Novel topoisomerase II/EGFR dual inhibitors: design, synthesis and docking studies of naphtho[2′,3′:4,5]thiazolo[3,2-a]pyrimidine hybrids as potential anticancer agents with apoptosis inducing activity

Figures & data

Figure 1. Examples for anticancer agents targeting topoisomerase II.

Figure 2. Clinically approved EGFR anticancer drugs.

Figure 3. Diagram represents design of novel hybrids based upon thiazolo[3,2-a] pyrimidine and 1,4-naphthoquinone moieties as dual topo II/EGFR inhibitor.

Scheme 1. Synthesis of the target hybrids 6a-i. Reagents and conditions: a) K2CO3, C2H5OH, reflux, 2h. b) DMF , rt., 12 h.

Figure 4. A representative example of ¹H NMR and ¹³C NMR of 6i hybrid.

Table 1. Cytotoxicity of the synthesised hybrids (6a-i), dox and erlotinib against MCF-7, A549 and HCT-116 cell lines.

Compound no.	IC50 (µM) ± S.E.
	MCF-7	A549	HCT-116
6a	3.34 ± 0.21	2.81 ± 0.18	3.58 ± 0.24
6b	4.78 ± 0.36	4.20 ± 0.41	11.54 ± 0.12
6c	4.42 ± 0.35	3.65 ± 0.25	3.66 ± 0.39
6d	5.66 ± 0.29	3.25 ± 0.31	11.77 ± 0.11
6e	5.14 ± 0.32	5.72 ± 0.37	9.81 ± 0.65
6f	29.11 ± 1.56	38.27 ± 3.21	26.82 ± 1.78
6 g	13.85 ± 1.12	42.17 ± 2.19	9.00 ± 0.66
6h	37.94 ± 2.25	21.23 ± 1.23	56.22 ± 4.11
6i	1.79 ± 0.11	1.55 ± 0.13	2.29 ± 0.18
Dox	5.62 ± 0.31	4.95 ± 0.29	6.78 ± 0.44
Erlotinib	7.26 ± 0.2	5.19 ± 0.36	8.64 ± 0.18

Data were expressed as mean ± standard error (SE) of three independent experiments.

Figure 5. Effect of the tested hybrids (6a, 6c and 6i) and Dox on topoisomerase IIα concentration in MCF-7 cell line.

Figure 6. DNA topo II inhibition assay of 6i, 6a and 6c.

Figure 7. IC₅₀ values of the tested hybrids (6a, 6c and 6i) and erlotinib against EGFR.

Figure 8. Effects of 6a, 6c and 6i hybrids and Dox on p53, Bax and Bcl-2 levels in MCF-7 cancer cell line.

Table 2. Effects of 6a, 6c and 6i hybrids on p53, Bax and Bcl-2 levels in MCF-7 cancer cell line.

Compound no.	IC50	p53		Bax		Bcl-2
		Conc.(pg/ml)	FLD	Conc.(pg/ml)	FLD	Conc.(ng/ml)	FLD
6a	3.34	922 ± 37.5	3.6	436.1 ± 5.1	5.24	3.99 ± 0.24	0.61
6c	4.42	678 ± 42.3	2.6	372.7 ± 9.4	4.48	2.17 ± 0.36	0.33
6i	1.79	1285 ± 29.9	5	581.5 ± 8.7	6.99	3.64 ± 0.08	0.55
Control	—	257 ± 19.1	1	83.16 ± 6.2	1	6.56 ± 0.22	1
Dox	5.62	1051 ± 22.6	4	511.2 ± 6.5	6.14	1.66 ± 0.03	0.25

Data were expressed as mean ± standard error (SE) of three independent experiments.

Table 3. Effects of 6a, 6c and 6i hybrids on caspase-7 and caspase-9 levels in MCF-7 cancer cell line.

