Molecular Docking, DFT Calculations, Effect of High Energetic Ionizing Radiation, and Biological Evaluation of Some Novel Metal (II) Heteroleptic Complexes Bearing the Thiosemicarbazone Ligand

New Pb(II), Mn(II), Hg(II), and Zn(II) complexes, derived from 4-(4-chlorophenyl)-1-(2-(phenylamino)acetyl)thiosemicarbazone, were synthesized. The compounds with general formulas, [Pb(H2L)2(OAc)2]ETOH.H2O, [Mn(H2L)(HL)]Cl, [Hg2(H2L)(OH)SO4], and [Zn(H2L)(HL)]Cl, were characterized by physicochemical and theoretical studies. X-ray diffraction studies showed a decrease in the crystalline size of compounds that were exposed to gamma irradiation (γ-irradiation). Thermal studies of the synthesized complexes showed thermal stability of the Mn(II) and Pb(II) complexes after γ-irradiation compared to those before γ–irradiation, while no changes in the Zn(II) and Hg(II) complexes were observed. The optimized geometric structures of the ligand and metal complexes are discussed regarding density functional theory calculations (DFT). The antimicrobial activities of the ligand and metal complexes against several bacterial and fungal stains were screened before and after irradiation. The Hg(II) complex has shown excellent antibacterial activity before and after γ-irradiation. In vitro cytotoxicity screening of the ligand and the Mn(II) and Zn(II) complexes before and after γ-irradiation disclosed that both the ligand and Mn(II) complex exhibited higher activity against human liver (Hep-G2) than Zn(II). Molecular docking was performed on the active site of MK-2 and showed good results.


Introduction
Thiosemicarbazones (TSCs) are a class of inorganic metal chelators that exhibit various complexes with transition metals, including Cu, Pd, Ni, and others [1,2]. TSCs and their metal complexes have interesting chemistry because of their variable bonding modes, promising biological implications, structural diversity, and ion-sensing ability [3]. In addition, they have been used as drugs and possess a wide range of biological activities, such as antibacterial [4], antifungal [5,6], antiviral [7], antiamoebic [8], antimalarial [9], and antitumor [10] effectiveness. Numerous literature studies have presented that the biological properties of metal complexes with NS, ONS, and ONN chelating thiosemicarbazones derivatives using various carbonyl compounds, and its complexes [11], are analogs to the metallo-salen compounds of the O,N,N,O-chelating set and have biologically active structures. Antimicrobial activity of vanillin-4-methyl-4-phenyl-3-thiosemicarbazone complexes with cobalt(II), nickel(II), copper(II), and zinc(II) metal ions were examined against All organic compounds and solvents were purchased from Fluka or Merck, Naser City, Egypt. The metal salts Pb(CH 3 COO) 2 , MnCL 2 , HgSO 4 , and ZnCL 2 were obtained from Fluka and then used for complex synthesis without further purification.

Synthesis of Metal Complexes
Different metal complexes were synthesized by stoichiometric addition of an ethanolic solution of metal salts (MX 2 ) (where M = Pb(II), Mn(II), Hg(II), or Zn(II), and X = Cl, SO 4 , or CH 3 COO) and ethanolic solution of 4-(4-chlorophenyl)-1-(2-(phenylamino)acetyl)thiosemicarbazone, which have been previously reported [18]. The mixture was magnetically stirred at 60 • C for 6-8 h. The obtained precipitates were filtered, washed with anhydrous diethyl ether, and then dried under a vacuum in the presence of P 4 O 10 to afford the complexes (B 1 -B 4 ), as shown in Scheme 1.

