Crystal structures, DNA interaction and antiproliferative activities of the cobalt(II) and zinc(II) complexes of 2-amino-1,3,4-thiadiazole with demethylcantharate

Article abstract of DOI:10.1016/j.ica.2010.06.039

Two new mixed-ligand complexes [M(atdz)(DCA)(H₂O) ₂]·2H₂O, (M = Co(II), Zn(II); atdz = 2-amino-1,3,4-thiadiazole, C₂H₃N₃S; DCA = demethylcantharate, 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylate, C ₈H₈O₅) were prepared and characterized by elemental analysis. The structures of the complexes were determined by X-ray diffraction. The crystals have empirical formulas CoC₁₀H ₁₉N₃O₉S (1) and ZnC₁₀H ₁₉N₃O₉S (2), respectively. Complex 1 and 2 are monoclinic systems with space group P2₁/m. The structures of the complexes assume severely distorted octahedral geometries. The DNA binding properties of the complexes were investigated by electronic absorption spectra, thermal denaturation studies, fluorescence quenching studies and viscosity measurements. All the results showed the interaction modes between the complexes and DNA were partial intercalation. The results of agarose gel electrophoresis indicated the complexes could cleave supercoiled DNA. The antiproliferative activities testing revealed that all the complexes showed weak to moderate activities against human hepatoma cells (SMMC7721) and human breast cells (MCF-7) in vitro.

Full text of DOI:10.1016/j.ica.2010.06.039

Inorganica Chimica Acta 363 (2010) 3399–3406
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Crystal structures, DNA interaction and antiproliferative activities of the cobalt(II)
and zinc(II) complexes of 2-amino-1,3,4-thiadiazole with demethylcantharate
*
Na Wang, Qiu-Yue Lin , Jie Feng, Yu-Ling Zhao, Yan-Jun Wang, Shi-Kun Li
Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces, Zhejiang Normal University, Jinhua, Zhejiang 321004, China
College of Chemical and Life Science, Zhejiang Normal University, Jinhua, Zhejiang 321004, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new mixed-ligand complexes [M(atdz)(DCA)(H2O)2]Á2H2O, (M = Co(II), Zn(II); atdz = 2-amino-1,3,4-
thiadiazole, C2H3N3S; DCA = demethylcantharate, 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylate, C8H8O5)
were prepared and characterized by elemental analysis. The structures of the complexes were deter-
mined by X-ray diffraction. The crystals have empirical formulas CoC10H19N3O9S (1) and ZnC10H19N3O9S
(2), respectively. Complex 1 and 2 are monoclinic systems with space group P21/m. The structures of the
complexes assume severely distorted octahedral geometries. The DNA binding properties of the com-
plexes were investigated by electronic absorption spectra, thermal denaturation studies, fluorescence
quenching studies and viscosity measurements. All the results showed the interaction modes between
the complexes and DNA were partial intercalation. The results of agarose gel electrophoresis indicated
the complexes could cleave supercoiled DNA. The antiproliferative activities testing revealed that all
the complexes showed weak to moderate activities against human hepatoma cells (SMMC7721) and
human breast cells (MCF-7) in vitro.
Received 24 January 2010
Received in revised form 13 June 2010
Accepted 18 June 2010
Available online 25 June 2010
Keywords:
Demethylcantharate
2-Amino-1,3,4-thiadiazole
Cobalt(II) complex
Zinc(II) complex
DNA binding
Antiproliferative activity
Crown Copyright Ó 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction
complexes against human hepatoma cells (SMMC7721) and hu-
man breast cells (MCF-7) in vitro were evaluated with MTT assay.
Cantharidin (exo,exo-2,3-dimethyl-7-oxabicyclo[2,2,1]heptane-
2,3-dicarboxylic acid anhydride) has been used as a remedy for
the treatment of hepatoma and oesophageal carcinoma [1]. Dem-
ethylcantharidin (NCTD) formed from cantharidin, possesses anti-
cancer activity and has less effect on the urine system and
digestive system [2]. Disodium demethylcantharate (Na2(DCA))
can be dissolved in water easily and it could inhibit protein ser-
ine/threonine phosphatases (PP1, PP2A and PP2B) [3]. It was re-
ported that the metal complexes containing demethylcantharidin
displayed high anti-tumour activity [4–7]. Thiadiazole is an impor-
tant class of S, N-containing heterocycles and has been reported to
be biologically compound [8]. Complexes containing demethyl-
cantharidin and thiadiazole have not been reported [9–10].
