Synthesis and in vitro antioxidant evaluation of new bis(α-aminoalkyl)phosphinic acid derivatives

GRAPHICAL ABSTRACT ABSTRACT Diamines were added to arylaldehydes in ethanol, which resulted in corresponding diimines. Novel bis-1-aminophosphinic acid compounds were synthesized through the interaction of diimines and hypophosphorous acid. The new compounds were characterized by elemental analyses, FT-IR and 1H, 13C and 31P NMR techniques. The in vitro antioxidant activity of the newly synthesized compounds were measured and found to exhibit significantly higher antioxidant activity than the standard.


Introduction
It is well known that amino acids are the main components of various proteins and that they generally play an important physiological role in the life process. 1-Aminophosphinic acids are phosphorus analogues of natural amino acids and are selective inhibitors of various proteolytic enzymes, particularly metallo-proteases. [1][2][3] For this reason, aminophosphinic acids have been researched and developed as potential antibacterial, antitumor, and antivirotic materials in recent years. [4][5][6] Much consideration has been given to aminophosphinic acid ligands and their complexes because of their novel structures and properties. [7][8][9][10][11][12][13] Aminophosphonic acids are also found as constituents of natural products. 14 In contrast to the widely studied 1-aminophosphonic acid derivatives, [15][16][17][18] relatively few papers have reported the chemistry of α-aminophosphinic acid derivatives.
Reactive oxygen species (ROS) such as O 2 − , H 2 O 2 , and • OH are generated in cells through aerobic metabolic processes or as a result of interaction with exogenous agents. Low ROS levels are essential for the proper cell function, but excess levels lead to "oxidative stress, " which has been linked to the progression of ageing and many human diseases, e.g., neurogenerative, cardiovascular, and cancer. Superoxide dismutases (SODs), catalase (CAT), and glutathione peroxidase (GPx) are enzymes which act as a primary cellular defence system against oxidative damage in living organisms. Organophosphorus compounds and P-heterocycles, in particular, have been recognized as antioxidant drugs. 19,20 Their mechanism and structure activity relationships (SAR) have been extensively studied. 21 Depending on their structure and scavenging properties, phosphites and phosphonates may act as both primary and secondary antioxidants. In general, phosphites are considered to be hydroperoxide decomposing (secondary antioxidants), but certain aryl phosphites should also be capable of acting as radical chainterminating (primary antioxidants) by trapping peroxyl radicals to produce aroxyl radicals. Recent studies have elucidated reaction modes and the relationship between structure, reaction mechanism, and antioxidant activity. 20,21 Over the past several years, our laboratories have reported the synthesis of 1-amino-H-phosphinic acids. 22,23 We have reported the synthesis and in vitro antimicrobial activity of novel aminophosphinic acids containing cyclobutane and 1.3thiazole. Building on previous studies, we have prepared a series of derivatives of new α-aminophosphinic acids ( Figure 1). In view of the importance of phosphinic acid derivatives, this study's goal was to find new biologically active molecules. Here, we report a new series of bis-1-amino-H-phosphinic acids and a preliminary biological study of their antioxidative activity.

Chemistry
Diimines are good precursors for synthesizing numerous organic compounds, especially heterocyclic compounds. 24,25 These easily accessible precursors can be produced by reacting aromatic aldehydes in ammonia.
Schiff bases 3a-i were prepared following the published procedure 22,23 by condensing the corresponding diamines (1) with arylaldehyde (2) in ethanol at room temperature. The corresponding imines were obtained in quantitative yields. The preparation of bis-aminophosphinic acids 4a-h was performed following Baylis et al. 26 Reactions were carried out in boiling ethanol for 24 hr, after which mixtures were left overnight to stir at room temperature ( Figure 1).
Because two stereogenic carbons bonded to a phosphorus atom, and due to the prototopic transfer of the acidic proton between the phosphoryl (P = O) and acidic (P-OH) sites, these compounds exist as two diastereomeric forms: one mesocompound (R * , S * -4) and one racemic pair (S * , S * or R * , R * -4), as shown in Figure 2.
The structures of 4a-h were determined by spectroscopy ( 1 H, 13 C and 31 P NMR, and FT-IR) and by comparison to data in the literature of similar compounds. 26-28 The proposed mechanism of formation of 4a-h is illustrated in Figure 3. The addition of hypophosphorous acid to the azomethine bond of Schiff bases 3a-i led to the formation of meso-compounds and racemic pairs.

