Mononuclear Tricoordinate Copper(I) and Silver(I) Halide Complexes of a Sterically Bulky Thiourea Ligand and a Computational Insight of Their Interaction with Human Insulin

Reaction of two equivalents of the bulky 1,3-bis(2,6-diethylphenyl)thiourea ligand (L) with MX (being M = Cu+, Ag+; and X = Cl−, Br−, I−) in acetonitrile afforded neutral complexes of the type [MXL2] [CuClL2].2CH3CN (1a); [CuBrL2].2CH3CN (1b); [CuIL2] (1c): [AgClL2] (2a); [AgBrL2] (2b) and [AgIL2] (2c). The two aromatic groups in free ligand were found to be trans with respect to the thiourea unit, which was a reason to link the ligand molecules via intermolecular hydrogen bonding. Intramolecular hydrogen bonding was observed in all metal complexes. The copper complexes 1a and 1b are acetonitrile solvated and show not only intra- but also intermolecular hydrogen bonding between the coordinated thiourea and the solvated acetonitrile molecules. Silver complexes reported here are the first examples of structurally characterized tricoordinated thiourea-stabilized monomeric silver(I) halides. Molecular docking studies were carried out to analyze the binding modes of the metal complexes inside the active site of the human insulin (HI) protein. Analysis of the docked conformations revealed that the electrostatic and aromatic interactions of the protein N-terminal residues (i.e., Phe and His) may assist in anchoring and stabilizing the metal complexes inside the active site. According to the results of docking studies, the silver complexes exhibited the strongest inhibitory capability against the HI protein, which possesses a deactivating group, directly bonded to silver. All compounds were fully characterized by elemental analysis, NMR spectroscopy, and molecular structures of the ligand, and five out of six metal complexes were also confirmed by single-crystal X-ray diffraction.


Introduction
Thiourea ligands are of interest mainly due to their ready accessibility and broad applications in catalysis, biological activity, and optical technology [1][2][3][4]. The presence of soft sulfur and harder nitrogen donor atoms makes these ligands show different bonding possibilities and thus exhibit versatility in coordination chemistry. Thioureas can act as neutral, monoanionic, or dianionic ligands [5][6][7]. Despite the fact that it is a mature field of research, it nevertheless continues to attract the attention of synthetic chemists due to the possible variation of steric and electronic effects at the nitrogen atoms. This, in turn, alters their physical as well as chemical properties and has thus led to the isolation of not only efficient but also air-and moisture-stable catalysts, for instance for azide-alkyne addition and Heck reactions [8][9][10]. One of the focuses in the past has been the synthesis of

Materials and Methods
Analytical grade solvents were used without any further purification. Deuterated solvents were obtained from Cambridge Isotope Laboratories and were degassed and dried prior to use. Thiourea ligand (1,3-bis(2,6-diethylphenyl)thiourea = L) was obtained from the reaction of CS 2 with a solution of 2,6-diethylaniline, trimethylamine, and water. Although the used ligand has been previously reported [21], we instead followed the synthetic methodology adopted by Cowley and co-workers [34]. NMR spectra were recorded on a Varian spectrometer at 300 MHz and 400 MHz at ambient temperature. The chemical shifts are reported in ppm relative to the internal TMS. Elemental analyses (CHN) were determined using a Vario EL III instrument. X-ray crystal structure analyses were performed by using a STOE-IPDS II and a STOE STADIVARI (λ(Mo-Kα) = 0.71073 Å) diffractometers equipped with Oxford Cryostream low-temperature units. Structure solution and refinement was accomplished using SIR97 [35], SHELXL2014 [36], WinGX [37], and Olex2 [38]. For L and 2c, we observed one B-alert each in the checkcif files due to the. missing of low-angle reflections as a result of beam stop and bad crystal quality, respectively. Selected crystallographic data are gathered in Table 1.

Synthesis of Compounds 1a-c and 2a-c
For the synthesis of 1a, acetonitrile (30 mL) was added to ligand (0.169 g, 0.5 mmol) and CuCl (0.025 g, 0.25 mmol) at room temperature. The resulting suspension was stirred overnight, resulting in a small amount of white precipitation. The precipitate was separated by filtration, and the filtrate was allowed to afford colorless crystals of 1a. Both precipitate and the crystalline material were identified to be the same material of the desired compound 1a. Following the same procedure, 1b was prepared by treating ligand (0.169 g, 0.5 mmol) with CuBr (0.36 g, 0.25 mmol). For 1c, ligand (0.169 g, 0.5 mmol) was treated with CuI (0.048 g, 0.25 mmol). Acetonitrile was evaporated and the crystalline material was re-dissolved in CHCl 3 and filtered. The filtrate was allowed to slowly evaporate to afford colorless crystals of 1c at room temperature. Following the synthetic procedure of 1a, compund 2a was prepared by reacting ligand (0.169 g, 0.5 mmol) with AgCl (0.036 g, 0.25 mmol). For 2b, ligand (0.228 g, 0.67 mmol) was reacted with AgBr (0.063 g, 0.33 mmol). Compound 2c was prepared by reacting ligand (0.169 g, 0.5 mmol) with AgI (0.059 g, 0.25 mmol). Acetonitrile was evaporated, and the crystalline material was re-dissolved in CHCl 3 and filtered. Slow evaporation of solvent from filtrate at room temperature afforded colorless crystals of 2c.

