Synthesis, antimicrobial screening, homology modeling, and molecular docking studies of a new series of Schiff base derivatives as prospective fungal inhibitor candidates

Article abstract of DOI:10.3390/molecules24183250

Twelve new Schiff base derivatives have been prepared by the condensation reaction of different amino substituted compounds (aniline, pyridin-2-amine, o-toluidine, 2-nitrobenzenamine, 4-aminophenol, and 3-aminopropanol) and substituted aldehydes such as n

Full text of DOI:10.3390/molecules24183250

molecules
Article
Synthesis, Antimicrobial Screening, Homology
Modeling, and Molecular Docking Studies of a New
Series of Schiff Base Derivatives as Prospective
Fungal Inhibitor Candidates
Yahya Toubi 1, Farid Abrigach 1,
*
, Smaail Radi 1,*, Faiza Souna 2, Abdelkader Hakkou 2,
Abdulrhman Alsayari 3, Abdullatif Bin Muhsinah 3 and Yahia N. Mabkhot 4
1
Laboratory of Applied Chemistry & Environment, Faculty of Sciences, Mohammed First University,
Oujda 60000, Morocco
2
Laboratory of Biochemistry, Faculty of Sciences, Mohammed First University, Oujda 60000, Morocco
Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha 61441, Saudi Arabia
Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Abha 61441,
3
4
Saudi Arabia
*
Correspondence: abrigach.farid@live.fr (F.A.); s.radi@ump.ac.ma (S.R.); Tel.: +212-536-500-601/02 (S.R.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 July 2019; Accepted: 2 September 2019; Published: 6 September 2019
Abstract: Twelve new Schiff base derivatives have been prepared by the condensation reaction of
different amino substituted compounds (aniline, pyridin-2-amine, o-toluidine, 2-nitrobenzenamine,
4-aminophenol, and 3-aminopropanol) and substituted aldehydes such as nicotinaldehyde,
o,m,p-nitrobenzaldehyde, and picolinaldehyde in ethanol using acetic acid as a catalyst. The
envisaged structures of the all the synthesized ligands have been confirmed on the basis of their
spectral analysis FT-IR, mass spectroscopy, 1H- and 13C-NMR. In vitro screening of their antibacterial
and antifungal potential against Escherichia coli bacterium and Fusarium oxysporum f.sp albedinis (F.o.a)
fungus, respectively, revealed that all the ligands showed no significant antibacterial activity, whereas
most of them displayed good antifungal activity. Homology modeling and docking analysis were
performed to explain the antifungal effect of the most and least active compound against two F.o.a
fungus proteins.
Keywords: Schiff bases; synthesis; antifungal; molecular docking; homology modeling;
pharmacophore site
1. Introduction
Nowadays, much attention is paid to the study of Schiff base compounds, not only for
their broad range of biological activities—such as antimalarial, antiviral, antitubercular, anticancer,
anti-inflammatory, antifungal, and antibacterial properties [
used as catalyst carriers, optical chemical sensors, thermo-stable materials, metal complexion agents,
corrosion inhibitors, and polymer stabilizers [ 10]. Such Schiff bases, also known as imines or
1–6]—but also because they are widely
7–
azomethines, can be obtained easily via the condensation reaction between primary amines and
carbonyl compounds. In addition, some works reveal that several natural compounds containing
azomethine groups form compounds of high pharmacological interest [11
,12]. Their critical biological
activities are due principally to the presence of the imine group (C=N) [13].
Molecules 2019, 24, 3250; doi:10.3390/molecules24183250
www.mdpi.com/journal/molecules

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In the case of infections, the search for potent and selective inhibitors has received particular
attention because of the increase of antimicrobial resistance, which has become one of the biggest
challenges to global health, food security, and development. In particular, and concerning the
agricultural area, Fusarium oxysporum f.sp albedinis (F.o.a) represents the leading dangerous agent among
all pathologies of date palms cultivation, particularly in North Africa [14]. In the last century, Bayoud
disease, which is caused by the telluric fungus F.o.a, has destroyed more than 15 million Moroccan
and Algerian date palm trees. Unfortunately, to this day, no curative treatment yet exists against this
fungus, except some limited methods such as disinfection of the soil, propagation. and use of resistant
varieties—which remains the first way to reduce the impact of this disease [15].
On the other hand, due to incomplete information on the pathogenesis of the F.o.a fungus, various
computational techniques can be used in order to better understand the mechanism of action of this
fungus. In this context, molecular docking simulation remains one of the most powerful tools that can
give an atomistic insight into molecular recognition by predicting the ability of a molecule to bind to
the active site of a protein. In our case, two F.o.a proteins were chosen as targets for our docking studies.
The first one was the Fusarium oxysporum Guanine nucleotide-binding protein (Fgb1), a membrane
protein of high importance in many biological processes of F. oxysporum fungal. The disruption of
this protein leads to pathogenicity reduction and alterations in physiological characteristics, like heat
resistance and germination frequency [16]. The second target was the Fusarium oxysporum phytase
domain (Fophy) enzyme, another protein that plays versatile roles in agricultural and feeding fields. It
catalyzes the degradation of phytate (an important constituent of grains, cereals, and oilseeds) into
inorganic phosphorus and myoinositol phosphate derivatives. Inhibition of the Fophy enzyme can
affect the growth of the fungus indirectly by stopping the degradation of phytate, which is well-known
as a strong chelating agent and readily binds covalent metal ions, rendering them insoluble and thus
unavailable for absorption [17,18]. In general, these facts make both of these proteins as prospective
potential targets to develop new anti-F.oxysporum inhibitors.
Accordingly, and keeping in mind the biological significance of the Schiff base derivatives, we
report in this paper the synthesis of new imines compounds as potent antimicrobial agents. In addition,
a molecular docking approach was performed for the most and the least antifungal derivatives in
the homology models we constructed for Fusarium oxysporum Guanine nucleotide-binding protein
(Fgb1) and Fusarium oxysporum phytase domain (Fophy), to gain better insight on the ligand–receptor
binding interactions and also to direct future syntheses.
2. Results and Discussion
2.1. Chemistry
The new Schiff bases 1–12 were synthesized by condensation reaction of primary amines (aniline,
pyridin-2-amine, o-toluidine,2-nitrobenzenamine, 4-aminophenol, and 3-aminopropan-1-ol) and
substituted aldehydes (nicotinaldehyde, o,m,p-nitrobenzaldehyde, and picolinaldehyde) in ethanol
using acetic acid as a catalyst to obtain the desired compounds in nearly 79–96% yield. The ligands
were quite stable at room temperature in the solid state. The spectroscopic data are in total agreement
with the proposed structures, as shown in Scheme 1.

