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Dina Farinha,1,2 Michael Migawa,3 Ana Sarmento-Ribeiro,1,4,5 Henrique Faneca1,2 1CNC - Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal; 2Institute for Interdisciplinary Research (IIIUC), University of Coimbra, Coimbra, Portugal; 3Ionis Pharmaceuticals Inc, Carlsbad, USA; 4Laboratory of Oncobiology and Hematology (LOH) and University Clinic of Hematology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 5Clinical Hematology Department, Centro Hospitalar Universitário de Coimbra (CHUC), Coimbra, PortugalCorrespondence: Henrique FanecaCNC - Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, 3004-504, PortugalTel +351-239-820-190Fax +351-239-853-607Email henrique@cnc.uc.ptBackground: Hepatocellular carcinoma (HCC) is one of the main causes of cancer-related death. Sorafenib, which is the first-line therapy for this disease, is associated with reduced therapeutic efficacy that could potentially be overcome by combination with selumetinib. In this context, the main goal of this work was to develop a new nanosystem, composed of a polymeric core coated by a lipid bilayer containing the targeting ligand GalNAc, to specifically and efficiently co-deliver both drugs into HCC cells, in order to significantly increase their therapeutic efficacy.Methods: The physicochemical characterization of hybrid nanosystems (HNP) and their components was performed by dynamic light scattering, zeta potential, matrix-assisted laser desorption ionization – time of flight mass spectroscopy, and transmission electron microscopy. Cellular binding, uptake and specificity of HNP were evaluated through flow cytometry and confocal microscopy. The therapeutic activity was evaluated namely through: cell viability by the Alamar Blue assay; cell death by flow cytometry using FITC-Annexin V; caspases activity by luminescence; mitochondrial membrane potential by flow cytometry; and molecular target levels by Western blot.Results: The obtained data show that these hybrid nanosystems present high stability and loading capacity of both drugs, and suitable physicochemical properties, namely in terms of size and surface charge. Moreover, the generated formulation allows to circumvent drug resistance and presents high specificity, promoting great cell death levels in HCC cells, but not in non-tumor cells. This potentiation of the antitumor effect of co-loaded drugs was carried out by an increased programmed cell death, being associated with a strong reduction in the mitochondrial membrane potential, a significant increase in the activity of caspases 3/7 and caspase 9, and much greater number of annexin V-positive cells.Conclusion: The developed formulation resulted in a high and synergistic antitumor effect, revealing a translational potential to improve therapeutic approaches against HCC.Keywords: hepatocellular carcinoma, hybrid nanosystems, drug delivery, GalNAc, sorafenib, selumetinib

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'- O -methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

The fact that only two pyrrolo[2,3-d][1,2,3]triazines heterocycles had been reported in the literature prompted us to initiate studies designed to provide additional members of this ring system. Initial attempts to prepare additional derivatives of the 7-unsubstituted pyrrolo[2,3-d][1,2,3]triazin-4-ones were limited by their low chemical reactivity. Subsequently, 7-benzyl-5-carboxamidopyrrolo[2,3-d][1,2,3]triazin-4-one (16) was prepared from diethyl 2-nitropyrrole-3,4,-dicarboxylate via an alkylation, ammonolysis, reduction and intramolecular diazocoupling sequence. Conversion of 16 into 7-benzyl-4-(1,2,4-triazol-1-yl)pyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile (17) was accomplished, and nucleophilic displacements of the 4-triazol-1-yl group were studied. Treatment of 17 with NH3/CH3CN gave a mixture of 4-amino-7-benzylpyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile (19) and 2-amino-1-benzylpyrrole-3,4-dicarbonitrile (21). A mechanism to account for the formation of this mixture is described along with studies on the effect that ammonia concentration and a TFA catalyst have on the product ratio. Compound 19 was converted into the 5-carboxamide and 5-thioamide derivatives of 19.