Compound no.	IC50	Caspase 7		Caspase 9
		Conc.(ng/ml)	FLD	Conc.(pg/ml)	FLD
6a	3.34	6.72 ± 0.23	14.93	21.17 ± 0.38	4.69
6c	4.42	1.89 ± 0.02	4.20	13.68 ± 0.42	3.03
6i	1.79	9.20 ± 0.87	20.41	32.29 ± 0.33	5.38
Control	—	0.45 ± 0.03	1	4.51 ± 0.29	1
Dox.	5.62	10.44 ± 0.92	23.2	29.48 ± 0.25	6.53

Data were expressed as mean ± standard error (SE) of three independent experiments.

Figure 9. (A) Cell cycle analysis in MCF-7 cell line treated with 6a and 6i hybrids. (B) Cell cycle analysis and apoptosis effect in MCF-7 cell line treated with 6a and 6i hybrids.

Table 4. Cell cycle analysis of 6a and 6i hybrids in MCF-7 cell line.

		DNA content
Compound no.	IC50 µM	%G0-G1	%S	%G2/M	%Pre-G1
6a	3.34	36.28	28.94	34.78	33.41
6i	1.79	45.94	51.17	2.89	45.26
Cont.MCF-7	–	57.23	35.09	7.68	1.89

Figure 10. (A) Percentage of apoptosis and necrosis for 6a and 6i hybrids in MCF-7 cell line. (B) Flow cytometric analysis of Annexin V-FITC/PI induced by 6a and 6i hybrids in MCF-7 cell line.

Table 5. Results of apoptotic assay of 6a and 6i hybrids in MCF-7 cell line.

Compound no.	Apoptosis			Necrosis
	Total	Early	Late
6a	33.41	5.26	18.92	9.23
6i	45.26	3.64	27.31	14.31
Cont.MCF-7	1.89	0.38	0.15	1.36

Table 6. Pharmacokinetic prediction of the synthesised hybrids (6a-i) by Molinspiration v2021.03.

Comp. no.	miLog P^a	TPSA^b	Mwt^c	nON^d	nOHNH^e	N violations^f	Nrotb^g	Volume^h
6a	3.34	101.55	413.41	7	0	0	2	331.29
6b	3.44	92.31	401.38	6	0	0	1	310.67
6c	2.93	110.78	443.44	8	0	0	3	356.83
6d	4.09	92.31	462.28	6	0	0	1	323.63
6e	3.96	92.31	417.83	6	0	0	1	319.28
6f	2.91	120.01	473.47	9	0	0	4	382.38
6g	3.28	92.31	383.39	6	0	0	1	305.74
6h	3.38	95.55	426.46	7	0	0	2	351.65
6i	3.24	138.14	428.38	9	0	0	2	329.08

^aLogarithm of partition coefficient between n-octanol and water (miLogP).

^bTopological polar surface area (TPSA)

^cMolecular weight (Mwt).

^dNumber of hydrogen bond acceptors (n-ON).

^eNumber of hydrogen bond donors (n-OHNH).

^fNumber of violation.

^gNumber of rotatable bonds (N-rotb).

^hMolecular Volume (Mol.Vol.)

Table 7. Bioactivity score of the synthesised hybrids (6a-i).

Comp. no.	GPCRL	ICM	KI	NRL	PI	EI
6a	−0.29	−0.43	−0.04	−0.42	−0.45	−0.20
6b	−0.25	−0.39	−0.02	−0.41	−0.44	−0.17
6c	−0.28	−0.41	−0.01	−0.42	−0.46	−0.18
6d	−0.34	−0.44	−0.04	−0.52	−0.52	−0.22
6e	−0.26	−0.38	−0.02	−0.45	−0.45	−0.19
6f	−0.28	−0.39	−0.01	−0.46	−0.45	−0.17
6g	−0.27	−0.39	−0.00	−0.44	−0.43	−0.16
6h	−0.25	−0.38	0.02	−0.39	−0.42	−0.17
6i	−0.37	−0.40	−0.12	−0.48	−0.52	−0.24

GPCRL: G protein-coupled receptor Ligand; ICM: Ion channel modulator; KI: Kinase Inhibitor; NRL: Nuclear receptor ligand; PI: Protease inhibitor; EI: Enzyme inhibitor

Table 8. Absorption properties of the synthesised hybrids (6a-i).

