Accepted Manuscript A Comparative Study of Cytotoxicity and Interaction with DNA/Protein of Five Transition Metal Complexes with Schiff Base Ligands Meiju Niu, Min Hong, Guoliang Chang, Xiao Li, Zhen Li PII: DOI: Reference:

S1011-1344(15)00146-3 http://dx.doi.org/10.1016/j.jphotobiol.2015.04.023 JPB 10020

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

26 December 2014 20 April 2015 20 April 2015

Please cite this article as: M. Niu, M. Hong, G. Chang, X. Li, Z. Li, A Comparative Study of Cytotoxicity and Interaction with DNA/Protein of Five Transition Metal Complexes with Schiff Base Ligands, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.04.023

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A Comparative Study of Cytotoxicity and Interaction with DNA/Protein of Five Transition Metal Complexes with Schiff Base Ligands Meiju Niu,a Min Hong,∗a Guoliang Chang,a Xiao Li,a and Zhen Lia

a

Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell Technology, School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, China

Abstract Five transition metal complexes NiL12 (1), CuL12 (2), ZnL12 (3), [MnL12(N3)]n·nCH2Cl2 (4), CuL22 (5) {HL1 = 3-{[2-(2-hydroxy-ethoxy)-ethylimino]-methyl}-naphthalen-2-ol,

HL2

=

2-{[2-(2-hydroxy-ethoxy)-ethylimino]

-methyl}-phenol} have been synthesized and fully characterized. In all of the complexes, the ligands coordinated to the metal ion in a negative fashion via O and N donor atoms. The X-ray structures of nickel complex 1 and copper complexes 2 and 5 are four-coordinated monomers and show slightly distorted square-planar geometry in the vicinity of the central metal atom. Zinc complex 3 exhibits a four-coordinated tetrahedral structure. Differently, manganese complex 4 reveals a six-coordinated octahedral structure, one-dimensional chain is linked by azide in the end-to-end mode. In vitro cytotoxicity of these complexes to various tumor cell lines was assayed by the MTT method. The results showed that most of these metal-Schiff base complexes exhibited enhanced cytotoxicity than Schiff base ligands, which clearly implied a positive synergistic effect. Moreover, these complexes appeared to be selectively active against certain cell lines. The interactions of these metal complexes with CT-DNA were investigated by UV-vis, fluorescence and CD spectroscopy, the results indicated that these complexes are metallointercalators and can interact with CT-DNA. The study of interaction between complexes and BSA indicated that all of the complexes could quench the intrinsic fluorescence of BSA in a static quenching process. Keywords: Transition metal complex；Crystal structure; Cytotoxicity activity; DNA-binding; BSA-binding.

1. Introduction Since the successful use of cisplatin and related platinum complexes as anticancer agents, developing other transition metal complexes with better efficiency and new mechanisms of action has become a central research theme in ∗

Corresponding author. Tel.:/Fax: +866358239121. E-mail address: [email protected] (M. Hong)

bioinorganic chemistry [1−5]. The discovery of nonplatinum metal-based anticancer complexes with potent anticancer activity, such as nickel(II) [6], copper(II) [7], zinc(II) [8], and manganese(II) [9] complexes, has been extensively investigated during the past two decades. Also, there are several literature reported that established organic drugs are coordinated to metal fragments with the purpose of enhancing their activity [10]. Salicylaldehyde-based Schiff base derivatives have been reported to have the antiviral and antitumor acivities [11,12]. Recently, some new coordination compounds based on Schiff base ligands were found to afford novel potential (pro) drugs. Therefore, transition metal complexes of Schiff base are extensively researched as promising alternatives to traditional cisplatin for anticancer drugs [13]. Intercalators are small organic molecules or metal complexes that unwind DNA to π-stack between two neighboring base pairs. Metallointercalators are metal complexes that bear at least one intercalating ligand. Since the preparation of the first platinum metallointercalator by S. J. Lippard and co-workers [14], there has been rising interest in octahedral transition metal complexes as metallointercalator [15] and new dual-function metal complexes that can interact with DNA by both coordination and intercalation [16], which may have potential diagnostic and therapeutic applications. Because naphthaldehyde Schiff base ligand is an aromatic planar ligand with N and carbonyl O donor atoms, it can coordinate to metal and form metal intercalator, which should exhibit stronger intercalation with DNA than salicylaldehyde Schiff base ligand for its bigger planarity. Another biomolecule proteins play an important role in transportation and deposition of endogenous and exogenous substances such as fatty acids, harmones and medicinal drugs. Bovine serum albumin (BSA) is often chosen as a target protein for the study of interactions with small molecules because of its low cost, ready availability and its similarity to human serum albumin (HSA) [17-19]. The investigations of the molecules with DNA and proteins are imperative and fundamental importance. Also the studies may provide information of structural features that determine the therapeutic effectiveness of drugs, and it will become an important research field in life sciences, chemistry, and clinical medicine. The nickel(II), copper(II), and cobalt(II)/(III) complexes with different Schiff base N/O donor ligands had shown potent anticancer activity in our previous studies[20,21]. To explore Schiff base-metal complexes as anticancer agents, 3-{[2-(2-hydroxy-ethoxy)-ethylimino]-methyl}-naphthalen-2-ol (HL1) was synthesized. Then it reacted with nickel(II), copper(II), zinc(II), manganese(II) salts to afford the anticipated metal complexes. Herein, we reported the synthesis and characterization of HL1 and its metal complexes: NiL12 (1), CuL12 (2), ZnL12 (3), and [MnL12(N3)]n·nCH2Cl2 (4). Their cytotoxicity and interactions with DNA/BSA were also investigated. In addition, to compare the effect of the ligand planarity on its bio-activity and interaction with DNA/BSA, complex 5 (CuL22) with salicylaldehyde Schiff base ligand was synthesized and studied.

