key: cord-0836600-fxw7jsqg
authors: Al‐Janabi, Ahmed S. M.; Saleh, Abdulrahman M.; Hatshan, Mohammad R.
title: Cytotoxicity, anti‐microbial studies of M(II)‐dithiocarbamate complexes, and molecular docking study against SARS COV2 RNA‐dependent RNA polymerase
date: 2021-02-11
journal: J Chin Chem Soc
DOI: 10.1002/jccs.202000504
sha: 0062f23b9f18fcf393de4771596495e7e7602c47
doc_id: 836600
cord_uid: fxw7jsqg

Ten transition metal dithiocarbamate (DTC)complexes of the type [M(κ (2)‐Et(2)DT)(2)] (1–5), and [M(κ (2)‐PyDT)(2)] (6–10) (where M = Co, Ni, Cu, Pd, and Pt; Et(2)DT = diethyl dithiocarbamate; PyDT = pyrrolidine dithiocarbamate) were synthesized and characterized by different methods. The dithiocarbamate acted as bidentate chelating ligands to afford a tetrahedral complexes with Co(II) ion and square planner with other transition metal ions. The dithiocarbamate complexes showed good activity against the pathogen bacteria species. The results showed the Pt‐dithiocarbamate complexes are more active against all the tested bacteria than the Pd‐dithiocarbamate complex. The dithiocarbamate complexes displayed the maximum inhibition zone against E. coli bacteria, whereas the lowest activity of the dithiocarbamate against Salmonella typhimurium bacteria. The cytotoxicity of the Pd(II) and Pt(II) complexes was screened against the MCF‐7 breast cancer cell line and the complexes showed moderate activity compared with the cis‐platin. The results indicated that the MCF7 cells treated with 500 μgml of ligands and Pd(II) and Pt(II) complexes after 24 hr exposure showed intercellular space and dead cells. Finally, molecular docking studies were carried out to examine the binding mode of the synthesized compounds against the proposed target; SARS COV2 RNA‐dependent RNA polymerase.

Metal complexes of dithiocarbamate ligands have been the topic of current and increasing concern because they possess a wide range of medical fields (such as antibacterial, anti-fungal, and anti-inflammatory) and industry, analytical, and organic chemistry. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] RNA-dependent RNA polymerase (RdRp) is the core enzyme in the replicative cycle of all positive-strand RNA (+RNA) viruses, which catalyzes the replication of RNA from a RNA template. [12] [13] [14] Coronaviruses (order Nidovirales) are a family of +RNA viruses with the largest single-stranded RNA genomes known to date. [15] [16] [17] [18] The unusual complex replication and transcription machinery of severe acute respiratory syndrome coronavirus (SARS-CoV) is composed of 16 nonstructural proteins (nsps), produced during co-and post-translational proteolytic processing of two replicase poly-proteins. [17] [18] [19] [20] 2 | RESULTS AND DISCUSSION

The reactions of dithiocarbamate salts {(NaEt 2 DT) or (NH 4 ) (PyDT)} with NiCl 2 .6H 2 O, CoCl 2 .6H 2 O, CuCl 2 .6H 2 O, K 2 PdCl 4 , or K 2 PtCl 4 in 2:1 (ligand: metal) molar ratio, gave complexes of the type [M(κ 2 -dithiocarbamate) 2 ] (Scheme 1) in good yield (77-91%). The dithiocarbamate ligands were bonded through the S atoms as bidentate mode.

The 1 H NMR spectra of complexes (2, 4, and 5) (Figure 1 ) displayed the protons of Et 2 DT − ligand as two separated peaks (quartate and triplate) within δH (1.17-1.24) ppm, and (3.81-3.85) ppm with coupling constant (6.85-7.92 Hz), due to the methyl and methylene groups, represents 12 protons and 8 protons, respectively.

