A.Z. Omar, T.M. Mosa, S.K. El-sadany et al.
Journal of Molecular Structure 1245 (2021) 131020
Table 3
In silico predicted pharmacokinetic properties of ligands 3–12 using pkCSM tool.
Absorption
Distribution
Metabolism
CYP1A2
Excretion
TC
Toxicity
Lig.
S
IS
SP
BBBP
CNSP
CYP2C19
CYP2C9
MTD
AMES
ORAT
HT
SS
3
3
4
5
6
6
7
8
9
1
1
1
a
−3.45
−4.18
−3.14
−4.40
−4.50
−4.88
−2.86
−4.72
−1.97
−2.97
−1.93
−1.96
91.88
91.04
62.84
100.0
95.77
94.74
54.77
66.53
90.46
61.70
90.71
92.70
−2.59
−2.42
−3.24
−2.94
−2.90
−2.96
−2.73
−2.74
−4.07
−2.90
−2.93
−1.86
0.39
−3.36
−3.06
−3.69
−3.22
−3.13
−3.10
−3.40
−2.67
−3.08
−2.73
−2.97
−2.27
No
Yes
No
Yes
Yes
Yes
No
No
No
No
No
Yes
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
0.53
0.51
−0.21
0.20
0.29
0.33
0.24
−0.09
0.67
1.09
0.48
0.82
0.57
0.44
1.37
0.28
0.17
0.19
0.65
−0.46
0.72
−0.58
0.23
0.13
No
No
No
No
No
No
No
Yes
No
No
Yes
No
3.03
3.16
3.33
2.64
3.19
3.09
2.96
2.57
2.65
2.65
2.53
2.78
Yes
No
Yes
Yes
Yes
No
No
No
No
No
No
No
No
Yes
b
0.57
−0.87
−0.32
0.05
No
Yes
Yes
Yes
Yes
Yes
No
a
b
0.01
−2.08
−1.05
−0.23
−0.33
−0.45
0.627
0
1
2
Yes
Yes
Yes
cerning blood brain permeability, the ability of the drug to cross
into the brain is important to decrease toxicities and side effects.
The predicted data revealed that ligands are lipophilic enough to
cross the blood brain barrier except for ligand 7 which exhib-
ited log BB less than −1. Additionally, most of the ligands having
no permeation to central nervous system (CNS) with blood brain
permeability-surface area product logPS less than −3. Regards to
the predicted Metabolism properties, it was found that the lig-
ands under study were predicted as inhibitors or non-inhibitors for
some cytochrome P450 isoenzymes such as CYP1A2, CYP2C19 and
CYP2C9. None of the assessed ligands was predicted as inhibitor
for CYP2C19 or CYP2C9 except ligands 3b and 12, respectively. On
the other hand, ligands 3b, 5, 6a, 6b and 12 were predicted as
CYP1A2 inhibitor. This inhibitory effect on CYP isoenzyme activity
may cause the probability of drug interactions. Moreover, excretion
properties expressed in total clearance log(CLtot) have been predi-
cated for all ligands under study which is important for determin-
ing dosing rates to reach steady state concentrations.
Asn142, Gly143, His163, Ser144, Met165, Glu166, Gln189, Thr26,
His41, Thr25, Cys145, Phe140, Met49 and Arg188’’ as the potential
drug binding sites, with more than one drug binding site identified
with eight inhibitors candidates. While for other ligands the struc-
ture had only one active site residue each. The best noted binding
energy value was obtained for ligand 8 (−6.87 kcal/mol). This lig-
and exhibited hydrogen bonding interactions between its thiosemi-
carbazide moiety and three amino acids residue at the backbone
of the enzyme namely, GLY143, ASN142 and THR26, as well as hy-
drogen bonding interaction between its nitro group and GLN189
residue. Moreover, ligand 10 with slightly lower protease inhibitory
activity than 8 (binding energy ≈ −6.81), interacted with the ac-
tive site at HIS43 and THR25 via H-p interactions and hydrogen
bonding interactions through its isatin keto group and HIS163. Al-
ternatively, ligand 12, which showed the lowest protease inhibi-
tion among the nominated ligands, only displayed p-H binding in-
teraction with the active site at MET165 via its phenyl rings but
failed to display any hydrogen bonding interactions. It could be
concluded that the appropriate substitution of the amino groups of
the piperazine scaffold (ligands 8, 10, 6a, 6b and 7), showed sig-
nificant hydrogen bonding interactions with SARS-CoV-2 protease
enzyme that possibly allowed improved inhibitory activity. Based
on the lowest dock energy value scored by 8 in relation to other
ligands, this appears to be the drug of choice for treating COVID-
19 infection. Since all the ligands have been docked with negative
dock energy onto the target protein, it will be practical to give
equal importance to all these protease inhibitor ligands.
The parameters related to toxicity including maximum tolerated
dose (MTD), AMES test, oral rat acute toxicity (ORAT), hepatotoxi-
city (HT), and skin sensitization (SS) have been predicted. AMES
test is employed to assess whether the drug is mutagenic or not
[
48]. All the synthesized ligands except 8 and 11 exhibited nega-
tive AMES tests indicating that they are non-mutagenic. Moreover,
ligands showed relatively high predicted lethal dose value LD50
(
2.53–3.33 mol/Kg) which is indicative of low acute toxicity.
The predicted hepatotoxicity showed that ligands 3a, 5, 6, 7, 8,
10, 11 and 12 would probably show hepatotoxicity associated with
3. Experimental
disrupted normal function of liver. However, most of the assessed
ligands don’t show skin sensitization
3
.1. Instruments and apparatus
2
.5. Molecular docking
Melting points were determined by MEL-TEMP II melting point
apparatus in open glass capillaries. The IR spectra were recorded as
potassium bromide (KBr) discs on a Perkin-Elemer FT-IR (Fourier-
Transform Infrared Spectroscopy), Faculty of Science, Alexandria
University. The NMR spectra were carried out at ambient tem-
perature (~25 °C) on a (JEOL) 500 MHz spectrophotometer using
tetramethylsilane (TMS) as an internal standard, NMR Unit, Faculty
of Science, Mansoura University. Elemental analyses were analyzed
at the Regional Center for Mycology and Biotechnology, Al-Azhar
University, Cairo, Egypt.
Considering the global threat posed by COVID-19, and with no
proven antiviral agent available for immediate relief, the current in
silico study provide structural insights about the protease of SARS-
CoV-2 and its molecular interactions with synthesized ligands 3–12
as protease inhibitors. Protease enzyme is essential for viral repli-
cation because it catalyzes the proteolytic process for the polypro-
teins that are translated from the viral RNA [49]. Thus, inhibition of
the protease activity would block viral replication and unlikely to
be toxic for human, which make protease one of the best drug dis-
covery targets in case of coronaviruses. The activity of protease en-
zyme is blocked by binding of inhibitor molecules to the active site
of the enzyme. Moreover, protease is suitable for designing wide-
spectrum inhibitors because it contains large number of amino
acids. All the eleven protease inhibitors candidates got docked onto
the predicted 3D model of protease of SARS-CoV-2 with a neg-
ative dock energy value as shown in Table 4. Molecular interac-
tion studies, Fig. 2, showed that protease model of SARS-CoV-2 had
3.2. Docking program
Molecular docking simulations were performed to achieve the
mode of interaction of prepared piperazines with the binding
pocket of SARS-CoV-2 protease. The newly released crystal struc-
ture of SARS-CoV-2 main protease as a receptor was retrieved from
ware version 2015.10 of Molecular Operating Environment (MOE)
5