Structural Properties and Biological Prediction of ({[(1e)-3-(1h-imidazol-1-yl)-1-phenylpropylidene] Amino}oxy)(4-methylphenyl)methanone: an in Silico Approach

Bioactive molecules are playing essential role in the field of drug discovery and various pharmaceutical applications. Vibrational spectral investigations of the anti-Candida agent


Introduction
Fungi are eukaryotic organisms and some of them are important human pathogens causing diseases such as aspergillosis, candidiasis and cryptococcosis.Actually, the incidence of severe fungal infections has increased in an alarming way over the past few decades.An antifungal drug is an agent that selectively attacks fungal pathogens with minimal toxicity to the host.Azoles are nitrogen-containing five-member heterocyclic ring system.They constitute the largest family of antifungal drugs and have been and are still widely used to treat superficial mucosal as well as deep and disseminated fungal infections.However, their extensive use gives rise to the development of resistance and resulted in therapeutic failure [1].Azole antifungal agents inhibit biosynthesis of cell membrane sterols [2,3].Single crystal X-ray structure and anti-Candida activity (minimum inhibitory concentration value = 0.3752 µmol/mL toward Candida albicans) of the titled azole-containing molecule, namely ({[(1E)-3-(1H-imidazol-1-yl)-1-phenylpropylidene]amino}oxy)(4-methylphenyl)methanone ((1E)-IPMM) were previously reported [4,5].
The present work deals with the FT-IR and FT-Raman spectral investigations of (1E)-IPMM to understand its structural geometry, inter-and intra-molecular interactions, hydrogen bonding, highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy and the natural bond orbital (NBO) analysis with the aid by density functional theory (DFT) calculations.The work also explores the biological activity of the title molecule by molecular docking approach.

General
Melting point was performed using Gallenkamp melting point device, and it is uncorrected.Crystallographic data have been deposited with the Cambridge Crystallographic Data Center (supplementary publication number CCDC-1006859).

Spectroscopic Measurements
The Fourier transform infrared spectrum of (1E)-IPMM was recorded using a Perkin Elmer RXL Spectrometer (Waltham, MA, USA) in the region 4000-400 cm ´1, with samples in the KBr using pellet press method.The resolution of the spectrum is 2 cm ´1.The FT-Raman spectrum of sample in the solid phase was recorded in the range 3500-50 cm ´1 using a Bruker RFS 100/S FT-Raman spectrophotometer (Ettlingen, Germany) with a 1064 nm Nd:YAG laser source of 100 mW power (Göttingen, Germany).

Quantum Chemical Calculations
The quantum chemical computations of (1E)-IPMM have been performed using Gaussian 09 Program Package [6] at the Becke3-Lee-Yang-Parr (B3LYP) level with standard 6-311++G(d,p) basis set [6].An empirical uniform scaling factor of 0.9673 was used to offset the systematic errors caused by basis set incompleteness, neglect of electron correlation and vibrational anharmonicity [7,8].The calculated Raman activities (S i ) have been converted to relative Raman intensities (I i ) using the following relationship derived from the intensity theory of Raman scattering [9,10].
where ν 0 is the exciting wavenumber, ν i is the vibrational wavenumber of the ith normal mode, h, c and k are fundamental constants, and f is a suitably chosen common normalization factor for all peak intensities.The simulated IR and Raman spectra have been plotted using pure Lorentzian band shape with a bandwidth of full width half maximum (FWHM) of 10 cm ´1.

Synthesis
The title molecule 3 was prepared using the commercially available acetophenone as illustrated in Scheme 1.

Synthesis
The title molecule 3 was prepared using the commercially available acetophenone as illustrated in Scheme 1.

