SYNTHESIS AND BIOLOGICAL EVALUATIONS OF A SERIES OF NOVEL AZOLYL, AZINYL, PYRANYL, CHROMONYL AND AZEPINYL PHOSPHONATES

– A facile synthetic methodology of novel azolyl, azinyl and azepinyl phosphonates


INTRODUCTION
α-Aminophosphonic acid diesters, as phosphorus analogues of α-aminocarboxylic acids, are of great interest due to their reported biological activities. 1 Some representatives of α-aminophosphonates have demonstrated promising enzyme inhibitory activities, as for example, HIV protease antagonists 2 and collagenase inhibitor. 3Also, they have an important anticancer, 4 antibacterial 5 and antiviral activities. 6ese biological properties are mostly associated with the tetrahedral structure of the phosphonyl group acting as ''a transition-state analogue''. 7−13 The Kabachnik−Fields method is the most noteworthy and remarkable, generally using amines, dialkyl phosphites and carbonyl compounds. 14,15Although, a number of different methods have been reported for the preparation of acyclic α-aminophosphonates, 16−19 there is still a need to search for new methods for the preparation of cyclic α-aminophosphonates which have found promising biological applications. 20,21As a part of our continuing interest in the preparation of acyclic and cyclic α-aminophosphonates, 22−25 we describe a facile methodology to prepare some novel acyclic and cyclic α-aminophosphonates and also γ-heterocyclic phosphonates.The method is based on the reaction of 6-methyl-3-formylchromone with nitrogen and carbon mono-and bi-nucleophiles in the presence of diethyl phosphite in one-pot three-components under solvent-free conditions.The antimicrobial activities and antioxidant properties of the synthesized compounds were also evaluated.

RESULTS AND DISCUSSION
6-Methyl-3-formylchromone (1) was allowed to react with some aliphatic and aromatic amines as nitrogen mono-nucleophiles namely ethylamine, benzylamine, 4-chloroaniline and 4-hydroxyaniline in the presence of diethyl phosphite at 70−80 C for 6 h under Kabachnik−Fields reaction conditions 26,27 to produce the corresponding chromonyl α-aminophosphonates 2−5 in 62−100% yields (Scheme 1).The IR spectra of the α-aminophosphonates 2−5 showed the presence of NH (3289−3443 cm -1 ), C=O pyrone (1640−1646 cm -1 ) and P=O (1216−1225 cm -1 ) groups.Moreover, their 1 H-NMR spectra recorded the characteristic ethoxy protons at the regions δ 0.93−1.29 ppm (CH 3 ) and δ 3.60−4.00ppm (CH 2 ).The peaks of CH−P protons appeared as doublets at the regions δ 4.39−5.21ppm with coupling constants in range 15−24 Hz.Furthermore, the protons H−2 of the chromone rings were displayed as singlets at the regions δ 8.20−8.95ppm.The 13 C-NMR spectral data of compounds 3 and 4 supported their structures due to the presence of the characteristic carbon atoms CH 3 , CH 2 , CH−P and C=O pyrone at the regions δ 16.0−16.5,60.4−62.6,45.2−45.5 (J PC = 148.5 and 158. 5 Hz) and 174.6−174.8ppm, respectively.Also, the 31 P-NMR spectrum of the α-aminophosphonate 5 displayed a singlet at δ 21. 6  Scheme 1 3-Formylchromones have attracted attention long ago as highly reactive compounds, which can serve as starting substances in the synthesis of a whole series of heterocycles.3-Formylchromones have useful chemical properties due to presence of three strong electrophilic centers at C−2, C=O pyrone and formyl group. 28,29In the present article, the synthetic utility of 6-methyl-3-formylchromone (1) is derived from its reaction with nitrogen and carbon bi-nucleophiles that starts predominately from the attack on the formyl group to give the nonisolable condensation product A then pyrone ring opening at the unsubstituted C−2 forming the intermediate B. The latter intermediate B undergoes addition of diethyl phosphite at the cyclic azomethine bond to form the target phosphonates (route a, Scheme 2).Also, these phosphonates may be formed via addition of diethyl phosphite at the acyclic azomethine bond of the intermediate A leading to the formation of the nonisolable intermediate C, which undergoes pyrone ring opening (route b, Scheme 2). 30The resulted compounds gave characteristic red, green and blue colors with an alcoholic ferric chloride solution which support pyrone ring opening.

