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Article

Synthesis and Antitumor Activity of 5-Trifluoromethyl-2,4- dihydropyrazol-3-one Nucleosides

1
Department of Chemistry, Faculty of Science, UAE University, Al-Ain P.O. Box 17551, UAE
2
Department of Biochemistry, Faculty of Medicine & Health Sciences, UAE University, Al-Ain P.O.Box 17666, UAE
*
Author to whom correspondence should be addressed.
Molecules 2004, 9(3), 109-116; https://doi.org/10.3390/90300109
Submission received: 13 January 2004 / Accepted: 20 January 2004 / Published: 28 February 2004
(This article belongs to the Special Issue Biologically Relevant Heterocyclic Compounds)

Abstract

:
2,4-Dihydropyrazole glucosides 3a-3c were prepared and tested for their antitumor activity. The structures of these compounds were established by 1H and 13C-NMR spectroscopy. Glucoside 3b shows an in vitro IC50 value of 16.4 µM against proliferation of the human promyelotic leukemia (HL60) cell line.

Introduction

Fluorinated compounds in general and fluorinated heterocycles in particular are the focus of much interest in modern medicinal chemistry. A variety of pharmacological agents bearing a trifluoromethyl group are of special interest as they exhibit wide ranging biological properties [1,2,3,4]. Many efficient methods for the introduction of a trifluoromethyl group into organic molecules have been developed. Trifluoromethyl-1,3-dicarbonyl compounds have been proven to be versatile building blocks for a wide variety of trifluoromethyl-substituted heterocyles. These include pyrazoles [5,6,7], isoxazoles [8,9], thiazoles [10], pyridines [11,12], pyrimidines [13,14], and pyrones [15]. The trifluoromethyl-substituted compounds have been reported to possess biological activities as herbicides [16], fungicides [17], analgesic agents [18], antipyretic agents [19], and inhibitors for platelet aggregation [20]. Recently, much attention has been focused on pyrazoles as antvirial and anticancer agents after the discovery of the pyrazole C-glycoside pyrazofurin [21,22]. This report describes the synthesis and anticancer evaluation of several trifluoromethyl-substituted pyrazole N-nucleosides.
Molecules 09 00109 i001

Results and Discussion

Chemistry

5-Trifluoromethyl-2,4-dihydropyrazol-3-one (1) was the chosen starting material for the synthesis of the new trifluoromethyl-substituted pyrazol-3-ones 2a-c and their corresponding glucosides 3a-c, 4 (Scheme 1) Compound 1 is readily available by the reaction of ethyl 4,4,4-trifluoro-3-oxobutanoate with hydrazine in ethanol in the presence of sulfuric acid [5,23]. Coupling reactions of 1 with aryldiazonium reagents were conducted by using a well-developed methodology [23,24,25] and the desired hydrazones 2a-c were obtained in high yields.
In order to obtain glucosides 3a-c as single nucleosides, compounds 2a-c were treated with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) in the presence of a catalytic amount of ammonium sulfate, and the resultant trimethylsilyl derivatives, without isolation, were allowed to react with β-D-glucose pentaacetate in 1,2-dichloroethane in the presence of tin(IV) chloride. Nucleosides 3a-c were thus obtained in good yield. Selected nucleoside 3a was then subjected to deacetylation at room temperature in a solution of triethylamine in anhydrous methanol to give 2-(β-D-glucopyranosyl)-4-(3'-trifluoro-methylphenylhydrazono)-5-trifluoromethyl-2,4-dihydropyrazol-3-one (4) in high yield.
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 1H-NMR from a large coupling constant (J >5 Hz) for a diaxial interaction between H-1" and H-2" [26,27].
Scheme 1.
Scheme 1.
Molecules 09 00109 g001

Anticancer activity.

The effects of nucleosides 3a-c on proliferation of human promyelotic leukemia (HL60) and mouse T lymphocytic leukemia (EL4) cell lines were evaluated. The IC50 values for compounds 3a-c indicate that HL60 cells are more sensitive than EL4 cells.
Table 1. Evaluation of pyrazole nucleosides
Table 1. Evaluation of pyrazole nucleosides
CompoundRIC50 (μM)
HL60 proliferation (human) EL4 proliferation (mouse)
3am-CF320.2±1.725.3±0.9
3bp-F16.4±1.819.4±2.0
3cm-F27.2±1.328.7±2.0
The results presented in Table 1 indicate that these newly synthesized compounds exhibit promising anticancer activities. The most potent compound 3b contains a fluorine atom in the para position of the aromatic ring. The less active analogues 3a and 3c are substituted with a meta-trifluoromethyl group and meta-fluoro atom, respectively.

