An optical urea biosensor was fabricated by stacking several layers of sol-gelfilms. The stacking of the sol-gel films allowed the immobilization of a Nile Bluechromoionophore (ETH 5294) and urease enzyme separately without the need of anychemical attachment procedure. The absorbance response of the biosensor was monitoredat 550 nm, i.e. the deprotonation of the chromoionophore. This multi-layer sol-gel filmformat enabled higher enzyme loading in the biosensor to be achieved. The urea opticalbiosensor constructed from three layers of sol-gel films that contained urease demonstrateda much wider linear response range of up to 100 mM urea when compared with biosensorsthat constructed from 1-2 layers of films. Analysis of urea in urine samples with thisoptical urea biosensor yielded results similar to that determined by a spectrophotometricmethod using the reagent p-dimethylaminobenzaldehyde (R² = 0.982, n = 6). The averagerecovery of urea from urine samples using this urea biosensor is approximately 103%.
In search of potent urease inhibitor indole analogues (1-22) were synthesized and evaluated for their urease inhibitory potential. All analogues (1-22) showed a variable degree of inhibitory interaction potential having IC50 value ranging between 0.60 ± 0.05 to 30.90 ± 0.90 µM when compared with standard thiourea having IC50 value 21.86 ± 0.90 µM. Among the synthesized analogues, the compounds 1, 2, 3, 5, 6, 8, 12, 14, 18, 20 and 22 having IC50 value 3.10 ± 0.10, 1.20 ± 0.10, 4.60 ± 0.10, 0.60 ± 0.05, 5.30 ± 0.20, 2.50 ± 0.10, 7.50 ± 0.20, 3.90 ± 0.10, 3.90 ± 0.10, 2.30 ± 0.05 and 0.90 ± 0.05 µM respectively were found many fold better than the standard thiourea. All other analogues showed better urease interaction inhibition. Structure activity relationship (SAR) has been established for all analogues containing different substituents on the phenyl ring. To understand the binding interaction of most active analogues with enzyme active site docking study were performed.Communicated by Ramaswamy H. Sarma.
Thiourea derivatives (1-38) were synthesized and evaluated for their urease inhibition potential. The synthetic compounds showed a varying degree of in vitro urease inhibition with IC50 values 5.53 ± 0.02-91.50 ± 0.08 μM, most of which are superior to the standard thiourea (IC₅₀ = 21.00 ± 0.11 μM). In order to ensure the mode of inhibition of these compounds, the kinetic study of the most active compounds has been carried out. Most of these inhibitors were found to be mixed-type of inhibitors, except compounds 13 and 30 which were competitive, while compound 19 was identified as non-competitive inhibitor with Ki values between 8.6 and 19.29 μM.
In this study 36 new compounds were synthesized by condensing barbituric acid or thiobarbituric acid and respective anilines (bearing different substituents) in the presence of triethyl orthoformate in good yields. In vitro urease inhibition studies against jack bean urease revealed that barbituric acid derived compounds (1-9 and 19-27) were found to exhibit low to moderate activity however thiobarbituric acid derived compounds (10-18 and 28-36) showed significant inhibition activity at low micro-molar concentrations. Among the synthesized compounds, compounds (15), (12), (10), (36), (16) and (35) showed excellent urease inhibition with IC50 values 8.53 ± 0.027, 8.93 ± 0.027, 12.96 ± 0.13, 15 ± 0.098, 18.9 ± 0.027 and 19.7 ± 0.63 μM, respectively, even better than the reference compound thiourea (IC50 = 21 ± 0.011). The compound (11) exhibited comparable activity to the standard with IC50 value 21.83 ± 0.19 μM. In silico molecular docking studies for most active compounds (10), (12), (15), (16), (35) and (36) and two inactive compounds (3) and (6) were performed to predict the binding patterns.
