Mixed-ligand manganese(II)-phenolate complexes: Study of DNA cleavage, cytotoxic activity, and induction of apoptosis

Article abstract of DOI:10.1080/00958972.2011.634910

Two manganese complexes, [Mn(L¹)₂(dibe) ₂](L¹) (1) and [Mn(L²)₂(dibe) ₂] (2) (where L¹= 2,2'- bipyridine (bipy), L ²=1,10-phenanthroline (phen)), were synthesized by using a carboxylic acid ligand (dibe= 2,2-dibenzylmalonate acid). The two complexes were characterized using IR, elemental analysis, and X-ray crystallography. Fluorescence analysis indicates that the two complexes can bind to HeLa cell DNA (HC-DNA), and gel electrophoresis assay demonstrates the ability of the complexes to cleave the HC-DNA. The two complexes exhibit cytotoxic specificity and significant cancer cell inhibitory rate. Furthermore, apoptotic tests demonstrate that these two complexes have apoptotic effects on HeLa cells. 2011 Taylor & Francis.

Full text of DOI:10.1080/00958972.2011.634910

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Mixed-ligand manganese(II)-phenolate
complexes: study of DNA cleavage,
cytotoxic activity, and induction of
apoptosis
a
a
a
a
a
En-Jun Gao , Ying Zhang , Lin Lin , Ren-Shu Wang , Lei Dai
a
a
a
a
,
Qi Liang , Ming-Chang Zhu , Mei-Lin Wang , Lei Liu , Wen-
a
a
Xuan He & Yan-Jin Zhang
a
Department of Coordination Chemistry, International Key
Laboratory of Shenyang Inorganic Molecule-Based Chemical,
Shenyang University of Chemical Technology, Shenyang 110142,
China
Version of record first published: 11 Nov 2011
To cite this article: En-Jun Gao, Ying Zhang, Lin Lin, Ren-Shu Wang, Lei Dai, Qi Liang, Ming-Chang
Zhu, Mei-Lin Wang, Lei Liu, Wen-Xuan He & Yan-Jin Zhang (2011): Mixed-ligand manganese(II)-
phenolate complexes: study of DNA cleavage, cytotoxic activity, and induction of apoptosis, Journal
of Coordination Chemistry, 64:22, 3992-4005
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Journal of Coordination Chemistry
Vol. 64, No. 22, 20 November 2011, 3992–4005
Mixed-ligand manganese(II)-phenolate complexes: study of
DNA cleavage, cytotoxic activity, and induction of apoptosis
EN-JUN GAO*, YING ZHANG, LIN LIN, REN-SHU WANG,
LEI DAI, QI LIANG, MING-CHANG ZHU, MEI-LIN WANG,
LEI LIU, WEN-XUAN HE and YAN-JIN ZHANG
Department of Coordination Chemistry, International Key Laboratory of Shenyang
Inorganic Molecule-Based Chemical, Shenyang University of Chemical Technology,
Shenyang 110142, China
(
Received 21 August 2011; in final form 14 October 2011)
1
Two manganese complexes, [Mn(L )
1
2
](L ) (1) and [Mn(L )
1
0
2
(dibe)
2
2
(dibe)
2
] (2) (where L ¼ 2,2 -
2
bipyridine (bipy), L ¼ 1,10-phenanthroline (phen)), were synthesized by using a carboxylic acid
ligand (dibe ¼ 2,2-dibenzylmalonate acid). The two complexes were characterized using IR,
elemental analysis, and X-ray crystallography. Fluorescence analysis indicates that the two
complexes can bind to HeLa cell DNA (HC-DNA), and gel electrophoresis assay demonstrates
the ability of the complexes to cleave the HC-DNA. The two complexes exhibit cytotoxic
specificity and significant cancer cell inhibitory rate. Furthermore, apoptotic tests demonstrate
that these two complexes have apoptotic effects on HeLa cells.
Keywords: Manganese; Fluorescence; Cleavage; Cytotoxicity; Apoptosis
1. Introduction
There has been substantial interest in the design and investigation of transition-metal
anticancer drugs [1–4]. Metal-based anticancer drugs, e.g. cisplatin, are widely used
in clinics. However, severe side effects occur after treatment [5–10]. Accordingly, it is
indispensable to develop less toxic, more-efficacious and target-specific metal-based
anticancer drugs in pharmaceutical research. Numerous transition-metal complexes are
synthesized and screened for their anticancer activities.
The design of these metal-based pharmaceuticals depends on the ligand framework,
the metal ion, and its oxidation state. Ligands can significantly alter the biological
activities of complex. Tailored, multifunctional ligands introduced into metal-based
medicinal agents limit the adverse effects of metal ion [11–13]. Carboxylic acids are
widely used as ligands as they can easily coordinate with metal by protonation [14].
Research on complexes with 1,10-phenanthroline (phen) and bipyridine (bipy) show
that the complexes have strong intercalation with DNA, high DNA cleavage efficiency
and cytotoxicity [15, 16]. We have reported palladium(II), platinum(II), copper(II),
*
Corresponding author. Email: ejgao@yahoo.com.cn
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2011.634910

Manganese(II)-phenolate
3993
CH (COOC H )
2
2
5 2
N
N
Ph
O
N
C H ONa
Bipy
CH Cl
O
2
O
N
(1)
HO
O
O
OC H
2 5
O
Ph
Ph
Ph
O
Ph
Ph
O
OC H
2
5
HO
NaOH
H O
2
HCl
N
7
Mn2+
N
O
OH
6'
N
4
2
5
1
5' 4'
Ph
6
3
3'
2'
N
Phen
O
HO
(
2)
1
'
O
O
O
HO
7
'
Ph
OH
O
O
dibe
Ph
Ph
O
1
Scheme 1. Schematic structure of the two complexes and the numbering scheme for H NMR spectroscopy.
cobalt(II), and zinc(II) complexes with prominent anticancer activities [17–25]. As an
important transition-metal element, manganese can also form complexes and some
manganese complexes exhibit excellent biological activities [26, 27].
1
1
In the present work, two Mn(II) complexes, [Mn(L ) (dibe) ](L ) (1) and
2
2
2
[
Mn(L ) (dibe) ] (2), were synthesized and characterized (dibe is 2,2-dibenzylmalonate
2 2
1
2
acid synthesized in our laboratory, shown in scheme 1). L and L are bipy and phen,
respectively. The DNA-cleaving and Hela cell apoptosis of the complexes have been
investigated by gel electrophoresis and MTT assay, respectively. The cytotoxic effects
of these complexes were examined on four cancer cell lines, HeLa, Hep-G2, KB, and
AGZY-83a.
2
2
. Experimental
.1. Materials
Chemicals purchased were of reagent-grade and used without purification unless
otherwise noted. All spectroscopic titration was carried out in aerated buffer
ꢀ
1
ꢀ1
(
5 mmol L Tris-HCl, 50 mmol L NaCl, pH ¼ 7.2) at room temperature. HC-DNA
applied here was extracted by us. The HeLa (human cervix epitheloid carcinoma) cells,
the Hep-G2 (human hepatocellular carcinoma) cells, the KB (human oral epithelial

3
994
E.-J. Gao et al.
carcinoma) cells and the AGZY-83a (human lung carcinoma) cells were obtained from
American Type Culture Collection.
2.2. Ligand synthesis
A total of 80 mL of sodium ethoxide and 30 mL of diethyl malonate were mixed, stirred
ꢁ
and heated to 80 C for 6 min, then 50 mL of benzyl chloride was dropwise added to the
mixture. The mixture was refluxed for 15 min, allowed to cool and washed with
deionized water. The organic phase was removed and the residue was extracted with
ethyl acetate which was distilled off and the fraction was acquired by vacuum
distillation. After dropwise adding the fraction to sodium hydroxide and cooling it to
room temperature, white crude solid appeared. Dissolving it to deionized water, heating
and acidifying its pH close to 1 ꢂ 2, white solid 2,2-dibenzylmalonic acid appeared after
ꢀ1
distilling. Yield: 34%. This step of reaction is shown in scheme 1. IR (cm , s, strong;
m, medium): ꢀ(–O–H) 3417(m), ꢀ(¼C–H) 3028(m), ꢀ(–C¼O) 1715(s), ꢀ(C¼C) 1591(s).
1
0
0
H NMR (DMSO-d , 300 MHz): ꢁ 9.27 (s, 2H, H7, H7 ), 7.40 (t, J ¼ 8.1 Hz, 4H, H2,
6
0
0
0
H2 , H4, H4 ), 7.29 (d, J ¼ 7.5 Hz, 4H, H1, H1 , H5, H5 ), 7.27 (t, J ¼ 7.8 Hz, 2H, H3,
0
0
H3 ), 3.13(s, 2H, H6, H6 ).
2
2
.3. Complex synthesis
.3.1. Synthesis of C H Mn N O . Complex 1 was prepared as follows: dibe was
6
4
54
1
6
8
dissolved in a KOH aqueous solution. Then a Mn(CH COO) aqueous solution
3
2
(
10 mmol, 10 mL) and an aqueous solution of L (10 mmol, 10 mL) were mixed, and the
pH adjusted to acidity, and the mixture stirred at room temperature. After reacting for
h, bipy ethanolic solution (10 mmol, 10 mL) was added and the mixture appeared
4
turbid; continuing stirring for 2 h, the clear solution was obtained after filtration under
atmospheric pressure. Several days later, golden yellow transparent crystals formed by
evaporating the solution at room temperature, were collected and found suitable for
X-ray crystallography. The crystals were collected by filtration. For C H Mn N O ,
6
4
54
1
6
8
Anal. Calcd (%): C, 70.52; H, 4.99; N, 7.71. Found (%): C, 70.49; H, 5.07; N, 7.64.
ꢀ
1
ꢀ
IR (cm , s, strong; m, medium): ꢀ(–O–H) 3417(m); ꢀ(COO ) 1678(s); ꢀ(C¼C) 1578(s);
(C–N) 1440(s); ꢁ(–C–H) 1610(m).
ꢀ
2
.3.2. Synthesis of C H Mn N O . Complex 2 was synthesized in an identical
58 46 1 4 8
manner as described for 1 with phen (10 mmol 10 mL) in place of bipy (shown
in scheme 1). The product was obtained as brown powder. Yield: 69%. For
C H Mn N O , Anal. Calcd (%): C, 70.94; H, 4.22; N, 5.71. Found (%): C, 70.90;
5
8
46
1
4
8
ꢀ
1
ꢀ
H, 4.31; N, 5.69. IR (cm , s, strong; m, medium): ꢀ(–O–H) 3385(m); ꢀ(COO ) 1610(s);
(C¼C) 1575(s); ꢀ(–C–N) 1328(s); ꢁ(–C–H) 1609(m).
ꢀ
2
.4. Physical measurements
Elemental analysis (C, H, and N) was performed on a model Finnigan EA 1112.
IR spectra were obtained as KBr pellets on a Nicolet FT-IR 470 spectrometer.

Manganese(II)-phenolate
.5. X-ray crystallographic determination for 1
3995
2
Data of 1 were collected on a Bruker Smart 1000 CCD X-ray single-crystal
˚
diffractometer with Mo-Ka radiation (ꢂ ¼ 0.71073 A) at 293(2) K, and intensity data
ꢁ
ꢁ
were obtained from 1.71 ꢃ ꢃ ꢃ 25.68 at 293(2) K by using an ! scan technique.
Corrections for the Lp factor and an empirical absorption correction were applied.
The structure was solved by direct methods using SHELXS-97. All non-hydrogen
atoms were determined with successive difference Fourier syntheses and refined by full
2
matrix least squares on F . All hydrogen atoms were located at theoretical positions.
CCDC reference number is 807131.
2
.6. Cell line and culture
The cell lines used in this experiment were routinely maintained in a RPMI-1640
1
ꢀ
medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mmol L
ꢀ
1
ꢀ1
of glutamine, 100 u mL of penicillin, and 100 mg mL of streptomycin in a highly
ꢁ
humidified atmosphere of 95% air with 5% CO at 37 C.
2
2
.7. Extraction of HC-DNA
ꢁ
HeLa cells were grown adherently on the culture flask at 37 C under sterile conditions
after 2–3 days of growth for extraction. According to our previous work [25], saturated
sodium chloride extraction method was used to extract DNA.
2
.8. Fluorescence spectroscopic studies
Evidence for the two complexes binding to HC-DNA via intercalation is given through
emission quenching experiment. The experiments of HC-DNA competitive binding
with ethidium bromide were carried out in buffer by keeping [DNA]/[EtBr] ¼ 2.5
ꢀ
1
ꢀ1
(
[DNA] ¼ 2.5 mmol L , [EtBr] ¼ 1 mmol L ) and varying the concentrations of the
ꢀ
1
metal complexes (0–17 mmol L ). The buffer used in the binding studies was
ꢀ1
ꢀ1
5
0 mmol L
Tris-HCl, pH ¼ 7.4, containing 10 mmol L
NaCl. The sample was
ꢁ
incubated 4 h at room temperature (20 C) before spectral measurements. For all
fluorescence measurements, both the entrance and exit slits were maintained at 10 nm.
The excitation wavelength was 526 nm and the emission range was set between 550 and
7
50 nm.
2
.9. Gel electrophoresis experiments
ꢁ
The HC-DNA was kept at ꢀ20 C. The sample was incubated at 310 K for an
appropriate time and a loading buffer was applied (0.25% bromphenol blue, 50%
ꢀ
1
glycerol). For gel electrophoresis experiments, HC-DNA (0.5 mg mL ) was treated
with the complexes. After incubation, the solution was subjected to electrophoresis
ꢀ
1
ꢀ1
on 0.8% agarose gel in a TAE buffer (40 mmol L Tris acetate 1 mm EDTA)
ꢀ1
at 120 V for 1 h. After electrophoresis, the gel was stained with 1.0 mg mL EB and
photographed under UV light.

3
996
.10. Cytotoxicity assay
E.-J. Gao et al.
2
The growth inhibitory effect of metal complexes on HeLa cells was measured using the
microculture tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide,
5
MTT] assay. In brief, cells were seeded into a 96-well culture plate at 2 ꢄ 10 cells/well in
a 100 mL culture medium. After incubation for 24 h, cells were exposed to the tested
complexes of serial concentrations. The complexes were dissolved in DMF and diluted
with RPMI 1640 or DMEM to the required concentrations prior to use (0.1% DMF
final concentration). The cells were incubated for 24 h and 72 h, followed by addition of
ꢀ
1
a 20 mL MTT solution (5 mg mL ) to each well and further cultivation for 4 h. The
media with MTT were removed and 100 mL of DMSO was added to dissolve the
formazan crystals at room temperature for 30 min. The absorbance of each cell
at 450 nm was determined by analysis with a micro plate spectrophotometer. The IC50
values were obtained from the results of quadruplicate determinations of at least three
independent experiments.
In another experiment, the effects on cell growth for the two complexes and cisplatin
were studied by culturing the cells in medium alone for 24 h, followed by 72 h treatment
ꢀ1
with 3 mg mL concentration. The viable cells remaining at the end of the treatment
period were determined by MTT assay and calculated as the percent of control, treated
with the vehicle alone (DMSO) under similar conditions.
2
.11. In vitro apoptotic assay
5
HeLa cells in usable condition were seeded in a 24-well culture plate at 2 ꢄ 10 cells/well
in a 1 mL culture medium, and 24 h later the medium including the cisplatin was given.
ꢀ
1
ꢀ1
The concentration of the two complexes was 1 mmol L mL . The level of apoptosis
was evaluated in at least three independent experiments. The hematoxyline–eosin stain
was the means of the apoptotic morphology observed by light microscopy. Under the
light microscope, the cytoplasm had been stained pink with blue-black in the nucleolus.
3
3
. Results and discussion
.1. Crystallographic structure of 1
The crystal structure of 1 was determined by X-ray crystallography (figure 1). Complex
belongs to the monoclinic system with space group P2(1)/c. A summary of data
1
collection and refinement for the complex is presented in table 1, and selected bond
distances and angles are given in table 2. As shown in figure 2, Mn is six coordinate with
two bipy molecules and two oxygens from two carboxyls of dibe; manganese forms an
˚
approximate octahedral geometry. The main bond lengths are: Mn–O(1) 2.1023(13) A,
˚
Mn–O(8) 2.1203(14) A, Mn–N(1) 2.3016(16) A, Mn–N(2) 2.2535(16) A, Mn–N(3)
˚
˚
˚
.3193(16) A, Mn–N(4) 2.2527(16) A. In the equatorial plane, the sum of their bond
˚
2
angles [O1–Mn–N4 ¼ 92.04(6), O1–Mn–N2 ¼ 102.04(6), N4–Mn–N1 ¼ 92.56(6),
ꢁ
ꢁ
ꢁ
N2–Mn–N1 ¼ 72.46(6)] is 359.10 , close to 360 . In the axial direction, apical
ꢁ
N3–Mn–O8 bond angle is 173.99(6) , close to 180 . Bond lengths among all Mn–O
are less than the bond lengths with all Mn–N, suggesting coordination of oxygen from