Scheme 3
In the present investigation, several subtypes of 1,3-bi-nucleophiles were used for recyclization of compound 1, in the presence of diethyl phosphite leading to the formation of some novel pyrimidinyl phosphonates as cyclic α-aminophosphonates.Thus, fusion of the aldehyde 1 with thiourea, guanidinium carbonate and cyanoguanidine in the presence of diethyl phosphite at 70−80 C yielded a first type of pyrimidinyl phosphonates 9−11, respectively (Scheme 4).The latter phosphonates showed absorption bands at the regions 3134−3452, 1641−1648 and 1206−1230 cm -1 corresponding to OH, NH, C=O and P=O functions, while the nitrile group in compound 11 appeared at 2225 cm -1 .The 1 H-NMR spectra of compounds 9−11 displayed CH 3 (δ 1.00−1.36ppm) and CH 2 (δ 3.60−4.00ppm) protons of ethoxy groups in each product, while H−6ˊ protons of the pyrimidine rings were found in the aromatic region.Also, the doublets that appeared at δ 5.10, 5.20 and 5.03 ppm (J = 20, 19 and 18 Hz) for compounds 9−11, respectively, correspond to the CH−P protons.The 13 C-NMR spectrum of compound 10 supported the presence of the carbonyl group which resonated at δ 186.1 ppm, while the carbon atoms of CH−P and C−2ˊ of the pyrimidine ring appeared at δ 47.0 and 163.6 ppm, respectively.Also, the 31 P-NMR spectrum of compound 10 displayed a singlet at δ 26.2 ppm for the phosphonate group.Further confirmation of the structures 9 and 10 was obtained from their mass spectral data, where both structures showed their molecular ion peaks at m/z 384 and 367, in addition to their base peaks at m/z 64 and 64, respectively.

Scheme 4
−33 The present study was extended to investigate the behavior of the aldehyde 1 with classical nitrogen 1,4-bi-nucleophiles in the presence of diethyl phosphite with a view to synthesize phosphonates beard on seven-membered heterocyclic systems.Thus, treatment of aldehyde 1 with each one of nitrogen 1,4-bi-nucleophiles such as ethanolamine, ethylenediamine, 2-aminophenol and 1,2-phenylenediamine in the presence of diethyl phosphite at 70−80 C for 4 hours furnished the corresponding phosphonate derivatives of 1,4-oxazepine 12, 1,4-diazepine 13, 1,5-benzoxazepine 14 and 1,5-benzodiazepine 15, respectively (Scheme 5).The IR spectra of the products 12−15 showed absorption bands at the regions 3232−3421, 1636−1646 and 1215−1228 cm -1 indicating the presence of NH, carbonyl and phosphonyl groups, respectively.The 1 H-NMR spectra of compounds 12 and 13 exhibited characteristic broad singlets at δ 3.46, 4.34 and 3.42 ppm for CH 2 CH 2 groups, respectively.Also, their 1 H-NMR spectra showed characteristic doublets at δ 4.94 (J = 20 Hz) and 5.00 (J = 21 Hz) ppm for the CH−P while H−7ˊ protons of the seven-membered rings were found in the aromatic region.Furthermore, the 1 H-NMR spectra of the benzo analogues 14 and 15 recorded coincident chemical shifts for CH−P protons at δ 5.10 and 5.18 ppm, respectively.In addition, the H−2ˊ protons of the 1,5-seven-membered rings appeared as singlets at δ 8.44 and 8.30 ppm for compounds 14 and 15, respectively.The 13 C-NMR spectra of compounds 13 and 14 revealed the carbonyl groups at δ 185.0 and 189.6 ppm, respectively.In addition, Moreover, its 31 P-NMR spectrum displayed one singlet at δ 24.5 ppm.The mass spectrum of 16 recorded the M+1 and M + peaks at m/z 393 and 392, respectively.The high point in this work was the synthesis of some novel fused pyran systems containing phosphonate substrate at γ-position that would expected to have biological properties.Thus, heating of 6-methyl-3-formylchromone (1) and each one of cyclic carbon nucleophiles such as dimedone, 1-phenylpyrazolidine-3,5-dione and barbituric acid in the presence of diethyl phosphite and few drops of as examples of Gramnegative bacteria.They were also examined against Candida albicans (ATCC 10231) as yeast and Aspergillus fumigatus as fungus.Agar diffusion technique was used for the determination of the preliminary antibacterial and antifungal activities. 34,35The test was performed on medium potato dextrose agar (PDA) which contained infusion of 200 g potatoes, 6 g dextrose and 15 g agar.Uniform size filter paper disks (3 disks per compound) were impregnated by equal volume (10 µL)   from the concentrations of 500 and 1000 g/mL dissolved compounds in dimethylformamide (DMF) and carefully placed on inoculated agar surface.After incubation for 36 hours at 27 °C in the case of bacteria and for 48 h at 24 °C in the case of fungi, the antimicrobial activities were determined by measuring the inhibition zones.Cephalothin, Chloramphenicol and Cycloheximide were used as reference drugs (30 µg/mL) for Gram positive bacteria, Gram negative bacteria and fungi, respectively.The minimum inhibitory concentration (MIC, g/mL) for some selected compounds against some species of microbes was also determined.The tube dilution technique 36 was applied for the determination of MIC of the tested compounds against microbes.Dilution series were set up with 250, 125, 62.5………3.25 g/mL of nutrient broth medium to each tube, 100 mL of standardized suspension of the test microbes (107 cell/ mL) were added and incubated at 37 C for 24 hours.The obtained results on the antimicrobial activities of the compounds and control drugs are given in Table 1.In general, the tested compounds recorded