Conclusions

New 2,4-dihydropyrazol-3-one nucleosides 3a-c, 4 were synthesized. Of these, the nucleosides 3a‑c were tested for their anticancer activities. The collective results clearly show the that nuceloside 2-(2",3",4",6"-tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-fluorophenylhydrazono)-5-trifluoromethyl-2,4-dihydropyrazol-3-one (3b), exhibits better biological activities than its structurally related analogues 3a and 3c.

Experimental

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 F254, 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. 1H-NMR and selected 13C-NMR spectra were recorded on Varian XL200 and Bruker AM 300 MHz instruments by using solutions in CDCl3 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 CDCl3 (3a-c) and MeOH (4), respectively.

5-Trifluoromethyl-2,4-dihydropyrazol-3-one (1).

This compound was obtained by the reaction of ethyl 4,4,4-trifluoro-3-oxobutanoate with hydrazine hydrate in ethanol in the presence of sulfuric acid by using a general literature procedure [5,23]: yield 85%; m.p. 218-220 °C; 1H-NMR δ 2.41 (s, 2H), 8.72 (bs, exchangeable with D2O); 13C‑NMR δ 14.2 (C-4), 114.0 (q, CF3), 143.0 (C-5), 159.7 (C-2); IR, 3300 (N-H), 1600 (C=O), 1500 cm-1(C=N).

General Method for the Preparation of 4-Arylhydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-ones (2a-c).

To a cold solution of 5-trifluoromethyl-2,4-dihydropyrazol-3-one (1, 0.76 g, 5 mmol) in ethanol (25 mL) containing sodium acetate (0.82 g, 10 mmol), a solution of an appropriate aryldiazonium chloride (5 mmol/2 mL H2O) was added dropwise with stirring at 0-5 oC. The reaction mixture was stirred at room temperature for 3 h and then the precipitated crude product was filtered, washed with cold H2O (2 x 10 mL), dried and crystallized from 95% ethanol.
4-(3'-Trifluoromethylphenylhydrazono)-5-trifluoromethyl-2,4-dihydropyrazol-3-one (2a): yield 80%; yellow crystals; m.p. 206-208 °C; Rf = 0.65 (EtOAc/hexanes, 1:4); 1H-NMR δ 7.51-7.68 (m, 4H), 8.26 (s, exchangeable with D2O), 13.68 (bs, exchangeable with D2O); 13C-NMR δ 113.5, 113.6, 116.6, 123.5, 123.6, 130.6, 140.9, 159.4; IR, 3410 (NH), 3060 (aromatic C−H), 1680 (C=O), 1600 (C=C),1559 (C=N), 1523 cm-1(N−N); Analysis. Calculated for C11H6F6N4O (324.04): C, 40.75; H, 1.87; N, 17.28. Found: C, 40.75; H, 1.86; N, 17.27.
4-(4'-Fluorophenylhydrazono)-5-trifluoromethyl-2,4-dihydropyrazol-3-one (2b): yield 75%; yellow crystals; m.p. 212-214 °C; Rf = 0.7 (EtOAc/hexanes,1:4); 1H-NMR δ 2.48 (s, 1H, H-4), 7.10-7.20 (m, 2H), 7.33-7.50 (m, 2H), 8.60 (s, exchangeable with D2O), 12.96 (bs, exchangeable with D2O); 13C‑NMR δ 116.8, 117.1, 118.3, 118.4, 160.0, 161.0, 164.0, 179.0; IR, 3450 (NH), 3070 (aromatic C−H), 2985 (aliphatic C−H), 1675 (C=O), 1600 (C=C),1550 (C=N), 1522 cm-1(N−N); Analysis. Calculated for C10H6F4N4O (274.05): C, 43.81; H, 2.21; N, 20.43. Found: C, 43.82; H, 2.19; N, 20.42.
4-(3'-Fluorophenylhydrazono)-5-trifluoromethyl-2,4-dihydropyrazol-3-one (2c): yield 78%; brown crystals; m.p. 201-203 °C; Rf = 0.72 (EtOAc/hexanes,1:4); 1H-NMR δ 6.90 (t, 1H), 7.17-7.29 (m, 2H), 7.36-7.40 (m, 1H), 7.80 (s, exchangeable with D2O), 13.56 (bs, exchangeable with D2O); 13C-NMR δ 103.8, 104.1, 112.6, 112.7, 114.1, 114.3, 131.2, 131.3, 141.0, 162.0; IR: 3450 (NH), 3060 (aromatic C−H), 2995 (aliphatic C−H), 1670 (C=O), 1560 (C=C),1550 (C=N), 1528 cm-1(N−N); Analysis. Calculated for C10H6F4N4O (274.05): C, 43.81; H, 2.21; N, 20.43. Found: C, 43.81; H, 2.20; N, 20.42.

