at SciVerse ScienceDirect

European Journal of Medicinal Chemistry 54 (2012) 697e708

Contents lists available

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Bimetallic complexes constructed from asymmetrical N,N0-bis(substituted)-oxamide: Cytotoxicities, and reactivities towardsDNA and protein

Xiao-Wen Li a, Lin Tao b, Yan-Tuan Li a,*, Zhi-Yong Wu b, Cui-Wei Yan c,*

aMarine Drug & Food Institute, Ocean University of China, 5 Yu shan Road, Qingdao 266003, PR ChinabKey Lab. Marine Drug, Chinese Minist. Educ., Ocean University of China, Qingdao, PR ChinacCollege of Marine Life Science, Ocean University of China, Qingdao 266003, PR China

a r t i c l e i n f o

Article history:Received 18 April 2012Received in revised form10 June 2012Accepted 12 June 2012Available online 19 June 2012

Keywords:Crystal structureBinuclear complexCytotoxicityDNA interactionProtein binding

* Corresponding authors. Fax: þ86 532 82033054.E-mail addresses: yantuanli@ouc.edu.cn (Y.-T.

(C.-W. Yan).

0223-5234/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2012.06.022

a b s t r a c t

A new asymmetrical N,N0-bis(substituted)oxamide ligand, N-(5-chloro-2-hydroxyphenyl)-N0-[3-(dime-thylamino)propyl]oxamide (C13H18N3O3Cl$H2O, H3L) and its two binuclear complexes [Cu2L(H2O)(bpy)](ClO4)$CH3OH (1) and [Ni2L(bpy)2](ClO4) (2) [bpy ¼ 2,20-bipyridine] have been synthesizedand characterized by X-ray single-crystal diffraction. In the crystal structure, H3L adopting a transoidconformation occurs as a neutral molecule linked with a water molecule by an intermolecular hydrogenbond. In the two complexes, the cis-L3� ligand bridges two metal ions with the corresponding separa-tions of 5.2032(15) Å (1) and 5.2466(7) Å (2), respectively. In vitro cytotoxic activities, and the reactivitiesof the three compounds towards DNA and protein are investigated.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Investigations on transition-metal complexes towards DNA andprotein are of current interest in connection with informationabout drug design and tools of molecular biology [1e9]. Modes ofDNA non-covalent interaction with metal complexes include elec-trostatic effect, groove binding, and intercalation. Many importantapplications of these metal complexes require that they can bind toDNA in an intercalative mode [10]. Both the planarity of ligand andthe coordination geometry of the metal ion play important roles indeciding the intercalating ability of complexes to DNA [11,12]. Byvarying the metal center, it is possible to modify the mode as wellas the extent of interaction of the complex with nucleic acids andfacilitate individual applications [13]. On the other hand, serumalbumins play an important and efficient role in drug delivery dueto their remarkable binding properties. With the advantages of lowcost and ready availability, bovine serum albumin (BSA) is a widelyused serum albumin. Many studies dealing with the interactionsbetween organic molecules and BSA have been carried out because

Li), cuiweiyan@ouc.edu.cn

son SAS. All rights reserved.

the structures of BSA are similar to human serum albumin (HSA) in76% [14]. Compared to the number of organic molecules [15e18],relatively few metal complexes [19,20] especially binuclearcomplexes [21] have been investigated the reactivities towards BSA.With the facts that copper and nickel elements are essentialelements to the life process, and binuclear copper and nickelcomplexes have been found to inhibit DNA replication and tumorcell proliferation [22e26], it is necessary to design and synthesizenew bicopper and binickel complexes to evaluate their cytotoxic-ities, and reactivities towards DNA and protein.

A promising strategy to design and synthesize binuclearcomplexes is the use of bridging ligand to react withmetal salts andterminal ligands. N,N0-bis(substituted)oxamides are well-known tobe good candidates as the binucleating bridging ligand in formingbinuclear complexes. Comparatively, due to the synthesis difficulty,the studies on the binuclear complexes involving the bridgingasymmetrical N,N0-bis(substituted)oxamides are very limited[27,28]. However, the fact that those complexes bridged by asym-metrical N,N0-bis(substituted)oxamides have shown predominantproperties [29e33] stimulate us to design and synthesize newbinuclear complexes with asymmetrical N,N0-bis(substituted)oxa-mides to gain some insight into the influence of metal ions in thiskind of complexes on structure, cytotoxic activities and reactivitiestowards DNA and protein.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708698

Recently,we reported several transition-metal complexes bridgedby asymmetricalN,N0-bis(substituted)oxamides and found they havesignificant DNA-binding and cytotoxic activities [34,35]. Enlightenedby the above-mentioned facts, and as a continuation of our on goingprogram, in this paper, a new asymmetrical N,N0-bis(substituted)oxamide ligand, N-(5-chloro-2-hydroxyphenyl)-N0-[3-(dimethyla-mino)propyl]oxalamide (H3L), and its two binuclear transition metalcomplexes, [Cu2L(H2O)(bpy)](ClO4)$CH3OH (1) and [Ni2L(bpy)2](ClO4) (2), were synthesized and structurally characterized by X-raysingle-crystal diffraction. In vitro cytotoxic activities of the threecompounds were tested by Sulforhodamine B (SRB) assays againsthuman hepatocellular carcinoma cell SMMC-7721 and human lungadenocarcinoma cell A549. The interactions of the three compoundswith herring sperm DNA (HS-DNA) are investigated by using UVabsorption and fluorescence spectra and viscometry. Furthermore,the protein binding ability has been monitored by using UV absorp-tion and tryptophan fluorescence quenching experiment in thepresence of the compounds using bovine serum albumin (BSA) asmodel protein. To the best of our knowledge, the binding of theasymmetrical N,N0-bis(substituted)oxamide bridged binuclearcomplexes to proteins has seldom been investigated.

2. Experimental

2.1. Materials and physical measurements

DNA and BSA were purchased from Sigma and were used assupplied. Other chemicals were of reagent grade. Carbon, hydrogenand nitrogen elemental analyses were performed with a Per-kineElmer elemental analyzer Model 240. Molar conductancewas measured with a Shanghai DDS-11A conductometer. Theinfrared spectra of the samples were recorded as KBr pellets ona Nicolet model Impact 470 FTIR spectrophotometer in the spectralrange 4000e400 cm�1. The mass spectra (ES-MS) were measuredwith a Waters Q-TOF GLOBLE mass spectrometer. The UVevisiblespectrum was recorded in a 1-cm-path length quartz cell ona Cary 300 spectrophotometer. Fluorescence was tested on an Fp-750w fluorometer. Viscosity measurement was carried out usingan Ubbelohde viscometer immersed in a thermostatic water bathmaintained at 289(�0.1) K.