Cellulose nanomaterial with rod-like structure and highly crystalline order, usually formed by elimination of the amorphous region from cellulose during acid hydrolysis. Cellulose nanomaterial with the property of biocompatibility and nontoxicity can be used for enzyme immobilization. In this work, urease enzyme was used as a model enzyme to study the surface modification of cellulose nanomaterial and its potential for biosensor application. The cellulose nanocrystal (CNC) surface was modified using 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation to introduce the carboxyl group at C6 primary alcohol. The success of enzyme immobilization and surface modification was confirmed using chemical tests and measured using UV-Visible spectrophotometer. The immobilization strategy was then applied for biosensor application for urea detection. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were used for electroanalytical characterization of the urea biosensor.
Urease is a potent metalloenzyme with diverse applications. This paper describes the scale up and purification of an extracellular urease from Arthrobacter creatinolyticus MTCC 5604. The urease production was scaled-up in 3.7 L and 20 L fermentor. A maximum activity of 27 and 27.8 U/mL and a productivity of 0.90 and 0.99 U/mL/h were obtained at 30 h and 28 h in 3.7 and 20 L fermentor, respectively. Urease was purified to homogeneity with 49.85-fold purification by gel filtration and anion exchange chromatography with a yield of 36% and a specific activity of 1044.37 U/mg protein. The enzyme showed three protein bands with molecular mass of 72.6, 11.2 and 6.1 kDa on SDS-PAGE and ~ 270 kDa on native PAGE. The cytotoxic effect of urease was assessed in vitro using cancer cell lines (A549 and MG-63) and normal cell line (HEK 293). Urease showed its inhibitory effects on cancer cell lines through the generation of toxic ammonia, which in turn increased the pH of the surrounding medium. This increase in extracellular pH, enhanced the cytotoxic effect of weak base chemotherapeutic drugs, doxorubicin (50 µM) and vinblastine (100 µM) in the presence of urease (5 U/mL) and urea (0-4 mM) significantly.
A series of hydrazinecarboxamide derivatives were synthesized and examined against urease for their inhibitory activity. Among the series, the 1-(3-fluorobenzylidene)semicarbazide (4a) (IC50 = 0.52 ± 0.45 µM), 4u (IC50 = 1.23 ± 0.32 µM) and 4h (IC50 = 2.22 ± 0.32 µM) were found most potent. Furthermore, the molecular docking study was also performed to demonstrate the binding mode of the active hydrazinecarboxamide with the enzyme, urease. In order to estimate drug likeness of compounds, in silico ADME evaluation was carried out. All compounds exhibited favorable ADME profiles with good predicted oral bioavailability.
A series of thiobarbituric acid derivatives 1-27 were synthesized and evaluated for their urease inhibitory potential. Exciting results were obtained from the screening of these compounds 1-27. Compounds 5, 7, 8, 11, 16, 17, 22, 23 and 24 showed excellent urease inhibition with IC50 values 18.1 ± 0.52, 16.0 ± 0.45, 16.0 ± 0.22, 14.3 ± 0.27, 6.7 ± 0.27, 10.6 ± 0.17, 19.2 ± 0.29, 18.2 ± 0.76 and 1.61 ± 0.18 μM, respectively, much better than the standard urease inhibitor thiourea (IC₅₀=21 ± 0.11 μM). Compound 3, 4, 10, and 26 exhibited comparable activities to the standard with IC₅₀ values 21.4 ± 1.04 and 21.5 ± 0.61 μM, 22.8 ± 0.32, 25.2 ± 0.63, respectively. However the remaining compounds also showed prominent inhibitory potential The structure-activity relationship was established for these compounds. This study identified a novel class of urease inhibitors. The structures of all compounds were confirmed through spectroscopic techniques such as EI-MS and (1)H NMR.
