Design and synthesis of novel phe-phe hydroxyethylene derivatives as potential coronavirus main protease inhibitors

Article abstract of DOI:10.1080/07391102.2021.1905549

In response to the current pandemic caused by the novel SARS-CoV-2, we design new compounds based on Lopinavir structure as an FDA-approved antiviral agent which is currently under more evaluation in clinical trials for COVID-19 patients. This is the first example of the preparation of Lopinavir isosteres from the main core of Lopinavir conducted to various heterocyclic fragments. It is proposed that main protease inhibitors play an important role in the cycle life of coronavirus. Thus, the protease inhibition effect of synthesized compounds was studied by molecular docking method. All of these 10 molecules, showing a good docking score compared. Molecular dynamics (MD) simulations also confirmed the stability of the best-designed compound in Mpro active site. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2021.1905549

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Design and synthesis of novel phe-phe
hydroxyethylene derivatives as potential
coronavirus main protease inhibitors
Zahra Khorsandi, Maral Afshinpour, Fatemeh Molaei, Rafee Habib Askandar,
Fariba Keshavarzipour, Maryam Abbasi & Hojjat Sadeghi-Aliabadi
To cite this article: Zahra Khorsandi, Maral Afshinpour, Fatemeh Molaei, Rafee Habib Askandar,
Fariba Keshavarzipour, Maryam Abbasi & Hojjat Sadeghi-Aliabadi (2021): Design and synthesis
of novel phe-phe hydroxyethylene derivatives as potential coronavirus main protease inhibitors,
Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2021.1905549
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2021.1905549
Design and synthesis of novel phe-phe hydroxyethylene derivatives as potential
coronavirus main protease inhibitors
Zahra Khorsandia, Maral Afshinpourb, Fatemeh Molaeic, Rafee Habib Askandard, Fariba Keshavarzipoure,
Maryam Abbasif
and Hojjat Sadeghi-Aliabadig
b
aDepartment of Chemistry, Isfahan University of Technology, Isfahan, Iran; Bioinformatics Lab., Department of Biology, School of Sciences,
c
Razi University, Kermanshah, Iran; Department of Anaesthesiology, Facility of Paramedical, Jahrom University of medical science, Jahrom,
d
e
Iran; Research Center, Sulaimani Polytechnic University, Sulaimani, Iraq; Isfahan Pharmaceutical Sciences Research Center, School of
f
Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran; Department of Medicinal Chemistry, Faculty of Pharmacy, Hormozgan
University of Medical Sciences, Bandar Abbas, Iran; Department of Medicinal Chemistry, Faculty of Pharmacy, Isfahan University of Medical
g
Sciences, Isfahan, Iran
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 18 October 2020
Accepted 15 March 2021
In response to the current pandemic caused by the novel SARS-CoV-2, we design new compounds
based on Lopinavir structure as an FDA-approved antiviral agent which is currently under more evalu-
ation in clinical trials for COVID-19 patients. This is the first example of the preparation of Lopinavir
isosteres from the main core of Lopinavir conducted to various heterocyclic fragments. It is proposed
that main protease inhibitors play an important role in the cycle life of coronavirus. Thus, the protease
inhibition effect of synthesized compounds was studied by molecular docking method. All of these 10
molecules, showing a good docking score compared. Molecular dynamics (MD) simulations also con-
firmed the stability of the best-designed compound in Mpro active site.
KEYWORDS
Covid-19; lopinavir;
molecular docking;
molecular dynamic
simulation; synthesis
Introduction
For instance, the commonly used HIV treatment is based
on main protease (Mpro/chymotrypsin-like protease (3CLpro))
inhibitors such as Lopinavir/Ritonavir which was applied as a
preliminary candidate for the treatment of COVID-19 infected
patients (Morales et al., 2020). This protease displays a poten-
tial target for the inhibition of CoV replication.
