Synthesis and biological evaluation of triazolones/oxadiazolones as novel urease inhibitors.

Urease is the main virulence factor of infectious gastritis and gastric ulcers. Urease inhibitors are regarded as the first choice for the treatment of such diseases. Based on the triazolone/oxadiazolone skeleton, a urea-like fragment being able to specifically bind the urease activity pocket and prevent urea from hydrolysis, we designed and synthesized 45 triazolones/oxadiazolones as urease inhibitors. Eight compounds were proved to show excellent inhibitory activity against Helicobacter pylori urease, being more potency than the clinically used urease inhibitor acetohydroxamic acid. The most active inhibitor with IC50 value of 1.2 µM was over 20-fold higher potent than the positive control. Enzymatic kinetic assays showed that these novel inhibitors reversibly inhibited urease with a mixed competitive mechanism. Molecular dockings provided evidence for the observations in enzyme assays. Furthermore, these novel inhibitors were proved as drug-like compounds with very low cytotoxicity to mammalian cells and favorable water solubility. These results suggested that triazolone and oxadiazolone were promising scaffolds for the design and discovery of novel urease inhibitors, and were expected as good candidates for further drug development.

Synergizing structure and function: Cinnamoyl hydroxamic acids as potent urease inhibitors.

The current investigation encompasses the structural planning, synthesis, and evaluation of the urease inhibitory activity of a series of molecular hybrids of hydroxamic acids and Michael acceptors, delineated from the structure of cinnamic acids. The synthesized compounds exhibited potent urease inhibitory effects, with IC50 values ranging from 3.8 to 12.8 µM. Kinetic experiments unveiled that the majority of the synthesized hybrids display characteristics of mixed inhibitors. Generally, derivatives containing electron-withdrawing groups on the aromatic ring demonstrate heightened activity, indicating that the increased electrophilicity of the beta carbon in the Michael Acceptor moiety positively influences the antiureolytic properties of this compounds class. Biophysical and theoretical investigations further corroborated the findings obtained from kinetic assays. These studies suggest that the hydroxamic acid core interacts with the urease active site, while the Michael acceptor moiety binds to one or more allosteric sites adjacent to the active site.

Epiberberine: a potential rumen microbial urease inhibitor to reduce ammonia release screened by targeting UreG.

Rumen microbial urease inhibitors have been proposed for regulating nitrogen emission and improving nitrogen utilization efficiency in ruminant livestock industry. However, studies on plant-derived natural inhibitors of rumen microbial urease are limited. Urease accessory protein UreG, plays a crucial role in facilitating urease maturation, is a new target for design of urease inhibitor. The objective of this study was to select the potential effective inhibitor of rumen microbial urease from major protoberberine alkaloids in Rhizoma Coptidis by targeting UreG. Our results showed that berberine chloride and epiberberine exerted superior inhibition potential than other alkaloids based on GTPase activity study of UreG. Berberine chloride inhibition of UreG was mixed type, while inhibition kinetics type of epiberberine was uncompetitive. Furthermore, epiberberine was found to be more effective than berberine chloride in inhibiting the combination of nickel towards UreG and inducing changes in the second structure of UreG. Molecular modeling provided the rational structural basis for the higher inhibition potential of epiberberine, amino acid residues in G1 motif and G3 motif of UreG formed interactions with D ring of berberine chloride, while interacted with A ring and D ring of epiberberine. We further demonstrated the efficacy of epiberberine in the ruminal microbial fermentation with low ammonia release and urea degradation. In conclusion, our study clearly indicates that epiberberine is a promising candidate as a safe and effective inhibitor of rumen microbial urease and provides an optimal strategy and suitable feed additive for regulating nitrogen excretion in ruminants in the future. KEY POINTS: • Epiberberine is the most effective inhibitor of rumen urease from Rhizoma Coptidis. • Urease accessory protein UreG is an effective target for design of urease inhibitor. • Epiberberine may be used as natural feed additive to reducing NH3 release in ruminants.

Molecular design of hydroxamic acid-based derivatives as urease inhibitors of Helicobacter pylori.

