Year : 2010  |  Volume : 2  |  Issue : 4  |  Page : 399-402 Table of Contents     

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

1 Department of Chemistry, Islamic Azad University, Young Researchers Club, Ardabil, Iran
2 Department of Chemistry, Islamic Azad University, Ardabil, Iran
3 Moravvej Student-Research Institute, Ardabil Branch, Ardabil, Iran
4 Department of Medicinal Sciences, Islamic Azad University, Ardabil, Iran

Date of Web Publication14-Oct-2010

Correspondence Address:
S Ghammamy
Department of Chemistry, Islamic Azad University, Ardabil
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DOI: 10.4103/0975-1483.71638

PMID: 21264102

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Using the water eliminated mechanism, reactions of 4-pyridinecarboxylic acid hydrazide and salicylaldehyde, benzaldehyde, cinnamaldehyde, and formaldehyde afforded the corresponding N 4 [(E)-1-(2-hydroxyphenyl) methylidene] (NHPM), N 4 -[(E)-2-phenylethylidene] (NPI), N 4 [(E,2E)-3-phenyl-2-propenylidene] (NPPI), and N 4 [(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).

Keywords: 4-pyridinecarboxylic acid hydrazide, isonicotinohydrazide, Jurkat, K562

How to cite this article:
Shabani F, Ghammamy S, Jahazi A, Siavoshifar F. 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. J Young Pharmacists 2010;2:399-402

How to cite this URL:
Shabani F, Ghammamy S, Jahazi A, Siavoshifar F. 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. J Young Pharmacists [serial online] 2010 [cited 2013 Apr 19];2:399-402. Available from: /text.asp?2010/2/4/399/71638

   Introduction Top

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 nitrogen-containing heterocyclic compounds, those containing bridgehead hydrazines have received considerable attention because of their pharmacological properties and clinical applications. [6],[7] For example, 1-arylamino-2, 3-dihydro-1Hpyrazolo [ 1, 2-b] phthalazine-5,10-dione derivatives were reported to possess anti-inflammatory, analgesic, anti hypoxic, and antipyretic properties. [8]

From these points of view, it is interesting to study different types of biologically active ligands. In this article, the synthesis, characterization, and anti-tumor properties of a number of the new ligands have been studied.

   Materials and Methods Top

4-pyridinecarboxylic acid hydrazide, salicylaldehyde, 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% heat-inactivated fetal bovine serum (FBS) (Gibco), 2.7% sodium bicarbonate, and 500 mg/L ampicillin.

Synthesis of the compounds; General method

To a magnetically stirred mixture of 4-Pyridinecarboxylic acid hydrazide (1.37 g, 10 mmol) in hot methanol (20 mL), one type of aldehyde, such as, salicylaldehyde (1.22 g,1 mmol), benzaldehyde (1.06 g, 1 mmol), cinnamaldehyde (1.39 g,1 mmol), or formaldehyde (0.321 g, 1 mmol) was added via a syringe and heated for 45 minutes at 60°C. After cooling to room temperature, the resulting precipitate was filtered and washed with hexane (20 mL) [Figure 1].
Figure 1: Synthesis route of compounds

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Typical procedure for preparation of N 4 [(E)-1-(2-hydroxyphenyl) methylidene] Isonicotinohydrazide (3a) . The solid residue was dried and crystallized from CH 3OH (1 : 2) to yield 3a as yellow crystals (1.5 g, 75%). Mp 244 - 246.6°C. IR (KBr) (υmax , cm−1 ): 3181 (O-H) 3003 (N-H), 1682 (C=O), 1612 (C=N), 1567 and 1489(C=C), 1289(C-N), 1157(N-N). 1 H NMR (CDCl 3 , Me 4 Si) : δH 7.1 and 7.28(5H, 2q, 3 J HH = 7.37, arom), 8.16(1H, s, NCH), 8.06 and 9.33(4H, 2d, 3 J HH = 5.28, pyridine), 11.04(1H, d, OH), 12.99(1H, s, NH). 13 C NMR (CDCl 3 , Me 4 Si) : δC 117.14, 119.65, 128.24, 130.62, and 155.74 (arom), 126.05, 138.92, and 148.79 (pyridine), 151.2(C 11 ), 165.28(OCN). Anal. calcd for C 13 H 11 N 3 O 2 (241.26) : C, 64.66; H, 4.55; N, 17.40%. Found: C, 64.98; H, 4.87; N, 17.73%.

