INTRODUCTION:

The application of microwave irradiations to the combinatorial chemistry becomes a powerful tool in accelerating the pace of library synthesis¹. Domestics microwave oven is most popularly used in synthesis because of its low cost and ready availability.

Major aim for the use of this technique is rapid synthesis of drug-like compounds as compared to conventional synthetic method which is tedious and time-consuming processes². Nucleic acids are vital to the life cycle of cells, because they are the carriers of species inheritance. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) both are polymers of nucleotide building blocks. Their molecular structures are based on two N-heterocyclic aromatic compounds namely, pyrimidine (C₄H₄N₂) and purine (C₅H₄N₄). Among the biological nucleobases three are pyrimidine- based, uracil (found in RNA only), thymine (in DNA only), and cytosine (in both RNA and DNA), and two are purine-based adenine and guanine (in both RNA and DNA)³.

Many nucleoside analogues substituted at the C-5 position of the heterocycle are known to have potent biological properties and have been investigated as antiviral, antimetabolite and anticancer agents.^4-6

5-halogen-substituted Pyrimidinedione, an interesting class of compounds biologically and chemically, has been prepared by direct halogenations of Pyrimidinedione⁷. 5-bromo-2,4-(1H,3H)Pyrimidinedione (5 BU) is brominated derivative of uracil that act as an antimetabolite or also called base analog, substituting for thymine in DNA and can induce DNA mutation⁸in the same way as 2-aminopurine. It is used mainly as an experimental mutagen, but its deoxyriboside derivative (5-bromo-2-deoxyuridine) is used to treat the neoplasm⁹

Among the 5-halogenopyrimidines, the most extensively studied compound is 5-fluoro-2,4(1H,3H)Pyrimidinedione. 5FU which inhibits thymidylate synthase by mimicking the structure of uracil, therefore result in depletion of thymidine and inhibition of DNA synthesis. In addition, 5-FU is a starting material for the production of certain types of prodrugs, which are metabolized to form 5-fluorouracil in the body such as the compound:1-(2-tetrahydrofurl)5-fluoro-uracil^10,11

EXPERIMENTAL:

Melting points were recorded by using Gallenkamp melting point apparatus Absorption spectra were taken on Hitachi U-2800 spectrophotometer. FTIR spectra were recorded on a Midac (USA) M-2000 by using KBr disk. Mass spectra of synthesized compounds were obtained on a GC-MS spectrometer Shimadzu (Japan) QP-2010. A Dawlance DW-180 domestic microwave oven (2450 MHz, 950 W) was used for all experiments. The purity of compounds was checked on silica gel coated aluminium plates (Merck).

Synthesis of 5-Fluoro-2, 4-(1H, 3H) Pyrimidinedione

Conventional:- 0.22 g 5-FluoroOrotic acid was dissolved in 10.0 mL THF. The solution was refluxed for two hrs. at 90^oC. The flask was cooled and ether was evaporated to give the product. Yield 88% mp.260 ^oC (decomposed), soluble in cold water.

Microwave:- 5-Fluorouracil was prepared by thermal decarboxylation of 5-fluoro Orotic acid in solvent which was not reacting with carboxyl group –COOH. Thus a mixture of 0.02g 5-fluoro Orotic acid and 5.0 mL tetrahydrofuran (THF) was irradiated by microwave for two minutes. The product was obtained after evaporation. Yield.71% mp.270 ^oC (decomposed), soluble in water.

Spectral Analysis was done by using UV, FTIR and GC–MS spectroscopy. The results are tabulated in Tables 2 to 4 respectively.

The Pharmacological evaluation was carried out by its antibacterial, antifungal and antiviral activities which are recorded in Tables 5 to 6 respectively.

