Synthesis, antibacterial screening and DFT studies of series of α-amino-phosphonates derivatives from aminophenols

Article abstract of DOI:10.1016/j.molstruc.2016.12.079

A series of α-aminophosphonate derivatives containing aminophenol moiety have been synthesized and evaluated for their antibacterial activities against two clinical strains Gram-positive and two Gram-negative applying Agar disc diffusion method, Minimal I

Full text of DOI:10.1016/j.molstruc.2016.12.079

Accepted Manuscript
Synthesis, antibacterial screening and DFT studies of series of α-amino-
phosphonates derivatives from aminophenols
Abdelkader Hellal, Salah Chafaa, Nadjib Chafai, Lasnouni Touafri
PII:
S0022-2860(16)31398-9
10.1016/j.molstruc.2016.12.079
MOLSTR 23277
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
20 September 2016
24 December 2016
26 December 2016
Please cite this article as: Abdelkader Hellal, Salah Chafaa, Nadjib Chafai, Lasnouni Touafri,
Synthesis, antibacterial screening and DFT studies of series of α-amino-phosphonates derivatives
from aminophenols, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.12.079
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form.
Please note that during the production process errors may be discovered which could affect the
content, and all legal disclaimers that apply to the journal pertain.

Graphical abstract
ACCEPTED MANUSCRIPT
4a
HOMO
LUMO
4b
4c
4d
HOMO
HOMO
HOMO
LUMO
LUMO
LUMO
4e
HOMO
LUMO
4f
HOMO
LUMO

ACCEPTED MANUSCRIPT
HIGHTLIGHTS
 Six α-aminophosphonates were synthezised via Kabachnik-Fields reaction.
 The biological property of threse synthesized α-aminophosphonates were screened in
vitro against some Gram-positive and Gram-negative pathogenic bacteria.
 The MIC and MBC were assessed by serial dilution method.

HOMO and LUMO energies, MEP distribution of the molecule were calculated.

The principal quantum chemical descriptors (HOMO, LUMO, MESP) have been correlated
with the antibacterial activity.

ACCEPTED MANUSCRIPT
Synthesis, antibacterial screening and DFT studies of series of α-amino-
phosphonates derivatives from aminophenols
Abdelkader Hellala,b*, Salah Chafaaa, Nadjib Chafaia, Lasnouni Touafrib,c
a*Laboratoire d’Electrochimie des Matériaux Moléculaires et des Complexes(LEMMC). Département Génie des
procédés, Faculté de Technologie, Université Ferhat Abbsas Sétif-1, Algeria, Email: haekpharm@yahoo.fr
bLaboratoire de Valorisation des Substances Naturelles(LVSN), Université Djilali Bounaâma de Khemis
Miliana, 44225, Algeria
cLaboratoire de Physique et Chimie des Matériaux (LPCM), Université Mouloud MAMMERI de Tizi-Ouzou,
15000, Algeria
Abstract
A series of α-aminophosphonate derivatives containing aminophenol moiety have been synthesized
and evaluated for their antibacterial activities against two clinical strains Gram-positive and two
Gram-negative applying Agar disc diffusion method, Minimal Inhibition Concentration (MIC) and
Minimal Bactericidal Concentration (MBC) methods. The chemical structures of the synthesized
compounds were verified on the basis of spectral data and elemental analyses. These compounds have
significant bactericid activity with totalities of bacterial strains used. DFT calculations with B3LYP/6-
31G (d,p) level have been used to analyze the electronic and geometric characteristics deduced for the
stable structure of the compounds. The principal quantum chemical descriptors (HOMO, LUMO,
MESP) have been correlated with the antibacterial activity.
Key words: α-aminophosphonates, Antibacterial activity, DFT, HOMO, LUMO, MESP.
1. Introduction
Aminophosphonate derivatives, structural analogues of the corresponding α-amino acids, constitute
an important class of organophosphorus compounds on account of their versatile biological activity
[1]. The general low mammalian toxicity of these compounds made them attractive for use in
agriculture and medicine [2-4]. α-aminophosphonates have attracted considerable attention since they
are considered as structural analogues of α-aminoacids and they act as potential antibiotic [5],
antimicrobial [6], antitumor [7] and antioxidants agents [8]. They also have several applications in the
agricultural industry [9]. α-aminophosphonates were found too be effective in inhibiting test-tube
growth of plasmodium falciparum, the parasite that causes malaria [10]. It has the same effect as
related types of single-celled parasites such as Toxoplasma and Cryptosporidium that cause
opportunistic infections in AIDS patients [11]. α-aminophosphonates have been found to act as
inhibitors of specific enzymes as HIV protease, thrombin and human collagenase and to suppress the
growth of various tumors and viruses [12,13]. For that reason, the synthesis of α-aminophosphonates
has received considerable attention and significant progress has been made to develop more efficient
methods for the synthesis of these compounds [14]. In this context, the “one-pot” three-component
reaction (Kabachnik-Fields reaction) is one of the most useful methods for the synthesis of α-
aminophosphonates due to its versatility and high yields.
In this work we have developed the synthesis of series of six α-aminophosphonates starting from
aminophenols as primary amine. To the best of our knowledge, neither quantum chemical applications
nor the antibacterial activity of the title compounds have been reported, as yet. The present
communication is aimed at comparing the antibacterial activity with the properties of the molecules
under investigation such as equilibrium energy, frontier orbital energy gap, dipole moment, softness
and hardness along with the electrostatic potential maps have also been used to understand the activity
1

