Design and synthesis of new 4-(2-nitrophenoxy)benzamide derivatives as potential antiviral agents: molecular modeling andin vitroantiviral screening

Article abstract of DOI:10.1039/d1nj02710g

Regarding the crucial role of deubiquitinase (DUB) enzymes in many viruses, in particular, Adenovirus, HSV-1, coxsackievirus, and SARS-CoV-2, DUB inhibition was reported as an effective new approach to find new effective antiviral agents. In the present study, a new wave of 4-(2-nitrophenoxy)benzamide derivatives was designed and synthesized to fulfill the basic pharmacophoric features of DUB inhibitors. The molecular docking of the designed compounds against deubiquitinase enzymes of the aforementioned viruses was carried out. Significant molecular docking results directed us to conductin vitroantiviral screening against the aforementioned viruses. The biological data showed very strong to strong antiviral activities with IC₅₀values ranging from 10.22 to 44.68 μM against Adenovirus, HSV-1, and coxsackievirus. Compounds8_c,8_d,10_b, and8_awere found to be the most potent against Adenovirus, HSV-1, coxsackievirus, and SAR-CoV-2, respectively. Also, the CC₅₀values of the examined compounds ranged from 72.93 to 120.50 μM. Finally, thein silicoADMET and toxicity studies demonstrated that the tested members have a good profile of drug-like properties. Furthermore, we concluded the structure-activity relationship (SAR) of the newly designed and synthesized compounds regarding theirin vitroresults, which may help medicinal chemists in further optimization to obtain more potential antiviral candidates in the near future as well.

Full text of DOI:10.1039/d1nj02710g

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Design and synthesis of new 4-(2-
nitrophenoxy)benzamide derivatives as
potential antiviral agents: molecular
modeling and in vitro antiviral screening†
a
Cite this: DOI: 10.1039/d1nj02710g
Abdallah E. Abdallah,*a Mohamed S. Alesawy,
Sally I. Eissa,bc
Esmail M. El-Fakharany,d Mohamed H. Kalaba,e Mohamed H. Sharaf,e
f
Noura M. Abo Shama,f Sara H. Mahmoud,f Ahmed Mostafa,
g
a
Ahmed A. Al-Karmalawy
and Hazem Elkady
*
Regarding the crucial role of deubiquitinase (DUB) enzymes in many viruses, in particular, Adenovirus,
HSV-1, coxsackievirus, and SARS-CoV-2, DUB inhibition was reported as an effective new approach to
find new effective antiviral agents. In the present study, a new wave of 4-(2-nitrophenoxy)benzamide
derivatives was designed and synthesized to fulfill the basic pharmacophoric features of DUB inhibitors.
The molecular docking of the designed compounds against deubiquitinase enzymes of the
aforementioned viruses was carried out. Significant molecular docking results directed us to conduct
in vitro antiviral screening against the aforementioned viruses. The biological data showed very strong to
strong antiviral activities with IC50 values ranging from 10.22 to 44.68 mM against Adenovirus, HSV-1, and
coxsackievirus. Compounds 8c, 8d, 10b, and 8a were found to be the most potent against Adenovirus,
HSV-1, coxsackievirus, and SAR-CoV-2, respectively. Also, the CC50 values of the examined compounds
ranged from 72.93 to 120.50 mM. Finally, the in silico ADMET and toxicity studies demonstrated that the
tested members have a good profile of drug-like properties. Furthermore, we concluded the structure–
activity relationship (SAR) of the newly designed and synthesized compounds regarding their in vitro
results, which may help medicinal chemists in further optimization to obtain more potential antiviral
candidates in the near future as well.
Received 2nd June 2021,
Accepted 6th August 2021
DOI: 10.1039/d1nj02710g
rsc.li/njc
process plays key roles in a wide range of essential cellular
functions as innate and adaptive immune responses.2–4 Deubi-
1. Introduction
Ubiquitin proteasome system (UPS) is a fundamental regulator quitinating enzymes are responsible for removing ubiquitin
for the degradation of targeted proteins.1 This degradative from the substrate proteins.5
The suppression of cellular ubiquitin-dependent immune
responses was reported as a common strategy of many viruses.
The mechanism discovered was that some viral proteases
showed deubiquitinating activity on the human ubiquitin
a Pharmaceutical Medicinal Chemistry & Drug Design Department,
Faculty of Pharmacy (Boys), Al-Azhar University, Cairo 11884, Egypt.
E-mail: Abdulla_emara@azhar.edu.eg, Hazemelkady@azhar.edu.eg
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls),
system. As a result, the viruses evade the host antiviral immune
responses and promote viral replication.6–9 This was supported
by the demonstration of in vitro deubiquitinating activities for
three viral enzymes, namely, papain-like protease (PLpro) of
severe acute respiratory syndrome coronavirus (SARS-CoV),10–12
adenovirus protease adenain,13 and a protease domain contained
in Herpes virus tegument ubiquitin specific protein (USP).14
Papain-like protease (PLpro) is a crucial enzyme for corona
replication and the ability to infect.15–18 It is one of the
deubquitinases that cleave ubiquitin from the target
protein.10,19,20 PLpro is a significant target for the development
of effective antiviral drugs. Such drugs would prevent viral
Al-Azhar University, Cairo 11884, Egypt
c Faculty of Pharmacy Al-Maareffa University for Science and Technology, Riyadh,
Kingdom of Saudi Arabia
d Protein Research Department, Genetic Engineering and Biotechnology Research
Institute, City of Scientific Research and Technological Applications, Egypt
e Botany and Microbiology Department, Faculty of Science, Al-Azhar University,
Egypt
f Center of Scientific Excellence for Influenza Viruses, National Research Centre,
Giza 12622, Egypt
g Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy,
Horus University-Egypt, New Damietta 34518, Egypt
† Electronic supplementary information (ESI) available: Appendix A. Supplemen-
tary data. See DOI: 10.1039/d1nj02710g
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replication and reduce mortality related to SARS-CoV derivatives having the basic crucial pharmacophoric features
outbreaks.21–24
of DUB inhibitors.
One of the fundamental factors of herpes virus invasion is
its deubiquitinase (Herpes virus tegument ubiquitin specific
1.2. The rational of molecular design
protein (USP)).25 In the Herpesviridae family, a variety of DUBs DUB inhibitors were shown to have antiviral activity. Many
were reported to play a pivotal role in the life cycle of the virus, viruses depend on deubiquitinating enzymes in invasion and
e.g., UL36USP of Herpes simplex virus 1 (HSV-1).26
replication. Thus, the design of the DUB inhibitor was suggested
Adenovirus L3 23K proteinase (Avp) was found to deubiqui- as a new approach for the introduction of promising antiviral
tinate many cellular proteins. Hence, adenovirus infection is candidates.29 Due to the structural similarity of deubiquitinating
associated with high deubiquitination activity in the host cell.13 enzymes of some viruses such as HSV and SARS-CoV, DUB
It was reported that the dysregulation of UPS suppresses inhibitors may show broad-spectrum antiviral activity. We found
coxsackie virus replication.27 RNA-dependent RNA polymerase structural similarities of some small molecule DUB inhibitors
3D, which is an essential enzyme for coxsackie virus, was that were reported to have antiviral activity. Four pharmacopho-
reported to be ubiquitinated. Such ubiquitination leads to the ric features can be defined for DUB inhibitors, namely, (1)
regulation of coxsackie replication.28
GRL0617 (1) was discovered as a non-covalent lead inhibitor hydrophobic region (Fig. 1).