Structural Geometry Analysis
The optimized geometry of the molecule is determined by minimizing its energy with respect to all geometrical parameters without imposing molecular symmetry constraints.The optimized molecular structure of the isolated molecule is shown in Figure 1.The optimized bond lengths, bond and dihedral angles are presented in Table 1 in comparison with the experimental values.The calculated bond lengths of C16=O1, and C16-O2 are found to be 1.1993 Å and 1.3828 Å, respectively.The shortening of C16=O1 is due to the presence of double bond character.The calculated bond lengths of C-N bonds are C3-N21 (1.2859 Å), C36-N4 (1.4568 Å), C4-N39 (1.3817 Å), C4-N43 (1.3692 Å), C5-N41 (1.3757 Å), and C5-N43 (1.3131 Å) and the lengthening of the bond C36-N4 is due the conjugation effect of the imidazole moiety.The bond length of C39-H40 (1.0774 Å) is found to be shorter than the other C-H bonds, which is due to the presence of intra-molecular C-H•••π bond interaction.In addition, the bond length of C33-C36 (1.5455 Å) appears to be longer than the other C-C bonds and it may be due to the charge transfer effect between the donor to acceptor group.The experimental and calculated value of bond length shows a good agreement in the correlation analysis (R 2 = 0.9945).The slight deviations in the bond angle and dihedral angles from the experimental XRD data are probably due to the intermolecular interactions in the crystalline state.

Structural Geometry Analysis
The optimized geometry of the molecule is determined by minimizing its energy with respect to all geometrical parameters without imposing molecular symmetry constraints.The optimized molecular structure of the isolated molecule is shown in Figure 1.The optimized bond lengths, bond and dihedral angles are presented in Table 1 in comparison with the experimental values.The calculated bond lengths of C16=O1, and C16-O2 are found to be 1.1993 Å and 1.3828 Å, respectively.The shortening of C16=O1 is due to the presence of double bond character.The calculated bond lengths of C-N bonds are C3-N21 (1.2859 Å), C36-N4 (1.4568 Å), C4-N39 (1.3817 Å), C4-N43 (1.3692 Å), C5-N41 (1.3757 Å), and C5-N43 (1.3131 Å) and the lengthening of the bond C36-N4 is due the conjugation effect of the imidazole moiety.The bond length of C39-H40 (1.0774 Å) is found to be shorter than the other C-H bonds, which is due to the presence of intra-molecular C-H¨¨¨π bond interaction.In addition, the bond length of C33-C36 (1.5455 Å) appears to be longer than the other C-C bonds and it may be due to the charge transfer effect between the donor to acceptor group.The experimental and calculated value of bond length shows a good agreement in the correlation analysis (R 2 = 0.9945).The slight deviations in the bond angle and dihedral angles from the experimental XRD data are probably due to the intermolecular interactions in the crystalline state.

Natural Bond Orbital (NBO) Analysis
The natural bond orbital (NBO) calculations were performed using NBO 3.1 program as implemented in the Gaussian 09 package [6] at the DFT level [11].The corresponding results have been presented in Table 2.A useful aspect of the NBO method is that it gives information about interactions in both filled and virtual orbital spaces, which could enhance the analysis of intra-and intermolecular interactions that will give a measure of the intermolecular delocalization or hyper conjugation.Delocalization of electron density between occupied Lewis-type (bond or lone pair) NBO orbital and formally unoccupied (anti-bond or Rydberg) non-Lewis NBO orbital corresponds to a stabilizing donor-acceptor interaction.The delocalization effect can be described as a charge transfer from the highest occupied bonding orbital into unoccupied anti bonding orbital and their importance can be more quantitatively characterized through a second order perturbative treatment that gives the energy lowering associated with such interaction.The magnitude of these delocalization effects can be determined from an analysis of the off diagonal elements in the Fock matrix of the NBO basis by taking into account all possible donor-acceptor interactions.The hyper conjugative interaction energy was deduced from the second-order perturbation approach as where <σ F σ> 2 or Fij 2 is the Fock matrix element between i and j NBO orbital, εσ and εσ* are the energies of σ and σ* NBO's, and nσ is the population of the donor σ orbital [12].The larger value of hyperconjugative interaction energy (E( 2)) implies that the interaction between electron donors and electron acceptors is more intensive and thus the greater the extent of the conjugation of the whole system.Here, the intra-molecular hyperconjugative interactions are formed by the orbital overlap between π(C-C) and π*(C-C) bond orbital which results in intramolecular charge transfer (ICT), causing stabilization of the system.These interactions are observed as an increase in electron density (ED) in C-C anti bonding orbital that weakens the respective bonds.The ED value of the phenyl rings

