Structure property relationship in two thiazole derivatives: Insights of crystal structure, Hirshfeld surface, DFT, QTAIM, NBO and molecular docking studies

Abstract Detailed structural and noncovalent interactions in two thiazole derivatives (N-(4-Bromophenyl)-2-(methylthio)thiazole-5-carboxamide and Ethyl-5-((4-bromophenyl)carbamoyl)thiazole-4-carboxylate) are investigated by single crystal X-ray diffraction study and computational approaches. The structure investigation revealed that various interactions like C-H…O, N-H…O, and N-H…N hydrogen bonds and Br…Br interactions are involved in constructing ring motifs to stabilize the crystal packing. Hirshfeld surface analysis and fingerprint plots were carried out to study the differences and similarities in the relative contribution of noncovalent interactions in both the molecules. The FMOs and other global reactive parameters are analyzed for thiazole derivatives. The strength and nature of weak interactions present in the molecule were characterized by RDG-based NCI and QTAIM analyses. Natural bond orbital (NBO) analysis unravels the importance of non-covalent and hyperconjugative interactions for the stability of the molecules in their solid state. Further, molecular docking of N-(4-Bromophenyl)-2-(methylthio)thiazole-5-carboxamide and Ethyl-5-((4-bromophenyl)carbamoyl)thiazole-4-carboxylate with SARS-Covid-19 have been carried out.


Introduction
Thiazoles have emerged as ubiquitous scaffolds with various biological and therapeutic activities, often found in drugs, agrochemicals, and functional materials [1,2]. Due to its potential use in pharmaceutical and medical applications, functionalized thiazoles have gathered attention. For example, antitumor [3], antiviral [4], antibacterial [5], anti-prion [6], anti-allergic [7], anti-hypertensive [8], anti-inflammatory, anti-fungal, anti-tubercular, anti-protozoal, antipyretic, anti-oxidative [9,10], analgesic and immunomodulatory [11] are predominant among the drugs based on thiazole moieties. In a recent development, thiazoles have been reported as a photo-stable voltage-sensitive dye with very high fluorescence quantum yield. Apart, they are also used as fluorescent intercalators to determine DNA binding affinity, modulators of cellular development fluorescent sensors for detecting bi-sulfite anions, as well as in the detection of organophosphate nerve agents, etc. [12].
Recently, efficient methods for the synthesis of 2-(methylthio)-N-aryl/alkyl-thiazole-5carboxamides and 4-(ethylcarboxy)-N-aryl/alkylthiazole-5-carboxamides were reported by cyclization of methyl-2-oxo-2-(amino)ethanedithioates with isocyanides as it takes advantage over the previously reported methods [13]. The structural studies of 4a and 4b was stabilized by various intermolecular interactions such as hydrogen bond interactions, N-H … O, C-H … N, and Cg … Cg interactions. Because of their synthetic significance, 2,5-and 4,5-disubstituted thiazoles are important in the research field, as part of our studies of these compounds was synthesized and characterized by various spectroscopic techniques. Single crystal X-ray diffraction measurements confirm the molecular structure of 4a and 4b Crystal structure analysis and Hirshfeld surface analysis were used for the intermolecular hydrogen bond interactions. Density functional theory (DFT) computations were also used to investigate molecular structure optimization, frontier molecular orbitals (FMOs), chemical reactive parameters, and molecular charge distribution in the title molecule.

Synthesis of N-(4-Bromophenyl)-2-(methylthio)thiazole-5-carboxamide (4a)
A solution of KOH (2.0 mmol) in EtOH was placed in an ice bath. A mixture of Methyl-2-((4-bromophenyl)amino)-2-oxoethanedithioate (1.0 mmol) and TosMIC (1.0 mmol) in EtOH was added to the cooled solution, and the reaction mixture was allowed to reach r.t. The reaction was quenched with ice-cooled water, and the mixture was extracted with EtOAc (3 Â 25 mL), washed with brine solution, dried over Na 2 SO 4 , and evaporated. The crude material was subjected to column chromatography using a hexane/EtOAc solvent system to give the desired compound.  13  A solution of K 2 CO 3 (2.0 mmol) in DMF was placed in an ice bath for 10 min. A mixture of Methyl-2-((4-bromophenyl)amino)-2-oxoethanedithioate (1.0 mmol) and ethyl isocyanoacetate (1.0 mmol) in DMF was added to the cooled solution, and the reaction mixture was allowed to reach r.t. The reaction was quenched with ice-cooled water, and the mixture was extracted with EtOAc (3 Â 25 mL), washed with brine solution, dried over Na 2 SO 4 , and evaporated. The crude material was subjected to column chromatography using a hexane/EtOAc solvent system to give the desired compound.

