A Novel Series of Dihydropyrimidines as Possible Antimicrobial Agents

 

Beena K.P.¹ and Akelesh T.²

¹Assistant Professor, Department of Pharmaceutical Chemistry, KMCH College of Pharmacy, Coimbatore

²Assistant Professor, Department of Pharmaceutics, R.V.S College of Pharmaceutical Sciences, Coimbatore

*Corresponding Author E-mail: beenaakelesh12@gmail.com

ABSTRACT:

Pyrimidines occupy a distinct and unique place in our life. This heterocyclic moiety has great biological and medicinal significance. A large array of pyrimidine drugs possess a variety of medicinal properties. A new series of dihydropyrimidines via biginelli reaction were prepared and condensed with hydrazine hydrazine to give carbohydrazido derivatives which was further condensed with phthalic anhydride in presence of ethanol and glacial acetic acid  to afford the titled compounds. The infra red spectrum was recorded on a JASCO 4100 FTIR using KBr pellet disc technique. NMR spectra of the synthesized compounds were recorded on a Brucker 300 MHz spectrometer. The spectra were in accordance with the synthesized compounds. All the synthesized compounds were evaluated against  Gram-positive bacteriae such as Bacillus lentus, Bacillus subtilis, Staphylococcus aureus and Gram-negative bacteria such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli  and fungi such as Candida albicans and  Aspergillus niger  to study the effect of introducing phthalic anhydride moiety into pyrimidine nucleus. Most of the novel synthesized compounds had lower antibacterial effects, but significant antifungal activity in comparison with standard drugs. Minimum inhibitory concentration of the titled compounds for fungi was determined by agar dilution method. The highest concentration to inhibit both Aspergillus niger and Candida albicans was found to be 62.5µg/ml.

 

KEYWORDS: Substituted dihydropyrimidines, phthalic anhydride, In-vitro antibacterial activity, In-vitro antifungal activity, Minimum inhibitory concentration

 

INTRODUCTION:

Pyrimidine^{1, 2} is a basic nucleus in DNA and RNA, it has been found to be associated with diverse biological activities. Pyrimidines and their derivatives are considered to be important for drugs and agricultural chemicals. Pyrimidine derivatives possess several interesting biological activities such as antimicrobial, antitumour³, anti tubercular⁴, antimalarial ⁵, antihistaminic activity⁶ and antifungal activities. Many Pyrimidine derivatives are used for thyroid drugs and leukemia.

 

The biological significance of the pyrimidine derivatives has led us to synthesize some substituted dihydropyrimidines by incorporating phthalic anhydride moiety and evaluating the targeted compounds for in-vitro bacterial activity and in-vitro antifungal activity.

 

MATERIAL AND METHODS:

Synthesis of  biginelli compound:

A mixture of 0.15 mole of thiourea, 0.1mole of ethylacetoacetate and 0.1mole of benzaldehyde were dissolved in 25ml of ethanol along with 3 drops of conc.HCl and refluxed for one and half an hour. The reaction mixture was then poured into 100ml ice cold water with stirring and left overnight at room temperature, filtered and dried. The products were recrystallised using ethanol. Similar procedure was followed for various substituted aldehydes.

 

Synthesis of carbohydrazido derivative:

A mixture of 0.1mole of biginelli compound and 0.1mole of hydrazine hydrate were dissolved in 20ml of ethanol along with 4 drops of conc.H₂SO₄ and refluxed for 3 hrs. The reaction mixture was then evaporated to obtain a residue which was further recrystallised from ethanol.

 

Synthesis of substituted dihydro pyrimidine derivatives (T-1 to T-6):

About 0.5gm of hydrazido product and 0.5gm of phthalic anhydride, 5ml of glacial acetic aceid were refluxed for one hour. The reaction mixture was then poured into ice cold water in a beaker, filtered and dried. The precipitate was then recrystallised from ethanol.

