Novel C3-Methylene-Bridged Indole Derivatives with and without Substituents at N1: The Influence of Substituents on Their Hemolytic, Cytoprotective, and Antimicrobial Activity.

Alkaloids are natural compounds useful as scaffolds for discovering new bioactive molecules. This study utilized alkaloid gramine to synthesize two groups of C3-substituted indole derivatives, which were either functionalized at N1 or not. The compounds were characterized by spectroscopic methods. The protective effects of the new compounds against in vitro oxidative hemolysis induced by standard oxidant 2,2'-azobis(2-amidinopropane dihydro chloride (AAPH) on human erythrocytes as a cell model were investigated. Additionally, the compounds were screened for antimicrobial activity. The results indicated that most of the indole derivatives devoid of the N1 substitution exhibited strong cytoprotective properties. The docking studies supported the affinities of selected indole-based ligands as potential antioxidants. Furthermore, the derivatives obtained exhibited potent fungicidal properties. The structures of the eight derivatives possessing indole moiety bridged to the imidazole-, benzimidazole-, thiazole-, benzothiazole-, and 5-methylbenzothiazoline-2-thiones were determined by X-ray diffraction. The C=S bond lengths in the thioamide fragment pointed to the involvement of zwitterionic structures of varying contribution. The predominance of zwitterionic mesomers may explain the lack of cytoprotective properties, while steric effects, which limit multiple the hydrogen-bond acceptor properties of a thione sulfur, seem to be responsible for the high hemolytic activity.

Synthesis and Hemolytic Activity of Bile Acid-Indole Bioconjugates Linked by Triazole.

New formyl and acetyl derivatives of bile acid propargyl esters and their bioconjugates with modified gramine molecules have been obtained using the click chemistry method to study their hemolytic potency. The structures of all compounds were confirmed by spectral (1H- and 13C NMR and FT-IR) analysis and mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. According to the results, the structural modification of formyl and acetyl bile acid derivatives, leading to the formation of new propargyl esters and indole bioconjugates, reduces their hemolytic activity. According to molecular docking studies, the tested ligands are highly likely to exhibit a similar affinity, as native ligands, for the active sites of specific protein domains (PDB IDs: 2Q85 and 5V5Z). The obtained results may be helpful for the development of selective bile acid bioconjugates as effective antibacterial, antifungal, or antioxidant agents.

Seasonal Variability of Volatile Components in Calypogeia integristipula.

Liverworts contain a large number of biologically active compounds that are synthesised and stored in their oil bodies. However, knowledge about the chemical composition of individual species is still incomplete. The subject of the study was Calypogeia integristipula, a species representing leafy liverworts. Plant material for chemotaxonomic studies was collected from various locations in Poland. The chemical composition was determined in 74 samples collected from the natural environment in 2021 and 2022 in three growing seasons: spring, summer and autumn, and for comparison with samples originating from in vitro culture. The plants were classified as Calypogeia integristipula on the basis of morphological characteristics, oil bodies, and DNA markers. The volatile organic compounds (VOCs) from the biological material were extracted by headspace solid phase microextraction (HS-SPME). The samples were then analysed by gas chromatography-mass spectrometry (GC-MS). A total of 79 compounds were detected, of which 44 compounds were identified. The remaining compounds were described using the MS fragmentation spectrum. Cyclical changes in the composition of compounds associated with the growing season of Calypogeia integristipula were observed. Moreover, samples from in vitro culture and samples taken from the natural environment were shown to differ in the composition of chemical compounds. In terms of quantity, among the volatile compounds, compounds belonging to the sesquiterpene group (46.54-71.19%) and sesqiuterpenoid (8.12-22.11%) dominate. A smaller number of compounds belong to aromatic compounds (2.30-10.96%), monoterpenes (0.01-0.07%), monoterpenoids (0.02-0.33%), and aliphatic hydrocarbons (1.11-6.12%). The dominant compounds in the analysed liverworts were: anastreptene (15.27-31.14%); bicyclogermacrene (6.99-18.09%), 4,5,9,10-dehydro-isolongifolene (2.00-8.72%), palustrol (4.95-9.94%), spathulenol (0.44-5.11%).

Novel gramine-based bioconjugates obtained by click chemistry as cytoprotective compounds and potent antibacterial and antifungal agents.

