Synthesis, Characterization and Cytotoxicity Evaluation of Some Novel Pyridine Derivatives

Reaction of isonicotinaldehyde with 2-cyanoacetohydrazide afforded (E)-2-cyano-N’-(pyridin-4-ylmethylene)acetohydrazide (1). Compound 1 was used as the precursor for the synthesis of novel pyridine derivatives by reaction with different arylidene malononitriles, malononitrile and acetylacetone to give pyridine derivatives 5a–e, 6 and 7, respectively. 4,4’-Bipyridine derivatives 9a–d were synthesized by a three-component reaction of isonicotinaldehyde, 2-cyanoacetohydrazide and activated nitriles 8a–d. Treatment of compound 9a with different aromatic aldehydes gave [1,2,4] triazolo[1,5-a]pyridine derivatives 11a–c. All reaction products were characterized by analytical and spectral data. For the novel compounds their bioactivity as antitumor agents was examined for in vitro cytotoxicity against HepG-2 and MCF-7. It was found that compounds 9a and 9b have high cytotoxic activity against both HepG-2 and MCF-7.


Introduction
Among the important class of azaheterocycles, pyridine derivatives constitute one of the most significant classes of compounds as they broadly occur as vital struc-tural subunits in many natural products, functional materials and pharmaceuticals 1 that exhibit many motivating biological activities. [2][3][4] For example atazanavir 5 and imatinib mesylate 6 (Figure 1) as two examples of drugs being prescribed for the treatment of HIV and chronic myelogenous leukemia, respectively.
Generally, pyridine derivatives have a huge spectrum of biological activities, like anti-leishmanial, 7 anti-diabetic, 8 anti-oxidant, 9 antitumor 10-12 and antiviral. 13 Recently, some of pyridine derivatives were shown to act as potential targets for the development of new drugs for the treatment of cancer, 14 as anti-platelet drugs, 15 and antiproliferative agents. 16

1. 1. Chemicals and Reagents
All the chemicals and solvents used in this study were obtained from Merck (Germany).

1. Instruments
Melting points were recorded on Gallenkamp electric melting point apparatus (Electronic Melting Point Apparatus, Great Britain, London) and are uncorrected. Infrared spectra were recorded on Pye Unicam SP 1000 IR spectrophotometer (Thermoelectron Co. Egelsbach, Germany) using a KBr wafer technique. The 1 H NMR spectra were determined by a Bruker 400 MHz spectrometer. DMSO-d 6 was used as the solvent, TMS was used as the internal standard and chemical shifts are given on δ scale in ppm. Mass spectra were determined on a GC-MS QP-100 EX Shimadzu (Japan). Microwave experiment was carried out using Milestone Start Microwave Lab Station. Elemental analyses were recorded on Perkin-Elmer 2400 Elemental analyzer at the Microanalytical Center at Cairo University, Cairo, Egypt.
In the absence of any catalyst and under solvent-free conditons or in the presence of triethylamine as the basic catalyst at room temperature the reaction did not proceed even after long reaction time (Table 1, entries 1, 2 and 4). However, in the presence of Et 3 N under reflux with the EtOH or THF as the solvents, the desired product was obtained in 39 or 92% yield, respectively (Table 1, entries 5  and 6). Moreover, when the synthesis of 1 was carried out under microwave irradiation under solvent-free conditions, afforded the desired reaction product in high yield (Table 1, entry 7). The solvent-free conditions are preferrable as they avoid the use of toxic, flammable, and expen-sive organic solvents. The main advantages of microwave irradiation synthesis are thus shorter reaction time, higher yield and better purity of the product.
The chemical structure of 1 was confirmed by its spectral and elemental analysis data. The IR spectrum of 1 showed the presence of three stretching frequencies at 3235, 2259 and 1704 cm -1 attributable to NH, CN and C=O groups, respectively. The 1 H NMR exhibited two singlet signals at δ 4.26 and 8.25 ppm due to CH 2 and CH=N, respectively. In addition, two doublet signals at δ 8.02 and 8.74 ppm due to pyridine protons are observed. The configuration around the double bond of the compound 1 could not be established by 1 H NMR spectroscopy. However, the steric effect enhances that the E isomer is more stable than Z isomer.
The spectral and analytical data of compounds 6 and 7 were in agreement with their proposed structures. 1 H NMR spectrum of 6 showed two singlet signals at δ 4.62 and 8.43 ppm owing to the C 5 -H of 2-pyridone ring and CH=N, respectively. Moreover, 1 H NMR of 6 exhibited two singlet signals (D 2 O-exchangable) at δ 4.65 and 6.68 ppm due to the two NH 2 groups. The IR analysis substantiated the results of 1 H NMR by the presence of four peaks in the region of 3321-4000 cm -1 for two NH 2 groups.
Nowadays, multicomponent reactions are gaining extensive economic and ecological importance as they conform to the fundamental principles of synthetic efficiency and reaction design. 19 We herein provide an efficient and facile procedure for the synthesis of 4,4'-bipyridine derivatives 9a-d via a one-pot three-components condensation of isonicotinaldehyde, 2-cyanoacetohydrazide and activated nitriles 8a-d 20,21 (Scheme 4).

2. 1. Cytotoxicity Against Hepatoma Cell
Line (HepG-2) and Human Breast Adenocarcinoma Cell Line (MCF-7) Cytotoxic activity. In order to investigate if the chemistry established here has led to possibly interesting nominees in cancer therapy, our primary aim was directed towards checking if the novel synthesized compounds possess any anticancer activities as predicted by this study. In vitro cytotoxic study was therefore performed against two mammalian cancer cell lines, HepG-2 (hepatoma cells or human liver hepatocellular carcinoma cell line) and MCF-7 (human breast adenocarcinoma cell line). This study indicated that compounds 9a and 9b showed very strong cytotoxic activity against HepG-2 cancer cells with IC 50 values of 8.83±0.30 and 10.08±0.66 µg/mL, respectively. Also, both 9a and 9b gave high cytotoxic effects against MCF-7 classifying these compounds as chemotherapeutically significant ( Table 2). The rest of other compounds showed a moderate to weak activity against the tested tumor cell lines. IC 50 is the concentration, which can reduce the growth of cancer cells by 50%.

Conclusion
In conclusion, herein we report a simple and convenient method for the synthesis of novel pyridine derivatives. All synthesized compounds were evaluated against two cancer cell lines (HepG-2 and MCF-7). Among all the synthesized compounds, compounds 9a,b have high cytotoxic activity against both HepG-2 and MCF-7. The rest of compounds showed a moderate to weak activity against the tested tumor cell lines.