2. Experimental section 2.1. Materials and reagents Salicylaldehyde, 2-hydroxy-1-naphthaldehyde and 4-phenoxy-phenylamine were obtained from aladdin-reagent company. CT-DNA and BSA were purchased from Sino-American Biotechnology Company. Ethidium bromide (EB) was obtained from Sigma-Aldrich. All other reagents were AR grade or biochemical quality. 10 mM Tris–HCl/50 mM NaCl, pH 7.2 buffer solution was prepared by using triple distilled water. Solution of CT-DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.8-1.9, indicating that the DNA was sufficiently pure and free of protein. Stock solutions of CT-DNA were stored at 4℃ and used within 5 days. The DNA concentration was determined by absorption spectroscopy using the molar absorption coefficient (6600 M-1 cm-1) at 258 nm. BSA stock solutions in 10 mM Tris-HCl/50 mM NaCl, pH 7.2 buffer solution was stored in the dark at 4℃. 2.2. Synthesis of ligand (HL1) and corresponding metal complexes (1-5) The reactions involved in the synthesis of Schiff base ligands and corresponding metal complexes are given in Scheme 1. 2.2.1 Synthesis of 3-{[2-(2-hydroxy-ethoxy)-ethylimino]-methyl}-naphthalen-2-ol (HL1). The ligand HL1 was prepared by refluxing a mixture of 2-hydroxy-1-naphthaldehyde (1.722 g, 10 mmol) and 4-phenoxy-phenylamine (2.433 g, 10 mmol) in 100 mL of absolute methanol for 5 h. The reaction mixture was cooled to room temperature and the solid obtained was filtered, washed several times with distilled methanol. The pure product were obtained by recrystallizing from ethanol with 87% yield. M.p.: 136-138 °C. Anal. Calc. for C23H16NO2 (MW = 338.38): C 81.63, H 4.76, N 4.14 %; Found: C 81.68; H 4.73; N 4.11 %. IR (KBr, cm-1): 1634.6 (s)，1484.2 (m)，1234.5 (m)，1163.1 (s)，1084.7 (s)，734.5 (m), 654.2 (m). 1H NMR（CDCl3，400 MHz, δ/ppm ）8.39 (s, 1H, -CH=N), 7.21-8.16 (m, 6H, -C10H6), 5.01 (s, 1H, -OH), 6.91-7.20 (m, 4H, -C6H4), 6.92-7.23 (m, 5H, -C6H5). 2.2.2 Synthesis of complex [NiL12] (1). Complex 1 was synthesized by refluxing a methanol solution of [Ni(CH3COO)2]·4H2O (0.2491 g, 1 mmol) and ligand HL1 (0.5181 g, 2 mmol) for 4 h. After cooling the reaction mixture to room temperature, the resultant solution was filtered, and the filteration was then kept aside for 7 days over which time green crystals were deposited. Yield: 82%. M.p.: 216-218 ℃. Anal.Calc. for C46H32N2NiO4 (MW = 735.45): C, 75.12; H, 4.38; N, 3.81, Found: C, 75.10; H, 4.35; N, 3.81. IR (KBr, cm-1): 1615.7 (s), 1601.6 (s), 1534.2(m), 1488.1 (m), 1235.6 (s), 1093.2 (s), 545.2 (w), 466.4 (w). ESI-MS: m/z 774.33 [NiL12 + K]+. UV-Vis (MeOH), λmax/nm: 318, 416. 2.2.3. Synthesis of complex [CuL12] (2).

