Design and synthesis of novel rigid dibenzo[b,f]azepines through ring closure technique as promising anticancer candidates against leukaemia and acting as selective topoisomerase II inhibitors and DNA intercalators

Figures & data

Figure 1. A group of previously reported drugs as topoisomerase II inhibitors and DNA intercalators, bear the same doxorubicin’s pharmacophoric features.

Figure 2. Schematic representation of main pharmacophoric features of the dibenzo[b,f]azepine derivatives (5a–g) as topoisomerase II inhibitors and DNA intercalators (like doxorubicin) produced by the rigidification of candidates (4a–g) using the ring closure principle.

Scheme 1. Synthesis of the novel open dibenzo[b,f]azepine analogues (4a–g) and their corresponding target closed ones (5a–g). (i) NaOH/H₂O/Reflux, (ii) Phosgene/Toluene/NH₂NH₂/24 h/RT, (iii) Acid chloride/CHCl₃/18 h/RT, and (iv) POCl3/30 min/Reflux.

Table 1. Growth inhibition % of the tested compounds (4a–g and 5a–g) against different 60 cell lines (NCI-60).

Subpanel tumour cell lines	Compounds
	4a	4b	4c	4d	4e	4f	4g	5a	5b	5c	5d	5e	5f	5g
Leukaemia
CCRF-CEM	–	–	–	–	–	–	–	26.09	37.32	30.43	26.62	–	28.17	15.64
HL-60(TB)	–	–	–	–	–	10.04	–	13.19	34.63	38.12	33.79	30.06	–	10.63
K-562	–	–	–	–	–	12.83	–	32.25	54.54	53.99	47.81	25.26	33.98	14.86
MOLT-4	–	–	–	–	–	–	–	16.54	35.64	20.40	20.98	19.35	16.32	10.86
RPMI-8226	–	–	–	–	–	–	–	67.73	17.48	19.19	52.00	–	12.08	27.84
SR	–	–	–	–	–	12.77	–	–	72.97	67.50	60.59	30.12	58.44	17.55
Non-small cell lung cancer
A549-ATCC	–	18.50	–	–	–	–	–	–	29.84	20.34	12.45	16.51	28.19	24.67
EKVX	–	–	–	–	–	11.01	13.76	–	27.55	20.83	–	–	28.86	2.24
HOP-62	–	–	–	–	–	–	–	15.34	22.73	15.39	–	–	–	18.40
HOP-92	–	–	–	–	–	18.58	–	18.28	33.48	23.99	14.33	22.82	20.92	–
NCI-H226	–	–	–	–	–	–	–	–	28.26	27.52	21.65	13.72	21.42	–
NCI-H23	–	–	–	–	–	–	–	–	–	–	–	–	12.36	–
NCI-H322M	–	–	–	–	–	–	–	50.19	–	10.54	–	4.97	9.52	–
NCI-H460	–	–	–	–	–	–	–	–	63.47	57.26	58.60	1.84	25.09	17.45
NCI-H522	–	–	–	–	–	14.06	–	–	26.33	22.24	17.10	15.03	24.20	–
Colon cancer
COLO 205	–	–	–	–	–	–	–	–	15.40	–	19.42	–	–	–
HCC-2998	–	–	–	–	–	–	–	33.39	–	–	–	–	–	–
HCT-116	–	–	–	–	–	–	–	54.24	52.18	47.74	45.78	–	27.26	31.40
HCT-15	–	–	–	–	–	–	–	–	66.47	63.52	61.14	30.73	57.04	4.03
HT29	–	–	–	–	–	11.44	–	–	18.42	–	–	–	–	–
KM12	–	–	–	–	–	–	–	–	21.42	18.46	13.08	–	–	–
SW-620	–	11.82	–	–	–	–	–	–	–	–	–	–	–	19.70
CNS cancer
SF-268	–	15.10	–	–	–	–	–	–	24.68	24.70	10.10	16.05	21.94	10.68
SF-295	–	–	–	–	–	–	–	–	30.39	29.55	22.01	–	19.93	–
SF-539	–	–	–	–	–	–	–	–	17.17	–	–	–	–	11.46
SNB-19	–	–	–	–	–	–	–	33.22	15.56	11.15	–	–	–	–
U251	–	–	–	–	–	–	–	20.15	47.02	42.56	31.80	––	32.33	10.12
Melanoma
LOX IMVI	–	–	–	–	–	–	–	–	38.76	35.74	25.60	–	18.73	–
MALME-3M	–	–	–	–	–	–	–	–	–	–	–	–	–	–
M14	–	–	–	–	–	–	–	12.06	–	–	–	–	–	–
MDA-MB-435	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SK-MEL-2	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SK-MEL-28	–	–	–	–	–	–	–	28.02	14.13	–	–	–	–	–
SK-MEL-5	–	–	–	–	–	–	–	–	53.17	47.97	34.61	13.66	15.84	–
UACC-257	–	14.38	12.55	–	–	–	–	13.62	–	–	–	–	–	19.15
UACC-62	–	–	–	–	–	18.93	–	–	32.21	31.29	20.91	29.97	12.77	10.82
Ovarian cancer
IGROV1	–	–	–	–	–	–	–	–	21.55	19.19	–	–	29.07	–
OVCAR-3	–	–	–	–	–	–	–	–	–	–	–	–	2.22	12.33
OVCAR-4	–	–	–	–	–	–	–	–	19.60	22.10	–	21.82	–	–
OVCAR-5	–	–	–	–	–	–	–	–	–	–	–	–	–	–
OVCAR-8	–	–	–	–	–	–	–	–	28.56	24.00	11.53	1.15	9.31	16.50
NCI/ADR-RES	–	–	–	–	–	–	–	–	23.41	14.54	–	–	12.05	–
SK-OV-3	–	–	–	–	–	–	–	–	–	–	–	–	–	–
Renal cancer
786-0	–	21.12	–	–	–	–	–	19.99	11.30	–	–	–	–	18.78
A498	–	–	–	–	–	–	–	–	13.39	12.95	–	14.79	–	–
ACHN	–	13.98	13.31	–	–	–	–	16.72	24.77	15.52	–	–	10.51	27.70
CAKI-1	–	–	–	–	–	–	–	–	31.88	28.47	16.19	29.70	33.13	–
RXF 393	–	–	–	–	–	–	–	–	24.94	17.92	–	–	10.44	–
SN12C	–	–	–	–	–	–	–	–	24.19	21.89	–	–	15.71	–
TK-10	–	–	13.25	10.32	10.01	–	–	16.05	–	–	–	–	20.26	21.15
UO-31	–	–	–	–	–	12.04	–	39.35	33.49	33.76	–	30.72	24.71	30.67
Prostate cancer
PC-3	–	–	–	–	–	25.11	–	–	48.54	44.08	41.40	24.58	40.89	–
DU-145	–	19.68	–	–	–	–	–	12.42	–	–	–	–	–	16.34
Breast cancer
MCF-7	–	–	–	–	–	13.97	–	14.45	30.47	35.65	15.73	17.44	24.40	–
MDA-MB-231-ATCC	–	–	–	–	–	–	–	–	22.27	19.78	9.21	15.31	–	–
HS 578 T	–	–	–	–	–	–	–	–	14.36	–	13.16	–	–	24.07
T-47D	–	–	–	–	–	12.61	–	–	21.46	–	–	18.42	18.29	12.00
MDA-MB-468	–	–	–	–	–	12.30	–	26.09	17.77	16.69	–	11.44	11.13	15.64

