Bimetallic Iron–Palladium Catalyst System as a Lewis-Acid for the Synthesis of Novel Pharmacophores Based Indole Scaffold as Anticancer Agents

The Friedel–Crafts reaction between substituted indoles as nucleophiles with chalcones-based benzofuran and benzothiophene scaffolds was carried out by employing a highly efficient bimetallic iron–palladium catalyst system. This catalytic approach produced the desired bis-heteroaryl products with low catalyst loading, a simple procedure, and with acceptable yield. All synthesized indole scaffolds 3a–3s were initially evaluated for their cytotoxic effect against human fibroblast BJ cell lines and appeared to be non-cytotoxic. All non-cytotoxic compounds 3a–3s were then evaluated for their anticancer activities against cervical cancer HeLa, prostate cancer PC3, and breast cancer MCF-7 cell lines, in comparison to standard drug doxorubicin, with IC50 values 1.9 ± 0.4 µM, 0.9 ± 0.14 µM and 0.79 ± 0.05 µM, respectively, and appeared to be moderate to weak anticancer agents. Fluoro-substituted chalcone moiety-containing compounds, 3b appeared to be the most active member of the series against cervical HeLa (IC50 = 8.2 ± 0.2 µM) and breast MCF-7 cancer cell line (IC50 = 12.3 ± 0.04 µM), whereas 6-fluroindol-4-bromophenyl chalcone-containing compound 3e (IC50 = 7.8 ± 0.4 µM) appeared to be more active against PC3 prostate cancer cell line.


A. PC3 cells (Prostate Cancer) assay
Cytotoxic activity of compounds was evaluated in 96-well flat-bottomed micro plates by using the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) colorimetric assay. For this purpose, PC3 cells (Prostate Cancer) were cultured in Dulbecco's Modified Eagle Medium, supplemented with 5% of fetal bovine serum (FBS), 100 IU/ml of penicillin and 100 µg/ml of streptomycin in 75 cm 2 flasks, and kept in 5% CO2 incubator at 37 o C. Exponentially growing cells were harvested, counted with haemocytometer and diluted with a particular medium. Cell culture with the concentration of 1x10 5 cells/ml was prepared and introduced (100 µL/well) into 96-well plates. After overnight incubation, medium was removed and 200 µL of fresh medium was added with different concentrations of compounds (1-30µM). After 48 hrs, 200 µL MTT (0.5 mg/ml) was added to each well and incubated further for 4 hrs. Subsequently, 100µL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 570 nm, using a micro plate reader (Spectra Max plus, Molecular Devices, CA, USA). The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC50) for PC3 cells. The percent inhibition was calculated by using the following formula: The results (% inhibition) were processed by using Soft-Max Pro software (Molecular Device, USA).

STANDARD DRUG:
Standard drug used in the MTT assay was doxorubicin.
Exponentially growing cells were harvested, counted with haemocytometer and diluted with a particular medium. Cell culture with the concentration of 6x10 4 cells/ml was prepared and introduced (100 µL/well) into 96-well plates. After overnight incubation, medium was removed and 200 µL of fresh medium was added with different concentrations of compounds (1-30µM). After 48 hrs, 200 µL MTT (0.5 mg/ml) was added to each well and incubated further for 3 hrs.
Subsequently, 100µL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 550 nm, using a micro plate reader (Spectra Max plus, Molecular Devices, CA, USA). The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC50) for BJ cells. The percent inhibition was calculated by using the following formula:

STANDARD DRUG:
Standard drug used in the MTT assay was doxorubicin.

C. HeLa cells (Cervical Cancer) assay
Cytotoxic activity of compounds was evaluated in 96-well flat-bottomed micro plates by using the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) colorimetric assay 40 . For this purpose, HeLa cells (Cervical Cancer) were cultured in Minimum Essential Medium Eagle, supplemented with 5% of fetal bovine serum (FBS), 100 IU/ml of penicillin and 100 µg/ml of streptomycin in 75 cm 2 flasks, and kept in 5% CO2 incubator at 37 o C. Exponentially growing cells were harvested, counted with haemocytometer and diluted with a particular medium. Cell culture with the concentration of 6x10 4 cells/ml was prepared and introduced (100 µL/well) into 96-well plates. After overnight incubation, medium was removed and 200 µL of fresh medium was added with different concentrations of compounds (1-30µM). After 48 hrs, 200 µL MTT (0.5 mg/ml) was added to each well and incubated further for 4 hrs. Subsequently, 100µL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 570 nm, using a micro plate reader (Spectra Max plus, Molecular Devices, CA, USA). The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC50) for HeLa. The percent inhibition was calculated by using the following formula: The results (% inhibition) were processed by using Soft-Max Pro software (Molecular Device, USA).

STANDARD DRUG:
Standard drug used in the MTT assay was doxorubicin.
D. MCF-7 breast cancer cell line assay.