ZEOLITE A/ZnCl 2 NANOPARTICLES AS A CATALYST FOR ECO-FRIENDLY SYNTHESIS OF PYRAZOLE-1-CARBOTHIOAMIDES WITH DOCKING VALIDATION AS COVID-19 MAIN PROTEASE (MPRO) INHIBITOR

. The synthesized Zeolite A/ZnCl 2 nanoparticles via the hydrothermal route were characterized using FTIR, XRD, and SEM/EDAX techniques. The characterized catalyst was used for the eco-friendly synthesis of 4,5-dihydro-pyrazole-1-carbothioamide derivatives. Under solvent-free conditions, a multi-component reaction between hydrazine, isothiocyanate, and chalcone was done with a prepared nano-catalyst as an inexpensive, recyclable, easy-to-get, and nontoxic catalyst. The molecular docking study explained that dihydro-1-carbothioamide pyrazoles can be considered COVID-19 main protease (M pro ) inhibitors. In order to investigate the 3D conformation of the compounds that were synthesized, the density functional theory (DFT) was applied with a B3LYP hybrid functional and a 6-311++ G(d,p) basis set. This allowed us to investigate the compounds' electronic and charge transfer properties. In this series of compounds, the derivative 30d showed the lowest HOMO–LUMO energy gap.


INTRODUCTION
Azoles are presented as important scaffolds with a five-membered ring in many natural and biological heterocyclic compounds. Natural organic compounds based on pyrazole are difficultly synthesized by living organisms and the formation of N-N bond is not an easy process in biosynthesis [1]. The pyrazole ring has been reported in 1966, then being essentially used as a part of pharmacophore in hypnotic drugs. The first cytotoxic pyrazole, phenylahistin, were isolated from natural sources in 1969 separated from the marine sponge Leucetta microraphis found on Australia's Great Barrier Reef, exhibited important biological activities, such as anticancer or neurotoxic effects [2,3]. Pyrazole ring contains the two main types of nitrogen, pyrrole like N which gives the acidic character, and aromaticity in addition to pyridine like N which gives the basic character. From this, the pyrazole ring has considered an electron-rich ring and has versatile chemical properties, and is employed in a lot of organic syntheses. Pyrazole ring exhibited two types of reactions; electrophilic and nucleophilic [4][5][6][7]. The nucleophilic nature was displayed from three positions (N1, N2, C4), in addition two electrophilic nature was displayed from two positions (C3, C5). Knowing that, depending on the reaction conditions, electrophilic addition takes place most often at C4 and/or to one of the two nitrogen atoms. Substitution at an annular carbon can only be accomplished via coupling reactions such as Suzuki couplings [8]. For boronic acid or ester cross-coupling to happen at the intended position, halogens must be present in C4 [9]. Pyrazole derivatives showed abroad spectrum of biological effects, for instance, antitubercular, antifungal, antimalarial, anticancer, and anti-AIDS [10][11][12][13][14][15][16]. Pyrazole and its derivatives are also considered as possible antimicrobial, antiepileptic, anti-inflammatory, antipsychotic, antidepressant, inhibitors of protein kinases, anti-aggregating, antiarthritic, cerebro protectors, reverse transcriptase inhibitor, a COX-2 inhibitor, nematocidal and soluble guanylate cyclase activity, etc [17]. Pyrazole and its derivatives have been found to be bioactive parts of commercially available therapies like deramaxx (NSAID), pyrazomycin and difenamizole (anticancer drugs), and floxan and difenamizole (anti-inflammatory drugs) ( Figure 1). Zeolites are famous aluminosilicate material which is commercial used as adsorbent, catalyst in organic synthesis [18] and petrochemical processes [19]. Zeolites are potentially attractive heterogeneous catalyst due to the easy recovery of product/substrate, catalyst recycling, and possible regioselectivity, easy to separate, environmentally friendly [20][21][22][23]. Zn-loaded zeolites showing increasing surface acidity [24] and are suitable catalyst for heterocyclization [25]. Zeolite nanoparticles showed improved catalytic performance because of the increase in mass diffusion; and could enhance the catalytic activity as a result of the increased accessibility of the active sites [26][27][28][29]. In this research, we introduce a zeolite A/ZnCl2 nanoparticles as new catalyst used to synthesis pyrazole-1-carbothioamides, candidate for covid-19 main protease (M pro ) inhibitor.

