Synthesis of some novel 2-(3-cyano -6-(thiophen- 2-yl)-4,4′- bipyridin-2- yloxy)acetohydrazide derivatives: assessment of their cytotoxic activity

A new series of pyrazole, bipyridine, N-amide derivatives and Schiff bases was synthesized using compound 2-(3-cyano-6- (thiophen-2-yl)-4,4′- bipyridin-2-yloxy) acetohydrazide (3) as a starting material. The compounds structures were confirmed depending on the spectroscopic methods and elemental analysis. Also, the compounds were evaluated as anticancer agents by the compounds screened towards adenocarcinoma breast cancer cell line (MCF-7). The compounds showed a promising cytotoxic effect against human breast cancer cells. Compound 7c showed the most effective activity compared to other compounds with (IC50 = 0.6 ± 0.01 μg mL−1) in comparison with the reference drug doxorubicin (IC50 = 1.6 ± 0.02 μg mL−1). While compound 3 is closely active with doxorubicin. Also compounds 2, 4, 6, 7a, 7b and 7d showed noticeable cytotoxic effect. Early and late apoptotic cells were detected using Acridine orange/Ethidium bromide staining technique. The results of biologically screening of the tested compounds give an idea about the importance in the compounds acting against breast cancer and may lead to the discovery of a potent anticancer agent.


Chemistry
In this research, a one-pot manner was used for the synthesis of compound2-oxo-4-(pyridin-4-yl)-6-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (1) where all the reaction components, 2-acetylthiophene, 4-pyridine carboxaldehyde, ammonium acetate, and ethyl cyanoacetate were added in the presence of ceric ammonium nitrate (CAN) and then refluxed in ethanol. The resulting compound 1 then alkylated with ethyl bromoacetate in ethanol and in the presence of a catalytic amount of potassium carbonate to give the alkylated derivative ethyl 2-(3-cyano -6-(thiophen-2-yl)-4,4′-bipyridin -2-yloxy) acetate (2). The structure of compound 2 was confirmed depending on the spectral data. For example, in the 1 H NMR spectrum, the characteristic signals of the ethoxy group appeared at 1.18 ppm for (CH 3 ) and at 4.15 ppm for (OCH 2 ) and the signal for (NH) group at 8.79 ppm was disappeared. Hydrazionlysis of compound 2 gave the acid hydrazide 3. In the acid hydrazide 1 H NMR spectrum the signals of the ethoxy groups at 4.15 and 1.18 ppm were disappeared and new signals appeared at 8.75 and 12. 48 for the (NH-NH 2 ) group. All other signals appeared at their expected position as illustrated in the experimental section. 2-(3-Cyano-6-(thiophen-2-yl)-4,4′bipyridin-2-yloxy)acetohydrazide (3), is used as a starting matter for the synthesis of all target compounds in this work (Scheme 1).
A new series of expectedly biologically active N-amide derivatives and Schiff bases was synthesized. Schiff bases 7a-d were obtained through condensation of compound 3 with different aldehydes namely 3-pyridine carboxaldehyde, 3,4-Diydroxy benzaldehyde, anisaldehyde and vanillin, in acetic acid. In all Schiff bases the signal characteristic to the (NH 2 ) group was disappeared and the signal of (NH) group at 12.48 ppm was shifted to new positions at 12.48, 9.99, 12.49 and 8.84 ppm. In compounds 7a, 7b, 7c, and 7d respectively. All the characteristic signals of the arylidine groups were appeared at their expected positions as shown in the experimental part, Compound 7c structure was confirmed based on the spectroscopic data in (Fig. 1) The reaction of 3 with p-toluenesulfonyl chloride in absolute ethanol afforded the corresponding 2-(3-cyano-6(thiophen-2-yl)-4,4″bipyridin-2-yloxy)N-(tosylmethylene)aceto hydrazide (8) (Scheme 3). Compound 8 structure was confirmed based on the spectroscopic data and the elemental analysis.

In vitro anticancer screening
The in vitro cytotoxic activities of compounds 1, 2, 3, 5, 6, 7a-d and 8 were determined using SRB assay towards breast cancer cell line (MCF-7) over concentration range of 0.01 to 1000 μg. The tested compounds exhibited a variable cytotoxicity profile against the tested human breast cancer cells. (Table 1 and Fig. 2). doxorubicin is a reference drug in this study The IC 50 : is the compounds concentrations reduce the cell viability to 50%. The data in Table 1 and Fig. 2 indicate the cytotoxicity profile of the newly synthesized compounds against breast cancer cells. The results showed considerable cytotoxicity against cancer cell, most of the compounds showed highly cell killing significant on MCF-7 cells; some of them were revealed a strong activity, others were found to be on par near the reference drug toxicity (IC 50 = 1.3 μg mL −1 ).
After staining cells using double stains AO/EtBr, cells appeared in the form of four colors as follows: living cells (normal green nuclei), early-programmed cell death (apoptotic) (bright green nucleus with segmented chromatin), late-programmed cell death (apoptotic) (orange nucleus with chromatin condensation or fragmentation) and necrotic cells (Kernel of uniformly colored orange cells).
The uniformly stained green cells with normal, round and intact nuclei that indicates the healthy cell control. Whereas, the highly cell killing with late apoptotic observed by treatment with compound 1 and some necrotic cell also observed with the compound itself; on the other hand there are no necrotic cells with compounds 2 and 3 compared to compound 1, and the derivative acetohydrazide 3 have high rate of late apoptotic compared to compounds 1 and 2 (Figs. 3 and 4).
The compound 5 killing the cells with early apoptotic way was more pronounced compared to compounds 3 and 6. Compound 6 has a necrotic cells after treatment compared to compounds 3 and 5. Also, compound 3 have cells with late apoptotic more than compounds 5 and 6 (Figs. 5 and 6).
Compound 7d has a highly late apoptotic effect on cancer cells compared to 3, 7a, 7b, 7d and compound 7c then 3 have early apoptotic more than 7a, 7b, and 7d is lower (Figs. 7 and 8).
Whereas, compound 8 has early apoptotic killing effect and cell necrotic against cancer cells more than compound 3, while compound 3 has a more cell late apoptotic effect than compound 8 (Figs. 9 and 10).
The biological activity of the tested compounds were indicated the promising cell killing effect of the 4,4′ bipyridine moiety in the compounds acting towards breast tumor cells.

