Synthesis, characterization, cytotoxic activities, and DNA-binding properties of the La(III) complex with Naringenin Schiff-base

Article abstract of DOI:10.1016/j.bmc.2005.10.031

A new Naringenin Schiff-base ligand (H₃L) and its complex, [La(H₂L)₂(NO₃)?3H₂O], have been synthesized and characterized on the basis of elemental analyses, molar conductivities, mass spectra, ^1<

Full text of DOI:10.1016/j.bmc.2005.10.031

Bioorganic & Medicinal Chemistry 14 (2006) 1880–1888
Synthesis, characterization, cytotoxic activities, and
DNA-binding properties of the La(III) complex
with Naringenin Schiff-base
Bao-dui Wang,a Zheng-Yin Yang,a,* Qin Wang,b Ti-kuan Caib and Patrick Crewdsonc
aCollege of Chemistry and Chemical Engineering and State Key Laboratory of Applied Organic Chemistry,
Lanzhou University, Lanzhou 730000, PR China
bCollege of life science, Department of Chemistry, Lanzhou University, Lanzhou 730000, PR China
cDepartment of Chemistry, University of Ottawa, Ottawa, Ont., Canada K1N 6N5
Received 9 August 2005; revised 20 October 2005; accepted 21 October 2005
Available online 28 November 2005
Abstract—A new Naringenin Schiff-base ligand (H3L) and its complex, [La(H2L)2(NO3)Æ3H2O], have been synthesized and charac-
terized on the basis of elemental analyses, molar conductivities, mass spectra, 1H NMR, thermogravimetry/differential thermal anal-
ysis (TG–DTA), UV spectra, and IR spectra. Spectrometric titrations, ethidium bromide displacement experiments, and viscosity
measurements indicate that the two compounds, especially the La(III) complex, strongly bind with calf-thymus DNA, presumably
via an intercalation mechanism. The intrinsic binding constants of the La(III) complex and ligand with DNA were 1.83 · 107 and
9.46 · 105 Mꢀ1, respectively. Comparative cytotoxic activities of the La(III) complex and ligand were also determined by MTT
[3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide] and SRB (sulforhodamine B) methods. The results showed that
the La(III) complex had significant cytotoxic activity against the tested cells.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
peutics but also to a greatly expand ability for chemists
to probe DNA and to develop highly sensitive diagnos-
tic agents.4
Numerous biological experiments have demonstrated
that DNA is the primary intracellular target of antican-
cer drugs due to the interaction between small molecules
and DNA, which cause DNA damage in cancer cells,
blocking the division of cancer cells and resulting in cell
death.1–3 Of these studies, the interaction of transition
metal complexes containing multidentate aromatic
ligands with DNA has gained much attention. This is
due to their possible application as new therapeutic
agents and their photochemical properties which make
them potential probes of DNA structure and conforma-
tion.4–9
Transition-metal complexes are being used at the fore-
front of many of these efforts. Stable, inert, and water-
soluble complexes containing spectroscopically active
metal centers are extremely valuable as probes of biolog-
ical systems. As both spectroscopic tags and functional
models for the active centers of proteins, metal complex-
es have helped elucidate the mechanisms by which
metalloproteins function.4
In order to develop new antitumor drugs which specifi-
cally target DNA, it is necessary to understand the
different binding modes a complex is capable of under-
taking. Basically, metal complexes interact with the dou-
ble helix DNA in either a non-covalent or a covalent
way. The former way includes three binding modes:
intercalation, groove binding, and external static elec-
tronic effects. Among these interactions, intercalation
is one of the most important DNA binding modes as
it invariably leads to cellular degradation. It was report-
ed that the intercalating ability increases with the pla-
narity of ligands.10,11 (Additionally, the coordination
The design of small complexes that bind and react at
specific sequences of DNA becomes important. A more
complete understanding of how to target DNA sites
with specificity will lead not only to novel chemothera-
Keywords: Naringenin Schiff-base; La(III) complex; Cytotoxic activi-
ties; DNA binding.