ACCEPTED MANUSCRIPT
of these. Structure–activity relationships allow a correct correlation with biological activity with some
appropriate quantum descriptors such as EHOMO, ELUMO, energy gap, dipolar moment, global
hardness…etc.
2 Experimental details
2.1 Chemicals/ Instrumentation
Chemicals were purchased from Merck and Fluka Chemical Companies. All of the products were
identified by their physical and spectral data. Infrared spectra were recorded on FT/IR JASCO 2400
(4000-400 cm-1). NMR spectra were recorded on a Bruker Avance 300 apparatus operating at 300
MHz with TMS as the internal standard. Chemical shifts are given in parts per million (ppm).
Multiplicities are shown as the abbreviations: s (singlet), brs (broad singlet), d (doublet), t (triplet), m
31
(multiplet). P NMR chemical shifts were referenced to external H3PO4 (85% w/w). The percentages
of carbon, hydrogen and nitrogen were determined by Elemental Analyses using Perkin Elmer 2400
CHN Elemental Analyzer. Melting points were determined on a melting point apparatus and were
uncorrected. The purity of the products and the progress of the reactions were accomplished by TLC
on silica-gel polygram SILG/UV 254 plates.
2.2 Typical procedure for the synthesis of α-aminophosphonate (4a).
A mixture of para aminophenol 1a (0.01 mol) and benzaldehyde 2a (0.01 mol) was stirred at room
temperature for 1h then diethylphosphite 3a (0.01 mol) was added dropwise. Stirring was continued
at room temperature for another 30 min, after which the mixture was added to the stirring
solution of oxalic acid as catalyst (10mol%) in water (20mL) and heated under reflux in an oil bath at
90°C for an appropriate time. Water (20 mL) was added to the cooled reaction mixture and extracted
with CH2Cl2 (3x20 mL). The combined organic extract was washed with water (2x10 mL), separated,
dried over MgSO4 and filtered. Product (4a) was isolated by preparative plate chromatography eluted
with n-hexane:EtOAc (1:1).It is important to note that the catalysts were recycled by simple extraction
of the product with water from the reaction mixture. The same procedure was carried in the absence of
catalysts.The other compounds 4b to 4f were prepared employing a procedure similar to that
described for compound 4a (Scheme 1) [15].
2.3 Evaluation of antimicrobial activity
2.3 .1 Antimicrobial Screening
The antibacterial activities of the Six diethyl α-aminophosphonates were screened in vitro for their
antimicrobial activity against two gram-negative and two gram-positive bacterial strains using disc
diffusion method [16,17]. Briefly, two calculated amount of the compounds were dissolved in DMSO
in three different vials for getting solutions having concentrations of 50 μg/ml and 100 μg/ml
respectively. They were then applied on filter paper disc. Standard chloramphenicol (100 μg/ml) was
used as positive control and DMSO as negative control. Both experimental and control discs were
placed in petri dishes seeded with organism in nutrient agar medium. The petri dishes were kept in a
refrigerator at 4°C for 24 hours to ensure diffusion of the test materials. Finally, they were incubated at
37±1°C for 24 hours and all experiments were done as triplicates. The antibacterial activity was
determined by measuring the diameter of zone of inhibition in mm.
2.3.2 Determination of MIC and MBC
Each bacteria has a level of antibiotic which will inhibit growth but not kill the organisms. This is
called the minimum inhibitory concentration. Related to this, a higher antibiotic concentration will kill
the organisms. This is called the minimum bactericidal concentration. By understanding the concepts
in determining antibiotic concentrations compared to the MIC and MBC, we can make rational
2

ACCEPTED MANUSCRIPT
decisions in determining how successful antibiotic treatment is likely to be [18]. The Minimum
Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of 4a-4f were
determined by serial dilution technique against the above mentioned pathogenic bacteria [18,19]. 4a-4f
were used from a concentration of 0.25 μg/ml to 256 μg/ml. A control test-tube containing only
medium (nutrient broth medium) was used to confirm the sterility of the medium. Bacterial suspension
(10μl) containing 107cells/ml was inoculated into all tubes. All of the test tubes were incubated at
37±1°C and observed for bacterial growth for 24 hours for MIC and 96 hours (4 days) for MBC
determinations after inoculation for 24 hours, the test tube with no visible growth of the
microorganism was taken to represent the MIC value of the sample in μg/ml. MBC, in which no viable
organism occurred was determined by keeping the test tubes which was used for MIC determination
for four days. After four days, bacterial growth was observed and MBC was determined at lowest
concentrations where no bacterial growth was observed.
2.3 .3 Evaluation of bactericidal and bacteriostatic capacity
The action of an antibacterial on the bacterial strains can be characterized with two parameters such
as Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC).
According to the ratio MBC/MIC, we appreciated antibacterial activity. If the ratio MBC/MIC≤4, the
effect was considered as bactericidal but if the ratio MBC/MICꢀ>4, the effect was defined as
bacteriostatic [20,21].
2.4 Computational studies
The molecules under investigation have been analyzed with density functional theory (DFT) [22],
employing Becke’s three parameter hybrid exchange functional [23] with Lee-Yang-Parr correlation
functionals (B3LYP) [24,25]. All the quantum chemical calculations in this study were performed
using the Gaussian 09 program [26]. The geometry was optimized by energy minimization with
respect to all the geometrical parameters without considering any molecular symmetry constraints.
Drawing the structure of the optimized geometry and visualization of the HOMO and LUMO
calculations have been done by GaussView 5.0.8 program [27].The chemical reactivity descriptors
were calculated using DFT. These are very important physical parameters to understand chemical and
biological activities of the synthesized compounds. The calculated HOMO-LUMO orbital energies can
be used to estimate the ionization energy [28], electron affinity [28], electronegativity [29], electronic
chemical potential [29], molecular hardness [30], molecular softness [31], and electrophilicity index
[31] using the following equations: Ionization Energy (I)=-EHOMO ,Electron Affinity (A)=-
ELUMO,Electronegativity (χ) = (I + A)/2,Electronic Chemical Potential (µ)= -χ ,Chemical Hardness (η =
(I− E)/2,Chemical Softness (S)=1/η ,Electrophilicity Index (ω)= µ2/2η.
3. Result and discussion
3.1 Chemistry
Six dialkyl α-aminophosphonates were obtained in good yield via Kabachnik-Fields reaction by the
reaction of various types of aromatic aldehydes electron donating or electron withdrawing functional
groups, aminophenols and diethylphosphite (Scheme 1) [15].Thin layer chromatography (TLC) was
employed to monitor reaction progress and to determine the purity of the products. All the title
compounds are readily soluble in polar organic solvents and melted in the temperature range of 104–
211ºC. The IR spectra of compounds (4a–4f) showed the NH band in the range of 3135–3488cm−1.
The sharp band observed in the range 1210–1290cm−1 is due to the νP=O, and a band for P–C
stretching occurred in the range 739–763cm−1. All the stretching frequencies are compiled in Table 2.
The 1H NMR spectra of the compounds (4a–4f) were recorded in the DMSO-d6 solvent. The aromatic
protons of α-aminophosphonic acid esters appeared as a multiplet in the region δ6.67–8.85. The P–C–
3

