Synthesis Of New P-substituted Iminophosphoranes Via Staudinger Raction And Antimicrobial Activity Evaluation

GRAPHICAL ABSTRACT Abstract A class of two series of new P-substituted iminophosphoranes, N-(1,3-benzothiazol-2-ylmethyl)-N-(alkyl subtituted-λ5-phosphanylidene)amine 5(a-e) and N-(alkyl substituted-λ5-phosphanylidene)-(3,5-difluorophenyl)methanamine 9(a-e) was accomplished via Staudinger reaction of sodium azide, 2-chloromethyl benzothiazole (1) or 3,5-difluorobenzylbromide (6) and various trivalent alkyl/aryl phosphines/ethyldiphenylphosphinite/dimethylphenylphosphonite 4(a-e). The structures of new products were elucidated from spectroscopic data and elemental analysis. In vitro antibacterial and antifungal activities of the title compounds were investigated against four bacterial strains and three fungal strains at 50 and 100 μg/mL concentrations, respectively. The minimum inhibitory concentration (MIC) of the title compounds were also determined. Among all the compounds, 3,5-difluorobenzyl core derivatives 9a and 9b, and 1,3-benzothiazol-2-ylmethyl constituent containing compounds, 5b and 5c showed promising antibacterial and antifungal activities, respectively, at lower MIC values in the range of 6.25–25.0 μg/mL and approximately closer to the standards.


Introduction
Phosphorus-nitrogen chemistry, particularly, trivalent and pentavalent phosphorus compounds, is enormous and has been used in wide variety of fields. 1 Pentacoordinated phosphorus derivatives are valuable substances in the molecular designing of bioorganic structures due to their unique biological properties. Also, compounds possessing P-N bond have been emerging as an important class of compounds in modern organic chemistry with numerous applications in catalytic synthetic transformations, 2 used as catalysts in organic synthesis 3 and inhibitors for the treatment of various diseases. 4 In general, the chemistry of iminophosphoranes formed by Staudinger reaction has been studied in considerable detail for more than five decades. 5 Staudinger reaction involves in the nucleophilic attack of the trivalent phosphorus at the terminal nitrogen of the azide leading to the phosphazide with retention of phosphorus center, which stabilizes via a four-membered transition state by the loss of nitrogen and then leads to the formation of iminophosphorane R 3 P D N-R. 6 Also, the unique synthetic potential of iminophosphoranes resulted from the presence of an electron-rich nitrogen atom and the nucleophilicity of nitrogen is a key factor of the necessary mechanistic significance in their applications as aza-Wittig reagents. 7 The distributions of electrons around the PHN bond have been investigated through theoretical, spectroscopic, and crystallographic studies. 8 Various hetero-cyclization reactions that involve iminophosphoranes have been reviewed and these compounds can be easily converted into hetero-cumulenes through anaza-Wittig type reaction. 9 The intramolecular aza-Wittig-type reaction has significant interest because of its high potential for the synthesis of a wide variety of nitrogen containing heterocycles, which can be formed from the rapid progress in the preparation of iminophosphoranes derivatives as starting materials. 10 The iminophosphoranes possess numerous applications in organic synthesis and coordination chemistry as ligands toward transition metal complexes due to the lone pair electrons on the N atom, and in organometallic chemistry. 11 Recently, more attention has been focused on iminophosphorane chemistry by researchers due to its high potential for the synthesis of natural products, 12 and incorporation of different substituents at the phosphorus.
In the last 10 years, many synthetic methods have been reported for the preparation of (N-isocyanimino)triphenylphosphorane and it has been used in the synthesis of metal complexes, 13 and the applications of these compounds have found utility in the preparation of various organic compounds. 14,15 The role of (N-isocyanimino)triphenylphosphorane in organic chemistry remains almost unexplored and it is expected to have unique synthetic potential because it provides a reaction system in which the iminophosphorane group can react with a reagent having a carbonyl functionality. 16 Iminophosphoranes react with carbonyl compounds to form Schiff's bases and phosphine oxides 17 and also react with carbon dioxide and carbon disulfide to form isocyanates and isothiocyanates, respectively. 18 Effective transformation of N-substituted iminophosphoranes by reaction with acids, 19 water, 20 alkyl halides, 21 oxiranes, 22 nitrosyl chloride, 23 acetylenecarboxylate, 24 and ozone 25 demonstrate their versatility in organic synthesis. Recently, some researchers have focused on the synthetic applications of iminophosphoranes, to develop efficient and robust methods for the synthesis of heterocyclic compounds. 26,27 In addition, benzothiazoles are significant fragments in medicinal chemistry due to their diversity of biological activities and occur broadly in biologically active natural products. 28 According to a literature survey, several compounds containing benzothiazole ring display a broad spectrum of biological activities such as antitumor, 29 antitubercular, 30 antimalarial, 31 anticonvulsant, 32 anthelmintic, 33 anti-inflammatory, 34 antifungal, 35 antibacterial, 36 and antiviral properties. 37 Various substituted benzothiazoles such as 2-aryl benzothiazole derivatives are used as radioactive amyloid imagining agents 38 and anticancer agents 39 , whereas 2-(4aminophenyl) benzothiazoles are novel potent, selective antitumor and cytotoxic agents. Also, fluorine containing organic molecules possesses promising importance in medicinal chemistry which led to valuable enhanced biological activity, metabolic stability, and liphophilicity. 40 The number of commercially significant life products and valuable drugs having antibacterial activity, antitumor activity, and antiinflammatory activity 41,42 have owed their activities to the presence of fluorine substituent in their structure.
Considering aforesaid facts, we have synthesized new series of P-substituted iminophosphoranes of 2-chloromethyl benzothiazole and 3,5-difluorobenzyl bromide by reacting with substituted phosphines. The antibacterial and antifungal activities were evaluated and the bio-screening data revealed that majority of the compounds showed potent antimicrobial activity.