*
Corresponding author. Fax: +86 931 8912582; e-mail: yangzy@
lzu.edu.cn
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.031

B. Wang et al. / Bioorg. Med. Chem. 14 (2006) 1880–1888
1881
geometry and ligand donor atom type also play key
roles in determining the binding extent of complexes
to DNA.)12,13 The metal ion type and its valence, which
are responsible for the geometry of complexes, also af-
fect the intercalating ability of metal complexes to
DNA.14,15
to 30 cmꢀ1. A weak band appearing at 413 cmꢀ1 is as-
signed to m(M–N). These data would indicate that the
nitrogen of the imino group also bonds to the rare earth
ion.33 The aqueous m(OH) and d(HOH) bands of the
complex appear at 3399 and 1594 cmꢀ1, respectively,
showing that there is some water in the complex.34
For the free ligand, m(NH) and d (NNH) appear at
3205 and 1520 cmꢀ1; the two bands for the complex shift
to 3228 and 1543 cmꢀ1, respectively, which demon-
strates that there is one hydrogen of the @NNH– in
the complex. All of these data confirm the fact that a
conjugate chelate ring formed by ligand-enolization ex-
ists in the complex. The absorption bands assigned to
the coordinated nitrates were observed at about 1489
(mas) and 893 (ms) cmꢀ1. The separation of the two high-
Flavonoids are a broadly distributed class of naturally
occurring pigments present in vascular plants and are
responsible for most of the colors in nature. These natu-
ral products are potentially antibacterial, anticancer,
antioxidant, anti-inflammatory, and antiallergenic
agents since they stimulate or inhibit a wide variety of en-
zyme systems as pharmacological agents.16–21 The best
known flavonoids, such as quercetin, morin, and rutin,
have been studied previously.22,23 Naringenin is one of
the lesser-known flavonoids expect for its use as antibac-
terial and anticancer agents.24 In addition, some hydraz-
ones and their complexes with metals often have diverse
biological and pharmaceutical activities.25–30 However,
up to now the biological activity and interactions of
naringenin hydrazones and their complexes with DNA
have not been reported. This aroused our interest in
the synthesis of a new ligand, 40,5,7-trihydroxy-flavanoe
benzoyl hydrazone (H3L), and its La(III) complex in
view of evaluating their pharmaceutical activities. The li-
gand was prepared by the reaction shown in Figure 1.
est frequency bands jm4 ꢀ m1j is approximately 176 cmꢀ1
,
ꢀ
and accordingly the coordinated NO3 ion in the com-
plex is a bidentate ligand.35
1
2.2. H NMR spectra
1
The H NMR spectra of the ligand (Fig. S1) and its
La(III) complex (Fig. S2) are assigned as follows: H2L
((CD3)2CO), d (ppm): 2.97 (1H, dd, J = 12.3, 17.0 Hz,
3(a)-H), 3.46 (1H, dd, J = 3.0, 17.0 Hz, 3(e)-H), 5.11
(1H, dd, J = 3.0, 12.3 Hz, 2(a)-H), 5.95 (1H, d,
J = 4.8 Hz, 6-H), 5.98 (1H, d, J = 4.8 Hz, 8-H) 6.90
(2H, d, J = 8.4 Hz, 30, 50-H), 7.36–7.55 (5H, m, PhH),
7.92 (2H, d, J = 8.4 Hz, 20, 60-H), 9.04 (1H, s, 4-OH),
9.50 (1H, s, 7-OH), 10.52 (1H, s, 5-OH), 13.08 (1H, s,
–NH–C@O). La (H2L)2(NO3)Æ3H2O (DMSO-d6), 2.89
(2H, d, J = 9.0 Hz, 3(a)-H), 3.53-3.80 (6H, br, H2O),
3.86 (2H, d, J = 9.0 Hz, 3(e)-H), 5.00 (2H, br, 2(a)-H),
5.49 (2H, J = 9.6 Hz, 6-H), 5.90 (2H, d, J = 9.6 Hz, 8-
H), 6.78 (4H, d, J = 10.5 Hz, 30, 50-H), 7.24–7.60 (10H,
m, PhH), 7.90 (4H, d, J = 10.5 Hz, 20, 60-H), 9.18 (2H,
s, 4-OH), 9.58 (2H, s, 7-OH), 13.16 (2H, s, –NH–
C@O–). In the complex, the –OH-5 hydrogen is replaced
by metal and the –NH– group of the complex shifts
downfield (9.58 ppm) compared to that of the free li-
gand. The hydrogen of the @NNH– group is still detect-
ed in the complex, which is also supported by the IR
spectra.