Helicobacter pylori is the main causative agent of gastric cancer, especially non-cardiac gastric cancers. This bacterium relies on urease producing much ammonia to colonize the host. Herein, the study provides valuable insights into structural patterns driving urease inhibition for high-activity molecules designed via exploring known inhibitors. Firstly, an ensemble model was devised to predict the inhibitory activity of novel compounds in an automated workflow (R2 = 0.761) that combines four machine learning approaches. The dataset was characterized in terms of chemical space, including molecular scaffolds, clustering analysis, distribution for physicochemical properties, and activity cliffs. Through these analyses, the hydroxamic acid group and the benzene ring responsible for distinct activity were highlighted. Activity cliff pairs uncovered substituents of the benzene ring on hydroxamic acid derivatives are key structures for substantial activity enhancement. Moreover, 11 hydroxamic acid derivatives were designed, named mol1-11. Results of molecular dynamic simulations showed that the mol9 exhibited stabilization of the active site flap's closed conformation and are expected to be promising drug candidates for Helicobacter pylori infection and further in vitro, in vivo, and clinical trials to demonstrate in future.

Global warming potential assessment under reclaimed water and livestock wastewater irrigation coupled with co-application of inhibitors and biochar.

The application of nitrification inhibitors (nitrapyrin) and urease inhibitors (N-(N-butyl) thiophosphoric triamide) under conventional water resources has been considered as an effective means to improve nitrogen utilization efficiency and mitigate soil greenhouse gas emissions. However, it is not known whether the inhibitors still have an inhibitory effect under unconventional water resources (reclaimed water and livestock wastewater) irrigation and whether their use in combination with biochar improves the mitigation effect. Therefore, unconventional water resources were used for irrigation, with groundwater (GW) control. Nitrapyrin and N-(N-butyl) thiophosphoric triamide were used alone or in combination with biochar in a pot experiment, and CO2, N2O, and CH4 emissions were measured. The results showed that irrigation of unconventional water resources exacerbated global warming potential (GWP). All exogenous substance treatments increased CO2 and CH4 emissions and suppressed N2O emissions, independent of the type of water, compared to no substances (NS). The inhibitors were ineffective in reducing the GWP whether or not in combination with biochar, and the combined application of inhibitors with biochar further increased the GWP. This study suggests that using inhibitors and biochar in combination to regulate the greenhouse effect under unconventional water resources irrigation should be done with caution.

Identification of (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids as novel urease inhibitors and the mechanism exploration.

Thirty-three (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids were synthesized in an effort to develop novel urease inhibitors. Among these compounds, 2-(N-(3-nitrophenyl)-N-(4-tert-butylphenylsulfonyl))aminoacetohydroxamic acid (e2) exhibited excellent inhibitory activity against Helicobacter pylori urease with no perceptible cytotoxicity to mammalian cells. Compound e2 showed over 690-fold higher potency than the clinical used urease inhibitor acetohydroxamic acid, reversibly inhibiting urease with a mixed mechanism. Molecular modeling revealed that (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids may possibly bind Ni ions and two hydrophobic regions with a 'Y'-like shape.

Effects of nitrification and urease inhibitors on ammonia-oxidizing microorganisms, denitrifying bacteria, and greenhouse gas emissions in greenhouse vegetable fields.

Soil microorganisms and N cycling are important components of biogeochemical cycling processes. In addition, the study of the effects of nitrification and urease inhibitors on N and microorganisms in greenhouse vegetable fields is essential to reducing N loss and greenhouse gas emissions. The effects of nitrification inhibitors [2-chloro-6-(trichloromethyl) pyridine (CP), dicyandiamide (DCD)], and urease inhibitor [N-(n-butyl) thiophosphoric triamide (NBPT)] on soil inorganic N (NH4+-N, NO2--N and NO3--N) concentrations and the production rates of greenhouse gases (N2O, CH4, and CO2) in greenhouse vegetable fields were investigated via indoor incubation experiments. Polymerase chain reaction amplification and high-throughput sequencing technology (Illumina Miseq) were used to explore the community structure and abundance changes of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and denitrifying bacteria (nirK and nirS). The results showed that CP and DCD obviously inhibited NH4+-N conversion, and NO2--N, and NO3--N accumulation, NBPT slowed down urea hydrolysis and NH4+-N production, and the apparent nitrification rates of soil were in the following order: NBPT > DCD > DCD + NBPT > CP + NBPT > CP. Compared with urea treatment, the peak N2O production rate of inhibitor treatment decreased by 73.30-99.30%, and the production rate of CH4 and CO2 decreased by more than 66.16%. DCD and CP reduced the abundance of AOA and AOB, respectively. Furthermore, NBPT hindered the growth of ammonia-oxidizing microorganisms and nirS-type denitrifying bacteria, and urea and nitrification inhibitors were detrimental to the growth of Ensifer and Sinorhizobium in the nirK community. Nitrification and urease inhibitors can effectively slow down nitrification and greenhouse gas emissions, reduce N loss and improve soil quality by inhibiting the growth of ammonia-oxidizing microorganisms and denitrifying bacteria.

