马正平陈秋云封如陈甜甜

(江苏大学化学化工学院，镇江212013)

SCN-桥联的铜配合物：抗肿瘤活性及对肿瘤细胞耗氧的影响

马正平陈秋云＊封如陈甜甜

(江苏大学化学化工学院，镇江212013)

合成和表征了一种新的N-苯基二吡啶甲基胺(phdpa)铜配合物[(phdpa)Cu(SCN)](ClO4)。X-衍射晶体结构数据显示配合物中铜原子与配体phdpa中3个N原子、SCN-中S原子和邻近分子中SCN-中N原子配位，形成扭曲八面体结构。生物活性数据显示该铜配合物抑制HepG-2细胞的生长，半数抑制率为10.4μmol·L-1。进一步机理研究数据显示这种SCN-桥联的铜配合物能诱导HepG-2细胞核的分裂，降低其耗氧量和ROS的含量，这表明该铜配合物是一种能影响HepG-2细胞氧代谢的多功能配合物。

铜配合物；抗肿瘤；ROS；耗氧量；晶体结构

0 Introduction

Copper plays a key role in biological processes, and its complexes are used as metallopharmacons or preferred molecules for cancer inhibition because copper could accumulate in tumors due to the selective permeability of cancer cell membranes to copper compounds[1].A number of copper complexes, which are artificial nucleases,have been screened for anticancer activity,and some of them were found to be active both in vitro and in vivo[2-3].

Except as artificial nucleases,copper(II) complexes are also reported as mimics of non-heme dioxygenases,which could induce apoptosis in cultured mammalian cells[4].Copper(Ⅱ)complexes of N-substituted di(picolyl)amines,mimics of non-heme dioxygenase,are active against the proliferation of cancer cells[5].The apoptosis mechanism of cancercells is influenced by the structure and conformation of these copper(Ⅱ)complexes[6-7].Oxygen scarceness limits the growth of tumors,because cancer cells-like most other cells in the organism-utilize oxygen to generate energy(at least in part)and as a substrate for a number of fundamental biochemical reactions including synthesis of macromolecules[7].Oxidative therapy is a relatively new anticancer strategy[8].The microenvironment of solid tumors is characterized by low pO2that is well below physiological levels. Intratumoral hypoxia is a major factor contributing to cancer progression and is exacerbated as a result of oxygen consumption by rapidly proliferating tumor cells near blood vessels,poor lymphatic drainage resulting in high interstitial pressure,and irregular blood supply through immature tumor.In solid tumors,energy production and synthesis of macromolecules are maximized withminimal reactive oxygen species(ROS) accumulation.Furthermore,a series of antioxidant enzymes are induced tomitigate the damaging effectsof ROS[9].The antitumor activities of the complex[(phdpa) Cu(H2O)(Ac)](Ac)(phdpa=N-benzyl di(pyridylmethyl) amine)are related to their interference on the oxygen metabolite of cancer cells[10].So,attenuation on oxygen related metabolic changes would be a synergistic approach to combination anti-cancer therapy and inhibition of cellular defenses against oxidative stress.We reporthere the synthesis,characterization of a new copper(Ⅱ)complex[(phdpa)Cu(SCN)](ClO4).In vitro essay resultsshow that the complex can inhibit the proliferation of cancer cells through interfering their O2consumption and ROSproduction.

1 Experimental

1.1 Chem ical reagents,analysis and physical measurements

All chemicals used in the syntheses were of reagent grade and were used without further purification.N-benzyl di(pyridylmethyl)amine(phdpa)was synthesized according to the reported procedures[11]. Tris(hydroxymethyl)aminomethane(tris)was obtained from Sigma USA.Water was purified with a Millipore MIlli-Q system.The C,H and N microanalyses were performed on Vario EL elemental analyzer.The electronic absorption spectrum was recorded in the 900～190 nm region using the VARIAN CARY 50-BIO UV-Vis spectrophotometer.Infrared spectrum was recorded on a Nicolet-470 spectrophotometer in the wavenumber range of 4 000～400 cm-1with KBr pellets.The fluorescence spectra were measured with Fp-750W Fluorometer.

