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  In the past few decades, Schiff bases and their metal complexes have been a focus of chemists and biologists because of their noteworthy antibacterial, antifungal, anticancer, urease inhibition, antioxidant and antiglycation activities^[1-8].It has been demonstrated that the presence of heterocyclic ring in the synthesized Schiff bases plays a major role in extending their pharmacological properties^[8].As a result, a sizable number of transition metal complexes with acylhydrazones and thiosemicarbazones derived from actyl-pyridine/pyrazine have been extensively investigated as potential anticancer agents^{[3, 8-10]}.However, as their structurally analogous, semicarbazones have been paid much less attention^[6].

  On the other hand, the previous studies revealed that Cu(Ⅱ) containing anticancer agents are promising leads for next generation metal-based anticancer agents because Cu(Ⅱ) plays a significant role in biological systems^{[1, 5]}. Therefore, in this paper, two Cu(Ⅱ) complexes with a semicarbazone ligand derived from 2-acetyl-3-methylpyrazine and 4-phenylsemicarbazide have been synthesized and structural determined by single-crystal X-ray diffraction.In addition, the interactions between three compounds and ct-DNA have been studied by ethidium bromide(EB) fluorescence probe.

  1    Experimental

  1.1    Materials and measurements

  Solvents and starting materials for synthesis were purchased commercially and used as received.Elemental analysis was carried out on an Elemental Vario EL analyzer.The IR spectra (ν=4 000~400 cm^-1) were determined by the KBr pressed disc method on a Bruker V70 FTIR spectrophotometer.¹H NMR spectra of L was acquired with Bruker AV400 NMR instrument in DMSO-d₆ solution with TMS as internal standard.The interactions between three compounds and ct-DNA are measured using literature method^[11] via emission spectra on a Varian CARY Eclipse spectrophotometer.

  1.2    Preparations of the ligand HL, complexes 1 and 2

  1: Green blocks.Anal.Calcd.for C₂₈H₂₈N₁₀O₂Cl₂Cu2 (%): C 45.78, H 3.84, N 19.07.Found(%): C 45.65, H 4.00, N 18.94.FTIR (cm^-1): ν(N=C-O) 1 619, ν(C=N) 1 594, ν(C=N)_pyrazine 1 548.

  The complexes 1 and 2 were generated by reaction of the ligand HL (5 mmol) with equimolar of CuCl₂·2H₂O and Cu(OAc)₂·H₂O in methanol solution (10 mL), respectively.Crystals suitable for X-ray diffraction analysis were obtained by evaporating the corresponding reaction solutions at room temperature.

  As shown in Scheme 1, the ligand HL was produced by condension of 2-acetyl-3-methylpyrazine (1.36 g, 0.01 mol) and 4-phenylsemicarbazide (1.51 g, 0.01 mol) in ethanol solution (30 mL) with continuous stirring at room temperature for 5 h.The white solid was filtered and washed three times by cold ethanol.Yield: 2.21g (85%).m.p.178~180 ℃.Elemental analysis Calcd.for C₁₄H₁₅N₅O(%): C 62.44, H 5.61, N 26.01.Found(%): C 62.56, H 5.39, N 25.89.FTIR (cm^-1): ν(C=O)_{semicarbazone} 1 702, ν(C=N) 1 604, ν(C=N)_pyrazine 1 593.¹H NMR (400 MHz, DMSO-d₆): δ 10.03(1H, s, NH), 8.77(1H, s, NH), 8.45~8.48 (2H, m, pyrazine-H), 7.54~7.56 (2H, m, phenyl-H), 7.23~7.27 (2H, m, phenyl-H), 6.95~6.99 (1H, m, phenyl-H), 2.75 (3H, s, CH₃), 2.28 (3H, s, CH₃).