Chemistry
The synthesis of P-substituted new iminophosphoranes 5(a-e)/ 9(a-e) was achieved in two steps. The schematic presentation was depicted in Scheme 1.
Structures of the newly synthesized compounds 5(a-e)/9(a-e) were confirmed by spectroscopic data. IR spectra of compounds 5(a-e)/9(a-e) showed intense bands in the region of 3080-3030, 1590-1530, 1452-1424, 1214-1272, 1033-1018, 978-940, and 690-675 cm ¡1 due to C-H aromatic , CHC aromatic , P-Ph, PHN, P-O-C (aliphatic), P-N, and C-H stretching frequencies respectively, confirmed the functionalities present in the title compounds. 43 1 H NMR spectra showed the chemical shifts in the region of d 2.54-2.63 as singlets due to PHN-CH 2and d 8.44-6.40 corresponding to aromatic protons confirming the formation of target compounds. 13 C NMR spectra showed the chemical shifts in the region of d 32.3-47.7 as singlet due to HN-CH 2and d 168.5-101.5 corresponding to aromatic carbons. In 31 P NMR spectra, appearance of the chemical shifts in the region of d 28.1-32.4 to phosphorus atom confirmed the iminophosphorane group in the title compounds. 44 In mass spectra, molecular and fragmented ion peaks were given further evidence to elucidate structures of the newly synthesized compounds. Elemental analysis also confirmed the structures of title compounds.

Biological activity
The title P-substituted iminophosphoranes 5(a-e)/9(a-e) were screened for their in vitro antibacterial activity against bacterial strains such as B. subtilis, S. aureus, E. coli, and P. carotovorum using the agar well-diffusion method. 45 Chloramphenicol was used as a reference standard for antibacterial study. In vitro antifungal activity of these compounds 5(a-e)/9(a-e) was also evaluated against fungi such as R. solani, A. niger, and S. rolfsii by using two-fold dilution method. 46 Ketoconazole was used as a reference standard for the antifungal study. The minimum inhibitory concentrations (MIC) of these compounds 5(a-e)/9 (a-e) were also determined by the tube dilution method. 47 The results of antibacterial, antifungal, and MIC screening of the tested compounds are given in Tables S1-S3, respectively (Supplemental Materials). Additional results and experimental details are also included in the Supplemental Materials.

