Synthesis of some novel 6′-(4-chlorophenyl)-3,4′-bipyridine-3′-carbonitriles: assessment of their antimicrobial and cytotoxic activity

Essam M. Hussein; Hossa F. Al-Shareef; Amany H. Aboellil; Heba A. Elhady

doi:10.1515/znb-2015-0065

Article Publicly Available

Synthesis of some novel 6′-(4-chlorophenyl)-3,4′-bipyridine-3′-carbonitriles: assessment of their antimicrobial and cytotoxic activity

, , and

Published/Copyright: October 7, 2015

Published by

Become an author with De Gruyter Brill

Author Information Explore this Subject

From the journal Zeitschrift für Naturforschung B Volume 70 Issue 11

Abstract

A series of novel substituted 6′-(4-chlorophenyl)-3,4′-bipyridine-3′-carbonitriles with incorporated pyrazole and/or triazole moieties have been synthesized using 2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetohydrazide (3) as starting material. Also, the key intermediate 3 reacted with aromatic aldehydes and tosyl chloride to give the corresponding Schiff bases and tosyl hydrazide derivatives, respectively. The antimicrobial of these newly synthesized compounds was evaluated against Bacillus subtilis as Gram-positive bacteria and Trichoderma viride as a fungus; some of these compounds such as 5, 6, 7, 8, 10, 12, and 14 showed excellent activities as antimicrobial agents. Moreover, the cytotoxic activity of the most active compounds was assessed in vitro against human tumor liver cancer cell line (HEPG2); compounds 8, 10, 13a, and 14 showed potent activities relative to Doxorubicin which was used as a reference standard drug in this study.

Keywords: antimicrobial activity; ceric ammonium nitrate (CAN); cytotoxic activity; HEPG2; pyridine

1 Introduction

A large number of heterocyclic compounds containing pyridine rings are associated with diverse pharmacological properties such as antimicrobial [1, 2], antiviral [3], anticonvulsant [4], anticancer [5] anti-HIV [6], and antimycobacterial activities [7]. Different substituents were introduced on the pyridine ring such as cyanoacetohydrazide and hydrazide bearing either thiazole, thiophene, benzothiophene, pyrazole, or triazole moieties, due to the well-documented anticancer activity of these moieties, to study their structure activity relationship (SAR) and their anticancer activity [8]. The ability of many compounds containing a pyridine moiety, especially those containing a cyano group, to exhibit antitumor activity has been demonstrated in numerous publications [9–14]. The view of our design of the newly synthesized compounds in this research point is based on other biologically active heterocycles reported in the field of microbial and cancer therapy such as pyrazoles [15, 16], triazoles [17, 18], and Schiff bases [19–21]. In continuation of our work on the synthesis of heterocyclic systems [22–25] and for biological evaluations [26, 27], we considered it attractive to synthesize some novel substituted 6′-(4-chlorophenyl)-3,4′-bipyridine-3′-carbonitriles and evaluation of their antimicrobial and cytotoxic activity. 2-(6′-(4-Chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetohydrazide (3) was prepared and used as a building block for the synthesis of the title compounds.

2 Results and discussion

2.1 Chemistry

One-pot, four-component reaction of 4-chloroacetophenone, nicotinaldehyde, ethyl cyanoacetate, and ammonium acetate in the presence of ceric ammonium nitrate (CAN) in ethanol at room temperature afforded 6-(4-chlorophenyl)-2-oxo-4-(pyridin-3-yl)-1,2-dihydropyridine-3-carbonitrile (1). Alkylation of compound 1 with ethyl chloroacetate in dimethyl sulfoxide (DMSO) at room temperature gave ethyl 2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetate (2) which condensed with hydrazine hydrate (99%) in ethanol to give 2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetohydrazide (3), a very useful starting material for the synthesis of all target compounds in this work (Scheme 1).

Scheme 1:

Synthesis of 2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetohydrazide (3).

Cyclization of compound 3 with ethyl acetoacetate and/or acetylacetone afforded the corresponding pyrazole derivatives 4 and/or 5, respectively. Also, the reaction of compound 3 with diethyl malonate or ethyl cyanoacetate afforded the corresponding pyrazole-3,5-dione derivative 6 (Scheme 2).

Scheme 2:

Synthesis of pyrazole derivatives (4–6). Reagents and conditions: (i) ethyl acetoacetate/AcOH, reflux; (ii) acetylacetone/AcOH, reflux; (iii) diethyl malonate/AcOH or ethyl cyanoacetate/AcOH, reflux.

To get a series of expectedly biologically active heterocycles with incorporated bipyridine and triazole moieties, compound 3 was allowed to react with carbon disulfide and methyl iodide to give the corresponding methyl hydrazinecarbodithioate derivative 7 which converted to the 4-aminotriazole-5-thione derivative 8 through condensation with hydrazine hydrate (99%). Also, the 4-phenyltriazole-5-thione derivative 10 was obtained on treatment of compound 3 with phenyl isothiocyanate in dimethylformamide to afford the corresponding N-phenyl hydrazinecarbothioamide derivative 9, which was cyclized with 5% alcoholic sodium hydroxide (Scheme 3). Moreover, the reaction of compound 3 with potassium thiocyanate in aqueous hydrochloric acid (10%) gave the corresponding hydrazinecarbothioamide derivative 11 which was cyclized in 5% alcoholic sodium hydroxide to give the triazole-5-thione derivative 12. Also, compound 12 was obtained in a single-step reaction through fusion of 3 with ammonium thiocyanate at 200 °C (Scheme 3).

Scheme 3:

Reagents and conditions: (iv) CS₂/KOH/DMSO, r.t.; (v) MeI, r.t.; (vi) N₂H₄ (99%)/EtOH, reflux; (vii) PhNCS/DMF, 60 °C; (viii) NaOH alc. (5%), reflux; (ix) KSCN/HCl-H₂O (10%, v/v), reflux; (x) NaOH alc. (5%), reflux; (xi) NH₄SCN/200 °C.

To get a new series of expectedly biologically active Schiff bases and N-amide derivatives, it was of interest to condense compound 3 with different aromatic aldehydes, namely 4-chlorobenzaldehyde, 4-bromobenzaldehyde, 4-methoxybenzaldehyde, and 4-nitrobenzaldehyde, in acetic acid to give the corresponding Schiff bases 13a–d. The reaction of 3 with tosyl chloride in absolute ethanol afforded the corresponding sulfonamide derivative 14 (Scheme 4).

Scheme 4:

Synthesis of Schiff bases 13a–d and compound 14.

The structure of the obtained compounds was ascertained by spectroscopic data and elemental analysis.