  2: Black blocks.Anal.Calcd.for C₃₂H₃₄N₁₀O₆Cu₂ (%): C 49.16, H 4.38, N 17.92.Found (%): C 49.36, H 4.52, N 17.74.FTIR (cm^-1): ν(N=C-O) 1 595, ν(C=N) 1 579, ν(C=N)_pyrazine 1 544, ν_as1(COO^-) 1 509, ν_as4(COO^-) 1 435 and 1 352.

  

  Figure Scheme 1. Synthesis route of HL

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  1.3.1    X-ray crystallography

  The structures were solved by direct methods and refined by full matrix least-square on F 2 using the SHELXTL-97 program^[13].All non-hydrogen atoms were refined anisotropically.All the H atoms were positioned geometrically and refined using a riding model.Details of the crystal parameters, data collection and refinements for complexes 1 and 2 are summarized in Table 1.

  The X-ray diffraction measurement for complexes 1 and 2 were performed on a Bruker SMART APEX Ⅱ CCD diffractometer equipped with a graphite monochromatized Mo Kα radiation (λ=0.071 073 nm) by using φ-ω scan mode.Semi-empirical absorption correction was applied to the intensity data using the SADABS program^[12].

  Table1. Crystal data and structure refinement for complexes 1 and 2

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  Table1. Crystal data and structure refinement for complexes 1 and 2

  CCDC: 1455421, 1; 1455422, 2.

  2    Results and discussion

  2.1    Crystal structure description

  

  Figure 1. Diamond drawing of 1 (a) and 2 (b) with 30% thermal ellipsoids and extend 2D supramolecular structure along a axis in complexes 1 (c) and 2 (d)

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  As shown in Fig.1a, complex 1 contains one discrete dimeric Cu (Ⅱ) molecule in the unit cell.Two Cu atoms of the dimer were separated by 0.352 4 nm and doubly bridged by two chloride anions to form an ideal planar four-membered Cu2Cl₂ core.Each of the Cu(Ⅱ) ions is penta-coordinated by one independent anionic ligand with N₂O donor set and two chloride anions, one of which acts as a μ²-bridge, thus giving a distorted square pyramid coordination geometry (τ=0.122)^[14].In the solid state, the discrete Cu (Ⅱ) dimers of 1 were further linked into a one-dimensional chain along a axis (Fig.1c) by intermolecular N-H…Cl (N5-H5A…Cl₁ⁱⁱⁱ, with D…A distance being 0.343 1(4) nm, D-H…A angle being 170.1°, Symmetry codes: ⁱⁱⁱ x+1, y, z) hydrogen bonds between the amine nitrogen atoms from one dimer and chloride anions from the adjacent one.

  The structure of 2 is similar as that of 1, while the chloride anion is replaced by monodentate acetate, in which one oxygen atom of the carboxyl group bridges two Cu(Ⅱ) ions to form μ-O.One-dimensional chain (Fig.1d) along a axis formed by intermolecular N-H…O (N5-H5A…O3^iv, with D…A distance being 0.288 6(3) nm, D-H…A angle being 157.8°, Symmetry codes: ^iv 2-x, 2-y, 2-z) hydrogen bonds are also present in the crystal of 2.

  A diamond drawing for complexes 1 and 2 is shown in Fig.1.Selected bond distances and angles are listed in Table 2.The lengths of C-O bond of the semicarbazone moiety are 0.127 9(5) and 0.126 9(3) nm in complexes 1 and 2, respectively, clearly showing that the ligand HL has enolizated and deprotonated in both complexes^[8].