A series of indole-1,4-disubstituted-1,2,3-triazole conjugates were synthesised by click chemistry. The haemolytic properties and cytoprotective activity of all the newly synthesised indole-triazole conjugates were tested in vitro. In addition, molecular docking was performed in silico for the selected conjugates to determine their antibacterial and antifungal properties. The results indicate that indole-triazole derivatives effectively protect human erythrocytes against free radical-induced haemolysis in a structure-dependent manner and that bis-indole-bis-triazole derivatives with alkyl linkers are excellent cytoprotective agents against oxidative haemolysis. The tested series of indole-1,4-disubstituted-1,2,3-triazole conjugates may have an affinity for the active sites of specific protein domains (PDB IDs: 2Q85 and 5V5Z) according to molecular docking studies.

Artificial Intelligence in Decrypting Cytoprotective Activity under Oxidative Stress from Molecular Structure.

Artificial intelligence (AI) is widely explored nowadays, and it gives opportunities to enhance classical approaches in QSAR studies. The aim of this study was to investigate the cytoprotective activity parameter under oxidative stress conditions for indole-based structures, with the ultimate goal of developing AI models capable of predicting cytoprotective activity and generating novel indole-based compounds. We propose a new AI system capable of suggesting new chemical structures based on some known cytoprotective activity. Cytoprotective activity prediction models, employing algorithms such as random forest, decision tree, support vector machines, K-nearest neighbors, and multiple linear regression, were built, and the best (based on quality measurements) was used to make predictions. Finally, the experimental evaluation of the computational results was undertaken in vitro. The proposed methodology resulted in the creation of a library of new indole-based compounds with assigned cytoprotective activity. The other outcome of this study was the development of a validated predictive model capable of estimating cytoprotective activity to a certain extent using molecular structure as input, supported by experimental confirmation.

Synthesis, Structure and Biological Activity of Indole-Imidazole Complexes with ZnCl2: Can Coordination Enhance the Functionality of Bioactive Ligands?

The ability of the indole-imidazole hybrid ligands to coordinate with the Zn(II) ion and the resulting structures of this new class of coordination compounds were analyzed in order to determine their structural properties and biological functionalities. For this purpose, six novel Zn(II) complexes, [Zn(InIm)2Cl2] (1), [Zn(InMeIm)2Cl2] (2), [Zn(IniPrIm)2Cl2] (3), [Zn(InEtMeIm)2Cl2] (4), [Zn(InPhIm)2Cl2] (5) and [Zn2(InBzIm)2Cl2] (6) (where InIm is 3-((1H-imidazol-1-yl)methyl)-1H-indole), were synthesized by the reactions of ZnCl2 and the corresponding ligand in a 1:2 molar ratio in methanol solvent at an ambient temperature. The structural and spectral characterization of these complexes was performed using NMR, FT-IR and ESI-MS spectrometry and elemental analysis, and the crystal structures of 1-5 were determined using single-crystal X-ray diffraction. Complexes 1-5 form polar supramolecular aggregates by utilizing, for this purpose, the N-H(indole)∙∙∙Cl(chloride) intermolecular hydrogen bonds. The assemblies thus formed differ depending on the distinctive molecular shape, which can be either compact or extended. All complexes were screened for their hemolytic, cytoprotective, antifungal, and antibacterial activities. The results show that the cytoprotective activity of the indole/imidazole ligand significantly increases upon its complexation with ZnCl2 up to a value comparable with the standard antioxidant Trolox, while the response of its substituted analogues is diverse and less pronounced.

New C8-substituted caffeine derivatives as promising antioxidants and cytoprotective agents in human erythrocytes.

New structurally diverse groups of C8-substituted caffeine derivatives were synthesized and evaluated for their chemical and biological properties. Mass spectrometry, FT-IR, and NMR characterizations of these derivatives were performed. The cytotoxic activity of the derivatives was estimated in vitro using human red blood cells (RBC) and in silico pharmacokinetic studies. The antioxidant capacity of the compounds was analyzed using a ferrous ion chelating activity assay. The ability of the derivatives to protect RBC from oxidative damage, including the oxidation of hemoglobin to methemoglobin, was assessed using a water-soluble 2,2'-azobis(2-methyl-propionamidine) dihydrochloride (AAPH) as a standard inducer of peroxyl radicals. The level of intracellular oxidative stress was assessed using the fluorescent redox probe 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). The results indicate that all derivatives are biocompatible compounds with significant antioxidant and cytoprotective potential dependent on their chemical structure. In order to explain the antioxidant and cytoprotective activity of the derivatives, a mechanism of hydrogen atom transfer (HAT), radical adduct formation (RAF), or single electron transfer (SET), as well as the specific interactions of the derivatives with the lipid bilayer of RBC membrane, have been proposed. The results show that selected modifications of the caffeine molecule enhance its antioxidant properties, which expands our knowledge of the structure-activity relationship of caffeine-based cytoprotective compounds.