2. Results
The complex is air stable for extended periods and solu-
ble in methanol, DMSO, and DMF; slightly soluble in
ethanol; insoluble in benzene, water, and diethyl ether.
The molar conductance value is 54.7 ohmꢀ1 molꢀ1 cm2
in DMF, showing that the complex is non-electrolytic
in DMF.31 The elemental analyses suggest that the for-
mula of the complex is [La(H2L)2(NO3)Æ3H2O]. The
complex exhibits its first endothermic peak at 58.4 ꢁC
in the TG–DAT (Fig. S3), which corresponds to
5.45%, a value that indicates three water molecules.
2.1. IR spectra
The m(C@O) vibration of the free ligand is at 1640 cmꢀ1
;
,
2.3. Electronic absorption titration
for the complex the peak shifts to 1609 cmꢀ1
Dm(ligand–complex) is equal to 31 cmꢀ1. A band, which ap-
pears at 470 cmꢀ1 after reaction with the metal, is as-
signed to m(M–O). These data indicate that the oxygen
of the carbonyl has likely formed a coordination bond
with the rare earth ion.32 The band at 1600 cmꢀ1 for
the free ligand is assigned to m(C@N); for the complex
the peak shifts to 1570 cmꢀ1, Dm(ligand–complex) is equal
The study of the electronic spectra in the ultraviolet and
visible ranges for the La(III) complex and ligand was
carried out in a buffer solution. The electronic spectra
of ligand had a strong band at kmax = 201 nm, a medium
band at
k
max = 323 nm and
a
weak band at
kmax = 223 nm. It follows from the literature that the
band at 323 nm is related to the absorbance of the B ring
6' 5'
O
4'
8
HO
O
O
H+
∆
1'
7
O
N
H N
OH
HN
C
+
2
B
HO
OH
2
A
6
3'
2'
3
5
4
H
N
OH
c
C
OH
O
Figure 1. Preparation route of the ligand (H3L).

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B. Wang et al. / Bioorg. Med. Chem. 14 (2006) 1880–1888
(cinnamoyl system), whereas the band at 201 nm is relat-
ed to the p ! p* transition absorbance (benzoyl system)
in the A ring. The band at 223 nm is the absorbance of
the C ring.36 The spectra of the La complex were the
same, except for the peak at 323 nm, which is shifted
to 335 nm.
Steady-state emission quenching experiments are also
used to observe the binding mode of the compounds
to DNA. Figure 4 shows the emission spectra of the
DNA–EB system with increasing amounts of La(III)
complex and free ligand. The emission intensity of the
DNA–EB system (k = 578 nm) decreased as the concen-
tration of the La(III) complex and ligand increased, and
an isobathic point appeared at 555 nm. The quenching
plots illustrate that the quenching of EB bound to
DNA by the ligand and the complex is in good agree-
ment with the linear Stern–Volmer equation.
The absorption spectra of the La(III) complex and li-
gand in the absence and presence of CT-DNA (at a con-
stant concentration of compound) are given in Figure 2.
In the presence of CT-DNA, the absorption bands of
La(III) complex at about 202 nm and 335 nm exhibited
hypochromism of about 47.28% and 17.51%, and batho-
chromism of about 13 and 3 nm, respectively. The li-
gand at 201 and 323 nm exhibits hypochromism of
about 37.53% and 11.42%, and bathochromism of about
7 and 1 nm. It is noteworthy that the hypochromicity of
the complex is much greater than that of the parent
ligand.
2.5. Viscosity studies
The effects of both compounds on the viscosity of DNA
are shown in Figure 5. The viscosities of the DNA in-
crease steadily with increasing concentration of ligand
and La(III) complex, and the extent of the increase
observed for the ligand is smaller than that for the
La(III) complex.
2.4. Fluorescence spectra
2.6. Cytotoxic activity
The La(III) complex and ligand can emit weak lumines-
cence in Tris buffer with a max wavelength of about
446 nm. The results of the emission titrations for the
La(III) complex and ligand with DNA are illustrated
in the titration curves (Fig. 3). Upon addition of
DNA, for the La(III) complex and ligand, the emission
intensities of the compounds grow to around 3.78 and
4.53 times larger, respectively, than those in the absence
of DNA.
