Spectroscopic, Solvation Effects and MD Simulation of an Adamantane-Carbohydrazide Derivative, a Potential Antiviral Agent

Abstract The spectroscopic, solvent effects, reactivity and MD simulations of N'-[(1E)-(2,6-dichlorophenyl-methylidene]adamantane-1-carbohydrazide (DMC) are reported. Solvation free energies of DMC in chloroform, ethanol and acetonitrile are −21.96, −24.39 and −12.31 kcal/mol and ethanol may be better for the solubilization. Electron donating and electron accepting powers are somewhat lower in solution, revealing a certain increase in the tendency to receiving electrons and a decrease in donate electrons in solution, except in acetonitrile, in which showed slightly higher values then in vacuum. ALIE surface show that regions where low energy is required to remove electrons are on benzene ring, chlorine atoms and N5. DMC is forming good contacts with protein's binding site residues and is important in the complex's stability showing antiviral activity.


Introduction
Early on, it was discovered that carboxylic acid hydrazides were effective chemotherapeutic agents for the treatment of mycobacterial infections. These were demonstrated to be above their prototype medications as a consequence of significant research. 1,2 Furthermore, adamantane-1-carbohydrazide and hydrazone derivatives have been examined in the past and found to have powerful antibacterial and antifungal properties. 3 Carbohydrazide derivatives have been described as corrosion inhibitors in addition to their biological applications. 4 Recently, a series of carbohydrazide compounds was developed, synthesized and evaluated for anti-proliferative efficacy as novel Nur77 modulators. 5 Hydrazones are a type of Schiff bases that contains the imine functionality obtained from the hydrazide group and has a wide range of biological applications, anti-tubercular, 6,7 antifungal, 8,9 antiviral, 10 anticancer 11,12 and anti-leishmanial 13,14 activities. In addition, the broad chemotherapeutic activities of adamantanes have been established as important groups with potent antiviral, 15,16 anticancer, 17,18 antibacterial, [19][20][21] antifungal 22 and anti-malarial 23 activities. Ekimov et al. described the characteristics of nanodiamonds. 24 Because of their numerous bioactivities, adamantane derivatives are significant components in medicinal chemistry and studies concentrating on these were done for discovering new chemical and biological functions. 3,19 The substitution of methyl groups on the carbon of adamantane improves oxidation stability. 25 Alkyl-adamantanes, which combine a dense polycyclic core with alkyl groups, show tremendous promise for enhancing the oxidation stability of jet fuel while keeping its high density. The adamantane core's symmetrical and compact structure can boost density, while the alkyl groups can lower the melting point substantially. 26 Use of adamantane-based derivatives is effective medicines for the treatment of a variety of pathological disorders. 27 When an adamantyl moiety is incorporated into numerous molecules, it almost results in compounds with a high lipophilicity. 28 El-Emam et al. reported the non covalent interactions in admantane derivatives recently. 29 In the solid state, di-, tri-and tetra aryl adamantanes have been shown to have a high inclination to construct various forms of supramolecular structures. 30 The adamantane skeleton served as a stiff linker between chromophoric units as well. 31,32 Wrona-Piotrowicz et al. reported the fluorescent effects adamantanes and the structures. 33 Because of its hydrophobic character, adamantane has been widely used in drug synthesis to modify pharmacological and pharmacokinetic properties of a variety of drugs. 34 Aljohar et al. reported the single crystal XRD of DMC. 35 Theoretical approaches to the study of the electronic structure are commonly utilized to predict and comprehend the properties of molecular systems. 36 These computational tools are commonly used in the research of organic molecules that could be used in pharmaceuticals. 37 The solvation free energy calculates the energy change in a molecule's transfer from gas to solvent and it is used to calculate a variety of properties like activity coefficients and solubilities. Spectroscopic investigation and docking with MD simulations are reported for DMC. Also effects of replacing chlorine by fluorine and bromine are discussed.

