Synthesis, characterization, and antitumor activity of rare earth metal complexes of benzoic acid nitrogen mustard

Article abstract of DOI:10.1007/s00044-012-0286-5

Benzoic acid nitrogen mustard and its rare earth metal complexes were synthesized and characterized by elemental analyses, IR, electronic spectrum, and EPR. The interaction of synthesized complexes with Ct-DNA was investigated and reviewed as a mixed manner of both intercalation and alkylation via fluorescence titration. Their biological activities were also evaluated in K₅₆₂ and Vero cell lines, indicating that complexes had a significant inhibitory effect; however, there was no synergistic effect instead of antagonistic effect compared to benzoic acid nitrogen mustard. The possible mechanism through cellular apoptosis was also explored by comet assay.

Full text of DOI:10.1007/s00044-012-0286-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2862–2869
DOI 10.1007/s00044-012-0286-5
ORIGINAL RESEARCH
Synthesis, characterization, and antitumor activity of rare earth
metal complexes of benzoic acid nitrogen mustard
•
Sufeng Zhou Yun Fu Xizhi Fan
•
•
•
Yanfang Zhang Changzheng Li
Received: 21 March 2012 / Accepted: 20 October 2012 / Published online: 2 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Benzoic acid nitrogen mustard and its rare
earth metal complexes were synthesized and characterized
by elemental analyses, IR, electronic spectrum, and EPR.
The interaction of synthesized complexes with Ct-DNA
was investigated and reviewed as a mixed manner of both
intercalation and alkylation via fluorescence titration. Their
biological activities were also evaluated in K562 and Vero
cell lines, indicating that complexes had a significant
inhibitory effect; however, there was no synergistic effect
instead of antagonistic effect compared to benzoic acid
nitrogen mustard. The possible mechanism through cellular
apoptosis was also explored by comet assay.
mutation, make its applications limited. In order to improve
its antitumor activity, many of its derivatives have been
synthesized; melphalan, cyclophosphamide, bendamustine,
and chlorambucil are good examples and have been widely
used in clinic (Leoni et al., 2008). Among massive deriva-
tives of nitrogen mustard, benzoic acid nitrogen mustard
(BANM) is effective against quiescent cell and tend to be less
susceptible to induced resistance than most anticancer drugs,
thereby its different derivatives were reported in many lit-
eratures (Beria and Nesi, 2002; Koutsourea et al., 2008). In
addition to its good fluorescence and noncell-cycle-specific
feature, BANM is widely used in prodrug design (Roger and
Roger, 1996), such as antibody-directed enzyme prodrug
therapy, which is designed to deliver it to a given site.
It is well known that rare earth metal is an important
chemical element and has significant biological feature.
Rare earth metal complexes have been widely used as
diagnosis reagent for pathological changes of lung, brain,
and kidney (Zhou et al., 2010). Some complex has ability
to promote blood circulation and protect cells from radia-
tion damage (Gschneidner et al., 1999). In addition, the
rare earth metal complexes have not been shown to have a
cumulative effect in vivo either as a result of oral admin-
istration or external use (Li et al., 2011). Their anti-
inflammation and anticancer activity indicate that rare
earth ions can stop bacterium metabolism (Qu et al., 2006)
or inhibit enzymatic activity (Achyuthan et al., 1989).
However, the complexes of rare earth with BANM are not
reported in literature. In order to evaluate whether a syn-
ergistic effect exists between them, BANM was synthe-
sized by multi-step and its rare earth complexes were also
prepared. Their biological activities were evaluated against
Vero and K562 cell lines, and the interactions with Ct-DNA
and cellular DNA in vitro were examined to probe their
interaction and the underlying biological mechanism.
Keywords Benzoic acid nitrogen mustard ꢀ Synthesis ꢀ
Antitumor activity ꢀ Rare earth metal complex ꢀ
Comet assay
Introduction
Nitrogen mustard is one of the most useful drugs in cancer
chemotherapy, lack of selectivity and its toxic-side effect as
well as resulting drug-resistance, carcinogenesis, and
Y. Fu ꢀ Y. Zhang ꢀ C. Li (&)
Department of Molecular Biology & Biochemistry, Xinxiang
Medical University, Xinxiang 453003, Henan,
People’s Republic of China
e-mail: changzhenl@yahoo.com
S. Zhou
Clinical Skill Training Center, Xinxiang Medical University,
Xinxiang 453003, Henan, People’s Republic of China
X. Fan
Department of Computer, Xinxiang Medical University,
Xinxiang 453003, Henan, People’s Republic of China
123

Med Chem Res (2013) 22:2862–2869
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Preparation of benzoic acid nitrogen mustard
Experimental
The synthetic scheme of BANM was as follows (Fig. 1):
4,4-Bis(2-chloroethyl)amino-benzaldehyde (benzalde-
hyde nitrogen mustard) was made according to our pub-
lished method (Li et al., 1999). BANM was prepared by
KMnO4 oxidation of benzaldehyde nitrogen mustard.
