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(II) metallates containing nitrogen heterocycles
Accepted Manuscript Research paper Synthesis, spectroscopic studies and biological evaluations of copper(I)/(II) metallates containing nitrogen heterocycles C. Elamathi, A. Madankumar, Werner Kaminsky, R. Prabhakaran PII: DOI: Article Number: Reference:
S0020-1693(19)30478-5 https://doi.org/10.1016/j.ica.2019.119039 119039 ICA 119039
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
9 April 2019 23 July 2019 23 July 2019
Please cite this article as: C. Elamathi, A. Madankumar, W. Kaminsky, R. Prabhakaran, Synthesis, spectroscopic studies and biological evaluations of copper(I)/(II) metallates containing nitrogen heterocycles, Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.119039
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Synthesis, spectroscopic studies and biological evaluations of copper(I)/(II) metallates containing nitrogen heterocycles C. Elamathia, A. Madankumarb, Werner Kaminskyc and R. Prabhakarana,* aDepartment bCancer
of Chemistry, Bharathiar University, Coimbatore 641 046, India.
biology Lab, Molecular and Nanomedicine research unit, Sathyabama institute of Science and Technology, Chennai 600 119, India.
cDepartment
of Chemistry, University of Washington, Washington, 98195-1700.
Abstract Nitrogen heterocyclic copper(I) complex (1) and copper(II) complex (2) were synthesized and characterized by various physicochemical methods. Single crystal X-ray diffraction analysis revealed the true coordination nature of ligands to the metal centre. The intercalative mode of binding between the compounds and nucleic acid was further confirmed by viscosity measurements. The interaction of complexes with serum albumin was studied by absorption and emission titration methods. The scavenging ability of the compounds against hydroxyl and DPPH radicals conveyed that the complexes have a better ability than their parent ligands. An in vitro anti cell-proliferation of the compounds revealed significant activity against human breast cancer cells (MCF-7) and found non-toxic against human normal keratinocyte cells (HaCaT). AO/EB dual staining assay of the cancer cells were pictured as the apoptotic pathway of cell death which is induced by the complexes. The result of our study found that complex 1 exhibited better activity than ligands (H2L1 and H2L2) and complex 2. Keywords: Heterocyclic copper(I)/(II) complexes, binding interactions, radical scavenging, cell-proliferation, AO/EB staining.
Corresponding author Tel.: +91-422-2428319; Fax: +91-422-2422387. E-mail address: [email protected] (R. Prabhakaran)
Introduction Thiosemicarbazones play a main role in the synthesis of certain biologically active compounds and hence inorganic and bioinorganic chemists have shown considerable interest in thiosemicarbazones and their ligands [1-8]. The structural and biological properties of thiosemicarbazones obtained from substituted N-heterocyclic quinoline have made the compound a centre of interest to the researchers [9-12]. The theoretical and practical significance of transition metal complexes including catalytic, photochromic properties and biological activity has made them attractive as well [13-15]. Quinolines are reacted with thiosemicarbazones to prepare versatile tridentate Schiff base ligands, which might bring promising biological function into play [16]. The copper(II) complexes of thiosemicarbazone ligands serve as an excellent substitute for fluorescent imaging agents [17,18]. Copper(II) complexes are easily available, generally less cytotoxic to cells and are conveniently monitored as well [19-21]. The adaptable coordinating behaviour of thiosemicarbazone ligands and the structural flexibility of copper(II) ions lead to the formation of a variety of copper (thiosemicarbazone) complexes with different coordination numbers and geometries [22-27]. In continuation with our earlier work, herein the current article deals with the chelating properties of the N-heterocyclic thiosemicarbazones H2L1 and H2L2 in order to improve our understanding of the behaviour of these ligand systems. To further investigate the substituent effects [28], we have synthesized and characterized some nitrogen based heterocyclic copper(II) complexes. In view of their possible biological properties, we have tested the ligands and complexes for their in vitro antiproliferative activity on human breast cancer cells (MCF 7) at five different concentrations and their non-toxic nature of these compounds were investigated by human keratinocyte cells (HaCaT). Results and discussion An equimolar reaction between CuCl2.2H2O and the ligands (H2L1 and H2L2) in a stoichiometric ratio resulted in the formation of new complexes as shown in Scheme 1. The new compounds are stable to air and moisture and soluble in ethanol, methanol, DMF, DMSO and water (complexes).
H N
O H 2N N H
H N
R
S MeOH, Reflux, 1hr
O
N H CuCl2.2H2O
Cu O O S HC
HC3
N
S O Cu N
CH3 H2N
O
N
H N
R
S R= H & Me
H H N N CH3 Cl Cu S O .H2O Cl N
CH O
H N
MeOH, Reflux, 1hr
H 3C CH3 NH2 N
N N N H
N
H N
N H
(2)
(1)
Scheme 1. Synthesis of heterocyclic copper(I)/(II) complexes Infrared Spectroscopy Vibrational spectroscopy has been used to assign the primary confirmation of the coordination mode of the ligands to copper ion. The IR spectrum of the ligands exhibited bands in the regions at 1650 and 1653 cm-1 was assigned for oxo (C=O) stretching vibration and this shifted to lower frequencies (1633 and 1631 cm-1) in complexes (1 and 2) indicating the bonding of oxo group to copper metal ion [29]. The vibrational frequency corresponding to the azomethine group was found at 1549 and 1561 cm-1 in the ligands and this was shifted to 1541 and 1543 cm-1 for complexes indicating the coordination of azomethine group to metal [30]. Further, a strong band appeared at 835 and 848 cm-1 in the spectra of ligands (H2L1 and H2L2) indicating the presence of thione group [31], and this band appeared at 830 cm-1 in complex 2 revealed the thione coordination to the metal ion. However, in complex 1 the band appeared at 722 cm-1 establishing thiol coordination of ligand after enolization and subsequent protonation on thiolate sulphur [32,33]. Absorption spectroscopy The electronic absorption spectra of all the ligands and complexes were recorded in DMSO at 10 μM concentration. The ligands exhibited two bands; one band at 256 nm and 264 nm assigned to л→л* transition. The other band observed at 354 and 359 nm corresponding to n→л* transitions [34]. In the copper complexes (1 and 2), two bands appeared in the regions (254 and 270 nm) and (375 and 380 nm) respectively, which has been assigned to intra ligand transition and ligand to metal charge transfer transition (LMCT) [35,36].
