M cm)
1.0 4

2
0.8 0.6 3

9
0.6 2
A

0.4
0.4 1
0 5 10 15 20 25
6

A
[DNA] x10 , (M)
0.2
0.2

0.0
250 300 350
λ (nm)
0.0
300 350 400 450 500
B 2.0 λ (nm)

{[DNA]/(εa-εf)} x10 , (M cm)

1.5 B

2
9
1.0 1.6
A

0.6
1.4
0.5 0.5
1.2
0.4
1.0
0.0 0 5 10 15 20 25
0.3
A
250 300 350 [DNA] x 10 , (M)
6

λ (nm)
0.2

C 2.0
0.1

0.0
1.5 300 350 400 450 500
λ (nm)

1.0
A

{[DNA]/(εa-εf)} x10 , (M cm)

C
2

2.4
1.5
0.5
9

2.0

0.0 1.0 1.6

250 300 350
λ (nm) 1.2
A

Figure 4. UV spectra of CT DNA in buffer solution (150 mM NaCl and 15 mM 5 10 15 20 25 30

0.5 6
trisodium citrate at pH 7.0) in the presence of (A) [Ni(i-MNT)(2a-5mt)2], 1 [DNA]x10 , (M)
([DNA] = 0.146 mM), (B) [Ni(i-MNT)(2a-2tzn)2], 2 ([DNA] = 0.164 mM) and (C)
[Ni(i-MNT)(Im)2], 3 ([DNA] = 0.264 mM) at diverse r values (r = [complex]/[DNA])
(r = 0–0.08). The arrows show the intensity changes upon increasing concentration
0.0
of complexes. 300 350 400 450 500
λ (nm)