All reactants and solvents were AR grade. 3-(4,5-Dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium
dodecyl sulfate (SDS), ethidium bromide (EB), Roswell Park
Memorial Institute 1640 (RPMI-1640), and agarose were pur-
chased from Sigma. Calf thymus DNA (Ct-DNA) was obtained
from Shanghai Lizhu Dong Feng Biotechnology Co. LTD,
Shanghai.
Preparation of the complexes
In a typical preparation, a solution of hydrated lanthanide
acetate (77.8 mg, 0.2 mmol) in 50 % water–ethanol was
added dropwise to a solution of BANM (78.7 mg, 0.6 mmol)
in 10 ml ethanol with stirring, the precipitate was formed.
The mixture was continuously stirred for 8 h. The product
was filtered off, washed with 50 % aqueous-ethanol, and
dried in vacua. The yield was *80 %. The analytical data of
BANM and the complexes are presented in Table 1.
Instrumentation and measurement
Stock solutions of nucleic acids were prepared by directly
dissolving commercial Ct-DNA in ddH2O, and the result-
ing solution was subjected to dialysis in the ddH2O at 4 °C.
The Ct-DNA sample purity was estimated by measurement
of A260 and A280; the ratio A260/A280 was above 1.8,
indicating that the nucleic acid was reasonably free of
protein contamination (Nelson and Cox, 2004). The molar
concentration of Ct-DNA was calculated according to the
following formula:
Fluorescence studies
The Scatchard plots were generated using following proce-
dure: in a 5-ml volumetric flask, 0.5 ml of Ct-DNA
(1.5 9 10-5 M), 2.0 ml Tris–HCl buffer (pH 7.2),
0.5 ml NaCl (0.5 M), and the solution of complexes with
different molar ratio (Rt = Ccomplex/CDNA) were added. After
diluted to 5 ml, the mixture was then titrated by 0.1 mg/ml
EB solution. The fluorescence intensity was determined at
kex/kem = 525/600 nm.
The effects of the complexes on the fluorescence spec-
trum of Ct-DNA-EB was evaluated based on the following
method: 0.5 ml of 50 lM Ct-DNA, 0.5 ml of 10 lM EB,
0.5 ml of 0.5 M NaCl, 2.0 ml Tris–HCl buffer (pH 7.2), and
an appropriate amount of Ln(III) complexes were mixed in a
5-ml volumetric flask and diluted to the giving volume
with ddH2O. The fluorescence intensity was determined at
kex/kem = 525/600 nm.
CDNA = KA260/6,600, here K is the dilution factor.
The solutions of complexes (5.0 9 10-4 M) were pre-
pared by dissolving them in DMSO-ethanol mixture sol-
vent (1:10, v/v).
Nitrogen, carbon, and hydrogen were determined using
an Elementar Vario EL. The IR spectra were recorded in
the 4,000–200 cm-1 region using KBr pellets and a
Nicolet-170-SX FTIR spectrometer. Electronic spectra in
DMSO were measured on a Shimadzu UV-240 spectro-
photometer. Conductance measurement was carried out
with a DDS-11A type conductivity bridge in DMSO at
10-3 M. EPR spectra (in solid and solution states) were
obtained from Bruker ER200D-SRC instrument at room
temperature. The magnetic field was varied from 0 to
6800 Gs. The spectra of fluorescence were recorded on
Hitachi 850 fluorescent spectrophotometer. Thermogravi-
metric analysis (TG) and differential thermal analysis
(DTA) was carried out using a diamond TG/DTA instru-
ment (PerkinElmer). Isothermally heated to 30 °C for
10 min under air flow (200 ml/min) and then heated from
30 to 800 °C in a static air atmosphere. The heating rate
was 10 °C/min. Al2O3 was used as the reference standard.
