Design, Synthesis, Crystal Structure, In Vitro and In Silico Evaluation of New N′-Benzylidene-4-tert-butylbenzohydrazide Derivatives as Potent Urease Inhibitors

Background: Hydrazides play a vital role in making biologically active compounds in various fields of chemistry. These determine antioxidant, antidepressant, antimalarial, anti-inflammatory, antiglycating, and antimicrobial activity. In the present study, twenty-three new N′ benzylidene-4-(tert-butyl)benzohydrazide derivatives (4–26) were synthesized by the condensation of aromatic aldehydes and commercially available 4-(tert-butyl)benzoic acid. All the target compounds were successfully synthesized from good to excellent yield; all synthesized derivatives were characterized via spectroscopic techniques such as HREI-MS and 1H-NMR. Synthesized compounds were evaluated for in vitro urease inhibition. All synthesized derivatives demonstrated good inhibitory activities in the range of IC50 = 13.33 ± 0.58–251.74 ± 6.82 µM as compared with standard thiourea having IC50 = 21.14 ± 0.425 µM. Two compounds, 6 and 25, were found to be more active than standard. SAR revealed that electron donating groups in phenyl ring have more influence on enzyme inhibition. However, to gain insight into the participation of different substituents in synthesized derivatives on the binding interactions with urease enzyme, in silico (computer simulation) molecular modeling analysis was carried out.


Introduction
Urease (EC 3.5.1.5) belongs to the family of amidohydrolase enzymes that possesses two nickel atoms in its core structure. The conversion of urea into ammonia and carbamate is catalyzed by the action of this enzyme. The excessive amount of ammonia released due to the hyperactivity of the urease enzyme leads to the alkalinity of the stomach, which in turn increases the gastric mucosa permeability [1]. The nitrogen metabolism of cattle and various other animals is controlled by the action of the urease enzyme [2]. The elevated levels of these enzymes lead to several pathogenic conditions, such as it helps in the survival of some bacterial pathogens, thus leading to some severe side effects [3]. In humans, the low pH of the stomach facilitates the survival of Helicobacter pylori (HP) which leads to the development of gastric and peptic ulcers, which may eventually cause cancer [4]. The increased level of ammonia is also responsible for several metabolic disorders and destroys the GIT epithelium. A number of compounds belonging to different classes are reported as urease inhibitors, such as thiolates that bind with the nickel atom of the enzyme. destroys the GIT epithelium. A number of compounds belonging to different classes are reported as urease inhibitors, such as thiolates that bind with the nickel atom of the enzyme. Hydroxamic acid and its derivatives act as competitive inhibitors and compete with urea, phosphorodiamidates, and a few peptide chains having a ligand that may chelate with nickel of urease. Unfortunately, these molecules have adverse side effects associated with them. Therefore, it is of crucial importance to identify more urease inhibitors having significant stability, bioavailability, and low toxicity [5].
Day by day, the chemistry of carbon-nitrogen double bond of hydrazone is fastly becoming the backbone of condensation reaction in benzo-fused N-heterocycles. Hydrazides are an important class of functional groups in organic chemistry possessing -NH-N=CH-groups with the availability of proton that aids in their pharmaceutical importance. The remedial possibilities of acid hydrazides gained momentum after the innovation of isonicotinic acid hydrazide (INH). The remarkable clinical value of INH [6,7] stimulated the study of other heterocyclic hydrazides possessing mono-cyclic nuclei such as furan, pyrrole, thiophene, and dicyclic nuclei such as quinoline and isoquinoline.

Chemistry
Twenty-three different N'-benzylidene-4-(t-Bu)benzohydrazide derivatives,  were synthesized by a routes outlined in Scheme 1. In the first step, methyl 4-tert-butylbenzoate (2) was synthesized from 4-(t-Bu)benzoic acid (1) by refluxing in methanol for two hours in the presence of concentrated H2SO4. Methyl 4-tert-butylbenzoate (2) was then reacted with hydrazine hydrate in methanol to give the corresponding hydrazide 3. The diversely substituted hydrazones  were obtained by refluxing different substituted aromatic aldehydes with 4-(tBu)benzohydrazide in the presence of methanol and glacial acetic acid with continuous stirring to get N'-benzylidene-4-(tert-butyl)benzohydrazide derivatives 4-26. TLC technique was used to monitor the reaction progress periodically. For the confirmation of newly synthesized benzohydrazide derivatives 4-26 (Table 1) [21], spectroscopic techniques such as 1 H-, 13 C-NMR, and HREI-MS were performed.

