Synthesis and Biological Evaluation of some Novel Pyrano[2,3- d ]Pyrimidine Derivatives

. The synthesis of novel Pyrano[2,3- d ]pyrimidine derivatives, had been synthesized by three component domino Knoevenagel hetero Diels-Alder reaction. The products were assayed for their in vitro biological assay antibacterial activity against with two Gram-positive bacteria Staphylococcus aureus MTCC-96, Streptococcus pyogenes MTCC 443, two Gram-negative bacteria Escherichia coli MTCC 442, Pseudomonas aeruginosa MTCC 441 and three fungal strains Candida albicans MTCC 227, Aspergillus Niger MTCC 282, Aspergillus clavatus MTCC 1323 taking ampicillin, chloramphenicol, ciprofloxacin, norfloxacin, nystatin, and griseofulvin as standard drugs. Among the various synthesized heterocyclic compounds, 1b, 1c and 1g are display broad spectrum antibacterial and antifungal activities against both gram-positive and gram-negative bacteria as compared with standard drugs.


Introduction
The development of a new strategy for the synthesis of complex organic molecules is an important aim in modern organic chemistry. L. F. Tietze introduced the domino-Knoevenagelhetero-Diels-Alder reaction as a powerful sequential transformation [1]. It has been proven that this reaction is a valuable method for the construction of annulated pyrans [2] and has already been used for the synthesis of several natural products [3]. A family of xanthone natural products with potent antiviral activity has been isolated from plants, and it is a potent inhibitor of human immunodeficiency virus-1 reverse transcriptase [4]. Pyrano [2,3-d]pyrimidine is unsaturated six member heterocycle which is formed by fusion of pyran and pyrimidine rings together, consisting of one oxygen atom at position number 8 and two nitrogen atoms at position number 1 and 3 respectively. If pyrano [2,3-d]pyrimidine moieties are clubbed into one molecule, then resultant derivative enhances its pharmaceutical activity as abundant in biologically active compounds such as antitumour [5], cardiotonic [6], antibronchitic [7] and antifungal activity [8]. Some of them exhibit antihypertensive activity [9], antimalarial [10], analgesic [10,11], and antimicrobial and antiviral evaluation [12,13] properties. Pyrano [2,3-d]pyrimidines are building blocks used to evaluate their antimicrobial activities and various derived natural products [14]. Therefore, for the preparation of these complex molecules large efforts have been directed toward the synthetic manipulation of pyrano[2,3-d]pyrimidine derivatives. As a result, a number of reports have appeared in the literature that usually requires forcing conditions, long reaction times and complex synthetic pathways. Pyrano [2,3-d]pyrimidine synthesis was reported under various conditions such as microwave irradiation [15] and [16], ultrasonic irradiation [17], solvent free condition and in aqueous medium in the absence of catalysts [18].
In order to develop an efficient synthesis of pyrano [2,3-d]pyrimidines is of considerable interest in this research to cater short reaction time, short reaction procedure and excellent yields by this proposed route. Methanol was used as a solvent and we describe here a rapid, viable and easy protocol for the synthesis of pyrano [2,3-d]pyrimidine derivatives by using piperidine as a catalyst (Scheme 1). The products 1a to 1o were screened for their antibacterial and antifungal evaluation.

Materials and Methods
Melting points were determined in open capillary tubes and are uncorrected. Formation of the compounds was routinely checked by TLC on silica gel-G plates of 0.5 mm thickness and spots were located by iodine. IR spectra were recorded on IRAffinity-1S FTIR Spectrophotometer-Shimadzu using 'neat' sample. Mass spectra were recorded on Shimadzu GC-MS-QP-2010 model using Direct Injection Probe technique. 1H NMR was determined in DMSO-d6 solution on a Bruker Ac 400 MHz spectrometer. Elemental analysis of the all the synthesized compounds was carried out on Elemental Vario EL III Carlo Erba 1108 model and the results are in agreements with the structures assigned.

