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

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.

www.nature.com/scientificreports/ acid derivatives such as compounds A display high inhibitory activity against urease ( Fig. 1) 14 . As can be seen in Fig. 1, the later compounds have a 5-aminomethylene-1,3-dimethylbarbituric moiety. On the other hand, Saeed et al. reported the synthesis of a series of arylmethylene hydrazine derivatives containing carbothioamide B as novel urease inhibitors 15 . Their biological data demonstrated that the most potent compound among the compounds B showing up to 36.2-fold higher inhibitory potency than standard inhibitor thiourea (Fig. 1). By considering the potent urease inhibitors A and B, in continuing our efforts to synthesize new urease inhibitors using simple chemical reactions, here, a new series of urease inhibitors 7a-o was designed by combination of 5-aminomethylene-1,3-dimethylbarbituric moiety and arylmethylene hydrazine derivatives ( Fig. 1) [16][17][18][19][20] . Compounds 7a-o were synthesized by a simple three-step procedure. All these compounds were evaluated for their in vitro urease inhibitory activities. Furthermore, molecular modeling and molecular dynamic studies of compounds 7a-o were also performed.

Results and discussion
Chemistry. Arylmethylene hydrazine-1,3-dimethylbarbituric derivatives 7a-o were synthesized by described method in Fig. 2. According to this method, reaction of 1,3-dimethylbarbituric acid 1 and trimethoxymethane 2 in ethanol under reflux condition afforded 5-(methoxymethylene)-1,3-dimethylbarbituric acid 3. The latter compound was reacted with hydrazine 4 in ethanol under reflux for produce 5-(hydrazineylmethylene)-1,3-dimethylbarbituric acid 5. In the final step, compound 5 and aromatic aldehydes 6a-o in the presence of catalytic amount of PTSA in ethanol at room temperature were converted to desired compounds 7a-o.   was determined using Jack bean (JB) urease in accordance to the reported protocols [18][19][20] . The obtained IC 50 values of title compounds against urease were presented in Table 1, in comparison with hydroxyurea and thiourea as reference inhibitors. Generally, all newly synthesized compounds 7a-o, with IC 50 values in the range of 0.61 ± 0.06-4.56 ± 0.18 μM, had significant inhibitory effect against urease. All these compounds were more potent than that of hydroxyurea (IC 50 = 100 ± 0.15 μM) and thiourea (IC 50 = 23 ± 1.7 μM). The 2-nitrophenyl derivative 7h and thiophen-2-yl derivative 7m with IC 50 values of 0.61 ± 0.06 and 0.86 ± 0.08 μM were found to be the most active compounds. In particular, compound 7h was 37.7 times more potent than strong urease inhibitor thiourea.
The second most potent compound among the synthesized compound was thiophen-2-yl derivative 7m. As can be seen in Table 1, introduction of 5-chloro substituent on thiophen ring, as in compound 7n, led to a significant decrease in inhibitory activity. Furthermore, naphthalen-1-yl derivative 7o was one of the strongest compounds among the synthesized compounds.
Urease kinetic study. To evaluate the mechanism inhibition of new arylmethylene hydrazine-1,3-dimethylbarbituric derivatives, kinetics study was performed on the most potent urease inhibitor 7h (Fig. 3). The inhibition mode and K i value were determined by Lineweaver-Burk plots and secondary re-plotting of the mentioned plots, respectively. As exhibited in Fig. 3a, with increasing concentrations of compound 7h, V max and K m increased. Therefore, this compound was a mixed-type inhibitor for urease (Fig. 3b, K i = 0.82 µM).
Docking study. Docking study was applied to distinguish interactions between the synthesized compounds and urease active site. The reliability of the applied docking protocol was assessed according to our previous  20 . This protocol was then similarly applied to all the synthesized compounds 7a-o.
The interaction mode of the top IFD scoring of all the synthesized compounds (7a-o) showed they successfully occupied in the bi-nickel active site cavity. Figure 4a shows the top IFD pose of the compounds over JB urease. The result depicts superimposing of barbituric acid ring of all the compounds pointed toward the bi-nickel center atoms through the carbonyl group at C 2 position of the barbiturate ring (Fig. 4b). The orientation of the mentioned carbonyl group is the same as the carbonyl oxygen in the AHA and thiourea as reference inhibitors. Furthermore, the arylmethylene hydrazine moieties oriented to the entering site of the active site and adapted by flexible conformation in the large hydrophobic opening of the active site flap pocket (Fig. 4a). The mentioned extended moiety in designing of the synthesized compounds has the superior inhibitory effect in comparison to the standard inhibitor (thiourea) through higher stabilization effect by implementing of various non-bonding interactions.
