Design, synthesis, docking, and anticancer evaluations of new thiazolo[3,2-a] pyrimidines as topoisomerase II inhibitors

Figures & data

Figure 1. Basic features of some reported Topo II inhibitors.

Figure 2. Rationale for designing new Topo II inhibitors.

Scheme 1. The synthetic pathway for the preparation of the starting material 1 and thiazolo[3,2-a]pyrimidines 2 and 3a–d

Figure 3. Plausible mechanism for synthesis of compounds 3b–d.

Scheme 2. The synthetic approach for the three sets of amide derivatives 4a–c, 5, 6a–c and 7a,b.

Table 1. IC₅₀ values of the designed targets against MCF7, A549, and A498 cell lines.

Ser	Cytotoxicity IC50 (nM)^a
	Compound	MCF7± SD	A549± SD	A498± SD
1	1	103 ± 1.62	210 ± 3.61	115 ± 2.3
2	2	41 ± 0.72	23 ± 0.45	332 ± 7.4
3	3a	107 ± 1.88	93 ± 1.79	73 ± 1.63
4	3b	22 ± 0.62	24 ± 0.61	46 ± 1.35
5	3c	16 ± 0.38	19 ± 0.52	9 ± 0.34
6	3d	3 ± 0.1	12 ± 0.38	4 ± 0.15
7	4a	111 ± 2.66	75 ± 1.97	47 ± 1.45
8	4b	56 ± 1.44	48 ± 1.35	113 ± 3.7
9	4c	2 ± 0.05	2 ± 0.07	1 ± 0.05
10	5	101 ± 2.11	31 ± 0.73	38 ± 1.02
11	6a	14 ± 0.36	8 ± 0.24	2 ± 0.07
12	6b	3 ± 0.08	6 ± 0.16	10 ± 0.32
13	6c	68 ± 1.6	19 ± 0.51	25 ± 0.75
14	7a	78 ± 1.93	54 ± 1.46	38 ± 1.19
15	7b	16 ± 0.47	19 ± 0.58	9 ± 0.33
*	Doxorubicin	9 ± 0.26	13 ± 0.42	7 ± 0.23

^aResults are the mean ± SD of two independent assays.

Figure 4. SAR study for the designed thiazolopyrimidines.

Figure 5. Agarose gel electrophoresis of compounds 3b-3d, 4c, 6a, 6b, 7a and 7b with Etoposide and Doxorubicin at 100, 10, 1, 0.1 μM concentrations.

Table 2. IC₅₀ of the tested compounds on Topo II in comparison with Etoposide and Doxorubicin.

Compounds	TOPO II IC50 (µM)± SD
3b	6.14 ± 0.36
3c	1.39 ± 0.08
3d	2.21 ± 0.13
4c	0.23 ± 0.01
6a	1.88 ± 0.11
6b	1.51 ± 0.09
7a	3.56 ± 0.21
7b	1.05 ± 0.06
Etoposide	0.32 ± 0.02
Doxorubicin	0.83 ± 0.05

Results are the mean ± SD of two independent assays.

Figure 6. In vitro cytotoxicity (IC₅₀) of compound 4c and Doxorubicin on WI-38 human cell line (Normal cell composed of fibroblasts and derived from lung tissue of a 3-month-gestation aborted female fetus).

Table 3. The degree of selectivity of the tested compound 4c and Doxorubicin.

Selectivity index (SI)^a
Compounds	MCF7	A549	A498
4c	7770	7770	15540
Doxorubicin	1151.11	796.92	1480

^aSI = IC₅₀ normal cell/IC₅₀ cancer cell.

Figure 7. (A) Cell cycle analysis of A549 treated with DMSO only, (B) Cell cycle analysis of A549 after treatment with 4c for 24 h and 48 h.

Table 4. Cell cycle analysis of A549 cells after treatment with 4c and DMSO control.

Compounds	%G0–G1	%S	%G2/M
4c	69.07	26.89	4.04
control	56.39	29.64	13.97

Figure 8. (A) Apoptosis and necrosis in A 549 cell line (control), (B) The effect of compound 4c on apoptosis and necrosis in A 549 cells, (C) Graphical representation of the effect on apoptosis and necrosis of 4c on A 549 cell line compared to control cells.

Figure 9. The (a) 3D and (b) 2D interaction mode of the ligand (Etoposide) within topoisomerase II binding pocket (PDB code 4GWK). The pink dot arrow represents H-bond.

Figure 10. The (a) 3D and (b) 2D interaction mode of Doxorubicin (reference drug) within topoisomerase II binding pocket. The pink dot arrow represents H-bonds and brown dot arrow represents H-π interaction.

Figure 11. The (a) 3D and (b) 2D interaction mode of compounds 3b-d within topoisomerase II binding pocket. The pink dot arrow represents H-bonds and brown dot arrow represents H-π interaction.

