Heterocyclic substituted thiosemicarbazones and their Cu(II) complexes: Synthesis, characterization and studies of substituent effects on coordination and DNA binding

Article abstract of DOI:10.1016/j.poly.2007.12.034

By reacting thiosemicarbazides substituted on the aminic nitrogen with 5-formyluracil, several new 5-formyluracil thiosemicarbazones (H₃ut) derivatives were synthesised and characterized. These ligands, treated with copper chloride and nitrate, afforded two different kinds of compounds. In the complexes derived from copper chloride the metal atom is pentacoordinated, being surrounded by the neutral ligand binding through SNO donor atoms and by two chlorines, while the nitrate derivatives consist of monocations and nitrate anions. The copper coordination (4 + 2) involves the SNO ligand atoms, two water oxygens and an oxygen atom of a monodentate nitrate group. On varying the substituents on the thiosemicarbazidic moiety, remarkable modifications of the coordination geometry are not observed for the complexes with the same counterion. For all the compounds, interactions with DNA (calf thymus) were studied using UV-Vis spectroscopy; the nuclease activity was verified on plasmid DNA pBR 322 by electrophoresis.

Full text of DOI:10.1016/j.poly.2007.12.034

Available online at www.sciencedirect.com
Polyhedron 27 (2008) 1361–1367
www.elsevier.com/locate/poly
Heterocyclic substituted thiosemicarbazones and their
Cu(II) complexes: Synthesis, characterization and studies
of substituent effects on coordination and DNA binding
Marisa Belicchi-Ferrari, Franco Bisceglie *, Giorgio Pelosi, Pieralberto Tarasconi
Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Viale G. P. Usberti 17/A, Campus Universitario,
`
Universita degli Studi di Parma, 43100 Parma, Italy
Received 4 July 2007; accepted 26 December 2007
Available online 7 March 2008
Abstract
By reacting thiosemicarbazides substituted on the aminic nitrogen with 5-formyluracil, several new 5-formyluracil thiosemicarbazones
(H3ut) derivatives were synthesised and characterized. These ligands, treated with copper chloride and nitrate, afforded two different
kinds of compounds. In the complexes derived from copper chloride the metal atom is pentacoordinated, being surrounded by the neu-
tral ligand binding through SNO donor atoms and by two chlorines, while the nitrate derivatives consist of monocations and nitrate
anions. The copper coordination (4 + 2) involves the SNO ligand atoms, two water oxygens and an oxygen atom of a monodentate
nitrate group. On varying the substituents on the thiosemicarbazidic moiety, remarkable modifications of the coordination geometry
are not observed for the complexes with the same counterion. For all the compounds, interactions with DNA (calf thymus) were studied
using UV–Vis spectroscopy; the nuclease activity was verified on plasmid DNA pBR 322 by electrophoresis.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Thiosemicarbazones; Copper(II) complexes; DNA interactions
1. Introduction
5-fluoro-20-deoxyuridine-50-O-monophosphate (F-dUMP),
inhibits thymidylate synthase, resulting in the depletion
of thymidine triphosphate (TTP), a necessary constituent
of DNA. Other fluorouracil metabolites incorporate into
both RNA and DNA; incorporation into RNA results in
major effects on both RNA processing and functions.
The study of prodrugs of 5-fluorouracil shows that also
its derivatives have a high potential antitumor effect [20]
and their study is important in order to overcome the limits
and side effects of the parent drug.
On this basis, it is supposed that other modified uracil
derivatives are molecules with potential biological proper-
ties. In particular in recent years we have already worked
on 5-formyluracil thiosemicarbazone, and we have found
that its complexes show appreciable biological activity
[21–23]. In these compounds in fact a cooperative effect
between the thiosemicarbazide and uracil moieties seems
to improve the properties of the individual molecules.
Heterocyclic aromatic thiosemicarbazones show a wide
range of biological properties [1–18]. Among the heterocy-
clic compounds, fluorouracil shows by itself a remarkable
antitumor activity and is in use in medical practice [19];
5-fluorouracil (5FU) is an antimetabolite fluoropyrimidine
analog of the nucleoside pyrimidine with antineoplastic
activity. Fluorouracil and its metabolites possess a number
of different mechanisms of action. In vivo it is converted
into the active metabolite 5-fluoroxyuridine monophos-
phate (F-UMP); replacing uracil, F-UMP incorporates
into RNA and inhibits RNA processing, thereby inhibiting
cell growth. Another among its active metabolites,
*
Corresponding author. Tel.: +39 0521 905 420; fax: +39 0521 905 557.
E-mail address: franco.bisceglie@unipr.it (F. Bisceglie).
0277-5387/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2007.12.034

1362
M. Belicchi-Ferrari et al. / Polyhedron 27 (2008) 1361–1367
S analyses were obtained with a Carlo-Erba 1108 instru-
Compound
Et-H3ut
R
ment. IR spectra were recorded using KBr pellets in the
4000–400 cmꢁ1 range and using CsI pellets in the 650–
150 cmꢁ1 range on a Nicolet 5PC FTIR spectrophotome-
ter. Calf thymus (CT) DNA was obtained from Serva
and was used as received. DNA samples were dissolved
in 50 mM NaCl/5 mM Tris, pH 7.2. A stock solution of
CT-DNA was prepared by dissolving the nucleic acid in
this buffer in order to obtain a ca. 10ꢁ5 M in base-pair,
[bp] solution that gave ratios of UV absorbance at 260
and 280 nm, A260/A280, of ca. 1.8, indicating that the
CT-DNA was sufficiently protein free. The concentration
of the nucleic acid solutions was determined by UV absor-
bance at 260 nm after 1:100 dilution of the stock solution.