Natural Bond Orbital (NBO) Analysis
The natural bond orbital (NBO) calculations were performed using NBO 3.1 program as implemented in the Gaussian 09 package [6] at the DFT level [11].The corresponding results have been presented in Table 2.A useful aspect of the NBO method is that it gives information about interactions in both filled and virtual orbital spaces, which could enhance the analysis of intra-and intermolecular interactions that will give a measure of the intermolecular delocalization or hyper conjugation.Delocalization of electron density between occupied Lewis-type (bond or lone pair) NBO orbital and formally unoccupied (anti-bond or Rydberg) non-Lewis NBO orbital corresponds to a stabilizing donor-acceptor interaction.The delocalization effect can be described as a charge transfer from the highest occupied bonding orbital into unoccupied anti bonding orbital and their importance can be more quantitatively characterized through a second order perturbative treatment that gives the energy lowering associated with such interaction.The magnitude of these delocalization effects can be determined from an analysis of the off diagonal elements in the Fock matrix of the NBO basis by taking into account all possible donor-acceptor interactions.The hyper conjugative interaction energy was deduced from the second-order perturbation approach as where <σ|F|σ> 2 or F ij 2 is the Fock matrix element between i and j NBO orbital, ε σ and ε σ˚a re the energies of σ and σ* NBO's, and n σ is the population of the donor σ orbital [12].The larger value of hyperconjugative interaction energy (E(2)) implies that the interaction between electron donors and electron acceptors is more intensive and thus the greater the extent of the conjugation of the whole system.Here, the intra-molecular hyperconjugative interactions are formed by the orbital overlap between π(C-C) and π*(C-C) bond orbital which results in intramolecular charge transfer (ICT), causing stabilization of the system.These interactions are observed as an increase in electron density (ED) in C-C anti bonding orbital that weakens the respective bonds.The ED value of the phenyl rings ("1.6e) shows strong charge delocalization.However, the two conjugated π bonds ("

Vibrational Spectral Analysis
The FT-IR and FT-Raman spectra of (1E)-IPMM have been analyzed on the basis of density functional theory calculations.The vibrational modes were assigned on the basis of potential energy distribution (PED) analysis using the VEDA 4 program [13].The combined experimental and simulated Infrared and Raman spectra are shown in Figures 2 and 3 respectively.The computed wavenumbers are compared with the experimental FT-IR and FT-Raman wavenumbers and their assignments are presented in Table 3. Vibrational analysis is based on the vibrational modes of the groups phenyl ring, methylene, methyl, imidazole ring and skeletal mode.Wavenumber (cm-1 )

Phenyl Ring Vibrations
In general, aromatic C-H stretching vibrations absorbs in the region 3080-3010 cm ´1 [14].The intense Raman band is observed 3066 cm ´1, which corresponds to C-H stretching vibration.In addition, aromatic C=C stretching vibrations occurs in the region 1625-1430 cm ´1 [15].The intense band is observed at 1597 cm ´1 in the Raman spectrum.The in-plane C-H deformation vibration generally appears in the region 1290-1000 cm ´1.The C-H out-of-plane deformation expected around 860-800 cm ´1 [14].A medium intense IR band is observed at 819 cm ´1 in IR spectrum, which is calculated to be at 821 cm ´1 with PED 50% can be attributed to C-H deformation mode.

Methylene Vibrations
The methylene symmetric vibrations expected around in the region 2865-2845 cm ´1 [15].The weak Raman band is observed at 2858 cm ´1 can be attributed to CH 2 symmetric stretching mode.The -CH 2 in-plane vibration expected around in the region 1480-1440 cm ´1 [14].The methylene scissoring mode is observed at 1443 cm ´1 (IR) and 1446 cm ´1 (Raman).The -CH 2 wagging appears around 1411-1174 cm ´1 [16].The Raman band appearing at 1349 cm ´1 is assigned to be methylene wagging vibration mode.

Methyl Vibrations
The methyl group asymmetric vibrations generally appear in the regions 2972-2952 cm ´1.The methyl asymmetric stretching band is observed at 2965 cm ´1, in both IR and Raman spectra.The methyl group symmetric bending modes expected to occur in the region 1365-1385 cm ´1 [15].The observed band at 1360 cm ´1 in IR spectrum is calculated to be at 1367 cm ´1 with PED 38% can be attributed to methyl bending modes.

Imidazole Ring Vibrations
The imidazole ring CH vibrations expected to occur at 3145-3115 cm ´1 [14].The medium Raman band is observed at 3136 cm ´1 assigned to imidazole C-H stretching mode.

