Synthesis and Cytotoxic Effect of Some Novel 1,2-Dihydropyridin-3-carbonitrile and Nicotinonitrile Derivatives

1-(2,4-Dichlorophenyl)-3-(4-fluorophenyl)propen-1-one (1) was prepared and reacted with an active methylene compound (ethyl cyanoacetate) in the presence of ammonium acetate to give the corresponding cyanopyridone 2. Compound 2 reacted with hydrazine hydrate, malononitrile, ethyl bromoacetate and phosphorous oxychloride to afford compounds 4 and 7–11, respectively. The 2-chloropyridine derivative 11 reacted with different primary amines, namely benzyl amine, piperonyl amine, 1-phenylethyl amine, and/or the secondary amines 2-methyl-pipridine and morpholine to give the corresponding derivatives 12–15. Hydrazinolysis of chloropyridine derivative 11 with hydrazine hydrate afforded the corresponding hydrazino derivative 17. Condensation of compound 17 with ethyl acetoacetate, acetylacetone, isatin and different aldehydes gave the corresponding derivatives 18–21. Some of newly synthesized compounds were screened for cytotoxic activity against three tumor cell lines. The results indicated that compounds 8 and 16 showed the best results, exhibiting the highest inhibitory effects towards the three tumor cell lines, which were higher than that of the reference doxorubicin and these compounds were non-cytotoxic towards normal cells (IC50 values > 100 μg/mL).


Introduction
Cancer is the second leading cause of death in both developing and developed countries [1]. The leading forms were lung cancer, colorectal cancer, liver cancer and breast cancer [2,3]. Cancer treatment has been a major research and development effort in academia and the pharmaceutical industry for numerous years [4,5]. Despite the fact that there is a large amount of information available dealing with the clinical aspects of cancer chemotherapy, we felt that there was a clear requirement for an updated treatment from the point of view of medicinal chemistry and drug design [6]. Another major goal for developing new anticancer agents is to overcome cancer resistance to drug treatment, which has made many of the currently available chemotherapeutic agents ineffective [7].
Motivated by the above recent literature observations and our own previous reports [20,21,[31][32][33], herein some new pyridine derivatives were synthesized, leading to interesting heterocyclic scaffolds that are mostly useful for the creation of varied chemical libraries of drug-like molecules for biological screening.

Chemistry
The synthesis of the designed target compounds was achieved as outlined in Schemes 1-3. During this investigation, the pyridin-3-carbonitrile starting material 2 was prepared by condensation of the corresponding enone 1 [34] with ethyl cyanoacetate in the presence of excess ammonium acetate (Scheme 1). Compound 2 can also be obtained in high yield through a four-component modified Hantzch reaction, in a one-step synthesis, by refluxing a mixture of 2,4-dichloro-acetophenone, 4-fluorobenzaldehyde, ethyl cyanoacetate and ammonium acetate in n-butanol. The structure of pyridin-3-carbonitrile 2 was supported by elemental analysis, IR, ( 1 H, 13 C) NMR and mass spectral studies. Its IR spectrum showed absorption bands at 3278, 2219, 1632 cm −1 indicating the presence of NH, CN and CO groups, respectively. Its 1 H-NMR spectrum displayed a broad D2O exchangeable singlet at δ 8.10 ppm for the NH proton, while its 13 C-NMR spectrum also revealed signals at δ 117. 6 and 161.8 ppm for CN and CO moieties, respectively. The mass spectrum showed a molecular ion peak at m/z 358 (M + , 98%), which tallies with its molecular formula C18H9Cl2FN2O.
Pyridin-3-carbonitrile 2 possesses several reactive sites, viz. CN, NH, and CO groups, which can play a great role in the synthesis of heterocyclic derivatives, most of which are interesting from both the chemical and biological point of view. Thus, hydrazinolysis of pyridin-3-carbonitrile 2 with hydrazine hydrate in absolute ethanol for 15 h affords the corresponding pyrazolo [3,4-b]pyridin-3amine derivative 4 through the elimination of a water molecule from the intermediate 3 (Scheme 1). Pyrazolo [3,4-b]pyridine derivative 4 was identified by the absence of the cyano and carbonyl groups signals in its IR and the presence of an amino group signal at δ 5.69 ppm and the broad band of the NH proton at δ 10.05 ppm in its 1 H-NMR spectrum. Its mass spectrum showed a molecular ion peak at m/z 372 (M + ; 72%), which conforms to its molecular formula C18H11Cl2FN4.

