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.

"Clicking" fragment leads to novel dual-binding cholinesterase inhibitors.

Cholinesterase inhibitors are potent therapeutics in the treatment of Alzheimer's disease. Among them, dual binding ligands have recently gained a lot of attention. We discovered novel dual-binding cholinesterase inhibitors, using "clickable" fragments, which bind to either catalytic active site (CAS) or peripheral anionic site (PAS) of the enzyme. Copper(I)-catalyzed azide-alkyne cycloaddition allowed to effectively synthesize a series of final heterodimers, and modeling and kinetic studies confirmed their ability to bind to both CAS and PAS. A potent acetylcholinesterase inhibitor with IC50 = 18 nM (compound 23g) was discovered. A target-guided approach to link fragments by the enzyme itself was tested using butyrylcholinesterase.

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.

Fentanyl Family at the Mu-Opioid Receptor: Uniform Assessment of Binding and Computational Analysis.

Interactions of 21 fentanyl derivatives with µ-opioid receptor (µOR) were studied using experimental and theoretical methods. Their binding to µOR was assessed with radioligand competitive binding assay. A uniform set of binding affinity data contains values for two novel and one previously uncharacterized derivative. The data confirms trends known so far and thanks to their uniformity, they facilitate further comparisons. In order to provide structural hypotheses explaining the experimental affinities, the complexes of the studied derivatives with µOR were modeled and subject to molecular dynamics simulations. Five common General Features (GFs) of fentanyls' binding modes stemmed from these simulations. They include: GF1) the ionic interaction between D147 and the ligands' piperidine NH⁺ moiety; GF2) the N-chain orientation towards the µOR interior; GF3) the other pole of ligands is directed towards the receptor outlet; GF4) the aromatic anilide ring penetrates the subpocket formed by TM3, TM4, ECL1 and ECL2; GF5) the 4-axial substituent (if present) is directed towards W318. Except for the ionic interaction with D147, the majority of fentanyl-µOR contacts is hydrophobic. Interestingly, it was possible to find nonlinear relationships between the binding affinity and the volume of the N-chain and/or anilide's aromatic ring. This kind of relationships is consistent with the apolar character of interactions involved in ligand⁻receptor binding. The affinity reaches the optimum for medium size while it decreases for both large and small substituents. Additionally, a linear correlation between the volumes and the average dihedral angles of W293 and W133 was revealed by the molecular dynamics study. This seems particularly important, as the W293 residue is involved in the activation processes. Further, the Y326 (OH) and D147 (Cγ) distance found in the simulations also depends on the ligands' size. In contrast, neither RMSF measures nor D114/Y336 hydrations show significant structure-based correlations. They also do not differentiate studied fentanyl derivatives. Eventually, none of 14 popular scoring functions yielded a significant correlation between the predicted and observed affinity data (R < 0.30, n = 28).

Donepezil-melatonin hybrids as butyrylcholinesterase inhibitors: Improving binding affinity through varying mode of linking fragments.

Hybrid inhibitors of acetyl- and butyrylcholinesterase are compounds that combine structural motifs of two different classical inhibitors, leading to a dual binding ligand. A rapidly growing collection of those compounds involves a wide diversity of structural motifs, but the way of linking two active fragments and its impact on the affinity toward cholinesterases usually remains beyond the extent of investigation. We present hereby a detailed analysis of this aspect using melatonin-donepezil hybrids. A new series of compounds, in which two fragments are connected using a carbamate linker, exhibits excellent activity and selectivity toward butyrylcholinesterase.

Some mechanistic aspects regarding the Suzuki-Miyaura reaction between selected ortho-substituted phenylboronic acids and 3,4,5-tribromo-2,6-dimethylpyridine.

Background: Atropisomers are very interesting stereoisomers having axial chirality resulting from restricted rotation around single bonds and are found in various classes of compounds. ortho-Substituted arylpyridines are an important group of them. A regio- and atropselective Suzuki-Miyaura cross-coupling reaction on 3,4,5-tribromo-2,6-dimethylpyridine was studied. Results: Reactions with various amounts of ortho-substituted phenylboronic acids with 3,4,5-tribromo-2,6-dimethylpyridine gave a series of mono- di- and triarylpyridine derivatives which allowed to draw conclusions about the order of substitution. Also, the observed selectivity in the case of ortho-methoxyphenylboronic acid suggested an additional metal O-chelation effect in the transition state, apparently not present in the ortho-chloro analogues. The rotational barrier in selected atropisomers was determined on the basis of HT NMR and thermal epimerisation experiments. The structure of most presented atropisomeric derivatives of 2,6-dimethylpyridine was confirmed by single-crystal X-ray analysis. Racemic chiral, differently substituted atropisomers were also examined by 1H NMR spectroscopy in the presence of a chiral solvating agent. Conclusion: This regio- and atropselectivity may be generally applicable to other arylpyridine systems. A regio- and atropselective Suzuki-Miyaura cross-coupling process has been observed, giving an efficient access to a class of atropisomeric compounds. An opposite selectivity using a differently ortho-substituted phenylbornic acid was observed.

