Copper_anticancer_DPA deriv__B_art%3A10.1007%2Fs11243-009-9200-5

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Synthesis, characterization, and bioactivities of copper complexeswith N-substituted Di(picolyl)amines

Lin-Yun Wang

Qiu-Yun Chen

Juan Huang

Kun Wang

Chang-Jian Feng

Zhi-Rong Gen

Received: 24 November 2008 / Accepted: 27 January 2009 / Published online: 24 February 2009 Springer Science+Business Media B.V. 2009

Abstract Three new Cu(II) complexes with ethyl bis(2-

pyridylmethyl)amino-2-propionate (Etdpa), or bis(2-pyri-dylmethyl)amino-2-propionate (Adpa), were synthesized

and characterized by physico-chemical and spectroscopic

methods. The X-ray crystal structure of [(Adpa)CuCl] shows

that the copper(II) atom is coordinated by three N atoms, one

oxygen atom from the ligand (Adpa) and one chloride anion,

forming a trigonal bipyramidal geometry. The spectropho-

tometric and fluorescence titration data indicate that the

interaction of square pyramidal [(Etdpa)CuCl2] with ct-DNA

is weak, but the trigonal bipyramidal complexes [(Adpa)

Cu(H2O)](ClO4) and [(Adpa)CuCl] interact with ct-DNA

with the mode of intercalation. The inhibition activities of the

three new copper(II) complexes on the four cancer cells (Mcf-

7, Eca-109, A549, and Hela) are in the order: [(Adpa)Cu

(H2O)](ClO4)[ [(Adpa)CuCl][ [(Etdpa)CuCl2], which

correlates with their DNA-binding properties. The results

show that the substituents introduced on the secondary amino

nitrogen atom of dpa have great contribution to the antitumor

activities of these copper(II) complexes. It is also found that

the coordination of copper(II) ions with AdpaH can decreasethe toxicity of AdpaH.

Introduction

Copper plays a key role in biological processes. The great

majority of the copper proteins are involved mainly in

oxidation/reduction reactions as well as in the dioxygen

transport and activation [1]. The development of mimic

systems for copper metalloenzymes has provided important

compounds which allowed scientists to understand both the

physicalchemical properties of the active site of the

copper enzymes as well as the reactivity exhibited by the

metalloenzymes [2]. Some of these mimetic compounds

show similar activities to those of natural metalloenzymes

[3]. It has been demonstrated that copper can accumulate in

tumors due to the selective permeability of cancer cell

membranes to copper compounds [4]. Copper(II) com-

plexes are the preferred over platinum (II) complexes for

cancer inhibition [5]. Di(picolyl)amine and its derivatives

are used as neutral, nondeprotonated chelating ligands to

complex copper(II) atoms to mimic non-heme dioxygenase

[6]. The reaction of dpa with Cu(ClO4)2 or CuCl2 lead to

hexa-coordinated [Cu(dpa)2](ClO4)2 [7] or the mononu-

clear complexes [Cu(dpa)Cl2] [8], respectively, in which

the geometry of reported coordinated Cu(II)-dpa com-

plexes is a distorted square pyramidal or trigonal

bipyramidal. The utility of these ligands is enhanced by the

ease with which substituents may be introduced on the

imino nitrogen atom, thus allowing the controlled modifi-

cation of solubility and molecular conformation through

the non-bonding interactions [9]. Recently N-substituted

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11243-009-9200-5) contains supplementarymaterial, which is available to authorized users.

