Synthesis and antibacterial activity of new aryl / alkyl phosphonates via Michaelis-Arbuzov rearrangement

Synthesis of new aryl / alkyl phosphonates 3a-j has been accomplished via a MichaelisArbuzov-type rearrangement by the reaction of aryl / alkyl halide (1a-j) with triethyl phosphite (2) in dry toluene at reflux temperature. Products 3a-j were characterized by IR, C and P NMR and their antibacterial activity was evaluated.


Introduction
Phosphorus compounds containing the P-C bond are not particularly abundant in nature.Their diverse biological activity, 1,2 has for a long time attracted considerable synthetic 3 and pharmacological interest. 4The Michaelis-Arbuzov rearrangement, also known as the Arbuzov reaction, is very versatile way to form P-C bond from the reaction of an aryl / alkyl halide and trialkyl phosphite. 5This reaction is one of the most extensively investigated and is widely used to prepare phosphonates, phosphinates and phosphine oxides. 61][12] Without catalyst, Michaelis-Arbuzov rearrangement can only be carried out with highly activated benzene compounds by heating them with phosphites to yield the corresponding phosphonates. 13In our work we synthesized aryl / alkyl phosphonates without catalyst and under the mild conditions.a Obtained viscous liquids that decompose on attempted vacuum distillation.b After separation from the column chromatography.
The 31 P NMR spectral data for 3a-j are given in the Table 2.The 31 P NMR signals for 3b, 3c, 3e and 3g-i appeared as two distinct signals in the range of -1.28 to -2.08 and 5.74 to 20.71 ppm.This may be due to the presence of two isomers 16b,16c in the solution state with sufficient internal energy difference and considerable stability that enable measurement of their 31 P NMR resonance.The other compounds 3a, 3d, 3f and 3j gave only one 31 P NMR signal in the range of -1.29 to -1.50 and 7.09 ppm. Compd.Compd.

Antibacterial activity
The compounds were diluted in DMSO for bioassay.Solvent control was included although no antibacterial activity has been noted for the solvent employed.Ciprofloxacin (Hi-media) controls were included to compare with compounds 3a-j.All samples were tested in triplicate and average results were recorded.The compounds were assayed for antibacterial activity against six registered bacterial isolates which were obtained from the NCIM (National Collection of Industrial Microorganisms, National Chemical Laboratories, Pune-411 003, India).General procedure for products 3a-j.In a flame-dried three-necked flask the appropriate aryl / alkyl halide (0.001 mol) was mixed with triethyl phosphite (0.249 g, 0.0015 mol) and stirred at reflux temperature for 6-8 hrs and protected with a CaCl 2 -tube, respectively.After the completion of reaction (monitored by TLC), the oily product was obtained.The product was purified by column chromatography on silica gel using petroleum ether-ethylacetate (7:3) as eluent.

Disc diffusion bioassay.
For bioassay suspension of approximately 1.5 x 10 8 bacterial cells per mL were used.In sterile normal saline was prepared as described by Forbes et al 17 and 1.5 mL of it was uniformly spread on Nutrient Agar (Hi-media) in 12 x 1.2 cm glass Petri dishes, left aside for 15 min and excess of suspension was then drained and discarded properly.For the agar disc diffusion method, the test compound was introduced onto the disc and then allowed to dry.Thus the disc was completely saturated with the test compound.Then the disc was introduced onto the upper layer of the medium with the bacteria.The petri dishes were incubated overnight at 37 °C for 24 hrs.Bioactivity was determined by measuring Diameter of Inhibition Zones (DIZ) in mm.The compounds' 3a-j concentrations were taken as 20 and 40 / mL were evaluated for disc method.Each test was done in triplicate and the mean of the diameter of the inhibition zones was calculated.Controls included the use of solvent without test compounds although no antibacterial activity was noted for the solvent employed in the test 18 (Table 3).

Determination of minimum inhibitory concentration (MIC).
Minimum inhibitory concentration (MIC) was determined for the compounds 3a-j.The concentration at which there was no visually detectable bacterial growth was taken as MIC.Compounds 3a-j concentrations of 0.1-5.6 mg / mL in steps of 100 µg / mL were evaluated.Specifically 0.1 mL of standardized inoculum (1-2 x 10 7 CFU / mL) was added to each tube.The tubes were incubated aerobically at 37 °C for 18-24 hrs.Two control tubes were maintained for each test batch.These included antibiotic control (tube containing compounds 3a-j and the growth medium without inoculum) and organism control (the tube containing the growth medium, physiological saline and the inoculum).The lowest concentration (highest dilution) of the compounds 3a-j that produced no visible bacterial growth (no turbidity) when compared with the control tubes was regarded as MIC 18 (Table 4).

Conclusions
We synthesized bioactive and novel phosphonates 3a-j in high yield by Michaelis-Arbuzov reaction without any catalyst.They showed moderate antibacterial activity against selected bacteria.Among all these compounds 3e showed highest antibacterial activity at lower ISSN 1424-6376 Page 134 © ARKAT concentration against both Gram negative and Gram positive bacteria.Compounds 3g, 3i showed lowest activity even at highest concentrations.Compound 3d showed highest activity against Gram positive bacteria when compared with Gram negative bacteria.

Table 1 .
Physical and IR spectral data of 3a-j

Table 3 .
Antibacterial activity of compounds 3a-j in terms of DIZ in mm