Next Article in Journal
Silymarin Constituents Enhance ABCA1 Expression in THP-1 Macrophages
Previous Article in Journal
The Activity of Cotinus coggygria Scop. Leaves on Staphylococcus aureus Strains in Planktonic and Biofilm Growth Forms
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 21
Issue 1
10.3390/molecules21010030
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Eman M. Flefel
1,2,
Hebat-Allah S. Abbas
2,3,*,
Randa E. Abdel Mageid
2 and
Wafaa A. Zaghary
4
1
Department of Chemistry, College of Science, Taibah University, Al-Madinah Al-Monawarah 1343, Saudi Arabia
2
Department of Photochemistry, National Research Centre, Dokki, Cairo 12622, Egypt
3
Department of Chemistry, College of Science, King Khalid University, Abha 9004, Saudi Arabia
4
Department of Pharmaceutical Chemistry, College of Pharmacy, Helwan University, Ain Helwan, Cairo 11795, Egypt
*
Author to whom correspondence should be addressed.
Molecules 2016, 21(1), 30; https://doi.org/10.3390/molecules21010030
Submission received: 9 November 2015 / Revised: 15 December 2015 / Accepted: 22 December 2015 / Published: 31 December 2015
(This article belongs to the Section Bioorganic Chemistry)
Browse Figures
Versions Notes

Abstract

:
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).

1. 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].
Chalcones, one of the major classes of natural products with widespread occurrence in vegetables, fruits, spices and soy-based foodstuffs, have been reported to possess several biological activities such as antibacterial [8,9], anti-fungal [10,11], anti-inflammatory [12], and anti-tumor activities [13,14]. An important feature of chalcones is their ability to act as an intermediate for the synthesis of biologically active heterocyclic compounds such as pyrimidine and pyridine derivatives [15,16]. The pyridine nucleus is an integral part of anti-inflammatory and anticancer agents [17,18]. On the other hand, cyanopyridone and cyanopyridine derivatives have shown to possess promising antimicrobial [19] antioxidant [20,21], antibiotic [22], antiinflamatory [23,24], analgesic, anticonvulsant [25] and anticancer [26,27,28,29] properties. 3-Cyano-2-pyridones are analogous to the alkaloid ricinine, the first known alkaloid containing a cyano group. The anticancer activity of 3-cyano-2-pyridone derivatives is of much interest owing to the different types of biological targets they might interfere with, e.g., PDE3, PIM1 kinase, and survivin (Figure 1) [30].
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.
Molecules 21 00030 g001 550
Figure 1. Various 3-cyano-2-oxopyridine derivatives with potential growth inhibitory and/or antiangiogenic actions through PDE3 inhibition (a); PIM-1 kinase inhibition (b); or survivin inhibition (c).
Figure 1. Various 3-cyano-2-oxopyridine derivatives with potential growth inhibitory and/or antiangiogenic actions through PDE3 inhibition (a); PIM-1 kinase inhibition (b); or survivin inhibition (c).
Molecules 21 00030 g001

2. Results and Discussion

2.1. Chemistry

The synthesis of the designed target compounds was achieved as outlined in Scheme 1, Scheme 2 and Scheme 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, (1H, 13C) 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 1H-NMR spectrum displayed a broad D2O exchangeable singlet at δ 8.10 ppm for the NH proton, while its 13C-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-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 1H-NMR spectrum. Its mass spectrum showed a molecular ion peak at m/z 372 (M+; 72%), which conforms to its molecular formula C18H11Cl2FN4.
Molecules 21 00030 g003 550
Scheme 1. General methods for the preparation of compounds 2–7. Reagents and conditions: (i) NaOH/EtOH, stirring; (ii) ethyl cyanoacetate/CH3COONH4/EtOH, reflux; (iii) hydrazine hydrate 98% (1 mL)/EtOH, reflux; and (iv) malononitrile/triethylamine (3 mL)/EtOH, reflux.
Scheme 1. General methods for the preparation of compounds 2–7. Reagents and conditions: (i) NaOH/EtOH, stirring; (ii) ethyl cyanoacetate/CH3COONH4/EtOH, reflux; (iii) hydrazine hydrate 98% (1 mL)/EtOH, reflux; and (iv) malononitrile/triethylamine (3 mL)/EtOH, reflux.
Molecules 21 00030 g003
Molecules 21 00030 g004 550
Scheme 2. General methods for the preparation of compounds 8–11. Reagents and conditions: (i) ethyl bromoacetate/anh. K2CO3/dry CH3COCH3, reflux; (ii) hydrazine hydrate 98% (2 mL)/EtOH, reflux; (iii) 4-flurobenzaldehyde/EtOH, reflux; and (iv) phosphorus oxychloride/EtOH, reflux.
Scheme 2. General methods for the preparation of compounds 8–11. Reagents and conditions: (i) ethyl bromoacetate/anh. K2CO3/dry CH3COCH3, reflux; (ii) hydrazine hydrate 98% (2 mL)/EtOH, reflux; (iii) 4-flurobenzaldehyde/EtOH, reflux; and (iv) phosphorus oxychloride/EtOH, reflux.
Molecules 21 00030 g004
Molecules 21 00030 g005 550
Scheme 3. General methods for the preparation of compounds 12–21a–e. Reagents and conditions: (i) benzylamine/EtOH, reflux; (ii) piperonylamine/EtOH, reflux; (iii) 1-phenylethylamine/EtOH, reflux; (iv) 2-methylpiperidine or morpholine/EtOH, reflux; (v) malononitrile/triethylamine (1 mL)/EtOH, reflux; (vi) hydrazine hydrate 98% (2 mL)/EtOH, reflux; (vii) ethyl acetoacetate/AcOH, reflux; (viii) acetylacetone/AcOH, reflux; (ix) DMF or AcOH, reflux; (x) isatin/3 drops AcOH/EtOH, reflux; and (xi) appropriate aromatic aldehyde, namely: 2-bromobenzaldhyde, 4-nitrobenzaldhyde, 2-methoxy-benzaldhyde, 3,4-dimethoxybenzaldhyde and/or1-naphthaldehyde/3 drops AcOH/EtOH, reflux.
Scheme 3. General methods for the preparation of compounds 12–21a–e. Reagents and conditions: (i) benzylamine/EtOH, reflux; (ii) piperonylamine/EtOH, reflux; (iii) 1-phenylethylamine/EtOH, reflux; (iv) 2-methylpiperidine or morpholine/EtOH, reflux; (v) malononitrile/triethylamine (1 mL)/EtOH, reflux; (vi) hydrazine hydrate 98% (2 mL)/EtOH, reflux; (vii) ethyl acetoacetate/AcOH, reflux; (viii) acetylacetone/AcOH, reflux; (ix) DMF or AcOH, reflux; (x) isatin/3 drops AcOH/EtOH, reflux; and (xi) appropriate aromatic aldehyde, namely: 2-bromobenzaldhyde, 4-nitrobenzaldhyde, 2-methoxy-benzaldhyde, 3,4-dimethoxybenzaldhyde and/or1-naphthaldehyde/3 drops AcOH/EtOH, reflux.
Molecules 21 00030 g005
Compound 2 was also refluxed with malononitrile to afford 4-amino-7-(2,4-dichlorophenyl)-5-(4-fluorophenyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carbonitrile (7) via the intermediates 5 and 6, as confirmed by elemental analysis, 1H- and 13C-NMR. The R spectrum of compound 7 showed bands at 3312, 3249, 3145 and 1688 cm−1 due to NH2, NH and CO groups, respectively; its 13C-NMR spectrum showed signals at δ 118.1 and 168.8 ppm corresponding to CN and CO groups, respectively. Its mass spectrum showed a molecular ion peak at m/z 424 (M+; 92%), which conforms to its molecular formula C21H11Cl2FN4O.
Moreover, when pyridin-3-carbonitrile 2 was alkylated with ethyl bromoacetate in acetone using anhydrous potassium carbonate as catalyst, the ester derivatives 8 was produced (Scheme 2). The 1H-NMR spectrum of 8 showed signals at δ 1.15, 4.13 and 5.11 ppm due to the presence of (CH3-ester), (OCH2-ester) and (O–CH2) respectively; and its 13C-NMR exhibited signals at δ 13.5, 43.8, 61.3 and 167.9 ppm due to (CH3), (2CH2) and (CO) groups, respectively.
Ester derivative 8 were condensed with hydrazine hydrate (98%) in ethanol to give 2-[3-cyano-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)pyridin-2-yloxy]acetohydrazide (9) (Scheme 2), confirmed by its IR and NMR spectra. Its IR spectrum showed strong peaks at 3314, 3282 and 3116 cm−1 indicating the presence of a –NHNH2 group, and the NMR (1H and 13C) and mass spectra were also in accordance with its structure.
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 (NH2) group absorption and its 1H-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 benzyl- amine, 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 1H-NMR of compound 15b for example, showed signals at δ 3.31 and 3.72 ppm due to the presence of (2N–CH2) and (2O–CH2) groups, respectively; and its 13C-NMR exhibited signals at δ 47.9, 49.1, 64.10 and 65.9 ppm due to the presence of (2N–CH2) and (2O–CH2), 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 (1H and 13C) 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 –NHNH2 group. Its mass spectrum showed a molecular ion peak at m/z 372 (M+; 39%), which conforms to its molecular formula C18H11Cl2FN4.
The 2-hydrazino-nicotinonitrile 17 is another key compound, which facilitates the synthesis of diverse heterocyclic compounds. Thus, it reacted with different active methylene (β-diketones), namely: ethyl acetoacetate and acetylacetone in glacial acetic acid, and thus the N-pyrazolo derivatives 18 and 19 were produced (Scheme 3). The IR spectrum of compound 19, for example, showed a characteristic band at 2210 cm−1 for the CN group and its 1H-NMR spectrum revealed singlets at δ 2.31, 2.45 and 6.15 ppm due to (2CH3) and the (CH-pyrazole) moieties, respectively. The 13C-NMR data displayed two characteristic signals at δ 18.4, 19.3 and 117.9 ppm for 2CH3 and CN groups, respectively. Also, on heating compound 17 with isatin in ethanol it afforded 6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)-2-(2-(2-oxoindolin-3-ylidene)hydrazinyl)nicotinonitrile (20) in good yield (Scheme 3). The structure of compound 20 gave correct elemental analyses values and spectral features.
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 NH2 group. In addition, the 1H-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 NH2 functional group at the CN group.