X-ray diffraction
The crystallographic data were collected on Bruker APEX II CCD diffractometer using MoKa radiation (k ¼ 0.71073 Å) for 4a and CuKa radiation (k ¼ 1.54178 Å) for 4b at room temperature. For data correction and data reduction, SADABS and SAINT softwares were used, respectively. The crystal structures of 4a and 4b were solved by direct method and refined by full matrix least-squares method against F 2 using SHELXS and SHELXL program to minimize the errors in the structures [14]. Anisotropic refinement was done for non-hydrogen atoms. PLATON [15] was used for exploring hydrogen bonding and for geometrical parameters calculations. Mercury 4.2.0 [16] was used for asymmetric unit representation and p-p stacking interaction.

Hirshfeld surface (HS) analysis
The Hirshfeld surface (HS) analysis is an efficient tool to investigate intermolecular interactions present in the crystal structure. The HS is mainly based on the electron distribution of a molecule and is calculated as the sum of spherical atom electron densities [17]. The HS is unique for a given crystal structure and set of spherical atomic electron densities. The d norm (normalized contact distance) calculated by the given equation was employed for interior and exterior intermolecular interactions simultaneously on a single HS. The two dimensional fingerprint plot furnishes a summary of intermolecular contacts present in the crystal [18]. The HS presented in this study was generated using CrystalExplorer 21.5 [19].

Theoretical methods
The energies of molecule 4a and 4b investigated in this study were performed using Gaussian 09 [20] software at the B3LYP functional with a large basis set 6-31 G(d,p). The visualization of the results is achieved with GuassView 6.0 [21]. The optimized coordinates were used for the theoretical analysis of noncovalent interactions present in the molecules. Bader's "quantum theory of atoms in molecules" has been utilized to analyze the interactions studied in this work employing Multiwfn 3.8 [22] package and the results are visualized in Visual Molecular Dynamics (VMD) software [23]. The topological properties of the charge density q(r) characterized by their critical points and its Laplacian (r 2 q(r)) were also calculated using aforesaid theory. The NCI is a visualization index based on the electron density [24]. Since NCI's are represented with the help of isosurfaces instead of bond critical points, it enables their strength and nature of interactions present in the molecule. The isosurfaces are differentiated by the sign of Hessian eigenvalues and defined by the isosurface color. However, the information obtained by the NCI plot is qualitative, i.e., which molecular regions are interaction [25]. The color scale is a red-green-blue with red for q þ (repulsive interaction) and blue for q À (attractive interaction). Green isosurfaces are related to weak interactions [26]. Further, natural bond orbitals (NBO) analysis has been carried out using NBO 3.1 program as incorporated in Gaussian09 to get the information regarding the population of electrons in subshells of atomic orbitals and delocalization of charge and electron densities of atoms in the 4a and 4b [27].