Skeletal Mode Vibrations
The C-N and C-C stretching vibrations generally arises in the region 1150-850 cm ´1 [17][18][19].The C-C and N-C stretching modes are observed at 1078 and 1010 cm ´1, respectively in the IR spectrum.

HOMO-LUMO Energy Analysis
The highest occupied molecular orbital (HOMO) energy characterizes the ability of electron giving, whereas the lowest unoccupied molecular orbital (LUMO) energy characterizes the ability of electron accepting.The energy difference between HOMO and LUMO orbitals is called as energy gap, which is an important stability factor for structures.They indicate the electron transport in molecular systems.It is worth noting that HOMOs have an overall π bonding character along with a considerable non-bonding character and LUMOs have an anti-bonding π* character.The strong charge transfer interaction through π conjugated bridge results in substantial ground state donor-acceptor mixing and the appearance of a charge transfer band in the electronic absorption spectrum.Therefore, an electron density (ED) transfer occurs from the more aromatic part of the π conjugated system in the electron-donor side to its electron-withdrawing part.The HOMO-LUMO energy gap for (1E)-IPMM was computed at the B3LYP/6-311++G(d, p) level basis set.The HOMO of the title molecule is located on the imidazole ring and LUMO on phenyl rings.This indicates that the charge transfer between the imidazole ring to phenyl ring system is through the C=N bond.The Eigen values of LUMO (´2.02 eV) and HOMO (´6.36 eV) and their energy gap (4.34 eV), explains the eventual charge transfer interactions taking place within the molecule.The frontier molecular orbital diagrams are shown in Figure 4.

NMR Spectral Analysis
The scaled and experimental chemical shift values are presented in Table 4.The computed chemical shifts were scaled down by the linear regression method for neglecting the systematic errors [20][21][22][23][24][25].The empirically scaling was carried out by following equation where σ is the calculated isotropic shielding value for a particular nucleus.

Molecular Docking Analysis
The structure of (1E)-IPMM was optimized based on the density functional theory using Gaussian 09 program [6].The molecular docking was performed using AutoDock Tools-1.5.4

NMR Spectral Analysis
The scaled and experimental chemical shift values are presented in Table 4.The computed chemical shifts were scaled down by the linear regression method for neglecting the systematic errors [20][21][22][23][24][25].The empirically scaling was carried out by following equation where σ is the calculated isotropic shielding value for a particular nucleus.

Molecular Docking Analysis
The structure of (1E)-IPMM was optimized based on the density functional theory using Gaussian 09 program [6].The molecular docking was performed using AutoDock Tools-1.5.4 interfaced with the MGL Tools-1.5.4 package [26].The antifungal target protein of Mycobacterium tuberculosis Cyp51 (PDB ID: 1EA1) in complex with the standard antifungal drug fluconazole, was selected for the present docking analysis [27].The three-dimensional (3D) coordinates of the protein file was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data bank [28], with a resolution of 2.21 Å.The protein preparation has been carried out by the following steps (i) all water molecules were removed (ii) hydrogen atoms were added to the crystal structure (iii) add Coulomb charges (iv) and previous docked inhibitor (fluconazole) was removed from the protein.The AutoGrid 4.2 [29,30] was used to create affinity grids centered on the active site with 126 ˆ126 ˆ126 grid size with a spacing of 0.42 Å.The rigid protein and flexible ligand dockings were performed by using AutoDock 4.2 with the Lamarckian genetic algorithm applying the following protocol: trials of 100 dockings, energy evaluations of 25,000,000, population size of 200, a mutation rate of 0.02, a crossover rate of 0.8, and an elitism value of 1.The docking results were evaluated by sorting the binding free energy predicted by docking confirmations.The predicted best confirmation binding energy was ´7.15 kcal/mol.The amino acid ARG96 present in the active site of the target protein binds with the ligand by N-H¨¨¨N hydrogen bonding.The protein-ligand interaction complex is given in Figure 5 supporting the symmetry between the observed antifungal activity of (1E)-IPMM and docking results.interfaced with the MGL Tools-1.5.4 package [26].The antifungal target protein of Mycobacterium tuberculosis Cyp51 (PDB ID: 1EA1) in complex with the standard antifungal drug fluconazole, was selected for the present docking analysis [27].The three-dimensional (3D) coordinates of the protein file was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data bank [28], with a resolution of 2.21 Å.The protein preparation has been carried by the following steps (i) all water molecules were removed (ii) hydrogen atoms were added to the crystal structure (iii) add Coulomb charges (iv) and previous docked inhibitor (fluconazole) was removed from the protein.The AutoGrid 4.2 [29,30] was used to create affinity grids centered on the active site with 126 × 126 × 126 grid size with a spacing of 0.42 Å.The rigid protein and flexible ligand dockings were performed by using AutoDock 4.2 with the Lamarckian genetic algorithm applying the following protocol: trials of 100 dockings, energy evaluations of 25,000,000, population size of 200, a mutation rate of 0.02, a crossover rate of 0.8, and an elitism value of 1.The docking results were evaluated by sorting the binding free energy predicted by docking confirmations.The predicted best confirmation binding energy was −7.15 kcal/mol.The amino acid ARG96 present in the active site of the target protein binds with the ligand by N-H•••N hydrogen bonding.The protein-ligand interaction complex is given in Figure 7 supporting the symmetry between the observed antifungal activity of (1E)-IPMM and docking results.