In case of the most active compound 7h (Fig. 5), the carboxyl groups in C 2 and C 4 position of barbituric acid ring created H-bond with His492 and Arg609, the hydrazone group formed H-bond interaction through Cys592 (SH) and the ortho nitro benzylidene group was able to form salt-bridge interaction toward Arg639 (Fig. 5a). As a result, compound 7h well occupied the active pocket of urease and tightly anchoring the helix-turn-helix motif through interacting with Cys592 and Arg609 over the active-site cavity (depicted in green cartoon) (Fig. 5b), which could reduce the flexibility of flap residue (590-609) and results in the inhibition of urease activity.
In order to reveal the effect of different aromatic ring instead of phenyl ring, the docked pose complex and the gibbs free binding energy (ΔG) of the compounds 7a, 7m and 7o were compare to each other. Figure 6a shows barbituric acid ring of compound 7a tightly coordinated along the metal bi-nickel center and further stabilized by H-bond interaction with His492. At the middle part of the molecule, the hydrazone group formed H-bond interaction through Cys592 (SH). In addition, the benzylidene group at the tail part of   (Table 2), which is in accordance with the experimental urease activity results (0.86, 1.94 and 4.43 μM, respectively, Table 1). In the case of compound 7m, the sulfur atom increased hydrophobic interaction by interacting with the mentioned hydrophobic pocket through sulfur atom of Met388, while in the case of compound 7o, the naphthyl ring rotated as a result of bigger size and interacted with the imidazole ring of His594 by π-π stacking interaction (Fig. 6b,c).
The result shows that the bioisoester substitution of phenyl group provides additional interactions for the compounds 7m and 7o to bind to the hydrophobic part of urease active site pocket.
This study confirms our previous investigation which outstands the key role of Cys592 and Arg609 in flexibility of mobile flap covering the active site entrance which consequently affect the inhibition activity of urease enzyme 21 . Table 2 shows the interaction part of the molecule, the involving residues and the calculated gibbs free binding energies of the compound with higher and lower urease inhibition activity over JB urease active site. It indicates that compounds with higher urease inhibitor activity (7h, 7m, 7c, 7l, 7i, and 7o, with IC 50 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), while compounds with lower urease activity (7f, 7n, 7g, and 7a with IC 50 of 3.56, 4.56, 3.62 and 4.43 μM, respectively) have insufficient interaction and could not provide the proper interaction with  www.nature.com/scientificreports/ Arg609, and Cys592 as the key interacting residues. Furthermore, this finding additionally supported by comparing the gibbs free binding energy of the mentioned compounds which are in accordance with the experimental urease inhibition activity.
Conformational analyses of the synthesized compounds over the urease active site. As the synthesized compounds hold benzylidene hydrazone moiety, they may adopt in four possible geometrical configurations including; the anti-periplanar (ap) and syn-planar rotameric (sp) forms for each Z and E isomers as shown in Fig. 7a. The superimposed structures of IFD compounds over the active site of JB urease have been shown in Fig. 7b. It represents that n-π conjugation result in a planar form of C=N-NH moiety. Considering Fig. 7a isomer E with anti-periplanar rotamer was the only configuration while no docked-structures got the Z configuration around C=N bond. This result is in accordance well with a previously reported study where there is no Z configuration around C=N of benzylidene hydrazone moiety due to the steric hindrance [22][23][24] . Therefore, according to the result it reveals that the E-ap form is the preferred geometrical isomer by the benzylidene hydrazone structure of barbituric acid derivatives.

Molecular dynamic (MD) investigation.