The 1 H NMR spectra of compounds 7, 9, and 10 ( Figure 2) show the protons of PyDT − ligand as two multiplet peaks within δH 2.00-2.04 ppm and 3.75-3.76 ppm, assigned to the methylene groups, represent eight protons for each peak, respectively. These results were supported by the 13 C NMR spectra of compounds 2, 4, 5, 7, 9, and 10. The 13 C NMR spectra of the compounds 2, 4, and 5 displayed the chemical shift of the CSS-, C A , and C B within δH = 200.61-203.63, 12.57-14.30, and 47.59-49.19 ppm, respectively ( Figures S1-3) , also the 13 C NMR spectra of the compounds 2, 4, and 5 displayed three peaks at δH = 194. 01-195.42, 14.59-15.60 , and 44.45-45.71 ppm, due to the chemical shift of the CSS-, C A , and C B , respectively ( Figures S4-6 ).

Selected IR bands of the complexes 1-10 are recorded in the experimental part (See Figure 3 ). In IR spectra which showed two distinguishing bands of dithiocarbamate ligand within 1,487-1,518 and 997-1,024 cm −1 refer to the ν(C-N) and ν(CSS) group, respectively. The band of (CSS) group looked as single band without splitting, designated that the dithiocarbamate ligands act as bidentate chelating mode. [24] [25] [26] [27] [28] Also the spectra showed frequency of the M-S group at 447-478 cm -1. [24] [25] [26] [27] [28] [29] [30] [31] [32] 

The anti-bacterial activity studies of the M(II)dithiocarbamate complexes are tabulated in Table 1 Figure 4 . The standard error for the test was ±0.03%, and the tests were frequent three periods at similar conditions. The diameter of the inhibitory zone (DIZ) was compared to that of Tetracycline as control positive. The relative (%) activity index was calculated as shown below: %Activity index = Inhibition zone of the test compounds Inhibition zone of the standard drug *100:

The Pd(II) and Pt(II) complexes showed good activity against the pathogen bacteria species. The obtained results can be summarized as following: 

The cytotoxicity effect of sodium N,N-diethyldithiocarbamate and ammonium pyrrolidine dithiocarbamate against MCF-7 cell line depended on the concentration in time of exposure 24 hr. The maximum inhibitory effect was observed at 500 μg/ml. Therefore, the cytotoxicity of Pd(II) and Pt(II) dithiocarbamate complexes was studied at 500 μg/ml only.

The results of the anti-cancer activity are listed in Table 2 . The Pd(II) and Pt(II) complexes show moderate activity compared with the cis-platin. The inhibitory effect against MCF-7 initiated with 23.71% for complex 4 and increased regularly to 23.89% for complex 9, 40.72%, and 47.76% for the Pt(II) complexes (5 and 10) at 

The binding mode of the reference (Remdesivir) exhibited an energy binding of −10.56 kcal/mol. By re-docking Remdesivir against SARS COV2 RNA-dependent RNA The binding mode of the candidate compound 1 exhibited an energy binding of −10.10 kcal/mol. Which a sulfur atoms and Ni metal atom interacted with Asp760, Asp623, Lys621, Cys622, MG1004, phosphate (pop) 1,003 and uracil nucleotide 20 by dipole-dipole and ionic interactions. While the hydrophobic ethyl group formed π-alkyl interactions with adenine nucleotide 11 ( Figure 8 (1)).

Where the binding style of the complex 2 exhibited an energy binding of −5.52 kcal/mol. Which a sulfur atoms and Co metal atom interacted with Val557 and Asp623 by dipole-dipole and ionic π-alkyl interactions. While the hydrophobic ethyl group formed π-alkyl interactions with Cys622, Lys545, Arg555, and adenine nucleotide 19. While sulfur groups formed one hydrogen bonding with adenine nucleotide 11 with distance of 2.90 Å (Figure 8 (2)).

