Synthesis, Biological Evaluation and In Silico Studies of Certain Oxindole–Indole Conjugates as Anticancer CDK Inhibitors

On account of their overexpression in a wide range of human malignancies, cyclin-dependent kinases (CDKs) are among the most validated cancer targets, and their inhibition has been featured as a valuable strategy for anticancer drug discovery. In this study, a hybrid pharmacophore approach was adopted to develop two series of oxindole–indole conjugates (6a–i and 9a–f) and carbocycle–indole conjugates (11a,b) as efficient antitumor agents with potential inhibitory action toward CDK4. All oxindole–indole conjugates, except 6i, 9b, and 9c efficiently affected the growth of the human breast cancer MCF-7 (IC50: 0.39 ± 0.05–21.40 ± 1.58 μM) and/or MDA-MB-231 (IC50: 1.03 ± 0.04–22.54 ± 1.67 μM) cell lines, whereas bioisosteric replacement of the oxindole nucleus with indane or tetralin rings (compounds 11a,b) diminished the anti-proliferative activity. In addition, hybrids 6e and 6f displayed effective cell cycle disturbance and proapoptotic capabilities in MCF-7 cells. Furthermore, the efficient anti-proliferative agents towards MCF-7 and/or MDA-MB-231 cell lines (6a–h, 9a, and 9e) were investigated for their potential inhibitory action toward CDK4. Hybrids 6a and 6e displayed good CDK4 inhibitory activity with IC50s equal 1.82 and 1.26 µM, respectively. The molecular docking study revealed that oxindole moiety is implicated in two H-bonding interactions via both (NH) and (C=O) groups with the key amino acids Glu94 and Val96, respectively, whereas the indole framework is stably accommodated in a hydrophobic sub-pocket establishing hydrophobic interactions with the amino acid residues of Ile12, Val20, and Gln98 lining this sub-pocket. Collectively, these results highlighted hybrids 6a and 6e as good leads for further optimization as promising antitumor drugs toward breast malignancy and CDK inhibitors.


Anti-proliferative action toward human breast cell lines
The two examined human breast cancer cell lines  have been obtained from American Type Culture Collection (ATCC). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (GIBCO), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37 o C in humidified atmosphere containing 5% CO2. Cells at a concentration of 0.50 x 10 6 were grown in a 25 cm 2 flask in 5 ml of culture medium.
The anti-proliferative activity of the tested hybrids 6a-i, 9a-f and 11a, b 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 compounds to allow attachment of cell to the wall of the plate. Tested compounds 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 compounds, doxorubicin and sorafenib were added to the cells (three wells were prepared for each individual dose). Cells were incubated with the compounds for 48 h at 37°C and in atmosphere of 5% CO2. 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 (IC50) was calculated.

Cell Cycle Analysis
Breast cancer MCF-7 cells were treated with hybrids 6e and 6f for 24 h (at their IC50 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).

ELISA Immunoassay
The levels of the pro-apoptotic markers (Bax, caspase-3 and p53) as well as the antiapoptotic marker Bcl-2 were determined using ELISA colorimetric kits per the manufacturer's instructions. Breast cancer MCF-7 cells were cultured as a monolayer in T-25 flasks and were seeded to attain 30% confluency prior to treatment. Cells were then treated separately with hybrids 6e and 6f at their IC50 concentrations for 48 h. At the end of treatment, cells were collected via trypsinization and centrifuged at 10,000 rpm. The pellet was then rinsed with PBS and lysed in RIPA lysis buffer at 4 °C for 45 min, then centrifuged at 14,000 rpm for 20 min to remove the cellular debris. Lysates were then collected and stored at −80 °C for later protein determination using Pierce BCA Protein Assay Kit according to manufacturer's recommendations.
The cell lysate was diluted 10 times, and 100 μL (50 mg protein) was added to the wells of four separate microtiter plates for the four ELISA kits that were pre-coated with primary antibodies specific to Bax, Bcl-2, caspase-3 and p53 proteins, respectively. A secondary biotin-linked antibody specific to the protein captured by the primary antibody was further added to bind the captured protein, forming a "sandwich" of specific antibodies around the desired protein in the cell lysate. The streptavidin-HRP complex was then used to bind the biotin-linked secondary antibody through its streptavidin portion. The HRP domain reacted with the added TMB substrate to form a colored product that measured at 450 nm by a plate reader ChroMate-4300 after the reaction was terminated via the addition of stop solution.

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 6e and 6f at their IC50 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).
Testing compounds were dissolved in 100% DMSO to specific concentration. The serial dilution was conducted by Integra Viaflo Assist in DMSO. The reaction mixture containing the examined compound and 33P-ATP was incubated at room temperature for 2 h and radioactivity was detected by filter-binding method. Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. Figure S1. 2D diagram for hybrid 6b showing its interaction with the CDK4 binding site.       (6a-i, 9a-f and 11a, b) 1H-indole-2-carbohydrazide 4