2.2. 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 IC50 values of the synthesized compounds compared to the reference drug are shown in Table 1.
Table
Table 1. Cytotoxic activity in (IC50, μg/mL) by the newly synthesized compounds against human cancer cell lines and normal cells.
Table 1. Cytotoxic activity in (IC50, μg/mL) by the newly synthesized compounds against human cancer cell lines and normal cells.
Comp. No.IC50 (μg/mL)
MCF-7NCI-H460SF-268WI 38
467.04 ± 6.23 c56.75 ± 8.20 c69.05 ± 9.15 c18.62 ± 1.21
736.22 ± 2.14 c74.03 ± 3.65 c62.13 ± 3.61 c22.97 ± 8.2
80.02 ± 0.002 a0.01 ± 0.002 a0.02 ± 0.045 anon-cytotoxic
92.41 ± 1.24 a2.30 ± 2.86 a0.46 ± 0.06 a62.19 ± 2.02
1030.58 ± 1.10 b30.67 ± 1.64 b28.18 ± 8.83 b19.80 ± 2.68
1316.26 ± 1.87 b18.92 ± 1.03 b23.24 ± 4.12 b20.38 ± 4.99
15a37.07 ± 7.34 c16.37 ± 2.32 b38.94 ± 2.63 c30.62 ± 6.21
160.01 ± 0.002 a0.02 ± 0.001 a0.01 ± 0.003 anon-cytotoxic
170.61 ± 0.082 a0.86 ± 0.02 a2.19 ± 0.83 a64.11 ± 1.22
1820.22 ± 2.26 b0.01 ± 0.003 a20.20 ± 3.26 b29.82 ± 4.88
1975.20 ± 13.86 c62.30 ± 10.35 c10.39 ± 4.19 a50.20 ± 10.22
200.66 ± 0.21 a0.90 ± 0.12 a2.34 ± 0.51 a72.45 ± 2.40
21d66.02 ± 8.25 c44.95 ± 10.46 c32.45 ± 6.04 bnon-cytotoxic
DMSO0000
Doxorubicin0.04 ± 0.0080.09 ± 0.0080.09 ± 0.007non-cytotoxic
MCF-7 (breast adenocarcinoma); NCI-H460 (non-small cell lung cancer); SF-268 (CNS cancer); WI 38 (normal fibroblast cells); Doxorubicin (anticancer positive control); DMSO (solvent, negative control); a highly active; b moderately active; c weakly active.
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 IC50 values ranged from 0.01 ± 0.002 to 0.02 ± 0.001 μg/mL) and these compounds were non-cytotoxic on the normal cells (IC50 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 (IC50 values ranged from 0.46 ± 0.006 to 2.43 ± 0.51 μg/mL) with weak cytotoxicity on the normal cells (IC50 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 (IC50 values ranged from 10.39 ± 4.19 to 75.20 ± 13.86 μg/mL) with cytotoxic effects on the human normal cell (IC50 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].
Molecules 21 00030 g002 550
Figure 2. Cytotoxic activity of some newly synthesized compounds against human cancer cell lines and normal cells.
Figure 2. Cytotoxic activity of some newly synthesized compounds against human cancer cell lines and normal cells.
Molecules 21 00030 g002

3. Experimental Section

3.1. 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. 1H-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. 13C-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.

