Synthesis and Characterization of 1-Substituted Tetra Hydro Pyrimidine Derivatives by Leuckart Reaction

 

Elavarasi. R, Tharabai. R, Vinitha. R, and  Dr. Abdul Hasan Sathali A.*

Department of  Pharmaceutical Chemistry, College of Pharmacy, Madurai Medical College, Madurai-625020, Tamilnadu, India.

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

 

ABSTRACT:

Ten (E1-E2) compounds of 1-Substituted tetra hydro pyrimidine derivatives synthesized by leuckart reaction using microwave irradiation. The structure of synthesized ten compounds were elucidated by IR, MASS, H’NMR Spectroscopy. Further these compounds were evaluated for invitro anticancer activity, antibacterial activity, antifungal activity. Invitro antioxidant activity was carried out DPPH (Diphenylpicrylhydrazyl) method. Invitro anticancer activity (osteosarcoma) was carried out by MTT assay (3-[4,5-dimethyl thiazol-2yl]2,5-diphenyl tetrazolium bromide) using human osteosarcoma cell line.  Antibacterial, Antifungal activity was carried out by disc- diffusion method, using muller-hinton agar medium and potato dextrose agarmedium. All compounds, were found to have good antibacterial activity and 4 compounds (E3) 2-amino-6-[4-(dimethyl amino)phenyl]-4-oxo-1-(1-phenyl ethyl)1,4,5,6-tetra hydro pyrimidine 5-carbonitril, (E4) 2-amino -4-oxo [2-phenyl ethenyl]-1-(1-phenyl ethyl) 1,4,5,6 tetra hydro pyrimidine 5- carbonitrile, (E8) 2 amino -6-[4-( dimethyl amino) phenyl] 1-diphenyl methyl-4-oxo 1,4,5,6 tetra hydro pyrimidine -5- carbonitrile,(E9) 2 amino-1-(diphenyl methyl)-4-oxo-6-[(E)-2-phenylethenyl] 1,4,5,6-tetra hydro pyrimidine 5-carbonitrile were found to be posses antibacterial activity as compared to standard drugs.  All compounds having anticancer activity and the 2 compounds (E3 and E4) showed the potent anticancer activity with least effect on normal cells.

 

KEYWORDS: Tetrahydropyrimidine, microwave irradiation, leuckart reaction, anticancer activity (osteosarcoma).

 


 

 

INTRODUCTION:

Pyrimidine is an important heterocyclic molecule is associated with several biological activities. Pyrimidines are heterocyclic compound that have an atom of nitrogen at 1’st  and 3’nd position. Pyrimidine is the useful intermediate for the development of many chemotherapeutic agents. It is one of the main pyrimidine nucleuses in anticancer and antiviral agent. Various potent drugs in market contains pyrimidine nucleus like pyrantelpamoate (anthelmintic), flucytosine (antifungal), minoxdil (antihypertension), fluorouracil and floxuridine (antineoplastic), pyrimethamine (antimalarial), idoxuridine and trifluridine (antiviral)[1,8]. The important pyrimidine compounds have diverse applications as bactericidal, fungicidal, analgesic, anti inflammatory, anticancer, antiviral, antimalarial, anthelmintic, antihypertension etc.1-Substituted tetra hydro pyrimidine derivatives are synthesized by the reaction of ketone with tetra hydro pyrimidine derivatives of secondary amine and formic acid undergoes the leuckart reaction.

 

This reaction is reductive amination process which converts primary or secondary amine to tertiary amine using protonated ketone and formic acid. This rection avoid the problem of quterization[2,5].

 

MATERIALS AND METHODS:

Melting point of synthesized compounds were determined in open capillary tube method. The I.R spectra were recorded on Shimadzu FTIR Spectrophotometer. The NMR spectra were recorded on Bruker-NMR 400 mhz using dimethyl sulphoxide (DMSO) and chemical shifts were recorded in parts per million (ppm) and Tetra methyl silane (TMS) used as reference standard in NMR spectroscopy. The MASS spectra were recorded on JEOL mass spectrometer. Purity of the synthesized compounds was checked by  Thin Layer Chromatography (TLC) using silica gel (G) as adsorbent and visualization was detected by iodine vapour.[3,13]

 

Chemistry: Scheme of reaction:

Step:1 Preparation of 2-amino - 4-oxo -6-aryl 1,4,5,6 –tetrohydropyrimidine -5-carbonitrile


 

                                                                                                1. P-Chlorobenzaldehyde

                                                                                                2. P-Methoxybenzaldehyde

                                                                                                3. p-Dimethylaminobenzaldehyde

                                                                                                4. Cinnamaldehyde

                                                                                                5. Bezaldehyde

 

 


A mixture of Aromatic aldehyde (1mmol), guanidine nitrate (1.5mmol), ethyl cyanoacetate (1.2mmol) and catalytic amount of potassium carbonate was taken in a round bottom flask (100ml) with water as a solvent and refluxed at 100◦C. The solution was poured into ice cold water. After the completion of the reaction the solid product was collected by filtration. The product was dried and recrystallized from hot ethanol to obtained pure product.