In this paper, the complexes [M(atdz)(DCA)(H2O)2]Á2H2O,
(M = Co(II), Zn(II); atdz = 2-amino-1,3,4-thiadiazole; DCA = dem-
ethylcantharate) were synthesized by metal acetate, 2-amino-
1,3,4-thiadiazole and disodium demethylcantharate. The interac-
tion between the complexes and DNA was studied by electronic
absorption spectra, thermal denaturation studies, fluorescence
quenching studies and viscosity measurements. The DNA cleavage
experiments were carried out. The antiproliferative activities of the
2. Experimental
2.1. Materials and instruments
All reagents and chemicals were obtained from commercial
sources. Demethylcantharidin (NCTD = 7-oxabicyclo[2,2,1]hep-
tane-2,3-dicarboxylic acid anhydride, C8H8O4) was obtained from
Nanjing Zelang Medical Technology Co. Ltd.; Ethidium bromide
(EB) and 2-amino-1,3,4-thiadiazole (atdz, C2H3N3S) were obtained
from Fluka Company; Calf thymus DNA (ct-DNA) was purchased
from Huamei Co. and was stored at 4 °C; plasmid DNA (pBR322)
was purchased from Shanghai Biologic Engineering Co. and was di-
luted to 25 l
g mLÀ1. Human hepatoblastoma cells (SMMC7721)
and human breast cells (MCF-7) were purchased from Shanghai
Institute of Cell Bank. Other chemical reagents in analytical reagent
grade were used without further purification.
Elemental analyses of C, H and N were carried out in a Vario EL
III elemental analyzer. Diffraction intensities for the complexes
were collected at 293 K on a Bruker SMART CCD area detector
diffractometer. Electronic absorption spectra were recorded on a
UV-2501 PC spectrophotometer. Fluorescence emission spectra
were recorded with a Perkin–Elmer LS-55 spectrofluorometer. Vis-
cosity experiments were carried on an Ubbelodhe viscometer. Gel
* Corresponding author at: Zhejiang Key Laboratory for Reactive Chemistry on
Solid Surfaces, Zhejiang Normal University, Jinhua, Zhejiang 321004, China.
E-mail address: sky51@zjnu.cn (Q.-Y. Lin).
0020-1693/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2010.06.039

3400
N. Wang et al. / Inorganica Chimica Acta 363 (2010) 3399–3406
image formation was obtained on UNIVERSAL HOOD 11-S.N. (BIO-
RAD Laboratories).
(cDNA = 3.72 Â 10À4 mol LÀ1) by microsyringe. Flow time was mea-
sured by a digital stopwatch. The average values of three replicated
measurements were used to evaluate the viscosity of the samples.
2.2. Synthesis
Data were presented as (
tion of compounds to DNA, where
presence of compound and 0 was the viscosity of DNA alone. Vis-
g/g0)
1/3 versus the ratio of the concentra-
was the viscosity of DNA in the
g
2.2.1. Synthesis of Na2(DCA)
g
Na2(DCA) was prepared in accordance with the literature tech-
nique [4]. Demethylcantharidin (NCTD) (16.8 g, 0.1 mol) and NaOH
(4.8 g, 0.12 mol) were dissolved in water, then the mixed solution
was evaporated. The crude production was filtered off and washed
with ethanol. The product was dried in vacuum. Anal. Calc. for Na2-
C8H8O5: C, 41.74; H, 3.48. Found: C, 41.96; H, 3.52%.
cosity values were calculated from the observed flow time of DNA
solution in presence of the compounds corrected from the flow
time of buffer alone (t0),
g
= (t À t0)/t0 [12].
2.3.5. Interaction with pBR322 plasmid DNA
Interaction between the complexes and pBR322 plasmid DNA
was studied by agarose gel electrophoresis. The samples were
incubated for 3 h at 37 °C, then 0.25% bromophenol blue and
1 mmol LÀ1 EDTA were added. The DNA cleavage products were
subjected to electrophoresis on 1.0% agarose gel containing
2.2.2. Synthesis of the complexes
Complex 1: the solution of Co(Ac)2Á4H2O (0.25 g, 1 mmol) and
Na2(DCA) (0.21 g, 1 mmol) was stirred at the room temperature.
After 1 h, the solution of atdz (0.20 g, 2 mmol) was added dropwise
in the mixed solution. The crystals suitable for single-crystal X-ray
diffraction were obtained as pink flakes over a period of 2 weeks.
Anal. Calc. for CoC10H19N3O9S (1): C, 28.85; H, 4.60; N, 10.09.
Found: C, 29.09; H, 4.25; N, 9.98%.
0.5 l
g mLÀ1 ethidium bromides. The gels were run at 110 V for
1.2 h in tris–acetic acid–ethylenediaminetetraacetic acid (TAE)
buffer and the bands were photographed.
