Microwave assisted synthesis of 4-aminophenol Schiff bases: DFT computations, QSAR/Drug-likeness proprieties and antibacterial screening

Article abstract of DOI:10.1016/j.molstruc.2021.130666

The present work is devoted to the synthesis and study of the structural and electronic properties of three Schiff bases prepared from para-aminophenol under green chemistry conditions. These compounds were successfully synthesized in excellent yields. The synthesized Schiff bases have been characterized by Elemental analysis, FTIR, and NMR (¹H and ¹³C) spectroscopic methods. The molecular geometry, Mulliken atomic charges and vibrational analysis of the studied compounds were investigated theoretically by performing density functional theory (DFT) using the B3LYP/6-31G++(d,p) method. Moreover, these compounds were screened for antibacterial activity. These compounds were found active to be the most active against all the bacteria investigated. The antibacterial activities have been correlated with chemical reactivity parameters and other electronic parameters looking for any relationship between electronic parameters and biological activity. This effort just provides preliminary data for some biological properties. The results are very encouraging, so we can say that these products are biologically active and can be used later in other applications towards drug development.

Full text of DOI:10.1016/j.molstruc.2021.130666

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Microwave assisted synthesis of 4-aminophenol Schiff bases: DFT
computations, QSAR/Drug-likeness proprieties and antibacterial
screening
Mersellam Mohamed , Hellal Abdelakder , Benhamou Abdellah
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S0022-2860(21)00799-7
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Please cite this article as: Mersellam Mohamed , Hellal Abdelakder , Benhamou Abdellah , Mi-
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Microwave assisted synthesis of 4-aminophenol Schiff bases: DFT computations,
QSAR/Drug-likeness proprieties and antibacterial screening
1
*
2,3
1
Mersellam Mohamed , Hellal Abdelakder , Benhamou Abdellah
1
Environmental Process Engineering Laboratory (EPEL), Faculty of Chemistry, Med BOUDIAF, University of
Science and Technology of Oran, B.P 1505 Oran El-M'naouer 31000, Algeria.
Laboratoire d’Electrochimie des Matériaux Moléculaires et des Complexes (LEMMC), Faculté de Technologie,
2
Université Ferhat Abbas-Sétif-1, Alegria.
3
Laboratoire de Valorisation des Substances Naturelles, Université Djilali Bounaama-Khemis Miliana, Alegria.
E-mail : med.mersellem@gmail.com, Abdelkader.hellal@univ-setif.dz, abdellah.benhamou@univ-usto.dz
Abstract
The present work is devoted to the synthesis and study of the structural and electronic properties of
three Schiff bases prepared from para-aminophenol under green chemistry conditions. These
compounds were successfully synthesized in excellent yields. The synthesized Schiff bases have been
1
13
characterized by Elemental analysis, FTIR, and NMR ( H and C) spectroscopic methods. The
molecular geometry, Mulliken atomic charges and vibrational analysis of the studied compounds were
investigated theoretically by performing density functional theory (DFT) using the B3LYP/6-
3
1G++(d,p) method. Moreover, these compounds were screened for antibacterial activity. These
compounds were found active to be the most active against all the bacteria investigated. The
antibacterial activities have been correlated with chemical reactivity parameters and other electronic
parameters looking for any relationship between electronic parameters and biological activity. This
effort just provides preliminary data for some biological properties. The results are very encouraging,
so we can say that these products are biologically active and can be used later in other applications
towards drug development.
Graphical Abstract
OH
O
NH2
Conventional heating
N
OH
R
R=-H; -CH ; -NO
3
2
Microwave heating
R
1

Highlights

Three Schiff bases derived from 4-aminophenol were synthesized by microwave and
reflux methods.




These Schiff bases are characterized by different spectroscopic techniques.
Antibacterial properties of the synthesized compounds have been screened.
Theoretical studies were carried using DFT calculations.
QSAR study was conducted.
Key words: Schiff bases, Para-aminophenol, Microwave, DFT, QSAR, Antibacterial activity.
1
. Introduction
Microwave-assisted synthesis is a branch of green chemistry. The most widely described
applications of microwave technology in organic materials processing have been microwave assisted
organic synthesis. All microwave-assisted synthesis techniques can be considered an alternative to
conventional methods because they reduce: reaction time; costs; solvent consumption [1], toxic
solvents, and drastic conditions of temperature. Microwave-assisted organic synthesis (MAOS) is
considered to be a promising green chemistry approach because it is simple, clean, fast, efficient, and
economic for the synthesis of a large number of organic molecules [2]. It reduces reaction time from
hours to minutes or even seconds, and it helps reduce side reactions and increase yields, uses fewer
solvents or is almost solvent-free [3].
Schiff bases having a great deal of attraction since they have been first synthesized in 1864 by
Hugo Schiff [4]. Schiff bases are compounds with a functional group which contains a carbon-
nitrogen double bond with the nitrogen atom connected to an aryl or alkyl group. Schiff bases form an
important class of compounds in medicinal, pharmaceutical, agriculture, cosmetic products etc. They
have several biological applications that include antibacterial [5], antifungal [6], antitumor [7] and
anticancer [8], antipyretic activities [9]. They are also used as liquid crystals [10] in analytical [11],
medicinal [12,13], and polymer chemistry [14,15]. Besides their utility in phosphorus chemistry as
started materials, Schiff’s bases have been used for the preparation of α-aminophosphonate esters [16-
2
1]. In view of these applications of Schiff bases and in continuation of our investigations in the fields
of Schiff bases and α-aminophosphonates [22-30], here we report the green synthetic approach
towards the synthesis of biologically active compounds, including conventional and microwave
assisted methods, spectral and microbial characterization of a class of Schiff bases by the reaction
between 4-aminophenol and substituted aromatic benzaldehyde.
2
. Experimental
2
.1 Material and methods
All the reagents and solvents used for the synthesis and spectroscopic studies were purchased from
Merck and were used without any further purification. Melting points were recorded in the Thermo
Fisher scientific apparatus. The monitoring of the progress of the reactions of the synthesized
compounds was carried out by thin layer chromatography (TLC), performed on silica gel plates. The
UV-Vis spectra were recorded on the Schimadzu UV spectrometer in the wavelength range 200–800
2

