Computer Aid Screening for Potential Antimalarial Choroquinone Compounds as Covid 19 Utilizing Computational Calculations and Molecular Docking Study

: Click reaction of 4,7-dichloroquinoline (1) with thiosemicarbazide (2) to afford the corresponding 2-(7-chloroquinolin-4-yl)thiosemicarbazide (3) utilized ultrasonic irradiation which can cyclized easily with ethylacetoacetate to give the 3-(7-chloroquinolin-4-ylamino)-tetrahydro-6-methyl-2-thioxopyrimidin-4(1 H )-one (4) via nucleophilic substitution reaction. The synthesized compounds was examined in vitro antimalarial activity with IC 50 = 11.92, 25.37 μg/mL against chloroquinone drug. Furthermore, the theoretical investigation of most active compounds CQT and CQP utilizing of DFT/B3LYP/6-311G(d) and HF/6-311G(d) basis’s set and evaluated their physical characters, bond length, bond angles, dihedral angles, also its HOMO-LUMO energy gap was 3.77 eV which indicate the reactivity of CQP. Moreover, the molecular docking studies of these synthesized compounds showed small energy affinity against SARS-CoV-2 main protease (PDBID: 6lu7) and crystal structure of thermoplasma acidophilum (PDBID: 1q2w) and shorter bond length. All these parameters could be considered with different extent to significantly affect the binding affinity of these compounds to the active protein sites for further biological evaluation on Covid-19.


Introduction
In December 2019, several cases of pneumonia of an unknown etiology appeared in Wuhan, Hubei Province, China. Later, a novel coronavirus was identified in a bronchoalveolar lavage fluid sample from the Wuhan Seafood Market through the use of metagenomic nextgeneration sequencing technology Wu et al., 2020a). On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) named the virus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). This is the seventh member of the coronavirus family that can infect humans after the appearance of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The World Health Organization classified the coronavirus pneumonia epidemic caused by SARS-CoV-2 as a public health emergency of international concern on January 30, 2020 (Zhao et al., 2010;Zhou et al., 2020). A thorough program, including surveillance, diagnostics, clinical treatment, research and the development of vaccines and drugs, is crucial to win the battle against COVID-19 and other infectious diseases . Etiology of SARS-COV-2 Corona viruses are not new infectious pathogens in the world. The first described coronavirus was isolated from chickens in 1937 (Ludwig and Zarbock, 2020). In the mid-1960s the first human coronaviruses were first identified (Steardo et al., 2020). The Corona virus family 208 can be divided into four genera: α, β, γ and δ, as per the genome structure and phylogenetic analysis of coronaviruses (Fehr and Perlman, 2015). The coronaviruses of the α and β mainly infect mammals and humans, while the coronaviruses of the γ and δ typically infect birds (Rodriguez-Morales et al., 2020;Guo et al., 2020). The pathogen that is causing the pandemic is related to the Acute Respiratory Syndrome Coronavirus (SARS-CoV), which caused another back outbreak in 2003 (Sjödin et al., 2020;Mirza and Froeyen, 2020). As of yet, there are no effective treatments or targeted therapeutics against the virus. Because of that, the scientific community is striving to investigate many different mechanisms to interfere with the virus' metabolism. Consequently, in recent clinical trials against COVID-19 several antiviral drugs used with patients with similar viral infections to antimalarial drugs, such chloroquine (I), quinine (II) and amodiaquine (III) in malaria treatment is common (dos Santos Chagas et al., 2019;Kwofie et al., 2020). Also the hydroxychloroquine (IV) has approved drug for malaria disease treatment via the FDA was explored as a medication for SARS-CoV-2. As displayed in Figure  1 (Yao et al., 2020;Liu et al., 2020). Previous reports have shown that, the chloroquine and hydroxychloroquine can inhibit the Coronavirus (COVID-19) by altering the pH at the surface of the cell membrane. This can inhibit the attachment of the virus to the cell membrane. Furthermore, 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 (Grimstein et al., 2019).
The theoretical DFT calculations and the conformational analyses can result in a great contribution to drug discovery by reducing financial costs and saving time (especially for emerging diseases such as COVID-19 and speeding up analyses of target interactions with drug candidates (Gimeno et al., 2019). As a result, different computational studies have been published to help better understand the mechanism of M-pro and try to inhibit its function (Mirza and Froeyen, 2020;Ton et al., 2020;Kong et al., 2020;Tang et al., 2020;Chen et al., 2020;Liu and Wang, 2020;Adem et al., 2020;Yoshino et al., 2020;Hosseini and Amanlou, 2020;Bzówka et al., 2020a;Khaerunnisa et al., 2020;Xu et al., 2020).

