Synthesis, crystal structures and biological evaluation of water-soluble zinc complexes of zwitterionic carboxylates

Article abstract of DOI:10.1016/j.ica.2011.06.054

Three water-soluble zinc complexes, [Zn(Cbp)₂Br₂] (1) (Cbp = N-(4-carboxybenzyl)pyridinium), {[Zn(BCbpy)₂(H ₂O)₄]₃Br₆·2(BCbpy) ·2(4,4′-bipy)} (2) (BCbpy = 1-(4-carboxybenzyl)-4,4

Full text of DOI:10.1016/j.ica.2011.06.054

Inorganica Chimica Acta 376 (2011) 389–395
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, crystal structures and biological evaluation of water-soluble zinc
complexes of zwitterionic carboxylates
Jin-Xiang Chen a, Wei-Er Lin a, Chun-Qiong Zhou a, Lee Fong Yau c, Jing-Rong Wang c, Bo Wang b,
Wen-Hua Chen a, , Zhi-Hong Jiang a,c,
⇑
⇑
a School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China
b School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, PR China
c School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
a r t i c l e i n f o
a b s t r a c t
Article history:
Three water-soluble zinc complexes, [Zn(Cbp)2Br2] (1) (Cbp = N-(4-carboxybenzyl)pyridinium),
{[Zn(BCbpy)2(H2O)4]3Br6ꢀ2(BCbpy)ꢀ2(4,40-bipy)} (2) (BCbpy = 1-(4-carboxybenzyl)-4,40-bipyridinium)
and {[Zn4(Bpybc)6(H2O)12](OH)8ꢀ9H2O}2n (3) (Bpybc = 1,10-bis(4-carboxybenzyl)-4,40-bipyridinium), were
synthesized and characterized by IR, elemental analysis and single-crystal X-ray crystallography. In com-
plex 1, the central Zn atom adopts a distorted tetrahedral coordination geometry that is formed from two
unidentate Cbp ligands and two Br atoms. For complex 2, the Zn atom in [Zn(BCbpy)2(H2O)4]2+ is strongly
coordinated by four water molecules and two N atoms from two BCbpy ligands, hence forming an octa-
hedral geometry. In complex 3, each Bpybc ligand bridges two [Zn(H2O)3]2+ units through two terminal
carboxylate groups in a monodentate coordination mode, thus forming a flowerlike two-dimensional net-
work. Agarose gel electrophoresis (GE) and ethidium bromide (EB) displacement experiments indicated
that complex 3 was capable of converting pBR322 DNA into open circular (OC) and linear forms, and
exhibited high binding affinity toward calf-thymus DNA. MTT assay showed that complex 3 displayed
inhibitory activities toward the proliferation of lung adenocarcinoma A549 and mouse sarcoma S-180
Received 12 March 2011
Received in revised form 14 June 2011
Accepted 30 June 2011
Available online 18 July 2011
Keywords:
Zwitterionic carboxylates
Zinc(II) complexes
Crystal structures
DNA cleavage
Biological evaluation
cells, with the IC50 values being 27.3 and 48.8 lM, respectively.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
Because zinc complexes having good water-solubility can find
wide potential applications [10], considerable efforts have been
spurred to develop their synthetic approaches [11–13]. One viable
approach to improve the water-solubility of the resulting zinc
complexes, is to incorporate carboxylate ligands with highly
hydrophilic groups [13,14]. Herein, we report the synthesis, crystal
structures and biological evaluation of water-soluble zinc
complexes 1–3 of three zwitterionic carboxylates having quater-
nary ammonium groups, including N-(4-carboxybenzyl)pyridini-
um bromide (HCbpBr), 1-(4-carboxybenzyl)-4,40-bipyridinium
bromide (HBCbpyBr) and 1,10-bis(4-carboxybenzyl)-4,40-bipyridi-
nium bromide (H2BpybcBr2) (Chart 1). Among them, it is reported
that HCbpBr and HBCbpyBr have diverse coordination modes
(Chart 2) [15–21].
