Novel Thiosemicarbazone Derivatives: In Vitro and In Silico Evaluation as Potential MAO-B Inhibitors

MAO-B inhibitors are frequently used in the treatment of neurodegenerative diseases such as Parkinson’s and Alzheimer’s. Due to the limited number of compounds available in this field, there is a need to develop new compounds. In the recent works, it was shown that various thiosemicarbazone derivatives show hMAO inhibitory activity in the range of micromolar concentration. It is thought that benzofuran and benzothiophene structures may mimic structures such as indane and indanone, which are frequently found in the structures of such inhibitors. Based on this view, new benzofuran/benzothiophene and thiosemicarbazone hybrid compounds were synthesized, characterized and screened for their hMAO-A and hMAO-B inhibitory activity by an in vitro fluorometric method. The compounds including methoxyethyl substituent (2b and 2h) were found to be the most effective agents in the series against MAO-B enzyme with the IC50 value of 0.042 ± 0.002 µM and 0.056 ± 0.002 µM, respectively. The mechanism of hMAO-B inhibition of compounds 2b and 2h was investigated by Lineweaver–Burk graphics. Compounds 2b and 2h were reversible and non-competitive inhibitors with similar inhibition features as the substrates. The Ki values of compounds 2b and 2h were calculated as 0.035 µM and 0.046 µM, respectively, with the help of secondary plots. The docking study of compound 2b and 2h revealed that there is a strong interaction between the active sites of hMAO-B and analyzed compound.

An Optimized Fluorometric Method to Test the Capacities of Cranberry Polyphenols and Metabolites to Inhibit the Adhesion of Type-P and Type-1 Fimbriated Uropathogenic E. coli

Objectives Adhesion of type-P and type-1 fimbriated uropathogenic E. coli to urinary tract epithelial cells initiates urinary tract infections. This research aimed to optimize and apply a fluorometric method to evaluate the capacities of cranberry polyphenols and metabolites to inhibit such adhesion in vitro. Methods BacLight Green labelled E. coli were incubated with cranberry polyphenols or microbial metabolites of cranberry polyphenols for 30 min at 37°C. Mixture was added to a 96-well microplate containing 1 × 105/well of human uroepithelial T24 cells and incubated for 1 h at 37°C. After incubation, E. coli not adhered were removed by phosphate buffer washing. Fluorescent intensity was measured on a microplate reader at 480 nm excitation and 516 nm emission. Results Stable and strong fluorescent readings were obtained with 800 μmol/L BacLight Green for E. coli labeling and an E. coli to T24 cells ratio of 400:1 for co-incubation. A standard curve was established using 0–63 μM myricetin. The half-maximal inhibitory concentrations (IC50) of myricetin were 13.2 μM against type-P E. coli adhesion and 5.5 μM against type-1 E. coli adhesion. A fraction enriched with procyanidin polymers had IC50 of 57.6 μg/mL against type-P E. coli and 19.3 μg/mL against type-1 E. coli, respectively. Its anti-adhesion activities were more potent than those of cranberry fractions enriched with procyanidin oligomers, flavonols, or anthocyanin. Procyanidin A2 had a maximal inhibition about 35% at 17.3 μM against type-P E. coli, but no anti-adhesion activity was observed against type-1 E. coli. Procyanidin B2 showed a plateaued inhibition about 15% at 173–691 μM against type-P E. coli. Its maximal inhibition against type-1 E. coli was around 25% at 346 μM. Hippuric acid, a major metabolite of cranberry polyphenols, had a maximal inhibition about 20% at 558 μM against type-1 E. coli adhesion, whereas its anti-adhesion activity against type-P E. coli was not detected. Conclusions The optimized fluorometric method showed that both structure and composition of cranberry polyphenols and metabolites affected their abilities to inhibit E. coli adhesion in vitro. Anti-adhesion activities of cranberry polyphenols also depend on type of E. coli fimbriae. Funding Sources University of Florida Research Foundation Seed Fund.