ACCEPTED MANUSCRIPT
H group proton resonated as a multiplet in the range δ3.08–4.30 due to coupling with phosphorus and
N–H. The N–H proton signal appeared at δ3.61–5.78 as a multiplet. The protons of P–O–CH2–C that
appeared as a quartet at δ3.56–3.62 and P–O–C–CH3 gave a triplet at δ1.12–1.19. The carbon
chemical shifts for P-C-H, P-O-CH2-CH3 and P-OCH2-CH3 in the title compounds observed in the
expected region.31P NMR signals appeared in the region 19.65-24.53ppm for all the compounds (4a-
4f).
Analytical data
[(4-Hydroxy-phenylamino)-phenyl-methyl]-phosphonic acid diethyl ester (4a). Mol.Wt: 335.1. mp
o
135-139 C. IR: 745 (P-C), 1268 (P=O), 3381 (NH)cm-1; 1H-NMR (DMSO-d6) δ (ppm): 7.86–6.90
(m, 9H, Ar-H), 3.41–3.08 (d, 1H, P-CH), 3.77–3.71 (t, 1H, N-Haliphatic), 3.01-2.88 (m, 4H, P-O-CH2),
1.23-1.11 (m, 6H, P-O-CH2-CH3); 10.60 (s, 1H, - OH); 13C-NMR (DMSO-d6) δ: 113.6-145(CHAr),
64.1 (d, (P-CH2-C)), 57.7 (s, (CH-P)), 14.1 (d, (P-C-CH3)), 31P-NMR (DMSO-d6) δ: 20.79; Anal. cald.
for C17H22NO4P: C, 60.89; H, 6.61; N, 4.18; O, 19.08; P, 9.24. Found: C, 60.90; H, 6.60; N, 4.15 [15].
[(4-Hydroxy-phenylamino)-(4-nitro-phenyl)-methyl]-phosphonic acid diethyl ester (4b). Mol.Wt:
1
380.1. mp 118–120oC. IR: 765 (P-C), 1263 (P=O), 3340 (NH)cm-1; H-NMR (DMSO-d6 ) δ (ppm):
7.84–6.95 (m, 8H, Ar-H), 5.66-5.18 (d, 1H, P-CH), 4.33–3.78 (t, 1H, N-Haliphatic), 3.46- 3.40 (m, 4H,
P-O-CH2), 1.24- 1.16 (m, 6H, P-O-CH2-CH3); 10.47 (s, 1H, -OH); 13C-NMR (DMSO-d6) δ: 112.6-
148(CHAr), 62.7 (d, (P-CH2-C)), 57.8 (s, (CH-P)), 13.1 (d, (P-C-CH3)), 31P-NMR (DMSO-d6 ) δ:
21.87; Anal. cald. for C17H21N2O6P: C, 53.69; H, 5.57; N, 7.37; O, 25.24; P, 8.14. Found: C, 53.66;
H, 5.54; N, 7.40 [15].
[(4-Hydroxy-phenylamino)-(4-methoxy-phenyl)-methyl]-phosphonic acid diethyl ester (4c). Mol.Wt:
1
365.1. mp 145–147oC. IR: 756 (P-C), 1266 (P=O), 3421 (NH)cm-1; H-NMR (DMSO-d6 ) δ (ppm):
8.85–7.22 (m, 8H, Ar-H), 5.66-5.18 (d, 1H, P-CH), 4.65–3.91 (t, 1H, N-Haliphatic), 3.56-3.51 (m, 4H,
13
P-O-CH2), 2.39 (s, 3H, Ar-OCH3), 1.13-1.10 (m, 6H, P-O-CH2-CH3); 10.58 (s, 1H, - OH); C-
NMR (DMSO-d6) δ: 113.9-148 (CHAr), 62.8 (d, (P-CH2-C)), 57.7 (s, (CH-P)), 13.9 (d, (P-C-CH3)),
31P-NMR (DMSO-d6 ) δ: 22.35; Anal. cald.for C18H24NO5P: C, 59.17; H, 6.62; N, 3.83; O, 21.90; P,
8.48. Found: C, 59.14; H, 6.60; N, 3.85 [15].
[(2-Hydroxy-phenylamino)-phenyl-methyl]-phosphonic acid diethyl ester (4d).Mol.Wt: 335.1. mp
o
109-111 C. IR: 740 (P-C), 1270 (P=O), 3377 (NH)cm-1; 1H-NMR (DMSO-d6) δ (ppm): 7.81–6.92
(m, 9H, Ar-H), 3.41–3.23 (d, 1H, P-CH), 3.71–3.66 (t, 1H, N-Haliphatic), 3.11-2.89 (m, 4H, P-O-
CH2), 1.25-1.21 (m, 6H, P-O-CH2-CH3); 10.64 (s, 1H, - OH); 13C-NMR (DMSO-d6) δ: 112.0-
140.09(CHAr), 64.6 (d, (P-CH2-C)), 57.5 (s, (CH-P)), 14.2 (d, (P-C-CH3)), 31P-NMR (DMSO-d6 ) δ:
21.80; Anal. cald. for C17H22NO4P: C, 60.89; H, 6.61; N, 4.18; O, 19.08; P, 9.24. Found: C, 60.91; H,
6.58; N, 4.17 [15].
[(2-Hydroxy-phenylamino)-(4-nitro-phenyl)-methyl]-phosphonic acid diethyl ester (4e).Mol.Wt:
380.1. mp 209-211oC. IR: 745 (P-C), 1263 (P=O), 3330 (NH)cm-1; 1H-NMR (DMSO-d6 ) δ (ppm):
7.81–6.91(m, 8H, Ar-H), 5.62-5.11 (d, 1H, P-CH), 4.23–3.98 (t, 1H, N-Haliphatic), 3.49- 3.39 (m, 4H,
P-O-CH2), 1.29- 1.15 (m, 6H, P-O-CH2-CH3); 10.41 (s, 1H, -OH); 13C-NMR (DMSO-d6) δ: 116.7-
31
148.90 (CHAr), 62.0 (d, (P-CH2-C)), 57.3 (s, (CH-P)), 13.1 (d, (P-C-CH3)), P-NMR (DMSO-d6 ) δ:
22.99; Anal. cald. for C17H21N2O6P: C, 53.69; H, 5.57; N, 7.37; O, 25.24; P, 8.14. Found: C, 53.66; H,
5.54; N, 7.40 [15].
[(2-Hydroxy-phenylamino)-(4-methoxy-phenyl)-methyl]-phosphonic acid diethyl ester (4f). Mol.Wt:
365.1. mp 104-107oC. IR: 750 (P-C), 1270(P=O), 3441 (NH)cm-1; 1H-NMR (DMSO-d6 ) δ (ppm):
8.71–7.20 (m, 8H, Ar-H), 5.60-5.11 (d, 1H, P-CH), 4.70–4.01 (t, 1H, N-Haliphatic), 3.71-3.57 (m, 4H,
P-O-CH2), 2.30 (s, 3H, Ar-OCH3), 1.23-1.17 (m, 6H, P-O-CH2-CH3); 10.46 (s, 1H, - OH); 13C-
4