aromatic planar system, (2) linker, (3) amide moiety, and (4)
for PLpro of SARS-CoV with no associated toxicity. It was proved
In line with these pharmacophoric features, we built a new
that GRL0619 binds within S4–S3 subsites PLpro, causing a scaffold, as illustrated in Fig. 2. The benzene ring was designed
unique mode of inhibition at the active site. These data provide as an aromatic planar system. This is because benzene is an
a proof of principle that potent non-covalent cysteine protease isostere to the pyridine of WP1130 3. It was found that the
inhibitors can be designed to target the DUB of the virus linker that contains a six-membered ring is better than the
without inhibiting the host DUB.22 Also, the DUB inhibitor, short open one. The IC50 of compounds GRL0617 1, 2, and 4
Wp1130 (3) was proved to have antiviral activity.29 In general, against PLpro were 0.6, 0.15, and 0.32 mM, respectively.32 Thus,
DUB inhibitors have antiviral activity22,29–31 and could provide we selected the linker to be a phenoxy group that has the same
a new approach for the development of effective antiviral length of the linker in compounds 2 and 4. Concerning the
drugs.29 Accordingly, DUB inhibitors are more likely to inhibit amide moiety, it was designed to be present in the new
viruses such as herpes, adeno, corona, and other viruses that derivatives. Finally, the hydrophobic moiety was designed in
depend on deubiquitination in its invasion and/or replication. this work to be phenyl or substituted phenyl ring. It may be
Thus, this new approach may have the advantage that the attached directly or via a spacer (one or two carbons) to the
developed DUB inhibitor could be active against more than amide moiety. Fig. 1 shows that the phenyl and substituted
one virus, in particular, coronavirus, which caused many out- phenyl groups attached directly or via the spacer to the
breaks with high mortality.
Based on the data mentioned above, we designed and cyclohexyl, to examine if the aliphatic moieties could serve as
synthesized new series of 4-(2-nitrophenoxy)benzamide good substituents for the aromatic groups (Fig. 2).
amide moiety. We also designed aliphatic groups, butyl and
a
Fig. 1 Common pharmacophoric features of some DUB inhibitors with antiviral activity.
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compound 7, the carboxylic proton appeared at 12.93 ppm.
This peak disappeared in all the charts of the final compounds
due to amide formation. Instead, the amidic proton exhibited a
peak at about 10.2 ppm for compounds 8a–d. On the other
hand, in compounds 9a,b this peak was upfield to be seen at
8.17 and 8.42 ppm; respectively. The amidic proton of com-
pounds 10a,b revealed peaks at 9.05 and 8.57 ppm, respectively.
2.2. Biological evaluation
2.2.1. In vitro antiviral assay. The eight newly synthesized
compounds (8a–10b) were tested for their cytotoxicity and virus-
inhibitory effect by determining the half-maximal cytotoxic
(CC50) and inhibitory (IC50) concentrations for each compound.
Similarly, ribavirin was tested as a positive control against
adenovirus, HSV-1, and coxsackievirus.
Fig. 2 A model for our designed compounds consistent with the DUB
inhibitor features.
2. Results and discussion
It was found that all the newly synthesized compounds
showed very strong to strong antiviral activities against
adenovirus, HSV-1, and coxsackievirus. On the other hand, they
exhibited non-significant activities against SARS-CoV-2. The
most promising candidate against SARS-CoV-2 was compound
8a with IC50 = 939.98 mM. Analyzing the antiviral activities of the
screened compounds against the aforementioned four viruses
(Fig. 3) revealed the following interesting results.
2.1. Chemistry
A mixture of equimolar amounts of 2-fluronitrobenzene 5 and
4-hydroxybenzoic acid 6 as well as 2 moles of K2CO3 was heated
at reflux temperature in isopropyl alcohol to give compound 7,
as illustrated in Scheme 1. To get the final compounds,
compound 7 was first treated with ethyl chloroformate and
triethylamine in DCM in an ice salt bath for 1 h. Then, an
appropriate amine, namely, aniline, 4-methoxyaniline, 4-
chloroaniline, 4-methylaniline, cyclohexylamine, butylamine,
benzylamine, and 2-phenylethan-1-amine, were added at the
same temperature. The reaction mixture was then stirred at
room temperature to afford the final compounds 8a–d, 9a,b, and
10a,b, as presented in Scheme 1. In the 1H NMR chart of
(1) Adenovirus. The descending order of the tested com-
pounds was found to be as follows: 8c (IC50 = 10.22 mM) 4
10b (IC50 = 15.42 mM) 4 8a (IC50 = 15.77 mM) 4 8b (IC50
=
19.78 mM) 4 10a (IC50 = 20.52 mM) 4 8d (IC50 = 21.01 mM) 4 9b
(IC50 = 29.87 mM) 4 9a (IC50 = 44.68 mM).
Scheme 1 General procedures for the preparation of the final target compounds; reagents and conditions: (i) isopropyl alcohol/K2CO3/reflux/4 h. (ii)
DCM/ClCOOEt/Et3N/ice salt bath/stirring/1 h. (iii) an appropriate amine/stirring, r.t.
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Fig. 3 The antiviral activities of the newly synthesized compounds and ribavirin against adenovirus, HSV-1, and coxsackievirus.
(2) HSV-1. The descending order of the tested compounds was
(4) SARS-CoV-2. Compound 8a was found to be the most
found to be as follows: 8d (IC50 = 12.68 mM) 4 9b (IC50 = 21.44 mM) 4 promising candidate as it showed IC50 of 940 mM while the
10b (IC50 = 21.77 mM) 4 10a (IC50 = 22.10 mM) 4 9a (IC50 = 22.59 mM) others showed far lower activities.
4 8b (IC50 = 22.61 mM) 4 8a (IC50 = 23.73 mM) 4 8c (IC50 = 26.33 mM).
It is worth mentioning that the cytotoxic concentrations of
the tested compounds, as indicated by their (CC50) values,
(3) Coxsackievirus. The descending order of the tested showed a promising safety profile. The CC50 values of the
compounds was found to be as follows: 10b (IC50 = 14.15 mM) examined compounds on adenovirus, HSV-1, and coxsackie-
4 10a (IC50 = 14.58 mM) 4 8b (IC50 = 15.89 mM) 4 8a (IC50
=
virus ranged from 72.93 to 120.50 mM.
19.63 mM) 4 8d (IC50 = 20.52 mM) 4 9a (IC50 = 21.27 mM) 4 8c
(IC50 = 22.48 mM) 4 9b (IC50 = 22.80 mM).
With regards to ribavirin, it showed IC50 = 105.94, 75.18, and
100.78 mM against adenovirus, HSV-1, and coxsackievirus,
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(iii) For coxsackievirus: the presence of a simple phenyl ring
attached to the amide group of compounds 10a and 10b
through the methylene or ethylene bridge achieved the best
activity.
(iv) For SARS-CoV-2: the plain phenyl moiety in compound
8a may be considered as a lead for further optimization.
(v) Overall, the direct attachment of the substituted phenyl
moiety to the amide group of the 4-(2-nitrophenoxy)benzamide
nucleus showed the most promising antiviral design for further
optimization.
2.3. In silico study
Fig. 4 Alignment of the co-crystallized pose (green) and the redocked
pose (turquoise) of the same ligand inside PLpro, PDB:3E9S.
2.3.1. Docking study. Molecular docking for the proposed
derivatives was performed against the active site of papain-like
protease as one of the deubiquitinase enzymes (PDB:3E9S).
respectively, and CC50 = 262.16 mM as well. It is apparent from Docking studies were carried out to recognize the binding
the data obtained that the new derivatives revealed much better modes and interactions with the crucial amino acids.33–37
results than ribavirin.