mism is due to stacking interaction between the chromophores of
the complexes and DNA base pairs probably consistent with the
intercalative binding mode.37,40 Additionally, the observed red-
shift of 8 nm is an evidence of the stabilization of the CT DNA
duplex.41
Figure 5 illustrates the spectral changes occurred in DMSO solu-
tion of complexes 1–3 during the titration upon addition of
increasing amounts of CT DNA. The initial spectrum refers to the
spectrum of a fixed concentration (4  105 M) of the free complex
in the absence of CT DNA. In the UV region, the intense absorption
bands observed in the spectra of the complexes are attributed to
the intraligand p?p* transition of the coordinated groups of corre-
sponding ligands.36
Even though no appreciable change in the position of the intral-
igand band of the complex is observed by addition of CT DNA, the
intensity of the band of 1 centered at 341 nm has been found to in-
Figure 5. UV spectra of (A) [Ni(i-MNT)(2a-5mt)2], 1, (B) [Ni(i-MNT)(2a-2tzn)2], 2
and (C) [Ni(i-MNT)(Im)2], 3 in DMSO solution in the presence of CT DNA at
increasing amounts. [Complex] = 40 lM, [CT DNA] = 0-40 lM. The arrows show the
intensity changes upon increasing concentration of CT DNA. Insets: plots of ð½DNA
ea ef Þ
versus [DNA].
crease in the presence of CT DNA, resulting in hyperchromism
(Fig. 5A). The hyperchromic effect observed might be ascribed to
external contact (electrostatic binding)42 or that the complex could
uncoil the helix structure of DNA and made more bases embedding
in DNA exposed.43 On the other hand, the intensity of the band of 1
centered at 455 nm has been found to initially decrease slightly in
the presence of DNA, resulting in a slight hypochromism (Fig. 5A).
Furthermore, two distinct isosbestic points at 445 nm and 465 nm
exist in the spectrum and accompany the hypochromism as the
DNA concentration increases.
 P. J. Cox et al. / Bioorg. Med. Chem. 17 (2009) 6054–6062
For complex 2, the intensity of all the observed bands (293 nm,
343 nm, 373 nm and 455 nm) increase in the presence of CT DNA,
resulting in hyperchromism (Fig. 5B). Additionally, a blue-shift of
5 nm is observed for bands centered at 293 nm (up to 288 nm)
and 373 nm (up to 368 nm) suggesting a conformational change
of the DNA duplex.41 In the case of 3, hyperchormism is observed
for both the observed bands at 279 nm and 374 nm (Fig. 5C), while
for the latter a slight blue-shift of 3 nm (to 371 nm) is also present.
The above experimental results derived from the UV titration
experiments suggest that all the complexes can bind to CT DNA,
maybe by electrostatic interaction.44 Nevertheless, the existing li-
gands in combination with the formed chelate rings provide an ex-
tended aromatic moiety to the complexes so their simultaneous
intercalative binding mode to the base pairs of DNA may not be ru-
led out.45
The binding strength of the complexes with CT DNA is mirrored
in the intrinsic binding constant Kb, which represents the binding
constant per DNA base pair and can be obtained by monitoring
the changes in the absorbance at 341 nm for 1, 343 nm for 2 and
374 nm for 3, with increasing concentrations of CT DNA, according
to the following equation (Eq. (1)):44
½DNA ½DNA 1
¼ þ ð1Þ
ðea  ef Þ ðeb  ef Þ K b ðeb  ef Þ
where [DNA] is the concentration of DNA in base pairs, the apparent
absorption coefficients ea, ef and eb correspond to Aobsd/[complex], the
extinction coefficient for the free complex and the extinction
coefficient for the complex in the fully bound form, respectively. In
plots ð½DNA
ea e Þ versus [DNA], Kb is given by the ratio of the slope to the y
f
intercept (insets in Fig. 5). The determined Kb values for complexes
1–3 are given in Table 4. These values suggest a relatively strong bind-
ing of the complexes to CT DNA and are lower than the EB binding
affinity for CT DNA, (Kb = 1.23 ± 0.07  105 M1 bp1),46 suggesting
that electrostatic and/or intercalative interaction may affect EB
partial displacement.33,34,46,47 The intrinsic binding constant (Kb)
obtained for complex 3 (=4.94 ± 0.05  104 M1) is higher than the
other two complexes indicating its stronger ability to bind to CT
DNA.38
2.4.2. Electrochemical study of the DNA-binding
The electrochemical investigations of metal–DNA interactions
can provide a useful complement to spectroscopic methods, for
example, for non absorbing species, and yield information about
interactions with both the reduced and oxidized form of the me-
tal.48 The electrochemical potential of a small molecule will shift
positively when it intercalates into DNA double helix, while a neg-
ative shift of the potential is observed for an electrostatic interac-
tion to DNA.49,50 Additionally, in the case of more potentials than
one, a positive shift of Ep1 and a negative shift of Ep2 could imply
that the molecule can bind to DNA by both intercalation and elec-
trostatic interaction.38,51
In Figure 3, the cyclic voltammograms of the complexes in the
presence of CT DNA in diverse r values are shown. The quasi-
reversible redox couple Ni(II)/N(I) for each complex in 1/2
DMSO/buffer solution has been studied upon addition of CT DNA
and the shifts of the corresponding cathodic Epc and anodic Epa
Table 4
The intrinsic binding constants (Kb) of complexes 1–3 with CT DNA and the Stern–
Volmer constants (KSV)
Complex Kb (M1) KSV (M1)
4
1 2.94(±0.28)  10 5.39(±0.32)  105
2 1.68(±0.20)  104 4.83(±0.29)  105
3 4.94(±0.05)  104 4.29(±0.28)  105
6059
potentials are given in Table 3. No new redox peaks appeared after
the addition of CT DNA to each complex, but the current intensity
of all the peaks decreased significantly, suggesting the existence of
an interaction between each complex and CT DNA. The decrease in
current intensity can be explained in terms of an equilibrium mix-
ture of free and DNA-bound complex to the electrode surface.52
It can be observed (Table 3) that complexes 1–3 exhibit similar
electrochemical behavior upon addition of CT DNA at diverse r val-
ues. For increasing amounts of CT DNA, the cathodic potential Epc
shows a positive shift (DEpc = +25 mV for 1, DEpc = +120 mV for 2
and DEpc = +5 mV for 3) while the anodic potential Epa shifts to
more negative values (DEpa = 70 mV for 1, DEpa = 75 mV for 2
and DEpa = 30 mV for 3) ( Fig. 3). These shifts of the potentials
show that complexes 1, 2 and 3 can bind to DNA by both interca-
lation and electrostatic interaction.33,34,50,51
2.4.3. Competitive DNA-binding studies with EB
A competitive ethidium bromide (EB) binding study has been
undertaken with fluorescence spectroscopic titration46,47,51 in or-
der to investigate if the complexes could displace EB from its EB-
DNA complex. EB (=3,8-diamino-5-ethyl-6-phenylphenanthridini-
um bromide), a phenenthridine fluorescence dye, is a typical indi-
cator of intercalation,53 forms soluble complexes with nucleic acids
and emits intense fluorescence in the presence of CT DNA due to
the intercalation of the planar phenenthridinium ring between
adjacent base pairs on the double helix.54
No fluorescence has been observed for the complexes at room
temperature in solution or in the presence of CT DNA and the bind-
ing of the complexes and DNA cannot be directly predicted through
the emission spectra. Thus, competitive EB binding studies have
been performed in order to gain support for the extent of binding
of each complex with DNA. EB does not show any appreciable
emission in buffer solution due to fluorescence quenching of the
free EB by the solvent molecules55 while in the presence of CT
DNA, the fluorescence intensity of EB is highly enhanced due to
its strong intercalation between the adjacent DNA base pairs.56
Addition of a second molecule, which can also bind to DNA, can de-
crease the DNA-induced EB emission.57 Two mechanisms have
been proposed to account for this reduction in the emission inten-
sity: the replacement of molecular fluorophores (EB in this case)
and/or electron transfer.58
The emission spectra of EB bound to CT DNA in the absence and
presence of each complex have been recorded for [EB] = 2
 105 M, [DNA] = 2.6  105 M and increasing amounts of each
complex. The emission band at 592 nm of the DNA-EB system
decreased in intensity upon addition of each complex at diverse r
values. This decrease of EB fluorescence (up to 20% of the initial
EB-DNA fluorescence intensity) (Fig. 6A) indicates the competition
of the complexes with EB in binding to DNA. The quenching of
DNA–EB fluorescence for complexes 1–3 suggests that they are
able to displace EB from the DNA–EB complex and they can inter-
act with CT DNA probably by the intercalative mode.47,59
The quenching efficiency for each complex is evaluated by the
Stern–Volmer constant KSV, which varies with the experimental
conditions according to Eq. (2):
Io
¼ 1 þ K SV ½complex ð2Þ
I
where Io and I are the emission intensities in the absence and the
presence of the complex, respectively. Figure 6B shows the Stern–
Volmer plots for the complexes and the KSV values calculated for
the complexes are shown in Table 4. The Stern–Volmer plot of
DNA–EB illustrates that the quenching of EB bound to DNA by each
complex is in good agreement (R = 0.99) with the linear Stern–Vol-
mer equation (Eq. (2)), which proves that the partial replacement of
EB bound to DNA by each complex results in a decrease in the fluo-
6060 P. J. Cox et al. / Bioorg. Med. Chem. 17 (2009) 6054–6062

