SYNTHESIS OF PYRIMIDINE CARBOXAMIDE DERIVATIVES CATALYZED BY URANYL NITRATE HEXA HYDRATE WITH THEIR ANTIBACTERIAL AND ANTIOXIDANT STUDIES

An efficient and simple method was developed for the synthesis pyrimidine-5-carboxamides using UO2(NO3)2.6H2O catalyst under conventional and microwave irradiation. The synthesis of dihydropyrimidine using uranyl nitrate had shown many advantages such as easy work up, shorter reaction times and higher yields using acetonitrile as a solvent. The structures of the synthesized compounds were confirmed by FT-IR, H NMR, C NMR and mass spectral data. All the synthesized compounds screened for in vitro antioxidant and antibacterial activity and the results are reported.


INTRODUCTION
Nitrogen based heterocycles provides broad range of biological activities; the lone pair present in the nitrogen atoms acts as donor facilitating the construction of various supramolecular blocks.Multi-component reactions (MCRs) are an important class of organic reactions, which is widely used to construct different target molecules in one pot reaction using three or more numbered starting materials.In 1893 an Italian chemist Pietro Biginelli has reported the synthesis of dihydropyrimidines by a simple one-pot cyclocondensation reaction of ethyl acetoacetate, benzaldehyde and urea [1].The literature reveals that Human kinesin Eg5, plays a crucial role in mitosis by establishing the bipolar spindle, which has been proved to be an interesting drug target for the development of cancer chemotherapeutics.In a similar way, monastrol, the first Biginelli compound, exhibited good anticancer property.Batzelladine A and B compounds of DHPMs derived [2] from natural marine sources, were the first low molecular weight natural products showing promising anti HIV activity and hence considered as potential molecules for treatment of AIDS.
The limited availability of the natural products renders them to be attractive targets for total synthesis [3].The dihydropyrimidinone (DHPM) is the most important core structure in the synthesis of different medicinally and pharmacologically valuable agents such as antibacterial [4], antiviral [5], antitumor [6], antihypertensive agents [7], calcium channel blockers [8], neuropeptide Y (NPY) antagonists [9], α-1a-antagonists [10] and anti-inflammatory drugs [11].In addition, the batzelladine alkaloids containing the DHPM ring structure inhibit the binding of HIV envelope protein gp-120 to human CD 4 cells and there are potential of new molecule for AIDS treatment [12][13].Therefore, dihydropyrimidinones (DHPMs) synthesis always shows the attraction to organic chemists.

EXPERIMENTAL General
All the reagents were purchased from Aldrich, SD Fine Chemicals and Qualigens and used without further purification.The 1 H NMR spectra were obtained on Bruker AV-500 MHz spectrometer with DMSO-d 6 as the solvent using tetramethylsilane (TMS) as the internal standard.Infrared (IR) spectra were recorded at room temperature from 4000-400 cm -1 with KBr pellets at a resolution of 4 cm -1 , using Avatar 330 equipped with DTGS detector.The microwave irradiation experiments were carried out using conventional (unmodified) household microwave oven equipped with a turntable was used (LG, MG-395 WA, 760 W) and operating at 2450 MHz.Mass spectra were obtained using HRMS (JEOL 1600 HRMS).Melting points were determined by open capillaries and are uncorrected.
Microwave irradiation.A mixture of aldehyde (1 mmol), acetoaetanilide (1 mmol), urea or thiourea (1.2 mmol), UO 2 (NO 3 ) 2 .6H 2 O (5 mol%) and acetonitrile (5 mL) were taken in a small beaker and then the reaction mixture was subjected to microwave irradiation at an interval of 3 min at 160 W for about 15-18 min; varying time periods as shown in Table 3.The completion of the reaction was monitored by TLC.After the completion of the reaction, the mixture was poured into ice cold water stirred well and the solid separated was filtered, dried and then recrystallized from hot methanol to afford pure compounds 4a-o.

Screening of antioxidant activity
The radical-scavenging activity of synthesized compound was evaluated and compared to that of butylated hydroxyl toluene (BHT) and ascorbic acid using the DPPH radical scavenging assay.This assay is based on the measurements of the scavenging ability of compounds towards the stable radical DPPH.The disappearance of absorption of this commercially available radical is measured spectrophotometrically at 517 nm in a dimethyl sulphoxide (DMSO) solution using a UV/Vis-spectrophotometer under thermo-static conditions at 25 ºC.DPPH has an odd electron and hence has a strong absorption band at 517 nm.When this electron becomes paired off, the absorption decreases stoichiometrically with respect to the number of electrons or hydrogen atoms taken up.Such a change in the absorbance by this reaction has been extensively adopted to test the capacity of several molecules to act as free radical scavengers.Hence, faster is the decrease of absorbance; more pronounced is the antioxidant activity of the compound.
3.0 mL of a freshly prepared DPPH solution of 6.02 x 10 -5 M in DMSO was placed in a test tube and 100 μL of a DMSO solution of each test compound added.After that the solution was kept at room temperature for 30 min in dark and the absorbance was measured at 517 nm.The control contained all the reaction reagents except the synthetic compound or positive control substance.The experiment was carried out in triplicate and average values are reported.The DPPH scavenging activity was expressed as the inhibition of free radical DPPH in percent (I %) as described by Tepe et al. [43].