The cytotoxicity assays of the La(III) complex and li-
gand against two kinds of tumor cells (human leukemia
HL-60 and lung adenocarinoma A-549) are shown in
Figures 6 and 7. The La(III) complex exhibited activity
higher than that of the free ligand at all concentrations.
At a concentration of 10ꢀ9 mol Lꢀ1, the La(III) complex
showed an inhibition rate of over 50% against human
leukemia HL-60. For the A-549 cell line the La(III)
complex demonstrated a stronger inhibitory effect than
even cisplatin37 over a range of concentrations from
10ꢀ5 to 10ꢀ8 mol Lꢀ1
.
2
3. Discussion
3.1. Structure of the La(III) complex
Since the crystal structure of the La(III) complex has not
been obtained yet, we characterized the complex and
determined its possible structure by elemental analyses,
molar conductivities, 1H NMR, IR data, TG–DTA,
and UV–vis measurements. The likely structure of the
complex is shown in Figure 8.
0
200
300
400
Wavelength (nm) (a)
2.5
2.0
1.5
1.0
0.5
0.0
3.2. DNA-binding constant and mode
The interactions of metal complexes with DNA have
been the subject of interest for the development of effec-
tive chemotherapeutic agents. Transition metal centers
are particularly attractive moieties for such research
since they exhibit well-defined coordination geometries
and also often possess distinctive electrochemical or
photophysical properties, thus enhancing the functional-
ity of the binding agent.5
200 250 300 350 400 450
Wavelength (nm) (b)
Figure 2. (a) Electronic spectra of ligand (10 lM) in the presence of
increasing amounts of CT-DNA. [DNA] = 0–22.25 lM. Arrow shows
the absorbance changes upon increasing DNA concentration. (b)
Electronic spectra of La(III) complex (10 M) in the presence of
increasing amounts of CT-DNA. [DNA] = 0–22.5 lM. Arrow shows
the absorbance changes upon increasing DNA concentration.
Electronic absorption spectroscopy was an effective
method in examining the binding mode of DNA with
the metal complex.38–40 If the binding mode was interca-
lation, the p* orbital of intercalated ligand can couple
with the p orbital of the base pairs, thus decreasing

B. Wang et al. / Bioorg. Med. Chem. 14 (2006) 1880–1888
1883
,
A
A
120
100
80
60
40
20
0
380
400
420
440
460
480
500
520
Wavelength λ /nm
,
B
B
300
200
100
0
400
420
440
460
480
500
520
Wavelength λ /nm
Figure 3. (A) The emission enhancement spectra of ligand (10 lM) in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, and 22.5 lM CT-DNA.
Arrow shows the emission intensity changes upon increasing DNA concentration. Inset: Scatchard plot of the fluorescence titration data of ligand,
K = 9.46 · 105 Mꢀ1. (B) The emission enhancement spectra of the La(III) complex (10 lM) in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5,
25, 27.5, 30, 32.5, 35, and 37.5 lM CT-DNA. Arrow shows the emission intensities upon increasing DNA concentration. Inset: Scatchard plot of the
fluorescence titration data of the La(III) complex, K = 1.83 · 107 Mꢀ1
.
the p ! p* transition energy and resulting in bathochro-
mism. If the coupling p orbital is partially filled by elec-
trons, it results in decreasing the transition probabilities
and concomitantly results in hypochromism.41
absorption of EB at 479 nm decreased and shifted to
515 nm in the presence of DNA, which is characteristic
of intercalation. Figure 9c is the absorption of a mixture
solution of EB, La(III) complex, and DNA. It was
found that the absorption at 515 nm increased com-
pared with Figure 9b. This could result from two rea-
sons: (1) EB bound to the La(III) complex strongly,
resulting in a decreased amount of EB intercalated into
DNA; (2) there exists competitive intercalation between
the La(III) complex and EB with DNA, thus releasing
some free EB from the DNA–EB system. However,
the former reason could be precluded since there were
no new absorption peaks.