Fig no:1  Synthesis of titled compounds (T-1) – (T-6)

where,

T-1    R=H, R¹=H, R²=H;                   T-2    R=Cl, R¹=H, R²=H;                  T-3    R=H, R¹=H, R²=OCH₃

T-4    R=NO₂, R¹=H, R²=H;               T-5    R=H, R¹=OCH₃, R²=OH;         T-6    R=H, R¹=H, R²=N(CH₃)₂

 

 

6-methyl-4 phenyl-5[(N-1,3 dioxo isoindolin-2yl)]amido-2-thio-3,4 dihydro pyrimidin-2(1H)one  T-1

Yield: 88.08%; m.p. 163-164ºC; IR (KBr, cm^-1) υ max: 1585(C=Cstretch), 3123(NHstretch), 1698(C=Ostretch), 1286(CNstretch), 2651(CH₃ stretch), 652(C-S stretch); ¹HNMR (DMSO-d6) δ: 7.17-7.67 (m,15H, aromatic), 2.16 (s, 3H, CH₃), 8.14 (s, 1H,NH), 9.20 (s, 1H,NH), 10.32 (s, 1H, CONH).

 

6-methyl-4-(o-chlorophenyl)-5[(N-1,3 dioxo isoindolin-2yl)]amido-2-thio-3,4 dihydro pyrimidin-2(1H)one  T-2

Yield: 85.06%; m.p. 150-160ºC; IR (KBr, cm^-1) υ max: 1571(C=Cstretch), 3127(NHstretch), 1618C=Ostretch), 1271(C-N stretch), 782(C-Cl stretch), 646(C-S stretch); ¹HNMR (DMSO-d6) δ: 7.18-7.53 (m,14H, aromatic), 2.23 (s, 3H, CH₃), 8.54 (s, 1H,NH), 9.22 (s, 1H,NH), 10.70 (s, 1H, CONH).

 

6-methyl-4 -(p-methoxyphenyl)-5[(N-1,3 dioxo isoindolin-2yl)]amido-2-thio-3,4 dihydro pyrimidin-2(1H)one  T-3

Yield: 94.73%; m.p. 165-170ºC; IR (KBr, cm^-1) υ max: 1586(C=Cstretch), 3126(NHstretch), 1684(C=Ostretch), 1281(C-Nstretch), 2645(CH₃stretch), 1070(C-O-C stretch), 662(C-S stretch); ¹HNMR (DMSO-d6) δ: 7.2-7.8 (m,17H, aromatic), 2.22 (s, 3H, CH₃), 8.56 (s, 1H,NH), 9.20 (s, 1H,NH), 10.31 (s, 1H, CONH). 3.24 (s, 3H, OCH₃)

 

6-methyl-4 -(o-nitrophenyl)-5[(N-1,3 dioxo isoindolin-2yl)]amido-2-thio-3,4 dihydro pyrimidin-2(1H)one  T-4

Yield: 92.33%; m.p. 168-170ºC; IR (KBr, cm^-1) υ max: 1525(C=Cstretch), 3125(NHstretch), 1699(C=Ostretch), 1285(C-Nstretch), 2651(CH₃stretch), 1401(Ar-NO₂ stretch), 653(C-S stretch); ¹HNMR (DMSO-d6) δ: 7.8-8.0 (m,14H, aromatic), 3.73 (s, 3H, CH₃), 8.14 (s, 1H,NH), 9.21 (s, 1H,NH), 10.36 (s, 1H, CONH).

 

6-methyl-4 -(p-hydroxy m-methoxyphenyl)-5[(N-1,3 dioxo isoindolin-2yl)]amido-2-thio-3,4 dihydro pyrimidin-2(1H)one  T-5

Yield: 60.31%; m.p. 170-180ºC; IR (KBr, cm^-1) υ max: 1515(C=Cstretch), 3536(NHstretch), 1644(C=Ostretch, 1276(C-Nstretch), 3121(OHstretch), 1031(C-O-C stretch) 663(C-S stretch); ¹HNMR (DMSO-d6) δ: 7.02-7.39 (m,17H, aromatic), 3.63 (s, 3H, CH₃), 8.55 (s, 1H,NH), 9.23 (s, 1H,NH), 10.24 (s, 1H, CONH). 11.30 (s, 1H, OH), 3.32 (s, 3H, OCH₃)

 

 

6-methyl-4 -(p-dimethyl aminophenyl)-5[(N-1,3 dioxo isoindolin-2yl)]amido-2-thio-3,4 dihydro pyrimidin-2(1H)one  T-6