It is well known that many metalloenzymes contain one or
more zinc(II) ions in the active sites [1–3]. These Zn(II) ions gener-
ally show high affinity toward the oxygen atom(s) of carboxyl
groups from carboxypeptidase [4]. Such zinc-containing active
sites play a crucial role in the catalytic processes and have been
characterized for their multiple activities [5,6]. Therefore, the
chemistry of zinc complexes of carboxylates has been receiving
an increasing attention. To date, a wide variety of model com-
pounds has been prepared with the aim to mimic the structures
and functions of the active sites of zinc metalloenzymes [7].
Remarkable among them are the biologically active compounds
that are synthesized from Zn2+ and amino acid derivatives, for
example, as anticonvulsant and antitumor agents and as synthetic
nucleases [8,9].
2. Experimental
2.1. General
⇑
Corresponding authors at: School of Pharmaceutical Sciences, Southern Medical
University, Guangzhou 510515, PR China. Fax: +86 20 61648533 (W.-H. Chen), +852
34112461 (Z.-H. Jiang).
IR spectra were recorded on a Nicolet MagNa-IR 550. Elemental
analyses for C, H, and N were performed on an EA1110 CHNS ele-
mental analyzer. Electrospray ionization mass (ESI-MS) spectra
E-mail addresses: whchen@smu.edu.cn
(Z.-H. Jiang).
(W.-H. Chen), zhjiang@hkbu.edu.hk
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.06.054

390
J.-X. Chen et al. / Inorganica Chimica Acta 376 (2011) 389–395
Br
Br
Br
N
N
N
COOH
HOOC
COOH
COOH
N
N
Br
HCbpBr
HBCbpyBr
H BpybcBr
2 2
Chart 1. Chemical structures of HCbpBr, HBCbpyBr and H2BpybcBr2.
N
N
N
O
O
M
O
O
O
O
O
M
O
O
O
N
N
M
M
N
O
M
M
O
O
M
M
O
N
N
M
Chart 2. Reported coordination modes of HBCbpyBr and H2BpybcBr2.
were measured on an Applied Biosystems Sciex API 4000 Qtrap
mass spectrometer. Agarose gel electrophoresis (GE) was con-
ducted on DYY-8C electrophoresis apparatus and DYCP-31DN elec-
trophoresis chamber, and detected on Alpha Hp 3400 fluorescence
and visible light digital image analyzer. Fluorescence spectra were
measured on a HITACHI F-2500 spectrofluorimeter.
Calf-thymus (CT) DNA and plasmid pBR322 DNA were obtained
from Sigma–Aldrich and Takara Chemical Co., respectively. Their
solutions were prepared in 5 mM Tris–HCl buffer (5 mM NaCl, pH
7.63). The concentration of CT DNA was determined spectrophoto-
metricallyusingthemolarextinctioncoefficientof13 200 Mꢁ1 cmꢁ1
per base pair (bp) at 260 nm [22]. HCbpBr, HBCbpyBr and H2Bpy
bcBr2 were prepared according to the reported protocols [16,23,
24]. All the other chemicals and reagents were obtained from com-
mercial sources and used without further purification. Buffer solu-
tions were prepared in triply distilled deionized water.
Elemental Anal. Calc. for C164H152Br6N20O28Zn3ꢀ26.7H2O: C, 49.15;
H, 5.16; N, 6.99. Found: C, 49.78; H, 4.74; N, 6.82%. IR (KBr disc,
cmꢁ1
) m 3446 (s), 3049 (w), 1614 (s), 1558 (w), 1456 (w), 1380
(s), 1158 (w), 811 (w), 744 (w).
