Clean Grinding Technique: A Facile Synthesis and In Silico Antiviral Activity of Hydrazones, Pyrazoles, and Pyrazines Bearing Thiazole Moiety against SARS-CoV-2 Main Protease (Mpro)

A novel series of some hydrazones bearing thiazole moiety were generated via solvent-drop grinding of thiazole carbohydrazide 2 with various carbonyl compounds. Also, dehydrative-cyclocondensation of 2 with active methylene compounds or anhydrides gave the respective pyarzole or pyrazine derivatives. The structures of the newly synthesized compounds were established based on spectroscopic evidences and their alternative syntheses. Additionally, the anti-viral activity of all the products was tested against SARS-CoV-2 main protease (Mpro) using molecular docking combined with molecular dynamics simulation (MDS). The average binding affinities of the compounds 3a, 3b, and 3c (−8.1 ± 0.33 kcal/mol, −8.0 ± 0.35 kcal/mol, and −8.2 ± 0.21 kcal/mol, respectively) are better than that of the positive control Nelfinavir (−6.9 ± 0.51 kcal/mol). This shows the possibility of these three compounds to effectively bind to SARS-CoV-2 Mpro and hence, contradict the virus lifecycle.


Introduction
Clean chemistry is a multidisciplinary concept for the judicious use of chemical reactions and prevents its pollution. It involves the designing of chemical products by using environmentally safe procedures that reduce the generation of hazardous substances, as well as the cost [1]. Grinding methods are appealing eco-friendly techniques for efficient organic synthesis with many advantages, such as: mild reaction conditions, easy separation and purifications [2,3], high efficiency and selectivity [4], and environmental acceptability [5]. In the meantime, hydrazone is an effective nucleus because hydrazine-containing compounds (R 1 R 2 C=NNH-R 3 ) offer a wide range of biological activities, and selectivity [4], and environmental acceptability [5]. In the meantime, hydrazone is an effective nucleus because hydrazine-containing compounds (R1R2C=NNH-R3) offer a wide range of biological activities, including anticholinesterase [6], antimycobacterial [7], anti-cancer [8], antibacterial, anti-inflammatory, anti-viral, antifungal, anti-tubercular, analgesic, muscle relaxants, and antihistamines [9][10][11][12]. Hydrazones possessing an azometine -NHN=CH-proton constitute an important class of compounds for new drug development. Both nitrogen atoms of the hydrazone group are nucleophilic and the carbon atom of the hydrazone group has both electrophilic and nucleophilic properties (Figure 1). These structural fragments are principally responsible for the physical and chemical properties of hydrazones. Therefore, many researchers have synthesized these compounds as target structures and evaluated their biological activities. These observations have been guiding the development of new hydrazones that possess varied biological activities [13][14][15]. Likewise, pyrazoline derivatives widely occur in nature in the form of alkaloids, vitamins, animal cells, and pigments. The unique structural array and pronounced pharmacological activities [16,17] of pyrazolines have made them attractive synthetic targets for many researchers. In continuation of our interest in green synthesis of heterocycles [2,[18][19][20][21][22][23], herein, we propose an easy, efficient, solid-state grinding method for the preparation of hydrazones. This method can also be extended to the reactions of hyrazide with active methylene and anhydride compounds.
SARS-CoV-2 represents the century challenge to human beings. It represents the primary agent for the novel, deadly, pandemic of COVID-19 worldwide [24]. The main viral protease (M pro ) of SARS-CoV-2 sheds drug designers due to its vital role in keeping the virus alive [25]. M pro is a crucial enzyme for the viral polypeptide processing into the different enzymes that maintain the life cycle of the SARS-CoV-2. Hence, blocking M pro activity is vital in combating COVID-19. Different compounds (natural and synthetic) are used as possible potential therapeutics against SARS-CoV-2 M pro . In this study, we tested some hydrazones, pyarzoles, and pyrazines derivatives synthesized using a clean grinding technique.
Molecular modeling represents an excellent method used to judge the chemical reactivity in silico [26]. The fast improvement in the last two decades makes molecular modeling the second hand for drug designers. It helps reduce time, money, and effort in the journey of drug design [27]. It was successful in helping find potent anti-viral agents against many viruses, like Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) [28][29][30]. Additionally, computer-aided drug design is used to find the potency of drugs and natural products against SARS-CoV-2 proteins during the last few months [31][32][33][34]. In this study, molecular docking combined with molecular dynamics simulation is used to test the different compounds against SARS-CoV-2 M pro .