The spectroscopic variations are strongly indicative of
the intercalation of the La(III) complex and the ligand
with CT DNA, involving a strong p-stacking interaction
between the complex (or ligand) and the DNA base
pairs. They can both insert into DNA base pairs, but
it appears that the complex inserts into DNA more
deeply. Since the hypochromism of the p ! p* transi-
tion absorbance (benzoyl system) in the A ring is greater
than those of B ring and C ring, we deduced that the A
ring intercalates deeply into the DNA base pairs,
whereas the B ring interacts with DNA through a
semi-intercalation or quasi-intercalation.42
The results of the emission titrations suggest that both
the compounds are protected from solvent water mole-
cules by the hydrophobic environment inside the DNA
helix, and that the complex can be protected more effi-
ciently than the free ligand. This implies that both the
compounds can insert between DNA base pairs deeply
and that the complex can bind to DNA more strongly
than just the free ligand. Since the hydrophobic environ-
In order to test if the La(III) complex could bind to
DNA by intercalation, ethidium bromide (EB) was em-
ployed, as EB interacts with DNA as a typical indicator
of intercalation.43 Figure 9 shows that the maximal

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B. Wang et al. / Bioorg. Med. Chem. 14 (2006) 1880–1888
,
35
30
25
20
15
10
A
A
540
560
580
600
620
640
Wavelength λ /nm
,
60
50
40
30
20
10
0
B
B
520
540
560
580
600
620
640
Wavelength λ /nm
Figure 4. (A) The emission spectra of DNA-EB system (10 lM and 0.32 lM EB), kex = 500 nm, kem = 520.0–650.0 nm, in the presence of 0, 5,10, 15,
20, 25, and 30 lM ligand. Arrow shows the emission intensity changes upon increasing ligand concentration. Inset: Stern–Volmer plot of the
fluorescence titration data of ligand, Kq = 3.54 · 103 Mꢀ1. (B) The emission spectra of DNA–EB system (10 and 0.32 lM EB), kex = 500 nm,
kem = 520.0–650.0 nm, in the presence of 0, 5, 10, 15, 20, 25, and 30 lM La(III) complex. Arrow shows the emission intensity changes upon increasing
La(III) complex concentration. Inset: Stern–Volmer plot of the fluorescence
ligand
La(III)complex
concentration of compounds (-LogC, M)
Figure 6. Cytotoxic activity of compounds against HL-60.
ment inside the DNA helix reduces the accessibility of
solvent water molecules to the compound and the com-
pound mobility is restricted at the binding site, a de-
crease of the vibrational modes of relaxation results.
The binding of the La(III) complex and ligand to
Figure 5. Effect of increasing amounts of La(III) complex and ligand
on the relative viscosity of caltf thymus DNA at 25.0 ꢁC.

B. Wang et al. / Bioorg. Med. Chem. 14 (2006) 1880–1888
1885
r/Cf versus r gave the binding constants 1.83 · 107 and
9.46 · 105 Mꢀ1 from the fluorescence data for the
La(III) complex and the free ligand, respectively. These
results show that the complex binds more strongly than
the free ligand. The higher binding affinity of complex is
probably attributed to the extension of the p system of
the intercalated ligand due to the coordination of La,
which also leads to a planar area greater than that of
the free ligand, which leads to the coordinated ligand pe-
netrating more deeply into, and stacking more strongly
with the base pairs of the DNA.
ligand
La(III)complex
The DNA-binding modes of both compounds were fur-
ther monitored by a fluorescent EB displacement as-
say.45 It is well known that EB can intercalate
nonspecifically into DNA which causes it to fluoresce
strongly. Competitive binding of other drugs to DNA
and EB will result in displacement of bound EB and a
decrease in the fluorescence intensity. This fluores-
cence-based competition technique can provide indirect
evidence for the DNA-binding mode. The emission
band at 578 nm of the DNA–EB system decreased in
intensity with an increase in the concentration of the
two compounds, which indicated that the compounds
could displace EB from the DNA–EB system. The
resulting decrease in fluorescence was caused by EB
changing from a hydrophobic environment to an aque-
ous environment.46 Such a characteristic change is often
observed in intercalative DNA interactions.47 The
quenching plots illustrate that the quenching of EB
bound to DNA by the ligand and complex is in good
agreement with the linear Stern–Volmer equation. In
the plots of F0/F versus [Q], Kq is given by the ratio of
the slope to the intercept. The Kq values for the La(III)
concentration of compounds (-LogC, M)
Figure 7. Cytotoxic activity of compounds against A-549.