General Procedure for the Synthesis of 2-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-aryl-hydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-ones (3a-c).

A mixture of a 4-arylhydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-one (2a-c, 10 mmol), HMDS (60 mL), and (NH4)2SO4 (125 mg) was heated under reflux for 6 h. Excess HMDS was removed by distillation and then residual HMDS was removed under a reduced pressure. A solution of the resultant silyl intermediate in anhydrous 1,2-dichloroethane (20 mL) was stirred and treated with a solution of β-D-glucose pentaacetate (9 mmol) in anhydrous 1,2-dichloroethane (20 mL). Following cooling to 5 °C and then addition of anhydrous SnCl4 (1.6 mL), the mixture was stirred at 23 °C until the reaction was completely as judged by TLC (EtOAc/hexanes, 1:4). The mixture was diluted with CHCl3 (150 mL), washed with a saturated solution of NaHCO3 (50 mL) and water (2 x 25 mL), and then dried (Na2SO4). Removal of solvent under a reduced pressure followed by column chromatography and then crystallization from 95% EtOH gave the analytically pure compounds 3a-c.
2-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-5-trifluoro-methyl-2,4-dihydropyrazol-3-one (3a): Yield 78% after silica gel chromatography (EtOAc/hexanes, 1:9); m.p. 235-238 oC; Rf = 0.3 (EtOAc/hexanes,1:4); [ α ] D 25 = 38.3; 1H-NMR δ 1.98-2.06 (4s, 12H), 3.90 (m, 2H), 4.09 (m, 1H), 5.12 (m, 1H), 5.36 (m , 1H), 5.50 (m, 1H), 6.26 (d, J = 6.0 Hz, 1H, H1'), 7.49-7.63 (m, 4H), 13C-NMR δ 20.3, 20.4, 20.5, 20.7, 61.6, 67.6, 68.3, 73.4, 74.3, 80.2, 113.5, 113.6, 119.6, 123.4, 123.7, 130.6, 132.1, 132.8, 140.6, 158.0, 168.6, 168.7, 169.2, 170.2, 170.5; IR, 3090 (aromatic C−H), 2998 (aliphatic C−H), 1755 (acetyl C=O), 1689 (C=O), 1620 (C=C), 1560 (C=N), 1528 (N−N), 1040 cm-1(C−O−C); Analysis: Calculated for C25H24F6N4O10•H2O (672.14): C, 44.63; H, 3.86; N, 8.33. Found: C, 44.60; H, 3.88; N, 8.35.
2-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(4'-fluorophenylhydrazono)-5-trifluoromethyl-2,4-dihydropyrazol-3-one (3b): Yield 65% after silica gel chromatography (EtOAC/hexanes, 1:9); m.p. 159 °C; Rf = 0.3 (EtOAc/hexanes, 1:4); [ α ] D 25 = +52.0; 1H-NMR δ 1.90-2.08 (4s, 12H), 3.82 (m, 2H), 4.09 (s, 1H, H-4), 4.22 (m, 1H), 5.15 (m , 2H), 5.53 (m, 1H), 5.96 (d, J = 5.4 Hz, 1H, H1°), 7.01-7.17 (m, 2H), 7.33-7.40 (m, 2H); 13C-NMR δ 20.3, 20.5, 20.7, 62.0, 68.0, 68.5, 73.8, 73.9, 80.8, 115.6, 121.3, 123.2, 129.7, 150.4, 159.0, 168.7, 169.3, 170.2, 170.5; IR, 3080 (aromatic C−H), 2995 (aliphatic C−H), 1750 (acetyl C=O), 1680 (C=O), 1610 (C=C), 1560 (C=N), 1526 (N−N), 1028 cm-1(C−O−C); Analysis. Calculated for C24H24F4N4O10•H2O (622.14): C, 46.29; H, 4.17; N, 9.00. Found: C, 46.25; H, 4.22; N, 9.02.
