Synthesis, electrochemical and classical evaluation of the antioxidant activity of three α-aminophosphonic acids: Experimental and theoretical investigation

Article abstract of DOI:10.1016/j.molstruc.2018.05.074

Three α-aminophosphonic acids; namely N-Ethyliminodimethylenediphosphonic acid (N-EDMPA), N-Propyliminodimethylenediphosphonic acid (N-PDMPA) and Ethylenediamine-tetrakis(methylenephosphonic acid) (EDTMP), were synthesized by the reaction of various primary amines, paraformaldehyde and phosphorous acid in acidic medium via Moedritzer-Irani reaction. The structures of the cited compounds were confirmed by UV–Vis, FT-IR, ¹H NMR, ¹³C NMR and ³¹P NMR spectra. The investigated compounds were screened for their antioxidant activity by mean of the free radical scavenging activity using 1,1-Diphenyl-2-Picryl Hydrazyl (DPPH) and Cyclic Voltammetry (CV) methods. The obtained values of the 50% inhibitory concentration (IC₅₀), show that N-PDMPA exhibit greater antioxidant activity and the order of the antioxidant activities of the studied compounds is: EDTMP < N-EDMPA < N-PDMPA. Furthermore, this biological activity is in good correlation with the density functional theory (DFT) calculations.

Full text of DOI:10.1016/j.molstruc.2018.05.074

Accepted Manuscript
Synthesis, electrochemical and classical evaluation of the antioxidant activity of three
α-aminophosphonic acids: Experimental and theoretical investigation
Mouna Mehri, Nadjib Chafai, Louiza Ouksel, Khalissa Benbouguerra, Abdelkader
Hellal, Salah Chafaa
PII:
S0022-2860(18)30648-3
10.1016/j.molstruc.2018.05.074
MOLSTR 25247
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 30 January 2018
Revised Date: 1 May 2018
Accepted Date: 23 May 2018
Please cite this article as: M. Mehri, N. Chafai, L. Ouksel, K. Benbouguerra, A. Hellal, S.
Chafaa, Synthesis, electrochemical and classical evaluation of the antioxidant activity of three α-
aminophosphonic acids: Experimental and theoretical investigation, Journal of Molecular Structure
(2018), doi: 10.1016/j.molstruc.2018.05.074.
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ACCEPTED MANUSCRIPT
60
40
80
PBMP
0.0 ml
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60
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0.1 ml
0.2 ml
0.3 ml
0.4 ml
0.5 ml
0.6 ml
0.7 ml
20
0
-20
-40
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-80
-100
R2
= 0,98349
4,4
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Potentiel(V)
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C oncen tr atio n(µ g/ml)