nm. FTIR spectra were recorded on Schimadzu FTIR 8400S spectrometer in the wavelength range
-
1
4
7
000- 400 cm using KBr pellet. For microwave irradiation a Sumsung microwave oven, operating at
1
13
00W was used. The H-NMR (400 MHz; CDCl ) and C- NMR (100 MHz; CDCl ) spectra were
3
3
recorded on a Bruker Ultra Shield Plus-400 MHz spectrometer, operating 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 (multiplet). The percentages of
carbon, hydrogen and nitrogen were determined by Elemental Analyses using Perkin Elmer 2400
CHN Elemental Analyzer.
2
.2 Synthesis
The main way of direct access to these compounds is a one-pot reaction of two reagents, one of
nucleophilic character and the other of electrophilic nature, this reaction leads to the formation of
Schiff's base compounds. It involves a carbonyl compound (aromatic aldehydes), an amine (4-
Aminophnol), according to the following sequence (Scheme 1).

Synthesis of Schiff bases by Conventional method (Heating)
Schiff bases are prepared by condensation of various aromatic aldehydes (4ꢀmmol) with para-
aminophenol (4ꢀmmol) in ethanol (10ꢀmL) and the mixture was refluxed for 3-6 hours with vigorous
stirring. The progress of the reaction was monitored by TLC. On completion of the reaction the
product was separated as yellow-colored amorphous product which was filtered, dried, and
recrystallized from ethanol.

Synthesis of Schiff bases by Microwave method
A mixture of various aromatic aldehydes (4mmol) and para-aminophenol (4mmol) in ethanol
1mL) were added in sample microwave tube. The contents were subjected to microwave irradiation at
(
1
00ꢀW for about 4-8ꢀmin. Progress of the reaction was monitored by TLC. After the completion of the
reaction, solid product was obtained in reaction mixture which was filtered and recrystallized with
ethanol. Recrystallization provides the title compounds as solid crystals.
2
.3 Computational Details
All quantum chemical calculations for Schiff bases were performed using DFT method in Gaussian
9 software program [31].The geometry of all prepared compounds was optimized at the B3LYP level
0
of theory along with standard 6-31++G(d,p) basis set [32,33]. DFT calculations were computed to
fully optimize the ground-state structure of the molecule. The geometry of Schiff bases was optimized
to minimize the molecular energies. The calculated vibrational frequencies ascertained that the
structure is stable (no imaginary frequencies). Geometrical parameters (bond lengths and angles) and
FMOs levels were calculated by basis set pointed before. Mulliken atomic charges have been
calculated at the same level of theory. To predict the chemical reactivity, some theoretical descriptors
were determined. In particular, the energy of the lowest unoccupied molecular orbital (ELUMO), the
energy of the highest occupied molecular orbital (EHOMO), the electronegativity (χ), the global softness
(
S) and the global electropilicity index (ω). These descriptors are all determined from the optimized
molecules. All the convergent precisions were the system default values, and all the calculations were
carried out on a personal computer.
2
.4 QSAR/Drug-likeness proprieties
The structures of SB1-SB3 were first pre-optimized with the Molecular Mechanics Force Field
(
MM+) procedure included in Hyperchem 7.5 [34] and the resulting geometries were further refined
by means of the semi empirical method PM3. We selected a gradient norm limit of 0.01 kcal/A˚ for
the geometry optimization. The descriptors studied were Surface Area Grid (SAG), Volume (VOL),
3

Hydration Energy (HE), Refractivity (REF), Polarisibility (POL). Also, the drug likeness score was
calculated. Lipinski rule of five is important and widely used in pharmaceutical industries as a first
step to determine the best compound for drugs. This rule predicts the bioavailability of the compounds
by determining its physiochemical properties [35].
2
.5 Antibacterial activity
The antibacterial studies are done to find the efficacy of the synthesized 4-aminophenol Schiff
bases. Antibacterial sensitivity of different compounds was studied by disc diffusion method [36].
Different concentration (50-100mg/ml) of the Schiff bases and standard, Amoxicillin, were prepared
in DMSO and tested for its antimicrobial strain such as Bacillus subtilis and Staphylococcus aureus
(
Gram positive bacteria) and Klebsiella pneumonia and Pseudomonas aeruginosa (Gram negative
bactetia). Two paper discs, one impregnated with Amoxicillin (100mg/ml) and other with DMSO were
used as positive and negative control, respectively. Triplicate plates for each microorganism strain
were prepared and incubated at 37◦C for 24 h. The inhibition zone was measured around the disc to
determine the activity and the average value was taken as a final result.
2
.6 Determination of MIC and CMB using the macro-dilution method
The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
values,as well as MBC/MIC ratios of the prepared Schiff bases were determined. The MIC is, the
lowest concentration of the agent that inhibits visible growth; and the MBC is the lowest concentration
of an agent which kills a defined proportion (usually 99.9%) of the population after incubation for a
set time [37]. Serial dilutions of the tested agents are made in a liquid microbial growth medium which
is inoculated with a standardized number of organisms and incubated for a prescribed time. At the end
of the incubation period (generally 18-24 hours), the tubes are visually examined for turbidity. BS1,
BS2 and BS3 were used from a concentration of 0.25μg/ml to 128μg/ml. A control test-tube
containing only medium (nutrient broth medium) was used to confirm the sterility of the medium.
7
Bacterial suspension (10 μl) containing 10 cells/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 for
MBC. 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 96 hours. After
four days, bacterial growth was observed and MBC was determined at lowest concentrations where no
bacterial growth was observed [38].
3
. Results and discussion
3
.1 Synthesis
Keeping in view the importance of Schiff bases, a series of simple and substituted 4-aminophenol
based Schiff bases were synthesized by two different methods (conventional and microwave irradiated
methods). For optimization of a chemical reaction, it is important to consider the various factors that
govern the overall reaction. Herein, nature of solvents, ratio of reactants and time were considered as
the three significant independent factors. The response would be dependent factor i.e. percentage
yield. All the reactions have optimized by changing reaction time and solvent, and here impact on %
yield presented for the compound (BS1) in (Table 1). It was found that the yield was up to 72% when
the reaction mixture was refluxed for 3h in ethanol. Similarly the synthesis of (BS2) and (BS3) was
also attempted under the same conditions. In microwave assisted synthesis, the mixture of 4-
aminophenol, with various aryl aromatic aldehydes are irradiated in an unmodified domestic
microwave oven (100W) for 4-8ꢀmin. to yield the range of desired product depicted in Table 2.
Optimal reaction time for the synthesis was 4-8 min. at 100W of microwave irradiation power.
4