MTT assay
The stock solutions of BANM complex were freshly pre-
pared in 10 % DMSO, and were diluted to the required
concentration with culture when used. Vero (African green
monkey kidney cell) and K562 human leukemia cells were
Fig. 1 Synthetic routes of
benzoic acid nitrogen mustard
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Med Chem Res (2013) 22:2862–2869
Table 1 Analytical data and general appearance of Ln(III) complexes
Compound
Color
Percent composition (%) found (calc.)
C
N
H
M
BANM
Colorless
White
50.1 (50.4)
41.9 (42.2)
41.6 (41.9)
42.0 (41.6)
42.0 (41.6)
41.9 (41.4)
41.0 (41.1)
41.2 (40.9)
40.6 (40.9)
5.7 (5.3)
4.4 (4.5)
4.6 (4.4)
4.6 (4.4)
4.5 (4.9)
4.8 (4.4)
4.7 (4.4)
4.7 (4.3)
4.7 (4.3)
4.8 (5.0)
4.2 (4.1)
4.3 (4.1)
4.5 (4.0)
4.5 (4.0)
4.3 (4.0)
3.9 (4.0)
4.2 (4.0)
4.0 (4.0)
La(BANM)3H2O
Nd(BANM)3H2O
Sm(BANM)3H2O
Eu(BANM)3H2O
Gd(BANM)3H2O
Dy(BANM)3H2O
Er(BANM)3H2O
Tm(BANM)3H2O
15.2 (14.8)
15.6 (15.3)
16.1 (15.8)
16.3 (15.9)
16.0 (16.4)
17.1 (16.9)
17.1 (17.3)
17.8 (17.4)
Slight blue
White
Pale yellow
White
Pale yellow
Slight red
Slight red
propagated continuously in RPMI 1640 medium supple-
mented with 10 % freshly inactivated fetal calf serum
(FCS) and antibiotics. The cells harvested from exponential
phase (2 9 105/ml) were seeded equivalently into 96-well
plate and the compounds studied were added in varied
concentrations. The plate was kept at 37 °C in a humidified
atmosphere of 5 % CO2 for 48 h, MTT solution of an
appropriate concentration (1 mg/ml) was added to each
well, and the plate was further incubated at 37 °C for 4 h.
Finally, 100 ll of 10 % SDS solution containing 1 % HCl
was added to each well and the plate was kept at 37 °C for
overnight. The measurement of absorbencies was per-
formed on an ELISA spectrophotometer at 570 nm.
with a cover-slip. The photography was taken on fluores-
cent microscopy.
Results and discussion
Characterization of the complexes
An Elementar Vario EL elemental analyzer was used to
analyze the carbon, hydrogen, and nitrogen content of
BANM and its complexes. The metal content was deter-
mined by EDTA complexometric titration. As presented in
Table 1, the composition of the complexes were found to
be 1:3 stoichiometry, corresponding to the formula
Ln(BANM)3H2O or Ln(C11H12Cl2O2)3H2O (where Ln =
La, Nd, Sm, Eu, Gd, Dy, Er, and Tm). The complexes are
soluble in DMF and DMSO, while molar conductance of
the complexes were in the range of 5–8/X/cm2/mol, indi-
cating their non-electrolyte nature (Geary, 1971).