Stability of the Schiff base ligands and complexes Stability of the ligands and complexes under physiological conditions were studied by spectrophotometric measurements for 24 h time interval. The UV–Vis spectrum of the Schiff base solution recorded in the range of 200–800 nm was quite stable for proving the stability of the solution about 24 h Fig. S1. Similar experiments were carried out to check the stability of the complexes and it was found that they were stable even after 24 h in water, DMSO, Tris-HCl (pH-7.2) and phosphate buffer (pH-7.2). Proton NMR spectroscopy The 1H-NMR spectra of the ligands H2L1&2 have been recorded in DMSO-d6 as shown in Fig S2-S4. The ligands exhibited a singlet at δ 11.71 and 11.92 ppm for N(1) proton [37]. Whereas, another singlet observed at δ 10.56 and 11.20 ppm analogous to quinoline imine N(3)H protons respectively [37]. The ligand H2L1 displayed a singlet at δ 8.53 ppm corresponding to azomethine (HC=N) proton. While a doublet at δ 8.60-8.61 ppm was assigned to azomethine proton of the ligand H2L2 [32,38]. A singlet appeared at δ 8.69 ppm in the ligand H2L2 was assigned to terminal N(4)H protons. However, a doublet appeared at δ 7.40–7.42 ppm analogous to terminal NH2 protons in the ligand H2L1 [38]. A sharp singlet was found at δ 8.12 ppm and 8.60 ppm corresponding to C(4)H protons of the ligands (H2L1 and H2L2) [28]. In addition, the ligands (H2L1 and H2L2) exhibited a doublet at δ 7.77-7.79 ppm and δ 7.54-7.56 ppm for C(5)H protons. Further, the additional doublet was found at δ 7.39-7.40 ppm range was attributed to C(6)H proton in ligand H2L2. While in ligand H2L1, a triplet was observed at δ 7.50-7.54 ppm corresponding to C(6)H proton. For C8(H) proton, a triplet at δ 7.17-7.20 ppm for H2L2 and a multiplet at δ 7.14-7.21 ppm for H2L1 were observed. A doublet was observed at δ 3.06-3.07 ppm corresponding to terminal methyl protons of the ligand H2L2 [39]. A sharp singlet was observed at δ 2.46 ppm in the ligands H2L2 were assigned to methyl protons of quinoline ring at C(7) position. Whereas, in H2L1 C(7) proton was observed as doublet at δ 2.47-2.49 ppm [40]. The new copper(II) complexes were found to be NMR inactive. The paramagnetic nature of copper(II) complexes was confirmed by EPR spectroscopy. X-band EPR spectra have been recorded for complex 2 in the solid state at room temperature, Fig. 1 showed a single isotropic peak. The ‘g’ value of the complex has been found to be 2.0731, and this value is in good agreement with the previously reported square pyramidal Cu(II) complex [41].
Fig. 1. EPR spectra for complex 2. X-ray crystallography study Crystal structure of complex 1 with atom numbering scheme is shown in Fig. 2, and refinement data are shown in Table. 1. The molecule is crystallized in P-1 space group with a triclinic crystal system. The asymmetric unit is formed by one half of the molecule and the other half is linked by a centre of inversion in the Cu(1)–S(1)–Cu(1)–S(1)#1 ring (Fig. 2. And Table. S1). The coordination geometry at each Cu(I) centre is square pyramidal. A tridentate copper(I) complex was formed by oxo oxygen (O1), azomethine nitrogen (N2) and thiolate sulphur (S1) atom to form a basal plane along with one oxygen atom from the solvent molecule of dimethylformamide. The two distorted square pyramids were formed by a thiolate sulphur atom in the axial position, with same bite angle (N(2)-Cu-S(1) and N(2)-Cu-S(1) #1), (85.82(8)°). The non-bonded Cu-Cu distance has been found to be 3.874 Å. The trans angles (N(2)-Cu(1)-O(2) and O(1)-Cu(1)-S(1)) were found to be 172.92(9)° and 173.76(7)° respectively, showing considerable deviation from 180° and significant distortion from the square pyramidal geometry [28]. The τ value of 0.014 at each metal centres exposed the slight distortion from the perfect square pyramidal geometry [41]. The molecular packing diagram is shown in Fig. S5. The observed bond angle and bond length values were compared to the already reported values [28]. The molecular structure of complex 2 is shown in Fig. 3. In this complex, copper ion is coordinated to O, N, S donor atoms of the thio ligand and two chloride ions, (axial Cl2 and equatorial Cl1). The O(1), N(2), S(1) and Cl1(equatorial) atoms occupy a square plane; whereas, the Cl2 ion occupies the axial site. The complex is crystallized as a monoclinic
Table 1. Structure refinement for complexes 1 and 2. Compounds
[Cu(μ-S-H-7MOQtscH)DMF]2 (1) 1906212 C30H36Cu2N10O4S2 791.89 100(2) 0.71073 Triclinic P -1 9.4019(9) 9.5456(8) 10.7981(10) 88.951(6) 85.826(6) 87.406(6) 965.42(15)
[Cu(7MOQtscMe)Cl2].H2O (2) 1906211 C13H16 Cl2CuN4O2S 426.80 100(2) 0.71073 Monoclinic P 21/n 13.6839(12) 9.4180(10) 13.9879(15) 90 113.055(5) 90 1658.73(3)
1 1.362
1 1.709
co-efficient 1.255
1.778
CCDC number Empirical formula Formula weight Temperature(K) Wavelength(Å) Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) Volume (Å3) Z Dx (Mg/m3) Absorption (mm-1) F(000) Crystal size (mm3) Theta range for collection (°) Index ranges
408 0.070 x 0.050 x 0.020 data 1.891 to 26.421
868 0.100 x 0.070 x 0.030 1.765 to 26.493
-11<=h<=11, -11<=k<=11, -13<=l<=13 Reflection collected 7605 Independent reflections 3924 [R(int) = 0.0515] Completeness of theta = 99.8 25.500° (%) Refinement method Full-matrix least-squares on F2
-17<=h<=17, -11<=k<=11, -17<=l<=17 6520 3393 [R(int) = 0.0579] 99.4
Data/restraints/parameters Goodness –of-fit on F2
3393 / 3 / 225 1.039
3924 / 0 / 221 0.952
Final R indices R1 = 0.0439, wR2 = 0.1063 [I>2sigma(I)] R indices (all data) R1 = 0.0725, wR2 = 0.1183 Largest diff. peak and hole 0.520 and -0.549 e.Å-3
Full-matrix least-squares on F2
R1 = 0.0450, wR2 = 0.0978 R1 = 0.0727, wR2 = 0.1109 0.660 and -0.630 e.Å-3
1
Fig. 2. ORTEP of the complex [Cu(μ-S-H-7MOQtsc-H)DMF]2 (1) with thermal ellipsoids at the 50% probability level. crystal system with a space group of P 21/n. The axial Cu-Cl bond length (Cl(2)-2.6382(12)) is longer than the equatorial chloride ion bond length (Cl(1)-2.2558(11)) which was described as Jahn-Teller distortion [41]. The trans bond angles in the square plane namely, N(2)-Cu(1)-Cl(1) 165.17(10) and O(1)-Cu(1)-S(1) 169.37(9) as well as the cis bond angles in the range of 85.82-92.50° and this revealed a significant distortion in a square pyramid geometry [28]. Further, the distortion value of a coordination polyhedron, τ = (β-α)/60, is calculated by the two largest bond angles in five coordination geometry (τ = 1 for ideal trigonal bipyramidal and 0 for square pyramidal environment) [41,42], and this value (τ) is equal to 0.07 for complex 2. The geometry of the complex was found to be distorted square pyramidal. Four different types of hydrogen bonds were found in complex 2, molecular packing and hydrogen bonding distances are given in Fig. S6-S7 and Table S1. An intramolecular hydrogen bonding between the hydrogen atoms of quinoline nitrogen N(1) to a basal chloride ion. An intermolecular hydrogen bonding was found between imine nitrogen N(3) and the oxygen atom of a water molecule, which is in the lattice unit. The second intermolecular hydrogen bond is formed between the hydrogen atom of a water molecule H1(W) and basal chloride ion in third structure. The third intermolecular hydrogen bond was formed between
the hydrogen atom of a water molecule H2(W) and axial chloride ion in third structure. There are three intermolecular and one intra molecular hydrogen bonding was noted in this complex to form a2D network.