Molecular cloning of melatonin 3-hydroxylase and its production of cyclic 3-hydroxymelatonin in rice (Oryza sativa).

Melatonin is metabolized in animals to cyclic 3-hydroxymelatonin (3-OHM) not by an enzymatic pathway, but by interaction with hydroxyl radicals. The production of 3-OHM in animals suggests the possible presence of 3-OHM in plants. Prior to the identification of 3-OHM in plants, we directly cloned the corresponding gene(s) responsible for 3-OHM synthesis using Escherichia coli library strains expressing genes belonging to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily from rice. Three of 35 E. coli library strains supplemented with 1 mmol/L melatonin were found to produce 3-OHM in their extracellular medium, suggestive of three 2-ODD genes involved in 3-OHM production. The purified recombinant 2-ODD 11, 2-ODD 26, and 2-ODD 33 proteins were shown to catalyze the metabolism of melatonin to 3-OHM, with 2-ODD 11 showing the highest melatonin 3-hydroxylase (M3H) catalytic activity. Consistent with the presence of M3H genes, rice leaves supplemented with 5 mmol/L melatonin produced 3-OHM [233 µg/g fresh weight (FW)], 2-hydroxymelatonin (21 µg/g FW), and N1 -acetyl-N2 -formyl-5-methoxykynuramine (5 µg/g FW). Three M3H transcripts were induced upon the treatment of rice leaves with cadmium followed by an increase in M3H enzyme activity. Cloning of M3H genes in plants has paved the way for the studies of melatonin in plants in terms of its multiple physiological roles.

Formal synthesis of (-)-podophyllotoxin through the photocyclization of an axially chiral 3,4-bisbenzylidene succinate amide ester--a flow photochemistry approach.

We have developed a strategy for the stereoselective synthesis of cyclolignans related to podophyllotoxin and its derivatives. The crucial step of the synthesis is the photocyclization of a chiral atropoisomeric 1,2-bisbenzylidenesuccinate amide ester, which can be prepared from suitable aromatic aldehydes, diethyl succinate and l-prolinol. The photocyclization was found to be more efficient when irradiation was performed in a home-built continuous flow photochemical reactor. The in-flow irradiation also allowed us to perform the reaction on a multigram scale. The chiral auxiliary was removed by reductive cleavage with the Schwartz's reagent to give the cytotoxic 1R,2R-cis-podophyllic aldehyde, which in turn could be easily reduced to the corresponding alcohol, completing the formal synthesis of (-)-podophyllotoxin.

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.

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.

The enantioselective synthesis of (S)-(+)-mianserin and (S)-(+)-epinastine.

A simple enantioselective synthetic procedure for the preparation of mianserin and epinastine in optically pure form is described. The key step in the synthetic pathway is the asymmetric reduction of the cyclic imine using asymmetric transfer hydrogenation conditions.

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.

Highly selective inhibition of butyrylcholinesterase by a novel melatonin-tacrine heterodimers.

Novel inhibitors of cholinesterases, especially butyrylcholinesterase (BuChE), were obtained by coupling melatonin-tacrine heterodimers via the carbamate bond. Compounds 14a-i possessed potent cholinesterase inhibitory activity (with IC50 values as low as 1.18 nM for acetylcholinesterase (AChE) and 0.24 nM for butyrylcholinesterase (BuChE)). These heterodimers exhibit selectivity toward BuChE, being from 4- to 256-fold more active toward BuChE than AChE, but still acting as better AChE inhibitors than tacrine 4.

Synthesis of imperatorin analogs and their evaluation as acetylcholinesterase and butyrylcholinesterase inhibitors.

In this study, we synthesized several imperatorin analogs using imperatorin and xanthotoxin as substrates. The anti-cholinesterase activities of all compounds were evaluated in in vitro experiments according to the modified Ellman's method. For each synthesized compound, IC50 values for both enzymes were established. Galantamine hydrobromide was used as a positive control in the enzymatic experiments. All active compounds showed selectivity toward butyrylcholinesterase (BuChE) rather than acetylcholinesterase. The most active ones were 8-(3-methylbutoxy)-psoralen and 8-hexoxypsoralen with IC50 values for BuChE of around 16.5 and 16.4 µM, respectively. The results of our study may be considered as the beginning of a search for potential anti-Alzheimer's disease drugs based on the structure of natural furocoumarins.

3-(2-Acetamido-eth-yl)-1H-indol-5-yl 4-nitro-phenyl carbonate.

In the title mol-ecule, C(19)H(17)N(3)O(6), the indole ring system is essentially planar (r.m.s. deviation = 0.009 Å) and forms a dihedral angle of 31.96 (9)° with the nitro-substituted benzene ring. In the crystal, mol-ecules are linked by pairs of N-H⋯O hydrogen bonds, forming inversion dimers which are connected by further N-H⋯O hydrogen bonds into a two-dimensional network parallel to (102).

Evaluation of different internal standardization approaches for the quantification of melatonin in cell culture samples by multiple heart-cutting two dimensional liquid chromatography tandem mass spectrometry.