The extinction coefficient e260 was taken as 13100
(1)
(2)
CH3-CH2-
CH2=CH-CH2-
H
Allyl-H3ut
R
N
H
N
Ph-H3ut
(3)
N
NH
S
O
N
H
O
MePh-H3ut (4)
Scheme 1. Chemical drawing of the R-H3ut ligands.
Moreover, uracil provides oxygens as additional potential
coordinating sites to the thiosemicarbazidic chain.
The present paper deals with the syntheses and charac-
terization of four 5-formyluracil thiosemicarbazones
(H3ut) with substituents on the aminic nitrogen of the
thiosemicarbazide chain. The introduction of the different
groups: ethyl-, allyl-, phenyl-, methylphenyl-groups (Scheme 1)
is made in order to vary the hydrophilic–lipophilic
character of the compounds with the aim of increasing
the absorption of the drugs in cells.
M
ꢁ1 cmꢁ1 [33]. Stock solutions were stored at 4 °C and
used after no more than 4 days. TRIS and TRIS–
acetate–EDTA 10ꢂ buffers and agarose were purchased
from Sigma. pBR322 DNA plasmid (Roche), DirectLoad
Wide-Range DNA marker, ethidium bromide and gel load-
ing solution type I 6ꢂ concentrate were from Sigma.
Chemical ionization-mass (m/z, 70 eV) fragmentation
patterns were obtained, using samples dissolved in metha-
nol, with a Finnegan 1020 6c mass spectrometer equipped
with a quadrupole mass selector MATSSQ 710. Melting
points were determined with a Gallenkamp instrument.
UV measurements were performed on a Perkin–Elmer
UV/Vis Lambda 25 spectrometer with quartz cuvettes.
Thermogravimetric analyses were performed on a Perkin–
Elmer TGA 7 from 30 °C to 450 °C with a 20 °C/min
gradient in N2 atmosphere. Conductivity measurements
were performed on a Mayo International Srl digital
conductimeter with samples dissolved in absolute EtOH.
NMR spectra were recorded on a Bruker AC300 Spectro-
meter.
Substitutions on the terminal N position can also affect
the coordination and biological properties [5,6,10,11,24–
29]. Moreover, as small molecules, they could react at spe-
cific sites along a DNA strand through a series of weak
interactions [30]. Many anticancer drugs exert their antitu-
mor effects through binding to DNA in one way or
another, thereby blocking the replication of DNA and
inhibiting the growth of tumor cells. The understanding
of DNA binding and cleavage is the basis for the design
of new, efficient antitumor drugs, their effectiveness
depending on the binding mode and affinity towards
DNA [31,32]. The biological action mechanism of thiosem-
icarbazone complexes is not clear or well defined so far.
One of the proposed pathways is DNA oxidative damage
induced by the copper ion [1]. In this view the presence
of a moiety with a potential high DNA affinity could act
as a Trojan horse in order to anchor the active metal com-
plex to the target site. Copper complexes are also synthe-
sized and identified by means of different spectroscopic
and analytical techniques.
2.2. Synthesis of the ligands
5-Formyluracil thiosemicarbazones (1), (3) and (4) (R-
H3ut, Scheme 1) were synthesized by dissolving ca.
0.80 mmol of aldehyde in 20 mL of water at reflux temper-
ature and adding dropwise an equimolar amount of thio-
semicarbazide previously dissolved by refluxing in 20 mL
of the same solvent. To the 5-formyluracil solution a cou-
ple of drops of HCl were added as a catalyst. The mixture
was left under magnetic stirring at reflux temperature for
3 h. All compounds afforded shining yellow powders with
cooling, which were filtered on Buchner funnel, washed
several times with H2O and allowed to dry in air. The same
method has already been used to synthesize ligand 2
[18,34].
To quantify the DNA binding capability in aqueous
solution, a spectroscopic investigation with calf thymus
DNA has been carried out. Nuclease activity has been ver-
ified on plasmid DNA pBR 322 by electrophoresis.
2. Experimental
2.1. Materials and physical measurements
5-Formyluracil (Aldrich, 98%), 4-ethylthiosemicarbazide
(Janssen), 4-allylthiosemicarbazide (Janssen), 4-(3-methyl)-
phenylthiosemicarbazide (Janssen), 4-phenylthiosemicar-
bazide (Janssen), Cu(NO3)2 ꢀ 3H2O (Carlo-Erba) and
CuCl2 ꢀ 2H2O (Carlo-Erba) were commercially available
and were used without further purification. C, H, N and
2.2.1. Et-H3ut (1)
Yield: 129 mg (67%). Anal. Calc. for C8H11N5O2S: C,
39.83; H, 4.59; N, 29.03; S, 13.29. Found: C, 40.15; H,
4.46; N, 28.47; S, 13.43%. Main IR peaks (KBr, cmꢁ1):
m(N–H) 3434 m and 3169 w; m(C–Haliphatic) 2973 w;

M. Belicchi-Ferrari et al. / Polyhedron 27 (2008) 1361–1367
1363
m(C@O) 1711 vs and 1675 vs; m(C@N) 1631 s; m(C@S) 772
2.3. Synthesis of the Cu(II) complexes
m. UV–Vis (DMF, nm, Mꢁ1 cmꢁ1) 270 (18900), 334
(21000). MS (m/z, %) MH+ (242, 85%); 1H NMR (CDCl3,
ppm): 11.55 (CSNH, 1H, br s), 9.95 (N3Hur, 1H, s), 9.55
(N1Hur, 1H, s), 8.51 (CHur, 1H, s), 8.03 (CH2NH, 1H, t),
7.86 (CH = N, 1H, s), 3.05 (CH2, 2H, m), 1.14 (CH3, 3H,
t). 13C NMR (CDCl3, ppm): 178.3 (CS), 175.9 (CH2N)
164.0 (C4ur), 151.4 (C2ur), 146.5 (C6ur), 113.7 (C5ur),
132.2 (CHN), 38.1 (CH3). M.p. 185 °C.
