Glycoconjugation of Quinoline Derivatives Using the C-6 Position in Sugars as a Strategy for Improving the Selectivity and Cytotoxicity of Functionalized Compounds.

Based on the Warburg effect and the increased demand for glucose by tumor cells, a targeted drug delivery strategy was developed. A series of new glycoconjugates with increased ability to interact with GLUT transporters, responsible for the transport of sugars to cancer cells, were synthesized. Glycoconjugation was performed using the C-6 position in the sugar unit, as the least involved in the formation of hydrogen bonds with various aminoacids residues of the transporter. The carbohydrate moiety was connected with the 8-hydroxyquinoline scaffold via a 1,2,3-triazole linker. For the obtained compounds, several in vitro biological tests were performed using HCT-116 and MCF-7 cancer cells as well as NHDF-Neo healthy cells. The highest cytotoxicity of both cancer cell lines in the MTT test was noted for glycoconjugates in which the triazole-quinoline was attached through the triazole nitrogen atom to the d-glucose unit directly to the carbon at the C-6 position. These compounds were more selective than the analogous glycoconjugates formed by the C-1 anomeric position of d-glucose. Experiments with an EDG inhibitor have shown that GLUTs can be involved in the transport of glycoconjugates. The results of apoptosis and cell cycle analyses by flow cytometry confirmed that the new type of glycoconjugates shows pro-apoptotic properties, without significantly affecting changes in the distribution of the cell cycle. Moreover, glycoconjugates were able to decrease the clonogenic potential of cancer cells, inhibit the migration capacity of cells and intercalate with DNA.

Synthesis and Preliminary Anticancer Activity Assessment of N-Glycosides of 2-Amino-1,3,4-thiadiazoles.

The addition of 2-amino-1,3,4-thiadiazole derivatives with parallel iodination of differently protected glycals has been achieved using a double molar excess of molecular iodine under mild conditions. The corresponding thiadiazole derivatives of N-glycosides were obtained in good yields and anomeric selectivity. The usage of iodine as a catalyst makes this method easy, inexpensive, and successfully useable in reactions with sugars. Thiadiazole derivatives were tested in a panel of three tumor cell lines, MCF-7, HCT116, and HeLa. These compounds initiated biological response in investigated tumor models in a different rate. The MCF-7 is resistant to the tested compounds, and the cytometry assay indicated low increase in cell numbers in the sub- G1 phase. The most sensitive are HCT-116 and HeLa cells. The thiadiazole derivatives have a pro-apoptotic effect on HCT-116 cells. In the case of the HeLa cells, an increase in the number of cells in the sub-G1- phase and the induction of apoptosis was observed.

The Effect of a New Glucose-Methotrexate Conjugate on Acute Lymphoblastic Leukemia and Non-Hodgkin's Lymphoma Cell Lines.

Patients with hematologic malignancies require intensive therapies, including high-dose chemotherapy. Antimetabolite-methotrexate (MTX) has been used for many years in the treatment of leukemia and in lymphoma patients. However, the lack of MTX specificity causes a significant risk of morbidity, mortality, and severe side effects that impairs the quality of patients' life. Therefore, novel targeted therapies based on the malignant cells' common traits have become an essential treatment strategy. Glucose transporters have been found to be overexpressed in neoplastic cells, including hematologic malignancies. In this study, we biologically evaluated a novel glucose-methotrexate conjugate (Glu-MTX) in comparison to a free MTX. The research aimed to assess the effectiveness of Glu-MTX on chosen human lymphoma and leukemia cell lines. Cell cytotoxicity was verified by MTT viability test and flow cytometry. Moreover, the cell cycle and cellular uptake of Glu-MTX were evaluated. Our study reveals that conjugation of methotrexate with glucose significantly increases drug uptake and results in similar cytotoxicity of the synthesized compound. Although the finding has been confined to in vitro studies, our observations shed light on a potential therapeutic approach that increases the selectivity of chemotherapeutics and can improve leukemia and lymphoma patients' outcomes.

The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues.

Non-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

In Vitro and In Vivo Efficacy of a Novel Glucose-Methotrexate Conjugate in Targeted Cancer Treatment.