Physical Measurement
Elemental analyses (C, H, and N) were carried out at the Microanalytical Unit, Cairo University, Egypt. Metal content complexometric titration was estimated using EDTA following the standard literature methods, as reported by Basset et al. [19]. The FT-IR spectra were noted as KBr pellets using a Nenexeus-Nicolidite-640-MSAFT-IR spectrometer (4000-400 cm −1 ). Mass spectra were acquired using the electron impact (EI) ionization technique at 70 eV using a Hewlett-Packard MS-5988 GC-MS instrument at the Microanalytical Center, National Research Centre, Dokki, Cairo, Egypt. The UV-visible absorption spectra were measured in an N, N-dimethylformamide (DMF) solution (10 −3 M) using a 4802 UV/vis double beam spectrophotometer (Dayton, NJ, USA). The molar conductivity measurements were recorded using a Tacussel conductometer type CD6N in DMF solution (10 −3 M). The magnetic properties of all complexes were recorded at room temperature by the modified Gouy method using a Magnetic Susceptibility Johnson Matthey Balance. The effective magnetic moments were calculated using the relation µeff = 2.828 (X m T) 1/2 B.M., where X m is the molar magnetic susceptibility corrected for diamagnetism of all atoms in the compounds using Selwood and Pascal's constants. Thermal analysis (TGA/DTG) was obtained by using a Shimadzu DTG-50 Thermal analyzer with a heating rate of 10 • C/min in a nitrogen atmosphere with the rate of 20 mL/min in a temperature range of 25-800 • C using platinum crucibles at the Central Lab, Faculty of Science, Menoufia University, Egypt. Using the Rigku Model ROTAFLEX Ru-200, X-ray diffractograms of the solid samples were measured at the National Research Centre, Cairo, Egypt. Structural analysis of the X-ray diffractograms given by computer control formally was finished using a Philips X'Pert Molecules 2021, 26, 5851 3 of 24 MPD X-ray diffractometer ready with Cu radiation Cu Ka (k = 1.540 56 Å). Usually, the most powder diffractometers use the Bragg-Brentano parafocusing geometry. The X-ray tube was utilized for a copper tube operating at 40 kV and 30 mA. The scanning range (2θ) was 20-80 • with a step size of 0.02 • and a counting time of 3 s/step. Quartz was used as the standard material to calibrate the instrumental extension. This identification of the complexes was done using the method described by Nair and Appukuttan [20] from the fit identified by the Scherrer formula; the average crystallite size, L, is measured as L = ňK = ßcosθ, where ňis the X-ray wavelength in the manometer, K is a constant equal to 0.9 related to crystallite shape, and ß is the peak width at half maximum height. The value of ß in the 2θ axis of the diffraction shape must be in radians. The θ is the Bragg angle in radians since Cos θ is compatible with the same number.

Physical Measurement
Elemental analyses (C, H, and N) were carried out at the Microanalytical Unit, Cairo University, Egypt. Metal content complexometric titration was estimated using EDTA following the standard literature methods, as reported by Basset et al. [19]. The FT-IR spectra were noted as KBr pellets using a Nenexeus-Nicolidite-640-MSAFT-IR spectrometer (4000-400 cm −1 ). Mass spectra were acquired using the electron impact (EI) ionization technique at 70 eV using a Hewlett-Packard MS-5988 GC-MS instrument at the Microanalytical Center, National Research Centre, Dokki, Cairo, Egypt. The UV-visible absorption spectra were measured in an N, N-dimethylformamide (DMF) solution (10 −3 M) using a 4802 UV/vis double beam spectrophotometer (Dayton, NJ, USA). The molar conductivity measurements were recorded using a Tacussel conductometer type CD6N in DMF solution (10 −3 M). The magnetic properties of all complexes were recorded at room temperature by the modified Gouy method using a Magnetic Susceptibility Johnson Matthey Balance. The effective magnetic moments were calculated using the relation µeff = 2.828 (XmT) 1/2 B.M., where Xm is the molar magnetic susceptibility corrected for diamagnetism

Computational Study
In this part, we tried to discover the optimized geometrical parameters, such as bond lengths, bond angles, and net charges, on the coordinated atoms. The total energies (highest occupied molecular orbital (HOMO) energies, lowest unoccupied molecular orbital (LUMO) energies, and the dipole moments) for the ligand and complexes were computed. Density Functional Theory (DFT) at levels B3LYP, 6-311G, and LANL2DZ as the basis sets are used in all ligands and complexes calculations, respectively [21]. These calculations were carried out using G09W software [22]. All docking steps were done by Molecular Operating Environment (MOE 2008) software (Chemical Computing Group Inc., Montreal, QC, Canada) [23] to simulate the binding model of these compounds into an MK-2 enzyme (3WI6).

