Medicinal Chemistry

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whereas bioisosteric conversion of the 2-one functionality to the 2-thione with subsequent .... O. H. S. N. O. O. H. R1. Scheme 2. Synthesis of sulfa-drugs 10a,b.

791

Send Orders for Reprints to [email protected] Medicinal Chemistry, 2018, 14, 791-808

RESEARCH ARTICLE ISSN: 1573-4064 eISSN: 1875-6638

Impact Factor: 2.631

Synthesis of Nucleosides and Non-nucleosides Based 4,6-disubstituted-2oxo-dihydropyridine-3-carbonitriles as Antiviral Agents

Tarek S. Ibrahim1,2, Hassan A. El-Sayed3, Maan T. Khayat1, Amany M.M. AL-Mahmoudy2, Ahmed H. Moustafa3,*, Ayat K.S. El-Deen3, Sherif A.F. Rostom4 and Siva S. Panda5,* 1

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 2Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt; 3Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt; 4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt; 5Department of Chemistry & Physics, Augusta University, Augusta, GA 30912, USA Abstract: Background: Viral diseases are considered main threats that face the humanity worldwide. The emergence of new viruses like influenza viruses emphasizes the significance of designing novel antiviral drugs. ARTICLE HISTORY Received: July 03, 2017 Revised: March 14, 2018 Accepted: April 12, 2018 DOI: 10.2174/1573406414666180522123524

Method: The aim of this work is to synthesize a new set of nucleoside and non-nucleoside cyanopyridine, characterized and evaluated for their in vitro antiviral properties against various strains. Conclusion: More of the compounds showed variable antiviral potential against a panel of eighteen DNA and RNA viruses. The screening data suggested that the order of activity of the active compounds are in the order of O-glycosyl > O-alkyl > N-alkyl > S-alkyl derivatives. In addition, the 4-fluoro substituted compounds are more effective among the O- and N-alkyl analogs, whereas remarkable antiviral activity was ascribed to the methoxylated O-glycosyl derivatives. Most of the active compounds proved to be more selective towards the inhibition of the replication of DNArather than the RNA-viruses. The analogs 1a, 2a, 12b, 14b and 16b possessed broad spectrum and noticeable antiviral potential against most of the tested DNA- and RNA-viruses (EC50  0.8-20 M), accompanied with considerably low cytotoxic margin (MCC  4-20 M), and comparable with reference standard antiviral agents.

Medicinal Chemistry

Keywords: Synthesis, pyridine, nucleosides, non-nucleosides, antiviral, influenza viruses. 1. INTRODUCTION The widespread of infectious diseases caused by various DNA and RNA viruses is becoming a dangerous health threat throughout the world [1]. Several double-stranded DNA viruses are responsible for various serious diseases such as oral stomatitis and ocular ulcerations caused by the herpes simplex virus-1 (HSV-1), skin and genital eruptions by HSV-2 [2], chickenpox and shingles are caused by varicella-zoster virus (VZV). Cytomegalovirus (HCMV) in one of the life-threatening virus and causes high mortality rates in immune-compromised patients [3, 4]. Similarly, many single-stranded RNA viruses are reported for their harmful effect in both human and animal populations including the send bis virus, vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), para-influenza virus and hepatitis A-C viruses [5-7]. Prominently, the acute viral in*Address correspondence to these authors at the Department of Chemistry & Physics, Augusta University, Augusta, GA 30912, USA; E-mails: [email protected], [email protected] 1875-6638/18 $58.00+.00

fection “Influenza” caused by the enveloped influenza RNA viruses (Types A-C) constitutes a serious public health problem owing to the induced severe fever and respiratory complications that may lead to death. Official statistics have revealed that about 500,000 deaths were reported worldwide out of 3 to 5 million severe influenza cases recorded annually. Among the type A influenza viruses stemmed the H1N1 and H3N2 subtypes as horrible health and economic burdens on human workforce [8]. Despite the remarkable advancements in the development of novel antiviral agents, yet there are some obstacles that hamper these efforts including the dangerousness of the clinically important viruses, the severe side effects and the frequent emergence of resistance [9, 10]. Although the development of nucleoside antiviral drugs has received a substantial interest and commonly used in the therapy of viral infections including HIV [11, 12], yet the non-nucleoside analogs have stemmed as significantly important antiviral agents [13, 14] (Fig. 1). Among the pharmaceutically-important heterocycles, the 3-cyanopyridine scaffold is widely found in several interesting nucleoside and © 2018 Bentham Science Publishers

792 Medicinal Chemistry, 2018, Vol. 14, No. 8

Ibrahim et al. O

O

O N

NH N

O

N

O

HN

N

N

O

N H

O

O

HO

OH

HO Didanosine

Stavudine

Telivudine

O N

NH N N

N N

N

NH N

NH2 NH2

N HO

NH2

HO

HO HO Abacavir

OH Entecavir

N

O P O

N

N

Adefovir

Fig. (1). Few examples of nucleosides and non-nucleosides based antiviral drugs.

non-nucleoside compounds endowed with versatile bioactivities including antimicrobial [15, 16], antifungal [17], antitubercular [18] antiviral [19], and anticancer [20] activities. Interest in the chemotherapeutic potential of pyridines has originated from their isosteric relevance to pyrimidines, which act as a vital building block in a number of essential cellular components including the of DNA and RNA [21, 22]. There are few reports that have described the synthesis and antiviral potential of 3-cyanopyridine derivatives [23, 24] which look promising for the development of new potential antiviral agents. From above-mentioned facts, herein we designed and synthesized various nucleoside and non-nucleoside pyridine analogs and evaluated their in vitro antiviral properties against a panel of virus strains. The targeted compounds were designed on the basis of 4,6-diaryl-2-oxo-1,2dihydropyridine-3-carbonitrile scaffold substituted with some functionalities which are reported to account for the biological significance of some relevant antiviral agents [25, 26]. For instance, O- and N-alkylation of the non-nucleoside analogs with different alkyl counterparts were considered, whereas bioisosteric conversion of the 2-one functionality to the 2-thione with subsequent S-alkylation was considered as an interesting structural modification. The pattern of substitution of the 4,6-diaryl moieties was selected so as to represent different electronic, lipophilic and steric environment, which would assist the aimed bioactivities. Finally, as an articular part in antiviral research, O-glycosylation of the parent 3-cyanopyridones with different sugar counterparts was taken into consideration as an important structure variation in order to investigate such structure modification on the anticipated biological activities. We have also studied the lipophilic properties of the synthesized compounds since lipophilicity plays important role in bioavailability and effectiveness of the desired activity. 2. RESULTS AND DISCUSSION 2.1. Chemistry The building blocks 2-oxo-4-aryl-6-(p-tolyl)-1,2dihydropyridine-3-carbonitrile (1a,b) were prepared by onepot multicomponent reaction of 4-methyl acetophenone,

ethyl cyanoacetate and appropriate aldehydes (4flourobenzaldehyde and 3,4-dimethoxybenzaldehyde) in the presence of excess ammonium acetate in absolute ethanol [27] (Scheme 1). Alkylation of 2-pyridone derivatives 1a,b was conducted with alkylating agents allyl/propargyl bromides dry acetone in the presence of anhydrous K2CO3 under refluxing condition for 12 h to obtain compounds 2a,b and 3a,b. Compounds 1a,b were also treated with 2-acetoxyethoxymethyl bromide and 4-bromobutylacetate in dry DMF under reflux condition for 12 h to yield the desired compounds 4a,b and 6a,b [28, 29]. The acetyl group of compound 5a,b and 6a,b was deprotected upon treatment with triethylamine in methanol at room temperature (Scheme 1). 4-(3,4-Dimethoxyphenyl)-2-thioxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (8) was obtained from the reaction of 1b with phosphorus pentasulphide in refluxing dry pyridine. Compound 8 was treated with chloroacetic acid in the presence of potassium hydroxide in refluxing absolute ethanol to afford 2-((3-cyano-4-(3,4-dimethoxyphenyl)-6-(ptolyl)pyridin-2-yl)thio)acetic acid (9). Compound 9 was further reacted with 4-amino benzenesulphonamide (sulphanilamide) and 4-amino-N-acetylbenzenesulfonamide (sulphacetamide) in the presence of tetrahydrofuran (THF), triethylamine (TEA) and ethylchloroformate [30] leading to the formation of sulphadrug 2-((3-cyano-4-(3,4-dimethoxyphenyl)-6-(p-tolyl)pyridin-2-yl)thio)-N-(4-sulfamoyl-phenyl) acetamide (10a) and N-(4-(N-acetylsulfamoyl)phenyl)-2-((3cyano-4-(3,4-dimethoxyphenyl)-6-(p-tolyl)pyridin-2-yl) thio)acetamide (10b) (Scheme 2). Reaction of 2-pyridone derivatives 1a and 1b with 2,3,4,6-tetra-O-acetyl--D-gluco/galactopyranosyl bromides in anhydrous DMF/K2CO3 afforded nucleoside 1(2`,3`,4`,6`-tetra-O-acetyl--D-glucopyranosyl-4-(4-fluorophenyl)-1-2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (11a), 1-(2`,3`,4`,6`-tetra-O-acetyl--D-glucopyranosyl4-(3,4-dimethoxyphenyl) -2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (11b) and glycosides 2-(2`,3`,4`,6`-tetraO-acetyl--D-galactopyranosyl)-4-(4-fluorophenyl)-6-(p-tolyl)nicotinonitrile (13a) and 2-(2',3',4',6'-tetra-O-acetyl--D-

Synthesis of Nucleosides and Non-nucleosides

Medicinal Chemistry, 2018, Vol. 14, No. 8 R1 R2 R1 R2 CN

O

N

CN

2a,b O

N ii 3a,b

iii R1 R1

R2

R1 R2

O

H

CN

+

+ O

O

R2

CN

i

CN

O

N

O

iv

O

N

O

H

OAc

4a,b 1a,b

a; R1 = F, R2 = H b; R1 = R2 = OCH3

2 v

vi R1 R1

R1

R2 R2

R2

CN vii

CN

CN N

O

N

O

OH 2

OAc

OH

5a,b

2

2 7a,b

O

O

N

6a,b

Reagents and reaction conditions; i: NH4OAc, abs. EtOH, reflux, 12 h; ii, iii: Allyl (or propargyl)bromide, anhyd. K2CO3, dry acetone, reflux, 12 h; iv, vi: 2-(bromomethoxy)ethyl acetate or (4-bromobutyl acetate) anhyd. K2CO3, dry DMF, reflux, 20 h; v, vii: TEA, MeOH/H2O, stirring at room temp., 24 h.