Comp.no.	Water solubility	Caco2 permeability	(HIA)%	Skin Permeability	P-glycoprotein substrate	P-glycoprotein I inhibitor	P-glycoprotein II inhibitor
6a	−4.336	1.207	100%	−2.723	No	Yes	Yes
6b	−4.382	1.227	100%	−2.722	No	Yes	Yes
6c	−4.232	0.569	100%	−2.732	No	Yes	Yes
6d	−4.718	0.566	100%	−2.716	No	Yes	Yes
6e	−4.678	1.123	100%	−2.715	No	Yes	Yes
6f	−4.230	0.578	100%	−2.737	No	Yes	Yes
6g	−4.618	1.282	99.7%	−2.713	No	Yes	Yes
6h	−4.529	0.583	97.3%	−2.715	No	Yes	Yes
6i	−4.723	0.434	96.5%	−2.733	No	Yes	Yes

Table 9. Distribution properties of the synthesised hybrids (6a-i).

Comp. no.	VDss	Fraction unbound (human)	BBB permeability	CNS permeability
6a	−0.227	0.275	−1.035	−2.084
6b	−0.299	0.290	−1.018	−1.958
6c	−0.272	0.270	−1.254	−3.008
6d	−0.201	0.266	−0.994	−1.756
6e	−0.220	0.267	−0.986	−1.779
6f	−0.422	0.253	−1.477	−3.188
6g	−0.291	0.255	−0.798	−1.888
6h	−0.155	0.267	−0.956	−1.984
6i	−0.396	0.236	−1.316	−2.097

Table 10. Metabolism and excretion properties of the synthesised hybrids (6a-i).

Comp. no.	CYP2D6 substrate	CYP3A4 substrate	CYP1A2 inhibitior	CYP2C19 inhibitior	CYP2C9 inhibitior	CYP2D6 inhibitior	CYP3A4 inhibitior	Renal OCT2 Substrate
6a	No	Yes	Yes	Yes	Yes	No	No	No
6b	No	Yes	Yes	Yes	Yes	No	No	No
6c	No	Yes	Yes	Yes	Yes	No	No	No
6d	No	Yes	Yes	Yes	Yes	No	No	No
6e	No	Yes	Yes	Yes	Yes	No	No	No
6f	No	Yes	No	No	No	No	No	No
6g	No	Yes	Yes	Yes	Yes	No	No	No
6h	No	Yes	Yes	Yes	Yes	No	No	No
6i	No	Yes	Yes	Yes	Yes	No	No	No

Table 11. Toxicity properties of the synthesised hybrids (6a-i) and Dox.

Comp. No.	Max. tolerated dose (human)	hERG I inhibitor	hERG II inhibitor	Oral Rat Acute Toxicity (LD50)	Hepatotoxicity
6a	0.337	No	Yes	2.523	No
6b	0.353	No	No	2.467	Yes
6c	0.366	No	Yes	2.653	No
6d	0.292	No	No	2.373	No
6e	0.289	No	No	2.368	No
6f	0.427	No	Yes	2.748	Yes
6g	0.424	No	No	2.322	No
6h	0.288	No	No	2.441	No
6i	0.207	No	No	2.980	No
Dox.	0.115	No	No	2.408	No

Figure 11. Docking and binding pattern of hybrids 6a (A&B), 6c (C&D), 6i (E&F) and Dox. (G&H) showing interactions with different amino acid residues found in the active site of human topo IIα ATPase (PDB code 1ZXM).

Table 12. Types of binding interactions and energy scores (kcal/mol) for hybrids (6a–i) and Dox at the human topo IIα ATPase/AMP-PNP active site.