Benzothiazole analogs (1-20) have been synthesized, characterized by EI-MS and (1)H NMR, and evaluated for urease inhibition activity. All compounds showed excellent urease inhibitory potential varying from 1.4±0.10 to 34.43±2.10μM when compared with standard thiourea (IC50 19.46±1.20μM). Among the series seventeen (17) analogs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, and 18 showed outstanding urease inhibitory potential. Analogs 15 and 19 also showed good urease inhibition activity. When we compare the activity of N-phenylthiourea 20 with all substituted phenyl derivatives (1-18) we found that compound 15 showed less activity than compound 20 having 3-methoxy substituent. The binding interactions of these active analogs were confirmed through molecular docking.
Despite of many diverse biological activities exhibited by benzimidazole scaffold, it is rarely explored for the urease inhibitory potential. For that purpose, benzimidazole analogues 1-19 were synthesized and screened for in vitro urease inhibitory potential. Structures of all synthetic analogues were deduced by different spectroscopic techniques. All analogues revealed inhibition potential with IC50 values of 0.90 ± 0.01 to 35.20 ± 1.10 μM, when compared with the standard thiourea (IC50 = 21.40 ± 0.21 μM). Limited SAR suggested that the variations in the inhibitory potentials of the analogues are the result of different substitutions on phenyl ring. In order to rationalize the binding interactions of most active compounds with the active site of urease enzyme, molecular docking study was conducted.
3,4-Dimethoxybenzohydrazide derivatives (1-25) have been synthesized and evaluated for their urease inhibitory potential. Among the series, compounds 2, 3, 4 and 5 with IC50 values 12.61 ± 0.07, 18.24 ± 0.14, 19.22 ± 0.21, and 8.40 ± 0.05 µM, respectively, showed excellent urease inhibitory potentials when compared with standard thiourea (IC50 value 21.40 ± 0.21 µM). Compounds 1, 6, 8, 18, 19 and 20 also showed good to moderate inhibition, while the remaining compounds were found to be completely inactive. The structures of compounds 6 and 25 were confirmed through X-ray crystallography while the structures of remaining compounds were confirmed through ESI-MS and 1H NMR. Molecular docking studies were performed understand the binding interactions with enzyme active site. The synthesized compounds were evaluated for cytotoxicity and found to be nontoxic.
The gastric biopsy urease test is an accurate and robust diagnostic test for Helicobacter pylori infection. Large endoscopy units use their own homemade unbuffered ultra-rapid urease test for diagnosis of H. pylori infection but several commercial rapid urease tests are available.
Sixteen 4-hydroxycoumarin derivatives were synthesized, characterized through EI-MS and (1)H NMR and screened for urease inhibitory potential. Three compounds exhibited better urease inhibition than the standard inhibitor thiourea (IC50=21±0.11μM) while other four compounds exhibited good to moderate inhibition with IC50 values between 29.45±1.1μM and 69.53±0.9μM. Structure activity relationship was established on the basis of molecular docking studies, which helped to predict the binding interactions of the most active compounds.
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.
In our previous report, we have identified 3,4-dihydropyrimidine scaffold as promising class of urease inhibitor in a structure based virtual screen (SBVS) experiment. In present study, we attempted to optimize the scaffold by varying C-5 substituent. The elongation of the C-5 chain was achieved by the reaction of C-5 ester with hydrazine leading to C-5 carbohydrazides which were further used as building blocks for the synthesis of fifteen new compounds having diverse moieties. A significantly higher in vitro urease inhibitory activity with IC50 values in submicromolar range was observed for semithiocarbazide derivatives (4a-c, 0.58-0.79µM) and isatin Schiff base derivative 5a (0.23µM). Docking analysis suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket and thus restrict the mobility of the flap by interacting with its key amino acid residues. The overall results of urease inhibition have shown that these compounds can be further optimized and developed as lead urease inhibitors.