3 Biological assessment

3.1 Antibacterial activity

The antibacterial activity results are summarized in Table 1. All target compounds 4–14 synthesized from compound 3 showed excellent antimicrobial activities. The results indicated that phenyltriazole-5-thione derivative 10 showed the best biological activity against Bacillus subtilis (inhibition zone = 2.9 cm). It also showed a better activity than its precursor compound 9 (inhibition zone = 2.2 cm). The modification of the functional group through cyclization of the latter, with 5% alcoholic sodium hydroxide, improves the activity.

Table 1

Antibacterial activity of the synthesized compounds (1–14).

Entry	Compound	Radius of inhibition zone (cm)	Degree of inhibition zone	Mean	SD	SE	Mean ± SE
1	4	1.3, 1.5, 1.7	+ + +	1.5	0.2	0.1156	1.5 ± 0.11
2	5	2.3, 2.5, 2.7	+ + + + +	2.5	0.2	0.1156	1.5 ± 0.11
3	6	1.6, 1.7, 1.8	+ + +	1.7	0.1	0.0578	2.5 ± 0.05
4	7	2.4, 2.5, 2.6	+ + + + +	2.5	0.1	0.0578	2.5 ± 0.05
5	8	1.0, 1.2, 1.4	+ + +	1.2	0.2	0.1156	1.5 ± 0.11
6	9	2.1, 2.2, 2.3	+ + + +	2.2	0.1	0.0578	2.5 ± 0.05
7	10	2.7, 2.9, 3.1	+ + + + + +	2.9	0.2	0.1156	1.5 ± 0.11
8	11	1.7, 2.0, 2.3	+ + + +	2.0	0.3	0.173	1.8 ± 0.17
9	12	2.2, 2.4, 2.6	+ + + + +	2.4	0.2	0.1156	1.5 ± 0.11
10	13a	2.0, 2.2, 2.4	+ + + +	2.2	0.2	0.1156	1.5 ± 0.11
11	13c	0.7, 0.8, 0.9	+ +	0.8	0.1	0.0578	2.5 ± 0.05
12	13d	2.0, 2.2, 2.4	+ + + + +	2.2	0.2	0.1156	1.5 ± 0.11
13	14	1.5, 1.8, 2.1	+ + +	1.8	0.3	0.1730	1.8 ± 0.17

SE, Standard error; SD, standard deviation. SD = 0.93, SE = 0.198, mean ± SE = 2.09 ± 0.19.

Cyclization of 3 afforded the three pyrazole derivatives 4, 5, and 6. Antibacterial activities of these derivatives did not exceed 2.5 cm for the inhibition zone (compound 5). A series of heterocycles with incorporated bipyridine and triazole moieties (compounds 7–12) generally presented excellent activities, especially the methyl hydrazinecarbodithioate derivative 7, but the inhibition zones were still at 2.5 cm for compound 7 and below for the others. Also a new series of Schiff bases (13a, c, and d) and sulfonamide derivatives 14 showed moderate activities, the inhibition zones of which ranged from 0.8 to 2.2 cm.

3.2 Antifungal activity

The antifungal activity results are summarized in Table 2. The results indicated that sulfonamide derivative 14 showed the best biological activity against Trichoderma viride (inhibition zone = 3.5 cm).

Table 2

Antifungal activity of the synthesized compounds (1–14).

Entry	Compound	Radius of inhibition zone (cm)	Degree of inhibition zone	Mean	SD	SE	Mean ± SE
1	4	1.3, 1.5, 1.7	+ + +	1.5	0.2	0.1156	1.5 ± 0.11
2	5	1.9, 2.0, 2.1	+ + + +	2.0	0.1	0.0578	2.0 ± 0.05
3	6	2.3, 2.5, 2.7	+ + + + +	2.5	0.2	0.1156	2.5 ± 0.11
4	7	2.4, 2.5, 2.6	+ + + + +	2.5	0.1	0.0578	2.5 ± 0.05
5	8	2.3, 2.5, 2.7	+ + + + +	2.5	0.2	0.1156	2.5 ± 0.11
6	9	1.9, 2.0, 2.1	+ + + +	2.0	0.1	0.0578	2.0 ± 0.05
7	10	2.7, 3.0, 3.3	+ + + + + +	3.0	0.3	0.1730	3.0 ± 0.17
8	11	1.8, 2.0, 2.2	+ + + +	2.0	0.2	0.1156	2.0 ± 0.11
9	12	1.0, 1.2, 1.4	+ +	1.2	0.2	0.1156	1.2 ± 0.11
10	13a	1.8, 2.0, 2.2	+ + + +	2.0	0.2	0.1156	2.0 ± 0.11
11	13c	1.4, 1.5, 1.6	+ + +	1.5	0.1	0.0578	1.5 ± 0.05
12	13d	1.4, 1.5, 1.6	+ + +	1.5	0.1	0.0578	1.5 ± 0.05
13	14	3.3, 3.5, 3.7	+ + + + + +	3.5	0.2	0.1156	3.5 ± 0.11

SE, Standard error; SD, standard deviation. SD = 0.88, SE = 0.187633, mean ± SE = 2.218 ± 0.188.

The phenyltriazole-5-thione derivative 10 showed an excellent antifungal activity (3.0 cm) compared with its precursor derivative 9 (2.0 cm for the inhibition zone). It is worth to mention that the pyrazole derivative 6 and triazoles 7 and 8 had considerable antifungal activities against T. viride; the inhibition zone recorded 2.5 cm in all cases. The biological activity of the Schiff base 13a was higher than that of 13c and 13d (2.0, 1.5, and 1.5 cm, respectively).

3.3 In vitro anticancer screening

Some of the newly synthesized compounds (5, 6, 8, 10, 13a, 13d, and 14) were evaluated for their in vitro cytotoxicity against human liver cancer cell line (HEPG2) (Table 3 and Fig. 1). Doxorubicin HCl is one of the most effective anticancer agents and was used as a reference drug in this study. The relationship between surviving fraction and drug concentration was plotted to obtain the survival curve of breast cancer cell line (HEPG2). The response parameter calculated was the IC₅₀ value, which corresponds to the concentration required for 50% inhibition of cell viability.

Table 3

Antitumor activity at different concentrations (0, 5, 12.5, 25, and 50 μg mL^–1) of some synthesized compounds (5, 6, 8, 10, 13a, 13d, and 14) and reference drug Doxorubicin (DOX).