2.2.3. {[Zn4(Bpybc)6(H2O)12](OH)8ꢀ9H2O}2n (3)
Similar procedures for the synthesis of 2 were employed, except
that H2BpybcBr2 (120 mg, 0.2 mmol) and Zn(NO3)2ꢀ6H2O (59 mg,
0.2 mmol) in H2O (5 mL) were used. Yield: 93 mg (82% based on
H2BpybcBr2). Elemental Anal. Calc. for C312H340N24O106Zn8: C,
56.39; H, 5.15; N, 5.06. Found: C, 55.91; H, 4.87; N, 4.92%. IR
(KBr disc, cmꢁ1
) m 3400 (s), 3047 (s), 1616 (s), 1597 (s), 1556 (s),
1395 (s), 1374 (s), 1164 (m), 1019 (w), 1808 (m), 770 (w), 512 (w).
2.3. X-ray structures of complexes 1–3
All the measurements were made on a Rigaku Mercury CCD
2.2. Synthesis of complexes 1–3
X-ray diffractometer by using graphite monochromated Mo K
a
(k = 0.71070 Å). Crystals of 1–3 were mounted with grease at the
top of a glass fiber, and cooled at 193 K in a liquid nitrogen stream.
Cell parameters were refined by using the program CrystalClear
(Rigaku and MSC, Ver. 1.3, 2001). The collected data were reduced
by using the program CrystalStructure (Rigaku and MSC, Ver. 3.60,
2004) while an absorption correction (multiscan) was applied.
The crystal structures of 1–3 were solved by direct methods and
refined on F2 by full-matrix least square methods with SHELXTL-97
program [25]. All non-hydrogen atoms were refined anisotropi-
cally, and all the hydrogen atoms were placed in geometrically ide-
alized positions. For 2, free 4,40-bipy was found to disorder over
two sites with an occupancy factor of 0.5/0.5 for N9/N9A, N10/
N10A, C73/C73A, C74/C74A, C75/C75A, C76/C76A, C77/C77A,
C78/C78A, C79/C79A, C80/C80A, C81/C81A and C82/C82A. The sol-
vent accessible void occupies a volume of 781.0 Å3 (8.3% of the to-
tal cell volume) and is filled with highly disordered H2O based on
the FT-IR spectra. Because the disorder models did not give satis-
factory results, the solvent contribution to the scattering factors
was taken into account with PLATON/SQUEEZE [26]. As a result, a
total of 267 electrons were found in each unit cell, corresponding
to 26.7H2O molecules per cell. Where relevant, the crystal data re-
ported in this paper contained no contribution from the disordered
2.2.1. [Zn(Cbp)2Br2] (1)
HCbpBr (118 mg, 0.4 mmol) was dissolved in H2O (5 mL), and
the pH was adjusted to 7 with 0.1 M NaOH solution. Then, a solu-
tion of Zn(NO3)2ꢀ6H2O (59 mg, 0.2 mmol) in H2O (5 mL) was added.
The resulting mixture was stirred for 30 min to give a clear solu-
tion, and then allowed to stand for 1 month to produce colorless
blocks. Subsequent washing with Et2O and drying under vacuum
yielded 1 (63 mg, 48% based on HCbpBr). Elemental Anal. Calc.
for C26H22Br2ZnN2O4: C, 47.92; H, 3.40; N, 4.30. Found: C, 47.45;
H, 3.37; N, 4.45%. IR (KBr disc, cmꢁ1
) m 1632 (w), 1594 (m), 1548
(w), 1507 (w), 1488 (w), 1416 (w), 1390 (w), 1374 (m), 1178 (w),
770 (m).
2.2.2. {[Zn(BCbpy)2(H2O)4]3Br6ꢀ2(BCbpy)ꢀ2(4,40-bipy)} (2)
To a solution of HBCbpyBr (149 mg, 0.4 mmol) in H2O (5 mL)
were added dropwise 0.1 M NaOH solution to adjust the pH 7,
and then a solution of Zn(NO3)2ꢀ6H2O (59 mg, 0.2 mmol) in H2O
(5 mL). The resulting mixture was stirred at room temperature for
1 h to give a clear solution, and then allowed to stand for 45 days
to produce colorless blocks. Subsequent washing with Et2O and
drying under vacuum yielded 2 (133 mg, 76% based on HBCbpyBr).

