Synthesis, Quantum Chemical Calculations, Molecular Docking and Studying the Effect of High Energetic Gamma Irradiation on Cu(I, II), Zn(II) and Cd(II) Complexes with their Antibacterial Activity.

New complexes of Cu(I,II), Zn(II) and Cd(II) of thiosemicarbazide ligand 1-(p-(methylanilinocetyl-4-phenyl-thiosemicarbazide)(H 2 L B ) have been prepared and characterized by 1 HNMR, Mass spectra, FT-IR, elemental analyses, molar conductance, UV-visible spectra, magnetic susceptibility measurements, thermogravimetric analysis (TGA/DTG) and X-ray diffraction pattern before and after irradiation. The results conrmed that gamma ray enhanced the stability of irradiated compounds as compared to non-irradiated compounds. XRD patterns proved that increasing the crystallinity of the samples and the particles in nano range after gamma irradiation. The obtained data indicated that the Cu(I) and Cd(II) ions coordinated to the ligand through the (C = O), N(2)H and (C = S), the ligand behaves as neutral tridentate. While in complexes Cu(II) and Zn(II)complexes (B 2 and B 3 ) the ligand behave as neutral tetradentate and coordination take place via (C = O) and two N(2)H. These studies revealed that, two kinds of stereochemical geometries; Cu(II) and Zn(II) complexes were predicted to be octahedral, Cu(I) and Cd(II)complexes were found to be tetrahedral. The theoretical conformational structure analyses were performed using density functional theory for ligand and complexes at B3LYP functional with 6-31G(++)d,p basis set for ligand and LANL2DZ basis set for complexes. The ligand and its metal complexes have been tested for their inhibitory effect on the growth of bacteria against gram-positive (Streptococcus pyogenes) and gram-negative (Escherichia coli). Results suggested that in case of 1µg/ml and 5µg/ml for Cu(II) and Zn(II) complexes have higher activity than other complexes. The chelation could facilitate the ability to cross the cell membrane of E. coli and can be explained by Tweedy’s chelation theory. Molecular docking investigation proved that; the Zn(II) complex had interesting interactions with active site amino acids of topoisomerase II DNA gyrase enzymes (code: 2XCT). was conrmed by different analytical and spectral techniques ( 1 H NMR, MS, FT-IR, UV-Vis, EPR and Powder X-ray diffraction), thermogravimetric studies as well as molecular modeling. FT-IR spectra showed that the compound behave as neutral or monobasic tetradentate. In case of complexes of Mn 2+ , Zn 2+ , Ag + and VO 2+ , through (N2-H), (C = O) or (C–O) groups. X-ray diffraction pattern of Mn 2+ , Pd 2+ and Ag + complexes before and after irradiation are recorded. XRD studies exhibited that decrease in the crystalline size of sample Mn 2+ as compared of samples Ag + and Pd 2+ upon irradiation and irradiation inuenced the crystallinity of the complexes. The possible structures of the ligand, Mn 2+ , Pd 2+ and Hg 2+ complexes have been computed by means of the molecular mechanic calculations using the hyper chem. 8.03 molecular modeling program. The effect of gamma irradiation was investigated by recording the new results of pervious spectroscopic techniques and other measurements. The TGA studies of unirradiated and irradiated complexes showed that irradiated complexes were more thermally stable than unirradiated. The compound and its metal complexes have been experienced for their inhibitory outcome on the growth of microorganisms against gram positive and gram negative. The results proved that the complexes B 1 - B 7 have potent antibacterial activity as compared to that of ligand [7–10]. In this work we described the synthesis, characterization, DFT, molecular docking as well as antibacterial activities of Cu(I,II), Zn(II) and Cd(II) of complexes of ligand