The impact of microwave irradiation and conventional heating in the synthesis of Schiff bases
compounds have been compared and summarized in Tables 2. The study clearly showed that all the
steps involved in this synthesis resulted in higher yields under microwave conditions than the
conventional method. The reaction carried out using conventional method required about 3–6hr, while
microwave irradiation method required only 8min. In conventional method, reaction mixture is heated
using an external heat source and therefore heat transferred through conductance. This method was
slow and inefficient method for transferring energy into the system because. However, the MW
method is simple and it gives good yields within a few minutes. The microwave irradiation produces
efficient internal heating by direct coupling of microwave energy with polar molecules. This method
was rapid, economic and gave better yields as compared to conventional method.
The synthesized compounds are amorphous solids, stable in air, soluble in DMSO, methanol,
ethanol, acetonitrile and chloroforme. The structures of the compounds were confirmed by elemental
analysis (quantity of carbon, hydrogen and nitrogen in each compound was determined) and FTIR
spectra (wave numbers of some specific groups were indicated). The proposed molecular formulas for
prepared compounds are in agreement with analytical data (C, H and N) together with spectroscopic
1
13
data namely FTIR, H-NMR and C-NMR. Infrared spectra considered a powerful technique to give
full information to elucidate the structure of the molecule. The FTIR spectra provide valuable
information regarding the formation of Schiff bases. The infrared spectra of Schiff bases shows band
-1
at 3400-3300cm assigned to OH vibrations of the functional (-OH). All compounds show strong
-
1
1
bands at 1610-1680 cm attributed azomethine (-C=N-) stretching vibration. H-NMR spectral data of
1
Schiff bases were recorded in DMSO-d . The H-NMR spectrum of BS1 showed two singlet signals
6
for methyl protons at δ 3.90 ppm and for azomethine proton at δ 8.60ppm along with multiplet in the
1
region δ 6.98-7.82ppm for aromatic protons. The H-NMR spectrum of BS2 and BS3 showed singlet
1
signals of the azomethine proton signals at δ8.86 and 8.94 ppm, respectively. H-NMR spectrums of
BS2 and BS3 showed multiplet signals in the region δ 7.00-8.90 ppm attributed to aromatic
1
3
protons.The C-NMR spectra data of Schiff bases were recorded in DMSO. The signals appeared at
δ160ppm and ~δ177ppm, respectively for the carbon atoms of aromatic and azomethine groups in the
~
Schiff bases.
The spectra data of the compounds are as follows:
-
1
4
-(Benzylidene-amino)-phenol: mp. 122-129°C, FTIR (KBr) cm : 3400-3300 (-OH str), 1610 (-C=N
1
13
str); H-NMR (DMSO-D , 400MHz): δ 8.60 (s, 1H, N=CH), 6.98-7.82 (m, 4H, Phenyl ring). C-NMR
6
(
DMSO-D , 300 MHz): δ 156.70 (N=C); Anal. calcd. (%) for C H NO %C:79.16; %H: 5.62;
6
13 11
%
N:7.10; found: %C:79.00; %H: 5.77; %N:6910.
0
-1
4
2
(
-[(4-Methyl-benzylidene)-amino]-phenol: mp.133-135 C, FTIR (KBr) cm : 3400-3300 (-OH str),
1
820 (-CH str) 1644 (-C=N str); H-NMR (DMSO-D , 400 MHz): δ 8.86 (s, 1H, N=CH), 7.84-7.40
m, 4H, Phenyl ring), 1.25-1.33(s, 3H, CH3). C-NMR (DMSO-D , 300 MHz): δ 158.20 (N=C);
6
3
6
13
Anal.calcd. (%) for C H NO %C:79.59; %H:6.20;%N:6.63; found: %C:80.02; %H:6.50;%N:6.55.
14
13
0
_1
-1
4
-[(4-Nitro-benzylidene)-amino]-phenol: mp.80-85 C, FTIR (cm ): FTIR (KBr) cm : 3400-3300 (-
1
OH str), 1678 (-C=N str); H-NMR (DMSO-D ): δ8.94 (s, 1H, N=CH), 7.87-7.60 (m, 4H, Phenyl
ring), 7.50-7.02 (m, 4H, Phenyl), C-NMR (DMSO-D , 300 MHz): δ δ 161.02 (N=C); Anal. calcd.
6
13
6
(
%) for C H N O %C:64.46; %H:4.16; %N:11.56, found: %C:63.96; %H:4.02; %N:11.76.
13 10 2 3
3
.2 Computational Results
Density Functional Theory (DFT) has become an effective tool for determining structure,
electronic properties of molecules, vibrational frequencies, atomization energies, ionization energies
5