J.-X. Chen et al. / Inorganica Chimica Acta 376 (2011) 389–395
391
Table 1
Crystallographic data for 1–3.
solvent molecules. Furthermore, the hydrogen atoms for the four
disordered coordinated waters (O1W, O2W, O3W, and O4W) were
not located. For 3, the solvent accessible void occupies a volume of
352.0 Å3 (1.2% of the total cell volume) and may be due to the dis-
order of the solvent, because the largest electron-density peak is
1.793 e/Å3 and there are only eight electron-density peaks beyond
1. A summary of the key crystallographic information for 1–3 was
tabulated in Table 1.
Complex
1
2
3
Molecular
formula
Formula weight 651.65
Crystal system tetragonal
Space group
a (Å)
b (Å)
c (Å)
b (°)
C
26H22Br2N2O4Zn C164H128Br6N20O28Zn3 C312H340N24O106Zn8
3502.43
monoclinic
P21/c
21.989(3)
20.632(2)
20.891(2)
97.761(2)
6645.04
hexagonal
R3
34.965(4)
34.965(4)
27.408(5)
ꢀ
I41cd
14.292(2)
14.292(2)
24.233(5)
2.4. DNA cleavage experiments
c
(°)
120.00
29018.0(7)
3
The cleavage of supercoiled pBR322 DNA by complexes 1–3 was
studied by agarose GE. The reaction was carried out by mixing DNA
V (Å3)
4949.9(14)
8
9390.9(18)
2
Z
(0.35 g/L, 0.7
buffer (5 mM NaCl, pH 7.63, 5.5
incubated at 50 °C for 5 h, followed by the addition of loading buf-
fer (4 L) containing 0.035% bromophenol blue, 36% glycerol,
l
L), the complex (4 mM, 9.8
l
L) and 5 mM Tris–HCl
T (K)
293(2)
1.749
) (Å) 0.71073
110(2)
1.239
0.71073
1.722
291(2)
1.141
0.71073
0.564
Dcalc (g cmꢁ3
)
lL). The reaction mixture was
k (Mo K
a
l
(cmꢁ1
) 4.257
l
Total
24 136
51 037
41 611
30 mM EDTA and 0.05% xylene cyanol FF to quench the reaction.
The solution was then loaded on 1% agarose gel containing ethi-
dium bromide (EB) (1.0 mg/L), and analyzed with electrophoresis
in Tris–acetate–EDTA (TAE) buffer (pH 7.37). Bands were visual-
ized by UV light and photographed. The probable cleavage mecha-
nism was studied in a similar way, except in the presence of
several scavengers, including H2O2 (1.0 mM), DMSO (0.4 M), glyc-
erol (0.4 M) and MeOH (2.5 M).
reflections
Unique
reflections
Number of
observations
Number of
parameters
2471
2870
159
15 064
20 371
977
10 560
31 210
720
Ra
wRb
0.0908
0.1604
0.0805
0.1261
1.132
0.0873
0.1584
1.010
Goodness-of-fit 1.164
(GOF) on F2c
D
D
qmax (e Åꢁ3
)
1.579
ꢁ1.454
2.766
ꢁ2.044
1.793
ꢁ1.702
2.5. DNA binding experiments
qmin (e Åꢁ3
)
a
b
c
2.5.1. EB displacement experiments
EB displacement experiments of complexes 1–3 were per-
formed at room temperature. As a typical example, to a solution
R =
wR = {
GOF = {
R
||Fo| ꢁ |Fc|/
R|Fo|.