Chemistry
A synthesis of annulated fused pyrano[2,3-d]pyrimidine 1a-1o as a domino Knoevenagel condensation reported in Scheme 1. The experimental procedure is straightforward. Solution of equimolar various aromatic aldehyde and barbituric stirred for 15-20 minutes in the presence of catalytic amount of piperidine in methanol as a solvent to gives knoevenagel adduct which further reacts with equimolar solution of ethyl vinyl ether under reflux condition for 5-6 hours afford pyrano [2,3-d]pyrimidinones in medium to excellent yield (Scheme 1).

General Procedures
Melting points of all the compounds are uncorrected and have been recorded by open capillary method. Silica gel-G was used for preparing the TLC plates using different solvent systems. Infra red spectra of all the compounds were scanned on SHIMADZU-FOURIER TRANSFORM INFRA RED (FTIR) -8400 Spectrophotometer using KBr pellet method. PMR Spectra were recorded on BRUKER Spectrophotometer (300 MHz) using TMS as an internal standard and DMSO-d 6 as solvent. Electron Impact (EI) mass spectra were recorded on GCMSQP2010 Mass Spectrometer.

Antimicrobial Evaluation
All of the synthesized compounds (1a to 1o) were tested for their antibacterial and antifungal activity (MIC) in vitro by broth dilution method [19][20]  The minimal inhibitory concentration (MIC) values for all the newly synthesized compounds, defined as the lowest concentration of the compound preventing the visible growth, were determined by using micro dilution broth method according to NCCLS standards [20]. Serial dilutions of the test compounds and reference drugs were prepared in Muellere-Hinton agar. Drugs (10 mg) were dissolved in dimethylsulfoxide (DMSO, 1 mL). Further progressive dilutions with melted Muellere-Hinton agar were performed to obtain the required concentrations of 1.56, 3.12, 6.25, 10, 12.5, 25, 50, 62.5, 100, 125, 250, 500 and 1000 µg mL -1 . The tubes were inoculated with International Letters of Chemistry, Physics and Astronomy Vol. 74 11 10 8 cfu mL -1 (colony forming unit/mL) and incubated at 37 ºC for 24 h. The MIC was the lowest concentration of the tested compound that yields no visible growth (turbidity) on the plate. To ensure that the solvent had no effect on the bacterial growth, a control was performed with the test medium supplemented with DMSO at the same dilutions as used in the experiments and it was observed that DMSO had no effect on the microorganisms in the concentrations studied. The results obtained from antimicrobial susceptibility testing are depicted in Table 1. Compounds 1c, 1g and 1b are display broad spectrum antibacterial activities against both gram-positive and gram-negative bacteria as compared with ciprofloxacin. Compounds 1b and 1g were found to be 4-fold more active against S. aureus and S. pyogens (MIC = 25 µg/mL) compared to the standard drug. While compound 1b showed equivalent activity against E. coli and P. aeruginosa and 4-fold more activity against S. pyogens (MIC = 25 µg/mL). Moreover, compound 1b exhibits 4-fold inhibition against S. aureus and equivalent inhibition against P. aeruginosa. As observed with high antibacterial potency of 1c and 1g a may be attributed to the presence of electron withdrawing substituents such as fluoro and bromo at 4 th position of aromatic ring. In comparison to the standard drug griseofulvin, the in vitro antifungal activity results indicated that compound 1g substituted with bromo group at 4 th position of phenyl ring was found to be the most potent against A. niger (MIC = 50 µg/mL), A. clavatus (MIC = 25 µg/mL) and C. albicans (MIC = 100 µg/mL) respectively.

Conclusions
In the present paper, we report the synthesis, spectral studies and antimicrobial activity of various pyran derivatives. The high bioactivity of these compounds makes them suitable tools for additional in vitro and in vivo evaluations, in order to develop new class of antimicrobial drugs or prodrugs with potential antimicrobial activity in the treatment of several dieses. Among the various synthesized heterocyclic compounds, 1b, 1c and 1g are found to be potent against both grampositive and gram-negative bacteria as compared with standard drugs. Further studies and modification in this area are in progress in our laboratory.