N 4 -[(E)-2-Phenylethylidene] Isonicotinohydrazide (3b). White crystals(1.76 g, 88%). Mp 184 - 187°C. IR (KBr) (υmax , cm−1 ) : 3198 (N-H), 1700 (C=O), 1600 (C=N), 1489 and 1447(C=C), 1283(C-N), 1148(N-N). 1H NMR (CDCl 3 , Me 4 Si) : δH 3.62(2H, d, 3 J HH = 6.64Hz, CHCH 2 ), 660(1H, t, tNCH), 6.72 and 7.13 (5H,2q, 3 JHH = 7.05 Hz, arom), 8.46 and 9.21(4H,2d, 3 J HH = 5.28, pyridine), 9.09(1H, s, NH).

13 C NMR(CDCl 3 , Me 4 Si): δC 34.31(C 12 ), 126.9, 127.23, 127.43, and 136.33 (arom), 122.82, 143.02, 151.06 (pyridine), 154.87(NCH), 165.44(OCN). Anal. calcd for C 13 H 11 N 3 O (225.27) : C, 69.25; H, 4.88; N, 18.64%. Found: C, 69.63; H, 5.11; N, 18.92%.

N 4 [(E,2E)-3-Phenyl-2-propenylidene] Isonicotinohydrazide (3c). Yellow crystals (1.84 g, 92%). Mp 200 - 203.3°C. IR (KBr) (υmax , cm−1 ) : 3233 (N-H), 1676(C=O), 1640 (C=N), 1451(C=C), 1308(C-N), 1174(N-N). 1 H NMR (CDCl 3 , Me 4 Si) : δH 6.06(1H, t, CHC), 6.94(1H, d, NCH), 7.51(1H, q, 3 J HH = 15.97, CHCH), 7.25 and 7.44(5H, 2q, 3 J HH = 2.12, 5.28, arom), 8.50 and 9.21(4H, 2d, 3 J HH =7.83, pyridine). 13 C NMR (CDCl 3 , Me 4 Si) : δC 124.76(C 13 ), 127, 128.92, 129.14, and 135.81(arom), 130.42(C 12 ), 144.62(C 11 ), 122.83, 143.55 and 151.06(pyridine), 165.05(OCN). Anal. calcd for C 15 H 13 N 3 O (251.30): C, 62.70; H, 5.17; N, 16.71%. Found: C, 63.02; H, 5.34; N, 17.07%.

N 4 [(E)ethylidene] Isonicotinohydrazide (3d). White crystals (1.3 g, 65%). Mp 244 - 246.6°C. IR (KBr) (υmax , cm−1 ): 3026 (N-H), 1660 (C=O), 1600 (C=N), 1410(C=C), 1300(C-N), 1164(N-N). 1 H NMR (CDCl 3 , Me 4Si): δH 7.55 and 6.52(2H, 2s, 2 J HH = 8.45, NCH), 8.45 and 9.21(4H, 2d, 3 J HH = 5.28, pyridine), 9.25(1H, s, NH). 13 C NMR (CDCl 3 , Me 4 Si) : δC 122.82, 143.6, and 151.06 (pyridine), 136.21(C 11 ), 165.6(OCN). Anal. calcd for C 7 H 7 N 3 O (149.14): C, 56.32; H, 4.69; N, 28.16%. Found: C, 56.65; H, 4.93; N, 28.43%.

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.

   Results and Discussion Top

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:

0.1 - 610 μM for NHPM, 0.1 - 680 μM for NPI, 0.1 - 450 μM for NPPI, and 0.1 - 300 μM for NEI.

The compounds were first dissolved in ethanol and then filtrated. The incubation continued up to 72 hours and at the end of this period, the IC 50 for each compound was measured using Trypan blue. The IC 50 value was the compound concentration needed for killing 50% of the tumor cells that were determined by comparison of the number of tumor cells in the control plate (blank plate : the sample plate without compound) and test plate. The corresponding IC 50 and IC 90 (50 and 90% inhibitory dose) values are shown in [Table 1].
Table 1: 72 hour IC50 values (μM) obtained for four compounds

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   Conclusion Top

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 Top

We gratefully acknowledge the financial support from the Research Council of Ardabil Islamic Azad University and the many technical supports that were provided by the Tarbiat Modarres University.