Comet assay
The single-cell gel electrophoresis (comet assay) was
adapted from the method of Singh and colleagues reported
in reference (Singh et al., 1988). K562 cells treated with
(50 lM) or without of lanthanide BANM after 48 h incu-
bation in a humidified atmosphere of 5 % CO2 were har-
vested by centrifugation at 1,000 rpm and then embedded
in 0.5 % low-melting-point agarose at a final concentration
of 104 cells/ml. 20 ll of this cellular suspension was then
spread onto duplicate frosted slides that had previously
been covered with 1 % normal-melting-point agarose as a
basal layer. Slides were allowed to solidify for 10 min at
4 °C before being placed in lysis buffer for 1 h (2.5 M
NaCl, 0.1 M ethylene diamine tetraacetic acid (EDTA),
0.01 M Tris, 1 % Triton X-1000, 10 % dimethyl sulph-
oxide (DMSO), pH 10). After lysis, the slides were trans-
ferred into alkaline buffer for 40 min (0.001 M EDTA,
0.3 M NaOH, pH[13) to allow the DNA to unwind before
migration at 0.66 V/cm and 300 mA for 30 min. All these
steps were performed in the dark. After neutralization in
0.4 M Tris–HCl pH 7.4, slides were stored at 4 °C until
analysis during the following 24 h period. Before analysis,
the slides were stained with EB (20 lg/ml) and covered
Infrared spectra
The eight rare earth metal complexes were similar in their
characteristic infrared absorption peaks as determined by
the method of KBr tablet, indicative of their similar struc-
tures. In the BANM spectrum, the absorbance of carboxylic
group occurred at 1,676 cm-1, while mOH appeared at
*3,000 cm-1 which was identical to the reported spectrum
(Sadlter Research Lab. Inc., 1970; Zhang et al., 2005). In
the spectra of complexes, the peaks at 1,676 cm-1 disap-
peared, and the new peaks were shown around 1574–1577,
1399–1407, and 1521–1514 cm-1, which can be assigned to
m
asy(COO-) and msym(COO-). A different coordination manner
of COO- in metal complexes was reported (Spinner, 1964);
IR spectroscopy is a useful tool in diagnosing the nature of
carboxylate coordination. The energy difference between
masy(COO-) and msym(COO-) is diagnostic of the geometry of
carboxylate coordination. Monodentate coordination is
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Med Chem Res (2013) 22:2862–2869
2865
Table 2 Electronic spectral data of Ln(III) complexes
Complex
Bond max (cm-1
)
S’L’J’
Oscillator strength (106)
Bonding parameter
Nujol
DMSO
Complex
Aqua ion
Nd(BANM)3H2O
11,534
12,579
13,514
14,750
17,182
19,048
11,764
15,456
18,939
20,618
4F3/2
4F5/2
4F7/2,2S3/2
4F9/2
4G5/2,2G7/2
4G7/2
4I9/2
4F9/2
(2H,4G)11/2
4F7/2
12.13
12.13
112.15
3.04
3.02
9.22
8.88
b = 0.9932
d % = 0.6847
b1/2 = 0.0583
g = 0.00341
13,420
14,800
16,984
72.39
32.75
41.97
46.28
71.08
125.59
10.50
6.58
2.37
2.37
2.91
2.22
Er(BANM)3H2O
b = 0.9962
d % = 0.0038
b1/2 = 0.0436
g = 0.0019
associated with an energy difference of 220–460 cm-1
bridging bidentate coordination with a difference at
140–170 cm-1, and bidentate coordination at 40–80 cm-1
,
occur as a result of the interaction of the central ion with
the surrounding ligand-field mixing into the fN configura-
tion states from configuration of opposite parity and thus
relaxing the parity restriction. Electric-dipole transitions
may also be induced as a result of the asymmetries pro-
duced by means of the vibronic coupling of the central ion
with its ligands. In contrast to the foregoing electric-
quadrupole and magnetic-dipole transition within the fN
configuration parity is allowed. In the Russell–Saunders
approximation, magnetic-dipole transitions are restricted
by the selection rules (Figgis et al., 1960): DJ B 1,
DL = 0, DS = 0, and Dl = 0. According to the selection
rules, some transitions are allowed to appear. Therefore,
the weak bands can be seen in higher concentration. In our
studied complexes, all the bands show a weak perturbation
due to the nephelauxetic effect. The absorption band
,
respectively (Nakamoto, 1978; Li et al., 2005). In the IR
spectra of the complexes, energy differences between
m
asy(COO-) and msym(COO-) were *175 and 60 cm-1; based
on this criterion, there were two coordination manners,
bridging bidentate and bidentate that existed in the com-
plexes. A dimmer structure involving bridging bidentate and
bidentate of COO- may be tentatively proposed for the rare
earth metal complexes. In addition, compared to BANM, the
new weak peaks around 498–503 cm-1 also appeared in the
spectra of the complexes, which can be assigned to mLn–O
(Zhao et al., 2009). The broad band in the spectra of the
complexes were observed at 3,365–3,420 cm-1 region,
which can be assigned to mO–H; however, water was not
involved in coordination as it’s other characteristic peaks
(600–650 cm-1) did not appear (Sigh et al., 1978). The
DTA-TG data further supported the conclusion for an
endothermic peak that appeared at 86 °C in DTA-TG
spectrum of lanthanide complex (2 % mass loss, calc.:
1.91 %), suggesting that water in the complexes was
absorption water. No more information could be used to
discriminate the coordination mode of BANM.