3.2. Synthetic Procedures

3.2.1. 6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-oxo-1,2-dihydropyridin-3-carbonitrile (2)

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).
Method B: A mixture of 2,4-dichloroacetophenone (1.88 g, 0.01 mol), 4-fluorobenzaldehyde (1.24 g, 0.01 mol), ethyl cyanoacetate (1.13 g, 0.01 mol) and ammonium acetate (6.16 g, 0.08 mol) in n-butanol (20 mL) was refluxed for 3 h, to give yellow crystals that were then filtered, washed with water, dried and recrystallized to give the title compound 2 (85% yield).
Compound 2: m.p. 276–277 °C; IR (KBr) νmax in cm−1: 3278 (NH), 2219 (CN), 1632 (C=O); 1H-NMR (DMSO-d6): 6.61 (s, 1H, pyridine H5), 7.41–7.85 (m, 7H, Ar–H), 8.10 (br. s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 117.6 (CN), 119.61, 120.23, 122.00, 122.54, 123.33, 123.59, 133.93, 135.87, 136,12, 142.45, 144.64, 146.21, 146.87, 151,32, 151.98 (16 Ar–C), 161.8 (C=O); MS, m/z (%): 358 [M]+ (98), 360 [M + 2]+ (59), 362 (M+ + 4; 11%). Anal. Calcd. for C18H9Cl2FN2O (359.18): C, 60.19; H, 2.53; N, 7.80%; found: C, 59.97 ; H, 2.74; N, 7.94%.

3.2.2. 6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-7H-pyrazolo[3,4-b]pyridin-3-amine (4)

Method A: A mixture of compound 2 (3.6 g, 0.01 mol) and hydrazine hydrate 98% (1 mL, 0.02 mol) in absolute ethanol (30 mL) was refluxed for 15 h. The reaction mixture was left at room temperature overnight and then poured into ice/cold water to complete precipitation. The product was filtered, dried and recrystallized from benzene to give compound 4 (75% yield).
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).
Compound 4: m.p. 309–311 °C; IR (KBr) νmax in cm−1: 3414, 3335, 3180 (NH2, NH); 1H-NMR (DMSO-d6): 5.69 (s, 2H, NH2, D2O exchangeable), 7.12–7.97 (m, 8H, Ar–H + pyridine H5), 10.05 (s, H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 109.76, 111.54, 112.32, 114.29, 115.21, 115.98, 120.54, 121.87, 123.34, 126.30, 132.64, 138.97, 144.54, 145.92, 148.40 (16 Ar–C), 157.1, 158.9 (2C=N); MS, m/z (%): 372 [M]+ (72), 374 [M + 2]+ (40), 376 [M + 4]+ (8). Anal. Calcd. for C18H11Cl2FN4 (373.21): C, 57.93; H, 2.97; N, 15.01%; found: C, 58.12 ; H, 2.70; N, 15.23%.

3.2.3. 4-Amino-7-(2,4-dichlorophenyl)-5-(4-fluorophenyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carbonitrile (7)

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) νmax in cm−1: 3312, 3249, 3145 (NH2, NH), 2223 (CN), 1688 (C=O); 1H-NMR (DMSO-d6): 5.46 (s, 2H, NH2, D2O exchangeable), 6.89 (s, 1H, pyridine H5), 7.10–7.41 (m, 7H, Ar–H), 8.20 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 118.1 (CN), 121.32, 122.87, 123.76, 125.32, 128.43, 132.65, 133.87, 134.12, 138.09, 139.56, 141.54, 147.65, 148.02, 149.53 (18 Ar–C), 158.6 (C=N), 168.9 (C=O); MS, m/z (%): 424 [M]+ (92), 426 [M + 2]+ (59), 428 [M + 4]+ (10). Anal. Calcd. for C21H11Cl2FN4O (425.24): C, 59.31; H, 2.61; N, 13.18%; found: C, 59.12; H, 2.79; N, 13.33%.

3.2.4. Ethyl 2-[3-cyano-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)pyridin-2-yloxy]acetate (8)

A mixture of compound 2 (3.6 g, 0.01 mol), ethyl bromoacetate (1.2 mL, 0.01 mol) and anhydrous potassium carbonate (2.10 g, 0.015 mol) in dry acetone (50 mL) was refluxed for 24 h. The reaction mixture was cooled and poured onto ice/cold water; the solid that separated out was filtered, dried and recrystallized from dioxane to give compound 8. Yield 75%; m.p. 119–121 °C; IR (KBr) νmax in cm−1: 2220 (CN), 1755 (C=O ester); 1H-NMR (DMSO-d6): 1.15 (t, J = 7.5 Hz, 3H, CH3-ester), 4.13 (q, J = 7.5 Hz, 2H, O-CH2-ester), 5.11 (s, 2H, O-CH2), 7.00–7.40 (m, 8H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 13.5 (CH3), 44.8, 61.3 (2CH2), 118.6 (CN), 123.30, 123.54, 127.10, 127.98, 131.34, 133.54, 137.07, 138.76, 141.99, 142.76, 144.65, 126.87, 148.43 (16 Ar–C), 159.1 (C=N), 167.9 (C=O); MS, m/z (%): 444 [M]+ (61), 446 [M + 2]+ (37), 448 [M + 4]+ (6), 371 [M–COOC2H5]+ (41). Anal. Calcd. for C22H15Cl2FN2O3 (445.27): C, 59.34; H, 3.40; N, 6.29%; found: C, 59.52 ; H, 3.71; N, 6.05%.

3.2.5. 2-[3-Cyano-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)pyridin-2-yloxy]acetohydrazide (9)

A mixture of compound 8 (4.5 g, 0.01 mol), hydrazine hydrate 98% (2 mL, 0.04 mol) and absolute ethanol (30 mL) was refluxed for 4 h. The reaction mixture was cooled and the formed solid was filtered, dried and recrystallized from acetic acid to give compound 9. Yield 76%; m.p. 197–199 °C; IR (KBr) νmax in cm−1: 3314, 3282, 3116 (NH2, NH), 2225 (CN), 1696 (C=O ester); 1H-NMR (DMSO-d6): 4.92 (s, 2H, CH2), 7.00–7.40 (m, 8H, Ar–H + pyridine H5), 8.2 (s, 1H, NH, D2O exchangeable), 9.8 (s, 2H, NH2, D2O exchangeable); 13C-NMR (DMSO-d6): 62.4 (CH2), 119.7 (CN), 121.65, 124.05, 124,90, 125.71, 128.23, 129.98, 133.76, 137.54, 142.12, 143.45, 145.86 (16 Ar–C), 158.6 (C=N), 169.4 (C=O); MS, m/z (%): 430 [M]+ (31), 432 [M + 2]+ (20), 434 [M + 4]+ (4), 371 [M–CONHNH2]+ (8). Anal. Calcd. for C20H13Cl2FN4O2 (431.25): C, 55.70; H, 3.04; N, 12.99%; found: C, 55.52; H, 3.21; N, 13.05%.

