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RESEARCH ARTICLE

Synthesis, Characterization and Biological Evaluation of 6-Ethoxy-2-oxo(thioxo)-4Phenyl-1,2-dihydropyridine-3,5-dicarbonitrile Nucleosides Hassan A. El-Sayed1,*, Alaa El-Din M. M. El-Torky1, Ahmed H. Moustafa1, * and Esraa A. Abd El Salam2 1

Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt; 2Lab. of Food Research, Ministry of Health, Sharkia, Egypt Abstract: Aim and Objective: This work concise on the synthesis of new pyridine nucleosides analogues using 6-ethoxy-2-oxo(thioxo)-4-phenyl-1,2-dihydropyridine-3,5-dicarbonitrile as nucleobases to evaluate their antimicrobial and anticancer bioactivity. Material and Method: Treating the heterocyclic base 1 or 2 with glycosyl bromides or other alkylating agents in the presence of K2CO3 or KOH in dry acetone or aq. acetone. Results: A series of cyclic and acyclic nucleosides of polyfunctionalized 2-oxo(thiooxo)nicotinonitrile derivatives 1 and 2 were synthesized by reaction of compounds 1 or 2 with glycosyl bromides or other alkylating agents in the presence of K2CO3 or KOH. The antimicrobial activity of some synthesized nucleosides has showed higher antibacterial and antifungal activity. Compounds 5, 6 and 10 have showed moderate anticancer activity virsus HepG2 and MCF-7 cell lines.

ARTICLEHISTORY Received: October 06, 2016 Revised: April 25, 2017 Accepted: April 30, 2017

Conclusion: We study of the uses of 2-oxo(thioxo)nicotinonitrile derivatives as nucleobases to synthesize a series of N-,S- and O-nucleoside derivatives and their acyclo analogues in mild reaction conditions. The antimicrobial and anticancer activities of the synthesized nucleosides and alkylated products showed higher to moderate activity.

DOI: 10.2174/1570179414666170927160806

Keywords: 2-Oxo(thiooxo)nicotinonitrile, cyclic nucleosides, acyclic nucleosides, glycosylation, antimicrobial activity, anticancer activity. 1. INTRODUCTION

pyridone derivatives act as phosphodiesterase 3 inhibitors (PDE3) [14]. The literature advertised that when the 2-pyridone nuclei used as aglycone part in the synthesis of nucleoside, the cytotoxicity towards various human cancer cell increase [15-17]. In principally, pyridone containing nucleosides I and II effective towards HL-60

The pharmacological importance of the synthesized polyfunctionalized 3-cyanopyridin-2(1H)-ones is a resuming field of the scientific attention, this is due to the existence of this class of pyriNH2 N

NH2 N

F

O OH

HO HO

O

O HO

HN O

HO (I)

N

F

O

O

O

(II)

O

O (III)

Fig. (1). Chemical structures of substituted-2(1H)-pyridone derivatives as potent anti-cancer.

dine nucleus in various biologically active compounds [1-3]. In the last 10 years, the natural compounds containing 2-pyridone moiety have emerged as an antimycotic [4], an effective antitumor [5, 6], antiviral [7, 8], anti-HIV [9] and mental disorder [10] drug. Some 2(1H)-pyridone-3-carbonitriles, like milrinone is quite-founded active vasodilatory and cardiotonic agents [11-13] and other 2-

*Address correspondence to this author at the Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt; Tel: +201112679779; E-mail: [email protected]; [email protected]

1570-1794/17 $58.00+.00

and L1210 lymphoid leukemia cells, respectively [18-20]. Likewise, 2-pyridone III [21] exhibited potent cytotoxicity in vitro against human colon cancer cells HCT-116 (Fig. 1). The S-glycoside linked heterocycles have noticed the attention of many chemists due to the biological significance of such compounds. For example several thioglycosides act as enzymes inhibitors [22], antiviral [23] and antimicrobial agents [24]. Previously, we focused on the design, synthesis and biological diversity evaluation of 2-pyridone nucleoside analogs [15, 25, 26]. Our efforts in this work focused on the synthesis of a new cyclo and acyclo nu-

© 2017 Bentham Science Publishers

2 Current Organic Synthesis, 2017, Vol. 14, No. 0

El-Sayed et al.