2.2. Synthesis

2.2.1. Synthesis of the ligand (C13H18N3O3Cl$H2O, H3L)The synthesis of N-(5-chloro-2-hydroxyphenyl)-N0-[3-(dime-

thylamino)propyl]oxalamide (H3L), consists of two steps. The firststep was the preparation of oxamido-2-amino-4-chlorophenol-ethyloxamate (H3oxch) according to the reported method [27]. Forthat, a 40 mmol (5.49 g) portion of ethyl oxalyl chloride in 10 mL oftetrahydrofuran (THF) was added dropwise into a 40 mL THF solu-tion of 40mmol (5.92 g) of 2-amino-4-chlorophenol (97%). The clearsolution was concentrated under vacuum and the H3oxch wasprecipitated as awhite powder thenwashedwith ethanol and driedunder vacuum. The second step was the synthesis of N-(5-chloro-2-hydroxyphenyl)-N0-[3-(dimethylamino)propyl]oxalamide (H3L). A5 mmol (1.22 g) amount of H3oxch in 20 mL absolute ethanol wasadded dropwise into 20 mL absolute ethanol solution containing6 mmol (0.76 mL) 3-(dimethylamino)propyl at 273 K. The resultingsolution was stirred for 2 h, and H3L was precipitated as a whitepowder and then recrystallized in ethanol and dried under vacuum.Well-shaped colorless single crystals of the ligandwere obtained byslow evaporation of the ethanol solution of the recrystallizedproduct. Yield: 72%. Analysis; Calc for C13H18N3O3Cl$H2O: C, 49.14;H, 6.34; N, 13.22. Found: C, 49.10; H, 6.32; N, 13.19. IR (KBr, cm�1):3356 [n(NH)]; 1686 [n(C]O)] (oxamidate group). UVevisible (in

DMF), lmax(nm) [ 3max (L mol�1 cm�1)]: 301(22 550). ES-MS, m/z:300.2 ([M þ H]þ).

2.2.2. Synthesis of [Cu2L(H2O)(bpy)](ClO4)$CH3OH (1)To a stirredmethanol solution (5mL) containing Cu(ClO4)2$6H2O

(0.0742 g, 0.2 mmol) was added dropwise a methanol solution(10 mL) of H3L (0.0299 g, 0.1 mmol) and piperidine (0.0256 g,0.3mmol) at roomtemperature.After stirring for 30min, amethanolsolution (5mL) of bpy (0.0156 g, 0.1mmol)was added dropwise. Themixture was stirred quickly at 333 K for 6 h, the resulting brownsolution was filtered and brown cube crystals of the complex suit-able for X-ray analysis were obtained by slow evaporation at roomtemperature. Yield: 82%. Analysis; Calc for Cu2C24H29N5O9Cl2: C,39.51; H, 4.01; N, 9.60. Found: C, 39.53; H, 3.98; N, 9.67. IR (KBr,cm�1): 1632 [n(C]O)]; 1442 [n(C]N)]; 1104, 622 [n(ClO4)]. Molarconductance,LM (DMF solution): 79 U�1 cm2 mol�1. UVevisible (inDMF), lmax(nm) [ 3max(L mol�1 cm�1)]: 612(800), 311(40 400),299(44 300), 243(75 700). ES-MS,m/z: 598.0 ([MeClO4eCH3OH]þ).

2.2.3. Synthesis of [Ni2L(bpy)2](ClO4) (2)Deep reddish purple crystals suitable for X-ray single-crystal

analysis were obtained by the same method of preparing complex 1except using Ni(ClO4)2$6H2O instead of Cu(ClO4)2$6H2O. Yield: 58%.Analysis; Calc forNi2C33H31N7O7Cl2: C, 47.99;H, 3.78; N,11.87. Found:C, 47.92; H, 3.75; N, 11.81. IR (KBr, cm�1): 1633 [n(C]O)]; 1472, 1443[n(C]N)]; 1094, 623 [n(ClO4)]. Molar conductance, LM (DMF solu-tion): 87 U�1 cm2 mol�1. UVevisible (in DMF), lmax(nm) [ 3max

(L mol�1 cm�1)]: 605(912), 420(623), 306(30 300), 295(29 500),241(30 500). ES-MS,m/z: 724.1 ([M � ClO4]þ).

2.3. X-ray crystallography

The X-ray diffraction experiments for three compounds weremade on a Bruker APEX area-detector diffractometer with graphitemonochromatic Mo Ka radiation (l ¼ 0.71073 Å). The crystalstructures were solved by the directed method followed by Fouriersyntheses. Structure refinements were performed by full matrixleast-squares procedures using SHELXL-97 on F2 [36].

2.4. In vitro cytotoxic activity evaluation by SRB assays

In vitro cytotoxic activities of the three compounds and cis-platin were evaluated against two cancer cell lines includingSMMC-7721 and A549 by using the Sulforhodamine B (SRB) assay.All cells were cultured in RPMI 1640 supplemented with 10% (v/v)fetal bovine serum, 1% (w/v) penicillin (104 U/mL) and 10 mg/mLstreptomycin. Cell lines are maintained at 310 K in a 5% (v/v) CO2atmosphere with 95% (v/v) humidity. Cultures were passagedweekly using trypsineEDTA to detach the cells from their cultureflasks. The three compounds and cis-platinwere dissolved in DMSOand diluted to the required concentration with culture mediumwhen used. The content of DMSO in the final concentrations did notexceed 0.1%. At this concentration, DMSOwas found to be non-toxicto the cells tested. Rapidly growing cells were harvested, counted,and incubated at the appropriate concentration in 96-well microplates for 24 h. The three compounds and cis-platin dissolved inculture medium were then applied to the culture wells to achievefinal concentrations ranging from 10�4 to 102 mg/mL. Control wellswere prepared by addition of culture medium without cells. Theplates were incubated at 310 K in a 5% CO2 atmosphere for 48 h.Upon completion of the incubation, the cells were fixed with ice-cold 10% trichloroacetic acid (100 mL) for 1 h at 277 K, washedfive times in distilled water and allowed to dry in the air andstained with 0.4% SRB in 1% acetic acid (100 mL) for 15 min.The cells were washed four times in 1% acetic acid and air-dried.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708 699

The stain was solubilized in 10 mM unbuffered Tris base (100 mL)and the OD of each well was measured at 540 nm on a microplatespectrophotometer. The IC50 values were calculated from thecurves constructed by plotting cell survival (%) versus thecompounds concentration (mg/mL).

2.5. DNA-binding studies

All experiments involving herring sperm DNA (HS-DNA) wereperformed in TriseHCl buffer solution (pH ¼ 7.12). Solutions of HS-DNA in TriseHCl buffer gave a ratio of UV absorbance at 260 and280 nm, A260/A280, of ca.1.9, indicating that the DNAwas sufficientlyfree of protein [37]. The concentration of DNA was determined byUV absorbance at 260 nm. The molar absorption coefficient, 3260,was taken as 6600 L mol�1 cm�1 [38]. Stock solution of DNA wasstored at 277 K and used after no more than 4 days. Concentratedstock solution of the two complexes and the free ligand wereprepared by dissolving the compounds in the TriseHCl buffersolution to required concentrations for all the experiments,respectively. Absorption spectral titration experiment was per-formed by keeping the concentration of the compounds constantwhile varying the HS-DNA concentration. Equal solution of HS-DNAwas added to the compounds solution and reference solution toeliminate the absorbance of DNA itself. In the ethidium bromide(EB) fluorescence displacement experiment, 5 mL of the EB TriseHClsolution (1 mmol L�1) was added to 1 mL of DNA solution (atsaturated binding levels) [39], stored in the dark for 2 h. Then thesolution of the compounds was titrated into the DNA/EB mixtureand then diluted in TriseHCl buffer to 5mL, producing the solutionswith the varied mole ratio of the compound to HS-DNA. Beforemeasurements, the mixture was shaken up and incubated at roomtemperature for 30 min. The fluorescence spectra were obtained atan excitationwavelength of 522 nm and an emissionwavelength of584 nm in the Fluorometer. In viscosity measurement, DNAsamples approximately 200 base pairs in length were prepared bysonication in order to minimize complexities arising from DNAflexibility [40]. Flow time was measured with a digital stopwatch,and each sample was measured three times, and an average flowtime was calculated. Relative viscosities for DNA in the presenceand absence of the compound were calculated from the relations ¼ (t � t0)/t0, where t is the observed flow time of DNA-containingsolution and t0 is that of TriseHCl buffer alone. Datawere presented

Scheme

as (s/s0)1/3 versus binding ratio [41], where s is the viscosity of DNAin the presence of complex and s0 is the viscosity of DNA alone.

