A Novel One-Pot and Efficient Procedure for Synthesis of New Fused Uracil Derivatives for DNA Binding

Hydrazinolysis of 6-chloro-1-methyluracil followed by condensation of the product with different aromatic aldehyde gives the respective hydrazones which undergoes oxidative cyclization using thionyl chloride to obtain pyrazolo[3,4-d]pyrimidines in good yields. On the other hand, nitrosation of 6-aminouracils followed by the reaction with different arylidineanilines gives new xanthine derivatives. Finally, indenopyrrolopyrimidine and indenopteridine are obtained in good yields via the reaction of 6-aminouracils and 5,6-diaminouracil with ninhydrin respectively. The newly synthesized compounds show binding, chelation and fragmentation of the nucleic acid DNA.


Introduction
The importance of fused pyrimidines, common source for the development of new potential therapeutic agents [1] [2], is well known.
Fused pyrimidines continue to attract considerable attention because of their great practical usefulness, primarily due to very wide spectrum of biological activities.This is evident especially from publications of regular reviews on the chemistry of systems where the pyrimidine ring is fused to various heterocycles such as purines, quinazolines, pyridopyrimidines, triazolopyrimidines, pyrazolopyrimidines, pyrimidoazepines, furopyrimidines and pyralopyrimidines.
Pyrido [2,3-d]pyrimidines possess dihydrofolate reductase inhibiting and antitumour activity [13].Similarly, in recent years, considerable attention has been focused on the development of new methodology to synthesize many kinds of pyrazolopyrimidine ring [14].Indeed, pyrazolopyrimidines [15] [16] and purines [17] represent an important class of heterocyclic compounds having wide range of pharmaceutical and biological activities.Therefore, versatile and widely applicable methods for the synthesis pyrazolopyrimidines and purines are of considerable interest.The existing methods for the preparation of pyrazolopyrimidines are based on heterocyclic hydrazones or hydrazine precursors.Pyrimidines and their derivatives are considered to be important for drugs.A large number of pyrimidine derivatives are reported to exhibit antimycobacterial [18], antitumor [19], antiviral [20], anticancer [21] [22] activities.In the present study, a series of new pyrimidine fused ring analogs have been synthesized and their biological effects are determined.

Chemistry
All melting points were determined with an Electrothermal Mel.-Temp.II apparatus and were uncorrected.Element analyses were performed at the Micro Analytical Unit, Chemistry Department, Mansoura University.The infrared (IR) spectra were recorded using potassium bromide disc technique on Nikolet IR 200 FT IR at Pharmaceutical Analytical Unit, Faculty of Pharmacy, Al-Azhar University.The proton nuclear magnetic resonance ( 1 H-NMR) spectra were recorded on Varian Gemini 300 MHz Spectrometer using DMSO-d 6 as a solvent and tetramethylsilane (TMS) as an internal standard (Chemical shift in δ, ppm), Faculty of Science, Chemistry Department, Cairo University.Mass spectra were recorded on DI-50 unit of Shimadzu GC/MS-QP 5050A at the Regional Center for Mycology and Biotechnology at Al-Azhar University.All reactions were monitored by TLC using precoted plastic sheets silica gel (Merck 60 F 254 ) and spots were visualized by irradiation with UV light (254 nm).The used solvent system was chloroform: methanol (9:1) & ethyl acetate: toluene (1:1).

Nucleic Acids Preparation
For extraction of genomic DNA, yeast cells were washed with cold phosphate borate sodium chloride (PBS) buffer and lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.2% Triton X-100 for 20 min at 4˚C.After centrifugation at 14,000 rpm for 15 min, the supernatant was treated with proteinase K (0.5 mg/ml) and 1% SDS for 1 h at 50˚C.DNA was extracted twice with buffered phenol/chloroform and precipitated with 140 mM NaCl and 2 volumes of ethanol at −20˚C overnight.DNA precipitates were washed twice with 70% ethanol, air-dried and dissolved in TE buffer, and treated for 1 h at 37˚C with RNase A according to reported method [24].Finally, DNA preparations were electrophoresed in 1% agarose gels.

Agrose Gel Preparation and Visualization of DNA
1% agarose gel was prepared by adding 1 gm ultra agarose to 100 ml Tris-Acetate-EDTA (TAE) buffer and heated in a microwave oven then cooled to ~60˚C before pouring in gel tray.
Examination of the gel was carried out using ultraviolet illuminated box.Ethidium bromide (0.1 mg/ml) solution was used to stain the nucleic acid (DNA bands) in the gel as it intercalates between DNA bases and give florescence.The gel was photographed using polarized camera.