Manganese(II)-phenolate
3997
Figure 1. Complex 1 with numbering scheme (H atoms were omitted for clarity) at 30% probability thermal
ellipsoids.
Table 1. Crystal data and reEnement for 1.
Parameter
Value
Formula weight
Crystal system
Space group
1090.11
Monoclinic
P2(1)/c
˚
ꢁ
)
Unit cell dimensions (A,
a
b
c
ꢄ
ꢅ
ꢆ
16.4351(10)
14.3731(9)
22.0276(13)
90.00(0)
103.2000(10)
90.00(0)
5066.0(5), 4
1.327
3
˚
Volume (A ), Z
ꢇ
ꢀ
3
)
Calcd (g cm1
ꢀ
ꢈ
Mo (mm
)
Crystal size (mm )
0.323
0.14 ꢄ 0.21 ꢄ 0.19
3
ꢁ
ꢃ range for data collection ( )
Limiting indices
F(000)
1.24 5 ꢃ 5 26.23
2ꢀ1 11 92 ꢃ.0h ꢃ 19; ꢀ15 ꢃ k ꢃ 17; ꢀ26 ꢃ l ꢃ 23
Reflections collected/unique [I 4 2ꢉ(I)]
Completeness (%)
Number of refined parameters
92 97 ., 39 25/9556 [R(int) ¼ 0.0346]
660
1.017
2
Goodness-of-fit on F
Final R indices [I 4 2ꢉ(I )]
R indices (all data)
R
R
1
1
¼ 0.0400, wR
2
2
¼ 0.0887
¼ 0.0980
¼ 0.0619, wR
ꢀ
3
˚
Dꢇ (max) and Dꢇ (min) (e A
)
0.33 and ꢀ0.213

3
998
E.-J. Gao et al.
˚
Table 2. Selected bond lengths (A) and angles ( ) for 1.
ꢁ
Mn(1)–O(1)
Mn(1)–O(8)
Mn(1)–N(4)
2.1023(14)
2.1203(13)
2.2527(16)
Mn(1)–N(2)
Mn(1)–N(1)
Mn(1)–N(3)
2.2535(16)
2.3016(16)
2.3193(16)
O(1)–Mn(1)–O(8)
O(1)–Mn(1)–N(4)
O(8)–Mn(1)–N(4)
O(1)–Mn(1)–N(2)
O(8)–Mn(1)–N(2)
N(4)–Mn(1)–N(2)
O(1)–Mn(1)–N(1)
O(8)–Mn(1)–N(1)
88.40(5)
92.04(6)
102.91(6)
102.02(6)
94.96(6)
157.57(6)
174.03(6)
94.28(6)
N(4)–Mn(1)–N(1)
N(2)–Mn(1)–N(1)
O(1)–Mn(1)–N(3)
O(8)–Mn(1)–N(3)
N(4)–Mn(1)–N(3)
N(2)–Mn(1)–N(3)
N(1)–Mn(1)–N(3)
92.56(6)
72.46(6)
94.88(6)
173.99(6)
71.98(6)
89.30(6)
82.93(6)
Figure 2. 1-D chain structure of 1, C–H ꢅ ꢅ ꢅ ꢊ and C–H ꢅ ꢅ ꢅ O interactions (shown by dotted lines).
Figure 3. 2-D framework diagram of 1 and the C–H ꢅ ꢅ ꢅ ꢊ interactions (shown by dotted lines).
dibe is stronger than nitrogen from bipy. The bond lengths mentioned above are in
accord with bond lengths of Mn–N and Mn–O in other coordination compounds [28].
˚
Two adjacent asymmetric complexes form a basic repeat unit via C–H ꢅ ꢅ ꢅ ꢊ (2.9205 A)
˚
and C–H ꢅ ꢅ ꢅ O (2.6468 A) interactions [29–31]. The basic units connect to form a 1-D
structure. The 1-D structure forms an undulated layer 2-D structure by C–H ꢅ ꢅ ꢅ ꢊ
˚
interactions (2.5094 A) with the dissociated bipy (shown in figure 3) [32]. The 2-D layers
˚
further form a 3-D framework via weak edge ꢊ ꢅ ꢅ ꢅ ꢊ stacking interactions (3.556 A) [33].

