Shedding Light on the Synthesis, Crystal Structure, Characterization, and Computational Study of Optoelectronic Properties and Bioactivity of Imine derivatives.

Two imine compounds named as (E)-2-(((3,4-dichlorophenyl)imino)methyl)phenol (DC2H) and (E)-4-(((2,4-dimethylphenyl)imino)methyl)phenol (DM4H) are synthesized, and their crystal structures are verified using the single-crystal X-ray diffraction (XRD) technique. The crystal structures of the compounds are compared with the closely related crystal structures using the Cambridge Structural Database (CSD). The crystal packing in terms of intermolecular interactions is fully explored by Hirshfeld surface analysis. Void analysis is carried out for both compounds to check the strength of the crystal packing. Furthermore, a state-of-the-art dual computational technique consisting of quantum chemical and molecular docking methods is used to shed light on the molecular structure, optoelectronic properties, and bioactivity of indigenously synthesized compounds. The optimized molecular geometries are compared with their counterpart experimental values. Based on previous reports of biofunctions of the indigenously synthesized imine derivatives, they are explored for their potential inhibition properties against two very crucial proteins (main protease (Mpro) and nonstructural protein 9 (NSP9)) of SARS-CoV-2. The calculated interaction energy values of DC2H and DM4H with Mpro are found to be -6.3 and -6.6 kcal/mol, respectively, and for NSP9, the calculated interaction energy value is found to be -6.5 kcal/mol. We believe that the current combined study through experiments and computational techniques will not only pique the interest of the broad scientific community but also evoke interest in their further in vitro and in vivo investigations.

Introduction

Schiff bases belong to an important class of ligands bearing an imine or azomethine (−C=N−) functional group. Hugo Schiff, a German scientist and Nobel Prize laureate, prepared the first of these condensation products from primary amines and carbonyl compounds in 1864.[1,2] The Schiff base is a structural counterpart of a ketone or aldehyde that has had the carbonyl group (C=O) replaced with an imine or azomethine group.[3] Schiff bases are very vital due to their ease of synthesis, availability, and electrical characteristics, particularly in the synthesis of metal complexes via azomethine nitrogen.[4,5] Schiff base coordination chemistry has gotten a lot of interest because of its important functions in analytical chemistry, metal refining, organic synthesis, metallurgy, electroplating, and photography.[6,7] Schiff bases not only play an important role in advanced coordination chemistry but also are equally significant in bioinorganic chemistry.[8] They have a wide range of medical uses due to their pharmacological characteristics because of the presence of an azomethine (C=N) linkage.[9] As a result, antibacterial, anticancer, antifungal, and diuretic properties have been described for many azomethines.[10,11]

In addition to this, Schiff bases have a wide range of uses in the food and dye industries and also in catalysis and agrochemical activities.[12] The relevance of metal complexes of Schiff bases in catalysis, supramolecular chemistry, separation and encapsulation processes, materials science, biological applications, and the creation of molecules with unique characteristics and molecular structures has long been recognized.[13] The azomethine nitrogen of Schiff bases offers a binding site for metal ions to be linked to different biomolecules such as proteins and amino acids for antigerm actions in biological systems.[14] Various studies have shown the enhanced biofunctionalities of Schiff bases as compared to their metal complexes. Depending on the transition metal ions present in Schiff bases, they exhibit antibacterial, antifungal, antiviral, antiulcer, and anticancer properties.[15,16] Using ring closure, cycloaddition, and replacement processes, Schiff bases have been used as synthons in the creation of a variety of commercial and physiologically active chemicals such as formazans, 4-thiazolidinines, benzoxazines, and so on.[17,18] The use of Schiff base derivatives in a variety of processes prompted researchers to create new Schiff bases for the development of environmentally friendly technologies.[19] Hirshfeld surface analysis[20] is an excellent way to explore the noncovalent interactions that are the key feature of the crystal packing of molecules in the solid state. We explore Hirshfeld surface analysis for DC2H and DM4H. Therefore, in continuation of our previous studies on Schiff bases and their metal complexes,[21−23] this time, we are reporting a completely experimental and computational study of two new Schiff bases named as DC2H and DM4H. A triple hybrid technique consisting of experimental, quantum chemical, and molecular docking methods will be used to investigate the above-synthesized molecules. The quantum chemical methods provide several fundamental insights into the structure–property relationship, which are incomprehensive with simple characterization techniques. Similarly, an in silico study through molecular docking highlights the potential of the studied ligands for their possible use as bioactive compounds against certain diseases, which we will assess for the inhibition of SARS-Co-V-2 in the current investigation.