1.2 Synthesis of[(phdpa)Cu(SCN)(ClO4)](1)

To a stirred ethanol solution(10 mL)of phdpa (289mg,1mmol),a green solution of Cu(ClO4)2·6H2O (370.5 mg,1 mmol)in ethanol(10 mL)was added dropwise.The mixture was stirred at 80℃for 2 h and then cooled to room temperature.NH4SCN(76.12 mg,1 mmol)in ethanol(10 mL)was added to the above solution.Themixture was refluxed at 75℃for 2 h then cooled to room temperature.After the solution was diffused with ethyl ether,blue crystals were obtained.Yield,80%.Anal.Calcd.(%)for C20H19ClCuN4O4S:C,47.06;H,4.75;N,10.98;Found (%):C,47.13;H,4.68;N,11.04.IR(KBr)ν/cm-1: ν(=CH)3 109 m,ν(-CH2-)2 935 m,ν(-SCN)2 106, ν(C=N)1 608 s,ν(C=C)1 574 s,ν(C=C)1 442 s, ν(C=C)1 452 m,ν(C=C)1 359 m,ν(ClO4-)1 165 s, 1083s,1000s,δ(=CH,Ar)765s,705s,ν(ClO4-)622s.

1.3 X-ray crystal structure determ inations

Crystallographic data for[phdpaCu(SCN)](ClO4) are listed in Table 1.The blue prism crystals of the complex were selected for lattice parameter determination and collection of intensity at 298 K on Rigaku Mercury2 CCD Area Detector with monochromatized Mo Kαradiation(λ=0.071 074 7 nm). The data was corrected for Lorenz and polarization effects during data reduction.A semi-empirical absorption correction from equivalents based on multi-scans was applied.The structure was solved by directmethods and refined on F2by full-matrix leastsquaresmethods using SHELTL version 5.10[12].All non-hydrogen atoms were refined anisotropically.All calculations were performed using the SHELX-97 programs.

CCDC:840495.

Table 1 Crystal data and structural refinements for[(phdpa)Cu(SCN)](ClO4)

1.4 Cytotoxicity testing

The cytotoxicity assay used two kinds of cell lines (A549 and HepG-2 cells).Cellswere cultured in RPMI-1640 medium containing 4.8 g·L-1of HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid),2.2 g· L-1NaHCO3and supplemented with penicillin/ streptomycin(1000 units·mL-1)and 10%calf serum. A549 cells were cultured in Dulbecco's Modified Eagle′sMedium(DMEM)containing 10%fetalbovine serum.All cells were grown at 37℃in a humidified atmosphere in the presence of5%CO2.Testcomplexes were dissolved in DMSO and diluted with culture media.After 24 h,complexeswere added and the same samples kept for 48 h.Cell viability was determined by the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenpyltetrazolium bromide(MTT)assay by measuring the absorbance at 590 nm with an ELISA reader.DMSO was used as blank control and 5-Fluorouracilwas used as reference control.IC50was calculated using software provided by Nanjing University.Each test was performed in triplicate.Comparisonsweremade by oneway analysisofvariance.Differenceswere considered to be significant when p＜0.05.All experiments were repeated at least three times.

1.5 Nuclei observation on H 2B-GFP-labled HepG-2 cells

H2B-labled HepG-2 cells were plated in 24-well plate at the density of 2.4×104.After incubating for 24 h with complexes,cells were stained with 4′,6-diamidino-2-phenylindole(DAPI)and nuclei changes were visualized under fluorescencemicroscope(Nikon TE2000 inverted microscope)

1.6 Oxygen consum ption and measurement of ROS in HepG-2 cells

HepG-2 cells were seeded at a density of 1×105cells·mL-1into sterile 96 well plates and grown in 5% CO2at 37℃for 24 h.Test compoundswere dissolved in H2O or DMSO and diluted with culture media. After 24 h,complexes were added.Samples were transferred into Clark cell.After an equilibration time of 2min(120 s),oxygen concentration wasmonitored by Clark oxygrtherm.Oxygen consumption rate was determined based on counting the cell numbers and density(nmol·(mL·min·105cells)-1).Each test was performed in triplicate.ROS levels were measured using the ROS Assay Kit from Beyotime(Beyotime, China),per themanufacturers protocol[13].ROS%was calculated as the geometricalmean of the total green fluorescence of the oxidation product,2,7-dichlorofluorescein(DCF)for per 104cells.