In recent months, the pandemic of novel Coronavirus
(COVID-19) is spreading around the world. The number of
confirmed cases at the time of writing this manuscript (4
January 2021) exceeded 83,910,386 and there were con-
firmed more than 1,839,660 deaths (WHO, 2020).
In recent months, numerous researches have been reported to
determine the effective inhibitors for SARS-CoV-2 through in-silico
docking models (Ghosh et al., 2020; Kadil et al., 2020). Also, several
antiviral medications such as Lopinavir, Zanamivir, Indinavir,
Saquinavir, and Remdesivir display potential as main proteases
and as a treatment for COVID-19 (Hall & Ji, 2020). More import-
antly, the beneficial of some compounds such as Lopinavir was
proven for the treatment of SARS-CoV-2 infections on clinical trials.
Most of known main proteases have a similar shape which
matches the capacity of the receptor active site. For example, the
core unit of Lopinavir and Ritonavir is L-phenylalanine (phe-phe
hydroxyethylene isostere) which in this study, their amino group is
functionalized with the different organic unit (Scheme 1).
COVID-19 is a member of human beta coronaviruses
which also include SARS and MERS (Elfiky et al., 2017). The
mortality rates for SARS and MERS HCoV are more than
COVID-19 (10% and 36%, respectively in comparison with
2–3%) but the spreading rate of the new virus is amazing in
a few months (Hemida & Alnaeem, 2019; WHO, 2016)
Screening of existing antiviral drugs was known as a fast
and useful strategy against SARS-CoV-2; thus, considering to
pandemic of COVID-19 and time-consuming of the drug dis-
covery process, finding a new compound against the virus
drug via repurposing seems like a logical and essential strat-
egy; however, drug discovery progression has to start some-
where. For further evaluations and founding new drugs for
the treatments of COVID-19, focusing on the chemical struc-
ture of available drugs against other viruses including the
similar SARS-CoV, middle east respiratory syndrome corona-
virus (MERS-CoV), human immunodeficiency virus (HIV), and
hepatitis C virus (HCV) have been suggested (Cunningham
et al., 2020; Ko et al., 2020; Zhou et al., 2020).
Considering to progressively application of these chemical
compounds as protease inhibitors, we design novel protease
inhibitors based on Lopinavir structure and introduce
a
short and efficient synthesis method for their preparation.
Although several methods have been developed for the synthe-
sis of these dipeptide derivatives, herein, we have afforded to
CONTACT Maryam Abbasi
Bandar Abbas, Iran; Hojjat Sadeghi-Aliabadi
mabbasi@hums.ac.ir
Department of Medicinal Chemistry, Faculty of Pharmacy, Hormozgan University of Medical Sciences,
sadeghi@pharm.mui.ac.ir Department of Medicinal Chemistry, Faculty of Pharmacy, Isfahan University of
Medical Sciences, Isfahan, Iran
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2021.1905549.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
Z. KHORSANDI ET AL.
Scheme 1. Chemical structures of Lopinavir and Ritonavir
the presented practical strategy which behind disadvantages of
commonly available approaches such as multi-complex steps,
harsh reaction condition and low yields of products (Bhaskar
et al., 2008; Damo et al., 2006). In continuous, the main pro-
teases-inhibiting potential of synthesized compounds was inves-
tigated using computational studies including molecular
docking and molecular dynamics simulation.
Fragment-based drug design is a general method to design
new compounds which have introduced as an impressive sub-
stitute to high throughput screening of compounds in drug dis-
covery (Kumar et al., 2012; Murray & Blundell, 2010). In this
approach, fragments as small organic moieties such as active
heterocyclic rings are fused to the main pharmacophore. Some
Scheme 2. Some heterocyclic fragments used in different pharmaceut-
ical products
fused heterocyclic systems contain pyrazole (Scheme 2f) are
among pharmacological importance compounds as inhibitors
of HIV-1, pesticides, fungicides, antihypertensive and anti-
cancer agents (Hassan et al., 1997; Min et al., 2006).
