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.

 

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

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,CMˉ¹)	¹H 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