4
2
associated nearly degenerate I9/2 ? G7/2,
4G5/2 transi-
tions of the Nd(III) ion is well known to exhibit strong
hypersensitive behavior (Choppin et al., 1966), making it
especially suitable for probing the coordinating environ-
ment around the metal ion. The hypersensitive band profiles
of Nd(III) complex resemble these of the six-coordinated
complex Nd(C6H5COCHCOC6H5) reported by Karraker
(Melby et al., 1964; Karraker, 1967), indicating that they
have similar coordination environment. The absorption
intensities were determined experimentally by the area
method (Kumer, 1998) and the experimental oscillator
strength (P 9 106) were calculated for the spectral band of
the Nd(III) and Er(III) complexes in solution state using the
reduced expression (Melby et al., 1964):
Electronic spectra
The absorption spectra of the Nd(III) and Er(III) were
recorded in the solid (Nujol mull) and in solution (0.01 M
in DMSO) state. The spectral data, along with the calcu-
lated bonding parameters are included in Table 2. The
electronic transitions of the trivalent lanthanides can be of
electric-dipole, magnetic-dipole, or electric-quadrupole
character; some transitions have appreciable contribution
from more than one mode. Electric-dipole transitions
within a pure fN configuration are strictly parity forbidden.
However, weak, induced electric-dipole transitions can
Z
9g
ðg2 þ 2Þ
p ¼ 4:318 ꢁ
eðvÞdv
2
where g is the refractive index of the solution, e is the
molar extinction, and m is the energy of the transition.
Compared to aqueous metal ion spectra, these values
showed an increased trend, which was mostly due to a
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Med Chem Res (2013) 22:2862–2869
lowering of molecular symmetry (Judd, 1966). This was
consistent with the result of IR. Various bonding parame-
ters, viz, nephelauxetic ratio (b, bonding parameter (b1/2),
Sinha’s parameter (d %), and covalency angular overlap
parameter (g) were calculated according to literature
method (Coutsolelos and Spyroulias, 1994). All these
parameters indicate that there were weak covalent bonds
between the metals and BANM.
in the presence of different complexes were not good.
These results indicated that a complicated binding mode
existed between all of the complex and Ct-DNA. The
complexes might bind to Ct-DNA with intercalation,
electrostatic action, and covalent binding mode (Lepecq
and Paotetti, 1967; Howe et al., 1976). In general, nitrogen
mustards can alkylate the N7 of guanine in DNA. The
BANM may also have similar function. The fluorescent
intensity decreased profoundly with an increase in the
amount of the complexes, indicating that the EB was
released from the grooves of DNA due to the cross-linkage
of the complexes. The linear relationship may be overall
behavior of covalent and electrostatic action, because there
is no suitable intercalative group in the complexes. The
fluorescence Scatchard plot of the binding of EB to
Ct-DNA in the presence of different concentration of
La(III) complex is shown as an example in Fig. 4. The n,
In order to confirm the proposed structure, the dyspro-
sium complex was studied by EPR spectroscopy in frozen
solution of DMSO. Only one signal was observed at room
temperature in solid state. However, in frozen solution
state, the spectrum of the complex exhibited its hyperfine
structure with six peaks (DG = 80) at the same time with
the peak at the region of free electron (nuclear spin of
Dy(III) is 5/2 for both isotopes). The appearance of super-
hyperfine coupling may be due to the interaction of the free
electron with its neighbor atoms and probably the dys-
prosium (Henrie and Choppin, 1968). The other informa-
tion was not obtained. Based on the electronic spectrum of
Nd(III) complex, and IR spectra, a dimer structure of the
complexes can be tentatively proposed as shown in Fig. 2.
KEB, and the relative coefficient of the Scatchard plots in
the presence of all the complexes are listed in Table 3.
Cytotoxicity effect
The growth inhibitory effects of the BANM and some of its
rare earth complexes were examined in Vero and human
K562 cell lines. Considering the cytotoxicity effect of
DMSO, the concentration of DMSO in the medium was
controlled below 1 %, so that DMSO did not affect the
growth and viability of the cell (DoDodoff et al., 1994).