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Synthesis, electrochemical and classical evaluation of the antioxidant
activity of three α-aminophosphonic acids: Experimental and theoretical
investigation
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Mouna Mehria, Nadjib Chafaia, Louiza Ouksela, Khalissa Benbouguerraa, Abdelkader
Hellala,b, Salah Chafaaa,*.
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aLaboratory of Electrochemical of Molecular Materials and Complex LEMMC. Department
of Process Engineering, Faculty of Technology, University Ferhat ABBAS Setif -1, El-
Maabouda 19000 Setif, Algeria
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bUniversité Djilali Bounaâma de Khemis Miliana, 44225, Algeria
*Corresponding author. E-mail address: schafaa@univ-setif.dz or chafaa_s@yahoo.fr
Abstract
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Three α-aminophosphonic acids; namely N-Ethyliminodimethylenediphosphonic acid
(N-EDMPA),
N-Propyliminodimethylenediphosphonic
acid
(N-PDMPA)
and
Ethylenediamine-tetrakis(methylenephosphonic acid) (EDTMP), were synthesized by the
reaction of various primary amines, paraformaldehyde and phosphorous acid in acidic
medium via Moedritzer-Irani reaction. The structures of the cited compounds were confirmed
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by UV-Vis, FT-IR, H NMR, C NMR and P NMR spectra. The investigated compounds
were screened for their antioxidant activity by mean of the free radical scavenging activity
using 1,1-Diphenyl-2-Picryl Hydrazyl (DPPH) and Cyclic Voltammetry (CV) methods. The
obtained values of the 50% inhibitory concentration (IC50), show that N-PDMPA exhibit
greater antioxidant activity and the order of the antioxidant activities of the studied
compounds is: EDTMP < N-EDMPA < N-PDMPA. Furthermore, this biological activity is in
good correlation with the density functional theory (DFT) calculations.
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Keywords: α-aminophosphonic acids; Antioxidant; Free radical; DPPH; Cyclic voltammetry;
DFT calculations.
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1. Introduction
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Free radicals are highly reactive and unstable compounds. Generally, are produced in
the human body during normal metabolic functions [1] or by exposure to different types of
radiations: chemicals, environmental stress and the sun [2]. These reactive species are capable
to oxidizing biomolecules and cause cell death and consequently cause tissue damage. Free
radical oxidative processes also play a significant pathological role in causing human
diseases. In medicine world, there are many illnesses are related to the oxidative tissue
damage, such as cancer, emphysema, cirrhosis, atherosclerosis and arthritis [3].
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Additionally, the antioxidant agents are utilized to protect human body against
oxidative damage of free radicals using some complex defense systems [4]. The antioxidant
behaviour of a compound is the result of its capacity to inhibit the initiation of free radical or
chain breaking in the process propagating oxidation [5]. Furthermore, various antioxidants
have been identified in human organism, such as endogenous enzymes (superoxide dismutase,
glutathione peroxidase, catalase...) and non-enzymatic (vitamins...) [6].
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Practically, there are several methods that can be used to evaluate the antioxidant
activity of compounds. Mainly, these methods may differ as function of the scavenged
species, the antioxidants nature, the reaction conditions and the detection methods. These
techniques involve different mechanisms of the determination of the antioxidant activity [7].
However, because the results in the measurement of the antioxidant capability depend on the
applied method, a single method cannot give a precise prediction of the antioxidant capacity
of compounds [8, 9], it is recommended to use more than one method to estimate the in vitro
antioxidant capability of a substance [10].
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Nature provides a diversity of antioxidants such as ascorbic acid (vitamin C),
tocopherols (vitamin E), ferritin and albumin [11]. Several works around the world have
carried out that many of plant extracts and different classes of phytochemicals have been
found to have quite prominent antioxidant activity [12, 13]. In some cases, the chemical
synthesis makes it possible to ameliorate the activity of these natural compounds by structural
modifications. Also, the organic synthesis makes it possible to obtain molecules with an
antioxidant activity similar to that of the natural compounds. In particular, it has been shown
that some α-aminophosphonic acids are a potential antioxidants [14-16]. The α-
aminophosphonic acids are considered an important category of organophosphorus
derivatives on reason of their exceptional biological activity. They have a structural analogues
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of the amino acids [17], that is why the α-aminophosphonic acids are found to compete
effectively with their amino acid counterparts for binding to the enzymes active centers or
other cellular targets. This, together with their low toxicity makes the α-aminophosphonic
acids an important class of antimetabolites and a potential source of medicinal compounds
[18].
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In our work, we report the synthesis of three α-aminophosphonic acids compounds
employing Moedritzer-Irani reaction [19]. The antioxidant activity of the synthesized acids
was evaluated by two antioxidant methodologies: cyclic voltammetry (CV) and 1,1-Diphenyl-
2-Picryl Hydrazyl (DPPH) methods. In order to understand the correlation between the
biological activity of the investigated compounds and their molecular structures, the different
quantum chemical parameters have been determined at the optimized geometries using the
density functional theory (DFT) method.
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2. Experimental and computational details
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All the chemical compounds utilized in this work were commercially available and
were purchased from Sigma-Aldrich and Fluka. Moreover, they have used as received without
any further purification.
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2.1. Procedure for the synthesis of the α-aminophosphonic acids
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- N-Ethyliminodimethylenediphosphonic acid (N-EDMPA): was synthesized according to
the method explained by Moedritzer-Irani [19] (Fig. 1). So, 0.1 mol of ethylamine and 0.2
mol of phosphorous acid 50% were dissolved in the 100 mL of water and 50 mL of HCl 37%.
The mixture was refluxed for 2 h under continuous stirring, then the paraformaldehyde (37%,
0.4 mol) was added drop by drop to the reaction mixture. Afterwards, the resulting mixture is
left under reflux for 4 h. Finally, the obtained solution is cooled at room temperature and it
remained clear without any formation of the precipitates. Subsequently, the solvent was
removed under reduced pressure using a rotary evaporator. To the obtained concentrated
solution, the ethanol has been added to obtain a white solid product, which is separated by
filtration, washed with ethanol, and then dried.
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- N-Propyliminodimethylenediphosphonic (N-PDMPA): was prepared by the same method
as for N-EDMPA, using 0.1 mol of propylamine with 0.2 mol of phosphorous acid and 0.4
mol of paraformaldehyde (Fig. 1).
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- Ethylenediamine-tetrakis(methylenephosphonic acid) (EDTMP): was synthesized
according to the same procedure than the previous ones, where we react 0.1 mol of
ethylenediamine with 0.4 mol of phosphoric acid and 0.8 mol of paraformaldehyde.
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The structures of the synthesized α-aminophosphonic acids were confirmed using IR,
UV-Vis, 1H, 13C and 31P NMR spectra.
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2.2. Characterization
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The melting points of the obtained α-aminophosphonic acids were measured by open
capillary method in BÜCHI melting point B-540, while the UV-Vis spectra in DMSO were
carried out between 200–800 nm using JASCO V-650 spectrophotometer with quartz cells of
1 cm path length. The FT-IR spectra were recorded in the region of 4000-600 cm-1 on a Jasco
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4200 spectrometer. The H NMR, 13C NMR and 31P NMR spectra of the investigated
compounds were recorded on a Bruker AV III 300 MHz in D2O with tetramethylsilane (TMS)
as an internal reference.
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2.3. Antioxidant activity
2.3.1. Free radical scavenging activity (DPPH)
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The free radical scavenging activity of N-EDMPA, N-PDMPA, EDTMP and butylated
hydroxytoluene (BHT as standard) is determined using DPPH assay. The DPPH assay is one
of the most common and relatively quick methods used for estimating radical scavenging
activity of a compound or a plant extract [20]. In this test, the antioxidants reduce the 1,1-
Diphenyl-2-Picryl Hydrazyl (DPPH•) having a violet to a yellow color of diphenylpicryl-
hydrazine (DPPH-H).
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In this context, we added 1.0 mL of the α-aminophosphonic acids (or standard)
dissolved in Dimethyl sulfoxide DMSO at different concentrations to 1.0 mL of the MeOH
solution of DPPH, the obtained mixture is left in the dark for 30 min at room temperature.
The decrease in absorbance was measured at 517 nm against blanks. The radical scavenging
activity (RSADPPH) of the various tested compounds was calculated using the following
equation [21]:
ꢆ
ꢊꢆ
ꢋꢌꢍꢎꢏꢐ
ꢇꢈꢈꢉ
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ꢀꢁꢂꢃꢄꢄꢅ % =
× 100
(1)
ꢆ
ꢇꢈꢈꢉ
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where ꢑꢒꢓꢓꢔ is the absorbance of the blank (1.0 mL of DPPH–methanol solution and
1.0 mL of DMSO) and ꢑꢕꢖꢗꢘꢙꢚ is the absorbance of the sample.
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The 50% inhibitory concentration value (IC50) expressed in ꢀg/ml, which was defined
as the effective concentration of the tested compounds, that is required to scavenge 50% of
the DPPH free radicals and compared with that of the BHT [22]. All tests were carried out in
three times and the IC50 values were reported as means SD of triplicates.
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2.3.2. Scavenging towards superoxide radical using cyclic voltammetric method
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Superoxide is an anionic free radical with the chemical formula O●ꢛꢊ. It is formed by
one-electron reduction of dioxygen (O2) [23], which is presented abundantly in nature.
Superoxide anion is the most dangerous radical among all oxygen radicals (ROS) because it
has longer half-life and thus can move to a longer distance [24], also it is capable of
generating other harmful radicals such as hydroxyl radical [25]. Some methods were
developed to determine specifically the superoxide scavenging [26]. Recently, the cyclic
voltammetry is used to determine the antioxidant activity towards this radical. This method is
based on the electrochemical generation of O●ꢛꢊ by the reduction of the dissolved oxygen in
aprotic solvents [27, 28].
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2.3.2.1. Apparatus and accessories
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The CV study was carried out employing a Voltalab 40 model PGZ301
potentiostat/galvanostat controlled by a personal computer through the voltamaster4 software.
All the experiences were executed in a double walled electrochemical cell includes three
electrodes engrossed in a solution including the analyte and a surplus of the supporting
electrolyte. The Platinum plate having an area of 1.28 cm2 was used as a counter electrode, a
saturated calomel electrode (SCE) was used as the reference electrode and glassy carbon (GC)
electrode (Ø=3.0 mm) was used as a working electrode and its surface was polished before
each measurement with silicon carbide 4000 paper, then rinsed with distilled water and with
the solvent used in the CV, the DMSO in our case.
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2.3.2.2. Procedures
a/ Cyclic voltammetry of oxygen
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In an electrochemical cell we put 20 mL of a solution containing the supporting
electrolyte Bu4NBF4 (0.1 M) dissolved in DMSO, this solution was saturated by dry air. The
solubility of oxygen in DMSO was 2.1 mM [29]. The Cyclic Voltammogram of the oxygen
reduction recorded at a scan rate of 20 mV/s, the potential range was - 1.01 to - 0.3 V/SCE
and at room temperature.
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b/ Cyclic voltammetry of oxygen in the presence of the tested compounds