Results and Discussion

The Cambridge Structural Database (CSD) search confirms that the crystal structures of the tilted compounds are new. Moreover, a CSD search is performed to find the crystal structures that are closely related to our crystal structure. The closely related crystal structures are compared with the crystal structures of the titled compounds in terms of molecular configuration and crystal packing. Table provides complete SC-XRD details of the titled compounds, whereas Table provides a comparison of selected bond lengths and bond angles of the titled compounds.

Table 1

SC-XRD Experimental Details of DC2H and DM4H

crystal data	DC2H	DM4H
CCDC	2102191	2102192
chemical formula	C13H9Cl2NO	C15H15NO
Mr	266.11	225.28
crystal system, space group	orthorhombic, P212121	orthorhombic, Pca21
temperature (K)	296	296
a, b, c (Å)	3.8954 (5), 11.0570 (13), 26.799 (4)	10.631 (2), 13.873 (3), 8.6997 (15)
α, β, γ (deg)	90, 90, 90	90, 90, 90
V (Å3)	1154.3 (3)	1283.1 (4)
Z	4	4
density (calculated) g/cm–3	1.531	1.166
F(000)	544	480
radiation type	Mo Kα	Mo Kα
wavelength (λ)	0.71073 Å	0.71073 Å
μ (mm–1)	0.542	0.073
crystal size (mm)	0.40 × 0.20 × 0.15	0.42 × 0.24 × 0.22
Data Collection
diffractometer	Bruker APEXII CCD diffractometer	Bruker APEXII CCD diffractometer
absorption correction	multiscan (SADABS; Bruker, 2007)	multiscan (SADABS; Bruker, 2007)
no. of measured, independent, and observed [I > 2σ(I)] reflections	10 413, 2760, and 1827	10 600, 1684, and 938
Rint	0.047	0.053
theta range for data collection (deg)	0.994–27.984	2.414–28.277
index ranges	–4≤ h ≤5, −12≤ k ≤14, and –33 ≤ l ≤35	–13≤ h ≤13, –18≤ k ≤16, and –11 ≤ l ≤11
(sin θ/λ)max (Å–1)	0.660	0.667
Data Refinement
R[F2 > 2σ(F2)], wR(F2), S	0.043, 0.083, 0.97	0.054, 0.136, 1.00
no. of reflections	2760	1684
no. of parameters	155	157
H-atom treatment	H-atom parameters constrained	H atoms treated by a mixture of independent and constrained refinement
Δρmax, Δρmin (e Å–3)	0.19, −0.23	0.19, −0.15

Table 2

Selected Bond Lengths (Å) and Bond Angles (deg) in DC2H and DM4H

selected bond lengths in DC2H		selected bond lengths in DM4H
O1-C1	1.360 (4)	O1-C13	1.352 (4)
N1-C7	1.278 (4)	N1-C1	1.432 (5)
N1-C8	1.409 (4)	N1-C9	1.267 (5)
C6-C7	1.440 (4)	C9-C10	1.453 (5)
Cl1-C10	1.729 (3)	C2-C7	1.513 (6)
Cl2-C11	1.726 (3)	C4-C8	1.514 (6)

