兩個dpa類配體的銅髤配合物的合成、結構、核酸酶活性及細胞毒性

岳愛琴 張宇婷 張鵬騫 高媛媛 張永坡王 敏 高春艷＊, 趙晉忠 杜維俊＊,

兩個dpa類配體的銅髤配合物的合成、結構、核酸酶活性及細胞毒性

岳愛琴1張宇婷2張鵬騫3高媛媛4張永坡2王 敏1高春艷＊,2趙晉忠2杜維俊＊,1

(1山西農業大學農學院，太谷 030801)
(2山西農業大學文理學院，太谷 030801)
(3北京麋鹿生態實驗中心，北京 100076)
(4內蒙古工業大學化工學院，呼和浩特 010051)

以dpa衍生配體4-methyl-N,N-bis(pyridin-2-ylmethyl)aniline(L)合成2個單核銅配合物[CuL(NO3)2](1)和[CuL(OAc)(H2O)]ClO4(2)，并對其進行了表征。單晶結構顯示，配合物1中的Cu中心可以描述為畸變的五角雙錐構型，而2的Cu中心為畸變的八面體構型。運用電子吸收和發射光譜法研究了配合物與CT-DNA的鍵合作用，結果表明2個配合物與DNA的相互作用均為部分插入模式。通過改變濃度、時間進一步檢測配合物切割DNA的能力，以及驗證其切割機理，結果表明在外界誘導劑存在下，2個配合物均表現出強的切割DNA的能力，其作用機理為氧化切割機理，其中活性氧可能為·OH和1O2。利用MTT法測定了配合物對體外HeLa、HepG-2和SGC-7901腫瘤細胞增殖的抑制能力。

銅配合物；dpa類配體；DNA鍵合；DNA切割；細胞毒性

0 Introduction

Medicinal inorganic chemistry hasbeen a research field of broad interest since the discovery of cisplatin as well as its anticancer activity in the 1960s[1-3].For the reason of severe side effects,general toxicity and drug resistance problems of cisplatin and its derivatives,metal-based therapeutics is a still expanding field up to now that has not stopped at the point of the discovery of new anticancer drug[4-5].Besides platinum,candidates of complexes for almost all transition metals are investigated in the past few years[6-8]for the purpose of developing new anticancer drugs with more efficient and less systemic toxicity.Among those transition metal compounds,copper complex is an interesting candidate showing much potential over cisplatin and its derivatives of reduced toxicity,novel action mechanism,various activity spectrum,and non-cross-resistance prospect[9].

Besides the choice of metal ions,purposeful design of ligand framework can significantly alter the biological properties by limiting the adverse effects of metal ion overload, modifying reactivity and lipophilicity,stabilizing specific oxidation states,and facilitating metal ion redistribution[10].Polypyridyl metal complexes have been exploited in a broad range of biological applications for their polydentate chelating structure and unique chemical and redox properties[11].In our previous works,we have reported the biological activity of Ni髤[12]and Zn髤[13-14]complexes containing polypyridyl ligands, the results suggested the complexes performed considerable cytotoxic activities.As a continuation of our interest,in this work,two new copper complexes with a mononuclear polypyridyl ligand were synthesized and structurally characterized.The DNA cleavage efficiencies and the cytotoxicity of the two complexes have been tested and analyzed.

1 Experimental

1.1 Materials and method

The reagents and solvents were purchased from commercial sources.Tridentate dpa-based ligand 4-methyl-N,N-bis(pyridin-2-ylmethyl)aniline(L)and L·xHClO4was synthesized according to previous work[12,14].Calf thymus(CT-DNA),Plasmid pBR322 DNA and ethidium bromide (EB)were purchased from Sigma-Aldrich.Stock solutions of Cu髤complexes(1.0 mmol·L-1in 10%(V/V)DMF aqueous solution)were stored at 4℃and prepared to series concentrations for all experiments.Tris-HCl was prepared using tripledistilled deionized sonicated water.