Indole Derivatives Bearing Imidazole, Benzothiazole-2-Thione or Benzoxazole-2-Thione Moieties-Synthesis, Structure and Evaluation of Their Cytoprotective, Antioxidant, Antibacterial and Fungicidal Activities.

In the search for new bioactive compounds, a methodology based on combining two molecules with biological properties into a new hybrid molecule was used to design and synthesize of a series of ten indole derivatives bearing imidazole, benzothiazole-2-thione, or benzoxazole-2-thione moieties at the C-3 position. The compounds were spectroscopically characterized and tested for their antioxidant, antibacterial, and fungicidal activities. The crystal structures were determined for five of them. Comparison of the closely related structures containing either benzothiazole-2-thione or benzoxazole-2-thione clearly shows that the replacement of -S- and -O- ring atoms modify molecular conformation in the crystal, changes intermolecular interactions, and has a severe impact on biological activity. The results indicate that indole-imidazole derivatives with alkyl substituent exhibit an excellent cytoprotective effect against AAPH-induced oxidative hemolysis and act as effective ferrous ion chelating agents. The indole-imidazole compound with chlorine atoms inhibited the growth of fungal strains: Coriolus versicolor (Cv), Poria placenta (Pp), Coniophora puteana (Cp), and Gloeophyllum trabeum (Gt). The indole-imidazole derivatives showed the highest antibacterial activity, for which the largest growth-inhibition zones were noted in M. luteus and P. fluorescens cultures. The obtained results may be helpful in the development of selective indole derivatives as effective antioxidants and/or antimicrobial agents.

Synthesis, antioxidant and cytoprotective activity evaluation of C-3 substituted indole derivatives.

A series of fifteen indole derivatives substituted at the C-3 position were synthesized and characterized. The antioxidant activity of all derivatives was investigated by three in vitro antioxidant assays, and the derivative with pyrrolidinedithiocarbamate moiety was the most active as a radical scavenger and Fe3+-Fe2+ reducer. It can be stated that possible hydrogen and electron transfer mechanism is suggested for the quenching of the free radical. Moreover, the indolyl radical stabilization and the presence of unsubstituted indole nitrogen atom are mandatory for the observed antioxidant activity, which strongly depends on the type of the substituent directly connected to the methylene group at the C-3 position. Human red blood cells (RBC) have been used as a cell model to study derivatives interaction with the cell membrane. Haemolytic activity and RBC shape transformation were observed for certain derivatives in a concentration-dependent manner. However, most of the derivatives at sublytic concentration showed high cytoprotective activity against oxidative haemolysis induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). The cytoprotective properties of derivatives can be explained mostly due to their interactions with the RBC membrane components. Taking together, theoretical estimations and experimental data confirm the beneficial interactions between the selected C-3 substituted indole derivatives and the RBC membrane under oxidative stress conditions. These results encourage us to further structural optimization of C-3 substituted indole derivatives as potent antioxidant compounds.

Chemical Fingerprinting of Cryptic Species and Genetic Lineages of Aneura pinguis (L.) Dumort. (Marchantiophyta, Metzgeriidae).

Aneura pinguis (L.) Dumort. is a representative of the simple thalloid liverworts, one of the three main types of liverwort gametophytes. According to classical taxonomy, A. pinguis represents one morphologically variable species; however, genetic data reveal that this species is a complex consisting of 10 cryptic species (named by letters from A to J), of which four are further subdivided into two or three evolutionary lineages. The objective of this work was to develop an efficient method for the characterisation of plant material using marker compounds. The volatile chemical constituents of cryptic species within the liverwort A. pinguis were analysed by GC-MS. The compounds were isolated from plant material using the HS-SPME technique. Of the 66 compounds examined, 40 were identified. Of these 40 compounds, nine were selected for use as marker compounds of individual cryptic species of A. pinguis. A guide was then developed that clarified how these markers could be used for the rapid identification of the genetic lineages of A. pinguis. Multivariate statistical analyses (principal component and cluster analysis) revealed that the chemical compounds in A. pinguis made it possible to distinguish individual cryptic species (including genetic lineages), with the exception of cryptic species G and H. The classification of samples based on the volatile compounds by cluster analysis reflected phylogenetic relationships between cryptic species and genetic lineages of A. pinguis revealed based on molecular data.