Chemistry
The nucleophilic substitution reaction 4,7dichloroquinoline (1) with thiosemicarbazide (2) utilizing ultrasonic irradiation for 30 min at room temperature to afford the corresponding 2-(7chloroquinolin-4-yl) thiosemicarbazide (3) as Click reaction as displayed in Fig. 2. The obtained compound was investigated via spectral data as; the spectrum of IR of the was displayed vibrational bands at amino and NH group at υ = 3390 cm 1 , υ = 3150 cm 1 ; respectively, while the NH bending vibration was showed at υ = 1585 cm 1 . Furthermore, the 1 HNMR of the investigated novel thiosemicarbazide was showed signals at δ8.89 due to N = CH, quinolone and singlet signal of NH2 at δ8.20. The mass spectrum showed a peak at m/z 252 corresponding to its molecular ion (Aboelnaga and EL-Sayed, 2018).
Absolute softness (σ) indicate the interaction of the compound, the difference of CQT (3) (740.1817 eV) while CQP (4) was (244.0135 eV), the large difference for CQT indicate that the activity of CQT to react with ethylacetoacetate .

Frontier Molecular Orbitals (FMO) and Molecular Electrostatic Potential Maps (ESP)
The electrical and optical properties can be inferred through chemical reactions and ultraviolet spectra, but FMO is an amazing guideline method for identifying these properties. Time-Dependent Functional Density Theory (TD-DFT) is used for studying FMO principles (Griffith and Orgel, 1957) The highest occupied HOMO molecular orbital acts as an electron donor and the LUMO lowest unoccupied molecular orbital acts as an electron acceptor ‫.مرجع‬ The energy difference between HOMOs and LUMOs related to the biological activity of the molecule (Dennington et al., 2009). Additionally, it 214 helps in describing the molecule reactivity and kinetic stability. The high kinetic stability is due to the large energy gap between HOMO-LUMO (Gaussian09, 2009). Figure 6 illustrates the distributions and energy levels of the HOMO, LUMO and orbitals computed at the B3LYP/6-311G (d) level for CQT and CQP. The positive and negative phases were symbolized in red and green colors, respectively. As shown in Fig. 4, the HOMO of compound CQT was localized in the fused of quinolone ring and its LUMO was localized in the Natom of quinolone moiety, the value of the energy gap between the HOMO -LUMO is (1.49 eV). Furthermore, the HOMO and LUMO of CQP was localized on Sulphur atom of tetrahydro-6-methyl-2-thioxopyrimidin-4(1H)-one ring with difference in band energy (3.77 eV). This energy gap between HOMO-LUMO indicates the high excitation energies for a lot of excited states and reactivity of these compounds. Moreover, molecular reactivity and biological recognition interactions can be studied by the molecular Electrostatic Potential (ESP) that the nuclei and electrons of a molecule create in the surrounding space (Fukui, 1982). So, the 3-(7chloroquinolin-4-ylamino)-tetrahydro-6-methyl-2thioxopyrimidin-4(1H)-one (4) (CQP) designate a certain point then gather this to remaining surface, indicates that there is a uniform distribution of surface contour to fused quinolone, whereas the methyl-2thioxopyrimidin-4(1H)-one moiety contains the C = O and C = S which acts as electrophilic centers which induced more protonation and gave more reactivity in the biological interaction and in the binding sites of proteins in molecular docking (Schlegel, 1982) as demonstrated in Fig. 4.

Antimalarial Activity
The investigated compounds CQT (3) and CQP (4) were previously treated against action of P. falciparum as displayed in Table 4. The CQT and CQP showed higher antimalarial activity in yield for CQT with (80% yield, IC50 = 25.37 μg/mL) and CQP (87% yield, IC50 = 11.92 μg/mL) as shown in Table 4 and Fig. 5. The preliminary SAR study of CQP has focused on the influence of occurrence methyl-2-thioxopyrimidin-4(1H)-one moiety attached to chloroquinoline and make more polarizable of electrons and enhancing their antimalarial activities, while the CQT with low activity due to presence of NH2 attached of C = S of thiosemicarbazide and more electrons center which increase the activity (Bawa et al., 2010).

