Novel benzo chromene derivatives: design, synthesis, molecular docking, cell cycle arrest, and apoptosis induction in human acute myeloid leukemia HL-60 cells

Figures & data

Figure 1. Multi-targeting synthetic small molecules for treatment of AML advanced to clinical trials.

Figure 2. Rationalised design of the proposed new cell cycle arrest and apoptotic agents relying on the CDK-2 inhibitor and Bcl-2 inhibitor.

Scheme A. Synthesis of compounds 1a–c to 4.

Scheme B. Synthesis of compounds 5a–c to 8a–c.

Scheme C. Synthesis of compounds 9a–d to 11a,b

Figure 3. Mean % growth of compounds 2–8c.

Figure 4. Mean % growth of compounds 9a–11b.

Figure 5. Inhibitory activity of 5a and 6a compounds on CDK-2/cyclin A2. *Significant from Flavoperidol at p < 0.0001.

Table 1. CDK-2/cyclin A2 inhibitory activity results of 5a and 6a synthesised compounds.

Compound ID	CDK-2/ cyclin A2 IC50 (µM) ± SD
Flavoperidol	0.052 ± 0.002
5a	0.171 ± 0.008*
6a	0.301 ± 0.013*

*Significant from Flavoperidol at p ˂ 0.0001.

Figure 6. Compounds 5a- and 6a-induced apoptosis in HL-60 cells; effect of benzocoumarin on apoptosis. Cells were treated with compounds 5a and 6a (IC⁵⁰=0.24, 0.29 respectively) for 48 h. Apoptotic cells were quantified by flow cytometry after staining with annexin V-fluorescein isothiocyanate (V-FITC)/propidium iodide (PI) (mean± SD, n = 3).

Figure 7. Compounds 5a- and 6a-induced apoptosis is mediated by both the extrinsic pathway and activation of the mitochondrial intrinsic apoptotic pathway. (A) Western blot detection of intrinsic pathway of apoptosis-associated protein expression. Cells were treated with compounds 5a and 6a (IC⁵⁰=0.24, 0.29 respectively) for 48 h and then harvested. β-actin was used as an internal reference. (B) Western blot detection of extrinsic apoptotic pathway related proteins. Cells were treated with compounds 5a and 6a (IC⁵⁰=0.24, 0.29 respectively) for 48 h and then harvested. β-actin was used as an internal reference. Data are presented as mean ± SD of three independent experiments. *p < 0.05 as compared to the control.

Figure 8. Benzocoumarin-induced cell cycle arrest in HL-60 cells. (A) Effect of benzocoumarin on cell cycle (mean± SD, n = 3). Cells were treated benzocoumarin (IC⁵⁰=0.24, 0.29 respectively) for 48 h. The images display a representative experiment from three independent experiments. Cells were quantitatively measured by flow cytometry. ***p < 0.001 as compared to the control. (B) Western blot detection of cell-cycle-associated protein expression. Cells were treated with benzocoumarin (IC⁵⁰=0.24, 0.29 respectively) for 48 h. β-actin was used as an internal reference.

Figure 9. Molecular docking analysis of the compounds 5a and 6a in the BH3-binding groove of Bcl-2. Bcl-2 proteins are presented as gray cartoons of solid surfaces. (A) Crystal structure of Bcl-2 in complex with a Bax BH3 peptide represented by a yellow ribbon (PDB: 2XA0). (B) Validation re-docking of crystallised ligand (green) (PDB ID: 1XJ) overlay of re-docked (yellow) conformations of navitoclax in Bcl-2 (PDB: 4LVT). 3D Docking model of compounds 5a and 6a occupy the binding site of Bcl-2, respectively (C) and (E). Key residues at the binding pocket (D) 5a (F) 6a.

Figure 10. (A) Validation process for redocking WG1 into (PDB ID: 7KJS). 3D Docking model of compounds 5a and 6a fit into the ATP-binding site of CDK-2 kinase, respectively. (B) 3D-structure of compound 5a; (C) 3D structure of compound 6a. Co-crystallised ligand with green colour WG1 and the docked molecule with yellow colour. (D), (E), and (F) interaction poses of CDK2-PF-06873600, 5a, and 6a, respectively.

Figure 11. predicted mechanism of benzo[h]chromene.