Chemistry
The synthesis of the designed target compounds was achieved as outlined in Schemes 1-3. During this investigation, the pyridin-3-carbonitrile starting material 2 was prepared by condensation of the corresponding enone 1 [34] with ethyl cyanoacetate in the presence of excess ammonium acetate (Scheme 1). Compound 2 can also be obtained in high yield through a four-component modified Hantzch reaction, in a one-step synthesis, by refluxing a mixture of 2,4-dichloro-acetophenone, 4-fluorobenzaldehyde, ethyl cyanoacetate and ammonium acetate in n-butanol. The structure of pyridin-3-carbonitrile 2 was supported by elemental analysis, IR, ( 1 H, 13 C) NMR and mass spectral studies. Its IR spectrum showed absorption bands at 3278, 2219, 1632 cm´1 indicating the presence of NH, CN and CO groups, respectively. Its 1 H-NMR spectrum displayed a broad D 2 O exchangeable singlet at δ 8.10 ppm for the NH proton, while its 13 C-NMR spectrum also revealed signals at δ 117.6 and 161.8 ppm for CN and CO moieties, respectively. The mass spectrum showed a molecular ion peak at m/z 358 (M + , 98%), which tallies with its molecular formula C 18 H 9 Cl 2 FN 2 O.
Pyridin-3-carbonitrile 2 possesses several reactive sites, viz. CN, NH, and CO groups, which can play a great role in the synthesis of heterocyclic derivatives, most of which are interesting from both the chemical and biological point of view. Thus, hydrazinolysis of pyridin-3-carbonitrile 2 with hydrazine hydrate in absolute ethanol for 15 h affords the corresponding pyrazolo [3,4-b]pyridin-3-amine derivative 4 through the elimination of a water molecule from the intermediate 3 (Scheme 1). Pyrazolo [3,4-b]pyridine derivative 4 was identified by the absence of the cyano and carbonyl groups signals in its IR and the presence of an amino group signal at δ 5.69 ppm and the broad band of the NH proton at δ 10.05 ppm in its 1 H-NMR spectrum. Its mass spectrum showed a molecular ion peak at m/z 372 (M + ; 72%), which conforms to its molecular formula C 18
Schiff base 10 can be produced via condensation of acetohydrazide 9 with an aromatic aldehyde, namely 4-flourobenzaldehyde, in ethanol (Scheme 2). The structure of Schiff base 10 was elucidated based on the spectral and analytical data. The IR spectrum revealed the absence of the absorption bands of (NH 2 ) group absorption and its 1 H-NMR spectrum showed a singlet at δ 8.10 ppm due to the presence of the (CH=N-) group.
In addition, chlorination of cyanopyridone 2 with phosphorous oxychloride afforded the 2-chloronicotinonitrile derivative 11 (Scheme 2) in good yield, after 8 h. The IR spectrum showed the absence of a characteristic CO group band.
It is known that position 2 in chloronicotinonitrile derivatives shows distinct activities toward nucleophiles, especially nitrogen nucleophiles. Thus, nucleophilic replacement of the chlorine atom of chloronicotinonitrile 11 was performed by refluxing with different primary amines, namely benzylamine, piperonylamine, 1-phenylethylamine and/or secondary amines, namely 2-methyl-piperidine and morpholine in boiling ethanol for 6-12 h to afford the corresponding 2-aminopyridine derivatives 12-15a,b; respectively (Scheme 3). The elemental analysis and spectral data of compounds 12-15a,b were in agreement with the proposed structures. The 1 H-NMR of compound 15b for example, showed signals at δ 3.31 and 3.72 ppm due to the presence of (2N-CH 2 ) and (2O-CH 2 ) groups, respectively; and its 13 C-NMR exhibited signals at δ 47.9, 49.1, 64.10 and 65.9 ppm due to the presence of (2N-CH 2 ) and (2O-CH 2 ), respectively. Furthermore, nucleophilic displacement was carried out by heating the chloropyridine derivative 11 with malononitrile in ethanol containing a few drops of triethylamine as a catalyst to give 2-[3-cyano-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)pyridin-2-yl]malononitrile (16). The structure of compound 16 was confirmed by its spectral data; the IR spectrum showed the presence of the CN group at 2218, 2225 cm´1. In addition, the NMR ( 1 H and 13 C) and mass spectral data were in accordance with its structure. Hydrazinolysis of the chloropyridine derivative 11 was performed by its reaction with excess hydrazine hydrate in refluxing ethanol to give the hydrazino derivative 17 (Scheme 3). The structure of 17 was confirmed by its spectral data. The IR spectrum exhibited the characteristic absorption bands at 3440, 3320, 3150 cm´1 indicating the presence of the -NHNH 2 group. Its mass spectrum showed a molecular ion peak at m/z 372 (M + ; 39%), which conforms to its molecular formula C 18 H 11 Cl 2 FN 4 .
In addition, to get a new series of Schiff bases expected to be biologically active, heating of 2-hydrazinonicotinonitrile 17 with different aromatic aldehydes, namely 2-bromobenzaldehyde, 4-nitrobenzaldeyde, 2-methoxybenzaldeyde, 3,4-dimethoxybenzaldeyde and/or 1-naphthaldeyde in ethanol gave the corresponding Schiff bases 21a-e, respectively. The structure of compounds 21a-e was characterized by the disappearance of the NH 2 group. In addition, the 1 H-NMR spectra showed a singlet at around δ 8.31-8.33 due to the presence of the azomethine group (CH=N-). Finally, reaction of 2-hydrazinonicotinonitrile 17 with acetic acid or DMF afforded the corresponding pyrazolo [3,4-b]pyridin-3-amine derivative 4 through intramolcular cyclization via the addition of the NH 2 functional group at the CN group.

