Synthesis, Structural Investigations, DFT Calculations, and Molecular Docking Studies of Novel 2-(Substituted-Aryloxymethyl)-5-(Pyridin-4-yl)-1, 3, 4-Oxadiazoles: Highly Potential InhA and Cytochrome c Peroxidase Inhibitors

Abstract We report derivatives of 2,5-disubstituted-1,3,4-oxadiazole as powerful anti-TB and antioxidant compounds. Using substituted aryloxy acetic acids (2a–f) and isoniazid (3) in the presence of phosphorus oxychloride, a series of new 2-(substituted-aryloxymethyl)-5-(pyridin-4-yl)-1,3,4-oxadiazoles (4a–f) are synthesized. IR, 1H NMR, and mass spectral data were used to physically and spectroscopically describe the synthesized molecules. Density Functional Theory (DFT) calculations were performed at the DFT/B3LYP level using 6-31 G++ (d, p) to reproduce the structure and geometry. The non-linear visual characteristic of compounds is determined by the first-order hyperpolarizability calculation. To analyze the charge transfer interface between the structures, HOMO and LUMO investigations were used. The in vitro anti-TB and antioxidant activity was carried out. The compound 4d exhibited excellent anti-TB activity with a MIC value of 3.12 µg/ml. The compounds 4b and 4c showed promising antioxidant activity at a concentration of 10 µg/ml with inhibition rates of 68.36% and 69.26% respectively. Furthermore, the docking studies for the newly synthesized molecules were carried out by Auto dock software with proteins InhA (4TZK) and Cytochrome c peroxidase (2X08). All the compounds showed a strong binding affinity for the docked proteins.


Instrumentation
On a Perkin-Elmer FT-IR spectrophotometer (Spectrum ONE), (vmax in per cm, KBr) were documented, 1 H (400 MHz) and 13 C (75 MHz) NMR spectra were documented on a Bruker AMX spectrophotometer using DMSO-d6 as solvent and TMS as an internal standard ESI-mass was recorded on a mass spectrometer equipped with an electrospray ionization source having mass a range of 4000 amu in quadruple and 20,000 amu in Tof.The purity of the compounds was checked using TLC (silica gel 60 G F254 plates) and iodine vapors were utilized as visualizing agents.The elemental analysis was carried out using the elemental analyzer Flash EA1112 series.

Drug likeness profile
The physiochemical characteristics, ADMET, drug-likeness, and bioactivity scores for molecules are critical in their fundamental unique proof as a synthetic lead and serve as a baseline against which integrated compounds are tested during lead development.The http://www.swissadme.ch/ was used to screen the stages of absorption, distribution, metabolism, and excretion of the ligand molecules.The main goal of this study is to estimate the pharmacokinetic characteristics of the substances under investigation.Table 1 shows the expected drug-likeness profiles of freshly synthesized compounds, as well as ADMET characteristics. 40ble 1.Drug-likeness profiles parameters of compounds (4a-f).

DFT calculations
The computations for the selected compounds (4a, 4b, 4c, and 4e) were performed on a personal computer at the DFT level using the GAUSSIAN 09 W application package for energy optimization.The B3LYP functional and the B3LYP/6-31Gþ(d, p) basis set were used to derive the optimum structural parameters for the molecules.At the 6-31 Gþþ (d, p) level, the potential energy surface was investigated.As shown in Figure 2, all the parameters were permitted to relax, and the calculation converged to an optimized shape that corresponded to a true minimum. 41,425.Biological evaluation