SC-XRD Description of the Crystal Structure of DC2H and DM4H

In DC2H (Figure a, Table ), o-cresol group A (C1-C7/O1) and 3,4-dichloroaniline group B (C8-C13/N1/Cl1/Cl2) are oriented at the dihedral angle of 6.8 (9)° with respect to each other, which indicates that the whole molecule is almost planar. The titled compound adopts a phenolic tautomeric form instead of a keto tautomeric form, as evident from the O1–C1 bond length of 1.360 (4) Å and the N1–C7 bond length of 1.278 (4) Å. Important bond lengths and bond angles of the titled compounds are given in Table . The molecular configuration is stabilized by the presence of intramolecular H-bonding of type O-H···N to form an S (6) H-bonded loop. The molecules are interlinked by the weak H-bonding of type C-H···Cl to form an infinite C12 zigzag chain that runs along the c crystallographic axis (Figure , Table S1). The neighboring chains are interlinked by a comparatively weak interaction of the off-set π···π stacking type. Phenyl rings (C1–C6) of symmetry-related molecules (x + 1, y, z) are involved in the off-set π···π stacking interaction with an intercentroid distance of 3.895 Å and a ring off-set value of 1.610 Å (Figure ). Phenyl rings (C8–C13) of symmetry-related molecules (x + 1, y, z) are involved in the off-set π···π stacking interaction with an intercentroid distance of 3.895 Å and a ring off-set value of 1.805 Å. A Cambridge Structural Database search provides a lot of crystal structures related to DC2H. The two most closely related crystal structures are found in the database with reference codes AWUSIV(24) and BANGOM03,[25] which are compared with the crystal structure of the titled compound. Both AWUSIV and BANGOM03 have one hydroxyl-substituted phenyl ring, while another phenyl ring in AWUSIV is trichloro-substituted, whereas in BANGOM03, another phenyl ring is pentafluoro-substituted. Like in DC2H, the molecular configuration of AWUSIV and BANGOM03 is also stabilized by intramolecular H-bonding of type O-H···N. The whole molecule of BANGOM03 is almost planar like the molecule of DC2H, but the molecule of AWUSIV is not planar as the dihedral angle between phenyl rings is 24.64 (11)°. The crystal packing of DC2H is different from the crystal packing of AWUSIV and BANGOM03. The crystal packing of DC2H is stabilized by C-H···O and off-set π···π stacking interactions, whereas the crystal packing of BANGOM03 is stabilized by O-H···O and off-set π···π stacking interactions. No significant intermolecular H-bonding is present in the crystal packing of AWUSIV.

Figure 1

ORTEP diagram of (a) DC2H and (b) DM4H. The diagrams are drawn at a probability level of 50%. H-atoms are shown by small circles of arbitrary radii.

Figure 2

Packing diagram of DC2H shows that the molecules form infinite C14 chains that run along the c crystallographic axis. Only selected H-atoms are shown for clarity.

Figure 3

Graphical representation of the off-set π···π stacking interaction in the crystal packing of DC2H. H-atoms are not shown for clarity. Distances shown are measured in Å.

In DM4H (Figure b, Table ), p-cresol group A (C9-C15/O1) and 2,4-dimethylaniline group B (C1-C7/N1) are oriented at a dihedral angle of 72.4 (8)° with respect to each other. The important bond lengths and bond angles of the titled compound are given in Table . The molecular configuration is stabilized by the presence of intramolecular H-bonding of type C-H···N to form an S (5) H-bonded loop. The molecules are interlinked by a strong O-H···N bonding to form a C8 zigzag chain that runs along the crystallographic a-axis, as given in Table S1 and shown in Figure . A comparatively weak H-bonding of type C-H···O is found in the crystal packing that interlinks the molecules in such a way that an infinite C5 chain is formed that runs along the c crystallographic axis, where CH is from the phenyl ring (C10–C15). No other type of weak intermolecular interaction is found in the crystal packing. A Cambridge Structural Database search provides two crystal structures that are most closely related to the crystal structure of the titled compound that contains one 4-hydroxyl-substituted phenyl ring, while the substitution on the other phenyl ring is different from the dimethyl-substituted phenyl ring in DM4H. Reference codes are SECXAB(26) (with one methyl and chloro-substituted phenyl ring) and TAMYOY(27) (with one methyl-substituted phenyl ring). DM4H is different from SECXAB and TAMYOY in terms of stabilization of the molecular configuration because the molecular configuration of DM4H is stabilized by intramolecular H-bonding of type C-H···N, whereas in SECXAB and TAMYOY, no intramolecular H-bonding is present. Like DM4H, the molecules of SECXAB and TAMYOY are nonplanar with the dihedral angles of 39.84 (7)° between aromatic rings in SECXAB and 77.44(8)° in TAMYOY. Like in DM4H, the molecules of TAMYOY are interlinked by O-H···N and C-H···O bonding, whereas the molecules of SECXAB are interlinked by O-H···N and C-H···π interactions.

Figure 4

Packing diagram of DM4H: a view along the ac crystallographic plane. Only selected H-atoms are shown for clarity.

Powder XRD patterns (PXRD) of the simulation-based single-crystal XRD using a cif file were generated to get more details about the crystallinity of both compounds. This simulated XRD pattern represents sharp peaks for both DC2H and DM4H compounds (Figure S1). The results showed that the obtained compound is highly crystalline. Xpert HighScore Plus software was used to obtain the simulated pattern. The five most intense peaks were hkl-indexed on a simulation basis.