The 1,2,4-triazoles (Scheme 2g) has also attracted wide-
spread attention due to their diverse applications as antibac-
terial, antidepressant, antiviral, antitumor, anti-inflammatory,
new anticancer, anti-alzheimer, and anti-malarial agents have pesticides, herbicides, dyes, lubricant, and analytical reagents
ꢀ
been developed via such process (Tanase et al., 2014).
(Dumas, 1999; Weng et al., 2012).
Herein, to design new structures, some heterocyclic frag-
ments were connected to the amino group of phe-phe
hydroxyethylene core. Through our basic knowledge of pro-
tease inhibitors, the hydrogen bond potential and a high
degree of hydrophobicity make more efficient in the block-
ing process (Sgrignani & Magistrat, 2012; Speck-Planche
et al., 2012; Suvannang et al., 2011). Therefore, we applied
non-toxic heterocyclic fragments which are known as the
most important fragments in medical chemistry; their struc-
ture was given in Scheme 2.
The reported fragments are seen in different compounds,
for example, guanine is one of the four main nucleobases
found in the nucleic acids, DNA and RNA and characterized
as potent immunosuppressive and chemotherapeutic agents
(Scheme 2b) (Chern et al., 1993).
8H-oxazolo[4,5-g]indole has been reported as potent cyto-
toxic activities towards cancerous cell lines in diffuse malig-
nant peritoneal mesothelioma (Scheme 2c,e). The
chromeno[3,4-b]pyrrol-4(3H)-one framework is often existing
in marine alkaloids such as ningalins and lamellarins (Scheme
2d). They also exhibit potent biological activities as immuno-
modulatory, anti-HIV-1, multidrug-resistant (MDR) reversal,
phosphodiesterase-5 inhibitory (PDE-5) activities, and anti-
Also, purine derivatives (Scheme 2h) were introduced as
antiviral agents and affect neuronal and muscle nicotinic acetyl-
ꢁ
ꢂ
choline receptors (Hrebabecky et al., 2012).
Pyranopyrazoles are other important class of heterocyclic
compounds which have used as pharmaceutical constitu-
ents. Pyrano[2,3c]pyrazoles (Scheme 2i,j) have shown anal-
gesic, anticancer, antitumor, and antiinflammatory activities
(Khoobi et al., 2015).
In conclusion, these new designed structures as a potential
of the COVID-19 main protease inhibition could be logical
because they consist of a combination of known bioactive mol-
ecules and phe-phe hydroxyethylene scaffold which is the core
of Lopinavir and Ritonavir as recommended drugs for the treat-
ment of COVID-19 infected patients (Scheme 3). To confirm this
opinion, the main interactions and binding energies of
designed compounds were studied by molecular docking and
molecular dynamics (MD) simulation.
Results and discussion
Synthesis
Herein, we would like to report a new green and economical
analgesic behavior (Fan et al., 2008; Imbri et al., 2014). The synthesis of new proposed compounds. The synthesis

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Scheme 3. Chemical structure of the new designed and synthesized compounds as COVID-19 main protease inhibitors.
process was shown in Scheme 4. Our general synthetic strat- Grubbs’ second-generation olefin metathesis catalyst.
egy is similar to that employed for Ritonavir according to the Although, some reports for the synthesis of this olefin are
literature (Stoner et al., 2000).
available; they suffer from serious limitations such as more
steps, using toxic and expensive reagents, and low yields of
products. Therefore, to large scale synthesis, presentation
more useful approach is valuable. The next step is hydrobor-
ation–oxidation reaction in presented Zn which gave final
alcohol in excellent selectivity; it can be explained by a che-
lation control model. The synthesis of this pharmacy import-
ant core from phenylalanine has been reported previously
which proceeds through a seven-step sequence using a large
amount of metal catalyst.