The Cu(II) complexes were synthesised following this
general procedure: ca. 0.30 mmol of the ligands were dis-
solved in EtOH (40 mL) with gentle heating. An equimolar
amount of Cu(NO3) ꢀ 3H2O or CuCl2 ꢀ 2H2O was dissolved
in the minimum quantity of the same solvent and added
dropwise to the pale yellow ligand solutions. All the result-
ing mixtures slowly became light green coloured and were
left under magnetic stirring at reflux temperature for 1 h.
By slow solvent evaporation dark green powders were
afforded and characterized.
2.2.2. Allyl-H3ut (2)
Yield: 135 mg (65%). Anal. Calc. for C9H11N5O2S: C,
42.68; H, 4.38; N, 27.65; S, 12.66. Found: C, 42.31; H,
4.48; N, 27.54; S, 12.92%. Main IR peaks (KBr, cmꢁ1):
m(N–H) 3163 m; m(C–Haromatic) 3014; m(C@O) 1713 vs
and 1672 vs; m(C@N) 1630 m; m(C@S) 793 m. UV–Vis
(DMF, nm, Mꢁ1cmꢁ1) 270 (20800), 335 (23300). MS (m/
2.3.1. [Cu(Et-H3ut)NO3(OH2)2]NO3 ꢀ 2H2O (5)
Anal. Calc. for C8H19CuN7O12S: C, 19.18; H, 3.82; N,
19.57; S, 6.40. Found: C, 19.21; H, 3.78; N, 19.48; S,
6.26%. Main IR peaks (KBr, cmꢁ1): m(O–H) 3583 m;
1
z, %) MH+ (254, 72%); H NMR (CDCl3, ppm): 11.61
m(N–H) 3479 m and 3237 m; m(C–Haromatic
) 3056;
m(C–Haliphatic) 2919 w; m(C@O) 1731 s and 1643 vs;
m(C@N) 1613 s; m(NO3ꢁ) 1385 vs and 1304 s; m(C@S) 775
m. Main Far IR peaks (CsI, cmꢁ1): m(Cu–N) 450 m;
m(Cu–O) 417 w; m(Cu–S) 348 m. UV–Vis (DMF, nm,
(CSNH, 1H, br s), 10.03 (N3Hur, 1H, s), 9.58 (N1Hur,
1H, s), 8.56 (CHur, 1H, s), 8.10 (CH2NH, 1H, t), 7.90
(CH = N, 1H, s), 5.88 (CH gem, 1H, m), 5.12 (CH2 cis +
trans, 2H, m), 4.22 (CH2NH, 2H, m). 13C NMR (CDCl3,
ppm): 179.1 (CS), 164.5 (C4ur), 152.1 (C2ur), 147.5 (C6ur),
135.5 (CH2 = CHCH2), 115.1 (CH2 = CHCH2), 113.9
(C5ur), 134.2 (CHN), 45.3 (CH2N). M.p. 192 °C.
M
ꢁ1 cmꢁ1) 257 (11700), 286 (13800), 335 (22000), 648
(165). K (Xꢁ1 cm2 molꢁ1) 50. TGA (°C, % weight loss)
105, 7; 195, 8; 230, 12; 295, 10; 340, 15.6.
2.3.2. [Cu(Et-H3ut)Cl2] ꢀ 4H2O (6)
2.2.3. Ph-H3ut (3)
Anal. Calc. for C8H19Cl2CuN5O6S: C, 21.46; H, 4.28; N,
15.64; S, 7.16. Found: C, 21.85; H, 3.99; N, 15.73; S, 7.31%.
Main IR peaks (KBr, cmꢁ1): m(O–H) 3424 m; m(N–H) 3344
m sh and 3248 m; m(C–Haromatic) 3050; m(C–Haliphatic) 2938
w; m(C@O) 1730 s and 1652 vs; m(C@S) 758 m. Main Far IR
peaks (CsI, cmꢁ1): m(Cu–N) 451 m; m(Cu–S) 352 m; m(Cu–
Cl) 315 w and 303 s. UV–Vis (DMF, nm, Mꢁ1 cmꢁ1) 253
(9200), 285 (11700), 335 (20300), 645 (129), 960 (25). K
(Xꢁ1 cm2 molꢁ1) 32. TGA (°C, % weight loss) 220, 12;
260, 11; 290, 20.
Yield: 162 mg (68%). Anal. Calc. for C12H11N5O2S: C,
49.82; H, 3.83; N, 24.21; S, 11.08. Found: C, 50.02; H,
4.03; N, 24.33; S, 10.98%. Main IR peaks (KBr, cmꢁ1):
m(N–H) 3391 s and 3204 w; m(C–Haromatic) 3045; m(C@O)
1726 mw and 1668 vs; m(C@N) 1621 m; m(C@S) 784 m.
UV–Vis (DMF, nm, Mꢁ1 cmꢁ1
) 270 (23900), 351
(26010). MS (m/z, %) MH+ (290, 78%); 1H NMR (CDCl3,
ppm): 11.60 (CSNH, 1H, br s), 10.10 (N3Hur, 1H, s), 9.52
(N1Hur, 1H, s), 8.54 (CHur, 1H, s), 8.10 (Ph-NH, 1H, s),
7.85 (CH = N, 1H, s), 7.54–7.00 (CHar, 5H, m). 13C
NMR (CDCl3, ppm): 178.8 (CS), 165.1 (C4ur), 151.8
(C2ur), 146.4 (C6ur), 114.2 (C5ur), 126.7–124.8 (Car), 134.2
(CHN). M.p. 201 °C.
