Uses of Cyanoacetylhydrazine in Heterocyclic Synthesis: Novel Synthesis of Pyrazole Derivatives with Anti-tumor Activities

The reaction of cyanoacetylhydrazine with chloroacetyl chloride gave N'-(2-chloroacetyl)-2-cyanoacetohydrazide. The latter underwent cyclization to afford 1-(5 amino-3-hydroxy-1H-pyrazol-1-yl)-2-chloroethanone, which underwent nucleophilic substitution to give 3-(5-amino-3-hydroxy-1H-pyrazol-1-yl)-3-oxopropanenitrile. The latter two compounds were used as key synthons to synthesize new thiophene, pyran, thiazole and some fused heterocyclic derivatives. The antitumor activity of the newly synthesized compounds was evaluated against three human tumor cells lines, namely breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268) and some of these compounds were found to exhibit much higher inhibitory effects towards the three tumor cell lines than the Gram positive control doxorubicin.


Introduction
Cancer is a major public health problem in the world. Chemotherapy is still one of the primary modalities for the treatment of cancer. However, the use of this method is limited mainly due to the small number of the available chemotherapeutic agents to choose among them and also because the use of these agents is often accompanied by undesirable side effects. This clearly underlies the urgent need for developing novel chemotherapeutic agents with more potent antitumor activities and reduced side effects.
The reaction of compound 5 with benzaldehyde (9) gave the phenylmethylidene derivative 10. The latter showed interesting reactivity towards cyanomethylene reagents, namely malononitrile (6a) and ethyl cyanoacetate (6b) and afforded the pyrazole-1-yl-pyran derivatives 11a and 11b, respectively. The latter products underwent ready cyclization in sodium ethoxide solution to give the dihydropyrazolo [1,5-  Finally, the reactivity of compound 4 as an α-halocarbonyl compound to produce thiazole derivatives was investigated. Thus, the reaction of the active methylene reagents 6a,b and 15a,b with  The effect of the newly synthesized products was evaluated on the in-vitro growth of three human tumor cell lines representing different tumor types, namely, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268), after a continuous exposure of 48 h. The results are summarized in Table 1. All the compounds were able to inhibit the growth of the human tumor cell lines in a dose-dependentmanner. 5,8-Diamino-7-cyano-2-hydroxy-6-phenyl-6,7-dihydro1,5α pyrano[2,3-d] pyrimidine (12a), 2-(4-(5-amino-3-hydroxy-1H-pyrazol-1-yl)-3-phenylthiazol-2(3H)-ylidene)malononitrile (18a) and 2-(4-(5-amino-3-hydroxy-1H-pyrazol-1-yl)-3-phenylthiazol-2(3H)-ylidene) pentane-2,4-dione (18c) showed the best results, exhibiting the highest inhibitory effects of the tested compounds towards the three tumor cell lines, which are higher than that of the reference compound doxorubicin. On the other hand, compounds 17a, 17d, and 18d showed high growth inhibitory effect but such activity are lower than the reference, doxorubicin. Comparing the activities of 11a and 11b indicated that the presence of the CN group in 11a resulted a stronger growth inhibitory effect than COOC 2 H 5 group in compound 11b. Similarly comparing the reactivity of compounds 12a and 12b indicated that the presence of the CN group in 12a also is responsible of its higher reactivity over 12b. On the other hand, compound 17a with the CN group is the most active compound towards the three cancer cell lines among the pyrazole derivatives 17a-d, of course the presence of the CN group in 17a is responsible for such high activity. On the other hand, compound 17c where X = Y = COCH 3 is of lower activity than 17d with X = COCH 3 & Y = COOC 2 H 5 . Finally it worthy to refer to observation that indicated also that compound 18a showed the highest reactivity towards the three cancer cell line among compound 8a-d. On the other hand, compounds 5, 7a, 7b, 10 and 14 exhibited the lowest reactivity towards the cancer cell lines.
Cell cultures: Three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer) were used. MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and NCI-H460 and SF-268 were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL), at 37 °C in a humidified atmosphere containing 5% CO 2 . Exponentially growing cells were obtained by plating 1.5 × 10 5 cells/mL for MCF-7 and SF-268 and 0.75 × 10 4 cells/mL for NCI-H460, followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.
Tumor cell growth assay: The effects of compounds 3-18a-d on the in-vitro growth of human tumor cell lines were evaluated according to the procedure adopted by the National Cancer Institute (NCI, Cairo, Egypt). In the 'in-vitro Anticancer Drug Discovery Screen' that uses the protein-binding dye sulforhodamine B to assess cell growth [28]. Briefly, cells growing exponentially in 96-well plates were then exposed for 48 h to five serial concentrations of each compound, starting from a maximum concentration of 150 M. Following this exposure period adherent cells were fixed, washed, and stained. The bound stain was solubilized and the absorbance was measured at 492 nm in a plate reader (Bio-Tek Instruments Inc., Powerwave XS, Wincoski, San Diego, CA, USA). For each test compound and cell line, a dose-response curve was obtained and the growth inhibition of 50% (GI 50 ), corresponding to the concentration of the compounds that inhibited 50% of the net cell growth, was calculated as described elsewhere [29]. Doxorubicin was used as a positive control and tested in the same manner.

Chemistry
All melting points are uncorrected. IR spectra were recorded for KBr discs on a Pye Unicam SP-1000 spectrophotometer. 1 H-NMR and 13 C-NMR spectra were measured on a Varian EM-390-200 MHz in DMSO as solvent using TMS as internal standard, and chemical shifts are expressed as δ. Analytical data were obtained from the Microanalytical Data Unit at Cairo University, Giza, Egypt and the Microanalytical Data Unit at Erlangen University, Erlangen, Germany.  (5). To a well stirred solution of compound 4 (1.75 g, 0.01 mol) in ethanol (30 mL) at 60 °C was added dropwise a solution of potassium cyanide (1.70 g, 0.02 mol in 5 mL water). Stirring was continued for 1 h and the resulting reaction mixture was poured onto crushed ice then acidified with concentrated hydrochloric acid (to pH 6). The formed solid product was collected by filtration. Crystallized from ethanol to give pale yellow crystals, yield 1.