Figure 8. The suggested structure of the complex.
complex and ligand are 7.48 · 104 and 3.54 · 103 Mꢀ1
,
respectively. The data show that the interaction of the
La(III) complex with DNA is stronger than that of the
free ligand, which is consistent with the above absorp-
tion spectral results. Since these changes indicate only
one kind of quenching process, it may be concluded that
the La(III) complex and free ligand bind DNA via the
same mode (intercalation mode).
Optical photophysical probes generally provide neces-
sary, but not sufficient, clues to support one binding
mode. Hydrodynamic measurements that are sensitive
to the length change (i.e., viscosity and sedimentation)
are regarded as the least ambiguous and most critical
tests for binding in solution in the absence of crystallo-
graphic structural data.48 A classical intercalation model
demands that the DNA helix lengthen as base pairs are
separated to accommodate the binding ligand, leading
to an increase in DNA viscosity. In contrast, a partial,
non-classical intercalation of compound could bend
(or kink) the DNA helix, reducing its effective length
and, concomitantly, its viscosity.49,50 Viscosity experi-
mental results clearly show that both the compounds
can intercalate between adjacent DNA base pairs, caus-
ing an extension in the helix, and thus increase the vis-
cosity of DNA; and that the La(III) complex can
intercalate more strongly and deeply than the free li-
gand, leading to the greater increase in viscosity of the
DNA with an increasing concentration of complex.
Figure 9. The visible absorption spectra of 1 · 10ꢀ5 M EB (a); (a)
+1 · 10ꢀ4 M DNA (b); (b) +3 · 10ꢀ4 La(III) complex; (c) in Tris–HCl
buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.1) solution.
DNA leads to a marked increase in emission intensity,
which also agrees with those observed for other interca-
lators.44 According to the Scatchard equation, a plot of

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B. Wang et al. / Bioorg. Med. Chem. 14 (2006) 1880–1888
The results obtained from viscosity studies validate
those obtained from the spectroscopic studies.
The ligand and the La(III) complex were dissolved in
a mixture solvent of 1% CH3OH and 99% Tris–HCl
buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.1) at con-
centration 1.0 · 10ꢀ5 M.
On the basis of all the spectroscopic studies together with
the viscosity measurements, we come to the conclusion
that the La(III) complex and ligand can bind to CT-
DNA in an intercalative mode and that the La(III) com-
plex binds to CT-DNA more strongly than the free ligand.
5.2. Measurements
Carbon, hydrogen, and nitrogen were analyzed on an
Elemental Vario EL analyzer. The metal contents of
the complex were determined by titration with EDTA.
Infrared spectra (4000–400 cmꢀ1) were determined with
KBr disks on a Therrno Mattson FTIR spectrometer.
The ultraviolet spectra were recorded on a Varian Cary
100 Conc spectrophotometer. The thermal behavior was
monitored on a Beijing Optics Instrument Factory PCT-
2 differential thermal analyzer. The molar conductance
values were determined in DMF on a Shanghai Leici
DSS-11A conductivity meter. 1H NMR spectra were
measured on a Varian VR 300-MHz spectrometer, using
TMS as a reference in (CD3)2CO for (H3L) and DMSO-
d6 for the La(III) complex. Mass spectra were performed
on a VG ZAB-HS (FAB) instrument. The fluorescence
3.3. Bioassay in vitro
The inhibitory rate of the La(III) complex is much higher
than that of the free ligand, which may be attributed to the
extended planar structure induced by the pꢀp* conjuga-
tion resulting from the chelation of the metal ion with the
ligand. Moreover, the inhibitory effect of the La(III) com-
plex was higher than that of cisplatin against the A-549
cell line at some experimental concentrations. These find-
ings of cytotoxic activity in vitro prompted us to explore
the DNA-binding studies on this compound.
4. Conclusion
spectra were recorded on
spectrofluorophotometer.
a
Hitachi RF-4500
Taken together, a new ligand, 40,5,7-trihydroxy-flavanoe
benzoyl hydrazone (H3L), and its La(III) complex have
been prepared and characterized. The DNA-binding
properties of the ligand and La(III) complex were inves-
tigated by absorption, fluorescence, and viscosity mea-
surements. The results support the fact that both the
compounds bind to CT-DNA via intercalation. The
binding constant shows that the DNA-binding affinity
increases in the order: free ligand < La(III) complex.