Screening of antibacterial activity
The in vitro antibacterial studies were carried out against three "Gram +ve" bacterial strains, Staphylococcus aureus (MTCC 3381), Pseudomonas aeruginosa (MTCC 2295) and Bacillus cereus (MTCC 8372) and two "Gram -ve" bacterial strains, Escherichia coli (MTCC 1302), Klebsiella pneumonia (MTCC 3384) by agar well diffusion method using Muller Hinton agar as the medium [44].A number of antimicrobial discs were placed on the agar for the sole purpose of producing zones of inhibition in the bacterial lawn.20 mL of agar media was poured into each Petri dish.Excess of suspension was decanted and plates were dried by placing them in an incubator at 37 o C for an hour.Using a punch, wells were made on these seeded agar plates and different concentrations of the test compounds in dimethylsulfoxide (DMSO) were added into each labeled well.A control was also prepared for the plates in the same way using DMSO as solvent.All the plates were incubated at 37 o C for 24 h.The degree of effectiveness was measured by determining the diameters of the zone of inhibition produced by the compounds.Activity of each compound was compared with standard drug ampicillin available in the market.Minimum inhibitory concentration (MIC) was calculated as the lowest concentration of the sample at which there is no visible growth of the bacteria.

RESULTS AND DISCUSSION
In the present work a simple, efficient and practical approach for the synthesis of 4-aryl pyrimidine-5-carboxamide by reacting actoacetanilide, urea or thiourea with substituted benzaldehyde using UO 2 (NO 3 ) 2 .6H 2 O under conventional and microwave irradiation methods are reported (Scheme 1).UO 2 (NO 3 ) 2 .6H 2 O is a cost effective catalyst for the synthesis of 5carboxamide-DHPM compounds compared to the other Lewis acid catalysts reported in the literature.
The optimized reaction condition was developed using condensation of benzaldehyde, urea and acetoacetanilide in the presence of catalytic amounts of UO 2 (NO 3 ) 2 .6H 2 O under conventional and microwave irradiations using different solvent system such as chloroform, dichloromethane, ethanol, methanol, acetonitrile and different catalytic mole percent was also examined and the results are reported (Table 1 and 2  Table 1 indicates that polar solvents such as methanol, ethanol and acetonitrile, gave better yields when compared to non polar ones, which indicate that acetonitrile is the best solvent for this conversion.Table 2. Effect of catalysts loading a . The results are shown that the reaction proceed more efficiently under microwave irradiation when compared to conventional heating.Furthermore, effect of catalyst loading was studied.The optimum catalyst mole percent was found to be 5 mol %, when we increasing the catalyst mole percent (1 and 2, Table 2) did not show any improvement in the yield percent.After optimization of the reaction conditions, this method was used for synthesis of DHPM using different substituted aldehydes, urea or thiourea and substituted acetoacetanilide under conventional heating and microwave irradiation conditions to synthesis of new DHPM derivatives by using UO 2 (NO 3 ) 2 .6H 2 O as a catalyst for this reaction (Table 3).
The catalyst which is highly soluble in water could be easily removed by washing with excess amount of cold water through suction filtration.All the synthesized compounds characterized through IR, 1 H NMR and HRMS mass spectra.

Antioxidant activity
Antioxidant activities of all the newly synthesized compounds 4a-o was carried out using DPPH free radical scavenging assay method.The antioxidant studies revealed that compound 4c, 4d, 4f and 4g show reasonable free radical scavenging activity (70.88, 71.45, 76.34, and 72.60℅, respectively) compared to that of BHT (butylated hydroxyl toluene) and ascorbic acid (100%), while that compound 4e, 4k and 4o shows good activity, while that compounds 4a, 4b, 4h, 4i,   4) and compared with those of standards BHT and ascorbic acid.The compounds 4d and 4o show moderate activity against P. aeruginosa and K. pneumonia and least activity against other organisms.Compounds 4a, 4b, 4e, 4f, 4i, 4j, 4l and 4n show least activity against all the micro organisms screened.The data on the antibacterial activity of these compounds is given in Table 5.

CONCLUSION
A simple and efficient procedure for the synthesis of 6-methyl-1,2,3,4-tetrahydro-N-aryl-2oxo/thio-4-arylpyrimidine-5-carboxamide derivatives by one-pot three-component condensation of different substituted aldehydes, urea or thiourea and substituted acetoacetanilides under conventional heating and microwave irradiation conditions using UO 2 (NO 3 ) 2 .6H 2 O as catalyst has been presented.The main advantages of this process such as mild reaction conditions, shorter reaction times, cost effective catalyst, easy work-up and high yields.The synthesized compounds 4a-o were screened for their in vitro antioxidant activity using DPPH free radical scavenging method.The compound 4e, 4k and 4o shows good antioxidant activity when compared to other synthesized compounds.Antibacterial activity of the synthesized compounds also carried out and it was observed that compounds 4c, 4g, 4h and 4k showed highest activity against S. aureus, P. aeruginosa, E. coli and K. pneumonia remaining compound showed moderate activity in comparison with standard drug.

Figure 1 .
Figure 1.Comparsion of antioxidant activity of compounds 4a-o, BHT and ascorbic acid using DPPH radical scavenging method after 30 min and 1 h incubation.Free radical scavenging capacities of the synthesized compounds 4a-o, BHT and ascorbic acid at 100 μg/mL concentration after half an hour and 1 hour incubation time in dark at room temperature are shown in Figure 1.The radical scavenging activity (RSA) for methanolic solutions of synthesized compounds 4a-o are presented in (Table4) and compared with those of standards BHT and ascorbic acid.

Table 1 .
Effect of solvent a. .

Table 4 .
Antioxidant activity of compounds 4a-o using DPPH radical scavenging method.BHT and ascorbic acid was used as a standard antioxidant.**DPPH in DMSO.E. coli and K. pneumonia and moderate activity against B. cereus while compounds 4d and 4j showed good activity against B. cereus and moderate activity against other bacterial strains. *