Irradiation Studies
For the irradiation studies, the solid samples of prepared compounds (B, B 1 , B 2 , B 3 , and B 4 ) were subjected to a 60 kGy γ-irradiation dose with a rate equal to 2.2 kGy h −1 [24]. The test was performed using the Indian 60 Co γ-ray cell type GE-4000 A at room temperature (at the Egyptian Atomic Energy Authority (EAEA), Nasr City, Egypt). After removing the samples from the radiation field, the FT-IR, absorption spectra, thermal analysis (TG/DTG), anti-bacterial, and anti-cancer activities of the irradiated samples were investigated. X-ray powder diffraction analyses of the un-irradiated (B, B 2 , and B 4 ) and irradiated (A 2 and A 4 ) samples of the synthesized compounds were conducted using a Rigku Model ROTAFLEX Ru-200 at the National Research Centre, Dokki, Cairo, Egypt. The divergence and receiving slits values were 1 and 0.1, respectively.

Antimicrobial Assay
The antimicrobial activity of the synthesized compounds was determined using the disc-agar diffusion method [25] at the microanalytical unit of Cairo University, Egypt. The antimicrobial activity was performed against the sensitive organisms, including Escherichia coli (Gram-negative bacteria), Staphylococcus aureus (Gram-positive bacteria), Aspergillus flavus (fungi), and Candida albicans (fungi). Standard discs of ampicillin (antibacterial agent) and amphotericin B (antifungal agent) were served as positive controls for antimicrobial activity, while the filter discs impregnated with 10 µL of the solvent dimethyl sulfoxide (DMSO) were used as a negative control. Briefly, 100 µL of the test bacteria/fungi was grown in 10 mL of fresh media until they reached a count of approximately 10 8 cells/mL for bacteria or 10 5 cells/mL for fungi [26]. A total of 100 µL of microbial suspension was spread onto agar plates corresponding to the broth, in which they were maintained. The plates were inoculated with filamentous fungi (Aspergillus flavus) at 25 • C for 48 h; Gram (+) bacteria (Staphylococcus aureus) and Gram (-) bacteria (Escherichia coli) were incubated at 35-37 • C for 24-48 h, and yeast (Candida albicans) incubated at 30 • C for 24-48 h. Subsequently, the diameters of the inhibition zones (in millimeters) were measured. Blank paper disks (Schleicher & Schuell, Spain, Sigma-Aldrich, St. Louis, MO, USA) with a diameter of 8.0 mm were impregnated with 10 µL of the tested concentration of the stock solutions. When a filter paper disc, impregnated with a tested chemical, is placed on agar, the chemical will diffuse from the disc into the agar. This diffusion will locate the chemical in the agar only around the disc. Inhibition of the organisms, which is evidenced by the clear zone surrounding each disk, was measured and used to calculate the mean of the inhibition zone.

