Synthesis and Antitumor Activity of 5-trifluoromethyl-2,4- Dihydropyrazol-3-one Nucleosides

2,4-Dihydropyrazole glucosides 3a-3c were prepared and tested for their antitumor activity. The structures of these compounds were established by 1 H and 13 C-NMR spectroscopy. Glucoside 3b shows an in vitro IC 50 value of 16.4 µM against proliferation of the human promyelotic leukemia (HL60) cell line.

The structures of all new compounds are fully consistent with the NMR data and elemental analysis.In particular, the β-configuration of the pyranosyl moieties was established by 1 H-NMR from a large coupling constant (J >5 Hz) for a diaxial interaction between H-1" and H-2" [26,27].
The effects of nucleosides 3a-c on proliferation of human promyelotic leukemia (HL60) and mouse T lymphocytic leukemia (EL4) cell lines were evaluated.The IC 50 values for compounds 3a-c indicate that HL60 cells are more sensitive than EL4 cells.

General
All reagents were purchased from commercial sources and used without further purification.Melting points were determined on Gallenkamp apparatus (Pyrex capillary) and are not corrected.All reactions were carried out under an inert atmosphere of nitrogen.Thin-layer chromatography (TLC) was performed on aluminum sheets coated with Kieselgel (silica gel,type 60 F 254 , Merck).Column chromatography was performed on Kieselgel 60.Elemental analyses were performed by the Central Laboratory Unit (CLU), UAE University.The IR spectra were recorded on a Shimadzu 940 spectrophotometer. 1 H-NMR and selected 13 C-NMR spectra were recorded on Varian XL200 and Bruker AM 300 MHz instruments by using solutions in CDCl 3 and TMS as an internal reference.Specific optical rotations (c = 0.1 g/100 mL in all cases, sodium D line, 25 °C) were obtained for solutions in CDCl 3 (3a-c) and MeOH (4), respectively.

MTT Cell Proliferation
All compounds tested were dissolved in DMSO (100 mM solution) and subsequently diluted in the culture medium before treatment of the cultured cells.Tested cells were plated in 96-well plates at a density 4×10 3 cells/well/100 µL of the proper culture medium and treated with the compounds at concentration 1.25 to 100 µM for 48h.In parallel, the cells were treated with 0.1% of DMSO as control.
A MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] Assay (Roche Molecular Biochemicals, 1 465 007) was performed 30 h later according to the instructions provided by Roche.This assay is based on the cellular cleavage of the tetrazolium salt, MTT, into a formazan that is soluble in cell culture medium and is measured at 550 nm directly in 96-well assay plates.Absorbance is directly proportional to the number of living cells in culture.Two types of cells were used in these studies, human promyelocytic leukemia HL60 and mouse T lymphocytic leukemia cell lines, provided by ATCC and cultivated in MEM (for HL60) or RPMI 1640 (for EL4) supplemented with 10% fetal bovine serum and 2 mM of L-glutamine.Tissue culture reagents were obtained from Gibco BRL.
The active analogues 3a and 3c are with a meta-trifluoromethyl group meta-fluoro atom, respectively.