Anti-TB activity
The anti-TB testing utilizes a published approach 43,44 of dispersion culture technique with Kirchner's media including Tween-80 against M. tuberculosis (Vaccine strain, H37 RV strain) strain.Add 0.5 ml sterile normal bovine serum to the sterile Kichner dispersion medium (4.5 ml) spread in the borosilicate test tube, inactivated by heating at 56 C for 30 min (150 Â 20 mm).The test samples were prepared in 100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.8 mg/ml concentrations in DMF.The inoculums were 0.1 ml of a standard culture of M. tuberculosis (ATCC 27294) containing 106 bacilli/ml.The tubes were tested for the presence or absence of test organism growth after an eight-day incubation period at 37 C.The endpoint will be the lowest concentration at which no growth is seen.The MIC was measured for all the compounds and the findings were compared with reference drug Isoniazid, Pyrazinamide, Streptomycin and a control tube containing no drug.
2.5.2.Antioxidant activity 2.5.2.1.1, 1-Diphenyl-2-picryl-hydrazil (DPPH) radical scavenging activity (RSA).The test compounds' RSA was performed in methanolic solution at concentrations of 10, 20, 40, 60, 80, and 100 mg/ml with freshly made DPPH solution (0.004% w/v) and compared with standard using the stated technique. 31,45All of the analyses were done in triplicates on average.The results are reported as a percentage of the difference between the absorbance of DPPH solution in the presence of test compounds and the absorption of DPPH solution in the absence of test compounds at 517 nm using Shimadzu UV-Visible spectrophotometer (Serial number: A114547).The DPPH free radical's percentage (%) scavenging activity was calculated using the equation:

Molecular docking studies
The primary protein structure of InhA (PDB code: 4TZK) and Cytochrome c peroxidase (PDB code: 2 Â 08) was obtained from Protein Data Bank.The 3D structure of Butylated hydroxyanisole (BHA) was obtained from PubChem (PubChem CID: 24667).Chemdraw was used to draw the newly synthesized compounds, which were then converted into 3D structures by optimizing partial charges.A molecular docking study of the 6 newly designed molecules, along with isoniazid and Butylated hydroxyanisole (BHA) the reference standard was performed using PyRX-0.9.2 software.The ligands were blindly docked using the AutoDock Vina program to identify the compound with the best binding properties.The grid box was set to the XYZ coordinates as 69.695, 50.504, and 63.187 with the centers of XYZ in the positions of À11.834, À5.496, and 7.491, respectively.The nine conformations of each compound along with the Vina score and RMSD value were used to screen for the best binding interaction.[48] 3. Results and discussion

Chemistry
The synthesis of 2-(substituted-aryloxymethyl)-5-(pyridin-4-yl)-1, 3, 4-oxadiazoles (4a-f) were represented in the Scheme 1.All the compounds (4a-f) were prepared by the reaction of substituted-aryloxy acetic acids (2a-f) and isoniazid (3) in the presence of phosphorous oxychloride in reflux condition for 6 h.The reaction was monitored by TLC (using mobile phase Chloroform: Methanol) and obtained products in good yield.Furthermore, the chemical structures of 4a-f were analyzed by spectral techniques like IR, NMR and mass spectrometry.In IR spectra, the appearance of two peaks at 1609 and 1583 cm À1 is responsible for -C ¼ N-C-stretching and a peak at 1099 cm À1 for -C-O-Cstretching of oxadiazole.The 1 H NMR spectra of compound 2a revealed signals at d 8.94 and 7.96 as two doublets for the 4 CH protons of the pyridine ring.
The two doublets at d7.17 and 6.91 for the 4 CH protons of the phenyl ring.Furthermore, two singlets appeared at d 5.43 and 2.24 due to the presence of -OCH2 and -CH3 functions respectively.The mass spectra of compound 4a illustrated the molecular ion peak at M. þ at 267 (27%) which is the molecular weight of the compound.The above spectral data indicate the formation of compound 4a from compounds 2a and 3.

Molecular geometry
The molecular structure of compounds (4a, 4b, 4c, and 4e), as well as the atom numbering, derived using the Gaussian 09 and GAUSSVIEW tools.Figure 2 shows the optimized structural parameters obtained using DFT (B3LYP) using the 6-31 Gþþ (d,p) basis set.

Molecular electrostatic potential (MEP)
The molecular electrostatic potential mapped surfaces show the charge distributions of molecules three-dimensionally which give the clear and special signatures of the interactions of the compounds (4a, 4b, 4c, and 4e).
The molecular electrostatic potential is related to the electronic density and is a very useful descriptor for determining sites for electrophilic attack and nucleophilic reactions as well as hydrogen-bonding interactions.
The MEP mapped surface of the compounds (4a, 4b, 4c, and 4e) was calculated by DFT/B3LYP at 6-31 Gþþ(d,p) basis set and the MEP surface are plotted in Figure 3.In Figure 3, red, blue and green colors represent regions of the most electronegative, most electropositive electrostatic and zero potential, respectively.

