Synthesis , biological evaluation and molecular docking studies of Mannich bases derived from 1 , 3 , 4-oxadiazole-2-thiones as potential urease inhibitors

Purpose: To design and synthesize a series of new structural motifs of urease inhibitors, 3[{(substituted phenyl) amino} methyl]-5-(3, 4, 5-trimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones and 3{[(pyridin-2-yl)amino]methyl}-5-(3,4,5-trimethoxy phenyl)-1,3,4-oxadiazole-2(3H)-thiones from 1, 3, 4oxadiazole-2-thione. Methods: Targeted Mannich base derivatives were synthesized by the reaction of 1, 3, 4-oxadiazole-2thione with formaldehyde and respective aromatic amines. These structural motifs were subjected to H–NMR, C–NMR and mass spectrometric analysis. Compound 4, i.e., 1,3,4-oxadiazole-2-thione and its corresponding Mannich bases (5-17) were subjected to in silico screening as urease inhibitors, using crystal structure of urease (Protein Data Bank ID: 5FSE) as a model enzyme. Furthermore, the targeted compounds were evaluated for their in vitro urease inhibition and anti-oxidant activities using thiourea and propyl gallate as standards, respectively. Results: The docking score of targeted compounds predicted that they are promising urease inhibitors. Subsequently, in vitro studies on Jack bean urease supported the results from virtual screening, and found compounds 4, 5, 9,10,12, 13, 14 and 15 very potent urease inhibitors with half-maximal inhibitory concentration (IC50) values in the range of 5.93 ± 0.13 to 9.76 ± 0.11, relative to thiourea (IC50 = 21.25 ± 0.15). Compounds 4 – 6, and compounds 12 17 also exhibited higher antioxidant activities than propyl gallate. Conclusion: In view of their potent urease inhibition and antioxidant activities, these structural motifs have potentials as new candidates for the development of anti-ulcer drugs.


INTRODUCTION
Compounds derived from 1,3,4-oxadiazole-2thione have received attention as new structural motifs for the design and development of novel drugs [1].Compounds with 1,3,4-oxadiazole-2thiones cores have occupied a specific place in medicinal and synthetic chemistry because of their extensive range of biological activities such as antibacterial [2], antifungal, anti-inflammatory, antiviral, anticancer [1], enzyme inhibitor [3], anticonvulsant and anti-diabetic properties [4].The 1,3,4-oxadiazole nucleus undergoes a variety of chemical reactions such as electrophilic and nucleophilic substitutions, as well as photochemical and thermal reactions [5].These properties make it a desirable medicinal backbone which can be used to construct biologically-useful molecules.A number of 1, 3, 4-oxadiazole-2-thione derivatives are used in clinical medicine as antiviral, anticancer, antihypertensive and antibiotic agents.These include Raltegravir ® , Zibotentan ® , Tiodazosin, Nesapidel and Furamizole [2].Urea is hydrolyzed to ammonia and carbon dioxide in the presence of urease (urea amidohydrolase) [6,7].The ammonia produced as by-product shows pathogenic-like behavior in animals and humans, leading to hepatic coma, peptic ulcer, hepatic encephalopathy, pyelonephritis and encrustation in the urinary tract [8].Helicobacter pylori is an acid-sensitive, gram-negative bacterium which survives only in the pH range of 7-8.This acidsensitive bacterium is able to thrive in the low pH environment of the stomach with the help of urease, which is produced in a highly active form and increases the pH of human stomach through the hydrolysis of urea [9].
Urease is a virulent factor for H. pylori and contributes to mucosal damage in the stomach, gastro-duodenal infection, peptic ulcers and gastric cancer [10].Thus, the inhibition of urease has attracted much attention as potential strategy for designing novel drugs against ulcer.More effective and more potent compounds with a whole new level of safety and specificity are still desired [11].In this regard, high throughput virtual and in vitro biological assay of molecules to understand their molecular behaviour in specific environments, will aid the identification of potent molecules from a crude cocktail [12].Recently, 1,3,4-oxadiazole-2-thiones derivatives have been investigated as active jack bean urease inhibitors with promising anti-urease activities [13].The aim of the current study was to synthesize Mannich bases derived from 1, 3, 4-oxadiazole-2-thione bearing 3, 4, 5-trimethoxy moiety and examine them as potential urease inhibitors and antioxidant agents.

