Synthesis, DNA interaction and antimicrobial activities of copper (II) complexes with Schiff base ligands derived from kaempferol and polyamines

Article abstract of DOI:10.1016/j.inoche.2012.08.010

Two novel copper (II) complexes 1 and 2 with Schiff base ligands derived from kaempferol and polyamines such as ethylenediamine and diethylenetriamine have been synthesized and characterized by elemental analysis, IR, UV-visible spectroscopy, ¹

Full text of DOI:10.1016/j.inoche.2012.08.010

Inorganic Chemistry Communications 25 (2012) 55–59
Contents lists available at SciVerse ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Synthesis, DNA interaction and antimicrobial activities of copper (II) complexes with
Schiff base ligands derived from kaempferol and polyamines
a
b
a
c
a
Xin-Bin Yang a, , Qin Wang , Yu Huang , Peng-Hui Fu , Jin-Sheng Zhang , Ren-Quan Zeng
⁎
a
Rongchang Campus, Southwest University, Chongqing 402460, PR China
Pharmacy College, Ningxia Medical University, Yinchuan 750004, PR China
School of Chemistry and Material Science, Guizhou Normal University, Guiyang 550001, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel copper (II) complexes 1 and 2 with Schiff base ligands derived from kaempferol and polyamines
such as ethylenediamine and diethylenetriamine have been synthesized and characterized by elemental
analysis, IR, UV–visible spectroscopy, 1H NMR, molar conductance measurements and molecular modeling
studies. The interactions of complexes with DNA have been studied by absorption spectra, viscosity measure-
ments and gel electrophoresis under physiological conditions. The experimental results indicated that two
complexes could bind to CT-DNA via an intercalative mode. Noticeably, cleavage DNA (pUC 19) activity of
the complex 2 is stronger than that of complex 1. The antimicrobial activities against Escherichia coli and
Staphylococcus aureus of two complexes were evaluated by the minimum inhibition concentrations (MIC),
which indicated that complex 2 possessed more active against E. coli.
Received 4 June 2012
Accepted 14 August 2012
Available online 21 August 2012
Keywords:
Kaempferol
Schiff base copper (II) complexes
DNA interaction
Antimicrobial activity
© 2012 Elsevier B.V. All rights reserved.
Flavonoids are a group of naturally occurring compounds that are
found in many high plants. Such compounds have evoked widespread
interest in biological and pharmacological activities including antioxi-
dant, anticancer, and antimicrobial, etc. [1–5]. Moreover, there has
been rapid growth in the development of metal complexes of flavonoids
that have better biological activity than flavonoids alone [6–10]. The
studies of molecular targets have also revealed that DNA could be affect-
ed by flavonoids and their metal complexes [11–13].
Schiff base metal complex is a kind of attractive reagents due to spe-
cific activities of pharmacology and physiology. People pay great interest
in synthesis, DNA interaction and biological activity of Schiff base metal
complexes in recent years [14–20]. However, to the best of our knowl-
edge, less attention was paid on the DNA interaction and biological ac-
tivity of Schiff base metal complexes derived from flavonoids. In this
work, we focus our attention on the synthesis, DNA binding and cleaving
abilities of two copper (II) complexes with Schiff base ligands derived
from kaempferol (3,3′,5,7-tetrahydroxyflavone) and different poly-
amines such as ethylenediamine and diethylenetriamine (Scheme 1).
Furthermore, the antimicrobial activities were also investigated in vitro.
Schiff base ligands L1 and L2 were synthesized by a typical procedure.
Polyamine (1 mmol) was added to kaempferol (0.578 g, 2 mmol) and
refluxed in ethanol (10 mL) for 3 h. The yellow precipitate was filtered
off, washed with ethanol, and dried. The structures of Schiff base ligands
were confirmed by 1H NMR, IR and elemental analysis, which were con-
sistent with the proposed structures (Scheme 1B) [21,22]. Schiff base
copper (II) complexes 1 and 2 were prepared by a following procedure.
To a solution of copper (II) chloride dehydrate (0.017 g, 0.10 mmol) in
ethanol (5 mL) was added a solution of the Schiff base ligands
(0.10 mmol) in ethanol (100 mL) and sodium bicarbonate (0.032 g,
0.3 mmol) at room temperature for 6 h. The solution was filtered, and
the filtrate was concentrated under reduce pressure. The resulting
green solid complex was filtered off, washed with diethyl ether and
dried in vacuo. We got the likely composition of complexes:
[C32H22CuN2O10]Cl2·2H2O (1) and [C34H27CuN3O10]Cl2·H2O (2) by IR,
elemental analysis and molar conductivity [23,24].
The interactions between Schiff base ligands and Cu (II) were
studied by absorption spectra. Kaempferol absorbed with maxima at
372 nm (band I) and 269 nm (band II). Band I is related to ring B
(cinnamoyl system) and band II to ring A (benzoyl system ) [25,26]. Ab-
sorption spectra of Schiff base ligand L1 in the ethanol solution with dif-
ferent concentration of CuCl2 are shown in Fig. 1A. Schiff base ligand L1
exhibits two strong absorption bands at 367 nm (band I) and 267 nm
(band II). The intensity of Schiff base ligand L1 (band I) decreased grad-
ually with addition of Cu (II) to the solution, a new absorbance peak
appeared at 429 nm. The results indicated that Schiff base ligand
could form complex with Cu (II). The appeared new peak at 429 nm
suggested that Cu (II) had bond to 3-hydroxyl of ring B and C_N of
ring C [10–12]. Band I bathochromic shift can be explained by the inter-
action of Cu (II) with the 3-hydroxyl group of ring B resulting in elec-
tronic redistribution between Schiff base ligand and Cu (II) to become
an extended π‐bonding system. However, the absorbance peak of com-
plex at 429 nm disappeared, and the new peak at 336 nm appeared
when the complex was dissolved in Tris–HCl buffer solution (pH=
7.4) containing ethanol (10%). The result demonstrated the presence
of one new complex form. When followed by UV–visible spectra,
⁎ Corresponding author.
E-mail address: yangxbqq@126.com (X.-B. Yang).
1387-7003/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.inoche.2012.08.010

