Synthesis and Biological Evaluation of Some Novel Pyrimidine Derivatives Derived from Chalcones

 

K. Ishwar Bhat, Abhishek Kumar*, Pankaj Kumar and Riyaz E.K

Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences ,Nitte University, Paneer, Deralakatte-575018, Mangalore, Karnataka

*Corresponding Author E-mail: abhi12bunty@gmail.com

 

ABSTRACT:

A series of novel 6-(substituted phenyl)-4-(nitrophenyl) pyrimidin-2-ol (RPY1-RPY8) have been synthesized upon refluxing with 3-(4-nitrophenyl)-1-phenyl substituted prop-2-en-1-one in presence of thiourea and glacial acetic acid. 3-(4-nitrophenyl)-1-phenyl substituted prop-2-en-1-one (RC1-RC8) were prepared by reaction with 4-nitrobenzaldehyde  and substituted acetophenones in presence of NaOH. The structures of the final synthesized compounds were confirmed by IR, 1H NMR and mass spectra.                                                                              

 

All the new compounds were screened for their in-vitro antimicrobial, antitubercular and cytotoxic activities. Most of the compounds have exhibited promising antibacterial, antifungal, antitubercular and cytotoxic activities.                       

 

KEYWORDS: Chalcones, pyrimidines, antibacterial activity, antifungal activity, antitubercular activity, cytotoxic activity

 


 

INTRODUCTION:

Pyrimidine are the most important six membered heterocyclic compounds having two nitrogen atoms. Pyrimidine occur in living systems in the form of nucleic acids and vitamins; since it is the basic nucleus in DNA and RNA and possess diverse biological activities. Pyrimidine derivatives are reported to possess analgesic1, anti-inflammatory2, antitumor3,4, antimicrobial5, antibacterial6, antifungal7, antiplatelet8 and antitubercular9 activities. Pyrimidine heterocycles possessing hydroxyl group has a unique place in medicinal chemistry and also plays a vital role in biological processes as well as synthetic drugs. Chalcones act as intermediates in the biosynthesis of various flavonoids and also have been used as intermediates in the synthesis of various pharmacologically significant heterocyclic molecules such as pyrimidines, pyrazolines, isoxazolines and benzodiazepines.                                  

 

By considering the above facts it was contemplated to synthesize a new series of pyrimidine derivatives (RPY1-RPY8). The final synthesized compounds were screened for their in-vitro antibacterial, antifungal, antitubercular and cytotoxic activities and compared with standard drugs.

  

MATERIALS AND METHODS:

All the chemicals were of analytical grade: 4-nitrobenzaldehyde, substituted    acetophenones, thiourea, glacial acetic acid, ethanol and sodium hydroxide.

 

Melting points were determined by open capillary method and are uncorrected. Purity of the intermediates and final compounds were monitored by thin layer chromatography (TLC) using silica gel G plates. The spots were visualized under UV light and by the exposure to iodine vapors. Ethylacetate: Acetone (1:9) was used as solvent for running the TLC of these compounds. All IR spectra were recorded in Alpha Bruker using ATR method. 1H NMR spectra were recorded on Bruker spectrophotometer (400 MHz) in DMSO-d6 solvent using tetra methyl silane (TMS) as an internal standard.. Mass spectra was recorded by LCMS method.

 

General Procedure:

Synthesis of 3-(4-nitrophenyl)-1-phenyl substituted prop-2-en-1-one10 (RC1-RC8):

A mixture of 4-nitrobenzaldehyde (0.01 mol) and substituted acetophenones (0.01 mol) in ethanol were stirred for 24 hrs in presence of 40% NaOH. The completion of the reaction was monitored by TLC. The mixture was poured into crushed ice and acidified with 5% HCl. The product was filtered and recrystallised from ethanol.

 

Synthesis of 6-(substituted phenyl)-4-(nitrophenyl) pyrimidin-2-ol11 (RPY1-RPY8):

A mixture of substituted chalcones (0.01 mol) in ethanol/glacial acetic acid and thiourea (0.01 mol) in 20% NaOH was refluxed for 20 hrs. After completion of the reaction, reaction mixture was poured into ice cold water, filtered and recrystallised from ethanol. Completion of the reaction was monitored by TLC using ethylacetate: acetone (1:9) as solvent.

 

Figure 1: Reaction scheme for Pyrimidine derivatives

 

Spectral data:

1-(4-aminophenyl)-3-(4-nitrophenyl)prop-2-en-1-one (RC3)

IR KBr (cm-1): 1722(C=O str), 1670(CH=CH str), 1590(Ar C=C str), 3100(Ar C-H str), 1355(Ar-NO2 C-H str), 3322 (NH2 str).  