Chemistry
An initial endeavor was to investigate the condensation reactions under solid-state conditions. Thus, grinding of 3-acetyl-7-amino-1-aryl- [1,2,4]triazolo [4,3-a]pyrimidin-5(1H)-one (1) [35] and 4-methyl-2-phenylthiazole -5-carbohydrazide (2) [36], in the presence of a few drops of acetic acid at Likewise, pyrazoline derivatives widely occur in nature in the form of alkaloids, vitamins, animal cells, and pigments. The unique structural array and pronounced pharmacological activities [16,17] of pyrazolines have made them attractive synthetic targets for many researchers. In continuation of our interest in green synthesis of heterocycles [2,[18][19][20][21][22][23], herein, we propose an easy, efficient, solid-state grinding method for the preparation of hydrazones. This method can also be extended to the reactions of hyrazide with active methylene and anhydride compounds. SARS-CoV-2 represents the century challenge to human beings. It represents the primary agent for the novel, deadly, pandemic of COVID-19 worldwide [24]. The main viral protease (M pro ) of SARS-CoV-2 sheds drug designers due to its vital role in keeping the virus alive [25]. M pro is a crucial enzyme for the viral polypeptide processing into the different enzymes that maintain the life cycle of the SARS-CoV-2. Hence, blocking M pro activity is vital in combating COVID-19. Different compounds (natural and synthetic) are used as possible potential therapeutics against SARS-CoV-2 M pro . In this study, we tested some hydrazones, pyarzoles, and pyrazines derivatives synthesized using a clean grinding technique.
Molecular modeling represents an excellent method used to judge the chemical reactivity in silico [26]. The fast improvement in the last two decades makes molecular modeling the second hand for drug designers. It helps reduce time, money, and effort in the journey of drug design [27]. It was successful in helping find potent anti-viral agents against many viruses, like Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) [28][29][30]. Additionally, computer-aided drug design is used to find the potency of drugs and natural products against SARS-CoV-2 proteins during the last few months [31][32][33][34]. In this study, molecular docking combined with molecular dynamics simulation is used to test the different compounds against SARS-CoV-2 M pro .
To verify the assigned structure of the products 3a-c, we have synthesized these compounds by alternative methods. Thus, treatment of aminothiouracil (4) with 2-[2-(4-methyl-2-phenylthiazole-5-carbonyl) hydrazono]-N-arylpropanehydrazonoyl chlorides (5a-c) [37] in dioxane, in the presence of a catalytic amount of triethylamine, under thermal conditions, gave the authentic products identical in all respects (mp, mixed mp, IR) with the isolated products 3a-c via Smiles rearrangement [38] and elimination of H 2 S (Scheme 2).

Molecular Simulations
The structures are tested for their anti-viral activity against SARS-CoV-2 main protease (M pro ) using molecular docking combined with molecular dynamics simulation (MDS) for up to 100 nanoseconds (see the Supplementary Figures S5 and S6). Figure 2 shows the average binding affinities of the compounds calculated using AutoDock Vina [42]. The anti-viral Nelfinavir (red column) is tested as a positive control. The error bars represent the standard deviation (SD) from all the docking trials using the different conformations of SARS-CoV-2 M pro . As reflected from Figure 2, the average binding affinities of the compounds 3a, 3b, and 3c (green columns) (−8.1 ± 0.33 kcal/mol, −8.0 ± 0.35 kcal/mol, and −8.2 ± 0.21 kcal/mol, respectively) are better (less) than that of the positive control Nelfinavir (−6.9 ± 0.51 kcal/mol). This shows the possibility of these three compounds to effectively bind to SARS-CoV-2 M pro and hence, contradict the virus lifecycle. Additionally, the compounds 9, 11a, 11b, 11c, 13, 15, 17, 19, 21, and 23 have average binding affinities comparable to Nelfinavir (−6.4 ± 0.09 kcal/mol for compound 13 down to −7.4 ± 0.13 kcal/mol for compound 23); hence, it may bind to SARS-CoV-2 M pro and stop the viral infection as well. To further analyze the binding mode, the docking complexes are analyzed by the aid of the Protein-Ligand Interaction Profiler (PLIP) software [43]. Table 1 shows the interaction profiles of the compounds 3a, 3b, 3c, 9, 11a, 11b, 11c, 13, 15, 17, 19, 21, and 23 docked into the active site residues of SARS-CoV-2 PL pro . The most reported types of interaction were the H-bonds and hydrophobic contacts. Noticeably, the most reported interacting residue in almost all complexes is E166. For the best three compounds (3a, 3b, and 3c), the interaction is almost identical with 7 H-bonds (N142, G143, S144, E166, D187, and Q189) and one hydrophobic contact (E166). These three compounds have the lowest binding energy values (-8.1 to -8.3 kcal/mol). For the other compounds, the number of formed interactions is fewer. This is reflected in the binding energy values (-6.4 to -7.3 kcal/mol). The interaction pattern for the best three thiazole compounds, 3a, 3b, and 3c, are depicted in Figure 3. These three compounds can interact with the same binding site pocket of the SARS-CoV-2 M pro as reflected in the superposition (Figure 4) of the compounds after docking. The compounds 3a, 3b, and 3c proved their ability to bind firmly to the active site of the protein, and hence could be successful candidates against COVID-19. Experimental validation of the binding potency of the three compounds against SARS-CoV-2 M pro is suggested as a future recommendation. Further in-depth MDS analysis of the best three compounds is suggested as future work. Additionally, we suggest testing these compounds against other enzymes for SARS-CoV-2, such as the papain-like protease (PL pro ) and RNA-dependent RNA polymerase (RdRp). It may bind firmly to these targets and may help inactivate SARS-CoV-2.

