DNA-binding study of nickel(II) and zinc(II) complexes with two novel chromenone-based Schiff-base ligands

Article abstract of DOI:10.1002/hlca.200800350

Two new chromenone-based Schiff-base ligands, 3-{[(1,5-dihydro-3-methyl-5- thioxo-4H-1,2,4-triazol-4-yl)imino]methyl}-6-hydroxy-4H-1-benzopyran-4-one (L¹) and 2,2′-bis[(6-hydroxy-4-oxo-4H-1-benzopyran-3-yl) methylene]carbonothioic dihydrazide (L²), and their Ni^II and Zn^II complexes were prepared. All the complexes were characterized by elemental analysis, IR data, and molar conductivity. The binding of these four complexes to calf-thymus DNA was carefully investigated by UV/VIS spectroscopy, fluorescence spectroscopy, viscosity measurements, and CD spectra. The experimental results indicate that the four complexes bind to calf-thymus DNA in an intercalative mode, with the intrinsic binding constants (K) of 3.94·10⁴ ([NiL¹]), 5.15·10 ³ ([ZnL¹]), 4.12·10⁴ ([NiL ²]), and 3.75·10⁴ m^-1 ([ZnL ²]). These data show that the complexes of L² can interact more strongly with DNA than complexes of L¹, and the Ni^II complexes have a higher binding constant than Zn^II complexes.

Full text of DOI:10.1002/hlca.200800350

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DNA-Binding Study of Nickel(II) and Zinc(II) Complexes with Two Novel
Chromenone-Based Schiff-Base Ligands
by Dong-Dong Qin, Zheng-Yin Yang*, Tian-Rong Li, and Gao-Fei Qi
College of Chemistry and Chemical Engineering and State Key Laboratory of Applied Organic
Chemistry, Lanzhou University, Lanzhou 730000, P. R. China
(phone: þ 86-931-8913515; fax: þ 86-931-8912582; e-mail: yangzy@lzu.edu.cn)
Two new chromenone-based Schiff-base ligands, 3-{[(1,5-dihydro-3-methyl-5-thioxo-4H-1,2,4-tria-
zol-4-yl)imino]methyl}-6-hydroxy-4H-1-benzopyran-4-one (L1) and 2,2’-bis[(6-hydroxy-4-oxo-4H-1-
benzopyran-3-yl)methylene]carbonothioic dihydrazide (L2), and their NiII and ZnII complexes were
prepared. All the complexes were characterized by elemental analysis, IR data, and molar conductivity.
The binding of these four complexes to calf-thymus DNA was carefully investigated by UV/VIS
spectroscopy, fluorescence spectroscopy, viscosity measurements, and CD spectra. The experimental
results indicate that the four complexes bind to calf-thymus DNA in an intercalative mode, with the
intrinsic binding constants (K) of 3.94 · 104 ([NiL1]), 5.15 · 103 ([ZnL1]), 4.12 · 104 ([NiL2]), and 3.75 · 104
mꢀ1 ([ZnL2]). These data show that the complexes of L2 can interact more strongly with DNA than
complexes of L1, and the NiII complexes have a higher binding constant than ZnII complexes.
Introduction. – Over the past decades, the interactions of small molecules with
DNA have attracted a great deal of attention since many anticancer drugs exert their
antitumor effects through binding to DNA in one way or another, thereby blocking the
replication of DNA and inhibiting the growth of tumor cells [1 – 3]. In many useful
applications of such small molecules, the compounds bind to DNA in an intercalation
mode. At present, tremendous interest has been aroused to explore the potential
applications of metal complexes as possible therapeutic agents and as nonradioactive
probes of nucleic acid structure [4 – 6]. However, most of these studies are focused on
RuII complexes of 2,2’-bipyridine (bpy), 1,10-phenanthroline (phen), and their
modified variants [7 – 10]. DNA-Binding studies of NiII and ZnII complexes of Schiff
bases, which have been intensively used in the medicinal and pharmaceutical field, are
rarely reported.