MD study has been investigated in order to reveal the effect of the most potent compound (compound 7h) on the urease structure and the active site environment in comparison to thiourea as the urease standard inhibitor.  RMSF define as the fluctuation of the protein's residues from its average position throughout the simulation, which represents the flexibility of protein structure. In this manner, helixes and sheets with organized structure  www.nature.com/scientificreports/ depict lower RMSF value while loops with loosely organized structure have high RMSF value. Comparing RMSF values of urease-compound 7h complex shows that it has obviously lower value through the whole part of the urease structure and specially the residues 590-606 showed significantly decreased RMSF value in the urease bound-state with compound 7h than urease-thiourea complex (Fig. 9a). The mentioned conserved residues with helix-turn-helix secondary structure belong to the α-subunit part of the enzyme known as mobile flap region covering the urease active site 25 . Figure 9b shows that compound 7h well occupied the active pockets of urease and tightly anchoring the helix-turn-helix motif over the active-site cavity (vertical green line), which considerably reduce the flexibility of the mobile flap residue (590-609) by interacting with key amino acid residues and results in the inhibition of urease activity. The mentioned interactions could not be observed for the urease-thiourea complex which proposed the role of rigidity of the mobile flap in higher urease inhibition activity of the synthesized compounds. Figure 10 represents different residues, types of interactions, and ligands functional group involvement during the whole MD simulation time.
So, because of the shifting orientation of compound 7h as a result of different interacting residues, the MD simulation time divided in two sections; 0 ns to 14.32 ns and 14.32 ns to 100 ns. Based on the cluster analysis of compound 7h, the percent of population in cluster 1 was 86.37% in the first section and 95% in the second section in which the representative frame from cluster 1 of the section 1 (Fig. 10b, up) and the section 2 (Fig. 10b, down) were selected for investigating the 3D complex interaction.
As it is obvious in Fig. 10b (up), at the first stage of MD compound 7h tightly coordinated toward the active site bi-nickel center through its barbituric acid. In addition, the ortho nitro benzylidene moiety provided ionbridge and π-cation interactions through Arg439. Otherwise, after about 14 ns the barbituric acid ring shifted from the bi-nickel center toward the active site flap and provided interactions with Cys592, His593 at one side and Arg609 and Arg611 at the other side of the active site flap while the ortho nitro benzylidene moiety still stabilized in its initial position faced to Arg439 through the electrostatic interaction (Fig. 10b, down) (the types of residue interactions during the MD simulation represents in different color by Fig. 10c). So, it can propose www.nature.com/scientificreports/ that compound 7h provides the stabilized interaction to Arg439 at the opening part of the active site through its polar nitro substitution. This finding is in accordance with our experimental results as the molecular level explanation on the higher urease activity of compound 7h and the compounds with polar substituent at the phenyl ring. Also, again the E-ap conformer, as the preferred geometrical isomer by the benzylidene hydrazone, recognized for both stages of MD simulation time.
Furthermore, Fig. 10d shows the detailed 2D-ligand atom interactions that occurred more than 30.0% of the simulation time during the equilibrated phase over urease complexed with thiourea and compound 7h. The interaction analysis suggests compound 7h stabilized by the ortho nitro group with Arg439 at the entrance of active site through salt-bridge interaction for about 43% of simulation time. Also, it formed water-mediated H-bond interaction with Arg609 and Arg611 at on side of the active site flap through the C4 carbonyl group of barbituric acid ring for 45% and 55% of simulation time, respectively. In addition, Cys592 on the other side of the active site flap provided the non-bonding interaction mostly through hydrophobic interaction for about 45% of MD simulation time (Fig. 10c, Cys592 bar chart).
It is noteworthy that Arg609 and Cys592 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 20 .
Comparing the results of compound 7h interaction with thiourea indicates thiourea did not show any interaction with these key residues at the active site flap region which can proposed the reason of higher urease inhibition activity of compound 7h rather than thiourea.
In silico prediction of pharmacokinetic properties. The main physico-chemical properties of the synthesized compounds, which represent drug-likeness, partition coefficient, solubility, cell permeation, were calculated. Aiming to discuss the reliability of predictions in a consensus way, the parameters calculated with two different software. The partition coefficient (Log P) predicted by Qikpro module of schrodinger and swissADME web server while solubility (Log S), cell permeation and the predicted % human absorption (oral; %HOA and intestinal; %HIA) were measured based on Qikpro and pkCSM web server. "Lipinski rule of five" was used to assess the drug-likeness of the synthesized compounds which includes calculating of the molecular weights, number of hydrogen bond donor, number of hydrogen bond acceptor and the predicted octanol/water partition co-efficient. According to Table 3, the number of violations of Lipinski's rule of five (ROF violations) for all the synthesized compounds was zero (0) and therefore all compounds (7a-o) meet the drug-likeness criteria 26 .