 

Step 2: preparation of 1-substituted tetrahydropyrimidine derivatives from 2-amino- 4-oxo - 6-aryl 1, 4, 5, 6 tetrahydropyrimidine -5-carbonitrile.


 

 


ketone-  Acetophenone, Benzophenone


COMPOUND

R1

R2

D-E1-E5

CH3

C6H5

D-E6-E10

C6H5

C6H5


Procedure:

Ketone (1.5ml), 2-amino-6-(4-chlorophenyl)-4-oxo1,4,5,6 tetrahydropyrimidine-5-carbonitrile (1.5gm) and formic acid (1.5ml) was irradiated in microwave at 80◦C for 4minutes in equimolar quantities. The solution was obtained and then cooled in ice bath until the crystals are formed. The crude product was obtained and washed with ice cold water and air dried.

 

Biological activity:

Invitro antioxidant activity:

The free radical scavenging activity of the synthesized compound is evaluated by assessing their ability to reduce the colour of DPPH (Diphenyl Picryl Hydrazyl Radical) in chloroform. DPPH stable free radical method is an easy, rapid sensitive way to survey the antioxidant activity of specific compound[14]. The data for the Antioxidant activity of synthesized compound were given in table no.4


Table No: 1 Physical Data Analysis

Compound code

Chemical name

Molecular formula

D – E1

2-amino -6-(4-chloro phenyl)-4-oxo -1-(1-phenylethyl)1,4,5,6-tetrahydro pyrimidine  -5-carbonitrile

C19H17ClN4O

D – E2

2-amin  -6-(4-methoxy phenyl) -4-oxo -1- (1-phenylethyl) 1,4,5,6-tetrahydropyrimidine -5-carbonitrile

C20H20N4O2

D – E3

2-amino -6-[4-(dimethyl amino) phenyl] -4-oxo -1-(1-phenylethyl) 1,4,5,6-tetrahydropyrimidine -5-carbonitrile

C21H23N5O

D – E4

2-amino -4-oxo [(E)-2-phenylethenyl] -1-(1-phenylethyl) 1,4,5,6-tetrahydropyrimidine -5-carbonitrile

C21H20N4O

D – E5

2-amino -4-oxo -6-phenyl -1-(1-phenylethyl- 1,4,5,6-tetrahydropyrimidine -5-carbonitrile

C19H18N4O

D – E6

2-amino -6-(4-chloro phenyl) -1-(diphenylmethyl) -4-oxo 1,4,5,6-tetrahydropyrimidine -5-carbonitrile

C24H19ClN4O

D – E7

2-amino -1-(diphenylmethyl) -6-(4-methoxy phenyl) -4-oxo 1,4,5,6-tetrahydropyrimidine -5-carbonitrile

C25H22N4O2

D – E8

2-amino -6-[4-(dimethyl amino) phenyl] -1-(diphenylmethyl) -4-oxo 1,4,5,6-tetrahydropyrimidine -5-carbonitrile

C26H27N5O

D – E9

2-amino -1-(diphenylmethyl) -4-oxo -6-[(E)-2-phenylethenyl] 1,4,5,6-tetrahydropyrimidine -5-carbonitrile

C26H22N4O

D – E10

2-amino -1-(diphenylmethyl) -4-oxo -6-phenyl 1,4,5,6-tetrahydro pyrimidine -5-carbonitrile

 C24H20N4O

 

Table no: 2

Compound code

Percentage yield

Melting point 0C

Molecular weight

Rf value

D – E1

75%

174

352.82

0.74

D – E2

68%

180

348.39

0.68

D – E3

82%

236

361.44

0.75

D – E4

62%

240

344.40

0.65

D – E5

55%

225

318.37

0.64

D – E6

80%

252

414.88

0.67

D – E7

64%

263

410.46

0.70

D – E8

74%

245

425.52

0.72

D – E9

58%

208

406.47

0.63

D – E10

67%

196

380.44

0.71

 

Table no:3 Spectral  data analysis

Compound code

IR(KBR,CMˉą)

ąH NMR

MS (M/Z M+)

D – E1

1612 (C=Ostr) 3433 (N-H)str1446 (C=N)  str 2223 (C≡N) str1309(C- N)str 2872(C-H) str 709(Ar-Cl)

δ2.5(2H)NH2δ1.2(3H)CH3δ2.9(1H)CH-CN δ2.2(1H)CH-N δ7.4(9H)Ar-H δ3.3(1H)CH-Cl

352.82

 