With similar way, complex
2
was synthesized with
2.4. Antiproliferative activity evaluation
Zn(Ac)2Á4H2O. Single crystals suitable for single-crystal X-ray dif-
fraction were obtained as block colourless crystals. Anal. Calc. for
ZnC10H19N3O9S (2): C, 28.41; H,4.53; N, 9.94. Found: C, 28.07; H,
4.38; N, 9.77%.
The antiproliferative activities of NCTD and complexes were
evaluated by using SMMC7721. The antiproliferative activities of
complexes were evaluated by using MCF-7 [13]. The antiprolifera-
tive activities were measured by the MTT assay. The compounds
were dissolved in DMSO as 100 mmol LÀ1 stock solutions and di-
luted with culture medium before used. The final concentration
of DMSO in the medium was less than 0.1% and it showed no inter-
ference with the biological activities tested [14]. After the cells
were seeded for 24 h, the complexes, NCTD were added and incu-
2.3. DNA binding and cleavage experiments
Calf thymus DNA (ct-DNA) was prepared with 0.1 mol LÀ1 NaCl.
The
concentration
of
ct-DNA
was
200 l
g mLÀ1
(cDNA = 3.72 Â 10À4 mol LÀ1). The ct-DNA solutions were stored at
4 °C and gave a ratio of UV–vis absorbance of 1.8 at 260 and
280 nm, indicating that DNA was sufficiently free of protein [11].
The buffer solution, (50 mmol LÀ1 NaCl, 5 mmolÁLÀ1 Tris–HCl
(tris(hydroxymethyl) aminomethane hydrochloride)) (pH 7.4)
was prepared with double-distilled water.
bated for 72 h. Then 100 l
L MTT (1 mg mLÀ1, dissolved in culture
medium) was added into each well and incubated for 4 h (37 °C).
The inhibition rate was calculated. The errors quoted standard
deviations and three replicates were used in the calculation of
these errors.
2.3.1. Electronic absorption spectra
Table 1
Electronic absorption spectra experiments were carried out at
room temperature by fixing the concentrations of the complexes
Crystal data and structure refinement.
(0.5
tion ranging from 0 to 23.3
l
mol LÀ1) while increasing amounts of DNA, over a concentra-
Complex
1
2
l
mol LÀ1. The complex-DNA solutions
Chemical formula
Formula weight
Crystal system
Space group
a (Å)
CoC10H19N3O9S
416.28
monoclinic
P21/m
6.36140(10)
9.5990(2)
12.4686(2)
90.00
95.8150(10)
90.00
ZnC10H19N3O9S
422.74
monoclinic
P21/m
6.3807(2)
9.6237(3)
12.4596(4)
90.00
95.966(2)
90.00
were allowed to pre-incubate for 5 min. Absorbencies were re-
corded after each successive addition of DNA solution and
equilibration.
b (Å)
c (Å)
2.3.2. Thermal denaturation experiments
Thermal denaturation experiments were carried out by moni-
toring the absorbance of 3.72 Â 10À5 mol LÀ1 DNA at 260 nm in
the absence and presence of 5 Â 10À6 mol LÀ1 complexes with
the temperature ranging from 25 to 100 °C.
a
(°)
b (°)
(°)
c
Volume (Å3)
Z
757.45(2)
2
760.95(4)
2
Crystal size (nm)
Shape
0.245 Â 0.136 Â 0.058 0.223 Â 0.122 Â 0.063
sheet
pink
block
2.3.3. Fluorescence quenching experiments
Colour
colourless
1.845
Fluorescence quenching experiments were carried out by add-
Dcalc (g/cmÀ3
)
1.825
ing complex solutions (0–25
l
mol LÀ1) to the samples containing
h rang for data collection (°) 1.64–27.65
Reflections collected/
2.68–27.57
6344/1858
0.13 l
l
mol LÀ1 EB and 37.2 mol LÀ1 DNA. The mixed solutions
12161/1873
were diluted by Tris–HCl buffer solution (pH 7.4). The final solu-
tions were incubated for 1 h at 4 °C. Then fluorescence was read
an excitation wavelength of 252 nm and an emission wavelength
between 520 and 700 nm.
unique
Rint
0.0363
1.326
0.0190
1.806
Absorption coefficient
(nmÀ1
)
F(0 0 0)
R/wR[I > 2
430
436
r(I)]
0.0334/0.1027
1873/6/133
1.049
0.511, À0.778
0.0244/0.0704
1858/6/133
1.051
0.392, À0.468
2.3.4. Viscosity measurements
Viscosity experiments were carried out on an Ubbelodhe vis-
cometer, immersed in a thermostatic water-bath maintained at
Data/restraints/parameters
Goodness-of-fit on F2
Largest difference in peak
and hole(e ÅÀ3
)
25 °C. The compounds were added into
a DNA solution

N. Wang et al. / Inorganica Chimica Acta 363 (2010) 3399–3406
3401
Table 2a
Table 3b
Selected bond lengths (Å) and angles (°) for complex (1), symmetry transformations
used to generate equivalent atoms: #1: x, Ày + 3/2, z.
