Molecular Spectroscopy and Molecular Docking Studies on (E)-1-(4-Bromobenzylidene) Thiourea

Equimolar amount of 4-bromobenzaldehyde and thiourea were dissolved in 30 ml of absolute ethanol. The mixture was shaken to make homogenous solution. Few drops of catalyst acetic acid was added to increase the rate of reaction. The content was refluxed at 90°C for 3 hours. The completion of the reaction was monitored by thin layer chromatography. After the reaction was completed, the content was cooled the mixture was poured into water. The solid product obtained was filtered and purified using absolute ethanol.

The FT-IR spectrum of the synthesized BBTU was measured in the 4000–400 cm⁻¹ region at the spectral resolution of 4 cm⁻¹ using on SHIMADZU FT-IR affinity Spectrophotometer (KBr pellet technique) in Faculty of Marine Biology, Annamalai University, Parangipettai. The FT-Raman spectrum was recorded on BRUKER: RFS27 spectrometer operating at laser 100 mW in the spectral range of 4000–50 cm⁻¹. FT-Raman spectral measurements were carried out from Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology (IIT), Chennai.

Melting point = 160˚, Yield = 85%

After the determination of the low-energy structures belonging to the compound at HF level calculations. Further, the different positions of the atoms are surveyed by the detailed potential energy surface (PES) scans at B3LYP density functional method with 6-31G(d,p) basis set in the ground state to obtain the most stable conformer of the BBTU molecule. In this PES scan process, the potential energy surface is built by varying the C3‒C4‒C12‒N13 dihedral angle from 0˚-360˚ in every 10˚ rotation, while all the other geometrical parameters have been simultaneously relaxed. From Figure 1 the title compound with the planar conformation exhibits global minimum energy conformers at 0˚, 180˚ and 360˚ (-3388.47966 hartree), respectively. Whereas, the global maximum energy conformers were identified at 90˚ and 270˚ (-3388.465013 and -3388.465002 hartree) in PES. This is due to constrain appeared between the phenyl ring and thiourea moiety. In the stable conformer, there is no ring strain and planarity disturbance were observed in the title molecule. It is worth to discuss, that the theoretical findings supposed from this calculation level are more reliable and taken for further discussions. The energies of the various possible conformers of BBTU listed in the Table 1. The optimized structure of BBTU was shown in Figure 2.

Figure 1.The potential energy surface scan curve of BBTU.

Figure 2.The optimized molecular structure of BBTU.

The observed FT-IR, FT-Raman, computed harmonic wavenumber along with intensities and their assignments for the present compound are illustrated in Table 2. The comparison of simulated and recorded FT-IR and FT-Raman spectra were shown in Figure 3 and Figure 4, respectively. In general, harmonic frequencies overestimate the experimental values mainly due to anharmonicity and therefore empirical scaling procedures are used to compare the experimental and theoretical spectra. The theoretical vibrational wavenumber obtained for compound BBTU is interpreted by means of Potential energy distribution (PED %) calculations using VEDA4 software. The normal modes assignment of the theoretical frequencies is visualized and substantiated with the help of the GaussView 5.0 visualization program. The synthesized BBTU consists of 19 atoms and hence has 51 normal modes of vibrations. The molecule BBTU belongs to C₁ symmetry.

Figure 3.The experimental and theoretical FT-IR spectra of BBTU.

Figure 4.The experimental and theoretical FT-Raman spectra of BBTU.

The νN-H stretching vibrations normally appear in the range of 3500-3300 cm^-128. The experimental wavenumber of 3419 cm^-1 in the FT-IR spectrum of BBTU is assigned for N-H stretching vibration. The theoretical N-H stretching wavenumber are visualized at 3579 cm^-1 for asymmetric and at 3439 cm^-1 for symmetric stretching wavenumber. Both the theoretical and experimental vibrational frequencies are pure vibrations with PED of 100%. The N-H scissoring vibrational mode appears in the 1638-1575 cm^–1 region with strong to very strong IR intensity, but exhibits weak or no Raman scattering. The NH₂ scissoring deformation is calculated at 1566 cm^–1 and the NH₂ wagging mode is calculated at 357 cm^–1.