Antioxidant activity
1,1-Diphenyl-2-picrylhydrazyl (DPPH) has often been used to characterize antioxidants.It is reversibly reduced and the odd electron in the DPPH free radical gives a strong absorption maximum at  517 nm, which is purple in color.This property makes it suitable for spectrophotometric studies.A radical scavenging antioxidant reacts with DPPH stable free radical and converts into 1,1-diphenyl-2-picrylhydrazine.The resulting decolorization is stoichiometric with respect to the number of electrons captured.The change in the absorbance produced in this reaction has been used to measure antioxidant properties.The solutions of tested compounds (150, 300 and 450 mol L -1 ) were added to DPPH (100 mol L -1 ) in DMSO/EtOH.The tubes were kept at an ambient temperature for 20 minutes and the absorbance was measured at  517 nm.The difference between the test and the control experiments was taken and expressed as the percent scavenging of the DPPH radical using the following formula % inhibition = (AB−AA/AB) x 100 where AB=absorption of blank and AA=Absorption of tested compound.The radical scavenging activity of ascorbic acid was also measured and compared with that of the different synthesized compounds. 37,38The observed data on the antioxidant activities of the compounds and control are shown in Table 2 and  recorded noticeable antioxidative properties more than the acyclic -aminophosphonates 25.This may due to the presence of free phenolic OH groups in compounds 619 which can scavenge the DPPH radical.The appearance of isoxazole unit in compound 8 exhibited greater activity than those having pyrazole units in compounds 6 and 7. Similarly, the thioxopyrimidinyl derivative 9 was more active than the other amino/cyanoiminopyrmidinyl derivatives 10 and 11.Amongst compounds having seven-membered rings 1215, the diazepinyl derivative 13 exhibited the highest inhibition activity.On the other hand, the pyridine system 16 did not record the hoped antioxidative properties.The pyrazolopyranyl phosphonate 18 was the most active one between the pyranyl phosphonates 1719.In this study, the systems 4, 9, 13 and 18 displayed the higher scavenging activities.However, the result exemplified that compound 13 having the diazepinyl unit in combination with phosphonic diester moiety is the most powerful antioxidant agent.