Molecular Docking Studies
A molecular modeling study was carried out to explain the cytotoxic activity profile demonstrated by the synthesized compounds. A conformational search using an implicit solvent model was accomplished for the prepared compounds; this was monitored by the refinement of the geometry of local minima through a Quantum-Mechanical (QM) (Morris et al., 2009). Consequently, adaptable docking of the compounds was cultivated in the crystal structure of the (PDBID: 6LU7 Version 2, 2.16 Å resolution, CQP (4) was stimulated with functions as a homo dimer  and leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6 (Jo et al., 2020). The docking simulation of CQT and CQP with (PDBID: 6lu7) as shown in Table 5 and Fig. 6 to evaluate the binding interaction energy and the distance between protein's and it was shown that the (PDBID: 6lu7) attached to CQP with (∆E = -14.4383 Kcal/mol, 2.337Å) with Hydrogen bonding with amino acid Pro132 of NH group rather than the CQT attched with binding energy (∆E = -12.2755 Kcal/mol, 3.576Å) and attched to chloroquinoline with Phe294. We identified a mechanismbased inhibitor (N3) through docking stimulation and determined that the crystal structure of Mpro of SARS-CoV-2 in complex with CQT and CQP. Through a combination of structure-based virtual and highthroughput screening, Furthermore, crystal structure of a protein of unknown function TA1206 from thermoplasma acidophilum (PDBID: 1qw2O) (Pathare et al., 2017) which contain of single unique chain 1qw2(A) (102 residues long) (Pathare et al., 2017). The compounds CQT and CQP were docked with (PDBID: 1qw2) with binding interaction energy (∆E = -6.66, -13.61 kcal/mol); respectively and with short bond distance og CQP with 2.51Å with NH between chloroquinone and tetrahydro-6methyl-2-thioxopyrimidin-4(1H)-one ring which confirmed this H-proton the most active in mobilization of electrons and increase the biological evaluation of Compound CQP rather than CQT which its docking ability with (PDBID: 1qw2) with NH2 groups and bond distance 3.21Å as shown in Table 5 and Fig. 6.

General Procedure
Melting points were measured with a Gallen Kamp melting point apparatus. Silica-gel-coated aluminum plates used to test the purity of the compounds. Infrared spectra (λ-cm 1 ) were recorded on Bruker Vector (Germany) and on Mattson FT-IR 1000 (Cairo University, Egypt), using KBr disks. 1 H NMR spectra were recorded on Gemini 300 MHz, 13 CNMR spectrometer, in DMSO-d6 using dimethyl sulfoxide as a solvent and tetramethylsilane (TMS) as an internal standard (Chemical shift in δ, ppm); 13 C NMR spectra were recorded on Gemini 50 MHz NMR spectrometer. Mass spectra were measured on GCQ Finnigan MAT and Elemental analyses were performed at the micro analytical Center, Cairo University, Giza, Egypt. Biological activity was determined in a laboratory by the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. All the chemicals were purchased from Sigma-Aldrich.