N CN

NC

O

NC

P2S5

+ N

CN

EtOH N H

O

OEt

ONa

O

Pyridine

CN

O

N H

1

S

2

Scheme 1. Synthetic pathway of pyridine-3,5-dicarbonitriles 1 and 2. Ph

Ph NC

CN

N

O OR OR

RO RO

RO

OAc OAc O

5 (R = Ac) Et3N

(1) AcO AcO

6 (R = H)

N

O OR

O

O RO

CN

NC

(2)

Ph NC

CN

Br

aq. KOH/ acetone

O

N H

X

OAc O

AcO AcO AcO

aq. KOH/ acetone

O RO 3 (R = Ac) Et3N

(1)

4 (R = H)

Br (2)

1,2

Ph

Ph CN

NC

N H

O OR

1; X = O 2; X = S

CN

NC

O

S

N H

S

OR

OR O

RO

O

RO RO

RO

O RO 7 (R = Ac)

Et3N

9 (R = Ac)

Et3N

8 (R = H)

10 (R = H)

Scheme 2. Glycosylation of pyridine-3,5-dicarbonitriles 1 and 2.

cleosides using the title compounds 1 and 2 as aglycones to evaluate their antimicrobial and anticancer bioactivity.

and 13C NMR evidenced the existence of the anomeric carbon for compound 3 at lower field ( = 89.08 ppm).

2. RESULTS AND DISCUSSION

The 1H NMR of nucleosides 3 and 5 showed the anomeric protons at chemical shift 6.09 and 6.0 ppm (J = 7.50 and 7.60 Hz) confirming the diaxial orientation H-2' (-configuration), respectively.

2.1. Chemistry 6-Ethoxy-2-oxo(thioxo)-4-phenyl-1,2-dihydronicotinonitrile-5carbonitrile (1) and (2) were obtained according to the literature [27] by base mediated Micheal addition reaction of malononitrile to ethyl 2-cyano-3-phenylacrylate, followed by sulfurization with phosphorous pentasulphide in refluxed dry pyridine (Scheme 1). Compounds 1 and 2 were glycosylated with both -D-glucosyl or / galactosyl bromide in acetone containing a catalytic potassium hydroxide leading to the production of the corresponding 2oxonicotinonitrile nucleosides 3 and 5 and nicotinonitrile-2-thioglycosides derivatives 7 and 8, respectively (Scheme 2). The spectral data evidenced the structures of the new product nucleosides. Where, formation of the N-nucleoside 3 and 5 was confirmed by the IR spectra (where the amidic C=O groups stretched at 1630 cm-1),

While, in the thioglycosides 7 and 9 the reaction complete on sulfur nucleophile better than the nitrogen nucleophile has been propped by appearing thier anomeric protons in the sugar moiety at higher field (appeared at  6.39 and 6.23 ppm, respectively). The C=S anisotropic effect lead to appearing the anomeric protons of N-nucleosides have a neighboring C=S at lower field (i.e. higher chemical shift; 6.9 - 7.2 ppm) [28]. The -configuration of thioglycosides 7 and 9 confirmed by coupling constant value (J1',2' = 8.20 Hz). Deprotection of nucleosides 3, 5, 7 and 9 were treated with triethyl amine in aqueous methanol solution, the deacetylated glycoside derivatives 4, 6, 8 and 10 were produced in high yields, re-

Synthesis, Characterization and Biological Evaluation

Current Organic Synthesis, 2017, Vol. 14, No. 0

3

Ph

Ph CN

NC

CN

NC Et3N/H2O

O

N

HO

MeOH, reflux

O

N 11

O

12

OAc

OH

Ph 2

AcO

Br Ph

CN

NC Et3N/H2O

OAc 2

O

Br

NC

Ph CN

NC

K2CO3 acetone

N

O

cetone CO 3/a

K2

O

13

O MeOH, reflux OAc

CN

N

HO 14

O

O

OH

Ph O

N H

O

OH

Cl K2 CO

NC OH

3 /acet

1

/ac 3 CO K2

K2CO3 acetone

one

O

N

O OH

15

Br

OH

ne eto

Br

CN

Ph NC

CN

Ph CN

NC

O

N

O

O

N

O

17

16 Scheme 3. Synthetic approach of nicotinonitrile acyclo nucleosides analogue 11-17.

spectively (Scheme 2). The IR spectral data of the deacetylated products 4 and 6 confirmed the formation of N-glycosides by presence of C=O amidic stretching band at 1634 and 1624 cm-1, respectively. Although, there are streching bands for free hydroxyl groups (OH) in between 3419-3437 cm-1, which confirm the deacetylation process and their 1H NMR agreed with the predictable structures. Nucleophilic alkylation of 6-ethoxy-2-oxo-4-phenyl-1,2dihydronicotinonitrile-5-carbonitrile (1) with 4-bromobutyl acetate, [2-acetoxyethoxy]methyl bromide, 1,2-dihydroxypropyl chloride, allyl bromide and propargyl bromide in dry acetone containing a catalytic equivalent amount of potassium carbonate afforded the target O-acyclic nucleoside analogs 11, 13 and 15-17 as O-alkylated products, respectively (Scheme 3). The regio spacifically of this O-alkylation reaction was evidenced from the absence of C=O (amide carbonyl) bands in the infra red spectra of such products 11, 13 and 15-17. 13 C NMR of compound 11 showed presence of carbons of glycon moiety at  = 20.59, 170.3 (CH3CO), 24.60, 24.72 (C-2' and C3') and 64.83 (C-1'), 68.21 (C-4'), while, the 1H NMR data of compound 13 gave signals at  2.01, 4.78, 3.81 and 4.13 ppm corresponding to CH3CO, 2H-1', 2H-3' and 2H-4' protons, respectively. The spectral data and elemental analysis of the compounds 15-17 are agreed with thier predictable structures.