Frontier molecular orbitals (FMOs)
The HOMO and LUMO (Highest Occupied Molecular Orbital and Lowest Unoccupied Molecular Orbital) are well-known as the major orbitals involved in chemical reactions, and they are referred to as the FMOs (Frontier Molecule Orbitals).The energy gap formed between HOMO and LUMO is a significant metric for evaluating molecular electrical transport capabilities and indicates molecular chemical stability.This energy gap can also be used to measure chemical reactivity, kinetic stability, polarizability, chemical hardness and softness, aromaticity, and electronegativity.The HOMO and LUMO orbitals, as well as their transition states, were calculated using the DFT/B3LYP method and the 6-31 Gþ (d,p) basis set for compounds (4a, 4b, 4c, and 4e), as shown in Figure 4.It is found that the filled p-orbital (HOMO) is mostly located on the Benzene ring of the compounds, while the unfilled anti p-orbital (LUMO) is on the Pyridine ring.When electron transitions take place from HOMO to LUMO electrons are mainly transferred from the Benzene ring to the Pyridine ring.

Global reactivity descriptors
The global chemical reactivity descriptors of molecules such as hardness (g), chemical potential (l), softness (S), electronegativity (v) and electrophilicity index (x) have been developed using HOMO and LUMO energy values for a molecule.These are derived using the formulae below based on E HOMO and E LUMO, and the calculated values are given in Table 2. Global hardness and softness are important properties to measure molecular stability and reactivity.The molecules which have more HOMO-LUMO energy band gap are called 'hard molecules', whereas the molecules with a small HOMO-LUMO energy band gap are called 'soft molecules'.Therefore, the molecules with the least HOMO-LUMO gap become more reactive.Evaluating the values of the Global hardness in Table 2 shows that compound 4c has the greatest.This means that compound 4c has the largest potential chemical resistance to change the number of electrons among the other molecules.It can be noted that the hardness of the molecules follows the order 4c > 4b > 4e > 4a.Also, it can be seen in Table 2 that 4a is the compound that displays the greater reactivity concerning others as a result of the high value of global softness.The softness of the molecules follows the order 4a > 4e > 4b > 4b.In addition, a large energy gap implies high stability for the molecule.Table 2 shows that the energy gap of compound 4c has the highest value (4.3585 eV) therefore it is less reactive than other structures.Also, the energy gap of structure 4a has the lowest value (4.0336 eV), which indicates that it is more reactive.It implies that the electronic transfer in molecule 4a is easier.This is due to the existence of a methyl group on the benzene ring reducing the energy of the HOMO.

Anti-TB assay
The anti-TB activity results for the synthesized compounds are presented in Table 3. Compound 4d, which had a MIC of 3.125 mg/ml and was equally efficient against M. tuberculosis bacteria as  the conventional drug Pyrazinamide, was found to be the most active of the studied compounds.
The results indicated that the nitro substitution at the aromatic ring is very much needed for the anti-TB activity.While the remaining compounds displayed low to moderate anti-TB activity, compounds 4b and 4c showed comparable anti-TB activity with a MIC value of 6.25 mg/ml.

Antioxidant activity
Amplified free radicals in the body are harmful to human life.The ability of antioxidants to combat free radical damage to food and biological systems is crucial.All of the synthesized compounds were tested for their in vitro antioxidant activity using the DPPH radical scavenging and the Metal-Chelating action (Fe 2þ ) techniques.
3.3.2.1.1, 1-Diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity (RSA).Free radicals are scavenged by antioxidants by providing DPPH electrons.The odd electron in DPPH makes it a static-free radical with challenging retention at 517 nm.The consumption lowers stoichiometrically as the amount of absorbing electrons or hydrogen particles decreases when the electrons are combined to saturation.This process is frequently used to examine a compound's capacity to operate as a free radical scavenger.The potential of synthetic derivatives to scavenge the DPPH radical was assessed.Compared to the reference BHA (Butylated hydroxyanisole), the actions were different.The RSA results showed that at a concentration of 10 mg/ml, the compounds 4b (68.3 ± 0.63) and 4c (69.2 ± 0.13) proved to be superior.The remaining compounds displayed minimal activity (Table 4).