Concentration (μg mL^–1)	Surviving fractions								SD
Concentration (μg mL^–1)	5	6	8	10	13a	13d	14	DOX	SD
0	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	0.0
5	0.816	0.649	0.540	0.348	0.473	0.749	0.452	0.537	0.157
12.5	0.645	0.448	0.414	0.328	0.364	0.565	0.422	0.433	0.104
25	0.406	0.419	0.362	0.329	0.519	0.423	0.450	0.378	0.058
50	0.361	0.385	0.343	0.335	0.532	0.377	0.389	0.341	0.064
SD	0.270	0.259	0.272	0.297	0.245	0.255	0.260	0.268

SD, Standard deviation.

Fig. 1:

The drug toxicity curves for some of the synthesized compounds (5, 6, 8, and 10).

Fig. 1 (continued):

The drug toxicity curves for of some synthesized compounds (13a, 13d, 14, and reference drug DOX).

Table 3 and Fig. 1 show the in vitro cytotoxicity of the newly synthesized compounds where some compounds revealed significant activity compared to Doxorubicin as a reference drug. It was observed from the results that most of the tested compounds showed significant anti-liver cancer activity; some of them were more potent and the others were found to be equipotent to the reference drug (IC₅₀ = 7.3 μg mL^–1).

It was found that the introduction of the 4-phenyltriazole moiety resulted in the best activity for compound 10 (IC₅₀ = 3.83 μg mL^–1). The sulfonamide derivative 14 (IC₅₀ = 4.58 μg mL^–1) and Schiff base 13a (IC₅₀ = 4.73 μg mL^–1) were the most potent compounds in this screening and they showed higher activity than Doxorubicin; then compounds 8 (IC₅₀ = 7.28 μg mL^–1) showed nearly the same activity as Doxorubicin.

The introduction of the other compounds showed a decrease in the activity with IC₅₀ values ranging from 10.6 to 20.0 μg mL^–1. From Table 3, statistical analysis showed the most significant similarity which occurred between the surviving cell counts for compound 8 and reference drug Doxorubicin (DOX) at all concentrations used (5, 12.5, 25, and 50 μg mL^–1). That compound had a slightly better activity than Doxorubicin. Moreover, compound 10 was more potent at all concentrations compared with Doxorubicin, while compounds 13a and 14 at concentrations 5 and 12.5 μg mL^–1 showed no significant difference compared with the reference drug but still have high effects.

These preliminary results of biologically screening of the tested compounds give an idea about the possible importance of the 3,4′-bipyridine moiety in the compounds acting against liver cancer and give an encouraging framework in this field that may lead to the discovery of a potent anticancer agent.

In conclusion, the synthesized compounds were evaluated for their in vitro cytotoxicity against human liver cancer cell line (HEPG2). Some of the tested compounds were equipotent, while the others were more potent compared with Doxorubicin. It is worthy to mention that compound 10 showed a significant cytotoxic activity which was even higher than that of the reference drug Doxorubicin in all concentrations, while compound 8 is nearly as active as Doxorubicin. Other compounds showed obvious activities but more or less lower than reference, but compounds 13a and 14 at 5 and 12.5 μg mL^–1 showed better activities.

4 Experimental section

4.1 Chemistry

The time required for completion of each reaction was monitored by TLC. All melting points are uncorrected and were measured on a Gallenkamp apparatus. The IR spectra were recorded on a Shimadzu 470 IR spectrometer (KBr, ν_max, cm^–1). The ¹H and ¹³C NMR spectra were measured on a Varian (¹H: 400 MHz, ¹³C: 100 MHz) spectrometer with TMS as an internal standard. Mass spectra were determined on a JEOL JMS-600 spectrometer. Elemental analyses (C, H, N, and S) were performed on an elemental analysis system Vario EL V_2.3. The results were found to be in good agreement with the calculated values.

4.1.1 Synthesis of 6-(4-chlorophenyl)-2-oxo-4-(pyridin-3-yl)-1,2-dihydropyridine-3-carbonitrile (1)

In a 50 mL round-bottomed flask, 4-chloroacetophenone (10 mmol), nicotinaldehyde (10 mmol), ethyl cyanoacetate (10 mmol), and ammonium acetate (15 mmol) were stirred in the presence of 5 mol% of CAN in ethanol (25 mL) at room temperature for 3 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water and solid product was collected by filtration, washed with water several times, dried, and recrystallized from ethanol to afford pure product 1 as pale buff crystals; yield 2.76 g (90%), m.p. 291–292 °C. – IR (KBr): ν = 3100 (NH), 2200 (CN), 1670 (C=O) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 7.25 (s, 1H, CH), 7.65 (d, 2H, 2CH), 8.10 (d, 2H, 2CH), 8.15 (m, 1H, CH), 8.25 (s, 1H, NH, exchangeable with D₂O), 8.90 (d, 1H, CH), 9.15 (d, 1H, CH), 9.35 (s, 1H, CH). – MS (EI, 70 eV): m/z (%) = 309.10 (35) [M+2]⁺, 307.51 (100) [M]⁺, 279.05 (25), 252.01 (17). – C₁₇H₁₀ClN₃O: calcd. C 66.35, H 3.28, Cl 11.52, N 13.65; found C 66.11, H 3.02, Cl 11.40, N, 13.46.

4.1.2 Synthesis of ethyl 2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetate (2)

A mixture of compound 1 (10 mmol), ethyl chloroacetate (10 mmol), and anhydrous potassium carbonate (15 mmol) in 25 mL of DMSO was stirred at room temperature for 2 h. The reaction mixture was poured onto the ice/water mixture; the solid product separated was collected by filtration, dried, and recrystallized from ethanol to give pale yellow needles, yield 3.22 g (82%), m.p. 120–121 °C. – IR (KBr): ν = 2200 (CN), 1740 (C=O), 1660 (C=N) cm^–1. – ¹H NMR (CDCl₃): δ = 1.25 (t, 3H, CH₃), 4.25 (q, 2H, CH₂), 5.05 (s, 2H, CH₂), 7.40 (d, 2H, 2CH), 7.50 (s, 1H, CH), 7.95 (d, 2H, 2CH), 8.75 (m, 3H, 3CH), 8.85 (s, 1H, CH) ppm. – MS (EI, 70 eV): m/z (%) = 395.03 (30) [M+2]⁺, 393.51 (100) [M]⁺, 347.97 (15), 319.84 (79) ppm. – C₂₁H₁₆ClN₃O₃: calcd. C 64.05, H 4.09, Cl, 9.00, N 10.67; found C 64.13, H 3.89, Cl 8.82, N 10.49.

