Synthesis, anti-bacterial evaluation, DFT study and molecular docking as a potential 3-chymotrypsin-like protease (3CLpro) of SARS-CoV-2 inhibitors of a novel Schiff bases

Article abstract of DOI:10.1016/j.molstruc.2020.129454

New Schiff bases {N'-(phenyl(pyridin-2-yl)methylene) isonicotinohydrazide (L¹H), N¹-(naphthalen-1-yl)-N²-(phenyl(pyridin-2-yl) methylidene) ethane-1,2-diamine (L²H), N-(6-chlorobenzo[d]thiazol-2-yl)-1-phenyl-1-(

Full text of DOI:10.1016/j.molstruc.2020.129454

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Synthesis, anti-bacterial evaluation, DFT study and molecular
docking as a potential 3-chymotrypsin-like protease (3CLpro) of
SARS-CoV-2 inhibitors of a novel Schiff bases
Ahmed S.M. Al-Janabi , Amin O. Elzupir , Tarek A. Yousef
PII:
S0022-2860(20)31769-5
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129454
MOLSTR 129454
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
29 August 2020
3 October 2020
13 October 2020
Please cite this article as: Ahmed S.M. Al-Janabi , Amin O. Elzupir , Tarek A. Yousef , Synthesis,
anti-bacterial evaluation, DFT study and molecular docking as a potential 3-chymotrypsin-like protease
(3CLpro) of SARS-CoV-2 inhibitors of a novel Schiff bases, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.129454
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Highlights
• Synthesis new Schiff bases and characterized by IR, NMR and elemental
analysis.
• In vitro antibacterial activity were investigated.
• Calculations of the density functional theory
• The synthetic compounds have also been docked with 3-chymotrypsin-like
protease (3CLpro).

Synthesis, anti-bacterial evaluation, DFT study and molecular
docking as a potential 3-chymotrypsin-like protease (3CLpro) of
SARS-CoV-2 inhibitors of a novel Schiff bases
Ahmed S. M. Al-Janabi1, Amin O. Elzupir2, Tarek A. Yousef 3, 4*
1 Department of Biochemistry, College of Veterinary Medicine, Tikrit University, Tikrit, Iraq.
2
Imam Mohammad Ibn Saud Islamic University (IMSIU), College of Science, Deanship of Scientific
Research, Riyadh, KSA
3 Department of Chemistry, Science College, Imam Mohammad Ibn Saud Islamic University, (IMSIU),
Riyadh, KSA, P.O. Box 90950, Riyadh 11623, Saudi Arabia
4Toxic and Narcotic drug, Forensic Medicine Department, Mansoura Laboratory, Medicolegal
organization, Ministry of Justice, Egypt
* Corresponding author. Tel.: +201222247712.
E-mail address: drroka78@yahoo.com
Abstract
New Schiff bases {N'-(phenyl(pyridin-2-yl)methylene) isonicotinohydrazide
(L1H), N1-(naphthalen-1-yl)-N2-(phenyl(pyridin-2-yl) methylidene) ethane-1,2-
diamine
(L2H),
N-(6-chlorobenzo[d]thiazol-2-yl)-1-phenyl-1-(pyridin-2-yl)
methanimine (L3H)}were synthesized by reaction of 2-benzoylpyridine with different
amines (2-amino-6-chlorobenzothiazole, isonicotinohydrazide and N1-(naphthalen-1-
yl)ethane-1,2-diamine) and characterized by 1H-NMR, 13C-NMR, IR mass
spectroscopy and elemental analysis. The compounds were assayed by the disc
diffusion method for anti-bacterial against five pathogenic bacteria species
(Staphylococcus aureus, Micrococcus luteus, Staphylococcus pyogenes, Bacillus
subtilis, and E. coli). All prepared Schiff bases showed good activity compared to
positive control (streptomycin), Moreover the L3H showed the highest activity against
S. aureus, and M. luteus than the other compounds and streptomycin. In additional
molecular docking studies with 3-chymotrypsin-like protease (3CLpro), the essential
enzyme for SARS-CoV-2 proliferation. The rest of compounds have shown promising
results as 3CLpro inhibitors interacting with the active sites of the enzymes. Finally,
DFT 's estimated electrostatic molecular potential results were used to illustrate the
molecular docking findings. The DFT calculations showed that L3H has the highest
dipole moment and electrophilicity index. Interestingly, L2H of the largest energy gap

