Cellular Distribution and Ultrastructural Changes in HaCaT Cells, Induced by Podophyllotoxin and Its Novel Fluorescent Derivative, Supported by the Molecular Docking Studies.

Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus's appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.

The Activity of Urolithin A and M4 Valerolactone, Colonic Microbiota Metabolites of Polyphenols, in a Prostate Cancer In Vitro Model.

The gut microbiota-derived metabolites of ellagitannins and green tea catechins, urolithin A (uroA) and 5-(3',4',5'-trihydroxyphenyl)-γ-valerolactone (M4), respectively, are among the main compounds absorbed into human system after ingestion of these polyphenols. The aim of this study was to establish the effects of M4, uroA, and their combinations on LNCaP cells, an androgen dependent prostate cancer in vitro model.. The LNCaP cells were incubated with increasing concentrations of tested metabolites. The cell proliferation was determined by measurement of DNA-bisbenzimide H 33 258 complexes fluorescence. The isobolographic analysis was used to establish the type of interaction between metabolites. The apoptosis, androgen receptor (AR) localization, and phosphorylation of Akt kinase were measured by flow cytometry. Prostate-specific antigen (PSA) secretion was determined by ELISA. M4 showed modest antiproliferative activity in LNCaP cells (IC50 = 117 µM; CI: 81 - 154). UroA decreased proliferation (IC50 = 32.7 µM; CI: 24.3 - 41.1) and induced apoptosis of LNCaP cells. The mixture of M4 with uroA had synergistic antiproliferative effect. Moreover, M4 potentiated inhibition of PSA secretion and enhanced retention of AR in cytoplasm caused by uroA. Interestingly, uroA increased levels of pSer473 Akt in LNCaP cells. These results show that colonic metabolites may contribute to chemoprevention of prostate cancer by varied polyphenol-rich diet or composite polyphenol preparations.

A convenient route to symmetrically and unsymmetrically substituted 3,5-diaryl-2,4,6-trimethylpyridines via Suzuki-Miyaura cross-coupling reaction.

A series of differently substituted 3,5-diaryl-2,4,6-trimethylpyridines were prepared and characterized using the Suzuki-Miyaura coupling reaction with accordingly selected bromo-derivatives and arylboronic acids. The reaction conditions were carefully optimized allowing high yield of isolated products and also the construction of unsymmetrically substituted diarylpyridines, difficult to access by other methods.

SYNTHETIC DERIVATIVES OF ISOQUINOLINE, DICARBOXYLIC ACID IMIDES AND THIOIMIDES AS BIOACTIVE COMPOUNDS.

This study is a continuation of a research program aimed at identifying potent drugs against bacterial infections, in which a series of organic compounds: dicarboxylic acid imides and thioimides, isoquinoline derivatives and open chain compounds, were examined for antimicrobial properties against Staphylococcus auneus and Escheiichia coli. In effect of this investigation, the most active compounds (35-40, 47) were selected for in vitiv tests against fourteen clinically important pathogenic isolates, the methicillin resistant Staphylococcus aueus (MRSA) and several reference Gram-negative bacteria: Prteus vulgaris, Pseudomonas aeruginosa, Klebsiella pneumonia, Stenonophoinonas inaltophilia, and Acinetobacter baumannii. The obtained data revealed that seven compounds (three dithioimides, 35, 39, 47, and four thioimides, 36-38, 40) exhibit effective antibacterial activity against the tested Staphylococcus auirus MSSA and MRSA strains. Among them, dicarboxylic acid thioimides 37 and 38 were proven to be the most active.

Chemical aspects of the primary ionization mechanisms in matrix-assisted laser desorption ionization.

It has been proposed that the primary ionization mechanism occurring in matrix-assisted laser desorption ionization (MALDI) experiments originates from the presence, in the solid-state matrix-analytes sample, of matrix dimers. These species are formed by the interaction of carboxylic groups present in the matrix molecules with the formation of strong hydrogen bonds. Theoretical calculations proved that the laser irradiation of these structures leads to one or two H-bridge cleavages, giving rise to an "open" dimer structure or to disproportionation with the formation of MH(+) and [M-H](-) species. The ions so formed can be considered highly effective in their reaction with analyte ions, leading to their protonation (or deprotonation). To achieve further evidence for these proposals, in the present study the energetics of the reactions of ions from different aromatic carboxylic acids with two amino acids (glycine and lysine) and three multipeptides (gly-gly, gly-gly-gly and gly-gly-gly-gly) was investigated. The lowest ∆G values were obtained for 2,5- dihydroxybenzoic acid, widely employed as the MALDI matrix. Also, for p-nitrobenzoic acid the reaction is slightly exothermic, while for the other aromatic carboxylic acids derivatives positives values of ∆G are present.

Identification and synthesis of by-products found in 4-methylthioamphetamine (4-MTA) produced by the Leuckart method.