56
X.-B. Yang et al. / Inorganic Chemistry Communications 25 (2012) 55–59
HO
,
OH
HO
4
B
OH
HO
OH
3
O
CH NHCH
(
)
2
n
2
n=0, L1
n=1, L2
N
N
O
O
O
C
A
OH
HO
OH
5
7
HO
OH
HO
A
B
Scheme 1. Kaempferol (A) and Schiff base ligands derived from kaempferol and different polyamines (B).
complexation of Cu (II) by Schiff base ligand L1 in Tris–HCl buffer solu-
tion (pH=7.4) was investigated and shown in Fig. 1B. The intensity of
two bands of Schiff base ligand L1 decreased gradually with addition
of Cu (II), and a new absorbance peak appeared at 336 nm. Hypso-
chromic shift of band I and disappearance of band II of Schiff base ligand
L1 can be explained by the interaction of Cu (II) with the 5-hydroxyl
group of ring A and C_N of ring C. These different approaches drew
the conclusion that the first site occupied by Cu in Schiff base ligand
L1 in the ethanol solution is 3-hydroxyl of ring B as the 3-hydroxyl has
a more acidic proton, and also because of the presence of a strong intra-
molecular hydrogen bond between 5-hydroxyl and C_N, it seems diffi-
cult for 5-hydroxyl to take part to the formation of complex, whereas
the destruction of intramolecular hydrogen bond by addition of Tris–
HCl buffer solution (pH=7.4) can be advantageous to form stable com-
plex by the coordination of Cu (II) to 5-hydroxyl of ring A [22]. Absorp-
tion spectra of the interaction of Cu (II) with Schiff base ligand L2 were
similar to those of the interaction of Cu (II) with Schiff base ligand L1
(Fig. 2).
Since no single crystals suitable for X-ray determination could be
isolated, structural information for complexes 1 and 2 was also obtained
from the B3LYP/6-311G** optimization calculations. As shown in Fig. 3
theory calculation result demonstrated that the coordination of Cu (II)
to 5-hydroxyl of ring A could be more stable than that of 3-hydroxyl
of ring B.
The mode of the two complexes bound to DNA was investigated
by absorption spectra. Generally, hypochromism is observed in the
absorption spectra of small molecules if they intercalate into DNA
base pairs [27]. The absorption spectra were obtained by titration of
50 μM complexes in Tris–HCl buffer solution (pH=7.4) with increas-
ing concentration of DNA, as shown in Fig. 4. The absorption bands of
complexes 1 and 2 at 336 nm exhibited hypochromism of about 31%
and 18%, respectively. The hypochromism suggested that complexes
may bind to DNA by an intercalative mode. The absorption spectra
of complexes had almost not changed when the ratio of [DNA]/[com-
plex] was 1.8. The results showed that the DNA-binding reached
Fig. 1. Absorption spectra of Schiff base ligand L1 in ethanol (A) and Tris–HCl buffer so-
lution (pH=7.4) containing ethanol (10%) (B) in the presence of Cu (II), respectively.
The molar ratios [CuCl2]/[L1]=0 (a), 0.25 (b), 0.50 (c), 0.75 (d), 1.0 (e).
Fig. 2. Absorption spectra of Schiff base ligand L2 in ethanol (A) and Tris–HCl buffer so-
lution (pH=7.4) containing ethanol (10%) (B) in the presence of Cu (II), respectively.
The molar ratios [CuCl2]/[L2]=0 (a), 0.25 (b), 0.50 (c), 0.75 (d), 1.0 (e).