∆E = 2.49 eV, there are several hydrophilic interactions that could facilitate the
binding with the receptors. All of these parameters could be shared to significantly
affect the protein sites of binding affinity with different extent
.
Keywords: COVID 19; DFT calculations; Schiff Bases; Molecular Docking
1. Introduction
Novel coronavirus (COVID-19) has arisen as an infectious disease and spread rapidly
throughout the world and is transmitted mainly through contact with contaminated
saliva droplets or by nose discharge while Patients diagnosed with cough or sneeze.
Human coronaviruses were first described in the mid-1960s [1,2]. Coronaviruses
belong to the Coronaviridae family, a family of single stranded enveloped- positive
sense RNA viruses. In addition, the Coronaviridae family was divided into four
genera: α, β, γ, and δ. Coronaviruses of ubiquitous and genera commonly infect
mammals and humans while birds are primarily infected by the form and generations.
That specification is in line with coronavirus phylogenetic analysis and genome
structure [3]. Computational features of the novel coronavirus [4,5] or more generally
new testable theories for standard drugs involved. A virtual screening technique has
recently been performed to identify the active site on the viral protease for the binding
of many natural compounds by molecular docking and cell-based assays [6]. Our
research group recently concentrated on determining the molecular geometry of
synthesized materials by comparing the desired properties from experimental
evaluation to estimated parameters from computational calculations [7].
Schiff bases have been reported to possess a wide range of biological properties such
as being anti-tumor, antiviral, anti-bacterial, anti-fungal and anti-inflammatory etc. [8]
Schiff bases containing N, S, and O atoms in structures show an important role in
biological systems because they have unusual electronic properties [9].
2-phenylquinazoline-4(3)H-one Schiff bases are confirmed to have antiviral activity
against certain strains of viruses, such as feline corona virus, influenza Viruses, and
type 1 and type 2 herpes simplex viruses [10]. From published literature, the antiviral
ability of these Schiff bases is evident and thus further focused work will help to
discover and improve new potential lead compounds to use them as drug candidates.

Recently a novel SARS-CoV-2 virus properly originated from bat was reported, cause
the severe acute respiratory syndrome, known as COVID-19 [11-13]. The enzyme 3-
chymotrypsin-like protease (3CLpro) cleaves at least 11 sites on the polyproteins
translated from the viral RNA of SARS-CoV-2. Thus, the compounds having ability
to inhibit this enzyme can be considering as an effective therapeutic agent for
COVID-19. This work describes the synthesis and spectroscopic characterization of
new Schiff bases and evaluated as anti-bacterial, besides, the synthetic compounds
have also been studied as 3CLpro inhibitors.
2. Experimental
2.1 Materials and Methods
2-amino-6-chlorobenzothiazole,
isonicotinohydrazide,
N1-(naphthalen-1-
yl)ethane-1,2-diamine, 2-benzoylpyridine and solvents were obtained through Sigma-
1
Aldrich, and used without any further purification. H and 13C NMR spectra were
recorded on Bruker Avance 400 MHz NMR spectrometer in DMSO as deuterated
solvent. The melting point was measured using SMP30 melting point apparatus. IR
spectra were recorded on a Shimadzu FT-IR 8400 spectrophotometer using KBr discs
in 400-4000 cm-1 range.
2.2 Preparation of Schiff bases
2.2.1 Preparation of N'-(phenyl(pyridin-2-yl)methylene)isonicotinohydrazide
(L1H)
A colorless solution of 2-benzoylpyridine (0.730 g, 3.985 mmol) in EtOH
(15mL) was added to an ethanol solution of isonicotinohydrazide (0.547g, 3.985
mmol) in (20 mL) with some drops of glacial acetic acid. The mixture was refluxed
for 6 h, the formed solution was filtered and left aside to cool slowly, then the dark
creamy ppt. was separated and recrystallized from hot ethanol and dried under
vacuum.
L1H. Dark creamy powder. Yield: (1.16 g, 91%). M.p. 182-183°C. Anal. Calc. for
C18H14N4O, (%):C, 71.51; H, 4.67; N, 18.53. Found C, 71.69; H, 4.53; N, 18.62. IR
(KBr, cm-1): 3110m(NH), 3049m, 2858m, 1668(C=O), 1633s (C=N), 1554s(C=C),
1487w, 1407s, 1330s, 1135s, 995s, 844s, 744s.1H NMR (, ppm, DMSO-d6): δ