3.2.6. 2-[3-Cyano-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)pyridin-2-yloxy]-N′-(4-fluorobenzylidene)-acetohydrazide (10)

A mixture of compound 9 (4.3 g, 0.01 mol) and 4-fluorobenzaldehyde (1.24 g, 0.01 mol) in ethanol (20 mL) was refluxed for 6 h. The solid formed after cooling was filtered, dried and recrystallized from acetic acid to give compound 10. Yield 79%; m.p. 219–221 °C ; IR (KBr) νmax in cm−1: 3218 (NH), 2226 (CN), 1665 (C=O); 1H-NMR (DMSO-d6): 5.1 (s, 2H, O–CH2), 7.32–7.87 (m, 12H, Ar–H + pyridine H5), 8.10 (s, 1H, CH=N (azomethine protone)), 11.08 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 64.1 (CH2), 117.5 (CN), 120.98, 121.01, 121.48, 123.40, 125.21, 127.83, 128.03, 128.99, 132.06, 133.56, 137.98, 138.43, 141.32, 142.65, 144.64, 146.10, 149.01, 149.97, 150.16, 150.63 (23 Ar–C), 157.2 (C=N), 159.4 (CH=N); MS, m/z (%): 536 [M]+ (97), 538 [M + 2]+ (64), 540 [M + 4]+ (11). Anal. Calcd. for C27H16Cl2F2N4O2 (537.34): C, 60.35; H, 3.00; N, 10.43%; found: C, 60.56 ; H, 3.21; N, 10.65%.

3.2.7. 2-Chloro-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)nicotinonitrile (11)

A mixture of compound 2 (3.6 g, 0.01 mol) and phosphorus oxychloride (4.6 mL, 0.03 mol) was refluxed for 8 h. The reaction mixture was poured into crushed ice and the separated solid was filtered, dried and recrystallized from dioxane to give compound 11. Yield 62%; m.p. 181–182 °C; IR (KBr) νmax in cm−1: 2223 (CN); 1H-NMR (DMSO-d6): 6.80 (s, 1H, pyridine H5), 7.62–8.01 (m, 7H, Ar–H); 13C-NMR (DMSO-d6): 119.2 (CN), 122.31, 125.86, 127.90, 128.54, 131.98, 133.32, 138.32, 139.09, 144.89, 145.07, 148.48 (16 Ar–C), 158.7 (C=N); MS, m/z (%): 376 [M]+ (62), 378 [M + 2]+ (60), 380 [M + 4]+ (18); 382 [M + 6]+ (2). Anal. Calcd. for C18H8Cl3FN2 (377.63): C, 57.25; H, 2.14; N, 7.42%; found: C, 57.10; H, 2.43; N, 7.65%.

3.2.8. General procedure for the synthesis of 2-(benzylamino)-6-(2,4-dichlorophenyl)-4-(4-fluoro-phenyl)-nicotinonitrile (12), 2-(benzo[d][1,3]dioxol-5-ylmethylamino)-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)nicotinonitrile (13), 6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)-2-(1-phenylethyl-amino)nicotinonitrile (14), and 6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)-2-(2-substituted-1-yl)nicotinonitriles 15a,b

A mixture of chloropyridine 11 (3.8 g, 0.01 mol) and the appropriate amine, namely benzyl-amine, piperonylamine, 1-phenylethylamine, 2-methylpiperidine and/or morpholine (0.01 mol) in absolute ethanol (30 mL) was refluxed for 6–12 h. The reaction mixture was poured onto ice/cold water, filtered, washed with petroleum ether 60–80 and finally crystallized from ethanol to give the desired derivatives 12–15a,b, respectively.
2-(Benzylamino)-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)nicotinonitrile (12): Yield 48%; m.p. 158–159 °C; IR (KBr) νmax in cm−1: 3218 (NH), 2220 (CN); 1H-NMR (DMSO-d6): 4.97 (s, 2H, CH2), 6.98 (br s, 1H, NH, D2O exchangeable), 7.62–8.31 (m, 13H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 51.45 (CH2), 118.7 (CN), 120.01, 120.96, 122.73, 124.65, 128.02, 129.90, 131.45, 133.63, 134.06, 136.81, 139.43, 141.45, 144.09, 146.12, 147.48, 148.18, 149.74, 150.23, 153.42 (22 Ar–C), 159.2 (C=N); MS, m/z (%): 447 [M]+ (30), 449 [M + 2]+ (17), 451 [M + 4]+ (3). Anal. Calcd. for C25H16Cl2FN3 (448.32): C, 66.98; H, 3.60; N, 9.37%; found: C, 67.10 ; H, 3.40; N, 9.55%.
2-(Benzo[d][1,3]dioxol-5-ylmethylamino)-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)nicotinonitrile (13): Yield 59%; m.p. 149–151 °C; IR (KBr) νmax in cm−1: 3229 (NH), 2218 (CN); 1H-NMR (DMSO-d6): 4.85 (s, 2H, CH2), 6.21 (s, 2H, CH2), 7.02 (br s, 1H, NH, D2O exchangeable), 7.68–8.22 (m, 11H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 49.5, 88.6 (2CH2), 117.2 (CN), 121.72, 122.98, 124.61, 128.03, 131.43, 133.92, 134.98, 137.38, 138.51, 142.26, 143.13, 144.06, 144.97, 148.26, 149.49 (22 Ar–C), 158.7 (C=N); MS, m/z (%): 491 [M]+ (19), 493 [M + 2]+ (13), 495 [M + 4]+ (2). Anal. Calcd. for C26H16Cl2FN3O2 (492.33): C, 63.43; H, 3.28; N, 8.53%; found: C, 63.66 ; H, 3.47; N, 8.41%.
6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-(phenylethylamino)nicotinonitrile (14): Yield 32%; m.p. 142–143 °C; IR (KBr) νmax in cm−1: 3203 (NH), 2228 (CN); 1H-NMR (DMSO-d6): 2.93 (m, 2H, CH2), 3.39 (m, 2H, CH2), 7.32 (br s, 1H, NH, D2O exchangeable), 7.76–8.36 (m, 13H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 39.1, 46.9 (2CH2), 119.1 (CN), 122.01, 122.86, 123.84, 124.62, 127.97, 128.12, 131.82, 133.27, 136.46, 139.65, 141.54, 144.87, 146.32, 147.07, 148.86, 149.41 (22 Ar–C), 159.0 (C=N); MS, m/z (%): 461 [M]+ (32), 463 [M + 2]+ (23), 465 [M + 4]+ (3). Anal. Calcd. for C26H18Cl2FN3 (462.35): C, 67.54; H, 3.92; N, 9.09%; found: C, 67.69 ; H, 3.71; N, 8.89%.
6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-(2-methylpiperidin-1-yl)nicotinonitrile (15a): Yield 47%; m.p. 98–100 °C; IR (KBr) νmax in cm−1: 2219 (CN); 1H-NMR (DMSO-d6): 1.36 (s, 3H, CH3), 1.59–1.73 (m, 6H, 3CH2-piperidine protons), 2.78 (m, 3H, (CH + CH2) piperidine protons), 7.48–8.01 (m, 8H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 18.6 (CH3), 22.4, 25.2, 36.9, 49.3 (4CH2), 56.7 (CH), 119.7 (CN), 120.76, 121.20, 125.85, 128.32, 129.04, 132.85, 133.25, 137.87, 138.13, 139.08, 141.24, 143.79, 143.9 (16 Ar–C), 157.9 (C=N). Anal. Calcd. for C24H20Cl2FN3 (440.34): C, 65.46; H, 4.58; N, 9.54%; found: C, 65.70 ; H, 4.32; N, 9.39%.
6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-morpholinonicotinonitrile (15b): Yield 32%; m.p. 112–114 °C; IR (KBr) νmax in cm−1: 2227 (CN); 1H-NMR (DMSO-d6): 3.31 (m, 4H, 2N-CH2), 3.72 (m, 4H, 2O-CH2), 7.52–7.78 (m, 8H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 47.9, 49.1 (2N-CH2), 64.10, 65.9 (2O–CH2), 119.1 (CN), 120.46, 122.04, 122.83, 126.23, 127.85, 129.27, 131.47, 133.56, 134.26, 138.93, 139.08, 142.86, 144.75, 146.12, 146.91, 147.15 (16 Ar–C), 158.9 (C=N). Anal. Calcd. for C22H16Cl2FN3O (428.29): C, 61.70; H, 3.77; N, 9.81%; found: C, 61.88 ; H, 3.42; N, 9.69%.