2 Results and discussion

2.1 General characterization

The copper(Ⅱ)complex was characterized by elemental analysis,IR and UV spectra.The results suggest that the composition of the complex is [(phdpa)Cu(SCN)](ClO4).The IR spectra of the free ligand(phdpa)show two pyridyl ring bands atapproximately 1 589 cm-1and 1 569 cm-1and theδ(CH) vibration of the pyridyl ring at 762 cm-1.The characteristic imineνC=N band at 1608 cm-1for the complex shifts to lower frequencies due to metal coordination.The pyridylbandsδ(CH)are found at765 cm-1.These shifts indicate that the nitrogen atom of the pyridyl rings donates a pair of electrons to the central metaland then formsa coordinatebond.The sharp peak at 2 106 cm-1indicates the presence of the SCN-.The peaksat1 083 cm-1indicate the existence ofClO4-.The electronic spectra in the ultraviolet and visible region for the complex were recorded in ethanol solution.The strong bands at 217 nm and 255 nm are attributed to theπ→π*and n→π*transition of the ligands, respectively.The broad band in the range of 600～700 nm,is characteristic ofa copper(II)d-d transition in a tetragonal ligand field,in which the copper(Ⅱ)has a distorted octahedral coordination environment.

2.2 Crystal structure of complex

The molecular structure of[(phdpa)Cu(SCN)] (ClO4)with the atomic labeling scheme is shown in Fig.1,and the selected bond lengths and angles are listed in Table 2.The copper atom is five coordinated by three N atoms of phdpa(N1,N2,N3),one sulfur atom of SCN-and one nitrogen atom of SCN-from neighboringmoleculeswith symmetry of(0.5+x,-0.5-y,z)as a coordinated polymer.The central copper atom in the complex has a distorted octahedron geometry,which is different from the trigonal bipyramidal complex[Cu(apme)(Cl)](BPh4)(apme=tris (2-pyridylmethyl)amine)[14].The N(1),N(2),N(3)and N(0A)form the equatorial plane with deviation 0.240 4,while the S2 occupies the apical positions. The coper(II)atom is located outside of the plane (shifted by 0.024 04 nm out of the equatorial plane towards N(3)).The bond distances of Cu(1)-N(0A)and Cu(1)-S(1)are 0.1949(4)nm and 0.265 9(9)nm, indicating that the S(1)is weakly coordinated with Cu (1).N(2)-Cu(1)-N(0A)and N(1)-Cu(1)-N(3)angles are 156.40(10)°,163.35(10)°,respectively.

Fig.1 Crystal structure of[(phdpa)Cu(SCN)]+,Thermal ellipsoids are drawn at30%probability,Hydrogen atoms are omitted

Table 2 Selected bond lengths(nm)and bond angles(°)for[(phdpa)Cu(SCN)](ClO4)

