Imparting aromaticity to 2-pyridone derivatives by O-alkylation resulted in new competitive and non-competitive PIM-1 kinase inhibitors with caspase-activated apoptosis

Figures & data

Figure 1. Previously discovered pyridone derivatives as PIM-1 kinase inhibitors.

Figure 2. Design of aromatic O-alkyl pyridine derivatives.

Scheme 1. Synthesis of O-alkylpyridine derivatives.

Figure 3. Proposed mechanism for formation of 3-aminopyrazolo[3,4-b]pyridine derivatives 6a–c.

Figure 4. IC₅₀ and EC₁₀₀ (μM) of the tested compounds against normal human cells (wi-38) after 72 h incubation.

Figure 5. In vitro anticancer activity, IC₅₀ (μM), of the tested compounds against human cancer cells after 72 h incubation.

Figure 6. Morphological alterations of the most active compounds (0.06 µM)-treated cancer cells lines in comparison with the untreated cancer cells after 72 h incubation.

Figure 7. Flowcharts of Annexin-PI analysis of 4c and 4f – treated cancer cell lines in comparison with the untreated cancer cells after 72 h incubation at concentrations equivalent to 0.013, 0.007, 0.005, and 0.006 µM, respectively.

Table 1. The total percentage of the apoptotic cell population in the most effective compounds-treated cancer cells lines.

Code	HepG-2	Caco-2	PC-3	NFS-60
The used IC50^a	0.013 µM	0.007 µM	0.005 µM	0.006 µM
Untreated	0.169 ± 0.02	0.005 ± 0.005	0.505 ± 0.015	0.495 ± 0.005
4c	46.78 ± 1.54	35.75 ± 4.25	29.26 ± 2.84	34.09 ± 2.59
4f	55.575 ± 0.6	55.9 ± 0.26	49.84 ± 1.5	50.055 ± 0.45
Dox	39.265 ± 0.81	36.08 ± 0.23	34.59 ± 0.21	37.89 ± 0.28

All values were expressed as mean ± SEM.

^a The apoptotic potential impact of the tested compounds was compared at the estimated lowest IC₅₀ value, at which 50% of cancer growth inhibition occurs, for each cancer cell lines.

Table 2. Relative fold increase in caspase activity by the most effective compounds relative to untreated HepG2 cancer cells.

Code	Fold increase in caspase activity
4c	1.92 ± 0.16
4f	2.59 ± 0.14
Dox	1.61 ± 0.088

All values were expressed as mean ± SEM.

Table 3. In vitro PIM-1 kinase inhibition data of the most active compounds.

Code	PIM-1 IC50 (µM)
4c	0.110 ± 0.0027
4f	0.095 ± 0.0023
Quercetin	1.17 ± 0.0294

All values were expressed as mean ± SEM.

Figure 8. Lineweaver–Burk double-reciprocal plot for PIM-1 kinase inhibition by 4c and 4f in comparison with quercetin as reference inhibitor in the presence of ATP.

Figure 9. Left: Co-crystallised ligand, ligand–enzyme interaction (2D). Right: Overlay of crystallised ligand (magenta) and docked ligand (yellow) with RMSD = 1.431 (3D) inside PIM-1 kinase active site.

Figure 10. 4c, ligand–enzyme interaction 2D (left) and 3D (right) inside PIM-1 kinase active site.

Figure 11. 4f, ligand–enzyme interaction 2D (left) and 3D (right) inside PIM-1 kinase active site.

Figure 12. Overlay of 4c (green), 4f (cyan), and ligand (magenta) inside PIM-1 kinase active site.

Table 4. Induced fit docking results of the active compounds inside PIM-1 kinase active site.

Code	Score (kcal/mol)	Hydrogen bonds interaction amino acids	Hydrophobic interaction amino acids	Polar interaction amino acids
Co-crystallised ligand (Figure 9)	−6.731	One H-bond between oxygen of C═O and Lys67 (key amino acid for inhibition of PIM-1 kinase).	Leu44, Phe49, Ala65, Ile104, Leu120, Val126, Leu174, Ile185	Gly45, Ser46, Glu89, Glu121, Arg122, Asp186 pi–H interaction between pyridone moiety and Val52
4c (Figure 10)	−8.18	One H-bond between nitrogen of CN and Lys67*.	Leu44, Phe49, Ala65, Ile104, Leu120, Val126, Ile185.	Gly45, Ser46, Gly47, Gly50, Ser51, Glu121, Arg122, Lys169, Glu171, Asn172, Asp128, Asp186 Three pi–H interactions: two pi–H interactions between pyridone moiety and Val52 and one pi–H interaction between phenolic moiety and Leu174.
4f (Figure 11)	−8.197	No H-bond.	Leu44, Phe49, Val52, Ala65, Ile104, Leu120, Val126, Phe130, Ile185.	Gly45, Ser46, Gly47, Gly50, Ser51, Lys67, Glu89, Arg122, Asp131, Glu171, Asn172, Asp128, Asp186 One pi–H interaction between phenolic moiety and Leu174.

*Amino acids interacted with the co-crystallised ligand were marketed in bold format and the key residue Lys67 was highlighted.

Table 5. LE and LLE values for the most active anticancer compounds against PIM-1 kinase enzyme.

Code	PIM-1 kinase
	LE	LLE
4c	0.264	2.418
4f	0.260	1.972