Conclusions
All DFT calculations of (1E)-IPMM have been performed at B3LYP/6-31++G(d,p) level basis set to predict the molecular geometry, vibrational wavenumbers and orbital energy analysis.The detailed vibrational assignments were unambiguously performed on the basis of PED analysis to predict the vibrational modes.The scaled wavenumbers are in good agreement with the experimental results.The decrease in C-H bond length reveals the presence of intra-molecular C-H•••π bond interaction.The NBO analysis also confirms the C-H•••π interactions.The HOMO-LUMO energy reveals the occurrence of the charge transfer interactions within the molecule.The molecular docking results indicated that the title molecule is an effective antifungal agent capable of interacting with its target protein (1EA1).Therefore, the title compound can be considered as an anti-Candida prodrug suitable for further investigations as new antifungal candidate.
1.8e) and π* bonds ("0.3e) of the imidazole ring clearly demonstrate a lesser degree of conjugation leading to dearomatization.The important interactions between filled (donors) Lewis type NBO and empty (acceptors)

Table 2 .
Second order perturbation theory analysis of Fock matrix in natural bond orbital (NBO) basis for (1E)-IPMM.
a E(2) means energy of hyperconjugative interactions; Equation (2); b Energy difference between donor and acceptor i and j NBO orbitals; c F(i,j) is the Fock matrix element between i and j NBO orbitals.
The phenyl ring carbon signals are observed at 125.80, 127.20, 144.64, 129.01, 129.54, 129.61, 130.09, 131.23 and 136.91 ppm.The two signals at 133.07 and 118.73 ppm are corresponding to the imidazole ring carbon atoms.Similarly, imidazole ring proton signals are observed at 6.84, 6.94 and 7.37 ppm.The methylene groups signals were observed at 31.04 and 43.65 ppm for carbons and at 3.36, 4.21 ppm for hydrogens.Their corresponding theoretical chemical shift values appeared at 30.68 and 44.04 ppm ( 13 C) and 3.24 and 4.21 ppm ( 1 H) in NMR spectra.The 13 C spectrum showed a signal at 21.79 ppm for the methyl carbon, which is calculated to be 19.86 ppm.The predicted chemical shift values showed good agreement with the experimental results.
The phenyl ring carbon signals are observed at 125.80, 127.20, 144.64, 129.01, 129.54, 129.61, 130.09, 131.23 and 136.91 ppm.The two signals at 133.07 and 118.73 ppm are corresponding to the imidazole ring carbon atoms.Similarly, imidazole ring proton signals are observed at 6.84, 6.94 and 7.37 ppm.The methylene groups signals were observed at 31.04 and 43.65 ppm for carbons and at 3.36, 4.21 ppm for hydrogens.Their corresponding theoretical chemical shift values appeared at 30.68 and 44.04 ppm ( 13 C) and at 3.24 and 4.21 ppm ( 1 H) in NMR spectra.The 13 C spectrum showed a signal at 21.79 ppm for the methyl carbon, which is calculated to be 19.86 ppm.The predicted chemical shift values showed good agreement with the experimental results.