We evaluate here different analytical strategies for the chromatographic separation and determination of N-acetyl-5-methoxytryptamine (MEL) and its oxidative metabolites N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N1-acetyl-5-methoxykynuramine (AMK) and cyclic 3-hydroxymelatonin (c3OHM) in cell culture samples. Two dimensional liquid chromatography (2D-LC) in the multiple heart-cutting mode was compared with regular 1D chromatographic separations of MEL and its oxidative metabolites. Our results showed that the use of trifluoroacetic acid (TFA) as mobile phase modifier was required to obtain a satisfactory resolution and peak shapes particularly for c3OHM. As TFA is not compatible with ESI ionization the application of the MHC mode was mandatory for a proper chromatographic separation. We evaluate also different internal standardization approaches based on the combined use of a surrogate standard (5-methoxytryptophol) and an internal standard (6-methoxytryptamine) for MEL quantification in cell culture samples obtaining unsatisfactory results both by 1D- and 2D-LC-ESI-MS/MS (from 9 ± 2 to 186 ± 38%). We demonstrate that only the application of isotope dilution Mass Spectrometry through the use of an in house synthesized 13C isotopically labelled analogue provided quantitative MEL recoveries both by using 1D- and 2D-LC-ESI-MS/MS (99±1 and 98±1. Respectively) in androgen-insensitive human prostate carcinoma PC3 cells.

Novel podophyllotoxin and benzothiazole derivative induces transitional morphological and functional changes in HaCaT cells.

Podophyllotoxin (PPT) is an antimitotic drug used topically in the treatment of anogenital warts. Due to its toxicity it cannot be administered systemically as an anticancer agent. However, modified PPT derivatives such as etoposide and teniposide are used clinically as systemic agents. Thus, we invented novel PPT derivative KL3 that was synthesized by photocyclization. Earlier we have shown that KL3 has an anticancer effect in various cell lines. Here we compared the toxicity of KL3 vs PPT on non-cancerous normal human keratinocytes (HaCaT) and peripheral blood mononuclear cells (PBMC) showing that KL3 is less toxic than PPT to non-cancerous cells. At concentrations that neither induced cell death, nor affected cell cycle, KL3 in HaCaT cells evoked transient ultrastructural features of ER stress, swelling of mitochondria and elongation of cytoplasmic processes. Those changes partially reversed with prolonged incubation while features of autophagy were induced. PPT in equivalent concentrations induced HaCaT cell death by cell cycle arrest, intrinsic apoptosis and finally disintegration of cell membranes followed by secondary necrosis. In conclusion, we show that the KL3 derivative of PPT in contrast to PPT allows repair of normal keratinocytes and triggers mechanisms that restore non-tumor cell homeostasis.

Selective inhibition of butyrylcholinesterase by singlet oxygen-generated melatonin derivatives.

The inhibition of cholinesterases plays a crucial role in a therapy of neurodegenerative diseases, including Alzheimer's disease. Especially, butyrylcholinesterase (BChE) has recently gained special interest. On the other hand, compounds having antioxidative properties may have a beneficial role in slowing down neurodegeneration processes. To combine these two effects, we synthesized a series of new derivatives of melatonin, which is a strong antioxidant, possessing structural elements essential for the inhibitory activity against cholinesterase. The structure of the new compounds was confirmed by NMR spectroscopy and mass spectrometry, and their activity against cholinesterases was measured in vitro using modified Ellman's method. The compounds obtained showed a high inhibitory activity, together with a strong selectivity against BChE. These results may point at new area of interest in a research on cholinesterase inhibitors.

Tetra-methyl 1,1,2-triphenyl-2H-1λ-phosphole-2,3,4,5-tetra-carboxyl-ate.

The title compound, C(30)H(27)O(8)P (1), was formed as one of two products {(1) and (2) [Krawczyk et al. (2010 ▶). Acta Cryst. E66 (cv2753)]} in the reaction of dimethyl acetyl-enedicarboxyl-ate with triphenyl-phosphine. The mol-ecule of (1) consists of a five-membered ring, in which the P atom is incorporated. One of the phenyl groups of the triphenyl-phosphine migrated to a vicinal C atom during the reaction. The five-membered ring of (1) is corrugated [r.m.s. deviation = 0.0719 (8) Å], whereas that in compound (2) is planar, the r.m.s. deviation being only 0.009 (2) Å.

Trimethyl-3-meth-oxy-4-oxo-5-triphenyl-phospho-ranyl-idene-cyclo-pent-1-ene-1,2,3-tricarboxyl-ate.

The title compound, C(30)H(27)O(8)P (2), was formed as one of two products {(1) [Krawczyk et al. (2010 ▶). Acta Cryst. E66 (cv2752)] and (2)} in the reaction of dimethyl acetyl-enedicarboxyl-ate with triphenyl-phosphine. The mol-ecule of (2) consists of a five-membered carbocyclic ring. The P atom is a part of a triphenylphosphoranylidene substituent. In contrast to (1), the five-membered ring of (2) is planar, the r.m.s. deviation being only 0.009 (2) Å.