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Measurement of superoxide radical scavenging activity was based on the method of
Le Bourvellec et al [30] with slight modification in experimental conditions. The effect of
various compounds was checked by the addition of the successive 0.1 mL of initial solution to
20 mL of oxygen solution and the cyclic voltammogram was recorded at the same
experimental conditions used before.
●ꢟ
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The percentage of radical scavenging activity towards superoxide radicals ꢜRSAꢝ ꢠof
ꢞ
the tested compounds was calculated using the following equation:
ꢣ
ꢢ
ꢊꢢ
ꢎꢌ ꢎꢌ
ꢣ
●ꢟ
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ꢀꢁꢂꢡ % =
× 100
(2)
ꢞ
ꢢ
ꢎꢌ
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where Iꢤꢝꢥ and Iꢤꢥ are the anodic peak current of O●ꢛꢊoxidation without and with the
tested compounds, respectively.
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2.4. Computational details
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The geometric optimizations of the synthesized compounds were performed using the
Gaussian program [31], based on the density functional theory (DFT), employing Becke’s
three parameter hybrid exchange functional with Lee-Yang-Parr correlation functionals
(B3LYP) at basis sets 6-31G (d,p) [32, 33].
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The quantum chemical parameters of the investigated compounds such as: energy gap
ꢁEGAP, the ionization energy, electron affinity, electronegativity, electronic chemical
potential, molecular hardness, molecular softness, and electrophilicity index which can be
calculated using the following equations [34]:
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Energy gap:
ꢀEGAP = EHOMO
̶
ELUMO (3)
Ionization Energy:
Electron Affinity:
Electronegativity:
Chemical Potential:
Chemical Hardness:
Chemical Softness:
Electrophilicity Index:
I =
̶
EHOMO
ELUMO
(4)
(5)
(6)
(7)
A =
̶
χ = (I + A)/2
ꢁ =
η = (ELUMO
̶
χ
̶
EHOMO)/2 (8)
S =1/η
ω = ꢁ2/2η
(9)
(10)
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These parameters are very important to understand the chemical and biological
activities of the synthesized compounds.
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3. Results and Discussion
3.1. Spectroscopic study
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In this work, we have prepared three α-aminophosphonic acids by Moedritzer-Irani
reaction, which is Mannich reaction analogous, it takes place by the nucleophilic attack of
phosphorous acid on the iminium salt formed by reaction between formaldehyde and the
amine in acidic medium. We obtained various aminophosphonic acids by refluxing three
aliphatic amines (ethylamine, propylamine and ethylenediamine) with phosphorous acid and
formaldehyde in hydrochloric media (Fig. 1). The resulting acids are very soluble in water
and therefore have to be recrystallized using ethanol. No byproducts were observed and the
compounds were isolated in good yields.
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The structures of the obtained compounds were identified and characterized by their
spectroscopic data (UV-Vis, FT-IR, and by 1H NMR, 13C NMR and 31P NMR spectroscopy).
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The obtained IR spectra of N-EDMPA, N-PDMPA and EDTMP, show bands in the
region 3000
bands occurred in the region 1400
̶
2838 cm-1 attributed to the stretching vibrations of C−H aliphatic. In addition, all
1230 cm-1 are assigned to the deformation vibrations of
̶
(C−H). The new absorption bands situated in the regions, 2550–2700, 1040–910 and 769–780
cm–1 are ascribed to the stretching vibration of (O=)PO-H, P=O and P-C aliphatic,
respectively, indicated the formation of phosphonic acid. The bands appeared at 1275, 1283
and 1207 cm-1 in N-PDMPA, N-PDMPA and EDTMP correspond to (C−N) linkage,
respectively. The absence of the characteristic bands of primary amine (NH2) and aldehyde
(C=O) confirms the formation of the proposed α-aminophosphonic acids framework.
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The electronic absorption spectra of the synthesized α-aminophosphonic acids
displayed intense bands in UV region which may be assigned to n → π* and π → π *
transitions in their spectra. The UV-Vis spectra of these three α-aminophosphonic acids
indicate that they are transparent in the visible region.
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The structures of N-EDMPA, N-PDMPA and EDTMP were confirmed by analysis of
NMR data. 1H NMR spectrum of these compounds showed a doublet peak for methylene, due
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to coupling of methylene protons with the phosphorus atom. The C NMR spectral data of
the synthesized compounds are also in accordance with the proposed structures and expected
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numbers of carbon were observed. The presence of phosphorus atoms in three compounds is
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confirmed by their P NMR spectra. In the P NMR spectra the signal of the phosphorus
atom appeared as triplet which results from the coupling of the phosphorus atoms with the
two protons of the methylene group.
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3.1.1. Analytical data
N-Ethyliminodimethylenediphosphonic acid (N-EDMPA)
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Mol.Wt: 233.097. Yield: 85% of white solid, mp 207 °C, UV–Vis (DMSO): λmax =
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282 nm (ε282= 210.253 l.mol-1.cm-1), P NMR (DMSO-d6) δ (ppm) = 8.57 (t, J= 12.7 Hz),
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1H NMR (DMSO-d6) δ (ppm) = 1.22
;
(t, 3H, J= 7.2 Hz CH3-CH2), 3.21 (s, 4H, N-CH2-P),
,
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3.45 (m, CH3-CH2), 4.72 (s, 4H, P-OH), C NMR (DMSO-d6), δ (ppm) = 8.32 (CH3-CH2),
49.81 (CH3-CH2), 51.21 (CH2-P), 52.39 (CH2-P), IR (Solid state) ν (cm-1): 2999 (C-Haliphatic),
2667 ((O=) P-OH), 1275 (C-N), 1106 (P=O), 937 (δ P-OH), 709 (P-C).
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N-Propyliminodimethylenediphosphonic acid (N-PDMPA)
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Mol.Wt: 247.123. Yield 83.4% of white solid, mp 184 °C, UV–Vis (DMSO): λmax=
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282 nm (ε282= 244.157 l.mol-1.cm-1), P NMR (DMSO-d6), δ (ppm) = 8.367 (t, J= 12.7 Hz),
1H NMR (D2O), δ (ppm) = 0.71 (t, 3H, J= 7.22 Hz
,
,
CH3-CH2), 1.51 (m, 2H, CH3-CH2-CH2),
N-CH2-P), 4.76 (s, 4H, P-OH), 13C NMR
3.20 (m, 2H, CH2-CH2-N), 3.38 (d, 4H, J= 12.6 Hz
(D2O), δ (ppm) = 9.72 (s, CH3-CH2), 16.75 (s, CH3-CH2), 49.95 (s, N-CH2-P), 51.7 (s, N-
CH2-P), 58.79 (s, CH2-CH2-N), IR (Solid state) ν (cm-1): 2999 (C-Haliphatic), 2975 (C-Haliphatic),
2535 ((O=)P-OH), 1283 (C-N), 1138 (P=O), 935 (δ P-OH), 715 (P-C).
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Ethylenediamine-tetrakis(methylenephosphonic acid) (EDTMP)
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Mol.Wt: 436.124. Yield 79% of white solid, mp 239 °C, UV–Vis (DMSO): λmax = 282
1
nm (ε282= 888.82 l.mol-1.cm-1), 31P NMR (DMSO-d6), δ (ppm) = 8.21(t, J= 12.4 Hz), , H
NMR (D2O), δ (ppm) = 3.55 (d, 8H, J= 12.6 Hz -N-CH2-P), 3.86 (s, 4H, N-CH2-CH2-N), 4.79
(s, 8H, P-OH), 13C NMR (D2O, δ ppm): 51.63 (N-CH2-CH2-N), 52.73 (4C, N-CH2-P), IR
(Solid state) ν (cm-1): 2965 (C-Haliphatic), 2593 ((O=) P-OH), 1207 (C-N), 1115 (P=O), 946 (δ
P-OH), 742 (P-C).
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3.2. Antioxidant activity
3.2.1. Free radical scavenging activity (DPPH)
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DPPH is a very stable free radical because of its spare electron delocalization over the
whole molecule. DPPH gives a maximum absorption at 517 nm with a deep violet color,
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when a solution of DPPH is mixed with a substrate acting as a hydrogen atom donor, a stable
non radical form of DPPH is obtained with simultaneous change of the violet to pale yellow
color and decrease in absorbance [35, 36]. DPPH may be also neutralized by direct reduction
via electron transfer [37].
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The obtained results from DPPH radical scavenging assay are illustrated in Fig. 2,
these data showed that, all the tested products show a gradual increase in activity with
increase of concentration.
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The DPPH results for the investigated compounds were expressed as IC50 and its
values are given in Fig. 3. The lower IC50 value, proves the higher free radical scavenging
activity of a sample, was indicating that a smaller amount is sufficient to decrease the
concentration of DPPH by 50%. The Table 1 shows that the N-PDMPA (IC50 = 30.423 µg/ml)
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was found to be the most active DPPH radical scavenger, followed by N-EDMPA (IC50
=
67.408 µg/ml). The EDTMP showed the highest IC50 (IC50 = 129.236 µg/ml), which indicates
that it has a moderate antioxidant capacity, the IC50 value of the standard in the order of
24.387 µg/ml.
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The results of this study show that the α-aminophosphonic acids are free radical
scavengers and primary antioxidants that reduced the DPPH to the corresponding hydrazine,
which may be attributable to its proton-donating ability.
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3.2.2. The capability of scavenging towards superoxide radical by cyclic voltammetric method
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In the development of the procedure, the cyclic voltammogram was firstly recorded in
the absence of the N-EDMPA, N-PDMPA and EDTMP to determine the anodic peak current
Iꢤꢝꢥ, this value corresponds to the concentration of O●ꢛꢊ at the electrode surface.
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3.2.2.1. Electrochemical generation and voltammetric behavior of superoxide radical
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The radical O●ꢛꢊ was generated according to the reduction reaction (i) (Fig. 4, peak C).
The presence of this radical is easily detected by its anodic current measured during the
reverse scan according to the oxidation reaction (Fig. 4, peak A).
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The cyclic voltammogram of the superoxide anion radical showed a reversible process
and the obtained value of ꢁEp is 70 mV which is the difference between the oxidation and
reduction peaks potentials [28].
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3.2.2.2. Voltammetric behavior of superoxide radical in the presence of N-EDMPA, N-
PDMPA and EDTMP
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In CV method, the tested compounds must be inactive in the potential range of the
couple Oꢛ/O●ꢛꢊ in order to avoid interference or a complex interpretation of the
voltammogram. For this reason, before investigating the radical scavenging of our compounds
towards superoxide radical, we conducted the voltammetry of the studied compounds in
potential range of - 1.01 to - 0.3 V/SCE, and we found that N-EDMPA, N-PDMPA and
EDTMP are inactive in this range of potential.
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The cyclic voltammogram of the super oxide anion radical in the presence of N-
EDMPA, N-PDMPA and EDTMP showed a proportional decrease in anodic current while the
intensity of the cathodic current is not significantly modified (Fig. 5).
10
11
12
13
The decrease of the anodic peak current of superoxide anion radical suggests that the
tested products react irreversibly with this radical and decrease its concentration around the
electrode surface. No change in the cathodic peak current imparts that there is no interaction
between the tested compounds and the molecular oxygen.
14
15
16
17
18
The observed changes in the shape of the cyclic voltammogram are identical in the
three cases, this behavior is analogous to that reported in literature for some commercial
flavonoids [30, 38]. Based upon this analogy, the proposed mechanism is thus H atom
abstraction from the compound by the superoxide radical and can be represented in the
followed mechanism:
ꢊ
19
20
21
Oꢛ + 1e
Oꢛ
(i)
ꢊ
Oꢛ + ROH
HOꢛ + ROH
RO − HOꢛ
HꢛOꢛ + RO
HOꢊꢛ + RO
(ii)
(iii)
22
23
24
25
26
27
28
This mechanism is a reversible electron transfer followed by an irreversible chemical
reaction (hydrogen atom transfer). The electrochemically formed superoxide anion radical
reacts with the antioxidant and forms an electro inactive species which is a radical and a
conjugate base of the hydrogen peroxide. In the other hand, the conjugate base of hydrogen
peroxide is not stable and reacts with antioxidant, taking a proton to form a relatively stable
product H2O2. The RO• produced is assumed to be of very low toxicity because of high
probability of its dimerization.
29
3.2.2.3. The scavenging activity and determination of IC50 values
30
31
The scavenging activity of the antioxidant was evaluated by its IC50 calculated from
the linear equation (The percentage of radical scavenging activity versus compound
10