Hirshfeld Surface Analysis

The Hirshfeld surface (HS) analysis tool is used by crystallographers and crystal engineers to see strong and weak intermolecular interactions that also exert influence on the molecular packing in crystals. This analysis was performed using Crystal Explorer version 21.5.[28] HS was mapped over dnorm (function of normalized distances), de (distance from a specific point on the mapped surface to the closest atom outside), and di (distances from a particular point on the surface to the nearest atom inside).[29,30] HS dnorm mapping uses blue, white, and red colors to distinguish between the interatomic contacts that are longer, at Van der Waals separations, and short interatomic contacts, respectively.[31,32]Figure a,b represents the HS of DC2H and DM4H mapped with dnorm to view the distinctive red spots, demonstrating specific points of contact in the crystal (see close contacts in a unit cell Figure S2a,b). The red spots (bright and faint) can be classified as potential hydrogen bonds. The shape index property was used to map the HS views of DC2H and DM4H, which represent the red and blue triangular regions shown in Figure c,d, respectively. Here, bright-red spots indicate the π···π stacking between the six-membered rings.[33,34] Similarly, HS curvedness mapping also shows the π···π stacking, which is obvious because of the flat regions around the carbon rings (Figure S3a,b). Figure e,f shows the electrostatic potential, while Figure g,h shows the deformation density for DC2H and DM4H, respectively. Deformation of the electron density is displayed with positive (blue) and negative (red) isosurfaces.

Figure 5

Hirshfeld surface (HS) of the DC2H mapped with dnorm (a). Hirshfeld surface (HS) of the DM4H mapped with dnorm (b), Hirshfeld surface of the DC2H and DM4H molecules drawn with shape index (c, d). Visualization of DM4H and DC2H molecules’ electrostatic potential (e, f). The deformation density (±0.008 e au–3) plot of DM4H and DC2H molecules (g, h).

The 2D fingerprint plots are a way to explore the crystal packing of the single crystals in terms of interatomic contacts and their contribution to the crystal packing.[35,36] The important 2D fingerprint plots of DC2H are compared with the 2D fingerprint plots of a very closely related crystal structure obtained from the literature with the reference code AWUSIV and CCDC no. 841140. Similarly, important 2D fingerprint plots of DM4H are compared with the 2D fingerprint plots of a closely related crystal structure obtained from the literature with the reference code TAMYOY and CCDC no. 1491358. A comparative study of the important 2D fingerprint plots is shown in Figure . The central triangular region of sky-blue color in 2D fingerprint plots for overall interactions of DC2H (Figure a) and AWUSIV (Figure f) indicates the presence of the π···π stacking interaction in the crystal packing. The most important interatomic contact for DC2H is H···H with a percentage contribution of 28.1% (Figure b), whereas for AWUSIV, the most important interatomic contact is Cl···H with a percentage contribution of 42.8% (Figure h). In AWUSIV, the H···H interatomic contact has a percentage contribution of 16.9% (Figure g). The Cl···H interatomic contact has a larger contribution in AWUSIV as compared to in DC2H because the crystal structure of AWUSIV contains a trichloro-substituted phenyl ring, whereas the crystal structure of DC2H contains a dichloro-substituted phenyl ring. The 2D fingerprint plots for the overall interactions of DM4H and TAMYOY contain two large spikes. These spikes show an N···H contact with a percentage contribution of 4.2% in DM4H (Figures o) and 5.2% in TAMYOY (Figure t). The most important interatomic contact for DM4H and TAMYOY is H···H with a percentage contribution of 56% in DM4H (Figures l) and 51.5% in TAMYOY (Figure q). The 2D plots of C···H and O···H for DM4H and TAMYOY are also compared in Figure with their contribution to the crystal packing.

Figure 6

(a–e) Important 2D fingerprint plots of DC2H, (a) overall interactions and (b–e) individual interatomic contacts, which are compared with important 2D plots (f–j) of a crystal structure closely related to the DC2H found from the literature with the reference code AWUSIV and CCDC no. 841140. Similarly, Important 2D fingerprint plots (k–o) of DM4H, (k) overall interactions and (l–o) individual interatomic contacts, which are compared with important 2D plots (p–t) of a crystal structure closely related to DM4H found from the literature with the reference code TAMYOY and CCDC no. 1491358.