Molecular docking studies
The conformation with the least binding energy and most stability based on docking analysis was identified.Bioinformatics analysis revealed that compound 4d had the best binding interactions with InhA.Compound 4d bound to carbohydrazide of InhA through three hydrogen bonds with Ile194, Ser94 and Ile21 amino acids (Figure 5).These binding patterns are similar to those of antioxidant molecules and the molecular docking study for the standard drug InhA is also illustrated in Figure 5.
Figure 5 shows that compound 4d was stabilized inside the binding pocket of InhA with a very promising binding score of À9.33 kcal/mol, compared to that of standard Isoniazid (-8.85 kcal/mol).The standard Isoniazid formed two hydrogen bonding with Ile194, and Gly192 with distances of 2.75, and 2.60 Å respectively but 4d formed only three hydrogen bonds with Ile194, Ser94 and Ile21 with distances of 2.98, 2.73, and 2.54 Å respectively.
The conformation with the least binding energy and most stability based on docking analysis was identified.Bioinformatics analysis revealed that compound 4c had the best binding interactions with BHA.Compound 4c bound to the c-heme edge of cytochrome c peroxidase through hydrogen bonds with amino acids (Figure 6).These binding patterns are similar to those of antioxidant molecules and the molecular docking study for the standard drug BHA is also illustrated in Figure 6.
Figure 6 shows that compound 4c was stabilized inside the binding pocket of BHA with a very promising binding score of À9.21 kcal/mol, compared to that of BHA (À8.81 kcal/mol).

Structure-activity relationship studies
The aforementioned synthetic compounds 4(a-f) showed comparable biological activity to conventional medicines.The anti-TB activity of synthesized compounds revealed that compound 4d showed outstanding activity against tested microbial pathogens, which was comparable to the standard drug.Studies on docking have supported this.The SAR study indicates that the compound containing strong electron-withdrawing groups like -NO 2 is necessary for anti-TB activity.Comparing compounds 4(a-f) to standard BHA (68.35%) at 10 lg/ml, compounds 4(a-f) showed antioxidant activity in the range of 52.74 to 69.25%.When compared to normal BHA, the compounds with halogen substitution provide high antioxidant activity, according to the results of the radical scavenging activity.
The MIC method was used to assess the compounds (4a-f) for their anti-TB activity against M. tuberculosis (ATCC 27294).Comparing its antimicrobial efficacy to that of the reference drugs isoniazid, pyrazinamide, and streptomycin, compound 4d showed good results.The results of docking studies supported the M. tuberculosis inhibition.Results showed that compounds 4b and 4c have excellent antioxidant activity when compared to the reference drug BHA.Furthermore, according to the docking studies, compounds 4b and 4c displayed an excellent dock for the protein cytochrome c peroxidase.

Figure 2 .
Figure 2. The geometrical optimized molecular structures with atomic labeling.

Percentageð%Þ
DPPH radical scavenging ¼ Absorbance of the control reaction ÀAbsorbance of the sample or standard drug=Absorbance of the control reaction

Figure 6 .
Figure 6.Molecular docking interaction representation of ligand 4c with 4X08.(A) 3D representation protein 4X08.(B) 3D interaction of ligand 4c with protein 4X08.(C) 2D representation of ligand 4c with protein 4X08.There are Van Der Waals forces of an attraction along with water hydrogen bonds.

Table 2 .
Quantum chemical descriptors of studied compounds.

Table 3 .
Anti-TB activity results of the novel compounds (4a-f) against M. tuberculosis.

Table 4 .
Anti-oxidant activity (by RSA method) results of the novel compounds (4a-f).All results expressed are the mean of three individual replicates (n ¼ 3 ± SD). a