(3+2)-Cycloadditions of Levoglucosenone (LGO) with Fluorinated Nitrile Imines Derived from Trifluoroacetonitrile: An Experimental and Computational Study.

The in situ-generated N-aryl nitrile imines derived from trifluoroacetonitrile smoothly undergo (3+2)-cycloadditions onto the enone fragment of the levoglucosenone molecule, yielding the corresponding, five-membered cycloadducts. In contrast to the 'classic' C(Ph),N(Ph) nitrile imine, reactions with fluorinated C(CF3),N(Ar) analogues lead to stable pyrazolines in a chemo- and stereoselective manner. Based on the result of X-ray single crystal diffraction analysis, their structures were established as exo-cycloadducts with the location of the N-Ar terminus of the 1,3-dipole at the α-position of the enone moiety. The DFT computation demonstrated that the observed reaction pathway results from the strong dominance of kinetic control over thermodynamic control.

(1-4)-Thiodisaccharides as anticancer agents. Part 5. Evaluation of anticancer activity and investigation of mechanism of action.

(1-4)-Thiodisaccharides, thiosugars with the 1-4-thio bridge, were recently shown to induce oxidative stress, as well as, apoptosis in cancer cells in the low micromolar range; however, the detailed mechanism of their anticancer action still remains unknown. In order to clarify the mechanism of (1-4)- thiodisaccharides action, we performed a series of tests including cytotoxic, clonogenic and apoptosis assays using an in vitro glioma cancer model with one ATCC cell line U87 and two novel glioma cell lines derived from cancer patients - H6PX and H7PX. We also evaluated the ability of (1-4)-thiodisaccharides to interfere with protein folding and synthesis processes, as well as, the thioredoxin system. (1-4)-thiodisaccharides induced glioma cell death, which were found to be accompanied with endoplasmic reticulum stress, inhibition of global protein synthesis, reduced overall cellular thiol level and thioredoxin reductase activity. We also performed a RT-PCR and Elisa analysis of (1-4)-thiodisaccharides-treated glioma cells to identify any changes within the pathway affected by (1-4)-thiodisaccharides. We observed a significant increase of expression in key markers of endoplasmic reticulum stress and pro-apoptotic protein, FASLG. We proposed that (1-4)-thiodisaccharides react with cellular thiols and disturb any cellular thiol-depended processes like thioredoxin system or protein folding.

Thio-functionalized carbohydrate thiosemicarbazones and evaluation of their anticancer activity.

Thiosemicarbazides and their analogs have shown potential medical applications as antiviral, antibacterial and anticancer drugs. We designed, synthesized and evaluated in vitro anticancer activity against ovarian (A2780), cervix (HeLa), colon (LoVo), breast (MCF-7) and brain (MO59J) human cancer cell lines of seven novel compounds -S-glycosylated thiosemicarbazones. We assessed the cyto- and genotoxic properties of all novel compounds using a variety of methods including comet assay, XTT assay, various fluorescent assays and toxicology PathwayFinder expression array. We tried to evaluate their possible mechanism of action with particular attention to induction of DNA damage and repair, apoptosis, oxidative stress analysis and cellular response in terms of changes in gene expression. The most sensitive cell line was human ovarian cancer. The results revealed that the major activity against A2780 cancer cell line displayed by our compounds is induction of DNA damage. This effect is not associated with apoptosis or oxidative stress induction and the resulting damage will not lead to cell cycle arrest. We also observed up-expression of heat shock related genes and NQO1 gene in response to our compounds. The second effect seems to be specific to glycosylated S-bond compounds as we observed it earlier. Upregulation of heat shock protein encoding genes suggest that our compounds induce stressful conditions. The nature of this phenomena (heat shock, pH shift or hypoxia) needs further study.

The oxidative induction of DNA lesions in cancer cells by 5-thio-d-glucose and 6-thio-d-fructopyranose and their genotoxic effects. Part 3.

