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Synthesis and investigation of anti-COVID19 ability of ferrocene Schiff base derivatives by quantum chemical and molecular docking

The recent outbreak of coronavirus disease (COVID-19) has rampaged the world with more than 236 million confirmed cases and over 4.8 million deaths across the world reported by the world health organization (WHO) till Oct 5, 2021. Due to the advent of different variants of coronavirus, there is an urgent need to identify effective drugs and vaccines to combat rapidly spreading virus varieties across the globe. Ferrocene derivatives have attained immense interest as anticancer, antifungal, antibacterial, and antiparasitic drug candidates. However, the ability of ferrocene as anti-COVID-19 is not yet explored. Therefore, in the present work, we have synthesized four new ferrocene Schiff bases (L1-L4) to understand the active sites and biological activity of ferrocene derivatives by employing various molecular descriptors, frontier molecular orbitals (FMO), electron affinity, ionization potential, and molecular electrostatic potential (MEP). A theoretical insight on synthesized ferrocene Schiff bases was accomplished by molecular docking, frontier molecular orbitals energies, active sites, and molecular descriptors which were further compared with drugs being currently used against COVID-19, i.e., dexamethasone, hydroxychloroquine, favipiravir (FPV), and remdesivir (RDV). Moreover, through the molecular docking approach, we recorded the inhibitions of ferrocene derivatives on core protease (6LU7) protein of SARS-CoV-2 and the effect of substituents on the anti-COVID activity of these synthesized compounds. The computational outcome indicated that L1 has a powerful 6LU7 inhibition of SARS-CoV-2 compared to the currently used drugs. These results could be helpful to design new ferrocene compounds and explore their potential application in the prevention and treatment of SARS-CoV-2.


Introduction
The outbreak of coronavirus disease  in Wuhan city of China in December 2019 was renamed severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) by the international committee on taxonomy [1] . Due to its rapid human-to-human transmission, the world health organization (WHO) declared COVID-19 as a pandemic on 12 March 2020 [2] . According to WHO, SARS-CoV2 has rampaged the world with more than 236 million confirmed cases and over 4.8 million deaths across the world till Oct 5, 2021. * Corresponding author.
In last week, the highest numbers of new cases were reported due to new variant of virus in India (70%) [3] . Public health has been affected the most because people do not have the opportunities to access a modern health system and medicine in developing countries of Africa, Latin America and the Caribbean [ 4 , 5 ]. Due to different variants of the coronavirus, there is an urgent need to identify effective drugs and vaccines to combat rapidly spreading virus varieties across the globe [6] .
Ferrocene derivatives have attained immense interest as anticancer, antifungal, antibacterial, and antiparasitic drug candidates [7] . Ferrocene was used for the treatment of anemia in the former USSR because of its low toxicity [8] . The lipophilicity of ferrocenyl groups allows ferrocene, to be administered orally for the treatment of gum diseases which is not the case for simple Fe(II) salts [9] . Furthermore, the activity of ferrocene compounds towards cancer was first investigated in 1978. This early work involved the synthesis of new compounds bearing an antigen that binds strongly to nucleic acids [10] . A ferrocenyl group was chosen as the antigenic moiety, which demonstrated that ferrocenyl polypeptides elicit a strong antigenic response [11] . Thus the ferrocene-based drugs have a marked effect on molecular properties e.g. redox activation and lipophilicity. Moreover, ferrocene compounds containing alcohol substituents have proved to be versatile precursors for effective anticancer and antimalarial agents [12][13][14] .
In the present work, we have synthesized ferrocene derivatives ( L1-L4 ) and characterized them by NMR spectroscopy and Mass spectrometry. The compound L4 was further characterised by single-crystal diffraction. Moreover, we have demonstrated the inhibition impact of ferrocene derivatives over SARS-CoV-2, providing an essential base for the resistance of ferrocene compounds against prime protease (6LU7) proteins of SARS-CoV-2 through docking simulations. We further highlighted the molecular descriptors, ionization potential (IP), frontier molecular orbitals (FMO), electron affinity (EA), and molecular electrostatic potential (MEP) analysis to explore the biological and pharmacological activities. The electronic characteristics along with global reactivity descriptors including electrophilicity index ( ω), electronegativity ( χ), chemical potential (μ), softness (S) as well as chemical hardness ( η) were carried out to understand the active sites of ferrocene derivatives. The outcome of the work demonstrated that the synthesized ferrocene derivatives have an appreciable ability to inhibit SARS-CoV-2 invasion within the human body.

