DESING AND SYNTHESIS OF NOVEL TRICATIONIC N-ACETYLGALACTOSAMINE PYRIDINIUM TRANSFECTION AGENTS FOR THE SELECTIVE DELIVERY OF TERAPEUTIC OLIGONUCLEOTIDES INTO HEPATIC CELLS

Transfection agents play a key role in oligonucleotide-based gene therapy by protecting genetic material from degradation and promoting its internalization into target cells. Despite the potential of these systems, their limited ability to ensure selective internalization still represents a significant obstacle. Among these, TRIFAPYs, a series of tricationic transfection agents recently developed at the Universitat de Barcelona, have shown good oligonucleotide transfection ability, although they still display limited selectivity in cellular internalization. To overcome this limitation and promote selectivity toward hepatic cells, the present work reports the design and synthesis of TRIGALPYs, in which the tricationic scaffold of the previously developed TRIFAPYs was retained and functionalized with three N- acetylgalactosamine rings, which are essential for interaction with the ASGPR receptor. For the preparation of TRIGALPYs, a synthetic route was developed aimed at obtaining derivatives characterized by alkyl chains of different lengths. This synthetic route highlighted the greater efficiency of nucleophilic aromatic substitution compared with Williamson etherification, and subsequent optimization led to the preparation of the pyridinic ether intermediates. The following transformations enabled the stereospecific introduction of the N-acetylgalactosamine ring and the formation of the final tricationic scaffold. Despite the successful outcome of the previous reactions, the issues encountered during the final deprotection step required a revision of the last stages of the initially proposed synthetic route. This modified approach made it possible to overcome the observed limitations and provided experimental evidence supporting the formation of TRIGALPYs. The 1H-NMR spectroscopic and HRMS spectrometric analyses of the crude reaction mixtures obtained at the end of the synthesis provided results consistent with the presence of TRIGALPYs. Although further purification and characterization studies are required for a more accurate structural confirmation, the collected data support the validity of the proposed synthetic route and confirm the presence of the expected products in the reaction mixture. Future studies will focus on the optimization of the final purification step and the evaluation of the biological properties of the synthesized compounds, including toxicity, hepatic selectivity of transfection, and binding affinity toward oligonucleotides.