The fluorescence spectral characteristics and binding
mode
The interaction of the complexes with the Ct-DNA was
examined by fluorescence method. The results showed that
the fluorescence intensity of the Ct-DNA-EB decreased
remarkably with increasing concentration of the complexes
added, which indicated that the complexes could bind to
Ct-DNA. The fluorescence excitation and emission spectra
of the Ct-DNA-EB system in the presence of different
concentration of La(III) complex are shown in Fig. 3. The
spectral characteristics of the Ct-DNA-EB system in the
presence of other complexes were similar. The fluores-
cence Scatchard plots of EB-Ct-DNA system in the pres-
ence of different concentrations of complexes showed that
the intercept on both the abscissa (n) and the slope (KEB
decreased with increasing amount of metal complexes.
However, the linear relationships of all the Scatchard plots
)
Fig. 2 Tentatively proposed
structure of rare earth
complexes with BANM
Fig. 3 Effects of the La(III) complex on the fluorescence excitation
(left) and emission (right) spectra of EB-Ct-DNA system. The
r (molar ratio of the complex to Ct-DNA) from top to bottom in the
spectra were 0, 2.5, 5, 10, and 15, respectively; the concentration of
EB and DNA in phosphorus were kept constant at 4.0 9 10-6 and
4.0 9 10-5 M, respectively
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Med Chem Res (2013) 22:2862–2869
2867
K562
Table 4 Cytotoxicity effect of some compounds (IC50 lM)
Compound
Vero
BANM
63
131
157
220
La(BANM)3H2O
Sm(BANM)3H2O
Eu(BANM)3H2O
207
307
176
from the complexes themselves. La(III), Sm(III), and
Eu(III) complexes had definite cytotoxicity effect on Vero
cell, but less on K562 cell line. Normally, the alkylating
drugs exhibit their antitumor activities through binding
cellular DNA; hence, the ability of passing through cellular
membrane is a very important factor. The difference
between them in antitumor activities may result from dis-
tinct difference in cellular recognition (receptor) or trans-
portation (transporter). In addition, we found that both the
BANM and the complexes had different ability causing
cellular mutation (data not shown). Compared with positive
group (absence of BANM and the complexes), the com-
plexes led to an increase in host cell nucleus larger than
that of BANM, which is characteristic of mutation in
morphology. It should be noted that although a lot of
compounds can interact with DNA via intercalation or
alkylation in vitro, the cytotoxicity exhibited by them on
cellular level or in vivo was varied significantly, which
depends on many factors, such as their permeability
through cellular membrane, stability against hydrolases
in vivo, and so on. The rare earth metal complexes of
BANM displayed less cytotoxic than the ligand, which was
beyond our expectation. The difference in cytotoxicity
between the complexes and ligand might reflect the dif-
ference in manner of crossing membrane, for some metal
complexes cross cellular membrane via specific trans-
porter, such as cisplatin, via copper transporter (Howell
et al., 2010). It is not clear that there is a difference in
transportation of BANM and its complexes to crossing
cellular membrane, but their distinct cytotoxicity could be
overall effects resulting from their interaction with bio-
logical components of host cell.
Fig. 4 The fluorescence Scatchard plots of DNA-EB system in the
presence of different concentration of La3? complex. Rt increases in
the order 0.5, 1.0, and 1.5 for line 1–3
Table 3 The characteristics of Scatchard plots of DNA-EB systems
in the presence of different concentrations of the complexes
Complex
La
R
Rt
n
KEB (106/mol/l)
0.9962
0.9961
0.9960
0.9915
0.9876
0.9891
0.8973
0.9125
0.9000
0.8865
0.9011
0.9201
0.9073
0.9121
0.8992
0.9215
0.9300
0.9179
0.5
1.0
1.5
0.5
1.0
1.5
0.5
1.0
1.5
0.5
1.0
1.5
0.5
1.0
1.5
0.5
1.0
1.5
0.215
0.205
0.195
0.212
0.198
0.185
0.214
0.203
0.191
0.217
0.201
0.189
0.212
0.197
0.185
0.215
0.201
0.187
1.66
1.59
1.39
1.67
1.60
1.51
1.65
1.57
1.42
1.64
1.58
1.50
1.66
1.58
1.49
1.62
1.56
1.41
Eu
Dy
Sm
Gd
Tm
Comet assay
The results are given in Table 4. It was evident that the
BANM had higher antitumor activities against the giving
cell lines, and its IC50 value is close to the data reported in
literature (Anastasiou et al., 1994). However, the com-
plexes did not have higher antitumor activity than the
BANM did. In general, the complexes in physiological
condition may undergo partial dissociation, releasing the
active BANM (as electrolyte). However, this situation may
not happen partly due to the non-electrolyte nature of the
complexes, so the cytotoxicity displayed was probably only
Comet assay has been reported to be a good method of
detecting apoptosis, and the amount of DNA damage in the
cell was estimated from tail length as the extent of the
migration of the genetic material in the direction of the anode
(Singh et al., 1988). In order to investigate the underlying
mechanism, we conducted comet assay. As shown in Fig. 5,
in control (Fig. 5, top), non-drug treated cells, the damaged
DNA was not detected, and the high-molecular-weight
supercoiled DNA remained intact. However, the cells treated
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Med Chem Res (2013) 22:2862–2869
between the complexes and ligand might reflect the dif-
ference in manner of crossing membrane, because some
metal complexes cross cellular membrane via specific
transporter, such as cisplatin, via copper transporter
(Howell et al., 2010). It is not clear that there is a differ-
ence in transportation of BANM and its complexes, so a
deep investigation was required in the future research.