2
Fig. 3. ORTEP diagram for complex [Cu(7MOQtsc-Me)Cl2].H2O (2) with thermal ellipsoids at the 50% probability level (Lattice water molecule is omitted in the above picture for clarity). Nucleic acid interactions DNA binding studies were performed using electronic absorption spectroscopy. In order to get the binding constants for ligands (H2L1&2) and complexes (1-2), electronic absorption titration experiments were performed with incremental amounts of calf-thymus DNA (CT-DNA) in 5 mM Tris–HCl buffer (pH 7.2) and by keeping the constant concentration of the individual ligands and complexes Fig. 4 and S8 [28,41]. The ligands (H2L1 and H2L2) exhibited two bands in the regions at 254-256 nm and 353-359 nm respectively. The first absorption band observed at 256 nm and 254 nm with the redshift of 2 nm and 3 nm respectively. However, the second absorption band at 353 nm and 359 nm with the redshift of 2 nm and 4 nm respectively. Whereas, in the complexes (1 and 2) two bands were observed in the regions at 253-255 nm and 381-384 nm with the redshift of 10 nm, 6 nm and 2 nm respectively. The л* orbital couples with the л orbital of the drug molecules in the nucleic acid base pairs, thus the л-л* transition probabilities diminished, and hence a hypochromism was observed, which designated that all the compounds bind to DNA via intercalative mode [28,41-45]. According to Wolfe-Shimer equation [46], a plot of [DNA] versus [DNA]/[εb_εf] gave a slope and an intercept equal to 1/[εa_εf] and (1/Kb) [εb_εf], respectively, Fig. 4. Inset and S8 Inset. The Kb values of the ligands and complexes are listed in Table. 2. From the Kb values, complexes are significantly high (of the order 105 M-1) showing that the complexes are tightly bound with CT-DNA and higher than that of the
classical interacalator EB (Kb = 1.23 (±0.07) x 105 M-1) [47]. Concerning the ligands and complexes, the Kb constants increase in the order 1 > 2 > H2L1 > H2L2. While in the case of complexes, binuclear copper(I) complex and found to be the order of 105, which was reported by stylidou et al [48]. However, the mononuclear copper(II) complex exhibited slightly less binding ability when compared to copper(I) complex and found to be 105, this value was compared to the previously reported literature [41,44,45,46,47].
Fig. 4. Electronic absorption spectra of the complexes (10 μM) in the absence and presence of gradual addition of the increasing concentration of nucleic acid (5-50 μM) at room temperature in 5 mM Tris-HCl/50 mM NaCl buffer (pH = 7.2). Ethidium bromide competition assay The binding of drug molecules with EB bound nucleic acid may give detailed information about the DNA binding affinity of the complexes. Fig. 5 and S9 showed the fluorescence spectra of DNA-EB system with increasing amounts of the compounds. The emission intensity of DNA-EB system decreased when the concentration of ligands and complexes were increased [49]. Further, the fluorescence quenching is described by the Stern-Volmer equation [41,50]. The slope of the linear plot of Io/I versus [Q] gave Ksv as shown in Fig. 5 Inset and S9 Inset. Singh et al. have reported the metallo intercalators and the values are found to be 10x105 [44-46]. The quenching constant (Ksv) values follow the same order as discussed in the absorption titrations and values are listed in Table. 2 and S2.
Fig. 5. Emission spectra of EB (5 μM) bound to DNA (5 μM) in the absence and presence of an incremental concentration of the complexes (10-100 μM) using Tris-HCl/50 mM NaCl buffer (pH = 7.2). Table. 2. The Kb (binding constant) and Ksv (Quenching constant) values for the interactions of the complexes (1-2) with CT-DNA. Compounds
Kb/M-1
Ksv/M-1
Complex 1
3.79 × 105±0.14
3.11 × 105±0.19
Complex 2
2.48 × 105±0.18
2.56 × 105±0.10
In most cases, the observed changes in the UV/emission spectra of the compounds are not as pronounced as lead to a clear conclusion regarding the possible DNA-interaction mode of the complexes. Subsequently, in order to clarify the DNA-binding mode of the complexes, DNA-viscosity measurements were carried out. Viscosity measurements In order to further confirm the mode of binding of the ligands and complexes to CT-DNA, viscosity measurements of DNA solutions were carried out in the presence and absence of the compounds. The viscosity of DNA is sensitive to changes in its length and is regarded as the least ambiguous and the most critical clues of a DNA binding mode in solution [49]. In general, intercalating agents are expected to elongate the double helix to accommodate the complexes between the base pairs, leading to an increase in the viscosity of DNA [49]. The effects of the complexes 1-2 on the viscosities of the CT-DNA solution are shown in Fig. 6, with increasing [complex]/[DNA] concentration, resulted in a significant change in the relative viscosity of CT-DNA solution was observed with intercalative binding
mode of complexes [44,45,51-53]. The relative viscosity of DNA increases steadily in the order of 2 > 1 > H2L1 > H2L2.
Fig. 6. Effect of the ligands and complexes on the viscosity of CT DNA. Protein interaction studies (BSA) UV-Vis spectral study The fluorescence quenching mechanisms are usually classified as either static or dynamic quenching. Static quenching usually resulted from the formation of a complex between quencher and fluorophore in the ground state, whereas in dynamic quenching the fluorophore and quencher get in touch with each other during the transient existence of the excited state [41]. One can have an idea about the type of quenching from UV-Vis absorption spectral studies. UV-Vis spectra of BSA in presence of compounds displayed (Fig. S10) an increase in absorption intensity of the BSA suggesting the static type of interaction due to the formation of ground state complex as reported earlier [41]. A constant concentration of BSA with increasing concentration of the compounds, fluorescence emission intensity falls gradually for ligands and complexes Fig. 7 and S11. The interaction which might take place between the compounds and BSA [41,44,45,54,55].
Fig. 7. Fluorescence titration spectra of BSA (10 μM) with increasing amounts of complexes (10-100 μM), at pH = 7.2. In order to express their quenching capacity between the drug molecules and BSA, the Stern–Volmer quenching plot was obtained by detecting the fluorescence quenching of BSA and drug molecules. According to the Stern–Volmer equation [56,57], Ksv (Stern-Volmer constant) was determined from the plot of Io/I vs [Q], Fig. 7 Inset and S11 Inset and the values are listed in Table 3 and S3. Moreover, the binding constant (Kb) have been determined by using the equation at room temperature log((Fo−F)/F) = ln(Kb) + n log [Q], where [Q] is the concentration of quencher that is the synthesized compounds here [58]. “Kb” is obtained from the plot of log((Fo−F)/F) versus log[Q] as a y-intercept, Fig. 8 and S12. Furthermore, “n” which is the number of the binding site per protein [59]. The value of “n” is nearly 1, representing the synthesized complexes bind to BSA with a molar ratio of 1: 1. The calculated results are shown in Table 3 and S3. Concerning that the binuclear copper(I) complex exhibited the significant interaction ability with bovine serum albumin and this value was similar to the previous literature, which was reported by stylidou et al [48]. Elamathi et al. reported the copper(II) complexes containing heterocyclic Schiff base ligands, and the values are found to be 10 X105 [41,44,45,59].
Fig. 8. Scat Chard plots of complexes with BSA. Table. 3. Quenching constant (Ksv), binding constant (Kbin) and a number of binding sites (n) for the interactions of complexes with BSA. Compounds
Ksv/M-1
Kb/M-1
n
Complex 1
3.17 × 105±0.17
7.93 × 105±0.16
0.92
Complex 2
1.29 × 105±0.22
4.61 × 105±0.18
1.13
Tryptophan fluorescence spectra of serum albumins To increase the concentration of the test compounds (10–100 μM) added to BSA (10 μM), the fluorescence intensity of tryptophan residue was reduced Fig. S13. The above results concluded that the microenvironment around tryptophan residue was altered by the addition of compounds [28,41]. Evaluation of radical scavenging ability Appreciable results obtained from the biomolecular interaction of the complexes, it was encouraged us to study the antioxidant activities of the ligands and complexes. Hence, the study on antioxidant properties of the complexes was carried out [60,61]. The antioxidant activities of the new complexes along with the standard, butylated hydroxytoluene (BHT) have been inspected with hydroxyl radicals, DPPH radicals and the IC50 values are given in Table. 4. The IC50 values indicated the compounds showed marked antioxidant activity in the order of 1 > 2 > H2L1 > H2L2 in both the experiments. The DPPH radical scavenging power of the compounds was the maximum and hydroxyl radical scavenging was the minimum, as shown in Fig. 9 and S14. These results are considerably better than that observed for standard antioxidant BHT. According to our obtained results, it can be concluded that the scavenging
effects of the free ligand are significantly less compared to their corresponding Cu(I) [62] and Cu(II) complex.[28] From the obtained IC50 values, copper(I) complex 1 exhibited better activity than the copper(II) complex 2. The highest activity of complex 1 may be due to copper(I) complex.