Thio-sugars have been described as potent inhibitors of cancer cell growth but the detailed mechanism of action remains unknown. Herein we investigated the mechanism of their anticancer action in the HeLa cell line. We investigated two thio-sugars: 5-thio-d-glucose (FCP1) and 6-thio-ß-d-fructopyranose (FCP2). We have observed that FCP1 as well as FCP2 clearly induced oxidative DNA lesions in cancer cells and increased the level of cellular ROS. A spin trap and antioxidants have decreased the level of DNA lesions induced by FCPs. FCPs also induced significant changes in the oxidative-stress gene expression. Therefore, we assume that ROS generation is correlated with the increased NOX5 expression by FCPs. Higher cyto- and genotoxicity of FCPs for HeLa cells in a low glucose environment suggested their role in the glucose metabolism. The data indicates that thio-sugars may become drug alternatives for the cancer treatment but such undertaking needs further studies.

The induction of oxidative stress in cervix carcinoma cells by levoglucosenone derived 4-S-salicyl derivative and (1-4)-S-thio-disaccharides. Part 4.

(1-4)-S-thiodisaccharides were shown to kill various cancer cell lines, including cervix, lung, mammary-gland and colon by unknown mechanisms. Here we identified two actions of levoglucosenone derived (1-4)-S-thiodisaccharides against cervix cancer cells: induction of oxidative stress and DNA damage. In consequence, (1-4)-S-thiodisaccharides lowered the cellular GSH level and changed the expression profile of genes encoding key proteins involved with oxidative stress response. We also observed that (1-4)-S-thiodisaccharides induced DNA damage and interfered with the thioredoxin (Trx) system. Both actions, as induced by FPC6, were stronger when dihedral angles of sulfur bridge were set to 110°, 100° and 109°, clearly indicating differences when compared to FPC8. These findings demonstrate that the 1-4-thio bridge of disaccharide is a powerful anticancer pharmacophore, and its potential use needs further studies.

Evaluation of the Mycobactericidal Effect of Thio-functionalized Carbohydrate Derivatives.

Sugars with heteroatoms other than oxygen have attained considerable importance in glycobiology and in drug design since they are often more stable in blood plasma due to their resistance to enzymes, such as glycosidases, phosphorylases and glycosyltransferases. The replacement of oxygen atoms in sugars with sulfur forms thio-sugars, which are potentially useful for the treatment of diabetes and some bacterial and viral infections. Here, we evaluated the antibacterial activity of thio-functionalized carbohydrate derivatives. A set of 21 compounds was screened against acid-fast Mycobacterium tuberculosis (Mtb), gram-negative Escherichia coli and gram-positive Staphylococcus aureus. The tested carbohydrate derivatives were most effective against tubercle bacilli, with as many as five compounds (thioglycoside 6, thiosemicarbazone 16A, thiosemicarbazone 20, aminothiadiazole 23, and thiazoline 26) inhibiting its growth with MIC50 ≤ 50 µM/CFU. Only two compounds (aminothiadiazole 23 and thiazoline 26) were able to inhibit the growth of E. coli at concentrations below 1 mM, and one of them, aminothiadiazole 23, inhibited the growth of S. aureus at a concentration ≤1 mM. The five compounds affecting the growth of mycobacteria were either thiodisaccharides (6, 16A, and 20) or thioglycosides (23 and 26). All of these compounds (6, 16A, 20, 23, and 26) were able to inhibit the growth of Mtb deposited within human macrophages. However, three of the five selected compounds (6, 23, and 26) exhibited relatively high cytotoxicity in mouse fibroblasts at micromolar concentrations. The selected thio-sugars are very promising compounds, thus making them candidates for further modifications that would decrease their cytotoxicity against eukaryotic cells without affecting their antimycobacterial potential.

[Thiosugars used as drugs]. / Tiocukry jako leki.

Thiosugars are carbohydrate analogs in which one or few of the oxygen atoms were replaced by sulfur. The sulfur atom which is present in the furan and pyran structures, changes biological properties of carbohydrates, as compared to their oxygen analogs. Among others, thiosugars are effective inhibitors of various cellular and enzymatic pathways and also have great therapeutic potential. They are used as a drugs in diabetes and infectious diseases treatment. Recent evidence suggests that these compounds may have therapeutic properties and be also used in the treatment of some pathological conditions, including cancer diseases. This research are aimed towards the development and improvement of the current methods of synthesis of new thiosugars through stabilization of sulfur bonds and in vitro and in vivo analysis of their potential therapeutic properties. In this work the summary of the latest reports about thiosugars and their application in the medicine is presented for the first time in the Polish language literature.

Thio-sugar motif of functional CARB-pharmacophore for antineoplastic activity. Part 2.