Cytotoxicity Assays
The cytotoxicity sample was prepared on cells by inoculating a 96-well tissue culture plate with 1 × 10 5 cells/mL (100 µL/well), incubated at 37 • C for 24 h, with washing media; the cell monolayer was washed twice to improve the complete monolayer sheet, and the growth medium was poured from 96-well microtiter plates after confluent sheet of cells were formed. Two-fold dilutions of the tested sample were prepared in Roswell Park Memorial Institute (RPMI) medium with 2% serum (maintenance medium). Then, 0.1 mL of each dilution was tested in different wells, leaving 3 wells as the control, receiving only a maintenance medium, and the plate was incubated at 37 • C and subsequently examined. Cells were checked for any actual indications of harmfulness, e.g., incomplete or complete loss of the monolayer, rounding, shrinkage, or cell granulation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was prepared (5 mg/mL in PBS) (Bio Basic Inc., Markham, ON, Canada), and then 20 µL MTT solution was added to each well. To thoroughly mix the MTT into the media, samples were incubated at 37 • C, 5% CO 2 , for 1-5 h to allow the MTT to be metabolized, and then placed on a shaking table at 150 rpm for 5 min, dumping off the media (to remove the dry residue on the paper towels if necessary). Formazan (MTT metabolic product) was resuspended in 200 µL DMSO solution and placed on a shaking table at 150 rpm for 5 min to mix the formazan into the solvent thoroughly. Finally, the optical density was recorded at 560 nm and the subtract background was measured at 620 nm. The optical density should be directly correlated with cell quantity [27,28].

Physicochemical Properties
The experimental results showed that all the synthesized metal complexes are colored, stable in air, and insoluble in most organic solvents, except DMF and DMSO. The elemental analysis and physical data are summarized in Table 1
Infrared spectra indicated that the intensity of the bands is sharper by using gamma rays [24].

Electronic Spectral Bands
The electronic spectral bands of the ligand and Pb(II), Mn(II), Hg(II), and Zn(II) complexes before (B, B 1 , B 2 , B 3 , and B 4 ) and after (A, A 1 , A 2 , A 3 , and A 4 ) γ-irradiation (λ max , nm) in DMF solution in the range of 200-800 nm at room temperature are listed in Table 3 and Figure 2. UV spectra of the ligand (B and A) were perceived as the existence of two absorption bands at 260, 300, and 266, 303 nm before and after γ-irradiation, respectively, assigned to the π-π* transition. While, the UV-visible spectra of the Mn(II) complexes (B 2 and A 2 ) before and after irradiation exhibited bands at 294, 292, 378, and 376 nm, respectively. The Zn(II) complexes (B 4 and A 4 ) before and after irradiation also displayed bands at 291, 294, 375, and 378 nm, which may be assigned to n-π* transitions, representing a square planar geometry [31,32]. The electronic spectra of the Hg(II) complexes before and after irradiation (B 3 and A 3 ) resulted in bands at 276, 281, 372, and 376 nm [33,34]. On the other side, the electronic spectra of the Pb(II) complexes (B 1 and A 1 ) show three-bands at 270, 300, 320, and 354 nm assigned to n-π* transitions in an octahedral geometry around the Pb(II) ion, which is further confirmed by its diamagnetic nature [35,36]. The complexes showed no d-d band; the complexes contained only paired electrons and were diamagnetic.
After γ-irradiation, all peaks presented in the spectral diagram were observed. The difference between the electronic spectra of the ligand and its complexes in changing the value of the λ max position and the value of absorbance were also investigated, indicating no change in the complexes' geometry; this result agrees with previous related studies. Irradiation can induce perturbation of energy levels as well as a deformity in the molecule [17]. Table 3. Electronic spectral data λ max (nm) at room temperature of the ligand (H 2 L) and their metal complexes before and after irradiation.

PXRD of the Ligand and Metal Complexes
The X-ray diffractograms of the ligand (B) and the Mn(II) (B 2 , A 2 ) and Zn(II) (B 4 , A 4 ) complexes were evaluated (Table 4 and Figure 3 and Figure S2). The powder diffraction patterns were recorded over the 2θ = 5-90 range lattice constants. The intensities of the powder lines and the corresponding 2θ values are different for the irradiated samples, indicating the amorphous nature of the complex, whereas, upon irradiation. the sample changed to crystalline materials. The average particle size of the crystalline structure of the ligand (B) and its complexes before and after irradiation was calculated using Scherer's equation [27,36]. The Scherer's constant (K) in the formula refers to the particle's shape, and it generally has a value of 0.9. It was found that the calculated crystalline size was in the nano range. The crystallite sizes were 35.12, 14.15, 32.59, 46.7, and 52.7 nm for the ligand, Mn(II), and Zn(II) unirradiated (B, B 2 , B 4 ) and irradiated (A 2 , A 4 ), respectively. The change in the size of the crystals may be due to the stress induced by the irradiation.
complexes before and after irradiation.