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R
R
N
X
N
N
NO2
1
2
3
: R = H
: R = 2-CH3 ; X = C
: R = 2-NO2 ; X = C
; X = C
5
: R = 4-NO2
6 : R = 3-NO2
7 : R = 2-NO2
4 : R = H
; X = N
N
OH
OH
R
N
N
NO2
11
8
9
10
: R = 4-NO2
: R = 3-NO2
: R = 2-NO2
N
N
12
Scheme 1. General structures of the prepared Schiff base derivatives.
The NMR spectra of all compounds were recorded on a Bruker AC 300 spectrometer. TMS was
used as the reference standard and the solvent was CDCl3. The 1H-NMR spectra clearly indicate the
presence of the characteristic peak of the alpha–hydrogen of CH=N as a singlet, which remained a
strong indication of the formation of the desired Schiff base derivatives
of these alpha–hydrogen were found to be ranged from 7.37 ppm for compound
compound . In general, the observed differences in the values of chemical shifts of the signal under
1–
12. The chemical shift values
1
to 8.75 ppm for
6
study can be explained by the shielding and deshielding effect of the CH=N proton, probably due
to the different electronic effects introduced by the substituent groups on the phenyl rings of each
compound. In fact, the presence of one or more electron withdrawing groups such as NO2 (compounds
5–12) leads to a decrease of the electron density on the C=N proton, which makes it more deshielded
than the proton in the case of compounds
1 and 2, which showed chemical shifts of 7.37 and 7.45 ppm,
respectively, which are less than that observed in the case of compounds 5–12 (up to 8 ppm).
2.2. Biological Activity
The antimicrobial potentials of the titled compounds were checked against the E.coli bacterium
strain and the Fusarium oxysporum f.sp albedinis fungus, utilizing the agar diffusion method.
Unfortunately, the results of the preliminary screening showed that none of the tested compounds
were found to exhibit antibacterial effect against the used bacterium. These results can be explained
by the high resistance of the E.coli strain to this kind of product, or also by the absence of active or
pharmacophore sites which can act as specific and potential features to inhibit the growth of this
bacterium [19].
On the contrary, the results of the in vitro antifungal assay revealed that most of the screened ligands
showed high to moderate activities against F.o.a based on their minimum inhibitory concentration
(MIC) values. The results obtained from the evaluation study are provided in Table 1. The maximum
activity was 0.02
values equal to 0.04
products also have considerable activities, with MICs varying from 0.08
to 0.30 . Comparing both the structures of and 3, we can conclude that
mL−1 for compound
µ
g
µ
·
mL−1, shown by compound
mL−1, while compound
showed the least MIC value (i.e., 0.9
mL−1 for compound 11
3
, followed by compounds
6,
7, and 12 with MIC
g
·
2
µg
·
mL−1). Other
µg
·
µg
·
5
2
the presence of a strong electron withdrawing group like nitro moiety (NO2) at the ortho position
of the phenyl ring is very suitable to increase the antifungal efficiency; in time the presence of an
electron donating group such as methyl moiety (CH3) is unfavorable for antifungal activity. However,