1H NMR (400 MHz, DMSO-d6): δ 7.91-8.12 (m, 10H, Ar-H), 6.63 (d, 1H, =CH), 6.95 (d, 1H, =CH), 2.56 (s, 2H, NH2). 

MS (M+): m/z 268.

4-(4-nitrophenyl)-6-phenylpyrimidin-2-thiol (RPY1)

IR KBr (cm-1): 1506(Ar C=C str), 836(Ar C-H bending), 3031(Ar C-H str), 1678(C=N), 2052(S-H str), 1360(Ar-NO2 C-H str). 

1H NMR (400 MHz, DMSO-d6): δ 7.21-7.76 (m, 10H, Ar-H), 9.37 (s, 1H, SH).

MS (M+): m/z 309.

 4-(4-fluorophenyl)-6-(4-nitrophenyl)pyrimidin-2-thiol (RPY2)

IR KBr (cm-1): 1508(Ar C=C str), 839(Ar C-H bending), 3028(Ar C-H str), 1676(C=N), 2056(S-H str), 1358(Ar-NO2 C-H str), 1220(C-F str).

1H NMR (400 MHz, DMSO-d6): δ 7.23-7.95 (m, 9H, Ar-H), 9.42 (s, 1H, SH).

MS (M+): m/z 327.

4-(4-nitrophenyl)-6-p-tolylpyrimidin-2-thiol (RPY7)

IR KBr (cm-1): 1512(Ar C=C str), 840(Ar C-H bending), 3025(Ar C-H str), 1672(C=N), 2046(S-H str), 1356(Ar-NO2 C-H str).

1H NMR (400 MHz, DMSO-d6): δ 7.13-7.78 (m, 9H, Ar-H), 4.63 (s, 6H, CH3), 9.54 (s, 1H, SH).

MS (M+): m/z 323.

 

Antimicrobial Activity:

All the synthesized compounds were evaluated for their antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis using cup plate method12. The synthesized test compounds were tested at a concentration of  100 µg/50µl and the standard compound i.e. Ciprofloxacin were tested at 25 µg/50µl. Dimethyl formamide (DMF) was used as control. In this technique, melted agar inoculated with microorganisms is poured into petridishes. Wells are made in the agar plate and a specific volume of the antimicrobial substances are placed in them, plates were incubated at a temperature of 37oC for 24 hrs. The antimicrobial substance diffuses through agar around its well and produces a clear zone of inhibition. The diameter of this zone (mm) gives an estimation of the degree of activity of the antimicrobial substance.

 

Antitubercular Activity:

The antitubercular activity of test compounds were assessed against Mycobacterium tuberculosis using microplate Alamar blue assay13. This methodology is non toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method. 200 µl of sterile deionized water was added to all outer perimeter wells of sterile 96 well plate to minimised evaporation of medium in the test wells during incubation. The 96 well plate received 100  µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate. The final drug concentrations of the tested compounds were 0.01 to 20.0 µl/ml. The plates were covered and sealed with parafilm and incubated at 37οC for 5 days. After this, 25  µl of freshly prepared 1:1 mixture of Alamar blue reagent and 10% tween 80 was added to the plate and incubated for 24 hours. A blue color in the well was interpreted as no bacterial growth and pink color was interpreted as growth. The minimum inhibitory concentration was defined as lowest drug concentration which prevented the color change from blue to pink.

 

Cytotoxicity Activity:

All the test compounds were studied for short term in vitro cytotoxicity against Ehrlich Ascites Carcinoma cells (EAC) cells. The tumor cells aspirated from peritoneal cavity of tumor bearing mice was washed thrice with normal saline and checked for viability using Tryphan blue exclusion method14. The cell suspension (1 million cells in 0.1 ml) was added to tubes containing various concentrations of the test compounds and volume was made upto 1 ml using phosphate buffered saline. Control tubes contained only cell suspension. The assay mixtures were incubated for 3 h, at 37οC and then percent of dead cells were evaluated by tryphan blue exclusion method.