L.-Y. Wang Q.-Y. Chen (&) J. Huang K. WangSchool of Chemistry and Chemical Engineering, Jiangsu

University, 212013 Zhenjiang, Peoples Republic of China

e-mail: [email protected]

C.-J. Feng

College of Pharmacy MSC09 5360, 1 University of New

Mexico, Albuquerque, NM 87131-0001, USA

Z.-R. Gen

State Key Laboratory of Coordination Chemistry, Nanjing

University, 212093 Nanjing, Peoples Republic of China

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Transition Met Chem (2009) 34:337345

DOI 10.1007/s11243-009-9200-5
http://dx.doi.org/10.1007/s11243-009-9200-5http://dx.doi.org/10.1007/s11243-009-9200-5


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Cytotoxicity testing

The cytotoxicity assay was in four kinds of cell lines (human

breast carcinoma cells Mcf-7, human esophageal cancer

cells Eca-109, human cervical cancer Hela cells, and human

lung adenocarcinoma A549 cells). Cells were cultured in

RMPI 1640 medium containing 4.8 g/L of Hepes, 2.2 g/L

NaHCO3 and supplemented with penicillin/streptomy-cin(1000 units/mL), and 10% calf serum. Hela, Mcf-7 cells

were cultured in DMEM medium containing 10% fetal

bovine serum. All cells were grown at 37 C in a humidified

atmosphere in the presence of 5%CO2. Eca-109, A549, Mcf-

7, Hela cells were seeded at a density of 4 9 104 cells/mL

into sterile 96 well plates and grown in 5% CO2 at 37 C.

Test compounds were dissolved in H2O and diluted with

culture media. After 24 h, compounds were added and

treated for 48 h. Cell viability was determined by the 3-[4,5-

Dimethylthiazol-2-yl]-2,5-diphenpyltetra-zolium bromide

(MTT) assay by measuring the absorbance at 570 nm with

ELISA reader. IC50was calculated by the software providedby Nanjing University. Each test was performed in triplicate.

Results and discussion

Synthesis and spectroscopic data

The synthesis route of the copper(II) complexes is shown in

Scheme1. The reaction of the ligand ethyl bis(2-pyridyl-

methyl)amino-2-propionate (Etdpa) with Cu(ClO4)2 and

CuCl2 in the presence of sodium hydroxide produced the

new complexes [(Adpa)Cu(H2O)](ClO4) (2) and [(Adpa)-CuCl] (3), respectively. The molar conductivities of the

complexes (1) and (3) in methanol are 15.6 and 18.1 S

cm2 mol-1, respectively, indicating that the complexes are

non-electrolytes. The molar conductivity (102 S cm2 mol-1)

of the complex (2) indicates this complexis a 1:1 electrolyte.

The IR spectra of the ligands show that there are two

pyridyl ring vibration bands at *1570 and 1590 cm-1 and

d(CH) vibration of pyridyl ring at *760 cm-1 [10]. These

vibrations in the copper complexes are all shifted. The

pyridyl ring vibrations bands were *1609 and 1573 cm-1

for [(Etdpa)CuCl2] (1) and 1612 and 1569 cm-1 for (2) and

(3). Thed(CH) vibration bands of pyridyl ring for all of the

copper complexes were found at *774, 779, and776 cm-1, respectively. These shifts indicate the pyridine

nitrogen atoms of the ligands donate a pair of electrons

each to the central metal forming coordinate bonds [17].

Them(C=O) band of the Etdpa and the complex (1) appears

at 1729 cm-1 indicating that the existence of ester group of

Etdpa. The infrared spectra of the complexes (2) and (3)

show mas(COO) stretching frequencies at 1643 cm-1 and

msym(COO) at 1388 cm-1, respectively. The difference

between mas(COO) and msym(COO) are about 255 cm-1,

suggesting that the carboxylate groups coordinate to the

copper(II) atoms only as monodentate ligands [18].

The Cu-pyridine charge transfer bands at ca. 254 nmdominated the UV spectra for the three complexes. The

copper atom may adopt geometries ranging from typical

trigonal bipyramidal to distorted square pyramidal depend-

ing on the nature of the ligands. The [(Etdpa)CuCl2] (1)

exhibits visible spectra with single broad bands at 650

700 nm, characteristic of a copper(II) dzx, dyz ? dx2-y2(2B1 ?

