New imidazole-2-thiones linked to acenaphythylenone as dual DNA intercalators and topoisomerase II inhibitors: structural optimization, docking, and apoptosis studies

Figures & data

Figure 1. (A) Reported imidazole-containing drugs I-III as anticancer agents^21–23, (B) Reported drugs bearing imidazole- polynuclear scaffold hybrids IV-V as anticancer agents^5,24,25.

Figure 2. Work approach describes a hypothesis for designing targeted compounds based on structure features required for DNA damage through double helix intercalation and topoisomerase II inhibition, as in standard DNA intercalator Doxorubicin.

Scheme 1. Synthesis of N-substituted-2-(2-oxoacenaphthylen-1(2H)-ylidene)hydrazine-carbothioamide derivatives 3a-f.

Scheme 2. Synthesis of imidazole-2-thiones 5a-n.

Figure 3. Fragmentation patterns A-C for mass spectra of compounds 5a-n.

Figure 4. Distinctive carbons of compound 5 g.

Scheme 3. Mechanism describes the formation of 5a-n.

Figure 5. Schematic diagram for the fluorimetric detection of the induced DNA damage using terbium chloride (Tb³⁺) luminescent biosensor.

Figure 6. Tb³⁺ luminescence enhancement at λ_max = 545 nm after excitation at λmax = 270 nm of the studied compounds compared to reference doxorubicin (DOX). Controls such as DNA-Tb³⁺ mixture, and Tb³⁺ alone are also presented.

Figure 7. Effect of increasing concentration of (a) 5b, (b) 5e, (c) 5h, (d) 5j, and (e) doxorubicin (DOX) as a reference of the luminescence of Tb- ctDNA complex. The insets show the fit to the linear region of the constructed plots.

Table 1. Analytical parameters for the fluorescence measurements of 5b, 5e, 5h, 5j, and doxorubicin as a reference with ctDNA mixtures.

Parameter	5b -DNA	5e -DNA	5h -DNA	5j -DNA	DOX-DNA
Linear dynamic range	0.01pM–100.0 μM	20.0–80.0 μM	0.01pM–100.0 μM	0.01pM–60.0 μM	0.01pM–100.0 μM
Correlation coefficient (r)	0.9915	0.9938	0.9964	0.9911	0.9912
Intercept (a)	6366.2	−263553	−1689	−5822.3	2167.3
Slope (b)	13283	7580	13306	8124.2	8923.2
LOD^a	7.78 nM	14.2 nM	7.14 nM	12.7 nM	4.09 nM
LOQ^b	25.9 nM	46.9 nM	23.8 nM	42.4 nM	13.7 nM

^a LOD:Limit of detection.

^b LOQ: Limit of quantitation.

Figure 8. In vitro anticancer activity, IC₅₀ (μM) of 5b, 5e, 5h, and 5j in comparison to Doxorubicin.

Table 2. Selectivity Index (SI) of compounds 5b, 5h, and doxorubicin against human breast adenocarcinoma (MCF-7).

Compound ID	MCF-7
5b	17.58
5h	49.27
Dox	3.9

Figure 9. (A) A graphical representation of apoptosis induction analysis of compounds 5b, 5h and doxorubicin as reference against MCF-7 at their IC₅₀(μM). (B) Apoptosis induction analysis of compounds 5b, 5h, and doxorubicin as reference against MCF- 7 at their IC₅₀ (μM).

Figure 10. (A) A graphical representation of cell cycle analysis of compounds 5b, 5h, and doxorubicin as a reference against MCF-7 at their IC₅₀ (μM). (B) Cell cycle analysis of compounds 5b, 5h, and doxorubicin as a reference against MCF-7 at their IC₅₀ (μM).

Figure 11. A graphical representation of 5b, 5h and DOX against Topo II inhibitory activity.

Figure 12. Structure-Activity relationship of DNA damage testing, antiproliferative activity and topoisomerase inhibitory activity for tested compounds.

Figure 13. 3D representation of the overlay of re-docked ligand (green) to the co-crystallized ligand EVP (red) (RMSD =1.06 Å).

Figure 14. The binding interaction of EVP (green) at the pocket of the active site of DNA-Topo IIβ complex in 2D (left panel) and 3D representation (right panel).

Figure 15. The binding interaction of doxorubicin (red) at the active site of DNA-Topo IIβ complex in 2D (left panel) and 3D representation (right panel).

Table 3. Molecular docking scoring and interactions of tested compounds at the active site of DNA-Topo IIβ (PDB ID: 3QX3) in comparison to reference drugs EVP and DOX.

