English

• 0.   Introduction

  Schiff bases are flexible imine ligands, their intrinsic properties originated from the ease of synthesis, wide-ranging complexing ability, structural diversities, and host of applications in different spheres^[1-4]. This ligand system played a vital role in the areas of coordination chemistry, bioinorganic chemistry, catalysis, material chemistry, sensor technology, and many other fields^[5-10]. The N, O donor Schiff base ligands have been widely studied due to their high coordinating ability to metal, and the resultant complexes exhibit significant structural diversities, catalytic activities, and unusual magnetic properties^[11-13]. The chemistry of transition metal Schiff base compounds has been a recurrent theme, still, the interest in this area continues unabated as many of these complexes show fascinating structural features, and interesting photochemical, electrochemical, fluorescence, non-linear optical, and magnetic properties^[14-16]. Such complexes also find potential use in magnetic materials, electronic devices, model compounds for important bioprocesses, and as catalysts in many organic transformations^[17-20]. Apart from these, metal Schiff base compounds are also important due to their relevance in the study of interactions with DNA, and in several important biological processes^[21]. The hydrazone-based Schiff base ligands are considered to be versatile multidentate ligands, able to coordinate with a host of metal ions^[22]. This ligand system and its metal complexes show prominent DNA binding, cleavage ability, and therapeutic as well as catalytic properties^[23]. Among the 3d transition metals, the complexes of Cu(Ⅱ) with Schiff bases are widely studied not only due to their ease of synthesis, possible applications in areas of catalysis, therapeutics, magnetic materials, etc. but also because many of them are used in mimicking the activity of several metalloenzymes^[24-25].

  Considering the immense importance of metal-Schiff base complexes as highlighted, we were interested in exploring Cu(Ⅱ)-Schiff base chemistry and endeavoured to synthesize newer hydrazone-based Schiff base complexes of Cu(Ⅱ) to ascertain some of their properties. The present report deals with the synthesis, characterization, and structure elucidation of hydrazone-based Schiff base complexes of Cu(Ⅱ) ion. To evaluate the biological potential of synthesized Cu(Ⅱ)-Schiff base complexes, their DNA binding ability and antibacterial activity were explored.

  1.   Experimental

  1.1   Materials and methods

  Reagent-grade chemicals were used. Calf thymus DNA (CT-DNA), tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl), ethidium bromide (EB), dimethyl sulfoxide (DMSO), and other chemicals used were analytical grade products and acquired from Sigma-Aldrich. The instruments used for recording FTIR and electronic spectra were Bruker Alpha Ⅱ FTIR spectrophotometer and Shimadzu 1800 spectro-photometer. Magnetic susceptibility was measured by the Gouy method using Hg[Co(SCN)₄] as standard. Fluorescence spectra were recorded on Fluorolog-3, Horiba Scientific Fluorescence spectrophotometer. C, H, and N contents in the obtained compounds were ascertained using a Perkin Elmer CHN analyzer (2400 series Ⅱ). The copper content in the compounds was determined by iodometric titration.

  1.2   Synthesis of the ligands HL¹, HL², L³

  To a methanolic solution (20 mL) of benzil (2.102 3 g, 10 mmol), hydrazine hydrate (0.500 6 g, 10 mmol) was added in small proportions. The solution was stirred for an hour when a white precipitate separated. Precipitated, benzil monohydrazone was collected by filtration, and washed with hexane several times to remove unreacted benzil. The ligands HL¹, HL², L³ were obtained by treating the freshly prepared benzil monohydrazone (0.224 2 g, 1 mmol) dissolved in methanol with 2-hydroxy benzaldehyde (0.122 1 g, 1 mmol) (HL¹)/4-hydroxy benzaldehyde (0.122 1 g, 1 mmol) (HL²)/2-methoxy benzaldehyde (0.1361 g, 1 mmol) (L³) under reflux at 90 ℃. Yellow crystalline compounds were obtained from the clear solution within 4-5 d. The single crystals suitable for X-ray crystallography were separated, washed with methanol, and dried in vacuo.

  HL¹. Yield: 70%. Anal. Calcd. For C₂₁H₁₆O₂N₂(%): C, 76.82; H, 4.87; N, 8.53. Found(%): C, 75.29; H, 4.60; N, 8.48. FTIR (KBr, cm^-1): 1 604 ν(C=O), 1 673 ν(C=N), 1 231 ν(C—O), 3 424 ν(O—H).

  HL². Yield: 73.45%. Anal. Calcd. For C₂₁H₁₆O₂N₂(%): C, 76.82; H, 4.87; N, 8.53. Found(%): C, 76.95; H, 4.82; N, 8.21. FTIR (KBr, cm^-1): 1 557 ν(C=O), 1 602 ν(C=N), 1 162 ν(C—O), 3424 ν(O—H).

  L³. Yield: 66.07%. Anal. Calcd. For C₂₂H₁₈O₂N₂(%): C, 77.19; H, 5.26; N, 8.18. Found(%): C, 77.21; H, 5.45; N, 8.15. FTIR (KBr, cm^-1): 1 598 ν(C=O), 1 673 ν(C=N), 1 230 ν(C—O).