Conclusions
In this paper I used compound 2-(3-cyano-6-(thiophen-2yl)-4,4′-bipyridin -2-yloxy)acetohydrazide 3 to synthesis a novel substituted pyrazole, bipyridine, N-amide derivatives and Schiff bases. The anticancer activity of the compounds was assessed against breast cancer cell line (MCF-7). The data obtained for the tested compounds shows the possible importance of these compounds to act as anticancer agents where compound 7c showed better activity than the standard drug itself. While other compounds such as compound 3 is equipotent with the standard drug. Compounds 2, 4, 6, 7a, 7b and 7d showed obvious activities but less than the reference.

Fig. 2
The dose response curves of the cytotoxicity of different compounds towards MCF-7tumor cell line. Cells were exposed to plant extract with different concentrations for 72 h. Cell viability was determined by SRB stain

Chemistry
Melting points were measured on a Gallenkamp apparatus, and are uncorrected. The desired time for completing the reaction was monitored by TLC. The IR spectra were recorded using (KBR) plates on a Shimadzu 470 IR spectrometer. The 1 H and 13 C NMR spectra were measured on a Bruker 400DRX-Avance NMR spectrometer at 400 MHz and chemical shifts (δ) are in ppm relative to TMS (tetramethylsilane). Mass spectra were measured on GC/MS with electron impact ionization by to (70 eV).
Elemental analyses were performed on Perkin-Elmer 2400 series П CHN elemental analyser.

General procedure for the synthesis of compounds 4-6
An equimolar amount of ethyl acetoacetate, acetylacetone and/or ethyl cyanoacetate (or diethyl malonate) and a mixture of compound 3 (0.01 mol) was refluxed in 15 mL acetic acid for 5 h. The produced product after cooling was filtered off, washed with water, dried, and crystallized with acetic acid to give compounds 4, 5, and 6 respectively.

General procedure for synthesis of Schiff bases 7a-d
A mixture of compound 3 (0.01 mol) and the appropriate aromatic aldehyde (3-pyridincarboxaldehyde, 3, 4 -dihydroxy benzaldehyde, anisaldehyde and vanillin (0.01 mol) in the presence of a catalytic amount of pipredine, in absolute ethanol (10 mL) was refluxed for 2 h. After cooling, the formed precipitate was filtered off, dried, and crystallized from acetic acid to afford the corresponding Schiff base 7a-d.

Cytotoxicity assay
The cytotoxicity of the chemical compounds was evaluated against (MCF-7) human tumor cell using Sulphorhodamine B assay (SRB) in King Khalid University, biology department. 80% confluency growing cells were trypsinized and cultured in a 96 well tissue culture plate for 24 h before treatment with the chemical compounds. Cells were exposed to the six different concentrations of each compound (0.01, 0.1, 1, 10, and 1000 µg/ml); untreated cells (control) were added. The cells were incubated with the concentrations for 72 h and subsequently fixed with TCA (10% w/v) for 1 h at 4 °C. After several washings, cells were stained by 0.4% (w/v) SRB solution for 10 min in dark place. Excess stain was washed with 1% (v/v) glacial acetic acid. After drying overnight, the SRBstained cells were dissolved with Tris-HCl and the color intensity was measured in microplate reader at 540 nm. The relation between viability percentage of each tumor cell line and compounds concentrations was analyzed to get the IC 50 (dose of the drug which reduces survival to 50%) using Sigma Plot 12.0 software [33].

Acridine orange/ethidium bromide staining for detection of early and late apoptotic cells
DNA binding dyes Acridine orange (AO) and Ethidium bromide (EtBr), were used for the morphological detection of viable, apoptotic and necrotic cells. AO is taken up by both non-viable and viable cells that emit green fluorescence when intercalated into DNA. EtBr is taken up only by nonviable cells whereas; it is excluded by viable cells and emits red fluorescence by intercalation into DNA. Cells were seeded on cover slide inside six well plates. Cells were incubated in CO 2 incubator with 37 °C temperature and 5% CO 2 for 24 h then treated with IC 50 s concentration of the chemical compounds and incubated for 48 h. Cells were washed with cold PBS 1× for three times. Cells were stained with a mixture Acridine Orange 100 μg/ml/Ethidium Bromide (AO/EB) 100 μg/ml in PBS 1x with 10% FBS on each well and then incubated for 5 min in RT. The cover slides with cultured stained cells were transfer immediately to new slides and the cells were ready to be visualized by the blue filter of the fluorescence microscope [34,35].