2.3.3. [Cu(Allyl-H3ut)NO3(OH2)2]NO3 ꢀ 3H2O (7)
Anal. Calc. for C9H21CuN7O13S: C, 20.36; H, 3.99; N,
18.47; S, 6.04. Found: C, 20.10; H, 4.17; N, 18.58; S,
6.42%. Main IR peaks (KBr, cmꢁ1): m(O–H) 3422 m;
m(N–H) 3185 m; m(C–Haromatic) 3024; m(C–Haliphatic) 2948
2.2.4. MePh-H3ut ꢀ H2O (4)
-
Yield: 195 mg (74%). Anal. Calc. for C13H15N5O3S: C,
48.59; H, 4.70; N, 21.79; S, 9.98. Found: C, 48.49; H,
4.68; N, 21.61; S, 10.19%. Main IR peaks (KBr, cmꢁ1):
w; m(C@O) 1730 s and 1650 s; m(C@N) 1600 m; m(NO3 )
1385 vs and 1304 s; m(C@S) 792 w. Main Far IR peaks
(CsI, cmꢁ1): m(Cu–N) 452 m; m(Cu–O) 418 w; m(Cu–S)
350 m. UV–Vis (DMF, nm, Mꢁ1 cmꢁ1) 250 (8950), 279
(12050), 339 (18300), 649 (108). K (Xꢁ1 cm2 molꢁ1) 54.
TGA (°C, % weight loss) 102, 10; 198, 7; 308, 14.
m(O–H) 3429 bs; m(N–H) 3251 m and 3200 w; m(C–Haromatic
)
3039; m(C–Haliphatic) 2999 w; m(C@O) 1718 s and 1676 vs;
m(C@N) 1632 m; m(C@S) 773 m. UV–Vis (DMF, nm,
M
ꢁ1 cmꢁ1) 272 (21800), 359 (24300). MS (m/z, %)
MH+ꢁH2O (304, 85%); H NMR (CDCl3, ppm): 11.58
(CSNH, 1H, br s), 10.10 (N3Hur, 1H, s), 9.54 (N1Hur,
1H, s), 8.54 (CHur, 1H, s), 8.08 (Ph-NH, 1H, s), 7.84
(CH = N, 1H, s), 7.55–7.00 (CHar, 4H, m), 2.31 (CH3,
3H, s). 13C NMR (CDCl3, ppm): 178.6 (CS), 165.4
(C4ur), 152.0 (C2ur), 146.5 (C6ur), 114.2 (C5ur), 128.0–
124.2 (Car), 134.2 (CHN), 17.9 (CH3). M.p. 210 °C.
1
2.3.4. [Cu(Allyl-H3ut)Cl2] ꢀ 3H2O (8)
Anal. Calc. for C9H17Cl2CuN5O5S: C, 24.47; H, 3.88; N,
15.85; S, 7.26. Found: C, 24.18; H, 4.12; N, 15.65; S, 7.17%.
Main IR peaks (KBr, cmꢁ1): m(O–H) 3433 m; m(N–H) 3250
m; m(C–Haromatic) 3047; m(C–Haliphatic) 2931 w; m(C@O)
1729 s and 1652 vs; m(C@N) 1604 m; m(C@S) 754 m. Main
Far IR peaks (CsI, cmꢁ1): m(Cu–N) 450 m; m(Cu–S) 355 m;

1364
M. Belicchi-Ferrari et al. / Polyhedron 27 (2008) 1361–1367
m(Cu–Cl) 316 w and 303 s. UV–Vis (DMF, nm, Mꢁ1 cmꢁ1
252 (9500), 278 (11620), 335 (21000), 644 (131), 652 (330).
K (Xꢁ1 cm2 molꢁ1) 22. TGA (°C, % weight loss) 103, 12;
258, 8.5; 288, 9.
)
[35]. These studies were carried out on the ligands and their
nitrate complexes because with their ionic character they
are expected to show more interactions towards DNA than
the neutral chloro complexes. The intrinsic binding con-
stant Kb for the interaction of the studied compounds with
CT-DNA was calculated by absorption spectral titration
data using the following equation: 1/Deap = 1/(DeKbD) +
1/De where Deap = |eA ꢁ ef|, De = |eB ꢁ ef|, D = [DNA],
and eA, eB and ef are the apparent, bound and free extinc-
tion coefficients of the compound, respectively. Kb is given
by the ratio of the slope to intercept from the plot of
[DNA]/(eA ꢁ ef) versus [DNA] and it is expressed in
2.3.5. [Cu(Ph-H3ut)(NO3)(OH2)2]NO3 (9)
Anal. Calc. for C12H15CuN7O10S: C, 28.10; H, 2.95; N,
19.12; S, 6.25. Found: C, 28.15; H, 2.63; N, 18.87; S, 6.55%.
Main IR peaks (KBr, cmꢁ1): m(O–H) 3475 m; m(N–H) 3294
m and 3217 m; m(C–Haromatic) 3024; m(C–Haliphatic) 2939 m;
-
m(C@O) 1739 s and 1639 s; m(C@N) 1606 m; m(NO3 ) 1385
vs; m(C@S) 769 w. Main Far IR peaks (CsI, cmꢁ1): m(Cu–
N) 452 m; m(Cu–O) 417 w; m(Cu–S) 352 m. UV–Vis
(DMF, nm, Mꢁ1 cmꢁ1) 265 (13850), 356 (14200), 630
(110). K (Xꢁ1 cm2 molꢁ1) 40. TGA (°C, % weight loss)
189, 8; 230, 15.