3.2.9. 2-[3-Cyano-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)pyridin-2-yl]malononitrile (16)

To a solution of compound 11 (3.8 g, 0.01 mol) in absolute ethanol (30 mL), triethylamine (1 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 ethanol to give compound 16. Yield 39%; m.p. 99–101 °C; IR (KBr) νmax in cm−1: 2218, 2225 (CN); 1H-NMR (DMSO-d6): 5.02 (s, 1H, CH), 7.35–8.13 (m, 8H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 32.8 (CH), 117.8, 119.5 (3CN), 120.76, 122.45, 123.26, 124.01, 124.81, 128.34, 129.27, 132.23, 133.84, 137.37, 139.06, 142.29, 144.52, 145.14, 146.17, 147.91 (16 Ar–C), 159.3 (C=N); MS, m/z (%): 406 [M]+ (96), 408 [M + 2]+ (62), 410 [M + 4]+ (11). Anal. Calcd. for C21H9Cl2FN4 (407.23): C, 61.94; H, 2.23; N, 13.76%; found: C, 62.05; H, 2.39; N, 13.63%.

3.2.10. 6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-hydrazinylnicotinonitrile (17)

A mixture of the chloropyridine derivative 11 (3.8 g, 0.01 mol) and hydrazine hydrate (98%, 2 mL, 0.04 mol) in ethanol (20 mL) was stirred under reflux for 6 h. The formed precipitate was filtered, dried and recrystallized from methanol to give the hydrazinyl derivative 17. Yield 85%; m.p. 223–224 °C; IR (KBr) νmax in cm−1: 3440, 3320, 3150 (NH2, NH), 2218 (CN); 1H-NMR (DMSO-d6): 5.40 (s, 2H, NH2, D2O exchangeable), 7.39–8.21 (m, 8H, Ar–H + pyridine H5), 9.40 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 119.5 (CN), 121.09, 122.58, 123.71, 125.35, 129.34, 132.98, 134.46, 138.47, 139.07, 142.32, 144.35, 145.07, 147.43, 148.54, 149.76, 150.12 (16 Ar–C), 158.9 (C=N); MS, m/z (%): 372 [M]+ (39), 374 [M + 2]+ (21), 346 [M + 4]+ (4). Anal. Calcd. for C18H11Cl2FN4 (373.21): C, 57.93; H, 2.97; N, 15.01%; found: C, 58.23; H, 2.80; N, 14.89%.

3.2.11. General procedure for the synthesis of 6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)-2-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)nicotinonitrile (18) and 6-(2,4-dichlorophenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-4-(4-fluorophenyl)nicotinonitrile (19)

A mixture of compound 17 (3.7 g, 0.01 mol) and ethyl acetoacetate or acetylacetone (0.01 mol) in acetic acid (15 mL) was refluxed for 8 h. The solid formed after cooling was filtered, dried and recrystallized from ethanol to give compounds 18, and 19 respectively.
6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl) nicotinonitrile (18): Yield 40%; m.p. 246–248 °C; IR (KBr) νmax in cm−1: 2227 (CN), 1701 (C=O); 1H-NMR (DMSO-d6): 1.94 (s, 3H, CH3), 2.26 (s, 2H, CH2), 7.52–7.78 (m, 8H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 19.3 (CH3), 41.5 (CH2), 118.3 (CN), 122.62, 123.42, 126.81, 129.17, 131.11, 133.91, 136.23, 137.05, 139.54, 14.13, 142.53, 143.06, 143.94 (16 Ar–C), 158.4, 159.3 (2C=N), 166.5 (C=O); MS, m/z (%): 438 [M]+ (16), 440 [M + 2]+ (11), 442 [M + 4]+ (2). Anal. Calcd. for C22H13Cl2FN4O (439.24): C, 60.15; H, 2.98; N, 12.75%; found: C, 60.25; H, 3.12; N, 12.54%.
6-(2,4-Dichlorophenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-4-(4-fluorophenyl)nicotinonitrile (19): Yield 32%; m.p. 287–289 °C; IR (KBr) νmax in cm−1: 2210 (CN); 1H-NMR (DMSO-d6): 2.31 (s, 3H, CH3), 2.45 (s, 3H, CH3), 6.15 (s, 1H, CH-pyrazole), 7.46–8.09 (m, 8H, Ar–H + pyridine H5); 13C-NMR (DMSO-d6): 18.4, 19.3 (2CH3), 108.4 (CH-pyrazole), 117.9 (CN), 122.94, 123.27, 124.18, 128.97, 129.46, 132.74, 133.24, 136.93, 138.08, 139.60, 143.84, 144.94, 148.23, 148.86 (17 Ar–C), 158.3, 159.5 (2C=N); MS, m/z (%): 436 [M]+ (12), 438 [M + 2]+ (8), 440 [M + 4]+ (1). Anal. Calcd. for C23H15Cl2FN4 (437.30): C, 63.17; H, 3.46; N, 12.81%; found: C, 62.98; H, 3.17; N, 12.61%.