Instrumentation
The elemental analysis (C H N) was performed using an Elementar Vario EL analyzer. Fourier transform IR spectra were measured on a Perkin-Elmer Spectrum One spectrometer with samples prepared as KBr discs. UV-Vis absorption spectroscopy of synthesized samples was carried out by Varian Cary 500 Scan UV-VIS NIR Spectrophotometer. Solution NMR spectra were recorded with Bruker Avance instruments operating at 1 H Larmor frequencies of 400 MHz, using DMSO-d 6 as solvent and TMS as an internal standard for 13 C and 1 H nuclei.

X-ray crystallography
A single crystal of the L4 with dimensions of 0.24 × 0.2 × 0.03 mm was mounted in a glass capillary and data were collected on a Stoe StadiVari diffractometer. Intensity data were collected with graphite monochromated Ga Ka radiation ( λ = 1.34143 nm) at 150 K. The structure was solved by direct methods using SHELXLS-2015 and refined against F 2 by full matrix least squares using SHELXL-2015 [15] . A summary of pertinent crystal data, experimental details and refinement results are shown in Table 1

Computational details
In biological systems, the density functional theory (DFT) is a fascinating method to analyze numerous important properties [17][18][19][20] . The DFT analysis has a significant contribution to the investigation of electronic characteristics of molecules [21][22][23][24][25] as well as optimize geometries within the ground state (S 0 ) [ 26 , 27 ]. B3LYP is a coherent functional for the S 0 geometries of numerous biologically active molecules. In current investigations regarding S 0 geometries optimizations as well as electronic characteristics, we adopted B3LYP/6-31G * * (LANL2DZ) within Gaussian16 software [28] . Furthermore, Autodock version 4.2 was endorsed through Autodock MGL tools for docking by employing the strategy of eliminating H 2 O followed by the addition of more polar hydrogen moiety. Autodock 4.2, the Autogrid resolute the native ligand location around the binding site by organizing the grid coordinates (X, Y, and Z-axis). The docking computations were executed with Autodock 4.2 along with Pymol version 1.7.4.5 Edu.

Synthesis of ferrocene derivatives (L1-L4)
Three new aromatic substituted ferrocene derivatives were synthesized ( L1-L4 ) in a good yield by reacting an equimolar amount of the phenyl-, nitro, sulfonyl-and L4 with chloro-substituted aminophenol (0.5 mmol) and ferrocenecarboxaldehyde (0.5 mmol) using ethanol as a solvent as shown in Scheme. 1 . The structure and composition of the ferrocene compounds were confirmed by 1 HNMR, mass spectrometry, FT-IR and UV-Vis spectroscopy. The structure of compound L4 was also characterized by single-crystal x-ray diffraction.
FT-IR spectra of L1-L4 were recorded in the frequency range of The signals of aromatic protons of L1 was seen as four singlets at 6.45, 6.54, 6.70, and 6.89 ppm whereas three multiplets for aromatic protons appeared at 7.24, 7.37 and 7.50 ppm. In the case of ligand L2 , two doublets with coupling constant of 8.5 Hz were observed at 6.75, 7.37 ppm, and a singlet of

Table 2
Selected bond distances and bond angles of L4.

Crystal structure of L4
The structure of L4 was confirmed using single-crystal X-ray crystallography. The structure and labeling scheme is depicted in Fig. 1 . Selected bond lengths and angles are presented in Table 2 .  [29] . The other bond lengths and angles of the ferrocenyl moiety agree with those re-ported for most ferrocene derivatives [30] The value of the torsion angle C(6)-C(11)-N(1)-C(12) (177.2 °) indicates that the imine adopts the anti-( E ) form which is good agreement with known Schiff bases derived from ferrocene [31] . Also, the advantage of the anti-( E ) form is to retain the co-planarity between the donor and acceptor group which is important for the charge transfer process [32] .