  Table2. Selected bond lengths(nm) and angles(°) in complexes 1 and 2

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  Table2. Selected bond lengths(nm) and angles(°) in complexes 1 and 2

  2.2    IR spectra

  The ν_{semicarbazone}(C=O) of the free ligand is 1 748 cm^-1, while it is disappeared in both complexes, meanwhile, new N=C-O stretching vibration absorption is observed at 1 619 and 1 595 cm^-1 in complexes 1 and 2, respectively, revealing that the C=O in O=C-N moiety has enolized and the oxygen atom coordinates to the metal ions in both complexes^[6-7].The ν(C=N) bands of the imine group and pyrazine ring in the ligand HL shift to lower frequency values in the complexes, indicating that the N atoms of both units take part in the coordination^[8].Furthermore, the bands at 1 509, 1 435 and 1 352 cm^-1 in complex 2 could be assigned to the split bands ν_as1(COO^-) and ν_as4(COO^-) of the acetate group, respectively, showing that there are bridged and monodentate carboxyl groups in the complex 2^[15].It is in accordance with the crystal structure study.

  2.3    EB-DNA binding study by fluorescencespectrum

  

  Figure 2. Emission spectra of EB-DNA system in the absence and presence of ligand HL (a),complexes 1(b) and 2 (c)

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  It is well known that EB can intercalate nonspecifically into DNA, which causes it to fluoresce strongly.Competitive binding of other drugs to DNA and EB will result in displacement of bound EB and a decrease in the fluorescence intensity^[16].The effects of the ligand and complexes on the fluorescence spectra of EB-DNA system are presented in Fig.2, the fluorescence intensities of EB bound to ct-DNA at about 600 nm show remarkable decreasing trends with the increasing concentration of the tested compounds, indicating that some EB molecules are released into solution after the exchange with the compounds.The quenching of EB bound to DNA by the compounds is in agreement with the linear Stern-Volmer equation: I₀/I=1+K_sqr^[11], where I₀ and I represent the fluorescence intensities in the absence and presence of quencher, respectively, K_sq is the linear Stern-Volmer quenching constant, r is the ratio of the concentration of quencher and DNA.In the quenching plots of I₀/I versus r, K_sq values are given by the slopes.The Ksq values are 0.268, 0.723 and 0.780 for the ligand HL, complexes 1 and 2, respectively.The results indicate that interactions of the complexes with DNA are stronger than that of the ligand HL, because the complexes have higher rigidity to bind the base pairs along DNA, which increases their binding abilities.In addition, the complexes 1 and 2 have similar Ksq values, showing that the coordination anions are almost irresponsible for the DNA interaction.

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     Ye X P, Zhu T F, Wu W N, et al. Inorg. Chem.Commun., 2014,47:60-62 doi: 10.1016/j.inoche.2014.07.022

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      沈伟,胡未极,吴小勇,等.无机化学学报, 2016,32:1101-1110 http://www.wjhxxb.cn/wjhxxbcn/ch/reader/view_abstract.aspx?flag=1&file_no=20160623&journal_id=wjhxxbcnSHEN Wei, HU Wei-Ji, WU Xiao-Yong , et al. Chinese J. Inorg. Chem. , 2016,32:1101-1110 http://www.wjhxxb.cn/wjhxxbcn/ch/reader/view_abstract.aspx?flag=1&file_no=20160623&journal_id=wjhxxbcn

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      Sheldrick G M. SADABS, University of Göttingen, Germany, 1996.

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• 

  Scheme 1  Synthesis route of HL

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  Figure 1  Diamond drawing of 1 (a) and 2 (b) with 30% thermal ellipsoids and extend 2D supramolecular structure along a axis in complexes 1 (c) and 2 (d)

  H atoms are omitted for clarity in (a) and (b); H atoms of C-H bonds are omitted for clarity in (c) and (d); Symmetry codes:ⁱ-x,-y,-z; ⁱⁱ 1-x,2-y,2-z; iii x+1,y,z; ^iv 2-x,2-y,2-z

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  Figure 2  Emission spectra of EB-DNA system in the absence and presence of ligand HL (a),complexes 1(b) and 2 (c)

  Arrow shows the fluorescence intensities change of EB-DNA system upon increasing tested compound concentration; Inset: plot of I₀/I versus r

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  Table 1.  Crystal data and structure refinement for complexes 1 and 2

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  Table 2.  Selected bond lengths(nm) and angles(°) in complexes 1 and 2

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