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2
3
concentration). From Table 1, it can be seen that the lowest value of IC50 (51.24 µg/ml) was
estimated for N-PDMPA and it corresponds to the highest antioxidant activity, while the
highest value of IC50 (87.55 µg/ml) was detected for EDTMP.
4
3.2.2.4. Thermodynamic parameters
5
6
7
8
The thermodynamic feasibility of the radical scavenging by the synthesized
α-aminophosphonic acids was estimated in terms of binding constant (Kb) and standard Gibbs
free energy (ꢁG°). The Kb values are determined using the curves of Fig. 6 plotted from the
following equation [39]:
ꢢ
ꢫ
ꢎꢌ
9
ꢧꢨꢩ ꢪ ꢬ ꢮ = ꢧꢨꢩ ꢯꢰ + ꢧꢨꢩ ꢱꢢꢲ
ꢳ
(11)
ꢊꢢ
ꢭꢐꢋꢭ
ꢎꢌ
ꢎꢌ
10
where Ctest is the concentration of the tested compounds.
11
12
13
From Table 1, it is apparent that the obtained Kb values of the tested α-
aminophosphonic acids are high values which confirm the strong interaction of the
ꢊ
investigated compounds with the radical Oꢛ .
14
15
ꢁG° can be calculated using Kb involving the following equation (12):
ꢁG° = − RT ln (Csolvent Kb)
(12)
16
17
where R is the gas constant (8.314 J K −1 mol −1 ), T (K) the absolute temperature and Csolvent
is the molar concentration of a solvent, which in this case is the DMSO (CDMSO = 14.08 M).
18
19
20
Negative values of ꢁG° (Table 1) indicates the spontaneity of the antiradical reaction,
but also points towards the stability of the newly formed species, which in turn is a strong
evidence of the effectiveness of the tested compounds.
21
3.3. Computational study
22
23
24
25
In the last few years, it was shown that the quantum-chemical calculations can be a
valuable tool in investigating the structure-activity relationships of various compounds and
the DFT study is one of the most widely used computational methods due to its accuracy and
less time consumption.
26
3.3.1. Structural and electronic properties
27
28
The optimized molecular structures calculated by B3LYP/6- 31G (d, p) method of the
most stable form of N-EDMPA, N-PDMPA and EDTMP are shown in Fig. 7.
11

