Discovery of 3,6-disubstituted pyridazines as a novel class of anticancer agents targeting cyclin-dependent kinase 2: synthesis, biological evaluation and in silico insights

Figures & data

Figure 1. Chemical structure of the clinically used (palbociclib, abemaciclib and ribociclib) or currently in clinical trials (roniciclib) CDK inhibitors.

Figure 2. Chemical structure for some 3,6‐disubstituted pyridazine derivatives reported as efficient anticancer small molecules.

Scheme 1. Preparation of ester 5; reagents and conditions: (i) Acetone, DMF, L-proline, r.t., 48 h; (ii) NH₂NH₂, AcOH, reflux, 6 h; (iii) K₂Cr₂O₇, Conc. H₂SO₄, 0 °C to r.t. overnight; (iv) EtOH, H₂SO₄, reflux, 4 h.

Scheme 2. Preparation of intermediateds 9a–e; reagents and conditions: (i) POCl₃, 100 °C, 5 h; (ii) LiOH, THF, H₂O, r.t., 1 h; (iii) SOCl₂, DMF (catalytic), 1,2-dichloroethane, reflux, 3 h; (iv) Methylene chloride, Et₃N, r.t., 4 h.

Scheme 3. Preparation of target pyridazines 11a–r; reagents and conditions: (i) 1,4-Dioxane, Hünig’s base, reflux, 6 h; (ii) Morpholine, 1,4-dioxane, Hünig’s base, reflux, 12 h; (iii) Primary amines 8a–e, EtOH, piperdine, reflux, 6 h.

Table 1. Anti-proliferative activities for the newly prepared pyridazines (11a–r) towards T-47D, MDA-MB-231 and SKOV-3 cell lines.

^aIC₅₀s are displayed as mean ± SD for three separate experiments.

^bNA: Pyridazines with IC₅₀ value more than 100 µM.

Table 2. Cytotoxic impact for pyridazines 11l and 11m against non-tumorigenic MCF-10A human breast cell line, as well as mean tumour selectivity index (S. I.) (MCF-10A/T-47D and MDA-MB-231).

Comp.	IC50 (µM)			Mean tumour selectivity
	MCF-10A	T-47D	MDA-MB-231
11l	19.63 ± 0.38	1.57 ± 0.05	1.30 ± 0.04	13.7
11m	11.44 ± 0.22	0.43 ± 0.01	0.99 ± 0.03	16.1

Figure 3. Influence of pyridazines 11l and 11 m on the distribution of cell cycle phases in breast cancer T-47D (A) and MDA-MB-231 (B) cells. Asterisk indicates a significant difference from control at p < 0.05.

Figure 4. Percentage of apoptotic and necrotic cells in T-47D (A) and MDA-MB-231 (B), with respect to untreated control cells, upon treatment with pyridazines 11l and 11m.

Figure 5. Influence of pyridazines 11l and 11m on the percentage of Annexin V⁺ apoptotic cells in T-47D cells.

Figure 6. Influence of pyridazines 11l and 11m on the percentage of Annexin V⁺ apoptotic cells in MDA-MB-231 cells.

Figure 7. (A) 2D, and (B) 3D diagram for pyridazine 11e demonstrating its interactions within the CDK2 active site.

Figure 8. (A) 2D, and (B) 3D diagram for pyridazine 11h demonstrating its interactions within the CDK2 active site.

Figure 9. (A) 2D, and (B) 3D diagram for pyridazine 11l demonstrating its interactions within the CDK2 active site.

Figure 10. (A) 2D, and (B) 3D diagram for pyridazine 11m demonstrating its interactions within the CDK2 active site.

Table 3. Docking energy scores (S) for pyridazines 11a–r and the co-crystallized ligand Roniciclib (in in kcal/mol).

Cpd.	Energy score (S) Kcal\mole	Cpd.	Energy score (S) Kcal\mole
11a	−8.1	11j	−8.3
11b	−8.0	11k	−8.7
11c	−8.2	11l	−9.5
11d	−7.9	11m	−10.2
11e	−9.0	11n	−8.6
11f	−8.3	11o	−8.0
11g	−7.9	11p	−8.7
11h	−10.1	11q	−8.2
11i	−8.5	11r	−8.4
Roniciclib	−9.2

Bold values are for pyridazines with best docking energy scores.

Table 4. The different bonding types and their distances (in Å) for pyridazines 11e, 11h, 11l, and 11m within the CDK2 active site.