etc. In present work, theoretical investigations were performed using the DFT at B3LYP level of
theory at 6- 31++G(d,p) in the Gaussian 09W program.
3
.2.1 Optimization of structures
For the calculations, the title compounds were first optimized by DFT methods with 6-31G++(d,p)
basis set by minimizing energy (The most stable structures correspond to the lowest energy). The
optimized structures with labeling of atoms are given in Fig.1. Optimized geometrical parameters like
bond lengths, bond angles were determined and listed in Table 3-4. The molecules were constructed
using the Gaussview software package. The optimization of the structures of the molecules (BS1, BS2
and BS3) allowed us to see and compare the effect of the substitution of the groups (hydrogen, methyl
and nitro) in the same position on the structural properties (geometry). The comparison of the values
of the bond lengths and some angles of the three molecules allowed us to make the following remarks:
It should be noted that the introduction of group CH (BS2) caused a negligible change with respect
3
to the radical H (BS1) for all the bonds. This can be explained by the similarity of effect between these
two substituents. For aromatic cycles; the introduction of the nitro group allows a reduction of the
bond lengths of with respect to the H and CH , this may be due to their electro-attractor character. For
3
⁰
⁰
⁰
the imine function, it is noted that the lengths of the C=N bonds are 1.2818A , 1.2823A and 1.2820A
for BS1, BS2 and BS3, respectively. So we notice that it is shorter for the BS1 and longer for the BS2.
This can be explained by the electro-donor effect which is more important in the case of CH .The
3
carbon-carbon bonds of phenyl ring are different from those of alkanes, alkenes and alkynes. The
lengths of the C-C bonds are equal and measure ~140pm. The carbon chain thus forms a regular
hexagon. In our case, there are no big differences between the values calculated by this method and the
values declared in literature. This justifies the good choice of the method). In the literature, the carbon-
hydrogen bond lengths are equal and measure 105μm. In our case, we find that the aromatic C-H have
lengths of 1.0848A. This difference may be due to the effects of the substituents. The nitro group
caused a slight increase in the O-H bond of 0.0003A relative to H and 0.0002A relative to CH3.
All bond angles C-C-C and C-C-H are equal to ~120°. The benzene ring is a regular plane
hexagon; the hydrogen atoms also belong to the plane of the cycle. The angle of the intermediate chain
C3-N13-C14 has variable values (120.7338° for BS1, 120.6489° for BS2 and 121.435° for BS3)
whereas the angle N13-C14-C16 has different values (122.5326° for BS1, 122.6041° for BS2 and
1
21.7752° for BS3). It is noted, on the one hand, that the molecules BS1 and BS2 have almost the
same values, on the other hand BS1 and BS2 have angles C3-N13-C14 less than BS3 (121.435°),
against the latter has an angle N13-C14-C16 less than the other two. This can be explained by the
electro-attracting effect of the Nitro group which is closer to this bond. In geometry, the angle between
two planes is called dihedral angle.
3
.2.2 Vibrational Spectral Analysis
After optimization, the vibrational frequencies of BS1-BS3 were calculated using the same method.
Vibrational frequencies calculations were carried out by the VEDA 4 (Vibrational Energy Distribution
Analysis) program [39].The basic modes of vibration of each compound that are calculated according
to the rule (3N-6 ). For the molecule BS1 we find 72 ((26*3)-6= 72) and the molecule BS2 we find
vib
8
1 ((29*3)-6= 81) and the molecule BS3 we find 78 ((28*3)-6= 78). All vibrations are active in IR. All
frequencies are assigned in terms of fundamental, harmonic and combination bands. The vibration
frequencies are calculated with this method, and then scaled by the factor 0.9679. Theoretical and
experimental infrared spectra of BS1, BS2 and BS3 are presented in Fig.2. Frequencies observed and
calculated using DFT / B3LYP with base 6-31G (d, p) with their likely attributions of BS1, BS2 and
BS3 are summarized in Table 5. Some discrepancies were observed between experimental and
6

calculated vibration frequency data and their attributions were also discussed. The objective of
vibration analysis is to find vibration modes related to specific molecular structures of computed
compounds. Table 5 shows the calculated and observed vibrational frequencies and their assignments
-
1
of BS1, BS2 and BS3 in the range 600-4000 cm .The normal mode assignments have been discussed
under the following sections:

Phenyl ring modes
-1
The Phenyl ring structure shows the presence of C-H stretching vibrations in the region3100cm -
-1
3
4
000 cm which is characteristic region for the ready identification of C-H stretching vibrations [40-
2]. In general the aromatic C-H stretching vibrations calculated theoretically are in good agreement
with the experimental values. The title molecules have out-of plane and in-plane aromatic C-H
bending vibrations. The out-of plane bending mode of C-H is found well with theoretical calculated
-
1
-1
values in the region 900–800 cm at B3LYP /6-31G(d,p). The observed value of 840cm is in
-
1
excellent agreement with 841 cm of B3LYP/6-31 G(d,p) results. The C-H in-plane bending
vibrations assigned in the region980-1260 cm even though found to be contaminated by C-CH (SB2)
-1
3
stretch are found in literature [43,44], while the experimentally observed values are at 1065 and1263
-
1
cm . The C-C stretching vibrations are significantly dominant in spectra of aromatic compound and it
has the characteristic of the aromatic ring. The C-C stretching vibrations of aromatic structures cover
-1
-1
the spectral range from 1500–1300 cm . The IR bands observed at 1495 and 1330 cm have been
assigned to C-C stretching vibrations. The calculated bands at B3LYP levels in the same region are in
excellent agreement with experimental observations. The ring in plane vibrations has given rise to
-1
-1
-1
weak bands across the low region, that is to say 1000 cm . The bands are 900cm and at 820 cm
have been assigned to C-C in plane bending vibrations. As is seen from Table 5, the predicted
frequencies by B3LYP agree well with the observed ones.

O-H group modes:
The hydroxyl stretching and bending bands can be identified by their broadness and strength of the
band which is dependent on the extend of hydrogen bonding. The O-H stretching vibration is pure and
highly localized mode. The O-H stretching vibrations are demonstrated by strong absorption bands in
-
1
-
3
.
700-3400 cm region [45]. In this study, the O-H stretching vibration was obtained as 3690-3600 cm
1
Generally, O-H in-plane bending stretching vibrations are found with C-H in-plane and C-C
stretching vibrations. O-H in-plane bending stretching vibrations demonstrated in 1617- 1114 cm
region [46,47]. In-plane bending stretching vibrations were obtained as 1566, 1505, 1460, 1385, 1374,
-
1
-1
1
318, 1274, 1230, 1150, 1123, 1066, 976 cm . O-H out-of-plane bending vibrations demonstrated as
-
1
-1
in 450-350 cm region [48,49]. Out-of-plane bending vibration was obtained as 359 cm . It has been
verified that the experimental and calculated O-H vibrations are in accordance with the previous
studies.

CH group modes (SB2)
3
The SB2 molecule possesses a CH group in the side substitution chain. For the assignments of
3
CH group, the C-H stretching’s at lower frequencies than those of the aromatic ring. For CH
3
3
-1
compound the mode appear in the region 3000 and 2850 cm are assigned to CH stretching mode of
3
vibrations. The theoretically computed value shows an excellent agreement with experimental results.
The CH deformations are found in FTIR spectra are in good agreement with the calculatedB3LYP
3
results. The torsion vibrations are not observed in the FTIR spectrum because these appear at very low
frequency.