11.53(s, 1H, NH), 8.92(d, J = 7.2 Hz, 2H, H15,16), 8.74 (dd, J = 7.4, 2.5 Hz, 1H, H1),
8.12 (dd, J = 7.4, 1.3 Hz, 1H, H3), 7.92(d, J = 7.2 Hz, 2H, H14,17), 7.71 (d, J = 7.6
Hz, 1H, H4), 7.62 (d, J = 7.5 Hz, 1H, H2), 7.39 (m, 5H, H8-12). 13C NMR (, ppm,
DMSO-d6): δ 169.53 (C=O), 161.92 (C6), 152.13(C5), 149.73(C1), 147.81(C15,16),
140.29(C7), 132.63(C3), 131.04(C8,12), 129.78(C10), 128.12(C9,11), 123.22
(C14,17), 119.39(C4). ESI-MS (m/z), calc. C18H14N4O, 302.116, found, 302.117.
2.2.2 Preparation of N1-(naphthalen-1-yl)-N2-(phenyl(pyridin-2-yl)methylidene)
ethane-1,2-diamine (L2H)
L2H was prepared by a similar method to that of L1H using N1-(naphthalen-1-
yl)ethane-1,2-diamine in place of isonicotinohydrazide.
L2H. Light brown-yellow powder. Yield: (1.20 g, 86%). M.p. 167-168°C. Anal. Calc.
for C24H21N3 (%):C, 82.02; H, 6.02; N, 11.96. Found C, 82.17; H, 6.23; N, 12.18. IR
(KBr, cm-1): 3131m (NH), 3087m, 2945w(C-H), 1639s (C=N), 1529s(C=C), 1452s,
1311s, 1164w, 927m, 811s, 690s, 607m. 1H NMR (, ppm, DMSO-d6): δ 8.72 (ddd, J
= 6.7, 1.4 Hz, 1H, H2), 8.07 (ddd, J = 7.7, 1.7 Hz, 1H, H3), 7.98 (m, 4H, H2,4,14,15),
7.67 (m, 3H, H9,10,11), 7.54 (t, J = 7.7 Hz, 2H, H8,12), 7.43 (s, 1H, NH), 7.32 (d, J =
6.8 Hz, 2H, H19,22), 7.21 (d, J = 8.1 Hz, 1H, H14), 7.01(dd, J = 7.7 Hz, 2H, H20,21),
13
2.302 (s, 4H, NH-CH2), 2.295 (s, 2H, CH2C=N). C NMR (, ppm, DMSO-d6): δ
165.28 (C6), 154.13(C13), 150.29(C5), 148.21(C1), 137.30(C7), 135.64(C17),
132.639(C3),
130.64(C18),
130.22(C8,12),
129.50(C10),
127.85(C9,11),
126.37(C16,19), 126.04(C15,22), 123.78(C20,21), 120.44(C4), 117.04(C14), 21.48
(CH2-N=C) 20.36 (CH2-NH). ESI-MS (m/z), calc. C24H21N3, 351.174, found,
351.176.
2.2.3 Preparation of N-(6-chlorobenzo[d]thiazol-2-yl)-1-phenyl-1-(pyridin-2-yl)
methanimine (L3H)
L3H was prepared by a similar method to that of L1H using 2-amino-6-
chlorobenzothiazole in place of isonicotinohydrazide.
L3H. brownish yellow powder. Yield: (0.93 g, 76%). M.p. 156-158°C. Anal. Calc. for
C19H12ClN3S, (%):C, C, 65.23; H, 3.46; N, 12.01; S, 9.16. Found C, 65.15; H, 3.28;

N, 12.31; S, 9.23. IR (KBr, cm-1): 3086m(CH), 1627s (C=N), 1542s(C=C), 1415s,
1315s, 1274s, 1141s, 1088s, 698s, 651m.1H NMR (, ppm, DMSO-d6): δ 8.80 (dd, J
= 7.8, 1.7 Hz, 1H, H1), 8.12 (s, 1H, H18), 7.98 (dd, J = 7.6, 1.5 Hz, 1H, H3), 7.72(d, J
= 7.4 Hz, 2H, H4), 7.68(d, J = 7.6 Hz, 1H, H2), 7.56 (m, 4H, H8,12,15,16), 7.36(m,
3H, H9-11). 13C NMR (, ppm, DMSO-d6): δ 171.78 (C13), 162.45 (C6), 153.40(C5),
148.92(C1), 147.81(C14), 141.55(C7), 134.07(C3), 133.65(C19), 131.73(C8,12),
128.96(C10), 128.03(C9,11), 126.56(15), 123.22(C16), 120.78(C18), 118.77(C4),
116.78(C17). ESI-MS (m/z), calc. C19H12ClN3S, 349.044, found, 349.033.
2.3 Antibacterial studies
The anti-bacterial activities of the complexes were tested by agar disc
diffusion method originally described by Bauer [14] against five bacteria types,
Staphylococcus aureus, Micrococcus luteus, Staphylococcus pyogenes, Bacillus
subtilis, and E. coli in (50, 100, and 200 g) of each compound compared with
streptomycin as positive control.
Minimum inhibitory concentration (MIC) of the Schiff bases were tested in in
Nutrient broth for bacteria by the two-fold serial dilution method [15]. The bacterial
suspension was attuned with sterile saline to a concentration of 1*10-5 - 10-6 CFU.
The tested compounds and standard control (Streptomycin) were prepared by two-fold
serial dilution to obtain the essential concentrations of 200, 100, 50, 25, 12.5 and 6.25
g/mL. The tubes were incubated in incubators at 37 C. The MICs were recorded by
visual observations after 24 h.
2.3 Optimization and molecular docking
The three-dimensional structures of the L1H, L2H, and L3H were generated and
optimized by Gaussian 09 program package [16] software. The crystal structure of
3CLpro of SARS-CoV-2 was obtained from the Protein Data Bank database (PDB ID:
6Y2E). Molecular docking experiments were carried out using the AutoDock Vina
tool plugin UCSF Chimera software (v 1.14), adopting the default values for the
parameters, and a grid box (-16 × -24.0 × 17) Å was centered at (35, 65, 65) Å. In
order to represents the real environment, water was added as a solvent, with
accessible surface area of 14358.5. The predicted binding affinity score was explored