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2
3
The electron donor distribution in the highest occupied molecular orbital (HOMO) and
the electron acceptor distribution in the lowest unoccupied molecular orbital (LUMO) are
calculated and the results are shown in Fig. 8.
4
5
6
7
8
9
The energy of HOMO corresponds to the ionization potential (I), whereas the energy
of LUMO corresponds to the electron affinity (A). On the other hand, the energy gap is the
minimum energy needed to excite an electron in a molecule and reflects the chemical activity
and the biological activity of the molecule. The larger HOMO – LUMO energy gap indicates
the harder and more stable/less reactive molecule. The decrease in the energy gap is
responsible for the bioactivity of the molecule [40].
10
11
12
It can be seen from Table 2 that the energy gap of the N-EDMPA, N-PDMPA and
EDTMP was found smaller, suggesting its good activity and the high antioxidant capacity has
been obtained for the N-PDMPA compound (ꢁEGAP = -7.207 eV).
13
14
15
Table 2 recapitulated the obtained values of the chemical reactivity descriptors of the
studied compounds determined by DFT calculations, such as total energy (E), chemical
hardness (η), electronic chemical potential (ꢁ) and electrophilicity (ω).
16
17
18
According to the calculated values of the total energy for the studied compounds
(Table 2), we observe that the EDTMP presents the least value of ETot, which shows that the
EDTMP is more stable than N-EDMPA and N-PDMPA.
19
20
21
22
23
24
25
26
27
The determined stable conformer of the stable compounds (EDTMP) was performed
using the conformational analysis. Therefore, the potential energy surface (PES) was
constructed by changing the selected dihedral angle N1 ̶ C2 ̶ C3 ̶
N4 from 0o to 180o with a
step of 10°. On the other hand, the remaining geometrical parameters have been
simultaneously relaxed. The obtained PES scan for the selected dihedral angle has presented
in Fig. 9. In principle, the minimum potential energy is due to the more stable conformation,
which is the geometry at equilibrium. From the results presented in Fig. 9, we observe
obviously that the minimum energy conformation is attributed to an angle of 180.16°, which
indicates the most stable conformation with an energy value of ̶ 71249.0587 eV.
28
3.3.2. Molecular electrostatic potential surfaces (MEP)
29
30
In order to find the active sites responsible for electrophilic and nucleophilic attacks,
we chose to use the molecular electrostatic potential surfaces (MEP) as a useful descriptor.
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2
3
4
5
6
7
MEP illustrates the charge distributions of molecules three dimensionally and
correlates the total charge distribution with dipole moment, electronegativity, partial charges
and site of chemical reactivity of a molecule [41]. The different values of the MEP at the
surface of the studied acids appear with the different colours (Fig. 10). In general, the red and
yellow colors indicate the negative regions of the MEP and correspond to nucleophilic
reactivity, while the blue color indicates the positive regions corresponding to electrophilic
reactivity.
8
9
The results of the MEP show that negative potentials are presented at electronegative
oxygen atoms, whereas positive potentials are presented at hydrogen atoms for all
compounds.
10
11
12
13
14
15
16
It has been proved by Aburas et al [42], that there are a high correlation between the
antioxidant activities values of the investigated derivatives and the dipole moment and charge
on oxygen atoms obtained by the natural charge distribution (NBO) method. All three
compounds are polar molecules, judging by their dipole moments (Table 2) and the most
polar molecule is N-PDMPA this confirms its higher antioxidant activity compared with two
other acids.
17
3.3.3. Atomic charges for N-EDMPA, N-PDMPA and EDTMP
18
19
20
21
22
23
24
25
26
27
28
29
30
Recently, the use of the allocation of positive and negative charges presents an
essential position in quantum chemical calculations; this is due to a several properties of
molecular system which is affected by the atomic charges, such as electronic structure,
molecular polarizability, dipole moment and acid-base behavior [43]. Generally, the charge
allocation on the atoms indicates the contributor and acceptor pairs including the charge
transport in the molecule [44, 45]. Additionally, in comparison with Mulliken charges the
NBO charges are very trustworthy, because they perform on the electron density
simultaneously. Moreover, the NBO examination offers a powerful technique for examining
intra and intermolecular bonding and interaction between bonds, and also offers a suitable
basis for examination of charge transport or conjugative interactions in molecular system. In
the other hand, the NBO analysis can be employed to evaluate the delocalization of electron
density among occupied Lewis-type orbitals and vacant non-Lewis NBOs, which indicates the
stabilization of the donor–acceptor interactions [46].
31
32
In this context, the investigated compounds have O, N and P heteroatoms capable to
interact with the reactive sites of the receptor. For the studied compounds, the natural atomic
13