  1.3   Synthesis of [Cu(L¹)₂], [Cu(HL²)₂(Ac)₂], and [Cu(L³)₂(Ac)₂]

  The ligand HL¹ (0.328 4 g, 1 mmol) was dissolved in 20 mL DMF to which a solution of copper acetate (0.199 6 g, 1 mmol) in DMF (10 mL) was added and the resulting solution was stirred at room temperature for 3-4 h. The green solution so obtained was filtered and left undisturbed at room temperature from which the microcrystalline product of complex [Cu(L¹)₂] (1) was separated within 6-7 d. The complexes, [Cu(HL²)₂(Ac)₂](2) and [Cu(L³)₂(Ac)₂](3) were prepared by following the same synthetic protocol. The ligand HL² (0.328 4 g, 1 mmol)/L³ (0.342 4 g, 1 mmol) reacted with Cu acetate monohydrate (0.199 6 g, 1 mmol) in DMF-water (1∶1, V/V), and the mixture was stirred for 5-6 h at room temperature, which afforded green microcrystalline products. The complexes were collected by filtration and washed several times with DMF followed by ethanol and dried in vacuo.

  [Cu(L¹)₂] (1). Yield: 63.21%. Anal. Calcd. For C₄₂H₃₀CuN₄O₄(%): C, 69.96; H, 4.44; N, 7.77; Cu, 8.81. Found(%): C, 69.79; H, 4.71; N, 7.96; Cu, 9.03. FTIR (KBr, cm^-1): 1 612 ν(C=O), 1 670 ν(C=N), 1 222 ν(C—O), 486 ν(Cu—N), 428-415 ν(Cu—O).

  [Cu(HL²)₂(Ac)₂] (2).Yield: 69.81%. Anal. Calcd. For C₄₆H₃₈CuN₄O₈(%): C, 65.84; H, 4.53; N, 6.68; Cu, 7.57. Found(%): C, 65.51; H, 4.43; N, 6.87; Cu, 7.76. FTIR (KBr, cm^-1): 1 605 ν_as(COO^-), 1557-1600 ν_s(COO^-), 1 557 ν(C=O), 1 557-1 600 ν(C=N), 3428 ν(O—H), 486 ν(Cu—N), 428-415 ν(Cu—O).

  [Cu(L³)₂(Ac)₂] (3). Yield: 72.21%. Anal. Calcd. For C₄₈H₄₂CuN₄O₈(%): C, 66.50; H, 5.01; N, 6.47; Cu, 7.33. Found(%): C, 66.89; H, 4.86; N, 6.33; Cu, 7.12. FTIR (KBr, cm^-1): 1 600 ν_as(COO^-), 1 350 ν_s(COO^-), 1 600 ν(C=O), 1 669 ν(C=N), 486 ν(Cu—N), 428-415 ν(Cu—O).

  1.4   Crystallographic data collection and refinement

  Single crystal X-ray diffraction data was recorded by the ω-scan technique using Cu Kα (λ =0.154 184 nm) or Mo Kα (λ=0.071 073 nm). The crystallographic studies of HL¹, HL², and L³ were conducted at 100 K using RIGAKU XtaLAB Synergy, Dualflex, and Pilatus 300K diffraction instruments along with Photon Jet micro-focus X-ray for source data collection, cell refinement, data reduction, and absorption correction were carried out using CrysAlis PRO software. The crystal structures were solved using the SHELXT 2018/2 program. Atomic scattering factors were taken from the International Tables for X-ray Crystallography. Positional coordinates of non-H-atoms were refined using the full-matrix least-squares method on F² with anisotropic thermal parameters by using the SHELXL 2018/3 program. All hydrogen atoms have been found in the difference electron density maps. The hydrogens bonded to carbon atoms were placed in calculated positions of (C—H: 0.093-0.098 nm), further included in the refinement as riding contributions with isotropic displacement parameters which were set to 1.2 times the U_eq of the parent atom. The H atoms of the phenolic—OH group were placed at a distance of O—H (0.806 nm) and then were refined without any restrictions. Molecular graphics were obtained by using the Mercury 2020.2.0 program. The details of the crystallographic data and refinement parameters are depicted in Table 1.

  Table 1

  

  Table 1.  Crystal data and structure refinement parameters for the ligands

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  Parameter	HL1	HL2	L3
  Empirical formula	C21H16N2O2	C21H16N2O2	C22H18N2O2
  Formula weight	328.36	328.36	342.38
  Crystal system	Monoclinic	Orthorhombic	Monoclinic
  Space group	P21/c	Fdd2	P21/n
  a/nm	1.688 86(8)	3.028 83(4)	1.546 51(3)
  b/nm	0.909 79(3)	2.761 76(9)	0.782 50(1)
  c/nm	1.107 49(3)	0.803 612(18)	1.585 04(3)
  β/(°)	99.377(3)		114.056(2)
  Volume/nm3	1.971 55(7)	6.722 1(3)	1.75153(6)
  Z	4	16	4
  Dc/(Mg·m-3)	1.299	1.298	1.298
  Absorption coefficient/mm-1	0.680	0.679	0.084
  F(000)	688	2 752	720
  Crystal size/mm	0.09×0.43×0.52	0.60×0.50×0.46	0.52×0.55×0.55
  θ range for data collection/(°)	2.7-66.6	4.3-66.6	2.8-25.0
  Index ranges	-20 ≤ h ≤ 20;	-36 ≤ h ≤ 30;	-18 ≤ h ≤ 18;
  	-10 ≤ k ≤ 10;	-32 ≤ k ≤ 32;	-9 ≤ k ≤ 9;
  	-13 ≤ l ≤ 13	-9 ≤ l ≤ 9	-18 ≤ l ≤ 18
  Reflection collected	59 891	40 530	45 286
  Independent reflection	2 961	2 962	2 978
  Goodness of fit on F2	1.05	1.09	1.06
  R indices (all data)	0.030 8	0.021 3	0.029 2
  Largest diff. peak and hole/(e·nm-3)	190 and -140	130 and -120	220 and -150