4.1.3 Synthesis of 2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetohydrazide (3)

A mixture of compound 2 (10 mmol), hydrazine hydrate (99%, 40 mmol), and 20 mL absolute ethanol was refluxed for 2 h. The reaction mixture was cooled; the formed product was filtered, dried, and recrystallized from ethanol to give pale yellow crystals, yield 3.30 g (87%), m.p. 185–186 °C. – IR (KBr): ν = 3450, 3300 (NH₂), 3200 (NH), 2200 (CN), 1680 (C=O), 1640 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ == 5.07 (s, 2H, CH₂), 7.55 (d, 2H, 2CH), 7.65 (s, 1H, CH), 8.00 (s, 2H, NH₂, exchangeable with D₂O), 8.25 (d, 2H, 2CH), 8.32 (d, 1H, CH), 8.70 (m, 2H, 2CH), 9.00 (s, 1H, CH), 9.50 (s, 1H, NH, exchangeable with D₂O) ppm. – ¹³C NMR (DMSO): δ = 65.3 (CH₂), 109.8 (2C), 116.8 (CN), 125.3 (2CH), 129.2 (CH), 130.2 (2CH), 132.3 (2C), 135.3 (2C), 140.5 (2CH), 148.3 (2CH), 151.0 (C), 170.0 (C=O) ppm. – MS (EI, 70 eV): m/z (%) = 381.10 (28) [M+2]⁺, 379.72 (100) [M]⁺, 306.15 (15), 277.92 (35). – C₁₉H₁₄ClN₅O₂: calcd. C 60.09, H 3.72, Cl 9.33, N 18.44; found C 60.17, H 3.60, Cl 9.25, N 18.20.

4.1.4 Typical procedure for the synthesis of compounds 4–6

A mixture of compound 3 (1 mmol) and an equimolar amount of ethyl acetoacetate or acetylacetone or diethyl malonate (or ethyl cyanoacetate) in 10 mL of acetic acid was refluxed for 5 h. The formed product after cooling was filtered off, washed with water, dried, and recrystallized with acetic acid to afford compounds 4, 5, and 6, respectively.

4.1.5 6′-(4-Chlorophenyl)-2′-(2-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)-3,4′-bipyridine-3′-carbonitrile (4)

Pale yellow crystals, yield 0.31 g (71%); m.p. 202–204 °C. – IR (KBr): ν = 2200 (CN), 1700 (C=O), 1660 (C=O), 1620 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 1.90 (s, 3H, CH₃), 1.95 (s, 2H, CH₂), 5.20 (s, 2H, CH₂), 7.60 (d, 2H, 2CH), 7.75 (d, 1H, CH), 8.05 (s, 1H, CH), 8.30 (d, 2H, 2CH), 8.85 (m, 2H, 2CH), 9.05 (s, 1H, CH) ppm. – ¹³C NMR (DMSO): δ = 14.1 (CH₃), 43.6 (CH₂), 64.2 (CH₂), 110.1 (2C), 117.3 (CN), 125.5 (2CH), 130.1 (3CH), 135.0 (4C), 139.9 (2CH), 148.0 (2CH), 151.8 (2C), 166.0 (C=O), 171.2 (C=O) ppm. – MS (EI, 70 eV): m/z (%) = 447.03 (5) [M+2]⁺, 445.01 (3) [M]⁺, 306.12 (20), 278.20 (100). – C₂₃H₁₆ClN₅O₃: calcd. C 61.96, H 3.62, Cl 7.95, N 15.71; found C 61.80, H 3.49, Cl 7.73, N 15.55.

4.1.6 6′-(4-Chlorophenyl)-2′-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-2-oxoethoxy)-3,4′-bipyridine-3′-carbonitrile (5)

Pale yellow crystals, yield 0.33 g (75%); m.p. 197–198 °C. – IR (KBr): ν = 2200 (CN), 1700 (C=O), 1680 (C=N), 1640 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 2.35 (s, 3H, CH₃), 2.66 (s, 3H, CH₃), 5.20 (s, 2H, CH₂), 6.30 (s, 1H, CH), 7.68 (d, 2H, 2CH), 8.10 (s, 1H, CH), 8.39 (d, 2H, 2CH), 8.75–9.25 (m, 4H, 4CH) ppm. – MS (EI, 70 eV): m/z (%) = 445.15 (8) [M+2]⁺, 443.02 (20) [M]⁺, 306.91 (33), 278.06 (100). – C₂₄H₁₈ClN₅O₂: calcd. C 64.94, H 4.09, Cl 7.99, N 15.78; found C 64.68, H 3.88, Cl 7.65, N 15.60.

4.1.7 6′-(4-Chlorophenyl)-2′-(2-(3,5-dioxopyrazolidin-1-yl)-2-oxoethoxy)-3,4′-bipyridine-3′-carbonitrile (6)

Pale yellow crystals, yield 0.30 g (69%); m.p. 216–217 °C. – IR (KBr): ν = 3200 (NH), 2200 (CN), 1700 (C=O), 1660 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 3.60 (s, 2H, CH₂), 5.15 (s, 2H, CH₂), 7.10 (m, 1H, CH), 7.60 (d, 2H, 2CH), 7.95 (s, 1H, CH), 8.20 (d, 2H, 2CH), 8.75 (d, 2H, 2CH), 8.98 (s, 1H, CH), 10.45 (s, 1H, NH, exchangeable with D₂O) ppm. – MS (EI, 70 eV): m/z (%) = 447.25 (5) [M]⁺, 306.75 (40), 278.79 (100). – C₂₂H₁₄ClN₅O₄: calcd. C 59.00, H 3.15, Cl 7.92, N 15.64; found C 59.12, H 2.97, Cl 7.73, N 15.50.

4.1.8 Synthesis of methyl 2-(2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetyl) hydrazinecarbodithioate (7)

To a vigorously stirred solution of 2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetohydrazide (3) (2 mmol) in dimethylsulfoxide (5 mL) at room temperature, carbon disulfide (2.5 mmol) and aqueous sodium hydroxide (0.6 mL, 20%) were added simultaneously over 0.5 h. The stirring was continued for further 30 min. Methyl iodide (2 mmol) was added dropwise to the reaction mixture with stirring at 5–10 °C; it was further stirred for 3 h and poured into ice water. The solid obtained was filtered, washed with water, dried, and crystallized from ethanol as pale yellow crystals. Yield 0.75 g (80%), m.p. 130–131 °C. – IR (KBr): ν = 3250 (NH), 2200 (CN), 1700 (C=O), 1645 (C=N), 1165 (C=S) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 2.05 (s, 1H, NH, exchangeable with D₂O), 2.19 (s, 3H, CH₃), 5.25 (s, 2H, CH₂), 5.90 (s, 1H, NH, exchangeable with D₂O), 7.65 (d, 2H, 2CH), 7.95 (s, 1H, CH), 8.15 (d, 1H, CH), 8.35 (d, 2H, 2CH), 8.85 (m, 2H, 2CH), 9.15 (s, 1H, CH) ppm. – ¹³C NMR (DMSO): δ = 16.6 (CH₃), 61.5 (CH₂), 109.6 (2C), 118.9 (CN), 125.3 (2CH), 128.6 (CH), 130.1 (2CH), 132.8 (2C), 135.3 (2C), 142.2 (2CH), 148.3 (2CH), 149.6 (C), 172.2 (C=O), 196.1 (C=S) ppm. – MS (EI, 70 eV): m/z (%) = 471.20 (3) [M+2]⁺, 469.57 (9) [M]⁺, 363.15 (17), 306.01 (30), 277.62 (100). – C₂₁H₁₆ClN₅O₂S₂: calcd. C 53.67, H 3.43, Cl 7.54, N 14.90, S 13.65; found C 53.72, H 3.26, Cl 7.50, N 14.72, S 13.51.