An overview: metal-based inhibitors of urease.

Urease is a kind of nickel-dependent metalloenzyme, which exists in the biological world widely, and can catalyse the hydrolysis of urea into ammonia and carbon dioxide to provide a nitrogen source for organisms. Urease has important uses in agriculture and medicine because it can catalyse the production of ammonia. Therefore, in this review, metal-based inhibitors of urease will be summarised according to different transition metal ions. Including the urease inhibition, structure-activity relationship, and molecular docking. Importantly, among these reviewed effective urease inhibitors, most of copper metal complexes exhibited stronger urease inhibition with IC50 values ranging from 0.46 µM to 41.1 µM. Significantly, the collected comprehensive information looks forward to providing rational guidance and effective strategies for the development of novel, potent, and safe metal-based urease inhibitors, which are better for practical applications in the future.

The fate of nitrification and urease inhibitors in simulated bank filtration.

The application of nitrification and urease inhibitors (NUI) in conjunction with nitrogen (N) fertilizers improves the efficiency of N fertilizers. However, NUI are frequently found in surface waters through leaching or surface runoff. Bank filtration (BF) is considered as a low-cost water treatment system providing high quality water by efficiently removing large amounts of organic micropollutants from surface water. The fate of NUI in managed aquifer recharge systems such as BF is poorly known. The aim of this work was to investigate sorption and degradation of NUI in simulated BF under near-natural conditions. Besides, the effect of NUI on the microbial biomass of slowly growing microorganisms and the role of microbial biomass on NUI removal was investigated. Duplicate sand columns (length 1.7 m) fed with surface water were spiked with a pulse consisting of four nitrification (1,2,4-triazole, dicyanodiamide, 3,4-dimethylpyrazole and 3-methylpyrazole) and two urease inhibitors (n-butyl-thiophosphoric acid triamide and n-(2-nitrophenyl) phosphoric triamide). The average spiking concentration of each NUI was 5 µg/L. Experimental and modeled breakthrough curves of NUI indicated no retardation for any of the inhibitors. Therefore, biodegradation was identified as the main elimination pathway for all substances and was highest in zones of high microbial biomass. Removal of 1,2,4-triazole was 50% and n-butyl-thiophosphoric acid triamide proved to be highly degradable and was completely removed after a hydraulic retention time (HRT) of 24 h. 50% of the mass recovery for nitrification inhibitors except for 3,4-dimethylpyrazole was observed at the effluent (4 days HRT). In addition, a mild effect of NUI on microbial biomass was noted. This study highlights that the degradation of NUI in BF depends on HRT and microbial biomass.

Influence of the urease inhibitor suspension (Atmowell®) on the fluorescent dye pyranine and its spray and drift behavior in wind tunnel measurements.

Too many ammonia emissions are released into the environment from cattle farming. These damage the environment and have an impact on animal and human health. Ammonia Emissions could be reduce by urease inhibitors. Before using the urease inhibitor suspension Atmowell® in cattle farming a risk assessment is required. This includes exposure data on the animal and human in the barn. As there is no method for exposure measurements yet the approach of fluorometry was taken. The fluorescent dye pyranine shall replace Atmowell® in later studies as a tracer. Before Atmowell® can be replaced, the interaction between Atmowell® and pyranine-according to the fluorescence and storage stability under the influence of ultraviolet light, has to be observed and excluded. Also, the spray and drift behavior must be examined in the wind tunnel with three different nozzles. The results show that Atmowell® has no effect on neither the fluorescence nor the degradation rate of a pyranine-solution. Furthermore, it is shown that a pyranine + Atmowell® mixture does not differ in drift behavior from a pure pyranine-solution. Because of these findings, an Atmowell®-solution can be substituted by a pyranine-solution without any effects on the results of an exposure measurement being expected.

