Copper(II) Chelates of Schiff Bases Enriched with Aliphatic Fragments: Synthesis, Crystal Structure, In Silico Studies of ADMET Properties and a Potency against a Series of SARS-CoV-2 Proteins

We report two complexes [Cu(LI)2] (1) and [Cu(LII)2] (2) (HLI = N-cyclohexyl-3-methoxysalicylideneimine, HLII = N-cyclohexyl-3-ethoxysalicylideneimine). The ligands in both complexes are trans-1,5-N,O-coordinated, yielding a square planar CuN2O2 coordination core. The molecule of 1 is planar with two cyclohexyl groups oriented to the opposite sites of the planar part of a molecule, while the molecule of 2 is significantly bent with two cyclohexyl groups oriented to the same convex site of a molecule. It was established that both complexes in MeOH absorb in the UV region due to intraligand transitions and LMCT. Furthermore, the UV-vis spectra of both complexes revealed two low intense shoulders in the visible region at about 460 and 520 nm, which were attributed to d–d transitions. Both complexes were predicted to belong to a fourth class of toxicity with the negative BBB property and positive gastrointestinal absorption property. According to the molecular docking analysis results, both complexes are active against all the applied SARS-CoV-2 proteins with the best binding affinity with Nsp 14 (N7-MTase), PLpro and Mpro. The obtained docking scores of complexes are either comparable to or even higher than those of the initial ligands. Complex 1 was found to be more efficient upon interaction with the applied proteins in comparison to complex 2. Ligand efficiency scores for the initial ligands, 1 and 2 were also revealed.