Initially, the docking process was validated through the running
Analyzing the previously mentioned antiviral results, we can of the docking procedure for the co-crystallized ligand against
conclude that the newly synthesized compounds exhibited very the active site of the pocket.38,39 It was found that the produced
strong to strong antiviral activities toward adenovirus, HSV-1, RMSD value between the generated pose of the docked molecule
and coxsackievirus in the IC50 value range of 10.22–44.68 mM. and the original one equals 1.06. This indicates the validity of
Moreover, compound 8a was the most promising candidate the docking process (Fig. 4). The results of the docking studies
against SARS-CoV-2.
against the papain-like protease (PDB:3E9S) revealed that the
Collectively, their antiviral activities toward a wide range of synthesized compounds have similar orientations inside the
different viruses, as indicated by the IC50 values, besides their active pocket. The designed compounds gave good binding
discussed predicted safety, as indicated by the CC50 values, give energies ranging from À16.22 to À19.65 Cdocker energy score
us a good idea for further modifications in the near future in (Table 1).
order to obtain a promising lead compound, especially against
the pandemic-causing SARS-CoV-2.
GRL0617 1 orientation with the amino acids of the pocket
has been studied and displayed in Fig. 5. The proposed binding
2.2.2. Structure–activity relationship (SAR) study. Regarding pattern revealed an affinity value of À18.50 Cdocker energy
the previously discussed antiviral activities of the newly designed score. As reported,22,40,41 the interaction between GRL0617 and
and synthesized candidates and in our way to optimize a PLpro is stabilized through a pair of hydrogen bonds and a
promising antiviral lead compound, we can conclude that:
series of hydrophobic interactions. The amide group of
(i) For Adenovirus: the p-chlorophenyl moiety of compound 8c GRL0617 forms hydrogen bonds with the side chain of
was found to be the most promising one, indicating the superior Asp165 (2.03Å) and the backbone nitrogen of Gln270 (1.75 Å).
activity of the electron withdrawing group at the para position.
Also, the 1-naphthyl group forms hydrophobic interactions
(ii) For HSV-1: the p-methylphenyl moiety of compound 8d, with the aromatic ring of Tyr 269 and with the side chains of
on the other hand, showed better activity, referring to the Pro248 and Pro249. The di-substituted benzene ring at the
importance of the electron donating group at the para position. opposite end of the inhibitor occupies the putative P3 position
Table 1 The calculated DG (Cdocker energy score) of the synthesized compounds and reference drug GRL0617 against wild (unmutant) and mutant
active pockets
Cdocker energy score
Mutant pockets
Asp165 to
Phe165
Pro249 to Glu249 &
Gln270 to Tyr270
Asp165 to phe165, Pro249
to glu249 & Gln270 to tyr270
Asp165 to Phe165, Pro249 to Glu249,
Tyr269 to Arg269 & Gln270 to Tyr270
Wild (unmutant
pocket)
Comp. no.
8a
8b
8c
8d
9a
9b
10a
10b
À17.71
À16.93
À16.96
À17.74
À19.60
À19.70
À17.71
À18.06
À19.54
À17.11
À9.88
À11.76
À11.96
À11.97
À9.81
À16.29
À15.55
À17.20
À16.38
À18.01
À18.85
À18.24
À14.46
À21.90
À18.27
À19.65
À17.43
À16.22
À18.59
À18.23
À18.11
À16.94
À18.50
À9.99
À11.44
À18.05
À17.02
À16.67
À10.34
À18.98
À12.48
À12.91
À12.08
À18.96
À15.10
GRL0617
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Fig. 6 (A) 3D binding mode of compound 8a into the PLpro active site. (B)
2D binding mode of compound 8a into the PLpro active site (hydrogen
bonds = green dashed lines, electrostatic interactions = orange dashed
lines, pi–pi interactions = deep pink dashed lines, and pi–alkyl interactions =
light pink dashed lines).
Fig. 5 (A) 3D binding mode of GRL0617 against the PLpro active site. (B)
2D binding mode of GRL0617 against the PLpro active site (hydrogen
bonds = green dashed lines, electrostatic interactions = orange dashed
lines, pi–pi interactions = deep pink dashed lines, and pi–alkyl interactions =
light pink dashed lines).
of the bound substrate. The benzene ring stacks against the
The docking results of compound 9a (affinity value of À18.59
aliphatic portions of Asp165 and Tyr269, whereas the ortho- Cdocker energy score) are almost similar to that of GRL0617.
methyl substituent at the R1 position points into the floor of The amide moiety formed two hydrogen bonds with Gln270
the cavity, which is lined by the side chains of Tyr265, Tyr274, (COOÀ, 2.10 Å) and Asp165 (NH, 2.18 Å). Moreover, the docked
and Leu163 (Fig. 5).
compound formed two hydrophobic interactions via its cyclo-
Compound 8a (affinity value of À18.27 Cdocker energy score) hexyl moiety with Pro248 and Pro249. Also, the central phenyl
combined with the receptor protein in a manner similar to that moiety formed one electrostatic and one pi–pi interactions with
of GRL0617. The amide moiety formed two hydrogen bonds Asp165 and Tyr269, respectively. Moreover, the 2-nitrophenyl
with the crucial residues Gln270 (distance: 3.02 Å) and Asp165 moiety formed three electrostatic interactions with Lys158 and
(distance: 2.11 Å). Moreover, the terminal phenyl moiety formed Glu162 (Fig. 8).
two hydrophobic interactions with Tyr269 and Pro249. Also, the
In order to support the docking results, we mutated the key
central phenyl ring moiety formed one hydrophobic interaction residues in the active pocket and compared the docking score/
with Tyr269 and one electrostatic interaction with Asp165. In orientation before and after mutations. Mutation was done at
addition, the 2-nitrophenyl moiety formed three electrostatic one, two, three, and/or four residues of the protein active site
interactions with Lys158 and Glu168 (Fig. 6).
including Asp165, Gln270, Pro249, and Tyr 269. The results of
The docking simulation of compound 8b revealed that it has the docking studies against the mutant residues revealed that
a good fitting into the enzyme active site with a docking score of the synthesized compounds are able to bind in the same
À19.65 Cdocker energy score. The amide moiety formed two manner as they do in the active pocket and have similar
hydrogen bonds with the essential residues Asp165 (2.28 Å) and orientations inside the binding site. Also, they showed energy
Gln270 (3.00 Å). Furthermore, hydrophobic interactions similar scores comparable to that in the active pocket (Table 1). It was
to that of GRL0617 were observed. The 4-methoxyphenyl moiety noticed that mutation at one and four residues showed better
formed three hydrophobic interactions with the aromatic rings results in orientation and scores in comparison with mutation
of Tyr 269 and Pro249. Also, the central phenyl ring formed two at two and three residues of the active site.
interactions: one electrostatic with Asp165 and one pi–pi inter-
Non-conservative and quite different mutation at one
action with Tyr269. In addition, the hydrophobic (2-nitrophenyl) essential site (Asp165 to Phe165) reveled that compounds 8d,
moiety formed three electrostatic interactions with Lys158 and 9a,b, 10b showed À17.74, À19.60, À19.70, À18.06 Cdocker
Glu168 (Fig. 7).
energy score, which were higher than the corresponding energy
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Fig. 7 (A) 3D binding mode of compound 8b into the PLpro active site. (B)
2D binding mode of compound 8b into the PLpro active site (hydrogen
bonds = green dashed lines, electrostatic interactions = orange dashed
lines, pi–pi interactions = deep pink dashed lines, and pi–alkyl interactions =
light pink dashed lines).