2
R
w(Fo ꢁ Fc2)2/
R .
w(Fo2)2}1/2
w((Fo ꢁ Fc2)2)/(n ꢁ p)}1/2
,
where n = number of reflections and
2
R
p = total numbers of parameters refined.
of CT DNA (1.5
pH 7.63) were added aliquots of complex 3 solution (0.2 M) in the
same buffer containing CT DNA (1.5 M) and EB (3.8 M). The cor-
lM) and EB (3.8 lM) in 5 mM Tris–HCl (5 mM NaCl,
l
l
perspective view of 1 is shown in Fig. 1. It can be seen that there is
a C2 axis through the central Zn atom that is strongly coordinated
by two unidentate Cbp ligands and two Br atoms, hence forming a
distorted tetrahedral ZnO2Br2 coordination geometry in which two
Cbp groups are oriented in the same directions. In addition, the Zn
atom has weak interactions with two O atoms from the same two
Cbp ligands. Such a structure closely resembles those of zinc
complexes of carboxylate ligands, such as {ZnBr2[HSCH(CH3)2
CH(NH3)COO]2} [28] and {ZnBr2[NH2COPy CH2COO]2} [29]. The
coordinated Zn–O, Zn–Br and the pendant Znꢀ ꢀ ꢀO bond lengths are
2.259(10), 2.634(2) and 2.620(8) Å, respectively. The bond angles
around the zinc(II) atom lie in the range from 93.1(3)° to 148.5(6)°.
responding fluorescence spectra were measured (kex = 490 nm) un-
til saturation was observed. The binding constants (Ka s) were
obtained by analyzing the relative fluorescence intensity (I/I0) as
a function of the concentrations of the added complexes 1–3 [27].
0
2.6. MTT assay
The cytotoxicities of complexes 1–3 were evaluated against hu-
man lung cancer cell line A549 and mouse sarcoma cancer cell line
S180, by using MTT assay. The cells were cultured in DMEM (for
A549) or RPMI (for S180) medium with 10% FBS. Exponentially
growing cells were seeded into a 96-well plate at a density of
1.5 ꢂ 104 cells/well and allowed to adhere for 16 h before treat-
ment (for A549 cells). Complexes 1–3 of varying concentrations
were added to each vial and incubated for another 48 h. Then,
3.1.2. Complex 2
It crystallizes in the monoclinic space group P21/c and the
asymmetric unit consists of one and half discrete {[Zn(BCbpy)2(-
H2O)4]} molecules, four Brꢁ anions (two of them having 0.5 occu-
pancies), one discrete BCbpy molecule and one disordered 4,40-
bipy molecule. As depicted in Fig. 2, there are two almost identical
but crystallographically independent molecules in complex 2. In
each molecular unit, the central Zn atom is strongly coordinated
by four water molecules and two N atoms from two BCbpy ligands,
thereby forming an octahedral geometry. Such a structure closely
resembles that of the corresponding cobalt complex of BCbpy li-
gand [15]. The O atoms from carboxylate groups of the two coordi-
nated BCbpy ligands have strong interaction with the O atoms from
the coordinated water molecules in the adjacent complex units.
Furthermore, the O atoms of the carboxylic groups from free BCbpy
ligands have strong interaction with the O atoms of the coordi-
nated water molecules from {[Zn(1)(BCbpy)2(H2O)4]} units, thus
generating one-dimensional centipede-like structure as shown in
MTT solution (5 mg/mL, 10
bated for 4 h at 37 °C. Then, lysing sodium dodecyl sulfate (SDS,
100 L) was added and the resulting solution was kept overnight
at room temperature. The optical densities were determined at
570 nm using a microplate reader. Dose–response curves were ob-
lL) was added to each well and incu-
l
tained and the results were expressed as IC50 values in
lM. Each
experiment was carried out for three times, and the mean values
were adopted.
3. Results and discussion
3.1. Crystal structures of complexes 1–3
3.1.1. Complex 1
It crystallizes in the tetragonal space group I41cd and the asym-
metric unit consists of half of the [Zn(Cbp)2Br2] molecules. The

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J.-X. Chen et al. / Inorganica Chimica Acta 376 (2011) 389–395
terminal carboxylic groups is ca. 18.82 Å [16]. The Zn–O bond
lengths are in the normal range.