X.-B. Yang et al. / Inorganic Chemistry Communications 25 (2012) 55–59
57
Fig. 3. The optimized structure of the complex 1 (A) and 2 (B). The pink ball stands for
copper atom, the red ball stands for oxygen atom, the blue balls for nitrogens, the gray
balls for carbons and the white balls for hydrogens.
saturation. In order to compare the binding abilities of the complexes
to DNA, the binding constant, Kb, was calculated from the spectro-
scopic titration data using the equation [28]:
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
½DNAꢀ= εa−εf ¼ ½DNAꢀ= εb−εf þ 1=Kb εb−εf
Fig. 4. Absorption spectra of copper complexes 1 (A) and 2 (B) in the presence of DNA
in Tris–HCl buffer solution (pH=7.4). [complex]=50 μM, [DNA]=0, 15, 30, 45, 60, 75,
90 μM, respectively. The arrow shows the intensity changes on increasing DNA
concentration.
Where εa,εb and εf are the apparent, bound, and free extinction co-
efficients respectively. Fit the plot of [DNA]/(εa−εf) vs. [DNA], the Kb
was obtained from the ratio of the slope to the Y intercept.
The binding constants obtained for complexes 1 and 2 are 2.9×104
and 1.3×104 M−1, respectively. From the binding constant values, it
is clear that the complexes are moderate binders and complex 1
shows a stronger binding ability towards DNA.
the relative cleavage efficiency of complexes 1 and 2. It is obvious that
complex 2 showed the stronger cleavage ability to DNA (Lane 3).
Therefore, our subsequent efforts focus on the reactivity of complex 2.
The cleavage of DNA by different concentrations of complex 2 was
studied for incubation time of 6 h. The amount of nicked DNA (Form
II) was observed in agarose gel electrophoresis diagram increased in
accord with the changed trend of the concentration of complex 2 in
the reaction system (Fig. 7). Increasing the concentration of complex
2 in the order of 75, 150, 225 and 300 μM resulted in 40%, 55%, 81%
and 96% of nicked DNA, respectively.
Minimum inhibition concentration (MIC) is the lowest concentra-
tion of an antimicrobial complex that will inhibit the visible growth of
microorganisms after overnight incubation. The MIC of Schiff base cop-
per (II) complexes and kaempferol (as a comparison drug) was tested
against bacterial strains by tube double dilution method. Ciprofloxacin
was used as the control. The MIC of ciprofloxacin, kaempferol, com-
plexes 1 and 2 was found to be 21, 100, 104 and 54 μM for Escherichia
coli, whereas the MIC of ciprofloxacin, kaempferol, complexes 1 and 2
was found to be 11, 120, 126 and 164 μM against Staphylococcus aureus,
respectively. The results indicated that the ciprofloxacin was highly ef-
fective against the studied bacteria. Moderate activity was observed for
kaempferol, complexes 1 and 2, and complex 2 possessed better antimi-
crobial activity for E. coli than kaempferol and complex 1.
Spectroscopic data are necessary, but not sufficient to support a
binding mode. Viscosity experiment is an effective tool to study the
binding mode of complexes to DNA in the absence of crystallographic
structural data and NMR. A classical intercalative mode causes a sig-
nificant increase in viscosity of DNA solution due to increase in sepa-
ration of base pairs at intercalation site and hence an increase in
overall DNA length. However, a partial and/or nonclassical intercala-
tion of complex may bend (or kink) the DNA helix, resulting in the
decrease in its effective length with a concomitant decrease in its vis-
cosity [29,30]. The effects of complexes on the viscosity of CT-DNA so-
lution were given in Fig. 5. It can be seen that the relative viscosity of
DNA increased with the addition of complexes. The results further re-
vealed that the complexes could bind to DNA by intercalation mode.
Besides the above methods, interactions between the copper (II)
complexes and DNA were also investigated by the cleavage assay of
plasmid DNA (pUC 19). The cleavage of the plasmid DNA was ana-
lyzed by monitoring the conversion of supercoiled circular DNA
(form I) to nicked DNA (Form II). The amounts of strand scission
were assessed by agarose gel electrophoresis [31].
Firstly we compared the cleavage abilities of the complexes at a
concentration of 225 μM and an incubation time of 6 h. Fig. 6 showed