ACCEPTED MANUSCRIPT
NMR (DMSO-d6) δ: 113.9-147.23(CHAr), 62.6 (d, (P-CH2-C)), 57.7 (s, (CH-P)), 13.7 (d, (P-C-CH3)),
31P-NMR (DMSO-d6 ) δ: 22.35; Anal. cald. for C18H24NO5P: C, 59.17; H, 6.62; N, 3.83; O, 21.90; P,
8.48. Found: C, 59.14; H, 6.60; N, 3.85 [15].
3.2 DFT Studies
3.2.1. Geometry Optimization
Nowadays computational based methods have become very popular to investigate the structure-
activity relationships of compounds and density functional theory (DFT) study is one of the most
widely used computational methods due to its accuracy and less time consumption. The energy
minima geometries of all products 4a–4f were optimized using the Gaussian 09 program at the
B3LYP/6-31G(d,p) level of DFT. Optimized geometries were further used for structural investigations
like frontier molecular orbitals analysis (FMOs), electrostatic potential (ESP) mapping and. The
optimized ground state structure of 4a–4f has been given in the Figure 1.
3.2.2 Frontier Molecular Orbital (FMO) Analysis
FMO analysis using computational methods is widely employed to explain the electronic as well as
the optical properties of organic compounds, for example, molecular reactivity and the capacity of a
molecule to absorb light. To clarify several types of reaction and for foreseeing the most reactive
position in conjugated systems, molecular orbitals and their properties like energy are utilized [32].
During molecular interactions, the highest occupied molecular orbital (HOMO) and lowest unoccupied
molecular orbital (LUMO) are the main participants. LUMO is the molecular orbital of the lowest
energy that is not occupied by electrons and speaks the capacity to obtain an electron; HOMO is
molecular orbital of the highest energy that is possessed by electrons and represents the ability to give
an electron. The HOMO donates electrons and its energy corresponds to the ionization potential (I),
whereas the LUMO accepts the electrons and its energy corresponds to the electron affinity (A) [33,
34]. FMO analysis of all products 4a–4f was performed at the same level as used for optimization.
The resulting HOMO-LUMO surfaces are shown in Figure 2, and their corresponding energies along
with the energy gaps are listed in Table 1.
The FMO analysis of the title compounds revealed that the isodensities in the HOMO for all
compounds, are mainly concentrated on aromatic moieties of aminophenol and phosphite group,
while, The isodensity in the LUMO is focused mainly around aromatic ring of aldehyde and
aminophenol for 4a, 4c, 4d and 4f, but it is located only on the phenyl ring of aldehyde moiety for 4b
and 4f. The energy gap of HOMO-LUMO clarify the charge exchange inside the molecule and reflect
the chemical activity and the biological activity of the molecule [34]. The larger the HOMO-LUMO
energy gap, the harder and more stable and less reactive the molecule [31,32]. The decrease in the
HOMO and LUMO energy gap explains the eventual charge transfer interaction taking place within
the molecule due to the strong electron-accepting ability of the electron acceptor group. The strong
charge transfer interaction is responsible for the bioactivity of the molecule. The HOMO-LUMO
energy gap for 4b and 4e is found to be the lowest and equal to 0.13738 and 0.13977, respectively,
whereas the highest band gap was observed for the other compounds. Among the synthesized
compound, 4b and 4e showed more stability and biological activity as it shows less energy gap, low
hardness, and more softness when compared with the other compounds.
Let us recall from experimental results that the compounds 4b and 4e have the best antibacterial
activity. We can thus conclude that the values of energy gap, softness and Ionization Potential are
proportional to the antibacterial activity whereas the values of dipolar moment, Electron affinity and
hardness are inversely proportional. The same results are obtained by comparing 4a, 4c, 4d, and 4f: the
decrease of the energy difference between the frontier molecule orbital and the total energy on one
5