2.4 Inhibition on the proliferation of cancer cells

Copper(Ⅱ)complexes were reported to inhibit the proliferation of cancer cells A549 and HepG-2[5,14,16]. The complex[(phdpa)Cu(SCN)](ClO4)was studied for their antitumor activity in vitro by determining the inhibitory percentage against growth of cancer cells A549 and HepG-2 using the method of MTT reduction.The IC50data for 5-fluorouracil(a reference compound)is 32.0μmolL-1.The IC50data for[(phdpa) Cu(SCN)](ClO4)on A549 and HepG-2 are 21.9 μmol·L-1and 10.4μmol·L-1respectively.The IC50values(10.4μmol·L-1)of[(phdpa)Cu(SCN)](ClO4)on HepG-2 was smaller than the reported[(phdpa)Cu (H2O)Ac)](Ac)(IC50,13.4μmol·L-1to HepG-2)[5]. However,the IC50data(21.9μmol·L-1)for this complex on A549 is larger than that of phen-based complexes[Cu(CH3COO)2(phen)](IC50,1.8μmol·L-1to A549)[14].These show that[(phdpa)Cu(SCN)](ClO4)(1) can inhibit selectively on the proliferation of HepG-2 cells.The dose and time-dependent anti-tumor effect for the complex(1)on HepG-2 cells is shown in Fig.2. The morphology of drug-treated cells was used to determine the extent of cytological effects.After incubating for 24 h with the complexes,HepG-2 cells was stained with DAPI,which is specific for visualisation of nuclear morphology and detection of DNA condensation.The untreated cells display a homogeneous morphology with nuclei evenly stained by DAPI.In contrast,after treatmentwith the complex for 24 h,most of the cell display fragment nuclei(Fig. 3).These indicate that the anticancer properties of [(phdpa)Cu(SCN)](ClO4)is nucleus relevant.

Fig.2 Dose and time-dependentanti-tumor effect of[(phdpa)Cu(SCN)](ClO4)(1)

Fig.3 Change in nucleusmorphology of HepG-2 cells. H2B-labled HeLa cellswere exposed to[(phdpa) Cu(SCN)](ClO4)(1)for 24 h,nuclei changewas observed.A,control;B,the complex(1), 11μmol·L-1

Fig.4 Effect on the O2consumption for[(phdpa)Cu(SCN)] (ClO4)(1)on the HepG-2 cells.Complex (11μmol·L-1),cccp:20μmol·L-16 h

Copper(Ⅱ)complexes of di(picolyl)amine derivatives are widely used as models of non-heme dioxygenase[15].Previously,we found that complex [(phdpa)Cu(H2O)(Ac)](Ac)interfere with themetabolite of cancer cells[10].Here,the effect on the O2consumption for[(phdpa)Cu(SCN)](ClO4)on the HepG-2 cells is shown in Fig.4.Experimental data show that this complex(11μmol·L-1)can decrease the O2consumption of HepG-2 cells in 6 h and the O2consumption does change with the time.These indicate that this complex could act on HepG-2 cell leading to the change of cell breath or oxygen involved metabolite.Oxygen scarceness limits the growth of tumors,because cancer cells-likemost other cells in the organism-utilize oxygen to generate energy (at least in part)and as a substrate for a number offundamental biochemical reactions including synthesis of macromolecules[9].So we deduce that strong interference on oxygenation of HepG-2 cells is a factor for the antitumor activity of[(phdpa)Cu(SCN)] (ClO4).Cancer cells use O2to generate excessive levels of the reactive oxygen species(ROS)and H2O2. This alteration in the metabolism of O2is a common feature of cancer cells and plays an important role in carcinogenesis.The effect of this complex on the level of ROS is shown in Fig.5.Extensive experimental results show that[(phdpa)Cu(SCN)](ClO4)could decrease ROS level in HepG-2 cells in 6 h,indicating that the complex may initiate ROS-mediated death of cancer cells.The increase of RO?S level in 12 h for the complex may be the result of the death of cancer cells signaling the production of ROS.So we deduce that this complex could induce cancer cells death through attenuation ROS signal.

Fig.5 Effecton the ROS content in HepG-2 for [(phdpa)Cu(SCN)](ClO4)(1,15μmol·L-1); ROSUP(20μmol·L-1)was used as control experimental.ROS%was calculated based on the DCF fluorescence per 104cell

3 Conclusions

A SCN-bridged copper(Ⅱ)complex of N-benzyl di (2-pyridylmethyl)amine,a non-planar aromatic heterocyclic rings ligand,could inhibit the proliferation of HepG-2 and cause the obvious nucleus fragmentation of HeLa cells.In addition,the complex could decrease the O2consumption and ROS level of HeLa cells in 6 h.The alteration in themetabolism of O2by copper(Ⅱ)complexes plays an important role in carcinogenesis.Experimental results show that attenuation on the metabolite of O2is an important factor for its anticancer activities.So,[(phdpa)Cu (SCN)](ClO4)is a new complex to attenuate the nucleus and interfere the metabolite of O2signaling apoptosis of cancer cells.