Flavonoids are ubiquitous in nature, especially in plants, and are known to be kinase
inhibitors. They also show activity in apoptosis, which is implicated in cancer
chemotherapy [11 – 13]. Chromenones, as one kind of flavonoids, have a structure
similar to those of flavonoids. Balbi and co-workers [14] and Ganguly and co-workers
[15][16] have reported a large amount of chromenone derivatives and investigated
their biological activities. However, their Schiff-base compounds and the correspond-
ing NiII and ZnII complexes have not been synthesized, and DNA-binding activities of
metal complexes have not been studied either. As known, triazole and thiourea
derivatives also display a broad range of biological activities, showing potential
applications as antitumor, antibacterial, antifungal, and antiviral agents [17][18].
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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With the endeavor to synthesize effective DNA-binding complexes for biological
use, we now report on the successful synthesis of the two new chromenone-based
Schiff-base ligands L1 and L2 containing a triazole or a thiourea group (see Scheme and
Exper. Part), and of their NiII and ZnII complexes. The intrinsic binding constants K of
the complexes with DNA vary from 5.15 · 103 mꢀ1 to 4.12 · 104 mꢀ1, and the quenching
constants Kq vary from 2.22 · 103 mꢀ1 to 1.30 · 105 mꢀ1. The K and Kq values for these
complexes decrease in the order [NiL2] > [NiL1] > [ZnL2] > [ZnL1]. These values are
similar to those of some known DNA intercalators, such as K ¼ 4.8 · 104 mꢀ1 for
[Ru(bpy)2(phi)]Cl2 (phi ¼ 9,l0-phenanthrenequinonediimine) [7] or K ¼ 2.1 · 104 mꢀ1
for [Ru(bpy)2(ddt)]2þ (ddt ¼ 3-(pyrazin-yl)-5,6-diphenyl-as-triazine) [8]. The large K
and Kq values are the direct evidence that the complexes have good ability to bind to
DNA in the intercalation mode.
Scheme. Synthesis of the Schiff-Base Ligands L1 and L2
Results and Discussion. – 1. Characterization of NiII and ZnII Complexes.
Compositions and Properties of the Complexes. The likely structures of the four
complexes are shown in Fig. 1. These assumptions are in accord with the results of
elemental analysis, IR data, TG/DTA (thermogravimetric/differential thermal analy-
sis), and molar conductivity. The results suggest that [NiL1] is composed of [NiL1 ·
1
)
Arbitrary atom numbering; for systematic names, see Exper. Part.

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527
H2O] · 2 NO3 · H2O and [ZnL1] of [ZnL1 · (H2O)3] · 2 NO3 · 0.5 H2O, whereas the com-
plexes of L2 are composed of [ML2 ] · 2 NO3 (M ¼ NiII or ZnII). The complexes are air-
2
stable for extended periods and soluble in DMSO, DMF, and MeOH, slightly soluble in
EtOH and H2O, and insoluble in benzene and Et2O. The molar conductivity value Lm is
166 and 172 S · cm2 · molꢀ1 for [NiL1 · H2O] · 2 NO3 · H2O and [ZnL1 · (H2O)3] · 2 NO3 ·
0.5 H2O in MeOH, and 154 and 162 S · cm2 · molꢀ1 for [NiL2 ] · 2 NO3 and [ZnL2 ] ·
2
2
2 NO3 in MeOH; these results show that the four complexes are 2 :1 electrolytes
[19].
Fig. 1. Proposed structures of the complexes [NiL1], [ZnL1], [NiL2], and [ZnL2]. For convenience, the
short forms [ML1] and [ML2] (M ¼ Ni or Zn) are used for these complexes.
IR Spectra. The ˜n(C¼N) vibration of the free ligands L1 and L2 appear at 1595 and
1590 cmꢀ1, whereas those of [NiL1] and [ZnL1] are shifted to 1578 and 1562 cmꢀ1, and
those of [NiL2] and [ZnL2] to 1575 and 1569 cmꢀ1, respectively, indicating that the N-
atom of the imino group C¼N of the two ligands is bound to a metal ion. The ˜n(C¼O)
vibrations of L1 and L2 are at 1630 and 1633 cmꢀ1 and shifted to 1635 and 1636 cmꢀ1 in
the case of [NiL1] and [ZnL1]; however, the absorption of L2 at 1633 cmꢀ1 is not shifted
in the case of [NiL2] and [ZnL2]. This indicates that the O-atom of the C¼O group of
L1 is involved in a coordination bond with the NiII or ZnII ion, in contrast to that of L2.