Deacetylation of compounds 11 and 13 in moderate base medium (Et3N) in aquoeus methanol produced the deacetylated derivatives 12 and 14, respectively (Scheme 3), with observation the hy-

drolysis of ethoxy group at position-6 for the dinicotinonitrile to hydroxyl group. This assigned by the absence of ethoxy protons signals in 1H NMR spectrum and presence of OH group at  = 10.56 and 10.59 ppm (exchange with D2O) beside that of glycon moiety, which appear at  = 3.34 and 4.71 ppm in both compounds 12 and 14, respectively. Their IR spectra showed broad absorption bands for free OH groups at 3426 and 3423 cm-1, respectively. 2.2. Pharmacological Evaluation 2.2.1. Antimicrobial Activity The antibacterial activities of 1, 2, 4, 8, 6, 10, 12, 14 and 15-17 samples were screened by the agar well diffusion method [29]. The experiment was reiterated 3 times and the average inhibition zones were elaborate. As reported in Table 1, it is obviously observed that the newely synthesized nucleosides have low to moderate effect versus Gram (+ve) (S. aureus and B. cereus) and (P. aeruginosa and E. coli) as Gram (-ve), which compared with Cefotaxime as control. While the Table 2, indicate moderate antifungal activity versus (A. flavus and A. niger) compared to the reference drug Dermatin as control. 2.2.2. Anticancer Activity Using MTT assay, the effects of the samples on the proliferation of HepG2 and MCF-7 cells were studied after 48 h of incubation. As shown in Figs. (2 and 3), to Table 3, the treatment of HepG2 and MCF-7cells with the samples 4, 5, 6, 8, 10, 12, 14 and

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Table 1.

El-Sayed et al.

Antibacterial effects of some new nucleosides (Inhibition zones mm). Diameter (mm) of inhibition zones against the corresponding standard microbial strains. Gm (+ve) bacteria

Gm (–ve) bacteria

Tested Compounds

Table 2.

S. aureus

B. Cereus

P. aeruginosa

E. coli

1

21

20

25

18

2

20

38

18

22

4

23

25

21

30

6

18

20

23

19

8

22

24

24

18

10

20

22

30

24

12

19

18

22

25

14

18

20

18

17

15

18

20

20

21

16

23

21

20

20

17

19

23

20

18

Cefotaxime

31

28

32

34

DMF

-

-

-

-

Antifungal activities of some new synthesized compounds (Inhibition zones mm). Diameter (mm) of Inhibition Zones Against the Corresponding Standard Microbial Strains Tested Compounds

Fungi Aspergillus flavus

Aspergillus niger

1

19

20

2

30

18

4

32

20

6

20

20

8

29

21

10

20

25

12

22

19

14

33

20

15

33

18

16

18

19

17

34

22

Dermatin

31

22

DMF

-

-

15 showed cytotoxic effects on both cell lines used, for HepG2 cells. Samples 5, 6, 10 showed higher cytotoxic effect than samples 4, 8, 12, 14 and 15 concluded from their IC50 values which were calculated from the graph to be 352, 212, 256 and 621, 566, 532, 617, 468 g/mL respectively. For MCF-7 cells the sensitivity of the cells was higher to the tested samples observed from the decrease in calculated IC50 values which were 298, 205, 200 g/mL for sample 2, 6, 10 and 326, 346, 652, 540, 614 g/mL for samples 4, 8, 12, 14 and 15, respectively. This activity attributed to the presence of the 4-phenyl substituted, free hydroxy and protect acetoxy groups the glycone moiety gave a good affinity against the enzyme, particularly the nucleoside containing galactose moiety. Due to the electrostatic attraction force between the planar phenyl and electron

withdrawn properity of acetoxy oxygens beside the presence of two nitrile groups in position 3 and 5, with the target site of the tumor cells. 4. EXPERIMENTAL The melting points are uncorrected and were measured using an Electro thermal IA 9100 apparatus. The IR spectra (KBr discs) were recorded on a Shimadzu FTIR 8101 PC infrared spectrophotometer. The operation frequency was 400 MHz for 1HNMR and 100 MHz for 13C NMR using BRUKER (400 MHz) spectrometer (at nucleic acid research center, Faculty of Science, Zagazig University). The coupling constants (J) are given in Hertz. The chemical shifts are

Current Organic Synthesis, 2017, Vol. 14, No. 0

Synthesis, Characterization and Biological Evaluation

Table 3.

