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.

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.

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.

Synthesis and Biological Evaluation of Dithiobisacetamides as Novel Urease Inhibitors.

Thirty-eight disulfides containing N-arylacetamide were designed and synthesized in an effort to develop novel urease inhibitors. Biological evaluation revealed that some of the synthetic compounds exhibited strong inhibitory potency against both cell-free urease and urease in intact cell with low cytotoxicity to mammalian cells even at concentration up to 250 µM. Of note, 2,2'-dithiobis(N-(2-fluorophenyl)acetamide) (d7), 2,2'-dithiobis(N-(3,5-difluorophenyl)acetamide) (d24), and 2,2'-dithiobis(N-(3-fluorophenyl)acetamide) (d8) were here identified as the most active inhibitors with IC50 of 0.074, 0.44, and 0.81 µM, showing 32- to 355-fold higher potency than the positive control acetohydroxamic acid. These disulfides were confirmed to bind urease without covalent modification of the cysteine residue and to inhibit urease reversibly with a mixed inhibition mechanism. They also showed very good anti-Helicobacter pylori activities with d8 showing a comparable potency to the clinical used drug amoxicillin. The impressive in vitro biological profile indicated their immense potential as therapeutic agents to tackle H. pylori caused infections.

Identification, potency evaluation, and mechanism clarification of α-glucosidase inhibitors from tender leaves of Lithocarpus polystachyus Rehd.

Lithocarpus polystachyus Rehd. known as Sweet Tea in China has attracted lots of interest for its good hypoglycemic effect and the potential as a hypoglycemic agent. Based on affinity separation-UPLC-Q-TOF-MS/MS, 54 potential α-glucosidase inhibitiors were identified and 44 were structurally determined. Out of them, 41 were identified for the first time from this plant including flavonoids, fatty acids, triterpenes, alkaloids, and coumarins. Enzyme assays revealed that flavonoids exhibited higher inhibitory activity against α-glucosidase than others with astilbin (IC50 = 6.14 µg·mL-1), morin (IC50 = 8.46 µg·mL-1), and naringenin (IC50 = 10.03 µg·mL-1) showing 2- to 4-fold higher potency than the positive control acarbose. They were proved as reversible inhibitors with mixed inhibition mechanism. Ki (Ki') values and molecular dockings strongly supported the potency order of astilbin, morin and naringenin that showed in the enzyme assays.

Recent Efforts in the Discovery of Urease Inhibitor Identifications.

Urease is an attractive drug target for designing anti-infective agents against pathogens such as Helicobacter pylori, Proteus mirabilis, and Ureaplasma urealyticum. In the past century, hundreds of medicinal chemists focused their efforts on explorations of urease inhibitors. Despite the FDA's approval of acetohydroxamic acid as a urease inhibitor for the treatment of struvite nephrolithiasis and the widespread use of N-(n-butyl)thiophosphoric triamide as a soil urease inhibitor as nitrogen fertilizer synergists in agriculture, urease inhibitors with high potency and safety are urgently needed. Exploration of novel urease inhibitors has therefore become a hot research topic recently. Herein, inhibitors identified worldwide from 2016 to 2021 have been reviewed. They structurally belong to more than 20 classes of compounds such as urea/thioure analogues, hydroxamic acids, sulfonamides, metal complexes, and triazoles. Some inhibitors showed excellent potency with IC50 values lower than 10 nM, having 10000-fold higher potency than the positive control thiourea.

Synthesis and Structure-Activity Relationship Studies of N-monosubstituted Aroylthioureas as Urease Inhibitors.