Methotrexate (MTX) is a commonly used antimetabolite, which inhibits folate and DNA synthesis to be effective in the treatment of various malignancies. However, MTX therapy is hindered by the lack of target tumor selectivity. We have designed, synthesized and evaluated a novel glucose-methotrexate conjugate (GLU-MTX) both in vitro and in vivo, in which a cleavable linkage allows intracellular MTX release after selective uptake through glucose transporter-1 (GLUT1). GLU-MTX inhibited the growth of colorectal (DLD-1), breast (MCF-7) and lung (A427) adenocarcinomas, squamous cell carcinoma (SCC-25), osteosarcoma (MG63) cell lines, but not in WI-38 healthy fibroblasts. In tumor cells, GLU-MTX uptake increased 17-fold compared to unconjugated MTX. 4,6-O-ethylidene-α-D-glucose (EDG), a GLUT1 inhibitor, significantly interfered with GLU-MTX induced growth inhibition, suggesting a glucose-mediated drug uptake. Glu-MTX also caused significant tumor growth delay in vivo in breast cancer-bearing mice. These results show that our GLUT-MTX conjugate can be selectively uptake by a range of tumor cells to cause their significant growth inhibition in vitro, which was also confirmed in a breast cancer model in vivo. GLUT1 inhibitor EDG interfered with these effects verifying the selective drug uptake. Accordingly, GLU-MTX offers a considerable tumor selectivity and may offer cancer growth inhibition at reduced toxicity.

Overcoming Hypoxia-Induced Chemoresistance in Cancer Using a Novel Glycoconjugate of Methotrexate.

The oxygen and nutrient-deprived tumor microenvironment is considered a key mechanism responsible for cancer resistance to chemotherapy. Methotrexate (MTX) is a widely incorporated chemotherapeutic agent employed in the treatment of several malignancies. However, drug resistance and systemic toxicity limit the curative effect in most cases. The present work aimed to design, synthesize, and biologically evaluate a novel glucose-methotrexate conjugate (Glu-MTX). Our study showed that Glu-MTX exerts an increased cytotoxic effect on cancer cells in comparison to MTX in hypoxia (1% O2) and glucose starvation conditions. Furthermore, Glu-MTX was found to inhibit the proliferation and migration of cancer cells more effectively than MTX does. Our results demonstrate that the conjugation of MTX to glucose led to an increase in potency against malignant cells under oxygen and nutrient stress. The observations shed light on a potential therapeutic approach to overcome chemoresistance in cancer.

Structure-bioavailability relationship study of genistein derivatives with antiproliferative activity on human cancer cell.

The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone. Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium. The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram'-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample. In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative's biological properties.

Glycoconjugation as a Promising Treatment Strategy for Psoriasis.

Despite the progress in the development of novel treatment modalities, a significant portion of patients with psoriasis remains undertreated relative to the severity of their disease. Recent evidence points to targeting the glucose transporter 1 and sugar metabolism as a novel therapeutic strategy for the treatment of psoriasis and other hyperproliferative skin diseases. In this review, we discuss glycoconjugation, an approach that facilitates the pharmacokinetics of cytotoxic molecules and ensures their preferential influx through glucose transporters. We propose pathways of glycoconjugate synthesis to increase effectiveness, cellular selectivity, and tolerability of widely used antipsoriatic drugs. The presented approach exploiting the heightened glucose requirement of proliferating keratinocytes bears the potential to revolutionize the management of psoriasis. SIGNIFICANCE STATEMENT: Recent findings concerning the fundamental role of enhanced glucose metabolism and glucose transporter 1 overexpression in the pathogenesis of psoriasis brought to light approaches that proved successful in cancer treatment. Substantial advances in the emerging field of glycoconjugation highlight the rationale for the development of glucose-conjugated antipsoriatic drugs to increase their effectiveness, cellular selectivity, and tolerability. The presented approach offers a novel therapeutic strategy for the treatment of psoriasis and other hyperproliferative skin diseases.

Isoflavones, their Glycosides and Glycoconjugates. Synthesis and Biological Activity.

