Exploring the Synthetic Chemistry of Phenyl-3-(5-aryl-2-furyl)- 2-propen-1-ones as Urease Inhibitors: Mechanistic Approach through Urease Inhibition, Molecular Docking and Structure–Activity Relationship

Abstract

Furan chalcone scaffolds belong to the most privileged and promising oxygen-containing heterocyclic class of compounds, which have a wide spectrum of therapeutic applications in the field of pharmaceutics, pharmacology, and medicinal chemistry. This research described the synthesis of a series of twelve novel and seven reported furan chalcone (conventional synthetic approach) analogues 4a–s through the application of microwave-assisted synthetic methodology and evaluated for therapeutic inhibition potential against bacterial urease enzyme. In the first step, a series of nineteen substituted 5-aryl-2-furan-2-carbaldehyde derivatives 3a–s were achieved in moderate to good yields (40–70%). These substituted 5-aryl-2-furan-2-carbaldehyde derivatives 3a–s were condensed with acetophenone via Claisen–Schmidt condensation to furnish 19 substituted furan chalcone scaffolds 4a–s in excellent yields (85–92%) in microwave-assisted synthetic approach, while in conventional methodology, these furan chalcone 4a–s were furnished in good yield (65–90%). Furan chalcone structural motifs 4a–s were characterized through elemental analysis and spectroscopic techniques. These nineteen (19)-afforded furan chalcones 4a–s were screened for urease inhibitory chemotherapeutic efficacy and most of the furan chalcones displayed promising urease inhibition activity. The most active urease inhibitors were 1-phenyl-3-[5-(2′,5′-dichlorophenyl)-2-furyl]-2–propen-1-one 4h with an IC₅₀ value of 16.13 ± 2.45 μM, and 1-phenyl- 3-[5-(2′-chlorophenyl)-2-furyl] -2-propen-1-one 4s with an IC₅₀ value of 18.75 ± 0.85 μM in comparison with reference drug thiourea (IC₅₀ = 21.25 ± 0.15 μM). These furan chalcone derivatives 4h and 4s are more efficient urease inhibitors than reference drug thiourea. Structure–activity relationship (SAR) revealed that the 2,5-dichloro 4h and 2-chloro 4s moiety containing furan chalcone derivatives may be considered as potential lead reagents for urease inhibition. The in silico molecular docking study results are in agreement with the experimental biological findings. The results of this study may be helpful in the future drug discovery and designing of novel efficient urease inhibitory agents from this biologically active class of furan chalcones.

1. Introduction

The urease (urea amidohydrolase, EC 3.5.1.5) is a nickel-containing enzyme that catalyzes the hydrolysis of urea to ammonia and carbamate in the final step of nitrogen metabolism [1,2,3]. The carbamate, which is thus formed, rapidly decomposes to yield a second molecule of ammonia. Urease is present in a variety of bacteria (pathogenic as well as soil bacteria), plants, algae, and fungi; therefore, urease is not a native human enzyme. In humans, urease comes from pathogenic bacterial strains such as Ureaplasma urealyticum, Proteus mirabilis, Klebsiella pneumoniae, bacteria from the Salmonella and Staphylococcus genera, etc., which are responsible for bacterial virulence. These bacterial strains are responsible for many diseases such as urinary tract infections, the formation of urinary stones, peptic ulcers, etc. [4,5]. Ureases are also involved in the development of struvite stone disease, urolithiasis, pyelonephritis, hepatic encephalopathy, hepatic coma, and urinary catheter encrustation [6]. Particularly notorious are the gastric ulcers due to the bacteria Helicobacter pylori, whereby urease activity results in an increased pH around the bacteria, aiding in its colonization and protecting it from the other harmful acidic environment of the stomach. Hence, urease inhibitors are effective antibacterial agents, and aid in enhancing the antibacterial effect of other antibiotics. Acetohydroxamic acid (Lithostat, Figure 1) is a well-known example of a clinically used urease inhibitor drug for the treatment of urinary tract infections and certain types of kidney stones.

Generally, natural and synthetic heterocycles are privileged derivatives [7,8], such as furans [9], quinoxalines [10], coumarins [11], benzimidazoles [12], heterocyclic sulfonamides [13], theophyllines [14], imidazoles [15], pyrazoles [16], thiadiazoles [17], etc., which demonstrate wide-spectrum biological activities in the fields of pharmacology, medicinal chemistry, pharmaceutical chemistry, and pharmaceutics. In the same way, various heterocyclic synthetic compounds have been designed to compete with the growing challenges related to ureolytic microorganisms, including thioureas [18], triazoles [19], thiadiazoles [20], benzimidazoles [21], hydroxamic acid [22], phosphoramidate, and thiazolacetamide [23].

Chalcones (Figure 1) are α- and β-unsaturated ketones in which two aromatic rings are joined by three carbon chains, which have a broad spectrum of biological activities such as cytotoxicity, antimitotic, anti-mutagenic, antitumor-promoting activities, antibacterial, antiviral, anti-inflammatory, enzyme inhibition, etc. [24,25,26]. Furan chalcones such as (E)-3-(furan-2-yl)-1-p-tolylprop-2-en-1-one, (E)-3-(furan-2-yl)-1-(3-hydroxyphenyl)pro-2-en-1-one, and aurones (2-(4-fluorobenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one) displayed excellent urease inhibition therapeutic efficacy as compared to the reference drug thiourea (Figure 1) [27,28].

In previously published studies, researchers explored the furan derivatives’ chemotherapeutic potential as bacterial tyrosinase inhibitors, human tyrosinase inhibitors, M. tuberculosis polyketide synthase 13 inhibitors, anti-oxidants, and anticancer agents [9,24,29,30,31,32,33]. In the present work, structural modifications were carried out to design the synthesis of novel furan chalcone derivatives due to the attractive and promising medicinal and pharmacological profiles of furan and chalcone moieties as cited in the literature [24,25,26,27,28,29,30,31]. The nineteen (19) furan chalcones were synthesized via Claisen–Schmidt condensation reaction by utilizing conventional synthetic approach [31] as well as microwave irradiation synthetic approach. These chalcones were screened against the urease enzyme to discover the inhibitory potential of these 19 furan chalcone derivatives.

Figure 1

Rationale design of furan chalcones as urease inhibitors [25,26,27,28].

[Figure omitted. See PDF]

2. Results and Discussion

2.1. Synthetic Chemistry of Furan-Based Chalcones 4a–s

A series of arylfuran-2-carbaldehydes (3a–s) and their respective furan chalcone derivatives (4a–s) were furnished according to the prescribed synthetic approach as depicted in Scheme 1. The arylfuran-2-carbaldehydes 3a–s were afforded in moderate to good yield (40–70%) through a catalytic Meerwein arylation of furfural with arenediazonium salts 2. The best yields in the arylation of furfural were obtained with diazonium salts containing a nitro group or two halogen atoms in the aromatic ring. The Claisen–Schmidt condensation reaction between arylfuran-2-carbaldehydes (3a–s) and acetophenone under basis (NaOH) catalytic conditions at ambient temperature was carried out in the presence of conventional synthetic approaches [31] as well as microwave irradiation synthetic methodology [33]. The different furan chalcones 4a–s were furnished in excellent yield (85–92%) and good yield (65–90%) by utilizing the microwave-assisted synthetic approach and conventional methodology, respectively. The structures of 19 furan chalcone molecules (4a–s), percentage yields of both synthetic methodologies, and melting points are displayed in Table 1.

2.2. Urease Inhibition Activity of Furan Chalcones 4a–s

Chalcones are important medicinal compounds to combat several pathological conditions associated with ureolytic enzymes (urease). In the literature, it has been reported that the high electron withdrawing effect of the nitro group and the moderate effect of the chloro group could possibly increase the activity of urease inhibitors [35,36]. Considering these facts, we prepared a series of furan chalcones, tested them for their urease inhibitory activities, and found promising urease inhibitory results. Urease inhibition assay was performed according to the reported protocol by Pervez et al. [37].

The furan chalcones 4a, 4i, and 4l displayed IC₅₀ values in the range of 90.81 ± 8.99 μM to 91.89 ± 2.24 μM, indicating that these compounds were the least effective against urease enzymes. The furan chalcone scaffolds 4b, 4c, 4d, 4g, and 4r were inactive and did not inhibit the urease enzyme as depicted in Table 2. The better urease inhibitory activities (IC₅₀ values in the range of 23.09 ± 3.65 μM to 33.96 ± 9.61 μM) were shown by the furan chalone derivatives 4e, 4j, 4k, 4m, and 4o, as displayed in Table 2. Furan chalcone derivatives 4n, 4p, and 4q exhibited moderate urease inhibition activity (IC₅₀ values in the range of 44.43 ± 6.91 μM to 52.64 ± 8.52 μM) as shown in Table 2. The most active urease inhibitors in our series included 2,5-dichloro functionality, containing 1-phenyl-3-[5-(2′,5′-dichlorophenyl)-2-furyl]-2-propen-1-one 4h, which has an IC₅₀ value of 16.13 ± 2.45 μM, and a 2-chloro moiety-based 1-phenyl-3-[5-(2′-chlorophenyl)-2-furyl]-2-propen-1-one 4s with an IC₅₀ value of 18.75 ± 0.85 μM. The 3,4-dichloro moiety containing 1-phenyl-3-[5-(3′,4′-dichlorophenyl) -2-furyl]-2- propen-1-one 4f displayed good urease inhibition activity with an IC₅₀ value of 21.05 ± 3.2 μM, as compared with the reference standard drug thiourea (Table 2).

2.3. Structure–Activity Relationship (SAR) Studies of Furan Chalcones 4a–s

Structural modifications were incorporated by substituting electron-donating and electron-withdrawing functional groups in aromatic rings of furan chalcones 4a–s, and their influences on the urease inhibitory potential were explored to develop structure–activity relationship (SAR).

In general, furan chalcone compounds containing -Cl and -COOH groups were found to be more active as compared to compounds containing -NO₂ and -Br groups. Furan chalcone compound 4h (Figure 2) was the most active inhibitor, having an IC₅₀ value of 16.13 ± 2.45 µM. Compounds 4a, 4d, and 4l are almost all in which the -NO₂ group is present at the ortho, meta, and para positions of the phenyl ring. However, for compounds in which the -Cl group is present along with the -NO₂ group, the activity was found to be enhanced, as in compounds 4j and 4m (IC₅₀ = 26.05 ± 2.25 µM and 26.71 ± 0.65 µM, respectively). In the same way, when the -Cl group is present at the para position of the phenyl ring, as in compound 4b, this decreases the urease inhibition therapeutic efficacy and the compound becomes inactive, but in compounds 4k and 4s (Figure 2), the urease inhibitory activity is enhanced (IC₅₀ = 23.09 ± 3.65 µM and 18.75 ± 0.85 µM, respectively) where the -Cl group is present at the ortho and meta positions of the phenyl ring. Compound 4c is inactive because the -Br group is present at the para position, but when it is replaced by the -COOH group, it shows promising urease inhibition as in compound 4e. Furan chalcone compound 4h is the most active inhibitor and has a 2,5-dichloro substituent at the phenyl ring (Figure 2), but if one chloro group is replaced by a -NO₂ group as in compound 4i, its activity decreases (IC₅₀ value increases to 90.81 ± 8.99 M). Furan chalcone molecule 4g is urease inactive, in which both chloro groups are meta to each other, while its other analogues, 3,4-dichloro containing compound 4f, 2,5-dichloro containing compound 4h, and 2,4-dichloro containing compound 4o, showed promising urease inhibition with IC₅₀ values of 21.05 ± 3.52 µM, 16.13 ± 2.45 µM, and 33.96 ± 9.61 µM, respectively. Compound 4e is inactive because a -NO₂ group is present at the meta-position of the phenyl group, but compound 4p, having a 2-methyl-5-nitro substituent on the phenyl group, exhibits an IC₅₀ value of 52.64 ± 8.52 µM, whereas its analogue 4r (Figure 2), having a 2-methyl-3-nitro substituent at the phenyl group, becomes inactive. Furan chalcones having one -NO₂ group exhibit an IC₅₀ of 91.43 ± 6.25 µM as in 4l; an introduction of a second nitro group on the same phenyl ring has the same effect on the IC₅₀ value as in compound 4q, which has 44.43 ± 6.91 µM, while the furan chalcones having chloro and nitro groups at the ortho position show moderate activity.