Electronic properties
The highest occupied / lowest unoccupied molecular orbitals (HOMOs / LUMOs) of studied compounds were probed at B3LYP/6-31G * * (LANL2DZ) level ( Fig. 3 ) and the optimized structure of L4 is given in figure S2. The spatial scattering of HOMO within dexamethasone was observed at C = O whereas LUMO over side ring of phenanthrene. The intramolecular charge transfer (ICT) was found for keto C = O (HOMO) to the side ring of phenanthrenee (LUMO). In remdesivir, the HOMO was at pyrrolotriazin unit whereas the LUMO at [ 1 , 2 , 4 ] triazin-7-yl aminopyrrolo [2,1-f] revealing the ICT from HOMO to LUMO. In hydroxychloroquine, ICT was also found from HOMO at amino group of (ethyl)amino]ethanol unit) to LUMO (quinolin) and HOMO → LUMO. In favipiravir, HOMO is primarily at 6-fluoro-3-hydroxypyrazine as well as oxygen of carboxamide moiety while the LUMO is pyrazine-2-carboxamide illuminating ICT from HOMO to LUMO. Similarly, in the synthesized ferrocene derivatives ICT was noticed from HOMOs to LUMOs. The activity of compounds was also strictly associated with the spatial distribution of occupied molecular orbitals enlightening the most credible locations in order that certainly attacked by reactive agents. The FMOs were superimposed considerably thus revealing the particularly reactive nature of the drugs. The energies of FMOs, for example, HOMO ( E HOMO ), LUMO ( E LUMO ), as well as HOMO-LUMO energy gaps ( E gap ) were significant parameters to probe molecular electronic characteristics. The E HOMO , E LUMO , and E gap of recently synthesized ferrocene derivatives along with reference drugs dexamethasone, remdesivir, hydroxychloroquine, and favipiravir are displayed in Table 3 . The molecular structures of the reference drugs are given below.
The measurements of global chemical reactivity descriptors (GCRD) are essential considerations to figure out the activity. Herein, the large number of GCRD parameters were estimated including the electrophilicity index ( ω), softness (S), electronegativity ( χ), chemical potential (μ) as well as chemical hardness ( η) with the help of HOMO/LUMO energies.
where I is the vertical ionization energy and A is vertical electron affinity.
As per Koopmans theorem [36] the ionization energy and electron affinity can be specified through HOMO and LUMO orbital energies as: Values of EA and IP of calculated are given in Table 3 . The higher energy of HOMO is corresponding to the more reactive molecule in the reactions with electrophiles, while lower LUMO energy is essential for molecular reactions with nucleophiles [37] . Hence, the hardness of any materials corresponds to the gap between the HOMO and LUMO orbitals. If the energy gap of HOMO-LUMO is large then the molecule becomes harder [35] .
The electronic chemical potential ( μ) of a molecule is calculated by:   The softness of a molecule is calculated by: The electronegativity of the molecule is calculated by: The electrophilicity index of the molecule is calculated by: The η is interconnected to aromaticity and the value of ω represents the stabilization energy for saturated compound by electrons from the exterior environment [ 38 , 39 ]. The value of μ conveys the electronic tendency to run away based on its electronic cloud whereas the η value provides a degree of hindrance of electronic cloud toward deformation. We have previously reported that Fig. 6. Docking simulation of the interaction between Ferrocene derivatives and reference drugs (green color) and 6LU7 protein. the better radical scavenging ability of a drug was a prerequisite to hinder viral infections [40] . The antioxidant compounds contribute an electron to the free radical and the resulting radical cation should be stable enough to demonstrate better radical scavenging ability in a one-electron transfer mechanism. In this way, the antioxidant ability can be evaluated by ionization potential (IP) and the physical parameter highlighting the electron transfer range can be estimated by IP = -E HOMO . It is anticipated that radical scavenging nature might be superior for those compounds which show smaller IP. The results in Table 3 revealed that L1 have the smallest IP value therefore, it might be a better radical scavenger having good antioxidant ability as compared to the reference drugs (dexamethasone, remdesivir, hydroxychloroquine, and favipiravir). The hole and electron injection energies (HIE and EIE) of these synthesized ferrocene derivatives were calculated and compared to Al and Au electrodes having work function (W) 4.08 and 5.10 eV respectively. The EIE of ferrocene derivatives were estimated as (eV = − E LUMO − ( −W )) and HIE as (eV = − W − ( −E HOMO )), see Table 3 . The hole and electron injection barriers of ferrocene derivatives were found to be smaller than most of the reference drugs.