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unfortunately, with these preliminary investigations, it is difficult to determine the right factors that
affect the antifungal potential of these derivatives. For this, more investigations are required using
other models and methods.
Table 1. Rate of inhibition of the growth of F.o.a according to the concentration of the tested compounds
(0.01–1.50 µg·mL−1) and the MIC values.
MIC
Rate of Inhibition (%)
Comp.
(µg·mL−1
)
0.01
0.05
0.10
0.15
0.25
0.50
0.75
1.00
1.50
1
2
3
4
5
6
7
8
9
10
11
12
(-)
0
0
8
0
0
0
0
0
0
0
0
0
-
0
0
22
0
7
0
53
0
26
0
82
0
55
0
100
7
78
0
100
7
10
46
67
14
7
15
62
48
-
86
0
100
7
100
15
100
12
9
100
100
23
13
17
79
69
-
100
27
100
15
0.10
0.90
0.02
0.25
0.30
0.04
0.04
0.12
0.25
0.20
0.08
0.04
-
0
0
0
0
9
15
13
10
0
0
0
0
12
-
13
33
0
0
0
15
12
-
17
49
9
0
0
33
24
-
23
55
12
7
12
47
26
-
72
89
14
8
14
66
61
-
100
100
37
15
23
90
82
-
(-): Negative control = DMSO/EtOH + distilled water.
2.3. Computational Studies
2.3.1. Molecular Electrostatic Potential (MEP) Maps
Molecular electrostatic potential (MEP) is a very powerful tool utilized to determine or predict the
reactive regions of nucleophilic and electrophilic attacks on a molecular system. It can be generated
by mapping the electrostatic potential on to the isoelectron density surface of the molecule, which
gives us the possibility to determine the distribution of the electronic charge over all the structure.
Actually, MEP mapping remains a useful way to understand the interaction of a molecule with its
environment and to study the hydrogen bonding interactions, as well as the biological recognition
processes [20]. In our case, the MEP maps of both compounds
DFT (Density Functional Theory) optimized geometries, and they are displayed in Figure 1. As can
be seen for compound , the negative electrostatic potential regions are mainly concentered over the
nitrogen atoms of the pyridine ring and the imine function with values of 1.349 and 0.761 eV (at an
isovalue of 0.0004 electrons/Å3), respectively. While for compound
, the negative charges are located
on the nitrogen atoms of the pyridine ring and the imine function, as well as the oxygen atoms of
the nitro group (NO2) at the ortho position of the phenyl ring, with values ranging from 1.404 to
0.741 eV. These results give us valuable information about the possible sites that can be involved in
interactions by hydrogen bonds with the amino acid residues of protein receptors.
2 and 3 were generated based on their
2
−
−
3
−
−

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Figure 1. Generated molecular electrostatic potential (MEP) maps of compound
2 (left) and compound
3
(
right). The negative (red, orange, and yellow) regions are related to electrophilic reactivity, whereas
positive (green and blue) regions accompany nucleophilic reactivity.
2.3.2. Homology Modeling
In order to gain better insights and to better understand the binding mode of the title compounds
2
and 3 towards both the Fusarium oxysporum Guanine nucleotide-binding protein beta subunit
(Fgb1) and the Fusarium oxysporum isolate FO 3-phytase-like (Fophy) proteins, it was planned to
perform molecular docking simulations. However, because of the unavailability of experimental crystal
structures of these proteins, homology modeling studies were carried out to construct three-dimensional
(3D) structures of Fgb1 and Fophy prior to use in our docking studies. For Fgb1 protein, the model was
generated and validated as described previously in our last works [21,22] using the crystal structure of
the Rattus norvegicus Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 (3SN6_chain
B) as a suitable template. Following the same procedure described for the Fgb1 model, the 3D structure
of the Fophy protein was also built. In brief, the DNA sequence of Fophy was selected as a target and
it was downloaded from the NCBI GenBank database with accession number KC708486, and then
converted to its equivalent amino acid sequence before running a BLASTp search. Aspergillus Niger
Phytase (3K4P_chain A) was found the best-matched template with a 95.2% similarity to the Fophy
sequence and with a resolution of 2.4 Å, making it an excellent template. The target and the template
protein sequences were then aligned with the aid of the Clustal Omega and visualized by DS programs
(Figure 2).The resulting sequence alignment shows a total conservation of 79.8% (399 residues), strong
similarity of 3% (15 residues), and weak identity of 0.6% (3 residues) of amino acid between the Fophy
sequence and that of the 3K4P_chain A—whilst 83 amino acids (16.6%) of Fophy do not match those
found in the selected template sequence.

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Figure 2. The alignment of Fusarium oxysporum phytase domain and Aspergillus Niger Phytase
template (PDB code: 3K4P) that yielded the homology model of Fophy. The identical residues are
represented by green color, residues with strong similarity are represented by blue, and residues with
weak identity are in orange, whereas the red color represents the non-matching residues.
Figure 3 shows the modeled 3D structure of Fophy based on the aligned sequence; the red color
indicates
α
-helices and the light blue color indicates
β-sheets, whereas
β-turns are indicated by green
color. All these elements are connected by loops (in light gray color). All
α
- helices, -sheets, -turns,
β
β
and random coils (loops) match the same alignment found in the template structure. The binding site
is shown as an orange transparent sphere.
Figure 3. Modeled structure of the Fophy protein based on the aligned sequence between Fophy and
Aspergillus Niger Phytase (3K4P_chain A) sequences.
Validation of the final model was performed by analyzing its Ramachandran plot (Figure 4A),
comparison plot (Figure 4B), and local quality plot (Figure 4C). The resulting Ramachandran plot
shows that more than 90% of amino acid residues are distributed in the most favored regions (red color)