3.2.12. 6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-[2-(2-oxoindolin-3-ylidene)hydrazinyl]nicotinonitrile (20)

A mixture of the compound 17 (3.7 g, 1 mmol) and isatin (1.5 g, 1 mmol) in ethanol (25 mL) containing 3 drops of acetic acid was refluxed for 2 h, then left overnight at room temperature. The formed precipitate was filtered, dried and recrystallized from benzene to give 20. Yield 79%; m.p. 268–269 °C; IR (KBr) νmax in cm−1: 3289, 3150 (2NH), 1723 (C=O), 2227 (CN); 1H-NMR (DMSO-d6): 6.98 (br s, 1H, NH, D2O exchangeable), 7.22–8.24 (m, 12H, Ar–H + pyridine H5), 10.02 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 120.01 (CN), 120.42, 121.07, 122.86, 123.07, 124.52, 129.05, 129.96, 131.46, 133.63, 135.64, 137,93, 139.75, 141.25, 143.86, 144.52, 145.28, 147.94, 148.6 149.25 (22 Ar–C), 157.9, 158.3 (2C=N), 167.23 (C=O). MS, m/z (%): 501 [M]+ (85), 503 [M + 2]+ (60), 505 [M + 4]+ (9). Anal. Calcd. for C26H14Cl2FN5O (502.33): C, 62.17; H, 2.81; N, 13.94%; found: C, 61.97; H, 2.60; N, 14.19%.

3.2.13. General procedure for the synthesis of 6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)-2-[2-(2-substiutedbenzylidene)hydrazinyl]nicotinonitriles (1a–e)

A mixture of compound 17 (3.7 g, 0.01 mol), an appropriate aromatic aldehyde namely 2-bromo-benzaldhyde, 4-nitrobenzaldhyde, 2-methoxybenzaldhyde, 3,4-dimethoxybenzaldhyde and/or 1-naphthaldehyde (0.01 mol) in ethanol (20 mL) containing 3 drops of acetic acid was refluxed for 6–8 h. The precipitate formed after cooling was filtered, dried and recrystallized to give compounds 21a–e, respectively.
2-[2-(2-Bromobenzylidene)hydrazinyl]-6-(2,4-dichlorophenyl)-4-(4-fluorophenyl)nicotinonitrile (21a): Yield 41%; m.p. 226–228 °C; IR (KBr) νmax in cm−1: 3299 (NH), 2217 (CN); 1H-NMR (DMSO-d6): 7.26–8.19 (m, 12H, Ar–H + pyridine H5), 8.32 (s, 1H, CH=N azomethine proton), 10.13 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 118.6 (CN), 121.83, 122.94, 123.10, 123.86, 127.39, 128.93, 129.27, 132.81, 135.23, 139.38, 141.04, 142.50, 144.52, 148.01, 148.87, 149.08, 150.65, 151.31 (22 Ar–C), 158.3 (C=N), 161.3 (CH=N); MS, m/z (%): 538 [M]+ (14), 540 [M + 2]+ (10), 542 [M + 4]+ (1). Anal. Calcd. for C25H14BrCl2FN4 (540.21): C, 55.58; H, 2.61; N, 10.37%; found: C, 55.68; H, 2.79; N, 10.51%.
6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-[2-(4-nitrobenzylidene)hydrazinyl]nicotinonitrile (21b): Yield 23%; m.p. 281–283 °C; IR (KBr) νmax in cm−1: 3253 (NH), 2223 (CN); 1H-NMR (DMSO-d6): 7.29–8.14 (m, 12H, Ar–H + pyridine H5), 8.31 (s, 1H, CH=N (azomethine protone)), 10.08 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 119.0 (CN), 121.52, 122.61, 123.48, 127.85, 128.03, 129.53, 130.21, 133.25, 134.56, 136.87, 137.04, 139.08, 139.96, 142.19, 143.15, 144.08, 148.60, 149.31 (22 Ar–C), 158.7 (C=N), 160.8 (CH=N); MS, m/z (%): 505 [M]+ (60), 507 [M + 2]+ (39), 509 [M + 4]+ (7). Anal. Calcd. for C25H14Cl2FN5O2 (506.32): C, 59.30; H, 2.79; N, 13.83%; found: C, 59.58; H, 2.63; N, 13.51%.
6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-[2-(2-methoxybenzylidene)hydrazinyl] nicotinonitrile (21c): Yield 34%; mp over 300 °C; IR (KBr) νmax in cm−1: 3258 (NH), 2218 (CN); 1H-NMR (DMSO-d6): 3.39 (s, 3H, OCH3), 7.49–8.09 (m, 12H, Ar–H + pyridine H5), 8.33 (s, 1H, CH=N (azomethine protone)), 10.16 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 51.9 (OCH3), 118.7 (CN), 121.09, 122.58, 123.71, 125.35, 129.34, 132.98, 134.46, 137.94, 138.47, 139.07, 142.32, 144.35, 145.07, 147.43, 148.54, 149.76, 150.12, 151.72 (22 Ar–C), 158.1 (C=N), 159.9 (CH=N). Anal. Calcd. for C26H17Cl2FN4O (491.34): C, 63.56; H, 3.49; N, 11.40%; found: C, 63.78; H, 3.26; N, 11.57%.
6-(2,4-Dichlorophenyl)-2-[2-(3,4-dimethoxybenzylidene)hydrazinyl]-4-(4-fluorophenyl) nicotinonitrile (21d): Yield 31%; m.p. 278–280 °C; IR (KBr) νmax in cm−1: 3294 (NH), 2220 (CN); 1H-NMR (DMSO-d6): 3.39 (2s, 6H, 2OCH3), 7.36–8.10 (m, 11H, Ar–H + pyridine H5), 8.31 (s, 1H, CH=N (azomethine protone)), 10.23 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 55.6, 56.01 (2OCH3), 119.3 (CN), 120.46, 121.08, 122.85, 123.05, 127.30, 128.74, 129.76, 130.64, 133.73, 134.05, 138.12, 138.98, 140.16, 143.54, 144.35, 145.97, 147.46, 148.02, 149.08 (22 Ar–C), 158.7 (C=N), 161.4 (CH=N). Anal. Calcd. for C27H19Cl2FN4O2 (521.37): C, 62.20; H, 3.67; N, 10.75%; found: C, 62.38; H, 3.43; N, 10.53%.
6-(2,4-Dichlorophenyl)-4-(4-fluorophenyl)-2-[2-(naphthalen-1-ylmethylene)hydrazinyl] nicotinonitrile (21e): Yield 24%; m.p. 296–298 °C; IR (KBr) νmax in cm−1: 3282 (NH), 2213 (CN); 1H-NMR (DMSO-d6): 7.24–8.26 (m, 15H, Ar–H + pyridine H5), 8.32 (s, 1H, CH=N (azomethine protone)), 10.18 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6): 119.0 (CN), 120.76, 122.45, 123.26, 124.01, 124.81, 128.34, 129.27, 132.23, 133.84, 137.37, 139.06, 142.29, 144.52, 145.14, 146.17, 147.91, 148.96, 149.24, 149,99, 150.65, 151.34, 152.61 (26 Ar–C), 158.3 (C=N), 161.0 (CH=N). Anal. Calcd. for C29H17Cl2FN4 (511.38): C, 68.11; H, 3.35; N, 10.96%; found: C, 67.96; H, 3.49; N, 11.06%.