Also, the bioavailability of a compound mainly depends on the absorption and metabolism process. The oral and gastrointestinal absorption procedures in turn relay on the solubility and permeability of the a compound 27 . The computed physico-chemical parameters used to assess absorption of the newly synthesized compounds 7a-o and hydroxyurea including; the predicted aqueous solubility (Log S), the predicted apparent Caco-2 cell permeability as a model for the gut-blood barrier (non-active transport) (PCaco-2 and LogCaco-2), the predicted % human absorption (oral; %HOA and intestinal; %HIA) which were measured based on two different methods Qikpro and pkCSM web server and presented in Table 4. Comparing the experimental solubility value of

Conclusion
In summary, a novel series of arylmethylene hydrazine-1,3-dimethylbarbituric derivatives 7a-o have been synthesized via simple chemical reactions, and their inhibitory activities against urease were evaluated. In the enzymatic assay, all the synthesized compounds 7a-o acted as potent inhibitors against urease (IC 50 values = 0.61 ± 0.06-4.56 ± 0.18 μM) and were more active than the standard inhibitors hydroxyurea and thiourea (IC 50 values = 100 ± 0.15 and 23 ± 1.7 μM, respectively). Furthermore, IFD study of the synthesized compounds in the urease active site showed that compounds with higher urease inhibitor activity could interact with higher number of residues, specially Arg609, Cys592 and showed higher computed free energy, while compounds with lower urease activity 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. Moreover, in silico pharmacokinetic study predicted that newly synthesized compounds are drug-likeness and can be orally active.  www.nature.com/scientificreports/ this mixture was allowed to stand overnight at room temperature for formation pure 5-(methoxymethylene)-1,3-dimethylbarbituric acid 3.

Synthesis of 5-(hydrazineylmethylene)-1,3-dimethylbarbituric acid 5.
A mixture of 5-(methoxymethylene)-1,3-dimethylbarbituric acid 3 (1 mmol) and hydrazine 4 (1 mmol) in ethanol (5 ml) was refluxed for 1 h. After that, the reaction mixture was allowed to cool at room temperature and poured into water, and the pure 5-(hydrazineylmethylene)-1,3-dimethylbarbituric acid 5 were filtered off.  Urease inhibitory activity and kinetic study. All used material and JB urease (EC 3.5.1.5) were purchased from Sigma-Aldrich (USA). Potassium phosphate buffer (PPB) solution with concentration 100 mM and pH = 7.4 was prepared in distilled water. Urease inhibition effects of the synthesized compounds 7a-o was determined using the modified Berthelot spectrophotometric method by a Synergy H1 Hybrid multi-mode microplate reader (BioTek, Winooski, VT, USA) at 625 nm [16][17][18][19][20] . The enzymatic reactions were performed in PPB solution which reach to the 985 μL by adding urea (850 μL) and the synthesized compound (100 μL, 0-10 mg/ mL). Then, urease (15 μL) was added and the concentration of liberated ammonia was measured after 60 min. The corresponding concentration of ammonia was determined by addition of the incubated solution (100 μL) to the mixture of 500 μL of solution I (5.0 g phenol and 25.0 mg sodium nitroprusside in 500 mL water) and 500 μL of solution II (2.5 g sodium hydroxide and 4.2 mL sodium hypochlorite (5% chlorine) in 500 mL water) which was incubated at 37 °C for 30 min. The absorbance was obtained by measuring indophenols. The activity of uninhibited JB urease was considered as the control activity of 100%. The inhibition assays were conducted according to this formula: I (%) = [1 − (T/C)] × 100; where I (%) is the enzyme inhibition, T (test) is the absorbance of the analyzed compounds in the presence of urease solution, and C (control) is the absorbance of the solvent in the presence of urease solution. Data were expressed as mean ± standard error (SD) and run in triplicate. The IC 50 values for the all compounds 7a-o were calculated using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA). Thiourea and hydroxyurea were used as the standard inhibitors for urease. For the kinetic study the urea concentrations were changed from 3.12 to 100 mM and concentrations 0, 0.5, and 1 µM of the most potent urease inhibitor was used.

