Synthesis and Anticancer and Antiviral Activities of C-2'-Branched Arabinonucleosides.

d-Arabinofuranosyl-pyrimidine and -purine nucleoside analogues containing alkylthio-, acetylthio- or 1-thiosugar substituents at the C2' position were prepared from the corresponding 3',5'-O-silylene acetal-protected nucleoside 2'-exomethylenes by photoinitiated, radical-mediated hydrothiolation reactions. Although the stereochemical outcome of the hydrothiolation depended on the structure of both the thiol and the furanoside aglycone, in general, high d-arabino selectivity was obtained. The cytotoxic effect of the arabinonucleosides was studied on tumorous SCC (mouse squamous cell) and immortalized control HaCaT (human keratinocyte) cell lines by MTT assay. Three pyrimidine nucleosides containing C2'-butylsulfanylmethyl or -acetylthiomethyl groups showed promising cytotoxicity at low micromolar concentrations with good selectivity towards tumor cells. SAR analysis using a methyl ß-d-arabinofuranoside reference compound showed that the silyl-protecting group, the nucleobase and the corresponding C2' substituent are crucial for the cell growth inhibitory activity. The effects of the three most active nucleoside analogues on parameters indicative of cytotoxicity, such as cell size, division time and cell generation time, were investigated by near-infrared live cell imaging, which showed that the 2'-acetylthiomethyluridine derivative induced the most significant functional and morphological changes. Some nucleoside analogues also exerted anti-SARS-CoV-2 and/or anti-HCoV-229E activity with low micromolar EC50 values; however, the antiviral activity was always accompanied by significant cytotoxicity.

Potential Original Drug for Aspergillosis: In Vitro and In Vivo Effects of 1-N,N-Dimethylamino-5-Isocyanonaphthalene (DIMICAN) on Aspergillus fumigatus.

As the recent outbreak of coronavirus disease 2019 (COVID-19) has shown, viral infections are prone to secondary complications like invasive aspergillosis with a high mortality rate, and therefore the development of novel, effective antifungals is of paramount importance. We have previously demonstrated that 1-amino-5-isocyanonaphthalene (ICAN) derivatives are promising original drug candidates against Candida strains (Patent pending), even against fluconazole resistant C. albicans. Consequently, in this study ICANs were tested on Aspergillus fumigatus, an opportunistic pathogen, which is the leading cause of invasive and systematic pulmonary aspergillosis in immunosuppressed, transplanted and cancer- or COVID-19 treated patients. We have tested several N-alkylated ICANs, a well as 1,5-naphthalene-diisocyanide (DIN) with the microdilution method against Aspergillus fumigatus strains. The results revealed that the diisocyanide (DIN) was the most effective with a minimum inhibitory concentration (MIC) value as low as 0.6 µg mL-1 (3.4 µM); however, its practical applicability is limited by its poor water solubility, which needs to be overcome by proper formulation. The other alkylated derivatives also have in vitro and in vivo anti-Aspergillus fumigatus effects. For animal experiments the second most effective derivative 1-N, N-dimethylamino-5-isocyanonaphthalene (DIMICAN, MIC: 7-8 µg mL-1, 36-41 µM) was selected, toxicity tests were made with mice, and then the antifungal effect of DIMICAN was tested in a neutropenic aspergillosis murine model. Compared to amphotericin B (AMB), a well-known antifungal, the antifungal effect of DIMICAN in vivo turned out to be much better (40% vs. 90% survival after eight days), indicating its potential as a clinical drug candidate.

Isocyanide Substitution in Acridine Orange Shifts DNA Damage-Mediated Phototoxicity to Permeabilization of the Lysosomal Membrane in Cancer Cells.

In cancer therapy, immunogenic cell death eliminates tumor cells more efficiently than conventional apoptosis. During photodynamic therapy (PDT), some photosensitizer (PS) targeting lysosomes divert apoptosis to the immunologically more relevant necrosis-like cell death. Acridine orange (AO) is a PS targeting lysosome. We synthesized a new compound, 3-N,N-dimethylamino-6-isocyanoacridine (DM), a modified AO, aiming to target lysosomes better. To compare DM and AO, we studied optical properties, toxicity, cell internalization, and phototoxicity. In addition, light-mediated effects were monitored by the recently developed QUINESIn method on nuclei, and membrane stability, morphology, and function of lysosomes utilizing fluorescent probes by imaging cytometry in single cells. DM proved to be a better lysosomal marker at 405 nm excitation and lysed lysosomes more efficiently. AO injured DNA and histones more extensively than DM. Remarkably, DM's optical properties helped visualize shockwaves of nuclear DNA released from cells during the PDT. The asymmetric polar modification of the AO leads to a new compound, DM, which has increased efficacy in targeting and disrupting lysosomes. Suitable AO modification may boost adaptive immune response making PDT more efficient.