2.4. Molecular Docking, ADMET, and Drug-Likeness Investigations 4a–s

To investigate the probable binding modes and mechanisms of these synthesized compounds, along with the molecular interactions by which these compounds interact with the urease enzyme active site, computational screening studies were performed utilizing the Autodock-Vina (v1.1.2) software. Molecular docking investigations of the top performing compounds in the in vitro studies 4h and 4s showed that both of these compounds bind with significantly strong affinities of −7.2 Kcal/mol (Table 3) with the urease enzyme compared to the control thiourea, which showed a binding affinity of −3.4 Kcal/mol (Table 3). The binding conformation and molecular contacts analysis of the compound 4h showed that the -2′,5′-dichloro phenyl moiety attached to the furan ring binds deep inside the urease active site and is oriented directly at the catalytic dinickel metal active center of this enzyme. Furthermore, the molecular contacts analysis of the compound 4h revealed that this compound exhibits multiple and diverse types of stronger interactions with the urease enzyme active site.

The molecular interaction analysis of the compound 4h showed that the -2,5-dichlorophenyl moiety of this compound plays an important role in the robust binding of this compound with the urease enzyme. The analysis showed that the chlorine atoms of the -phenyl ring present at the 2′ and 5′ positions exhibit stronger interactions with important active-site residues of the urease enzyme. One of the chlorine atoms was able to engage with the important catalytic nickel ions via metal-acceptor bonding, along with engaging the HIS138, HIS248, and HIS274 amino acid residues through the alkyl hydrophobic type interaction. Similarly, the second chlorine atom of the -phenyl ring also made an important conventional hydrogen bond interaction with the CYS321 residue, along with an alkyl hydrophobic interaction with the ALA19 urease active site amino acid. Moreover, the -phenyl ring of the compound 4h also made multiple interactions by engaging the ALA169 and ALA365 via Pi–alkyl-type interactions and the ARG338 via Pi–cation interactions.

Furthermore, the -furan moiety of 4h also showed multiple interactions with important active-site residues of the urease enzyme. This moiety made a Pi–donor hydrogen bond with the CYS321 amino acid residue, along with a Pi–sulfur-type molecular contact with MET366 and an amide–Pi-stacked interaction with the ALA365 amino acid of the urease active site. The other -phenyl moiety of this compound also contributed two interactions of the Pi–alkyl hydrophobic type by engaging the MET317 and VAL320 outer pocket residues of the urease enzyme active site. Figure 3 contains the two-dimensional and three-dimensional conformational orientations and interaction summary of compound 4h inside the urease enzyme active site, while Table 3 contains the binding affinities and structures of the investigated compounds.

Previously in the literature, it has been reported that urease inhibitors hamper or inhibit the activity of this enzyme by utilizing different mechanisms [38]. All types of urease enzymes contain an important cysteine amino acid residue. This cysteine residue, which is a part of the flap and is situated on a protein loop that faces the flap at the entry to the active site cavity, is particularly crucial to the way the urease enzyme functions [39,40]. Several inhibitors have been reported that inhibit the urease enzyme by targeting this specific cysteine residue; similarly, several other inhibitors have been reported to directly inhibit this enzyme by targeting and modifying the two nickel ions, which result in a complete inhibition of this enzyme [38].

Keeping in view these previously reported inhibitors and their mode of action and the robust bindings and interactions of the compound 4h, which was able to interact directly with the important catalytic amino acids, i.e., the flap cysteine and other important histidine residues, and the direct molecular contacts of 4h with the urease enzyme catalytic nickel ions, it can be inferred from these studies that compound 4h is a promising candidate for urease inhibition.

ADMET Studies

Moreover, ADME and toxicity investigations of compounds 4h and 4s were also performed to evaluate their pharmacokinetics and other drug-likeness-related properties. These investigations revealed that these compounds possess favorable gastrointestinal tract absorption if taken orally. These compounds were non-inhibitors of the CYP3A4 and CYP2D6 metabolic enzymes and showed good lipophilicity scores (Log P_o/w (iLOGP)) along with favorable Log S (ESOL) water solubility scores. These compounds belong to the moderately soluble class of compounds. These compounds also exhibit satisfactory TPSA scores along with good bioavailability values; they also completely followed the Lipinski and Veber drug rules. In the toxicity studies, it was found that these compounds are non-inhibitors of the hERG channel and show no human hepatotoxicity (H-HT); they also show lower probabilities of being AMES and respiratory toxic; moreover, these compounds are also compliant with the Acute Toxicity Rule (ATR) and show zero alerts of toxicity. Table 4 contains some of the discussed ADMET properties and their parameters/values.

3. Materials and Methods

All reagents and solvents were obtained from the supplier or recrystallized or redistilled as necessary. Thin-layer chromatography was performed using aluminum sheets (Merck) coated with silica gel 60 F254. Urease enzyme was purchased from Canavalia ensiformis (Jack bean), CAS Number: 9002-13-5. IR spectra were recorded using an IR PerkinElmer Spectrum 1 FTIR spectrophotometer, and peaks were reported as ax (neat)/cm⁻¹, which refer to the minimum wave numbers. Proton magnetic resonance spectra were recorded in CDCl₃ with a Bruker AM 300 spectrometer (Rheinstetten-Forchheim, Germany) operating at 300 MHz, respectively. The ¹³C NMR spectra were recorded in CDCl₃ with a Bruker AM 100 spectrometer operating at 100 MHz. Tetramethylsilane was used as an internal standard. Elemental analyses for C, H, and N were recorded with the PerkinElmer 2400 Series II CHN Analyzer. Melting points were recorded on a GallenKamp apparatus and are uncorrected.