M
ꢁ1. The previous equation, originally used to calculate
the binding constants for hydrophobic derivatives, is
now broadly used to investigate a wide variety of metal
complexes containing phenanthroline and its derivatives
and it has been adopted to obtain binding constant values
from metal complexes with different ligands [23,30,36–39].
Fixed amounts of the ligands and of the complexes
were dissolved in DMSO because the high solubility of
the compounds in this solvent allowed us to prepare
concentrated solutions and therefore to utilize reduced
volumes (5 lL) for titrations. It was also verified that
the DMSO percentage (0.7%) added to the DNA solution
did not interfere with the nucleic acid; in fact, the 260 nm
absorption band is not subjected to modifications in
intensity and position. Concentrated solutions of NaCl,
Tris–HCl (pH 7.2) buffer and DNA were prepared. Calcu-
lated amounts of the described stock solutions were taken
to final concentration values of 50 mM NaCl, 5 mM
Tris–HCl and increasing amounts of DNA over a range
of DNA concentrations from 10ꢁ5 to 10ꢁ3 M. These
solutions were then added to the 5 lL solution of the
considered compounds in order to maintain the final vol-
ume of the solutions as fixed to 700 lL. The compounds
were titrated at room temperature. The changes in absor-
bance of DNA of an intraligand (IL) band upon each
addition were monitored at the maximum wavelengths
334, 335, 351 and 359 nm for 1, 2, 3 and 4, respectively
for the ligands, and at the maximum wavelengths 336,
339, 356 and 360 nm for complexes 5, 7, 9 and 11,
respectively.
2.3.6. [Cu(Ph-H3ut)Cl2] ꢀ H2O (10)
Anal. Calc. for C12H13Cl2CuN5O3S: C, 32.62; H, 2.96;
N, 15.85; S, 7.26. Found: C, 33.06; H, 2.64; N, 15.57; S,
7.47%. Main IR peaks (KBr, cmꢁ1): m(O–H) 3429 m;
m(N–H) 3309 w and 3200 m; m(C–Haromatic) 3062 m; m(C–
H
aliphatic) 2935 w; m(C@O) 1739 s and 1670 vs; m(C@N)
1597 m; m(C@S) 762 m. Main Far IR peaks (CsI, cmꢁ1):
m(Cu–N) 448 m; m(Cu–S) 349 m; m(Cu–Cl) 318 w and 303
s. UV–Vis (DMF, nm, Mꢁ1 cmꢁ1) 273 (12920), 360
(14130), 661 (135). K (Xꢁ1 cm2 molꢁ1) 24. TGA (°C, %
weight loss) 105, 4; 255, 8.5; 292, 9.
2.3.7. [Cu(MePh-H3ut)NO3(OH2)2]NO3 ꢀ 3H2O (11)
Anal. Calc. for C13H15CuN7O13S: C, 26.88; H, 3.99; N,
16.88; S, 5.52. Found: C, 27.11; H, 3.87; N, 17.05; S, 5.47%.
Main IR peaks (KBr, cmꢁ1): m(O–H) 3446 m; m(N–H) 3290
w and 3095 m; m(C–Haromatic) 3032; m(C–Haliphatic) 2947 vw;
-
m(C@O) 1737 ms and 1642 ms; m(C@N) 1607 m; m(NO3 )
1385 vs; m(C=S) 789 w. Main Far IR peaks (CsI, cmꢁ1):
m(Cu–N) 450 m; m(Cu–O) 419 w; m(Cu–S) 350 m. UV–Vis
(DMF, nm, Mꢁ1 cmꢁ1) 264 (13100), 360 (14050), 611
(195). K (Xꢁ1 cm2 molꢁ1) 45. TGA (°C, % weight loss)
105, 10; M.p. (°C) 164.
2.3.8. [Cu(MePh-H3ut)Cl2] ꢀ 3H2O(12)
Anal. Calc. for C13H19Cl2CuN5O5S: C, 31.75; H, 3.89;
N, 14.24; S, 6.52. Found: C, 32.05; H, 3.75; N, 14.37; S,
6.29%. Main IR peaks (KBr, cmꢁ1): m(O–H) 3436 br;
2.5. Nuclease activity
m(N–H) 3255 m; m(C–Haromatic) 3057 m; m(C–Haliphatic
)
The DNA cleavage was carried out on double stranded
plasmid DNA pBR 322 by electrophoresis. Buffers were
prepared using sterile distilled water. Solutions composed
of 1.6 lL of plasmid (40 lg/mL), 4 lL of the studied
compounds dissolved in DMSO (0.1 mM) and 8.4 lL Tris
buffer solution (10 mM) were incubated for 1 h at 37 °C.
This was followed by addition of a gel loading solution,
and each sample was loaded directly into different wells
on a 1.5% agarose gel for analysis by electrophoresis
at 75 V for 3 h. The gel was stained with ethidium bro-
mide and photographed under UV light in a transil-
luminator.
2927 w; m(C@O) 1723 s and 1650 vs; m(C@S) 770 w. Main
Far IR peaks (CsI, cmꢁ1): m(Cu–N) 465 w; m(Cu–S) 351 m;
m(Cu–Cl) 318 w and 303 m. UV–Vis (DMF, nm,
M
ꢁ1 cmꢁ1
) 272 (1050), 373 (12500), 640 (250). K
(Xꢁ1 cm2molꢁ1) 22. TGA (°C, % weight loss) 105–130,
11; 240, 8; 295, 9.