2E) [19] transition in a tetragonal ligand field, in

which the copper(II) ion has a distorted square-pyramidal

coordination environment. Because the dd transition bands

of [(Adpa)Cu(H2O)](ClO4) (2) and [(Adpa)CuCl] (3) in

aqueous solution were 866 and 863 nm, respectively, rather

than 650 nm, we conclude that the copper(II) atoms in the

complexes (2) and (3) mainly adopt a trigonal bipyramidal

rather than a distorted square-pyramidal geometry [20].

Crystal structure of [(Adpa)CuCl] (3)

The molecular structure of [(Adpa)CuCl] (3) with the atomic

labeling scheme is shown in Fig.1, and the selected bond

lengths and angles are listed in Table2. The monodepro-

nated Adpa acts as a tetradentate ligand toward a copper(II)

ion. The copper atom is coordinated by three N atoms (N1,

N2, N3, one oxygen atom (O2) of the (Adpa) and one chlo-

ride anion (Cl2), resulting a five-coordinated mononuclear

copper(II) complex, which is different from the reported syn-

anticarboxylate bridged polymeric one-dimensional chain

copper(II) complex {[Cu(l-pmea)](ClO4) H2O} (pmea =bis(2-pyridylmethyl)amino-2-ethanoic acid) [19]. The five-

coordinated copper(II) complex [(Adpa)CuCl] forms a

trigonal bipyramidal, similar to the geometry of reported

[Cu(apme)(Cl)](BPh4) (apme = tris(2-pyridylmethyl) amine)

[21]. The N1, N2, and O2 form the equatorial trigonal plane,

while the N3 and Cl2 occupy the apical positions. The

N

NH3CH2COOC

N

CuCl

Cl

N

N

N

CuClN

N

COOCH2CH3

NEtdpa

N

N

COO

N

Cu

CuCl2

Cu(ClO4)2

CuCl2

OH2

2

+

NaOH

NaOH

1 3

O

O

Scheme 1 Synthesis route of copper(II) complexes [(Etdpa)CuCl2]

(1), [(Adpa)Cu(H2O)](ClO4) (2), and [(Adpa)CuCl] (3)

340 Transition Met Chem (2009) 34:337345

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copper(II) atom is shifted by 0.326 Aoutof theequatorial plane

toward the tertiary amino ligand. The bond distances of Cu1

N3 and Cu1O2 are 2.012(2) and 2.028(2) A. The N3Cu1

Cl2 angle is 178.29(6). Intermolecular hydrogen bonds

involving the carbon atoms, oxygen atoms (O1, O2), and the

chloride atom (Cl2) result in networks in 3. The Cl2 is linked to

the hydrogen atoms H15 (Cl(2)H15 [-1/2 ? x, 1.5 - y,

-1/2 ? z] of 2.653 A) of the neighboring molecule. The car-

boxyl oxygen atoms (O1, O2) bond to hydrogen atoms from

carbon atoms of aromatic rings C11 [1 ? x, y, z] and C9

[1/2 ? x, 1.5 - y, -1/2? z] with interatomic distances

O1H11 of 2.709 (4) A, O2H9 of 2.446 (4) A. The addi-

tional interactionsarep-p stackinginteractionsbetweenthe two

adjacent pyring rings (N1B/C1B-C5B) [-1/2? x, 1.5 - y,

-1/2 ? z] and (N1AA/C1AA-C5AA) [1/2 ? X, 1.5 - y,

-1/2 ? z] or the (N1/C1-C5) [x, y, z] and (N1A/C1A-C5A)

[1 ? x, y, z], with the interplannar distance of ca. 3.687 A

(Fig. S1). The molecules are linked through intermolecular

hydrogen bonds of CHO,CHCl andpacked throughp-p

stacking interaction forming network structures (Fig. S2).