5

Anticancer activities of some new synthesized compounds sample. IC50 (μg/ML)

Tested Compounds HepG2

MCF-7

4

621

326

5

352

298

6

212

205

8

566

346

10

256

200

12

532

652

14

617

540

15

468

614

Doxorubicin

0.467

0.750

expressed on the  (ppm) scale using TMS as the standard reference. Antimicrobial activity was carried out at Research laboratory of bacteriology, Faculty of Science, Zagazig University. The anticancer activity was carried at Cancer Biology Laboratory, National Research Center, Giza, Egypt. Elemental analysis was determined on a Perkin Elmer 240, Mass Spectrometry was determined on DI Analysis Shimadzu QP-2010 Plus (Micro Analytical Center, Cairo University, Giza, Egypt). HRMS (EI) was carried in Bioorganic Chemie, Institut fur Chemie, Universty of Hohenheim, Stuttgart, Germany. 4.1. 6-Ethoxy-2-oxo-4-phenyl-1,2-dihydronicotinonitrile-5-carbonitrile (1) The preparation method present in the literature [27]. Yield 50 %, mp 218-220°C. IR:  3320, 2228, 1640 and 1149 cm-1; 1H NMR (DMSO/d6): 1.38 (t, Hethyl, 3H, 3J = 6.60 Hz), 4.50 (q, Hethyl, 2H, 3J = 6.60 Hz), 7.54-7.60 (m, Hphenyl, 5H) and 10.49 (s, NH, 1H). 4.2. 6-Ethoxy-4-phenyl-2-thioxo-1,2-dihydronicotinonitrile-5carbonitrile (2) Phosphorus pentasulfide (P2S5) (0.01 M) was added to a quik fit flask containing a solution of compound 1 (0.01 M) in pyridine (20 mL). After refluxing the reaction mixture 16 hours; was cooled, neutralized with diluted HCl and filtered. Yield 91 %, mp 260262°C (Brown powder from ethanol). IR:  3431, 2209 and 1231 cm-1. Anal. for C15H11N3OS (MWt:281.33); Calcd: C, 64.04; H, 3.94; N, 14.94; Found: C, 63.93; H, 4.00; N, 14.92. 4.3. General Synthesis of Nucleosides 3, 5 and Thioglycosides 7 and 9 To a stirred mixture of ethoxy pyridine derivative 1 or/ 2 (0.01 M) and potassium hydroxide (0.011 M) in aq. Acetone [10 mL dis. H2O + 30 mL acetone], the glucosyl or / galactosyl bromide (0.011 M) in acetone (10 mL) was added. After completing the stirring for 24 hours (TLC) at r.t. followed by evaporating under vaccum, the residue was separated by column chromatography (200-400 mesh, eluent: CH2Cl2 100%). 4.3.1. 1-(2',3',4',6'-Tetra-O-acetyl--D-glucopyranosyl)-6-ethoxy2-oxo-4-phenyl-1,2-dihydronicotinonitrile-5-carbonitrile (3) Yellow powder, mp 110-112°C; yield 70 %. IR:  2229 and 1754 cm-1. 1H NMR (DMSO/d6):  1.52 (t, Hethyl, 3H, 3J = 7.50 Hz), 2.02, 2.03, 2.05, 2.06 (4s, 4CH3CO, 12H), 3.95 (2d, H-6', 1H, 2J 6',6" = 12.08 Hz, 3J 5',6' = 4.63 Hz), 4.18 (m, H-6", 1H), 4.25 (m, H-5', 1H,), 4.42 (2d, H-4', 1H, 3J 3',4' = 8.0 Hz), 4.55 (q, Hethyl, 2H, 3J = 6.8 Hz), 4.98 (t, H-2', 1H, 3J 1',2' = 8.0 Hz), 5.22 (t, H-3', 1H, 3J 2',3' =

9.30 Hz), 6.09 (d, H-1', 1H, 3J 1',2' = 7.5 Hz), 7.54 (m, Hphenyl, 5H). C NMR (DMSO-d6):  = 14.07 (CH3CH2), 20.18, 20.24, 20.27, 20. 32 (4CH3CO), 56.2 (CH2CH3), 61.79, 68.21, 70.54, 71.69, 72.13, 89.08 (sugar-C), 94.23, 100.14, 112.7, 113.6 (2 CN), 128.5, 128.8, 130.8, 132.9, 162.0, 163.2 (Ar-C), 165.2, 169.1, 169.3, 169.7 and 170.0 (C=O). Anal. for C29H29N3O11 (MWt:595.55); Calcd: C, 58.49; H, 4.91; N, 7.06. Found: C, 58.38; H, 4.87; N, 7.13. 13