(–) Growth inhibition % for samples that is not active.

Table 2. Topoisomerase II activity of the tested candidates (5a–g) against doxorubicin.

Ser	Candidate	Topoisomerase II (IC50) µM	SD±
1	5a	15.63	0.86
2	5b	10.24	0.58
3	5c	31.76	1.78
4	5d	16.83	0.94
5	5e	6.362	0.36
6	5f	9.561	0.53
7	5g	18.95	1.06
–	Doxorubicin	3.445	0.19

Figure 3. Topoisomerase II inhibitory activity of the tested compounds (5a–g) against doxorubicin. (A) Percentage of topoisomerase II inhibition at concentration (100 µM) of the tested compounds; (B) DNA-fragmentation gel images of the most active compounds (5e and 5f) at various concentrations (100–1.3 µg/ml).

Table 3. The cytotoxicity results of compound 5e against SR and normal cell lines.

Compound	Cytotoxicity IC50 µg/ml		SI
	SR	PSC-800-011
5e	13.05 ± 0.62	43.86 ± 2.68	3.36
Doxorubicin	3.78 ± 0.14	12.83 ± 0.79	3.39

Figure 4. Analysis of cell cycle for 5e-treated SR cells. *P < 0.05 compared to control by GraphPad prism using unpaired t-test.

