Synthesis, antibacterial and antifungal activity of 5-aryl amido-6-isopropyl-2-(mercapto/ hydroxy)-4-[(4′-difluoro methoxy) (3′-hydroxy) phenyl]-1,4-dihydro pyrimidine

. Various isopropylacetoacetanilides were treated with 4-(difluoromethoxy)-3-hydroxy benzaldehyde and thiourea/urea in the presence of acid catalyst to produce the 1,4-dihydropyrimidine derivatives ( 4a-5i and 5a-5i ) with good yields. All the synthesized compounds were characterized by mass, 1 H-NMR and IR spectrum, also evaluated for antibacterial and antifungal activity against two Gram +ve and two Gram -ve bacterial and fungi strains. The compounds 4e , 4g and 5d were found comparatively promising active against all the bacterial and fungal strains.


INTRODUCTION
Multi-component coupling reactions (MCR's), involving the inherent formation of several bonds in one step, have proven to be efficient and powerful tool for the rapid formation of complex heterocyclic compounds in recent years [1]. The unique features of these reactions are their operational simplicity, structural diversity, versatility, high synthetic efficiency, atom-economy, and single step synthesis without isolating the intermediates. The synthesis of 1,3-dihydropyrimidines via one-pot condensation of aldehyde, -dicarbonyl compound, and thiourea/urea known as Biginelli reaction [2] is one of the most recognized and often used MCR's for the synthesis of these valuable heterocyclic compounds.
Microbial infections are a growing problem in contemporary medicine. According to statistical evidence provided by WHO, many of the drugs treatment breakthroughs of the last century could be lost through the spread of antimicrobial resistance [3]. For instance, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae are important pathogens causing invasive diseases such as sepsis, meningitis, necrotizing fasciitis, pneumonia [4], nosocomial pneumonia [5], cystic fibrosis, acute leukemia, organ transplants, and intravenous-drug addiction [6]. Some of these pathogens have been reported to develop resistance [7] to the wellknown commercially available drugs. As a result, many infectious diseases may one day become uncontrollable and could rapidly spread throughout the world. Consequently, the discovery of potent antibiotic drugs is considered to be one of the greatest scientific and medical goals.
There is a growing interest pertaining to the synthesis of bioactive heterocyclic compounds in pharmacy. Among various heterocyclic compounds, pyrimidine derivatives have apparently gained considerable importance owing to their varied biological activities such as adenosine receptor antagonist [8], anti-inflammatory [9], CDK inhibitor [10], calcium channel antagonist [11] and antitumor [12] activities. On the other hand, a promising diverse pharmacological activity is shown by the pyrimidine nucleus such as anti-biotic, anti-fungal [13], anti-cancer [14], HIV protease inhibitor [15] and chemotherapeutic activities [16].
In the present work, we synthesized new several compounds via a one-pot, multi-component reaction of 4-(difluoromethoxy)-3-hydroxy benzaldehyde with various 1-Aryl amino-4methylpantane-1:3-dione and thiourea/urea (Scheme 2). The biological activities (MIC) of the synthesized compounds were compared with known standard drugs.

EXPERIMENTAL
Thin layer chromatography was accomplished on 0.2-mm precoated plates of silica gel G60 F254 (Merck). Visualization was made with UV light (254 and 365 nm). IR spectra were recorded on a FT-IR-8400 spectrophotometer using DRS prob. 1 H-NMR (400 MHz) spectra were recorded on a Bruker AVANCE II spectrometer in DMSO-d6. Chemical shifts are expressed in δ ppm downfield from TMS as an internal standard. Mass spectra were determined using direct inlet probe on a GCMS-QP 2010 mass spectrometer (Shimadzu). Melting points were measured in open glass capillaries and are uncorrected. Elemental analysis of the all the synthesized compounds were carried out on Elementar Vario EL III Carlo Erba 1108 model, and the results are in agreements with the structures assigned. International Letters of Chemistry, Physics and Astronomy Vol. 51

Procedure for synthesis of substituted 1-Aryl amino-4-methylpantane-1:3-dione (2a-2i)
A mixture of Aniline (0.5gm, 5.37 mol) and 4-methyl-3-oxopentanoate (0.83, 6.4 mol) in 1,4dioxane was refluxed in the presence of KOH for 14 hrs. The reaction mixture is monitored by TLC. After the completion of reaction, the product was poured into crushed ice, neutralize with dil. HCl make pH 4-5 and the separated solid was filtered out and crystallized from ethanol m.p 127 O C.

Antibacterial and antifungal activity
The newly synthesized compounds were screened for their antibacterial activity against Grampositive (S. aureus ATCC 6538, M. luteus ATCC 9345), Gram negative (E. coli ATCC 4230, S.thyphi ATCC 14028) bacteria, as described by the guidelines in NCCLS-approved standard document M7-A4, using the micro dilution broth procedure [17]. Ampicillin trihydrate was used as the reference antibacterial agent. The antifungal activities of the newly synthesized chemical compounds were tested against yeast strain (C. albicans ATCC 14053) according to the guidelines in NCCLS-approved standard document M27-A2, using the micro dilution broth procedure [18]. Fluconazole was used as the reference antifungal agent. The solutions of test compounds and reference drug were prepared by dissolving in DMSO at a concentration of 2560 μg/mL. The 2-fold dilutions of the compounds and the reference drug were prepared (1280, 640, 320, 160, 80, 40, 20, International Letters of Chemistry, Physics and Astronomy Vol. 51 10 μg/mL). Antibacterial activities of the newly synthesized chemical compounds were performed in Mueller-Hinton broth medium at a pH of 7.2 with an inoculum of (1-2) × 10 3 cells/mL by the spectrophotometric method, and an aliquot of 100μL solution was added to each tube of serial dilution. The chemical compounds-broth medium serial tube dilutions inoculated with each bacterium were incubated on a rotary shaker at 37°C for 18 hrs at 150 rpm. The minimum inhibitory concentration (MIC) of each chemical compound was recorded as the lowest concentration of each chemical compound in the tubes with no growth (i.e., no turbidity) of inoculated bacteria. Minimum inhibitory concentration (MIC, μg/mL) was measured and compared with control; the MIC values of the compounds (4a-4i and 5a-5i) are represented in table no. II.

Table no. II
From the result of biological evaluation, it has been observed that the compounds exhibited interesting biological activity, however with a degree of variation. Most of the compounds tested were found to have comparable antibacterial and exhibit low antifungal activity. From the Table-

CONCLUSION
In summary, we have synthesized a series of new 1,4-dihydropyrimidine derivatives. All the newly synthesized compounds were confirmed with spectroscopic data like 1 H-NMR, Mass, IR Spectra, elemental analysis and evaluated antibacterial and antifungal activity. The antibacterial and antifungal study shows that the compounds 4e, 4g, 4e, 5a, 5d, 5e and 5i have promising activity with MICs between 40 and 80 µg/mL. The importance of such work lies in the possibility that the new compounds might be more efficacious drugs against bacteria, which could be helpful in designing more potent antibacterial agent for therapeutic use.