INTRODUCTION:

Free radicals are unstable molecules which have an odd number of electrons and therefore try to capture another electron from another molecule to become stable. While the molecule is now stable, the molecule which had its electron stolen is now a free radical itself due to not having the desired number of electrons to make the molecule stable. This situation obviously results in a chain reaction in which each newly created free radical seeks an electron to steal to make it stable, whilst creating a free radical itself¹.

This process occurs naturally, and the human body performs this task in some situations for our benefit, such as when fighting off infection. Free radical formation and its damaging chain reaction can also be because of negative factors such as smoking, radiation and intense exercise. When this damage is excessive it is said antioxidants should be consumed to curb the free radical damage. For those who seek optimal health, antioxidants are often supplemented to reduce free radicals, with damage caused by free radicals also ever increasing with age.

The syntheses of pyrimidine have attracted the attention of chemists because of their potential pharmocodynamic properties. Recently much interest has been focused on the synthesis of pyrimidines possessing anti-inflammatory², analgesic², antimalarial³ and antioxidant⁴ and antiulcer⁵ activity.

Pyrimidine does not exist in nature but in the form of its different derivatives are found as a part of more complex systems and widely distributed. Therefore, the research on the synthesis of the pyrimidine and its analogues is an ongoing search.

EXPERIMENTAL:

The melting points of the synthesized compounds were determined by open capillary tubes and Thiel’s melting point apparatus. The reaction was monitored by TLC. The IR Spectras of the compounds were recorded on Shimadzu 1320 FT-IR spectrometer (KBr pellets method) and values of Vmax are reported in cm^-1. Nuclear magnetic resonance spectra were obtained on DMM X-200 MHz Brookfield from Astrazeneca Pharma Ind. Ltd. Using cm^-1 and chemical shift (δ) were reported in parts per million downfield from standard reference Tetramethylsilane (TMS).

Preparation of 3-acetyl coumarin:⁶

To a cooled suspension of a mixture of salicylaldehyde (0.5mol) and ethyl acetoacetate (0.5mol) piperidine (2ml) was added with shaking. The mixture was then maintained at freezing temperature for 2-3 hrs. Yellow coloured lumps were formed. These lumps were broken in cold ethanol. The precipitate obtained was filtered and recrystallized from warm glacial acetic acid to give needle shaped crystals.

IR KBr (cm^-1): 3032 (Str, aromatic CH), 1739 (lactone C=O), 1678 (methyl C=O), 1552 (aromatic C=C), 1213 (-C-O-C-), 754 (B, aromatic CH)

Preparation of chalcone:⁶

Conventional method:

A mixture of 3-acetyl coumarin (2.67g, 0.01mol) and substituted aromatic aldehyde (1.49g, 0.01 mol) in 25ml ethanol, piperidine (2g) in 5ml ethanol was added dropwise. The mixture was heated and refluxed for 12 hours. After cooling, the product was separated out and washed with ethanol (20ml) .The product was recrystllized from glacial acetic acid.

Preparation of pyrimidine derivatives:⁷

Chalcone (0.01 mol) and Guanidine Nitrate (0.015mol) in absolute ethanol (10ml) were refluxed on water bath for 6 hours. The solvent was completely evaporated and residue was poured into ice cold water. The precipitate solid was collected by filtration.

IR KBr (cm^-1): 3063 (-NH str), 3018 (Str, aromatic CH), 1649 (C=O), 1587 (C=N), 1440 (C=C), 1242 (-C-O-C-) 763 (B, aromatic CH), 688 (-C-Cl)

¹H NMR (δ ppm): 7.9 (2H, S, NH₂), 7.13 (1H, S, CH=C), 7.3 (1H, S, CH=C, pyran), 7.38-7.5 (m, 8H aromatic)

Preparation of chloroacetylated pyrimidine derivatives:⁸

To the above prepared derivatives (0.01mol), chloroacetyl chloride (0.012mol) was added and heated under reflux in dry benzene (20ml) for 3 hours on steam bath. Benzene was distilled off to a possible extent and cooled. The product resulted was filtered, washed with small portions of petroleum ether and dried. The product was purified by recrystallization from alcohol to get a crystalline solid.

IR KBr (cm^-1): 3063 (-NH str), 3022 (Str, aromatic CH), 1739 (C=O), 1653 (C=O), 1585 (-C=N), 1444 (C=C), 1244 (-C-O-C-), 763 (B, aromatic CH), 690 (-C-Cl)

¹H NMR (δ ppm): 7.9 (1H, S, NH), 7.1 (1H, S,-CH=C),7.3 (1H, S,CH=C, pyran), 7.38-7.5 (m, 8H aromatic ), 1.6 (2H, S, -CH2Cl)

SCHEME OF WORK

Table No.1: Physicochemical Properties of the synthesized pyrimidine derivatives

Compound No.	Substituent R	Molecular Formula	Melting Point(⁰C)	Yield (%)
F1	C₆H₄Cl	C₂₁H₁₃Cl₂N₃O₃	145-148	58
F2	C₆H₄Br	C₂₁H₁₃N₃O₃BrCl	162-165	55
F3	C₆H₄ NO₂	C₂₁H₁₃ClN₄O₅	109-112	57
F4	C₆H₄F	C₂₁H₁₃N₃O₃FCl	112-116	45
F5	C₆H₄OH	C₂₁H₁₄ClN₃O₄	140-144	48
F6	C₆H₄ NO₂	C₂₁H₁₃ClN₄O₅	152-156	56
F7	C₆H₃Cl₂	C₂₁H₁₂Cl₃N₃O₃	142-146	64