4.3.2. 1-(2',3',4',6'-Tetra-O-acetyl--D-galactopyranosyl)-6-ethoxy-2-oxo-4-phenyl-1,2-dihydronicotinonitrile-5-carbonitrile (5) Yellow powder, mp 98-100ºC; yield 36 %. IR:  2227, 1752 and 1630 cm-1. 1H NMR (CDCl3):  1.53 (t, Hethyl, 3H, 3J = 7.50 Hz), 1.98, 2.03, 2.15, 2.21, (4s, 4CH3CO, 12H), 3.91 (2d, H-6', 1H, 2 J 6',6" = 12.31 Hz, 3J 5',6' = 5.20 Hz), 4.13 (2d, H-6", 1H, 3J 5',6' = 6.0 Hz, 2J6',6" = 12.31 Hz), 4.18 (m, H-5', 1H), 4.58 (q, Hethyl, 2H, 3J = 7.50 Hz), 5.00 (2d, H-3', 1H, 3J 3',4' = 2.61 Hz, 3J 2',3' = 10.4 Hz), 5.18 (2d, H-2', 1H, 3J 2',3' = 10.4 Hz, 3J 1',2' = 7.6 Hz), 5.61 (2d, H-4', 1H, 3 J 3',4' = 2.61 Hz, 3J 4',5' = 8.38 Hz), 6.0 (d, H-1', 1H, 3J = 7.6 Hz), 7.54 (m, Hphenyl, 5H). 13C NMR (CDCl3): 14.07 (CH3CH2), 20.20, 20.22, 20.25, 20.33 (4 CH3CO), 56.30 (CH3CH2), 61.9, 68.0, 68.1, 70.7, 71.8, 89.7 (sugar-C), 114.9, 115.6 (2CN), 128.9, 129.6, 129.8, 130.5, 131.0, 132.3, 135.6, 142.4 (Ar-C) and 165.1, 169.4, 169.9, 170.3, 170.5 (C=O). Anal. for C29H29N3O11 (MWt: 595.55); Calcd: C, 58.49; H, 4.91; N, 7.06. Found: C, 58.57; H, 4.84; N, 7.11. 4.3.3. 2-(2,3,4,6-Tetra-O-acetyl--D-glucopyranosylsulfanyl)-6ethoxy-4-phenyl-pyridine-3,5-dicarbonitrile (7) Brown powder, mp 100-102ºC; yield 57 %. IR:  2215 and 1756 cm-1. 1H NMR (CDCl3):  1.53 (t, Hethyl, 3H, 3J = 7.50 Hz), 2.00, 2.02, 2.04, 2.08 (4s, 4CH3CO, 12H), 3.69 (d, H-6', 1H, 3J 5', 6' = 8.4 Hz), 4.13 (d, H-6'', 1H, 3J 5', 6' = 12.0 Hz), 4.28 (q, Hethyl, 2H, 3 J = 7.50 Hz), 4.42 (d, H-5', 1H, 3J 3',4' = 8.0 Hz), 4.96 (t, H-4', 1H, 3 J 3',4' = 9.2 Hz), 5.06 (t, H-2', 1H, 3J 1',2' = 9.6 Hz), 5.20 (t, H-3', 1H, 3 J 2',3' = 9.6 Hz), 6.39 (d, H-1', 1H, 3J 1',2' = 8.2 Hz), 7.15 (s, Hphenyl, 5H). 13C NMR (DMSO-d6): 14.06 (CH3CH2), 20.22, 20.26, 20.29, 20.34 (4 CH3CO), 56.28 (CH3CH2), 61.5, 68.0, 68.9, 71.4, 72.2, 87.9 (sugar-C), 114.8, 115.4 (2CN), 128.9, 129.0, 130.5, 131.0, 131.9, 134.1, 141.9, 152.8 (Ar-C) and 166.3, 169.0, 169.3, 170.0 170.1 (C=O), Anal. for C29H29N3O10S (MWt:611.62); Calcd: C, 56.95; H, 4.78; N, 6.87. Found: C, 57.08; H, 4.85; N, 6.80. 4.3.4. 2-(2',3',4',6'-Tetra-O-acetyl--D-galactopyranosylsulfanyl)6-ethoxy-4-phenyl-pyridine-3,5-dicarbonitrile (9) Brown powder, mp 108-110ºC; yield 45.6 %. IR:  2217and 1745 cm-1. 1H NMR (CDCl3):  1.25 (t, Hethyl, 3H, 3J = 7.50 Hz), 2.00, 2.05, 2.08, 2.10 (4s, 4CH3CO, 12H), 3.50 (d, H-6', 1H, 3J 5', 6’

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El-Sayed et al.

= 5.80 Hz), 3.75 (d, H-6'', 1H, 3J 5', 6' = 6.2 Hz), 4.10 (m, H-5', 1H), 4.28 (q, Hethyl, 2H, 3J = 7.50 Hz), 4.94 (2d, H-3', 1H, 3J 3',4' = 2.8 Hz, 3 J 2',3' = 10.6 Hz), 5.07 (d, H-2', 1H, 3J 1',2' = 10.2 Hz), 5.23 (2d, H-4', 1H, 3J 4',5' = 8.3 Hz, 3J 3',4' = 2.81 Hz), 6.23 (d, H-1', 1H, 3J 1',2'= 8.2 Hz), 7.25 (m, Hphenyl, 5H). Anal. for C29H29N3O10S (MWt: 611.62); Calcd: C, 56.95; H, 4.78; N, 6.87. Found: C, 57.02; H, 7.70; N, 6.80.