Introduction
Copper is of great importance for living organisms since it plays a pivotal role in some biological processes. Particularly, a series of proteins comprise copper ions as prosthetic groups and are thus known as copper proteins [1,2], of which the metal-containing centers, in turn, are classified into several types. Of these types, Type II copper centers, abbreviated as T2Cu, contain a square planar coordination core formed either by the nitrogen-or mixed nitrogen/oxygen donor ligands [1]. T2Cu centers in the copper proteins are usually involved in redox processes [2].
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Results and Discussion
A one pot in situ reaction of a solution of Cu(OAc) 2 in ethanol with a solution of cyclohexylamine and 3-methoxy-or 3-ethoxysalicylaldehyde in the same solvent has facilitated the production of mononuclear discrete complexes [Cu(L I ) 2 ] (1) and [Cu(L II ) 2 ] (2) (HL I = Ncyclohexyl-3-methoxysalicylideneimine, HL II = N-cyclohexyl-3-ethoxysalicylideneimine), respectively ( Figure 1). The isolated compounds were characterized by the means of the IR and UV-vis spectroscopy data. Their composition and structure were established by microanalysis, and single crystal and powder X-ray diffraction.
The IR spectra of both complexes are very similar and contain a set of bands at about 2750-3100 cm −1 (Figure 2), corresponding to CH stretching vibrations of the aromatic and aliphatic fragments. An intense band at about 1620 cm −1 and a band at about 1600 cm −1 correspond to C=N and C=C bending. Bands at about 1360 and 1470-1480 cm −1 were attributed to CH stretching vibrations of the aliphatic groups. Vibrations of the C-O-C functionalities are shown as bands at about 1220-1250 cm −1 . According to single crystal X-ray diffraction, 1 crystallizes in monoclinic space group P21/c and the structure is the same as reported before [27]. Complex 2 crystallizes in orthorhombic space group Pbcn. The asymmetric unit cell of both complexes contains a half of a molecule [Cu(Lig)2] with the copper(II) cation lying on the inversion center. The ligands in both molecules are trans-coordinated through the imine nitrogen atom and phenolic oxygen atom, yielding a square planar CuN2O2 coordination core with two Cu1~N1~C1~C2~C7~O1 six-membered chelate rings ( Figure 3). All five atoms of the coordination core are perfectly lying on the same least square plane in the structure of 1, while in the structure of 2 these atoms are slightly deviated from the least square plane (Cu1~0.02 Å, N1~0.04 Å, O1~0.05 Å). The Cu1-N1/O1, C1-N1 and C7-O1 bond lengths and bond angles around the metal cation are similar in both structures (Table 1). However, a minor deviation from linearity was observed for the O1-Cu1-O1′ bond angle in the structure of 2 (Table 1). Notably, the cyclohexyl fragments in both structures adopt a chair conformation ( Figure 3). The IR spectra of both complexes are very similar and contain a s 2750-3100 cm −1 (Figure 2), corresponding to CH stretching vibrations aliphatic fragments. An intense band at about 1620 cm −1 and a band correspond to C=N and C=C bending. Bands at about 1360 and 1470 tributed to CH stretching vibrations of the aliphatic groups. Vibrations tionalities are shown as bands at about 1220-1250 cm −1 . According to single crystal X-ray diffraction, 1 crystallizes in mon P21/c and the structure is the same as reported before [27]. Complex thorhombic space group Pbcn. The asymmetric unit cell of both comp of a molecule [Cu(Lig)2] with the copper(II) cation lying on the inver ands in both molecules are trans-coordinated through the imine nitro nolic oxygen atom, yielding a square planar CuN2O2 coordinat Cu1~N1~C1~C2~C7~O1 six-membered chelate rings ( Figure 3). All fiv dination core are perfectly lying on the same least square plane in the in the structure of 2 these atoms are slightly deviated from the (Cu1~0.02 Å, N1~0.04 Å, O1~0.05 Å). The Cu1-N1/O1, C1-N1 and C and bond angles around the metal cation are similar in both structures a minor deviation from linearity was observed for the O1-Cu1-O1 structure of 2 (Table 1). Notably, the cyclohexyl fragments in both stru conformation ( Figure 3). According to single crystal X-ray diffraction, 1 crystallizes in monoclinic space group P2 1 /c and the structure is the same as reported before [27]. Complex 2 crystallizes in orthorhombic space group Pbcn. The asymmetric unit cell of both complexes contains a half of a molecule [Cu(Lig) 2 ] with the copper(II) cation lying on the inversion center. The ligands in both molecules are trans-coordinated through the imine nitrogen atom and phenolic oxygen atom, yielding a square planar CuN 2 O 2 coordination core with two Cu1~N1~C1~C2~C7~O1 six-membered chelate rings ( Figure 3). All five atoms of the coordination core are perfectly lying on the same least square plane in the structure of 1, while in the structure of 2 these atoms are slightly deviated from the least square plane (Cu1~0.02 Å, N1~0.04 Å, O1~0.05 Å). The Cu1-N1/O1, C1-N1 and C7-O1 bond lengths and bond angles around the metal cation are similar in both structures (Table 1). However, a minor deviation from linearity was observed for the O1-Cu1-O1 bond angle in the structure of 2 (Table 1). Notably, the cyclohexyl fragments in both structures adopt a chair conformation ( Figure 3).    (5) 155.80 (16) Interestingly, the most crucial difference between the molecular structures of the described complexes was observed for the overall geometry of the molecules. Particularly, the molecule of 1 is essentially planar with two cyclohexyl groups oriented to the opposite sites of the planar part of a molecule ( Figure 3). However, the molecule of 2 is significantly bent with two cyclohexyl groups oriented to the same convex site of a molecule ( Figure  3). This is also clearly reflected from the corresponding dihedral angles (Table 1). From one side, such a dramatic difference in the molecular structures of complexes can be explained by a repulsion of the bulky cyclohexyl fragment and the methyl group of the ethoxy fragment in the structure. However, from the other side, closer inspection and comparison of the molecular structures of 1 and 2 has allowed us to reveal that the cyclohexyl fragments tend to form C-H•••H-C homopolar dihydrogen bonding with the imine hydrogen atoms, methyl hydrogen atoms and oxygen atoms of the methoxy fragment in 1 and ethoxy fragment in 2. Recently, we have reported on the influence of C-H•••H-C homopolar dihydrogen bonding on the overall stabilization of the molecular structure of coordination compounds and even on the crucial influence of this interaction on coordination geometry [30]. In-depth studies of intramolecular interactions in the molecular structures of complexes 1 and 2 will be performed using computational approaches and the obtained results will be published elsewhere.  