3.1. Synthesis of Furan Chalcone Molecules 4a–s

3.1.1. Synthesis of 5-aryl-2-furaldehydes

Substituted aniline (4.5 g) was dissolved in a mixture of conc. hydrochloric acid and 20 mL of water under stirring and cooled in an ice bath at −5 °C. A solution of sodium nitrite (2 g in 10 mL of water) was added portion-wise, keeping the temperature below 7–8 °C. The reaction mixture was left for an hour for the completion of diazotization, then filtered with the help of glass wool (if any turbidity was observed). Then, a solution of furfural was taken (2 mL in 10 mL of acetone and water), and the diazonium solution was added to it drop by drop. A solution of copper chloride (2 g in 10 mL of water) was also added. The reaction mixture was heated to 30 °C (if necessary) and stirred for 4–6 h, then left for 24 h at room temperature. The precipitates obtained were filtered, dried, and recrystallized with ethanol. By using the above method, various 5-arylfuran-2-carbaldehydes were prepared [41,42,43,44,45].

3.1.2. Synthesis of Furan Chalcones 4a–s

Method A: Conventional Synthetic Approach

Equimolar quantities of aryl furan-2-carbaldehyde (0.001 mol) and appropriate acetophenone (0.001 mol) were taken in an ethanol and water mixture (10 mL ethanol + 10 mL water) in the presence of NaOH as a catalyst in an ice bath (−5 °C), and the mixture was stirred for 4 h. The solid products formed were filtered, dried, and recrystallized from ethanol [31].

Method B: Microwave-Assisted Synthetic Approach

Equimolar quantities (0.001 mol) of 2-acetyl heterocyclic derivatives and respective aldehydes (0.001 mol) were mixed and dissolved in a minimum amount (3 mL) of alcohol. To this, an aqueous sodium hydroxide solution (0.003 mol) was slowly added and mixed. The entire reaction mixture was microwave-irradiated for about 2–6 min at 180 watts [33].

By following Method “A” and Method “B”, various chalcone derivatives (4a–s) were prepared and their analytical data are given in Supplementary Files.

3.1.3. Furan Chalcones 4a–s Urease Inhibition Assay

The enzyme assay is a modified form of the commonly known Berthelot assay. A total volume of 85 µL assay mixture contained 10 µL of phosphate buffer at pH 7.0 in each well of the 96-well plate, to which 10 µL of sample solution and 25 µL of enzyme solution (0.1347 units) were added. The contents were pre-incubated at 37 °C for 5 min. Then, 40 µL of urea stock solution (20 mM) was added to each well, and incubation continued at 37 °C for another 10 min. After the given time, 115 µL phenol hypochlorite reagent was added to each well (freshly prepared by mixing 45 µL phenol reagent with 70 µL of alkali reagent). For color development, incubation was performed at 37 °C for another 10 min. Absorbance was measured at 625 nm using the 96-well plate reader Synergy HT. The percentage enzyme inhibition was calculated using the following formula:

Inhibition (%) = 100 − (Absorbance of test sample/Absorbance of control) × 100

IC₅₀ values (concentration at which 50% enzyme catalyzed reaction occurs) of compounds were calculated using EZ-Fit Enzyme Kinetics Software version 5.03 (Perrella Scientific Inc. Amherst, MA, USA) [46].

3.1.4. Molecular Docking Studies

Molecular docking investigations were performed using the Autodock Vina (v1.1.2) software [47]. The structures of the compounds 4a–s were prepared using the ChemDraw Professional (v16.0) software, and then these structures were energy-minimized using the MOE (v2009.10) software’s MMFF94x forcefield and then saved in the (.mol2) format. These structures were then imported into Autodock Vina, where the necessary Gasteiger charges were added, and then they were saved in the PDBQT format for further screening studies. The protein structure of E. coli urease used in this investigation was accessed from the RCSB website with PDB ID 1E9Y [48]. Protein preparation and optimization were performed via MGL Tools (v1.5.7), whereby the H₂O molecules were removed from the protein molecule, and polar hydrogens and Kollman charges were added. The protein’s binding site (active site) was chosen by setting a grid box located at coordinates (X = 127.83, Y = 129.52, Z = 86.18) with XYZ grid dimensions around the active site of 35 angstroms, and then proceeded with the docking using the AutoDock Vina; all the other parameters were set to default. The binding conformations and molecular interaction analysis of the docked compounds with the urease enzyme were performed using the Biovia Discovery Studio (v2017) software.

3.1.5. ADMET Studies

The ADME and drug-likeness studies were conducted using the SwissADME [49] server, while the toxicity investigations were performed using the ADMETlab 2.0 [50] online server.

4. Conclusions

In the present study, a series of twelve novel and seven reported chalcone derivatives (4a–s) were achieved via an improved microwave-assisted synthetic methodology and evaluated for urease inhibitory chemotherapeutic efficacy. In this series, furan-based chalcone compounds 4h and 4s exhibited a comparatively higher urease inhibition chemotherapeutic potential, while others showed moderate urease inhibitory activity as compared to reference standard thiourea. The nature of the substituent on the benzene ring of the aryl group controls the urease inhibition. The most active urease inhibitors were 1-phenyl-3-[5-(2′,5′-dichlorophenyl)-2-furyl]-2-propen-1-one 4h, having an IC₅₀ value of 16.13 ± 2.45 μM, and 1-phenyl-3-[5-(2′-chlorophenyl)-2-furyl]-2-propen-1-one 4s, with an IC₅₀ value of 18.75 ± 0.85 μM in comparison with the reference drug thiourea (IC₅₀ = 21.25 ± 0.15 μM). The order of urease inhibition is 4h > 4s > 4f > 4k > 4ae > 4m > 4o > 4aq > 4n > 4p > 4i > 4l > 4a, and it may act as a potential leading molecule in the drug discovery program. Detailed structure–activity relationship (SAR) and molecular docking studies were carried out to identify the most probable binding site interactions that may lead to the design of even more effective urease inhibitors from this biologically active furan chalcone class of compounds.