Bromine compounds absorb strongly in the region 650-485 cm^-1 due to the C-Br stretching vibrations ²⁹. The theoretical wavenumbers of C–Br stretching vibration coupled with other group vibrations. According to PED, there is no pure C–Br band vibration. In our present study, the C-Br vibration was calculated at 659 cm^-1 along with CCC and CNC bending vibrations. The 90% of PED contribution confirms the corresponding wavenumber. The C–Br out-of-plane bending and in-plane bending vibrations are assigned to the Raman bands at 298 and 218 cm⁻¹, respectively.

The C=S is less polar than the C=O group and encountered great difficulty due to vibrational mixings between C=S stretching the other vibrational modes. The C=S group is found over the wide range of 1035-245 cm^–1 whereas, C–S stretching vibration results strong bands in Raman spectra which are normally easy to identify and is difficult in Infrared region ²⁹. In consequence, the band is not intense, and it falls at lower frequencies, where it is much more susceptible to coupling effects and identification is therefore difficult and uncertain ³⁰. In our present study the C=S band is observed in FT-IR at 740 cm^–1 and in FT-Raman spectrum at 733 cm^–1 as strong bands. Its theoretical wavenumber calculated at 750 cm^–1 with 67% of PED contribution.

The C=N stretching skeletal bands are observed in the range 1650–1550 cm^–131. For BBTU, the C=N observed at 1658 cm^–1 in FT-IR spectrum and at 1636 cm^–1 in FT-Raman spectrum. The DFT prediction for the corresponding mode is calculated at 1611 cm^–1 with 72% contribution in PED. The C-N stretching vibrations are observed in combination with other vibration modes and are dispersed in the range of 1062-1407 cm^{–1 32}. In this study, the C15-N14 and C15-N13 were observed at 1334 and 1207 cm^–1 in FT-IR spectrum, besides with bending vibrations of βCNH. The corresponding theoretical wavenumber for C-N vibrations are calculated at 1324 and 1239 cm^–1.

The aromatic ring structure shows the presence of C−H stretching vibrations in the region 3100–3000 cm^–1, which is the characteristic region of C−H stretching vibrations ³³. The wavenumbers computed in the region 3087–3054 cm^–1 is assigned to C−H stretching vibrations of BBTU. The strong band observed at 3062 cm^–1 in FT-IR spectrum was assigned to C−H stretching vibrations of the phenyl ring. From Table 2, it is evident that the calculated wavenumber at 3087, 3084, 3074 and 3054 cm^–1 are pure modes, they almost contributing more than 90% of PED. The C−H in-plane ring bending vibrations are normally occurs as a number of strong to weak intensity bands in the region 1000–1300 cm^–134. The bands observed at 1166 cm^–1 in FT-IR spectrum and at 1093 cm^–1 inFT-Raman spectrum are assigned for the C−H in-plane bending vibrations of the aromatic ring. The theoretical wavenumber at 1320, 1193 and 1129 cm^–1 are in good agreement with the experimental values. The C−H out-of-plane bending vibration is expected in the range 750–1000 cm^–1 in the FT-IR spectra of substituted benzenes ³⁵. The position of C−H out-of-plane vibration is determined almost extensively by the relative position of the substituents and is independent of nature ³⁶. The vibrational bands observed at 933 and 812 cm^–1 in FT-IR spectrum were assigned for the C−H out-of-plane bending vibrations of the phenyl ring.

The ring stretching vibrations are very much important in the spectrum of aromatic compounds and are highly characteristic of the aromatic ring itself. In general, the C−C bands are of variable intensity and observed at 1625–1590, 1590–1575, 1540–1470, 1460–1430 and1380–1280 cm^–1 from the frequency ranges given by Varsanyi for the five bands in the finger print region ³⁶. In BBTU, the strong vibrational bands at 1433, 1373, 1278 cm^–1 in FT-IR spectrum and at 1383 cm^–1 in FT-Raman spectrum were assigned for C-C vibrations of the phenyl ring. The C-C theoretical wavenumber at 1536, 1459, 1377 and 1276 cm^–1 have shown good agreement with the experimental wavenumber. The phenyl ring breathing mode and trigonal bending vibrations are calculated at 1039 and 987 cm^–1. The weak band at 1028 cm^–1 assigned to the phenyl ring breathing mode vibration.