BACKGROUND: Thiourea is a classical urease inhibitor which is usually used as a positive control, and many N,N'-disubstituted thioureas have been determined as urease inhibitors. However, due to steric hindrance, N,N'-disubstituted thiourea motif could not bind urease as thiourea. On the contrary, N-monosubstituted thiourea with a tiny thiourea motif could theoretically bind into the active pocket as thiourea. OBJECTIVE: A series of N-monosubstituted aroylthioureas were designed and synthesized for evaluation as urease inhibitors. METHODS: Urease inhibition was determined by the indophenol method and IC50 values were calculated using computerized linear regression analysis of quantal log dose-probit functions. The kinetic parameters were estimated via surface plasmon resonance (SPR) and by nonlinear regression analysis based on the mixed type inhibition model derived from Michaelis-Menten kinetics. RESULTS: Compounds b2, b11, and b19 reversibly inhibited urease with a mixed mechanism, and showed excellent potency against both cell-free urease and urease in the intact cell, with IC50 values being 90- to 450-fold and 5- to 50-fold lower than the positive control acetohydroxamic acid, respectively. The most potent compound b11 showed an IC50 value of 0.060 ± 0.004µM against cell-free urease, which bound to urea binding site with a very low KD value (0.420±0.003nM) and a very long residence time (6.7 min). Compound b11 was also demonstrated to have very low cytotoxicity to mammalian cells. CONCLUSION: The results revealed that N-monosubstituted aroylthioureas bound to the active site of urease as expected, and represent a new class of urease inhibitors for the development of potential therapeutics against infections caused by urease-containing pathogens.

N-monosubstituted thiosemicarbazide as novel Ure inhibitors: synthesis, biological evaluation and molecular docking.

Background: Identification of novel Ure inhibitors with high potency has received considerable attention. Methodology & results: Ure inhibition was determined using the indophenol method, the affinities to Ure were estimated via surface plasmon resonance. Seventeen new plus ten known N-monosubstituted thiosemicarbazides were synthesized and identified as novel Ure inhibitors. Out of these compounds, compound b5 shows excellent activity against both crude Ure from Helicobacter pylori (IC50 = 0.04 µM) and Ure in living cell (IC50 = 0.27 µM), with the potency being over 600-fold higher than clinical used drug acetohyroxamic acid, respectively. Surface plasmon resonance demonstrated the high affinity (Kd.#x00A0;= 6.32 nM) of b5 to Ure. Conclusion: This work provides a class of novel and promising Ure inhibitors.

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.

The synthesis and evaluation of phenoxyacylhydroxamic acids as potential agents for Helicobacter pylori infections.

Two series of ω-phenoxy contained acylhydroxamic acids as novel urease inhibitors were designed and synthesized. Biological activity evaluations revealed that ω-phenoxypropinoylhydroxamic acids were more active than phenoxyacetohydroxamic acids. Out of these compounds, 3-(3,4-dichlorophenoxy)propionylhydroxamic acid c24 showed significant potency against urease in both cell free extract (IC50 = 0.061 ±â€¯0.003 µM) and intact cell (IC50 = 0.89 ±â€¯0.05 µM), being over 450- and 120-fold more potent than the clinically prescribed urease inhibitor AHA, repectively. Non-linear fitting of experimental data (V-[S]) suggested a mixed-type inhibition mechanism and a dual site binding mode of these compounds.

Arylamino containing hydroxamic acids as potent urease inhibitors for the treatment of Helicobacter pylori infection.

A novel series of aniline-containing hydroxamic acids were designed, synthesized and evaluated as anti-virulence agents for the treatment of gastritis and gastric ulcer caused by Helicobacter pylori. In vitro enzyme-based screen together with in vivo assays and structure-activity relationship (SAR) studies led to the discovery of three potent urease inhibitors 3-(3,5-dichlorophenylamino)N-hydroxypropanamide (3a), 3-(2-chlorophenylamino)N-hydroxypropanamide (3d) and 3-(2,4-dichlorophenylamino)N-hydroxypropanamide (3n). Compounds 3a, 3d and 3n showed excellent urease inhibition with IC50 values 0.043 ±â€¯0.005, 0.055 ±â€¯0.008 and 0.018 ±â€¯0.002 µM, and significantly depressed gastritis developing at the dose of 32 mg/kg b. i.d with eradication rates of H. pylori reaching 92.3, 84.6 and 100%, respectively. Preliminary safety studies (acute toxicity in mice) disclosed that 3a, 3d and 3n was well-tolerated in KM mice with LD50s of 2982.8, 3349.4 and 3126.9 mg/kg, respectively. Collectively, the data obtained in this study indicate that 3a, 3d and 3n, in particular 3n, could considered as promising candidates for the potential treatment of H. pylori caused gastritis and gastric ulcer, and hence merit further studies.