Elemental analyses and IR spectra were obtained on the Perkin-Elmer analyzer and Perkin-Elmer FT-IR spectrometer,respectively.Electronic spectra and fluorescence spectral data were collected on the JASCO V-570 spectrophotometer and MPF-4 fluorescence spectrophotometer at room temperature.The gel imaging and documentation DigiDoc-It System were assessed using Labworks Imaging and Analysis Software (UVI,England).TheMTT assaywas determined by measuring the absorbance of each well at 570 nm using a Bio-Rad 680 microplate reader(Bio-Rad,USA).

1.2 Preparation of the complexes

1.2.1 Synthesis of[CuL(NO3)2](1)

A methanol solution (10 mL)with 0.2 mmol L·xHClO4(x was counted as 1)was added to the ethanol solution(10 mL)of Cu(NO3)2·3H2O(0.2 mmol,48 mg).The resulting mixture was stirred for 10 h at room temperature.After filtration,green prism crystals suitable for X-ray diffraction were obtained by slow evaporation of the filtrate after a week,which were collected by filtration,washed with diethyl ether and dried in air(Yield:45%).Anal.Calcd.for C19H19CuN5O6(%):C,47.85;H,4.02;N,14.68.Found(%):C,48.32;H,3.29;N,14.76.FT-IR (KBr,cm-1):3 445,2 924,2 362,1 613,1 516,1 475,1 386,1 294,1 120,1 031,818,782,624.

1.2.2 Synthesis of[CuL(OAc)(H2O)]ClO4(2)

Complex 2 was prepared using a similar procedure with that of 1 except of adding an aqueous solution(5 mL)of Cu(OAc)2·H2O(0.2 mmol,40 mg)to the reaction mixture.Blue prism crystals suitable forX-ray diffraction were precipitated by slow evaporation of the filtrate after a week,which were collected by filtration,washed with cold diethyl ether and dried in vacuum(yield:42%).Anal.Calcd.for C21H24ClCuN3O7(%):C,47.64;H,4.57;N,7.94.Found(%):C,47.71;H,4.63;N,7.85.FT-IR (KBr,cm-1):3 490,1 582,1 512,1 400,1 342,1 286,1 099,972,929,832,773,929,832,773,687,624,560,421.

1.3 X-ray crystallography

Single crystals of the complexes with suitable size (0.40 mm×0.25 mm×0.12 mm for 1 and 2)were selected.X-ray diffraction data were collected on a Bruker Smart 1000 CCD diffractometer using Mo Kα radiation(λ=0.071 073 nm)with the ω-2θ scan technique.Diffraction data were collected at 293(2)K.Both the crystal structures were solved using direct methods(SHELXS-97)[15]and refined with full-matrix leastsquares technique on F2using the SHELXL-97[16].The hydrogen atoms were added theoretically,and riding on the concerned atoms and refined with fixed thermal factors.Crystallographic data details and structure refinement parameters are presented in Table 1.Selected bond lengths and angles are listed in Table S1.

CCDC:1521098 for 1;1521097 for 2.

Table 1 Crystallographic data for complexes 1 and 2

1.4 DNA binding,DNA cleavage and cytotoxicity experiments

The chemical nuclease activity and cytotoxicity experiments were conducted using the similar methods described previously[12-14,17]. Detailed experimental methods can be found in the supporting information.

2 Results and discussion

2.1 Description of the crystal structures

Both of the mononuclear Cu髤complexes have been structurally characterized by X-ray crystallography(Fig.1).Complex 1 crystallizes in a triclinic cell with P1 space group.The metal center is heptacoordinated with N3O4donor sets,and weak coordinated interactions(Cu1-O2 0.257 5(5)nm and Cu1-O4 0.247 4(5)nm)exist in the[CuL(NO3)2]unit.The atoms O1,O2,N1,O4 and O6 occupy the corners of the pentagonal basal plane,and the angles around the copper ion within the basal plane vary from 46.28(7)°to 99.04(13)°and the sum of angles spanning these five bonds is 360.07°(Table S1),underscoring the flat nature of this equatorial plane.In addition,the atoms N2 and N3 occupy the axial positions(Cu1-N2 0.195 1(7)nm;Cu1-N3 0.195 9(6)nm and N2-Cu1-N3 164.7(3)°).Therefore,the geometry around copper center can be described as a distorted pentagonal bipyramidal.