Fig. 8 (A) 3D binding mode of compound 9a into the PLpro active site. (B)
2D binding mode of compound 9a into the PLpro active site (hydrogen
bonds = green dashed lines, electrostatic interactions = orange dashed
lines, pi–pi interactions = deep pink dashed lines, and pi–alkyl interactions =
light pink dashed lines).
scores in the original active pocket. Taking compound 8a as a 8a, 9a, 9b, and 10a and a great decrease in the affinities of
representative example for such mutation revealed that compounds 8b, 8c, 8d, and 10b compared to the wild (unmutant)
compound 8a has an affinity value of À17.71 Cdocker energy pocket. The detailed interactions and figures described that the
score and combined with the mutant PLpro in a manner as that two sites mutation was provided in the ESI† (Fig. S4–S6).
of the active pocket. The carbonyl group of the amide moiety
It can be also seen from Table 1 that mutation at three
formed one hydrogen bond with Gln270 (distance: 1.80 Å). essential amino acid residues (Asp165, Pro249, and Gln270 to
Moreover, the terminal phenyl moiety formed three hydrophobic totally different amino acids, Phe165, Glu249, and Tyr270)
interactions with Pro248, Pro249, and Phe165. Also, the central revealed lower affinity binding for all the new compounds, with
phenyl ring formed one hydrophobic interaction with Phe165. the exception of compound 10b, which displayed higher affinity
Likewise, the phenoxy oxygen formed one hydrogen bond with than that of the original pocket.
Phe165 (distance: 3.04 Å). In addition, the 2-nitrophenyl moiety
Interestingly, mutation at four different sites (including
formed two hydrogen bonds with Phe165 (distance: 2.64 Å) and Asp165, Pro249, Tyr269, and Gln270 to Phe165, Glu249,
Lys158 (distance: 2.41 Å), besides two electrostatic interactions Arg269, and Tyr270) revealed that compounds 8d, 9b, and 10a
with Lys158 and Glu168 (Fig. 9).
displayed À16.38, À18.45, and À18.24 Cdocker energy score,
The docking results of compound 9a against the mutant respectively, which were higher than the corresponding energy
pocket revealed an affinity value of À19.60 Cdocker energy scores in the original active pocket. The detailed interactions
score. The carbonyl group of the amide moiety formed one and figures describing the four sites mutation are provided in
hydrogen bond with Gln270 (2.52 Å). In addition, compound 9a the ESI† (Fig. S1–S3).
formed three hydrophobic interactions with Pro248, Pro249,
The obtained results indicated that all the synthesized
and Tyr269 through its terminal phenyl moiety. Furthermore, compounds have similar positions and orientations inside
the central phenyl moiety formed one pi–pi interaction the recognized pocket (Fig. 11).
with Phe165. Moreover, the 2-nitrophenyl moiety formed four
electrostatic interactions with Lys158 and Glu162 (Fig. 10).
2.3.2. Correlation between the binding affinity of the newly
synthesized compounds and their biological results against
Meanwhile, non-conservative mutation at the essential adenovirus, HSV-1, and coxsackievirus. To explain our results,
residues Pro249 and Gln270 to Glu249 and Tyr270 showed we make a correlation between the binding affinity of each
the ability of the new derivatives to bind to the mutant pocket. compound in each virus (against its active pocket) and its
Table 1 revealed a small decrease in the affinities of compounds biological activity. At first, the docking studies of the newly
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Fig. 10 (A) 3D binding mode of compound 9a into the mutant PLpro
active site. (B) 2D binding mode of compound 9a into the mutant PLpro
active site (hydrogen bonds = green dashed lines, electrostatic interactions =
orange dashed lines, pi–pi interactions = deep pink dashed lines, and
pi–alkyl interactions = light pink dashed lines).
Fig. 9 (A) 3D binding mode of compound 8a into the mutant PLpro active
site. (B) 2D binding mode of compound 8a into the mutant PLpro active
site (hydrogen bonds = green dashed lines, electrostatic interactions =
orange dashed lines, pi–pi interactions = deep pink dashed lines, and
pi–alkyl interactions = light pink dashed lines).
designed compounds against the deubiquitinating enzyme of
adenovirus (PDB:4PID), HSV-1 (4TTI), and coxsackievirus (IZ8R)
were performed, respectively.
Regarding adenovirus, it is worth mentioning that all our
new compounds showed superior binding scores (À5.87 to
À6.46 Cdocker energy score) compared to the co-crystallized
native inhibitor (À5.23 Cdocker energy score). Also, compounds 8c
and 10b showed the most promising biological activities achieved
higher binding score ranking fourth and first, respectively.
Concerning HSV-1, the biological results were matched with the
binding affinity results, where compound 8d was the most active
candidate against HSV-1, which displayed the highest binding
score (À22.56 Cdocker energy score). With respect to coxsackie-
virus, the highest docking affinity obtained was for compound 10b,
which displayed the best biological results. Meanwhile, compound
9b, which showed the lowest docking affinity, was the weakest in
the biological results. Also, the biological results of the new
derivatives showed a narrow range of IC50 from 14.15 to 22.80
mM. Similarly, the binding scores obtained were of narrow range
from À4.33 to À4.92 Cdocker energy score (Table 2). The detailed
interactions and figures described the docking of the representative
examples of the synthesized compounds on the three viruses,
which is provided in the ESI† (Fig. S6–S11).
Fig. 11 Superimposition of some docked compounds inside the binding
pocket of 3E9S.
the physicochemical, drug-like parameters, and pharmacoki-
netics in the drug discovery process.42
Lipinski suggested that the absorption of an orally administered
compound is more likely to be better if the molecule satisfies at
least three out of four of the following rules: (1) H bond donors
(OH, NH, and SH) r 5; (2) H bond acceptors (N, O, and
S atoms) r 10; (3) molecular weight o 500; (4) log P o 5.
Compounds violating more than one of these rules could not
have good bioavailability. Moreover, reduced molecular
flexibility, as measured by the number of rotatable bonds,
and low polar surface area are found to be important predictors
of good oral bioavailability.43 Compounds that meet the criteria
of 10 or fewer rotatable bonds and polar surface area equal to or
2.3.3. Pharmacokinetic profiling study. To identify the
potential drug candidates, drug-likeness profile and the
ADMET were developed for the preliminary estimation of
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Table 2 Correlation between the binding affinity of the newly synthesized compounds and their biological results against adenovirus, HSV-1, and
coxsackievirus
8a
8b
8c
8d
9a
9b
10a
10b
Adenovirus
Docking score
Affinity order
Biology order
À5.87
À6.30
À6.19
À6.09
À6.07
À5.92
À6.43
À6.46
8
3
3
4
4
1
5
6
6
8
7
7
2
5
1
2
HSV-1
Docking score
Affinity order
Biology order
À14.80
À17.06
À17.88
À22.56
À18.80
À18.16
À18.36
À17.24
8
6
7
7
5
8
1
1
2
3
4
2
3
4
6
5
Coxsackievirus
Docking score
Affinity order
Biology order
À4.60
À4.47
À4.91
À4.92
À4.40
À4.33
À4.51
À4.92
4
4
6
3
3
7
2
5
7
6
8
8
5
2
1
1
less than 140 A will have a high probability of good oral predicts cytochrome P450 2D6 enzyme inhibition; (v) hepato-
bioavailability.43
toxicity, which predicts the potential hepatotoxicity of a given
In the present study, an in silico computational study of the compound; (vi) plasma protein binding, which predicts the
representative compounds given in Table 3 was conducted to fraction of drug that could be bound by plasma proteins.45 The
determine the number of rotatable bonds, topology polar surface predicted descriptors are listed in Table 4.
