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

Abstract In this research, two novel series of dibenzo[b,f]azepines (14 candidates) were designed and synthesised based on the rigidification principle and following the reported doxorubicin’s pharmacophoric features. The anti-proliferative activity was evaluated at the NCI against a panel of 60 cancer cell lines. Further, the promising candidates (5a–g) were evaluated for their ability to inhibit topoisomerase II, where 5e was noticed to be the most active congener. Moreover, its cytotoxicity was evaluated against leukaemia SR cells. Also, 5e arrested the cell cycle at the G1 phase and increased the apoptosis ratio by 37.34%. Furthermore, in vivo studies of 5e showed the inhibition of tumour proliferation and the decrease in its volume. Histopathology and liver enzymes were examined as well. Besides, molecular docking, physicochemical, and pharmacokinetic properties were carried out. Finally, a SAR study was discussed to open the gate for further optimisation of the most promising candidate (5e). Highlights Two novel series of dibenzo[b,f]azepines were designed and synthesised based on the rigidification principle in drug design. The anti-proliferative activity was evaluated at the NCI against a panel of 60 cancer cell lines. 5e was the most active anti-topo II congener (IC50 = 6.36 ± 0.36 µM). 5e was evaluated against leukaemia SR cells and its cytotoxic effect was confirmed (IC50 = 13.05 ± 0.62 µM). In vivo studies of 5e significantly inhibited tumour proliferation by 62.7% and decreased tumour volume to 30.1 mm3 compared to doxorubicin treatment.


Supporting information SI 1. General chemistry protocol
All melting points were determined using the Electrothermal Capillary melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded as KBr pellets with JASCO FT/IR-6100 Spectrometer and values are represented in cm -1 . 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were carried out on Jeol ECA 400 MHz spectrometer using TMS as the internal standard and chemical shift values were recorded in ppm on the δ scale. The 1 H NMR data were represented as follows: chemical shifts, multiplicity (s. singlet, d. doublet, dd. doublet of doublet, t. triplet, m. multiplet), and the number of protons. The 13 C NMR data were represented as chemical shifts. Mass spectral data were obtained by expression-coupled with express-plate Compact Mass Spectrometer (ESI, APCI/TLC/MS), Advion, USA. Silica gel TLC (thin layer chromatography) cards from Merck (silica gel precoated aluminum cards with the fluorescent indicator at 245 nm) were used for thin layer chromatography.
Visualization was performed by illumination with a UV light source (254 nm). Column chromatography was carried out on silica gel 60 (0.063-0.200 mm) obtained from Merck. SI 2. Spectral data of compounds (4a-g and 5a-g), (IR, 1 H NMR, 13 C NMR, and Mass spectroscopy) Figure S1: 1 H-NMR spectra of compound 4a Figure S2: 1 H-NMR spectra of compound 4b Figure S3: 1 H-NMR spectra of compound 4c Figure S4: 1 H-NMR spectra of compound 4d  Solid Ehrlich carcinoma (SEC) were got from the National Cancer Institute (Cairo University, Egypt). The tumor cell line was proliferated in mice through serial intraperitoneal (I.P.) transplantation of a volume of 0.2 mL physiological saline contains 1 × 10 6 viable cells for 24 h. SEC cells were collected 7 days after I.P. implantation. The harvested cells were diluted with saline to obtain a concentration of 5 × 10 6 viable SEC cells/mL. A volume of 0.2 mL saline contains 1 × 10 6 SEC cells that were I.P. implanted into each normal mouse. SEC cells (1 × 10 6 tumor cells/mouse) were implanted subcutaneously into the right thigh of the hind limb.
The experimental animals were randomly divided into four groups. Group 1 served as the normal saline control. Group 2 served as the SEC control (1 × 10 6 cells/mouse). Group 3 served as the compound-treated group (6 mg/kg B.Wt., I.P.). Group 4 received the standard anticancer drug of Doxorubicin (DOX) (6 mg/kg BW, I.P.) and is considered as a reference control. Body weight and survival were recorded daily until the 24 th day in both treated and control groups. At the end of experiment, the blood of each group was collected under light anesthesia for estimation of hematological and biochemical assays. The anesthetized animals were then sacrificed for evaluation of the antitumor activity and histopathological examination.

SI 5.3. Blood assays
At the end of the experiment, animals from different groups were sacrificed, and blood samples were collected for determination of liver enzymes ALT, AST levels. Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated using commercial kits (ELITech clinical systems, France). Serum albumin level was determined by kit purchased from STANBIO Company (USA).

SI 5.4. Histopathological study
Specimens of liver-sacrificed mice were fixed in 10% saline formalin. The fixed liver specimens were dehydrated in ascending series of ethyl alcohol and embedded in paraffin. Sections at 5 mm thicknesses were stained with hematoxylin and eosin and examined under light microscopy.