2.6. BSA-binding studies

All experiments involving bovine serum albumin (BSA) wereperformed in 50 mM NaCl/TriseHCl buffer solution (pH ¼ 7.12).And solutions of BSA and the three compounds were prepared bydissolving them in the NaCl/TriseHCl buffer solution to requiredconcentrations, respectively. For UV absorption experiment, a 5 mLsolution of BSA (10 mM) was titrated with various concentrations ofthe compounds. Equal solutions of compounds were added to thereference solutions to eliminate the absorbance of the compoundsthemselves. In the tryptophan fluorescence quenching experiment,quenching of the tryptophan residues of BSA [42] was done bykeeping the concentration of the BSA constant while varying thecompounds (quenchers) concentration, producing the solutionswith the varied mole ratio of the quenchers to BSA. The fluores-cence spectra were recorded at an excitationwavelength of 295 nmand an emission wavelength of 347 nm in the Fluorometer aftereach addition of the quencher.

3. Results and discussion

3.1. Synthesis and general property of the binuclear complexes

The aim of this study was to obtain binuclear complexes withdifferent metal ions. Therefore, heterobinucleating ligand, N-(5-chloro-2-hydroxyphenyl)-N0-[3-(dimethylamino)propyl]oxalamide(H3L), was chosen as bridging ligand, which can coordinate tometalions through not only the oxamido oxygens and nitrogens but alsophenolate oxygens [43]. Simultaneously, 2,20-bipyridine (bpy) wasused as the terminal ligands. In the course of preparing thecomplexes the use of piperidine as base makes the bridging ligand(H3L) coordinate to copper(II)/nickel(II) center through the depro-tonated oxamido nitrogen and phenolate oxygen atoms. Indeed,elemental analyses indicate that the reaction of H3L withCu(ClO4)2$6H2O/Ni(ClO4)2$6H2O and bpy in 1:2:1 mol ratio yieldedthe binuclear complexes, [Cu2L(H2O)(bpy)](ClO4)$CH3OH (1) and[Ni2L(bpy)2](ClO4) (2). The synthetic pathway for the bridgingligand (H3L) and the two complexes 1 and 2may be represented byScheme 1. For the two binuclear complexes, the molar conductance

1.

Fig. 1. An ORTEP view of H3L with the thermal ellipsoids at 30% probability level. Hatoms are shown as small spheres of arbitrary radii. Hydrogen bonds are shown asdotted lines.

Fig. 2. An ORTEP view of complex 1 with the thermal ellipsoids at 30% probabilitylevel. H atoms are shown as small spheres of arbitrary radii. Hydrogen bonds areshown as dotted lines.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708700

values (79 and 87 U�1 cm2 mol�1) in DMF solution fall in theexpected range for 1:1 electrolytes [44]. The results suggest that thetwo complexes both consist of a binuclear complex cation and anuncoordinated perchlorate anion in solutions. The stability ofcomplexes 1 and 2 existed as a whole binuclear entity in solutionwas supported by the ES-MS results which can be found insynthesis part.

3.2. IR and electronic spectra

The IR spectra of the free ligand and the two binuclearcomplexes taken in the region of 4000e400 cm�1 provide someinformation regarding the mode of coordination in the binuclearcomplexes and are analyzed in a careful comparison with that ofthe free ligand (H3L). In the IR spectrum of the free ligand, a sharpstrong band observed at 3286 cm�1 is the result of overlappingbetween n(OeH) and n(NeH), which is absent in the spectra of thetwo complexes, indicating that the phenolic oxygen atom andamido nitrogen atom take part in the coordination. In addition, thecarbonyl (C]O) stretching vibration of H3L (1686 cm�1) is found tobe shifted toward lower wave-numbers of 1632 and 1633 cm�1,respectively. This shift has often been used as a diagnostic indicatorfor oxamido-bridged structures [45]. Besides, the eC]Nestretching vibration for the terminal ligands (bpy) is observed atabout 1450 cm�1, indicating that these terminal ligands areinvolved in the coordination in the two binuclear complexes [46].Furthermore, a broad and intense band centered at ca. 1100 cm�1,and a strong sharp band at about 620 cm�1, typical for a non-coordinated perchlorate group [46,47], are observed for the twobinuclear complexes. This is consistent with the conductance dataof the binuclear complexes.

In order to obtain further structural information, the electronicspectra of the two complexes were recorded in the UVevisibleregion (200e800 nm) using DMF as solvent. For both complexes1 and 2, four main absorption bands with varied intensities can beobserved. The intense bands about 240 nm are assignable to inter-or intra-ligand (pep*) transition. Meanwhile the bands around300 nm in the complexes can be assigned to the charge transfertransition between ligand and metal. Besides, broad bands areobserved at the lower frequency in the spectra, corresponding tothe ded transition of copper(II) or nickel(II) [48]. The structures ofthe two binuclear complexes are further supported by thefollowing determination of the crystal structures.

3.3. Crystal structures

The ORTEP views of the free ligand (H3L) and complexes 1 and 2were displayed in Figs. 1e3, respectively. Table 1 summarizes thecrystal data and structural refinement parameters for the threecompounds and their selected bond lengths and angles are listed inTable 2. Non-covalent interactions found in the crystal structuresare described in supporting information (Table S1, Figs. S1-S3).

3.3.1. Crystal structure of the ligand (C13H18N3O3Cl$H2O, H3L)As shown in Fig. 1a, the crystal structure of the ligand occurs as

a neutral molecule linked with a solvent water molecule by anintermolecular hydrogen bond between the phenolic hydroxyloxygen atom (O1) and the water molecule (O4). The ligand hasa transoid conformation, and the six non-H atoms of oxamido groupare almost coplanar, which is similar to other oxamido compounds[49]. The oxamido plane (r.m.s. deviation 0.0688 Å) is nearlyparallel to the benzene ring, with a dihedral angle of 7.18(10)�.

Hydrogen bonds dominate the crystal structure. Themolecule ofthe ligand and water molecule are connected by classical hydrogenbonds listed in Table S1 into a one-dimensional chain parallel to

b axis (Fig. S1). These chains are linked bywater molecules (O4) andoxamido oxygen atoms (O31/2þx, �1/2þy, z) into a two-dimensionalhydrogen bonding network extending along (0 0 1) plane.