utilizing the View Dock tool. The binding to active sites and images were processed
by the UCSF Chimera [17-19].
3. Results and Discussion
3.1 Synthesis of Schiff bases
The Schiff bases have been synthesized by condensation of 2-benzoylpyridine with 2-
amino-6-chlorobenzothiazole or isonicotinohydrazide or N1-(naphthalen-1-yl)ethane-
1,2-diamine to afford a dark creamy with L1H, light brown-yellow in L2H, and a
brownish yellow with L3H (Scheme 1). The prepared Schiff bases are stable in air and
soluble in EtOH, DMSO and DMF. The compounds were characterized by using
1
elemental analysis, H, 13C NMR, and IR techniques. All attempts to get crystals
suitable for X-ray diffraction studies were unsuccessful.
Scheme 1: Preparation of Schiff bases L1H, L2H and L3H
3.2 Characterization of Schiff bases
3.2.1 IR Spectra
The IR spectra of the prepared Schiff bases (Fig. 1) displayed a strong band
within the 1627-1639 cm-1 range for the azomethine group (C=N) [20-22]. And
disappeared the (C=O) of 2-phenylpyridine, and (NH2)sy, asy stretching vibration of
amine, indicates the formation of the proposed compounds. The (C=C) vibration of
the aromatic rings showed within the 1529-1554 cm-1 range [22]. The spectra of L1H

and L2H displayed a medium band at 3110cm-1 and 3131cm-1assigned to the (N-H).
And the L1H spectrum appeared the (C=O) of isonicotine group at 1668cm-1 [23]
Also the spectrum of L2H showed the stretching vibration of aliphatic group at
2945cm-1[22]. Other vibrations are listed in experimental section.
Fig. 1: IR spectra of the prepared Schiff bases
3.2.2 1H and 13CNMR Spectra
The 1H-NMR spectrum of L1H in DMSO-d6 displayed the NH proton at 11.53
ppm as a singlet peak. And four doublet peaks at 8.92ppm, 7.92ppm, 7.71ppm and
7.62ppm, assigned to the H15,16, H14,17, H4, and H2, corresponding to 2,1,2,1
protons respectively. Also the spectrum showed two doublet of doublets peaks at
8.74ppm, and 8.12ppm for the H1 and H3, respectively. A multiplet peak was
showed at 7.39ppm assigned to protons in position 8-12 (phenyl ring) (Fig. SI1,
supporting information).
1
The H-NMR spectrum of L2H (Fig 2), clearly display the phenyl protons
belong to the pyridyl, phenyl and naphthyl rings. A two doublet peaks displayed at
7.21ppm and 7.32ppm due to a H14 and H19,22, corresponding to one and two
protons, respectively. And two doublet of doublet of doublets at 8.72ppm, and