area (TPSA), and other physicochemical properties according to
the directions of Lipinski’s rule of five.44
The results revealed that all the tested compounds showed
medium BBB penetration levels except compound 8c and ligand
We found that all the tested compounds and ligands showed (GRL0617) displayed high BBB penetration levels. These results
no violation of Lipinski’s rule and hence displayed a drug-like indicate that most compounds were anticipated to be safe to
molecular (DLM) nature. The log P, molecular weight, number the CNS. It is well known that numerous drug candidates have
of H-bond donors, and number of H-bond acceptors of all the failed during clinical tests because of problems related to their
examined compounds are within the accepted values of less absorption properties.46 Intestinal absorption is the percentage
than 5, 500, 5, and 10, respectively. Besides, the number of of a drug that is absorbed across the gut wall.47 A well-absorbed
rotatable bonds and TPSA of all the tested compounds are drug is absorbed at least 90% into the human bloodstream.48
within the admissible values of less than 10 and 140 Å2, According to the in silico ADMET studies, it was found that all of
respectively.
them have good absorption behavior. Moreover, the solubility
2.3.4. In silico ADME analysis. Discovery studio 2.5 was level of the tested compounds is expected to be better than or
used to predict the ADMET descriptors for all the synthesized even similar to that of the reference drug, which showed a low
compounds compared with co-crystallized ligand as a reference solubility level. The cytochrome P450 2D6 (CYP2D6) model
drug. ADMET studies include many descriptors: (i) blood–brain predicts the potential of a compound to inhibit the CYP2D6
barrier penetration, which predicts blood–brain barrier enzyme using the 2D chemical structure as the input. CYP2D6 is
penetration of a molecule; (ii) intestinal absorption, which an essential enzyme involved in the metabolism of a wide range
predicts human intestinal absorption (HIA) after oral admini- of substrates in the liver. Therefore, CYP2D6 inhibition is
stration; (iii) aqueous solubility, which predicts the solubility of needed as part of the regulatory procedures in the drug discovery
each compound in water at 25 1C; (iv) CYP2D6 binding, which process.49 All the examined members together with the reference
drug were predicted to be CYP2D6 inhibitors. The hepatotoxicity
prediction of such compounds revealed that all the new
Table
3
Physicochemical properties of the newly synthesized
compounds and ligand shared the drawback of unwanted
hepatotoxic effects except compound 9a, which is anticipated
to be safer than the ligand (GRL0617). The plasma protein
binding model predicts whether a compound is likely to be
highly bound (Z90% bound) to the carrier proteins in the
blood.50 All the compounds and ligand are expected to bind
the plasma proteins more than 90% (Fig. 12).
2.3.5. Toxicity studies. The prediction of toxicity was
carried out in the Discovery Studio 2.5 software based on the
validated and assembled models as follows.51,52 (i) FDA rat
carcinogenicity test, which calculates a substance’s potential of
becoming a carcinogen. (ii) Carcinogenic potency TD50, which
predicts the rate of the tumorigenic doses 50 (TD50) of a drug in
compounds
Lipinski’s rule of 5
Veber’s rule
Violation of Number
Lipinski’s of rotatable
Compound Log P Mole. wt HBD HBA rule
bonds
TPSA
8a
8b
8c
8d
9a
9b
10a
10b
4.069 334.32
4.052 364.35
4.733 368.77
4.555 348.35
4.351 340.37
3.821 314.33
4.076 348.35
4.397 362.37
1
1
1
1
1
1
1
1
2
4
5
4
4
4
4
4
4
2
0
0
0
0
0
0
0
0
0
5
6
5
5
5
7
6
7
3
84.15
93.38
84.15
84.15
84.15
84.15
84.15
84.15
55.12
GRL0617 3.647 304.38
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Table 4 Computed ADMET for the designed compounds and the reference ligand
Comp. no.
BBB levela
Absorption levelb
Solubility levelf
CYP2D6d
Hepatotoxicity probabilityc
PPBe
8a
8b
8c
8d
9a
9b
10a
10b
2
2
1
2
2
2
2
2
1
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
0.986
0.980
0.973
0.960
0.331
0.807
0.960
0.834
0.768
1
1
1
1
1
2
2
2
2
GRL0617
a
b
BBB level, blood brain barrier level, 0 = very high, 1 = high, 2 = medium, 3 = low, 4 = very low. Absorption level, 0 = good, 1 = moderate, 2 = poor,
c
d
3 = very poor. Solubility level, 0 = extremely low, 1 = very low, 2 = low, 3 = good, 4 = optimal. CYP2D6, cytochrome P2D6, 0 = non inhibitor, 1 =
e
f
inhibitor. Hepatotoxicity probability, value 4 0.5 means toxic, value o 0.5 means non-toxic. PBB, plasma protein binding, 0 means less than
90%, 1 means more than 90%, 2 means more than 95%.
non-carcinogens except compound 8b and the co-crystalized
ligand were predicted to be carcinogens. For carcinogenic
potency TD50 rat model, all the compounds showed TD50 values
ranging from 1.29 to 36.63 mg per kg body weight dayÀ1, which
are higher than the co-crystalized ligand (7.02 mg per kg body
weight dayÀ1) except compounds 8b and 9a, which showed
TD50 values of 2.27 and 1.29 mg per kg body weight dayÀ1
,
respectively, which are lower than that of the co-crystalized
ligand. Regarding the rat maximum tolerated dose model, all
the compounds revealed maximum tolerated dose with a range
of 0.045 to 0.097 g per kg body weight lower than that of the
co-crystalized ligand (2.54 g per kg body weight), except
compounds 8c and 9b, which demonstrated maximum tolerated
Fig. 12 The expected ADMET study of the tested compounds and the
reference ligand.
a rat chronic exposure toxicity test of carcinogenic potency.53 dose of 0.125 and 0.183 g per kg body weight, which is higher
(iii) Rat maximum tolerated dose (MTD).54,55 (iv) Rat oral LD50, than that of the co-crystalized ligand. With respect to the rat
which predicts the rat oral acute median lethal dose (LD50) of a oral LD50 model, compounds 8d showed oral LD50 value of
chemical.56 (v) Rat chronic LOAEL, which predicts the rat (1.14 mg per kg body weight dayÀ1) that the higher than co-
chronic lowest observed adverse effect level (LOAEL) crystalized ligand (0.85 mg per kg body weight dayÀ1), while the
value.57,58 (vi) Ocular irritancy, which predicts whether a other compounds revealed oral LD50 values ranging from 0.13
particular compound is likely to be an ocular irritant and to 0.64 mg per kg body weight dayÀ1), which is lower than that
how severe the irritation is in the Draize test.59 (vii) Skin of the co-crystalized ligand. For the rat chronic LOAEL model,
irritancy, which predicts whether a particular compound is the compounds showed LOAEL values ranging from 0.003 to
likely to be a skin irritant and how severe it is in a rabbit skin 0.025 g per kg body weight. These values are lower than the
irritancy test.59
co-crystalized ligand (0.124 g per kg body weight). Moreover, all
As shown in Table 5, most compounds demonstrated low the compounds and GRL0617 were predicted to be mild
toxicity against the tested models in silico. FDA rat carcinogenicity irritants against the ocular irritancy model. On the other
model indicated that all of the tested compounds are hand, the tested compounds and ligand were predicted to be
Table 5 Toxicity properties of the prepared compounds
Carcinogenic
Rat maximum
Rat chronic
FDA rat carcinogenicity potency TD50 (rat)a tolerated dose (feed)b Rat oral LD50 LOAELb
Ocular irritancy Skin irritancy
b
Comp.