Glycosylation of small biologically active molecules, either of natural or synthetic origin, has a profound impact on their solubility, stability, and bioactivity, making glycoconjugates attractive compounds as therapeutic agents or nutraceuticals. A large proportion of secondary metabolites, including flavonoids, occur in plants as glycosides, which adds to the molecular diversity that is much valued in medicinal chemistry studies. The subsequent growing market demand for glycosidic natural products has fueled the development of various chemical and biotechnological methods of glycosides preparation. The review gives an extensive overview of the processes of the synthesis of isoflavones and discusses recently developed major routes towards isoflavone-sugar formation processes. Special attention is given to the derivatives of genistein, the main isoflavone recognized as a useful lead in several therapeutic categories, with particular focus on anticancer drug design. The utility of chemical glycosylations as well as glycoconjugates preparation is discussed in some theoretical as well as practical aspects. Since novel approaches to chemical glycosylations and glycoconjugations are abundant and many of them proved suitable for derivatization of polyphenols a new body of evidence has emerged, indicating that sugar moiety can play a much more significant role, when attached to a pharmacophore, then being a mere "solubilizer". In many cases, it has been demonstrated that semisynthetic glycoconjugates are much more potent cytostatic and cytotoxic agents than reference isoflavones. Moreover, the newly designed glycosides or glycoside mimics can act through different mechanisms than the parent active molecule.

Synthesis and preliminary biological assay of uridine glycoconjugate derivatives containing amide and/or 1,2,3-triazole linkers.

A series of UDP-sugar analogues was synthesized and their preliminary biological activity was evaluated. Glycoconjugates of uridine 1 and 2 were synthesized by condensation of uridine-5'-carboxylic acid and 1-amino sugars derivatives of d-glucose and d-galactose, glycoconjugates 3 and 4 were synthesized by azide-alkyne 1,3-dipolar cycloaddition (CuAAC) of 1-azido sugars and propargylamide derivatives of uridine while glycoconjugates 5 and 6 were synthesized by CuAAC of propargyl ß-O-glycosides and 5'-azido uridine. Evaluation of inhibitory activity of compounds 1-6 against commercially available ß-1,4-galactosyltransferase I (ß4GalT) show that compound 5 inhibited the enzyme in µmolar range. Additionally, the antitumor activity of the obtained glycoconjugates 1-6 were tested using MTT assay.

Insulin and novel thioglycosides exert suppressive effect on human breast and colon carcinoma cells.

The rationale for the implementation of novel therapies should be based on hallmarks of cancer. Two novel compounds labelled as thioglycoside A and B were designed and evaluated on breast and colon cancer cell lines. We assessed their cytotoxic effect after sensitizing cancer cells with insulin. In order to explore the underlying mechanisms, we performed tests to assess cell migration and motility, apoptosis, expression of glucose transporter 1 and proapoptotic proteins. Both compounds proved to have an antitumor effect which was significantly enhanced in combination with insulin. Linking glucose and anticancer agent presents an approach that exploits the Warburg effect. Targeting dysfunctional glycometabolism and increased glucose absorption is emerging as a promising anticancer strategy.

Synthetic genistein glycosides inhibiting EGFR phosphorylation enhance the effect of radiation in HCT 116 colon cancer cells.

The need to find new EGFR inhibitors for use in combination with radiotherapy in the treatment of solid tumors has drawn our attention to compounds derived from genistein, a natural isoflavonoid. The antiproliferative potential of synthetic genistein derivatives used alone or in combination with ionizing radiation was evaluated in cancer cell lines using clonogenic assay. EGFR phosphorylation was assessed with western blotting. Genistein derivatives inhibited clonogenic growth of HCT 116 cancer cells additively or synergistically when used in combination with ionizing radiation, and decreased EGFR activation. Our preclinical evaluation of genistein-derived EGFR inhibitors suggests that these compounds are much more potent sensitizers of cells to radiation than the parent isoflavonoid, genistein and indicate that these compounds may be useful in the treatment of colon cancer with radiation therapy.

Synthesis and cytotoxicity of 2,3-enopyranosyl C-linked conjugates of genistein.

A series of glycoconjugates, derivatives of genistein containing a C-glycosylated carbohydrate moiety, were synthesized and their anticancer activity was tested in vitro in the human cell lines HCT 116 and DU 145. The target compounds 15-17 were synthesized by treating ω-bromoalkyl C-glycosides derived from L-rhamnal (1) with a tetrabutylammonium salt of genistein. The new, metabolically stable analogs of previously studied O-glycosidic genistein derivatives inhibited proliferation of cancer cell lines through inhibition of the cell cycle.

