Indolamine 2,3-dioxygenase 1 (IDO1) is a heme-containing cytosolic enzyme that catalyzes the first step of tryptophan metabolism, which leads to the formation of kynurenine as the major toxic metabolite. Lack of tryptophan and increased concentration of kynurenine in the tumor microenvironment affects three signaling pathways in the cell, leading to the immune escape of tumor cells and disease progression. IDO1-induced neovascularization also contributes to disease progression. For these reasons, IDO1 represents an interesting potential target in cancer immunotherapy. There are currently quite a few IDO1 inhibitors in clinical trials. So far the findings show a synergistic effect in combination with established treatments for cancer. The aim of our thesis was to synthesize and biochemically evaluate novel IDO1 inhibitors on the basis of 3-phenylisoxazolo[5,4-d]pyrimidin-4(5H)-one in order to obtain at least one inhibitor in the micromolar range. Furthermore, we also wanted to optimize the yields and purity of intermediates of the most problematic step of the general synthetic pathway, which is the synthesis of isoxazole-4-carboxamide. The synthesis of new IDO1 inhibitors was based on the multi-step synthetic route, which was used to prepare nine final compounds. Their identity and purity was confirmed by various analytical techniques. Biochemical evaluation based on fluorescence measurement was used to determine their inhibitory activity on IDO1. Three compounds (31, 32 and 33) had their IC50 values in low micromolar range. The most potent inhibitor of IDO1 was 2-(3-(3,4-difluorophenyl)-4-oxoisoxazolo[5,4-d]pyrimidin-5(4H)-yl)-N-(4-nitrophenyl)acetamide (31) with an IC50 value of 68,48 μM. Based on the molecular docking of compound 31 into the enzyme's active site we predicted potential interactions of 31 with amino acid residues. According to results obtained for 31 it represents an important starting point for further research and development of more potent new IDO1 inhibitors as potential anticancer agents.