A 100 4. Experimental

80 4.1. Materials and instrumentation

1
% EB fluorescence

2
60 4.1.1. Materials
3
All chemicals were purchased from Aldrich, EDH Chemicals and
40 Merck and were used as received. CT DNA and EB were purchased
from Sigma, NaCl and all solvents were purchased from Merck, tri-
20 sodium citrate was purchased from Riedel-de Haen. All the chem-
icals and solvents were reagent grade and were used as purchased.
0 Tetraethylammonium perchlorate (TEAP) was purchased from Car-
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 lo Erba and, prior to its use, it was recrystallized twice from ethanol
r and dried under vacuum.
DNA stock solution was prepared by dilution of CT DNA to buffer
B 6 (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0)
followed by exhaustive stirring at 4 °C for three days, and kept at
5 4 °C for no longer than a week. The stock solution of CT DNA gave
a ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.89, indi-
4 cating that the DNA was sufficiently free of protein contamination.60
The DNA concentration was determined by the UV absorbance at
Io/I

3 1 260 nm after 1:20 dilution using e = 6600 M1 cm1.61

2 3
1 Elemental analysis was performed on a Perkin–Elmer 240B ele-
0 2 4 6
6 with a Perkin–Elmer model 403 Atomic Absorption Spectrometer.
[Complex] x 10 , (M)
Figure 6. (A) Plot of EB relative fluorescence intensity (%) versus r (=[complex]/
[DNA]) for complexes 1–3 in buffer solution (150 mM NaCl and 15 mM trisodium
citrate at pH 7.0). (B) Stern–Volmer quenching plot of EB bound to CT-DNA by
complexes 1–3. [EB] = 20 lM, [DNA] = 26 lM, kem = 592 nm.
rescence intensity.52 The high KSV values of the complexes show
that they can be bound very tightly to the DNA.33,34,45–47 Among
the three complexes, 1 has the highest Ksv suggesting its stronger
ability to displace EB from the EB–DNA complex than the other
two complexes.
3. Conclusions
The mixed-ligands Ni(II) complexes afforded by the reaction of
[Ni(L)4Cl2] with dianion of 1,1-dicyano-2,2-ethylenedithiolate li-
gand (i-MNT). The crystal and molecular structures of the studied
Ni(II) compounds reveal the same distorted square-planar geome-
try for all complexes. The (i-MNT) ligand is coordinated with the
two sulfur atoms and the endocyclic nitrogen atom of the two
2a-5mt or 2a-2tzn or Im ligands. The isolated neutral, diamagnetic
and of square-planar geometry complexes are of interest, since
they meet the necessary properties for various biological tests. In
the cyclic voltammograms of the complexes recorded in DMSO
solution and in 1/2 DMSO/buffer solution, the expected waves
attributed to redox couples and the corresponding potentials, char-
acteristic for Ni(II) complexes have been recorded.
The study of the interaction of the complexes with CT DNA has
been performed with UV spectroscopy and cyclic voltammetry and
has revealed that the complexes can bind to DNA. UV spectroscopic
titrations have been used in order to calculate the binding strength
of the complexes with CT DNA which is mirrored in the intrinsic
binding constant Kb. Cyclic voltammetric studies have shown that
complexes 1–3 can bind to CT DNA by both intercalation and elec-
trostatic interaction. Competitive binding studies with EB with
fluorescence spectroscopy have shown that the interaction be-
tween DNA–EB complex and the complexes can release EB from
its DNA complex, indicating that they can bind to DNA probably
via the intercalative mode.
2
4.1.2. Instrumentation—physical measurements
mental analysis instrument, whereas Ni analyses were performed
8 10
Molar conductivities were measured on a WTW conductivity bridge
employing a calibrated dip type cell and 103 M solutions in DMSO.
Magnetic susceptibility measurements of powdered samples were
performed at room temperature (25 °C) utilizing a Johnson–Mat-
they balance, calibrated with Hg[Co(NCS)4]. Infrared spectra were
recorded throughout the 4000–225 cm1 region, with samples pre-
pared as KBr or polyethylene discs, with a Perkin–Elmer FT-IR Spec-
trum One Spectrophotometer equipped with a CsI beam splitter.
UV–vis (UV–vis) spectra were recorded in DMSO solution at con-
centrations in the range 105–103 M on a Hitachi U-2001 dual
beam spectrophotometer. Fluorescence spectra were recorded in
solution on a Hitachi F-7000 fluorescence spectrophotometer.
Cyclic voltammetry studies were performed on an Eco chemie
Autolab Electrochemical analyzer. Cyclic voltammetric experi-
ments were carried out in a 30 mL three-electrode electrolytic cell.
The working electrode was platinum disk, a separate Pt single-
sheet electrode was used as the counter electrode and a Ag/AgCl
electrode saturated with KCl was used as the reference electrode.
The cyclic voltammograms of the complexes were recorded in
0.4 mM DMSO solutions and in 0.4 mM 1/2 DMSO/buffer solutions
at m = 100 mV s1 where TEAP and the buffer solution were the
supporting electrolytes, respectively. Oxygen was removed by
purging the solutions with pure nitrogen previously saturated with
solvent vapors. All electrochemical measurements were performed
at 25.0 ± 0.2 °C.
4.2. Synthesis of the dithiolate ligands
The disodium salt of 1,1-dicyano-2,2-ethylenedithiolate trihy-
drate Na2(i-MNT)3H2O (Scheme 1) was prepared by established
procedures.62–64
NaS C N
C C 3H2O
NaS C N
Na2(i–MNT)·3H2O
Scheme 1. Structure of disodium salt of 1,1-dicyano-2,2-ethylenedithiolate trihy-
drate ligand.
 P. J. Cox et al. / Bioorg. Med. Chem. 17 (2009) 6054–6062 6061