(t, 2H-1', 2H,3J = 6.42 Hz), 4.57 (m, Hethyl and 2H-4', 4H), 7.56 (m, Hphenyl, 5H); 13C NMR (DMSO/d6):  14.00 (CH3CH2), 20.59 (CH3CO), 24.60, 24.72 (C-2' and C-3'), 63.29 (OCH2CH3), 64.83 (C-1'), 68.21 (C-4'), 88.65, 88.70, 113.7, 113.8 (2CN), 128.4, 128.7, 130.6, 133.1, 161.3, 165.5, 165.6, 170.3 (sp2-C). Anal. for C21H21N3O4 (MWt: 379.41); Calcd: C, 66.48; H, 5.58; N, 11.08; Found: C, 66.37; H, 5.63; N, 10.99.

4.4. General Synthesis of Compounds 4, 6, 8 and 10

4.5.2. 2-(((3,5-Dicyano-6-ethoxy-4-phenylpyridin-2-yl)oxy)methoxy)ethyl acetate (13)

These compound were synthesized as previous reported method [25, 26] 4.4.1. 6-Ethoxy-1-(-D-glucopyranosyl)-2-oxo-4-phenyl-1,2-dihydronicotinonitrile-5-carbonitrile (4) Yellow powder, mp 138-140ºC; yield 90 %. IR:  3431, 2216 and 1634 cm-1. 1H NMR (DMSO/D2O):  1.32 (t, H ethyl, 3H, 3J = 6.40 Hz), 2.89-3.29 (m, H-6'-2', 6H), 3.96 (q, Hethyl, 2H, 3J = 6.40 Hz), 5.69 (d, H-1', 1H, 3J 1',2' = 8.4 Hz), 7.23-7.48 (m, Hphenyl, 5H). Mass Spectrometry (M+1) m/e = 428.14 (5.80%), 382.14 (23.20%), 264.8 (16.40%), 220.05 (89.40%) and 164.07 (18.40%). Anal. for C21H21N3O7 (MWt: 427.41); Calcd: C, 59.01; H, 4.95; N, 9.83. Found: C, 59.00; H, 4.93; N, 9.85. 4.4.2. 6-Ethoxy-1-(-D-galctopyranosyl)-2-oxo-4-phenyl-1,2-dihydronicotinonitrile-5-carbonitrile (6) Yellow powder, mp 158-160ºC; yield 92 %. IR:  3422, 2215 and 1624 cm-1. 1H NMR (DMSO/D2O):  1.28 (t, Hethyl, 3H, 3J = 6.40 Hz), 3.22 (m, H-3',6',6", 3H), 3.83 (m, H-2',4',5', 3H,), 4.35 (q, Hethyl, 2H, 3J = 6.40 Hz), 6.04 (d, H-1', 1H, 3J 1',2' = 7.9 Hz), 7.537.78 (m, Hphenyl, 5H). Anal. for C21H21N3O7 (MWt: 427.41); Calcd: C, 59.01; H, 4.95; N, 9.83. Found: C, 59.00; H, 4.93; N, 9.80. 4.4.3. 6-Ethoxy-2-(-D-glucopyranosylsulfanyl)-4-phenylpyridine3,5-dicarbonitrile (8) Brown powder, mp 130-135 ºC; yield 85 %. IR:  3419, 2210 cm-1. 1H NMR (DMSO/D2O):  1.30 (t, Hethyl, 3H, J = 6.60 Hz), 2.98-3.36 (m, H-6'-2', 6H), 3.89 (q, Hethyl, 2H, 3 J = 6.6 Hz), 5.78 (d, H-1', 1H, 3J 1',2' = 8.20 Hz), 7.03-7.41 (m, Hphenyl, 5H). Anal. for C21H21N3O6S (443.47); Calcd: C, 56.87; H, 4.77; N, 9.48. Found: C, 56.95; H, 4.81; N, 9.53. HRMS (EI) calcd for C21H21N3O6S [M+]: 445.1304; found: 445.1302 4.4.4. 6-Ethoxy-2-(-D-galactopyranosylsulfanyl)-4-phenylpyridine-3,5-dicarbonitrile (10) Brown powder, mp 138-140ºC; yield 85 %. IR:  3437 and 2222 cm-1. 1H NMR (DMSO/D2O):  1.29 (t, 3H, H ethyl, 3J = 6.50 Hz,), 3.30 (m, H-3',6',6", 3H), 3.69 (m, H-2',4',5', 3H), 4.36 (q, Hethyl, 2H, 3J = 6.50 Hz), 6.08 (d, H-1', 1H, 3J 1',2' = 7.8 Hz), 7.637.78 (m, Hphenyl, 5H). Anal. for C21H21N3O6S (MWt: 443.47); Calcd: C, 56.87; H, 4.77; N, 9.48. Found: C, 56.79; H, 4.80; N, 9.44.