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2
charges of the selected atoms are listed in Table 3. We observe clearly that the highest
negative charge to be found on the O7, O8, O9, O11, O12 and O13 for N-EDMPA and O7,
O8, O12, O13 and O14 for N-PDMPA and O10, O11, O12, O14, O15, O16, O18, O19 and
O20 for EDTMP. On the other hand, the highest positive charge to be found on the P6 of N-
EDMPA, N-PDMPA and EDTMP. The positive part of the receptor is interacted more
probably with the negatively charged atomic sites of the investigated compounds (O and N),
while the negative part of the receptor will interact more facilely with the most positively
charged atoms (P and H). These interactions maybe play an essential role in bioactivity of the
investigated molecules [47]. Finally, from the NBO analysis we conclude that the acidity of
the hydrogen atoms of hydroxyl group is very significant for the antioxidant activity.
3
4
5
6
7
8
9
10
11
4. Conclusions
12
13
14
15
16
17
18
19
20
21
22
In the present work, we have synthesized three bioactive α-aminophosphonic acids
using various primary amines as the starting materials. The obtained compounds are stable
1
solids characterized by high melting points and their structures were established by IR, H,
13C and 31P NMR spectra. Moreover, the antioxidant activity of the target compounds was
examined by both chemical DPPH radical-scavenging activity and electrochemical method.
Cyclic voltammetry method developed in this study measures the scavenging effect toward
superoxide anion radical and indicates the strong interaction between the O●ꢛꢊand N-EDMPA,
N-PDMPA and EDTMP, this is quantified in terms of the high obtained values of Kb. In the
other hand, the negative sign of ꢁG° suggests the spontaneity of the antiradical reaction.
According to this method, we can also propose that the antiradical reaction mechanism is the
hydrogen atom transfer.
23
24
25
26
27
28
Both methods indicate that the studied α-aminophosphonic acids may act as a
moderate antioxidant. Also, they indicate that the difference in the chemical structures of the
investigated α-aminophosphonic acids affect the antioxidant activity. Consequently, the
highest activity of N-PDMPA is probably attributed to the increase in the number of carbon
atoms. The presence of several adjacent hydroxide groups in the EDTMP molecular structure
increase the intermolecular hydrogen bonds, this will decrease their antioxidant activity.
29
30
31
For a better understanding of the antioxidant activity, the quantum chemical
parameters were calculated employing DFT method. A good correlation was visible between
the experimentally reported values and the calculated quantum chemical parameters such as
14