The crystals of complex 2 were obtained in similar manner to complex 1 by replacing CuCl2·2H2O with [Ni(CH3COO)2]·4H2O. Yield: 72%. M.p.: 208-209 ℃. Anal.Calc. for C46H32N2CuO4 (MW = 740.30): C, 74.63; H, 4.36; N, 3.78, Found: C, 74.59; H, 4.27; N, 3.78. IR (KBr, cm-1): 1616.3 (s), 1601.0 (m), 1537.1 (m), 1488.7 (m), 1234.1 (s), 1092.1 (s), 553.1(w), 464.4 (w). ESI-MS: m/z 763.52 [CuL12 + Na]+. UV-Vis (MeOH), λmax/nm: 317, 416. 2.2.4. Synthesis of complex [ZnL12] (3). The crystals of complex 3 were obtained in similar manner to complex 1 by replacing Ni(CH3COO)2·2H2O with Zn(CH3COO)2·2H2O. Yield: 80 %. M.p.: 232-235 ℃. Anal. Calc. for C46H32N2O4Zn (MW = 742.16): C, 74.44; H, 4.34; N, 3.77, Found: C, 74.39; H, 4.31; N, 3.77. IR (KBr, cm-1): 1613.7 (s), 1598.6 (m) 1535.6 (m), 1489.0(m), 1234.4 (s), 1078.3 (s), 545.3 (w), 449.9 (w). ESI-MS: m/z 765.36 [ZnL12 + Na]+. UV-Vis (MeOH), λmax/nm: 320, 406. 2.2.5. Synthesis of complex [MnL12(N3)] n·nCH2Cl2 (4). At

room

temperature,

Mn(CH3COO)2·4H2O

(0.2496

g,

1

mmol)

was

added

to

a

stirred

methanol-dichloromethane solution (20 mL) of ligand HL1 (0.5181 g, 2 mmol), and the stirring was continued for about 0.5 h. To the resulting solution, NaN3 (0.13 g, 2 mmol) dissolved in a minimum amount of water was added. Then the solution was refluxed for about 3.5 h to complete the air oxidation of Mn(II) to Mn(III). The filtered solution was then kept aside for 10 days over which time black crystals were deposited. Yield: 71%. M.p. 240-241 ℃. Anal. Calc. for C47H34Cl2MnN5O4 (MW = 858.64): C, 65.60; H, 3.96; N, 8.18, Found: C, 65.54; H, 4.05; N, 8.16. IR (KBr, cm-1): 2041.4(s), 1618.0 (s), 1597.3 (m), 1540.5 (m), 1485.7(m), 1229.5 (s), 1080.0 (s), 547.9 (w), 482.9 (m). ESI-MS: m/z 689.82 [MnL12]+. UV-Vis (MeOH), λmax/nm: 324, 404. 2.2.6. Synthesis of complex [CuL22] (5). Due to the difficulty of the purification and seperation of liguid HL2, complex 5 was prepared by the one-pot method with two ligand precursors and metal salt. Briefly, the methanol (15 mL) solution of salicylaldehyde (0.1220 g, 1 mmol) was added to a methanol solution (5mL) of 4-phenoxy-phenylamine (0.105 g, 1 mmol). The mixture was stirred for 2 h at 60 ℃, then a methanol solution (5 mL) of Cu(CH3COO)2·H2O (0.1996 g, 1 mmol) was added dropwise to it. After refluxing for 4 h, the resultant solution was cooled to room temperature and filtered. The filtration was then kept aside for 7 days over which time green crystals were deposited. Yield: 78%. M.p. 237-238 °C. Anal. Calc. for C38H28CuN2O4 (MW = 640.18): C, 71.29; H, 4.41; N, 4.37, Found: C, 71.25; H, 4.37; N, 4.38. IR (KBr, cm-1): 1615.9 (s), 1589.7 (m), 1532.7 (m), 1487.6 (m), 1232.8 (s), 1085.8 (s), 533.2 (w), 478.3 (w). ESI-MS: m/z 646.76. [CuL22 + Na]+. UV-Vis (MeOH), λmax/nm: 329, 436. 2.3. Physical measurements and instrumentation

Elemental analyses (C, H, N) were performed on Perkin-Elmer 2400 II analyzer. IR spectra (4000−400 cm−1) were recorded on a Nicolet Avatar Model FT-IR spectrophotometer. Melting points (M.p.) of the compounds were determined with a Lab India instrument. Electrospray ionization mass spectroscopic (ESI-MS) analyses are performed with a Bruker microTOF-Q mass spectrometer (Bruker Daltonics Inc., Billerica, MA), and the mass spectra are obtained in the positive mode. Electronic absorption spectra were recorded using a HP-8453A diode array spectrophotometer. Emission spectra were measured with LS55 spectrofluorometer. Circular dichroism (CD) spectra measurements were measured on a Jasco J-810 spectropolarimeter. 2.4. X-ray crystallography Diffraction data for the title compounds were obtained on a Bruker Smart 1000 CCD diffractometer (graphite monochromized Mo Kα radiation, λ = 0.71073 Å). All data were corrected using SADABS method and the final refinement was performed by full-matrix least-square methods with anisotropic thermal parameters for non-hydrogen atoms on F2 using SHELX-97 program. The hydrogen atoms were added theoretically, riding on the concerned atoms and refined with fixed thermal factors. Crystal data and experimental details of the structure determinations of the ligand HL1 and complexes 1-5 are listed in Table S1 and S2. 2.5. Cytotoxicity In

