Synthesis, Molecular Docking, In Vitro and In Vivo Studies of Novel Dimorpholinoquinazoline-Based Potential Inhibitors of PI3K/Akt/mTOR Pathway

Abstract

A (series) range of potential dimorpholinoquinazoline-based inhibitors of the PI3K/Akt/mTOR cascade was synthesized. Several compounds exhibited cytotoxicity towards a panel of cancer cell lines in the low and sub-micromolar range. Compound 7c with the highest activity and moderate selectivity towards MCF7 cells which express the mutant type of PI3K was also tested for the ability to inhibit PI3K-(signaling pathway) downstream effectors and associated proteins. Compound 7c inhibited the phosphorylation of Akt, mTOR, and S6K at 125-250 nM. It also triggered PARP1 cleavage, ROS production, and cell death via several mechanisms. Inhibition of PI3Kα was observed at a concentration of 7b 50 µM and of 7c 500 µM and higher, that can indicate minority PI3Kα as a target among other kinases in the titled cascade for 7c. In vivo studies demonstrated an inhibition of tumor growth in the colorectal tumor model. According to the docking studies, the replacement of the triazine core in gedatolisib (8) by a quinazoline fragment, and incorporation of a (hetero)aromatic unit connected with the carbamide group via a flexible spacer, can result in more selective inhibition of the PI3Kα isoform.

Keywords: PI3K/Akt/mTOR inhibitor; S6K; cancer; dimorpholinoquinazoline; kinase inhibition activity.

Figure 1

FDA-approved inhibitors of PI3K/Akt/mTOR pathway [2,30,31].

Figure 2

General structure of the target compounds.

Scheme 1

Synthesis of 7-aryl-2,4-bis(morpholino)quinazoline 6. Reagents and conditions: a-SnCl₂*2H₂O, H₂O-HCl (1:1), 90 °C; b-urea, 150 °C, no solvent; c-POCl₃, Et₃N, 95 °C; d-morpholine, DCM, 0 °C → rt; e-N-Boc-protected p-aminophenylpinacolborane, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane-H₂O, 100 °C; f-TFA, DCM, rt.

Scheme 2

Synthesis of a small library of quinazoline-based asymmetric carbamides.

Figure 3

Characteristics of cytotoxicity. (a) Average IC₅₀ for the compounds 7a–7k. The most active compounds are shown in blue. (b) Synergistic effect of 1 mM metformin (MF) and 4 nM compound 7c on the growth of MCF7 cells. (c) Effect of 300 µM MF on MCF7 proliferation. (d) IC₅₀ for 7c and 7c + MF 300 µM for Eahy926, Colon26, and MCF7.

Figure 4

Compound 7c suppresses the PI3K/Akt/mTOR signaling pathway. (a–i) MCF7 cells were treated with different concentrations of 7c or 500 nM wortmannin (W) for 24 h. Cell lysates were immunoblotted with antibodies against PI3K/Akt/mTOR-related total or phosphorylated (p) proteins (a–f) and PARP1/cleaved PARP1 (g,h). (i) Control α-tubulin. (j,k) The ratios of p-proteins to total ones for compound 7c (circles) or wortmannin (triangles) (j) and cleaved PARP1 to full PARP1 (k) as measured by ImageJ program from two independent experiments.

Figure 5

Inhibition of PI3Kα enzymatic activity with compounds 7b (blue) or 7c (red).

Figure 6

Mechanisms of cell death induced by the compounds 7. (a,b) Representative data on the cell cycle of SkBr-3 cells after 24 h incubation with 10 μM of 7b (green), 7c (orange), and 7j (red). Control cells are shown in blue color. (c–e) Apoptosis of SkBr-3 cells: (c) control cells, (d) cells incubated with 7c for 24 h cells. (e) Apoptosis induction level for 7b,c,j at 5 μM (gray) and 10 μM (violet). Apoptosis level in control cells is shown with a black line. Statistical significance is shown by asterisks.

Figure 7

Reactive oxygen species production in the presence of compounds 7. (a,b) SkBr-3 (a) and MCF7 (b) ROS production expressed as the ratios of intracellular ROS production by the stimulated cells to the control ones. (c–f) Microphotographs of SkBr-3 cells in the presence of compound 7c. Black arrows show dying cells, blue arrows show unable to duplicate DNA cells.

Figure 8

Biological effects induced by compound 7c in vitro. Confocal microphotographs of control (a,e) and compound 7c treated (5 μM) (b,c,f,g) for 24 h MCF7 cells stained with Hoechst33342 (a–f), LysoTrackRed (e,f), and DCF (g). Release of nuclear material into cytoplasm (c,d, red arrows), disassembling of chromosomes (green arrow), large vesicles in the cytoplasm of the cells (white arrows), depolarization of lysosomes (f), and ROS production (g) show different types of cell death under compounds 7 influence. Scale bar is 10–15 μm.

Figure 9

Confocal microphotographs of control (a) and the compound 7c treated (5 μM) (b–d) for 24 h MCF7 cells stained with Hoechst33342 (blue) and WGA-AlexaFluor555 (red). Enlargement of organelles (green arrows) and autophagolysosome formation (white arrows) are found. Scale bar is 10–23 μm.

Figure 10

Effect of compound 7c on tumor growth. (a,b) BALB/c mice were s.c. inoculated with colorectal Colon-26 cells. Tumor growth in control and at early (a) or late (b) treatment with compound 7c. Dates of the treatment with 7c are shown with arrows, significant differences—with asterisks (p < 0.05). Data are shown as average ± SEM.

Figure 11

Structure of the molecules used for docking studies.

Figure 12

Docking of the compounds 7b (a,b) and 7c (c,d) into the catalytic site of PI3Kα (a,c) and PI3Kγ (b,d). The gray dotted line is hydrophobic interactions, the blue solid line is hydrogen bonds, the light green dotted line is parallel π-π stacking, dark green dashed—normal π-π stacking. yellow dotted line—salt bridge. Pictures 5E and 5F represent a superposition of 7b, 7c and Gedatolisib 8 in the catalytic site of PI3Kα (e) and PI3Kγ (f). Gedatolisib (8) is colored in blue, 7b—red and 7c—green. Visualization of the docking results (protein-ligand complexes) was carried out using the PLIP service. The superposition of ligands was visualized via UCSF Chimera program.