Scheme 1. Synthesis of non-nucleosides based 4,6-disubstituted-2-oxo-dihydropyridine-3-carbonitriles. O

O O

O O

CN

CN i

N

CN ii

O

N

H 1b

O

OH

S

S

N

H

O

9

8

iii

O O

CN

H

a; R1 = H b; R1 = COCH3

N N

S

H O

N S

10a,b

O

O

Reagents and reaction conditions: i: P2S5, dry pyridine, reflux, 6 h; ii: ClCH2COOH, KOH, abs. EtOH, reflux, 12 h; iii: sulphanilamide or sulphacetamide, THF, TEA/ethyl chloroformate, r.t., 2 h.

Scheme 2. Synthesis of sulfa-drugs 10a,b.

R1

793

794 Medicinal Chemistry, 2018, Vol. 14, No. 8

Ibrahim et al. R1 R2

CN

O

N a; R1 = F, R2 = H b; R1 = R2 = OCH3

11a,b; R = Ac

OR O

iv 12a,b; R = H

RO RO OR R1

R1 R2

i

R2

R1 R2 CN

N OR

OR

O

CN ii

iii

OR O

N

OR

O OR

O

H RO

15a,b; R = Ac iv

RO

1a,b

OR

OR O

O O

RO

N

CN

OR 13a,b; R = Ac

a; R1 = F, R2 = H b; R1 = R2 = OCH3

16a,b; R = H

iv 14a,b; R = H

Reagents and reaction conditions: i, ii, iii: Glucosyl, Galactosyl or Lactosyl bromide (respectively), anhyd. K2CO3, dry DMF, stirring at r.t. 48 h; iv: TEA, MeOH/H2O, stirring at r.t., 24 h. Scheme 3. Synthesis of nucleosides based 4,6-disubstituted-2-oxo-dihydropyridine-3-carbonitriles.

galactopyranosyloxy)-4-(3,4-dimethoxy-phenyl)-6-(p-tolyl) nicotinonitrile (13b) (Scheme 3). Deprotection of nucleosides and glycosides 11a,b, and 13a,b was carried out in the presence of TEA/ MeOH and few drops of water at room temperature to afford the corresponding free nucleosides and glycosides 12a,b and 14a,b, respectively (Scheme 3). Treatment of 2-pyridone derivatives 1a,b with lactosyl bromide in the same above conditions at room temperature yielded lactoside 15a,b in 58 and 68 % yields, respectively (Scheme 3). Deacetylation of lactoside 15a,b in the presence of TEA / MeOH and few drops of water gave the corresponding deprotected lactoside 16a,b in high yields 95-97 % (Scheme 3). 2.2. Biology 2.2.1. In vitro Antiviral Activity All the newly synthesized compounds 1-16 were evaluated for their in vitro antiviral activity against a panel of eighteen strains belonging to DNA- and RNA-virus types in various cell cultures employing the cytopathicity (CPE) assay [31]. Among these, eight strains of DNA-virus types were used including two strains of herpes simplex virus type 1 [HSV-1 (KOS) and HSV-1 (TK- KOS ACVr)], herpes simplex virus type 2 [HSV-2 (G)] vaccinia virus (VV), vesicular

stomatitis virus (VSV), two strains of cytomegalovirus [CMV (AD-169) and (Davis)] and two strains of varicella zoster virus [TK+ VZV OKA and TK- VZV 07-1]. In addition, ten strains of RNA-virus were utilized including vesicular stomatitis virus [VSV], coxsackie virus B4 [CV-B4], respiratory syncytial virus [RSV], parainfluenza-3 virus [PIV3], reovirus-1 [RV-1], sindbis virus [SV], punta toro virus [PTV], beside three influenza virus subtypes [influenza A H1N1, H2N3 and influenza B]. The obtained antiviral and cytotoxic potentials (EC50 and MCC; M) of twenty five compounds were recorded in Tables 1-6 in comparison with known reference antiviral agents (brivudin, cidofovir, acyclovir, ganciclovir, ribavirin, zanamivir, amantadine, and rimantadine). Concerning the antiviral potential against individual virus strains, the results recorded in Tables 1-3 described the activity against DNA-viruses in the human embryonic lung (HEL) cell culture. Table 1 illustrated that the glycosides 14b and 16a displayed considerable antiviral potential against the four tested virus strains (EC50 range 9-20 M), which was more potent than the standard brivudin against HSV-1 (TK- KOS ACV r), HSV-2 (G) and vaccinia virus (VV), with remarkably better cytotoxic effect (MCC  100 and 20 M vs 250 M, respectively). The analogs 1a, 2a,b, 3b, 5a, 7a,b, 10a, 12a, 13a,b, 14a, 15a and 16b exhibited the same range of moderate antiviral activity (EC50  20 M)

Synthesis of Nucleosides and Non-nucleosides

b

795

Antiviral activity against DNA-viruses (EC50; M)a and minimum cytotoxic concentration (MCC; M)b of the investigated compounds in HEL cell culture.

Table 1.

a

Medicinal Chemistry, 2018, Vol. 14, No. 8

Compound No.

MCC

Herpes Simplex Virus-1 (KOS)

Herpes Simplex Virus-1 TK- KOS ACV r

Herpes Simplex Virus-2 (G)

Vaccinia Virus

1a

100

20

20

20

20

1b

100

100

100

100

100

2a

100

20

20

20

20

2b

100

20

20

20

20

3a

100

100

100

100

100

3b

100

20

20

20

20

5a

100

20

20

20

20

5b

100

100

100

100

100

7a

100

20

20

20

20

7b

100

20

20

20

20

9

100

100

100

100

100

10a

100

20

20

20

20

10b

100

100

100

100

100

11a

4

4

4

4

4

11b

100

100

100

100

100

12a

100

20

20

20

20

12b

20

20

20

20

20

13a

100

20

20

20

20

13b

100

20

20

20

20

14a

100

20

20

20

20

14b

100

9

20

9

9

15a

100

20

20

20

20

15b

100

73

100

100

100

16a

20

9

20

10

9

16b

100

20

20

20

20

Brivudin

250

0.016

50

50

7.3

Cidofovir

250

2

2

1.2

17

Acyclovir

250

0.4

10

0.2

250

Ganciclovir

100

0.03

0.2

0.03

100

Concentration that reduces virus-induced cytopathogenicity by 50 % (M). Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology (M).

against the four virus types tested. Whereas the rest of the tested compounds were effective at the concentration that results in microscopically detectable alterations in normal cell morphology, therefore they were considered inactive owing to high cytotoxicity and poor selectivity. In Table 2, the analogs 2a,b, 5a, 7a,b, 12b and 16a revealed an observable antiviral activity (EC50  4 M) against the cytomegalovirus AD-169 and Davis strains in the HEL cell culture,

together with a remarkably low cytotoxic effect (EC50 20 M), when compared with ganciclovir and cidofovir (the reference standard antiviral agents). Meanwhile, the methoxylated derivatives 7b and 14b proved to be equipotent against the AD-169 strain (EC50 9 M), whereas they showed noticeable activity against the Davis strain (EC50 8 and 11 M, respectively). However, their potential is lower than both reference standard antiviral agents. Regarding

796 Medicinal Chemistry, 2018, Vol. 14, No. 8

Table 2.

a b c

Ibrahim et al.

Antiviral activity (EC50; M) a against cytomegalovirusb (two strains) and minimum cytotoxic concentration (MCC; M)c of the investigated compounds in HEL cell culture. Compound No.

MCC

AD-169 Strain

Davis Strain

1a

100

20

20

1b

100

100

100

2a

20

4

4

2b

20

4

4

3a

100

100

100

3b

100

20

20

5a

20

4

4

5b

100

20

20

7a

100

20

20

7b

100

9

8

9

100

20

20

10a

100

20

20

10b

100

20

20

11a

100

20

20

11b

100

63

100

12a

100

20

20

12b

20

4

4

13a

100

20

20

13b

100

20

20

14a

100

20

20

14b

100

9

11

15a

100

20

20

15b

100

20

20

16a

20

4

4

16b

100

20

20

Ganciclovir

100

1.5

1

Cidofovir

100

0.2

0.2

Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU). DNA-virus. Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology (M).

Table 3, compounds 1a, 2a,b, 5a, 7a,b, 11a, 12b, 13a,b, 14a,b, 15b and 16a exhibited an observable activity against both varicella zoster virus strains (VZV TK+ OKA and TK07-1) (EC50 range 3.35-20 M) and significantly low cytotoxicity (MCC range 4-20 M). In particular, the fluorinated analog 2a showed equipotent high antiviral activity against both VZV strains (EC50 1 M), together with a very low cytotoxicity (MCC 4 M). This compound proved to possess nearly two-fold and 36 times the potency of acyclovir against the TK+ OKA and TK- 07-1 strains (EC50 1 and 29 M; respectively).

Shifting to the antiviral activity against RNA-viruses in immortal HeLa cell culture, the results listed in Table 4 showed that compounds 1a, 12b, 13a, 14a and 14b exhibited a low cytotoxic effect (MCC 20 M) and a considerable antiviral activity (EC50  4 M) against vesicular stomatitis, coxsackie B4 and respiratory syncytial viruses, which was obviously better than ribavirin; the reference standard antiviral agent. Meanwhile, the antiviral potential against four RNA-viruses (parainfluenza-3, reovirus-1, sindbis and punta toro viruses) in Vero cell culture revealed that compound 2a possessed a reliable activity against the four tested viruses

Synthesis of Nucleosides and Non-nucleosides

Table 3.

Medicinal Chemistry, 2018, Vol. 14, No. 8

797

Antiviral activity (EC50; M)a against varicella zoster virusb (two strains) and minimum cytotoxic concentration (MCC; M)c of the investigated compounds in HEL cell culture.

Compound No.

MCC

TK+ VZV Strain OKA

TK- VZV Strain 07-1

1a

20

11

12

1b

100

100

20

2a

4

1

1

2b

20

4

4

3a

100

100

20

3b

100

20

20

5a

20

4

4

5b

100

20

20

7a

20

4

4

7b

20

20

4

9

100

20

20

10a

100

20

100

10b

100

100

100

11a

20

3.4

4

11b

100

53

46

12a

100

20

20

12b

20

4

4

13a

20

4

20

13b

20

4

4

14a

20

4

4

14b

20

20

4

15a

100

20

20

15b

100

63

20

16a

20

4

4

16b

100

20

20

Acyclovir

100

1

29

Brivudin

100

0.004

49

a

Effective concentration required to reduce virus plaque formation by 50%. Virus input was 20 plaque forming units (PFU). b DNA-virus. c Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.