This six-step procedure was applied to synthesized prod-
ucts in acceptable yields. Utilizing commonly available
reagents and good reaction conditions, this process is suffi-
cient for large-scale production and has been used to pre-
pare these types of compounds in high yields (more
than 85%).
As illustrated in Scheme 4, the synthesis was started from
commercially available L-phenyl alanine. L-phenyl alanine
was protected with Boc and converted to its corresponding
Boc-amino alcohol. The alcohol was oxidized to crossponding
After de-protection from amines, they were reacted with
aldehyde and one carbon extension to olefin compound was methyl 2-chloroacetate and various introduced amino hetero-
occurred using standard Wittig reaction. These reactions cycles. The isolated yields of final products (which are shown
were taken continuously without purification. The cross- in Scheme 4) were given in Table 1. Details experimental
metathesis reaction of olefin was done using Hoveyda- were given in supplemental data. Herein, we report a short

4
Z. KHORSANDI ET AL.
Scheme 4. Reaction of each step: (a) (i) AcCl, MeOH, reflux, 3 h; (ii) (Boc)2O, TEA, THF, r.t, 7 h; (b) LiCl, NaBH4, EtOH, THF, r.t, 16 h; (c) (i) PCC/charcoal, CH2Cl2, r.t,
2 h; (ii) CH3P(C6H5)3Br, NaHMDS, ꢀ20 ꢁC, 40 min and at 20 ꢁC, 12 h (d) 10 mol % Hoveyda-Grubbs’ second-generation catalyst, CH2Cl2, 40 ꢁC 14 h; (e) Zn(BH4)2,
H2O2, NaOH, THF, 0 to r.t, 5 h; (f) ClCOCOCl, CH3OH, 40 ꢁC, 4 h; (ii) CH3ONa, CH3OCH2COCl, Toluene, 50 ꢁC, 2 h (g) 1-phenyl-1H-imidazole, THF 0 ꢁC, 1 h, r.t, 24 h.
the poses of each ligand were ordered in terms of binding
energy and clusters (Table 2).
Table 1. Yields of final desired products.
Entry
Compounda
Yieldb (%)
Entry
Compounda
Yieldb (%)
All compounds were perfectly placed in the active site.
Among the ten mentioned compounds, structure A demon-
strated the lowest binding energy, thus it was chosen for fur-
ther studies. To confirm the stability of compound A in the
active site of Mpro protein, the MD simulation was per-
formed and also to compare its interaction modes with
Lopinavir as a standard drug. Two and three-dimensional
analysis for synthesized compounds were shown in Figures
S1 and S2.
The compound A, Lopinavir and N3 were superimposed
and were shown in Figure S3. It can be seen that compound
A similar to Lopinavir and N3 was completely perched into
the active site.
1
2
3
4
5
A
B
C
D
E
55
61
56
48
57
6
7
8
9
10
F
G
H
I
53
69
49
49
51
J
aCrossponding to Scheme 4.
bIsolated yield.
and efficient synthesis of novel molecular scaffolds contain-
ing Ritonavir and Lopinavir core and heterocycles as a syn-
thon in the hope to get lead compounds as antiviral agents.
Molecular docking study
The top 3 proposed inhibitors were chosen in terms of
energy and main interaction in active site (Figure 2). Figure 2
shows hydrogen bonds between Lopinavir (reference stand-
ard) and Ser144, Cys145, Glu166 and Arg188 and pi-alkyl
interactions with Met165, Leu167, and Pro168.
Compound A forms H-bonds with Mpro protein amino
acids Cys145, His164, Glu166 and Gln188 and pi-alkyl interac-
tions with Cys145 and Met165. Compound B forms H-bonds
with the MPro protein amino acids Asn142, Cys145, Glu166,
Gln189, and two sigma and pi interactions with His41.
Compound C forms H bonds with the MPro protein amino
acids Cys145, Glu166 and Gln189, and pi interaction with
His41, also all calculated binding energies using Autodock
are shown in Table 2.