Yield: 60.28%; m.p. 175-180ºC; IR (KBr, cm^-1) υ max: 1524(C=Cstretch), 3125(NHstretch), 1607(C=Ostretch), 1289(C-N str),1367(C-H stretch), 643(C-S stretch); ¹HNMR (DMSO-d6) δ: 7.09-8.3 (m, 20H, aromatic), 2.26 (s, 3H, CH₃), 8.56 (s, 1H, NH), 9.21 (s, 1H, NH), 10.32 (s, 1H, CONH). 2.15 (s, 6H, N(CH₃)₂)

 

ANTIMICROBIAL ACTIVITY:

Antibacterial activity

The synthesized compounds were screened for antibacterial activity against gram positive organisms such as Bacillus lentus, Bacillus subtilis and Staphylococcus aureus and gram negative organisms such as Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae by agar diffusion method. Dimethyl sulphoxide was used as the control. Trimethoprim was used as standard control. The culture medium was nutrient agar.

Compounds T-1 to T-6 showed only mild activity against gram positive and gram negative bacteriae compared to that of standard.

 

Antifungal activity:

The synthesized compounds were screened for antifungal activity against Candida albicans and  Aspergillus niger  by agar diffusion method. Dimethyl sulphoxide was used as control. Voriconazole was used as standard control. The culture medium was Sabouraud dextrose agar medium

 

Compound T-3 and T-6 exhibited same activity as that of standard against Candida albicans.Compounds T-1, T-2, T-4 and T-5 exhibited significant activity. Compound T-1, T-3 and T-4 exhibited highly significant activity against Aspergillus niger compared to that of standard. Compound T-6 exhibited similar activity as that of standard against Aspergillus niger. Compounds T-2 and T-5 exhibited significant activity. The datas of antibacterial and antifungal activity were summarized in Table no.1

 

Table no.1 Antimicrobial activity data of synthesized compounds by agar diffusion method

ZONE OF INHIBITION (mm)
ORGANISMS	T-1	T-2	T-3	T-4	T-5	T-6	Standard
Bacillus subtilis	10	11	10	10	10	10	25
Bacillus lentus	12	12	10	11	10	11	22
Staphylococcus aureus	10	9	9	11	10	10	25
Escherichia coli	11	9	9	12	11	10	25
Pseudomonas aeruginosa	10	10	10	13	12	9	27
Klebsiella pneumoniae	11	11	11	14	12	15	22
Candida albicans	13	14	15	11	13	15	15
Aspergillus niger	17	14	17	16	13	15	15

 

Determination of the minimum inhibitory concentration (MIC)

The MIC of the synthesized compounds (T-1 to T-6) was determined by conventional agar dilution method with respect to different microorganism including Candida albicans and Aspergillus niger. The targeted compounds were dissolved in dimethyl sulfoxide and diluted to highest concentration (250µg/ml) to be tested, and then fold serial dilutions were made in a concentration range from 250µg/ml to 3.9µg/ml in sterile test tubes containing standardized inoculums. All the tubes were incubated at 25^oC for 24 hours, after incubation minimum inhibitory concentration values were determined.

 

The highest dilution of extract that shows no turbidity was observed and recorded. This dilution was considered to have the concentration of the drug equivalent to MIC.

 

RESULTS AND DISCUSSION:

The main aim of the present work was to synthesize some new dihydro pyrimidine derivatives. The structures of the synthesized compounds were assigned on the basis of IR spectra and NMR spectra⁷. All the compounds were screened for their invitro antimicrobial activity⁸. Most of the compounds were found to exhibit moderate activity against all bacterial strains when compared to standard. Compounds T-1, T-3, T-4, T-6 displayed greater zone of inhibition against fungi strains in comparison to standard. The antifungal screening data revealed that all the compounds showed excellent activity against Aspergillus niger and Candida albicans. The minimum inhibitory concentration for both the fungi was evaluated to be 62.5μg/ml.

In conclusion almost all the titled compounds were found to exhibit a mild antibacterial activity and a more pronounced antifungal activity.

 

ACKNOWLEDGEMENT:

The authors express their deep thanks to Kovai Medical Centre Research and Educational Trust, Coimbatore, Tamilnadu, India for their constant support and encouragement.

 

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Received on 04.06.2011       Modified on 13.06.2011

Accepted on 23.06.2011      © RJPT All right reserved