Crystal Explorer 21.5 was used to calculate the interaction energy between pairs of molecules[37] (Figure S4a,b). The interaction energies were calculated based on two models: CE-B3LYP/6–31G(d,p) and CE-HF/3–21 G; furthermore, the 6–31 G basis set was used for both compounds. The total energy E was calculated in kJ/mol. Etot is the individual sum of four factors: electrostatic (Eele), polarization (Epol), dispersion (Edis), and exchange repulsion (Erep). All calculations for the pairs of molecules are provided in Table S2 for DM4H and in Table S3 for DC2H. The total energy stabilizes the crystal packing with interaction energies of −32.8 kJ/mol (DC2H) and −57.6 kJ/mol (DM4H) for both molecules. Our analysis for these two molecules shows that they have the following components: electrostatic (4.5 kJ/mol), polarization (−3.2 kJ/mol), dispersion (−66.3 kJ/mol), and repulsion (+33.1 kJ/mol) for the DC2H molecule and electrostatic (−69.7 kJ/mol), polarization (−23.7 kJ/mol), dispersion (−34.6 kJ/mol), and repulsion (+74.0 kJ/mol) for the DM4H molecule. Based on the analysis of interactions in the crystal structure, it is implied that DM4H can be preferred at a supramolecular level.

The energy frameworks were simulated to give information about the contribution of the total energy.[38]Figure S5 shows the energy framework that gives visual understanding as cylinders joining the molecules (interaction pairs). Here, we fixed the radius of the cylinder at 100 kJ/mol. The color of the cylinders represents the specific type of energy, such as red cylinders for Eele (Coulomb’s interaction energy), green cylinders for Edis (dispersion energy), and blue cylinders for Etot (total energy). This approach is very useful for crystal engineers to deeply understand the mechanical behavior of the compounds at a molecular level. The energy frameworks revealed that the packing topology in terms of electrostatics is predominantly guided by DM4H and the dispersion energy topology is supported by π···π stacking.

Computational Results

Computational Methodology

All the quantum chemical calculations have been performed using the Gaussian 16 suite of programs.[39] The density functional theory method has been used to optimize the geometries of compounds DC2H and DM4H with the M06/6-31G* method. After optimizations, frequency calculations have been performed to get the global minimum structures with all positive frequencies for the compounds DC2H and DM4H. The AutoDock Vina program[40] is used for performing molecular docking studies with all default parameters. MGL tools[41] and Discovery Studio Visualizer[42] were used for input file preparation and output result visualization, respectively. Several attempts were made to get the most suitable docking models by doing several replicated docking calculations. Further essential details for protein and ligand preparations and other default parameters for docking studies can be seen in the Supporting Information of the article and also in our previous studies.[43]

Optimized Molecular Geometries of DC2H and DM4H

The optimized geometries of compounds DC2H and DM4H are given in Figure , where their respective experimental values are given in parenthesis. A careful analysis of Figure shows that calculated and experimental geometries are in reasonable agreement. For DC2H, the experimental and calculated C18-N5 bonds are found to be 1.409 and 1.404 Å, respectively. Similarly, the experimental and calculated C16-N5 bonds are found to be 1.278 and 1.289 Å, respectively, in DC2H. The two calculated (experimental) halogen carbon bonds, C21-Cl1 and C22-Cl2, are illustrated as 1.729 Å (1.747 Å) and 1.726 (1.746) Å, respectively, in DC2H. For DC2H, the experimental and calculated C6-O30 bonds are found to be 1.360 Å and 1.339 Å, respectively. A somewhat similar trend can also be seen among the calculated and experimental bond lengths of compound DM4H where only two heteroatoms, i.e., N and O, are involved in molecular geometry. For DM4H, its calculated (experimental) three crucial bonds, C4-N3, N3-C21, and C28-O1 are elucidated as 1.431 Å (1.404 Å), 1.277 Å (1.266 Å), and 1.361 Å (1.351) Å, respectively. Overall, most of the calculated bond distances are seen as larger than the experimental bond distances (values in parenthesis). This trend is justifiable because the current geometry optimizations were performed, in a vacuum while experimental crystal structures are in the solid state.

Figure 7

Optimized molecular geometries of compounds DC2H and DM4H where values in parenthesis are experimentally taken from crystal data.