Crystal Structure Description
The crystal structure of 4-tert-butyl-N′-[(E)-(4-fluoro-3-methoxyphenyl)methylidene]benzohydrazide (4+) has been reported previously [22].  (5) The 4-(tBu)-N'-(2-hydroxy-3-methoxybenzylidene)benzohydrazide (5) crystallized in the monoclinic, Cc space group. The asymmetric unit of 5 is shown in Figure 1. The crystal data of 5 is shown in Table 2, whereas the selected bond lengths and angles are shown in Table 3. The O3-C8 bond length is negligibly shorter than a similar bond length   (5) The 4-(tBu)-N -(2-hydroxy-3-methoxybenzylidene)benzohydrazide (5) crystallized in the monoclinic, Cc space group. The asymmetric unit of 5 is shown in Figure 1. The crystal data of 5 is shown in Table 2, whereas the selected bond lengths and angles are shown in Table 3. The O3-C8 bond length is negligibly shorter than a similar bond length in compound 14. Similarly, the N-N bond length in compound 5 is 1.364 Å, comparably shorter than a similar bond length in 14. The reason coined for this effect is the substitution of hydroxyl and methoxy groups at ortho and meta (C1 and C2) positions of the benzene ring. Similar behavior is observed in bond lengths as well. Therefore, we will restrict the crystal description to 14 only. in compound 14. Similarly, the N-N bond length in compound 5 is 1.364 Å, comparably shorter than a similar bond length in 14. The reason coined for this effect is the substitution of hydroxyl and methoxy groups at ortho and meta (C1 and C2) positions of the benzene ring. Similar behavior is observed in bond lengths as well. Therefore, we will restrict the crystal description to 14 only. The hydrogen bonding is unique in 5, as shown in Table 4. Overall, there is a single intramolecular hydrogen bonding interaction shown by O1-H1O···N1 with a length of 2.581 (4) Å showing strong interaction. Similarly, there are intermolecular hydrogen bonds as well, which lead to the accumulation of a co-crystallized water molecule in the crystal lattice. The hydrogen bonding interaction is shown in Table 4. The crystal packing diagram of 5 is shown in Figure 2.

Complex 5 14
Limiting indices The hydrogen bonding is unique in 5, as shown in Table 4. Overall, there is a single intramolecular hydrogen bonding interaction shown by O1-H1O···N1 with a length of 2.581 (4) Å showing strong interaction. Similarly, there are intermolecular hydrogen bonds as well, which lead to the accumulation of a co-crystallized water molecule in the crystal lattice. The hydrogen bonding interaction is shown in Table 4. The crystal packing diagram of 5 is shown in Figure 2.

Structure-Activity Relationship (SAR)
To explain the structure-activity relationship (SAR) of these analogs, we have classified the top eight compounds of the series into two groups according to their nature of substituents. Limited SAR study was established for synthesized derivatives 4-26. The most active compounds among the series were compounds 6 and 25, with IC 50 values of 13.33 ± 0.58 and 13.42 ± 0.33 µM when compared with standard thiourea (IC 50 = 21.14 ± 0.425 µM) ( Table 7). The potency of compounds 6 and 25 might be due to the presence of anthracene and di-methoxy moiety, respectively. In both cases, the substituents are electron rich in nature. Compounds 17, 24, 22, 12, 26, 20, and 5 are the second group of active compounds having hydroxyl groups as substituents. The activity of these analogs might be due to the hydroxyl group ( Figure 5). It seems that the remaining compounds showed much lower activity than the standard. Results of the enzyme inhibition showed that the nature of the substituents played an important role in enhancing the inhibition potential of the core structure. Apart from it, both compounds 5 and 14 studied crystallographically also reveal activity. Compound 5 with IC 50 = 56.57 ± 3.18 µM is revealing better activity than the compound 14 (IC 50 = 81.21 ± 7.4 µM). The possible explanation for this behavior may be linked to the presence and absence of methoxy moiety. Compound 5 revealed almost identical activity to other similar analogs such as 20. Both the derivatives possess ortho-substituted hydroxyl group, which is involved in hydrogen bonding with hydrazide moiety, as may be seen in the crystal structure of compound 5.