PXRD of the Ligand and Metal Complexes
The X-ray diffractograms of the ligand (B) and the Mn(II) (B2, A2) and Zn(II) (B4, A4) complexes were evaluated (Table 4 and Figures 3 and S2). The powder diffraction patterns were recorded over the 2θ = 5-90 range lattice constants. The intensities of the powder lines and the corresponding 2θ values are different for the irradiated samples, indicating the amorphous nature of the complex, whereas, upon irradiation. the sample changed to crystalline materials. The average particle size of the crystalline structure of the ligand (B) and its complexes before and after irradiation was calculated using Scherer's equation

Mass Spectra
The mass spectra were mainly performed to confirm the composition and support the structure of the synthesized chelates. The molecular ion peaks for the Hg(II) complex were examined at m/z = 849.1 (Table 1 and Figure 4), which suggests the stoichiometry of the metal and ligand in metal chelates as a 2:1 ratio. The observed data of the complexes are in agreement with their formula as designated from the microanalytical data.

Mass Spectra
The mass spectra were mainly performed to confirm the composition and support the structure of the synthesized chelates. The molecular ion peaks for the Hg(II) complex were examined at m/z = 849.1 (Table 1 and Figure 4), which suggests the stoichiometry of the metal and ligand in metal chelates as a 2:1 ratio. The observed data of the complexes are in agreement with their formula as designated from the microanalytical data.   The proposed structures of the complexes were established from analytical and multi spectroscopic methods (Scheme 1). The proposed structures of the complexes were established from analytical and multi spectroscopic methods (Scheme 1).

Thermal Analysis
The thermal behavior of the ligand (B, A) and Pb(II) (B 1 , A 1 ), Mn(II) (B 2 , A 2 ), Hg(II) (B 3 , A 3 ), and Zn(II) (B 4 , A 4 ) complexes before and after γ-irradiation are listed in Table 5 and Figure 5a-e. The TG curves of B and A in Figure 5a revealed thermal stability till 190 • C and 240 • C, and also showed two decomposition steps in a temperature range of 190-633 • C and 240-680 • C, with the total weight loss of calc. 99.9% (found 100%) and calc. 100% (found 100%) before and after γ-irradiation, respectively. After irradiation, the thermogravimetric analyses (TG) curves of the ligand revealed that γ-irradiation stimulated more thermal stability of the substance than those that were un-irradiated. These results are consistent with the structure of the ligand resolute as determined by the elemental analysis and IR spectroscopy.  Figure 5b shows that the TG curves of the Pb(II) complexes before B 4 and after A 4 γ-irradiation exhibited weight loss calc. 6.047% (found 6.75%) at a temperature range of 30-125 • C, attributed to losing one molecule of water and ethanol on one step. On further heating, the complexes decomposed at 245-548 • C. The final stage ended with the remaining Pb as the final residue. After γ-irradiation, the TG curve of (A 4 ) was similar to (B 4 ) before γ-irradiation. Figure 5c shows TG curves for the Mn(II) complexes before B 1 and after A 1 γirradiation. The TG curves of B 1 exhibited three degradation stages; the first stage in a temperature range of 170-402 • C exhibited weight loss (calc./found % 62.34/62.31) allocated to the loss of the C 24 H 22 Cl 3 N 4 moiety. The second stage in a temperature range of 436-553 • C (calc./found % 13.19/13.22) was correlated with the loss of the C 3 H 4 N 2 S moiety. The third stage in a temperature range of 719-791 • C (calc./found % 11.35/11.32) corresponded to the loss of the C 2 H 2 N 2 S moiety, leaving species MnO + CO in the fourth stage as the final remainder over at 791 • C. Moreover, the TG curve of A 1 displayed three degradation stages; the first stage in a temperature range of 172-329 • C exhibited weight loss (calc./found % 77.64/77.62) that was allocated to the loss of the C 28 H 26 Cl 3 N 4 S 2 moiety. The second stage in a temperature range of 350-652 • C (calc./found % 9.23/9.21) corresponded to the loss of the CH 2 N 4 moiety, leaving species of MnO + CO in the third stage as the final remainder over at 652 • C.  Figure 5e shows TG curves for the Zn(II) complexes before B2 and after A2 γ-irradiation. It observed that the TG curves of B2 were similar to that of A2, and both of them exhibited four degradation stages; the first stage exhibited weight loss (calc./found % 55.76/55.74) in a temperature range of 152-356 °C, which was allocated to the loss of the