CONCLUSION
In conclusion, we have explored one-pot three component's reaction, which furnished novel classes of functionalized heterocyclic analogues of acyclic and cyclic α-aminophosphonates and phosphonates from readily available 6-methyl-3-formylchromone, nitrogen and carbon mono-and bi-nucleophiles and diethyl phosphite.The procedure is efficient and general.The reactions have been shown to display relatively good functional group tolerance and good yields.We hope that this approach may be value to others seeking novel synthetic fragments with unique properties for medicinal chemistry.

EXPERIMENTAL
The melting point was determined in an open capillary tube on a digital Stuart SMP-3 apparatus.Infrared spectra were measured on FT-IR (Nicolet IS10) spectrophotometer using KBr disks. 1 H-NMR spectra were measured on Gemini-300BB spectrometer (300 MHz), using DMSO-d 6 as a solvent and TMS (δ) as the internal standard. 13C-NMR spectra were measured on Mercury-300BB (75 MHz using DMSO-d 6 as a solvent) and Bruker-600 (150 MHz using CDCl 3 as a solvent) spectrometer and TMS (δ) as the internal standard. 31P-NMR spectra were registered on a Bruker-600 (242 MHz) spectrometer at room temperature using DMSO-d 6 as a solvent and TMS as internal standard and 85% H 3 PO 4 as external reference.Mass spectra recorded on a Gas Chromatographic GCMSqp 1000 ex Shimadzu instrument at 70 ev.Elemental microanalyses were performed Perkin-Elmer 2400 II at the Chemical War department, Ministry of Defense.The purity of the synthesized compounds was checked by thin layer chromatography (TLC).

General procedure for the preparation of target compounds 2-19
A mixture of 6-methyl-3-formylchromone (1) (5 mmol, 0.94 g), nucleophile (5 mmol) and diethyl phosphite (10 mmol, 1.38 mL) was heated under reflux at 70−80 C for 2−8 h (in case of 2, 8 and 16−19 few drops of triethylamine were added).The reaction mixture was cooled then poured into ice and left for complete precipitation.The precipitate formed was filtered off, dried and crystallized from the proper solvent.

2 When 6 - 6 .
Scheme 2 Scheme 5 Scheme 6 Scheme 7 variable antimicrobial activities towards the used microorganism.The most compounds recorded low to moderate inhibitory effects towards all the microorganisms.The antimicrobial spectrum of the synthesized compounds against Gram-negative bacteria demonstrated very low inhibitions.Compound 16 exhibited moderate inhibition against Staphylococcus aureus with high MIC value 250 g/mL.Similarly, compounds 8 and 11 recorded moderate inhibitions against Bacillus subtilis with high MIC value 250 g/mL.All the tested compounds except 2 and 4 exhibited relatively low to high inhibitory activities against Candida albicans.Furthermore, compounds 59, 11 and 16 exhibited relatively moderate inhibitory activities against Candida albicans especially compounds 8 and 9 which recorded MIC values at 250 g/mL.Compound 17 is the most effective one against Staphylococcus aureus and Bacillus subtilis with MIC values of 125 g/mL.For activity against Candida albicans and Aspergillus fumigatus, compound 19 recorded the best activity with MIC values of 62.5 and 125 g/mL, respectively, in comparison to the other synthesized compounds.

illustrated in Figure 1 .
The results of scavenging the stable DPPH radical recorded variable antioxidant activities towards the synthesized compounds at the different concentrations 150, 300 and 450 mol L -1 .Compounds 2, 5, 7, 11, 12 and 15 showed moderate activities.In the meantime, compounds 3, 6, 8, 10, 14, 16, 17 and 19 displayed good activities.On the other hand, compounds 4, 9, 13 and 18 proved to exhibit potent antioxidative activities.The structure activity relationships of the tested compounds demonstrated that all the synthesized compounds recorded remarkable inhibition activities in range 37.8178.35%at the different concentrations due to the presence of 4-methylphenol group in all the compounds except compounds 25 which have other free NH groups attached to phosphonate moieties.The cyclic -aminophosphonates 615 and phosphonates 1619

Table 1 .
In vitro antimicrobial activities of the synthesized compounds 2-19 at 500 and 1000 g/mL and the MIC values for some selected compounds.No inhibition or inhibition less than 5 mm.