Molecular Docking
The synthesized metal-based ligand complexes were studied using a molecular docking simulation with the crystal structure of human insulin (HI) as a target protein. The HI was retrieved from the RCSB protein data bank (https://www.rcsb.org/ accessed on 27 February 2022). The co-crystallized macromolecule cucurbit [7] uril is included in the HI protein structure. In order to elucidate the molecular mechanism of inhibition, the metal complexes (1a-c and 2a-c) were docked in the active sites of the HI protein using the molecular operating environment (MOE) v2020 tool [39] and PDB code 3Q6E [40]. The target was prepared using the protein preparing module of the MOE. All solvent molecules were removed before the docking studies could begin. The cleft-like active site was mapped around the cognate ligand. Highly stiff complexes were found to benefit from the HI protein's availability of a cleft-like pocket. As an active site, we leveraged that cleft. Finally, refined crystal structure was used for further docking study using the default parameters of MOE (i.e., Placement: Triangle Matcher, Rescoring 1: London dG, Refinement: Forcefield, Rescoring 2: GBVI/WSA). For the ligand, a total of 50 conformations were chosen before executing the docking approach. For the protein-ligand interaction (PLI) study, the top-ranked conformations by docking score and binding energy were chosen. Additionally, we have evaluated the specificity of the target with the metal complex using dynamut and WEBnm [41,42].
Compound L crystallized in monoclinic crystal system in P2 1 /c space group with six molecules in the unit cell. The two aromatic groups are in trans position with respect to the thiourea unit ( Figure 1). This configuration favors dimer formation between the molecules, and in this case, intermolecular N-H· · · S hydrogen bonds (3.266 and 3.328 Å) link the molecules into dimers (Figure 2). One of the ethyl groups shows disorder, and this is the reason for the relatively large displacement parameter and an unusual C8-C9 bond distance (1.428(6) Å). The short C-S distance (1.682(3) Å) clearly shows its double bond character (for comparison, C-S single bond is 1.82 Å) [57]. The S1-C1-N1-C2 and N2-C1-N1-C2 torsion angles are 1.58 and 178.42 • , respectively. Experimental details of the X-ray single crystal structure analyses are summarized in Table 1.  To isolate the metal complexes, acetonitrile was added to L and the corresponding metal (I) halide (metal = Cu, Ag; halide = Cl, Br, I) in 2:1 ratio at room temperature (Scheme 1). The reaction mixture was then stirred overnight at room temperature. After filtration, the filtrate was concentrated and stored at room temperature to afford the corre-  , and δ = 6.43 and 9.31 ppm (2c), respectively). The shift to lower field could be attributed to the possible presence of intramolecular hydrogen bonding between thiourea and halide ligands [58,59]. For comparison, a singlet appears at 6.42 ppm in CDCl 3 for the two N-H protons of the free ligand [21]. The 13 C NMR spectra of 1 and 2 show characteristic peaks at 178.9 ppm (1a), 178.6 ppm (1b), and 178.0 ppm (1c); and 178.7 ppm (2a), 179.1 ppm (2b), and 179.7 ppm (2c), respectively, for C=S moiety (see Supplementary Materials). The other aliphatic and aromatic chemical shifts for both bulky thiourea ligands are in the typical regions with very less variation. The NMR data of 1a/b show that these complexes are stable even if the co-crystallized acetonitrile molecules are removed by drying the samples in high vacuum.
All complexes except 2b were further characterized by X-ray structural and elemental analyses. All compounds are mononuclear three-coordinated metal(I) halide complexes, in which metal is coordinated by two sulfur atoms of each bulky thiourea ligand, and one halogen atom, thus exhibiting trigonal planar molecular configurations with a butterfly structure. The driving force for this structure might be the intramolecular hydrogen bonds between halogen and the two nitrogen atoms of the two ligands to form pseudo-sixmembered rings (NHClCuSC). Intermolecular hydrogen bonds observed were as a result of the solvated molecules. Experimental details of the X-ray single-crystal structure analyses are summarized in Table 1.
The use of coordination compounds as metal-based drugs is known to lower the blood sugar levels in diabetic patients [68]. To develop a new drug, the fundamental quest is to understand their interaction and efficiencies with the receptor site. The availability of the 3D structure of the target protein makes molecular docking one of the best screen options. Molecular docking simulation was performed in order to evaluate the binding pattern of the synthesized metal complexes inside the active site of the human insulin (HI), using the default settings built in the MOE modelling tool.