Hydrogen bonds in the title complex(2) [nm and (o)] symmetry transformations used
to generate equivalent atoms: #1: x, Ày + 1/2, z; #2: x À 1, y, z; #3: Àx + 2, y + 1/2,
Àz + 1; #4: x + 1, Ày + 1/2, z; #5: Àx + 1, Ày + 1, Àz.
Complex 1
D–HÁ Á ÁA
Complex 2
N(3)–H(3B)Á Á ÁO(1)
d(D–H)
d(HÁ Á ÁA)
d(DÁ Á ÁA)
<(DHA)
Co(1)–O1W
Co(1)–O(2)#1
Co(1)–O(1)
O(1W)–Co(1)–
2.0631(16) Co(1)–O1W#1
2.1061(15) Co(1)–O(2)
2.0631(16)
2.1061(15)
2.095(2)
2.139(2)
Co(1)–N(2)
0.86
0.86
0.86
2.47
2.47
2.33
3.079(2)
3.079(2)
3.162(3)
128.9
128.9
163.4
94.56(10)
O(1W)#1–Co(1)–
O(2)#1
91.48(7)
N(3)–H(3B)Á Á ÁO(1)#1
N(3)–H(3C)Á Á ÁN(1)#2
O(1W)–H(1WA)Á Á ÁO(1)#3
O(1W)–H(1WB)Á Á ÁO(2)#4
O(2W)–H(2WA)Á Á ÁO(2)#1
O(1W)#1
O(2)#1–Co(1)–O(2)
O(1W)–Co(1)–O(1)
O(2)#1–Co(1)–O(1)
O(1W)–Co(1)–N(2)
O(2)#1–Co(1)–N(2)
O(1W)–Co(1)–O(2)#1
N(2)–Co(1)–O(1)
82.30(9)
86.64(6)
90.33(6)
90.98(7)
92.32(7)
173.07(6)
176.48(8)
O(2)–Co(1)–O(1W)
O(1W)#1–Co(1)–O(1)
O(2)–Co(1)–O(1)
O(1W)#1–Co(1)–N(2)
O(2)–Co(1)–N(2)
O(1W)#1–Co(1)–O(2)
91.48(7)
86.64(6)
90.33(6)
90.98(7)
92.32(7)
173.07(6)
0.811(15) 2.001(16) 2.8080(17) 173(2)
0.819(16) 1.978(16) 2.7785(17) 165(2)
0.822(16) 2.082(19) 2.8759(19) 162(3)
O(2W)–H(2WB)Á Á ÁO(2W)#5 0.727(16) 2.086(18) 2.808(3)
172(3)
3. Results and discussion
3.1. Structure description of the complexes
Table 2b
The X-ray structural analysis showed the types and the positions
of atoms were quite similar except for a replacement of the Co(II)
atom in 1 with a Zn(II) atom in 2. They had the same space group
(P21/m) and quite similar unit cell dimensions. In addition, there were
extensive hydrogen-binding interactions for the two complexes.
Selected bond lengths (Å) and angles (°) for complex (2) symmetry transformations
used to generate equivalent atoms: #1: x, Ày + 1/2, z.
Complex 2
Zn(1)–O(1W)
Zn(1)–O(1)#1
Zn(1)–O(3)
2.0783(12) Zn(1)–O(1W)#1
2.1341(11) Zn(1)–O(1)
2.1506(16) Zn(1)–N(2)
2.0783(12)
2.1341(12)
2.0652(19)
O(1W)#1–Zn(1)–O(1W)
O(1)#1–Zn(1)–O(1)
O(1W)#1–Zn(1)–O(3)
O(1)#1–Zn(1)–O(3)
O(1W)#1–Zn(1)–N(2)
O(1)#1–Zn(1)–N(2)
94.30(8)
81.78(7)
85.62(4)
88.98(4)
92.36(5)
93.28(5)
O(1W)–Zn(1)–O(1)#1 91.69(5)
O(1)–Zn(1)–O(1W)#1 91.69(5)
3.1.1. Crystal structure of the complex 1
Molecular structure of complex 1 is shown in Fig. 1a. Each Co(II)
ion was six-coordinated by two oxygen atoms O1W, O1WA from
water, one bridge oxygen O1, two carboxylate oxygen atoms O2,
O2A in two different carboxylate groups from demethylcantharate
and one nitrogen atom N2 from atdz. O2, O1W, O1WA and O2A lay
in the equatorial plane with the torsion angle 0.000(59)°. The
bridge oxygen atom O1 and the nitrogen atom N2 were in the axial
positions. The bond angle of O1–Co1–N2 was 176.48(8)°, so it
formed a distorted octahedral structure. Owing to the binding of
the bridge oxygen atom O1 with Co(II), two six-membered rings
(Co1–O1–C2–C3A–C4A–O2A) and (Co1–O1–C2A–C3–C4–O2) were
created. In addition, a seven-membered ring (Co1–O2–C4–C3–
C3A–C4A–O2A) was formed because of the coordination of carbox-
ylate oxygen atoms O2 and O2A, which could make the complex
more stable.
