Synthesis and Biological Activity of Some new 2-Heterocyclic/acyclic amino/4'-acetamidophenoxy-3- (4-chloro-phenyl)-3, 4-dihydrobenzo[e]- [1,3,2]oxazaphosphinine 2-sulfides

2-Heterocyclic/acyclicamino/4'-acetamidophenoxy-3-(4-chlorophenyl)3,4-dihydrobenzo-[e][1,3,2]oxazaphosphinine 2-sulfides (4a-j) were synthesized through a two steps process. In the first step, 2-chloro-3-(4-chlorophenyl)-3, 4dihydrobenzo[e] [1,3,2]-oxazaphosphinine 2-sulfide (2) was prepared by the reaction of 2-[(4-chlorophenylamino)methyl]phenol (1) with thiophosphoryl chloride in the presence of triethylamine in dry toluene-tetrahydrofuran. In the second step, 2 was treated with various heterocyclic/acyclicamines/4'acetamidophenol in presence of triethylamine-/sodium hydride in toluenetetrahydrofuran at 45-50°C. All the synthesized compounds (4a-j) were characterized by elemental analysis, IR, NMR (H, C and P) and mass spectra and their biological activity was evaluated for antimicrobial activity.


Introduction
Organophosphorus compounds being ubiquitous in nature have found multifaceted applications.Oxazaphosphinine derivatives, cyclophosphamide and its analogues, isophosphamides are clinically useful anticancer drugs 1,2 organophosphorus esters are used as pesticides and insecticides 3 Organophosphoramides are used as ligands in lewis based catalysed allylation and aldol addition reactions 4 Some of the benzoxazaphosphinines posses significant microbial activity 5,6 Several organophosphorus fungicides such as iprobenfos and edifenphos are used for the control of fungal diseases in plants 7 .Application of pesticides in soil has become a necessity for the control of both soil-borne and foliar diseases.Modern agricultural practices depend on synthetic chemical to maintain insect pest population in economic threshold. 8.The results obtained in our present study indicated that some of the microorganisms are able to degrade the pesticide compounds which were synthesized.The ease with which they are able to degrade depends on the complexity of their structure.More complex structure degrade the required quantity more slowly either because fewer microorganisms in the soil to produce enzyme capable of degrading them or because of their inaccessibility to microbes 9 .Phosphorus being a part of important biomolecules plays a fundamental role in microbial cell physiology and biochemistry 10 .In view of this, a series of 2heterocyclic/acyclic amino/4'-acetamidophenoxy-3-(4-chlorophenyl)-3, 4-dihydro-benzo[e] [1,3,2]oxazaphosphinine 2-sulfides (4a-j) have been synthesized.

Results and Discussion
The synthetic route involves preparation of the title compounds through a two-step process.[1,3,2]oxa-zaphosphinine 2-sulfide (2) was obtained from the cyclocondensation of 2-[(4-chlorophenylamino) methyl]phenol (1) with thiophosphoryl chloride in equimolar quantities in the presence of triethylamine in dry toluene-tetrahydrofuran at 35°C for 2 h.Subsequent nucleophilic substitution of heterocyclic/acyclic amines/4'-acetamidophenol (3a-j) on this monochloride (2) at 45-50 °C for 3h, yielded title compounds (4a-j).They were characterized by IR, 1 H, and 31 P NMR spectroscopic data. 13C NMR data for 4a, 4b, 4d and 4f and mass spectral data for 4b, 4d, 4e and 4f are given in experimental section.Compounds 4a-j showed IR absorption bands at 803-825, 949-975, 1082-1101 and 1211-1234 cm -1 for P=S, P-O, N-C and O-C arom respectively 3,11 .Their aromatic protons show a multiplet in their 1 H NMR spectra at slightly downfield (δ 6.43-7.62)when compared with those of the starting compound 1 (δ 6.29-7.32)owing to the deshielding effect of the 2-thio-benzoxazaphosphinine ring.From 1 H NMR data of 4i, the amidic proton (-NH) signal amidic -NH proton is less reactive 3 than phenolic group towards 2. The C-4 methylene protons resonated as two doublets of doublet at δ 4.32-4.48( 2 J HH = 10.6-11.2, 3 J PH = 5.2-5.3Hz) and 4.78-4.98( 2 J HH = 10.6-11.2, 3 J PH = 11.1-12.8Hz) indicating their non-equivalence and coupling with phosphorus in the six-membered chair conformation of the 2-thio-benzoxazaphosphinine system 3,12 C-4 resonated at δ 44.5-46.0 in the 13 C NMR spectra 3,6 .Other NMR data were also consistent for the substituents attached to the nitrogen, oxygen atom and aromatic protons and are presented in experimental section.The mass spectral data of 4a, 4d, 4e and 4f exhibited [M+2] + , M + and daughter ions with substituted benzoxazaphosphinine and heterocyclic/acyclic moieties at appropriate m/z values.Their mass spectral behavior is in good agreement with that of similar organophosphorus compounds 3 .Thus the combined elemental analysis, IR, NMR and mass spectral data conclusively agree with the proposed structures for the title compounds.

Biological activity
Antimicrobial activity of the title compounds 4a-j revealed interesting information.Even though the starting compound 2-[(4-chlorophenylamino)-methyl]-phenol 1 has phenolic, chloro, N-alkyl and NH functions, it does not show any antimicrobial activity.But when this compound is cyclized with thiophosphoryl chloride and followed by nucleophilic substitution at phosphorus resulting the compounds, 4a-j containing substituted oxazaphosphonin-2-yl amine/ phenyl systems showed promising antimicrobial activity.-XR'R'' Compd. -XR'R''

Antimicrobial activity
The antimicrobial activity of the compounds was comparable with that of the commercial antibiotics.The results indicated that compounds 4a-j exhibited significant antibacterial and antifungal activities.The compounds with nitrogen atom in the five membered aromatic rings showed the greatest inhibitory effect against one or more types of microorganisms (Table 1).The compound 4i showed the maximum zone of inhibition (25 mm) against Staphylococcus aureus.

General synthetic procedures
All reactions were carried out in anhydrous conditions under nitrogen atmosphere.Melting points were determined with open capillary tubes using Mel-temp apparatus and were corrected.IR spectra (ῡ max cm -') were recorded on a Perkin Elmer 1600 instrument as KBr pellets.Mass spectra were recorded on a LC-MSD-Trap-SL instrument at 70 eV.The 31 P, 1 H And 13 C NMR spectra were taken on a Bruker AMX-300 MHz spectrometer operating at 121.5 MHz for 31 P NMR, 300 MHz for 1 H NMR and 75 MHz for 13 C NMR.
All the compounds were dissolved in CDCl 3 and chemical shifts were referenced to 85% H 3 PO 4 ( 31 P NMR) or TMS ( 1 H NMR and 13 C NMR).Elemental analyses were performed using Perkin Elmer 2400 instrument at the Central Drug Research Institute (CDRI), Lucknow, India.2-[(4-Chlorophenylamino) methyl] phenol (1) was prepared using a reported procedure 13 .

Bioassay
The experiments were carried out on the cultures Escherichia coli, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus and fungal cultures Aspergillus niger and Candida albicans.

Microbial testing
The filter paper disc method 14,15 was fillowed using Sabouraud dextrose broth and Mueller Hinton broth.These agar media were inoculated with 0.5 mL of the 24h liquid cultures containing 10 7 microorganisms/mL.Whatman No. 1 filter paper discs containing 10 µg, 20 µg and 30 µg of test compounds were placed on the nutrient agar already plated with test bacteria.The incubation time was 24h at 37°C for bacteria and