In Vitro Anticancer Screening
The in vitro cytotoxic activity the newly synthesized compounds against human breast cell line (MCF7), non-small cell lung cancer NCI-H460, CNS cancer SF-268 and WI 38 (normal fibroblast cells) were evaluated using doxorubicin as the reference drug, according to the method reported by Skehan et al. [35]. The IC 50 values of the synthesized compounds compared to the reference drug are shown in Table 1. From the results presented in Table 1 and Figure 2, it is evident that some of the compounds were active against the three human cancer cell lines. Compounds 8 and 16 displayed high cytotoxic activity against the tested cell lines (most of the IC 50 values ranged from 0.01˘0.002 to 0.02˘0.001 µg/mL) and these compounds were non-cytotoxic on the normal cells (IC 50 values > 100 µg/mL) and exhibited better cytotoxicity against most of cancer cell lines than doxorubicin as standard drug. Moreover, compounds 9, 17 and 20 exhibited high growth inhibitory activity on the various cancer panel cell lines (IC 50 values ranged from 0.46˘0.006 to 2.43˘0.51 µg/mL) with weak cytotoxicity on the normal cells (IC 50 values ranged from 62.19˘2.02 to 72.45˘2.40 µg/mL). In addition, other compounds showed moderate to weak cytotoxicity against all cancer cell lines (IC 50 values ranged from 10.39˘4.19 to 75.20˘13.86 µg/mL) with cytotoxic effects on the human normal cell (IC 50 values ranged from non-cytotoxic to 50.20˘10.22 µg/mL) in comparison with doxorubicin. The resultant data can be analyzed with respect to the chemical structures of the examined compounds; thus it can be noticed that the derivatives 8 and 16 that bear ester or malononitrile side chains on the parent cyanopyrine nucleus showed the highest potency as growth inhibiting agents against the three human cancer cell lines, which might be due to their lipophilicity that allows their accumulation inside tumor tissues inducing growth inhibition effects [36].

General Information
All melting points are uncorrected and were determined on a Stuart electric melting point apparatus. The microanalyses were within ±0.4% of the theoretical values and were carried out at the Microanalytical Centre, National Research Centre, Cairo, Egypt. IR spectra (KBr) were recorded on a FT-IR 400D infrared spectrometer (Shizmadu-series, Kyoto, Japan) using the OMNIC program and are reported as frequency of absorption in cm −1 . 1 H-NMR spectra were recorded on a Bruker (Rheinstetten, Germany) spectrophotometer at 400 MHz using TMS as internal standard and with residual signals of the deuterated solvent δ = 7.26 ppm for CDCl3 and δ 2.51 ppm for DMSO-d6. 13 C-NMR spectra were recorded on the same spectrometer at 100 MHz and referenced to solvent signals δ = 77 ppm for CDCl3 and δ 39.50 ppm for DMSO-d6. The mass spectra were recorded on a Shimadzu GCMS-QP-1000 EX mass spectrometer (Kyoto, Japan) at 70 eV using the electron ionization technique. Homogeneity of all compounds synthesized was checked by TLC which was performed on Merck 60 (Munich, Germany) ready-to-use silica gel plates to monitor the reactions and test the purity of the new synthesized compounds. The chemical names given for the prepared compounds are according to the IUPAC system. Method A: A mixture of 1-(2,4-dichlorophenyl)-3-(4-fluorophenyl)prop-2-en-1-one (1, 2.95 g, 0.01 mol), ethyl cyanoacetate (1.13 g, 0.01 mol) and ammonium acetate (6.16 g, 0.08 mol) in ethanol (40 mL) was refluxed for 10 h. After cooling, the precipitate was filtered, dried and recrystallized from dioxane to give compound 2 (35% yield).