Electrochemistry

Cyclic voltammograms for the copper(II) complexes at a

glassy carbon electrode in 0.05 M NaClO4and 0.05 M NaF

were shown in Fig.2. The electrochemical behavior of the

[(Adpa)CuCl] and [(Etdpa)CuCl2] at a glassy carbon elec-

trode in 0.05 M NaClO4and 0.05 M NaF was characteristic

of quasi-reversible one-electron Cu(II)/Cu(I) redox pro-

cesses and a adsorptive stripping peak from deposition of

copper on the electrode, which is scan rate dependent. The

[(Adpa)Cu(H2O)](ClO4) exhibits a reversible one-electron

redox process with the half-wave potential of -0.403 V

involving the CuII/CuI couple (Epc = -0. 448 V, Epa =

-0.358 V, the ratio of anodic and cathodic peak currentsIpa/Ipc are *1) and a irreversible one-electron Cu

II/CuIII

oxidation processes with EPC = -0.001 V. The one-elec-

tron CuII/CuI oxidation and reduction half-wave potentials

(E1/2) for the three complexes are in the range of-0.403 to

0.445 mV at 25 mV s-1, which are more negative than those

of the square-pyramidal complex [Cu(dpa)Cl2] [8]. The

ranking of the CuII/CuI potentials for the [(Adpa)

Cu(H2O)](ClO4) is near to that observed for the trigonal

bipyramidal Cu(II) complex of dpa (E1/2 = -0.39 V) [22].

Fig. 1 Crystal structure

for the complex [(Adpa)CuCl].

Thermal ellipsoids are

drawn at 50% probability

Table 2 Selected bond lengths (A) and bond angles () for the

complex [(Adpa)Cu(Cl)]

Bond distances

Cu(1)N(3) 2.012(2) Cu(1)N(2) 2.082(3)

Cu(1)N(1) 2.026(2) Cl(2)Cu(1) 2.2184(15)

Cu(1)O(2) 2.028(2)

Bond angles

N(3)Cu(1)N(1) 82.10(10) N(2)Cu(1)Cl(2) 99.86(8)

N(3)Cu(1)O(2) 80.35(10) N(3)Cu(1)N(2) 81.80(9)

N(1)Cu(1)O(2) 124.25(8) N(1)Cu(1)N(2) 121.12(9)

N(3)Cu(1)Cl(2) 178.29(6) O(2)Cu(1)N(2) 108.01(9)

N(1)Cu(1)Cl(2) 96.65(9) O(2)Cu(1)Cl(2) 99.45(9)

Transition Met Chem (2009) 34:337345 341

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Because a positive shift in the half-wave potential reflects

a less stable Cu(II) complex [23], the stability of the

Cu(II) complexes can be ranked as follows: [(Adpa)

CuCl][ [(Etdpa)CuCl2] [ [(Adpa)Cu(H2O)](ClO4).

Binding characteristics of complex with DNA

The spectrophotometric titration spectra of the [(Adpa)-

Cu(H2O)](ClO4) (2), [(Adpa)CuCl] (3) are shown in Fig. 3a

and b, respectively. It is observed that the absorption bandsof (2) and (3) at 255 nm exhibited hypochromism of 17.2

and 13.1%, and bathochromism shift of about 5 nm when

the ct-DNA was added to the solution of the complexes.

Figure3a and b shows that the absorption spectra of com-

plexes increase on increasing the concentration of ct-DNA.

This is a typical hyperchromic effect, which was caused

possibly by the intercalation binding mode between the

complexes and ct-DNA. Hypochromic effect indicates the

complexes (2) and (3) induce the change of DNA double-

helix structure [24]. Bathochromism shift indicates thep*

orbital of intercalated ligand couple with theporbital of the

base pairs, thus reducing the pp* transition energy [25].Therefore, we speculate that complexes (2) and (3) inter-

acting with ct-DNA have the mode of intercalation.