Fig. 9. Hydroxyl (OH) and 2-2’-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity at various concentrations of the complexes and standard butylatedhydroxytoluene (BHT). Table. 4. The Radical scavenging activity of the ligands and complexes. Compounds H2L1 H2L2 Complex 1 Complex 2 BHT
IC50 values in (μΜ) Hydroxyl radical 187.54±2.64 290.41±1.13 18.11±1.14 20.45±1.53 313.52±1.23
DPPH radical 63.72±1.13 71.16±0.69 4.92±0.79 7.46±1.16 10.88±1.67
Anticancer activity in vitro Cancer cell growth inhibition The positive results obtained from the previous biological studies for the N-heterocyclic Schiff bases and their copper(I)/(II) complexes cheered us to test their cytotoxicity against human breast cancer cell line (MCF7) and one normal human keratinocyte (HaCaT) cells with cisplatin as a standard drug. The complexes were dissolved in DMSO and blank samples having the same volume of DMSO are taken as a control to find the activity of the solvent in this cytotoxicity experiment. The results were examined by means of cell inhibition expressed as (50% activity) IC50 values Fig. 10. It is to be noted that the ligands (H2L1-2) displayed significant activity at low concentration on cancer cells. The
copper complexes showed better activity as compared to the ligands and standard drug molecule. The results of in vitro cytotoxic activity studies have shown that the IC50 value of the ligands and complexes against HaCaT human keratinocyte (normal cells) is found to be above 100 μΜ, which confirmed that the compounds are very specifically active on cancer cells Fig. 10. The highest cytotoxic properties of copper(I) complexes over the ligands and copper(II) complex [38,41,44,45,62,63].
Fig. 10. The ligands (H2L1&2) copper(I)/(II) complexes (1 and 2) inhibit MCF-7 cells and HaCaT cells proliferation in a dose-dependent manner. Both cells were treated with dissimilar concentrations of compounds for 24 h, the cell viability was determined and the results were expressed as a percentage of cell viability with control. Results shown are mean, which are three separate experiments performed in triplicate. The anticancer activity of the compounds on MCF-7 cells follows the order: Cisplatin (21.58±0.14) μM < Complex 2 (13.58±0.52) μM < Complex 1 (12.88±0.31) μM for MCF-7 cells respectively. Acridine orange/ethidium bromide staining assay Apoptosis studies were done by using dual staining method like acridine orange (AO) and ethidium bromide (EB) to find the changes in membrane integrity between apoptosis and necrosis [64,65,66]. Ligands did not show significant activity, complexes exhibited significant activity. The apoptotic study for AO/EB staining assay was carried out for complexes (1 and 2). AO can pass through the cell membrane of both living and apoptotic cells, while the cells were stained by EB designate the loss of membrane integrity. Visualization under a fluorescence microscope, living cells appear as green, necrotic cells stained as a red and apoptosis cells appear as greenish yellow or orange colour morphological changes were observed. From Fig. 11, DMSO (MCF-7) cells have both green and light orange fluorescence, while the control (standard drug) (MCF-7) cells have a normal
morphology showed a bright green fluorescence. However, the MCF-7 cells treated with IC50 concentration of complexes 1 and 2 revealed morphological changes as compared to the positive (standard drug) and negative (DMSO) controls, and were characterised as early apoptotic (greenish yellow fluorescence), and late apoptotic with condensed and fragmented nuclei (orange fluorescence). This revealed that complexes 1 and 2 induced cell death in MCF-7 cells [64-67]. A similar type of thiosemicarbazone containing copper complexes were tested against human epidermoid carcinoma cells (A431) was reported in our earlier literature [41], results strongly suggested that induction of apoptotic pathways for the anticancer activity of these complexes maybe necrosis. However, in this case, the complexes were treated with human breast cancer cells and this showed the induction of apoptotic pathway for the activity of the complexes may be apoptosis. DMSO
Control
Complex 1
Complex 2
Fig. 11. Fluorescence images of acridine orange/ethidium bromide stained MCF-7 cells were treated with IC50 concentration of complexes for 24 h. Conclusion The new heterocyclic copper(I)/(II) complexes derived from nitrogen heterocyclic Schiff bases have been synthesized and characterized by various analytical and spectroscopic techniques. The solid-state structures of complexes (1 and 2) were confirmed by single crystal X-ray analysis. In complex 1 ligand coordinated to metal through oxo oxygen, azomethine nitrogen and thiolate sulphur to form a binuclear nature of distorted square pyramidal copper(I) complex. While complex 2, ligand coordinated to the metal via ONS fashion to form a mononuclear copper(II) complex having distorted square pyramidal geometry. The DNA binding properties of the ligands and their corresponding copper complexes were investigated by absorption and fluorescence measurements. The results supported the fact that the compounds interactively bound with the CT-DNA. Fluorescence quenching experiments with BSA confirmed the static quenching mechanism may be followed when fluorophore interacts with quencher. The radical scavenging power of the compounds towards hydroxyl and DPPH radical exposed that the complexes have better
scavenging ability as compared to ligands and the butylated hydroxy toluene (BHT). The in vitro cytotoxicity of the compounds was evaluated against the MCF-7 (human breast cancer) cell lines by comparing with cisplatin, the complexes exhibited significant cytotoxicity. Morphological analysis was determined by fluorescence microscopes using AO/EB staining assay showed that the induction of apoptotic pathway for the anticancer activity of these complexes may be apoptosis. There is a correlation between the DNA/protein binding and cytotoxicity of the complexes proving that complexes can be better candidates as anticancer drugs. According to our obtained biological results, the order of binding capacity of the compounds were directly proportionately to the radical scavenging and cytotoxicity of the compounds and the order is 1 > 2 > H2L1 > H2L2. This order showed complex 1 has better activity compared to the ligand and corresponding copper(II) complex. The higher activity of complex 1 may be due to the presence of the two active metal canters in it. Acknowledgement The author C. E gratefully acknowledges to the Department of Science and Technology (DST) and Ministry of Science and Technology, New Delhi, INDIA for an INSPIRE Fellowship, New Delhi, India [IF150603 dated. [10.04.2015]. Supplementary data Crystallographic
data
for
[Cu(μ-S-H-7MOQtsc-H)DMF]2
(1)
and
[Cu(7MOQtsc-
Me)Cl2].H2O (2) have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication (CCDC No.1906212 and 1906211). The data can be obtained free of
charge
at
www.ccdc.cam.ac.uk/conts/retrieving.html
Crystallographic Data Centre.
or
from
the
Cambridge
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A new water soluble N-heterocyclic copper(I)/(II) complexes were synthesized and characterized. Further, new complexes were subjected to binding interactions, antioxidant and anti-proliferation. Among this, copper(I) complex 1 showed better activity compare to copper(II) complex and their ligands.
Highlights
Water soluble Cu(I)/(II) complexes containing N-heterocyclic Schiff bases.
Ligands coordinated to Cu(II) complexes coordinated through ONS- and ONS chelation.