Complex 2 crystallizes in a monoclinic cell with P21/c space group.The metal center is hexa-coordinated with N3O3donor sets,and weak coordinated interaction(Cu1-O2 0.278 9(3)nm)also exist in the[CuL(OAc)(H2O)]ClO4unit,and the geometry around metal center can be described as a distorted octahedron.The atoms O2 and O3 occupy the axial positions(Cu1-O2,0.278 9(3)nm and Cu1-O3,0.245 7(3)nm),the atoms O1,N1,N2 and N3 occupy the corners of the basal plane,the angles around the copper ion within the basal plane vary from 82.26(12)to 97.49(12)°and the sum of angles spanning these five bonds is 359.76°,underscoring the flat nature of this equatorial plane.

Fig.1 ORTEP view of the molecular structure and atom-labeling scheme of complexes 1(a)and 2(b)with 30%probability ellipsoid

2.2 DNA-binding and cleavage activities

2.2.1 DNA-binding study

Electronic absorption spectroscopy was an effective method in examining the binding mode and strength of the complex with CT-DNA[18].Small molecules binding with DNA through intercalation usually result in the changes in the absorbance and shift in wavelength.The typical titration curve for 1 and 2 are shown in Fig.S1(a～b),and a plot of(εa-εf)/(εb-εf)versus cDNAfor the titration of DNA to complex is presented in corresponding inset.As given in Table 2,the observed absorption peaks at 207 and 214 nm for complexes 1 and 2 are attributed to intraligand π-π*transition.As increasing the concentration of CTDNA,the ligand-based bands exhibit hypochromism(For 1,hypochromism was about 78.8%and for 2 it was 66.7%)with red shifts(10 and 7 nm for 1 and 2,respectively) in band position,which indicatesintercalation between the complexes and DNA[19].To confirm the binding strength of the complexes with CT-DNA,the intrinsic binding constants Kbwere calculated according to the equation[20]:cDNA/(εa-εf)=cDNA/(εb-εf)+1/[Kb(εb-εf)], where cDNAis the DNA concentration in nucleotides;εais the extinction coefficient observed for the charge transfer absorption band at a given DNA concentration;εfis the extinction coefficient of the free complex in solution;εbis the extinction coefficient of the complex when fully bound to DNA.The binding constant Kbvalues (Table 2)follow the order:2(9.60×104L·mol-1)gt;1(4.21×104L·mol-1),which suggest that complex 2 has slightly stronger binding affinity than 1.The Kbvalues are smaller than reported for typical classical intercalators(EB-DNA,3.3×105mol·L-1in 50 mmol·L-1Tris-HCl/1.0 mol·L-1NaCl buffer,pH 7.5)[21],which suggests that the binding strength of the two complexes with DNA is a medium intercalative mode.

Table 2 Absorption spectral and fluorescence spectral properties of complexes 1 and 2 bound to CT-DNA

In order to further clarify the CT-DNA binding activity,fluorescence spectralmeasurementswere carried out.No luminescence is observed for both complexes and CT-DNA at room temperature,therefore the binding activity is evaluated by the fluorescence emission intensity of EB bound to DNA as a probe.EB-DNA emits intense fluorescent due to their strong intercalation between the adjacent DNA base pairs[22],which could be quenched by the addition of another compound.The relative binding propensity of the complexes toEB-DNA studied in buffer solution (5 mmol·L-1Tris-HCl/50 mmol·L-1NaCl,pH=7.2)is shown in Fig.S2(a～b),and the plots of I0/I versus ccomplexfor the quenched intensity of 1 and 2 to EB-DNA are shown in the insets,respectively.Fluorescence intensities of EB-DNA at 602 nm(510 nm excitation)were measured,and the extent of reduction of the emission intensity by varying the concentration of the complexes gives a measure of the binding propensity.In the Stern-Volmer equation I0/I=1+KcQ[23],I0and I represent the fluorescence intensities in the absence and presence of quencher,respectively;K is the Stern-Volmer quenching constant,and cQis the concentration of the quencher.The quenching plot indicates the quenching of EB bound to CT-DNA by complex is in agreement with the linear Stern-Volmer equation.In the equation KEBcEB=Kappccomplex,KEBis a constant of 1.0×107mol·L-1(cEB=2.4 μmol·L-1),Kappis the calculated apparent binding constant values,and ccomplexis the concentration at a half reduction of the fluorescence intensity of EB.The Kappvalues(Table 2)are nearly equal and follow the order:1(7.13×105L·mol-1)gt;2(6.83×105L·mol-1).The apparent binding constants values are less than that of the classical intercalators and metallointercalators(1.0×107L·mol-1)[24],indicating medium binding strength of the complexes with CT-DNA.On the whole,the result of fluorescence spectral measurements is consistent with obtained Kbvalues by UV spectroscopy.2.2.2 DNA Cleavage Studies

