Cocrystals of Ethenzamide With 2-Nitrobenzoic Acid - Conformational Analysis, MD Simulations And DFT Investigations


 In crystal engineering and pharmaceutical chemistry, cocrystals have a wide range of applications. Ethenzamide (EA) is found to form cocrystal with 2-nitrobenzoic acid (NBA). Geometry properties like stability energy, charge distribution, bond length, electronic properties and thermodynamic characteristics have been analyzed. The C-H…O hydrogen bond involves C-H of EA and oxygen of NBA. Configuration with the angle, N3-C4-C5-C6 gives the lowest energy conformation. Partition coefficient value suggests that EA-NBA has pharmaceutics behavior. RMSD values show the simulation’s relative stability and the complexes, remained stable throughout.


Introduction
Cocrystals have grown in popularity over the last 20 years as a result of their superior medicinal properties relative to the parent drug. Cocrystals are multi-component structures in which each compound is neutral and solid at room temperature [1,2]. Cocrystallization is particularly important in the pharmaceutical industry because cocrystals of an API have better biopharmaceutical qualities than pure drug [3,4]. Because of the variations in solubility of the individual components, standard solution based approaches are not always effective in cocrystal preparation. Cocrystal processing has demonstrated high e ciency using solid state methods [5,6]. Various approaches have been used to better understand cocrystallization [7][8][9]. Du et al. reported the cocrystals of piracetam with hydroxybenzoic acids [10,11].
Ethenzamide is an analgesic and antipyretic NSAID [12] and used in conjunction with active ingredients and its crystal structure was only recently reported [13,14]. Kozak et al. reported the characterization of cocrystals of ethenzamide [15]. The effect of external factors on ethenzamide-glutaric acid cocrystal was studied spectroscopically by Kozak and Pindelska [16]. Using ethenzamide, technologies in tablet production was recently reported [17]. Experimentally and theoretically, Aravinthraj et al. investigated the molecular interactions of 2-nitrobenzoic acid with other compounds [18]. 2-Nitrobenzoic acid can be used as a bacterial strain's growth supplement [19]. Hariprasad et al. reported the synthesis of cocrystals of ethenzamide [20]. DFT and MD simulations of EA-NBA are reported in the present work due to the importance of cocrystals in medicinal chemistry. Methods Geometry ( Fig. 1) properties like stability energy, charge distribution, bond length, electronic properties (µ, α, σ, χ, η, ω, µ) and thermodynamic characteristics such as (E, C v , S and ∆G) have been calculated by B3LYP/6-311++(2d,2p) with Gaussian and Gaussview software [21,22]. Some of pharmo-kinetic properties also have been calculated. The selected protein-drug and complex was subjected to MD simulation in Gromacs-2019.4 and as in literature [23][24][25][26].

Conformational analysis
To nd lowest energy con guration, potential energy scans are performed through ve torsion angles (Table 1 and Fig.S1) out of which the con guration τ(5) (N3-C4-C5-C6) gives the lowest one at 0.0º. All the conformations give another global minimum at 120.0, 100.0, 80.0, 130.0 and 130.0º for τ(1) to τ (5) conformations, with higher energy. For further analysis, τ(5) conformation is considered [27].  [20]. Correlation coe cient (R) and standard deviations were calculated for statistical validation between the geometrical parameters (table S1) obtained from DFT optimization and crystal structure data. The correlation coe cient for bond lengths and bond angles were found to be 0.9961 and 0.9541, respectively. From the curve tting analysis (Fig.S2), the standard deviations for bond lengths and angles are 0.0201 and 1.4562, respectively. It is found that the structural parameters are statistically closed to the experimental results [28].
The important functional groups of vibrational modes (table S2)  Research on subjects of crystal engineering and supra molecular synthesis of cocrystals is large and growing [33,34]. In order to complement these themes, we would like to discuss the preliminary actions that should be addressed (before any reaction take place!) when aiming to maximize the experimental e cacy of developing co-crystalline materials. The FDA de nes co-crystals as "crystalline materials composed of two or more different molecules, typically active pharmaceutical ingredient (API) and cocrystal formers ('coformers'), in the same crystal lattice" [34]. Co-crystallization is a promising method for modifying and improving an API's physicochemical properties without causing covalent modi cations to the drug molecule. Co-crystals are frequently used to address the poor solubility and bioavailability of BCS class II and IV medications, which account for 70% of all therapeutic candidates in development [33]. Chemical stability, hygroscopicity, mechanical characteristics, and ow qualities have been enhanced as a result of cocrystal formation. Co-crystallization can also be utilized for puri cation and enantiomeric separation [33].
In order to recognize the bio-pharmaceutic behavior of this compound, the optimization process has been done by B3LYP/6-311++(2d,2p). In Fig.S4  By consideration chemical geometry of chemical compound, the structure analysis will be obtained for recognition the biochemical manner. When the electron structure of chemical compounds demonstrated, the electronics and thermodynamic and pharmaceutics properties will get and calculated in good manner and the researcher can result the bioactive behavior of molecules. In order to recognize the chemical reactivity and pharmaceutical behavior, Table 2 obtained the necessary parameters that lead to get best results to show pharmaceutics behavior. In Table 2 the softness, hardness, electronegativity, electropositivity, chemical potential are the electronic parameters for determining the chemical reactivity. Log p, surface area, volume, hydration energy and polarizability demonstrate the pharmaceutical properties. Energy and C v are thermodynamic parameters. A partition coe cient (P) or distribution coe cient (D) is the ratio of a compound's concentrations in a mixture of two immiscible solvents. This ratio represents a comparison of the solute's solubility in two liquids. The partition coe cient describes the concentration ratio of unionized compound species, whereas the distribution coe cient describes the concentration ratio of all compound species (ionized plus un-ionized). Both phases are often solvent in the chemical and medicinal sciences. One of the solvents is usually water, whereas the other is hydrophobic, such as 1-octanol. As a result, the partition coe cient determines whether a chemical substance is hydrophilic ("water-loving") or hydrophobic ("water-fearing"). Hydrophobic medicines with high octanol-water partition coe cients are found in cell lipid bilayers. Hydrophilic medicines on the other hand, are found largely in aqueous environments such as blood serum (low octanol/water partition coe cients). According to data in Table 2 cocrystal compound logP = 5.17 obtained this cocrystal compound has pharmaceutics behavior.