Figure 12. The (a) 3D and (b) 2D interaction mode of compound 4c and its metabolite within topoisomerase II binding pocket. The pink dot arrow represents H-bonds and brown dot arrow represents H-π interaction.

Figure 13. The (a) 3D and (b) 2D interaction mode of compounds 6a, 6b and 7a, 7b within topoisomerase II binding pocket. The pink dot arrow represents H-bonds and brown dot arrow represents H-π interaction.

Table 5. Docking study outcomes of the selected compounds showing the binding interactions with topoisomerase II enzyme binding site.

Compound	Binding energy score ΔG (Kcal/mol)	RMSD (A^ο)	Interactions bond length (Å)
Etoposide (Ligand)	–10.23	0.75	H. Bonds: Asp 463 (2.63 Å, 2.94 Å), Met 766 (3.88 Å)
Doxorubicin	–7.61	1.40	H. Bonds: Asp 463 (2.98 Å), Ser 464, (3.08 Å), Asp 543 (2.80 Å), Arg 617 (3.06 Å). π Stacking: Arg 487 (4.19 Å), DT9 (3.85 Å). Ionic interaction: Asp 545 (3.61 Å)
3b	–7.20	1.52	π Stacking: Arg 487 (3.90 Å), DT9 (4.22 Å)
3c	–7.30	1.47	π Stacking: Arg 487 (3.84 Å), DT9 (4.30 Å)
3d	–7.21	1.81	H. Bonds: Gly 760 (3.29 Å) π Stacking: Arg 487 (3.94 Å)
4c	–5.88	1.25	H. Bonds: Asp 463 (4.23 Å), His 459 (3.69 Å), Arg 713 (3.75 Å). π Stacking: Arg 487 (3.94 Å)
Metabolite of 4c	–6.89	1.34	H. Bonds: Asp 463 (3.57 Å), Lys 489 (3.08 Å). π Stacking: Lys 489 (4.72 Å), Ionic interaction: Arg 713 (3.75 Å).
6a	–4.67	1.72	Ionic interaction: Asp 463 (3.61 Å). H. Bonds: Lys 489 (3.49 Å).
6b	–4.76	1.84	H. Bonds: Arg 487 (2.79 Å), DT9 (3.06 Å)
7a	–7.13	1.53	H. Bonds: Asp 463 (3.98 Å), π Stacking: DT9 (3.93 Å)
7b	–5.45	1.60	H. Bonds: Asp 463 (4.40 Å), His 759 (3.75 Å). π Stacking: DT9 (4.08 Å).

Table 6. The in silico predicted pharmacokinetics for most active compounds compared to Doxorubicin and Etoposide.

Molecule	GI absorption	BBB permeable	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor
(3c)	High	No	No	No	Yes	No	Yes
(4c)	Low	No	Yes	Yes	Yes	No	Yes
(6a)	High	No	No	No	No	No	Yes
(6b)	High	No	No	No	Yes	No	Yes
(7b)	Low	No	Yes	Yes	Yes	No	Yes
DOX	Low	No	No	No	No	No	No
ETP	Low	No	No	No	No	Yes	No

Table 7. In silico physicochemical properties for most active compounds in comparison to doxorubicin and etoposide.

Molecule	^aMW ≤500	^bLog Po/w ≤5	^cHBA ≤10	^dHBD ≤5	^eTPSA Ǻ^2 ≤140	^fNRB ≤10	^gLog S
(3c)	524.59	2.45	8	0	125.82	9	–5.6**
(4c)	582.88	5.55	6	1	119.39	9	–6.62***
(6a)	529.65	1.76	8	1	125.87	10	–3.97*
(6b)	502.58	1.23	8	1	131.86	10	–3.53*
(7b)	597.01	6.61	9	2	131.42	10	–6.15***
DOX	543.52	−0.32	12	6	206.07	5	–3.91*
ETP	588.56	1.01	13	3	160.83	5	–3.75*

^aMW: molecular weight; ^bLog P_o/w: partition coefficient octanol/water; ^cHBA: number of H-bond acceptors; ^dHBD: number of H-bond donors; ^eTPSA: topological polar surface area; ^fNRB: number of rotatable bonds; ^gLog S: Aqueous solubility (*soluble;**moderately soluble; ***poorly soluble).

Table 8. The drug-likeness of most active compounds as well as the references Doxorubicin and Etoposide drugs.

Molecule	Lipinski #violations	Veber #violations	PAINS #alerts	Brenk #alerts
(3c)	1	0	0	0
(4c)	1	0	0	0
(6a)	1	0	0	0
(6b)	1	0	0	0
(7b)	1	0	0	0
DOX	3	1	1	1
ETP	2	1	0	0