The absorptions at 1257 and 1237 cmꢀ1 of L1 and L2 are assigned to the ˜n(C¼S) stretch

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[17], which are shifted to 1233 and 1230 cmꢀ1 in the case of [NiL1] and [ZnL1], and to
1215 and 1219 cmꢀ1 in the case of [NiL2] and [ZnL2]. The ˜n(NꢀH) vibration of L1 is at
3191 cmꢀ1, and after coordinating to the metal, the NꢀH vibration still appears at 3196
in the case of [NiL1] and at 3120 in the case of [ZnL1], suggesting that L1 forms the
complexes in its thione form [17][18]. For the four complexes, the bands at 1383 and
1384 cmꢀ1 are assigned to the free NO3ꢀ ion [20].
1
1H-NMR Spectra. The H-NMR spectra ((D6)DMSO, 200 MHz) of L1 and [ZnL1]
are assigned as follows: L11): d(H) 2.34 (s, Me); 7.28 (dd, J ¼ 2.9, 9.0 Hz, HꢀC(7)); 7.39
(d, J ¼ 2.9 Hz, HꢀC(5)); 7.62 (d, J ¼ 9.0 Hz, HꢀC(8)); 9.05 (s, HꢀC(2)); 10.22 (s,
CH¼N); 10.26 (s, exchangeable, OH); 13.73 (s, exchangeable, NH). [ZnL1]1): d(H)
2.34 (s, Me); 7.28 (dd, J ¼ 2.9, 9.0 Hz, HꢀC(7)); 7.37 (d, J ¼ 2.9 Hz, HꢀC(5)); 7.62 (d,
J ¼ 9.0 Hz, HꢀC(8)); 9.01 (s, HꢀC(2)); 10.18 (s, CH¼N); 10.26 (s, exchangeable,
OH); 13.73 (s, exchangeable, NH). Thus, the NꢀH of the 1H-1,2,4-triazole moiety of
both L1 and [ZnL1] is observed, indicating that both the ligand and the complex exist as
in their thione form (see 4 in the Scheme) [17][18]. This is consistent with the results
obtained from the IR spectra.
UV/VIS Spectra. The study of the electronic spectra of the complexes in the UV
region was carried out in buffer solution (containing 1% MeOH). [NiL1] and [ZnL1]
give rise to two strong bands at lmax 202 and 246 nm, and 204 and 247 nm, respectively.
[NiL2] and [ZnL2] also exhibit a strong band at 202 nm. The band at 202 or 204 nm is
assigned to the p ! p* transition absorbance of the chromenone moiety. The band at
246 or 247 nm is due to the absorbance of the triazole ring. The VIS spectra of [NiL1]
and [NiL2] were measured in MeOH solution (concentration 1 · 10ꢀ3 m). [NiL1] shows
one absorption band at 525 nm (e ¼ 248), but [NiL2] exhibits two absorption bands at
603 (e ¼ 73) and 903 nm (e ¼ 63). The different d – d transitions of the Ni2þ ion suggest a
different coordination configuration (Fig. 1).
2. DNA-Binding Interaction. Absorption Titration and Ethidium Bromide (EB)
Replacement Experiment. The interaction of [NiL1], [ZnL1], [NiL2], and [ZnL2] with
DNA were investigated by means of spectrometric titration and viscosity measure-
ments to evaluate their binding affinities and modes [21]. Electronic-absorption
spectroscopy is one of the most useful techniques for DNA-binding studies of metal
complexes. The UV titration spectra of [NiL1] and [NiL2] are given in Fig. 2 as
examples (those of [ZnL1] and [ZnL2] are not shown). In the presence of DNA, the
absorption band of [NiL1] at 202 nm exhibits hypochromism of ca. 27.1% and
bathochromism of 16 nm ([ZnL1] at 204 nm: ca. 11.1% and 3 nm, resp.); the
corresponding values of [NiL2] at 202 nm are ca. 37.6% and 9 nm (ZnL2 at 202 nm:
36.5% and ca. 8 nm, resp.). These results suggest an association of all four metal
complexes with DNA. Hypochromism and bathochromism are both the spectral
features of DNA concerning its double-helix structure, hypochromism meaning that
the DNA binding mode of the complex is electrostatic or by intercalation which can
stabilize the DNA duplex [22]. The remarkable hypochromism (27.1, 11.1, 37.6, and
36.5%) and redshift (16, 3, 9, and 8 nm) for [NiL1], [ZnL1], [NiL2], and [ZnL2] are
direct evidences of the intercalation mode. After binding of the complex to the base
pairs of DNA, the p* orbital of the intercalated ligand can couple with the p orbital of
the base pairs, thus, decreasing the p ! p* transition energies, which results in
hypochromism and bathochromism.