  1.5   Computational details

  The Gaussian 09 Revision C.01 program package was used for all calculations. The gas phase geometries of the molecular species (1, 2, 3) were optimized fully by unrestricting symmetry in singlet ground states with B3LYP functionals. The basis set LANL2DZ with effective core potential (ECP) was employed for Cu following the associated valence double ζ basis set of Hay and Wadt.

  1.6   DNA binding experiments

  1.6.1   Preparation of stock solution

  The CT-DNA stock solution was prepared by dissolving a suitable amount of CT-DNA in tris(hydroxy-methylaminomethane)hydrochloride (Tris-HCl) buffer (0.01 mol·L^-1, pH 7.4) at ambient conditions. An EB (50 µmol·L^-1) stock solution was prepared by dissolving an appropriate amount of the dye in deionized double-distilled water. The solutions (1 mmol·L^-1) of the complexes were prepared by dissolving them in DMSO.

  1.6.2   Absorption spectroscopy

  The UV-Vis spectroscopic study was carried out on a Shimadzu 1800 spectrophotometer. The DNA binding absorption experiments were performed in a wavelength range of 240-300 nm by the addition of increasing concentration of the solutions of complexes 1-3 (1 mmol·L^-1) to a fixed amount of CT-DNA (50 µmol·L^-1).

  1.6.3   Emission spectroscopy

  The competitive binding of the complexes with ethidium bromide (EB) was studied by fluorescence spectroscopy. The CT-DNA-EB complex was prepared by adding 20 µmol·L^-1 EB and 26 µmol·L^-1 CT-DNA in buffer (150 mmol·L^-1 NaCl and 15 mmol·L^-1 trisodium citrate at pH 7.0). The intercalating effect of complexes 1-3 with the DNA-EB adduct was studied by successive addition of a fixed amount of complex solution to the solution of the DNA-EB system. The changes in the addition of the respective complex to the DNA-EB adduct solution were ascertained by recording the variation in fluorescence spectral emission of the DNA-EB system.

  1.7   Antibacterial activity test

  Antibacterial activity assay was tested on human pathogenic Gram-negative bacteria Klebsiella pneu-moniae (K. pneumoniae), Escherichia coli (E. coli), and Shigella boydii (S. boydii), and gram-positive bacteria Staphylococcus aureus (S. aureus). The strains were tested with complexes 1-3 dissolved in DMSO. Overnight cultured stock bacteria in nutrient broth were used to test the antibacterial efficacy of the complexes. In 100 mm petri dish nutrient agar was poured and after 10 min 50 µL fresh bacterial culture was spread on the agar plate. For determination of the zone of inhibition, 5 mm Whatman filter paper discs were placed on prespread bacteria in a nutrient agar plate. Different concentrations (1, 10, 100 µg in each disc) of the complexes were poured on the discs which were diffused on an agar plate. After 14 h of incubation at 37℃, the clear zone of inhibition was measured by scale. DMSO was used in the measurement as the negative control.

  2.   Results and discussion

  2.1   Synthesis

  The strategy involved in the synthesis of the ligands consists of two steps. Condensation of benzil with hydrazinehydrate in equimolar proportion resulted in the formation of benzil monohydrazone which was further condensed separately with aldehydes viz. salicylaldehyde, 4-hydroxy benzaldehyde, and 2-methoxy benzaldehyde to obtain hydrazone-based Schiff base ligands abbreviated as HL¹, HL² and L³. The synthesized ligands were structurally characterized by eleental analyses, and spectral studies including FTIR, UV-Vis, and single-crystal X-ray crystallographic meaurements.

  Three new mononuclear Cu(Ⅱ) Schiff base complexes were obtained from the direct reaction of the synthesized Schiff base ligands with copper acetate in a 1∶1 molar ratio in DMF-water (2∶1, V/V) medium. The synthesized complexes are green micro-crystalline solids, stable for prolonged periods, and insoluble in most organic solvents except DMSO. The complexes were comprehensively characterized and their structures were assessed from the results of elemental analyses, molar conductance measurements, FTIR, UV-Vis spectral, room temperature magnetic moment measurements, and density functional theory (DFT) studies. Single crystals of the complexes suitable for X-ray diffraction studies could not be obtained even after several attempts. The optimized structures of the complexes were ascertained by DFT studies.

  The measured molar conductances of 24.3, 30.5, and 22.5 S·cm²·mol^-1 for complexes 1, 2, and 3 indicate their non-electrolytic nature.