Straightforward and rapid determination of acrylamide in coffee beans by means of HS-SPME/GC-MS.

A straightforward and rapid preparation procedure for the extraction of the acrylamide from coffee beans, by means of the volatile silylated derivatives of acrylamide (AA) and headspace solid phase microextraction (HS-SPME) is described. Commercially available SPME fibre coated with polydimethylsiloxane (PDMS) was used. The silylation reactions of the AA were executed with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The deuterium labelled d3-acrylamide was used as an internal standard. The acrylamide level was quantified using gas chromatography coupled with the mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. The limit of quantification (LOQ) for measuring acrylamide was 3 µg/kg. The described method demonstrates satisfactory repeatability (RSD = 2.6%), with an intermediate precision (RSD = 9.4%) and recovery (99-105%).

7-Deacetyl-10-alkylthiocolchicine derivatives - new compounds with potent anticancer and fungicidal activity.

A series of new semi-synthetic 7-deacetyl-10-alkylthiocolchicne derivatives with ethyl, n-propyl, i-propyl and n-butyl substituents were synthesised and characterised by spectroscopic methods, elemental analysis, DFT calculations and molecular docking simulations. All the synthesized compounds have been tested for fungicidal and anticancer activities against SKOV-3, LoVo, MCF-7, MDA-MB-231 and the lung-derived fibroblast CCD39Lu. All the new colchicine derivatives exhibit significantly higher cytotoxicity towards the SKOV-3 tumour cell line than the natural product - colchicine. The most effective cytotoxic agents were 7-deacetyl-10-n-buthylthiocolchicine and 7-deacetyl-10-i-propylthiocolchicine. Among all the compounds tested, 7-deacetyl-10-n-buthylthiocolchicine exhibited the highest fungicidal activity. Molecular modeling indicated that several mutations found in the ß-tubulin unit of the tested fungal strains are crucial for antifungal activity and selectivity of 7-deacetyl-10-n-buthylthiocolchicine. The obtained results may be useful for the development of selective colchicine derivatives as effective fungicidal and/or anticancer drugs.

High correlation of chemical composition with genotype in cryptic species of the liverwort Aneura pinguis.

Chemical constituents of cryptic species detected within the liverwort Aneura pinguis were identified using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). Fibre coating with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) was used. A total of 48 samples of A. pinguis were analysed. The studied plants were identified genetically based on barcode DNA sequences and represented three cryptic species (A, B and F) of A. pinguis. Cryptic species A and B are genetically diverse; both represent three evolutionary lineages: A1, A2, A3 and B1, B2, B3, respectively. The cryptic species F that was recently detected is not diverse. The most characteristic compounds in analysed samples were sesquiterpene hydrocarbons (up to 17.7% for A1; 15.7% for A2; 20.6% for A3; 7.7% for B1; 2.0% for B2; 3.7% for B3; 10.2% for F), oxygenated sesquiterpenoids (up to 68.0% for A1; 54.7% for A2; 52.6% for A3; 63.5% for B1; 88.7% for B2; 82.7% for B3; 78.8% for F), and linear aliphatic hydrocarbons (up to 14.8% for A1; 1.1% for A2; 12.1% for A3; 6.9 for B1; 5.2% for B2; 1.1% for B3; 7.0% for F). The dominant compound in the studied samples was pinguisone. The second dominant compound present in the tested plant material was deoxopinguisone, except for lineage B2, where only a small relative concentration of this compound was found. A high content of deoxopinguisone in cryptic species A (lineages A1, A2 and A3) was accompanied by the presence of isopinguisone and methyl norpinguisonate, whereas these two compounds were not detected in cryptic species B (lineages B1 and B3) and F. The chemical compounds detected in the studied samples of A. pinguis were subjected to multivariate statistical analysis. The results showed that the chemical composition depends mainly on the genotype of the plant and slightly on the habitat. However, there was no clear correlation between the volatile compounds and the date of collection of the studied plants.