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Fig. 2. UV Spectra of the complexes [NiL1] and [NiL2] (10 mm) in the presence of increasing amounts of
DNA [DNA] ¼ 0 – 40 mm. The arrow indicates the absorbance changes upon increasing DNA
concentration.
To test the complex-binding to DNA by intercalation, ethidium bromide (EB) was
employed, as EB interacts with DNA as a typical indicator of intercalation [23]. Fig. 3
shows the VIS spectra of EB, EB/DNA, and EB/DNA/[NiL1] as an example. Thus, the
maximal absorption of EB at 479 nm (Fig. 3,a) decreases and simultaneously shifts to
509 nm in the presence of DNA (Fig. 3,b), which is characteristic of intercalation. A
mixture EB/DNA/[NiL1] shows an absorption increase at 509 nm and a redshift
(Fig. 3,c), compared to Fig. 3,b. These results suggest that a competitive intercalation
of [NiL1] and EB in DNA takes place, thus releasing some free EB from the EB/DNA
system when [NiL1] is added. The other complexes show a similar behavior,
establishing that the four complexes bind to DNA mainly by intercalation.
Fig. 3. VIS Spectra of a) EB (1 · 10ꢀ5 m), b) EB (1 · 10ꢀ5 m)/DNA (2.5 · 10ꢀ5 m), and c) EB (1 · 10ꢀ5 m)/
DNA (2.5 · 10ꢀ5 m)/[NiL1] (2.5 · 10ꢀ5 m)
Spectrofluorimetric Titration and Ethidium Bromide (EB) Replacement Experi-
ment. Upon addition of DNA, the emission intensity of [NiL1], [ZnL1], [NiL2], and
[ZnL2] increases by a factor of ca. 1.21, 1.11, 1.3, and 1.15 ([DNA]/[complex] ¼ 2). The
results of the emission titrations suggest that the four complexes can be protected from

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solvent H2O molecules by the hydrophobic environment inside the DNA helix, since
the latter reduces the accessibility of solvent H2O molecules to the complex, and the
complex mobility is restricted at the binding site, leading to a decrease of the
vibrational modes of relaxation [24 – 26]. Hence, the fluorescence emission intensity of
the complexes is enhanced after the DNA is added. A spectrofluorimetric titration of
[NiL1] is shown in Fig. 4.
Fig. 4. Emission-enhancement spectra of [NiL1] (10 mm) in the presence of 0 – 20 mm DNA. The arrow
indicates the emission-intensity changes upon increasing DNA concentration.
The DNA-binding modes of the four complexes were further monitored by a
fluorescent EB displacement assay [27]. EB is a conjugate planar molecule. Its
fluorescence intensity is very weak, but it is greatly increased when EB is specifically
intercalated into the base pairs of double-stranded DNA. When EB is expelled by
another intercalator, the fluorescence of the DNA – EB complex is quenched [28].
Therefore, a fluorescence-based competition technique can provide indirect evidence
for the DNA-binding mode [29][30]. The experimental results show that the
fluorescence intensity of the DNA – EB system decreases upon the addition of each
of the four complexes. The results also imply that the complexes bind to DNA by
intercalation, and they show that the ZnII complexes exhibit a weaker interaction with
the DNA – EB system than the NiII complexes [31]. According to the linear Stern –
Volmer equation, the quenching constants Kq for [NiL2], [NiL1], [ZnL2], and [ZnL1]
are 1.30 · 105, 2.03 · 104, 7.44 · 103, and 2.22 · 103 mꢀ1, respectively (Fig. 5). This order is
entirely consistent with the results obtained from the spectrofluorimetric titrations.
