In Silico and Chromatographic Methods for Analysis of Biotransformation of Prospective Neuroprotective Pyrrole-Based Hydrazone in Isolated Rat Hepatocytes.

In the current study, chromatographic and in silico techniques were applied to investigate the biotransformation of ethyl 5-(4-bromophenyl)-1-(2-(2-(2-hydroxybenzylidene) hydrazinyl)-2-oxoethyl)-2-methyl-1H-pyrrole-3-carboxylate (11b) in hepatocytic media. The initial chromatographic procedure was based on the employment of the conventional octadecyl stationary phase method for estimation of the chemical stability. Subsequently, a novel and rapid chromatographic approach based on a phenyl-hexyl column was developed, aiming to separate the possible metabolites. Both methods were performed on a Dionex 3000 ThermoScientific (ACM 2, Sofia, Bulgaria) device equipped with a diode array detector set up at 272 and 279 nm for analytes detection. An acetonitrile: phosphate buffer of pH 3.5: methanol (17:30:53 v/v/v) was eluted isocratically as a mobile phase with a 1 mL/min flow rate. A preliminary purification from the biological media was achieved by protein precipitation with methanol. A validation procedure was carried out, where the method was found to correspond to all ICH (Q2) and M10 set criteria. Additionally, an in silico-based approach with the online server BioTransformer 3.0 was applied in an attempt to predict the possible metabolites of the title compound 11b. It was hypothesized that four CYP450 isoforms (1A2, 2C9, 3A4, and 2C8) were involved in the phase I metabolism, resulting in the formation of 12 metabolites. Moreover, docking studies were conducted to evaluate the formation of stable complexes between 11b and the aforementioned isoforms. The obtained data indicated three metabolites as the most probable products, two of which (M9_11b and M10_11b) were synthesized by a classical approach for verification. Finally, liquid chromatography with a mass detector was implemented for comprehensive and summarized analysis, and the obtained results revealed that the metabolism of the 11b proceeds possibly with the formation of glucuronide and glycine conjugate of M11_11b.

Synthesis, DFT Study, and In Vitro Evaluation of Antioxidant Properties and Cytotoxic and Cytoprotective Effects of New Hydrazones on SH-SY5Y Neuroblastoma Cell Lines.

A series of ten new hydrazide-hydrazone derivatives bearing a pyrrole ring were synthesized and structurally elucidated through appropriate spectral characteristics. The target hydrazones were assessed for radical scavenging activity through 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tests, with ethyl 5-(4-bromophenyl)-1-(2-(2-(4-hydroxy-3,5-dimethoxybenzylidene)hydrazine-yl)-2-oxoethyl)-2-methyl-1H-pyrrole-3-carboxylate (7d) and ethyl 5-(4-bromophenyl)-1-(3-(2-(4-hydroxy-3,5-dimethoxybenzylidene) hydra zine-yl)-3-oxopropyl)-2-methyl-1H-pyrrole-3-carboxylate (8d) highlighted as the best radical scavengers from the series. Additional density functional theory (DFT) studies have indicated that the best radical scavenging ligands in the newly synthesized molecules are stable, do not decompose into elements, are less polarizable, and with a hard nature. The energy of the highest occupied molecular orbital (HOMO) revealed that both compounds possess good electron donation capacities. Overall, 7d and 8d can readily scavenge free radicals in biological systems via the donation of hydrogen atoms and single electron transfer. The performed in vitro assessment of the compound's protective activity on the H2O2-induced oxidative stress model on human neuroblastoma cell line SH-SY5Y determined 7d as the most perspective representative with the lowest cellular toxicity and the highest protection.

Structure-Based Design of Novel MAO-B Inhibitors: A Review.

With the significant growth of patients suffering from neurodegenerative diseases (NDs), novel classes of compounds targeting monoamine oxidase type B (MAO-B) are promptly emerging as distinguished structures for the treatment of the latter. As a promising function of computer-aided drug design (CADD), structure-based virtual screening (SBVS) is being heavily applied in processes of drug discovery and development. The utilization of molecular docking, as a helping tool for SBVS, is providing essential data about the poses and the occurring interactions between ligands and target molecules. The current work presents a brief discussion of the role of MAOs in the treatment of NDs, insight into the advantages and drawbacks of docking simulations and docking software, and a look into the active sites of MAO-A and MAO-B and their main characteristics. Thereafter, we report new chemical classes of MAO-B inhibitors and the essential fragments required for stable interactions focusing mainly on papers published in the last five years. The reviewed cases are separated into several chemically distinct groups. Moreover, a modest table for rapid revision of the revised works including the structures of the reported inhibitors together with the utilized docking software and the PDB codes of the crystal targets applied in each study is provided. Our work could be beneficial for further investigations in the search for novel, effective, and selective MAO-B inhibitors.

Development of Hyphenated Techniques and Network Identification Approaches for Biotransformational Evaluation of Promising Antitubercular N-pyrrolyl hydrazide-hydrazone in Isolated Rat Hepatocytes.

Novel, rapid and precise RP-HPLC-DAD method was developed, validated and successfully applied for determination of metabolic changes of ethyl 5-(4-bromophenyl)-1-(3-(2-(2-hydroxybenzylidene)hydrazinyl)-3-oxopropyl)-2-methyl-1H-pyrrole-3-carboxylate (12b) in isolated rat hepatocytes. The analytes were detected by a simple DAD detector at 279 nm wavelength. A single-step extraction method was implemented to enable fast purification and extraction from cellular culture, resulting in a complete recovery. Thereafter, the method was adequately transferred to a LC-MS system for identification of unknown products. Additionally, network metabolism evaluation was performed to predict the structures of major metabolites with their isotope mass through BioTransformer 3.0. The data from the LC-MS analysis and the online server were compared for comprehensive identification. The results indicated formation of four metabolic products, obtained through processes of hydrolysis (12 and b), hydroxylation in the structure 12b (M1) and O-dealkylation (M2).

Simultaneous analysis of water-soluble and fat-soluble vitamins through RP-HPLC/DAD in food supplements and brewer's yeast.

The current study is focused on investigation and quantitation of seven commercially available on the Bulgarian market food supplements, containing multivitamin mixtures of water-soluble and fat-soluble vitamins. In addition, a second fermentation brewer's yeast is also analyzed. The analytical procedures are performed on a RP-HPLC/DAD using Purospher STAR C18 (Merck Millipore, Germany) 5 µm, 25 × 0.46 cm column, conditioned at 25 °C in a column oven. Dionex UltiMate 3000 high performance liquid chromatograph was carried out in diode array detector, set up at 270 nm for water-soluble vitamins, except for vitamin B5, where 210 nm was applied as analytical wavelength. The fat-soluble vitamins were detected at 325 nm and 265 nm for vitamin A and vitamin E, respectively. Two general methods were developed where Method 1 was based on gradient elution and Method 2 was based on isocratic elution. Both methods identified stated by the manufacturer labeled amounts. The developed methods are applicable for routine analysis of vitamin contents both in multivitamin preparations and in brewer's yeast from secondary fermentation.