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X.-B. Yang et al. / Inorganic Chemistry Communications 25 (2012) 55–59
Fig. 5. Effects of increasing amount of copper complexes 1 (■) and 2 (▲) on the viscos-
ity of CT-DNA, [DNA]=50 μM.
In summary, the Schiff base copper (II) complexes 1 and 2 derived
from kaempferol and polyamines were synthesized and characterized.
The interactions of complexes with DNA were studied by UV spectra,
viscosity and gel electrophoresis under physiological conditions. The re-
sults indicate that complexes 1 and 2 are capable of binding DNA by an
intercalative mode and cleaving DNA. Moreover, complex 2 shows the
considerably high cleavage DNA abilities. In addition, the antimicrobial
activities of kaempferol, complexes 1 and 2 were evaluated by the min-
imum inhibition concentrations (MIC) and the results indicated that
complex 2 exhibited more antimicrobial activity against E. coli. These
studies reveal that the complexes can be used as potential chemothera-
peutic agents.
Fig. 7. Effect of concentration of complex 2 on the cleavage of pUC19 DNA (10 μg/mL)
in a Tris–HCl buffer (100 mM, pH 7.4) at 37 °C for 6 h. (A) Agarose gel electrophoresis
diagram: lane 1, DNA control; lanes 2–5, [complex 2]=75, 150, 225 and 300 μM.
(B) Quantitation of % plasmid relaxation (Form II %) relative to plasmid DNA per lane.
Acknowledgments
the Central Universities (XDJK2010C004) and Chunhui of Ministry of
Education project (Z2011048).
This work was financially supported by the National Science Foun-
dation of China (No. 21172182), the Fundamental Research Funds for
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[31] Plasmid DNA (pUC 19) cleavage activity of complexes was monitored by using
agarose gel electrophoresis. In a typical experiment, supercoiled DNA (pUC 19)
(10 μg/mL, 10 μL) in Tris–HCl (100 mM, pH 7.4) was treated with different con-
centrations of complexes, followed by dilution with the Tris–HCl buffer to a
total volume of 30 μL. The samples were then incubated at 37 °C for 6 h, and load-
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[21] L1: IR (KBr, cm−1): 3291, 3115, 1644, 1570, 1501, 1309, 1173, 838. 1H NMR
(600 MHz, DMSO, TMS) δ (ppm): 2.63–2.66 (m, 4H), 6.05 (s, 2H), 6.26 (s, 2H),
6.89–6.91(d, 4H), 8.02–8.03 (d, 4H). Anal. Calcd. For C32H24N2 O10·2H2O: C,
60.76; H, 4.46; N, 4.43%. Found: C, 60.35; H, 4.56; N, 4.38%.
[22] L2: IR (KBr, cm−1): 3318, 3116, 1659, 1569, 1505, 1312, 1177, 823. 1H NMR
(600 MHz, DMSO, TMS) δ (ppm): 2.61–2.70 (m, 8H), 5.99 (s, 2H), 6.20 (s, 2H),
6.88–6.89(d, 4H), 8.02–8.03 (d, 4H). Anal. Calcd. For C34H29N3 O10·2H2O: C,
60.44; H, 4.92; N, 6.22%. Found: C, 60.15; H, 4.96; N, 6.16%.