Diverse functionalized representatives of (1-4)-S-thiodisaccharides, 6-9 were synthesized and assessed for cytotoxicity and apoptosis against human cancer cell lines (A549, LoVo, MCF-7 and HeLa). The FCP 6 was more active against MCF-7 cells (i.e., an estrogen-dependent breast cancer line), whereas other (1-4)-S-thiodisaccharides showed strongest activity against A549 cells (i.e., a lung adenocarcinoma line). We propose to use a concept of functional 'CARB-pharmacophores' when evaluating a potential for the compounds' general antineoplastic activity. Future studies will determine the reasons for cell-type specificity of these compounds. The thio-sugar motif appears to be a promising lead for future developments.

A potential CARB-pharmacophore for antineoplastic activity: part 1.

Diverse functionalized representatives of various classes of sugars, such as thio-, anhydro-, and sulfamido-sugars and myo-inositol oxide, were synthesized and assessed for cytotoxicity against human cancer cell lines (A549, LoVo, MCF-7 and HeLa). The inositol oxide (4) was more active against MCF-7 cells (i.e., an estrogen-dependent breast cancer line), whereas all 3 sulfur-containing compounds showed strongest activity against A549 cells (i.e., a lung adenocarcinoma line). We propose to use a concept of functional 'CARB-pharmacophores' when evaluating a potential for the compounds' general antineoplastic activity. Future studies will determine the reasons for cell-type specificity of these compounds. The thio-sugar motif appears to be a promising lead for future developments.

New N-Adducts of Thiadiazole and Thiazoline with Levoglucosenone and Evaluation of Their Significant Cytotoxic (Anti-Cancer) Activity.

The conjugate N-adducts of thio-1,3,4-diazole and 2-thiazoline with levoglucosenone were synthesized via a stereoselective, base-catalyzed conjugate N-Michael addition to levoglucosenone at C-4. Structural assignments were established using 1H and 13C NMR analysis, and X-ray single-crystal analysis for one of the compounds. The biological properties of the novel compounds were tested on a cell model. Cytotoxicity was analyzed via colorimetric assay. Two distinct types of cell death, apoptosis and necrosis, were analyzed by determining the phosphatidylserine levels from the outer leaflet of the plasma membrane, caspase activation, and lactate dehydrogenase release. We also evaluated DNA damage using an alkaline comet assay. The level of oxidative stress was measured with a modified comet assay and an H2DCFDA probe. The thio-1,3,4-diazole adduct (FCP23) and the 2-thiazoline adduct (FCP26) exhibit similar cytotoxicity values for cancer cells (ovarian (A2780), breast (MCF-7), cervix (HeLa), colon (LoVo), and brain (MO59J and MO59K)), but their mechanism of action is drastically different. While FCP23 induces oxidative stress, DNA damage, and necrosis, FCP26 induces apoptosis through caspase activation.

Carbohydrate-Based Micro/Nanocapsules With Controlled External Surface for Medical Applications.

Micro/nanocapsules would have many more applications if we were able to controllably populate their surface with various chemical moieties. The present review introduces a novel variant of interfacial polymerization (IP) as a very robust method of manufacturing reservoir micro/nanocapsules equipped with several different functionalities on the capsules' surface. We call the method-IPCESCO (Interfacial Polymerization for Capsules' External Surface Control). As always in IP, the capsules' forming reaction is between monomers dissolved in opposite phases (oil or water) and takes place at the interface. Each monomer carries two or more functionalities reacting with functional groups of the monomer dissolved in the other phase. IPCESCO requires that one or both monomers are additionally equipped with (protected) functional groups interfering neither with the payload nor with the polymer formation. These additional groups end up everywhere in the polymeric shell but most importantly they are present on the external surface of capsules. These "handles" allow for the introduction of various moieties onto the capsules' surface. Since carbohydrate chemists developed a plurality of protecting and deprotecting methods for various functional groups such as aldehyde and hydroxyl, modified mono, and oligosaccharides are particularly well-suited to act as monomers in IPCESCO. The article discusses possible monomers and their synthesis, the transformation of protected reactive groups on the external capsules' surface into the desired functionalities, the control of the number of moieties on the surface and the capsules surface's architecture. The most important application of the novel encapsulation technology is in drug delivery. Possible surface units facilitating capsules' transport in the body, delivery to specific locations and mechanisms of capsules rupture are also addressed. Other applications of novel capsules include an ultra-sensitive quantitation and removal of pathogens, transport of nutrients in plants, detection of various antigens and other parameters in single cells.

