Ultrasound-promoted solvent-free synthesis of some new α-aminophosphonates as potential antioxidants

Ultrasonic irradiation has been adopted in order to achieve an efficient synthesis of some novel α-aminophosphonates by Pudovik reaction. Major benefits of this method are as follows: eco-friendly, free of catalyst, high yielding, uncomplicated work-up procedure, short reaction time, and solvent free condition. Spectral characterisation and elemental analysis of the synthesized samples was carried out. In vitro antioxidant activity of the title compounds was screened by DPPH ̇, O2̇ and NO ̇ scavenging methods. Good activity was shown by most of the compounds as compared with the standards.

Alternatively, synthesis of organic compounds by means of ultrasound irradiation is a successful protocol which is an alternate energy source for organic reactions typically accomplished by normal heating (Juarez et al., 2009(Juarez et al., , 2010. One advantage of using cavitation as an energy source to promote organic reactions includes shorter reaction times (Srivastava et al., 2009). In the cavitation process, the molecules of the liquid are split, produces bubbles which further collapse in the compression cycle during the rarefaction cycle. These quick and aggressive implosions produce tiny-lived regions by means of temperatures of approximately 5000°C, pressures of about 1000 atm and heating and cooling rates above 10 7°C per second. Such localized hot spots can be considered as micro reactors where the sound energy is distorted into a helpful chemical form (Duarte et al., 2010;Stefani et al., 2005). This method is found to be a hygienic, convenient and helpful etiquette in organic synthesis compared with traditional methods (Srivastava et al., 2009). In an effort to achieve higher yields with selectivity, this method is determined to be excellent alternative in organic synthesis (Gallardo and Nakayama, 2010;Heydari et al., 2007). Currently, some researchers have reported the solventfree synthesis of AAPs using ultrasound from amines, aldehydes and and diethylphosphite by diverse catalysts (Agawane et al., 2010;Shinde et al., 2011). On the basis of previous reports and present need of merits on the synthesis of AAPs and in extension of our research to increase new protocols (Ravikumar et al., 2018), we focussed at the synthesis of AAPs via Pudovik reaction under solvent-free condition using ultrasonication. DPPH, O 2and NO methods had been chosen to test their antioxidant activity.

Chemistry
A series of AAPs, 7a-e and 8a-e were synthesized in two steps by Pudovik reaction. At first, a series of imines (4a-e and 5a-e) were prepared by reacting equimolar quantities of amines (1a-e) and aldehydes (2 and 3) in ethanol at 40°C for approximately 1h. Then, the corresponding imine was treated with diethylphosphite (6) to give AAPs, 7a-e and 8a-e using both conventional approach as well as ultrasonication methods. Table 1 and Scheme 1 represent the results and the synthetic protocol of this process.
The chemical structures of all the title compounds 7a-e and 8a-e were characterized by IR, 1 H, 13 C, 31 P NMR, mass spectral data and elemental analyses and their data were presented in the experimental section. IR absorptions in the regions 3345-3308, 1236-1210 and 1018-1012 cm -1 were assigned to NH, P=O and P-O-C stretching vibrations respectively for the title compounds (Subramanyam et al., 2013). The 1 H NMR spectra gave signals due to Ar-H in the range of δ 8.13-6.82 ppm. The proton signal at δ 4.55 was assigned to -NH stretching vibrations of all the title compounds. The proton signal in the range δ 3.90-3.84 as doublet was assigned to P-CH stretching vibrations of all the title compounds. The methylene and methyl protons of P-O-CH 2 CH 3 resonated as multiplet and triplet respectively at δ 4.05 and 1.18 cm -1 . 13 C NMR chemical shift for methylene and methyl carbons were observed at 61.9 ppm and 16.2 ppm respectively for the title compounds 7a-e and 8a-e. The chemical shift for P-CH carbons was observed at δ 57.5 ppm respectively for the title compounds. 31 P NMR signals were observed in the region 20.3-15.2 ppm for 5a-j . In their mass spectra, M +. ions were observed in the expected m/z values. The representative spectra of compound 7a were provided in Supplementary material as Figures S1-S6.

Antioxidant activity
Antioxidant activity of newly prepared compounds become screened at concentrations 50 μg/mL and 100 μg/mL by DPPH, Superoxide and Nitric oxide (NO) radical scavenging activities. The investigational results be obtainable in Table 2.
Out of all, the compounds; 7b and 7c showed the highest DPPH radical scavenging activity with % inhibition 64.2, 81.5 μg/mL, and 63.6, 80.3 μg/mL, respectively at 50 and 100 μg/mL. The remaining compounds showed moderate activity while compared with the antioxidant, Ascorbic acid.

Materials and methods
The chemicals were purchased from Sd. Fine Chem. Ltd., India and some of them were purified using standard procedures. The purity of the compounds was checked by TLC on silica gel coated Al sheet. In conventional technique, the reaction was carried out on magnetic stirrer cum hot plate. Ultrasonicator, BANDELIN SONOREXR (Germany) (35 kHz, 200 W) with inbuilt heating 30°C-80°C was used for ultrasonic irradiation technique. J (coupling constants) and δ (chemical shift) values were reported in Hz and ppm, respectively. Bruker AMX spectrometer was used to record 31 P (161.9 MHz), 1 H (400 MHz) and 13 C (100 MHz) NMR spectra. The symbols 's' for singlet, 'd' for doublet, 't' for triplet and 'm' for multiplet were used to represent peaks in NMR spectra. L.C. MS were recorded on SHIMADZU 2010A. Thermo Finnigan Flash 1112 apparatus was used for CHN analysis. Bruker IFS 55 (Equinox) FTIR spectrometer in KBr was used to record IR spectra.

Synthesis of AAPs, 7a-e and 8a-e by conventional heating method
The imine (0.01 mole) become reacted with diethylphosphite (6) (0.015 mole) in EtOH using tetramethylguanidine (TMG) at 50°C-60°C for 3-5 h to yield the corresponding AAPs (7a-e and 8a-e). TLC (ethylacetate: n-hexane, 6:4) was used for verifying the development of the process. As soon as the reaction was finished, as confirmed via TLC, the aggregate became chilled to room temperature. The pure compounds (7a-e and 8a-e) were obtained by column chromatography by means of n-hexane:ethyl acetate (2:3) as eluent. The yield become located in the range of 68-78%.

Antioxidant activity DPPH˙ Scavenging activity
We followed the usual approach of Cotelle et al. (1996) with some adjustments for screening DPPH . scavenging activity of synthesized compounds at concentrations 50 and 100 μg/mL. The screening was carried out in triplicate and the common values were taken as final result. The % inhibition of DPPH . was tested by taking the results of the test with those of the control.

O ·̄2 scavenging activity
We have used the same old method of Robak and Gryglewski (1988) with minor variations in incubation period and concentration of PMS for screening O ·̄2 scavenging activity of synthesized compounds at concentrations 50 and 100 μg/mL. Butylated hydroxyl toluene (BHT) was used as standard.

NO˙ Scavenging activity
NO˙ Scavenging activity of the title compounds was carried out using standard method of modified protocol of Green et al. (1982) and Marcocci et al. (1994) with minor modifications at concentrations 50 and 100 μg/mL. BHT was used as standard drug for the study. The experiment was carried out in triplicate and the results are presented in Table 2.