Frontier Molecular Orbitals (FMOs) and Molecular Electrostatic Potentials (MEPs)

The FMOs play a very vital role in the reactivity and stability of molecular compounds. To comprehend the insights into the distribution of FMOs, we depict the HOMO and LUMO of the entitled compounds as shown in Figure a. There is a uniform distribution of electron density for the respective HOMO and LUMO orbitals over the surface of both entitled molecules. The dimethyl phenyl group of DM4H shows a slight donor behavior owing to its two methyl groups, where electron density moved away from the dimethyl phenyl moiety as seen in the HOMO and LUMO of DM4H. The HOMO–LUMO energy gaps of DC2H and DM4H compounds are also shown along with their individual orbital energies. The orbital energy gap of the DM4H compound is 4.59 eV, which is slightly larger (0.1 eV) as compared to that of DC2H (4.49 eV). The slight difference is due to the similar skeleton of both molecules. Furthermore, the electrostatic molecular potentials are also depicted to see the distributions of the local potential over the total electron density surface as color coding (Figure b). The red and blue colors indicate the negative and positive regions of electron density of the total electron density surface, respectively. In DC2H and DM4H molecules, the negative potentials are seen around the Cl and OH groups, making them vulnerable to electrophilic attacks, while positive regions can be seen as less prominent.

Figure 8

(a) HOMO and LUMO orbitals for compounds DC2H and DM4H and (b) molecular electrostatic potentials for compounds DC2H and DM4H, where positive and negative maxima are represented with blue and red color coding. The isovalues of 3D density surfaces are ±0.002 a. u.

Molecular Docking Analysis

Computational chemistry has emerged significantly as a tool of trust with recent progress in algorithm developments. Molecular docking, a subfield of computational chemistry and bioscience, is providing several quick insights into the modern drug designing process. The cornerstone of molecular docking is based on the fact that most biological functions are controlled by specific proteins, and if a molecule shows the potential to interact with such specific proteins, it may also have the potential to inhibit the function of such proteins. Recently, several cutting-edge research reports from nature and science journals have also employed molecular docking methods to get insights into their research results.[44,45] Along these lines, two very crucial proteins, Mpro and NSP9 of SARS-CoV-2, are selected to study their interactions with indigenously synthesized ligands. Phenols constitute the largest class of bioactive compounds on the earth, which are usually formed through the secondary metabolites of plants. The antioxidant and anti-inflammatory properties of phenols are well-reported. Recent studies have highlighted their potential for possible antiviral potential as demonstrated by their efficacy against several pathogens, including influenza virus, enterovirus, and a class of coronaviruses.[46] As the COVID-19 disease (caused by SARS-CoV-2) has divested the world health sector and created a pandemic, it is very imperative to turn every stone to find a potential therapeutic drug candidate. Along the abovementioned lines, using molecular docking techniques, the current study also explores a few promising pieces of evidence for the antiviral potential of the above-entitled compounds.

Calculation of Binding Energy

The binding energy of docked ligands and protein molecules is a combination of many kinds of interaction energies, which are calculated during the docking of the ligand with a protein. Binding energy plays a crucial role in establishing the stability of a ligand–protein complex. More negative binding energy reflects a more stable protein–ligand complex. Table discloses the binding energy and inhibition constant values for both proteins (Mpro and NSP9). The binding energy values of DC2H and DM4H with Mpro are −6.3 and −6.6 kcal/mol, respectively (see Table ). The inhibition constant values of these two compounds with Mpro and NSP9 are 23.18 and 13.94 μmol, respectively. DC2H and DM4H have similar binding energies and inhibition constant values with the NSP9 protein, which are −6.5 kcal/mol and 16.52 μmol, respectively (see Table ). The negative binding energies indicate the favorable interactions of the synthesized compounds with Mpro and NSP9 proteins of SARS-CoV-2. Recently, Burkhanova et al. studied aniline dyes through molecular docking, which showed binding affinity towards Mpro with a binding energy value of about −7.6 kcal/mol.[47] A more relevant molecular docking study was about hydroxystilbamidine as Mpro inhibitors, where the imine group in the compound forms hydrogen bonds with aspartic acid moieties of Mpro as our current DC2H compound with histidine.[48] Furthermore, a semiquantitative comparison of binding energies of DC2H and DM4H, so-called standard drug molecules, shows that the binding energy with Mpro is better than the previously calculated value of chloroquine (−5.20 kcal/mol) and hydroxyl chloroquine (−5.60 kcal/mol) and comparable to that of Remdesivir (−7.00 kcal/mol),[49] which is a recently used drug against SARS-CoV-2.