(EC50  4 M), whereas the analog 12b proved to be highly selective to sindbis and punta toro viruses with EC50 values 1 and 2 M, respectively. Both analogs 2a and 12b were significantly more potent than ribavirin against the same viral panel (Table 5). Finally, the results depicted in Table 6 showed that compounds 5a and 16a showed the highest antiviral activity against three RNA influenza virus subtypes namely; influenza A H1N1, H2N3 and influenza B (EC50  1 M) in median Darby canine kidney (MDCK) cell culture, with a very low cytotoxicity level (MCC 4 M). Meanwhile, both analogs 5a and 16a proved to be equipotent with zanamivir against influenza A H1N1 virus, whereas they were remarkably more potent than ribavirin, amantadine, and rimantadine against the same virus. Furthermore, the same analogs showed significantly better antiviral profile than

zanamivir, ribavirin, amantadine and rimantadine against the influenza A H2N3 and influenza B viruses. Whereas, compounds 7a,b, 10a, 12b, 13a and 15a moderate antiviral activity (EC50 range 6-20 M) against the three influenza virus subtypes, with a sufficiently low cytotoxic effect (MCC 20 M). With regard to the antiviral spectrum of the investigated compounds, it could be recognized that most of the active compounds are more selective towards the inhibition of the replication of DNA- rather than the RNA-viruses. In this view, the parent fluorinated cyanopyridone 1a exhibited moderate activity against most of the tested DNA-viruses (EC50  20 M; Tables 1, 2), however it showed particular efficiency against both strains of varicella zoster virus (EC50 11 and 12 M, respectively) with a considerably low cyto-

798 Medicinal Chemistry, 2018, Vol. 14, No. 8

Antiviral activity against RNA-viruses (EC50; M)a and minimum cytotoxic concentration (MCC; M)c of the investigated compounds in HeLa cell culture.

Table 4.

a b

Ibrahim et al.

Compound No.

MCC

Vesicular Stomatitis Virus

Coxsackie Virus B4

Respiratory Syncytial Virus

1a

20

4

4

4

1b

100

100

100

100

2a

100

20

20

20

2b

100

20

20

20

3a

100

100

100

100

3b

100

20

20

20

5a

20

20

20

20

5b

100

100

100

100

7a

100

20

20

20

7b

20

20

20

20

9

100

20

20

20

10a

100

20

20

20

10b

100

100

100

100

11a

20

20

20

20

11b

100

100

100

100

12a

100

20

20

20

12b

20

4

4

4

13a

20

4

4

4

13b

20

20

20

20

14a

20

4

4

4

14b

20

4

4

4

15a

100

100

100

100

15b

100

20

20

20

16a

100

20

20

20

16b

100

20

20

20

Ribavirin

250

29

250

10

Concentration that reduces virus-induced cytopathogenicity by 50 % (M). Minimum cytotoxic concentration required to cause a microscopically detectable alteration of normal cell morphology (M).

Synthesis of Nucleosides and Non-nucleosides

Table 5.

a b

Medicinal Chemistry, 2018, Vol. 14, No. 8

799

Antiviral activity against RNA-viruses (EC50; M)a and minimum cytotoxic concentration (MCC; M)c of the investigated compounds in Vero cell culture.

Compound No.

MCC

Para-influenza 3 virus

Reovirus-1

Sindbis Virus

Punta Toro Virus

1a

100

20

20

20

20

1b

100

100

100

100

100

2a

20

4

4

4

4

2b

100

20

20

20

20

3a

100

100

100

100

100

3b

100

100

100

100

100

5a

100

20

20

20

20

5b

100

100

100

100

100

7a

100

20

20

20

20

7b

100

100

100

100

100

9

100

100

100

100

100

10a

100

100

100

100

100

10b

100

100

100

100

100

11a

100

20

20

20

20

11b

100

20

20

20

20

12a

100

20

20

20

20

12b

20

4

4

1

2

13a

20

20

20

20

20

13b

100

20

20

20

20

14a

20

20

20

20

20

14b

20

20

20

20

20

15a

100

100

100

100

100

15b

100

20

20

20

20

16a

100

20

20

20

20

16b

100

100

100

100

100

Ribavirin

250

250

250

250

250

Concentration that reduces virus-induced cytopathogenicity by 50 % (M). Minimum cytotoxic concentration required to cause a microscopically detectable alteration of normal cell morphology (M).

toxicity (MCC 20 M) (Table 3). Whereas, the allyloxypyridine derivatives 2a,b revealed considerable activity against most of the tested DNA-viruses (EC50 range 4-20 M; Tables 1-3), where the fluorinated analog 2a exhibited remarkable potency against both strains of varicella zoster virus (EC50 1 M) with a markedly low cytotoxicity (MCC 4 M) (Table 3). Meanwhile, both analogs showed moderate to weak activity against most of the tested RNA-viruses (Tables 4, 5), together with marginal or even no efficacy against the three influenza viruses (Table 6). Moreover, the fluorinated acyclic glycoside 5a showed moderate activity against most of the tested DNA-viruses (EC50 range 4-20 M; Ta-

bles 1-3). In addition, this analog showed moderate activity against the parainfluenza-3, reovirus-1, sindbis, punta toro RNA-viruses (EC50 20 M; Table 5), while it proved to be highly effective against the three types of influenza viruses tested (EC50 1 M; Table 6). However, it was deprived of any antiviral activity against the vesicular stomatitis, coxsackie virus B4, respiratory syncytial viruses (Table 4). Furthermore, although the methoxylated O-glucoside 12b lacked any antiviral activity against the three herpes simplex and vaccinia viruses (Table 1), yet it displayed considerable activity against both strains of cytomegalovirus and varicella zoster virus (EC50  4 M; Tables 2-3). Meanwhile, 12b

800 Medicinal Chemistry, 2018, Vol. 14, No. 8

Table 6.

Ibrahim et al.

Antiviral activity against the influenza RNA-viruses (EC50; M)a and minimum cytotoxic concentration (MCC; M)b of the investigated compounds in MDCK cell culture.

Compound No.

MCC

Influenza A H1N1 Subtype

Influenza A H3N2 Subtype

Influenza B

1a

100

20

20

20

1b

100

100

100

100

2a

20

20

20

20

2b

100

20

20

20

3a

100

100

100

100

3b

100

20

20

20

5a

4

1

1

1

5b

100

100

100

100

7a

20

4

4

4

7b

20

4

4

4

9

100

100

100

100

10a

20

4

4

4

10b

100

100

100

100

11a

100

20

20

20

11b

100

100

100

100

12a

100

100

100

100

12b

20

4

4

4

13a

20

4

4

4

13b

100

20

20

20

14a

100

100

100

100

14b

100

100

100

100

15a

20

9

20

20

15b

20

20

20

20

16a

4

1

1

1

16b

4

4

4

4

Zanamivir

100

1

4

2

Ribavirin

100

7

10

10

Amantadine

200

200

1

200

Rimantadine

200

4

0.06

200

a

Concentration that reduces virus-induced cytopathogenicity by 50 % (M). c Minimum cytotoxic concentration required to cause a microscopically detectable alteration of normal cell morphology (M).

showed moderate activity against most of the tested RNAviruses (Tables 4-6), with particular effectiveness against sindbis and punta toro viruses (EC50 1 and 2 M, respectively; Table 5). On the other hand, the methoxylated O-glalactoside 14b exhibited special activity against the three herpes simplex and vaccinia viruses (EC50 range 9-20 M; Table 1), two

cytomegalovirus subtypes (EC50 8.94 and 10.94 M, respectively; Table 2), in addition to the varicella zoster virus TK07-1 strain (EC50  4 M; Table 5). Meanwhile, 14b showed moderate activity against three RNA viruses namely; vesicular stomatitis, coxsackie B4 and respiratory syncytial viruses (Table 4). Finally, the methoxylated O-lactoside 16b revealed special high activity against two herpes simplex and

Synthesis of Nucleosides and Non-nucleosides

Table 7.

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801

Calculated logP and logS of nucleosides and non-nucleosides based 4,6-disubstituted-2-oxo-dihydropyridine-3carbonitriles. Compound No.

logP

logS

1a

4.35

-4.70

1b

4.70

-4.88

2a

5.55

-5.24

2b

4.90

-5.14

3a

4.70

-4.88

3b

4.35

-4.70

5a

3.62

-4.86

5b

3.51

-4.83

7a

3.24

-4.79

7b

2.84

-4.86

9

4.25

-5.03

10a

4.73

-5.28

10b

4.60

-5.32

11a

4.05

-5.36

11b

3.94

-5.21

12a

1.67

-4.01

12b

1.70

-3.86

13a

4.59

-5.25

13b

4.72

-5.13

14a

2.22

-4.02

14b

2.19

-3.95

15a

3.41

-4.83

15b

3.25

-5.22

16a

0.89

-3.48

16b

0.82

-3.26

vaccinia viruses (EC50 range 9-10 M; Table 1), two cytomegalovirus subtypes (EC50  4 M; Table 2), in addition to the two varicella zoster virus strains (EC50  4 M; Table 3). Meanwhile, 16b showed moderate activity against three RNA viruses namely; vesicular stomatitis, coxsackie B4 and respiratory syncytial viruses (Table 4) and a significant potential against the three influenza virus subtypes (EC50 1 M; Table 6). A deep insight into the structures of the active compounds it could be recognized that the target compounds represent four series namely; the O-alkyl 2-5 and N-alkyl 6-7 series (Scheme 1), the S-alkyl series 8-10 (Scheme 2), and the O-glycosyl derivatives 11-16 (Scheme 3). In general, the order of activity goes mostly in the following order: Oglycosyl > O-alkyl > N-alkyl > S-alkyl derivatives. Additionally, among the O- and N-alkyl derivatives, the 4-fluoro

atom (R1 = F, R2 = H; Scheme 1) sounds to be the most favorable substituent, whereas remarkable antiviral activity was ascribed to the methoxylated O-glycosyl derivatives (R1 = R2 = OCH3; Scheme 3). In this context, regarding scheme 1, the parent fluorinated cyanopyridone 1a (R1 = F, R2 = H) showed moderate broad spectrum against most of the tested DNA- and RNA-viruses. O-alkylation with allyl moiety furnished the fluorinated allyloxy derivative 2a (R1 = F, R2 = H) with better antiviral activity against DNA-viruses especially the varicella zoster virus and a markedly low cytotoxicity. Whereas, O-alkylation with propargyl counterpart as in 3a,b resulted in a dramatic reduction or even total abolishment in the overall antiviral activity. However, although the fluorinated acyclic O-glycoside 5a exhibited a limited improvement in the antiviral spectrum when compared with the parent cyanopyridone 1a, yet a remarkable effectiveness against the three types of influenza viruses tested was achieved. On