Antioxidant and cytotoxic activity of new di- and polyamine caffeine analogues.

A series of new di- and polyamine-caffeine analogues were synthesised and characterised by NMR, FT-IR, and MS spectroscopic methods. To access the stability of the investigated caffeine analogues, molecular dynamic simulations were performed in NAMD 2.9 assuming CHARMM36 force field. To evaluate the antioxidant capacity of new compounds, three different antioxidant assays were used, namely 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH•) scavenging activity, ferrous ions (Fe2+) chelating activity, and Fe3+→Fe2+reducing ability. In vitro, the ability of new derivatives to protect human erythrocytes against oxidative haemolysis induced by free radical from 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) was estimated. The cytotoxic activity was tested using MCF-7 breast cancer cells and human erythrocytes. All compounds showed the antioxidant capacity depending mostly on their ferrous ions chelating activity. In the presence of AAPH, some derivatives were able to effectively inhibit the oxidative haemolysis. Two derivatives, namely 8-(methyl(2-(methylamino)ethyl)-amino)caffeine and 8-(methyl(3-(methylamino)propyl)amino)caffeine, showed cytotoxic activity against MCF-7 breast cancer cells but not against human erythrocytes. Therefore, it is concluded that the selected di- and polyamine caffeine analogues, depending on their chemical structure, were able to minimise the oxidative stress and to inhibit the tumour cell growth. The confirmed antioxidant and cytotoxic properties of some caffeine derivatives make them attractive for potential applications in food or pharmaceutical industries.

Mass Spectrometry Study of New Polyamine Derivatives of Caffeine.

The mass spectrometric fragmentations of ten new 8-alkylaminocaffeine derivatives were investigated. The fragmentation pathways of new polyamine derivatives of caffeine on the basis of low and high-resolution electron ionization (EI) mass spectra were discussed. In the case of new compounds, classical fragmentation of purine skeleton related to the elimination of a neutral molecule CH3N(1)C(2)O from a molecular ion was not observed. Nevertheless, new, interesting fragmentation of described caffeine derivatives was observed. Moreover, heats of formation of odd-electron ions of 8-alkylaminocaffeine derivatives were calculated.

Non-covalent interactions between thio-caffeine derivatives and water-soluble porphyrin in ethanol-water environment.

To determine the binding interactions and ability to form the non-covalent systems, the association process between 5,10,15,20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine tetra-p-tosylate (H2TTMePP) and a series of five structurally diverse thio-caffeine analogues has been studied in ethanol and ethanol-water solutions, analyzing its absorption and steady-state fluorescence spectra. The porphyrin fluorescence lifetimes in the systems studied were established as well. During the titration with thio-caffeine compounds the slight bathochromic effect and considerable hypochromicity of the porphyrin Soret band maximum can be noted. The fluorescence quenching effect observed for interactions in H2TTMePP - thio-caffeine derivative systems, as well as the order of binding and fluorescence quenching constants (of 105-103mol-1) suggest the existence of the mechanism of static quenching due to the formation of non-covalent and non-fluorescent stacking complexes. In all the systems studied the phenomenon of the fractional accessibility of the fluorophore for the quencher was observed as well. Additionally, the specific binding interactions, due to the changes in reaction environment polarity, can be observed. It was found that thio-caffeine compounds can quench the porphyrin fluorescence according to the structure of thio-substituent in caffeine molecule. The obtained results can be potentially useful from scientific, therapeutic or environmental points of view.

Haemolytic activity of formyl- and acetyl-derivatives of bile acids and their gramine salts.

Bile acids (lithocholic: LCA, deoxycholic: DCA and cholic: CA) and their formyl- and acetyl-derivatives can be used as starting material in chemical synthesis of compounds with different biological activity strongly depended on their chemical structures. Our previous studies showed that biological activity of bile acids salts with gramine toward human erythrocytes was significantly different from the activity of bile acids alone. Moreover, gramine effectively modified the membrane perturbing activity of other steroids. As a continuation of our work, the haemolytic activity of formyl- and acetyl-substituet bile acids as well as their gramine salts was studied in vitro. The structures of new compounds were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. The results shown that the haemolytic activity of formyl- and acetyl-LCA and DCA was significantly higher in comparison with their native forms at the whole concentration range. At high concentration, formyl derivative of CA was as effective as LCA and DCA derivatives whereas at lower concentration its haemolytic activity was at the level of original acid. The acetyl-CA was not active as membrane perturbing agents. Furthermore, gramine significantly decreased the membrane-perturbing activity of hydrophobic bile acids derivatives. The results obtained with the cellular system are in line with physicochemical calculation.