Synthesis and antiviral activity of a novel glycosyl sulfoxide against classical swine fever virus.

A novel compound-2″,3″,4″,6″-tetra-O-acetyl-ß-d-galactopyranosyl-(1→4)-2',3',6'-tri-O-acetyl-1-thio-ß-d-glucopyranosyl-(5-nitro-2-pyridyl) sulfoxide-designated GP6 was synthesized and assayed for cytotoxicity and in vitro antiviral properties against classical swine fever virus (CSFV) in this study. We showed that the examined compound effectively arrested CSFV growth in swine kidney cells (SK6) at a 50% inhibitory concentration (IC50) of 5 ± 0.12 µg/ml without significant toxicity for mammalian cells. Moreover, GP6 reduced the viral E2 and E(rns) glycoproteins expression in a dose-dependent manner. We have excluded the possibility that the inhibitor acts at the replication step of virus life cycle as assessed by monitoring of RNA level in cells and culture medium of SK6 cells after single round of infection as a function of GP6 treatment. Using recombinant E(rns) and E2 proteins of classical swine fever virus produced in baculovirus expression system we have demonstrated that GP6 did not influence glycoprotein production and maturation in insect cells. In contrast to mammalian glycosylation pathway, insect cells support only the ER-dependent early steps of this process. Therefore, we concluded that the late steps of glycosylation process are probably the main targets of GP6. Due to the observed antiviral effect accompanied by low cytotoxicity, this inhibitor represents potential candidate for the development of antiviral agents for anti-flavivirus therapy. Further experiments are needed for investigating whether this compound can be used as a safe antiviral agent against other viruses from unrelated groups.

Absorption and metabolism of biologically active genistein derivatives in colon carcinoma cell line (Caco-2).

Several genistein derivatives comprising an isoflavonoid skeleton substituted with an alkyl chain and a sugar moiety show ability to inhibit proliferation of cancer cells in vitro at the concentration several-fold lower than genistein. In our previous studies we shown that these compounds influenced the mitotic spindle, blocked the cell cycle and induced apoptosis. The purpose of this study was to determine the relationship between structural modifications of genistein molecule and the intestinal disposition of its derivatives. Transport and metabolism of these compounds were studied in the human intestinal Caco-2 model. The results of our study indicate that transport and metabolism of genistein derivatives depend both, on the structure of the carbonyl linker and position of genistein molecule substitution. All new compounds showed higher permeability coefficient in comparison to genistein. Moreover, genistein derivatives described in this work were transformed in Caco-2 cells into glucuronide and sulfate metabolites.

Development of novel molecular probes of the Rio1 atypical protein kinase.

The RIO kinases are essential protein factors required for the synthesis of new ribosomes in eukaryotes. Conserved in archaeal organisms as well, RIO kinases are among the most ancient of protein kinases. Their exact molecular mechanisms are under investigation and progress of this research would be significantly improved with the availability of suitable molecular probes that selectively block RIO kinases. RIO kinases contain a canonical eukaryotic protein kinase fold, but also display several unusual structural features that potentially create opportunity for the design of selective inhibitors. In an attempt to identify structural leads to target the RIO kinases, a series of pyridine caffeic acid benzyl amides (CABA) were tested for their ability to inhibit the autophosphorylation activity of Archeaoglobus fulgidus Rio1 (AfRio1). Screening of a small library of CABA molecules resulted in the identification of four compounds that measurably inhibited AfRio1 activity. Additional biochemical characterization of binding and inhibition activity of these compounds demonstrated an ATP competitive inhibition mode, and allowed identification of the functional groups that result in the highest binding affinity. In addition, docking of the compound to the structure of Rio1 and determination of the X-ray crystal structure of a model compound (WP1086) containing the desired functional groups allowed detailed analysis of the interactions between these compounds and the enzyme. Furthermore, the X-ray crystal structure demonstrated that these compounds stabilize an inactive form of the enzyme. Taken together, these results provide an important step in identification of a scaffold for the design of selective molecular probes to study molecular mechanisms of Rio1 kinases in vitro and in vivo. In addition, it provides a rationale for the future design of potent inhibitors with drug-like properties targeting an inactive form of the enzyme. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Novel small molecular inhibitors disrupt the JAK/STAT3 and FAK signaling pathways and exhibit a potent antitumor activity in glioma cells.

