Syntheses and structural characterization of divalent metal complexes (Co, Ni, Pd and Zn) of sterically hindered thiourea ligand and a theoretical insight of their interaction with SARS-CoV-2 enzyme

Reacting two equivalents of sterically hindered 1,3-bis(2,6-diethylphenyl)thiourea ligand (L) with CoCl2, NiBr2, PdX2 (X = Cl; Br) and ZnI2 in acetonitrile afforded the corresponding bulky thiourea ligand stabilized four coordinated monomeric [L2CoCl2] (1), [L2NiBr2] (2), [L2PdX2] (3a: X = Cl; 3b: X = Br) and [L2ZnI2] (4.2CH3CN) complexes. Compound 1, 2 and 4.2CH3CN are tetrahedral whereas Pd complexes (3a and 3b) are square planar. In solution, palladium complexes are dominated by cis-isomers. Structural characterization shows inter- and intramolecular hydrogen bonding. Hirshfeld surface and fingerprint plots indicated significant intermolecular interactions in the crystal network. Molecular docking analysis revealed relatively higher SARS-CoV-2 enzyme interacting abilities of the synthesized complexes compared to the free ligand. All compounds have been characterized by elemental analyses, NMR spectroscopy and single-crystal X-ray diffraction.


Introduction
The coordination chemistry of thiourea ligands has always been of central focus not only because of their variety of coordination modes but also due to their broad applications as organocatalysts and potent pharmaceutical agents [1][2][3][4][5]. The sulphur and nitrogen donor atoms present in the parent thiourea enable these ligands to show different bonding possibilities and versatility in coordination chemistry [6][7][8]. Other coordinating donor moieties may also be added but it doesn't necessarily always lead to metal binding. Thus, this ligand class can act as monodentate, bidentate or polydentate ligands, due to the presence of multi-donor atoms [9]. Thioureas may behave as neutral, monoanionic or dianionic ligands [10][11][12]. Steric and electronic variations at the nitrogen atoms have always been of interest as it not only alters their coordination to metal centers but also their physical and chemical properties [13]. For instance, the air and moisture stable Pd complexes of the sterically bulky N,Nˊ-disubstituted thiourea are highly active catalysts for Heck and Suzuki coupling reactions [14][15][16]. The steric hindrance not only prevents the formation of polynuclear complexes but also blocks the coordination of solvent molecules to the metal centre. For example, the reaction of N,Nˊ-dicyclohexylthiourea with Co II , Ni II , and Zn II salts leads selectively to four coordinated complexes [17]. Since long thioureas are known for intramolecular and intermolecular hydrogen bonding interactions due to the presence of the N-H and C=S groups [18,19]. Such hydrogen bonds as well as secondary interactions play a vital role in biological systems and compounds having such short contacts have been found to be better biologically active candidates [20]. Coordination compounds and their biological applications are of particular importance to us [21][22][23][24]. Specifically, there has been a growing demand of metalbased compounds due to their bioinorganic applications. Studies have shown that the presence of metal ions can considerably improve the results than that shown by thiourea alone [25]. The presence of hydrogen bonding in thioureas alone favors not only such interactions bus also increases their solubility in an aqueous medium for a better cellular uptake. Since, thiourea is widely used in pharmaceutical industry, its selection as a ligand poses no questions. Cobalt, nickel, zinc and palladium complexes have received special attention in bioinorganic applications due to their binding ability to human proteins [26].
Since, the outbreak of the coronavirus disease in 2019 , the development of an effective and safe antiviral agent, specifically to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the focus of current research [27,28]. Spike (S) protein of the virus is responsible for interacting and binding to the host cell receptors [29,30]. X-ray crystallographic studies have shown that the human angiotensin-converting enzyme 2 (ACE2) is the specific receptor for the spike (S) protein's receptor binding domain (RBD) [31][32][33]. The emergence of the new variants of the virus is challenging the durability of the available vaccines for permanent treatment. Thus, novel antiviral agents are critical for controlling the pandemic and decreasing the mortality. The major challenges in drug development process are the substantial amount of time and money needed to move through all the phases. Theoretical analyses describing their interaction with such sites have provided an alternative approach to synthetic protocols to efficiently screen millions of compounds in short time [34][35][36][37]. Molecular docking is among the most popular virtual screening techniques that not only predicts the binding affinity but also the structure of protein-target complex, which is necessary for lead optimization at the atomic level [38]. In silico and molecular docking studies have shown that divalent metal complexes being active antiviral agents and immunity enhancers can be very effective against SARS-CoV-2. [39][40][41] Considering the potential bioactivities of the thiourea derivatives [42,43], we have extended our findings to divalent metals (Co, Ni, Pd and Zn) using symmetrically substituted bulky ligand, N,N′-bis(2,6-diethylphenyl)thiourea [44]. In addition to their syntheses, structural characterization and Hirshfeld analyses, we have applied molecular docking approach to evaluate the binding and interaction abilities of the reported compounds as a starting point for the in silico drug discovery.