8a
8b
8c
8d
9a
9b
10a
10b
Non-carcinogen
Carcinogen
36.63
2.27
7.90
20.93
1.29
32.09
17.19
34.31
7.02
0.086
0.045
0.125
0.051
0.075
0.183
0.045
0.097
0.038
0.41
0.52
0.64
1.14
0.13
0.41
0.43
0.35
0.85
0.015
0.015
0.019
0.021
0.025
0.017
0.003
0.008
0.124
Mild-irritant
Mild-irritant
Mild-irritant
Mild-irritant
Mild-irritant
Mild-irritant
Mild-irritant
Mild-irritant
Mild-irritant
Non-irritant
Non-irritant
Non-irritant
Non-irritant
Mild -irritant
Mild -irritant
Mild -irritant
Non-irritant
Non-irritant
Non-carcinogen
Non-carcinogen
Non-carcinogen
Non-carcinogen
Non-carcinogen
Non-carcinogen
GRL0617 Carcinogen
a
b
Unit: mg per kg body weight dayÀ1
.
Unit: g per kg body weight.
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non-irritant against the skin irritancy model, except com- 7.2, 1.1 Hz, 1H, Ar–H), 7.76–7.81 (m, 3H, Ar–H), 8.01 (d, J =
pounds 9a and 9b were anticipated to be mild irritants.
8.8 Hz, 2H, Ar–H), 8.13 (dd, J = 8.2, 1.6 Hz, 1H, Ar–H), 10.23 (s,
1H, NH); 13C NMR (DMSO-d6, 100 MHz) d (ppm): 117.78,
120.86, 122.91, 124.15, 125.90, 126.34, 129.08, 130.54, 130.95,
135.86, 139.58, 142.12, 148.44, 159.26, 165.14.
3. Conclusion
In an attempt to find promising antiviral agents, eight deriva-
tives of 4-(2-nitrophenoxy)benzamide were designed and
synthesized in line with the DUB inhibitors’ identified features.
The synthesized derivatives showed significant results against
Adenovirus, HSV-1, and coxsackievirus with IC50 ranging from
10.22 to 44.68 mM. Hence, this work suggests that 4-(2-
nitrophenoxy)benzamide can serve as a scaffold for the future
development of effective antiviral agents. Moreover, these new
derivatives should be considered for further evaluation in the
way of discovery of new antiviral drugs. Despite the new
derivatives showing non-significant activity against SARS-CoV-
2, compound 8a emerged as the best candidate. Hence, it can
serve as a lead for the development of more effective agents for
combating COVID-19.
4.1.2.2. N-(4-Methoxyphenyl)-4-(2-nitrophenoxy)benzamide (8b).
Gray solid (yield 84.82%); m.p. = 207 1C; 1H NMR (DMSO-d6, 400
MHz) d (ppm): 3.75 (s, 3H, CH3), 6.92 (d, J = 9.0 Hz, 2H, Ar–H),
7.14 (d, J = 8.8 Hz, 2H, Ar–H), 7.31 (dd, J = 8.3, 0.9 Hz, 1H, Ar–H),
7.47 (ddd, J = 8.3, 8.3, 1.1 Hz, 1H, Ar–H), 7.65 (d, J = 9.0 Hz, 2H,
Ar–H), 7.78 (ddd, J = 8.4, 8.4, 1.6 Hz, 1H, Ar–H), 8.00 (d, J =
8.8 Hz, 2H, Ar–H), 8.12 (dd, J = 8.2, 1.5 Hz, 1H, Ar–H), 10.11 (s,
1H, NH); 13C NMR (DMSO-d6, 400 MHz) d (ppm): 55.63, 114.20,
117.80, 122.47, 122.82, 125.81, 126.33, 130.40, 131.07, 132.66,
135.82, 142.10, 148.51, 156.02, 159.09, 164.68.
4.1.2.3. N-(4-Chlorophenyl)-4-(2-nitrophenoxy)benzamide (8c).
1
Gray solid (yield 82.53%); m.p. = 167 1C; H NMR (DMSO-d6,
400 MHz) d (ppm): 7.16 (d, J = 8.8 Hz, 2H, Ar–H), 7.33(dd, J = 8.3,
0.9 Hz, 1H, Ar–H), 7.40 (d, J = 8.8 Hz, 2H, Ar–H), 7.48 (ddd, J =
8.3, 7.2, 1.1 Hz, 1H, Ar–H), 7.77–7.82 (m, 3H, Ar–H), 8.00 (d, J =
8.8 Hz, 2H, Ar–H), 8.13 (dd, J = 8.2, 1.5 Hz, 1H, Ar–H), 10.36 (s,
1H, NH); 13C NMR (DMSO-d6, 400 MHz) d (ppm): 117.78,
122.32, 122.96, 125.94, 126.36, 127.72, 128.98, 130.59, 130.65,
135.86, 138.61, 142.14, 148.39, 159.40, 165.21.
4. Experimental
4.1. Chemistry
All melting points were obtained by the open capillary method
on a Gallen lamp Melting point apparatus and were uncorrected.
1H NMR and 13C NMR spectra were recorded on a BRUKER
400 MHZ-NMR spectrophotometer. TMS was used as an internal
standard in deuterated DMSO, and the chemical shifts were
measured in d ppm.
4.1.1. General method for the synthesis of 4-(2-nitro-
phenoxy)benzoic acid (7). Equimolar amounts of 1-fluoro-2-
nitrobenzene 5 and 4-hydroxybenzoic acid 6 were refluxed
in isopropyl alcohol for 8 h. The obtained precipitate was
collected, dried, and crystallized from ethanol to afford
compound 7.
4.1.2.4. 4-(2-Nitrophenoxy)-N-(p-tolyl)benzamide (8d). White
solid (yield 83.46%); m.p. = 200 1C; 1H NMR (DMSO-d6,
400 MHz) d (ppm): 2.28 (s, 3H, CH3), 7.15–7.17 (m, 4H, Ar–H),
7.31 (dd, J = 8.3, 1.0 Hz, 1H, Ar–H), 7.47 (ddd, J = 8.3, 7.2, 1.1 Hz,
1H, Ar–H), 7.63 (d, J = 8.4 Hz, 2H, Ar–H), 7.78 (ddd, J = 8.4, 6.8,
1.6 Hz, 1H, Ar–H), 8.00 (d, J = 8.8 Hz, 2H, Ar–H), 8.12 (dd, J = 8.2,
1.6 Hz, 1H, Ar–H), 10.15 (s, 1H, NH); 13C NMR (DMSO-d6,
400 MHz) d (ppm): 20.95, 117.79, 120.88, 122.85, 125.84,
126.33, 129.45, 130.47, 131.06, 133.07, 135.83, 137.08, 142.11,
148.49, 159.16, 164.90.