Tested compound	Binding score	Type of interaction	Binding pattern representation
	(Kcal/mol)	(amino acid residue)
5a	−6.80	H-acceptor (Gln 778)	SI Figure 95
		H-acceptor (H2O molecule)
5b	−6.59	H-donor (Glu 477)	Figure 16
		H-donor (Asp 557)
		H-acceptor (H2O molecule)
		2 H-Pi (DG13)
		4 Pi-H (Arg503)
		2 Pi-H (DT9)
		2 Mg metal interaction
5c	−7.40	2 Pi-H (Arg503)	SI Figure 96
5d	−7.69	H-acceptor (DT9)	SI Figure 97
		H-acceptor (DG 10)
		H-acceptor (H2O molecule)
		Pi-Pi (DG13)
5e	−7.76	H-acceptor (DT9)	SI Figure 98
		H-acceptor (H2O molecule)
5f	−7.16	2 H-acceptor (H2O molecule)	SI Figure 99
5 g	−7.01	Pi-H (Arg503)	SI Figure 100
		Pi-H (DT9)
5h	−7.00	2 H-donor (DT9)	Figure 17
		H-donor (Arg503)
		H-acceptor (Gly478)
		H-acceptor (Asp479)
		H-acceptor (H2O molecule)
		H-acceptor (DA12)
		Pi-H (Arg503)
		Pi-H (Gly504)
		Pi-H (DT9)
5i	−7.64	Pi-H (Arg503)	SI Figure 101
		H-donor (DT9)
5j	−7.93	2 H-acceptor (H2O molecule)	SI Figure 102
		H-acceptor (Asp479)
		H-acceptor (DT9)
		H-acceptor (DG10)
5k	−7.53	H-acceptor (H2O molecule)	SI Figure 103
		Pi-Pi interaction (DT9)
5l	−7.78	H-acceptor (DT9)	SI Figure 104
		H-acceptor (H2O molecule)
5m	−8.07	H-acceptor (DG13)	SI Figure 105
		2 Pi-Pi interaction (DG13)
		Pi-H (DA12)
5n	−7.75	Pi-H (Arg503)	SI Figure 106
		H-Pi (DG13)
		H-Pi (DT9)
Dox^a	−6.41	2 H- donor (DG13)	Figure 15
		H- donor (Asp479)
		H-donor (DG 10)
		2 H- acceptor (DT9)
		H- acceptor (Arg503)
		H-acceptor (H2O molecule)
		2 H-Pi (DA12)
		Pi-H (DT9)
		Pi-H (H2O molecule)
EVP^b	−10.092	2 H- donor (DG13)	Figure 14
		H- donor (Arg503)
		2 H- donor (DT9)
		H-acceptor (Gln778)
		H- donor (Met782)
		H-acceptor (Met782)
		H-acceptor (H2O molecule)
		2 H-Pi (DT9)
		H-Pi (DA12)

^a Doxorubicin.

^bCo-crystallized ligand etoposide.

Figure 16. The binding interaction of compound 5b (blue) at the active site of DNA-Topo IIβ complex in 2D (left panel) and 3D representation (right panel).

Figure 17. The binding interaction of compound 5h (yellow) at the active site of DNA-Topo IIβ complex in 2D (left panel) and 3D representation (right panel).

Figure 18. (A) Cartoon representation of the molecular surface of DNA-Topo IIβ complex (PDB:3QX3) showing EVP (green), 5b (blue), 5h (yellow), doxorubicin (red) in the binding pocket. (B) Focused view of the active site. (C) Transparent mode of the cartoon representation illustrating the binding pocket with the essential aminoacids and nucleotides.

Table 4. In silico predictions of the pharmacokinetics and drug-likeness properties for 5b, 5e, 5h, and 5j.

Code	MW	GIA	BBB	LogP	Veber #violations	PAINS #alerts
5b	431.51	High	No	5.79	0	1
5e	461.53	High	No	5.76	0	1
5h	465.95	Low	No	6.42	0	1
5j	495.98	Low	No	6.39	0	1
Code	Pgp substrate	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP3A4 inhibitor
5b	No	Yes	Yes	Yes	No
5e	No	Yes	Yes	Yes	Yes
5h	No	Yes	Yes	No	No
5j	No	No	Yes	Yes	No

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 descriptor ⁶¹. Veber #violationscounts the number of violations of Veber rule summarised as: NRB ≤ 10 and TPSA ≤ 140 Ų. PAINS #alertscounts the number of pan-assay interference compounds/substrucutres. All calculations were performed using SwissADME⁵⁹.

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Data availability statement

Data set generated during and/or analysed during the current study are available from the corresponding author on reasonable request.