Design and efficient synthesis of pyrazoline and isoxazole bridged indole C-glycoside hybrids as potential anticancer agents

C-glycosides are important class of molecules exhibit diverse biological activities and present as structural motif in many natural products. Two series of new pyrazoline and isoxazole bridged indole C-glycoside molecular hybrids (n = 36) were efficiently synthesized starting from diverse indole 3-carboxaldehydes derived α, β-unsaturated ketone derivatives of β-D-glucosyl-propan-2-one, β-D-galactosyl-propan-2-one and β-D-mannosyl-propan-2-one, reacting with hydrazine hydrate and hydroxyl amine hydrochloride in shorter reaction time (15 min) under microwave assisted condition. Anticancer activity of these newly synthesized pyrazoline and isoxazole bridged indoles C-glycoside hybrids were determined in details through cellular assays against MCF-7, MDA-MB-453 and MDA-MB-231 cancer cell lines. The selected library members displayed low micromolar (IC50 = 0.67–4.67 µM) and selective toxicity against breast cancer cell line (MCF-7). Whereas these compounds were nontoxic towards normal cell line (MCF-10A). Mechanistic studies showed that, active compounds inhibit COX-2 enzyme, which was also supported by molecular docking studies. These findings are expected to provide new leads towards anticancer drug discovery.

The results summarized in Table 2 showed moderate growth inhibition at 10 μM concentration in all compounds except for 11, 26, 27, 33, 35, 36, 38, 39, 42 and 43. Better inhibition of cell viability was observed with compounds 9, 14, 20, 25 and 34 in MDA-MB-231 (breast cancer cells). These compounds show almost 50% inhibition at 25 µM concentration. These five compounds were further tested in serial dilution to find out IC 50 and results are shown in Fig. 2.
The IC 50 values for most active compounds 9, 25 and 34 were calculated and are presented in Fig. 3. The IC 50  MDA-MB-231 represents a specific subtype, known as the triple negative breast cancer (TNBC). We extended our investigation with these five hit compounds (9, 14, 20, 25, 34 and 37) in two other different cancer cell lines (MCF-7 and MDA-MB-453), each representing a separate class of breast cancers (MCF-7: hormone receptor/ HR-positive; MDA-MB-453: human epidermal growth factor 2 receptor/HER2 positive). We also included normal mammary epithelial MCF-10A cells in our experiments to confirm if the growth-inhibitory activities of the selected compounds are truly cancer cell-specific. The activity data from three different cell lines (tested at 1, 10 and 25 μM) are summarised in Table 3.
The IC 50 values for most active compounds 9, 20, 25, 34 and 37 were calculated using five different concentration (1.0, 5.0, 10.0, 25 and 50 µM) and are presented in Table 4. The IC 50 values for compound 9, 20, 25, 34 and 37 against MCF-7 breast cancer cells line were found 30.0, 20.99, 4.67, 0.71 and 0.67 µM, respectively. These compounds did not reach up to IC 50 value against MDA-MB-453 cells. Furthermore, none of these compounds did have any growth inhibitory activity against normal breast epithelial cell (MCF-10A). The IC 50 values for these compounds against MDA-MB-231 cells (Fig. 2) are also included in Table 4 for comparison.
When living cells treated with a cytotoxic compound, they could either stop growing and dividing, or die through either of two distinct processes i.e. necrosis or apoptosis 58 . Basically, cells undergoing necrosis (accidental cell death) swell and lose membrane integrity before shutting down and releasing their intracellular contents into the surrounding environment. This type of cell death is usually triggered by external factors such as toxic chemical or traumatic physical events. When the cell membranes are damaged or compromised in any way, lactate dehydrogenase (LDH), a soluble yet stable enzyme found inside every living cell, gets released into the surrounding extracellular space. The presence of this enzyme in the culture medium can be used as a cell death marker. The relative amounts of live and dead cells within the medium can then be quantitated by measuring the amount of released LDH using a colorimetric or fluorometric LDH cytotoxicity assay. Herein we confirmed cytotoxicity of active compounds 9, 20, 25, 34 and 37 by fluorometric LDH cytotoxicity assay using maximum LDH control (MLC) which shows % of cytotoxicity as 13,8,22,31 and 36 respectively at 25 µM concentration (Fig. 4).
In order to understand the cytotoxicity mechanism of the active compounds, COX-2 assay was performed. The in-vitro COX-2 inhibitory activity of the active compounds (9, 20, 25, 34 and 37) was evaluated using celecoxib as a reference with a fluorescence-based COX-2 assay ("COX-2 Fluorescent Inhibitor Screening Assay Kit", Item no. K547, Bio Vision, USA). In this method we detect the fluorescence compound PGG2 which can easily be analysed with an excitation wavelength of 540 nm and emission wavelength of 590 nm. The result of COX-2 inhibitory activity has been summarised in Table 5 Table 2. Anticancer activity results. Table 5 that active compounds showing some inhibition and maximum COX-2 inhibition shown by compound 37 which is in consistence with its cytotoxic activity against MCF-7 cells (Table 4).
To undertand the COX-2 inhibition and molecular interaction of the active compounds with the COX-2 enzyme (PDB code: 1CX2), molecular docking studies were performed using Autodock-Vina software 59 (for conversion to supported docking format) and PyMol software (for visualization and analysis of the interaction of ligands with the protein after docking). The three-dimensional orientation of the docked compounds showing important binding interaction through hydrogen bonds with carbohydrate moiety and hydrophobic interactions as shown Fig. 5.