Molecular docking
Molecular docking is a structure based drug design method to identify and explore the amino acid interactions between the macromolecule and ligands with low energy conformation. In silico molecular docking studies were performed to investigate the binding affinity and their modes of two thiazole derivatives (4a and 4b) against the main protease of SARS-Covid-19 (Mpro) using AutoDock Vina with MGL tools 1.5.6 [28]. The binding sites were defined using the grid box with an energy range of 4. Each amino acid's value is defined by its Kollman charges generated from the associated electrostatic potential and the polar hydrogen atoms were added to the protein. The protein had a total Kollman charge was four and the ligand is zero. The default settings in Autodock Vina were used to energy minimization of the protein and ligand preparation. The protein structure (PDB ID:6m2q) was downloaded from the protein data bank https:// www.rcsb.org/. Biovia Discovery Studio 2019 Client [29] visualization software was employed to present the output files. Two-dimensional diagram of the receptor-ligand interactions in the molecule is generated to represent the type of and active interactions.
In 4a, the 4-bromophenyl and thiazole moieties exist in the planar form having rmsd values of 0.007 Å and 0.003 Å, respectively, and the same two moieties are bridged by carbamoyl group having -anti-periplanar conformation with torsion angle of À176.71 .
In 4b, the 4-bromophenyl group and thiazole moieties exist in planar form with rmsd values of 0.005 Å and 0.006 Å, respectively, and bridged by carbamoyl group having -anti-periplanar conformation with dihedral angle of À177.44 . These conformation analysis also indicated that the hydrogen bonding interactions for both entitled molecules. In 4a, the molecules are connected with each other via intermolecular interactions    of the type N10A-H3A … O9B, C6A-H14A … O9B, and C6B-H11 … O9B. These interactions are mainly responsible for the existence of dimeric nature in the molecule and form R 2 2 (7) supramolecular synthon, where the acceptor N-atom and donor O-atom is from carbamoyl group of the two individual molecules (Fig. 2). Also, N10B-H2 … N7A, C6B-H3 … N7A, and C13B-H8 … O9A intermolecular interactions are involved in the molecular packing of the structure. In 4b, C3-H3 … O10 and C13-H13 … N14 intermolecular interactions are involved in the crystal packing and form R 2 2 (6) and R 6 6 (48) supramolecular synthon formed by neighboring molecules (Fig. 2). The details of hydrogen bond geometry are listed in Table 2.
Besides, p-p stacking interactions are present in the between the rings which helps in stabilization of crystal packing in both 4a and 4b are shown in Fig. 3. In 4a, Cg1 and Cg2 represent the thiazole and bromophenyl rings of the molecule A and Cg3 and Cg4 represents the thiazole and bromophenyl rings of the molecule B. The parallel offset stacking interactions are found between thiazole rings (Cg1 … Cg1) of molecule A having a centroid distance of 3.882(2) Å. There also exist a stacking interactions between thiazole and bromophenyl rings of molecule B and bromophenyl rings of the molecule B with a centroid distance of 4.131(2) Å and 3.753(2) Å, respectively (Fig. 3a). In 4b,  Table 2. Hydrogen bond geometric parameters of 4a and 4b.

Molecule
Interactions Cg1 and Cg2 represents the thiazole and bromophenyl rings of the molecule. p … p stacking interactions are found between two thiazole rings of the molecule (Cg … C1) and between thiazole ring and bromophenyl rings of the molecule with a centroid distance of 3.676(3) Å and 3.714(3) Å, respectively (Fig. 3b). The complete information regarding p … p interactions are summarized in Table 3.

