Development of 3-methyl/3-(morpholinomethyl)benzofuran derivatives as novel antitumor agents towards non-small cell lung cancer cells

Abstract As one of the most lethal malignancies, lung cancer is considered to account for approximately one-fifth of all malignant tumours-related deaths worldwide. This study reports the synthesis and in vitro biological assessment of two sets of 3-methylbenzofurans (4a–d, 6a–c, 8a–c and 11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b and 18) as potential anticancer agents towards non-small cell lung carcinoma A549 and NCI-H23 cell lines, with VEGFR-2 inhibitory activity. The target benzofuran-based derivatives efficiently inhibited the growth of both A549 and NCI-H23 cell lines with IC50 spanning in ranges 1.48–47.02 and 0.49–68.9 µM, respectively. The three most active benzofurans (4b, 15a and 16a) were further investigated for their effects on the cell cycle progression and apoptosis in A549 (for 4b) and NCI-H23 (for 15a and 16a) cell lines. Furthermore, benzofurans 4b, 15a and 16a displayed good VEGFR-2 inhibitory activity with IC50 equal 77.97, 132.5 and 45.4 nM, respectively.


Anti-proliferative action toward human breast cell lines
The two examined human non-small cell lung carcinoma cell lines (A549 and NCI-H23) 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% CO 2 . 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 3-methylbenzofurans (4a-d, 6a-c, 8a-c and 11) and 3-(morpholinomethyl)benzofurans (15a-c, 16a-b, 17a-b and 18) measured in vitro using the MTT assay. Briefly, cells were plated out in 96-well microtiter plate (0.5X10^4 cells/well) and incubated for 24 h before treatment with the tested benzofurans to allow cells to attach to the bottom of the well of the plate. Tested benzofuranswere 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 benzofurans, and staurosporine were added to the cells (three wells were prepared for each individual dose). Cells were incubated with the benzofurans for 48 h at 37°C and in atmosphere of 5% CO 2 . 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 microplate reader.
The relation between percent of surviving fraction and log 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 and the results are presented in Table 1. 3

Cell Cycle Analysis
Lung cancer A549 cells were treated with benzofuran 4b, whereas NCI-H23 cells were treated with benzofurans 15a and 16a 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. Lung cancer A549 and NCI-H23 cells were cultured to a monolayer then treated with benzofurans (4b, 15a and 16a) 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).

VEGFR-2 Kinase Inhibitory Activity
The VEGFR-2 kinase inhibitory activity was determined in vitro for benzofurans (4b, 15a and

Anti-tubercular activity
Microplate alamar blue assay (MABA) was used to determine MICs of the test compounds against M. tuberculosis ATCC 27294. Isoniazid was used as a reference drug. Preparation of the inoculum was done using fresh Lowenstein Jensen (LJ) medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone, 0.5% glycerol, supplemented oleic acid, albumin, dextrose, and catalase (OADC), adjusted to a McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Tested compounds were dissolved in DMSO. Drug-free controls containing broth with DMSO were included in the experiment. The final concentration of DMSO in the test medium did not exceed 0.5% (v/v) of the total solution composition, which had no effect on the growth of M.
tuberculosis. The 96 wells plates were treated with 100 µl of two-fold serial dilution of each compound. Final concentrations of the tested compounds in wells were 1000-0.006 µg/mL. Sterile deionized water (200 µl) was added to all outer-perimeter wells of sterile 96 well plates to decrease evaporation of the medium in the test wells during incubation. A growth control without antibiotic and a sterile control were also set on each plate. The plate was then covered, sealed in plastic bags and incubated at 37 °C in normal atmosphere. After 7 days of incubation, each well was supplied with 30 µl of the Alamar blue solution, and the plate was re-incubated overnight. Color change from blue (oxidized state) to pink (reduced) highlighted the growth of bacteria, and the MIC was expressed as the minimum concentration of compound which prohibited blue to pink color change.
MIC values were calculated in µg/ml.