Yellow syrup; yield 40 %. IR:  2227 and 1741 cm-1. 1H NMR (DMSO-d6):  1.39 (t, 3H, Hethyl, 3J = 7.30 Hz), 2.01 (s, CH3CO, 3H), 3.81 (t, 2H-3', 2H, 3 J = 5.60 Hz), 4.13 (t, 2H-4', 2H, 3J = 5.60 Hz), 4.58 (q, Hethyl, 2H, 3J = 7.30 Hz), 4.78 (s, 2H-1', 2H) and 7.59 (m, Hphenyl, 5H). Mass Spectrometry (M+1) m/e = 382.14 (22.0%), 264.08 (16.40%), 220.05 (89.8%), and 118.06 (5.60%). Anal. for C20H19N3O5 (MWt: 381.38); Calcd: C, 62.99; H, 5.02; N, 11.02. Found: C, 63.09; H, 4.98; N, 10.94. 4.5.3. 2-(2,3-Dihydroxypropoxy)-6-ethoxy-4-phenylpyridine-3,5dicarbonitrile (15) Brown powder, mp 98-100°C; yield 30 %. IR:  3457, 3372, 2214 cm-1. 1H NMR (DMSO/D2O):  1.18 (t, Hethyl,3H, 3J = 7.30 Hz), 3.94 (d, 2H-1', 2H, 3J = 5.8 Hz), 4.12 (q, Hethyl, 2H, 3J = 7.30 Hz), 4.25 (m, 2H-3', 2H), 4.90 (m, H-2', 2H), 5.30 (s, OH, 1H), 5.83 (s, OH, 1H,) and 7.13-7.55 (m, Hphenyl, 5H). Anal. for C18H17N3O4 (MWt: 339.35); Calcd: C, 63.71; H, 5.05; N, 12.38; Found: C, 63.63; H, 6.11; N, 12.43. 4.6. General Procedure for Deacetylation of Ayclo Nucleosides 11 and 13 A mixture of synthesized acyclo nucleoside 11 or 13 (0.01 M), triethylamine (0.5 mL), water (3 drops) and methanol (25 mL) was refluxed for 6 hours (TLC). The mixture was evaporated under vacumm several times with methanol and crystallized from methanol. 4.6.1. 2-Hydroxy-6-(4-hydroxybutoxy)-4-phenylpyridine-3,5-dicarbonitrile (12) Pale brown powder, mp 170-172°; yield 85.4 %. IR:  3426 and 2210 cm-1. 1H NMR (DMSO/D2O):  1.17-1.21 (m, 2H-2' and 2H3', 4H), 3.25 (t, 2H-1', 2H-4', 4H, 3J = 7.2 Hz), 3.34 (t, OH, 1H, 3J = 5.4 Hz), 7.37-7.46 (m, Hphenyl, 5H) and 10.56 (s, OH, hydrolysis of ethoxy group at position-6, exchange with D2O, 1H). 13C NMR (DMSO-d6)  = 25.57, 29.35 (2CH2), 60.84 (CH2OH), 67.72 (OCH2), 114.9, 115.3 (2CN), 129.1, 129.3, 129.5, 130.9, 131.8, 135.5, 143.0, 153.4 and 164.4 (Ar-C). Anal. for C17H15N3O3 (MWt: 309.32); Calcd: C, 66.01; H, 4.89; N, 13.58. Found: C, 66.11; H, 4.85; N, 13.64.

4.5. General Synthesis of Acyclic Nucleosides 11, 13 and 15

4.6.2. 2-Hydroxy-6-((2-hydroxyethoxy)methoxy)-4-phenylpyridine-3,5-di-carbonitrile (14)

To a mixture of 2-pyridone derivative 1 (0.01 M) in acetone (20 mL), potassium carbonate (K2CO3) (0.011 M) and an appropriate acyclic sugar (namely, 4-bromobutyl acetate, (2-acetoxyethoxy) methyl bromide or 3-chloropropane-1,2-diol) (0.011 M) were added. After refluxing the reaction mixture for 10-12 hours (TLC), filter off the inorganic salts and the filterate was evaporated under vaccum and seperated by column chromatography (200-400 mesh) using CH2Cl2 as eluent.