8.07ppm for the H2 and H3, respectively. Also the spectrum displayed two
multiplets peaks at 7.98ppm and 7.67ppm, due to the protons in position (1,4,15,16)
and (9-11), respectively. The protons in position 20 and 21 appeared as doublet of
doublets at 7.01ppm (JHH = 7.8 Hz). And the H8,12 showed as triplet peak at
7.54ppm with coupling constant to the neighboring protons (JHH = 7.7 Hz). The NH
proton was showed at 7.43 ppm as a singlet peak, whereas the methylene groups
displayed as two singlet at 2.302 and 2.295 ppm, due to the NH-CH2 and CH2N=C,
respectively, corresponding to four protons.
Fig. 2: 1H NMR spectrum of the L2H in DMSO-d6
1
The H-NMR spectrum of L3H in DMSO-d6 showed two doublet of doublets
peaks at 8.80ppm, and 7.98ppm for the H1 and H3, respectively. And two doublet
peaks at 7.72ppm, and 7.56ppm, due to the H4 and H2, corresponding to one
proton of each peak, respectively. Also the spectrum showed two multiplet peaks
were showed at 7.56ppm and 7.36ppm due to protons in position (8,12,15,16) and
(9-11). The H18 was showed as a singlet peak at 8.12ppm (Fig. SI 3, supporting
information).
13
The above results were supported by C NMR and elemental analysis data (Fig.
3). The C of the azomethine group (C6) of the new Schiff bases appeared at
161.92ppm, 165.28ppm, and 162.45ppm for the L1H, L2H and L3H respectively.

Whereas the C1 displayed at 148.21ppm, 149.73ppm, and 148.92ppm, respectively.
And that the methylene group of the L2H appeared at 21.48ppm, 20.36ppm assigned
to (CH2-N=C) and (CH2-NH) respectively. The spectrum of L1H displayed the
chemical shift of carbonyl group at δ 169.53ppm. Other 13C chemical shifts are listed
in experimental section(Fig. SI 2 and 4, supporting information).
Fig. 3: 13C NMR spectrum of the L2H in DMSO-d6
3.3 Antibacterial Activity
Antibacterial activity of the Schiff bases (L1H, L2H and L3H) are summarized
in Table 1. The data were obtained against five types of the pathogen bacteria
(Staphylococcus aureus, Micrococcus luteus, Staphylococcus pyogenes, Bacillus
subtilis, and Escherichia coli in (50, 100, and 200 g/Disc) of each compound
compared with streptomycin as positive control. The diameter of the inhibitory zone
(DIZ) was compared to that of streptomycin, which is the positive control. The
compounds displayed good activity against the bacteria. The Schiff base L3H is more
active for the tested bacteria compared with other Schiff bases (L1H, and L2H),
whereas the L2H, exhibits less activity against all the tested bacteria. Also designate

the increasing concentration of Schiff bases from 50 to 200 g/disc, the inhibition
effect is increased.
Table 1: Antibacterial activity (diameter of inhibition zone (cm)) of Schiff bases
against five four different bacterial species
Conc.
Micrococcus
luteus
Staphylococ
cus aureus
Staphylococcus
pyogenes
Bacillus
subtilis
Escherich
ia coli
Comp.
g/disc
50
9
12
11
8
10
L1H
100
200
14
19
15
19
14
18
12
17
13
17
50
9
8
10
9
9
L2H
100
200
13
16
12
17
12
15
12
17
11
14
50
12
14
13
10
11
L3H
100
200
16
19
20
24
17
18
15
19
14
18
Streptomycin
100
22
19
21
19
23
The MICs are given in Table 2. Schiff base L3H displayed the highest effective
against the tested bacteria S. aureus, M. luteus, S. pyogenes, B. subtilis with MIC
values of 6.25, 25, 25, and 25g/mL, respectively, but less active against E. coli, with
MIC value 100g/mL. The antibacterial activity of L3H is more active than the other
Schiff bases (L1H, and L2H), suggesting the L3H compound has a (Cl and S) atoms in
the structure, these atoms possibly via enhanced membrane transport into the cell or
some other mode of action [24,25].
Table 2: Minimum inhibitory concentration (MIC) in (g/mL) of Schiff bases.
Micrococcus Staphylococcus Staphylococcus
Bacillus
subtilis
Escherichia
coli
Compounds
luteus
aureus
pyogenes
L1H
L2H
50
25
100
50
100
25
100
50
25
25
50
50
25
L3H
12.5
3.12
100
6.25
Streptomycin
12.5
6.25
12.5