The two compounds have shown considerable cytotoxic
activity against two tumor cell lines, and the suppression
rate of the La(III) complex is higher than that of the li-
gand itself. The La(III) complex against the A-549 cell
line was more potent than cisplatin at most experimental
concentrations. These findings can shed some light on
designing new potent anticancer drugs.
5.3. Preparation of ligand (H3L)
Naringenin (2.72 g, 10 mmol) and benzoyl hydrazine
(1.37 g, 11 mmol) were dissolved in ethanol (40 mL).
Acetic acid (1.0 mL) was added to this solution. The
solution was refluxed on a water bath for one day with
stirring. After that, the yellow solution was concentrat-
ed and cooled, a yellow solid separating out. The yel-
low precipitate was filtered, washed with water, and
recrystallized from ethanol to give the ligand. Yield:
2.34 g (60%). Mp 231–232 ꢁC. FAB-MS: m/z = 391
[M+H]+. Anal. Calcd for C22H18O5N2: C, 67.69; H,
4.62; N, 7.18. Found: C, 67.29; H, 4.59; N, 7.20. IR
m
d
max(cmꢀ1): mC@O: 1640, mC@N: 1600 cmꢀ1, mNH: 3205,
NNH: 1520.
5.4. Preparation of complex
5. Experimental
5.1. Materials
The ligand (1.0 mmol, 0.390 g) was dissolved in ethanol
(20 mL) and triethylamine (1.0 mmol, 0.102 g) was then
added. After 5 min, La(NO3)3Æ6H2O (0.5 mmol, 0.217 g)
was added quickly and the solution was stirred for 10 h
at room temperature. A yellow precipitate, the La(III)
complex, was separated from the solution by suction fil-
tration, purified by washing several times with ethanol,
and dried for 24 h in vacuo. Anal. Calcd for LaC44H42-
N5O16: C, 51.01; H, 4.09; N, 6.76; La, 13.42. Found: C,
DNA and EB (ethidium bromide) were purchased from
Sigma Chemical Co. Naringenin was produced in Hui
Ke Co. (P.R.C.). Benzoyl hydrazine was produced by
the Shang Hai Reagent Co (P.R.C.). All chemicals used
were of analytical grade. The La(III) nitrate was pre-
pared from its oxide (99.9%) acquired from Nong Hua
(P.R.C.). All the experiments involving interaction of
the La(III) complex and the ligand with DNA were car-
ried out in doubly distilled water buffer containing
5 mM Tris [Tris(hydroxymethyl)-aminomethane] and
50 mM NaCl, and adjusted to pH 7.1 with hydrochloric
acid. A solution of CT-DNA in the buffer gave a ratio of
UV absorbance of about 1.8–1.9:1 at 260 and 280 nm,
indicating that the DNA was sufficiently free of pro-
tein.51 The DNA concentration per nucleotide was
determined by absorption spectroscopy using the molar
absorption coefficient (6600 Mꢀ1 cmꢀ1) at 260 nm.52
50.94; H, 3.99; N, 6.56; La, 13.22. IR mmax(cmꢀ1): mC@O
1609, mC@N: 1570 cmꢀ1, mNH: 3228, dNNH: 1543, dHOH
:
:
1594, mNO3: 1489, 1311, 1158, 893. Thermal analysis:
T = 58.4 ꢁC (H2O loss: 5.45, calcd: 5.21).
5.5. Electronic spectra
Absorption titration experiments were performed with
fixed concentrations of the drugs (10 lM) while gradual-
ly increasing the concentration of DNA. While measur-
ing the absorption spectra, an equal amount of DNA

B. Wang et al. / Bioorg. Med. Chem. 14 (2006) 1880–1888
1887
was added to both the compound solution and the refer-
ence solution to eliminate the absorbance of DNA itself.
5.8. Cytotoxicity assay
Tumor cell lines used in this work were grown in RPMI-
1640 medium supplemented with 10% (vol/vol) calf ser-
um, 2 mmolꢀ1 glutamine, 100 U mLꢀ1 penicillin (U = 1
unit of activity), and 100 lg mLꢀ1 streptomycin (GIB-
CO, Grand Island, NY) at 310 K under 5% CO2. Cells
in 100 lL culture medium were seeded into 96-well
plates (Falcon, CA).
