Design, Synthesis and In Vitro Mechanistic Investigation of Novel Hexacyclic Cage-Like Hybrid Heterocycles

Novel hexacyclic cage-like hybrid heterocycles have been synthesized in excellent yields employing a relatively less explored non-stabilized azomethine ylides derived from acenaphthenequinone and tyrosine with functionalized dipolarophiles using [3 + 2] cycloaddition strategy. The synthesized hexacyclic cage-like hybrid heterocycles were characterized by spectroscopic analysis. Following the physical characterization, these cage-like hybrid heterocycles were tested for their biological activity by means of different cancer (A549 and Jurkat cells) and non-cancer (BRL-3A and PCS-130) in vitro cell culture systems. The results of the study under tested concentrations (up to 100 μM) indicated that these compounds are not affecting any viability to the cell growth of non-cancer cells, while providing significant anticancer activity against both of the cancer cells. Further analysis of in-depth mechanistic study for the cell death indicated that these compounds are exhibiting late apoptosis or early necrosis pathway to the cells where it is operated by the induction of caspases.


Preparation of Stock Solutions
For the treatment, the testing compounds at different concentrations (5,25,50,75, and 100 µ M) and a positive control of camptothecin (CPT) (40 µ M) were prepared in the dimethyl sulfoxide media. Further dilutions to the required concentrations of the testing compounds were made in either of F-12K (A549 cells), RPMI-1640 (Jurkat cells), DMEM (MCF10 cells), or primary smooth muscle cell medium  in order to treat during the in vitro molecular biology studies.

Cell Culture and Maintenance
For the investigation of in vitro biological response of the synthesized compounds labelled in the range of 4(a-k), different assays such as cell viability and proliferation, apoptotic assay, caspasedependent pathway, and so forth, were performed. For the testing, we have selected two cancerous (A549 cells of human adenocarcinoma alveolar basal epithelial origin and Jurkat cells of human acute T-cell lymphoma) and two non-cancerous cell lines (MCF-10 normal breast cell line and PCS-130-010 human primary lung smooth muscle cells) where they all obtained from the National Centre for Cell Science (NCCS, Pune, Maharashtra, India). In order to culture the cells, we used the respective mediums consisting of 10% FBS, penicillin (100 I.U./mL), streptomycin (100 µ g/mL) at 37 °C in a humidified atmosphere of 5% CO2 in air. For the testing, each time when the cell growth reaches to 80-90% confluency, the cells were separated out by tripsinization, counted, and transferred to a 96well plate containing the complete growth medium at a density of 2 × 10 4 cells/well. Now the cells are allowed to adhere for at least 8 h before the final treatment and further, the samples are added to the cells containing 10% FBS of respective mediums.

Cell Viability and Proliferation Assay
The applied cell viability and proliferation assay for the testing of the synthesized compounds is based on the reduction of MTT agent, that is, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. For the assay, the cells seeded into the 96-well plate (2 × 10 4 cells/well) when reach to their confluence levels were treated with the synthesized compounds 4(a-k) at different concentrations in the range of 5-100 µ M and incubated further for 24 and 48 h period in an incubator. Following the period, the cell medium was replaced with a fresh medium and the assay was followed according to the manufacturer's instruction where the absorbance was recorded at 570 nm wavelength. In this, CPT (40 µ M) was selected as the positive control and the cells undergone without any treatment was taken as the negative control. Further, the IC50 values (concentration needed for 50% loss of cell viability) for all the synthesized compounds and CPT were investigated at all the incubation times and cell lines.

Apoptosis Assay
The cell death mechanism of the synthesized compound-treated A549 cells was analyzed by the Annexin V/FITC apoptotic kit, where the fluorescence intensity exhibited by the FITC-conjugated Annexin-V and PI-treated cells were analyzed by making use of the flow cytometry. Briefly, the A549 cells at a density of 10 6 cells/well were seeded in a 6-well plate and when the cell growth reaches to its confluence levels, were treated with the testing compound (4a) at its IC50 concentration as investigated by the cell viability and proliferation assay. After the incubation period (either of 24 or 48 h), the cells were undergone trypsinization, centrifugation to obtain the pellet which was resuspended gently in 100 μL of AnnexinV-FITC binding buffer, and then incubated further in 5 μL of AnnexinV-FITC in the dark for 10 min at 25 °C. Now the cells were subjected to centrifugation (2000 rpm for 5 min) and the obtained pellet was re-suspended in 500 μL of AnnexinV-FITC binding buffer and 5 μL of PI was added in an ice bath, followed by the flow cytometric analysis. The Cell Quest software was applied for the investigation and analysis of apoptotic cells.

Cell Cycle Analysis
The A549 cells at a density of 10 6 cells/well were first seeded into a six-well plate and following the incubation for 12 h, the cells were treated with the compound 4a at IC50 concentrations in a serumfree media and incubated for additional 24 or 48 h. After the period, the cells were undergone trypsinization and centrifugation (1000× g for 10 min) to obtain the pellet, which was re-suspended in PBS, followed by the fixation with the addition of 70% ethanol at least for 2 h. Now the cells were centrifuged (1000× g for 10 min) and the obtained pellet was re-suspended in PBS; after 1 min, the cells were centrifuged, pellet was suspended again in PI (1 mL) staining solution. After the incubation of cells with PI for 15 min, the cells were subjected to the flow cytometric analysis at an excitation and emission wavelengths of 488 nm and 670 nm respectively using the Cell Quest software (San Jose, CA, USA).

Caspase-3 Expression
In order to test the role of caspase-3 for the compound 4a treated A549 cells, the cells were first treated with IC50 concentrations and incubated for either 24 or 48 h periods (similar to earlier assays). After the period, the cells were harvested and then fixed with the ice cold ethanol (70%), followed by the incubation for additional 30 min at −20 °C. Now the cells were washed twice with PBS and then treated with Caspase-3 FITC reagent (20 µ L), followed by the incubation at room temperature in dark for 60 min. Following the incubation in dark, the cells were washed with PBS and re-suspended in 0.5 mL of PBS and then analyzed for caspase-3 expression by the EVOS TM FL Auto fluorescence microscopy (Thermo Scientific, India) where the Image J software was applied.

Statistical Analysis
The results of in vitro cell culture systems presented are the mean ± SD of 3-5 individual experiments where the statistical analysis was based on student's t-test. For the statistical analysis, the significant value represented by * (p < 0.05) and highly significant value represented by ** (p < 0.01) against the control ran using the Graphpad prism software version 6 (San Diego, CA, USA).