ACCEPTED MANUSCRIPT
hand and the increase of the dipolar moment while passing from 4a to 4f means that antibacterial
activity of 4a is better than that of 4c, 4d, and 4f, respectively. From these theoretical calculations
carried out for studied molecules 4a-4f, it can be concluded that the electronic properties are correlated
with their experimental results. On the other hand, it is important to examine the HOMO and the
LUMO for these molecules because the relative ordering of occupied and virtual orbital provides a
reasonable qualitative indication of electronic properties and the ability of electron hole transport. In
general, as plotted in Figure 2, the HOMO possesses an antibonding character between the consecutive
subunits; whereas the LUMO generally shows a bonding character between the subunits.
3.2.3 Molecular Electrostatic Potential Surface
In this study, molecular electrostatic potential (MESP) maps have been mapped for 4a-4f as shown
in Figure 3. Electrostatic potential characteristics are considered to be key features of molecules
through which it recognizes its receptor. The molecular electrostatic potential (MESP), the force acting
on a positive test charge located at point through the electrical charge cloud generated through the net
charge of molecule (electrons and nuclei), has been a widely used entity in the chemical literature,
generally employed as a tool for probing electron rich regions. MESP correlates the total charge
distribution with dipole moment, electronegativity, and partial charges and site of chemical reactivity
of a molecule. It simultaneously displays molecular size, shape as well as positive, negative and
neutral electrostatic potential regions in terms of color grading and is very useful in research of
molecular structure with its physiochemical property relationship. It provides a visual method to
understand the relative polarity of a molecule and serves as a useful quantity to explain hydrogen
bonding, reactivity and structure-activity relationship of molecules including biomolecules and drugs
[35-37]. Nam et al. reported their discovery that electrostatic interactions accounted for the majority of
the rate acceleration in the mechanism of RNA transphosphorylation in solution catalyzed by the
hairpin ribozyme [38]. Different values of the electrostatic potential at the surface of a molecule
appear with the different colours. In general the attractive potential appears in red coloured regions
(nucleophilic reactivity) and those of repulsive potential (electrophilic reactivity) appear in blue. The
predominance of green region in the MESP surfaces corresponds to a potential halfway between the
two extremes that are indicated in red and blue colors, respectively. Negative electrostatic potential
corresponds to an attraction of the proton by the concentrated electron density in the molecules (from
lone pairs, pi-bonds, etc.). Positive electrostatic potential corresponds to repulsion of the proton by the
atomic nuclei in regions where low electron density exists and the nuclear charge is incompletely
shielded. MESP for 4b and 4c compounds showed the presence of localized most electronegative
potential region (in red color) near the oxygen atoms of the nitro group, while the rest of compounds
have only one localized most electronegative potential region in the vicinity of oxygen atoms of
phosphite, hydroxy, and methoxy groups (for 4c and 4f). The positive potentials are located near the
hydrogen atoms attached with heteroatom like oxygen and nitrogen. Moreover, a gradual depletion of
both red and blue areas and an increase of green color around the aromatic rings were also observed.
On the whole, appearance of both most electronegative and electropositive regions along with
moderate section in synthesized compounds demonstrates that these regions can act as electron donors
or acceptors to the active site thus making these compounds very reactive.
3.3. Antibacterial activity
All the compounds were screened against Gram-positive bacteria and Gram-negative bacteria at
two concentrations, 50 and 100μg/ml by the disc diffusion method and the results were compared with
the standard antibiotic (Chloramphenicol) were used as positive control and DMSO used as negative
control. The results for the antibacterial activity of 4a-4f as well as the standard and DMSO have been
presented in Table 2. DMSO had no effect on the bacteria in the concentrations studied. All the tested
6