5.6. Fluorescence spectra
To compare quantitatively the affinity of the compound
bound to DNA, the intrinsic binding constants Kb of the
two compounds to DNA were obtained by the lumines-
cence titration method. Fixed amounts of compound
were titrated with increasing amounts of DNA, over a
range of DNA concentrations from 2.5 to 37.5 lM.
An excitation wavelength of 352 nm was used, and total
fluorescence emission intensity was monitored at
446 nm. The concentration of the bound compound
was calculated using Eq. 1:44
For human leukemia HL-60 cell, the microculture MTT
[3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazoli-
um bromide] assay57 was conducted as follows. The HL-
60 cells were treated with compounds in a concentration
gradient to give final concentrations at 1 · 10ꢀ5
,
1 · 10ꢀ6, 1 · 10ꢀ7, 1 · 10ꢀ8, 1 · 10ꢀ9, and 1 · 10ꢀ10 M,
respectively. A 20 lL aliquot of MTT solution (5 mg/
mL) was added directly to all the appropriate wells.
The culture was then incubated for 4 h. A 50 lL of mix-
ture solution composed of SDS [CH3(CH211OSO3Na]
50(%), isobutyl acohol 5(%), and hydrochloride
(0.01 mol mLꢀ1) was added to the system. After the
plates were incubated overnight, the optical densities
were read on a plate reader (model VERSA Max,
Molecular Devices) at 570 nm.
Cb ¼ Ct½ðF ꢀ F 0Þ=ðF max ꢀ F 0Þꢁ;
ð1Þ
where Ct is the total compound concentration, F is the
observed fluorescence emission intensity at given DNA
concentration, F0 is the intensity in the absence of
DNA, and Fmax is the fluorescence of the totally bound
compound. Binding data were cast into the form of a
Scatchard plot53 of r/Cf versus r, where r is the binding
ratio Cb/[DNA]t and Cf is the free ligand concentration.
All experiments were conducted at 20 ꢁC in a buffer con-
taining 5 mM Tris–HCl (pH 7.1) and 50 mM NaCl
concentrations.
For lung adenocarinoma A-549 cell, the growth inhibi-
tion was analyzed by the SRB (sulforhodamine B) as-
say.58 Simply, following the treatment with the
compound for 72 h, the cell cultures were fixed with
10% trichloroacetic acid and incubated for 60 min at
277 K. Then, the plates were washed and dried, SRB
solution (0.4% wt/vol in 1% acetic acid) was added
and the culture was incubated for an additional
15 min. After the plates were washed and dried, bound
stain was solubilized with Tris buffer, and the optical
densities were read on the same plate reader at
515 nm. The growth inhibitory rate of treated cells was
calculated by (ODcontrol ꢀ ODtest)/ODcontrol · 100%.
Further support for the La(III) complex and ligand
binding to DNA via intercalation is given through the
emission quenching experiment. EB is a common fluo-
rescent probe for DNA structure and has been employed
in examinations of the mode and process of metal com-
plex binding to DNA.54 A 2-mL solution of 10 lM
DNA and 0.33 lM EB (at saturating binding levels38)
was titrated by 5–30 lM La(III) complex and ligand
(kex = 500 nm,
kem = 520.0–650.0 nm). According to
the classical Stern–Volmer equation:55
F 0=F ¼ Kq½Qꢁ þ 1;
where F0 is the emission intensity in the absence of
quencher, F is the emission intensity in the presence of
quencher, Kq is the quenching constant, and [Q] is the
quencher concentration. The shape of Stern–Volmer
plots can be used to characterize the quenching as being
predominantly dynamic or static. Plots of F0/F versus
[Q] appear to be linear and Kq depends on temperature.
Acknowledgment
This work is supported by the National Natural Science
Foundation of China 20475023) and Gansu NSF.
Supplementary data
5.7. Viscosity measurements
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmc.2005.10.031.
Viscosity experiments were conducted on an Ubbdlodhe
viscometer, immersed in a thermostatic water bath
maintained to 25.0 ꢁC. Titrations were performed for
the La(III) complex and the ligand (0.5–3 lM), and each
compound was introduced into a DNA solution (5 lM)
References and notes
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