3.3.2. Crystal structure of [Cu2L(H2O)(bpy)](ClO4)$CH3OH (1)As illustrated in Fig. 2a, the structure of 1 consists of a binuclear

copper(II) complex cation [Cu2L(H2O)(bpy)]þ, one uncoordinatedperchlorate anion and one solvent methanol molecule. The Cu/Cudistance through the oxamide-bridge is 5.2032(15) Å. The cis-oxamide coordinates to Cu1 and Cu2 in the usual chelating modewith bite angles of 82.27(13) and 86.08(11)�, respectively.

The environment around Cu1 atom can be best described asa distorted square-pyramidal geometry with the s value of 0.05[50]. Atoms N1, N2, N3 and O1 from the bridging ligand (L) form thebasal coordination plane, while an oxygen atom (O4) from thecoordinate water molecular occupied the apical position with

Fig. 3. An ORTEP view of complex 2with the thermal ellipsoids at 30% probability level. H atoms are shown as small spheres of arbitrary radii. Hydrogen bonds are shown as dottedlines.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708 701

Cu1eO4 bond length of 2.423(3) Å. The maximum displacement ofthem from the coordination plane is 0.1138(17) Å (N1). Cu1 atomdisplaces 0.2303(15) Å out of the plane. The behavior of L3� resultsin the formation of two five- and one six-membered rings aroundCu1 atom. The two five-membered rings Cu1eO1eC1eC2eN1 andCu1eN1eC7eC8eN2 are almost planar. The puckering parameters[51] of the six-membered ring Cu1eN2eC9eC10eC11eN3 areQ¼ 0.508(10) Å, q¼ 33.4(9)� and 4¼ 210.4(14)�. The Cu1eN3 bond[2.042(3) Å] is longer than the Cu1eN1 bond [1.939(3) Å] andCu1eN2 bond [1.991(4) Å], which is consistent with the strongerdonor ability of the deprotonated amido nitrogen compared withthe amino nitrogen atoms [52]. Cu2 atom has a distorted square-

Table 1Crystal data and details of the structure determination for the ligand H3L andcomplexes 1 and 2.

Compounds H3L 1 2

Formula C13H20ClN3O4 C24H29Cl2Cu2N5O9

C33H31Cl2Ni2N7O7

M 317.77 729.52 825.93Crystal System Monoclinic Triclinic MonoclinicSpace group C2/c P-1 C2a (Å) 12.2386(18) 8.369(4) 21.6780(14)b (Å) 11.1168(18) 12.302(5) 12.1165(8)c (Å) 23.919(4) 14.606(6) 15.1668(10)a (�) 90 93.333(5) 90b (�) 98.898(3) 102.715(6) 114.7520(10)g (�) 90 100.987(3) 90V (Å3) 3215.1(9) 1432.1(11) 3617.7(4)Z 8 2 4D(calc)(g cm3) 1.313 1.692 1.516m (Mo Ka)(mm�1) 0.256 1.732 1.245F(000) 1344 744 1696Temperature (K) 296 296 296Radiation (Å) Mo Ka

0.71073Mo Ka 0.71073 Mo Ka 0.71073

Tot., uniq. data, R(int) 9338, 3703,0.024

10 966, 6356,0.036

10 851, 7199,0.028

q range 1.8 to 27.7 2.1 to 27.5 2.0 to 27.6Observed data [I > 2s(I)] 2651 4702 5209R, wR2, S 0.0470, 0.1425,

1.0460.0445, 0.1693,1.088

0.0422, 0.0847,0.968

Goodness-of-fit 1.046 1.088 0.968Max., av. shift/error 0.00, 0.00 0.00, 0.00 0.00, 0.00

planar geometry, which coordinates to the exo-O atoms of thecis-L3� ligand (O2 and O3). The other two atoms of the plane (N4,N5) are both from the bpy molecule. The deviations of four definedatoms (N4, N5, O2, O3) from the coordinated plane are in the rangeof 0.0589 (14)e0.0630(16) Å, and Cu2 atom displaces 0.0556(16) Åout of the plane.

The complex cations and solvent methanol molecules are con-nected by hydrogen bonds listed in Table S1 into a one-dimensionalchain parallel to the a axis. These chains are linked by perchlorateanions into a two-dimensional hydrogen bonding networkextending along (0 0 1) plane (Fig. S2). Furthermore, as shown in

Table 2Selected bond distances (Å) and bond angles (�) for the ligand H3L and complexes 1and 2.

Bond lengths Bond angles

H3LCl1eC4 1.743(2) O1eC1eC2 116.59(17)O1eC1 1.354(2) O1eC1eC6 124.44(18)N1eC2 1.408(3) Cl1eC4eC3 118.37(17)N1eC7 1.339(3) Cl1eC4eC5 119.56(17)O2eC7 1.218(2) N1eC2eC1 116.26(17)C7eC8 1.531(3) N1eC2eC3 123.67(18)O3eC8 1.231(2) C2eN1eC7 128.05(17)N2eC8 1.315(3) O3eC8eN2 126.10(18)Complex 1Cu1eN1 1.939(3) N1eCu1eN2 82.27(13)Cu1eN2 1.991(4) N1eCu1eO1 82.41(13)Cu1eN3 2.042(3) N2eCu1eN3 97.11(14)Cu2eN4 1.972(3) N3eCu1eO1 95.72(12)Cu2eN5 1.960(4) N4eCu2eO3 179.29(13)Cu1eO1 1.974(3) N4eCu2eN5 82.01(13)Cu2eO2 1.940(3) N5eCu2eO2 171.69(14)Cu2eO3 1.944(3) O2eCu2eO3 86.08(11)Complex 2Ni1eN1 1.846(4) N1eNi1eN2 84.49(14)Ni1eN2 1.902(4) N1eNi1eO1 85.33(13)Ni1eN3 1.939(4) N2eNi1eN3 98.87(16)Ni1eO1 1.865(3) N3eNi1eO1 91.41(15)Ni2eN4 2.090(4) N4eNi2eN5 78.73(15)Ni2eN5 2.052(4) N4eNi2eN6 98.47(16)Ni2eN6 2.069(4) N4eNi2eO2 90.20(13)Ni2eN7 2.084(3) N4eNi2eN7 177.12(16)Ni2eO2 2.061(3) N5eNi2eO3 90.74(15)Ni2eO3 2.087(3) N5eNi2eO2 166.91(15)

88

19

45

5.4

75

17

38

7.6

0

20

40

60

80

100

(ng/mL)Compounds

IC50

(µ

g/m

L)

SMMC-7721

A549

1 2 cis-platin H3L

Fig. 4. In vitro cytotoxic activities of the three compounds and cis-platin againsthuman hepato-cellular carcinoma cell line SMMC-7721 and human lung adenocarci-noma cell A549.