Author Contributions

Conceptualization S.A.; Methodology, M.F. and S.A.; Software, S.F.; Formal analysis, M.F., S.A. and M.A.; Writing—original draft. S.A. and A.M.Z.; Data curation, S.N. and B.R.S.; Investigation, S.A. and M.A.K.; Supervision S.A. and M.A.K., Project administration S.A. and M.A.K., Investigation. S.F.; Validation, M.F. and S.A.; Visualization. S.N.; Writing—review and editing M.A.K. and S.A.; Formal analysis, Funding acquisition, Visualization, Writing—review and editing; A.I.; G.A.S.: Data curation; Funding acquisition, Writing—review and editing; Y.A.B.J.: All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data are contained in the manuscript and Supplementary Materials.

Acknowledgments

The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2023R457), King Saud University, Riyadh, Saudi Arabia. Authors would like to acknowledge the financial support to the Scholar Miraj Fatima by HEC Pakistan in the form of an indigenous PhD fellowship. The author is also thankful to H.E.J Research Institute of Chemistry, University of Karachi, Pakistan for carrying out spectroscopic and elemental analysis.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures, Scheme and Tables

Scheme 1. Synthesis of furan moiety-based chalcone derivatives 4a–s.

Figure 2. SAR of furan chalcones as urease inhibitors.

Figure 3. Compound 4h interacting with urease enzyme active site, upper panel (3D conformation) and lower panel (2D conformation).

Table 1

Synthetic data of furan chalcone derivatives 4a–s.

Structures of Compounds	Product Yield (%)		M.P (°C)
	Method A: Conventional SyntheticApproach [31]	Method B:Microwave Irradiation Synthetic Approach [33]		Lit M.P (°C)
[Image omitted. Please see PDF.]	71	85	172–174	172 [31]
[Image omitted. Please see PDF.]	68	90	126–128	126 [31]
[Image omitted. Please see PDF.]	77	90	140–141	140 [31]
[Image omitted. Please see PDF.]	72	88	136–138	136 [31]
[Image omitted. Please see PDF.]	69	85	196–198	200 decompose[31]
[Image omitted. Please see PDF.]	67	90	197–200	-
[Image omitted. Please see PDF.]	70	88	166–168	-
[Image omitted. Please see PDF.]	65	82	130–132	-
[Image omitted. Please see PDF.]	75	90	195–198	-
[Image omitted. Please see PDF.]	90	92	202–204	-
[Image omitted. Please see PDF.]	80	90	248–251	-
[Image omitted. Please see PDF.]	78	90	106–108	106 [34]
[Image omitted. Please see PDF.]	82	88	197–200	-
[Image omitted. Please see PDF.]	75	80	Decompose ˂ 300	-
[Image omitted. Please see PDF.]	86	z90	116–118	-
[Image omitted. Please see PDF.]	84	92	166–68	-
[Image omitted. Please see PDF.]	82	90	181–182	-
[Image omitted. Please see PDF.]	72	86	130–132	-
[Image omitted. Please see PDF.]	88	90	100–102	96–98 [34]

Table 2

Urease inhibition activity of furan chalcone derivatives 4a–s.

Compounds	%Age Urease Inhibition Activity(0.1 mM)	Urease Inhibition IC50(µM)
4a	50.71 ± 0.62	91.89 ± 2.24
4b	27.15 ± 0.17	-
4c	39.41 ± 0.34	-
4d	46.55 ± 0.91	-
4e	65.46 ± 0.77	24.95 ± 1.77
4f	69.71 ± 1.86	21.05 ± 3.52
4g	39.72 ± 2.46	-
4h	66.24 ± 1.36	16.13 ± 2.45
4i	51.72 ± 1.62	90.81 ± 8.99
4j	68.24 ± 0.11	26.05 ± 2.25
4k	69.52 ± 0.16	23.09 ± 3.65
4l	51.47 ± 0.41	91.43 ± 6.25
4m	63.14 ± 0.86	26.71 ± 0.65
4n	62.28 ± 1.28	46.77 ± 5.44
4o	54.88 ± 1.98	33.96 ± 9.61
4p	57.37 ± 3.02	52.64 ± 8.52
4q	54.74 ± 0.73	44.43 ± 6.91
4r	12.64 ± 3.37	-
4s	71.41 ± 1.77	18.75 ± 0.85
Thiourea		21.25 ± 0.15

Table 3

Binding affinities and structures of the investigated compounds against the urease enzyme.

SN	Name	Structure	Binding Affinities with Urease Enzyme
1	4h	[Image omitted. Please see PDF.]	−7.2 Kcal/mol
2	4s	[Image omitted. Please see PDF.]	−7.2 Kcal/mol
3	Thiourea (control)	[Image omitted. Please see PDF.]	−3.2 Kcal/mol

Table 4

ADME and Toxicity profiles along with drug-likeness parameters of compounds 4h and 4s.

SN	Compound Name	GI-Tract Absorption	Bioavailability Scores	Lipinski Rule	Water Solubility	Acute Toxicity	Log Po/w (iLOGP)
1	4h	High	0.55	Accepted	4.87 × 10−4 mg/mL	None	3.67
2	4s	High	0.55	Accepted	1.69 × 10−3 mg/mL	None	3.11

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biomedicines11092428/s1. NMR and FTIR Spectra are available as supplementary materials.