2-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-fluoro-phenylhydrazono)-5-trifluoromethyl-2,4-dihydropyrazol-3-one (3c): yield 68% after silica gel chromatography (EtOAc/hexanes, 9:1); m.p. 123-125 oC; Rf = 0.3 (EtOAC/hexanes,1:4); [ α ] D 25 = +36.8; 1H-NMR δ 1.90-198 (4s, 12H), 3.81 (m, 2H), 4.12 (m, 1H), 5.02 (m, 1H), 5.36 (m , 1H), 5.65 (m, 1H), 5.70 (d, J = 6.2 Hz, 1H, H1°), 7.15-7.40 (m, 3H); 13C-NMR δ 20.4, 20.5, 20.6, 20.8, 76.6, 68.3, 69.7, 73.5, 74.3, 80.3, 103.7, 104.2, 112.7, 114.2, 114.6, 122.9, 128.3, 131.2, 141.6, 158.1, 168.6, 169.3, 170.2, 170.6; IR, 3070 (aromatic C−H), 2996 (C−H), 1750 (acetyl C=O), 1682 (C=O), 1604 (C=C), 1558 (C=N), 1528 (N−N), 1026 cm-1(C−O−C); Analysis. Calculated for C24H24F4N4O10•H2O (622.14): C, 46.29; H, 4.17; N, 9.00. Found: C, 46.27; H, 4.20; N, 9.10.
2-(β-D-glucopyranosyl)-4-(3'-trifluoromethyl-phenylhydrazono)-5-trifluoromethyl-2,4-dihydropyrazol-3-one (4): yield 88% after silica gel chromatography (EtOAc/MeOH, 9:1); m.p. 196-199 °C (decomp.); [ α ] D 25 = +25.3; 1H-NMR δ 3.83-4.58 (m, 6H), 4.17 (m, exchangeable with D2O, 4H), 5.22 (m, 1H), 5.53 (d, J = 8.4 Hz, 1H, H-1°), 7.44-7.71 (m, 4H), IR, 3070 (aromatic C−H), 2995 (aliphatic C−H), 1680 (C=O), 1612 (C=C), 1560 (C=N), 1528 (N−N), 1023 cm-1(C−O−C); Analysis. Calculated for C17H16F6N4O6•2H2O (522.10): C, 39.07; H, 3.83; N, 10.72. Found: C, 38.96; H, 3.87; N, 10.70.

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 4×103 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.

Acknowledgements

The authors wish to thank the Research Affairs Committee at the UAE University for the financial support under contract no 09-03-2-11/03.

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MDPI and ACS Style

Abdou, I.M.; Saleh, A.M.; Zohdi, H.F.. Synthesis and Antitumor Activity of 5-Trifluoromethyl-2,4- dihydropyrazol-3-one Nucleosides. Molecules 2004, 9, 109-116. https://doi.org/10.3390/90300109

AMA Style

Abdou IM, Saleh AM, Zohdi HF. Synthesis and Antitumor Activity of 5-Trifluoromethyl-2,4- dihydropyrazol-3-one Nucleosides. Molecules. 2004; 9(3):109-116. https://doi.org/10.3390/90300109

Chicago/Turabian Style

Abdou, Ibrahim M., Ayman M. Saleh, and Hussein F.. Zohdi. 2004. "Synthesis and Antitumor Activity of 5-Trifluoromethyl-2,4- dihydropyrazol-3-one Nucleosides" Molecules 9, no. 3: 109-116. https://doi.org/10.3390/90300109

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