Synthesis of Novel Indolyl-Pyrimidine Antiinflammatory, Antioxidant and Antibacterial Agents

A number of chalcones were synthesized by reacting indole-3-aldehyde, prepared by Vilsemeir Haack reaction with 4-substituted acetophenone in NaOH solution in ethanol. These chalcones were immediately reacted with urea, thiourea and guanidine hydrochloride in presence of concentrated hydrochloric acid as reagent to obtain the corresponding hydroxy, thio and amino pyrimidines. The synthesized heterocyclics were characterized on the basis of physical, chemical tests and spectroscopic data and were tested for the acute antiinflammatory activity, antioxidant, antibacterial activity using carragenan-induced rat paw oedema method, DPPH (diphenylpicrylhydrazyl) radical scavenging method and cup plate method using Muller-Hinton agar media respectively. Evaluation of the compounds revealed remarkable antiinflammatory activity reflected by their ability to reduce the carragenan-induced inflammation in rats, appreciable antioxidant activity and also antibacterial activity was observed.

Due to interesting activity of various substituted pyrimidines as biological agents 1-3 , considerable attention has focused on this class. The pharmaceutical importance of these compounds lies in the fact that they can be effectively used as analgesics, antiinflammatory, anticonvulsant, insecticidal, herbicidal, antitubercular, anticancer and antidiabetic agents. The ability of indole to exhibit antiinflammatory, antimicrobial, antifungal activities 4-6 prompted the selection of indole as starting compound.
In the light of these interesting biological activities, it appeared of interest to synthesize some new hydroxy/ thio/amino pyrimidine derivatives. Indole-3-aldehyde (2) prepared by Vilsemeir Haack reaction from indole (1) was reacted with 4-substituted acetophenones (a-j) in ethanolic NaOH to obtain chalcones (3a-j), which were condensed with urea, thiourea and guanidine hydrochloride in presence of cyclising agent, concentrated hydrochloric acid to obtain hydroxy pyrimidines (4a-j) thio pyrimidines (5a-j) amino pyrimidines (6a-j) respectively. The synthetic sequence leading to the formation of targeted compounds is depicted in scheme 1.
The constitution of the products has been supported by element analyses, chemical tests, IR, NMR and Mass spectral data. All the compounds were evaluated for antiinfl ammatory activity using the carrageenaninduced rat paw edema model, antioxidant activity using DPPH and antibacterial activity against four different strains of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli at different concentration of 5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml.

MATERIALS AND METHODS
All the melting points were determined using open capillary tubes in scientifi c melting point apparatus and are uncorrected. UV values were obtained by using Shimadzu UV/Vis Spectrophotometer, UV-1601PC, at Manipal College of Pharmaceutical Sciences, Manipal. IR spectra of the synthesized compounds were scanned using a Shimazdu-Fourier Transform Infrared Spectrophotometer-8300 and KBr press at Manipal College of Pharmaceutical Sciences. PMR spectra of the synthesized compounds were recorded on 300 MHz spectrophotometer at IISC Bangalore. Mass spectra of the synthesized compounds were recorded on Shimazdu-GC-MS QP 5050A at Manipal College of Pharmaceutical Sciences. Progress, purity of the reaction and the intermediates of the aldehyde was confirmed by the Tollen's test and 2,4-dinitrophenylhydrazine test. The compound was characterized by comparing the malting point and PMR with that reported in literature 7 .

1-(4-substituted phenyl)-3-indolyl-2-propen-1-one (3a-j):
Equimolar quantities (0.01 mol) of indole-3-aldehyde and substituted acetophenones were taken in 100 ml conical fl ask and dissolved in 20 ml of ethanol to this (0.03 mol) of NaOH in minimum quantity of water was added. The mixture was stirred on a magnetic stirrer and the reaction was monitored with TLC. Reaction mixture was diluted with water and acidifi ed with concentrated hydrochloric acid. The precipitated chalcone was fi ltered and recrystallized from absolute ethanol. The purity of chalcones was tested with thin layer chromatography using solvent system: petroleum ether (60-80º):ethyl acetate [70:30] 4-(Indol-3-yl)-6-(4-substituted phenyl)-2-substituted pyrimidines: Chalcone (0.01 mol) and urea, thiourea or guanidine hydrochloride (0.01 mol) were dissolved in absolute were analyzed using precoated TLC plate and using UV chamber. Male Wistaar rats (150-200 g) were procured and the antiinflammatory activity was carried out on approval of Institutional Animal Ethics Committee, KMC, Manipal.

RESULTS AND DISCUSSION
All the physical characteristics of the synthesized copound are presented in Table 1. The purified products were screened for antiinflammatory, antioxidant and antibacterial activity. Six compounds were screened for antiinflammatory activity using carrageenan-induced rat paw oedema method. Out of them 4f, 5band 6c have shown better antiinfl ammatory activity than the standard drug, ibuprofen ( Table 2). All products were screened for antioxidant activity using DPPH radical scavenging method. Compounds 4b, 4f, 4h, 5b, 5h, 6e and 6h have exhibited good antioxidant activity which is comparable with that of standard drug, ascorbic acid (Table 3). All products were screened for antibacterial activity towards different strains of Staphylococcus aureus, Bacillus subtilis Pseudomonas aeruginosa and Escherichia coli at different concentration of 5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml compared to standard drug, ciprofl oxacin. The compounds 4b, 4c, 4d, 4f, 4h 4j, 5b, 5c, 5d, 5f, 5h, 5j, 6d, 6f, 6h and 6j showed very good zone of inhibition at the concentration of 5-10 µg/ml against different bacteria (Table 4). Based on the structure activity relationships, it can be concluded that the presence of halogen group at 4 positions i.e. 4c, 4d, 4f, 5c, 5d, 5f, 6d and 6f showed good activity towards gram negative bacteria. Presence of nitro group and methoxy group at 4 position i.e. 4h, 4j, 5h, 5j, 6h, and 6j displayed good activity towards Gram positive bacteria.