O(1W)–Zn(1)–O(3)
O(1)–Zn(1)–O(3)
O(1W)–Zn(1)–N(2)
O(1)–Zn(1)–N(2)
O(1W)–Zn(1)–O(1)
85.62(4)
88.98(4)
92.36(5)
93.28(5)
171.60(5)
O(1W)#1–Zn(1)–O(1)#1 171.60(5)
N(2)–Zn(1)–O(3) 177.02(6)
Table 3a
Hydrogen bonds in the title complex (1) [nm and (o)] symmetry transformations used
to generate equivalent atoms: #1: x, Ày + 3/2, z; #2: x + 1, y, z; #3: x À 1, y, z; #4: Àx,
Ày + 1, Àz; #5: x, Ày + 1/2, z.
D–HÁ Á ÁA
Complex 1
N(3)–H(3B)Á Á ÁO(2)
d(D–H)
d(HÁ Á ÁA)
d(DÁ Á ÁA)
<(DHA)
0.86
0.86
0.86
0.819(16)
0.822(16)
0.822(17)
0.808(18)
2.47
2.47
2.31
1.967(16)
1.993(17)
2.18(2)
2.042(19)
3.079(3)
3.079(3)
3.144(4)
2.779(2)
2.807(2)
2.891(3)
2.848(5)
128.8
128.8
162.9
171(3)
170(3)
145(3)
176(3)
Fig. 2a is the packing diagram of the complex 1. Structure
inspection indicated the intermolecular hydrogen bond N(3)–
H(3C)Á Á ÁN(1)#2, O(1W)-H(1WA)Á Á ÁO(3)#3 formed 1D chains. The
linear one-dimensional chained propagate along the b axis. Viewed
N(3)–H(3B)Á Á ÁO(2)#1
N(3)–H(3C)Á Á ÁN(1)#2
O(1W)–H(1WA)Á Á ÁO(3)#3
O(1W)–H(1WB)Á Á ÁO(2)#4
O(2W)–H(2WA)Á Á ÁO(3)#5
O(2W)–H(2WB)Á Á ÁO(2W)#5
down the
b
axis, hydrogen bonds N(3)–H(3B)Á Á ÁO(2), N(3)–
H(3B)Á Á ÁO(2)#1, O(1W)–H(1WB)Á Á ÁO(2)#4, O(2W)–H(2WA)Á Á Á
O(3)#5, O(2W)–H(2WB)Á Á ÁO(2W)#5 linked the 1D chained into a
3D network.
2.5. Crystal structure determination
The complexes
[Co(atdz)(DCA)(H2O)2]Á2H2O
3.1.2. Crystal structure of the complex 2
(1)
and
Fig. 1b was molecular structure of complex 2. Each Zn(II) ion
was six-coordinated by O1W, O1WA from water, one bridge oxy-
gen O3 and two carboxylate oxygen atoms O1, O1A from demeth-
ylcantharate, N2 from atdz. O1, O1A, O1W and O1WA lay in the
equatorial plane with the torsion angle 0.000(45)°. The O3 and
the N2 were in the axial positions. It also formed a distorted octa-
hedral structure because the bond angle of O3–Zn1–N2 was
177.02(6)°. For the binding of the bridge oxygen atom O3 with
Zn(II), two six-membered rings (Zn1–O1A–C4A–C3A–C6–O3) and
(Zn1–O1–C4–C3–C6A–O3) and a seven-membered ring (Zn1–O1–
C4–C3–C3A–C4A–O1A) were created.
As shown in Fig. 2b, further inspection showed intermolecular
hydrogen bonds N(3)–H(3B)Á Á ÁO(1), N(3)–H(3B)Á Á ÁO(1)#1, N(3)–
H(3C)Á Á ÁN(1)#2, O(1W)–H(1WA)Á Á ÁO(1)#3, O(1W)-H(1WB)Á Á Á
O(2)#4, O(2W)–H(2WA)Á Á ÁO(2)#1, O(2W)-H(2WB)Á Á ÁO(2W)#5
linked the complexes into a 3D network.