Manganese(II)-phenolate
3999
50
40
30
20
10
0
1
1
5
5
40
545
2
550
555
560
565
570
575
Wavelength (nm)
5
4
3
2
1
0
1
0
5
0
0
0
0
5
40
545
550
555
560
565
570
575
Wavelength (nm)
ꢀ
1
Figure 4. Emission spectrum of EB bound to DNA in the presence of 1 and 2, ([EB] ¼ 1 mmol L
,
ꢀ1
ꢀ1
[
increasing complex concentration.
DNA] ¼ 2.5 mmol L , [complex] ¼ 0–17 mmol L , ꢂex ¼ 526 nm). The arrow shows the intensity changes on
3.2. Fluorescence spectroscopic properties
Fluorescence quenching measurements can be used to monitor metal binding [34]. The
molecular Fuorophore EtBr emits intense Fuorescence in the presence of HC-DNA due
to its strong intercalation between adjacent DNA base pairs. It was previously reported
that quenching of DNA-EtBr Fuorescence by addition of complexes causes a reduction
in the emission intensity, indicating competition between the complex and EtBr in
binding to DNA [35]. The study involves addition of the complexes to DNA pretreated
with EtBr ([DNA]/[EtBr] ¼ 2.5) and measurement of the intensity of emission [36].
The emission spectra of EtBr bound to DNA in the absence and presence of the two
complexes are shown in figure 4 [37–39]. Fluorescence intensity of DNA-EtBr complex
gradually reduces with the addition of 1 or 2. The more the DNA-EtBr complex added,
the lower was its fluorescence intensity. It can also be seen from figure 4 that the

4
000
E.-J. Gao et al.
2
1
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25
r
Figure 5. Stern–Volmer quenching plots of 1 and 2 with the value of slope 0.4352 for 1 and 0.5751 for 2.
intercalation of the complexes into DNA is different with 2 being higher than that of 1
due to the presence of an extended aromatic planar ring.
Efficiencies of the two complexes binding to DNA are compared quantitatively,
according to the classical Stern–Volmer equation: I /I ¼ 1 þ K r, where I and I
0
sq
0
represent the Fuorescence intensity in the absence and presence of the complex,
respectively, and r is the concentration ratio of the complex to DNA. Ksq is a linear
Stern–Volmer quenching constant dependent on the ratio of the bound concentration of
EtBr to the concentration of DNA [40]. The Ksq value is obtained as the slope of I /I
0
versus r linear plot. The Fuorescence-quenching curves of DNA-bound EtBr in the
absence and presence of the two complexes are given in figure 5. The Ksq value of 2 is
higher than that of 1. The values of Ksq are 0.5751 and 0.4352, respectively.
Accordingly, the intercalation ability of 2 into DNA is stronger than that of 1 [41].
3
.3. Gel electrophoresis study
The influence of the complexes on the structure of DNA is evaluated by their DNA-
cleavage ability [42–44], which can be monitored by agarose gel electrophoresis [45–47].
In the electrophoresis experiments, intact supercoiled DNA migrates fast (Form I),
open circular DNA migrates slowly (Form II) and linear DNA occurs between Form I
and II (Form III) [11]. The DNA used in the present studies is extracted from HeLa
cells in our laboratory [48, 49]. The results of the electrophoresis experiments for the
two complexes with HC-DNA are shown in figure 6. These complexes induce obvious
ꢀ1
cleavage of the HC-DNA at 13.2, 6.6 and 3.3 mmol L ; HC-DNA is cut into small
molecular DNA. Lanes 2 and 5 show that supercoiled HC-DNA (Form I) gradually
decreased about 70%, open circular form (Form II) gradually increased, and linear
form (Form III) appeared. Capability of cleavage is stronger at high concentration than
that at low concentration, which is consistent with the tendency in fluorescence
quenching experiments.
To further illustrate cleaving efficiency of the two complexes, the experimental results
of optical density scanning are shown in figure 7, obtained through optical density

Manganese(II)-phenolate
4001
Figure 6. DNA strand break in HeLa cells treated with 1 and 2. Lane 0: Marker; Lane 1: DNA alone; Lanes
–7: different concentration of the complex: (2) 13.2; (3) 6.6; (4) 3.3; (5) 13.2; (6) 6.6; (7) 3.3 mm (lanes 2–4: 1,
lanes 5–7: 2).
2
Figure 7. DNA-cleavage activity of 1 and 2 by the optical density scanning experiment.
scanning experiment of figure 6. Figure 7 shows the effect of HC-DNA cleavage varies
in different concentrations. The peak value of lane decreases with gradual increase of
concentration of complexes, showing the remainder of DNA base pair is sparse with
increase of concentration of complexes. The two complexes in the present work exhibit
strong cleaving ability for the HC-DNA.
3.4. Cytotoxicity study
The prepared complexes were tested for their cytotoxic activity in vitro by MTT assay.
IC50 values were estimated for four human tumor cell lines, HeLa, Hep-G2, KB, and

