Synthesis of Oxadiazole-Based-Thiourea, Evaluation of Their β-Glucuronidase Inhibitory Potential, and Molecular Docking Study

Abstract Oxadiazole-based-thiourea analogs (1–18) were synthesized and evaluated for their β-glucuronidase inhibitory activity. All analogs showed extensive β-glucuronidase inhibitory potential ranging between (IC50 = 2.20 ± 0.01 µM) to (IC50 = 52.25 ± 1.20 µM) by comparing with the standard D-Saccharic acid 1,4-lactone (IC50 = 48.30 ± 1.25 µM). Among the series, analogue 7 having IC50 value (IC50 = 2.20 ± 0.01 µM)), 16 (IC50 = 4.40 ± 0.10 µM) and 4 (IC50 = 9.20 ± 0.20 µM) showed an excellent inhibitory potential greater than that of standard D-saccharic acid-1,4- lactone. Analogue 7 (IC50 = 2.20 ± 0.01 µM) of this series having flouro group at ortho-position of phenyl ring was recognized to be most active analogue due to involvement of flouro group in hydrogen bonding with the enzyme active site. In addition, all synthesized derivatives characterized by using High Resolution Mass Spectrometry (HR-MS), 13C-NMR and Nuclear Magnetic Resonance Spectroscopy (1H-NMR proton). For better understanding of binding mode of interactions of these active analogs molecular docking study was performed. Structure Activity relationship (SAR) was also discussed.


Introduction
The b-Glucuronidase is member of enzyme of glycosidase, which catalyzes the breakup of oxygen bond through b-glucuronidase. It is found in Clostridia, Bacteroides, Escherichia, and Peptostreptococcus genera. 1 In human body, these enzymes are present in organs such as bile, spleen, kidney, and serum. 2 In varied pathological conditions, b-glucuronidase over expression causes numerous ailments such as colon, lungs, renal carcinoma, leukemia, urinary tract infections, HIV, renal diseases, and pancreatic cancer [3][4][5][6][7] . In inflammatory joint disease, this enzyme has been released in the synovial fluid just as rheumatoid arthritis. 8 Inhibition of b-glucuronidase enzyme is very critical in colon cancer because of a greater intestinal level of this enzyme consistent with the occurrence of colon carcinoma. 9 Azoles are heterocyclic compounds used as anti-fungal, also show inhibitory activity against b-Glucuronidase. 10 Oxadiazole is an essential class of heterocyclic compounds mainly used as a scaffold in lead molecules and different drugs. Most of the researchers use 1,3,4-oxadiazole and 1,2,3-oxadiazole as effective bioisosteres to remove esters and amide functionalities from compounds which are resisting to enzyme-catalyzed hydrolysis, thus advancing their biological and pharmacokinetic properties. 11,12 Oxadiazole based derivatives showed in-vitro anti-cancer activity against prostate, pancreas, and breast cancer, 9 as well as anti-proliferative activity against gastric cell lines. 13 The derivatives of oxadiazole also showed numerous activity such as anti-inflammatory, 14,15 antimalarial, anti-TB, anti-schistosomiasis, 16 anti-convulsant, 17,18 anti-mitotic, 19,20 anti-diarrheal, 21 anti-emetic and acting as hypnotic, sedative and as analgesic agents. 22 Oxadiazole analogs also showed an herbicidal effect without affecting the product. 23 Literature survey revealed the biological importance of oxadiazole 24 as well as thiourea analogues with other moieties. Keeping in view the importance, here is in this study we have plan to synthesize hybrid analogues of oxadiazole-based-thiourea, their b-glucuronidase inhibition, SAR, and molecular docking studies.
In the continuation of our work on the b-glucuronidase inhibitors, we have synthesized various classes of compounds like benzimidazole, benzothiazole, indole, oxadiazole, and oxathiazole 25 as shown in Figure 1. From our previous experiences, we synthesized oxadiazole derivatives as potent inhibitors of b-glucuronidase.
All analogs showed good inhibition against b-glucuronidase if compared with the standard D-Saccharic acid 1,4-lactone (IC 50 ¼ 48.30 ± 1.25 mM). Structure activity relationship (SAR) has been established for some potent compounds mainly based on bringing different substituents on phenyl part of isothiocyanate Figure 2.
Looking to the general structure of oxadiazole based thiourea newly synthesized scaffolds (1-18) analogue 7 (IC 50 ¼ 2.20 ± 0.01 mM) of this series having flouro group at ortho-position of phenyl ring was recognized to be most active analogue, if compared with the standard Figure 3.   Same pattern of inhibition was also observed in chloro substituted analogs, like compound 10, a 2-chloro substituted analog (IC 50 ¼ 9.40 ± 0.20 lM), compound 12, a 4-chloro substituted analog (IC 50 ¼ 13.97 ± 0.20 lM) and 11, a 3-chloro substituted analog (IC 50 ¼ 29.35 ± 0.40 lM). The little bit difference in the potential of these analogs seems due to difference in the position of substituent ( Figure 4).
It was concluded from the whole study that ortho-substituted analog showed more potency than para and meta and para substituted analogs showed more potency than meta-substituted analog.

