Synthesis, in vitro urease inhibitory potential and molecular docking study of benzofuran-based-thiazoldinone analogues

In continuation of our work on enzyme inhibition, the benzofuran-based-thiazoldinone analogues (1–14) were synthesized, characterized by HREI-MS, 1H and 13CNMR and evaluated for urease inhibition. Compounds 1–14 exhibited a varying degree of urease inhibitory activity with IC50 values between 1.2 ± 0.01 to 23.50 ± 0.70 µM when compared with standard drug thiourea having IC50 value 21.40 ± 0.21 µM. Compound 1, 3, 5 and 8 showed significant inhibitory effects with IC50 values 1.2 ± 0.01, 2.20 ± 0.01, 1.40 ± 0.01 and 2.90 ± 0.01 µM respectively, better than the rest of the series. A structure activity relationship (SAR) of this series has been established based on electronic effects and position of different substituents present on phenyl ring. Molecular docking studies were performed to understand the binding interaction of the compounds.

www.nature.com/scientificreports/ Urease enzyme is involved to function by using urea as nitrogen source 12,13 . Urease is responsible for the one of the major diseases induced by Helicobacter pylori, allowing them to survive inside the stomach at low pH thus play a vital role in peptic and gastric ulcer pathogenesis, apart from cancer too 12 . Urease play a key role in the infection stones formation that equally take part in pathogenesis of pylenephritis, hepatic encephalopathy, urolithiasis, urinary catheter encrustation and hepatic coma 14 . Certain secondary complication like ulcer, pus formation and infectious diseases can be treated by inhibiting urease enzyme with help of specific potent inhibitors 15 . Previously published urease inhibitors are 2-acylated and sulfonated 4-hydroxycoumarins, bis-indolylmethane thiosemicarbazides and benzimidazole analogues [16][17][18] .
Scaffold 1 (IC 50 = 1.2 ± 0.01 μM) that has 4-chloro moiety as substituent on phenyl ring was most potent scaffold among the whole series. An excellent potential of this scaffold might be due to the existence of 4-chloro moiety on phenyl ring which is electron withdrawing in nature. www.nature.com/scientificreports/ If we compare scaffold 3 (IC 50 = 2.2 ± 0.01 μM) that has NO 2 moiety at positon-2 on phenyl ring with scaffold 6 (IC 50 = 5.70 ± 0.02 μM) that has NO 2 moiety at position-4 on phenyl ring and scaffold 14 (IC 50 = 15.60 ± 0.50 μM) that has NO 2 moiety at position-3 on phenyl ring. All these three NO 2 substituted scaffolds showed that the potential difference may be due to the different positions of NO 2 moiety.
If we compare scaffold 2 (IC 50 = 6.10 ± 0.05 μM) that have hydroxyl at 3-position and methoxy moiety at 4-position on the phenyl ring with scaffold 7 (IC 50 = 3.10 ± 0.01 μM) that have hydroxyl at 4-position and methoxy group at 3-position on the phenyl ring. The potential difference between these two scaffolds may be due to different positions of hydroxyl and methoxy moiety on the phenyl ring.
Overall it has been concluded that either electron withdrawing groups (EWG) or electron donating groups (EDG) on phenyl ring exhibited good potency but slightly difference in their potency was also mostly affected due to positions of substituents.