Viscosity Measurements. To further clarify the interactions between the four
complexes and DNA, viscosity measurements were carried out. Hydrodynamic
measurements that are sensitive to length change (i.e., viscosity and sedimentation)
are regarded as the least ambiguous and the most critical tests of a binding model in
solution in the absence of crystallographic structural data. A classical intercalation
model demands that the DNA helix must lengthen as base pairs are separated to
accommodate the binding molecule, leading to an increase of DNA viscosity. In
contrast, a partial and/or nonclassical intercalation of a molecule could bend (or kink)
the DNA helix, reducing its effective length and, concomitantly, its viscosity. The effects
of the four complexes on the viscosity of DNA at 25.08 are shown in Fig. 6. The

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Fig. 5. Stern – Volmer plot of the fluorescence titration data of the complexes [NiL1], [NiL2], [ZnL1], and
[ZnL2]
viscosities of the DNA increase steadily with increasing amounts of each of the four
complexes. This behavior is consistent with that of other intercalators, e.g., EB. Thus,
the viscosity results clearly show that the four complexes can intercalate between
adjacent DNA base pairs, causing an extension in the helix [32].
Fig. 6. Effect of increasing amounts of the complexes on the relative viscosity of calf thymus DNA at 25.08
CD Spectra Studies. The foregoing studies suggest that the two NiII complexes
[NiL1] and [NiL2] bind more strongly to DNA than the ZnII complexes. We wanted to
find out if this strong interaction can change the conformation of DNA, so the intrinsic
CD spectra of calf-thymus DNA were examined. As shown in Fig. 7, the CD spectrum
of free DNA exhibits a negative band at 241 nm due to the helicity and a positive band
at 275 nm due to the base stacking, which are the characteristics of DNA in the right-
hand B form [33]. On addition of the complexes to DNA, there is an increase in

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ellipticity of the positive band at 275 nm, which is, however, accompanied by a redshift
of 4 and 7 nm for [NiL1] and [NiL2], respectively, of the negative band at 241 nm. This
result shows that the two NiII complexes can effectively interact with the DNA base
pairs and thus slightly change the DNA helicity of the B form [34][35].
Fig. 7. CD Spectra of DNA (6.0 · 10ꢀ5 m) in the presence (– – –) and absence (—) of NiII complex. [NiII
complex]/[DNA] 0.5.
Conclusion. – Two new chromenone-based Schiff bases and their NiII and ZnII
complexes were successfully synthesized. Electronic absorption spectra, fluorescence
spectra, and viscosity measurements indicate that the four complexes can bind to calf-
thymus DNA, presumably via an intercalation mechanism. The DNA-binding
constants K and the EB quenching constants Kq were determined. The primary reason
that the four complexes can intercalate into DNA may due to the planar structure of
the chromenone moiety and the enhancement of rigidity of the complexes. The
presented results may contribute to the development of transition-metal complexes
with chromenone derivatives of pharmaceutical value, and may also provide some
guidance for the development of intercalation compounds.
This work was supported by the National Natural Science Foundation of China (20475023) and the
Gansu NSF (0710RJZA012).
Experimental Part
1. General. Acetic anhydride, hydroquinone, and transition-metal nitrates were produced in China.
Calf-thymus DNA and ethidium bromide (EB) were purchased from Sigma Chemicals Co., USA. All the
experiments involving interaction of the complexes with DNA were carried out in doubly dist. H2O
buffer containing 5 mm Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol) and 50 mm NaCl, and
adjusted to pH 7.1 with aq. HCl soln. A soln. of calf-thymus DNA in the buffer gave a ratio of UV
absorbance of ca. 1.8 – 1.9 :1 at 260 and 280 nm, indicating that the DNA was sufficiently free of protein
[36]. The DNA concentration per nucleotide was determined by absorption spectroscopy and the molar
absorption coefficient (6600 mꢀ1 cmꢀ1) at 260 nm [37]. CD Spectra: Olos RSM 1000. UV/VIS Spectra:
Varian-Cary-100-Conc spectrophotometer; lmax (e) in nm. Fluorescence spectra: Hitachi-RF-4500
spectrofluorophotometer. IR Spectra: Thermo-Mattson-FT-IR spectrometer; KBr disks; ˜n in cmꢀ1
.