Acknowledgments This study was supported by Xinxiang Medical
University (ZD2011-6). The authors would like to thank Dr. qiu-e
Cao (Yun Nan University, China) for help in fluorescence experiment.
References
Achyuthan KE, Mary A, Greenberg CS (1989) Tb(III)-ion-binding-
induced conformational changes in platelet Factor XIII. Biochem
J 257:331–338
Anastasiou A, Catsoulacos P, Papageorgiou A, Margariti E (1994) On
the formation of homo-azasteroidal esters of N,N-bis(2-chloro-
ethyl)aminobenzoic acid isomers and their antitumor activity.
J Heterocycl Chem 31:367–374
Beria I, Nesi M (2002) Syntheses of brostallicin starting from
distamycin A. Tetrahedron Lett 43:7323–7327
Fig. 5 Typical comet images from K562 cells treated with
La(BANM)3. Top part (untreated as control); and low part (treatment
with 50 lM followed by 48 h incubation), comet tails were visible as
arrow pointed. All images stained with EB
Choppin GR, Henrie DE, Buijs K (1966) Environmental effects on f–f
transitions I. Neodymium(III). Inorg Chem 5:1743–1748
Coutsolelos AG, Spyroulias GA (1994) Dysprosium metallation to
substituted tetraaryl-porphyrin complexes; synthesis and spec-
troscopic characterization. Polyhedron 13:647–653
with lanthanide complex had a comet tail (Fig. 5, bottom),
indicating that the genomic DNA of host cells were broken
and fragmentized, which was a general characteristic of
apoptotic cell. So we presume that the cytotoxicity exhibited
by lanthanide complex may be via interaction (alkylation or
intercalation) with cellular DNA, leading to cross-linking of
intra or inter-strand of DNA, and initiating apoptosisprocess.
However, the details of the interaction of lanthanide complex
with host cell was not clear; a further investigation on
apoptotic molecules, such as, how does p53, bcl, bax, or
caspases response on mRNA and protein level was required
in the future study.
DoDodoff N, Grancharow K, Gugova R (1994) Platinum (II)
complexes of benzoic- and 3-methoxybenzoic acid hydrazides.
Synthesis, characterization, and cytotoxic effect. J Inorg Bio-
chem 54:221–233
Figgis BN, Lewis J, Wilkins RG (eds) (1960) Modern coordination
chemistry. Interscience, New York
Geary WJ (1971) The use of conductivity measurements in organic
solvents for the characterisation of coordination compounds.
Coord Chem Rev 7:81–122
Gschneidner KA, Jr., LeRoy E (1999) Handbook on the physics and
chemistry of rare earths. North-Holland, Amsterdam
Henrie DE, Choppin GR (1968) Environmental effects on f–f
transitions. II. ‘‘hypersensitivity’’ in some complexes of trivalent
neodymium. J Chem Phys 49:477–482
Howe GM, Wu KC, Bauer WR (1976) Binding of platinum and
palladium metallointercalation reagents and antitumor drugs to
closed and open DNAs. Biochemistry 15:4339–4346
Howell SB, Safaei R, Larson CA, Sailor MJ (2010) Copper
transporters and the cellular pharmacology of the platinum-
containing cancer drugs. Mol Pharmacol 77:887–894
Judd BR (1966) Hypersensitive transitions in rare earth ions. J Chem
Phys 44:839–840
Karraker DG (1967) Hypersensitive transitions of six-, seven-, and
eight-coordinate neodymium, holmium, and erbium chelates.