[Zn(atdz)(DCA)(H2O)2]Á2H2O (2) were structurally characterized
using single-crystal X-ray diffraction technique. Crystal data and
experimental details for structural analyses are listed in Table 1.
Selected bond lengths and bond angles are presented in Table 2
and the hydrogen bond lengths and bond angles are given in Table
3. The crystals were placed on the glass fibre for data collection
which performed on a Bruker SMART CCD area detector diffrac-
tometer. Data were collected with a graphite monochromatic Mo
Ka radiation (k = 0.071073 nm) using the x-2h scan technique at
296(2) K. The structures were solved by direct methods and ex-
panded with Fourier techniques. The non-hydrogen atoms were re-
fined anisotropically and partial hydrogen atoms were located by
geometrical calculation, while the positions and thermal parame-
ters of water H atoms were fixed in a difference Fourier map. All
calculations were carried out with SHELXTL-97 package [15].

3402
N. Wang et al. / Inorganica Chimica Acta 363 (2010) 3399–3406
Fig. 1. Molecular structure of complex (1) (a) and complex (2) (b).
3.2. DNA binding and cleavage studies
where [DNA] was the concentration of DNA, eA, eF and eB corre-
sponded to the apparent extinction coefficient, the extinction coef-
3.2.1. Electronic absorption spectra
ficient for the free compound and its fully DNA-bound
Electronic absorption spectroscopy is usually used to determine
the binding strength and mode of DNA with the complexes [16].
The absorption spectra of the title complexes with increasing con-
centrations of ct-DNA are given in Fig. 3. As increasing concentra-
tion of ct-DNA, the absorption bands of the complexes at 207 nm
(1), 208 nm (2) were affected, resulting in hypochromicity and
slight red shift.
combination, respectively. In the plots of [DNA]/(eA
[DNA], Kb was given by the ratio of the slope to intercept. The bind-
ing constants Kb for the complexes (a) and (b) were
À
eF) versus
1
2
2.26 Â 102 L molÀ1 and 1.85 Â 103 L molÀ1, respectively. The bind-
ing strength of the classical intercalator ethidium bromide (EB)
with DNA was two and three orders of magnitude higher than that
of complex 1 and complex 2, respectively [18]. So the binding
mode between the complexes and DNA were not classical
intercalative.
The intrinsic binding constant was determined according to this
equation: [DNA]/(eA
À
eF) = [DNA]/(eB
À
eF) + 1/[Kb(eB
À
eF)] [17],

N. Wang et al. / Inorganica Chimica Acta 363 (2010) 3399–3406
3403
Fig. 2. Packing diagram of the complexes, showing hydrogen bonding interactions (dashed lines). Complex (1) (a) and complex (2) (b).
3.2.2. Thermal denaturation studies
3.2.3. Fluorescence quenching studies
Thermal denaturation studies of ct-DNA with the complexes
provide evidence for the ability of the complexes to stabilize the
double-stranded DNA [19]. The melting temperature Tm of DNA
characterizes the transition from double-stranded to single-
stranded nucleic acid [20]. The interaction between DNA and small
molecules results in the increasing helix melting temperature [21–
23].The effect of the complexes on the melting temperature (Tm) of
calf thymus DNA in buffer is shown in Fig. 4. In this experiment,
the Tm of ct-DNA alone was 74 °C. After added the complexes,
Fig. 5 shows that the emission spectra of EB bounded to DNA
with complex (1) (a), complex (2) (b) or not. As the increasing con-
centrations of the complexes, the emission intensity at 592 nm of
EB-DNA system decreased in different degrees.
According to the classical Stern–Volmer equation [26]: I0/
I = 1 + Ksqr, where I0 and I represented the fluorescence intensities
in the absence or presence of the complexes, respectively. r was the
concentration ratio of the complexes to DNA. Ksq was a linear
Stern–Volmer quenching constant. The Ksq value was obtained as
the slope of I0/I versus r linear plot. From the inset in Fig. 5, the
Ksq value were 0.61, 3.41 and 0.54 for the complex 1, 2 and atdz,
which suggested that the interaction of the compounds with
DNA were strong and could release some free EB from EB-DNA, be-
cause of the present of planar thiadiazole rings. NCTD could not
quench the emission intensities of EB-DNA system obviously
[27]. The crystal data (in Table 2) indicated that the complexes
could partially insert into the DNA double helix, because of their
distorted octahedral structures and the planer thiadiazole rings
of the complexes. The torsion angle of the equatorial plane of com-
D
Tm increased by 2.7 and 2.9 °C for complex 1 and complex 2,
respectively. The factors within a DNA double helix may account
for the equilibrium structure, the dynamics of helix formation
and distortion, and even the high fidelity of replication, including:
hydrogen bonding interaction between complementary base pairs,
hydrophobic interaction and base stacking interaction. The interac-
tion of DNA with complexes 1 and 2 led to the stabilization of the
double helix with the increasing Tm, because of the intercalation of
the planer thiadiazole rings and hydrogen bonds between oxygen,
nitrogen, sulfur atoms of atdz and DNA base pairs.