NO group modes (SB3)
2
7

There are six modes of vibrations due to the NO group , namely, asymmetric NO stretching -
2
2
ν (NO ),symmetric NO stretching-ν (NO ), NO rocking-ρ(NO ), NO wagging-ω(NO ), NO
2
as
2
2
s
2
2
2
2
2
-
1
-1
scissoring -δ(NO ) and NO torsion-τ(NO ),which correspond to the frequencies 1511 cm , 1226 cm ,
5
2
-1
2
2
-1
-
1
-1
42 cm ,683 cm , 819 cm and 74 cm respectively. The theoretically computed value shows an
excellent agreement with experimental results.

C-N vibrations
−
1
−1
Frequency at 1600 cm indicates C=N double bond while frequency 1300 cm indicates the
presence of CN single bond [50]. The CN bands, which are imine groups, were observed at 1640 and
1
−
1
−1
590 cm . Similarly the out-of-plane bending vibrations were calculated at 460 cm .The calculated
value are in excellent agreement with the observed value forthe corresponding mode of vibrations.
3
.2.3 Mulliken atomic charges
The atomic charge in molecules is fundamental to chemistry. Mulliken atomic charge calculation
plays an important role in the application of quantum chemical computations to molecular systems,
because the atomic charges affect the dipole moment, polarizability, electronic structure, and much
more properties of the molecular systems [51]. The charge distribution in atoms suggests donor and
acceptor formation involving charge transfer in the molecule. Atomic charge has been used to describe
electronegativity and charge transfer in chemical reactions [52,53]. Mulliken atomic charges (in unit of
e) at various atomic sites of BS1-BS3 calculated by the B3LYP/(d,p) method, are collected in Table 6
and represented in Fig.3, from which it can be noticed that all the carbon atoms, except C3, C6, C14
and C16, have negative Mulliken atomic charges. However, it can be see an electronegative atom like
N13 and O11 atoms. Oxygen (O11) has a maximum negative charge value of about -0.6 in the OH
group. The presence of large negative charge on O and N atoms and net positive charge on H atom
may suggest the formation of intermolecular interaction in solid forms [54]. All the H atoms possess
positive Mulliken atomic charge. The maximum positive atomic is obtained for C14 which is a carbon
present in the C=N functional group.
3
.2.4 HOMO-LUMO Analyses
The frontier molecular orbital’s (FMO) plays a significant role in the electrical and optical
properties and also in chemical reactions. The HOMO represents the ability to donate electrons, while
LUMO as an electron acceptor [55]. This electronic absorption corresponds to the transition from the
ground to the first excited state. Highest occupied molecular orbital (HOMO) and lowest unoccupied
molecular orbital (LUMO) are been computed using B3LYP level 6-31++(d,p) of DFT. The 3D orbital
diagrams of the HOMO and LUMO frontier for the three molecules are shown in Fig.4. The green and
red regions show MO with totally opposite phases. The positive phase of the molecule is represented
in red color and negative phase in green color. In BS1-BS3, it is found that the highest occupied
molecular orbital is localized mainly on all atoms, whereas the LUMO is also relocated on the whole
of the p-conjugate system, which also indicate that, the electron density transfer from the HOMO to
the LUMO are mainly derived from the electronic transitions of n →π*. The computed energy data of
these molecular orbitals is shown in Table 7. Theoretical calculations showed that the highest
molecular energy among compounds BS1, BS2 and BS3 was shown by the unsubstituted compound
BS1(-631.989au) and that there was relationship between the electronegativity of substituent groups
and the molecular energies of the compounds. Compound BS3 bearing the nitro substituent, had the
lowest molecular energy (-836.489au).
The difference between HOMO and LUMO orbital is called as energy gap ΔE that is an important
parameter of stability for structures. The energy gap (ΔE) represents the chemical reactivity of the
compounds. For a system, the lower value of ΔE makes it more reactive or less stable. The
8

calculations show that the energy gap (ΔE) of BS1, BS2 and BS3 are, respectively, 0.14768au,
.14788au and 0.11861au. As shown in Table 7, compound BS3 has a lower energy gap than BS1 and
0
BS2, respectively. The order of reactivity is: BS3>BS2>BS1. So BS3 is more reactive and less stable
than the other two molecules, this may be due to the existence of the nitro group.
3
.2.5 Global Reactivity Descriptors
In order to investigate the influence of electronic effect of substituents on the global reactivity
–
descriptors, such as global electrophilicity (ω), electrodonating power (ω ), electroaccepting power
+
±
(
ω ), and net electrophilicity (Δω ), we were interested in the effects of the CH electrodonating and
3
NO electrowithdrawing substituents.
2
Using the HOMO and LUMO energy values for a molecule, the descriptors of the global chemical
reactivity of molecules such as hardness (η), chemical potential (μ), softness (S), electronegativity (χ)
and the electrophile index (ω) can be measured. Considering the Koopman’s theorem, the following
expressions can be used to define the global reactivity descriptors, like the ionization potential (IP) and
electron affinity (EA) are the Eigen value of the HOMO and LUMO with change of sign [56]:
IP = -EHOMO
EA = -ELUMO
(1)
(2)
The Ionization Potential of a molecule is the energy that must be supplied to a neutral atom to pull
out an electron and form a positive ion. While, Electronic Affinity refers to the ability of a neutral
atom or molecule to capture an extra electron. Molecule with a low (IP) value has a better electron
donor character, those with a high (AE) value a good electron acceptor character. The ionization
potential (IP) of the BS1, BS2 and BS3 molecules calculated by the B3LYP / 6-31G++(d, p) method is
0
.20177, 0.19888 and 0.21796a.u., respectively. The ionization potential value of the BS2 is lower
than those of the BS1 and BS3. This indicates that BS2 (electrodonating) is likely to give up electrons.
The electronic affinity (AE) for the three molecules is 0.05753, 0.05144 and 0.10095a.u. respectively
for BS1, BS2 and BS3. The value of electron affinity calculated for BS3 is significantly greater than
those of BS2 and BS1. This indicates that this latter one (with NO electrowithdrawing) is the unique
2
good electroacceptor. But all the BS2 and BS1 compounds studied have shown to be bad electron
acceptor.
Absolute hardness (η) and Softness (σ) are essential properties to measure the molecular stability
and reactivity. Chemical hardness measures the resistance to change in the electron distribution or
charge transfer. In this sense, chemical hardness corresponds to the gap between the HOMO and
LUMO. The larger the HOMO–LUMO energy gap, the molecule is harder and more stable/less
reactive [57,58]. The chemical hardness fundamentally indicates the resistance towards the
deformation or polarization of the electron cloud of the atoms, ions or molecules under small
perturbation of chemical reaction. Softness is a property of molecules that measures the extent of
chemical reactivity. It is the reciprocal of hardness. Softness and hardness are calculated by:
η = ELUMO-EHOMO
(3)
(4)
σ = 1/η
Table 8 contains the computed chemical hardness and softness values for compounds BS1-BS3. By
analyzing the results, it can be seen that BS1 has more hardness, low softness and less reactivity than
the other compounds.
Electrophilicity index (ω) is the measure of the electrophilic tendency of a molecule; it measures
the capacity of a species to accept electrons. It is a measure of the stabilization in energy after a system
9