3.1.4. Characterization of 1–3
Complexes 1–3 were further characterized by elemental analy-
sis and IR. Firstly, complexes 1 and 3 gave elemental compositions
that were consistent with their chemical formula. However, com-
plex 2 afforded a slightly high error in carbon elemental analysis,
possibly due to the loss of two solvated H2O molecules during
the course of sample preparation. In the IR spectra of 1–3, the ab-
sence of any strong bands around 1700 cmꢁ1 indicated that the
carboxylic acid groups of three ligands were deprotonated. In addi-
tion, the asymmetric and symmetric stretching vibrations of the
COO groups were observed at 1594/1390 cmꢁ1 for 1 and 1597/
1395 cmꢁ1 for 3, respectively. The difference in
s(COO) (
= 204 cmꢁ1 for 1 and 202 cmꢁ1 for 3) suggests that
mas(COO) and
m
D
Fig. 1. Molecular structure of 1 with 50% thermal ellipsoids. All the hydrogen atoms
are omitted for clarity. Symmetry transformations were used to generate equivalent
atoms: A: ꢁx, ꢁy + 2, z + 1.
the carboxylate groups coordinated to the metal ions only in a
monodentate bridging mode. The stretching vibrations of the
COO groups of 1 and 3 were shifted towards higher frequency com-
pared with that (observed at 1558/1380 cmꢁ1) of the carboxylate
group of 2, suggesting that the carboxylate group of 2 was not
coordinated to zinc ion [30].
Fig. 3. Interestingly, its difference from the corresponding cobalt
complex is that such chains are formed by alternating of every
two {[Zn(1)(BCbpy)2(H2O)4]} units and one {[Zn(2)(BCbpy)2(-
H2O)4]} unit. The contacts of Zn(1)ꢀ ꢀ ꢀZn(1A) and Zn(1)ꢀ ꢀ ꢀZn(2A)
are 16.898 and 17.144 Å, respectively. The Zn–N and Zn–O dis-
tances are in the normal range of 2.135(6)–2.153(5) Å and
2.074(4)–2.115(4) Å, respectively.
3.2. Cleavage of pBR 322 DNA
It is well known that many metal complexes are capable of cat-
alyzing the hydrolysis of DNA [7], therefore we investigated the
cleaving activities of complexes 1–3 toward pBR322 DNA, by using
agarose GE. Fig. 6 shows the GE patterns for the cleavage of pBR322
DNA by complexes 1–3 and their corresponding ligands at pH 7.63
and 50 °C. It can be seen that complex 3 could relax the supercoiled
form of DNA (CCC) to an open circular form (OC) (Lane 4), indicat-
ing that 3 was capable of cleaving pBR322 DNA. However, com-
plexes 1 and 2 (Lanes 2 and 3), the three ligands (Lanes 5–7) and
4,40-bipyridine (Lane 9) showed no cleaving activities. It should
be noted that compared with pBR322 DNA itself (Lane 1), less Form
II (OC) was observed in the presence of the three ligands and 4,40-
bipyridine. To gain insight into this phenomenon, we examined the
morphological changes induced by heating pBR322 DNA from 37
to 50 °C, as it is reported that thermal conversion of Form I (CCC)
into Form II (OC) may occur [31]. As shown in Fig. S1, the amount
of Form II (OC) increased with the increase in the temperatures
(Lanes 1–3, Fig. S1), whereas slightly decreased with the increase
in the concentrations of H2BpybcBr2 at 50 °C (Lanes 4–6, Fig. S1).
This result strongly suggested that the three ligands and 4,40-bipyr-
idine significantly retarded the thermal transition of pBR322 DNA
from Form I (CCC) to Form II (OC) at 50 °C, thus leading to the less
presence of Form II.