N N Table 5
N
Crystal data and structure refinement for complex 1
H3C NH2 NH2
S S NH Molecular formula C12H12N6NiS4
Formula weight 427.23
2a–5mt 2a–2tzn Im Temperature (K) 120(2)
Wavelength (Å) 0.71073
Scheme 2. Structural formulae of 2-amino-5-metyl-thiazole, 2-amino-2-thiazoline Crystal system Monoclinic
and Imidazole. Space group P21/n
a (Å) 8.1524(2)
4.3. Synthesis of the starting compounds b (Å) 14.0906(3)
c (Å) 15.5424(2)
a (°) 90
The starting compounds used [Ni(L)4Cl2] were prepared by the b (°) 95.3390(10)
reaction of NiCl26H2O with L = 2a-5mt, 2a-2tzn and Im in 1:4 mo- c 90
lar ratio.65 The formulae of the 2-amino-5-methyl-thiazole, 2-ami- Volume (Å3) 1777.64(6)
no-thiazoline and imidazole are given in Scheme 2. Z 4
Density (calcd) (Mg/m3) 1.596
The mixed-ligand chelates of the general type [Ni(i-MNT)(L)2]
Absorption coefficient (mm1) 1.566
were prepared by treating the starting compounds [Ni(L)4Cl2] with F(0 0 0) 872
dithiolate i-MNT in 1:1 molar ratio in methanol.19 A representative Crystal size (mm3) 0.10  0.04  0.02
reaction of the synthesis of [Ni(i-MNT)(2a-5mt)2] is given in h range for data collection (°) 3.00–27.48
Index ranges 10 6 h 6 10, 18 6 k 6 18,
Scheme 3.
20 6 l 6 20
Reflections collected 27,062
4.4. Synthesis of [Ni(i-MNT)(2a-5mt)2], 1 Independent reflections 4069 [R(int) = 0.0482]
Completeness to h = 27.48° 99.8%
A solution of [Ni(2a-5mt)4Cl2] (0.699 g, 1 mmol) in 20 mL MeOH Max. and min. transmission 0.9693 and 0.8591
Refinement method Full-matrix least-squares on F2
was mixed slowly with a solution of Na2(i-MNT) (0.24 g, 1 mmol) in
Data/restraints/parameters 4069/0/210
10 mL MeOH. After 1 h of stirring the resulting dark-brown solid was Goodness-of-fit on F2 1.017
collected by filtration, washed with methanol three times and ether Final R indices [I > 2r(I)] R1 = 0.0278, wR2 = 0.0571
and dried in air. Weight: 0.304 g. Yield: 72%. Slow evaporation of R indices (all data) R1 = 0.0428, wR2 = 0.0612
Final weighting scheme calcd w ¼ 1=½r2 ðF 2o Þ þ ð0:0250PÞ2
[Ni(i-MNT)(2a-5mt)2] solution in CH3CN/CH2Cl2 at low temperature
þ0:7523P, where P ¼ ðF 2o þ 2F 2c Þ=3
(4 °C) yielded brown crystals suitable for X-ray diffraction analysis. Largest diff. peak and hole (e Å3) 0.290 and 0.299
Anal. Calcd for C12H12N6S4Ni: C, 33.74; H, 2.83; N, 19.67; Ni, 13.74.
Found: C, 33.68; H, 2.83; N, 19.65; Ni, 13.68. IR: mmax/cm1;
m(C„N): 2211(vs); m(C@CS2): 1431(vs); mas(CS2): 941(m); ms(CS2):
813(m); m(Ni–N): 478(w); m(Ni–S): 392(w) (KBr disk); UV–vis: C10H8N6S2Ni: C, 35.85; H, 2.41; N, 25.08; Ni, 17.52. Found: C,
kmax/nm (log e) (in DMSO): 622 (1.64), 457 (4.10), 340 (4.40). 35.81; H, 2.44; N, 24.95; Ni, 17.49. IR: mmax/cm1; m(C„N):
2228(s); m(C@C): 1376(vs); mas(CS2): 916(w); ms(CS2): 898(w);
4.5. Synthesis of [Ni(i-MNT)(2a-2tzn)2], 2 m(Ni–N): 486(w); m(Ni–S): 387(w) (KBr disk); UV–vis: kmax/nm
(log e) (in DMSO): 634 (1.25), 456 (4.12), 340 (4.41).
Complex 2 was obtained by slow addition of Na2(i-MNT)3H2O
(0.24 g, 1 mmol) in MeOH (20 mL) to a solution of [Ni(2a-2tzn)4Cl2] 4.7. DNA-binding studies
(0.538 g, 1 mmol) in MeOH (20 mL). The brown solid was filtered,
washed successively with 5 mL of MeOH and Tt2J and dried in The interaction of complexes 1–3 with CT DNA has been studied
air. Weight: 0.26 g, Yield: 65%. Anal. Calcd for C10H12N6S4Ni: C, with UV spectroscopy and CV in order to investigate the possible
29.79; H, 3.00; N, 20.84; Ni, 14.56. Found: C, 29.81; H, 2.91; N, binding modes to CT DNA and to calculate the binding constants
20.80; Ni, 14.35%. IR: mmax/cm1; m(C„N): 2203(vs); m(C@CS2): to CT DNA (Kb). In UV titration experiments, the spectra of CT
1434(vs); mas(CS2): 936(w); ms(CS2): 901(w); m(Ni–N): 481(w); DNA in the presence of each complex have been recorded for a con-
m(Ni–S): 396(w) (KBr disk); UV–vis: kmax/nm (log e) (in DMSO): stant CT DNA concentration in diverse [complex]/[CT DNA] mixing
625 (1.57), 457 (4.26), 341 (4.53). ratios (r). The intrinsic binding constants, Kb, of the complexes with
CT DNA have been determined using the UV spectra of the com-
4.6. Synthesis of the complex [Ni(i-MNT)(Im)2], 3 plexes recorded for a constant concentration in the absence or
presence of CT DNA for diverse r values. Control experiments with
Complex 3 was prepared by gradual addition of Na2(i- DMSO were performed and no influence of the spectra was
MNT)3H2O (0.24 g, 1 mmol) in MeOH (20 mL) to a solution of observed.
[Ni(Im)4Cl2] (0.40 g, 1 mmol) in MeOH (20 mL). The brown solid The interaction of the complexes with CT DNA has been also
was isolated by filtration, washed with 5 mL of MeOH and Tt2J investigated by monitoring the changes observed in the cyclic vol-
and dried in air. Weight: 0.20 g, Yield: 60%. Anal. Calcd for tammogram of a 0.40 mM 1/2 DMSO/buffer solution of the com-