Molecular Docking, Interactions Report
To predict the inhibition mechanism of isolated compounds shown by the kinetics study, molecular docking of the active compounds was carried out with the crystal structure of the urease enzyme. The most favorable docking conformations of all compounds were observed inside the active site with proper orientation. The active site consists of both the hydrophobic and hydrophilic amino acids.
The hydrophilic amino acids included Glu166, 223, Arg339, His323, 324, Asp224, 363, and Asp 494, while the hydrophobic part was composed of Lys169, Ala170, 366, Leu319, Cys322, and Met 637. The two Ni ions also played a vital role by linking the key amino acid and ligands. It has been observed that almost all the conformations of all the ligands showed interactions with key residues inside the pocket. The docked poses were ranked by the scores from the GBVI/WSA binding free energy calculation. The most promising docked conformation of each compound was further evaluated for binding mode. Detailed docking results are listed in Table 8.  Figure 5. Active inhibitors of the synthesized series.

Molecular Docking, Interactions Report
To predict the inhibition mechanism of isolated compounds shown by the kinet study, molecular docking of the active compounds was carried out with the crystal str ture of the urease enzyme. The most favorable docking conformations of all compoun were observed inside the active site with proper orientation. The active site consists both the hydrophobic and hydrophilic amino acids.
The hydrophilic amino acids included Glu166, 223, Arg339, His323, 324, Asp224, 3 and Asp 494, while the hydrophobic part was composed of Lys169, Ala170, 366, Leu3 Cys322, and Met 637. The two Ni ions also played a vital role by linking the key ami acid and ligands. It has been observed that almost all the conformations of all the ligan showed interactions with key residues inside the pocket. The docked poses were rank by the scores from the GBVI/WSA binding free energy calculation. The most promisi docked conformation of each compound was further evaluated for binding mode. D tailed docking results are listed in Table 8.   The binding mode of the active compounds with the active site residues showed a healthy assignation with the backbone of the enzyme using hydrogen bonds, polar bonds, pi-pi, and pi-H interactions. Three-dimensional interactions of some favorable inhibitors are shown in Figure 6. This showed that ligands occupy the active site residues, using hydrogen bonds, polar bonds, arene-cation, and metal ion interactions to engage the backbone of the enzyme tightly.

Materials and Methods
NMR experiments were performed on Avance-Bruker (400.15 MHz for 1 H). The spectra were recorded by HRESI-MS spectrometer (LCQ-DECA XP Plus, Thermo-F gan, San Diego, CA, USA). Thin layer chromatography (TLC) was performed on coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Darmstadt, Germ Chromatograms were visualized by UV visible light at 254 nm or iodine vapors. Highly pure analytical grade chemicals and solvents were procured from (D stadt, Germany) and used as received. Aryl benzaldehyde derivatives and 4-(t-Bu)be acid were purchased from Sigma (St Louis, MO, USA). Sodium hydroxide, soluble s maltose, and other chemicals were obtained from Merck (Darmstadt, Germany).
3.1.1. General Procedure for the Synthesis of methyl-4-(t-Bu)benzoate (2) and 4-(tert tyl) benzohydrazide (3) In the case of compound 10, the chloronitrotoluene part, NO 2 and NH are found actively interacting with the active site residues Cys 322, Met 367, and Ni 798 through Hydrogen bonding, π-Hydrogen, and metal ion interactions, respectively. The phenolic substituted derivative 24 is observed with lesser interactions compared with the chloronitrobenzene derivative 10 by performing two interactions with Lys 169 and Asp 363. Asp 363 established a Hydrogen donor interaction with the OH group of the ligand while Lys 169 formed the H-π interaction with the aromatic π system of the ligand. The hydrazine moiety presented an inert behavior that might be attributed to the negative inductive effect of these groups.
The dimethoxy substituted derivative, compound 25, showed better interactions and docking scores in accordance with the biological activity. Figure 5 reflects that hydrazine moiety in compound 25 performed Hydrogen bonding with Cys 322, and the substituted methoxy group established metal/ionic contact with Ni 798 of the target protein. Moreover, the two aromatic rings pi system interact through π-H bonding to Lys 169 and His 222.
The docking study complemented the experimental results based on the multiple interactions of ligands with key residues of the urease enzyme. The docking pose of the most active compounds, 10, 24, and 25, inhibited the catalytic activities of the urease by binding firmly through strong hydrogen bonding, hydrophobic, polar interactions with key residues, and metal/ion contact.
The molecular docking study of these compounds revealed that the ligands with polar, light, and electron-rich groups such as hydroxyl, alkoxy, and nitro groups showed better interaction mode and high docking scores against the target protein and therefore had good inhibitory activities. On the other hand, ligands with electron-withdrawing groups, non-polar such as methyl or benzene, and bulky groups have shown poor interactions and low docking scores.