Methods
DMC was prepared with adamantne-1-carboxylic acid 1 via esterification with methanol and sulfuric acid to get methyl ester 2, reacted with hydrazine hydrate for corresponding carbohydrazide 3. The reaction of carbohydrazide 3 with 2,6-dichlorobenzaldehyde via heating under reflux in ethanol yielded the title compound 4 in 87% yield 35,38,39 (Scheme 1-supporting). Vibrational spectra (Figures S1 and S2) were on a DR/Jasco FT-IR and Bruker RFS spectrometers.
The theoretical quantum calculations of DMC ( Figure 1) were done with the help of Gaussian 09 and Gaussview 5 40,41 via wB97XD/6-311 þ G Ã , 42,43 where FMOs, MEP, average local ionization energy (ALIE) surfaces and solvation free energies were analyzed. The default settings were used for the convergence criteria regarding SCF and optimization procedure. Frequency analyses present no imaginary frequencies, corresponding to local minima and the assignments were done through PED analysis on optimized structures using GAR2PED software package. 44 MEP and ALIE were generated by Multiwfn, 45 solvation free energies in chloroform, ethanol and acetonitrile were obtained via Truhlar's SMD model 46 with IEF-PCM algorithm.
To investigate the stability and physical movements for the docking complex, MD simulations have been performed with 100 ns simulation time. The simulation is done with the help of Desmond module (Schrodinger) software. 47,48 For protein-ligand docked complex, TIP3P model and Gromos9643a1 force field was applied to provide a larger coverage of organic functionality. NaCl are added to neutralize the charges in the system and Simple Point Charge (SPC) is also used to fetch correct density and permittivity. MD Simulation continued till 100 ns at the conditions of (NVT and NPT). The ambient temperature and pressure are 310 K and 1.013 bar. For better results, simulation was run three times. Different results were analyzed for RMSD, RMSF, H-bonds, and Rg etc. which helps us to check the thermodynamic stability.

Vibrational spectra
For DMC, adamantyl group modes are assigned at (Table S2) 53 and due to hydrogen bonding there is a split for NH mode at downshifted from computational value by an amount 312 cm À1 .

Solvent effects, chemical and electronic properties
The solvation free energy in chloroform, ethanol and acetonitrile estimates the energy change in the transfer of a molecule between gas and solvent, being important for determine a wide spectrum of properties such as activity coefficients and solubilities. 45,62 For DMC, the solvation free energieswere calculated via SMD model in chloroform, ethanol and acetonitrile, which provided values equal À21.96, À24.39 and À12.31 kcal/mol respectively. Although the values obtained be negative for all solvents tested, the comparison of the calculated solvation free energies values suggests that ethanol may be better for the solubilization of DMC. The value of the solvation free energy indicates good solubility of DMC in an aqueous medium, factor that corroborates the biological activity, in as much as that bioorganic processes occur in aqueous medium.
Another important descriptors are the electron donating (x À ) and accepting x þ ð Þ powers, which are defined as: Where a larger value of x þ ð Þ corresponds to a great accepting charge capacity, whereas a smaller value of x À ð Þ indicates a better electron donor molecular system. 