Synthesis of 5-chloro-2, 4-(1H, 3H)Pyrimidinedione

Conventional:- To 1.00g uracil was added 10.0 mL cold superoxol (30% hydrogen peroxide and 10.0 mL cold concentrated HCl). Within five to ten minutes effervescence was started. The effervescence continued until all the uracil had dissolved. After the reaction had subsided, the reaction mixture was covered and left overnight. The solid product was recovered by evaporation and recrystallized to get white crystals of 5-chloro-2, 4-(1H, 3H) Pyrimidinedione. Yield 74%, mp. 290 ^oC (decomposed), soluble in water.

Microwave:- In dry 100ml pyrex flask 0.1g uracil, 1.00ml hydrogenperoxide and 1.00mL concentrated HCl were placed and the contents were heated under microwave radiations for thirty sec. the solid product was recovered by concentrating the solution. Yield 86% m.p. 285 ^oC (decomposed), soluble in water.

Spectral Analysis was done by using UV, FTIR and GC–MS spectroscopy. The results are tabulated in Tables 2--4 respectively.

The Pharmacological evaluation was carried out by its antibacterial, antifungal and antiviral activities which are recorded in Tables 5--6 respectively.

Synthesis of 5-bromo-2,4-(1H,3H) Pyrimidinedione

Conventional:- 2.00 g of 2, 4-(1H, 3H) Pyrimidinedione was dissolved in 8.0 mL distilled water. 2.00 mL liquid bromine was added to the suspension of 2,4-(1H,3H) Pyrimidinedione in water and the mixture was agitated vigorously. The resulting yellow coloured solution was heated to dryness on a steam bath for one hour. The crude product was recrystallized at least twice to obtain fine crystals. Yield 65%, mp.296 ^oC (decomposed), soluble in hot water.

Microwave:- 1.00g of 2, 4-(1H, 3H)-Pyrimidinedione was added to 8.0 mL distilled water. 1.50 mL liquid bromine was added to the suspension of uracil in water and the mixture was agitated vigorously. The resulting yellow coloured solution was heated under microwave radiations for sixty seconds. The crude product was recrystallized at least twice to obtain fine crystals. Yield 75%, mp.290 ^oC(decomposed), soluble in hot water.

Spectral Analysis was done by using UV, FTIR and GC–MS spectroscopy. The results are tabulated in Tables 2-4 respectively.

The Pharmacological evaluation was carried out by its antibacterial, antifungal and antiviral activities which are recorded in Tables 5-6 respectively.

Synthesis of 5-iodo-2,4(1H,3H)Pyrimidinedione

Conventional: 1.12 g uracil, 1.27 g solid iodine and 0.8 g sodium hydroxide were dissolved in 50.0 mL distilled water. Reaction mixture was stirred at 70 ^oC for two hrs. After stirring, mixture was cooled at 20^oC and aqueous 12% sodium hypochlorite solution was added to reaction mixture. The resulting mixture was stirred at 20⁰C for one hour. A white product was obtained which was filtered, washed and dried. Yield 76%, mp.275^od, soluble in hot water.

Microwave:- 0.1g uracil, 0.2g sodium hydroxide and 0.1g iodine were dissolved in 50.0 mL distilled water. After stirring reaction mixture was heated in microwave for sixty seconds. The flask was cooled at 20 ^oC and then 12% sodium hypochlorite solution was added, the resulting mixture was further stirred at 20 ^oC for 1 hour. A white product was obtained which was filtered, washed and dried. Yield 92%, mp.280 ^oC (decomposed), soluble in hot water.

Spectral Analysis was done by using UV, FTIR and GC–MS spectroscopy. The results are tabulated in Tables 2--4 respectively.

The Pharmacological evaluation of synthesized compounds was carried out by its antibacterial, antifungal and antiviral activities which are recorded in Tables 5--6 respectively.