[1]Garbutcheon-singh K B,GrantM P,Harper B P,etal.Curr. Top.Med.Chem.,2011,11:521-542

[2]Dong X D,Wang X Y,Lin M X,etal.Inorg.Chem., 2010,49:2541-2549

[3]Ramakrishnan S,Shakthipriya D,Suresh E,etal.Inorg. Chem.,2011,50:6458-6471

[4]HUANG Juan(黄娟),CHEN Qiu-Yun(陈秋云),WANG Lin-Yun(王玲昀),et al.Chinese J.Inorg.Chem.(WujiHuaxue Xuebao),2009,25(6):1077-1089

[5]CHEN Qiu-Yun(陈秋云),HUANG Juan(黄娟),LIJun-Fen (李军峰),etal.Chinese Inorg.Chem.(WujiHuaxue Xuebao), 2008,24(11):1789-1793

[6]Drummond G R,Selemidis S,Griendling K K,etal.Nature Rev.Drug.Discov.,2011,10:453-471

[7]Selvakumar B,Rajendiran V,Uma M P,etal.J.Inorg. Biochem.,2006,100:316-330

[8]Deegan C,McCann M,Devereux M,et al.Cancer Lett., 2007,247:224-233

[9]Nagasawa H.J.Pharm.Sci.,2011,115:446-452

[10]Chen Q Y,Fu H J,Zhu W H,etal.Dalton Trans.,2011,40: 4414-4420

[11]Chen Q Y,Fu H J,Huang J,etal.Spectrochim.Acta A., 2010,75:355-360

[12]Sheldrick GM,SHELXTL-97,Program for Crystal Structure Solution and Refinement,University of Gottingen,Germany, 1997.

[13]Liu H L,Xu JJ,Dai X M,etal.J.Appl.Toxicol.,2009,29: 489-495

[14]Utz D,Kisslinger S,Hampel F,etal.J.Inorg.Biochem., 2008,102:1236-1245

[15]Kruppa M,Konig B.Chem.Rev.,2006,106:3520-3560

[16]Li XW,Zheng Y J,Li Y T,etal.Eur.J.Med.Chem., 2011,46:3851-3857

SCN-bridged Cu(Ⅱ)Com plexes:Anticancer Activity and Effect on Attenuation of O2Consumption in HepG-2 Cells

MA Zhen-Ping CHEN Qiu-Yun＊FENG Ru CHEN Tian-Tian
(School of chemistry and chemical engineer,Jiangsu University,Zhenjiang,Jiangsu 212013,China)

A new copper(Ⅱ)complex of N-benzyl di(2-pyridylmethyl)amine(phdpa)was synthesized and characterized.X-ray crystal structure shows that the copper atom in[(phdpa)Cu(SCN)](ClO4)is coordinated with a phdpa and a SCN-forming a coordinated polymer.In vitro essay results show that this copper(Ⅱ)complex exhibits good inhibition on the proliferation of HepG-2 cells with IC50of 10.4μmol·L-1.In addition,[(phdpa)Cu(SCN)] (ClO4)can induce the obvious nucleus fragmentation of HepG-2 cells,decrease the O2consumption and reactive oxygen species(ROS)level in HepG-2 cells,indicating that this SCN-bridged copper(Ⅱ)complex is a multifunctional copper(Ⅱ)complex.CCDC:840495.

copper(Ⅱ)complex;antitumor;ROS;O2consumption;crystal structure

book=0,ebook=76

O627.41

A

1001-4861(2012)11-2395-06

2012-02-26。收修改稿日期：2012-05-07。

国家自然基金(No.20971059)和江苏大学高级人才基金(06JDG050)资助项目。

＊通讯联系人。E-mail：chenqy@ujs.edu.cn;会员登记号：S06N0814M1009。