  2.2   Infrared spectra and electronic spectra

  The FTIR spectra of free Schiff base ligands HL¹ and HL² showed a broad medium intensity band at ca. 3 424 cm^-1 attributable to the O—H stretching mode of the phenolic —OH group. However, this absorption is absent in L³ due to the presence of the o-methoxy group as the substituent instead of the phenolic —OH group in the aromatic ring of the aldehyde. Strong absorption bands observed in 1 602-1 673 cm^-1 are assigned to ν(C=N). The C=O stretching vibration of the ligands was observed in the region of 1 598-1 654 cm^-1. The band that appeared at 1 162-1 231 cm^-1 for HL¹ and HL² in the free ligands is attributed to C—O vibration^[26]. Medium intensity band observed at ca. 1 170 cm^-1 in all the ligands originated from N—N stretching vibration. Aromatic ring vibrations of the free ligands were observed as medium-intensity bands in the region of 800-500 cm^-1. Coordination of azomethine moiety through the nitrogen atom of Schiff bases to the metal ion is expected to lower the C=N stretching frequency. On complexation, C=N vibration in the complexes shifted to a lower wavenumber of 1 602-1 673 cm^-1 in comparison to the free ligands, confirming the participation of the azomethine group in coordination with the metal center through N-atom^[27-28]. In addition, the participation of the phenolic —OH group in coordination with the metal center in complex 1 was ascertained from the shift of ν(C—O) (1 222 cm^-1) to a lower wavenumber compared to the free ligand. The unaltered position of the free carbonyl group (—C=O) of the benzil moiety of the ligands suggests non-participation of the carbonyl group in coordination with the metal center.

  The above observations suggest that in complex 1 the ligand is coordinated to the metal center in a bidentate fashion through its phenolic oxygen and azomethine nitrogen atom. The square planer coordination environment around the metal center in complex 1 is attained through the bidentate coordination of two ligand moieties involving their imine nitrogen and phenolic oxygen atoms.

  In complexes 2 and 3 the Schiff base ligands HL² and L³ are coordinated to the metal center in an unidentate fashion only involving their azomethine nitrogen atom. In HL², the —OH group of the aldehyde moiety sterically is not in a favorable position to coordinate with the metal center and hence acts as a monodentate ligand. In the case of L³, the substituent —OCH₃ moiety on the aldehyde ring does not participate in coordination rendering the ligand to coordinate only via azomethine nitrogen atom. The ν(C=N) (1 557-1 669 cm^-1) in complexes 2 and 3 are lower in value than the free ligand as expected due to the coordination of azomethine moiety to the metal center. In complexes 2 and 3 the square planer coordination geometry is attained through the participation of two acetato ligands in coordination with the metal center in a monodentate manner. The use of cupric acetate as metal salt acted as the source of coordinated acetate ligands.

  The appearance of bands for complexes 2 and 3 in the region of 1 428-1 350 cm^-1 and 1 557-1 604 cm^-1 are attributable to symmetric and asymmetric stretching vibrations of the —COO group of acetato ligand. The difference (Δν) between ν_as(COO^-) and ν_s(COO^-) larger than 200 cm^-1 in each case supports monodentate coordination of the acetate ligands^[29].

  Furthermore, medium-intensity vibrations in the lower frequency region (ca. 500 cm^-1) in the spectra of the complexes are assignable to metal-oxygen and metalnitrogen vibrations respectively.

  The electronic absorption spectra of the Schiff base ligands HL¹, HL², and L³ and their metal complexes (1, 2, and 3) under investigation were recorded in DMSO solution (10 µmol·L^-1) (Fig.S3 and S4, Supporting information). Electronic spectra of the ligands HL¹, HL², and L³ show absorption bands at 280-300 nm and 330-343 nm. The spectral band at 280-300 nm is assignable to intraligand charge transfer (ILCT) transition and those observed at the region of 330-343 nm correspond to n-π* transition of azomethine group (— C=N)^[30]. The high energy intraligand charge transfer and the n-π* transitions of the azomethine chromophore (—C=N) for the complexes, observed at 270-275 nm were shifted to a lower frequency. The lowering of frequency of the above transition indicates the involvement of the azomethine nitrogen atom in coordination with the Cu(Ⅱ) center^[31].

  2.3   Room temperature magnetic moment

  The room temperature magnetic moments of complexes 1, 2, and 3 were found to be 1.68 B.M., 1.63 B.M., and 1.64 B.M. respectively. The observed values for the complexes are close to the spin-only magnetic moments of the d⁹ system of Cu(Ⅱ) ion.

  2.4   Description of crystal structures

  The crystals of benzil monohydrazone-based Schiff bases were grown in a methanol solution and suitable crystals of HL¹, HL², and L³ were characterized structurally by single-crystal X-ray diffraction measurements. An ORTEP drawing of analyzed structures with an atom numbering scheme is illustrated in Fig. 1-3, respectively. Selected bond distances and angles for all structures are given in Table 2 whereas the details of the hydrogen bond contacts are listed in Table 3. The average bond length and bond angle parameters of phenyl rings are in the expected ranges. The data of crystallographic measurements demon-strates that HL¹ and L³ crystallize in a monoclinic system with space groups P2₁/c and P2₁/n respectively while the HL² crystallizes in the orthorhombic system with space groups Fdd2. The N—N (hydrazone) distances are in the range of typical N—N single bond distances of 0.140 39(12)-0.141 00(14) nm. The observed C—N double bonds in hydrazone units lie between 0.128 42(13) and 0.128 80(13) nm. The torsion angles involving the —N=C— units exhibit values in a range of 178.40(9)°-178.67(8)°. Generally, the geometrical parameters in the presented structures do not differ significantly from those previously reported in the literature^[32-34]. The structure of HL¹ is stabilized by a short intramolecular O2—H1…N2 hydrogen bond and weak C—H…O interactions, while in HL², an intermolecular hydrogen bond of the type O—H···O exists. The crystal packing in L³ is only controlled by non-classical weak C—H···O interactions (Table 3, Fig.S5-S7).