Yellow solid (yield 81.35%); m.p. = 163 1C; 1H NMR (DMSO-
d6, 400 MHz) d (ppm): 7.08 (d, J = 8.8 Hz, 2H, Ar–H), 7.34 (dd, J =
8.2, 0.8 Hz, 1H, Ar–H), 7.48 (ddd, J = 8.4, 8.4, 1.0 Hz, 1H, Ar–H),
7.78 (ddd, J = 8.5, 8.5, 1.6 Hz, 1H, Ar–H), 7.97 (d, J = 8.8 Hz, 2H,
Ar–H), 8.12 (dd, J = 8.2, 1.5 Hz, 1H, Ar–H), 12.93 (s, 1H, COOH);
13C NMR (DMSO-d6, 100 MHz) d (ppm): 117.66, 123.30, 126.12,
126.34, 126.69, 132.22, 135.88, 142.19, 148.08, 160.42, 167.06.
4.1.2. General method for the synthesis of the final
compounds (8a–d, 9a,b, and 10a,b). Ethyl chloroformate (0.076 mL)
was added dropwise to a mixture of 4-(2-nitrophenoxy)benzoic acid
(7) (0.20 g) and Et3N (0.123 mL) in DCM. The reaction mixture was
stirred for 1 h in an ice salt bath. Then, an equimolar amount of an
appropriate amine was added, and the reaction mixture was
allowed to stir at r.t. for a further 1 h. The obtained precipitate
was filtered off, and washed with water and methanol to furnish
the corresponding benzamide derivative.
4.1.2.5. N-Cyclohexyl-4-(2-nitrophenoxy)benzamide (9a). White
solid (yield 80.74%); m.p. = 165 1C; 1H NMR (DMSO-d6,
400 MHz) d (ppm): 1.11–1.82 (m, 10H, 5CH2 cyclohexyl), 3.75
(m, 1H, CH cyclohexyl), 7.08 (d, J = 8.8 Hz, 2H, Ar–H), 7.25 (dd,
J = 8.3, 1.0 Hz, 1H, Ar–H), 7.44 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H,
Ar–H), 7.75 (ddd, J = 8.4, 7.4, 1.6 Hz, 1H, Ar–H), 7.89 (d, J =
8.8 Hz, 2H, Ar–H), 8.10 (dd, J = 8.2, 1.6 Hz, 1H, Ar–H), 8.17 (d, J =
7.9 Hz, 1H, NH); 13C NMR (DMSO-d6, 400 MHz) d (ppm): 25.41,
25.72, 32.88, 48.85, 117.80, 122.47, 125.57, 126.26, 130.06,
131.06, 135.74, 141.97, 148.71, 158.60, 164.97.
4.1.2.6. N-Butyl-4-(2-nitrophenoxy)benzamide (9b). White
solid (yield 73.15%); m.p. = 134 1C; 1H NMR (DMSO-d6,
400 MHz) d (ppm): 0.90 (t, J = 7.4 Hz, 3H, CH3), 1.31 (m, 2H,
CH2CH3), 1.50 (m, 2H, CH2CH2CH3), 3.25 (dt, J = 6.9 Hz, 2H,
À ÀÀ
CH2NH), 7.08 (d, J = 8.8 Hz, 2H, Ar–H), 7.26 (dd, J = 8.3, 0.7 Hz,
4.1.2.1. 4-(2-Nitrophenoxy)-N-phenylbenzamide (8a). White 1H, Ar–H), 7.44 (ddd, J = 8.3, 8.3, 0.9 Hz, 1H, Ar–H), 7.75 (ddd,
solid (yield 84.75%); m.p. = 195 1C; 1H NMR (DMSO-d6, J = 8.8, 8.8, 1.5 Hz, 1H, Ar–H), 7.88 (d, J = 8.7 Hz, 2H, Ar–H),
400 MHz) d (ppm): 7.10 (t, J = 7.4 Hz, 1H, Ar–H), 7.15 (d, J = 8.10 (dd, J = 8.2, 1.5 Hz, 1H, Ar–H), 8.42 (t, J = 5.5 Hz, 1H, NH);
8.8 Hz, 2H, Ar–H), 7.32–7.37 (m, 3H, Ar–H), 7.48 (ddd, J = 8.4, 13C NMR (DMSO-d6, 400 MHz) d (ppm): 14.17, 20.12, 31.73,
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39.34, 117.82, 122.58, 125.60, 126.25, 129.91, 130.91, 135.73, dissolved using DMSO (200 mL). A multi-well plate reader was
142.02, 148.67, 158.68, 165.67.
used to measure the absorbance of formazan solutions at lmax
of 540 nm with 620 nm as the reference wavelength. The
cytotoxicity percentage for each tested compound was calculated
relative to the untreated cells. Finally, Graph Pad Prism 5 (Graph
Pad Software Inc., San Diego, CA, USA) was used to plot the
%cytotoxicity curve of each tested candidate, and the half-
maximal cytotoxic concentration (CC50) was extracted from the
linear equation.
4.1.2.7. N-Benzyl-4-(2-nitrophenoxy)benzamide (10a). White
solid (yield 81.85%); m.p. = 183 1C; 1H NMR (DMSO-d6,
400 MHz) d (ppm): 4.48 (d, J = 6.0 Hz, 2H, CH2NH), 7.11 (d,
J = 8.8 Hz, 2H, Ar–H), 7.22–7.33 (m, 6H, Ar–H), 7.45 (ddd, J = 8.3,
8.3, 1.1 Hz, 1H, Ar–H), 7.76 (ddd, J = 8.4, 8.4, 1.6 Hz, 1H, Ar–H),
7.96 (d, J = 8.8 Hz, 2H, Ar–H), 8.11 (dd, J = 8.2, 1.5 Hz, 1H, Ar–H),
9.05 (t, J = 6.0 Hz, 1H, NH); 13C NMR (DMSO-d6, 400 MHz) d
(ppm): 43.10, 117.85, 122.73, 125.70, 126.28, 127.19, 127.65,
128.74, 130.08, 130.45, 135.77, 140.15, 142.06, 148.58, 158.96,
165.82.
4.2.1.3. Plaque reduction assay. Vero HSV-1cells were seeded
in six-well culture plates (105 cells/ml) at 37 1C and 5% CO2 for
24 h. Previously titrated hCoV-19/Egypt/NRC-03/2020 SARS-
CoV-2, HSV-1, Adeno-7, and CB-4 viruses were diluted to
optimal dilution (10À4) to allow countable plaques and then
mixed with a non-cytotoxic concentration (NCTC) of each tested
derivative. The mixture of the tested derivative/virus was kept at
37 1C for 1 h before being added to the previously mentioned
cells after the removal of the growth medium and then the
virus-compound mixtures were inoculated twice. 3 mL of
DMEM with 2% agarose, 1% antibiotic antimycotic mixture,
and 2% FBS were added to the cell monolayer after 1 h of
contact for the adsorption of the viruses. The plates were kept
for 4 days at 37 1C to solidify and 1 mL of formalin (10%)
was added to each well for 2 h for cell fixation and virus
inactivation. The removal of the overlayer by water and the
fixed cells were stained with 1% crystal violet (1 mL) for 10 min
for the visualization of the plaques. The dye was discarded and
the plates were washed with water and dried. Also, a control
was used for each tested virus by the inoculation of the
untreated virus in each plate. The viral inhibition curve% of
each tested candidate against adenovirus, HSV-1, coxsackie-
virus, and SARS-CoV-2 were plotted using Graph Pad Prism
5 and the IC50 values (50% inhibitory concentrations) were
calculated from the non-linear regression curve-fit analysis.
