The conjugates of 5'-deoxy-5-fluorocytidine and hydroxycinnamic acids - synthesis, anti-pancreatic cancer activity and molecular docking studies.

New amide conjugates 1-6 of hydroxycinnamic acids (HCA) and 5'-deoxy-5-fluorocytidine (5-dFCR), the prodrug of 5-fluorouracil (5-FU), were synthesized and tested in vitro against pancreatic cancer lines (PDAC). The compounds showed slightly higher efficacy against primary BxPC-3 cells (IC50 values of 14-45 µM) than against metastatic AsPC-1 (IC50 values of 37-133 µM), and similar to that of 5-FU for both PDAC lines. Compound 1, which has a para-(acetyloxy)coumaroyl substituent, was found to be the most potent (IC50 = 14 µM) with a selectivity index of approximately 7 to normal dermal fibroblasts (IC50 = 96 µM). The potential pharmacological profiles were discussed on the basis of the ADME data. Docking to the carboxylesterase CES2 showed that the synthesized compounds have the ability to bind via hydrogen bonding between a specific acetate group of the sugar moiety and Ser228, which belongs to the catalytic triad that causes hydrolysis. Docking to albumin, a major transport protein in the circulatory system, revealed a strong interaction of the conjugates at the binding site which is native to warfarin and responsible for its transport in the body.

Synthesis, Biological Activity, ADME and Molecular Docking Studies of Novel Ursolic Acid Derivatives as Potent Anticancer Agents.

A series of new ursolic acid (UA) derivatives substituted with various amino acids (AAs) or dipeptides (DP) at the C-3 position of the steroid skeleton was designed and synthesized. The compounds were obtained by the esterification of UA with the corresponding AAs. The cytotoxic activity of the synthesized conjugates was determined using the hormone-dependent breast cancer cell line MCF-7 and the triple-negative breast cancer cell line MDA. Three derivatives (l-seryloxy-, l-prolyloxy- and l-alanyl-l-isoleucyloxy-) showed micromolar IC50 values and reduced the concentrations of matrix metalloproteinases 2 and 9. Further studies revealed that for two compounds (l-seryloxy- and l-alanyl-l-isoleucyloxy-), a possible mechanism of their antiproliferative action is the activation of caspase-7 and the proapoptotic Bax protein in the apoptotic pathway. The third compound (l-prolyloxy- derivative) showed a different mechanism of action as it induced autophagy as measured by an increase in the concentrations of three autophagy markers: LC3A, LC3B, and beclin-1. This derivative also showed statistically significant inhibition of the proinflammatory cytokines TNF-α and IL-6. Finally, for all synthesized compounds, we computationally predicted their ADME properties as well as performed molecular docking to the estrogen receptor to assess their potential for further development as anticancer agents.

New, Low-Molecular Weight Chemical Compounds Inhibiting Biological Activity of Interleukin 15.

Chronic overproduction of IL-15 contributes to the pathogenesis of numerous inflammatory and autoimmune disorders. Experimental methods used to reduce the cytokine activity show promise as potential therapeutic approaches to modify IL-15 signaling and alleviate the development and progression of IL-15-related diseases. We previously demonstrated that an efficient reduction of IL-15 activity can be obtained by selective blocking of the specific, high affinity subunit alpha of the IL-15 receptor (IL-15Rα) with small-molecule inhibitors. In this study, we determined the structure-activity relationship of currently known IL-15Rα inhibitors in order to define the critical structural features required for their activity. To validate our predictions, we designed, analyzed in silico, and assessed in vitro function of 16 new potential IL-15Rα inhibitors. All newly synthesized molecules were benzoic acid derivatives with favorable ADME properties and they efficiently reduced IL-15 dependent peripheral blood mononuclear cells (PBMCs) proliferation, as well as TNF-α and IL-17 secretion. The rational design of IL-15 inhibitors may propel the identification of potential lead molecules for the development of safe and effective therapeutic agents.