ACCEPTED MANUSCRIPT
compounds showed higher zone of inhibition when tested with higher doses (Table 2). A low activity
was shown by 4a, 4c, 4d and 4f against Gram positive bacteria and Gram negative bacteria compared
to the reference compound and the synthesized compounds (4b, 4e). The synthesized compound (4b,
4e) presenting the strongest antimicrobial activity than 4a, 4c, 4d and 4f and broadest range of action
were, in descending order, 4b, 4e, 4a, 4c, 4d and 4f . For the 4b and 4e products, the highest activity
(diameter of zone of inhibition >27mm) was demonstrated against all tested bacteria at high
concentration (100µg/ml), while the lowest activity (diameter of zone of inhibition <16mm) was
demonstrated by the products at low concentration (50µg/ml) against gram-negative bacteria.
MIC and MBC values of these compounds against pathogenic bacteria are shown in Table 3. For
the synthesized compounds, MIC values ranged between 8 and 32µg/ml against gram-positive and
gram-negative, and MBC values, between 32 and 256 µg/ml. Almost as a rule, MBC values were
several folds higher than those of MIC, suggesting a better inhibitory than bactericidal activity. The
exception was the case of 4a, 4c, 4d and 4f compounds where the MICs against all tested strains were
lowest values. 4b and 4e presented higher MIC and MBC values than the other evaluated.
Antibacterial potency of 4b and 4e against these bacteria expressed in MIC indicated the synthesized
products are more effective against gram-positive at lower concentration than that against gram-
negative bacteria. The strong antibacterial activity of 4b was confirmed by the lowest MIC and MBC
values (8-16µg/ml and 16-128 µg/ml respectively) observed against all tested microorganisms. The
MIC values of 4e were found to be between 8-32 μg/ml and the MBC values for S1 were 32-128
μg/ml. The bactericidal and bacteriostatic effect of the tested compounds was determined using the
ratio MBC/MIC. Antimicrobial substances are considered as bacteriostatic agents when the ration
MBC/MIC>4 and bactericidal agents when the ration MBC/MIC≤4. The summary of the microcide
and the microbiostatic effects were given in Table 3. In the present study, 4b and 4e and the standard
showed the ratio MBC/MIC≤4, suggesting that these compounds act as bactericidal agents on these
strains. Most other products showed MBC/MIC ratio of more than 4, which may be classified as
bacteriostatic agents.
In classifying the antibacterial activity as Gram-positive or Gram-negative, it would be expected
that a much greater number of drugs would be active against Gram-positive than Gram-negative
bacteria [39]. However, in this study, the title compounds are active against both types of the bacteria,
which may indicate broad-spectrum properties. It is seen that the activity of the title compounds is
affected by the nature of the substituents on the aromatic ring, this in relation to the lipophilicity of the
compounds and their membrane permeability, a key factor in determining their entry inside the cells.
The introduction of substituent (NO2) in the para-position of the benzaldehyde ring decreases
markedly the antibacterial activity. The antibacterial effect declines on going from unsubstituted
aminophenol to methoxy and finally nitro derivative. On the other hand, the variation in the
effectiveness of the studied compounds against different organisms depends either on the
impermeability of the cells of the microbes or on differences in ribosome of microbial cells.
4. Conclusion
In the present study, the synthesis of series of six aminophosphonates from Ortho and Para
aminophenol via Kabachnik-Fields reaction. Frontier Molecular Orbital (FMO) Analysis and
molecular electrostatic potential map of six α-aminophosphonates derivative from Ortho and Para
aminophenol, has been studied using DFT calculations. Antibacterial activity is also discussed. In vitro
antimicrobial screening of the synthesized of α-aminophosphonates showed good antibacterial activity
against some pathogenic Gram-positive and Gram-negative bacteria. The results also indicate that the
difference in the chemical structure of the α-aminophosphonates affects the microbiological activity.
The good activity of synthesized compounds 4b and 4e is probably attributed to the presence of
7