2.4. DNA interaction studies
Binding constants for the interaction of the studied com-
pounds with nucleic acid were determined as described in

M. Belicchi-Ferrari et al. / Polyhedron 27 (2008) 1361–1367
1365
3. Results and discussion
decrease in the stretching frequency of the C@S bond from
ca. 770–790 cmꢁ1 in the thiosemicarbazones upon com-
plexation indicates coordination via the sulfur atom. The
presence of a new band in the 345–355 cmꢁ1 range assign-
able to m(Cu–S), is another indication of the involvement of
sulfur coordination. The presence of an absorption at ca.
420 cmꢁ1 in the spectra of the complexes is assignable to
m(Cu–O). The IR spectra of the chloro complexes also dis-
play bands at ca. 315 and 300 cmꢁ1, characteristic of coor-
dinated m(Cu–Cl).
The electronic spectra of the complexes in DMF solu-
tion are presented in Section 2. Each thiosemicarbazone
and its copper(II) complexes have ring p ? p* bands in
the range 260–312 nm and n ?p* bands in the range
312–357 nm. The ligand field strength of the sulfur atom
has been found to vary more than that of any other donor
atom, with variations of up to 50% being known [40]. This
is probably due to the variation in p-bonding effects
according to whether the sulfur atom has two or three lone
pairs, as in thioketones (>C@S) or mercaptide ions („C–
Sꢁ), which allows ample scope for M-S p-bonding. The
complex spectra have bands in the range 330-357 nm which
must be due to L ? M transfer from sulfur to copper [41].
3.1. Synthesis and spectroscopy
The ligands were synthesised in water in good yields.
The products absorbed water giving rise to a gel-like tex-
ture and therefore it was necessary to pay attention to
the drying step on the Buchner funnel and in air.
The copper complexes were synthesised by the direct
reaction of the inorganic salt solutions with the ligand solu-
tions. Under the experimental conditions used both mono-
valent anions are always present and therefore no ligand
deprotonation is observed. The synthesis of the complexes
is reproducible and the thiosemicarbazones coordinate as
tridentate ligands in their neutral form (H3ut). The elemen-
tal analyses are in agreement with the general formula
M(R-H3ut)X2 ꢀ nH2O, where X is the counterion. Conduc-
tivity measurements show that complexes bearing nitrate as
counterions behave as 1:1 electrolyte type compounds and
therefore are monocationic [40], while complexes bearing
chlorine as counterions behave as neutral molecular com-
plexes. Thermogravimetric analyses show that two water
molecules are directly bound to the metal center in the
nitrate derivatives. Therefore, as a consequence of the inor-
ganic anionic nature, two types of stoichiometries are
exhibited, namely [Cu(R-H3ut)NO3(OH2)2]NO3 and
[Cu(R-H3ut)Cl2] (see Scheme 2).
3.3. Absorption spectral features of DNA binding
The binding constants obtained for ligands 1, 2, 3 and 4
are 2.1 ꢂ 103, 1.0 ꢂ 103, 2.9 ꢂ 102 and 5.6 ꢂ 103, respec-
tively. The binding constants obtained for complexes 5, 7,
9 and 11 are 2.5 ꢂ 102, 5.9 ꢂ 102, 2.3 ꢂ 101 and 2.3 ꢂ
103, respectively. In Fig. 1 the plot of [DNA]/(eA ꢁ ef) ver-
sus [DNA] for absorption titration of the studied com-
plexes is reported, useful for obtaining Kb by the ratio of
the slope to intercept.
These observations agree with previous results [23].
3.2. IR and electronic spectra
The infrared spectral data of the ligands and their metal
complexes are reported in Section 2 with their tentative
assignments. It is interesting to note that the uracyl moiety
owns two carboxyl groups but only the one whose m(C@O)
is assigned at ca. 1670 cmꢁ1 is involved in coordination.
Upon metal binding this carboxyl stretching is shifted
downwards by 20–30 cmꢁ1 while m(C@O) not directly
linked to the metal is shifted to higher frequencies because
of inductive effects arising from the metal coordination. On
coordination of the azomethine nitrogen, m(C@N) shifts to
lower wavenumbers by 15–25 cmꢁ1, as the band shifts from
ca. 1630 cmꢁ1 in the uncomplexed thiosemicarbazone spec-
tra to ca. 1600 cmꢁ1 in the spectra of the complexes. Coor-
dination of the azomethine nitrogen is confirmed with the
presence of bands at ca. 450 cmꢁ1 (at 465 cmꢁ1 for complex
12) assignable to m(Cu–N) for these complexes. The
Our experimental Kb values are lower than those
observed for classical intercalators (ethidium-DNA, 1.4 ꢂ
106 in 25 mM Tris–HCl/40 mM NaCl buffer, pH 7.8 [42];
3.0 ꢂ 106 in 5 mM Tris–HCl/50 mM NaCl buffer, pH 7.2
[this work]), indicating that the compounds bind DNA
with less affinity, as already noticed for copper(II) com-
plexes with macrocyclic ligands [36]. It is interesting to note
that the metal complexes reported in this work bind to
DNA with less affinity than copper complexes with ali-
phatic thiosemicarbazones [37], complexes synthesised with
formyluracil thiosemicarbazones bearing less bulky substit-
uents on the side chain [23] and copper complexes with thi-
osemicarbazide [43]. Since upon complexation no
significant increase of Kb was observed, the constant values
can probably be related to the role played by the ligand in
the binding to DNA. The terminal NH group seems to play
an important function in the binding process: as the access
to the NH becomes more difficult with the presence of
bulky substituents, the association to the nucleic acid
becomes weaker. Compounds 4 and 11, having the bulkier
methylphenyl substituent, show a major affinity towards
the nucleic acid with respect to the phenyl analogues 3
and 9 (Kb 5.6 ꢂ 103 (4) versus 2.9 ꢂ 102 (3) and Kb
O
O-
H
N
N+
H
H
N
Cu+
N
O
HN
N
NH
Cl
Cu
Cl
S
N
HN
O
S
O
O
N
H
O
N
H
OH2
H2O
Scheme 2. Hypothesized structure for complex 9 (left) and 10 (right)
deduced from spectroscopic data.

