Synthesis 2-Bromo-6-(4,5-Diphenyl-1-((1-Phenyl-1H-1,2,3-Triazol-4-yl) Methyl)-1H-Imidazol-2-yl) Pyridine Derivatives: Evaluation of Anticancer Activity and Molecular Docking Studies


 A series of novel 2-bromo-6-(4,5-diphenyl-1-((subtituted-phenyl-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazol-2-yl)pyridine (7a-l) were synthesized and characterized by various analytical techniques. All the synthesized compounds were evaluated for their in vitro anticancer activity against two human cancer cell lines such as HT-1080 (Human fibro sarcoma cells) and Caco-2(Human colorectal adenocarcinoma cells) and IC50values were compared against standard anticancer drug doxorubicin. Among all the synthesized compounds 7b, 7c and 7f has shown potent activity against HT-1080. Similarly compounds 7b and 7c shown significant activity among all the synthesized compounds against Caco-2. Furthermore, the results were supported by molecular docking studies.


Introduction
Cancer has become one of the major global health concerns Since it is the second leading cause of death after cardiovascular diseases, it remains a major health problem around the world 1 . Cancer is a collection of related diseases that involve abnormal cell growth with the potential to invade or spread to other parts of the body. Since it involves the conversion of any normal cells to a cancerous cell showing tandem replication and cell divisions at much faster rate in comparison to the normal cells and thus provides a potential target for the development of chemotherapeutic agents. The worldwide occurred estimated deaths and new cancer cases in 2020 were 10.0 million and 19.3 million. The cancer burden globally is expected to be raise 47% from 2020 to 2040 with 28.4 million cases 2 . Chemo resistance is an important phenomenon associated with the cell division. In addition, safety pro le and side effects are the major concerns with anticancer drugs. Therefore, the development of novel agents with increased e cacy while reducing the side effects will encourage the researchers towards the drug design and development.
1,2,3-Triazoles, are the most important nitrogen containing heterocycles, and these derivatives possess diverse biological properties Moreover, some of 1,2,3-triazole containing anticancer agents such as Derivatives of Pyridine are the privileged scaffolds with wide range of pharmacological properties, which plays an important role by inhibition of CDK, EGFR, PI3K, and RGGT etc in different cancers 3,4 . Anti-cancer agents like Masitinib and ABT-751 (E7010) containing pyridine as a core moiety have already been under clinical trials for the treatment of cancers, indicating the importance of pyridine derivatives in the discovery of anticancer agents 5 .All 1,2,3-triazole-pyridine hybrids Fig-2  A series of 1,2,3-triazole-pyridine hybrids Fig-3 was highly active against the two enzymes NAMPT and HDAC with IC 50 value 18-190 nM 8,9 . Hybrid molecules have the potential to reduce side effects and overcome the drug resistance since hybrids with two or more different pharmacophores may also own multiple action mechanism Considering the anti-cancer activity exhibited by both 1,2,3-triazole derivatives and the pyridine derivatives from literature search and that fusion of 1,2,3-triazole and pyridine could result in molecules having greater anti-cancer activity due to synergistic effect of both 1,2,3-triazole-pyridine scaffolds.
Hence, a series of 1,2,3-triazole-pyridine hybrids have been synthesized and evaluated for their in vitro anti-cancer activity using human cell lines with results supported by docking studies.

Biological Evaluation and Structural Activity Relationship (SAR):
The newly synthesized compounds (7a-l) were screened for their in vitro anti-cancer activity using two human cancer cell lines such as HT-1080 (Human bro sarcoma cells) and Caco-2 (Human colorectal adenocarcinoma cells) by MTT assay. Doxorubicin was used as a reference anti-cancer drug and the results were expressed in terms of IC 50 (µM) values as summarized in Table 1.
Among the synthesized compounds, 7b, and 7c shown more potent anti-cancer activity against HT-1080 and Caco-2 cell lines, compared to control drug. It showed that compound (7b) with 4-Bromo group on the phenyl ring exhibited more potent activity than the control doxorubicin. Similarly substitution with strong electron withdrawing agent like chloro group at ortho postion on the phenyl ring resulted compound 7c shown slightly decrease in activity compared to 7b. whereas Replacement of 4-bromo on the phenyl ring with 4-chloro Resulted compound 7d which showed lower activity than 7b. The unsubstituted and alkyl substitutions on the phenyl ring were not tolerated. Compound 7g with electron donating agent, 4methoxy substitution shown slightly decrease in activity compared to control drug. Shifting of methoxy substitution from ortho position to para position compound resulted in loss of activity. Molecular docking studies: The ligands were sketched in chemdraw and saved it in mol2 format. All the sketched molecules were converted to energy minimized 3D structures by using ligprep module for in-silico protein -ligand docking using Schrödinger 11.4. Each molecule was docked separately. Initially the molecule was loaded; torsions were set and saved it in PDB format. All the heteroatoms were removed from the 5FGK. PDB (CDK8-CYCC IN COMPLEX WITH 8-[3-(3-Amino-1H-indazol-6-yl)-5-chloro-pyridine-4-yl]-2,8-diaza-spiro [4.5] decan-1-one). The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene to make complex receptor free of any ligand before docking. Receptor grid generated using glide module. The best conformation was chosen with the lowest docked energy after the docking search was completed. The interactions of 5FGK protein and ligand conformations, including hydrogen bonds and the bond lengths were analyzed. Molecular docking study was performed by using maestro (Schrödinger 11.4) which was a suite of automated docking tools and was used to predict the a nity, activity, binding orientation of ligand with the target protein and to analyze best conformations, the protein with all the compounds (7a-l) were loaded individually evaluated. We observed that in the compound 7b molecule two aromatic rings showing Π-Π stacking interactions with aminoacids histidine and tryptophan, Triazole & Imidazole rings showing cation pi interaction with amino acid arginine, pyridine moiety showing Π-Π stacking interactions with aminoacid Tyrosineand showed best t, potent dock score then compared with doxorubicin.

Conclusion
In conclusion, a series of 1,2,3-triazole linked pyridine hybrids derivatives (7a-l) were synthesized and characterized by 1 HNMR, 13 CNMR, and mass spectral analysis. Further, these synthesized compounds were tested for their anticancer activity against two human cancer cell lines, HT-1080(Human bro sarcoma cells), Caco-2 (Human colorectal adenocarcinoma cells). Doxorubicin is used as a control.
Among them, compounds 7b, 7c and 7f showed potent anticancer activity compared to control drug. Docking results shown that compounds exhibited potent dock score then compared with doxorubicin.
This MTT assay is a colorimetric assay that measures the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it gets reduced and forms insoluble dark purple formazan crystals. The crystals are then solubilized with an organic solvent DMSO and the soluble formazan product is measured by reading absorbance at 570 nm with spectrophotometer. Since reduction of MTT can only occur in metabolically active cells, the level of activity is the measure of the viability of the cells.