The binding mode of the candidate compound 3 exhibited an energy binding of −10.79 kcal/mol. Which a sulfur atoms and Cu metal atom interacted with Asp760, Cys622, MG1004, phosphate (pop) 1,003 and uracil nucleotide 20 by dipole-dipole and ionic π-alkyl interactions. While the hydrophobic ethyl group formed π-alkyl interactions with adenine nucleotide 11, Asn691, and Val 554 ( Figure 8 [3] ). Whereas the complex 4 exhibited an energy binding of −9.50 kcal/mol. Which a sulfur atoms and Pd metal atom interacted with Asp760, Arg555, MG1004, and phosphate (pop) 1,003 by dipole-dipole and ionic π-alkyl interactions. While the hydrophobic ethyl group formed π-alkyl interactions with adenine nucleotide 11, Pro620 and Val 554 (Figure 8 (4) ).

T A B L E 1 Diameter inhibition zone (mm) and activity index (%) of dithiocarbamate complexes against bacterial species (5)). The binding mode of the candidate complexes 6, 7, and 8 have the same pattern in binding with targeted receptor and exhibited an energy binding of −9.33, −9.37, and −5.51 kcal/mol, respectively. Which a sulfur atoms and Ni, Co and Cu metal atoms interacted with Asp760, Asp623, Arg555, Lys621, Lys551, Asp761, Ser814, MG1004, phosphate (pop) 1,003 and uracil 20 by dipoledipole and ionic interactions. (Figure 9 (1-3) ).

The binding mode of the candidate compound 9 exhibited an energy binding of −7.93 kcal/mol. Which a sulfur atoms and Pd metal atom interacted with Asp760, Cys622, Asp623, MG1004, phosphate (pop) 1,003, adenine nucleotide 11 and uracil nucleotide 20 by dipole-dipole and ionic interactions. While the pyrrolidine ring formed π-alkyl interaction with Ala558. The sulfur groups formed one hydrogen bonding Asp760 with distance of 2.65 Å (Figure 9 (4) ).

The binding mode of the candidate compound 10 exhibited an energy binding of −8.53 kcal/mol). Which a sulfur atoms and Pt metal atoms interacted with Asp760, Arg555, Lys551, Asp761, Ser814, MG1004, phosphate (pop) 1,003, and uracil 20 by dipole-dipole and ionic interactions. The SH group formed one hydrogen bonding Asp761 with distance of 2.85 Å (Figure 9 (5)).

Molecular mapping surface was done to expect how the candidate occupying the critical targeted pocket. Compounds 1, 3, 4 and 7 are showing good occupying the critical space inside the target protein ( Figure 10 ). [ ,974 w, 2,834 w, 1,517 s, 1,434 s, 1,006 

The antibacterial activity of the Pd(II) and Pt(II)dithiocarbamate complexes was examined against six pathogenic bacteria, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, and Vibrio Parahemolyticus. in DMSO as a solvent at 1 × 10 −3 M concentration. The activity of the Pd(II) and Pt(II) complexes was tested by agar disc diffusion method designated by Molder et al. [33] and the results were compared with Tetracycline as standard antibiotic.

The cytotoxicity of Pd(II) and Pt(II)complexes was screened against MCF7 breast cancer cell line by adding trypsin-version solution to the tissue culture flask (size 25 cm 2 ), then 20 ml of the RPMI-1640 medium was added with fetal calf serum (FCS). The cells were mixed and 0.2 ml was transferred from each mixture into each of the 96 wells using a fine automatic pipette. The plates were left in the incubator at 37 C for 18-24 hr until the adhesion of cells in the well, then the old media from wells were removed, 0.2 ml of the prepared concentrations of dithiocarbamate ligands were added using the Serum-free media (15.1, 31.2, 62.5, 125, 250, 400 , and 500 μg/ml) with three replicates per a concentration. In addition, 0.2 ml of the prepared concentrations of each complexes were added using the Serum-free media (500 μg/ml) only. Four replicates were performed using DMSO (dimethyl sulfoxide) as a negative control and four replicates were added to PBS supplemented with 0.2 ml of serum free medium assay positive control and plates were incubated at 37 C for 24 hr according to the method mentioned. The plate was taken out from the incubator and 50 μl of crystal violet stain per well was added then returned to the incubator for 20 min. After that, the contents were removed and washed with PBS until the excess dye was removed and the cells were allowed to dry. The results were read using the optical spectroscopy device at a wave length of 492 nm. The rate was determined to inhibit the growth of cancer cells according to the following equation:

where A is the control reading and B is the treatment reading for each concentration.