EXPERIMENTAL Analytical procedures
Chemicals and reagents used for the synthesis of target compounds were purchased from Sigma Aldrich and Merck (Lahore, Pakistan).The purity of the synthesized structures and progress of the reactions were monitored using pre-coated silica gel 60 F 254 aluminium TLC plates (Merck).Their melting points were measured with Gallenkamp apparatus, while IR spectra were obtained on Bio-Red Merlin or Bruker.The 1 H -NMR of each synthesized compound was recorded at a frequency 300 MHz on Bruker AM 300 or at a frequency of 500 MHz on Agilent Technologies spectrophotometer using CDCl 3 or DMSO-d 6 as solvent.The J values of the analyzed compounds are presented in Hertz (Hz).The spectra of 13 C -NMR were recorded at a frequency of 75.5 MHz or 125 MHz.EIMS spectra were obtained using Agilent TOF-6220 analyzer, while the elements were analyzed on a Perkin Elmer 2400 CHN instrument.

Synthesis of structural motifs
The strategy for the synthesis of compounds 2 -17 is summarized in Scheme Formalin (37 %, 0.002 moles) and 0.002 moles of relevant amine were reacted with 0.002 moles 5-(3, 4, 5-trimethoxyphenyl)-1, 3, 4-oxadizole-2thiones in ethanol (about 40 ml), and vortexed for 1 -6 h at 30 °C.The progress of the reaction was monitored by TLC, and at the end, the mixture was cooled overnight.Thereafter, the resultant sediments were recovered by filtration, rinsed in an appropriate solvent and subjected to re-crystallisation in alcohol.Compounds 5 -17 were identified through IR spectroscopy by broad bands in the range 3387 -3410 cm -1 which is unique to -NH group.Peaks were seen between 1615 to 1665 cm -1 obviously from stretches in C=N, and also between 1145 to1225 cm -1 because of C=S stretches.Product formation was depicted in 1 H -NMR spectroscopy by NH proton peak between 5.4 and 5.9 ppm and a -CH 2 peak close to 5.4 ppm.
For compounds 6 -7 and 15 -17, the -NH proton peak appeared near 8.4 ppm (when electron withdrawing group i.e.NO 2 was attached to phenyl ring at ortho position with respect to -NH proton).The CH 2 and NH protons coupled with each other to give rise to doublet and triplet (J = 6.8-7.5 Hz) respectively.
Product formation was confirmed by the appearance of amino methyl signals, since amino methylene is formed at the last stage of the reaction.Aromatic phenyl protons appeared at 6.45 -8.16 ppm and de-shielded by the ring current effect.Protons from the OCH 3 group resonated between 3. 81 and 3.95 ppm, whereas methyl protons were found between 2.38 and 2.51 ppm up the field.In 13 C -NMR analysis, CH 2 peak at 57.6 -58.4-ppm confirmed the amino methyl function.
However, this was absent in the reactant 4.These results were strengthened by DEPT spectra which showed different peaks (same or opposite sides of the solvent signal) for different types of carbons.Two significant peaks observed at 176 and 159 ppm due to carbon linkage with sulphur and nitrogen atoms, respectively.The signals of all carbon atoms of the aromatic ring were observed in region 101 -153 ppm downfield.The carbon atoms of methyl and methoxy groups attached to aromatic ring appeared at 17.08 -21.29 ppm and 56.46 -61.14 respectively.Unambiguous confirmation of the structure (4) was obtained by X-ray crystallographic analysis [14].

Urease assay and assessment of inhibition
Compounds 4 -17 were screened for ability to inhibit urease in vitro by the phenol hypochlorite method described by Weatherburn [15].In 96well plate, 25 μl jack bean urease enzyme solution was incubated with reaction mixture containing 55 μl of phosphate buffer, 5 μl of test compound followed by 15 μl of 100 mM urea at 37°C for 15 min.For colour appearance, 100 μl of phenol-hypochlorite reagent was added in each well followed by incubation at 37°C for 30 min.The results were evaluated by calculating change in absorbance taken by 96 wells plate reader at 620 nm and by using formula 100 -[(OD of test / OD of control) × 100.The entire assay was performed in triplicate at pH 7 and using thiourea as standard inhibitor of urease.

Determination of antioxidant activity
DPPH scavenging activity was measured using a slight modification of the method in the literature [16,17].