  Figure 1

  

  Figure 1.  An ORTEP drawing of HL¹ with the atom-numbering scheme

  Displacement ellipsoids are drawn at the 50% probability level.

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  Figure 2

  

  Figure 2.  An ORTEP drawing of HL² with the atom-numbering scheme

  Displacement ellipsoids are drawn at the 50% probability level.

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  Figure 3

  

  Figure 3.  An ORTEP drawing of L³ with the atom-numbering scheme

  Displacement ellipsoids are drawn at the 50% probability level.

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  Table 2

  

  Table 2.  Selected bond lengths (nm) and bond angles (°) of the complexes

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  Bond	HL1	HL2	L3
  O1—C2	0.121 83(13)	0.122 48(18)	0.121 75(14)
  N1—N2	0.140 39(12)	0.140 98(17)	0.141 00(14)
  N1—C1	0.128 80(13)	0.128 8(2)	0.128 49(13)
  N2—C15	0.128 42(13)	0.128 0(2)	0.128 52(13)
  O2—C17(—C19)	0.135 36(12)	0.136 34(19)	0.136 37(14)
  N2—N1—C1	112.00(8)	110.82(13)	113.17(9)
  N1—N2—C15	114.24(8)	111.88(13)	109.43(9)
  N1—C1—C2	121.14(9)	119.44(13)	122.74(10)
  N1—C1—C9	119.93(9)	120.92(13)	118.16(10)
  N2—C15—C16	120.95(9)	121.86(13)	123.01(10)

  Table 3

  

  Table 3.  Hydrogen bonding parameters of the complexes

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  Compound	D—H···A	d(D—H)/nm	d(H···A)/nm	d(D···A)/nm	∠DHA/(°)
  HL1	O2—H1···N2	0.092 6(17)	0.176 1(17)	0.260 08(11)	149.3(15)
  	C19—H19···O2ⅰ	0.095	0.251	0.343 34(14)	164
  	C20—H20···O2ⅱ	0.095	0.245	0.329 54(13)	149
  HL2	O2—H1···O1ⅲ	0.086(2)	0.195(2)	0.280 78(16)	175(2)
  	C15—H15···O1ⅳ	0.095	0.245	0.333 54(18)	155
  L3	C20—H20···O1ⅴ	0.095	0.256	0.344 72(15)	156
  	C22—H22B···O1ⅵ	0.098	0.256	0.346 97(14)	154
  Symmetry codes: ⅰ 1-x, -1/2+y, 1/2-z; ⅱ x, -1/2-y, -1/2+z; ⅲ -x, 1-y, z; ⅳ -1/4-x, -1/4+y, 1/4+z; ⅴ 1/2-x, -1/2+y, 1/2-z; ⅵ 1-x, 1-y, 1-z.

  2.5   DNA binding ability and interaction mode

  2.5.1   Electronic absorption titration

  The interaction between CT-DNA and metal complexes can be investigated by electronic absorption titration experiments^[35]. When the complexes bind to DNA, a DNA-drug adduct ("drug"refers to the metal complexes) is formed in the ground state. Generally, a bathochromic shift along with a hypochromic effect is observed in such cases^[34]. The possibility of complexes 1-3 binding to CT-DNA was investigated by absorption spectral studies. The absorption spectra for the interaction of a constant concentration of CT-DNA solution with different concentrations of complexes 1-3 were recorded and presented in Fig. 4. In the present case, a hyperchromic effect was noticed on the gradual addition of the complexes to CT-DNA solution. The changes as observed in the absorbance for the interaction of CT-DNA with complexes without any change in the location of the peak at 258 nm indicate that the synthesized complexes 1-3 bind with CT-DNA probably through electrostatic interaction, van der Walls force, and/or hydrogen bonding^[36].

  Figure 4

  

  Figure 4.  Absorption spectra of CT-DNA (50 µmol·L^-1) in the presence of increasing amounts of complexes 1, 2, and 3

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  Benesi and Hildebrand′s method (Eq. 1) ^[37] was used in determining the apparent binding constant (K_app) for the interaction of the complexes with CT-DNA.