Agarose gel electrophoresis was used to explore the supercoiled (SC)pBR322 plasmid DNA cleavage activity of the two complexes in a medium of 50 mmol·L-1Tris-HCl/NaCl buffer for 4 h.In the absence of external agents,the concentration-dependent DNA cleavage activities were observed under the nearly physiological conditions(pH=7.2,37℃)(Fig.S3),and both of 1 and 2 could not induce obvious DNA cleavage with the increase of concentration (10～130 μmol·L-1).The ratios of SC DNA (Form Ⅰ)for complex 1 gradually reduce with the increase of concentration,while NC DNA (Form Ⅱ )doesn′t increase,which suggests that 1 partially degraded SC DNA into undetectable minor fragments[25].When the concentration of 2 increase to 130 μmol·L-1,no obvious change for the ratios of FormⅠand FormⅡwere observed.The above suggest that 1 showed slightly better concentration-dependent activities than 2.The concentration-dependent DNA cleavage experidments by complex were also performed in the presence of H2O2(Fig.2)and GSH(glutathione)(Fig.3),respectively.Notably,the DNA cleavage efficiencies of both complexes exhibit remarkable enhancement.In Fig.2,at the concentration of 10 μmol·L-1Cu2+,both complexes are efficient cleavers of SC DNA(FormⅠ)and produce more than 90%of NC DNA(FormⅡ),which implies that H2O2as a revulsant or an activator plays a vital role.When the concentration of complex increase to 40 μmol·L-1,obvious LC DNA(FormⅢ)is produced and the ratios of which followed the order of 1(57.3%)gt;2(31.0%).In order to further clarify the vital role of external revulsant,the GSH (glutathione)instead of H2O2were added.Similarly,as shown in Fig.3 (lane 2～5),the DNA cleavage efficiencies of both complexes also exhibit remarkable increases.At the concentration of 50 μmol·L-1Cu2+,the DNA cleavage efficiencies(the ratios of FormⅢ)follow the order of 1(61.8%)gt;2(45.2%).Both H2O2and GSH showed similar behavior in DNA cleavage reactions,although H2O2was slightly more active than GSH.

Fig.2 Gel electrophoresis diagrams showing the cleavage of pBR322 DNA(0.1 μg·μL-1)with complex 1(a)and 2(b)in Tris-HCl/NaCl buffer(pH=7.2)at 37℃

Fig.3 Gel electrophoresis diagrams showing the cleavage of pBR322 DNA(0.1 μg·μL-1)with complex 1(a)and 2(b)in Tris-HCl/NaCl buffer(pH=7.2)at 37℃

Fig.4 Time-dependence of pBR322 DNA cleavage by complexes 1(a)and 2(b)according to the inset