3.4 Theoretical studies
3.4.1 DFT Calculations Studies
At B3LYP 6-311G (d,p) basis set, the theoretical DFT calculations were carried
out in gas phase by DFT method. The results of the theoretical DFT calculations for
all the compounds under investigation (Figs 4-6) showed the non-planarity. Table 3
summarizes the approximate calculations of DFT for Electronic Energy, Heat
Capacity, Entropy (S), Thermal Energy, polarizability, and dipole moment of L1H,
L2H, and L3H.
Table 3: Electronic Energy (Hartree/Particle), Heat Capacity (Cv), Entropy (S)
(cal/mol-kelvin), Thermal Energy polarizability α (a.u.), and dipole moment (Debye)
of L1H, L2H, and L3H
Parameter
L1H
-988.49
7.99
L2H
-1091.40
2.59
L3H
-1745.44
3.33
Electronic Energy
Total Dipole Moment
Polarizability (α)
E (Thermal)
235.49
192.294
69.839
142.655
285.79
261.24
86.79
268.28
69.58
Heat Capacity (Cv)
Entropy (S)
67.89
166.53
134.04
DFT calculated data showed that the L1H, L2H, and L3H dipole moment are in
the order of L2H < L3H < L1H. The high dipole moment L1H may demonstrate their
binding pose within a specific target protein and its results from the predicted binding
affinity to be discussed in the next molecular docking section. The polarizability of
the materials depends on how a charge approach influences the resistance of the
molecular system electron cloud. Additionally, it depends on the nature of the
compounds and the scale of the molecular structure. Large-sized molecules are more
polarizable compounds. L1H is the smallest in size and has the least polarizability
(235.49 a.u.), but L2H with the greatest complexity is expected to have the greatest
polarizability, (285.79 a.u.). Ultimately, heat capacity is the one with the most

complex set of concepts and the richest set of consequences for protein folding and
binding, of all the major thermodynamic variables calculated for proteins. It gives
entropy and enthalpy a temperature dependency which will alter their signs and
determine which of them will dominate. The heat capacity order is as follows: L3H <
L1H < L2H. The unfolding protein typically has a positive Cp, which results in
optimum stability and often cold denaturation [26].
Fig. 4: Optimized geometrical structures of L1H, L2H, and L3H with atomic
numbering.
3.4.2. Frontier Molecular Orbitals
The frontier molecular orbitals (FMO) can provide objective qualitative
information about the HOMO electrons being susceptible to transfer to the LUMO.
In addition, HOMO and LUMO are very useful quantum chemical parameters to
assess the molecules' reactivity and are used to measure other parameters, such as
the descriptors for chemical reactivity. The energies of the studied compounds'
HOMOs and LUMOs were measured using DFT method at the base set of B3LYP
6-311 G (d, p) and are tabled in Table 5. The isodensity surface plots of HOMO and
LUMO for L1H, L2H, and L3H are shown in Fig. 5.

The results of the FMOs energy analysis revealed that the energies of HOMOs of
L3H is higher compared with L1H and L2H. However, the destabilization of the
LUMO level is found to be higher in L3H than the others. Consequently, the energy
gap is in the order of L1H < L3H < L2H.
Fig. 5: HOMO and LUMO plots of the L1H, L2H, and L3H compounds
Recently, many reports showed that the FMOs have to be taken into
consideration in investigation of the structure activity relationships [27-29]. The
FMOs theory showed that the energy level of the HOMO and the LUMO are the most
significant aspects that impact the bioactivities of small structural drugs. Mainly
HOMOs that offer electrons, however, the LUMOs accept electrons. Obviously, the
level of energy of HOMOs are different for all studied compounds. L3H showed the
most lying HOMO than the other compounds and consequently it could be a better
electron donor drug. Interestingly, L2H of the largest energy gap ∆E = 2.49 eV, there
are several hydrophilic interactions that could facilitate the binding with the receptors.
This suggests that such hydrophilic interactions considerably impact the binding
affinity of such small drugs to the receptors. The HOMO of a certain drug and the

LUMO with the adjacent residues could share the orbital interactions during the
binding process.
Table 5: Calculated EHOMO (EH), ELUMO (EL), energy band gap (EH – EL), chemical potential (μ),
electronegativity (χ), global hardness (η), global softness (S), global electrophilicity index (ω) and
softness (σ) for L1H, L2H, and L3H.
EH /
eV
EL /
eV
(EL-EH)
/Ev
Comp.
χ / eV μ / eV η / eV S / eV-1 ω / eV σ / eV-1
-6.6
-6.16
0.44
6.38
-6.38
0.22
0.11
92.51
5.74
3.56
2.27
0.40
1.49
L1H
L2H
L3H
-7.32
-5.39
-4.83
-4.72
2.49
0.67
6.075 -3.78 1.245
5.055 -1.55 0.335
0.6225
0.1675
3.4.3. Chemical Reactivity Descriptors
Calculations, such as the energy of the highest occupied molecular orbital,
EHOMO, energy of the lowest unoccupied molecular orbital, ELUMO, obtain
quantium chemical parameters of organic compounds. Additional parameters, such as
separation energies (ΔE), absolute electro-negativities (v), chemical potentials (Pi),
absolute hardness (g), absolute softness (r), global electrophilicity (x), global and
softness (S) were calculated by equations ( 1-3)[30,31].
χ = -1/2 (ELUMO + EHOMO
)
(1)
(2)
(3)
(4)
(5)
µ= - χ =1/2 (ELUMO + EHOMO
)
η =1/2 (ELUMO - EHOMO
)
S =1/2 η
ω = µ2/2 η
The inverse value of the global hardness is designed as the softness (σ) as follow:
σ =1/ η (6)

