Towards Covid-19 TMPRSS2 enzyme inhibitors and antimicrobial agents: Synthesis, antimicrobial potency, molecular docking, and drug-likeness prediction of thiadiazole-triazole hybrids

1,3,4-Thiadiazole analogues 3 and 4 were synthesised via the reaction of 1-(5-methyl-1-(5-(methylthio)-1,3,4-thiadiazol-2-yl)-1H-1,2,3-triazol-4-yl)ethan-1one 2 with vanillin or thiophene-2-carboxaldhyde, respectively through chalcone reaction. Compounds 3 and 4 were submitted to react with thiosemicarbazide affording 5-(4‑hydroxy-3-methoxyphenyl)-3-(5-methyl-1-(5-(methylthio)-1,3,4-thiadiazol-2-yl)-1H-1,2,3-triazol-4-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (5) give 3-(5-methyl-1-(5-(methylthio)-1,3,4-thiadiazol-2-yl)-1H-1,2,3-triazol-4-yl)-5-(thiophen-2-yl)-4,5 dihydro-1H-pyrazole-1-carbothioamide (6), respectively. The letters were reacted with N-(4-chlorophenyl)-2-oxopropanehydrazonoyl chloride to give compounds 7 and 8. The chemical compositions of the novel compounds were affirmed by spectral and microanalytical data. Meanwhile, all the newly synthesized compounds have been screened for their ability to prevent the proliferation of different pathogens named Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, and Candida albicans in vitro. Additionally, the potency of the newly synthesized compounds to be anti-COVID-19 candidates was studied through a molecular docking study. The newly prepared molecules 2–8 were studied in silico against transmembrane serine protease 2 (TMPRSS2) to identify their potential therapeutic activity against Coronavirus. Moreover, the drug-likeness of the compounds was tested theoretically by ADMET studies. Compound 8 exhibited a better binding affinity (-9.1 kcal/mol) against the target enzyme TMPRSS2. Additionally, it respects Lipinski's rule of five and has acceptable ADMET properties, indicating that compound 8 could be interesting for the treatment of Covid-19.


Introduction
Coronavirus 2019 (Covid-19) is the major cause of global public health problems [1 , 2] . Coronavirus is an infectious disease caused by SARS-Cov-2 virus [3] . The present need is to identify new antiviral therapeutic agents for Covid-19, which is why TMPRSS2 was selected as a drug target for Covid-19 as it plays a critical role in the propagation of disease. Heterocycles have always been regarded as the basic bedrock of the most modern biomedical and medicinal chemical research. Moreover, microbial infections are still among the major causes of death among the world's population due to increased bacterial antibiotic resistance phenomena and the development of resistance to chemotherapeutics. For these reasons, there is an urgent need to design and synthesize new antimicrobial agents.

Antimicrobial activity
The up growing chemotherapeutic resistance of various microbial pathogens had been considered the most significant clinical problem globally. Consequently, investigation of new organic compounds which could be derived from active groups to serve as wide-spectrum antimicrobials could contribute to compacting such challenges. In the current study, the antimicrobial potency of five newly synthesized compounds against some microbes was detected. The results revealed that compound 8 exhibited antimicrobial activity against all the tested microbes. The toxicity of the other tested compounds was noticed and there were different considerable activities observed against gram-negative bacteria and yeast. The MIC of the tested compound was also listed, as shown in Table 1 .
It was worth noting that MIC is defined as the average of the lowest concentrations with no observable growth of microorganisms. Compound 8 revealed a considerable broad spectrum of antimicrobial activities against all strains of the tested pathogens with low concentrations which ranged from 5 to 10 μg/mL; Compound 8 exhibited significant antimicrobial activity against Pseudomonas aeruginosa at the concentration lower than the standard drug Ciprofloxacin. Likewise, Compound 2 provided MIC value at low concentration toward Pseudomonas aeruginosa and Staphylococcus aureus while it showed a relatively higher MIC concentration against Escherichia coli . Although compound 3 had a MIC value at low concentrations toward Bacillus subtilis, Staphylococcus aureus, and Candida albicans , it showed a relatively higher MIC concentration against E. coli and Pseudomonas aeruginosa . Compound 4 provided MIC value at low concentration toward Candida albicans and high MIC concentration against Bacillus subtilis, Staphylococcus aureus, and Candida albicans . Other tested organisms could be resistant to it even at high concentrations. Further, MIC was observed at high concentrations against Pseudomonas aeruginosa, and at low concentrations against E. coli Bacillus subtilis, Staphylococcus aureus, and Candida albicans for compound 6. This study revealed that the antimicrobial activity of the tested new derivatives displayed potent antibacterial and antifungal activities.