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and 9.4% of residues are in additional allowed regions (yellow color)—while none or few residues
are located in generously allowed regions (light yellow color) and disallowed regions (white color),
respectively. In the comparison plot, every dot (black, gray, or light gray) represents one experimental
protein structure, where the analyzed model is represented as a red star. From this plot, it can be
concluded that our model is comparable to the experimental protein structures because the red star is
on the experimental line. In addition, the local quality plot shows, for each residue of the generated
model, the predictable similarity to the native structure. Typically, residues showing a score up to 0.6
are expected to be of good quality. In our case, as shown in Figure 4C, all residues indicate similarities
to the target above 0.6, which indicates that the generated model is in the norms of good structures.
Finally, by considering all these results of the Ramachandran, comparison, and local quality plots,
we can suggest that the built model is reasonably good in geometry and stereochemistry, and is very
appropriate to examine the next step, which is the ligand–protein docking studies of our compounds.
Figure 4. Validation of the generated homology model of Fophy. (A) Ramachandran plot, (B) comparison
plot, and ( ) local quality plot. For more information about Ramachandran plot descriptions the reader
C
should be referred to the web site at (https://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/
GetPage.pl?doc=TRUE&pdbcode=n/a&template=doc_procheck01.html).
2.3.3. Molecular Modeling Study
Molecular docking calculations were performed to identify selective compounds that could be
interacting with the catalytic sites of the Fgb1 and the Fophy proteins. In this study, flexibility was
allowed in all the rotable bonds of the ligand, while the protein was used as a rigid structure.
Docking against the Fgb1 Protein
The binding modes of the selected molecules (compounds
the modeled Fgb1 are depicted in Figure 5. The binding energy
the binding affinity of each molecule to the Fgb1 protein. From the docking study, it was found that
the ligand shows the best affinity towards the Fgb1 protein with a Gbinding 7.34 kcal/mol, when
compared to the compound , which shows a Gbinding of 6.94 kcal/mol. As can be observed from
3
and
2) in the protein active pocket of
∆
Gbinding has been used to evaluate
3
∆
=
−
2
∆
−

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Figure 5, compound
3
forms five H-bond interactions with the amino acid residues of the receptor—one
between the nitrogen atom of the imine function and the hydrogen atom of Arg167 with a distance
of 2.32 Å, and with a distance of 2.40 Å, the nitrogen atom of the pyridine ring made the second
H-bond with amino acid Thr118. Moreover, the pharmacophoric nitro group at the ortho position of
the phenyl ring interacts via three H-bonds, two with the residue Val294 (2.47 and 3.37 Å) and one with
Ser206 residue with a distance of 3.29 Å. In contrast, compound
2 forms only two H-bond interactions
between the residues Arg167 and Thr118 and nitrogen atom of the imine function and the pyridine
ring, respectively. From these findings, we can see that the ligand–protein complex is stabilized mainly
by hydrogen bonds, especially those that the nitro group forms with Val294 and Ser206, which mean
that this nitro moiety helps to maintain the molecule in the binding pocket and makes it more active
against the studied target.
Figure 5. Binding mode of compound
For both and , gray color for carbon atoms, blue for nitrogen, red for oxygen and write color for
hydrogen atoms.
2 (left) and compound 3 (right) in the active site of Fgb1 protein.
2
3
Docking Against the Fophy Protein
As second target protein, Fusarium oxysporum isolate FO 3-phytase-like, was chosen to perform
a docking study of the most and the least active compounds, as mentioned previously. The parameters
that determine the binding affinity of each compound to the active pocket of the Fophy protein were
calculated and are reported in the Table 2.
Table 2. Docking results against the homology model of the Fophy protein.
∆Gbinding (kcal/mol)
−5.84
Compound
2
Interaction
Bond Length (Å)
N(pyridine)–(H)Ser431
N(pyridine)–(H)Ser431
O(NO2)–(O)Glu422
O(NO2)–(O)Asp329
O(NO2)–(HN)Ile326
2.33
2.15
3.16
3.41
3.54
3
−6.78
The most active compounds, i.e., 3, gave a calculated docking score (
∆
Gbinding) of 6.78 kcal/mol,
−
higher than that shown by compound 2, which is equal to −5.84 kcal/mol. This finding indicates that
compound
compound
3
2
, in its optimal docked pose, is more attached (more active) to the Fophy protein than
, which is in total agreement with our experimental results. The optimal binding of
3
appears to arise from four principal interactions, specifically, one of the nitro group oxygen interacts
with Glu422 of the Fophy active domain via the formation of a medium–strong halogen bonding
interaction (3.16 Å). The other oxygen of the nitro group forms a halogen bond with Asp329 (3.41 Å),
as well as a hydrogen bond with Ile326 (3.54 Å). The interactions are also stabilized by another H-bond