280 320 360 400 4400.0

0.1

0.2

0.3

0.4a

Abs

Wavelength(nm)

3.7

4.1

4.5

4.9

1 2 3 4 5 6

[DNA]×105 mol/L

[DN

A]/

( εa–

ε f)×

109 m

ol/L

220 240 260 280 300 320

0.3

0.4

0.5

0.6

0.7

b

Abs

Wavelength(nm)

0.5

0.7

0.9

1.1

1 2 3 4 5 6 7

[DNA]×105 mol/L

[DN

A]/

( εa–

ε f)×

108 m

ol/L

0.10

0.15

0.20

0.25

0.30

0.35

240 270 300 330 360 390

c

Abs

Wavelength(nm)

0.5

0.6

0.7

0.8

0.9

1

1.1

1 2 3 4 5 6

[DNA]×105 mol/L

[DN

A]/

( εa –

εf)×

108 m

ol/L

Fig. 5. Absorption spectra of compounds (a forH3L, b for1 and c for2) upon the titration ofHS-DNA. Arrow indicates the change upon increasing the DNA concentration. Inset: Plot of[DNA]/( 3a � 3f) vs. [DNA] for the absorption titration of HS-DNAwith the compound.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708702

Fig. S3, there are two types of offset pep stackings in complex 1. Oneis observed between the benzene ring and the N5 pyridine ring at(�1 þ x, y, z) [symmetry operation (iii)]; the closest distancebetween atomC23iii and the benzene ring is 3.307(5) Å. Another oneis between the pyridine ring containing atoms N5 and the benzenering at (1 þ x, y, z), with a closest separation of 3.389(4) Å (C2v).

3.3.3. Crystal structure of [Ni2L(bpy)2](ClO4) (2)As illustrated in Fig. 3, complex 2 consists of a [Ni2L(bpy)2]þ

cation and two halves of uncoordinated perchlorate anions ofwhich the occupancies are constrained to 0.5 because of thedisordered. Some atoms of the two perchlorate anions are disor-dered of higher inversion symmetry [symmetry code: (i) �x, y, �z,O4i, O5i, O6i, O7i; (ii) �x þ 1, y, �z þ 1, Cl3ii] than the cations. Thedeprotonated L3� ligand exhibits a cisoid conformation and bridgestwo Ni atoms, with a Ni/Ni distance of 5.2466 (7) Å.

Ni1 atom has a distorted square-planar geometry, formed by N1,N2, N3 and O1 from the L3�. The maximum displacement from thecoordination plane is 0.040(2) Å (N1). Ni1 lies exactly in the plane[0.0059(18) Å]. The behavior of L3� results in the formation of twofive- and one six-membered rings around Ni1 atom. The five-membered ring Ni1eN1eC7eC8eN2 has a twist conformationwith puckering parameters of Q ¼ 0.083(4) Å and 4 ¼ 230(2)�,while the other five-membered rings Ni1eO1eC1eC2eN1 isalmost planar. The puckering parameters [51] of the six-memberedring Ni1eN2eC9eC10AeC11AeN3 are Q ¼ 0.618(12) Å,q ¼ 121.4(8)� and 4 ¼ 27.8(9)�. Similar to complex 1, complex 2 hasa longer Ni1eN3 bond [1.939(4) Å] than Ni1eN1 [1.846(4) Å] andNi1eN2 [1.902(4) Å] [52]. Ni2 atom is coordinated by four N-atomdonors of two bpymolecules and two O atoms of the L3� ligand. Thehexa-coordinated atom Ni2 has a distorted octahedral geometry.Atoms N4 and N7 are axially coordinated, with an approximatelylinear NeNieN angle [177.12(16)�]. The equatorial plane is definedby the other four atoms and themean displacement from this planeis 0.1815 Å. Atom Ni2 lies exactly in the plane [0.0087(18) Å]. Thebpy terminal ligands are present in the usual chelated ring on Ni2with bite angles of 78.73(15) and 78.92(16)�, respectively. Theintermolecular interactions in the structure of complex 2 are notstudied, since there are no classic hydrogen bonds, no significantpep stacking interactions are found in its three-dimensionalsupramolecular structure.

Comparing the crystal structures of the two binuclearcomplexes, we find that the two complexes have the same bridgingligand (L3�) and terminal ligand (bpy). The main differencebetween them is the metal ion which affects their coordinated

Table 3The Kb and Ksv values for the free ligand H3L and complexes 1 and 2.

H3L 1 2

HS-DNA Kb � 10�4 (M�1) 0.73 3.2 2.6Ksv � 10�4 (M�1) 2.6 5.1 3.8

BSA Ksv � 10�4 (M�1) 4.2 2.1 2.5Kq � 10�12 (M�1 s�1) 4.2 2.1 2.5n 1.09 0.72 0.94K � 10�4 (M�1) 11.0 0.96 1.02

560 580 600 620 6400

20

40

60

80

100

120

1

1.1

1.2

1.3

0 2 4 6 8 10 12

Ι0/Ι

a

Fluo

resc

ence

em

issi

on in

tens

ity

Wavelength(nm)

560 580 600 620 6400

20

40

60

80

1

1.2

1.4

1.6

0 2 4 6 8 10 12

Ι0/Ι

b

Fluo

resc

ence

em

issi

on in

tens

ity

Wavelength(nm)

560 580 600 620 6400

20

40

60

80

100

120

1

1.1

1.2

1.3

1.4

1.5

0 2 4 6 8 10 12

Ι0/Ι

c

Fluo

resc

ence

em

issi

on in

tens

ity

Wavelength(nm)

L[compound]×106mol/

L[compound]×106mol/

L[compound]×106mol/

Fig. 6. Emissionspectraof theHS-DNA-EBsystemuponthetitrationofcompounds(a forH3L,b for 1 and c for 2). Arrow shows the change upon the increasing compound concentration.Inset: Plot of I0/I vs. [compound] for the titration of the compound to HS-DNA-EB system.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708 703

environment. Furthermore, they affect the cytotoxic activities andDNA binding properties of the binuclear complexes (vide infra).

3.4. In vitro cytotoxic activities

In vitro cytotoxic activities of the three compounds and cis-platin against two cancer cell lines human hepatocellular carci-noma cell line SMMC-7721 and human lung adenocarcinoma cellline A549 were conducted in our study. The results, shown in Fig. 4,indicated that all of the three compounds have rather cytotoxicitiesagainst the two cancer cell lines, and complex 1 demonstrated thebest activities among the three compounds. Although themeasured cytotoxic activities are less than those of cis-platin (IC50values of 5.4 and 7.6 ng/mL, respectively), inhibition of cell prolif-eration produced by the three compounds on the same batch of celllines and under identical experimental conditions is still ratheractive. As it has been indicated that the cytotoxicities of the threecompounds would vary with the metal ions and the extent of theirintercalation into DNA base pairs [13], the significant cytotoxicity ofcomplex 1 may be partially attributed to its strong intercalative-property (vide infra). These findings of the cytotoxic activitiesprompt us to explore the DNA and protein binding properties of thethree compounds.

3.5. DNA-binding properties

3.5.1. Electronic absorption titrationThe application of electronic absorption spectroscopy is one of

the most useful techniques for DNA-binding properties [53]. Theabsorption spectra of the free ligand H3L and the two binuclearcomplexes in the absence and presence of herring sperm DNA (HS-DNA) are recorded, and the absorption spectra of the threecompounds are given in Fig. 5. As shown in these figures, whentitrated by HS-DNA, all of them presented significant hypochrom-ism, accompanied the slightly red-shifts in the absorbancemaxima.In general, the hypochromism and red-shift are associated with thebinding of the metal complexes to the DNA helix, due to theintercalative mode involving a strong stacking interaction betweenthe aromatic chromophore of compounds and the base pairs ofDNA [53]. These results suggest that all of the three compoundsinteract with HS-DNA through the intercalation mode.

