Discovery of 3,6-disubstituted pyridazines as a novel class of anticancer agents targeting cyclin-dependent kinase 2: synthesis, biological evaluation and in silico insights

Abstract Human health in the current medical era is facing numerous challenges, especially cancer. So, the therapeutic arsenal for cancer should be unremittingly enriched with novel small molecules that selectively target tumour cells with minimal toxicity towards normal cells. In this context, herein a new series of 3,6-disubstituted pyridazines 11a–r has been synthesised and evaluated for in vitro anticancer activity. They possessed good anti-proliferative action towards human breast cancer T-47D (IC50 range: 0.43 ± 0.01 − 35.9 ± 1.18 µM) and MDA-MB-231 (IC50 range: 0.99 ± 0.03 − 34.59 ± 1.13 µM) cell lines, whereas they displayed weak activity against the tested ovarian cancer cell line SKOV-3. Among the studied compounds, the methyltetrahydropyran-bearing pyridazine 11m emerged as the unique submicromolar growth inhibitor herein reported towards both T-47D (IC50 = 0.43 ± 0.01 µM) and MDA-MB-231 (IC50 = 0.99 ± 0.03 µM) cell lines. In addition, the biological results indicated that pyridazines 11l and 11m exerted an efficient alteration within the cell cycle progression as well as induction of apoptosis in both T-47D and MDA-MB-231 cells. Moreover, pyridazines 11l and 11m displayed good mean tumour S. I. values of 13.7 and 16.1 upon assessment of their cytotoxicity towards non-tumorigenic breast MCF-10A cells. Furthermore, an in silico study proposed CDK2 as a probable enzymatic target for pyridazines 11, and explored their binding interactions within the vicinity of CDK2 binding site. Subsequently, pyridazines 11e, 11h, 11l, and 11m were selected to be evaluated for their ability to inhibit CDK2, where they exerted good inhibitory activity (IC50 = 151, 43.8, 55.6 and 20.1 nM, respectively). Finally, the in silico study implied that target pyridazines 11 exhibited not only an efficient anticancer activity but also an acceptable ADME, physicochemical and druglikeness properties, specifically pyridazines 11l and 11m. Overall the obtained results from this study quite sustained our strategy and gave us a robust opportunity for further development and optimisation of 3,6-disubstituted pyridazine scaffold to enrich therapeutic arsenal with efficient and safe anticancer CDK inhibitors.

The anti-proliferative activity of the tested pyridazines 11a-r was measured in vitro using the Sulfo-Rhodamine-B stain (SRB) assay. Briefly, Cells were inoculated in 96-well microtiter plate (5X10 4 cells/ well) for 24 h before treatment with the tested pyridazines to allow attachment of cell to the wall of the plate. Tested pyridazines were dissolved in DMSO at 1 mg/ml immediately before use and diluted to the appropriate volume just before addition to the cell culture. Different concentrations of tested pyridazines and staurosporine were added to the cells (three wells were prepared for each individual dose). Cells were incubated with the pyridazines for 48 h at 37°C and in atmosphere of 5% CO 2 . After 48 h cells were fixed, washed, and stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader. The relation between percent of surviving fraction and drug concentration is plotted to get the survival curve for each cell line. The concentration required for 50% inhibition of cell viability (IC 50 ) was calculated.

Cell Cycle Analysis
Breast cancer T-47D and MDA-MB-231 cells were treated with pyridazines 11l and

Annexin V-FITC Apoptosis Assay
Phosphatidylserine externalization was assayed using Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, USA) according to the manufacturer's instructions. Breast cancer T-47D and MDA-MB-231 cells were cultured to a monolayer then treated with pyridazines 11l and 11m at their IC 50 concentration. Briefly, cells were then harvested via trypsinization, and rinsed twice in PBS followed by binding buffer. Moreover, cells were resuspended in 100 μL of binding buffer with the addition of 1 μL of FITC-Annexin V followed by an incubation period of 30 min at 4 °C. Cells were then rinsed in binding buffer and resuspended in 150 μL of binding buffer with the addition of 1 μL of DAPI (1 μg/μL in PBS). Cells were then analyzed using the flow cytometer BD FACS Canto II and the results were interpreted with FlowJo7.6.4 software (Tree Star, Ashland, OR, USA).

Statistical Analysis
Data are presented as means ± S.D. Individual groups were compared using the twotailed independent Student's t-test. Multiple group comparisons were carried out using oneway analysis of variance (ANOVA) followed by the Tukey-Kramer test for post-hoc analysis. Statistical significance was accepted at a level of p < 0.05. All statistical analyses were performed using GraphPad InStat software, version 3.05 (GraphPad Software, Inc., La Jolla, CA, USA). Graphs were sketched using GraphPad Prism software, version 5.00 (GraphPad Software, Inc., La Jolla, CA, USA).
13 Figure S1. Different target classes suggested by SwissTargetPrediction online tool (for compound 5J), showing the protein kinases as the most probable targets