  $ A_{\text {obs }}=(1-\alpha) c_0 \varepsilon_{\mathrm{DNA}} l+\alpha c_0 \varepsilon_{\mathrm{c}} l $

  (1)

  Here, A_obs is the observed absorbance of the solution, containing different concentrations of Cu(Ⅱ) complexes at 258 nm, α is the degree of association between CT-DNA and complex, ε_DNA and ε_c are the molar extinction coefficients at 258 nm for CT-DNA and the complex, respectively, l is the optical path length, and c₀ is the initial concentration of CT-DNA. Eq. 1 can be expressed as follows:

  $ A_{\text {obs }}=(1-\alpha) A_0+\alpha A_{\mathrm{c}} $

  (2)

  where A₀ and A_c are the absorbances of CT-DNA and the complexes at 258 nm at a concentration of c₀. The parameter α is equated to K_appc_complex/(1+K_appc_complex). Eq.1 can be rearranged to^[38]:

  $ 1 /\left(A_{\text {obs }}-A_0\right)=1 /\left(A_{\mathrm{c}}-A_0\right)+1 /\left[K_{\text {app }}\left(A_{\mathrm{c}}-A_0\right)\right] \times 1 / c_{\text {complex }} $

  (3)

  On increase of absorbance at 258 nm due to absorption of the DNA-drug complex, a linear relationship between the reciprocal concentration of the drug, 1/c_complex vs the reciprocal of difference in concentration in the absence and presence of the drug, 1/(A_obs-A₀) existed. From the above relationship, slope 1/[K_app(A_c-A₀)] and intercept 1/(A_c-A₀) values were obtained (Fig. 5). The apparent binding constants (K_app) are shown in Table 4. The K_app values for the complexes are in the order of 103-104 L·mol^-1, which is comparable to reported values for copper(Ⅱ) complexes^[39]. The results of the studies suggest a moderate binding of complexes 1-3 to CT-DNA.

  Figure 5

  

  Figure 5.  Plots of 1/(A_obs-A₀) vs 1/c_complex for absorption spectrum titration of CT-DNA with complexes 1, 2, and 3

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  Table 4

  

  Table 4.  Binding constants for complexes 1, 2 and 3

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  Complex	Binding constant/(L·mol-1)	R2
  1	1.27×104	0.929 66
  2	9.10×103	0.990 22
  3	1.51×104	0.929 66

  2.5.2   Competitive interaction with EB

  Fluorometric measurements utilizing EB as a fluorescent probe are generally undertaken to determine the binding nature of chemical species with target biomolecules such as DNA. The molecular fluorophore EB emits intense fluorescence emission when it binds to CT-DNA^[40]. The changes in the emission intensity when it binds DNA are often employed to investigate the interaction between DNA and the metal complexes^[41]. The enhanced emission intensity of EB-DNA may be quenched by the interaction of another chemical species that can bind with DNA by displacing EB probably via intercalative mode^[42]. To determine the capability of the complexes to displace EB from its EB-bound DNA adduct, a competitive EB binding study was carried out with fluorescence spectral measurements^[43]. The fluorescence spectra of EB-DNA adduct in the absence and presence of the complexes are shown in Fig. 6.

  Figure 6

  

  Figure 6.  (a) Emission spectra of DNA-EB system in the presence of increasing concentrations of complexes 1-3 along with the magnified images on the right; (b) Stern-Volmer plots of the fluorescence titration of 1-3

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  Successive addition of increasing concentration of complexes 1-3 to DNA-EB adduct led to the quenching of the fluorescence emission intensity at 592 nm of the DNA-EB moiety. The observed emission spectra suggest that the copper(Ⅱ) complexes 1-3 compete with EB to bind with CT-DNA which resulted in quenching of fluorescence intensity. On increasing the concentration of complexes 1-3 gradually, the quenching of the fluorescence intensity of the EB-DNA complex suggests the displacement of EB from the CT-DNA-EB adduct. The observed changes in the fluorescence intensity are suggestive of a probable intercalating mode of interaction of complexes 1-3 with CT-DNA^[44]. Fluorescence quenching of CT-DNA-EB adduct in the presence of the complex solution was further analyzed with the help of the Stern-Volmer equation:

  $ F_0 / F=K_{\mathrm{q}} c_{\mathrm{Q}}+1 $

  (4)

  where F and F₀ are the emission intensity in the pres-ence and absence of a quencher, c_Q is the concentration of the quencher and K_q is the Stern-Volmer quenching constant which can be obtained from the slope of the plot of F₀/F vs c_Q. The obtained values of K_q were of the order of 103 (Table 5), which are in good agreement with the results obtained from absorption spectral studies.

  Table 5

  

  Table 5.  Quenching constants of complexes 1-3

  下载: 导出CSV

  Complex	Quenching constant/(L·mol-1)	R2
  1	3.97×103	0.929 66
  2	3.50×103	0.990 22
  3	8.71×103	0.963 2

  2.6   Antibacterial activity

  The antibacterial activity data obtained in terms of zone of inhibition measurements are presented in Fig. 7. The synthesized Cu(Ⅱ) complexes were screened against Gram-negative bacteria including K. pneumoniae, E. coli, S. boydii, and Gram-positive S. aureus using disc diffusion method. The results obtained from the antibacterial screening suggest that complex 2 exhibited an appreciable activity. The zone of inhibition data showed that complex 2 was most active against K. pneumoniae, followed by S. boydii. However, complex 2 did not exhibit any antibacterial activity towards E. coli and gram-positive S. aureus. In the case of K. pneumoniae and S. boydii, the maximum inhibition was found in 100 µg per disc for both strains. Complex 2 probably binds with the cell membrane or DNA of the Gram-negative bacterial strains K. pneumoniae and S. boydii to arrest cell growth. Among the complexes studied, complexes 1 and 3 did not show any significant antibacterial activity against the bacterial strains under study.

  Figure 7

  

  Figure 7.  Histogram of zone diameter at different concentrations of complex 2 on strains K. pneumoniae (A), E. coli (I), S. aureus (J), and S. boydii (Q)

  Data are represented as mean±SD of three concentrations in three different experiments; *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance, P > 0.05.