Cpd	Bond type (Involved A. A.)	Distance	Cpd	Bond type (Involved A. A.)	Distance
11e	Hydrogen bond (Lys33)	2.69	11l	Hydrogen bond (Leu83)	2.61
	Hydrogen bond (Lys33)	2.81		Halogen interaction (Leu83)	3.39
	Halogen interaction (Asp145)	3.31		Halogen interaction (His84)	3.14
	Halogen interaction (Asp145)	3.35		Halogen interaction (His84)	3.02
	Halogen interaction (Gly13)	2.39		Pi-Alkyl (Ile10)	4.35
	Halogen interaction (Gly16)	3.62		Non-classical hydrogen bond (Ile10)	3.60
	Halogen interaction (Glu12)	3.26		Ionic bond (Asp86)	3.37
	Pi-Anion (Asp145)	3.65		Pi-Alkyl (Leu134)	5.13
	Pi-Alkyl (Leu134)	4.57		Pi-Alkyl (Phe80)	4.53
	Alkyl-Alkyl (Leu134)	4.64		Alkyl-Alkyl (Val18)	4.92
	Alkyl-Alkyl (Val18)	4.99		Alkyl-Alkyl (Val64)	5.26
	Alkyl-Alkyl (Val18)	4.59		Alkyl-Alkyl (Ala144)	4.08
	Pi-Alkyl (Ile10)	5.02		Alkyl-Alkyl (Lys33)	5.37
	Alkyl-Alkyl (Lys89)	5.37		Non-classical hydrogen bond (Gln85)	2.25
	Alkyl-Alkyl (Lys89)	5.48
	Alkyl-Alkyl (Lys89)	5.49
11h	Hydrogen bond (Asp86)	2.13	11m	Hydrogen bond (Leu83)	2.02
	Hydrogen bond (Leu83)	2.59		Alkyl-Alkyl (Lys89)	5.34
	Halogen interaction (Asp145)	3.15		Halogen interaction (Ile10)	2.94
	Halogen interaction (Asp145)	3.19		Halogen interaction (Ile10)	3.10
	Halogen interaction (Asn132)	3.14		Halogen interaction (Asp86)	3.00
	Halogen interaction (Leu83)	2.86		Halogen interaction (Asp86)	3.27
	Halogen interaction (Leu83)	3.17		Halogen interaction (Gln131)	2.80
	Pi-Alkyl (Ile10)	4.30		Pi-Alkyl (Ile10)	3.94
	Alkyl-Alkyl (Ile10)	4.71		Alkyl-Alkyl (Ile10)	5.42
	Pi-Alkyl (Leu134)	5.04		Non-classical hydrogen bond (Ile10)	3.48
	Alkyl-Alkyl (Leu134)	4.37		Alkyl-Alkyl (Lys33)	5.28
	Pi-Alkyl (Phe82)	5.02		Non-classical hydrogen bond (Phe82)	2.66
	Pi-Alkyl (Val18)	4.76		Non-classical hydrogen bond (Glu81)	3.74
	Alkyl-Alkyl (Val18)	4.43		Pi-Sigma interaction (Phe80)	3.50
	Pi-Alkyl (Ala144)	3.97		Alkyl-Alkyl (Val18)	5.02
	Pi-Pi interaction (Phe80)	5.14		Alkyl-Alkyl (Val64)	5.03
	Non-classical hydrogen bond (Lys33)	2.79		Alkyl-Alkyl (Ala144)	4.29
				Alkyl-Alkyl (Ala31)	5.00
				Pi-Alkyl interaction (Leu134)	4.96

Table 5. IC₅₀ values for the CDK2 inhibitory action of pyridazines 11e, 11h, 11l, and 11m.

Comp.	IC50 (nM)
	CDK2
11e	151 ± 6.16
11h	43.8 ± 1.79
11l	55.6 ± 2.27
11m	20.1 ± 0.82
Staurosporine	12.4 ± 0.51

Figure 11. Boiled-Egg chart for pyridazines 11e, 11h, 11l, and 11m.

Figure 12. The oral bioavailability radar chart for pyridazines 11e (A), 11h (B), 11l (C) and 11m (D), produced by swissADME online web tool.

Table 6. The in silico predicted ADME for pyridazines 11e, 11h, 11l, and 11m.

Cpd.	BBB	GIA	P-gP substrate	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor
11e	No	Low	Yes	No	No	No	Yes	Yes
11h	No	High	No	No	Yes	No	No	Yes
11l	Yes	High	Yes	Yes	No	No	Yes	No
11m	Yes	High	Yes	Yes	No	No	No	No

GIA: gastrointestinal absorption; BBB: Blood Brain Barrier; PgP: P-glyco protein transporter.

Table 7. Compliance with Druglikeness rules (Lipinski, Veber, Egan, Ghose and Muegge), as well as PAINS and Brenk filters for target pyridazines 11e, 11h, 11l, and 11m.

Cpd.	Lipinski violations	Veber violations	Muegge violations	Egan violations	Ghose violations	PAINS alerts	Brenk alerts
11e	Yes	Yes	No	No	No	0	0
11h	Yes	Yes	Yes	No	No	0	0
11l	Yes	Yes	Yes	Yes	Yes	0	0
11m	Yes	Yes	Yes	Yes	Yes	0	0

PAINS: Pan Assay Interference Structures.