XRD experimental data
The XRD pattern of the studied zeolite sample is shown in (Figure 2). Obtained spectra show the crystalline nature of the sample with comparable diffraction to published data of zeolite A (JCPDS 38-0237) revealing intense bands at Braggs angles 2θ = 6.  [30]. The zeolite A showed the same previously assigned pattern indicating that the structure of the studied material is well retained even after the mailing process.   Captured image mapping shows a homogenous distribution of all constituting elements (Na, Mg, Al, Si, Ca, and O atoms) present inside the chemical structure listed in (Table 1), as well as their atomic and weight percentages. The data also approved that silica is the main constituent along the studied network structure combined with alumina, lime, magnesium, and sodium via oxygen linkages. It was noticed also that the weight fractions of both analyzed silica and alumina are nearly equal.  High-resolution transmission electron microscopy/selected area electron diffraction (HRTEM/SAED) (Figures 3a and 3b) show high-resolution transmission electron microscope images combined with their selected area electron diffraction (HRTEM/SAED). Studied samples show nearly homogenous morphology with a size ranging between 20-30 nm. In addition, the selected area diffraction pattern (SAED) of prepared nanocrystals reveals a collective pattern of concentric rings with bright spots around a circular path, pointing to a crystalline structure coherent with XRD data.

Fourier transform infrared (FTIR)
( Figure 3c) reveals FTIR optical absorption spectral data of the studied zeolite A sample. Obtained data reveals the following spectral features in correlation with their vibrational groups within the spectral range extending from 4000-400 cm -1 . The bands centered at about 3460, and 1665 cm -1 attributed to the presence of OH groups resulting from moisture attack when mixing the sample with hygroscopic potassium bromide powder during measurements. Broad, strong band at 1010 cm -1 is typically attributed to asymmetric stretching vibrations of silicon and aluminium atoms connected to oxygen atoms inside the network structure [31]. Deconvolution analysis within the spectral range extending between 2000 and 400 cm -1 shown in ( Figure 5) reveals overlapping peaks attributed to such vibrations. The bands at 554 and 455 cm -1 were assigned to external vibrations of double four-rings, and Si-O or Al-O bending vibrations, respectively. Scheme 1. Synthesis of pyrazole-1-carbothioamides 3a-d.

Catalyzed synthesis of pyrazole-1-carbothioamides 3a-d
Utilizing catalysis in organic reactions is an endlessly fascinating and ever-changing phenomenon. My research team is continuously looking for new catalysts to characterise and use in the advancement of organic synthesis [32][33][34][35]. In this paper, we present a new type of catalyst that can be used to synthesis pyrazole-1-carbothioamides, an important class of organic compounds. We must begin with the optimization step, as is common in this type of reaction for the best reaction condition choice. The reactants (hydrazine hydrate, phenyl isothiocyanate, and unsubstituted chalcone) were mixed as a reference step in the presence of zeolite alone and zinc chloride alone, as well as in the absence of a catalyst. The product was not detected in any of the three cases. That made it obvious that the catalyst, which was present in the form of a zeolite-ZnCl2 mixture, was essential. We started the reaction procedure by mixing the catalyst with hydrazine hydrate, phenyl isothiocyanate for 30 min. Then, the addition of chalcone to the separated product yielded the open structure 2-(3-oxo-1,3-diphenylpropyl)-N-phenylhydrazine-1-carbothioamide (m.p. = 140 °C).When the reaction was repeated with polar solvents like H2O or ethanol, mixed products-cyclic pyrazole and open structure-was formed. Under solvent-free conditions, the best reaction conditions was initiated by adding 1 mole of chalcone to 1.2 moles of hydrazine hydrate (98%) and 1 mole of phenyl isothiocyanate in the presence of 10 mg of zinc chloride@Zeolite catalyst (Scheme 1). Table 2 shows the application of the reaction on different chalcones and phenyl isothiocyanates.

Computational studies
The geometries of the pyrazole-1-carbothioamides 3a-d were optimised using density functional theory (DFT) at the B3LYP/6-311 ++ G (d, p) level [36][37][38] and implemented in the programme Gaussian 09 W [39]. Frequency calculations show that the optimised geometries are stable, with positive values for all obtained frequencies. Figure 4 depicts the optimised structures, while Figure  5 depicts the patterns of distribution of frontier molecular orbitals; the highest occupied molecular orbitals (HOMOs); and the lowest unoccupied molecular orbitals (LUMOs). In 3a-d most of the HOMO is localized mainly on thioamide moiety and the pyrazole ring nitrogen atoms with a slight contribution of the phenyl ring-connected to the thioamide. The 3a-c LUMO has consisted of the π*-orbitals of the 1-thioamide-3-phenyl pyrazole substituents and nitrogens of pyrazole. In 3d, although the HOMO is similar to other derivatives, its LUMO showed a completely different composition, where it is localized only on the 5-phenyl pyrazole substituent. The HOMO and LUMO energies (EHOMO, ELUMO), besides the HOMO-LUMO energy gap (Egap) are shown in (   Electronegativity (χ), which indicates the acidic or basic character, global hardness (η) which measures the resistance in charge transfer, and global softness (δ) which describes the molecule's ability to receive electrons. Additionally, energy reduction due to HOMO-LUMO electron flow can be measured by electrophilicity (ω). Table 1 shows that the 3d compound had the lowest global hardness, which was 1.22 eV. On the other hand, the softness is shown in the opposite order, with 3d being the softest at 0.82 eV.