Inorg Chem 6:1863–1868
Koutsourea AI, Fousteris MA, Arsenou ES, Papageorgiou A, Pairas
GN, Nikolaropoulos SS (2008) Synthesis, in vivo antileukemic
evaluation and comparative study of novel 5ꢀ-7-keto steroidal
esters of chlorambucil and its active metabolite. In Vivo
22:345–352
Conclusion
In the present study, BANM and its rare earth metal
complexes had been synthesized. The aim was to determine
whether the rare earth metals had synergistic effect once
forming complexes due to important physiological function
of the rare earth metals. Beyond our expectation, the rare
earth metal complexes exhibited less cytotoxicity than
BANM did. Although the complexes had a definite anti-
tumor activity, the metal ions did not display synergistic
instead of antagonistic effect once forming complexes with
BANM. We presume that the characteristic of non-elec-
trolyte of the complexes may be partial reason for the less
cytotoxicity. In addition, the difference in cytotoxicity
Kumer PA (1998) Synthesis and spectral characterization of
lanthanide(III) metal complexes of an amino acid hydrazide,
N-(2-acetyltbiophenoimno)-2-benzamidoethan amide. Synth
React Inorg Met-Org Chem 28(5):699–713
123

Med Chem Res (2013) 22:2862–2869
2869
Leoni LM, Bailey B, Reifert J et al (2008) Bendamustine (treanda)
displays a distinct pattern of cytotoxicity and unique mechanistic
features compared with other alkylating agents. Clin Cancer Res
14:309–317
complexes with 2 (((4,6-dimethyl)-2-pyrimidinyl)thio)-acetic
acid. J Rare Earths 24:15–19
Roger GM, Roger FS (1996) Antibody-enzyme conjugates for cancer
therapy. J Natl Cancer Inst 88(3/4):153–165
Lepecq JB, Paotetti C (1967) A fluorescent complex between
ethidium bromide and nucleic acids: physical—chemical char-
acterization. J Mol Biol 27:87–106
Li C, Wang L, Meng X, Zhao H (1999) Synthesis, characterization
and antitumor activity of benzaldehyde nitrogen, mustard
picolinoyl hydrazone complexes. Transition Met Chem
24:206–209
Li MX, Shao M, Dai H, An BL, Lu WC, Zhu Y, CX Du (2005)
Synthesis and crystal structure of a novel copper(II) complex
with acetylenedicarboxylate and 2,20-bipyridine. Chin Chem
Lett 16(10):1405–1408
Sadlter Research Lab. Inc. (1970) Sadlter standard Infrared grating
spectra, 171162K
Sigh M, Misra SN, Verma RD (1978) Spectral and magnetic
properties of lanthanon fluoroacetates. J Inorg Nucl Chem
40:1939–1940
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple
technique for quantitation of low levels of DNA damage in
individual cells. Exp Cell Res 175:184–191
Spinner E (1964) The vibration spectra of some substituted acetate
ions. J Chem Soc 4217–4226
Zhang ZW, Jiang T, Ren SM, Zhang YX, Yu JS (2005) Synthesis,
crystal structure and in vitro antitumor activity of di-n-butyltin
p-[N,N-bis(2-chloroethyl)amino]benzoates. Chin J Chem 23(12):
1655–1758
Zhao L, Chen Y, Zhang H, Li C, Sun R, Yang Q (2009) Study of
structure and two-dimension correlation infrared spectroscopy
on three rare-earth/3-methylbenzoic acid complexes. J Mol
Struct 920:441–449
Li J, Wang L, Bai H, Yang B, Yang
H (2011) Synthesis,
characterization, and anti-inflammatory activities of rare earth
metal complexes of luteolin. Med Chem Res 20:88–92
Melby LR, Rose NJ, Abamson E, Caris JC (1964) Synthesis and
fluorescence of some trivalent lanthanide complexes. J Am
Chem Soc 68:5117–5125
Nakamoto K (1978) Infrared and Raman spectra of inorganic
coordination compounds, 4th edn. Interscience, New York
Nelson DL, Cox MM (2004) Lehninger principles of biochemistry.
W. H. Freeman, New York
Zhou M, Fei H, Liu Y, Li WF (2010) Metal complexes and their
applications in bioassay bio-imaging and protein staining. Prog
Chem 22(1):201–209
Qu J, Wang L, Liu Y, Yumin S, Wang Y, Jia X (2006) Syntheses,
characterization and antitumour activities of rare Earth metal
123