Design, synthesis and biological evaluation of novel morpholinopyrimidine-5-carbonitrile derivatives as dual PI3K/mTOR inhibitors

Abstract

In this study, novel morpholinopyrimidine-5-carbonitriles were designed and synthesized as dual PI3K/mTOR inhibitors and apoptosis inducers. The integration of a heterocycle at position 2, with or without spacers, of the new key intermediate 2-hydrazinyl-6-morpholinopyrimidine-5-carbonitrile (5) yielded compounds 6-10, 11a-c and 12a-h. The National Cancer Institute (USA) tested all compounds for antiproliferative activity. Schiff bases, 12a-h analogs, were the most active ones. The most promising compounds 12b and 12d exhibited excellent antitumor activity against the leukemia SR cell line, which is the most sensitive cell line, with IC₅₀ 0.10 ± 0.01 and 0.09 ± 0.01 μM, respectively, along with significant effects on PI3Kα/PI3Kβ/PI3Kδ with IC₅₀ values of 0.17 ± 0.01, 0.13 ± 0.01 and 0.76 ± 0.04 μM, respectively, for 12b and 1.27 ± 0.07, 3.20 ± 0.16 and 1.98 ± 0.11, respectively, for 12d compared to LY294002. Compared to Afinitor, these compounds inhibited mTOR with IC₅₀ values of 0.83 ± 0.05 and 2.85 ± 0.17 μM, respectively. Annexin-V and propidium iodide (PI) double labeling showed that compounds 12b and 12d promote cytotoxic leukemia SR apoptosis. Compounds 12b and 12d also caused a G2/M cell cycle arrest in the leukaemia SR cell line. The findings of this study indicate that the highest effect was observed for 12b, which was supported by western blot and docking analysis.

Fig. 1. Pyrimidines and morpholinopyrimidines as anticancer agents.

Fig. 2. Dual PI3K/mTOR inhibitors.

Scheme 1. Synthesis of compounds 4–8.

Scheme 2. Synthesis of compounds 9–11a–c and 12a–h.

Fig. 3. Postulated mechanism for the synthesis of compound 6.

Fig. 4. Effect of 12b and 12d on % DNA content in leukemia SR cells.

Fig. 5. Effect of 12b and 12d on apoptosis and necrosis in leukemia SR cells.

Fig. 6. Effects of substance 12b on PI3K and mTOR in leukemia SR cells. The IC₅₀ value of compound 12b was applied to leukemia SR cells before they underwent a western blot analysis.

Fig. 7. (A and B) Binding mode of native ligand X6K with PI3K-α (PDB ID: 4L23): 3D and 2D. (C and D) Binding mode of the target compound 12b with PI3K-α (PDB ID: 4L23): 3D and 2D.

Fig. 8. (A and B) Binding mode of native ligand X6K with mTOR (PDB code 4JT6): 3D and 2D. (C and D) Binding mode of the target compound 12b with mTOR (PDB code 4JT6): 3D and 2D.