Synthesis, Characterization, and Cytotoxicity of Some New 5-Aminopyrazole and Pyrazolo[1,5-a]pyrimidine Derivatives

5-Amino-N-aryl-3-[(4-methoxyphenyl)amino]-1H-pyrazole-4-carboxamides 4a–c were synthesized by the reaction of N-(aryl)-2-cyano-3-[(4-methoxyphenyl)amino]-3-(methylthio)acrylamides 3a–c with hydrazine hydrate in ethanol. The reaction of 5-amino-N-aryl-1H-pyrazoles 4a–c with acetylacetone 5 or 2-(4-methoxybenzylidene)malononitrile 8 yielded the pyrazolo[1,5-a]pyrimidine derivatives 7a–c and 10a–c, respectively. The structures of the synthesized compounds were established based on elemental analysis and spectral data (IR, MS, 1H-NMR, and 13C-NMR). Representative examples of the new synthesized products were screened for their in vitro cytotoxic activity against Ehrlich Ascites Carcinoma (EAC) cells.


Introduction
The design and synthesis of novel mono-, di-, and polycyclic fused nitrogen heterocyclic compounds is among the active principles in chemical materials, particularly those displaying strategic roles in the development of different industries, especially from the biological point of view. Pyrazoles and related fused heterocyclic derivatives have great importance in the medicinal field as biological agents such as antimicrobial, anti-inflammatory, and anticancer agents [1][2][3][4][5][6]. The importance of pyrazole and pyrazolopyrimidine in the pharmacological industry as antitumor agents [7,8] promoted us to synthesize new derivatives that may serve as new chemotherapeutic drugs.
In light of these facts and as a continuation of our previous work in the synthesis of novel compounds with promising biological applications [14][15][16], we report herein the synthesis of new 5-aminopyrazole and pyrazolo [1,5-a]

Results and Discussion
Chemistry N-substituted cyanoacetamide derivatives 1a-c [17] were utilized as key starting materials in the synthesis of novel heterocyclic compounds (Schemes 1, 3, and 4). Cyanoacetamide derivatives 1a-c were reacted with 4-methoxyphenylisothiocyanate in absolute ethanol in the presence of an equimolar amount of potassium hydroxide to give the corresponding intermediates (2a-c); when the latter was alkylated with methyl iodide in ethanol, it afforded the novel ketene N,S-acetals 3a-c. The structures of 3a-c were established on the basis of their elemental analysis and spectral data (MS, IR, 1 H-NMR, and 13 C-NMR).

Sch. 2. Mechanism for the formation of 5-amino-N-aryl-3-[(4-methoxyphenyl)amino]-1Hpyrazole-4-carboxamides 4a-c
Compounds 4a-c were reacted with acetylacetone 5 in boiling glacial acetic acid to afford the corresponding new pyrazolo[1,5-a]pyrimidines 7a-c. The formation of compounds 7a-c was therefore assumed to proceed via an initial attack of the exocyclic amino group of 4a-c on the keto group of the 1,3-dicarbonyl compound 5, followed by intramolecular cyclization via the elimination of water (Scheme 3). The structures of 7a-c were established on the basis of their elemental analysis and spectral data (MS, IR, 1 H-NMR, and 13 C-NMR

Experimental
All melting points were measured on a Gallenkamp melting point apparatus and are uncorrected. The IR spectra were recorded (KBr disk) on a Perkin Elmer 1650 FTIR instrument. The 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) spectra were recorded on a Varian spectrometer using DMSO-d 6 or CDCl 3 as the solvent and TMS as an internal standard. Chemical shifts are reported in ppm. Mass spectra were recorded on a Varian MAT 112 spectrometer at 70 eV. Elemental analyses were performed at the Microanalytical Center, Cairo University, Egypt.
The progress of the reactions was monitored by thin-layer chromatography (TLC) using aluminum sheets coated with silica gel F 254 (Merck) with viewing under short-wavelength UV lamp detection. All evaporations were carried out under reduced pressure at 40 o C.
Reagents and solvents used in the synthesis were purchased from Sigma-Aldrich. Compounds 1a-c were prepared according to the reported procedure [17].

Biological Experiments
In Vitro Cytotoxic Activity Doxoroubicin, the reference drug which was used in this study, is one of the most effective antitumor agents used to produce regressions in acute leukemia, Hodgkin disease, and other lymphoma. The relationship between the survival ratio and drug concentration was plotted to obtain the survival curve of the Ehrlich Ascites Carcinoma (EAC) cells. The parameter IC 50 is the concentration of the drugs inducing 50% inhibition of cell growth of the treated cells in comparison with the growth of the control cells.

Procedure
The EAC cells were obtained by needle aspiration of the ascetic from preinoculated mice under aseptic conditions. The tumor cells suspension (2.5×10 6 cells/ml) was prepared in RPMI-1640 media. The tested compounds were prepared with various dilutions by dissolving: 100, 50, 25, and 10 µg in DMSO (1 ml

Conclusion
In conclusion, N-substituted cyanoacetamide derivatives 1a-c were used as starting materials for the synthesis of some new N-(aryl)-2-cyano-3-(methylthio)acrylamides 3a-c, 5-amino-1H-pyrazoles 4a-c, and pyrazolo[1,5-a]pyrimidines 7a-c and 10a-c. The new synthesized compounds were characterized by analytical and spectroscopic data. Some selected new compounds were screened for their potential cytotoxic activity. The results of the cytotoxicity for the tested compounds against Ehrlich Ascites Carcinoma (EAC) cells indicated that the pyrazolo[1,5-a]pyrimidine derivatives 7a (IC 50 =10 µg/ml) and 10c (IC 50 =25 µg/ml) were found to have the most potent growth inhibitory activity against EAC cells in comparison with the reference drug, Doxorubicin (IC 50 =37.4 µg/ml). Accordingly, this class of compounds could be considered as useful templates for future development, derivatization, or modification to obtain more potent and selective antitumor agents.