Synthesis and antitumor activity of 5-trifluoromethyl-2,4-dihydropyrazol-3- one nucleosides

Article abstract of DOI:10.3390/90300109

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.

Full text of DOI:10.3390/90300109

Molecules 2004, 9, 109–116
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis and Antitumor Activity of 5-Trifluoromethyl-2,4-
dihydropyrazol-3-one Nucleosides
Ibrahim M. Abdou*,1, Ayman M. Saleh 2 and Hussein F. Zohdi 1
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; e-mail: i.abdou@uaeu.ac.ae; Homepage:
http://faculty.uaeu.ac.ae/~iabdou/
Received: 13 January 2004 / Accepted: 20 January 2004 / Published: 28 February 2004
Abstract: 2,4-Dihydropyrazole glucosides 3a-3c were prepared and tested for their
1
13
antitumor activity. The structures of these compounds were established by H and C-
NMR spectroscopy. Glucoside 3b shows an in vitro IC50 value of 16.4 µM against
proliferation of the human promyelotic leukemia (HL60) cell line.
Keywords: Trifluoromethylpyrazole; glucopyranose; antitumor agents.
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 [1-4]. 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 [5-7], isoxazoles [8,9],

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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.
O
H
N
NH
2
N
OH
OH
O
OH
OH
Pyrazofurin
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 [23-25] 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].

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Scheme 1
F C
CF
3
3
N Cl
2
N
N
ethanol
R
N
N
N
H
+
o
O
AcONa/0 C
N
O
R
H
H
2a-c
1
HMDS/(NH ) SO
4 2
4
OAc
O
AcO
AcO
OAc
OAc
(CH )Cl / SnCl
2
2
4
CF
3
CF
CF
3
3
OH
OAc
N
N
N
N
N
H
N
H
O
O
Et N/MeOH
3
HO
HO
AcO
AcO
N
N
R
OH
O
O
OAc
4
3a-c
Compound
2a, 3a
R
m-CF3
p-F
2b, 3b
2c , 3c
m-F
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
IC50 (µM)
HL60 proliferation (human)
20.2±1.7
Compound
R
m-CF3
p-F
EL4 proliferation (mouse)
25.3±0.9
3a
3b
3c
16.4±1.8
27.2±1.3
19.4±2.0
28.7±2.0
m-F
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

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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.
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.
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
13
spectrophotometer. 1H-NMR and selected C-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,
°
1
23]: yield 85%; m.p. 218-220 C; H-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).

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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
o
chloride (5 mmol/2 mL H2O) was added dropwise with stirring at 0-5 C. 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
13
(s, exchangeable with D2O), 13.68 (bs, exchangeable with D2O); C-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
°
1
crystals; m.p. 212-214 C; Rf = 0.7 (EtOAc/hexanes,1:4); H-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),
13
7.36-7.40 (m, 1H), 7.80 (s, exchangeable with D2O), 13.56 (bs, exchangeable with D2O); C-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

Molecules 2004, 9
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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,
25
D
o
1
1:9); m.p. 235-238 C; Rf = 0.3 (EtOAc/hexanes,1:4);
[
α
]
= 38.3; H-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), C-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,
13
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.
25
D
°
1
159 C; Rf = 0.3 (EtOAc/hexanes, 1:4);
[
α
]
= +52.0; H-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
13
(m, 2H), 7.33-7.40 (m, 2H); C-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.
25
D
o
1
123-125 C; Rf = 0.3 (EtOAC/hexanes,1:4);
[
α
]
= +36.8; H-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
13
(m, 3H); C-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.);
25
D
[
α
]
= +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

Molecules 2004, 9
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(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.
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