Synthesis of Novel Halogenated Heterocyclic compounds and their uses as Target SARS-CoV-2 main Protease (M pro ) and Potential Anti-Covid-19

: Since the first appearance of the coronavirus disease-2019 (COVID-19) in Wuhan, China, in December 2019, it has been spreading globally with devastating ramifications. The lack of anti-COVID-19 treatment to date warrants urgent research into potential therapeutic targets. Virtual drug screening techniques enable the identification of novel compounds that are capable of targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ). The latter plays a fundamental role in mediating viral replication and transcription, rendering it an attractive drug target. In this study, twenty six novel halogenated, heterocyclic compounds, which can inhibit M pro , were tested by molecular docking combined with molecular dynamics simulation. Three compounds showed the highest binding affinity to the protein active site and their binding modes coincide with that of Nelfinavir. The binding of the halogenated compounds to M pro may inhibit the replication and transcription of SARS-CoV-2 the virallife cycle. In times of dire need for anti-COVID-19 treatment, this study lays the groundwork for further experimental research to investigate the efficacy and potential medical uses of these compounds to treat COVID-19. Novel compounds including fused thiophene, pyrimidine and pyran derivatives were tested against human RNA N7-MTase (hRNMT) and selected viral N7-MTases such as SARS-CoV nsp14 and Vaccinia D1-D12 complex to evaluate their specificity and their molecular modeling was also studied in the aim of producing anti covid-19 target molecules.


Introduction
Medicinal chemistry had its beginning when chemists, pharmacist and physicians isolated and purified active principles of plants and animals' tissues and taken from microorganism and their fermentation products. Some of these chemicals has been associated with therapeutic properties: Medicinal chemistry which has leaned on the classical fields of chemistry, especially organic chemistry, biology and some area of physics [1][2][3][4][5][6][7][8]. A limited number of natural and synthetic products and serve directly as therapeutic agents although lack of specificity frequently limits their application in human and veterinary medicines and in analogous pesticidal and other uses in agriculture [9][10][11][12]. By dissecting the structure of these products chemically, one arrives at its therapeutically significant molecular sections, the pharamacophores, the portion that can be deleted are of no interest as components of drug action; they are regarded as the result of the biosynthetic efforts on the parent organism to construct materials for its own matebolic or defensive purposes. Most of the drugs belong to the class of heterogenius compounds. Heterocyclic compounds played a vital role in the metabolism of all living cells; large number of them are five and six membered heterocyclic compounds having one to three heteroatoms in their nucleus [13][14][15][16][17][18]. The compounds may be thiophene, pyran derivatives that were basis of genetic material DNA, and these heterocyclic compounds may be isolated or fused heterocyclic systems. Some of the common heterocyclic compounds used in the medicines are as amino acids like proline, histidine and tryptophan, the vitamins and coenzymes precursors such as thiamine, riboflavin, pyridoxine, folic acid, biotin, B12 and E families of the vitamins. There is a vast number of pharmacologically active heterocylic compounds, many of which are in regular clinical use. The thiophene and its derivatives have a vital role in biological properties [19][20][21][22]. In the present work, we report the synthesis of new heterocyclic compounds with high chloro content together with their studying as potential anti-corona virus. The current pandemic coronavirus disease-2019 (COVID-19) is a new infectious pneumonia-like illness caused by a novel virusstrain, so-called severe acute respiratory syndrome coronavirus 2or SARS-CoV-2 [23,24]. The hydroxychloroquine an approved drug for malaria disease by FDA was explored as a medication for SARS-CoV-2 [25,26]. Previous reports revealed that, the chloroquine and hydroxychloroquine can inhibit the coronavirus (COVID-19) by changing the pH at the surface of the cell membrane. This action can inhibit the attachment of the virus to the cell membrane. In addition, it can prevent nucleic acid replication, glycosylation of viral proteins, virus assembly, new virus particle delivery, virus release, and other mechanisms to obtain its antiviral effects [27]. Heterocycles are widely investigated for possible medicinal applications [28,29]. Favipiravir (1) [30], amodiaquine (2) [31], 20-fluoro-20-deoxycytidine (3) [32], and (4) [33] are known as antiviral drugs. In biological systems, the halogenation of organic molecules is catalyzed by enzymes haloperoxidases such as MPO, EPO and LPO in normal physiological processes, which combine the inorganic substrates Xand H2O2 to produce RHS. RHS in turn oxidize the hydro-carbon or-nitrogen substrate RH/RNH to synthesize many ) (2) By reaction (1), many halogenated organic compounds are biosynthesize they inhibit various RNA and DNA viruses. For that reason, in the present work we concerned with heterocyclic compounds that are halogen rich and studying their potential anti-covide 19 inhibitions. Hence, we aim to determine whether the protease of COVID-19 can be a target protein of these nucleotides in silico. Moreover, a comparative study between these drugs with the FDA approved remdesivir and hydrocloroquine antiviral drugs against a broad range of RNA viruses [34] has been established to investigate the effectiveness of the drugs as inhibitors for COVID 19. Demonstrations for the synthesis of halogen rich heterocyclic compounds together with their potentialities for corona virus were demonstrated through this work.