vitro

cytotoxicities

of

the

complexes

were

studied

using

standard

MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] bioassay in different cancer cells at 24 h of drug administration. Stock solutions of the studied compounds were prepared in dimethyl sulfoxide (DMSO, Sigma Aldrich) at concentrations of 10 mg/mL and diluted by cell culture medium to various working concentration. Human colon carcinoma cell lines (HCT-116) and human lung carcinoma cell line (A549) were cultured in 96-well culture plate in RPMI-1640 medium containing 10% FBS and 1% antibiotics, maintain culture at 37 ℃, 5% CO2 and 95% air in the CO2 incubator for 24 h. Various concentrations of prepared complexes were added to the cells and incubation were continued for 48 h. Then the media was removed, MTT was dissolved in medium and added to each well, and incubated for another 4 h. The purple formazan crystals were solubilized by the addition of 100 µL DMSO. A reading was taken on a plate reader, and the absorbance was measured at 570 nm by the ELISA reader after the plate was shaken for 5 min. The values are the averages from at least three independent experiments, which were measured as the percentage ratio of the absorbance of the treated cells to the untreated controls. The IC50 values were determined by non-linear regression analysis. 2.6. DNA binding studies All the DNA-binding experiments were carried out in buffer solutions containing 10 mM Tris, 50 mM NaCl and the pH was adjusted to 7.2 with hydrochloric acid. The absorption titration with CT-DNA was by keeping the

concentration of the complex constant while varying the DNA concentrations. Due correction was made for the absorbance of CT-DNA itself. The complex-DNA mixing solutions were incubated for 1 h before the absorption spectra were recorded. For fluorescence quenching experiments, EB solution was added to the prepared buffer solution of DNA (CT-DNA) for 2h, then added to methanol solution of the complex from 0 to 36 µM. All samples were excited at 258 nm and emission spectra were recorded at 500–700 nm. The spectrum was recorded at the scan speed of 200 nm/min and slit width of 10.0 nm for both the excitation and emission monochromators. CD spectra of CT-DNA were carried out on J- 810 spectropolarimeter at room temperature with a quartz cell of 1cm path length by increasing complex ratio in the buffer. Each sample solution was scanned in the range of 220– 320 nm with a scan speed of 100 nm/min and 1 s response time. Each spectrum was the average of three accumulations from which the buffer background had been subtracted. 2.7. Protein binding studies BSA-binding experiments were carried out in buffer solutions containing 10 mM Tris, 50 mM NaCl and the pH was adjusted to 7.2 with hydrochloric acid. The fluorescence emission spectra were carried out in the range of 290–430 nm with an excitation wavelength at 280 nm and the excitation and emission slits with a band pass of 5 nm were used at a scan speed of 200 nm/min for all the measurements. The synchronous fluorescence spectra were recorded in the range of 250-320 nm. The concentration of BSA was kept at 1 µM and the complex was varied from 0 to 14 µM. The UV-visible spectra were scanned in the range of 200–300 nm and the concentration of BSA was kept at 1 µM the complex was kept at 1 µM.

3. Results and discussion 3.1. Description of solid state structures Since complexes 1, 2 and 5 possess general formula [ML2] (M =Ni, Cu) and similar structure (four-coordinated square-planar geometry), except for their different metal center and ligand (HL1 or HL2), herein only the structure of 2 is discussed. As shown in Figure 1 (Figure S2 for complex 1 and Figure S3 for complex 5), the copper ion exhibits a distorted square-planar structure surrounded by the two O and two N atoms from the two bidentate Schiff base ligands. Selected bond lengths and bond angles for complexes 1, 2 and 5 are given in the Table S3, and the geometric parameters of L1 or L2 cation are comparable to those of Schiff base ligands (Figure S1 and Tabele S3). Such structrual characteristics was also observed in the nickel(II) [22] or copper(II) [23] complexes, which retains an approximate planarity and can behave like a metallointercalator to intercalate between the neighboring base pairs of DNA to give high cytotoxicity. Although with the same ligand, zinc(II) complex 3 exhibits a different coordination geometry (shown as Figure