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  2.7   Theoretical study

  In the absence of suitable single crystals, X-ray diffraction studies for structural analysis of the complexes could not be done. The optimized structures of the complexes were ascertained based on DFT studies and are shown in Fig. 8. The calculated bond distances and angles for the complexes are presented in Table S1. Theoretical study reveals that complex 1 is mononuclear and possesses a distorted square planar geometry where the Cu(Ⅱ) center is coordinated by two imine nitrogen and two oxygen atoms from two ligand moieties. The calculated Cu1—N1, Cu1—N1#, Cu1—O1, and Cu1—O1# bond lengths were found to be 0.204 693, 0.201 975, 0.203 000, and 0.190 478 nm. The determined O1 —Cu1—N1#, N1 —Cu1—O1, N1—Cu1—O1#, and N1# —Cu1—O1# bond angles are 92.878 37°, 91.208 10°, 91.360 78°, and 91.401 83° respectively.

  Figure 8

  

  Figure 8.  Geometry-optimized structures of complexes 1-3

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  In complex 2 the mononuclear Cu(Ⅱ) center is coordinated by two imine nitrogen (N1 and N1#) of two HL² ligands and oxygen atoms (O1 and O1#) from two acetate ligands resulting in a square planar coordination polyhedra around the metal center. The Cu1—N1, Cu1—N1#, Cu1—O1, and Cu1—O1# optimized bond lengths were found to be ca. 0.206 383, 0.212 627, 0.191 620, and 0.194 152 nm respectively. The optimized O1—Cu1—N1#, N1—Cu1—O1, N1—Cu1— O1# and N1#—Cu1—O1# bond angles are 97.179 06°, 88.345 37°, 87.956 52°, and 85.580 85° respectively.

  Complex 3 also contains a Cu(Ⅱ) mononuclear unit coordinated by two imine nitrogen (N1 and N1#) of two L³ ligand moieties and oxygen atoms (O1 and O1#) from two acetato ligands forming a square planar geometry around the Cu(Ⅱ) center. The Cu1—N1, Cu1— N1#, Cu1—O1, and Cu1—O1# optimized bond lengths are ca. 0.210 451, 0.208 506, 0.194 281, and 0.199 474 nm respectively, and the calculated O1— Cu1—N1#, N1— Cu1— O1, N1 —Cu1—O1#, and N1# —Cu1—O1# bond angles are 92.895 09°, 92.841 67°, 87.242 98°, and 89.350 90° respectively. The deviation of metal-ligand bond angles from the ideal value of a square planar geometry for the complexes suggests distorted square planar structures for the complexes reported herein.

  The results of elemental analyses and spectroscopic measurements are also in good agreement with the DFT-calculated optimized structures of the complexes.

  3.   Conclusions

  To summarize it may be inferred that, three new mononuclear Cu(Ⅱ) complexes, [Cu(L¹)₂] (1), [Cu(HL²)₂ (Ac) ₂] (2), and [Cu(L³)₂ (Ac)₂] (3) have been synthesized with hydrazone based Schiff base ligands HL¹, HL², and L³ derived from the condensation of benzil monohy-drazone and aldehydes, viz. salicylaldehyde, 4-hydroxy benzaldehyde, and o-anisaldehyde. Characterization methods including FTIR, UV-Vis, ¹H NMR, elemental analyses, and molar conductance measurements were used in the comprehensive characterization of the ligand and complexes. In addition to spectral character-ization, ligands were also structurally characterized by single-crystal X-ray diffraction studies.

  In complex 1, Schiff base (HL¹) is coordinated to the metal center in a bidentate fashion involving azomethine nitrogen and phenolic oxygen atoms, whereas in complex 2 and 3 Schiff base ligands HL², and L³ are coordinated in a monodentate manner through their imine nitrogen. The coordination polyhedra in complexes 2 and 3 were completed by the coordination of two unidentate acetato ligands. Biological relevance, viz. the DNA binding ability of the synthesized complexes was evaluated by spectroscopic methods. Absorption and emission spectral studies suggest moderately strong interaction of complexes 1, 2, and 3 with CT-DNA. Emission spectral study suggests the partial displacement of EB from CT-DNA-EB adduct and a proba-ble intercalative mode of binding of complexes to CT-DNA. Results of antibacterial screening indicate that complex 2 exhibits an appreciable antibacterial activity against the pathogenic bacterial strains screened.

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

  
  Acknowledgments: Dutta Purkayastha R. N., Bhattacharjee Maitri, and Boruah Smriti Rekha are thankful to the authority of Tripura University, Suryamaninagar, for providing an infrastructure facility. Bhattacharjee Maitri and Boruah Smriti Rekha are thankful to Tripura University for providing a university fellowship.
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• 

  Figure 1  An ORTEP drawing of HL¹ with the atom-numbering scheme

  Displacement ellipsoids are drawn at the 50% probability level.

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  Figure 2  An ORTEP drawing of HL² with the atom-numbering scheme

  Displacement ellipsoids are drawn at the 50% probability level.

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  Figure 3  An ORTEP drawing of L³ with the atom-numbering scheme

  Displacement ellipsoids are drawn at the 50% probability level.