The synthesis of the designer drug 4-methylthioamphetamine (4-MTA) has been carried out using the well-known Leuckart reaction in four versions. The treatment of 4-methylthiophenylacetone with formamide, mixture of formamide/formic acid, ammonium formate, and mixture of ammonium formate and formic acid followed by acid hydrolysis brought about the formation of 4-MTA contaminated with a number of impurities. The gas chromatography mass-spectrometry (GC-MS) analysis of the reaction mixtures allowed identification of the most prominent impurities, such as diasteromers of N,N-di-[ß-(4-methylthiophenyl)isopropyl]amine, N,N-di-[ß-(4-methylthiophenyl)isopropyl]methylamine, N,N-di-[ß-(4-methylthiophenyl)isopropyl]formamide, the Schiff bases derived from 4-MTA and 4-methylbenzaldehyde (benzaldimine) and 4-methylthiophenylacetone (ketimine) as well as some heterocycles: 4-methyl-5-(4'-methylthiophenyl)pyrimidine, 4-(4'-methylthiobenzyl)pyrimidine, 2,6-dimethyl-3,5-di-(4'-methylthiophenyl)pyridine, 2,4-dimethyl-3,5-di-(4'-methylthiophenyl)pyridine. The correctness of identification was confirmed by independent synthesis of these compounds. Each synthesized reference compound was characterized by means of MS, (1)H and (13)C NMR, and IR methods. The stereochemistry of (RR/SS) diasteromer of N,N-di-[ß-(4-methylthiophenyl)isopropyl]amine was confirmed by a crystallographic method.

Identification of common impurities present in the synthetic routes leading to 4-methylthioamphetamine (4-MTA). Part II: Reductive amination and nitropropene route.

In this paper the by-products arising during the synthesis of 4-methylthioamphetamine (4-MTA) by LiAlH(4) reduction of 1-(4-methylthiophenyl)-2-nitropropene (nitropropene route) and reductive amination of 4-methylthiophenyl-2-propanone in the presence of NaCNBH(4) are investigated. The identification of 4-methylthio derivatives of N-(ß-phenylisopropyl)benzaldimine, 4-methylthio derivative of N-(ß-phenylisopropyl)benzyl methyl ketimine, 1-(4-methylthiophenyl)-N-(4-methylthiobenzyl)-2-propanamine, (RS) and (SS/RR)-N,N-di-[ß-(4-methylthiophenyl)isopropyl]amine, 4-methylthiobenzyl ether and methylthiobenzoic acid methyl ester as most prominent impurities in crude 4-MTA synthesised by reductive amination of 4-methylthiophenyl-2-propanone, is reported. Methylthio derivatives of 2-methyl-3-phenylaziridine, 2-benzylaziridine, and 4-methylthio derivative of BMK oxime as route-specific markers of nitropropene route leading to 4-MTA, were also characterized. The identity of these compounds was confirmed by their independent synthesis.

The synthesis, mass spectrometric properties and identification of some N,N-di-(ß-arylisopropyl)formamides related to the synthesis of ring-modified amphetamines.

This study examines the electron impact (EI) induced mass spectrometric behavior of several N,N-di-(ß-arylisopropyl)formamides, which are connected to the Leuckart synthesis of some amphetamine analogues. Emphasis is laid on the fragmentation paths, which are common for all compounds under investigation and may be used in construction of the prediction scheme useful for identification of similar impurities, especially in absence of desirable authentic material. On the basis of this scheme several new N,N-di-(ß-arylisopropyl)formamides have been identified in selected amphetamine analogues synthesized by the Leuckart method, including 4-methylthioamphetamine (4-MTA), 4-fluoroamphetamine (4-FA), 4-methylamphetamine, 3-trifluoromethylamphetamine, 3,4-methylenedioxyamphetamine (MDA), 2,5-dimethoxyamphetamine (2,5-DMA), 2,4,5- and 3,4,5-trimethoxyamphetamines (2-TMA and 3-TMA).

The Suzuki-Miyaura reaction in the chemical transformations of vindoline.

Vindoline and its analogues are important constituents of the Madagascan periwinkle Catharanthus roseus, and some of them are valuable chemotherapy drugs used in treatment for some types of cancer, including leukaemia, lymphoma, breast and lung cancer. The search for semi-synthetic congeners of natural substances is still an important task for organic chemistry. In this communication we report the synthesis of five new vindoline derivatives, 15-(2-methoxyphenyl)vindoline 11, 15-(3-methoxyphenyl)vindoline 12, 15-(2-nitrophenyl)vindoline 13, 15-(3-cyanophenyl)vindoline 15, and 15-(4-cyanophenyl)vindoline 16 using the Suzuki-Miyaura reaction as the key step. X-Ray analysis of compound 16 is also reported.