802 Medicinal Chemistry, 2018, Vol. 14, No. 8

the contrary, N-alkylation with hydroxy butyl group as in 7a,b resulted in a marked reduction in the overall antiviral potential and spectrum, except the methoxylated analog 7b which proved to be highly active against both strains of cytomegalovirus. Shifting to scheme 2, bioisosteric conversion of the 2-one functionality to the 2-thione group as in 8 and further S-alkylation with an acetic acid moiety as in 9 or the introduction of a sulphonamido moiety as in 10a,b resulted in a dramatic drop or total loss of activity. On the other hand, O-glycosylation of the parent cyanopyridones 1a,b with different sugar counterparts 11-16 (Scheme 3) led to a noticeable improvement if the antiviral potential and/or spectrum. In particular, the methoxylated glycosides 12b, 14b and 16b (R1 = R2 = OCH3) proved to be the most active members of this family. Compared with the O-glucoside 12b and the Ogalactoside 14b, the O-lactoside congener 16b proved to the most active member as it possessed high antiviral potential and spectrum against most of the tested DNA-viruses, beside a distinctive activity against the three influenza RNAviruses. The lipophilic and hydrophilic characters of a drug play important role in producing a biological effect. Hydrophobic drugs with high partition coefficients are preferentially distributed to hydrophobic lipid bilayers of cells while hydrophilic drugs with low partition coefficients, preferentially are found in hydrophilic blood serum. Hydrophobicity/lipophilicity helps in determining the distribution of the drug in the biological system. In general hydrophobic scaffolds are important in biological activity [32-34]. The lipophilicity and water solubility of the compounds expressed as logP and logS are the main predictors of the activity. The logP and logS are the measure of hydrophobicity/lipophilicity and was calculated using ALOGPS 2.1 program. The results are given in Table 7. The calculated values of logP and logS for the synthesized nucleosides and nonnucleosides based 4,6-disubstituted-2-oxo-dihydropyridine3-carbonitriles looks promising and could be utilized to develop potential antiviral agents. 3. EXPERIMENTAL 3.1. Chemistry All melting points are uncorrected and were measured using an Electro thermal IA 9100 apparatus. The IR spectra (KBr discs) were recorded on a Pye Unicam Sp-3 - 300 or a Shimadzu FTIR 8101 PC infrared spectrophotometer (Cairo University, Cairo, Egypt). The operation frequency was 400 MHz for 1H and 100 MHz for 13C NMR using BRUKER 400 MHz spectrometer at Zagazig University, Nucleic Acid Center Research. For some compounds, the operation frequency was 500 MHz for 1H and 125 MHz for 13C NMR using JEOL Delta-2 NMR Spectrometer 500 MHz at Mansoura University, Egypt. The coupling constants (J) are given in Hertz. The chemical shifts are expressed on the  (ppm) scale using TMS as the standard reference. Elemental analyses were determined on a Perkin-Elmer 240 (Microanalysis Center, Cairo University, Egypt).

Ibrahim et al.

3.2. General Procedure for the Synthesis of Heterocyclic Bases 1a,b A mixture of p-methyl acetophenone (10 mmol), ethyl cyanoacetate (10 mmol), appropriate aldehydes, namely (4fluorobenzaldehyde and 3,4-dimethoxybenzaldehyde (10 mmol), and ammonium acetate (80 mmol) in absolute ethanol (20 mL) was heated under reflux for 12 hours, the formed product was filtered off and recrystallized from ethanol / acetic acid (1:2) ratio. 3.3. 4-(4-Fluorophenyl)-2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (1a) Colorless crystals; Yield 36 %; m. p. 284-286 oC. IR (KBr): 3441 cm-1 (NH), 2222 cm-1 (CN) and 1699 cm-1 (C=O, amide). 1H NMR (DMSO-d6):  = 2.37 (s, 3H, CH3), 6.81 (s, 1H, pyridone-5), 7.33 (d, 2H, J = 8.0 Hz, Ar-H), 7.39 (d, 2H, J = 8.8 Hz, Ar-H), 7.81-7.86 (m, 4H, Ar-H), 12.40 (s, 1H, NH). 13C NMR (DMSO-d6):  21.1 (CH3), 105.5, 111.5, 111.8, 117.0 (CN), 121.4, 127.6, 128.1, 129.4, 141.2, 148.5, 150.7, 159.4, 162.2 and 172.0 (Ar-C and C=O). Anal. Calcd for C19H13FN2O (304.32): C, 74.99; H, 4.31; N, 9.21. Found: C, 75.06; H, 4.26; N, 9.25. 3.4. 4-(3,4-Dimethoxyphenyl)-2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (1b) Yellow powder; Yield 34 %; m. p. 224-226 oC. IR (KBr): 3253 cm-1 (NH), 2217 cm-1 (CN) and 1694 cm-1 (C=O, amide). 1H NMR (DMSO-d6):  = 2.38 (s, 3H, CH3), 3.84 (s, 6H, 2OCH3), 6.81 (s, 1H, pyridone-5), 7.13 (d, 1H, J = 8.40 Hz, Ar-H), 7.34-7.39 (m, 4H, Ar-H), 7.80 (d, 2H, J = 7.60 Hz, Ar-H), 12.33 (s, 1H, NH). 13C NMR (DMSO-d6):  21.0 (CH3), 55.7 (2 OCH3), 105.5 (Ar-C), 117.1 (CN), 121.5, 127.7, 128.2, 129.5 (Ar-C), 141.3 (Ar-C), 150.7, 150.9 (Ar-C), 159.5 (C=O), 162.2 (Ar-C). Anal. Calcd for C21H18N2O3 (346.38): C, 72.82; H, 5.24; N, 8.09. Found: C, 72.75; H, 5.30; N, 8.13. 3.5. General Procedure for Alkylation (2a,b and 3a,b) A mixture of 1a,b (10 mmol) with appropriate alkylating agents namely, allyl/propargyl bromides was added to dry acetone (20 mL) in the presence of anhydrous K2CO3 (11 mmol), the reaction mixture was heated under reflux for 12 hours, cooled, and pour onto ice water, the precipitate was collected, dry and crystallized from methanol. 3.6. 2-(Allyloxy)-4-(4-fluorophenyl)-6-(p-tolyl)nicotinonitrile (2a) Yellow powder; Yield 87.5 %; m. p. 160-162 oC. IR (KBr): 2218 cm-1 (CN). 1H NMR (DMSO-d6):  = 2.38 (s, 3H, CH3), 5.12 (d, 2H, J = 5.20 Hz, OCH2), 5.32 (d, 1H, J = 10.8 Hz, H-3'), 5.51 (d, 1H, J = 17.2 Hz, H-3"), 6.16-6.20 (m, 1H, H-2'), 7.34 (d, 2H, J = 8.0 Hz, Ar-H), 7.44-7.49 (m, 2H, Ar-H), 7.79 (s, 1H, pyridone-5), 7.83-7.88 (m, 2H, ArH), 7.15 (d, 2H, J = 8.0 Hz, Ar-H). 13C NMR (DMSO-d6):  21.0 (CH3), 67.3 (CH2), 91.9 (Ar-C), 113.4 (CN), 115,7 (Ar-C), 115.9, 118.1 (=CH), 127.4, 129.5, 132.9, 140.7 (=CH), 155.2 (Ar-C), 157.3, 162.2, 163.5, 164.1. Anal. Calcd for C22H17FN2O (344.38): C, 76.73; H, 4.98; N, 8.13. Found: C, 76.73; H, 4.98; N, 8.13.

Synthesis of Nucleosides and Non-nucleosides

3.7. 2-(Allyloxy)-4-(3,4-dimethoxyphenyl)-6-(p-tolyl)nicotinonitrile (2b) Colorless powder; Yield 52 %; m. p. 166-167 oC. IR (KBr): 2211 cm-1 (CN). 1H NMR (DMSO-d6):  = 2.38 (s, 3H, CH3), 3.84 (3H, OCH3), 3.86 (3H, OCH3), 5.10 (d, 2H, J = 4.40 Hz, OCH2), 5.32 (d, 1H, J = 10.8 Hz, H-3'), 5.50 (d, 1H, J = 17.2 Hz, H-3"), 6.13 - 6.19 (m, 1H, H-2'), 7.15 (d, 1H, J = 8.0 Hz, Ar-H), 7.34-7.38 (m, 4H, Ar-H), 7.77 (s, 1H, pyridone-5), 8.14 (d, 2H, J = 7.60 Hz, Ar-H). 13C NMR (DMSO-d6):  21.01(CH3), 55.7 (2 OCH3), 67.2 (CH2), 91.5(Ar-C), 113.2 (CN), 118.0 (=CH), 121.6 (CN), 127.4, 129.5, 133.1, 134.0, 140.6 (=CH), 148.7 (Ar-C), 150.4, 156.1, 157.0, 163.7. Anal. Calcd for C24H22N2O3 (386.44): C, 74.59; H, 5.74; N, 7.25. Found: C, 74.68; H, 5.69; N, 7.18. 3.8. 4-(4-Fluorophenyl)-2-(prop-2-yn-1-yloxy)-6-(p-tolyl) nicotinonitrile (3a) Colorless powder; Yield 76 %; m. p. 175-176 oC. IR (KBr): 2218 cm-1 (CN). 1H NMR (DMSO-d6):  = 2.38 (s, 3H, CH3), 3.62 (d, 1H, J = 2.0 Hz, CH), 5.28 (s, 2H, OCH2), 7.34-7.46 (m, 4H, Ar-H), 7.83-7.94 (m, 3H, Ar-H and pyridone-5), 8.19-8.26 (m, 2H, Ar-H). 13C NMR (DMSO-d6): 21.0 (CH3), 54.7 (CH2), 78.1 (CCH), 78.9, 92.0 (Ar-C), 114.0 (CN), 115.1 (Ar-C), 115.8, 116.0, 127.6, 129.6, 131.2, 131.2, 155.4, 157.2, 162.2, 162.6, 164.2. Anal. Calcd for C22H15FN2O (342.37): C, 77.18; H, 4.42; F, 5.55. Found: C, 77.24; H, 4.38; F, 5.52. 3.9. 4-(3,4-Dimethoxyphenyl)-2-(prop-2-yn-1-yloxy)-6-(ptolyl)nicotinonitrile (3b) Colorless powder; Yield 68 %; m. p. 156-158 oC. IR (KBr): 2214 cm-1 (CN). 1H NMR (DMSO-d6):  = 2.38 (s, 3H, CH3), 3.61 (s, 1H, CH), 3.85 (3H, OCH3), 3.86 (3H, OCH3), 5.26 (s, 2H, OCH2), 7.14 (d, 1H, J = 8.0 Hz, Ar-H), 7.34-7.39 (m, 4H, Ar-H), 7.81 (s, 1H, pyridone-5), 8.17 (d, 2H, J = 7.60 Hz, Ar-H). 13C NMR (DMSO-d6):  21.0 (CH3), 54.5 (CH2), 55.7 (2 OCH3), 77.9 (CCH), 78.8, 92.0 (Ar-C), 111.7, 112.2, 114.0 (CN), 115.5 (Ar-C), 121.5, 127.5, 127.9, 129.5, 133.8, 140.7, 148.7, 150.4, 156.3, 156.9, 162.8. Anal. Calcd for C24H20N2O3 (384.43): C, 74.98; H, 5.24; N, 7.29. Found: C, 74.91; H, 5.19; N, 7.35. 3.10. General Procedure for the Synthesis of Acyclic Nucleosides (4a,b, and 6a,b) A mixture of 1a,b (10 mmol) with an appropriate acyclic sugar namely, 4-bromobutylacetate bromide and (2acetoxyethoxy)methyl bromide and was added to dry DMF (20 mL) in the presence of anhydrous K2CO3 (11 mmol), the reaction mixture was heated under reflux for 20 hours and followed by TLC, cooling then poured onto ice water the formed precipitate was collected by filtration, and separated by silica gel chromatography (200-400 mesh) using CH2Cl2 / pot. ether 40/60 (in ratio 80: 20) as eluent. 3.11. 2-(((3-Cyano-4-(4-fluorophenyl)-6-(p-tolyl)pyridin2-yl)oxy)methoxy)ethyl acetate (4a) Colorless powder; Yield 75 %; m. p. 102-104 oC. IR (KBr): 2216 cm-1 (CN), 1732 cm-1 (C=O, CH3 CO). 1 H

