Discovery of new pyridine-quinoline hybrids as competitive and non-competitive PIM-1 kinase inhibitors with apoptosis induction and caspase 3/7 activation capabilities

Figures & data

Figure 1. A schematic representation of pyridone derivative I complexed with PIM-1 kinase. Dashed lines indicated hydrogen bonds.

Figure 2. Quinoline and quinolone having PIM-1 kinase inhibitory activity.

Figure 3. Design of pyridine-quinoline hybrids.

Scheme 1. Synthesis of 2-pyridone/2-thioxopyridine – quinoline hybrids. (a) Ac₂O; (b) POCI₃/DMF; (c) morpholine or piperdiine/ K₂CO₃/DMF; (d) 3-hydroxyacetophenone / ammonium acetate/ CNCH₂COOC₂H₅/ ethanol, reflux; (e) 3-hydroxyacetophenone /ammonium acetate / CNCH₂CSNH₂/ ethanol, reflux.

Scheme 2. Synthesis of 2-O-substitutedpyridine – quinoline hybrids. (a) Ac₂O, reflux; (b) BrCH₂COOC₂H₅/K₂CO₃, reflux; (c) NaHCO₃ / reflux; (d) K₂CO₃, reflux; (e) NaHCO₃/ reflux.

Scheme 3. Synthesis of 2-pyridone/2-thiopyridine/2-aminopyridine – quinolone hybrids. (a) CH₃COOH, reflux; (b) 3-hydroxyacetophenone / ammonium acetate/ CNCH₂COOC₂H₅/ ethanol, reflux; (c) 3-hydroxyacetophenone /ammonium acetate / CNCH₂CSNH₂/ ethanol, reflux; (d) 3-hydroxyacetophenone /ammonium acetate / CNCH₂CN/ ethanol, reflux.

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

Figure 5. In vitro anticancer activity, IC₅₀ (μM), of the tested compounds against Caco-2.

Figure 6. In vitro anticancer activity, IC₅₀ (μM), of the tested compounds against HepG-2.

Figure 7. In vitro anticancer activity, IC₅₀ (μM), of the tested compounds against NFS-60.

Figure 8. In vitro anticancer activity, IC₅₀ (μM), of the tested compounds against PC-3.

Figure 9. Morphological alterations of the most active compounds-treated cancer cells lines in comparison with the untreated cancer cells.

Figure 10. Flow charts of Annexin-PI analysis of 5b, 5c, and 6e – treated cancer cell lines in comparison with the untreated cancer cells.

Figure 11. Flow charts of Annexin-PI analysis of 13a, 13c, and 14a – treated cancer cell lines in comparison with the untreated cancer cells.

Figure 12. Lineweaver–Burk double-reciprocal plot for PIM kinase inhibition by 5c, 5b, and 6e in comparison with quercetin as reference inhibitor.

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
Untreated	0.169 ± 0.02	0.005 ± 0.005	0.505 ± 0.015	0.495 ± 0.005
5b	58.9 ± 0.88	44.045 ± 0.185	50.7 ± 0.58	54.2 ± 0.47
5c	54.14 ± 0.42	33.89 ± 0.175	47.26 ± 0.92	51.53 ± 0.09
6e	69.02 ± 0.18	67.715 ± 0.1.15	68.81 ± 0.49	68.385 ± 0.54
13a	70.59 ± 1.07	58.28 ± 0.26	59.71 ± 0.82	59.23 ± 0.04
13c	66.83 ± 1.32	51.17 ± 0.33	57.91 ± 0.34	58.83 ± 0.36
14a	42.155 ± 0.16	42.17 ± 0.82	56.545 ± 0.98	42.005 ± 0.32
Dox	39.265 ± 0.81	36.08 ± 0.23	34.59 ± 0.21	37.89 ± 0.28

Note: All values were expressed as mean ± SEM.

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

Code	Fold increase in caspase activity
5b	2.60 ± 0.031
5c	2.21 ± 0.04
6e	3.54 ± 0.24
13a	3.39 ± 0.03
13c	2.90 ± 0.07
14a	1.86 ± 0.11
Dox	1.61 ± 0.088

Note: All values were expressed as mean ± SEM.

Table 3. In-vitro PIM-1 and PIM-2 kinase inhibition data of the most active compounds.

Code	PIM-1 IC50 (µM)	PIM-2 IC50 (µM)
5b	1.31	0.67
5c	1	1.66
6e	1.10	1.49
13a	1.27	–
13c	7.78	–
14a	0.385	–
Quercetin	1.17	0.61

Figure 13. Lineweaver–Burk double-reciprocal plot for PIM kinase inhibition by 13a and 14a in comparison with quercetin as reference inhibitor.