The present study focused on the main protease in COVID-
19 (PDB ID 6LU7) as the potential for COVID-19 inhibition
(Jin et al., 2020).
To find interactions and binding energy between
COVID19-Mpro protein and the predicted compounds, a
molecular docking was done by AutoDock 4.2 program
(Morris et al., 2009). Among the experimental X-ray structures
of COVID19-Mpro protein, the crystallographic structure with
a PDB entry code of 6LU7 was selected. The first, validation
docking on COVID19-Mpro protein and the ligand in X-ray
crystallography (N3) was done. The analysis of all docked
poses showed that the N3 ligand was located in the binding
pocket. The main residues in this pocket were His41, Phe140,
Leu141, Asn142, Gly143, Cys145, His163, His164, Met165,
Glu166, Leu167, Pro168, Asp187, Arg188, Gln189 and Thr190.
The initial coordinates of the ligand were used as the refer-
ence and a root-mean-square deviation (RMSD) was obtained
Molecular dynamics simulation
between docked ligand and reference at less than 2 Å. Two According to molecular docking results, compound A was
and three-dimensional analysis for the N3 ligand is shown in chosen for MD simulation. A 100 ns MD simulation was car-
Figure 1.
ried out to corroborate the stability of the A compound in
According to standard drugs (Ritonavir and Lopinavir), the Mpro protein active site. Also, interaction modes of com-
novel inhibitors were designed. The molecular docking of pound A were compared with a Mpro protein inhibitor as a
the designed ligands with MPro protein was carried out and reference (Lopinavir).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Figure 1. The binding mode of N3 in the active site of COVID19-Mpro protein, obtained from AutoDock4, (a and b) 3D structure, (c) 2D structure.
Table 2. The calculated free binding energies of the designed compounds with COVID19-Mpro protein using Autodock.
Binding energy (DG)
Inhibition
constant
Intermolecular
energy
Entry
Structures
(kcal/mol)
Hydrogen bonding and p-p interactions
1
2
3
4
5
6
7
8
N3
lopinavir
ꢀ8.19
ꢀ7.81
ꢀ9.61
ꢀ9.54
ꢀ9.32
ꢀ9.20
ꢀ9.19
ꢀ9.00
ꢀ8.88
ꢀ8.41
ꢀ8.01
ꢀ7.31
990.78 nM
1.90 uM
ꢀ13.86
ꢀ13.18
ꢀ13.79
ꢀ13.72
ꢀ13.50
ꢀ13.38
ꢀ13.37
ꢀ13.18
ꢀ13.65
ꢀ13.78
ꢀ12.78
ꢀ13.28
Phe140, Leu141, His163, Glu166, His41, Asp187
Ser144, Cys145, Glu166, Arg188, His41
Cys145, His164, Glu166, Gln188, His41
Asn142, Cys145, Glu166, Gln189, His41
Cys145, Glu166, Gln189, His41
Asn142,Gly143,Gln189, His163
Gly143, Cys145, Glu166, Thr190
Cys145, Thr190
A
B
C
D
E
F
G
H
I
90.10 nM
101.59 nM
146.33 nM
180.00 nM
182.58 nM
251.28 nM
310.51 nM
686.67 nM
1.35 uM
9
Gly143, Glu166
Gly143, Glu166
Thr26, His164, Thr25
Phe140, Glu166, Gln189, His41, Leu141
10
11
12
J
4.31 uM
Root mean square deviation (RMSD), root-mean-square
fluctuation (RMSF), and gyration radius were investigated as
the time-dependent behaviors of MD trajectories. To esti-
mate the conformational stability of Mpro protein during the
simulation, RMSD of backbone complexes and RMSD of two
compounds (compound A and Lopinavir) were studied.