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803

NMR (DMSO-d6):  = 2.02 (s, 3H, CH3CO), 2.38 (s, 3H, CH3), 3.32 (t, 2H, J = 5.80 Hz, OCH2 (H-3')), 4.46 (t, 2H, J = 5.80 Hz, CH2OCO (H-4')), 4.79 (s, 2H, NCH2O (H-1')), 7.33-8.16 (m, 9H, Ar-H and pyridone-5). 13C NMR (DMSOd6):  20.57, 20.91 (2CH3), 62.0 (C-3'), 64.9 (C-4'), 91.9 (C1'), 113.4, 115.1, 115.6, 115.8, 127.3, 129.4, 131.0, 132.1, 133.6, 140.7, 155.1, 157.1, 161.9, 164.3, 170.2 (Ar-C, CN and C=O). Anal. Calcd for C24H21FN2O4 (420.43): C, 68.56; H, 5.03; N, 6.66. Found: C, 68.49; H, 5.06; N, 6.70. 3.12. 2-(((3-Cyano-4-(3,4-dimethoxyphenyl)-6-(p-tolyl) pyridin-2-yl)oxy)methoxy)ethyl acetate (4b) Pale yellow powder; Yield 70 %; m. p. 160-162 oC. IR (KBr): 2217 cm-1 (CN), 1733 cm-1 (C=O, CH3CO). 1 H NMR (DMSO-d6):  = 2.01 (s, 3H, CH3CO), 2.38 (s, 3H, CH3), 3.32 (t, 2H, J = 5.76 Hz, OCH2 (H-3')), 3.85 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.45 (t, 2H, J = 5.76 Hz, CH2OCO (H-4')), 4.79 (s, 2H, NCH2O (H-1')), 7.33-8.16 (m, 9H, Ar-H and pyridone-5). 13C NMR (DMSO-d6):  20.57, 20.91 (2 CH3), 56.4 (2 OCH3), 62.2 (C-3'), 64.9 (C-4'), 91.9 (C-1'), 95.3, 113.4 (CN), 115.1, 115.6, 115.8, 127.3, 129.4, 131.0, 132.1, 133.6, 140.7, 155.1, 157.1, 161.9, 164.3, 170.2 (Ar-C and C=O). Anal. Calcd for C26H26N2O6 (462.49): C, 67.52; H, 5.67; N, 6.06. Found: C, 67.46; H, 5.72; N, 6.10. 3.13. 4-(3-Cyano-4-(4-fluorophenyl)-2-oxo-6-(p-tolyl)pyridin-1(2H)-yl)butylacetate (6a) Colorless powder; Yield 65 %; m. p. 140-142 oC. IR (KBr): 2214 cm-1 (CN), 1726 cm-1 (C=O, CH3CO), 1645 cm-1 (C=O, amide). 1H NMR (DMSO-d6):  = 1.78-1.88 (m, 2H, H-3'), 1.88-1.96 (m, 2H, H-2'), 1.98 (s, 3H, CH3CO), 2.38 (s, 3H, CH3), 4.09 (t, 2H, J = 6.0 Hz, NCH2 (H-1')), 4.60 (t, 2H, J = 5.6 Hz, CH2OCO (H-4')), 7.33-7.45 (m, 4H, Ar-H), 7.76 (s, 1H, pyridone-5), 7.81-7.98 (m, 2H, Ar-H), 8.14 (d, 2H, J = 8.0 Hz, Ar-H). 13C NMR (DMSO-d6):  = 20.65, 20.92 (CH3CO and CH3), 24.8, 24.9 (2CH2), 63.4 (NCH2), 66.5 (CH2OCO), 91.8, 113.1, 115.6, 127.3, 129.4, 130.9, 131, 0, 132.2, 133.8, 140.6, 155.0, 157.2, 161.8, 164.3, 170.3 (Ar-C, CN and 2C=O). Anal. Calcd for C25H23FN2O3 (418.46): C, 71.76; H, 5.54; N, 6.69. Found: C, 71.68; H, 5.59; N, 6.73. 3.14. 4-(3-Cyano-4-(3,4-dimethoxyphenyl)-2-oxo-6-(ptolyl)pyridin-1(2H)-yl)butyl acetate (6b) Colorless powder; Yield 72 %; m. p. 100-102 oC. IR (KBr): 2215 cm-1 (CN), 1733 cm-1 (C=O, CH3CO), 1635 cm-1 (C=O, amide). 1H NMR (DMSO-d6):  = 1.79-1.87 (m, 2H, H-3'), 1.88-1.97 (m, 2H, H-2'), 1.99 (s, 3H, CH3CO), 2.38 (s, 3H, CH3), 3.85 (s, 6H, 2OCH3), 4.10 (t, 2H, J = 6.20 Hz, NCH2 (H-1')), 4.58 (t, 2H, J = 5.80 Hz, CH2OCO (H4')), 7.14 (d, 1H, J = 8.0 Hz, Ar-H), 7.35-8.15 (m, 7H, Ar-H and pyridone-5-H). 13C NMR (DMSO-d6):  20.9 (CH3), 20.7 ((CH3 CO), 24.9, 25.0 (2CH2), 55.7, 56.0 (2 OCH3), 63.6 (NCH2), 115.5 (CN), 121.5 (Ar-C), 121.8, 127.7, 129.5, 141.3, 147.5, 148.6, 162.2, 170.4 (Ar-C, CN and 2C=O). Anal. Calcd for C27H28N2O5 (460.52): C, 70.42; H, 6.13; N, 6.08. Found: C, 70.35; H, 6.09; N, 6.13.

804 Medicinal Chemistry, 2018, Vol. 14, No. 8

Ibrahim et al.

3.15. General Procedure for Deacetylation of Acyclo Nucleosides (5a,b, and 7a,b) A mixture of synthesized acyclo nucleosides (4a,b, and 6a,b) (10 mmol) in methanol (20 mL), triethylamine (1 mL) and few drops of water were stirred overnight at room temperature, and then the solvent was removed under reduced pressure. The residue was crystallized from methanol to produced (5a,b, and 7a,b). 3.16. 4-(4-Fluorophenyl)-2-((2-hydroxyethoxy)methoxy)6-(p-tolyl)nicotinonitrile (5a) o

Colorless powder; Yield 80 %; m. p. 122-123 C. IR (KBr): 3412 cm-1 (OH), 2218 cm-1 (CN). 1H NMR (DMSOd6):  = 2.34 (s, 3H, CH3), 3.32 (t, 2H, CH2, H-3’), 3.83 (t, 2H, CH2, H-4’), 4.61 (s, 2H, CH2, OCH2O, H-1'), 4.93 (t, 1H, OH, exchange with D2O), 7.34-7.43 (m, 4H, Ar-H), 7.77 (s, 1H, pyridone-5), 7.81-7.93 (m, 2H, Ar-H), 8.14 (d, 2H, J = 8.20 Hz, Ar-H). 13C NMR (DMSO-d6):  21.0 (CH3), 29.0 (CH2), 59.1 (CH2), 68.7 (CH2), 92.0, 113.3 (CN), 115.8 (Ar-C), 116.0, 127.4, 129.6, 131.0, 131.2, 140.7, 155.3, 157.3, 164.2. Anal. Calcd for C22H19FN2O3 (378.40): C, 69.83; H, 5.06; N, 7.40. Found: C, 69.77; H, 5.09; N, 7.35. 3.17. 4-(3,4-Dimethoxyphenyl)-2-((2-hydroxyethoxy)methoxy)-6-(p-tolyl)nicotine-nitrile (5b) Colorless powder; Yield 94 %; m. p. 140-142 oC. IR (KBr): 3423 cm-1 (OH), 2213 cm-1 (CN). 1H NMR (DMSOd6):  = 2.33 (s, 3H, CH3), 2.90 (t, 2H, CH2, H-3’), 3.85-3.87 (m, 8H, CH2 (H-4’), 2 OCH3,), 4.60 (s, 2H, OCH2O, H-1'), 4.93 (t, 1H, OH, exchange with D2O), 7.16-8.15 (m, 8H, ArH and pyridone-5). 13C NMR (DMSO-d6):  21.3 (CH3), 56.1 (2OCH3), 59.1 (CH2), 68.7 (CH2), 92.0, 114.3 (CN), 115.7 (Ar-C), 116.0, 127.3, 129.6, 131.0, 131.2, 140.7, 155.3, 157.3, 164.2. Anal. Calcd for C24H24N2O5 (420.46): C, 68.56; H, 5.75; N, 6.66. Found: C, 68.49; H, 5.78; N, 6.61. 3.18. 4-(4-Fluorophenyl)-1-(4-hydroxybutyl)-2-oxo-6-(ptolyl)-1,2-dihydropyridine-3-carbonitrile (7a) Pale yellow powder; Yield 90 %; m. p. 160-162 oC. IR (KBr): 3429 cm-1 (OH), 2219 cm-1 (CN), 1631 cm-1 (C=O, amide). 1H NMR (DMSO-d6):  = 1.78-1.86 (m, 2H, H-3'), 1.89-1.92 (m, 2H, H-2'), 2.38 (s, 3H, CH3), 4.50 (t, 2H, CH2OH (H-4')), 4.62 (t, 2H, NCH2 (H-1')), 5.34 (t, 1H, OH, exchange with D2O), 7.34-7.45 (m, 4H, Ar-H), 7.77 (s, 1H, pyridone-5), 7.80 (m, 2H, Ar-H), 8.13 (d, 2H, J = 8.20 Hz, Ar-H). 13C NMR (DMSO-d6):  21.3 (CH3), 26.2 (CH2), 31.3 (CH2), 60.7 (CH2), 92.0, 113.3 (CN), 115.8 (Ar-C), 116.0, 127.4, 129.6, 131.0, 131.2, 141.8, 156.4, 158.0, 163.4. Anal. Calcd for C23H21FN2O2 (376.42): C, 73.39; H, 5.62; N, 7.44. Found: C, 73.31; H, 5.59; N, 7.39. 3.19. 4-(3,4-Dimethoxyphenyl)-1-(4-hydroxybutyl)-2-oxo6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (7b) Yellow powder; Yield 90 %; m. p. 160-162 oC. IR (KBr): 3417 cm-1 (OH), 2216 cm-1 (CN) and 1635 cm-1 (C=O, amide). 1H NMR (DMSO-d6/D2O):  = 1.60-1.69 (m, 2H, H-3'), 1.82-1.91 (m, 2H, H-2'), 2.35 (s, 3H, CH3), 3.46 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.83 (t, 2H, CH2OH (H-4')), 4.54 (t, 2H, N-CH2 (H-1')), 5.34 (t, 1H, OH, exchange with