Mesoporous Aerogel Microparticles Injected into the Abdominal Cavity of Mice Accumulate in Parathymic Lymph Nodes.

Mesoporous aerogel microparticles are promising drug delivery systems. However, their in vivo biodistribution pathways and health effects are unknown. Suspensions of fluorescein-labeled silica-gelatin hybrid aerogel microparticles were injected into the peritoneum (abdominal cavity) of healthy mice in concentrations of 52 and 104 mg kg-1 in a 3-week-long acute toxicity experiment. No physiological dysfunctions were detected, and all mice were healthy. An autopsy revealed that the aerogel microparticles were not present at the site of injection in the abdominal cavity at the end of the experiment. The histological study of the liver, spleen, kidneys, thymus and lymphatic tissues showed no signs of toxicity. The localization of the aerogel microparticles in the organs was studied by fluorescence microscopy. Aerogel microparticles were not detected in any of the abdominal organs, but they were clearly visible in the cortical part of the parathymic lymph nodes, where they accumulated. The accumulation of aerogel microparticles in parathymic lymph nodes in combination with their absence in the reticuloendothelial system organs, such as the liver or spleen, suggests that the microparticles entered the lymphatic circulation. This biodistribution pathway could be exploited to design passive targeting drug delivery systems for flooding metastatic pathways of abdominal cancers that spread via the lymphatic circulation.

MTT Test and Time-lapse Microscopy to Evaluate the Antitumor Potential of Nucleoside Analogues.

BACKGROUND/AIM: Conventional viability tests, help to screen the cellular effects of candidate molecules, but the endpoint of these measurements lacks sufficient information regarding the molecular aspects. A non-invasive, easy-to-setup live-cell microscopic method served to in-depth analysis of mechanisms of potential anticancer drugs. MATERIALS AND METHODS: The proposed method combining the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test with time-lapse scanning microscopy (TLS), provided additional data related to the cell-cycle and the dynamic properties of cell morphology. Apoptotic and necrotic events became detectable with these methods. RESULTS: Quantification of the results was assisted by image analysis of the acquired image sequences. After demonstrating the potential of the TLS method, a series of experiments compared the in vitro effect of a known and a newly synthesized nucleoside analogue. CONCLUSION: The proposed approach provided a more in-depth insight into the cellular processes that can be affected by known chemotherapeutic agents including nucleoside analogues rather than applying repeated individual treatments.

Synthesis and Cytostatic Effect of 3'-deoxy-3'-C-Sulfanylmethyl Nucleoside Derivatives with d-xylo Configuration.

A small library of 3'-deoxy-C3'-substituted xylofuranosyl-pyrimidine nucleoside analogues were prepared by photoinduced thiol-ene addition of various thiols, including normal and branched alkyl-, 2-hydroxyethyl, benzyl-, and sugar thiols, to 3'-exomethylene derivatives of 2',5'-di-O-tert-butyldimethylsilyl-protected ribothymidine and uridine. The bioactivity of these derivatives was studied on tumorous SCC (mouse squamous carcinoma cell) and immortalized control HaCaT (human keratinocyte) cell lines. Several alkyl-substituted analogues elicited promising cytostatic activity in low micromolar concentrations with a slight selectivity toward tumor cells. Near-infrared live-cell imaging revealed SCC tumor cell-specific mitotic blockade via genotoxicity of analogue 10, bearing an n-butyl side chain. This analogue essentially affects the chromatin structure of SCC tumor cells, inducing a condensed nuclear material and micronuclei as also supported by fluorescent microscopy. The results highlight that thiol-ene chemistry represents an efficient strategy to discover novel nucleoside analogues with non-natural sugar structures as anticancer agents.

Metastatic Spread from Abdominal Tumor Cells to Parathymic Lymph Nodes.

Metastatic studies on rats showed that after subrenal implantation of tumor cells under the capsule of the kidney or subhepatic implantation under Glisson's capsule of the liver generated primary tumors in these organs. It was assumed that tumor cells that escaped through the disrupted peripheral blood vessels of primary tumors entered the peritoneal cavity, crossed the diaphragm, and appeared in the thoracal, primarily in the parathymic lymph nodes. This explanation did not answer the question whether distant lymph nodes were reached via the blood stream from the primary tumor or through the thoracal lymphatic vessels. In this work, we investigated the metastatic pathway in C3H/HeJ mice, after direct intraperitoneal administration of murine SCC VII cells bypassing the hematogenic spread of tumor cells. The direct pathway was also mimicked by intraperitoneal injection of Pelican Ink colloidal particles, which appeared in the parathymic lymph nodes, similarly to the tumor cells that caused metastasis in the parathymic lymph nodes and in the thymic tissue. The murine peritoneal-parathymic lymph node route indicates a general mechanism of tumor progression from the abdominal effusion. This pathway starts with the growth of abdominal tumors, continues as thoracal metastasis in parathymic lymph nodes and may proceed as mammary lymph node metastasis.