Figure 5. Induction of apoptosis by compound 5e in SR cells shows both apoptotic (late and early) and necrotic cell death. **P < 0.001 and *P < 0.05 compared to control by GraphPad prism using unpaired t-test.

Figure 6. Solid tumour growth was inhibited in SEC-bearing mice by administration of compound 5e (6 mg/Kg B.W., IP) (SEC model). (A) The tumour mass of different treated groups (morphological representation); “normal control, SEC-group, SEC+ 5e, and SEC + Dox.” (B) Anti-tumour activity of tumour mass, volume, and TIR% in different treated groups. (C) Comparative morphological examinations of the tumour tissues tested groups. “Values are expressed as Mean ± SEM values of mice in each group (n = 7).” Values denoted by sign **(P ≤ 0.001) were found to be statistically different from the SEC control when compared with an unpaired t-test in GraphPad Prism. (H&E stain, magnification ×400).

Figure 7. (A): ALT and AST in different SEC-treated groups. “Values are expressed as Mean ± SD of independent trials (n = 7),” “*(P ≤ 0.05) significantly different between SEC control and normal control, while ^#(P ≤ 0.05) is significantly different between treated groups compared to SEC control.” (B) Histopathological examinations of liver tissues in different treated groups of SEC-bearing mice. (H&E stain, magnification ×400). Normal mice with uniform hepatocytes, and portal tract with uniform portal tract (Black arrows). SEC group show portal tract expansion with chronic inflammatory cells (Black arrows), area of lytic necrosis (Arrowhead), and hydropic degeneration of hepatocytes (Red arrows). Treated 5e group show portal tract inflammation (Black arrow), with spillage of inflammatory cells into the limiting plate (Red arrowhead), foci of lytic necrosis, and mild lobular inflammation (Black arrowheads). Hepatocytes show mild hydropic degeneration (Red arrows).

Figure 8. Re-docking of the EVP (green) and its docked pose (yellow).

Figure 9. Binding of doxorubicin within the active pocket of topoisomerase II-DNA complex.

Figure 10. Binding of EVP within the active pocket of topoisomerase II-DNA complex.

Figure 11. Binding of compound 5e within the active pocket of topoisomerase II-DNA complex.

Figure 12. Binding of compounds 5b (A) and 5f (B) inside the topoisomerase II-DNA complex.

Table 4. Physicochemical parameters of the synthesised compounds 5a–g.

Comp.	M.wt	NRB	HBA	HBD	TPSA (Å^2)	Lipinski	Log P	Water solubility	MR
5a	371.82	2	3	0	42.16	Yes, 1 violation	5.09	PS	111.29
5b	351.40	2	3	0	42.16	Yes, 1 violation	4.89	PS	111.25
5c	382.37	3	5	0	87.98	Yes, 1 violation	3.98	MS	115.10
5d	387.43	2	3	0	42.16	Yes, 1 violation	5.38	PS	123.79
5e	351.40	3	3	0	42.16	Yes, 1 violation	4.66	PS	110.30
5f	387.43	2	3	0	42.16	Yes, 1 violation	5.38	PS	123.79
5g	351.40	3	3	0	42.16	Yes, 1 violation	4.66	PS	110.30

Num. of rotatable bonds (NRB); H. bond Acceptor (HBA); H. bond donor (HBD); Topological polar surface area (TPSA); Molar Refractivity (MR).

Table 5. Pharmacokinetic and ADMET parameters of the synthesised candidates 5a–g.

Comp.	ADMET parameters					PAINS	Synthetic accessibility
	Log KP (Skin permeation)	BBB permeant	CY1A2 inhibitor	GI absorption	p-gp substrate
5a	–4.23 cm/s	Yes	Yes	High	No	0 alert	3.50
5b	–4.30 cm/s	Yes	Yes	High	No	0 alert	3.67
5c	–4.86 cm/s	Yes	Yes	High	No	0 alert	3.64
5d	–3.89 cm/s	No	Yes	High	No	0 alert	3.76
5e	–4.35 cm/s	Yes	Yes	High	Yes	0 alert	3.57
5f	–3.89 cm/s	No	Yes	High	No	0 alert	3.76
5g	–4.35 cm/s	Yes	Yes	High	Yes	0 alert	3.57

BBB, blood-brain barrier; GI absorption, gastrointestinal absorption.

Figure 13. SAR for the newly synthesised derivatives 4a–g and 5a–g as topoisomerase II inhibitors and DNA intercalators.