Global evaluation of inhibitor impacts on ammonia and nitrous oxide emissions from agricultural soils: A meta-analysis.

Inhibitors are widely considered an efficient tool for reducing nitrogen (N) loss and improving N use efficiency, but their effectiveness is highly variable across agroecosystems. In this study, we synthesized 182 studies (222 sites) worldwide to evaluate the impacts of inhibitors (urease inhibitors [UI], nitrification inhibitors [NI] and combined inhibitors) on crop yields and gaseous N loss (ammonia [NH3 ] and nitrous oxide [N2 O] emissions) and explored their responses to different management and environmental factors including inhibitor application timing, fertilization regime, cropping system, water management, soil properties and climatic conditions using subgroup meta-analysis, meta-regression and multivariate analyses. The UI were most effective in enhancing crop yields (by 5%) and reducing NH3 volatilization (by 51%), whereas NI were most effective at reducing N2 O emissions (by 49%). The application of UI mitigates NH3 loss and increases crop yields especially in high NH3 -N loss scenarios, whereas NI application would minimize the net N2 O emissions and the resultant environmental impacts especially in low NH3 -N loss scenarios. Alternatively, the combined application of UI and NI enables producers to balance crop production and environmental conservation goals without pollution tradeoffs. The inhibitor efficacy for decreasing gaseous N loss was dependent upon soil and climatic conditions and management practices. Notably, both meta-regression and multivariate analyses suggest that inhibitors provide a greater opportunity for reducing fertilizer N inputs in high-N-surplus systems and presumably favor crop yield enhancement under soil N deficiency situations. The pursuit of an improved understanding of the interactions between plant-soil-climate-management systems and different types of inhibitors should continue to optimize the effectiveness of inhibitors for reducing environmental losses while increasing productivity.

Synthesis, evaluation and mechanism exploration of 2-(N-(3-nitrophenyl)-N-phenylsulfonyl)aminoacetohydroxamic acids as novel urease inhibitors.

Thirteen 2-(N-(3-nitrophenyl)-N-phenylsulfonyl)aminoacetohydroxamic acids which were reported for the first time were designed and synthesized as novel urease inhibitors. Most of them showed higher potency than the positive control acetohydroxamic acid, with 2-(N-(3-nitrophenyl)-N-(4-bromophenylsulfonyl)aminoacetohydroxamic acid (d7) being the most active (IC50 = 0.13 ± 0.01 µM). Compound d7 reversibly inhibits urease with mixed mechanism showing excellent binding affinity to urease active site (KD = 0.34 nM, Ki=0.065 ± 0.003 µM andKi' = 1.20 ± 0.09 µM) and very low cytotoxicity against mammalian cells (cell viability of 91.4 % against HepG2 at 250 µg/mL). These positive results indicated that d7 may be used as the lead for further research to develop urease inhibitors with promising properties.

Chelerythrine Chloride: A Potential Rumen Microbial Urease Inhibitor Screened by Targeting UreG.

Inhibition of ruminal microbial urease is of particular interest due to its crucial role in regulating urea-N utilization efficiency and nitrogen pollution in the livestock industry. Acetohydroxamic acid (AHA) is currently the only commercially available urease inhibitor, but it has adverse side effects. The urease accessory protein UreG, which facilitates the functional incorporation of the urease nickel metallocentre, has been proposed in developing urease inhibitor through disrupting urease maturation. The objective of this study was to screen natural compounds as potential urease inhibitors by targeting UreG in a predominant ruminal microbial urease. In silico screening and in vitro tests for potential inhibitors were performed using molecular docking and an assay for the GTPase activity of UreG. Chelerythrine chloride was selected as a potential urease inhibitor of UreG with an inhibition concentration IC50 value of 18.13 µM. It exhibited mixed inhibition, with the Ki value being 26.28 µM. We further explored its inhibition mechanism using isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy, and we found that chelerythrine chloride inhibited the binding of nickel to UreG and induced changes in the secondary structure, especially the α-helix and ß-sheet of UreG. Chelerythrine chloride formed a pi-anion interaction with the Asp41 residue of UreG, which is an important residue in initiating the conformational changes of UreG. In conclusion, chelerythrine chloride exhibited a potential inhibitory effect on urease, which provided new evidence for strategies to develop novel urease inhibitors targeting UreG to reduce nitrogen excretion from ruminants.