3.1.3. Complex 3
ꢀ
It crystallizes in the hexagonal space group R3 and the asym-
metric unit consists of one third of the discrete [Zn4(Bpyb-
c)6(H2O)12] molecules, four OHꢁ (one of them having one third
occupancies and two of them having two third occupancies) and
seven solvated H2O molecules (two of them having 0.5 occupancies
and four of them having 0.25 occupancies). As shown in Fig. 4,
there is a 3-fold axis located on the central Zn atom that is coordi-
nated by three water molecules, thereby forming a [Zn(H2O)3] unit.
Each Bpybc ligand bridges two [Zn(H2O)3] units through two ter-
minal carboxylic groups in a monodentate coordination mode,
thereby forming a flowerlike two-dimensional network as shown
in Fig. 5. The Zn atoms adopt an octahedral coordination geometry.
Because the methylene groups are used as knots to link the pyridyl
and phenyl rings, the whole Bpybc ligand is not linear but exhibits
a zigzag conformation. The two pyridyl rings of the 4,40-bipyridyl
group of Bpybc ligand are almost coplanar, whereas the phenyl
rings are twisted significantly from the adjacent pyridyl ring, with
a dihedral angle of 75.1(1)°. The linear distance between the two
Fig. 2. Molecular structure of 2 showing two almost identical mononuclear building blocks. All the hydrogen atoms are omitted for clarity. Symmetry transformations were
used to generate equivalent atoms: A: ꢁx + 1, ꢁy + 2, z + 1.

J.-X. Chen et al. / Inorganica Chimica Acta 376 (2011) 389–395
393
Fig. 3. One-dimensional double-stranded centipede-like structure formed by Oꢀ ꢀ ꢀO contacts in 2.
Fig. 4. Coordination environment of Zn(1) and Zn(2) in complex 3 with 50% thermal ellipsoids. All the hydrogen atoms are omitted for clarity. Symmetry transformations
were used to generate equivalent atoms: A: ꢁx ꢁ y + 1, x + 1, ꢁz; B: ꢁx + y + 1, ꢁx + 1, z.
Then, we carried out the concentration-dependent DNA cleav-
age by 3 (Fig. 7). It is clear that the conversion of the supercoiled
pBR322 DNA (Form I) to the nicked DNA (Form II) and linear
DNA (Form III) was enhanced with the increase of the concentra-
tions of complex 3 (Lanes 2–6), strongly suggesting that the cleav-
age was due to the presence of complex 3.
It is known that nucleic acid can be cleaved through an oxida-
tive or hydrolytic pathway. Therefore, to gain insight into the prob-
able mechanism of action, we conducted the cleavage reactions in
the presence of hydroxyl radical scavengers (DMSO, MeOH and
glycerol) and an oxidant (H2O2) (Fig. S2) [32]. As a result, none of
them had any influence on the DNA cleavage, strongly suggesting
that hydroxyl radical was not involved in the DNA cleavage and
that the cleavage might proceed via a hydrolytic rather than oxida-
tive mechanism.
The aforementioned observation may be rationalized by taking
into consideration the difference in the structures of complexes 1–
3. It is known that binding is a crucial step for DNA cleavage [13],
and that polynuclear metal complexes generally have potent cata-
lytic activity in DNA hydrolysis [7]. Complex 3 is composed of Bpybc
ligands having two positive charges, and thus, should show higher
DNA binding affinity than complexes 1 and 2, because of its stronger
electrostatic interaction with DNA. This and the polynuclear nature
may be the predominant factors for the higher DNA cleaving activity
of complex 3.
To verify these hypotheses, we firstly detected complex 3 in
aqueous solution by means of ESI mass spectrometry. As shown
in Fig. S3, one ion peak at m/z at 705.7 was observed, which was
assignable to [Zn4(Bpybc)6(H2O)-4H]4+. For further characteriza-
tion of the dissociation patterns of this abundant fragment ion,
the multiple stage mass spectrometric measurements were carried
out (Fig. S4). This ion was dissociated dominantly to the ion peaks
at m/z 425.5 and 292.1, which were assignable to ([HBpybc]+) and
([HBCbpy+H]+), respectively. These results strongly suggested that
the cluster 3, or the core [Zn4(Bpybc)6] fragment remained in water
solution.