References

1. Karplus, P.A.; Pearson, M.A.; Hausinger, R.P. 70 Years of Crystalline Urease: What Have We Learned?. Acc. Chem. Res.; 1997; 30, pp. 330-337. [DOI: https://dx.doi.org/10.1021/ar960022j]

2. Li, M.; Ding, W.; Baruah, B.; Crans, D.C.; Wang, R. Inhibition of protein tyrosine phosphatase 1B and alkaline phosphatase by bis(maltolato)oxovanadium (IV). J. Inorg. Biochem.; 2008; 102, pp. 1846-1853. [DOI: https://dx.doi.org/10.1016/j.jinorgbio.2008.06.007]

3. Mobley, H.L.; Hausinger, R.P. Microbial ureases: Significance, regulation, and molecular characterization. Microbiol. Rev.; 1989; 53, pp. 85-108. [DOI: https://dx.doi.org/10.1128/mr.53.1.85-108.1989]

4. Collins, C.M.; D’Orazio, S.E.F. Bacterial ureases: Structure, regulation of expression and role in pathogenesis. Mol. Microbiol.; 1993; 9, pp. 907-913. [DOI: https://dx.doi.org/10.1111/j.1365-2958.1993.tb01220.x]

5. Montecucco, C.; Rappuoli, R. Living dangerously: How Helicobacter pylori survives in the human stomach. Nat. Rev. Mol. Cell Biol.; 2001; 2, pp. 457-466. [DOI: https://dx.doi.org/10.1038/35073084]

6. Williams, R.J.P. Metallo-enzyme catalysis. Chem. Commun.; 2003; 10, pp. 1109-1113. [DOI: https://dx.doi.org/10.1039/b211281g]

7. Ibrar, A.; Khan, I.; Abbas, N. Structurally Diversified Heterocycles and Related Privileged Scaffolds as Potential Urease Inhibitors: A Brief Overview. Archiv der Pharmazie; 2013; 346, pp. 423-446. [DOI: https://dx.doi.org/10.1002/ardp.201300041]

8. Kafarski, P.; Talma, M. Recent advances in design of new urease inhibitors: A review. J. Adv. Res.; 2018; 13, pp. 101-112. [DOI: https://dx.doi.org/10.1016/j.jare.2018.01.007]

9. Irfan, A.; Faiz, S.; Rasul, A.; Zafar, R.; Zahoor, A.F.; Kotwica-Mojzych, K.; Mojzych, M. Exploring the Synergistic Anticancer Potential of Benzofuran–Oxadiazoles and Triazoles: Improved Ultrasound- and Microwave-Assisted Synthesis, Molecular Docking, Hemolytic, Thrombolytic and Anticancer Evaluation of Furan-Based Molecules. Molecules; 2022; 27, 1023. [DOI: https://dx.doi.org/10.3390/molecules27031023]

10. Irfan, A.; Tahir, O.A.; Umer, M.; Ahmad, S.; Kousar, H. A review on biological studies of Quinoxaline derivative. World J. Pharm. Pharm. Sci.; 2017; 6, pp. 11-30.

11. Srikrishna, D.; Godugu, C.; Dubey, P.K. A Review on Pharmacological Properties of Coumarins. Mini Rev. Med. Chem.; 2018; 18, pp. 113-141. [DOI: https://dx.doi.org/10.2174/1389557516666160801094919]

12. Hernández-López, H.; Tejada-Rodríguez, C.J.; Leyva-Ramos, S. A Panoramic Review of Benzimidazole Derivatives and their Potential Biological Activity. Mini Rev. Med. Chem.; 2022; 22, pp. 1268-1280. [DOI: https://dx.doi.org/10.2174/1389557522666220104150051] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34983345]

13. Irfan, A.; Batool, F.; Irum, S.; Ullah, S.; Umer, M.; Shaheen, R.; Chand, A.J. A Therapeutic Journey of Sulfonamide Derivatives as Potent Anti-Cancer Agents: A Review. WJPR; 2018; 7, pp. 257-270.

14. Shahzadi, I.; Zahoor, A.F.; Tüzün, B.; Mansha, A.; Anjum, M.N.; Rasul, A.; Irfan, A.; Kotwica-Mojzych, K.; Mojzych, M. Repositioning of acefylline as anti-cancer drug: Synthesis, anticancer and computational studies of azomethines derived from acefylline tethered 4-amino-3-mercapto-1,2,4-triazole. PLoS ONE; 2022; 17, e0278027. [DOI: https://dx.doi.org/10.1371/journal.pone.0278027] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36520942]

15. Alghamdi, S.S.; Suliman, R.S.; Almutairi, K.; Kahtani, K.; Aljatli, D. Imidazole as a Promising Medicinal Scaffold: Current Status and Future Direction. Drug Des. Dev. Ther.; 2021; 29, pp. 3289-3312. [DOI: https://dx.doi.org/10.2147/DDDT.S307113]

16. Aziz, H.; Zahoor, A.F.; Shahzadi, I.; Irfan, A. Recent Synthetic Methodologies Towards the Synthesis of Pyrazoles. Polycycl. Aromat. Compd.; 2019; 41, pp. 698-720. [DOI: https://dx.doi.org/10.1080/10406638.2019.1614638]

17. Irfan, A.; Ullah, S.; Anum, A.; Jabeen, N.; Zahoor, A.F.; Kanwal, H.; Kotwica-Mojzych, K.; Mojzych, M. Synthetic Transformations and Medicinal Significance of 1,2,3-Thiadiazoles Derivatives: An Update. Appl. Sci.; 2021; 11, 5742. [DOI: https://dx.doi.org/10.3390/app11125742]

18. Li, W.-Y.; Ni, W.-W.; Ye, Y.-X.; Fang, H.-L.; Pan, X.-M.; He, J.-L.; Zhou, T.-L.; Yi, J.; Liu, S.-S.; Zhou, M. et al. N-monoarylacetothioureas as potent urease inhibitors: Synthesis, SAR, and biological evaluation. J. Enzym. Inhib. Med. Chem.; 2020; 35, pp. 404-413. [DOI: https://dx.doi.org/10.1080/14756366.2019.1706503]

19. Ghomi, M.K.; Noori, M.; Montazer, M.N.; Zomorodian, K.; Dastyafteh, N.; Yazdanpanah, S.; Sayahi, M.H.; Javanshir, S.; Nouri, A.; Asadi, M. et al. [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as new therapeutic candidates against urease positive microorganisms: Design, synthesis, pharmacological evaluations, and in silico studies. Sci. Rep.; 2023; 13, 10136. [DOI: https://dx.doi.org/10.1038/s41598-023-37203-z] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37349372]

20. Khan, I.; Ali, S.; Hameed, S.; Rama, N.H.; Hussain, M.T.; Wadood, A.; Uddin, R.; Ul-Haq, Z.; Khan, A.; Ali, S. et al. Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole derivatives. Eur. J. Med. Chem.; 2010; 45, pp. 5200-5207. [DOI: https://dx.doi.org/10.1016/j.ejmech.2010.08.034]