The natural bond orbital (NBO) calculations were performed in order to investigate the intra- and inter-molecular bonding interactions among bonds in BBTU. The intra-molecular charge transfer and hyperconjugative interactions of BBTU were also analyzed. The NBO analysis gives information about energy of the interactions between filled Lewis type NBOs and empty non-Lewis type NBOs and estimating their energy by second order perturbation theory. A filled bonding or lone pair orbital can act as a donor and a vacant anti-bonding or lone pair orbital can act as an acceptor. These intra-molecular interactions between orbitals can strengthen and weaken bonds in the molecular system. The interaction between a lone pair donor and anti-bonding acceptor orbital will weaken the bond associated with the anti-bonding orbital. In contrast, the interaction with a bonding pair as the acceptor orbital will strengthen the bond ^{37, 38, 39}. The second order Fock matrix was performed to evaluate the donor (i) – acceptor (j) interactions in the NBO basis. The stabilization energies of the title compound have been investigated using second order perturbation theory. The stabilization energy E⁽²⁾ associated with each donor NBO(i) and acceptor NBO(j) delocalization is calculated from the second order perturbation approach ⁴⁰ by the following equation.

(2)

Where, qi is the donor orbital occupancy, εi and εj are diagonal elements (orbital energies) and F(i, j) is off diagonal NBO Fock matrix elements.

NBO analysis was performed by the B3LYP level of theory with 6-31G(d,p) basis set for the molecule BBTU. NBO theory allows the assignment of hybridization of atomic lone pairs and of the atoms involved in bond orbitals. These are important data in spectral interpretation as the frequency ordering is related to the bond hybrid composition. The second order perturbation energy values E⁽²⁾ collected in Table 3 reveals the important interactions between the Lewis and non-Lewis type NBO orbitals of BBTU molecular system consisting para bromo-substituted phenyl ring fused with thiourea. In the studied molecule, the strong intra-molecular hyperconjugative interactions were observed between lone pair electrons of N, S with σ and π anti-bonding electrons with C‒C, C‒N and C‒S resulting in intra-molecular charge transfer (ICT) causing stabilization of the BBTU molecular system. Enormous stabilization energy of 332.17 KJ/mol was found for LP(1)N14→C15-S16 interactions, this larger stabilization show the larger delocalization of electrons in the title molecule. Lone pair electrons form S16 distributed stabilization energy to σ* (N13‒C15), (N14‒C15) leads to the stabilization of 52.93, 46.36 KJ/mol for BBTU. The magnitude of charges transferred from bonding lone pair of N13 with ED (1.9041) to σ* (C12‒H17), (C15‒S16) shows 43.43 and 46.19 KJ/mol stabilization energy. The π(C12‒ N13)→π*(C4‒C5), (C15‒S16) leads to the stabilization of system with energy of 34.48 KJ/mol to phenyl ring system and 98.99 KJ/mol to C=S of the thiourea moiety.

The electron density of the six conjugated double bonds of the phenyl ring (~1.66e) clearly demonstrates the strong delocalization through the interactions π(C1‒C6)→π*(C2‒ C3), (C4‒C5), π(C2‒C3)→π*(C1‒C6), (C4‒C5) and π(C4‒C5)→π*(C1‒C6), (C2‒C3) with the stabilization energies of about 72.38, 83.18, 93.26, 77.7 and 87.95, 82.84 KJ/mol, respectively. The donor LP(3) Br11→ π*(C1‒C6) transfers the hyperconjugative interaction energy of about 43.51 KJ/mol to acceptor anti-bond. The bonding π(C4‒C5)→anti-bonding π*(C12‒N13) interaction leads to the stabilization of 86.23 KJ/mol to the thiourea side chain of the molecular system. These types of intra-molecular stability of BBTU are responsible for pharmaceutical and biological properties. Hence, the BBTU structure is stabilized by these orbital interactions.