2.1.General
The melting points obtained for the synthesized compounds were uncorrected and were recorded using an Electrothermal digital melting point apparatus. IR spectra (KBr discs) were measured using a FTIR plus 460 or PyeUnicam SP-1000 spectrophotometer. 1
In addition most of the tested compounds were not active toward hRNMT. Interestingly some compounds like 10a, 10f, 10g and 10h showed IC50's < 0.05 against hRNMT.

Structure retrieval and preparation:
Subsequently, SCIGRESS3.2 software was used to optimize the molecules using the implemented molecular mechanics force field (MM3) and the semiempirical parameterization method 6 (PM6) [36,37]The optimized structures are checked for any transition state by calculating the Infrared (IR) spectra [38]. The optimized structures were prepared for the docking experiment using the AutoDock Tools software [39] and the AutoDockVina software was utilized in the docking of the halogenated compounds into the active site of SARS-CoV-2M pro [40]. The best resolution structure for SARS-CoV-2 M pro was used in this study (PDB ID: 6Y84), and it was subjected to equilibration and Molecular Dynamics Simulation (MDS) run for up to 100 ns using Nanoscale Molecular Dynamics (NAMD) software [41]. Following the MDS, the SARS-CoV-2 M pro trajectories were clustered using Chimera software into five different clusters [42] and a representative structure from each cluster was used in the docking study.

Molecular Docking study:
The

Results and Discussion
The effective treatment options for SARS-CoV-2 can either be the use of broad-spectrum antiviral drugs or specific therapeutic molecules thatcan disrupt the viral lifecycle. Since the rapid transmission of coronavirus has proven to be devastating worldwide, several preventive approaches have been suggested by the health care authorities.The implementation of quarantine for infected patients, the monitoring and timely diagnosis of suspected cases, the use of protective masks, andthorough hand washing can help to control disease dissemination [45].
Unfortunately,infection and mortality rates are rising, and no specific COVID-19 treatment has been confirmed to be successful.Thus,extensive research aimed at discovering and developing COVID-19 treatment drugs is urgently warranted.Studies have investigated the therapeutic targeting of the main protease of SARS-CoV-2 (M pro ) to combat COVID-19 [46]. In this study, the halogenated compounds exhibit the potential to inhibit the M pro , albeit without guaranteed activity. Nevertheless, these insightful findings provide a foundation for computational drug discovery of novel compounds to combat SARS-CoV-2.
Generally, halogen bonding interactions in biological systems have been widely acknowledged [47][48][49][50][51][52][53][54][55][56][57][58][59][60][61]. [Not only did halogen-containing compounds attract attention through a fundamental research perspective, but also through drug evolution to meet clinical endpoints [62]. This is exemplified by the formulation of a series of halogencontaining inhibitors of cathepsin after the discovery of hydrogen bonding between selected ligands and its active site [57]. In addition, Xu et al studied the interaction between sildenafil and its receptor, PDE5, with the goal of improving the binding efficiency between its successors and the receptor [59]. Following the analysis of the atomic details ofreceptor-drug contacts, a molecular docking program was used to replace hydrogen atoms in the drug with halogen atoms that, theoretically, could produce halogen bonds. By utilizing a hybrid method including both quantum and molecular mechanics, Xu et al were capable of refining the search for successors which were subsequently synthesized [59,62]. The progeny's binding energies with the receptor have been discovered to be in remarkable accordance with the computational predictions. This proves thathalogen bonding could be rationally exploited to impact drug discovery [62].  7a, 7c, 7d, 10a, 10b, 10d, 10e, 10f, 10h, 10i, 10k and 10m.
Conversely, compounds 3a, 3b, 5a, 5b, 5c, 5d and7b (blue columns) show higher average binding energies, with less affinity, compared to Nelfinavir against SARS-CoV-2 M pro (-5.92 ±0.34 up to -4.76 ±0.17 kcal/mol). The AutoDockVina scores are listed among the number of H-bonds and hydrophobic contacts and the residues that interact. The green residue is the one that interacted with halogen bonds with the ligands. Bold residues are the most common residues.