Resolution and evaluation of 3-chlorophenyl-3-hydroxypropionylhydroxamic acid as antivirulence agent with excellent eradication efficacy in Helicobacter pylori infected mice.

The continuing emergence of drug-resistant Helicobacter pylori (HP) drives the ongoing need for the development of new and effective anti-HP drugs. Urease inhibitor has now gained strong interest as an alternative approach for HP infections. 3-Chlorophenyl-3-hydroxypropionylhydroxamic acid (CPH), a novel urease inhibitor identified in our group, shows impressive potency, which was optically separated for a further exploration. Here, we report in vitro/in vivo pharmacological evaluation of (±)-CPHs and the enantiomers. The raceme and the individual enantiomers significantly suppress gastritis at 32 mg/kg b.i.d dose with lower toxicity to mammalian cells (with CC50s ≥ 3.16 mM) and mice (LD50s ≥ 2338 mg/kg) than the clinically used agent acetohydroxamic acid. Furthermore, a significant increase of eradication of HP is observed for the combination of (±)-CPHs or the enantiomers with an antimicrobial. These studies revealed that CPH is a promising candidate for an alternative treatment of HP-dependent conditions by targeting virulence factor urease, and CPH may be used as a raceme.

Synthesis and evaluation of adenosine containing 3-arylfuran-2(5H)-ones as tyrosyl-tRNA synthetase inhibitors.

Tyrosyl-tRNA synthetase (TyrRS) is an aminoacyl-tRNA synthetase family protein that possesses an essential role in bacterial protein synthesis. The synthesis, structure-activity relationship, and evolution of a novel series of adenosine-containing 3-arylfuran-2(5H)-ones as TyrRS inhibitors are described. Advanced compound d3 from this series exhibited excellent affinity for TyrRS with IC50 of 0.61 ± 0.04 µM. Bacterial growth inhibition assays demonstrated that d3 showed submicromolar antibacterial potency against Escherichia coli and Pseudomonas aeruginosa, and compared to the marketed antibiotics ciprofloxacin.

3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease inhibitors: Synthesis, molecular docking and biological evaluation.

Helicobacter pylori urease is involved in several physiologic responses such as stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas. Thus, inhibition of urease is taken for a good chance to treat H. pylori-caused infections, we have therefore focused our efforts on seeking novel urease inhibitors. Here, a series of arylpropionylhydroxamic acids were synthesized and evaluated for urease inhibition. Out of these compounds, 3-(2-benzyloxy-5-chlorophenyl)-3-hydroxypropionylhydroxamic acid (d24) was the most active inhibitor with IC50 of 0.15±0.05µM, showing a mixed inhibition with both competitive and uncompetitive aspects. Non-linear fitting of kinetic data gives kinetics parameters of 0.13 and 0.12µg·mL(-1) for Ki and Ki', respectively. The plasma protein binding assays suggested that d24 exhibited moderate binding to human and rabbit plasma proteins.

Synthesis of Perfluorinated Isoquinolinediones through Visible-Light-Induced Cyclization of Alkenes.

A novel visible-light-induced carboperfluoroalkylation of alkenes using perfluoroalkyl iodides and bromides as Rf sources, leading to isoquinoline-1,3-diones, was developed. This method offers rapid entry to perfluorinated isoquinoline-1,3(2H,4H)-diones from N-alkyl-N-methacryloyl benzamides under mild reaction conditions, allowing for the incorporation of a wide variety of perfluorinated groups such as CF3, C3F7, C4F9, C6F13, C8F17, C10F21, and CF2CO2Et.

Adenosine analogs as inhibitors of tyrosyl-tRNA synthetase: Design, synthesis and antibacterial evaluation.

Herein we describe the synthesis and evaluation of a series of adenosine analogs for in vitro antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Out of these compounds, compound c6 has much stronger antibacterial potency against Pseudomonas aeruginosa than ciprofloxacin, and was determined to target tyrosyl-tRNA synthetase with IC50 of 0.8±0.07 µM. Structure-activity relationship analysis suggested that introduction of a fluorine atom at the 3'-position of benzene ring of the phenylacetyl moiety significantly increased affinities to the enzyme. In comparison with isopropylidene analogs, 2',3'-deprotected compounds displayed higher inhibitory activity. Molecular dockings provided an explanation for observations in biological assays.

Synthesis and evaluation of new tyrosyl-tRNA synthetase inhibitors as antibacterial agents based on a N2-(arylacetyl)glycinanilide scaffold.

Tyrosyl-tRNA synthetase (TyrRS), an essential enzyme in bacterial protein biosynthesis, is an attractive therapeutic target for finding novel antibacterial agents, and a series of N2-(arylacetyl)glycinanilides has been herein synthesized and identified as TyrRS inhibitors. These efforts yielded several compounds, with IC50 in the low micromolar range against TyrRS from Staphylococcus aureus. Out of the obtained compounds, 3ap is the most active and exhibits excellent activity against both Gram-positive (S. aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains. In comparison with the parent scaffold 3-arylfuran-2(5H)-one, N2-(arylacetyl)glycinanilide significantly improved the potency against Gram-negative bacterial strains, indicating that this scaffold offers a significant potential for developing new antibacterial drugs.

Synthesis and evaluation of N-analogs of 1,2-diarylethane as Helicobacter pylori urease inhibitors.

Therapies based on urease inhibition are now seriously considered as the first line of treatment for infections caused by Helicobacter pylori. However, the present inhibitors are ineffective or unstable in highly acidic gastric juice. Here, we report a series of benzylanilines as effective inhibitors of H. pylori urease. Out of the obtained twenty-one compounds, N-(3,4-dihydroxybenzyl)-4-nitroaniline (4) was evaluated in detail and shows promising features for development as anti-H. pylori agent. Excellent potency against urease in both cell-free extract and intact cell was observed at low concentrations of 4 (IC50=0.62 ± 0.04 and 1.92 ± 0.09 µM), which showed over 29- and 54-fold increase in potency with respect to the positive control AHA. The SAR analysis revealed that protection of 3,4-dihydroxy group of 4 as methoxy or changes of 4-NO2 will result in a moderate to dramatic decrease in potency.

Synthesis, molecular docking and biological evaluation of 3-arylfuran-2(5H)-ones as anti-gastric ulcer agent.

3-Arylfuran-2(5H)-one derivatives show good antibacterial activity and were determined as tyrosyl-tRNA synthetase (TyrRS) inhibitors. In a systematic medicinal chemistry exploration, we demonstrated chemical opportunities to treat infections caused by Helicobacter pylori. Twenty 3-arylfuran-2(5H)-ones were synthesized and evaluated for anti-H. pylori, antioxidant and anti-urease activities which are closely interconnected with H. pylori infection. The results displayed that some of the compounds show excellent antioxidant activity, and good anti-H. pylori and urease inhibitory activities. Out of these compounds, 3-(3-methylphenyl)furan-2(5H)-one (b9) showed the most potent antioxidant activity (IC50=8.2 µM) and good anti-H. pylori activity (MIC50=2.6 µg/mL), and it can be used as a good candidate for discovering novel anti-gastric ulcer agent.