DNA Double Strand Breaks Repair Inhibitors: Relevance as Potential New Anticancer Therapeutics.

DNA double-strand breaks are considered one of the most lethal forms of DNA damage. Many effective anticancer therapeutic approaches used chemical and physical methods to generate DNA double-strand breaks in the cancer cells. They include: IR and drugs which mimetic its action, topoisomerase poisons, some alkylating agents or drugs which affected DNA replication process. On the other hand, cancer cells are mostly characterized by highly effective systems of DNA damage repair. There are two main DNA repair pathways used to fix double-strand breaks: NHEJ and HRR. Their activity leads to a decreased effect of chemotherapy. Targeting directly or indirectly the DNA double-strand breaks response by inhibitors seems to be an exciting option for anticancer therapy and is a part of novel trends that arise after the clinical success of PARP inhibitors. These trends will provide great opportunities for the development of DNA repair inhibitors as new potential anticancer drugs. The main objective of this article is to address these new promising advances.

A click chemistry approach to glycomimetics: Michael addition of 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose to 4-deoxy-1,2-O-isopropylidene-L-glycero-pent-4-enopyranos-3-ulose--a convenient route to novel 4-deoxy-(1-->5)-5-C-thiodisaccharides.

The base catalyzed conjugate Michael addition of the 1-thiosugar, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose, 1, to a new highly reactive enone 4-deoxy-1,2-O-isopropylidene-L-glycero-pent-4-enopyranos-3-ulose, 2, proceeds steroselectively with formation of adduct 3 in 94% yield. Convenient stereoselective reduction of the C-3 keto function of 3 with L-Selectride followed by in situ acetylation produces thiodisaccharide 4 in good 82% yield. Cleavage of the 1,2-O-isopropylidene protecting group with p-toluenesulfonic acid in methanol, followed by de-O-acetylation, produced an inseparable anomeric mixture of methyl 4-deoxy-5-C-(beta-D-glucopyranosyl)-thio-alpha/beta-L-ribo-pyranoside 5 in 72% overall yield. This approach constitutes a new general two-step click chemistry route to the previously unknown class of 4-deoxy-(1-->5)-5-C-thiodisaccharides as stable and biologically important glycomimetics.

A novel carbohydrate derived compound FCP5 causes DNA strand breaks and oxidative modifications of DNA bases in cancer cells.

1,5-Anhydro-6-deoxy-methane-sulfamido-D-glucitol (FCP5) is a functionalized carbohydrate containing functional groups that render it potentially therapeutically useful. According to our concept of 'functional carb-pharmacophores' (FCPs) incorporation of the methanesulfonamido pharmacophore to 1,5 glucitol could create a therapeutically useful compound. Our previous studies revealed that FCP5 was cytotoxic to cancer cells. Therefore, in this work we assessed the cytotoxic mechanisms of FCP5 in four cancer cell lines - HeLa, LoVo, A549 and MCF-7, with particular focus on DNA damage and repair. A broad spectrum of methods, including comet assay with modifications, DNA repair enzyme assay, plasmid relaxation assay, and DNA fragmentation assay, were used. We also checked the potential for FCP5 to induce apoptosis. The results show that FCP5 can induce DNA strand breaks as well as oxidative modifications of DNA bases. DNA lesions induced by FCP5 were not entirely repaired in HeLa cells and DNA repair kinetics differs from other cell lines. Results from molecular docking and plasmid relaxation assay suggest that FCP5 binds to the major groove of DNA with a preference for adenosine-thymine base pair sequences and directly induces DNA strand breaks. Thus, FCP5 may represent a novel lead for the design of new major groove-binding compounds. The results also confirmed the validity of functional carb-pharmacophores as a new source of innovative drugs.

Functionalized S-thio-di- and S-oligosaccharide precursors as templates for novel SLex/a mimetic antimetastatic agents.