The first order hyperpolarizabilities (β₀, α₀ and Δ_α) of BBTU is calculated using DFT/B3LYP/6-31G(d,p) basis set, based on the finite-field approach. In the presence of an applied electric field, the energy of a system is a function of the electric field. First hyperpolarizability is a third rank tensor that can be described by a 3x3x3 matrix. The 27 components of the 3D matrix can be reduced to 10 components due to Kleinman symmetry ⁴¹. It can be given in the lower tetrahedral format. It is obvious that the lower part of the 3x3x3 matrix is a tetrahedral. The components of β are defined as the coefficients in the Taylor series expansion of the energy in the external electric field. When the external electric field is weak and homogeneous, this expansion becomes:

(3)

Where E⁰ is the energy of the unperturbed molecules, F_α is the field at the origin, and µ_α, α_αβand β_αβγ are the components of the dipole moment, polarizability and the first hyperpolarizabilities, respectively. The total static dipole moment μ, the mean polarizability α0, the anisotropy of polarizability Δ_α and the mean first hyperpolarizability β₀, using the x, y, z components ⁴² are defined as

(4)

(5) (6)

(7)

Many organic molecules, containing conjugated π electrons are characterized by large values of molecular first hyperpolarizabilities, were analyzed by means of vibrational spectroscopy ^{43, 44, 45, 46}. The intra molecular charge transfer from the donor to acceptor group through a single-double bond conjugated path can induce large variations of both the molecular dipole moment and the molecular polarizability, making IR and Raman activity strong at the same time ⁴⁷.

Theoretical investigation plays an important role in understanding the structure – property relationship, which is able to assist in designing novel NLO materials. It is well known that the higher values of dipole moment, molecular polarizability and hyperpolarizability are important for more active NLO properties. It is evident from Table 4, the molecular dipole moment (μ), molecular polarizability and hyperpolarizability are calculated about 1.2315 (D), 3.78 and 23.70 x10^-30esu, respectively. The β₀ value of the title compound is sixtyfour times greater than that of urea. Hence, our title molecule is an interesting object for Non-linear Optics.

Frontier molecular orbitals are nothing but, the highest occupied molecular orbitals and lowest unoccupied molecular orbitals of the molecule. The LUMO as an electron acceptor represents the ability to obtain an electron; donor represents the ability to donate an electron. The energy gap of HOMO-LUMO explains the eventual charge transfer interaction within the molecule, which influences the biological activity of the molecule. The positive and negative phases are represented in red and green colour respectively. The energy of the two important FMOs such as the highest occupied molecular orbitals (HOMO), the lowest unoccupied molecular orbitals (LUMO) have been calculated. The frontier molecular orbitals of the title molecule are mapped in Figure 5.

Figure 5.The frontier molecular orbitals of BBTU

As seen from Figure 5, the HOMO is localized over thiourea side chain. LUMO is localized over entire region of the title molecule. The analysis of the wave function indicates that the electron absorption corresponds to the transition from the ground to the first excited state and is mainly described by one electron excitation from the HOMO to LUMO. The HOMO→LUMO transition implied that an electron density transfer from thiourea to bromo substituted phenyl ring. The values of the energy separation between the HOMO→LUMO is identified as 3.2860 eV. This HOMO–LUMO gap leads to high excitation energies, a good stability for BBTU. In addition, it is noted that there is an overlap between the HOMO and LUMO, which is necessary for obtaining large second-order response ⁴⁹. The energies of the FMOs are used for the determination of global reactivity descriptors. The frontier molecular orbital energies are tabulated in the Table 5.

MEP and electrostatic potential are useful quantities to illustrate the charge distributions of molecules and used to visualize variably charged regions of a molecule. Therefore, the charge distributions can give information about how the molecules interact with another molecule. The reactive behavior of the BBTU molecule is visualized with the help of three dimensional MEP surface. The molecular electrostatic potential and the contour map of the BBTU are shown in Figure 6. The MEP surface of the molecule under investigation is constructed by using B3LYP/ 6-31G(d,p) method. MEP surface describes the charge distribution in the molecule and helps in predicting the sites for nucleophilic and electrophilic attack in the molecule. The region of negative charge is pictured out by red color, which indicates the electrophilic attack sites of our molecule. The red region localized over the ‒CH=N imine linkage and in core region of phenyl ring of the title molecule. The region of positive charge is pictured out by blue color, which indicates the nucleophilic attack sites were localized over NH₂ group of the thiourea moiety and around bromine atom attached with phenyl ring of the BBTU. The green color corresponds to a potential half way between the red and blue regions, also represents the neutral charge. The contour map of BBTU describes the electron density mapped surface of the BBTU.