Biological Activity ¹²

All the synthesized compounds were screened for antibacterial, antifungal and antiviral activities against B.subtilis, S.aureus, E.coli, A.niger, A.flavus and ND(new castle disease/rani khait) virus at 10,20,30 µg/ml concentrations. None of these compounds showed significant activity against A. niger and A. flavus while all 5-halogen substituted Pyrimidinedione showed significant activity against NDV. Only 5-Bromo-substituted derivatives exhibited better activity against all strain of bacteria at all concentration, while 5-chlorouracil exhibited better activity against S.aureus only at high concentration.

The antibacterial activity was performed by using two methods: well-diffusion and disk-diffusion method, using nutrient agar as the culture medium. The agar media were inoculated by swabbing overnight cultures onto the surface of agar plates. Well of appropriate diameter were punched in the media with the help of a gel puncher. Bottom of the wells were sealed by placing one drop of melted N-agar. The three different concentrations (10, 20,30µg/mL) in N-saline of each sample were introduced in respective wells. Inhibitory activity was measured (in mm) as diameter of the observed zone of inhibition. Inhibition zones and data are presented in Table-5

Antiviral Activity: was performed by applying spot agglutination method. Inoculated 0.1 ml of virus mixed sample solution containing 100ul of saline solution, 100ul NDV and 100ul of 10% sample solution into allantoic cavity of nine-days old embryonated chicken eggs and then incubated for 24-48 hrs. at 37⁰C. All 5-halogens-pyrimidinedione showed significant activity against NDV which is reported in Table 6.

RESULT AND DISCUSSION:

A series of 5- halogen substituted Pyrimidinedione were synthesized by using two methods, Conventional and Microwave-assisted methods. It was observed that the reaction time and yield manifested by the microwave technique were much better compared to the conventional method as shown in Table-1.

Table-1 Comparison between Conventional and Microwave-Assisted method of synthesis of 5-Halogen-2,4-(1H,3H) Pyrimidinedione in terms of yield and time

Sr. No.	Sample Number	X	Conventional		Microwave
Sr. No.	Sample Number	X	Time (min)	Yield (%)	Time (sec)	Yield (%)
1	I	F	120	88	120	71
2	II	Cl	30	74	30	86
3	III	Br	60	65	60	75
4	IV	I	120	76	60	92

The time required for conventional synthesis was reduced from days to hours, hours to minutes and minutes to seconds by using microwave irradiation. In case of 5- iodo uracil, the reaction time was greatly reduced from 2hrs to 60s. The yield of all synthesized compounds except 5- flouro uracil was high as compared to that in conventional method.

The compounds synthesized by both methods showed the similar melting points and solubility which are given in Table 2. All compounds were partially soluble in cold water but fairly soluble in hot water and they melted with decomposition. Spectral data of UV, FTIR and GC-MS supported the structure assigned. The UV absorption spectra matched with standard spectra in both methods. FTIR spectral data further confirmed the structure of synthesized compounds which is given in Table 3. The N-H stretching vibrations at 3218-2690cm^-1 were observed in compounds synthesized by conventionally and microwave methods. The (C=O) carbonyl stretching vibrational region was similar in 5-fluoro, chloro, bromo and iodouracil synthesized by both technique. This region contains two main bands, a weak one placed above 1700cm^-1 and an intense one located below 1700cm^-1. The presence of halogens further confirmed by their influence on the stretching vibration of carbonyl group and C₅=C₆ bond. The frequencies of these vibrations are decrease from 5-FU to 5-IU due to halogen mass. Same effect of halogen influence was observed in those compounds which were synthesized by microwave technique.

The C-X vibration usually appears in the region v 1200-572 cm^-1 due to presence of halogens (F--I) in general. The vibrational frequencies for all the bonds: v (N-H) str. v (C=O) str. v(C=C) and (C-X) decreases from F to I with some negligible exceptions in 5-IU (C-X) stretching. This is due to decrease in halogen electronegativity as well as increase in halogen mass.