2). The central Zn(II) ion is tetrahedrally coordinated by two deprotonated Schiff base ligands. The dihedral angles between two naphthanyl ring-ZnNO planes of about 80.125(38)° indicate an obvious distortion from an ideal tetrahedral geometry for complex 3 [24]. The N-Zn-N and O-Zn-O angles [121.59(17)° and 112.5118°] are typical for a tetrahedral geometry. The Zn-O bond lengths of 1.911(3) Å are obviously shorter than those documented in the literature, whereas the Zn-N bond lengths of 1.997(3) Å are slightly longer than the literature values [24,25]. The coordination of Mn(III) is to two Schiff base ligands binding in the equatorial mode and two N3¯ ions in axial positions (Figure 3). Each azide functions as a trans-µ-(1,3) bridge, resulting in a one-dimensional polymer (Figure 4). The chain structure belongs to a modification of the type I azide chain based on a classification by Escuer et al [26]. The unique feature of the present chain is that each monomeric unit is related to its adjacent ones by a 2-fold screw axis, leading to a zigzag propagating along the crystallographic c axis. The equatorial atoms Mn(1), N(1), N(1A), O(1), and O(1A) are nearly coplanar (rms deviation = 0.4902 Å). The two halves of the naphthanyl rings of two ligands, excluding the diphenyl ether groups, are also individualy parallel planar. The two Mn-O distances are equal and comparable to those in the mono-ligand polymeric compound, Mn(salpn)N3, where salpn is the dianion of N,N-bis(salicylidene)-1,3-diaminopropane [27]. However, at variance with the mono-ligand compound, the two Mn-N distances in 4 are also equal [2.033(4) Å], which is different from those of Mn(salpn)N3. As expected for the Jahn-Teller ion, the axial Mn-N(2) and Mn-N(2A) distances [2.266(4) Å] are considerably longer. 3.2. In vitro cytotoxicity assay In vitro cell culture studies are valuable tools for screening of chemotherapy agents and provide preliminary data for further relative studies [7]. The in vitro cytotoxicities of Schiff base ligand HL1 and its nickel(II), copper(II), zinc(II) and manganese(III) complexes 1-4, as well as complex 5 were evaluated by the MTT method against four typical human tumor cell lines (using cisplatin as the positive control) involving colon, lung, promyelocytic leukemia, and erythroleukemra cancers. The effects of the complexes on the viability of these cells evaluated after an exposure period of 48 h showed antitumour activity and their corresponding IC50 values, related to inhibition of tumour cell growth at the 50% level, are shown in Table 1. In addition, treatment of cancer cells with an IC50 concentration of the tested compound for different times (12, 24, 36, 48 h) resulted in gradually decrease in the percentage of cell viability (see Figure 5). These results reveal that compounds 3 and 4 display a time-dependent increase in the inhibition of the growth of cancer cell. Coordination of metal ions was essential for cytotoxicity of novel complexes. Indeed, IC50 values for the free ligand were much higer than the values for corresponding complexes, for in cytotoxicity not only does the coordination of metal ions play an important role but also for the nature of ring substituents. Among the complexes with the same ligand, complexes (3 and 4) contained zinc(II) or manganese(III) ions show the most active. Nickel(II) complex was

less active, which is related to its poor solubility in biological media. Two copper(II) complexes with naphthanyl or phenyl ring substituents present different cytotoxic activities. These results clearly suggested that the Schiff base ligand alone did not play a key role in the antitumor function, and the combination of Schiff base with metal produced appriable synergetic effect. In addition, for comparison purposes, the cytotoxicity of cisplatin was evaluated under the same experimental conditions, and the IC50 value was higher than 50 µM in all the cancer cell lines studied. As shown in Figure 6, after the tumor cells were incubated with 20.0 µg/mL test complexes for 48 h, each complex exhibited different inhibition effect against the four cell lines, which further showed various cytotoxicity of these Schiff base-metal complexes against the tested cell lines even though complexes 1, 2 and 5 possess similar structure (inner-sphere). Such phenonmenon is difficult to explain. In fact, except for platin-derivatives, there is relatively little mechanistic information on how metal antitumour drugs function, but it is clear that different metal ions can work through different routes that lead to different cellular responses [5]. 3.3. DNA binding studies Despite the presence of other biological targets in tumor cells, including RNA, enzyme, and protein, it is generally accepted that DNA is the primary target for many metal-based anticancer drugs such as cisplatin [5]. Thus, the mode and propensity for binding of complexes 1-5 to CT-DNA were studied with the help of electronic absorption, fluorescence and circular dichroism (CD) techniques. 3.3.1 UV-visible absorption studies The complexation of ligands to metal ion in solution was confirmed by ESI-MS. Electronic absorption spectroscopy is usually employed to determine the binding ability of metal complex with DNA [28,29]. Representative electronic absorption spectra of complex 5 in the absence and presence of CT-DNA is illustrated in Figure 7 (Spectra of complexes 1, 2, 3 and 4 are presented as Figure S4 in the ESI†). The absorption spectra of complexes 1-5 displayed two well-resolved bands in the range of 300 to 430 nm, out of which the high energy absorption bands appeared between 300–330nm is assigned to the intra-ligand charge transfer transitions (ILCT) of the type π→π* and n→π*. And the bands appeared around 430 nm are assigned to metal-to-ligand charge transfer (MLCT) transitions [30]. With the increasement of the concentration of CT-DNA, the absorption band of complexes 1 , 2, 3, 4 and 5 show hypochromicity also with the obvoius bathochromic shift suggesting that these complexes bind to DNA in a characteristic intercalative mode [31]. In order to compare the binding affinity of the complex with CT-DNA, the intrinsic binding constant (Kb) was calculated by the equation of [DNA]/(εa−εf)=[DNA]/(εb−εf)+1/Kb(εb−εf) [32], where [DNA] is the concentration of CT-DNA in base pairs, εa corresponds to the apparent extinction coefficient for the complex in the presence of CT-DNA, εf represents the extinction coefficient for the free complex and εb is the extinction coefficient of the fully