Introduction
Thiosemicarbazones are compounds which have increased importance over the decades as prospective medicine candidates. When coordinated to elements, they have evidenced as good antitumour, antimicrobial, antioxidant and antiprotozoal managers. Transition element created complexes hold several advantages over other metal chelated because of their good acceptability and decrease toxicity in living organisms [1].
Thiosemicarbazone compound are of diverse importance for the reason that of their useful for living and pharmacological activities. Thiosemicarbazone derivatives have found application in drug development for the treatment of central nervous system disorders, of bacterial infection, as well as analgesic and antiallergic agent.
Thiosemicarbazones are potent intermediates for the synthesis of pharmaceutical and bioactive materials and thus, they are used extensively in the eld of medicinal chemistry. Moreover, thiosemicarbazones have found their way into almost every branch of chemistry; commercially they are used as dyes, photographic lms, plastic and in textile industry [2] The biological activity of these compounds depends upon the starting materials and their reaction conditions, also related to molecular conformation in particular, which can also be signi cantly affected by the presence of intra-and intermolecular hydrogen bonding [3]. Thiosemicarbazones commonly performance such as chelating compound for metal ions, attachment through (C = S) and (C = N-) groups, although in several cases they behave as mono dentate compound where they bind through (C = S) only [4]. A series of copper(II) complexes of 2-phenylamino acetyl-N-phenyl hydrazine carbthioamide (H 2 L) have been prepared and characterized by chemical and physical studies. The thermal behaviors of these chelates before and after γirradiation show that the complexes have induced more thermal stability after γ-irradiation. Solid state dc electrical conductivity for complexes was investigated before and after γ-irradiation [5,6]. Recently, complexes of VO 2+ , Mn 2+ , Zn 2+ , Ru 3+ , Pd 2+ , Ag + and Hg 2+ have been prepared by reacting their metal salts with ligand, named (4-(4-chlorophenyl)-1-(2-(phenylamino) acetyl) thiosemicarbazone). Structure of synthesized metal complexes was con rmed by different analytical and spectral techniques ( 1 H NMR, MS, FT-IR, UV-Vis, EPR and Powder X-ray diffraction), thermogravimetric studies as well as molecular modeling. FT-IR spectra showed that the compound behave as neutral or monobasic tetradentate. In case of complexes of Mn 2+ , Zn 2+ , Ag + and VO 2+ , through (N2-H), (C = O) or (C-O) groups. X-ray diffraction pattern of Mn 2+ , Pd 2+ and Ag + complexes before and after irradiation are recorded. XRD studies exhibited that decrease in the crystalline size of sample Mn 2+ as compared of samples Ag + and Pd 2+ upon irradiation and irradiation in uenced the crystallinity of the complexes. The possible structures of the ligand, Mn 2+ , Pd 2+ and Hg 2+ complexes have been computed by means of the molecular mechanic calculations using the hyper chem. 8.03 molecular modeling program. The effect of gamma irradiation was investigated by recording the new results of pervious spectroscopic techniques and other measurements.
The TGA studies of unirradiated and irradiated complexes showed that irradiated complexes were more thermally stable than unirradiated. The compound and its metal complexes have been experienced for their inhibitory outcome on the growth of microorganisms against gram positive and gram negative. The results proved that the complexes B 1 -B 7 have potent antibacterial activity as compared to that of ligand [7][8][9][10]. In this work we described the synthesis, characterization, DFT, molecular docking as well as antibacterial activities of Cu(I,II), Zn(II) and Cd(II) of complexes of ligand 1-(p-(methylanilinocetyl-4-phenyl-thiosemicarbazide)(H 2 L B ).

Materials
All chemicals used in this study were of analytically reagent grade, commercially available from Fulka and used without previous puri cation as CuI (anhydrous), Cu(ClO 4 ) 2 .3H 2 O, ZnCl 2 .3H 2 O compounds which represent the metal ions used in and CdCl 2 (anhydrous), complexation process. All solvents were used as it is without previous puri cations.

Synthesis of thiosemicarbazide ligand (H 2 L B )
The organic ligand 1-(p-(methylanilinocetyl-4-phenyl-thiosemicarbazide) (H 2 L B ) was prepared by mixing equimolar amount of desired hydrazide (0.01mol) in 10 ml of absolute ethanol and the appropriate amount of phenyl isothiocynate in 10 ml of absolute EtOH. The reaction mixture was re ux for 6 hrs. After cooling, the resulting precipitate was ltered off, washed several times with ethanol and diethyl ether and dried in vacuum in presence of P 4 O 10 .

Synthesis of metal complexes
The metal complexes were prepared by adding to hot absolute ethanol solution (~ 20 mL) MX 2 .nH 2 O where M = Cu(I and II), Zn(II), Cd(II), and X = I − , ClO 4 -,SO 4 −− and Cl − , − , n = 0-1 in appropriate molar ratio. Where: λ is the X-ray wavelength in the nanometer, K is factor related to crystallite shape, and with a value about 0.9 and ß is the peak width at half maximum height. The value of ß in the 2θ axis of diffraction shape must be in radians. The θ is the Bragg angle and be able to in radians since the Cos θ compatible with the same number.