Docking study was applied to identify the possible binding interactions between the active candidates and SARS COV2 RNA-dependent RNA polymerase-targeted site. Docking study will provide some insights into the additional structural alterations and development of new more potent and selective SARS COV2 RNA-dependent RNA polymerase inhibitors. In the present research, a molecular modeling study which relied on SARS COV2 RNA-dependent RNA polymerase crystal structure (PDB code 7BV2) was executed applying "molecular operating environment (MOE) version 2019.01" Chemical Computing Group Inc. software. [34] At first, water molecules have been removed from the complex. Then, crystallographic disorders and unfilled valence atoms were corrected using protein report and utility and clean protein options. Protein energy was minimized by applying MMFF94 force fields. The rigid of binding Site was structure of protein was obtained by applying fixed atom constraint. The protein essential amino acids were defined and prepared for docking process. Two-dimensional structures of tested compounds were drawn using Chemo-Bio Draw Ultra16.0 and saved in MDL-SD file format, from MOE 19.012 software, the saved file was opened, 3D structures were protonated and energy was minimized by applying.05 RMSD kcal/mol. MMFF94 force field. Then, the minimized structures were prepared for docking using prepared ligand protocol. Molecular docking process was carried out using the CDOCKER protocol. The receptor was held rigid while the ligands were allowed to be flexible during the refinement each molecule was allowed to produce 10 different interaction poses with the protein.

Then docking scores (-CDOCKER interaction energy) of the best-fitted poses with the active site at SARS COV2 RNA-dependent RNA polymerase was recorded and 3D view was generated by Discovery Studio 2016 Client software. We use all these processes to predict the proposed binding mode, affinity, preferred orientation of each docking pose, and binding free energy (ΔG) of the tested compounds with SARS COV2 RNA-dependent RNA polymerase and the binding free energy (ΔG) of the tested compounds are listed in Table 3 .

The active site was predicted by using the site finder option of using MOE 19.01 software. [34] The site finder option was used to calculate possible active sites in SARS COV2 RNA-dependent RNA polymerase. Calculations were made to determine potential sites for ligand binding and docking, and restriction sets for rendering partial molecular surfaces. In SARS COV2 RNA-dependent RNA polymerase critical active site contain Asp623, Lys621, Cys622, Asp760, MG1004, and phosphate (pop) 1,003, Asn691, adenine 11 and uracil 20 amino acid and nucleotides residues. showed good activity against the tested bacteria species, and displayed the maximum inhibition zone against E. coli bacteria, whereas the lowest activity against Salmonella typhimurium bacteria, and the inhibition order of the DTC complexes are as following: 9 > 4 > 5 > 10:

Increase of inhibition zone ! :

The cytotoxicity of the Pd(II) and Pt(II) complexes was screened against MCF-7 breast cancer cell line. The inhibitory effect against MCF-7 which initiated with 23.71% for (4) and increased regularly to 23.89% for complex (9), 40.72% and 47.76% for the Pt(II) complexes (5 and 10) at the same concentration. Additionally, the tested compounds showed promising activities against SARS COV2 RNA-dependent RNA polymerase, in which the most of candidates have a good binding mode and excellent interaction with critical amino acids in targeted pocket, moreover candidate 1, 3, 4, and 7 have the score-10.10, −10.79, −9.50, and − 9.37, respectively, which compared with remdesivir −10.56, compound 3 may be better than remdesivir.

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