DNA-bound complex, respectively. The binding constant Kb is given as the ratio of the slope to the intercept, both of which are from the plot of –[DNA] / (εa−εf) versus [DNA]. The binding constant Kb for these complexes were 1.40×104, 1.78×104, 2.73×104, 1.03×104 and 6.4×103 M-1, corresponding to complexes 1–5, respectively. This binding constant value clearly showed that zinc(II) complex 3 bound more strongly with CT-DNA than other metal complexes with the same ligand through an intercalative mode, and the magnitude order is 3(Zn2+) > 2(Cu2+) > 1(Ni2+) > 4(Mn3+). But with the same metal ion, the planarity of the ligand plays an important role for their intercalation with CT-DNA, just as copper(II) complex 2 that possess a more planar naphthanyl ring shows bigger Kb value than complex 5 coordinated with phenyl ring Schiff base ligands. Just due to the bad planarity of HL2 than HL1, compound 5 displays the least binding affinity among these five compounds. This is consistent with the previous reports [10]. 3.3.2. Competitive binding between EB and complexes for CT DNA No fluorescence was observed for these Schiff base complexes, neither in buffer solution nor in the presence of CT-DNA. Therefore, ethidium bromide (EB) was used as a probe for DNA structure detection to study the binding mode of the complex with CT-DNA [33]. EB is a weak fluorescent compound. However, the luminescence of EB was significantly enhanced when intercalated into DNA. The interaction of the complex with DNA were evaluated by the EB–DNA adduct, which can be used to distinguish intercalating and nonintercalating compounds [34]. If EB was replaced by the metal complex, fluorescence intensity of EB–DNA system will be obviously quenched [35]. The fluorescence spectra of EB–DNA system in the absence and presence of the complex 3 is shown in Figure 8 (spectra of complexes 1, 2, 4 and 5 are presented as Figure S5 in the ESI†), the insert plots are the I0/I vs. r (C[complex]/C[DNA]). According to the classical Stern-Volmer equation [36]: I/I0 = 1 + Ksq·r the values of Ksq for complexes 1-5 are 1.33, 1.69, 2.98, 1.17 and 0.92, which are higher than that of some other reported tansition metal complexes (Ksq = 0.41 and 0.53) [37,38]. Also, the size of these values follow the order: 3 > 2 > 1 > 4 > 5, which is consistent with the UV-visible spectral titration results. And the studies show that these five complexes can quench the fluorescence of EB-DNA system through an intercalative mode. 3.2.3. Dichromism spectroscopy of interaction between complexes and CT DNA Circular dichroism (CD) has been widely used to examine the non-covalent DNA binding interactions of metal complexes, owing to its ability to provide binding interaction as well as information about nucleic acid conformation. The CD spectrum of DNA exhibits a positive peak at 273 nm because of base stacking and a negative peak at 246 nm due to right handed helicity, this is a characteristic of the B-form of CT-DNA [36,37]. The changes in the CD spectra can be attributed to the corresponding changes in the CT-DNA structure. The CD spectra show almost no change in