Biomolecular interactions and cell-proliferation were carried out for all the compounds.
MCF-7 staining pictured the most of the cell death via apoptotic and seems to be orange fluorescence.
S0020-1693(19)30478-5 https://doi.org/10.1016/j.ica.2019.119039 119039 ICA 119039
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
9 April 2019 23 July 2019 23 July 2019
Please cite this article as: C. Elamathi, A. Madankumar, W. Kaminsky, R. Prabhakaran, Synthesis, spectroscopic studies and biological evaluations of copper(I)/(II) metallates containing nitrogen heterocycles, Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.119039
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Synthesis, spectroscopic studies and biological evaluations of copper(I)/(II) metallates containing nitrogen heterocycles C. Elamathia, A. Madankumarb, Werner Kaminskyc and R. Prabhakarana,* aDepartment bCancer
of Chemistry, Bharathiar University, Coimbatore 641 046, India.
biology Lab, Molecular and Nanomedicine research unit, Sathyabama institute of Science and Technology, Chennai 600 119, India.
cDepartment
of Chemistry, University of Washington, Washington, 98195-1700.
Abstract Nitrogen heterocyclic copper(I) complex (1) and copper(II) complex (2) were synthesized and characterized by various physicochemical methods. Single crystal X-ray diffraction analysis revealed the true coordination nature of ligands to the metal centre. The intercalative mode of binding between the compounds and nucleic acid was further confirmed by viscosity measurements. The interaction of complexes with serum albumin was studied by absorption and emission titration methods. The scavenging ability of the compounds against hydroxyl and DPPH radicals conveyed that the complexes have a better ability than their parent ligands. An in vitro anti cell-proliferation of the compounds revealed significant activity against human breast cancer cells (MCF-7) and found non-toxic against human normal keratinocyte cells (HaCaT). AO/EB dual staining assay of the cancer cells were pictured as the apoptotic pathway of cell death which is induced by the complexes. The result of our study found that complex 1 exhibited better activity than ligands (H2L1 and H2L2) and complex 2. Keywords: Heterocyclic copper(I)/(II) complexes, binding interactions, radical scavenging, cell-proliferation, AO/EB staining.
Corresponding author Tel.: +91-422-2428319; Fax: +91-422-2422387. E-mail address: [email protected] (R. Prabhakaran)
Introduction Thiosemicarbazones play a main role in the synthesis of certain biologically active compounds and hence inorganic and bioinorganic chemists have shown considerable interest in thiosemicarbazones and their ligands [1-8]. The structural and biological properties of thiosemicarbazones obtained from substituted N-heterocyclic quinoline have made the compound a centre of interest to the researchers [9-12]. The theoretical and practical significance of transition metal complexes including catalytic, photochromic properties and biological activity has made them attractive as well [13-15]. Quinolines are reacted with thiosemicarbazones to prepare versatile tridentate Schiff base ligands, which might bring promising biological function into play [16]. The copper(II) complexes of thiosemicarbazone ligands serve as an excellent substitute for fluorescent imaging agents [17,18]. Copper(II) complexes are easily available, generally less cytotoxic to cells and are conveniently monitored as well [19-21]. The adaptable coordinating behaviour of thiosemicarbazone ligands and the structural flexibility of copper(II) ions lead to the formation of a variety of copper (thiosemicarbazone) complexes with different coordination numbers and geometries [22-27]. In continuation with our earlier work, herein the current article deals with the chelating properties of the N-heterocyclic thiosemicarbazones H2L1 and H2L2 in order to improve our understanding of the behaviour of these ligand systems. To further investigate the substituent effects [28], we have synthesized and characterized some nitrogen based heterocyclic copper(II) complexes. In view of their possible biological properties, we have tested the ligands and complexes for their in vitro antiproliferative activity on human breast cancer cells (MCF 7) at five different concentrations and their non-toxic nature of these compounds were investigated by human keratinocyte cells (HaCaT). Results and discussion An equimolar reaction between CuCl2.2H2O and the ligands (H2L1 and H2L2) in a stoichiometric ratio resulted in the formation of new complexes as shown in Scheme 1. The new compounds are stable to air and moisture and soluble in ethanol, methanol, DMF, DMSO and water (complexes).
H N
O H 2N N H
H N
R
S MeOH, Reflux, 1hr
O
N H CuCl2.2H2O
Cu O O S HC
HC3
N
S O Cu N
CH3 H2N
O
N
H N
R
S R= H & Me
H H N N CH3 Cl Cu S O .H2O Cl N
CH O
H N
MeOH, Reflux, 1hr
H 3C CH3 NH2 N
N N N H
N
H N
N H
(2)
(1)
Scheme 1. Synthesis of heterocyclic copper(I)/(II) complexes Infrared Spectroscopy Vibrational spectroscopy has been used to assign the primary confirmation of the coordination mode of the ligands to copper ion. The IR spectrum of the ligands exhibited bands in the regions at 1650 and 1653 cm-1 was assigned for oxo (C=O) stretching vibration and this shifted to lower frequencies (1633 and 1631 cm-1) in complexes (1 and 2) indicating the bonding of oxo group to copper metal ion [29]. The vibrational frequency corresponding to the azomethine group was found at 1549 and 1561 cm-1 in the ligands and this was shifted to 1541 and 1543 cm-1 for complexes indicating the coordination of azomethine group to metal [30]. Further, a strong band appeared at 835 and 848 cm-1 in the spectra of ligands (H2L1 and H2L2) indicating the presence of thione group [31], and this band appeared at 830 cm-1 in complex 2 revealed the thione coordination to the metal ion. However, in complex 1 the band appeared at 722 cm-1 establishing thiol coordination of ligand after enolization and subsequent protonation on thiolate sulphur [32,33]. Absorption spectroscopy The electronic absorption spectra of all the ligands and complexes were recorded in DMSO at 10 μM concentration. The ligands exhibited two bands; one band at 256 nm and 264 nm assigned to л→л* transition. The other band observed at 354 and 359 nm corresponding to n→л* transitions [34]. In the copper complexes (1 and 2), two bands appeared in the regions (254 and 270 nm) and (375 and 380 nm) respectively, which has been assigned to intra ligand transition and ligand to metal charge transfer transition (LMCT) [35,36].
Stability of the Schiff base ligands and complexes Stability of the ligands and complexes under physiological conditions were studied by spectrophotometric measurements for 24 h time interval. The UV–Vis spectrum of the Schiff base solution recorded in the range of 200–800 nm was quite stable for proving the stability of the solution about 24 h Fig. S1. Similar experiments were carried out to check the stability of the complexes and it was found that they were stable even after 24 h in water, DMSO, Tris-HCl (pH-7.2) and phosphate buffer (pH-7.2). Proton NMR spectroscopy The 1H-NMR spectra of the ligands H2L1&2 have been recorded in DMSO-d6 as shown in Fig S2-S4. The ligands exhibited a singlet at δ 11.71 and 11.92 ppm for N(1) proton [37]. Whereas, another singlet observed at δ 10.56 and 11.20 ppm analogous to quinoline imine N(3)H protons respectively [37]. The ligand H2L1 displayed a singlet at δ 8.53 ppm corresponding to azomethine (HC=N) proton. While a doublet at δ 8.60-8.61 ppm was assigned to azomethine proton of the ligand H2L2 [32,38]. A singlet appeared at δ 8.69 ppm in the ligand H2L2 was assigned to terminal N(4)H protons. However, a doublet appeared at δ 7.40–7.42 ppm analogous to terminal NH2 protons in the ligand H2L1 [38]. A sharp singlet was found at δ 8.12 ppm and 8.60 ppm corresponding to C(4)H protons of the ligands (H2L1 and H2L2) [28]. In addition, the ligands (H2L1 and H2L2) exhibited a doublet at δ 7.77-7.79 ppm and δ 7.54-7.56 ppm for C(5)H protons. Further, the additional doublet was found at δ 7.39-7.40 ppm range was attributed to C(6)H proton in ligand H2L2. While in ligand H2L1, a triplet was observed at δ 7.50-7.54 ppm corresponding to C(6)H proton. For C8(H) proton, a triplet at δ 7.17-7.20 ppm for H2L2 and a multiplet at δ 7.14-7.21 ppm for H2L1 were observed. A doublet was observed at δ 3.06-3.07 ppm corresponding to terminal methyl protons of the ligand H2L2 [39]. A sharp singlet was observed at δ 2.46 ppm in the ligands H2L2 were assigned to methyl protons of quinoline ring at C(7) position. Whereas, in H2L1 C(7) proton was observed as doublet at δ 2.47-2.49 ppm [40]. The new copper(II) complexes were found to be NMR inactive. The paramagnetic nature of copper(II) complexes was confirmed by EPR spectroscopy. X-band EPR spectra have been recorded for complex 2 in the solid state at room temperature, Fig. 1 showed a single isotropic peak. The ‘g’ value of the complex has been found to be 2.0731, and this value is in good agreement with the previously reported square pyramidal Cu(II) complex [41].