Compound No.	Types of interactions	Energy scores
6a	- One hydrogen bonding interaction with Asn 120 (3.30 Å). - One hydrogen bonding interaction with Ser 149 (3.35 Å). - One hydrogen bonding interaction with Asn 91 (3.21 Å). - One hydrogen bonding interaction with Ala 167 (3.18 Å).	−8.16
6b	- One hydrogen bonding interaction with Ser 149 (3.19 Å).	−7.52
6c	- One hydrogen bonding interaction with Ser 149 (2.79 Å). - One hydrogen bonding interaction with Gly 166 (2.47 Å).	−8.42
6d	- One hydrogen bonding interaction with Arg 98 (3.41 Å).	−6.39
6e	- One hydrogen bonding interaction with Lys 157 (3.01 Å). - One hydrogen bonding interaction with Thr 215 (2.89 Å).	−7.86
6f	- One hydrogen bonding interaction with Arg 98 (3.05 Å). - One π-hydrogen bonding interaction with Ser 148 (4.64 Å).	−5.58
6g	- One hydrogen bonding interaction with Asn 120 (3.17 Å).	−7.01
6h	- One hydrogen bonding interaction with Thr 215 (3.44 Å).	−5.84
6i	- One hydrogen bonding interaction with Ala 167 (3.35 Å). - One hydrogen bonding interaction with Lys 168 (3.09 Å). - One metal-ion interaction with Mg^+2 (903) (2.47 Å).	−9.51
Dox.	- Two hydrogen bonding interaction with Ser 149 (2.57 and 3.12 Å). - One hydrogen bonding interaction with Asn 91 (3.32 Å). - One hydrogen bonding interaction with Asn 120 (2.47 Å). - One hydrogen bonding interaction with Asn 150 (2.58 Å).	−8.35

Table 13. Types of binding interactions and energy scores (kcal/mol) for hybrids (6a–i) and erlotinib at the EGFR kinase active site.

Compound no.	Types of interactions	Energy scores
6a	- One hydrogen bonding interaction with Met 793 (3.61 Å). - Two hydrogen bonding interactions with Asp 855 (3.62 and 2.92 Å). - One hydrogen bonding interaction with Lys 745 (3.16 Å). - Two π-hydrogen bonding interaction with Val 726 (3.98 and 4.42 Å).	−8.72
6b	- One hydrogen bonding interaction with Asp 855 (3.28 Å). - Two π-hydrogen bonding interaction with Val 726 (3.96 and 4.01 Å).	−6.41
6c	- One hydrogen bonding interaction with Asp 855 (3.51 Å). - One hydrogen bonding interaction with Lys 745 (3.60 Å). - One π-hydrogen bonding interaction with Val 726 (4.03 Å).	−7.32
6d	- One π-hydrogen bonding interaction with Leu 844 (3.86 Å).	−5.89
6e	- One π-hydrogen bonding interaction with Leu 844 (3.86 Å).	−5.78
6f	- One π-hydrogen bonding interaction with Leu 718 (4.56 Å). - Two π-hydrogen bonding interaction with Val 726 (4.41 and 4.00 Å).	−4.95
6 g	- Two π-hydrogen bonding interaction with Val 726 (4.44 and 4.05 Å).	−6.01
6h	- One hydrogen bonding interaction with Lys 745 (3.10 Å). - One π-hydrogen bonding interaction with Leu 718 (4.58 Å).	−6.67
6i	- One hydrogen bonding interaction with Met 793 (3.66 Å). - Two hydrogen bonding interactions with Asp 855 (2.91 and 3.61 Å). - One hydrogen bonding interaction with Lys 745 (3.17 Å). - Two π-hydrogen bonding interaction with Val 726 (3.98 and 4.41 Å).	−8.25
Erlotinib	- One hydrogen bonding interaction with Met 793 (3.48 Å). - Two π-hydrogen bonding interaction with Leu 718 and Thr 854, Thr 790, Gln 791 through H2O (4) mediated bond (3.94 Å and 3.79 Å). - One hydrogen bonding interaction with H2O (22) (2.93 Å)	−7.33

Figure 12. Docking and binding pattern of compounds 6a (A&B), 6c (C&D), 6i (E&F) and erlotinib (G&H) showing interactions with different amino acid residues found in the ATR-active site of EGFR (PDB code: 1XKK).