Figure 1: The average binding energies (kcal/mol) of 22 halogenated compounds and Nelfinavir against SARS-CoV-2 M pro active site residue H41 and C145. Error bars represent the standard deviation (SD). The halogenated compounds are classified into top compounds (dark green), compounds with comparable binding energywithNelfinavir (light green), and compounds that bind SARS-CoV-2 M pro with less affinity compared with Nelfinavir (blue), while Nelfinavir is red-colored.
A representative docking complex was selected for further analysis of each compound. Table 2 shows the PLIP analysis of the compounds after docking when the main interactions established were Hydrogen, halogen bonds and hydrophobic contacts. It is noteworthy that the most common residues that interactedwith the halogenated compounds were T25, N142, E166, D187 and Q189.The best three compounds in terms of binding affinity (compounds 13, 17, and 21) are depicted in figure 2 with stick representation. As reported from both Table 2 and Figure 2, the most common residues that contributed to the interaction with the halogenated compounds wereT25, E166, D187, and Q189. halogen-containing compounds as potential antiviral agents [63,64].

Figure 3
The superposition of the docking complexes of Nelfinavir (gray), and the compounds 10c(green), 10g(blue), and 10l (orange) docked into the active site of SARS-CoV-2 M pro . The enlarged panel is depicted to show how the compounds are lying in the active site cavity of the protein.
Compounds 3a and 3b were capable for thiophene synthesis through the well known Gewals's thiophene synthesis [65][66][67]. Thus, the reaction of either 3a or 3b with each of elemental sulfur and either malononitrile (4a) or ethyl cyanoacetate (4b) gave the 2,2,2trichloroethylidene)-6,7-dihydrobenzo[b]thiophen-5(4H)-one5a and 5b, respectively. The analytical and spectral data of compounds 5a-d were in agreement of their strutuctures.   In recent years, multicomponent reactions (MCRs) have become essential, efficient, bond-forming methods for expedient synthesis of a wide range of active organic compounds and natural products without separation and purification of intermediates.
The MCRs, which are important classes of chemical transformations, have recently attracted much attention owing to their high efficacy, shorter reaction times, mild conditions, simplicity, and environmental friendliness [68][69][70][71]. During the last several years, the diverse applications of such2-amino-4H-pyran heterocyclic scaffolds in medicinal chemistry have drawn appreciable attention among synthetic chemiststo explore useful synthetic routes to these heterocycles of potentialinterest with antimicrobial and antitumor activities [72,73]. Compounds 5a,c were ready for multicomponent reactions due to the presence of α-oxomethlene moiety within such molecules. Therefore, the reaction of either of 5aor 5cwith either benzaldehyde (9a), 4chlorobenzaldehyde (9b) or 4-methoxybenzaldehyde (9c) and either malononitrile (4a) or ethyl cyanoacetate (4b) in 1,4-dioxane containing a catalytic amount of triethylamine gave the 8,9-dihydro-4H-thieno[2,3-g]chromene derivatives 10a-m, respectively (Scheme 3). The analytical and spectral data of the latter compounds were in agreement with their respective structures.

Conclusion:
Halogenated compounds have been widely acknowledged in the field of medicinal chemistry owing to their binding potentials. that 10a, 10b, 10c, 10e, 10f, 10g and 10h showed high % inhibitions against SARs-Cov nsp 14. Whereas, compounds 5a, 7a, 8b,   10a, 10b, 10c and 10i showed high inhibitions on hRNMT.this study explored the binding affinity of 22 halogenated compounds to the SARS-CoV-2 M Pro and discovered 15 compounds with higher binding affinity thanNelfinavir, of which 3 showed remarkable results. In times of dire need for anti-COVID-19 treatment, our results lay the foundation for further exploratory research on these candidate compounds to examine their mechanism of action and efficacy against corona virus.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
This study was approved by the local Research Ethics Committee at the University of Tabuk, Kingdom of Saudi Arabia.

FUNDING
This study was funded by a research grant ( 1441-0190 ) from the University of Tabuk, Kingdom of Saudi Arabia.