21. Moghadam, E.S.; Al-Sadi, A.M.; Talebi, M.; Amanlou, M.; Amini, M.; Abdel-Jalil, R. Novel benzimidazole derivatives; synthesis, bioactivity and molecular docking study as potent urease inhibitors. DARU J. Pharm. Sci.; 2022; 30, pp. 29-37. [DOI: https://dx.doi.org/10.1007/s40199-021-00427-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35040104]

22. Mishra, H.; Parrill, A.L.; Williamson, J.S. Tree-dimensional quantitative structure-activity relationship and comparative molecular feld analysis of dipeptide hydroxamic acid Helicobacter pylori urease inhibitors. Antimicrob. Agents Chemother.; 2002; 46, pp. 2613-2618. [DOI: https://dx.doi.org/10.1128/AAC.46.8.2613-2618.2002]

23. Dastyafteh, N.; Noori, M.; Montazer, M.N.; Zomorodian, K.; Yazdanpanah, S.; Iraji, A.; Ghomi, M.K.; Javanshir, S.; Asadi, M.; Dianatpour, M. et al. New thioxothiazolidinyl-acetamides derivatives as potent urease inhibitors: Design, synthesis, in vitro inhibition, and molecular dynamic simulation. Sci. Rep.; 2023; 13, 21. [DOI: https://dx.doi.org/10.1038/s41598-022-27234-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36593349]

24. Irfan, A.; Faisal, S.; Ahmad, S.; Al-Hussain, S.A.; Javed, S.; Zahoor, A.F.; Parveen, B.; Zaki, M.E.A. Structure-Based Virtual Screening of Furan-1,3,4-Oxadiazole Tethered N-phenylacetamide Derivatives as Novel Class of hTYR and hTYRP1 Inhibitors. Pharmaceuticals; 2023; 16, 344. [DOI: https://dx.doi.org/10.3390/ph16030344] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36986444]

25. Zhuang, C.; Zhang, W.; Sheng, C.; Zhang, W.; Xing, C.; Miao, Z. Chalcone: A Privileged Structure in Medicinal Chemistry. Chem. Rev.; 2017; 117, pp. 7762-7810. [DOI: https://dx.doi.org/10.1021/acs.chemrev.7b00020]

26. Rajendran, G.; Bhanu, D.; Aruchamy, B.; Ramani, P.; Pandurangan, N.; Bobba, K.N.; Oh, E.J.; Chung, H.Y.; Gangadaran, P.; Ahn, B.-C. Chalcone: A Promising Bioactive Scaffold in Medicinal Chemistry. Pharmaceuticals; 2022; 15, 1250. [DOI: https://dx.doi.org/10.3390/ph15101250] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36297362]

27. Sultan, A.; Shajahan, S.; Ahamad, T.; Alshehri, S.M.; Sajjad, N.; Nisa, M.; Rehman, M.H.U.; Torun, L.; Khalid, M.; Acevedo, R. Silica-supported heterogeneous catalysts-mediated synthesis of chalcones as potent urease inhibitors: In vitro and molecular docking studies. Monatsh. Chem.; 2020; 151, pp. 123-133. [DOI: https://dx.doi.org/10.1007/s00706-019-02534-z]

28. Olleik, H.; Yahiaoui, S.; Roulier, B.; Courvoisier-Dezord, E.; Perrier, J.; Pérès, B.; Hijazi, A.; Baydoun, E.; Raymond, J.; Boumendjel, A. et al. Aurone derivatives as promising antibacterial agents against resistant Gram-positive pathogens. Eur. J. Med. Chem.; 2019; 165, pp. 133-141. [DOI: https://dx.doi.org/10.1016/j.ejmech.2019.01.022] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30665143]

29. Irfan, A.; Zahoor, A.F.; Kamal, S.; Hassan, M.; Kloczkowski, A. Ultrasonic-Assisted Synthesis of Benzofuran Appended Oxadiazole Molecules as Tyrosinase Inhibitors: Mechanistic Approach through Enzyme Inhibition, Molecular Docking, Chemoinformatics, ADMET and Drug-Likeness Studies. Int. J. Mol. Sci.; 2022; 23, 10979. [DOI: https://dx.doi.org/10.3390/ijms231810979]

30. Irfan, A.; Faisal, S.; Zahoor, A.F.; Noreen, R.; Al-Hussain, S.A.; Tuzun, B.; Javaid, R.; Elhenawy, A.A.; Zaki, M.E.A.; Ahmad, S. et al. In Silico Development of Novel Benzofuran-1,3,4-Oxadiazoles as Lead Inhibitors of M. tuberculosis Polyketide Synthase 13. Pharmaceuticals; 2023; 16, 829. [DOI: https://dx.doi.org/10.3390/ph16060829]

31. Aslam, S.; Asif, N.; Khan, M.N.; Khan, M.A.; Munawar, M.A.; Nasrullah, M. Synthesis of Novel Arylfurfurylchalcones. Asian J. Chem.; 2013; 25, pp. 7738-7742. [DOI: https://dx.doi.org/10.14233/ajchem.2013.14590A]

32. Irfan, A.; Zahoor, A.F.; Rasul, A.; Al-Hussain, S.A.; Faisal, S.; Ahmad, S.; Noor, R.; Muhammed, M.T.; Zaki, M.E.A. BTEAC Catalyzed Ultrasonic-Assisted Synthesis of Bromobenzofuran-Oxadiazoles: Unravelling Anti-HepG-2 Cancer Therapeutic Potential through In Vitro and In Silico Studies. Int. J. Mol. Sci.; 2023; 24, 3008. [DOI: https://dx.doi.org/10.3390/ijms24033008] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36769327]

33. Ahmad, M.R.; Sastry, V.G.; Bano, N.; Anwar, S. Synthesis of novel chalcone derivatives by conventional and microwave irradiation methods and their pharmacological activities. Arab. J. Chem.; 2016; 9, pp. S931-S935. [DOI: https://dx.doi.org/10.1016/j.arabjc.2011.09.002]