Computational studies
The input les of ligand and its metal complexes were prepared with GaussView 5.0.8 [12] Gaussian 09 rev. A.02 [13] was used to make the all calculations by the DFT/B3LYP method. 6-31G (++)d, p and LANL2DZ are the standard basis sets for the synthesized ligand and its metal complexes, respectively.

Irradiation studies
For irradiation studies of solid samples of ligand and complexes were subjected to γ-irradiation [14] to a dose of 60 kGy using Indian 60 Co γ-ray cell type GE-4000 A (at room temperature at the Egyptian Atomic Energy Authority Nasr City, Egypt) at a dose rate of 2.2 kGy h − 1 . After removing the samples from the radiation eld the FT-IR, absorption spectra ,XRD and thermal analysis (TG/DTG) and biological activity of the irradiated samples were investigated by the same methods used for before irradiated compound .

Antibacterial activity
The in vitro antibacterial activity studies were carried out at Genetic Engineering and Biotechnology Research Institute, Department of Microbial Biotechnology at Sadat City University, Egypt, by using Broth Dilution Method [15,16] with some alterations, to investigate the inhibitory effect of some synthesized complexes before (B 1 -B 4 ) and after (A 1 -A 4 ) irradiation on the sensitive organisms Streptococcus pyogenes as Gram-positive bacteria and Escherichia coli as Gram-negative bacteria. Nutrient broth medium was prepared by using Brain Heart Infusion (BHI) broth and distilled water. The test compounds in measured quantities were dissolved in DMSO which has no inhibition activity to get two different concentrations (1mg/mL, 5mg/mL) of compounds. The strains selected for the study were prepared in (BHI) broth medium with shaking and autoclaved for 20 min 15 pounds of pressure and at 121 o C before inoculation. The bacteria were then cultured for 24 h at 37 o C in an incubator.. One ml of the standard bacterial culture was used as inoculation in a nutrient broth. For growth studies, culture of microbial cells were inoculated and grown aerobically in BHI broth for control and along with various concentrations of the test compounds in individual asks. Growth was calculated turbidometrically at 650 nm using conventional Spectrophotometer, in which turbidity produced is measured by taking absorbance and compared with turbidity produced by control. The growth rate of different bacteria in absence as well as in presence of test compounds was performed for each concentration. Absorption measurements were accomplished by spectrophotometer after 24 and 48 h of incubation to determine the number of viable organisms per milliliter of sample and were used to the calculated the % inhibition.

Molecular docking
All docking steps were done by MOE 2008 (Molecular Operating Environment) software to simulate the binding model of these compounds into topoisomerase II DNA gyrase enzymes (2XCT). The protein crystal structure was obtained from the Protein Data Bank (PDB).

Elemental analysis and molar conductance
The analytical and physical data of the prepared ligand and metal complexes are collected in Table (1

Nuclear magnetic resonance spectroscopy
The 1 H NMR is a helpful tool for the preparation of organic compounds in conjugation with other spectrometric information, nuclear magnetic resonance is a physical phenomenon based upon the magnetic properties of an atom's nucleus.. The most commonly used nuclei are hydrogen l and carbon 13 , although certain isotopes of many other elements nuclei can also be observed. Comparison of the proton nuclear magnetic resonance of 1-(p-(methylanilinocetyl-4-phenyl-thiosemicarbazide) ligand before and after γ-irradiation (H 2 L B and H 2 L A ) recorded in DMSO-d 6 solution is found in (Fig. 1). The 1 H NMR spectrum of the ligand before γ -irradiation(H 2 L B ) in DMSO-d 6 exhibited a chemical shift (δ \ppm) = 2.5ppm for (DMSO) before and after γ-irradiation, the N(4)H signal appears with 9.44 ppm and the N(2)H signal appears with 9.51, 10.05 ppm indicating the involvement of these hydrogen through intra-molecular hydrogen bonding with the carbonyl oxygen, the peak of N(1)H appeared at 9.62 ppm for ligand after gamma irradiation .The singlet signal appears with 2.14, 3.9, 3.7 ppm attributed to the protons of methyl CH 3 , singlet signal appears with 3.7 ppm attributed to the protons of CH 2 , the multiplet signal appears with 5.85-7.1, 6.5-7.4 ppm attributed to the aryl protons. The intensity of the bands after irradiation are higher than before irradiation and some bands disappear upon irradiation [19,20].