DFT Calculations of the Ligand and Metal Complexes
The natural charges, obtained from Natural Bond Orbital Analysis (NBO), showed that the more negative active sites were in the following order: S14 (−0.29189) < N12  In addition, the third stage in a temperature range 296-421 • C (calc./found % 11.30/11.32) was correlated with the loss of SO 4 . The fourth stage in a temperature range of 429-615 • C (calc./found % 3.53/3.55) corresponded to the loss of NO, leaving metal oxide (2HgO) as the final residue over at 615 • C. The TG curve after irradiation A 3 was similar to before irradiation B 3 ,with a different extent of dissociation. Figure 5e shows TG curves for the Zn(II) complexes before B 2 and after A 2 γ-irradiation. It observed that the TG curves of B 2 were similar to that of A 2 , and both of them exhibited four degradation stages; the first stage exhibited weight loss (calc./found % 55.76/55.74) in a temperature range of 152-356 • C, which was allocated to the loss of the C 23 H 18 N 2 Cl 3 moiety. The second stage in a temperature range of 482-652 • C (calc./found % 22.94/22.92) corresponds to the loss of the C 4 H 8 N 4 S 2 moiety. While, the third stage in a temperature range of 733-799 • C (calc./found % 5.47/5.50) was correlated with the loss of the CH 2 N 2 moiety, leaving Zn + 2CO in the fourth stage as the final remainder over at 799 • C.

DFT Calculations of the Ligand and Metal Complexes
The natural charges, obtained from Natural Bond Orbital Analysis (NBO), showed that the more negative active sites were in the following order: S14 (−0.29189) < N12 (−0.40986) < O10 (−0.65733). Thus, the metal ions preferred the coordination through O10, N12, or S14, forming membered rings. Figure S3 shows the optimized structures of the ligand and metal complexes as the lowest energy configurations. The lead atom is six-coordinate in an octahedral geometry by using the S14, N12, and O-acetate donor atoms. However, the other metal complexes were optimized in tetrahedral shapes using the most electron-donating atoms S14 and N12. Many bond lengths were elongated after coordination that supported the coordination through the previously mentioned donor sites. Some of these bond lengths were elongated, such as R(C13-N15), R(N12-C13), R(N12-N11), R(C9-O10), and R(C8-N7) [37]. The polarity of ligand increased after complexation by its coordination with metal ions, as indicated from the magnitude of their dipole moments. The ionic complexes have higher polarity than the non-electrolytic complexes. The natural charges computed from the NBO analysis on the coordinated atoms are observed in Table 6 Table 7 shows some of the optimized bond lengths (Å) and bond angles (degrees) for H 2 L and the complexes using B3LYP/6-311G. The coordinates of the optimized ligand and complexes are tabulated in Tables S1-S5.
14241 - Table 7. Some of the optimized bond lengths (Å) and bond angles (degrees) for H 2 L and the complexes using B3LYP/6-311G.
The lower value of the energy gap explains the charge transfer interactions taking place within the molecule, which influences the molecule's biological activity. The energy gap reflects the chemical activity of the molecule. A molecule with a small frontier orbital gap is generally associated with a high chemical reactivity and is defined as a soft molecule. Table 8. Ground state properties of the H 2 L ligand using B3LYP/6-31G and its metal complexes using B3LYP/LANL2DZ.  The thiosemicarbazide ligand and chelate compounds before and after irradiation had varying degrees of inhibitory effect on the growth of Gram-negative bacteria, such as