the case of groove binding and electrostatic binding, whereas the intercalative binding affects both the positive and negative bands, as observed for classical intercalators, such as ethidium bromide and methylene blue [39]. The observed CD spectra of CT-DNA in the absence and presence of complex 1 are shown in Figure 9 (respective spectra of complexes 2, 3, 4 and 5 are presented as Figure S6 in the ESI†). In the presence of every complex, both the positive (ca. 278 nm) and negative (ca. 246 nm) peaks increased in intensity, this behavior is characteristic of intercalation between complexes 1-5 and CT-DNA [40]. Further, we can see that the binding of the complexes 1-5 to CT-DNA lead to a significant change in the base stacking but can not unwind the DNA helix conformation. Based on the above analysis (UV-visible, fluorescence and circular dichroism spectra), we can draw the conclusion that all complexes could bind to CT-DNA in the classical intercalation mode due to the planarity of the chelating Schiff base ligand. Both the binding constant obtained by UV-vis and fluorescence phenomenon reveals the stronger DNA-binding for zinc(II) complex 3 than others, which is in accordance with their in vitro cytotoxicities. 3.4. Protein binding studies 3.4.1. Fluorescence quenching of BSA by metal complexes (1–5) Fluorescence spectroscopy is an effective method to qualitatively analyze the binding of complex with BSA. Generally, the fluorescence of a protein is caused by three intrinsic characteristics of the protein, namely tryptophan, tyrosine, and phenylalanine residues [40]. The quenchment of fluorescence is usually caused by the environmental alteration around the fluorophore and that can reveal the nature of BSA-binding reaction. The effect of complex 1 on BSA fluorescence intensity is shown in Figure 10 (for complexes 2, 3, 4 and 5, see Figure S7, ESI†). Fluorescence quenching process can be analyzed by the Stern–Volmer equation [41]: I0/I = 1 + Ksv[Q] = 1 + kqτ0[Q] where I0 and I are the fluorescence intensities of fluorophore at 347 nm in the absence and presence of the title complexes, respectively, Kq represents the apparent quenching rate constant of biomolecular fluorescence, τ0 is the average lifetime of the fluorophore, [Q] is the concentration of quencher and Ksv is the Stern–Volmer constant. The insets plot I0/I versus [Q] exhibits a good linear relationship with the linear correlation coefficient R = 0.998, 0.990, 0.989, 0.996 and 0.998. And the Ksv values obtained from the slope of the linear are 5.78×103, 5.87×103, 5.98×103, 5.28×103 and 6.09×103 M-1 for complexes 1-5. 3.4.2. UV-Vis absorption spectroscopy of BSA by metal complex 1 Generally, the fluorescence quenchment can be explained by various mechanisms, which are mainly classified as dynamic quenching and static quenching. The dynamic quenching refers to the process that the fluorophore and the quencher come into contact during the transient existence of the excited state, while the static quenching refers to the fluorophore and quencher forms a fluorophore-quencher ground state [40]. In order to differentiate the types of

quenching, representative UV-Vis absorption spectroscopy of BSA in the absence and presence of the complex 1 is shown in Figure 11 (for complexes 2, 3, 4 and 5, see Figure S8, ESI†). The weak absorption peak at 287 nm showed an increase in intensity in the absence of metal complex, which revealed that the fluorescence quenching mainly due to static quenching procedure by forming a complex-BSA ground state [42,43]. 3.4.3. Binding constant and number of binding sites For static quenching interaction, fluorescence intensity data can also be calculated using the equilibrium constant (Kb) and the number of binding sites (n) for the complex-BSA system by the following equation [44]: log ((I0-I)/I) =log Kb+nlog [Q] Kb and n can be calculated from the intercept and the slope in Figure 12 (log ((I0-I)/I) versus log [Q]) according to the above equation. The values of Kb were obtained to be 2.73×104, 2.95×104, 8.90×104, 1.28×104 and 1.66×104 M-1, and n was found to be 0.93, 0.94, 1.04, 0.86 and 0.88. This value of n is approximately equal to 1, suggests that there is only one binding site for the complex on BSA molecule. The results suggest that a strong binding ability of complex 3 compared to the other complexes under investigation. 3.4.4. Characteristics of synchronous fluorescence spectra Synchronous fluorescence spectrum provides information on the molecular microenvironment, particularly in the vicinity of the fluorophore functional groups. For BSA, the synchronous fluorescence ∆λ value is 15 nm which is the characteristic of the tyrosine residues, and a larger ∆λ value of 60 nm is the characteristic of the tryptophan residues [43,44]. In order to research the structural changes of the BSA in present of complex, synchronous fluorescence spectra with addition of complex were recorded at both ∆λ = 15 nm and 60 nm. The synchronous fluorescence spectroscopy of complex 2 was given in Figure 13 (for complexes 1, 3, 4 and 5, see Figures S9, S10, S11 and S12, ESI†). Upon increasing the concentration of the complexes 1, 2, 4 and 5 the fluorescence intensity of emission corresponding to tryptophan and the tyrosine decreased, accompanied with a bathochromic shift of emission wavelength. This indicated the complex affected both the micro-environments of tryptophan and tyrosine residues. What’s more, the polarity around the tryptophan and tyrosine residues is increased and the hydrophily is strengthened. However, a significant decrease of fluorescence intensity of tyrosine residues with a slight hypsochromic shift of emission wavelength was found after the addition of complex 3. This result suggested that the tyrosine residue was located in a more hydrophobic environment [45].

4. Conclusions Five transition-metal complexes containing double negative bidentate O,N chelating Schiff base ligands have been synthesized and characterized by single-crystal X-ray diffraction. The nickel complex 1 and copper complexes 2 and

5 show a slightly distorted square-planar geometry in the vicinity of the central metal atom, while the zinc(II) center in complex 3 exhibits a tetrahedral structure. But manganese complex 4 reveals a one-dimensional chain linked by azide in the end-to-end mode, and the Mn(III) ion present a six-coordinated octahedral geometry. The in vitro cytotoxicity of all complexes against four selected human tumor cell lines is different. In some cases, they exhibited significant enhanced antitumor to those Schiff base ligand. From the bio-inorganic chemistry point of view, binding interactions of the five new complexes with CT-DNA and BSA have been carried out. Binding experiments revealed that the type of metal and the planarity of the chelating ligands showed a pronounced effect on their binding ability with both DNA and BSA. From the results obtained in DNA/BSA interaction studies, it revealed that, the Zn(II) complex showed a more significant effect on their binding ability, which is consistent with its stronger cytotoxicity. Also, we can see that copper complexes 2 possessing a more planar naphthanyl unit instead of the phenyl ring available in complex 5 showed stronger binding. However, in contrast, complex 5 exhibited higher cytotoxicity agaist HCT116, HL-60 and K562 cell lines except for A549. So the exact molecular mechanism requires further detailed investigation.