In silico docking study and ADMET analysis
In the current study, the ability of the newly prepared molecules to inhibit Covid-19 TMPRSS2 was calculated by the molecular docking approach. The compounds have high binding affinities against TMPRSS2 enzyme ranging from -9.1 to -5.5 kcal/mol, which is better achieved using compound 8  2  160  40  ND  40  ND  3  120  160  20  40  40  4  ND  ND  80  80  20  5  40  60  ND  ND  ND  6  40  160  40  20  20  7  30  40  120  ND  20  8  10  5  10  7  5  Ciprofloxacin  5  7 2.5 1.25 ND Nystatin ND ND ND ND 5 Ciprofloxacin and Nystatin were used as standard drugs as control, ND: not determined. ARG130, and GLN283 though three π -cation and one π -sigma interactions, respectively. Finally, compound 8, with the highest binding affinity ( −9.1 kcal/mol), exhibited three π -stacked interactions with PHE66, LYS68, and GLN283. The ADMET predictions were used to calculate the pharmacokinetics properties of the synthesized molecules. As observed in Table 3 , all compounds respected the conditions mentioned in Lipinski's rule of five (Ro5) and had acceptable ADMET properties.

Instruments
The reagents used in this work were used without purification. Melting points of the prepared compounds were determined using Griffin apparatus and are uncorrected. The completion of the reactions was mentioned by Thin Layer Chromatography (TLC) technique on aluminium plates coated with silica gel. IR spectra were recorded on Shimadzu 408 and Bruker Vect. 22. NMR spectra were run at 500 MHz using TMS as the internal standard. The chemical shifts were measured in ppm ( δ) related to TMS (0.00 ppm). MS were recorded on an HP model, Mass 5988 Mass spectrometer at 70 eV. All new compounds were analysed for C, H, and N at Cairo University, Egypt.

In silico molecular docking, drug-likeness and toxicity predictions
PyRx-a virtual screening tool with the standard protocol [23] was used to perform the docking process between the target enzyme TMPRSS2 and the newly synthesized compounds 2-8. The crystal structure of the target was retrieved from the RCSB Protein Data Bank web server (PDB ID: 1z8g) [24] and then prepared and energy minimized using CHARMM Force Field [25] . Additionally, the chemical structures (2D) of the compounds were drawn using ChemOffice tool (ChemDraw 16.0), then converted to three-dimensional (3D) using open Babel GUI [26] . Moreover, compounds were energetically minimized using UFF Force Field [27] . The energy-minimized ligand molecules were used as input for PyRx-tool to perform the docking simulation. Nine conformers were obtained for each docked compound, and the conformer with the highest binding affinity was selected for further study [4 , 15 , 28-30] . Finally, ADMET predictions of the molecules were calculated using free-access tools, such as Molinspiration, admetSAR, and SwissADME.

Conclusion
In search of anti-Covid-19 therapeutics, a new series of thiadiazole-triazole hybrids were synthesized. Meanwhile, the newly synthesized compounds showed potent antimicrobial activities against the tested pathogens, especially compound 8, which showed the highest activity against all the tested microbes at low MIC concentration. Additionally, the ability of these compounds to inhibit Covid-19 TMPRSS2 enzyme was investigated through a structure-based docking study to identify potent anti-Covid-19 drug candidates. It has been observed that compound 8 has the highest binding affinity toward the target enzyme. Therefore, it could be used as a potential therapeutic agent to combat Coronavirus.

Credit authors statement
All the authors designed the manuscript, synthesized the new compounds which were tested for their Covid-19 TMPRSS2 Enzyme Inhibition potency and their antimicrobial potency. Additionally, the authors revised the manuscript and designed it in its final form.

Declaration of Competing Interest
None.

Data for reference
Data will be made available on request.

Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.molstruc.2022.133659 .