Chemistry
An electrothermal Gallenkamp apparatus was operated to measure the melting points for the newly synthesized compounds (Bibby Sci. Lim. Stone, Staffordshire, UK). Pye-Unicam SP300

Chemistry
An electrothermal Gallenkamp apparatus was operated to measure the melting points for the newly synthesized compounds (Bibby Sci. Lim. Stone, Staffordshire, UK). Pye-Unicam SP300 instrument in potassium bromide discs was used to measure Infra-Red, IR spectra (Shimadzu, Tokyo, Japan). A Varian Mercury VXR-300 spectrometer (300 MHz for 1 H-NMR and 75 MHz for 13 C-NMR) (Varian, Inc., Karlsruhe, Germany) was manipulated to measure the 1 H NMR, and 13 C-NMR spectra and the chemical shifts were related to that of the solvent. GCMS-Q1000-EX Shimadzu spectrometers (Tokyo, Japan) were conducted to record the mass spectra of the samples on the ionizing voltage at 70 eV. Elemental analyses were carried out at the Microanalytical Centre of Cairo University, Giza, Egypt.

Molecular Dynamics Simulation
Nanoscale Molecular Dynamics (NAMD) and Visualizing Molecular Dynamics (VMD) software were used in the molecular dynamics study (MDS) using the standard CHARMM 36 forcefield, and the proteins were solvated with the TIP3P water model [44][45][46][47]. The temperature of 310 K and the 154 mM NaCl concentration were adjusted during the simulation period to simulate the protein at its physiological conditions. The MDS calculations were done on the Bibliotheca Alexandrina supercomputer, Alexandria, Egypt. Chimera software was used to cluster the trajectories after MDS using default parameters [48]. Five representative structures of SARS-CoV-2 M pro were used in the docking study after clustering the trajectories of the MDS of the M pro (PDB ID: 6Y84).

Molecular Docking
AutoDock Vina 1.1.2 software was used to test the thiazole compounds against SARS-CoV-2 main protease different conformations, while Nelfinavir was used as a positive control. The ligands were prepared using the PyMOL 2.4 and AutoDock Tools 1.5.6 software [49]. Any missed hydrogen atoms were added, followed by charges addition before input files generation. The docking utilizes flexible active site (H41 and C145) and flexible ligand protocol. The AutoDock Vina grid box size used in the docking experiments is 40 × 40 × 40 Å 3 , while the box centers were set to be at the dyads H41 and C145 (−3.9, 19.

Conclusions
In this context, a series of novel hydrazones (3a-c, 9, 11a-c), pyarzoles (13, 15, 17, 19), and pyrazines (21,23) were synthesized using a solvent-drop, eco-friendly, environmental grinding method. The structure of the newly prepared compounds was established based on both elemental analysis and spectroscopic data and by an alternative method wherever possible. Also, the anti-viral activity of all the products was tested against SARS-CoV-2 main protease (M pro ) using molecular docking combined with molecular dynamics simulation (MDS). The average binding affinities of the compounds 3a, 3b, and 3c were better than that of the positive control Nelfinavir. So, these compounds can bind effectively to SARS-CoV-2 M pro and hence, contradict the virus lifecycle.