Materials and Methods
NMR experiments were performed on Avance-Bruker (400.15 MHz for 1 H). The mass spectra were recorded by HRESI-MS spectrometer (LCQ-DECA XP Plus, Thermo-Finnigan, San Diego, CA, USA). Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Darmstadt, Germany). Chromatograms were visualized by UV visible light at 254 nm or iodine vapors.
Highly pure analytical grade chemicals and solvents were procured from (Darmstadt, Germany) and used as received. Aryl benzaldehyde derivatives and 4-(t-Bu)benzoic acid were purchased from Sigma (St Louis, MO, USA). Sodium hydroxide, soluble starch, maltose, and other chemicals were obtained from Merck (Darmstadt, Germany).
3.1.2. General Procedure for the Synthesis of N-Acylhydrazones 4-(t-But)benzohydrazide  Twenty-three different novel hydrazones (4-26) of 4-(t-But)benzohydrazide 3 were synthesized in good to excellent yields. In a typical reaction, 4-(tert-butyl)benzohydrazide 3 (0.192 g, 0.001 mmol) was dissolved in methanol (10 mL) containing a catalytic amount of acetic acid, then various substituted aromatic aldehydes (0.001 mmol) were added to the flask and refluxed it for 4-6 h. The reaction mixture was decanted into a beaker containing crushed ice. Precipitates were formed, filtered, and washed with an excess of water and hot n-hexane, and the products were dried and collected [21]. All the synthesized derivatives were characterized by HRESI-MS and 1 H-NMR spectroscopy. Furthermore, we have already reported the crystal structure of compound 4 [22].

Urease Inhibition Assay
The measurement of urease inhibitory activity was carried out according to the literature method [33]. The assay mixture containing 75 µL of Jack bean urease and 75 µL of tested compounds with various concentrations (dissolved in DMSO) was pre-incubated for 15 min on a 96-well assay plate. Acetohydroxamic acid was used as a reference. Then 75 µL of phosphate buffer at pH 6.8 containing phenol red (0.18 mmol·L −1 ) and urea (400 mmol·L −1 ) were added and incubated at room temperature. The reaction time re-quired for enough ammonium carbonate to form to raise the pH phosphate buffer from 6.8 to 7.7 was measured by a microplate reader (560 nm), with the end-point being determined by the color change of the phenol-red indicator.

Docking Methodology
A molecular docking study was performed according to the reported methodology [34] to predict the binding mode of the active synthesized compounds in the active site of the urease enzyme using MOE (Molecular Operating Environment). The three-dimensional structures of the isolated compounds were built using the builder tool implemented in MOE software. The generated compounds were 3D protonated, and energy minimized using the default parameters of the MOE (gradient: 0.05, Force Field: MMFF94X). 3D structure of the target protein was retrieved from the protein databank (https://www.rcsb.org/ accessed on 4 August 2022) (PDB ID 4ubp), the solvent molecules were removed, and 3D protonation was carried out. To get a stable conformation of the protein molecule, 3D protonation of the protein was energy minimized using the default parameters of MOE. For docking studies, the parameters of MOE used were Placement: Triangle Matcher, Rescoring 1: London dG, Refinement: Forcefield, Rescoring 2: GBVI/WSA. For each ligand, 10 conformations were allowed to be formed, and the top-ranked conformations on the basis of docking score were selected for further analysis.

Crystal Structure Determination
Crystals 1, 5, and 14 were mounted on a glass fiber in inert paraffin oil. Data were recorded at 170 K on an STOE-IPDS 2T diffractometer with graphite-monochromated Mo-K α -radiation (λ = 0.71073 Å). The program XArea was used to integrate diffraction profiles; numerical absorption corrections were carried out with the programs X-Shape and X-Red32, all from STOE© version 2010. The structures were solved by dual space methods (SHELXT-2016) [35] and refined by full-matrix least-squares techniques using the WingX GUI [36] and SHELXL-2018 [37]. All non-hydrogen atoms were refined with anisotropic displacement parameters. The hydrogen atoms were refined isotropically on calculated positions using a riding model with their U iso values constrained to 1.5 U eq of their pivot atoms for terminal sp 3 carbon atoms and 1.2 times for all other carbon atoms.