D2O), 7.13 (d, 1H, J = 8.0 Hz, Ar-H), 7.13-8.09 (m, 7H, ArH and pyridone-5-H). 13C NMR (DMSO-d6):  21.0 (CH3), 25.2 (CH2), 29.0, 55.7 (OCH3), 55.8, 60.4 (CH2O), 112.7 (Ar-C), 112.2, 112.9, 115.8 (CN), 121.5 (Ar-C), 121.5, 127.4, 128.0, 129.5, 140.5, 148.7, 164.2 (C=O). Anal. Calcd for C25H26N2O4 (418.48): C, 71.75; H, 6.26; N, 6.69. Found: C, 71.78; H, 6.29; N, 6.68. 3.20. 4-(3,4-Dimethoxyphenyl)-2-thioxo-6-(p-tolyl)-1,2dihydropyridine-3-carbonitrile (8) A mixture of 4-(3,4-dimethoxyphenyl)-2-oxo-6-(ptolyl)-1,2-dihydro-pyridine-3-carbonitrile (1b) (0.01mol) and P2S5 (0.01mol) was refluxed in dry pyridine (20 ml) for 6 hrs. The solvent was evaporated and the residue was treated with dil. acetic acid. The precipitate was filtered off and crystallized from absolute ethanol to give (8). As a yellow powder; Yield 51%; m. p. 198-200˚C. IR (KBr): 3431cm-1 (NH), 2217 cm-1 (CN) and 1263 cm-1 (C=S). Anal. Calcd for C21H18N2O2S (362.44); C, 69.59; H, 5.01; N, 7.73. Found C, 69.60; H, 5.11; N, 7.75. 3.21. 2-((3-Cyano-4-(3,4-dimethoxyphenyl)-6-(p-tolyl)pyridin-2-yl)thio)acetic acid (9) This intermediate was prepared through the S-alkylation reaction using chloroacetic acid to alkylate the sulfhydroxyl group of (8). A solution of (8) (14.7 mmol) in ethanol (30 mL), KOH (14.7 mmol) and chloroacetic acid (14.7 mmol) was added and the mixture was refluxed for 12 hours. The hot mixture was filtered and the ethanolic solution was evaporated under reduced pressure. The residue was dissolved in distilled water, acidified with diluted hydrochloric acid to (pH = 3). The precipitate was collected by filtration, washed with cold distilled water and dried in an oven at 4045oC to provide (9), which crystallized from ethanol. Yellow powder; Yield 62%; m. p. 120 ˚C. IR (KBr): 3427 cm-1 (OH), 2214 cm-1 (CN) and 1710 cm-1 (C=O, acid). 1H NMR (DMSO-d6):  = 1.90 (s, 3H, CH3), 3.86, 3.86 (2s, 6H, 2OCH3), 4.15 (s, 2H, SCH2), 7.14 (d, 2H, J = 8.4 Hz, Ar-H), 7.33 (d, 1H, J = 7.8 Hz, Ar-H), 7,78 (d, 1H, J = 7.69 Hz, ArH), 7.85 (s, 1H, Pyridone-H-5), 8.15 (d, 2H, J = 8.0 Hz, ArH), 12.51(br, 1H, OH, exchange with D2O). Anal. Calcd for C23H20N2O4S (420.48): C, 65.70; H, 4.79; N, 6.66. Found: C, 65.73; H, 4.80; N, 6.63. 3.22. General Procedure for the Synthesis of Sulpha Drugs (10a,b) An anhydrous solution of alkylated (9) (11.4 mmol.) and TEA (11.4 mmol.) in THF (30 mL) was cooled to -10 °C. To this solution, an aliquot of ethyl chloroformate (11.4 mmol.) was added dropwise with continuous stirring. The resulting mixture was left for 30 min., with continuous stirring at 0°C. A cold aqueous solution (10 mL) of sulfa drug (sulphanilamide, sulfacetamide) (11.4 mmol.) and TEA (11.4 mmol.) was added to the above mixture. The final mixture was vigorously stirred for 2 hrs at room temperature, diluted with water (30 mL) and then was extracted with diethyl ether (2 20 mL). The aqueous phase was acidified with dilute HCl to pH = 3, and extracted with ethyl acetate (3  20 mL). The extracted were pooled together, dried over anhydrous sodium sulfate and then evaporated under reduced pressure. The

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residue was treated with petroleum ether (60 / 80), the precipitate was collected, dried and crystallized from ethanol to give (10a,b).

162.0, 164.5, 169.3, 169.7, 169.8 and 170.1 (Ar-C, CN and 5 C=O). Anal. Calcd for C33H31FN2O10 (634.61): C, 62.46; H, 4.92; N, 4.41. Found: C, 62.53; H, 4.97; N, 4.37.

3.23. 2-((3-Cyano-4-(3,4-dimethoxyphenyl)-6-(p-tolyl)pyridin-2-yl)thio)-N-(4-sulfamoyl-phenyl)acetamide (10a)

3.27. 2-(2',3',4',6'-Tetra-O-acetyl--D-glucopyranosyloxy)-4-(3,4-dimethoxyphenyl)-6-(p-tolyl) nicotinonitrile (11b)

Yellow powder; Yield: 67 %; m. p. 170-172˚C. IR (KBr): 3445 cm-1 (br, NH and NH2), 2215 cm-1 (CN), 1711 cm-1 (C=O) and 1263 cm-1 (asym. SO2). 1H NMR (DMSO-d6):  = 2.38 (s, 3H, CH3), 3.85, 3.86 (2s, 6H, 2OCH3), 4.16 (s, 2H, SCH2), 4.26 (s, 2H, NH2), 7.16-8.16 (m, 12H, Ar-H), 12.89 (br, 1H, NH). Mass Spectrometry: M+1 (m/e) = 574.0 (3.8%) as parent ion and m/e = 245.05 (22.8%), 375.05 (100%) base peak, 376.0 (28.5%). Anal. Calcd for C29H26N4O5S2 (574.67): C, 60.61; H, 4.56; N, 9.75. Found: C, 60.48; H, 4.49; N, 9.70. 3.24. N-(4-(N-Acetylsulfamoyl)phenyl)-2-((3-cyano-4-(3,4dimethoxyphenyl)-6-(p-tolyl)pyridin-2-yl)thio)acetamide (10b) Brown powder; Yield: 55 %; m. p.148-150˚C. IR (KBr): 3488 cm-1 (2NH), 2216 cm-1 (CN), 1712 cm-1 (2C=O) and 1260 cm-1 (asym. SO2). 1H NMR (DMSO-d6):  = 2.28 (s, 3 H, COCH3 ), 2.38 (s, 3H, CH3), 3.85, 3.86 (2s, 6H, 2OCH3), 4.16 (s,2H, SCH2), 7.16 (d, 2H, J = 8.0 Hz, Ar-H), 7.34 (m, 5H, Ar-H), 7.87-7.92 (m, 1H, Ar-H), 7.96 (d, 1H, J = 8.2 Hz, Ar-H), 8.16-8.21 (m, 3H, Ar-H) and 12.89 (br, 2H, 2NH). Mass Spectrometry: M+1 (m/e) = 617.0 (7.16%) as parent ion and m/e = 91.05 (50.79 %), 345.05 (53.43%), 361.05 (54.76%), 362.05 (70.55%), 375.0 (85.44%) as base peak, 376.0 (48.69%). Anal. Calcd for C31H28N4O6S2 (616.71): C, 60.37; H, 4.58; N, 9.08. Found: C, 60.29; H, 4.53; N, 9.03. 3.25. General Procedure for Synthesis Nucleosides (11a,b, 13a,b and 15a,b)

of

Cyclic

A solution of 2,3,4,6-tetra-O-acetyl--D-gluco/galactopyranosyl bromides, and (10 mmol) in dry DMF (20 mL) was added to a solution of compounds 1a,b (10 mmol) in dry DMF (10 mL) in the presence of KOH (11 mmol), the reaction mixture was stirred at room temperature 48 hrs and followed by TLC and poured onto ice water, the precipitate was collected, dried and then the formed solid product was separated by silica gel chromatography (200-400 mesh) using CH2Cl2 as eluent. 3.26. 4-(4-Fluorophenyl)-2-(2',3',4',6'-tetra-O-acetyl--Dglucopyranosyloxy)-6-(p-tolyl) nicotinonitrile (11a) Yellow powder; Yield 42 %; m. p. 160-162 oC. IR (KBr): 2223 cm-1 (CN), 1752 cm-1 (C=O, CH3CO). 1H NMR (DMSO-d6):  = 1.95, 1.98, 2.0, 2.02 (4s, 12H, 4CH3CO), 2.37 (s, 3H, CH3), 4.01 (dd, 1H, J5',6' = 4.8, J6,6 = 12.80 Hz, H-6'), 4.13 (dd, 1H, J5,6 = 4.0, J6,6 = 12.80 Hz, H-6''), 4.71 (m, 1H, H-5'), 4.89 (t, 1H, J = 9.6 Hz, H-4'), 5.21 (dd, 1H, J2,3 = 9.2, J1,2 = 8.4 Hz, H-2'), 5.35 (dd, 1H, J2,3 = 9.2, J3,4 = 9.6 Hz, H-3'), 6.84 (d, 1H, J1,2 = 8.4 Hz, H-1'), 6.81 (s, 1H, pyridone-5), 7.32 (d, 2, J = 7.6 Hz, Ar-H), 7.40-7.79 (m, 6H, Ar-H). 13C NMR (DMSO-d6):  = 20.4, 20.5, 20.6, 20.9, 21.0 (4CH3CO and CH3), 62.1 (C-6), 66.3 (C-4), 69.3 (C3), 70.8 (C-2), 72.3 (C-5), 89.0 (C-1), 105.6, 115.7, 115.9, 116.6, 127.6, 129.5, 130.7, 130.8, 132.5, 141.4, 151.6, 158.6,

