Synthesis of diindolylmethane (DIM) bearing thiadiazole derivatives as a potent urease inhibitor.

The current study describes synthesis of diindolylmethane (DIM) derivatives based-thiadiazole as a new class of urease inhibitors. Diindolylmethane is natural product alkaloid reported to use in medicinal chemistry extensively. Diindolylmethane-based-thiadiazole analogs (1–18) were synthesized and characterized by various spectroscopic techniques 1HNMR, 13C-NMR, EI-MS and evaluated for urease (jack bean urease) inhibitory potential. All compounds showed excellent to moderate inhibitory potential having IC50 value within the range of 0.50 ± 0.01 to 33.20 ± 1.20 µM compared with the standard thiourea (21.60 ± 0.70 µM). Compound 8 (IC50 = 0.50 ± 0.01 µM) was the most potent inhibitor amongst all derivatives. Structure-activity relationships have been established for all compounds. The key binding interactions of most active compounds with enzyme were confirmed through molecular docking studies.

The most active compound among the series is analog 8 (IC 50 = 0.50 ± 0.01 µM) having two-nitro groups at ortho and para position on phenyl ring. The greater inhibition shown by this compound is seemed due to electron-withdrawing group on phenyl ring. The second most active analog among the series is compound 6 (IC 50 = 0.70 ± 0.01 µM) having three chloro groups on phenyl ring at 2,4,5-position. The greater potential of this analog is also seeming due to having EWG at phenyl ring.
Similarly the decline in inhibition was observed when EWG is replaced by methyl group as shown in analog 16 (IC 50 = 23.80 ± 1.00) having methyl at the ortho position of phenyl ring with analog 17 (IC 50 = 28.60 ± 1.20) having methyl at meta position and analog 18 (IC 50 = 20.40 ± 1.20) having methyl at the para position. All of the three analogs contain methyl groups attached at different positions showed a different kind of inhibition, which might be due to attachment of substituents at a different position on phenyl ring. In the current study, we have found that inhibitory potential was greatly affected by the nature, position, and number of substituents. All those analogs having electron-withdrawing groups (EWG) on phenyl ring showed greater potential as compared to those analogs having electron-donating groups (EDG). The binding interaction was confirmed through molecular docking studies. Molecular docking. The IC 50 values diindolylmethane bearing thiadiazol derivatives as a potent urease inhibitor are presented in Table 1. The urease inhibition by the synthesized derivatives may strongly related to the type, number, positions of the functional group in the aromatic ring of basic skeleton of diindolylmethane bearing thiadiazol derivatives and to the strength of the intermolecular interaction that may have formed these functional groups and the residues of the active of urease (Table 1). To understand the urease inhibition by the synthesized derivatives, a molecular docking study has been carried out to determine the binding modes of all synthesized derivatives 1-18 from one side and the active residues of the urease from another side. These compounds differ by the number and position of the substituted functional groups in the aromatic ring (Table 1). For instance, compounds 2, 3 and 10 are substituted by a mono nitro in the group in para and ortho positions, and di-nitro groups in ortho and para positions, respectively (Table 1). Compounds 6, 7 and 10 also differ by the number and positions of substituted chloro groups (Table 1). 16-18 are monosubstituted by a methyl group at ortho, meta and para positions respectively (Table 1). Table 2 summarized the calculated binding energies of the stable complexes ligand-urease, the number of established intermolecular hydrogen bonding between the synthesized compounds (1-18) and active site residues of urease.
The formed complexes between diindolylmethane bearing thiadiazol derivatives (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and the active residues of urease displayed negative bending energies, which indicates that the inhibition of urease by the selected diindolylmethane bearing thiadiazol derivatives is thermodynamically favorable ( Table 2). From the docking results in Table 2, binding energies of the stable complexes vary slightly from −10.55 to −8.20 kcal/mol. Such variation is low enough to be considered as a potent descriptor in rationalizing the observed inhibition of urease by the selected derivatives. However, the number of hydrogen bonding, its distances and intermolecular interactions between the substitute groups of the selected derivatives and the active residues may strongly help in understanding the observed urease inhibition by these selected compounds. For instance, the higher urease inhibition of 8 compared with 2 and 3 may refer to the number of hydrogen bonding formed between the nitro groups in the former and latter (Fig. 2). Indeed, in the 8-urease complex two hydrogen bonds are formed between the nitro groups at ortho and para positions with ARG 366 and VAL 367 amino acids of distances 2.46 and 2.93 Å, respectively. While in 2-urease and 3-urease complexes, the hydrogen bonds are formed between the nitro group at para (2) and ortho (3) positions with ARG 336 amino acid of distances 2.76 and 2.67 Å, respectively. The higher urease inhibition of 3 compared with 2 may also refer to the stronger hydrogen bond formed with the former (2.76Å) compared with the latter (2.67 Å).
Similarly, the higher urease inhibition of 6 compared with 7 and 10 may refer to the number of residues that interact with chloro groups in the former and to the strength of these interactions ( Table 2).

Urease inhibition assay.
Urease is an enzyme (jack bean urease) that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The production of ammonia was measured by the indophenol method and used to determine the urease inhibitory activity 57,58 . The percentage remaining activity was calculated from the formula % Remaining Activity = [(ODtest)/(ODcontrol) × 100]. Thiourea was used as a standard inhibitor. To calculate IC 50 values, different concentrations of synthesized compounds and standards were assayed at the same reaction conditions. Docking studies. The binding modes between selected bis-indole bearing thiadiazol derivatives and the active residues of urease have been investigated using Autodock package 59 . The staring geometries of urease and the original docked acetohydroxamic acid were download from the RCSB data bank web site (PDB code 1FWE) 60 . Water molecules were removed; polar hydrogen atoms and Kollman charge were added to the extracted receptor using the automated tool in AutoDock Tools 4.2. The active site is identified based on co-crystallized receptor-ligand complex structure of urease. The re-docking of the original ligand acetohydroxamic acid into the active site is well reproduced with a RMSD value less than 0.717 Å. Molecular geometries of selected diindolylmethane bearing thiadiazol derivatives were minimized at Merck molecular force field 94 (MMFF94) level44. The optimized structures were saved as PDB files. Nonpolar hydrogens were merged and rotatable bonds were defined for each docked ligand. Docking studies were performed by Lamarckian genetic algorithm, with 500 as total number of run for binding sites for original ligand the synthesized derivatives. In each respective run, a population of 150 individuals with 27000 generations and 250000 energy evaluations were employed. Operator weights for crossover, mutation, and elitism were set to 0.8, 0.02, and 1, respectively. The docking calculations have been carried out using an Intel Core i5-3770 CPU 3.40 GHz workstation.