CH3
S
H 2N
N S C N
[Ni(2a-5mt)4Cl2] + Na2(i-MNT).3H2O Ni C C
N S C N

H3C + 2NaCl + 2a-5mt +3H2O

S NH2

Scheme 3. Representative reaction of the synthesis of [Ni(i-MNT)(2a-5mt)2].

6062 P. J. Cox et al. / Bioorg. Med. Chem. 17 (2009) 6054–6062
plexes upon addition of CT DNA at diverse r values. The buffer was
also used as the supporting electrolyte and the cyclic voltammo-
grams were recorded at m = 100 mV s1.
The competitive studies of each complex with EB have been
investigated with fluorescence spectroscopy in order to examine
whether it is able to displace EB from its CT DNA–EB complex.
The CT DNA–EB complex was prepared by adding 20 lM EB and
26 lM CT DNA in buffer (150 mM NaCl and 15 mM trisodium cit-
rate at pH 7.0). The intercalating effect of complexes 1–3 with
the DNA–EB complex was studied by adding a certain amount of
a solution of the compound step by step into the solution of the
DNA–EB complex. The influence of the addition of each compound
to the DNA–EB complex solution has been obtained by recording
the variation of fluorescence emission spectra.
4.8. Crystal structure determination of [Ni(i-MNT)(2a-5mt)2]
Crystal data, data collection and refinement details are given in
Table 5. X-ray intensity data were collected at 120 K on a Bruker-
Nonius diffractometer graphite monochromated MoKa radiation.
A total of 27062 reflections were measured (3.00° < h < 27.48°)
which were further merged to 4069 independent reflections
(Rint = 0.0482), of which 3304 were considered as observed
[I > 2r(I)]. Multi-scan absorption corrections were made with the
program SADABS.66 The structure was solved with direct methods
using the program67 SIR97 and refined by full-matrix least-squares
refinement with the program SHELXL97,68 while geometric calcula-
tions were performed with the program PLATON.69
Acknowledgements
We thank Professor P.C. Christidis for his valuable discussion.
Supplementary data
Complete lists of atomic coordinates, anisotropic displacement
parameters, bond lengths and angles, hydrogen bonds have been
deposited at the Cambridge Crystallographic Data Centre [CCDC
No. 716372], 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44
123 336033; e-mail: deposit@ccdc.cam.ac.uk or www://www.ccdc.
cam.ac.uk). Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2009.06.058.
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