71,72  Both FMOs comprise ( Figure 2) the benzene ring, chlorine, C13, N5, N6, C12 and O3 atoms. Table 1 shows that the theoretical FMOs energies and HOMO-LUMO energy gap values calculated for DMC are slightly higher in solution, indicating that the dielectric constant of solvent slightly affects FMOs energies, and consequently affecting the reactivity indexes, revealing higher values for g K in solution and lower values for v K and x K in vacuum, except in acetonitrile, where DMC showed slightly higher values for v K and x K : Electron donating and electron accepting powers are somewhat lower in solution, revealing a certain increase in the tendency to donate electrons and a decrease tendency in accepting electrons in solution, except in acetonitrile, in which showed slightly higher values compared to those in vacuum. Furthermore, the calculated global reactivity indices compared to calculated ones for other bioactive molecules, [73][74][75][76] indicates the studied molecule as a moderate soft molecule with moderate attractive electron power and good donating electron power. When chlorine atoms are replaced by fluorine and bromine, there is no significant change for the chemical descriptors and HOMO and LUMO energy values are, À6.52 and À1.99 eV for fluorine substitution and À6.54 and À1.91 eV for bromine substitution and due to the higher electrophilicity index, fluorine and bromine substituted derivatives show higher bioactivity. 77,78 Other important parameters obtained from quantum mechanics calculations, specifically in DFT, are MEP and ALIE. They consist in colored 3 D surfaces, ranging from red to blue, passing through green and yellow. MEP surfaces allow visualize the charge distributions of molecules, being very useful for evaluating electrostatic interactions between molecules, such as hydrogen bonds (H-bonds). 79-81 ALIE show energy values required to remove electrons over surface, helping to predict reactive sites sensitive to electrophilic attack. 82 Regarding MEP (Figure 3), positive potentials occur over H48 (0.027), H35 (0.031), H22 (0.024) and H5 (0.055 a.u.) and negative over O3 (-0.066) and N6 (-0.069). ALIE surface (Figure 3) shows that regions where low energy is required to remove electrons are on benzene ring ($0.40a.u.), chlorine atoms ($0.38a.u.) and N5 ($0.40a.u.). It's noteworthy the surfaces over O3 atom show high values in ALIE (0.45 a.u.), indicating O3 is more prone to interact with positively charged species than to suffer electrophilic attacks.
Fukui function is a derivative of electronic energy, E, used to predict reactive regions of a molecule, allowing us determine sites prone to receive nucleophilic, electrophilic or radical attack 83,84 and ,allows definition of three local reaction descriptors (LRD), by different approximations, for this function. Among them, the finite difference approximations (FDA) is the most used and are defined as: qN þ 1(r), qN(r) and qN-1(r) indicates respectively, electronic density of the anion, neutral and cationic chemical species. In this sense, positive values of f þ , f À and f 0 gives areas prone to receive nucleophilic, electrophilic and free radical attacks respectively.
Morell et al. 85 proposed a dual descriptor giving reactive sites of a molecule as: But can be obtained approximately as follows: The dual descriptor is very useful for predicting the reactive sites due to the no ambiguity, as f 2 ðrÞ>0 indicates sites predisposed to suffer nucleophilic attacks and f 2 ðrÞ<0 reveals sites prone to suffer electrophilic attacks. 86 From Fukui indices surfaces for DMC (Figure 4), concerning nucleophilic attacks, the studied molecule shows prone reactive sites on C34, C38, C26, N6 and C12 atoms and on C13 ¼ C8 double bond. Zones susceptible to suffer electrophilic attacks are located on N6 ¼ C13 and N4-C12bonds and on O3, C34, C26 and C38 atoms. f 2 r ð Þ isosurfaces reveal that C38, C34, C26 and N6 atoms are predominantly electrophilic sites (prone to receive nucleophilic attacks) due to the positive isovalues presented (pink regions). Furthermore, confirmed that N4, C13 and O3 atoms are nucleophilic centers (prone to receive electrophilic attacks) due to negative isovalues revealed (green regions). In MEP plot, all molecules, C ¼ O group and phenyl ring of all compounds are strongest repulsion areas and NH is the strongest attractive region. 87