Molecular electrostatic potential
The molecular electrostatic potential (MEP) was measured experimentally by diffraction approaches and calculated computationally to explore the reactivity of various compounds and/or species [ 41 , 42 ]. The MEP is an important feature to understand the reactivity of various species [43] . The MEP mapped for ferrocene derivatives and reference drugs are illustrated in color visualizations in Fig. 3 . The red color indicates the higher negative potential regions which are favorable for an electrophilic attack, whereas the blue color identifies the higher positive potential regions promising for nucleophilic attack. The MEP decreases in the order blue > green > yellow > orange > red. The maps of molecular electrostatic potential (MEP) were essential to visualize charged regions within the compounds. The MEP mapped with respect to ferrocene derivatives and reference drugs are shown in color displays in Fig. 4 . The red and blue color band identifies negative as well as positive potential areas that would be favorable for electrophilic as well as nucleophilic attack, respectively. In dexamethasone, the negative potential can be seen on O-atoms while positive potential is formed at the H atoms of hydroxyl groups. In remdesivir, negative potential can be seen on oxygen atoms while positive potential at hydrogen atoms of the amino group. In hydroxychloroquine, negative potential can be seen on the oxygen atom of quinolin and oxygen atom of ethanol group while positive potential at hydrogen atoms of hydroxyl and -NH. In favipiravir, negative potential can be seen on the oxygen atoms while positive potential at hydrogen atoms carboxamide moiety. The negative potential can be seen on the O-atom of -OH in L1 , on the nitro group in L2 , and oxygen atoms of -SO 3 H in L3 . However, a positive potential is concentrated within hydrogen atoms. The negative potential (red region) on oxygen atoms indicated further the possible sites for the electrophilic attack. Whereas, blue color (positive potential) on hydrogen atoms revealed that these sites would be promising for nucleophilic attack.

Molecular docking
The structure of the 6LU7 proteins of SARS-CoV-2 is deposited in the Worldwide Protein Data Bank. The virus core protease crystal structure in the complex (6LU7) without water molecules and inhibitor are shown in Fig. 5 . The 6LU7 protein structure was   Table 4 and Fig. 5 . The binding energy values between ligands and 6LU7 proteins of SARS-CoV-2 (active sites in the title compounds and 6LU7 proteins of the SARS-CoV-2) were given in Table 4 and Fig. 6

Conclusion
Since COVID-19 has acquired pandemic status and new variants of corona require the attention of academicians to discover a possible safe and effective drug to ameliorate its effects worldwide. Due to various biological applications of ferrocene derivatives, their structural properties like lipophilicity of ferrocenyl groups, redox potential activation energy, low toxicity, strong antigenic response and oral administration make them potential candidates against COVID-19. In the present study, four new ferrocene derivatives were successfully synthesized and characterized by NMR spectroscopy, mass spectrometry, and single-crystal x-ray diffraction technique. A theoretical insight on synthesized ferrocene derivatives was accomplished by docking score, frontier molecular orbitals energies, active sites, and molecular descriptors which were further compared with drugs being currently used against COVID-19, i.e. , dexamethasone, hydroxychloroquine, favipiravir, and remdesivir. Moreover, the inhibitions of ferrocene derivatives were recorded on the core protease (6LU7) protein of SARS-CoV-2 and the effect of substituents on the anti-COVID activity through the molecular docking approach. The docking results for L1-L4 highlighted that the L1 might have better anti-oxidant and anti-COVID19 ability than other currently used drugs. The computational outcome indicated that such compounds have powerful 6LU7 inhibition of SARS-CoV-2. These findings could be helpful for further exploration of new ferrocene derivatives and their potential applications in the prevention and treatment of SARS-CoV-2. The experimental investigation of biological and anti-Covid activity of these compounds is in progress and will be published separately.

CRediT authorship contribution statement
Ghulam Abbas : Conceptualization, Methodology, writing and reviewing Ahmad Irfan : Computational studies and reviewing, Ishtiaq Ahmed : spectroscopic analysis, Firas Khalil Al-Zeidaneen : Uv-Vis and Mass spectrometry. S. Muthu : Molecular docking, Olaf Fuhr : Single crystal X-ray diffraction and Renjith Thomas Molecular docking and reviewing.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.