4
002
E.-J. Gao et al.
Table 3. Cytotoxicity of the complexes against selected human tumor
cells after 72 h of incubation.
ꢀ
1 ꢀ1
Complex
Tumor
IC50 (mmol L
)
HeLa
Hep-G2
KB
AGZY-83a
Complex 1
Complex 2
Cisplatin
9.45 ꢆ 1.36
7.81 ꢆ 1.11
0.79 ꢆ 0.21
3.13 ꢆ 0.91
2.81 ꢆ 0.69
1.73 ꢆ 0.47
4.53 ꢆ 1.24
3.68 ꢆ 1.03
1.42 ꢆ 0.29
20.79 ꢆ 4.21
16.53 ꢆ 3.74
2.05 ꢆ 0.49
Complex 1
Complex 2
Cisplatin
48
5
4
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
5
0
41
39
38
33
32
31
26
24
22
20
20
HelaHep-G
2KBAG
ZY-83a
ꢀ1
Figure 8. Effect of 3.0 mg mL of the complexes on breast cancer cells viability after 72 h of incubation. All
determinations are expressed as percentage of the control (untreated cells).
AGZY-83a, summarized in table 3. The complexes both have certain anti-tumor
activities which are inferior to those of cisplatin. The activity of 2 is stronger than that
of 1 and the activity of the two complexes on Hep-G2 cells is stronger than on other
cells. Figure 8 reveals the effect on cell growth after a treatment period of 72 h at
ꢀ1
3
(
.0 mg mL
untreated cells) is observed for treatment of complexes. The viability rate for the
treatment of 2 is smaller than that of 1, which coincides with the IC50 values.
concentration. A viability rate less than 50% of the control values
3
.5. In vitro apoptotic study
Cell apoptosis is the process of programmed cell death. When the cells are treated with
the complexes, cell apoptosis may occur. The changes of size and morphology of the
cells can be observed by dying them with hematoxylin-eosin. The normal intact HeLa

Manganese(II)-phenolate
4003
Figure 9. Morphological changes of HeLa cells. (a) normal HeLa cells for 24 h; (b) HeLa cells treated with
cisplatin for 24 h; (c) HeLa cells treated with 1 for 24 h; (d) HeLa cells treated with 2 for 24 h.
cells exhibit dense state and intercellular tight junction; single cell is polygonal in shape
or irregular cell morphology and cytoplasm is a clear appearance (shown in figure 9a).
Figure 9(b), (c), and (d) are HeLa cells treated with cisplatin, 1 and 2 for 24 h. These
figures are different from figure 9(a), where the cell nucleus became pyknotic (shrunken
and dark) and condensed chromatin located on the nuclear membrane. As stated
previously, the two complexes have apoptosis on the HeLa cells, but lower than
cisplatin. In the present work, morphological changes of the HeLa cells have been
observed under the optical microscope.
4
. Conclusions
A carboxylic acid ligand (dibe ¼ 2,2-dibenzylmalonate acid) was synthesized. We report
1
1
synthesis and characterization of two complexes, [Mn(L ) (dibe) ](L ) (1) and
[
2
2
2
Mn(L ) (dibe) ] (2). The DNA-binding properties of the complexes were examined
2 2
by fluorescence spectra, which suggested their involvement in intercalative DNA
interaction with different binding affinities. The cleavage of HC-DNA by the complexes
was investigated using agarose gel electrophoresis, and the results indicate that the

4
004
E.-J. Gao et al.
complexes exhibit efficient DNA cleavage. Cytotoxic studies show that the two
complexes exhibit cytotoxic activity against different cell lines tested in general and are
especially effective against Hep-G2 cell lines. Additionally, the apoptotic tests indicate
that the complexes had an apoptotic effect on HeLa cells.
Acknowledgements
We gratefully acknowledge the Natural Science Foundation of China (No. 20971090,
2
2
1171118), the science and technology projects fund of Shenyang City (No. F10-
15-1-00).
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