Hirshfeld surface (HS) analysis and fingerprint (FP) analysis
The HS of the molecules 4a and 4b are shown in Fig. 4, showing surfaces that have been mapped over normalized contact distance (d norm ). The given surfaces are shown as transparent to allow clear visualization of the molecular moiety. The HS clearly revealed that the pattern of intermolecular interactions are different in both the structures, which prompted us to explore the contributions of the weak noncovalent forces in the crystal packing and the importance of p … p interactions in establishing the organization of the structures. In An analysis of the HS mapped over shape index and curvedness properties are useful to investigate the influence of p … p stacking on the molecular assembly. The structures of 4a and 4b, is used an example as the crystal features p(bromophenyl) … p(thiazole)   (2) 3.425 (2) 1.436 i : -x, -y, 1-z. ii : 1-x, -y, 1-z. iii : -x, 1-y, 1-z. iv : 1Àx, 1-y, 1-z. v : À1 þ x, y, z. stacking interactions. From Fig. 5a clearly revealed that the pattern of red and blue triangle in the same area of the shape index surface is indicative of p … p stacking interactions. The blue triangles are represent convex regions due to the presence of ring carbon atoms of the molecules inside the surface, whereas red triangles represent concave regions caused by carbon atoms of the rings above the surface. The existence of p … p stacking is also evident by the flat region toward the bottom of both sides of the molecules 4a and 4b, and is clearly seen on the curvedness surface mapped over HS (Fig. 5b). The HS mapped over electrostatic potential for 4a and 4b as shown in Fig. 5c. Here, the blue and red regions around the different atoms are associated with positive and negative electrostatic potential, respectively. The red regions are around the oxygen atoms involved in the C-H … O and N-H … O contacts discussed above. Blue regions are appear around the hydrogen atoms which are connected to the most electronegative atom nitrogen.
2D FP analysis was carried out to investigate the effect of thiazole and bromophenyl substituents on the intermolecular interactions observed in the crystal structure. The FP plots for different intermolecular interactions shown in Fig. 6. The summary of these  individual contacts including reciprocal contacts and their contributions are listed in Table 4.
As can be seen from Table 4 that the major contributions to the total HS is from H … H contacts with 28.9% in 4a and 29.2% in 4b. The strong hydrogen bond interactions between O … H/H … O are indicated by two large spikes at bottom of FP and accounted for 9.9% and 16.7% of 4a and 4b, respectively. Besides, the contact between hydrogen and bromine (H … Br/Br … H) is another major interaction consists of 12.5% and 13.6% to the total HS for 4a and 4b, respectively. The other minor contacts include H … S, C … C, C … H, C … S, and N … H all together accounts for remaining percentage of interactions to the total HS.

Frontier molecular orbital (FMO) analysis
The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are known as frontier molecular orbitals. The energy of HOMO represents electron donating capacity whereas energy of LUMO represents electron acceptor capacity of the molecule. The investigation of FMOs of molecules and their energy levels is one of decisive factor in determining molecular electrical properties viz., conductivity, chemical reactivity, kinetic stability, biological properties and their applications in optoelectronic devices [30]. Generally, molecules having lower HOMO-LUMO energy  gap are more polarizable, conductor, and display high chemical reactivity with low kinetic stability whereas molecules with high energy gap are stable and low chemical reactivity [31]. Figure 7 shows the energy level diagram of the FMOs of the molecules 4a and 4b. The calculated values of FMOs for studied molecules are listed in Table 5. The calculated energy value of HOMO-LUMO is 4.194 eV for 4a and 3.521 eV for 4b, respectively, and explains eventual charge transfer interactions taking place within the molecules. The other reactive parameters are calculated with the help of E HOMO and E LUMO values. As can be seen from Table 5, the chemical hardness value of 4b is lesser than 4a hence it is clearly indicates that 4b is more reactive than 4a. Molecule 4b shows good electrophile in nature since it shows a higher value than 4a. DN max gives the amount of charge that an electrophile system may accept. Therefore, the maximum charge transfer is obtained for the molecule 4b whose energy gap is low compared to another system.

Molecular electrostatic potential (MEP) analysis
The MEP surface analysis was carried out to investigate the reactive behavior of chemical systems in both electrophilic and nucleophillic reactions. MEP at a point around a molecule gives a magnitude of the net electrostatic effect produced at that point by the total charge distribution (electron þ nuclei) of the molecule. Also, MEP map provides visual aid to understand the relative polarity of the molecules. Electron density mapped with electrostatic potential surface gives the information about size, shape, density of charges, and chemical sites present in the molecules. Colored scale were used to represent the different values of electrostatic potential at the surface [32]. Red colored regions represent the most negative electrostatic potential; blue indicate the regions of positive electrostatic potential. 3D MEP maps of the two thiazole derivatives were shown in Fig. 8a.
As can be seen from Fig. 8a, maximum negative electrostatic potential region is observed around the oxygen atom of the carbamoyl group in 4a and 4b, carboxylate group of 4b. Also, negative potential region appears around the nitrogen atom of the  thiazole ring of both the molecules. A maximum positive potential region is localized on the hydrogen atoms present in both the molecules indicating a possible site for nucleophillic attack. These sites give information regarding the region from where the molecule can have weak interactions. MEP map of 4a and 4b with isosurface value of 0.002 show the presence of r-hole around the Br atom (Fig. 8b). On these maps, the r-hole is clearly specified by the presence of positive electrostatic potential on the Br atom in the C-Br bond. This r-hole involved in the weak interaction to stabilize the molecule.