To further assess the cleavage rate of chemical nuclease,the kinetic parameters for complexes 1 and 2 promoted DNA cleavage were determined.The timedependence ofDNA cleavage experimentswere carried out(Fig.4)under the same condition(pH=7.2,37 ℃,cGSH=250 μmol·L-1,cCu2+=10 μmol·L-1).The ratios of FormⅠgradually disappeared and FormⅡincreased with reaction time increase (3～15 min).More than 90%NC DNA(FormⅡ)was observed within 9 min for complex 1 (Fig.4a Inset)and within 15 min for complex 2 (Fig.4b Inset).The decrease of FormⅠor increase of FormⅡwas fitted to a single exponential decay curve (pseudo-first-order kinetics)by the equation[26-27]:y=(100-y0)[1-exp(-kobsx)],where y0is the initial ratio of a form of DNA;y is the ratio of a specific form of DNA at time x;kobsis the apparent rate constant.The reaction profile for the complex displayed approximately pseudo-first-order kinetic behavior(Fig.4(a～b))with kobsfollow the order of 1(0.35 min-1)gt;2(0.22 min-1),showing better than the result of the kobsfor copper-ATCUN complexes(0.07 and 0.14 min-1)obtained by Cowan′s group[28].

In order to obtain the information about the active oxygen species(ROS)which was responsible for the DNA cleavage,the potential mechanism of DNA cleavage mediated by the complex was investigated in the presence of GSH.DNA cleavage experiments(Fig.5)were carried out using reagents like KI as hydroxyl radical scavenger(·OH),NaN3as singlet oxygen(1O2)quencher,EDTA as the chelator of complex,catalase ashydrogen peroxide scavenger,and superoxide dismutase(SOD)as O2-radical scavenger.In Fig.5(a～b),addition of KI(lane 3)to SC DNA partly inhibited the DNA cleavageactivity,which suggested the possible involvement of hydroxyl radial(·OH)as the reactive species.Also,the complexes showed partial inhibition of DNA-cleavage in the presence of the NaN3(lane 4),and D2O (lane 8)enhanced the DNA cleavage[29],indicating possible involvement of singlet oxygen as the reactive species.No obvious inhibition was observed for other radical scavengers.Therefore,the data suggest the involvement of both hydroxyl radicals (·OH)and singlet oxygen (1O2)as ROS.In addition,The EDTA (lane 5),a Cu髤-specific chelating agent that strongly bind to Cu髤forming a stable complex,can efficiently inhibit DNA cleavage,indicating metalion playsthekeyrolein the cleavage.The addition of methyl green(lane 9),which is known to interact to DNA at major groove[30],effectively inhibited DNA cleavage by complex.The result suggests that the complex mainly has interaction with DNA through major groove.

Fig.5 Cleavage of plasmid pBR322 DNA(0.1 μg·μL-1)in presence of 20 μmol·L-1complex 1(a)～2(b)(0.04%DMF)and different inhibitors after 4 h incubation at 37℃

2.3 MTT assay

MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay is a colorimetric assay based on the conversion of the yellow tetrazolium salt to purple formazan crystals by metabolically active cells,which has been done to test the ability of complexes to inhibit cell growth and induce cell death in HeLa(human cervical carcinoma),HepG-2 (human liver hepatocellularcarcinoma)and SGC-7901 (human gastric carcinoma)cancer cell lines.In Table 2,both1 and 2 exhibit significant cytotoxic activities toward tested tumor cells and inhibit the growth of cells in a dose-dependent manner,and 2 shows slightly better antitumor effect than 1.Complex 2 exhibits strong anti-proliferative effect on HepG-2 cells with the IC50value of(34.2±2.3)μmol·L-1,which is close to the cisplatin(IC50=(25±3.1)μmol·L-1)and probably has the potential to act as an effective metal-based anticancer drug.

Table 2 IC50of complexes 1 and 2 obtained with different cell lines for 48 h

3 Conclusions

Two new mononuclear Cu髤complexes have been synthesized and characterized.Crystal structure showed that the metal center of 1 is hepta-coordinated with N3O4donorsets,existing weak coordinated interactions,and can be described as a distorted pentagonal bipyramidal.Weak coordinated interaction also exists in the crystal unit of 2 where the metal center is hexa-coordinated and the geometry can be described as a distorted octahedron. Partial intercalation and medium binding strength between the complexes and CT-DNA has been demonstrated.The DNA cleavage efficiencies of both complexes exhibit remarkable enhancement in the presence of H2O2or GSH,and H2O2was slightly more active than GSH.The oxidative cleavage mechanism was confirmed via a pathway involving formation of both·OH and1O2as ROS.The in vitro cytotoxicity of the complexes has been assessed by MTT on tumor cells lines(HeLa,HepG-2 and SGC-7901),both 1 and 2 exhibit significant cytotoxic activities and inhibit the proliferation of cells.