has accepted additional amount of electronic charge from the environment. In 1999, Parr defined the
electrophilicity (ω) index as a measure of energy lowering due to maximal electron flow between
donor and acceptor. The Electrophilicity index is calculated using the formula [59]:
2
ω = μ /2η ...................(5)
Results from Table 8 shows that BS3 with high electrophilicity index value (0.21222a.u.) than the
other compounds. High values of electrophilicity index increases electron accepting abilities of the
molecules. Thus, electron accepting abilities of compounds 8-12 are arranged in following order: BS3
>
BS1 > BS2. Substitution of a hydrogen atom of BS1 by an electron-withdrawing nitro group
increases the electrophilicity ω index to 0.21222a.u. Domingo et al. established an electrophilicity (ω)
scale for the classification of organic molecules as strong electrophiles with ωꢀ>ꢀ1.5ꢀeV, moderate
electrophiles with 0.8ꢀ<ꢀωꢀ<ꢀ1.5ꢀeV, and marginal electrophiles with ωꢀ<ꢀ0.8ꢀeV [60]. From Table 8, all
molecules, BS3 (ω=5.774eV), BS1 (ω=3.015eV) and BS2 (ω= 2.872eV), presenting an
electrophilicity (ω) index higher than 2.0 eV. They are Strong electrophiles and participate easily in
polar processes.
The chemical potential is used to determine the direction of the electron transfer during a
condensation between two molecules; it is calculated by the formula:
μ = (EHOMO + ELUMO)/2
(6)
The greater the electronic chemical potential, then the compound is more stable or less reactive
than the other compounds. Results of chemical hardness and electronic chemical potential showed that
BS1 and BS2 are harder and less reactive than BS3.
Electronegativity is the measure of an atom, molecule or solid substance to attract electrons to
itself. The first connection of the electronegativity (χ) concept with quantum mechanics within
density-functional theory (DFT) is assigned to Parr et al. [61], where:
χ = −µ =(IP+EA)/2
(7)
Where µ is the chemical potential in DFT [62].
Higher is the electronegativity of the species, greater is its electron accepting power or rather the
electrophilicity. The Table 8 shows the order of electronegativity as BS3> BS1 >BS2. According to
these results, BS3 have the greater electron accepting power.
From theoretical calculations established, it was found that the BS3 possessed the lowest energetic
gap (ΔEgap =0.11861a.u.), so it is the softest molecule (S=8.4309a.u.) and it is the best to be easily
excited by against. Conversely, compound BS2 has the highest energy gap (ΔE =0.14788a.u.), so it
gap
is the hardest molecule (η =0.0739a.u.).
3
.2.6 Electric dipole moments
Density functional theory (DFT) calculation has had huge success as a tool capable of predicting
important physical and chemical properties. We calculate the electric dipole moment of a molecules
BS1-BS3. Dipole moments are important to understand the intermolecular interactions. The dipole
moment is mainly used to study intermolecular interactions such as dipole-dipole forces of Van der
Waals type, etc. The magnitude of the dipole moment is a description of the degree of polarity of the
molecule and, as such, is an important quantity in intermolecular interactions, including those
associated with hydrogen-bond formation and other related dipole-dipole and ion-dipole interactions.
The moment of the total dipole can be calculated using the following equation.
2
2
2 1/2
μ = (μ + μ + μ ) ........................ (8)
tot
x
y
z
1
0