General Procedures
Analytical grade solvents were used without any further purification and all manipulations were carried out under air without any particular precautions to prohibit oxygen or moisture.

Hirshfeld Surface Analysis
Crystal Explorer 17.50 was used to generate the Hirshfeld Surfaces and 2D fingerprint plots by using the crystallographic information files as input files [48]. The default settings applied for generating the Hirshfeld Surface/fingerprint plots are: property: none; resolution: High (standard). For the generation of fingerprint plots (di vs de plot) we applied; range: standard, filter: by elements and fingerprint filter options are both inside-outside elements including reciprocal contacts. The interactions shorter than the sum of the corresponding van der Waals radii of the atoms, are shown as red spots and the longer contacts with the positive d norm value are highlighted as blue spots ( Figure S1-S5).

Molecular Docking and Normal Mode Analysis
The 3-D structures of the synthesized ligand and its metal complexes were drawn by Molecular Operating Environment (MOE-2016) software [49]. The test compounds were protonated and the energy was minimized by using MOE-2016 software. The Angiotensin converting enzyme of human body, neucleocapsid and spike proteins of SARS-CoV-2 (PDB id: 1R42, 6M3M, 6CS2) were retrieved from the protein data bank [www.rcsb.org/pdb]. All the water molecules from the retrieved crystal structures were removed by using MOE-2016 software (www.chemcomp.com).
For Pd complexes, the calculations were performed for the cis-isomers as energy minimization in the docking procedure favored the cis-isomer formation. All the herein reported compounds were docked with pdb files using default parameters of MOE-2016 i.e. Triangle Matcher, Rigid receptor, score-London dG, and poses-10. We further evaluated the specificity of the docked targets with our synthesized compounds using normal mood analysis (NMA) via WEBnm [50].
These analyses predict the stability and shows the changes in the dynamic nature of the target due to binding of the compound.

NMR Spectroscopy
NMR spectroscopy was used to determine the coordination site of thiourea ligand to be an N or S donor. In comparison to paramagnetic 1 and 2 with broad signals, the diamagnetic complexes 3 and 4 show well resolved signals. Chloroform (CDCl 3 ) solution shows that square planar palladium complexes 3a and 3b coexist as a mixture of cis-and trans-isomers in the ratio of ca.
75%:25% and 56%:44%, respectively at room temperature [assignment is based on the relative resonance integral ratio of the N-H peaks involved in intramolecular hydrogen bonding at δ 10.90 and 10.54 ppm (3a) 10.27 and 9.95 ppm (3b)]. It has been previously investigated and presumed that the unrecrystallized such complexes exist as single isomers [52][53][54]. We didn't observe any variation in the 1 H NMR spectra for 24 hours, suggesting that the isomer distribution rapidly reaches equilibrium in solution and were irrespective of the NMR solvent of crystallization used.
The 1 H NMR spectrum of tetrahedral 4.2CH 3 CN in CDCl 3 exhibit only two peaks for N-H protons, one at higher and the other at lower fields [δ = 6.52 and 9.72 ppm]. The shift to lower field is possibly due to the intramolecular hydrogen bonding between thiourea and halide ligands [55,56]. On the other hand, a singlet appears at 6.42 ppm in CDCl 3 for the two N-H protons of the free thiourea ligand [44]. ppm, respectively. These data indicate the bond formation through the S atom.