Conclusion
In summary, we reported the synthesis of P-substituted iminophosphoranes of 2-chloromethylbenzothiazole/3,5-difluorobenzylbromide via Staudinger reaction. Antibacterial activity against four bacterial strains and antifungal activity against three fungal strains were evaluated at two concentrations, 50 and 100 mg/mL including their MIC values. Most of the newly synthesized compounds exhibited good to promising growth of inhibition of microbial pathogens. Among all the compounds, two 3,5-difluorobenzyl core derivatives 9a and 9b against all the tested bacterial strains and two 1,3-benzothiazol-2-ylmethyl constituent containing compounds, 5b and 5c against all the tested fungi showed potential activity at lower MIC values in the range of 6.25-12.5 mg/mL and which are approximately closer to the standards.

Experimental
All reagents and chemicals are purchased from Sigma-Aldrich, Merck and S.d. fine. Reagent grade solvents are used for spectroscopic studies. All the reactions were conducted in nitrogen atmosphere and glassware was dried in hot air oven at 150 C. Melting points were recorded in an open capillary tube by GUNA digital apparatus and are uncorrected. IR spectroscopic data was recorded on Bruker Alpha-Eco ATR-FTIR (attenuated total reflection-Fourier transform infrared) interferometer with single reflection sampling module equipped with ZnSe crystal. 1 H, 13 C, and 31 P NMR spectra were recorded on a BRUKER AV 400 spectrometer operating at 400 MHz for 1 H NMR, 100 MHz for 13 C NMR, and 161.9 MHz for 31 P NMR. Tetramethylsilane (TMS) in 1 H, 13 C NMR, and 85% H 3 PO 4 in 31 PNMR were used as internal and external standards, respectively, in DMSO-d 6 solvent. E.S.I-Mass spectra were recorded on Aglient 1000 mass spectrometer. Elemental analysis was performed on Thermo Finnigan FLASH EA 1112 instrument. Chemical shifts were recorded in parts per million (ppm) and multiplicities are represented as abbreviations: s (singlet), brs (broad singlet), d (doublet), and m (multiplet).
General procedure for the synthesis of N-(1,3benzothiazol-2-ylmethyl)-N-(1,1,1-triphenyl-λ 5phosphanylidene)amine (5a) 2-Chloromethylbenzothiazole (1) (0.183 g, 0.001 mol) and potassium iodide (KI) (0.166 g, 0.001 mol) were taken into a round bottomed flask containing 20 mL of THF. The reaction mixture was stirred for 3 h at 40 C. After completion of the reaction as checked by TLC, cooled the reaction mass to RT and then filtered to remove the salt (KCl), resulted in iodo compound, 2-(iodomethyl) benzo[d]thiazole (2). The filtrate was transferred into a flask and sodium azide (NaN 3 ) (0.065 g, 0.001 mol) was added. The reaction mixture was stirred at 25-30 C for 3 h to form an intermediate, 2-azidomethylbenzothiazole (3). The reaction mixture was filtered to remove the salt, NaI, and filtrate was taken for the next step. Triphenylphosphine (4a) (0.262 g, 0.001mol) was added to 3 under N 2 atmosphere. The reaction mixture was stirred at 65-70 C for 4 h and the progress of the reaction was monitored by TLC using ethylacetate: n-hexane (2:3) as an eluent. After completion of the reaction, the solvent was removed from the reaction mixture in a rotaevaporator to get the crude product and it was purified by column chromatography using ethylacetate:nhexane (1:4) to obtain pure product, N-(1,3-Benzothiazol-2ylmethyl)-N-(1,1,1-triphenyl-λ 5 -phosphanylidene) amine (5a). The same procedure was adopted for the synthesis of remaining title compounds and the physical data of these compounds are summarized in Table 1.

Funding
The authors G. Madhava and SK. Thaslim Basha express their grateful thanks to University Grants Commission (UGC), New Delhi, India for providing financial assistance by awarding Senior Research Fellowship (SRF).