Figure 6.The MEP mapped surface and contour map of BBTU.

According to Koopmans theorem ⁵⁰, the first ionization energy of the molecular system is equal to the negative of the orbital energy of the HOMO and electron affinity as the energy of the LUMO of respective system. In the framework of energy gap analysis, the ionization energy and electron affinity can be expressed as I = −E_HOMO and A = −E_LUMO by HOMO-LUMO orbital energies. The combination of electron affinity and ionization energy gives the electronic chemical potential, μ = 1/2(E_HOMO+E_LUMO). Global hardness expressed by η = 1/2(E_LUMO-E_HOMO), global electrophilicity index by ω = μ²/2η and softness s = 1/η ⁵¹. Chemical potential and hardness are the better descriptor of global chemical reactivity. For the title compound, E_HOMO = −6.022964 eV, E_LUMO = −2.736882 eV, Energy gap = HOMO– LUMO = 3.286082 eV, Ionization potential I = −6.022964, Electron affinity A = −2.736882 global hardness η = 1.643041, chemical potential μ = 4.379923, global electrophilicity ω = μ²/2η = 5.83787182eV and softness ζ=1/η = 0.60862754. The electrophilicity indexmeasures the stabilization in energy when the system acquires an additional electronic charge from the environment. Electrophilicity encloses both the ability of an electrophile to gain additional electronic charge and the resistance of the system to exchange electronic charge with the environment.

Protein–ligand interactions play a critical role in the distribution, metabolism and transport of small molecules in biological systems and processes ⁵². AutoDock is a suite of automated docking tools designed to predict how small molecules, such as substrates or drug candidates, bind to a receptor of known three-dimensional structure. With the aim to investigate the binding mode, molecular modeling study was performed using AutoDock Tools for docking ⁵³. 5NI was chosen to be docked into the active site of different receptors 1JIJ and 3U2D of antimicrobial proteins which was obtained from Protein Data Bank (PDB). Ligands were docked into the functional sites of the respective proteins individually and the docking energy was monitored to achieve a minimum value. AutoDock results indicate the binding position and bound conformation of the peptide, together with a rough estimate of its interaction. Docked conformation which had the minimum binding energy was selected to analyze the mode of binding. The Lamarckian genetic algorithm ⁵⁴ was applied to search for protein-ligand interaction with and for visualization performed using PyMOL ⁵⁵.

The objective of present study was to evaluate the drug activity and binding affinity of BBTU with a target protein using the program of AutoDock. The resulting free energy of binding and inhibition constant of BBTU with protein 3U2D and IJIJ observed at the end of docking simulation is shown in Table 6. Pictorial representations of best possible binding sites of ampicillin and BBTU with 3U2D receptor were shown in Figure 7. From Figure 8 it is shown that BBTU ligand was focused in the groove of the protein and hydrogen bonding interactions are highlighted. The BBTU was docked deeply with the binding pocket of 3U2D forming two hydrogen bonds with SER55 and ILE51. The NH₂ group of BBTU forms hydrogen bonding with the distances of 2.1 and 2.2 Å with oxygen atoms of the residues SER55 and ILE51, respectively. The best binding score of BBTU was obtained as -5.53kcal/mol and the estimated inhibition constant of BBTU was found to be 87.90 μM against DNA gyrase. Hydrophobic and van der Waals interactions were also detected in the lead molecule of BBTU with DNA gyrase are with the residues of PRO87, ILE 175, VAL52, GLY83 and GLY85. The pi-anion and pi-alkyl interactions are identified with the residues of GLU58 and ILE86 from the 2D model interactions. These type of π-interactions are due to the electron density in the core of the phenyl ring are clearly demonstrated by the contour map of the BBTU. This result indicates the antibacterial activity of the title compound.

Figure 7.The Binding pose of Ampicillin (a) and BBTU (b) with 3U2D receptor.

Figure 8.The binding poses and hydrogen bonding interactions of BBTU with 3U2D.