Nucleic Acid Affinity, Binding and Fragmentation Assay
The test compounds were dissolved in DMSO at 20 µg/µl concentrations, mixed with 2 µg/µl DNA and incubated at room temperature for 2 hrs.The mixtures were mixed with the gel loading buffer and then electrophoresed in the agarose gel (1% w/v) at 80 V for 1.5 hrs.As positive control for affinity, binding and fragmentation, methotrexate (20 µg/µl) was mixed with DNA, and as negative control DMSO was mixed with equal amount of DNA.After running, agarose gels were stained with ethidium bromide and visualized using polarized camera.
Thus, refluxing of compounds 4a-f with thionyl chloride resulted in intramolecular cyclization affording pyrazolopyrimidines 5a-f presumably via the formation of the 5-chlorosulfinyl derivatives A which loses (SO) group and HCl to form Xa-f. The structure of target compounds was confirmed by element analysis in addition to IR, 1 H-NMR spectral data.Compounds 6a,b were prepared in good yields by the condensation of ethyl cyanoacetate with N-benzyluea [29] or N-[(2-chlorophenyl)methyl]urea [30] in sodium ethoxide or methoxide.In this work, it was in need to prepare first the unavailable starting material, 6-amino-1-[(2-chlorophenyl)methyl]-5-nitrosouracil (7).Reaction of 6-amino-1-[(2-chlorophenyl)methyl]uracil (6b) with aqueous sodium nitrite in the presence of acetic acid afforded a high yield of the coloured nitroso derivative 7 [31].Thus, reaction of 7 with different arylidene aniline in acetic acid took place by the elimination of aniline to give 8a-d.An expected mechanism might be as follows: On the other hand, the reaction of aminouracils 6a,b by refluxing with ninhydrin in DMF resulted in the formation of indenopyrrolopyrimidines 9a,b in a moderate yields.It was reported that the 2-position of the ninhydrin is more reactive towards nitrogen [32], oxygen [32] [33] and carbon based nucleophiles [32]- [34].The cyclization affording 9a,b presumably occurred via the formation of nonisolable acyclic intermediate.The latter might be formed via the attack of the more nucleophilic carbon at 5-position of uracil to the more reactive center at 2-position of ninhydrin.Cyclization could be affected via the addition of the amino group to the carbonyl at 1-position of ninhydrin moiety affording the final product 9a,b. 1 H-NMR showed the two benzylic hydrogens as two doublets at δ = 4.94 -4.67 ppm which indicated that they were not magnetically equivalent.This observation may be attributed to the presence of stereoisomers resulted from the two asymmetric carbons at 4b and 9b positions.The project now directed towards the possible utility of diaminouracils for the synthesis of the title compound 11.Thus, the reaction of 5,6-diamino-1,3-dimethyluracil hydrochloride (10) [29] [35]- [37] with ninhydrin in the presence of triethylamine or ammonium hydroxide afforded the title compound 11.The formation of 11 from the aminouracil 10 and ninhydrin may be proceed through first condensation between the more reactive NH 2 at 5-position with the more electrophilic center at C-2 of ninhydrin.Attack of the less reactive NH 2 group at 6-position to one of the C = O groups of the reagent afforded the cyclized tautomer B which was stabilized by loss of H 2 O to give 11.

Biological Evaluation
The newly synthesized compounds were subjected to nucleic acid binding assay using agarose gel electrophoresis method.

Nucleic Acids Binding Assay
Different synthetic drugs induced DNA damage was evaluated by measuring the level of genomic DNA fragmentation and detecting DNA ladders on agarose gel electrophoresis (Figure 1).Compared with the vehicle control group (lane 2 negative control and lane 3 positive control), there was no significant change in genomic DNA fragmentation in some treated groups.There were major differences in the response of extracted DNA (from Lanes 4-17 in Figure 1).It is possible that drugs exert its effect solely by indirect mechanisms.This contrast may have been due to different enzyme(s) being with differing susceptibilities to drugs.

Conclusion
Our results describe a simple and efficient method for the synthesis of different novel fused uracils.Heteroannulation on the C-5 of uracil usually requires forcing conditions and complex synthetic pathways.Our synthetic compounds concern with the reactions of uracils with different benzylideneaniline, araldehydes and ninhydrin which have a biological screen.