4.1.2.8. 4-(2-Nitrophenoxy)-N-phenethylbenzamide (10b). White
solid (yield 79.35%); m.p. = 174 1C; 1H NMR (DMSO-d6,
400 MHz) d (ppm): 2.85 (t, J = 7.2 Hz, 2H, CH2CH2NH), 3.47
(dt, J = 7.2, 6.2 Hz, 2H, CH2NH), 7.02 (d, J = 8.7 Hz, 2H, Ar–H),
7.18–7.32 (m, 6H, Ar–H), 7.45 (ddd, J = 8.2, 7.3, 1.0 Hz, 1H,
Ar–H), 7.76 (ddd, J = 8.5, 6.9, 1.6 Hz, 1H, Ar–H), 7.87 (d, J =
8.8 Hz, 2H, Ar–H), 8.10 (dd, J = 8.2, 1.5 Hz, 1H, Ar–H), 8.57 (t, J =
5.4 Hz,1H, NH); 13C NMR (DMSO-d6, 400 MHz) d (ppm): 35.58,
41.37, 117.81, 122.69, 125.68, 126.27, 126.54, 128.80, 129.12,
129.93, 130.73, 135.78, 140.01, 142.05, 148.60, 158.80, 165.77.
À ÀÀ
4.2. Biological testing
4.2.1. In vitro virological studies60
4.2.1.1. Virus and cells. Dulbecco’s modified Eagle’s medium
(Lonza, Verviers, Belgium) enriched with fetal bovine serum
(10% Gibco, New York, NY, USA) and antibiotic antimycotic
mixture (1% Lonza) was used to cultivate Vero cells. Then, the
aforementioned cells were kept at 5% CO2 and 37 1C. Vero cells
were used to propagate an hCoV-19/Egypt/NRC-3/2020 SARS-
CoV-2 virus (Accession Number on GSAID: EPI_ISL_430820),
HSV-1 (human herpes simplex virus type 1), Adeno-7 (Adeno-
virus type 7), and CB-4 (Coxsackie virus type B4), respectively,
which were harvested after the appearance of the cytopathic
effects (CPE). Finally, the viral stocks were titrated by the
median tissue culture infectious dose (TCID50) using both
the MTT method and the plaque infectivity assay, and stored
at À80 1C until further use.
4.3. In silico studies
4.3.1. Docking studies. The crystal structures of the targeted
protein were downloaded from the Protein Data Bank, http://
www.rcsb.org/pdb (PDB ID: 3E9S, resolution: 2.01 Å) for PLpro,
(PDB ID: 4PID, resolution: 1.59 Å) for adenovirus, (PDB ID: 4TTI,
4.2.1.2. In vitro cytotoxicity assay. Cytotoxicity assays for the resolution: 1.89 Å) for HSV-1, and (PDB ID: IZ8R, resolution:
newly synthesized derivatives (8a–10b) in the Vero cells were 1.95 Å) for coxsackievirus. Discovery Studio 2.5 software was used
examined in vitro using the MTT method61,62 with minor in the docking studies. At first, the targeted molecules were
modifications. On the first day, Vero cells were cultured for prepared by the removal of the water molecules. Then, the
24 h at 37 1C and 5% CO2 in 96-well plates with a cell density protein structures were subjected to energy minimization by
E1 Â 104 per well. To determine the antiviral response for each applying CHARMm force fields for charge and MMFF94 force
derivative, fresh DMEM media containing 100 TCID50/0.1 mL field for partial charge.63–66 The binding sites of the proteins
from each virus was substituted with FBS-containing media were defined and prepared for docking. The reference ligand and
after washing the cells three times with phosphate-buffered the designed compounds 2D structures were sketched using
saline (PBS). After incubation for 2 h, the prepared cells were ChemBioDraw Ultra 14.0 and saved in the MDL-SD file format.
washed with 1Â PBS and treated with different concentrations Next, the SD file was opened, 3D structures were protonated, and
(2.5–160 mg mLÀ1) of the tested compounds (8a–10b) in triplicate. energy was minimized by applying CHARMm force fields for
After 72 h of incubation, 20 mL of 5 mg mLÀ1 stock solution charge and MMFF94 force field for partial charge. The docking
(MTT solution) was added for each well and incubated for a process was achieved using the CDOCKER-CHARMm-based
further 4 h at 37 1C. Then, the formed formazan crystals were technique. A maximum of 10 conformers was considered for
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respiratory syndrome coronavirus has deubiquitinating
activity, J. Virol., 2005, 79(24), 15189–15198.
each molecule in the docking analysis. Next, the docking scores
of the best-fitted conformer of each molecule with the amino
acids at the binding pocket were recorded.67–72
11 R. Alnajjar, A. Mostafa, A. Kandeil and A. A. Al-Karmalawy,
Molecular docking, molecular dynamics, and in vitro
studies reveal the potential of angiotensin II receptor
blockers to inhibit the COVID-19 main protease, Heliyon,
2020, 6(12), e05641.
12 A. A. Al-Karmalawy and I. H. Eissa, Molecular docking and
dynamics simulations reveal the potential of anti-HCV
drugs to inhibit COVID-19 main protease, Pharm Sci, 2021,
10.
13 M. Y. Balakirev, M. Jaquinod, A. L. Haas and J. Chroboczek,
Deubiquitinating function of adenovirus proteinase,
J. Virol., 2002, 76(12), 6323–6331.
14 L. M. Kattenhorn, G. A. Korbel, B. M. Kessler, E. Spooner
and H. L. Ploegh, A deubiquitinating enzyme encoded by
HSV-1 belongs to a family of cysteine proteases that is
conserved across the family Herpesviridae, Mol. Cell, 2005,
19(4), 547–557.
4.3.2. ADMET studies. ADMET descriptors (absorption,
distribution, metabolism, excretion, and toxicity) of the tested
candidates were determined using Discovery studio 2.5.
Initially, the CHARMM force field was utilized and the prepared
compounds were minimized according to the preparation
of small molecule protocol. Then, the ADMET descriptors
protocol was used to accomplish these studies.65,68,73,74
4.3.3. Toxicity studies. The toxicity parameters of the
synthesized compounds were computed using Discovery studio
2.5. In the beginning, the CHARMM force field was applied
then the prepared compounds were minimized according to
the preparation of a small molecule protocol. Then, different
parameters were calculated from the toxicity prediction
(extensible) protocol.75
Conflicts of interest
15 B. H. Harcourt, D. Jukneliene, A. Kanjanahaluethai,
J. Bechill, K. M. Severson, C. M. Smith, P. A. Rota and
S. C. Baker, Identification of severe acute respiratory syn-
drome coronavirus replicase products and characterization
of papain-like protease activity, J. Virol., 2004, 78(24),
13600–13612.
16 R. Yu, L. Chen, R. Lan, R. Shen and P. Li, Computational
screening of antagonists against the SARS-CoV-2 (COVID-
19) coronavirus by molecular docking, Int. J. Antimicrob.
Agents, 2020, 56(2), 106012.
17 I. Eissa, A. Al-Karmalawy, M. A. Dahab, A. M. Metwaly,
S. S. Elhady, E. B. Elkaeed and K. M. Darwish, Molecular
docking and dynamics simulation revealed the potential
inhibitory activity of ACEIs against SARS-CoV-2 targeting
hACE2 receptor, Front. Chem., 2021, 9, 227.
18 A. A. Elmaaty, R. Alnajjar, M. I. Hamed, M. Khattab,
M. M. Khalifa and A. A. Al-Karmalawy, Revisiting activity
of some glucocorticoids as a potential inhibitor of SARS-
CoV-2 main protease: theoretical study, RSC Adv., 2021,
11(17), 10027–10042.
This work was funded by the authors and there is no conflict of
interest.
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