Facile synthesis and antiproliferative activity of new 3-cyanopyridines

Background Pyridines have been reported to possess various pharmacological activities. Results Sodium 3-oxo-3-(2-oxo-2H-chromen-3-yl)prop-1-en-1-olate (2) and sodium 3-oxo-3-(3-oxo-3H-benzo[f]chromen-2-yl)prop-1-en-1-olate (7) were prepared and reacted with 2-cyano-N’-(1-aryl(heteryl)ethylidene)acetohydrazides 3a–d to produce 2-oxo-1,2-dihydropyridine-3-carbonitrile derivatives 5a–d and 9a–d, respectively, in good yields. Also, 3a–d reacted with sodium (2-oxocyclopentylidene)methanolate (11a) or sodium (2-oxocyclohexylidene) methanolate (11b) to yield 2-oxo-tetrahydro-1H-cyclopenta[b]pyridine-3-carbonitriles 13a–d and 2-oxo-hexahydroquinoline-3-carbonitriles 13e–h, respectively. The mechanisms that account for the formation of the products are discussed. Additionally, the structures of all the newly synthesized products are confirmed, based on elemental analysis and spectral data. Several of the newly synthesized compounds are evaluated for their antitumor activity against HEPG2 and their structure activity relationship (SAR) was studied. Conclusions The results revealed that the pyridine derivatives 5c and 5d (IC50 = 1.46, 7.08 µM, respectively) have promising antitumor activity against liver carcinoma cell line (HEPG2), compared to the reference drug, doxorubicin.


Introduction
The pyridine core is a key constituent in a scope of bioactive compounds which occur artificially and naturally. It has been appeared to have a wide scope of biological applications [1][2][3]. Among these, substituted cyanopyridines were found to have antihypertensive [4], antipyretic, anti-inflammatory and analgesic properties [5]; cardiotonic [6], antimicrobial [7], and anticancer activities [8,9]. Among the successful examples as drug candidates possessing the pyridine core are streptonigrone, lavendamycin and streptonigrin, which are depicted in the literature as anticancer agents. Some pyridine derivatives were contemplated for their topoisomerase inhibitory action and cytotoxicity against a few human malignant growth cell lines, thus marking them as novel anticancer agents [10]. Accordingly, it has been accounted those different pyridine derivatives, as bioisosteres of α-terthiophene (protein kinase C inhibitor) [11], have significant topoisomerase I and II inhibitory activity and cytotoxicity against many human cancer cell lines [12][13][14][15].
Early reports on the ability of α-terpyridine to form a metal complex [16] and to bind with DNA/RNA [17] have been the reason for the investigation of pyridine derivatives as antitumor agents. In light of the above discoveries and in continuation of our endeavors to synthesize new antitumor compounds [18][19][20][21][22][23][24][25][26][27], the aim of this report is to synthesize a new series of 3-pyridinecarbonitriles, which are anticipated to be active as antitumor agents.
The structures of the reaction products 5a-d and 9a-d were established and confirmed by their elemental analysis and spectral data (MS, IR, 1 HNMR, 13 CNMR). Thus, the structure of 5a is supported by its mass spectrum, which showed a molecular ion corresponding to the formula C 23  To account for the formation of the products 5a-d and 9a-d, it is suggested that the studied reactions started with a nucleophilic attack by the methylene group of compound 3 at the formyl group of compound 2 or 7, which formed in situ due to the reaction of the formyl salts with water. This resulted in the formation of the non-isolable intermediate 4 or 8, followed by cyclization through the elimination of the water molecule, leading to the formation of the final pyridine derivatives 5 or 9 (Schemes 1 and 2).
As depicted in Scheme 3, the formation of 10 seems to start with an initial attack by a carbanion of the active methylene compound 3 to the formyl group of the salt 11, which formed in situ due to the reaction of the formyl salts 11 with water, forming. Subsequent enolization followed by elimination of water led to product 13.

Antitumor activity
The antitumor activity of compounds 5a-d, 9a-d and 13a-d was determined against a liver carcinoma cell line, HEPG2. Doxorubicin was utilized as a reference drug and showed IC50 = 0.72 μM against this liver carcinoma cell line. Collected data were used to plot a dose-response curve, of which the concentration (μM) of the tested compounds required to kill of 50% of the cell population (IC 50 ). Antitumor activity was expressed as the mean IC 50 of three different experiments.
The outcomes showed that the vast majority of the tested compounds demonstrated extraordinary variable activity contrasted with the reference drug, as shown in Table 1 and Fig. 1. The descending order of activity of the new compounds was as follows: 5c > 5d > 5a > 13c > 5b > 9a > 9b > 9d > 13d > 13a > 13b.
Examination of the SAR leads to the following conclusions.
The pyridine derivatives having coumarine ring 5a-d exhibited more anticancer activity than pyridines having naphthocoumarine ring 9a-d while the latter pyridines 9a-d exhibited more activity than cyclopenta[b]pyridines 13a-d.

Experimental section
Melting points were recorded in open capillaries using an electrothermal Gallenkamp apparatus and are uncorrected. Elemental analyses were carried out by the microanalytical center at Cairo University. The 1 H and 13 C NMR spectra were recorded in DMSO-d 6 on a Bruker DRX NMR spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C NMR. Chemical shift (δ) values are expressed in ppm and are referenced to the residual solvent signals of DMSO-d 6 .
The mass spectra were recorded on GCMSQ1000-EX Shimadzu spectrometers. The IR spectra were measured on a Pye-Unicam SP300 instrument.

Synthesis of sodium salt of cycloalkanones 11a, b
In a three-necked flask (250 mL), sodium methoxide (0.054 g, 10 mmol) and ether (20 mL) were poured through a separating funnel, the appropriate cyclopentanone (10a) or cyclohexanone (10b) (10 mmol of each) with ethyl formate (0.74 g, 10 mmol) were added, and then stirred. The formed solid products 11a and 11b were collected and used directly in the following reactions.

Synthesis of 2-oxo-1,2-dihydropyridine-3-carbonitrile derivatives 13a-h
A solution of 11a or 11b (10 mmol of each), the appropriate cyanoacid hydrazones 3a-d (10 mmol) and piperidine acetate (1 mL) in water (3 mL) was refluxed for 10 min. Acetic acid (1.5 mL) was added to the hot solution. The solid product was filtered off and recrystallized from the proper solvent to give products 13a-h. The physical constants and spectral data of the obtained products 13a-h are listed below: