A First Microwave-Assisted Synthesis of a New Class of Purine and Guanine Thioglycoside Analogs

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

, VOL. , NO. , –



http://dx.doi.org/./..

A First Microwave-Assisted Synthesis of a New Class of

Purine and Guanine Thioglycoside Analogs

Galal Elgemeie , Mamdouh Abu-Zaied , Ali Hebishy , Nermen Abbas ,

a

and Mai Hamed

a

b

Chemistry Department, Faculty of Science, Helwan University, Helwan, Cairo, Egypt; Green Chemistry

c

Department, National Research Centre, Dokki, Giza, Egypt; Chemistry Department, Faculty of Science,

Taibah University, Saudi Arabia

ABSTRACT

ARTICLE HISTORY

A ﬁrst microwave-assisted synthesis of a new class of novel

purine thioglycoside analogs from readily available starting

materials has been described. The key step of this protocol

is the formation of sodium pyrazolo[1,5-a]pyrimidine-7-thiolate

and 7-mercaptopyrazolo[1,5-a]pyrimidine derivatives via conden-

sation of 5-amino-1H-pyrazoles with sodium 2,2-dicyanoethene-

Received  January 

Accepted  June 

KEYWORDS

Microwave synthesis; purine

thioglycoside analogs;

thioglycosides; ketene

dithiolate salts;

antimetabolites; nucleoside

analogs

1

,1-bis(thiolate) salts or 2-(dimercaptomethylene)malononitrile,

respectively, under microwave irradiation, followed by coupling

with halo sugars to give the corresponding purine thioglycoside

analogs. The obtained purines and purines thioglycosides deriva-

tives were evaluated in vitro against lung (A549), colon (HCT116),

liver (HEPG2), and prostate (PC3) cancer cell lines. Some of these

compounds (5b, 5d, 5f, and 9a–d) exhibited little potency toward

thefourcelllines. Ontheotherhand, compound5aelicitedhigher

cytotoxicity on both prostate (PC3) and colon (HCT116), respec-

tively, while it was found moderate on lung (A549), and inactive

on liver (HEPG2). Moreover, compound 5c was found moderate

with LC50 values 52.0–88.9 μM for almost all the cell lines.

1

. Introduction

Purines are important in biological systems since two of the four DNA bases are

purine derivatives, and many modern pharmaceutical agents contain purine frag-

ments. Several examples of biologically important purine derivatives have been

identiﬁed, such as antagonists of nucleic acid synthesis (6-mercaptopurine) which

is highlighted as one of the most signiﬁcant new purine derivatives to have been

[1]

identiﬁed in the last years, Also 6-mercaptopurine is used an antileukemic

[2–4]

agent.

fungal, antimicrobial, antiviral, and antitumor activities.

purine derivatives exhibited cytotoxic eﬀects against cancer cell lines, but also the

in addition, the pharmacological activity of thiopurines includes anti-

[5,6]

Not only do some

CONTACT Galal Elgemeie

sity, Helwan, Cairo, Egypt.

elgemeie@hotmail.com

Chemistry Department, Faculty of Science, Helwan Univer-

Color versions of one or more of the ﬁgures in the article can be found online at www.tandfonline.com/lncn .

©

 Taylor & Francis Group, LLC

2

G. ELGEMEIE ET AL.

antiproliferative eﬀect of many derivatives was evaluated on a panel of tumor cell

[7]

lines. Recently, it has been reported that mercaptopurine (6MP) and thiogua-

nine (6TG) derivatives, bearing a purine moiety possess many types of biological

[8,9]

[10]

activities,

including anticancer activity. Consequently, many mercaptopurine

derivatives were designed and synthesized as antimetabolic agents. We have recently

reported diﬀerent successful approaches for the synthesis of purine and pyrimi-

[

11–14]

dine nucleoside analogs.

Derivatives of these ring systems are interesting as

[15–21]

antimetabolites in biochemical reactions.

Continuing our eﬀorts for the devel-

opment of simple, eco-friendly and cost-eﬀective methodologies, we report herein

a novel microwave-assisted synthesis of thioguanine and mercaptopurine analogs

and their thioglycosides as antimetabolic agents. All examples reported in the lit-

erature are for N-glycosides and very few examples are reported for guanine or

[22–24]

purine thioglycosides.

As far as we know, this is the ﬁrst method to be reported

for the preparation of this ring system. In this study, a number of derivatives have

been found to possess antitumor activity of suﬃcient interest to warrant further

investigation.

2

. Results and discussion

2.1. Chemistry

It has been found that reaction of malononitrile with carbon disulﬁde in the pres-

ence of sodium ethoxide gives sodium 2,2-dicyanoethene-1,1-bis(thiolate) 2. Com-

pound 2 is readily reacted with one equivalent of 5-aminopyrazole derivatives 1a–f

in the presence of dimethylformamide under microwave activation 600 W for 10

minutes at 154°C, to give the corresponding sodium pyrazolo[1,5-a]pyrimidine-

7

-thiolate derivatives 3a–f in good yields. Compounds 3a–f reacted with halo-

sugars 4 in dimethylformamide at room tempe rature to give the corresponding

S-glycosides 5a–l with high yields (Scheme 1 ). It has been suggested that the

cis-(α) sugars react by a simple SN reaction to give the β-glycoside products.

The structures of 5a-l were established on the basis of their elemental analysis

and spectral data (IR, H NMR, C NMR). For example, the analytical data for

5

2

[25]

1

13

1

a revealed a molecular formula C H N O S, its H NMR spectrum showing

22

6

9

the anomeric proton as a doublet at δ 5.55–5.56 ppm with a spin-spin coupling

constant of 8.70 Hz indicating the β-conﬁguration. The other six glucose pro-

tons resonated at δ 4.21–5.23 ppm. When glycosides 5b and 5i were treated with

methanolic ammonia at room temperature for 10 minutes, the deprotected deriva-

tives 6a and 6b were obtained in almost quantitative yields (Scheme 2 ), the struc-

tures of which were established on the basis of elemental analysis and spectral data.

Thus, the analytical data for 6a reveal the molecular formula C H N O S. The

1

9

20

8

5

1

H NMR spectrum shows the anomeric proton as a doublet at δ 5.56–5.58(J₁

ꢀ

,2 =

9.95 Hz), indicating the presence of only the β -D-conﬁguration. Encouraged by

these results, we decided to synthesize the S-glycosides 5 using the reaction of 1 with

bis(mercapto)methylene]malononitrile 7 and comparing the resulting products for

[

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

3

Scheme . Synthetic pathway for purine thioglycoside analogs.

stereochemical considerations. Thus, in a simple experimental procedure, treatment

of compounds 1b–f with 7 in DMF solvent under microwave activation 600 W

for 10 minutes at 154°C, aﬀorded the corresponding 7-mercaptopyrazolo[1,5-

a]pyrimidine derivatives 9a–e. The latter compounds were treated with sodium

ethoxide followed by addition of the peracetylated sugars 4a,b in DMF at room

temperature to aﬀord the S-glycosyl compounds 5b–f,h–l. The latter were shown

to be the same as those obtained from the reaction of 3 with 4 by comparing the

obtained melting points and spectral data. Methylation of compounds 9a–e with

methyl iodide in sodium ethoxide gave the 7-methylthiopyrazolo[1,5-a]pyrimidine

products 10a–e. Compounds 10a–e can also be prepared by reaction of 1b–f

with 2-[bis(methylthio)methylene]malononitrile 8 in dimethylformamide under

microwave activation 600 W for 10 minutes at 154°C. When compounds 5b–f,h–l

4

G. ELGEMEIE ET AL.

Scheme . Synthetic pathway for purine thioglycoside free sugar.

ꢀ

and 10a–e were subjected to the reaction with hydrazine, the dipyrazolo[1,5-a:4 ,3 -

e]pyrimidine derivatives 11a–e were obtained (Scheme 3 ).^[The structure of com-

pounds 11 were established on the basis of elemental analysis and spectral data (MS,

26]

1

H NMR and IR). Thus, the IR spectrum of 11a revealed the absence of a CN band,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

5

Scheme . Synthetic pathway for mercaptopyrazolo[,-a]pyrimidines and dibyrazolopyrimidines.

1

and H NMR spectrum contained a broad band at δ = 8.74 ppm assignable to NH

group, a multiplet at δ = 7.32–7.59 ppm assigned to the aromatic protons and three

broad singlets at δ 6.61 and 7.71 ppm assignable for three NH groups.

2

.2. Antitumor evaluation

Potential cytotoxicity eﬀect of the ten of the newly synthesized compounds and

doxorubicin drug were evaluated in the National Research Centre (Bio-assay–Cell

Culture Laboratory) using MTT assay. The synthesized compounds 5a–f, 9a–d

6

G. ELGEMEIE ET AL.

Table . Cytotoxicity of the synthesized candidates on lung (A), colon (HCT), liver (HEPG), and

prostate (PC) cancer cell lines.

Cytotoxic activity for

A cell line

Cytotoxic activity for

HCT cell line

Cytotoxic activity for

HEPG cell line

Cytotoxic activity for

PC cell line

Compound

No.

 μM (%) LC ( μM)  μM (%) LC ( μM)  μM (%) LC ( μM)  μM (%) LC ( μM)





a

b

c

d

e

f

a

b

c

.

.

.

.

.

.

.

.

.

.

68.5

—

55.4

—

.

.

.

.

.

.

.

.

.

.

59.4

—

88.9

—

.

.

.

.

.

.

.

.

.

.

—

52.0

—



.

.

.

.

.

.

.

.

.

32.7

—-

70.2

—

d

Doxorubicin

.

.

.

.

LC: Lethal concentration of the sample which causes the death of % of cells in  hours.

were subjected to in vitro antitumor screening against human cancer cell lines

[27–31]

using cell- based disease.

The compounds were evaluated for their cytotox-

icity against four human tumor cell lines namely hepatocellular carcinoma HEPG2,

colon carcinoma HCT116, prostate carcinoma PC3, and lung carcinoma A549.

Cell viability was assessed by the mitochondrial dependent reduction of yellow

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple

[25,27]

formazan.

A probit analysis was carried for LC₅₀determination using SPSS

1

1 program. The antitumor drug doxorubicin was used as positive control. In

regard to the antitumor selectivity among the used tumor cell lines, the chemi-

cal compounds showed weak, moderate as well as strong inhibition ability against

most of the tested cancer cells as revealed by the LC .The tested compounds

50

5

b, 5d, 5e, 5f, and 9a–d exhibited low antitumor activity toward both the four

cell lines (at 100 μ M the values of mortality of cancer cell lines range between

2% and 59%) (Table 1 ). However compound 5c proved to be slightly active

toward all cancer cell lines with LC₅₀values of 55,4 μ M for lung cell line (A549)

Figure 1 ), 88.9 μM for colon cell line (HCT116) (Figure 2 ), 52.0 μM for hepa-

tocellular cell line (HEPG2) (Figure 4 ) and 70.2 μM for prostate cell line (PC3)

Figure 3 ) respectively. On the other hand, compound 5a exhibited slight activ-

1

(

ity against the lung cell line with an LC₅₀value of 68.5 μM and higher activity

against the colon cell line HCT116, and the prostate cell line PC3 with LC₅₀val-

ues of 59.4 and 32.7 μM, respectively, compared to the standard drug doxorubicin.

Looking at the structure-activity relationships of the tested compounds which

contain a purine nucleus, it was found that the substance 5c, possessing a p-

methoxyphenyl ring appended to the C-3 position of the purine nucleus via a

diazenyl linker and a thioglycosidic linkage at C-7 exhibited a minor amount of

antitumor activity. In contrast, linking a phenyl, p-methyl, p-chloro, p-bromophenyl

ring to the purine nucleus (5b, 5d, 5e, 5f, and 9a–d) leads to essentially no antitu-

mor activity compared with the standard drug. The loss of potency may be due to

a decrease of cellular penetration or an inability to making eﬀective contacts with

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

7

Figure . Representative graph showing observed responses of A549 cell in the presence of increas-

ing concentrations of compounds 5a and 5c.

the active site. On the other hand, the corresponding purine nucleus carrying two

substituents especially with a thioglycosidic linkage at C-7 and cyano group at posi-

tion 3 (5a) do have some activity. Such thioglycosides may act in the same way as

antimetabolites by inhibiting key enzymes in the biosynthesis of purine and pyrim-

idine that are incorporated in the DNA molecule, resulting in cell death.

Figure . Representative graph showing observed responses of HCT116 cell in the presence of

increasing concentrations of compounds 5a and 5c.

8

G. ELGEMEIE ET AL.

Figure . Representative graph showing observed responses of PC3 cell in the presence of increasing

concentrations of compounds 5a and 5c.

3

. Experimental

3.1. Chemistry

1

All melting points were measured on a Gallenkamp melting point apparatus. The H

NMR spectra were measured on a Jeol-500 MHz spectrometer for solutions DMSO-

d and CDCl using Si(CH ) as an internal standard at National Research Cen-

6

3

3 4

ter, Cairo, Egypt. Progress of the reactions was monitored by TLC using aluminum

sheets coated with silica gel F254 (Merck). Viewing under a short-wavelength UV

lamp aﬀected detection. Microwave reactions were conducted using Microwave.

Figure . Representative graph showing observed responses of HEPG2 cell in the presence of

increasing concentrations of compound 5c.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

9



... General procedure for the synthesis of a–f

A mixture of 4-(aryldiazenyl)-1H-pyrazole-3,5-diamine or 5-amino-1H-pyrazole-

-carbonitrile 1a–f (10 mmol) and sodium 2,2-dicyanoethene-1,1-bis(thiolate) 2

10 mmol) in DMF (20 mL) irradiated in a microwave oven 600 W for 10 minutes at

54°C. The reaction mixtures were cooled to room temperature and triturated with

4

(

1

MeOH to aﬀord compounds 3a–f which was collected by ﬁltration and recrystal-

lized from the appropriate solvent. Yield (92%).

3

.1.1.1. Sodium 5-amino-3,6-dicyanopyrazolo[1,5-a]pyrimidine-7-thiolat(3a).

−

1

Brown solid; (MeOH); yield (92%); mp >300°C; IR (KBr, cm ) υ 3465, 3284,

2

213, 2216, 1607, 1598. C H N NaS.

8 3 6

3

.1.1.2. Sodium 2,5-diamino-6-cyano-3-(phenyldiazenyl)pyrazolo[1,5-a]

pyrimid-ine-7-thiolate(3b). Brown solid; (MeOH); yield (90%); mp >300°C; IR

−

1

(KBr, cm ) υ 3466, 3439, 3025, 2206, 1609, 1595. C H N NaS.

13 9 8

3

.1.1.3. Sodium 2,5-diamino-6-cyano-3-((4-methoxyphenyl)diazenyl)pyrazolo[1,

5

-a]pyrimidine-7-thiolate (3c). Brown solid; (MeOH); yield (95%); mp >300°C;

IR (KBr, cm ) υ 3465, 3433, 3286, 2927, 2879, 2205, 1606, 1596. C H N NaOS.

−

1

14

11

8

3

.1.1.4. Sodium 2,5-diamino-6-cyano-3-(p-tolyldiazenyl)pyrazolo[1,5-a]

pyrimid-ine-7-thiolate(3d). Brown solid; (MeOH); yield (95%); mp >300°C; IR

−

1

(KBr, cm ) υ 3455, 3427, 3065, 2956, 2206, 1719, 1601. C H N NaS.

14 11 8

3

.1.1.5. Sodium 2,5-diamino-6-cyano-3-((4-chlorophenyl)diazenyl)-6-cyanopyr-

azolo[1,5-a]pyrimidine-7-thiolate(3e). Brown solid; (MeOH); yield (95%); mp

>

−

1

300°C; IR (KBr, cm ) υ 3468, 3452, 3036, 2221, 1638, 1596. C H ClN NaS.

13 8 8

3

.1.1.6. Sodium 2,5-diamino-6-cyano-3-((4-bromophenyl)diazenyl)-6-cyanopyr-

azolo[1,5-a]pyrimidine-7-thiolate(3f.). Brown solid; (MeOH); yield (93%); mp

>

−

1

300°C; IR (KBr, cm ) υ 3435, 3389, 3028, 2209, 1645, 1613. C H BrN NaS.

13 8 8

... General procedures for the synthesizing of (a–l)

Method A.

To a solution of 3a–f (10 mmol) in dry DMF (20 mL) a solution of halosugars 4

in dry DMF (10 mL) was dropped within 30 minutes and the reaction mixture was

stirred at room temperature until completion (TLC. 6–8 hours). After completion,

the reaction mixture was poured on ice water. A solid product precipitate was ﬁltered

oﬀ and washed with water, dried and puriﬁed by column chromatography using an

appropriate solvent system giving compounds 5a–l.

1

0

G. ELGEMEIE ET AL.

Method B.

To a solution of 9 (10 mmol) in equivalent sodium ethoxide was stirred for

0 minutes at room temperature. Then the reaction mixture was evaporated under

3

reduced pressure and it dissolved in dry DMF (20 mL) followed by addition of the

activated cyclic sugars of (2,3,4,6-tetra-O-acetyl-α-D-gluco or galacto)pyronosyl

bromide 4 in dry DMF (10 mL) within 30 minutes and the reaction mixture was

stirred at room temperature until completion (TLC, 3–8 hours). After completion,

the reaction mixture was poured on water. The aqueous phase was extracted with

ethyl acetate (3 × 20 mL), the combined organic phase was washed with water,

dried over anhydrous sodium sulphate. Removal of solvent gave a residue which

was puriﬁed by column chromatography using an appropriate solvent system to give

compounds 5.

ꢀ

3

.1.2.1. 5-Amino-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-glucopyranosylthio)-pyrazolo

[1,5-a]pyrimidine-3,6-dicarbonitrile(5a). Compound 5a was puriﬁed by column

chromatography (petroleum. ether /ethyl acetate 6:1, R = 0.35–0.37 region); yellow

f

1

solid; (EtOH); yield (89%); mp154°C; H NMR (500 MHz, CDCl ): δ 2.05–2.09

4s, 12H, 4xOAc), 4.21, 4.23 (m, 2H, 2H-6 ), 4.24 (m, 2H, H-4 , H-5 ), 4.62 (t,

3

ꢀ

(

1

ꢀ

H, H-3 ), 5.23 (t, 1H, H-2 ), 5.55–5.56 (d, 1H, J

ꢀ

8.70 Hz, H-1 ), 6.63 (s, 2H,

1

–2 =

13

ꢀ

NH ), 8.21 (s, 1H, pyrazole H-2); C NMR: δ 20.18 (4CH CO), 62.72 (C-6 ), 68.82

2

ꢀ

3

ꢀ

(

1

C-4 ), 70.24 (C-2 ), 72.62 (C-3 ), 76.51 (C-5 ), 81.46 (C-1 ), 69.15 (C-3), 90.35

C-6), 115.25 (CN), 116.42 (CN), 131.63 (C-3 ), 154.34 (C-2), 161.82 (C-5), 170.76

4CO), 172.44 (C-7). Anal. Calcd. For C H N O S (546.51): C, 48.35; H, 4.06; N,

5.38; S, 5.87%. Found: 48.28; H, 4.02; N, 15.25; S, 5.72%.

a

2

22

6

9

ꢀ

3

.1.2.2. 2,5-Diamino-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-glucopyranosylthio)-3-

(phenyldiazenyl) pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5b). Compound 5b

was puriﬁed by column chromatography (petroleum. ether/ethyl acetate 8:2, R =

f

1

0

.74–0.76 region); yellow solid; (EtOH; yield (85%); mp165°C; H NMR (500 MHz,

DMSO-d ) δ 1.94–2.09 (4s, 12H, 4xOAc), 4.01–4.09 (m, 3H, 2H-6 and H-5 ), 4.99

ꢀ

6

ꢀ

(

t, 1H, H-4 ), 5.13 (t, 1H, H-3 ), 5.40 (t,1H, H-2 ), 5.66–5.68 (d, 1H, J

ꢀ

9.95 Hz,

1

–2 =

ꢀ

13

H-1 ), 6.66 (s, 2H, NH ), 6.86 (s, 2H, NH ), 7.34–7.89 (m, 5H, C H ); C NMR;

δ 21.22 (4CH CO), 62.62 (C-6 ), 69.64 (C-4 ), 70.43 (C-2 ), 74.21 (C-3 ), 77.11

C-5 ), 84.21 (C-1 ), 91.21 (C-6), 93.51 (C-3), 117.23 (CN), 128.51 (Ar-C), 133.51

C-3 ), 156.12 (C-2), 163.53 (C-5), 171.51 (4CO), 173.31 (C-7). Anal. Calcd. For

2

6

5

ꢀ

3

ꢀ

a

ꢀ

(

C H N O S (640.62): C, 50.62; H, 4.41; N, 17.49; S, 5.01%. Found: C, 50.51; H,

27

28

8

9

4.36; N, 17.40; S, 5.11%.

ꢀ

3

.1.2.3. 2,5-Diamino-3-((4-methoxyphenyl)diazenyl)-7-(2 ,3 ,4 ,6 -tetra-O-

acetyl-β-D-glucopyranosylthio) pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5c).

Compound 5c was puriﬁed by column chromatography (petroleum. ether/ethyl

acetate 8:2,R = 0.45–0.47region); yellow solid; (EtOH); yield (90%); mp177°C;

f

−

1

IR (KBr, cm )υ 3455, 3397, 3029, 2223, 1734, 1649; H-NMR (500 MHz,CDCl )

3

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

11

ꢀ

δ 1.95–2.08 (4s, 12H, 4xOAc), 3.68 (s, 3H, OCH ), 4.15 (m, 2H, 2H-6 ), 4.23 (m,

3

ꢀ

1

H, H-5 ), 4.42 (t, 1H, H-4 ), 4.56 (t, 1H, H-3 ), 5.18–5.20 (t, 1H, H-2 ), 5.23–5.24

5.00 Hz, H-1 ), 6.32 (s, 2H, NH ), 6.52 (s, 2H, NH ), 6.99–7.68 (m,

H, C H ). Anal. Calcd. For C H N O S (670.65): C, 50.15; H, 4.51; N, 16.71; S,

ꢀ

(d, 1H, J₁

ꢀ

–2 =

2

4

6 4 28 30 8 10

4.78%. Found: C, 50.25; H, 4.55; N, 16.62; S, 4.62%.

ꢀ

3

.1.2.4. 2,5-Diamino-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-glucopyranosylthio)-3-(p-

tolyldiazenyl) pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5d). Compound 5d was

puriﬁed by column chromatography (petroleum ether/ethyl acetate 8:2,R = 0.70–

f

1

−

0

3

1

.72 region); yellow solid; (EtOH); yield (82%); mp180°C; IR (KBr, cm )υ 3452,

1

410, 3380, 2947, 2221, 1751, 1620. H-NMR (500 MHz, CDCl ) δ 2.01–2.04 (4s,

3

ꢀ

2H, 4xOAc), 2.80 (s, 3H, CH ), 4.14–418 (m, 2H,2 H-6 ,), 4.25 (m, 1H, H-5 ), 5.22

3

ꢀ

(t, 1H, H-4 ), 5.35 (t,1H, H-3 ), 5.58 (t, 1H, H-2 ), 5.82–5.84 (d, 1H, J

ꢀ

14.15 Hz,

1

-2 =

6

ꢀ

H-1 ), 6.61 (brs, 2H, NH ), 6.81 (brs, 2H, NH ), 7.22–7.36 (m, 4H, C H ). Anal.

2

4

Calcd. For C H N O S (654.65): C, 51.37; H, 4.62; N, 17.12; S, 4.90%. Found: C,

28

30

8

9

51.40; H, 4.53; N, 17.15; S, 4.85%.

ꢀ

3

.1.2.5. 2,5-Diamino-3-((4-chlorophenyl)diazenyl)-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-

β-D-glucopyranosylthio)pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5e). Com-

pound 5e was puriﬁed by column chromatography (petroleum. ether/ethyl acetate

8

:2,R = 0.45–47 region); yellow solid; (EtOH); yield (81%); mp185°C; IR (KBr,

f

1

−

1

cm )υ 3475, 3416, 3242, 2210, 1745, 1604; H-NMR (500 MHz, CDCl )δ 2.02–

3

ꢀ

2

.07 (4s, 12H, 4xOAc), 3.98–4.26 (m, 1H, H-5 ), 4.27–4.28 (m, 2H, 2H-6 ), 4.80–5.00

ꢀ

(t, 1H, H-4 ), 5.22 (t, 1H, H-3 ), 5.38 (t,1H, H-2 ), 5.79–5.80 (d, 1H, J

ꢀ

5.12 Hz,

1

–2 =

ꢀ

H-1 ), 6.83 (brs, 2H, NH ), 6.99 (brs, 2H, NH ), 7.45–7.70 (m, 4H, C H ). Anal.

2

6

4

Calcd. For C H ClN O S (675.07): C, 48.04; H, 4.03; N, 16.60; S, 4.75%. Found:

27

8

9

C, 48.01; H, 4.01; N, 16.52; S, 4.71%.

ꢀ

3

.1.2.6. 2,5-Diamino-3-((4-bromophenyl)diazenyl)-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-

β-D-glucopyranosylthio)pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5f). Com-

pound 5f was puriﬁed by column chromatography (petroleum. ether/ethyl acetate

8

:2,R = 0.65–67 region); yellow solid; (EtOH); yield (90%); mp19°C; IR (KBr,

f

1

−

1

cm )υ 3455, 3401, 2218, 1751, 1620; H-NMR (500 MHz, CDCl )δ 1.95–2.08

3

ꢀ

(

4

4s, 12H, 4xOAc), 3.92–394 (m, 2H, H-6 ), 4.15 (m, 1H, H-5 ), 4.73 (t,1H, H-4 ),

.95 (t, 1H, H-3 ), 5.35 (t, 1H, H-2 ), 5.55–5.57 (d, 1H, J

ꢀ

9.2 Hz, H-1 ), 6.62

1

–2 =

(brs, 2H, NH ), 6.81 (brs, 2H, NH ), 7.41–7.68 (m, 4H, C H ). Anal. Calcd. For

2 2 6 4

C H BrN O S (719.52): C, 45.07; H, 3.78; N, 15.57; S, 4.46 5. Found: C, 45.20; H,

27

8

9

3.68; N, 15.60; S, 4.40%.

ꢀ

3

.1.2.7. 5-Amino-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-galactopyranosylthio)-

pyrazolo[1,5-a]pyrimidine-3,6-dicarbonitrile(5g). Compound 5g was puriﬁed

by column chromatography (petroleum. ether/ethyl acetate 8:2,R = 0.70–72

f

1

2

G. ELGEMEIE ET AL.

1

region); yellow solid; (EtOH); yield (89%); mp154°C; H-NMR (500 MHz, CDCl ):

3

ꢀ

δ 2.03–2.11 (4s, 12H, 4xOAc), 4.11 (m, 2H, 2H-6 ), 4.29–4.30 (m, 1H, H-5 ),

ꢀ

5

1

(

.15 (t, 1H, H-4 ), 5.23 (t, 1H, H-3 ), 5.31 (t, 1H, H-2 ), 5.74–5.76 (d, 1H, J

0.70 Hz, H-1 ), 6.73 (brs, 2H, NH ), 8.34 (s, 1H, pyrazole H-2); C NMR: δ 21.25

4CH CO), 62.54 (C-6 ), 68.22 (C-4 ), 71.44 (C-2 ), 73.32 (C-3 ), 77.42 (C-5 ), 81.22

C-1 ), 92.25 (C-6), 93.12 (C-3), 116.33 (CN), 117.24 (CN), 132.52 (C-3 ), 155.44

C-2), 160.46 (C-5), 170.66 (4CO), 172.22 (C-6). Anal. Calcd. For C H N O S

ꢀ

1

–2 =

ꢀ

13

2

ꢀ

3

ꢀ

a

22

6

9

546.51): C, 48.35; H, 4.06; N, 15.38; S, 5.87%. Found: C, 48.45; H, 4.16; N, 15.40; S,

5.85%.

ꢀ

3

.1.2.8. 2,5-Diamino-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-galactopyranosylthio)-3–

(phenyldiazenyl)pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5h). Compound 5h

was puriﬁed by column chromatography (petroleum. ether/ethyl acetate 8:2,R =

f

1

−

0

.45–47 region); yellow solid; (EtOH); yield (80%); mp172°C; IR (KBr, cm )υ

1

3

470, 3436, 3029, 2207, 1743, 1596; H-NMR (500 MHz, DMSO-d ): δ 1.94–2.19

6

ꢀ

(4s, 12H, 4xOAc), 4.06–4.15 (m, 3H,2H-6 and H-5 ), 4.94–4.97 (t, 1H, H-4 ), 5.28–

ꢀ

5.38 (m, 2H, H-3 and H-2 ), 5.52–5.54 (d, 1H, J

ꢀ

10.3 Hz, H-1 ), 6.10 (s, 2H,

1

–2 =

NH ), 6.43 (s, 2H, NH ), 7.37–7.89 (m, 5H, C H ). Anal. Calcd. For C H N O S

2

6

5

27 28

8

9

(

5

640.62): C, 50.62; H, 4.41; N, 17.49; S, 5.01%. Found: C, 50.50; H, 4.35; N, 17.37; S,

.11%.

ꢀ

3

.1.2.9. 2,5-Diamino-3-((4-methoxyphenyl)diazenyl)-7-(2 ,3 ,4 ,6 -tetra-O-

acetyl-β-D-galactopyranosylthio)-pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5i).

Compound 5i was puriﬁed by column chromatography (petroleum. ether/ethyl

acetate 8:2,R = 0.62–64 region); yellow solid; (EtOH); yield (85%); mp201°C;

f

1

H-NMR (500 MHz, DMSO-d ): δ 1.92–2.10 (4s, 12H, 4xOAc), 3.41 (s, 3H,

OCH ), 3.98–4.10 (m, 2H, 2H-6 ), 4.11–4.12 (m, 1H, H-5 ), 4.92–4.94 (t, 1H, H-4 ),

.11–5.13 (t, 1H, H-3 ), 5.38 (t, 1H, H-2 ), 5.79–5.81 (d, 1H, J

.35 (s, 2H, NH ), 6.64 (s, 2H, NH ), 7.31–7.54 (m, 4H, C H ); C NMR: δ 20.17

4CH CO), 62.12 (C-6 ), 68.62 (C-4 ), 70.31 (C-2 ), 73.32 (C-3 ), 76.32 (C-5 ), 82.21

C-1 ), 89.41 (C-6), 90.21 (C-3), 115.45 (2C, Ar-C), 117.11 (CN), 123.51 (Ar-C),

27.68 (2C, Ar-C), 134.52 (C-3 ), 158.10 (C-2), 167.68 (C-5), 169.82 (4CO), 171.17

6

ꢀ

3

ꢀ

5

6

(

ꢀ

10.7 Hz, H-1 ),

1

–2 =

1

3

2

ꢀ

6

4

ꢀ

3

ꢀ

a

1

(

4

C-6). Anal. Calcd. For C H N O S (670.65): C, 50.15; H, 4.51; N, 16.17; S,

.78%. Found: C, 50.35; H, 4.45; N, 16.78; S, 4.62%.

28 30 8 10

ꢀ

3

.1.2.10. 2,5-Diamino-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-galactopyranosylthio)-3-

(

p-tolyldiazenyl)pyrazolo [1,5-a]pyrimidine-6-carbonitrile(5j). Compound 5j

was puriﬁed by column chromatography (petroleum ether/ethyl acetate 8:2,R =

f

1

0

.61–63 region); yellow solid; (EtOH); yield (82%); mp186°C; H-NMR (500 MHz,

DMSO-d )δ 2.05–2.09 (4s, 12H, 4xOAc), 2.69–2.70 (s, 3H, CH ), 4.12 (m, 1H,

6

3

ꢀ

H-5 ), 4.23 (t, 1H, H-4 ), 4.33–4.40 (m, 2H, 2H-6 ), 4.67 (t,1H, H-3 ), 5.23 (t, 1H,

H-2 ), 5.55–5.56 (d, 1H, J₁

ꢀ

3.8 Hz, H-1 ), 6.53 (s, 2H, NH ), 6.65 (s, 2H, NH ),

–2 =

2

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

13

7

.25–7.44 (m, 4H, C H ). Anal. Calcd. For C H N O S (654.65): C, 51.37; H,

6 4 28 30 8 9

4.62; N, 17.12; S, 4.90%. Found: C, 51.20; H, 4.70; N, 17.22; S, 4.76%.

ꢀ

3

.1.2.11. 2,5-Diamino-3-((4-chlorophenyl)diazenyl)-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-

β-D-galactopyranosylthio) pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5k). Com-

pound 5k was puriﬁed by column chromatography (petroleum ether/ethyl acetate

1

8

(

:2,R = 0.55–57 region); yellow solid; (EtOH); yield (86%); mp188°C; H-NMR

f

ꢀ

500 MHz, CDCl ): δ 2.02–2.36 (4s, 12H, 4xOAc), 3.89 (m, 1H, H-5 ), 4.12 (t, 1H,

H-4 ), 4.24–4.26 (t, 1H, H-3 ), 5.11–5.31 (t, 1H, H-2 ), 5.33–5.34 (m, 2H, 2H-6 ),

10.7 Hz, H-1 ), 6.20 (s, 2H, NH ), 6.34 (s, 2H, NH ),

.25–7.71 (m, 4H, C H ). Anal. Calcd. For C H ClN O S (675.07): C, 48.04; H,

3

ꢀ

5

7

4

.49–5.51 (d, 1H, J₁

ꢀ

–2 =

2

6

4

27 27

8

9

.03; N, 16.60; S, 4.75%. Found: C, 48.24; H, 4.27; N, 16.25; S, 4.80%.

ꢀ

3

.1.2.12. 2,5-Diamino-3-((4-bromophenyl)diazenyl)-7-(2 ,3 ,4 ,6 -tetra-O-acetyl-

β-D-galactopyranosylthio) pyrazolo[1,5-a]pyrimidine-6-carbonitrile(5l). Com-

pound 5l was puriﬁed by column chromatography (petroleum. ether/ethyl acetate

1

8

(

J₁

:2, R = 0.65–0.67 region); yellow solid; (EtOH); yield (80%); mp191°C; H-NMR

f

ꢀ

500 MHz, CDCl ): δ 1.99–2.05 (4s, 12H, 4xOAc), 3.71 (m, H, 2H-5 ), 4.12–4.23

m, 2H, H-6 ), 5.13 (t, 1H, H-2 ), 5.32–5.34 (m, 2H, H-3 , H-4 ), 5.49–5.51 (d, 1H,

9.9 Hz, H-1 ), 6.29 (s, 2H, NH ), 6.64 (s, 2H, NH ), 7.25–7.48 (m, 4H, C H ).

Anal. Calcd. For C H BrN O S (719.52): C, 45.07; H, 3.78; N, 15.57; S, 4.46%.

3

ꢀ

–2 =

2

6

4

27

8

9

Found: C, 45.25; H, 3.58; N, 15.45; S, 4.32%.



... Ammonolysis of b and i

Dry gaseous ammonia was passed through a solution of protected nucleoside 5b

or 5i (10 mmol)in dry methanol (20 mL) at room temperature for 10 minutes the

reaction mixture was stirred until the reaction was judged complete by TLC (10–

1

2 hours) using (CHCl / MeOH 9:1) (Rf, 0.76–0.78). The resulting mixture was then

3

concentrated under reduced pressure to aﬀord (6a or 6b) as solid residue that was

crystallized from appropriate solvent.

3

.1.3.1. 2,5-Diamino-7-(β-D-glucopyranosylthio)-3-(phenyldiazenyl)pyrazolo

[

1,5-a]pyrimidine-6-carbonitrile(6a). Yellow solid; (EtOH); yield (85%);

−

1

mp178°C; IR (KBr, cm ) υ 3631, 3407, 3331, 3102, 2220, 1620, 1573. H NMR

ꢀ

(

2

500 MHz, CDCl ) δ 3.85–3.92 (m, 2H, 2H-6 ), 4.11 (m, 1H, H-5 ), 4.50–4.60 (m,

H, H-4 , H-3 ), 4.70 (s, 1H, 2 -OH), 5.12 (m, 3H, 3 -OH, 4 -OH, and 6 -OH), 5.32

3

ꢀ

(

2

t, 1H, H-2 ), 5.56–5.58 (d, 1H, J

H, NH ), 7.42–7.68 (m, 5H, C H ). Anal. Calcd. For C H N O S (472.48): C,

ꢀ

9.95 Hz, H-1 ), 6.45 (s, 2H, NH ), 6.75 (s,

1

–2 =

2

2 6 5 19 20 8 5

48.30; H, 4.27; N, 23.72; S, 6.79%. Found: C, 48.40; H, 4.16; N, 23.65; S, 6.72%.

3

.1.3.2. 2,5-Diamino-3-((4-methoxyphenyl)diazenyl)-7-(β-D-galactopyranosyl-

hio)pyrazolo[1,5-a] pyrimidine-6-carbonitrile(6b). Yellow solid; (EtOH); yield

14

G. ELGEMEIE ET AL.

1

ꢀ

(

3

(

1

85%); mp185°C; H NMR (500 MHz, CDCl ) δ 3.70–374 (m, 3H, 2H-6 , H-5 ),

3

ꢀ

.82 (s, 3H, OCH ), 4.34 (m, 2H, H-4 , H-3 ), 4.61 (d, 2H, 2 -OH, 3 -OH), 4.84

3

ꢀ

d, 1H, 4 -OH), 5.47 (d, 1H, 6 -OH), 5.52 (t, H, H-2 ), 5.61–5.62 (d, 1H, J

0.95 Hz, H-1 ), 6.62 (s, 2H, NH ), 6.84 (s, 2H, NH ), 7.43–7.67 (m, 4H, C H ).

ꢀ

1

–2 =

ꢀ

2

6

4

Anal. Calcd. For C H N O S (502.14): C, 47.80; H, 4.41; N, 22.30; 6.38%. Found:

20

22

8

6

C, 47.72; H, 4.35; N, 22.28; S, 6.35%.



... General procedure for the synthesizing of a–e

A mixture of each of 4-(aryldiazenyl)-1H-pyrazole-3,5-diamine 1 (10 mmol) and 2-

dimercaptomethylene)malononitrile) 7 (10 mmol) was dissolved in DMF (20 mL)

(

in small beaker. The reaction mixture was put in a domestic microwave oven 600 W

and irradiate for 10 minutes at 154°C. The progress of the reaction was monitored

by TLC until the reactants disappeared, then the solution was concentrated and the

remaining residue was triturated with MeOH to aﬀord 9 which was puriﬁed by col-

umn chromatography (3:1 petroleum ether –EtOAc) and recrystallized from the

appropriate solvent.

3

.1.4.1. 2,5-Diamino-7-mercapto-3-(phenyldiazenyl)pyrazolo[1,5-a]pyrimidine-

−

1

6

-carbonitrile(9a). Brown solid; (MeOH); yield (82%); mp 284°C; IR (KBr, cm )

υ 3417, 3309, 3207, 3147, 3109, 2206, 1620, 1563; H NMR (500 MHz, DMSO-d ):

1

6

δ 6.47 (s, 2H, NH ), 6.75 (s, 2H, NH ), 7.24–7.67 (m, 5H, C H ), 12.21 (s, 1H, SH);

2

6

5

1

3

C NMR: δ 89.39 (C-6), 94.21 (C-3), 117.24 (CN), 128.61(4C, Ar-C), 128.82 (2C,

Ar-C), 135.14 (C-3 ), 156.45 (C-2), 166.36 (C-5), 191.51 (C-7); MS. m/z (%) 307

20) [M ]. Anal. Calcd. For C H N S (310.34): C, 50.31; H, 3.25; N, 36.11; S,

a

+

3

(

13 10 8

10.33%. Found: C, 50.30; H, 3.12; N, 36.21; S, 10.20%.

3

.1.4.2. 2,5-Diamino-7-mercapto-3-((4-methoxyphenyl)diazenyl)pyrazolo[1,5-

a]pyrimidine-6-carbonitril (9b). Brown solid; (MeOH); yield (85%); mp 272°C;

IR (KBr, cm ) υ 3433, 3406, 3296, 2909, 2210, 1673, 1649. H NMR (500 MHz,

CDCl )δ 3.80 (s, 3H, OCH ), 6.51 (s, 2H, NH ), 6.68 (s, 2H, NH ), 7.25–7.33 (m,

−

1

3

2

13

4H, C H ), 7.61 (s, 1H, SH); C NMR: δ 56.71 (CH ), 88.75 (C-6), 93.23 (C-3),

6

4

3

1

14.53 (2C, Ar-C), 117.62 (CN), 122.12 (4C, Ar-C), 129.61 (2C, Ar-C), 133.14

a

+2

(C-3 ), 155.13 (C-2), 166.26 (C-5), 194.53 (C-7); MS. m/z (%) 341 (18) [M ]. Anal.

Calcd. For C H N OS (340.36): C, 49.40; H, 3.55; N, 32.92; S, 9.42%. Found: C,

14

12

8

49.35; H, 3.40; N, 32.85; S, 9.25%.

3

.1.4.3. 2,5-Diamino-7-mercapto-3-(p-tolyldiazenyl)pyrazolo[1,5-a]pyrimidine-

6

-carbonitrile(9c). Yellow solid; (MeOH); yield (88%); mp 298–300°C; IR (KBr,

cm )υ 3407, 3306, 3117, 3062, 2206, 1596, 1696. H NMR (500 MHz, DMSO-d )

−

1

6

δ 2.69 (s, 3H, CH ), 6.23 (s, 2H, NH ), 6.62 (s, 2H, NH ), 7.15–7.55 (m, 4H, C H ),

3

2

6

4

13

7

.91 (s, 1H, SH); C NMR: δ 40.22 (CH ), 89.35 (C-6), 93.21 (C-3), 117.12 (2C,

Ar-C), 126.27 (CN), 128.36 (4C, Ar-C), 131.61 (2C, Ar-C), 132.31 (C-3 ), 156.33

3

a

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

15

(

C-2), 165.21 (C-5), 190.58 (C-7). Anal. Calcd. For C H N S (324.36): C, 51.84;

14 12 8

H, 3.73; N, 34.55; S, 9.89%. Found: C, 51.80; H, 3.61; N, 34.46; S, 9.77%.

3

.1.4.4. 2,5-Diamino-3-((4-chlorophenyl)diazenyl)-7-mercaptopyrazolo[1,5-

a]pyrimidine-6-carbonitrile (9d). Brown solid; (MeOH); yield (75%); mp 286°C;

1

H NMR (500 MHz, DMSO-d ): δ 6.61 (s, 2H, NH ), 6.75 (s, 2H, NH ), 7.45–

6

2

13

7

.58 (m, 4H, C H ), 11.99 (SH); C NMR: δ 89.35 (C-6), 93.22 (C-3), 117.12

6 4

a

(CN), 126.27 (Ar-C), 128.36 (2C, Ar-C), 131.61 (2C, Ar-C), 132.31 (C-3 ), 156.33

+

3

(C-2), 165.21 (C-5), 190.58 (C-7); MS. m/z (%) 347 (25) [M ]. Anal. Calcd. For

C H ClN S (344.78): C, 45.29; H, 2.63; N, 32.50; S, 9.30%. Found: C, 45.20; H,

13

9

8

2.55; N, 32.43; S, 9.25%.

3

.1.4.5. 2,5-Diamino-3-((4-bromophenyl)diazenyl)-7-mercaptopyrazolo[1,5-

a]pyrimidine-6-carbonitrile (9e). Brown solid; (MeOH); yield (86%); mp 282°C;

1

H NMR (500 MHz, DMSO-d ) δ 6.61 (s, 2H, NH ), 6.81 (s, 2H, NH ), 7.25–7.40

6

2

13

(

m, 4H, C H ), 11.12 (s, 1H, SH); C NMR: δ 89.35 (C-6), 93.22 (C-3), 117.12

CN), 123.24 (Ar-C), 130.13 (4C, Ar-C), 130.78 (C-3 ), 128.11 (C, Ar-C), 157.13

C-2), 163.22 (C-5), 191.33 (C-7). Anal. Calcd. For C H BrN S (389.23): C, 40.11;

6 4

a

13

9

8

H, 2.33; N, 28.79; S, 8.24%. Found: C, 40.06; H, 2.30; N, 28.65; S, 8.18%.



... General procedure for the synthesizing of (a-e)

Method A:

A solution of compounds 1 (10 mmol) and 2-(bis(methylthio)methylene) mal-

ononitrile 8 (10 mmol) dissolved in DMF (20 mL) and boiled at 154°C under

microwave 600 W for 10 minutes. The reaction was monitored by TLC (petroleum

ether–EtOAc 3;1) until the reactants disappeared. The solvent was then evaporated

and the remaining residue was triturated with MeOH to aﬀord compounds 10 which

was separated by column chromatography and recrystallized from the appropriate

solvent. Yield (86%).

Method B:

A solution of compounds 9 (10 mmol) in sodium ethoxide(10 mmol) was

reﬂuxed for 30 minutes allowed to cool to room temperature and methyl iodide

(

10 mmol) then was added slowly to this mixture, then the reaction mixture was

reﬂuxed with stirred for 3 hours. The formed solid product was collected by ﬁltra-

tion and recrystallized from appropriate solvent.

3

.1.5.1. 2, 5-Diamino-7-(methylthio)-3-(phenyldiazenyl)pyrazolo[1,5-a]pyrimid-

ine-6-carbonitrile(10a). Brown solid; (MeOH); yield (95%); mp >300°C; IR (KBr,

cm ) υ 3468, 3397, 2978, 2220, 1620, 1611. H NMR (500 MHz, CDCl ): δ 2.81 (S,

−

1

3

13

3

H, SCH ), 6.62 (s, 2H, NH ), 6.85 (s, 2H, NH ) 7.33–7.49 (m, 5H, C H ); C NMR:

3 2 2 6 5

δ 14.12 (CH ), 91.52 (C-6), 93.21 (C-3), 117.52 (CN), 128.91 (4C, Ar-C), 130.23

3

a

(

2C, Ar-C), 133.51 (C-3 ), 156.57 (C-2), 165.25 (C-5), 173.52 (C-6). Anal. Calcd.

1

6

G. ELGEMEIE ET AL.

For C H N S (324.36): C, 51.84; H, 3.73; N, 34.55; S, 9.89%. Found: C, 51.75; H,

14

12

8

3.70; N, 34.52; S, 9.80%.

3

.1.5.2. 2,5-Diamino-3-((4-methoxyphenyl)diazenyl)-7-(methylthio)pyrazolo

[1,5-a]pyrimidine-6-carbonitrile(10b). Brown solid; (MeOH); yield (95%); mp

1

>

300°C. H NMR (500 MHz, CDCl ): δ 2.33 (S, 3H, SCH ), 3.85 (s, 3H, OCH ),

3

13

6.30 (s, 2H, NH ), 6.51 (s, 2H, NH ), 7.33–7.61 (m, 4H, C H ); C NMR: δ 14.53

2 2 6 4

(

1

SCH ), 57.21 (OCH ), 91.22 (C-6), 95.11 (C-3), 115.12 (2C, Ar-C), 117.63 (CN),

22.4 (Ar-C), 128.79 (2C, Ar-C), 134.22 (C-3 ), 157.11 (C-2), 161.53 (Ar-C), 165.65

3 3

a

(

3

C-5), 174.24 (C-6). Anal. Calcd. For C H N OS (354.39): C, 50.84; H, 3.98; N,

1.62; S, 9.05%. Found C, 50.77; H, 3.78; S, 9.01%.

15 14 8

3

.1.5.3. 2,5-Diamino-7-(-methylthio)-3-(p-tolyldiazenyl)pyrazolo[1,5-a]pyrimid-

ine-6-carbonitrile(10c). Brown solid; (MeOH); yield (90%); mp >300°C; IR (KBr,

cm ) υ 3452, 3412, 3382, 2933, 2893, 2220, 1604, 1596. H NMR (500 MHz,

CDCl ): δ 2.51 (s, 3H, CH ), 2.90 (S, 3H, SCH ), 6.64 (s, 2H, NH ), 6.86 (s, 2H,

−

1

3

2

13

NH ), 7.32–7.79 (m, 4H, C H ); C NMR: δ 13.13 (SCH ), 21.23 (CH ), 90.82

2

6

4

3

(

C-6), 96.12 (C-3), 117.33 (CN), 125.71 (Ar-C), 128.72 (2C, Ar-C), 129.22 (2C,

a

Ar-C), 134.11 (C-3 ), 138.23 (Ar-C), 154.11 (C-2), 164.45 (C-5), 172.34 (C-7).

Anal. Calcd. For C H N S (324.36): C, 53.24; H, 4.17; N, 33.11; S, 9.48%. Found:

15

14

8

C,53.35; H, 4.20; N, 33.25; S, 9.35%.

3.1.5.4. 2,5-Diamino-3-((4-chlorophenyl)diazenyl)-7-(methylthio)pyrazolo[1,5-

a]pyrimidine-6-carbonitrile(10d). Brown solid; (MeOH); yield (90%); mp 300°C;

−

1

IR (KBr, cm ) υ 3472, 3417, 3018, 2943, 2218, 1614, 1598. H NMR (500 MHz,

DMSO-d ): δ 2.68 (s, 3H, SCH ), 6.66 (s, 2H, NH ), 6.87 (s, 2H, NH ), 7.32–7.76

6

3

2

(

m, 4H, C H ). Anal. Calcd. For C H ClN S (358.81): C, 46.86; H, 3.09; N, 31.23;

6 4 14 11 8

S, 8.94%. Found: C,46.70; H, 3.05; N, 31.20; S, 8.85%.

3

.1.5.5. 2,5-Diamino-3-((4-bromophenyl)diazenyl)-7-(methylthio)pyrazolo[1,5-

a]pyrimidine-6-carbonitrile(10e). Brown solid; (MeOH); yield (90%); mp

>

−

1

300°C; IR (KBr, cm ) υ 3458, 3402, 2943, 2883, 2219, 1620, 1596. H NMR

(

7

500 MHz, DMSO-d )δ 2.83 (s, 3H, SCH ), 6.65 (s, 2H, NH ), 6.88 (s, 2H, NH )

.34–7.78 (m, 4H, C H ). Anal. Calcd. For C H BrN S (403.26): C, 41.70; H,

6 4 14 11 8

6

3

2

2.75; N, 27.79; S, 7.95%. Found: C,41.76; H, 2.60; N, 27.68; S, 7.82%.