4.1.9 Synthesis of 2′-((4-amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy)-6′-(4-chlorophenyl)-3,4′-bipyridine-3′-carbonitrile (8)

To a suspension of compound 7 (1 mmol) in ethanol (10 mL) was added hydrazine hydrate (2 mmol, 99%), and the mixture was heated under reflux until the methylmercaptan evolution ceased (5 h). The reaction mixture was refluxed for further 3 h. After cooling, the solid obtained was filtered off, dried, and recrystallized from acetic acid as yellow crystals; yield: 0.28 g (65%), m.p. 201–202 °C. – IR (KBr): ν = 3350, 3300 (NH₂), 3200 (NH), 2200 (CN), 1640 (C=N), 1160 (C=S) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 5.25 (s, 2H, CH₂), 5.79 (s, 1H, NH, exchangeable with D₂O), 6.50 (br, 2H, NH₂, exchangeable with D₂O), 7.55 (d, 2H, 2CH), 7.65 (s, 1H, CH), 8.05 (d, 1H, CH), 8.30 (d, 2H, 2CH), 9.00 (m, 2H, 2CH), 9.20 (s, 1H, CH) ppm. – ¹³C NMR (DMSO): δ = 62.9 (CH₂), 110.6 (2C), 117.5 (CN), 125.3 (2CH), 128.6 (CH), 130.1 (2CH), 132.6 (2C), 135.3 (2C), 142.2 (2CH), 145.3 (2CH), 147.6 (2C), 194.2 (C=S) ppm. – MS (EI, 70 eV): m/z (%) = 437.01 (2) [M+2]⁺, 435.87 (5) [M]⁺, 346.71 (100), 306.41 (22). – C₂₀H₁₄ClN₇OS: calcd. C 55.11, H 3.24, Cl 8.13, N 22.49, S 7.36; found C 55.19, H 3.01, Cl 8.00, N 22.52, S 7.10.

4.1.10 Synthesis of 2-(2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetyl)-N-phenylhydrazinecarbothioamide (9)

A mixture of compound 3 (2 mmol) and phenyl isothiocyanate (2 mmol) in 10 mL of dimethylformamide was stirred at 60 °C for 5 h, then cooled to room temperature, poured onto ice/water, and acidified with dilute hydrochloric acid. The formed precipitate was collected by filtration. It was washed with water several times, then dried, and recrystallized from ethanol to give pale yellow crystals; yield: 0.68 g (67%), m.p. 215–216 °C. – IR (KBr): ν = 3150 (NH), 2210 (CN), 1700 (C=O), 1650 (C=N), 1645 (C=N), 1180 (C=S) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 3.15 (s, 1H, NH, exchangeable with D₂O), 5.10 (s, 2H, CH₂), 6.05 (s, 1H, NH, exchangeable with D₂O), 6.86–7.21 (m, 3H, 3CH), 7.55 (m, 5H, 5CH), 7.90 (s, 1H, CH), 8.15–8.25 (m, 3H, 3CH), 8.60 (d, 1H, 1CH), 9.15 (s, 1H, CH), 10.95 (s, 1H, NH, exchangeable with D₂O) ppm. – MS (EI, 70 eV): m/z (%) = 516.80 (1) [M+2]⁺, 349.15 (20), 321.61 (10), 306.20 (7), 278.04 (100). – C₂₆H₁₉ClN₆O₂S: calcd. C 60.64, H 3.72, Cl 6.88, N 16.32, S 6.23; found C 60.59, H 3.49, Cl 6.63, N 16.05, S 6.15.

4.1.11 Synthesis of 6′-(4-chlorophenyl)-2′-((4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy)-3,4′-bipyridine-3′-carbonitrile (10)

Compound 9 (1 mmol) in 20 mL of ethanolic sodium hydroxide (5%) was heated under reflux for 2 h. After cooling the reaction mixture was poured onto ice/water and acidified with dilute hydrochloric acid. The formed product was collected by filtration, washed with cold water, dried, and recrystallized from ethanol to give a yellow powder; yield: 0.35 g (72%), m.p. 278–280 °C. – IR (KBr): ν = 3400 (NH), 2205 (CN), 1660 (C=N), 1615 (C=N), 1185 (C=S) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 5.15 (s, 2H, CH₂), 6.55 (s, 1H, NH, exchangeable with D₂O), 6.91–7.30 (m, 6H, 6CH), 7.60 (d, 2H, 2CH), 7.80 (s, 1H, CH), 8.36–8.42 (m, 3H, 3CH), 8.62 (d, 1H, 1CH), 9.15 (s, 1H, CH) ppm. – MS (EI, 70 eV): m/z (%) = 498.55 (3) [M+2]⁺, 346.20 (26), 306.35 (15), 277.84 (100). –C₂₆H₁₇ClN₆OS: calcd. C 62.84, H 3.45, C 7.13, N 16.91, S 6.45; found C 62.59, H 3.21, Cl 6.86, N 16.70, S 6.20.

4.1.12 Synthesis of 2-(2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetyl)hydrazine carbothioamide (11)

A mixture of 3 (2 mmol), 10% aq. HCl (10 mL, v/v), and potassium thiocyanate (2.5 mmol) was heated under reflux for 4 h. The reaction mixture was allowed to cool to room temperature. The solid formed was collected by filtration, washed with water, dried, and crystallized from ethanol to afford pale yellow crystals; yield: 0.74 g (84%), m.p. 189–190 °C. – IR (KBr): ν = 3400, 3350 (NH₂), 3250 (NH), 2200 (CN), 1700 (C=O), 1620 (C=N), 1180 (C=S) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 2.45 (s, 1H, NH, exchangeable with D₂O), 5.05 (s, 2H, CH₂), 7.10–7.30 (m, 3H, 3CH), 7.90 (s, 1H, CH), 8.25 (d, 1H, CH), 8.35 (d, 2H, 2CH), 8.90 (d, 1H, CH), 9.20 (s, 1H, CH), 10.35 (s, 2H, NH₂, exchangeable with D₂O), 10.90 (s, 1H, NH, exchangeable with D₂O) ppm. – ¹³C NMR (DMSO): δ = 59.8 (CH₂), 110.2 (2C), 117.8 (CN), 125.3 (2CH), 128.6 (CH), 130.3 (2CH), 131.8 (2C), 135.3 (2C), 142.2 (2CH), 147.6 (2CH), 149.1 (C), 175.1 (C=O), 195.3 (C=S) ppm. – MS (EI, 70 eV): m/z (%) = 440.67 (5) [M+2]⁺, 438.57 (2) [M]⁺, 348.97 (87), 320.80 (15), 306.62 (65), 278.79 (100); – C₂₀H₁₅ClN₆O₂S: calcd. C 54.73, H 3.44, Cl 8.08, N 19.15, S 7.31; found C 54.51, H 3.15, Cl 7.81, N 19.00, S 7.10.

4.1.13 Synthesis of 6′-(4-chlorophenyl)-2′-((5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy)-3,4′-bipyridine-3′-carbonitrile (12)

Method A: A mixture of compound 3 (1 mmol) and ammonium thiocyanate (5 mmol) was fused in an oil bath at 150 °C for 1 h. The solid mass was dissolved in hot water, and then filtered to separate the insoluble materials. The filtrate was cooled and then acidified with dilute hydrochloric acid; the pure product that formed as yellow crystals was filtered off and dried; yield: 0.25 g (60%), m.p. 261–262 °C.

Method B: Compound 11 (1 mmol) in 20 mL of ethanolic sodium hydroxide (5%) was heated under reflux for 2 h. After cooling, the reaction mixture was poured onto ice/water, and then acidified with dilute hydrochloric acid. The formed solid product was collected by filtration, washed with cold water, dried, and recrystallized from ethanol to give yellow crystals; yield: 0.30 g (71%), m.p. 260–262 °C. – IR (KBr): ν = 3300 (NH), 2200 (CN), 1640 (C=N), 1625 (C=N), 1180 (C=S) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 5.15 (s, 2H, CH₂), 5.95 (s, 1H, NH, exchangeable with D₂O), 7.10–7.29 (m, 3H, 3CH), 7.86 (s, 1H, CH), 8.05 (d, 1H, CH), 8.35 (d, 2H, 2CH), 8.93 (d, 1H, CH), 9.25 (s, 1H, CH), 10.90 (s, 1H, NH, exchangeable with D₂O) ppm. – ¹³C NMR (DMSO): δ = 60.3 (CH₂), 110.2 (2C), 117.5 (CN), 123.3 (2CH), 128.6 (CH), 130.3 (2CH), 132.2 (2C), 135.3 (2C), 142.0 (2CH), 147.6 (2CH), 148.6 (2C), 196.7 (C=S) ppm. – MS (EI, 70 eV): m/z (%) = 422.58 (2) [M+2]⁺, 348.08 (35), 320.80 (15), 306.02 (65), 277.98 (100). – C₂₀H₁₃ClN₆OS: calcd. C 57.07, H 3.11, Cl 8.42, N 19.97, S 7.62; found C 57.23, H 2.88, Cl 8.30, N 19.70, S 7.45.

4.1.14 General procedure for the synthesis of N′-(arylidene)-2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy) acetohydrazides 13a–d

A mixture of compound 3 (1 mmol) and the appropriate aromatic aldehyde (4-chlorobenzaldehyde, 4-bromobenzaldehyde, 4-methoxybenzaldehyde, and/or 4-nitrobenzaldehyde, 1 mmol each) in absolute ethanol (10 mL) was heated under reflux in the presence of glacial acetic acid (1–2 drops) for 2 h. After cooling, the formed precipitate was filtered off, dried, and recrystallized from acetic acid to afford the corresponding Schiff base (13a–d).

4.1.15 N′-(4-Chlorobenzylidene)-2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy) acetohydrazide (13a)

Yellow crystals, yield: 0.37 g (75%); m.p. 189–190 °C. – IR (KBr): ν = 3300 (NH), 2200 (CN), 1690 (C=O), 1645 (C=N), 1620 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 5.30 (s, 2H, CH₂), 7.50–7.75 (m, 5H, 5CH), 7.95 (d, 2H, 2CH), 8.15 (s, 1H, CH), 8.20–8.45 (m, 4H, 4CH), 9.00 (d, 1H, CH), 9.15 (s, 1H, CH), 12.15 (s, 1H, NH, exchangeable with D₂O) ppm. – MS (EI, 70 eV): m/z (%) = 503.89 (12) [M+2]⁺, 501.97 (26) [M]⁺, 348.20 (20), 319.86 (35), 306.61 (100). – C₂₆H₁₇Cl₂N₅O₂: calcd. C 62.16, H 3.41, Cl 14.11, N 13.94; found C 61.86, H 3.26, Cl 13.93, N 13.70.

4.1.16 N′-(4-Bromobenzylidene)-2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy) acetohydrazide (13b)

Yellow crystals, yield: 0.38 g (71%); m.p. 198–199 °C. – IR (KBr): ν = 3400 (NH), 2200 (CN), 1690 (C=O), 1645 (C=N), 1625 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 5.20 (s, 2H, CH₂), 7.48–7.72 (m, 5H, 5CH), 7.90 (d, 2H, 2CH), 8.15 (s, 1H, CH), 8.20–8.40 (m, 4H, 4CH), 8.90 (d, 1H, CH), 9.15 (s, 1H, CH), 11.40 (s, 1H, NH, exchangeable with D₂O) ppm. – MS (EI, 70 eV): m/z (%) = 548.91 (19) [M+2]⁺, 546.77 (23) [M]⁺, 349.76 (15), 322.02 (40), 79.51 (100). – C₂₆H₁₇BrClN₅O₂: calcd. C 57.11, H 3.13, Br 14.61, Cl 6.48, N 12.81; found C 56.83, H 2.90, Br 14.46, Cl 6.15, N 12.70.

4.1.17 2-(6′-(4-Chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)-N′-(4-methoxybenzylidene) acetohydrazide (13c)

Yellow crystals, yield: 0.42 g (85%); m.p. 180–181 °C. – IR (KBr): ν = 3400 (NH), 2200 (CN), 1690 (C=O), 1645 (C=N), 1620 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 3.90 (s, 3H, CH₃), 5.10 (s, 2H, CH₂), 7.55–7.75 (m, 5H, 5CH), 7.85 (d, 2H, 2CH), 8.25 (s, 1H, CH), 8.30–8.45 (m, 4H, 4CH), 9.05 (d, 1H, CH), 9.20 (s, 1H, CH), 12.30 (s, 1H, NH, exchangeable with D₂O) ppm. – MS (EI, 70 eV): m/z (%) = 499.05 (7) [M+2]⁺, 497.88 (16) [M]⁺, 349.16 (22), 320.15 (45), 277.90 (100). – C₂₇H₂₀ClN₅O₃: C 65.13, H 4.05, Cl 7.12, N 14.06; found C 65.00, H 3.80, Cl 6.90, N 13.85.