Figure 14. (A) Overlay of the X-ray co-crystal (simon sticks) on its docked pose (cyan sticks) in the binding site of PIM-1 kinase (PDB ID: 2OBJ). (B) Overlay of the docking poses of 5b, 5c, and 6e as green, cyan, and magenta sticks, respectively, in the binding site of PIM-1 kinase. (C) Interaction pattern of 5c with PIM-1 residues in 2D depictions. Polar and non-polar regions of the binding site were presented by red and green coloured molecular surface, respectively. Dashed lines indicate favourable interactions. Non-polar hydrogen atoms were omitted for clarity.

Table 4. LE and LLE values for the most active anticancer compounds against the four cancer cell lines (HepG-2, Caco-2, PC-3, NFS-60).

Code	HepG-2		Caco-2		PC-3		NFS-60
	LE	LLE	LE	LLE	LE	LLE	LE	LLE
5b	0.34	4.22	0.30	3.28	0.31	3.52	0.33	3.97
5c	0.33	4.68	0.30	4.00	0.30	4.12	0.31	4.30
6e	0.32	3.07	0.30	2.73	0.30	2.53	0.29	2.43
13a	0.44	6.29	0.39	5.31	0.39	5.37	0.41	5.71
13c	0.40	6.06	0.35	5.11	0.36	5.29	0.38	5.69
14a	0.40	5.23	0.40	5.18	0.39	5.10	0.39	4.96

Figure 15. (A) Overlay of the docking poses of 13a, 13c, and 14a as magenta, green, and cyan sticks, respectively, in the binding site of PIM-1 kinase. (B) Interaction pattern of 13a, 13c, and 14a with PIM-1 residues in 2D depictions. Polar and non-polar regions of the binding site were presented by red and green coloured molecular surface, respectively. Dashed lines indicate favourable interactions. Non-polar hydrogen atoms were omitted for clarity.

Table 5. The docking score distribution of 5b, 5c, 6e, 13a, 13c, 14a and Quercetin against PIM-1 and PIM-2 kinases.

Code	PIM-1	PIM-2
5b	−9.43^a	−6.91
5c	−9.43	−5.04
6e	−9.85	−5.2
13a	−12.73	ND^b
13c	−11.37	ND
14a	−13.81	ND
Quercetin	−10.33	−11.92

^aThe docking score of FRED.

^bND stands for not determined.

Figure 16. (A) Overlay of the X-ray co-crystal (grey sticks) on its docked pose (cyan sticks) in the binding site of PIM-2 kinase (PDB ID: 4X7Q). (B) Overlay of the docking pose of 5b, 5c, and 6e as green, cyan, and magenta sticks, respectively, in the binding site of PIM-2 kinase. (C) Interaction pattern of 5b with PIM-2 residues in 2D depictions. Polar and non-polar regions of the binding site were presented by red and green coloured molecular surface, respectively. Dashed lines indicate favourable interactions. Non-polar hydrogen atoms were omitted for clarity.

Table 6. In-silico predictions of the pharmacokinetics and drug-likeness properties for 5b, 5c, 6e, 13a, 13c and 14a.

Code	MW	GIA	BBB	LogP	Lipinski #violations	Veber #violations	PAINS #alerts
5b	458.9	High	No	3.57	0	0	0
5c	454.5	High	No	2.91	0	0	0
6e	468.6	Low	No	4.73	0	0	0
13a	355.4	High	No	2.01	0	0	0
13c	385.4	High	No	1.98	0	0	0
14a	371.4	Low	No	3.41	0	0	0
Code	Pgp substrate	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor
5b	Yes	No	Yes	Yes	No	No
5c	Yes	No	No	Yes	No	Yes
6e	Yes	No	Yes	Yes	No	No
13a	No	No	No	No	No	No
13c	No	No	No	Yes	No	No
14a	No	Yes	No	Yes	Yes	Yes

Notes: GIA: human gastrointestinal absorption; BBB: blood-brain barrier permeation; P-gp: permeability glycoprotein; CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4 are the five major isoforms of cytochromes P450 (CYP). LogP is calculated as XLOGP3 descriptor⁴¹. Lipinski #violations counts the number of violations of Lipinski rule summarised as: lipophilicity (logP) ≤ 5, molecular weight ≤ 500, number of hydrogen bond donors ≤ 5 and number of hydrogen bond acceptors ≤ 10. Veber #violations counts the number of violations of Veber rule summarised as: NRB ≤ 10 and TPSA ≤ 140 Ų. PAINS #alerts counts the number of pan-assay interference compounds/substructures. All calculations were performed using SwissADME³⁶.

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