To evaluate how strongly these complexes interact with DNA, we
measured the binding affinities toward CT DNA of complexes 1–3 by
means of EB displacement experiment. It is well known that EB can
intercalate non-specifically into DNA. Competitive binding of other
drugs to DNA leads to the displacement of bound EB and a decrease
in the fluorescence intensity [33]. Addition of complex 1 led to slight
quenching, suggesting that its interaction with CT DNA was very
weak (Fig. 8). Both complexes 2 and 3 were capable of substituting
EB bound to CT DNA, however, the latter was much more effective.
Nonlinear fitting analyses of the relative fluorescence intensity

394
J.-X. Chen et al. / Inorganica Chimica Acta 376 (2011) 389–395
Fig. 5. Extended flowerlike two-dimensional network structure of 3.
1.0
0.9
Fig. 6. Agarose GE patterns for the cleavage of pBR322 DNA by 1–3 and their
corresponding ligands (3.85 mM) at pH 7.63 and 50 °C. Lane 1: DNA alone; Lanes 2–
9: DNA with 1, 2, 3, HCbpBr, H2BpybcBr2, HBCbpyBr, Zn2+ and 4,40-bipy,
,
respectively.
0.8
0.7
0
20
40
60
80
100
Concentration (µM)
Fig. 8. Fluorescence decrease of EB (3.8
lM) induced by the competitive binding of
1 (j), 2 (N) and 3 (d) to CT DNA (1.5
l
M) in 5 mM Tris–HCl buffer (5 mM NaCl, pH
7.63) at room temperature (kex = 490 nm, kem = 594 nm).
Fig. 7. Agarose GE patterns for the cleavage of pBR322 DNA by 3 of increasing
concentrations at 50 °C and pH 7.63. Lane 1: DNA alone; Lanes 2–6: 0.3, 0.6, 1.2, 2.4
and 3.6 mM of 3, respectively.
Table 2
Cytotoxicities (IC50, lM) of complexes 1–3 and their corresponding ligands.
Complex
S180
A549
Complex
S180
A549
(I/I0) as a function of the concentrations of added 2 and 3 afforded
their binding constants being (2.55 0.50) ꢂ 104 Mꢁ1 and
(4.28 2.79) ꢂ 105 Mꢁ1, respectively.
1
2
3
>100
>100
27.3
>100
98.8
48.8
HCbpBr
HBCbpyBr
H2BpybcBr2
>100
>100
>100
>100
>100
>100
3.3. Biological activity
To exploit the potential application of complexes 1–3, we eval-
uated their biological activities against human lung cancer A549
and mouse sarcoma cancer S180, by using MTT assay [34,35]. Table
2 lists the IC50 values of complexes 1–3 and their corresponding li-
gands. It can be seen that complexes 1 and 2 and all the ligands
were inactive under our assay conditions, whereas complex 3
exhibited moderate inhibitory activity with the IC50 values being
27.3 lM toward lung adenocarcinoma A549 cells and 48.8 lM to-
ward mouse sarcoma S-180 cells, respectively. This result was in
agreement with their above-mentioned DNA cleavage activity.
4. Conclusions
In summary, three water-soluble zinc complexes of zwitterionic
carboxylates have been synthesized and characterized by IR, ele-
mental analysis and single-crystal X-ray crystallography. EB dis-
placement and GE experiments indicated that complex
3
displayed moderate ability to bind CT DNA and to cleave plasmid
pBR322 DNA from supercoiled to nicked and linear forms. In addi-
tion, biological study indicated that complex 3 could inhibit the
proliferation of lung adenocarcinoma A549 and mouse sarcoma

J.-X. Chen et al. / Inorganica Chimica Acta 376 (2011) 389–395
395
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This work was financially supported by the Medical Scientific
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