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1H-NMR Spectra: Varian-VR-300-MHz and Bruker-Avance-DRX-200-MHz spectrometer; in
(D6)DMSO; d in ppm, J in Hz. Elemental analyses: Vario-EL analyzer.
2. Ligands. L1 and L2 Hydroquinone Diacetate (¼ Benzene-1,4-diol Diacetate; 1). The mixture of
hydroquinone (22 g, 0.20 mol), Ac2O (42.0 g, 0.41 mol), and one drop of 98% H2SO4 soln. was stirred for
5 min. The gel-like product was poured into crushed ice, and the mixture left for 4 h. After filtration and
washing with H2O, the solid was recrystallized from 50% EtOH: 20.0 g (52%) of 1. White crystals. M.p.
120 – 1228. ([38] 1218).
2,5-Dihydroxyacetophenone (¼1-(2,5-Dihydroxyphenyl)ethanone; 2) [39]. The mixture of dry 1
(20.0 g) and dry AlCl3 (50.0 g) was heated to 110 – 1208 for 30 min (drying tube packed with CaCl2 and
HCl absorption unit), and then heated at 160 – 1658 for 3 h. After cooling, 36% HCl soln. (10 ml) was
added, and then the mixture poured into crushed ice. After filtration and several washings with H2O, the
solid was recrystallized from 95% EtOH: 10.0 g (64%) of 2. Green needles. M.p. 202 – 2038.
6-Hydroxy-4-oxo-4H-1-benzopyran-3-carboxaldehyde (3). POCl3 (20 ml) was added slowly and
dropwise into the soln. of dried 2 (3.0 g, 19.7 mmol) in dry DMF (40 ml) in an ice-water bath. Then the
mixture was stirred at r.t. overnight and then heated at 408 for 1 h. After cooling, the mixture was poured
into crushed ice and left for 4 h. Filtration and recrystallization of the solid from DMF/H2O gave pure 3
(2.4 g, 65%). Brown powder. M.p. 230 – 2318. IR: 1697 (C¼O), 1632 (CH¼O). 1H-NMR (300 MHz)1):
7.27 (dd, J ¼ 2.7, 9.0, HꢀC(7)); 7.39 (d, J ¼ 2.7, HꢀC(5)); 7.61 (d, J ¼ 9.0, HꢀC(8)); 8.84 (s, HꢀC(2));
10.1 (CHO); 10.24 (OH, exchangeable).
3-{[(1,5-Dihydro-3-methyl-5-thioxo-4H-1,2,4-triazol-4-yl)imino]methyl}-6-hydroxy-4H-1-benzopyr-
an-4-one (4; L1). A mixture of 3 (0.190 g, 1 mmol) and 4-amino-2,4-dihydro-5-methyl-3H-1,2,4-triazol-3-
thione (0.130 g, 1 mmol) in MeOH (40 ml) was refluxed for 10 min. Then, AcOH (0.5 ml) was added. The
yellow precipitate was washed several times with MeOH and dried in vacuo: 4 (¼ L1; 80%). M.p. 277 –
2788. IR: 1630 (C¼O), 1595 (C¼N), 1257 (C¼S), 3191 (NH), 2951 (Me). 1H-NMR (200 MHz)1): 2.34
(s, Me); 7.28 (dd, J ¼ 2.9, 9.0, HꢀC(7)); 7.39 (d, J ¼ 2.9, HꢀC(5)); 7.62 (d, J ¼ 9.0, HꢀC(8)); 9.05 (s,
HꢀC(2)); 10.22 (s, CH¼N); 10.26 (s, exchangeable, OH); 13.73 (s, exchangeable, NH).
2,2’-Bis[(6-hydroxy-4-oxo-4H-1-benzopyran-3-yl)methylene]carbonothioic Dihydrazide (5; L2). A
soln. of carbonothioic dihydrazide (0.053 g, 0.5 mmol) in MeOH (50 ml) was added dropwise to the soln.
of 3 (0.190 g, 1.0 mmol) in MeOH (30 ml) at 708. The resulting soln. was refluxed for 4 h, and then the
yellow precipitate was washed several times with MeOH: 5 (¼ L2; 75%). M.p. 235 – 2378. IR: 1633
(C¼O), 1590 (C¼N), 1237 (C¼S). 1H-NMR (300 MHz)1): 7.26 (dd, J ¼ 3.0, 8.7, HꢀC(7)); 7.32 (d, J ¼
3.0, HꢀC(5)); 7.48 (d, J ¼ 8.7, HꢀC(8)); 9.23 (s, HꢀC(2)); 10.16 (s, CH¼N).