Mass spectroscopy
Mass spectral data con rm the structure of the ligand as indicated by the molecular ion peak (M + ) corresponding to their molecular weight. MS of H 2 L B (Fig. 4)   shift as compared with before γ-irradiation [21]. After γ-irradiation the intensity of the peaks are more sharper than before γ-irradiation. Table 2 Infrared spectral bands (cm − 1 ) for ligand and its metal complexes before and after irradiation  The IR spectra of Cu(I, II) complexes before and after γ-irradiation display new bands at 603, 614 and 529,545 cm − 1 assigned to υ(Cu-O) and υ(Cu-N) respectively [22,23]. IR spectra of the complexes (A 1 and A2) showed that the intensity of the IR bands became more intense than before γ-irradiation [24].
3.4.3. IR spectra of Zinc(II) complexes before and after γirradiation The IR spectra of Zn(II) complexes (Fig. 9)

UV-Vis spectra and magnetic moment properties
The electronic spectral bands of the ligand (H 2 L B ) and Cu(I, II), Zn(II) and Cd(II) complexes in DMF solution within the range 200-800 nm are tabulated in Table 3 and depicted in Figs. (11)(12)(13)(14). The electronic spectrum of the ligand exhibits bands at 314, 292 and 279 nm, respectively.
3.5.1. The electronic absorption spectra of copper complexes before and after γ-irradiation.
The electronic spectra of Cu(I) complexes before and after irradiation (B 1 and A 1 ) display bands at 300 and 409 nm, respectively. Cu(I) ions have the d 10 con guration and therefor the Cu(I) complexes should not exhibit any d-d transition and have tetrahedral geometry [27].
398 and 653 nm in DMF refer to L→M charge transfer and d→d transitions, respectively in octahedral geometry [28]. Diamagnetic behavior of complex (B 1 ) and the magnetic suitability value of complex (B2) is 1.78 B.M., which is an indicative of tetrahedral and octahedral geometry [27,29].

Zinc(II) complexes before and after γ-irradiation
The electronic absorption spectra of Zn(II) complexes before and after γ-irradiation displayed bands at 300, 282; 448 and nm in DMF solution, octahedral structure of Zn(II) complex is suggested which is diamagnetic in nature [30].

Cadium(II) complexes before and after γ-irradiation
The electronic absorption spectra of Cd(II) complexes before and after γ-irradiation displayed three bands at 284,390, 610 nm ; 282,385and 608 nm in DMF solution attributed to charge transfer transition which assigned to tetrahedral geometry around Cd(II) ion [31,32]. The Cd(II) complexes are diamagnetic because of d 10 electronic con guration of Cd(II) ion [33]. . The XRD patterns of the synthesized compounds were carried out in order to give an insight about the lattice dynamics of the compounds. The X-ray diffraction were recorded by using (Cu Kα ) radiation (1.5406 Å). The intensity were collected over a 2h range of 5-90 o . The average grain size of the samples was estimated using the diffraction intensity peak. The pattern found re ects a tracker on the fact that each solid describes a de nite compound of a de nite construction which is not contaminated with initial materials. This identi cation of the complexes was done by a known method [34].The mean grain size (D) of the particles was determined from the XRD line broadening measurement using the sized complexes may serve strongly in different application elds in between the biological one [35].
Figures (15)(16)(17) show that Cu(I,II), Zn(II) complexes new peaks appear and some peaks displaced to longer interplanar spacings. The major factors tending to in uence the intensity of powder patterns are structure factor, polarization factor, atomic scattering factor, multiplicities and preferred orientations. Upon irradiation, the position of atoms in the lattice changes and consequently, the scattering power also changes, leading to changes in intensity which display high resistance [36]. It should be noted that the Zn(II) complex (A 3 ) after irradiation increases the crystalline size than B 3 before irradiation.

Thermal behavior of ligand and metal complexes before and after γ-irradiation
The thermal behavior of the ligand and Cu(I, II), Zn(II) and Cd(II) complexes before and after γ-irradiation was investigated by thermogravimetric technique in temperature range 25-800°C. The thermal behavior data of the ligand and Cu(I,II), Zn(II) and Cd(II) complexes (B 1 , B 2 , B 3 , B 4 and A 1 ,A 2 , A 3, A 4 ) before and after γ-irradiation are tabulated in Table 5 and depicted in Figs. (18-20).