Antimicrobial Activity
The thiosemicarbazide ligand and chelate compounds before and after irradiation had varying degrees of inhibitory effect on the growth of Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa, Gram-positive bacteria (Staphylococcus aureus and Streptococcus mutans), and fungi (Candida albicans and Asperagillus Nigar); the inhibitory effect results are presented in Table S6 and Figure 7. Furthermore, C. albicans was affected by complex (B 3 , B 4 ) before irradiation, with inhibition ranges of 10.3 ± 0.5 and 23.6 ± 0.6, respectively. In turn, complex (A 3 ) after irradiation showed higher activity, with an inhibition range of 31.6 ± 0.6. A. nigar was affected by complex (B 3 ) before irradiation, with an inhibition range of 29.3 ± 0.6, and the higher activity was for complex (A 3 ) after irradiation, with an inhibition range of 30.6 ± 0.6; this is compared with the positive control drug used for both fungi. The in vitro antimicrobial activity exhibited by the synthesized compounds before and after irradiation is in Table S6. The results showed that complex (B 3 ) before irradiation towards Gram-negative bacteria (E. coli, K. pneumonia, and P. aeruginosa) had higher activity than other complexes, with an inhibition range of 23.3 ± 0.6, 22.6 ± 0.6, and 21.3 ± 0.6, respectively. The complex (A 3 ) after irradiation had the highest activity, with an inhibition range of 29.6 ± 0.6, 20.6 ± 0.6, and 27.6 ± 0.6, respectively, compared with other complexes before and after irradiation. In addition, the results reported that complex (A 3 ) after irradiation had the highest activity with an inhibition range of 33.3 ± 0.6 and 19.6 ± 0.6, while complex (B 3 ) before irradiation towards Gram-positive bacteria (S. aureus and S. mutans) had higher activity than other complexes, with an inhibition range of 36.6 ± 0.6 and 28.6 ± 0.6, respectively. Therefore, the [Hg 2 (H 2 L)(OH)SO 4 ] complex after irradiation had a higher activity than other complexes. These results can be demonstrated according to the basis of Overtone's concept and Tweedy's chelation theory [38][39][40][41], as the chelation increases the delocalization of p-electrons over the whole ring. Hence, this enhances the compounds' penetration into lipid membranes. In addition, the oxidation state of the metal ion, type, and number of donor sites besides their relative presence within the ligand, solubility, conductivity, particle size, and bond length between the metal and ligand are also important in determining the antimicrobial activity of compounds [42,43].

Cytotoxicity
The cytotoxic activities of the ligand and their complexes before and after irradiation were evaluated against the human liver cancer cell line (HepG2) and normal cell line (HEK-293), as presented in Table 9, Figure 8, and Figure S4. The results are expressed as the IC 50 , which is the concentration of a drug that causes a 50% reduction in the proliferation of cancer cells when compared to the growth of the control cells. The thiosemicarbazide ligand before irradiation (B) was more biologically active than after irradiation (A), where the IC 50 value of B is 20.45, while A is 29.25. The Mn(II) and Zn(II) complexes after irradiation (A 2 , A 4 ) against the human liver HepG2 cancer are more effective than the Mn(II) and Zn(II) complexes before irradiation (B 2 , B 4 ). Moreover, (A 2 , A 4 ) had lower IC 50 values than (B 2 , B 4 ), respectively. size, and bond length between the metal and ligand are also important in determining the antimicrobial activity of compounds [42,43].

