Anti-tumor Activity of N4 [(E)-1-(2-hydroxyphenyl) Methylidene], N4-[(E)-2-Phenylethylidene], N4 [(E,2E)-3-Phenyl-2-propenylidene], and N4 [(E)ethylidene] Isonicotinohydrazide on K562 and Jurkat Cell Lines

Using the water eliminated mechanism, reactions of 4-pyridinecarboxylic acid hydrazide and salicylaldehyde, benzaldehyde, cinnamaldehyde, and formaldehyde afforded the corresponding N4[(E)-1-(2-hydroxyphenyl) methylidene] (NHPM), N4-[(E)-2-phenylethylidene] (NPI), N4[(E,2E)-3-phenyl-2-propenylidene] (NPPI), and N4[(E) ethylidene] (NEI) isonicotinohydrazide, in high yields, after several minutes, as reported. These new compounds have shown antitumor activity against two kinds of cancer cells, which are K562 (human chronic myeloid leukemia) and Jurkat (human T lymphocyte carcinoma).


INTRODUCTION
Nitrogen-containing heterocyclic compounds are widespread in nature, and their applications to biologically active pharmaceuticals, agrochemicals, and functional materials are becoming more and more important. [1] Due to the existence of the pyridine ring in the structure of many biologically active compounds, for example herbicides such as, nicotinic acid, vitamin B 5 , nicotinamide vitamin B 6 , pyridonal and pyridonamine, and nicotinamide adenine dinucleotide, the pyridine ring has been studied extensively, both experimentally and theoretically. [2] The development of new efficient methods to synthesize N-heterocycles with structural diversity is one major interest of modern synthetic organic chemists. [3][4][5] Among a large variety of nitrogencontaining heterocyclic compounds, those containing benzaldehyde, cinnamaldehyde, and formaldehyde were Merck chemicals, and were used without further purification. Organic solvents were of reagent grade. Electronic spectra were recorded by Camspec UV-Visible spectrophotometer model Wpa bio Wave S2 100. The IR spectra were recorded using the FT-IR Bruker Tensor 27 spectrometer. 1 HNMR was recorded on a Bruker AVANCE DRX 500 spectrometer. All the chemical shifts were quoted in ppm using the high-frequency positive convention; 1 H NMR spectra was referenced to external SiMe 4 . The percent composition of elements was obtained from the Microanalytical Laboratories, Department of Chemistry, OIRC, Tehran.

Cell culture
The human chronic myeloid leukemia: K562 cell line and the human T lymphocyte carcinoma: Jurkat cell line, used for treatment with the drugs, was provided. K562 and Jurkat cells were grown at 37°C in an atmosphere containing 5% CO 2 , with RPMI-1640 MEDIUM HEPES Modification, with L-glutamine and 25 mM HEPES (SIGMA-ALDRICH CHEMIE GmbH), supplemented with 10% heatinactivated fetal bovine serum (FBS) (Gibco), 2.7% sodium bicarbonate, and 500 mg/L ampicillin.

In vitro activities
The compounds were assayed for cytotoxicity in vitro against K562 (human chronic myeloid leukemia) cells and Jurkat (human T lymphocyte carcinoma) cells.

The two cell lines were provided by the Pasteur Institute
Laboratory of Natural and Biomimetic in Iran. The procedure for cytotoxicity studies was similar to that reported earlier. [9] Briefly, in order to calculate the concentration of each drug that produced a 50% inhibition of cell growth (IC 50 ), 190 mL of cell suspension (5 × 10 4 cell/mL) was exposed to various concentrations of compounds dissolved in sterile Ethanol. The final concentration of Ethanol in the growth medium was 2% (v/v) or lower; the concentrations were without effect on cell replication. [10,11] After incubation periods of 72 hours for all cell lines, the cell concentrations were determined both in the control and in drug-treated cultures. All experiments were carried out six times and six series.

Preparation of NHPM, NPI, NPPI, and NEI compounds
The reaction of aldehydes with 4-pyridinecarboxylic acid hydrazide, results in the formation of Isonicotinohydrazide compounds by the water eliminated mechanism. These compounds are quite stable and can be stored without any appreciable change. These are insoluble in common organic solvents, such as, dichloromethane, chloroform, hexane, and benzene. However, they are soluble in ethanol, THF, DMSO, and DMF. Their structures have been characterized by elemental analysis, 1 H NMR, 13 CNMR, and IR. Their elemental analyses are in agreement with their proposed formula. The spectral data of the compounds have a good relationship with the literature data.
NHPM, NPI, NPPI, and NEI compounds have been tested against two human cancer cell lines : K562 and Jurkat. The IC 50 cytotoxicity values of the compounds were compared with those found for starting organic bases as well as for some of the anti-cancer agents used nowadays. [10][11][12] Cytotoxicity studies The general method used for testing on anti-tumor properties of these compounds is the standard testing method that has been previously described in greater detail: After 12 hours of pre-incubation at 37°C in 5% CO2 and 100% humidity atmosphere, the new compounds were added in the following concentration ranges: 0.  Table 1.

CONCLUSION
It is clear from the earlier discussion that NHPM, NPI, NPPI, and NEI compounds offer a new outlook for chemotherapy. The results of antitumor activity show that the compounds exhibit antitumor properties, and it is important to note that they show enhanced inhibitory activity. The mechanism by which these compounds act as antitumor agents is apoptosis. It has also been proposed that concentration plays a vital role in increasing the degree of inhabitation. [13][14][15][16]

ACKNOWLEDGMENTS
We gratefully acknowledge the financial support from the Research Council of Ardabil Islamic Azad University and the