Brown syrup; Yield 63 %. IR (KBr): 2218 cm-1 (CN), 1747 cm-1 (C=O, CH3CO). 1H NMR (DMSO-d6):  1.98, 2.0, 2.02, 2.04 (4s, 12H, 4CH3CO), 2.37 (s, 3H, CH3), 3.86 (3H, OCH3), 3.88 (3H, OCH3), 4.14 (dd, 1H, J5',6 = 5.40, J6,6 = 12.23 Hz, H-6), 4.26 (dd, 1H, J5,6 = 4.95, J6,6 = 12.23 Hz, H-6), 4.91 (m, 1H, H-5), 5.10 (t, 1H, J = 9.21 Hz, H-4), 5.27 (dd, 1H, J2,3 = 9.0, J1,2 = 8.20 Hz, H-2), 5.55 (dd, 1H, J2,3 = 9.0, J3,4 = 9.21 Hz, H-3), 6.70 (d, 1H, J1,2 = 8.20 Hz, H-1), 6.99 (s, 1H, pyridone-5), 7.01-7.74 (m, 7H, Ar-H). 13C NMR (DMSO-d6):  = 20.3, 20.4, 20.6, 20.7, 20.8 (4CH3CO and CH3), 54.9, 55.7 (2CH3O), 62.2 (C-6), 66.4 (C-4), 69.5 (C-3), 70.9 (C-2), 72.4 (C-5), 89.1 (C-1), 91.0, 93.9, 111.7, 111.9, 112.4, 117.3, 120.4, 121.5, 127.7, 128.5, 129.6, 131.0, 148.7, 150.8, 156.2, 162.5, 169.5, 169.8, 169.9 and 170.2 (Ar-C, CN and 5 C=O). HRMS (EI): m/z [M+] calcd for C35H36N2O12: 676.2301; found: 676.2310. Anal. Calcd for C35H36N2O12: C, 62.12; H, 5.36; N, 4.14. Found: C, 62.06; H, 5.32; N, 4.18. 3.28. 4-(4-Fluorophenyl)-2-(2',3',4',6'-tetra-O-acetyl--Dgalactopyranosyloxy)-6-(p-tolyl) nicotinonitrile (13a) Brown syrup; Yield 61 %. IR (KBr): 2222 cm-1 (CN), 1747 cm-1 (C=O, CH3CO). 1H NMR (DMSO-d6):  = 1.96, 1.99, 2.02, 2.08 (4s, 12H, 4CH3 CO), 2.37 (s, 3H, CH3), 4.02 (dd, 1H, J5,6 = 6.80, J6,6 = 12.57 Hz, H-6), 4.13 (dd, 1H, J5,6 = 5.98, J6,6 = 12.57 Hz, H-6), 4.71-4.92 (m, 1H, H5), 5.13 (t, 1H, J3,2 = 10.0, J3,4 = 2.86 Hz, H-3), 5.42 (t, 1H, J2,1 = 8.10, J2,3 = 10.0 Hz, H-2), 5.50 (t, 1H, J4,3 = 2.86, J4,5 = 3.10 Hz, H-4), 6.64 (d, 1H, J1,2 = 8.10 Hz, H1), 6.82 (s, 1H, pyridone-5), 7.35-7.73 (m, 8H, Ar-H). 13C NMR (DMSO-d6):  20.5, 20.6, 20.7, 20.8, 21.0 (4CH3 CO and CH3), 61.9 (C-6), 65.3 (C-4), 66.9 (C-3), 67.7 (C-2), 68.3 (C-5), 69.7 (C-1), 115.8 (CN), 127.68 (Ar-C), 129.5, 130.8, 141.3, 169.7, 169.96, 170.04, 170.11 (5 C=O). HRMS (EI): m/z [M+] calcd for C33H31FN2O10: 634.2002; found: 634.2010. Anal. Calcd for C33H31FN2O10: C, 62.46; H, 4.92; N, 4.41. Found: C, 62.40; H, 4.96; N, 4.38. 3.29. 2-(2',3',4',6'-Tetra-O-acetyl--D-galactopyranosyloxy)-4-(3,4-dimethoxy-phenyl)-6-(p-tolyl) nicotinonitrile (13b) Brown powder; Yield 68%; m. p. 105-107 oC. IR (KBr): 2226 cm-1 (CN), 1750 cm-1 (C=O, CH3 CO). 1H NMR (DMSO-d6):  = 1.89, 1.99, 2.03, 2.04 (4s, 12H, 4CH3CO), 2.38 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.19 (dd, 1H, J5,6 = 6.34, J6,6 = 12.80 Hz, H-6), 4.13 (dd, 1H, J5,6 = 5.95, J6,6 = 12.80 Hz, H-6), 4.71-4.78 (m, 1H, H-5), 5.21 (t, 1H, J3,2 = 9.87, J3,4 = 2.86 Hz, H-3), 5.42 (t, 1H, J2,1 = 8.40, J2,3 = 9.87 Hz, H-2), 5.50 (t, 1H, J4,3 = 2.86, J4,5 = 3.0 Hz, H-4), 6.28 (d, 1H, J1.2 = 8.40 Hz,H-1), 6.99 (s, 1H, pyridone-5), 7.31-7.71 (m, 7H, Ar-H). 13C NMR (DMSO-d6):  20.40, 20.46, 20.50, 20.6, 20.7 (4CH3CO and CH3), 55.7, 55.7(2 OCH3), 61.9 (C-6), 65.3

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(C-4), 66.9 (C-3), 67.6 (C-2), 68.3 (C-5), 68.4 (C-1), 114.8 (CN), 121.7 (Ar-C), 127.7, 133.5, 141.2, 169.6, 169.9, 170.1 (5 C=O). Anal. Calcd for C35H36N2O12 (676.67): C, 62.12; H, 5.36; N, 4.14. Found: C, 62.17; H, 5.40; N, 4.09.

J1,2 = 8.4 Hz, H-1), 6.85 (s, 1H, pyridone-5), 7.32 (d, 2, J = 7.6 Hz, Ar-H), 7.40-7.79 (m, 6H, Ar-H). HRMS (EI): m/z [M+] calcd for: C25H23FN2O6 466.1512; found: 466.1501. Anal. Calcd for C25H23FN2O6: C, 64.37; H, 4.97; N, 6.01. Found: C, 64.39; H, 5.02; N, 5.97.

3.30. 1-(2,3,4,6-Tetra-O-acetyl--D-galactopyranosyl(14)-(2,3,6-tri-O-acetyl--D-glucopyranosyl)-4-(4fluorophenyl)-2-oxo-6-(p-tolyl)-1,2-dihydro-pyridine-3carbonitrile (15a)

3.34. 2-(-D-Glucopyranosyloxy)-4-(3,4-dimethoxyphenyl)-6-(p-tolyl)nicotinonitrile (12b)

Brown syrup; Yield 58%. IR (KBr): 2221cm-1 (CN), 1750 cm-1 (C=O, CH3CO), 1644 cm-1 (C=O, amide). 1 H NMR (DMSO-d6):  = 1.93, 1.95, 1.98, 2.0, 2.02, 2.05, 2.10 (7s, 21H, 7CH3CO), 2.36 (s, 3H, CH3), 4.01–4.05 (m, 3H, H2b, H-6a, H-6b), 4.89 (dd, 1H, J5a,6a = 6.05, J6a,6a = 11.74 Hz, H-6a), 4.91-5.10 (m, 1H, H-5b), 5.23-5.28 (m, 2H, H5a, H-6b), 5.26 (d, 1H, J1b,2b = 7.80 Hz, H-1b), 5.33-5.39 (m, 1H, H-4b), 5.45-5.52 (m, 1H, H-2a), 5.62 (dd, 1H, J3a,4a = 9.50, J4a,5a = 9.82 Hz, H-4a), 5.65-5.71 (m, 1H, H3b), 5.75-5.84 (m, 1H, H-3a), 6.23 (d, 1H, J1a,2a = 8.80 Hz, H-1a), 6.86 (s, 1H, pyridone, H-5), 7.19 (d, 2H, J = 7.60 Hz, Ar-H), 7.26, 7.86 (2m, 6H, Ar-H). HRMS (EI): m/z [M+] calcd for C45H47FN2O18: 922.2802; found: 922.2810. Anal. Calcd for C45H47FN2O18: C, 58.57; H, 5.13; N, 3.04. Found: C, 58.55; H, 5.10; N, 3.00. 3.31. 1-(2,3,4,6-Tetra-O-acetyl--D-galactopyranosyl(14)-(2,3,6-tri-O-acetyl--D-glucopyranosyl)-4-(3,4dimethoxyphenyl)-2-oxo-6-(p-tolyl)-1,2-dihydro-pyridine3-carbonitrile (15b) Brown syrup; Yield 68%. IR (KBr): 2212 cm-1 (CN), 1743 cm-1 (C=O, CH3CO), 1640 cm-1 (C=O, amide). 1 H NMR (DMSO-d6):  = 1.83, 1.95, 1.97, 1.99, 2.01, 2.07, 2.10 (7s, 21H, 7CH3 CO), 2.35 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.98–4.02 (m, 3H, H-2b, H-6a, H-6b), 4.85 (dd, 1H, J5a,6a = 5.95, J6a,6a = 12.01 Hz, H-6a), 4.844.90 (m, 1H, H-5b), 5.18-5.22 (m, 2H, H-5a, H-6b), 5.23 (d, 1H, J1b,2b = 7.82 Hz, H-1b), 5.27-5.32 (m, 1H, H-4b), 5.39 (m, 1H, H-2a), 5.61 (dd, 1H, J3a,4a = 9.32, J4a,5a = 9.90 Hz, H-4a), 5.66-5.73 (m, 1H, H-3b), 5.64-5.69 (m, 1H, H3a), 6.81 (d, 1H, J1a,2a = 8.82 Hz, H-1a), 6.81 (s, 1H, pyridone, H-5), 7.09-7.94 (m, 7H, Ar-H). Anal. Calcd for C47H52N2O20 (964.92): C, 58.50; H, 5.43; N, 2.90. Found: C, 58.49; H, 5.40; N, 2.87. 3.32. General Procedure for Deacetylation of Nucleosides (12a,b, 14a,b and 16a,b) A mixture of synthesized nucleosides (11a,b, 13a,b, and 15a,b) (10 mmol) in methanol (20 mL), triethylamine (1 mL) and few drops of water were stirred overnight at room temperature, and then the solvent was removed under reduced pressure. The residue was crystallized from methanol to produced (12a,b, 14a,b and 16a,b). 3.33. 4-(4-Fluorophenyl)-2-(-D-glucopyranosyloxy)-6-(ptolyl)nicotinonitrile (12a) Yellow powder; Yield 98 %; m. p. 200-202oC. IR (KBr): 3419 cm-1 (4 OH), 2220 cm-1 (CN) and 1642 cm-1 (CN). 1 H NMR (DMSO-d6 / D2O):  = 2.33 (s, 3H, CH3), 2.98-3.36 (m, 6H, H-6', H-6", H-5', H-4', H-3' and H-2'), 6.81 (d, 1H,

