Intracellular concentrations represent a key parameter in assessing pharmacological efficacy. They reflect the amount of active compound that reaches its target within the cell. The passage of an active compound across the cellular membrane is influenced by various factors including the properties of the cell membrane, the presence of transport mechanisms, intracellular binding, and the compound’s metabolic stability. Consequently, measurement of extracellular concentrations alone does not provide an accurate estimate of the active compound’s true intracellular uptake. Determining intracellular concentrations requires a sensitive and selective analytical technique, as the amounts of the active compound in cell lysates are often very low, and the matrix is complex, containing numerous endogenous compounds that can interfere with the analysis. The most used technique for measuring intracellular concentrations of active compounds is high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS), which allows for sensitive, selective, and reliable analysis of a wide range of compounds. In the scope of this master’s thesis, we developed an LC-MS analytical method for determining intracellular concentrations of the compound Osmi-4b and its six analogues, which act as inhibitors of the enzyme O-β-N-acetylglucosaminyl transferase (OGT). These compounds share a common heterocyclic scaffold but differ in the substituent (-R) on the nitrogen atom of the amide group. OGT is involved in the process of O-GlcNAc glycosylation, in which N-acetylglucosamine (GlcNAc) is added to serine or threonine residues of proteins. Inhibiting OGT has potential therapeutic value for Alzheimer’s disease, certain types of cancer, and other disorders associated with altered glycosylation. During the development of the analytical method, we validated its suitability for determining intracellular concentrations for each compound by testing selected verification parameters. Subsequently, we analyzed cell lysates from the AMO-1 cell line after incubation with the selected compounds. The results showed that the intracellular concentrations of the selected compounds were proportional to their logD values at pH 7.5. The lowest intracellular concentrations were observed for the more polar compounds DY-38 and DY-39, while DY-34 exhibited a distribution similar to that of Osmi-4b. The highest concentrations were measured for AVS-18 and AVS-20, which exceeded the values of Osmi-4b.
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