Molecules 2019, 24, 3250
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formed between the nitrogen atom of the pyridine ring and the hydrogen atom of Ser431 residue with
a distance of 2.15 Å. On the other hand, compound was found to be attached to the active pocket of
Fophy with a single strong H-bond between the nitrogen atom of the pyridine ring and Ser431 residue
2
with a distance of 2.15 Å. The 3D binding models of both compounds
model are shown in Figure 6.
2 and 3 in the Fophy homology
Figure 6. Binding mode of compound
2 (left) and compound 3 (right) in the active site of the Fophy
protein. For both and , light blue color for carbon atoms, blue for nitrogen, red for oxygen and write
2
3
color for hydrogen atoms.
For comparison, the disappearance or the absence of interactions between the nitrogen atom of
the imine function and the active site residues in the case of the Fophy protein, in comparison with the
case of the Fgb1 protein, can be explained by the absence of amino acid residues close to this atom,
which makes the formation of such interactions impossible or more difficult. Also, it is important to
mention that all the interactions formed between the ligands and the amino acids of the binding pocket
of both studied proteins occurred by the ligand’s atoms with high electron densities, as mentioned by
the generated MEP maps.
Finally, in general, all these important findings and theoretical observations open up the way for
more chemical investigations, in order to make our compounds even more efficient, and also present a
guideline to facilitate a better understanding of the mode of action of the ligand enzyme complex and
−
give a clear insight into the design of new molecules in the future. Furthermore, within the general
context of development of new anti-Fusarium agents targeting proteins and specially Fgb1, additional
suitable experimental assays are required to confirm the good capacity of these active compounds.
3. Materials and Methods
3.1. General Information
All solvents and other chemicals, obtained from usual commercial sources, were of analytical
grades and used without further purification. Melting points were determined by using a BUCHÏ
510 m.p. apparatus (FSO, Oujda, Morocco). The NMR spectra were obtained with a Bruker AC 300
spectrometer (CNRST, Rabat, Morocco). Infrared (FT-IR) spectra were acquired on a Shimadzu infrared
spectrophotometer (FSO, Oujda, Morocco) using the KBr disc technique. Molecular weights were
determined by a JEOL JMS DX-300 Mass Spectrometer (CNRST, Rabat, Morocco).
3.2. General Procedure for the Synthesis of Compounds 1–12
An equal molar solution of a primary amine (0.01 mol) and an aldehyde (0.01 mol) in dry methanol
(50 mL) was refluxed for 3 h using a few drops of acetic acid as a catalyst. The reaction mixture was
then cooled to room temperature and the formed precipitate was filtered, washed with methanol