Antioxidant properties of thio-caffeine derivatives: Identification of the newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine as antioxidant and highly potent cytoprotective agent.

A series of nine thio-caffeine analogues were synthesized and characterised by NMR, FT-IR and MS spectroscopic methods. Molecular structures of four of them were determined using single crystal X-ray diffraction methods. The antioxidant properties of all compounds, at concentration ranges from 0.025 to 0.1mg/mL, were evaluated by various chemical- and cell-based antioxidant assays. Human erythrocytes were used to examine in vitro haemolytic activity of all compounds and their protective effect against oxidative haemolysis induced by AAPH, one of the commonly used free radical generator. All compounds studied showed no effect on the human erythrocytes membrane structure and permeability with the exception of 8-(phenylsulfanyl)caffeine. Among the nine caffeine thio-analogues tested, the newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine possessed exceptionally high antioxidant properties. Moreover, it protects human erythrocytes against AAPH-induced oxidative damage as efficiently as the standard antioxidant Trolox. Therefore, 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine may have a significant cytoprotective potential caused by its antioxidant activity.

Nicotine alkaloids as antioxidant and potential protective agents against in vitro oxidative haemolysis.

The capacity of eleven nicotine alkaloids to reduce oxidative stress was investigated. In order to provide a structure-activity relationships analysis, new nicotine derivatives with a substituent introduced into the pyrrolidine ring were synthesized and investigated together with nicotine and its known analogs. All newly synthesized compounds were characterized by (1)H, (13)C NMR and EI-MS technique. The antioxidant properties of nicotine, its known analogs and newly produced derivatives, were evaluated by various antioxidant assays such 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH(•)) scavenging, ferrous ions (Fe(2+)) chelating activity and total reducing ability determination by Fe(3+) â†’ Fe(2+) transformation assay. The protective effects of all compounds tested against 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and tert-butyl hydroperoxide (t-BuOOH)-induced oxidative haemolysis and morphological injury of human erythrocytes, were estimated in vitro. The results showed that nicotine alkaloids exhibited various antiradical efficacy and antioxidant activity in a structure- and a dose-dependent manner. In addition, the capacity of nicotine alkaloids to protect erythrocytes from AAPH- and t-BuOOH-induced oxidative haemolysis, was dependent on its incubation time with cells. Our findings showed that chemical and biological investigations conducted simultaneously can provide comprehensive knowledge concerning the antioxidant potential of nicotine alkaloids. This knowledge can be helpful in better understanding the properties of nicotine alkaloids under oxidative stress conditions.

Synthesis, spectroscopy, theoretical and biological studies of new gramine-steroids salts and conjugates.

New gramine connections with bile acids (lithocholic, deoxycholic, cholic) and sterols (cholesterol, cholestanol) were synthesized. The structures of products were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. Unexpectedly, the products of the reaction of gramine with cholesterol and cholestanol were symmetrical compounds consisting of two molecules of sterols connected by N(CH3)2 group. All new synthesized compounds interact in vitro with the human erythrocyte membrane and alter discoid erythrocyte shape inducing stomatocytosis or echinocytosis. Increase in the incorporation of the fluorescent dye merocyanine 540 (MC540) into the erythrocyte membrane indicates that new compounds at sublytic concentrations are capable of disturbing membrane phospholipids asymmetry and loosening the molecular packing of phospholipids in the bilayer. Gramine significantly decreases the membrane partitioning properties as well as haemolytic activity of lithocholic acid in its new salt. Moreover, both deoxycholic and cholic acids completely lost their membrane perturbing activities in the gramine salts. On the other hand, the capacity of new gramine-sterols connections to alter the erythrocyte membrane structure and its permeability is much higher in comparison with sterols alone. The dual effect of gramine on the bile acid and sterols cell membrane partitioning activity observed in our study should not be neglected in vivo.