Antioxidant Activity:⁵

DPPH method:-

Chemicals:

DPPH (1, 1 diphenyl-2-picryl-hydrazyl), DMSO (dimetyl sulphoxide), Methanol

Preparation of different concentrations of compounds:

30mg of the drug was dissolved in 30 ml DMSO (1 mg/ml or 1000mg/ml solution).From the above solution, different dilutions such as 500mg/ml, 250mg/ml, 125mg/ml, 62.5mg/ml, 31.25mg/ml, 15.62mg/ml, 7.81mg/ml and 3.9mg/ml were prepared.

Preparation of DPPH stock solution:

21 mg of DPPH was placed in 100ml volumetric flask and methanol was added up to the mark.

Preparation of DPPH working solution:

18 ml of DPPH stock solution was pipetted out into a 100ml volumetric flask and diluted with methanol.

Procedure:

Sample:

0.25 ml of above dilutions (sample) were taken in a test tube and 5ml DPPH working solution was added into each test tube.

Sample blank:

0.25 ml of above dilutions (sample) were taken in a test tube and 5 ml of methanol was added into each test tube.

Control:

0.25ml of DMSO was taken in a test tube and then 5ml of DPPH working solution was added.

Control blank:

0.25ml of DMSO was taken in a test tube and then 5ml of methanol was added.

These test tubes were incubated at 37°C for 20min and the absorbance was determined by using UV spectrophotometer at 517nm.The % inhibition was calculated by using formula,

Control – Sample

% Inhibition = ------------------------------------------ X 100

Control

The IC₅₀value was obtained by plotting the graph, taking % inhibition on y axis and concentration on x axis.

Ascorbic acid:

Ascorbic acid was used as a standard and above procedure was followed to compare the antioxidant activity of different synthesized compounds with ascorbic acid.

Antimicrobial Studies:⁹

In our current study, the antimicrobial activity was carried out by the Filter disk method. Here responses of microorganisms to the synthesized compounds were measured and compared with the response of the standard reference drug. The standard reference drug used in the present work was Amoxicillin trihydrate.

Table No.2: Antioxidant activity of synthesized compounds

Sl. No	Mole. Formula	% Inhibition	IC₅₀ ± SEM DPPH Method
1.	C₂₁H₁₃Cl₂N₃O₂	1.05 - 50.39	446.43 ± 2.830
2.	C₂₁H₁₃N₃O₂BrCl	5.31-43.19	> 500
3.	C₂₁H₁₃ClN₄O₄	6.20 - 66.22	224.63 ± 1.097
4.	C₂₁H₁₃N₃O₂FCl	7.69 - 46.24	> 500
5.	C₂₁H₁₄ClN₄O₄	4.61 - 56.59	475.23 ± 1.798
6.	C₂₁H₁₃ClN₄O₄	2.24 - 54.08	374.93 ± 1.545
7.	C₂₁H₁₃Cl₂N₃O₂	8.92 - 48.02	>500
STD.	Ascorbic acid	45 - 98	13.33 ± 0.88

Preparation of test solutions:

Each test compound was dissolved in DMSO to get a concentration of 5mg/ml and 10 mg/ml. This concentration was used for testing antibacterial activity.

Procedure:

The petridishes were thoroughly washed and sterilized. Prepared Agar media was added into sterilized petridish and allowed it to get solidified. After solidification of media, 0.1ml of inoculum was added over it and spreaded for even distribution of organism. Here both high and low strength disks are applied for each antibiotic to be tested. The organism is reported as being sensitive if a clear zone appears around both disks. If the zone appears around the high concentration alone, the organism is called moderately susceptible. If zones are taking in both the disks, the organism is considered resistant to drug. 5µl of the sample was placed on the disk. Using the concentration of 5mg/ml and 10 mg/ml, samples were prepared in duplicate in each petridish. A standard (Amoxicillin trihydrate for antibacterial activity) was maintained with same concentration in another plate and a control having only DMSO in one plate. Then the petridishes were incubated at 37ºC for 24hrs and zones of inhibition were observed and measured.

Table No.3: Antimicrobial activity of the synthesized compounds.

Comp.	Zone of Inhibition(mm)
Comp.	*S. aureus*	*S. pneumococci*	*E. coli*	*P. aeruginosa*	*Klebsiella pneumoniae*
1	18	11	13	12	15
2	13	13	11	13	14
3	20	16	16	17	18
4	19	15	11	18	17
5	14	11	12	13	12
6	15	13	14	14	17
7	12	13	12	12	14
Amoxicillin trihydrate	23	25	21	24	26

RESULTS AND DISCUSSION

The Physicochemical Properties of the synthesized pyrimidine derivatives is shown in table-1. The results (Table-3) indicates that in general Nitro, Flouro and Chloro derivatives out of seven prepared compounds showed good antibacterial activity against both Gram positive and Gram negative bacteria which were used for screening. None of the compounds showed significant antioxidant activity compared to Ascorbic acid (Table-2).

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