Table 9 presents the components of the moment {μi (i = x, y, z)} and the total dipole moment μtot
for the three compounds BS1, BS2 and BS3. Table 9 shows that the calculated values of dipole
moments in the case of BS3 (6.0987Debye (D)) are quite superior to BS2 (0.9527D) and BS1
(
0.6667D). The reason is that NO is very strong acceptor (-M, -I) group and creates bigger opposite
2
charges on molecule. The electron-withdrawing behavior of nitro group in BS3 reflected in dipole
moments. In contrast, the 휋-donor character of methyl group (+R) is required in order to explain the
raise of dipole moments of BS2 through methyl substitution (Fig.5). The highest value of the dipole
moment is observed for the μ component. In this direction, this value is equal to 6.0532D for BS3.
x
Comparison between compounds BS2 and BS3 showed that the methyl substituent decreased the
dipole moment.
3
.3 Antibacterial activity
All compounds were screened in vitro for their antibacterial activity against 4 important Gram-
positive and Gram-negative pathogens bacteria; B. subtilis (ATCC 6633), S. aureus (ATCC 25923), K.
pneumonia (ATCC 33499) and P. aeruginosa (ATCC 27853). Using disc standard drugs such as
Amoxicillin. DMSO was used as standard and the activity is expressed as zone of inhibition in mm. It
o
is reported as the average of three readings. The plates were incubated for 24h at 37 C and zones of
inhibition for each microbial growth were measured relative to control DMSO.
The results of the antibacterial assessments of the BS1-BS3, with positive and negative controls,
are presented in Table 10. In this research, results showed that all the tested compounds could be
characterized by antibacterial properties with the acceptable concentration-dependent inhibition effect
against all bacteria used in study. A comparative zone of inhibition (mm) for Schiff bases and standard
drug are reported, from which it is clear that BS1-BS3 showed good to moderate antibacterial activity
in comparison with standard drug. However, BS2, compound with the methyl group in their structure,
showed a higher antibacterial activity against the all bacterial strains as compared to BS1 and BS3.
BS2 had strong activity against S. aureus, B. subtilis, K. pneumonia, and P. aeruginosa, respectively.
None of the tested compounds showed activity against Pseudomonas aeruginosa except one
compound (SB2) showed low activity at 100μg/ml. It should be noted that the compound (BS2) was
found to be the most active of test compounds against the microbial cultures tested. Comparison of the
structure and activity of compounds BS3, BS1 and BS2 revealed that by incorporating the methyl
group into the structure (BS1), activity against bacteria was ~2.45(B. subtilis), 2.25 (K. pneumonia),
2
.65 (S. aureus), and 3.25(P. aeruginosa) times higher. All compounds showed the most prominent
effect on Gram-positive bacteria, while the lowest inhibitory activity was noticed for Gram-positive
bacteria. In antibacterial research, a pronounced distinction can be made between the sensitivity of
Gram-negative and Gram-positive bacteria, where Gram-negative organisms tend to be less sensitive
on average. This is due to the presence of an outer membrane (OM) and a hydrophilic periplasmic
space in Gram-negative bacteria. The permeability properties of this barrier, therefore, have a major
impact on the susceptibility of the microorganism to antibiotics [63].
An antibacterial molecule requires special physicochemical properties to access the site of action
inside the bacterial cell. The cell wall represents a complex barrier to the entrance of compounds.
Gram-positive bacteria possess a thick peptidoglycan layer and, in the case of Gram-negative bacteria,
in addition to the inner membrane and the peptidoglycan layer, an outer membrane represents an
additional barrier for many small molecules [64] is correlated with their solubility in nonpolar solvents
and their solubility in water [65]. To cross the bacterial membrane, the small molecule is first solvated
in the extracellular media, immersed in the alkylated part of the lipid bilayer, finally diffusing to the
cytoplasmic side, where it is again solvated. This behavior can be related experimentally with the
partition coefficient of the compound in a non-polar/aqueous phase, and theoretically with the
1
1

lipophilicity. There is a strong possibility that the antibacterial activity in this series of compounds
BS1-BS3) can be correlated with their ability to penetrate biological membranes, which is dependent
(
on lipophilicity factors. A correlation between biological activity and hydrophobic character is
frequently observed. A compound whose site of action is inside the cell must interact with the cell
membrane to gain access to its target in the required critical concentration, and a lipophilic moiety is
important for this interaction [66]. The partition coefficient (logP) of a compound in the n-
octanol/water system represents the hydrophobic properties of this compound. The replacement, in an
active molecule, of a hydrogen atom by a substituent or functional group can deeply modify the nature
of the pharmacological effect. Generally, a change of functional group or a substituent on a phenyl
ring has a major effect on the lipophilicity of the molecule and therefore influences the absorption,
distribution or excretion of compounds. Substitution of hydrogen by methyl groups (methylation)
significantly reduces hydrophilicity and increases the lipophilicity and thus increases the rate of cell
penetration, which is an important feature of antimicrobial drug efficiency [67].
3
.4 Determination of MIC and MBC
MIC and MBC values of BS1-BS3 compounds against all tested pathogenic bacteria are shown in
Table 12. Conventionally, MIC is determined using a series of dilutions of the samples in liquid
culture medium, to produce a range of concentrations in test tubes (macrodilution). After inoculation
of the test strain into each sample concentration, bacterial growth is determined by visible turbidity
after 18–24 h of incubation. For the synthesized Schiff bases, MIC values ranged between 2 and
3
2μg/ml against gram-positive and gram-negative, and MBC values, between 16 and 128μg/ml.
Almost as a rule, MBC values were several folds higher than those of MIC, suggesting a better
inhibitory than bactericidal activity. BS3 presented higher MIC and MBC values than the other
evaluated. This compound showed weak to moderate activity against both Gram-positive and Gram-
negative bacteria (MIC between 8 and 128μg/ml and MBC between 16 and 128μg/ml). Antibacterial
potency of BS1 and BS2 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 BS2 was confirmed by the lowest MIC and MBC values
(
2-16μg/ml) observed against all tested microorganisms. The CMI and CMB are two parameters for
calculating the CMB/CMI ratio. This report makes it possible to characterize a bactericidal,
bacteriostatic action or to determine the "tolerance" of a bacterial strain. Antibacterial substances are
considered as bacteriostatic agents when the ration MBC/MIC>4 and bactericidal agents when the
ratio MBC/MIC≤4. The microcide and microbiostatic effects of BS1-BS3 are summarized in Table 11.
In the present study, BS1 and BS2 showed the ratio MBC/MIC≤4, suggesting that these compounds
act as bactericidal agents on these strains. BS3 showed MBC/MIC ratio of more than 4, which may be
classified as bacteriostatic agent.
3
.5 Drug-likness proprieties
Based on Lipinski rule, an oral active drug is better to have Log P < 5 with the molecular weight <
00 and molar reactivity from 40 to 130. In this research, the structures possess the required
5
parameters and might be considered as orally active compounds. Careful examinations of Table 12,
suggests that our synthesized structures have the proper characteristics such that the molar mass are <
5
00 and the molar reactivity are between 40 and 130 for our synthesized structures. As a conclusion,
our synthesized structures would meet the requirements as orally active compounds.
3
.6 Correlation physicochemical parameters Antibacterial activity
This study was used to correlate antibacterial activity of the obtained compounds into their
physicochemical parameters. With regard to molecular properties, further investigations by the
1
2

