Arylmethylene hydrazine derivatives containing 1,3-dimethylbarbituric moiety as novel urease inhibitors

Abstract

A new series of arylmethylene hydrazine derivatives bearing 1,3-dimethylbarbituric moiety 7a-o were designed, synthesized, and evaluated for their in vitro urease inhibitory activity. All the title compounds displayed high anti-urease activity, with IC₅₀ values in the range of 0.61 ± 0.06-4.56 ± 0.18 µM as compared to the two standard inhibitors hydroxyurea (IC₅₀ = 100 ± 0.15 μM) and thiourea (IC₅₀ = 23 ± 1.7 μM). Among the synthesized compounds, compound 7h with 2-nitro benzylidene group was found to be the most potent compound. Kinetic study of this compound revealed that it is a mix-mode inhibitor against urease. Evaluation of the interaction modes of the synthesized compounds in urease active site by molecular modeling revealed that that compounds with higher urease inhibitor activity (7h, 7m, 7c, 7l, 7i, and 7o, with IC₅₀ of 0.61, 0.86, 1.2, 1.34, 1.33, 1.94 μM, respectively) could interact with higher number of residues, specially Arg609, Cys592 (as part of urease active site flap) and showed higher computed free energy, while compounds with lower urease activity (7f, 7n, 7g, and 7a with IC₅₀ of 3.56, 4.56, 3.62 and 4.43 μM, respectively) and could not provide the proper interaction with Arg609, and Cys592 as the key interacting residues along with lower free binding energy. MD investigation revealed compound 7h interacted with Arg609 and Cys592 which are of the key residues at the root part of mobile flap covering the active site. Interacting with the mentioned residue for a significant amount of time, affects the flexibility of the mobile flap covering the active site and causes inhibition of the ureolytic activity. Furthermore, in silico physico-chemical study of compounds 7a-o predicted that all these compounds are drug-likeness with considerable orally availability.

Figure 1

Design strategy for new arylmethylene hydrazine-1,3-dimethylbarbituric derivatives as urease inhibitors.

Figure 2

Synthesis of arylmethylene hydrazine-1,3-dimethylbarbituric derivatives 7a–o.

Figure 3

Kinetic analysis of urease inhibition by compound 7h. (a) The Lineweaver–Burk plot in the absence and presence of different concentrations of compound 7h; (b) The secondary plot between 1/V_max and various concentrations of compound 7h.

Figure 4

Representation of the compounds docking poses over the active site (a) close-up illustration of barbiturate ring relative to the bi-nuclear center (b), the active site flap colored in green color. The molecular graphic in this figure was generated using VMD 1.9.3.

Figure 5

Close up representation of the best energy value of the most active compound 7h complexed with JB urease (a), 90-degree anti-clockwise rotation view (b). The molecular graphic in this figure was generated using VMD 1.9.3.

Figure 6

3D representation of ligand-residue interactions of compound 7a (a), compound 7m (b), compound 7o (c) over urease active site. Active site flap colored in green. The molecular graphic in this figure was generated using VMD 1.9.3.

Figure 7

Four possible configurations of benzylidene hydrazone moiety of the synthesized compounds (a). Superimposition of the docked structures in the active site of urease (b). The 2D structure representation was drawn by ChemAxon Marvin 15.10.12.0.

Figure 8

RMSD representation of the compound 7h (in yellow) and urease backbone in complex with thiourea (in green) and in complex with compound 7h (in red) for over 100 ns MD simulation time. The plot in was generated using Microsoft Excel (https://www.office.com/).

Figure 9

RMSF of the urease backbone in complexed with thiourea (in green) and compound 7h (in red) (a), ligand binding location for over 100 ns MD simulation time; (b) α-helical and ß-strand regions are highlighted in light red and blue backgrounds, respectively. The molecular graphic in this figure was generated using VMD 1.9.3.

Figure 10

The timeline representation of the interactions shows the residues interact with compound 7h in each trajectory frame (more than one specific contact with the ligand is represented by a darker shade of orange) (a). The 3D representation of urease in bound-state with compound 7h in two different orientations related to 0–14.32 ns and 14.32 to the rest of simulation time (active site flap are depicted in green cartoon mode) (b). The simulation interactions diagram panel. The stacked bar charts are normalized over the course of the trajectory: some protein residues may make multiple contacts with the ligand (c). 2D representation of ligand-residue interactions that occur at least 30% of simulation time of urease bound-state of thiurea and compound 7h (d). The molecular graphic in this figure was generated using VMD 1.9.3.