Determination of Antimalarial Activity
Cultures of P. falciparum were measured according to a protocol from (Moloney et al., 1990;Trager and Williams, 1992). The percentage of Inhibition was studied the 50% inhibitory concentrations (IC50) was measured and the data got from the inhibitordependent concentration growth curves were registered into plots with nonlinear retreating analysis from y-axis (inhibition %) to x-axis (inhibitor concentration) (Singh et al., 1997) P. falciparum isolate NF54 was maintained in continuous culture with gentamicin (40 μg/mL) in Petri dishes (5 cm in diameter) with a gaseous phase of 90% N2, 5% O2 and 5% CO2, according to a protocol from (Moloney et al., 1990;Trager and Williams, 1992) P. falciparum parasites were cultured in human erythrocytes (blood group A+ at 10% (v/v) hematocrit) in RPMI 1640 medium (Sigma) supplemented with 25 mM HEPES, 20 mM sodium bicarbonate and 10% heat-inactivated human A+ plasma. The culture was routinely monitored through Geimsa staining of the thin blood smears. The parasitemia of the infected erythrocytes was determined in Giemsa-stained smears by light microscopy. Parasitemias and morphological changes detected in the cultures were scored visually with a 100-fold oilimmersion objective, counting at least 1000 erythrocytes to determine the percentage of the infected erythrocytes (Kaiser et al., 2003). Antimalarial activity assay: The experiments were performed in 96-well culture plates (Nunc); compounds were tested at two-fold dilutions in a dose-titration range of 500 to 2 μM. One hundred microliters of infected human red blood cell suspension (1% parasitemia, 4% hematocrit), with more than 90% of ring forms, were added to each well containing 100 mL of extracts pre-diluted in RPMI-1640. Test plates were incubated for 48 h. Parasite multiplication was determined microscopically after Giemsa staining and expressed as a percentage of the controls without test compounds. A drug-free control (methanol/water 50:50% v,v) was used in all experiments and CQ (0.01 μM) was used as the standard reference drug. Parasitemia and stage distribution were estimated as triplicates daily from Giemsa-stained smears by counting 1000 erythrocytes (Noedl et al., 2002).

Computational Procedures
Calculations of DFT with a hybrid functional B3LYP (Becke's three-parameter hybrid functional using the BLYP correlation functional) with the 6-311G(d) basis set and Hartree-Fock calculations with the 6-311G(d) basis set using the Berny method (Jamróz, 2013), were performed with the Gaussian 09 W program (Ditchfield, 1972). No symmetry constraints were applied during the geometry optimization. The harmonic vibrational frequencies were calculated at the same level of theory for the optimized structures to prove the optimized structures as true minimums and confirm that no imaginary frequency occurs. The wideranging assignments of the vibrational modes were accomplished on the basis of the Potential Energy Distribution (PED), calculated using Vibrational Energy Distribution Analysis (VEDA) program (Foresman and Frish, 1996).

Molecular Docking
The molecular model of innovative sulfonamide derivatives was fabricated using standard bond lengths and angles, with the AutoDock Vina and detected by Discovery Studio Client (version 4.2). Following geometry optimization, a systematic conformational examine was supported out to an RMS gradient of 0.01 Å, with energy minimization of the resultant conformations employing the Confirmation Examination module implemented in Auto Dock Vina. The experimental Structure of Mprofrom SARS-CoV-2 and discovery of its inhibitors (PDBID: 6lu7) (Jo et al., 2020) and Crystal structure of a protein of unknown function ta1206 from thermoplasma acidophilum (PDBID: 1qw2) (Pathare et al., 2017). Missing hydrogens were added to the enzyme and partial charges were considered. After removing the co-crystallized inhibitor, validation monitored by docking of the compounds were carried out by AutoDock Vina and viewed by Discovery Studio Client (version 4.2) (Morris et al., 2009). The goal protein was kept inflexible, while the ligands were disappeared permitted to determine the conformational space exclusive the enzyme cavity; Twenty dispersed docking simulations were run via default parameters and the confirmations were designated constructed on the arrangement of total statistics, E conformation and appropriate with the relevant amino acids in the binding pocket.

Conclusion
In this study, the synthesis of some novel chloroquinoline derivative using green methodology, the synthesized compounds were exhibited high antimalarial activities. The SAR relationship related to the most active compound was CQP (4) with IC50 = 11.92 μg/ml and due to cyclized tetrahydro-6-methyl-2-thioxopyrimidin-4(1H)one ring attached to chlorine ring. The optimized molecular structure of compounds CQT and CQP utilizing of DFT/B3LYP/6-311G(d) and HF/6-311G(d) basis's set, approves their stability and the geometric parameters suggestions between the calculated and the experimental data values indicate that B3LYP basis set is better than the HF method in approximating bond lengths and in evaluating energy, Mullikan and NBO charges. Furthermore, the CQT and CQP were docked against (PDB ID: 6lu7) and (PDBID: 1q2w) and showed the NHhydrogen: 2.337Å of CQP with (Mpro) of SARS-CoV-2. So for further biological investigation we will test these compounds and other quinolone derivatives in treatment of SARS-Covid-19.