1366
M. Belicchi-Ferrari et al. / Polyhedron 27 (2008) 1361–1367
1.6
0.8
0.1
15
25
35
45
55
65
75
85
[CT-DNA] x 105 / M b. p.
5
7
9
11
Fig. 1. Typical plots of [DNA]/(eA ꢁ ef) vs [CT-DNA] for absorption titration with CT-DNA in 50 mM NaCl/5 mM Tris buffer, pH 7.2 at 25 °C.
2.3 ꢂ 103 (11) versus 2.3 ꢂ 101 (9)). This effect can proba-
bly be ascribed to the major deviation from planarity of
the overall thiosemicarbazonic system due to the methyl
substituent on the aromatic ring, as observed in N terminal
substituted thiosemicarbazones [28] that decreases the ste-
ric hindrance on increasing the accessibility to the aminic
group. The weak interactions between our compounds
and the nucleic acid are also confirmed by a negligible hyp-
ochromism and bathochromism in the electronic absorp-
tion spectra of DNA bound to different compounds.
These phenomena are generally attributed to intercalation,
involving a strong stacking interaction between the aro-
matic chromophores and the base pairs of DNA. The ratio
r (r = [CT-DNA]/[complex]) varied between 0 and 15 in
5 mM Tris–HCl buffer (pH 7.2), 50 mM NaCl at 25 °C.
Our results are in agreement with the fact that the glyco-
sidic bond of a formyluracil residue derivative in DNA is
about 3 orders of magnitude more labile than that of the
parent pyrimidine, thymine and is assignable to the elec-
tron-withdrawing effect of the 5-formyl substituent [44].
Additionally, the electron-withdrawing substituent destabi-
lizes the N-glycosidic bond, rendering oligonucleotides
containing formyluracil, highly susceptible to hydrolysis.
The thiosemicarbazidic chain in the five position of the ura-
cil moiety could additionally form potentially irreversible
covalent cross-links with an amino group of DNA-binding
proteins, resulting in locking the binding instead of block-
ing binding [44]. The presence of substituents on the termi-
nal nitrogen seems to weaken the capability of bonding
towards DNA.
millimolar concentrations for all tested compounds the
supercoiled was the predominant form of DNA followed
by a slight amount of the open circular form, indicating
that there is no significant breaking of plasmid-DNA due
to oxidative stress or phosphodiester hydrolysis.
4. Conclusions
As hypothesized in the introduction, the presence of a
substituent on the thiosemicarbazidic chain strongly affects
the DNA interactions of thiosemicarbazones and their
metal complexes. The variation of the hydrophilic/lipo-
philic character due to the different alkylic or aromatic
groups could vary the uptake by the cells but this feature
has no consequence on the direct effects of the compounds
towards DNA; on the contrary, with respect to the unsub-
stituted analogous compounds, the free NH2 thiosemicar-
bazone moiety seems to play an important role in DNA
binding. The presence of the substituents, on the other
hand, has no consequence on the coordination mode that,
on the contrary, seems to be more influenced by the
counterion.
References
[1] R.W. Byrnes, M. Mohan, W.E. Antholine, R.X. Xu, D.H. Petering,
Biochemistry 29 (1990) 7046.
[2] K.C. Agrawal, A.C. Sartorelli, J. Med. Chem. 12 (1969) 771.
[3] W.E. Antholine, J. Knight, H. Whelan, D.H. Petering, Mol. Pharm.
13 (1977) 89.
[4] D.L. Klayman, J.F. Bartosovich, T.S. Griffin, C.J. Mason, J.P.
Scovill, J. Med. Chem. 22 (1979) 855.
[5] D.L. Klayman, J.P. Scovill, J.F. Bartosovich, C.J. Mason, J. Med.
Chem. 22 (1979) 1367.
3.4. Nuclease activity
[6] D. Ronen, L. Sherman, S. Bar-Nun, Y. Teitz, Antimicrob. Agents
Chemother. 31 (1987) 1798.
[7] R.C. Condit, R. Easterly, R.F. Pacha, Z. Fathi, R.J. Meis, Virology
185 (1991) 857.
[8] M. Belicchi Ferrari, G. Gasparri Fava, P. Tarasconi, R. Albertini, S.
Pinelli, R. Starcich, J. Inorg. Biochem. 53 (1994) 13.
In addition to the studies carried out with CT-DNA, a
gel electrophoresis experiment using pBR322, a circular
plasmid DNA, was performed with all our compounds.
From the results (data not shown) it was verified that at

M. Belicchi-Ferrari et al. / Polyhedron 27 (2008) 1361–1367
1367
[9] J.G. Cory, A.H. Cory, G. Rappa, A. Lorico, M.-C. Liu, T.-S. Lin,
A.C. Sartorelli, Biochem. Pharmacol. 48 (1994) 335.
[10] D. Kovala-Demertzi, P. Nath Yadav, M.A. Demertzis, M. Coluccia,
J. Inorg. Biochem. 78 (2000) 347.
[26] U. Abram, K. Ortner, R. Gust, K. Sommer, J. Chem. Soc., Dalton
Trans. (2000) 735.