34. Holla, B.S.; Veerendra, B.; Shivanandaa, M.K. Non-linear optical properties of new arylfuranylpropenones. J. Cryst. Growth; 2004; 263, pp. 532-535. [DOI: https://dx.doi.org/10.1016/j.jcrysgro.2003.11.070]

35. Ibrar, A.; Kazmi, M.; Khan, A.; Halim, S.A.; Saeed, A.; Mehsud, S.; AlHarrasi, A.; Khan, I. Robust therapeutic potential of carbazole-triazine hybrids as a new class of urease inhibitors: A distinctive combination of nitrogen-containing heterocycles. Bioorg. Chem; 2020; 95, pp. 103479-103482. [DOI: https://dx.doi.org/10.1016/j.bioorg.2019.103479] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31901517]

36. Wahid, S.; Jahangir, S.; Versiani, M.A.; Khan, K.M.; Salar, U.; Ashraf, M.; Farzand, U.; Wadood, A.; Taha, M.; Perveen, S. Atenolol Thiourea Hybrid as Potent Urease Inhibitors: Design, Biology-Oriented Drug Synthesis, Inhibitory Activity Screening, and Molecular Docking Studies. Bioorg. Chem.; 2020; 94, pp. 103359-103362. [DOI: https://dx.doi.org/10.1016/j.bioorg.2019.103359] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31640931]

37. Pervez, H.; Chohan, Z.H.; Ramzan, M.; Nasim, F.-U.-H.; Khan, K.M. Synthesis and biological evaluation of some new N4-substituted isatin-3-thiosemicarbazones. J. Enzym. Inhib. Med. Chem.; 2009; 24, pp. 437-446. [DOI: https://dx.doi.org/10.1080/14756360802188420] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18629680]

38. AL-Hazimi, H.M.A.; Al-Alshaikh, M.A. Microwave assisted synthesis of substituted fu-ran-2-carboxaldehydes and their reactions. J. Saudi Chem. Soc.; 2010; 14, pp. 373-382. [DOI: https://dx.doi.org/10.1016/j.jscs.2010.04.009]

39. Brain, C.T.; Hallett, A.; Ko, S.Y. Thioamide Synthesis:  Thioacyl-N-phthalimides as Thio-acylating Agents. J. Org. Chem.; 1997; 62, pp. 3808-3809. [DOI: https://dx.doi.org/10.1021/jo970528v]

40. Holla, B.S.; Akberali, P.M.; Shivananda, M.K. Studies on nitrophenylfuran derivatives: Part XII. Synthesis, characterization, antibacterial and antiviral activities of some nitro-phenylfurfurylidene-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines. Farmaco; 2002; 56, pp. 919-927. [DOI: https://dx.doi.org/10.1016/S0014-827X(01)01124-7] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11829111]

41. Holla, B.S.; Akberali, P.M.; Shivananda, M.K. Studies on arylfuran derivatives: Part X. Synthesis and antibacterial properties of arylfuryl-Δ2-pyrazolines. Farmaco; 2000; 55, pp. 256-263. [DOI: https://dx.doi.org/10.1016/S0014-827X(00)00030-6]

42. Puterová, Z.; Krutošíková, A.; Lyčka, A.; Ďurčeková, T. Reactions of Substituted Furan-2-carboxaldehydes and Furo[b]pyrrole Type Aldehydes with Benzothiazolium Salts. Molecules; 2004; 9, pp. 241-255. [DOI: https://dx.doi.org/10.3390/90400241]

43. Svane, S.; Sigurdarson, J.J.; Finkenwirth, F.; Eitinger, T.; Karring, H. Inhibition of Urease Activity by Different Compounds Provides Insight into the Modulation and Association of Bacterial Nickel Import and Ureolysis. Sci. Rep.; 2020; 10, 8503. [DOI: https://dx.doi.org/10.1038/s41598-020-65107-9] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32444844]

44. Zambelli, B.; Mazzei, L.; Ciurli, S. Intrinsic Disorder in the Nickel-Dependent Urease Network. Prog. Mol. Biol. Transl. Sci.; 2020; 174, pp. 307-330. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32828469]

45. Martin, P.R.; Hausinger, R.P. Site-Directed Mutagenesis of the Active Site Cysteine in Klebsiella Aerogenes Urease. J. Biol. Chem.; 1992; 267, pp. 20024-20027. [DOI: https://dx.doi.org/10.1016/S0021-9258(19)88659-3]

46. Ansari, F.L.; Wadood, A.; Ullah, A.; Iftikhar, F.; Ul-Haq, Z. In silico studies of urease inhibitors to explore ligand-enzyme interactions. J. Enzym. Inhib. Med. Chem.; 2009; 24, pp. 151-156. [DOI: https://dx.doi.org/10.1080/14756360801945598] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18608771]

47. Trott, O.; Olson, A.J. AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J. Comput. Chem.; 2009; 31, pp. 455-461. [DOI: https://dx.doi.org/10.1002/jcc.21334] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19499576]

48. Ha, N.C.; Oh, S.T.; Sung, J.Y.; Cha, K.A.; Lee, M.H.; Oh, B.H. Supramolecular Assembly and Acid Resistance of Helicobacter Pylori Urease. Nat. Struct. Biol.; 2001; 8, pp. 505-509. [DOI: https://dx.doi.org/10.1038/88563]

49. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A Free Web Tool to Evaluate Pharmacokinetics, Drug-Likeness and Medicinal Chemistry Friendliness of Small Molecules. Sci. Rep.; 2017; 7, 42717. [DOI: https://dx.doi.org/10.1038/srep42717]

50. Xiong, G.; Wu, Z.; Yi, J.; Fu, L.; Yang, Z.; Hsieh, C.; Yin, M.; Zeng, X.; Wu, C.; Lu, A. et al. ADMETlab 2.0: An Integrated Online Platform for Accurate and Comprehensive Predictions of ADMET Properties. Nucleic Acids Res.; 2021; 49, pp. W5-W14. [DOI: https://dx.doi.org/10.1093/nar/gkab255]