To compare quantitatively the binding affinity of the threecompounds toward HS-DNA, the intrinsic binding constants Kb ofthe three compounds with HS-DNA were determined by moni-toring the changes in absorbance at 301 nm (H3L), 299 nm (1) and295 nm (2) with increasing concentration of HS-DNA according tothe following equation [53]:

½DNA�=�

3a � 3f

�¼ ½DNA�=

�3b � 3f

�þ 1=Kb

�3b � 3f

�(1)

where 3a, 3f and 3b correspond to the extinction coefficient,respectively, for each addition of DNA to the tested compounds, forthe free tested compounds and for the tested compounds in thefully bound form. The binding constants for three compounds can

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708704

be estimated from the plots of [DNA]/( 3a � 3f) vs. [DNA] (inset inFig. 5). As shown in Table 3, the intrinsic binding constants Kb of thethree compounds with HS-DNA follow the order 1 > 2 > H3L. Thehigher Kb value observed for the two complexes may be due to thecomplexation of the oxamido-bridge and the terminal ligandsproviding more planar structures than the free ligand, which couldincrease the degree of intercalation. Besides, electrostatic interac-tion may be another factor for complexes 1 and 2 to enhance thebinding strength to DNA, which is lack in H3L. The lower bindingaffinity of complex 2 to HS-DNA than that of complex 1may be dueto the type of metal ions, which are responsible for the coordinatedgeometry of the complexes and have a profound effect on the DNA-binding properties, as revealed by the different binding affinities.

3.5.2. Fluorescence titrationThe ethidium bromide (EB) fluorescence displacement experi-

ment has been widely used to investigate the interaction of smallmolecule with DNA. In order to further investigate the interactionmodes of the three compounds with HS-DNA, the EB fluorescencedisplacement experiments were used, and the principle can bedescribed as follows [54].

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

0 0 .0 2 0 .0 4 0 .0 6 0 .0 8 0 .1

[complex]/[DNA]

(η/ η

0)1/

3

Fig. 7. Effect of the increasing amount of the compounds (- for H3L,A for 1 and: for2) on the relative viscosity of herring sperm DNA at 289(�0.1) K, [DNA] ¼ 0.1 mM.

DNAðlow fluorescence intensityÞ þ EBðlow fluorescence intensityÞ#EB� DNAðenhanced fluorescence intensityÞ#Fluorescence intensity of EB can be quenched

It can be seen from Fig. 6, the fluorescence emission intensitiesof EB bound to HS-DNA at 584 nm show remarkable decreasingtrend with increasing concentration of the ligand and the twocomplexes, indicating some EB molecules were released intosolution after an exchange with the three compounds which resultin the fluorescence quenching of EB. This observation is oftenconsidered as the characteristic of intercalation [55].

In order to understand quantitatively the magnitude of thebinding strength of the three compounds with HS-DNA, the linearSterneVolmer equation is employed [56]:

I0=I ¼ 1þ Ksv½Q � (2)

where I0 and I represent the fluorescence intensities in the absenceand presence of quencher, respectively. Q is the concentration ofquencher. Ksv is a linear SterneVolmer quenching constant. In thequenching plot of I0/I versus [compound] (Fig. 6), Ksv of the threecompounds are given by the slope. The results, shown in Table 3,support the fact that the Ksv values for the ligand and the twocomplexes follow the order 1>2>H3L, which is in agreement withthat derived by UV absorption spectra measurements.

3.5.3. Viscosity measurementsTo further clarify the interaction mode of the three compounds

and HS-DNA, the relative specific viscosity measurements werecarried out by varying the concentration of the added compounds.Viscosity measurement is a classical means to study the bindingmode of small moleculewith DNA in solution and provides strongerarguments for intercalative binding mode [57,58]. In classicalintercalation, the DNA helix lengthens as base pairs are separated toaccommodate the bound compound leading to increased DNAviscosity whereas a partial, non-classical compound intercalationcauses a bend in DNA helix reducing its effective length and therebyits viscosity. The effects of the three compounds on the viscosity ofHS-DNAare shown in Fig. 7. As illustrated in thisfigure, on increasingtheamountof the three compounds, the relative viscosityofHS-DNA

increasing steadily, which is proved that the compounds bound toDNA by intercalation. The increased degree of viscosity, which maydepend on their affinity to DNA follows the order of 1 > 2 > H3L.

3.6. BSA-binding properties

3.6.1. UV absorption spectra of BSA in the presence of thecompounds

UV absorption spectrum is a very simple and applicable methodto explore the structural change and to know the complex forma-tion in solution [59]. Fig. 8 shows the UV absorption spectra of BSAin the presence of different concentrations of the three compounds.As can be seen from this figure, free BSA has maximum absorptionat 278 nm and it increases with the addition of the threecompounds. This phenomenon indicates the interaction of BSAwith the compounds [60].

3.6.2. Tryptophan quenching experimentIn order to further investigate the interaction of the three

compounds with protein, the tryptophan emissionequenchingexperiments were carried out using bovine serum albumin (BSA)

in the presence of the three compounds. The emission intensitydepends on the degree of exposure of the two tryptophan sidechains [61], 134 and 212, to polar solvent. It can be seen from Fig. 9,the fluorescence emission intensities of BSA at 347 nm showremarkable decreasing trend with increasing concentration of thethree compounds, indicating that the interaction of the compoundswith BSA could cause changes in protein secondary structureleading to changes in tryptophan environment of BSA [62]. In orderto understand quantitatively the magnitude of the compounds toquench the emission intensity of BSA, the linear SterneVolmerequation (3) is also employed:

250 260 270 280 290 3000.2

0.3

0.4

0.5a

Abs

Wavelength(nm)

250 260 270 280 290 300

0.20

0.25

0.30

0.35

0.40

b

Abs

Wavelength(nm)

250 260 270 280 290 3000.20

0.25

0.30

0.35

0.40

c

Abs

Wavelength(nm)

Fig. 8. Absorption spectra of BSA upon the titration of H3L (a), 1 (b) and 2 (c). Arrow indicates the change upon increasing the compound concentration.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708 705

I0=I ¼ 1þ Ksv½Q � ¼ 1þ Kqs0½Q � (3)

I0 and I represent the fluorescence intensities in the absence andpresence of quencher, respectively. Q is the concentration ofquencher. Ksv is a linear SterneVolmer quenching constant. Kq isthe biomolecular quenching rate constant, s0 is the average lifetime

of the fluorophore in absence of the quencher as 10�8 S. From thequenching plot of I0/I versus [compound] (inset in Fig. 9), Ksv and Kqof the three compounds are obtained from the slope(Kq ¼ w1012 M�1 s�1, Table 3). However, maximum collisionquenching constant of various kinds of quenchers to bio-macromolecule is 2.0 � 1010 M�1 s�1 [63]. Obviously, the rate

320 340 360 380 400 4200

50

100

150

200

250

300

1

1.1

1.2

1.3

1.4

1 3 5 7 9 11

Ι0/Ι

a

Fluo

resc

ence

em

issi

on in

tens

ity

Wavelength(nm)