   References Top

1.Teimouri MB. One-pot three-component reaction of isocyanides, dialkyl acetylenedicarboxylates and phthalhydrazide: Synthesis of highly functionalized 1H-pyrazolo[1,2-b]phthalazine-5,10-diones. Tetrahedron 2006;62:10849-53.  Back to cited text no. 1      
2.Ogretir C, Tay NF, Ozturk II. A theoretical study on protonation of some halogen substitutedpyridine derivatives. J Mol Graph Model 2007;26:740-7.  Back to cited text no. 2      
3.Padwa A, Waterson AG. Curr. Synthesis of nitrogen heterocycles using the intramolecularpummerer reaction. Org Chem 2000;4:175.   Back to cited text no. 3      
4.Orru RV, de Greef M. Recent advances in solution-phase multicomponent methodology for the synthesis of heterocyclic compounds. Synthesis; 2003. p. 1471.   Back to cited text no. 4      
5.Kirsch G, Hesse S, Comel A. Synthesis of five- and six-membered heterocycles throughpalladium-catalyzed reactions. Org Chem 2004;1:47.  Back to cited text no. 5      
6.Clement RA. The oxidation of 2, 3-dihydrophthalazine-1,4-dione with lead tetraacetate. phthalazine-1,4-dione and 1,4-dihydropyridazino[1,2-b]-phthalazine-6,11-dione. Org Chem 1960;25:1724.   Back to cited text no. 6      
7.Heine HW, Henrie R, Heitz L, Kovvali SR. Diaziridines. III. Reactions of some 1-alkyl- and1, 1-dialkyl-1H-diazirino[1,2b]phthalazine-3,8-diones. J Org Chem 1974;39:3187.  Back to cited text no. 7      
8.Al-Assar F, Zelenin KN, Lesiovskaya EE, Bezhan IP, Chakchir BA. Synthesis and pharmacological activity of 1-hydroxy-, 1-amino-, and 1-hydrazino-substituted 2,3-dihydro-1H- pyrazolo[1,2-a]pyridazine-5,8-diones and 2,3-dihydro-1H-pyrazolo[1,2-b]phthalazine-5,10-diones.J Pharm Chem 2002;36:598.   Back to cited text no. 8      
9.Zhao G, Lin H, Zhu S, Sun H, Chen Y. Dinuclear palladium(II) complexes containing two monofunctional [Pd(en)(pyridine)Cl]+ units bridged by Se or S. Synthesis, characterization, cytotoxicity and kinetic studies of DNA binding. J Inorg Biochem 1998;70:219-26.  Back to cited text no. 9      
10.Ishida J, Wang HK, Bastow KF, Hu CQ, Lee KH. Antitumor agents 201. Cytotoxicity of harmine and β-carboline analogs. Bioorg Med Chem Lett 1999;9:3319-24.  Back to cited text no. 10      
11.Son JK, Zhao LX, Basnet A, Thapa P, Karki R, Na Y, et al. Synthesis of 2,6-diaryl-substituted pyridines and their antitumoractivities. Eur J Med Chem 2008;43:675-82.   Back to cited text no. 11      
12.Kim YS, Song R, Chung HC, Jun MJ, Sohn YS. Coordination modes vs. antitumor activity: Synthesis and antitumor activity of novel platinum(II) complexes of N-substituted aminodicarboxylic acids. J Inorg Biochem 2004;98:98-104.  Back to cited text no. 12      
13.Shabani F, Ghammamy S, Mehrani K, Teimouri MB, Soleimani M, Kaviani S. Antitumor activity of 6-(cyclohexylamino)-1,3-dimethyl-5(2-pyridyl) furo[2,3-d]pyrimidine-2,4(1H,3H)-dione and Its Ti(IV), Zn(II), Fe(III), and Pd(II) Complexes on K562 and jurkat cell lines. Bioinorg Chem Appl 2008:501021 [In Press].  Back to cited text no. 13      
14.Ghammam SH, Shabani F. Synthesis, characterization and anti-tumor activity of Ti (IV) and Mn (II) complexes of N4[(E,2E)-3-Phenyl-2-propenylidene] isonicotinohydrazide. Der PharmaChemica 2009;1:30-6.  Back to cited text no. 14      
15.Ghammam SH, Shabani F. Synthesis, characterization and anti-tumour activity of VOF3 and Cu(II)porphyrine complexes. Der Pharma Chemica 2009;1:124-9.  Back to cited text no. 15      
16.Shabani F, Saghatforoush LA, Ghammamy SH, Shakeri R, Shabani M. Synthesis, characterization and anti-tumour activity of Iron (III) Schiff base complexes with unsymmetric tetradentate ligands. Bull Chem Soc Ethiopia 2010 [In Press].  Back to cited text no. 16      


  [Figure 1]

  [Table 1]


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