Interestingly, the most crucial difference between the molecular structures of the described complexes was observed for the overall geometry of the molecules. Particularly, the molecule of 1 is essentially planar with two cyclohexyl groups oriented to the opposite sites of the planar part of a molecule ( Figure 3). However, the molecule of 2 is significantly bent with two cyclohexyl groups oriented to the same convex site of a molecule ( Figure 3). This is also clearly reflected from the corresponding dihedral angles (Table 1). From one side, such a dramatic difference in the molecular structures of complexes can be explained by a repulsion of the bulky cyclohexyl fragment and the methyl group of the ethoxy fragment in the structure. However, from the other side, closer inspection and comparison of the molecular structures of 1 and 2 has allowed us to reveal that the cyclohexyl fragments tend to form C-H···H-C homopolar dihydrogen bonding with the imine hydrogen atoms, methyl hydrogen atoms and oxygen atoms of the methoxy fragment in 1 and ethoxy fragment in 2. Recently, we have reported on the influence of C-H···H-C homopolar dihydrogen bonding on the overall stabilization of the molecular structure of coordination compounds and even on the crucial influence of this interaction on coordination geometry [30]. Indepth studies of intramolecular interactions in the molecular structures of complexes 1 and 2 will be performed using computational approaches and the obtained results will be published elsewhere. The bulk samples of 1 and 2 were examined by means of powder X-ray diffraction analysis ( Figure 4). The experimental X-ray powder pattern is in full agreement with the calculated powder pattern obtained from single crystal X-ray diffraction, showing that the bulk material is free from phase impurities. The bulk samples of 1 and 2 were examined by means of powder X-ray diffractio analysis ( Figure 4). The experimental X-ray powder pattern is in full agreement with th calculated powder pattern obtained from single crystal X-ray diffraction, showing that th bulk material is free from phase impurities.  . The former high-energy bands correspond to intraligand π → π* and n → π* transition arising from the benzene and imine fragments, while the latter band was assigned to lig and-to-metal charge transfer (LMCT). Furthermore, a closer inspection of the UV-vis spe tra of both complexes revealed two low intense shoulders in the visible region at abou 460 and 520 nm, which were attributed to d-d transitions ( Figure 5). According to ProTox-II, a virtual lab for the prediction of toxicities of small molecule [31,32], both complexes belong to a fourth class of toxicity with the predicted LD50 of abou 1200 mg/kg ( Figure 6). As evidenced from the SwissADME [33] bioavailability radar, th discussed compounds are preferred in the three parameters, namely polarity, insaturatio and flexibility, and less preferred in lipophilicity, size and insolubility ( Figure 6).
The BOILED-Egg method was found to be efficient to predict the human blood-brai barrier (BBB) penetration and gastrointestinal absorption [34]. This approach is based o lipophilicity (WLOGP) and polarity (topological polar surface area, TPSA) (Figure 6 The former high-energy bands correspond to intraligand π → π* and n → π* transitions arising from the benzene and imine fragments, while the latter band was assigned to ligand-to-metal charge transfer (LMCT). Furthermore, a closer inspection of the UV-vis spectra of both complexes revealed two low intense shoulders in the visible region at about 460 and 520 nm, which were attributed to d-d transitions ( Figure 5). The bulk samples of 1 and 2 were examined by means of powder X-ray diffractio analysis ( Figure 4). The experimental X-ray powder pattern is in full agreement with th calculated powder pattern obtained from single crystal X-ray diffraction, showing that th bulk material is free from phase impurities.  . The former high-energy bands correspond to intraligand π → π* and n → π* transition arising from the benzene and imine fragments, while the latter band was assigned to lig and-to-metal charge transfer (LMCT). Furthermore, a closer inspection of the UV-vis spec tra of both complexes revealed two low intense shoulders in the visible region at abou 460 and 520 nm, which were attributed to d-d transitions ( Figure 5). According to ProTox-II, a virtual lab for the prediction of toxicities of small molecule [31,32], both complexes belong to a fourth class of toxicity with the predicted LD50 of abou 1200 mg/kg ( Figure 6). As evidenced from the SwissADME [33] bioavailability radar, th discussed compounds are preferred in the three parameters, namely polarity, insaturatio and flexibility, and less preferred in lipophilicity, size and insolubility ( Figure 6).
The BOILED-Egg method was found to be efficient to predict the human blood-brai barrier (BBB) penetration and gastrointestinal absorption [34]. This approach is based o lipophilicity (WLOGP) and polarity (topological polar surface area, TPSA) ( Figure 6 Points located in the yellow region (BOILED-Egg's yolk) are molecules predicted t According to ProTox-II, a virtual lab for the prediction of toxicities of small molecules [31,32], both complexes belong to a fourth class of toxicity with the predicted LD 50 of about 1200 mg/kg ( Figure 6). As evidenced from the SwissADME [33] bioavailability radar, the discussed compounds are preferred in the three parameters, namely polarity, insaturation and flexibility, and less preferred in lipophilicity, size and insolubility ( Figure 6). passively permeate through the BBB, while points located in the white region (BOILED Egg's white) are molecules predicted to be passively absorbed by the gastrointestinal tract. Blue (PGP+) and red (PGP−) dots are for molecules predicted to be effluated and not to be effluated from the central nervous system by the P-glycoprotein, respectively. As evidenced from the blue dots' positions for both complexes, the BBB penetration property is negative and gastrointestinal absorption property is positive with the positive PGP effect on the molecule ( Figure 6). We have further applied a molecular docking approach for both complexes with a series of the SARS-CoV-2 proteins. Furthermore, initial ligands were also redocked for a proper comparison of the obtained results. The target structures were primarily selected in accordance with the structural features of the virus [35,36] as well as based on biological mechanisms and functions that can be utilized to reduce, prevent or treat the virus [37] ( Table 2). Table 2. Ligand efficiency scores for the initial ligands, and complexes 1 and 2 inside the binding sites of the listed proteins. The BOILED-Egg method was found to be efficient to predict the human blood-brain barrier (BBB) penetration and gastrointestinal absorption [34]. This approach is based on lipophilicity (WLOGP) and polarity (topological polar surface area, TPSA) ( Figure 6). Points located in the yellow region (BOILED-Egg's yolk) are molecules predicted to passively permeate through the BBB, while points located in the white region (BOILED Egg's white) are molecules predicted to be passively absorbed by the gastrointestinal tract. Blue (PGP+) and red (PGP−) dots are for molecules predicted to be effluated and not to be effluated from the central nervous system by the P-glycoprotein, respectively. As evidenced from the blue dots' positions for both complexes, the BBB penetration property is negative and gastrointestinal absorption property is positive with the positive PGP effect on the molecule ( Figure 6).

