Design, synthesis, anticancer evaluation and molecular docking studies of 1,2,3-triazole incorporated 1,3,4-oxadiazole-Triazine derivatives

A new library of 1,2,3-triazole-incorporated 1,3,4-oxadiazole-triazine derivatives (9a-j) was designed, synthesized, and tested in vitro for anticancer activity against PC3 and DU-145 (prostate cancer), A549 (lung cancer), and MCF-7 (breast cancer) cancer cell lines using the MTT assay with etoposide as the control drug. The compounds exhibited remarkable anticancer activity, with IC50 values ranging from 0.16 ± 0.083 μM to 11.8 ± 7.46 μM, whereas the positive control ranged from 1.97 0.45 μM to 3.08 0.135 μM. Compound 9 d with a 4-pyridyl moiety shown exceptional anticancer activity against PC3, A549, MCF-7, and DU-145 cell lines, with IC50 values of 0.17 ± 0.063 μM, 0.19 ± 0.075 μM, 0.51 ± 0.083 μM, and 0.16 ± 0.083 μM, respectively.

Furthermore, these compounds are examined for anticancer activity in vitro against four different cancer cell lines, including PC3 and DU-145 (prostate cancer), A549 (lung cancer), and MCF-7 (melanoma) (breast cancer).

Molecular docking studies
In total ten compounds, 9h (− 9.6 kcal/mol), 9f (− 9.4 kcal/mol), 9j (− 9.4 kcal/mol), showed good activity in the tubulin CNN (colchicine) binding site ( Fig. 2: A good activity on tubulin GDP binding site ( Fig. 3: E, F, G, and H). Nevertheless, none of the compounds showed good activity when compared to GDP (− 9.4 kcal/mol) at GDP binding site ( Table 2). The amino acid interactions of each compound with respective binding sites given in Table 3.

General
All the solvents, catalysts, fine chemicals, salts, and reagents were purchased from AVRA PVT Ltd, Hyderabad. The progress of the organic reactions justified by Merck TLC plates with the utilization of UV light. Both 1 H & 13 C spectra were taken from BRUKER NMR (300 MHz, 400 MHz) instrument. Melting points were measured with locally manufactured melting point apparatus.

MTT assay
100 μL medium was employed to inoculate the micro titer 96 well tissue culture plates having 1 × 104 cells. 37 • C temperature was maintained to incubate these plates in 5% CO 2 humidified incubator over 18 h' time before the starting of experiment. After the removal of old medium, 100 μL fresh medium provided for our test compound derivatives and standard drug at various concentrations like 0.5 μM, 1 μM, and 2 μM respectively. Now it was added to each well and allowed for incubation at 37 • C over 24 h' time. Now the medium was removed and restore with 10 μL MTT dye. Again, the platers were allowed for incubation at 37 • C over 2h time. The outcoming formazan crystals were dissolved in 100 μL extracted buffer. Optical density (O.D) was recorded at 570 nm with Multi-mode Varioskan Instrument-Themo Scientific micro plate reader. In medium, % of DMSO not exceeded to 0.25%.

Ligand preparation and docking studies
Accelrys Discovery studio version 4.0 was utilized to visualize the ligand structures, receptors, and hydrogen-bonding networks. It was also used to render images. The ligand structures were drawn using the Chemsktech software and were converted to 3 d format saved to mol2 format for further processing. All ligands were Energy minimized by chimera applying 'AMBER' force field with steepest descent algorithm. Protein was collected from RCSB bank (www.rcsb.org) in PDB ID: 1SA0 (Crystal structures of tubulin complexed with colchicine binding site and GDP binding site, both binding sites selected for this study). The Crystal Structure of the human Tubulin complex with colchicine Protein [1SA0] was resolved using X-ray diffraction method with a resolution factor of 3.58 Å was retrieved from PDB Retrieved structure, which has been further modified for docking calculations. Autodock 4.0 was the primary docking program used for semi-flexible docking studies. Preparation of the ligands and protein receptors in pdbqt file and determination of the grid box size was carried out using Autodock Tools version 1.5.6. A grid box with the dimensions X:30, Y:30, Z:30 Å and a grid spacing of 1.0 Å focused at X: 113, Y:89.289, Z: 7.212 was identified as the protein target colchicine docking site. A grid box with the dimensions X:30, Y:30, Z:30 Å and a grid spacing of 1.0 Å focused at X:97.758 Y:74.4.34 Z: 0.001 was identified as the protein target gdp docking site. The protocol used for performing protein and ligand preparation, along with docking studies, were described elsewhere.