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2
ꢁEGAP, ꢁ and NBO charges. It was found for N-EDMPA, N-PDMPA and EDTMP that the
antioxidant activity increased with the smaller values of ꢁEGAP, and higher values of ꢁ.
3
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Figure Captions
Fig.1. Synthesis route of α- aminophosphonic acids derivatives:
3
4
5
N-Ethyliminodimethylenediphosphonic acid (N-EDMPA), n=2
N-Propyliminodimethylenediphosphonic acid (N-PDMPA), n=2
Ethylenediamine tetra-methylenephosphonic acid (EDTMP), n=4
6
7
Fig.2. Radical Scavenging Activity of N-EDMPA, N-PDMPA, EDTMP and BHT with
different concentration.
8
9
Fig.3. IC50 for the RSADPPH displayed by N-EDMPA, N-PDMPA, EDTMP and BHT, Values
are Mean ± S.D (n=3 for test compounds and standards).
10
11
Fig.4. Cyclic voltammograms of (a) DMSO - Bu4NBF4, 0.1 M (b) O●ꢛꢊ in DMSO - Bu4NBF4,
0.1 M medium with scan rate = 20 mV/s:
12
13
14
Glassy carbon (GC) as a electrode working
Platinum Plate (1.28 cm2) as a counter electrode
Saturated calomel electrode (SCE) as a reference
ꢊ
15
16
17
Fig.5. Evolution of the cyclic voltammograms of Oꢛ in the presence of different volumes of
N-EDMPA, N-PDMPA and EDTMP, in DMSO - Bu4NBF4 0.1 M medium with scan rate =
20mV/s:
18
19
20
Glassy carbon (GC) as a working electrode
Platinum Plate (1.28 cm2) as a counter electrode
Saturated calomel electrode (SCE) as a reference
21
22
Fig.6. log (Iꢤꢥ /Iꢤꢝꢥ − Iꢤꢥ ) versus log (1/Cꢴꢵꢶꢴ ) for N-EDMPA, N-PDMPA and EDTMP.
Fig.7. Optimized molecular structures of N-EDMPA, N-PDMPA and EDTMP.
23
24
Fig.8. The frontier molecular orbitals density distributions for N-EDMPA, N-PDMPA and
EDTMP.
25
26
Fig.9. Scan of total energy of the more stable compounds (EDTMP) with the selected dihedral
angle (N1 ̶ C2 ̶ C3 ̶ N4).
27
28
29
Fig.10. Molecular electrostatic potential of N-EDMPA, N-PDMPA and EDTMP.
20

