The doctoral thesis research was focused on three targets of the immune system - indoleamine 2,3-dioxygenase 1 (IDO1) and Toll-like receptors 7 and 8 (TLR7 and TLR8). While IDO1 is responsible for immunomodulation through the metabolism of tryptophan, TLR7 and TLR8 act as sensors for nucleic acids of different pathogens. All three targets are involved in the pathology of several diseases, among them also in different cancers. Our research work on IDO1 started with the synthesis of a focused library of analogs based on the previously discovered hit compound. Therefore, a multistep synthetic procedure for the preparation of isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold was developed, enabling substitutions on the main scaffold in two different positions. Inhibitory IDO1 activity of all synthesized compounds was evaluated in a fluorescence-based assay, with selectivity over two isoforms, i.e., indoleamine 2,3-dioxygenase 2 and tryptophan 2,3-dioxygenase, being further assessed. Prepared compounds selectively inhibited IDO1 in low micromolar range, with the most potent among them possessing aniline fragment, which was substituted with the trifluoromethyl, cyclohexyl or methoxycarbonyl group on the para position. The cytotoxicity profile of inhibitors was further determined and for the most potent compound 23 (IC50 of 23 [micro]M) we also evaluated its cellular activity. Two series of low micromolar and selective TLR7 agonists were prepared based on three hit compounds with chromeno[3,4-d]imidazol-4(1H)-one and 2-(trifluoromethyl)quinoline/ quinazoline-4-amine scaffolds. Substitutions on the chromenoimidazolone scaffold led to the shift from antagonist to agonist activity, with compound 26 representing the best-in-class agonist with EC50 value of 1.8 [micro]M. The agonists from the second series possessed secondary or tertiary amines with smaller alkyl substituents or bulkier rigid aliphatic ring on the position 4 of the main scaffold. Furthermore, 46 as the most potent compound from this series significantly induced the expression of proinflammatory cytokine IL8 from peripheral blood mononuclear cells. A systematic structure -activity relationship study of TLR8 negative modulators was further performed. Two synthetic pathways were developed for the preparation of a focused library of analogs based on the hit compound with pyrimidine scaffold, which were modified in three different positions. We confirmed the imperative presence of 6-trifluoromethyl group and other two substituents on positions 2 and 4 of the main 4-(furan-2-yl)-6-(trifluoromethyl)pyrimidine scaffold of our TLR8 modulators. Furthermore, para hydroxy or hydroxymethyl groups on benzylamine substituent at position 2 were found to be essential for potent modulation of TLR8. Several of the prepared compounds exhibited concentration-dependent inhibition of TLR8-mediated signaling in HEK293 cells in low micromolar range. The most perspective compounds 4-(furan-2-yl)-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine (15a) and (4%(((4%(furan-2-yl)-6-(trifluoromethyl)pyrimidin-2-yl)amino)methyl)phenyl)methanol (53) represent promising chemical probes for further optimization to a lead compound with potent immunomodulatory properties. During our research work on TLR8 we also performed computer-aided design of novel modulators. The potency of purchased hit compounds was evaluated on TLR8, with selectivity over TLR7 also being determined. A dual TLR7 and TLR8 agonist 3%(1-hydroxyhexan-2-yl)-5-methylquinazolin-4(3H)-one (T91) was discovered and its ability to induce the expression of proinflammatory cytokines on peripheral blood mononuclear cells was also assessed. The compound T91 significantly induced the expression of IL8, thus confirming its immunomodulatory activity. Research work was concluded with the optimization of conditions for Suzuki coupling, which is one of the most common reactions for the formation of C-C bonds and was used for the synthesis of some TLR8 negative modulators. We focused on the coupling between different 2,4-dichloropyrimidines and aryl- or heteroaryl boronic acids. A thorough screening of reaction conditions (e.g., solvent, catalyst, temperature, and time) and microwave irradiation led to a very efficient and straightforward synthetic procedure for the preparation of C4-substituted pyrimidines. Due to short reaction time (15 min) and extremely low catalyst loading (0,5 mol %) of vii tetrakis(triphenylphosphine)palladium (0), the established procedure could be used for quick and low-cost regioselective preparation of substituted pyrimidine rings. During our research work we successfully designed and synthesized low-molecular IDO1 inhibitors, as well as TLR7 and TLR8 modulators with low micromolar potency. For the preparation of substituted pyrimidines we developed a method for Suzuki coupling using a microwave reactor. Several TLR7 and TLR8 modulators induced the expression of proinflammatory cytokines. Prepared series of compounds crucially contributed to the development of lead compounds with immunomodulatory properties.
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