-1.21

-1.11

-1.01

-0.91

-0.81

-0.71

-0.61

-0.51

-0.41

-5.7 -5.5 -5.3 -5.1 -4.9

Log[Q]

Log

[(F0

-F)/

F]

320 340 360 380 400 4200

50

100

150

200

250

300

1

1.05

1.1

1.15

1.2

0 2 4 6 8

Ι0/Ι

b

Fluo

resc

ence

em

issi

on in

tens

ity

Wavelength(nm)

-1.7

-1.5

-1.3

-1.1

-0.9

-0.7

-6 -5.8 -5.6 -5.4 -5.2 -5

Log[Q]

Log

[(F0

-F)/

F]

320 340 360 380 400 4200

50

100

150

200

250

300

1

1.1

1.2

1.3

0 2 4 6 8 10 12

[compound]×106 mol/L

[compound]×106 mol/L

[compound]×106 mol/L

Ι0/Ι

c

Fluo

resc

ence

em

issi

on in

tens

ity

Wavelength(nm)

-1.1

-1

-0.9

-0.8

-0.7

-0.6

-0.5

-5.7 -5.5 -5.3 -5.1 -4.9

Log[Q]

Log

[(F0

-F)/

F]

Fig. 9. Emission spectra of BSA upon the titration of H3L (a), 1 (b) and 2 (c). Arrow shows the change upon the increasing compound concentration. Inset: Plot of I0/I vs. [compound]for the titration of the compound to BSA; Right: Plot of log [(F0 � F)/F] vs. log [Q] for the titration of the compound to BSA.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708706

constant of protein quenching initiated by the three compounds isgreater than 2.0� 1010 M�1 s�1. This shows that above quenching isnot initiated by dynamic collision but via the combination of thecompound and BSA. Therefore, the quenching of BSA fluorescenceby the three compounds depended on the formation of thecomplex between the three compounds and BSA. The binding

constant (K) was calculated by the method given in the followingsection.

3.6.3. Binding constant and number of binding sitesWhen small molecules bind independently to a set of equiv-

alent sites on a macromolecule, then the equilibrium between the

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708 707

free and bound molecule is given by the following equation[64,65]:

log½ðF0 � FÞ=F� ¼ logK þ nlog½Q � (4)

F0 and F represent thefluorescence intensities in the absence andpresence of quencher, respectively. K is the binding constant of thethree compounds with BSA, n is the binding sites. From thequenching plot of log [(F0� F)/F] versus log [Q] (right in Fig. 9), K andn of the three compounds can be obtained. The K and n values arelisted in Table 3. As shown in this table, the binding constantbetween the three compounds and BSA follows the orderH3L> 2z1, and the binding sites are all about 1. The higher bindingconstant for the free ligandH3L reveals the stronger protein-bindingability of the neutral ligand with better hydrophobicity.

4. Conclusion

In order to examine the effect of the metal ions in binuclearcomplexes upon the structure, cytotoxic activities and reactivitiestowards DNA and protein, in this paper, an asymmetrical N,N0-bis(substituted)oxamide ligand, N-(5-chloro-2-hydroxyphenyl)-N0-[3-(dimethylamino)propyl]-oxalamide (H3L) and its two binuclearcomplexeswith formulas of [Cu2L(H2O)(bpy)](ClO4)$CH3OH (1) and[Ni2L(bpy)2](ClO4) (2) were synthesized and characterized by X-raysingle-crystal diffraction. In vitro cytotoxic activities of the threecompounds were investigated, and complex 1 displayed the bestcytotoxic activities, which are consistent with DNA-binding abili-ties. In agreement with the electronic absorption spectroscopy,fluorescence spectroscopy and viscosity measurement, all of thethree compounds, especially the two binuclear complexes couldinteract with HS-DNA through the intercalation mode and bothfollow the binding affinity order of 1 > 2 > H3L. As for the proteinbinding abilities, the free ligand H3L exhibits a higher ability thanthat of complexes 1 and 2. The results implied that the cytotoxicactivities of the compounds may be associated with or originatefrom their ability to intercalate the base pairs of DNA and thatthrough modifying the nature of the metal ions the DNA-bindingand cytotoxic activities could possibly be tuned. This strategyshould be valuable in understanding the cytotoxic activities oftransition metal complexes as well as laying a foundation for therational design of new, powerful agents for probing and targetingnucleic acids.

Acknowledgments

This project was supposed by the National Natural ScienceFoundation of China (No. 21071133), the Program for Science andTechnology of Shandong Province (2011GHY11521), and theNatural Science Foundation of Qingdao City [No. 11-2-4-1-(9)gchand 12-1-3-52-(1)-nsh].

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ejmech.2012.06.022.

References

[1] M. Chauhan, K. Banerjee, F. Arjmand, Inorg. Chem. 46 (2007) 3072e3082.[2] F. Arjmand, M. Aziz, Eur. J. Med. Chem. 44 (2009) 834e844.[3] M.J. Clarke, Coord. Chem. Rev. 236 (2003) 209e233.[4] H.T. Chifotides, K.R. Dunbar, Acc. Chem. Res. 38 (2005) 146e156.[5] P.T. Selvi, H. StoecklieEvans, M. Palaniandavar, J. Inorg. Biochem. 99 (2005)

2110e2118.

[6] V. Rajendiran, R. Karthik, M. Palaniandavar, H. StoecklieEvans, V.S. Periasamy,M.A. Akbarsha, B.S. Srinag, H. Krishnamurthy, Inorg. Chem. 46 (2007)8208e8221.

[7] A. Su1kowska, J. Równicka, B. Bojko, W. Su1kowski, J. Mol. Struct. 651e653(2003) 133e140.

[8] K. Moebus, J. Siepmann, R. Bodmeier, Eur. J. Pharm. Biopharm. 72 (2009)42e53.

[9] P. Sevilla, J.M. Rivas, F. García-Blanco, J.V. García-Ramos, S. Sánchez-Cortés,Biochim. Biophys. Acta 1774 (2007) 1359e1369.

[10] H. Xu, K.C. Zheng, Y. Chen, Y.Z. Li, L.J. Lin, H. Li, P.X. Zhang, L.N. Ji, J. Chem. Soc.Dalton Trans. 11 (2003) 2260e2268.

[11] H. Xu, K.C. Zheng, H. Deng, L.J. Lin, Q.L. Zhang, L.N. Ji, New J. Chem. 27 (2003)1255e1263.

[12] M. Asadi, E. Safaei, B. Ranjbar, L. Hasani, New J. Chem. 28 (2004) 1227e1234.[13] J. Jiang, X.L. Tang, W. Dou, H.H. Zhang, W.S. Liu, C.X. Wang, J.R. Zheng, J. Inorg.