Molecules 2019, 24, 3250
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(35 mL) and diethyl ether (2
methanol/dioxane (1:4) mixture gave pure products (1–12).
×
5 mL), and then dried under vacuum. The recrystallization from
◦
N-((E)-pyridin-3-ylmethylidene)aniline ( ): White Powder. Yield 80%; m.p. 123–126 C; IR (KBr,
1
cm−1): 3082 (C-H, arom); 1602 (C=N, arom); 1597 (C=N); 1570 (C=C); 1H-NMR (300 MHz, CDCl3,
ppm): 7.3 (m, 1H, benzene), 7.37 (s, CH=N, imine), 8.8 (m, 1H, pyridine); 13C-NMR (75 MHz, CDCl3,
ppm): 122.1 (C14-benzene), 122.3 (C10-benzene), 123.9 (C5-pyridine), 127.3 (C12-benzene), 130.1 (C13,
C11-benzene), 130.2 (C3-pyridine), 130.4 (C4-pyridine), 137.2 (C9-benzene), 149 (C2-pyridine), 151.5
(C6-pyridine), 152 (C7-imine). Mass spectrum (EIMS) [M]+ = 183.
◦
2-methyl-N-((E)-pyridin-3-ylmethylidene)aniline (
2
): White powder. Yield 92%; m.p. 176–181 C;
IR (KBr, cm−1): 3080 (C-H, arom); 2965(C-H, CH3); 1609 (C=N, arom); 1592 (C=N); 1568 (C=C);
1H-NMR (300 MHz, CDCl3, ppm): 7.4 (m, 1H, methylbenzene), 7.45 (s, CH=N, imine), 8.8 (m, 1H,
pyridine), 2.4 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3, ppm): 15.7 (C15- methylbenzene), 122.2 (C14-
methylbenzene), 123.9 (C5-pyridine), 127.1 (C13-methylbenzene), 127.2 (C12-methylbenzene), 130.4
(C11-benzene, C3-pyridine), 130.6 (C10-methylbenzene), 137.2 (C4-pyridine), 147 (C9- methylbenzene),
151.5 (C2-pyridine), 152 (C6-pyridine), 155.92 (C7-imine). Mass spectrum (EIMS) [M]+ = 167.
2-nitro-N-((E)-pyridin-3-ylmethylidene)aniline (
3080 (C-H, arom); 1609 (
C=N, arom); 1592 (C=N); 1568 (C=C); 1360 (C-NO2, arom); 1H-NMR (300 MHz,
3
): Crystal. Yield 85%; m.p. 145–150 ◦C; IR (KBr, cm−1):
ν
CDCl3, ppm): 6.8 (m, 1H, nitrobenzene), 7.48 (s, CH=N, imine), 8.8 (m, 1H, pyridine); 13C-NMR
(75 MHz, CDCl3, ppm): 122.4 (C11-nitrobenzene), 123.2 (C14-nitrobenzene), 123.9 (C12-nitrobenzene),
128.2 (C3-pyridine), 130.4 (C13-nitrobenzene), 136.2 (C4-pyridine), 137.2 (C10-nitrobenzene), 141.9
(C9-nitrobenzene), 144.1 (C2-pyridine), 151.5 (C6-pyridine), 154.69 (C7-imine). Mass spectrum (EIMS)
[M]+ = 228.
◦
N-((E)-pyridin-3-ylmethylidene)pyridin-2-amine (
4
): White powder. Yield 96%; m.p. 119–125 C;
IR (KBr, cm−1): 3084 (C-H, arom); 1610 (C=N, arom); 1597 (C=N); 1570 (C=C); 1H-NMR (300 MHz,
CDCl3, ppm): 7.8 (m, 1H, pyridine), 8.50 (s, CH=N, imine), 8.8 (m, 1H, pyridine); 13C-NMR (75 MHz,
CDCl3, ppm): 116.2 (C3-pyridine), 122.4 (C5-pyridine), 123.9 (C13-pyridine), 130.4 (C9-pyridine), 137.2
(C14-pyridine), 137.3 (C4-pyridine), 150.4 (C6-pyridine), 151.5 (C10-pyridine), 152 (C12-pyridine), 156.5
(C2-pyridine), 160 (C7-imine). Mass spectrum (EIMS) [M]+ = 184.
2-nitro-N-((E)-(4-nitrophenyl)methylidene)aniline (5
): Yellow powder. Yield 85%; m.p. 145–150 ◦C;
IR (KBr, cm−1): 3080 (C-H, arom); 1609 (C=N, arom); 1592 (C=N); 1568 (C=C); 1360 (C=NO2, arom);
1H-NMR (300 MHz, CDCl3, ppm): 8.2 (m, 1H, nitrobenzene), 8.10 (s, CH=N, imine); 13C-NMR (75 MHz,
CDCl3, ppm): 121.1 (C13, nitrobenzene), 121.2 (C11, nitrobenzene), 122.4 (C3, nitrobenzene), 123.2 (C6,
nitrobenzene), 128.2 (C4, nitrobenzene), 130.1 (C14, nitrobenzene), 130.2 (C10, nitrobenzene), 136.2 (C5,
nitrobenzene), 139.9 (C9, nitrobenzene), 141.9 (C2, nitrobenzene), 148.3 (C1, nitrobenzene), 150.35 (C8,
imine), 150.7(C12, nitrobenzene). Mass spectrum (EIMS) [M]+ = 272.
2-nitro-N-((E)-(3-nitrophenyl)methylidene)aniline (6
): Yellow powder. Yield 96%; m.p. 181–183 ◦C;
IR (KBr, cm−1): 3080 (C-H, arom); 1609 (C=N, arom); 1592 (C=N); 1568 (C=C); 1360 (C=NO2, arom);
1H-NMR (300 MHz, CDCl3, ppm): 8.2 (m, 1H, nitrobenzene), 8.75 (s, CH=N, imine); 13C-NMR (75 MHz,
CDCl3, ppm): 122.4 (C13, nitrobenzene), 123.2 (C11, nitrobenzene), 123.4 (C3, nitrobenzene), 124.1 (C6,
nitrobenzene), 128.2 (C4, nitrobenzene), 129.8 (C14, nitrobenzene), 134.7 (C10, nitrobenzene), 135.3 (C5,
nitrobenzene), 136.2 (C9, nitrobenzene), 141.8 (C2, nitrobenzene), 148.3 (C1, nitrobenzene), 148.42 (C8,
imine), 148.5(C12, nitrobenzene). Mass spectrum (EIMS) [M]+ = 272.
2-nitro-N-((E)-(2-nitrophenyl)methylidene)aniline (
7
): Yellow powder. Yield 83%; m.p. 181–183 ◦C;
C=C); 1360 (C-NO2, arom);
IR (KBr, cm−1): 3080 (C-H, arom); 1609 (C=N, arom); 1592 (C=N); 1568 (
ν
1H-NMR (300 MHz, CDCl3, ppm): 8.2 (m, 1H, nitrobenzene), 8.05 (s, CH=N, imine); 13C-NMR (75 MHz,
CDCl3, ppm): 122.4 (C13, nitrobenzene), 123.2 (C11, nitrobenzene), 123.4 (C3, nitrobenzene), 124.1 (C6,
nitrobenzene), 128.2 (C4, nitrobenzene), 129.8 (C14, nitrobenzene), 134.7 (C10, nitrobenzene), 135.3 (C5,