Development of One-Step Non-Solvent Extraction and Sensitive UHPLC-MS/MS Method for Assessment of N-(n-Butyl) Thiophosphoric Triamide (NBPT) and N-(n-Butyl) Phosphoric Triamide (NBPTo) in Milk.

N-(n-butyl) thiophosphoric triamide (NBPT) is a urease inhibitor utilised in urea-based fertilizers. In Ireland, fertilizer treated with NBPT is applied to pasture to mitigate both ammonia and nitrous oxide emissions, but concerns arise as to the potential for residues in milk products. A quick ultrafiltration extraction and ultra-high performance liquid chromatography coupled with mass spectrometry triple quadrupole (UHPLC-MS/MS) quantitation method was developed and validated in this study. The method was applied in the analysis of samples collected from a field study investigating potential transfer of NBPT residues into milk. NBPT and NBPTo residues, were extracted from fortified milk samples and analysed on a UHPLC-MS/MS with recoveries ranging from 74 to 114%. Validation of the UHPLC-MS/MS method at low (0.0020 mg kg-1) and high (0.0250 mg kg-1) concentration levels in line with SANTE/12682/2019 showed overall trueness in the range of 99 to 104% and precision between 1 and 10%, RSD for both compounds. The limit of quantitation (LOQ) was 0.0020 mg kg-1 and other tested parameters (linearity, sensitivity, specificity, matrix effect, robustness, etc.) satisfied acceptance criteria. Stability assessment using spiked samples revealed the compounds were stable in raw and pasteurised milk for 4 weeks at -80 °C storage temperature. Maintaining samples at pH 8.5-9.0 further improved stability. Analysis of 516 milk samples from the field study found that NBPT and NBPTo concentrations were below the LOQ of 0.0020 mg kg-1, thus suggesting very low risk of residues occurring in the milk. The method developed is quick, robust, and sensitive. The method is deemed fit-for-purpose for the simultaneous determination of NBPT and NBPTo in milk.

Effects of boric acid on urea-N transformation and 3,4-dimethylpyrazole phosphate efficiency.

BACKGROUND: 3,4-Dimethylpyrazole phosphate (DMPP) is a nitrification inhibitor which can restrict nitrate (NO3 - ) production. Boric acid is a substance which inhibits urease activity. However, few studies have focused on the inhibitory effect of boric acid on urea hydrolysis and the possible synergistic effect with DMPP. Thus, an incubation trial was conducted to determine the impact of boric acid and DMPP addition on urea-N transformation, and their synergistic effects, in chernozem soil (Che) and red soil (RS). Four treatments were set up in each soil: urea only (U); urea combined with DMPP (UD); urea combined with boric acid (UB); and urea combined with both DMPP and boric acid (UDB). RESULTS: Compared to U, adding DMPP (UD) increased NH3 emissions by 11% and 13% and decreased soil NO3 - -N concentration by 38% and 13% in Che and RS, respectively. Boric acid addition (UB) effectively prolonged the half-life time of urea by 0.8 and 0.4 days, reduced NH3 volatilizations by 11% and 16% and delayed the occurrence of NH3 emission peaks for 3 and 4 days in contrast to U treatment in Che and RS, respectively. UDB treatment mitigated the NH3 volatilizations caused by the addition of DMPP (UD) by 16% and 29% in Che and RS, respectively. Additionally, a better nitrification inhibition rate was found in the UDB treatment compared to other treatments in both soils. CONCLUSIONS: There is potential to develop a new N transformation inhibition strategy with the use of a combination of boric acid and DMPP. © 2020 Society of Chemical Industry.

Novel thiobarbiturates as potent urease inhibitors with potential antibacterial activity: Design, synthesis, radiolabeling and biodistribution study.