Fig. 1. EPR spectra for complex 2. X-ray crystallography study Crystal structure of complex 1 with atom numbering scheme is shown in Fig. 2, and refinement data are shown in Table. 1. The molecule is crystallized in P-1 space group with a triclinic crystal system. The asymmetric unit is formed by one half of the molecule and the other half is linked by a centre of inversion in the Cu(1)–S(1)–Cu(1)–S(1)#1 ring (Fig. 2. And Table. S1). The coordination geometry at each Cu(I) centre is square pyramidal. A tridentate copper(I) complex was formed by oxo oxygen (O1), azomethine nitrogen (N2) and thiolate sulphur (S1) atom to form a basal plane along with one oxygen atom from the solvent molecule of dimethylformamide. The two distorted square pyramids were formed by a thiolate sulphur atom in the axial position, with same bite angle (N(2)-Cu-S(1) and N(2)-Cu-S(1) #1), (85.82(8)°). The non-bonded Cu-Cu distance has been found to be 3.874 Å. The trans angles (N(2)-Cu(1)-O(2) and O(1)-Cu(1)-S(1)) were found to be 172.92(9)° and 173.76(7)° respectively, showing considerable deviation from 180° and significant distortion from the square pyramidal geometry [28]. The τ value of 0.014 at each metal centres exposed the slight distortion from the perfect square pyramidal geometry [41]. The molecular packing diagram is shown in Fig. S5. The observed bond angle and bond length values were compared to the already reported values [28]. The molecular structure of complex 2 is shown in Fig. 3. In this complex, copper ion is coordinated to O, N, S donor atoms of the thio ligand and two chloride ions, (axial Cl2 and equatorial Cl1). The O(1), N(2), S(1) and Cl1(equatorial) atoms occupy a square plane; whereas, the Cl2 ion occupies the axial site. The complex is crystallized as a monoclinic
Table 1. Structure refinement for complexes 1 and 2. Compounds
[Cu(μ-S-H-7MOQtscH)DMF]2 (1) 1906212 C30H36Cu2N10O4S2 791.89 100(2) 0.71073 Triclinic P -1 9.4019(9) 9.5456(8) 10.7981(10) 88.951(6) 85.826(6) 87.406(6) 965.42(15)
[Cu(7MOQtscMe)Cl2].H2O (2) 1906211 C13H16 Cl2CuN4O2S 426.80 100(2) 0.71073 Monoclinic P 21/n 13.6839(12) 9.4180(10) 13.9879(15) 90 113.055(5) 90 1658.73(3)
1 1.362
1 1.709
co-efficient 1.255
1.778
CCDC number Empirical formula Formula weight Temperature(K) Wavelength(Å) Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) Volume (Å3) Z Dx (Mg/m3) Absorption (mm-1) F(000) Crystal size (mm3) Theta range for collection (°) Index ranges
408 0.070 x 0.050 x 0.020 data 1.891 to 26.421
868 0.100 x 0.070 x 0.030 1.765 to 26.493
-11<=h<=11, -11<=k<=11, -13<=l<=13 Reflection collected 7605 Independent reflections 3924 [R(int) = 0.0515] Completeness of theta = 99.8 25.500° (%) Refinement method Full-matrix least-squares on F2
-17<=h<=17, -11<=k<=11, -17<=l<=17 6520 3393 [R(int) = 0.0579] 99.4
Data/restraints/parameters Goodness –of-fit on F2
3393 / 3 / 225 1.039
3924 / 0 / 221 0.952
Final R indices R1 = 0.0439, wR2 = 0.1063 [I>2sigma(I)] R indices (all data) R1 = 0.0725, wR2 = 0.1183 Largest diff. peak and hole 0.520 and -0.549 e.Å-3
Full-matrix least-squares on F2
R1 = 0.0450, wR2 = 0.0978 R1 = 0.0727, wR2 = 0.1109 0.660 and -0.630 e.Å-3
1
Fig. 2. ORTEP of the complex [Cu(μ-S-H-7MOQtsc-H)DMF]2 (1) with thermal ellipsoids at the 50% probability level. crystal system with a space group of P 21/n. The axial Cu-Cl bond length (Cl(2)-2.6382(12)) is longer than the equatorial chloride ion bond length (Cl(1)-2.2558(11)) which was described as Jahn-Teller distortion [41]. The trans bond angles in the square plane namely, N(2)-Cu(1)-Cl(1) 165.17(10) and O(1)-Cu(1)-S(1) 169.37(9) as well as the cis bond angles in the range of 85.82-92.50° and this revealed a significant distortion in a square pyramid geometry [28]. Further, the distortion value of a coordination polyhedron, τ = (β-α)/60, is calculated by the two largest bond angles in five coordination geometry (τ = 1 for ideal trigonal bipyramidal and 0 for square pyramidal environment) [41,42], and this value (τ) is equal to 0.07 for complex 2. The geometry of the complex was found to be distorted square pyramidal. Four different types of hydrogen bonds were found in complex 2, molecular packing and hydrogen bonding distances are given in Fig. S6-S7 and Table S1. An intramolecular hydrogen bonding between the hydrogen atoms of quinoline nitrogen N(1) to a basal chloride ion. An intermolecular hydrogen bonding was found between imine nitrogen N(3) and the oxygen atom of a water molecule, which is in the lattice unit. The second intermolecular hydrogen bond is formed between the hydrogen atom of a water molecule H1(W) and basal chloride ion in third structure. The third intermolecular hydrogen bond was formed between
the hydrogen atom of a water molecule H2(W) and axial chloride ion in third structure. There are three intermolecular and one intra molecular hydrogen bonding was noted in this complex to form a2D network.