Table 3

Binding Energy (Kcal/mol) and Inhibition Constant (Ki, μmol) of Docked Compounds DC2H and DM4H with Mpro and NSP9 Proteins

ligands	targeted protein	binding affinity	inhibition constant
DC2H	Mpro	–6.3	23.18
DM4H		–6.6	13.94
DC2H	NSP9	–6.5	16.52
DM4H		–6.5	16.52

Details of Intermolecular Interactions

The Mpro and NSP9 proteins show various intermolecular interactions, including H-bonds and hydrophobic and electrostatic interactions. All these interactions with amino acid residues and their bond lengths are given in Table and Figure . Figure shows 3D orientations, while an overview of 2D interactions is provided in Figure S6 of the Supporting Information for ligand–protein interactions where both types of visualizations are commonly used in molecular docking studies. DC2H exhibited two conventional H-bond interactions with His41 (2.41 Å) and Gln189 (2.22 Å) amino acid residues of Mpro. It also shows two electrostatic interactions with His41 (2.90 Å) and Cys145 (4.99 Å) amino acid residues. DC2H exhibits three hydrophobic interactions, which are alkyl, π-alkyl, and π···π stacking interactions with Met49 (5.12 Å), Met165 (4.82 Å), and His41 (4.83 Å) amino acid residues, respectively. In interactions of DC2H with Mpro, the amino acid residue His41 has an active role as it revealed all H-bonds and hydrophobic and electrostatic interactions. DC2H with NSP9 showed two types of interactions: conventional H-bonding and hydrophobic interactions. The conventional H-bonding interactions are with Gly38 (2.36 Å) and Gly39 (2.54 Å) amino acid residues. The hydrophobic interactions are with Phe41 (4.06 Å), Phe57 (4.20 Å, 5.18 Å), Ile92 (4.34 Å), Ile66 (4.21 Å), and Val42 (3.90 Å, 3.70 Å, and 5.43 Å). Figure shows the 3D interactions of DC2H with Mpro and NSP9 proteins. DM4H also revealed various interactions with Mpro and NSP proteins. With Mpro, it showed one conventional H-bond and two electrostatic and four hydrophobic interactions. The conventional H-bond interactions are with Phe140 (2.53 Å), electrostatic interactions are with Cys145 (5.60 Å) and Met165 (5.68 Å), hydrophobic interactions are with His41 (3.97 Å and 4.35 Å) and Met49 (3.96 Å and 4.71 Å). It showed seven hydrophobic and two conventional H-bond interactions with the NSP9 protein. The conventional H-bond interactions are with Pro58 (2.72 Å) and Arg40 (2.66 Å) amino acid residues. The hydrophobic interactions are with Phe41 (4.98 Å, and 5.29 Å), Val42 (5.20 Å), Ile92 (4.87 Å, and 5.33 Å), Phe57 (4.31 Å), and Ile66 (5.25 Å) amino acid residues.

Table 4

All Interactions, along with Associated Residues and Respective Bond Lengths, between Receptor Mpro and Spike Proteins of SARS-CoV-2 and Compounds DC2H and DM4H with the Highest Binding Energies

	interactions with associated residues and bond length
ligands	hydrogen bond	hydrophobic interactions	electrostatic and others
DC2H-Mpro	(1) His41 (2.41)	(1) Met49 (5.12)	(1) Cys145 (4.99)
	(2) Gln189 (2.22)	(2) Met165 (4.82)	(2) His41 (2.90)
		(3) His41 (4.83)
DC2H-NSP9	(1) Gly38 (2.36)	(1) Phe41 (4.06)
	(2) Gly39 (2.54)	(2) Ile92 (4.34)
		(3) Phe57 (4.20)
		(4) Phe57 (5.18)
		(5) Ile66 (4.21)
		(6) Val42 (3.90)
		(7) Val42 (3.70)
		(8) Val42 (5.43)
DM4H-Mpro	(1) Phe140 (2.53)	(1) His41 (3.97)	(1) Cys145 (5.60)
		(2) His41 (4.35)	(2) Met165 (5.68)
		(3) Met49 (3.96)
		(4) Met49 (4.71)
DM4H-NSP9	(1) Pro58 (2.72)	(1) Phe41 (4.98)
	(2) Arg40 (2.66)	(2) Phe41 (5.29)
		(3) Val42 (5.20)
		(4) Ile92 (4.87)
		(5) Ile92 (5.33)
		(6) Phe57 (4.31)
		(7) Ile66 (5.25)