JAK (Janus kinase)/STAT (signal transducers and activators of transcription) signaling is involved in the regulation of cell growth, differentiation and apoptosis. Constitutive activation of STATs, in particular STAT3, is observed in a large number of human tumors, including gliomas and may contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis, thus it emerges as a promising target for anti-cancer therapy. To investigate the therapeutic potential of blocking STAT3 in glioma cells a set of small synthetic molecules - caffeic acid derivatives, structurally related to AG490 was screened for its ability to inhibit STAT3. Inhibitor 2 (E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide was the most effective in inhibition of JAK/STAT3 signaling and at doses ≥ 25 µM significantly reduced the level of phosphorylated JAK1, JAK2 and STAT3 (at Tyr705) and downregulated the expression of known STAT3 targets. In treated cells we observed rapid detachment and rounding of cells associated with reduction of focal adhesion kinase phosphorylation and activity, followed by upregulation of phosphorylated p38, JNK and ERK1/2 levels. Accumulation of cells with fragmented DNA, increases of the cleaved caspase 3 and fragmented PARP levels were detected 24 h after the treatment suggesting ongoing apoptotic cell death. Three human malignant glioblastoma cell lines defective in tumor suppressors TP53 and/or PTEN were susceptible to inhibitor 2 that induced the programmed cell death. Global gene expression profiling revealed modulation of numerous genes in cells treated with inhibitor 2 revealing novel, potential JAK/STAT targets. Our study demonstrates that suitably modified caffeic acid molecules exhibit significant cytotoxic potential toward glioma cells.

Synthesis of genistein 2,3-anhydroglycoconjugates -- potential antiproliferative agents.

The title compounds, variously protected 2.3-anhydrosugars linked with genistein through an alkyl chain, were synthesized in a sequence of reactions. First step involved Ferrier rearragement of 3,4-di-O-acetyl-L-rhamnal with 3-bromopropanol to obtain 2,3-unsaturated bromoalkylglycosides. The next step was epoxidation with m-CPBA and finally these compounds were connected with genistein in reaction of 7-O-genistein tetra-butylamonium salt with 2,3-anhydro bromoalkylglycosides. Obtained glycoconjugates differ in orientation of an oxirane ring and the protecting group in a sugar moiety. All compounds were tested in vitro for antiproliferative potential in cancer cells.

Synthetic conjugates of genistein affecting proliferation and mitosis of cancer cells.

This paper describes the synthesis and antiproliferative activity of conjugates of genistein (1) and unsaturated pyranosides. Constructs linking genistein with a sugar moiety through an alkyl chain were obtained in a two-step synthesis: in a first step genistein was converted into an intermediate bearing an ω-hydroxyalkyl substituent, containing two, three or five carbon atoms, at position 7, while the second step involved Ferrier glycosylation reaction, employing glycals. Antiproliferative activity of several genistein derivatives was tested in cancer cell lines in vitro. The most potent derivative, Ram-3 inhibited the cell cycle, interacted with mitotic spindles and caused apoptotic cell death. Neither genistein nor the sugar alone were able to influence the mitotic spindle organization. Our results indicate, that conjugation of genistein with certain sugars may render the interaction of derivatives with new molecular targets.

Synthetic derivatives of genistein, their properties and possible applications.

Genistein, the principal isoflavone constituent of soybean, attracts much attention as a natural molecule with significant affinity towards targets of potential medicinal interest, but also as a food supplement or prospective chemopreventive agent. Since its physicochemical properties are considered suboptimal for drug development, much effort has been invested in designing its analogs and conjugates in hope to obtain compounds with improved efficacy and selectivity. The aim of this article is to summarize current knowledge about the properties of synthetic genistein derivatives and to discuss possible clinical application of selected novel compounds. Some basic information concerning chemical reactivity of genistein, relevant to the synthesis of its derivatives, is also presented.