Urease enzyme is a virulence factor that helps in colonization and maintenance of highly pathogenic bacteria in human. Hence, the inhibition of urease enzymes is well-established to be a promising approach for preventing deleterious effects of ureolytic bacterial infections. In this work, novel thiobarbiturate derivatives were synthesized and evaluated for their urease inhibitory activity. All tested compounds effectively inhibited the activity of urease enzyme. Compounds 1, 2a, 2b, 4 and 9 displayed remarkable anti-urease activity (IC50 = 8.21-16.95 µM) superior to that of thiourea reference standard (IC50 = 20.04 µM). Moreover, compounds 3a, 3g, 5 and 8 were equipotent to thiourea. Among the tested compounds, morpholine derivative 4 (IC50 = 8.21 µM) was the most potent one, showing 2.5 folds the activity of thiourea. In addition, the antibacterial activity of the synthesized compounds was estimated against both standard strains and clinical isolates of urease producing bacteria. Compound 4 explored the highest potency exceeding that of cephalexin reference drug. Moreover, biodistribution study using radiolabeling approach revealed a remarked uptake of 99mTc-compound 4 into infection induced in mice. Furthermore, a molecular docking analysis revealed proper orientation of title compounds into the urease active site rationalizing their potent anti-urease activity.

Flavonoid analogues as urease inhibitors: Synthesis, biological evaluation, molecular docking studies and in-silico ADME evaluation.

A series of novel flavonoid analogues were designed and synthesized. The aimed compounds for urease inhibitory activities were clearly superior to the control drug thiourea (more than 10 times). Among these compounds, L2 (IC50 = 1.343 µM) and L12 (IC50 = 1.207 µM) exhibited the most excellent urease inhibitory activity in vitro. The molecular dockings of L2, L12 and L22 into urease were performed to explore the binding modes and their structure-activity relationship. Furthermore, these aimed compounds showed good druggable properties.

Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies.

A new series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and evaluated as Helicobacter pylori urease inhibitors. All the synthesized compounds (IC50 = 10.37 ± 1.0-77.52 ± 2.7 µM) were more potent than standard inhibitor hydroxyurea against urease (IC50 = 100.00 ± 0.2 µM). Furthermore, comparison of IC50 values of the synthesized compounds with the second standard inhibitor thiourea (IC50 = 22.0 ± 0.03 µM) revealed that compounds 7a-b and 7f-h were more potent than thiourea. Molecular modeling study of the most potent compounds 7a, 7b, 7f, and 7g was also conducted. Additionally, the drug-likeness properties of the synthesized compounds, based on Lipinski rule and other filters, were evaluated.

N-monoarylacetothioureas as potent urease inhibitors: synthesis, SAR, and biological evaluation.

A urease inhibitor with good in vivo profile is considered as an alternative agent for treating infections caused by urease-producing bacteria such as Helicobacter pylori. Here, we report a series of N-monosubstituted thioureas, which act as effective urease inhibitors with very low cytotoxicity. One compound (b19) was evaluated in detail and shows promising features for further development as an agent to treat H. pylori caused diseases. Excellent values for the inhibition of b19 against both extracted urease and urease in intact cell were observed, which shows IC50 values of 0.16 ± 0.05 and 3.86 ± 0.10 µM, being 170- and 44-fold more potent than the clinically used drug AHA, respectively. Docking simulations suggested that the monosubstituted thiourea moiety penetrates urea binding site. In addition, b19 is a rapid and reversible urease inhibitor, and displays nM affinity to urease with very slow dissociation (koff=1.60 × 10-3 s-1) from the catalytic domain.

Utility of anthranilic acid and diethylacetylenedicarboxylate for the synthesis of nitrogenous organo/organometallic compounds as urease inhibitors.

Fumarate diester 3 was synthesized upon reacting anthranilic acid with diethylacetylenedicarboxylate. Compound 3 was reacted with different nucleophiles in mild reaction conditions. Selected reaction routes that afforded products 6, 9, 10, 11, and 12 were explained. The estimated mechanism for the reaction of 3 with ethylenediamine to afford 9 was proved by X-ray single-crystal and retro-synthetic reaction. Acetyl anthranilic acid was utilized with zinc and copper to afford the organometallic compounds 14a and 14b, respectively. Three single crystals were afforded for 3, 9 and the organocopper complex 14b. Target compounds were screened for their inhibitory potential against urease enzyme. Most compounds were more potent than thiourea as standard inhibitor, considering that oxopiperazine 9 exhibited double the activity: IC50 = 8.16 ± 0.65 µM (thiourea IC50 = 20.04 ± 0.33 µM). Docking studies were in agreement with the in vitro enzyme assay.