ACCEPTED MANUSCRIPT
pharmacologically active nitro groups attached to phenyl group of aromatic aldehyde. When the
substitution of these groups is replaced by hydrogen or methoxy groups, a sharp decrease in activity
against most of strains was observed. Compounds 4b and 4e exhibited better activity compared to that
of standard Chloramphenicol against all the bacterial strain. Further the result showed that Gram-
positive exhibited better activity than Gram-negative organism. The theoretical calculations carried out
for the studied molecules 4a-4f, are also conducted for analysis of electrostatic characteristics of
compounds. Inspection of the molecular electrostatic potential surfaces (MESP) and frontier molecular
orbitals (HOMO, LUMO) successfully explained their significant role in driving bioactivity. In
general, this study is used as example to illustrate how the electronic and geometric characteristics can
be useful in identifying all possible bioactivity of organic compounds. From the theoretical
calculations carried out for the studied molecules 4a-4f, it can be concluded that the electronic
properties are correlated well with their experimental results.
References
[1] R.A. Cherkasov, V.I. Galkin, Usp. Khim, 67, 940(1998).
[2] Yu. E. Vel’trishchev, E.A. Yur’eva, A.N. Kudrin, A.M. Koritmyi, O.G. Arhipova, N.V. Alekseeva, L.V.
Krinitskaya, V.K. Shcherbakov, E.A. Varsamovich Khim. Farm, Zh, 17, 282(1983).
[3] J. Oleksyszyn, V.P. Kukhar, H.R. Hudson (Eds.), Aminophosphonic and Aminophosphinic Acids
Chemistry and Biological Activity, John Wiley & Sons, Chichester, 537 (2000).
[4] D.D. Baird, Proc. North Cent, Weed Control Conf, 26, 64(1971).
[5] F.R. Atherton, C. H. Hassall, R.W. Lambert, J. Med. Chem., 29, 20(1986).
[6] A.M. Zomova, Zh.V. Molodykh, L.A. Kudryavtseva, L.V. Teplyakova, S.V. Fedorov, B.E. Ivanov, Pharm.
Chem. J., 20, 774 (1986).
[7] L.H. Jin, B.A. Song, G.P. Zhang, R.Q. Xu, S.M. Zhang, X.W. Gao, D.Y. Hu,; S. Song Yang, Bioorg. Med.
Chem. Lett., 16, 1537(2006).
[8] H. Kleszczyńska, J. Sarapuk, Cell.& Mol. Biol Lett., 6, 83-91(2001).
[9] (a) L. Maier, Phosphorus, Sulfur, Silicon Relat. Elem., 53, 43(1990). (b) Maier, L.; Diel, P. J. Phosphorus,
Sulfur, Silicon Relat. Elem., 57, 57(1991).
[10] R. Rawls, Chem. Eng News June 29th, 13(1998).
[11] F. Roberts, C.W. Roberts, J.J. Johnson, D.E. Kyle, T. Krell, J.R. Coggins, G.H. Coombs, W.K. Milhous, S.
Tzipori, D.J.P. Freguson, D. Chakrabarti, R. McLeod, Nature, 393, 801(1998).
[12] J. Oleksyszyn, J.C. Powers, Biochemistry, 30, 485(1991).
[13] D. Green, G. Patel, S. Elgendy, J.A. Baban, G. Claeson, V.V. Kakkar, J. Deadman, Tetrahedron Lett, 34,
6917(1993).
[14] (a) M. Ordóñez, H.Rojas-Cabrera, C. Cativiela, Tetrahedron, 65, 17-49(2009); (b) Z.H. Kudzina, M. H.
Kudzinb, J. Drabowiczc, C.V. Stevens, Curr. Org. Chem., 15, 2015-2071(2011); (c) M. Ordóñez,
J.L.Viveros-Ceballos, C. Cativiela, A. Arizpe, Curr. Org. Synth., 9, 310-341(2012); (d) M. Ordóñez, F.
J.Sayago, Cativiela, C. Tetrahedron, 68, 6369-6412(2012).
[15] A. Hellal, S. Chafaa, L. Touafri, Korean J. Chem. Eng., 33,2366(2016).
[16] K.R. Cruickshank , Medical Microbiology, A Guide to Diagnosis and Control of Infection, 2nd Ed. E.S.
Livingston Ltd., Edinburgh and London, (1968).
[17] A.W. Beuer, M. M. Kirby, J.C. Sherries and A.Truck, Am J Clin Pathol., 45, 493(1969).
[18] JR. Starke, Chapter 32: Infective Endocarditis. In: R.D. Feigin, J.D. Cherry (eds). Textbook of Pediatric
Infectious Diseases, 4th edition., Philadelphia: W.B. Saunders, pp. 315-338(1998).
[19] T. Yamauchi, J. Kamon, H. Waki, Nat. Med., 7,941–946(2001).
[20] P. Berche, J.L. Gaillard, M. Simonet, Flammarion Medicine Sciences, 64-71(1988).
[21] D. Gatsing, V. Tchakoute, D. Ngamga, J.R. Kuiate, J.D.D. Tamokou, Iran. J. Med. Sci. 34 :126-136(2009).
[22] W. Kohn and L.J. Sham, Phys. Rev., (140) 1133–1138 (1965).
[22] A.D. Becke. Density functional thermochemistry, The role of exact exchange. J.Chem.Phys.; 98: 5648-5652
(1993).
[24] C. Lee, W.Yang, R.G. Parr, Phys. Rev., (37), 785–789 (1998).
[25] B. Miehlich, A. Savin, A. Stoll, H. Preuss, Chem. Phys. Lett., (157), 200–206(1989).
[26] M.J. Frisch et. al., Gaussian 09, Revision A.1, Gaussian, Inc., Wallingford CT(2009).
[27] R.D. Dennington, T.A. Keith, J.M. Millam, GaussView 5.0. 8. Gaussian Inc.(2008).
[28] J.B. Foresman, A. Frisch, Exploring Chemistry with Electronic Structure Methods, Gaussian, Pittsburg, Pa,
USA, (1995).
[29] L. Pauling, The Nature of the Chemical Bond, Cornell University Press, Ithaca, NY, USA, (1960).
8

ACCEPTED MANUSCRIPT
[30] P. Senet, Chemical Physics Letters, 275, 5-6, 527–532 (1997).
[31] R.G. Parr, R.G. Pearson, Journal of the American Chemical Society, 105, 26, 7512–7516(1983).
[32] N. Choudhary, S. Bee, A. Gupta, P. Tandon, Comput Theor Chem., 1016: 8-21(2013).
[33] E. Kavitha, N. Sundaraganesan, S. Sebastian. Indian J. Pure Ap. Phy.,; 48: 20-30(2010).
[34] J. Fleming, Frontier Orbitals and Organic Chemical Reactions, John Wiley and Sons. New York. (1976).
[35] S. Chidangil, M.k. Shkla, P.C.Mishra, J.Mol. Model, 4, 250(1998).
[36] G. Pepe, D.Siri, J.P. Reboul, J.Mol.Struc. (Theochem), 256, 175(1992).
[37] S.Anubha, T.Poonam, AP. Ayala, J.Sudha, Vib.Spec., 56, 82(2011).
[38] K.Nam, J.Gao, D.M. York (2008).
[39] A.R. Mcutcheon, S.M. Ellis, R.E.W. Hancock, G.H.N. Towers, J. Ethnopharmacol. 37, 213–223(1992).
9

ACCEPTED MANUSCRIPT
Figure captions
R2
O
H
C
NH2
H
OC2H5
P O
R1
O P OC2H5
R2
OC2H5
NH
OC2H5
1
2
3
4
R1
4a: R1=p-OH, R2=H
4b: R1=p-OH, R2=NO2
4c: R1=p-OH, R2=OCH3
4d: R1=o-OH, R2=H
4e: R1=o-OH, R2= NO2
4f: R1=o-OH, R2= OCH3
Scheme 1. Synthesis of α-aminophosphonates (4a-4f) via Kabachnik-Fields reaction.