3.4.5. Molecular Electrostatic Potential (MEP)
The molecular electrostatic potential (MEP) is important to quantify in order to
validate the evidence regarding the reactivity of the compounds studied as inhibitors.
Even though the MEP provides an indication of the molecular size and shape of both
the positive, negative and neutral electrostatic potential. This may be a method for
predicting relationships of physicochemical properties with the molecular structure of
the drugs being investigated. In addition, the electrostatic molecular potential is a
valuable method for estimating drug reactivity against electrophilic and nucleophilic
attacks.
Under the same base sets, the molecular electrostatic potential of the L1H, L2H,
and L3H is determined using the same process and is seen in Fig. 6. The maximum
negative area within the MEP is the chosen electrophilic attack sites, indicated as red
color. So, the negatively charged sites, and the reverse satiation for the blue regions,
should draw an attacking electrophile. It is apparent that the molecular size and shape
as well as the orientation of the negative, positive, and neutral electrostatic potential
differed by product due to the type of atoms and their electronic existence. The
difference in mapping the electrostatic potential around the compound may be
primarily responsible for variation of its binding receptor affinities.
Fig.6: Molecular electrostatic potentials (MEP) of the L1H, L2H, and L3H
compounds

3.4.6. Mulliken Atomic Charges
The Mulliken atomic charges of the estimated compounds (1–3) were
calculated the DFT using B3LYP 6-311G (d,p) at a basis set, the data were tabulated
in Table 6. It showed that the C5 is the most positive and O6 have the most negative
charge for L1H. In case of L2H it is observed that the most nucleophilic centers are
N4, N5 and N17 which are the most electrophilic susceptibility positions. On the
other hand, it is obvious that the nucleophilic susceptibility of the L2H is recognized
on C16 sites. However, N4, N5 and N16 are the most negative charges of L3H, while
its respective positively charged atoms are C5 and S19. The positively charged
centers are the most susceptible sites for nucleophilic attacks i.e., electron donation.
However, the most negatively charged centers are the most susceptible sites for
electrophilic one [32].
Table 6. The Mulliken atomic charges of L1H, L2H, and L3H.
L1H
L2H
L3H
1
2
3
4
5
6
7
8
9
C -0.111215
1
2
3
4
5
6
7
8
9
C -0.015149
1
2
3
4
5
6
7
8
9
C -0.201304
C
C
0.106601
0.134528
C
C
0.003903
0.130135
C
C
0.280357
0.068197
N -0.167052
0.511645
N -0.263679
N -0.334325
N -0.350220
N -0.328298
C
O -0.397335
C -0.091149
N -0.485201
C
0.205425
C
C
C
C
0.106955
0.039048
0.026534
0.060093
C -0.027529
C
C
C
0.005259
0.051085
0.067703
C
0.051185
10 C -0.202889
11 C -0.098940
12 C -0.094946
13 C -0.108201
14 C -0.165764
15 C -0.126124
16 C -0.164606
17 C -0.089722
18 C -0.177462
19 C -0.000692
20 N -0.330006
21 C -0.031671
22 C -0.080192
23 N -0.561293
10
10 C -0.036389
11 C 0.026019
12 C -0.022731
13 C 0.096392
14 C 0.137059
15 C 0.177593
16 N -0.650109
17 C 0.285513
18 C -0.486679
19 S 0.552361
20 C 0.167863
21 C 0.035451
22 C -0.238069
23 C 0.136350
24 Cl 0.118015
11 C -0.033723
12 0.028768
13 C -0.077589
14
15
16
17 N -0.381044
18
19
20 C -0.010492
21 0.034302
22 C -0.058167
23
24
25
C
C
C
C
0.106634
0.123458
0.192665
C
C
0.119451
0.056393
C
C
C
C
0.008089
0.005574
0.000038
26 C -0.014334
27 0.077149
C