Ligand Efficiency
We have further applied a molecular docking approach for both complexes with a series of the SARS-CoV-2 proteins. Furthermore, initial ligands were also redocked for a proper comparison of the obtained results. The target structures were primarily selected in accordance with the structural features of the virus [35,36] as well as based on biological mechanisms and functions that can be utilized to reduce, prevent or treat the virus [37] ( Table 2). Table 2. Ligand efficiency scores for the initial ligands, and complexes 1 and 2 inside the binding sites of the listed proteins.     According to the docking analysis results, both complexes were found to be active against all the applied SARS-CoV-2 proteins with the best binding affinity with Nonstructural protein 14 (N7-MTase), Papain-like protease (PLpro) and Main protease (Mpro) (Figure 7, Table 3). Furthermore, the obtained docking scores of complexes are either comparable to or even higher of those of the initial ligands (Table 3). Moreover, complex 1 was found to be more efficient upon interaction with the applied proteins in comparison to complex 2 (Table 3). Interactions responsible for binding of 1 and 2 with Nonstructural protein 14 (N7-MTase), Papain-like protease (PLpro) and Main protease (Mpro) are shown in Figure 7 and collected in Table 2. According to the obtained results, hydrophobic interactions of the alkyl and π···alkyl types are main contributors for binding the ligands to proteins (Table 3).   We have also established additional ligand efficiency scores to shed more light on the bioactivity of 1 and 2 towards the applied SARS-CoV-2 proteins. As such, for all complexes of 1 and 2 with the studied proteins, we have calculated inhibition constant (K i ), miLogP, ligand efficiency (LE), ligand efficiency_scale (LE_Scale), fit quality (FQ) and ligand-efficiency-dependent lipophilicity (LELP) [38][39][40][41][42][43] (Table 2). Furthermore, for comparison we have also calculated the same ligand efficiency scores for complexes of the studied proteins with initial ligands (Table 2). Notably, the K i value must be as low as possible for a more efficient inhibition and should fall in the µM range for a compound to be considered as a Hit, and >10 nM for a drug [42]. Furthermore, for a compound to be considered as a Hit the LE, FQ and LELP parameters are recommended as ≥0.3, ≥0.8 and from −10 to 10, respectively [42].