The ligand before and after γ -irradiation
The TG curves of the ligand before and after γ-irradiation show that it is thermally stable till 140°C,125°C, respectively. Also the TG curves show three decomposition steps in the temperature range 140-550 o C ;125-510°C with total weight loss of Calc.100% (Found 100%) before and after γ-irradiation, respectively.

Zinc (II) Complex
The  From all of the above, the suggested chemical structure of metal complexes are shown in Scheme 1

Structure characterization with DFT study
The geometric structures of H 2 L B ligand and its metal complexes were optimized as shown in (Fig. 21). Upon coordination of H 2 L B to the metal atom, some bond lengths become slightly longer than in the free ligand accompanied with changes in angles that were clari ed in Table 6

Antibacterial Activity
The synthesized ligand and its metal complexes were separately exposed to gamma irradiation to test their improvement as active antibacterial drugs [39]. Results in Table (8), Figs. 23 and 24 showed antibacterial activity against the tested microbes. Generally, it was found that antibacterial activity of both the synthetic ligand and metal complexes before and after γ-irradiation was proportionally increased with increased concentration. The tested compounds before and after γ-irradiation are found to have remarkable biological activity. The results in >B 2 >B 1 for before and after irradiation with1µg/ml concentration [23]. Antibacterial activity of 5µg/ml concentration for both the free acyclic ligand and its complexes before and after irradiation followed the order: B 2 = B 3 > H 2 L A > A 2 > A 1 > A 3 > H 2 L B >B 1 when compounds were used with S. pyogenes [40]. Results suggested that in case of 1µg/ml and 5µg/ml Cu(II) and Zn(II) complexes have higher activity than other complexes The chelation could facilitate the ability to cross the cell membrane of E. coli and can be explained by Tweedy's chelation theory. Chelation/complexation could enhance the lipophilic nature of the central metal atom which in turn, favors its permeation through the lipoid layer of the membrane thus causing the metal complex to cross the bacterial membrane more effectively thus increasing the activity of the complexes. Besides from this many other factors such as solubility, dipole moment, conductivity in uenced by metal ion may be possible reasons for remarkable antibacterial activities of these complexes [41]. Exposure to gamma irradiation remarkably enhanced the antibacterial activity for both the ligand and its complexes when it was used in case of E.coli. The activity also increased after irradiation in case of S. pyogenes. This may be attributed to the different nature of the cell wall for both microbes which may be correlated with other factors such as solubility, dipole moment, and conductivity in uenced by metal ion. Additionally, exposure to gamma irradiation increased the antibacterial activity of both the free a cyclic ligand and their complexes when used with both concentrations (1µg/ml and 5µg/ml) in case of the Gram positive S. pyogenes bacterium. It also has been observed that some moieties such as N(2)H linkage introduced into such compounds exhibits extensive biological that may be responsible for increase in hydrophobic character and liposolubility of the molecules in crossing the cell membrane of the microorganism and enhance biological utilization ratio and activity of complexes activity [42]. The antibacterial studies of the prepared compounds screened against both Gram positive and Gram negative bacteria proved that these compounds exhibit remarkable antibacterial activity and can be used in the future as therapeutic drugs for pathogenic bacterial diseases. The molecular docking To understand the interaction of all the synthesized molecules with topoisomerase II DNA gyrase enzymes, the crystal structure of topoisomerase II was downloaded from Protein Data Bank (PDB ID: 2XCT) and the molecular docking studies were performed using the Moe program. The protein ligand interaction plays a signi cant role in structural based drug designing. The different types of interactions are mentioned in Table 9 and seen in Fig. 25.
The preferred compounds Cd(II) Cu(II) and Zn(II) complexes had a scoring value of − 5.02, − 9.41 and − 10.87 ,respectively. The Zn(II) complex showed the highest binding a nity and interaction with topoisomerase II DNA gyrase enzymes (2XCT) by using most types of protein binding interactions. The binding a nity of our compounds achieved higher or the same values numerous previous works against the same type of protein [43,44]. The molecular docking of our work supported that the chelates are more active than their parent ligand against the same microorganism as mentioned also in many of our previous works [45][46][47][48].   Antibacterial activity for ligand and copper, zinc and cadium (B1, B2,B3,B4 and A1,A2,A3, A4) complexes before and after irradiation against E. coli Antibacterial activity for ligand and copper, zinc and cadium (B1, B2,B3, B4 and A1,A2,A3, A4) complexes before and after irradiation against S. pyogenes