Yellow powder, mp 158-160°C; yield 84.5 %. IR:  3423, 2211 cm-1. 1H NMR (DMSO/D2O):  4.25 (t, 2H-3', 2H, 3J = 6.4 Hz), 4.42 (t, 2H-4', 2H, 3J = 6.4 Hz), 4.71 (t, OH, D2O exchange, 1H, 3J = 5.4 Hz), 4.90 (s, 2H-1', 2H,); 7.38-7.45 (m, Hphenyl, 5H) and 10.59 (s, 1H, pyridone-6-OH, exchange with D2O). Anal. for C16H13N3O4 (MWt: 311.29); Calcd: C 61.73; H 4.21; N, 13.50 Found: C 61.66; H, 4.25; N, 13.48. HRMS (EI) calcd for C16H13N3O4 [M+]: 311.0910; found: 311.0912.

4.5.1. 4-((3,5-Dicyano-6-ethoxy-4-phenylpyridin-2-yl)oxy)butyl acetate (11)

4.7. Synthesis of Alkylated Compounds 16 and 17

Buff powder, mp 112-114°C, yield 40%. IR:  2228, 1731 and 1159 cm-1. 1H NMR (DMSO-d6):  1.38 (t, Hethyl, 3H, 3J = 7.21 Hz), 1.72 (m, 2H-3', 2H), 1.84 (m, 2H-2', 2H), 2.0 (s, CH3CO, 3H), 4.05

To a mixture of 2-pyridone derivative 1 (0.01 M) in acetone (20 mL), potassium carbonate (K2CO3) (0.011 M) and allyl and propargyl bromides (0.011 M) were added. After refluxing the reaction

Synthesis, Characterization and Biological Evaluation

mixture for 15 hours (TLC), filter off the inorganic salts; evaporate under vaccum; then crystalization from ethanol. 4.7.1. (16)

Current Organic Synthesis, 2017, Vol. 14, No. 0

[4]

2-(Allyloxy)-6-ethoxy-4-phenylpyridine-3,5-dicarbonitrile [5]

Colorless crystal, mp 124-126°C; yield 45 %. IR:  2227, 1166 cm-1. 1H NMR (DMSO-d6):  1.38 (t, Hethyl, 3H, 3J = 6.9 Hz), 4.56 (q, Hethyl, 2H, 3J = 6.9 Hz), 5.07 (d, 2H-1', 2H, 3J = 5.4 Hz), 5.37 (d, H-3', 1H, 3 J = 9.98 Hz), 5.51 (d, H-3", 1H, 3J = 16.90 Hz), 6.14 (m, H -2', H-1), 7.36-7.59 (m, Hphenyl, 5H). HRMS (EI) calcd for C18H15N3O2 [M+]: 305.1213; found: 305.1211. Anal. for C18H15N3O2 (MWt: 305.33): Calcd: C, 70.81; H, 4.95; N, 13.76, Found: C, 70.89; H, 4.91; N, 13.80. 4.7.2. 2-Ethoxy-4-phenyl-6-(prop-2-yn-1-yloxy)pyridine-3,5-dicarbonitrile (17) White powder, mp 175-178°C; yield 45 %. IR:  2229 and 1165 cm-1. 1H NMR (DMSO-d6):  1.40 (t, Hethyl, 3H, 3J = 6.9 Hz), 3.70 (s, H-3', 1H), 4.63 (q, Hethyl, 2H, 3J = 6.9 Hz), 5.26 (t, H-1', 2H, 2J = 1.20 Hz) and 7.60 (m, Hphenyl, 5H). 13C NMR (DMSO/d6):  14.06 (CH3CH2), 56.12 (OCH2CC), 65.19 (CH3CH2), 77.7, 78.81 (CC), 88.68, 89.60, 113.49 (CN), 113.60 (CN), 128.4, 128.7, 130.7, 132.9, 161.6, 164.4, 165.3 (Ar-C). Anal. for C18H13N3O2 (MWt: 303.31), Calcd: C, 71.28; H, 4.32; N, 13.85. Found: C, 71.35; H, 4.29; N, 13.78. CONCLUSION In summary, we study of the uses of 2-oxo(thioxo)nicotinonitrile derivatives as nucleobases to synthesize a series of N-,Sand O-nucleoside derivatives and their acyclo analogs in mild reaction conditions. The antimicrobial activity of the synthesized nucleosides and alkylated products showed higher antibacterial and antifungal activity. In addition to, compounds 5, 6 and 10 showed moderate anticancer activity virsus HepG2 and MCF-7 cell lines. ETHICS APPROVAL AND CONSENT TO PARTICIPATE

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[13]

[14]

[15]

[16]

Not applicable. [17]

HUMAN AND ANIMAL RIGHTS No Animals/Humans were used for studies that are base of this research.

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CONSENT FOR PUBLICATION

[19]

Not applicable. [20]

CONFLICT OF INTEREST

[21]

The authors declare no conflict of interest, financial or otherwise.

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ACKNOWLEDGEMENTS The authors would like to extend their sincere appreciation to microbiology lab. Faculty of Science, Zagazig University for biological activity, and organic chemistry lab. Chemistry Dep. Faculty of Science, Zagazig University for supporting this work.

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