X-ray Single Crystal and Hirshfeld Surface Analysis
All compounds were further characterized by X-ray structural and elemental anal-yses. Single crystals of 1.CH 2 Cl 2 were grown from methylene chloride, whereas of 2 and 4.2CH 3 CN were obtained from acetonitrile by slow evaporation at room temperature. Crystals of 3a/b.2CHCl 3 were grown from chloroform. All compounds were found to be mononuclear four-coordinated metal(II) halide complexes, in which the corresponding metal ion is coordinated by two sulphur atoms of the two bulky thiourea ligands, and two halogen atoms. Compounds   In contrast to predominant tetrahedral arrangement around Co the situation for the analogous Ni II compounds is more complicated as it can adopt octahedral, tetrahedral or square planar geometries with thiourea ligands [17,[57][58][59][60][61][62][63][64][65][66][67]. In solvate free complex 2, nickel is located on two fold symmetry axis with pseudo-tetrahedral geometry (Figure 4). The distortion is mainly due to small S1-Ni-   Other member of group 10, Pd led to expected square planar complexes. X-ray analysis showed both 3a.2CHCl 3 and 3b.2CHCl 3 to be trans-isomers (Figure 6 and Figure 7, respectively).
Complexes 3a.2CHCl 3 [69]. Intermolecular interactions also exit due to the solvated CHCl 3 molecules. Attempts to isolate crystals of cis-isomers using other solvents or at low temperature were not successful.         H⋯S/S⋯H (2.3%) (Figure 12). The H⋯N contacts due to the intermolecular N-H···N hydrogen bonding (2.018 and 2.048 Å) between the co-crystallized acetonitrile and amine H-atom of the ligand can be viewed as prominent red spots ( Figure S5). The H⋯S/S⋯H interactions make a marginal contribution to the packing of the title compound. Experimental details of the X-ray single crystal structure analyses are summarized in Table 3.

Magnetic Studies
The magnetic susceptibility experiments show the magnetic moments of µ eff (298 K) of 3.9 and 3.6 µB, for 1 and 2, respectively. The observed magnetic moments are consistent with the solid state structures of the tetrahedral geometries around the cobalt and nickel ions and are comparable to literature values [70,71].

Molecular Docking and Normal Mode Studies
The free ligand and its metal complexes were screened theoretically by molecular docking  docking score = -7.4919) has also shown one polar interaction but H-acceptor with GLN 340 active residue (Figure 14 bottom left). The Co complex (1) with docking score of -6.9505 has shown four polar interactions including one H-acceptor, one π-H and two π-cation with ASN 49, ASN 58 and ARG 357 (Figure 14 top right). The Ni complex (2) with a docking score of -6.8385 has developed three interactions including one H-donor, one H-acceptor and one π-H interaction with GLU 57, THR 55 (Figure 14 middle right). The free ligand (docking score = -6.7015) has two polar interactions in the form of H-donor interactions with ASN 49 (Figure 14 bottom right).

Conclusion
The reaction of the divalent metal salts of the general formula MX 2 (where M = Co, Ni, Pd or Zn and X = Cl, Br, and I) with sterically hindered thiourea ligand (L) leads to the formation of the corresponding monomeric MX 2 L 2 complexes. The thiourea ligand acts as unidentate ligand through the sulfur atom forming air and moisture stable complexes. All complexes except those for Pd adopt tetrahedral geometry. In solution, square planar palladium complexes coexist as a mixture of cis-and trans-isomers (major and minor components, respectively), however, crystallization always led to the isolation of trans-isomers. The complexes are dominated by intramolecular hydrogen bonds which lock the thiourea N-H moiety and the halide ion.
Intermolecular H-bonds were also observed in case of solvated molecules and metal coordinated thiourea ligands. Hirshfeld analyses show that the large number of H⋅⋅⋅H, C⋅⋅⋅H/H⋅⋅⋅C and X⋅⋅⋅H/H⋅⋅⋅X interactions due to van der Waals interactions and hydrogen bonding play major roles in crystal packing. The encouraging binding modes and docking scores suggest that the synthesized compounds, especially Zn complex (4) due to the lack of Jahn Teller effect and Pd complex (3a) due to electronegativity/relative orientation interact better with the receptors of the bio-molecules. These complexes might be used as potent corona virus inhibitors after in vitro as well as in vivo investigations.

Graphical Abstract
Mononuclear bis(thiourea) complexes of divalent Co, Ni, Pd and Zn have been synthesized by applying neutral sterically hindered ligand. Metal complexes show inter-and intramolecular hydrogen bonding. Cis-and trans-isomers were identified for Pd complexes in solution. The encouraging binding mode and docking score of Zn suggest its potential to be used as potent corona virus inhibitors after in vitro as well as in vivo investigations