As shown in Figure 3(A), in the first 20 ns, the RMSD profile
of the Mpro-Lopinavir complex was seen as more stable than
Mpro-A complex but in remaining simulation time, the profile
of Mpro-A complex was more stable than the Mpro-Lopinavir
complex. Generally, RMSD profile did not alter more than 0.28
and 0.38 nm in Mpro-A and Mpro-Lopinavir complexes, respect-
ively. By analysis of the RMSD plots of the two ligands (Figure
3(B)), it can be identified that compound A and Lopinavir were
superimposed in the second 50 ns simulations. The RMSD pro-
file results show that both ligands had significant stability in
the active site during MD simulation.
The compactness of the protein (Rg) was displayed in
Figure 4(A). The Rg value of A and the Lopinavir were super-
imposed and the continuity of both complexes was saved
during the simulation. The alteration of protein flexibility
(RMSF) was investigated during the MD simulation. As shown
in Figure 4(B), the RMSF profiles of both complexes were
superimposed in all amino acids. The main residues, Cys145
and His163, were seen more stable during MD simulation.
Average values of RMSD, RMSF and Rg were calculated
0.333, 0.092 and 2.191, respectively for COVID19-Mpro-A
complex. The binding free energy has been also computed
for Mpro-Lopinavir and Mpro-A complexes using the
g_mmpbsa. The obtained binding energy components are
reported in Table 3. As regards of the stability of compound
Experimental section
Chemistry
General synthetic methods: 1H NMR and 13C NMR spectra
were recorded by 400 MHz spectrometer instrument. EI mass
spectral analyses were recorded on Shimadzu Japan
QP2010 S model spectrometer. Thin-layer chromatography
(TLC) on silica gel plate was used to checking compounds
purity by using hexane and ethyl acetate. The purification
process was performed using column chromatography on sil-
ica gel (60–120 mesh) by ethyl acetate and hexane mixture
as eluent. Details procedures for synthesis and characteriza-
tion data of products were given in supplementary data.
Molecular docking methods
To find the main interactions and the binding energy of
designed compounds with the COVID-19 main protease as a
receptor, molecular docking was done by AutoDock 4.2 pro-
gram (Morris et al., 2009). The pdb file of COVID-19 main
proteases was taken from the Protein Data Bank (PDB ID:
6LU7). All water molecules, ligand, and ions were removed
from pdb file. Then polar hydrogens were added and the
partial atomic charge was calculated by Kollman method.
Then the prepared file was saved in pdbqt format to use in
the following steps. Three-dimensional structures of designed
compounds were depicted in Marvin Sketch Ver. 5.7,
ChemAxon (Cosconati et al., 2010). The partial charges of
atoms were determined according to the Gasteiger-Marsili
procedure, and non-polar hydrogens of the compounds were
merged (Morris et al., 1998). A 50 ꢂ 50 ꢂ 50 Å (x,y, and z) grid
A in active site of Mpro-protein, this compound could be box was centered on the protease binding pocket with a
propose as COVID-19 Mpro inhibitor. 0.375 nm spacing for each dimension. Docking was

6
Z. KHORSANDI ET AL.
Figure 2. The binding mode of Lopinavir, A, B and C (1, 2, 3 and 4 respectively) in the active site of COVID19-Mpro protein, obtained from AutoDock4; 3D struc-
tures and 2D structure.
performed by Lamarckian genetic algorithm and empirical by Discovery Studio visualizer version 17.2 and Pymol version
ꢃ
scoring function by using a flexible method. The program
was run for a total number of 50 Genetic algorithm runs.
Eventually, the docking procedure was carried out by
AutoDock 4.2. All of the runs were ranked in term of the
binding energy and were analyzed to obtain the best con-
formation and orientation of the ligand in the active site of
1.1evel (Dassault Systemes BIOVIA, 2016).