General
Molecular modeling procedure. In order to find out the interactions mode of designed molecules over urease enzyme, Maestro Molecular Modeling platform (version10.5) by Schrödinger, LLC was performed 29 . The X-ray crystallographic structure of Jack bean urease (JB urease) (in complex with acetohydroxamic acid, AHA) was downloaded from the Protein Data Bank (PDB ID; 4h9m) (www. rcsb. org). As urease is reported to be functionally active in monomeric state, all the docking studies were performed on single monomer. In addition, prosthetic group and co-factors are not directly involved in urease inhibition, so they totally removed before docking investigation. Water molecules and co-crystallized ligands were removed from the enzymes crystallographic structures. The 2D structures of all synthesized compounds were drawn in Marvin 15.10.12.0 program (http:// www. chema xon. com) and converted into pdb file 30  www.nature.com/scientificreports/ module were used to prepare protein and ligand structure properly 31,32 . The missing side chains of the proteins were filled using the Prime tool and missing residues were updated. The accurate side-chain, backbone conformational changes or both during ligand binding at the active site of urease enzyme were predicted by IFD method using Glide software (Schrödinger LLC 2018, USA) 33 . The AHA binding site was used to generate the grid for IFD calculation. The maximum 20 poses with receptor and ligand van der Waals radii of 0.7 and 0.5, respectively considered. Residues within 5 Å of the AHA at the active site were refined followed by side-chain optimization. Structures whose Prime energy is more than 30 kcal/mol are eliminated based on extra precious Glide docking.
The ligand binding energies (ΔG bind) were calculated for each synthesized compound using Molecular mechanics/generalized born surface area (MM-GBSA) modules (Schrödinger LLC 2018) based on the following equation; where ΔG Bind is the calculated relative free energy which includes both ligand and receptor strain energy 33 . E Complex is the MM/GBSA energy of the minimized complex, and E Ligand is the MM/ GBSA energy of the ligand after removing it from the complex and allowing it to relax. E Receptor is the MM-GBSA energy of relaxed protein after separating it from the ligand. The MM-GBSA calculation was performed based on the best pose structure obtained from IFD complexes. Molecular dynamic simulation. Molecular simulations of this study were performed using the Desmond v5.3 using Maestro interface (from Schrödinger 2018-4 suite) 34 . The appropriate pose for MD simulation procedure of the compound was achieved by IFD method.
In order to build the system for MD simulation, the protein-ligand complexes were solvated with SPC explicit water molecules and placed in the center of an orthorhombic box of appropriate size in the periodic boundary condition. Sufficient counter-ions and a 0.15 M solution of NaCl were also utilized to neutralize the system and to simulate the real cellular ionic concentrations, respectively. The MD protocol involved minimization, pre-production, and finally production MD simulation steps. In the minimization procedure, the entire system was allowed to relax for 2500 steps by the steepest descent approach. Then the temperature of the system was raised from 0 to 300 K with a small force constant on the enzyme in order to restrict any drastic changes. MD simulations were performed via NPT (constant number of atoms, constant pressure i.e. 1.01325 bar and constant temperature i.e. 300 K) ensemble. The Nose-Hoover chain method was used as the default thermostat with 1.0 ps interval and Martyna-Tobias-Klein as the default barostat with 2.0 ps interval by applying isotropic coupling style. Long-range electrostatic forces were calculated based on particle-mesh-based ewald approach with the he cut-off radius for columbic forces set to 9.0 Å. Finally, the system subjected to produce MD simulations for 20 ns for each protein-ligand complex. During the simulation every 1000 ps of the actual frame was stored. The dynamic behavior and structural changes of the systems were analyzed by the calculation of the root mean square deviation (RMSD) and RMSF. Subsequently, the representative frames of the simulation extracted based on the clustering method from the equilibrated trajectory system for investigating of ligand-protein complex interaction.
In silico pharmacokinetic properties of synthesized compounds. QikProp module of Schrodinger 35 , swissADME 36 , and pkCSM 37 were used to calculate the important physico-chemical properties of the synthesized compounds like drug-likeness, partition coefficient, solubility, cell permeation.