 


 

RESULTS AND DISCUSSION:

Table 1: Physicochemical data of the synthesized Pyrimidine derivatives (RPY1-RPY8)

Comp. code

R

Mol. formula

Mol. wt

M.P oC

Rf Value

% Yield

RPY-1

H

C16H11N3O2S

309

211-213

0.62

78

RPY-2

4-F

C16H10FN3O2S

327

206-208

0.76

70

RPY-3

4-NH2

C16H12N4O2S

324

220-222

0.56

69

RPY-4

4-Cl

C16H10ClN3O2S

343

237-239

0.66

72

RPY-5

4-CH3

C17H13N3O2S

323

260-262

0.70

75

RPY-6

4-OCH3

C17H13N3O3S

339

201-203

0.60

68

RPY-7

2-CH3

C17H13N3O2S

323

252-254

0.72

80

RPY-8

2-OCH3

C17H13N3O3S

339

216-218

0.68

72

 

Table 2: Antimicrobial activity of the compounds (RPY1-RPY8) by cup plate method

Compound

Diameter of zone of inhibition (mm)

S.aureus

B.subtilis

E.coli

P.aureginosa

C.albicans

A.niger

RPY1

15

16

17

10

09

07

RPY2

18

20

11

13

08

08

RPY3

16

18

15

15

08

07

RPY4

10

09

09

08

07

09

RPY5

11

10

09

12

10

11

RPY6

16

21

20

18

12

09

RPY7

07

09

-

10

09

08

RPY8

18

17

17

15

07

06

Ciprofloxacin

26

26

22

23

-

-

Fluconazole

-

-

-

-

20

21

Control

-

-

-

-

-

-

 

Table 3: Antitubercular activity of compounds (RPY1-RPY8) by Microplate Alamar blue assay

Compounds

Conc. (µg/ml)

100

50

25

12.5

6.25

3.125

1.6

0.8

0.4

0.2

RPY1

S

S

S

S

R

R

R

R

R

R

RPY3

S

S

S

R

R

R

R

R

R

R

RPY5

S

S

S

S

S

R

R

R

R

R

RPY7

S

S

S

S

S

R

R

R

R

R

RPY8

S

S

S

R

R

R

R

R

R

R

Streptomycin

S

S

S

S

S

R

R

R

R

R

Pyrazinamide

S

S

S

S

S

S

R

R

R

R

 

 


Table 4: Cytotoxicity activity of compounds (RPY1-RPY8) by Trypan Blue exclusion method

Compounds

No. of dead cells (%) at different concentrations (µg/ml)

10

20

50

100

200

Control

-

-

-

-

-

RPY2

9

19

24

45

67

RPY3

5

15

21

32

46

RPY7

3

9

15

22

38

RPY8

11

21

30

48

69

5-Fluorouracil

20

36

51

85

96

 

Antimicrobial activity:

The in vitro antibacterial and antifungal activity of the synthesized compounds were determined by using cup-plate method. The results of antibacterial and antifungal activity of newly synthesized compounds are reported  against Bacillus subtilis, Staphylococcus aureus, Escherichia  coli, Pseudomonas  aeruginosa and Candida albicans and Aspergillus niger respectively. Compounds RPY1, RPY2,

 

RPY3, RPY6 and RPY8 showed good antibacterial activity compared to the standard drug ciprofloxacin. All the test compounds RPY1-RPY8 showed moderate antifungal activity compared to the standard drug fluconazole. The results of the antimicrobial activity are summarized in Table 2.

 

Antitubercular Activity:

The test compounds were evaluated for their antitubercular activity against M.tuberculosis using Microplate Alamar Blue assay. A blue colour in the well was interpreted as no bacterial growth and pink colour was scored as growth. This indicates that the test compound has potent antitubercular activity in in-vitro condition. Compound RPY5 and RPY7 induced the greatest effect on bacterial growth with an activity at a concentration of 6.25µg/ml compared to the standard drug streptomycin and pyrazinamide. The results of the antitubercular activity are summarized in Table 3.

 

Cytotoxicity Activity:

The test compounds were subjected to in vitro cytotoxicity against Ehrlich Ascites Carcinoma (EAC) cells using “Tryphan Blue exclusion method”. The damaged cells are stained blue by Tryphan blue stain and can be distinguished from viable cells. Compounds RPY2 and RPY8 induced the greatest effect on EAC cells with an activity more than 60% at a concentration of 200µg/ml. The results of the cytotoxicity activity are summarized in Table 4.

 

CONCLUSION:

This study reports the successful synthesis of substituted pyrimidine derivatives with moderate yields and most of the synthesized compounds showed potent antimicrobial, antitubercular  and cytotoxicity activity.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Nitte University for providing the necessary facilities to carry out this research. The authors are grateful to Head, SAIF, Punjab University, Chandigarh for providing spectroscopic data, Amala Cancer Research Centre, Thrissur.

 

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Received on 12.07.2014                Modified on 20.07.2014

Accepted on 10.08.2014                © RJPT All right reserved

Research J. Pharm. and Tech. 7(9): Sept. 2014  Page 995-998