The present article describes the synthesis, in vitro urease inhibition and in silico molecular docking studies of a novel series of bi-heterocyclic bi-amides. The synthesis of title compounds was initiated by benzoylation, with benzoyl chloride (1), of the key starter ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (2) in weak basic aqueous medium followed by hydrazide formation, 4, and cyclization with CS2 to reach the parent bi-heterocyclic nucleophile, N-{4-[(5-sulfanyl-1,3,4-oxadiazol-2-yl)methyl]-1,3-thiazol-2-yl}benzamide (5). Various electrophiles, 8a-l, were synthesized by a two-step process and these were finally coupled with 5 to yield the targeted bi-heterocyclic bi-amide molecules, 9a-l. The structures of the newly synthesized products were corroborated by IR, 1H NMR, 13C NMR, EI-MS and elemental analysis. The in vitro screening of these molecules against urease explored that most of the compounds exhibit potent inhibitory potential against this enzyme. The compound 9j, with IC50 value of 2.58 ± 0.02 µM, exhibited most promising inhibitory activity among the series, relative to standard thiourea having IC50 value of 21.11 ± 0.12 µM. In silico studies fully augmented the experimental enzyme inhibition results. Chemo-informatics analysis showed that synthesized compounds (9a-l) mostly obeyed the Lipinski's rule. Molecular docking study suggested that ligand 9j exhibited good binding energy value (-7.10 kcal/mol) and binds within the active region of target protein. So, on the basis of present investigation, it was inferred that 9j may serve as a novel scaffold for designing more potent urease inhibitors.
On the basis of previous report on promising α-glucosidase inhibitory activity of 5-bromo-2-aryl benzimidazole derivatives, these derivatives were further screened for urease inhibitory and cytotoxicity activity in order to get more potent and non-cytotoxic potential dual inhibitor for the patients suffering from diabetes as well as peptic ulcer. In this study, all compounds showed varying degree of potency in the range of (IC50=8.15±0.03-354.67±0.19μM) as compared to standard thiourea (IC50=21.25±0.15μM). It is worth mentioning that derivatives 7 (IC50=12.07±0.05μM), 8 (IC50=10.57±0.12μM), 11 (IC50=13.76±0.02μM), 14 (IC50=15.70±0.12μM) and 22 (IC50=8.15±0.03μM) were found to be more potent inhibitors than standard. All compounds were also evaluated for cytotoxicity towards 3T3 mouse fibroblast cell line and found to be completely non-toxic. Previously benzimidazole 1-25 were also showed α-glucosidase inhibitory potential. In silico studies were performed on the lead molecules i.e.2, 7, 8, 11, 14, and 22, in order to rationalize the binding interaction of compounds with the active site of urease enzyme.
Bisindolylmethane thiosemicarbazides 1-18 were synthesized, characterized by 1H NMR and ESI MS and evaluated for urease inhibitory potential. All analogs showed outstanding urease inhibitory potentials with IC50 values ranging between 0.14 ± 0.01 to 18.50 ± 0.90 μM when compared with the standard inhibitor thiourea having IC50 value 21.25 ± 0.90 μM. Among the series, analog 9 (0.14 ± 0.01 μM) with di-chloro substitution on phenyl ring was identified as the most potent inhibitor of urease. The structure activity relationship has been also established on the basis of binding interactions of the active analogs. These binding interactions were identified by molecular docking studies.
4-Thiazolidinone analogs 1-20 were synthesized, characterized by (1)H NMR and EI-MS and investigated for urease inhibitory activity. All twenty (20) analogs exhibited varied degree of urease inhibitory potential with IC50 values 1.73-69.65μM, if compared with standard thiourea having IC50 value of 21.25±0.15μM. Among the series, eight derivatives 3, 6, 8, 10, 15, 17, 19, and 20 showed outstanding urease inhibitory potential with IC50 values of 9.34±0.02, 14.62±0.03, 8.43±0.01, 7.3±0.04, 2.31±0.002, 5.75±0.003, 8.81±0.005, and 1.73±0.001μM, respectively, which is better than the standard thiourea. The remaining analogs showed good to excellent urease inhibition. The binding interactions of these compounds were confirmed through molecular docking studies.