Docking studies
The interactions of 1,3,4-oxadiazole-2-thione derivatives with urease were studied theoretically through docking experiments, which were performed using AutoDockTools version 1.5.6 (ADT) software [18].Default ten docking runs were set for all compounds to observe their interactions with active site of urease molecule.Ligand and receptor binding energy/affinity were calculated with ADT software package by using search parameter Genetic Algorithm.Ligand structure was optimized using Avogadro software [19].
Sporosarcina pasteurii (formerly Bacillus pasteurii) urease (SPU) [20] file with PDB code: 5FSE (contain three protein chains designated as A, B, and C) was downloaded in PDB format from RCSB Protein Data Bank, which was further processed for docking studies by isolating chain-C which play vital role in urease activity and contain active pockets [21].Chain-A and chain-B along with non-protein fragments e.g.ligand and solvent molecules were removed using BIOVIA Discovery Studio Visualizer v16.1.0.15350.Docking analysis was done using ADT.Hydrogens were added and non-polar hydrogen were merged in receptor molecule (5FSE chain-C).Moreover, the Kollman charges were also added to the receptor molecule.For docking, the receptor macromolecule was considered as a rigid structure.AutoGrid program was used for generating 58 × 60 × 70 Å grid points and 0.254 Å spacing for affinity (grid) maps, while ADT default parameter and functions, along with all possible torsions of the ligand molecule were used in the electrostatic, bonding and energy calculations.Lamarckian genetic algorithm (LGA 4.2) was used for docking simulations.Ten different binding conformations of ligand with receptor were obtained with their respective binding energies/affinities.The pose with strongest binding affinity to receptor (out of the ten) was chosen as the most stable one and further employed in the post-docking analysis.

Statistical analysis
Biological studies were performed at five different concentrations in order to calculate IC 50 values using linear regression method with Graphpad Prism 5.The results are shown as mean ± SEM (n = 3).

Docking analysis data
Docking scores (Table 1) i.e. kinase inhibition values (KI), binding energy and interactions of the synthesized compounds 4 -17 in active pocket of receptor urease enzyme (PDB ID: 5FSE Chain-C) predicted these compounds as promising urease inhibitors, which was further supported by the experimental results (Table 2).In docking studies, it was observed that compound 5 (Figure 1 -2) and 13 form hydrogen bond with HIS323 (Table 3) which is next to CYS322 blocked by quinone known as urease inhibitor [21].

Urease inhibition
The virtual screening of compounds 4 -17 as promising urease inhibitors was further supported by their in vitro urease inhibition against Jack bean urease in an assay using standard inhibitor thiourea, in which thiourea had an IC 50 value of 21.25 ± 0.15 μM.Almost all the synthesized derivatives (twelve out of fourteen) of this series exhibited remarkable urease inhibitory potency superior to that of thiourea (Table 2).The interaction of compound 5 (representative) with the active site of urease is depicted in Figure 1, while the active site groups involved in hydrogen bonding are shown in Figure 2.

Antioxidant activity
The DPPH radical scavenging activity is a standard assay in antioxidant activity measurements.In the current study, the standard antioxidant compounds propyl gallate and quercetin were used as positive controls for comparison with the tested compounds.The antioxidant activities of the synthesized compounds are shown in Table 4.

DISCUSSION
In silico studies showed that all the ligands interact with active site and have ability to occupy it like quinone.Predicted hydrogen bonding (Table 3) also forecast that these molecules have ability to inhibit urease activity permanently by bonding to the active site, as depicted by representative molecule 5.

Figure 1 :
Figure 1: Ligand 5 represented with stick structure inside the active site of urease (PDB code: 5FSE) depicted in MMS form [22].

Figure 2 :
Figure 2: Hydrogen bonding (represented by dotted line) and interacting sites of urease (PDB code: 5FSE Chain-C) with ligand 5 represented by structure contain magenta colour stick[18]

Table 3 :
[18]ptor molecule (PDB code: 5FSE Chain-C) active site interactions with compounds 4 -17 as predicted through ADT[18]The urease inhibition activities of all the synthesized compounds ranged in IC 50 values from 5.93 μM to 27.64 μM.The results indicate that 3, 4, 5-trimethoxy-substituted benzene next to oxadiazole ring shows excellent urease inhibition.Among the investigated compounds, compound 4 bearing methoxy groups at 3,4,5 positions in the benzene ring, was observed as the most effective urease inhibitor, with IC 50 value of 5.93 ± 0.13 μM.Compound 5 bearing methyl group at position 2, and nitro group at position 5 of the benzene ring showed stronger inhibitory activity close to 6.35 μM, when compared with compound 7 with a methyl group at position 4 and nitro group at position 2, which was less active.
Compound 9 bearing electron-donating methoxy group at p-position and electron-withdrawing nitro group at o-position was slightly more active (IC 50 value of 8.54 ± 0.17 μM) than compounds 8 and 11 having electron-donating methoxy group