Acknowledgements:This work was supported by the National Natural Science Foundation of China (Grant No.31171580),Scientific and Technologial Innovation Programs of Higher Education Institutions in Shanxi (Grant No.2015148),Natural Science Foundation of Shanxi (Grants No.201601D011076,201701D221157),Shanxi Key Research and Development Program (Grants No.201703D221008-4,201703D221004-5),Shanxi Agricultural University youth topnotch innovative personnel support program(Grant No.201203),the PhD Research Startup Foundation of Shanxi Agricultural University (Grant No.2013YJ40),The Key Scientific Research Projects of Coal Fund in Shanxi(Grant No.FT201402-01).The Natural Science Foundation of Inner Mongolia (Grant No.2016BS0206),and The Inner Mongolia Autonomous Region Higher Scientific Research Project(Grant No.NJZY088).

Supporting information is available at http://www.wjhxxb.cn

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Two Copper髤Complexes with dpa-Based Ligand:Syntheses,Structures,Nuclease Activity and Cytotoxicity

YUE Ai-Qin1ZHANG Yu-Ting2ZHANG Peng-Qian3GAO Yuan-Yuan4ZHANG Yong-Po2
WANG Min1GAO Chun-Yan＊,2ZHAO Jin-Zhong2DU Wei-Jun＊,1
(1College of Agronomy,Shanxi Agricultural University,Taigu,Shanxi 030801,China)
(2College of Arts and Sciences,Shanxi Agricultural University,Taigu,Shanxi 030801,China)
(3Beijing Milu Ecological Research Center,Beijing 100076,China)
(4Chemical Engineering College,Inner Mongolia University of Technology,Hohhot 010051,China)

Two new mononuclear copper髤complexes,[CuL(NO3)2](1)and[CuL(OAc)(H2O)]ClO4(2),with dpabased ligand(L=4-methyl-N,N-bis(pyridin-2-ylmethyl)aniline)have been synthesized and characterized by various physico-chemical techniques.The crystal structure of complex 1 displays a distorted pentagonal bipyramidal geometry,and the geometry around copper center of 2 can be described as a distorted octahedron.Interaction of the complexes with CT-DNA has been explored by using absorption and emission spectral methods,and the result suggests that the binding strength of the two complexes with DNA is a medium intercalative mode.Theconcentration-dependent and time-dependent DNA cleavage activity and the mechanism of DNA cleavage have been investigated,which suggest the DNA cleavage efficiencies of both complexes exhibit remarkable enhancement in the presence of external revulsants,and oxidative mechanism has been demonstrated via the pathway involving both hydroxyl radicals(·OH)and singlet oxygen(1O2)as ROS.The in vitro cytotoxic activity of the complexes has been examined by MTT on three cell lines such as HeLa,HepG-2 and SGC-7901.CCDC:1521098,1;1521097,2.

copper髤complexes;dpa-based ligand;DNA binding;DNA cleavage;cytotoxicity

O614.121

A

1001-4861(2017)12-2287-09

10.11862/CJIC.2017.273

2017-05-15。收修改稿日期：2017-09-28。

國家自然科學基金(No.31171580)、山西省高等學校科技創新項目(No.2015148)、山西省自然科學基金(No.201601D011076，201701D221157)、山西省重點研發計劃項目(No.201703D221008-4，201703D221004-5)、山西農業大學中青年拔尖創新人才支持計劃(No.201203)、山西農業大學引進人才科研啟動金(No.2013YJ40)、山西省煤基重點項目(No.FT201402-01)、內蒙古自然科學基金(No.2016BS0206)和內蒙古自治區高等學校科學研究項目(No.NJZY088)。

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