Molecular docking and simulation studies
Ethenzamide is a NSAID with analgesic and antipyretic properties and, title molecule is docked [35][36][37] with the PDBs, 2ABZ, 5JCL, 3SDP and 2V7B corresponding to mutant of leech carboxypeptidase inhibitor [38], monodehydroascorbate reductase [39], iron superoxide dismutase [40], and benzoate CoA ligase inhibitor [41]. The binding energy and amino acids interactions at the active sites are given in table S5 and Fig.S9. 2ABZ gives maximum global and atomic contact energies and based on this, MD simulations are also carried with this PDB and the ligand.
In our study, crystal structure of C19A/C43A mutant of leech carboxypeptidase inhibitor(2ABZ), APO and complex with selected ligand from docking drug was subjected to molecular dynamics simulation analysis. MD simulation for 100 ns were done to understand stability of above-mentioned protein-ligand complexes RMSD, RMSF, Rg, H-Bonds (hydrogen bonds), Ligand RMSD, SASA, Secondary structure element analysis and MMPSA calculations were made.
Root mean square deviation is a term to determine differences between the two con rmations. Higher RMSD gives more deviation and the RMSD values are calculated against the simulation timescale of 40 to 100ns. Average RMSDs from 0 to 100 ns for APO and drug protein were 0.91nm and 0.89nm. This represents stability of APO and ligand complex in simulation [42]. RMSD results for APO and its complex with ligand are depicted in Fig. 3(a) and (b). During the 100ns simulation, it was observed that the APO and complexes are equilibrated after 40ns of time. The RMSD mean for APO and complexes were calculated from 40ns to 100ns. The amino acids involved in bringing the overall structural deviation are explored in the RMSF plots. The ligand RMSD is used to nd the differences between the two con rmations. The ligand RMSD values are calculated against the simulation timescale of 0 to 100ns.
Average drug RMSDs from 0 to 100 ns for inhibitors were 0.03 nm which gives the stability of the drug with the protein.
RMSF analysis determines which amino acids of the protein make more vibrations, resulting in the destabilization of protein in presence/absence of the ligands. The RMSF values are calculated against the simulation timescale of 0 to 100ns. The RMSF results for APO and its complex with ligand, chain A and chain B as depicted in Fig. 3(c) and (d). The average RMSF-A and RMSF-B from 0 to 100 ns for APO and drug complex were 0.3 nm and 0.4, respectively [43]. Compactness of the protein can be determined by the radius of gyration. Folding and unfolding of the protein was analyzed by the Rg values against the simulation timescale of 0 to 10,000ps for APO and its complex with ligand. The average Rg from 0 to 100 ns for APO and ligand complexes were 1.6 and 1.05 nm. The RG result of the APO and its complex with ligand as depicted in Fig. 4(a) [44].
The terms " exibility" and "compactness" are often used interchangeably. To understand the modulation of inhibitors on the protein, SASA changes the compactness of protein. SASA were determined, though the variations in SASA values in all of the complexes were quite small. SASA values range from 0 to 100 represented in Fig. 4(b). Average SASAs from 0 to 100 ns for APO and inhibitors ligand were 80 and 81 respectively [45].
The creation of hydrogen bonds stabilizes protein-drug complexes. In our research, hydrogen bonds ( Fig.  5) formed in the molecular docking analysis are con rmed by the simulation analysis. The ability to investigate the structural behavior of a protein requires an understanding of secondary structural content. As in Fig.S10(a-d), we evaluated changes in secondary structure in apo and ligand complexes. How much energy is required for the ligand to bind to protein is determined by MMPBSA. The binding energy of DRG was − 135.238 +/-21.157 kJ/mol ( Table 3)