Antioxidant activity
All phosphinic acid derivatives 4a-h and standards were prepared at a range of concentrations from 50 to 500 µg/mL. Table S1 (Supplemental Materials) shows the results of antioxidant activity. Various methods were used to compare the antioxidant activity of the new compounds against the standards. Experimental details are given in the supplemental materials available online.
Hydroxyl radical (•OH) scavenging activity. All compounds, BHT and ascorbic acid standards showed similar hydroxyl radical scavenging activity. Nonetheless, compounds 4c and 4f exhibited the highest values. The hydroxyl radical (•OH) scavenging activity of test compounds were in the following order: ascorbic acid (Table S1).
Superoxide radical scavenging activity. Superoxide anions are oxygen-centred radicals and indicators of active free radicals with the potential of reacting with biological macromolecules and thus causing tissue damage. 29 All compounds showed nearly equal activity levels. Compounds 4c, 4e, and 4f exhibited the greatest values, which were higher than α-tocopherol. Compound 4b exhibited the lowest activity. The superoxide radical scavenging activity of the standards and test compounds were in the following order: (Table S1).
Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. The power of an antioxidant to transfer one electron to reduce a compound may be a significant indicator of potential antioxidant activity. 30 The ability to scavenge stable DPPH radicals is a widely preferred and important method of evaluating antioxidant activity. 31 In this study, scavenging DPPH radicals was variable. Compound 4a exhibited the lowest activity, while compounds 4e and 4g had the greatest values. Scavenging efficiency on DPPH radicals was in following order: ascorbic acid (Table S1).
Metal chelating activity. Chelating reactives are useful as secondary antioxidants as they reduce redox potential and thus stabilize a metal ion's oxidized form. Ferrozine can form complexes with Fe 2+ . This process is obstructed when chelating agents are present, which decreases the red color of the complex. Thus, measuring the reduction of color makes it possible to calculate chelating activity. 32 The production of these radicals may cause lipid peroxidation, protein modification, and DNA damage. Chelating agents may not trigger metal ions and possibly prevent metal-dependent processes. 33 In this study, 4c demonstrated the most activity, while other compounds showed a low ion chelating activity. In terms of chelating activity, they were in the following order: (Table S1).
Hydrogen peroxide scavenging activity. Hydrogen peroxide exhibits powerful oxidizing effects and can be generated in vivo by various oxidizing enzymes, including superoxide dismutase. The activity of hydrogen peroxide scavenging was in the following order: ascorbic acid > 4f > 4c > BHT > 4h > 4g > 4d > 4e > 4a > 4b. Compound 4f (IC50: 60.07µg/mL) and 4c (IC50: 60.87µg/ml) scavenged hydrogen peroxide much more than other compounds and the BHT standard. It appears that levels of hydrogen peroxide at or below around 20-50 mg have restricted cytotoxicity for many cell types. Hence, the elimination of hydrogen peroxide is crucial for the protection of pharmaceutical and food systems (Table S1). 34

Experiments
All solvents and reagents were purchased from commercial sources and used without further purification. Melting points were determined on an Electrothermal 9100 melting point apparatus and uncorrected, but checked with a differential scanning calorimeter (DSC). The IR spectra were measured with a Perkin-Emler Spectrum One FT-IR spectrophotometer. The 1 H, 13 C, and 31 P spectra were taken on a Bruker AC-400 NMR spectrometer operating at 400 MHz for 1 H, 100 MHz for 13 C, and 162 MHz for 31 P. Compounds were dissolved in NaOD/D 2 O and chemical shifts were referenced to TMS ( 1 H and 13 C NMR) and 85% H 3 PO 4 ( 31 P NMR). Elemental analyses were performed on a LECO-CHNS-938. The Supplemental Material contains 1 H, 13 C, and 31 P NMR spectra for compounds 4a-h ( Figures S1-S21).

General procedure for the synthesis of 3a-h
A solution of diamine (20 mmol) in absolute ethanol (30 mL) was slowly added to a solution of aldehyde (40 mmol) in absolute ethanol (20 mL). The stirred reaction mixture was refluxed for 4 hr. After cooling, a precipitate was formed which was collected by filtration, washed with cold ethanol, and recrystallized from a 9:1 mixture of ethanol and water.

General procedure for the synthesis of 4a-h
Hypophosphorous acid (50%, 2 mol equivalent) in ethanol was added to the imine in ethanol (50 mL for 20 mmol). The mixture was refluxed for 24 hr and then cooled. The solid product was filtered off and washed with a solvent mixture of ethanol and water and air dried at room temperature.

Conclusion
In this study, we synthesized a new series of bis-1-amino-Hphosphinic acid derivatives and used different methods to compare their antioxidative activity. The novel phosphonous acids were obtained in moderate yields varying from 40 to 60%, which was more than expected, since several authors [40][41][42] reported much lower conversion rates for additions to two azomethine groups. Among various bis-1-amino-H-phosphinic acids derivatives, compounds 4c and 4f showed higher antioxidant activity than the other derivatives and the standard BHT compound. The lowest activity values were generally from compounds 4a and 4b.

Statistical evaluation
Data analyses were done using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Linear regression was conducted to provide mathematical models of determinism between dependency (concentration) and independency (inhibition percentage). IC50 values were calculated with both linear and cubical models. Data were evaluated at 95% confidence intervals.