Biochem. 104 (2010) 583e591.[14] N. Wang, L. Ye, F.F. Yan, R. Xu, Int. J. Pharm. 351 (2008) 55e60.[15] X.D. Li, H. Li, M. Liu, G.Q. Li, L.W. Li, D.Z. Sun, Thermochim. Acta 521 (2011)

74e79.[16] B. Ojha, G. Das, Chem. Phys. Lipids 164 (2011) 144e150.[17] A. Mallick, S.C. Bera, S. Maiti, N. Chattopadhyay, Biophys. Chem. 112 (2004)

9e14.[18] M. Obata, S. Hirohara, K. Sharyo, H. Alitomo, K. Kajiwara, S. Ogata, M. Tanihara,

C. Ohtsuki, S. Yano, Biochim. Biophys. Acta 1770 (2007) 1204e1211.[19] R. Tang, C.H. Tang, C.Q. Tang, J. Organomet. Chem. 696 (2011) 2040e2046.[20] E.G. Ferrer, A. Bosch, O. Yantorno, E.J. Baran, Bioorg. Med. Chem. 16 (2008)

3878e3886.[21] D.S.Raja,N.S.P. Bhuvanesh,K.Natarajan, Eur. J.Med. Chem.46 (2011) 4584e4594.[22] K.D. Karlin, Z. Tyeklar, Bioinorganic Chemistry of Copper, Chapman & Hill,

New York, 1993.[23] Q.X. Wang, K. Jiao, F.Q. Liu, X.L. Yuan, W. Sun, J. Biochem. Biophys. Methods 70

(2007) 427e433.[24] K.R. Rupesh, S. Deepalatha, M. Krishnaveni, R. Venkatesan, S. Jayachandran,

Eur. J. Med. Chem. 41 (2006) 1494e1503.[25] Z. Boulsourani, G.D. Geromichalos, K. Repana, E. Yiannaki, V. Psycharis,

C.P. Raptopoulou, D. Hadjipavlou-Litina, E. Pontiki, C. Dendrinou-Samara,J. Inorg. Biochem. 105 (2011) 839e849.

[26] P.G. Avaji, C.H.V. Kumar, S.A. Patil, K.N. Shivanandad, C. Nagaraju, Eur. J. Med.Chem. 44 (2009) 3552e3559.

[27] Z.D. Matovi�c, V.D. Mileti�c, G. Samard�z�c, G. Pelosi, S. Ianelli, S. Trifunovi�c, Inorg.Chim. Acta 358 (2005) 3135e3144.

[28] S.Q. Zang, R.J. Tao, Q.L. Wang, N.H. Hu, Y.X. Cheng, J.Y. Niu, D.Z. Liao, Inorg.Chem. 42 (2003) 761e766.

[29] J.P. Costes, F. Dahan, A. Dupuis, J.P. Laurent, Inorg. Chem. 39 (2000) 169e173.[30] A. Erxleben, Inorg. Chem. 40 (2001) 208e213.[31] R.J. Tao, S.Q. Zang, Y.X. Cheng, Q.L. Wang, N.H. Hu, J.Y. Niu, D.Z. Liao, Poly-

hedron 22 (2003) 2911e2916.[32] R.J. Tao, S.Q. Zang, N.H. Hu, Q.L. Wang, Y.X. Cheng, J.Y. Niu, D.Z. Liao, Inorg.

Chim. Acta 353 (2003) 325e331.[33] L.N. Zhu, N. Xu, W. Zhang, D.Z. Liao, K. Yoshimura, K. Mibu, Z.H. Jiang, S.P. Yan,

P. Cheng, Inorg. Chem. 46 (2007) 1297e1304.[34] Y.L. Song, Y.T. Li, Z.Y. Wu, J. Inorg. Biochem. 102 (2008) 1691e1699.[35] X.W. Li, Y. Yu, Y.T. Li, Z.Y. Wu, C.-W. Yan, Inorg. Chim. Acta 367 (2011) 64e72.[36] G.M. Sheldrick, SHSLXL97, Program for Crystal Structure Refinement,

University of Göttingen, Germany, 1997.[37] J. Marmur, Mol. J. Biol. 3 (1961) 208e212.[38] M.E. Reichmann, S.A. Rice, C.A. Thomas, P.J. Doty, J. Am. Chem. Soc. 76 (1954)

3047e3053.[39] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro, J. Am. Chem. Soc. 108 (1986)

2081e2088.[40] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933e3940.[41] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1993)

2573e2584.[42] N.S. Quiming, R.B. Vergel, M.G. Nicolas, J.A. Villanueva, J. Health Sci. 51 (2005)

8e15.[43] H.H. Lu, Y.T. Li, Z.Y.Wu, K. Zheng, C.W. Yan, J. Coord. Chem. 64 (2011) 1360e1374.[44] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81e122.[45] H. Ojima, K. Nonoyama, Coord. Chem. Rev. 92 (1988) 85e111.[46] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination

Compounds, fifth ed., Wiley, New York, 1997.[47] Y.T. Li, C.W. Yan, H.S. Guan, Polyhedron 22 (2003) 3223e3230.[48] A.B.P. Lever, Inorganic Electronic Spectroscopy, Elsevier Publishing Co.,

Amsterdam, 1984.[49] C.Y. Su, W.J. Zhang, B.S. Kang, Acta Crystallogr. Sect. C 55 (1999) 636e637.[50] A.W. Addison, T.N. Rao, J. Reedijk, J. Van Rijin, G.C. Verschoor, J. Chem. Soc.

Dalton Trans. (1984) 1349e1356.[51] D. Cremer, J.A. Pople, J. Am. Chem. Soc. 97 (1975) 1354e1358.[52] J.K. Tang, Y. Ou-Yang, H.B. Zhou, Y.Z. Li, D.Z. Liao, Z.H. Jiang, S.P. Yan, P. Cheng,

Crystal Growth Des. 5 (2) (2005) 813e819.[53] S.E. Evans, M.A. Mendez, K.B. Turner, L.R. Keating, R.T. Grimes, S. Melchoir,

V.A. Szalai, J. Biol. Inorg. Chem. 12 (2007) 1235e1249.[54] K. Dhara, P. Roy, J. Ratha, M. Manassero, P. Banerjee, Polyhedron 26 (2007)

4509e4517.[55] S. Anbu, M. Kandaswamy, P. Suthakaran, V. Murugan, B. Varghese, J. Inorg.

Biochem. 103 (2009) 401e410.

X.-W. Li et al. / European Journal of Medicinal Chemistry 54 (2012) 697e708708

[56] O. Stern, M. Volmer, Z. Phys. 20 (1919) 183e188.[57] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 31 (1992)

9319e9324.[58] L. Jin, P. Yang, J. Inorg. Biochem. 68 (1997) 79e83.[59] Y.J. Hu, Y. Liu, J.B. Wang, X.H. Xiao, S.S. Qu, J. Pharm. Biomed. Anal. 36 (2004)

915e919.[60] Y.Y. Yue, X.G. Chen, J. Qin, X.J. Yao, Dyes Pigm. 79 (2008) 176e182.

[61] B.F. Pan, F. Gao, L.M. Ao, J. Magn. Magn. Mater. 293 (2005) 252e258.[62] S.S. Bhat, A.A. Kumbhar, H. Heptullah, A.A. Khan, V.V. Gobre, S.P. Gejji,

V.G. Puranik, Inorg. Chem. 50 (2011) 545e558.[63] W.R. Ware, J. Phys. Chem. 66 (1962) 455e458.[64] V. Anbazhagan, R. Renganathan, J. Lumin. 128 (2008) 1454e1458.[65] P. Banerjee, S. Ghosh, A. Sarkar, S.C. Bhattacharya, J. Lumin. 131 (2011)

316e321.