Molecular docking and dynamics
PASS analysis of DMC predicts antiviral activity 88,89 and antiviral proteins of PDBs, 1EAH, 1PIV, 1R08, 3UZV,6VYO, 6YB7 are used for docking which give binding energies of À8.6, À8.2, À7.9, À7.4, À8.1, À7.3 kcal/mol, respectively. [90][91][92] The proteins were prepared for docking by removing the co-crystallized ligands, waters and co-factors. The AutoDock Tools package was used to find the Kollman charges and polar hydrogen's and the ligand was prepared for docking by minimizing its energy. Partial charges were also calculated and the active site of the enzyme was defined to include residues of the active site within a grid of 40 Å Â 40Å Â 40Å. The protocol was tested by extracting co-crystallized inhibitor from the protein and then docking the same and docking protocol predicted the same conformation as was present in the crystal structure with RMSD in the range 2 Å. Amongst the docked conformations, one which bonded well at the active site was analyzed for detailed interactions.
The amino acid interactions with all selected PDBs are shown in Figure S3 and from that DMC exhibit inhibitory activity against 1EAH with a high binding affinity which is a polio virus type 2. 93 For DMC with 1EAH: amino acid Asp236 and Ser206 form H-bond with NH and C ¼ O; Also, His207, Lys113, Met140, Gly230, Ala202, Ala232, Ala231, Tyr205, Phe237 interacts with DMC with an affinity value of À8.5 kcal/mol. For bromine substitution in DMC: amino acids of the receptor Asp218 interacts with NH group and Tyr209 with phenyl ring; Ala213, ala232 interacts with bromine atom, adamantine ring respectively; Tyr205, Phe237 interacts with adamantine ring while Tyr209, Lys222 interacts with phenyl ring with energy À8.6 kcal/mol. For Fluorine substitution: the residues of the amino acids Asp236 interacts with NH group and fluorine; Ser206 forms two H-bond with fluorine atom and C ¼ O whereas Ser214 forms H-bond with C ¼ O; Ala202, Ala232, Leu215, Tyr209 shows alkyl interaction with adamantane ring but Phe237 interacts with phenyl ring with energy of À8.6 kcal/mol.
From the docking results, PDB 1EAH, which giving maximum binding affinity was selected for MD simulations. RMSD values are found for apo protein and DMC for the time trajectory of 100 ns. For the full-time trajectory of 100 ns, the RMSD for protein ranges from 2.2 Å to 3.4 Å (Figure 5a). The average value of RMSD for protein is observed as 3.2 Å (Figure 5a) and for DMC is 4 Å (Figure 5a). Unlike RMSD of protein, RMSD of DMC keeps fluctuating at different time intervals throughout the simulation time. The value of RMSD for DMC increases proportionally for the initial 20 ns of the simulation time and shows rapid rise in value from 2 Å to 7.5 Å. The graph of RMSD shows gradual decrease in values between 20 to 40 ns of trajectory and then constantly fluctuates from 1.2 Å to 2.4 Å. After 60 ns, the RMSD graph again shows a rise to 3.2 Å and fluctuate constantly between values 2.4 Å to 3.2 Å. The high values of RMSD (say more than 2.0 Å) validate the good docking of protein and DMC. Thus, we can say that the docked complex stays stable throughout the simulation time.
MD simulations 94,95 investigate insight dynamic changes and according to several studies, any change in protein properties could be linked to RMSF. 96,97 Flexibility is an important thermodynamic property for proteins to maintain their best functions. 98 The RMSF graph shows high fluctuations repeatedly throughout the simulation time. The average of the RMSF values is observed to be 1.2 Å (Figure 5b). The highest value of RMSF for complex is observed to be 5.4 Å. During the whole simulation, the protein remains unaltered when complexed with ligand. The RMSF values validate the stability and heterogeneity of all the active and flexible protein residues.
The main factors in protein-drug interactions are Hydrogen bonding (HB), van der Waals, and electrostatic forces. 98 Among them, HB plays a vital part in folding, stability, and recognition. Figure S4 shows the protein interactions with the ligand as stacked bar chart plot as: HBs, hydrophobic, ionic, and water bridges. For protein and ligand complex, water bridges, HBs and hydrophobic interactions play a crucial part in interactions. The amino acids ALA232, ASN235, and ASP236 exhibited HB contact with the protein, form HB interactions. This indicated that the protein ligand complex is stable throughout the simulation time.
To determine the degree of protein folding stability, we normally used radius of gyration (Rg). Rg fluctuations over time suggest unstable folding, whereas a constant value indicates stable folding. [99][100][101] Variation of Rg for protein docked ligand complex is shown in the Figure S5. Variation in R g value reveals for complex structure shows quite stable behavior throughout the simulation time trajectory except 0 ns to 30 ns time period. The lower the value of Rg, the more compact the complex will be, even if it will be more stable. Average R g value for complex structure is found to be 4.2 Å ( Figure S6) exhibits the stability of protein-ligand complex structure. To investigate the interactions between protein residues and ligand, a total contacts timeline diagram was created ( Figure S7). The various energy values obtained from MD simulations are presented in Table 2. During simulations, a maximum of 6 unique connections were created between the protein and the ligand, as seen in the top paneland the bottom panel shows that the residues ALA_232, ASN_235, and ASP_236 showed interactions with DMC (darker shade) ( Figure S7). As a result, we can deduce that ligand is forming good contacts with protein's binding site residues and is important in the complex's stability 102,103

Conclusion
The chemical, electronic and solvation effects of DMC are presented in the work. The NH stretching mode shows a splitting in IR, at 3458, 3241 cm À1 while the computational value is 3553 cm À1 and the downshift in the IR spectrum is 312 cm À1 which is due to hydrogen bond interaction in the system. The value of the solvation free energy indicates good solubility of DMC in an aqueous medium, factor that corroborates the biological activity, in as much as that bioorganic processes occur in aqueous medium. Zones susceptible to suffer electrophilic attacks are on N6 ¼ C13 and N4-C12bonds and on O3, C34, C26 and C38 atoms. PASS analysis of DMC predicts antiviral activity and MD simulation studies exhibited that the ligand is thermodynamically feasible and binds stably with the active site of protein and complex were strong and stable throughout 100 ns. Contacts timeline analysis of ligand exhibited that ALA232, ASN235, and ASP236 were the crucial interactions.

Declarations
Author's contributions

Disclosure statement
No potential conflict of interest was reported by the authors.