General Information
All melting points are uncorrected and were determined on a Stuart electric melting point apparatus. The microanalyses were within˘0.4% of the theoretical values and were carried out at the Microanalytical Centre, National Research Centre, Cairo, Egypt. IR spectra (KBr) were recorded on a FT-IR 400D infrared spectrometer (Shizmadu-series, Kyoto, Japan) using the OMNIC program and are reported as frequency of absorption in cm´1. 1 H-NMR spectra were recorded on a Bruker (Rheinstetten, Germany) spectrophotometer at 400 MHz using TMS as internal standard and with residual signals of the deuterated solvent δ = 7.26 ppm for CDCl 3 and δ 2.51 ppm for DMSO-d 6 . 13 C-NMR spectra were recorded on the same spectrometer at 100 MHz and referenced to solvent signals δ = 77 ppm for CDCl 3 and δ 39.50 ppm for DMSO-d 6 . The mass spectra were recorded on a Shimadzu GCMS-QP-1000 EX mass spectrometer (Kyoto, Japan) at 70 eV using the electron ionization technique. Homogeneity of all compounds synthesized was checked by TLC which was performed on Merck 60 (Munich, Germany) ready-to-use silica gel plates to monitor the reactions and test the purity of the new synthesized compounds. The chemical names given for the prepared compounds are according to the IUPAC system.
Method B: Compound 17 (3.7 g, 0.01 mol) in DMF or AcOH (20 mL) was refluxed for 8 h. The reaction mixture was cooled; the solid product that precipitated was filtered, dried and recrystallized from ethanol to give compound 4 (43% yield). To a solution of compound 2 (3.6 g, 0.01 mol) in absolute ethanol (30 mL), triethylamine (3 mL), and malononitrile (0.7 g, 0.01 mol) were added. The reaction mixture was refluxed for 6 h., then left to cool to room temperature, poured into cold water and neutralized with diluted hydrochloric acid to complete precipitation. The solid obtained was filtered, washed with water, dried and recrystallized from methanol to give compound 7. Yield 70%; m.p. 188-189˝C; IR (KBr) ν max in cm´1: 3312, 3249, 3145 (NH 2

Cell Cultures
The newly synthesized compounds were evaluated in vitro against three human cancer cell lines; which are MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and SF-268 (CNS cancer), and WI 38 (normal fibroblast cells) were used in this study. MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) but NCI-H460, SF-268 and WI 38 were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayers routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated fetal bovine serum (FBS), 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL), at 37˝C in a humidified atmosphere containing 5% CO 2 . Exponentially growing cells were obtained by plating 1.5ˆ105 cells/mL for MCF-7 and SF-268, and 0.75ˆ104 cells/mL for NCI-H460 followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.

Cancer Cell Growth Assay
The effect of compounds on the in vitro growth of human tumor cell lines were evaluated according to the procedure adopted by the National Cancer Institute (NCI, Austin, TX, USA) in the "In vitro Anticancer Drug Discovery Screen" that uses the protein-binding dye sulforhodamine B (SRB) to assess cell growth [35]. In the assay protocol, all cells were incubated at 37˝C under humidified atmosphere containing 5% CO 2 . Briefly, exponentially cells growing in 96-well plates were then exposed for 48 h to five serial concentrations of each compound, starting from a maximum concentration of 150 µg/mL. Following this exposure period, adherent cells were fixed, washed and stained. The bound stain was solubilized and the absorbance was measured at 492 nm in a Power Wave XS plate reader (Bio-Tek Instruments Inc., Winston, NC, USA). For each test compound and cell line, a dose response curve was obtained and the inhibitory concentration of 50% (IC 50 ), corresponding to the concentration of the compounds that inhibited 50% of the net cell growth was calculated as described elsewhere [37]. Doxorubicin was used as a positive control and tested in the same manner.

Conclusions
This study focused on the synthesis of a new 1,2-dihydropyridin-3-carbonitrile and nicotinonitrile derivatives as potential anticancer agents. Some of newly synthesized derivatives were examined in vitro as cytotoxic agents against three human cancer cell lines. It could be noticed that the ester functionality-bearing derivative 8 and the derivative 16 carrying a malononitrile side chain attached to the parent cyanopyridine nucleus showed the best results, exhibiting the highest inhibitory effects towards the three tumor cell lines, which were higher than that of the reference compound doxorubicin and these compounds were non-cytotoxic towards normal cells (IC 50 values >100 µg/mL). In addition, compounds 9, 17 and 20 exhibited high growth inhibitory activity on the various cancer panel cell lines, with weak cytotoxicity on the normal cells.