Several methods for functionalization of the 4-position of imidazo[4,5-d][1,2,3]triazin-4-one were investigated. These investigations were successful and led to the preparation of 4-amino, 4-triazol-1-yl, 4-methoxy, 4-methylthio, 4-methylamino, 4-thio, 4-nitrobenzyl, and 4-unsubstituted 9-(beta-D-ribofuranosyl)-imidazo-[4,5-d][1,2,3]triazine (2-azapurine ribosides). The 4-unsubstituted compound (19) was slightly active against HCMV in plaque and yield reduction experiments and was not cytotoxic at 100 microM. The methylamino (15), hydrazino (16), and p-nitrobenzylthio (20) were inactive against HCMV but slightly cytotoxic. The thiomethyl-substituted analog (21) was the most active with activity comparable to ganciclovir but with greater cytotoxicity. We conclude that even though none of the tested compounds had antiviral activity superior to ganciclovir, the new synthetic methods will provide a route to more interesting compounds.

[formula: see text] In the course of our work on novel pyrrolo[2,3-d][1,2,3]triazines we have discovered that 1 undergoes an elimination of nitrogen at 250 degrees C to give 2. We have conducted 15N labeling studies that establish that the loss of N-2 and N-3 from 1 had occurred rather than N-1 and N-2, presumably via a retro Diels-Alder reaction of the imino tautomer 7. This study provides an alternative to the commonly accepted mechanism which involves the loss of N-1 and N-2 via the transient formation of a diazonium compound generated from 4-amino- or 4-oxo-substituted 1,2,3-triazines condensed with carbocycles or heterocycles.

Several 2-substituted alpha-D- and alpha-L-lyxofuranosyl and 5-deoxylyxofuranosyl derivatives of 5,6-dicholro-2-(isopropylamino)-1-(beta-L-ribofuranosyl) benzimidazole (1263W94) and 2,5,6-trichloro-1(beta-D-ribofuranosyl)benzimidazole (TCRB) were synthesized and evaluated for activity against two herpesviruses (HSV-1 and HCMV) and for their cytotoxicity against HFF and KB cells. Condensation of 1,2,3,5-tetra-O-acetyl-L-lyxofuranose (2a) with 2,5,6-trichlorobenzimidazole (1) yielded the alpha-nucleoside 3a. The 2-bromo derivative and 2-methylamino derivative were prepared by treatment of 3a with HBr followed by deprotection or from methylamine, respectively. Compound 3a was deprotected and the resultant nucleoside used to prepare the 2-cyclopropylamino and 2-isopropylamino derivatives. The 2-alkylthio nucleosides were prepared by condensing 2a with 5,6-dichlorobenzimidazole-2-thione followed by deprotection. Alkylation of this adduct gave the 2-methylthio and 2-benzylthio derivatives. Condensation of 5-deoxy-1,2,3-tri-O-acetyl-L-lyxofuranosyl, prepared from L-lyxose, with 1 or 2-bromo-5,6-dichlorobenzimidazole (15), followed by deprotection, gave the 2-chloro or 2-bromo-5'-deoxylyxo-furanosyl derivative, respectively. The cyclopropylamino derivative was prepared from the 2-chloro derivative. All D-isomers were prepared in an analogous fashion from D-lyxose. Either compounds were inactive against HSV-1 or weak activity was poorly separated from cytotoxicity. In contrast, the 2-halogen derivatives in both the alpha-lyxose and 5-deoxy-alpha-lyxose series were active against the Towne strain of HCMV. The 5-deoxy alpha-L analogues were the most active, IC50's = 0.2-0.4 microM, plaque assay; IC90's = 0.2-2 microM, yield reduction assay. All of the 2-isopropylamino or 2-cyclopropylamino derivatives were less active (IC50's = 60-100 microM, plaque assay; IC90's = 17-100 microM, yield reduction assay) and were not cytotoxic. The methylamino, thio, and methylthio derivatives were neither active nor cytotoxic. The benzylthio derivatives were weakly active, but this activity was poorly separated from cytotoxicity. The alpha-lyxose L-isomers were more active in a plaque assay against the AD169 strain of HCMV compared to the Towne strain, thereby providing additional evidence of antiviral specificity.