Natural bond orbital (NBO) analysis
The NBO analysis were carried out to get better understanding of the chemical bonding features like intra-and inter-molecular contacts, hyperconjugation, stability, and correlation between donor and acceptor of the two thiazole derivatives. Regarding the interactions in both filled and virtual orbitals, NBO offers useful information. Additionally, it determines the distribution of the highest plausible percentage of electron density in the atoms and in their bonds, resulting in the most precise interpretation of the wave function's "natural Lewis structure" [33]. The second-order perturbation technique was used to determine the hyper-conjugative interaction energy.
where q i is the occupancy of the donor orbitals, j and i are diagonal elements and F i, j ð Þ is the off diagonal NBO Fock matrix elements. In light of these findings, Table 6 presents NBO results demonstrating the formation of Lewis and non-Lewis orbitals by valence hybrids corresponds to intramolecular interactions. Large E (2) values in NBO analyses indicate active interaction between electron-donors and electron-acceptors as well as a larger degree of conjugation across the system.
As can be seen from Table 6, in 4a, the molecular orbital overlap between LP(1)N10 ! p Ã O9-C8 has the highest stabilization energy of 64.93 kcal/mol results in the intramolecular charge transfer (ICT) in the molecule. The other second order perturbation energies associated with hyperconjugative interactions of 4a such as p Ã N7-C3 !p Ã C5-C6 , are considerable very large. These hyperconjugative interactions are more responsible for the stability of the molecule 4a. In the case of molecule 4b, the most significant hyperconjugative interactions of energy 116.45 kcal/mol obtained for p Ã N14-C13 ! p Ã C15-C11 . The other hyperconjugative interactions of 4b are LP(1)N8!p Ã O10-C9 , have the stabilization energy of 75.03, 58.43, 22.89, and 21.18, respectively. It is clearly observed that the p-type interactions in both the molecules are found to have higher interaction energies than other type of interactions. Also, most of the occupancies of the orbitals are of p-character, and the strong hyperconjugative interactions are responsible for the stabilization of both the molecules.

QTAIM and NCI-RDG analysis
Detailed information regarding the strength and nature of intra-and inter-molecular interactions are present in the molecular system obtained from topological analyses of their electron densities. QTAIM analysis allowed us to find the BCPs (bond critical points) and helps to analyze their properties viz., electron density (q BCP ), Laplacian (r 2 BCP ), Lagrangian kinetic energy (G BCP ), and potential energy (V BCP ). Table 7 summarizes the properties related to the bond critical points (Fig. 9)  Understanding the functionality of molecular systems requires a thorough explanation of the attractive and repulsive noncovalent interactions present in those systems. The QTAIM analysis does not include some weak noncovalent interactions since it only considers critical points in the electron density. Since the NCI index is based on the reduced density gradient (RDG), which is a function of the electron density (r) and its first derivative (r), it is used to obtain the complete information about weak interactions present in the molecular systems. The NCI-RDG analysis furnishes the visualization of the regions where the noncovalent interactions occur. The 3D RDG isosurface (e and f in Fig. 9) and 2D scattered plots (c and d in Fig. 9) with color bar blue-green-red signify the nature of interactions present in the molecules. The value of sign(k 2 )q(r) is the key indication of the strength and nature of the interactions, sign(k 2 )q(r) > 0 indicates the repulsive or strong hindrance in the molecule, sign(k 2 )q(r) < 0 for attractive interactions, and sign(k 2 )q(r) near to zero corresponds to weak (vdW) interactions [34].
The red spikes in the 2D scattered plot indicate the steric repulsion observed in the center of bromophenyl and thiazole moieties, which arises due to the steric repulsive force that existed between the carbon atoms in the rings. The green spikes indicate the weak van der Waals interactions observed in both the molecules. The blue spike between N-H … O interaction clearly indicates the strong hydrogen bond interactions present in 4b. Since the carboxylate substituent is attached to the 4 th position of the thiazole ring, it is easy to exist strong hydrogen bond interaction between N-H … O. The green colored isosurface present in the molecule validates the different intramolecular interactions in the experimental structure. Table 7. The interactions and binding affinities between the 4a and 4b with SARS-CoV-2 3CL protease (3CL pro) (PDB ID: 6m2q).