It has been reported that
D
Tm for metallointercalators (>10 °C)
plex 2 (0.000(45)°) was smaller than that of complex 1
[23–25] and the organic intercalator EB (13 °C) [21] were higher
than complex 1 and 2. These results indicated the binding strength
between the complexes and DNA was only moderate [19].
(0.000(59)°). The bond lengths of Zn(1)–N(2) (2.0652(19) Å) was
shorter than that of Co(1)–N(2) (2.095(2) Å). The symmetry and
the complexing ability of atdz with M(II) in complex 2 were better

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N. Wang et al. / Inorganica Chimica Acta 363 (2010) 3399–3406
Fig. 3. Absorption spectra of the complexes in the presence of increasing amount of
ct-DNA. Arrows show the absorption changes upon increasing DNA concentration.
For (1) (a) and (2) (b): ccomplex = 0.5 Â 10À6 mol LÀ1; from a to e: cDNA Â 105 = 0, 9.3,
14, 18.6, and 23.3 mmol LÀ1, respectively.
Fig. 5. Emission spectra of EB-DNA system in the absence and presence of complex
(1) (a), complex (2) (b) and atdz (c); insert in fig 5: fluorescence quenching curve of
DNA-bound EB by the complexes kex = 252 nm, kem = 520–700 nm. cEB = 1.3 Â 10À7
mol LÀ1, from (a) to (e): r = ccompound/cDNA = 0, 0.13, 0.27, 0.54, and 0.67, respectively.
Fig. 4. Plots of the changes of absorbance at 260 nm of ct-DNA on heating in the
absence and the presence of the complexes. cDNA = 3.72 Â 10À5 mol LÀ1 and
ccomplex = 5.0 Â 10À6 mol LÀ1 in 1 mmol LÀ1 Tris–HCl with 10 mmol LÀ1 NaCl.
3.2.4. Viscosity measurements
To further investigate the binding abilities of the compounds
with DNA, viscosity measurements were carried out on ct-DNA
by varying the concentrations of the added compounds. Intercala-
tive DNA binding results in an increase in viscosity, because this
binding mode causes elongation of DNA polymer by effecting
than that in complex 1. The factors facilitated higher intercalation
strength with DNA for complex 2.

N. Wang et al. / Inorganica Chimica Acta 363 (2010) 3399–3406
3405
Table 4
separation of DNA base pairs. A partial or non-classical intercalation
Selected cleavage data of pBR322 DNA by the complexes.
of the compounds can bend or kink DNA resulting in a decrease in its
effective length with a concomitant decreasing in its viscosity [28].
The effects of the complexes, atdz and NCTD on the viscosity of
ct-DNA at 25 °C are studied (Fig. 6). The relative viscosity of DNA
had no obvious change in the present of NCTD. It could not insert
the DNA double helix, because of the rigid non-planarity of the
six-membered ring and the hindering effect of ether oxygen atom
in NCTD [27]. The complexes with planer thiadiazole rings could
partially insert into the DNA base pair and form partial stacking
of the complexes with the base pairs [29]. The decreasing degrees
of DNA viscosity for the complexes were higher than that for atdz.
The interaction of DNA with complexes 1 and 2 led to the stabiliza-
tion of the double helix, because of hydrogen bonding interaction
between oxygen, nitrogen, sulfur atoms of atdz and DNA base pairs
Serial no.
Reaction condition
Form (%)
I
II
1
2
3
4
5
6
7
8
DNA control
82.28
77.88
75.24
74.32
82.28
77.35
68.35
60.12
17.72
22.12
24.76
25.68
17.72
22.65
31.65
39.88
DNA + 1 (100
DNA + 1 (200
DNA + 1 (300
DNA control
DNA + 2 (100
DNA + 2 (200
DNA + 2 (300
l
l
l
M)
M)
M)
lM)
lM)
lM)
[30]. The results showed that the binding modes of the complexes
with DNA were partial intercalation.