Cytotoxicity
The cytotoxic activities of the ligand and their complexes before and after irradiation were evaluated against the human liver cancer cell line (HepG2) and normal cell line (HEK-293), as presented in Table 9, Figure 8, and Figure S4. The results are expressed as the IC50, which is the concentration of a drug that causes a 50% reduction in the proliferation of cancer cells when compared to the growth of the control cells. The thiosemicarbazide ligand before irradiation (B) was more biologically active than after irradiation (A), where the IC50 value of B is 20.45, while A is 29.25. The Mn(II) and Zn(II) complexes after irradiation (A2, A4) against the human liver HepG2 cancer are more effective than the Mn(II) and Zn(II) complexes before irradiation (B2, B4). Moreover, (A2, A4) had lower IC50 values than (B2, B4), respectively.
The attained IC50 values of vinblastine, the ligand, and Mn(II) and Zn(II) complexes before and after irradiation are in the following order: Vinblastine    Various thiosemicarbazide derivatives have been used as starting materials for compounds with better biological activities. Molecular modeling tools are used to explore their mechanism of action. One of the most important enzymes that control signal transduction and cell proliferation is mitogen-activated protein kinase-activated protein kinase  The attained IC 50 values of vinblastine, the ligand, and Mn(II) and Zn(II) complexes before and after irradiation are in the following order: Vinblastine (4.58) > B (20.45) > A 2 (23.95) > A (29.45) > B 2 (32.6) > A 4 (86.24) > B 4 (189.96) µg/mL. These obtained results concluded that the synthesized ligand and its complexes have a good anticancer effect on the HepG2 cell line, except the Zn(II) complex before irradiation (B 4 ) and Mn(II) complex after irradiation (A 2 ), which are the most active ones. Moreover, vinblastine had an enhanced anticancer effect on the selected cancer cell lines.

Molecular Docking Studies
Various thiosemicarbazide derivatives have been used as starting materials for compounds with better biological activities. Molecular modeling tools are used to explore their mechanism of action. One of the most important enzymes that control signal transduction and cell proliferation is mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK-2 or MK-2) [44]. Discovering new inhibitors of this key enzyme has received attention as a strategy for discovering novel anticancer agents [45]. The ligand and its metal complexes were docked on the active site of the MK-2 enzyme in a trial to suggest a mechanism of action for their cytotoxicity. The protein data bank file is PDB: 3WI6). The file contains an MK-2 enzyme co-crystallized with an inhibitor. All docking procedures were achieved by MOE software. The inhibitor interacts with the MK-2 active site with Glu-145 hydrogen bonds involving Glu-145 and Asp 207 ( Figure 9). The docking protocol was validated by redocking the Mn, Zn, and Pb complexes on the active site of the MK-2 enzyme with the highest energy score for the Pb complex (−7.28 kcal/mol). All the docked compounds were fitted on the active site of the MK-2 enzyme. The docking scores and amino acid interactions for the docked compounds are summarized in Table 10. The types of interactions involved were side-chain acceptor, metal contact receptor, and solvent contact. The Hg compound displayed the best docking score (−8.16 kcal/mol), which may explain its promising cytotoxic activity. Finally, we can conclude that the molecular docking of our compounds on the active site of the mitogen-activated kinase (MK-2) revealed good amino acid interactions. We observed good agreement between the experimental IC 50 values and the molecular docking of the selected enzyme target relative to the Pb and Mn complexes, but the ligand scoring energy sequence was not compatible with its IC 50 value. amino acid interactions. We observed good agreement between the experimental IC50 values and the molecular docking of the selected enzyme target relative to the Pb and Mn complexes, but the ligand scoring energy sequence was not compatible with its IC50 value.  Figure 9. Cont. and Zn(II) (B4, A4) complexes before and after irradiation against HEK-293 cell line compared to Vinblastine; Tables S1-S5: The coordinates of the optimized ligand (B) and their metal complexes; Table S6: Antimicrobial activity of unirradiated and irradiated complexes.