Molecular docking.
In catalytic pocket of urease enzyme to explore the binding modes of the synthesized scaffolds the Molecular Operating Environment (MOE) package 33 was used to study molecular docking study. With the help of builder tool executed in MOE package, 3D structural coordinates of scaffolds were generated. By using default parameter of MOE, energy of the synthesized scaffolds was minimized, and all 3D coordinates of the scaffolds were protonated. Using PDB code 4UBP, from the online free server protein databank (www. rcsb.org), the crystallographic 3D structure of urease enzyme was retrieved. Next, the structure was added to MOE for protonation, energy to get the stable conformation of protein with the help of default parameter of MOE package. Finally, using the default parameters of MOE package to perform molecular docking studies i.e., Placement: Rescoring 1, Triangle Matcher, Refinement, London dG: Rescoring 2, Forcefield: GBV1/WSA. For each synthesized scaffold, total 10 conformations were allowed to be form. Later, for addition analysis the top ranked conformations were selected.
Docking study. In order to explain the synthesized scaffolds binding pattern, molecular docking studies was conducted in the urease enzyme catalytic pocket (PDB code 4UBP). The urease enzyme catalytic site contains both hydrophobic and hydrophilic site residues ( Fig. 2A). The hydrophilic site includes 223, 494, 323, 324, 249, G166, D224, R339 and H315, while hydrophilic site composed of 366, A170, C322, L319 and K169. Furthermore, these two Nickle ions (Ni799 and Ni798) conjointly compete a key role by linking the ligands and key residues. Though, the results of docking showed a well fit sketch of binding in catalytic site and with catalytic residues adopt the most favorable interaction (Fig. 2). Usually, through the binding mode analysis it showed not only that the inhibitory potential of those scaffold was excellent that possess electron withdrawing groups (EWG's) at para position, but also with catalytic residues showed favorable interactions. In contrast, among the series in our current study some scaffolds which possess -di-(EWG's) at para and ortho position that exhibited less potency against urease enzyme. The reason for the decrease in enzyme activity might be that the halides at para, ortho directing groups most likely prefer to deactivate the benzene ring that result in potency decrease 34 . Furthermore, the scaffolds among the series that has electron donating groups (EDG's) possess good inhibitory potential against urease enzyme. Though binding mode analysis of most potent scaffold 1 (1.2 ± 0.01 μM) showed the fit well pattern of binding in catalytic site, therefore the scaffold was unable to adopt favorable ionic and other interactions (i.e. hydrophobic, hydrogen bond etc.) with catalytic residues like `D363, E223, L365, R339 and with the modified residue KCX220. Furthermore, the two Ni ions (Ni799 & Ni798) accept ionic bond with O13 and S19 of the corresponding scaffold, and additionally improve the potential against urease enzyme (Fig. 2B). High potential of this scaffold might be due to the reason that the attached EWG-1 decreases the Scheme 1. Synthesis of benzofuran bearing thiazoldinone analogues. www.nature.com/scientificreports/ π-system electronic density thus making the π-system more electrophilic and hence initiate the partial positive charge on benzene, which make this benzene unable to adopt π-interaction with catalytic residues (R339). In same manner, the second most potent scaffold 5 that has electron donating group (EDG-OH) among series through binding modes showed the similar binding pattern (Fig. 2C) upon comparison with most potent scaffold 1. In both scaffolds (1 and 5) the difference perceived through binding modes analysis are not only ionic interaction of both Ni ions and modified residues KCX220 but also due to variation in EDG and EWG. Scaffold 5 showed ionic interaction only with single Ni ion while in case of scaffold 1 interaction was with both Ni ions as well as KCX220 residues.
In same manner, the scaffolds 3 and 8 through binding mode analysis showed similar binding mode (Fig. 2D, E). With experimental results the docking results are related well based on key residues with ligands multiple interactions of the urease enzyme. Docking poses of all active scaffolds were computatiionally reserved the catalytic potentials of urease by firmly binding through hydrogen bonding, strong hydrophobic and polar interactions with key residues. conclusion Benzofuran bearing thiazolidinone scaffolds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) have synthesized in excellent to moderate yield (68-88%). These synthesized scaffolds were evaluated for urease inhibitory potential. All the synthesized scaffolds showed excellent to good inhibitory potential against urease inhibition with IC 50 value ranging between 1.2 ± 0.01 to 23.50 ± 0.70 µM as compare to standard thiourea having IC 50 value 21.40 ± 0.21 µM. SAR of potent scaffolds was established and were confirmed through molecular docking studies. Among the series, scaffold 1 was found potent urease inhibitor which can act as lead scaffold for further development of drug.