3.5 Molecular docking:
The synthetic novel Schiff bases have shown a significant rule against CLpro, the
important enzyme for proliferation of SARS-COV-2 virus. The inhibitory effect was
investigated based on their interaction to the catalytic residues of His41 and Cys145, the
essential residues of Glu166 and Ser1 for maintaining CLpro on the correct conformation
[33]. The synthetic compounds have been docked with 3CLpro. They form hydrogen
bonds (blue stripes) and Van der Waal’s interactions (yellow stripes) as depicted in
Figs 7 to 9. L2H and L3H have shown the best binding affinity of -7.6 for both. Of
them, L3H had a better RMSD as presented it Table 7. the synthetic Schiff bases L1H
OPT has shown a tendency to form hydrogen bonds with the active residue of Glu166.
Whereas, they are all interact with others catalytic residues of of His41 and Cys145 of
CLpro. Our results suggest these compounds as a potent CLpro inhibitors, particularly
L3H and thus could be used for the treatment of COVID-19 after a suitable in vitro
and in vivo validation as well as clinical trials.
Table 7. The binding interactions of L1H, L2H, and L3H to 3-chymotrypsin-like
protease of SARS-CoV-2.
Pharmaceutical
name
Score
(kcal/mol)
Hydrogen bond
(distance)
RMSD
Van der Waal’s (distance)
Glu166 (2.275 Å) His41 (3.664 Å/ 3.777 Å).
L1H
L2H
L3H
-6.6
-7.6
-7.6
27.120 –
29.360
Cys145 (3.470 Å/ 3.490 Å).
Glu166 (14 side contacts, distance
range: 2.275 Å to 3.826Å).
-
-
His41 (5 side contacts, distance
range 3.464 Å to 3.785 Å).
Cys145 (3.707 Å)
28.639 –
31.280
Glu166 (6 side contacts, distance
range 3.440 Å to 3.780 Å).
His41 (4 side contacts, distance
range 3.624 Å to 3.826 Å).
Cys145 (3.779 Å)
1.524 -
6.174
Glu166 (4 side contacts, distance
range 3.390 Å to 3.955 Å).

Fig. 7: L1H docked with 3-chymotrypsin-like protease (3CLpro) of SARS-CoV-2.
Hydrocarbon skeleton of L1H is cyan, nitrogen atoms are blue, oxygens red. Below, different
side of view of magnified images of its contact sites to HIS41, CYS145 and GLU166.
Fig. 8: L2H docked with 3-chymotrypsin-like protease (3CLpro) of SARS-CoV-2.
Hydrocarbon skeleton of L2H is cyan, nitrogen atoms are blue, oxygens red. Below, different
side of view of magnified images of its contact sites to HIS41, CYS145 and GLU166.

Fig. 9: L3H docked with 3-chymotrypsin-like protease (3CLpro) of SARS-CoV-2.
Hydrocarbon skeleton of L3H is cyan, nitrogen atoms are blue, oxygens red. Below, different
side of view of magnified images of its contact sites to HIS41, CYS145 and GLU166.
Conclusion:
New Schiff bases, N'-(phenyl(pyridin-2-yl)methylene)isonicotinohydrazide (L1H), N-
(2-((phenyl(pyridin-2-yl)methylene)amino)ethyl)naphthalen-1-amine (L2H), and N-
(6-chlorobenzo[d]thiazol-2-yl)-1-phenyl-1-(pyridin-2-yl)methanimine (L3H) were
synthesized by condensation of 2-benzoylpyridine with three different amines and
characterized by different physicochemical studies. The antimicrobial activities of all
complexes were tested at three concentrations 50, 100 and 200 g/mL, against five
bacterial types. L3H is more active for the tested bacteria compared with other Schiff
bases (L1H, and L2H) and has highest effective against the tested bacteria S. aureus,
M. luteus, S. pyogenes, B. subtilis with MIC values of 6.25, 25, 25, and 25g/mL,
respectively, whereas a less active against E. coli, with MIC value 100g/mL. The
L3H compound is more active than the other Schiff bases because it has a (Cl and S)
atoms in the structure, these atoms possibly via enhanced membrane transport into the
cell or some other mode of action. The compounds have also been docked with
3CLpro. They have shown good interaction with the catalytic sites of 3CLpro, thus
could be consider as a potent inhibitors and therapeutic agents for COVID-19 after

suitable in vitro and in vivo validation as well as clinical trials. The molecular docking
results were illustrated in terms of the DFT calculations. The results of the DFT
showed that L3H is the most lying HOMO and therefore it may be the best to serve as
an electron donor. In addition, N4, N5 and N16 are the most electrophilic centers of
L3H and this may be due to the presence of the lone pair of electrons on these atoms.
Finally, performing further in vitro and small animal models is very promising in vivo
studies establishing solid experimental evidence of its activity as COVID-19
inhibitors.
Conflict of interest statement
The authors declare no conflict of interest.
Credit authorship contribution statement
Ahmed S. Al-Janabi, Supervision, Conceptualization, Methodology, Investigation,
Writing - review & editing.
Tarek A. Yousef, Writing - review & editing, Data curation, Formal analysis,
Amin O. Elzupir Methodology, Investigation, Software, Writing - review & editing.
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