2
Fig. 3. ORTEP diagram for complex [Cu(7MOQtsc-Me)Cl2].H2O (2) with thermal ellipsoids at the 50% probability level (Lattice water molecule is omitted in the above picture for clarity). Nucleic acid interactions DNA binding studies were performed using electronic absorption spectroscopy. In order to get the binding constants for ligands (H2L1&2) and complexes (1-2), electronic absorption titration experiments were performed with incremental amounts of calf-thymus DNA (CT-DNA) in 5 mM Tris–HCl buffer (pH 7.2) and by keeping the constant concentration of the individual ligands and complexes Fig. 4 and S8 [28,41]. The ligands (H2L1 and H2L2) exhibited two bands in the regions at 254-256 nm and 353-359 nm respectively. The first absorption band observed at 256 nm and 254 nm with the redshift of 2 nm and 3 nm respectively. However, the second absorption band at 353 nm and 359 nm with the redshift of 2 nm and 4 nm respectively. Whereas, in the complexes (1 and 2) two bands were observed in the regions at 253-255 nm and 381-384 nm with the redshift of 10 nm, 6 nm and 2 nm respectively. The л* orbital couples with the л orbital of the drug molecules in the nucleic acid base pairs, thus the л-л* transition probabilities diminished, and hence a hypochromism was observed, which designated that all the compounds bind to DNA via intercalative mode [28,41-45]. According to Wolfe-Shimer equation [46], a plot of [DNA] versus [DNA]/[εb_εf] gave a slope and an intercept equal to 1/[εa_εf] and (1/Kb) [εb_εf], respectively, Fig. 4. Inset and S8 Inset. The Kb values of the ligands and complexes are listed in Table. 2. From the Kb values, complexes are significantly high (of the order 105 M-1) showing that the complexes are tightly bound with CT-DNA and higher than that of the
classical interacalator EB (Kb = 1.23 (±0.07) x 105 M-1) [47]. Concerning the ligands and complexes, the Kb constants increase in the order 1 > 2 > H2L1 > H2L2. While in the case of complexes, binuclear copper(I) complex and found to be the order of 105, which was reported by stylidou et al [48]. However, the mononuclear copper(II) complex exhibited slightly less binding ability when compared to copper(I) complex and found to be 105, this value was compared to the previously reported literature [41,44,45,46,47].
Fig. 4. Electronic absorption spectra of the complexes (10 μM) in the absence and presence of gradual addition of the increasing concentration of nucleic acid (5-50 μM) at room temperature in 5 mM Tris-HCl/50 mM NaCl buffer (pH = 7.2). Ethidium bromide competition assay The binding of drug molecules with EB bound nucleic acid may give detailed information about the DNA binding affinity of the complexes. Fig. 5 and S9 showed the fluorescence spectra of DNA-EB system with increasing amounts of the compounds. The emission intensity of DNA-EB system decreased when the concentration of ligands and complexes were increased [49]. Further, the fluorescence quenching is described by the Stern-Volmer equation [41,50]. The slope of the linear plot of Io/I versus [Q] gave Ksv as shown in Fig. 5 Inset and S9 Inset. Singh et al. have reported the metallo intercalators and the values are found to be 10x105 [44-46]. The quenching constant (Ksv) values follow the same order as discussed in the absorption titrations and values are listed in Table. 2 and S2.
Fig. 5. Emission spectra of EB (5 μM) bound to DNA (5 μM) in the absence and presence of an incremental concentration of the complexes (10-100 μM) using Tris-HCl/50 mM NaCl buffer (pH = 7.2). Table. 2. The Kb (binding constant) and Ksv (Quenching constant) values for the interactions of the complexes (1-2) with CT-DNA. Compounds
Kb/M-1
Ksv/M-1
Complex 1
3.79 × 105±0.14
3.11 × 105±0.19
Complex 2
2.48 × 105±0.18
2.56 × 105±0.10
In most cases, the observed changes in the UV/emission spectra of the compounds are not as pronounced as lead to a clear conclusion regarding the possible DNA-interaction mode of the complexes. Subsequently, in order to clarify the DNA-binding mode of the complexes, DNA-viscosity measurements were carried out. Viscosity measurements In order to further confirm the mode of binding of the ligands and complexes to CT-DNA, viscosity measurements of DNA solutions were carried out in the presence and absence of the compounds. The viscosity of DNA is sensitive to changes in its length and is regarded as the least ambiguous and the most critical clues of a DNA binding mode in solution [49]. In general, intercalating agents are expected to elongate the double helix to accommodate the complexes between the base pairs, leading to an increase in the viscosity of DNA [49]. The effects of the complexes 1-2 on the viscosities of the CT-DNA solution are shown in Fig. 6, with increasing [complex]/[DNA] concentration, resulted in a significant change in the relative viscosity of CT-DNA solution was observed with intercalative binding
mode of complexes [44,45,51-53]. The relative viscosity of DNA increases steadily in the order of 2 > 1 > H2L1 > H2L2.
Fig. 6. Effect of the ligands and complexes on the viscosity of CT DNA. Protein interaction studies (BSA) UV-Vis spectral study The fluorescence quenching mechanisms are usually classified as either static or dynamic quenching. Static quenching usually resulted from the formation of a complex between quencher and fluorophore in the ground state, whereas in dynamic quenching the fluorophore and quencher get in touch with each other during the transient existence of the excited state [41]. One can have an idea about the type of quenching from UV-Vis absorption spectral studies. UV-Vis spectra of BSA in presence of compounds displayed (Fig. S10) an increase in absorption intensity of the BSA suggesting the static type of interaction due to the formation of ground state complex as reported earlier [41]. A constant concentration of BSA with increasing concentration of the compounds, fluorescence emission intensity falls gradually for ligands and complexes Fig. 7 and S11. The interaction which might take place between the compounds and BSA [41,44,45,54,55].
Fig. 7. Fluorescence titration spectra of BSA (10 μM) with increasing amounts of complexes (10-100 μM), at pH = 7.2. In order to express their quenching capacity between the drug molecules and BSA, the Stern–Volmer quenching plot was obtained by detecting the fluorescence quenching of BSA and drug molecules. According to the Stern–Volmer equation [56,57], Ksv (Stern-Volmer constant) was determined from the plot of Io/I vs [Q], Fig. 7 Inset and S11 Inset and the values are listed in Table 3 and S3. Moreover, the binding constant (Kb) have been determined by using the equation at room temperature log((Fo−F)/F) = ln(Kb) + n log [Q], where [Q] is the concentration of quencher that is the synthesized compounds here [58]. “Kb” is obtained from the plot of log((Fo−F)/F) versus log[Q] as a y-intercept, Fig. 8 and S12. Furthermore, “n” which is the number of the binding site per protein [59]. The value of “n” is nearly 1, representing the synthesized complexes bind to BSA with a molar ratio of 1: 1. The calculated results are shown in Table 3 and S3. Concerning that the binuclear copper(I) complex exhibited the significant interaction ability with bovine serum albumin and this value was similar to the previous literature, which was reported by stylidou et al [48]. Elamathi et al. reported the copper(II) complexes containing heterocyclic Schiff base ligands, and the values are found to be 10 X105 [41,44,45,59].
Fig. 8. Scat Chard plots of complexes with BSA. Table. 3. Quenching constant (Ksv), binding constant (Kbin) and a number of binding sites (n) for the interactions of complexes with BSA. Compounds
Ksv/M-1
Kb/M-1
n
Complex 1
3.17 × 105±0.17
7.93 × 105±0.16
0.92
Complex 2
1.29 × 105±0.22
4.61 × 105±0.18
1.13
Tryptophan fluorescence spectra of serum albumins To increase the concentration of the test compounds (10–100 μM) added to BSA (10 μM), the fluorescence intensity of tryptophan residue was reduced Fig. S13. The above results concluded that the microenvironment around tryptophan residue was altered by the addition of compounds [28,41]. Evaluation of radical scavenging ability Appreciable results obtained from the biomolecular interaction of the complexes, it was encouraged us to study the antioxidant activities of the ligands and complexes. Hence, the study on antioxidant properties of the complexes was carried out [60,61]. The antioxidant activities of the new complexes along with the standard, butylated hydroxytoluene (BHT) have been inspected with hydroxyl radicals, DPPH radicals and the IC50 values are given in Table. 4. The IC50 values indicated the compounds showed marked antioxidant activity in the order of 1 > 2 > H2L1 > H2L2 in both the experiments. The DPPH radical scavenging power of the compounds was the maximum and hydroxyl radical scavenging was the minimum, as shown in Fig. 9 and S14. These results are considerably better than that observed for standard antioxidant BHT. According to our obtained results, it can be concluded that the scavenging
effects of the free ligand are significantly less compared to their corresponding Cu(I) [62] and Cu(II) complex.[28] From the obtained IC50 values, copper(I) complex 1 exhibited better activity than the copper(II) complex 2. The highest activity of complex 1 may be due to copper(I) complex.