Figure 9

3D interactions of DC2H and DM4H with amino acid residues of Mpro (pink) and Nsp9 (blue) proteins; (a and b) 3D interactions of DC2H and DM4H with the Mpro protein, respectively; and (c and d) 3D interactions of DC2H and DM4H with the Nsp9 protein, respectively.

Interestingly, as all the interactions between ligands and proteins can be broadly divided as polar and nonpolar, we have presented the density surface representations of polar and nonpolar interacting residues of the binding pockets as shown in Figure S7. The interactions of DC2H and DM4H with the Mpro protein are represented in Figure S7a,b, respectively, where the total density surface of the protein is illustrated in cyan, polar areas are symbolized in magenta, and nonpolar areas are illustrated in yellow. The Nsp9 interactions with DC2H and DM4H are illustrated in Figure S7c,d, where the total density surface of the protein is shown in a warm-pink color, the polar area is denoted in green, and the nonpolar area are shown in yellow. An overview of polar and nonpolar surfaces signifies their respective contributions to assessing the nature of interactions in a semiquantitative way.

Conculsions and Perspectives

Two crystalline imine compounds, DC2H and DM4H, are synthesized efficiently, and their crystal structures are confirmed using the single-crystal X-ray diffraction technique. The crystal structures of these compounds are fully explored in terms of molecular configuration, intramolecular H-bonding, and intermolecular H-bonding. Moreover, a Cambridge Structural Database (CSD) search is carried out to find closely related crystal structures, and then, these crystal structures are compared with those of DC2H and DM4H. Intermolecular interactions that are the main aspect of the crystal packing of both compounds are further explored by Hirshfeld surface analysis. The quantum chemical and molecular docking calculations were performed to study the optoelectronic and antiviral potential of the synthesized compounds. The possible inhibition properties against two very crucial proteins (Mpro and NSP9) of SARS-CoV-2 were studied through molecular docking. The calculated binding energies of DC2H and DM4H with Mpro are found to be -6.3 and -6.6 kcal/mol, respectively, where negative energies indicate the favorable interactions of the synthesized compounds with the above-studied proteins of SARS-CoV-2. Thus, we have successfully not only synthesized these compounds but also investigated their potential functional properties.

Experimental Section

Material and Methods

The different solvents and precursors employed in the present experimental work were highly pure and purchased from Acros Organics, Alfa Aesar, and Sigma-Aldrich/Merck and were used as received.

Synthesis of Schiff Bases DC2H and DM4H

The precursors, 2,4-dimethylaniline or 3,4-dichloroaniline (5 mmol), were dissolved in 25 mL of ethanol, to which an ethanolic solution (25 mL) of equimolar quantities (5 mmol) of 2-hydroxybenzaldehyde or 4-hydroxybenzaldehyde was added dropwise. The reactants were refluxed for 2 h, and then, the clear solution was left for crystallization until the light-yellow needle-shaped crystals of DC2H and colorless rod-like crystals of DM4H were grown at the bottom and along the sidewall of the container. The crystals were separated by decantation and then dried gently under the folds of filter paper (Scheme ).

Scheme 1

Synthesis of Schiff bases DC2H and DM4H

X-ray Data Collection and Refinement Details

We collected the X-ray data of DC2H and DM4H on a Bruker Kappa Apex-II CCD diffractometer. The diffractometer contains an X-ray tube to generate monochromatic rays (λ = 0.71073 Å). Bruker APEX-II,[50] SHELXS-97,[51] and SHELXL 2018/3[52] are employed for data collection, structure solution, and refinement, respectively. Refinement of all nonhydrogen atoms is performed using ADP (anisotropic displacement parameters), while the refinement of H-atoms is performed using isotropic displacement parameters and using the riding model. The H-atoms are inspected by the keen observation of residual peaks using refinement of data. Mercury 4.0[53] and Platon[54] software tools are employed for the sake of graphics related to XRD.