Molecules 2019, 24, 3250
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nitrobenzene), 136.2 (C9, nitrobenzene), 141.8 (C2, nitrobenzene), 148.3 (C1, nitrobenzene), 145 (C8,
imine), 148.5(C12, nitrobenzene). Mass spectrum (EIMS) [M]+ = 272.
N-((E)-(4-nitrophenyl)methylidene)pyridin-2-amine (
ν
8
): Crystal. Yield 85%; m.p. 150–155 ◦C; IR (KBr,
C=C); 1364 ( C=NO2, arom);
cm−1): 3084 (C-H, arom); 1601 (C=N, arom); 1590 (C=N); 1562 (
ν
ν
1H-NMR (300 MHz, CDCl3, ppm): 8.2 (m, 1H, nitrobenzene), 8.10 (s, CH=N, imine), 7.8 (m, 1H,
pyridine); 13C-NMR (75 MHz, CDCl3, ppm): 116.2 (C11-C13, nitrobenzene), 121.2 (C10, nitrobenzene),
121.3 (C14, nitrobenzene), 130.1 (C2, pyridine), 137.3 (C3-C5, pyridine), 139.9 (C4, pyridine), 148.42 (C8,
imine), 150.4 (C6, pyridine),150.7 (C9, nitrobenzene), 160.7 (C12, nitrobenzene). Mass spectrum (EIMS)
[M]+ = 228.
N-((E)-(3-nitrophenyl)methylidene)pyridin-2-amine (
cm−1): 3085 (C-H, arom); 1606 (C=N, arom); 1597 (C=N); 1565 (C=C); 1364 (C-NO2, arom); 1H-NMR
9
): Crystal. Yield 79%; m.p. 142–145 ◦C; IR (KBr,
ν
(300 MHz, CDCl3, ppm): 8.5 (m, 1H, nitrobenzene), 8.15 (s, CH=N, imine), 8.00 (m, 1H, pyridine);
13C-NMR (75 MHz, CDCl3, ppm): 116.2 (C11-C13, nitrobenzene), 121.2 (C10, nitrobenzene), 121.3
(C14, nitrobenzene), 130.1 (C2, pyridine), 137.3 (C3-C5, pyridine), 139.9 (C4, pyridine), 148.42 (C8,
imine), 150.4 (C6, pyridine),150.7 (C9, nitrobenzene), 160.7 (C12, nitrobenzene). Mass spectrum (EIMS)
[M]+ = 228.
N-((E)-(2-nitrophenyl)methylidene)pyridin-2-amine (10): Yellow powder. Yield 95%; m.p. 208–212 ◦C;
IR (KBr,
ν
cm−1): 3085 (C-H, arom); 1606 (C=N, arom); 1597 (C=N); 1565 (C=C); 1364 (C=NO2, arom);
1H-NMR (300 MHz, CDCl3, ppm): 8.2 (m, 1H, nitrobenzene), 8.6 (s, CH=N, imine), 8.05 (m, 1H,
pyridine); 13C-NMR (75 MHz, CDCl3, ppm): 116.2 (C11-C13, nitrobenzene), 121.2 (C10, nitrobenzene),
121.3 (C14, nitrobenzene), 130.1 (C2, pyridine), 137.3 (C3-C5, pyridine), 139.9 (C4, pyridine), 148.42 (C8,
imine), 150.4 (C6, pyridine),150.7 (C9, nitrobenzene), 160.7 (C12, nitrobenzene). Mass spectrum (EIMS)
[M]+ = 228.
3-(((Z)-(2-nitrophenyl)methylidene)amino)propan-1-ol (11): Yellow powder. Yield 85%; m.p.
187–190 ◦C; IR (KBr,
ν
cm−1): 3500 (O-H); 3078 (C-H, arom); 2870 (C-H2); 1597 (C=N); 1565 (C=C);
1364 (C-NO2, arom); 1070 (C-O); 1H-NMR (300 MHz, CDCl3, ppm): 7.8 (m, 1H, nitrobenzene), 8.7 (s,
CH=N, imine), 3.55(N-CH2-CH2-CH2-O), 1.84 (N-CH2-CH2-CH2-O), 3.53(N-CH2-CH2-CH2-O), 2.1 (s,
OH); 13C-NMR (75 MHz, CDCl3, ppm): 34.2 (C12, propanol), 57.9 (C11, propanol), 60.5(C13, propanol),
122.4(C6, nitrobenzene), 123.2 (C3, nitrobenzene), 128.2 (C5, nitrobenzene), 136.2 (C2, nitrobenzene),
141.9 (C4, nitrobenzene), 148.3 (C1, nitrobenzene), 157.20 (C9, nitrobenzene). Mass spectrum (EIMS)
[M]+ = 209.
4-(((E)-pyridin-2-ylmethylidene)amino)phenol (12): White powder. Yield 80%; m.p. 185–188 ◦C; IR
(KBr,
ν
cm−1): 3550 (O-H); 3068 (C-H, arom); 1587 (C=N); 1568 (C=C); 1072 (C-O); 1H-NMR (300 MHz,
DMSO, ppm): 7.8 (m, 1H, phenol), 7.8 (m, 1H, pyridine), 8.13 (s, CH=N, imine), 13C-NMR (75 MHz,
CDCl3, ppm): 117.3 (C11-C13, phenol), 123.8 (C10-C14, phenol), 123.9 (C3, pyridine), 126.2 (C5,
pyridine), 136.1 (C4, pyridine), 141.6 (C9, phenol), 149.2 (C6, pyridine), 137.83 (C7, imine), 153.6 (C2,
pyridine), 157 (C12, phenol). Mass spectrum (EIMS) [M]+ = 199.
3.3. Biological Evaluation
3.3.1. In Vitro Antifungal Assay
The in vitro antifungal activities of all the new compounds were determined by the agar diffusion
technique against Fusarium oxysporum f.sp albedinis (F.o.a) fungus, as previously described [23,24]. For
tests, the agar media (potato dextrose agar) were inoculated with a solution of the tested compound in
DMSO/EtOH (50/50) at different concentrations, and then mycelial discs of 6 mm diameter of the F.o.a
fungus were placed into the middle of each Petri plate. Thereafter, all the plates were incubated at 28 ◦C
for 7 days. The growth of the microorganism was followed by a measurement of mycelium diameter.
The inhibition percentage of a molecule is equal to the ratio of the mycelium diameter of the culture in