D – E2

1637(C=O)str3368(N-H)str1502(C=N) str2216(C≡N)str1275(C-N) str2943(C-H) str 2834(OCH3)

δ3.4(2H)NH2δ2.5(3H)CH3 δ2.5(1H)CH-CN   δ3.5(1H)CH-N  δ7.3(9H)Ar-H)δ3.7(3H)OCH3

348.39

 

D – E3

1660(C=O)str  3730 (N-H) str) 1431(C=N) str 2208(C≡N) str  1371(C-N) str 2860(C-H) str  1327(N(CH3)2)

δ2.2(2H)NH2  δ1.3(3H)CH3   δ2.5(1H) CH-CN  δ4.2(1H) CH-N   δ7.6(9H)Ar-H δ6.5 N(CH3)2

361.44

 

 

D – E4

1612(C=O) str  3300(N-H) str 1448(C=N) str 2220(C≡N) str 1085(C-N)str 2852(C-H) str  1658(C=C)str  3134(=CH) str

δ2.7(2H)NH2  δ1.2(3H)CH3   δ2.4(1H)CH-CN  δ3.9(1H) CH-N  δ7.4(10H) Ar- H  δ2.1( CH =CH)

344.40

 

 

D – E5

1598(C=O)str 3311(N-H str 1444(C=N) str 2223(C≡N) str 1010(C-N)str 2850(C-H) str  2850(Ar-H) str

δ2.5(2H)NH2   δ1.3(3H)CH3   δ2.7(1H)CH-CN  δ2.5(1H) CH-N δ7.8(10H) Ar- H

318.37

 

D –E6

1658(C=O) str 3315(N-H) str 1481(C=N) str 2223(C≡N) str  1001(C-N) str 638(Ar-Cl)

δ2.6(2H)NH2   δ2.8(1H)CH-CN  δ2.1(1H) CH-N   δ7.3(14H)Ar-H    δ3.2(1H)CH-Cl

414.88

 

D – E7

1654(C=O) str 3414(N-H) str 1598(C=N) str 2216(C≡N) str 1018(C-N) str846(OCH3)

 δ3.3(2H)NH2   δ2.6(1H)CH-CN  δ2.2(1H) CH-N   δ7.3(14H)Ar-H    δ 3.7(3H)OCH3

410.46

 

D – E8

1548(C=0) str 3458(N-H) str 1562(C=N) str 2250(C≡N) str 1064(C-N) str 1317(N(CH3)2)

δ2.1(2H)NH2   δ2.6(1H)CH-CN  δ4.3(1H) CH-N   δ7.5(14H)Ar-H   δ6.5(6H)N(CH3)2

425.52

 

D – E9

1610(C=O) str 3380(N-H) str 1597(C=N) str 2220(C≡N) str  1085(C-N) str 1658(C=C) str  3028(=CH) str

  δ2.5(2H)NH2    δ2.8(1H)CH-CN  δ3.8(1H) CH-N   δ7.2(15H)Ar-H   δ2.1(2H)CH=CH

406.47

 

D – E10

1658(C=O)str 3309(N-H)str 1442(C=N) str 2223(C≡N) str  1010(C-N) str 3001(Ar-H) str

  δ3.1(2H)NH2   δ2.6(1H)CH-CN   δ2.2(1H)CH-N  δ7.7(14H)Ar- H

380.44

 

 

Table no.4 Antioxidant activity of synthesized compounds

Compound code

Absorbance of different concentration

50µg/ml

100µg/ml

150µg/ml

D – E1

0.910±0.0095

0.776±0.0049

0.585±0.0083

D – E2

0.947±0.0066

0.815±0.0052

0.560±0.0092

D – E3

0.747±0.0069

0.533±0.0052

0.355±0.0050

D – E4

0.950±0.0084

0.805±0.0080

0.513±0.0053

D – E5

0.870±0.0037

0.759±0.0025

0.467±0.0049

D – E6

0.808±0.0083

0.744±0.0078

0.402±0.0083

D – E7

0.791±0.0063

0.603±0.0087

0.397±0.0060

D – E8

0.782±0.0038

0.586±0.0036

0.383±0.0084

D – E9

0.761±0.0049

0.568±0.0073

0.364±0.0043

D – E10

0.977±0.0069

0.758±0.0060

0.534±0.0034

Standard

1.288±0.0221

0.829±0.0094

0.516±0.0043

The in vitro antioxidant activity for all the compounds showed positive results. The compounds E2, E3, E4, E6, E7, E9 showed more potent activity.