However, there is no obvious bathochromic shifts and

hypochromicities when the ligand Etdpa and the [(Etdpa)

CuCl2] (1) were used in the same condition. The complex

(1) shows weak binding to the ct-DNA, which is similar

to the reported square-pyramidal ternary(L-leucine)-bpy

(2,20-bipyridine) copper(II) complex [26].

It is well known that EB can intercalate nonspecifically

into DNA. Competitive binding of other drugs to DNA and

EB will result in displacement of bound EB and a decrease

in the fluorescence intensity [27,28]. When [(Etdpa)CuCl2]

(1) was added into the solution of DNAEB complex,

respectively, the change of the fluorescence intensity of

DNAEB complex was small and ruleless, which indicatesthat there is nearly no intercalated interaction between the

complex (1) with DNA. However, when the complexes (2)

and (3) were added into the solution of DNAEB complex,

the fluorescence intensity of DNAEB complex decreased

with the increasing concentration of (2) a n d (3). The

fluorescence spectra for the complexes [(Adpa)Cu(H2O)]

(ClO4) (2) and [(Adpa)CuCl] (3) were shown in Fig.4a and

b. Since intercalated EB is the only fluorescent species, the

1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5

d

a

[(Adpa)CuCl]

[(Adpa)Cu(H2O](ClO4)

[(Etdpa)CuCl2]

Potential/V

Fig. 2 Cyclic voltammogram of [(Etdpa)CuCl2] (1), [(Adpa)

Cu(H2O)](ClO4) (2), and [(Adpa)CuCl] (3) in water containing

50 mM NaClO4 and 50 mM NaF. Scan rate: 25 mV s-1 (a),

50 mV s-1 (b), 75 mV s-1 (c), 100 mV s-1 (d)

200 250 300 350 400

200 250 300 350 400

0.5

1.0

0.0

1.5

f

a

Abso

rbance

/nm

0.2

0.4

0.6

0.8

0.0

1.0

f

a

Absorbance

/nm

(a)

(b)

Fig. 3 a Electronic spectra of [(Adpa)Cu(H2O)](ClO4) (2) (50 lM)

in the presence of increasing amounts of ct-DNA (af); DNA

concentrations are 0, 23.8, 71.5, 95.3, 119, and 142.9 lM for spectra

(af), respectively.b Electronic spectra of [(Adpa)CuCl] (3) (50 lM)

in the presence of increasing amounts of ct-DNA (af); DNA

concentrations are 0, 23.8, 71.5, 95.3, 119, and 142.9 lM for spectra

(af), respectively

342 Transition Met Chem (2009) 34:337345

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observed fluorescence decrease indicates that the com-

plexes (2) and (3) can replace EB inside the DNA cavities.

Such a characteristic change is often observed in the

intercalative DNA interaction [29].

The binding constantsKAof (2) and (3) with DNA in the

presence of EB were determined using the following

relationship [30]

log F0 F =F n log KA n log

Dt n Nt

F0F =F0;

2

where [Nt] and [Dt] are the total concentration of DNAEB

complex and the complex, respectively. The plots of

log(F0 - F)/F versus log([Dt] - n[Nt](F0 - F)/F0) for

DNAEB complex in the presence of complexes are shown

in Fig.5 at 8 C, and the binding constants are listed in

Table3. The binding constants KA of (2) and (3) are

3.03 9 104 and 1.399 103, which indicate that the

interaction of [(Adpa)Cu(H2O)](ClO4) (2) to ct-DNA is

stronger than that of the [(Adpa)CuCl] (3). This order is

well consistent with the results of spectrophotometric

titration. The different DNA-binding constants for com-

plexes (2) and (3) may due to their different total charge

[30].

Inhibition on the proliferation of cancer cells

Three copper(II) complexes (1)(3) and the reported com-

plex [Cu(dpa)Cl2] [8] were studied for their antitumor

activity in vitro by determining the inhibitory percentage

against growth of cancer cells Mcf-7, A549, Hela, and Eca-

109 using the method of 3-[4,5-Dimethylthiazol-2-yl]-2,

5-diphenpyltetrazolium bromide reduction (MTT method).