Fig. 9. Hydroxyl (OH) and 2-2’-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity at various concentrations of the complexes and standard butylatedhydroxytoluene (BHT). Table. 4. The Radical scavenging activity of the ligands and complexes. Compounds H2L1 H2L2 Complex 1 Complex 2 BHT
IC50 values in (μΜ) Hydroxyl radical 187.54±2.64 290.41±1.13 18.11±1.14 20.45±1.53 313.52±1.23
DPPH radical 63.72±1.13 71.16±0.69 4.92±0.79 7.46±1.16 10.88±1.67
Anticancer activity in vitro Cancer cell growth inhibition The positive results obtained from the previous biological studies for the N-heterocyclic Schiff bases and their copper(I)/(II) complexes cheered us to test their cytotoxicity against human breast cancer cell line (MCF7) and one normal human keratinocyte (HaCaT) cells with cisplatin as a standard drug. The complexes were dissolved in DMSO and blank samples having the same volume of DMSO are taken as a control to find the activity of the solvent in this cytotoxicity experiment. The results were examined by means of cell inhibition expressed as (50% activity) IC50 values Fig. 10. It is to be noted that the ligands (H2L1-2) displayed significant activity at low concentration on cancer cells. The
copper complexes showed better activity as compared to the ligands and standard drug molecule. The results of in vitro cytotoxic activity studies have shown that the IC50 value of the ligands and complexes against HaCaT human keratinocyte (normal cells) is found to be above 100 μΜ, which confirmed that the compounds are very specifically active on cancer cells Fig. 10. The highest cytotoxic properties of copper(I) complexes over the ligands and copper(II) complex [38,41,44,45,62,63].
Fig. 10. The ligands (H2L1&2) copper(I)/(II) complexes (1 and 2) inhibit MCF-7 cells and HaCaT cells proliferation in a dose-dependent manner. Both cells were treated with dissimilar concentrations of compounds for 24 h, the cell viability was determined and the results were expressed as a percentage of cell viability with control. Results shown are mean, which are three separate experiments performed in triplicate. The anticancer activity of the compounds on MCF-7 cells follows the order: Cisplatin (21.58±0.14) μM < Complex 2 (13.58±0.52) μM < Complex 1 (12.88±0.31) μM for MCF-7 cells respectively. Acridine orange/ethidium bromide staining assay Apoptosis studies were done by using dual staining method like acridine orange (AO) and ethidium bromide (EB) to find the changes in membrane integrity between apoptosis and necrosis [64,65,66]. Ligands did not show significant activity, complexes exhibited significant activity. The apoptotic study for AO/EB staining assay was carried out for complexes (1 and 2). AO can pass through the cell membrane of both living and apoptotic cells, while the cells were stained by EB designate the loss of membrane integrity. Visualization under a fluorescence microscope, living cells appear as green, necrotic cells stained as a red and apoptosis cells appear as greenish yellow or orange colour morphological changes were observed. From Fig. 11, DMSO (MCF-7) cells have both green and light orange fluorescence, while the control (standard drug) (MCF-7) cells have a normal
morphology showed a bright green fluorescence. However, the MCF-7 cells treated with IC50 concentration of complexes 1 and 2 revealed morphological changes as compared to the positive (standard drug) and negative (DMSO) controls, and were characterised as early apoptotic (greenish yellow fluorescence), and late apoptotic with condensed and fragmented nuclei (orange fluorescence). This revealed that complexes 1 and 2 induced cell death in MCF-7 cells [64-67]. A similar type of thiosemicarbazone containing copper complexes were tested against human epidermoid carcinoma cells (A431) was reported in our earlier literature [41], results strongly suggested that induction of apoptotic pathways for the anticancer activity of these complexes maybe necrosis. However, in this case, the complexes were treated with human breast cancer cells and this showed the induction of apoptotic pathway for the activity of the complexes may be apoptosis. DMSO
Control
Complex 1
Complex 2
Fig. 11. Fluorescence images of acridine orange/ethidium bromide stained MCF-7 cells were treated with IC50 concentration of complexes for 24 h. Conclusion The new heterocyclic copper(I)/(II) complexes derived from nitrogen heterocyclic Schiff bases have been synthesized and characterized by various analytical and spectroscopic techniques. The solid-state structures of complexes (1 and 2) were confirmed by single crystal X-ray analysis. In complex 1 ligand coordinated to metal through oxo oxygen, azomethine nitrogen and thiolate sulphur to form a binuclear nature of distorted square pyramidal copper(I) complex. While complex 2, ligand coordinated to the metal via ONS fashion to form a mononuclear copper(II) complex having distorted square pyramidal geometry. The DNA binding properties of the ligands and their corresponding copper complexes were investigated by absorption and fluorescence measurements. The results supported the fact that the compounds interactively bound with the CT-DNA. Fluorescence quenching experiments with BSA confirmed the static quenching mechanism may be followed when fluorophore interacts with quencher. The radical scavenging power of the compounds towards hydroxyl and DPPH radical exposed that the complexes have better
scavenging ability as compared to ligands and the butylated hydroxy toluene (BHT). The in vitro cytotoxicity of the compounds was evaluated against the MCF-7 (human breast cancer) cell lines by comparing with cisplatin, the complexes exhibited significant cytotoxicity. Morphological analysis was determined by fluorescence microscopes using AO/EB staining assay showed that the induction of apoptotic pathway for the anticancer activity of these complexes may be apoptosis. There is a correlation between the DNA/protein binding and cytotoxicity of the complexes proving that complexes can be better candidates as anticancer drugs. According to our obtained biological results, the order of binding capacity of the compounds were directly proportionately to the radical scavenging and cytotoxicity of the compounds and the order is 1 > 2 > H2L1 > H2L2. This order showed complex 1 has better activity compared to the ligand and corresponding copper(II) complex. The higher activity of complex 1 may be due to the presence of the two active metal canters in it. Acknowledgement The author C. E gratefully acknowledges to the Department of Science and Technology (DST) and Ministry of Science and Technology, New Delhi, INDIA for an INSPIRE Fellowship, New Delhi, India [IF150603 dated. [10.04.2015]. Supplementary data Crystallographic
data
for
[Cu(μ-S-H-7MOQtsc-H)DMF]2
(1)
and
[Cu(7MOQtsc-
Me)Cl2].H2O (2) have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication (CCDC No.1906212 and 1906211). The data can be obtained free of
charge
at
www.ccdc.cam.ac.uk/conts/retrieving.html
Crystallographic Data Centre.
or
from
the
Cambridge
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A new water soluble N-heterocyclic copper(I)/(II) complexes were synthesized and characterized. Further, new complexes were subjected to binding interactions, antioxidant and anti-proliferation. Among this, copper(I) complex 1 showed better activity compare to copper(II) complex and their ligands.
Highlights
Water soluble Cu(I)/(II) complexes containing N-heterocyclic Schiff bases.
Ligands coordinated to Cu(II) complexes coordinated through ONS- and ONS chelation.
Biomolecular interactions and cell-proliferation were carried out for all the compounds.
MCF-7 staining pictured the most of the cell death via apoptotic and seems to be orange fluorescence.