... General procedure for the synthesizing of (a–e)

A solution of 5 or 10 (10 mmol) and hydrazine hydrate (10 mmol) in ethanol

20 mL) containing a few drops of triethylamine was reﬂuxed for 5 hours,

(

cooled, the precipitate was ﬁltered oﬀ and crystallized from the appropriate

solvent.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

17

ꢀ

3

.1.6.1. 6-(Phenyldiazenyl)-1H-dipyrazolo[1,5-a:4 ,3 -e]pyrimidine-3,4,7-

−

1

triamine(11a). Yellow solid; (EtOH); yield (80%); mp >300°C; IR (KBr, cm )υ

480, 3390 (NH , NH); H NMR (DMSO)δ 6.61 (brs, 4H, 2NH ), 7.32–7.59 (m,

H, C H ), 7.71 (brs, 2H, NH ), 8.74 (s, 1H, NH); MS. m/z (%) 308 (22) [M ].

1

3

5

2

+

6

5

2

Anal. Calcd. for C H N (308.30): C, 50.64; H, 3.92, N, 45.43%. Found: C, 50.49;

13

12 10

H, 3.87; N, 45.36%.

ꢀ

3

.1.6.2. 6-((4-Methoxyphenyl)diazenyl)-1H-dipyrazolo[1,5-a:4 ,3 -e]pyrimidine-

3

,4,7-triamine(11b). yellow solid; (EtOH); yield (83%); mp >300°C; IR (KBr,

−

1

cm )υ 3577, 3408, 3314; H NMR (DMSO) δ 3.81 (s, 3H, OCH ), 6.36 (brs, 2H,

3

NH ), 6.48 (brs, 2H, NH ), 6.92 (m, 2H, phenyl protons), 7.38 (brs, 2H, NH ), 7.61

2

+

(

m, 2H, phenyl protons), 8.44 (brs, 1H, NH); MS. m/z (%) 338 (12) [M ]. Anal.

Calcd. for C H N O (338.33): C,49.70; H, 4.71; N, 41.40%. Found: C, 49.62; H,

14

14 10

4.57; N, 41.36%.

ꢀ

3

.1.6.3. 6-(p-Tolyldiazenyl)-1H-dipyrazolo[1,5-a:4 ,3 -e]pyrimidine-3,4,7-

−

1

triamine(11c). yellow solid; (EtOH); yield (76%); mp >300°C; IR (KBr, cm )υ

327. H NMR (DMSO) δ 3.91 (s, 3H, OCH ), 6.56 (s, 4H, 2NH ), 7.23–7.26 (m,

1

3

2

H, C H ), 7.36 (s, 2H, NH ), 7.54–7.57(m, 2H, C H ), 8.44 (s, 1H, NH). Anal.

6

2

6

2

Calcd. for C H N (322.33): C, 52.17; H, 4.38; N, 43.45%. Found: C, 52.12; H,

14

14 10

4.25; N, 43.35%.

ꢀ

3

.1.6.4. 6-((4-Chlorophenyl)diazenyl)-1H-dipyrazolo[1,5-a:4 ,3 -e]pyrimidine-

3

,4,7-triamine(11d). yellow solid; (EtOH); yield (82%); mp >300°C; IR (KBr,

−

1

cm )υ 3530, 3440 (NH , NH). Anal. Calcd. for C H ClN (342.75): C, 45.56;

2

13 11

10

H, 3.23, N, 40.87%. Found: C, 45.42; H, 3.15; N, 40.77%.

ꢀ

3

.1.6.5. 6-((4-Bromophenyl)diazenyl)-1H-dipyrazolo[1,5-a:4 ,3 -e]pyrimidine-

3

,4,7-triamine(11e). yellow solid; (EtOH); yield (75%); mp >300°C; IR (KBr,

−

1

cm ) υ 3458, 3400, 2220. H NMR (DMSO)δ 7.09 (brs, 4H, 2NH ), 7.28–7.49 (m,

2

4

H, C H ), 7.67 (brs, 2H, NH ), 10.01 (s, 1H, NH). Anal. Calcd. for C H BrN

6 4 2 13 11 10

(387.20): C, 40.33; H, 2.86, N, 36.17%. Found: C, 40.25; H, 2.78; N, 36.10%.

3.2. Biological assays

... Antitumor screening

Cell viability was assessed by the mitochondrial dependent reduction of yellow

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple

[23]

formazan.

Procedure: All the following procedures were done in a sterile area using a Lam-

inar ﬂow cabinet biosafety class II level (Baker, SG403INT, Sanford, ME, USA).

1

8

G. ELGEMEIE ET AL.

Cells were suspended in RPMI 1640 medium for HePG2- PC3 and HCT116–

DMEM for A549. The media are supplemented with 1% antibiotic-anti mycotic

mixture(10,000U/ml Potassium Penicillin, 10,000 μg/ml Streptomycin Sulfate and

2

5 μg/ml Amphotericin B), 1% L-glutamine and 10% fetal bovine serum and kept at

3

7°C under 5% CO . Cells were batch cultured for 10 days, then seeded at concentra-

tion of 10 × 10 cells/well in fresh complete growth medium in 96-well microtiter

2

3

plastic plates at 37°C for 24 hours under 5% CO using a water jacketed Carbon

2

dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated,

fresh medium (without serum) was added and cells were incubated either alone

(

negative control) or with diﬀerent concentrations of sample to give a ﬁnal con-

centration of (100–50-25–12.5–6.25–3.125–0.78 and 1.56 ug/ml). After 48 hours

of incubation, medium was aspirated, 40ul MTT salt (2.5 μg/ml) were added to

each well and incubated for further four hours at 37°C under 5% CO . To stop

2

the reaction and dissolve the formed crystals, 200 μL of 10% Sodium dodecyl sul-

phate (SDS) in deionized water was added to each well and incubated overnight at

3

7°C. A positive control which composed of 100 μg/ml was used as a known cyto-

[19,24]

toxic natural agent who gives 100% lethality under the same conditions.

The

absorbance was then measured using a microplate multi-well reader (Bio-Rad Lab-

oratories Inc., model 3350, Hercules, California, USA) at 595 nm and a reference

wavelength of 620 nm. A statistical signiﬁcance was tested between samples and

negative control (cells with vehicle) using independent t-test by SPSS 11 program.

DMSO is the vehicle used for dissolution of plant extracts and its ﬁnal concentration

on the cells was less than 0.2%. The percentage of change in viability was calculated

according to the formula: ((Reading of extract / Reading of negative control) -1) x

1

00.A probit analysis was carried for LC and LC determination using SPSS 11

50 90

program.

4

. Conclusion

We have developed a new and simple method for the synthesis of the purine and

guanine thioglycoside analogs under microwave activation. The mild reaction con-

ditions, clean reaction proﬁles, zero side product and cost eﬃciency render this

approach as a useful and innovative to the existing methods for glycoside formation.

Further studies on the application of this method for the synthesis of other highly

functionalized biologically active glycosides are underway. The antitumor activities

of the synthesized compounds were tested against human tumor cell lines: (lung

(A549), colon (HCT116), liver (HEPG2), and prostate (PC3)).

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Article Doi

A First Microwave-Assisted Synthesis of a New Class of Purine and Guanine Thioglycoside Analogs

DOI: 10.1080/15257770.2016.1202964

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