Acknowledgements We acknowledge the financial support of the Natural Science Foundation of Shandong Province (Nos. ZR2013BM017), the Natural Science Foundation of China (No. 20971063 and 21105042).

Supplementary material †CCDC No. 1031356, 1031161, 1031162, 1031163, 1031164 and 1031165 for ligand HL1, complexes 1, 2, 3, 4 and 5. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Crystal and structure refinement data are shown in Table S1 and S2, selected bond lengths (Å) and angles (°) are shown in Table S3, molecular structure of ligand HL1, complex 1 and 5 are presented in Figures S1-3, UV–vis absorption spectra of complexes 1, 2, 3 and 4 are presented as Figure S4, the fluorescence spectra of EB–DNA system in the absence and presence complexes 1, 2, 4 and 5 are presented as Figure S5, CD spectra of CT-DNA in the absence and presence of complexes 2, 3, 4 and 5 are presented as Figure S6, the effect of complex complexes 2, 3, 4 and 5 are presented as Figure S7, UV–vis absorption spectra of BSA in the absence and presence of complexes 2, 3, 4 and 5 are presented as Figure S8, the synchronous fluorescence spectroscopy of complexes 2, 3, 4 and 5 are presented as Figures S9, S10, S11 and S12, respectively. See DOI: 10.1039/b000000x/

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Scheme Captions Scheme 1. Synthesis of complexes 1-5

Figure Captions Fig.1. Molecular structure of complex 2. Hydrogen atoms are omitted for clarity. Fig.2. Molecular structure of complex 3. Hydrogen atoms are omitted for clarity. Fig.3. Molecular structure of complex 4. Hydrogen atoms and dichloromethane solvent molecule are omitted for clarity. Fig.4. Perspective view of polymeric chain in complex 4. Hydrogen atoms and dichloromethane solvent molecule are omitted for clarity. Fig.5. Effects of complexes 3 and 4 [dose level of 20 µM] on the viability (%) of HCT-116 cell line at different times. Fig.6. Inhibition [%] of complexes 1, 2, 3, 4 and 5 [dose level of 20.0 µg/mL] against human tumor cells. Fig.7. UV–vis absorption spectra of complex 5 in the absence and presence of CT-DNA, [VOL] ＝10 µM, from 1 to 6, [DNA] ＝0, 2, 4, 6, 8 and 10 µM, respectively; Inset: plots of -[DNA]/(εa−εf) vs. [DNA]. Fig.8. Effects of complex 3 on the fluorescent spectra of EB-DNA system (λex= 258nm); CDNA= 30 µM; CEB= 3 µM; from 1 to 7 CVOL =0, 6, 12, 18, 24, 30, 36 µM respectively; Inset: plot of I0/I vs r ( r = CVOL/ CDNA). Fig.9. CD-spectra of CT-DNA in the absence and presence of the complex 1, [DNA] =100 µM, [VOL] = 0 and 40 µM, respectively. Fig.10. Fluorescence emission spectra of BSA in the absence and presence of the complex 1. [BSA] = 1 µM, [Complex] = 0, 2, 4, 6, 8, 10, 12, 14 µM, respectively; λex = 280 nm. (Inset: Plot of [Q] vs. I0/I). Fig.11. UV–vis absorption spectra of BSA in the absence and presence of the complex 1. [BSA] = 1 µM, [complex] = 0 and 1 µM. Fig.12. The plot of log[(I0-I)/I] vs. [Q]. Fig.13. Synchronous spectra of BSA as a function of concentration of the complex 1 with wavelength difference of ∆λ = 15 nm and ∆λ = 60 nm.

Table Captions Table 1. IC50 (µM) of all compounds against HCT-116, A549, HL-60 and K562 for 48 h treatment.

Table 1. IC50 (µM) of all compounds against HCT-116, A549, HL-60 and K562 for 48 h treatment. Cell lines

1

2

3

4

5

HL1

Cisplatin

HCT-116

46.18

15.55

19.57

15.90

13.31

>50

53

A549

>50

22.68

11.14

22.78

> 50

>50

>50

HL-60

>50

>50

8.35

14.26

12.39

>50

>50

K562

>50

>50

23.13

39.72

13.97

30.52

>50

Highlight 1. Five transition metal complexes were synthesized and characterized. 2. In vitro cytotoxicities of all compounds were tested against five cancer cell lines. 3. DNA/BSA interactions of all compounds were studied.