Yellow powder; Yield 92 %; m. p. 120-122 oC. IR (KBr): 3393 cm-1 (4OH) and 2220 cm-1 (CN). 1H NMR (DMSO-d6 / D2O):  = 2.38 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.41-3.72 (m, 6H, H-6', H-6'', H-5', H-4', H-3' and H-2'), 3.83 (t, 1H, OH-6'), 4.25 (t, 1H, OH-4'), 4.26 (t, 1H, OH-3'), 4.89 (t, 1H, OH-2'), 6.20 (d, 1H, J = 8.0 Hz, H1'), 6.80 (s, 1H, pyridone-5), 7.13-7.80 (m, 7H, Ar-H). Anal. Calcd for C27H28N2O8 (508.52): C, 63.77; H, 5.55; N, 5.51. Found: C, 63.82; H, 5.50; N, 5.55. 3.35. 4-(4-Fluorophenyl)-2-(-D-galactopyranosyloxy)-6(p-tolyl)nicotinonitrile (14a) Brown powder; Yield 84 %; m. p. 150-152 oC. IR (KBr): 3400 cm-1 (4 OH) and 2224 cm-1 (CN). 1H NMR (DMSOd6 / D2O):  = 2.36 (s, 3H, CH3), 3.36-3.98 (m, 3H, H-3', H-6' and H-6"), 3.47-3.51 (m, 3H, H-2', H-4' and H-5'), 6.14 (d, 1H, J1',2' = 7.9 Hz, H-1'), 7.35-7.73 (m, 9H, Ar-H, pyridine H-5). 13C NMR (DMSO-d6):  20.4 (CH3), 60.6 (C-6), 68.8 (C-4), 68.9 (C-3), 69.5 (C-2), 70.4 (C-5), 72.1 (C-1), 114.8 (CN), 121.7 (Ar-C), 123.3, 129.5, 127.8, 133.5, 141.2, 156.4, 163.4. Anal. Calcd for C25H23FN2O6 (466.46): C, 64.37; H, 4.97; N, 6.01. Found: C, 64.33; H, 5.02; N, 5.98. 3.36. 2-(-D-Galactopyranosyloxy)-4-(3,4-dimethoxyphenyl)-6-(p-tolyl)nicotine-nitrile (14b) Brown powder; Yield 98 %; m. p. 128-130 oC. IR (KBr): 3423 cm-1 (4 OH) and 2225 cm-1 (CN). 1H NMR (DMSOd6 / D2O):  = 2.35 (s, 3H, CH3), 3.32-3.99 (m, 3H, H-3', H-6' and H-6"), 3.40-3.46 (m, 3H, H-2', H-4' and H-5'), 3.51, 3.37 (2s, 6H, 2 OCH3), 6.09 (d, 1H, J1',2' = 8.0 Hz, H-1'), 7.12 (d, 1H, J = 8.4 Hz, Ar-H), 7.31-7.37 (m, 4H, Ar-H), 7.76 (s, 1H, Ar-H, pyridine H-5), 8.10 (d, 2H, J = 7.2 Hz, Ar-H). 13C NMR (DMSO-d6):  21.3 (CH3), 56.1 (2 OCH3), 62.6 (C-6), 68.8 (C-4), 71.5 (C-3), 73.4 (C-2), 76.4 (C-5), 92.5 (C-1), 101.8 (CN), 121.7, 123.3 (Ar-C), 127.8, 129.5, 130.7, 133.5, 136.0, 141.2, 150.2, 156.3, 160.2, 164.4. Anal. Calcd for C25H23FN2O6 (466.46): C, 64.37; H, 4.97; N, 6.01. Found: C, 64.33; H, 5.02; N, 5.98. HRMS (EI): m/z [M+] calcd for C27H28N2O8: 508.1812; found: 508.1810. Anal. Calcd for C27H28N2O8: C, 63.77; H, 5.55; N, 5.51. Found: C, 63.74; H, 5.57; N, 5.50. 3.37. 4-(4-Fluorophenyl)-1-(-D-Galactopyranosyl-(14) -(-D-glucopyranosyl)-2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (16a) Brown syrup; Yield 95 %; IR (KBr): 3424 cm-1 (OH), 2219 cm-1 (CN), 1636 cm-1 (C=O, amide). 1H NMR (DMSO-d6):  = 2.37 (s, 3H, CH3), 3.35 - 3.76 (m, 12H, H2'b , H-3'b, H-4'b, H-5'b, H-6'b, H-6"b, H-2'a , H-3'a, H-4'a, H-5'a, H-6'a, H-6"a), 4.18 (d, 1H, OH-4'b), 4.45 (d, 1H, OH6'b), 4.64 (d, 1H, J = 4.28 Hz, OH-3'b), 4.90 (d, 1H, OH-

Synthesis of Nucleosides and Non-nucleosides

Medicinal Chemistry, 2018, Vol. 14, No. 8

2'b), 5.10 (d, 1H, OH-6'a), 6.0 (d, 1H, OH-3'a), 6.09 (d, 1H, OH-2'a), 6.19 (d, 1H, J1'b,2'b = 7.75 Hz, H-1'b), 6.32 (d, 1H, J1'a,2'a = 8.80 Hz, H-1'a), 6.86 (s, 1H, pyridone, H-5), 7.18 (d, 2H, Ar-H), 7.23-7.70 (2m, 6H, Ar-H). Anal. Calcd for C31H33FN2O11 (628.60): C, 59.23; H, 5.29; N, 4.46. Found: C, 59.20; H, 5.25; N, 4.49. 3.38. 1-(-D-Galactopyranosyl-(14)-(-D-glucopyranosyl)-4-(3,4-dimethoxy-phenyl)-2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (16b) -1

Yellow syrup; Yield, 97 %; IR (KBr): 3416 cm (7 OH), 2210 cm-1 (CN), 1633 cm-1 (C=O, amide). 1H NMR (DMSO-d6):  = 2.35 (s, 3H, CH3), 3.14 - 3.37 (m, 12H, H2'b , H-3'b, H-4'b, H-5'b, H-6'b, H-6"b, H-2'a , H-3'a, H-4'a, H-5'a, H-6'a, H-6"a), 3.82 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.18 (d, 1H, OH-4'b), 4.26 (d, 1H, OH-6'b), 4.32 (d, 1H, J = 4.23 Hz, OH-3'b), 4.88 (d, 1H, OH-2'b), 5.67 (d, 1H, OH-6'a), 5.84 (d, 1H, OH-3'a), 6.03 (d, 1H, OH-2'a), 6.22 (d, 1H, J1'b,2'b = 7.60 Hz, H-1'b), 6.65 (d, 1H, J1'a,2'a = 8.76 Hz, H1'a), 6.77 (s, 1H, pyridone, H-5), 7.09-7.79-7.84 (m, 7H, ArH). HRMS (EI): m/z [M+] calcd for C33H38N2O13: 670.2412; found: 670.2410. Anal. Calcd for C33H38N2O13: C, 59.10; H, 5.71; N, 4.18. Found: C, 59.13; H, 5.69; N, 4.16. 3.39. Biology 3.39.1. Antiviral Activity Assays The novel compounds were tested against a broad panel of viruses, namely, herpes simplex virus type 1 (KOS), herpes simplex virus type 2 (G), vaccinia virus, VSV, and thymidine kinase-deficient herpes simplex virus type 1 (TKKOS ACVr), in HEL cell culture [35, 36]. Moreover, HEL cell culture was also used to test such compounds against either cytomegalovirus (HCMV) strains AD-169 and Davis or VZV; TK VZV strain Oka, TK-VZV strain 07-1. The antiviral assays were based on inhibition of virus-induced cytopathogenicity or plaque formation in HEL. Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or virus input was 100 plaque forming units (PFU) in case of cytomegalovirus or 20 plaque forming units (PFU) for VZV in the presence of varying concentrations of the test compounds [37]. Viral cytopathogenicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 or compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%. The minimal cytotoxic concentration (MCC) of the compounds was defined as the compound concentration that caused a microscopically visible alteration of cell morphology. Alternatively, cytotoxicity of the test compounds was measured based on inhibition of cell growth. HEL cells were seeded at a rate of 5x103 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at 37°C, the cell number was determined with a Coulter counter. The cytostatic concentration was calculated as the CC50, or the compound concentration required for re-

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ducing cell proliferation by 50% relative to the number of cells in the untreated controls. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Not applicable. HUMAN AND ANIMAL RIGHTS No Animals/Humans were used for studies that are the basis of this research. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS The authors would like to thank Professors Jan Balzarini, Robert Snoeck, and Graciela Andrei at Rega Institute for Medical Research, Faculty of Medicine, K.U. Leuven, Leuven, Belgium. REFERENCES [1]

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