Molecular dynamics simulation methods
The best-ranked nominee from docking results was consid-
the protein. Visualization of docking results has been done ered for evaluating their thermodynamic behavior and

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 3. RMSD plots of COVID19-Mpro protein inhibitors as a function of simulation time. (A) RMSD of backbone atoms of COVID19-Mpro-A (purple) and
COVID19-Mpro-Lopinavir (green) complexes. (B) RMSD plot of Compound A and Lopinavir.
Figure 4. (A) The gyration radius plot of Mpro-Lopinavir (green) and A (purple). (B) The RMSF plot of Mpro- Lopinavir (green) and A (purple).
Table 3. The binding energy components obtained from g_mmpbsa.
a
b
c
d
e
Complex
DGbinding
DGpolar
DGnonpolar
DEelec
DEvdW
Mpro-Lopinavir
Mpro-A
ꢀ90.310 7.565
ꢀ75.878 9.078
162.973 8.611
134.881 6.582
ꢀ21.976 0.783
ꢀ20.441 0.611
ꢀ47.603 6.208
ꢀ7.741 5.286
ꢀ200.705 11.742
ꢀ182.577 9.024
aBinding energy.
bPolar solvation energy.
cNon-polar solvation energy.
dElectrostatic component to the binding energy in kcal/mol.
eVan der Waals component to the binding energy in kcal/mol.
stability of binding mode in the Mpro binding pocket using ensemble at a constant number of particles, volume, and tem-
molecular dynamics (MD) simulation studies. All MD simulations
were accomplished by GROMACS-2019.3 package (Abraham
et al., 2015). The Amber 99.sb force field was engaged in MD
simulations (Cornell et al., 1995). Drug topology parameters
were ready by the AnteChamber Python Parser InterfacE
(ACPYPE) (Da Silva & Vranken, 2012). To characterize which resi-
due adopts non-standard ionization states, was obtained pKa
values using PROPKA 3.1 webserver (Søndergaard et al., 2011).
A TIP3P water model was select (Jorgensen, 1983) and the
complex of ligand-protein was soaked in a dodecahedron water
box. Some cations, Naþ ions, were substituted with solvent
water molecules to neutralizing the system. The energy mini-
mization was performed and MD simulation was commenced
perature; 2) 1 ns simulation in the NPT ensemble at a constant
number of particles, pressure, and temperature. Finally, MD
simulation was run at 300 K temperature for 100 ns. The Particle
Mesh Ewald (PME) method and the linear constraint (LINCS)
algorithm were carried out to computing long-range electro-
static interactions and covalent bond constraints, respectively.
Structure visualization was done using VMD 1.8.6 (Humphrey
et al., 1996) and PyMOL Tcl.
Conclusion
In summary, we designed and synthesized novel potential
by two stages of the process: 1) 500ps simulation in the NVT COVID-19 main protease inhibitors that contain the main

8
Z. KHORSANDI ET AL.
Damo, I. A., Benedetti, F., Berti, F., & Campaner, P. (2006). Stereoselective
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core of Lopinavir connected to various heterocyclic frag-
ments. Lopinavir is one of the rare successful clinical trial
agents for the treatment of COVID-19 patients. First, molecu-
lar docking was carried out to identify the interactions of
these compounds with the main protease protein. Our find-
ings revealed that all designed structures were docked suc-
cessfully but compound A exhibited the lowest binding
energy within the protein pocket and could be considered
as a potential of COVID-19 main protease inhibition. The
molecular dynamic simulation was also confirmed our claim.
Next, these structures were synthesized through facile and
efficient six-step reactions instead of some available strat-
egies which contain more than ten harsh reaction steps.
Because of the similarity of these synthesized compounds to
Lopinavir and Ritanivir scaffold, they could be introduced as
potential of main protease inhibition. However, further
research is needed to investigate the validation of these
compounds using in vitro and in vivo methods to pave a
way for these compounds in drug discovery.
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Disclosure statement
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No potential conflict of interest was reported by the author(s).
Hrebabecky, H., Dejmek, M., Dracınsky, M., Sala, M., Leyssen, P., Neyts, J.,
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