4.1.18 2-(6′-(4-Chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)-N′-(4-nitrobenzylidene) acetohydrazide (13d)

Yellow crystals, yield: 0.40 g (80%); m.p. 240–241 °C. – IR (KBr): ν = 3250 (NH), 2200 (CN), 1695 (C=O), 1640 (C=N), 1630 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 5.35 (s, 2H, CH₂), 7.60–7.80 (m, 5H, 5CH), 7.95 (d, 2H, 2CH), 8.35 (s, 1H, CH), 8.40–8.60 (m, 4H, 4CH), 9.15 (d, 1H, CH), 9.30 (s, 1H, CH), 12.00 (s, 1H, NH, exchangeable with D₂O) ppm. – MS (EI, 70 eV): m/z (%) = 514.05 (14) [M+2]⁺, 466.98 (10), 348.86 (25), 306.76 (100). – C₂₆H₁₇ClN₆O₄: calcd. C 60.88, H 3.34, Cl 6.91, N 16.39; found C 60.70, H 3.13, Cl 6.66, N 16.16.

4.1.19 Synthesis of N′-(2-(6′-(4-chlorophenyl)-3′-cyano-3,4′-bipyridin-2′-yloxy)acetyl)-4-methylbenzenesulfonohydrazide (14)

A mixture of compound 3 (1 mmol) and p-toluenesulfonyl chloride (1 mmol) in 10 mL of absolute ethanol was refluxed for 3 h. The formed precipitate was filtered, washed with water, dried, and recrystallized from dioxane to give yellow crystals. Yield: 0.32 g (60%), m.p. 279–280 °C. – IR (KBr): ν = 3350 (NH), 2200 (CN), 1700 (C=O), 1645 (C=N), 1620 (C=N) cm^–1. – ¹H NMR ([D₆]DMSO): δ = 1.95 (s, 1H, NH, exchangeable with D₂O), 2.30 (s, 3H, CH₃), 5.10 (s, 2H, CH₂), 7.10–7.30 (m, 3H, 3CH), 7.60 (d, 2H, 2CH), 8.05 (d, 2H, 2CH), 8.20 (s, 1H, CH), 8.40 (d, 2H, 2CH), 8.80 (d, 1H, CH), 9.10 (d, 1H, CH), 9.35 (s, 1H, CH), 9.95 (br, 1H, NH, exchangeable with D₂O) ppm. – MS (EI, 70 eV): m/z (%) = 536.01 (15) [M+2]⁺, 533.91 (8), 379.06 (30), 349.16 (100), 321.91 (54). – C₂₆H₂₀ClN₅O₄S: calcd. C 58.48, H 3.78, Cl 6.64, N 13.12, S 6.00; found C 58.19, H 3.60, Cl 6.40, N 12.88, S 5.73.

4.2 Biological screening

4.2.1 Antibacterial activity

The newly synthesized compounds were screened for their antibacterial activity against bacterial isolate, namely B. subtilis, by the cup-plate method [28]. The sterilized nutrient agar medium was distributed 100 mL each in two 250 mL conical flasks and allowed to cool to room temperature. To these media, bacterial subcultures grown for 18–24 h were added and shaken thoroughly to ensure uniform distribution of the organism throughout the medium. Then, this agar medium was distributed in equal portions, in sterilized Petri dishes, ensuring that each Petri dish contains about 20 mL of the medium. The medium was then allowed for solidification. Then, cups were made with the help of a sterile cork borer (6 mm diameter) punching into the set of agar media.

The solutions of required concentration (50 μg mL^–1) of test compounds were prepared by dissolving the compounds in DMSO and filled into the cups with 1 mL of the respective solution. Then, the Petri dishes were kept for incubation in an inverted position for 24–48 h at 37 °C in an incubator. When growth inhibition zones were developed surrounding each cup, their diameter in cm was measured and compared with that of the distilled water.

4.2.2 Antifungal activity

The newly synthesized compounds were screened for their antifungal activity against the fungus T. viride at the concentration levels of 50 μg mL^–1 by the cup-plate method, using distilled water as the standard. To the sterilized potato dextrose agar medium incubated for 72 h, subcultures of fungus were added and shaken thoroughly to ensure uniform distribution. Then, this mixture was poured into previously sterilized and labeled Petri dishes and allowed to solidify. Then, with the help of a borer four cups were made in each plate. Two cups were filled with 0.1 mL of two test dilutions and the other two cups with respective concentrations of standard dilutions. Then, the plates were left as it is for 2–3 h for diffusion and then they were kept for incubation at 37 °C for 24 h. Then the diameter of the zones of growth inhibition was measured and compared with that of the standard.

4.2.3 Anticancer activity

The synthesized compounds (5, 6, 8, 10, 13a, 13d, and 14) were evaluated for their in vitro anticancer activity against human tumor liver cancer cell line (HEPG2). The cytotoxicity was measured in vitro for the newly synthesized compounds using the sulforhodamine B stain (SRB) assay protocol [29]. The in vitro anticancer screening was done by the pharmacology unit at Pharmacology Unit, Cancer Biology Department, the National Cancer Institute, Cairo University, Giza, Egypt. Cells were placed in a 96-multiwell plate (104 cells per well) for 24 h, before treatment with the compound(s) to allow attachment of the cells to the wall of the plate. Tested compounds were dissolved in DMSO. Different concentrations of the compound under test (0.0, 5.0, 12.5, 25.0, and 50.0 μg mL^–1) were added to the cell monolayer. Triplicate wells were prepared for each individual concentration. Monolayer cells were incubated with the compound(s) for 48 h at 37 °C and in an atmosphere of 5% CO₂. After 48 h, the cells were fixed, washed, and stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Excess unbound dye was removed by four washes with 1% acetic acid and attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an enzyme-linked immunoabsorbent assay reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve for the liver tumor cell line after the specified time. The molar concentration required for 50% inhibition of cell viability (IC₅₀) was calculated and compared to the reference drug Doxorubicin (CAS, 25316-40-9). The surviving fractions were expressed as means, and the results are given in Table 3 and Fig. 1.

