Synthesis of podophyllotoxin-glycosyl triazoles via click protocol mediated by Silver (I) N -heterocyclic carbenes and their anti-cancer evaluation as Topoisomerase-II inhibitors

Herein we report the regioselective synthesis of podophyllotoxin-Glycosyl triazole hybrids catalysed by Ag(I)- N-heterocyclic carbene (Ag(I)-NHC) in a short reaction time (~30 min) at ambient conditions. In principle, it is the first report of Click alkyne-azide cycloaddition catalysed by Ag(I)-NHC catalyst and moreover, this new methodology yielded good results when compared with traditional CuAAC in terms of reaction time and selectivity. The synthesised compounds were further explored for in vitro anticancer activity against four human cancer cell lines Du145, HeLa, A-549, and MCF-7 and found that these synthesised compounds possess significant anticancer activity. Further, the compounds 5a and 5e were identified as promising leads due to their better activity across all cell lines than that of the standard drug etoposide. Molecular docking studies of 5a & 5e with DNA Topoisomerase-II were revealed that the free energy calculations of active compounds were in good agreement with observed IC 50 values.


3.1.General procedure for the synthesis of 4β-(prop-2-ynyloxy) epipodophyllotoxin (3)
Podophyllotoxin (1) (1 mmol) and propargyl alcohol (2) (1 mmol) was dissolved in dichloromethane (10 ml) and added slowly Boron trifloride etharate dropwise over a period of 10 minutes while maintaining the temperature at -20ºC and stirring was continued for 2 hours at the same temperature. After conversion was complete, the mixture is quenched by addition of pyridine and diluting with dichloromethane (40 ml). The organic layer was separated and the aqueous layer extracted with dichloromethane (2 x 20 ml). The combined organic layers were washed consecutively with cold dilute HCl and brine solution. The extracted organic solution was dried (anhydrous Na 2 SO 4 ) and evaporated under reduced pressure to afford a crude product which was subjected to column chromatography (silica gel, 60-120 mesh, eluent; nhexane/EtOAc gradient) to afford pure product (3). Yield: 45%; White powder solid; mp: 152-154 0 C;

3.2.General procedure for the synthesis of Sugar Azides (4a-l)
Sugar azides were synthesised by the reported procedures in the literature based on Bertho's method. Firstly, Sugars are acetylated adding an excess amount of acetic anhydride in anhydrous pyridine and the reaction mixture is allowed to stir overnight at room temperature. Concentrate the reaction mixture under reduced pressure and co-evaporate several times with Toluene to remove (traces of) Pyridine. Purify the residue with column chromatography with hexane:EtOAc (6:4). This should give you 95-97 % yield of your Alfa/beta product in 3:1 ratio. Pentacetylated sugars made to react with HBr in AcOH (33 %) at 0 °C. Then, stir the reaction mass at room temperature for 2 hours. The reaction mass was diluted with dichloromethane (40 ml) and water.
The organic layer was separated and the aqueous layer extracted with dichloromethane (2 x 20 ml). The combined organic layers were dried (anhydrous Na 2 SO 4 ) and evaporated under reduced pressure to afford a desired product glycosyl bromide in very high yield (~98 %). Pentacetylated glycosyl bromide was then refluxed for 2h at 100 °C with NaN 3 in the presence of PTC to yield corresponding glycosyl azides, which was subjected to column chromatography (silica gel, 60-120 mesh, eluent; n-hexane/EtOAc gradient) to afford pure products (4a-l).

3.3.General procedure for the synthesis of Podophyllotoxin-triazole hybrids (5a-l)
O-Propargylated podophyllotoxin (terminal alkyne) 3 (1 mmol) and Ag (I)-NHC (5 mol %) was dissolved in dry EtOH (10 ml) and added sugar azide (4a-l) (1 mmol). The reaction mass was stirred for 30 minutes. Complete consumption of starting material as judged by TLC and GC analysis. The reaction mass was evaporated under reduced pressure and diluting with dichloromethane (40 ml) and water. The organic layer was separated and the aqueous layer extracted with dichloromethane (2 x 20 ml). The combined organic layers were dried (anhydrous Na 2 SO 4 ) and evaporated under reduced pressure to afford a crude product which was subjected to column chromatography (silica gel, 60-120 mesh, eluent; n-hexane/EtOAc gradient) to afford pure products (5a-l).

in vitro Cytotoxicity assay
An in vitro cytotoxicity study was performed by adopting MTT assay. In this assay the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced by metabolically active cells by the action of dehydrogenase enzymes. The resulting intracellular purple formazan can be solubilised and quantified by spectrophotometric means.
The MTT reagent yields low background absorbance values in the absence of cells. For each cell type the linear relationship between cell number and signal produced is established, thus allowing an accurate quantification of changes in the rate of cell proliferation.