computed reactivity descriptors (See Table 8) were performed for these structures. It is well known
that the antibacterial activity is related to intermolecular interactions between the drug and receptor.
Table 8 shows the values of the calculated global chemical reactivity parameters for the synthesized
compounds. The chemical reactivity values vary with the molecular structure and the substituent. In
this investigation, the Inhibition zone values of synthesized Schiff bases were computationally related
to their physicochemical descriptors.
Furthermore, the EHOMO is a quantum parameter which reflects the ability of molecule to electron
donation. The ranking of the studied compounds according to increasing ELUMO and EHOMO as well as
increasing their biological activity is BS3<BS1<BS2; (EHOMO). On other hand, the ELUMO determines
the reactivity of molecule as electron acceptor species where the low LUMO energy indicates the
higher ability to accept electrons. The ranking of the studied compounds according to increasing
ELUMO is BS3< BS1<BS2; (ELUMO). Being related to polarity, the dipole moment is a component of the
partitioning energy, which is directly proportional to log P. The dipoles with the cell membrane to
induce electromechanical changes in the cell membrane which facilitate permeation. Cafiso [68], has
suggested that a significant proportion of changes to the dipole potential of lipid membranes may
involve electrodynamic alignment of the dipoles of bound water molecules, as well as the dipoles of
charged drugs. Total dipole moment (µ ) imitates the ability of interaction of compounds with the
tot
surrounding medium. In this series of compounds the dipole moment of (BS3) is greater than other
tested compounds (µ =6.1467D) revealing that it would have higher ability of interaction with the
tot
surrounding medium. It also showed that (BS3) has more binding ability resulting in less antibacterial
activity.
4
. Conclusion
In this study, three Schiff base derivatives from 4-aminophenol were synthesized by using the
microwave and reflux methods. These compounds were obtained in a short time and good yields by
using microwave irradiation technique. Structure of all the compounds was confirmed by
determination of elemental composition, NMR and infrared spectra. The prepared compounds
primarily investigated by performing density functional theory calculations. DFT calculations were
applied successfully to predict the structural geometry and confirm the IR data. Results show that the
BS3 has low energy gap, low Hardness, greater softness and higher electrophilicity and the BS1 has
high energy gap, high hardness and low softness. So, it clear that non-substituted Schiff base (BS1) is
the most stable, the least reactive that other molecules. Therefore substitution with CH or NO groups
3
2
has a positive effect for increasing chemical reactivity. In addition, antibacterial activity investigation
of the compounds shows the acceptable prevention of these compounds against some bacteria. The
compounds containing a methyl group (BS2) showed higher activity. Theoretical calculations were
performed in order to investigate physicochemical properties that may be related to the antimicrobial
action of the studied compounds. The results demonstrated that the antimicrobial activity of the
investigated compounds was influenced by some physicochemical properties of the type of substituent
on the aromatic ring. A good correlation was obtained between the lipophilicity values (LogP) that
were theoretically determined and the structures of the compounds. The results revealed that the
presence of methyl group, on the phenyl ring situated in position 4 increase lipophilicity. Mean while,
the substitution of the same phenyl ring with polar groups (Nitro) led to lower values of the
lipophilicity parameters. Schiff base BS2 containing CH substituent could inhibit the growth of all
3
tested bacteria. The shift of the nitro group in compound BS3 led to a significant reduction in
antibacterial effects. These results can be inspired by researchers for the synthesis of new potential
bioactive compounds.
1
3

Credit Author Statement
Author
Credit Statement
Mohamed Mersellem
Conceptualization, Methodology, Resources Writing -
Original Draft
Abdelkader Hellal
Benhamou Abdellah
Conceptualization, Methodology, Validation Software,
Writing - Original Draft, Supervision
Supervision
Conflict of Interest Statement
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
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8

Figure captions
O
NH2
HO
N
CS/Mw
R
OH
R
R=-H; -CH ; -NO2
3
Scheme 1. Synthesis route of BS1, BS2 and BS3 Schiff bases under microwave (MW) and
conventional heating (CS).
(
BS1)
(
BS2)
(
BS3)
Fig.1. Optimized structures and the numbering of atoms for BS1, BS2 and BS3.
1
9

0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0
0
1
2
3
4
5
6
7
5
1
1
2
2
0 0
5 0
0 0
5 0
B S 1 -T H E O
B S 1 -E X P
4
0 0 0
3 5 0 0
3 0 0 0
2 5 0 0
2 0 0 0
1 5 0 0
1 0 0 0
5 0 0
4 0 0 0
3 5 0 0
3 0 0 0
2 5 0 0
2 0 0 0
1 5 0 0
1 0 0 0
5 0 0
-
1
-1
W N (cm
)
W N (cm )
0
0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1
2
3
4
5
6
7
5 0
1 0 0
1
2
2
5 0
0 0
5 0
B S 2 -E X P
B S 2 -T H E O
3 0 0
4
0 0 0
3 5 0 0
3 0 0 0
2 5 0 0
2 0 0 0
1 5 0 0
1 0 0 0
5 0 0
4
0 0 0
3 5 0 0
3 0 0 0
2 5 0 0
2 0 0 0
-1
1 5 0 0
1 0 0 0
5 0 0
-
1
W N (cm
)
W N (cm )
0
0
5
0 0
2
4
6
8
0 0
1
0 0 0
5 0 0
0 0 0
0 0
0 0
0 0
1
2
B S 3 -E X P
B S 3 -T H E O
4
0 0 0
3 5 0 0
3 0 0 0
2 5 0 0
2 0 0 0
1 5 0 0
1 0 0 0
5 0 0
4
0 0 0
3 5 0 0
3 0 0 0
2 5 0 0
W N (cm
2 0 0 0
-1
)
1 5 0 0
1 0 0 0
5 0 0
-
1
W N (cm
)
Fig.2. Theoretical and experimental IR spectrum of BS1, BS2 and BS3.
2
0

(BS1)
(BS2)
(BS3)
Fig.3. Mulliken atomic charges for BS1, BS2 and BS3 at B3LYP with 6–31++G(d,p) basis set.
.
2
1

LUMO/BS1
HOMO/BS1
HOMO/BS2
HOMO/BS3
LUMO/BS2
LUMO/BS3
Fig.4. Molecular orbital surfaces of BS1, BS2 and BS3 at B3LYP/6–31++G(d,p) basis set.
.
2
2

(BS1)
(BS2)
(BS3)
Fig.5. Dipole moments for the optimized geometry of the title compounds at B3LYP with 6–
1++G(d,p) basis set.
3
2
3