Newly Synthesized Melphalan Analogs Induce DNA Damage and Mitotic Catastrophe in Hematological Malignant Cancer Cells.

Myeloablative therapy with highdoses of the cytostatic drug melphalan (MEL) in preparation for hematopoietic cell transplantation is the standard of care for multiple myeloma (MM) patients. Melphalan is a bifunctional alkylating agent that covalently binds to nucleophilic sites in the DNA and effective in the treatment, but unfortunately has limited therapeutic benefit. Therefore, new approaches are urgently needed for patients who are resistant to existing standard treatment with MEL. Regulating the pharmacological activity of drug molecules by modifying their structure is one method for improving their effectiveness. The purpose of this work was to analyze the physicochemical and biological properties of newly synthesized melphalan derivatives (EE-MEL, EM-MEL, EM-MOR-MEL, EM-I-MEL, EM-T-MEL) obtained through the esterification of the carboxyl group and the replacement of the the amino group with an amidine group. Compounds were selected based on our previous studies for their improved anticancer properties in comparison with the original drug. For this, we first evaluated the physicochemical properties using the circular dichroism technique, then analyzed the zeta potential and the hydrodynamic diameters of the particles. Then, the in vitro biological properties of the analogs were tested on multiple myeloma (RPMI8226), acute monocytic leukemia (THP1), and promyelocytic leukemia (HL60) cells as model systems for hematological malignant cells. DNA damage was assessed by immunostaining γH2AX, cell cycle distribution changes by propidium iodide (PI) staining, and cell death by the activation of caspase 2. We proved that the newly synthesized derivatives, in particular EM-MOR-MEL and EM-T-MEL, affected the B-DNA conformation, thus increasing the DNA damage. As a result of the DNA changes, the cell cycle was arrested in the S and G2/M phases. The cell death occurred by activating a mitotic catastrophe. Our investigations suggest that the analogs EM-MOR-MEL and EM-T-MEL have better anti-cancer activity in multiple myeloma cells than the currently used melphalan.

Antioxidant activity of novel diosgenin derivatives: Synthesis, biological evaluation, and in silico ADME prediction.

A series of novel diosgenin (DSG) derivatives has been synthesized and tested in vitro for their antioxidant activity. Initially, four analogues have been evaluated for their cytotoxicity using normal human skin fibroblast (NHDF) as model cells. As a result, 84% of NHDF cells were still alive at 5 µM, so these compounds can be considered as innoxious to fibroblasts at this concentration. Then, hemolytic activity against human erythrocytes was studied in order to evaluate the potential impact of tested compounds against normal host cells. The result < 5% of hemolysis rates suggest no lytic activity for most compounds. After that, the main test - evaluation the antioxidant effect of DSG and its new derivatives against lipid peroxidation in the o/w emulsion model - was performed. The most promising compound (8) exhibited the significant antioxidant activity and the biocompatibility towards normal human dermal fibroblasts and red bloods cells. This p-aminobenzoic derivative revealed 61.6% blocking of induced lipid oxidation. Furthermore, eleven predicted ADME properties were predicted for all tested compounds and revealed that they are in compliance with drug-likeness criteria.

Synthesis and In Vitro Activity of Novel Melphalan Analogs in Hematological Malignancy Cells.