3.3. Anticancer Activity

3.3.1. 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% CO2. Exponentially growing cells were obtained by plating 1.5 × 105 cells/mL for MCF-7 and SF-268, and 0.75 × 104 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.

3.3.2. 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% CO2. 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% (IC50), 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.

4. 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 (IC50 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.

Author Contributions

The listed authors contributed to this work as described in the following: Eman M. Flefel and Hebat-Allah S. Abbas developed the concept of the work, carried out the synthetic work, interpreted the results and prepared the manuscript, Randa E. Abdel Magid, carried out the synthetic work, interpreted the results and prepared the manuscript and Wafaa A. Zafgary interpreted the results and cooperated in the preparation of the manuscript. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Zhang, J.Y. Apoptosis-based anticancer drugs. Nat. Rev. Drug Disccov. 2002, 1, 101–102. [Google Scholar] [CrossRef]
  2. Ali, A.; Fergus, K.; Wright, F.C.; Pritchard, K.I.; Kiss, A.; Warner, E. The impact of a breast cancer diagnosis in young women on their relationship with their mothers. Breast 2014, 23, 50–55. [Google Scholar] [CrossRef] [PubMed]
  3. Lam, S.W.; Jimenez, C.R.; Boven, E. Breast cancer classification by proteomic technologies: Current state of knowledge. Cancer 2014, 40, 129–138. [Google Scholar] [CrossRef] [PubMed]
  4. Hassan, G.S.; Kadry, H.H.; Abou-Seri, S.M.; Ali, M.M.; Mahmoud, A.E.E. Synthesis and in vitro cytotoxic activity of novel pyrazolo[3,4-d]pyrimidines and related pyrazole hydrazones toward breast adenocarcinoma MCF-7 cell line. Bioorg. Med. Chem. 2011, 19, 6808–6817. [Google Scholar] [CrossRef] [PubMed]
  5. Taher, A.T.; Georgey, H.H.; El-Subbagh, H.I. Novel 1,3,4-heterodiazole analogues: Synthesis and in vitro antitumor activity. Eur. J. Med. Chem. 2012, 47, 445–451. [Google Scholar] [CrossRef] [PubMed]
  6. Carmen, A.J.; Carlos, M. Medicinal Chemistry of Anticancer Drugs, 1st ed.; Elsevier: Amsterdam, The Netherlands, 2008; pp. 1–8. [Google Scholar]
  7. Borowski, E.; Bontemps-Gracz, M.M.; Piwkowska, A. Strategies for overcoming ABC-transporters-mediated multidrug resistance (MDR) of tumor cells. Acta Biochim. Pol. 2005, 52, 609–627. [Google Scholar] [PubMed]
  8. Avila, H.P.; Smania, E.F.; Monache, F.D.; Smania, A. Structure-activity relationship of antibacterial chalcones. Bioorg. Med. Chem. 2008, 16, 9790–9794. [Google Scholar] [CrossRef] [PubMed]
  9. Liu, Y.; Sun, X.; Yin, D.; Yuan, F. Syntheses and biological activity of chalcones-imidazole derivatives. Res. Chem. Intermed. 2013, 39, 1037–1048. [Google Scholar] [CrossRef]
  10. Sortino, M.; Delgado, P.; Juarez, S.; Quiroga, J.; Abonia, R.; Insuasty, B.; Nogueras, M.; Rodero, L.; Garibotto, F.M.; Enriz, R.D.; et al. Synthesis and antifungal activity of (Z)-5-arylidenerhodanines. Bioorg. Med. Chem. 2007, 15, 484–494. [Google Scholar] [CrossRef] [PubMed]
  11. Lopez, S.N.; Castelli, M.V.; Zacchino, S.A.; Dominguez, J.N.; Lobo, G.; Charris-Charris, J.; Cortes, J.C.; Ribas, J.C.; Devia, C.; Rodriguez, A.M.; et al. In vitro antifungal evaluation and structure-activity relationships of a new series of chalcone derivatives and synthetic analogues with inhibitory properties against polymers of the fungal cell wall. Bioorg. Med. Chem. 2001, 8, 1999–2013. [Google Scholar] [CrossRef]
  12. Cheng, J.H.; Hung, C.F.; Yang, S.C.; Wang, J.P.; Won, S.J.; Lin, C.N. Synthesis and cytotoxic, anti-inflammatory, and anti-oxidant activities of 2′,5′-dialkoxylchalcones as cancer chemopreventive agents. Bioorg. Med. Chem. 2008, 16, 7270–7276. [Google Scholar] [CrossRef] [PubMed]
  13. Katsori, A.M.; Hadjipavlou-Litina, D. Chalcones in cancer: Understanding their role in terms of QSAR. Curr. Med. Chem. 2009, 16, 1062–1081. [Google Scholar] [CrossRef] [PubMed]
  14. Modzelewska, A.; Pettit, C.; Achanta, G.; Davidson, N.E.; Huang, P.; Khan, S.R. Anticancer activities of novel chalcone and bis-chalcone derivatives. Bioorg. Med. Chem. 2006, 14, 3491–3495. [Google Scholar] [CrossRef] [PubMed]
  15. Abdelhafez, O.M.; Abdel-Latif, N.A.; Badria, F.A. DNA, Antiviral activities and cytotoxicity of new furochromone and benzofuran derivatives. Arch. Pharm. Res. 2011, 34, 1623–1632. [Google Scholar] [CrossRef] [PubMed]
  16. Abdel-Latif, N.A. Synthesis and antidepressant activity of some new coumarin derivatives. Sci. Pharm. 2005, 74, 173–216. [Google Scholar]
  17. Son, J.K.; Zhao, L.X.; Basnet, A.; Thapa, P.; Karki, R.; Na, Y.; Jahng, Y.; Jeong, T.C.; Jeong, B.S.; Lee, C.S.; et al. Synthesis of 2,6-diaryl-substituted pyridines and their antitumor activities. Eur. J. Med. Chem. 2008, 43, 675–682. [Google Scholar] [CrossRef] [PubMed]
  18. Amr, A.G.; Abdulla, M.M. Anti-inflammatory profile of some synthesized heterocyclic pyridone and pyridine derivatives fused with steroidal structure. Bioorg. Med. Chem. 2006, 14, 4341–4352. [Google Scholar] [CrossRef] [PubMed]
  19. Hammam, A.G.; Abdel Hafez, N.A.; Midura, W.H.; Mikolajczyk, M.Z. Chemistry of seven-membered heterocycles, VI. Synthesis of novel bicyclic heterocyclic compounds as potential anticancer and anti-HIV agents. Z. Naturforsch. 2000, 55, 417–424. [Google Scholar] [CrossRef]
  20. Kotb, E.R.; Anwar, M.M.; Abbas, H.A.S.; Abd El-Moez, S.I. A concise synthesis and antimicrobial activity of a novel series of naphthylpyridine-3-carbonitrile compounds. Acta Pol. Pharm. Drug Res. 2013, 70, 667–679. [Google Scholar]
  21. Sayed, H.H.; Morsy, E.M.; Flefel, E.M. Synthesis and reactions of some novel nicotinonitrile, thiazolotriazole, and imidazolotriazole derivatives for antioxidant evaluation. Synth. Commun. 2010, 40, 1360–1370. [Google Scholar] [CrossRef]
  22. Akira, M.; Aya, N.; Shigeki, I.; Motoki, T.; Kazuo, S. JBIR-54, a new 4-pyridinone derivative isolated from Penicillium daleae Zaleski fE50. J. Antibiot. 2009, 62, 705–706. [Google Scholar]
  23. Al-Omar, M.A.; Amr, A.E.; A.l-Salahi, R.A. Anti-inflamatory, analgesic, anticonvulsant and antiparkinsonian activities of some pyridine derivatives using 2,6-disubstituted isonicotinic acid hydrazides. Archiv. Phaem. 2010, 343, 648–656. [Google Scholar]
  24. Martin, C.; Göggel, R.; dal Piaz, V.; Vergelli, C.; Giovannoni, P.; Ernst, M.; Uhlig, S. Airway relaxant and anti-inflammatory properties of a PDE4 inhibitor with low affinity for the high-affinity rolipram binding site. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2002, 365, 284–289. [Google Scholar] [CrossRef] [PubMed]
  25. Amr, A.E.; Sayed, H.H.; Abdulla, M.A. Synthesis and reactions of some new substituted pyridine and pyrimidine derivatives as analgesic, anticonvulsant and antiparkinsonian agents. Arch. Pharm. Chem. Life Sci. 2005, 338, 433–440. [Google Scholar] [CrossRef] [PubMed]
  26. Al-Abdullah, E.S. Synthesis and anticancer activity of some novel tetralin-6-yl-pyrazoline, 2-thioxopyrimidine, 2-oxopyridine, 2-thioxo-pyridine and 2-iminopyridine derivatives. Molecules 2011, 16, 3410–3419. [Google Scholar] [CrossRef] [PubMed]
  27. Abo-Ghalia, M.; Abdulla, M.M.Z.; Amr, A.E. Synthesis of some new (Nα-dipicolinoyl)-bis-l-leucyl-dl-norvalyl linear tetra and cyclic octa bridged peptides as new antiinflammatory agents. Z. Naturforsch. 2003, 58b, 903–910. [Google Scholar]
  28. Kotb, E.R.; El-Hashash, M.A.; Salama, M.A.; Kalf, H.S.; Abdel Wahed, N.A.M. Synthesis and reactions of some novel nicotinonitrile derivatives for anticancer and antimicrobial evaluation. Acta Chim. Slov. 2009, 56, 908–919. [Google Scholar]
  29. Kumar, S.; Das, S.; Dey, S.; Maity, P.; Guha, M.; Choubey, V.; Panda, G.; Bandyopadhyay, V. Antiplasmodial activity of [(aryl)arylsulfanylmethyl]pyridine. Antimicrob. Agents Chemother. 2008, 52, 705–715. [Google Scholar] [CrossRef] [PubMed]
  30. Ghosh, P.S.; Manna, K.; Banik, U.; Das, M.; Sarkar, P. Synthetic strategies and pharmacology of 2-oxo-3-cyanopyridine derivatives: A review. Int. J Pharm. Pharm. Sci. 2014, 6, 39–42. [Google Scholar]
  31. Abbas, H.-A.S.; El Sayed, W.A.; Fathy, N.M. Synthesis and antitumor activity of new dihydropyridine thioglycosides and their corresponding dehydrogenated forms. Eur. J. Med. Chem. 2010, 45, 973–982. [Google Scholar] [CrossRef] [PubMed]
  32. Al-Mutairi, M.S.; Al-Abdullah, E.S.; Haiba, M.E.; Khedr, M.A.; Zaghary, W.A. Synthesis, molecular docking and preliminary in vitro cytotoxic evaluation of some substituted tetrahydronaphthalene (2′,3′,4′,6′-Tetra-O-Acetyl-β-d-Gluco-/Galactopyranosyl) derivatives. Molecules 2012, 17, 4717–4732. [Google Scholar] [CrossRef] [PubMed]
  33. Kotb, E.R.; Abbas, H.-A.S.; Flefel, E.M.; Sayed, H.H.; Abdel Wahed, N.A.M. Utility of hantzsch ester in synthesis of some 3,5-bisdihydropyridine derivatives and studying their biological evaluation. J. Heterocycl. Chem. 2015, 52, 1531–1539. [Google Scholar] [CrossRef]
  34. Sayed, H.H.; Flefel, E.M.; Abd El-Fatah, A.M.; El-Sofany, W.I. Focus on the synthesis and reactions of some new pyridine carbonitrile derivatives as antimicrobial and antioxidant agents. Egypt J. Chem. 2010, 53, 17–35. [Google Scholar]
  35. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenne, S.; Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82, 1107–1112. [Google Scholar] [CrossRef] [PubMed]
  36. Lee, P.; Zhang, R.; Li, V.; Liu, X.; Sun, R.W.Y.; Che, C.M.; Wong, K.K.Y. Enhancement of anticancer efficacy using modified lipophilic nanoparticle drug encapsulation. Int. J. Nanomed. 2012, 7, 731–737. [Google Scholar] [Green Version]
  37. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paul, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J. Natl. Cancer Inst. 1991, 83, 757–766. [Google Scholar] [CrossRef] [PubMed]
  • Sample Availability: Samples of the compounds are available from the authors.