On the Amine-Catalyzed Suzuki-Miyaura Coupling Using a Catalysis-Based Fluorometric Method

<p>The Suzuki-Miyaura coupling is one of the most frequently used reactions in organic synthesis. Recent work by others suggested that an arylamine, prepared by palladium catalysis and tricyclohexylphosphine, could catalyze Suzuki-Miyaura coupling reactions without transition metals. Herein, we used a fluorometric quantification method for palladium previously developed in our laboratory to unambiguously conclude that there is a correlation between the palladium content in the arylamine and the rate of a Suzuki-Miyaura coupling. Also, our mass spectroscopic analysis of the arylamine revealed the presence of a palladium-phosphine complex. When Pd(OAc)₂ was used as a catalyst for the same Suzuki-Miyaura coupling, tricyclohexylphosphine was detrimental to the coupling and that the arylamine played negligible role. This study demonstrates the utility of the fluorometric technology for catalysis research.<br></p>

On the Amine-Catalyzed Suzuki-Miyaura Coupling Using a Catalysis-Based Fluorometric Method

<p>The Suzuki-Miyaura coupling is one of the most frequently used reactions in organic synthesis. Recent work by others suggested that an arylamine, prepared by palladium catalysis, could catalyze Suzuki-Miyaura coupling reactions without transition metals. Herein, we used a fluorometric quantification method for palladium previously developed in our laboratory to unambiguously conclude that there is a correlation between the palladium content in the arylamine and the rate of a Suzuki-Miyaura coupling. Also, our mass spectroscopic analysis of the arylamine revealed the presence of a palladium-phosphine complex. We discovered that the phosphine was detrimental to the palladium catalysis and that the arylamine played negligible role. This study demonstrates the utility of the fluorometric technology for catalysis research.</p>

On the Amine-Catalyzed Suzuki-Miyaura Coupling Using a Catalysis-Based Fluorometric Method

<p>The Suzuki-Miyaura coupling is one of the most frequently used reactions in organic synthesis. Recent work by others suggested that an arylamine, prepared by palladium catalysis, could catalyze Suzuki-Miyaura coupling reactions without transition metals. Herein, we used a fluorometric quantification method for palladium previously developed in our laboratory to unambiguously conclude that there is a correlation between the palladium content in the arylamine and the rate of a Suzuki-Miyaura coupling. Also, our mass spectroscopic analysis of the arylamine revealed the presence of a palladium-phosphine complex. We discovered that the phosphine was detrimental to the palladium catalysis and that the arylamine played negligible role. This study demonstrates the utility of the fluorometric technology for catalysis research.</p>

Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues

Antimicrobial compounds have been successfully used to control many plant and animal diseases. Recently, oxytetracycline (OTC) and streptomycin have been approved for the treatment of Huanglongbing in citrus. Since the application of OTC is under strict regulations, several methods have been developed to determine and monitor its levels in the environment including high-performance liquid chromatography, ELISA, colorimetric, and fluorometric assays. In this study, we developed a fluorometric method for the determination of OTC in plant tissues based on its complexation with europium. Our preliminary trials showed that phenols and flavonoids interfere with the europium assay by reacting with the sensitizing reagent, cetyltrimethylammonium chloride. Consequently, we used the 60 mg hydrophilic–lipophilic balanced (HLB) cartridges to purify the OTC from the plant matrix. The recovery of OTC from spiked leaf samples was 75 ± 7.6%. Using the 500 mg HLB, we were able to detect 0.3 ppm OTC in the final sample extract, which corresponds to 3 µg g−1 fresh weight (FWT). The developed method was successfully used to measure the level of OTC in leaves obtained from trunk-injected trees. The results obtained by the europium method were similar to those obtained using the ELISA assay. We also tested the cross-reactivity of OTC metabolites with the europium method. The 4-epi-OTC showed a high cross-reactivity (50.0 ± 3.6%) with europium assay, whereas α-apo-OTC and β-apo-OTC showed small cross-reactivity. We showed that the europium-sensitized fluorescence-based method can be successfully used to assess OTC in citrus plant tissues after a cleanup step. Our results showed that this method was sensitive, reproducible, and can be used to analyze many samples simultaneously.