 

 


In vitro anticancer activity:

The human osteosarcoma cell line (MG 63) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS). The cells were maintained at 370C, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. The anticancer activity done by MTT assay [14,15]. 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore,the amount of formazan produced is directly proportional to the number of viable cells.After 48 h of incubation, 15µl of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and then measured the absorbance at 570 nm using micro plate reader. The percentage cell viability was then calculated with respect to control as follow % Cell viability = [A] Test / [A]control x 100The result of anticancer activity of the compounds were shown in Table no.5.

 

Antibacterial and antifungal activity:

The antibacterial and antifungal activities of the synthesized compounds were studied by disc diffusion method. The antibacterial activity of the compounds evaluated against E.coli, Staphylococcus epidermidis and Streptococcus pyrogenes [4]. Antifungal activity of the compounds evaluated  against Candida albicans and Aspergillus parasiticus [7]. The results were shown in table no.6 and 7.

 

RESULTS AND DISCUSSION:

The compounds were synthesized by “Leuckart reaction” which shows good percentage yield and their physical data is given in Table no.1and 2. The structure of the synthesized compounds were elucidated by IR, NMR, MASS spectroscopy and their spectral data is given in Table no. 3. The purity of the compounds evaluated by TLC method.

Table no.5 In vitro anticancer activity  of synthesized compounds

Compound code

Concentration

(µM)

℅ Cell viability

D – E2

0.1

1

10

50

100

101

100

95

92

80

D – E3

0.1

1

10

50

100

94

92

88

69

57

D – E4

0.1

1

10

50

100

100

97

94

73

70

D – E7

0.1

1

10

50

100

102

100

96

91

86

D – E8

0.1

1

10

50

100

91

89

85

67

54

D – E9

0.1

1

10

50

100

101

97

90

85

60

 

Antibacterial and antifungal activity:

All synthesized compounds showed antibacterial and antifungal activity. Compounds E3, E4, E8 and E9 have more active as compared to standard drug.

 


 

Table no:6 Antibacterial activity of synthesized compounds

Compound

Code

Zone of Inhibition in mm

E. coli

Staph. epidermidis

Strep. pyogenes

150µg/ml

300µg/ml

150µg/ml

300µg/ml

150µg/ml

300µg/ml

D – E1

4

11

R

5

12

12

D – E2

5

8

9

12

4

7

D – E3

12

15

7

15

11

14

D – E4

9

12

10

14

8

16

D – E5

8

7

12

14

R

6

D – E6

9

10

R

R

9

11

D – E7

6

6

4

7

6

9

D – E8

7

9

8

11

8

14

D – E9

9

12

12

16

7

13

D – E10

8

10

6

9

4

10

Control

R

R

R

R

R

R

Standard

17

18

18

 

 


Table no:7 Antifungal activity of synthesized compounds

Compound code

Zone of inhibition in mm

Candida albicans

Aspergillus paraciticus

150µg/ml

300µg/ml

150µg/ml

300µg/ml

D – E1

17

21

14

15

D – E2

16

19

12

16

D – E3

14

21

10

14

D – E4

17

18

11

17

D – E5

10

13

14

13

D –E6

14

19

9

14

D – E7

18

16

13

16

D – E8

15

19

11

18

D – E9

14

19

15

23

D – E10

12

17

16

18

Control

R

R

R

R

Standard

20

17

 

 

CONCLUSION:

The present study describes the synthesis of 1-substituted tetrahydropyrimidine derivatives by Leuckart reaction. This methodology offers the spirited advantages having lesser time reaction and yield higher percentage of products. The structures of the synthesized compounds were elucidated by IR, NMR and Mass spectroscopy. The synthesized compounds were screened for antioxidant, anticancer, antibacterial, antifungal activity. The in-vitro anti oxidant property for all the compounds showed positive results. The compounds D – E2, D – E3, D – E4, D – E7, D – E8, D – E9 showed more potent activity. These six compounds were selected and evaluated for anticancer activity. The results obtained showed that synthesized compounds D – E3, D – E4,D – E8, D – E9 showed anticancer activity against cancer cells. These four compounds exhibit best antibacterial activity as compared to standard drug of ofloxacin. It proves the suitable structural modification will have to be carried to get novel compound having potent anticancer activity with least effect on normal cells. The antimicrobial activities of synthesized compounds were to obtain zone of inhibition by disc diffusion method. Among the synthesized compounds were found to be good antifungal activity as compared to standard drug of Flucconazole.

 

 

ACKNOWEDGEMENT:

Authors express special thanks to Mr. Aadhirajan, KMCH College, Coimbatore for anticancer activity studies and Mr. R. Murugesan, IIT, Chennai for NMR and MASS spectral studies.

 

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Received on 18.10.2014          Modified on 27.10.2014

Accepted on 05.11.2014         © RJPT All right reserved

Research J. Pharm. and Tech. 7(12): Dec. 2014; Page 1433-1437