Share and Cite

MDPI and ACS Style

M. Flefel, E.; S. Abbas, H.-A.; E. Abdel Mageid, R.; A. Zaghary, W. Synthesis and Cytotoxic Effect of Some Novel 1,2-Dihydropyridin-3-carbonitrile and Nicotinonitrile Derivatives. Molecules 2016, 21, 30. https://doi.org/10.3390/molecules21010030

AMA Style

M. Flefel E, S. Abbas H-A, E. Abdel Mageid R, A. Zaghary W. Synthesis and Cytotoxic Effect of Some Novel 1,2-Dihydropyridin-3-carbonitrile and Nicotinonitrile Derivatives. Molecules. 2016; 21(1):30. https://doi.org/10.3390/molecules21010030

Chicago/Turabian Style

M. Flefel, Eman, Hebat-Allah S. Abbas, Randa E. Abdel Mageid, and Wafaa A. Zaghary. 2016. "Synthesis and Cytotoxic Effect of Some Novel 1,2-Dihydropyridin-3-carbonitrile and Nicotinonitrile Derivatives" Molecules 21, no. 1: 30. https://doi.org/10.3390/molecules21010030

APA Style

M. Flefel, E., S. Abbas, H. -A., E. Abdel Mageid, R., & A. Zaghary, W. (2016). Synthesis and Cytotoxic Effect of Some Novel 1,2-Dihydropyridin-3-carbonitrile and Nicotinonitrile Derivatives. Molecules, 21(1), 30. https://doi.org/10.3390/molecules21010030

Article Metrics

Back to TopTop