Novel furo[2,3- d]pyrimidine derivatives as PI3K/AKT dual inhibitors: design, synthesis, biological evaluation, and molecular dynamics simulation

Abstract

The current study aimed to synthesize a series of innovative improved anticancer chemical entities by combining the unique advantages of 1,3,4-thiadiazole as an established anticancer pharmacophore with the furopyrimidine scaffold which is a key component of many reported cytotoxic agents. Sixteen furopyrimidine derivatives were designed and evaluated by several biological tests including antiproliferative activity against 60 human cancer cell lines, measurement of GI₅₀, TGI, and LC₅₀ values, MTT and selectivity index (SI) calculation, in vitro enzymatic PI3Kα/β and AKT inhibitory assay, cell cycle analysis and apoptosis evaluation. The results indicated that the designed compound 10b revealed potent anticancer activity with a mean GI of 108.32%, remarkable anticancer activity with GI₅₀ values ranging from 0.91 to 16.7 μM and strong cytostatic action (TGI range: 2.32-15.0 μM) against 38 cancer cell lines. It also exhibited a potent and selective antiproliferative activity against the breast cancer HS 578T cell line (GI₅₀ = 1.51 μM and TGI = 4.96 μM). Furthermore, compound 10b demonstrated the highest inhibitory activity against PI3Kα/β and AKT enzymes with IC₅₀ = 0.175 ± 0.007, 0.071 ± 0.003 and 0.411 ± 0.02 μM, respectively. Additionally, it could strongly induce cell cycle arrest in breast cancer HS 578T cells at the G0-G1 phase and trigger apoptosis. Molecular docking and dynamics were also performed in this study which revealed that compound 10b provided an improved binding pattern with the key amino acids in the PI3K and AKT-1 binding sites. According to the findings, the designed compound 10b has potent antiproliferative and apoptotic activities with a wide therapeutic index particularly against breast cancer.

Fig. 1. Chemical structures of the reported PI3K and AKT inhibitors.

Fig. 2. A. Simple diagram describing the pharmacophore for PI3K p110α inhibitors. B. A diagram describing the possible binding interactions for AKT1 inhibitors.

Fig. 3. 1,3,4-Thiadiazole-based derivatives (III and IV) and furopyrimidine derivatives (V–VII) with anticancer activity.

Fig. 4. The design of the target compounds based on PI3K inhibitor PI-103.

Scheme 1. Reagents and conditions: i) formic acid/acetic anhydride, reflux 48 h, 130 °C; ii) absolute ethanol, hydrochloric acid, reflux 2 h, 90 °C; iii) absolute ethanol, dps of acetic acid, reflux 3 h, 90 °C; iv) absolute ethanol, sodium acetate, reflux 8 h, 90 °C. Synthesis of furopyrimidine derivatives 2–5, starting from 4-acetyl-2-amino-5-methylfuran-3-carbonitrile (1).

Scheme 2. Reagents and conditions: i) absolute ethanol, reflux 18 h, 90 °C; ii) absolute ethanol, triethylamine, reflux 6 h, 90 °C. Synthesis of furopyrimidine derivatives 6a–c, 8a–d, and 10a–f.

Fig. 5. Graph illustrating the effect of compound 10b on mTOR phosphorylation in breast cancer HS 578T cells, compared to staurosporine and untreated control cells. Statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparisons test. Differences were considered significant at ****p < 0.0001.

Fig. 6. Compound 10b induces cell cycle arrest in breast cancer HS 578T cells. The graphical illustration shows the cell cycle distribution analysis in control and treated cells. Statistical significance was assessed using two-way ANOVA followed by Šídák's multiple comparisons test. Differences from the control were considered significant at probability levels *p < 0.05, **p < 0.002, and ***p < 0.0008.

Fig. 7. Influence of compound 10b on the percentage of Annexin V-positive staining in HS 578T cells. The percentages of apoptotic and necrotic cells in both control and treated groups are depicted graphically.

Fig. 8. Caspase-9 activity in breast cancer HS 578T cells treated with compound 10b compared to staurosporine. Statistical significance was assessed using one-way ANOVA followed by Tukey's multiple comparisons test. Differences from controls were considered significant at ****p < 0.0001.

Fig. 9. 3D representations of the superimposition of the co-crystallized ligands (red) and the docking pose (green) of co-crystalized ligands (PI-103 and IX) interacting with the key amino acids in the PI3K (A) and AKT-1 (B) binding sites, respectively.

Fig. 10. (A) 2D diagram and (B) 3D representation of compound 10b in the PI3K binding site.

Fig. 11. (A) 2D diagram and (B) 3D representation of compound 10b in the AKT-1 binding site.

Fig. 12. Molecular dynamics simulation analysis of compound 10b binding to human AKT1 and PI3K (PDB IDs: 3O96 and 4L23; respectively). (A) The most populated binding pose of 10b in the AKT1 active site. (B) The most populated binding pose of 10b in the PI3K active site. (C) RMSD profiles over 200 ns simulation show higher stability of 10b within AKT1 (brick red) compared to PI3K (blue).

Fig. 13. The forecasted Boiled-egg plot for compounds 10a–d, as generated by the SwissADME online tool, illustrates their projected pharmacokinetic properties.

Fig. 14. The bioavailability radar chart for compounds 10a–d displays their properties, including saturation (INSATU), flexibility (FLEX), lipophilicity (LIPO), size (SIZE), polarity (POLAR), and solubility (INSOLU). The pink region indicates the optimal range for oral bioavailability, while the red lines show the predicted values for these compounds.