3.2.5. Cleavage of pBR322 plasmid DNA
When circular pBR322 plasmid DNA is subjected to agarose gel
electrophoresis, relatively fast migration can be observed for the
intact supercoil Form I. The cleavage of Form I is processed to pro-
duce slower moving open-circular Form II [31]. The cleavage abil-
ities of the complex (1) (a) and the complex (2) (b) to circular
plasmid pBR322 DNA were monitoring by gel electrophoresis
(Fig. 7). Selected DNA cleavage data are given in Table 4. The sam-
ples containing plasmid DNA and different concentrations of the
complexes (100–300 l
mol LÀ1) were incubated at physiological
condition. Complex 2 promoted the cleavage of plasmid DNA more
efficiently than complex 1. The structures of the complexes
showed that specific binding existed between the small molecules
containing imidazole and AT base pairs of DNA [30]. Complexes
containing benzimidazole could promote the hydrolysis of phos-
phate diester and the hydrolytic cleavage of pBR322DNA [32].
Complex 1 and 2 containing thiadiazole which had similar struc-
ture with imidazole could promote the hydrolytic cleavage of
pBR322DNA.
3.3. Antiproliferative activity evaluation
Fig. 6. Effect of increasing amounts of the compounds on the relative viscosity of
DNA, at 25 °C. cDNA = 3.72 Â 10À4 mol LÀ1; ccompound = 0, 0.005, 0.010, 0.015, 0.020,
and 0.025 mmol LÀ1, respectively.
As shown in Fig. 8a, the antiproliferative activities of the NCTD,
complex 1 and complex 2 at concentrations of 25, 50, 100,
200 l
mol LÀ1 against human hepatoma cells (SMMC7721)
in vitro viability were investigated. The results indicated NCTD
and the complexes had strong antiproliferative effect, but atdz al-
most had no inhibition activity against SMMC7721 cell lines in the
range of tested concentration. Complex 1 and 2, which exhibited
almost equal efficiency, were more active than NCTD. At
50
2 (33 2.9%) were much higher than that of NCTD (8.2 1.1%). At
lower concentrations (25
mol LÀ1), inhibition rates of the com-
l
mol LÀ1, inhibition rate of complex 1 (35 7.9%) and complex
l
plex 1 were much higher than that of the complex 2.
The IC50 value for NCTD, the complexes 1 and 2 against
SMMC7721 cell lines were 116 9.5, 87 12 and 79 4.7 lmol
LÀ1, respectively. Complex 1 and 2 exhibited almost equal effi-
ciency and were all more active than NCTD.
As shown in Fig. 8b, the antiproliferative activities of complex 1
and complex 2 at concentrations of 18.75, 37.5, 75, 150 and
300 l
mol LÀ1 against human breast cells (MCF-7) in vitro viability
were investigated. The inhibition rate of complex 1 were higher
than that of complex 2. The IC50 value for the complexes 1 and 2
against MCF-7 cell lines were 54 7.3 and 66 9.2
respectively.
l ,
mol LÀ1
The results indicated that the structure of heterocycle contain-
ing nitrogen and sulfur atoms was favourable to norcantharidin
antiproliferative activity. There was the difference in antiprolifera-
tive activity because of the different central ion M(II).
Fig. 7. Electrophoretic separation of pBR322 DNA induced by the complex (1) (a),
the complex (2) (b). [DNA] = 3 l
g mLÀ1. Lane 1: DNA alone; lane 2: DNA + complex
(100
l
l
mol LÀ1); lane 3: DNA + complex (200
mol LÀ1).
l
mol LÀ1); lane 4: DNA + complex
(300

3406
N. Wang et al. / Inorganica Chimica Acta 363 (2010) 3399–3406
proliferative activity against human hepatoma cells (SMMC7721)
and human breast cells (MCF-7). The experiments will be signifi-
cant for the further development of demethylcantharidin drugs
and improvement of the medicinal value of demethylcantharidin.
All the results will be helpful to understand the relation among
the binding ability of the complex-DNA, the cleaving activity and
the antiproliferative activities.
Acknowledgement
We thank Institute of matria medica of Zhejiang Academy of
Medical Sciences for help with anti-proliferative activity test.
Financial support from Natural Science Foundation of Zhejiang
Province, China (Grant No. Y407301) is gratefully acknowledged.
Appendix A. Supplementary material
CCDC 715017 and 713142 contains the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.ica.2010.06.039.
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4. Conclusions
The crystals of the target complexes [M(atdz)(DCA)
(H2O)2]Á2H2O, (M = Co(II), Zn(II); atdz = 2-amino-1,3,4-thiadiazole;
DCA = demethylcantharate) were obtained and determined by
X-ray diffraction. The results of electronic absorption spectra, ther-
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complexes and DNA were partial intercalation. Zn(II) complex
had the higher binding ability with DNA than Co(II) complex. Zn(II)
complex promoted the cleavage of plasmid DNA more efficiently
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