Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design, synthesis, biological evaluation, and in silico studies

Abstract The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N1-unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC50 of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC50 of 4.81 and 4.34 μM, respectively. On the other hand, the N1-substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC50 of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N1-unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N1-substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.


Anti-proliferative activity toward human breast cell lines
The two examined human breast cancer cell lines (T-47D and MCF-7) have been obtained from American Type Culture Collection (ATCC). Cells lines were maintained as monolayers in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100µg/ml streptomycin sulfate. Cells were sub-cultured with trypsine /EDTA solution, counted with haemocytometer and plated onto 96-well plates (5000 cells/well) and left overnight to form a semi-confluent monolayer. Cell monolayers were treated in quadrates with vehicle (DMSO, 0.1% v/v), test samples or Adriamycin as positive control for an exposure time of 48 h. At the end of exposure, MTT solution in PBS (5 mg/ml) was then added to all well including no cell blank and left to incubate for 90 min. The formation of formazan crystals were visually confirmed using phase contract microscopy. DMSO (100 µl/well) was added to dissolve the formazan crystals with shaking for 10 min after which the absorbance was read at 590 nm against no cell blanks on a FLuo Star Optima microplate reader (BMG technologies, Germany).
Cell proliferation was calculated comparing the OD values of the DMSO control wells and those of the samples represented as % proliferation to the control. Dose-response experiment was performed on samples producing > or =50% loss of cell proliferation using five serial 2-fold dilutions (50, 25, 12.5, 6.25 and 3.125 µM) of the sample. IC 50 values (concentration of sample causing 50% loss of cell proliferation of the vehicle control) were calculated using non-linear regression curve fitting of the dose response plots on GraphPad Prism V.6.0 software.

Cell Cycle Analysis
Breast cancer MCF-7 cells were treated with hybrids (5d, 5e and 5f) for 24 h (at their IC 50 concentration), and then cells were washed twice with ice-cold phosphate buffered saline (PBS).
Subsequently, the treated cells were collected by centrifugation, fixed in ice-cold 70% (v/v) ethanol, washed with PBS, re-suspended with 100 μg/mL RNase, stained with 40 μg/mL PI, and analyzed by flow cytometry using FACS Calibur (Becton Dickinson, BD, Franklin Lakes, NJ, USA). The cell cycle distributions were calculated using CellQuest software 5.1 (Becton Dickinson).

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 MCF-7 cells were cultured to a monolayer then treated with hybrids (5d, 5e and 5f) at their IC 50 concentration. Briefly, cells were then harvested via trypsinization, and rinsed twice in PBS followed by binding buffer. Moreover, cells were re-suspended 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). Figure S1. 2D diagram for hybrid 5a showing its interaction with the CDK2 binding site.