GC-MS Spectral analysis:

The mass fragmentation data of the 5-halogendioxypyrimidione is given in Table 4. In case of 5-FU, the mass spectrum showed the molecular ion peak at m/z 136 corresponding to molecular formula C₄H₃F N₂O_{2 (}130). Molecular ion peak at 136 attributed due to protonation of oxygen and nitrogen atoms.Since oxygen and nitrogen containing compounds form fairly stable oxonium and ammonium ions. Ion molecular collisions take place at pressure higher than 0.5mm and peaks in the mass spectrum that appear higher than the mass of the molecular ion. In case of 5-chloro uracil the molecular ion peak appear at m/z 168 corresponding to molecular formula C₄Cl₂ N₂O₂. This shows the fragmentation of the molecular ion. In case of 5-bromo and 5- iodouracil , molecular ion peak appear at m/z 190 and 238 corresponding to molecular formula C₄H₃BrN₂O₂ and C₄H₃IN₂O_2.

CONCLUSION:

Microwave-assisted method provides an excellent approach for the safe, rapid, inexpensive and simple environmental-friendly synthesis of medicinally important 5-halogen substituted 2,4-(1H,3H) Pyrimidinedione. The present work is an important addition to microwave-assisted organic synthetic methodologies. It is concluded that by selecting such novel synthetic method, a large number of bioactive important compounds can be synthesized under safe, simple, clean, environmental-friendly conditions with the increased yield, save of time and energy within the seconds only.

Table-3: FTIR Analysis Vibrational Frequencies in cm^-1

Compounds	N₁-H	N₃-H	C₆-H₁₂	C₂=O	C₄=O	C₅=O₆	C₅-H/X
5-F uracil Microwave	3444.6	3159.3	2848.7	1706.08	1773	1649	1237.6
Conventional	3458.8	3170.12	2848.78	1705.83	1702.13	1605	1238.08
5-Chloro uracil Microwave	2718	2718	3062	1666	1711	1631	669
Conventional	3472.81	3074.007	2858.56	1780.17	1760.23	1483.25	683.44
5-bromo uracil Microwave	2462.18	2462.18	2812	1687	1626	1412.10	543.50
Conventional	3074.94	2918.84	2765	1672.19	1672.19	1434.62	645.56
5-iodouracil Microwave	3166.07	3064.23	2358.74	1699.9	1631.2	1491.09	603.04
Conventional	3205-2811	3205	3062	1711	1711	1631	669

X = F, Cl, Br, I

Table-4: GC-MS Analysis 5-Halogen-2,4-(1H,3H)Pyrimidinedione

Sr.No.	Formula	Base Peak	m/z Value
1.	C₄H₃ F N₂O₂	18	136
2.	C₄H₃ Cl N₂O₂	112	167
3.	C₄H₃Br N₂O₂	112	190
4.	C₄ H₃ IN₂O₂	18	238

Table-5: Antibacterial activity by well-diffusion method

Sr. No.

Compound

name

Concentration

in µg/ml

Organism name with inhibition of zone (mm)

B. subtilis

S. aureus

E. Coli

5-FU I

5-ClU II

11R

5-BrU III

5-IU IV

Where R = resistant

Table-6: Antiviral Activity

Sr. No.	Sample Name	Conc. in mg/ml	Name of Virus ND reduction in growth
1.	5-FU I	0.1	+ve
2.	5-ClU II	0.1	+ve
3.	5-BrU III	0.1	+ve
4.	5-IU IV	0.1	+ve

Compound No.	Compound Name	Solubility	Melting Point ^oC	UV-absorption Spectra λ_maxnm
I	5-flourourcil	Soluble in cold water	260 ^oC Decomposed	206, 281
II	5-chlorouracil	Soluble in hot water	285 ^oC Decomposed	203, 248
III	5-bromouracil	Soluble in hot water	290^oC Decomposed	197.5, 258.5
IV	5-iodouracil	Soluble in hot water	275^oC Decomposed	214, 282