Ligand Efficiency
Of all the complexes of the applied proteins with 1 and 2, the ligand efficiency scores for complexes with Nonstructural protein 14 (N7-MTase) are close to be within the recommended ranges for a Hit and even close to values for a drug, although the LELP values are clearly out of the recommended range ( Table 2). These results are preferable for complex of Nonstructural protein 14 (N7-MTase) with 1 in comparison to 2 and even to the initial ligand, although the K i value for a latter ligand is about two times lower but with a less preferable LELP value ( Table 2). For complexes of 1 with Papain-like protease (PLpro) and Main protease (Mpro), the ligand efficiency scores are also within the recommended ranges and even superior to those of the initial ligand except for the LELP values (Table 2).

Physical Measurements
The IR spectra in KBr pellets were recorded with a FT-IR FSM 1201 spectrometer in the range 400-4000 cm −1 . UV-vis spectra from the 10 −4 M freshly prepared solutions in freshly distilled MeOH were recorded on an Agilent 8453 instrument. Powder X-ray diffraction was carried out using a Rigaku Ultima IV X-ray powder diffractometer. The parallel beam mode was used to collect the data (λ = 1.54184 Å). Elemental analyses were performed with a Thermo Scientific FLASH 2000 CHNS analyzer (Waltham, MA USA).

Synthesis
A hot solution of Cu(OAc) 2 (1 mmol, 0.182 g) in ethanol (10 mL) was added dropwise to a hot solution of cyclohexylamine (2 mmol, 0.198 g) and 3-methoxysalicylaldehyde or 3-ethoxysalicylaldehyde (2 mmol, 0.304 and 0.332 g) in the same solvent (20 mL) under vigorous stirring. The resulting mixture was left undisturbed under ambient conditions for slow evaporation of the solvent to give green prism-like crystals suitable for single crystal X-ray diffraction.

Single Crystal X-ray Diffraction
The X-ray diffraction data for 1 and 2 were collected on Bruker SMART Apex-II and Bruker D8 Venture diffractometers, respectively, equipped with a CCD detector (Mo-Kα, λ = 0.71073 Å, graphite monochromator). Semi-empirical absorption correction was applied by the SADABS program [44]. The structures were solved by direct methods and refined by the full-matrix least squares in the anisotropic approximation for non-hydrogen atoms. The structure of 1 was refined as a two-component twin. The calculations were carried out by the SHELX-2014 program package [45] using Olex2 1.2 [46]. CCDC 2235309 and 2235310 contain the crystallographic data for 1 and 2, respectively. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures (accessed on 7 February 2023) or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)-1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.

Molecular Docking
Molecular docking simulations of complexes 1 and 2 with a series of the SARS-CoV-2 proteins were carried using the CB-Dock2 server [47,48], which reveals protein cavities to guide blind docking by the algorithm of AutoDock Vina [49]. The targeted protein structures were subtracted from the RCSB PDB database [50] and were pretreated before the docking, including water removing and inserting hydrogen atoms and missing residues and charges.

Conclusions
We have synthesized complexes [Cu(L I ) 2 ] (1) and [Cu(L II ) 2 ] (2) (HL I = N-cyclohexyl-3methoxysalicylideneimine, HL II = N-cyclohexyl-3-ethoxysalicylideneimine), which were confirmed by IR spectroscopy, single crystal and powder X-ray diffraction, and elemental analysis. The ligands in both complexes are trans-1,5-N,O-coordinated, yielding a square planar CuN 2 O 2 coordination core. The molecule of 1 is essentially planar with two cyclohexyl groups oriented to the opposite sites of the planar part of a molecule, while the molecule of 2 is significantly bent with two cyclohexyl groups oriented to the same convex site of a molecule. Complexes in MeOH absorb in the UV region due to intraligand transitions and LMCT. Furthermore, the UV-vis spectra of 1 and 2 revealed two low intense shoulders in the visible region at about 460 and 520 nm due to d-d transitions.
Both complexes were predicted to belong to a fourth class of toxicity with the negative BBB property and positive gastrointestinal absorption property and the positive PGP effect on the molecule. Complexes were also found to be active against all the applied SARS-CoV-2 proteins with the best binding affinity with Nsp 14 (N7-MTase), PLpro and Mpro. The obtained docking scores of complexes are either comparable to or even higher of those of the initial ligands. Finally, complex 1 was found to be more efficient upon interaction with the applied proteins in comparison to complex 2. Ligand efficiency scores for complexes of 1 and 2 with Nsp 14 (N7-MTase) are close to being within the recommended ranges for a Hit and even close to the values required for a drug.