Compounds
Compound hydrophobic interaction with nucleic acid

Electron localized function (ELF) and localized orbital locator (LOL) analysis
The QTAIM and NCI-RDG analyses discussed in section 3.6 were appended by topological analyses of the ELF (g(r)) and LOL (v(r)). The use of ELF and LOL to perform covalent bonding analysis reveals areas of molecular space where the probability of discovering an electron pair is high. These ELF and LOL maps were plotted using Multiwfn 3.8 software. The ELF, LOL and ELF relief map figures of both 4a and 4b molecules are shown in Fig. 10a-c, respectively. Generally, the values of ELF range from 0 to 1. The values between 0.5 and 1.0 interval of the ELF indicate that the electron involved in bonding and nonbonding localized electrons, while smaller values (less than 0.5) indicates that electrons are delocalized [35]. Also, the values of LOL between o to 1.0 is identical to the range of ELF, meaning that both have similar chemical contents. However, LOL provides more conclusive and clear information than the ELF. The LOL accomplishes large values (greater than 0.5) in regions where the electron density is dominated by electron localization. Figure 10 clearly shows that the covalent regions with large LOL values indicated by the red color and blue color represents the electron depletion regions between the valence shell and inner shell. The ELF and LOL values corresponds to the BCPs of all noncovalent interactions discussed in QTAIM and NCI analyses.

Molecular docking studies:
In silico docking studies were performed to understand the activity of the crystallized compounds for the rational design of new potent molecules. In this work, molecular  docking of the most active compounds has been applied to study the different type of interactions and clarify the probable binding modes between thiazole derivatives and SARS-CoV-2 3CL protease (3CL pro) (PDB ID: 6m2q). Therefore, we used Autodock 4.2.6 software to dock the target moieties with SARS-CoV-2 3CL protease protein. The protein crystal structure was optimized and minimized using the protein preparation wizard's default setting for correcting PDB structure for docking. Table 1 shows the molecular docking scores and interaction residues of proteins with target drugs. Docking scores for 4a and 4b were À6.3 and À6.2, respectively, according to the docking studies (Fig. 11).

Conclusion
In the present investigation, the authors explored the conformational features of two thiazole derivatives and the role of various noncovalent interactions involved in crystal packing. The single crystal structure analysis of 4a and 4b revealed that the thiol group (-SMe) is attached to 2 nd position of the thiazole moiety in 4a whereas carboxylate chain is attached to 4 th position of the thiazole ring in 4b. Due to these conformational changes, the analysis of supramolecular self-assembly of the molecular structure shows that hydrogen bond interactions are involved mainly in crystal packing. Fingerprint plots show that the major contribution is from H … H contacts (28.9% in 4a and 29.2% in 4b). The energy gap of the frontier molecular orbitals are 4.194 eV in 4a and 3.521 eV in 4b, respectively. MEP analysis clearly revealed the electronegative nature around the oxygen and nitrogen atoms and electropositive nature around the hydrogen atoms present in the molecules and the presence of r-hole around the Br atoms in both the molecules. The NBO analysis show LP(1)N10 ! p Ã O9-C8 and p Ã N14-C13 ! p Ã

C15-C11
interactions having stabilization energy with 64.93 kcal/mol and 116.45 kcal/mol in 4a and 4b, respectively compared to all other bonding and anti-bonding orbitals. The QTAIM and NCI-RDG analyses validate the weak interactions involved in the molecules. The ELF and LOL analyses highlight the chemically significant regions in both the molecules. In addition, the designed derivatives were investigated as an inhibitor for COVID-19 using in-silico molecular docking analysis. Both the compounds 4a and 4b shown good binding affinity toward the main protease of COVID-19 with À6.3 and À6.2 kcal/mol. The results are clearly revealed that the ligands have comparable binding affinity for the SARSCov-19 main protease to those of approved medicines like favipiravir (À4.06 kcal/mol) and remdesivir (À6.96 kcal/mol), respectively.