Adhesive interactions between molecules expressed on vascular endothelium and circulating tumor cells are key early events in cancer metastasis. Best characterized to date is the selectin family of cell adhesion molecules, which can bind to and stabilize blood-borne cells on organ vasculature, facilitating the cell-cell and cell-substratum interactions leading to tumor seeding and proliferation. Major ligands of E-selectin, the selectin family member expressed on vascular endothelial cells, include sialylated, fucosylated glycans such as Sialyl Lewis type carbohydrate complexes (SLe(x) and SLe(a)). These carbohydrate antigens are ubiquitously expressed on tumor cells with high metastatic potential, including colon and pancreatic carcinomas, and have been found to selectively and avidly bind E-selectin. Compounds that prevent E-selectin-SLe(x/a)binding represent an attractive tool in the prevention of cancer dissemination. Review of preclinical in vitro and in vivo studies suggest that SLe(x/a) 'mimetics' may serve as a potent class of anti-metastatic compounds. These agents are designed to outcompete SLe(x/a) antigens expressed on tumor cell surfaces to prevent initial vascular adhesion. Critical in generating exogenous oligosaccharides as SLe(x/a) mimetics is the stereoselective joining of specific mono- and di- saccharides that express functional groups integral in E-selectin-SLe(x/a) binding. Employing sulfur linkages to couple saccharide units enhances the biological stability of these complex carbohydrates. The synthesis of novel S-thiodisaccharides and C-disaccharides as SLe(x/a) precursors using the chiral sugars levoglucosenone, isomeric isolevoglucosenone and their functionalized analogs is described. The highly stereoselective functionalization of both enones at the C-4, C-3 and C-2 positions by the set of Michael addition reactions of reactive 1-thiosugars is reviewed. These functionalized S-thio di- and S-oligosaccharide precursors have direct application for use as templates in the synthesis of novel SLe(x/a)mimetics.

Thio-sugars VII. Effect of 3-deoxy-4-S-(beta-D-gluco- and beta-D-galactopyranosyl)-4-thiodisaccharides and their sulfoxides and sulfones on the viability and growth of selected murine and human tumor cell lines.

The first conversion of (1-->4)-thiodisaccharides into corresponding sulfoxides and sulfones by conventional oxidation with m-chloroperoxybenzoic acid (MCPBA) is reported. The effects of alpha-(1-->4)-3'-deoxythiodisaccharides (8-9) and their sulfoxide (14-15) and sulfone (16-17) derivatives on murine leukemia and human colon and pancreatic carcinoma cell viability were studied. Concentrations of thio-sugars that decreased tumor cell line viability by 50% (IC(50)), measured via the MTT assay, ranged from 6.4 to 38.3 microg/mL. The effect of alpha-(1-->4)-3'-deoxythiodisaccharide derivatives were most profound on human pancreatic epithelial carcinoma (PANC-1) cells with compounds 8 and 9 having IC(50) values of 6.4 microg/mL and 8.2 microg/mL, respectively. Sulfone derivatives 16 and 17 also had pronounced effects on PANC-1 cell viability (IC(50)=10.2 microg/mL and 9.6 microg/mL, respectively). These results indicate that deoxythio-disaccharide analogs generated by functionalization of the universal chiral precursor levoglucosenone may have cytotoxic properties and therapeutic potential as anticancer agents.

Mycalamides, pederin and psymberin as natural carbohydrates and potential antitumor agents: past and future perspectives.

The mycalamide class of potent antiviral and antitumor natural compounds originally isolated from marine sponges in 1988 is a new interdisciplinary approach to molecular recognition. We review new synthetic approaches to this new family of natural products with remarkable biological activity. Some biological evaluation data are compiled and compared to other structurally similar molecular targets.

Thiosugars: new perspectives regarding availability and potential biochemical and medicinal applications.

Thiosugars, containing a sulfur atom as heteroatom or a disaccharide linked via a sulfur bridge, possess unique physicochemical properties such as water solubility, which differs from conventional functionalized monosaccharides. The differences in biological activities between thiosugars and their oxygen analogs depend on geometric, conformational, and flexibility differences. They depend also on their electronic differences, the sulfide function being less electronegative and more polarizable than the ethereal moiety. Many functionalized thiosugars occur naturally and are potential targets for the development of carbohydrate-based therapeutics. Among the few new examples of the potential new targets are salacinol and kotalanol, tagetitoxin, thiolactomycin and analogues, mycothiol and analogues, and S-nitrosothiols. These new developments and representative examples of functionalized thiosugar prototypes as potential new targets are presented in this mini review.