[27] M. Belicchi Ferrari, S. Capacchi, F. Bisceglie, G. Pelosi, P. Tarasconi,
Inorg. Chim. Acta 312 (2001) 81.
[11] J.P. Scovill, D.L. Klayman, C.F. Franchino, J. Med. Chem. 25 (1982)
1261.
[28] M. Belicchi Ferrari, F. Bisceglie, G. Pelosi, P. Tarasconi, R. Albertini,
S. Pinelli, J. Inorg. Biochem. 87 (2001) 137.
[12] Z. Lakovidou, A. Papageorgiou, M.A. Demertzis, E. Mioglou, D.
Mourelatos, A. Kotsis, P.N. Yadav, D. Kovala-Demertzi, Anticancer
Drugs 12 (2001) 65.
[13] K. Hussain Reddy, P. Sambasiva Reddy, P. Ravindra Babu, Trans.
Met. Chem. 25 (2000) 154.
[14] M. Belicchi-Ferrari, F. Bisceglie, C. Casoli, S. Durot, I. Morgenstern-
Badarau, G. Pelosi, E. Pilotti, S. Pinelli, P. Tarasconi, J. Med. Chem.
48 (2005) 1671.
[15] S. Adsule, V. Barve, D. Chen, F. Ahmed, Q.P. Dou, S. Padhye, F.H.
Sarkar, J. Med. Chem. 49 (2006) 7242.
[29] M. Belicchi Ferrari, F. Bisceglie, E. Leporati, G. Pelosi, P. Tarasconi,
S. Pinelli, Bull. Chem. Soc. Jpn. 75 (2002) 781.
[30] Mudasir, N. Yoshioka, H. Inoue, J. Inorg. Biochem. 77 (1999)
239.
[31] A.E. Friedman, C.V. Kumar, N.J. Turro, J.K. Barton, Nucl. Acids
Res. 19 (1991) 2595.
[32] A.M. Pyle, T. Morii, J.K. Barton, J. Am. Chem. Soc. 112 (1990) 9432.
[33] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem.
Soc. 76 (1954) 3047.
[34] F. Andreeff, M. Privat de Garilhe, Bull. Soc. Chim. Biol. 50 (2) (1968)
435.
[35] A. Wolfe, G.H. Shimer Jr., T. Meehan, Biochemistry 26 (1987) 6392.
[36] J. Liu, T. Zhang, T. Lu, L. Qu, H. Zhou, Q. Zhang, L. Ji, J. Inorg.
Biochem. 91 (2002) 269.
[16] C.R. Kowol, R. Berger, R. Eichinger, A. Roller, M.A. Jakupec, P.P.
Schmidt, V.B. Arion, B.K. Keppler, J. Med. Chem. 50 (2007) 1254.
[17] F. Karatas, M. Koca, H. Kara, S. Servi, Eur. J. Med. Chem. 41 (2006)
664.
[18] J. Boissier, P. Lepine, J. De Rudder, M. Privat de Garilhe, F Patent
(1968), 1512549.
[37] F. Bisceglie, M. Baldini, M. Belicchi-Ferrari, E. Buluggiu, M. Careri,
G. Pelosi, S. Pinelli, P. Tarasconi, Eur. J. Med. Chem. 42 (2007)
627.
[38] V. Uma, M. Kanthimathi, T. Weyhermuller, Balachandran Unni
Nair, J. Inorg. Biochem. 99 (2005) 2299.
[39] M. Baldini, M. Belicchi-Ferrari, F. Bisceglie, S. Capacchi, G. Pelosi,
S. Pinelli, P. Tarasconi, J. Inorg. Biochem. 99 (2005) 1504.
[40] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81.
[41] M.J.M. Campbell, Coord. Chem. Rev. 15 (1975) 279.
[42] W.D. Wilson, F.A. Tanious, M. Fernandez-Saiz, C.T. Rigl, in: K.R.
Fox (Ed.), Methods in Molecular Biology, vol. 90, Humana, Clifton,
NJ, 1997.
[19] N. Wolmark, H. Rockette, J. Clin. Oncol. 17 (1999) 3553.
[20] W.A. Denny, Eur. J. Med. Chem. 36 (2001) 577.
[21] M. Belicchi Ferrari, G. Gasparri Fava, E. Leporati, G. Pelosi, R.
Rossi, P. Tarasconi, R. Albertini, A. Bonati, P. Lunghi, S. Pinelli, J.
Inorg. Biochem. 70 (1998) 145.
[22] M. Belicchi Ferrari, F. Bisceglie, G. Pelosi, P. Tarasconi, R. Albertini,
A. Bonati, P. Lunghi, S. Pinelli, J. Inorg. Biochem. 83 (2001) 169.
[23] M. Baldini, M. Belicchi-Ferrari, F. Bisceglie, G. Pelosi, S. Pinelli, P.
Tarasconi, Inorg. Chem. 42 (2003) 2049.
´
´
´
[24] D.X. West, J.K. Swearingen, J. Valdes-Martınez, S. Hernandez-
Ortega, A.K. El-Sawaf, F. van Meurs, A. Castineiras, I. Garcia, I.
Bermejo, Polyhedron 18 (1999) 2919.
[43] P. Murali Krishna, K. Hussain Reddy, P.G. Krishna, G.H. Philip,
Indian J. Chem. 46A (2007) 904.
[25] M. Belicchi Ferrari, S. Capacchi, G. Reffo, G. Pelosi, P. Tarasconi, R.
Albertini, S. Pinelli, P. Lunghi, J. Inorg. Biochem. 81 (2000) 89.
[44] D.K. Rogstad, J. Heo, N. Vaidehi, W.A. Goddard III, A. Burdzy,
L.C. Sowers, Biochemistry 43 (2004) 5688.