ACCEPTED MANUSCRIPT
1
2
Table Captions
Table 1
3
4
IC50 values of N-EDMPA, N-PDMPA and EDTMP obtained by the DPPH and superoxide
anion inhibition tests.
5
Table 2
6
7
Global chemical reactivity descriptors for N-EDMPA, N-PDMPA and EDTMP calculated by
B3LYP/6- 31G (d, p).
8
9
Table 3
Atomic charges of N-EDMPA, N-PDMPA and EDTMP calculated by B3LYP/6- 31G (d, p).
10
21

Table 1
ACCEPTED MANUSCRIPT
IC50 values of N-EDMPA, N-PDMPA and EDTMP obtained by the DPPH and superoxide anion
inhibition tests
Compound Methods IC50(µg/ml)
Kb (l/mol) -ꢀG°(kJ/mol)
DPPH
67.408± 0,8
65.668
30.423± 6,5
57.635
N-EDMPA
N-PDMPA
ꢀ●ꢁꢂ
3451.437
4036,454
26.736
27.212
DPPH
ꢀ●ꢁꢂ
DPPH
ꢀ●ꢁꢂ
129.236± 3,9
EDTMP
87.55
3162.27
26.519

Table 2
ACCEPTED MANUSCRIPT
Global chemical reactivity descriptors for N-EDMPA, N-PDMPA and EDTMP calculated by
B3LYP/6- 31G (d, p)
Paramètres quantiques
ETot (eV)
N-EDMPA
N-PDMPA
EDTMP
– 36713.80374 –37783.56306
̶
71255.34237
EHOMO (eV)
– 6.34461
0.52355
– 6.86816
6.34461
– 0.52355
2.91053
– 2.91053
3.43408
0.29112
1.2334
– 6.59604
0.611440
– 7.20748
6.59604
– 0.61144
2.99230
– 2.99230
3.60374
0.27749
1.24230
4.9175
̶
5.85671
0.37552
ELUMO (eV)
ꢀEGAP (eV)
– 6.23223
5.85671
– 0.37552
2.74059
–2.74059
3.11611
0.32091
1.20516
2.9445
Ionization Energy (I)
Electron Affinity (A)
Electronegativity (χ)
Chemical Potential (ꢁ)
Chemical Hardness (η)
Chemical Softness (S)
Electrophilicity Index (ω)
Dipole Moment (Debye)
4.7193

Table 3
ACCEPTED MANUSCRIPT
Atomic charges of N-EDMPA, N-PDMPA and EDTMP calculated by B3LYP/6- 31G (d, p)
N-EDMPA N-PDMPA EDTMP
Atom NBO Charge Atom NBO Charge Atom NBO Charge
C1
C2
N3
C4
C5
P6
O7
O8
O9
P10
O11
O12
O13
H14
H15
H16
H17
H18
H19
H20
H21
H22
H23
H24
H25
H26
-0.6948400
-0.2637600
-0.5351900
-0.6658700
-0.6705400
2.3891700
-1.0715200
-1.0310900
-1.0419400
2.3742500
-1.0697000
-1.0357400
-1.0201000
0.2332600
0.2374100
0.2451500
0.2496500
0.2065300
0.2520900
0.2773400
0.2492100
0.2672000
0.5239400
0.5303700
0.5355900
0.5291100
N1
C2
C3
C4
C5
C6
P7
-0.5433800
-0.6647600
-0.6764000
-0.6887400
-0.4804200
-0.2595500
2.3593500
-1.0672900
-1.0282700
N1
C2
C3
N4
C5
C6
C7
C8
P9
-0.5172700
-0.2801700
-0.2702500
-0.5460600
-0.6624900
-0.6636600
-0.6481700
-0.6604000
2.3830400
-1.0561700
-1.0526900
-1.0370900
2.4109200
-1.0990500
-1.0146400
-1.0469400
2.3353700
-1.0730700
-1.0288200
-1.0268300
2.3854000
-1.0784500
-1.0646500
-1.0304600
0.2564300
0.2298600
0.2086900
0.2582100
0.2760200
0.2591500
0.2748800
0.2490500
0.2688900
0.2668400
0.2632800
0.2539400
0.5361800
0.5323700
0.5350100
0.5505900
0.5275300
0.5236300
0.5475900
0.5244700
O8
O9
O10 -1.0272300 O10
P11 2.3486100 O11
O12 -1.0687200 O12
O13 -1.0298700 P13
O14 -1.0230800 O14
H15 0.2753800 O15
H16 0.2753900 O16
H17 0.2761200
P17
H18 0.2678800 O18
H19 0.2312600 O19
H20 0.2300500 O20
H21 0.2440400
H22 0.2448200 O22
H23 0.2360500 O23
H24 0.2266100 O24
H25 0.2386700 H25
H26 0.5242600 H26
P21
H27 0.5216000 H27
H28 0.5312500 H28
H29 0.5263800 H29
H30
H31
H32
H33
H34
H35
H36
H37
H38
H39
H40
H41
H42
H43
H44

ACCEPTED MANUSCRIPT
Fig.1. Synthesis route of α- aminophosphonic acids derivatives:
-
-
-
N-Ethyliminodimethylenediphosphonic acid (N-EDMPA), n=2
N-Propyliminodimethylenediphosphonic acid (N-PDMPA), n=2
Ethylenediamine-tetrakis(methylenephosphonic acid) (EDTMP), n=4

ACCEPTED MANUSCRIPT
80
70
60
50
40
30
20
10
0
N-EDMPA
N-PDMPA
EDTMP
BHT
0
50
100
150
200
250
Concentration(µg/ml)
Fig.2. Radical Scavenging Activity of N-EDMPA, N-PDMPA, EDTMP and BHT with different
concentration

ACCEPTED MANUSCRIPT
BHA
N-PDMPA
N-EDMPA
EDTMP
24.387
30.423
67.408
129.236
0
50
100
150
I5O (µg/ml)
Fig.3. IC50 for the RSADPPH displayed by N-EDMPA, N-PDMPA, EDTMP and BHT, Values are
Mean ± S.D (n=3 for test compounds and standards)

ACCEPTED MANUSCRIPT
60
A
40
.-
O
O + 1e
2
2
20
(b)
(a)
0
-20
-40
-60
-80
-100
.-
O
O + 1e
2
2
C
-1,1
-1,0
-0,9
-0,8
-0,7
-0,6
-0,5
-0,4
-0,3
-0,2
Potentiel(V)
Fig.4. Cyclic voltammograms of (a) DMSO - Bu4NBF4, 0.1 M (b) O●ꢀꢁ in DMSO - Bu4NBF4, 0.1 M
medium with scan rate = 20 mV/s:
- Glassy carbon (GC) as a electrode working
- Platinum Plate (1.28 cm2) as a counter electrode
- Saturated calomel electrode (SCE) as a reference