3. Complexes. 3.1. Ligand L1 (0.151 g, 0.5 mmol) in MeOH (30 ml) was heated to 708, then
Ni(NO3)2 · 6 H2O (0.146 g, 0.5 mmol) in MeOH (5 ml) was added. After 30 min stirring, a clear light
green soln. was obtained. The clear soln. was concentrated, and then AcOEt was added. The resulting
yellow green solid was collected by centrifugation and washed with AcOEt including a small portion of
EtOH. The product [NiL1] was dried in vacuo. [ZnL1], a yellow solid, was prepared in the same way.
Aqua{3-{{[1,5-dihydro-3-methyl-5-(thioxo-kS)-4H-1,2,4-triazol-4-yl]imino-kN}methyl}-6-hydroxy-
4H-1-benzopyran-4-one-kO4}nickel(2þ) Nitrate Hydrate (1:2 :1) ([NiL1] or [NiL1 · H2O] · 2 NO3 · H2O):
Yield 65%. Thermal analysis: T 758 (H2O loss 3.35; calc. 3.46). Lm ¼ 166 S · cm2 · molꢀ1 (10ꢀ3 m in MeOH
at 258). UV: 202 (55100), 246 (52900) (1 · 10ꢀ5 m in MeOH/buffer soln. 1:99); d – d transition 525 (248)
(1 · 10ꢀ3 m in MeOH). IR: 1635 (C¼O), 1578 (C¼N), 1233 (C¼S), 3196 (NH), 1384 (NO3). Anal. calc.
for C13H14N6NiO11S (521.04): C 29.97, H 2.71, N 16.13; found: C 30.05, H 3.02, N 16.48.
Triaqua{3-{{[1,5-dihydro-3-methyl-5-(thioxo-kS)-4H-1,2,4-triazol-4-yl]imino-kN}methyl}-6-hy-
droxy-4H-1-benzopyran-4-one-kO4}zinc(2þ) Nitrate Hydrate (2 :4 :1) ([ZnL1] or [ZnL1 · 3(H2O)] ·
2 NO3 · 0.5 H2O): Yield 70%. Thermal analysis: T 728 (H2O loss 8.47; calc. 8.12). Lm ¼ 172 S · cm2 ·
molꢀ1 (10ꢀ3 m in MeOH at 258). UV (1 · 10ꢀ5 m in MeOH/buffer soln. 1:99): 204 (30900), 247 (23010).
IR: 1636 (C¼O), 1562 (C¼N), 1230 (C¼S), 3220 (NH), 1384 (NO3). Anal. calc. for C13H17N6O12.5SZn
(554.78): C 28.14, H 3.09, N 15.15; found: C 28.41, H 2.92, N 15.84.
3.2. Ligand L2 (0.135 g, 0.3 mmol) in MeOH (30 ml) was heated to 708, then Ni(NO3)2 · 6 H2O
(0.146 g, 0.5 mmol) in MeOH (5 ml) was added. After 30 min stirring, a clear light green soln. was
obtained. The clear soln. was concentrated, and then AcOEt was added. The resulting green solid was

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Helvetica Chimica Acta – Vol. 92 (2009)
collected by centrifugation and washed with AcOEt including a small portion of EtOH. The product
[NiL2] was dried in vacuo [ZnL2], a yellow solid, was prepared in the same way.
Bis{2,2’-bis[(6-hydroxy-4-oxo-4H-1-benzopyran-3-yl)methylene]carbonothioic
dihydrazide-
kN2,kN2’,kS}nickel(2þ) Nitrate (1:2) ([NiL2] or ([NiL2 ] · 2 NO3): Yield 75%. Lm ¼ 154 S · cm2 · molꢀ1
2
(10ꢀ3 m in MeOH at 258). UV: 202 (68000), 326 (38100) (1 · 10ꢀ5 m in MeOH/buffer soln. 1:99); dꢀd
transition 603 (73), 903 (63) (1 · 10ꢀ3 m in MeOH). IR: 1632 (C¼O), 1575 (C¼N), 1215 (C¼S), 1383
(NO3). Anal. calc. for C42H28N10NiO18S2 (1083.55): C 46.55, H 2.59, N 12.93; found: C 46.74, H 2.52, N
12.68.