Despite the continuous developments in pharmacology and the high therapeutic effect of new treatment options for patients with hematological malignancies, these diseases remain a major health issue. Our study aimed to synthesize, analyze in silico, and determine the biological properties of new melphalan derivatives. We obtained three methyl esters of melphalan having in their structures amidine moieties substituted with thiomorpholine (EM-T-MEL), indoline (EM-I-MEL), or 4-(4-morpholinyl) piperidine (EM-MORPIP-MEL). These have not yet been described in the literature. The in vitro anticancer properties of the analogs were determined against THP1, HL60, and RPMI8226 cells. Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (alkaline comet assay), and their ability to induce apoptosis (Hoechst33342/propidium iodide double staining method; phosphatidylserine translocation; and caspase 3/7, 8, and 9 activity measurements). Changes in mitochondrial membrane potential were examined using the specific fluorescence probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine). The EM-T-MEL derivative had the highest biological activity, showing higher cytotoxic and genotoxic properties than the parent drug. Moreover, it showed a high ability to induce apoptosis in the tested cancer cells. This compound also had a beneficial effect in peripheral blood mononuclear cells (PBMC). In conclusion, we verified and confirmed the hypothesis that chemical modifications of the melphalan structure improved its anticancer properties. The conducted study allowed the selection of the compound with the highest biological activity and provided a basis for chemical structure-biological activity analyses.

Unexpected Reaction Products of Uracil and Its Methyl Derivatives with Acetic Anhydride and Methylene Chloride.

New acetyl derivatives of uracil, 6-methyluracil, and thymine were obtained in the course of an unconventional synthesis in methylene chloride. It was shown that products with the acetyloxymethyl fragment are formed according to a mechanism different from that for products with the acetyloxyethyl group. In particular, for uracil it was proven that the reaction with Ac2O, TEA, and CH2Cl2 leads to 1-acetyloxymethyluracil, where the N1 substituent is composed of the -CH2- fragment that originated from CH2Cl2 and the 1-acetyloxy moiety from Ac2O. The reaction of uracil with Ac2O, TEA, CH2Cl2, and DMAP leads to an acetyloxyethyl derivative in which the -CH2-CH2- fragment originates from TEA and the 1-acetyloxy moiety from Ac2O. A possible mechanism for the formation of new compounds was suggested and supported by the density functional theory/B3LYP quantum mechanical calculations. New compounds (39 in total, including seven deuterated) were fully characterized by nuclear magnetic resonance and high-resolution mass spectrometry techniques.

Enigmatic Histamine Receptor H4 for Potential Treatment of Multiple Inflammatory, Autoimmune, and Related Diseases.

The histamine H4 receptor, belonging to the family of G-protein coupled receptors, is an increasingly attractive drug target. It plays an indispensable role in many cellular pathways, and numerous H4R ligands are being studied for the treatment of several inflammatory, allergic, and autoimmune disorders, including pulmonary fibrosis. Activation of H4R is involved in cytokine production and mediates mast cell activation and eosinophil chemotaxis. The importance of this receptor has also been shown in inflammatory models: peritonitis, respiratory tract inflammation, colitis, osteoarthritis, and rheumatoid arthritis. Recent studies suggest that H4R acts as a modulator in cancer, neuropathic pain, vestibular disorders, and type-2 diabetes, however, its role is still not fully understood.

A diversity of alkylation/acylation products of uracil and its derivatives: synthesis and a structural study.

tert-Butyl dicarbonate (Boc2O) and ethyl iodide (EtI) reactions with uracil (U), thymine (T) and 6-methyluracil (6-MU) were performed following routine procedures in pyridine/DMF solvents and with DMAP as the catalyst. Among 20 synthesized compounds, a derivative of 6-methyluracil substituted by the Boc-pyridine moiety at the C5 position appeared unexpectedly. The NMR spectra confirmed the molecular structure of all uracil derivatives. Parallel quantum mechanical DFT calculations supported the experimental findings.

Multinuclear NMR Measurements and DFT Calculations for Capecitabine Tautomeric Form Assignment in a Solution.

The molecular structure of capecitabine (a widely applied prodrug of 5-fluorouracil) was studied by multinuclear NMR measurements and DFT quantum mechanical calculations. One or two tautomeric forms in a solution were detected depending on the solvent used. In the organic solvents, a mixture of two forms of capecitabine was observed: carbamate and imine tautomers. In the aqueous solution, only the carbamate form was found. The methylation of capecitabine yields mainly two products in different proportions: N³-methylcapecitabine and N7-methylcapecitabine. The protonation of capecitabine in organic solvents with perchloric acid occurs at the N3 nitrogen atom. DFT calculations strongly support the results coming from the analysis of the NMR spectra.

Pharmacophore guided discovery of small-molecule interleukin 15 inhibitors.

Upregulation of interleukin 15 (IL-15) contributes directly i.a. to the development of inflammatory and autoimmune diseases. Selective blockade of IL-15 aimed to treat rheumatoid arthritis, psoriasis and other IL-15-related disorders has been recognized as an efficient therapeutic method. The aim of the study was to identify small molecules which would interact with IL-15 or its receptor IL-15Rα and inhibit the cytokine's activity. Based on the crystal structure of IL-15Rα·IL-15, we created pharmacophore models to screen the ZINC database of chemical compounds for potential IL-15 and IL-15Rα inhibitors. Twenty compounds with the highest predicted binding affinities were subjected to in vitro analysis using human peripheral blood mononuclear cells to validate in silico data. Twelve molecules efficiently reduced IL-15-dependent TNF-α and IL-17 synthesis. Among these, cefazolin - a safe first-generation cephalosporin antibiotic - holds the highest promise for IL-15-directed therapeutic applications.

Crystal structure and tautomerism of capecitabine.

The crystal and molecular structure of capecitabine, an anticancer pharmaceutical substance, was solved and refined using single-crystal X-ray diffraction. The compound was synthesized from a derivative of cytidine by a modified method. The crystal of capecitabine for X-ray study was grown by seedless crystallization from a single solvent. The low and room temperature single-crystal X-ray crystallographic study revealed that capecitabine exists in the solid state exclusively in one of the two possible prototropic tautomers. In the molecular structure of this tautomer, the hydrogen atom is attached to the N3 nitrogen atom of the pyrimidine ring (imine tautomer) and not to the N(4) nitrogen of the carbamate (carbamate tautomer), as has been widely reported up to the present. The imine tautomer was also found to be thermodynamically preferred in the ab initio calculations. This finding indicates that the reported structural formula of capecitabine, as well as its systematic chemical name, must be revised.

Cytostatic and cytotoxic activity of synthetic genistein glycosides against human cancer cell lines.

Genistein, the principal soy isoflavone, is a molecule of great interest as an innovative chemotherapeutic agent or as a lead-compound in anticancer drug design. To enhance intrinsic activity of genistein and to explore its pharmacophoric potential, its glycosidic derivatives were synthesized. On the basis of structural features and calculated lipophilicity coefficient (ClogP) the derivatives were classified as hydrophilic (i.e. those containing free sugar moiety) or lipophilic (i.e. those with alkylated or acylated sugar hydroxyls). The in vitro cytostatic and cytotoxic studies showed hydrophilic glycosides to be practically inactive against human cancer cell lines when compared to the free aglycone. On the contrary, lipophilic glycosides were significantly more active than the parent isoflavone although the correlation between ClogP and the activity was not clear. On the basis of GI50 and LC50 values two of the most active glycosides were found to be several times more potent in their cytostatic and cytotoxic effect than genistein. Additionally all lipophilic glycosides were revealed to exhibit different mode of action in comparison to genistein. It may suggest that these compounds do not undergo rapid biodegradation, either in culture media or inside cells, and exert their biological effects primarily as intact molecules.

Determination of new derivatives of genistein in culture media by liquid chromatography.

Methods for determination of genistein and its four new analogues in culture media have been developed to support studies on their potential anticancer activities. The investigated compounds were extracted from the media using liquid-liquid extraction with appropriate solvent. After evaporation of organic solvents each of the dry extracts was reconstituted in appropriate mobile phase. Reversed-phase HPLC was applied to quantitative determining of tested compounds. The methods are specific, sensitive and technically simple. They were used to evaluate concentration level of investigated compounds in experiments with human promyelocytic leukemia cells (HL-60 cell line).