Molecules 2019, 24, 3250
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the presence of a dose of the tested compound over the mycelium diameter of the reference culture
multiplied by 100. The minimum inhibition concentration (MIC) is the least dose of the compound
which caused inhibition of the microorganism growth.
ꢀ
ꢁ
Do − Dx
Inhibitionpercentage =
× 100
Do
where Do and Dx are the diameters of the mycelial growth of the F.o.a in the absence and in the presence
of the tested compound, respectively. Due to lack of a known antibiotic that can treat this infection
directly, (DMSO/EtOH + distilled water) mixture was used as negative control without any standard
reference drug.
3.3.2. Antibacterial Test
The prepared compounds were screened for their in vitro antibacterial potential against Escherichia
coli bacterium strain in accordance with the National Committee for Clinical Laboratory Standards
(NCCLS) recommendations [25] using the disc diffusion method. Briefly, overnight culture of E.coli
strain was subcultured on Muller–Hinton agar into Petri plates. Sterile WHATMAN paper discs of
6 mm diameter were put in the middle of each plate and then soaked up by DMSO/EtOH solution of
the tested compounds at different concentrations. The incubation was carried out at 37 ◦C for 24 h.
The zones of inhibition around each disc were noted.
3.4. Computational Studies
3.4.1. Ligand’s Preparation
The structures of all Schiff base derivatives were drawn using ACD/ChemSketch software (version
12.01(free version), FSO, Oujda, Morocco) [26]. Geometry optimization was done with the aid of the
Gaussian 09 package (FSO, Oujda, Morocco) [27] at DFT/B3LYP level with 6-31G(d,p) basis. GaussView
06 software was used to calculate and generate the molecular electrostatic potential (MEP) maps of the
optimized structures.
3.4.2. Homology Modeling
Both the Fusarium oxysporum Guanine nucleotide-binding protein beta subunit (Fgb1) and the
Fusarium oxysporum isolate FO 3-phytase-like (Fophy) proteins were used in the present work. The
target primary amino acid sequence of Fgb1 was retrieved from the UniProtKB database (accession
number Q96VA6), while the DNA sequence of Fophy was received from the NCBI GenBank database
with accession number KC708486, and then translated to the equivalent amino acid sequence with the
aid of Discovery Studio client v16 software (DS) (version 16 (academic version), Oujda, Morocco) [28].
To find suitable homologues sequences (templates), with known experimental 3D structures, of the
studied targets (Fgb1 and Fophy), similarity searches against PDB were performed using the BLAST
server (free web service, NCBI, Bethesda, MD, USA) [29]. The Clustal Omega program server (free web
service, EMBL-EBI, Hinxton, UK) [30] was used to align the proteins’ sequences of the best selected
templates and the studied targets. For each target protein, a homology model was generated by
the automated protein structure homology-modeling server SWISS-MODEL (free web service, SIB,
Lausanne, Switzerland) [31]. The Ramachandran plot generated by PROCHECK [32] via PDBsum
database (free web service, EMBL-EBI, Hinxton, UK) [33], and local quality and comparison plots via
SWISS-MODEL, were utilized to check the validity of the built models. DS (version 16 (academic
version), FSO, Oujda, Morocco) was used to visualize the three-dimensional structure and to predict
the active site of the resultant models.

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3.4.3. Molecular Docking
Molecular docking studies of the most and the least active antifungal compounds against the
homology models of the Fgb1 and Fophy proteins were performed using the SwissDock server (free
web service, SIB, Lausanne, Switzerland) [34]. The CHARMM force field was used in the calculations,
keeping the default parameter settings. The analysis of docking results was carried out using the UCSF
Chimera molecular viewer (version 1.12, FSO, Oujda, Morocco) [35].
4. Conclusions
A series of twelve Schiff base derivatives were synthesized and characterized by FT-IR, mass
1
spectroscopy, H- and 13C-NMR. The preliminary antibacterial and antifungal screening against
E. coli bacterium and Fusarium oxysporum f.sp albedinis (F.o.a) fungus, respectively, proved that most of
these compounds exhibited good to excellent antifungal activity, but very weak antibacterial effect.
Furthermore, theoretical investigations by homology modeling and docking studies were undertaken
in order to illustrate and understand the possible binding mode of the most and least active compounds
towards the Fgb1 and Fophy proteins. The results obtained were found to be in good agreement
with the experimental findings in both cases. However, more appropriate in vitro experiments on the
inhibition of Fgb1 or Fophy with the novel Schiff base compounds should be carried out, and their
interactions should be confirmed by others, performing methods such as free-energy calculations and
molecular dynamic simulations.
Author Contributions: Formal analysis, F.A.; Funding acquisition, S.R., A.A., A.B.M. and Y.N.M.; Investigation,
Y.T., F.A. and F.S.; Resources, S.R. and A.H.; Supervision, S.R.; Visualization, F.A.; Writing–original draft, F.A.;
Writing–review & editing, S.R.
Funding: This research was funded by the Deanship of Scientific Research at King Khalid University, no.
(R.G.P.2/23/40/2019) (Saudi Arabia) and was supported by the PPR2-MESRSFC-CNRST-P10 project (Morocco).
Acknowledgments: This work was supported by the PPR2-MESRSFC-CNRST-P10 project (Morocco). The authors
also extend their appreciation to the Deanship of Scientific Research at King Khalid University for its funding this
prolific research group no. (R.G.P.2/23/40/2019).
Conflicts of Interest: The authors declare that they have no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).