ACCEPTED MANUSCRIPT
4a
4b
4c
4c
4f
4e
Figure 1. Optimized structures of 4a–4f at the B3LYP/6-31G(d,p) level of theory..

ACCEPTED MANUSCRIPT
4a
HOMO
LUMO
4b
4c
4d
HOMO
HOMO
HOMO
LUMO
LUMO
LUMO
4e
HOMO
LUMO
4f
HOMO
LUMO
Figure 2. HOMO and LUMO surfaces of the 4a-4f at the B3LYP/6-31G(d,p) level of theory.

ACCEPTED MANUSCRIPT
4a
4b
4c
4d
4f
4b
Figure 3. Molecular electrostatic potential mapped on the isodensity surface for 4a-4f molecules
calculated at the B3LYP/6-31G(d,p) level of theory.

ACCEPTED MANUSCRIPT
TABLE CAPTIONS
Table 1.
Global chemical reactivity indices of 4a–4f.
Molecular Energy
ELUMO (a.u)
4a
4b
4c
4d
4e
4f
-0.00962
-0.21668
0.20706
0.21668
0.00962
0.10353
4.82951
-0.11315
0.06183
0.11315
-0.09118
-0.22856
0.13738
0.22856
0.09118
0.06869
7.27907
-0.15987
0.18604
0.15987
-0.00709
-0.21655
0.20946
0.21655
0.00709
0.10473
4.774181
-0.11182
0.05969
0.11182
-0.00606
-0.21536
0.2093
-0.08786
-0.22763
0.13977
0.22763
0.08786
0.069885
7.154611
-0.00362
-0.21535
0.21173
0.21535
0.00362
0.105865
4.722996
EHOMO (a.u)
Energy gap (∆)
Ionization Potential (I)
Electron affinity (A)
Global Hardness (η)
Global Softness (S)
Chemical potential (µ)
Electrophilicity (ω)
Electronegativity (χ)
0.21536
0.00606
0.10465
4.77783
-0.11071 -0.157745 -0.109485
0.05856
0.11071
0.178031
0.157745
0.056614
0.109485
A= -ELUMO , I= -EHOMO, η=1/2(ELUMO-EHOMO), µ=1/2(EHOMO + ELUMO), ω = µ2/2η, S = 1/2η, χ=(I+A)/2

ACCEPTED MANUSCRIPT
Table 2.
Antibacterial activity of 4a, 4b, 4c, 4d, 4e, 4f and Chloramphenicol.
Gram positive
Gram negative
E. coli P. aeruginosa
B. subtilis
S.aureus
DMSO
100
µg/ml
50
µg/ml
100
µg/ml
50
µg/ml
100
µg/ml
50
µg/ml
100
µg/ml
50
µg/ml
100
µg/ml
4a
11.6±0.4 21.4±0.6 12.7±0.0 18.4±0.8 11.0±0.7 16.4±0.0 10.4±0.8 15.9±0.8
20.4±0.5 32.6±0,4 19.6±0.9 31.0±0.7 13.9±0.7 29.4±0.8 15.9±0,5 28.0±0.5
09.0±0.1 17.8±0.5 11.4±0.0 14.0±0.8 10.4±0.0 16.3±0.0 12.9±0.2 14.8±0.5
09.4±0.6 16.8±0.5 10.4±0.0 13.3±0.0 11.0±0.7 15.0±0.1 11.2±0.0 15.6±0,5
19.9±0,4 31.8±0.0 18.4±0.0 30.4±0.1 13.4±0.9 28.3±0.0 15.0±0.8 27.4±0.6
10.4±0.8 15.4±0.0 10.4±0.0 .0±0.5 12.8±0.5 15.9±0,4 12.4±0.8 16.4±0.8
R
R
R
R
R
R
R
4b
4c
4d
4e
4f
Chloramphenicol
R= Resistance
30.8±0.0
27.5±0.9
31.8±0.0
27.5±0.9

ACCEPTED MANUSCRIPT
Table 3.
Minimum Inhibitory Concentration MIC and MBC of 4a, 4b, 4c, 4d, 4e and 4f.
B. subtilis S.aureus E. coli
MBC/MIC MBC/MIC MBC/MIC
P. aeruginosa
MBC/MIC
MIC MBC
MIC MBC
MIC MBC
MIC MBC
(Effect)
4 (+)
2 (+)
4 (+)
8(-)
(Effect)
(Effect)
(Effect)
4a
4b
4c
4d
4e
4f
16
8
128
16
16
8
128
16
8(-)
32
16
32
64
64
32
256
64
8(-)
32
32
16
32
32
64
256
64
8(-)
2 (+)
8(-)
4 (+)
8(-)
4 (+)
8(-)
32
8
128
64
16
32
8
128
128
32
256
256
128
128
128
256
64
4 (+)
4 (+)
8(-)
4 (+)
4 (+)
4 (+)
8(-)
8
16
2 (+)
8(-)
2 (+)
4(+)
32
256
32
256
256
(+): bactericidal effect, (-) bacteriostatic effect