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  Figure 4  Absorption spectra of CT-DNA (50 µmol·L^-1) in the presence of increasing amounts of complexes 1, 2, and 3

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  Figure 5  Plots of 1/(A_obs-A₀) vs 1/c_complex for absorption spectrum titration of CT-DNA with complexes 1, 2, and 3

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  Figure 6  (a) Emission spectra of DNA-EB system in the presence of increasing concentrations of complexes 1-3 along with the magnified images on the right; (b) Stern-Volmer plots of the fluorescence titration of 1-3

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  Figure 7  Histogram of zone diameter at different concentrations of complex 2 on strains K. pneumoniae (A), E. coli (I), S. aureus (J), and S. boydii (Q)

  Data are represented as mean±SD of three concentrations in three different experiments; *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance, P > 0.05.

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  Figure 8  Geometry-optimized structures of complexes 1-3

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  Table 1.  Crystal data and structure refinement parameters for the ligands

  Parameter	HL1	HL2	L3
  Empirical formula	C21H16N2O2	C21H16N2O2	C22H18N2O2
  Formula weight	328.36	328.36	342.38
  Crystal system	Monoclinic	Orthorhombic	Monoclinic
  Space group	P21/c	Fdd2	P21/n
  a/nm	1.688 86(8)	3.028 83(4)	1.546 51(3)
  b/nm	0.909 79(3)	2.761 76(9)	0.782 50(1)
  c/nm	1.107 49(3)	0.803 612(18)	1.585 04(3)
  β/(°)	99.377(3)		114.056(2)
  Volume/nm3	1.971 55(7)	6.722 1(3)	1.75153(6)
  Z	4	16	4
  Dc/(Mg·m-3)	1.299	1.298	1.298
  Absorption coefficient/mm-1	0.680	0.679	0.084
  F(000)	688	2 752	720
  Crystal size/mm	0.09×0.43×0.52	0.60×0.50×0.46	0.52×0.55×0.55
  θ range for data collection/(°)	2.7-66.6	4.3-66.6	2.8-25.0
  Index ranges	-20 ≤ h ≤ 20;	-36 ≤ h ≤ 30;	-18 ≤ h ≤ 18;
  	-10 ≤ k ≤ 10;	-32 ≤ k ≤ 32;	-9 ≤ k ≤ 9;
  	-13 ≤ l ≤ 13	-9 ≤ l ≤ 9	-18 ≤ l ≤ 18
  Reflection collected	59 891	40 530	45 286
  Independent reflection	2 961	2 962	2 978
  Goodness of fit on F2	1.05	1.09	1.06
  R indices (all data)	0.030 8	0.021 3	0.029 2
  Largest diff. peak and hole/(e·nm-3)	190 and -140	130 and -120	220 and -150

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  Table 2.  Selected bond lengths (nm) and bond angles (°) of the complexes

  Bond	HL1	HL2	L3
  O1—C2	0.121 83(13)	0.122 48(18)	0.121 75(14)
  N1—N2	0.140 39(12)	0.140 98(17)	0.141 00(14)
  N1—C1	0.128 80(13)	0.128 8(2)	0.128 49(13)
  N2—C15	0.128 42(13)	0.128 0(2)	0.128 52(13)
  O2—C17(—C19)	0.135 36(12)	0.136 34(19)	0.136 37(14)
  N2—N1—C1	112.00(8)	110.82(13)	113.17(9)
  N1—N2—C15	114.24(8)	111.88(13)	109.43(9)
  N1—C1—C2	121.14(9)	119.44(13)	122.74(10)
  N1—C1—C9	119.93(9)	120.92(13)	118.16(10)
  N2—C15—C16	120.95(9)	121.86(13)	123.01(10)

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  Table 3.  Hydrogen bonding parameters of the complexes

  Compound	D—H···A	d(D—H)/nm	d(H···A)/nm	d(D···A)/nm	∠DHA/(°)
  HL1	O2—H1···N2	0.092 6(17)	0.176 1(17)	0.260 08(11)	149.3(15)
  	C19—H19···O2ⅰ	0.095	0.251	0.343 34(14)	164
  	C20—H20···O2ⅱ	0.095	0.245	0.329 54(13)	149
  HL2	O2—H1···O1ⅲ	0.086(2)	0.195(2)	0.280 78(16)	175(2)
  	C15—H15···O1ⅳ	0.095	0.245	0.333 54(18)	155
  L3	C20—H20···O1ⅴ	0.095	0.256	0.344 72(15)	156
  	C22—H22B···O1ⅵ	0.098	0.256	0.346 97(14)	154
  Symmetry codes: ⅰ 1-x, -1/2+y, 1/2-z; ⅱ x, -1/2-y, -1/2+z; ⅲ -x, 1-y, z; ⅳ -1/4-x, -1/4+y, 1/4+z; ⅴ 1/2-x, -1/2+y, 1/2-z; ⅵ 1-x, 1-y, 1-z.

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  Table 4.  Binding constants for complexes 1, 2 and 3

  Complex	Binding constant/(L·mol-1)	R2
  1	1.27×104	0.929 66
  2	9.10×103	0.990 22
  3	1.51×104	0.929 66

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  Table 5.  Quenching constants of complexes 1-3

  Complex	Quenching constant/(L·mol-1)	R2
  1	3.97×103	0.929 66
  2	3.50×103	0.990 22
  3	8.71×103	0.963 2

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