Docking study
The IC 50 values of oxadiazole-based-thiourea derivatives as b-glucuronidase inhibitors are shown in (Table 1). The inhibitory potential of all synthesized compounds is mainly related to the number, type and position of the substituents in the substitute group R of the synthesized derivatives (Table 1). For a better understanding of enzyme inhibition of the synthesized compounds, molecular docking study has been carried out to know the established binding interaction of the three selected synthesized compounds 7, 8 and 9. These compounds differ by the substitution of the fluorine group in the aromatic ring (Scheme 2). Compound 9 shows higher activity than 7, which shows higher activity than 8. Table 2 summarized the binding energies calculated, established intermolecular hydrogen bonding number between synthesized compounds (7, 8 and 9) and b-glucuronidase active site res The complexes formed between the docked selected compounds (7, 8 and 9) and amino acids of the active binding site of b-glucuronidase displayed negative bending energies, which indicates that the inhibition of b-glucuronidase by the selected compounds is thermodynamically favorable ( Table 2). As shown in the docking results in Table 2 and Figure 7, the higher potential of 7 and 9 compared with 8 is mostly referable to (i) the complex that formed stability between the b-glucuronidase and the docked compounds and (ii) to the involvement of fluorine in intermolecular interactions with the b-glucuronidase active residues. The higher activity of 7 compared with 9 may refer to the stronger intermolecular hydrogen bonding and intermolecular of fluorine group with the amino acids of b-glucuronidase active site. In one hand, the hydrogen bond formed between Glu 245, Tyr 334 and Asp 105 with 7 (9)

Conclusion
Oxadiazole-based-thiourea analogs (1-18) were synthesized, characterized through 1 H-NMR, 13 C-NMR, HREI-MS and screened against b-glucuronidase inhibitory potential. All analogs showed a varied degree of b-glucuronidase inhibitory potential ranging between 2.20 ± 0.01 to 52.25 ± 1.20 mM when compared with the standard drug D-saccharic acid-1,4-lactone (IC 50 value 48.30 ± 1.20 mM). Among the series analog 7,16 and 4 showed an excellent inhibitory potential many folds better than the standard b-glucuronidase inhibitor. The binding interactions of these active analogues were confirmed through molecular docking.

Molecular docking details
The intermolecular binding modes between docked synthesized oxadiazole derivatives and active residues of b-glucuronidase have been investigated using Autodock package. 26 The starting b-glucuronidase geometries and the docked ligand N-alkyl cyclophellitol-aziridine were uploaded from the RCSB data bank web site (PDB code 5G0Q). 27 Removed water molecules; Kollman charge and polar hydrogen atoms were added to the structure of extracted receptor by using the automated Auto Dock Tools 4.2. The identified active site is based on the structure of the b-glucuronidase co-crystallized receptor-ligand complex. The re-docked original ligand N-alkyl cyclophellitol-aziridine into the active site is well repeated with an RMSD value less than 2 Å. The molecular structure geometries of synthesized oxadiazole derivatives were minimized at Merck molecular force field 94 (MMFF94) level44. The structures optimized were saved as pdb files. Non-polar hydrogens were incorporated, and rotatable bonds were defined for each docked ligand. Through Lamarckian genetic algorithm docking studies were carried out, with 500 as the total number of a run for a binding site for the original ligand. In every run, a population of 150 individuals with 27000 generations and 250000 energy evaluations were used. Operator weights for mutation, crossover, and elitism were set to 0.02, 0.8 and 1 respectively. The binding sites were defined by a grid of 40 Â 40 Â 40 points each with a grid spacing of 0.375 Å. The docking calculation has been carried out using an Intel (R) Core (TM) i5-3770 CPU @ 3.40 GHz workstation.