Bis{2,2’-bis[(6-hydroxy-4-oxo-4H-1-benzopyran-3-yl)methylene]carbonothioic
dihydrazide-
kN2,kN2’,kS}zinc(2þ) Nitrate (1:2) ([ZnL2] or [ZnL2 ] · 2 NO3): Yield 70%. Lm ¼ 162 S · cm2 · molꢀ1
2
(10ꢀ3 m in MeOH at 258). UV (1 · 10ꢀ5 m in MeOH/buffer soln. 1:99): 202 (61900), 320 (20100). IR: 1632
1
(C¼O), 1569 (C¼N), 1219 (C¼S), 1384 (NO3). H-NMR (300 MHz): 7.32 (br., HꢀC(7)); 7.56 (br.,
HꢀC(5)); 7.88 (br., HꢀC(8)); 9.17 (br., HꢀC(2)); 10.13 (br., CH¼N). Anal. calc. for C42H28N10O18S2Zn
(1090.27): C 46.26, H 2.57, N 12.85; found: C 46.30, H 2.62, N 12.61.
4. Absorption Titration. Absorption titration experiments were performed with fixed conc. of the
complexes (10 mm) while gradually increasing the conc. of DNA (1 mm) at 258. While measuring the
absorption spectra, an equal amount of DNAwas added to both the complex soln. and the reference soln.
to eliminate the absorbance of DNA itself. To further compare quantitatively the affinity of the
complexes bound to DNA, based on the absorption titrations, the intrinsic binding constants K were
determined with Eqn. 1 [40], where [DNA] is the conc. of DNA in base pairs, ea, ef, and eb correspond to
the apparent absorption coefficient Aobsd/[complex], the extinction coefficient for the free complex, and
the extinction coefficient for the complex in the fully bound form, resp.
[DNA]/(ea ꢀ ef) ¼ [DNA]/(eb ꢀ ef) þ 1/[K(eb ꢀ ef)]
(1)
5. Fluorescence Spectra. Fixed amounts of complexes were titrated with increasing amounts of DNA
at 258. An excitation wavelength of 323 nm for [NiL1] and [ZnL1], and of 318 nm for [NiL2] and [ZnL2]
was used, and the fluorescence-emission intensity was monitored at 447 nm for L1 complexes and at
451 nm for L2 complexes.
EB is a common fluorescent probe for DNA structure and has been employed in examinations of the
mode and process of metal-complex binding to DNA. A soln. (2 ml) of 10 mm DNA and 0.025 mm EB [41]
was titrated by 5 – 25 mm complex (lex 500 nm, lem 520 – 650 nm) at 258. The data were plotted according
to the classic Stern – Volmer Eqn. 2 [42], where F0 is the emission intensity in the absence of quencher, F
is the emission intensity in the presence of quencher, Kq is the quenching constant, and [Q] is the
quencher conc. (Fig. 5). The shape of the Stern – Volmer plots can be used to characterize the quenching
as being predominantly dynamic or static. Plots of F0/F vs. [Q] appear to be linear, and Kq depends on
temperature.
F0/F ¼ Kq[Q] þ 1
(2)
6. Viscosity Measurements. Viscosity experiments were conducted on an Ubbelodhe viscometer,
immersed in a thermostatic water bath maintained at 25.08. Each complex was introduced into a DNA
soln. (10 mm) present in the viscometer. Data was presented as (h/h0)1/3 vs. the ratio of the conc. of the
complexes and DNA, where h is the viscosity of DNA in the presence of complexes and h0 is the viscosity
of DNA alone [43] (Fig. 6).
7. CD Spectra. The CD spectra were recorded at r.t. by increasing the complex/DNA ratio (r¼ 0.0,
0.5). Each sample soln. was scanned in the range 220 – 320 nm. The conc. of DNA was 6.0 · 10ꢀ5 m.
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Received July 2, 2008