The IC50data of the copper(II) complexes (1)(3), [Cu(dpa)

Cl2], the ligand Etdpa, and AdpaH were shown in Table4.

The complexes (1)(3) can inhibit the proliferation of the

Mcf-7 cell with IC50in the range of 37.1224.13 lM, which

is smaller than that (96.23lM) of [Cu(dpa)Cl2].This may be

due to the solubility, and molecular conformation of com-

plexes (1)(3) were different from [Cu(dpa)Cl2]. The

complexes (2) and (3) were more active against the cancer

cell Eca-109 than [(Etdpa)CuCl2] and [Cu(dpa)Cl2] with

IC50 in the range 23.2131.09 lM (Table4). These data

indicate that the substituents introduced on the secondary

amino nitrogen atom of dpa have great contribution to the

antitumor activities of these copper(II) complexes. It is also

found that the AdpaH was more active against the

560 580 600 620 640 660 680 700

560 580 600 620 640 660 680 700

0

50

100

150

200

250

300

f

a

F

/nm

0

50

100

150

200

250

300

f

a

F

/nm

(a)

(b)

Fig. 4 a Fluorescence spectra of DNAEB in the presence of

[(Adpa)Cu(H2O)](ClO4) at 8 C. The total concentrations of [(Adpa)-

Cu(H2O)](ClO4) are (a) 0, (b) 10.0, (c) 20.0, (d) 30.0, (e) 40.0,

(f) 50.0lmol L-1. EB and DNA concentration are 0.68 and

20 lmol L-

1. b Fluorescence spectra of DNAEB in the presenceof [(Adpa)CuCl] at 8 C. The total concentrations of [(Adpa)CuCl]

are (a) 0, (b) 10.0, (c) 20.0, (d) 30.0, (e) 40.0, (f) 50.0 lmol L-1. EB

and DNA concentration are 0.68 and 20 lmol L-1

-5.1 -5.0 -4.9 -4.8 -4.7 -4.6 -4.5 -4.4 -4.3 -4.2

-2.0

-1.8

-1.6

-1.4

-1.2

-1.0

-0.8

log((

F0-F)/F)

log([Dt]) - n[N

t](F

0- F)/F

0

[(Adpa)Cu(H2O)](ClO

4)

[(Adpa)CuCl]

Fig. 5 The plots of log((F0 - F)/F) versus log([Dt] - n [Nt]

(F0 - F)/F0) for [(Adpa)Cu(H2O)](ClO4) and [(Adpa)CuCl] at 8 C

Table 3 The binding constants and binding site of the complexes (2)

and (3) with DNA at 8 C

Complex KA (L mol-1) n r

[(Adpa)Cu(H2O)](ClO4) (2) 3.03 9 104 1.25 0.992

[(Adpa)CuCl] (3) 1.39 9 103 1.01 0.997


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27. Boger DL, Fink BE, Brunette SR, Tse WC, Hedrick MP (2001)

J Am Chem Soc 123:5878. doi:10.1021/ja010041a

28. Wang BD, Yang ZY, Wang Q (2006) Bioorgan Med Chem 14:

1880

29. Biver T, Secco F, Tine MR, Venturini M (2004) J Inorg Biochem

98:33. doi:10.1016/j.jinorgbio.2003.08010

30. Ware WR (1962) J Phys Chem 66:455. doi:10.1021/j100809a020


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http://dx.doi.org/10.1021/ja010041ahttp://dx.doi.org/10.1016/j.jinorgbio.2003.08010http://dx.doi.org/10.1021/j100809a020http://dx.doi.org/10.1021/j100809a020http://dx.doi.org/10.1016/j.jinorgbio.2003.08010http://dx.doi.org/10.1021/ja010041a

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Copper_anticancer_DPA deriv__B_art%3A10.1007%2Fs11243-009-9200-5