Molecular docking study
During the past three years, the Corona pandemic has terrified the world. It was necessary to search for an urgent drug to reduce the effects of this epidemic on humans. This is why my research group, [41][42][43][44][45], has made great efforts to search for a cure for this disease. Initially, a theoretical study should be conducted using drug design programs (MOE) on the suitability of the prepared compounds with the protease enzyme.  (Figure 6) showed the 2D and 3D interaction diagrams of M pro . Figure 6. Top: 2D of the (3b-M pro active side). Bottom: 3D distance measurements of (3b-M pro active side).

The measuring distance between the drug-ligand
There are two electrostatic bonds between the sulfur atom and the amino acid residues Gly143 and Asn142 with distances of 2.61 and 3.11 Å, respectively also there is another bond between the nitrogen atom in the pyrazole ring and Asn142 with a 2.59 Å distance. This compound exhibited 7 intramolecular forces, indicating a high drug-ligand interaction.

General remarks
Melting points were determined with Gallenkamp melting point apparatus and are uncorrected. The infrared (IR) spectra were recorded on Thermo Scientific Nicolet iS10 FTIR. 1 H NMR and 13 C NMR spectra were recorded DMSO-d6 as a solvent using JEOL's spectrometer at 500 MHz using tetramethylsilane (TMS) as internal standard. Chemical shifts are expressed in δ, ppm. 1 H NMR data are reported in order: multiplicity (br, broad; s, singlet; d, doublet; t, triplet; dd, doublet of doublet; m, multiplet), approximate coupling constant in Hertz, number of protons and type of protons. The purity of the compounds was checked by 1 H NMR and thin layer chromatography (TLC) on silica gel plates using a mixture of (dichloromethane/methanol) or (petroleum ether/ethyl acetate) as eluent. UV lamp was used as a visualizing agent. Elemental analyses were recorded on Thermo DSQ II spectrometer at Faculty of Science, Alazhar University.

Catalyst preparation
Zeolite A powder combined with ZnCl2 catalyst was synthesized using the hydrothermal method. ZnCl2 (1.36 g) aqueous solution was added to 4.0 g zeolite suspension in a 100 mL Teflon-lined autoclave. The sealed autoclave was then placed in a regulated furnace adjusted at 100 °C for 6 h. The furnace is turned off and left to be cooled at a rate of about 10 °C/h. The dried powder was then kept in a desiccator until use.
General procedure for the synthesis of N,3,5-triaryl-4,5-dihydro-1H-pyrazole-1-carbothioamides 3a-d The reaction was started by mixing chalcone (1a-c, 10 mmol) and hydrazine hydrate 80% (1 mL, 20 mmol), the zeolite/ZnCl2 (0.2 g, 20 mol%) was added and the mixture was allowed to stir at 70-80 °C, The reaction was monitored by TLC until 3,5-diphenyl-4,5-dihydro-1H-pyrazoles 2a-c were formed. Then, phenyl isothiocyanate derivative (10 mmol) was added and continued stirring until the reaction completion and this was demonstrated by using TLC. The product was extracted with ethyl acetate (20 mL). Then, the mixture was filtered off and the extract was vaporized. The remaining residue was recrystallized using ethanol to give a pure product.

CONCLUSION
Zinc chloride@Zeolite catalyst was successfully synthesized via ordinary hydrothermal technique. XRD approves the crystalline nature of synthesized nanoparticles while both SEM/EDAX and HRTEM/SAED show the homogenous morphology with a size ranging between 20-30 nm. The dihydro pyrazole-1-carbothioamide derivatives were successfully synthesized by an eco-friendly method using synthesized and characterized nano Zinc chloride@Zeolite catalyst under solvent-free conditions. The 3D conformation, electronic and charge transfer properties of the synthesized derivatives was investigated by, the density functional theory (DFT) where, the derivative 30d showed the lowest HOMO-LUMO energy gap. Using drug design software, the synthesized pyrazoles can be considered COVID-19 main protease (Mpro) inhibitors.