In general, hydrophobic residues have been found to have a strong interaction with the ligand essential moiety. The best docking positions with the most interactions were those that were ranked first based on the least amount of energy (calculated as a negative value by MOE), as listed in Table 2. In order to further study the interactions of the metal-based docked conformations with the key residues, the most favorable docking poses of the 50 docked conformations for each metal complex were analyzed. Both the hydrophobic and hydrophilic portions were used to create the active site cleft (see in Figure 9a). Catalytic residues are mostly found in the hydrophilic region of the HI protein, and they may play an effective role in increasing or decreasing its activity.  Molecular docking studies showed that the metal complexes stabilize themselves inside the active site cleft through electrostatic and aromatic interactions. The N-terminal Phe and His residues may stabilize the molecules and prevent their dissociation from the active site residues. The hydrophobic and hydrophilic amino acid residues formed a stable host-guest complex by interacting via π-stacking and hydrogen bonding. The protein-ligand interaction profiles of the complexes are shown in Figure 9b-g. In all the complexes, the sulfur atom of the thiourea ligand was ligated with the corresponding metal atom of the complexes. Despite the rigid phenyl ring, all the complexes managed to attain the docking table conformation inside the active site cleft, as shown in Figure 9a. In addition to that, the phenyl established significant aromatic interactions with the active site residues, which played a significant role in anchoring the metal complexes inside the pocket of HI. The present docking study found that the silver-based complexes showed the strongest inhibitory potential against the HI protein, which possesses a deactivating group that is directly connected to silver. The deactivating group might withdraw some of the electronic density from the reaction center, leaving the moiety with a partial positive charge, which drives the moiety to form crucial contacts with critical residues, resulting in greater stability. Metal complexes 1a and 2a, containing chlorine, both exhibited interactions with the active site residue through the halogen atom, which can be attributed to the higher electronegative nature of chloride ion. Complexes having bromine and iodine did not show any type of electrostatic interactions with the active site residues. Thus, it can be argued that the electronegativity may play an important role in designing novel metal-based HI inhibitors. Since the halogen group withdraws electronic density from the compound, it needs to stabilize itself by adopting interaction with the active residues. In general, the hydrophobic residues preferred to establish the interactions with the important phenyl moiety of the complex. Aromatic moieties adopted interactions particularly with the sulfur and metal atoms, directly in the metal-based complexes. Interestingly, these hydrophobic residues bonded with high potential, and anchored the complex inside the active site of the protein with binding energy of around −10.7 to −7.1 kcal/mol, which is quite significant for stabilization of the metal complex inside the active site cavity. The impact of withdrawing the electronic density from the compound further compelled the compound to manage possible interaction for the ease of gaining a stable environment inside the active site cleft.
Additionally, we have evaluated the normal mode analysis (NMA) for 2a in order to check the dynamic nature/equilibrium modes of the complex ( Figure 10). The NMA can model amino acids using Cα atoms to reduce computing cost. Normal mode analysis shows that the 2a and protein complex is stabilized by harmonic potentials. The results indicate that upon binding with 2a, the overall protein conformation remains compact and had a strong positive correlation instead of negative correlation, which further indicates that this complex remains in the active site and will need enough energy to be dissociated from the pocket. It was observed that the residues' fluctuation is much higher than that of the complex state, revealing the impact of the complex in the binding site, to rescue the protein from high conformation. Thus, it can be suggested that these complexes might be used as a stand-in surrogate for developing and designing novel drugs.

Conclusions
The reaction of salts of univalent metals MX (where M = Cu or Ag and X = Cl, Br, and I) with sterically hindered thiourea ligand leads selectively to corresponding monomeric MXL 2 complexes. All compounds are neutral, and air and moisture stable. Intermolecular H-bonds were responsible for forming dimers in free ligand. The tricoordinate monomeric complexes of silver halides reported here are the first structurally characterized examples of the vastly studied thiourea ligands. Intramolecular H-bonds were observed between N-H· · · X atoms, whereas intermolecular H-bonds were evident between solvated acetonitrile and metal-coordinated thiourea ligands. The docking results demonstrated that the silver-based ligand complex 2a has a strong inhibitory potential against the HI protein, which contains a deactivating group that is directly linked to silver. Based on the current docking simulations, it is suggested that these complexes may further be investigated for their inhibitory potential against the HI. These may serve as a surrogate for discovery of novel HI inhibitors.