Corresponding author: Essam M. Hussein, Faculty of Applied Sciences, Department of Chemistry, Umm Al-Qura University, Makkah, Saudi Arabia; and Faculty of Science, Department of Chemistry, Assiut University, Assiut 71516, Egypt, Fax: +0020-882342708, E-mail: essam.hussein78@yahoo.com

References

[1] N. B. Patel, S. N. Agravat, F. M. Shaikh, Med. Chem. Res. 2011, 20, 1033.10.1007/s00044-010-9440-0Search in Google Scholar

[2] N. B. Patel, S. N. Agravat, Chem. Heterocycl. Compd. 2009, 45, 1343.10.1007/s10593-010-0432-2Search in Google Scholar

[3] A. M. R. Bernardino, A. R. de Azevedo, L. C. da S. Pinheiro, J. C. Borges, V. L. Carvalho, M. D. Miranda, M. D. F. de Meneses, M. Nascimento, D. Ferreira, M. A. Rebello, V. A. G. G. da Silva, I. C. P. P. de Frugulhetti, Med. Chem. Res. 2007, 16, 352.Search in Google Scholar

[4] E. G. Paronikyan, A. S. Noravyan, I. A. Dzhagatspany, I. M. Nazaryan, R. G. Paronikyan, Pharm. Chem. J. 2002, 36, 465.10.1023/A:1021836504036Search in Google Scholar

[5] A. Srivastava, S. N. Pandeya, J. Curr. Pharm. Rev. Res. 2011, 4, 5.Search in Google Scholar

[6] T. J. Tucker, J. T. Sisko, R. M. Tynebor, T. M. Williams, P. J. Felock, J. A. Flynn, M.-T. Lai, Y. Liang, G. McGaughey, M. Liu, M. Miller, G. Moyer, V. Munshi, R. Perlow-Poehnelt, S. Prasad, J. C. Reid, R. Sanchez, M. Torrent, J. P. Vacca, B.-L. Wan, Y. Yan, J. Med. Chem. 2008, 51, 6503.Search in Google Scholar

[7] M. G. Mamolo, D. Zampieri, V. Falagiani, L. Vio, M. Fermeglia, M. Ferrone, S. Pricl, E. Banfi, G. Scialino, Arkivoc2004, 5, 231.Search in Google Scholar

[8] M. S. Al-Said, M. S. Bashandy, S. I. Al-Qasoumi, M. M. Ghorab, Eur. J. Med. Chem. 2011, 46, 137.10.1016/j.ejmech.2010.10.024Search in Google Scholar

[9] M. I. El-Zahar, S. S. Abd El-karim, M. E. Haiba, World J. Chem. 2009, 4, 182.Search in Google Scholar

[10] V. Beneteau, T. Besson, J. Guillard, S. Leonce, B. Pfeiffer, Eur. J. Med. Chem. 1999, 43, 1053.10.1016/S0223-5234(99)00130-0Search in Google Scholar

[11] A. M. Badawi, H. El-Sharkawy, D. A. Ismail, Aust. J. Basic Appl. Sci. 2008, 2, 301.Search in Google Scholar

[12] M. Pannala, S. Kher, N. Wilson, J. Gaudette, I. Sircar, Shao-Hui Zhang, A. Bakhirev, G. Yang, P. Yuen, F. Gorcsan, N. Sakura, M. Barbosa, J.-F. Cheng, Bioorg. Med. Chem. Lett. 2007, 17, 5978.10.1016/j.bmcl.2007.07.071Search in Google Scholar

[13] B. M. Frost, G. Lonnerholm, P. Nygren, R. Larsson, E. Lindhagen, Anti-Cancer Drugs2002, 13, 735.10.1097/00001813-200208000-00008Search in Google Scholar

[14] F. A. French, E. J. Blanz, S. C. Shaddix, R. Brockman, J. Med. Chem. 1974, 17, 172.10.1021/jm00248a006Search in Google Scholar

[15] I. Bouabdallah, L. A. M’Barek, A. Zayed, A. Ramadani, I. Zidane, A. Melhaoui, Nature Prod. Res. 2006, 20, 1024.10.1080/14786410600921441Search in Google Scholar

[16] A. H. Abadi, A. A. Eissa, G. S. Hassan, Chem. Pharm. Bull. 2003, 51, 838.10.1248/cpb.51.838Search in Google Scholar

[17] J. Bonte, Eur. J. Cancer2000, 36, 114.10.1016/S0959-8049(00)00269-0Search in Google Scholar

[18] P. E. Lønning, The Breast1996, 5, 202.10.1016/S0960-9776(96)90094-6Search in Google Scholar

[19] A. T’ang, E. J. Lien, M. M. Lai, J. Med. Chem. 1985, 28, 1103.10.1021/jm00146a022Search in Google Scholar

[20] J. D. Modi, S. S. Sabnis, C. V. Deliwala, J. Med. Chem. 1970, 13, 935.10.1021/jm00299a031Search in Google Scholar

[21] N. A. Abd El-Latif, E. A. Amr, A. I. Alusein, Monatsh. Chem. 2007, 138, 559.10.1007/s00706-007-0628-zSearch in Google Scholar

[22] E. M. Hussein, Z. Naturforsch. 2012, 67b, 231.10.1016/B978-0-12-387777-2.00014-8Search in Google Scholar

[23] E. M. Hussein, A. M. El-Khawaga, J. Heterocycl. Chem. 2012, 49, 1296.Search in Google Scholar

[24] E. M. Hussein, Monatsh. Chem. 2013, 144, 1691.10.1007/s00706-013-1047-ySearch in Google Scholar

[25] E. M. Hussein, Russ. J. Org. Chem. 2015, 51, 54.10.1134/S1070428015010091Search in Google Scholar

[26] E. M. Hussein, M. I. Abdel-Monem, Arkivoc2001, 10, 85.10.21608/jbes.2023.372263Search in Google Scholar

[27] Sh. A. Abdel-Mohsen, E. M. Hussein, Russ. J. Bioorg. Chem. 2014, 40, 343.Search in Google Scholar

[28] H. G. Vogel (Ed.), Drug Discovery and Evaluation. Pharmacological Assays, 2nd edition, New York, Springer, 2002, p. 670.Search in Google Scholar

[29] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H. Bokesch, S. Kenney, M. R. Boyd, J. Nat. Cancer Inst. 1990, 82, 1107.10.1093/jnci/82.13.1107Search in Google Scholar

Received: 2015-4-1

Accepted: 2015-6-17

Published Online: 2015-10-7

Published in Print: 2015-11-1

Articles in the same Issue

https://doi.org/10.1515/znb-2015-0065

Keywords for this article

antimicrobial activity; ceric ammonium nitrate (CAN); cytotoxic activity; HEPG2; pyridine